Murray et al.
Supplementary Methods
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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. (Clare.Murray@astrazeneca.com)
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Supplementary Methods
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Supplementary Methods
I.
Synthetic Materials and Methods
II.
Synthesis of Compound 1
III.
Synthesis of Compound 2
IV. Synthesis of Compound 3H3-3
V.
Synthesis of Compound 125I-4
VI. Synthesis of Compound 5
VII. Synthesis of Compound 6
VIII. Synthesis of Compound 8
IX. Synthesis of Compound 9
X.
Synthesis of Compound 10
XI. Synthesis of Compound 11
XII. References
………………………………………………………………………………………….
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
Murray et al.
Supplementary Methods
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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 highperformance liquid chromatography (HPLC).
II. Synthesis of 5-[(3-hydroxypropyl)thio]-3-methyl-1-(2-methylpropyl)-6-(1naphthalenylmethyl)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-(1naphthyl)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]+.
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Supplementary Methods
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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 2methylpropanoic 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-(1naphthalenylmethyl)-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]+.
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Supplementary Methods
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3-Methyl-1-(2-methylpropyl)-6-(1-naphthalenylmethyl)thieno[2,3-d]pyrimidine2,4(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-(1naphthalenylmethyl)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] 4methylbenzenesulfonothioate1 (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%).
Murray et al.
Supplementary Methods
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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-(2methylpropyl)-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 (10.0 g, 0.06 mol, 1.0 equiv)
and (para-toluenesulphonyl)methyl isocyanide (11.5 g, 0.06 mol, 1.0 equiv) in a
mixture of dry DMSO (30 ml) and diethyl ether (30 ml) was added over 1 h to sodium
hydride (2.75 g of a 60% oil dispersion, 0.068 mol, 1.0 equiv) stirred in dry diethyl
ether (90 ml). 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 (3.30 g, 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-3carboxylate (17)
To sodium hydride (0.42 g of a 60% oil dispersion, 10.5 mmol, 1.0 equiv), freed from
oil, stirred in dry DMF (15 ml) was added compound (16) (2.2 g, 10.5 mmol, 1.0
Murray et al.
Supplementary Methods
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equiv) in portions over 20 min. After 10 min, potassium iodide (0.01 g) and (1naphthalenyl) methyl chloride (1.85 g, 10 mmol, 1.0 equiv) in DMF (20 ml) 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 (2.6 g, 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)-1Hpyrrolo[3,4-d]pyridazin-1-one (18)
Compound (17) (0.35 g, 1 mmol, 1.0 equiv) and methyl hydrazine (0.10 ml, 2.0
mmol, 2.0 equiv) in ethanol (15 ml) 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 (0.16 g, 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-(1naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyridazin-1-one (2)
To compound (18) (0.345 g, 1.0 mmol, 1.0 equiv) and S-[3-[[(1,1dimethylethyl)dimethylsilyl]oxy]propyl] 4-methylbenzenesulfonothioate1 (0.72 g, 2.0
Murray et al.
Supplementary Methods
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mmol, 2.0 equiv) in tetrahydrofuran (10 ml) stirred at -78 C was added lithium
diisopropylamide in tetrahydrofuran (0.39M, 5.1 ml, 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 (10 ml) was added 40% hydrofluoric acid (0.07 ml). 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 (0.17 g, 39%).
mp: 152 C; H-NMR (300 MHz, CDCl3):  7.95-7.89 (m, 2H), 7.85 (d, 1H), 7.617.53 (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-2,3t2]-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-5pyrimidinecarbonitrile (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
Murray et al.
Supplementary Methods
Page 9
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[3,4d]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 isohexane:ethyl acetate) to give compound (20) as a solid (1.18 g, 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+.
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3-Methyl-6-(1-naphthalenylmethyl)-1-(2-oxopropyl)-1H-pyrrolo[3,4d]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 isohexane:ethyl acetate) to give compound (21) as a solid (0.66 g, 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[3,4d]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 (0.47 g, 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+.
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Supplementary Methods
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5-[(3-Hydroxypropyl)thio]-3-methyl-1-(2-methyl-2-propenyl)-6-(1naphthalenylmethyl)-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-[[(1,1dimethylethyl)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) (0.26 g,
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-(1naphthalenylmethyl)-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).
Murray et al.
Supplementary Methods
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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) (0.7 g, 3.3 mmol, 1.0 equiv) and methyl hydrazine (0.60 ml, 11.3
mmol, 3.4 equiv) in ethanol (20 ml) 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 (0.65 g, 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)-1Hpyrrolo[3,4-d]pyridazin-1-one (4)
Murray et al.
Supplementary Methods
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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-(2methylpropyl)-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-(2methylpropyl)-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.
Murray et al.
Supplementary Methods
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The iodo-azide product (125I-4) was purified using preparative HPLC. The
radiochemical purity was typically >99%. The radioactive concentration was
determined by liquid scintillation counting and was normally found to be in the range
of 2 to 3 MBq ml-1. The radiochemical yield was typically between 20 - 30%.
VI. Synthesis of 1-[[1,2,3,4-tetrahydro-1-(2-methylpropyl)-2,4-dioxo-6-[[2(trifluoromethyl)phenyl]methyl]thieno[2,3-d]pyrimidin-5-yl]carbonyl]azetidine
(5)
Diethyl 2-amino-3,4-thiophenedicarboxylate (27)
Triethylamine (85.4 ml, 0.61 mol, 1.0 equiv) was added dropwise over 15 min to a
stirred suspension of ethyl pyruvate (69.3 g, 0.61 mol, 1.0 equiv), ethylcyanoacetate
(65.2 ml, 0.61 mol, 1.0 equiv) and sulfur (19.6 g, 0.61 mol, 1.0 equiv) in DMF (300
ml). After the addition was complete the reaction was heated at 50°C for a further 3 h
and then cooled to room temperature. The black solution was poured into water (2 L)
and extracted with diethyl ether (x 3). The combined organic extracts were washed
with brine and dried (MgSO4). The solution was then filtered through a large pad of
silica, washing with diethyl ether (approx. 4 L). The filtrate was concentrated under
reduced pressure to give compound (27) as an orange solid (86.9 g, 58%).
H-NMR
(300 MHz, CDCl3):  6.60 (s, 1H), 5.97 (s, 2H), 4.34-4.22 (m, 4H), 1.38-
1.21 (m, 6H); ESI-MS m/z: (pos) 244 [M+H]+.
