Synthesis of radiopharmaceutical agents

Abstract

Synthesis of radiopharmaceutical agents It is disclosed a method to synthesize a compound of formula (I), or a pharmaceutically acceptable salt thereof: Formula (I), (I) said process comprising contacting a trialkylstannane precursor of formula (II), or a pharmaceutically acceptable salt thereof: Formula (II), (II) with an alkali metal salt of a radionuclide selected from the group consisting of 123I, 124I, 125I, 131I, 211At, in appropriate acidic conditions and in presence of a peroxide ROOH in an 10 appropriate concentration. Said method is particularly suited for synthesizing [131I]N-(2- diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide, providing a notable reduction in the quantity of side products, allowing a much purer finished product, while making use of reagents which are adapted to the constraints which need to be met when developing a product for clinical use and, more in particular, to the constraints to be met in order to 15 adapt the process for being fully automated.

Description

DescriptionTitle: Synthesis of radiopharmaceutical agents

[0001] The present invention describes a method to synthesize a radiopharmaceutical agent for the internal radiotherapeutic treatment of melanoma.Technical Field

[0002] This disclosure pertains to the field of pharmaceutical chemistry and more in particular refers to the synthesis of radiopharmaceutical compounds comprising an aromatic nitrogen containing core.Background Art

[0003] Despite advances in treatment, malignant melanoma remains a major public health problem in many countries. It displays a strong tendency to metastasize, and patients with metastases at stage IV have a five-year survival rate of only 32%. There is therefore an urgent need to find effective ways to diagnose and treat disseminated melanoma.

[0004] Radiopharmaceutical products have been of particular interest since make possible, on one hand, the detection of the product in the context of diagnosis, and in addition they constitute an active agent for therapeutic use.

[0005] Benzamide-based radiopharmaceutical products targeting melanin were first developed for imaging melanoma and then for therapeutic purposes with targeted radionuclide therapy.

[0006] In that connection, a family of halobenzamide derived compounds has been developed for use in diagnostic and therapeutic methods for the detection and treatment of melanoma. In particular, said halobenzamide derived compounds are (hetero)aromatic analogues of / \ / -(2-diethylaminoethyl)iodobenzamide which feature a bi- or tricyclic aromatic nucleus including a benzene ring fused with a heteroaryl ring comprising one or more nitrogen atoms. (Chezal et al., 2008, J Med Chem, W02008012782A2, W02009095872A2). Among those compounds, [131l] / V-(2-diethylaminoethyl)-6- iodoquinoxaline-2-carboxamide (Ia1) presents a highly favorable pharmacokinetic profile in vivo for therapy.

[0007] However, the methods disclosed until now for many of said radiopharmaceutical products have limitations. Indeed, the known synthetic methods may lead to the formation of side products or may require the use of reagents not suitable for the pharmaceutical constraints which need to be met when developing a product for clinical use. Indeed, evenif some methods have been disclosed for the synthesis of this family of compounds, and in particular for [131l] / V-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide, there is no guidance on how to provide a method which is best adapted for automated procedures.

[0008] Indeed, the synthetic methods of these radiopharmaceutical products need to be adapted to be implemented in fully automated procedures, such as those using cassettebased automated synthesizers, used in radiopharmaceutical compagnies and nuclear medicine departments in the context of theragnostic procedures. Accordingly, there is still a need for synthetic methods which provide the desired radiopharmaceutical product in high yields and with a high degree of chemical and radiochemical purities, while avoiding the presence of side products in the final product and, at the same time, making use of reagents which are adequate for clinical use.Summary

[0009] The invention relates to a method to obtain a compound of formula (I)comprising a radionuclide R3 selected from the group consisting of123l,124l,125l,131l,211At, with remarkable results compared to the synthesis described in the prior art.

[0010] In particular, this method allows obtaining [131l] / V-(2-diethylaminoethyl)-6- iodoquinoxaline-2-carboxamide (Ia1), as well as other iodobenzamide derivatives, with remarkable results compared to the synthesis described in the prior art: providing a notable reduction (of more than 98%) in the quantity of some nonradioactive side products, thus allowing a much purer finished product (more than 99% of radiochemical purity), while making use of reagents which are adapted to the constraints which need to be met when developing a product for clinical use and, more in particular, to the constraints to be met in order to adapt the process for being fully automated.

[0011] One aspect of the present invention relates, therefore, to a process to synthesize a compound of formula (I), or a pharmaceutically acceptable salt thereof:said process comprising: contacting a trialkylstannane precursor of formula (II), or a pharmaceutically acceptable salt thereof:with an alkali metal salt of a radionuclide selected from the group consisting of123l,124l, 125| 131| 2n^tjn anappropriate solvent at a pH lower than 1 , and in presence of a peroxide ROOH, and wherein the molar concentration of the peroxide ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1and the molar amount of the trialkylstannane precursor of formula (II) is between 0.2 nmol.GBq'1and 10.0 pmol.GBq'1; whereinm is an integer varying from 2 to 4; R1 and R2 are each, independently of one another, a hydrogen atom, a (Ci-C6)alkyl group or a (C2-Ce)alkenyl group;R3 is a radionuclide selected from the group consisting of123l,124l,125l,131l,211At;R4, Rs and Re are each, independently of one another a (Ci-Ce)alkyl group; andR is H or Ry-C(O)- and R? is C1-6 alkyl.

