A Chemical Probe for Dark Kinase STK17B Derives Its Potency and High Selectivity through a Unique P-Loop Conformation

Article abstract of DOI:10.1021/acs.jmedchem.0c01174

STK17B is a member of the death-associated protein kinase family and has been genetically linked to the development of diverse diseases. However, the role of STK17B in normal and disease pathology is poorly defined. Here, we present the discovery of thieno[3,2-d] pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe for this understudied "dark"kinase. 11s is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of 11s and related thieno[3,2-d]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor. Molecular dynamic simulations of STK17B revealed the flexibility of the P-loop and a wide range of R41 conformations available to the apo-protein. The isomeric thieno[2,3-d]pyrimidine SGC-STK17B-1N (19g) was identified as a negative control compound. The >100-fold lower activity of 19g on STK17B was attributed to the reduced basicity of its pyrimidine N1.

Full text of DOI:10.1021/acs.jmedchem.0c01174

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Article
A Chemical Probe for Dark Kinase STK17B Derives Its Potency and
High Selectivity through a Unique P‑Loop Conformation
Alfredo Picado, Apirat Chaikuad, Carrow I. Wells, Safal Shrestha, William J. Zuercher, Julie E. Pickett,
Frank E. Kwarcinski, Parvathi Sinha, Chandi S. de Silva, Reena Zutshi, Shubin Liu, Natarajan Kannan,
Stefan Knapp, David H. Drewry, and Timothy M. Willson*
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ABSTRACT: STK17B is a member of the death-associated protein kinase family and has been genetically linked to the
development of diverse diseases. However, the role of STK17B in normal and disease pathology is poorly defined. Here, we present
the discovery of thieno[3,2-d] pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe for this understudied “dark” kinase.
11s is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-
ray crystallography of 11s and related thieno[3,2-d]pyrimidines bound to STK17B revealed a unique P-loop conformation
characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor. Molecular dynamic simulations of STK17B
revealed the flexibility of the P-loop and a wide range of R41 conformations available to the apo-protein. The isomeric thieno[2,3-
d]pyrimidine SGC-STK17B-1N (19g) was identified as a negative control compound. The >100-fold lower activity of 19g on
STK17B was attributed to the reduced basicity of its pyrimidine N1.
and DAPK3 share many of these biological functions as
integrators of cell death signaling.
INTRODUCTION
■
Protein kinases are a large family of enzymes that catalyze the
transfer of the γ-phosphate from the cofactor ATP to a serine,
threonine, or tyrosine residue in their substrate proteins.
Structural studies show that these enzymes contain a β-sheet-
rich N-lobe and α-helix-rich C-lobe that is connected by a
flexible hinge into which ATP is bound.1 Sequence analysis of
the human genome has identified over 500 members of the
kinase protein family2 that have been organized into nine
groups based on sequence similarity. One of these groups is
the calcium/calmodulin regulated kinases (CAMK), which
contains the death-associated protein kinase (DAPK) family of
five serine/threonine kinases: namely, the closely related
DAPK1−3 and the more distant STK17A and STK17B (also
known as DRAK1 and DRAK2, respectively).3 The five
members of the DAPK family are characterized by a highly
conserved N-terminal catalytic domain but showed large
differences in their flanking regulatory domains. DAPK1 is a
regulator of caspase-dependent and independent cell death. It
is necessary for apoptosis and is annotated as a tumor
suppressor whose expression is lost in many cancers. DAPK2
Much less is known about the roles of STK17A and STK17B
in cell signaling. STK17A expression was associated with an
increase in head and neck squamous cell carcinoma through
inhibition of TGFβ1-mediated tumor suppression.4 STK17A
was also overexpressed in glioblastoma multiforme (GBM) and
associated with decreased patient survival. Knockdown of
STK17A decreased cellular proliferation, motility, and invasion
in multiple GBM cell lines.4 STK17B may also have a role in
cancer. It is overexpressed in hepatocellular carcinoma and
breast cancer, and silencing of STK17B in cells suggested that
it may have utility in treatment of these malignancies.4
Notably, STK17B is abundant in lymphoid organs and
predominantly expressed in mature T- and B-cells.5 STK17B
Received: July 7, 2020
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knock-out mice were remarkably resistant to autoimmune
challenge when crossed onto a non-obese diabetic mouse
strain6 (a model of type-1 diabetes) or challenged with
experimental autoimmune encephalomyelitis7 (a model of
multiple sclerosis). This autoimmune protection appeared to
be driven by reduction in the survival of autoreactive T-cells
that were responsible for cell and tissue damage. Thus,
STK17B may have a unique role within the DAPK family as an
immune-regulatory target.
Although STK17B may be an interesting therapeutic target
in oncology and autoimmunity, there have been no selective
inhibitors available to probe the sequelae of decreasing its
catalytic activity in cells or animals. The only reported STK17B
inhibitors are either promiscuous, show poor selectivity within
the DAPK family, or lack activity in cells.4 Recent efforts in
broad screening have surfaced a range of scaffolds that bind to
STK17B, but none with all the properties required for a
chemical probe. Among the nonselective STK17B inhibitors
are quercetin 1, which is a family-wide CAMK inhibitor,7,8 and
the receptor tyrosine kinase inhibitor dovitinib 2, which has
micromolar cross-reactivity with both STK17A/B9 (Figure 1).
of highly selective kinase inhibitors a challenge. Nonetheless,
medicinal chemists have been successful in exploiting some
distinctive features in the ATP binding site among protein
kinases.14,15 Approaches commonly used to gain selectivity
include (a) targeting a hydrophobic back pocket that is not
penetrated by ATP, but which is accessible in those kinases
with a small gate-keeper residue (V, T, A, or G); (b) exploiting
the intrinsic flexibility of the hinge region between the N- and
C-lobes that contains several glycine residues; (c) development
of covalent inhibitors that capture cysteine residues that are
found in a subset of kinases; or (d) binding into an extended
pocket formed when kinases adopt an inactive conformation,
called DFG-out, thus blocking ATP from access to the catalytic
domain. Using one or more of these approaches, several high-
quality chemical probes have been developed for protein
kinases.
The kinase N-lobe contains a highly flexible phosphate-
binding loop (P-loop; also referred to as the glycine-rich loop)
that has received less attention in the development of selective
inhibitors. The P-loop makes critical contacts with the γ-
phosphate of ATP to support catalysis. It was observed in a
kinked conformation in the structure of imatinib with ABL that
supported a hydrophobic cage around the drug.16 A rare P-
loop conformation was seen in MAP4K4 bound to a selective
inhibitor and extended analysis of crystal structures in the
Protein Data Bank (PDB) revealed an unusual conformation in
seven additional kinases.17 We now report the discovery of a
series of potent, selective, and cell active STK17B inhibitors
based on the thieno[3,2-d]pyrimidine PFE-PKIS 43 (4), which
was uncovered through deep profiling of the kinase inhibitor
set known as PKIS2.18 Co-crystal structures of 4 and related
thieno[3,2-d]pyrimidines with STK17B uncovered a unique P-
loop flip that appears to be the molecular basis of their
remarkable potency and selectivity. Optimization of the
thieno[3,2-d]pyrimidine chemotype resulted in the first high-
quality chemical probe for STK17B.
Figure 1. Chemical structures of STK17B inhibitors.
Several other inhibitors have shown up to 20-fold selectivity for
STK17B within the DAPK family, but none of them have been
reported to be cell active.10−12 Benzofuranone 3 is the only
inhibitor with modest STK17B selectivity that was also
reported to be active in cells. In a phenotypic assay, 3
protected islet β-cells from apoptosis induced by free fatty acid
palmitate.13
Almost all known kinase inhibitors are heterocycles that are
competitive with the cofactor ATP. The targeting of a
conserved cofactor binding site can make the development
RESULTS
■
Identification of an STK17B Inhibitor. Several thieno-
[3,2-d]pyrimidines, previously published as inhibitors of Tpl2
(MAP3K8, also known as COT),19 were donated by Pfizer to
the Structural Genomics Consortium (SGC) kinase chemo-
genomic set,18 now known as KCGS.20 The compounds were
screened at 1 μM through the DiscoverX scanMAX panel of
403 wild-type human kinases, and three compounds, PFE-
PKIS 43 (4), PFE-PKIS 14 (5), and PFE-PKIS 9 (6), were
Table 1. Thieno[3,2-d]pyrimidines with STK17B Activity
a
DiscoverX
b
entry
compound
X
R1
R2
scanMAX S10 (1 μM)
STK17A KD (nM)
STK17B KD (nM)
Tpl2 IC50 (nM)
1
2
3
PFE-PKIS 43 (4)
PFE-PKIS 14 (5)
PFE-PKIS 9 (6)
S
S
O
SCH3
CONH2
NHNCH
H
H
0.005
0.020
0.012
230
270
250
3.8
11
30
3400
1500
1000
a
DiscoverX KINOMEscan. scanMAX, S10 (1 μM) = number of kinases with 10% or less of control activity remaining at 1 μM / number of wild type
b
kinases tested (403), n = 1. KD, determined by dose response, n = 2. Reported by Ni et al. (ref 19).
B
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identified as narrow spectrum kinase inhibitors with S10 (1
μM) ≤ 0.020 (Table 1).18 Full KD determinations performed
for all kinases inhibited >75% at 1 μM revealed that 4−6 were
potent inhibitors of STK17B with 8−60-fold selectivity over
STK17A (Table 1). Notably, 4−6 were each reported to be
only weak inhibitors of Tpl2.19 4 was the most potent and
selective of the analogues. To probe the molecular basis of its
remarkable kinase selectivity, a co-crystal structure was
obtained with the catalytic domain of STK17B (Figure 2).
thienopyrimidine analogues targeting three regions of the
molecule, as follows: the carboxylic acid, heterocyclic core, and
6-aryl group.
Syntheses of Analogues. The synthesis 6-arylthieno[3,2-
d]pyrimidines 4 and 11a−11ab was adapted from the
abbreviated procedures reported by Pfizer (Scheme 1).19
Scheme 1. Synthesis of 6-Arylthieno[3,2-d]pyrimidines 4
and 11a−11ab and Primary Amide 12.
