Structure-based design and synthesis of potent benzothiazole inhibitors of interleukin-2 inducible T cell kinase (ITK)

Article abstract of DOI:10.1016/j.bmcl.2013.09.069

Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demons

Full text of DOI:10.1016/j.bmcl.2013.09.069

Accepted Manuscript
Structure-based Design and Synthesis of Potent Benzothiazole Inhibitors of In-
terleukin-2 Inducible T Cell Kinase (ITK)
Colin H. MacKinnon, Kevin Lau, Jason D. Burch, Yuan Chen, Jonathon Dines,
Xiao Ding, Charles Eigenbrot, Alexander Heifetz, Allan Jaochico, Adam
Johnson, Joachim Kraemer, Susanne Kruger, Thomas M. Krülle, Marya
Liimatta, Justin Ly, Rosemary Maghames, Christian A.G.N. Montalbetti, Daniel
F. Ortwine, Yolanda Pérez-Fuertes, Steven Shia, Daniel B. Stein, Giancarlo
Trani, Darshan G. Vaidya, Xiaolu Wang, Steven M. Bromidge, Lawren C. Wu,
Zhonghua Pei
PII:
S0960-894X(13)01143-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.09.069
BMCL 20921
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 July 2013
17 September 2013
23 September 2013
Please cite this article as: MacKinnon, C.H., Lau, K., Burch, J.D., Chen, Y., Dines, J., Ding, X., Eigenbrot, C.,
Heifetz, A., Jaochico, A., Johnson, A., Kraemer, J., Kruger, S., Krülle, T.M., Liimatta, M., Ly, J., Maghames, R.,
Montalbetti, C.A.G., Ortwine, D.F., Pérez-Fuertes, Y., Shia, S., Stein, D.B., Trani, G., Vaidya, D.G., Wang, X.,
Bromidge, S.M., Wu, L.C., Pei, Z., Structure-based Design and Synthesis of Potent Benzothiazole Inhibitors of
Interleukin-2 Inducible T Cell Kinase (ITK), Bioorganic & Medicinal Chemistry Letters (2013), doi: http://
dx.doi.org/10.1016/j.bmcl.2013.09.069
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Graphical Abstract
Structure-based Design and Synthesis of Potent
Benzothiazole Inhibitors of Interleukin-2 inducible T
Cell Kinase (ITK)
Leave this area blank for abstract info.
Colin H. MacKinnona, Kevin Laub, Jason D. Burchb, Yuan Chenb, Jonathon Dinesa, Xiao Dingb, Charles
Eigenbrotb, Alexander Heifetza, Allan Jaochicob, Adam Johnsonb, Joachim Kraemerc, Susanne Krugerc, Thomas
M. Krüllea, Marya Liimattab, Justin Lyb, Rosemary Maghamesa, Christian A. G. N. Montalbettia, Daniel F.
Ortwineb, Yolanda Pérez-Fuertesa, Steven Shiab, Daniel B. Steinc, Giancarlo Trania , Darshan G. Vaidyaa, Xiaolu
Wangc, Steven M. Bromidgea, Lawren C. Wub and. Zhonghua Peib,∗
13b
ITK Ki = 0.7 nM
Clp = 12 ml/min
F = 35%

