Murray et al.Supplementary MethodsPage 1

Monocarboxylate transporter MCT1 is a novel target for immunosuppression

Clare M. Murray1, Raymond Hutchinson1, John R. Bantick2, Graham P. Belfield3, Amanda D. Benjamin1, Diana Brazma1a, Robert V. Bundick1, I. David Cook1b, Robert I. Craggs1, Susan Edwards1, Leslie R. Evans3c, Richard Harrison2, Elain Holness1, Andrew P. Jackson3, Clive G. Jackson3, Lee P. Kingston2, Matthew W.D. Perry2, Andrew R. J. Ross3, Paul A. Rugman1, Sasvinder S. Sidhu1, Michael Sullivan3d, David A. Taylor-Fishwick1e, P. Craig Walker3, Yvonne M. Whitehead1, David J. Wilkinson2, Andrew Wright4, David K. Donald2

Departments of 1Discovery BioScience, 2Medicinal Chemistry, 3Molecular Biology and 4Physical and Metabolic Science, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, UK

Present addresses: a, Dept. of Academic Haematology, Royal Free and University College Medical School, London; b, Global Discovery Enabling Capabilities and Sciences, AstraZeneca R&D Alderley Park, Cheshire; c, Delta Biotechnology Ltd., Nottingham; d, Advanced Science and Technology Lab, AstraZeneca R&D Charnwood; e, Cell and Molecular Biology, Diabetes Research Institute, Eastern Virginia Medical School, Norfolk VA.

Correspondence should be addressed to C.M. ()

Supplementary Methods

  1. Synthetic Materials and Methods
  2. Synthesis of Compound 1
  3. Synthesis of Compound 2
  4. Synthesis of Compound 3H3-3
  5. Synthesis of Compound 125I-4
  6. Synthesis of Compound 5
  7. Synthesis of Compound 6
  8. Synthesis of Compound 8
  9. Synthesis of Compound 9
  10. Synthesis of Compound 10
  11. Synthesis of Compound 11
  12. References

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I. Synthetic Materials and Methods

Reagents were obtained from Aldrich Chemical or Lancaster Chemical companies and used without purification. High-performance liquid chromatography (HPLC) grade solvents were obtained from Fisher Scientific. Unless otherwise stated, reactions were carried out at ambient temperature (18-25C) and under positive nitrogen pressure with magnetic stirring. TLC was performed on Merck silica gel 60 F245 plates and visualised under UV light (254 nm) or by staining with potassium permanganate (KMnO4). Flash chromatography was performed on E. Merck 230-400 mesh silica gel 60. Preparative reverse phase (RP)HPLC separations were performed using a Waters Symmetry, Novapak or Xterra column. NMR spectra were recorded on a Varian Unity spectrometer at a proton frequency of either 300 or 400 MHz. Chemical shifts are expressed in ppm relative to TMS (1H, 0 ppm) or CDCl3 (13C, 77.0 ppm); coupling constants are expressed in Hz. Mass spectra were measured on either a VG 70-250S spectrometer using electron impact ionisation (EI) or on an Agilent 1100 MSD G1946D spectrometer using electrospray ionisation (ESI) or atmospheric pressure chemical ionisation (APCI); generally only ions which indicate the parent mass are reported.

Intermediates were characterised by mass spectrometry (MS) and/or NMR analysis and purity was assessed by either thin layer chromatography (TLC) and/or high-performance liquid chromatography (HPLC).

II. Synthesis of 5-[(3-hydroxypropyl)thio]-3-methyl-1-(2-methylpropyl)-6-(1-naphthalenylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (1)

Ethyl 2-amino-5-(1-naphthalenylmethyl)-3-thiophenecarboxylate (12)