Reagents and conditions: (a) (i) LDA 1.2 equiv, THF, −78 °C, 20
min, (ii) I2 1.2 equiv, −78 °C, 20 min, rt., 12 h, 68%; (b) (i) ethyl 2-
mercaptoacetate 1.5−2.0 equiv, MeOH, 0 °C, (ii) Et3N 3-4 equiv, 0
°C to rt., 12 h, 65%; (c) (i) 1 M LiOH (aq) 2.0 equiv, THF:EtOH
1:1, 70 °C, 1 h, (ii) 1 M HCl (aq) until pH 2−3, 0 °C, 80%; (d) (i)
ArB(OH)2 1.1 equiv, PdCl2(PPh3)2 5 mol %, K2CO3 2.2 equiv, 1,4-
dioxane:water 3:1, 95 °C, 3−12 h, (ii) 1 M HCl (aq) until pH 2−3, 0
°C 30−65%; (e) HATU 1.0 equiv, ammonia (aq) excess, N,N-
diisopropylethylamine 2.2 equiv, DMF, rt., 12 h, 25%.
Figure 2. X-ray structure of the catalytic domain of STK17B. (A)
STK17B/4 complex (PDB: 6Y6F). STK17B protein backbone is
shown in green. The side chains of R41, K62, and the backbone amide
of A113 are highlighted. 4 is shown as sticks with atoms colored C
pink, N blue, O red, and S yellow. (B) STK17B/4 complex (PDB:
6Y6F). Alternate view showing the interaction of the carboxylic acid
of 4 with R41 and K62. The unusual conformation of the P-loop is
further stabilized by a salt bridge between R41 and E117. (C)
STK17B/ADP complex (PDB: 6QF4). STK17B protein backbone is
shown in light blue. The side chains of R41, K62, E111, and E117 and
the backbone amide of A113 are highlighted. ADP is shown as sticks
with atoms colored C pink, N blue, O red, and P orange.
The first step involved the deprotonation and iodination of 4-
chlorothieno[3,2-d]pyrimidine (7), resulting in 4-chloro-6-
iodothieno[3,2-d]pyrimidine (8) in good yield. Intermediate 8
underwent an SNAr reaction with ethyl 2-mercaptoacetate to
obtain 9, which was saponified to 10 in good yield. Compound
10 was a key intermediate that allowed synthesis of 6-
arylthieno[3,2-d]pyrimidines 4, 11a−11ac by Suzuki coupling
with a range of arylboronic acids. The primary amide 12 was
generated by reaction of 4 with ammonia.
Syntheses of side-chain-modified analogues (Scheme 2)
utilized two additional intermediates that were obtained by the
As expected, 4 occupied the ATP-binding pocket of STK17B
(Figure 2A). However, the orientation of the inhibitor was
markedly different to the proposed binding mode in Tpl2.19 In
the STK17B structure, the carboxylic acid of 4 was directed
toward the catalytic lysine (K62) with the hydrophobic 6-aryl
substituent extended toward the solvent (Figure 2A,B).
Notably, it was the N1 of the thieno[3,2-d]pyrimidine that
was observed to bind to the amide backbone of A113 in the
hinge, not the N3 as previously proposed for Tpl2.19 In
addition to the interaction with K62, the carboxylic acid of 4
formed a second salt bridge with R41 of the P-loop (Figure
2A,B). The salt bridge with R41 was accommodated by a
remarkable conformational change in the P-loop in which R41
had flipped a 180° from its orientation in the structure of
STK17B bound to ADP (Figure 2C).21
Although, 4 showed outstanding potency and selectivity as a
potential chemical probe for STK17B, additional testing
revealed that it had very low stability in liver microsomes.
After 30 min, 100% of the compound was consumed in mouse
liver microsomes and its t1/2 was determined to be <5 min
(vide infra). To gain insights into the low metabolic stability of
4, its P450 metabolism was modeled by SMARTCyp,22 which
identified the sulfur atom of the methyl thioether moiety as the
most reactive part of the molecule (SI Figure 1). To improve
the metabolic stability of 4 and define the structure activity for
STK17B inhibition, we initiated the synthesis of a series of
Scheme 2. Syntheses of Side-Chain-Modified Analogues.
Reagents and conditions: (a) 4-Chlorophenylboronic acid 1.0 equiv
or 4-(methylthio)phenylboronic acid 1.0 equiv, Pd(PPh3)4 5 mol %,
NaHCO3 1.0 equiv, CsF 1.0 equiv, 1,4-dioxane:water 3:1, 95 °C, 3 h,
55%; (b) ethyl 2-hydroxyacetate 2.0 equiv, NaH 2.6 equiv (60%
dispersion in mineral oil), anhydrous DMF, 100 °C, 30 min
microwave, 45%; or ethyl glycinate hydrochloride 2.5 equiv, TEA
10 equiv, EtOH, 90 °C, 18 h, 78% (c) (i) LiOH 2.2 equiv, THF, 70
°C, 4 h, (ii) 1 M HCl (aq) until pH 4−5, 0 °C, 82%.
regioselective Suzuki coupling of 8, yielding 4-chloro-6-
arylthieno[3,2-d]pyrimidines 13a and 13b. These intermedi-
ates were further derivatized by SNAr reaction with ethyl 2-
hydroxyacetate and ethyl glycinate, yielding esters 14 and 16a,
which were saponified to yield compounds 15 and 16b,
respectively.
C
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A series of regioisomeric thieno[2,3-d]pyrimidines were
synthesized from the commercially available 6-bromo-4-
chlorothieno[2,3-d]pyrimidine (17) (Scheme 3). SNAr reac-
gave an IC50 of 20 nM on STK17B (Table 2), slightly weaker
than the 3.8 nM KD value obtained in the DiscoverX binding
assay. 4 also demonstrated robust activity in the live cell
NanoBRET assay with an IC50 of 210 nM (Table 2, entry 1).
Substitution of −SMe for −Cl in the 6-aryl ring (11a) retained
STK17B activity and selectivity in the binding assay (Table 2,
entry 2) and showed only a small decrease in activity in cells.
The primary amide analogue 12 of acid 4 showed a reduced
STK17B activity in the binding assay and a lack of activity in
cells (Table 2, entry 3). Replacement of sulfur for oxygen in
the sidechain in 15 reduced the STK17B activity in the binding
assay, and the analogue was inactive in cells (Table 2, entry 5).
Surprisingly, its ester analogue 14 had equivalent (albeit low)
activity in the STK17B binding assay (Table 2, entry 4), but
was also inactive in cells. Double substitution of nitrogen for
sulfur and ester for the acid in the side chain resulted in an
analogue 16a that was devoid of STK17B activity (Table 2,
entry 6). The binding affinity for amino acid 16b (Table 2,
entry 7) could not be determined due to interference of the
compound with the split luciferase assay. However, it was
shown to be inactive when screened in cells (IC50 > 10 μM).
The regioisomeric thieno[2,3-d]pyrimidine analogues 19a and
19b (scaffold B) showed only very low activity in both the
STK17A and STK17B binding assays and were inactive in the
cell-based assay (Table 2, entries 8 and 9). Overall, these initial
results confirmed the importance of the α-thioacetic acid side
chain for potent STK17B activity and revealed a rather narrow
structure−activity. Only 4 and its 6-chloro analogue 11a
showed activity in cells, and only one regioisomer of the
thienopyrimidine scaffold was active. The sensitivity of the
acetic acid side chain to the nature of the α-hetero substituent
was surprising since in COT/Tpl2 substitution of sulfur for
oxygen was tolerated,19 although with reduced activity.
Scheme 3. Synthesis of Regioisomeric Thieno[2,3-
d]pyrimidines.
Reagents and conditions: (a) (i) 2-mercaptoacetic acid 1.25 equiv,
EtOH or CH3CN, 0 °C, (ii) TEA 2.5 equiv, 0 °C to rt., 12 h, (iii) 1 M
HCl (aq) until pH 2−3, 0 °C, 75%; (b) (i) ArB(OH)2 1.0 equiv,
Pd(PPh3)4 5 mol %, K2CO3 3−4 equiv, 1,4-dioxane:water 3:1, 95 °C,
3 h, (ii) 1 M HCl (aq) until pH 2−3, 0 °C 48−65%.
tion with 2-mercaptoacetic acid gave 18 in good yield.
Thieno[2,3-d]pyrimidine 17 showed higher reactivity in this
substitution reaction than the corresponding thieno[3,2-
d]pyrimidine regioisomer 8, which did not react directly
with 2-mercaptoacetic acid. Suzuki coupling of 18 with several
boronic acids resulted in products 19a−19g in moderate
yields.
Structure−Activity Relationships. 4 and its analogues
were initially screened for their STK17A and STK17B activities
using a split luciferase KinaseSeeker binding assay23 at a
concentration of 1 μM. Any analogue showing >70%
competition at STK17B was subjected to dose response to
determine its IC50 in the binding assay. In addition, all
analogues were tested in dose response up to 10 μM by a
NanoBRET assay24 in HEK293 cells to determine intracellular
STK17B target engagement. In the thieno[3,2-d]pyrimidine
scaffold A, initial exploration focused on the α-thioacetic acid
side chain because of the key interactions observed in the co-
crystal structure of 4 with STK17B (Figure 2). In the binding
assay, 4 was selective for STK17B over STK17A at 1 μM and
Since substitution of the 6-aryl ring appeared to be tolerated
in STK17B, a second round of analogues sought to explore
structure activity in this part of the molecule. Analogues were
synthesized in the regioisomeric thienopyrimidines C and D
with the α-thioacetic acid functionality in place (Table 3).
Analogues in the thieno[3,2-d]pyrimidine scaffold C generally
Table 2. Initial SAR
KinaseSeekera
NanoBRETb
entry
compound
scaffold
X
R1
R2
STK17A (% I)
STK17B (% I)
STK17B IC50 (nM)
STK17B IC50 (nM)
1
2
3
4
5
6
7
8
9
4
11a
12
14
15
16a
16b
19a
19b
A
A
A
A
A
A
A
B
B
S
S
S
O
O
NH
NH
S
OH
OH
NH2
OEt
OH
OEt
OH
OH
OH
SCH3
Cl
SCH3
Cl
15
3
1
4
0
90
90
61
36
36
20 3.9
210 40
680 190
39 8.2

c
>10,000
>10,000
>10,000
>10,000
c

c
Cl

c
SCH3
SCH3
SCH3
OCH3
4
d
0
d

c

>10,000
0
14
26
18

>10,000c
>10,000
S

a
KinaseSeeker split luciferase binding assay. % I, inhibition compared to control (DMSO) at 1 μM, n = 2, data 10%. IC50, determined by dose
b
c
d
response, n = 2 SD.  not determined. Target engagement determined by the NanoBRET assay in HEK293 cells, n = 3 SE. n = 1. Not
determined due to assay interference.
