11C -labeled 06 -benzylguanine, pet probe capable of visualizing 06-methyl guanine methyl-transferase activity, and production method of the same

Abstract

An object of the invention is to provide a .sup.11C-labeled O.sup.6-benzylguanine capable of obtaining a PET image and a process for producing the same. The .sup.11C-labeled O.sup.6-benzylguanine of the invention is represented by the following chemical formula (a). ##STR00001##
The .sup.11C-labeled O.sup.6-benzylguanine is produced by: a coupling step of cross-coupling a methyl iodide labeled with .sup.11C and the following organotin compound (b) (R.sup.1 represents an alkyl group, and R.sup.2 and R.sup.3 represent a leaving group which can be eliminated with a base) in the presence of a palladium complex, a phosphine ligand, and cuprous halide in an aprotic lactam; and a desorption step of desorbing the leaving groups R.sup.2 and R.sup.3 of the coupling product obtained by the coupling step with a base. ##STR00002##

Claims

1. A PET probe comprising a .sup.11C-labeled O.sup.6-benzylguanine represented by the following chemical formula (a): ##STR00013## which has a chemical purity of 99% or more and a radiochemical purity is 99% or more.

2. The PET probe comprising a .sup.11C-labeled O.sup.6-benzylguanine according to claim 1, wherein a methyl group labeled with .sup.11C is bonded to a meta position of a benzene ring.

3. A method for imaging an MGMT, comprising: administering the PET probe of claim 1 to a living body; and capturing a PET image of said probe in said living body.

4. A method for producing the PET probe comprising a .sup.11C-labeled O.sup.6-benzylguanine according to claim 1, comprising: a coupling step of cross-coupling a methyl iodide labeled with .sup.11C and the following organotin compound (b) (R1 represents an alkyl group, and R.sup.2 and R.sup.3 represent a leaving group which can be eliminated with a base) in the presence of a palladium complex, a phosphine ligand, and cuprous halide in an aprotic lactam, and a desorption step of desorbing the leaving groups R.sup.2 and R.sup.3 of the coupling product obtained by the coupling step with a base: ##STR00014##

5. The method for producing the .sup.11C-labeled O.sup.6-benzylguanine according to claim 4, wherein SnR.sup.1.sub.3 is bonded to a meta position of a benzene ring.

6. The method for producing the .sup.11C-labeled O.sup.6-benzylguanine according to either claim 4 or claim 5, wherein R.sup.2 is a trifluoroacetyl group and R.sup.3 is a tert-butoxycarbonyl group.

7. The method for producing the .sup.11C-labeled O.sup.6-benzylguanine according to claim 4, wherein the aprotic lactam is N-alkyl-2-pyrrolidinone.

8. The method for producing the .sup.11C-labeled O.sup.6-benzylguanine according to claim 4, wherein in the coupling step, at least one of a carbonate salt, a fluoride of an alkali metal and an alkali metal salt of ascorbic acid is added.

9. The method of claim 3, wherein the PET image is of the brain of the living body.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a graph showing the MGMT inhibitory effect of the compounds (1) to (4).

(2) FIG. 2 is a diagram showing a synthetic route of methylation of organotin compound 5 having no protecting group for amino group and organotin compound 6 having amino group protected with trifluoro group by [.sup.11C] CH.sub.3I.

(3) FIG. 3 is a labeling synthesis reaction formula and an HPLC chart in the case where the organotin compound 5 not protected with an amino group is methylated by [.sup.11C] CH.sub.3I.

(4) FIG. 4 is a labeling synthesis reaction formula and an HPLC chart in the case where the organotin compound 6 having the amino group at the 2-position protected with a trifluoroacetyl group is methylated by [.sup.11C]CH.sub.3I.

(5) FIG. 5 is a labeling synthesis reaction formula and an HPLC chart in the case where an organotin compound 7 in which the amino group at the 2-position is protected with a trifluoroacetyl group and the amino group at the 9-position is protected with a tert-butoxycarbonyl (BOC) group is methylated by [.sup.11C] CH.sub.3I (An added base is sodium ascorbate).

(6) FIG. 6 is a labeling synthesis reaction formula and an HPLC chart in the case where an organotin compound 7 in which the amino group at the 2-position is protected with a trifluoroacetyl group and the amino group at the 9-position is protected with a tert-butoxycarbonyl (BOC) group is methylated by [.sup.11C] CH.sub.3I (K.sub.2CO.sub.3 is added as a base).