A solution of oxalyl chloride (7.40 ml, 85.0 mmol, 2 equiv) in anhydrous dichloromethane (50 ml) was added dropwise to a stirred suspension of 3-(1-naphthyl)propanoic acid (8.50 g, 42.5 mmol, 1.0 equiv) in anhydrous dichloromethane (100 ml) and DMF (0.1 ml). After a further 2 h, the resulting solution was concentrated under reduced pressure and the residual oil dried in vacuo at 50C for 4 h. The oil was redissolved in anhydrous tetrahydrofuran (45 ml) and added to a mixture of 10% palladium on carbon (0.50 g) and anhydrous 2,6-lutidine (5.82 ml, 50.0 mmol, 1.2 equiv) in tetrahydrofuran (30 ml). The mixture was hydrogenated at 2 atmospheres for 4 days and then filtered through a kieselguhr pad. The filtrate was concentrated under reduced pressure and the residual oil dried under reduced pressure to give a solid. This solid was redissolved in anhydrous DMF (20 ml) then ethyl cyanoacetate (4.53 ml, 42.0 mmol, 1.0 equiv) and sulfur (1.35 g, 42.0 mmol, 1.0 equiv) were added and the mixture stirred at 50C for 2 h. Water (300 ml) followed by saturated sodium chloride solution (50 ml) was added and the mixture was extracted with diethyl ether (3300 ml). The combined organic extracts were dried (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (elution with 2:3 diethyl ether:hexane) to give compound (12) as a yellow solid (11.0 g, 82%).

H-NMR (300 MHz, DMSO-d6): 8.09-8.13 (m, 1H), 7.90-7.96 (m, 1H), 7.84 (d, 1H), 7.56-7.41 (m, 4H), 7.08 (s, br, 2H), 6.56 (s, 1H), 4.35 (s, 2H), 4.12 (q, 2H), 1.20 (t, 3H); APCI-MS m/z: (pos) 312 [M+H]+.

Ethyl 2-(2-methylpropyl)amino-5-(1-naphthalenylmethyl)-3-thiophenecarboxylate (13)

Sodium borohydride (1.30 g, 34.4 mmol, 2 equiv) was added in 10 portions over 5 h to a stirred solution of compound (12) (5.50 g, 17.6 mmol, 1 equiv) in 2-methylpropanoic acid (40 ml, 431 mmol, 25 equiv) at 0C. The mixture was stirred for 16 h then further sodium borohydride (1.80 g, 47.6 mmol, 2.7 equiv) was added in 10 portions over 8 h and stirring continued for a further 16 h. The solution was poured into water (1 L), neutralized with sodium bicarbonate and extracted with ethyl acetate (2500 ml). The organic extracts were dried (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (elution with 1:3 diethyl ether:hexane) to give compound (13) as a colourless solid (6.20 g, 89%).

mp: 57-59 C; H-NMR (300 MHz, DMSO-d6): 8.11-8.14 (m, 1H), 7.93 (m, 1H, m), 7.84 (dd, 1H), 7.5 (m, 4H), 6.70 (s, 1H), 4.40 (s, 2H), 4.14 (q, 2H), 2.91 (dd, 2H), 1.92-1.66 (m, 1H, m), 1.22 (t, 3H)0.86 (d, 6H); APCI-MS m/z: (pos) 368 [M+H]+.

Ethyl 2-[[(acetylamino)carbonyl](2-methylpropyl)amino]-5-(1-naphthalenylmethyl)-3-thiophenecarboxylic acid (14)

Acetyl chloride (1.08 ml, 15.8 mmol, 1.2 equiv) was added to a stirred suspension of silver cyanate (2.37 g, 15.8 mmol, 1.2 equiv) in anhydrous toluene (50 ml). After 1 h, compound (13) (4.65 g, 12.7 mmol, 1.0 equiv) was added and stirring was continued for 16 h. The mixture was filtered and the solid residue was washed with diethyl ether (50 ml). The combined liquors were concentrated under reduced pressure and the residue purified by flash chromatography (elution with 1:1 diethyl ether:hexane) to give compound (14) as an oil (5.05 g, 100%).

H-NMR (300 MHz, CDCl3): 7.99 (dd, 1H), 7.90 (dd, 1H), 7.84 (d, 1H), 7.58-7.41 (m, 4H), 7.30 (s, 1H,), 7.09 (s, 1H, s), 4.53 (s, 2H), 4.24 (q, 2H), 3.06-3.80 (2H, br), 2.44 (s, 3H), 1.78-1.92 (m, 1H), 1.29 (t, 3H), 0.87 (d, 6H); APCI-MS m/z: (pos) 453 [M+H]+.

