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

Bioorganic & Medicinal Chemistry 24 (2016) 2215–2234

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.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

^aDepartment of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA

^bDepartment of Molecular Structure and Characterization, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA

^cDepartment of Molecular Structure and Characterization, Amgen Inc., 360 Binney St., Cambridge, MA 02142, USA

^dDepartment of Medicinal Chemistry, Amgen Inc., 360 Binney St., Cambridge, MA 02142, USA

^eDepartment 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-Raf^V600Ewith 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

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-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^WTbiochemical (^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-Raf^V600Eselectivity.

Wild-type

Sulfonamide

1. Introduction

the B-Raf protein (B-Raf^V600E), accounts for 90% of the B-Raf muta-

tions.²Relative to wild type B-Raf (B-Raf^WT), the B-Raf^V600Eprotein

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.¹Additionally, 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).³Thus, B-Raf^V600Erepre-

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.⁵Our

own work toward this objective resulted in the identiﬁcation of

aminoisoquinoline 1 as a potent and selective B-Raf inhibitor

(Fig. 1).⁶The 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

2216

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234

Table 1

H

N

Comparison of the activity and selectivity of compounds 1, 2, and 3^\

H

N

Compd B-Raf^WT

no.

B-Raf^{V600E b}S^*MIA PaCa-2 A375

^cS^{^}

^pA^à(%)

413

Me

H

N

F

IC₅₀(nM) IC₅₀(nM)

EC₅₀(nM)

IC₅₀(nM)

Cl

1

2

3

2

20

0.4

2

4

1

5

22

2290

2

17

1

11

134 674

529 755

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-Raf^WTIC₅₀/B-Raf^V600EIC₅₀

.

^

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

MIA PaCa-2 cellular assay relative to control.

H₂N

N

F

N

H

N

F

cal to be employed in the early stages of SAR exploration.⁷Concep-

tually, a desirable B-Raf^V600Einhibitor would be a compound that

O

S

N

F

S

N

F

H

O

Me

exhibits low binding afﬁnity for the B-Raf^WTprotein (high IC₅₀

)

HN

F

S

Me

R

relative to its afﬁnity for the target B-Raf^V600E. Such a compound

must also, besides potently inhibiting the down-stream ERK phos-

phorylation in the A375 cells that harbor the B-Raf^V600Eprotein,

demonstrate minimal or reduced potency (high EC₅₀) in eliciting

down-stream ERK phosphorylation in the MIA PaCa-2 cells

harboring the B-Raf^WTprotein. 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

IC₅₀values derived from the kinase assays using B-Raf^WTand

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-2.⁶Compound 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).⁷Further 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-Raf^V600Eprotein, it also activated the signal-

ing pathway in the human pancreatic carcinoma MIA PaCa-2 cells

which carry the B-Raf^WTprotein (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.⁸Concurrent to our ﬁndings, similar results with other

second-generation B-Raf inhibitors appeared in the literature that

also suggestedthat inhibitionofB-Raf^WTmayresult 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-Raf^WT-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,10The dimerization is required for

normal Ras-dependent Raf activation but not for the function of

mutants with high catalytic activity, such as Raf^V600E. Further, a

dimer-interface peptide inhibitor can block both the Raf

dimerization and down-stream signaling.¹¹Based on this theory, it

follows that inhibitors that disfavor dimerization of the B-Raf^WTꢂin-

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-Raf^V600Eassociated cancers. Indeed, a recent study reported the

design of PLX-7904/PB04 with increased selectivity for B-Raf^V600E

over B-Raf^WTand impaired hyper-activation of normal MAPK

signal.¹²

B-Raf^V600Eenzymes (IC / IC

);¹³(2) the cellular selectivity

WT

V600E

50

(^cS), as measured by the ratio of the EC₅₀value derived from the

MIA PaCa-2 cellular assay and the IC₅₀value 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-Raf^V600Ecellular assay

(i.e., same A375 IC₅₀values) 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 EC_maxvalues are not included but are partly reﬂected

in the estimated EC₅₀values.

