K. Zimmermann et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 2
versus bulkier and more polar, positively charged Arg in JAK1
and TYK2).
Kinase and cell potency data for C-7 amides 11–14
Compd R0R00N
R
JAK2 JAK1 JAK3 TYK2 SET-2a A549b
(nM) (nM) (nM) (nM) (nM) (nM)
Introduction of a pyridine ring as replacement for the phenyl C-
ring gave compounds with low selectivity versus JAK1 (e.g., com-
pound 20). However, the N-methyl-indazole C-ring replacement
was well tolerated when combined with the preferred C-7 sub-
stituent. Compound 21 has excellent kinase selectivity and good
antiproliferative activity against JAK2-dependent SET-2 cells.
Metabolic stability of 21 is comparable to 14. (75% and 59%
remaining compound after 10 min incubation with human and
rat liver microsomes resp.). While the solubility of 21 at acidic
O
O
O
N
N
N
11
12
13
H
7.4
5.7
31
49
67
246
193
311
70
171
201
170
210
110
2640
3-OMe
3080
3-F-4-OMe 5.5
3-OMe 3.5
10,000
N
14
158
225
243
80
3840
N
a
SET-2 cell proliferation IC50 (JAK2 dependent cell line).
A549 cell proliferation IC50 (JAK2 independent cell line, used as counter-screen
b
pH was improved compared to the initial screening hit 1
for non-selective cytotoxic compounds).
(0.044 mg/ml at pH 1.0), its solubility at neutral pH and permeabil-
ity were too low to achieve sufficient oral exposure and were infe-
rior to 14 (<0.001 mg/ml at pH 6.5, PAMPA = 37 nm/s).
Compounds 14 and 21 were further tested in the AMBIT
KinomeScan panel of 380 kinases to assess kinase selectivity.
Compounds 14 and 21 bind to <6% and <3% of kinases in the panel,
respectively.14
O
NH2
H
N
3
7
O
R
N
R"
R'
11-14
In summary, we herein report hit optimization of carbazole 1
leading to the identification of novel potent JAK2 inhibitors (14
and 21), with greater than 45-fold selectivity over other JAK family
members (JAK1, JAK3, TYK2). These analogs also displayed excel-
lent cellular potency against JAK2-dependent SET-2 cells (80 and
127 nM; respectively). In addition to having the desired kinase
selectivity profile, compound 14 also provided improved pharma-
ceutics properties over 21. Further studies towards improving the
ADME properties of this series of JAK2 inhibitors will be published
in due course (Fig. 4).
The syntheses of compounds 1–9 was carried out as per
Scheme 1.9 Para-amino-benzoic amides 22a and 22b undergo
SNAr reaction with 5-bromo-2-fluoro-benzonitrile 23 to give
biphenylamines 24a,b. Standard Suzuki conditions were used to
install the ring C 25. Cyclization to the carbazole and hydrolysis
of the CN group to a primary amide was achieved upon heating
25 with palladium acetate in acetic acid. These harsh conditions
also resulted in partial cleavage of the amide, leading to isolation
of acid 26 as a byproduct, which was utilized to access amide 7
Figure 3. Structures of compounds 11–14.
(S)-dimethylamino-pyrrolidine amide, 14, demonstrated greater
than 45-fold selectivity against other members of the JAK kinase
family. The compound also displayed good cell potency (SET-2
IC50 = 80 nM) and cellular selectivity.13 Compound 14 was further
evaluated for its in-vitro ADME properties. The solubility of 14 is
significantly higher than that of compound 1. (>1.9 mg/mL at pH
1.0; 0.41 mg/mL at pH 6.5; compared to <0.001 mg/mL across pH
range for 1). The permeability of 14 is modest (PAMPA = 292
compared to >600 for e.g., 2, 4, 11, 13), as is metabolic stability
(79% and 46% remaining compound after 10 min incubation with
human and rat liver microsomes resp.) (Fig. 3).
We further explored 7-amino-carbazoles (Table 3). The ‘reverse
amide’ 15 displayed poor JAK family selectivity. Several other
amides of the 7-amino-carbazole (not shown) displayed a similar
trend.
Tertiary amines (e.g., 16) exhibited a significant improvement
in the selectivity versus JAK3, however showed modest selectivity
versus JAK1 and TYK2 and also reduced cellular potency. The intro-
duction of a basic amine into the C7-substituent (e.g., 17–19) fur-
ther improved selectivity over JAK1 and TYK2. The effects on JAK
family selectivity observed for various substituents at C7 are con-
sistent with residue differences in its vicinity (Gln853 in JAK2
O
NH2
R
H
N
R"
N
R'
15-21
Figure 4. Structures of compounds 15–21.
Table 3
Kinase and cell potency data for C-7 amines 15–21
Compd
R0R00N
R
JAK2 (nM)
10.2
JAK1 (nM)
77
JAK3 (nM)
59
TYK2 (nM)
87
SET-2a (nM)
100
A549b (nM)
1310
H
O
N
15
O
O
O
N
16
17
18
7.4
2.1
2.9
218
141
195
990
264
197
158
71
500
370
470
10,000
3510
N
N
Cl
Cl
N
N
N
N
160
2730
O
19
20
5.2
6.1
432
100
242
442
197
117
260
120
930
N
N
O
N
2000
N
N
N
21
5.5
339
324
247
130
1410
N
a
SET-2 cell proliferation IC50 (JAK2 dependent cell line).
A549 cell proliferation IC50 (JAK2 independent cell line, used as counter-screen for non-selective cytotoxic compounds).
b