3-Methyl-1-(2-methylpropyl)-6-(1-naphthalenylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (15)

Sodium ethoxide (0.18 g, 2.60 mmol, 3.5 equiv) was added to a stirred solution of compound (14) (0.30 g, 0.76 mmol, 1.0 equiv) in ethanol (6 ml). After 6 h, iodomethane (0.17 ml, 2.73 mmol, 1.0 equiv) was added. After a further 16 h iodomethane (0.17 ml, 2.73 mmol, 1.0 equiv) was added. After a further 24 h, the reaction mixture was poured onto dilute hydrochloric acid (30 ml) and extracted with ethyl acetate (230 ml). The organic extracts were dried (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (elution with 1:1 diethyl ether:hexane) and then triturated with diethyl ether to give compound (15) as a colourless solid (0.24 g, 87%).

mp:137-138 C; H-NMR (300 MHz, CDCl3 ): 8.02-7.95 (m, 1H), 7.90-7.86 (m, 1H), 7.82 (d, 1H), 7.52-7.4 (m, 4H), 7.04 (t, 1H), 4.52 (s, 2H), 3.68 (d, 2H), 3.38 (s, 3H), 2.32-2.18 (m, 1H), 0.93 (d, 6H); APCI-MS m/z: (pos) 379 [M+H]+.

5-[(3-Hydroxypropyl)thio]-3-methyl-1-(2-methylpropyl)-6-(1-naphthalenylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (1)

n-BuLi (2.0 M solution in hexanes, 0.32 ml, 0.64 mmol, 1.2 equiv) was added dropwise to a solution of diisopropylamine (0.093 ml, 0.52 mmol, 1.0 equiv) in tetrahydrofuran (5ml) at 0C. The solution was stirred for 5 minutes then cooled to -78C and a solution of compound (15) (0.20 g, 0.52mmol, 1.0 equiv) in tetrahydrofuran (5 ml) was added. After 15 min, a solution of S-[3-[[(1,1-dimethylethyl)dimethylsilyl]oxy]propyl] 4-methylbenzenesulfonothioate1 (0.19 g, 0.52 mmol, 1.0 equiv) in anhydrous tetrahydrofuran (2 ml) was added. The mixture was stirred for a further 1 h at -78C then allowed to warm to room temperature. After 16 h the reaction mixture was poured onto saturated aqueous sodium bicarbonate solution (30 ml) and then extracted with diethyl ether (230 ml). The organic extracts were dried (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (elution with diethyl ether) and was then recrystallised from ethyl acetate/hexane to give compound (1) as colourless crystals (0.098 g, 40%).

mp: 130-131C; H-NMR (300 MHz, CDCl3 ): 8.07-8.01 (m, 1H), 7.92-7.84 (m, 1H), 7.82 (d, 1H), 7.56-7.45 (m, 2H), 7.44 (t, 1H), 7.35 (d, 1H), 4.78 (s, 2H), 3.89 (q, 2H), 3.63 (d, 2H), 3.42 (s, 3H), 3.17 (t, 2H), 2.84 (t, 1H), 2.24-2.10 (m, 1H), 1.90 (quin, 2H), 0.88 (d, 6H); C NMR (75 MHz, DMSO-d6): 157.05, 152.69, 150.64, 137.22, 135.32, 133.50, 131.07, 128.68, 127.73, 126.78, 126.48, 126.03, 125.65, 124.44, 123.48, 113.30, 59.45, 54.96, 32.54, 32.43, 31.02, 27.99, 26.43, 19.73; APCI-MS m/z: (pos) 469 [M+H]+; analysis (calcd,found for C25H28N2O3S2):C (64.08,63.86), H (6.02,5.99), N (5.98,6.11), S (13.68,13.55).

