Optimization of potent, selective, and orally bioavailable pyrrolodinopyrimidine-containing inhibitors of heat shock protein 90. Identification of development candidate 2-amino-4-{4-chloro-2-[2-(4-fluoro-1h- pyrazol-1-yl)ethoxy]-6-methylphenyl}-n-(2,2-difluoropropyl)-5, 7-dihydro-6h-pyrrolo[3,4-d]pyrimidine-6-carboxamide

Article abstract of DOI:10.1021/jm200128m

Figure Presented. A novel class of heat shock protein 90 (Hsp90) inhibitors was discovered by high-throughput screening and was subsequently optimized using a combination of structure-based design, parallel synthesis, and the application of medicinal chemistry principles. Through this process, the biochemical and cell-based potency of the original HTS lead were substantially improved along with the corresponding metabolic stability properties. These efforts culminated with the identification of a development candidate (compound 42) which displayed desired PK/PD relationships, significant efficacy in a melanoma A2058 xenograft tumor model, and attractive DMPK profiles.

Full text of DOI:10.1021/jm200128m

Supporting Information
Optimization of Potent, Selective, and Orally
Bioavailable
Pyrrolodinopyrimidine-containing
Inhibitors of Heat Shock Protein 90 (Hsp90).
Identification of Development Candidate 2-amino-4-{4-
chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-
methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-6H-
pyrrolo[3,4-d]pyrimidine-6-carboxamide
Luke Zehnder, Michael Bennett, Jerry Meng, Buwen Huang, Sacha Ninkovic, Fen
Wang, John Braganza, John Tatlock, Tanya Jewell, Joe Zhongxiang Zhou, Ben
Burke, Jeff Wang, Karen Maegley, Pramod Mehta, Min-Jean Yin, Ketan S.
Gajiwala, Michael J. Hickey, Shinji Yamazaki, Evan Smith, Ping Kang, Anand
Sistla, Elena Dovalsantos, Michael R. Gehring, Robert Kania, Martin Wythes, and
Pei-Pei Kung*
Pfizer Worldwide Research and Development, La Jolla Laboratories, 10770, Science
Center Dr., San Diego, CA 92121, USA
Content of Supporting Information:
I.
Chemistry
S2-S19
II.
III.
IV.
Compound characterization table
Biology
X-ray crystallographic Studies of 6, 10, 16 and 35
S18-S19
S19-S21
S21-S22
S
1

I.
Chemistry
General experimental
Starting materials and solvents were purchased from commercial suppliers and were used
without further purification. The reported yields are for purified material and are not
1
optimized. H-NMR spectra were recorded on a Bruker instrument operating either at
300 MHz, or 400 MHz. Chemical shifts (δ) are in part per million referenced to TMS.
Compound 2a, 2b, 2c, and 3 were synthesized using the same procedure described in
reference 9. All compounds which were tested in the biochemical assay were analyzed
by both microanalysis (CHN) and LC/MS to confirm the purity ≥95%.
Synthesis of compounds 63 and 64.
I
I
Cl
O
O
O
O
O
O
Cl
O
O
Cl
a
b
B
B
d
c
Cl
O
Cl
O
O
Cl
Cl
Cl
Cl
Cl
61
Reagents and conditions: (a) NaH, I2, toluene, 81 °C, 12 h, 56%; (b)
chloromethoxyethane, K2CO3, DMF, 23 °C, 12 h; (c) 4,4,5,5-tetramethyl-
[1,3,2]dioxaborolanein, Et3N, Pd(OAc)2, biphenyl-2-yl-dicyclohexyl-phosphane,
dioxane, 80 °C, 2 h, 49%. (d) BBr3, DCM, 0 °C, 20 min., 44%.
N
N
I
I
I
I
NO2
a
NO2
NO2
N
b
O
c
d
O
e
Cl
Cl
Cl
Cl
Cl
f
O
O
B
O
O
B
O
g
O
Cl
Cl
63
Reagents and conditions: (a) NCS, CH3OH, 60 °C, 4 h, 98%; (b) NaNO2, H2SO4, 23 °C,
0.5 h, 95%; (c) Fe, NH4Cl, EtOH, 50 °C, 0.5 h, 83%; (d) NaNO2, H2SO4, 23 °C, 0.5 h,
quantitative yield; (e) CH3I, K2CO3, CH3CN, 23 °C, 12 h, 74%; (f) 4,4,5,5-tetramethyl-
[1,3,2]dioxaborolanein, Et3N, PdCl2(dppf)2, CH3CN, 90 °C, 3 h, 81%. (g) BBr3, DCM, 0
°C, 10 minutes, 80%.
S
2

