Discovery of Pyridazinone and Pyrazolo[1,5- a]pyridine Inhibitors of C-Terminal Src Kinase

Article abstract of DOI:10.1021/acsmedchemlett.9b00354

C-terminal Src kinase (CSK) functions as a negative regulator of T cell activation through inhibitory phosphorylation of LCK, so inhibitors of CSK are of interest as potential immuno-oncology agents. Screening of an internal kinase inhibitor collection identified pyridazinone lead 1, and a series of modifications led to optimized compound 13. Compound 13 showed potent activity in biochemical and cellular assays in vitro and demonstrated the ability to increase T cell proliferation induced by T cell receptor signaling. Compound 13 gave extended exposure in mice upon oral dosing and produced a functional response (decrease in LCK phosphorylation) in mouse spleens at 6 h post dose.

Full text of DOI:10.1021/acsmedchemlett.9b00354

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Letter
Discovery of Pyridazinone and Pyrazolo[1,5-
a]pyridine Inhibitors of C-terminal Src Kinase
Daniel P. O'Malley, Vijay Ahuja, Brian Fink, Carolyn Cao, Cindy Wang, Jesse
Swanson, Susan Wee, Ashvinikumar V Gavai, John S Tokarski, David Critton,
Anthony A. Paiva, Benjamin M. Johnson, Nicolas Szapiel, and Dianlin Xie
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00354 • Publication Date (Web): 25 Sep 2019
Downloaded from pubs.acs.org on September 28, 2019
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Discovery of Pyridazinone and Pyrazolo[1,5-a]pyridine Inhibitors of
C-terminal Src Kinase
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Daniel P. O’Malley*, Vijay Ahuja, Brian Fink, Carolyn Cao, Cindy Wang, Jesse Swanson, Susan Wee,
Ashvinikumar V. Gavai, John Tokarski, David Critton, Anthony A. Paiva, Benjamin M. Johnson,
Nicolas Szapiel, Dianlin Xie
Bristol-Myers Squibb Company, Research and Development, Route 206 and Province Line Road, Princeton, New Jersey
08543.
Keywords: CSK inhibitor. Kinase inhibitor. Immuno-oncology. Metabolite ID.
F
O
O
Hinge binder exchange
Metabolic soft spot blocking
HN
HN
HN
CSK HTRF IC50: 5600 nM
Jurkat ZAP70 EC50: 7900 nM
CSK HTRF IC50: <3 nM
CSK Caliper IC50: 4 nM
Jurkat ZAP70 EC50: 41 nM
 Tcell proliferation in vitro
N
N
N
N
O
N
Cl
Cl
N
Me
Me
Me
O
CN
HN
N
NH
O
Me
Me  LCK phosphorylation in vivo
N N
O
MeO
1
13
ABSTRACT: C-terminal Src kinase (CSK) functions as a negative regulator of T cell activation through inhibitory phosphorylation
of LCK, so inhibitors of CSK are of interest as potential immuno-oncology agents. Screening of an internal kinase inhibitor collection
identified pyridazinone lead 1, and a series of modifications led to optimized compound 13. Compound 13 showed potent activity in
biochemical and cellular assays in vitro and demonstrated the ability to increase T cell proliferation induced by T cell receptor
signaling. Compound 13 gave extended exposure in mice upon oral dosing and produced a functional response (decrease in LCK
phosphorylation) in mouse spleens at six hours post dose.
The development of antibodies targeting CTLA-4, PD-1, and
PD-L1 to activate the immune system holds great promise for
the treatment of cancer. Although these treatments offer
substantial therapeutic benefit, as reflected by their recent
approval for the treatment of melanoma, non-small cell lung
cancer, renal cell carcinoma, and a number of other indications,
there remains a population of patients that do not respond to
chemical inhibition have demonstrated that this inhibition
increases the response of T cells to weak antigens.9 In addition,
both CSK and LCK have been shown to associate with PD-1,
and PD-1 activation has been shown to reduce ZAP-70
phosphorylation, indicating that CSK inhibition could
synergize with anti-PD-1/PD-L1 therapy.10,11 To date, there
have been no reports of potent small molecule CSK inhibitors.12
The development of suitable tool molecules to evaluate the
potential to activate T cells through CSK inhibition is therefore
of great interest.
currently available immuno-oncology agents.