D
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Table 3. SAR of 6-Aryl Substituents
a
KinaseSeeker split luciferase binding assay. % I, inhibition compared to control (DMSO) at 1 μM, n = 2, data 10%. IC50, determined by dose
b
c
response, n = 2 SD.  not determined. Target engagement determined by the NanoBRET assay in HEK293 cells, n = 3 SE. n = 1.
showed good STK17B binding with selectivity over STK17A
(Table 3, entries 1−9). Potent activity (>70% inhibition at 1
μM) was seen with electron-donating groups o-OMe, m-SMe,
p-SiPr, p-OBn (11b−11d, 11j), alkyl groups p-Et, p-vinyl, p-iPr,
p-cPr (11e−11h), and m,p-dichloro substitution (11i). In the
cell-based NanoBRET assay, the most potent compounds were
11b and 11e−11i (Table 3, entries 1 and 4−8), which bear
substituents in para and meta positions. Surprisingly, analogues
11d and 11j with a p-SiPr and p-OBn substituents were
inactive in cells (Table 3, entries 3 and 9) despite having
potent binding to STK17B. Compound 11c, substituted in the
ortho position, showed moderate activity in cells (Table 3,
entry 2). The dichloro analogue 11i showed good activity in
cells (Table 3, entry 8). Most notably, 11b, the m-SMe
analogue of 4, was about 2.5-fold less potent in the STK17B
binding and cell assays (Table 3, entry 1). Analogues of the
regioisomeric thieno[2,3-d]pyrimidine D (Table 3, entries 10−
13) again showed low activity on STK17A and STK17B and
showed no activity against STK17B in cells. This last result
confirmed the initial finding in Table 2, which pointed to the
lack of STK17B activity in the thieno[2,3-d]pyrimidine
scaffold.
Table 4. SAR of Polar 6-Aryl Substituents
a
KinaseSeeker split luciferase binding assay. % I, inhibition compared
to control (DMSO) at 1 μM, n = 2, data 10%. IC50, determined by
To explore the tolerance for polarity in the 6-aryl ring,
additional analogues were synthesized in the thieno[3,2-
d]pyrimidine scaffold C (Table 4). In the binding assay,
these compounds showed robust STK17B activity with good
b
dose response, n = 2 SD. Target engagement determined by the
c
NanoBRET assay in HEK293 cells, n = 3 SE. n = 1.
E
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selectivity over STK17A regardless of the type and position of
the substituent around the phenyl ring (Table 4, entries 1−7).
In the cell-based assay, amides and sulfoxide (11 k−11m) were
inactive (Table 4, entries 1−3). Analogues with a p-Ac (11n)
and m-morpholine (11o) showed moderate activity in cells
(Table 4, entries 4−5). Interestingly, compound 11p bearing a
p-morpholine demonstrated submicromolar cell potency
(Table 4, entry 6). In contrast, the acetylpyridine 11q was
inactive in cells (Table 4, entry 7).
Table 6. SAR of Fluorine Substituted 6-Aryl Substituents
The next series of analogues explored the reduction of
electron density in the 6-aryl ring using a thiophene
modification or addition of a mildly electron-withdrawing
group in the para position (Table 5). Two analogues where the
Table 5. SAR of Lipophilic 6-Substituents
a
KinaseSeeker split luciferase binding assay. % I, inhibition compared
to control (DMSO) at 1 μM, n = 2, data 10%. IC50, determined by
b
dose response, n = 2
SD. Target engagement determined by
NanoBRET assay in HEK293 cells, n = 3 SD.
(11p), or m-trifluromethoxyphenyl (11ab) showed good cell
potency.
a
KinaseSeeker split luciferase binding assay. % I, inhibition compared
Metabolic Stability. Since initial evaluation of 4 had
shown it to possess poor metabolic stability likely due to P450
metabolism of the p-SMe substituent, a series of the cell active
6-arylthieno[3,2-d]pyrimidines were selected for metabolic
stability studies. In parallel, the compounds were tested for
aqueous solubility and their LogD at pH 7.0 was calculated.
Table 7 shows the results of this study with compounds sorted
by their metabolic stability after 30 min of incubation in mouse
liver microsomes. 4 (Table 7, entry 1), which showed no
compound remaining after a 30 min incubation in mouse liver
microsomes, had a calculated LogD in the drug-like range25
and modest aqueous solubility. Replacement of p-SMe by p-
CO2Et (11u) did not improve metabolic stability (Table 7,
entry 2). Addition of a m-F substituent (11y) to the ring
resulted in a small improvement in metabolic stability (Table
7, entry 3). Removal of the p-SMe substituent further
improved metabolic stability as seen with 11c, 11g, 11f,
11w, and 11r (Table 7, entries 4−8). 11p, 11s, 11h, and 11o
bearing p-morpholino, benzothiophene, p-cyclopropyl, and m-
morpholino group, respectively, showed even greater metabolic
stability, each with >50% of the compound remaining after 30
min (Table 7, entries 9−12). The fluorinated analogues 11z,
11aa, and 11x showed the best metabolic stability in the series
(Table 7, entries 13−15). None of the analogues showed
improved aqueous solubility over 4 despite changes in their
calculated LogD. The benzothiophene 11s had lower
solubility, but this did not affect its activity in cells.
to control (DMSO) at 1 μM, n = 2, data 10%. IC50, determined by
b
dose response, n = 2
SD. Target engagement determined by
NanoBRET assay in HEK293 cells, n = 3 SD.
6-phenyl ring was replaced by thiophene (11r) or
benzothiophene (11s) (Table 5, entries 1 and 2) yielded
compounds with potent and selective STK17B binding and
robust activity in cells. In contrast, the more electron negative
p-CN (11t) and p-CO2Et (11u) derivatives were less potent in
cells (Table 5, entries 3 and 4).
For the final series of analogues, the effect of fluorine
substitution on the 6-aryl ring was explored with a goal to
identify modifications that would be less prone to P450
oxidation (Table 6). The mono- and di-fluorophenyl analogues
(11v−11x) were active in the binding assay but showed only
modest activity in cells (Table 6, entries 1−3). Reintroduction
of the p-SMe substituent in 11y resulted in a boost in cell
activity (Table 6, entry 4). The p-OMe analogue 11z (Table 6,
entry 5) showed a 1.3-fold drop in cell potency. Compounds
11aa and 11ab bearing a trifluoromethyl and a trifluorome-
thoxy substituent, respectively, were found to have good
binding to STK17B and sub-μM cell potency (Table 6, entries
6−7).
In summary, the structure−activity study identified a series
of 6-arylthieno[3,2-d]pyrimidine-4-thioacetic acids with good
binding selectivity for STK17B over STK17A. Analogues with
the best cell potency as measured by NanoBRET target
engagement were often substituted on the 6-aryl group with
small para-substituents, which could be either lipophilic (4,
11e, 11f, 11g, 11 h, 11i, and 11y) or mildly electron
withdrawing (11a, 11u, and 11aa). In addition, analogues with
thiophene (11r), benzothiophene (11s), p-morpholinophenyl
Selection of a Chemical Probe. After consideration of
the binding, cell potency, and metabolic stability data, 11s was
selected as a potential chemical probe. Despite its lower kinetic
aqueous solubility (5 μg/mL), 11s demonstrated the highest
cell potency in the NanoBRET target engagement assay (IC50
= 190 nM) among the analogues that showed <50%
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Table 7. Microsomal Stability and Physiochemical
Properties
Table 8. Kinase Selectivity of Chemical Probe (11s)
a
KINOMEscan
Enzymeb
NanoBRETc
Kinase
MET
NEK6
PIM2
WEE1
STK17B
CAMKK2
AURKB
CAMKK1
STK38L
STK17A
% I
KD
IC50 (nM)
IC50 (nM)
100
100
100
100
99
92
86
86
71
>10,000
>10,000
1500
>10,000
5.6
>10,000
>10,000
>10,000
>10,000
34
>10,000
8900
5200




190
21
2400
>10,000

110
49
>10,000
440
>10,000
4700
>10,000
>10,000
27
a
DiscoverX KINOMEscan. % I, scanMAX % inhibition of control at 1
b
μM, n = 1. KD, determined by dose response, n = 2. IC50 for enzyme
inhibition at Km ATP determined at Eurofins, n = 2. Target
c
engagement IC50 determined by NanoBRET assay in HEK293 cells.
 not determined.
on STK17A, CAMKK2, AURKB, and STK38L (Table 8). 11s
showed an IC50 = 2.4 μM on CAMKK2 but >10 μM on the
other kinases. Thus, 11s is a potent STK17B inhibitor with
remarkable selectivity over STK17A across multiple assay
formats. It had outstanding kinome-wide selectivity, with cross-
reactivity on CAMKK1 and CAMKK2 as the only potential
confounding activities noted in the binding assays. Impor-
tantly, 11s is the most potent and selective cell active STK17B
inhibitor reported to date and will be highly useful as a
chemical probe for this dark kinase.
Identification of a Negative Control Analogue. To
augment the utility of 11s as a chemical probe, a close
analogue was designed as an inactive control26 (Table 9).
