Discovery of 4-morpholino-6-aryl-1H-pyrazolo[3,4-d]pyrimidines as highly potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR): Optimization of the 6-aryl substituent

Article abstract of DOI:10.1021/jm9013828

Design and synthesis of a series of 4-morpholino-6-aryl-1H-pyrazolo[3,4-d] pyrimidines as potent and selective inhibitors of the mammalian target of rapamycin (mTOR) are described. Optimization of the 6-aryl substituent led to the discovery of inhibitors carrying 6-ureidophenyl groups, the first reported active site inhibitors of mTOR with subnanomolar inhibitory concentrations. The data presented in this paper show that 6-arylureidophenyl substituents led to potent mixed inhibitors of mTOR and phosphatidylinositol 3-kinase α (PI3K-α), whereas 6-alkylureidophenyl appendages gave highly selective mTOR inhibitors. Combination of 6-alkylureidophenyl groups with 1-carbamoylpiperidine substitution resulted in compounds with subnanomolar IC₅₀ against mTOR and greater than 1000-fold selectivity over PI3K-α. In addition, structure based drug design resulted in the preparation of several 6-arylureidophenyl-1H-pyrazolo[3,4-d]pyrimidines, substituted in the 4-position of the arylureido moiety with water solubilizing groups. These compounds combined potent mTOR inhibition (IC₅₀<1 nM) with unprecedented activity in cellular proliferation assays (IC ₅₀<1 nM). 2009 American Chemical Society.

Full text of DOI:10.1021/jm9013828

Supporting Information
Jeroen C. Verheijen,1* David J. Richard,1 Kevin Curran,1 Joshua Kaplan,1 Mark Lefever,1
Pawel Nowak,1 David J. Malwitz,1 Natasja Brooijmans,1 Lourdes Toral-Barza,2 Wei-Guo
Zhang,2 Judy Lucas,2 Irwin Hollander,2 Semiramis Ayral-Kaloustian,1 Tarek S. Mansour,1 Ker
Yu2 and Arie Zask1
1Chemical Sciences and 2Oncology Research, Wyeth Research, 401 N. Middletown Rd, Pearl
River, NY 10965
Table of Contents
S1
S2
S3
Title Page
Biological Methods
Preparation of analogs
S12 Preparation of additional compounds
S20 Table S1
S22 Table S2
S23 Table S3
S24 Table S4
S25 Table S5
S27 Table S6
S28 References
S1

Biological Methods
Assays of other kinases
Assays of 22 protein kinases were performed by the Wyeth Screening Sciences Kinase
Profiling group (Wyeth Research, Collegeville, PA). The assays employed the commercially
purchased enzymes ABL1, Aurora B, CDK1, CDK2, CHK1, CK1gamma1, ERK2, FYN,
GCK, HCK, LYN, MET, MK2, p38alpha, PDGFRalpha, PKA, PKCalpha, ROCK1, RSK1,
SRC, VEGFR2 (Invitrogen) and IKKalpha (Upstate Biotechnology) and various fluorescein-
labeled substrate peptides. The assays were performed in 384-well plate format with enzyme,
1.5 ꢀM substrate peptide, ATP (2x Km value), and various inhibitors for 1 h, and results were
detected by the Caliper LabChip 3000 chip-based mobility shift electrophoresis assay platform
(Caliper LifeSciences).
Other tumor cell lines growth assays
Cell proliferation inhibition assays were performed as previously described. 1
S2

Preparation of analogs
6-(1H-indol-5-yl)-4-morpholin-4-yl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6a): Yield:
29 mg (0.049 ꢀmol, 41%). LCMS: 93% pure, RT 2.52 min, m/z 397.2 ([M+H]+). HRMS: m/z
397.17685 ([M+H]+). For [M+H]+ mass error = -0.29 mmu.
3-(4-morpholino-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)aniline (6b): Yield: 32 mg
(0.087 ꢀmol, 72%). LCMS: 87% pure, RT 2.19 min, m/z 373.2 ([M+H]+).
6-(4-morpholino-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)pyridin-2-amine (6e): Yield:
12 mg (0.031 ꢀmol, 31%). LCMS: 99.8% pure, RT 1.90 min, m/z 374.2 ([M+H]+).
6-(4-morpholino-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)pyridin-3-amine (6f): Yield:
14 mg (0.037 ꢀmol, 37%). LCMS: 100% pure, RT 1.93 min, m/z 374.2 ([M+H]+).
N'-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin- 6-
yl]phenyl}-N,N-dimethylurea (16b):
To commercially available 4-isocyanatophenyl boronic acid, pinacol ester (49 mg, 0.2 mmol)
in a microwave vial was added a 2N solution of dimethylamine in THF (1 mL) and the mixture
was stirred for 0.5 h. The mixture was concentrated and to the resulting ureidophenyl boronic
acid, pinacol ester 15b was added 4-(1-(1-benzylpiperidin-4-yl)-6-chloro-1H-pyrazolo[3,4-
d]pyrimidin-4-yl)morpholine hydrochloride (12, 50 mg, 0.12 mmol) and 0.25 mL of a 2M
aqueous solution of sodium carbonate. Tetrakis(triphenylphosphine)palladium(0) (14 mg, 0.1
eq) was added and the mixture was heated for 10 min at 185 oC under microwave irradiation.
The mixture was concentrated, dissolved in DMSO, filtered and purified by reversed phase
S3