III. Synthesis of 2,6-dihydro-7-[(3-hydroxypropyl)thio]-2-methyl-4-(2-methylpropyl)-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyridazin-1-one (2)

Methyl 4-(3-methyl-1-oxobutyl)-1H-pyrrole-3-carboxylate (16)

A solution of (E)-methyl 6-methyl-4-oxo-2-heptenoate2 (10.0g, 0.06 mol, 1.0 equiv) and (para-toluenesulphonyl)methyl isocyanide (11.5g, 0.06 mol, 1.0 equiv) in a mixture of dry DMSO (30ml) and diethyl ether (30ml) was added over 1 h to sodium hydride (2.75g of a 60% oil dispersion, 0.068mol, 1.0 equiv) stirred in dry diethyl ether (90ml). After a further 1 h, saturated ammonium chloride solution was added and the mixture was extracted with ethyl acetate, which was washed well with water, dried (MgSO4), and concentrated under reduced pressure to a gum. The gum was purified by flash chromatography (elution with 2:3 ethyl acetate:iso-hexane) to afford a solid (3.30g, 27%) which was crystallised from ethyl acetate/cyclohexane to give compound (16) as colourless crystals.

mp: 136C; H-NMR (300 MHz, CDCl3): 8.8 (br, 1H), 7.37 (t, 1H), 7.26 (t, 1H), 3.83 (s, 3H), 2.78 (d, 2H), 2.22 (m, 1H), 0.96 (d, 6H); APCI-MS m/z: (pos) 210 [M+H]+.

Methyl 4-(3-methyl-1-oxobutyl)-1-(1-naphthalenylmethyl)-1H-pyrrole-3-carboxylate (17)

To sodium hydride (0.42g of a 60% oil dispersion, 10.5mmol, 1.0 equiv), freed from oil, stirred in dry DMF (15ml) was added compound (16) (2.2g, 10.5 mmol, 1.0 equiv) in portions over 20 min. After 10 min, potassium iodide (0.01g) and (1-naphthalenyl) methyl chloride (1.85g, 10 mmol, 1.0 equiv) in DMF (20ml) were added. The mixture was stirred for 4 h and then poured into 0.5 M hydrochloric acid and extracted with ethyl acetate. The organic layer was washed well with water and then brine, dried (MgSO4), and concentrated under reduced pressure to a gum, which was purified by flash chromatography (elution with 1:3 ethyl acetate: iso-hexane) to give an oil. The oil was crystallised from cyclohexane to yield compound (17) as colourless crystals (2.6g, 71%).

mp: 81-82C; H-NMR (300 MHz, CDCl3):  7.95 (m, 2H), 7.80 (m, 1H), 7.55 (m, 2H), 7.46 (dd, 1H), 7.25 (m, 1H), 7.23 (d, 1H, J=2.7 Hz), 7.17 (d, 1H, J=2.7 Hz), 5.50 (s, 2H), 3.79 (s, 3H), 2.76 (d, 2H), 2.2 (m, 1H), 0.94 (d, 6H); APCI-MS m/z: (pos) 350 [M+H]+.

2,6-Dihydro-2-methyl-4-(2-methylpropyl)-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyridazin-1-one (18)

Compound (17) (0.35g, 1 mmol, 1.0 equiv) and methyl hydrazine (0.10ml, 2.0 mmol, 2.0 equiv) in ethanol (15ml) were heated at reflux for 16 h. The mixture was poured into dilute hydrochloric acid and extracted with ethyl acetate, which was washed with brine, dried (MgSO4), and concentrated under reduced pressure to a gum. The gum was purified by flash chromatography (elution with 1:1 ethyl acetate: iso-hexane) to afford a solid, which was recrystallised from cyclohexane to give compound (18) as colourless crystals (0.16g, 46%).

mp: 110-112C; H-NMR (300 MHz, CDCl3):  7.91 (m, 2H), 7.83 (m, 1H), 7.53 (m, 3H), 7.52 (d, 1H, J=2.1 Hz), 7.22 (d, 1H), 7.04 (d, 1H, J=2.1 Hz), 5.73 (s, 2H), 3.71 (s, 3H), 2.53 (d, 2H), 2.11 (m, 1H), 0.94 (d, 6H); APCI-MS m/z: (pos) 346 [M+H]+.

