Identification of a novel and selective series of Itk inhibitors via a template-hopping strategy

Article abstract of DOI:10.1021/ml400206q

Inhibition of Itk potentially constitutes a novel, nonsteroidal treatment for asthma and other T-cell mediated diseases. In-house kinase cross-screening resulted in the identification of an aminopyrazole-based series of Itk inhibitors. Initial work on this series highlighted selectivity issues with several other kinases, particularly AurA and AurB. A template-hopping strategy was used to identify a series of aminobenzothiazole Itk inhibitors, which utilized an inherently more selective hinge binding motif. Crystallography and modeling were used to rationalize the observed selectivity. Initial exploration of the SAR around this series identified potent Itk inhibitors in both enzyme and cellular assays.

Full text of DOI:10.1021/ml400206q

Letter
pubs.acs.org/acsmedchemlett
Identification of a Novel and Selective Series of Itk Inhibitors via a
Template-Hopping Strategy
Catherine M. Alder, Martin Ambler,† Amanda J. Campbell, Aurelie C. Champigny, Angela M. Deakin,
John D. Harling, Carol A. Harris, Tim Longstaff, Sean Lynn, Aoife C. Maxwell,* Chris J. Mooney,
Callum Scullion,‡ Onkar M. P. Singh, Ian E. D. Smith, Donald O. Somers, Christopher J. Tame,
Gareth Wayne, Caroline Wilson,§ and James M. Woolven
Respiratory Therapy Area Unit, GlaxoSmithKline Pharmaceuticals, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1
2NY, U.K.
S
* Supporting Information
ABSTRACT: Inhibition of Itk potentially constitutes a novel,
nonsteroidal treatment for asthma and other T-cell mediated
diseases. In-house kinase cross-screening resulted in the
identification of an aminopyrazole-based series of Itk
inhibitors. Initial work on this series highlighted selectivity
issues with several other kinases, particularly AurA and AurB.
A template-hopping strategy was used to identify a series of
aminobenzothiazole Itk inhibitors, which utilized an inherently
more selective hinge binding motif. Crystallography and
modeling were used to rationalize the observed selectivity.
Initial exploration of the SAR around this series identified potent Itk inhibitors in both enzyme and cellular assays.
KEYWORDS: Interleukin-2 inducible tyrosine kinase, Itk, kinase inhibitors, aminobenzothiazole, template hopping, kinase selectivity
nterleukin-2 inducible tyrosine kinase (Itk) is a nonreceptor
Iprotein tyrosine kinase that is expressed in T cells, mast cells,
and NK cells. Itk plays an important role in signaling,
downstream of the T cell receptor in response to antigen
presentation by MHC proteins, and its inhibition leads to
reduced levels of key inflammatory cytokines.1 In vivo
experiments with Itk knockout mice suggest a role for Itk
inhibitors in the treatment of asthma.2
A number of Itk inhibitor series have been disclosed in the
literature with a focus on achieving broad kinase selectivity as
well as good levels of cellular activity; both of which have been
relatively challenging for this tyrosine kinase.3−6 Despite these
publications, there have been no reports of an Itk inhibitor
entering clinical trials and hypotheses regarding its clinical
potential remain untested.7
Figure 1. Structure and activity of aminopyrazole-based Itk inhibitors.
Activity data presented as pKi.13
In-house cross screening resulted in the identification of a
series of aminopyrazoles as inhibitors of Itk. This series was of
particular interest to us as, in contrast to previous series
investigated, compounds in this series displayed a promising
level of ligand efficiency (LE = 0.36, compound 1).8 An initial
X-ray crystal structure of compound 1 (Figure 1) in Itk
confirmed the aminopyrazole group was binding to the hinge
region of Itk and utilizing a three-point hinge binding motif.
Initial optimization work focused on the pyrimidine 2-position
and the pendant group of the pyrazole. However, these
modifications did not produce compounds with the desired
100-fold selectivity margin over key kinases, namely, LCK,
AurA, and AurB. Compound 2 (Figure 1) represents the best
combination of potency and selectivity achieved with this series.