HPLC (TFA buffers) to give the target compound as the trifluoroacetate salt (50 mg, 0.076
mmol, 63%). LCMS: 100% pure, RT 1.98 min, m/z 541.3 ([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-methoxyurea (16c): methoxylamine.HCl (167 mg, 2.0 mmol) was stirred in 2
mL DCM and 2 mL of a 1N aqueous solution of sodium hydroxide prior to addition of 4-
isocyanatophenylboronic acid, pinacol ester. The mixture was diluted with dichloromethane
and water and the organic phase was transferred to a microwave vial and concentrated. The
ureidophenyl boronic acid, pinacol ester was dissolved in dimethoxyethane (2 mL) prior to
Suzuki-Miyaura coupling. Yield: 12 mg (18 ꢀmol, 15%). LCMS: 93% pure, RT 1.94 min, m/z
543.3 ([M+H]+).
N-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-2,2-dimethylhydrazinecarboxamide (16d): 1,1-dimethylamine (152 ꢀL, 2.0
mmol) was stirred in 2 mL DCM with 4-isocyanatophenylboronic acid, pinacol ester for 1.5 h.
The mixture was diluted with dichloromethane and water and the organic phase was transferred
to a microwave vial and concentrated. The ureidophenyl boronic acid, pinacol ester was
dissolved in dimethoxyethane (2 mL) prior to Suzuki-Miyaura coupling. Yield: 15 mg (19
ꢀmol, 15%). LCMS: 98% pure, RT 1.99 min, m/z 556.3 ([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}urea (16e): 2 mL of a 0.5N solution of ammonia in dioxane was used. Excess amine
and solvent were removed under a stream of nitrogen and the ureidophenyl boronic acid,
pinacol ester was dissolved in dimethoxyethane (2 mL) prior to Suzuki-Miyaura coupling.
Yield: 5 mg (8 ꢀmol, 7%). LCMS: 100% pure, RT 1.81 min, m/z 513.3 ([M+H]+).
S4

1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-ethylurea (16f): 1 mL of a 2N solution of ethylamine in THF was used. Excess
amine and solvent were removed under a stream of nitrogen and the ureidophenyl boronic acid,
pinacol ester was dissolved in dimethoxyethane (2 mL) prior to Suzuki-Miyaura coupling.
Yield: 60 mg (92 ꢀmol, 76%). LCMS: 98% pure, RT 1.91 min, m/z 541.3 ([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-(2-fluoroethyl)urea (16g): 2-fluoroethylamine.HCl (199 mg, 2.0 mmol) was
stirred in 2 mL DCM and 2 mL of a 1N aqueous solution of sodium hydroxide prior to addition
of 4-isocyanatophenylboronic acid, pinacol ester. The mixture was diluted with
dichloromethane and water and the organic phase was transferred to a microwave vial and
concentrated. The ureidophenyl boronic acid, pinacol ester was dissolved in dimethoxyethane
(2 mL) prior to Suzuki-Miyaura coupling. Yield: 7 mg (11 ꢀmol, 9%). LCMS: 100% pure, RT
1.94 min, m/z 559.3 ([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-isopropylurea (16h): isopropylamine (42 ꢀL, 0.5 mmol) in 1 mL
dimethoxyethane was used. Excess amine and solvent were removed under a stream of
nitrogen and the ureidophenyl boronic acid, pinacol ester was dissolved in dimethoxyethane (2
mL) prior to Suzuki-Miyaura coupling. Yield: 25 mg (35 ꢀmol, 29%). LCMS: 87% pure, RT
1.97 min, m/z 555.3 ([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-(2-hydroxyethyl)urea (16i): A 1N stock solution of amine was prepared by
diluting 1 mmol of ethanolamine (60 ꢀL) with dimethoxyethane to a total volume of 1.0 mL.
0.2 mL of the stock solution (0.2 mmol amine), diluted with 1 mL of dimethoxyethane, was
S5

used to react with the isocyanatophenylboronate. The crude solution of ureidophenyl boronic
acid, pinacol ester was used in the Suzuki-Miyaura coupling. Yield: 40 mg (59 ꢀmol, 49%).
LCMS: 100% pure, RT 1.79 min, m/z 557.3 ([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-(2-methoxyethyl)urea (16j): A 1N stock solution of amine was prepared by
diluting 1 mmol of 2-methoxyethylamine (87 ꢀL) with dimethoxyethane to a total volume of
1.0 mL. 0.2 mL of the stock solution (0.2 mmol amine), diluted with 1 mL of
dimethoxyethane, was used to react with the isocyanatophenylboronate. The crude solution of
ureidophenyl boronic acid, pinacol ester was used in the Suzuki-Miyaura coupling. Yield: 38
mg (56 ꢀmol, 47%). LCMS: 99% pure, RT 1.88 min, m/z 571.3 ([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-[2-(dimethylamino)ethyl]urea (16k): A 1N stock solution of amine was
prepared by diluting 1 mmol of N,N-dimethylethylenediamine (109 ꢀL) with dimethoxyethane
to a total volume of 1.0 mL. 0.2 mL of the stock solution (0.2 mmol amine), diluted with 1 mL
of dimethoxyethane, was used to react with the isocyanatophenylboronate. The crude solution
of ureidophenyl boronic acid, pinacol ester was used in the Suzuki-Miyaura coupling. Yield:
39 mg of the bis-trifluoroacetate salt (48 ꢀmol, 40%). LCMS: 100% pure, RT 1.65 min, m/z
584.3 ([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-(1-methylpiperidin-4-yl)urea (16l): A 1N stock solution of amine was prepared
by diluting 1 mmol of 4-amino-1-methylpiperidine (114 mg) with dimethoxyethane to a total
volume of 1.0 mL. 0.2 mL of the stock solution (0.2 mmol amine), diluted with 1 mL of
dimethoxyethane, was used to react with the isocyanatophenylboronate. The crude solution of
S6