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-Raf^V600Epathway (hence anti-tumor

activity) and the activation of the B-Raf^WTpathway (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-Raf^WTcells. 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-Raf^WTpathway 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-Raf^V600Eselectivity. 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 EC₅₀may 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 EC₅₀verses IC₅₀(dark-blue and dark-red curves). Compound X appears to be less

selective based on its lower EC₅₀value (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-Raf^WT

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.¹⁹The 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.²⁰The ﬁnal couplings between anilines 9–10

3. Design principle

At the time of our investigation, little was known about how to

rationally engineer B-Raf^V600Eselectivity 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-Raf^V600E, as

exempliﬁed by the approval of vemurafenib (2)¹⁴and dabrafenib

(3).¹⁵A 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-Raf^WTcells. 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.¹⁶We

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-Raf^V600Epathway (i.e.,

high ^bS in biochemical assays and high ^cS in cellular assays) and

have minimal activation via the B-Raf^WTpathway (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 2^14dshowed 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.¹⁷The aminoquinoline moiety in 1 forms

and

6-(2-ﬂuoropyridin-3-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-

purine 12⁶were achieved by nucleophilic aromatic substitution

(S_NAr) 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 13.²⁰S_NAr 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). S_NAr 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,²¹which 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-Raf^V600Eprotein 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.^14aIt has been hypothesized

2218

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234

Figure 3. (A) Crystal structure of B-Raf^V600E+ 1 showing DFG-out,

and dashed lines indicate hydrogen bonds. (B) Crystal structure of B-Raf^V600E+ 2 showing DFG-in,

(C) Overlay of 1 and 2 based on protein superposition; protein is not depicted for clarity. (D) Overlay of B-Raf^V600E+ 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-Raf^V600E+ 2 (gray

aC-helix is orange, the activation loop is red,

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-Raf^V600Ebiochemical (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-Raf^V600Eactivity and selectivity, aryl sulfon-

amides 4a–k were evaluated in comparison with aryl amine 1

(Table 2).²²In the biochemical assays, phenyl sulfonamide 4a

showed IC₅₀values of 17 and 7 nM toward B-Raf^WTand B-Raf^V600E

,

respectively. Compared to 1, the larger drop of B-Raf^WTpotency

(ꢀ8-fold) compared to B-Raf^V600Epotency (ꢀ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

F

N

THPN

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

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

H₂N

F

j, c

12

d, h, or k

N

HN

NH₂

F

N

THPN

N

NHCbz

13

14

R (4, aryl):

Me

N

∗

F ∗

∗

S

∗

S

N

Me

N

H

N

F

N

Me

H

a

b

c

d

e

f

g

h

i

j

k

R (5, alkyl):

Me

∗

Me

∗

CF₃

∗

Me

a

b

c

d

e

f

g

h

∗

Me ∗

∗

Me

O

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

O

R

F

H₂N

F

S

NR¹R²

12, f, g

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

N

HN

NH₂

O

F

h

S

i, g

N

THPN

N

HN

N

6f,g

O

N

H

F

N

THPN

14

N

17

NR¹R²:

∗

Me

∗

OH

∗

N

Me

N

Me

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

F

H

N

F

O

HN

S

Aryl

O

Compd no.

Aryl

B-Raf^WTIC₅₀(nM)

2

B-Raf^V600EIC₅₀(nM)

2

^bS^*

1

MIA PaCa-2 EC₅₀(nM)

22

A375 IC₅₀(nM)

2

^cS^{^}

11

^pA^à(%)

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

3

1510

5650

15

101

23

1470

600

∗

83

36

242

4e

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

4g

4h

∗

1

783

31

N

∗

N

4i

1

0.2

8

5

807

2220

—

6

135

3

1210

1170

—

N

H

∗

4j

19

—

2

710

—

N

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-Raf^WTIC₅₀/B-Raf^V600EIC₅₀

.

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-Raf^V600Ebiochemical 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

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-Raf^WTIC₅₀and the MIA PaCa-2 EC₅₀values than

the cellular shifts seen in the B-Raf^V600Eassays. 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 EC₅₀values 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 IC₅₀values 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

F

H

N

F

O

HN

S

Alkyl

O

Compd no.