Building on the structure activity data that showed the
regioisomeric thieno[2,3-d]pyrimidines were inactive as
STK17B inhibitors, the analogue 19g was synthesized (Scheme
3). When profiled across the DiscoverX scanMAX panel at 1
μM no kinase was identified where 19g showed >75%
displacement of the immobilized ligand (SI File 1). However,
a
% compound remaining in mouse liver microsomes at 30 min.
b
c
Kinetic solubility at pH 7.4. Calculated LogD at pH 7.0.
metabolism in the liver microsome assay. To further
characterize its kinome-wide selectivity, 11s was profiled at 1
μM across the DiscoverX scanMAX panel of 403 wild-type
human kinase assays (SI File 1). Eight kinases, including
STK17B, were annotated with >75% displacement of the
immobilized ligand at 1 μM (Table 8). KD determinations
were performed by dose response for these eight kinases plus
STK38L (71% I at 1 μM) and STK17A (27% I at 1 μM). 11s
gave a KD = 5.6 nM on STK17B in the DiscoverX binding
assay but showed some cross-activities with CAMKK2,
CAMKK1, AURKB, STK17A, and PIM2. Annotations for
MET, NEK6, WEE1, and STK38L were shown to be false
positives in the initial scanMAX since they did not confirm in
the dose-response experiment. To further explore the
selectivity profile of 11s, enzyme inhibition assays were
performed in the presence of ATP for all 10 kinases annotated
in the initial scanMAX experiment. In the enzyme assays, 11s
showed potent inhibition of STK17B with an IC50 = 34 nM
and > 100-fold selectivity over STK17A. It was also >100-fold
selective over all of the other kinases tested, with only
STK17A, CAMKK1, and AURKB generating a measurable
dose−response (IC50 5−9 μM). Robust inhibition was not
seen on CAMKK2 in the enzyme assay despite its activity in
the DiscoverX binding assay. As a final confirmation of the
kinase selectivity of 11s, live-cell NanoBRET assays were run
Table 9. STK17B Potency and Relative Basicity of Chemical
Probe (11s) and Negative Control (19g)
11s
19g
STK17B
a
binding KD (nM)
5.6
34
190
910
4800
>10 000
b
enzyme IC50 (nM)
c
cell IC50 (nM)
basicity
d
pKa (N1)
2.8
1.1
0.1
0.5
d
pKa (N3)
a
b
KD, determined by dose response at DiscoverX, n = 2. IC50 for
c
enzyme inhibition at Km ATP determined at Eurofins, n = 2. Target
engagement IC50 determined by NanoBRET assay in HEK293 cells, n
= 3. pKa was obtained from density functional theory (DFT)
d
calculations using the molecular electrostatic potential as the
descriptor.
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Figure 3. X-ray structure of the catalytic domain of STK17B. (A) STK17B/11s complex (PDB: 6Y6H). STK17B protein backbone is shown with
the P-loop conformation A in green and the P-loop conformation B in light blue. The side chains of R41, K62, and the backbone amide of A113 are
highlighted. 11s is shown as sticks with atoms colored C pink, N blue, O red, and S yellow. (B) STK17B/11s complex (PDB: 6Y6H) conformation
A showing the interaction of 11s carboxylic acid with R41 and K62. Conformation A of the P-loop is further stabilized by a salt bridge between R41
and E117. (C) STK17B/11s complex (PDB: 6Y6H) conformation B showing the interaction of 11s carboxylic acid with only K62 and A113. (D)
STK17B/quercetin complex (PDB: 3LM5). STK17B protein backbone is shown in light blue. The side chains of R41, E80, and D179 and the
backbone amides of E111 and A113 are highlighted. Quercetin is shown as sticks with atoms colored C pink and O red. (E) STK17B/dovitinib
complex (PDB: 7AKG). STK17B protein backbone is shown in light blue. The side chains of R41 and K62 and the backbone amides of E111 and
A113 are highlighted. Dovitinib is shown as sticks with atoms colored C pink, N blue, F pale blue, and O red.
19g did displace >65% of the immobilized ligand on five
kinases. KD determinations indicated that four of these were
false positives, and only HIPK4 gave a measurable KD = 290
nM. In addition, KD on STK17B and STK17A were measured
as 0.91 and 9.8 μM, respectively. In the enzyme inhibition
assay, 19g had an IC50 = 4.8 μM on STK17B and IC50 > 10
μM on STK17A. 19g was, therefore, >100-fold less active as an
inhibitor of STK17B than the chemical probe 11s (Table 9).
Furthermore, in cells, 19g was inactive at 10 μM in the
STK17B NanoBRET cellular target engagement assay. Based
on these activity data and its chemical similarity to the
chemical probe, 19g is a suitable negative control for using in
biological assays.
The relative inactivity of all thieno[2,3-d]pyrimidine
analogues on STK17B was somewhat surprising. Specifically,
the >100-fold decreased STK17B activity of thieno[2,3-
d]pyrimidine 19g compared to thieno[3,2-d]pyrimidine 11s
was not easily explained by a simple analysis of their shape
complementarity within the ATP binding pocket of STK17B
(data not shown). To further understand the molecular basis
of the dramatic difference in activity between the regioisomers,
density functional theory (DFT) calculations were performed
to evaluate the molecular electrostatic potential as the
descriptor of protonation propensity and predict the pKa
value for the respective heterocycles.27,28 A benchmark
calculation was first performed on the four regioisomers of
thienopyridine. The calculated pKa values were in very close
agreement with the potentiometric pKa values reported in
literature (SI Table 1),29 highlighting the dramatically lower
basicity of the thieno[2,3-b]pyridine isomer. When applied to
the thieno[3,2-d]pyrimidine 11s, DFT calculated that N1 was
more basic than N3 (Table 9). However, for thieno[2,3-
d]pyrimidine 19g, both nitrogens were found to be much less
basic and the relative basicity was reversed between N1 and
N3. Remarkably, thieno[3,2-d]pyrimidine N1 of the chemical
probe 11s was calculated to be 500-fold more basic than
thieno[2,3-d]pyrimidine N1 of the negative control 19g. In the
active thieno[3,2-d]pyrimidine series, N1 forms the key H-
bond with the hinge motif of the ATP-binding pocket. Our
computational results suggest that N1 in the negative control
thieno[2,3-d]pyrimidine series may lack sufficient basicity to
support a similar strength of molecular recognition.30
Structural Studies. The STK17B chemical probe 11s
demonstrated remarkable kinase selectivity for an ATP-
competitive inhibitor. Most notably, the amino acids lining
the ATP-binding pocket of STK17A are 100% identical except
for a conservative mutation of K37 (STK17B) to R65
(STK17A) on the outside edge of the solvent accessible
region. To explore whether the unusual conformation flip of
the P-loop seen in the 4/STK17B co-crystal structure (Figure
2) could contribute to kinase selectivity, the co-crystal
structures of STK17B in complexes with the chemical probe
11s and close analogue 11h were solved. The structures of
11s/STK17B and 11h/STK17B revealed electron density for
the P-loop in two distinct conformations (Figure 3 and SI
Figure 2). In the folded conformation A, the P-loop resembled
that seen in the 4/STK17B structure in which R41 formed a
salt bridge with the carboxylic acid of the inhibitor (Figure
3B). In the extended conformation B, the P-loop was in an
orientation with R41 rotated away from the inhibitor binding
pocket similar to that observed in the ADP/STK17B structure
(Figure 3C). Detailed inspection of the 11s/STK17B structure
revealed only a single moderate (3.0 Å) H-bond interaction
between the backbone amide of A113 in the hinge region and
N1 of the thieno[3,2-d]pyrimidine inhibitor. In folded
conformation A, the carboxylic acid of 11s was held in a
sandwich of salt bridges with R41 of the P-loop and catalytic
lysine (K62) and also made tight (2.5 Å) H-bonds to a water
molecule and ethylene glycol solvent molecule (Figure 3B).
The 6-benzothiophene substituent sat in a hydrophobic pocket
that was open to the solvent and appeared to derive most of its
binding affinity through shape complementarity. The phenol of
Y112 and terminal amine of K37 were the only polar residues
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Figure 4. MD simulation of apo-STK17B (PDB: 6Y6H). R41 and key residues forming interactions are shown as sticks and colored in magenta and
salmon, respectively. (A) R41 starting in conformation A. After 1000 ns of the simulation, R41 flips to conformation B. (B) R41 starting in
conformation B where it remains throughout the simulation.
in this region but did not interact with 11s. Y112 also did not
appear to form a π-stacking interaction with the inhibitor.
The STK17B co-crystal structures of two nonselective
DAPK family inhibitors, quercetin and dovitinib (Figure 3
and SI Table 2) were also solved. Quercetin is a broad
spectrum inhibitor of the CAMK group,8 while dovitinib
inhibits the DAPK family with IC50 in the 1−5 μM range.7
Quercetin formed H-bonds to several STK17B residues
including the hinge region, D179 of the DFG motif, and E80
of the αC helix. A similar network of H-bonds was observed in
the co-crystal structure of quercetin with DAPK1 (PDB:
5AUW).4 For dovitinib, the only polar interaction with
STK17B was two H-bonds from the pyridinone to E111 and
A113 of the hinge region (2.9 and 2.6 Å, respectively).
Notably, in both the quercetin/STK17B (Figure 3D) and
dovitinib/STK17B (Figure 3E) complexes, the P-loop
exhibited extended conformation B where neither inhibitor
interacted with R41, which was rotated away from the inhibitor
pocket. Thus, only in the co-crystal structures with thieno[3,2-
d]pyrimidine-4-thioacetic acids 4, 11s, and 11h were the P-
loop observed in folded conformation A.
Molecular Dynamics. The folded P-loop conformation of
STK17B seen with the thieno[3,2-d]pyrimidines-4-thioacetic
acids 4, 11s, and 11h provided an explanation for their
remarkable kinome-wide selectivity. However, the near 100-
fold selectivity over STK17A was still surprising given the high
sequence identity of its kinase domain including the critical P-
loop. A docking study with 11s was performed to probe the
origin of the STK17B/A selectivity in more detail. Since no X-
ray crystal structure of STK17A was available, a homology
model was generated with the 3D structure-comparative
modeling software MODELLER31 using the structure of
STK17B/11s (PDB: 6Y6H) as a template (SI File 5). The
resulting homology model, which directly compared the
binding sites of STK17A and STK17B in complex with the
chemical probe 11s, showed that the identity and con-
formation of the ATP-binding site residues contacting the
inhibitor were nearly identical (SI Figure 4). The only
difference observed in the model was the conservative change
of STK17B K37 to STK17A R16 at the solvent accessible
mouth of the pocket, which lies 3.1 Å from the terminal
benzothiophene ring of 11s. Since multiple analogues of 11s
with a range of 6-substituents also showed similar STK17B/A
selectivity (e.g., 4−6 in Table 1), we concluded that residues
outside of the primary binding pocket must be responsible.
