Pyrrole-3-carboxamides as potent and selective JAK2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.06.025

We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Comp

Full text of DOI:10.1016/j.bmc.2014.06.025

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrrole-3-carboxamides as potent and selective JAK2 inhibitors
⇑
Maria Gabriella Brasca , Marcella Nesi, Nilla Avanzi, Dario Ballinari, Tiziano Bandiera, Jay Bertrand,
Simona Bindi, Giulia Canevari, Davide Carenzi, Daniele Casero, Lucio Ceriani, Marina Ciomei,
Alessandra Cirla, Maristella Colombo, Sabrina Cribioli, Cinzia Cristiani, Franco Della Vedova,
Gabriele Fachin, Marina Fasolini, Eduard R. Felder, Arturo Galvani, Antonella Isacchi, Danilo Mirizzi,
Ilaria Motto, Achille Panzeri, Enrico Pesenti, Paola Vianello, Paola Gnocchi, Daniele Donati
Nerviano Medical Sciences S.r.l., Oncology, Viale Pasteur 10, 20014 Nerviano (MI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel
class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally
bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition
in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of
known biomarkers, and with a favourable pharmacokinetic and safety profile.
Received 6 May 2014
Revised 11 June 2014
Accepted 12 June 2014
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
JAK2
Myeloproliferative disorders
Protein kinase inhibitor
Tumour cell proliferation inhibition
Anti-cancer agents
1. Introduction
besides the STATs and exerting epigenetic actions, for example, by
phosphorylating histone H3.5 The Janus kinases are characterized
JAK2 is a member of the Janus kinase (JAK) family of intracellular
tyrosine kinases, which also include JAK1, JAK3 and TYK2. The JAK
kinases are involved in signal transduction pathways mediated by
cytokines, JAK2 in particular is a critical mediator for hormone-like
cytokines such as growth hormone (GH), prolactin (PRL), erythro-
poietin (EPO), thrombopoietin (TPO), and cytokine receptor ligands
involved in hematopoietic cell development, such as interleukin-3
(IL-3) or granulocyte-macrophage colony-stimulating factor (GM-
CSF).1 Moreover, deletion of JAK2 in mice resulted in embryonic
lethality by day 12.5 (E12.5) due to a lack of hematopoiesis.2,3 Upon
ligand binding, JAKs phosphorylate tyrosine residues in the recep-
tor cytoplasmic domains and in JAKs themselves leading to recruit-
ment and activation of downstream signaling proteins such as
signal transducer and activator of transcription (STAT). STAT phos-
phorylation leads to their dimerization and translocation to the
nucleus where they regulate expression of specific genes involved
in proliferation, apoptosis and differentiation.4 Recently, a non
canonical nuclear action of JAK1 and JAK2 has been described to
affect gene expression by activating other transcription factors
by 7 distinct JAK homology regions (JH1 to JH7). The catalytically
active kinase domain (JH1) is located at the carboxy-terminus,
immediately adjacent to the pseudo-kinase domain (JH2). The func-
tion of the pseudo-kinase domain is to negatively regulate the
activity of the kinase domain. An activating point mutation of
JAK2 (JAK2-V617F, valine to phenylalanine substitution) in the
pseudo-kinase domain has been identified in hematopoietic cells
of patients with myeloproliferative disorders (MPD).6 In particular,
the JAK2-V617F mutation is found in >95% of patients with polycy-
themia vera (PV, characterized by overproduction of red blood
cells), in ꢀ50% of patients with essential thrombocythemia (ET,
characterized by overproduction of platelets), and in ꢀ50% of
patients with myelofibrosis (MF, characterized by fibrosis of the
bone marrow).7 Moreover, disregulated JAK-STAT signaling has
been identified in various inflammatory diseases and in a variety
of cancers.8 These findings together with several in vitro and
in vivo data supported the hypothesis that inhibition of JAK2
presents an opportunity of targeted therapy for these myeloprolif-
erative diseases.9
Accordingly, inhibitors with varying levels of JAK2 selectivity
have been reported10 and have entered clinical trials,11 notably
SAR302503,12 lestaurtinib (CEP-701),13 momelotinib (CYT387),14
pacritinib,15 gandotinib (LY2784544),16 BMS-911543,17 and
⇑
Corresponding author. Tel.: +39 0331 581533; fax: +39 0331 581347.
E-mail address: gabriella.brasca@nervianoms.com (M.G. Brasca).
http://dx.doi.org/10.1016/j.bmc.2014.06.025
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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2
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
AZD-1480.18 Recently, ruxolitinib,19 which displays equivalent
activity against JAK1 and JAK2, with decreased activity against
JAK3 and TYK2, has been approved by the FDA for the treatment
of patients with MF, validating this target. However, the chronic
dosing required for MPD treatment and the potential risk of
immunosuppressive side effects related to inhibition of JAK1,
JAK3, or TYK2 suggest to pursue the identification of a JAK2
selective inhibitor in order to increase safety.20
40a–b and subsequent amidation of carboxylic acid 41a–b under
usual coupling condition. Amino-pyrimidine 38e and 40a were
converted into the corresponding iodo derivatives 42a–b
(Scheme 2). Palladium catalyzed coupling with 4-(4-methyl-pip-
erazin-1-yl)-phenylamine furnished the arylamino derivatives
43a–b. Hydrolysis of carboxylic esters 43a–b gave the correspond-
ing acids 44a–b, which were submitted to the coupling reaction to
provide amides 23–24.
Due to high homology in the adenosine triphosphate (ATP)
binding pocket among the JAK family kinases, the discovery of
JAK2 inhibitors having JAK family selectivity has presented a signif-
icant challenge. Several interesting amino acid differences are
found between the active sites of JAK kinases,21 and indeed
compounds with reasonable JAK family selectivity profile have
been reported in recent years by different groups. However, it is
not a trivial task to pin down the key elements underlying JAK
family selectivity in order to rationally exploit them in the drug
discovery process. Despite this difficulty, we initiated a program
aimed at identifying potent inhibitors of JAK2 and possibly gaining
a better insight into JAK family selectivity.
In Scheme 3, the preparation of pyrroles 20–22 is described
using a completely different synthetic approach. Ketonitrile 46
was obtained by reacting commercially available methyl ester 45
with acetonitrile in the presence of potassium tert-pentoxide as
base, in toluene at room temperature. Reaction of 46 with amino
acetaldehyde diethylacetal followed by treatment with trifluoro-
acetic acid at room temperature afforded the pyrrole 47. Its acetyl
derivative 48 was reacted with N,N-dimethylformamide diisopro-
pyl acetal and the enaminone was condensed with guanidine car-
bonate or formamidine acetate or methylguanidine hydrochloride
to afford compounds 49–51, respectively. Finally, conversion of
nitriles 49–51 to amides 6, 20–21 was performed under acidic con-
dition. Alkylation of the amino group of 6 with 4–methoxybenzal-
dehyde under reductive amination condition furnished its
benzylamino derivative 22.
2. Chemistry
Compounds 2–34 were synthesized22 according to Schemes
1–3. Preparation of amides 2–17, and 25–34 was accomplished
according to Scheme 1, starting from bromoketone 35,23 which
3. Results and discussion
was transformed into
a
-cyano-
c
-ketoester 36. Reaction of 36 with
Recently, we reported on a new chemical class of 5-heteroaryl-
3-carboxamido-2-aryl pyrroles as Cdc7 inhibitors.24 This work led
to the identification of the potent Cdc7 inhibitor 1, which also
hydrobromic acid in acetic acid provided the 2-bromopyrrole 37,24
which was subjected to the Suzuki reaction in the presence of
bis(triphenylphosphine) palladium(II) dichloride as catalyst with
either commercially available or in-house synthesized22a aryl
boronic acids, thus providing the corresponding 2-aryl pyrroles
38a–o. Saponification of the carboxylic esters 38a–o afforded
39a–o, which were reacted with suitable amines under usual cou-
pling condition to give 2–17, and 25–34. In Scheme 2, the prepara-
tion of compounds 18–19 and 23–24 is described. Alkylation of
carboxylic ester 38e with alkyl halides and cesium carbonate in
N,N-dimethylformamide provided the corresponding derivatives
40a–b. Final compounds 18–19 were obtained by hydrolysis of
showed an encouraging activity against JAK2 (IC50 = 3.2 lM,
Table 1). In the absence of the crystallographic data of Cdc7, a
homology model based on the structure of CDK2 was used. The
docking studies and the SAR of the 5-heteroaryl-3-carboxamido-
2-aryl pyrroles correlate well with a modeled binding mode of 1
in the Cdc7 kinase homology model, where the nitrogen of the
pyrimidinyl ring interacts with the hinge region, the amino group
is directed towards the solvent, the carbonyl oxygen of the amide
interacts with the conserved Lys and the aromatic group in posi-
tion 2 of the pyrrole fits diagonally under the glycine-rich loop.
O
O
O
O
CN
Br
c
a
b
N
Br
N
O
N
N
N
N
HBr
H
N
O
H2N
H2N
H2N
35
36
37
O
O
O
R3
OH
O
R2
R2
R2
d
e or f
N
H
N
N
H
N
N
N
H
N
N
N
R1
R1
R1
H2N
H2N
H2N
38a-o
39a-o
2-17, 25-34
a R1 = Me, R2 = H
b R1 = Cl, R2 = H
c R1 = H, R2 = Cl
d R1 = Cl, R2 = Me
e R1 = Me, R2 = Cl
f R1 = Cl, R2 = Cl
g R1 = OMe, R2 = Cl
h R1 = Me, R2 = CF3
i R1 = Cl, R2 = CF3
j R1 = Cl, R2 = OCF3
k R1 = CF3, R2 = CF3
l R1 = CF3, R2 = Cl
m R1 = Et, R2 = Cl
n R1 = Et, R2 = CF3
o R1 = i-Pr, R2 = Cl
Scheme 1. Reagents and conditions: (a) ethyl cyanoacetate, NaOEt, DIPEA, THF, rt, overnight; (b) 33% HBr/AcOH, DCM, 0 °C to rt; (c) substituted boronic acid, LiCl, 1 M
Na2CO3, Pd(Ph3P)2Cl2, EtOH, toluene, 100 °C, 5 h; (d) 1.5 M KOH in 95% EtOH, reflux, 20 h; (e) HOBTꢁNH3, EDCI, DIPEA, THF/DMF, rt, 3 h; (f) primary or secondary amines, EDCI,
DIPEA, THF/DMF, rt, 3 h.
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
O
O
O
O
NH2
O
OH
O
Cl
Cl
Cl
Cl
b
a
c
N
R4
N
R4
N
R4
N
H
N
N
N
N
N
N
N
N
H2N
H2N
H2N
H2N
38e
18-19
41a R4 = Me
41b R4 = Et
40a R4 = Me
40b R4 = Et
d
d
O
O
O
O
O
OH
O
NH2
Cl
Cl
Cl
Cl
e
b
c
N
N
N
N
N
N
N
N
N
N
R4
N
N
R4
R4
R4
HN
HN
I
HN
44a R4 = H
43a R4 = H
42a R4 = H (from 38e)
42b R4 = Me (from 40a)
23-24
44b R4 = Me
43b R4 = Me
N
N
N
N
N
N
Scheme 2. Reagents and conditions: (a) R4X, Cs2CO3, DMF, rt, 3 h; (b) 2 N NaOH, EtOH, reflux, 3 h; (c) HOBTꢁNH3, EDCI, DIPEA, THF/DMF, rt, 3 h; (d) CsI, CuI, I2, iso-
pentylnitrite, THF, reflux, 2 h; (e) Pd(OAc)2, ( )-BINAP, K2CO3, DMF, 80 °C, 3 h.
CN
NC
O
CN
O
e, f for 49
e, g for 50
e, h for 51
Cl
Cl
Cl
Cl
O
b, c
a
d
N
H
N
H
O
45
47
46
48
O
O
CN
NH2
NH2
Cl
Cl
Cl
N
H
N
H
j (from 6)
i
N
H
N
N
N
N
N
N
R5
R5
HN
49 R5 = NH2
50 R5 = H
6 R5 = NH2
20 R5 = H
22
51 R5 = NHCH3
21 R5 = NHCH3
O
Scheme 3. Reagents and conditions: (a) acetonitrile, potassium tert-pentoxide 1.7 M in toluene, rt, 20 min; (b) amino acetaldehyde diethylacetal, toluene, reflux, 5 h; (c) TFA,
rt, 30 min; (d) acetyl chloride, anhydrous aluminum trichloride, DCM, rt, 30 min; (e) N,N-dimethylformamide diisopropyl acetal, DMF, 90 °C, overnight; (f) guanidine
carbonate, DMF, 5 h, 110 °C; (g) formamidine acetate, DMF, 5 h, 150 °C; (h) methylguanidine hydrochloride, K2CO3, DMF, 5 h, 110 °C; (i) TFA, H2O, 98% H2SO4, 70 °C, 5 h; (j) 4-
methoxybenzaldehyde, NaBH(OAc)3, TFA, DMF, rt, overnight.