Diethyl 2-[(2-methylpropyl)amino]-3,4-thiophenedicarboxylate (28)
Sodium borohydride (20.0 g, 0.53 mol, 2.5 equiv) was added portionwise over 2 h to a
stirred suspension of compound (27) (50.0 g, 0.2 mol, 1 equiv) in 2-methylpropanoic
acid (300 ml, 3.23 mol, 6 equiv) and stirred for 16 h. Further sodium borohydride
(2.0 g, 5.30 mmol, 0.25 equiv) was added and the mixture was stirred for 2 h. The
reaction mixture was diluted with water and neutralised with solid sodium
bicarbonate, then extracted with diethyl ether (x3). The combined organic extracts
were dried (MgSO4) and concentrated under reduced pressure to give an oil. The
Murray et al.
Supplementary Methods
Page 15
residue was purified by flash chromatography (elution with 5:95 ethyl acetate: isohexane) to give compound (28) as a yellow oil (55.5 g, 90%).
H-NMR
(300 MHz, CDCl3):  7.70 (br t, J=5 Hz, 1H), 6.94 (m, 1H), 4.30 (q, J=7.1
Hz, 2H), 4.24 (q, J=7.1 Hz, 2H), 3.06-3.02 (m, 2H), 1.98 (m, 1H), 1.35 (t, J=7.1 Hz,
3H), 1.30 (t, J=7.1 Hz, 3H), 1.07-0.90 (m, 6H); ESI-MS m/z: (pos) 300 [M+H]+.
1-[[1,2,3,4-Tetrahydro-1-(2-methylpropyl)-2,4-dioxothieno[2,3-d]pyrimidin-5yl]carbonyl]azetidine (29)
Acetyl chloride (15.8 ml, 0.22 mol, 1.2 equiv) was added dropwise to a stirred
suspension of compound (28) (55.5 g, 0.19 mol, 1.0 equiv) and silver cyanate (34.8 g,
0.23 mol, 0.25 equiv) in toluene (200 ml). The reaction mixture was stirred
overnight. Diethyl ether (1 L) was added and the resulting suspension was filtered.
The filtrate was washed with saturated aqueous sodium bicarbonate solution, brine,
dried (MgSO4) and concentrated under reduced pressure to give a yellow oil. Sodium
(12.8 g, 0.56 mol, 3.0 equiv) was added portionwise to ethanol (250 ml) and stirred
for 5 h. This solution was then added portionwise to the above oil in ethanol (50 ml)
and the mixture was stirred for a further 5 h. The solvent was concentrated under
reduced pressure to approx 50 ml, then poured onto saturated aqueous sodium
bicarbonate solution (300 ml) and extracted with ethyl acetate (x3). The combined
organic extracts were dried (MgSO4) then concentrated under reduced pressure to
give an oil. To a suspension of this oil (4.45 g, 16 mmol) in dichloromethane (100
ml) was added oxalyl chloride (4.1 mmol, 47 mmol, 3.0 equiv), followed by DMF
(catalytic) and stirred for 2 h then concentrated under reduced pressure. The residue
was redissolved in dichloromethane (50 ml). Azetidine hydrochloride (3.0 g, 32
mmol, 2.0 equiv) was added followed by triethylamine (8.8 ml, 63 mmol, 4.0 equiv)
and then stirred for 1 h. The reaction mixture was concentrated under reduced
pressure. The residue was purified by flash chromatography (elution with 2:98
methanol:dichloromethane ) to give an oil which was triturated with diethyl ether to
give compound (29) as a colourless solid (4.71 g, 97%).
H-NMR
(300 MHz, CDCl3):  8.3 (s, 1H), 6.98 (s, 1H), 4.24 (t, 1H), 3.99 (t, 2H),
3.77 (d, 2H), 2.33 (m, 3H), 1.00 (d, 6H); ESI-MS m/z: (pos) 308 [M+H]+.
Murray et al.
Supplementary Methods
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1-[[1,2,3,4-Tetrahydro-6-[hydroxy[2-(trifluoromethyl)phenyl]methyl]-1-(2methylpropyl)-2,4-dioxothieno[2,3-d]pyrimidin-5-yl]carbonyl]azetidine (30)
n-BuLi (2.0 M solution in hexanes, 16.2 ml, 32 mmol, 3.0 equiv) was added dropwise to
a solution of diisopropylamine (5.3 ml, 38 mmol, 3.5 equiv) in tetrahydrofuran (20 ml)
at 0 C. The solution was stirred for 20 minutes then added to a stirred suspension of
compound (29) (3.32 g, 11 mmol, 1.0 equiv) in anhydrous tetrahydrofuran (80 ml) at 78°C. The mixture was stirred for a further 1 h at -78C then allowed to warm to 0°C
over approximately 1.5 h. The reaction mixture was cooled to -78°C and 2(trifluoromethyl)benzaldehyde (5.65 g, 32 mmol, 3.0 equiv) in tetrahydrofuran (30 ml)
was added dropwise. The reaction was allowed to reach room temperature overnight.
Methanol (2 ml) was added, followed by sat. NH4Cl solution and the mixture was then
extracted with ethyl acetate (x2). The organic extracts were dried (MgSO4), filtered and
concentrated under reduced pressure. The residue was purified by flash
chromatography (elution with 15:85 iso-hexane:ethyl acetate) and was then triturated
with diethyl ether to give compound (30) as a colourless solid (3.52 g, 68%).
H-NMR
(300 MHz, CDCl3):  8.13-8.05 (m, 2H), 7.75-7.69 (m, 2H), 7.53 (t, 1H), 6.36
(s, 1H), 4.58 (m, 1H), 4.35-4.08 (m, 3H), 3.84-3.70 (m, 2H), 3.54-3.47 (m, 1H), 2.392.32 (m, 2H), 2.20-2.10 (m, 1H), 0.94-0.89 (m, 6H) - complex due to atropisomerism;
APCI-MS m/z: (pos) 464 [M-OH]+.
