Phenylaminopyrimidines as inhibitors of Janus kinases (JAKs)

Article abstract of DOI:10.1016/j.bmcl.2009.08.071

A series of phenylaminopyrimidines has been identified as inhibitors of Janus kinases (JAKs). Development of this initial series led to the potent JAK2/JAK1 inhibitor CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide).

Full text of DOI:10.1016/j.bmcl.2009.08.071

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5887–5892
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phenylaminopyrimidines as inhibitors of Janus kinases (JAKs)
a
a
b
Christopher J. Burns a, David G. Bourke a, , Laura Andrau , Xianyong Bu , Susan A. Charman ,
Andrew C. Donohue a, Emmanuelle Fantino a, Michelle Farrugia a, John T. Feutrill a, Max Joffe a,
Marcel R. Kling a, Margarita Kurek a, Tracy L. Nero a, Thao Nguyen a, James T. Palmer a, Ian Phillips a,
David M. Shackleford b, Harrison Sikanyika a, Michelle Styles a, Stephen Su a, Herbert Treutlein a,
Jun Zeng a, Andrew F. Wilks a
*
a Cytopia Research Pty Ltd, 576 Swan St, Richmond, VIC 3121, Australia
b Centre for Drug Candidate Optimisation, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Pde, Parkville, VIC 3052, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phenylaminopyrimidines has been identified as inhibitors of Janus kinases (JAKs). Develop-
ment of this initial series led to the potent JAK2/JAK1 inhibitor CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-
morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide). Details of synthesis and SAR studies of these
compounds are reported.
Received 10 June 2009
Revised 19 August 2009
Accepted 20 August 2009
Available online 23 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
JAK2
Kinase
Inhibitor
Janus
CYT387
Myeloproliferative
The Janus kinases (JAKs), consisting of JAK1, JAK2, JAK3, and
TYK2, are an important family of cytoplasmic tyrosine kinases as
a consequence of their essential role in cytokine signal transduc-
tion.1 Association of individual JAK proteins to activated cytokine
receptors leads to autophosphorylation and subsequent phosphor-
ylation of specific STAT (Signal Transducer and Activation of Tran-
scription) proteins. The phosphorylated STAT proteins then
dimerize and translocate to the cell nucleus, which leads to DNA
transcription. Overactivation of JAK-STAT signaling, through genet-
ic mutations or increased localized concentration of cytokines, has
been identified in various inflammatory diseases and in a variety of
cancers.2 The discovery that a constitutively activating mutation in
JAK2 (V617F) is central to the pathogenesis of myeloproliferative
disorders (MPDs), including Polycythemia Vera3 (PV), has acceler-
ated the search for JAK2 inhibitors for the treatment of these and
other diseases.4
arylpyrimidin-2-amine class. Table 1 illustrates examples of this
chemotype with corresponding biochemical and cellular potency.
Table 1 indicates that compounds possessing an H-bond donor
in the para-position showed a greater potency against JAK2. A
model (Fig. 1) of the most active inhibitor (compound 12) in the
ATP binding site of the JAK2 enzyme, shows that the 2-amino NH
and pyrimidine N1 of the compound most likely form H-bond
interactions with the hinge region of JAK2 (Leu932). In the mod-
eled binding mode, the para-hydroxy substituent of the 4-phenyl
ring is located in a pocket formed by Lys857 to Ser862 of the
Gly-rich loop, Gly993 and Asp994 of the DFG motif and residue
Asn981. There are a number of potential interaction sites within
7 Å of the 4-phenyl para-position, including Asp994, which is
within H-bond distance of the para-hydroxy group. The modeled
binding mode of compound 12 suggested that larger 4-phenyl
para-substituents would be accommodated within this pocket,
under the Gly-rich loop, and allow exploration of additional inter-
action sites. Accommodation of larger para-substituents in this
binding pocket is consistent with the data presented in Table 1
(compounds 5, 7, and 8). However, the decreased inhibition of
JAK2 for these compared to compound 12 indicated an opportunity
for further optimization of this substituent.
Screening of Cytopia’s internal kinase-focused compound li-
brary against isolated JAK2 enzyme and a JAK2 dependent engi-
neered cell line (Baf3TEL-JAK2)5,6 led to the identification of
several sub-micromolar hits of the N-(4-morpholinophenyl)-4-
* Corresponding author. Tel.: +61 3 9208 4243; fax: +61 3 9208 4299.
E-mail addresses: cytopia@cytopia.com.au, bourkda213@hotmail.com (D.G.
Bourke).
As phenols are known to be rapidly glucuronidated in vivo, the
isosteric replacement of phenol with sulfonamide led us to explore
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.071