3,5-Dichloro-2-iodo-phenol
I
Cl
O
Cl
Sodium hydride (368 g, 9.2 mol, 60% dispersion in minerol oil) was added to a solution
o
of 3,5-dichloro-phenol (500 g, 3.067 mol) in dry toluene (5 L) at 0 C under N2
atmosphere. After the addition, the resulting mixture was allowed to warm up to room
o
temperature and stirred for 20 minutes. The suspension was then cooled back to 0 C,
and iodine (779 g, 3.067 mol) was added slowly. Then the reaction mixture was stirred at
room temperature overnight. TLC (petroleum ether/DCM 1:1) indicated complete
consumption of 3,5-dichloro-phenol. The reaction mixture was quenched with 1N HCl (5
L) and diluted with ether (5.0 L). The separated organic layer was washed with brine
(500 mL), dried over Na2SO4, and concentrated in vacuo to give crude compound 2,
which was basified to pH = 10 with 3N NaOH (3 L) and extracted with DCM (1.5 L x 2),
the separated aqueous layer was acidified to pH = 3 with HCl (2N, 3 L) . The mixture
was filtrated and the cake was dried in vacuo to give the title compound as a white solid
(550 g, 56%).
1,5-Dichloro-3-ethoxymethoxy-2-iodo-benzene
I
Cl
O
O
Cl
Chloromethoxy-ethane (206 g, 2.2 mol) was added to a solution of 3,5-dichloro-2-iodo-
phenol (500 g, 1.7 mol) and K2CO3 (284 g, 2.1 mol) in DMF (5 L). The reaction mixture
was stirred at room temperature for 12 h. TLC (petroleum ether/EtOAc 2:1) indicated
complete consumption of 3,5-dichloro-2-iodo-phenol. Most of the DMF was removed in
vacuum and H2O (5.0 L) was added. The resulting mixture was extracted with Et2O (2.5
L x 3) and washed brine (1 L), dried over Na2SO4, and concentrated in vacuo to give
crude title compound (543 g, 90%) as a white solid, which was used for the next step
directly.
2-(2,4-Dichloro-6-ethoxymethoxy-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane
S
3

O
O
B
Cl
O
O
Cl
A solution of 1,5-dichloro-3-ethoxymethoxy-2-iodo-benzene (250 g, 0.72 mol), 4,4,5,5-
tetramethyl-[1,3,2]dioxaborolanein (223 mL, 1.5 mol), Et3N (300 mL, 2.4 mol) in
dioxane (1500 mL) was purged with N2 for 30 minutes. Then Pd(OAc)2 (8.7 g, 0.04 mol)
and biphenyl-2-yl-dicyclohexyl-phosphane (27 g, 0.08 mol) were added to the resulting
o
mixture. After the addition, the reaction mixture was stirred at 80 C for 2 h. TLC
(petroleum ether/EtOAc 50:1) indicated complete consumption of 1,5-dichloro-3-
ethoxymethoxy-2-iodo-benzene. The resulting mixture was filtered and the filtrate was
concentrated in vacuo to give crude title compound. The title compound was crystalized
from petroleum ether (2.0 L) as a white solid (120 g, yield: 48.5%).
2-(2,4-Dichloro-6-hydroxy-phenyl)-boronic acid (61)
HO
OH
B
Cl
OH
Cl
Boron tribromide (524 g, 2.1 mol) was added to a stirred solution of 2-(2,4-Dichloro-6-
ethoxymethoxy-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (150 g, 0.43 mol) in
dry CH2Cl2 (1200 mL) was added dropwise at 0 oC under N2 atmosphere. The resulting
solution was stirred for 20 minutes. TLC (petroleum ether/EtOAc 50:1) indicated
complete
consumption
of
2-(2,4-Dichloro-6-ethoxymethoxy-phenyl)-4,4,5,5-
tetramethyl-[1,3,2]dioxaborolane. The reaction mixture was poured into ice water and
the aqueous solution was basified to pH ~ 10 by adding 3N NaOH (750 mL). The
aqueous layer was collected and adjusted to pH ~ 3 with 1N HCl (2000 mL). The
aqueous solution was extracted with EtOAc (3 x 2000 mL). The combined organic layers
were washed with brine (1.0 L), dried over Na2SO4, and concentrated in vacuo to give
1
compound 61 as a white solid (39.2 g, 43.7%). H NMR (400 MHz, CD3OD) δ ppm:
6.88 (d, J = 1.2, 1 H), 6.73 (d, J = 1.2, 1 H). MS: ([2M-2H2O]- m/z = 377).
S
4

4-Chloro-2-methyl-6-nitro-phenylamine
N
O
N
O
Cl
N-chlorosuccinimide (365g, 2.6 mol) was added to a solution of 2-Methyl-6-nitro-
o
phenylamine (375 g, 2.4 mol) in MeOH (4000 mL) at 60 C. The mixture was refluxed
for 4 h and TLC (petroleum ether EtOAc = 10:1) showed the reaction was complete. The
mixture was concentrated and the residue was dissolved in EtOAc (4L) and washed with
5% of aqueous NaOH solution for three times. The organic layer was dried and
concentrated to give the title compound (450 g, 98%) as a brown solid.
5-Chloro-2-iodo-1-methyl-3-nitro-benzene
I
O
N
O
Cl
A solution of sodium nitrite (146 g, 2.9 mol) in H2SO4 (750 mL) was added dropwise into
a solution of compound 4-chloro-2-methyl-6-nitro-phenylamine (250 g, 1.4 mol) in
acetic acid (2000 mL). The mixture was allowed to stir at room temperature for 0.5 h and
TLC (petroleum ether EtOAc = 10:1) showed the reaction was complete. Water (4 L)
and urea (146g) were added and the mixture was stirred at room temperature for 10 min,
A solution of KI (320 g) in H2O (2 L) was added dropwise. The mixture was stirred at
room temperature for 30 minutes and was filtered. The filter cake was washed with more
water and dried in vacuum to give the title compound (400 g, 95%) as a brown solid.
5-Chloro-2-iodo-3-methyl-phenylamine
I
N
Cl
A solution of NH4Cl (356 g, 2.9 mol) in H2O (4 L) was added into a solution of 5-
chloro-2-iodo-1-methyl-3-nitro-benzene (400 g, 1.34 mol) in EtOH (4 L), The mixture
was heated to 50 oC and Fe (350 g, 6.25mol) was added at one portion. The mixture was
allowed to stir at reflux for 30 min and TLC (petroleum ether EtOAc = 10:1) showed the
S
5