1-[[1,2,3,4-Tetrahydro-1-(2-methylpropyl)-2,4-dioxo-6-[[2(trifluoromethyl)phenyl]methyl]thieno[2,3-d]pyrimidin-5-yl]carbonyl]azetidine
(5)
Trifluoroacetic acid (15 ml) and triethylsilane (15 ml) were added to a stirred solution of
compound (30) (3.52 g, 7.31 mmol, 1.0 equiv) in dichloromethane (50 ml) and stirred
overnight. The reaction mixture was poured on to dilute sodium hydroxide solution and
the pH was adjusted to 6 by adding dilute hydrochloric acid. The aqueous was extracted
with dichloromethane (x3), the combined organic extractes were dried (Na2SO4) and
concentrated under reduced pressure. The residue was purified by flash
chromatography (elution with 15:85 iso-hexane: ethyl acetate) and was then triturated
with diethyl ether to give compound (5) as a colourless solid (2.73 g, 80%).
Murray et al.
Supplementary Methods
Page 17
mp: 146°C; H-NMR (300 MHz, CDCl3):  8.08 (s, 1H), 7.68 (d, 1H), 7.53 (m, 2H),
7.40 (m, 1H), 4.21-4.39 (m, 3H), 4.17-4.00 (m, 2H), 3.84-3.72 (m, 2H), 3.58 (m, 1H),
2.37-2.16 (m, 3H), 0.93 (d, 6H) - complex due to atropisomerism; HRMS m/z: (pos)
[M+H]+ calcd for C22H23F3N3O3S, 466.1412; found, 466.1410.
VII. Synthesis of (3R)-1-[[1,2,3,4-tetrahydro-3-methyl-1-(2-methylpropyl)-2,4dioxo-6-(4-quinolinylmethyl)thieno[2,3-d]pyrimidin-5-yl]carbonyl]-3pyrrolidinol (6)
6-Chloro-3-methyl-1-(2-methylpropyl)-2,4(1H,3H)-pyrimidinedione (31)
A mixture of 6-chloro-3-methyl-1H-pyrimidine-2,4(1H,3H)-dione5 (27.9 g, 174
mmol, 1.0 equiv), 1-iodo-2-methylpropane (21.9 ml, 174 mmol, 1.0 equiv) and
potassium carbonate (26.4 g, 190 mmol, 1.2 equiv) in DMF (110 ml) was stirred at
90°C for 40 h. The reaction was cooled to room temperature and diluted with water
(800 ml) and brine (100 ml). The product was extracted with diethyl ether (x 2). The
organic extracts were dried (MgSO4) and concentrated under reduced pressure. The
residual oil was triturated with diethyl ether and the crystals were filtered, washed
with diethyl ether and dried under reduced pressure to give the desired compound
(7.38 g, 20%). The mother liquors were purified by flash chromatography (elution
with 1:1 diethyl ether: iso-hexane) to give compound (31) as an oil (6.90 g, 18%).
H-NMR
(300 MHz, CDCl3):  5.92 (s, 1H), 3.9 (d, 2H), 3.34 (s, 3H), 2.2 (m, 1H),
0.96 (d, 6H).
6-Mercapto-3-methyl-1-(2-methylpropyl)-2,4(1H,3H)-pyrimidinedione (32)
To a stirred solution of compound (31) (31.5 g, 140 mmol, 1.0 equiv) in ethanol (120
ml) was added sodium hydrogen sulfide hydrate (11.8 g, 160 mmol, 1.2 equiv) and
the mixture was stirred for 16 h. Further sodium hydrogen sulfide hydrate (5.92 g, 80
mmol, 0.6 equiv) was added and stirring was continued for 5 h. The reaction mixture
was diluted with water and extracted with ethyl acetate (x2). The aqueous layer was
acidified by addition of concentrated hydrochloric acid and then extracted with ethyl
Murray et al.
Supplementary Methods
Page 18
acetate (x3). The combined organic extracts were dried (MgSO4) and concentrated
under reduced pressure to give compound (32) as a solid (25.4 g, 85%).
H-NMR
(300 MHz, CDCl3):  4.3 (d, 2H), 4.16 (s, 2H), 3.32 (s, 3H), 2.32 (m, 1H),
0.94 (d, 6H); APCI-MS m/z: (pos) 215 [M+H]+.
Ethyl 1,2,3,4-tetrahydro-3-methyl-1-(2-methylpropyl)-2,4-dioxothieno[2,3d]pyrimidine-5-carboxylate (33)
Compound (32) (49.5 g, 0.13 moles, 1.0 equiv) was dissolved in dry DMF (900 ml)
and ethyl bromopyruvate (30 ml, 0.15 moles, 1.1 equiv) was added, and then with
stirring anhydrous potassium carbonate (16.0 g, 0.12 moles, 2 equiv) was also added.
The mixture was stirred for 5 h and then poured into water (5 L). The aqueous
solution was acidified with dilute hydrochloric acid, and then extracted thoroughly
with ethyl acetate. The organic extract was dried (MgSO4) and concentrated under
reduced pressure to leave a semisolid mass (42.3 g). A portion of this semisolid mass
(24.0 g) was dissolved in dichloromethane (500 ml) and cooled at 0 C then titanium
tetrachloride (13.5 ml, 0.14 mol, 1.2 equiv) was slowly added with efficient stirring.
The reaction mixture was stirred for 1 h at 0 C and then 3 h at room temperature.
The reaction mixture was poured slowly into vigorously stirred ice-water (1.5 L), and
then the resulting suspension was extracted into dichloromethane . After drying
(MgSO4) the organic solvent was concentrated under reduced pressure, and the
residue was purified by flash chromatography (elution with 1:1 ethyl acetate: isohexane) to afford compound (33) as a pale yellow solid (15.0 g, 37%).
H-NMR (300 MHz, CDCl3):  7.28(s, 1H), 4.4(q, 2H), 3.8(d, 2H), 3.4(s, 3H), 2.312.45(m, 1H), 1.4(t, 3H), 1.0(d, 6H); APCI-MS m/z: (pos) 311 [M+H]+.
Ethyl 1,2,3,4-tetrahydro-6-(hydroxy-4-quinolinylmethyl)-3-methyl-1-(2methylpropyl)-2,4-dioxothieno[2,3-d]pyrimidine-5-carboxylate (34)
A solution of lithium diisopropylamide (50 mmol, 1.0 equiv) in anhydrous
tetrahydrofuran (80 ml) was added dropwise over 1h to a stirred solution of
compound (33) (8.02 g, 50 mmol, 1.0 equiv) and 4-quinolinecarboxaldehyde (8.12 g,
50 mmol, 1.0 equiv) in anhydrous tetrahydrofuran (80 ml) at -78C. The mixture was
Murray et al.