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C. J. Burns et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5887–5892
Table 1
SAR about the 4-aryl substituent
H
N
R
N
N
N
O
Compd
R
JAK2 IC50 (nM)
533
Baf3TEL-JAK2 IC50 (nM)
>20,000
N
1
2
3
467
326
ND
O
5559
H2N
4
322
202
88
83
81
67
33
16
3
8518
H2N
O
O
5
5220
3525
ND
Figure 1. Model of compound 12 in the ATP binding site of JAK2.7
N
H
HO
6
selectivity, Table 2 ranks compounds by decreasing inhibition of
the Baf3TEL-JAK3 cell line. Comparative data is included for CP-
690,550, a JAK inhibitor currently in clinical trials for transplant
rejection.12 Both 3- and 4-substituted anilines are tolerated and
appear to afford a similar degree of selectivity over JAK3. While
biochemical selectivity and potency values for the sulfonamides
were promising as compared to compound 12, they were not as
profound in measures of corresponding cellular selectivity, sug-
gesting possible non-JAK3-related activity in the Baf3TEL-JAK3
and CTLL-2-driven assays.
O
H2N
S
7
O
N
8
3730
3110
7134
5624
380
N
H
N
9
O
Despite slightly reduced cellular selectivity we selected com-
pound 21 for further investigation. Preliminary pharmacokinetic
profiling of compound 21 in male Sprague Dawley rats (21 mg/
H
N
10
11
12
kg) indicated a low Cmax (0.27 lM) and a low absolute oral bio-
H2N
O
availability of 5.4%. The total blood clearance after IV administra-
tion was determined to be 36.6 mL/min/kg. As this value is only
66% of the nominal value for hepatic blood flow in the rat (i.e.,
55.2 mL/min/kg),13 it is unlikely that hepatic first pass elimination
is the only contributor to the low oral bioavailability. Another po-
tential factor limiting the oral bioavailability of compound 21 may
be poor absorption (vide infra).
HO
O
ND—not determined.
As compound 21 displayed low oral bioavailability we chose to
investigate other phenyl substitutions. From our initial screening
hit panel, carboxamides attached to the 4-phenyl ring (Table 1,
compounds 3, 5, and 9) also were active. In combination with the
carboxamide as an H-bond donor, we chose a nitrile group as a
lipophilic probe with the potential to act as an H-bond acceptor.
A series of compounds with this functionality allowed us to inves-
tigate the same binding pocket that the phenol of compound 12
and the sulfonamide of compound 21 occupy. We accessed this
series in an analogous manner to that shown in Scheme 1.5 Table
3 illustrates the progression of compounds from this series. Rever-
sal of the carboxamide orientation (compound 25) or removal of
the carbonyl itself (compound 26) did not significantly change
binding versus the meta-substituted example (compound 24).
The preferred regiochemical substitution placed the cyanometh-
ylamide in the para-position (27 and 28). Substitution ortho to
the cyanomethylamide of compound 28 (compounds 29 and 30)
or methylation on this group (compounds 31 and 32) reduced
potency toward JAK2. Finally we modified the pyrimidine ring at
C5 to explore the effect of this substitution, as there appears to
be available space in the JAK2 ATP binding site (Fig. 1). The
a series of N-phenylmethanesulfonamide analogues (Table 2). We
prepared this series8 via a two-step procedure (Scheme 1); first,
a regioselective Suzuki reaction9, followed by aniline displacement
under acidic conditions10 (Scheme 1, path b) or a palladium cata-
lyzed Buchwald reaction11 (Scheme 1, path c). The route taken
for each example was dependent on the basicity of the substituent
on the aniline used; anilines with weakly basic groups were pref-
erably condensed with the 2-chloropyrimidine under acidic condi-
tions. The modeled binding mode of compound 12 (Fig. 1)
suggested that substitution in the 3- or 4-position of the N-phenyl
ring would be tolerated and solvent exposed; consequently, we
investigated substitutions at both positions.
We assessed the sulfonamide series for JAK2 inhibition and also
for selectivity against the homologous JAK3 enzyme, as JAK3 inhi-
bition has the functional consequence of immune supression.2
Table 2 indicates biochemical potency for the most potent mem-
bers of this series, along with inhibition of proliferation of both a
JAK3 dependent cell line (Baf3TEL-JAK3) and a JAK1/JAK3 depen-
dent cell line (IL-2 stimulated CTLL-2).5 As a measure of functional

