Purinylpyridinylamino-based DFG-in/αC-helix-out B-Raf inhibitors: Applying mutant versus wild-type B-Raf selectivity indices for compound profiling

Article abstract of DOI:10.1016/j.bmc.2016.03.055

One of the challenges for targeting B-Raf^V600E with small molecule inhibitors had been achieving adequate selectivity over the wild-type protein B-Raf^WT, as inhibition of the latter has been associated with hyperplasia in normal tissues. Recent studies suggest that B-Raf inhibitors inducing the 'DFG-in/αC-helix-out' conformation (Type IIB) likely will exhibit improved selectivity for B-Raf^V600E. To explore this hypothesis, we transformed Type IIA inhibitor (1) into a series of Type IIB inhibitors (sulfonamides and sulfamides 4-6) and examined the SAR. Three selectivity indices were introduced to facilitate the analyses: the B-Raf^V600E/B-Raf^WT biochemical (^bS), cellular (^cS) selectivity, and the phospho-ERK activation (^pA). Our data indicates that α-branched sulfonamides and sulfamides show higher selectivities than the linear derivatives. We rationalized this finding based on analysis of structural information from the literature and provided evidence for a monomeric B-Raf-inhibitor complex previously hypothesized to be responsible for the desired B-Raf^V600E selectivity.

Full text of DOI:10.1016/j.bmc.2016.03.055

Bioorganic & Medicinal Chemistry 24 (2016) 2215–2234
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Purinylpyridinylamino-based DFG-in/ C-helix-out B-Raf inhibitors:
a
Applying mutant versus wild-type B-Raf selectivity indices
for compound profiling
Longbin Liu a, , Matthew R. Lee b,y, Joseph L. Kim c, Douglas A. Whittington c, Howard Bregman d,
⇑
Zihao Hua d, Richard T. Lewis d, Matthew W. Martin d, Nobuko Nishimura a, Michele Potashman d,
Kevin Yang a, Shuyan Yi d, Karina R. Vaida d, Linda F. Epstein c, Carol Babij e, Manory Fernando e,
Josette Carnahan e, Mark H. Norman a
a Department of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Department of Molecular Structure and Characterization, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Department of Molecular Structure and Characterization, Amgen Inc., 360 Binney St., Cambridge, MA 02142, USA
d Department of Medicinal Chemistry, Amgen Inc., 360 Binney St., Cambridge, MA 02142, USA
e Department of Oncology Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
a r t i c l e i n f o
a b s t r a c t
One of the challenges for targeting B-RafV600E with small molecule inhibitors had been achieving
adequate selectivity over the wild-type protein B-RafWT, as inhibition of the latter has been associated
with hyperplasia in normal tissues. Recent studies suggest that B-Raf inhibitors inducing the
Article history:
Received 6 February 2016
Revised 20 March 2016
Accepted 27 March 2016
Available online 13 April 2016
‘DFG-in/a
C-helix-out’ conformation (Type IIB) likely will exhibit improved selectivity for B-RafV600E. To
explore this hypothesis, we transformed Type IIA inhibitor (1) into a series of Type IIB inhibitors
(sulfonamides and sulfamides 4–6) and examined the SAR. Three selectivity indices were introduced
to facilitate the analyses: the B-RafV600E/B-RafWT biochemical (bS), cellular (cS) selectivity, and the
Keywords:
B-Raf
Inhibitor
DFG
C-helix
Selectivity
v600e
phospho-ERK activation (pA). Our data indicates that
a-branched sulfonamides and sulfamides show
higher selectivities than the linear derivatives. We rationalized this finding based on analysis of structural
information from the literature and provided evidence for a monomeric B-Raf-inhibitor complex
previously hypothesized to be responsible for the desired B-RafV600E selectivity.
Ó 2016 Elsevier Ltd. All rights reserved.
Wild-type
Sulfonamide
1. Introduction
the B-Raf protein (B-RafV600E), accounts for 90% of the B-Raf muta-
tions.2 Relative to wild type B-Raf (B-RafWT), the B-RafV600E protein
The Ras-mitogen activated protein kinase signal pathway Ras #
Raf # MEK # ERK regulates cell growth, proliferation, and differ-
entiation. Frequent oncogenic mutations within members of this
pathway are closely associated with a number of human cancers.
In particular, mutations of the BRAF gene occur in approximately
50–70% malignant melanomas, and at lower frequency in thyroid
(3%), ovarian (30%), and colon (10%) cancers.1 Additionally, among
the >100 different mutant BRAF genes identified, the one with the
T1799A mutation, which results in the replacement of valine-600
with glutamic acid in the activation loop in the kinase domain of
exhibits significantly higher kinase activity (ꢀ500ꢁ) and elevated
ability (ꢀ5ꢁ) to activate down-stream kinases as measured by
the formation of phospho ERK (p-ERK).3 Thus, B-RafV600E repre-
sents a viable target for the treatment of cancers whose genetic ori-
gin stems from activating mutations of the BRAF gene, such as
malignant melanoma where limited options exist for this extre-
mely aggressive disease.4
Since the discovery of the first generation Raf inhibitor sorafenib,
much effort has been focused on the design of B-Raf inhibitors with
improved intrinsic activity and selectivity over other kinases.5 Our
own work toward this objective resulted in the identification of
aminoisoquinoline 1 as a potent and selective B-Raf inhibitor
(Fig. 1).6 The key elements for the enhanced kinase selectivity of 1
reside in: (1) the slightly bulky isoquinoline ring that is better
accommodated by the pocket near the X-DFG motif at the beginning
⇑
Corresponding author. Tel.: +1 805 447 3442.
E-mail address: lliu@amgen.com (L. Liu).
Current address: Lilly Biotechnology Center San Diego, 10300 Campus Point
y
Drive, Suite 200, San Diego, CA 92121, USA.
http://dx.doi.org/10.1016/j.bmc.2016.03.055
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

2216
L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
Table 1
H
N
N
Comparison of the activity and selectivity of compounds 1, 2, and 3\
H
N
N
N
N
N
Compd B-RafWT
no.
B-RafV600E bS* MIA PaCa-2 A375
cS^
pAà (%)
413
Me
H
N
F
IC50 (nM) IC50 (nM)
EC50 (nM)
IC50 (nM)
Cl
1
2
3
2
20
0.4
2
4
1
1
5
22
2290
2
17
1
11
134 674
529 755
O
O
S
O
F
Me
HN
N
NH
Cl
0.4 529
\
For n P 2, the values are reported as an average and the standard deviations are
1
2
included in Supporting information.
(Vemurafenib)
*
Biochemical selectivity index (bS) = B-RafWT IC50/B-RafV600E IC50
.
^
Cellular selectivity index (cS) = MIA PaCa-2 EC50/A375 IC50
.
à
p-ERK activation index (pA) = the maximum percentage of p-ERK activation in the
H
N
N
N
MIA PaCa-2 cellular assay relative to control.
H2N
N
N
F
N
H
N
F
cal to be employed in the early stages of SAR exploration.7 Concep-
tually, a desirable B-RafV600E inhibitor would be a compound that
O
S
N
F
S
N
N
F
H
O
O
Me
Me
exhibits low binding affinity for the B-RafWT protein (high IC50
)
HN
F
S
Me
R
relative to its affinity for the target B-RafV600E. Such a compound
must also, besides potently inhibiting the down-stream ERK phos-
phorylation in the A375 cells that harbor the B-RafV600E protein,
demonstrate minimal or reduced potency (high EC50) in eliciting
down-stream ERK phosphorylation in the MIA PaCa-2 cells
harboring the B-RafWT protein. Based on these considerations,
two selectivity indices were introduced in our SAR studies: (1)
the biochemical selectivity (bS), as measured by the ratio of the
IC50 values derived from the kinase assays using B-RafWT and
O
3
4: R = Aryl
5: R = Alkyl
6:
(Dabrafenib)
R = Amino
Figure 1. Structures of B-Raf inhibitors (1–3) and the proposed hybrids (4–6).
of the activation loop of the B-Raf protein where X is glycine, while in
most other kinases a larger amino acid (X) is present; and (2) the 4-Cl
substitution in the extended hydrophobic pocket that is less toler-
ated in some kinases, such as Lck and Tie-2.6 Compound 1 was
equally potent toward both wild-type and mutant B-Raf in the
biochemical assays where phosphorylation of MEK1 by the B-Raf
proteins were measured (IC50 = 2 nM, Table 1).7 Further investiga-
tions revealed that, although compound 1 potently inhibited
down-stream ERK phosphorylation (p-ERK, IC50 = 2 nM) in A375
cells which harbor the B-RafV600E protein, it also activated the signal-
ing pathway in the human pancreatic carcinoma MIA PaCa-2 cells
which carry the B-RafWT protein (p-ERK, EC50 = 22 nM). Extensive
in vivo studies revealed that, while administration of 1 resulted in
tumor growth inhibition in animal models bearing the human mel-
anoma A375 tumor cells, hyperplasia of normal epithelial cells were
also observed.8 Concurrent to our findings, similar results with other
second-generation B-Raf inhibitors appeared in the literature that
also suggestedthat inhibitionofB-RafWT mayresult in the seemingly
paradoxical activation of the MAPK pathway by B-Raf inhibitors.9
One hypothesis that has been put forth is that, in thepresence of high
levels of activated Ras protein, the B-RafWT-inhibitor complex may
induce hetero-dimer formation with C-Raf or other RAF family
members. The hetero-dimer then trans-activates the unoccupied
paralogous RAF protomer, resulting in elevated down-stream
signaling such as p-ERK.9c–e,10 The dimerization is required for
normal Ras-dependent Raf activation but not for the function of
mutants with high catalytic activity, such as RafV600E. Further, a
dimer-interface peptide inhibitor can block both the Raf
dimerization and down-stream signaling.11 Based on this theory, it
follows that inhibitors that disfavor dimerization of the B-RafWTꢂin-
hibitor complexes should pose a lower risk of MAPK pathway activa-
tion in normal cells, which is associated with hyperplasia in normal
tissues, and offer greater therapeutic benefit in the treatment of
B-RafV600E associated cancers. Indeed, a recent study reported the
design of PLX-7904/PB04 with increased selectivity for B-RafV600E
over B-RafWT and impaired hyper-activation of normal MAPK
signal.12
B-RafV600E enzymes (IC / IC
);13 (2) the cellular selectivity
WT
V600E
50
50
(cS), as measured by the ratio of the EC50 value derived from the
MIA PaCa-2 cellular assay and the IC50 value derived from the
A375 cellular assay (EC50/ IC50). It turns out that in the MIA
PaCa-2 cellular assay, the inhibitor-induced maximum p-ERK
increase can vary significantly between compounds. Therefore,
the percentage of p-ERK activation (pA), defined as the maximum
increase of p-ERK relative to baseline control in the MIA PaCa-2 cell
assay was also introduced.
While the concept for the biochemical selectivity index (bS) is
straightforward, the relationship between the two cellular selectiv-
ity indices pA and S can be complicated and is illustrated graphi-
c
cally in Figure 2. By using these selectivity parameters we can
compare and contrast different compound profiles to aid in our
SAR investigations. For example, if two compounds, X (blue) and
Y (red), were similarly potent in the B-RafV600E cellular assay
(i.e., same A375 IC50 values) with compound X showing a steeper
activation slope and smaller pA than compound Y in the MIA
PaCa-2 assay, the latter would appear to be more specific [cS(Y)
> cS(X)] even though it caused more p-ERK activation [pA(Y) > pA
(X)]. It should be noted that, since the activation curves (hence
maximum p-ERK) in the MIA PaCa-2 assay are often not well
defined at the top concentrations used in our assay, the corre-
sponding ECmax values are not included but are partly reflected
in the estimated EC50 values.
The value of the selectivity indices is illustrated with the data of
compound 1 (Table 1). For example, the biochemical selectivity
index bS for compound 1 is ꢀ1, since it was equally potent inhibiting
both the wild-type and the V600E-mutant enzymes; however, the
cellular selectivity index (cS) for 1 is 11. Functionally, cS may be more
relevant than bS, since the former reflects the degree of separation
between the inhibition of the B-RafV600E pathway (hence anti-tumor
activity) and the activation of the B-RafWT pathway (cell growth).
c
Thus, in essence, S is a reflection of the ‘cellular’ safety index of a
compound and may be inversely related to a compound’s ability to
drive Raf protein dimerization. The maximum p-ERK level (pA) for
compound 1 was found to be 413% above the baseline control,
meaning there was 4-fold pathway activation in B-RafWT cells. With
the selectivityindices for compound1 established, our goal was then
to design compounds with improved selectivity profiles.
2. Definition of selectivity indices
Although the direct measure of inhibitor-induced Raf-dimer
formation had been described in the mechanistic studies of the
paradoxical B-RafWT pathway activation, it was deemed impracti-

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
2217
p-ERK
that the shift of the
a
C-helix might be responsible for deterring
(POC%)
the formation of B-Raf heterodimers that promote down-stream
p-ERK formation. We reasoned that, if the aminoisoquinoline
moiety in 1 was replaced with a sulfonamide or sulfamide group,
as represented by the generic structures 4, 5, and 6, it may be
possible to switch the binding mode from ‘DFG-out/
(Type IIA binder) to ‘DFG-in/ C-helix-out’ (Type IIB binder) and
~
~
~
~
200
150
100
aC-helix-in’
a
thereby achieve enhanced B-RafV600E selectivity. Additionally, we
hoped to probe how structural changes in the sulfonamide tail
piece would affect each of the selectivity parameters bS, cS, and
pA. In this paper, we report the structure–activity relationships
and structure–selectivity relationships of the hybrid series. We
note that concurrent to our work, other groups had also
synthesized compounds identical or similar to some of the
examples described here.18
cS (Y)
cS (X)
50
0
A375 IC50
EC50(X)
EC50(Y)
[c]
Figure 2. Illustration of how EC50 may vary as a function of the slope and the
maximum activation in the MIA PaCa-2 assay (pA) for compounds X (blue) and Y
(red) with similar A375 IC50. The cellular selectivity (cS) is determined by the ratio
of EC50 verses IC50 (dark-blue and dark-red curves). Compound X appears to be less
selective based on its lower EC50 value (i.e., smaller cS) compared to Y. On the other
hand, Y induces a higher percentage of p-ERK (i.e., higher pA) than X in the B-RafWT
cells.
4. Synthesis
The sulfonamides 4–5 were readily prepared by the methods
shown in Scheme 1. In one approach, commercially available
2,6-difluoroaniline (7) was protected as the acetamide prior to
nitration at the 3-position.19 The nitro group was reduced under
catalytic hydrogenation conditions (H2, Pd/C) to aniline 8. The
latter was treated with either aryl or alky sulfonyl chlorides in
the presence of catalytic amounts of dimethylamino pyridine to
furnish the corresponding sulfonamides that were subsequently
treated with hydrogen chloride in ethanol to cleave the acetamide
group furnishing anilines 9 and 10, respectively. Alternatively, ani-
lines 9–10 were prepared from commercially available 2,4-difluo-
roaniline (11) by first introducing the C-1 sulfonamide
functionality followed by installing the C-3 amino group via
sequential lithiation–carboxylation, Curtius rearrangement, and
Boc-deprotection.20 The final couplings between anilines 9–10
3. Design principle
At the time of our investigation, little was known about how to
rationally engineer B-RafV600E selectivity in an ATP-competitive
B-Raf inhibitor since the site of mutation at Val-600 in B-Raf lie
outside the ATP binding site. Recently however, sulfonamide-based
B-Raf inhibitors have emerged as a class of effective agents in treat-
ing patients with malignant melanoma harboring B-RafV600E, as
exemplified by the approval of vemurafenib (2)14 and dabrafenib
(3).15 A comparison of the sulfonamides with 1 using the biochem-
ical selectivity index (bS, Table 1) showed that while vemurafenib
(2) was 5ꢁ more selective, dabrafenib (3) showed inversed selec-
tivity. Although both 2 and 3 were more selective (>10ꢁ) than 1
in the cellular setting (cS), they also showed higher levels of p-
ERK induction (pA) in B-RafWT cells. Thus, considering all three
parameters (bS, cS, and pA), the selectivity profiles of 2 and 3 are
suboptimal and may be responsible for the development of skin
lesions in patients after treatment with both therapeutics.16 We
felt that the use of bS, cS, and pA in SAR analysis would facilitate
our efforts to improve the selectivity profiles of 1 by identifying
compounds that selectively inhibit the B-RafV600E pathway (i.e.,
high bS in biochemical assays and high cS in cellular assays) and
have minimal activation via the B-RafWT pathway (i.e., low pA).
Given the superior cS exhibited by the sulfonamides 2 and 3, we
decided to investigate the sulfonamide pharmacophore within the
scaffold defined by 1. Molecular modeling studies comparing the
bound conformations of 1 and 214d showed that the hinge-
binding domains (purine in 1 and pyrrolopyridine in 2) of both
series occupy the same space in the binding pocket of the B-Raf
protein (Fig. 3). The mid-section of the molecules link the hinge-
binders with the tail groups docking in the extended
hydrophobic region, where the binding mode diverges between
the two structural classes.17 The aminoquinoline moiety in 1 forms
and
6-(2-fluoropyridin-3-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-
purine 126 were achieved by nucleophilic aromatic substitution
(SNAr) under strongly basic conditions (lithium or sodium hexam-
ethyl disilylazide). The tetrahydropyranyl (THP) protecting group
of the purine moiety was cleaved with aqueous hydrogen chloride
to give the final products 4a,b,d,g and 5b,d,h,j,k,m,o. We also
developed a more divergent approach that utilized a common
late-stage intermediate for the final sulfonamide analog synthesis.
Thus, aniline 11 was first protected as the benzyloxy carbamide
(Cbz) prior to the installation of the C-3 amino group as described
earlier (e.g., 11 to 9 and 10), furnishing aniline 13.20 SNAr coupling
between 13 and 12, followed by hydrogenolysis of the Cbz group,
afforded the penultimate intermediate aniline 14 that was then
functionalized with various aryl or alkyl sulfonyl chlorides to give
4c,e,f,h–k and 5a,c,e–g,i,l,n,p after cleavage of the THP group.
The syntheses of the sulfamide analogs (6a–i) are shown in
Scheme 2. In the first approach, the mono-protected 2,4-difluoro-
1,3-diaminobenzene 8 or 15 was treated with sulfuryl chloride
and various amines (R1R2NH) to install the C-1 sulfamide function-
ality. Removal of the Boc or Ac protecting group afforded aniline
16. Alternatively, sulfamide groups were introduced on aniline
11 prior to the introduction of the C-3 amino group using the direc-
ted lithiation–carboxylation–Curtis sequence as described earlier
for the transformation of 11 to 9 or 10 (Scheme 1). SNAr coupling
between 16 and 12 followed by THP-cleavage afforded analogs
6a,b,e,h. In the third approach, aniline 14 was directly modified
with sulfonyl chloride and amines. This approach was used to pre-
pare analogs 6c,d,i. Lastly, aniline 14 was treated with 2-oxooxazo-
lidine-3-sulfonyl chloride,21 which was generated in situ from the
reaction of 2-bromoethanol with chlorosulfonylisocyanate, leading
to intermediate 17. Reaction of the latter with 3-hydroxypyrrolidi-
nes gave analogs 6f and 6g.
a hydrogen-bond with Glu501 of the
aC-helix and directs the
chlorophenyl group towards the extended hydrophobic pocket of
the B-RafV600E protein that, in turn, assumes the inactive DFG-out
conformation, despite the activating phospo-mimetic nature of
Glu600 on the activation loop. In contrast, the sulfonamide moiety
in 2 interacts with the backbone amides of both Asp594 and
Gly596 and steers the propyl group into the ‘Raf-selective’ pocket
produced by a shift of the
a
C-helix.14a It has been hypothesized

