(19)
(11)EP 3 663 307 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
10.06.2020 Bulletin 2020/24

(21)Application number: 18842323.0

(22)Date of filing:  03.08.2018
(51)Int. Cl.: 
C07F 13/00  (2006.01)
C07B 59/00  (2006.01)
(86)International application number:
PCT/JP2018/030006
(87)International publication number:
WO 2019/027059 (07.02.2019 Gazette  2019/06)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
KH MA MD TN

(30)Priority: 04.08.2017 JP 2017151632

(71)Applicant: Osaka University
Suita-shi, Osaka 565-0871 (JP)

(72)Inventors:
  • SHIRAKAMI, Yoshifumi
    Suita-shi Osaka 565-0871 (JP)
  • IKEDA, Hayato
    Suita-shi Osaka 565-0871 (JP)
  • KANAI, Yasukazu
    Suita-shi Osaka 565-0871 (JP)
  • SHIMOSEGAWA, Eku
    Suita-shi Osaka 565-0871 (JP)
  • HATAZAWA, Jun
    Suita-shi Osaka 565-0871 (JP)
  • WATABE, Tadashi
    Suita-shi Osaka 565-0871 (JP)
  • KANEDA, Kazuko
    Suita-shi Osaka 565-0871 (JP)

(74)Representative: De Clercq & Partners 
Edgard Gevaertdreef 10a
9830 Sint-Martens-Latem
9830 Sint-Martens-Latem (BE)

  


(54)PRODUCTION METHOD FOR RADIOLABELED ARYL COMPOUND


(57) The present invention aims to produce a radiolabeled aryl compound by a method that enables an easy labelling with a high radiochemical yield in a short time, and that enables formulation immediately after the labelling.
The present invention relates to a method of producing the radiolabeled aryl compound (I), which comprises reacting the aryl boronic acid compound (II) with a radionuclide selected from 211At, 210At, 123I, 124I, 125I and 131I, in the presence of an oxidizing agent selected from an alkali metal iodide, an alkali metal bromide, N-bromosuccinimide, N-chlorosuccinimide and hydrogen peroxide, in water.

        Ar―X     (I)

        Ar―Y     (II)

wherein each symbol is as defined in the description.


Description

Technical Field



[0001] The present invention relates to a production method of radiolabeled aryl compound applicable to RI internal therapy or diagnosis for cancer.

Background Art



[0002] RI internal therapy or diagnosis for cancer by use of radionuclides that emit α-ray, β-ray, γ-ray and the like utilizes specific bindings of radionuclide-labeled drugs to target molecules, i.e., molecules specifically expressed or overexpressed in cancer cells, and it has been applied in a clinical practice. For example, Na131I has been applied to therapy for thyroid cancer, and 223RaCl2 has been applied to therapy for prostate cancer bone metastasis.

[0003] Application of 211At, one of radionuclides, is expected as new RI internal therapy for cancer (e.g., 4-211At-L-phenylalanine (Non-Patent Document 1), Na211At, etc.). 211At is a radionuclide produced by an accelerator such as cyclotron and the like, and has a short half-life of 7.2 hours. Therefore, a sequence of processes containing production of 211At, labelling of a drug with 211At, formulation of the drug, administration of the drug to patient with cancer, and RI internal therapy by the drug should be promptly carried out. In particular, since the labelling and the subsequent formulation should be easily carried out in a short time, the formulation is desirably carried out immediately after the labelling. Moreover, since the labeled drug is to be formulated into an injection for intravenous administration, the labeling is desirably carried out without using toxic reagent, under an organic solvent-free condition composed only of water, and the like, in terms of prompt formulation after the labelling. 123I for diagnosis also has a short half-life of 13.23 hours, and therefore, the labelling and the subsequent formulation should be easily carried out in a short time, as in the case in 211At.

[0004] Non-Patent Document 1 discloses that 4-211At-L-phenylalanine can be applied to RI internal therapy for brain tumor, and a precursor, N-Boc-4-tributylstannyl-L-phenylalanine is produced in a radiochemical yield of 35-50%, by electrophilic destannylation according to a method described in Non-Patent Document 2. However, since the precursor is an N-Boc form, it cannot be dissolved in a solvent composed only of water, and use of an organic solvent for dissolution requires evaporation. In addition, the above method requires de-Boc step after the electrophilic destannylation. Moreover, Non-Patent Document 2 does not disclose specific methods for the de-Boc step, only specifically discloses labelling of 4-iodo-L-phenylalanine with 211At by halogen exchange reaction in the presence of CuSO4, SnSO4 and an acid, at 120°C for 60 minutes. The method requires removal of toxic Cu and Sn, and the reaction at 120°C for 60 minutes is not easy method in a short time. The labelling by the above-mentioned methods is not desirable, and labelling and the subsequent formulation cannot be easily carried out in a short time. Moreover, the radiochemical yield is low and most-unsatisfactory.

[0005] Non-Patent Document 3 discloses that aryl boronic acid or an ester thereof is labeled with Na123I by electrophilic substitution reaction in the presence of 1,10-phenanthroline and a Cu catalyst such as Cu2O, Cu(OCOCF3)2 and the like, at 80°C, in water/methanol. However, the method is not desirable in terms of use of methanol and a Cu catalyst, and reaction at high temperature. In addition, the radiochemical yield is at most 87% and unsatisfactory. Moreover, the document also discloses that aryl boronic acid or an ester thereof is labeled with Na123I by electrophilic substitution reaction in the presence of chliramine-T, in water/tetrahydrofuran. However, the method is not desirable in terms of use of tetrahydrofuran, and the method cannot be applied to an electron deficient arene.