[0012] As shown in the examples of the present application (see table 7), the selection of an appropriate acid and maintaining a pH lower than 1 are key factors allowing obtaining the desired product with improved radiochemical yield from a stannane precursor of formula (II). The inventors of the present application have shown that the development of an automated synthetic method with the use of a stannane precursor was possible, eliminating stannane compounds from the final formulation, without the use of strong oxidants. In that regard, the synthetic conditions may impact the presence and quantity of unwanted side products (overoxidation of the precursor, competing reactions, etc.) which would significantly affect the development of an automated synthesis for clinical purposes. The inventors were able to show that the selection of a peroxide as the oxidant allows obtaining the desired product with high (radio)chemical purity while avoiding the formation of side products in a very effective manner (see tables 8, 9, 10 and 12). On the other hand, other known oxidation conditions (such as the use of chloramine-T) unexpectedly resulted in the formation of very significant amounts of unwanted side products, making said oxidation conditions unsuitable for automated purposes. Moreover, the inventors also identified that the selection of the amount of peroxide is also a key factor in obtaining a high chemical purity, since it allows for an effective oxidation, with good conversion yields while, at the same time avoiding overoxidation of the aromatic core which would result in the presence of additional side products and, thus, be prejudicial to the chemical purity of the desired product. Finally, the selection of the amount of the stannane precursor in relation to the amount of the radionuclide allows to obtain the final product with an excellent radiochemical purity while allowing its implementation in an automated process avoiding a surplus of starting materials at the end of the process, which would result in purification issues.

[0013] One aspect of the present invention relates to a process to synthesize compound of formula (I) as described above herein, whereinare each ethyl; R3 is1311, R4, Rs and Re are each butyl, the compound of formula (I) being [131l] / V-(2-diethylaminoethyl)-6- iodoquinoxaline-2-carboxamide has a formula (Ia1):the trialkylstannane precursor of formula (II) is a tributylstannane of formula (I Ia1 ):and wherein said process comprises contacting the tributylstannane precursor of formula (Ila1), or a pharmaceutically acceptable salt thereof, with an alkali metal salt of1311, in an appropriate solvent at a pH lower than 1 , and in presence of a peroxide ROOH, wherein R is H or R?-C(O)- and R? is C1-6 alkyl, preferably wherein R is H; and wherein the molar concentration of the peroxide ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1, preferably wherein R is H and wherein the molar concentration of H2O2 is between 0.015 mmol.mL'1and 1.0 mmol.mL'1; and the molar amount of the tributylstannane precursor of formula (I Ia1 ) is between 0.002 pmol.GBq'1and 10.0 pmol.GBq'1.

[0014] In some aspects, the processes disclosed in the present invention further comprise a subsequent step of adding a reducing agent in the presence of a base.Brief Description of the Figures

[0015] Figure 1 : Order of radiosynthesis steps used for automation production of the compound of formula (Ia1).Detailed description

[0016] The present invention refers to a process to synthesize radiopharmaceutical compounds comprising a nitrogen-containing heteroaromatic core which allows obtainingsaid compounds with remarkable (radio)chemical purity and using ingredients which are well adapted for the implementation of said process in a fully automated procedure, such as a cassette- based automated synthesizer, for use in radiopharmaceutical companies and nuclear medicine departments in the context of theranostic procedures.

[0017] In particular the present invention discloses a process to synthesize a compound of formula (I), or a pharmaceutically acceptable salt thereof:said process comprising: contacting a trialkylstannane precursor of formula (II), or a pharmaceutically acceptable salt thereof:with an alkali metal salt of a radionuclide selected from the group consisting of123l,124l, 125| 131| 2n^tjn anappropriate solvent at a pH lower than 1 , and in presence of a peroxide ROOH, and wherein the molar concentration of ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1; and the molar amount of the trialkylstannane precursor of formula (II) is between 0.2 nmol.GBq'1and 10.0 pmol.GBq'1; whereinm is an integer varying from 2 to 4;Ri and R2 are each, independently of one another, a hydrogen atom, a (Ci-Ce)alkyl group or a (C2-Ce)alkenyl group;R3 is a radionuclide selected from the group consisting of123l,124l,125l,131l,211At;R4, Rs and Re are each, independently of one another a (Ci-Ce)alkyl group; and R is H or R?-C(O)- and R? is C1-6 alkyl.

[0018] In a preferred embodiment Ar is. In said preferred embodiment, the compound of formula (I) has formula (la), or a pharmaceutically acceptable salt thereof:the trialkylstannane precursor of formula (II) has formula (Ila), or a pharmaceutically acceptable salt thereof:and the process according to the present invention comprises: contacting the trialkylstannane precursor of formula (Ila), or a pharmaceutically acceptable salt thereof: with an alkali metal salt of a radionuclide selected from the group consisting of123l,124l, 125| 131 | 211^ jn anappropriate solvent at a pH lower than 1 , and in presence of a peroxide ROOH, andwherein the molar concentration of ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1and the molar amount of the trialkylstannane precursor of formula (Ila) is between 0.2 nmol.GBq'1and 10.0 pmol.GBq'1; and wherein m, R, R1, R2, R3, R4, Rs, Rs and R? are as defined in the claims and in the present description.