Bioorganic & Medicinal Chemistry Letters
jo u rn al ho me p ag e : w ww .e lse vie r .c om
Structure-based Design and Synthesis of Potent Benzothiazole Inhibitors of
Interleukin-2 Inducible T Cell Kinase (ITK)
Colin H. MacKinnona, Kevin Laub, Jason D. Burchb, Yuan Chenb, Jonathon Dinesa,
Xiao Dingb, Charles Eigenbrotb, Alexander Heifetza, Allan Jaochicob, Adam Johnsonb,
Joachim Kraemerc, Susanne Krugerc, Thomas M. Krüllea, Marya Liimattab, Justin Lyb,
Rosemary Maghamesa, Christian A. G. N. Montalbettia, Daniel F. Ortwineb, Yolanda
Pérez-Fuertesa, Steven Shiab, Daniel B. Steinc, Giancarlo Trania , Darshan G. Vaidyaa,
Xiaolu Wangc, Steven M. Bromidgea, Lawren C. Wub and Zhonghua Peib,∗
aEvotec (UK) Ltd., 114 Milton Park, Abingdon, Oxfordshire, OX14 4SA, United Kingdom
bGenentech Inc., 1 DNA Way, South San Francisco, CA 94080, United States
cEvotec AG, Manfred Eigen Campus, Essener Bogen 7, D-22419, Hamburg, Germany
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling
cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based
optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory
potency against ITK with good cellular activity and kinase selectivity. We also elucidate the
binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK
complexes.
Keywords:
2012 Elsevier Ltd. All rights reserved.
ITK inhibitor;
TCR signalling;
Benzothiazole;
Anti-inflammatory;
Asthma
SAR
———
∗ Corresponding author. Tel.: 1-650-467-1542; e-mail: pei.zhonghua@gene.com

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec kinase family of non-receptor tyrosine kinases, plays a major
role in T-cell signaling.1 Along with other T-cell specific tyrosine kinases (including LCK and ZAP-70), ITK serves to amplify the
signal associated with the T-cell receptor (TCR) cascade. Aberrant signaling behavior in this cascade is known to lead to
autoimmune disorders and inflammation.2 In studies with ITK knockout mice, levels of Th2 cytokines such as IL-4, IL-5 and IL-13
have been reduced.3 Moreover, in such mice, the immunological symptoms of allergic asthma were attenuated. Additionally, lung
inflammation, eosinophil infiltration and mucous production were drastically reduced in response to challenge with the allergen
ovalbumin.4 These and other reports suggest that selective inhibition of ITK may represent a novel therapy for the treatment of
asthma.5
Herein we report the optimization of a series of benzothiazole amides starting from a HTS hit 1, resulting in inhibitors with sub-
nanomolar biochemical and sub-micromolar cellular potencies. We also elucidate the binding mode of these inhibitors by solving
the X-ray crystal structures of the complexes.
Figure 1. Potency and orientation of HTS hit 1 docked in the ATP-binding pocket of ITK.
Acid intermediates 3a-d were obtained from the α, β-unsaturated esters 2a-d by cyclopropanation with the ylide formed from
deprotonation of trimethylsulfoxonium iodide by NaH in DMSO to afford esters 3a-d (Scheme 1). 6, 7 In order to synthesize 3a, the
bromine atom was converted to the cyano group in 77% yield. Hydrolysis of the esters afforded the corresponding acids, which
were then coupled with the amine to give the desired amides 4a-d.
a
R
Et
R
OEt
O
O
(±)-3a', 3a-d
R
for 3a' R = Br
b
2a 4-bromophenyl
2b 2-pyridyl
3a R= CN
2c 4-methoxyphenyl
2d cyclohexyl
c, d
H
R
N
N
O
S
N
OMe
H2N
S
(±)
OMe
R
4a 4-cyanophenyl
4b 2-pyridyl
4c 4-methoxyphenyl
4d cyclohexyl
Scheme 1. Reagents and conditions: (a) Me3SO+I-, NaH, DMSO, 50 °C, 20-63%; (b) CuCN, DMF, 153°C, 77%; (c) 2M
NaOH, MeOH, 50 °C then 2M HCl (aq), 75-92%; (c) HATU, DIPEA, DMF, 80 °C, 24-71%.
Analogs of 6-heteroaryl benzothiazoles 6a-h were prepared via Suzuki couplings with commercially available aryl boronic esters
or through boronic ester 7 (Scheme 2), both starting from intermediate 5, itself synthesized according to Scheme 1. Tetrazole 6i was
prepared by cycloaddition of azidotributyl tin and nitrile 5 (R= OMe, X = CN).