Alkyl

B-Raf^WTIC₅₀(nM)

412

B-Raf^V600EIC₅₀(nM)

360

^bS^*

1

MIA PaCa-2 EC₅₀(nM)

6690

A375 IC₅₀(nM)

356

^cS^{^}

19

^pA^à(%)

∗

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

1

1450

2140

202

17

33

63

85

65

3

1100

1070

500

∗

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

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-Raf^WTIC₅₀/B-Raf^V600EIC₅₀

.

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 IC₅₀values in the B-Raf^V600Eassays 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-Raf^WTactivation. 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-Raf^V600Ecellular 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-Raf^WTpathway (^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-Raf^V600Einhibi-

tors over the wild-type protein B-Raf^WTsince 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-Raf^V600Einhi-

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

F

H

N

F

O

HN

S

NR₂

O

Compd no.

NR₂

B-Raf^WTIC₅₀(nM)

90

B-Raf^V600EIC₅₀(nM)

8

^bS^*

11

MIA PaCa-2 EC₅₀(nM)

1780

A375 IC₅₀(nM)

54

^cS^{^}

33

^pA^à(%)

389

∗

Me

N

6a

Me

∗

N

Me

6b

6c

157

165

22

57

7

3

1780

9910

87

20

16

262

600

N

603

Me

∗

N

6d

6e

31

38

2

16

3

2070

1220

44

65

47

19

401

220

15

∗

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

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-Raf^WTIC₅₀/B-Raf^V600EIC₅₀

.

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-Raf^WTpathway 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-Raf^V600Especiﬁc. 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 Raf^WT-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-Raf^WT+ 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-Raf^WTdimer and an inhibitor-bound B-Raf^WTmonomer (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-Raf^WT+ 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-Raf^WT+ 6a structure colored as in A. The second protein molecule is

visible faintly in green. (C) Kinase domain from the crystallographic monomer of B-Raf^WT+ 6a showing the greater number of disordered loops (gray dotted lines). (D) Overlay

of B-Raf^WT+ 6a (colored as in A) with B-Raf^WT+ 1 (beige; PDB 3IDP). (E) Overlay of the crystallographic monomer of B-Raf^WT+ 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.²⁴One 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.²⁷In conjunction with our SAR results, these

structural analyses suggest that incorporating elements in an inhi-

a

The dimer of B-Raf^WT+ 6a observed in the crystal lattice is very

similar to published structures of other sulfonamide-containing

a

inhibitors in B-Raf,²⁵including 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

pointed out by others, this protein conformation resembles that of

the inactive conformation of the EGFR, ErbB3, and CDK2 kinase

domains.²⁶A novel aspect of the B-Raf^WT+ 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-Raf^WTactivation.

7. Summary

Whereas the shifted aC-helix in the dimer is well ordered, the corre-

sponding portion of the B-Raf^WT+ 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-Raf^V600Ecan be signiﬁcantly

altered. Along with the SAR analyses, we discussed the

B-Raf^V600E/B-Raf^WTselectivity based on both the biochemical and

the cellular readouts. Our results underscore the complex nature

of B-Raf^V600Einhibition and B-Raf^WTactivation, 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-Raf^WT(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-Raf^WTnot

only conﬁrmed the association between the C-helix shift in the

B-Raf protein and the high selectivity of the inhibitor for B-Raf^V600E

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.²⁸The co-crystal

structure of 6a in B-Raf^WTdemonstrated 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-Raf^WTmonomer/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,26The 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-

Raf^WTshowed 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.^14aClose 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 His₆afﬁnity tag was expressed in insect cells (Hi-5; Invitro-

gen) and puriﬁed as described previously.^3,6Puriﬁed 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-

Raf^WT(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 HKL2000.²⁹The structure was solved

by molecular replacement using EPMR³⁰and 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 REFMAC5³¹as imple-

mented in CCP4³²and 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 2^14dand 3¹⁵were 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 N₂was 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 CO₂was 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

N₂for 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),

2230

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 N₂was 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

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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

L. Liu et al. / Bioorg. Med. Chem. 24 (2016) 2215–2234

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10

lM

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1

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Article Doi

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

DOI: 10.1016/j.bmc.2016.03.055

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