ureidophenyl boronic acid, pinacol ester was used in the Suzuki-Miyaura coupling. Yield: 33
mg of the bis-trifluoroacetate salt (39 ꢀmol, 33%). LCMS: 100% pure, RT 1.68 min, m/z 610.4
([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-phenylurea (16m): aniline (18 ꢀL, 0.2 mmol) in 1 mL dimethoxyethane was
used. The crude ureidophenyl boronic acid, pinacol ester was diluted with dimethoxyethane (1
mL) prior to Suzuki-Miyaura coupling. Yield: 15 mg (22 ꢀmol, 18%). LCMS: 92% pure, RT
2.10 min, m/z 589.3 ([M+H]+).
1-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}-3-pyridin-3-ylurea (16n): 3-aminopyridine (19 mg, 0.2 mmol) in 1 mL
dimethoxyethane was used. The crude ureidophenyl boronic acid, pinacol ester was diluted
with dimethoxyethane (1 mL) prior to Suzuki-Miyaura coupling. Yield: 4 mg (6 ꢀmol, 5%).
LCMS: 92% pure, RT 1.81 min, m/z 590.3 ([M+H]+).
2-hydroxyethyl (4-{4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-
pyrazolo[3,4-d]pyrimidin-6-yl}phenyl)carbamate (22b): Prepared using the same method as
described for 22a, using 0.77 mmol of 4-(4-morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-
yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)aniline hydrochloride with 20 eq (15 mmol) of ethylene
glycol. The product was obtained after HPLC purification (TFA buffers) as the TFA salt. Yield
63 mg. LCMS: 100% pure, RT 1.68 min, m/z 559.3 ([M+H]+).
1-methyl-3-(4-(4-morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)phenyl)urea (22c):
S7

To 4-(4-Morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)aniline hydrochloride (21, 685 mg, 1.35 mmol) was added dichloromethane (10 mL) and
triethylamine (0.75 mL) to give a yellow solution. Triphosgene (201 mg) was added and the
mixture was stirred at ambient temperature for 5 min. A 2N solution of methylamine in THF
was added (13.5 mL, 27 mmol) and the mixture was stirred for 30 min. The solvents were
concentrated. The crude product was applied to a silica gel column and eluted with a gradient
of methanol (0-20%) in ethyl acetate containing 1% triethylamine to give the title compound as
216 mg (0.41 mmol, 30%) of an off-white solid. LCMS: 100% pure, RT 1.77 min, m/z 528.3
([M+H]+). HRMS: m/z 528.28165 ([M+H]+). Exptl - Calc'd= -1.35 mmu. 1H-NMR (DMSO-
d6): δ 11.62 (1H, bs), 9.18 (1H, bs), 9.10 (1H, bs), 8.97 (1H, d, J=4.4 Hz), 8.75 (1H, d, J=7.2
Hz), 8.32 (3H, t, J=8.0 Hz), 8.06 (1H, t, J=6.6 Hz), 7.52 (2H, d, J=8.8 Hz), 5.09 (1H, m), 4.60
(2H, s), 3,99 (4H, m), 3.78 (4H, m), 3.59 (2H, m), 3.36 (2H, m), 2.66 (3H, s), 2.60 (2H, d,
J=12.8 Hz), 2.16 (2H, d, J=12.8Hz).
1-methoxy-3-(4-{4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-
pyrazolo[3,4-d]pyrimidin-6-yl}phenyl)urea (22d): Prepared using the same method as
described for 22a, using 49 mg (0.11 mmol) of 4-(4-morpholino-1-(1-(pyridin-3-
ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)aniline (21) and 160 mg (1.92
mmol) methoxylamine.HCl in 1.92 mL 1N NaOH. The product was obtained after HPLC
purification (TFA buffers) as the TFA salt. Yield: 44 mg. LCMS: 100% pure, RT 1.81 min,
m/z 544.3 ([M+H]+).
S8