Supplementary Methods
Page 19
stirred for a further 1h at -78C then quenched with glacial acetic acid (10 ml),
allowed to warm to room temperature, diluted with saturated aqueous sodium
bicarbonate solution (100 ml) and extracted into ethyl acetate (2x100 ml). The
combined extracts were dried (MgSO4), filtered and concentrated under reduced
pressure. The residue was purified by flash chromatography (elution with 3:2 ethyl
acetate: iso-hexane) to give compound (34) as a colourless solid (7.35 g, 91%).
H-NMR
(300 MHz, CDCl3):  9.02 (d, 1H), 8.17 (d, 1H), 7.90 (d, 1H), 7.83 (d, 1H),
7.72 (t, 1H), 7.52 (t, 1H), 6.78 (s, 1H), 4.48 (q, 2H), 3.71 (dd, 1H), 3.61 (s, br, 1H),
3.49 (dd, 1H), 3.38 (s, 3H), 2.10-2.16 (m, 1H), 1.43 (t, 3H), 0.88 (d, 3H), 0.85 (d,
3H); ESI-MS m/z: (pos) 468 [M+H]+.
Ethyl 1,2,3,4-tetrahydro-3-methyl-1-(2-methylpropyl)-2,4-dioxo-6(4-quinolinylmethyl)thieno[2,3-d]pyrimidine-5-carboxylate (35)
Trifluoroacetic anhydride (4.57 ml, 32.7 mmol, 1.5 equiv) was added to a solution of
compound (34) (10.2 g, 21.8 mmol, 1.0 equiv) and triethylamine (9.12 ml, 65.5 mmol,
3.0 equiv) in anhydrous tetrahydrofuran (200 ml) and the mixture stirred for 15 min.
10% palladium on charcoal (700 mg) was added and the mixture hydrogenated at 3
bar for 5 h. The reaction mixture was filtered through Celite, washing with saturated
aqueous sodium bicarbonate solution then ethyl acetate. The organic material was
extracted into ethyl acetate, dried (MgSO4), filtered and concentrated under reduced
pressure. The residue was purified by flash chromatography (elution with 1:1 ethyl
acetate: iso-hexane) to give compound (35) as a colourless solid (7.68 g, 78%).
H-NMR
(300 MHz, CDCl3):  8.89 (d, 1H), 8.16 (d, 1H), 8.11 (d, 1H), 7.75 (t, 1H),
7.60 (t, 1H), 7.29 (d, 1H), 4.61 (s, 2H), 4.45 (q, 2H), 3.64 (d, 2H), 3.39 (s, 3H), 2.102.16 (m, 1H), 1.37 (t, 3H), 0.90 (d, 6H); ESI-MS m/z: (pos) 452[M+H]+.
Sodium 1,2,3,4-tetrahydro-3-methyl-1-(2-methylpropyl)-2,4-dioxo-6-(4quinolinylmethyl)thieno[2,3-d]pyrimidine-5-carboxylate (36)
A solution of compound (35) (7.68 g, 17.0 mmol, 1.0 equiv) in tetrahydrofuran (200
ml) and methanol (25 ml) was deoygenated by repeated evacuation and flushing with
nitrogen. 1M sodium hydroxide (24.0 ml, 24.0 mmol, 1.4 equiv) was added and the
Murray et al.
Supplementary Methods
Page 20
mixture stirred for 18 h. The resulting precipitated solid was collected by filtration
and washed with tetrahydrofuran to give compound (36) as a colourless solid (6.75 g,
88%).
H-NMR
(300 MHz, DMSO-d6):  8.83 (d, 1H ), 8.60 (m, 1H), 8.00 (dd, 1H), 7.74
(td, 1H), 7.57 (td, 1H), 7.52 (dd, 1H), 4.56 (s, 2H), 3.56 (d, 2H), 3.20 (s, 3H), 2.102.15 (m, 1H), 0.81 (d, 6H); ESI-MS m/z: (pos) 424 [M+H]+.
(3R)-1-[[1,2,3,4-Tetrahydro-3-methyl-1-(2-methylpropyl)-2,4-dioxo-6-(4quinolinylmethyl)thieno[2,3-d]pyrimidin-5-yl]carbonyl]-3-pyrrolidinol (6)
To a suspension of compound (36) (5.75 g, 11.7 mmol, 1.0 equiv) in dichloromethane
(100 ml) was added 1-hydroxybenzotriazole hydrate (2.37 g, 17.5 mmol, 1.5 equiv)
and the mixture was stirred for 15 min. 1-Ethyl-3-(3’dimethylaminopropyl)carbodiimide hydrochloride (3.36 g, 17.51 mmol, 1.5 equiv)
was then added and stirring continued for 30 min. (R)-3-Hydroxypyrrolidine (1.45
ml, 17.5 mmol, 1.5 equiv) was added and the mixture was stirred for a further 3 h.
The mixture was poured onto sodium bicarbonate solution (100 ml) and extracted
with ethyl acetate, dried (MgSO4) and concentrated under reduced pressure. The
residue was purified by flash chromatography (elution with 8% methanol:ethyl
acetate). The residue was further purified by recrystallisation from ethyl acetate/ isohexane to give compound (6) as a colourless solid (5.41 g, 97%).
mp: 134-144°C; H-NMR (300 MHz, DMSO-d6):  8.87-8.85 (m, 1H), 8.30-8.21 (m,
1H), 8.06-8.04 (m, 1H), 7.80-7.76 (m, 1H), 7.67-7.61 (m, 1H), 7.48-7.43 (m, 1H),
5.20-3.88 (m, 3H), 3.80-2.43 (m, 9H), 2.33-1.24 (m, 3H), 0.95-0.90 (m, 6H) complex due to atropisomerism; APCI-MS m/z: (pos) 493 [M+H]+; analysis
(calcd,found for C26H28N4O4S.0.5C4H8O2 (ethyl acetate)): C (62.39,62.67), H
(5.87,6.01), N (10.76,10.44), S (6.01,5.97).
VIII. Synthesis of 1-[[1,2,3,4-tetrahydro-3-methyl-1-(2-methylpropyl)-2,4-dioxo6-[[2-(trifluoromethyl)phenyl]methyl]thieno[2,3-d]pyrimidin-5-yl]carbonyl]pyrrolidine (8)
Murray et al.
