Identification of potent ITK inhibitors through focused compound library design including structural information

Article abstract of DOI:10.1016/j.bmcl.2010.09.119

A series of novel compound libraries inhibiting interleukin-2 inducible T cell kinase (ITK) were designed, synthesized and evaluated. In the first design cycle two library scaffolds were identified showing low micromolar inhibition of ITK. Further iterative design cycles including crystal structure information of ITK and structurally related kinases led to the identification of indolylindazole and indolylpyrazolopyridine compounds with low nanomolar ITK inhibition.

Full text of DOI:10.1016/j.bmcl.2010.09.119

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6998–7003
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of potent ITK inhibitors through focused compound library
design including structural information
Matthias Herdemann , Isabelle Heit , Frank-Uwe Bosch, Gianluca Quintini, Claudia Scheipers à,
⇑
Alexander Weber §,
⇑
Nycomed GmbH, Byk-Gulden-Str. 2, D-78467 Konstanz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel compound libraries inhibiting interleukin-2 inducible T cell kinase (ITK) were designed,
synthesized and evaluated. In the first design cycle two library scaffolds were identified showing low
micromolar inhibition of ITK. Further iterative design cycles including crystal structure information of
ITK and structurally related kinases led to the identification of indolylindazole and indolylpyrazolopyri-
dine compounds with low nanomolar ITK inhibition.
Received 8 August 2010
Revised 23 September 2010
Accepted 24 September 2010
Available online 29 September 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
ITK
Interleukin-2 inducible T cell kinase
ITK inhibitors
Library design
Kinases
Drug design
Docking
Crystal structure
X-ray
Lead finding
Lead optimisation
Focused kinase library
Ligand efficiency
Interleukin-2 inducible T cell kinase (ITK) is a member of the Tec
family of non-receptor tyrosine kinases and is mainly expressed in
T cells, mast cells and natural killer cells.1 In T cells, ITK is activated
after T cell receptor (TCR) stimulation leading to IL-2 production,
proliferation and differentiation.2 In studies with ITKꢀ/ꢀ mice
reduced lung inflammation and impaired T cell response were
observed in an ovalbumin-induced allergic asthma model.3 Similar
results were reported with a selective ITK inhibitor4 indicating the
important role of ITK kinase activity in inflammatory processes.
Therefore, inhibiting ITK should be an attractive approach to inter-
fere with the pathogenesis of T cell-mediated diseases.
approach included structural information provided by X-ray struc-
ture of ITK in complex with Staurosporine,5 as well as in-depth anal-
ysis of published ITK inhibitors (Fig. 1) which were docked into the
ATP binding pocket of ITK. The aim of the project was the design of
novel hit structures which would represent a promising starting
point for subsequent lead finding and lead optimisation. The newly
designed scaffolds should possess high potency on enzyme inhibi-
tion combined with favourable physicochemical properties. Fur-
thermore, the expected synthetic feasibility of the new libraries
were taken into consideration to ensure that subsequent chemical
modifications should be easy to address in following optimisation
steps. In addition, within the first design cycle we focus on small
lead-like compounds6 and therefore restricted the molecular weight
of the synthesized compounds to 350 Da. Expecting low micromolar
inhibition within this first series, the ligand efficiency (LE, calculated
binding energy per non-hydrogen atom)7 of these compounds will
be greater than 0.3, serving as a good starting point for further opti-
misations.8 Furthermore, the published X-ray structure of ITK in
complex with Staurosporine5 was used to support the library design
concept to identify most favourable interactions within the protein.
Reference compounds9 with known ITK inhibitory activity (Fig. 1)
were docked and minimised in the ITK ATP binding pocket (PDB
Here, we report the design of new ITK inhibitors obtained within
an ITK hit identification project. Our initial focused library design
⇑
Corresponding authors. Tel.: +49 7531 84 3130; fax: +49 7531 84 93130 (I.H.).
E-mail addresses: isabelle.heit@nycomed.com (I. Heit), alexander.weber@boeh-
ringer-ingelheim.com (A. Weber).