Scheme 2. Reagents and conditions: (a) ArBR2 or ArX, Na2CO3, Pd(dppf)2Cl2.DCM, 1,4-dioxane, 85 °C, 11-64%; (b) bis
(pinacol boron ester), Na2CO3, Pd(dppf)2Cl2.DCM, 1,4-dioxane, 100 °C, 7-53%; (c) Azidotributyltin, THF, 80 °C, 23%. For
the exact structures, see Table 2.
Analogs containing basic amines required somewhat more elaborate syntheses. Protection of the aldehyde and ester formation of
4-formyl cinnamic acid 8 in one step using trimethylorthoformate and acetyl chloride in methanol, followed by cyclopropanation
provided ester 9 (Scheme 3). Selective deprotection of acetal 9 was achieved by with 2M HCl in methanol and the amino group was
installed by standard reductive amination with the resulting aldehyde. Saponification afforded acid 10 which was coupled to
benzothiazole aminea 12a-b, to provide 13a directly or 13b after removal of the Boc group. Amines 12a-b were prepared in one
step from commercially available bromide 11 by a Suzuki coupling.
N
N
f
H2N
H2N
S
Br
S
Ar
12
11
12a
Ar =
N
NBoc
12b
Ar =
N
Scheme 3. Reagents and conditions: (a) (MeO) 3CH, AcCl, MeOH, 80 °C; (b)Me3SO+I-, NaH, DMSO, 50 °C, 59% (two
steps); c) 2M HCl, dioxane; (d) Me2NH, AcOH, NaBH(OAc)3, THF, rt; (e) 2M NaOH, MeOH, 50 °C then 2M HCl (aq), 96%
(three steps); (f) ArB(OH)2, Na2CO3, Pd(dppf)2Cl2.DCM, 1,4-dioxane, 85 °C, 26-50%; (g) 12, HATU, DIPEA, DMF, 80 °C,
15-20%; (h) conc.HCl, MeOH, rt, quant., for 13b only.
Meta isomer 16 was accessed in a similar fashion, where the required cinnamate ester 15 was prepared by a Heck reaction on 3-
bromobenzaldehyde 14 (Scheme 4).8 Finally, trisubstituted benzene analogue 19 was prepared from cinnamic ester 18, obtained by
Horner-Wadsworth-Emmons olefination of the commercially available aldehyde 17 (Scheme 5).

All targets were initially synthesized as racemates. Key compounds, namely 6f, 13 and 19, were subsequently resolved using
supercritical fluid chromatography (SFC) on a chiral stationary phase.
Scheme 4. Reagents and conditions: (a) Ethyl acrylate, Pd(OAc)2, NaHCO3, Et3N, PPh3, DMF, 100 °C, 52%.
Scheme 5. Reagents and conditions: (a) MeO2CCH2P(O)(OMe)2, NaH, THF, rt, quant.
High-throughput screening (HTS) of the Genentech compound library identified racemic benzothiazole amide 1 as a moderately
potent inhibitor of ITK (Figure 1). Molecular modeling of 1 bound in the ATP-binding pocket of ITK based on a published ITK
crystal structure9 was used to generate a binding hypothesis (Figure 1). In the model, two hydrogen bonds are formed between the
hinge residue Met438 and the two nitrogen atoms of the benzothiazole amide. The cyclopropyl moiety directs the attached benzene
ring to achieve a π-π interaction with Phe437.
Based on the hypothesized binding mode, we first did a quick survey of the left-hand substitution (LHS). When the phenyl ring
was substituted with an electron-withdrawing cyano group, the resulting compound 4a had potency similar to that of 1 (Table 1).10
When the phenyl group was replaced with a 2-pyridyl group, the resulting compound 4b displayed decreased potency. When an
electron-donating methoxy group was introduced, the resulting analog 4c maintained potency, but more importantly the aqueous
solubility at pH 7.4 improved from < 2 µM of 1 to 11 µM. Attempts to reduce planarity by replacing the phenyl group with a
cyclohexyl group resulted in significant reduction of potency (4d). The above SAR is consistent with the hypothesis that the LHS
phenyl ring is engaged in a π-π interaction with Phe437.
Table 1. LHS SAR of benzothiazole amides