reaction was complete. The mixture was filtered and EtOH was removed by evaporation.
The aqueous solution was extracted with EtOAc (5 L) and the organic layer was dried,
filtered, and concentrated to give the title compound (300 g, 83%) as a brown solid.
5-Chloro-2-iodo-3-methyl-phenol
I
O
Cl
A solution of sodium nitrite (146 g, 2.9 mol) in H2SO4 (750 mL) was added dropwise into
a solution of 5-chloro-2-iodo-3-methyl-phenylamine (250 g, 0.94 mol) in acetic acid
(2000 mL). The mixture was allowed to stir at room temperature for 0.5 h and TLC
(petroleum ether EtOAc = 20:1) showed the reaction was complete, The mixture was
added dropwise into boiling water (5 L) after stirred at reflux for 10 min. The mixture
was extracted with EtOAc (5 L) and the organic layer was dried, filtered, and
concentrated to give crude title compound (300 g, 120%) as a brown solid. The crude
product was used direct to the next step.
5-Chloro-2-iodo-1-methoxy-3-methyl-benzene
I
O
Cl
Iodomethane (238 g, 1.6 mol) was added to a solution of 5-chloro-2-iodo-3-methyl-
phenol (300g, 1.12 mol) and K2CO3 (185 g, 1.33 mol) in CH3CN (6 L). The mixture was
stirred at room temperature for 12 h and TLC (petroleum ether EtOAc = 20:1) showed
the reaction was complete. The mixture was concentrated and the residue was dissolved
in EtOAc (4 L) and washed with H2O (2 L). The organic layer was dried, filtered, and
concentrated to give the crude product which was purified through a silica gel to give the
title compound (233 g, 74%) as an orange solid.
2-(4-Chloro-2-methoxy-6-methyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane
S
6

O
O
B
O
Cl
PdCl2(dppf)2 (13 g, 0.02 mol ) and Et3N (470 mL, 3.4 mol) were added to a solution of 5-
chloro-2-iodo-1-methoxy-3-methyl-benzene (150 g, 0.53 mol) in CH3CN (1600 mL).
4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (240 mL, 1.6 mol) was added dropwise at 23 oC.
The mixture was heated to reflux for 3 h and TLC (petroleum ether EtOAc = 20:1)
showed the reaction was complete. The mixture was concentrated and the residue was
dissolved in EtOAc (1 L) and washed with H2O (1 L). The organic layer was dried,
filtered, and concentrated to give the crude product which was purified through a silica
gel chromatography to give the title compound (140 g, 81%) as an off yellow solid.
2-(4-Chloro-2-hydroxy-6-methyl-phenyl)-boronic acid (63)
HO
OH
B
OH
Cl
Boron tribromide (90 mL, 0.94 mol) was added dropwise to a solution of 2-(4-chloro-2-
methoxy-6-methyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (100 g, 0.35
mol) in DCM (1.2 L) at 0 oC. The mixture was allowed to stir at 0 oC for 10 minutes and
TLC (petroleum ether EtOAc = 20:1) showed the reaction was complete. The mixture
was poured into ice water and extracted with EtOAc (3 x 1 L). The organic layer was
dried, filtered, and concentrated to give a solid residue. The residue was washed with
1
petroleum ether to give compound 63 (50 g, 80%) as an off white solid. H NMR (400
MHz, CD3OD) δ 6.67 (s, 1H), 6.58 (d, J = 1.0, 1H), 2.19 (s, 3H). MS: ([2M-2H2O]- m/z
= 335).
S
7

Synthesis of compounds 9, 10, and 11.
F
F
F
N
O
N
O
N
O
a
b
N
N
N
N
N
N
Cl
Cl
Cl
8
9
10
Reagents and conditions: (a) BBr3, DCM, 0 °C, 24 h, 68%; (b) 1-iodo-4,4,4-
trifluorobutane, K2CO3, DMF, 60 °C, 12 h, 69%.
F
F
F
N
O
N
N
O
a
b
N
N
N
N
Cl
61
N
N
Cl
Cl
Cl
Cl
11
Reagents and conditions: (a) 61, Pd(PPh3)4, Na2CO3, 85 °C, 2 h, 76%; (b) 1-iodo-4,4,4-
trifluorobutane, K2CO3, DMF, 60 °C, 12 h, 50% (two steps).
2-(2-amino-6-methylpyrimidin-4-yl)-3-chlorophenol (9)
N
O
N
N
Cl
Boron tribromide (1 M in heptane; 10 mL, 10 mmol) was added to a suspension of
o
compound 8 (2.46 g, 9.9 mmol) in DCM (35 mL) at 0 C. The resulting mixture was
warmed to ambient temperature and stirred for 24 h. An additional boron tribromide (20
o
mL) and DCM (65 mL) were carefully added at 0 C and the resulting mixture was
o
warmed to ambient temperature and stirred for 24 h. After stirring at 23 C for 72 h,
LCMS indicated less than 5% starting material remaining. The reaction mixture was
o
cooled at 0 C prior to adding water (0.5 mL every 2 minutes for a total of 5 mL)
carefully. The mixture was then poured into ice water/diethylether (1:1, 200 mL total).
Sodium bicarbonate was added portion wise until the mixture was neutralized to pH = 7.
EtOAc (200 mL) was added. The mixture was stirred for 30 minutes, the two phases were
S
8