2,6-Dihydro-7-[(3-hydroxypropyl)thio]-2-methyl-4-(2-methylpropyl)-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyridazin-1-one (2)

To compound (18) (0.345g, 1.0 mmol, 1.0 equiv) and S-[3-[[(1,1-dimethylethyl)dimethylsilyl]oxy]propyl] 4-methylbenzenesulfonothioate1(0.72g, 2.0 mmol, 2.0 equiv) in tetrahydrofuran (10ml) stirred at -78C was added lithium diisopropylamide in tetrahydrofuran (0.39M, 5.1ml, 2.0 mmol, 2 equiv). The reaction mixture was allowed to warm to room temperature overnight and then saturated ammonium chloride solution was added. The mixture was extracted with ethyl acetate, which was then washed with brine, dried, and concentrated under reduced pressure to a gum. The gum was purified by flash chromatography (elution with 1:1 ethyl acetate: iso-hexane) to give a solid. To a stirred suspension of this solid in acetonitrile (10ml) was added 40% hydrofluoric acid (0.07ml). After 16 h, aqueous sodium bicarbonate solution was added and the mixture was partially concentrated under reduced pressure to leave a residue. The residue was extracted with ethyl acetate, which was washed with brine, dried (MgSO4), and concentrated under reduced pressure to a solid. The solid was purified by flash chromatography (elution with 3:2 ethyl acetate: iso-hexane) to give, after triturating with diethyl ether: iso-hexane, compound (2) as a colourless solid (0.17g, 39%).

mp: 152C; H-NMR (300 MHz, CDCl3):  7.95-7.89 (m, 2H), 7.85 (d, 1H), 7.61-7.53 (m, 2H), 7.39 (dd, 1H), 7.05 (s, 1H), 6.75 (d, 1H), 5.97 (s, 2H), 4.01-3.87 (m, 3H), 3.75 (s, 3H), 3.12 (t, 2H), 2.48 (d, 2H), 1.98-2.12 (m, 1H), 1.77 (quin, 2H), 0.90 (d, 6H);C NMR (75 MHz, DMSO-d6): 156.72, 142.84, 133.43, 133.09, 129.73, 128.68, 127.97, 126.71, 126.25, 125.58, 123.19, 122.77, 122.55, 120.10, 119.73, 117.71, 59.18, 48.72, 41.05, 37.61, 33.66, 32.16, 27.27, 22.46; APCI-MS m/z: (pos) 436 [M+H]+; analysis (calcd,found for C25H29N3O2S):C (68.90,68.97), H (6.70,6.80), N (9.65,9.72), S (7.36,7.18).

IV. Synthesis of 5-[(3-hydroxypropyl)thio]-3-methyl-1-[2-(methyl-t)propyl-2,3-t2]-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione (3H3-3)

1,2,3,4-Tetrahydro-1-(2-hydroxypropyl)-3,6-dimethyl-2,4-dioxo-5-pyrimidinecarbonitrile (19)

To a solution of ethyl N-(2-cyano-3-ethoxy-1-oxo-2-butenyl)-carbamate3 (5.0 g, 22.1 mmol, 1.0 equiv) in ethanol (50 ml) at reflux was added DL-1-amino-2-propanol (1.88 ml, 23.6 mmol, 1.1 equiv). The reaction mixture was stirred for 5h at reflux then cooled and concentrated under reduced pressure. The resulting gum was suspended in water (50 ml), treated with sodium hydroxide (1.46 g, 36.5 mmol, 1.7 equiv) and then stirred for 1 h. Dimethyl sulphate (3.45 ml, 36.4 mmol, 1.6 equiv) was added and stirring was continued for 1 h. The precipitate was collected and the aqueous solution was concentrated then extracted into dichloromethane. The organic solution was dried (MgSO4) and concentrated under reduced pressure. The residue was combined with the precipitate (above) to afford compound (19) as a solid (4.35g, 88%).

H-NMR (300 MHz, CDCl3):  4.24-4.16 (m, 1H), 4.10 (dd, 1 H), 3.70 (dd, 1 H), 3.34 (s, 3H), 2.70 (s, 3H), 2.67 (d, 1H), 1.31 (d, 3H); EI-MS m/z: (pos) 223 M+.