methyl 4-[6-(4-{[(2-fluoroethyl)carbamoyl]amino}phenyl)-4-morpholin-4-yl-1H-
pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carboxylate (25b): a solution of 2-
fluoroethylamine.HCl (99 mg) in 1N NaOH (1 mL) was used. Yield: 22 mg (42 ꢀmol, 35%).
LCMS: 100% pure, RT 2.19 min, m/z 527.2 ([M+H]+).
methyl 4-(6-{4-[(cyclopropylcarbamoyl)amino]phenyl}-4-morpholin-4-yl-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (25c): a solution of
cyclopropylamine (70 ꢀL) in dichloromethane (1 mL) was used. Yield: 5 mg (10 ꢀmol, 8%).
LCMS: 100% pure, RT 2.23 min, m/z 521.3 ([M+H]+).
methyl 4-(6-{4-[(anilinocarbonyl)amino]phenyl}-4-morpholin-4-yl-1H-pyrazolo[3,4-
d]pyrimidin-1-yl)piperidine-1-carboxylate (25d): a solution of aniline (93 ꢀL) in
dichloromethane (1 mL) was used. Yield: 15 mg (27 ꢀmol, 23%). LCMS: 100% pure, RT 2.43
min, m/z 557.3 ([M+H]+).
methyl 4-(6-(4-(3-(4-(4-methylpiperazin-1-yl)phenyl)ureido)phenyl)-4-morpholino-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (25e): Using the general conditions
described above, methyl 4-(6-(4-aminophenyl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)piperidine-1-carboxylate (24a) was treated with 4-(4-methylpiperazin-1-yl)aniline to
provide 25e. Yield: 0.012 g, 14%, white solid. LCMS: 99% pure, RT 1.97 min, m/z 655.3
([M+H]+).
methyl 4-(6-(4-(3-(4-(2-hydroxyethyl)phenyl)ureido)phenyl)-4-morpholino-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (25f): Using the general conditions
described above, methyl 4-(6-(4-aminophenyl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)piperidine-1-carboxylate (24a) was treated with 2-(4-aminophenyl)ethanol to provide 25f.
Yield: 0.052 g, 34%, white solid. LCMS: 99% pure, RT 2.22 min, m/z 601.3 ([M+H]+).
S9

isopropyl 4-[6-(4-{[(2-fluoroethyl)carbamoyl]amino}phenyl)-4-morpholin-4-yl-1H-
pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carboxylate (26b): a solution of 2-
fluoroethylamine.HCl (99 mg) in 1N NaOH (1 mL) was used. Yield: 45 mg (80 ꢀmol, 67%).
LCMS: 100% pure, RT 2.38 min, m/z 555.3 ([M+H]+).
isopropyl 4-(6-{4-[(cyclopropylcarbamoyl)amino]phenyl}-4-morpholin-4-yl-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (26c): a solution of
cyclopropylamine (70 ꢀL) in dichloromethane (1 mL) was used. Yield: 49 mg (89 ꢀmol, 74%).
LCMS: 100% pure, RT 2.43 min, m/z 549.3 ([M+H]+).
tert-butyl 4-[6-(4-{[(2-fluoroethyl)carbamoyl]amino}phenyl)-4-morpholin-4-yl-1H-
pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carboxylate (27b): a solution of 2-
fluoroethylamine.HCl (99 mg) in 1N NaOH (1 mL) was used. Yield: 45 mg (78 ꢀmol, 65%).
LCMS: 94% pure, RT 2.46 min, m/z 569.3 ([M+H]+).
tert-butyl 4-(6-{4-[(cyclopropylcarbamoyl)amino]phenyl}-4-morpholin-4-yl-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (27c): a solution of
cyclopropylamine (70 ꢀL) in dichloromethane (1 mL) was used. Yield: 49 mg (87 ꢀmol, 73%).
LCMS: 95% pure, RT 2.52 min, m/z 563.3 ([M+H]+).
methyl 4-(6-(4-(3-(4-(2-(4-methylpiperazin-1-yl)ethyl)phenyl)ureido)phenyl)-4-
morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (25h): A
procedure analogous to that used for the preparation of methyl 4-(4-morpholino-6-(4-(3-(4-(2-
(pyrrolidin-1-yl)ethyl)phenyl)ureido)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-
carboxylate (25g) was used, substituting N-methyl-piperazine for pyrrolidine. Purification by
S10

HPLC provided 25h. Yield: 0.011 g, 22%, off-white solid. LCMS: 100% pure, RT 1.95 min,
m/z 683.4 ([M+H]+).
methyl 4-(4-morpholino-6-(4-(3-(4-(2-morpholinoethyl)phenyl)ureido)phenyl)-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (25i): A procedure analogous to
that used for the preparation of methyl 4-(4-morpholino-6-(4-(3-(4-(2-(pyrrolidin-1-
yl)ethyl)phenyl)ureido)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate
(25g) was used, substituting morpholine for pyrrolidine. Purification by HPLC provided 25i.
Yield: 0.010 g, 20%, off-white solid. LCMS: 100% pure, RT 2.00 min, m/z 670.3 ([M+H]+).
S11