(7) FIG. 7 is an HPLC chart showing changes in blood concentration after administration of pMeBG (3) to SD rats.

(8) FIG. 8 is a graph showing the time course of the average plasma concentration of mMeBG (2) and its metabolites after administration of Compound 2 to rats.

(9) FIG. 9 is a graph showing the time course of the mean plasma concentration of pMeBG (3) and its metabolites after administration of Compound 3 to rats.

MODE FOR CARRYING OUT THE INVENTION

(10) Study on Methylation of O.sup.6-benzylguanine

(11) ##STR00006##

(12) The introduction position of the [.sup.11C] methyl group of the benzene ring present in the above-mentioned O.sup.6-benzylguanine (1) was investigated. For this purpose, unlabeled O.sup.6-(3-methylbenzyl) guanine (2), O.sup.6-(4-methylbenzyl) guanine (3) and O.sup.6-(3,5-dimethylbenzyl) guanine (4) were synthesized by reaction of corresponding alcohols with 2-amino-6-chloropurine according to the method described in the following literature of Liu et al. Then, the inhibitory activity of these MGMTs was investigated. X. Liu, Q.-H. Zheng, G. D. Hutchins, X. Fei, L. C. Erickson, K. D. Miller, B. H. Mock, B. E. Glick-Wilsob, W. L. Winkel, K. L. Stone, K. A. Carlson, Synth. Commun. 2003, 33, 941-952.

(13) The enzyme inhibitory activity against MGMT of O.sup.6-(3-methylbenzyl) guanine (2), O.sup.6-(4-methylbenzyl) guanine (3) and O.sup.6-(3,5-dimethylbenzyl) guanine (4), were investigated using fluorescent quantitatively labeled oligonucleotide substrates with DNA damage specific to MGMT for quantification. As a result, these three compounds all had MGMT inhibitory effect similar to O.sup.6-benzylguanine (1), particularly O.sup.6-(3-methylbenzyl) guanine (2) and O.sup.6-(4-methylbenzyl) guanine 3) had a slightly higher inhibitory activity than O.sup.6-BG (1) (see FIG. 1). Based on this result, the inventors used O.sup.6-(3-methylbenzyl) guanine (2) as a target for labeling by .sup.11C.

(14) Specifically, we decided to utilize the sp.sup.3-sp.sup.2 (heteroaromatic ring) type cross-coupling reaction between [.sup.11C] CH.sub.3I and the heteroaromatic ring organotin compound using the Pd.sup.0 catalyst we have already developed (Patent document 1). In consideration of the effect of using bulky phosphine ligand in excess on the Pd.sup.0 complex, the effect of using an aprotic polar solvent, and the effect of addition of copper ions and base to prevent progress of tinning reaction by acidic protons, the reaction was carried out under the condition of [Pd.sub.2(dba).sub.3]/P(o-CH.sub.3C.sub.6H.sub.4).sub.3/CuCl/K.sub.2CO.sub.3/NMP solvent. However, almost no desired .sup.11C-labeled O.sup.6-benzylguanine was obtained (see Route 1 in FIG. 2 and FIG. 3). Therefore, a two-step synthesis method was tried in which the amino group contained in the guanine structure was protected with trifluoroacetate with trifluoroacetic anhydride and deprotection reaction was carried out after the reaction. Then, introduction of the protecting group occurred only at the 2-amino group of O.sup.6-benzylguanine, and an organotin compound 6 was obtained. This compound was subjected to [.sup.11C] CH.sub.3 conversion under the same conditions and deprotected with lithium hydroxide, whereby O.sup.6-(3-methylbenzyl) guanine (2) labeled with .sup.11C was obtained with 72% HPLC analysis yield (See Route 2 in FIG. 2 and FIG. 4).

(15) Furthermore, an organotin compound 7 in which the amino group at the 2-position of O.sup.6-benzylguanine was protected with a TFA group and the amino group at the 9-position was protected with a tert-butoxycarbonyl (BOC) group was prepared and deprotected with a strong base, O.sup.6-(3-methylbenzyl) guanine (2) labeled with the objective .sup.11C-labeled compound was obtained with high HPLC analysis yield of 97±1% (see FIG. 6). The total time for synthesis and purification in this case is 47 minutes, the total radiation is 1.34 GBq, the specific activity is 95-99 GBq/mmol, the disintegration-corrected yield is 19%, the chemical purity is 99% or more, the radiochemical purity is 99% or more, which was found to be sufficiently usable as a PET probe.