Substantial SAR knowledge had been built up around the other
parts of the template at this stage, and it was hypothesized that
replacing the aminopyrazole motif with an inherently more
selective hinge-binder could be an efficient method of accessing
novel and selective Itk inhibitors.
A robust Itk crystallography system was not available at this
time, and therefore a fragment based approach9−11 using
crystallography to identify new hinge-binding groups was not
Received: May 28, 2013
Accepted: August 12, 2013
© XXXX American Chemical Society
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feasible. Instead, two different methods for selecting alternative
hinge-binders were utilized. The first of these used an in-house
set of compounds specifically chosen for their potential to bind
to the hinge region of a kinase.12 This set was screened against
Itk and any hits selected for use in this work.
Additionally, a set of low molecular weight hits from an
historical high-throughput screen, together with ongoing cross-
screening hits that had not yet been followed-up, were re-
examined. Hits of interest were rescreened at higher compound
concentration if necessary. This set of hits was analyzed to
identify hinge-binders of interest. Together, these methods
enabled us to select a set of aminoheterocycles to act as
replacement hinge-binders.
The potency and selectivity criteria set at the start of this
work were a pKi of at least 7.5 in the Itk enzyme assay
combined with a minimum 100-fold selectivity over AurA and
AurB (due to their fundamental role in cell cycle regulation16).
Potential for further optimization was also a desirable quality.
Some key results from this set of compounds are shown in
Table 1. A variety of different heterocycles were targeted as
replacement hinge-binders. Most of these had some activity at
Itk although many were at least as active at Aurora (e.g., 3a,
Table 1). Interestingly, it was observed that the SAR from the
aminopyrazole series was not always transferable to this set of
compounds. Compounds 3d and 4d (Table 1) are direct
analogues that differ only in the group at the 2-position. The
trans-aminocyclohexanol analogue meets the potency criteria,
while the prolineamide analogue is inactive in the Itk assay.
More encouraging results were obtained with benzothiazole-
based inhibitors. Both the benzothiazole 3f and aza-
benzothiazole 3e analogues fulfilled the potency and selectivity
criteria that were set at the start of the work. The L-
prolineamide analogue 4f was 100-fold less active.
A set of compounds was designed to rapidly evaluate the
potential of these new hinge-binders. The central pyrimidine
ring of the original template was retained although the 6-benzyl
group was replaced with the fluorinated derivative (Figure 2)
From our work on the aminopyrazole series, it seemed likely
that the des-fluoro analogue of compound 3f would
demonstrate an increase in Itk potency. This was indeed the
case with a 5-fold increase in potency observed (compound 5,
Table 1), and gratifyingly, it also retained the selectivity window
over AurA and AurB.17 Although selectivity over Lck was not
ideal at this stage, it was perceived to be a solvable issue. The
Aurora selectivity was the key attribute that had not been
demonstrated with previous series. These benzothiazole-based
inhibitors also demonstrated encouraging cellular data:
compound 5 achieved a pIC50 of 7.6 in a cell assay measuring
inhibition of IFNγ production from PBMCs.
Figure 2. General structure of hinge-binding replacement set.
which was tolerated with minimal loss of potency and greatly
contributed to synthetic ease. The aminoheterocycles chosen to
act as replacement hinge-binders were introduced in the 4-
position and each hinge-binder was synthesized as both the
trans-aminocyclohexanol and L-prolineamide analogues (Figure
2). These moieties had produced the best overall results in the
aminopyrazole series.