[0019] The term “alkyl” refers to a monovalent or divalent, linear or branched, saturated hydrocarbon chain, comprising 1-6 carbon atoms (also named (Ci-e)alkyl), such as methyl, ethyl, propyl, isopropyl, n-butyl, / so-butyl, sec-butyl and tert-butyl. In some aspects of the invention, m=2.

[0020] The term “alkenyl” refers to a monovalent radical of a linear or branched hydrocarbon chain comprising 2 to 6 carbon atoms and at least one carbon-carbon double bond. Examples of C2-6 alkenyl groups include, among others, groups such as ethenyl, n- propenyl, / so-propenyl, n-butenyl, / so-butenyl, sec-butenyl or tert-butenyl.

[0021] In some aspects, R1 and R2 may be both a C1-6 alkyl, in particular R1 and R2 may be both selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, / so- butyl, sec-butyl and tert-butyl; and preferably R1 and R2 are both methyl or ethyl.

[0022] In some aspects of the invention, R3 is an iodine radionuclide selected from the group consisting of123l,124l,125l and1311, more preferably the radionuclide R3 is1311.

[0023] In one aspect, m is 2; R1 and R2 are each ethyl and R3 is131l, the compound of formula (I) has a formula (Ia1), or a pharmaceutically acceptable salt thereof:the trialkylstannane precursor of formula (II) has a formula (Ila’):and the process comprises:contacting a trialkylstannane precursor of formula (Ila’), or a pharmaceutically acceptable salt thereof with an alkali metal salt of1311, in an appropriate solvent at a pH lower than 1, and in presence of a peroxide ROOH; wherein the molar concentration of ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1, for example between 0.008 mmol.mL'1and 0.8 mmol.mL'1, preferably between 0.014 mmol.mL'1and 0.5 mmol.mL'1, more preferably between 0.020 mmol.mL'1and 0.3 mmol.mL'1; and the molar amount of the trialkylstannane precursor of formula (Ila) is between 0.002 pmol.GBq'1and 10.0 pmol.GBq'1, for example between 0.025 pmol.GBq'1and 5.0 pmol.GBq'1, preferably between 0.050 pmol.GBq'1and 2.5 pmol.GBq'1, more preferably between 0.070 pmol.GBq'1and 1.0 pmol.GBq'1; and wherein R, R4, Rs and Re are as defined in the claims and in the present description.

[0024] In some aspects of the invention, R4, Rs and Re are linear and branched alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, / so-butyl, sec-butyl, terf-butyl, tert-butyl-methyl. In a preferred embodiment of the invention, R4, Rs and Re are butyl, more preferably n- butyl.

[0025] In a particular aspect, m is 2; R1 and R2 are each ethyl; R3 is131l, R4, Rs and Re are each butyl; the compound of formula (I) has a formula (Ia1), or a pharmaceutically acceptable salt thereof:the trialkylstannane precursor of formula (II) has a formula (Ila1), or a pharmaceutically acceptable salt thereof:and the process comprises:contacting the tributylstannane precursor of formula (Ila1), or a pharmaceutically acceptable salt thereof, with an alkali metal salt of1311 in an appropriate solvent at a pH lower than 1 , and in presence of a peroxide ROOH, wherein R is H or R?-C(O)- and R? is C1-6 alkyl; and wherein the molar concentration of ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1, for example between 0.008 mmol.mL'1and 0.8 mmol.mL'1, preferably between 0.014 mmol.mL'1and 0.5 mmol.mL'1, more preferably between 0.020 mmol.mL'1and 0.3 mmol.mL'1; and the molar amount of the tributylstannane precursor of formula (I Ia1) is between 0.002 pmol.GBq'1and 10.0 pmol.GBq'1, for example between 0.025 pmol.GBq'1and 5.0 pmol.GBq'1, preferably between 0.050 pmol.GBq'1and 2.5 pmol.GBq'1, more preferably between 0.070 pmol.GBq'1and 1.0 pmol.GBq'1.

[0026] The pharmaceutically acceptable salts of the compounds of formulae (la), (Ia1), (I), (Ila), (Ila’), (I Ia1) and (II) include the addition salts with pharmaceutically acceptable acids, such as inorganic acids, for example hydrochloric, hydrobromic, phosphoric or sulphuric acid, and organic acids, such as acetic, trifluoroacetic, propionic, oxalic, succinic, fumaric, malic, tartaric, citric, ascorbic, maleic, glutamic, benzoic, toluenesulphonic, methanesulphonic, stearic and lactic acid.

[0027] In some aspects of the invention the pharmaceutically acceptable salt is a hydrochloride salt.

[0028] In addition, when the compounds of formulae (la), (Ia1), (I), (Ila), (Ila’), (Ila1) and (II) or their pharmaceutically acceptable salts can form solvates (namely hydrates); the invention may also include such solvates.