Since the benzothiazole moiety is in the vicinity of the gatekeeper Phe435 according to our binding model, it was envisioned that
an aromatic ring attached to C6 position could potentially engage in a π-π interaction with this residue. When a 3- or 4-pyridyl
group was introduced, the resulting compounds 6a and 6b had indeed improved potency while a 2-pyridyl group (6c) displayed
decreased potency compared to 1 (Table 2), presumably because the polar nitrogen atom has to fit in an hydrophobic pocket. A
hydroxymethyl group on the pyridine was introduced to interact with the vicinal Ser499 that is unique to ITK. The resulting analog
6d displayed improved potency compared to the unsubstituted pyridine analog 6b. Pyridone 6e, which was designed to pick up
hydrogen-bond interactions with adjacent lysine and aspartic acid residues, also showed improved potency. 6d and 6e were the first
analogs to achieve Ki < 10 nM.
We then investigated smaller 5-membered aromatic rings attached to the C6 position of the benzothiazole ring. The resulting
analog 6f showed a potency of 6.4 nM. Inhibitory potency decreased when either the pyrazole nitrogen (6g) or the adjacent carbon
(6h) was methylated. Analog 6i with an acidic tetrazole moiety, designed to interact the nearby Lys391, displayed markedly
reduced potency, perhaps because the tetrazole is deprotonated at physiological pH.
Table 2. Biochemical and cellular potency of C6-substituted benzothiazole amides

All compounds summarized in Tables 1 and 2 demonstrated little or no cellular potency, as measured by the inhibition of
phosphorylation of ITK’s natural substrate phospholipase C-γ1 (PLC γ−1) in Jurkat cells (IC50 > 14 µM).11 In addition, the majority
of the these compounds displayed poor aqueous solubility. In an attempt to address these two issues, we identified the LHS phenyl
ring as a promising site to add solubilizing groups in order to improve solubility. A number of analogs with polar substituents on the
LHS phenyl group were prepared and tested. From this work, 4-dimethylaminomethyl analogue 13a (Table 3) displayed ~10-fold
potency improvement relative to compound 6b, so did compound 13b relative to 6f. This improved biochemical potency was
translated into good cell potency: for example, compound 13b had a p-PLCγ IC50 of 89 nM.
A selected number of racemic compounds was resolved by chiral SFC. The (S, S) enantiomer was generally more active than its
enatomer. For instance, (S, S)-13b has a Ki of 0.7 nM, while its enantiomer has a Ki of 6 nM. This is consistent with the binding
model as later confirmed by X-ray structure (vide infra).
Analogs 16 and 19 with the amine group at the meta-position also displayed improved potency compared to 6f (Table 3). Both
compounds displayed improved aqueous solubility compared to 13a and 13b, presumably due to reduced symmetry and/or an
increased fraction of sp3 atoms (Fsp3, the ratio of sp3 carbon atoms to total number of carbon atoms). Both strategies are known to
disrupt crystal packing and therefore improve solubility.12 It is worth to note that these optimized inhibitors display from 52-fold
(13a) to 680-fold (S, S-6f) selectivity over lymphocyte-specific protein tyrosine kinase (LCK).13 Because LCK operates upstream of
ITK in the TCR cascade and also phosphorylated other Tec family members, inhibition of LCK is expected to affect a broader
spectrum of T cells and thus may be undesirable.13
Table 3. Potency, selectivity and selected physicochemical properties of benzothiazole amidesa
ITK
Ki (nM)
LCK Ki
(µM)
0.89
pPLC-γ1 LogD
Solubilityb
(µM)
<1
(pH 7.4)
IC50 (µM)
>14
6.4
2.5
4.4
4.4
( )-6f
(S,S)-6f
1.7
>20
<1
8.4
0.6
0.7
0. 44
0.046
0.055
10
0.089
0.091
2.7
3.0
3.0
<1
<1
<1
( )-13a
( )-13b
(S,S)-13b
1.9
0.5
0.15
0.11
0.80
2.8
2.6
11
( )-16
0.048
180
(S,S)-19