[0006] Patent Documents 1 to 4 discloses the aryl trialkyltin is labeled with Na123I, Na211At and the like by electrophilic destannylation reaction. However, none of the reactions is not desirable in terms of use of an organic solvent and toxic Sn.

Document List


Patent Document



[0007] 

Patent Document 1: US 4826672

Patent Document 2: US 5077035

Patent Document 3: JP 2001-503412

Patent Document 4: JP 2009-521469


Non-Patent Document



[0008] 

Non-Patent Document 1: Nuklearmedizin, 2013, vol.52, pp.212-21

Non-Patent Document 2: Applied Radiation and Isotopes, 2010, vol.68, pp.1060-1065

Non-Patent Document 3: Chem. Commun., 2016, vol.52, pp.13277-13280


Summary of the Invention


Problems to be Solved by the Invention



[0009] The present invention aims to produce a radiolabeled aryl compound by a method that enables an easy labelling with a high radiochemical yield in a short time, and that enables formulation immediately after the labelling.

Means of Solving the Problems



[0010]  The present inventors have conducted intensive studies in an attempt to solve the above-mentioned problems and found that, by the following method, a radiolabeled aryl compound can be easily produced in a high radiochemical yield in a short time, and the formulation can be carried out immediately after the labelling, which resulted in the completion of the present invention.

[0011] Accordingly, the present invention provides the following.
  1. [1] A method of producing a radiolabeled aryl compound represented by the formula (I):


            Ar―X     (I)

    wherein

    Ar is a C6-14 aryl group optionally having substituent(s), and

    X is 211At, 210At, 123I, 124I, 125I or 131I,

    or a salt thereof (hereinafter, sometimes to be referred to as radiolabeled aryl compound (I)), which comprises reacting an aryl boronic acid compound represented by the formula (II):


            Ar―Y     (II)

    wherein

    Ar is as defined above, and

    Y is a borono group (-B(OH)2) or an ester group thereof,

    or a salt thereof (hereinafter, sometimes to be referred to as radiolabeled aryl compound (II)), with a radionuclide selected from 211At, 210At, 123I, 124I, 125I and 131I, in the presence of an oxidizing agent selected from an alkali metal iodide, an alkali metal bromide, N-bromosuccinimide, N-chlorosuccinimide and hydrogen peroxide, in water.
  2. [2] The method according to the above-mentioned [1], wherein the reaction is carried out in an organic solvent-free system.
  3. [3] The method according to the above-mentioned [1] or [2], wherein the reaction is carried out at room temperature.
  4. [4] The method according to any of the above-mentioned [1] to [3], wherein the radionuclide is 211At or 210At, and the oxidizing agent is selected from sodium iodide, sodium bromide, N-bromosuccinimide, N-chlorosuccinimide and hydrogen peroxide.
  5. [5] The method according to any of the above-mentioned [1] to [3], wherein the radionuclide is 123I, 124I, 125I or 131I, and the oxidizing agent is selected from N-bromosuccinimide and N-chlorosuccinimide.
  6. [6] The method according to any of the above-mentioned [1] to [5], wherein Y is a borono group (-B(OH)2).
  7. [7] The method according to any of the above-mentioned [1] to [6], wherein the substituent of the C6-14 aryl group optionally having substituent(s) represented by Ar is a group capable of binding specifically to a target molecule.
  8. [8] The method according to the above-mentioned [7], wherein the target molecule is an antigen, a transporter, a receptor, an enzyme or a gene, which is specifically expressed or overexpressed in a cancer cell.
  9. [9] The method according to any of the above-mentioned [1] to [8], wherein Ar is
    a group represented by the formula:

    wherein

    R2 is a halogen atom, m is 0 or 1,

    n is 0 or an integer of 1 to 4, and

    * is a binding site to X or Y, or

    a group derived from a peptide having a partial structure represented by the formula:

    wherein

    R2 is a halogen atom,

    m is 0 or 1,

    n is 0 or an integer of 1 to 4, and

    * is a binding site to X or Y.

  10. [10] The method according to any of the above-mentioned [1] to [8], wherein Ar is
    a group represented by the formula:

    wherein

    R3 is a hydrogen atom or a halogen atom,

    m is 0 or 1, and

    * is a binding site to X or Y, or

    a group derived from a peptide having a partial structure represented by the formula:

    wherein

    R3 is a hydrogen atom or a halogen atom,

    m is 0 or 1, and

    * is a binding site to X or Y.

  11. [11] The method according to any of the above-mentioned [1] to [8], wherein the aryl boronic acid compound represented by the formula (II) is 4-boronophenylalanine, 4-borono-2-fluorophenylalanine or 3-boronophenylalanine, and the radiolabeled aryl compound represented by the formula (I) is 4-astato (211At) phenylalanine, 4-astato (211At) -2-fluorophenylalanine or 3-astato(211At)phenylalanine.
  12. [12] 3-Astato (211At) phenylalanine or a salt thereof.

Effect of the Invention



[0012] According to the production method of the present invention, the radiolabeled aryl compound (I) can be easily produced in a high radiochemical yield in a short time, and the formulation can be carried out immediately after the labelling. Therefore, the labelling and formulation can be easily carried out in a short time, and a sequence of processes from preparation of a radionuclide to RI internal therapy or diagnosis for cancer can be promptly carried out.

Brief Description of the Drawings



[0013] 

[Figure 1]
Figure 1 shows a thin layer chromatography (TLC) of the aqueous 211At solution prepared in Reference Example 1.

[Figure 2]
Figure 2 shows a thin layer chromatography (TLC) of the reaction solution of Example 1.