[0029] In some aspects of the invention, in the peroxide ROOH, R is H or R?-C(O)- wherein R? is C1-6 alkyl, preferably R is H and the peroxide ROOH is H2O2. In a preferred embodiment of the invention, the peroxide ROOH is H2O2. In some aspects of the invention, R? is a linear or a branched alkyl such as methyl, ethyl, propyl, isopropyl, n- butyl, / so-butyl, sec-butyl, tert-butyl, tert-butyl-methyl, preferably methyl.

[0030] In some aspects the molar concentration of ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1, for example between 0.008 mmol.mL'1and 0.8 mmol.mL'1, preferably between 0.014 mmol.mL'1and 0.5 mmol.mL'1, more preferably between 0.020 mmol.mL'1and 0.3 mmol.mL'1.

[0031] In some preferred aspects, ROOH is H2O2, and the molar concentration of H2O2 is between 0.015 mmol.mL'1and 1.0 mmol.mL'1, preferably between 0.018 mmol.mL'1and 0.5 mmol.mL'1, more preferably between 0.020 mmol.mL'1and 0.3 mmol.mL'1.

[0032] In some aspects, R3 is123l and the molar amount of the trialkylstannane precursor of formula (II), (Ila) or (Ila’), or of the tributylstannane of formula (Ila1), is between 0.3 nmol.GBq'1and 10.0 pmol.GBq'1, for example between 0.005 pmol.GBq'1and 5.0 pmol.GBq'1, preferably between 0.010 pmol.GBq'1and 2.5 pmol.GBq'1, more preferably between 0.030 pmol.GBq'1and 1.0 pmol.GBq'1.

[0033] In some aspects, R3 is124l and the molar amount of the trialkylstannane precursor of formula (II), (Ila) or (Ila’), or of the tributylstannane of formula (Ila1), is between 0.001 pmol.GBq'1and 10.0 pmol.GBq'1, for example between 0.005 pmol.GBq'1and 5.0 pmol.GBq'1, preferably between 0.010 pmol.GBq'1and 2.5 pmol.GBq'1, more preferably between 0.030 pmol.GBq'1and 1.0 pmol.GBq'1.

[0034] In some aspects, R3 is125l and the molar amount of the trialkylstannane precursor of formula (II), (Ila) or (Ila’), or of the tributylstannane of formula (Ila1), is between 0.013 pmol.GBq'1and 10.0 pmol.GBq'1, for example between 0.015 pmol.GBq'1and 5.0 pmol.GBq'1, preferably between 0.020 pmol.GBq'1and 2.5 pmol.GBq'1, more preferably between 0.030 pmol.GBq'1and 1.0 pmol.GBq'1.

[0035] In some aspects, R3 is1311 and the molar amount of the trialkylstannane precursor of formula (II), (Ila) or (Ila’), or of the tributylstannane of formula (Ila1), is between 0.002 pmol.GBq'1and 10.0 pmol.GBq'1, for example between 0.025 pmol.GBq'1and 5.0 pmol.GBq'1, preferably between 0.050 pmol.GBq'1and 2.5 pmol.GBq'1, more preferably between 0.070 pmol.GBq'1and 1.0 pmol.GBq'1.

[0036] In some aspects, R3 is211At and the molar amount of the trialkylstannane precursor of formula (II), (Ila) or (Ila’), or of the tributylstannane of formula (Ila1), is between 0.002 pmol.GBq'1and 10.0 pmol.GBq'1, for example between 0.010 pmol.GBq'1and 5.0 pmol.GBq'1, preferably between 0.020 pmol.GBq'1and 2.5 pmol.GBq'1, more preferably between 0.030 pmol.GBq'1and 1.0 pmol.GBq'1.

[0037] Thus, when ROOH is H2O2, m is 2; R1 and R2 are each ethyl; R3 is1311; R4, Rs and Re are each butyl, one particular aspect refers to the process to synthesize a compound of formula (Ia1), or a pharmaceutically acceptable salt thereof:said process comprising:contacting a tributylstannane precursor of formula (I Ia1 ) , or a pharmaceutically acceptable salt thereof:with an alkali metal salt of1311 in an appropriate solvent at a pH lower than 1, and in presence of H2O2, and wherein the molar concentration of H2O2 is between 0.015 mmol.mL'1and 1.0 mmol.mL'1, preferably between 0.018 mmol.mL'1and 0.5 mmol.mL'1, more preferably between 0.020 mmol.mL'1and 0.3 mmol.mL'1, and the molar amount of the tributylstannane precursor of formula (Ila1) is between 0.002 pmol.GBq'1and 10.0 pmol.GBq'1, for example between 0.025 pmol.GBq'1and 5.0 pmol.GBq'1, preferably between 0.050 pmol.GBq'1and 2.5 pmol.GBq'1, more preferably between 0.070 pmol.GBq'1and 1.0 pmol.GBq'1.

