Discovery and optimization of pyrazolopyrimidine sulfamates as ATG7 inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115681

Autophagy is postulated to be required by cancer cells to survive periods of metabolic and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation pathways involved in autophagosome formation. This article describes the design and optimization of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these compounds.

Full text of DOI:10.1016/j.bmc.2020.115681

Journal Pre-proofs
Discovery and Optimization of Pyrazolopyrimidine Sulfamates as ATG7 In-
hibitors
Shih-Chung Huang, Sharmila Adhikari, James E. Brownell, Emily F.
Calderwood, Jouhara Chouitar, Natalie Roy D'Amore, Dylan B. England,
Klaudia Foley, Sean J. Harrison, Patrick J. LeRoy, David Lok, Anna
Lublinsky, Li-Ting Ma, Saurabh Menon, Yu Yang, Ji Zhang, Alexandra E.
Gould
PII:
S0968-0896(20)30511-3
https://doi.org/10.1016/j.bmc.2020.115681
BMC 115681
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
6 January 2020
27 July 2020
28 July 2020
Please cite this article as: S-C. Huang, S. Adhikari, J.E. Brownell, E.F. Calderwood, J. Chouitar, N. Roy
D'Amore, D.B. England, K. Foley, S.J. Harrison, P.J. LeRoy, D. Lok, A. Lublinsky, L-T. Ma, S. Menon, Y.
Yang, J. Zhang, A.E. Gould, Discovery and Optimization of Pyrazolopyrimidine Sulfamates as ATG7 Inhibitors,
Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115681
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Discovery and Optimization of
Pyrazolopyrimidine Sulfamates as ATG7
Inhibitors
Shih-Chung Huang,* Sharmila Adhikari, James E. Brownell, Emily F.
Calderwood, Jouhara Chouitar, Natalie Roy D'Amore, Dylan B. England, Klaudia
Foley, Sean J. Harrison, Patrick J. LeRoy, David Lok, Anna Lublinsky, Li-Ting
Ma, Saurabh Menon, Yu Yang, Ji Zhang and Alexandra E. Gould
Takeda Pharmaceuticals International Co., 40 Landsdowne Street, Cambridge,
Massachusetts 02139, United States
*
Corresponding author.
E-mail address: sampson.huang@takeda.com
1

Keywords: E1 enzyme, ATG7, ATG7 inhibitor, autophagy,
ABSTRACT
Autophagy is postulated to be required by cancer cells to survive periods of metabolic
and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation
pathways involved in autophagosome formation. This article describes the design and optimization
of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular
levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these
compounds.
1
. Introduction
Autophagy and the ubiquitin-proteasome pathway (UPP) are two major protein
degradation systems of the cell that play important roles in protein homeostasis and cellular
1
metabolism. The UPP is responsible for degrading 80-90% of proteins including many regulated,
short-lived, abnormal, or damaged proteins, whereas autophagy is primarily responsible for the
degradation of protein aggregates, cellular organelles and infectious organisms. Both systems
degrade proteins into small peptides and help maintain amino acid pools and energy balance in the
cell. The clinical significance of proteasome inhibition, particularly in hematologic malignancies,
2
has been well documented. The potential benefits of autophagy manipulation, however, are less
well established with both induction and inhibition considered to be of therapeutic value.3,4
However, autophagy inhibition has been demonstrated to be toxic to tumor cell lines.4
The key steps of the autophagic process include the assembly of dual-membrane vesicles
called autophagosomes which sequester elements of the cytosol for degradation, and subsequent
lysosomal fusion to form catabolically active autolysosomes.5,6 ATG7 is a ubiquitin-like protein
(Ubl) activating enzyme (aka E1) required for the activation of two Ubl conjugation pathways
7
involved in autophagosome formation. ATG12 is a Ubl that forms a conjugate with ATG5 in a
manner dependent on both ATG7 and the E2 conjugating enzyme ATG10.4,6,8 The ATG5-ATG12
conjugate potentiates the second Ubl pathway in which the human orthologs of yeast ATG8
(LC3A, LC3B, LC3C, GATE16, GABARAP, and GABARAPL1) form conjugates with the lipid
9
phosphatidylethanolamine (PE) in a manner dependent on the E2 ATG3. Accumulation of
2

lipidated ATG8 orthologs is considered a hallmark of autophagy and is commonly used as a marker
of active autophagy in cells and tissues.4,6,10
Autophagy inhibitors in clinical development, such as chloroquine and its derivatives,
4
interrupt lysosomal activity and fusion. Our strategy was to assess autophagy pathway modulation
further upstream via inhibition of autophagosome formation.11,12 Since E1 enzymes such as ATG7
function in the first step of their corresponding Ubl conjugation pathways, E1 inhibition should
specifically modulate the downstream biological consequences of that pathway. As such,
inhibition of E1s represents a method for interfering with the integrity of several aspects of cellular
physiology which may be functional in several disease mechanisms and thus represent potentially
important therapeutic targets. Herein, we describe the identification and optimization of ATG7
inhibitors that impact autophagy pathways in cells and in vivo.
We have had a longstanding interest in Ubl pathway inhibition and have recently described
our success in designing inhibitors of the E1s of the NEDD8,13 ubiquitin,14 and SUMO15
pathways. A key to this work is our demonstration that certain analogs of adenosine sulfamate can
selectively inhibit specific E1s by a mechanism called substrate-assisted inhibition in which the
enzyme catalyzes the formation of a covalent Ubl-inhibitor adduct which resembles the first
intermediate of the E1 reaction cycle.16,17,18 Briefly, the adenosine sulfamate binds to the ATP
binding pocket of the E1~Ubl thioester form of the enzyme which then catalyzes the conjugation
of the sulfamate to the C-terminus of the Ubl to form the inhibitor adduct.
Figure 1. Structure of compound 1
NH2
O
N
N
N
N
O
OH
O
O
S
H2N
O
OH
1
ATG7 IC50
UAE IC50
NAE IC50
SAE IC50
0
.089 ± 0.036 μM
> 1.00 μM
> 1.00 μM
> 50.0 μM
3

2
. Results and discussion
2
.1. Structure-activity relationships
Compound 1 (Figure 1), an inhibitor of ATG7, was initially identified as the sole hit from
a focused high-throughput screen of our library of adenosine sulfamate analogs. Despite the
scarcity of actionable chemical matter to pursue, we were quite interested in this initial hit due to
its potency and selectivity against other E1s. An in vitro enzyme assay was employed to evaluate
compound activity against the target as well as other E1s. ATG7 inhibition in whole cells was
assessed by examining multiple points in this E1 enzyme’s mechanism of UBL activation.
Inhibition of the formation of the ATG7-ATG8 thioester is our most direct assessment of cellular
inhibition of ATG7. Lipidated LC3B is one of the products of the final step in the
ATG7/ATG3/ATG5-12 cascade, in which LC3B, an ATG8, is shuttled from E1 to E2 and then
conjugated with phosphatidylethanolamine in the presence of the ATG5-12, the E3. Measuring the
loss of lipidated LC3B using an IF readout was another important cellular assay for our
1
9
compounds. General off-target effects were conveniently assessed by incubation of compounds
2
0
with H1650 cells, a lung cancer derived ATG7-null cell line. It was quickly determined that the
pyrazolopyrimidine nucleobase was required for maintaining ATG7 activity (data not shown) and
that substitution at the 7-position was key for the observed E1 selectivity pattern. Thus
optimization of 1, with respect to enzyme and cellular potency, began with the exploration of
numerous analogs varying the substitution at the 7-position (Table 1) of the pyrazolopyrimidine
ring. Removing the methyl group provided compound 2, which is ATG7-active, but not selective
over other E1s and suffers from chemical instability. Further excision of the entire 7-methoxy
moiety, as in compound 3, results in significantly less activity in the enzyme assay. The similarly-
sized difluoromethoxy substituent 4 showed slightly improved activity, while further increases in
steric bulk diminished potency (cf. ethoxy (5), isopropoxy (6) and benzyloxy (7) analogs).
We next explored the nature of the linkage joining the 7-position substituent and
nucleobase. We observed that a carbon linkage was as well tolerated as the corresponding oxygen
linkage (cf. 8 and 7). We were pleased to find that a sulfur linkage not only improved potency but
allowed for improved tolerance for a range of 7-substituent sizes (methylthio (9), ethylthio (10)
and isopropylthio derivative (11) showed similar activity). Even aromatic substitutions with a
sulfur linkage, including pyrazolothio (12), m-tolylthio (13), benzylthio (14) and phenylthio
4