Supplementary Methods
Page 21
1,2,3,4-Tetrahydro-3-methyl-1-(2-methylpropyl)-2,4-dioxothieno[2,3d]pyrimidine-5-carboxylic acid (37)
Compound (33) (6.0 g, 19.4 mmol, 1.0 equiv) was dissolved in ethanol (50 ml) and
sodium hydroxide (1.0 g, 25.0 mmol, 1.3 equiv) in water (10 ml) and stirred for 2 h.
The reaction mixture was concentrated under reduced pressure and then acidified with
dilute hydrochloric acid. A precipitate formed, which was collected by filtration to
give compound (37) as a beige solid (5.06g, 94%).
H-NMR
(300 MHz, DMSO-d6):  14.84 (s, 1H), 8.08 (s, 1H), 3.83 (d, 2H), 3.33 (s,
3H), 2.2 (m, 1H), 0.94 (d, 6H); APCI-MS m/z: (pos) 283 [M+H]+.
1,2,3,4-Tetrahydro-6-[hydroxy[2-(trifluoromethyl)phenyl]methyl]-3-methyl-1-(2methylpropyl)-2,4-dioxothieno[2,3-d]pyrimidine-5-carboxylic acid (38)
Lithium diisopropylamide (17.7 ml, 17.7 mmol, 2.5 equiv) was added dropwise to a
stirred solution of compound (37) (2.0 g, 7.1 mmol, 1.0 equiv) in tetrahydrofuran (20
ml) at -78°C, and stirred for 5 min. 2-(Trifluoromethyl)benzaldehyde (2.0 ml, 14.2
mmol, 2.0 equiv) was added at -78°C and the reaction mixture was stirred at this
temperature for 2.5 h. The reaction was quenched with dilute hydrochloric acid and
allowed to reach room temperature, then extracted with ethyl acetate. The organic
layer was washed with dilute hydrochloric acid (x 2) and brine, dried (MgSO4) and
concentrated under reduced pressure. The residue was purified by flash
chromatography (elution with 1:1 ethyl acetate: iso-hexane) to give compound (38) as
an oil (3.23 g, 100%).
H-NMR
(300 MHz, DMSO-d6):  7.99 (d, 1H), 7.7 (m, 2H), 7.55 (t, 1H), 6.88 (s,
1H), 3.64 (m, 2H), 3.50 (s, 3H), 2.15 (m, 1H), 0.90 (t, 6H); APCI-MS m/z: (pos) 457
[M+H]+.
1,2,3,4-Tetrahydro-3-methyl-1-(2-methylpropyl)-2,4-dioxo-6-[[2(trifluoromethyl)phenyl]methyl]thieno[2,3-d]pyrimidine-5-carboxylic acid (39)
Trifluoroacetic acid (5 ml) and triethylsilane (2.5 ml) were added to a stirred solution
of compound (38) (3.23 g, 7.07 mmol, 1 equiv) and stirred for 2 days. Water was
Murray et al.
Supplementary Methods
Page 22
added and the reaction extracted with ethyl acetate, the organic phase was washed
with brine, dried (MgSO4) and concentrated under reduced pressure. The residue was
purified by flash chromatography (elution with 4:1 ethyl acetate: iso-hexane and 1%
acetic acid) to give compound (39) as an oil (1.0 g, 32%).
H-NMR
(300 MHz, CDCl3):  7.72 (d, 1H), 7.53 (t, 1H), 7.43 (t, 1H), 7.34 (d, 1H),
4.95 (s, 2H), 3.73 (d, 2H), 3.51 (s, 3H), 2.24 (m, 1H), 0.92 (d, 6H); APCI-MS m/z:
(pos) 441 [M+H]+.
1-[[1,2,3,4-Tetrahydro-3-methyl-1-(2-methylpropyl)-2,4-dioxo-6-[[2(trifluoromethyl)phenyl]methyl]thieno[2,3-d]pyrimidin-5-yl]carbonyl]pyrrolidine (8)
Oxalyl chloride (0.25 ml, 2.26 mmol, 2.0 equiv) was added to a stirred solution of
compound (39) (0.5 g, 1.13 mmol, 1.0 equiv) in dichloromethane (13 ml). DMF
(catalytic) was added and the reaction was stirred for 45 min then concentrated under
reduced pressure. The residue was dissolved in tetrahydrofuran (15 ml), pyrrolidine
(0.27 g, 2.26 mmol) was added dropwise and stirred for 3 h. The reaction was
concentrated under reduced pressure then purified by reverse phase HPLC to give
compound (8) as a colourless solid (0.32 g, 57%).
H-NMR
(300 MHz, CDCl3):  7.74 (d, J=8.0 Hz, 1H), 7.66 (t, J=7.6 Hz, 1H), 7.53
(d, J=7.6 Hz, 1H), 7.49 (t, J=8.0 Hz, 1H), 4.25-4.11 (m, 2H), 3.80-1.55 (m, 14H), 0.88
(d, J=6.6 Hz, 6H) - complex due to atropisomerism; C NMR (75 MHz, DMSO-d6):
162.17, 157.09, 152.40, 150.29, 136.71, 132.84, 132.49, 132.00, 128.96, 127.72,
126.78 (q, J=30.1 Hz), 125.95 (q, J=5.0 Hz), 124.36 (q, J=273.1 Hz), 111.39, 55.53,
46.69, 44.98, 29.50, 27.90, 26.52, 25.17, 23.92, 19.77; HRMS m/z: (pos) [M+H]+
calcd for C24H27F3N3O3S, 494.1725; found, 494.1725.
IX. Synthesis of 5-[(3-hydroxypropyl)thio]-3-methyl-1-(2-methylpropyl)-6-[[2(trifluoromethyl)phenyl]methyl]thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (9)
3-Methyl-1-(2-methylpropyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (40)
Murray et al.
Supplementary Methods
Page 23
Sodium acetate (38.9 g, 0.47 mol, 4.0 equiv) was added to a stirred suspension of
compound (32) (25.4 g, 0.12 mol, 1.0 equiv) in water (1 L). The reaction mixture was
stirred for 5 h, then filtered. Aqueous chloroacetaldehyde solution (142 ml, 50% wt.,
0.12 mol, 1.0 equiv) was added to the filtrate and stirred for 16 h. The mixture was
acidified with concentrated hydrochloric acid and extracted with ethyl acetate (x 3).