The screening of our corporate collection of compounds belong-
ing to this class led to the identification of a small set of submi-
cromolar inhibitors of JAK2 (2–6, Table 1). Ortho (2–3) and meta
(4) substitution on the phenyl ring improved the affinity for JAK2
compared to 1, while 2, 5 substitution pattern on the phenyl ring
(5–6) appeared to be the best combination. In particular, com-
pound 6 demonstrated high affinity for JAK2 and reduced potency
two hydrogen bonds to the backbone carbonyl of Gly993 and the
side-chain of Asp994 (DFG motif). The carbonyl oxygen of the
amide is directed towards the solvent and it interacts with the car-
boxylic residue of Asp939 via two other molecules of water (W3
and W4). W3 also establishes a hydrogen bond with the backbone
NH of Ser936. The phenyl ring in position 2 is perpendicular to the
pyrrole ring, adopting an orientation that allows it to direct the
chlorine atom up towards the hydrophobic interior of the gly-
cine-rich loop, while the methyl group is directed downwards into
a shallow cavity defined by Arg980, Asn981, Leu983, Gly993, and
Asp994 (Fig. 1B).
Based on the binding mode of compound 6, we started an
exploration of this class with the goal of generating compounds
with potency against JAK2, selectivity for JAK2 over JAK1, JAK3,
and TYK2, and in vivo efficacy with favourable pharmacokinetic
and safety profile.
against Cdc7 (IC50 = 0.360 lM).
To better understand the binding of these compounds, the crys-
tal structure of 6 bound to the kinase domain of JAK2 was deter-
mined (Fig. 1A). Interestingly, the binding mode of compound 6
is significantly different from the model proposed for 1, requiring
a 180 degree rotation of the molecule. The nitrogen of the pyrimi-
dine forms a hydrogen bond with the backbone NH of the hinge
residue Leu932 and the amino group interacts with the backbone
carbonyl of Glu930. Moreover, compound 6 is involved in a net-
work of water mediated interactions with key residues in the
active site: water molecule W1 mediates the interaction between
the pyrrole nitrogen and water molecule W2, which, in turn, makes
Our initial efforts were focused on analysis of R3, R4, and R5
substituents. Based on the structural analysis of the carboxamide
moiety of 6, exposed to the solvent area, a larger series of residues
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 1
Preliminary exploration: R1, R2 substitution
O
NH2
R2
N
N
H2N
H
N
R1
Compd
R1
R2
JAK2 IC50, l
Ma
1
2
3
4
5
6
H
Me
Cl
H
Cl
H
H
H
Cl
Me
Cl
3.2
0.695
0.615
0.617
0.187
0.020
Me
a
Values are means of three experiments.
linked to the pyrrole ring was investigated. Data concerning activity
of analogues of 6 on JAK family enzymes are summarized in Table 2.
Condensation of carboxylic acid 39e with different amines gave rise
to a series of amides 7–17. The nature of the amide was essential to
affinity, as deduced from the detrimental effect of a tertiary amide
function on compound 8. Small residues such as methyl (7) or linear
alkyl bearing hydrophilic moieties (10–11) were tolerated, while
ethyl residue (9) or straight alkyl bearing hydrophobic moieties
(12–13) as well as bulkier and hydrophobic residues such as iso-
butyl, benzyl and phenyl (14–16) caused a decrease of the inhibitory
activity of JAK2. Instead, the presence of a bulky and hydrophilic
moiety (17) was still tolerated. The biochemical selectivity of the
most interesting compounds 6, 7, 11 and 17, showing promising
JAK2 potency (IC50 <50 nM, Table 2), was greater than six, five and
threefold for JAK1, JAK3 and TYK2, respectively.
Introduction of methyl group as R4 substituent (18) showed a
slight decrease in affinity for JAK2, when compared to 6, probably
due to the displacement of water molecule W1 and subsequent
loss of the above described polar interactions between the pyrrole
nitrogen of 6 and residues Gly993 and Asp994 (DFG) (Fig. 1A).
More dramatic was the introduction of ethyl group (19), which
caused a drop in affinity for JAK2.
From the SAR data in Table 2, it was clear that removal of the
pyrimidinyl ring amino group (20) was deleterious due to the loss
of interaction with the hinge region, while a small substituent on
the amino group like a methyl (21) was tolerated. A benzyl moiety
(22) led to a significant decrease in affinity. According to the
observed binding mode of 6, a further extension of the benzyl moi-
ety into the active site is not likely to be tolerated due to a steric
clash with the side-chain of the gatekeeper Met929. Interestingly,
compounds 23 and 24, bearing a bulky substituent as R5, showed
appreciable affinity for JAK2.
In order to investigate this matter, the JAK2 structure with 24
was solved (Fig. 2), revealing the alternative binding mode of com-
pound 1, modelled into Cdc7. The aminopyrimidine moiety forms
two hydrogen bonds with the backbone NH and the carbonyl of
hinge residue Leu932. The carbonyl oxygen of the amide makes a
direct hydrogen bond with the side-chain nitrogen of Lys882 and
a water mediated interaction with the backbone NH of Asp994
(DFG), while the NH of the amide binds one of the side-chain oxy-
gens of Asp994.
The superposition of the JAK2 structures with 6 and 24 shows
the similarities and differences between the two inhibitor com-
plexes (Fig. 3). In the hinge region, both inhibitors take advantage
of the potential hydrogen bonds although they do so with different
spatial orientation of the pyrimidine ring. Compound 6 directs the
pyrimidine amino group towards the back of the pocket while 24
directs it towards the solvent. Both compounds interact with the
Figure 1. (A) Compound 6 bound to the active site of JAK2. Compound 6 is shown
with yellow carbon atoms while protein is colored in white. Water molecules
participating in the hydrogen bond network (W1, W2, W3, W4) are shown as red
spheres and hydrogen bonds as red dashed lines. (B) Surface diagram showing how
the aromatic group in position 2 is oriented perpendicular to the pyrrole ring, with
the chlorine atom pointing up towards the glycine-rich loop and the methyl
reaching down towards the cavity defined by Arg980, Asn981, Leu983, Gly993 and
Asp994 (showed with green carbon atoms). The surface of the cavity is highlighted
in green.
side-chain of Asp994, but 6 does so through two bridging water
molecules, while 24 establishes a direct hydrogen bond between
its amide nitrogen and the terminal oxygen of the DFG aspartate.
The water molecules involved in the hydrogen bonding network
for the two compounds are not equivalent. In particular, W2, found
in the structure of JAK2 in complex with 6 and rather conserved in
the published JAK2 structures, is not present in the structure of
JAK2 in complex with 24. Interestingly, both inhibitors place the
5-chloro-2-methylphenyl group underneath the glycine-rich loop
although 6 manages to fit perfectly underneath the loop while 24
shifts the loop upwards, probably in order to avoid steric clashes
with the substituted phenyl moiety. Finally, both inhibitors take
advantage of the solvent exposed region although they do so from
different points: 24 extends towards the solvent with the 4-(4-
methylpiperazin-1-yl)phenyl moiety while 6 does so with its
amide group.
Disappointingly, compounds 23 and 24 showed poor selectivity
versus JAK3 and several other kinases of our panel (data not shown).
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 2
Further exploration: R3, R4, R5 substitution
O
R3
Cl
N
R4
N
N
R5
Compd R3
R4
R5
JAK2 IC50
,
l
Ma JAK1/JAK2
selectivityb
JAK3/JAK2
selectivityc
TYK2/JAK2
selectivityd
SET-2 IC50, l
Ma,e
6
7
8
9
NH2
H
H
H
H
NH2
NH2
NH2
NH2
0.020
0.035
3.056
0.109
13
10
ND
7
6
9
ND
7
4
8
ND
4
0.43
0.48
ND
NHMe
N(Me)2
NHEt
ND
H
N
10
11
12
H
H
H
NH2
NH2
NH2
0.057
0.044
0.114
13
7
7
8
5
7
6
5
ND
OH
H
N
>3.0
ND
N
H
H
N
4
F
H
N
13
14
H
H
NH2
NH2
0.268
0.384
7
2
6
6
3
ND
ND
O
H
N
10
15
16
NHBz
NHPh
H
H
NH2
NH2
0.154
0.247
5
0.5
8
14
9
2
ND
ND
H
N
17
H
NH2
0.046
>30
13
6
>3.0
N
18
19
20
21
NH2
NH2
NH2
NH2
Me NH2
0.066
1.537
1.235
0.043
13
ND
ND
6
7
4
ND
ND
ND
1.01
Et
H
H
NH2
H
NHMe
ND
ND
3
ND
ND
4
O
22
NH2
H
0.394
12
4
4
ND
HN
HN
23
NH2
H
0.059
22
0.3
1
ND
N
N
N
N
HN
24
NH2
Me
0.022
32
0.2
3
0.25
ND = not determined.
a
Values are means of three experiments.
b
c
JAK1 IC50/JAK2 IC50
JAK3 IC50/JAK2 IC50
.
.
d
e
TYK2 IC50/JAK2 IC50
.
Coefficient of variation (CV) <30%.
Compounds showing promising JAK2 potency (IC50 <50 nM,
activity, likely due to decreased cell permeability. In fact only mod-
erate Caco2 permeability was observed for compounds 11 and 17
(data not shown), while compound 21 was not tested.
In order to characterize the selectivity within the JAK family of
compounds 6 and 7 in cells, the IL-2 dependent T cell lymphoma
DERL-7 cell line was used, since IL-2 induces JAK1 and JAK3, but
not JAK2 activation in this line.26 Moreover, the JAK independent
Table 2) were tested in JAK2 dependent human megakaryoblastic
leukemic SET-2 cell line, derived from a patient with essential
thrombocythemia and harboring the V617F mutation of JAK2.25
Interesting potency in the SET-2 cell proliferation assay was
observed for compounds 6 and 7, confirming activity as JAK2
inhibitors, whereas 11, 17 and 21 showed lower antiproliferative
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
the methoxy group as R1 (26) caused a drastic reduction in binding
affinity. The replacement of trifluomethyl of 28 with trifluometh-
oxy as R2 substituent (29) or substitution of chloro of 28 with tri-
fluomethyl as R1 substituent (30) was still tolerated, but inversion
of the substituents as in 31 (R1 = CF3, R2 = Cl) caused a decrease in
JAK2 affinity compared to 28.
An improvement of biochemical selectivity for JAK2 over JAK1
was observed for 25, 28, and 30 compared to 6, while only 25
showed enhanced selectivity for JAK2 over JAK3. Greater than fifte-
enfold biochemical selectivity for JAK2 over TYK2 was observed for
25 and 28–30. Of particular interest was compound 28 since it
exhibited a good potency in SET-2 antiproliferative assay and an
improvement in the selectivity in DERL-7 and in A2780 cell prolif-
eration assay (more than threefold).
The crystal structure of compound 28 bound to the kinase
domain of JAK2 was solved (Fig. 4). The observed compound bind-
ing mode closely resembles that observed for compound 6. The
aminopyrimidine establishes the same two hydrogen bonds with
the backbone of hinge residues Glu930 and Leu932 and the net-
work of water mediated interactions described earlier is also con-
served. The aromatic group is perpendicular to the pyrrole ring,
and it directs the chlorine atom downwards into the cavity defined
by Arg980, Asn981, Leu983, Gly993 and Asp994. The trifluoro-
methyl substituent, instead, points up towards the hydrophobic
interior of the glycine-rich loop and forms two multipolar interac-
tions with the carbonyl of Lys857 and Gly856.
Figure 2. Compound 24 bound to the active site of JAK2. Compound 24 is shown
with cyan carbon atoms while the protein is colored in white. Water molecule W10
is shown as a red sphere and red dotted lines represent intermolecular hydrogen
bonds.
Compound 28 shows better selectivity profile than compound 6
against JAK1 and TYK2 (Table 3). Most residues in close contact
(monitored distance 65 Å) with the inhibitor are identical or
highly similar in the three kinases. The main aminoacid differences
are found in the solvent exposed region (JAK2 Asp939 ? JAK1
Glu966), in the C-lobe side of the active site (JAK2 Ile982 ? JAK1
Val1009, TYK2 Val1029) and in the glycine-rich loop (JAK2
Lys857 ? JAK1 Glu883, TYK2 Glu905; JAK2 Asn859 ? JAK1
His885, TYK2 His907; JAK2 Ser862 ? JAK1 Lys888, TYK2 Lys910).
Since compounds 6 and 28 differ in the kind of R1 and R2 substit-
uents, we can hypothesize that the gain in selectivity against JAK1
and TYK2 derives from differences in the polarity and flexibility of
the glycine-rich loop region that affect the van der Waals interac-
tions network.