1-(2-Hydroxypropyl)-3-methyl-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione (20)

Compound (19) (4.24g, 19.0 mmol, 1.0 equiv) was suspended in 75% formic acid (80ml) and Raney Nickel (50% dispersion in 8ml water) was added. The mixture was heated at 90oC for 15 min. After cooling the suspension was filtered through a kieselguhr pad and concentrated under reduced pressure. The residue was dissolved in water (100ml) and extracted into ethyl acetate, each aliquot of extraction was washed with sodium bicarbonate solution. The combined organic extracts were dried (MgSO4) and concentrated under reduced pressure to yield a colourless foam which was dissolved in chloroform (20ml) and heated to 50oC. A solution of bromine (0.4 ml, 7.81 mmol, 0.4 equiv) in chloroform (5 ml) was added and after stirring for 10 min at 50oC the mixture was concentrated under reduced pressure. The residue was dissolved in ethanol (25 ml), treated with triethylamine (2.96 ml, 19.0 mmol, 1.0 equiv) and then 1-naphthalenylmethylamine (1.55 ml, 10.5 mmol, 0.55 equiv) was added. After 20 h at room temperature the reaction was poured into 2M HCl (100 ml) and extracted with ethyl acetate, dried (MgSO4) and then concentrated under reduced pressure. The residue was purified by flash chromatography (elution with 2:1 iso-hexane:ethyl acetate) to give compound (20) as a solid (1.18g, 17%).

H-NMR (300 MHz, CDCl3):  7.92-7.89 (m, 2H), 7.83-7.80 (m, 1H), 7.56-7.49 (m, 2H), 7.47 (t, 1H), 7.27-7.24 (m, 2H), 6.44 (s, 1H), 5.56 (s, 2H), 4.26-4.18 (m, 1H), 3.80 (d, 2 H), 3.37 (s, 3H), 2.80 (d, 1H), 1.25 (d, 3H); EI-MS m/z: (pos) 363 M+.

3-Methyl-6-(1-naphthalenylmethyl)-1-(2-oxopropyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione (21)

A solution of DMSO (0.42 ml, 5.9 mmol, 2.2 equiv) in dichloromethane (10 ml) was added dropwise to a solution of oxalyl chloride (0.26 ml, 2.7 mmol, 1.0 equiv) in dichloromethane (20 ml) at -78oC. After 15 min a solution of compound (20) (970 mg, 2.7 mmol, 1.0 equiv) in dichloromethane (20 ml) at -78oC was added. After 5 min triethylamine (0.9 ml, 6.5 mmol, 2.4 equiv) was added, the reaction was stirred for 10 min then allowed to warm to 0oC. Water (100 ml) was added and the mixture was extracted with dichloromethane, dried (MgSO4) and concentrated under reduced pressure. The residue was purified by flash chromatography (elution with 2:3 iso-hexane:ethyl acetate) to give compound (21) as a solid (0.66g, 68%).

H-NMR (300 MHz, CDCl3):  7.93-7.89 (m, 2H), 7.81-7.78 (m, 1H), 7.56-7.50 (m, 2H), 7.47 (t, 1H), 7.30-7.24 (m, 2H), 6.21 (s, 1H), 5.53 (s, 2H), 4.49 (s, 2H), 3.38 (s, 3H), 2.19 (s, 3H); EI-MS m/z: (pos) 361 M+.

3-Methyl-1-(2-methyl-2-propenyl)-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione (22)

To a stirred suspension of methylenetriphenylphosphonium bromide (1.22 g, 3.41 mmol, 2.2 equiv) in dry tetrahydrofuran (20 ml) at -78oC was added sodium hexamethyldisilazide (1M solution in tetrahydrofuran; 3.1ml, 3.1 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature for 1 h. The resulting solution was added to a solution of compound (21) (560 mg, 1.55 mmol, 1.0 equiv) in dry tetrahydrofuran (30 ml) at 0oC, and stirred at 5oC for 2 h then at room temperature for 20 min. The mixture was poured into water (50 ml) and extracted into ethyl acetate, dried (MgSO4) and concentrated under reduced pressure. The residue was purified by flash chromatography (elution with 3:1 iso-hexane:ethyl acetate) to give compound (22) as a solid (0.47g, 84%).