The combined organic extracts were washed with saturated aqueous sodium
bicarbonate solution, dried (MgSO4) and concentrated under reduced pressure. The
residue was purified by flash chromatography (elution with 1:1 diethyl ether: isohexane) to give compound (40) as a solid (26.8 g, 94%).
H-NMR
(300 MHz, CDCl3):  7.36 (d, 1H), 6.48 (d, 1H), 3.81 (d, 2H), 3.43 (s, 3H),
2.3 (m, 1H), 1.00 (d, 6H); APCI-MS m/z: (pos) 239 [M+H]+.
5-Bromo-6-[hydroxy[2-(trifluoromethyl)phenyl]methyl]-3-methyl-1-(2methylpropyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (41)
To a solution of compound (40) (20g, 84 mmol, 1.0 equiv) in dichloromethane (160
ml) was added a solution of bromine (4.65 ml, 90 mmol, 1.1 equiv) in
dichloromethane (20 ml). The reaction mixture was stirred for 20 min then
concentrated under reduced pressure, dissolved in ethyl acetate and washed with 50%
aqueous sodium bicarbonate solution, aqueous sodium thiosulfate, brine and then
dried (MgSO4) and concentrated under reduced pressure to give a pale yellow solid.
The solid was dissolved in tetrahydrofuran (480 ml) and cooled to -78°C. Lithium
diisopropylamide (45 ml, 90 mmol, 2M solution in hexanes/tetrahydrofuran, 1.1
equiv) was added dropwise. After complete addition the reaction mixture was stirred
for 30 min before a solution of 2-trifluoromethyl benzaldehyde (16.61 ml, 95 mmol,
1.1 equiv) in THF (20 ml) was added and the resultant solution stirred at -78°C for 3
h. Saturated ammonium chloride solution (500 ml) was added and the cooling bath
removed. Once the reaction mixture had reached room temperature it was
concentrated under reduced pressure and partitioned between ethyl acetate and water.
The ethyl acetate layer was dried (MgSO4) and concentrated under reduced pressure
to give, after trituration with ethyl acetate/ iso-hexane, compound (41) (24.7 g, 60%)
as an off-white solid.
Murray et al.
H-NMR
Supplementary Methods
Page 24
(400 MHz, CDCl3):  7.74-7.71 (m, 2H), 7.62 (t, 1H), 7.50 (t, 1H), 6.56 (s,
1H), 3.82 (dd, 1H), 3.67 (dd, 1H), 3.40 (s, 3H), 2.77 (br s, 1H), 2.27 (sept, 1H), 0.96
(d, 6H); APCI-MS m/z: (pos) 491/493 [M+H]+.
5-Bromo-3-methyl-1-(2-methylpropyl)-6-[2-(trifluoromethyl)benzoyl]thieno[2,3d]pyrimidine-2,4(1H,3H)-dione (42)
Compound (41) (2.5 g, 51 mmol, 1.4 equiv), manganese (IV) oxide (3.19 g, 37 mmol,
1.0 equiv) and dichloromethane were charged to a flask and stirred for 6 days. The
resulting suspension was filtered through celite and the filtrate was concentrated
under reduced pressure. The residue was purified by flash chromatography (elution
with 2:1 ethyl acetate: iso-hexane) to give compound (42) as a colourless solid (0.68
g, 28%).
H-NMR
(300 MHz, CDCl3):  7.78 (m, 1H), 7.65 (m, 2H), 7.42 (m, 1H), 3.96 (s,
3H), 3.86 (d, 2H), 2.35 (m, 1H), 1.01 (d, 6H); APCI-MS m/z: (pos) 489/491 [M+H]+.
5-[(3-Hydroxypropyl)thio]-3-methyl-1-(2-methylpropyl)-6-[2(trifluoromethyl)benzoyl]thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (43)
3-Mercaptopropanol (1.62g, 1.63 mmol, 1.6 equiv) was added dropwise to a
suspension of sodium hydride (0.076 g, 1.8 mmol, 1.9 equiv) in tetrahydrofuran (50
ml) and stirred for 30 min. Compound (42) (0.5 g, 1.0 mmol, 1.0 equiv) in
tetrahydrofuran was added dropwise and the reaction mixture was stirred for 1 h then
poured onto water and extracted with ethyl acetate. The organic extracts were washed
(brine), dried (MgSO4) and concentrated under reduced pressure to give a yellow oil.
The oil was purified by flash chromatography (elution with 1:1 iso-hexane:ethyl
acetate) to give compound (43) as a yellow oil (0.5 g, 98%).
H-NMR
(300 MHz, CDCl3):  7.78 (m, 1H), 7.63 (m, 2H), 7.39 (m, 1H), 3.88 (d,
2H), 3.78 (m, 1H), 3.62 (q, 2H), 3.42 (s, 3H), 2.92 (t, 2H), 2.42 (m, 1H), 1.57 (m,
2H), 1.05 (s, 3H), 1.03 (s, 3H); APCI-MS m/z: (pos) 501 [M+H]+.
5-[(3-Hydroxypropyl)thio]-6-[hydroxy[2-(trifluoromethyl)phenyl]methyl]-3methyl-1-(2-methylpropyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (44)
Murray et al.
Supplementary Methods
Page 25
Sodium borohydride (0.015 g, 0.4 mmol, 1.04 equiv) was added to a stirred solution
of compound (43) (0.19g, 0.39 mmol, 1.0 equiv) in ethanol (30 ml). The solution was
stirred for 6 h and then concentrated under reduced pressure. The residue was
partitioned between ethyl acetate and water. The ethyl acetate layer was washed with
brine, dried (MgSO4) and concentrated under reduced pressure. The residue was
purified by flash chromatography (elution with 0-60% ethyl acetate: iso-hexane) to
give compound (44) as a colourless foam (0.10 g, 50%).
H-NMR
(300MHz, CDCl3):  7.86 (d, 1H), 7.68 (m, 2H), 7.49 (t, 1H), 6.83 (d, 1H),
3.9 (m, 1H), 3.87 (m, 1H), 3.83 (m, 2H), 3.64 (m, 1H), 3.41 (s, 3H), 3.16 (m, 1H),
3.04 (m, 1H), 3.29 (d, 1H), 2.28 (m, 1H), 0.96 (dd, 6H); APCI-MS m/z: (pos) 503
[M+H]+.