(16) Hereinafter, embodiments of the present invention will be described in detail.

(17) (Analyzer and Conditions Used in Examples)

(18) .sup.1H, .sup.13C, .sup.19F NMR was measured by ECS 400 manufactured by JEOL Ltd. The chemical shift was set to 0.00 ppm when TMS was used as a standard, this was set to 7.26 for .sup.1H NMR and 77.0 for .sup.13C NMR when CHCl.sub.3 was used as a standard, and was set at −76.55 ppm for .sup.19F NMR with CF.sub.3COOH as a standard. Also, the abbreviations s, d, t, q and m represent singlet, doublet, triplet, quartet, and multiplet, respectively. For high resolution mass spectrometry (HRMS), PE Biosystems, Mariner system or JEOL JMS-700/GI was used. All [.sup.11C] methylation reactions were performed remotely in a draft shielded with lead. [.sup.11C] CO.sub.2 was produced by a .sup.14N (p, α) .sup.11C nuclear reaction using a CYPRIS HM-18 cyclotron (manufactured by Sumitomo Heavy Industries, Ltd.). Heating, dilution of the reaction mixture, injection of sample into HPLC, collection and concentration of fractions were carried out by a radiopharmaceutical automatic synthesizer to obtain .sup.11C-labeled compound. Radioactivity was quantified with ATOMLAB™ 500 dose calibrator (Biodex Medical Systems, Inc.). The synthesized [.sup.11C]methylated compound was analyzed by Shimadzu HPLC system combining bias supply 925-SCINT (AMETEK Co., Ltd.) and linear count rate meter 7131-1 (Ohyo Koken Kogyo Co., Ltd.). The Shimadzu HPLC system consists of system controller (SPD—10 Avp), online degasser (DGU—12 A), solvent delivery unit (LC—10 ATvp), column oven (CTO—10 A), photodiode array detector (SCL—10 Avp) and software (LC-solution).

Synthesis of O.SUP.6.-[3-(tributylstannyl) benzyloxy]guanine (5)

(19) ##STR00007##

(20) 3-(Tributylstannyl)benzyl alcohol (8.5 g, 21 mmol) was placed in a 30 mL round bottom flask and heated to 130° C. Then, under a nitrogen stream, sodium (197 mg, 8.6 mmol) was added. After confirming that all the sodium was dissolved, the temperature was lowered to 115° C. and 2-amino-6-chloropurine (8, 1.2 g, 7.1 mmol) was added under an argon stream. And the mixture was stirred at the same temperature overnight. The obtained solution was cooled to room temperature, the undissolved solid was removed by filtration, the filtrate was concentrated under reduced pressure, and the resulting material was subjected to silica gel column chromatography using a mixed solution of dichloromethane:methanol=9:1 as an eluent, 51.15 g (2.17 mmol, 30.4%) of the compound was obtained as a pale yellow solid.

(21) δ.sub.H (400 MHz; CDCl.sub.3) 0.86 (9H, t, J=7.4 Hz, CH.sub.3), 0.95-1.11 (6H, m, CH.sub.2), 1.26-1.35 (6H, m, CH.sub.2), 1.48-1.56 (6H, m, CH.sub.2), 5.02 (2H, s, NH.sub.2), 5.54 (2H, s, CH.sub.2), 7.28-7.64 (4H, m, Ph), 7.70 (1H, s, NCH), 11.4-12.3 (1H, brs, NH); δ.sub.C (100 MHz; CDCl.sub.3) 9.6 (3C), 13.7 (3C), 27.4 (3C), 29.1 (3C), 68.7, 128.0, 128.3, 128.7, 135.5, 136.4, 136.5, 142.3, 159.2).

Synthesis of O.SUP.6.-[3-(tributylstannyl)benzyloxy]-N.SUP.2.-(trifluoroacetyl)guanine (6)

(22) ##STR00008##

(23) Triethylamine (53 μL, 37 mg, 0.38 mmol) was added to a solution (5 mL) of Compound 5 (192 mg, 0.363 mmol) in dry dichloromethane, and the mixture was cooled to 0° C. Trifluoroacetic anhydride (53 μL, 80 mg, 0.38 mmol) was added to the mixture and the mixture was stirred for 2 hours. The reaction mixture solution was poured into 10 ml of water and extracted three times with 10 ml of dichloromethane and then the organic layer was combined and washed with 15 ml of water and 15 ml of brine and dried with anhydrous sodium sulfate. After filtration, concentration was carried out. The obtained crude product was purified by column chromatography (silica gel (50 g), dichloromethane:methanol=20:1), Compound 6 (125 mg, 0.201 mmol, 55.3%) was obtained.