There is
consequently a critical need for additional agents to help extend
the benefits of immunotherapy to additional patients and
indications.1-3
Screening of an internal kinase inhibitor collection identified
compound 1 (Figure 1) as a starting point for optimization.
Despite its modest potency in a CSK HTRF binding assay
(Table 1), this compound increased ZAP-70 phosphorylation in
Jurkat cells and had no measureable LCK activity. As the
ultimate aim of inhibiting CSK was the enhancement of LCK
activity, selectivity for inhibition of CSK over LCK was
considered highly important. Although the two proteins are
only 44% identical in their kinase domains, only two of the
residues facing the ATP-binding pocket differ (Leu251 and
Ala381 in Lck), complicating attempts to obtain selective
inhibitors. The initial goals for optimization were to improve
potency in the binding and cellular assays and to improve the
In T cells, the SRC-family kinase LCK plays a key role in
initiating the proximal
T
cell receptor pathway by
phosphorylating the ξ and CD3 chains of the T cell receptor, as
well as downstream kinases such as ZAP-70.4 LCK is in turn
negatively regulated by phosphorylation on Tyr505 by c-
terminal SRC kinase (CSK), which causes LCK to adopt its
closed, inactive conformation.5,6,7 Inhibition of CSK could
therefore augment T cell activation in response to antigen
recognition by the T cell receptor by retaining LCK in an
activated form. Indeed, siRNA knockdown of CSK in primary
T cells and Jurkat cells increases LCK signaling and response
to T cell receptor stimulation.8 Studies in transgenic mice
expressing a variant of CSK engineered to be sensitive to
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ACS Medicinal Chemistry Letters
low metabolic stability in human and mouse liver microsomes,
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while maintaining selectivity against LCK.
O
O
O
O
4'
HN
HN
HN
HN
HN
N
N
N
N
N
N
N
N
O
3'
3'
X
Me
N
Cl
Cl
H
N
H
N
H
N
N
N
N
Me
Me
Me
4"
4"
HN
X
Y
O
Me
Me
Me
Me
Me
Me
N
N
N
O
O
O
Me
Me
Me
5"
X
NH
NH
NH
6"
1"
2"
O
O
O
N
N
6"
9
1"
Y
7:
8:
9:
10
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1
2:
3:
4:
5:
6:
X=H
X=Me
X=H, Y=Me
X=Me, Y=H
X=Me, Y=Me
X=Cl, Y=H
X=Me, Y=H
X=H, Y=H
10:
X=Me, Y=OMe
F
F
5
O
O
O
O
3
HN
HN
HN
HN
HN
N
N
N
N
N
N
N
N
Me
3'
Me
Me
Cl
Cl
H
N
N
N
N
4'
HN
O
O
HN
O
HN
O
CN
Me
CN
Me
Me
Me
O
O
O
Me
Me
Me
2"
N
N
N
N
N
N
N N
1"
MeO
MeO
MeO
7"
11
12
13
14
Figure 1. Structures of CSK inhibitors 1-14.
Since structural information regarding the binding of 1 to
CSK was not available at the outset of the project, initial
optimization efforts focused on introducing conformational
biases as a means to enhance potency. The azetidinylmethyl
group in 1 was replaced with a piperidine (compound 2) based
on the hypothesis that this would reduce conformational
flexibility and favor the necessary orientation of the urea group.
This resulted in a substantial increase in CSK potency and
improvement in human metabolic stability, although the
increase in cellular potency was modest and mouse metabolic
stability remained low.