To explore the possibility that residues outside of the ATP-
binding pocket could influence the conformational flexibility of
the P-loop and explain the remarkable potency and selectivity
of 4 and 11s, we performed molecular dynamic (MD)
simulations of STK17B in the apo and inhibitor bound
forms starting from R41 in both A and B conformations.
Microsecond timescale simulation (1.25 μs) of the apo-
STK17B with R41 in folded conformation A revealed
exceptional flexibility of the P-loop. Starting from folded
conformation A, after 1 μs, R41 switched to extended
conformation B (Figure 4A, SI Movie 1). This switch was
characterized by an intermediate state (not observed in crystal
structures) in which R41 formed a transient network of
hydrogen bonds with E117, S120, and E125 in the region
encompassing the helix αD as well as packing against the
aromatic ring of F119 (Figure 4A). In the process, the distance
between E125 and R41 was reduced from 17.1 Å in the crystal
structure to within the H-bond distance in the intermediate
state. On the other hand, a 1.25 μs long simulation of the apo-
STK17B with R41 starting in extended conformation B did not
show a switch back to folded conformation A. Instead R41
maintained the salt bridge with E38, as observed in the crystal
structure (Figure 4B, SI Movie 2).
In the inhibitor bound forms, both 4 and 11s stayed bound
to the protein during the 250 ns simulation time scale (Figure
5A and SI Movies 3−5). The results showed that 4 adopted
two distinct conformations due to twisting of the thienopyr-
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Figure 5. MD simulation of inhibitor bound STK17B (PDB: 6Y6F and 6Y6H). (A) RMSD plot of ligands (4 or 11s) during the course of
simulation. GROMACS 2018.1 built-in tool was used to calculate RMSD of the ligand after least square fit using the Cα atoms. (B) Total number
of hydrogen bonds between the ligand and STK17B during the simulation. (C) Total number of hydrogen bonds between the ligand and R41
during the simulation.
imidine substituents, as shown by the increase in root-mean-
square deviation (RMSD) at around 85 ns (Figure 5A, upper
panel and SI Movie 3) while 11s adopted only a single distinct
conformation (Figure 5A, middle and lower panels and SI
Movies 4 and 5). In STK17B folded conformation A, the
inhibitors formed more hydrogen bonds with the protein
(Figure 5B, top and middle panels) than in STK17B extended
conformation B (Figure 5B, lower panel). The higher number
of hydrogen bonds can be attributed to the interaction of R41
with the inhibitors (Figure 5C, top and middle panels). In
STK17B extended conformation B, 11s adopted only a single
inhibitor conformation in which it was bound to K62 of the
catalytic pocket (SI Movie 5) despite the lack of interaction
with R41 (Figure 5C, lower panel). Thus, 11s was bound
stably within the ATP-pocket independent of the interaction
with R41. However, the additional salt bridge between the
inhibitor and R41 in folded conformation A of the P-loop
would be expected to increase the enthalpy of binding.
chemical probes to study their pharmacology.18,32 Many of
these dark kinases are known to play an important role in cell
signaling as shown by genetic studies.33 STK17B is one such
example. The phenotype of knock-out mice shows that the loss
of STK17B protects against autoimmune challenge.5,34,35
However, prior to our work, no potent, cell active, selective
STK17B inhibitor was available to study the role of this dark
kinase in immune cell signaling.4
ATP-competitive kinase inhibitors generally show cross-
activity with multiple kinases beyond their primary target. As a
community project to create a comprehensive collection of
inhibitors for every human kinase, we sought donations of
molecules from pharmaceutical companies and academic
investigators.18,32 The output of this project, known as the
Kinase Chemogenomic Inhibitor Set (KCGS), currently
contains selective inhibitors of over 200 kinases.20 The
identification of 4 as a potent and selective inhibitor of
STK17B is a powerful example of this approach. Several
thieno[3,2-d]pyrimidines, originally synthesized as inhibitors
of the kinase COT/Tpl2,19 were donated by Pfizer to the
project. Deep profiling of these molecules through the
DiscoverX scanMAX panel identified activity on STK17B
DISCUSSION
■
Despite the track record for development of kinase inhibitors
as drugs for oncology, inflammation, and fibrosis, the majority
of human kinases lack selective inhibitors that can be used as
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that was generally 100 times greater than on the original Tpl2
target.
more plausible explanation for the divergent activity of the
thieno[3,2-d]pyrimidine- and thieno[2,3-d]pyrimidine-4-α-
thioacetic acids in STK17B was revealed by analysis of their
electrostatic potentials. DFT calculations based on the
molecular electrostatic potential of the heterocycles unveiled
dramatic differences between the regioisomers as reflected by
their predicted pKa values. It was already documented that
related thienopyridine regioisomers showed differences in their
measured pKa values; thieno[2,3-b]pyridine has a much lower
pKa compared to the other three regioisomers due to the
inductive effect of sulfur adjacent to the pyridine nitrogen.29
Likewise, in the thieno[2,3-d]pyrimidine 19g, electron density
on N1 was much lower than in thieno[3,2-d]pyrimidine 11s
and there was a 500-fold difference in the calculated pKa. The
extremely weak basicity of N1 in the thieno[2,3-d]pyrimidine
regioisomers may decrease their ability to be recognized by
STK17B. Notably, there are some thieno[2,3-d]pyrimidines
that have been reported as kinase inhibitors,41 and in one case,
a thieno[2,3-d]pyrimidine-α-thiopropanoic acid was a potent
inhibitor of CSNK2. However, it was also found that changing
the salt concentration was sufficient to invert this inhibitor
binding pose such that N1 no longer interacted with the
hinge.42 Thus, while not all thieno[2,3-d]pyrimidines are
devoid of kinase activity,41 in the case of STK17B, it appears
that the more basic thieno[3,2-d]pyrimidine regioisomers are
much more active as inhibitors. Importantly, the identification
of an inactive analogue (19g) that is a simple regioisomer of
the chemical probe (11s) will be useful to control for off-target
activity in the study of STK17B biology.26
The 6-aryl group on the thieno[3,2-d]pyrimidine sits in the
region of the ATP-pocket that reaches toward the solvent.
Structure−activity studies confirmed that substitution of this
ring was tolerated and could be used to optimize the cell
activity and increase the metabolic stability of the series.
Analogues with small lipophilic substituents on the para-
position demonstrated some of the best cell activity, whereas
the ortho- and meta-substituents were much less active. The
larger thiophene and benzothiophene analogues (11r and 11s)
also showed potent cell activity. The most surprising result was
seen with the para-morpholine substituent (11p), which had
robust cell activity. In contrast, substituents that added an H-
bond acceptor or donors were generally less active, as were
substitutions that were electron withdrawing (e.g., −CN and
−CO2Et). Our ability to measure STK17B target engagement
in cells by NanoBRET assay24 was critical in being able to
navigate the subtle balance of lipophilicity and polarity
required to offset the intrinsic charge of the carboxylic acid.
It is notable that none of the acidic thieno[3,2-d]pyrimidines
previously optimized as COT/Tpl2 inhibitors were reported to
be active in cells.19
The results of a structure−activity study showed the critical
role of the α-thioacetic acid for potent activity on STK17B.
Replacement of the sulfur with nitrogen was not tolerated and
even the switch to oxygen led to a loss in STK17B activity. In
contrast, the switch of sulfur for oxygen was tolerated in COT/
Tpl2.19 Notably, the orientation of 4 was flipped 180°
compared to the proposed binding mode in Tpl2. A co-crystal
structure of 4 with STK17B revealed the key role of the
carboxylic acid in binding to the catalytic pocket of the kinase.
The carboxylic acid was sandwiched between two residues:
R41 from the P-loop and the catalytic lysine K62. The
interaction with the catalytic lysine is not uncommon with
polar or acidic kinase inhibitors,36,37 but the salt bridge with
R41 was remarkable, given that it required a large conforma-
tional change in the P-loop. This conformational change was
also seen in the co-crystal structure of the chemical probe 11s
with STK17B. Could the P-loop flip explain the remarkable
selectivity of thieno[3,2-d]pyrimidines 4 and 11s for STK17B
over its close neighbor STK17A?
Several lines of evidence support the critical role of the P-
loop in the kinase selectivity of the thieno[3,2-d]pyrimidines.
First, the P-loop is known to be one of the most flexible
regions of the kinase catalytic domain and has been implicated
previously in selective inhibition of ABL and MAP4K4.16,17,38
Likewise, molecular dynamic simulation showed that apo-
STK17B can easily access both folded conformation A and
extended conformation B of the P-loop. Second, quercetin and
dovitinib, which bind with P-loop conformation B in their
STK17B structures, show no selectivity over STK17A. The co-
factor ADP also crystallized in P-loop conformation B with the
position of the ribose such that it would block R41 from
adopting folded conformation A (Figure 2C). Finally, all of the
residues lining the ATP-binding pocket are identical between
STK17A and STK17B making it even more likely that the
remarkable selectivity of the thieno[3,2-d]pyrimidine inhib-
itors is driven by differences in conformational flexibility of the
kinases. STK17A and STK17B differ in regions outside of the
ATP binding region that might impact inhibitor specificity by
altering the conformational flexibility of R41 in the P-loop, as
revealed in the molecular dynamic simulation. In particular,
E125 and L126 in STK17B are substituted to an aspartate
(D153) and arginine (R154), respectively (SI Figure 3). These
STK17A specific variations are expected to destabilize the
intermediate R41 conformation observed in STK17B (Figure
4A). Thus, while the thieno[3,2-d]pyrimidines can access both
conformations, it is only in the unique folded conformation A
that the salt bridge with R41 would provide increased binding
affinity.