Present address: EPO, Patentlaan 3-9, 2288 EE The Hague, Netherlands.
Present address: Intervet Innovation GmbH, Zur Probstei, 55270 Schwabenheim,
à
Germany.
§
Present address: Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer
Straße 65, 88397 Biberach an der Riß, Germany.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.119

M. Herdemann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6998–7003
6999
O
N
O
O
N
O
N
N
O
N
S
N
H
O
S
N
S
N
H
N
1
2
NH2
O
N
N
NH
S
O
S
NH2
O
3
Figure 1. Published ITK inhibitors included in focused library design approach.
code: 1sm2),10 leading to a superposition of the most interesting
structural classes that are known to influence ITK activity. Both
the crystal structure of ITK in complex with Staurosporine and the
docked conformations of reference ligands in ITK lead to the identi-
fication of main protein–ligand interactions: (1) hydrogen-bond
interactions with backbone carbonyl oxygen of Glu-436 and back-
bone nitrogen and oxygen of Met-438 in the hinge region (HR), (2)
polar interactions with Ser-499 and Asp-500 side chains and (3)
aromatic and hydrophobic interactions with the gatekeeper (GK)
Phe-435 and a small solvent exposed hydrophobic pocket (Ile-369,
Leu-379, Phe-437). Especially, the hydrogen bonding network of
ligands towards the hinge region in ITK should be similar compared
to structurally related protein–ligand kinase complexes.11 Pub-
lished crystal structures of CDK2 with aminothiazole (PDB code:
2btr, 2bts) and aminopyrazole inhibitors (PDB code: 1vyw, 1vyz)
show that both inhibitor classes bind to CDK2 forming hydrogen
bonds to amino acid backbone atoms in the CDK2 hinge region
(Glu-81, Leu-83).12 A protein-based superposition of those CDK2
X-ray structures with ITK (PDB code: 1sm2) indicate that the hydro-
gen bonding network of both inhibitor classes towards the hinge
region in CDK2 could be formed in ITK, too.
*
*
O
*
a: R =
b: R =
N
e: R =
N
O
N
R
O
N
*
N
N
f: R =
N
N
H
N
c: R =
d: R =
*
*
4a-g
g: R =
N
N
N
O
*
R
O
a: R = H
N
b: R = NO2
N
N
H
5a-b
Figure 2. Indazole and pyrazole derivatives identified after first design cycle.
The potency of the library compounds was tested in an in vitro
Itk kinase assay using the full-length protein.15 After the first syn-
thesis cycle indazole and pyrazole derivatives prepared previously
by amide coupling of the corresponding commercially available
acids were identified with low micromolar ITK inhibition (Fig. 2).
The proposed binding mode of both classes based on docking stud-
ies in ITK is shown in Figure 3. Both lead-like classes possess excel-
lent physicochemical properties and represent good starting points
for further optimisation (MW <350 Da, LE >0.3). A crystal structure
of compound 4a in ITK was not available after the first design cycle,
instead a co-crystal structure of indazole 4a in CDK2 could be ob-
served (data not shown), indicating hydrogen bonding with both
indazole nitrogen atoms to the CDK2 hinge region. Compound 4a
similar size and ligand efficiency. Such compounds would very
likely have a planar structure. This approach is illustrated in
Scheme 1. The transformation of the amide function of compounds
4 to a five-membered ring of their derivatives 6 and 7 should alter
the angle at the amides nitrogen atom. Consequently, the para-
substituents of initial hits 4a–g would have to be attached at posi-
tion 6 of the indole in order to occupy the same space within the
ATP binding pocket. Therefore, the next design cycles were focused
on indazole derivatives of structures 6, as well as on analogous pyr-
azolopyridines 7, attempting to increase the affinity towards the
target kinase ITK while gaining selectivity towards CDK2.