(24) δ.sub.H (400 MHz; CDCl.sub.3) 0.87 (9H, t, J=7.4 Hz, CH.sub.3), 0.97-1.11 (6H, m, CH.sub.2), 1.25-1.34 (6H, m, CH.sub.2), 1.48-1.56 (6H, m, CH.sub.2), 5.66 (2H, s, CH.sub.2), 7.33-7.56 (4H, m, Ph), 8.33 (1H, s, NCH), 8.62 (1H, s, NHCOCF.sub.3), 12.7 (1H, s, NH); δ.sub.C (100 MHz; CDCl.sub.3) 9.5 (3C), 13.6 (3C), 27.3 (3C), 27.9 (3C), 29.0 (3C), 70.1, 87.6, 120.1, 128.0, 128.9, 134.5, 136.7, 137.0, 141.0, 142.5, 145.8, 151.3, 152.0, 153.5 (q, .sup.1J(.sup.13C-.sup.19F)=38.3 Hz), Ph 161.4, 153.171.1; δ.sub.F (MHz; CDCl.sub.3) −76.80 (s, CF.sub.3).

Synthesis of N-(tert-butoxycarbonyl)-O.SUP.6.-(3-bromobenzyl)guanine (11)

(25) ##STR00009##

(26) O.sup.6-(3-Bromobenzyl) guanine (10, 1.41 g, 4.42 mmol) was added to a solution of potassium tert-butoxide (496 mg, 4.42 mmol) in ethanol (5 mL) at 0° C. under an argon stream. After 30 minutes, ethanol was removed under reduced pressure. The residue was dissolved in DMF (5 mL) and brought to 0 C, and a solution DMF (2 mL) of (Boc).sub.2O (978 μL, 964 mg, 4.42 mmol) was added. The mixture was stirred overnight at room temperature, quenched with water and extracted with ethyl acetate. The crude product was purified by column chromatography (silica gel (50 g), dichloromethane:methanol=20:1), Compound 11 (125 mg, 0.201 mmol, 55.3%) as a pale yellow solid was obtained.

(27) δ.sub.H (400 MHz; CDCl.sub.3) 1.67 (9H, s, CH.sub.3), 5.16 (2H, s, NH.sub.2), 5.51 (2H, s, CH.sub.2), 7.23 (1H, t, J=8.8 Hz, Ph), 7.41 (1H, d, J=8.8 Hz, Ph), 7.47 (1H, d, J=8.8 Hz, Ph), 7.64 (1H, s, Ph), 8.01 (1H, s, NCH); δ.sub.C (100 MHz; CDCl.sub.3) 28.0 (3C), 67.2, 86.3, 115.9, 122.5, 130.0, 131.2 (2C), 137.8, 138.4, 146.1, 153.9, 160.5, 161.0.

Synthesis of N.SUP.9.-(tert-butoxycarbonyl)-O.SUP.6.-[3-(tributylstannyl)benzyl]guanine (12)

(28) ##STR00010##

(29) Compound 11 (576 mg, 1.36 mmol), hexabutyl distannane (2.0 mL, 2.4 g, 4.1 mmol), tetrakis (triphenylphosphine) palladium (0) (157 mg, 0.136 mmol) was added to a dried 50 ml Schlenk tube in an argon atmosphere, and freshly distilled 1,4-dioxane (10 mL) was added. The mixture was heated to 80° C., stirred overnight, then cooled to room temperature and a palladium component was removed by passing the reaction through celite. The filtrate was concentrated in vacuo and the residue was purified by silica gel column chromatography using an eluent of hexane:ethyl acetate=3:1, 629 mg (73% yield) of Compound 12 as a pale yellow solid was obtained.