As said before, the crystal structure of 6 bound to the kinase
domain of JAK2 shows that the methyl group (R1 substituent),
linked to the aromatic ring, is directed downwards into a shallow
cavity (Fig. 1B) formed by the Arg980, Asn981 Leu983, Gly993
and Asp994. While Gly993 is the residue immediately preceding
the conserved DFG motif and is present in JAK2, JAK1, and TYK2,
it is replaced by an alanine (Ala966) in JAK3.21b,c Targeting this area
has proven useful in order to pursue selectivity towards JAK3,16,18
where the presence of the bulkier and less flexible Ala966 instead
of Gly993 reduces the size of the cavity and does not allow an opti-
mal accommodation of the methyl group. To further explore this
cavity, ethyl analogues of 6 and 27 were synthesized (32 and 33,
respectively). Gratifyingly, insertion of ethyl group to the 2 posi-
tion of the phenyl ring, which would extend deeper into this
hydrophobic pocket, improved not only JAK2 biochemical activity
but also cellular activity in SET-2 antiproliferative assay compared
to the parent compounds 6 and 27. Furthermore, 32 and 33
showed an improvement in the selectivity in DERL-7 (more than
fourfold) and in A2780 cell proliferation assay (more than sixfold).
A larger R1 substituent, such as iso-propyl (34), displayed fiftyfold
weaker JAK2 affinity with respect to 32, due to the shape and size
of this pocket in JAK2.
Figure 3. Overlay of the crystal structures of compounds 6 (yellow carbon atoms)
and 24 (light blue carbon atoms) bound to JAK2 (shown in white) highlighting their
remarkably different binding modes.
cell line A2780, which we have observed to be highly sensitive to
antiproliferative agents, was used to assess general off target cyto-
toxicity (Table 3).
Compound 6 showed modest selectivity on DERL-7 cell line
(less than twofold) and none in A2780 cell proliferation assay,
while 7 seemed to be slightly more selective in these antiprolifer-
ative assays compared to 6.
With the aim at improving the selectivity parameter both in
biochemical assay and in cells, maintaining affinity for JAK2 and
potency on SET-2 cell line, we decided to further investigate the
2, 5 substitution pattern on the phenyl ring (R1 and R2 groups).
It was observed that replacement of the substitution pattern of
6 (R1 = Me, R2 = Cl) was tolerated in several cases (Table 3). For
example, 25 (R1, R2 = Cl), 27 (R1 = Me, R2 = CF3) and 28 (R1 = Cl,
R2 = CF3) maintained good affinity for JAK2, while introduction of
In order to assess the potential of the most promising com-
pounds 28, 32, and 33 further studies were performed. All com-
pounds showed good solubility in neutral buffer and
a
preliminary pharmacokinetic study was evaluated in healthy nude
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Table 3
Optimization: R1, R2, R3 substitution
O
R3
R2
N
N
H2N
H
N
R1
Compd
R1
R2
R3
JAK2 IC50
Ma
,
JAK1/JAK2
selectivityb
JAK3/JAK2
selectivityc
TYK2/JAK2
selectivityd
SET-2 IC50
Ma,e
,
DERL-7 IC50
Ma,e
,
A2780 IC50
l
Ma,e
,
l
l
l
6
7
Me
Me
Cl
OMe
Me
Cl
Cl
Cl
Cl
Cl
CF3
CF3
OCF3
CF3
Cl
Cl
CF3
Cl
NH2
NHMe
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
0.020
0.035
0.012
0.308
0.010
0.008
0.024
0.021
0.053
0.003
0.002
0.150
13
10
64
32
17
39
18
36
32
8
6
9
20
11
9
8
4
2
3
4
8
0.43
0.48
0.70
ND
0.76
0.48
1.10
3.07
ND
0.74
2.07
1.66
ND
0.64
1.88
ND
ND
ND
0.88
1.47
ND
0.38
0.98
2.46
ND
0.44
1.47
ND
ND
ND
2.00
2.21
ND
25
26
27
28
29
30
31
32
33
34
16
ND
6
17
18
22
11
3
Cl
CF3
CF3
Et
Et
iso-Pr
11
11
11
0.21
0.32
ND
10
27
8
7
ND = not determined.
a
Values are means of three experiments.
b
c
JAK1 IC50/JAK2 IC50
JAK3 IC50/JAK2 IC50
.
.
d
e
TYK2 IC50/JAK2 IC50
.
Coefficient of variation (CV) <30%.
around 1 h, except for 32, which showed high clearance and low
AUC. Compound 28, after oral administration at a dose of 10 mg/
kg, showed the best pharmacokinetic parameters: interesting oral
bioavailability (31%), with an AUC of 9.5 lM h and half life of 2.3 h.
Compound 28 displayed a combination of potency on JAK2,
selectivity over related JAK family enzymes in biochemical and cel-
lular assay, and physicochemical properties that made it suitable
for further profiling as orally active JAK2 inhibitor. It was subse-
quently screened against an in-house panel of 52 serine-threonine
and tyrosine kinases, showing more than twentyfold selectivity
against all the unrelated kinases. Asides from internal profiling
data, compound 28 was also tested in the KINOMEscanÒ screening
platform that employs an active site directed competition binding
assay to quantitatively measure interactions between test com-
pound and 442 human kinases (including clinically relevant
mutant kinases), covering >80% of the human catalytic protein
kinome (KINOMEscanÒ, a division of DiscoveRx Corporation, San
Diego, California, USA).27 Results for primary screen binding inter-
actions are reported as % Ctrl, where lower numbers indicate stron-
Figure 4. Overlay of 6 (yellow carbon atoms) and 28 (teal carbon atoms) bound to
JAK2 (white ribbon). 28 maintains the same binding mode observed for 6, but it is
able to form two multipolar interactions with the backbone carbonyl of Gly856 and
Lys857 of the glycine-rich loop.
ger hits in the matrix. At 0.1 lM, only JAK2, out of 442 kinases
tested had % Ctrl <1. Outside the JAK family, only six kinases
showed a binding between 1% and 10% Ctrl (CLK1, CLK2, CLK4,
DYRK1A, DYRK1B, TAOK1). In agreement with that observed in
our biochemical assays, the selectivity against the JAK family mem-
bers was of five, twentyfour and one hundred and tenfold for JAK3,
TYK2 and JAK1, respectively, (TREEspot™ image is included in the
mice (Table 4). When administered intravenously (iv) at 10 mg/kg,
all compounds showed good exposure levels, as determined by the
area under the curve (AUC), moderate clearance and a half life
Table 4
Solubility in neutral buffer and in vivo pharmacokinetic parameters in Balb nu/nu micea of selected compounds
Compd
Solubility pH 7 (
lM)
PK data (iv), 10 mg/kgb
PK data (po), 10 mg/kgc
Cmax
(lM)
AUC1
(l
M.h)
CL (mL/min/kg)
Vss (L/kg)
t1/2 (h)
Cmax
(l
M)
AUC1
(
lM h)
t1/2 (h)
F %
28
32
33
159
200
182
17.6
4.5
38.9
26.4
2.0
20.7
16.2
94.0
21.6
0.9
1.8
0.6
1.1
0.2
1.0
4.2
0.6
4.4
9.5
0.7
4.6
2.3
0.8
1.8
31
17
24
a
b
c
n = 3 animals per study.
Dosed in 10% Tween 80 in 5% dextrose.
Dosed in 0.5% methocel.
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8
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Supporting information). All these data confirmed the high selec-
tivity of 28.
vehicle
1 h
3 h
6 h
12 h
24 h
P-STAT5
P-STAT3
GAPDH
To further confirm the JAK2 selectivity of the compound 28
within the JAK family, we determined inhibition of the phosphor-
ylation of constitutive STAT5 and STAT3 in the JAK2 dependent
SET-2 cell line and inhibition of JAK1/3 induced STAT5 phosphory-
lation in IL-2 dependent DERL-7 cell line. In JAK2 dependent cell
line (SET-2) compound 28 induced a dose-dependent reduction
in the phosphorylated forms of STAT5 and STAT3 at doses consis-
tent with inhibition of the proliferation (P0.2 lM), without affect-
ing the total level of the respective proteins (Fig. 5). Instead,
inhibition of JAK1/3 induced STAT5 phosphorylation in IL-2 depen-
Figure 6. Ex-vivo biomarkers modulation in lysates from SET-2 xenograft model.
Tumours (3 tumours/group) were isolated at different post-treatment times after
single po administration of 50 mg/kg of compound 28. Inhibition of P-STAT5 and
P-STAT3 was analysed by Western Blot. Analysis of GAPDH was used as loading
control.
dent DERL-7 cell line was observed at higher doses (P2 lM), sup-
porting the JAK2 selectivity also in cells.
on, 5 days off, for 3 cycles). This optimized schedule showed high
efficacy (TGI = 95%), tumour regression and significant but revers-
ible body weight loss (BWL = 15%).
The efficacy of 28 was evaluated in vivo in the SET-2 xenograft
mouse model. As shown in Table 5 and Fig. 2 in Supporting infor-
mation, 28 was orally administered at 15 and 30 mg/kg, twice a
day, for 10 consecutive days in SCID mice. Both treatments resulted
in significant tumour growth inhibition (TGI = 50% and 71%,
respectively), associated with a moderate and reversible body
weight loss (BWL = 6% at higher dose). A better efficacy profile
for 28 was observed at higher dose (50 mg/kg, twice a day, 5 days
After single administration of 28 in the SET-2 xenograft mouse
model at 50 mg/kg, inhibition of the phosphorylation of STAT5 and
STAT3 was observed up to 12 h post treatment, with maximal inhi-
bition at 3 h (Fig. 6). At the dose of 30 mg/kg the tumours were
analyzed at 1 and 3 h post treatment, and inhibition of phosphor-
ylation of STAT proteins was confirmed (Fig. 3, Supporting infor-
mation). These findings supported the mechanism of action of 28
as JAK2 inhibitor also in vivo and demonstrated the potent and
durable in vivo activity of the compound.
A
µM, compd 28
0.08 0.4
2
10
-
4. Conclusion
P-STAT5-Y694
The screening of our chemical collection led to the identification
of compound 6, displaying activity against JAK2. A structure
based and medicinal chemistry driven optimization led to the
identification of the potent and orally bioavailable JAK2 inhibitor
28 (NMS-P953), with selectivity over JAK family enzymes in
biochemical and cellular assays. In vivo data in mice harboring
SET-2 xenograft tumours demonstrated that the antitumour activity
of compound28 is related to the inhibitionof JAK2, with a favourable
pharmacokinetic and safety profile. NMS–P953 was selected for
further characterization in additional tumour models that will be
presented in due course.
Total STAT5
P-STAT3-Y705
Total STAT3
B
5. Experimental
5.1. Chemistry
µM, compd 28
-
-
-
0.4
+
2
10
+
+
+
20 ng/ml, human IL-2
P-STAT5 -Y694
All solvents and reagents, unless otherwise stated, were com-
mercially available, of the best grade and were used without fur-
ther purification. All experiments dealing with moisture-sensitive
compounds were carried out under dry nitrogen or argon atmo-
sphere. Thin-layer chromatography was performed on Merck Silica
Gel 60 F254 pre-coated plates. Column chromatography was con-
ducted either under medium pressure on silica (Merck Silica Gel
Total STAT5
Figure 5. In vitro biomarkers modulation of compound 28. (A) Inhibition of
P-STAT5 and P-STAT3 in JAK2 dependent SET-2 cell line after 2 h of treatment with
different concentrations of 28. (B) Inhibition of P-STAT5 in DERL-7 cell line (JAK1/
JAK3 dependent) activated by IL-2.
40–63 lm) or performed by using a Biotage SP1 flash purification
system with prepacked silica gel cartridges (Biotage or Varian).
Components were visualized by UV light (k: 254 nm) and by iodine
vapor. 1H NMR spectra were recorded at a constant temperature of
28 °C on
a Varian INOVA 400 spectrometer (operating at
Table 5
400.5 MHz for 1H) and equipped with a 5 mm 1H{13C,15N} z axis
PFG indirect detection probe. Chemical shifts were referenced with
respect to the residual solvents signals. Data are reported as fol-
lows: chemical shift (d), multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, br s = broad signal, td = triplet of doublet,
dd = doublet of doublets, ddd = doublet of doublets of doublets,
m = multiplet), coupling constants (Hz), and number of protons.
HPLC–MS/UV analyses were performed on a LCQ DecaXP (Thermo,
San Jose, US) ion trap instrument, equipped with an electrospray
(ESI) ion source. The mass spectrometer is connected to a Surveyor
In vivo activity of compound 28 in the SET-2 xenograft tumour model in SCID micea
Treatment
Dose
mg/kg
%TGIb (end of the
treatment)
%BWLc (end of the
treatment)
Bid 1-10 day
Bid 1-10 day
Bid 5 days on, 5 days
off, for 3 cycles
15
30
50
50
71
95
2
6
15
a
b
c
n = 7 animals per study.