[Figure 3]
Figure 3 shows a thin layer chromatography (TLC) of the reaction solution of Example 2.

[Figure 4]
Figure 4 shows a thin layer chromatography (TLC) of the reaction solution of Example 3.

[Figure 5]
Figure 5 shows a thin layer chromatography (TLC) of the reaction solution of Example 4.

[Figure 6]
Figure 6a shows a thin layer chromatography (TLC) of the aqueous Na123I solution, and Figure 6b shows a thin layer chromatography (TLC) of the reaction solution of Example 5.

[Figure 7]
Figure 7 shows a cellulose acetate membrane electrophoresis of the reaction solution of Example 6.

[Figure 8]
Figure 8 shows a thin layer chromatography (TLC) of the reaction solution of Example 7.

[Figure 9]
Figure 9 shows comparison of 4-211At-Phe or 3-211At-Phe amount taken up by C6 glioma derived from rat in Example 10.

[Figure 10]
Figure 10 shows SPECT imaging of C6 glioma-transplanted rat by 4-211At-Phe in Example 11 (30 minutes and 3 hours after administration).


Description of Embodiments



[0014] The present invention is explained in detail in the following.

[0015] In the present specification, examples of the "C6-14 aryl group" include phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl and 9-anthryl.

[0016] In the present specification, examples of the "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

[0017] In the present specification, examples of the "C1-6 alkyl group" include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neo-pentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl.

[0018] Each symbol in the formulas (I) and (II) is explained below.

[0019] In the formulas (I) and (II), Ar is a C6-14 aryl group optionally having substituent(s).

[0020] The "C6-14 aryl group" of the "C6-14 aryl group optionally having substituent(s)" represented by Ar is preferably phenyl.

[0021] Examples of the "substituent" of the "C6-14 aryl group optionally having substituent(s)" represented by Ar include groups capable of binding specifically to a target molecule. Examples of the target molecule include antigens, transporters, receptors, enzymes, genes and the like, which are specifically expressed or overexpressed in cancer cells. Specific examples of such "substituent" include C1-6 alkyl groups (preferably methyl, ethyl) substituted by a carboxy group and an amino group; a carboxy group; an amino group; a guanidino group; groups having a tropane skeleton; fatty-acid residues (groups obtained by removing any one hydrogen atom from fatty-acids); residues of biologically related substances such as peptides, proteins, antibodies, nucleic acids and the like (groups obtained by removing any one hydrogen atom from biologically related substances); and the like.

[0022] Ar is preferably a C6-14 aryl group having substituent(s), more preferably a phenyl group having substituent(s), still more preferably a residue derived from an amino acid having phenyl group(s), or a residue derived from a peptide having phenyl group(s).

[0023] As used herein, the above-mentioned "residue derived from an amino acid having phenyl group(s)" means a group obtained by removing, from an amino acid having phenyl group(s) (e.g., phenylalanine or phenylglycine optionally substituted by halogen atom(s), etc.), any one hydrogen atom on the phenyl ring.

[0024] Preferable example is a group represented by the formula:

wherein

R2 is a halogen atom,

m is 0 or 1,

n is 0 or an integer of 1 to 4, and

* is a binding site to X or Y.



[0025] More preferable example is a group represented by the formula:

wherein

R3 is a hydrogen atom or a halogen atom,

m is 0 or 1, and

* is a binding site to X or Y.



[0026] In another embodiment, more preferable example is a group represented by the formula:

wherein

R3 is a hydrogen atom or a halogen atom,

m is 0 or 1, and

* is a binding site to X or Y.



[0027] The above-mentioned "residue derived from a peptide having phenyl group (s)" means a group obtained by removing, from a peptide having phenyl group(s) (e.g., a peptide containing phenylalanine or phenylglycine optionally substituted by halogen atom(s), etc.), any one hydrogen atom on the phenyl ring.

[0028] Preferable example is a group derived from a peptide having a partial structure represented by the formula:

wherein

R2 is a halogen atom,

m is 0 or 1,

n is 0 or an integer of 1 to 4, and

* is a binding site to X or Y.



[0029] More preferable example is a group derived from a peptide having a partial structure represented by the formula:

wherein

R3 is a hydrogen atom or a halogen atom,

m is 0 or 1, and

* is a binding site to X or Y.



[0030] In another embodiment, more preferable example is a group derived from a peptide having a partial structure represented by the formula:

wherein

R3 is a hydrogen atom or a halogen atom,

m is 0 or 1, and

* is a binding site to X or Y.



[0031] The "halogen atom" represented by R2 or R3 is preferably a fluorine atom.

[0032] R2 is preferably a fluorine atom.

[0033] n is preferably 0 or 1.

[0034] R3 is preferably a hydrogen atom or a fluorine atom.

[0035] m is preferably 1.

[0036] In the formula (II), Y is a borono group (-B(OH)2) or an ester group thereof.

[0037] Examples of the "ester group of borono group" represented by Y include the following ester groups.



wherein R1 is a C1-6 alkyl group,
Y is preferably a borono group (-B(OH)2).

[0038] In the formula (I), X is a radionuclide 211At, 210At, 123I, 124I, 125I or 131I.

[0039] When the radiolabeled aryl compound (I) or aryl boronic acid compound (II) is in the form of a salt, examples of such salts include metal salts (e.g., alkali metal salts such as sodium salt, potassium salt etc.; alkaline-earth metal salts such as calcium salt, magnesium salt, barium salt etc.), an ammonium salt, salts with an organic base (e.g., trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine), salts with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid), salts with an organic acid (e.g., formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid), and the like.