(30) δ.sub.H (400 MHz; CDCl.sub.3) 0.87 (9H, t, J=7.4 Hz, CH.sub.3), 0.98-1.11 (6H, m, CH.sub.2), 1.27-1.36 (6H, m, CH.sub.2), 1.49-1.57 (6H, m, CH.sub.2), 1.66 (9H, s, CH.sub.3), 5.14 (2H, s, NH.sub.2), 5.55 (2H, s, CH.sub.2), 7.32 (1H, t, J=7.3 Hz, Ph), 7.41 (1H, d, J=7.3 Hz, Ph), 7.45 (1H, d, J=7.3 Hz, Ph), 7.54 (1H, s, Ph), 8.00 (1H, s, NCH); δ.sub.C (100 MHz; CDCl.sub.3) 9.6 (3C), 13.6 (3C), 27.3 (3C), 28.0 (3C), 29.0 (3C), 68.7, 86.2, 116.0, 127.9, 128.4, 135.3, 136.3, 136.6, 137.5, 142.2, 146.2, 153.8, 160.5, 161.4.

Synthesis of N.SUP.9.-(tert-butoxycarbonyl)-O6-[3-(tributylstannyl)benzyl]-N2-(trifluoroacetyl)guanine (7)

(31) ##STR00011##

(32) Trimethylamine (153 μL, 101 mg, 962 μmol) was added to a dry dichloromethane (5 mL) solution of Compound 12 (629 mg, 0.998 mmol), after cooling the mixed solution to 0° C., trifluoroacetic anhydride (153 μL, 231 mg, 1.10 mmol) was added and the mixture was stirred for 2 hours. The reaction solution was poured into about 10 ml of water and extracted with dichloromethane (15 ml×3). The organic layers were combined, washed with water (15 mL) and brine (15 mL), and then dried over anhydrous sodium sulfate. The crude product obtained by filtration and concentration of the solution was purified by column chromatography (silica gel (5 g), hexane/acetone=3:1), Compound 7 (330 mg, 0.454 mmol, 45.5%) as a colorless solid was obtained.

(33) δ.sub.H (400 MHz; CDCl.sub.3) 0.87 (9H, t, J=7.4 Hz, CH.sub.3), 0.98-1.11 (6H, m, CH.sub.2), 1.26-1.34 (6H, m, CH.sub.2), 1.48-1.54 (6H, m, CH.sub.2), 1.71 (9H, s, CH.sub.3), 5.69 (2H, s, CH.sub.2), 7.33 (1H, t, J=7.3 Hz, Ph), 7.43 (1H, d, J=7.3 Hz, Ph), 7.53 (1H, d, J=7.3 Hz, Ph), 7.63 (1H, s, Ph), 8.31 (1H, s, NCH), 8.61 (1H, s, NH); δ.sub.C (100 MHz; CDCl.sub.3) 9.5 (3C), 13.6 (3C), 27.3 (3C), 27.9 (3C), 29.0 (3C), 70.1, 87.6, 120.1, 128.0, 128.9, 134.5, 136.7, 137.0, 141.0, 142.5, 145.8, 151.3, 152.0, 153.5 (q, .sup.1J(.sup.13C-.sup.19F)=38.3 Hz), Ph 161.4, 153.171.1; δ.sub.F (MHz; CDCl.sub.3) −76.75 (s, CF.sub.3).

(34) HRMS (ESI) m/z:

(35) [M+H].sup.+ calcd for C.sub.31H.sub.45F.sub.3N.sub.5O.sub.4 .sup.120Sn 728.2446; found 728.2464

(36) [M+Na].sup.+ calcd for C.sub.31H.sub.45F.sub.3N.sub.5O.sub.4 .sup.120Sn 750.2265; found 750.2267

Synthesis of O.SUP.6.-(3-methylbenzyl)-guanine (2)