(ITK IC50 = 5
ligand also inhibits CDK2 in the low micromolar range (CDK2
IC50 = 3 M). Interestingly, the results from docking and minimisa-
l
M) was tested against CDK2 which revealed that this
In order to allow an independent variation of substituents at both
moieties of compounds 6 and 7 (Scheme 1), it was decided to com-
bine both heterocyclic fragments in the very last steps of the synthe-
sis. This was done by Suzuki coupling of iodo-indazole 8 or iodo-
pyrazolopyridine 9 respectively with the indol-2-ylboronic acids
10 using the [1,10-bis(diphenylphosphino)ferrocene]palladium(II)
complex Pd(dppf)Cl2–DCM13 as catalyst and cesium carbonate as a
base (Scheme 2). The crude products were then directly deprotected
with potassium carbonate in a mixture of water/methanol/THF to
give the final compounds 6 and 7.16 Compound 6g (R1 = H, R2 = 6-
CO2H) was isolated in 83% overall yield after refluxing the purified
l
tion of 4a in ITK (Fig. 3) indicated that 4a would not be of entirely
planar structure. The docking studies predicted a twist of the ani-
line’s phenyl ring with regard to the indazole-amide moiety. How-
ever, it was assumed that for the binding to ITK, but not for CDK2, a
more planar inhibitor scaffold would be preferred. We considered
that the replacement of the amide function by a five-membered
ring, that is, the replacement of the anilide moiety by an indole,
would be the most effective approach to obtain compounds of

7000
M. Herdemann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6998–7003
BR
BR
GK
Phe435
GK
Phe435
PBR
PBR
Glu436
Phe437
Glu436
O
O
O
HR
HR
HN
HN
H
H
N
Phe437
N
O
N
O
NH
Met438
O
N
NH
Met438
O
H
N
N
H
O
SP
SP
O
SAR
SAR
HR = hinge region, GK = gatekeeper, BR = burried region, PBR = phosphate binding region, SP = sugar pocket,
SAR = solvent accessible region
Figure 3. Proposed binding mode of indazole- and pyrazole derivatives in ITK identified after the first design cycle. Hydrogen bonding interactions of compound 4a (left) and
5a (right) with the hinge region are shown in dotted lines.
was synthesized starting from commercial 3-fluoropyridine
R
R
O
(Scheme 3). After regioselective deprotonation with nBuLi/TMEDA
which was prepared in situ, formylation has been achieved with
DMF. Intermediate 11 was then converted without purification in
two steps to 1H-pyrazolo[4,3-b]pyridine (12). After condensation
of the aldehyde with hydrazine the ring closure was achieved at
130 °C. The yield for the entire sequence from 3-fluoropyridine to
1H-pyrazolo[4,3-b]pyridine (12) was 26–30%. Building block 9 was
then obtained by regioselective iodination of heterocycle 12 under
basic conditions followed by Boc protection (81%). The same reac-
tion conditions as for the transformation of 12 to 9 were applied to
commercially available indazoles for the preparation of Boc pro-
tected iodo-indazoles 8 (80–95% yield). Those boronic acids 10
which were not commercially available were prepared starting from
the corresponding indoles 13 (Scheme 4). After Boc protection the
intermediate 14 was reacted with triisopropoxyborane in THF using
the base lithium diisopropylamide (LDA). Following work-up, indol-
2-ylboronic acids 10 were isolated in 70–80% yield.
A
N
H
N
N
N
N
N
H
H
4
6, 7
A
R2
R1
N
H
N
N
H
6: A = CH
7: A = N
Scheme 1. Second synthesis cycle approach.
The prepared indolylindazoles 6 and indolylpyrazolopyridines 7
were first evaluated in an enzymatic assay using an ITK full-length
enzyme.15 Subsequently, selected compounds were tested for their
inhibition of anti-CD3/anti-CD28 induced interleukin-2 (IL-2)
intermediate of Suzuki coupling with KOH in ethanol. Under these
conditions the methyl carboxylate group was readily cleaved to give
the desired acid function after acidic work-up. The building block 9
R2
I
A
(i), (ii)
NH
R2
R1
B(OH)2
BOC
N
+
A
N
R1
N
N
BOC
N
H
8: A = CH
9: A = N
10
6: A = CH
7: A = N
Scheme 2. Reagents and conditions: (i) Pd(dppf)Cl2–DCM, Cs2CO3, dioxane, 80 °C, 2–4 h, argon; (ii) 4 M K2CO3aq, MeOH, THF, 60 °C, 3–4 h or KOH, EtOH, reflux, 1 h.

