Supplemental Data
Experimental Procedures for PPD-1 and PPD-2 Synthesis
ethyl 2,4-dioxohexanoate (Int-2)
2-Butanone (12.3 mL, 0.14 mol) and diethyl oxalate (18.6 mL, 0.14 mol) were added successively to a
stirred solution of NaOEt/EtOH (44.4 mL of a 21wt.% solution, 0.14 mol) at 0 ºC under Ar. The resulting
brown solution was warmed to room temperature and heated at reflux for 2 h, then cooled to room
temperature overnight. The resulting orange/brown suspension was concentrated under reduced
pressure to give a thick brown gum, which was partitioned between water (200 mL) and EtOAc (100 mL).
The layers were separated and the aqueous layer was extracted with EtOAc (2 x 50 mL). The aqueous
layer was acidified to pH 2 with 2N HCl(aq) and extracted with Et2O (3 x 100 mL). The combined Et2O
layers were washed with brine (100 mL), dried (MgSO4), filtered and concentrated under reduced
pressure to give the crude product (16.3 g) as a brown oil. Purification by Kügelrohr distillation gave ethyl
2,4-dioxohexanoate (12.56 g, 53%) as a pale yellow liquid.
1H
NMR (400 MHz, CDCl3) 1.16 (t, 3H), 1.36 (t, 3H), 2.52 (q, 2H), 4.34 (q, 2H), 6.36 (s, 1H)
ethyl 1-(cyclobutylmethyl)-5-ethyl-1H-pyrazole-3-carboxylate (Int-3)
A solution of ethyl 2,4-dioxohexanoate (4.56 g, 26.5 mmol) in AcOH (20 mL) was added to a stirred
suspension of (cyclobutylmethyl)hydrazine (3.8 g, 27.8 mmol) in AcOH (30 mL) at room temperature. The
resulting pale yellow mixture was stirred at room temperature for 6 h, the AcOH was then removed by
rotary evaporation. The resulting orange/brown residue was dissolved in EtOAc (50 mL) and washed with
sat. NaHCO3(aq) (2 x 50 mL) and brine (50 mL), dried (MgSO4), filtered and concentrated under reduced
pressure to give the crude product (6.6 g) as an orange/brown oil. Purification by flash column
chromatography on silica with heptane-EtOAc (4:1) as eluent gave ethyl 1-(cyclobutylmethyl)-5-ethyl-1Hpyrazole-3-carboxylate (4.97 g, 79%) as the major regioisomer. Regiochemistry was confirmed by
NOESY experiments.
1H
NMR (CDCl3, 400 MHz) 1.28 (t, 3H), 1.37 (t, 3H), 1.91-1.73 (m, 4H), 2.05-1.97 (m, 2H), 2.62 (q, 2H),
2.84 (septet, 1H), 4.10 (d, 2H), 4.37 (q, 2H), 6.56 (s, 1H)
NOESY: 2.62 (q, 2H, CH2CH3) has NOE to 4.10 (d, 2H, CH2N).
ethyl 1-(cyclobutylmethyl)-5-ethyl-4-nitro-1H-pyrazole-3-carboxylate (Int-4)
To a solution of ethyl 1-(cyclobutylmethyl)-5-ethyl-1H-pyrazole-3-carboxylate (4.76 g, 20.1 mmol) in
H2SO4 (20 mL) was added HNO3 (20 mL) at room temperature. The resulting yellow/orange solution was
heated at 40 ºC for 3 h, then cooled to room temperature. The resulting solution was poured into
vigorously stirred ice-water (400 mL) and extracted with EtOAc (200 mL). The EtOAc layer was washed
with water (100 mL), sat. NaHCO3(aq) (2 x 100 mL) and brine (100 mL), dried (MgSO 4), filtered and
concentrated to give the crude product (4.95 g) as a brown oil. Purification by flash column
chromatography on silica with heptane-EtOAc (3:1) as eluent gave compound ethyl 1-(cyclobutylmethyl)5-ethyl-4-nitro-1H-pyrazole-3-carboxylate (4.07 g, 72%) as a yellow oil.