(37) ##STR00012##

(38) [Pd.sub.2(dba).sub.3] (1.0 mg, 1.1 μmol), P(o-tolyl).sub.3 (5.4 mg, 18 μmol), CuCl (0.4 mg, 4 μmol), and K.sub.2CO.sub.3 (0.8 mg, 5 μmol) were added to a 1 mL Schlenk tube filled with dried argon gas. A NMP (300 μL) solution of organotin compound 7 (8.7 mg, 12 μmol) was introduced into a Schlenk tube cooled to −10° C. through a stainless steel cannula. Further, after adding a methyl iodide solution (12 μL, 0.10 M in DMF, 1.2 μmol), the mixture was reacted at 80° C. for 4 minutes. Then, a 1.0 M LiOH aqueous solution (600 μL) was added and after stirring at 80° C. for 3 minutes, the reaction solution was quickly cooled in an ice bath. The mixture obtained was introduced into a short column of silica gel (1 g), eluted with acetonitrile (2 mL), and a naphthalene solution (0.10 M DMF, 10 μL, 1.0 μmol) was added as an internal standard. The solution obtained was analyzed by HPLC (mobile phase, acetonitrile/20 mM sodium dihydrogenphosphate=40:60; column, CAPCELL PAK C18 MG 5 μm, 4.6 (id)×250 mm flow rate 1 mL/min UV detection 280 nm Retention time 3.4 min). As a result, the yield of Compound 2 was 100%. Identification of compound 2, using organotin compound 7 (8.7 mg, 12 μmol) and methyl iodide (120 μL, 0.10 M in DMF, 12 μmol) [Pd.sub.2(dba).sub.3]/P(o-CH.sub.3C.sub.6H.sub.4).sub.3/CuCl/K.sub.2CO.sub.3 (1:16:2:5 molar ratio) and reacted at 80° C., was carried out by .sup.1H NMR.

(39) .sup.1H NMR (DMSO-d.sub.6) δ=2.32 (s, 3H, CH.sub.3), 5.44 (s, 2H, CH.sub.2), 6.26 (s, 2H, NH.sub.2), 7.16-7.29 (m, 4H, Ar), 7.82 (s, 1H, guanine H8), 12.51 (broad s, 1H, guanine H9).

Synthesis of O.SUP.6.-(3-[.SUP.11.C]methylbenzyl)guanine ([.SUP.11.C](2)) when K.SUB.2.CO.SUB.3 .is Used as a Base

(40) A mixture of [Pd.sub.2(dba).sub.3] (1.0 mg, 1.1 μmol), P(o-tolyl).sub.3 (3.4 mg, 11 μmol), CuCl (0.4 mg, 4 μmol), K.sub.2CO.sub.3 (1.4 mg, 11 μmol), and Organotin Compound 7 (0.8 mg, 1.1 μmol) dissolved in NMP (300 μL) was cooled to keep the temperature below −10° C. until [.sup.11C]CH.sub.3I was prepared. [.sup.11C]CH.sub.3I was prepared from [.sup.11C]CO.sub.2 by the conventionally known LiAlH.sub.4 method, and in the state where cooling was stopped, [.sup.11C]CH.sub.3I gas was introduced into the reaction solution containing palladium, and rapidly heated to 100° C., the reaction solution was bubbled with nitrogen gas for 2 minutes after 2 minutes. After bubbling, the reaction solution was cooled for 10 seconds, 0.5 M LiOH (600 μL) was added to the mixture, and bubbling with nitrogen at 100° C. for 3 minutes was performed. Then the reaction solution was diluted with a CH.sub.3CN/H.sub.2O (35:65 v/v, 600 μL) solution containing sodium ascorbate (2.2 mg, 11 μmol), then passed through a fine filter F (F 162, Forte Grow Medical co., Ltd.) filled with quartz glass wool (Tosho co., Ltd.) and separated by HPLC (mobile phase, CH.sub.3CN/20 mM sodium phosphate (pH 4.9) 5:95 and 35:65 column, CAPCELL PAK C 18 MG 120 20 (id)×250 mm flow rate 10 ml/min UV detection 280 nm retention time 21 min). From the peak area in radio-HPLC, the HPLC analysis yield was 97±1% (n=3) (see FIG. 6). The fraction containing the desired product was placed in a flask and the organic solvent was removed under reduced pressure. The residue was dissolved in physiological saline (3.0 mL) in which 0.25% polysorbate was dissolved, and a formulation for intravenous administration was prepared. Total synthesis time from completion of irradiation to preparation of injection containing purification by preparative HPLC totaled 47 minutes. After completion of the synthesis, the radioactivity was 1.76±0.42 GBq (n=2), and the specific activity was 95 to 99 GBq/μmol (n=2). The disintegration-corrected yield calculated on the trapped [.sup.11C]CH.sub.3I was 20±1% (n=2). .sup.11C labeled Compound 2 was confirmed by co-injection of unlabeled Compound 2 on HPLC (mobile phase, CH.sub.3CN and 20 mM sodium phosphate=40:60, CAPCELL PAK C 120, 20 (id)×250 mm flow rate 1 ml/min, UV detection 280 nm, retention time 6.1 min). The chemical purity measured at 280 nm wavelength was >92% and the radiochemical purity was >97%. Higher radioactive formulations tended to have lower radiochemical purity.