2218
L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
Figure 3. (A) Crystal structure of B-RafV600E + 1 showing DFG-out,
and dashed lines indicate hydrogen bonds. (B) Crystal structure of B-RafV600E + 2 showing DFG-in,
(C) Overlay of 1 and 2 based on protein superposition; protein is not depicted for clarity. (D) Overlay of B-RafV600E + 1 (blue protein, orange
protein, green C-helix) highlighting the pronounced difference in the C-helix conformations.
a
C-helix-in (Type IIA) binding of the inhibitor (PDB 3IDP). The
C-helix-out (Type IIB) binding of the inhibitor, colored as in A (PDB 3OG7).
C-helix) and B-RafV600E + 2 (gray
aC-helix is orange, the activation loop is red,
a
a
a
a
With the higher cS seen from simple phenyl sulfonamides, the
effects of heteroaryl sulfonamides were probed. The 3-pyridyl sul-
fonamide 4e was more potent than 1 in the biochemical assays but
was no more selective. Its cellular potencies and cS (79) were sim-
ilar to that of the 2-fluorophenyl analog 4b but with a higher pA.
Compared to 4e, the 2-pyridyl analog 4f was more than 10-fold
less potent and showed modest cellular selectivity (cS = 45). The
2-thiophene sulfonamide 4g was more potent than 1 in both the
biochemical and the cellular assays. Its cellular selectivity index
was modest (cS = 57) yet its p-ERK activation in MIA PaCa-2 cells
was very high (pA = 1870). In comparison, the corresponding thia-
zole analog 4h was less potent and selective in the cellular assays
(cS = 25). Pair-wise analyses of 4h/4g and 4a/4f suggested that
aryls containing a nitrogen atom adjacent to the sulfone function-
ality tended to show eroded cellular potencies and specificities.
The 1H-pyrazole-4-sulfonamide 4i exhibited the best potency in
both the B-RafV600E biochemical (IC50 = 0.2 nM) and cellular
(IC50 = 6 nM) assays as well as highest selectivities (bS = 5 and
cS = 135). In contrast, its N-methyl homolog (4j) suffered a signifi-
cant drop in cellular potency (A375 IC50 = 710 nM) and selectivity
(cS = 3). Even more dramatic was the >1000-fold loss in the bio-
chemical potency seen with the 3,5-dimethyl pyrazole analog 4k.
This was likely due to unfavorable conformational penalties
imposed by the two methyl groups flanking the sulfonamide
group.
5. Results and discussion
To gauge the impact of replacing an amine with a sulfonamide
functionality on B-RafV600E activity and selectivity, aryl sulfon-
amides 4a–k were evaluated in comparison with aryl amine 1
(Table 2).22 In the biochemical assays, phenyl sulfonamide 4a
showed IC50 values of 17 and 7 nM toward B-RafWT and B-RafV600E
,
respectively. Compared to 1, the larger drop of B-RafWT potency
(ꢀ8-fold) compared to B-RafV600E potency (ꢀ3.5-fold) resulted in
a 2-fold increase in selectivity index (bS = 2) in 4a. In the cellular
assays, 4a was over 100-fold less active than 1 in the wild-type
cells (MIA PaCa-2 EC50 = 2290 nM) and only 14-fold less potent
in the mutant cells (A375 IC50 = 28 nM). Therefore, the resulting
cellular selectivity (cS = 82) was improved by >7-fold over 1. How-
ever, in the MIA PaCa-2 cells, sulfonamide 4a also induced higher
levels of p-ERK activation (pA = 1540%) than 1 (pA = 413%). Thus,
the gain of cS (7-fold) was off-set by the increased pA (ꢀ4-fold)
for sulfonamide 4a. A fluorine substitution at the 2-position of
the phenyl ring (4b) resulted in slightly higher bS (3-fold), better
cellular potency (but not selectivity), and lower pA than 4a. A 3-flu-
orine substituted analog (4c) was as potent and non-selective as 1
in the biochemical assays, although its cS and pA were similar to
those of 4a. The 4-fluoro analog (4d) showed significant losses in
both the biochemical (>18-fold) and the cellular (>100-fold)
potencies as well as marginal improvements in bS and cS (ꢀ2-fold)
compared to 1. Interestingly, the pA of 4d was the lowest among
the four sulfonamides examined, albeit still higher than that of 1.
The data in Table 2 indicates that, for the aryl sulfonamides
(with exceptions for thiazole 4h and pyrazole 4j), there is a good

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
2219
N
N
F
N
THPN
F
F
F
N
AcHN
F
H2N
F
a, b, c
d, e
j, k
12
H2N
F
NH2
NHSO2R
7
8
9: R = aryl
10: R = alkyl
F
O
S
R
O
F
N
N
HN
N
H
F
i, f, g, h
d, f, g, h
N
HN
N
F
NH2
4a-k: R = aryl
5a-p: R = alkyl
11
F
F
H2N
F
j, c
12
d, h, or k
N
N
HN
NH2
F
N
THPN
N
NHCbz
13
14
R (4, aryl):
Me
N
∗
F ∗
∗
∗
∗
∗
∗
∗
∗
S
∗
∗
S
N
N
N
Me
N
N
N
H
N
F
F
N
Me
H
a
b
c
d
e
f
g
h
i
j
k
R (5, alkyl):
Me
Me
Me
∗
Me
Me
Me
∗
∗
∗
CF3
∗
∗
∗
∗
Me
Me
Me
a
b
c
d
e
f
g
h
∗
∗
Me ∗
∗
∗
∗
∗
∗
Me
O
Me
Me
O
i
j
k
l
n
o
p
m
Scheme 1. Synthesis of sulfonamides 4–6. Reagents and conditions: (a) Ac2O, DCM, rt, 16 h; (b) HNO3, H2SO4, 0 °C ? rt, 18 h; (c) H2, Pd/C (10%), EtOH or EtOAc, rt; (d) RSO2Cl,
Pyr, DMAP, DCM, rt; (e) HCl (aq), EtOH, 90 °C, 16 h; (f) nBuLi, THF, ꢂ78 °C, CO2; (g) (PhO)2P(O)N3, Et3N, tBuOH, 85 °C; (h) TFA, DCM, rt, or 65 °C; (i) CbzCl, Pyr, DCM, rt, 16 h; (j)
NaHMDS or LiHMDS, THF or DMF, 0 °C; (k) HCl (aq), THF, rt.
F
F
F
O
O
R
F
H2N
F
S
NR1R2
12, f, g
N
N
HN
N
H
F
N
HN
NHSO2NR1R2
NH2
N
a, b
a, c, d, e
a, e
16b,e
16h
16a
8:
R = AcNH
6a,b,e,h
11: R = H
15:
R = BocNH
a, b or e
6c,d,i
F
F
N
N
HN
NH2
O
O
O
F
h
S
i, g
N
THPN
N
N
HN
N
N
6f,g
O
N
H
F
N
THPN
14
N
17
NR1R2:
∗
∗
∗
Me
∗
∗
∗
∗
OH
∗
∗
N
Me
Me
N
Me
Me
N
N
N
N
N
N
N
OH
Me
O
NH
a
b
c
d
e
f
g
h
i
Scheme 2. Synthesis of sulfamides (6). Reagents and conditions: (a) R1R2NH, SO2Cl2, Pyr, DMAP, DCM, ꢂ30 °C ? rt; (b) HCl (aq), EtOH; (c) nBuLi, THF, ꢂ78 °C, CO2; (d) (PhO)2P
(O)N3, Et3N, tBuOH, 85 °C; (e) TFA, DCM, rt, or 65 °C; (f) NaHMDS or LiHMDS, THF or DMF, 0 °C; (g) HCl (aq), THF, rt; (h) Cl(SO2)NCO, Br(CH2)2OH, DCM, 0 °C; Et3N, rt; (i)
R1R2NH, MeCN, Et3N, 110 °C.

2220
L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
Table 2
Comparison of the activity and selectivity of aryl sulfonamides 4a–k\
H
N
N
N
N
N
F
H
N
F
O
HN
S
Aryl
O
Compd no.
Aryl
B-RafWT IC50 (nM)
2
B-RafV600E IC50 (nM)
2
bS*
1
MIA PaCa-2 EC50 (nM)
22
A375 IC50 (nM)
2
cS^
11
pAà (%)
413
1
—
∗
4a
4b
4c
4d
17
10
4
7
2
3
1
2
2290
873
28
82
1540
989
∗
3
12
73
F
∗
F
F
3
1510
5650
15
101
23
1470
600
∗
∗
∗
83
36
242
4e
1
1
1
786
10
79
1430
N
4f
41
0.5
2
21
1
2
1
2
7940
402
177
7
45
57
25
1110
1870
1660
N
∗
S
S
4g
4h
∗
1
783
31
N
∗
N
N
4i
1
0.2
8
5
807
2220
—
6
135
3
1210
1170
—
N
H
∗
4j
19
—
2
710
—
N
Me
Me
∗
4k
216
—
—
NH
N
Me
A dash (—) sign indicates either non-applicable (e.g. Ar group for 1) or data unavailable.
\
For n P 2, the values are reported as an average and the standard deviations are included in Supporting information.
*
^
à
Biochemical selectivity index (bS) = B-RafWT IC50/B-RafV600E IC50
.
Cellular selectivity index (cS) = MIA PaCa-2 EC50/A375 IC50
p-ERK activation index (pA) = the maximum percentage of p-ERK activation in the MIA PaCa-2 cellular assay relative to control.
.
correlation of potencies between the B-RafV600E biochemical and
the A375 cellular assays. In general, the cell-to-enzyme shifts were
larger (>5-fold) compared to aryl amine 1. Most of the aryl sulfon-
amide analogs exhibited improved cellular selectivities relative to
we continued our investigation by examining a series of alkyl
sulfonamides and the results are shown in Table 3.
The methyl sulfonamide (5a) was only modestly active in the
biochemical assays, possibly reflecting the lack of adequate contact
c
c
1, as judged by the higher S values. The increased S for the aryl
sulfonamides were a direct consequence of the larger separation
between the B-RafWT IC50 and the MIA PaCa-2 EC50 values than
the cellular shifts seen in the B-RafV600E assays. It is worth noting
that there were no correlations between the biochemical (bS) and
the cellular (cS) selectivities, as was also shown from the data in
Table 1 for the two benchmark compounds (2 and 3). Lastly, the
maximum level of activation of p-ERK, as indicated by pA, varied
widely (600–1870%) for the aryl sulfonamides and were much
higher than for 1. Since the derived EC50 values in the
MIA PaCa-2 assay were dependent not only on the slope of the acti-
vation curves, but also on the degree of the activation (pA), the
value of the cellular selectivity (cS) can be significantly affected
by the magnitude of pA as illustrated in Figure 2. With this in mind,
in the aC-helix pocket. With a bulkier propyl group as in com-
pound 5b, low nano-molar IC50 values were realized in both the
biochemical and the cellular assays. However, 5b showed no bio-
chemical selectivity (bS = 1), modest cellular selectivity (cS = 39),
and high p-ERK activation (pA = 1300). The inferior selectivity of
5b compared to that of 2 (cS = 134) suggested that structural vari-
ation at the hinge-binding region can have significant impact on
the B-Raf selectivity. The higher substituted ethyl sulfonamide
homologs such as trifluoroethyl (5c), isobutyl (5d), and cyclopropyl
methyl (5e) derivatives maintained good A375 cellular potencies
(IC50 = 17–40 nM) while exhibiting increased bS and cS (with the
exception of 5c). These compounds also induced high p-ERK activa-
tion in the MIA PaCa-2 cells (1070–1200%). Further increasing the
bulkiness to a neo-pentyl group (5f) resulted in a loss of selectivity