Preparation of additional compounds
The following compounds were all prepared according to Scheme 2 – 4.
5-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]pyridin-2-amine (S1a): Yield: 33 mg (57 ꢀmol, 47%). LCMS: 100% pure, RT 1.62 min,
m/z 471.3 ([M+H]+).
5-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]pyrimidin-2-amine (S1b): Yield: 33 mg (56 ꢀmol, 46%). LCMS: 100% pure, RT 1.82 min,
m/z 472.2 ([M+H]+).
5-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]pyridin-2-ol (S1c): Yield: 33 mg (56 ꢀmol, 46%). LCMS: 100% pure, RT 1.79 min,
M+H=472.2. IR: 1653 cm-1: carbonyl C=O stretch. 1H-NMR (acetone-d6): δ 8.63 (1H, d, J=2.0
Hz), 8.47 (1H, dd, J=2.4 Hz, 9.6Hz), 8.16 (1H, bs), 7.65 (2H, m), 7.47 (3H, t, J=3.0 Hz), 6.41
(1H, d, J=9.6 Hz), 5.09 (1H, m), 4.41 (2H, bs), 4.05 (4H, t, J=5.0 Hz), 3.83 (4H, t, J=5.0 Hz),
3.64 (2H, bs), 3.26 (2H, m), 2.74 (2H, m), 2.09 (2H, m).
1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-6-pyridin-3-yl-1H-pyrazolo[3,4-
d]pyrimidine (S1d): Yield: 30 mg (53 ꢀmol, 44%). LCMS: 100% pure, RT 1.78 min, m/z
456.2 ([M+H]+).
3-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6- yl]phenol
(S1e): purified using NH4OH buffers to give the free base of the target compound. Yield: 23
mg (49 ꢀmol, 41%). LCMS: 94% pure, RT 1.97 min, m/z 471.2 ([M+H]+). 1H-NMR (DMSO-
d6): δ 9.51 (1H, s), 8.30 (1H, s), 7.87 (2H, m), 7.35 (4H, m), 7.27 (2H, m), 6.87 (1H, ddd,
S12

J=1.2Hz, 2.8 Hz, 8.0 Hz), 4.78 (1H, m), 4.00 (4H, m), 3.79 (4H, t, J=4.8 Hz), 3.57 (2H, s), 2.97
(2H, m), 2.21 (4H, m), 1.90 (2H, m).
4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6- yl]phenol
(S1f): Yield: 32 mg (54 ꢀmol, 45%). LCMS: 100% pure, RT 1.90 min, m/z 471.2 ([M+H]+).
3-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6- yl]aniline
(S1g): Yield: 40 mg (69 ꢀmol, 57%). LCMS: 100% pure, RT 1.80 min, m/z 470.3 ([M+H]+).
N-{3-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}formamide (S1h): Yield: 36 mg (59 ꢀmol, 49%). LCMS: 100% pure, RT 1.98 min,
m/z 498.3 ([M+H]+).
N-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}formamide (S1i): Yield: 46 mg (74 ꢀmol, 62%). LCMS: 100% pure, RT 1.89 min,
m/z 498.3 ([M+H]+).
N-{3-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}acetamide (S1j): Yield: 8 mg (12 ꢀmol, 10%). LCMS: 98% pure, RT 2.00 min, m/z
512.3 ([M+H]+).
N-{4-[1-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl]phenyl}acetamide (S1k): Yield: 42 mg (67 ꢀmol, 55%). LCMS: 99% pure, RT 1.98 min,
m/z 512.3 ([M+H]+).
5-[1-(1-Benzyl-piperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-yl]-1,3-
dihydro-benzoimidazol-2-one (S1l): 53 mg (0.2 mmol) 4-amino-3-nitrophenylboronic acid,
pinacol ester was suspended in 2 mL DME. A catalytic amount of Pd/C was added and the
nitro group was reduced under an atmosphere of hydrogen over 4 days. 130 ꢀL NEt3 was
added followed by 30 mg triphosgene. The mixture was stirred for 15 min and the resulting
S13

1,3-dihydro-benzoimidazol-2-one boronate was reacted with 50 mg 1-(1-Benzyl-piperidin-4-
yl)-6-chloro-4-morpholin-4-yl-1H-pyrazolo[3,4-d]pyrimidine according to the general
procedure. Yield: 35 mg (55 ꢀmol, 55%). LCMS: 100% pure, RT 1.84 min, m/z 511.2
([M+H]+).
Scheme S1 a
O
N
O
N
a
N
N
N
N
N
N
O
N
N
R
N
H2N
H
S2a-e
6c
a Reagents and conditions: (a) RCOOH, IIDQ
General procedure for IIDQ acylation of 4-(4-morpholin-4-yl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)aniline 6c:
To a solution of 4-(4-morpholin-4-yl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)aniline (6c,
40 mg, 0.1 mmol) in DMF (600 µL) was added 0.2 – 0.6 mmol of carboxylic acid and 0.2 – 0.6
mmol of IIDQ (60 – 180 µL). The reaction mixture was stirred for 72 h at RT or 50° C. The
reactions were worked up by concentration followed by HPLC purification.
N-[4-(4-morpholin-4-yl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6- yl)phenyl]glycinamide
(S2a): N-(tert-butoxycarbonyl)glycine (35 mg, 0.2 mmol) and IIDQ (60 ꢀL, 0.2 mmol) were
used at room temperature. The Boc group in the resulting product was removed by addition of
S14