aFor structure of 6f, see Table 2. For structures of 13, 16 and 19, see Schemes 3-5. b Solubility measured at pH 7.4.
We were able to solve the crystal structures of several inhibitors complexed to ITK as the project progressed. The X-ray crystal
structure of 13a bound to ITK confirmed our hypothesized binding mode (Figure 2a). The nitrogen atom of the benzothiazole and
the amide NH form two H-bonds with the backbone NH and the carbonyl of hinge residue Met438, respectively. A non-classical
hydrogen bond between the carbonyl group of adjacent residue Glu436 and the hydrogen atom on the C4 of the benzothiazole ring
is apparent. The RHS pyridyl ring indeed occupies the nearby pocket and lies underneath gatekeeper Phe435 to form a face-to-face
π-π interaction. Furthermore, the salt bridge normally observed between the conserved Lys391 and Asp500 residues (vide infra) is
disrupted due to the movement of the side-chain of Lys391 in order for it to avoid a steric clash with the pyridyl ring of the ligand.
Co-crystallization of 13b with the kinase domain of ITK was solved to 2.1 Å resolution (Figure 2b). The overall binding mode is
very similar to that of 13a. However, the pyrazole NH now forms a hydrogen bond with the side chain of Lys391 in addition to
displaying a π-π stacking interaction with the gatekeeper Phe435. This explains why the corresponding N-methylated analog 6g is
less potent, where the H-bond is impossible. This smaller pyrazole ring is well accommodated such that the salt bridge between the
conserved Lys391 and Asp500 residues is maintained. These factors combined may explain why 5-membered analog 13b is about
12-fold more potent than the corresponding 6-membered analog 13a. The LHS phenyl group is well resolved in this complex, and is
shown to be engaged in an edge-to-face π-π interaction with Phe437.
Figure 2. X-ray crystal structures of ITK kinase domain complexed with (a) 13a and (b) (S,S)-13b. Ligands are shown as
ball-and-stick while protein residues are shown in stick. Nitrogen atoms of ITK are in blue, oxygen in red. Only selected
residues of ITK are shown for clarity. Interactions between ligand and ITK are indicated by the dashed lines in gold. PDB
codes are 4MF0 and 4MF1 for 13a and 13b, respectively.
With the single enantiomer of 13b in hand, we determined its PK profile in rat (Table 4) and were pleased to observe a low
clearance (about 20% of hepatic blood flow), acceptable oral bioavailability (F=35%) and a half-life of about 3h. Presumably partial
protonation of the weakly basic amine at gastric pH helps solubilize the compound and thus absorption in vivo. Compound 13b also
has a low-to-moderate clearance in human hepatocytes of 8.6 ml/min/kg.
Table 4. Rat pharmacokinetic profile of amine (S, S)-13b
Route
IV
Dose
AUC
last
(h*uM)
2.8
F
CL
T1/2
Vss
(%) (ml/min (h) (L/kg)
/kg)
1 mpk
NA
12
3.4
3.2