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
2221
Table 3
Comparison of the activity and selectivity of alkyl sulfonamides 5a–p\
H
N
N
N
N
N
F
H
N
F
O
HN
S
Alkyl
O
Compd no.
Alkyl
B-RafWT IC50 (nM)
412
B-RafV600E IC50 (nM)
360
bS*
1
MIA PaCa-2 EC50 (nM)
6690
A375 IC50 (nM)
356
cS^
19
pAà (%)
∗
5a
1000
Me
∗
∗
5b
5c
12
10
9
3
1
3
936
713
24
40
39
18
1300
1200
Me
CF3
Me
5d
5e
5f
16
26
5
6
8
6
3
3
1
1450
2140
202
17
33
63
85
65
3
1100
1070
500
∗
Me
∗
∗
Me
Me
Me
5g
5h
5i
111
66
186
14
7
1
5080
1540
778
>1000
45
—
517
356
274
400
723
574
410
628
∗
∗
Me
5
34
29
62
12
44
164
48
Me
Me
Me
∗
∗
Me
Me
38
5
27
5j
112
132
46
18
41
4
6
8090
2070
1790
1800
1480
130
173
41
∗
∗
∗
5k
5l
3
12
11
5
5m
5n
21
2
11
∗
26
5
31
O
∗
∗
5o
5p
51
18
31
3
6
4370
—
40
109
—
244
—
179
356
O
A dash (—) sign indicates data unavailable.
\
For n P 2, the values are reported as an average and the standard deviations are included in Supporting information.
*
^
à
Biochemical selectivity index (bS) = B-RafWT IC50/B-RafV600E IC50
.
Cellular selectivity index (cS) = MIA PaCa-2 EC50/A375 IC50
p-ERK activation index (pA) = the maximum percentage of p-ERK activation in the MIA PaCa-2 cellular assay relative to control.
.
(cS = 3) and a noticeable reduction of p-ERK activation (pA = 500).
The overall profile of 5f resembled aryl amine 1 more than
sulfonamides, raising the possibility that the bulky neo-pentyl
inferior selectivity (2–3-fold in bS, cS, and pA) when compared to
the isopropyl derivative (5h). However, as the ring size increased
as in the 4-, 5-, and 6-membered ring analogs (5l,m,o), both the
potency and the selectivity improved. The cyclopentyl analog
(5m) showed the best combination of bS (11-fold) and cS (164-fold)
while the cyclohexyl analog (5o) showed the best combination of
cS (ꢀ110-fold) and pA (244%). Cyclic ethers were also examined
to explore potential polar interactions as well as to impart
improved solubility. It was found that the presence of an oxygen
atom led to either reduced cellular selectivity (see, 5n) or cellular
potency (see, 5p) relative to their corresponding carbocyclic coun-
terparts. The data from Table 3 showed that, like the aryl sulfon-
amides, there was a good correlation between the biochemical
and the cellular IC50 values in the B-RafV600E assays for the alkyl
sulfonamides. Similarly, there were no obvious correlations
group might prefer a switch from a DFG-in/
aC-helix-out to a
DFG-out/ C-helix-in binding mode. Although the benzyl
a
sulfonamide (5g) showed modest biochemical potency, it was
inactive in the cellular assays. The isopropyl sulfonamide (5h)
showed higher bS (5-fold) and modest cS (34-fold) as compared
to the non-
a
-branched alkyl sulfonamides (5bꢂ5e). Additionally,
this compound also had the lowest level of activation (pA 356%)
in the MIA PaCa-2 cells. Up to this point, 5h was the only potent
B-Raf inhibitor from the purinylpyridinylamino-2,4-difluorophenyl
sulfonamide series that displayed acceptable selectivity based on
all three selectivity indices (bS, cS, and pA). This desirable profile
seemed to hold for 2-butyl (5i) and 2-pentyl (5j) derivatives with
a slight erosion of the A375 cellular potency from the latter. The
cyclopropyl analog (5k) showed reduced potency (by 3-fold) and
c
between bS and S. However, in contrast to the aryl sulfonamides,
lower activation of p-ERK via the wild-type B-Raf pathway was

2222
L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
observed with a majority of the
a
-branched alkyl sulfonamides
bition while imparting minimal B-RafWT activation. To help under-
stand the complex structure–selectivity relationships, the
biochemical and the cellular selectivity indices (bS and cS) of the
inhibitors from Tables 1–4 were each plotted against the p-ERK
activation (pA) in a scatter chart (Fig. 4). The shape of each com-
pound denotes the B-RafV600E cellular activity (A375 IC50: circle,
<55 nM; diamond, >56 nM) and the color codes for its structural
(pA < 700%). In this regard, several of these alkyl sulfonamides
(5h,i,l,n,o) performed better than either 2 or 3.
The favorable pA profile exhibited by the
a-branched alkyl sul-
fonamides prompted us to examine bis-alkyl sulfamides. We antic-
ipated that the N-substituents of the sulfamides may function as
isosteres of the
a-branched alkyl groups of the sulfonamides and
may also provide structural features inaccessible from either the
aryl or the alkyl sulfonamides. As shown in Table 4, N,N-dimethyl
sulfamide 6a showed cellular potency and selectivity similar to
that of isopropyl sulfonamide 5h. Increasing the sizes of the sul-
famide group resulted in reduced cellular potency and selectivity
(see, 6b and 6c). Cyclic sulfamides seemed to be preferred when
compared to the acyclic ones, as suggested by the data for com-
pounds 6c and 6d. The five-membered cyclic sulfamide (6d) was
more potent and selective than the 6-membered sulfamide (6e).
Heteroatoms were not well tolerated as indicated by the activities
of hydroxyprolinyl, morpholinyl, and piperazinyl analogs (6f,g,h,i).
type (i.e., compound 1—orange; aryl sulfonamides—red; a-
branched sulfonamides—blue; linear sulfonamides—yellow; sul-
famides—green). For the purpose of comparison, the profiles for
vemurafenib 2 and dabrafenib 3 were also included in the plots.
Additionally, the data points in Figure 4 were divided into four
quadrants using aryl amine 1 as the reference point. It is clear that
the majority of the analogs showed better biochemical selectivities
than 1. Interestingly, dabrafenib 3 was more potent toward the
wild-type than to the mutant B-Raf proteins and was located in
the southeast quadrant in Figure 4A whereas vemurafenib 2 occu-
pied the northeastern quadrant due to its high bS value. Both the
aryl and linear alkyl sulfonamides tended to induce high levels of
p-ERK (pA) in the wild-type MIA PaCa-2 cells (>600%) and, as a
result, occupied the eastern quadrants. By contrast, most of the
As was observed with the
a-branched alkyl sulfonamides, lower
levels of pERK activation in the B-RafWT pathway (pA 6 600%) were
observed with these sulfamides.
Collectively, the data presented for compounds 4–6 highlight
the challenges of evaluating selectivity profiles of B-RafV600E inhibi-
tors over the wild-type protein B-RafWT since the three selectivity
indices (bS, cS, and pA) each seem to follow their own course of
dependency on the structures of the inhibitors. Thus, it became
clear that no single selectivity parameter was sufficient for the
selection of compounds that would offer maximal B-RafV600E inhi-
a
-branched analogs showed pA values <600%. Furthermore, only
members from these groups occupy the northwestern quadrant
defined by higher bS and lower pA with respect to aryl amine 1.
Compounds located within this ‘selective quadrant’ represent
improved selectivity against biochemical affinity and cellular acti-
vation and, as shown in Figure 4A, also rank better than 2 and 3 in
these regards. With a cellular potency cut-off of 55 nM, compounds
Table 4
Comparison of the activity and selectivity of sulfamides 6a–i\
H
N
N
N
N
N
F
H
N
F
O
HN
S
NR2
O
Compd no.
NR2
B-RafWT IC50 (nM)
90
B-RafV600E IC50 (nM)
8
bS*
11
MIA PaCa-2 EC50 (nM)
1780
A375 IC50 (nM)
54
cS^
33
pAà (%)
389
∗
Me
N
6a
Me
∗
∗
N
Me
Me
Me
6b
6c
157
165
22
57
7
3
1780
9910
87
20
16
262
600
N
603
Me
∗
∗
N
N
6d
6e
31
38
2
16
3
2070
1220
44
65
47
19
401
220
15
∗
∗
∗
N
N
N
OH
6f
122
68
9
14
4
—
201
233
—
—
OH
6g
16
7190
31
376
6h
6i
102
62
2
3580
—
293
12
—
300
—
O
∗
N
>1000
>1000
—
1000
NH
A dash (—) sign indicates data unavailable.
\
For n P 2, the values are reported as an average and the standard deviations are included in Supporting information.
*
^
à
Biochemical selectivity index (bS) = B-RafWT IC50/B-RafV600E IC50
.
Cellular selectivity index (cS) = MIA PaCa-2 EC50/A375 IC50
p-ERK activation index (pA) = the maximum percentage of p-ERK activation in the MIA PaCa-2 cellular assay relative to control.
.

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
2223
5h,i,m,o and 6a,d were readily identified as potent and selective
from analogs 4–6. A similar analysis of the cellular selectivity index
(cS) as a function of pA (Fig. 4B) also revealed the same set of potent
compounds in the corresponding selective quadrant, albeit in a
different rank order along the y-axis (cS). In this setting, the two
marketed B-Raf inhibitors (2 and 3) showed excellent cellular
selectivity compared to most of the analogs in Figure 4B. However,
in terms of cellular activation (pA), they seem to ranked poor
relative to those from the selective quadrant. Thus, we believe that
B-Raf inhibitors identified from the selective quadrant may poten-
tially offer larger therapeutic window regarding the off-target
safety concerns.
Although we have identified the northwestern quadrant in
Figure 4 as the selective quadrant for the type IIB inhibitors
described herein, it should be noted that the southwestern quad-
rant also represents inhibitors with lower activation (pA) of the
B-RafWT pathway albeit also low biochemical (bS) and cellular
(cS) selectivity. This set of compounds are interesting in that as
the pA diminishes toward baseline value (0%), both bS and cS would
become less relevant. As a result, such a compound would still be
highly B-RafV600E specific. Compounds that bind to B-Raf with a
slow-off rate and long residence time tend to occupy the south-
western quadrant. An alternative hypothesis to the dimer-transac-
tivation pathway was that fully occupied RafWT-dimers could not
activate the down-stream signaling. Therefore, tighter, pan-Raf
binders will induce the formation of dimers whereby both pro-
tomers would be completely populated by the inhibitors and thus
prevent the undesired activation of the wild type pathway.23
6. X-ray crystallography and molecular modeling studies
The intriguing favorable potency and selectivity measures
exhibited by the sulfonamides and the sulfamides with branching
alpha to the sulfone functionality prompted us to investigate their
binding to the B-Raf protein. To this end, an X-ray cocrystal struc-
ture of B-RafWT + 6a was obtained. The crystal structure confirmed
the expected mode of binding for the compound based on our ini-
tial design considerations, but it also presented an unexpected sur-
prise in that the crystal lattice contained both an inhibitor-bound
B-RafWT dimer and an inhibitor-bound B-RafWT monomer (Fig. 5).
In both the monomer and the dimer, the purine ring of 6a forms
two hydrogen bonds to Cys532 in the hinge region of the kinase.
Figure 4. Scatter plot (log scales) of (A): biochemical selectivity (bS) verses p-ERK activation (pA); (B) cellular selectivity (cS) verses p-ERK activation (pA) for analogs from
Tables 2–4. The compound-type are color-coded and the A375 cellular potency are represented with either diamonds (0–55 nM) or circles (>56 nM), as shown in the legend in
panel (A).

2224
L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
Figure 5. (A) Crystal structure of B-RafWT + 6a showing DFG-in/
aC-helix-out (Type IIB) binding. The activation loop (red) contains the short AS-H1 helix that helps maintain
the a
C-helix (orange) in an inactive state. (B) Kinase domain from the crystallographic dimer in the B-RafWT + 6a structure colored as in A. The second protein molecule is
visible faintly in green. (C) Kinase domain from the crystallographic monomer of B-RafWT + 6a showing the greater number of disordered loops (gray dotted lines). (D) Overlay
of B-RafWT + 6a (colored as in A) with B-RafWT + 1 (beige; PDB 3IDP). (E) Overlay of the crystallographic monomer of B-RafWT + 6a with monomeric structure (PDB: 4WO5,
pink).
The difluorophenyl central ring sits in a hydrophobic pocket
between gatekeeper residue Thr529 and catalytic Lys483 and occu-
pies approximately the same space as the isoquinoline ring in 1.
The sulfamide nitrogen-atom, in its deprotonated form, serves as
a hydrogen bond acceptor for the backbone-NH of Asp594 from
the DFG sequence, which assumes the ‘in’-conformation.24 One of
the sulfamide oxygen atoms engages in hydrogen bond interac-
tions with the backbone-NHs of both Phe595 and Gly596. The sec-
ond sulfamide oxygen atom forms a hydrogen bond to an ordered
water molecule that in turn interacts with catalytic Lys483 and the
backbone carbonyl oxygen atom of Gly596. The dimethyl-nitrogen
portion of the sulfamide functionality is oriented ‘down’ into the
Raf-selective pocket at the base of the
aC-helix, resulting in the
shift of the C-helix relative to that observed in the DFG-out com-
a
plex from aryl amine 1 (see the overlay in Fig. 5D). The branched
sulfamide is accommodated by a slight enlargement of this pocket
resulting from a small shift in the position of the Leu505, Leu514,
and Phe595 side chains relative to structures containing inhibitors

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
2225
bearing a linear propyl sulfonamide group (see Supporting Fig. S1).
Overall, the binding mode of 6a is similar to that of 2.
sulfonamide group would result in unfavorable steric clashes
and, as a result, disfavor binding to this DFG-out/ C-helix-in, the
‘on’ state of the protein. We think that the shifting away from
DFG-out/ C-helix-in toward DFG-in/ C-helix-out conformation
imposed by the -branched sulfonamides and sulfamides on B-
Raf protein is responsible for the better overall selectivity profiles
of these inhibitors.27 In conjunction with our SAR results, these
structural analyses suggest that incorporating elements in an inhi-
a
The dimer of B-RafWT + 6a observed in the crystal lattice is very
similar to published structures of other sulfonamide-containing
a
a
inhibitors in B-Raf,25 including the presence of an
a
-helical segment
of the activation loop (termed AS-H1) that inserts between the out-
ward shifted C-helix and the rest of the N-terminal lobe (Fig. 5). As
a
a
pointed out by others, this protein conformation resembles that of
the inactive conformation of the EGFR, ErbB3, and CDK2 kinase
domains.26 A novel aspect of the B-RafWT + 6a dimer is that it is
formed by a crystallographic two-fold rotation axis (e.g., the two
halves of the dimer are identical). In other published structures an
entire dimer is contained within one asymmetric unit of the crystal.
bitor that favor the
aC-helix out-shift and are incompatible with
the DFG-in conformation would likely lead to highly selective inhi-
bitors with minimal risk associated with B-RafWT activation.
7. Summary
Whereas the shifted aC-helix in the dimer is well ordered, the corre-
sponding portion of the B-RafWT + 6a monomer is much more poorly
In the present work, we have shown that, by replacing an aryl
amine with various sulfonamides in our previous B-Raf inhibitor,
the selectivity profiles towards B-RafV600E can be significantly
altered. Along with the SAR analyses, we discussed the
B-RafV600E/B-RafWT selectivity based on both the biochemical and
the cellular readouts. Our results underscore the complex nature
of B-RafV600E inhibition and B-RafWT activation, and illustrate the
defined by the electron density. In fact, amino acids from Asn486 to
Gln496 in the
final model and the side chain of Arg509, a residue at the base of
the C-helix that makes important contacts in the B-Raf dimer, is
aC-helix and the preceding loop are missing in the
a
also largely disordered. Additional disorder is observed in the Gly-
rich (or P-loop) region of the kinase and in the activation loop
beyond the DFG sequence. Overall, despite the disordered regions,
the conformation of the monomeric, inhibitor-bound kinase domain
aligns quite closely to a recently reported monomer structure of pro-
pane-1-sulfonic acid [3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-ca-
rbonyl)-2,4-difluorophenyl]amide (PLX4720) + B-RafWT (Fig. 5E).26
This similarity includes a slightly larger displacement of the
c
value of using multiple selectivity indices (bS, S, and pA) for com-
pound evaluation and selection. From this study, we were able to
identify several a-branched sulfonamide and sulfamide inhibitors
(5h,i,m,o, 6a,d) that seem to show overall superior selectivity pro-
files relative to vemurafenib and dabrafenib as well as the original
lead (1). In addition, these compounds are also selective against
other kinases. For example, in the Ambit kinase selectivity scan
against a panel of 100 kinases, compound 6a showed selectivity
scores of S(10) = 0.02 and S(35) = 0.03 (see Support information for
detail). Thus, the hits identified in this work will serve as useful scaf-
folds and strategy for future efforts aimed toward optimization of
physicochemical properties. A recent report reached similar conclu-
a
C-helix relative to its position in the B-Raf dimer structures. Thus,
the unique structural features from 6a complexed to B-RafWT not
only confirmed the association between the C-helix shift in the
B-Raf protein and the high selectivity of the inhibitor for B-RafV600E
a
,
they also provided structural evidence for the existence of mono-
meric inhibitor–protein complexes that were postulated for the
desired inhibition pathway.
sions concerning the utility of
effective inhibitors of paradoxical RAF activation.28 The co-crystal
structure of 6a in B-RafWT demonstrated the expected DFG-in/
C-
a-branched sulfamide inhibitors as
However, the
not be the only reason for the higher selectivity and lower ERK acti-
vation observed from the -branched sulfonamides and sulfamides
aC-helix-out conformation induced by 6a may
a
helix-out binding mode, but furthermore, revealed an unexpected
mixed B-RafWT monomer/dimer packing arrangement in the crys-
tals. Both the monomer and the dimer showed inhibitor binding,
but the monomeric B-Raf showed extensive disorder in the
N-terminal lobe of the kinase that is consistent with a growing body
of evidence whereby Raf dimerization and pathway activation are
facilitated by an ordered, active kinase conformation.10b,26 The set
of selectivity indices allowed for analysis of mutant/wild-type
B-Raf selectivity with respect to both target engagement and
target activation. This in turn facilitated the advancement of
working hypothesis during the stage of inhibitor design. Finally,
X-ray crystallography studies provided structural information for
further optimization under the guidance of the selectivity indices.
In a broader sense, the concepts presented in this work may be
applicable for the design of inhibitors targeting other tumorigenic
pathways that deploy compensatory activation mechanisms.
a
since linear sulfonamides also bind to B-Raf protein in this form.
An earlier study of a n-propyl sulfonamide bound to dimeric B-
RafWT showed that in addition to the DFG-in/
a
C-helix-out confor-
mation, a second protomer with a DFG-out/
aC-helix-in (Type IIA)
conformation corresponding to the kinase state permissive to
dimerization was also present.14a Close examination of this
alternative DFG-out binding mode shows that the
the propyl group is surrounded by Ile527, Leu505, and Lys483
within 3.5–4.3 Å (Fig. 6). Therefore, an -substitution at the
a-carbon of
a
8. Experimental section
8.1. Biological assays
The protocols for the biochemical and cellular assays have been
described previously.7
8.2. Crystal structure determination
The kinase domain of wild-type B-Raf (433–726) with an N-ter-
minal His6 affinity tag was expressed in insect cells (Hi-5; Invitro-
gen) and purified as described previously.3,6 Purified protein was
concentrated to ꢀ3.5 mg/mL using an Ultrafree-0.5 concentrator
Figure 6. Alternate binding mode of a n-propyl sulfonamide inhibitor bound to B-
RafWT (chain B, PDB 3C4C) showing close contacts of the propyl group with the
protein.