substituent (15) were well tolerated. All of these compounds showed good selectivity over the
other E1 enzymes. The tolerability of the 7-substituent sulfur linkage might be explained by unique
2
1
noncolvalent sulfur interactions which have been well documented. It is also known that the
thioether provides access to a unique dihedral angle that may allow the thio-substituent to fit into
the binding pocket more optimally.22,23
Table 1. Enzymatic and cellular potencies of ribose analogs
NH2
R
7
N
N
N
N
O
OH
O
O
S
H2N
O
OH
ATG7 IC
Thioester IC
LC3B IC
(μM)
H1650 EC
5
0
50
50
50
R
a
a,b,c
a
a
(μM)
(μM)
(μM)
0.335 ± 0.049b
n.d.e
2.6d
n.d.e
n.d.e
2.3d
5.94d
> 50.0d
0.075d
1
2
3
4
0.089 ± 0.036
0.084 ± 0.043
0.457 ± 0.111
0.029 ± 0.019
O
HO
n.d.e
H
F
0.162 ± 0.070b
10.02 ± 3.51
F
O
O
.479 ± 0.117c
0
5
6
0.255 ± 0.072
2.60 ± 0.83
n.d.e
n.d.e
n.d.e
49.4d
n.d.e
> 50.0d
O
7
8
9
0.264 ± 0.103
0.351 ± 0.127
0.014 ± 0.004
1.28b,d
n.d.e
n.d.e
n.d.e
1.50d
> 50.0d
> 50.0d
Ph
Ph
O
0.083b,d
1.54 ± 0.38
S
0
.431c,d
0.160b,d
1
0
1
0.022d
n.d.e
15.4d
S
S
0.284 ± 0.185b
.25c,d
4.23d
> 50.0d
1
0.017 ± 0.005
1
5

HN
N
1.28c,d
0.178b,d
0.322b,d
n.d.e
n.d.e
> 50.0d
> 50.0d
> 50.0d
1
1
2
3
0.016 ± 0.007
0.018 ± 0.006
S
S
1
1
4
5
0.023d
n.d.e
n.d.e
Ph
S
0
.604c,d
0.201b,d
> 50.0d
Ph
0.029 ± 01.006
S
a
b
Unless otherwise noted, IC values are shown as mean values of three or more determinations. 8 hours
5
0
c
d
of compound incubation. 4 hours of compound incubation. IC values are shown as one determination
5
0
e
or means of two determinations. Not determined.
Exploration of the SAR of the 2’-position of the sugar moiety is summarized in Table 2.
2
’-Deoxyribose 7-methoxy (16) and 7-difluoromethoxy (17) analogs showed comparable activity
with their ribose analogs (compounds 1 and 4), and improved selectivity was observed as
determined by lack of activity in the H1650 (ATG7 null) cell viability assay for the 7-alkyl
thioether deoxyriboses 18 (methylthio), 19 (ethylthio) and 20 (isopropylthio) when compared with
their ribose analogs. Aryl deoxyribose thioether substituents were tolerated (compounds 21-24);
although these modifications did not improve inhibitory activity in the enzyme or cellular assays.
Optimization of the furanose portion of our nucleoside analogs continued with an
exploration of 2’-fluorodeoxy-arabinose derived compounds. In this series, both methoxy (25) and
difluoromethoxy (26) substitutions on the nucleobase maintain ATG7 activity whereas thioethers,
including methylthio (27), isopropylthio (28), m-tolylthio (29) and 3-chlorophenylthio (30)
analogs, provide some of our most potent ATG7 inhibitors. Despite the attractive potency of these
fluoro-arabinose analogs, they exhibit poor chemical stability at room temperature and in aqueous
solution resulting from intramolecular nucleophilic attack of the 3-N onto the 5’-sulfamate (Figure
2
2
). We suspect that the increased chemical reactivity may be due to the greater proclivity of the
’-fluorodeoxy arabinose/pyrazolopyrimidine to exist in a pseudo-axial orientation, which is
stabilized by a 2’-fluoro-3’-hydroxy gauche interaction.24
6

Table 2. Enzymatic and cellular potencies of 2’-deoxyribose and of 2’-fluorodeoxy-arabinose
analogs
NH2
R
7
N
N
N
N
O
2'
X
O
O
S
H N
2
O
OH
ATG7 IC
Thioester IC
LC3B IC
(μM)
H1650 EC
Xa
H
50
50
50
50
R
b
b,c,d
b
b
(μM)
(μM)
(μM)
1
1
6
7
0.249e
n.d.f
n.d.f
31.5e
O
0.100e
0.545c,e
1.77e
> 50.0e
F
H
F
O
1
8
H
0.048 ± 0.022
0.086c,e
0.938 ± 0.231
10.4e
S
0
.280 ± 0.051d
1
2
9
0
H
H
0.052 ± 0.011
0.367d,e
1.36 ± 0.07
> 50.0e
> 50.0e
S
S
0.027e
0.150c,e
1.09e
.438d,e
0
2
2
2
1
2
3
H
H
H
0.095 ± 0.051
0.104e
0.422d,e
0.499d,e
n.d.f
1.26e
1.15e
> 50.0e
> 50.0e
> 50.0e
S
Cl
S
F
S
0.080 ± 0.029
0.533 ± 0.116
2
4
H
0.066 ± 0.026
n.d.f
0.71 ± 0.104
> 50.0e
S
2
2
5
6
-F(S)
-F(S)
0.177e
0.317c,e
0.453d,e
n.d.f
n.d.f
33.9e
18.3e
O
F
0.043 ± 0.019
F
O
2
7
-F(S)
0.041 ± 0.004
0.047c,e
0.977e
5.79e
S
7

2
2
8
9
-F(S)
-F(S)
0.020 ± 0.007
0.056c,e
0.727e
0.483e
22.4e
49.1e
S
.199 ± 0.042d
0
0.035e
0.051c,e
0.140d,e
S
3
0
-F(S)
0.031e
0.056c,e
0.534e
39.7e
0.198 ± 0.016d
Cl
S
a
b
Stereoconfiguration of the 2’ carbon listed in parentheses. Unless otherwise noted, IC values are shown
5
0
c
d
as mean values of three or more determinations. 8 hours of compound incubation. 4 hours of compound
e
f
incubation. IC values are shown as one determination or means of two determinations. Not determined.
5
0
Figure 2. Proposed mode of degradation of fluoro-arabinose sulfamates
H
N
H
N+
S
S
NH2
N
N
N
O
S
O
S
O O
N
N
N
H
H N
2
N
3
O-
H N
2
F
O
O
F
O
5'
H
OH
H
H
OH
28
A few tactics were designed to address this chemical instability. First, we removed the 3-
N of the adenosine-like base but this resulted in a significant loss of enzyme activity (data not
shown). Subsequently, we prepared L--lyxose analogs, inverting the 4’ stereo center to prevent
the intramolecular cyclization (Table 3). Unfortunately, unsatisfactory losses in potency were
observed (compounds 31 and 32). Next, L-α-2’-deoxylyxose was examined as a replacement, with
the methythio (33) and isopropylthio (34) derivatives showing improved activity compared to
lyxose. Additional analogs were prepared including 3-chlorophenylthio (35), 5-indanethio (36)
and 2-fluoro-4-methylphenylthio (37) substituents. The disubstituted phenyl derivative 37
maintains potency of the F-arabinose analogs while improving on the chemical stability. The 7-
methylthio L-α-2’-fluorodeoxy-xylose 38 was prepared based on the increased activity found in
the fluoro-arabinose series but it did not bring the expected improvement in activity.
Table 3. Enzymatic and cellular potencies of lyxose, deoxylyxose and fluoro-xylose analogs
8

NH2
R
N
N
N
N
O
X
O
O
S
OH
H N
2
O
ATG7 IC
Thioester IC
LC3B IC
(μM)
H1650 EC
Xa
50
50
50
50
R
b
b,c,d
b
b
(μM)
(μM)
(μM)
3
3
1
2
-OH(R)
-OH(R)
0.325e
1.28d,e
6.00e
> 50.0e
S
0.129e
0.882c,e
20.1e
> 50.0e
S
5.00d,e
>
3
3
3
4
H
H
0.124 ± 0.055
0.057 ± 0.028
0.295d,e
1.26 ± 0.301
> 50.0e
> 50.0e
S
0.468d,e
1.92e
S
3
3
3
5
6
7
H
H
H
0.190d
0.156d
n.d.f
n.d.f
n.d.f
1.37e
0.996e
0.659e
> 50.0e
> 50.0e
> 50.0e
Cl
S
S
F
0.062 ± 0.021
S
3
8
-F(S)
0.145d
n.d.f
2.53e
> 50.0e
S
a
b
Stereoconfiguration of the 2’ carbon listed in parentheses. Unless otherwise noted, IC values are shown
5
0
c
d
as mean values of three or more determinations. 8 hours of compound incubation. 4 hours of compound
e
f
incubation. IC values are shown as one determination or means of two determinations. Not determined.
5
0
Our exploration of the ATG7 potency of pyrazolopyrimidine sulfamates led us to a few
conclusions. First, the 7-position substituent of the pyrazolopyrimidine in compound 1 is important
for ATG7 activity and attempts to increase the size of the substituent while maintaining the oxygen
ether linkage led to a decrease in activity. Introduction of a thioether linkage allowed further
substitution and provided improved potency and selectivity. The ribose and deoxyribose analogs
have similar potency, and while the fluoro-arabinose analogs offer improved activity; their
9