The co-crystal structure of the thieno[3,2-d]pyrimidine-4-α-
thioacetic acids 4 and 11s with STK17B showed a single 3.0 Å
H-bond interaction between the pyrimidine N1 with the
backbone NH of A113 in the hinge loop, which is in the range
of distances typically seen for Type I kinase inhibitors.39,40 The
observation of only a single H-bond suggests it is critical for
molecular recognition of the thieno[3,2-d]pyrimidines by the
STK17B hinge region. Notably, all of the regioisomeric
thieno[2,3-d]pyrimidine-4-α-thioacetic acids, including the
negative control 19g, were inactive as inhibitors of STK17B
and almost all other kinases (SI File 1). The shape of the ATP-
binding pocket in STK17B provides no insights as to why the
regioisomers showed such dramatic differences in activity. A
Modification of the 6-aryl substituent also addressed the
poor metabolic stability of 4. The p-SMe group was a likely site
of oxidative metabolism, being predicted to be a P450 hot spot
by the SMARTCyp algorithm. Replacement of the −SMe
moiety with groups less prone to oxidation yielded analogues
with much improved stability in liver microsomes. From the
series, we selected 11s as the analogue with the best properties
to be a high-quality chemical probe,26 given its balance of
potency, selectivity, cell activity, and metabolic stability. 11s is
a nanomolar STK17B inhibitor that has 100-fold selectivity
over other DAPK family members including STK17A. It shows
robust target engagement of STK17B in cells at concentrations
below 1 μM and has good stability in liver microsomes. The
K
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performance liquid chromatography (UPLC) system equipped with
a photodiode array (PDA) detector running acetonitrile/water
gradient. Samples for high-resolution mass spectrometry (HRMS)
were analyzed with a ThermoFisher Q Exactive HF-X mass
spectrometer coupled with a Waters Acquity H-class liquid chromato-
graph system. Samples were introduced through a heated electrospray
source (HESI) at a flow rate of 0.6 mL/min. Electrospray source
conditions were set as follows: spray voltage 3.0 kV, sheath gas
(nitrogen) 60 arb, auxiliary gas (nitrogen) 20 arb, sweep gas
(nitrogen) 0 arb, nebulizer temperature 375 °C, capillary temperature
380 °C, and RF funnel 45 V. The mass range was set to 150−2000 m/
z. All measurements were recorded at a resolution setting of 120,000.
All compounds were > 95% pure by analytical LC.
4-Chloro-6-iodothieno[3,2-d]pyrimidine (8). An oven dried
250 mL three-neck round-bottom flask provided with a pressure-
equalizing addition funnel, it was charged with 4-chlorothieno[3,2-
d]pyrimidine 7 (1.0 equiv, 20.0 mmol, 3.40 g); it was added under
positive N2 pressure, followed by the addition of anhydrous THF (65
mL). The solution was cooled to −78 °C, LDA (2.0 M, 1.2 equiv,
24.0 mmol, 12.0 mL) was added portionwise over 2−3 min, and the
mixture was stirred for 20 min at −78 °C. A solution of I2 (1.2 equiv,
24.0 mmol, 6.08 g) in anhydrous THF (15 mL) was added over 10
min using the equalizing addition funnel. This mixture was stirred for
30 min at −78 °C, allowed to warm up to room temperature over the
curse of 1 h, and then stirred again for 2 more hours at this
temperature. It was quenched with saturated NH4Cl(aq)/ice water,
CHCl3 (200 mL) was added, and the layers were separated. The
aqueous layer was extracted with CHCl3 two times; organic layers
were combined and rinsed with aq Na2S2O3 two times and brine two
times, dried over Na2SO4, concentrated in vacuo, and purified over
silica gel with hexanes/EtOAc in a gradient up to 100% of the polar
solvent giving a clear brown solid (4.5 g, 65%). 1H NMR (400 MHz,
CDCl3) δ 8.90 (s, 1H), 7.84 (s, 1H).
modest aqueous solubility does not impact its use in cell-
culture experiments.
Like other members of the thieno[3,2-d]pyrimidine series,
11s showed outstanding selectivity across a panel of over 400
human kinases. However, two kinases that emerged as a
potential off-targets were CAMKK1 and CAMKK2. The
chemical probe showed <10-fold selectivity over these kinases
in the DiscoverX binding assay, although the selectivity
window was greater in enzyme inhibition and cell-based
NanoBRET assays (Table 8). Both the DAPK and CAMKK
families belong to the CAMK branch of the kinome. Although
sequence analysis of their ATP-binding pockets predicted that
cross-reactivity of Type-I inhibitors would not be seen,14 our
recent chemocentric review highlights carboxylic acid contain-
ing compounds GSK650394 and STO6090 as potent
CAMKK2 inhibitors,43 suggesting that there are commonalities
in the ATP-binding pockets that might support the binding of
acidic kinase inhibitors. Using NanoBRET target engagement
assays, 11s showed >10-fold selectivity for STK17B over
CAMKK2 in cells. Thus, the cumulative binding, enzyme
inhibition, and target engagement data (Table 8) indicate that
at a 1 μM concentration 11s will be a selective STK17B
inhibitor in cell-based assays.
CONCLUSIONS
■
SGC-STK17B-1 (11s) has been identified as a high-quality
chemical probe for the dark kinase STK17B that demonstrated
submicromolar cellular activity and remarkable selectivity over
other kinases including STK17A. The thieno[3,2-d]-
pyrimidine-4-α-thioacetic acid chemotype bound to STK17B
in a unique conformation that sandwiched the inhibitors
between the catalytic lysine and P-loop arginine. The kinase
selectivity demonstrated by these inhibitors was likely due to
the interaction with unique conformational states sampled by
STK17B, which allowed access to the folded P-loop observed
in multiple X-ray co-crystal structures. The public availability
of SGC-STK17B-1 (11s) as the first truly potent and selective
chemical probe of STK17B and its negative control analogue
SGC-STK17B-1N (19g) will aid in the illumination of the cell
biology of this dark kinase.
Ethyl 2-((6-iodothieno[3,2-d]pyrimidin-4-yl)thio)acetate (9).
In a 25 mL round-bottom flask provided with a rubber septum, a
magnetic stir bar, and under positive N2 pressure, 4-chloro-6-
iodothieno[3,2-d]pyrimidine 8 (1.0 equiv, 3.5 mmol, 1.05 g) was
suspended in EtOH (6.0 mL) at 0 °C (ice bath), followed by the
dropwise addition of ethyl 2-mercaptoacetate (1.6 equiv, 5.6 mmol,
0.62 mL) and triethylamine (3.2 equiv, 11.2 mmol, 1.6 mL). The
reaction mixture was stirred overnight with slow warm up from 0 °C
to room temperature. Dichlomethane was added to the reaction
crude, solvents evaporated in vacuo, and purified over silica gel with
hexanes/EtOAc in a ramp up to 15% EtOAc (the main by-product of
this reaction is the des-iodopyrimidine, which elutes right after the
main product). After purification, a whitish solid was recovered (800
mg, 62%): 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 7.78 (s, 1H),
4.24 (q, J = 7.1 Hz, 2H), 4.18 (s, 2H), 1.29 (t, J = 7.1 Hz, 3H).
2-((6-Iodothieno[3,2-d]pyrimidin-4-yl)thio)acetic acid (10).
In a 100 mL round-bottom flask provided with a reflux condenser and
magnetic stir bar, ethyl 2-((6-iodothieno[3,2-d]pyrimidin-4-yl)thio)-
acetate 9 (1.0 equiv, 2.1 mmol, 800 mg) was dissolved in a mixture of
EtOH (15 mL) and THF (15 mL), followed by the addition of LiOH
(2.0 equiv, 4.2 mmol, 100 mg) predissolved in water (5 mL). This
mixture was refluxed at 70 °C for 1 h. Solvents were removed under
reduced pressure, the white solid obtained was taken into ice/water,
and the pH adjusted to 2−3 with 2 M aq HCl. The resulting solid was
filtrated, thoroughly rinsed with water, and air and vacuum dried
yielding a white solid (590 mg, 80%): mp 183.5−186.0 °C. 1H NMR
EXPERIMENTAL SECTION
■
General Chemistry. Solvents were used as ACS or HPLC grade.
Anhydrous tetrahydrofuran (THF), lithium diisopropylamide 2.0 M
solution (THF/heptane/ethylbenzene), commercial reagents, and
catalyst were used as received. For air sensitive reactions, the
glassware was oven dried and cooled under nitrogen. Low-
temperature baths (−78 °C) were made from dry ice and 2-propanol.
All the reactions were carried out under positive pressure of N2. NMR
spectra were recorded as CDCl3 and DMSO-d6 solutions on a 400,
500, or 700 MHz instrument. The 1H NMR chemical shits are
reported as δ values in parts per million (ppm) relative to
tetramethylsilane (TMS, δ = 0.00). For the residual chloroform
signal, CHCl3 (δ = 7.26) was used as a reference, and for the other,
DMSO signal (quintet, centerline δ = 2.50) was used as a reference.
13C NMR chemical shifts are reported as δ values in parts per million
(ppm) relative to TMS and DMSO signal (septet, centerline δ =
39.52) as a reference. Analytical thin-layer chromatography (TLC)
was performed on silica gel plates, 200 μm with an F254 indicator,
visualization was accomplished by UV light (254/365 nm) or 3%
ethanol solution of phosphomolybdic acid. Flash column chromatog-
raphy was performed with 50 μm irregular silica or 60 μm spherical
silica-prepacked cartridges. Yields are reported as a pure material after
isolation by column chromatography or recrystallization. Analytical
LC/MS data was obtained using a Waters Acquity Ultrahigh-
(400 MHz, DMSO-d6) δ 8.79 (s, 1H), 7.92 (s, 1H), 3.90 (s, 2H); 13
C
NMR (176 MHz, DMSO) δ 168.5, 161.4, 159.2, 154.0, 133.8, 132.3,
93.1, 33.5. HRMS (HESI) calcd for C8H6N2O2S2I [M + H]+
352.8837, found 352.8917.