Synthesis of O.SUP.6.-(3-[.SUP.11.C] methylbenzyl)guanine ([.SUP.11.C](2)) when Sodium Ascorbate is Used as a Base

(41) A mixture of [Pd.sub.2(dba).sub.3] (1.0 mg, 1.1 μmol), P(o-tolyl).sub.3 (3.4 mg, 11 μmol), CuCl (0.4 mg, 4 μmol), sodium ascorbate (2.2 mg, 11 μmol), and Organotin Compound 7 (0.8 mg, 1.1 μmol) dissolved in NMP (300 μL) was cooled to keep the temperature below −10° C. until [.sup.11C]CH.sub.3I was prepared. [.sup.11C]CH.sub.3I gas was introduced into the reaction solution and rapidly heated to 100° C., then bubbled with nitrogen gas for 2 minutes after 2 minutes. After bubbling, the reaction solution was cooled for 10 seconds, 0.5 M LiOH (600 μL) was added to the mixture, and bubbling with nitrogen at 100° C. for 3 minutes was performed. Then the reaction solution was diluted with a CH.sub.3CN/H.sub.2O (35:65 v/v, 600 μL) solution containing sodium ascorbate (2.2 mg, 11 μmol), then passed through a fine filter F (F 162, Forte Grow Medical co., Ltd.) filled with quartz glass wool (Tosho co., Ltd.) and separated by HPLC (mobile phase, CH.sub.3CN/20 mM sodium phosphate (pH 4.9) 5:95 and 35:65 column, CAPCELL PAK C 18 MG 120 20 (id)×250 mm flow rate 10 ml/min UV detection 280 nm retention time 21 min). From the peak area in radio-HPLC, the HPLC analysis yield was 86±9% (n=3) (see FIG. 5). The fraction containing the desired product was placed in a flask and the organic solvent was removed under reduced pressure. The residue was dissolved in physiological saline (3.0 mL) in which 0.25% polysorbate was dissolved, and a formulation for intravenous administration was prepared. Total synthesis time from completion of irradiation to preparation of injection containing purification by preparative HPLC totaled 48 to 51 minutes. After completion of the synthesis, the radioactivity was 0.92±0.32 GBq (n=3), and the specific activity was 74 to 76 GBq/μmol (n=3). The disintegration-corrected yield calculated on the trapped [.sup.11C]CH.sub.3I was 15±8% (n=3). .sup.11C labeled Compound 2 was confirmed by co-injection of unlabeled Compound 2 on HPLC (mobile phase, CH.sub.3CN and 20 mM sodium phosphate=40:60, CAPCELL PAK C 120, 20 (id)×250 mm flow rate 1 ml/min, UV detection 280 nm, retention time 6.1 min). The chemical purity measured at 280 nm wavelength was >97% and the radiochemical purity was >93%.

(42) In the .sup.11C-labeled O.sup.6-benzylguanine of the above example, a methyl group labeled with .sup.11C was bonded to the meta position of the benzene ring, but it is also possible to bind a methyl group labeled with .sup.11C to the ortho position or the para position of the benzene ring by changing the bonding position of the organotin substituent in the organotin compound as a raw material.

(43) <Pharmacokinetics Test Using Rats>

(44) Regarding O.sup.6-(3-methylbenzyl) guanine (2) and O.sup.6-(4-methylbenzyl) guanine (3) not labeled with .sup.11C synthesized by the above method (that is, cold), brain permeability was evaluated by the following method.

(45) After intravenous administration (10 mg/kg) of O.sup.6-(3-methylbenzyl) guanine (2) or O.sup.6-(4-methylbenzyl) guanine (3) to Sprague-Dawley rats, blood and brain tissues were collected after a predetermined time, and analyzed by HPLC. The results are shown in Table 1 and FIGS. 1 to 3.