inherent instability makes them of limited utility in cellular and in vivo contexts. Moving the 5’-
sulfamate to the other face of the sugar as in deoxylyxose analogs achieves the desired level of
potency and stability.
2
.2. Autophagy pathway regulation
Significant enzymatic potency was observed with these compounds and warranted cell-based
evaluation. Table 4 summarizes the results of both enzymatic and cell-based profiling of three
inhibitors. Compounds 18, 19 and 37 inhibit the formation of endogenous LC3B puncta in the
neuroglioma cell line, H4. Lipidated LC3B localizes to autophagosome membranes after
processing by ATG7. Decreases in the number of puncta is consistent with inhibition of LC3B
lipidation, and thus, a cell-based readout of autophagy pathway blockade. Concentration response
curves were generated by calculating the decrease of LC3B puncta in compound-treated samples
relative to the DMSO-treated controls; IC50 values were determined from those curves and a
representative figure is shown below (Figure 3a).
Figure 3. Visualization of autophagy pathway regulation with selected compounds in cells. a)
Decrease in endogenous LC3B puncta (green) in H4 cells following a 6 hour treatment with
compound 18 at multiple concentrations. Nuclei are stained with Dapi (blue). b) Accumulation of
p62 (red) in SKOV-3 cells, after 48 hours treatment with 20 μM of compounds 18, 19 and 37 is
observed. Significant changes in NBR1 (green) were not observed. Nuclei are stained with Dapi
(blue). c) p62 (green and viability (blue)) and NBR1 quantification for compound 37.
a)
DMSO
20M
5M
1.25M
0.31M
0.08M
LC3B
Merge
1
0

b)
DMSO
Compound 18
Compound 19
Compound 37
p62
NBR1
Merge
c)
Further characterization of additional autophagy markers was performed in the ovarian
adenocarcinoma cell line, SKOV-3. An accumulation of p62, a ubiquitin-binding protein
associated with autophagosome formation, was observed (representative images and quantification
shown in Figure 3b and c) and is indicative of autophagy pathway inhibition at potencies similar
to those observed in the LC3B assay. Our compounds were not effective in regulating the
formation of NBR1 puncta in the cytoplasm of these cells at concentrations up to 20 M (Figure
3
b). NBR1 has both LC3 and ubiquitin binding domains and is an autophagy receptor.25
1
1

Furthermore, compounds were unable to prevent the formation of the ATG5-12 complex, a stable
conjugate whose formation is mediated by ATG7 and ATG10 (data not shown).
While our compounds do not appear to fully inhibit the autophagy pathway in cell culture,
all three compounds were found to regulate autophagy more effectively in vivo following a 150
mg/kg subcutaneous dose, as can be seen from Figure 4. Compounds 18 and 19 consistently
modulate LC3B and NBR1, in contrast to our cellular results. Compound 37 shows slower onset
of the inhibition of LC3B lipidation and little effect on NBR1 accumulation despite higher
exposure.
1
2

Table 4. Selected E1 Selectivity and Cellular Data for 18, 19 and 37a
Compound 18
Compound 19
Compound 37
Structure
S
NH2
NH2
S
NH2
N
S
N
N
N
N
F
N
N
N
N
N
N
N
O
O
O
O
O
S
O
O
O
O
S
O
O
S
O
H N
2
OH
OH
OH
H N
2
H N
2
0.048 ± 0.022b
0.052 ± 0.011b
> 1
0.062 ± 0.021b
> 1
ATG7 IC (μM)
5
0
UAE IC (μM)
> 1
50
NAE IC (μM)
> 1
> 1
> 1
> 1
50
SAE IC (μM)
> 1
> 1
5
0
H1650 EC (μM)
10.4
> 50.0
0.367
> 50.0
n.d.d
5
0
c
0.280 ± 0.051b
0.938 ± 0.231b
> 20
Thioester IC (μM)
5
0
1.36 ± 0.07b
> 20
0.659
19.4
LC3B IC (μM)
5
0
e
NBR1 EC (μM)
5
0
f
p62 EC (μM)
3.0
2.3
3.0
5
0
a
b
d
Unless otherwise noted, IC values are shown as one determination or means of two determinations.
5
0
c
IC values are shown as mean values of three or more determinations. 4 hours of compound incubation.
5
0
e
f
Not determined. Accumulation of NBR1 in SKOV-3 cells. Accumulation of p62 in SKOV-3 cells
1
3

Figure 4. Selected pyrazolopyrimidine sulfamates regulate autophagy in vivo† a) Fold change of
LC3B. b) Fold change of NBR1 accumulation. c) IHC images of NBR1 accumulaiton 4 hours after
dosing. †HCT-116 xenografted mice were dosed (150 mg/kg subcutaneous injection) with
compounds 18, 19 and 37 and plasma and tumors were harvested at the noted intervals. Plasma
exposures at 4 hours for each compound are listed below the x axis in (a) and (b).
a)
b)
c)
3
. Chemistry
The compounds described herein were synthesized as described in Schemes 1 to 5. The
substituted pyrazolopyrimidine intermediates were prepared following reported procedures26,27
and then glycosylated with an appropriately protected sugar intermediate. The resulting
1
4

nucleosides allowed for the further modification at the pyrazolopyrimidine as well as sugar
moiety.28
3
.1. Synthesis of ribose analogs
Scheme 1 illustrates the synthesis of ribose analog 12 specifically and serves as a
representative synthetic route for other ribose analogs. Methylthio-pyrazolopyrimidine was
glycosylated in the presence of the boron trifluoride ethyl etherate-activated ribofuranose ester29
and then deprotected to provide the triol 40. Selective protection of the two secondary alcohols as
the corresponding acetonide followed by silyl protection of the primary alcohol provided
compound 42. Sulfide 43 was prepared by treating methyl sulfide 42 with sodium tert-butyl
thiolate, and this relatively unstable sulfide was quickly coupled with an aryl halide such as an
iodopyrazole to give 44. Selective deprotection of the silyl protecting group provided 45 which
was then treated with chlorosulfonamide.30 The synthesis was completed by removal of the
acetonide protecting group under acidic conditions.
Scheme 1. Synthesis of ribose analogsa
NH2
N
NH2
N
NH2
N
NH2
N
S
S
S
S
NH2
N
S
N
N
N
N
a
b
c
d
N
N
N
N
N
N
N
N
N
O
O
O
O
N
N
OAc
OH
H
O
O
AcO
HO
HO
TIPSO
OAc
OH
O
O
39
40
41
42
N
HN
HN
N
NH2
N
HS
NH2
N
NH2
N
NH2
N
N
S
N
S
N
S
THP
N
N
N
e
f
g
h
N
N
N
N
N
N
N
N
O
O
O
O
O
OH
TIPSO
O
O
O
TIPSO
HO
O
O
S
O
O
H N
OH
2
O
43
44
45
12
a
.
Reagents and conditions: (a) 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose, BF OEt , CH NO , reflux,
3
2
3
2
5
9 %. (b) NH , MeOH, rt, quant. (c) (MeO) CMe , p-TsOH, acetone, rt, 46 %. (d) TIPSCl,
3 2 2
imidazole, DMAP, DMF, rt, 79 %. (e) NaSt-Bu, DMF, 165 °C, 66 %. (f) 4-iodo-1-(tetrahydro-2H-
.
pyran-2-yl)-1H-pyrazole, XPhos Pd G1, Cs CO , DMF, 120 °C, 30 %. (g) HF pyridine, MeOH, rt,
2
3
quant. (h) chlorosulfonamide, DMF, rt and then TFA/water, rt, 34 %.
.2. Synthesis of deoxyribose analogs
3
1
5

In a similar manner, Scheme 2 illustrates the synthesis of 2’-deoxyribose analog 19 and is
representative of the synthesis of the deoxyribose compounds. Substituted pyrazolopyrimidine 48
3
1
was prepared from malononitrile following similar reported procedures. The glycosylation was
achieved by treating the nucleobase 48 with Hoffer’s chloro-sugar in the presence of a strong
3
2
base. Protecting group manipulations allowed for selective sulfamate functionalization of the 5’-
alcohol and removal of the 3’-silyl ether under fluoride conditions to provide the final sulfamate
analog 19.
Scheme 2. Synthesis of deoxyribose analogsa
NH2
S
N
N
N
N
NH2
N
N
a
b
c
S
d
N
N
S
N
O
N
NH2
O
N
S
S
N
N
H
N
N
O
H
O
46
47
48
49
O
NH2
N
NH2
N
NH2
N
NH2
N
S
S
S
S
N
e
N
f
N
g
h
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
HO
TIPSO
HO
S
OH
OH
OTIPS
OTIPS
52
H2N
O
5
0
51
19
a
Reagents and conditions: (a) carbon disulfide, potassium carbonate, ethyl iodide, TBAI, DMF,
0 °C, 40 %. (b) hydrazine hydrate, MeOH, 50 °C, quant. (c) formamide, 200 °C, 86 %. (d)
5
(2R,3S,5R)-5-chloro-2-{[(4-methylbenzoyl)oxy]methyl}tetrahydrofuran-3-yl 4-methylbenzoate,
t-BuOK, DMF, MeCN, THF, rt, 44 %. (e) NaOMe/MeOH, rt, quant. (f) TIPSCl, imidazole,
DMAP, DMF, rt, 47 % (g) HCl, EtOH rt, 62 % (h) chlorosulfonamide, DMF and then triethylamine
trishydrofluoride, THF, rt, 81 %.
Scheme 3. Synthesis of fluoro-arabinose analogsa
1
6