General Procedure A: Synthesis of 6-Arylthieno[3,2-d]-
pyrimidines (4, 11a−11ab). In a 25 mL round-bottom flask
provided with a reflux condenser, a rubber septum, a magnetic stir bar,
and under positive N2 pressure, the following reagents were added: 6-
iodothieno[3,2-d]pyrimidin-4-yl)thio)acetic acid 10 (1.0 equiv, 0.21
mmol, 75 mg), K2CO3 (2.2 equiv, 0.47 mmol, 65 mg), Pd(PPh3)Cl2
(5−6 mol %, 8−10 mg), and the corresponding arylboronic acid (1.1
L
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1
equiv, 0.23 mmol). A previously N2 degassed solution of 1,4-dioxane/
water (3/1, 2.5 mL) was added, and the reaction mixture was stirred
under mild reflux for 8−12 h. After this period, the solvent was
removed under reduced pressure, solids taken into ice/water, and the
pH adjusted to 2−3 with 2 M HCl(aq). The resulting solid was
filtrated, thoroughly rinsed with water, air and vacuum dried, and
purified over silica gel using CH2Cl2/MeOH/HOAc (95/4/1) in a
gradient up 5−10% of the polar solvent.
obtained (43 mg, 62%): mp 198.0−201.0 °C. H NMR (500 MHz,
DMSO-d6) δ 12.95 (s, 1H), 8.95 (s, 1H), 8.13 (s, 1H), 7.96 (d, J =
8.4 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 6.82 (dd, J = 17.7, 10.9 Hz,
1H), 5.99 (d, J = 17.7 Hz, 1H), 5.39 (d, J = 10.9 Hz, 1H), 4.26 (s,
2H); 13C NMR (176 MHz, DMSO) δ 169.3, 161.0, 159.4, 154.1,
151.2, 139.0, 135.7, 131.2, 127.0, 127.0, 126.8, 120.0, 116.1, 31.5.
HRMS (HESI) calcd for C16H12N2O2S2 [M + H]+ 329.0418, found
329.0401.
2-((6-(4-(Methylthio)phenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (PFE-PKIS 43) (4). Prepared using general
procedure A, with 4-(methylthio)phenylboronic acid. After purifica-
tion, a pale yellow solid was obtained (30 mg, 40%): mp 194.0−197.0
2-((6-(4-Isopropylphenyl)thieno[3,2-d]pyrimidin-4-yl)thio)-
acetic Acid (11g). Prepared using general procedure A, with 4-
isopropylphenylboronic acid. After purification, a whitish solid was
obtained (40 mg, 55%): mp 154−159 °C. 1H NMR (400 MHz,
DMSO-d6) δ 12.94 (s, 1H), 8.94 (s, 1H), 8.05 (s, 1H), 7.92−7.87 (m,
2H), 7.45−7.40 (m, 2H), 4.26 (s, 2H), 3.00−2.94 (sept, J = 6.9 Hz,
1H), 1.24 (d, J = 6.9 Hz, 6H); 13C NMR (176 MHz, DMSO) δ 169.4,
161.0, 159.5, 154.0, 151.9, 151.1, 129.6, 127.4, 126.8, 126.6, 119.5,
33.3, 31.5, 23.6. HRMS (HESI) calcd for C17H16N2O2S2 [M + H]+
345.0731, found 345.0707.
1
°C. H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 8.93 (s, 1H),
8.07 (s, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 4.25
(s, 2H), 2.55 (s, 3H); 13C NMR (176 MHz, DMSO) δ 169.4, 160.9,
159.6, 154.1, 151.3, 141.8, 128.1, 127.1, 126.5, 126.0, 119.4, 31.5,
14.2. HRMS (HESI) calcd for C15H12N2O2S3 [M + H]+ 349.0139,
found 349.0121.
2-((6-(4-Chlorophenyl)thieno[3,2-d]pyrimidin-4-yl)thio)-
acetic acid (11a). Prepared using general procedure A, with (4-
chlorophenyl)boronic acid. After purification, a white solid was
obtained (40 mg, 55%): mp >250.0 °C. 1H NMR (400 MHz, DMSO-
d6) δ 12.95 (s, 1H), 8.96 (s, 1H), 8.16 (s, 1H), 8.01 (d, J = 8.4 Hz,
2H), 7.62 (d, J = 8.4 Hz, 2H), 4.26 (s, 2H); 13C NMR (176 MHz,
DMSO) δ 169.3, 161.3, 159.3, 154.1, 150.1, 135.0, 130.8, 129.4,
128.5, 127.0, 120.8, 31.5.HRMS (HESI) calcd for C14H9ClN2O2S2
[M + H]+ 336.9872, found 336.9849.
2-((6-(4-Cyclopropylphenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11h). Prepared using general procedure A, with 4-
cyclopropylphenylboronic acid. After purification, a whitish solid was
1
obtained (35 mg, 48%): mp 184.0−187.0 °C. H NMR (700 MHz,
DMSO-d6) δ 12.94 (s, 1H), 8.93 (s, 1H), 8.03 (s, 1H), 7.84 (d, J =
8.3 Hz, 2H), 7.23 (d, J = 8.3 Hz, 2H), 4.25 (s, 2H), 2.03−1.98 (m,
1H), 1.05−1.01 (m, 2H), 0.79−0.75 (m, 2H); 13C NMR (176 MHz,
DMSO) δ 169.3, 160.8, 159.5, 154.0, 151.8, 146.9, 128.9, 126.6,
126.5, 126.2, 119.2, 31.4, 15.1, 10.1. HRMS (HESI) calcd for
C17H14N2O2S2 [M + H]+ 343.0574, found 343.0558.
2-((6-(3-(Methylthio)phenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11b). Prepared using general procedure A, with 3-
(methylthio)phenylboronic acid. After purification, a pale yellow solid
2-((6-(3,4-Dichlorophenyl)thieno[3,2-d]pyrimidin-4-yl)thio)-
acetic Acid (11i). Prepared using general procedure A, with 3,4-
dichlorophenylboronic acid. After purification, a white solid was
1
was obtained (48 mg, 65%): mp >250.0 °C. H NMR (400 MHz,
DMSO-d6) δ 8.88 (s, 1H), 8.11 (s, 1H), 7.78 (t, J = 1.8 Hz, 1H), 7.68
(dt, J = 7.5, 1.3 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.41−7.35 (m, 1H),
3.92 (s, 2H), 2.58 (s, 3H); 13C NMR (176 MHz, DMSO) δ 171.0,
158.7, 154.1, 150.3, 144.4, 140.0, 132.8, 129.8, 127.4, 127.0, 123.3,
123.1, 120.7, 28.9, 14.5. HRMS (HESI) calcd for C15H12N2O2S3 [M
+ H]+ 349.0139, found 349.0124.
1
obtained (31 mg, 40%): mp 222.0−226.0 °C. H NMR (500 MHz,
DMSO-d6) δ 12.96 (s, 1H), 8.98 (s, 1H), 8.32 (d, J = 2.2 Hz, 1H),
8.27 (s, 1H), 7.95 (dd, J = 8.4, 2.2 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H),
4.27 (s, 2H); 13C NMR (176 MHz, DMSO) δ 169.2, 161.5, 159.1,
154.1, 148.5, 132.8, 132.5, 132.3, 131.4, 128.4, 127.2, 126.8, 121.9,
31.5. (HESI) calcd for C14H8Cl2N2O2S2 [M + H]+ 372.9453, found
372.9438.
2-((6-(2-Methoxyphenyl)thieno[3,2-d]pyrimidin-4-yl)thio)-
acetic Acid (11c). Prepared using general procedure A, with 2-
methoxyphenylboronic acid. After purification, a whitish solid was
2-((6-(4-(Benzyloxy)phenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11j). Prepared using general procedure A, with 4-
(Benzyloxy)phenylboronic acid. After purification, a pale yellow solid
1
obtained (35 mg, 50%): mp 219.0−221.5 °C. H NMR (400 MHz,
DMSO-d6) δ 12.93 (s, 1H), 8.93 (s, 1H), 8.11 (s, 1H), 8.07−8.04 (m,
1H), 7.51 (ddd, J = 8.6, 7.5, 1.7 Hz, 1H), 7.28 (dd, J = 8.6, 1.1 Hz,
1H), 7.13 (td, J = 7.5, 1.1 Hz, 1H), 4.26 (s, 2H), 4.02 (s, 3H); 13C
NMR (176 MHz, DMSO) δ 169.4, 160.8, 158.1, 156.1, 153.7, 147.3,
131.6, 129.1, 127.4, 121.2, 121.0, 120.2, 112.6, 56.0, 31.4. HRMS
(HESI) calcd for C15H12N2O3S2 [M + H]+ 333.0367, found 333.0345.
2-((6-(4-(Isopropylthio)phenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11d). Prepared using general procedure A, with 4-
(isopropylthio)phenylboronic acid. After purification, a pale yellow
1
was obtained (26 mg, 30%): mp 192.0−195.0 °C. H NMR (500
MHz, DMSO-d6) δ 12.93 (bs, 0.5H), 11.95 (bs, 0.5H), 8.92 (s, 1H),
7.98 (s, 1H), 7.94−7.91 (m, 2H), 7.48 (d, J = 7.5 Hz, 2H), 7.42 (dd, J
= 8.3, 6.8 Hz, 2H), 7.36 (d, J = 7.5 Hz, 1H), 7.19−7.16 (m, 2H), 5.22
(s, 2H), 4.25 (s, 2H); 13C NMR (176 MHz, DMSO) δ 169.3, 160.6,
160.1, 159.7, 154.0, 151.7, 136.6, 128.5, 128.3, 127.9, 127.7, 126.4,
124.6, 118.5, 115.6, 69.4, 31.4. HRMS (HESI) calcd for
C21H16N2O3S2 [M + H]+ 409.0680, found 409.0671.
1
2-((6-(4-Acetamidophenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11k). Prepared using general procedure A, with 4-
acetamidophenylboronic acid. After purification, a pale yellow solid
solid was obtained (36 mg, 45%): mp 234.0−237.0 °C. H NMR
(400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.02 (s, 1H), 7.89 (d, J = 8.1
Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 3.95 (s, 2H), 3.67 (sept, J = 6.8 Hz,
1H), 1.30 (d, J = 6.8 Hz, 6H); 13C NMR (176 MHz, DMSO) δ 163.5,
158.9, 154.1, 153.6, 150.3, 138.8, 135.8, 129.6, 129.4, 127.1, 126.7,
124.3, 119.8, 40.0, 36.1, 22.7. HRMS (HESI) calcd for C17H16N2O2S3
[M + H]+ 377.0452, found 377.0433.
1
was obtained (23 mg, 30%): mp 232.0−235.0 °C. H NMR (400
MHz, DMSO-d6) δ 12.94 (s, 1H), 10.23 (s, 1H), 8.92 (s, 1H), 7.99
(s, 1H), 7.94−7.88 (m, 2H), 7.77−7.72 (m, 2H), 4.25 (s, 2H), 2.09
(s, 3H); 13C NMR (176 MHz, DMSO) δ 169.4, 168.7, 160.8, 159.6,
154.0, 151.7, 141.3, 127.4, 126.4, 126.3, 119.2, 118.7, 31.5, 24.2.