TGI = tumour growth inhibition.
BWL = body weight loss.
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
HPLC system (Thermo, San Jose, US) with an UV photodiode array
detector (UV detection 215–400 nm). A Phenomenex Gemini C18
being charged with Pd(Ph3P)2Cl2 (470 mg, 0.67 mmol), degassed
four times back filling with argon each time and then heated at
100 °C for 5 h. After cooling to room temperature, the precipitate
was filtered and the filtrate was evaporated under reduced pres-
sure, dissolved in DCM and washed with water. The organic layer
was then dried over sodium sulfate and concentrated. The crude
material was chromatographed on silica gel (DCM/EtOAc 50:50)
to afford ethyl 5-(2-aminopyrimidin-4-yl)-2-(5-chloro-2-methyl-
phenyl)-1H-pyrrole-3-carboxylate (38e, 1.99 g, 87%). 1H NMR
(400 MHz, DMSO-d6) d 12.18 (br s, 1H), 8.21 (d, J = 5.2 Hz, 1H),
7.40 (dd, J = 8.2, 2.2 Hz, 1H), 7.27–7.34 (m, 3H), 7.01 (d, J = 5.2 Hz,
1H), 6.41 (s, 2H), 4.04 (q, J = 7.1 Hz, 2H), 2.11 (s, 3H), 1.09 (t,
J = 7.1 Hz, 3H); LC–MS (ESI): m/z 357 [M+H]+; HRMS (ESI): m/z
calcd for C18H17ClN4O2+H+ 357.1113, found 357.1115.
The intermediate 38e (1.0 g, 2.80 mmol) was treated with 1.5 M
KOH in 95% EtOH (37.3 mL, 20 equiv) under reflux for 20 h. After
cooling, the residue was concentrated, dissolved in water and
washed with DCM. A solution of 2 N HCl was added, under agita-
tion, to the aqueous phase cooled to 5 °C. The resultant precipitate
was collected by filtration to give 5-(2-aminopyrimidin-4-yl)-2-(5-
chloro-2-methylphenyl)-1H-pyrrole-3-carboxylic acid (39e, 0.92 g,
95%). 1H NMR (400 MHz, DMSO-d6) d 12.54 (br s, 1H), 12.06 (s, 1H),
8.28 (d, J = 6.5 Hz, 1H), 7.77 (br s, 1H), 7.59 (d, J = 2.6 Hz, 1H), 7.40–
7.45 (m, 1H), 7.28–7.37 (m, 3H), 2.12 (s, 3H); LC–MS (ESI): m/z 329
[M+H]+; HRMS (ESI): m/z calcd for C16H13ClN4O2+H+ 329.0800,
found 329.0799.
110 Å, LC column 50 ꢂ 4.6 mm, 3
lm particle size was used.
Mobile phase A was ammonium acetate 5 mM buffer (pH 4.5 with
acetic acid)/acetonitrile 95:5, and mobile phase B was ammonium
acetate 5 mM buffer (pH 4.5 with acetic acid)/acetonitrile 5:95.
Gradient from 0% to 100% B in 7 min, hold 100% B 2 min. Flow rate
1 mL/min. Injection volume 10 lL. Full scan, mass range from 50 to
1200 amu. Heated capillary temperature was 275 °C and spray
voltage value was set at 4 kV. Mass are given as m/z ratio. Instru-
ment control, data acquisition and processing were performed by
using Xcalibur 2.1 software (Thermo). As formerly reported,28
ESI(+) high–resolution mass spectra (HRMS) were obtained on a
Q-Tof Ultima (Waters, Manchester, UK) mass spectrometer directly
connected with a Agilent 1100 micro–HPLC system (Palo Alto, CA,
US). GC–MS analyses were performed on a GCQ (FinniganMAT) ion
trap instrument, equipped with an electron (EI) ion source. The
mass spectrometer is connected to a Trace CG system (Thermo,
San Jose, US) through a transfer line. A DB-5MS (J&W Scientific),
30 m ꢂ 0.25 mm I.D., film 0.25
lm was used. Temperature gradient
from 60 °C (hold 1 min) to 280 °C (hold 10 min) in 12 min. Injector
temperature was 280 °C and the transfer line temperature was
250 °C. Full scan, mass range from 35 to 650 amu. Source temper-
ature was 200 °C. Electron energy was 70 eV.
5.1.1. 5-(2-Aminopyrimidin-4-yl)-2-phenyl-1H-pyrrole-3-carbo-
xamide (1)
From ethyl 3-oxo-3-phenyl-propionate and 1-(2-aminopyrimi-
din-4-yl)-2-bromoethanone hydrobromide (35) according to a
described procedure.24
A solution of 39e (470 mg, 1.43 mmol) in DMF/THF (1/1, 5 mL)
and N,N-diisopropylethylamine (1.02 mL, 5.86 mmol) was stirred
at 0 °C. EDCI (594 mg, 3.1 mmol) and HOBTꢁNH3 (430 mg,
2.79 mmol) were added and the reaction mixture was stirred for
3 h at room temperature. The mixture was dropped into a satu-
rated solution of NaHCO3 and ice and the resulting precipitate
was collected by filtration to afford 6 (384 mg, 82%). 1H NMR
(400 MHz, DMSO-d6) d 11.84 (br s, 1H), 8.19 (d, J = 5.2 Hz, 1H),
7.32–7.40 (m, 2H), 7.26–7.30 (m, 2H), 7.22 (br s, 1H), 6.93 (d,
J = 5.2 Hz, 1H), 6.73 (br s, 1H), 6.32 (br s, 2H), 2.12 (s, 3H);
LC–MS (ESI): m/z 328 [M+H]+; HRMS (ESI): m/z calcd for
5.1.2. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
1H-pyrrole-3-carboxamide (6)
Ethyl cyanoacetate (5.3 mL, 50 mmol) was added to a suspen-
sion of sodium (1.15 g, 50 mmol) in 150 mL of anhydrous EtOH
at 0 °C. After 20 min at room temperature, the reaction mixture
was concentrated and the resultant solid was added to a solution
of 1-(2-aminopyrimidin-4-yl)-2-bromoethanone hydrobromide
(35, 14.85 g, 50 mmol) and N,N-diisopropylethylamine (8.8 mL,
50 mmol) in anhydrous THF (300 mL). The reaction mixture was
stirred overnight at room temperature, concentrated and the resi-
due was suspended in water and extracted with DCM. The organic
extracts were dried over sodium sulfate and concentrated. The
crude was purified by flash chromatography (DCM/MeOH 95:5)
to give ethyl 4-(2-aminopyrimidin-4-yl)-2-cyano-4-oxobutanoate
C
16H14ClN5O+H+ 328.0960, found 328.0959.
The following compounds 2–5, and 25–34 were prepared
according to the method described above using the suitable aryl
boronic acid.
5.1.3. 5-(2-Aminopyrimidin-4-yl)-2-(2-methylphenyl)-1H-pyr-
role-3-carboxamide (2)
1H NMR (400 MHz, DMSO-d6) d 12.29 (br s, 1H), 8.24 (d,
J = 6.5 Hz, 1H), 7.74 (br s, 2H), 7.62 (d, J = 2.1 Hz, 1H), 7.24–7.39
(m, 3H), 7.22 (d, J = 6.5 Hz, 1H), 7.04 (br s, 1H), 6.87 (br s, 1H),
2.16 (s, 3H); LC–MS (ESI): m/z 294 [M+H]+; HRMS (ESI): m/z calcd
for C16H15N5O+H+ 294.1350, found 294.1350.
(36, 5.16 g, 41%). 1H NMR (400 MHz, DMSO-d6)
d 8.52 (d,
J = 4.9 Hz, 1H), 6.97 (d, J = 4.9 Hz, 1H), 4.56 (t, J = 5.6 Hz, 1H), 4.19
(q, J = 7.1 Hz, 2H), 3.74 (d, J = 5.6 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H);
LC–MS (ESI): m/z 249 [M+H]+; HRMS (ESI): m/z calcd for
C11H12N4O3+H+ 249.0982, found 249.0975.
A suspension of 36 (5.0 g, 20.14 mmol) in DCM (40 mL) was
added dropwise to 33% HBr in AcOH (40 mL) at 0 °C. The mixture
was left at 0 °C for 30 min and then at room temperature until
disappearance of the starting material. The solid was filtered,
washed with DCM, neutralized with 7 N NH3 in MeOH to afford
ethyl 5-(2-aminopyrimidin-4-yl)-2-bromo-1H-pyrrole-3-carboxyl-
ate (37, 5.5 g, 88%). 1H NMR (400 MHz, DMSO-d6) d 12.84 (br s, 1H),
8.22 (d, J = 5.4 Hz, 1H), 7.23 (s, 1H), 6.99 (d, J = 5.1 Hz, 1H), 6.42 (br
s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H); LC–MS (ESI):
m/z 312 [M+H]+; HRMS (ESI): m/z calcd for C11H11BrN4O2+H+
311.0138, found 311.0139.
5.1.4. 5-(2-Aminopyrimidin-4-yl)-2-(2-chlorophenyl)-1H-pyr-
role-3-carboxamide (3)
1H NMR (400 MHz, DMSO-d6) d 11.89 (br s, 1H), 8.19 (d,
J = 5.2 Hz, 1H), 7.49–7.54 (m, 1H), 7.35–7.46 (m, 3 H), 7.33 (d,
J = 2.6 Hz, 1H), 7.19 (br s, 1H), 6.93 (d, J = 5.2 Hz, 1H), 6.69 (br s,
1H), 6.33 (br s, 2H); LC–MS (ESI): m/z 314 [M+H]+; HRMS (ESI):
m/z calcd for C15H12ClN5O+H+ 314.0803, found 314.0804.
5.1.5. 5-(2-Aminopyrimidin-4-yl)-2-(3-chlorophenyl)-1H-pyr-
role-3-carboxamide (4)
1H NMR (400 MHz, DMSO-d6) d 11.79 (br s, 1H), 8.22 (d,
J = 5.2 Hz, 1H), 7.73 (t, J = 1.8 Hz, 1H), 7.62 (td, J = 1.7, 7.1 Hz, 1H),
7.48 (br s, 1H), 7.46 (br s, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.28 (d,
J = 2.4 Hz, 1H), 7.01 (d, J = 5.2 Hz, 1H), 6.90 (br s, 1H), 6.38 (br s,
2H); LC–MS (ESI): m/z 314 [M+H]+; HRMS (ESI): m/z calcd for
To a solution of 37 (2.0 g, 6.43 mmol) dissolved in EtOH (20 mL)
and toluene (20 mL), LiCl (408 mg, 9.64 mmol), 1 M aq Na2CO3
(17 mmol),
5-chloro-2-methylphenylboronic
acid (1.423 g,
8.35 mmol) were added. The resulting reaction mixture was
degassed three times back filling with argon each time before
C
15H12ClN5O+H+ 314.0803, found 314.0807.
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.025

10
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5.1.6. 5-(2-Aminopyrimidin-4-yl)-2-(2-chloro-5-methylphenyl)-
1H-pyrrole-3-carboxamide (5)
5.1.14. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-ethylphenyl)-
1H-pyrrole-3-carboxamide (32)
1H NMR (400 MHz, DMSO-d6) d 11.85 (br s, 1H), 8.19 (d,
J = 5.2 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 2.6 Hz, 1H),
7.25 (m, 1H), 7.20–7.24 (m, 1H), 7.14 (br s, 1H), 6.93 (d,
J = 5.2 Hz, 1H), 6.68 (br s, 1H), 6.33 (br s, 2H), 2.32 (s, 3H);
LC–MS (ESI): m/z 328 [M+H]+; HRMS (ESI): m/z calcd for
1H NMR (400 MHz, DMSO-d6) d 11.87 (br s, 1H), 8.19 (d,
J = 5.4 Hz, 1H), 7.40 (dd, J = 2.3, 8.3 Hz, 1H), 7.34 (d, J = 2.7 Hz,
1H), 7.32 (d, J = 8.3 Hz, 1H), 7.25 (d, J = 2.2 Hz, 1H), 7.16 (br s,
1H), 6.92 (d, J = 5.2 Hz, 1H), 6.71 (br s, 1H), 6.32 (br s, 2H), 2.46
(q, J = 7.6 Hz, 2H), 0.97 (t, J = 7.6 Hz, 3H); LC–MS (ESI): m/z 342
[M+H]+; HRMS (ESI): m/z calcd for C17H16ClN5O+H+ 342.1116,
found 342.1116.
C
16H14ClN5O+H+ 328.0960, found 328.0965.