TFA (0.6 mL). HPLC purification was done using NH4OH buffers. Yield: 15 mg (0.035 ꢀmol,
35%). LCMS: 75% pure, RT 2.07 min, m/z 430.2 ([M+H]+).
N2,N2-dimethyl-N-[4-(4-morpholin-4-yl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin- 6-
yl)phenyl]glycinamide (S2b): N,N-dimethylglycine (63 mg, 0.6 mmol) and IIDQ (180 ꢀL, 0.6
mmol) were used in DMA at 50 oC. HPLC purification was done using TFA buffers. Yield: 22
mg (0.048 ꢀmol, 48%). LCMS: 100% pure, RT 2.04 min, m/z 458.2 ([M+H]+).
N-[4-(4-morpholin-4-yl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenyl]-beta-
alaninamide (S2c): Boc-beta-alanine (38 mg, 0.2 mmol) and IIDQ (60 ꢀL, 0.2 mmol) were
used at room temperature. The Boc group in the resulting product was removed by addition of
TFA (0.6 mL) and heating to 50 oC. HPLC purification was done using NH4OH buffers. Yield:
8 mg (0.019 ꢀmol, 19%). LCMS: 85% pure, RT 2.05 min, m/z 444.2 ([M+H]+).
1-methyl-N-[4-(4-morpholin-4-yl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)phenyl]piperidine-4-carboxamide (S2d): 1-Me-piperidine-4-carboxylic acid.HCl (36 mg,
0.2 mmol) and IIDQ (60 ꢀL, 0.2 mmol) were used at 50 oC. HPLC purification was done using
NH4OH buffers. Yield: 50 mg (0.10 ꢀmol, 100%). LCMS: 93% pure, RT 2.20 min, m/z 498.3
([M+H]+).
3-methoxy-N-[4-(4-morpholin-4-yl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)phenyl]propanamide (S2e): 3-methoxypropionic acid (21 mg) and IIDQ (60 ꢀL, 0.2
mmol) were used at room temperature. HPLC purification was done using NH4OH buffers.
Yield: 16 mg (0.036 ꢀmol, 36%). LCMS: 99.5% pure, RT 2.42 min, m/z 459.2 ([M+H]+).
Preparation of urea isosteres is outlined in Schemes S2 and S3. As shown in Scheme S2,
condensation of aniline 21 with diphenyl cyanocarbonimidate, followed by reaction of
S15

resulting S3 with ammonia, gave cyanoguanidine S4. Condensation of 21 with
methylisothiocyanate gave thiourea S5. As shown in Scheme S3, methylsulfamoyl chloride S8
was prepared by adaptation of known methods.2,3 Thus, acylation of methylamine with
chlorosulfonic acid, followed by chlorination of resulting S6 led to methylsulfamoyl chloride
S7. Acylation of 24c with methylsulfamoyl chloride S7 gave sulfamate S8.
Scheme S2a
O
N
O
N
O
N
a
b
N
N
N
N
N
N
CN
CN
N
N
N
N
N
N
N
N
H2N
H2N
N
H
21
PhO
N
N
H
N
S4
N
S3
N
O
N
N
N
c
N
N
N
S
N
N
N
H
H
S5
N
N
a Reagents and conditions: (a) NEt3, PhOC(NCN)OPh; (b) NH3; (c) MeNCS.
2-cyano-1-(4-{4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-
pyrazolo[3,4-d]pyrimidin-6-yl}phenyl)guanidine (S4):
4-(4-morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)aniline (21, 50 mg, 0.09 mmol) was dissolved in DCM (1 mL). 1 equivalent of diphenyl
cyanocarbonimidate (22 mg, 0.09 mmol) and 2 equivalents (26 ꢀL) triethylamine were added.
S16

The reaction was stirred at room temperature until LCMS revealed complete conversion into
S3. The mixture was concentrated and a 0.5 N solution of ammonia in dioxane (10 mL) was
added. The mixture was heated in a sealed vial at 70 oC overnight. The solvents were
evaporated and the product was purified by RP-HPLC (TFA buffers) to give the title
compound as the TFA salt. Yield: 28 mg (43 ꢀmol, 48%). LCMS: 100% pure, RT 1.74 min,
m/z 538.3 ([M+H]+).
1-methyl-3-(4-{4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-
pyrazolo[3,4-d]pyrimidin-6-yl}phenyl)thiourea (S5):
4-(4-morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)aniline (23, 50 mg) was dissolved in dichloromethane (1 mL) and triethylamine was added
(65 ꢀL). Methylisothiocyanate (15 ꢀL, 0.2 mmol) was added and the mixture was heated at 40
oC overnight. The solvents were evaporated and the crude product was dissolved in DMSO,
filtered and purified by HPLC (TFA buffers) to give the title compound as TFA salt. Yield: 26
mg (0.039 mmol, 39%). LCMS: 84% pure, RT 1.78 min, m/z 544.3 ([M+H]+).
Scheme S3a
O
N
N
O
N S O-Na+
O
N
a
c
b
NH2
N
N S Cl
H
O
N
H
O
O
N S N
H
O
S7
S6
H
S8
N
Boc
a Reagents and conditions: (a) 1. NEt3, ClSO3H, 2. NaOH, H2O; (b) POCl3; (c) 24c.
S17