[0040] In the present invention, the radiolabeled aryl compound (I) is produced by reacting the aryl boronic acid compound (II) with a radionuclide selected from 211At, 210At, 123I, 124I, 125I and 131I in the presence of an oxidizing agent selected from an alkali metal iodide, an alkali metal bromide, N-bromosuccinimide, N-chlorosuccinimide and hydrogen peroxide, in water.

[0041] The aryl boronic acid compound (II) is preferably an amino acid having a borono-substituted phenyl group(s), or a peptide containing the amino acid, more preferably an amino acid having a borono-substituted phenyl group(s). The borono-substituted phenyl group optionally has additional substituent(s) such as a halogen and the like.

[0042] The aryl boronic acid compound (II) is more preferably 4-boronophenylalanine, 4-borono-2-fluorophenylalanine, 4-boronophenylglycine or 4-borono-2-fluorophenylglycine, particularly preferably 4-boronophenylalanine or 4-borono-2-fluorophenylalanine.

[0043] In another embodiment, the aryl boronic acid compound (II) is more preferably 4-boronophenylalanine, 4-borono-2-fluorophenylalanine, 4-boronophenylglycine, 4-borono-2-fluorophenylglycine, 3-boronophenylalanine or 3-boronophenylglycine, particularly preferably 3-boronophenylalanine, 4-borono-2-fluorophenylalanine or 3-boronophenylalanine.

[0044] The aryl boronic acid compound (II) is not limited to the compounds exemplified above, and the radiolabelling method of aryl compound of the present invention can also be applied to various aryl boronic acid compounds, for example, boronohydroxybenzene and boronocarboxybenzene (carboxyphenylboronic acid).

[0045] When the aryl boronic acid compound (II) is the above-mentioned amino acid having a borono-substituted phenyl group (s), it is used generally in the form of an aqueous solution, preferably in the form of a solution dissolved in an aqueous alkali solution such as an aqueous sodium hydrogencarbonate solution and the like.

[0046] Examples of the alkali metal iodide include sodium iodide, potassium iodide and the like. Among them, preferred is sodium iodide.

[0047] Examples of the alkali metal bromide include sodium bromide, potassium bromide and the like. Among them, preferred is sodium bromide.

[0048] The combination of the radionuclide and the oxidizing agent is preferably
  1. (1) a combination of the radionuclide of 211At or 210At, and the oxidizing agent selected from sodium iodide, sodium bromide, N-bromosuccinimide, N-chlorosuccinimide and hydrogen peroxide; or
  2. (2) a combination of the radionuclide of 123I, 124I, 125I or 131I, and the oxidizing agent selected from N-bromosuccinimide and N-chlorosuccinimide. The oxidizing agent may be used alone or in combination of two or more kinds thereof. The oxidizing agent is used generally in the form of an aqueous solution.


[0049] The oxidizing agent is used in an amount sufficient to oxidize the radionuclide, generally in a large excess amount relative to the radionuclide. It is used preferably in a concentration of 0.0001 to 0.2 mol/L, more preferably in a concentration of 0.001 to 0.1 mol/L, in terms of reaction efficiency and economic efficiency.

[0050] The radionuclide is used in the reaction generally in the form of an aqueous solution, preferably in the form of a solution prepared by dissolving in an aqueous alkali solution such as an aqueous sodium hydrogencarbonate solution and the like, in terms of stability.

[0051] In cases where the radionuclide is 211At, first, bismuth is irradiated with helium particles accelerated to 28 MeV by cyclotron, and 211At is generated by the resulting 209Bi (α, 2n) 211At nuclear reaction. Next, by heating, the target substance 209Bi is melted, but the 211At is vaporized, and then the vaporized 211At is trapped in liquid nitrogen, and dissolved in water to prepare an 211At undiluted solution. Then, for the purpose of stabilization of 211At, an aqueous alkali solution such as an aqueous sodium hydrogencarbonate solution and the like is added to the undiluted solution to prepare an aqueous 211At alkali solution.

[0052] In cases where the radionuclide is 210At, first, bismuth is irradiated with helium particles accelerated to 29 MeV or more by cyclotron, and 210At is generated by the resulting 209Bi (α,3n)210At nuclear reaction. Next, by the same procedure mentioned above, an aqueous 210At solution is prepared.

[0053] In cases where the radionuclide is 123I, it is available as an aqueous Na123I solution.

[0054] In cases where the radionuclide is 124I, first, tellurium is irradiated with proton particles accelerated by cyclotron, and 124I is generated by the resulting 124Te(p,n) 124I nuclear reaction. Next, the target substance 124Te is melted, and the remaining 124I is dissolved in an aqueous sodium hydroxide solution to prepare an aqueous 124I sodium hydroxide solution.

[0055] In cases where the radionuclide is 125I, it is available as an aqueous Na125I solution.

[0056] In cases where the radionuclide is 131I, it is available as an aqueous Na131I solution.

[0057] Since 211At has a short half-life of 7.2 hours, 210At has a short half-life of 8.3 hours, and 123I has a short half-life of 13.2 hours, these radionuclides should be used in the subsequent reaction immediately after the preparation. While 124I has a relatively long half-life of 4.2 days, 125I has a relatively long half-life of 59.4 days, and 131I has a relatively long half-life of 8.04 days, these radionuclides are also preferably used in the subsequent reaction immediately after the preparation.

[0058] The aryl boronic acid compound (II) is used generally in a large excess amount relative to the radionuclide, preferably in a concentration of 0.0001 mol/l to 0.5 mol/l, more preferably in a concentration of 0.001 mol/l to 0.2 mol/l, per 1 Bq to 1,000 GBq of the radionuclide, in terms of reaction efficiency and economic efficiency.