M. Herdemann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6998–7003
7001
Table 1
CHO
F
N
N
N
(i), (ii)
(iii), (iv)
Results of selected indole substitutions
N
Compound
A
R1 R2
IC50
(l
M) enzymea IC50 M) cell, IL-2a
(l
N
F
H
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
7f
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.14
0.12
0.13
22
0.35
0.11
2.3
4.6
1.2
—
4.1
0.7
—
2.2
3.2
1.4
—
5.6
0.9
—
11
12
5-OMe
6-OMe
7-OMe
5-Me
6-Me
I
I
N
N
(vi)
(v)
N
N
BOC
N
6-CO2H 3.98
H
N
0.20
0.28
0.06
27
0.30
0.07
0.24
0.23
H
5-OMe
6-OMe
7-OMe
5-Me
6-Me
4-Br
9
Scheme 3. Reagents and conditions: (i) nBuLi/TMEDA, Et2O, ꢀ75 °C, 4 h, argon; (ii)
DMF, Et2O, ꢀ75 °C, 2 h under argon; (iii) N2H4–H2O, rt, 18 h; (iv) 130 °C, 5 h; (v) I2,
KOH, DMF, 0 °C to rt, 2.5 h; (vi) Boc2O, NEt3, DMAP, DMF, 0 °C to rt, 1 h.
N
N
N
7g
7h
6-Br
—
a
IC50 values are means from two to three independent experiments.
release in primary human CD4+ T cells as relevant cellular sys-
tem.17 Within the indolylindazole series, the unsubstituted com-
pound (6a) inhibited ITK with an IC50 of 140 nM in the
Table 2
enzymatic assay and showed an IC50 of 2.3 lM in the cellular assay
Results of selected indazole substitutions
(Table 1). Similar potency was observed for the derivatives having
a methoxy substituent in either position 5 or 6 (6b and 6c). How-
ever, as predicted by docking studies, the introduction of a meth-
oxy substituent at position 7 resulted in a dramatic loss of
activity. Due to steric restrictions at that position in the ITK binding
pocket (Phe-437) the binding affinity of 6d was significantly re-
duced (IC50 = 22 lM). The potencies observed for 6a–c correspond
to a more than 10-fold activity increase compared to those of ini-
tial indazole hits 4 (Fig. 2). In particular, while first generation
compound 4a had an enzymatic IC50 of 5 lM, the corresponding
Compound
R1
R2
IC50
(
l
M) enzymea
IC50 (lM) cell, IL-2
6k
6l
5-Me
6-Me
5-F
H
H
H
H
H
H
0.25
0.16
0.17
0.19
0.08
0.08
0.31
0.09
0.14
0.07
0.08
—
—
—
—
—
—
—
—
—
—
—
6m
6n
6o
6p
6q
6r
6s
6t
6u
6-F
5-CN
6-CN
5-Me
6-Me
6-F
6-Me
6-Me
6-Me
6-Me
6-OMe
6-CN
6-F
second generation compound 6c inhibited ITK enzyme with an
IC50 of 130 nM (Table 1). A methyl substituent at the indole’s posi-
tions 5 or 6 led to similar enzymatic activities as methoxy groups
a
IC50 values are means from two to three independent experiments.
(6e and 6f). In contrast, with an IC50 of about 4 lM compound 6g
unsubstituted compound 6a (6o and 6p). As a further exploration
of the initial SAR, as well as a validation of our docking model,
substituted indoles were combined with substituted indazole frag-
ments. Both, a 6-methyl and a 6-cyano substituent at the indazole
heterocycle enhanced the activity of 6-methyl indolyl compounds
(6r and 6t) compared to parent compound 6f. The same was ob-
served for 6-methoxy indolyl compound (6u) with regard to 6c.