methylsulfamoyl chloride (S7):
A solution of methylamine hydrochloride (2.00 g, 29.62 mmol) in triethylamine (41.3 mL,
296.2 mmol) was stirred at room temperature for 25 min. Chloroform (60 mL) was then added
and the solution was cooled to 0 oC. Chlorosulfonic acid (1.98 mL, 29.62 mmol) was slowly
added over 15 min. The suspension was concentrated under reduced pressure, then treated with
2N aqueous sodium hydroxide (45 mL, 90 mmol). Water was removed under reduced pressure
and warm ethanol (100 mL) was added. The suspension was filtered and the filtrate was
concentrated to provide the sodium salt of methylsulfamic acid (S6). The crude product was
dissolved in 1,2-dichloroethane (200 mL), and phosphorus oxychloride (5.42 mL, 59.24 mmol)
was added. The solution was heated to 80 oC for 64 h, then cooled to room temperature and
concentrated under reduced pressure to provide methylsulfamoyl chloride S7 (1.42 g, 11.01
mmol) as a light yellow oil.
tert-butyl
4-(6-(4-(N-methylsulfamoylamino)phenyl)-4-morpholino-1H-pyrazolo[3,4-
d]pyrimidin-1-yl)piperidine-1-carboxylate (S8)
tert-Butyl 4-(6-(4-aminophenyl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-
1-carboxylate 24c was treated with methyl sulfamoyl chloride (S7, 2 eq) and pyridine (4 eq) in
DMF and stirred at RT for 3.5 h. Concentration and purification by reverse phase preparative
high performance liquid chromatography provided tert-butyl 4-(6-(4-(N-
methylsulfamoylamino)phenyl)-4-morpholino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-
S18

carboxylate S8. Yield: 0.068 g, 0.12 mmol, 16%. HPLC: 74% pure, RT 15.3 min. 1H-NMR
(CDCl3): δ 8.40 (2H, d, J=9.2 Hz), 7.95 (1H, s), 7.3 (2H, m), 6.8 (1H, bs), 5.04 (1H, m), 4.30
(2H, m), 4.07 (4H, t, J=4.6 Hz), 3.91 (4H, t, J=4.8 Hz), 2.99 (2H, m), 2.72 (3H, s), 2.23 (2H,
m), 2.00 (2H, m), 1.50 (9H, s).
S19

Table S1. Aminopyridine analogs.
O
N
N
N
N
R
N
N
Bn
mTOR
PI3K-α
Cmpd.
R
Selectivityb
IC50 (nM)a
IC50 (nM)a
17 +/- 3.0
107 +/- 30
18 +/- 3.0
6
S1a
H2N
H2N
HO
N
N
21 +/- 2.5
0.9
1.0
0.4
S1b
S1c
S1d
N
8,000 +/- 200 7,810 +/- 589
N
695 +/- 25
244 +/- 64
N
aMean +/- SEM. bSelectivity (IC50 PI3K-α/IC50 mTOR).
Similar activity was observed for aminopyridine S1a and aminopyrimidine S1b. The latter
compound was an equipotent inhibitor of mTOR and PI3K and may function as an interesting
lead for the development of dual mTOR/PI3K inhibitors. Replacement of the amine in S1a
with a hydroxyl group (leading to S1c) led to a more than 400-fold decrease in potency. This
S20

observation may reflect the fact that S1c exists predominantly in the pyridinone tautomeric
form,4 which will not be able to form the same H-bonds as the aminopyrimidine. Finally, the
requirement for a 4-amine group was confirmed by the greatly decreased activity of pyridine
S1d, reflecting the loss of the interaction between the exocyclic amine and Asp2195 and
Glu2190.
S21

Table S2. Comparison of meta vs. para-substitution.
O
N
N
N
N
R
N
N
Bn
mTOR
PI3K-α
Cmpd.
R
m/p
Selectivityb
IC50 (nM)a
83 +/- 4.4
170 +/- 30
IC50 (nM)a
31 +/- 6.8
720 +/- 194
HO
m
p
0.4
4
S1e
S1f
m
p
325 +/- 32
200 +/- 20
989 +/- 79
3
7
S1g
13a
H2N
1,368 +/- 137
H
N
m
p
1,230 +/- 270 2,862 +/- 360
106 +/- 15 684 +/- 40
2
7
S1h
S1i
H
O
H
N
m
p
2,450 +/- 450 2,005 +/- 162
13 +/- 3.1 420 +/- 122
0.8
32
S1j
S1k
O
aMean +/- SEM. bSelectivity (IC50 PI3K-α/IC50 mTOR).
S22

Table S3. Introduction of water-solubilizing groups through acylated anilines.
O
N
N
N
N
N
R
mTOR
PI3K-α
Cmpd.
S2a
R
Selectivityb
IC50 (nM)a IC50 (nM)a
O
H2N
19 +/- 3.8
49 +/- 15
3
5
3
N
H
O
N
62 +/- 7.5 325 +/- 83
24 +/- 5.5 64 +/- 4.0
S2b
N
H
O
S2c
H2N
N
H
O
O
77 +/- 5.5 209 +/- 10
34 +/- 2.0 303 +/- 38
3
9
S2d
S2e
N
H
N
O
N
H
aMean +/- SEM. bSelectivity (IC50 PI3K-α/IC50 mTOR).
S23