[0059] The above-mentioned reaction is carried out by mixing the aryl boronic acid compound (II), an oxidizing agent and a radionuclide, and the mixing order is not particularly limited. The reaction is preferably carried out by adding an aqueous radionuclide alkali solution and an aqueous oxidizing agent solution, in this order, to an aqueous solution (preferably an aqueous sodium hydrogencarbonate solution) of the aryl boronic acid compound (II), or by adding an aqueous oxidizing agent solution and an aqueous radionuclide alkali solution, in this order, to an aqueous solution (preferably an aqueous sodium hydrogencarbonate solution) of the aryl boronic acid compound (II), more preferably by adding an aqueous radionuclide alkali solution and an aqueous oxidizing agent solution, in this order, to an aqueous solution (preferably an aqueous sodium hydrogencarbonate solution) of the aryl boronic acid compound (II).

[0060] The above-mentioned reaction is carried out in water, i.e., in an organic solvent-free system.

[0061] The above-mentioned reaction is carried out at room temperature, specifically at 0°C - 40°C, preferably 10°C - 35°C. In the production method of the present invention, the reaction proceeds rapidly in a short time, even at room temperature. For example, the reaction is completed for 1 minute to 3 hours, particularly 1 minute to 30 minutes.

[0062] The completion of the reaction is confirmed by thin layer chromatography (TLC) analysis, based on the disappearance of a free radionuclide.

[0063] In the production method of the present invention, the radiolabeled aryl compound (I) can be obtained in a high radiochemical yield of 75% or more, particularly 80% or more, especially 90% or more.

[0064] Since the reaction solution contains neither an organic solvent nor a toxic reagent, the reaction solution can be formulated into an injection and the like immediately after the completion of the reaction, without isolation of the radiolabeled aryl compound (I).

[0065] As explained above, in the production method of the present invention, the labeling can be easily carried out in a high radiochemical yield in a short time, without use of an organic solvent and a toxic reagent. Therefore, a sequence of processes from preparation of a radionuclide to RI internal therapy or diagnosis for cancer can be promptly carried out.

[0066] The radiolabeled aryl compound (I) produced by such method is preferably

4-astato(211At)phenylalanine,

3-astato(211At)phenylalanine,

4-astato(211At)-2-fluorophenylalanine,

4-astato(210At)phenylalanine,

3-astato(210At)phenylalanine,

4-astato(210At)-2-fluorophenylalanine,

4-iodo(123I)phenylalanine,

3-iodo(123I)phenylalanine,

4-iodo(123I)-2-fluorophenylalanine,

4-iodo(124I)phenylalanine,

3-iodo(124I)phenylalanine,

4-iodo(124I)-2-fluorophenylalanine,

4-iodo(125I)phenylalanine,

3-iodo(125I)phenylalanine,

4-iodo(125I)-2-fluorophenylalanine,

4-iodo(131I)phenylalanine,

3-iodo(131I)phenylalanine,

4-iodo(131I)-2-fluorophenylalanine,

4-astato(211At)phenylglycine,

3-astato(211At)phenylglycine,

4-astato(211At)-2-fluorophenylglycine,

4-astato(210At)phenylglycine,

3-astato(210At)phenylglycine,

4-astato(210At)-2-fluorophenylglycine,

4-iodo(123I)phenylglycine,

3-iodo(123I)phenylglycine,

4-iodo(123I)-2-fluorophenylglycine,

4-iodo(124I)phenylglycine,

3-iodo(124I)phenylglycine,

4-iodo(124I)-2-fluorophenylglycine,

4-iodo(125I)phenylglycine,

3-iodo(125I)phenylglycine,

4-iodo(125I)-2-fluorophenylglycine,

4-iodo(131I)phenylglycine,

3-iodo(131I)phenylglycine, or

4-iodo(131I)-2-fluorophenylglycine,
more preferably

4-astato(211At)phenylalanine,

3-astato(211At)phenylalanine,

4-astato(211At)-2-fluorophenylalanine,

4-astato(210At)phenylalanine,

3-astato(210At)phenylalanine,

4-astato(210At)-2-fluorophenylalanine,

4-iodo(123I)phenylalanine,

3-iodo(123I)phenylalanine,

4-iodo(123I)-2-fluorophenylalanine,

4-iodo(124I)phenylalanine,

3-iodo(124I)phenylalanine,

4-iodo(124I)-2-fluorophenylalanine,

4-iodo(125I)phenylalanine,

3-iodo(125I)phenylalanine,

4-iodo(125I)-2-fluorophenylalanine,

4-iodo(131I)phenylalanine,

3-iodo(131I) phenylalanine, or

4-iodo(131I)-2-fluorophenylalanine,

particularly preferably

4-astato(211At)phenylalanine,

3-astato(211At)phenylalanine, or

4-astato(211At)-2-fluorophenylalanine.



[0067] Among the radiolabeled aryl compound (I), 3-astato(211At)phenylalanine is a novel compound. The compound is taken up in large amounts by cancer cells, and therefore, it is particularly expected to be applied to RI internal therapy for cancer.

Examples



[0068] The present invention is explained in detail by referring to the following Examples, which are not to be construed as limitative, and the invention may be changed within the scope of the present invention.

[0069] In the following Examples and Reference Examples, the radiochemical yield is calculated by the following formula. radiochemical yield (%) = (radioactivity of the desired compound on thin-layer plate or electrophoretic membrane/total radioactivity on thin-layer plate or electrophoretic membrane)×100

[0070] The thin-layer plate and electrophoretic membrane was exposed on BAS imaging plate (GE Healthcare), and the BAS imaging plate was analyzed by an image analyzer (Tyhoon FLA7000, GE Healthcare). The data processing was performed using ImageQuantTL Analysis Toolbox (GE Healthcare).