NH2
N
NH2
N
NH2
N
NH2
N
I
I
BnO
N
BnO
N
NH2
N
I
N
N
a
b
c
d
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
F
F
F
F
H
BzO
HO
HO
TIPSO
OBz
OH
OH
55
OTIPS
53
54
56
F
F
NH2
N
HO
N
NH2
N
NH2
N
O
O
F
F
N
e
f
N
g
N
N
N
N
N
N
O
F
O
O
F
TIPSO
F
O
O
HO
OTIPS
S
OH
OH
H2N
O
57
58
26
a
Reagents and conditions: (a) [(2R,3R,4S,5R)-3-(Benzoyloxy)-5-bromo-4-fluorotetrahydrofuran-
2
-yl]methyl benzoate, t-BuOK, DMF/MeCN, rt, 64 %. (b) NH , MeOH, rt, 74 %. (c) benzyl
3
alcohol, CuI, 3,4,7,8-tetramethyl-1,10-phenanthroline, Cs CO , 80 °C, 45 %. (d) TIPSCl,
2
3
imidazole, DMAP, DMF, rt, 58 %. (e) H , Pd/C, MeOH, rt, 81 %. (f) sodium chlorodifluoroacetate,
2
K CO , DMF, 80 °C, 88 %. (g) chlorosulfonamide, MeCN, 0 °C, 6 %.
2
3
3
.3. Synthesis of fluoro arabinose analogs
Fluoro-arabinose 26 serves as a representative synthesis of the fluoro-arabinose derivatives
and is described in Scheme 3. Reaction of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine with a 2’-
fluoro-sugar derivative in the presence of potassium tert-butoxide provided the desired nucleoside
3
3
5
3. This versatile iodo-intermediate 54 was converted into a number of different intermediates,
allowing for selective and late stage modification at the 7-position. Metal-mediated carbon-sulfur,
carbon-nitrogen and carbon-carbon coupling reactions were used to provide intermediates. For
example, alcohols could be coupled with iodo-intermediate 54 to provide benzylether substituted
pyrazolopyrimidine 55. Protection of the sugar alcohols and debenzylation provided free alcohol
5
7 which was treated with sodium chlorodifluoroacetate to install the difluoromethoxy group. To
reduce the number of synthetic steps, direct sulfamate formation at the primary alcohol 58 was
achieved, but with lower yield.
Scheme 4. Synthesis of deoxylyxose analogsa
1
7

NH2
N
NH2
N
I
NH2
N
S
I
NH2
N
S
NH2
N
I
N
N
N
a
b
c
N
d
N
N
N
N
N
N
N
N
N
O
O
O
OH
N
N
OAc
OAc
OH
O
H
OH
AcO
HO
HO
O
OH
60
O
iPr)2Si
OH
(
Si(iPr)2
O
5
9
61
62
NH2
N
NH2
S
S
NH2
N
S
N
N
N
N
e
N
f
N
g
N
N
N
N
O
O
O
HO
O
O
O
HO
(iPr)2Si
O
S
O
H2N
OH
O
(iPr)2Si
Si(iPr)2
Si(iPr)2
O
O
63
64
36
a
.
Reagents and conditions: (a) 1,2,3,5-tetra-O-acetyl-L-lyxofuranose, BF OEt , CH NO , rt, 36 %.
3
2
3
2
(
b) 7N ammonia in MeOH, rt, 89 %. (c) indane-5-thiol, XPhos Pd G2, Cs CO , DMF, 100 °C, 48
2 3
%. (d) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, DMAP, imidazole, DMF, rt, 59 %. (e) TCDI,
THF, reflux then TTMSS, AIBN, 1,4-dioxane, reflux, 55 %. (f) 1M HCl, ethanol, 0 °C, quant. (g)
chlorosulfonamide, DMF and then triethylamine trishydrofluoride, THF, rt, 58 %.
3
.4. Synthesis of deoxylyxose analogs
The preparation of lyxose derivative 36 is described in Scheme 4. Glycosylation of 3-iodo-
H-pyrazolo[3,4-d]pyrimidin-4-amine with 1,2,3,5-tetraacetate-L-lyxofuranose34 gave the
1
3
5
pyrazolopyrimidine lyxose derivative predominantly as the desired alpha-anomer 59. Selective
manipulation of the 2’-alcohol of 61 was achieved through protection of the 3’,5’-diol as its
tetraisopropyldisiloxane (TiPDS) ether 62. We found starting the reaction at low temperature
allows the 5’- primary alcohol to react first. Warming to room temperature results in reaction at
the 3’-alcohol to complete the protection. Subsequent radical 2’-dehydroxylation gave the
3
6
deoxyribose derivative 63. Selective deprotection of the primary alcohol under acidic conditions
at low temperature provided alcohol 64 which was converted to the sulfamate and fully deprotected
to provide 36. In order to more efficiently diversify a later synthetic stage, a synthetic route in
which deoxygenation occurred prior to glycosylation was required. The route developed is
summarized in Scheme 5. Compound 65 was prepared from commercially available L-xylose in
3
7
two steps, following literature procedures. Radical deoxygenation was immediately followed by
3
8
acetonide removal to provide 67. The resulting diol was much easier to handle than the volatile
deoxygenated acetonide. Compound 68, the glycosylation substrate, was prepared by
1
8

demethylation followed by acylation. Subsequent reaction with iodo-pyrazolopyrimidine formed
intermediate 69. We were pleased that the desired alpha-anomer predominated, despite not having
a directing group in the 2’-position. Deprotection, protection and selective 5’-deprotection gave
key intermediate 72 which was elaborated to the final compound 37.
Scheme 5. Alternative synthesis of deoxylyxose analogsa
NH2
N
NH2
N
I
I
O
O
O
O
S
OAc
OBz
O
S
N
N
O
O
a
b
O
c
O
d
e
OH
O
N
N
N
N
O
O
O
O
OH
OBz
BzO
HO
OH
OBz
OH
70
65
66
67
68
69
NH2
N
NH2
I
I
NH2
NH2
N
S
S
N
N
N
F
N
F
N
f
g
h
N
N
i
N
N
N
N
N
N
N
O
O
O
O
TIPSO
HO
O
HO
O
OTIPS
OTIPS
72
S
OTIPS
73
H2N
OH
O
71
37
a
Reagents and conditions: (a) NaH, CS , MeI, THF, rt, 97 %. (b) Bu SnH, AIBN, toluene, reflux
2
3
and then pTsOH, MeOH, rt, 55 %. (c) benzoyl chloride, Et N, DCM, rt then TFA, H O, rt then
3
2
.
Ac O, pyridine, rt, 34 %. (d) 3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine, BF OEt , CH NO ,
2
3
2
3
2
rt, 45 %. (e) 7N ammonia in MeOH, rt, quant. (f) TIPSCl, DMAP, imidazole, DMF, 40 °C, 70 %.
g) EtOH, HCl, rt, 45 %. (h) 2-fluoro-4-methylbenzenethiol, CuI, K CO , iPrOH, 1,2-ethanediol,
(
2
3
reflux, 70 %. (i) chlorosulfonamide, DMF and then triethylamine trishydrofluoride, THF, rt, 72 %.
. Conclusion
We have discovered a series of novel inhibitors of ATG7 which exhibit both cellular and
4
in vivo modulation of autophagy pathway markers. The modular structure of the
pyrazolopyrimidine scaffold lent itself well to analog synthesis and selectivity was achieved by
elaboration of the 7-position substituent while modification of the sugar ring system improved
compound stability and cellular activity. These new compounds are selective ATG7 inhibitors and
are useful in vitro and in vivo tools to investigate ATG7-dependent biology.
5
. Experimental Section
.1. Chemistry
5
1
9