As observed for 6k and 6n with regard to 6a, neither the 5-methyl
nor the 6-fluoro group changed the activities of 6q and 6s com-
pared to 6f. According to the previously discussed criterion of li-
gand efficiency the values of representative compounds of the
first design cycle (LE = 0.30–0.35) were further improved for com-
pounds of the following cycles, that is, indolylindazoles 6 and indo-
lylpyrazolopyridines 7. They reached LE values in the range of up to
0.40–0.50.
In view of its promising potency in the enzymatic assay
(IC50 = 60 nM) compound 7c was used for in-depth molecular mod-
elling analyses. Figure 4 shows the binding modes of the known
benzimidazole ITK inhibitor 2 and pyrazolopyridine 7c based on
docking studies10 in the ITK binding pocket, as well as an alignment
of both compound classes. The main interactions with the protein,
that is, the hydrogen-bond interactions with backbone carbonyl
was significantly less active, due to its carboxylic acid function. A
similar SAR was observed within the pyrazolopyridine series.
Unsubstituted compound 7a had an enzymatic IC50 of 200 nM
and exhibited the same potency in the functional assay as 6a (Table
1). Surprisingly, with an enzymatic IC50 of 60 nM 7c was twofold
more potent than the analogous indolylindazole 6c. However, both
compounds possess the same cellular potency of 1.2 lM. Likewise,
the enzymatic potency of the 5-methyl derivative 7f was slightly
higher than that of 6f. Both compounds 6f and 7f inhibited IL-2
in the functional assay with an IC50 below 1 lM. Furthermore, a
bromo substituent was also tolerated in position 6 of the indole,
as well in position 4 (7g and 7h). With regard to the indazole moi-
ety of compounds 6, our initial work was focussed on the positions
5 and 6 (Table 2). Docking studies10 performed with 4a (Fig. 3)
indicated that the ITK binding pocket should allow substitutions
at both positions. A substituent in position 6 could make favour-
able interactions with gatekeeper Phe-435. However, it should be
limited in size to avoid a steric clash with the gatekeeper residue.
Testing for the affinity to the target enzyme revealed that methyl
and fluoro substituents are tolerated in both positions and do not
alter the enzymatic activity (6k–n). A cyano group, either in posi-
tion 5 or in 6, increases slightly the enzymatic activity compared to
(i)
R2
(ii)
R2
R2
B(OH)2
N
BOC
N
H
N
BOC
14
13
10
Scheme 4. Reagents and conditions: (i) Boc2O, NEt3, DMAP, DMF, 0 °C to rt, 1 h; (ii) B(OiPr)3, LDA, THF, 0 °C, 2 h, argon.

7002
M. Herdemann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6998–7003
BR
GK
Phe435
O
PBR
Glu436
Phe437
HR
S
HN
O
O
NH
NH2
Met438
HN
N
O
O
N
SP
O
SAR
BR
GK
Phe435
O
PBR
Glu436
HR
HN
Phe437
HN
N
O
N
NH
Met438
HN
O
SP
O
SAR
Figure 4. Minimisation of published ITK inhibitor 2 (magenta) and indolylindazole derivative 7c (cyan) in ITK binding pocket result in the shown alignment of both classes.
oxygen of Glu-436 and backbone nitrogen and oxygen of Met-438 in
the hinge region and aromatic and hydrophobic interactions with
the gatekeeper Phe-435 are identical for both ITK inhibitors. As the
alignment indicates, the methoxy group of 7c occupies apparently
the same space as the methyl amide moiety of benzimidazole 2.14
Based on these docking results it appeared that the methoxy group
of 7c could be replaced in further optimisation cycles with larger,
sterically more demanding substituents. In order to verify our initial
hypothesis that a planar inhibitor scaffold might not only increase
ITK binding affinity, but also enhance the selectivity of ITK against
CDK2 inhibition, 7c has also been tested against CDK2 enzyme. In
view of initial hit 4a which inhibited both enzymes almost equipo-
(Table 3). However, 4 out of 32 kinases tested in the panel were
inhibited in the submicromolar range. A modest selectivity was ob-
served with regard to Aurora A, Aurora B and VEGF-R2 (3–5-fold). In
addition, the receptor tyrosine kinase FLT3 was inhibited equipo-
tently. Nevertheless, taking into account that 7c is substituted with
one single methoxy group only the observed selectivity profile was
considered as encouraging.