separated and the organic phase was washed with brine (50 mL). The aqueous phase was
extracted with more EtOAc (100 mL). The combined organic phase was dried over
MgSO4, filtered, and concentrated until ~ 10 mL of solvent left. The solid was filtered
and washed with Et2O (10 mL) and dried to afford the title compound as a white solid
1
(1.57 g, 68%). H NMR(400 MHz, CDCl3) δ 10.24 (s, 1H), 7.20 (dd, J = 8.1, 1H), 6.94
(d, J = 7.8, 1H), 6.87 (d, J = 8.3, 1H), 6.60 (s, 2H), 6.49 (s, 1H), 2.26 (s, 3H). MS: (M+H
m/z = 236.0, 238.0). Anal. Calcd for C11H10N3O: C, 56.06%; H, 4.28%; N, 17.83%.
Found: C, 55.91%; H, 4.25%; N, 17.56%.
4-[2-Chloro-6-(4,4,4-trifluoro-butoxy)-phenyl]-6-methyl-pyrimidin-2-ylamine (10).
F
F
F
N
O
N
N
Cl
1-Iodo-4,4,4-trifluorbutane (121 mg, 0.51 mmol) was added to a solution of 9 (100 mg,
0.42 mmol) and K2CO3 (117 mg, 0.85 mmol) in DMF (5 mL). The resulting mixture was
o
stirred at 60 C for 12 h. After cooling down to rt, the reaction mixture was partitioned
between ethyl acetate (20 mL) and water (20 mL). The organic phase was dried, filtered,
and concentrated to dryness. The desired product was purified by silica gel column and
eluted at 60-70% EtOAc in hexanes to afford the title compound as a solid (101 mg, 69%
yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.38 (t, J = 8.3, 1 H), 7.10 (dd, J = 13, 8.0,
2 H), 6.55 (s, 2 H), 6.41 (s, 1 H), 4.01 (t, J = 5.8, 2 H), 2.24 (s, 3 H), 2.10 - 2.22 (m, 2 H),
1.70 - 1.82 (m, 2 H); MS: (M+H m/z) = 346.20/348.20; HRMS: Calcd. For
C15H16ClF3N3O: 347.095843; Found: 347.094902; Error = -2.71 ppm; Anal. calcd. for
C15H15ClF3N3O●0.03HOAc●0.1H2O: C: 51.78; H: 4.42; N: 12.03; Found: C: 51.58; H:
4.31; N: 12.00. HPLC Purity (254nm): 98.0%, RT = 9.825 min.
4-[2,4-Dichloro-6-(4,4,4-trifluoro-butoxy)-phenyl]-6-methyl-pyrimidin-2-ylamine
(11)
F
F
F
N
O
N
N
Cl
Cl
S
9

Sodium carbonate (0.85 mL, 1.71 mmol, 2 N) was added to a solution of 61 (147 mg,
0.73 mmol), 2-amino-4-chloro-6-methyl pyridine (112 mg, 0.8 mmol) in dioxane (12
mL). The mixture was bubbled with N2 for 15 min. Pd(PPh3)4 (82 mg, 0.071 mmol) was
o
then added to this solution. The resulting reaction mixture was heated at 85 C for 2 h.
After cooling down to rt, the reaction mixture was concentrated to dryness, and the
resulting residue was partitioned between water (20 mL) and EtOAc (2 x 30 mL). The
organic phase was separated and collected. The aqueous phase was acidified with 1 M
HCl to pH = 3, and extracted with more ethyl acetate (2 x 20 mL). The combined organic
layer was dried, filtered, and concentrated to dryness. This residue was used for the next
step reaction without further purification.
1-Iodo-4,4,4-trifluorbutane (338 mg, 1.42 mmol) was added to a solution of K2CO3 (196
mg, 1.42 mmol) and the above residue in DMF (6 mL). The resulting reaction mixture
o
was heated at 60 C for 12 h. After cooling down to rt, the reaction mixture was diluted
with 10 mL water, and then extracted with ethyl acetate (2 x 50 mL). The combined
organic layer was washed with brine (10 mL), dried, filtered, and concentrated to
dryness. The desired product was purified by silica gel column and eluted at 40-50%
EtOAc in hexanes to afford the title compound as a solid (136 mg, 50% over two steps).
1H NMR (400 MHz, DMSO-d6) δ ppm: 7.36 (t, J = 8.2, 1 H), 7.08 (dd, J = 8.2, 3.2, 2 H),
6.55 (s, 2 H), 6.39 (s, 1 H), 3.96 - 4.06 (m, 6 H), 2.24 (s, 3 H), 1.82 (s, 3 H), 1.66 - 1.76
(m, 2 H), 1.03 (s, 9 H); MS: (M+H m/z) = 380.05/382.00; HRMS: Calcd. for
C15H15Cl2F3N3O (M+H): 381.056869; Found: 381.056361; Error = -1.33 ppm; Anal.
calcd for C18H25ClN4O●1.14HOAc●0.97H2O: C: 56.02; H: 7.30; N: 12.89. Found: C:
56.07; H: 7.04; N: 12.89; HPLC Purity (254nm): 99.0%, RT = 10.525 min.
Synthesis of compounds 24 and 25.
S
10