PO
5mpk
4.8
35
NA
3.0
NA
These optimized inhibitors generally demonstrate fair to good kinase selectivity. For instance, only three off-target kinases [Abl,
Musk and RAF (Y340D, Y341D)] of the 65 tested, were inhibited >50% by hydroxypyridine 6d at 1 µM.14 When (S, S)-13b was
tested in the same panel at 0.1 µM, eight off-target kinases were inhibited with >50% (Abl, CDK2, CK1α, Flt3, KDR, PAK4, Txk,
YES). We believe that 6d is more selective than 13b because of interaction between the hydroxyl group of 6d and the unique
Ser442 of ITK.
In summary, we have discovered a new class of benzothiazole-based ITK inhibitors and were able to improve the potency by
920-fold from a HTS hit through rational design. We were able to elucidate the binding mode of these inhibitors by solving the X-
ray crystal structures of representative analogs complexed to the kinase domain of ITK. Advanced leads such as 13b and (S, S)-19,
possesses sub-nanomolar inhibitory potency, double-digit nanomolar cell potency, triple-digit micromolar aqueous solubility and
good selectivity over LCK.
References & Notes
1.
Felices, M,; Falk, M.; Kosaka, Y.; Berg, L.J. Adv. Immunol. 2007, 93, 145.
2. Sakaguchi, S.; Benham, H.; Cope, A.P.; Thomas R.; Immunol. Cell Biol.; 2012, 90, 277
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Schaeffer, E. M.; Debnath, J.; Yap, G.; McVicar, D.; Liao, X. C.; Littman, D. R.;Sher, A.; Varmus, H. E.; Lenardo, M. J.; Schwartzberg, P. L.
Science 1999, 284, 638.
5.
For small-molecule inhibitors of ITK, see (a) Das, J.; Liu, C.; Moquin, R.V.; Lin, J.; Furch, J. A.; Spergel, S. H.; McIntyre, K. W.; Shuster, D. J.;
O’Day, K. D.; Penhallow, B.; Huang, C-Y.; Kanner, S. B.; Lin, T-A.; Dodd, J. H.; Barrish, J. C.; Wityak, J. Bioorg. Med. Chem. Lett. 2006,16,
2411; (b) Lin, T. A.; McIntyre, K..; Das, J.; Liu, C.; O’Day, K. D.; Penhallow, C-Y.; Whitney, G. S.; Shuster, D. J.; Xia, X.; Towsend, 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. Biochem. 2004, 43, 11056; (c) Snow, R.J.; Abeywardane. A.; Campbell , S.;
Lord, J.; Kashem, M. A.; Khine, H. H.; King, J.; Kowalski, J. A.; Pullen, S. S.; Roma, T.; Roth, G. P.; Sarko, C. R.; Wilson, N. S.; Winter, M. P.;
Wolak, J. P.; Cywin, C. L. Bioorg. Med. Chem. Lett. 2007, 17, 3660; (d) Moriarty, K. J.; Winters M, Qiao, L.; Ryan, D.; DesJarlis, R.; Robinson,
D.; Cook, B. N.; Kashen, M. A.; Kaplita, P. V.; Liu, L. H.; Farrell, T. M.; Khine, H. H.; King, J.; Pullen, S. S.; Roth, G. P.; Magolda, R.
Takahashi, H. Bioorg. Med. Chem. Lett. 2008,18, 5537; (e) Winters, M.P.; Robinson, D. J.; Khine, H. H.; Pullen, S. S; Woska Jr.; J. R.;
Raymond, E. L.; Sellati, R.; Cywin, C. L.; Snow, R. J.; Kashem, M. A.; Wolak, J. P.; King, J.; Kaplita, P. V.; Liu., L. H.; Bioorg. Med. Chem.
Lett. 2008, 18, 5541; (f) Riether, D.; Zindell. R.; Kowalski A, et al. Bioorg. Med. Chem. Lett. 2009, 19,1588; (g) Lo H.Y.; Bentzien, J.; Fleck, R.
W.; Pullen, S. S.; Khine, H. H.; Woska, J. R.; Kugler, S. Z.; Kashen, M. A.; Takahashi, H. Bioorg. Med. Chem. Lett. 2008, 18, 6218; (h)
Charrier, J. D.;Miller, A.;Kay, D. P.;Brenchley, G.;Twin, H. C.;Collier, P. N.;Ramaya, S.;Keily, S. B.;Durrant, S. J.;Knegtel, R. M.;Tanner, A.
J.;Brown, K.; Curnock, A. P.;Jimenez, J. M. J. Med.Chem. 2011, 54, 2341; (i) Herdemann. M.; Weber, A.; Jonveaux, J; Jonveaux, J.; Schwoebel,