5.1.7. 5-(2-Aminopyrimidin-4-yl)-2-(2,5-dichlorophenyl)-1H-
pyrrole-3-carboxamide (25)
5.1.15. 5-(2-Aminopyrimidin-4-yl)-2-[2-ethyl-5-(trifluoromethyl)
phenyl]-1H-pyrrole-3-carboxamide (33)
1H NMR (400 MHz, DMSO-d6) d 12.35 (br s, 1H), 8.26 (d,
J = 6.1 Hz, 1H), 7.43–7.58 (m, 7H), 7.09 (d, J = 6.1 Hz, 1H), 6.87 (br
s, 1H); LC–MS (ESI): m/z 348 [M+H]+; HRMS (ESI): m/z calcd for
1H NMR (400 MHz, DMSO-d6) d 11.93 (br s, 1H), 8.19 (d,
J = 5.2 Hz, 1H), 7.69 (dd, J = 1.5, 8.1 Hz, 1H), 7.53 (d, J = 8.1 Hz,
1H), 7.50 (d, J = 1.6 Hz, 1H), 7.37 (d, J = 2.6 Hz, 1H), 7.27 (br s,
1H), 6.91 (d, J = 5.2 Hz, 1H), 6.71 (br s, 1H), 6.32 (br s, 2H), 2.56
(q, J = 7.5 Hz, 2H), 1.01 (t, J = 7.6 Hz, 3H); LC–MS (ESI): m/z 376
[M+H]+; HRMS (ESI): m/z calcd for C18H16F3N5O+H+ 376.1380,
found 376.1382.
C
15H11Cl2N5O+H+ 348.0414, found 348.0419.
5.1.8. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methoxyphenyl)-
1H-pyrrole-3-carboxamide (26)
1H NMR (400 MHz, DMSO-d6)
d 11.63 (br s, 1H), 8.19
(d, J = 5.2 Hz, 1H), 7.36–7.41 (m, 2H), 7.25 (d, J = 2.6 Hz, 1H),
7.20 (br s, 1H), 7.08–7.12 (m, 1H), 6.92 (d, J = 5.2 Hz, 1H), 6.74
(br s, 1H), 6.35 (s, 2H), 3.75 (s, 3H); LC–MS (ESI): m/z 344
[M+H]+; HRMS (ESI): m/z calcd for C16H14ClN5O2+H+ 344.0909,
found 344.0915.
5.1.16. 5-(2-Aminopyrimidin-4-yl)-2-[5-chloro-2-(propan-2-yl)
phenyl]-1H-pyrrole-3-carboxamide (34)
1H NMR (400 MHz, DMSO-d6) d 11.89 (br s, 1H), 8.18 (d,
J = 5.2 Hz, 1H), 7.44 (dd, J = 2.3, 8.3 Hz, 1H), 7.38 (d, J = 8.3 Hz,
1H), 7.34 (d, J = 2.7 Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.11 (br s,
1H), 6.91 (d, J = 5.2 Hz, 1H), 6.71 (br s, 1H), 6.32 (br s, 2H), 2.79
(spt, J = 6.9 Hz, 1H), 1.06 (d, J = 6.8 Hz, 6H); LC–MS (ESI): m/z 356
[M+H]+; HRMS (ESI): m/z calcd for C18H18ClN5O+H+ 356.1273,
found 356.1271.
5.1.9. 5-(2-Aminopyrimidin-4-yl)-2-[2-methyl-5-(trifluoromethyl)
phenyl]-1H-pyrrole-3-carboxamide (27)
1H NMR (400 MHz, DMSO-d6) d 11.91 (br s, 1H), 8.20 (d,
J = 5.2 Hz, 1H), 7.64 (dd, J = 1.5, 8.1 Hz, 1H), 7.53 (d, J = 1.5 Hz,
1H), 7.49 (d, J = 8.1 Hz, 1H), 7.37 (d, J = 2.4 Hz, 1H), 7.32 (br s,
1H), 6.92 (d, J = 5.2 Hz, 1H), 6.74 (br s, 1H), 6.32 (br s, 2H), 2.23
(s, 3H); LC–MS (ESI): m/z 362 [M+H]+; HRMS (ESI): m/z calcd for
5.1.17. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N-methyl-1H-pyrrole-3-carboxamide (7)
C
17H14F3N5O+H+ 362.1223, found 362.1225.
To a solution of 39e (142 mg, 0.43 mmol) in DMF/THF (1:1,
3 mL) DIPEA (0.301 mL, 1.72 mmol) and MeNH2 2 M solution in
THF (0.432 mL, 0.86 mmol) were added and the solution was
stirred at 0 °C. EDCI (157 mg, 0.86 mmol) and HOBT (117 mg,
0.86 mmol) were added and the reaction mixture was stirred for
3 h at room temperature. The mixture was diluted with water
and extracted with DCM (4 ꢂ 10 mL). The organic phase was
washed with brine, water and then dried over sodium sulfate
and concentrated. The crude material was chromatographed on
silica gel (DCM/MeOH 90:10) to afford 7 (124 mg, 84%).
5.1.10. 5-(2-Aminopyrimidin-4-yl)-2-[2-chloro-5-(trifluoromethyl)
phenyl]-1H-pyrrole-3-carboxamide (28)
1H NMR (400 MHz, DMSO-d6) d 12.07 (br s, 1H), 8.22 (d,
J = 5.3 Hz, 1H), 7.71–7.81 (m, 3H), 7.40–7.44 (m, 1H), 7.37 (d,
J = 2.4 Hz, 1H), 6.90 (d, J = 5.3 Hz, 1H), 6.76 (br s, 1H), 6.35 (br s,
2H); LC–MS (ESI): m/z 382 [M+H]+; HRMS (ESI): m/z calcd for
C16H11ClF3N5O+H+ 382.0677, found 382.0677.
5.1.11. 5-(2-Aminopyrimidin-4-yl)-2-[2-chloro-5-(trifluoro-
methoxy)phenyl]-1H-pyrrole-3-carboxamide (29)
1H NMR (400 MHz, DMSO-d6) d 12.04 (br s, 1H), 8.21 (d,
J = 5.2 Hz, 1H), 7.64 (ddd, J = 1.1, 1.5, 8.8 Hz, 1H), 7.41–7.46 (m,
2H), 7.39 (br s, 1H), 7.30 (d, J = 2.4 Hz, 2H), 6.90 (d, J = 5.1 Hz,
1H), 6.76 (br s, 1H), 6.35 (b s, 2H); LC–MS (ESI): m/z 398 [M+H]+;
HRMS (ESI): m/z calcd for C16H11ClF3N5O2+H+ 398.0626, found
398.0624.
1H NMR (400 MHz, DMSO-d6) d 11.84 (br s, 1H), 8.19 (d,
J = 5.4 Hz, 1H), 7.80 (q, J = 4.8 Hz, 1H), 7.35 (dd, J = 2.3, 8.3 Hz,
1H), 7.31 (d, J = 2.6 Hz, 1H), 7.27 (d, J = 2.3 Hz, 1H), 7.28 (d,
J = 8.0 Hz, 1H), 6.93 (d, J = 5.2 Hz, 1H), 6.32 (br s, 1H), 2.62 (d,
J = 4.5 Hz, 3H), 2.10 (s, 3H); LC–MS (ESI): m/z 342 [M+H]+; HRMS
(ESI): m/z calcd for C17H16ClN5O+H+ 342.1116, found 342.1118.
The following compounds 8–17 were prepared according to the
method described above using the suitable amine.
5.1.12. 5-(2-Aminopyrimidin-4-yl)-2-[2,5-bis(trifluoromethyl)
phenyl]-1H-pyrrole-3-carboxamide (30)
5.1.18. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N,N-dimethyl-1H-pyrrole-3-carboxamide (8)
1H NMR (400 MHz, DMSO-d6) d 12.08 (br s, 1H), 8.21 (d,
J = 5.2 Hz, 1H), 7.79 (br s, 1H), 7.37 (d, J = 2.4 Hz, 1H), 6.85 (d,
J = 5.2 Hz, 1H), 6.34 (br s, 2H); LC–MS (ESI): m/z 416 [M+H]+; HRMS
(ESI): m/z calcd for C17H11F6N5O+H+ 416.0941, found 416.0945.
1H NMR (400 MHz, DMSO-d6) d 11.84 (br s, 1H), 8.19 (d,
J = 5.2 Hz, 1H), 7.35 (dd, J = 2.3, 8.3 Hz, 1H), 7.29 (d, J = 2.2 Hz,
1H), 7.30 (d, J = 8.1 Hz, 1H), 7.00 (s, 1H), 6.99 (d, J = 5.2 Hz, 1H),
6.36 (s, 1H), 2.84 (s, 6H), 2.17 (s, 3H); LC–MS (ESI): m/z 356
[M+H]+; HRMS (ESI): m/z calcd for C18H18ClN5O+H+ 356.1273,
found 356.1278.
5.1.13. 5-(2-Aminopyrimidin-4-yl)-2-[5-chloro-2-(trifluoro-
methyl)phenyl]-1H-pyrrole-3-carboxamide (31)
1H NMR (400 MHz, DMSO-d6) d 12.03 (br s, 1H), 8.21 (d,
J = 5.4 Hz, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.70 (dd,
J = 1.4, 8.5 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.35 (d, J = 2.6 Hz, 1H),
7.34 (br s, 1H), 6.86 (d, J = 5.2 Hz, 1H), 6.68 (br s, 1H), 6.36 (br s,
2H); LC–MS (ESI): m/z 382 [M+H]+; HRMS (ESI): m/z calcd for
5.1.19. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N-ethyl-1H-pyrrole-3-carboxamide (9)
1H NMR (400 MHz, DMSO-d6) d 11.83 (br s, 1H), 8.19 (d,
J = 5.2 Hz, 1H), 7.80 (t, J = 5.7 Hz, 1H), 7.35 (dd, J = 2.3, 8.3 Hz,
1H), 7.33 (d, J = 2.6 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.28 (d,
J = 2.3 Hz, 1H), 6.94 (d, J = 5.2 Hz, 1H), 6.32 (br s, 2H), 3.12 (dq,
C
16H11ClF3N5O+H+ 382.0677, found 382.0679.
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.025

M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
J = 5.7, 7.2 Hz, 2H), 2.11 (s, 3H), 1.02 (t, J = 7.2 Hz, 3H); LC–MS (ESI):
m/z 356 [M+H]+; HRMS (ESI): m/z calcd for C18H18ClN5O+H+
356.1273, found 356.1277.
5.1.26. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N-phenyl-1H-pyrrole-3-carboxamide (16)
1H NMR (400 MHz, DMSO-d6) d 12.05 (br s, 1H), 9.74 (s, 1H),
8.23 (d, J = 5.2 Hz, 1H), 7.65 (dd, J = 1.0, 8.6 Hz, 2H), 7.57 (d,
J = 1.3 Hz, 1H), 7.37 (dd, J = 2.3, 8.0 Hz, 1H), 7.34 (d, J = 2.3 Hz,
1H), 7.30 (d, J = 8.0 Hz, 1H), 7.26 (m, 2H), 7.00 (m, 1H), 6.99 (d,
J = 5.2 Hz, 1H), 6.37 (br s, 2H), 2.14 (s, 3H); LC–MS (ESI): m/z 404
[M+H]+; HRMS (ESI): m/z calcd for C22H18ClN5O+H+ 404.1273,
found 404.1274.
5.1.20. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N-(2-hydroxyethyl)-1H-pyrrole-3-carboxamide (10)
1H NMR (400 MHz, DMSO-d6) d 11.86 (br s, 1H), 8.20 (d,
J = 5.4 Hz, 1H), 7.71 (t, J = 5.8 Hz, 1H), 7.32–7.38 (m, 2H),
7.23–7.30 (m, 2H), 6.94 (d, J = 5.2 Hz, 1H), 6.34 (br s, 2H), 4.61 (t,
J = 5.4 Hz, 1H), 3.38–3.44 (m, 2H), 3.17 (q, J = 6.1 Hz, 2H), 2.10 (s,
3H); LC–MS (ESI): m/z 372 [M+H]+; HRMS (ESI): m/z calcd for
5.1.27. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N-(1-methylpiperidin-4-yl)-1H-pyrrole-3-carboxamide (17)
1H NMR (400 MHz, DMSO-d6) d 11.85 (br s, 1H), 8.19 (d,
J = 5.2 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.33–7.38 (m, 2H), 7.29 (d,
J = 2.0 Hz, 1H), 7.28 (d, J = 8. 7 Hz, 1H), 6.94 (d, J = 5.2 Hz, 1H),
6.32 (br s, 2H), 3.55 (m, 1H), 2.63 (m, 2H), 2.12 (s, 3H), 2.10 (s,
3H), 1.88 (dt, J = 2.1, 11.4 Hz, 2H), 1.65 (m, 2H), 1.45 (dq, J = 3.9,
11.6 Hz, 2H); LC–MS (ESI): m/z 425 [M+H]+; HRMS (ESI): m/z calcd
for C22H25ClN6O+H+ 425.1851, found 425.1846.
C
18H18ClN5O2+H+ 372.1222, found 372.1230.