The next step was to rationalize the Aurora selectivity
observed with the aim of increasing Itk potency while
maintaining Aurora selectivity. Without the benefit of a crystal
structure of a compound from this series at the time, we used
computational models derived from available Itk and Aurora
crystal structures to guide us. Compound 3f was docked into
Itk (GLIDE SP, Schrodinger Inc.19), showing clearly that the
C4−C5 region of the benzothiazole/thiazolopyridine ring fitted
well against Val419, adjacent to the gatekeeper residue, Phe435,
while maintaining a strong hinge-binding motif. It was observed
that the equivalent residue in Aurora (Leu194) protrudes into
the space occupied by C4−C5 as docked into Itk. Examination
of previous models of nonselective inhibitors showed that
hitherto this critical space in the receptor had not been
occupied. It was hypothesized that the steric clash between the
benzothiazole/thiazolopyridine C4−C5 and Aurora’s Leu194
side chain could explain the observed selectivity of this series.
The model was used to design a small set of aminothiazole
analogues to test our hypothesis. Compounds that presented a
methyl group at position 4 of the thiazole (e.g., compound 6),
equivalent to the benzothiazole/thiazolopyridine C4, exhibited
the same significant selectivity for Itk over Aurora. Absence of a
4-position substituent abolished all selectivity (e.g., compound
7).
This set was synthesized by the general method shown in
Scheme 1. Methyl 2,6-dichloropyrimidine-4-carboxylate 18 was
a
Scheme 1
a
Reagents and conditions: (a) 4-FPhMgBr (2 M), THF, −78 °C, N2,
30 min. (b) DAST, DCM, RT, N2, 20 h. (c) aminoheterocycle, NaH,
THF, 50 °C. (d) amine, DIPEA, IPA, 160−170 °C, 1−3 h, microwave.
It was now apparent that an inherently more selective hinge-
binder had been identified. The next step was to explore
substitution around the benzothiazole ring and scope out the
initial SAR. At this time, because of the increasing Itk activity of
this series, it was necessary to reconfigure the assay to run with
a higher concentration of the substrate ATP.20,21 This increased
competition with inhibitors of the ATP binding site and
treated with 4-fluorophenylmagnesium bromide, and the
resulting ketone 7 reacted with DAST to yield the general
pyrimidine intermediate 19.14 Reaction with the chosen
aminoheterocycles was typically carried out in the presence of
sodium hydride, and each intermediate 21 was reacted
separately with both trans-aminocyclohexanol and L-prolinea-
mide using microwave heating in the presence of DIPEA.15
B
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Table 1. Replacement Hinge-Binder Compound Set, Activity, and Selectivity Data
effectively shifted the pKi range of the assay upward making it
possible to differentiate between our most potent compounds.
Substitution at the 5-position resulted in a drop-off in potency
at Itk (Table 2, compound 8), adding further weight to our
theory that the benzothiazole core is a close fit against the
kinase surface in Itk. Limited work was carried out at the 7-
position at this time, although it was observed that replacement
of hydrogen with bromine did not result in any significant
change in Itk potency. The 6-position was explored in more
detail, and it was observed that a range of substituents were
tolerated at this position (compounds 10−14) the highlight
being the 6-ethyl analogue 13 (LE = 0.38), which was inactive
against AurA and highly selective over AurB. Switching to the
thiazolopyridine core (compounds 15−17) generally resulted
in compounds that maintained their Itk potency and selectivity
over Lck, AurA, and AurB.
Compound 13 had demonstrated the best selectivity against
a limited in-house panel of kinases and hence was chosen as the
series exemplar for profiling against a wider panel of kinases
(Table 3). After further in-house screening and profiling at
Upstate,22 compound 13 was found to inhibit only 3 other
kinases at a pIC50 greater than 7. Importantly, in addition to the
desired selectivity over Aurora,23 this compound was also
sufficiently selective over the kinase Btk, which is closely related
to Itk and part of the same TEC family of kinases. The main
selectivity issue with this compound (and this series) was the
activity observed against IRAK4, Lck, and related kinase Src.
Compound 13 also achieved a pIC50 of 7.3 in the cell assay,
measuring inhibition of IFNγ production from PBMCs.