1H
NMR (400 MHz, CDCl3) 1.26 (t, 3H). 1.38 (t, 3H), 1.98-1.76 (m, 4H), 2.12-2.03 (m, 2H), 2.85 (septet,
1H), 2.97 (q, 2H), 4.09 (d, 2H), 4.42 (q, 2H)
2-(cyclobutylmethyl)-3-ethyl-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (PPD-1)
2-(cyclobutylmethyl)-3-ethyl-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione was prepared in several
steps from ethyl 1-(cyclobutylmethyl)-5-ethyl-4-nitro-1H-pyrazole-3-carboxylate via a route analogous to
that described for synthesis of PPD-2 below. Ethyl 1-(cyclobutylmethyl)-5-ethyl-4-nitro-1H-pyrazole-3-
carboxylate can be converted to the corresponding primary amide by treatment with ammonia/MeOH
solution. Reduction of the nitro group by hydrogentation, followed by ring closure using CDI gave 2(cyclobutylmethyl)-3-ethyl-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (PPD-1). An alternative route to
PPD-1 involved coupling of 3-bromo-2-(cyclobutylmethyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione
with vinyl boronic acid, followed by hydrogenation.
1H
NMR (CDCl3, 400 MHz): 1.05 (t, 3H), 1.65-1.9 (m, 4H), 1.9-2.0 (m, 2H), 2.7-2.8 (m, 3H), 4.15 (d, 2H),
10.75 (s, 2H)
Methyl 4-nitro-1H-pyrazole-3-carboxylate (Int-7)
To a solution of 4-nitro 1H-pyrazole-3-carboxylic acid (150 g, 955.41 mmol) in MeOH (1 L) at 0oC was
added acetylchloride (100 mL), the reaction mixture was slowly warmed to rt. The reaction mixture was
stirred at the room temperature for 48 h. The solvent was removed completely under reduced pressure
and the reaction quenched with 10% NaHCO 3. The aqueous layer was extracted with EtOAc, dried
(Na2SO4), filtered and concentrated under reduced pressure to give methyl 4-nitro-1H-pyrazole-3carboxylate as a white solid (162 g, 99%). The product was used without further purification.
1H NMR (300MHz, d-DMSO) ppm 3.9 (s, 3H), 8.95 (s, 1H), 14.4 (bs, 1H)
LC MS [M+H]+ 170.0
Methyl 1-(cyclobutylmethyl)-4-nitro-1H-pyrazole-3-carboxylate (Int-8)
To a solution of methyl 4-nitro-1H-pyrazole-3-carboxylate (162 g, 0.946 mol) in DMF (1L) was added
K2CO3 (261.7 g, 1893 mol) and NaI (14.19 g, 94.69 mmol). (bromomethyl)cyclobutane was added to the
reaction mixture and the mixture stirred at 60oC for 48 h. The reaction mixture was filtered and quenched
with water. The aqueous layer was extracted with EtOAc, dried (Na2SO4), filtered and concentrated. The
crude residue was purified by silica gel column chromatography eluting with 10% EtOAc in pet ether to
give Methyl 1-(cyclobutylmethyl)-4-nitro-1H-pyrazole-3-carboxylate as an off-white solid (80 g, 35%). The
regiochemistry of the isomer was confirmed using NOE experiments.
1H NMR (300MHz, d-DMSO) ppm 1.65-2.1 (m, 6H), 2.8 (m, 1H), 3.90 (s, 3H), 4.25 (d, 2H), 9.0 (s, 1H)
LC MS [M+H]+ 180.1
1-(cyclobutylmethyl)-4-nitro-1H-pyrazole-3-carboxamide (Int-9)
To methyl 1-(cyclobutylmethyl)-4-nitro-1H-pyrazole-3-carboxylate was added a solution of ammonia in
MeOH (800 mL, 7M) at 0-5oC. The reaction mixture was then heated at 80oC in an autoclave for 8h.The
reaction mixture was distilled off completely to give 1-(cyclobutylmethyl)-4-nitro-1H-pyrazole-3carboxamide as an off-white solid (70 g, 75%). The product was used in the next step without further
purification.