H-NMR (300 MHz, CDCl3):  7.92-7.88 (m, 2H), 7.82-7.80 (m, 1H), 7.56-7.50 (m, 2H), 7.46 (t, 1H), 7.22-7.20 (m, 2H), 6.39 (s, 1H), 5.55 (s, 2H), 4.90 (s, 1H), 4.81 (s, 1H), 4.39 (s, 2H), 3.39 (s, 3H), 1.72 (s, 3H); EI-MS m/z: (pos) 359 M+.

5-[(3-Hydroxypropyl)thio]-3-methyl-1-(2-methyl-2-propenyl)-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione (23)

Compound (22) (350 mg, 0.97 mmol, 1.0 equiv) and S-[3-[[(1,1-dimethylethyl)dimethylsilyl]oxy]propyl] 4-methylbenzenesulfonothioate1 (527 mg, 1.46 mmol, 1.5 equiv) were dissolved in dry tetrahydrofuran (10 ml) at -78oC. LDA (1.95 mmol, 2.0 equiv) in dry tetrahydrofuran (5 ml) was added and after 1 h the temperature was raised to 0oC. The reaction was quenched by addition of sodium bicarbonate solution (30 ml) and was extracted with diethyl ether. Drying and evaporation gave a residue which was dissolved in acetonitrile (10 ml) and treated with 40% hydrofluoric acid (0.4 ml) for 30 min. The reaction mixture was poured into sodium bicarbonate solution and extracted into ethyl acetate, dried (MgSO4) and concentrated under reduced pressure. The residue was purified by flash chromatography (elution with 3:1 iso-hexane:acetone) to give compound (23) (0.26g, 59%) which was subsequently recrystallised from iso-hexane /ethyl acetate to give colourless crystals.

mp: 151-152C; H-NMR (300 MHz, CDCl3):  7.79 (m, 2H), 7.83 (d, 1H), 7.58 (m, 2H), 7.39 (t, 1H), 6.76 (d, 1H), 6.37 (s, 1H), 5.83 (s, 2H), 4.83 (s, 1H), 4.72 (s, 1H), 4.35 (s, 2H), 3.83 (dd, 2H), 3.44 (s, 3H), 3.12 (t, 2H), 1.80 (m, 2H), 1.67 (s, 3H); APCI-MS m/z: (pos) 450 [M+H]+.

5-[(3-Hydroxypropyl)thio]-3-methyl-1-[2-(methyl-t)propyl-2,3-t2]-6-(1- naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione (3H3-3)

Compound (23) (2.28 mg, 0.0051 mmol), 10% Pd/carbon (2.35 mg) and ethanol (0.5 ml) were placed in a 1 ml round-bottomed flask which was attached to a tritium manifold. The contents of the flask were frozen in liquid nitrogen and the flask then evacuated before tritium gas (241 GBq, 2.6 ml, 0.113 mmol) was introduced. The flask was allowed to warm to room temperature and the contents left to stir for 22 hours. The flask was removed from the apparatus and the catalyst removed by filtration. The filtrate was diluted with ethanol (5 ml) and the solvent removed under reduced pressure. This was repeated with a further portion of ethanol (5 ml).

Compound (3H3-3) was purified using preparative HPLC. The volume of the (3H3-3) containing fraction was made up to 10 ml by the addition of 50% w/v aqueous sodium thiosulphate (0.1 ml) and ethanol. The radiochemical purity was typically >97%. The radioactive concentration was determined by liquid scintillation counting and was normally found to be in the range of 24 to 35 MBq ml-1.

V. Synthesis of 6-[[4-azido-3-(iodo-125I)phenyl]methyl]-2,6-dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one (125I-4)

1-Azido-4-chloromethyl-2-iodo-benzene (24)

To a solution of 4-azido-3-iodo-benzenemethanol4(350 mg, 1.27 mmol, 1.0 equiv) in dichloromethane (20 ml) was added triethylamine (0.185 ml, 1.33 mmol, 1.0 equiv) and methanesulphonyl chloride (0.10 ml, 1.29 mmol, 1.0 equiv). The mixture was stirred for 20 h then concentrated under reduced pressure. The residue was purified by flash chromatography (elution with 1:4 ethyl acetate: iso-hexane) to give compound (24) (210 mg, 56%) as a pale orange oil.