C. J. Burns et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5887–5892
5889
Table 2
SAR of the N-phenylmethanesulfonamide series
H
O
N
S
O
H
N
N
R
N
Compd
R
JAK2 IC50 (nM)
JAK3 IC50 (nM)
Baf3TEL-JAK2 IC50 (nM)
Baf3TEL-JAK3 IC50 (nM)
CTLL-2 IC50 (nM)
CP-690,550
12
NA
NA
33
3
5
62
1574
380
570
2553
132
<160
N
13
14
15
6
110
538
930*
234
580
812
240
569
11
8
730
735
N
N
1730
2663
O
O
N
N
16
17
18
12
9
636
771
896
892
1944
1944
2196
6764
1669
5607
OH
OH
>1000*
1619
16
N
N
19
20
14
18
960*
941
3143
3433
5177
1552
N
N
>1000*
1002
N
N
O
S
21
22
2
4
587
351
802
396
3557
3945
2220
489
O
N
N
O
23
6
149
933
6551
1487
OH
*
Data is from a 3-point screen (5, 1, and 0.2
l
M).
H
N
O
5-methylpyrimidine (compound 33) retained JAK2 potency but
showed lower functional selectivity, relative to compound 28,
measured in the CTLL-2 assay. In contrast, the 5-methoxypyrimi-
dine (compound 34) reduced potency toward JAK2 by 10-fold,
indicating a size limit for substitution at this position.
H
O
O
N
O
S
S
a
+
Cl
N
Cl
N
Cl
OH
B
N
N
N
OH
H2N
O
Further modification of compound 28 allowed us to investigate
the effect of replacement of the morpholinoaniline moiety. Table 4
shows analogues of compound 28 wherein some compounds bear-
ing a more basic amine group (compounds 36 and 37) are 10-fold
less potent against JAK2. This is in contrast to data from the earlier
series (Table 2, compound 17) where the different electronics of
the sulfonamide series may be a contributing factor. Attachment
of electron-withdrawing groups ortho to the morpholine group
(compounds 35 and 38) also cost biochemical or JAK2-driven cel-
lular potency. Likewise, analogues of compound 28 with other N-
phenyl substituent’s did not afford an increase in JAK2 biochemical
potency. Therefore, we considered the profile of compound 28 to
be sufficiently optimized to proceed into pharmacokinetic studies.
We first assessed the permeability of compounds 28 and 21
across Caco-2 cell monolayers, as a model of human intestinal per-
meability (Table 5).14 Compound 21 displayed directional non-uni-
form permeability across the monolayer, suggestive of efflux. This
finding may in part explain the measured absolute oral bioavail-
ability for compound 21 of only 5.4%. In contrast, compound 28
H2N
S
O
c
N
b
OH
OH
H
N
O
H
O
O
N
O
S
S
H
N
H
O
N
N
N
N
S
O
N
N
N
23
21
Scheme 1. (a) Toluene/n-PrOH, Pd(PPh3)4, 2 M aq Na2CO3, 100 °C (61%); (b)
TsOHÁH2O, dioxane, reflux (38%); (c) DME, Pd2(dba)3, K3PO4, (o-biphenyl)P(t-Bu)2,
100 °C (42%).
displayed similar apparent permeability values in both directions,
indicating the compound was not a substrate for efflux. The phar-
macokinetic properties of compound 28, following both IV and oral

5890
C. J. Burns et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5887–5892
Table 3
SAR of the nitrile carboxamide series
H
R1
N
N
N
R2
N
O
Compd
R1
R2
H
JAK2 IC50 (nM)
545
JAK3 IC50 (nM)
>5000*
Baf3TEL-JAK2 IC50 (nM)
>20,000
Baf3TEL-JAK3 IC50 (nM)
>20,000
CTLL-2 IC50 (nM)
>20,000
H
N
N
24
O
O
N
N
25
26
27
H
H
H
303
132
10
1760
>1000*
66
10,347
9824
720
>20,000
>20,000
4205
18,891
8031
915
N
H
H
N
H
N
N
N
N
O
O
O
28
29
H
H
18
65
155
798
2425
7567
1249
2043
N
H
>1000*
2631
N
H
O
N
H
30
31
32
33
34
H
30
9
205
275
245
ND
N
O
O
H
549
298
8
3910
1418
85
3725
6340
433
4250
N
H
N
N
N
N
O
O
O
H
16,880
5959
4170
240
N
Me
OMe
N
H
186
2310
8505
>20,000
6160
N
H
*
Data is from a 3-point screen (5, 1 and 0.2
lM); ND—not determined.
Table 4
SAR of the N-cyanomethylbenzamide series
O
N
H
N
H
N
N
R
N
Compd
R
JAK2 IC50 (nM)
374
JAK3 IC50 (nM)
Baf3TEL-JAK2 IC50 (nM)
1237
Baf3TEL-JAK3 IC50 (nM)
1709
CTLL-2 IC50 (nM)
3993
F
F
F
35
2020
N
O
N
36
37
166
163
362
840
1105
9505
3675
5070
O
O
1450
2805
N