2226
L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
(Millipore) in the presence of 0.2 mM compound 6a. Inhibitor con-
centration was supplemented to 0.5 mM in the concentrated pro-
tein sample prior to cocrystallization experiments. Crystals were
grown at room temperature by vapor diffusion in hanging drops
8.3.1.3. (c) N-(3-Amino-2,6-difluorophenyl)acetamide (8).
In a 150-mL round-bottomed flask under N2, N-(2,6-diflu-
oro-3-nitrophenyl)acetamide (4.10 g, 19.0 mmol) and palladium
on carbon (10%, 0.40 g) were mixed with EtOH (119 mL). The reac-
tion mixture was evacuated and refilled with hydrogen (3-times).
The mixture was stirred under a balloon pressure of hydrogen at
rt for 2 h. The mixture was filtered through a pad of diatomaceous
earth and the filtrate was concentrated. The resulting dark residue
was absorbed on a plug of silica gel and was purified on silica with
NH3–MeOH (1 M) in DCM (10–100%) to afford the title compound
(3.22 g, 17.3 mmol, 91%) as tan solid. LCMS (ESI, pos. ion) calcd for
C8H8F2N2O: 186.1; found: 187.1 (M+1). 1H NMR (400 MHz, DMSO-
d6) d ppm 9.47 (br s, 1H), 6.72–6.87 (m, 1H), 6.56–6.69 (m, 1H),
4.98 (s, 2H), 2.03 (s, 3H).
consisting of 0.8 lL of protein solution mixed with an equal vol-
ume of well solution (16% (w/v) PEG 8000, 10% tacsimate, 0.1 M
Tris, pH 8.5). Prior to data collection, crystals were transferred
sequentially into mother liquor containing 5–30% ethylene glycol
before flash-cooling in liquid nitrogen. X-ray diffraction data were
collected at beamline CMCF1-08ID of the Canadian Light Source
using a Rayonix 300 CCD detector and k = 0.9795 Å. Data were
indexed and scaled using HKL2000.29 The structure was solved
by molecular replacement using EPMR30 and the coordinates of a
proprietary B-RAF structure as a search model. Importantly, only
monomeric kinase domain search models produced a correct solu-
tion. Refinements were performed with REFMAC531 as imple-
mented in CCP432 and model building was done with COOT.33
Data collection and refinement statistics appear in Table S3.
8.3.2. Method A: General procedure for the synthesis of aryl
sulfonamides (9) from 8
To
a
stirred solution of N-(3-amino-2,6-difluorophenyl)
acetamide (8) in pyridine was added aryl sulfonyl chloride
(1.1 equiv) slowly at rt. After being stirred at rt for 1.5 h, the mix-
ture was diluted with water and acidified with aqueous HCl (4 N).
The resulting slurry was filtered and the solids were washed with
water to afford the crude acetamide intermediate. The intermedi-
ate was treated with concentrated hydrogen chloride (15 equiv)
in EtOH. The mixture was stirred at 90 °C for 14 h and was concen-
trated in vacuo. The residue was quenched with saturated sodium
bicarbonate and the resulting solid was collected via filtration and
washed with water to afford the title product.
8.3. Chemistry
Unless otherwise noted, all reagents and materials were
obtained from commercial suppliers and used without further
purification. Reactions involving air- or moisture-sensitive
reagents were performed under a nitrogen or argon atmosphere.
Silica gel chromatography was performed using either glass col-
umns packed with silica gel (200–400 mesh, Aldrich Chemical) or
prepacked silica gel cartridges (Biotage or Redisep). NMR spectra
were recorded with Bruker spectrometers. Chemical shifts are
reported in parts per million (ppm, d units). All final compounds
were purified to P95% purity unless otherwise noted as deter-
mined by LCMS (LCAP at 215 nm) obtained on an Agilent 1100 or
1200 spectrometer using one of the following methods: Method
A, a gradient of 5–95% acetonitrile in water (containing 0.1% TFA)
over 3.5 min on an Agilent SB-C18 column (50 mm ꢁ 3.0 mm,
8.3.3. N-(3-Amino-2,4-difluorophenyl)benzenesulfonamide (9a)
This material was prepared from 8 and benzenesulfonyl chloride
according to the procedure described in Method A as brown solid
(2.88 g, 94%). Compound 9a was also prepared from 11 as described
in Scheme 1. LCMS (ESI, pos. ion) calcd for C12H10F2N2O2S: 284.0;
found: 285.0 (M+1). 1H NMR (400 MHz, CDCl3) d ppm 7.77
(d, J = 7.4 Hz, 2H), 7.60–7.54 (m, 1H), 7.50–7.41 (m, 2H), 6.88
(dt, J = 5.5, 8.7 Hz, 1H), 6.81–6.72 (m, 1H), 6.47 (br s, 1H), 3.66
(br s, 2H).
2.5 lm) at 40 °C with a flow rate of 1.5 mL/min; Method B, a gradi-
ent of 5–95% acetonitrile in water (containing 0.1% TFA) over
3.5 min on a Waters XBridge C18 column (50 mm ꢁ 3.0 mm,
3 lm) at 40 °C with a flow rate of 1.5 mL/min. Low-resolution mass
spectrometry (MS) data were obtained using ES ionization mode
8.3.4. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluorop-
henyl)benzenesulfonamide (4a)
8.3.4.1. (a) N-(2,4-Difluoro-3-(3-(9-(tetrahydro-2H-pyran-2-yl)-
9H-purin-6-yl)pyridin-2-ylamino)phenyl)benzenesulfonamide.
A solution of lithium bis(trimethylsilyl)amide (1.00 M in THF,
(positive).
Compounds 214d and 315 were synthesized according to the lit-
erature described methods.
8.3.1. N-(3-Amino-2,6-difluorophenyl)acetamide (8)
1.30 mL, 1.30 mmol) was added dropwise to
N-(3-amino-2,4-difluorophenyl)benzenesulfonamide (9a, 0.150 g,
0.528 mmol) and 6-(2-fluoropyridin-3-yl)-9-(tetrahydro-2H-
a solution of
8.3.1.1. (a) N-(2,6-Difluorophenyl)acetamide.
To a solution
of 2,6-difluoroaniline (16.1 mL, 160 mmol) in DCM (213 mL) was
added acetic anhydride (15.8 mL, 170 mmol). The mixture was stir-
red at rt for 16 h. The reaction mixture was washed with saturated
sodium bicarbonate and brine. The organic phase was dried over
anhydrous sodium sulfate, and concentrated to give the title com-
pound (26.9 g, 160 mmol, 98%) as a white solid. LCMS (ESI, pos.
ion) calcd for C8H7F2NO: 171.0; found: 172.2 (M+1).
pyran-2-yl)-9H-purine (12, 0.174 g, 0.580 mmol) in THF (9.00 mL).
After being stirred at rt for 16 h, the reaction mixture was quenched
by the addition of HCl (aq, 1 N). The solution was partitioned
between ethyl acetate and saturated sodium bicarbonate. The aque-
ous phase was separated and extracted with ethyl acetate. The com-
bined organic phases were washed with brine, dried over anhydrous
sodium sulfate, filtered, and concentrated to afford an orange brown
oil. This material was purified via column chromatography on silica
gel eluting with ethyl acetate–hexane (0–50%) to afford the title
compound (0.178 g, 0.316 mmol, 60%) as tan solid. LCMS (ESI, pos.
ion) calcd for C27H23F2N7O3S: 563.2; found: 564.2 (M+1).
8.3.1.2. (b) N-(2,6-Difluoro-3-nitrophenyl)acetamide.
Nitric
acid (25.6 mL, 610 mmol) was added to sulfuric acid (53.3 mL,
760 mmol) cooled in an ice-water bath. N-(2,6-Difluorophenyl)ac-
etamide (13.0 g, 76.0 mmol) was added. The resulting mixture was
stirred at rt for 18 h. The mixture was poured into ice-water and
the precipitate was filtered. The solid residue was washed with
water and air-dried to give the title compound (11.2 g, 51.8 mmol,
68%) as a yellow solid. LCMS (ESI, pos. ion) calcd for C8H6F2N2O3:
216.0; found: 217.2 (M+1). 1H NMR (300 MHz, DMSO-d6) d ppm
10.04 (br s, 1H), 8.02–8.28 (m, 1H), 7.44 (td, J = 9.1, 1.8 Hz, 1H),
2.11 (s, 3H).
8.3.4.2. (b) N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-diflu-
orophenyl)benzenesulfonamide (4a).
A solution of N-(2,4-
difluoro-3-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)pyrid-
in-2-ylamino)phenyl)benzenesulfonamide (0.178 g, 0.316 mmol)
in trifluoroacetic acid (3.16 mL) was heated at 65 °C for 2.5 h. The
reaction mixture was partitioned between DCM and saturated

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
2227
sodium bicarbonate. The aqueous phase was extracted with addi-
tional DCM. The combined organic phases were washed with brine,
dried over anhydrous sodium sulfate, filtered, and concentrated to
afford the title compound (4a, 0.148 g, 0.309 mmol, 98%) as a tan
solid. LCMS (ESI, pos. ion) calcd for C22H15F2N7O2S: 479.1; found:
480.2 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm 11.44 (br s,
1H), 9.64 (br s, 1H), 9.00 (s, 1H), 8.71 (s, 1H), 8.15 (dd, J = 4.7,
1.9 Hz, 1H), 7.49–7.84 (m, 6H), 7.05–7.19 (m, 2H), 7.01 (dd,
J = 7.8, 4.7 Hz, 1H), 4.38 (br s, 1H).
through the solution for 1 h. The mixture was stirred at the same
temperature for 1 h before the cooling bath was removed. The
mixture was poured into ice-water and the organic solvent was
evaporated. The aqueous phase was treated with NaOH (1 N) to pH
ꢀ10. The basified solution was washed with ethyl acetate. The
aqueous layer was treated with HCl (aq, 6 N) to pH ꢀ1. The acidified
solution was then extracted with ethyl acetate. The combined
extracts were washed with water, brine and dried over anhydrous
sodium sulfate. The solvent was evaporated under vacuum and the
residue was triturated with hexane to give the title compound
(6.99 g, 22.7 mmol, 56%) as a brown solid. LCMS (ESI, pos. ion) calcd
for C15H11F2NO4: 307.1; found: 330.0 (M+Na).
8.3.4.3.
N-(3-Amino-2,4-difluorophenyl)-2-fluorobenzenesul-
This material was from 8 and 2-fluoroben-
fonamide (9b).
zene-1-sulfonyl chloride prepared according to the procedure
described in Method A (0.280 g, 61% over two steps). LCMS (ESI,
pos. ion) calcd for C12H9F3N2O2S: 302.0; found: 303. (M+1). 1H
NMR (400 MHz, DMSO-d6) d ppm 10.17 (s, 1H), 7.59–7.79 (m,
2H), 7.39–7.52 (m, 1H), 7.27–7.37 (m, 1H), 6.73–6.86 (m, 1H),
6.33 (dt, J = 5.4, 8.6 Hz, 1H), 5.24 (s, 2H).
8.3.6.3. (c) Benzyl 3-amino-2,4-difluorophenylcarbamate (13).
To a suspension of 3-(benzyloxycarbonylamino)-2,6-difluo-
robenzoic acid (1.67 g, 5.44 mmol) in tert-butanol (27.2 mL,
5.44 mmol) was added triethylamine (1.06 mL, 7.61 mmol). The
mixture was stirred for 5 min before diphenyl phosphorazide
(1.52 mL, 7.07 mmol) was added. The resulting mixture was stirred
at 85 °C overnight. The solvent was evaporated and the residue was
dissolved in DCM and cooled to 0 °C. To this cooled solution was
added TFA (2.09 mL, 27.2 mmol) and the mixture was stirred at
rt for 2.5 h. Additional TFA (3.0 mL) was added and the mixture
was stirred at rt overnight. The mixture was diluted with DCM
and washed with saturated sodium bicarbonate and brine. The
organic phase was dried over anhydrous sodium sulfate, filtered
and concentrated. The crude material was absorbed onto a plug
of silica gel and purified by chromatography with EtOAc in hexane
(0–30%) to afford the title compound (13, 0.602 g, 2.17 mmol, 40%)
as a yellow solid. LCMS (ESI, pos. ion) calcd for C14H12F2N2O2:
278.1; found: 279.2 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm
9.21 (br s, 1H), 7.39–7.47 (m, 4H), 7.28–7.38 (m, 1H), 6.84 (ddd,
J = 1.9, 9.0, 10.6 Hz, 1H), 6.60–6.76 (m, 1H), 5.13 (s, 2H).
8.3.5. N-(3-((3-(9H-Purin-6-yl)pyridin-2-yl)amino)-2,4-difluoro-
phenyl)-2-fluorobenzenesulfonamide (4b)
This compound was prepared from 6-(2-fluoropyridin-3-yl)-9-
(tetrahydro-2H-pyran-2-yl)-9H-purine (12, 0.213 g, 0.713 mmol)
and crude N-(3-amino-2,4-difluorophenyl)-2-fluorobenzenesulfon-
amide (9b, 0.280 g, 0.926 mmol) using sodium bis(trimethylsilyl)
amide (2.85 mL, 2.85 mmol) according to the procedure described
for THP-4a. The crude intermediate N-(2,4-difluoro-3-(3-(9-
(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)pyridin-2-ylamino)ph-
enyl)-2-fluorobenzenesulfonamide (0.500 g, 121%) was dissolved in
THF (4 mL) and was treated with HCl (aq, 6 N, 0.594 mL, 3.56 mmol)
at rt for 16 h. The reaction mixture was diluted with ethyl acetate
and quenched with saturated sodium bicarbonate solution. The
aqueous phase was extracted with ethyl acetate. The combined
organic phases were washed with brine, dried over anhydrous
sodium sulfate, filtered, and concentrated. The crude material was
purified by chromatography on silica eluting with NH3–MeOH
(1 M) in DCM (1–5%) to provide the title compound (4b, 0.082 g,
0.165 mmol, 23%) as yellow solid. LCMS (ESI, pos. ion) calcd for
8.3.7. 2,6-Difluoro-N1-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-6-yl)pyridin-2-yl)benzene-1,3-diamine (14)
8.3.7.1. (a) Benzyl 2,4-difluoro-3-(3-(9-(tetrahydro-2H-pyran-2-
yl)-9H-purin-6-yl)pyridin-2-ylamino)phenylcarbamate.
To
C
22H14F3N7O2S: 497.1; found: 498.0 (M+1). 1H NMR (400 MHz,
a stirred solution of 6-(2-fluoropyridin-3-yl)-9-(tetrahydro-2H-
pyran-2-yl)-9H-purine (12, 2.78 g, 9.29 mmol) and benzyl 3-
amino-2,4-difluorophenylcarbamate (13, 2.84 g, 10.2 mmol) in
THF (50 mL) was added sodium bis(trimethylsilyl)amide (1 M in
THF, 27.9 mL, 27.9 mmol) at 0 °C dropwise. The dark red mixture
was stirred at 0 °C for 30 min. The reaction mixture was parti-
tioned between ethyl acetate and a buffer solution (pH ꢀ5). The
aqueous phase was extracted with ethyl acetate. The combined
organic phases were washed with brine, dried over anhydrous
sodium sulfate, filtered, and concentrated. The crude material
was absorbed onto a plug of silica gel and purified by chromatog-
raphy eluting with EtOAc in hexane (20–60%) to provide the title
compound (4.10 g, 7.35 mmol, 79%) as yellow solid.
DMSO-d6) d ppm 13.86 (br s, 1H), 11.48 (br s, 1H), 10.46 (br s, 1H),
9.66 (d, J = 5.2 Hz, 1H), 9.01 (s, 1H), 8.72 (s, 1H), 8.14 (dd, J = 1.8,
4.7 Hz, 1H), 7.67–7.81 (m, 2H), 7.46 (t, J = 9.4 Hz, 1H), 7.36
(t, J = 7.7 Hz, 1H), 7.14 (d, J = 7.8 Hz, 2H), 7.02 (dd, J = 4.7, 7.9 Hz, 1H).
8.3.6. Benzyl 3-amino-2,4-difluorophenylcarbamate (13)
8.3.6.1. (a) Benzyl 2,4-difluorophenylcarbamate.
To a 250-
mL round-bottomed flask was added 2,4-difluoroaniline (11,
4.62 mL, 45.4 mmol), DCM (103 mL) and pyridine (7.40 mL,
91 mmol). The solution was stirred at rt and was treated with benzyl
chloroformate (8.15 mL, 54.5 mmol) drop wise via an addition fun-
nel. After being stirred at rt overnight, the mixture was poured into
water and was extracted with DCM. The combined extracts were
washed with water, brine, dried with anhydrous sodium sulfate, fil-
tered, and concentrated to give the title compound (11.1 g,
42.2 mmol, 93%) as a white solid which was used without further
purification. LCMS (ESI, pos. ion) calcd for C14H11F2NO2: 263.1; found:
286.1 (M+Na). 1H NMR (400 MHz, DMSO-d6) d ppm 9.43 (br s, 1H),
7.51–7.66 (m, 1H), 7.24–7.46 (m, 6H), 7.01–7.12 (m, 1H), 5.15 (s, 2H).
8.3.7.2. (b) 2,6-Difluoro-N1-(3-(9-(tetrahydro-2H-pyran-2-yl)-
9H-purin-6-yl)pyridin-2-yl)benzene-1,3-diamine (14).
To a
round-bottomed flask with benzyl 2,4-difluoro-3-(3-(9-(tetrahy-
dro-2H-pyran-2-yl)-9H-purin-6-yl)pyridin-2-ylamino)phenylcar-
bamate (0.630 g, 1.13 mmol) was added palladium on carbon (10%,
0.120 g), methanol (10 mL) and ethyl acetate (10 mL). The resulting
mixture was evacuated-back filled with hydrogen and was stirred
at rt under a hydrogen balloon overnight. The solid was filtered and
the solution was concentrated. The crude material was absorbed
onto a plug of silica gel and purified by chromatography eluting
with NH3–MeOH (1 M) in DCM (0–3%) to provide the title com-
pound (14, 0.274 g, 0.647 mmol, 57%) as yellow solid. LCMS (ESI,
pos. ion) calcd for C21H19F2N7O: 423.1; found: 424.2 (M+1). 1H
NMR (400 MHz, DMSO-d6) d 11.31 (s, 1H), 9.59 (dd, J = 1.9,
8.3.6.2. (b) 3-(Benzyloxycarbonylamino)-2,6-difluorobenzoic
acid.
To a 250-mL round-bottomed flask under N2 was added
benzyl 2,4-difluorophenylcarbamate (10.7 g, 40.8 mmol) and
tetrahydrofuran (100 mL). The solution was cooled at ꢂ78 °C and
was treated with n-butyllithium solution (2.5 M in hexanes,
32.6 mL, 82 mmol) via an addition funnel. The resulting pink
solution was stirred at ꢂ78 °C for 0.5 h. Dry CO2 was bubbled