Reference Example 1 Preparation of an aqueous 211At solution



[0071] 211At was generated by 209Bi (α, 2n) 211At nuclear reaction, irradiating bismuth with helium particles accelerated (28 MeV) by cyclotron. After the irradiation, by heating, the target substance 209Bi was melted, but the 211At was vaporized, and then the vaporized 211At was trapped in liquid nitrogen, and dissolved in a small amount of water to give an 211At undiluted solution. To the obtained 211At undiluted solution was added a 7% aqueous sodium hydrogencarbonate solution to prepare an aqueous 211At solution having a radioactive concentration of about 5 MBq/ml (immediately after the production). The thin layer chromatography (TLC) of the aqueous 211At solution are shown in Figure 1 (thin-layer plate: G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)). The spots of 211At were detected on Rf=1.0 (80%) and 0.89 (8%).

Example 1 Synthesis of 4-211At-L-phenylalanine (oxidizing agent: NCS)



[0072] 4-Borono-L-phenylalanine (Bpa) was dissolved in a 7% aqueous sodium hydrogencarbonate solution to prepared a 10 mg/ml of solution. The solution (0.2 ml) was put into a small glass vial, and the aqueous 211At solution (5 MBq/ml, 0.2 ml) prepared in Reference Example 1 was added thereto, and then an aqueous N-chlorosuccinimide (NCS) solution (4 mg/ml, 0.04 ml) was slowly added dropwise thereto at room temperature. After 30 minutes, the reaction solution was analyzed by thin layer chromatography (TLC) (thin-layer plate: G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)) (Figure 2). The radiochemical yield of 4-211At-L-phenylalanine (Rf=0.73) was 95%.

Example 2 Synthesis of 4-211At-L-phenylalanine (oxidizing agent: NBS)



[0073] 4-Borono-L-phenylalanine (Bpa) was dissolved in a 7% aqueous sodium hydrogencarbonate solution to prepared a 10 mg/ml of solution. The solution (0.2 ml) was put into a small glass vial, and the aqueous 211At solution (5 MBq/ml, 0.2 ml) prepared in Reference Example 1 was added thereto, and then an aqueous N-bromosuccinimide (NBS) solution (4 mg/ml, 0.04 ml) was slowly added dropwise thereto at room temperature. After 30 minutes, the reaction solution was analyzed by thin layer chromatography (TLC) (thin-layer plate: G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)) (Figure 3). The radiochemical yield of 4-211At-L-phenylalanine (Rf=0.68) was 75.3%.

Example 3 Synthesis of 4-211At-L-phenylalanine (oxidizing agent: NaI)



[0074] 4-Borono-L-phenylalanine (Bpa) was dissolved in a 7% aqueous sodium hydrogencarbonate solution to prepared a 10 mg/ml of solution. The solution (0.2 ml) was put into a small glass vial, and the aqueous 211At solution (5 MBq/ml, 0.2 ml) prepared in Reference Example 1 was added thereto, and then an aqueous sodium iodide (NaI) solution (10 mg/ml, 0.1 ml) was slowly added dropwise thereto at room temperature. After 30 minutes, the reaction solution was analyzed by thin layer chromatography (TLC) (thin-layer plate: G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)) (Figure 4). The radiochemical yield of 4-211At-L-phenylalanine (Rf=0.79) was 90%.

Example 4 Synthesis of 4-211At-L-phenylalanine (oxidizing agent: NaBr)



[0075] 4-Borono-L-phenylalanine (Bpa) was dissolved in a 7% aqueous sodium hydrogencarbonate solution to prepared a 10 mg/ml of solution. The solution (0.2 ml) was put into a small glass vial, and the aqueous 211At solution (5 MBq/ml, 0.2 ml) prepared in Reference Example 1 was added thereto, and then an aqueous sodium bromide (NaBr) solution (10 mg/ml, 0.1 ml) was slowly added dropwise thereto at room temperature. After 30 minutes, the reaction solution was analyzed by thin layer chromatography (TLC) (thin-layer plate: G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)) (Figure 5). The radiochemical yield of 4-211At-L-phenylalanine (Rf=0.63) was 84.7%.

Example 5 Synthesis of 4-123I-L-phenylalanine (oxidizing agent: NBS)



[0076] 4-Borono-L-phenylalanine (Bpa) was dissolved in a 7% aqueous sodium hydrogencarbonate solution to prepared a 10 mg/ml of solution. The solution (0.2 ml) was put into a small glass vial, and an aqueous Na123I solution (74 MBq/ml, 5 mmol/1 aqueous NaOH solution, 0.2 ml) was added thereto, and then an aqueous N-bromosuccinimide (NBS) solution (4 mg/ml, 0.04 ml) was slowly added dropwise thereto at room temperature. After 30 minutes, the reaction solution was analyzed by thin layer chromatography (TLC) (thin-layer plate: G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)) (Figure 6b). The radiochemical yield of 4-123I-L-phenylalanine (Rf=0.70) was 93%. The thin layer chromatography (TLC) (thin-layer plate: G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)) of the aqueous Na123I solution is shown in Figure 6a.