49 nM in the cellular ZAP-70 assay (Table 2); since compounds
in this series often had IC50 values below 3 nM in the CSK
HTRF assay (the lower limit of the assay), they were also tested
in a Caliper assay to give a more precise measurement of their
potency.
Table 1. Characterization of CSK inhibitors 1-6.a,b
Cpd
CSK IC50 LCK IC50
(nM)
ZAP-70
EC50 (nM)
HLM/MsLM
%Rem.
(nM)
(Ymax
)
1
2
3
4
5
6
5600
>50000
7900(73%)
2/33
65/15
68/9
An attempt to bias the conformation of the biaryl junction to
favor a non-planar orientation by introduction of a methyl group
at C4” of the pyridazinone (3) resulted in a similar profile, as
did replacement of the C3’ chloro substituent on the phenyl ring
with a larger ethyl group (4). Movement of the C4” methyl to
C5” (5) caused a slight drop in potency; incorporation of methyl
groups at both positions (6) gave a moderate increase in both
binding and cellular potency. Interestingly, compounds
containing both the C3’ ethyl and C4” methyl groups (4 and 6)
showed substantially increased mouse metabolic stability,
whereas compounds containing only one of the two groups (3
and 5) did not.
79
80
70
430
8
24000 5700(180%)
31000 4050(350%)
43000 2000(350%)
34000 2700(280%)
53/78
72/1
26000
420(270)%
61/59
aFor assay conditions and replicate numbers, see supporting
information bHLM: Human liver microsomes. MsLM: Mouse
liver microsomes. %Rem: Percent remaining at 10 minutes.
Table 2. Characterization of CSK inhibitors 7-14.a,b
Unfortunately, efforts to further increase potency by
straightforward modification of compound 6 proved unfruitful,
so attempts were made to replace the pyridazinone with a series
of other groups that had the potential to maintain a similar
pattern of hydrogen bonding interactions. This approach was
validated by compound 7, which was designed based on the
hypothesis that N1” and the C2” hydrogen of the
pyrazolopyridine would occupy a similar location to the C6”
carbonyl and N1” hydrogen of pyridazinone 6. This compound
had an IC50 value of 5 nM in the CSK HTRF assay and EC50 of
Cpd
CSK IC50 (nM)
HTRF Caliper
LCK
IC50
(nM)
ZAP-70
EC50 (nM)
(Ymax)
HLM/
MsLM
%Rem
7
8
5
4
300
120
3100
26
49(250%)
42(190%)
1400(240%)
28(220%)
>20000
84/24
72/10
71/21
80/2
<3
26
<3
42
4
21
2
9
10
11
13
42
NT/59
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amide 12. Analysis of the metabolism of compound 12 revealed
12
13
14
<3
<3
4
4
4
6
230
260
110
88(370%)
41(360%) 91/100
56(210%) 85/99
85/9
1
2
3
4
5
6
7
8
a new site of metabolism, oxidation of the indazole ring.
Attempts were made to block this by fluorination of the
indazole ring; the C-5 fluoro derivative 13 showed a dramatic
improvement in metabolic stability combined with good
biochemical and cellular potency. Interestingly, compound 14,
which contains the fluoroindazole but maintains the tert-butyl
urea that had proved to be metabolically labile in other analogs,
also showed improved metabolic stability. This suggests that
the role of the fluorine on the indazole extends beyond simply
preventing oxidation of the indazole through local steric or
electronic effects, possibly by creating an unfavorable
interaction with the metabolizing enzyme.
aFor assay conditions and replicate numbers, see supporting
information NT: not tested.
b
O
HN
9
N
N
10
11
12
13
14
15
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Me
H
N
N
Scheme 1. Synthesis of compound 8.a
HN
O
Me
O
Me Me
O
MsO
N
Boc
+O
N
N
8
MeO
16
N
N
NH
a, b
O
15
HN
HN
+O
N
N
O
H2N
NH2
HO
Me
N
Me
N
18
c
O
CN
Me
O
Me
N
Boc
N
N
17
HO
O
12
O
Figure 2. Sites of metabolism of compounds 8 and 12.