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Table 2. Representative SAR and Benzothiazole Ring System
compd
R1
R2
X
Itk pKi
Lck pKi
AurB pKi
a
8
Me
H
Me
H
H
H
H
H
H
H
H
H
C
C
C
C
C
C
C
N
N
N
<5
<4.6
6.5
6.6
7.1
7.3
7.0
7.2
7.1
7.0
7.0
4.7
6.4
6.5
6.6
6.6
6.4
7.1
6.5
7.0
7.2
9
9.0
9.4
9.5
10
11
12
13
14
15
16
17
Me
Cl
a
CF3
Et
7.9
9.2
9.6
9.7
9.6
8.8
CN
OMe
H
Et
a
pKi measured at low ATP concentration.21
Table 3. Kinase Selectivity Profile of Compound 13
kinase
enzyme pIC50
kinase
enzyme pIC50
IRAK4
Src
8.4
7.4
7.1
6.9
6.8
6.8
CAMKK2
Tec
6.7
6.6
6.5
6.4
6.4
6.2
TXK
Lck
Btk
LRRK2
AurB
Bmx
IR
EGFR
At this time, the 2.0 Å resolution cocrystal structure of Itk in
complex with compound 3e, one of our initial thiazolopyridine
hits, was obtained (Figure 3a).24 Compound 3e binds in the
ATP-binding domain of Itk; the aminothiazolopyridine core
makes two H-bonding interactions with the backbone carbonyl
and NH of Met438 in the hinge region and is flanked by the
side chains of Ala389 and Leu489. Compound 3e appears to
adopt a conformation stabilized by a water-bridged hydrogen-
bonding network between the methoxy, pyridinyl, and
cyclohexanol hydroxyl groups. The 6-methoxy group is located
in a predominately hydrophobic subsite formed by gatekeeper
Phe435, Val419, Ser499, and Asp500 of the DFG motif.
The coplanar pyrimidinyl moiety resides within the space
formed by Gly441 and the side chains of Ile369 and Phe437,
directing the aminocyclohexanol moiety to a region where it
stacks against the glycine-rich loop and Cys442. Additionally,
there are long-range water mediated hydrogen bonds to C-
terminal domain Asp445 side chain and Arg486 main chain
carbonyl and the (moderately disordered) side chains of
catalytic residues Lys391 and Asp500.
Modeling predicted this binding mode of compound 3e, and
an overlay of Aurora A (Figure 3b) corroborated our view of
the beneficial role of Val419 in Itk versus the larger Leu194
residue found at that position in AurA. It suggested that Leu194
would clash with the inhibitor, thus impeding binding and
leading to the observed selectivity.
The crystal structure of 3e in Itk also highlighted the
importance of Ser499, which is involved in a series of water
mediated hydrogen bonds to the trans-aminocyclohexanol
moiety. Analysis of 491 protein kinases revealed that serine is
Figure 3. (a) Cocrystal structure of compound 3e bound to Itk kinase
domain.24 Omit map electron density (at 3σ) is shown. (b) Connolly
surfaces of compound 3e (green) and neighboring residue (Val419) of
cocrystal structure overlaid with the equivalent AurA residue (Leu194,
colored purple) defined from selected AurA crystal structures.25,26
This overlay highlights a possible reason for the improved selectivity of
the Itk aminobenzothiazole inhibitor series with respect to Aurora.
relatively rare at this position (less than 18% of kinases
analyzed). Targeting this residue in order to further increase
selectivity and potency was the next strategy that we explored,
and the results of this work will form the basis of forthcoming
publications.
In summary, we have identified a novel and selective series of
Itk inhibitors using a template-hopping strategy. Excellent
selectivity against key selectivity kinases AurA and AurB was
achieved. Modeling and crystallography were instrumental in
understanding the selectivity achieved against AurA and AurB
and formulating strategies to further improve potency against
Itk while retaining selectivity.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental data for the synthesis and characterization of new
compounds, assay protocols, and X-ray crystallography data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
D
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ACS Medicinal Chemistry Letters
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(9) Erlanson, D. A.; McDowell, R. S.; O’Brien, T. Fragment-based
drug discovery. J. Med. Chem. 2004, 47, 3463−3482.