H-NMR (300 MHz, CDCl3): 7.82(d, 1H), 7.42(dd, 1H), 7.11(d, 1H), 4.51(s, 2H).

2,6-Dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one (25)

Compound (16) (0.7g, 3.3 mmol, 1.0 equiv) and methyl hydrazine (0.60ml, 11.3 mmol, 3.4 equiv) in ethanol (20ml) were heated at reflux for 16 h. The mixture was poured into dilute hydrochloric acid and extracted with ethyl acetate, which was washed with brine, dried (MgSO4), and concentrated under reduced pressure to give compound (25) as a gum (0.65g, 95%).

H-NMR (300 MHz, CDCl3):  11.56 (br s, 1H), 7.57 (t, 1H), 7.24 (t, 1H), 3.78 (s, 3H), 2.62 (m, 2H), 2.32-2.08 (m, 1H), 0.97 (d, 6H); APCI-MS m/z: (pos) 206 [M+H]+.

6-[(4-Azido-3-iodophenyl)methyl]-2,6-dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one (4)

Compound (25) (25 mg, 0.12 mmol, 1.0 equiv), compound (24) (40 mg, 0.14 mmol, 1.2 equiv) and caesium carbonate (40 mg, 0.12 mmol, 1.0 equiv) were mixed in dry DMF (5 ml). After stirring for 3 days the reaction was poured into water and extracted into ethyl acetate. The organic solution was washed with brine, dried (MgSO4) and concentrated under reduced pressure. The residue was purified by flash chromatography (elution with 1:3 ethyl acetate: iso-hexane) to give compound (4) (50 mg, 89%) as a colourless solid.

mp: 138-139C; H-NMR (300 MHz, CDCl3):  7.62 (d, 1H), 7.47 (d, 1H), 7.17 (dd, 1H), 7.11 (d, 1H), 6.98 (d, 1H), 5.20 (s, 2H), 3.72 (s, 3H), 2.55 (d, 2H), 2.13 (m, 1H), 0.96 (d, 6H); APCI-MS m/z: (pos) 463 [M+H]+.

6-[(4-Azido-3-(trimethylstannyl)phenyl)methyl]-2,6-dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one (26)

Compound (4) (10 mg, 0.02 mmol, 1.0 equiv), hexamethylditin (0.10 ml, 0.48 mmol, 24 equiv) and tetrakis(triphenylphosphine)palladium(0) (2 mg, 0.001 mmol, 0.1.0 equiv) were combined in dry toluene (5 ml) and heated at 100oC for 4 h. After cooling, the mixture was concentrated under reduced pressure and the residue was purified by preparative thin layer chromatography (elution with ethyl acetate) to afford compound (26) as a colourless oil (10 mg, 92%).

H-NMR (300 MHz, CDCl3):  7.46 (d, 1H), 7.22-7.10 (m, 3H), 7.01 (d, 1H), 5.22 (s, 2H), 3.72 (s, 3H), 2.55 (d, 2H), 2.13 (m, 1H), 0.96 (d, 6H), 0.3 (s, 9H (+Sn satellites)); APCI-MS m/z: (pos) 501 [M+H]+.

6-[[4-azido-3-(iodo-125I)phenyl]methyl]-2,6-dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one (125I-4)

To a solution of sodium [125I]iodide (Amersham Pharmacia Biotech, IMS30; 2000 Ci mmol-1, 1 mCi; 0.5 nmol, 10 µl) was added a solution of compound (26) (10 µl, 3.0 nmol) followed by chloramine-T in water (2 µl, 3.6 nmol). The vial was sealed, shaken and left to stand for 10 mins. An aliquot of sodium metabisulphite (2 µl, 16 nmol) was added to the reaction followed by methanol (25 µl) and the vial shaken.