5.1.21. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N-[2-(methylamino)ethyl]-1H-pyrrole-3-carboxamide (11)
Compound 11 was obtained from tert-butyl [2-({[5-(2-amino-
pyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-1H-pyrrol-3-yl]car-
bonyl}amino)ethyl]methylcarbamate (prepared according to the
method described above) after treatment with 50% CF3COOH in
DCM.
1H NMR (400 MHz, DMSO-d6) d 11.86 (br s, 1H), 8.19 (d,
J = 5.2 Hz, 1H), 7.66 (t, J = 5.7 Hz, 1H), 7.36 (dd, J = 2.2, 8.2 Hz,
1H), 7.33 (s, 1H), 7.29 (d, J = 2.3 Hz, 1H), 7.29 (d, J = 8.2 Hz, 1H),
6.94 (d, J = 5.2 Hz, 1H), 6.33 (br s, 2H), 3.18 (q, J = 6.3 Hz, 2H),
2.54 (t, J = 6.5 Hz, 2H), 2.26 (s, 3H), 2.11 (s, 3H); LC–MS (ESI): m/z
385 [M+H]+; HRMS (ESI): m/z calcd for C19H21ClN6O+H+
385.1538, found 385.1541.
5.1.28. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
1-methyl-1H-pyrrole-3-carboxamide (18)
To a solution of 38e (105.2 mg, 0.295 mmol) in DMF (2 mL),
Cs2CO3 (101 mg, 0.31 mmol) and MeI (28 lL, 0.43 mmol) were
added. The mixture was stirred at room temperature for 3 h, the
solvent was then removed. EtOAc and water were added to the res-
idue, the layers were separated, the aqueous layer was extracted
with EtOAc and the combined organic layers were washed with
water, dried over sodium sulfate, filtered and concentrated. The
crude material was purified by flash chromatography (DCM/MeOH
98:2) affording ethyl 5-(2-aminopyrimidin-4-yl)-2-(5-chloro-2-
methylphenyl)-1-methyl-1H-pyrrole-3-carboxylate (40a, 74 mg,
68%). 1H NMR (400 MHz, DMSO-d6) d 8.20 (d, J = 5.2 Hz, 1H), 7.45
(dd, J = 2.3, 8.4 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 2.3 Hz,
1H), 7.24 (s, 1H), 6.97 (d, J = 5.2 Hz, 1H), 6.58 (s, 2H), 3.98 (q,
J = 7.1 Hz, 2H), 3.67 (s, 3H), 2.00 (s, 3H), 1.00 (t, J = 7.1 Hz, 3H);
LC–MS (ESI): m/z 371 [M+H]+; HRMS (ESI): m/z calcd for
5.1.22. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N-(2-fluoroethyl)-1H-pyrrole-3-carboxamide (12)
1H NMR (400 MHz, DMSO-d6) d 11.90 (br s, 1H), 8.20 (d,
J = 5.2 Hz, 1H), 8.03 (t, J = 5.5 Hz, 1H), 7.38 (d, J = 1.9 Hz, 1H), 7.35
(dd, J = 2.3, 8.3 Hz, 1H), 7.28 (d, J = 2.3 Hz, 1H), 7.28 (d, J = 8.3 Hz,
1H), 6.94 (d, J = 5.2 Hz, 1H), 6.33 (br s, 1H), 4.43 (td, J = 5.2,
47.5 Hz, 2H), 3.44 (q, J = 5.2 Hz, 2H), 2.10 (s, 3H); LC–MS (ESI): m/
z
374 [M+H]+; HRMS (ESI): m/z calcd for C18H17ClFN5O+H+
374.1179, found 374.1185.
C
19H19ClN4O2+H+ 371.1270, found 371.1268.
5.1.23. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N-(2-methoxyethyl)-1H-pyrrole-3-carboxamide (13)
The intermediate 40a (70 mg, 0.19 mmol) was treated with 2 N
NaOH (1.0 mL) in EtOH (1 mL) under reflux for 3 h. After cooling, a
solution of citric acid was added. The resultant precipitate was col-
lected by filtration to give 5-(2-aminopyrimidin-4-yl)-2-(5-chloro-
2-methylphenyl)-1-methyl-1H-pyrrole-3-carboxylic acid (41a,
62 mg, 95%). 1H NMR (400 MHz, DMSO-d6) d 8.15 (d, J = 5.2 Hz,
1H), 7.36 (dd, J = 2.2, 8.2 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.20 (d,
J = 2.1 Hz, 1H), 7.10 (s, 1H), 6.88 (d, J = 5.4 Hz, 1H), 6.47 (s, 2H),
3.61 (s, 3H), 2.02 (s, 3H); LC–MS (ESI): m/z 343 [M+H]+; HRMS
(ESI): m/z calcd for C17H15ClN4O2+H+ 343.0957, found 343.0966.
To a solution of 41a (34 mg, 0.1 mmol) in DMF/THF (1:1, 2 mL)
1H NMR (400 MHz, DMSO-d6) d 11.87 (br s, 1H), 8.19 (d,
J = 5.2 Hz, 1H), 7.69 (t, J = 5.5 Hz, 1H), 7.36 (dd, J = 2.2, 8.2 Hz,
1H), 7.34 (d, J = 2.6 Hz, 1H), 7.28 (d, J = 2.3 Hz, 1H), 7.29 (d,
J = 7.9 Hz, 1H), 6.94 (d, J = 5.2 Hz, 1H), 6.33 (br s, 2H), 3.22 (s,
3H), 2.10 (s, 3H); LC–MS (ESI): m/z 386 [M+H]+; HRMS (ESI): m/z
calcd for C19H20ClN5O2+H+ 386.1379, found 386.1384.
5.1.24. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
N-(2-methylpropyl)-1H-pyrrole-3-carboxamide (14)
1H NMR (400 MHz, DMSO-d6) d 11.84 (br s, 1H), 8.19 (d,
J = 5.2 Hz, 1H), 7.72 (t, J = 5.9 Hz, 1H), 7.35 (dd, J = 2.3, 8.3 Hz,
1H), 7.35 (d, J = 2.3 Hz, 1H), 7.29 (d, J = 2.2 Hz, 1H), 7.28 (d,
J = 8.3 Hz, 1H), 6.95 (d, J = 5.2 Hz, 1H), 6.33 (br s, 2H), 2.92 (t,
J = 6.4 Hz, 2H), 2.11 (s, 3H), 1.71 (spt, J = 6.8 Hz, 1H), 0.81 (d,
J = 6.7 Hz, 6H); LC–MS (ESI): m/z 384 [M+H]+; HRMS (ESI): m/z
calcd for C20H22ClN5O+H+ 384.1586, found 384.1592).
and DIPEA (36 lL, 0.2 mmol) was added. EDCI (29 mg, 0.15 mmol)
and HOBTꢁNH3 (23 mg, 0.15 mmol) were added and the reaction
mixture was stirred for 3 h at room temperature. The mixture
was dropped into a saturated solution of NaHCO3 and ice and
extracted with DCM. The combined organic layers were washed
with water, dried over sodium sulfate, filtered and concentrated.
The crude material was purified by flash chromatography (DCM/
MeOH 95:5) affording 18 (21 mg, 63%). 1H NMR (400 MHz,
DMSO-d6) d 8.21 (d, J = 5.4 Hz, 1H), 7.40 (dd, J = 2.2, 8.2 Hz, 1H),
7.33 (s, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 2.2 Hz, 1H), 7.04
(br s, 1H), 6.81 (d, J = 5.4 Hz, 1H), 6.71 (br s, 1H), 6.54 (s, 2H),
3.61 (s, 3H), 2.00 (s, 3H); LC–MS (ESI): m/z 342 [M+H]+; HRMS
(ESI): m/z calcd for C17H16ClN5O+H+ 342.1116, found 342.1122.
The following compound 19 was prepared according to the
method described above using ethyl bromide instead of methyl
iodide.
5.1.25. 5-(2-Aminopyrimidin-4-yl)-N-benzyl-2-(5-chloro-2-
methylphenyl)-1H-pyrrole-3-carboxamide (15)
1H NMR (400 MHz, DMSO-d6) d 11.89 (br s, 1H), 8.38 (t,
J = 6.0 Hz, 1H), 8.20 (d, J = 5.2 Hz, 1H), 7.40 (d, J = 2.6 Hz, 1H),
7.34 (m, 1H), 7.17–7.32 (m, 7H), 6.94 (d, J = 5.2 Hz, 1H), 6.33 (br
s, 2H), 4.32 (d, J = 6.1 Hz, 2H), 2.09 (s, 3H); LC–MS (ESI): m/z 418
[M+H]+; HRMS (ESI): m/z calcd for C23H20ClN5O+H+ 418.1429,
found 418.1429.
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.025

12
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5.1.29. 5-(2-Aminopyrimidin-4-yl)-2-(5-chloro-2-methylphenyl)-
1-ethyl-1H-pyrrole-3-carboxamide (19)
of NaHCO3 and extracted with DCM/MeOH (9:1, 5 ꢂ 5 mL). The
combined organic layers were washed with water, dried over
Na2SO4, filtered and concentrated. The crude material was purified
by flash chromatography (DCM/MeOH 9:1) affording 23 (21 mg,
70%, 2 steps). 1H NMR (400 MHz, DMSO-d6) d 11.90 (d, J = 2.2 Hz,
1H), 9.11 (s, 1H), 8.35 (d, J = 5.2 Hz, 1H), 7.68 (d, J = 9.0 Hz, 2H),
7.41 (d, J = 2.7 Hz, 1H), 7.37 (dd, J = 2.3, 8.2 Hz, 1H), 7.29–7.34
(m, 2H), 7.10 (d, J = 5.2 Hz, 1H), 6.91 (d, J = 9.1 Hz, 2H), 6.79 (br s,
1H), 3.07 (m, 4H), 2.47 (m, 4H), 2.23 (s, 3H), 2.15 (s, 3H); LC–MS
(ESI): m/z 502 [M+H]+; HRMS (ESI): m/z calcd for C27H28ClN7O+H+
502.2117, found 502.2118.
1H NMR (400 MHz, DMSO-d6) d 8.20 (d, J = 5.2 Hz, 1H), 7.41 (dd,
J = 2.2, 8.2 Hz, 1H), 7.36 (s, 2H), 7.35 (d, J = 8.2 Hz, 1H), 7.25 (d,
J = 2.3 Hz, 1H), 7.03 (br s, 1H), 6.81 (d, J = 5.4 Hz, 1H), 6.70 (br s,
1H), 6.52 (s, 2H), 4.43 (qd, J = 6.9, 13.5 Hz, 1H), 4.00 (qd, J = 6.9,
13.5 Hz, 1H), 2.01 (s, 3H), 0.99 (t, J = 6.9 Hz, 3H); LC–MS (ESI): m/
z
356 [M+H]+; HRMS (ESI): m/z calcd for C18H18ClN5O+H+
356.1273, found 356.1271.
5.1.30. 2-(5-Chloro-2-methylphenyl)-5-(2-{[4-(4-methylpiperazin-
1-yl)phenyl]amino}pyrimidin-4-yl)-1H-pyrrole-3-carboxamide
(23)
The following compound 24 was prepared according to the
method described above starting from 40a.
To a well stirred suspension of 38e (416 mg, 1.16 mmol) in THF
(15 mL) maintained in an inert atmosphere of argon, cesium iodide
(302 mg, 1.16 mmol), iodine (296 mg, 0.58 mmol), copper(I) iodide
(74 mg, 0.39 mmol) and iso-pentyl nitrite (0.236 mL, 1.75 mmol)
were added. The reaction mixture was stirred vigorously under
reflux for 2 h. After cooling, the reaction mixture was concentrated
to small volume, poured into water (100 mL) and extracted with
DCM (5 ꢂ 20 mL). The combined organic layers were washed with
33% ammonium hydroxide (2 ꢂ 10 mL), 5% solution of sodium
thiosulphate (2 ꢂ 50 mL), brine (2 ꢂ 20 mL) and dried over anhy-
drous sodium sulfate, filtered and concentrated. The crude material
was purified by flash chromatography (EtOAc/hexane 40:60)
affording ethyl 2-(5-chloro-2-methylphenyl)-5-(2-iodopyrimidin-
4-yl)-1H-pyrrole-3-carboxylate (42a, 130 mg, 24%). 1H NMR
(400 MHz, DMSO-d6) d 12.58 (br s, 1H), 8.43 (d, J = 5.4 Hz, 1H),
7.91 (d, J = 5.4 Hz, 1H), 7.53 (d, J = 2.7 Hz, 1H), 7.41–7.45 (m, 1H),
7.32–7.36 (m, 2H), 4.05 (m, 2H), 2.11 (s, 3H), 1.10 (m, 3H);
LC–MS (ESI): m/z 467 [M+H]+; HRMS (ESI): m/z calcd for
5.1.31. 2-(5-Chloro-2-methylphenyl)-1-methyl-5-(2-{[4-(4-
methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)-1H-
pyrrole-3-carboxamide (24)
1H NMR (400 MHz, DMSO-d6) d 9.19 (s, 1H), 8.37 (d, J = 5.2 Hz,
1H), 7.51 (m, 2H), 7.40–7.44 (m, 2H), 7.37 (m, 1H), 7.26 (d,
J = 2.3 Hz, 1H), 7.08 (br s, 1H), 6.99 (d, J = 5.2 Hz, 1H), 6.88 (m,
2H), 6.75 (br s, 1H), 3.62 (s, 3H), 3.05 (m, 4H), 2.44 (m, 4H), 2.22
(s, 3H), 2.01 (s, 3H); LC–MS (ESI): m/z 516 [M+H]+; HRMS (ESI):
m/z calcd for C28H30ClN7O+H+ 516.2273, found 516.2273.