[0038] In particular, the inventors have found that, the choice of a peroxide, and in particular of hydrogen peroxide as an oxidant, and more in particular, in the molar ratios defined in the present invention allows the introduction of a radionuclide, such as1311, for obtaining the compound of formula (I), and in particular the compound of formula (la) and the compound of formula (Ia1), almost quantitatively, with a remarkable chemical purity as shown in example 2 (table 7) which allows the use of said process in a fully automated procedure, such as a cassette- based automated synthesizer. In addition, the ingredients and conditions of the process of the invention are available with a pharmaceutical grade and are thus suitable to be implemented in radiopharmaceutical companies and nuclear medicine departments in the context of theranostic procedures. In addition, all the ingredients, and in particular, hydrogen peroxide, are available with a pharmaceutical grade. The choice of the acidic conditions (type of acid and pH), a peroxide ROOH, and in particular of hydrogen peroxide, as an oxidant, in the molar ratios defined in the present invention, allows performing mild labeling conditions, minimizing the risk of over oxidation and overall minimizing the side products from chlorination as shown in example 2 (table 9).

[0039] Accordingly, the processes of the invention are adapted to be implemented in a fully automated synthesis, i.e. , in cassette-based synthesizers.

[0040] The oxidation process is carried out at a pH lower than 1. The skilled person would know how to select an acid suitable for use in the process of the invention to provide a pH lower than 1. In particular, examples of acids useful in the process of the invention are hydrochloric acid and sulphuric acid.

[0041] In particular, the inventors have found that, the choice of hydrochloric acid and sulphuric acid allows obtaining the compound of formula (I), and in particular the compound of formula (Ia1) with a quasi-quantitative radiochemical yield as it can be seen in the example 2 (table 7).

[0042] In some aspects of the invention, the process is carried out in the presence of an acid selected from the group consisting of hydrochloric acid and sulphuric acid. In some aspects of the invention the acid used is hydrochloric acid.

[0043] Alkali metal salts of the iodine radionuclide and in particular of1311, suitable for use in the process of the present invention include lithium salts, sodium salts and potassium salts. In a preferred embodiment of the invention, alkali metal salts of the iodine radionuclide, and in particular of1311, suitable for use in the process of the present invention is sodium salts.

[0044] In some aspects of the invention, the alkali metal salt is a sodium salt of a radionuclide.

[0045] In some aspects of the invention, the processes disclosed in the present invention further comprise a subsequent step of adding a reducing agent in the presence of a base.

[0046] The base used in said subsequent step may be sodium hydroxide.

[0047] The reducing agent may be citrate, ascorbate, sodium metabisulfite, sodium sulfite, potassium sulfite, ammonium sulfite, sodium bisulfite, ammonium bisulfite, and potassium metabisulfite. Additional Suitable reducing agents may include ammonium thioglycolate, tris (2-carboxyethyl)phosphine hydrochloride, 2-mercaptoethanol, and dithiothreitol. Combinations of reducing agents may also be used in various embodiments. In some aspects, the reducing agent is sodium metabisulphite.Examples

[0048] The objective of the assays was to provide a radiosynthesis method for compound (Ia1) which would meet pharmaceutical requirements in terms of yield and (radio)chemical purity to develop a fully automatized process.Example 1. Development of a chromatographic method

[0049] A liquid chromatographic method (HPLC) has been developed and validated to measure the chemical and radiochemical purities of synthesis batches. On the one hand, it can verify the absence of the stannane precursor (I Ia1) in the finished product. It can also be used to identify the presence of side products that are chemically related to the (Ia1) molecule.Analytical Processes

[0050] Instrumentation: HPLC SHIMADZU - Software LAURA

[0051] Column: Kinetex 5 pm C18 (Phenomenex): 150 x 3 mm

[0052] Mobile Phase: A: ammonium formate 10 mM pH 3; B: acetonitrileTO: 30% BT2 min: 30% BT5 min: 90% BT10 min: 90% BT11 min: 30% BT15 min: 30% B

[0053] Time: 15 min

[0054] Flow: 0.5 mL.min'1

[0055] Detector: UV detector (248 and 254 nm) and gamma detector

[0056] Injection volume: 100 pL

[0057] Temperature: 40°C

[0058] Retention time:Non-radioactive equivalent of compound (Ia1) : 2.9 min (UV detection)Compound (Ia1) : 3.0 min (gamma detection)Stannane precursor (Ila1): 11.9 min (UV detection)Validation of analytical procedures

[0059] The reference guidelines used are:- ICH Harmonised Tripartite Guideline "Text on validation of analytical Procedures" step 4 (November 2005)- ICH Harmonised Tripartite Guideline "Validation of Analytical Procedures: Text and Methodology" step 4 (November 2005)I.1. Validation of the HPLC method for determining chemical puritvSpecificity

[0060] Specificity was tested by HPLC analysis of a mixture containing / \ / -(2- diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide which is the non-radioactive equivalent of compound (Ia1) and the stannane precursor (I Ia1). UV detection retention times of around 2.9 min for the non-radioactive equivalent of compound (Ia1) and aroundI I .8 min for the stannane precursor show that the two products are well separated (resolution well in excess of 1.5).Linearity of HPLC method of A / -(2-diethylaminoethyl)-6-iodoquinoxaline-2- carboxamide.