N
HN
N
20
N
N
Investigation of the C3’ chloro substituent revealed that
replacement with a methyl group (compound 8) maintained
potency, while its removal (9) resulted in a decrease in both
biochemical and cellular potency. This result suggested that
this group plays an important role through direct interaction
with CSK and/or influence on the conformation of the C4’
amide bond. An attempt to improve potency by introduction of
a methoxy group at C6” (10) had minimal effect on CSK
potency, but did cause an undesired increase in LCK potency.
Despite this, compound 10 maintained activity in the cellular
assay, indicating that even a relatively low level of selectivity
for CSK over LCK may be sufficient in a cellular context.
d, e, f
Me
N
Boc
NH2
19
O
HN
N
N
Me
H
N
N
HN
O
Me
Me
O
Me
Despite the exciting levels of potency shown by these
compounds, their poor mouse metabolic stability prevented
their use in animal studies. In an attempt to remove potential
sites of metabolism on the substituted piperidine, compound 11,
in which this substituent has been truncated to a methyl group,
was prepared. Although 11 did show an increase in mouse
metabolic stability, it also had lower CSK potency and did not
show cellular activity.
N
N
8
aReagents and conditions: (a) 16, Cs2CO3, DMF, 80 ºC, 39%.; (b)
LiOH, MeOH, H2O, quant.; (c) 18, PyBOP, Hünig’s base, DMF,
79%; (d) 20, PyBOP, Hünig’s Base, THF, rt to 60 ºC, 47%; (e)
HCl, dioxane, quant.; (f) tert-butyl isocyanate, Hünig’s Base,
DMF, 41%.
In order to ascertain the location of its metabolic
vulnerabilities, metabolism of compound 8 by mouse liver
microsomes was studied and oxidation of the tert-butyl group
was identified as the sole metabolite (Figure 2).13
In an attempt to understand the binding mode of these
chemotypes, efforts were made to obtain crystal structures with
several compounds. Although no usable crystal structures were
obtained with CSK, a structure of 11 in complex with LCK was
obtained (Figure 3). The structure reveals that 11 sits in the
ATP binding site and makes hydrogen bonds from N1” of the
pyrazolopyridine ring to the hinge backbone NH of Met319 and
from the amide NH at C4’ to the side chain hydroxyl oxygen of
Thr316. In addition, the distance between C7” of the
This knowledge spurred attempts to block metabolism of the
tert-butyl group by replacing it with a series of groups
containing polar and electron-withdrawing substituents.
Although this yielded compounds that maintained the high level
of potency shown by 8, these compounds did not show the
expected increase in metabolic stability, as typified by cyano
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Page 4 of 8
pyrazolopyridine and the backbone carbonyl oxygen of Met319
is 3.1 Å and the distance between C2” of the backbone oxygen
of Glu317 is 3.5 Å, indicating the presence of favorable binding
interactions between these residues and 11. The amide carbonyl
oxygen at C3 of the indazole ring makes a hydrogen bond with
the side chain of Lys273.
1
2
3
4
5
6
O
3
HN
N
N
Me
3'
Me
4'
HN
O
7
8
9
2"
N N
1"
MeO
7"
11
10
11
12
13
14
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59
60
Figure 3. X-ray structure of LCK and 11. Hydrogen bonds and favorable interactions are denoted with dashes. The carbons and ribbon
representation of LCK are colored green except for the loop between β-strand3 and helix-C which is colored blue. The carbons of 11 are
colored pink, oxygen is red, and nitrogen is blue. Residues in the binding pocket that differ between LCK and CSK have red labels. Bottom
right: Close-up of the surface of LCK around the indazole. The surface of LCK is colored green except for the loop between β-strand3 and
helix-C which is colored blue.