Accession Codes
The PDB accession code for the X-ray cocrystal structure of Itk
+ 3e is 4L7S.
AUTHOR INFORMATION
Corresponding Author
*(A.C.M.) Tel: +44 1438763791. E-mail: aoife.c.maxwell@gsk.
com.
■
(10) Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J. Recent
developments in fragment-based drug discovery. J. Med. Chem. 2008,
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(11) Scott, D. E.; Coyne, A. G.; Hudson, S. A.; Abell, C. Fragment-
based approaches in drug discovery and chemical biology. Biochemistry
2012, 51, 4990−5003.
Present Addresses
†(M.A.) MRC Technology, 1−3 Burtonhole Lane, Mill Hill,
London, NW7 1AD, U.K.
(12) Bamborough, P.; Brown, M. J.; Christopher, J. A.; Chung, C.;
Mellor, G. W. Selectivity of kinase inhibitor fragments. J. Med. Chem.
2011, 54, 5131−5143.
‡(C.S.) University of Strathclyde, 295 Cathedral Street,
Glasgow, G1 1XL, U.K.
§(C.W.) School of Life Sciences, Sir James Black Centre,
University of Dundee, Dow Street, Dundee, DD1 5EH, U.K.
(13) Hancock, A. A.; Bush, E. N.; Stanisic, D.; Kyncl, J. J.; Lin, C. T.
Data normalization before statistical analysis: keeping the horse before
the cart. Trends Pharmacol. Sci. 1998, 9, 29−32.
Author Contributions
(14) Fauq, A. H.; Singh, R. P.; Meshri, D. T. Encyclopedia of Reagents
for Organic Synthesis; Wiley & Sons: New York, 2006.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(15) Alder, C. M.; Baldwin, I. R.; Barton, N. P.; Campbell, A. J.;
Champigny, A. C.; Harling, J. D.; Maxwell, A. C.; Simpson, J. K.;
Smith, I. E. D.; Tame, C. J. Preparation of Pyrimidine Derivatives As
Itk Kinase Inhibitors. WO 2010/106016 A1.
Notes
The authors declare no competing financial interest.
(16) Kelly, K. R.; Ecsedy, J.; Mahalingam, D.; Nawrocki, S. T.;
Padmanabhan, S.; Giles, F. J.; Carew, J. S. Targeting Aurora kinases in
cancer treatment. Curr. Drug Targets 2011, 12, 2067−2078.
(17) AurA and AurB share 71% identity within the catalytic domain18
and the key Leu residue is common to both. AurA and AurB activity
tracked closely for this chemotype; AurB activity data was used to
guide the medicinal chemistry strategy and hence is quoted here.
(18) Carmena, M.; Earnshaw, W. C. The cellular geography of aurora
kinases. Nat. Rev. Mol. Cell. Biol. 2003, 4, 842−854.
ABBREVIATIONS
■
ATP, adenosine triphosphate; AurA, Aurora A; AurB, Aurora B;
Btk, Bruton’s tyrosine kinase; DAST, diethylaminosulfur
trifluoride; DCM, dichloromethane; DIPEA, N,N-diisopropy-
lethylamine; IKKβ, inhibitor of nuclear factor kappa-β kinase
subunit beta; IPA, isopropyl alcohol; IRAK4, Interleukin-1
receptor associated kinase 4; Itk, interleukin-2 inducible
tyrosine kinase; LCK, lymphocyte-specific protein tyrosine
kinase; MHC, major histocompatibility complex; PBMC,
peripheral blood mononuclear cell; SAR, structure−activity
relationships; TEC, tyrosine kinase expressed in hepatocellular
carcinoma; THF, tetrahydrofuran
(19) Glide, version 4.5; Schrodinger, LLC: New York, 2007.
̈
(20) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition
constant (K1) and the concentration of inhibitor which causes 50%
inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973,
22, 3099−3108.
(21) Table 2 includes data from both standard (low ATP) and
desensitized (high ATP) Itk assay formats as described in the
Supporting Information. All other data described was generated using
the standard assay format.
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