All solvents and chemicals were used as purchased without further purification unless
noted. The progress of all reactions was monitored on Merck precoated silica gel plates (with
fluorescence indicator UV ). Spots were visualized by irradiation with ultraviolet light (254 nm).
2
54
Column chromatography was performed with Teledyne ISCO silica gel (230–400 mesh) with the
1
13
solvent mixtures specified in the corresponding experiment. Proton ( H) and carbon ( C) NMR
spectra were recorded in the solvent reported on Bruker 300 MHz Avance and 400 MHz Avance
III HD spectrometers (5 mm QNP or BBFO Smart Probe) using residual solvent peaks as the
reference. Compound purity was determined by analysis of the diode array UV trace of an LC-MS
spectrum using the following procedure. Compounds were dissolved in DMSO, methanol, or
acetonitrile, and the solutions were analyzed using an Agilent 1100 LC interfaced to a micromass
Waters Micromass Zspray Mass Detector (ZMD). One of two gradients was used to elute the
compounds either a formic acid (FA) gradient (acetonitrile containing 0-100% 0.1% formic acid
in water) or an ammonium acetate (AA) gradient (acetonitrile containing 0-100% 10 mM
ammonium acetate in water). All compounds were determined to be >95% pure unless otherwise
noted. High-resolution mass spectrometry (HRMS) analysis was performed using Agilent 6545 Q-
ToF MS equipped with Agilent 1260 Infinity II Bio-inert Multsampler, Bio-inert Column
compartment, bio-inert Quat. Pump, and DAD MW system. About 5 μL (1 mM DMSO solution)
of each compound was injected into Waters Xselect C18 CSH 3.5u, 2.1mm ID x 30mm column.
The pump flow rate was 0.5 mL/min. Buffer A is the aqueous solvent and consisted of 0.1% formic
acid (Sigma, St. Louis, MO) in HPLC grade water (VWR Scientific, West Chester, PA). Buffer B,
the organic phase, consisted of 0.1% formic acid in acetonitrile (VWR Scientific). The gradient
was set as follows: 0 min, 5% buffer B; 0.5 min, 5% buffer B; 1.5 min, 100% buffer B; 2.5 min,
1
00% buffer B; 2.6 min, 5% buffer B, and 3.5 min, 5% buffer B. The compounds were ionized
using a Dual Jet Stream Electric Spray Ionization with VCap 4.5 kV voltage and nozzle 2.0 kV
voltage. The mass spectrometer was set to acquire masses from 0 to 1700 D in positive mode.
MS/MS spectra of each compound were also acquired under Agilent MassHunter Workstation
Data Acquisition and Agilent MassHunter Qualitative Analysis to confirm the chemical structure
of the compounds.
5
.1.1. [(2R,3S,4R,5R)-5-(4-amino-3-methoxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-
dihydroxytetrahydrofuran-2-yl]methyl sulfamate (1)
2
0

[(3aR,4R,6R,6aR)-6-(4-amino-3-methoxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]methanol (0.100 g, 0.296 mmol) was dissolved in
DMF (0.9 mL). Chlorosulfonamide (0.103 g, 0.889 mmol) was added and the mixture was stirred
at room temperature for 2 h. The mixture was poured into saturated aqueous sodium bicarbonate
solution and extracted with ethyl acetate. The extracts were combined, washed with water and
brine and then dried over sodium sulfate, filtered and concentrated under reduced pressure. To the
crude material was added hydrochloric acid (6 M, 0.741 mL) and the resulting mixture was stirred
overnight at room temperature. The mixture was neutralized with saturated aqueous sodium
bicarbonate solution and then twice diluted with toluene (100 mL) and concentrated to ¼ volume.
Subsequently, the moist mixture was coevaporated from acetonitrile until dry. The resulting white
solid was dissolved in acetonitrile, adsorbed onto Celite and eluted through a C18 cartridge (86 g,
9
5/5 to 30/70 water/acetonitrile gradient, with 10 mM ammonium acetate). Product-containing
fractions were concentrated under reduced pressure. Water was removed through concentration
1
from acetonitrile then toluene to afford 1 (0.082 g, 74 %) as a white solid. H NMR (400 MHz,
DMSO-d ): δ 8.15 (s, 1H), 7.49 (br s, 2H), 6.07 (d, J=2.81 Hz, 1H), 5.49-5.58 (m, 1H), 5.25-5.38
6
(m, 1H), 4.41-4.49 (m, 1H), 4.31-4.39 (m, 1H), 4.17-4.27 (m, 1H), 4.03-4.12 (m, 2H), 3.98 (s, 3H).
1
3
C NMR (101 MHz, DMSO-d ): δ 157.5, 157.3, 155.7, 155.4, 88.6, 87.9, 80.7, 73.0, 70.9, 69.5,
6
+
5
5.9. HRMS (ESI): m/z calculated for C H N O S [M + H ]: 377.0874. Found: 377.0876.
1
1
16
6
7
5
.1.2. [(2R,3S,4R,5R)-5-(4-amino-3-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-
dihydroxytetrahydrofuran-2-yl]methyl sulfamate (2)
Compound 2 was prepared in a manner similar to that described for 1 using 4-amino-1-
[(3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]-1H-
1
pyrazolo[3,4-d]pyrimidin-3-ol in 46 % yield as a white solid. H NMR (400 MHz, CD OD): δ 8.12
3
(s, 1H), 6.14 (d, J=3.76 Hz, 1H), 4.54-4.62 (m, 1H), 4.41-4.50 (m, 1H), 4.27-4.37 (m, 1H), 4.12-
+
4
3
.27 (m, 2H). HRMS (ESI): m/z calculated for C H N O S [M + H ]: 363.0717. Found:
1
0
14
6
7
63.0716.
5
.1.3. [(2R,3S,4R,5R)-5-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-
dihydroxytetrahydrofuran-2-yl]methyl sulfamate (3)
Compound 3 was prepared in a manner similar to that described for 1 using
[(3aR,4R,6R,6aR)-6-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-
2
1

1
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]methanol in 62 % yield as a white solid. H NMR
(
400 MHz, DMSO-d ): δ 8.20 (s, 2H), 6.07-6.26 (m, 1H), 4.46-4.57 (m, 1H), 4.30-4.39 (m, 1H),
6
1
3
4
1
.20-4.27 (m, 1H), 4.08-4.16 (m, 1H), 3.96-4.06 (m, 1H). C NMR (101 MHz, DMSO-d ): δ
6
58.5, 156.8, 154.6, 134.2, 100.7, 88.9, 81.4, 73.7, 71.4, 70.1. HRMS (ESI): m/z calculated for
+
C H N O S [M + H ]: 347.0768. Found: 347.0776.
1
0
14
6
6
5
.1.4. {(2R,3S,4R,5R)-5-[4-amino-3-(difluoromethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-
dihydroxytetrahydrofuran-2-yl}methyl sulfamate (4)
Compound 4 was prepared in a manner similar to that described for 5 using
{
(3aR,4R,6R,6aR)-6-[4-amino-3-(difluoromethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2,2-
1
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol in 44 % yield as a white solid. H NMR
(
400 MHz, DMSO-d ): δ 8.23 (s, 1H), 7.29-7.72 (m, 3H), 6.11 (d, J=2.81 Hz, 1H), 5.63 (br s, 1H),
6
5
4
2
.39 (br s, 1H), 4.38-4.46 (m, 1H), 4.29-4.37 (m, 1H), 4.15-4.26 (m, 1H), 4.08-4.15 (m, 1H), 4.00-
1
3
.07 (m, 1H). C NMR (101 MHz, DMSO-d ): δ 157.7, 157.1, 154.9, 148.7, 115.2 (dd, J=260.1,
6
60.1 Hz, 1C), 88.8, 88.1, 81.0, 73.2, 70.7, 69.4. HRMS (ESI): m/z calculated for C H F N O S
1
1
14
2
6
7
+
[
M + H ]: 413.0686. Found: 413.0683.
5
.1.5. {(2R,3S,4R,5R)-5-[4-amino-3-ethoxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-
dihydroxytetrahydrofuran-2-yl}methyl sulfamate (5)
(3aR,4R,6R,6aR)-6-(4-Amino-3-ethoxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-
[
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]methanol (0.360 g, 1.02 mmol) was dissolved in
DMF (3.5 mL). Chlorosulfonamide (0.355 g, 3.07 mmol) was added and the mixture was stirred
at room temperature for 2 h. The mixture was poured into saturated aqueous sodium bicarbonate
solution (75 mL) and extracted with ethyl acetate (100 +75 mL). The extracts were combined,
washed with water and brine and then dried over sodium sulfate, filtered and concentrated under
reduced pressure. The crude material (0.453 g, 1.05 mmol) was dissolved in a mixture of formic
acid (10 mL) and water (10 mL) and stirred overnight at room temperature. The mixture was twice
diluted with toluene (100 mL) and concentrated to ¼ volume. Subsequently, the moist mixture was
coevaporated from acetonitrile until dry. The resulting white solid was dissolved in acetonitrile,
adsorbed onto 3.5g Celite and eluted through a C18 cartridge (86 g, 95/5 to 30/70 water/acetonitrile
gradient, with 10 mM ammonium acetate). Product-containing fractions were concentrated under
reduced pressure. Water was removed through acetonitrile then toluene azeotrope to afford 5
2
2