2228
L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
7.8 Hz, 1H), 9.07 (s, 1H), 8.94 (s, 1H), 8.20 (dd, J = 1.9, 4.7 Hz, 1H),
6.97 (dd, J = 4.7, 7.8 Hz, 1H), 6.83 (dt, J = 1.7, 9.3 Hz, 1H), 6.61 (dt,
J = 5.5, 9.3 Hz, 1H), 5.86 (dd, J = 2.1, 11.0 Hz, 1H), 4.96 (s, 2H),
3.98–4.11 (m, 1H), 3.67–3.81 (m, 1H), 2.26–2.42 (m, 1H), 1.92–
2.10 (m, 2H), 1.70–1.84 (m, 1H), 1.50–1.68 (m, 2H).
ion) calcd for C21H14F2N8O2S: 480.1; found: 481.0 (M+1). 1H NMR
(400 MHz, DMSO-d6) d ppm 11.45 (br s, 1H), 9.64 (br s, 1H), 9.00
(s, 1H), 8.87 (d, J = 1.8 Hz, 1H), 8.81 (dd, J = 1.4, 4.7 Hz, 1H), 8.72
(s, 1H), 8.15 (dd, J = 1.9, 4.7 Hz, 1H), 8.11 (td, J = 2.0, 8.1 Hz, 1H),
7.62 (dd, J = 4.6, 7.7 Hz, 1H), 7.07–7.20 (m, 2H), 7.02 (dd, J = 4.7,
7.9 Hz, 1H).
8.3.8. Method B: General procedure for the synthesis of sulfon-
amides (4 and 5) from aniline 14 and sulfonyl chlorides
8.3.13. N-(3-((3-(9H-Purin-6-yl)pyridin-2-yl)amino)-2,4-difluo-
rophenyl)pyridine-2-sulfonamide (4f)
To a solution of 2,6-difluoro-N1-(3-(9-(tetrahydro-2H-pyran-2-
yl)-9H-purin-6-yl)pyridin-2-yl)benzene-1,3-diamine (14, 0.150 g,
0.354 mmol) in pyridine (5.0 mL) was added 4-(dimethylamino)
pyridine (0.022 g, 0.177 mmol) and aryl or alkyl sulfonyl chloride
(0.531 mmol). The resulting mixture was heated at 80 °C under
N2 for 2 h (LCMS was taken to monitor the progress of the reac-
tion). Longer reaction times or more sulfonyl chlorides were neces-
sary in some cases for complete consumption of the aniline. Upon
completion of the reaction, the mixture was allowed to cool to rt
and was poured into a mixture of EtOAc and ice water. The aque-
ous layer was extracted with EtOAc and the combined organic
phases were dried over anhydrous sodium sulfate and concen-
trated in vacuo. The crude product was purified by column chro-
matography to obtain the THP-protected intermediate. Cleavage
of the THP was accomplished either with 10% TFA in DCM at rt
or with HCl (5 N in THF, 4 equiv) at rt for 2 h. The mixture was neu-
tralized with saturated sodium bicarbonate and was extracted
with CHCl3. The organic phase was dried over anhydrous sodium
sulfate and concentrated in vacuo. The crude product was purified
by column chromatography to afford the final product.
This compound was prepared from 14 and pyridine-2-sulfonyl
chloride according to the procedure described in Method B as a
light-yellow powder (25.0 mg, 0.0520 mmol, 22%). LCMS (ESI,
pos. ion) calcd for C21H14F2N8O2S: 480.1; found: 481.0 (M+1). 1H
NMR (400 MHz, DMSO-d6) d ppm 12.08 (s, 1H), 9.79 (dd, J = 1.8,
7.7 Hz, 1H), 8.59 (s, 1H), 8.52 (d, J = 4.50 Hz, 1H), 8.07 (s, 1H),
8.01 (dd, J = 1.8, 4.6 Hz, 1H), 7.79–7.90 (m, 2H), 7.30–7.41 (m,
1H), 7.09 (dt, J = 6.1, 9.1 Hz, 1H), 6.82 (dd, J = 4.7, 7.6 Hz, 1H),
6.61 (t, J = 8.8 Hz, 1H).
8.3.14. N-(3-Amino-2,4-difluorophenyl)thiophene-2-sulfonamide
(9g)
This material was prepared from 8 and thiophene-2-sulfonyl
chloride according to the procedure described in Method A as yel-
low solid (0.330 g, 1.14 mmol, 97%). LCMS (ESI, pos. ion) calcd for
C
10H8F2N2O2S2: 290.0; found: 291.0 (M+1). 1H NMR (400 MHz,
DMSO-d6) d ppm 10.03 (s, 1H), 7.93 (dd, J = 1.4, 5.0 Hz, 1H), 7.47
(dd, J = 1.4, 3.7 Hz, 1H), 7.15 (dd, J = 3.7, 5.0 Hz, 1H), 6.83 (ddd,
J = 2.0, 8.90, 10.7 Hz, 1H), 6.35 (dt, J = 5.5, 8.6 Hz, 1H), 5.24 (s, 2H).
8.3.9. N-(3-((3-(9H-Purin-6-yl)pyridin-2-yl)amino)-2,4-difluo-
rophenyl)-3-fluorobenzenesulfonamide (4c)
This compound was prepared in a one-pot variation from 14
and 3-fluorobenzene-1-sulfonyl chloride according to the proce-
8.3.15. N-(3-((3-(9H-Purin-6-yl)pyridin-2-yl)amino)-2,4-difluo-
rophenyl)thiophene-2-sulfonamide (4g)18a
This compound was prepared from 9g and 12 according to the
sequence described for 4b as an amorphous solid (0.180 g,
0.371 mmol, 43%). LCMS (ESI, pos. ion) calcd for C20H13F2N7O2S2:
485.1; found: 486.0 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm
13.90 (br s, 1H), 11.57 (s, 1H), 10.39 (br s, 1H), 9.74 (d, J = 7.9 Hz,
1H), 9.09 (s, 1H), 8.79 (s, 1H), 8.25 (dd, J = 1.9, 4.7 Hz, 1H), 8.03
(dd, J = 1.4, 5.0 Hz, 1H), 7.59 (dd, J = 1.4, 3.7 Hz, 1H), 7.16–7.29
(m, 3H), 7.09 (dd, J = 4.7, 7.9 Hz, 1H).
dure described in Method
B as a white powder (13.0 mg,
0.0262 mmol, 19%). LCMS (ESI, pos. ion) calcd for C22H14F3N7O2S:
497.1; found: 498.0 (M+1). 1H NMR (500 MHz, DMSO-d6) d ppm
11.46 (d, J = 11.9 Hz, 1H), 9.65 (br s, 1H), 8.99 (s, 1H), 8.70 (s,
1H), 8.14 (dd, J = 1.9, 4.6 Hz, 1H), 7.61–7.69 (m, 1H), 7.59 (d,
J = 7.8 Hz, 1H), 7.50–7.56 (m, 2H), 7.09–7.17 (m, 2H), 7.01 (dd,
J = 4.8, 7.8 Hz, 1H).
8.3.16. N-(2,4-Difluoro-3-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-6-yl)pyridin-2-ylamino)phenyl)thiazole-2-sulfonamide
(4h)
8.3.10. N-(3-Amino-2,4-difluorophenyl)-4-fluorobenzenesulfon-
amide (9d)
This material was prepared from 8 and 4-fluorobenzene-1-sul-
fonyl chloride prepared according to the procedure described in
Method A as yellow solid (0.38 g, 88% over two steps). LCMS (ESI,
pos. ion) calcd for C12H9F3N2O2S2: 302.0; found: 303.0 (M+1). 1H
NMR (400 MHz, DMSO-d6) d ppm 9.86 (s, 1H), 9.81–9.91 (m, 1H),
7.60–7.74 (m, 2H), 7.60–7.74 (m, 2H), 7.60–7.74 (m, 2H), 7.30–
7.40 (m, 2H), 6.74 (ddd, J = 10.6, 8.9, 1.9 Hz, 1H), 6.24 (td, J = 8.6,
5.5 Hz, 1H), 5.17 (s, 2H).
This compound was prepared from 14 and thiazole-2-sulfonyl
chloride according to the procedure described in Method B as an
amorphous solid (0.062 g, 0.13 mmol, 69%). LCMS (ESI, pos. ion)
calcd for C19H12F2N8O2S2: 486.1; found: 486.8. (M+1). 1H NMR
(300 MHz, DMSO-d6) d ppm 13.85 (br s, 1H), 11.52 (s, 1H), 10.89
(s, 1H), 9.67 (dd, J = 7.8, 1.5 Hz, 1H), 9.02 (s, 1H), 8.72 (s, 1H),
8.14–8.24 (m, 2H), 8.11 (d, J = 3.1 Hz, 1H), 7.11–7.26 (m, 2H),
7.02 (dd, J = 7.9, 4.7 Hz, 1H).
8.3.11. N-(3-((3-(9H-Purin-6-yl)pyridin-2-yl)amino)-2,4-difluo-
rophenyl)-4-fluorobenzenesulfonamide (4d)18d
8.3.17. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)-1H-pyrazole-4-sulfonamide hydrochloride (4i)
This compound was prepared from 14 and 1H-pyrazole-4-sul-
fonyl chloride according to the procedure described in Method B as
an amorphous solid (40 mg, 0.079 mmol, 22%). LCMS (ESI, pos. ion)
calcd for C19H13F2N9O2S: 469.1; found: 470.0. (M+1). 1H NMR
(400 MHz, DMSO-d6) d ppm 11.50 (br s, 1H), 9.92 (s, 1H), 9.64 (d,
J = 7.2 Hz, 1H), 9.03 (s, 1H), 8.73 (s, 1H), 8.20 (dd, J = 1.9, 4.7 Hz,
1H), 7.98 (br s, 2H), 7.10–7.26 (m, 2H), 7.04 (dd, J = 4.7, 7.7 Hz, 1H).
This compound was prepared from 9d and 12 according to the
sequence described for 4b (61.0 mg, 0.123 mmol, 13%). LCMS (ESI,
pos. ion) calcd for C22H14F3N7O2S: 497.1; found: 498.0 (M+1). 1H
NMR (400 MHz, DMSO-d6) d ppm 11.44 (d, J = 6.2 Hz, 1H), 9.63
(br s, 1H), 9.00 (s, 1H), 8.71 (s, 1H), 8.15 (dd, J = 1.9, 4.8 Hz, 1H),
7.74–7.89 (m, 2H), 7.42 (t, J = 8.8 Hz, 2H), 7.07–7.14 (m, 2H), 7.02
(dd, J = 4.7, 7.8 Hz, 1H).
8.3.12. N-(3-((3-(9H-Purin-6-yl)pyridin-2-yl)amino)-2,4-difluo-
rophenyl)pyridine-3-sulfonamide (4e)18a
This compound was prepared from 14 and pyridine-3-sulfonyl
chloride according to the procedure described in Method B as a yel-
low solid (TFA salt, 37.0 mg, 0.0620 mmol, 22%). LCMS (ESI, pos.
8.3.18. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)-1-methyl-1H-pyrazole-4-sulfonamide(4j)18a
This compound was prepared from 14 and 1-methyl-1H-pyra-
zole-4-sulfonyl chloride according to the procedure described in