(46) TABLE-US-00001 TABLE 1 Concentration in the brain and brain/plasma ratio after intravenous administration (10 mg/kg) of mMeBG (2) (or pMeBG (3)) to rats Conc. Time.sup.a Conc. HPLC.sup.b Brain Weight Conc. brain.sup.c Plasma.sup.d min μg/mL g μg/mL μg/mL Brain/Plasma ratio.sup.h mMeBG (2) Under 30 0.507 Ave. S.D. 1.860 Ave. S.D. 1.325 Ave. S.D. 4.450 e 0.298 Ave. S.D. anaesthesia, 0.568 0.513 0.052 1.840 1.845 0.013 1.495 1.348 0.137 4.450 0.336 0.303 0.031 fasting 0.464 1.835 1.223 4.450 0.275 Under 30 0.536 Ave. S.D. 1.861 Ave. S.D. 1.400 Ave. S.D. 5.192 f 0.270 Ave. S.D. nonanaesthesia, 0.635 0.523 0.067 1.909 1.829 0.086 1.634 1.379 0.152 5.192 0.315 0.265 0.029 fasting 0.474 1.677 1.323 5.192 0.255 0.481 1.785 1.291 5.192 0.249 0.454 1.854 1.188 5.192 0.229 0.555 1.888 1.436 5.192 0.277 Under 30 0.784 Ave. S.D. 2.121 Ave. S.D. 1.893 Ave. S.D. 4.450 e 0.425 Ave. S.D. anaesthesia, 0.684 0.663 0.132 1.885 1.992 0.120 1.772 1.661 0.303 4.450 0.398 0.373 0.068 nonfasting 0.522 1.970 1.318 4.450 0.296 pMeBG (3) Under 44 0.118 2.020 0.470 2.481 g 0.189 anaesthesia, 32 0.231 1.900 0.959 2.481 0.386 fasting Abbreviations: average (Ave.), standard deviation (S.D.) .sup.aTime is blood removal time after i.v. administration. .sup.bConc. HPLC is concentration (micro g/mL) of compound in brain homogenate determined by HPLC analysis. .sup.cConc. brain (micro g/mL) was calculated from [conc. HPLC (micro g/mL)] × [volume of brain homogenate (mL)]/[brain weight (g)]. Here, the specific gravity of brain is set to 1, and micro g/g is converted to micro g/mL. .sup.dPlasma sample was collected from another rat different from brain sample. .sup.eAverage (n = 5) of the plasma concentration 30 min after i.v. administration (10 mg/kg) of 2 in rat under anaesthesia and fasting. .sup.fAverage (n = 3) of the plasma concentration 30 min after i.v. administration (10 mg/kg) of 2 in rat under nonanaesthesia and fasting. .sup.gAverage (n = 3) of the plasma concentration 30 min after i.v. administration (10 mg/kg) of 3 in rat under anaesthesia and fasting. .sup.hBrain/Plasma ratio was calculated from [conc. brain (micro g/mL)]/[conc. plasma (micro g/mL)].

(47) The results of collecting samples of brain tissue and blood 30 minutes after administration of Compound (2) (or Compound (3)) are shown in Table 1. The amount of the compound in the brain is calculated from the brain weight and the brain concentration in Table 1, from the ratio of the amount of the compound in the brain to the dose, the concentration of the compound in the brain of the compound (2) and the compound (3) were calculated to be 0.1% based on the total dose. In addition, the ratio to the concentration in the plasma was 0.3. From the above results, it was evaluated that the compound (2) and the compound (3) are substances having blood-brain barrier permeability.

(48) Compound (2) and compound (3) stably exist in the plasma, but as shown in FIG. 7 and FIG. 9, compound (3) is quickly metabolized to unknown, more polar compounds within 240 minutes after intravenous administration to rats, as shown in FIG. 8, it was found that the compound (2) was more stable than the compound (3) and was present for a long time.

(49) It was clarified by the above body pharmacokinetics test that O.sup.6-(3-methylbenzyl)guanine (2) and O.sup.6-(4-methylbenzyl)guanine (3) are substances with blood-brain barrier permeability and are accumulated in the brain. Since these compounds have the property of binding to MGMT in the brain, O.sup.6-benzylguanine of the present invention labeled with .sup.11C of these compounds is useful as a PET probe, and it became possible to image MGMT in the brain.

(50) The present invention is not limited to the embodiments and examples of the invention. Various modifications are also included in the present invention as long as those skilled in the art can easily devise without departing from the scope of the claims.

INDUSTRIAL APPLICABILITY

(51) Since the present invention can be used for imaging of MGMT using a PET apparatus, it can be used for research on in vivo dynamics of MGMT, cancer diagnosis in brain cancer, and the like.