1
(
0.253 g, 60 %) as a white solid. H NMR (400 MHz, DMSO-d ): δ 8.15 (s, 1H), 7.49 (br s, 2H),
6
6
1
.06 (d, J=2.81 Hz, 1H), 5.53 (br d, J=5.14 Hz, 1H), 5.30 (br d, J=6.36 Hz, 1H), 4.39-4.47 (m,
1
3
H), 4.29-4.38 (m, 3H), 4.22 (br d, J=7.09 Hz, 1H), 4.00-4.11 (m, 2H), 1.36-1.47 (m, 3H). C
NMR (101 MHz, DMSO-d ): δ 157.6, 157.3, 155.1, 155.1, 88.8, 87.9, 80.6, 73.1, 70.9, 69.6, 64.5,
6
+
1
4.4. HRMS (ESI): m/z calculated for C H N O S [M + H ]: 391.1030. Found: 391.1027.
1
2
18
6
7
5
.1.6. [(2R,3S,4R,5R)-5-(4-amino-3-isopropoxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-
dihydroxytetrahydrofuran-2-yl]methyl sulfamate (6)
Compound 6 was prepared in a manner similar to that described for 1 using
[
(3aR,4R,6R,6aR)-6-(4-amino-3-isopropoxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]methanol in 58 % yield as a white solid. H NMR
400 MHz, DMSO-d ): δ 8.15 (s, 1H), 6.03-6.10 (m, 1H), 4.92-5.10 (m, 1H), 4.37-4.42 (m, 1H),
1
(
6
4
6
7
4
.27-4.35 (m, 1H), 4.19-4.26 (m, 1H), 4.05-4.12 (m, 1H), 3.97-4.04 (m, 1H), 1.39 (d, J=4.40 Hz,
1
3
H). C NMR (101 MHz, DMSO-d ): δ 157.6, 157.3, 154.9, 154.5, 89.2, 87.9, 80.6, 73.2, 72.1,
6
+
1.0, 69.7, 21.7. HRMS (ESI): m/z calculated for C H N O S [M + H ]: 405.1187. Found:
1
3
20
6
7
05.1187.
5
.1.7. {(2R,3S,4R,5R)-5-[4-amino-3-(benzyloxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-
dihydroxytetrahydrofuran-2-yl}methyl sulfamate (7)
Compound 7 was prepared in a manner similar to that described for 1 using
{
(3aR,4R,6R,6aR)-6-[4-amino-3-(benzyloxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol in 64 % yield as a white solid. H NMR
400 MHz, DMSO-d ): δ 8.16 (s, 1H), 7.56 (d, J=7.09 Hz, 2H), 7.50 (s, 2H), 7.31-7.46 (m, 3H),
1
(
6
6
.06 (d, J=2.93 Hz, 1H), 5.51 (d, J=5.26 Hz, 1H), 5.40 (s, 2H), 5.29 (d, J=6.36 Hz, 1H), 4.37-4.45
m, 1H), 4.29-4.37 (m, 1H), 4.23 (dd, J=3.06, 10.15 Hz, 1H), 4.06 (dd, J=3.18, 5.75 Hz, 1H), 3.97-
(
1
3
4
1
.04 (m, 1H). C NMR (101 MHz, DMSO-d ): δ 157.5, 157.3, 155.2, 154.8, 136.3, 128.3, 128.2,
6
28.0, 88.8, 87.9, 80.6, 73.1, 70.9, 70.0, 69.6. HRMS (ESI): m/z calculated for C H N O S [M +
1
7
20
6
7
+
H ]: 453.1187. Found: 453.1186.
5
.1.8. {(2R,3S,4R,5R)-5-[4-amino-3-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-
dihydroxytetrahydrofuran-2-yl}methyl sulfamate (8)
2
3

To a solution of {(3aR,4R,6R,6aR)-6-[4-amino-3-(phenylethynyl)-1H-pyrazolo[3,4-
d]pyrimidin-1-yl]-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol (0.112 g, 0.275
mmol) in ethanol (4.0 mL) was added palladium (10 wt. % on carbon, 0.011 g, 0.82 mmol) and
the reaction mixture was stirred at room temperature under an atmosphere of hydrogen overnight.
The reaction mixture was diluted with ethyl acetate and filtered through a pad of Celite. The filter
pad was washed with ethyl acetate (3 x 50 mL) and the filtrate was collected and concentrated
under reduced pressure. The crude product was purified by silica gel chromatography
(dichloromethane to 95/5 dichloromethane/methanol gradient) to afford {(3aR,4R,6R,6aR)-6-[4-
amino-3-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2,2-dimethyltetrahydrofuro[3,4-
+
1
d][1,3]dioxol-4-yl}methanol as a white solid (132 mg, quant.). LC-MS (m/z): 412 [M + H] . H
NMR (400 MHz, CDCl ): δ 8.29 (s, 1H), 7.33-7.22 (m, 3H), 7.19-7.15 (m, 2H), 6.50 (d, J=3.2 Hz,
3
1
H), 5.63 (s, 2H), 5.16 (dd, J=5.9, 3.2 Hz, 1H), 5.10-5.07 (m, 1H), 4.57-4.53 (m, 1H), 3.94-3.89
(m, 1H), 3.76 (dd, J=12.8, 2.7 Hz, 1H), 3.27-3.09 (m, 5H), 1.65 (s, 3H), 1.39 (s, 3H).
The intermediate from the above step, {(3aR,4R,6R,6aR)-6-[4-amino-3-(2-phenylethyl)-1H-
pyrazolo[3,4-d]pyrimidin-1-yl]-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol,
1
was used to prepare 8 in a manner similar to that described for 1 in 66 % yield as a white solid. H
NMR (400 MHz, DMSO-d ): δ 8.17 (s, 1H), 7.51 (s, 2H), 7.15-7.30 (m, 5H), 6.12 (d, J=2.81 Hz,
6
1
H), 5.54 (d, J=5.14 Hz, 1H), 5.33 (d, J=6.36 Hz, 1H), 4.40-4.46 (m, 1H), 4.32-4.39 (m, 1H), 4.24
(
(
dd, J=3.00, 10.21 Hz, 1H), 4.06-4.16 (m, 1H), 3.95-4.06 (m, 1H), 3.25-3.32 (m, 2H), 2.95-3.09
1
3
m, 2H). C NMR (101 MHz, DMSO-d ): δ 158.2, 156.0, 155.1, 145.7, 141.2, 128.6, 128.1, 125.9,
6
9
8.7, 88.1, 80.7, 73.3, 71.0, 69.8, 33.4, 29.4. HRMS (ESI): m/z calculated for C H N O S [M +
18 22 6 6
+
H ]: 451.1394. Found: 451.1395.
5
.1.9. {(2R,3S,4R,5R)-5-[4-amino-3-(methylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-
dihydroxytetrahydrofuran-2-yl}methyl sulfamate (9)
Compound 9 was prepared in a manner similar to that described for 1 using
{
(3aR,4R,6R,6aR)-6-[4-amino-3-(methylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2,2-
1
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol in 54 % yield as a white solid. H NMR
(
400 MHz, DMSO-d ): δ 8.20 (s, 1H), 7.49 (br s, 2H), 6.13 (d, J=3.18 Hz, 1H), 5.58 (d, J=5.26
6
Hz, 1H), 5.34 (br d, J=6.11 Hz, 1H), 4.45-4.53 (m, 1H), 4.34-4.41 (m, 1H), 4.24 (dd, J=2.87, 9.84
1
3
Hz, 1H), 4.09-4.15 (m, 1H), 4.02-4.09 (m, 1H), 2.62 (s, 3H). C NMR (101 MHz, DMSO-d ): δ
6
2
4