5-[(3-Hydroxypropyl)thio]-3-methyl-1-(2-methylpropyl)-6-[[2(trifluoromethyl)phenyl]methyl]thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (9)
Trifluoroacetic acid (3 ml) and triethylsilane (1.5 ml) were added to a stirred solution
of compound (44) (0.21 g, 0.41 mmol, 1.0 equiv) and stirred for 16 h. Saturated
aqueous sodium bicarbonate solution was added and the mixture was then extracted
with ethyl acetate (x 3). The combined organic extracts were washed with brine,
dried (MgSO4) and concentrated under reduced pressure to give an oil. The oil was
dissolved in methanol (20 ml) and sodium bicarbonate (0.12 g, 1.43 mmol, 3 equiv)
was added. The mixture was stirred for 1 h then concentrated under reduced pressure.
The residue was partitioned between ethyl acetate and water. The organic layer was
washed with brine, dried (MgSO4) and concentrated under reduced pressure. The
residue was purified by normal phase HPLC (elution with 0-80% ethyl acetate: isohexane) to give compound (9) as a colourless solid (0.11 g, 41%).
mp: 102-104°C; H-NMR (300 MHz, CDCl3):  7.69 (d, 1H), 7.49 (m, 1H), 7.39 (m,
1H), 7.18 (d, 1H), 4.54 (s, 2H), 3.85 (t, 2H), 3.72 (d, 2H), 3.43 (s, 3H), 3.1 (t, 2H),
2.85 (s, 1H), 2.24 (m, 1H), 1.89-1.83 (m, 2H), 0.95 (s, 3H), 0.93 (s, 3H);C NMR
(75 MHz, DMSO-d6): 157.03, 153.01, 150.09, 137.25, 134.90, 132.97, 130.94,
127.60, 126.97 (q, J=32.3 Hz), 126.16 (q, J=5.2 Hz), 125.64, 124.38 (q, J=274.6 Hz),
113.40, 59.34, 55.12, 32.39, 32.17, 29.96, 27.99, 26.51, 19.77; APCI-MS m/z: (pos)
Murray et al.
Supplementary Methods
Page 26
487 [M+H]+; analysis (calcd,found for C22H25N2O3S): C (54.31,54.06), H (5.18,4.95),
N (5.76,5.71), S (13.18,12.83).
X. Synthesis of 2,5-dihydro-1-[[1,2,3,4-tetrahydro-3-methyl-1-(2-methylpropyl)2,4-dioxo-6-[[2-(trifluoromethyl)phenyl]methyl]thieno[2,3-d]pyrimidin-5yl]carbonyl]-1H-pyrrole (10)
Compound (39) (0.2 g, 0.45 mmol, 1.0 equiv) in dichloromethane was treated with
oxalyl chloride (0.2 ml, 2.29 mmol, 5.0 equiv) followed by DMF (catalytic). The
reaction mixture was stirred for 1 h, then concentrated under reduced pressure. The
residue was dissolved in dichloromethane, triethylamine (0.5 ml, 3.46 mmol, 7.7
equiv) and 3-pyrroline (0.09 ml, 1.17 mmol, 2.6 equiv) were added and stirred for 16
h, then concentrated under reduced pressure. The residue was purified by flash
chromatography (elution with 3:1 iso-hexane:ethyl acetate). The residue was further
purified by reverse phase HPLC to give compound (10) as a colourless solid (0.21 g,
38%).
mp: 75-78°C; H-NMR (400 MHz, CDCl3):  7.66 (d, 1H), 7.55 (d, 1H), 7.5 (t, 1H),
7.36 (t, 1H), 5.91-5.89 (m, 1H), 5.73-5.71 (m, 1H), 4.57-1.70 (m, 12H), 0.95-0.93 (m,
6H) - complex due to atropisomerism; APCI-MS m/z: (pos) 492 [M+H]+; analysis
(calcd,found for C24H24F3N3O3S): C (58.65,58.59), H (4.92,5.08), N (8.55,8.51), S
(6.52,7.19).
XI. Synthesis of (3R)-1-[[1,2,3,4-tetrahydro-6-(1H-indol-3-ylmethyl)-3-methyl-1(2-methylpropyl)-2,4-dioxothieno[2,3-d]pyrimidin-5-yl]carbonyl]-3-pyrrolidinol
(11)
Methyl 1,2,3,4-tetrahydro-3,6-dimethyl-1-(2-methylpropyl)-2,4-dioxothieno[2,3d]pyrimidine-5-carboxylate (45)
To a suspension of compound (32) (9.31 g, 44.0 mmol, 1.0 equiv) in water (300 ml)
was added sodium acetate (17.8 g, 218 mmol, 5.0 equiv). The mixture was stirred for
20 min then filtered. To the filtrate was added methyl 3-bromo-2-ketobutyrate6 (8.91
g, 46.0 mmol, 1.0 equiv). The mixture was stirred for 1 h then extracted with ethyl
acetate (3x100 ml), dried (MgSO4) and concentrated under reduced pressure. The
Murray et al.
Supplementary Methods
Page 27
residue was dissolved in dichloromethane (150 ml) and treated with titanium
tetrachloride (5 ml, 41.0 mmol, 0.9 equiv). The reaction mixture was stirred for 30
min then further titanium tetrachloride (5 ml, 41.0 mmol, 0.9 equiv) was added and
stirring was continued for 1 h. The mixture was poured slowly into water (100 ml),
extracted into dichloromethane (3x100 ml), dried (MgSO4) and concentrated under
reduced pressure. The residue was purified by flash chromatography (elution with 1:3
ethyl acetate: iso-hexane) to afford, after trituration with diethyl ether, compound (45)
as an off-white solid (9.24 g, 69%).
H-NMR
(300MHz, CDCl3):  3.96 (s, 3H), 3.74 (d, 2H), 3.46 (s, 3H), 2.45 (s, 3H),
2.30 (m, 1H), 0.98 (d, 6H); APCI-MS m/z: (pos) 311 [M+H]+.