S.; Heit, I. Bioorg. Med. Chem. Lett. 2011, 21, 1852; (j) McLean, L. R.; Zhang, Y.; Zaidi, N.; Bi, X.; Wang, R.; Dharanipragada, R.; Jurcak, J.
G.; Gillespy, T. A.; Zhao, Z.; Musick, K. Y.; Choi, Y. M.; Barrague, M.; Peppard, J.; Smicker, M.; Duguid, M.; Parkar, A.; Fordham, J.;
Kominos, D. Bioorg. Med. Chem. Lett. 2012, 22, 3296; (k) Zapf, C. W.;Gerstenberger, B. S.;Xing, L.;Limburg, D. C.;Anderson, D. R.;Caspers,
N.;Han, S.;Aulabaugh, A.; Kurumbail, R.;Shakya, S.;Li, X.;Spaulding, V.;Czerwinski, R. M.;Seth, N.;Medley, Q. G. J. Med. Chem., 2012, 55,
10047. (l) For a recent review on ITK inhibitors, see: Charrier, J-D.; Knegtel, R. MA. Expert Opin. Drug Discov. 2013, 8, 369.
Newcomb, M.; Choi S, Toy, P.H. Can. J. Chem. 1999, 77, 1123.
6.
7.
8.
Hamdouchi, C.; Topolski, M.; Goedkin, V.; Walborsky, H. M. J. Org. Chem. 1993, 58, 3148.
Biedeger, R.J.; Decker, R.E.; Dupre, B.; Hamaker, L.K.; Holland, G.W.; Kassir, J.M.; Li, W.; Market, R.V.; Nguyen, N.; Scott, I. L.; Wu, C.
European Patent: EP 1213288 A1, 2002.
9.
(a) Brown, K; Long, J.M.; Vial, S.C.M.; Dedi, N.; Dunster, N.J.; Renwick, S.B.; Tanner, A.J.; Frantz, J.D.; Fleming, M.A.; Cheetham, G.M.T.;
J. Biol. Chem. 2004, 279, 18727; (b) Kutach, A.K.; Villasenor, A.G.; Lam, D.; Belunis, C.; Janson, C.; Lok, S.; Hong, L.N.; Liu,
C.M.; Deval, J.; Novak, T.J.; Barnett, J.W.; Chu, W.; Shaw, D.; Kuglstatter, A. Chem. Biol. Drug Des. 2010, 76, 154.
10. Biochemical assay: GST-ITK full length enzyme was from Invitrogen (PV3875) and substrate was BLK peptide (Ac-EFPIYDFLPAKKK-NH2).
Reactions were carried out in a final volume of 51 µl with 50 mM HEPES (pH 7.2), 15 mM MgCl2, 2 mM DTT, 0.015% Brij-35, 1 nM ITK, 2µM
substrate, 20 µM ATP, and 2% DMSO. After 35 min incubation at RT, reactions were stopped upon addition of 10 µl of 30% TCA. Samples
were centrifuged (4350 rpm, 4 °C, 5 min) and subjected to LC/MS analysis on a Waters Acquity UPLC/TQD system equipped with a Waters
Acquity UPLC BEH C18 (2.1 × 50 mm) 1.7 µm column (injection volume: 5 µl, column temperature: 60 °C, flow rate: 1 ml/min, solvent A:
0.1% formic acid in LC/MS grade water, solvent B: 0.1% formic acid in LC/MS grade ACN). Analytes were separated by applying a gradient
from 15 to 32% solvent B within 0.7 min and detected in positive mode ESI-MS/MS by MRM (multiple reaction monitoring) of transitions
819.8/84.8 (BLK substrate) and 859.0/84.8 as well as 859.0/120.7 (phosphorylated BLK product).
11. The ability of compounds to inhibit phosphorylation of PLCg1 in Jurkat cells was determined using an immuno-detection assay that will be
described in detail in a separate communication.
12. Ishikawa, M.; Hashimoto, Y. J. Med. Chem. 2011, 54, 1539.
13. (a) For a benzothiazole-based LCK inhibitor, see Das, J.; Lin, J.; Moquin, R. V.; Shen, Z.; Spergel,S. H.; Wityak, J.; Doweyko, A. M.; DeFex, H.
F.; Fang, Q.; Pang, S.; Pitt, S.; Shen, D. R.; Schieven, G. L.; J. C. Barrish. Bioorg. Med. Chem. Lett. 2003, 13,2145; (b) We did not have in-house
X-ray crystal structure of LCK, so do not have structure-based explanation of selectivity over LCK.
14. Selectivity screening was performed by SelectScreen® Kinase Profiling Services (Invitrogen-Life Technologies, Madison, USA).

Graphical Abstract
Structure-based Design and Synthesis of Potent
Benzothiazole Inhibitors of Interleukin-2 Inducible T
Cell Kinase (ITK)
Leave this area blank for abstract info.
13b
ITK Ki = 0.7 nM
Clp = 12 ml/min
F = 35%