O
N
O
N
O
N
O
N
O
O
N
H
O
O
O
O
Cl
R
R
a
N
N
O
b
c
N
N
N
N
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
55
Cl
Cl
Cl
24, R= N (CH2)2CF2
Reagents and conditions: (a) BrCH2CH2Cl, K2CO3, DMF, 70 oC, 3 h, 67%; (b)
azetidine-HCl, KI/K2CO3, 120oC, microwave, 30 min; (c) TFA, 23 oC, 3 h; (d) CDI,
cyclobutylamine, TEA, THF, 60 oC, 2 h, 48% over three steps.
2-Amino-4-[2,4-dichloro-6-(2-chloro-ethoxy)-phenyl]-5,7-dihydro-pyrrolo[3,4-
d]pyrimidine-6-carboxylic acid tert-butyl ester
O
O
N
Cl
N
O
N
N
Cl
Cl
A mixture of tert-butyl 2-amino-4-(2,4-dichloro-6-hydroxyphenyl)-5,7-dihydro-6H-
pyrrolo[3,4-d]pyrimidine-6-carboxylate (200 mg, 0.5 mmol), 1-bromo-2-chloroethane
(144 mg, 1 mmol), cesium carbonate (492 mg, 1.5 mmol), and 4 mL anhydrous DMSO
was stirred at 70 °C for 2 h. After cooling down to rt, the reaction mixture was partitioned
between ethyl acetate (30 mL) and water (30 mL). The organic phase was separated,
washed with water (1 x 30mL) and brine (1 x 30mL), dried over sodium sulfate, and
concentrated to dryness, and purified by column with 40% EtOAc/heptane to afford the
title compound as a solid (156 mg, 67.4% yield). MS: (M+H m/z) = 459.10/461.05.
2-Amino-4-{2,4-dichloro-6-[2-(3,3-difluoro-azetidin-1-yl)-ethoxy]-phenyl}-5,7-
dihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic acid tert-butyl ester
S
11

O
N
O
O
F
N
F
N
N
N
Cl
Cl
3,3-Difluoroazetinine hydrochloride (79 mg, 0.61 mmol) was added to a solution of tert-
butyl 2-amino-4-[2,4-dichloro-6-(2-chloroethoxy)phenyl]-5,7-dihydro-6H-pyrrolo[3,4-
d]pyrimidine-6-carboxylate (140 mg, 0.31 mmol), K2CO3 (126 mg, 0.92 mmol), and KI
(76 mg, 0.46 mmol) in DMF (4 mL). The mixture was heated at 120 °C in a microwave
for 30 minutes. After cooling down to 23 °C, the reaction mixture was partitioned
between EtOAc (20 mL) and water (20 mL). The organic phase was separated, washed
with water (1 x 20 mL) and brine (1 x 20 mL), dried, and purified by silica gel
chromatography. The desired product was eluted with 60% EtOAc/Heptanes to afford
the title compound as a solid (84 mg, 53% yield). MS: (M+H m/z) = 516.10/518.0.
4-{2,4-Dichloro-6-[2-(3,3-difluoro-azetidin-1-yl)-ethoxy]-phenyl}-6,7-dihydro-5H-
pyrrolo[3,4-d]pyrimidin-2-ylamine
N
F
N
F
N
O
N
N
Cl
Cl
Trifluoroacetic acid (2 mL) was added to a mixture of tert-butyl 2-amino-4-{2,4-
dichloro-6-[2-(3,3-difluoroazetidin-1-yl)ethoxy]phenyl}-5,7-dihydro-6H-pyrrolo[3,4-
d]pyrimidine-6-carboxylate (84 mg, 0.163 mmol). The mixture was stirred at 23 °C for 3
h. After removing excess TFA, the resulting residue was dissolved in a small amount of
CH3CN, neutralized with NaHCO3, and concentrated to afford a solid. This solid residue
was then suspended in ethyl acetate, and filtered. The resulting filtrate was concentrated
to afford the title compound. MS: (M+H m/z) = 416.05/418.00.
2-Amino-4-{2,4-dichloro-6-[2-(3,3-difluoro-azetidin-1-yl)-ethoxy]-phenyl}-5,7-
dihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic acid cyclobutylamide (24).
S
12

O
N
N
O
F
N
F
N
N
N
Cl
Cl
Cyclobutylamine (23 mg, 0.326 mmol, in 1 mL of THF) was added to a solution of CDI
(53 mg, 0.326 mmol) in THF (2 mL) at 0 °C. After stirring at 23 °C for 3 h, a solution of
4-{2,4-dichloro-6-[2-(3,3-difluoroazetidin-1-yl)ethoxy]phenyl}-6,7-dihydro-5H-
pyrrolo[3,4-d]pyrimidin-2-amine (68 mg, 0.163 mmol) in THF (2 mL) and TEA (0.07
mL, 0.502 mmol, neat) was added to the cyclobutylamine/CDI mixture, and the resulting
reaction mixture was stirred at 60 °C for 2 h. After concentrating to dryness, the
resulting residue was concentrated to dryness, and purified prep HPLC to afford the title
1
compound as a solid (40 mg, 48% yield over three steps). H NMR (400 MHz, DMSO-
d6) δ ppm: 7.34 (d, J = 1.5, 1 H), 7.25 (d, J = 1.8, 1 H), 6.85 (s, 2 H), 6.47 - 6.57 (m, 2 H),
4.26 - 4.48 (m, 2 H), 4.09 - 4.17 (m, 3 H), 3.98 - 4.08 (m, 2 H), 3.36 - 3.47 (m, 2 H), 2.74
(br. s., 2 H), 2.08 (dd, J = 6.8, 3.5, 2 H), 1.85 - 1.97 (m, 2 H), 1.54 (dd, J = 9.9, 7.3, 2 H).
MS: (M+H m/z) = 513.10/515.05; HRMS: Cacld for C22H25Cl2F2N6O2 (M+H): 513.1379;
Found: 513.1378; HPLC Purity (254nm): 98.4%, RT = 11.425 min.
2-Amino-4-[2,4-dichloro-6-(2-ethylamino-ethoxy)-phenyl]-5,7-dihydro-pyrrolo[3,4-
d]pyrimidine-6-carboxylic acid cyclobutylamide (25)
O
N
N
N
N
O
N
N
Cl
Cl
Following the procedure for the preparation of 24 but substituting 55 with 48e and
substituting azetidine hydrochloride with ethyl amine provided the title compound with
14% yield.
1H NMR (400 MHz, DMSO-d6) δ ppm: 7.34 (s, 1 H), 7.31 (s, 1 H), 6.82 (s, 2 H), 6.51 (d,
J = 7.8, 1 H), 4.40 (br. s., 2 H), 4.01 - 4.19 (m, 4 H), 2.73 (t, J = 4.9, 2 H), 2.38 - 2.45
(m, 2 H), 2.02 - 2.14 (m, 2 H), 1.83 - 1.96 (m, 4 H), 1.44 - 1.61 (m, 2 H), 0.87 (t, J =
S
13