Reference Example 2 Synthesis of 4-iodo-L-phenylalanine using non-radioactive iodine



[0077] 4-Borono-L-phenylalanine (Bpa) was dissolved in a 7% aqueous sodium hydrogencarbonate solution to prepared a 10 mg/ml of solution. The solution (0.3 ml) was put into a small glass vial, and an aqueous NaI solution (10 mg/ml, 0.3 ml) was added thereto, and then an aqueous N-bromosuccinimide (NBS) solution (10 mg/ml, 0.3 ml) was slowly added dropwise thereto at room temperature. After 30 minutes, the reaction solution was analyzed by thin layer chromatography (TLC) (thin-layer plate: G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)), and the single spot was detected on Rf=0.7 (ultraviolet radiation and coloration in iodine bath). Then, the reaction solution was 1000 fold-diluted with water, and analyzed by LC-MS for amino acid analysis, compared to commercially available 4-iodo-L-phenylalanine as a control sample. The product was detected at the same retention time as in the control sample 4-iodo-L-phenylalanine, and the extraction mass was also the same as that of the control sample (theoretical mass = 290.9756, extraction mass = 291.9835). The purity of the product was 98.9%. The impurity was L-phenylalanine (1.1%) alone, and 4-bromo-L-phenylalanine and the like were not detected.

Example 6 Synthesis of 4-211At-2-fluoro-L-phenylalanine (oxidizing agent: NBS)



[0078] 4-Borono-2-fluoro-L-phenylalanine (FBpa) was dissolved in a 7% aqueous sodium hydrogencarbonate solution to prepared a 5 mg/ml of solution. The solution (0.2 ml) was put into a small glass vial, and the aqueous 211At solution (5 MBq/ml, 0.1 ml) prepared in Reference Example 1 was added thereto, and then an aqueous N-bromosuccinimide (NBS) solution (4 mg/ml, 0.04 ml) was slowly added dropwise thereto at room temperature. After 15 minutes, the reaction solution was analyzed by cellulose acetate membrane electrophoresis method (Figure 7). The radiochemical yield of 4-211At-2-fluoro-L-phenylalanine (Rf=0.68) was 92.2%.

Example 7 Synthesis of 3-211At-D,L-phenylalanine



[0079] 3-Borono-D,L-phenylalanine (3-Bpa) was dissolved in a 1.4% aqueous sodium hydrogencarbonate solution to prepared a 10 mg/ml of solution. The solution (0.2 ml) was put into a small glass vial, and the aqueous 211At solution (5 MBq/ml, 0.2 ml) prepared in Reference Example 1 was added thereto, and then an aqueous N-bromosuccinimide (NBS) solution (4 mg/ml, 0.04 ml) was slowly added dropwise thereto at room temperature. After 30 minutes, the reaction solution was analyzed by thin layer chromatography (TLC) (thin-layer plate: silica gel G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)) (Figure 8). The radiochemical yield of the produced 3-211At-D,L-phenylalanine (Rf=0.76) was 93.3%.

Example 8 1-211At-2-hydroxybenzene



[0080] 2-Boronohydroxybenzene was dissolved in a 7% aqueous sodium hydrogencarbonate solution to prepared an aqueous solution having a concentration of 5 mg/ml. The aqueous solution was put into a small glass vial, and the aqueous 211At solution (5 MBq/ml, 0.2 ml) prepared in Reference Example 1 was added thereto, and then an aqueous N-bromosuccinimide (NBS) solution (4 mg/ml, 0.04 ml) was slowly added dropwise thereto at room temperature. After 30 minutes, an aqueous ascorbic acid solution (3 mg/ml, 0.03 ml) was added to the reaction solution to quench the reaction. The reaction solution was analyzed by thin layer chromatography (TLC) (thin-layer plate: silica gel G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)). The radiochemical yield of the produced 1-211At-2-hydroxybenzene (Rf=0.91) was 98.1%. This result demonstrates that the method of radiolabelling an aryl compound of the present invention can be applied not only to boronophenylalanine but also to boronohydroxybenzene.

Example 9 1-211At-4-carboxybenzene



[0081] 4-Barboxyphenylboronic acid was dissolved in a 7% aqueous sodium hydrogencarbonate solution to prepared an aqueous solution having a concentration of 16 mg/ml. The aqueous solution was put into a small glass vial, and the aqueous 211At solution (5 MBq/ml, 0.2 ml) prepared in Reference Example 1 was added thereto, and then an aqueous N-bromosuccinimide (NBS) solution (4 mg/ml, 0.04 ml) was slowly added dropwise thereto at room temperature. After 30 minutes, an aqueous ascorbic acid solution (3 mg/ml, 0.03 ml) was added to the reaction solution to quench the reaction. The reaction solution was analyzed by thin layer chromatography (TLC) (thin-layer plate: silica gel G60 (Merck), developing solvent: ACN:water:TFA (66:33:1)). The radiochemical yield of the produced 1-211At-4-carboxybenzene (Rf=0.81) was 87.5%. This result demonstrates that the method of radiolabelling an aryl compound of the present invention can be applied not only to boronophenylalanine but also to boronocarboxybenzene (carboxyphenylboronic acid).