In conclusion, ITK inhibitors were designed and synthesized in a
focused kinase library approach within iterative cycles. Structural
information of the ITK ATP binding pocket supported both the
identification of main interactions of first generation libraries with
the protein and the successful optimisation of these initial hits in
following syntheses. Representative compounds, such as 7c, are
potent against the target enzyme and in a cellular assay. Moreover,
they exhibit an excellent ligand efficiency and 7c showed promis-
ing initial selectivity in a panel of about 30 kinases. The identified
compound cluster was therefore selected as a main starting point
for the ITK lead finding project.
tently (ITK IC50 = 5 lM vs CDK2 IC50 = 3 lM), the selectivity ob-
served for 7c was significantly higher than expected. Compound
7c is almost 70-fold more potent against ITK compared to CDK2
(ITK IC50 = 0.06 lM versus CDK2 IC50 = 4 lM). Subsequent external
profiling of 7c in a kinase panel applying similar assay conditions18
confirmed the selectivity with regard to cyclin dependent kinases

M. Herdemann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6998–7003
7003
Table 3
11. (a) Ghose, A. K.; Herbertz, T.; Pippin, D. A.; Salvino, J. M.; Mallamo, J. P. J. Med.
Chem. 2008, 51, 5149; (b) McInnes, C. Curr. Opin. Drug Discov. Devel. 2006, 9,
339.
Selectivity profiling of compound 7c in a panel of 32 kinases
Kinase
IC50
(l
M) enzymea,b
Kinase
IC50 (l
M) enzymea,b
12. (a) Kim, K. S.; Kimball, S. D.; Misra, R. N.; Rawlins, D. B.; Hunt, J. T.; Xiao, H. Y.;
Lu, S.; Qian, L.; Han, W. C.; Shan, W.; Mitt, T.; Cai, Z. W.; Poss, M. A.; Zhu, H.;
Sack, J. S.; Tokarski, J. S.; Chang, C. Y.; Pavletich, N.; Kamath, A.; Humphreys, W.
G.; Marathe, P.; Bursuker, I.; Kellar, K. A.; Roongta, U.; Batorsky, R.; Mulheron, J.
G.; Bol, D.; Fairchild, C. R.; Lee, F. Y.; Webster, K. R. J. Med. Chem. 2002, 45, 3905;
(b) Misra, R. N.; Xiao, H. Y.; Kim, K. S.; Lu, S.; Han, W. C.; Barbosa, S. A.; Hunt, J.
T.; Rawlins, D. B.; Shan, W.; Ahmed, S. Z.; Qian, L.; Chen, B. C.; Zhao, R.; Bednarz,
M. S.; Kellar, K. A.; Mulheron, J. G.; Batorsky, R.; Roongta, U.; Kamath, A.;
Marathe, P.; Ranadive, S. A.; Sack, J. S.; Tokarski, J. S.; Pavletich, N. P.; Lee, F. Y.;
Webster, K. R.; Kimball, S. D. J. Med. Chem. 2004, 47, 1719; (c) Pevarello, P.;
Brasca, M. G.; Orsini, P.; Traquandi, G.; Longo, A.; Nesi, M.; Orzi, F.; Piutti, C.;
Sansonna, P.; Varasi, M.; Cameron, A.; Vulpetti, A.; Roletto, F.; Alzani, R.;
Ciomei, M.; Albanese, C.; Pastori, W.; Marsiglio, A.; Pesenti, E.; Fiorentini, F.;
Bischoff, J. R.; Mercurio, C. J. Med. Chem. 2005, 48, 2944; (d) Pevarello, P.;
Brasca, M. G.; Amici, R.; Orsini, P.; Traquandi, G.; Corti, L.; Piutti, C.; Sansonna,
P.; Villa, M.; Pierce, B. S.; Pulici, M.; Giordano, P.; Martina, K.; Fritzen, E. L.;
Nugent, R. A.; Casale, E.; Cameron, A.; Ciomei, M.; Roletto, F.; Isacchi, A.;
Fogliatto, G.; Pesenti, E.; Pastori, W.; Marsiglio, A.; Leach, K. L.; Clare, P. M.;
Fiorentini, F.; Varasi, M.; Vulpetti, A.; Warpehoski, M. J. Med. Chem. 2004, 47,
3367.