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
2229
Method B as an amorphous solid (60.0 mg, 0.124 mmol, 35%).
LCMS (ESI, pos. ion) calcd for C20H15F2N9O2S: 483.1; found: 484.1
(M+1). 1H NMR (400 MHz, DMSO-d6) d ppm 11.54 (br s, 1H),
9.84–10.17 (m, 1H), 9.74 (br s, 1H), 9.07 (s, 1H), 8.77 (s, 1H), 8.25
(td, J = 1.8, 3.1 Hz, 2H), 7.74 (d, J = 0.8 Hz, 1H), 7.15–7.30 (m, 2H),
7.09 (dd, J = 4.7, 7.8 Hz, 1H), 3.91 (s, 3H).
ppm 6.73–6.98 (m, 2H), 6.28 (br s, 1H), 3.83 (br s, 2H), 2.98 (d,
J = 6.6 Hz, 2H), 2.31 (dt, J = 13.4, 6.7 Hz, 1H), 1.10 (d, J = 6.7 Hz, 6H).
8.3.25. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)-2-methylpropane-1-sulfonamide (5d)18a
This compound was prepared from 9 and 10h according to the
sequence described for 4b as an amorphous solid (0.110 g,
0.239 mmol, 87%). LCMS (ESI, pos. ion) calcd for C20H19F2N7O2S:
459.1; found: 460.1 (M+1). 1H NMR (300 MHz, DMSO-d6) d ppm
11.57 (s, 1H), 9.70 (dd, J = 7.8, 1.7 Hz, 1H), 9.64 (s, 1H), 9.03 (s,
1H), 8.73 (s, 1H), 8.20 (dd, J = 4.7, 1.8 Hz, 1H), 7.29 (td, J = 8.7,
5.8 Hz, 1H), 7.13–7.22 (m, 1H), 7.04 (dd, J = 7.8, 4.8 Hz, 1H), 3.01
(d, J = 6.4 Hz, 2H), 2.19 (dt, J = 13.3, 6.6 Hz, 1H), 1.03 (d, J = 6.7 Hz,
6H).
8.3.19. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluor-
ophenyl)-3,5-dimethyl-1H-pyrazole-4-sulfonamide (4k)
This compound was prepared from 14 and 3,5-dimethyl-1H-
pyrazole-4-sulfonyl chloride according to the procedure described
in Method B (30 mg, 0.060 mmol, 20%) as a light-yellow powder.
LCMS (ESI, pos. ion) calcd for C21H17F2N9O2S: 497.1; found: 498.0
(M+1). 1H NMR (400 MHz, DMSO-d6) d ppm 13.65–14.06 (bs,
1H), 12.86 (s, 1H), 11.46 (br s, 1H), 9.50–9.95 (m, 2H), 9.02 (s,
1H), 8.72 (s, 1H), 8.19 (dd, J = 1.9, 4.7 Hz, 1H), 7.07–7.25 (m, 2H),
7.02 (dd, J = 4.7, 7.9 Hz, 1H), 2.15 (br s, 6H).
8.3.26. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)-1-cyclopropylmethanesulfonamide (5e)
This compound was prepared from 14 and cyclopropyl-
methanesulfonyl chloride according to the procedure described
in Method B as an amorphous solid (27.8 mg, 0.060 mmol, 20%).
LCMS (ESI, pos. ion) calcd for C20H17F2N7O2S: 457.1; found: 458.0
(M+1). 1H NMR (500 MHz, DMSO-d6) d ppm 9.52 (s, 1H), 9.04 (s,
1H), 8.72 (s, 1H), 8.20 (dd, J = 4.6, 1.9 Hz, 1H), 7.26 (td, J = 8.8,
5.4 Hz, 1H), 7.10–7.19 (m, 1H), 7.02–7.09 (m, 1H), 3.86–3.95 (m,
1H), 3.50 (s, 2H), 3.17 (s, 1H), 2.21–2.36 (m, 4H), 1.85–1.98 (m, 2H).
8.3.20. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4 difluo-
rophenyl)methanesulfonamide (5a)15,18a
This compound was prepared from 14 and methanesulfonyl
chloride according to the procedure described in Method B as an
amorphous solid (17 mg, 0.041 mmol, 68%). LCMS (ESI, pos. ion)
calcd for C17H13F2N7O2S: 417.1; found: 417.8 (M+1). 1H NMR
(300 MHz, MeOH-d4) d ppm 9.53 (br s, 1H), 9.00 (s, 1H), 8.52 (s,
1H), 8.15 (dd, J = 4.8, 1.8 Hz, 1H), 7.32–7.42 (m, 1H), 7.07 (td,
J = 9.2, 2.0 Hz, 1H), 6.98–7.03 (m, 1H), 3.03 (s, 3H).
8.3.27. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)-2,2-dimethylpropane-1-sulfonamide (5f)
8.3.21. N-(3-Amino-2,4-difluorophenyl)propane-2-sulfonamide
(10b)
This compound was prepared from 14 and 2,2-dimethyl-
propane-1-sulfonyl chloride according to the procedure described
in Method B as an amorphous solid (2.9 mg, 0.006 mmol, 2%).
LCMS (ESI, pos. ion) calcd for C21H21F2N7O2S: 473.1; found: 474.2
(M+1). 1H NMR (500 MHz, DMSO-d6) d ppm 11.51 (br s, 1H), 9.64
(br s, 1H), 9.03 (s, 1H), 8.71 (s, 1H), 8.19 (dd, J = 4.8, 1.9 Hz, 1H),
7.25–7.35 (m, 1H), 7.13–7.22 (m, 1H), 7.03 (dd, J = 7.8, 4.6 Hz,
1H), 3.08 (s, 2H), 1.10 (s, 9H).
This material was prepared from 8 and propane-2-sulfonyl
according to the procedure described in Method A as white solid
(0.430 g, 1.72 mmol, 57% overall). LCMS (ESI, pos. ion) calcd for
C9H12F2N2O2S: 250.1; found: 251.2 (M+1). 1H NMR (400 MHz,
DMSO-d6) d ppm 9.35 (s, 1H), 6.87 (ddd, J = 2.0, 8.9, 10.7 Hz, 1H),
6.52 (dt, J = 5.5, 8.7 Hz, 1H), 5.31 (s, 2H), 2.96–3.07 (m, 2H),
1.66–1.83 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H).
8.3.28. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)-1-phenylmethanesulfonamide (5g)
8.3.22. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)propane-1-sulfonamide (5b)18a,18d
This compound was prepared from 14 and phenylmethanesul-
fonyl chloride according to the procedure described in Method B
as an amorphous solid (130 mg, 0.263 mmol, 44%). LCMS (ESI,
pos. ion) calcd for C23H17F2N7O2S: 493.1; found: 494.0 (M+1). 1H
NMR (400 MHz, DMSO-d6) d ppm 11.56 (br s, 1H), 9.70 (br s, 2H),
9.05 (s, 1H), 8.73 (s, 1H), 8.21 (dd, J = 4.7, 1.8 Hz, 1H), 7.31–7.44
(m, 5H), 7.21 (td, J = 8.7, 5.8 Hz, 1H), 7.09–7.17 (m, 1H), 7.04 (dd,
J = 7.8, 4.7 Hz, 1H), 4.48 (s, 2H).
This compound was prepared from 10b and 12 according to the
sequence described for 4b as an amorphous solid (0.203 g,
0.456 mmol, 62%). LCMS (ESI, pos. ion) calcd for C19H17F2N7O2S:
445.1; found: 446.0 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm
13.87 (br s, 1H), 11.58 (s, 1H), 9.61–9.75 (m, 2H), 9.04 (s, 1H),
8.74 (s, 1H), 8.20 (dd, J = 1.8, 4.7 Hz, 1H), 7.29 (dt, J = 5.7, 8.7 Hz,
1H), 7.12–7.23 (m, 1H), 7.04 (dd, J = 4.7, 7.9 Hz, 1H), 3.01–3.13
(m, 2H), 1.67–1.83 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H).
8.3.29. N-(3-Amino-2,4-difluorophenyl)propane-2-sulfonamide
(10h)
8.3.23. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)-2,2,2-trifluoroethanesulfonamide (5c)18d
This material was prepared from 8 and propane-2-sulfonyl
chloride according to the procedure described in Method A as
light-yellow solid (0.760 g, 3.04 mmol, 32%). This compound was
also prepared starting from 11 as described in Scheme 1. LCMS
(ESI, pos. ion) calcd for C9H12F2N2O2S: 250.1; found: 251.0 (M+1).
1H NMR (400 MHz, CDCl3) d ppm 6.92 (dt, J = 5.5, 8.8 Hz, 1H),
6.74–6.84 (m, 1H), 6.29 (br s, 1H), 3.81 (br s, 2H), 3.27 (spt,
J = 6.9 Hz, 1H), 1.40 (d, J = 6.8 Hz, 6H).
This compound was prepared from 14 and 2,2,2-trifluo-
roethanesulfonyl chloride according to the procedure described
in Method B as an amorphous solid (65 mg, 0.13 mmol, 54%). LCMS
(ESI, pos. ion) calcd for C18H12F5N7O2S: 485.1; found: 486.0 (M+1).
1H NMR (300 MHz, DMSO-d6) d ppm 11.58 (br s, 1H), 10.33 (br s,
1H), 9.70 (d, J = 7.4 Hz, 1H), 9.03 (s, 1H), 8.73 (s, 1H), 8.20 (dd,
J = 4.7, 1.9 Hz, 1H), 7.33 (td, J = 8.7, 5.7 Hz, 1H), 7.14–7.27 (m,
1H), 7.04 (dd, J = 7.8, 4.8 Hz, 1H), 4.52 (q, J = 9.8 Hz, 2H).
8.3.30. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)propane-2-sulfonamide (5h)18a
8.3.24. N-(3-Amino-2,4-difluorophenyl)-2-methylpropane-1-sul-
fonamide (10d)
This material was prepared from 8 and isopropylsulfonyl chlo-
ride according to the procedure described in Method A as colorless
oil (0.430 g, 1.63 mmol, 61%). LCMS (ESI, pos. ion) calcd for C10H14
F2N2O2S: 264.1; found: 265.1 (M+1). 1H NMR (300 MHz, CDCl3) d
This compound was prepared from 12 and 10h according to the
sequence described for 4b as an amorphous solid (0.583 g,
1.31 mmol, 43%). LCMS (ESI, pos. ion) calcd for C19H17F2N7O2S:
445.1; found: 446.1 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm
13.86 (br s, 1H), 11.56 (br s, 1H), 9.50–9.84 (m, 2H), 9.05 (s, 1H),

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L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
8.74 (s, 1H), 8.17–8.25 (m, 1H), 7.27–7.39 (m, 1H), 7.13–7.24 (m,
1H), 7.00–7.10 (m, 1H), 3.19–3.30 (m, 1H), 1.31 (d, J = 6.8 Hz, 6H).
(m, 2H), 9.00–9.05 (m, 1H), 8.71 (s, 1H), 8.19 (dd, J = 4.7, 1.9 Hz,
1H), 7.32 (td, J = 8.9, 5.6 Hz, 1H), 7.09–7.21 (m, 1H), 6.97–7.07
(m, 1H), 4.19 (br s, 1H), 3.09 (d, J = 7.0 Hz, 2H), 1.05–1.12
(m, 1H), 0.54–0.59 (m, 2H), 0.33–0.40 (m, 2H).
8.3.31. N-(3-((3-(9H-Purin-6-yl)pyridin-2-yl)amino)-2,4-difluo-
rophenyl)butane-2-sulfonamide (5i)
This compound was prepared from 14 and butane-2-sulfonyl
chloride according to the procedure described in Method B as an
amorphous solid (46 mg, 0.10 mmol, 34%). LCMS (ESI, pos. ion)
calcd for C20H19F2N7O2S: 459.1; found: 460.2 (M+1). 1H NMR
(400 MHz, DMSO-d6) d ppm 13.31–14.01 (m, 1H), 11.39 (br s,
1H), 9.52 (br s, 2H), 8.90 (s, 1H), 8.59 (s, 1H), 8.06 (d, J = 2.4 Hz,
1H), 7.18 (d, J = 5.7 Hz, 1H), 6.98–7.10 (m, 1H), 6.91 (dd, J = 4.7,
7.4 Hz, 1H), 2.87 (br s, 1H), 1.85 (d, J = 4.7 Hz, 1H), 1.25–1.47 (m,
1H), 1.17 (d, J = 6.6 Hz, 3H), 0.82 (t, J = 7.1 Hz, 3H).
8.3.37. N-(3-Amino-2,4-difluorophenyl)cyclopentanesulfon-
amide (10m)
This material was prepared from 8 and cyclopentanesulfonyl
chloride according to the procedure described in Method A as
yellow oil (64.0 mg, 0.232 mmol, 17%). LCMS (ESI, pos. ion) calcd
for C11H14F2N2O2S: 276.1; found: 277.2 (M+1).
8.3.38. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)cyclopentanesulfonamide (5m)
This compound was prepared from 10m and 12 according to the
sequence described for 4b as an amorphous solid (59.0 mg,
0.125 mmol, 54%). LCMS (ESI, pos. ion) calcd for C21H19F2N7O2S:
471.1; found: 472.2 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm
11.44 (br s, 1H), 9.57 (br s, 2H), 8.96 (s, 1H), 8.64 (s, 1H), 8.12
(dd, J = 1.6, 4.6 Hz, 1H), 7.18–7.31 (m, 1H), 7.09 (t, J = 8.7 Hz, 1H),
6.96 (dd, J = 4.7, 7.8 Hz, 1H), 3.48 (t, J = 7.7 Hz, 1H), 1.77–1.95 (m,
4H), 1.59 (d, J = 6.6 Hz, 2H), 1.50 (d, J = 6.8 Hz, 2H).
8.3.32. N-(3-Amino-2,4-difluorophenyl)pentane-2-sulfonamide
(10j)
This material was prepared from 8 and pentane-2-sulfonyl
chloride according to the procedure described in Method A as color-
less oil (30 mg, 0.11 mmol, 10%). LCMS (ESI, pos. ion) calcd for
C
11H16F2N2O2S: 278.0; found: 279.0 (M+1). 1H NMR (400 MHz,
CDCl3) d ppm 6.92 (td, J = 8.9, 5.4 Hz, 1H), 6.74–6.85 (m, 1H), 6.25
(br s, 1H), 3.81 (br s, 2H), 3.10 (ddd, J = 9.9, 6.6, 3.4 Hz, 1H), 1.96
(ddd, J = 9.9, 6.6, 2.7 Hz, 1H), 1.59–1.66 (m, 1H), 1.45–1.54 (m, 1H),
1.38 (d, J = 6.8 Hz, 3H), 1.24–1.35 (m, 1H), 0.92 (t, J = 7.2 Hz, 3H).
8.3.39. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)tetrahydrofuran-3-sulfonamide (5n)
This compound was prepared from 14 and tetrahydrofuran-3-
sulfonyl chloride according to the procedure described in Method
B as an amorphous solid (39 mg, 0.082 mmol, 54%). LCMS (ESI,
pos. ion) calcd for C20H17F2N7O3S: 473.1; found: 474.0 (M+1). 1H
NMR (400 MHz, DMSO-d6) d ppm 11.60 (br s, 1H), 9.72 (br s, 1H),
9.09 (s, 1H), 8.78 (s, 1H), 8.26 (dd, J = 1.8, 4.7 Hz, 1H), 7.36 (dt,
J = 6.0, 8.6 Hz, 1H), 7.17–7.29 (m, 1H), 7.10 (dd, J = 4.7, 7.8 Hz,
1H), 4.02–4.09 (m, 1H), 3.95–4.02 (m, 2H), 3.83–3.94 (m, 1H),
3.67–3.82 (m, 1H), 2.16–2.34 (m, 2H).
8.3.33. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)pentane-2-sulfonamide (5j)
This compound was prepared from 10j and 12 according to the
sequence described for 4b as an amorphous solid (15 mg,
0.032 mmol, 49%). LCMS (ESI, pos. ion) calcd for C21H21F2N7O2S:
473.1; found: 474.0 (M+1). 1H NMR (300 MHz, DMSO-d6) d ppm
11. 53 (br s, 1H), 9.67 (br s, 2H), 9.03 (s, 1H), 8.72 (s, 1H), 8.19
(dd, J = 4.7, 1.9 Hz, 1H), 7.30 (td, J = 8.8, 5.8 Hz, 1H), 7.09–7.22
(m, 1H), 7.03 (dd, J = 7.9, 4.8 Hz, 1H), 2.96–3.16 (m, 1H),
1.80–1.96 (m, 1H), 1.35–1.52 (m, 2H), 1.21–1.33 (m, 4H), 0.86
(t, J = 7.2 Hz, 3H).
8.3.40. N-(3-Amino-2,4-difluorophenyl)cyclohexanesulfonamide
(10o)
This material was prepared from 8 and cyclohexanesulfonyl
chloride according to the procedure described in Method A as a
white solid (0.161 g, 0.555 mmol, 34%). LCMS (ESI, pos. ion) calcd
8.3.34. N-(3-Amino-2,4-difluorophenyl)cyclopropanesulfon-
amide (10k)
This material was prepared from 8 and cyclopropanesulfonyl
chloride according to the procedure described in Method A as
yellow solid (0.278 g, 1.15 mmol, 68%). LCMS (ESI, pos. ion) calcd
for C9H10F2N2O2S: 248.0; found: 249.2 (M+1). 1H NMR (400 MHz,
DMSO-d6) d ppm 9.33 (s, 1H), 6.87 (ddd, J = 2.0, 8.9, 10.7 Hz, 1H),
6.54 (dt, J = 5.5, 8.6 Hz, 1H), 5.31 (s, 2H), 2.52–2.67 (m, 1H),
0.90–0.97 (m, 2H), 0.81–0.89 (m, 2H).
for
C
12H16F2N2O2S: 290.1; found: 291.1 (M+1). 1H NMR
(400 MHz, DMSO-d6) d ppm 9.34 (br s, 1H), 6.78–6.94 (m, 1H),
6.53 (dt, J = 5.5, 8.7 Hz, 1H), 5.31 (s, 2H), 2.81–2.95 (m, 1H), 2.02–
2.16 (m, 2H), 1.72–1.82 (m, 2H), 1.61 (d, J = 11.8 Hz, 1H), 1.32–
1.48 (m, 2H), 1.17–1.31 (m, 2H), 1.09–1.15 (m, 1H).
8.3.41. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)cyclohexanesulfonamide (5o)
8.3.35. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)cyclopropanesulfonamide (5k)18a
This compound was prepared from 10o and 12 according to the
sequence described for 4b as an amorphous solid (0.117 g,
0.241 mmol, 53%). LCMS (ESI, pos. ion) calcd for C22H21F2N7O2S:
485.1; found: 486.2 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm
13.72–14.00 (m, 1H), 11.57 (br s, 1H), 9.52–9.85 (m, 2H), 9.03 (s,
1H), 8.73 (s, 1H), 8.20 (dd, J = 2.0, 4.8 Hz, 1H), 7.31 (dt, J = 5.7,
8.8 Hz, 1H), 7.13–7.23 (m, 1H), 7.04 (dd, J = 4.7, 7.9 Hz, 1H), 2.96
(t, J = 11.8 Hz, 1H), 2.11 (d, J = 11.6 Hz, 2H), 1.79 (d, J = 13.5 Hz,
2H), 1.55–1.70 (m, 1H), 1.34–1.49 (m, 2H), 1.18–1.32 (m, 2H),
1.13 (d, J = 11.7 Hz, 1H).
This compound was prepared from 10k and 12 according to the
sequence described for 4b as an amorphous solid (36 mg,
0.081 mmol, 16%). LCMS (ESI, pos. ion) calcd for C19H15F2N7O2S:
443.1; found: 444.2 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm
11.58 (br s, 1H), 9.66 (d, J = 6.2 Hz, 1H), 9.03 (s, 1H), 8.71 (s, 1H),
8.21 (dd, J = 1.8, 4.7 Hz, 1H), 7.31 (d, J = 6.0 Hz, 1H), 7.20 (t,
J = 9.3 Hz, 1H), 7.04 (dd, J = 4.8, 7.8 Hz, 1H), 2.65 (s, 1H), 0.94–
1.05 (m, 2H), 0.85–0.92 (m, 2H).
8.3.36. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)cyclobutanesulfonamide (5l)18a
8.3.42. N-(3-((3-(9H-Purin-6-yl)pyridin-2-yl)amino)-2,4-difluo-
rophenyl)tetrahydro-2H-pyran-4-sulfonamide (5p)
This compound was prepared from 14 and cyclobutanesulfonyl
chloride according to the procedure described in Method B as an
amorphous solid (63.2 mg, 0.138 mmol, 46%). LCMS (ESI, pos. ion)
calcd for C20H17F2N7O2S: 457.1; found: 458.0 (M+1). 1H NMR
(500 MHz, DMSO-d6) d ppm 11.44–11.54 (m, 1H), 9.57–9.67
This compound was prepared from 14 and tetrahydro-2H-
pyran-4-sulfonyl chloride according to the procedure described
in Method B as an amorphous solid (21.7 mg, 0.045 mmol, 15%).
LCMS (ESI, pos. ion) calcd for C21H19F2N7O3S: 487.1; found: 488.0
(M+1). 1H NMR (500 MHz, DMSO-d6)
d ppm 9.69 (s, 1H),