5.1.32. 2-(5-Chloro-2-methylphenyl)-5-(pyrimidin-4-yl)-1H-
pyrrole-3-carboxamide (20)
Potassium tert-pentoxide 1.7 M in toluene (86.4 mL,
147.9 mmol) was added drop wise to a solution of methyl 5-
chloro-2-methylbenzoate (45, 18.2 g, 98.6 mmol) and acetonitrile
(15.4 mL, 296 mmol) in anhydrous toluene (150 mL). The mixture
was stirred at room temperature for 20 min, then diluted with
1 N HCl (60 mL), and extracted with EtOAc (3 ꢂ 150 mL). The
organic layer was collected, washed with water and brine, dried
over sodium sulfate and concentrated. Column chromatography
on silica gel (0–20% EtOAc/hexane) provided 3-(5-chloro-2-meth-
ylphenyl)-3-oxopropanenitrile (46, 13.7 g, 72%). 1H NMR
(400 MHz, DMSO-d6) d 7.88 (d, J = 2.1 Hz, 1H), 7.57 (dd, J = 1.9,
8.4 Hz, 1H), 7.38 (m, 1H), 4.69 (s, 2H), 2.41 (s, 3H); GC–MS (EI):
C
18H15ClIN3O2+H+ 467.9971, found 467.9966.
Pd(OAc)2 (5.5 mg, 0.025 mmol), (+)-BINAP (15.5 mg, 0.025
mmol) and DMF (5 mL) were charged in a round-bottom flask
flushed with argon. The flask was evacuated and backfilled with
argon. The mixture was stirred under argon for 30 min and 42a
(117 mg, 0.25 mmol), 4-(4-methyl-piperazin-1-yl)-phenylamine
(143 mg, 0.75 mmol), potassium carbonate (518 mg, 3.75 mmol)
were added. The flask was evacuated and backfilled with argon.
The resulting mixture was stirred at 80 °C for 3 h under argon.
After cooling to room temperature, the reaction mixture was
filtered on a pad of celite. The solvent was concentrated, the
crude solid was purified by flash chromatography on silica gel
(DCM/MeOH 90:10) to afford ethyl 2-(5-chloro-2-methylphenyl)-
5-(2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)-
1H-pyrrole-3-carboxylate (43a, 40 mg, 30%). 1H NMR (400 MHz,
m/z 193.0294; HRMS (ESI): m/z calcd for
C
10H8ClNO+Na+
216.0186, found 216.0184.
A mixture of 46 (13.0 g, 67.1 mmol), aminoacetaldehyde diethyl
acetal (11.7 mL, 80.5 mmol) and toluene (150 mL) was stirred
under reflux for 5 h under nitrogen atmosphere in the Dean-Stark
apparatus. The mixture was evaporated in vacuo and added to TFA
(50 mL) at 5 °C. After stirring at room temperature for 30 min, the
reaction mixture was concentrated and then diluted with EtOAc
and saturated solution of sodium hydrogen carbonate. The organic
layer was separated, washed with water and brine, dried over
sodium sulfate and concentrated. Column chromatography on sil-
ica gel (0–20% EtOAc/hexane) afforded 2-(5-chloro-2-methyl-
phenyl)-1H-pyrrole-3-carbonitrile (47, 8.1 g, 56%, 2 steps). 1H
NMR (400 MHz, DMSO-d6) d 11.99 (br s, 1H), 7.34–7.47 (m, 2H),
7.38 (d, J = 2.2 Hz, 1H), 7.04 (t, J = 2.8 Hz, 1H), 6.59 (t, J = 2.6 Hz,
1H), 2.26 (s, 3H); LC–MS (ESI): m/z 217 [M+H]+; HRMS (ESI): m/z
calcd for C12H9ClN2+H+ 217.0527, found 217.0526.
To a mixture of 47 (1.80 g, 8.28 mmol) in DCM (40 mL) was
added acetyl chloride (0.936 mL, 13.14 mmol) at room tempera-
ture, under nitrogen. The resultant mixture was cooled to 0 °C
and anhydrous aluminum trichloride (2.62 g, 19.8 mmol) was
added in small portions during a period of 10 min, keeping the
internal temperature below 5 °C. Upon complete addition, the mix-
ture was brought to room temperature and allowed to stir for
30 min. Then, the mixture was slowly poured in a solution of ice-
cooled 1 N HCl solution (18 mL). The aqueous layer was separated
and extracted twice with DCM (40 mL). The combined organic
DMSO-d6)
d 12.24 (d, J = 2.2 Hz, 1H), 9.15 (s, 1H), 8.38 (d,
J = 5.2 Hz, 1H), 7.63 (d, J = 9.0 Hz, 2H), 7.42 (dd, J = 2.3, 8.2 Hz,
1H), 7.35 (d, J = 8.2 Hz, 1H), 7.33–7.39 (m, 2H), 7.16 (d, J = 5.2 Hz,
1H), 6.89 (d, J = 9.1 Hz, 2H), 4.06 (q, J = 7.0 Hz, 2H), 3.07 (m, 4H),
2.46 (m, 4H), 2.22 (s, 3H), 2.14 (s, 3H), 1.09 (t, J = 7.1 Hz, 3H);
LC–MS (ESI): m/z 531 [M+H]+; HRMS (ESI): m/z calcd for
C
29H31ClN6O2+H+ 531.2270, found 531.2274.
The carboxylate 43a (35 mg, 0.06 mmol) was treated with 2 N
NaOH (0.3 mL) in EtOH (1 mL) under reflux for 3 h. After cooling,
acetic acid was added and after centrifugation, the resultant
precipitate was collected by filtration to give 2-(5-chloro-2-
methylphenyl)-5-(2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}
pyrimidin-4-yl)-1H-pyrrole-3-carboxylic acid (44a), which was
used directly in the next synthetic step.
A solution of the crude 44a in DMF/THF (1:1, 1 mL) and DIPEA
(46 lL, 0.26 mmol) was stirred at 0 °C. EDCI (25 mg, 0.13 mmol)
and HOBTꢁNH3 (10 mg, 0.13 mmol) were added and the reaction
mixture was stirred for 3 h at room temperature. THF was
evaporated, the mixture was dropped into a saturated solution
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.025

M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
13
extracts were washed with brine, dried over sodium sulfate and
concentrated under reduced pressure. The crude material was
chromatographed on silica gel (10–20% EtOAc/hexane) to afford
5-acetyl-2-(5-chloro-2-methylphenyl)-1H-pyrrole-3-carbonitrile
(48, 2.0 g, 86%). 1H NMR (400 MHz, DMSO-d6) d 12.89 (br s, 1H),
7.60 (d, J = 2.4 Hz, 1H), 7.49 (dd, J = 2.4, 8.4 Hz, 1H), 7.43 (d,
J = 2.3 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 2.43 (s, 3H), 2.24 (s, 3H);
LC–MS (ESI): m/z 259 [M+H]+; HRMS (ESI): m/z calcd for
1H NMR (400 MHz, DMSO-d6) d 11.87 (br s, 1H), 8.20 (d,
J = 5.4 Hz, 1H), 7.39 (s, 1H), 7.34–7.38 (m, 1H), 7.26–7.32 (m, 2H),
7.19 (br s, 1H), 6.88 (d, J = 5.1 Hz, 1H), 6.67–6.83 (m, 1H), 2.88 (d,
J = 4.1 Hz, 3H), 2.12 (s, 3H); LC–MS (ESI): m/z 342 [M+H]+; HRMS
(ESI): m/z calcd for C17H16ClN5O+H+ 342.1116, found 342.1118.
5.1.34. 2-(5-Chloro-2-methylphenyl)-5-{2-[(4-methoxybenzyl)
amino]pyrimidin-4-yl}-1H-pyrrole-3-carboxamide (22)
C
14H11ClN2O+Na+ 281.0452, found 281.0452.
To a suspension of 48 (225 mg, 0.87 mmol) in DMF (2.5 mL) was
added N,N-dimethylformamide diisopropyl acetal (0.54 mL,
2.61 mmol). The mixture was allowed to stir overnight at 90 °C,
evaporated in vacuo, suspended in DMF (2.5 mL) and treated with
guanidine carbonate (127 mg, 1.05 mmol). The mixture was heated
to 110 °C for 5 h under efficient stirring. The resulting mixture was
concentrated and chromatographed on silica gel (10–30% EtOAc/
hexane) to afford 5-(2-aminopyrimidin-4-yl)-2-(5-chloro-2-meth-
ylphenyl)-1H-pyrrole-3-carbonitrile (49, 148 mg, 55%, 2 steps).
To a suspension of 48 (225 mg, 0.87 mmol) in DMF (2.5 mL) was
added N,N-dimethylformamide diisopropyl acetal (0.54 mL,
2.61 mmol). The mixture was allowed to stir overnight at 90 °C,
evaporated in vacuo, suspended in DMF (2.5 mL) and treated with
formamidine acetate (208 mg, 2.0 mmol). The mixture was heated
to 150 °C for 5 h under efficient stirring. The resulting mixture was
diluted by drop wise addition of water and extracted with EtOAc.
The organic phase was washed with brine, water and then dried
over sodium sulfate and concentrated. The crude material was
chromatographed on silica gel (hexane/EtOAc 90:10) to afford
2-(5-chloro-2-methylphenyl)-5-(pyrimidin-4-yl)-1H-pyrrole-3-car-
bonitrile (50, 77 mg, 30%, 2 steps). 1H NMR (400 MHz, DMSO-d6) d
12.98 (br s, 1H), 9.13 (d, J = 1.2 Hz, 1H), 8.79 (d, J = 5.4 Hz, 1H), 7.90
(dd, J = 1.3, 5.4 Hz, 1H), 7.61 (s, 1H), 7.48 (m, 2H), 7.43 (m, 1H), 2.30
(s, 3H); LC–MS (ESI): m/z 295 [M+H]+; HRMS (ESI): m/z calcd for
1H NMR (400 MHz, DMSO-d6)
d 12.79 (br s, 1H), 8.30 (d,
J = 5.7 Hz, 1H), 7.46–7.54 (m, 3H), 7.44 (m, 1H), 7.14 (d,
J = 5.7 Hz, 1H), 7.05 (br s, 1H), 2.28 (s, 3H); LC–MS (ESI): m/z 310
[M+H]+; HRMS (ESI): m/z calcd for C16H12ClN5+H+ 310.0854, found
310.0853.
To a solution of 49 (142 mg, 0.46 mmol) in TFA (2.0 mL) were
sequentially added water (0.3 mL) and 98% sulfuric acid (0.6 mL)
under efficient stirring. The mixture was allowed to stir for 8 h at
70 °C and then was diluted by drop wise addition of water
(6 mL). The reaction mixture was made basic (pH 10-12) by adding
30% aqueous ammonia (2 mL) under stirring. The precipitated solid
was collected by filtration, washed with water and finally dried in a
vacuum oven at 50 °C affording 6 (128 mg, 85%).
To a stirred solution of 6 (100 mg, 0.305 mmol), TFA (0.28 mL,
3.66 mL) and 4-methoxybenzaldehyde (0.074 mL, 0.61 mmol) in
DMF (2 mL) was added portionwise sodium triacetoxyborohydride
(129 mg, 0.61 mmol). After stirring at room temperature overnight,
the solution was taken up in EtOAc and thoroughly washed with
brine. After drying over sodium sulfate, the solvent was removed
and the residue was purified by flash chromatography (DCM/
MeOH, 96:4) to provide 22 (86 mg, 63%). 1H NMR (400 MHz,
DMSO-d6) d 11.84 (br s, 1H), 8.19 (d, J = 5.2 Hz, 1H), 7.39 (d,
J = 2.4 Hz, 1H), 7.26–7.40 (m, 7H), 7.19 (br s, 1H), 6.84 (d,
J = 8.8 Hz, 2H), 6.78 (br s, 1H), 4.55 (d, J = 4.1 Hz, 2H), 3.70 (s,
3H), 2.13 (s, 3H); LC–MS (ESI): m/z 448 [M+H]+; HRMS (ESI): m/z
calcd for C24H22ClN5O2+H+ 448.1535, found 448.1543.