1
57.7, 156.8, 155.6, 141.2, 99.1, 88.3, 81.0, 73.3, 71.0, 69.5, 14.5. HRMS (ESI): m/z calculated
+
for C H N O S [M + H ]: 393.0646. Found: 393.0650.
1
1
16
6
6 2
5
.1.10. {(2R,3S,4R,5R)-5-[4-amino-3-(ethylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-
dihydroxytetrahydrofuran-2-yl}methyl sulfamate (10)
Compound 10 was prepared in a manner similar to that described for 1 using
{
(3aR,4R,6R,6aR)-6-[4-amino-3-(ethylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2,2-
1
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol in 80 % yield as a white solid. H NMR
(
400 MHz, DMSO-d ): δ 8.21 (s, 1H), 7.51 (s, 2H), 6.13 (d, J=3.06 Hz, 1H), 5.61 (br s, 1H), 5.38
6
(br s, 1H), 4.43-4.54 (m, 1H), 4.32-4.40 (m, 1H), 4.24 (dd, J=3.18, 10.27 Hz, 1H), 4.08-4.16 (m,
1
3
1
H), 3.97-4.08 (m, 1H), 3.13 (q, J=7.21 Hz, 2H), 1.33 (t, J=7.27 Hz, 3H). C NMR (101 MHz,
DMSO-d ): δ 157.8, 156.8, 155.3, 139.9, 99.7, 88.3, 80.9, 73.4, 71.0, 69.6, 26.9, 14.7. HRMS
6
+
(
ESI): m/z calculated for C H N O S [M + H ]: 407.0802. Found: 407.0800.
1
2
18
6
6 2
5
3
.1.11. {(2R,3S,4R,5R)-5-[4-amino-3-(isopropylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-
,4-dihydroxytetrahydrofuran-2-yl}methyl sulfamate (11)
Compound 11 was prepared in a manner similar to that described for 1 using
{
(3aR,4R,6R,6aR)-6-[4-amino-3-(isopropylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2,2-
1
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol in 92 % yield as a white solid. H NMR
(
400 MHz, DMSO-d ): δ 8.22 (s, 1H), 6.16 (d, J=2.81 Hz, 1H), 5.63 (br s, 1H), 5.41 (br s, 1H),
6
4
4
.45-4.51 (m, 1H), 4.37 (t, J=5.07 Hz, 1H), 4.25 (dd, J=3.00, 10.21 Hz, 1H), 4.09-4.19 (m, 1H),
1
3
.02 (dd, J=7.52, 10.21 Hz, 1H), 3.60-3.69 (m, 1H), 1.31-1.38 (m, 6H). C NMR (101 MHz,
DMSO-d ): δ 158.4, 157.2, 155.4, 139.3, 101.0, 88.8, 81.4, 73.9, 71.5, 70.1, 39.2, 23.5. HRMS
6
+
(
ESI): m/z calculated for C H N O S [M + H ]: 421.0959. Found: 421.0960.
1
3
20
6
6 2
5
.1.12. {(2R,3S,4R,5R)-5-[4-amino-3-(1H-pyrazol-4-ylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-
yl]-3,4-dihydroxytetrahydrofuran-2-yl}methyl sulfamate (12)
(3aR,4R,6R,6aR)-6-[4-Amino-3-(1H-pyrazol-4-ylsulfanyl)-1H-pyrazolo[3,4-
{
d]pyrimidin-1-yl]-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol (45, 0.053 g, 0.13
mmol) was dissolved in DMF (1.0 mL). Chlorosulfonamide (0.022 g, 0.196 mmol) was added and
the mixture was stirred at rt for 2h. The mixture was poured into saturated aqueous sodium
bicarbonate solution and extracted with ethyl acetate. The extracts were combined, washed with
2
5

water and brine and then dried over sodium sulfate, filtered and concentrated under reduced
pressure. The crude material was added to a solution of TFA and water (0.112 mL, 10:1
TFA:water). The reaction mixture was allowed to stir for 2.5 h and then concentrated under
reduced pressure. The crude material was purified by preparative reverse phase HPLC to afford
1
1
1
1
1
2 (0.020 g, 34 %) as a white solid. H NMR (400 MHz, DMSO-d ): δ 13.18 (br s, 1H), 8.22 (s,
6
H), 8.07 (br s, 1H), 7.68 (br s, 1H), 7.51 (s, 2H), 6.10 (d, J=3.18 Hz, 1H), 5.58 (d, J=5.26 Hz,
H), 5.34 (d, J=6.36 Hz, 1H), 4.39-4.48 (m, 1H), 4.24-4.30 (m, 1H), 4.21 (dd, J=3.30, 10.51 Hz,
1
3
H), 4.10 (dt, J=3.55, 6.48 Hz, 1H), 3.97 (dd, J=7.40, 10.45 Hz, 1H). C NMR (101 MHz, DMSO-
d6): δ 157.7, 156.8, 155.2, 139.7, 104.9, 99.8, 88.5, 81.0, 73.1, 70.8, 69.6. HRMS (ESI): m/z
+
calculated for C H N O S [M + H ]: 445.0707. Found: 445.0701.
1
3
16
8
6 2
5
1
.1.13. [(2R,3S,4R,5R)-5-{4-amino-3-[(3-methylphenyl)sulfanyl]-1H-pyrazolo[3,4-d]pyrimidin-
-yl}-3,4-dihydroxytetrahydrofuran-2-yl]methyl sulfamate (13)
Compound 13 was prepared in a manner similar to that described for 1 using
[(3aR,4R,6R,6aR)-6-{4-amino-3-[(3-methylphenyl)sulfanyl]-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-
1
2
,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl]methanol in 72 % yield as a white solid. H
NMR (400 MHz, DMSO-d ): δ 8.26 (s, 1H), 7.53 (s, 2H), 7.16-7.30 (m, 2H), 7.05-7.13 (m, 2H),
6
6
4
.19 (d, J=3.30 Hz, 1H), 5.65 (d, J=5.26 Hz, 1H), 5.42 (d, J=6.24 Hz, 1H), 4.48-4.58 (m, 1H),
.30-4.36 (m, 1H), 4.24 (dd, J=3.42, 10.51 Hz, 1H), 4.14 (dt, J=3.55, 6.54 Hz, 1H), 4.00 (dd,
1
3
J=7.40, 10.45 Hz, 1H), 2.26 (s, 3H). C NMR (101 MHz, DMSO-d ): δ 157.7, 157.0, 155.3, 139.1,
6
1
36.4, 133.2, 129.5, 129.2, 128.1, 125.8, 101.2, 88.5, 81.1, 73.2, 70.9, 69.6, 20.9. HRMS (ESI):
+
m/z calculated for C H N O S [M + H ]: 469.0959. Found: 469.0962.
1
7
20
6
6 2
5
.1.14. {(2R,3S,4R,5R)-5-[4-amino-3-(benzylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-
dihydroxytetrahydrofuran-2-yl}methyl sulfamate (14)
Compound 14 was prepared in a manner similar to that described for 1 using
{
(3aR,4R,6R,6aR)-6-[4-amino-3-(benzylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2,2-
1
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol in 40 % yield as a white solid. H NMR
(
400 MHz, DMSO-d ): δ 8.19 (s, 1H), 7.53 (br s, 2H), 7.35-7.45 (m, 2H), 7.22-7.35 (m, 3H), 6.13
6
(d, J=2.93 Hz, 1H), 5.60 (br d, J=5.14 Hz, 1H), 5.38 (br d, J=6.24 Hz, 1H), 4.44-4.51 (m, 1H),
1
3
4
.32-4.44 (m, 3H), 4.20-4.31 (m, 1H), 4.09-4.17 (m, 1H), 3.99-4.09 (m, 1H). C NMR (101 MHz,
2
6

DMSO-d ): δ 157.7, 156.8, 155.3, 139.6, 137.1, 129.0, 128.4, 127.3, 99.5, 88.2, 80.9, 73.3, 70.9,
6
+
6
9.6, 36.5. HRMS (ESI): m/z calculated for C H N O S [M + H ]: 469.0959. Found: 469.0958.
1
7
20
6
6 2
5
.1.15. {(2R,3S,4R,5R)-5-[4-amino-3-(phenylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-
dihydroxytetrahydrofuran-2-yl}methyl sulfamate (15)
Compound 15 was prepared in a manner similar to that described for 12 using
{
(3aR,4R,6R,6aR)-6-[4-amino-3-(phenylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2,2-
1
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}methanol in 40 % yield as a white solid. H NMR
(
400 MHz, DMSO-d ): δ 8.26 (s, 1H), 7.52 (br s, 2H), 7.26-7.38 (m, 5H), 6.19 (d, J=3.48 Hz, 1H),
6
5
.63 (br d, J=3.55 Hz, 1H), 5.40 (br d, J=4.77 Hz, 1H), 4.50-4.56 (m, 1H), 4.29-4.35 (m, 1H), 4.23
1
3
(
dd, J=3.42, 10.39 Hz, 1H), 4.10-4.17 (m, 1H), 4.01 (dd, J=7.40, 10.45 Hz, 1H). C NMR (101
MHz, DMSO-d ): δ 157.6, 157.0, 155.4, 136.3, 133.5, 129.6, 128.7, 127.3, 101.2, 88.5, 81.1, 73.2,
6
+
7
0.8, 69.6. HRMS (ESI): m/z calculated for C H N O S [M + H ]: 455.0802. Found: 455.0803.
1
6
18
6
6 2
5
.1.16. [(2R,3S,5R)-5-(4-amino-3-methoxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-
hydroxytetrahydrofuran-2-yl]methyl sulfamate (16)
(2R,3S,5R)-5-(4-Amino-3-methoxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-{[tert-
[
butyl(dimethyl)silyl]oxy}tetrahydrofuran-2-yl]methanol (84.0 mg, 0.21 mmol) was dissolved in
DMF (2.0 mL) and the solution was cooled to 0 °C. Chlorosulfonamide (49.1 mg, 0.42 mmol) was
added and the reaction was allowed to warm slowly to room temperature and allowed to stir
overnight. Water was added and the reaction mixture was extracted with ethyl acetate. The organic
solutions were combined, washed with water (2x) and brine, dried over sodium sulfate, filtered
and concentrated. Ethanol (8.0 mL) was added followed by HCl (1.0 M, 0.63 mL, 0.64 mmol) and
the reaction mixture was allowed to stir at room temperature overnight. The precipitated solid was
removed by filtration, washed with ether and dried under vacuum. Pyridine (0.103 mL, 1.27 mmol)
was added to the filtrate and the solvents were evaporated. The residue was combined with the
solid and the crude material was purified by preparative reverse phase HPLC to afford 16 (8.3 mg,
1
1
4
1 %) as a white solid. H NMR (400 MHz, CD OD): δ 8.16 (s, 1H), 6.64 (dd, J=6.8, 5.5 Hz, 1H),
3
.69-4.74 (m, 1H), 4.31-4.37 (m, 1H), 4.19-4.26 (m, 1H), 4.12-4.17 (m, 1H), 4.11 (s, 3H), 2.95
(dt, J=13.4, 5.9 Hz, 1H), 2.38 (ddd, J=13.6, 7.0, 4.6 Hz, 1H). HRMS (ESI): m/z calculated for
+
C H N O S [M + H ]: 361.0925. Found: 361.0923.
1
1
16
6
6
2
7