7.1, 3 H). MS: (M+H m/z) = 465.10/467.05; HRMS: Calcd. for C21H27Cl2N6O2 (M+H):
465.1567; Found: 465.1568; HPLC Purity (254nm): 96.9%, RT = 8.201 min.
Synthesis of compounds 36 and 37.
O
O
N
O
N
N
O
N
O
N
N
N
c
a
b
N
46
N
N
61
N
N
N
N
N
N
N
I
Cl
Cl
Cl
Cl
36
o
Reagents and conditions: (a) amine, CDI, TEA, DMF, 0 oC to rt; add 46, rt to 45 C, 2
h, 43%; (b) 61, Na2CO3, Pd(PPh3)4, dioxane, 85 °C, 24 h, 51%; (c) 1-(2-Chloro-ethyl)-4-
methyl-1H-pyrazole, Cs2CO3, KI, DMSO, 100 °C, 6 h, 35%.
2-Amino-4-iodo-5,7-dihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic
bicyclo[1.1.1]pent-1-ylamide
acid
O
N
N
N
N
N
I
Following the procedure for the preparation of 47e but substituting cyclobutylamine with
1
bicyclo[1.1.1]pent-1-ylamine provided the title compound in 43% yield. H NMR (300
MHz, DMSO-d6) δ 7.09 (br. s., 1H), 7.02 (br. s., 2H), 4.38 (s, 2H), 4.23 (s, 2H), 2.37 (s,
1H), 1.96 (s, 6H). MS: (M+H m/z = 372.2).
2-Amino-4-(2,4-dichloro-6-hydroxy-phenyl)-5,7-dihydro-pyrrolo[3,4-d]pyrimidine-
6-carboxylic acid bicyclo[1.1.1]pent-1-ylamide
S
14

O
N
N
O
N
N
N
Cl
Cl
Following the procedure for the preparation of 48e but substituting 47e with 2-Amino-4-
iodo-5,7-dihydro-pyrrolo[3,4-d]pyrimidine-6-carboxylic acid bicyclo[1.1.1]pent-1-
ylamide provided the title compound in 51% yield. MS: (M+H m/z = 406.2).
1-(2-Chloro-ethyl)-4-methyl-1H-pyrazole
N
N
Cl
A solution of 4-methyl pyrazole (0.177 g, 2.16 mmol) and 1-chloro-2-iodoethane (1.20 g,
6.30 mmol) in 1,4-dioxane (5.0 mL) was treated with cesium carbonate (1.10 g, 3.38
o
mmol) and heated to 95 C. After 24 h, the rxn mixture was cooled to rt, diluted with
DCM (10 mL), filtered, and concentrated. The resulting residue was purified on silica
gel eluting with 0-70% EtOAc in hexanes to provide the title compound as a clear oil (49
1
mg, 16%). H NMR (300 MHz, CDCl3) δ 7.46 (s, 1H), 7.33 (s, 1H), 4.49 (t, J = 5.93 Hz,
2H), 3.92 (t, J = 5.84 Hz, 2H), 2.12 (s, 3H).
2-Amino-4-{2,4-dichloro-6-[2-(4-methyl-pyrazol-1-yl)-ethoxy]-phenyl}-5,7-dihydro-
pyrrolo[3,4-d]pyrimidine-6-carboxylic acid bicyclo[1.1.1]pent-1-ylamide (36)
O
N
N
N
N
N
O
N
N
Cl
Cl
A solution of 2-Amino-4-(2,4-dichloro-6-hydroxy-phenyl)-5,7-dihydro-pyrrolo[3,4-
d]pyrimidine-6-carboxylic acid bicyclo[1.1.1]pent-1-ylamide (59 mg, 0.15 mmol) in
DMSO (1.5 mL) was treated with cesium carbonate (177 mg, 0.543 mmol), potassium
iodide (7 mg), and 1-(2-Chloro-ethyl)-4-methyl-1H-pyrazole (49 mg, 0.34 mmol) and
heated to 100oC. After 6h, the reaction mixture was cooled to rt and filtered. Preparative
S
15