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
2231
7.15–7.19 (m, 1H), 7.01–7.08 (m, 1H), 4.08 (br s, 1H), 3.87–4.01 (m,
5H), 3.80 (s, 3H), 3.82 (s, 3H), 3.67 (d, J = 10.7 Hz, 2H), 3.39 (d,
J = 11.5 Hz, 1H), 1.93–2.04 (m, 1H), 1.64–1.76 (m, 1H).
extracted with CHCl3. The combined organic layers were dried over
MgSO4, filtered, and concentrated. The crude product was purified
by column chromatography using MeOH in DCM (3–8%) to obtain
the desired product (0.105 g, 0.235 mmol, 85%) as yellow solid.
LCMS (ESI, pos. ion) calcd for C18H16F2N8O2S: 446.1; found: 447.0
(M+1). 1H NMR (300 MHz, DMSO-d6) d ppm 11.55 (br s, 1H), 9.69
(d, J = 7.4 Hz, 1H), 9.58 (br s, 1H), 9.04 (s, 1H), 8.72 (s, 1H), 8.19
(dd, J = 4.8, 1.8 Hz, 1H), 7.32 (td, J = 8.7, 5.8 Hz, 1H), 7.11–7.22
(m, 1H), 7.03 (dd, J = 7.8, 4.8 Hz, 1H), 2.73 (s, 6H).
8.3.43. N-(3-Amino-2,4-difluorophenyl)-N,N-dimethylsulfamide
(16a)
8.3.43.1. (a) tert-Butyl 3-(N,N-dimethylsulfamoyl)-2,6-difluo-
rophenylcarbamate.
To a solution of tert-butyl 3-amino-
2,6-difluorophenylcarbamate34 (15, 0.600 g, 2.46 mmol) in pyri-
dine (10.0 mL) was added dimethylamine (1.60 mL, 3.19 mmol)
and N,N-dimethylpyridin-4-amine (0.075 g, 0.614 mmol). The
reaction mixture was cooled to ꢂ30 °C and stirred for 10 min
before sulfuryl dichloride (0.299 mL, 3.68 mmol) was added drop-
wise. After an additional 20 min, the mixture was allowed to warm
to rt. The reaction mixture was poured into EtOAc (30 mL) and vig-
orously stirred for 10 min. The EtOAc layer was separated. The
tarry residue was triturated with DCM (3 mL) and EtOAc (20 ml).
The combined organic layers were concentrated in vacuo. The
crude product was purified by column chromatography using
MeOH in DCM (3–5%) to afford the desired product (0.500 g,
1.42 mmol, 58%) as light-brown solid. LCMS (ESI, pos. ion) calcd
8.3.45. N-(3-Amino-2,4-difluorophenyl)-N-ethyl-N-methylsul-
famide (16b)
8.3.45.1. (a) N-(3-(N-Ethyl-N-methylsulfamoylamino)-2,6-diflu-
orophenyl)acetamide.
To a solution of N-(3-amino-2,6-diflu-
orophenyl)acetamide (8, 0.500 g, 2.69 mmol) in pyridine (10.0 mL)
was added N,N-dimethylpyridin-4-amine (0.082 g, 0.67 mmol) and
N-ethylmethylamine (0.300 mL, 3.49 mmol) under N2. The reaction
mixture was cooled to ꢂ30 °C and stirred for 10 min before sulfuryl
chloride (0.327 mL, 4.03 mmol) was added dropwise. After 20 min,
the mixture was allowed to warm to rt and continued to stir for
1 h. The reaction mixture was poured into with EtOAc (30 mL)
and vigorously stirred for 10 min. The EtOAc layer was separated.
The tarry residue was dissolved in DCM (3 mL) followed by addi-
tion of EtOAc (20 mL). This mixture was vigorously stirred for
10 min and the organic layer was decanted. The combined organic
layers were concentrated and the crude product was purified by
column chromatography using acetone in hexanes (10–40%) to
obtain the desired product (0.400 g, 1.30 mmol, 49%) as yellow
solid. LCMS (ESI, pos. ion) calcd for C11H15F2N3O3S: 307.1; found:
308.0 (M+1). 1H NMR (300 MHz, CDCl3) d ppm 7.45 (td, J = 8.8,
5.6 Hz, 1H), 6.96 (td, J = 9.1, 2.0 Hz, 1H), 6.75 (br s, 1H), 6.36 (br
s, 1H), 3.22 (q, J = 7.1 Hz, 2H), 2.83 (s, 3H), 2.16–2.32 (m, 3H),
1.11 (t, J = 7.09 Hz, 3H).
for
C
13H19F2N3O4S: 351.1; found: 374.0 (M+Na). 1H NMR
(300 MHz, CDCl3) d ppm 7.42 (td, J = 8.9, 5.5 Hz, 1H), 6.87–7.00
(m, 1H), 6.33 (br s, 1H), 5.97 (br s, 1H), 2.84 (s, 6H), 1.52 (s, 9H).
8.3.43.2. (b) N-(3-Amino-2,4-difluorophenyl)-N,N-dimethylsul-
famide (16a).
To a solution of tert-butyl 3-(N,N-dimethylsul-
famoyl)-2,6-difluorophenylcarbamate (0.500 g, 1.42 mmol) in
DCM (5.0 mL) was added trifluoroacetic acid (2.11 mL, 28.5 mmol).
The resulting mixture was capped and stirred at rt for 2 h. The reac-
tion mixture was neutralized using NaOH (aq, 5 N). The aqueous
layer was extracted with DCM. The combined organic layers were
dried over MgSO4, filtered, and concentrated. The crude product
was purified by column chromatography using MeOH in DCM (3–
8%) to obtain the desired product (0.240 g, 0.955 mmol, 67%) as yel-
low solid. LCMS (ESI, pos. ion) calcd for C8H11F2N3O2S: 251.2; found:
252.1 (M+1). 1H NMR (300 MHz, CDCl3) d ppm 6.84–6.94 (m, 1H),
6.75–6.84 (m, 1H), 6.28 (br s, 1H), 3.80 (br s, 2H), 2.77–2.90 (m, 6H).
8.3.45.2. (b) N-(3-Amino-2,4-difluorophenyl)-N-ethyl-N-methyl-
sulfamide (16b).
To a 20-mL microwave vial was added N-(3-
(N-ethyl-N-methylsulfamoylamino)-2,6-difluorophenyl)acetamide
(0.400 g, 1.30 mmol), concentrated HCl (1.60 mL, 19.5 mmol), and
EtOH (10.0 mL). The vial was capped and heated at 90 °C for 4 h.
The reaction mixture was concentrated and the residue was parti-
tioned between EtOAc and saturated sodium bicarbonate. The
aqueous layer was extracted with EtOAc. The combined organic
layers were dried over MgSO4, filtered, and concentrated. The
crude product was purified by column chromatography using ace-
tone in hexanes (10–30%) to afford the desired product (0.275 g,
1.04 mmol, 80%) as a white solid. LCMS (ESI, pos. ion) calcd for C9-
8.3.44. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluor-
ophenyl)-N,N-dimethylsulfamide (6a)
8.3.44.1. (a) N-(2,4-Difluoro-3-(3-(9-(tetrahydro-2H-pyran-2-yl)
-9H-purin-6-yl)pyridin-2-ylamino)-N,N-dimethylsulfamide.
To a solution of 6-(2-fluoropyridin-3-yl)-9-(tetrahydro-2H-
pyran-2-yl)-9H-purine (12, 0.120 g, 0.400 mmol) and N-(3-
amino-2,4-difluorophenyl)-N,N-dimethylsulfamide (16a, 0.121 g,
0.480 mmol) in THF (5.0 mL) was added sodium bis(trimethylsi-
lyl)amide (1.0 M in THF, 1.60 mL, 1.60 mmol) at 0 °C in one portion.
The dark-red mixture was stirred at 0 °C for 10 min and at rt for
30 min. The reaction mixture was partitioned between EtOAc and
a buffer solution (sodium citrate, pH 5). The aqueous layer was
extracted with EtOAc. The combined organic layers were dried over
MgSO4, filtered, and concentrated. The crude product was purified
by column chromatography using acetone in hexane (20–30%) to
afford the THP-protected product (0.145 g, 0.273 mmol, 68%) as a
yellow solid. LCMS (ESI, pos. ion) calcd for C23H24F2N8O3S: 530.2;
found: 531.2 (M+1).
H
13F2N3O2S: 265.1; found: 266.0 (M+1). 1H NMR (300 MHz, CDCl3)
d ppm 6.69–6.95 (m, 2H), 6.29 (br s, 1H), 3.79 (br s, 2H), 3.22 (q,
J = 7.2 Hz, 2H), 2.83 (s, 3H), 1.11 (t, J = 7.2 Hz, 3H).
8.3.46. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)-N-ethyl-N-methylsulfamide (6b)
This material was prepared in a similar procedure as described
for 6a as an amorphous solid (0.080 g, 0.17 mmol, 79%). LCMS (ESI,
pos. ion) calcd for C19H18F2N8O2S: 460.1; found: 461.0 (M+1). 1H
NMR (300 MHz, DMSO-d6)
d ppm 11.54 (s, 1H), 9.69 (d,
J = 6.7 Hz, 1H), 9.54 (br s, 1H), 9.04 (s, 1H), 8.72 (s, 1H), 8.19 (dd,
J = 4.8, 1.8 Hz, 1H), 7.30 (td, J = 8.8, 5.8 Hz, 1H), 7.11–7.22 (m,
1H), 7.03 (dd, J = 7.8, 4.8 Hz, 1H), 3.11 (q, J = 7.2 Hz, 2H), 2.73 (s,
3H), 1.03 (t, J = 7.2 Hz, 3H).
8.3.44.2. (b) N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-
difluorophenyl)-N,N-dimethylsulfamide (6a).
To a solution
of N-(2,4-difluoro-3-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-
6-yl)pyridin-2-ylamino)-N,N-dimethylsulfamide (0.145 g, 0.270
mmol) in THF (2.0 mL) was added hydrogen chloride (aq, 5 N,
1.09 mL, 5.47 mmol). The resulting mixture was stirred at rt in a
closed system for 1 h. The mixture was partitioned between a
buffer solution (pH ꢀ7) and CHCl3. The aqueous layer was
8.3.47. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)-N,N-diethylsulfamide (6c)
A pressure-resistant vial was charged with 2,6-difluoro-N1-(3-
(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)pyridin-2-yl)benzene-
1,3-diamine (14, 0.140 g, 0.33 mmol), pyridine (80 lL, 0.99 mmol),