C
16H11ClN4+H+ 295.0745, found 295.0750.
To a solution of 50 (67 mg, 0.23 mmol) in TFA (1.0 mL) were
sequentially added water (0.15 mL) and 98% sulfuric acid
(0.30 mL) under efficient stirring. The mixture was allowed to stir
for 5 h at 70 °C and then was diluted by drop wise addition of
water (1 mL). The reaction mixture was made basic (pH 10-12)
by adding 30% aqueous ammonia (3 mL) under stirring. The precip-
itated solid was collected by filtration, washed with water and
finally dried in a vacuum oven at 50 °C affording 20 (72 mg, 83%).
1H NMR (400 MHz, DMSO-d6) d 12.22 (br s, 1H), 9.04 (d,
J = 1.1 Hz, 1H), 8.70 (d, J = 5.5 Hz, 1H), 7.74 (dd, J = 1.4, 5.4 Hz,
1H), 7.57 (d, J = 2.7 Hz, 1H), 7.36 (m, 1H), 7.12–7.33 (m, 3H), 6.81
(br s, 1H), 2.13 (s, 3H); LC–MS (ESI): m/z 313 [M+H]+; HRMS
(ESI): m/z calcd for C16H13ClN4O+H+ 313.0851, found 313.0853.
5.1.33. 2-(5-Chloro-2-methylphenyl)-5-[2-
(methylamino)pyrimidin-4-yl]-1H-pyrrole-3-carboxamide (21)
To a suspension of 48 (225 mg, 0.87 mmol) in DMF (2.5 mL) was
added N,N-dimethylformamide diisopropyl acetal (0.54 mL,
2.61 mmol). The mixture was allowed to stir overnight at 90 °C,
evaporated in vacuo, suspended in DMF (2.5 mL) and treated with
methylguanidine hydrochloride (115 mg, 1.05 mmol) and K2CO3
(159 mg, 1.14 mmol). The mixture was heated to 110 °C for 5 h
under efficient stirring. The resulting mixture was concentrated
and chromatographed on silica gel (10–30% EtOAc/hexane) to
afford 2-(5-chloro-2-methylphenyl)-5-[2-(methylamino)pyrimi-
din-4-yl]-1H-pyrrole-3-carbonitrile (51, 135 mg, 48%, 2 steps). 1H
NMR (400 MHz, DMSO-d6) d 12.53 (br s, 1H), 8.27 (d, J = 4.6 Hz,
1H), 7.46–7.53 (m, 3H), 7.44 (m, 1H), 7.38 (m, 1H), 6.98 (d,
J = 5.2 Hz, 1H), 6.91 (br s, 1H), 2.88 (d, J = 4.5 Hz, 3H) 2.28 (s, 3H);
LC–MS (ESI): m/z 324 [M+H]+; HRMS (ESI): m/z C17H14ClN5+H+
324.1011, found 324.1013.
To a solution of 51 (74 mg, 0.23 mmol) in TFA (1.0 mL) were
sequentially added water (0.15 mL) and 98% sulfuric acid
(0.30 mL) under efficient stirring. The mixture was allowed to stir
for 8 h at 70 °C and then was diluted by drop wise addition of
water (3 mL). The reaction mixture was made basic (pH 10-12)
by adding 30% aqueous ammonia (1 mL) under stirring. The
precipitated solid was collected by filtration, washed with water
and finally dried in a vacuum oven at 50 °C affording 21 (66 mg,
88%).
5.2. Crystallographic methods
Crystallization studies were performed using
a construct
encompassing the C-terminal kinase domain (aa 835-1132). Crys-
tals of the kinase domain of JAK2 in complex with compounds 6,
24 and 28 were grown from a solution of 1.5–2.2 M sodium malon-
ate pH 7. The protein was concentrated at 10 mg/mL and compound
was added to a nominal concentration of 1 mM. For data collection,
the crystals were transferred to drops containing the equivalent
mother liquor with 20% glycerol. Diffraction data for compounds
6 and 28 were collected at the ESRF (Grenoble, France) on beam-
lines ID23-1 and ID29, respectively, while diffraction data for com-
pound 24 were collected in house using a Rigaku Micromax-007 HF
X-ray generator and Mar345 Image Plate Detector (Marresearch).
Data were processed using the HKL2000 package.29 The three struc-
tures were solved by molecular replacement with Phaser30 using a
published structure of JAK2 (PDB code 2B7A) as a search model.
Model building was done using Coot31 and refinement was done
with RefMac.32 The coordinates and the corresponding structure
factors were deposited in the Protein Data Bank with code 4D0W,
4D1S and 4D0X for 6, 24 and 28, respectively.
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.025

14
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5.3. Biochemical assay
5.5. Biomarker evaluation in cells
Enzyme assays were performed using a radiometric assay in
which specific JAK2, JAK1, JAK3 and TYK2 peptide substrates are
trans-phosphorylated by JAK kinases in the presence of ATP traced
For the mechanism of action the SET-2 cell line was treated with
compound at the indicated concentration for 2 h at 37 °C or with
just DMSO as a control. DERL-7 cell line was starved for 18 h and
then treated with the compound for 2 h before activation with
human IL-2 (20 ng/ml, 15 min at 37 °C). At the end of treatment
both the cell lines were centrifuged, washed with cold PBS and
lysed in RIPA buffer. The lysates were centrifuged at 13,000 rpm
for 15 min at 4 °C. Equal amounts of cell lysates were resolved by
SDS–PAGE and then transferred to Wathman Nitrocellulose trans-
fer membrane (GE Healthcare UK Limited, Buckinghamshire, UK).
The membranes were incubated in blocking solution consisting
of 5% powered milk in TBST at room temperature for 1 h and then
overnight at 4 °C with the antibodies specific for P-STAT5, P-STAT3,
STAT5 and STAT3 total protein (Cell Signaling Technology, Danvers,
MA). Protein bands were visualised by using an enhanced chemilu-
minescence detection system (LiteablotÒ Plus, Euroclone, Milan,
Italy) and autoradiography.
with 33P-c-ATP and recombinant epitope tagged kinase domains
JAK2 (residues 808-1132, Invitrogen, Eugene, OR), JAK1 (residues
861-1152, P23458 UniProtKB/Swiss-Prot), JAK3 (residues 781-1124,
P52333 UniProtKB/Swiss-Prot) and TYK2 (residues 833-1187,
P29597 UniProtKB/Swiss-Prot). The kinase assays were run with a
final enzyme concentration of 1 nM for JAK2 and JAK3, 2.5 nM for
JAK1 and 3 nM for TYK2. On the contrary of the other JAKs, an
auto-phosphorylation step was requested for JAK1, due to not linear
kinase reaction (1 lM JAK1 in presence of 750 lM ATP was kept at
28 °C for 60 min). After this preactivation step the JAK1 kinetic
resulted linear over time. The peptidic substrate LPLDKDYYVVREPGQ
was used at 64 and 40
tididic substrate KKHTDDGYMPMSPGVA was used at 154 and
71 M for JAK1 and TYK2, respectively. The ATP, in the presence
of 1–2 nM 33P- -ATP, was used at concentration twofold the
Km for each enzyme. The reaction was performed in 50 mM
HEPES pH 7.5 containing 10 mM MgCl2, 2.5 mM DTT, 10
lM for JAK2 and JAK3, respectively. The pep-
l
c
a
5.6. High throughput solubility
lM
Na3VO4 and 0.2 mg/mL BSA. At the end of the kinase reaction,
all the unreacted ATP is captured by an excess of Dowex ion-
exchange resins (SIGMA, DOWEX 1 ꢂ 8, 200–400 mesh) previously
prepared by three overnight washes with 150 mM sodium
formate, pH 3.0. The supernatant is subsequently withdrawn
and transferred into a counting plate and analysed by b-counting.
Data per each compound was analyzed by an internally
customized version of the SW package ‘Assay Explorer’ which
provides sigmoidal fittings of the ten dilution curves for IC50
determination.
Selectivity against other kinases was evaluated on an internally
developed Kinase Selectivity Screening (KSS) panel, designed to
represent the overall diversity of the kinome, as described in detail
in a recent publication.33 The panel includes: ABL, ACK1, AKT1, Alk,
AUR1, AUR2, BRK, CDC7, CDK2/CYCA, CHK1, CK2alpha/beta, eEF2K,
EGFR1, ERK2, FAK, FGFR1, Flt3, GSK3beta, Haspin V473-K798,
IGFR1, IKK2, IR, JAK1, JAK2, JAK3, KIT, LCK, MELK, MET, MK2,
MPS1, MST4, NEK6, NIM, P38alpha, PAK4, PDGFRb, PDK1, PERK,
PIM1, PIM2, PKAalpha, PKCbetaII, PLK1, RET, SULU1, SYK, TRKA,
TYK2, VEGFR2, VEGFR3, ZAP70.
Solubility at pH 7 was performed as previously described.34
5.7. In vivo pharmacokinetics
The pharmacokinetic profiles of compounds were investigated
in overnight fasted male Nu/Nu mice following a single dose given
intravenously (iv) and orally (po). The vehicle used was 10% Tween
80 in 5% dextrose solution for iv administration and 0.5% methocel
for os administration. A total of three mice were treated for each
administration. Blood samples of each mouse were collected from
the saphenous vein at predose, 0.083, 0.5, 1, 6, and 24 h postdosing.
Samples were centrifuged at 10,000g for 3 min at 4 °C and the
plasma was stored at ꢃ80 °C until analysis. Samples were analyzed
by LC/MS/MS technique.
5.8. In vivo pharmacology
Acute megakaryoblastic leukemia cell line SET-2 (107 cells) was
inoculated s.c. in 5–6 weeks old female severe combined immuno-
deficient (SCID) mice (Charles River) previously exposed to gamma
irradiation (200 Rads of whole body gamma irradiation). Mice
bearing a palpable tumour (100–200 mm3) were treated with the
vehicle (0.5% methocel) or the compound. Tumour dimensions
were measured regularly using Vernier calipers and tumour vol-
ume was calculated according to the following formula: tumour
volume (mm3) = length ꢂ width2/2. The tumour growth inhibition
5.4. Cells proliferation assay
JAK2 dependent human megakaryoblastic leukemia SET-2 cell
line (DSMZ, Braunschweig GERMANY), human IL-2 dependent T
cell lymphoma DERL-7 cell line (DSMZ, Braunschweig GERMANY)
and human ovarian carcinoma A2780 (ECACC, Salisbury, UK) were
cultured in RPMI 1640 medium Glutamax (Gibco BRL, Gaithesburg,
MD, USA), supplemented with 10% fetal bovine serum (FBS) at
37 °C and 5% CO2. Human IL-2 (20 ng/ml, Sigma-Aldrich, St. Louis,
MO) was added at medium for DERL-7. Approximately 5 ꢂ 103
(% TGI) was calculated according to the equation:
%
TGI = 100 ꢃ (Mean tumour volume of treated group/Mean tumour
volume of control group) ꢂ 100. All procedures adopted for hous-
ing and handling of animals were in strict compliance with Italian
and European guidelines for Laboratory Animal Welfare.
For in vivo mechanism of action studies, mice bearing tumour
were treated with single dose of compound and sacrificed at differ-
ent post treatment times (3 animals/time), the tumours were lysed
and analyzed by Western Blot as described for cells.
cells were plated into 384 microtiter plate wells in 50
l
L of growth
media with different concentrations of inhibitors. The cells were
incubated at 37 °C and 5% CO2 for 72 h, then the plates were
processed using Cell Titer-Glo assay (Promega, Madison, WI,
USA), following the manufacturer’s instruction. Briefly, 25 lL/well
reagent solution is added to each well and after 5 min shacking
micro-plates are read by Envision luminometer (PerkinElmer,
Waltham, MA, USA). Data are analyzed by Symix Assay Explorer
software (Symix Technologies Inc.) that provides sigmoidal
Acknowledgments
We thank Nicoletta Colombo for skillful assistance in NMR
spectra recording and interpretation, Nadia Amboldi and the group
of Cell Screening for cell proliferation assay, and the Experimental
Therapy group for in vivo experiments.
fitting algorithm of the
determination.
8 points dilutions curves for IC50
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.025

M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
15
Lee, F. Y.; Woolfson, A.; Lasho, T. L.; Pardanani, A.; Tefferi, A.; Lorenzi, M. V.
Leukemia 2012, 26, 280.
Supplementary data
18. Ioannidis, S.; Lamb, M. L.; Wang, T.; Almeida, L.; Block, M. H.; Davies, A. M.;
Peng, B.; Su, M.; Zhang, H.-J.; Hoffmann, E.; Rivard, C.; Green, I.; Howard, T.;
Pollard, H.; Read, J.; Alimzhanov, M.; Bebernitz, G.; Bell, K.; Ye, M.; Huszar, D.;
Zinda, M. J. Med. Chem. 2011, 54, 262.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.06.025.
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Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.025