1H NMR (300MHz, d-DMSO) ppm 1.65-2.05 (m, 6H), 2.7-2.8 (m, 1H), 4.2 (d, 2H), 7.75 (bs, 1H), 8.0
(bs, 1H), 8.9 (s,1H)
LCMS [M+H]+ 225.1
4-amino-1-(cyclobutylmethyl)-1H-pyrazole-3-carboxamide (Int-10)
To a solution of 1-(cyclobutylmethyl)-4-nitro-1H-pyrazole-3-carboxamide (70 g, 312.19 mmol) in EtOH
(1L) was added to slurry of 10% Pd/C (10 g) in EtOH (500 ml) at rt. The reaction mixture was stirred at rt
under hydrogen atmosphere at 60 psi for 8 h. The reaction mixture was filtered through celite pad and
concentrated to give a crude solid which was washed with Et 2O to give 4-amino-1-(cyclobutylmethyl)-1Hpyrazole-3-carboxamide (55 g, 83%).
1H
NMR (300MHz, d-DMSO) ppm 1.8-2.0 (m, 6H), 2.6-2.8 (m, 1H), 4.0 (s, 2H), 4.6 (s, 2H), 6.95 (s,1H),
7.1 (m, 2H).
LCMS [M+H]+ 195.1
2-(cyclobutylmethyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (Int-11)
To a solution of 2-(cyclobutylmethyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (55 g, 283.15 mol) in
CH3CN (200 mL) was added Et3N (149 mL, 1.132 mol) and carbonyldiimidazole (55 g, 339.78 mmol). The
reaction mixture was stirred at 90oC for 16 h. The reaction mixture was cooled to rt and filtered. The solid
was dried to give 2-(cyclobutylmethyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (40g, 64%). The
compound was used in the next step without further purification.
1H NMR (300MHz, d-DMSO) ppm 1.65-2.0 (m, 6H), 2.65 (m, 1H), 4.2 (d, 2H), 7.62 (s, 1H), 10.7 (s, 1H),
10.85 (s, 1H)
LCMS [M+H]+ 221.1
3-bromo-2-(cyclobutylmethyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (Int-12)
To a solution 2-(cyclobutylmethyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (40 g, 181.63 mmol) in
CH3CN and AcOH (10:1, 330 mL) was added N-bromosuccinimide (38.79 g, 217.95 mmol). The reaction
mixture was stirred at 100oC for 16 h. The reaction mixture was cooled to rt and the solid filtered off. The
solid was washed with water and dried to give 3-bromo-2-(cyclobutylmethyl)-2H-pyrazolo[4,3d]pyrimidine-5,7(4H,6H)-dione (35 g, 65%)
1H NMR (300MHz, d-DMSO) ppm 1.7-2.0 (m, 6H), 2.8 (m, 1H), 4.20 (d, 2H), 11.05 (s, 1H), 11.15 (s,
1H). LCMS [M+H]+ 301.1
2-(cyclobutylmethyl)-3-(pyridin-4-yl)-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (PPD-2)
A mixture of 3-bromo-2-(cyclobutylmethyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (2.06 g, 6.88
mmol), pyridine-4-boronic acid (2.06 g, 16.7 mmol), 1,4-dioxane (1 L), water (280 mL) and K3PO4 (6.36 g,
27.6 mmol) were de-gassed with argon for 10 min. Pd2(dba)3 (628 mg, 0.688 mmol) and PCy3 (384 mg,
1.67 mmol) were added and the mixture heated to reflux and stirred for 1.5 h.
The solvent was
evaporated under reduced pressure to leave ca. 30 mL remaining. The pH of the solution was adjusted
to pH 6-7 with 10% citric acid and extracted with EtOAc (3 × 100 mL). The pH was then adjusted to pH 45 with 10% citric acid, the aqueous layer was salted with NaCl, then extracted with MEK (3 × 100 mL).
The combined extracts were evaporated under reduced pressure. Purification by flash chromatography
(eluting with 1% MeOH:dichloromethane to 5% MeOH:dichloromethane gradient) gave product as a
yellowish solid at ca. 80% purity. The solid was slurried in methanol (5 mL), filtered off and dried in vacuo
to give product 2-(cyclobutylmethyl)-3-(pyridin-4-yl)-2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione as a
white solid (950 mg, 35%).
1H
NMR (400MHz, d-DMSO) ppm 1.5-1.9 (m, 6H), 2.5-2.7 (m, 1H), 4.1-4.3 (m, 2H), 7.48 (d, 2H), 8.69
(d, 2H), 10.82 (s, 1H), 10.98 (s, 1H).
LCMS: [M+H]+ 298.2