The C3’ methyl of the phenyl ring points into a small pocket
below β-strand 3 and contributes to the non-planarity of the
phenyl ring relative to the amide. This likely explains the
decrease in potency observed in compound 9 as compared to 7
and 8. The indazole ring forms an edge-to-face interaction with
Phe285 and has a lipophilic interaction with Met280.
Comparison of the structure of LCK with 11 bound to the apo
structure (Figure 4) shows that 11 binds in an inactive DFG-out
conformation, with significant distortion of helix C.
the less hindered amine to give 19, which was then acylated a
second time with acid 20.
It is noteworthy that the N1 methyl group in compound 11
points toward the loop between β-strand 3 and α-helix C.
Analysis of the protein surface in this area (Figure 1 inset)
shows relatively little space to accommodate substituents larger
than this methyl group, and compounds containing a substituted
piperidine at this position show increased selectivity for CSK
relative to 11. This region of LCK shares little homology with
CSK, so this increased selectivity is likely attributable to an
increased ability of the corresponding region of CSK to make
favorable interactions with substituents larger than methyl.
The N1 substituent is not in proximity to Leu251 or Ala381, the
two residues in the binding pocket that are different in CSK
(corresponding to Ile201 and Ser331, respectively), so the
sequence differences in the loop between β-strand 3 and α-helix
C are likely the major contributor to selectivity between LCK
and CSK.
Figure 4. X-ray structures of LCK and 11 (green) superposed with
apo LCK (yellow, PDB code 3LCK) highlighting the residues of
the DFG motif. The carbons of 11 are colored pink.
Removal of the Boc group and installation of the tert-butyl urea
with tert-butyl isocyanate completed the synthesis of compound
8. For the preparation of 4 (Scheme 2), bromide 21 was
converted to boronic ester 22, which was then coupled to
choloropyridazinone 23 to give 24. Coupling with acid 17 was
Illustrative syntheses of compounds 8 and 4 are shown in
Schemes 1 and 2. Alkylation of indazole 15 with mesylate 16
followed by ester hydrolysis gave carboxylic acid 17.
Acylation of bis-aniline 18 with this acid occurred primarily at
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ACS Medicinal Chemistry Letters
effected with PyBOP; choice of coupling reagents was
important, as reagents such as HATU had a tendency to form
adducts with unprotected pyridazinones such as 24 and its
coupled product. Synthesis of 4 was completed by removal of
the Boc group and formation of the urea with tert-butyl
isocyanate.
6
14
12
>120
88
0.5%
<0.1%
0.4%
1.0%
0.3%
3.0
6.7
8.9
1
2
3
4
5
6
7
8
13
14
>120
>120
<0.2%
aPB: protein binding. bFSI(100): percent of 238 kinases tested in
an internal panel with an IC50 value within 100 fold of the
compound’s CSK HTRF IC50.
Scheme 2. Synthesis of compound 4.a
At a 100 mg/kg oral dose, compound 4 showed a relatively
high initial exposure (Table 4) with a T1/2 of 1.9 hours and
negligible exposure at 24 hours. In contrast, compound 13 had
an extended absorption period with a maximum concentration
at 7 hours, a 5.3 hour T1/2, and substantial exposure even at 24
hours after dosing. Spleens from the mice were harvested and
the level of LCK Y505 phosphorylation was measured. Mice
treated with 4 showed an 87% decrease in LCK phosphorylation
compared to vehicle-treated mice at 3 hours, when plasma
concentration was 8.7 µM. The decrease for mice treated with
13 was 88% at 6 hours (close to the Tmax of 7 hours). Thus, both
4 and 13 show the anticipated effect of CSK inhibition upon
oral dosing in mice.