Example 10 Glioma cell uptake experiment



[0082] The concentration of the 4-211At-L-phenylalanine (4-211At-Phe) solution prepared in Example 2 and the 3-211At-D,L-phenylalanine (3-211At-Phe) solution prepared in Example 7 were each adjusted to 5 MBq/2mg·ml. C6 glioma cell derived from rat was seeded on each well of 24-well plate by 5×105 cells/well, and the cells were used in the next day's experiment (cell number: about 1×106 cells/well). Before the experiment, the medium was replaced with an amino acid-free HBSS medium (0.5 ml/well). 4-211At-Phe or 3-211At-Phe was added to each well of the plate by 10 µl, and the wells were divided into three groups of inhibitor-free group, 1% Phe addition group (competitive inhibitor) and 100 mM BCH addition group (LAT1 inhibitor), and incubated at 37°C for 30 minutes or 60 minutes. After the incubation, the culture solution was removed, and the cells were washed with PBS, and lysed, and the amount of radioactivity taken up intracellularly was measured. The results are shown in Figure 9. 4-211At-Phe and 3-211At-Phe were both taken up by the cell, and the cell uptake of 3-211At-Phe was slightly more than that of 4-211At-Phe. Moreover, the cell uptake after 60 minutes was reduced, compared to that after 30 minutes. The competitive inhibitor (Phe) reduced the cell uptake of 4-211At-Phe and 3-211At-Phe to 1/2 to 1/3, and LAT1 inhibitor (BCH) reduced the cell uptake of 4-211At-Phe and 3-211At-Phe to 1/4 to 1/8. That is to say, the results demonstrated that 4-211At-Phe and 3-211At-Phe were both specifically taken up by the cells via LAT1, and therefore, 4-211At-Phe and 3-211At-Phe can be expected to be applied to RI internal therapy for cancer. Especially, the cell uptake of 3-211At-Phe was more than that of 4-211At-Phe, and therefore, 3-211At-Phe can be further expected for the above-mentioned application.

Example 11 SPECT imaging of C6 glioma-transplanted rat by 4-211At-L-phenylalanine



[0083] The 4-211At-L-phenylalanine (4-211At-Phe) (1 MBq) prepared in Example 2 was administered to the tail vein of the glioma-transplanted rat, and the rat was imaged by SPECT camera (E-cam, Siemens). The SPECT images 30 minutes and 3 hours after the administration are shown in Figure 10. In both images, the accumulation of 4-211At-Phe in the tumor (both flanks, indicated by arrows in the figure) was observed. Therefore, 4-211At-Phe can be expected to be applied to RI internal therapy or diagnosis for cancer.

Industrial Applicability



[0084] According to the production method of the present invention, the radiolabeled aryl compound (I) can be easily produced in a high radiochemical yield in a short time, and the formulation can be carried out immediately after the labelling. Therefore, the labelling and formulation can be easily carried out in a short time, and a sequence of processes from preparation of a radionuclide to RI internal therapy or diagnosis for cancer can be promptly carried out.

[0085] This application is based on patent application No. 2017-151632 filed on August 4, 2017 in Japan, the contents of which are encompassed in full herein.


Claims

1. A method of producing a radiolabeled aryl compound represented by the formula (I):

        Ar―X     (I)

wherein

Ar is a C6-14 aryl group optionally having substituent (s), and

X is 211At, 210At, 123I, 124I, 125I or 131I,

or a salt thereof, which comprises reacting an aryl boronic acid compound represented by the formula (II):

        Ar—Y     (II)

wherein

Ar is as defined above, and

Y is a borono group (-B(OH)2) or an ester group thereof,

or a salt thereof, with a radionuclide selected from 211At, 210At, 123I, 124I, 125I and 131I, in the presence of an oxidizing agent selected from an alkali metal iodide, an alkali metal bromide, N-bromosuccinimide, N-chlorosuccinimide and hydrogen peroxide, in water.
 
2. The method according to claim 1, wherein the reaction is carried out in an organic solvent-free system.
 
3. The method according to claim 1 or 2, wherein the reaction is carried out at room temperature.
 
4. The method according to any of claims 1 to 3, wherein the radionuclide is 211At or 210At, and the oxidizing agent is selected from sodium iodide, sodium bromide, N-bromosuccinimide, N-chlorosuccinimide and hydrogen peroxide.
 
5. The method according to any of claims 1 to 3, wherein the radionuclide is 123I, 124I, 125I or 131I, and the oxidizing agent is selected from N-bromosuccinimide and N-chlorosuccinimide.
 
6. The method according to any of claims 1 to 5, wherein Y is a borono group (-B(OH)2).
 
7. The method according to any of claims 1 to 6, wherein the substituent of the C6-14 aryl group optionally having substituent(s) represented by Ar is a group capable of binding specifically to a target molecule.
 
8. The method according to claim 7, wherein the target molecule is an antigen, a transporter, a receptor, an enzyme or a gene, which is specifically expressed or overexpressed in a cancer cell.
 
9. The method according to any of claims 1 to 8, wherein Ar is a group represented by the formula:

wherein

R2 is a halogen atom,

m is 0 or 1,

n is 0 or an integer of 1 to 4, and

* is a binding site to X or Y, or

a group derived from a peptide having a partial structure represented by the formula:

wherein

R2 is a halogen atom,

m is 0 or 1,

n is 0 or an integer of 1 to 4, and

* is a binding site to X or Y.


 
10. The method according to any of claims 1 to 8, wherein Ar is a group represented by the formula:

wherein

R3 is a hydrogen atom or a halogen atom,

m is 0 or 1, and

* is a binding site to X or Y, or

a group derived from a peptide having a partial structure represented by the formula:

wherein

R3 is a hydrogen atom or a halogen atom,

m is 0 or 1, and

* is a binding site to X or Y.


 
11. The method according to any of claims 1 to 8, wherein the aryl boronic acid compound represented by the formula (II) is 4-boronophenylalanine, 4-borono-2-fluorophenylalanine or 3-boronophenylalanine, and the radiolabeled aryl compound represented by the formula (I) is 4-astato (211At) phenylalanine, 4-astato (211At) -2-fluorophenylalanine or 3-astato (211At) phenylalanine.
 
12. 3-Astato (211At) phenylalanine or a salt thereof.
 




Drawing




























REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description