Table S4. Antiproliferative activity (IC50’s, nM) of ATP-competitive mTOR inhibitors against
a broad panel of tumor cell lines.
Sensitive cell lines
LNCap
prostate
(PTEN -/-)
500
U87MG
glioma
(PTEN -/-)
1,000
600
PC3MM2
prostate
(PTEN -/-)
800
MDA361
breast
(PIK3CA)
1,050
240
MCF7
breast
(PIK3CA)
280
Cmpd.
13o
24a
18a
24c
213
180
13
1.5
30
1.2
8
<0.7
3.4
12
0.7
2.8
0.7
Less sensitive lines
MDA435 MDA231 DU145 HT29
Cmpd.
breast
2,700
1,050
65
breast
3,800
1,600
320
prostate colon
4,000 5,000
1,250 10,000
13o
24a
18a
24c
120
90
2,800
3,000
40
180
S24

Table S5. Ureidophenyl analogs and isosteres.
O
N
N
N
R1
N
N
N
R2
mTOR
PI3K-α
LNCaP cell
IC50 (nM)c
Cmpd.
R1
R2
Selectivityb
IC50 (nM)a IC50 (nM)a
S
1.8 +/-0.05 124 +/- 9.5
69
70
S5
N
H
N
H
N
N
CN
N
4.6 +/- 1.3 260 +/- 50
56
23
1,400
4,000
S4
H2N
N
H
H
N
O
195 +/- 5.0
85 +/- 3.5
4,394
3,423
S1l
N
H
O
41
4,000
S8
O
HN S N
H
O
O
aMean +/- SEM. bSelectivity (IC50 PI3K-α/IC50 mTOR). cThe average error for LNCaP IC50
determinations was <25%.
A further investigation of isosteric replacements for the ureidophenyl group is presented in
Table S5. Methylthiourea S5 was less potent and selective than the corresponding methyl urea.
S25

A more pronounced decrease in activity is observed for cyanoguanidine S4. Compound S1k, in
which the ureido group was cyclized onto the phenyl ring, was much less potent than the
corresponding methyl urea. Finally, sulfamate S8 was significantly less potent than the
corresponding methyl urea.
S26

Table S6. Activity and selectivity profile of ATP-competitive mTOR inhibitors against a panel
of lipid and protein kinases. IC50’s (ꢀM) against the various kinases are listed.
PIKKs
mTOR
PI3Kα
PI3Kγ
ATR
Cmpd.
0.0046 0.801 6.224 >50
0.0004 0.041 0.495 11.4
0.0007 0.080 0.409 11.7
24a
24c
29a
Non-PIKKs
ABL1 AuroraB CDK1
CDK2
CHK1 CK1γ1 ERK2 FYN
GCK HCK
LYN A
MET
Cmpd.
>50 >50
>50 45.1
>50 >50
>50
>50
>50
>50
>50 17.9 >50 >50 >50 >50
>50 >50 >50 >50 >50 >50
>50 >50 >50 >50 >50 >50
>50 >50
>50 >50
>50 >50
24a
>50
>50
24c
29a
Non-PIKKs (cntd.)
VEGFR2
MK2 P38α
PDGFRα PKA ROCK1 RSK1
SRC
PKCα IKKα
Cmpd.
>50
>50 28.9 >50
>50 >50
>50 >50
>50 >50
>50
>50
>50
>50 >50 >50
>50 >50 >50
>50 >50 >50
24a
>50
>50
>50 >50
>50 >50
>50
>50
24c
29a
S27

References
1. Yu, K.; Toral-Barza, L.; Discafani, C.; Zhang, W. G.; Skotnicki, J.; Frost, P.; Gibbons,
J. mTOR, a Novel Target in Breast Cancer: The Effect of CCI-779, an mTOR Inhibitor,
in Preclinical Models of Breast Cancer. Endocr.-Relat. Cancer 2001, 8, 249–258.
2. Wiley, R.A.; Pearson, D.A.; Schmidt, V.; Wesche, S.B.; Roxon, J.J. Synthesis and
bacterial metabolism of cis- and trans-2-alkyl analogues of sodium cyclamate. J. Med.
Chem. 1983, 26, 1077-1079.
3. Dow, R.L.; Paight, E.S.; Schneider, S.R.; Hadcock, J.R.; Hargrove, D.M.; Martin, K.A.;
Maurer, T.S.; Nardone, N.A.; Tess, D.A.; DaSilva-Jardine, P. Potent and selective,
sulfamide-based human b3-adrenergic receptor agonists. Bioorg. Med. Chem. Lett.
2004, 14, 3235-3240.
4. LigPrep (version 2.2, Schrödinger, LLC, New York, NY, 2005) was used to predict the
predominant tautomeric state(s) for 13c. Using LigPrep with Epik, states were
determined at pH 7.4 +/- 1.0 and at pH 7.4. In both cases, the only state returned by
LigPrep was that of the keto-form. In addition, the IR spectrum for S1c showed a strong
absorbtion at a wavenumber of 1653 cm-1, ascribed to a carbonyl C=O stretch,
consistent with the keto-form.
S28