9
NH2
N
NH
Cl
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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NH2
Me
23
Me
Me
B
a
b
O
O
Br
21
NH2
22
O
N
Me
Me
N
17
Table 4. PK and PD parameters for compounds 4 and 13.a
N
Boc
HO
N
NH
Cpd
Cmax
(µM)
Tmax
(h)
C24h
AUC
T1/2
(h)
LCK
pY505
c, d, e
(µM)
(µM•h)
O
4
17.3
19.3
0.3
7
0.001
1.3
51.5
1.9
5.3
87%
88%
24
13
221.5
O
aLCK pY505: percent change in LCK pY505 compared to
vehicle, t= 3 h for 4, t=6 h for 13. C57bl/6 mice were used.
HN
N
N
Me
Me
IL-2 Response (pg/ml)
O
N
2500
Me
Me
[13] (nM)
HN
N
NH
Me
2000
1500
1000
500
0
0
O
4
4
12
37
111
333
aReagents
and
conditions:
(a)
PdCl2(dppf)•DCM,
bis(pinacolato)diboron, KOAc, dioxane, 100 ºC, 39%; (b) 23,
PdCl2(dppf)•DCM, Na2CO3, DMF, H2O, 35%; (c) 17, PyBOP,
Hünig’s Base, DMF, 67%; (d) HCl, dioxane, quant.; (e) tert-butyl
isocyanate, Hünig’s Base, DCM, 67%.
CHO
CHO-OKT3
Based on their favorable potency and metabolic stability
profiles, pyridazinones 4 and 6 and pyrazolopyridines 13 and
14 were subjected to further in vitro profiling (Table 3). All
compounds showed high protein binding. Compounds 13 and
14 showed long T1/2 values in the presence of both human and
mouse liver microsomes, while the T1/2 of compound 4 was
moderate for mouse and short for human, and the T1/2 of
compound 6 was low for both species. Pyrazolopyridine 13 was
chosen for advancement into mouse PK/PD studies due to its
high level of potency and metabolic stability; 4 was chosen to
represent the pyridazinone series due to its greater metabolic
stability relative to 6.
Proliferation (MTS cell count)
200000
150000
100000
50000
0
[13] (nM)
0
4
12
37
111
333
Table 3. Further profiling of compounds 4, 6, 13, and 14.a,b
Cpd
4
HLM
T1/2
(min)
MsLM
T1/2
(min)
Human
PB
%Free
Mouse
PB
%Free
FSI(100)
5.5
CHO
CHO-OKT3
Figure 5. Top: CD4+ T cell IL-2 secretion in response to 13.
Bottom: CD4+ T cell proliferation in response to 13.
10
31
0.4%
0.9%
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To test whether compound 13 was able to activate T cells in
ABBREVIATIONS
1
2
3
4
5
6
7
8
a context-appropriate manner, an assay was utilized in which
Chinese hamster ovary (CHO) cells were mixed with CD4+ T
cells.14 Two types of cells were used: parental CHO cells and
CHO cells expressing OKT, a cognate antibody for the T cell
receptor. These cells were treated with compound 13 and
monitored for IL-2 secretion and proliferation. The results from
this experiment are summarized in Figure 5. As expected, the
parental CHO cells induced a minimal response which was not
increased by compound 13. CHO cells expressing OKT
induced a stronger response, which was increased in a dose-
dependent fashion by 13. At 333 nM of compound 13, IL-2
secretion was increased by 5.7 fold; under the conditions
employed, proliferation reached a maximum increase of 2 fold
at 37 nM. Taken together, these results confirm the hypothesis
that CSK inhibition can enhance the response of T cells to
antigen stimulation.