ABL1
7.68
0.19
0.27
—
IKK-beta
MEK-1
NEK-2
p38-alpha
PAK2
—
Aurora A
Aurora B
B-RAF
2.45
3.22
—
—
—
3.20
—
22.40
—
CDC42 BpB
CDK1/CycB
CDK2/CycA
CDK4/CycD1
CDK5/p25
CDK7/CycH1
CDK9/CycT
CHK1
—
5.23
5.38
1.45
7.41
1.78
—
5.94
—
—
PBK
PDGFR-beta
PKC-beta1
PKC-theta
PLK1
S6-Kinase
SAK
SRC
TTK
VEGF-R2
WEE1
—
3.82
1.31
8.75
0.31
5.72
CK2-alpha1
DAPK1
FAK
5.49
0.06
FLT3
a
IC50 values calculated from a single experiment.
No data: IC50 >30 lM.
13. (a) Jiang, N.; Ragauskas, A. J. Tetrahedron Lett. 2006, 47, 197; (b) Beletskaya, I. P.;
Ganina, O. G.; Tsvetkov, A. V.; Fedorov, A. Y.; Finet, J.-P. Synlett 2004, 2797.
14. Cook, B. N.; Bentzien, J.; White, A.; Nemoto, P. A.; Wang, J.; Man, C. C.;
Soleymanzadeh, F.; Khine, H. H.; Kashem, M. A.; Kugler, S. Z.; Wolak, J. P.; Roth,
G. P.; De Lombaert, S.; Pullen, S. S.; Takahashi, H. Bioorg. Med. Chem. Lett. 2009,
19, 773.
b
Acknowledgements
15. Protein kinase scintillation proximity assay (enzymatic assay): 150 ng purified
recombinant baculovirus-expressed GST-Itk full-length was incubated in 50 ll
We thank J. Wilsberg, T. Piper and Dr. I. Schlemminger for skilful
synthetic support, M. Beschle and K.-M. Nagy for LC–MS purifica-
tions, Dr. H.-C. Holst and T. Kottysch for NMR analyses, A. Borkowski,
S. Braun, M. Nickel, Dr. M. Schäfer and Dr. T. Ciossek for biological
testing.
kinase assay buffer (2 mM MOPS/NaOH, pH 7.0, 5 mM MgCl2, 5 mM MnCl2, 0.5%
glycerol, 0.1 mM EDTA, pH 7.4, 1 mM DTT, 0.0001% Brij35, 0.3 mg/ml BSA)
containing 0.1 mM ATP, 2 mCi [33P]-ATP and 1,5
lM of biotinylated JAK2 peptide
(Biotin-ßAla-ßAla-KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC-NH2) in
Wallac Isoplates (Perkin–Elmer). Substrate phosphorylation was measured by
incorporation of 33Pi from
c
-[33P]-ATP (Amersham). After 30 min at room
temperature, the reaction was stopped by adding 150 ml stop solution (10 mM
ATP, 1.33 mg/ml streptavidin-coated yttrium silicate beads (Amersham), 5 mM
EGTA, pH 7.5, 0.1% Triton X-100). Radioactivity was counted with the Wallac
Micro b1450 (Perkin–Elmer).
References and notes
1. (a) Yamada, N.; Kawakami, Y.; Kimura, H.; Fukamachi, H.; Baier, G.; Altman, A.;
Kato, T.; Inagaki, Y.; Kawakami, T. Biochem. Biophys. Res. Commun. 1993, 192,
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638; (b) Fowell, D. J.; Shinkai, K.; Liao, X. C.; Beebe, A. M.; Coffman, R. L.;
Littman, D. R.; Locksley, R. M. Immunity 1999, 11, 399.