[0061] The statistical function used in these cases is linear regression with least squares. The curve equation, the correlation coefficient and the determination coefficient (R2) are then calculated.Equation: y = ax + b where: a = slope b = intercept y = peak area in mAU.s (milli-Absorbance Unit per second) x = non-radioactive equivalent of compound (Ia1) concentration in pg.mL'1

[0062] Six solutions of the non-radioactive equivalent of compound (Ia1) with different concentrations from 0.1 to 10 pg.mL'1were prepared by serial dilution starting from a "mother” solution with the highest concentration (100 pg.mL'1). These six standard solutions were prepared and analyzed on three different days. Results for linearity are presented below in Table 1:Table 1 : Concentrations tested and areas obtained at 254 nm

[0063] The linear regression equation is: y = 852.09 x - 2.3695 where: x = non-radioactive equivalent of compound (Ia1) concentration in pg.mL'1y = peak area in rnAll.s at 254 nmR2= 0.9991

[0064] A Fischer value calculation was done for the slope with one degree of freedom using the theoretical values to obtain Fcalc and the experimental values to obtain Ftab: Fcaic= 16944Ftab= 4.493998

[0065] In conclusion, Fcaic > Ftab the slope exists significantly (a = 5%).Accuracy

[0066] Precision is verified under repeatability and intermediate precision conditions.Repeatability

[0067] It is determined on 6 samples of the same concentration (1 pg.mL'1). The statistical parameter concerned, the coefficient of variation (CV%) is determined using the following equation: 100With parameters presented in the Table 2 below:Table 2: Parameters used during each day of the test

[0068] The CV% are below 2%, so the method is repeatable.

[0069] Intermediate precision: Three series of 6 samples of the same concentration (1 pg.mL'1) were analyzed on different days. An analysis of variance (ANOVA) was performed to determine Fisher's value. The results are presented in Table 3 below.

[0070] Table 3: Statistical analysis

[0071] The value of Fcaicuiated = 1.3135 is lower than Ftabuiated = 3.68232, the results obtained are not significantly different at the 5% risk.

[0072] The accuracy of the non-radioactive equivalent of compound (Ia1) assay method has been validated.1.2. Limit of quantification (LOQ) of the non-radioactive equivalent of compound

[0073] Eight samples (0.1 pg.rnL'1) are analyzed on eight different days. Results are present in next table (Table 4). LOQ is assessed by a coefficient of variation (CV%) value less than 15%.Table 4: Limit of quantification of the non-radioactive equivalent of compound (I a1 )

[0074] The CV% of 13.7% is less than 15%, the concentration of 0.1 pg.rnL'1is determined as the limit of quantification for the non-radioactive equivalent of compound(Ia1).Linearity of HPLC method for the stannane precursor (Ila1)

[0075] The statistical function used in these cases is linear regression with least squares. The curve equation, the correlation coefficient and the determination coefficient (R2) are then calculated.Equation: y = ax + b where: a = slope b = intercept y = peak area in rnAll.s x = quantity of compound (I Ia1 ) injected in ng

[0076] A solution of stannane precursor (Ila1) at 5 pg.rnL-1were prepared by dilution in water / ethanol 1:1 starting from a "mother” solution 100 pg.rnL'1in ethanol. Three standard solutions were prepared and analyzed on three different days. Injected volumes are 1 , 2, 3, 4, 5 and 10 pL.

[0077] The linear regression equation is: y = 3.1952 x + 8.5147where: x = quantity of compound (I Ia1 ) injected in ng y = peak area in rnAll.s at 248 nmR2= 0.9943Limit of detection of the stannane precursor (Ila1)

[0078] The purification method on a C18 cartridge eliminates the stannane derivative. This is why only the detection limit of the stannane precursor is determined.

[0079] Initially, a range was carried out by injecting quantities of 5 to 50 ng of stannane in 100 pL of saline solution. Analysis of this range, coupled with analysis of eight blanks, resulted in a proposed detection limit of 50 ng.mL'1. To validate this detection limit, repeatability over 6 injections was carried out with a coefficient of variation of 27%. The detection limit for stannane is 50 ng.mL'1.Theoretical limit of detection of the stannane precursor (Ila1)

[0080] The theoretical LOD has been determined by eight injections of the matrix solution (100 pL of Saline+7.5% ethanol) for retention times between 11 and 13 minutes. Results are present in next table (Table 5):Table 5: Theoretical limit of detection of the stannane precursor [Ila1]

[0081] The theoretical LOD is M+3o = 7 rnAll.s at 248 nm.Experimental limit of detection of the stannane precursor

[0082] Based on theoretical limits of detection (LOD), the validation of the experimental LOD has been done by six injections of 5 ng of the stannane precursor (I la 1 ) in the matrix solution (100 pL of Saline+7.5% ethanol). Results are present in next table (Table 6):Table 6: Experimental limit of detection of the stannane precursor (I Ia1)

[0083] The limit of detection retained at 248 nm is 5 ng of injected stannane precursor (Ila1).1.3. Validation of the HPLC method for determining radiochemical purityIdentification

[0084] The retention times of compound (Ia1) and the non-radioactive equivalent of compound (Ia1) are identical in UV detection with an average shift of 0.07 min between the gamma radioactivity detector and the UV detector connected in series.Specificity

[0085] Specificity was determined by HPLC analysis of the finished product containing radioactive iodides and compound (Ia1). Calculation of the resolution (R) greater than 1.5 shows the specificity of the method. Compound (Ia1): retention time = 2.9 min (gamma detection)1311-: retention time = 1.7 min (gamma detection)R = 1.84Example 2. Application of the chromatographic method for the selection of radiosynthesis conditions2.1 : Analysis of the process yield / Selection of radiosynthesis conditions

[0086] Conditions for manual radiosynthesis:

[0087] Set-up: 100 pg precursor compound (Ila1) 1 100 pL ethanol + 50 pL acid solution + 5 pL [131l]Nal + [10-40] pL oxidant ; Reaction conditions: 15 min at room temperature.