CHO, Chinese hamster ovary; CTLA-4, cytotoxic T lymphocyte-
associated protein 4; dppf, 1,1’-bis(diphenylphosphino)ferrocene;
HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxide hexafluorophosphate; HTRF, homogeneous
time-resolved fluorescence; LCK, lymphocyte-specific protein
tyrosine kinase; PD, pharmacodynamics; PD-1; programmed cell
death protein 1; PD-L1, programmed death-ligand 1; PK,
pharmacokinetics;
yloxy)tripyrrolidinophosphonium hexafluorophosphate; ZAP-70,
zeta-chain-associated protein kinase 70
PyBOP,
(Benzotriazol-1-
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(12) For a report of a CSK inhibitor with an IC50 of 1.9 µM in an
ELISA assay, see Kilimnik, A.; Kostjukova, M. N.; Pyatkin, I.
H; Pronin, A. M.; Strelnikova, S. R.; Fedotov, Y. A.;
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terminal Src Kinsase (Csk). Cell. Mol. Biol. Lett. 2003, 8, 588.
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Josephs, J.; Zvyaga, T; Zamora, I.; Shou, W. Z. Development,
optimization, and implementation of a centralized metabolic
soft spot assay. Bioanal. 2017, 9, 541-552.
In summary, we have developed a series of small molecule
inhibitors of CSK that can serve as in vitro and in vivo tools to
evaluate the potential of this target as an immuno-oncology
therapy. Switching from a pyridazinone to pyrazolopyridine
hinge binder gave a substantial increase in cellular potency.
Metabolite identification studies enabled the strategic blocking
of metabolic soft spots by the exchange of a tert-butyl urea for
a cyano amide and fluorination of an indazole, culminating in
the discovery of compound 13. Compound 13 reduced
inhibitory LCK phosphorylation in vivo upon oral dosing and
showed the ability to enhance T cell activation in response to
antigen stimulation. Notably, this increase was not observed in
the absence of an antigen. These findings support further
evaluation of the potential of CSK inhibition to enhance
antitumor immune response. The molecules described in this
manuscript provide suitable tools to evaluate the relationship
between the extent and duration of CSK inhibition and the
efficacy and tolerability of treatment in animal models, an
important consideration given the toxicity observed in knockout
mice.15,16
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Experimental procedures and characterization data for the
compounds described (PDF)
AUTHOR INFORMATION
Corresponding Author
* Email: daniel.o’malley@bms.com
Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding Sources
This work was funded by Bristol-Myers Squibb Company.
Notes
The authors declare no competing financial interest.
(14) For application of a similar assay, see Englehardt, J.J.; Selby,
M, J.; Korman, A.J.; Feingersh, M, D.; Stevens, B. L. Anti-Icos
agonist antibodies and uses thereof. WO 2018/187613 A2,
October 11, 2018.
(15) For additional studies on a different series of CSK inhibitors
from this group, see Wang, C; Cao, C; Berman-Booty, L;
Eraslan, R; Sanjuan, M; Vite, G; Hunt, J; Fink, B; Wee, S.
Targeting CSK kinase activity to enhance antitumor immunity
ACKNOWLEDGMENT
The authors wish to acknowledge the contributions of the CSK
Discovery Working Group.
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ACS Medicinal Chemistry Letters
[abstract]. Proceedings of the AACR Special Conference on
inflammation in conditional knockout mice. Vet. Pathol. 2018,
55, 331-340.
Tumor Immunology and Immunotherapy; 2017 Oct 1-4;
Boston, MA. Philadelphia (PA): AACR; Cancer Immunol. Res.
2018;6(9 Suppl):Abstract nr B02.
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(16) Berman-Booty, L. D.; Eraslan, R.; Hanumegowda, U.; Cantor,
G. H.; Bounous, D. I.; Janovitz, E.B.; Jones, B. K.; Buiakova,
O.; Hayward, M.; Wee, S. Systemic loss of C-terminal Src
kinase
expression
elicits
spontaneous
suppurative
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F
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O
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CSK Caliper IC50: 4 nM
HN
N
N
Jurkat ZAP70 EC50: 41 nM
 Tcell proliferation in vitro
 LCK phosphorylation in vivo
Cl
N
O
HN
N
O
NC
13
N
O
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