HPLC provided the title compound as a white powder (26 mg, 35%). 1H NMR (300
MHz, DMSO-d6) δ 7.33 (d, J = 1.13 Hz, 1H), 7.25 (d, J = 1.51 Hz, 1H), 7.10 (s, 1H),
6.97 (s, 1H), 6.91 (br. s., 1H), 6.80 (br. s., 2H), 4.19 - 4.40 (m, 6H), 3.95 (d, J = 12.81
Hz, 1H), 3.68 (d, J = 13.75 Hz, 1H), 2.35 (s, 1H), 1.94 (s, 6H), 1.90 (s, 3H). MS: (M+H
m/z = 514.2). Anal. Calcd. for C24H25N7O2Cl2●2.0 H2O: C, 52.37%; H, 5.31%; N,
17.81%. Found: C, 52.48%; H, 4.99%; N, 17.63%.
Cl
Cl
Cl
O
N
O
N
O
O
N
O
H
N
N
N
N
N
N
N
b
c
a
53
N
N
N
O
H2N
N
H2N
N
H2N
N
Cl
Cl
Cl
Cl
Cl
Cl
37
Reagents and conditions: (a) Methanesulfonic acid 2-(4-chloro-pyrazol-1-yl)-ethyl
o
ester, K2CO3, KI, DMF, 120 °C, 40 minutes, 48%; (b) HCl, DCM, 23 C, 12 h, quant.;
(c) bicyclo[1.1.1]pent-1-ylamine, CDI, TEA, DMF, 0 to 60 oC, 3 h, 39%.
Methanesulfonic acid 2-(4-chloro-pyrazol-1-yl)-ethyl ester
N
O
Cl
N
O
S O
Sodium hydride (136 mg, 60% dispersion in mineral oil) was added to a solution of 4-
chloro pyrazole (0.34 g, 3.3 mmol) in DMF (3 mL) under nitrogen atmosphere. The
reaction was stirred at 23 oC for 1 h and was canulated into a solution of Methanesulfonic
acid 2-bromo-ethyl ester (810 mg, 4 mmol) in DMF (5 mL). The solution was heated at
70 oC for 3 h. The reaction was quenched with H2O (20 mL) and extracted with EtOAc
(2 x 40 mL). The combined organic layer was dried, filtered, and concentrated to get a
brown oil. This curde mixture was purified by column chromatography to provide the
title compounds as a brown oil (59 mg, 8%).
2-Amino-4-{2,4-dichloro-6-[2-(4-chloro-pyrazol-1-yl)-ethoxy]-phenyl}-5,7-dihydro-
pyrrolo[3,4-d]pyrimidine-6-carboxylic acid tert-butyl ester
S
16

Cl
O
N
O
O
N
N
N
N
N
Cl
Cl
Following the procedure for the preparation of 57 but substituting 65 with
methanesulfonic acid 2-(4-chloro-pyrazol-1-yl)-ethyl ester provided the title
compound in 48% yield. MS: (M+H m/z = 525.1).
1H NMR (400 MHz, DMSO-d6) δ 7.55 (s, 1H), 7.39 (s, 1H), 7.32 - 7.34 (m, 1H), 7.27 (s,
1H), 6.81 (br. s., 2H), 4.40 (d, J = 3.54 Hz, 4H), 4.28 - 4.34 (m, 2H), 3.91 (dd, J = 13.39,
24.00 Hz, 1H), 3.63 (t, J = 13.89 Hz, 1H), 1.38 - 1.48 (m, 9H).
4-{2,4-Dichloro-6-[2-(4-chloro-pyrazol-1-yl)-ethoxy]-phenyl}-6,7-dihydro-5H-
pyrrolo[3,4-d]pyrimidin-2-ylamine
Cl
N
N
N
N
O
N
N
Cl
Cl
Following the procedure for the preparation of 60 but substituting 57 with 2-Amino-4-
{2,4-dichloro-6-[2-(4-chloro-pyrazol-1-yl)-ethoxy]-phenyl}-5,7-dihydro-pyrrolo[3,4-
d]pyrimidine-6-carboxylic acid tert-butyl ester provided the title compound in
quantitative yield. MS: (M+H m/z = 425.1).
2-Amino-4-{2,4-dichloro-6-[2-(4-chloro-pyrazol-1-yl)-ethoxy]-phenyl}-5,7-dihydro-
pyrrolo[3,4-d]pyrimidine-6-carboxylic acid bicyclo[1.1.1]pent-1-ylamide (37)
Cl
O
N
N
N
N
N
O
N
N
Cl
Cl
S
17

Following the procedure for the preparation of 42 but substituting 60 with 4-{2,4-
Dichloro-6-[2-(4-chloro-pyrazol-1-yl)-ethoxy]-phenyl}-6,7-dihydro-5H-pyrrolo[3,4-
d]pyrimidin-2-ylamine and substituting 2,2-difluoro-propylamine hydrochloride with
bicyclo[1.1.1]pent-1-ylamine provided the title compound as a pale yellow solid residue
1
(23 mg, 39%). MS: (M+H m/z = 534.0). H NMR (400 MHz, DMSO-d6) δ 7.49 (s, 1H),
7.36 (s, 1H), 7.35 (d, J = 1.5, 1H), 7.28 (d, J = 1.8, 1H), 6.90 (s, 1H), 6.78 (s, 2H), 4.33 -
4.48 (m, 4H), 4.27 - 4.33 (m, 2H), 3.96 (d, J = 13.1, 1H), 3.72 (d, J = 13.1, 1H), 2.35 (s,
1H), 1.94 (s, 6H).
Anal. Calcd. for C23H22N7O2Cl3●1.0 H2O: C, 49.97%; H, 4.38%; N, 17.73%. Found: C,
50.36%; H, 4.23%; N, 17.40%.
II. Compound characterization table
Compound characterization table for novel compounds:
compd
LC/MS
(M+1)
236.0
346.2
380.1
479.2
503.2
492.2
490.1
504.2
436.2
498.0
499.2
461.2
489.2
452.2
452.0
513.1
465.1
488.1
435.2
448.2
463.1
474.1
Purity (UV 1HNMR
254 nm)
93%
Elemental
Analysis
HRMS
9
√
√
√
√
√
98%
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
99%
95%
96%
√
√
√
98%
95%
95%
√
√
95%
√
√
√
98%
96%
95%
97%
√
√
√
√
√
√
95%
99%
98%
97%
99%
√
√
√
√
√
√
95%
99%
√
√
99%
√
√
98%
√
S
18