Methyl 1,2,3,4-tetrahydro-6-(1H-indol-3-ylmethyl)-3-methyl-1-(2-methylpropyl)2,4-dioxothieno[2,3-d]pyrimidine-5-carboxylate (46)
A solution of compound (45) (1.0 g, 3.22 mmol, 1.0 equiv) and N-bromosuccinimde
(0.63 g, 3.54 mmol, 1.1 equiv) in chloroform (20 ml) was irradiated with a halogen
lamp for 2 h. To the cooled solution was added saturated sodium bicarbonate solution
(20 ml) and indole (0.76 g, 6.45 mmol, 2.0 equiv) and the mixture was stirred for 5
days then extracted with dichloromethane (2x30 ml), dried (MgSO4) and concentrated
under reduced pressure. The residue was purified by flash chromatography (elution
with 1:2 ethyl acetate:iso-hexane) to afford compound (46) as a pale yellow foam
(0.86 g, 63%).
H-NMR
(300MHz, DMSO-d6):  7.47 (d, J=7.9 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H),
7.30 (d, J=2.5 Hz, 1H), 7.08 (ddd, J=8.1, 7.1, 1.2 Hz, 1H), 6.96 (ddd, J=7.9, 7.1, 1.0
Hz, 1H), 4.21 (s, 2H), 3.87 (s, 3H), 3.61 (d, J=7.5 Hz, 2H), 3.20 (s, 3H), 2.15-2.01 (m,
1H), 0.83 (d, J=6.7 Hz, 6H); APCI-MS m/z: (pos) 426 [M+H]+.
1,2,3,4-Tetrahydro-6-(1H-indol-3-ylmethyl)-3-methyl-1-(2-methylpropyl)-2,4dioxothieno[2,3-d]pyrimidine-5-carboxylic acid (47)
To a solution of compound (46) (2.1 g, 4.94 mmol, 1.0 equiv) in tetrahydrofuran (25
ml) and methanol (10 ml) was added sodium hydroxide solution (1 M; 5 ml, 5 mmol,
1.0 equiv) and the mixture was stirred for 5 days. The mixture was concentrated
Murray et al.
Supplementary Methods
Page 28
under reduced pressure, diluted with water (300 ml), acidified to pH 2 with dilute
hydrochloric acid then extracted with chloroform (3x200 ml), dried (MgSO4) and
concentrated under reduced pressure. The residue was purified by flash
chromatography (elution with 4:1 ethyl acetate:dichloromethane) to afford, after
trituration with diethyl ether, compound (47) as a pale pink solid (1.6 g, 79%).
H-NMR
(300MHz, DMSO-d6):  14.04 (s, 1H), 11.01 (s, 1H), 7.83 (d, J=2.3 Hz,
1H), 7.48 (d, J=7.7 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H), 7.08 (t, J=7.1 Hz, 1H), 6.96 (t,
J=7.1 Hz, 1H), 4.38 (s, 2H), 3.62 (d, J=7.7 Hz, 2H), 3.25 (s, 3H), 2.20-1.97 (m, 1H),
0.82 (d, J=6.7 Hz, 6H); APCI-MS m/z: (pos) 412 [M+H]+.
(3R)-1-[[1,2,3,4-Tetrahydro-6-(1H-indol-3-ylmethyl)-3-methyl-1-(2methylpropyl)-2,4-dioxothieno[2,3-d]pyrimidin-5-yl]carbonyl]-3-pyrrolidinol
(11)
A mixture of compound (47) (0.41 g, 1.0 mmol, 1.0 equiv), 1-ethyl-3-(3’dimethylaminopropyl)carbodiimide hydrochloride (0.38 g, 2.0 mmol, 2.0 equiv), 1hydroxybenzotriazole hydrate (0.27 g, 2.0 mmol, 2.0 equiv), (R)-3hydroxypyrrolidine (0.17 g, 2 mmol, 2 equiv) and 4-dimethylaminopyridine
(catalytic) in dichloromethane were stirred for 4 h. The mixture was washed with
dilute hydrochloric acid, saturated aqueous sodium bicarbonate solution, brine, then
dried (MgSO4) and concentrated under reduced pressure. The residue was purified by
flash chromatography (elution with 4:6 methanol:dichloromethane ) to give
compound (11) as a colourless solid (0.29 g, 62%).
mp: 225-226°C; H-NMR (300 MHz, DMSO-d6):  10.98 (s, 1H), 7.58-7.47 (m, 1H),
7.37-7.26 (m, 2H), 7.10-7.05 (t, 1H), 6.98-6.93 (t, 1H), 5.06-4.82 (m, 1H), 4.38-1.00
(m, 15H), 0.85 (m, 6H) - complex due to atropisomerism; APCI-MS m/z: (pos) 481
[M+H]+; analysis (calcd,found for C25H28N4O4S): C (62.48,62.41), H (5.87,5.79), N
(11.66,11.65), S (6.67,7.10).
XIII. References
1. Michne, W. F., Schroeder, J. D., Guiles, J. W., Treasurywala, A. M., Weigelt,
C. A., Stansberry, M. F., McAvoy, E., Shah, C. R., Baine, Y., Sawutz, D. G.,
Murray et al.
Supplementary Methods
Page 29
Miller, P. B., Stankunas, B. M., Reid, J., Bump, E. & Schlegel, D. Novel
Inhibitors of the Nuclear Factor of Activated T Cells (NFAT)-Mediated
Transcription of beta-Galactosidase: Potential Immunosuppressive and
Antiinflammatory Agents J. Med. Chem. 38, 2557-2569 (1995).
2. Dumont, W., Vermeyen, C. & Krief, A. Novel synthetic route -oxoacrylates.
Application to the synthesis of pyrenophorin antibiotic. Tetrahedron Lett. 25,
2883-2886 (1984).
3. Senda, S., Hirota, K. & Notani, J. Pyrimidine derivatives and related
compounds. XVI. Synthesis of 1,3-disubstituted 5-cyanouracil derivatives
and related compounds. Chem. Pharm. Bull. 20, 1380-1388 (1972).
4. Shu, A.Y.L., Yamashita, D.S., Holt, D.A. & Heys, J.R. Synthesis of I-125
labeled photoaffinity rapamycin analogs. J. Labelled Cpd. Radiopharm. 38,
227-237 (1996).
5. Iwata, M., Bruice, T.C., Carrell, H.L. & Glusker, J.P. Reactions of 4aPeroxides and 4a-Pseudobases of N10- and N5-Phenethylflavins. J. Am. Chem.
Soc. 102, 5036-5044 (1980).
6. Lee, W.S., Nam, K.D., Hahn, H.G. & Mah, H.D. Conversion of 1,3thiazolidine and its sulfoxide to dihydro-1,4-thiazine. J. Heterocyclic Chem.
30, 1105-1109 (1993).