[0088] Radiochemical yield (RCY) was determined using different acids and oxidants: the only parameter studied in these assays included in table 7 below was the radiochemical yield, not the chemical purity.Table 7: Conditions and radiochemical yields (RCY)

[0089] Conclusion: pH must not be higher than 1 to achieve acceptable RCY. Surprisingly, some acids, even when pH is kept lower than 1 , promoted the degradation of the stannane precursor (Ila1) (see trifluoroacetic acid) and no detectable RCY was obtained. The two preferred acids are thus HCI and H2SO4. Based on the results, all / V- chlorosuccinimide, chloramine-T, peracetic acid, and hydrogen peroxide are able to achieve high RCY of the product.2.2: Analysis of the product chemical purity / Selection of radiosynthesis conditions

[0090] Table 8 below shows the molecules found in the solutions during a synthesis using Chloramine-T as an oxidant (and using HCI as the chosen acid). The chromatographic method described allows coupling with positive-mode mass spectrometry (ESI / MS) detection.Table 8: Details of the products obtained. Nd: not determined; rt: retention time; Relative quantities refer to (HLPC / UV) areas measured in relation to 100 for the desired product; (*) Due to the intensity of the peak of molecule B that overlaps the peak of molecule D, it is not possible to quantify the brominated derivative D.

[0091] The use of chloramine T, as shown in the table above (Table 8), leads to the formation of several side products, the most important being the chlorinated molecule B (ratio relative to compound (I a1 ) of the order of 50).

[0092] The aim of improving the radiosynthesis stage is to significantly reduce the quantities of these compounds present in the finished product.

[0093] Table 9 below presents some evaluated oxidants. The AUG ratio of the compounds Ia1 / B is the parameter studied (results provided as an average of 1 to 7 different experiments):Table 9: UV-AIIC ratio - presence of chlorinated side product. Acid used: hydrochloric acid

[0094] In view of the finished product / chlorinated impurities ratio, the results show that when a peroxide reagent is used as oxidant, the quantity of chlorinated impurities is reduced compared to a process using chloramine-T or / V-chlorosuccinimide as oxidant.

[0095] Table 10 below shows an example of chemical purity results obtained after radiosynthesis (including purification and formulation steps) of the molecule (Ia1) in the presence of H2O2 and hydrochloric acid.Table 10: Details of the products obtained, rt: retention time; Relative quantities refer to (HLPC / UV) areas measured in relation to 100 for the desired product

[0096] None of the impurities initially present using Chloramine-T as oxidant increased in the finished product when H2O2 was used as oxidant. In addition, the amount of impurity A (de-halogenated derivative of Ia1) decreased by 98% and the amount of the chlorinated impurity C decreased by 99.7%. Using H2O2 as oxidant in hydrochloric acid allows to obtain the finished product with a remarkable chemical purity.Example 3: Radiosynthesis of compound (Ia1): automatization process

[0097] Compound (Ia1) is synthesized in an AlllnOne® (Trasis) radiochemistry synthesizer. Figure 1 presents the layout of the cassette. Positioning of reagents and materials is detailed in the table below (Table 11).

[0098] Compound (Ia1) is synthesized using a radioiodo-destannylation reaction with an organometallic precursor. This electrophilic substitution method is used for radiolabeling with iodine radioisotopes, with an oxidized radioactive iodine species (+1 charge) acting as an electrophile and reacting with electron rich species. A covalent carbon-radioiodine bond is generated with the loss of a leaving group. Organometallic species like tributylstannane derivatives are particularly useful for this reaction. The oxidized species is generated in situ by the oxidation of radioactive iodide (primary as a sodium salt). The oxidizing agent employed for the automatized synthesis of compound (Ia1) is hydrogen peroxide. Hydrogen peroxide is available with a pharmaceutical grade and performed mild labeling conditions (minimizing the risk of over oxidation and overall minimizing the side products from chlorination when chloramine-T for example was used). The reaction is initiated in acidic conditions with hydrogen peroxide added to the precursor mixture (in ethanol) and to the 131 -iodide salt precursor (in aqueous NaOH solution). The reaction proceeds at room temperature for 15 min and is stopped with the addition of a quenchsolution (aqueous mixture of NaOH and Na2S20s). The purification step is performed using a SPE cartridge (preconditioning: 5 mL ethanol followed by 5 mL water). The unreacted stannane precursor (Ila1) and the product (Ia1) remain bound to the resin support while1311' traces are eluted with the reaction solvents followed by water. The elution step with an ethanol / water solution removed only compound (Ia1). Final formulation is performed with a saline solution (NaCI 0.9%). The compound (Ia1) is obtained with a radiochemical purity higher than 97%.Table 12: One example of reagent quantities used during automated radiosynthesis[H2O2] = 0.18 mmol.mL’1; [Ila1] = 0.071 pmol.GBq-1

[0099] Table 13 below shows the result of chemical purity obtained after 21 automated radiosynthesis according to procedure described in this Example 3, above. Mean radiochemical purity of Ia1 is 97.4±1.0%.Table 13: Details of the products obtained, rt: retention time; Relative quantities for compounds A, X, B, D, E and Ila1 refer to (HLPC / UV) areas measured in relation to 100 for the desired product. Relative quantity for lodine-131, [131I]C and for compound Ia1 obtained from the radioactive activity measured. Starting activity: 0.5-12 GBq; n.d. : not detected.