2232
L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
DCM (3.3 mL), and diethylsulfamoyl chloride (0.80 mL,
4.65 mmol). The mixture was heated at 60 °C for 2 d. The mixture
was cooled to rt and was diluted with DCM and water. The
organic layer was concentrated and the residue was suspended
in HCl (aq, 6 N, 0.280 mL, 1.65 mmol) and THF (3.3 mL). The
resulting mixture was stirred at rt for 1 h and concentrated under
reduced pressure to afford a yellow residue. The resulting crude
product was purified by HPLC (10–70% CH3CN/H2O; 0.1% TFA) to
afford the desired product as TFA salt (0.015 g, 0.025 mmol,
7.7%). LCMS (ESI, pos. ion) calcd for C20H20F2N8O2S: 474.1; found:
475.2 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm 11.50 (br s, 1H),
9.64 (br s, 1H), 9.49 (s, 1H), 9.05 (s, 1H), 8.73 (s, 1H), 8.20 (dd,
J = 1.9, 4.7 Hz, 1H), 7.30 (d, J = 5.9 Hz, 1H), 7.18 (t, J = 9.2 Hz, 1H),
7.04 (dd, J = 4.7, 7.9 Hz, 1H), 3.18 (q, J = 7.1 Hz, 4H), 1.03
(t, J = 7.1 Hz, 6H).
0.944 mmol, 35%). LCMS (ESI, pos. ion) calcd for C11H15F2N3O2S:
291.1; found: 292.1 (M+1). 1H NMR (300 MHz, CDCl3) d ppm
6.68–6.97 (m, 2H), 6.27 (br s, 1H), 3.79 (br s, 2H), 3.13–3.27 (m,
4H), 1.49–1.63 (m, 6H).
8.3.50. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)piperidine-1-sulfonamide (6e)
This compound was prepared from 16e and 12 according to the
sequence described for 6a as an amorphous solid (95 mg,
0.20 mmol, 50%). LCMS (ESI, pos. ion) calcd for C21H20F2N8O2S:
486.1; found: 487.1 (M+1). 1H NMR (300 MHz, DMSO-d6) d ppm
11.52 (br s, 1H), 9.66 (br s, 2H), 9.03 (s, 1H), 8.72 (s, 1H), 8.18
(dd, J = 4.8, 1.8 Hz, 1H), 7.32 (td, J = 8.8, 5.8 Hz, 1H), 7.16 (td,
J = 9.3, 1.6 Hz, 1H), 7.03 (dd, J = 7.8, 4.8 Hz, 1H), 3.09 (d,
J = 5.3 Hz, 4H), 1.48 (d, J = 4.7 Hz, 6H).
8.3.48. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difluo-
rophenyl)pyrrolidine-1-sulfonamide (6d)
8.3.48.1. (a) N-(2,4-Difluoro-3-(3-(9-(tetrahydro-2H-pyran-2-
yl)-9H-purin-6-yl)pyridin-2-ylamino)phenyl)pyrrolidine-1-sul-
8.3.51. N-(2,4-Difluoro-3-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-6-yl)pyridin-2-ylamino)phenyl)-2-oxooxazolidine-3-
sulfonamide (17)
To
a
mixture of chlorosulfonyl isocyanate (0.154 mL,
fonamide.
To a solution of 2,6-difluoro-N1-(3-(9-(tetrahydro-
1.77 mmol) in DCM (5.89 mL) cooled at 0 °C under N2 was added
2-bromoethanol (0.125 mL, 1.77 mmol). After 1 h, a suspension of
2,6-difluoro-N1-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)
pyridin-2-yl)benzene-1,3-diamine (14, 0.748 g, 1.77 mmol) and
triethylamine (0.492 mL, 3.53 mmol) in DCM (6 mL) was added
dropwise. The resulting yellow suspension was stirred at 0 °C for
20 min and at rt for 1 h. The solution was cooled to 0 °C and was
quenched with HCl (aq, 10%, 3.5 mL) followed by DCM (20 mL).
The layers were separated and the organic layer was washed with
water, dried over sodium sulfate, filtered, and concentrated to
afford 17 (0.900 g, 1.57 mmol, 89%) as a yellow solid. LCMS (ESI,
pos. ion) calcd for C24H22F2N8O5S: 572.1; found: 573.2 (M+1).
2H-pyran-2-yl)-9H-purin-6-yl)pyridin-2-yl)benzene-1,3-diamine
(14, 0.150 g, 0.35 mmol) in pyridine (5.0 mL) was added
4-(dimethylamino)pyridine (0.022 g, 0.18 mmol) and pyrrolidine
(0.039 mL, 0.46 mmol). The reaction mixture was cooled to
ꢂ30 °C and was stirred for 10 min. Sulfuryl chloride (0.043 mL,
0.53 mmol) was added dropwise. After the addition, the mixture
was allowed to stir for an additional 20 min before it was stirred
at rt for 1 h. The reaction mixture was poured into EtOAc (20 ml)
and stirred for 10 min. The organic layer was separated and the
residue was dissolved into DCM (3 mL) followed by addition of
EtOAc (20 mL). The mixture was stirred for 10 min and the organic
layer was separated. The combined organic layers were
concentrated and the crude product was purified by column
chromatography using DCM/EtOAc/MeOH (70%/27%/3%) to obtain
the desired product (0.090 g, 0.16 mmol, 46%) as yellow solid. LCMS
(ESI, pos. ion) calcd for C25H26F2N8O3S: 556.2; found: 556.8 (M+1).
1H NMR (300 MHz, CDCl3) d ppm 11.58 (s, 1H), 9.66 (dd, J = 7.9,
1.8 Hz, 1H), 9.02 (s, 1H), 8.39 (s, 1H), 8.23 (dd, J = 4.7, 1.9 Hz, 1H),
7.45 (td, J = 8.8, 5.4 Hz, 1H), 6.84–7.09 (m, 2H), 6.47 (s, 1H), 5.90
(dd, J = 10.2, 2.5 Hz, 1H), 4.16–4.32 (m, 1H), 3.84 (td, J = 11.7,
2.6 Hz, 1H), 3.20–3.45 (m, 4H), 2.06–2.27 (m, 3H), 1.67–1.95
(m, 7H).
8.3.52. (3S)-N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-difl-
uorophenyl)-3-hydroxypyrrolidine-1-sulfonamide (6f)
8.3.52.1. (a) (3S)-N-(2,4-Difluoro-3-(3-(9-(tetrahydro-2H-pyran-
2-yl)-9H-purin-6-yl)pyridin-2-ylamino)phenyl)-3-hydroxypyrr-
olidine-1-sulfonamide.
3-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)pyridin-2-yla-
mino)phenyl)-2-oxooxazolidine-3-sulfonamide (17, 0.340 g,
To a suspension of N-(2,4-difluoro-
0.590 mmol) in acetonitrile (1.2 mL) was added 4Å molecular
sieves and (S)-pyrrolidin-3-ol (0.480 mL, 5.94 mmol) followed by
triethylamine (0.858 mL, 5.94 mmol). After being stirred at
110 °C overnight, the reaction mixture was allowed to cool to rt
and was diluted with DCM. The suspension was filtered, and the
residue was rinsed with MeOH in DCM (10%). The combined fil-
trates were concentrated and purified on silica gel with MeOH in
DCM (2%) to afford the product (0.035 g, 0.061 mmol, 10%). LCMS
(ESI, pos. ion) calcd for C25H26F2N8O4S: 572.2; found: 573.2 (M+1).
8.3.48.2. (b) N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-
difluorophenyl)pyrrolidine-1-sulfonamide (6d).
To a solu-
tion of N-(2,4-difluoro-3-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-6-yl)pyridin-2-ylamino)phenyl)pyrrolidine-1-sulfonamide
(0.080 g, 0.14 mmol) in THF (2.0 mL) was added HCl (aq, 5 N,
0.575 mL, 2.87 mmol). The resulting mixture was stirred at rt for
2 h. The mixture was partitioned between a buffer solution
(KH2PO4–Na2HPO4, pH 7) and CHCl3. The aqueous layer was
extracted with CHCl3. The combined organic layers were dried over
MgSO4, filtered, and concentrated. The crude product was purified
by column chromatography using MeOH in DCM (3–6%) to afford
the 6d (0.0550 g, 0.116 mmol, 81%) as a yellow solid. LCMS (ESI,
pos. ion) calcd for C20H18F2N8O2S: 472.1; found: 473.0 (M+1). 1H
NMR (300 MHz, DMSO-d6) d ppm 11.52 (br s, 1H), 9.65 (br s, 2H),
9.04 (s, 1H), 8.73 (s, 1H), 8.18 (dd, J = 4.7, 1.9 Hz, 1H), 7.35 (td,
J = 8.8, 5.7 Hz, 1H), 7.17 (td, J = 9.2, 1.6 Hz, 1H), 7.04 (dd, J = 7.8,
4.8 Hz, 1H), 3.09–3.26 (m, 4H), 1.64–1.90 (m, 4H).
8.3.52.2. (b) (3S)-N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,
4-difluorophenyl)-3-hydroxypyrrolidine-1-sulfonamide hydro -
chloride (6f).
(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)pyridin-2-ylamino)
phenyl)-3-hydroxypyrrolidine-1-sulfonamide (0.030 g, 0.052
To a suspension of (3S)-N-(2,4-difluoro-3-(3-
mmol) in THF (0.21 mL) was added HCl (aq, 6 N, 0.044 mL,
0.26 mmol). After being stirred at rt for 2 h, the mixture was concen-
trated to dryness and the residue was triturated with acetonitrile.
The solids were collected and was dried under high vacuum to afford
(3S)-N-(3-(3-(9H-purin-6-yl)pyridin-2-ylamino)-2,4-difluorophen-
yl)-3-hydroxypyrrolidine-1-sulfonamide hydrochloride as an amor-
phous solid (6f, 15 mg, 0.029 mmol, 54%). LCMS (ESI, pos. ion) calcd
for C20H18F2N8O3S: 488.1; found: 489.2 (M+1). 1H NMR (400 MHz,
DMSO-d6) d ppm 11.53 (s, 1H), 9.65 (d, J = 6.2 Hz, 1H), 9.52 (s, 1H),
9.04 (s, 1H), 8.73 (s, 1H), 8.21 (dd, J = 1.9, 4.8 Hz, 1H), 7.36 (dt,
J = 5.7, 8.8 Hz, 1H), 7.10–7.23 (m, 1H), 7.05 (dd, J = 4.7, 7.9 Hz, 1H),
8.3.49. N-(3-Amino-2,4-difluorophenyl)piperidine-1-sulfonamide
(16e)
This compound was prepared from 8 and piperidine according
to the sequence described for 16b as a white solid (0.275 g,

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234
2233
4.24–4.37 (m, 1H), 3.23–3.39 (m, 3H), 3.06 (dd, J = 2.2, 9.9 Hz, 1H),
1.85–2.00 (m, 1H), 1.68–1.81 (m, 1H).
(300 MHz, DMSO-d6) d ppm 11.61 (br s, 1H), 9.96 (br s, 1H), 9.69
(d, J = 7.2 Hz, 1H), 9.04 (s, 1H), 8.73 (s, 2H), 8.22 (dd, J = 4.8,
1.8 Hz, 1H), 7.33 (td, J = 8.7, 5.8 Hz, 1H), 7.13–7.25 (m, 1H), 7.05
(dd, J = 7.8, 4.8 Hz, 1H), 3.34 (br s, 4H, overlapping with water),
3.17 (br s, 4H).
8.3.53. (3R)-N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-
difluorophenyl)-3-hydroxypyrrolidine-1-sulfonamide (6g)
This compound was prepared from (R)-pyrrolidin-3-ol accord-
ing to the procedure described for 6f as an amorphous solid (HCl
salt, 15 mg, 0.029 mmol, 63%). LCMS (ESI, pos. ion) calcd for
Abbreviations
C
20H18F2N8O3S: 488.1; found: 489.2 (M+1). 1H NMR (400 MHz,
MAPK, mitogen activated protein kinases; ERK, extracellular
signal-regulated kinases; bS, biochemical selectivity; cS, cellular
selectivity; pA, percent of ERK activation as measured by the
formation of phospho ERK (p-ERK); SAR, structure–activity
relationship; ATP, adenosine-50-triphosphate. Abbreviations for
chemical reagents and experimental descriptions are adopted from
the convention of the Journal of Organic Chemistry.
DMSO-d6) d ppm 11.57 (br s, 1H), 9.70 (d, J = 7.4 Hz, 1H), 9.57 (s,
1H), 9.09 (s, 1H), 8.78 (s, 1H), 8.26 (dd, J = 1.9, 4.7 Hz, 1H), 7.41
(dt, J = 5.7, 8.7 Hz, 1H), 7.21 (dt, J = 1.5, 9.2 Hz, 1H), 7.09 (dd,
J = 4.7, 7.8 Hz, 1H), 4.29–4.41 (m, 1H), 3.28–3.45 (m, 3H), 3.11
(dd, J = 2.1, 10.0 Hz, 1H), 1.91–2.04 (m, 1H), 1.73–1.87 (m, 1H).
8.3.54. N-(3-Amino-2,4-difluorophenyl)morpholine-4-
sulfonamide (16h)
8.3.54.1. (a) tert-Butyl 2,6-difluoro-3-(morpholine-4-sulfon-
Acknowledgements
amido)phenylcarbamate.
To a solution of tert-butyl 3-
We thank Yunxin Bo, Debbie Choquette, Fang-Tsao Hong,
Roxanne Kunz, Markian Stec, Seifu Tadesse, Nuria Tamayo, and
Yong-Jae Kim for providing synthetic intermediates and for
contributing to studies (unpublished) that extended the scope of
the current series; Gina Ranieri, Richard Walter, and the staff of
Shamrock Structures LLC for performing X-ray data collection. We
also acknowledge Randy Hungate, Rick Kendall, and Philip Tagari
for supporting this project and Margaret Chu-Moyer for providing
helpful suggestions regarding the preparation of this manuscript.
amino-2,6-difluorophenylcarbamate (15, 0.600 g, 2.457 mmol) in
pyridine (10.0 mL) was added 4-(dimethylamino)-pyridine
(0.075 g, 0.614 mmol) and morpholine (0.278 mL, 3.19 mmol).
The reaction mixture was cooled to ꢂ30 °C and sulfuryl chloride
(0.299 mL, 3.68 mmol) was added dropwise. After the addition,
the mixture was allowed to stir for an additional 20 min and then
at rt for 2 h. The mixture was poured into cold EtOAc (30 mL) and
was vigorously stirred for 10 min. The organic layer was decanted
and the dark residue was dissolved in DCM (3 mL) followed by
dilution with EtOAc (20 mL). The combined organic layers were
concentrated in vacuo and the crude product was purified by col-
umn chromatography using NH3–MeOH (2 N) in DCM (3–5%) to
give the desired product as light-brown solid (730 mg, 1.85 mmol,
76%). LCMS (ESI, pos. ion) calcd for C15H21F2N3O5S: 393.1; found:
416.0 (M+Na).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.03.055.
References and notes
8.3.54.2. (b) N-(3-Amino-2,4-difluorophenyl)morpholine-4-sul-
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fonamide (16h).
To a 100-mL round-bottomed flask, tert-
butyl 2,6-difluoro-3-(morpholine-4-sulfonamido)phenylcarba-
mate (0.496 g, 1.261 mmol) and TFA (5.0 mL, 64.9 mmol) were
mixed in DCM (5 mL). The dark mixture was stirred at rt for
30 min and was neutralized with sodium bicarbonate to pH ꢀ7.
The mixture was extracted with DCM and 10% IPA/CHCl3. The com-
bined organic phases were dried over sodium sulfate, filtered, and
concentrated. The crude product was purified on silica with EtOAc
in hexanes (10 –80%) to afford 16h as light-brown film (150 mg,
0.511 mmol, 41%). LCMS (ESI, pos. ion) calcd for C10H13F2N3O3S:
293.1; found: 294.0 (M+1). 1H NMR (400 MHz, CDCl3) d ppm
6.75–6.94 (m, 2H), 6.28 (br s, 1H), 3.82 (br s, 2H), 3.62–3.73 (m,
4H), 3.17–3.29 (m, 4H).
8.3.55. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-
difluorophenyl)morpholine-4-sulfonamide (6h)
This compound was prepared from 16h and 12 according to the
procedure described for 6a as an amorphous solid (47 mg,
0.097 mmol, 20%). LCMS (ESI, pos. ion) calcd for C20H18F2N8O3S:
488.1; found: 489.1 (M+1). 1H NMR (400 MHz, DMSO-d6) d ppm
11.56 (br s, 1H), 9.68 (br s, 2H), 9.04 (s, 1H), 8.73 (s, 1H), 8.21 (d,
J = 3.5 Hz, 1H), 7.13–7.42 (m, 2H), 7.04 (dd, J = 7.4, 4.9 Hz, 1H),
3.60 (br s, 4H), 3.10 (br s, 4H).
8.3.56. N-(3-(3-(9H-Purin-6-yl)pyridin-2-ylamino)-2,4-
difluorophenyl)piperazine-1-sulfonamide (6i)
This compound was prepared from tert-butyl piperazine-1-car-
boxylate and 14 according to the procedure described for 6d as an
amorphous solid (68 mg, 0.139 mmol, 30%). LCMS (ESI, pos. ion)
calcd for C20H19F2N9O2S: 487.1; found: 488.1 (M+1). 1H NMR

2234
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