524.0
500.2
498.2
517.0
514.0
514.2
534.0
518.0
534.0
514.2
531.0
510.0
99%
99%
95%
99%
99%
95%
99%
95%
99%
99%
95%
99%
31
32
33
34
35
36
37
38
39
40
41
42
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
All LC/MS data were gathered on an Agilent 1100 LC with MSD (Agilent model
G1946B upgraded to D model) single-quadrupole mass spec detectors running with
electrospray spray ionization source. The LC instrument includes a binary pump (Agilent
model G1312A) with upper pressure limit of 400 bar attached to autosampler (Agilent
model G1313A) which uses external try for sample submission. The column
compartment (Agilent model G1316A) which is attached to diode array (Agilent model
G1315A). The instrument acquisition and data handling was done with ChemStation rev.
B.02.01.
Elution Conditions:
Column: Waters Xbridge C18, 2.1x30 mm, 2.5 µm particle size; Column Temperature 80 °C
Solvent A: Water (0.1% formic acid and 0.05% ammonium formate)
Solvent B: Methanol (0.1% formic acid and 0.05% ammonium formate)
Gradient: 5-95 %B in 2.5 min, 95%B 2.5-3.0 min; Flow rate 1.2 mL/min
III.
Hsp90 Biochemical Assay
Compounds were evaluated for potency against HSP-90 using a SPA (scintillation
Biology
proximity assay) competition binding assay. Briefly, either full length or N-terminal
HSP-90 that contains a polyhistine (6-His) tag on its C-terminus binds to copper on
Yttrium-silicate scintillant beads via chelation to the His-tag. A tritiated proprietary
small molecule ligand is used as the radiolabeled ligand in the competition assay.
3
Binding of H-ligand to HSP-90 brings the isotope into proximity with the beads. Test
S
19

3
compounds which bind to HSP-90 can compete with H-ligand at the ATP-binding site
on the N-terminus of HSP-90.
The SPA assay for binding to HSP-90 was performed in 96-well flat bottom white
plates (Corning #3604). 5 nM HSP-90 was incubated in binding buffer (100 mM Hepes,
pH 7.5 and 150 mM KCl), 50 nM 3H-ligand, various concentrations of inhibitors, and 100
ꢀg/well SPA beads at room temperature for 30 minutes with shaking. Prior to adding the
labeled ligand to the assay mix it was first diluted to 40% label with unlabeled ligand.
For Ki determinations, inhibitors were diluted in 100% DMSO to 22 different
concentrations prior to addition to the HSP-90/3H-ligand solution. The final DMSO in
the assay was 4% v/v. Copper-Ysi SPA beads (Perkin Elmer Health Sciences,
#RPNQ0096) were diluted in binding buffer prior to addition. The beads were allowed to
settle for 30 minutes after which the plates were counted using a TopCount NXT
instrument (Perkin Elmer Inc.).
For Ki determinations, corrected CPM were transformed into % inhibition using
the background wells (no enzyme) and DMSO only controls. The % inhibition values
were plotted vs. log inhibitor concentration and fit to four parameter logistic equation:




top − bottom
Y = bottom +




(
log IC − X × slope
)
50
1+10
Where Y is % Inhibition, top is the upper plateau, bottom is the lower plateau, X is the
log of the competing compound concentration, and slope is the Hill Slope. The IC50 is
then used to calculate the Ki by using the Cheng-Prusoff equation:







L





Ki = IC50/ 1+

Kd



Where Ki is the inhibition constant, L is the total concentration of 3H-ligand (50 nM), and
Kd is the dissociation constant for the ligand (40 nM). The synthesis of the precursor of
this ligand is described in Kung, P.-P. et al. J. Med. Chem. 2010, 53, 499-503
tritium-labeled ligand
S
20

T
T
HN
O
OH
O
N
HO
Cl
Cell based assay
Akt Luminex assay: non-small-cell lung carcinoma cell line NCI-H1299 (ATCC
Number: CRL-5803) was cultured in DMEM+10%FBS, 10,000cells/well were cultured
in 96-well plate. Compounds were added to each well starting at 10 uM with 3 fold serial
dilution of 11 points of resulting concentrations. 24 hours post adding compounds, cell
lysates were prepared and subjected to Luminex 100 system using the Beadlyte Akt/PKB
kit – Upstate #46-605.
IV.
X-ray crystallographic Studies of 10, 16 and 35
Crystallization conditions:
Compound stocks were made in 100 mM stock solutions using DMSO. For complex
formation a ratio of 5:1 ligand: protein was used. Protein/ligand complexes were set up
at 13 °C in the hanging drop method. These crystals were grown in 10-25% ethylene
glycol, 0.1M phosphate citrate pH 4.2-4.6, and 0-20 mM TCEP pH 7. Crystals would
appear between 1 and 3 days and harvested a week after set up.
S
21

Crystallographic data and refinement statistics.
Compound #
6
10
16
35
Data collection
P21
54.2, 44.1,
53.3, β =
115.7o
P212121
45.9, 79.4,
118.5
P212121
43.1, 79.5,
130.1
P212121
46.2, 79.6,
118.6
Space group
Unit cell (Å)
1.7
2.0
2.65
1.7
Resolution (Å)
Observations
Unique
74,217
20,999
125,213
29,909
57,618
12,218
162,391
48,645
reflections
Rsym
0.047 (0.083)
27.2 (13.4)
0.059 (0.229)
23.5 (7)
0.074 (0.221)
18.3 (6.5)
0.052 (0.373)
20.5 (3.2)
I/σ
0.827 (0.934)
0.994 (0.984)
Refinement
0.186
0.898 (0.796)
0.994 (0.987)
Completeness
0.197
0.228
0.211
0.285
0.187
0.212
R factor
Rfree
0.220
RMSD
0.005
1.2
0.005
0.9
0.006
0.9
0.004
1.0
Bond length
(Å)
Bond angle (o)
S
22