Claims

Claims

1. A process to synthesize a compound of formula (I), or a pharmaceutically acceptable salt thereof:said process comprising: contacting a trialkylstannane precursor of formula (II), or a pharmaceutically acceptable salt thereof:with an alkali metal salt of a radionuclide selected from the group consisting of123l,124l, 125| 131| 2n^tjn anappropriate solvent at a pH lower than 1 , and in presence of a peroxide ROOH, wherein the molar concentration of ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1, and the molar amount of the trialkylstannane precursor of formula (II) is between 0.2 nmol.GBq'1and 10.0 pmol.GBq'1; and whereinm is an integer varying from 2 to 4;Ri and R2 are each, independently of one another, a hydrogen atom, a (Ci-C6)alkyl group or a (C2-C6)alkenyl group;R3 is a radionuclide selected from the group consisting of123l,124l,125l,131l,211At;R4, Rs and Re are each, independently of one another a (Ci-Ce)alkyl group; andR is H or RyC(O)- and R? is C1-6 alkyl.

2. The process according to claim 1 , wherein;the compound of formula (I) has a formula (la):the compound of formula (II) has a formula (Ila):and the values of R1, R2, R3, R4, Rs, Rs and m are those defined in claim 1.

3. The process according to any one of claims 1 or 2, wherein Ri and R2 are each, independently of one another selected from the list consisting of methyl, ethyl and propyl, preferably ethyl.

4. The process according to any one of claims 1 to 3, wherein m=2.

5. The process according to claim 4, wherein R3 is1311, and the molar amount of the trialkylstannane precursor of formula (II) is between 0.002 pmol.GBq'1and 10.0 pmol.GBq'1.

6. The process according to claim 1 , wherein R1 and R2 are both ethyl, R3 is131l, m= 2; the compound of formula (I) has a formula (Ia1), or a pharmaceutically acceptable salt thereof:the compound of formula (II) has a formula (Ila’), or a pharmaceutically acceptable salt thereof:and the process comprises: contacting the trialkylstannane precursor of formula (Ila’), or a pharmaceutically acceptable salt thereof, with an alkali metal salt of1311, in an appropriate solvent at a pH lower than 1 , and in presence of a peroxide ROOH; wherein the molar concentration of ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1, and the molar amount of the trialkylstannane precursor of formula (Ila) is between0.002 pmol.GBq'1and 10.0 pmol.GBq'1; and wherein R is H or R?-C(O)- and R? is C1-6 alkyl, and wherein R4, Rs and Re are each, independently of one another a (Ci-Ce)alkyl group.

7. The process according to any one of claims 1 to 6, wherein R4, Rs and Re are butyl.

8. The process to synthesize a compound of formula (I) according to claim 1 , whereinare each ethyl; R3 is1311, R4, Rs and Re are each butyl, the compound of formula (I) has a formula (Ia1):and the trialkylstannane precursor of formula (II) has a formula (I Ia1 ):and the process comprises: contacting the trialkylstannane precursor of formula (Ila), or a pharmaceutically acceptable salt thereof, with an alkali metal salt of1311, in an appropriate solvent at a pH lower than 1 , and in presence of a peroxide ROOH; wherein the molar concentration of ROOH is between 0.003 mmol.mL'1and 1.0 mmol.mL'1, and the molar amount of the trialkylstannane precursor of formula (Ila) is between0.002 pmol.GBq'1and 10.0 pmol.GBq'1; and wherein R is H or R?-C(O)- and R? is C1-6 alkyl, and wherein R4, Rs and Re are each, independently of one another a (Ci-Ce)alkyl group.

9. The process according to any of claims 1 to 8 wherein the peroxide ROOH is H2O2 and the molar concentration of H2O2 is between 0.015 mmol.mL'1and 1.000 mmol.mL'1.

10. The process according to any of claims 1 to 9 wherein the process is carried out in the presence of an acid selected from the group consisting of hydrochloric acid and sulphuric acid.

11. The process according to any of claims 1 to 10 wherein the alkali metal salt is a sodium salt.

12. The process according to any of claims 1 to 11 further comprising a subsequent step of adding a reducing agent in the presence of a base.

13. The process according to claim 12 wherein the base is sodium hydroxide.

14. The process according to any of claims 12 or 13 wherein the reducing agent is sodium metabisulphite.

15. The process according to any one of the preceding claims wherein the pharmaceutically acceptable salt is a hydrochloride salt.

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