5
.1.17. {(2R,3S,5R)-5-[4-amino-3-(difluoromethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3-
hydroxytetrahydrofuran-2-yl}methyl sulfamate (17)
Compound 17 was prepared in a manner similar to that described for 19 using 3-
{
[(2R,3S,5R)-5-[4-amino-3-(difluoromethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2-
(hydroxymethyl)tetrahydrofuran-3-yl]oxy}-1,1,3,3-tetraisopropyldisiloxan-1-ol in 4 % yield as a
1
white solid. H NMR (400 MHz, CD OD): δ 8.23 (s, 1H), 7.21-7.69 (m, 1H), 6.68 (dd, J=7.0, 5.0
3
Hz, 1H), 4.69-4.77 (m, 1H), 4.25-4.31 (m, 1H), 4.12-4.23 (m, 2H), 2.92 (dt, J=13.6, 5.8 Hz, 1H),
+
2
3
.42 (ddd, J=13.6, 7.1, 5.1 Hz, 1H). HRMS (ESI): m/z calculated for C H F N O S [M + H ]:
1
1
14
2
6
6
97.0736. Found: 397.0740.
5
.1.18. {(2R,3S,5R)-5-[4-amino-3-(methylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3-
hydroxytetrahydrofuran-2-yl}methyl sulfamate (18)
Compound 18 was prepared in a manner similar to that described for 16 using [(2R,3S,5R)-
5
-[4-amino-3-(methylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3-{[tert-
butyl(dimethyl)silyl]oxy}tetrahydrofuran-2-yl]methanol in 31 % yield as a white solid. H NMR
400 MHz, DMSO-d ): δ 8.20 (s, 1H), 7.48 (s, 2H), 6.57 (dd, J=5.07, 7.03 Hz, 1H), 5.49 (d, J=4.77
1
(
6
Hz, 1H), 4.51-4.57 (m, 1H), 4.18-4.25 (m, 1H), 3.98-4.05 (m, 2H), 2.75-2.83 (m, 1H), 2.62 (s,
1
3
3
H), 2.29-2.38 (m, 1H). C NMR (101 MHz, DMSO-d ): δ 157.7, 156.7, 155.2, 140.9, 99.2, 83.7,
6
+
8
3.6, 71.1, 69.2, 37.9, 14.5. HRMS (ESI): m/z calculated for C H N O S [M + H ]: 377.0696.
1
1
16
6
5 2
Found: 377.0698.
5
.1.19. {(2R,3S,5R)-5-[4-amino-3-(ethylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3-
hydroxytetrahydrofuran-2-yl}methyl sulfamate (19)
To a round bottom flask containing a solution of [(2R,3S,5R)-5-[4-amino-3-
ethylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3-{[tert-
(
butyl(dimethyl)silyl]oxy}tetrahydrofuran-2-yl]methanol (52, 6.57 g, 15.4 mmol) in DMF (100
mL) was added chlorosulfonamide (3.57 g, 30.9 mmol) at 0 °C and the mixture was stirred at 0 °C
for 30 min. The mixture was poured into saturated aqueous sodium bicarbonate solution and
extracted with ethyl acetate. The extracts were combined, washed with water and brine and then
dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material in
THF (100 mL) was added triethylamine trishydrofluoride (4.95 mL, 30.4 mmol). The reaction
mixture was allowed to stir at rt overnight. The reaction mixture was concentrated and purified by
2
8

1
preparative reverse phase HPLC to afford 19 (4.38 g, 81 %) as a white solid. H NMR (400 MHz,
DMSO-d ): δ 8.20 (s, 1H), 7.50 (s, 2H), 6.57 (dd, J=5.01, 6.97 Hz, 1H), 5.51 (d, J=4.89 Hz, 1H),
6
4
.50-4.57 (m, 1H), 4.21 (dd, J=3.61, 9.23 Hz, 1H), 4.00-4.05 (m, 1H), 3.94-4.00 (m, 1H), 3.13 (q,
1
3
J=7.25 Hz, 2H), 2.77 (td, J=5.85, 12.87 Hz, 1H), 2.30-2.40 (m, 1H), 1.33 (t, J=7.27 Hz, 3H). C
NMR (101 MHz, DMSO-d ): δ 157.8, 156.7, 154.9, 139.5, 99.9, 83.7, 83.6, 71.0, 69.3, 38.0, 27.0,
6
+
1
4.7. HRMS (ESI): m/z calculated for C H N O S [M + H ]: 391.0853. Found: 391.0856.
1
2
18
6
5 2
5
.1.20. {(2R,3S,5R)-5-[4-amino-3-(isopropylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3-
hydroxytetrahydrofuran-2-yl}methyl sulfamate (20)
Compound 20 was prepared in a manner similar to that described for 19 using [(2R,3S,5R)-
5
-[4-amino-3-(isopropylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3-{[tert-
butyl(dimethyl)silyl]oxy}tetrahydrofuran-2-yl]methanol in 75 % yield as a white solid. H NMR
400 MHz CD OD): δ 8.22 (s, 1H), 6.71 (dd, J=6.7, 5.3 Hz, 1H), 4.67-4.82 (m, 1H), 4.29-4.38 (m,
1
(
3
1
1
4
H), 4.12-4.24 (m, 2H), 3.74 (spt, J=6.7 Hz, 1H), 3.00 (dt, J=13.5, 5.8 Hz, 1H), 2.41-2.50 (m, 1H),
+
.43 (t, J=6.6 Hz, 6H). HRMS (ESI): m/z calculated for C H N O S [M + H ]: 405.1009. Found:
1
3
20
6
5 2
05.1004.
5
.1.21. [(2R,3S,5R)-5-{4-amino-3-[(3-methylphenyl)sulfanyl]-1H-pyrazolo[3,4-d]pyrimidin-1-
yl}-3-hydroxytetrahydrofuran-2-yl]methyl sulfamate (21)
Compound 21 was prepared in a manner similar to that described for 19 using {(2R,3S,5R)-
5
-{4-amino-3-[(3-methylphenyl)sulfanyl]-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-3-
1
[
(triisopropylsilyl)oxy]tetrahydrofuran-2-yl}methanol in 64 % yield as a white solid. H NMR
(
400 MHz, CD OD): δ 8.24 (s, 1H), 7.20-7.33 (m, 2H), 7.13 (dd, J=15.2, 7.7 Hz, 2H), 6.74 (dd,
3
J=7.0, 5.0 Hz, 1H), 4.63-4.77 (m, 1H), 4.23-4.39 (m, 1H), 4.11-4.22 (m, 2H), 2.95-3.07 (m, 1H),
.47 (ddd, J=13.5, 7.1, 5.3 Hz, 1H), 2.32 (s, 3H). HRMS (ESI): m/z calculated for C H N O S
2
1
7
20
6
5 2
+
[
M + H ]: 453.1009. Found: 453.1015.
5
.1.22. [(2R,3S,5R)-5-{4-amino-3-[(3-chlorophenyl)sulfanyl]-1H-pyrazolo[3,4-d]pyrimidin-1-
yl}-3-hydroxytetrahydrofuran-2-yl]methyl sulfamate (22)
Compound 22 was prepared in a manner similar to that described for 26 using (2R,3S,5R)-
5
-{4-amino-3-[(3-chlorophenyl)sulfanyl]-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-2-
1
(
hydroxymethyl)tetrahydrofuran-3-ol in 20 % yield as a white solid. H NMR (400 MHz, DMSO-
2
9