C. J. Burns et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5887–5892
5891
Table 4 (continued)
Compd
R
JAK2 IC50 (nM)
116
JAK3 IC50 (nM)
274
Baf3TEL-JAK2 IC50 (nM)
1143
Baf3TEL-JAK3 IC50 (nM)
3280
CTLL-2 IC50 (nM)
F
38
1265
3770
1510
7895
1852
1832
1249
N
O
O
N
N
39
40
41
42
43
28
113
69
52
47
41
18
1876
364
364
152
192
155
2565
2205
2045
423
7450
6245
9975
1230
2050
2425
S
O
S
O
N
N
N
525
N
N
N
O
798
Table 5
Apparent permeability (Papp) values
Table 6
Compound 28 (CYT387) inhibition of V617F mutant JAK2 enzyme and associated
cellular proliferation
Compound
Papp A À B (Â106 cm/s)
Papp B À A (Â106 cm/s)
Ratio
O
Mannitol
Propranolol
21
0.7
38.7
4.6
1.1
48.8
56.4
27.3
1.5
1.3
12.3
0.9
N
H
H
N
N
N
28
30.4
N
N
O
Enzyme or cell
IC50 (nM)
administration to male Sprague Dawley rats (2.5 and 18 mg/kg,
respectively) were assessed. After oral dosing, compound 28 exhib-
ited high plasma concentrations (Cmax = 40.4 lM; Tmax = 4 h), with
JAK2V617F (JH1-JH2)
BaF3 wt (+IL-3)
SET-2
11
1430
232
quantitative absolute oral bioavailability and an apparent half life
of 2.4 h. The high oral bioavailability, can likely be partly ascribed
to the low blood clearance of compound 28 (6.3 mL/min/kg) and
therefore low susceptibility to hepatic first pass metabolism.
Kinase profiling demonstrated, that in addition to its JAK2 activ-
ity, compound 28 is also an inhibitor of JAK1 (IC50 = 11 nM). Potent
inhibition of both JAK1 and JAK2 is a common feature of other JAK2
inhibitors currently in clinical development.15 Compound 28 dis-
played little activity against a broader panel of other kinases with
only 2 of >150 kinases showing an IC50 < 100 nM.15b Furthermore,
we investigated inhibition of the clinically relevant mutant JAK2
enzyme (V617F) in biochemical assays and cellular screens (Table
6). Compound 28 was equipotent on the mutant JAK2 enzyme and
inhibited proliferation of cell lines dependant on wild type JAK2
(BaF3 wt) or JAK2V617F (SET-2 and HEL 92.1.7) but did not affect
a cell line lacking such a dependence (K562).
Compound 28 (CYT387) possesses excellent pharmaceutical
and pharmacokinetic properties. In addition CYT387 induced a
profound reversal of symptoms in a murine model of MPDs involv-
ing transplantation of JAK2V617F-transduced bone marrow16 and
inhibited the growth of erythropoietin-independent erythroid col-
onies from PV patients.15a The data obtained from these and other
preclinical studies supports progression of CYT387 into the clinic
HEL 92.1.7
K562
1804
>20,000
for the treatment of diseases associated with aberrant JAK activity,
such as MPDs.
References and notes
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3. Tefferi, A. Leukemia Lymphoma 2008, 49, 388.
4. Pardanani, A. Leukemia 2008, 22, 23.
5. Enzyme screening was performed using GST-tagged kinase domain in an
AlphaScreenTM assay with [ATP] = 80
l
M. Cell-based proliferation assays were
run over 72 h with Alamar Blue staining. Dose response measurements were
performed using 1:2 serial dilutions 8-point from 20 M for cell-based assays
and 8, 12 or 16-point from 5 M for enzyme assays. All enzyme and cell data
l
l
are n = 1 or average of duplicates or average of independent n = 2, except for
CP-690,550 and compound 28 where n > 20. Acceptable variances for
duplicates or n > 1 are values within one dilution point. For a description of
biochemical and cellular assay conditions, along with synthetic examples, see:
Burns, C. J.; Donohue, A. C.; Feutrill, J. T.; Nguyen, T. L. T.; Wilks, A. F.; Zeng, J.,
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structure (PDB code 2B7A).7b Docking of the inhibitor into the JAK2 ATP
binding site was performed with Cytopia’s ‘structure based drug design’ in-
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D. R.; Court, N.; Wilks, A. F., submitted for publication.
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their purity determined to be P90% except for compounds 8, 27, 30, 34, and 42
which were P85%.
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