16. General procedure for Suzuki coupling and Boc deprotection: Under argon
indazole building block 8 or 9 (0.30 mmol) and boronic acid 10 (0.39 mmol)
were dissolved in dioxane (3 mL). Subsequently, 2 M Cs2CO3aq (1.2 mmol) and
then palladium catalyst Pd(dppf)2Cl2–DCM (0.015 mmol) were added. The
reaction mixture was stirred at 80 °C for 2–4 h. The organic and aqueous layers
were separated, the aqueous phase was extracted with AcOEt (2 ꢁ 2 mL). The
organic phases were filtered over aluminum oxide. After evaporation of the
filtrate under reduced pressure the residue was dissolved in MeOH (2 mL). THF
(2 mL) and 4 M K2CO3aq (2 mL) were added and the mixture was stirred
vigorously at 60 °C over 3–4 h. The mixture was condensed under reduced
pressure and AcOEt (5 mL) was added. The layers were separated and the
aqueous layer was extracted with AcOEt (3 ꢁ 5 mL). The combined organic
layers were dried over Na2SO4 and evaporated under reduced pressure. The
crude products were purified either by preparative HPLC (C18, acetonitrile/
water at pH 3.75, ammonium formate buffer) or by flash chromatography
(silica gel, cyclohexane/AcOEt).
3. Mueller, C.; August, A. J. Immunol. 2003, 170, 5056.
4. Lin, T. A.; McIntyre, K. W.; Das, J.; Liu, C.; O’Day, K. D.; Penhallow, B.; Hung, C.
Y.; Whitney, G. S.; Shuster, D. J.; Yang, X.; Townsend, R.; Postelnek, J.; Spergel, S.
H.; Lin, J.; Moquin, R. V.; Furch, J. A.; Kamath, A. V.; Zhang, H.; Marathe, P. H.;
Perez-Villar, J. J.; Doweyko, A.; Killar, L.; Dodd, J. H.; Barrish, J. C.; Wityak, J.;
Kanner, S. B. Biochemistry 2004, 43, 11056.
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17. CD4+ T cell assay: CD4+ T cells were isolated from human whole blood (citrate
0.31% w/v) by Percoll gradient centrifugation followed by negative selection
with CD4+ T cell Isolation Kit II (Miltenyi) according to the manufacturer’s
instructions. CD4+ T cells were seeded 2 ꢁ 105 cells/well in 96-MTP in RPMI
6. (a) Stocks, M. J.; Wilden, G. R.; Pairaudeau, G.; Perry, M. W.; Steele, J.;
Stonehouse, J. P. ChemMedChem 2009, 4, 800; (b) Hann, M. M.; Oprea, T. I. Curr.
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Bembenek, S. D.; Tounge, B. A.; Reynolds, C. H. Drug Discovery Today 2009, 14,
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W.; Shuster, D. J.; O’Day, K. D.; Penhallow, B.; Hung, C. Y.; Doweyko, A. M.;
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D. J.; O’Day, K. D.; Penhallow, B.; Hung, C. Y.; Kanner, S. B.; Lin, T. A.; Dodd, J. H.;
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www.moloc.ch.
1640 medium containing 10% heat-inactivated FBS, 2 mM L-Glutamine and 1%
penicillin/streptomycin (Gibco). The cells were preincubated with test
compounds for 30 min (0.1% DMSO final concentration) and stimulated with
0.3
lg/well plate bound a-CD3 (OKT3, Janssen&Cilag)) and 3
lg/ml a-CD28
(Beckman) for 48 h at 37 °C. 150
ll of the supernatants were collected. The IL2
release was measured by electro-chemiluminescence (Bioveris) according to
the manufacturer’s protocol using anti-IL2 antibodies from R&D Systems.
18. Kinase selectivity profiling: The selectivity of compound 7c was characterised in
a kinase panel test (ProQinase GmbH, Freiburg, Germany). Thirty two kinases
were tested in a radiometric [33P] in vitro kinase assay (FlashPlate™) at an ATP
concentration of 100 nM. Compound 7c was applied in dose response curves
from 10 nM to 30 lM to calculate IC50 values.