HRMS (HESI) calcd for C16H13N3O3S2 [M + H]+ 360.0476, found
360.0463.
2-((6-(4-Ethylphenyl)thieno[3,2-d]pyrimidin-4-yl)thio)acetic
Acid (11e). Prepared using general procedure A, with 4-ethyl-
phenylboronic acid. After purification, a whitish solid was obtained
(28 mg, 40%): mp 175.0−177.5 °C. 1H NMR (400 MHz, DMSO-d6)
δ 12.94 (s, 1H), 8.94 (s, 1H), 8.05 (s, 1H), 7.89 (d, J = 8.0 Hz, 2H),
7.39 (d, J = 8.0 Hz, 2H), 4.26 (s, 2H), 2.68 (q, J = 7.5 Hz, 2H), 1.21
(d, J = 7.5 Hz, 3H); 13C NMR (176 MHz, DMSO) δ 169.3, 160.9,
159.5, 154.0, 151.9, 146.6, 129.4, 128.8, 126.8, 126.6, 119.4, 31.4,
27.9, 15.3. HRMS (HESI) calcd for C16H14N2O2S2 [M + H]+
331.0574, found 331.0561.
2-((6-(3-Acetamidophenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11l). Prepared using general procedure A, with 3-
acetamidophenylboronic acid. After purification, a pale yellow solid
1
was obtained (26 mg, 35%): mp 221.0−225.0 °C. H NMR (500
MHz, DMSO-d6) δ 12.96 (s, 1H), 10.18 (s, 1H), 8.96 (s, 1H), 8.21−
8.17 (m, 1H), 8.01 (s, 1H), 7.68 (ddd, J = 11.1, 7.9, 1.9 Hz, 2H), 7.48
(t, J = 7.9 Hz, 1H), 4.27 (s, 2H), 3.32 (s, 3H); 13C NMR (176 MHz,
DMSO) δ 169.4, 168.7, 161.2, 159.3, 154.1, 151.6, 140.2, 132.2,
130.0, 126.7, 121.4, 120.6, 120.1, 116.7, 31.5, 24.1. HRMS (HESI)
calcd for C16H13N3O3S2 [M + H]+ 360.0476, found 360.0454.
2-((6-(4-Vinylphenyl)thieno[3,2-d]pyrimidin-4-yl)thio)acetic
Acid (11f). Prepared using general procedure A, with 4-vinyl-
phenylboronic acid. After purification, a pale yellow solid was
M
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2-((6-(4-(Methylsulfinyl)phenyl)thieno[3,2-d]pyrimidin-4-
yl)thio)acetic Acid (11m). Prepared using general procedure A,
with 4-(methylsulfinyl)phenylboronic acid. After purification, a
whitish solid was obtained (34 mg, 45%): mp 220.0−222.5 °C. H
DMSO-d6) δ 12.97 (s, 1H), 8.99 (s, 1H), 8.33 (s, 1H), 8.19−8.17 (m,
2H), 8.03−7.99 (m, 2H), 4.27 (s, 2H); 13C NMR (176 MHz,
DMSO) δ 169.6, 162.1, 159.1, 154.4, 149.6, 136.4, 133.5, 128.0,
127.8, 122.6, 118.6, 112.6, 31.8. HRMS (HESI) calcd for
C15H9N3O2S2 [M + H]+ 328.0214, found 328.0190.
1
NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 8.98 (s, 1H), 8.24 (s,
1H), 8.20−8.16 (m, 2H), 7.87−7.83 (m, 2H), 4.27 (s, 2H), 2.82 (s,
3H); 13C NMR (176 MHz, DMSO) δ 169.3, 161.4, 159.2, 154.2,
150.2, 148.5, 133.9, 127.5, 127.1, 124.7, 121.4, 43.1, 31.5. HRMS
(HESI) calcd for C15H12N2O3S3 [M + H]+ 365.0088, found 365.0071.
2-((6-(4-Acetylphenyl)thieno[3,2-d]pyrimidin-4-yl)thio)-
acetic Acid (11n). Prepared using general procedure A, with 4-
acetylphenylboronic acid. After purification, a whitish solid was
2-((6-(4-(Ethoxycarbonyl)phenyl)thieno[3,2-d]pyrimidin-4-
yl)thio)acetic Acid (11u). Prepared using general procedure A, with
4-ethoxycarbonylphenylboronic acid. After purification, a pale orange
1
solid was obtained (39 mg, 50%): mp 182.0−185.0 °C. H NMR
(400 MHz, DMSO-d6) δ 12.96 (s, 1H), 8.97 (s, 1H), 8.25 (s, 1H),
8.12 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 4.33 (q, J = 7.1 Hz,
2H), 4.25 (s, 2H), 1.34 (t, J = 7.1 Hz, 3H); 13C NMR (176 MHz,
DMSO) δ 169.3, 165.0, 161.6, 159.1, 154.1, 149.8, 136.0, 130.9,
130.0, 127.3, 127.0, 121.9, 61.0, 31.6, 14.1. HRMS (HESI) calcd for
C17H14N2O4S2 [M + H]+ 375.0473, found 375.0445.
1
obtained (40 mg, 55%): mp 193.0−197.0 °C. H NMR (500 MHz,
DMSO-d6) δ 12.97 (s, 1H), 8.99 (s, 1H), 8.29 (s, 1H), 8.14 (d, J =
8.5 Hz, 2H), 8.10 (d, J = 8.5 Hz, 2H), 4.28 (s, 2H), 2.65 (s, 3H); 13C
NMR (176 MHz, DMSO) δ 197.3, 169.3, 161.5, 159.2, 154.2, 150.0,
137.6, 135.9, 129.2, 127.4, 127.0, 121.9, 31.5, 26.9. HRMS (HESI)
calcd for C16H12N2O3S2 [M + H]+ 345.0367, found 345.0357.
2-((6-(3-Morpholinophenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11o). Prepared using general procedure A, with 3-
morpholinophenylboronic acid. After purification, a pale orange solid
2-((6-(3-Fluorophenyl)thieno[3,2-d]pyrimidin-4-yl)thio)-
acetic Acid (11v). Prepared using general procedure A, with 3-
fluorophenylboronic acid. After purification, a white solid was
1
obtained (39 mg, 58%): mp 194.0−196.5 °C. H NMR (400 MHz,
DMSO-d6) δ 12.96 (s, 1H), 8.97 (s, 1H), 8.22 (s, 1H), 7.91 (dt, J =
10.2, 2.2 Hz, 1H), 7.80 (dd, J = 7.7, 1.6 Hz, 1H), 7.60 (td, J = 8.0, 6.0
Hz, 1H), 7.38 (dd, J = 8.6, 2.6 Hz, 1H), 4.27 (s, 2H); 13C NMR (176
MHz, DMSO-d6) δ 169.2, 162.5 (d, J = 246.3 Hz), 161.5, 159.1,
154.1, 149.9 (d, J = 2.8 Hz), 134.1 (d, J = 8.4 Hz), 131.5 (d, J = 8.5
Hz), 127.1, 122.9 (d, J = 2.5 Hz), 121.3, 117.1 (d, J = 21.3 Hz), 113.6
(d, J = 23.7 Hz), 31.6; 19F NMR (376 MHz, DMSO-d6) δ −111.82 to
−111.92 (m, 1F). HRMS (HESI) calcd for C14H9FN2O2S2 [M + H]+
321.0167, found 321.0151.
1
was obtained (29 mg, 35%): mp 229.0−233.0 °C. H NMR (500
MHz, DMSO-d6) δ 12.94 (s, 1H), 8.95 (s, 1H), 8.13 (s, 1H), 7.46 (t,
J = 2.1 Hz, 1H), 7.41−7.34 (m, 2H), 7.12−7.07 (m, 1H), 4.26 (s,
2H), 3.77 (dd, J = 5.8, 3.8 Hz, 4H), 3.26−3.23 (m, 4H); 13C NMR
(176 MHz, DMSO) δ 169.3, 161.0, 159.4, 154.0, 152.4, 151.7, 132.6,
130.1, 126.7, 120.0, 117.3, 116.9, 112.7, 66.0, 48.0, 31.4. HRMS
(HESI) calcd for C18H17N3O3S2 [M + H]+ 388.0789, found 388.0780.
2-((6-(4-Morpholinophenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11p). Prepared using general procedure A, with 4-
morpholinophenylboronic acid. After purification, an orange solid was
2-((6-(2-Fluorophenyl)thieno[3,2-d]pyrimidin-4-yl)thio)-
acetic Acid (11w). Prepared using general procedure A, with 2-
fluorophenylboronic acid. After purification, a white solid was
1
obtained (29 mg, 35%): mp 205.0−208.0 °C. H NMR (500 MHz,
1
obtained (40 mg, 60%): mp 184.0−187.0 °C. H NMR (400 MHz,
DMSO-d6) δ 12.92 (s, 1H), 8.88 (s, 1H), 7.89 (s, 1H), 7.82 (d, J =
8.9 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 4.24 (s, 2H), 3.78−3.72 (m,
4H), 3.28−3.23 (m, 4H); 13C NMR (176 MHz, DMSO) δ 169.5,
160.2, 159.9, 154.0, 152.5, 152.3, 127.8, 125.9, 121.8, 117.1, 114.6,
65.9, 47.2, 31.4. HRMS (HESI) calcd for C18H17N3O3S2 [M + H]+
388.0790, found 388.0773.
DMSO-d6) δ 12.96 (s, 1H), 8.99 (s, 1H), 8.13−8.07 (m, 2H), 7.58 (s,
1H), 7.49 (dd, J = 11.7, 1.2 Hz, 1H), 7.43−7.39 (m, 1H), 4.27 (s,
2H); 13C NMR (176 MHz, DMSO-d6) δ 169.3, 161.5, 158.9 (d, J =
149.4 Hz), 158.4, 154.1, 144.3 (d, J = 3.5 Hz), 132.3 (d, J = 8.8 Hz),
129.8, 127.2 (d, J = 5.3 Hz), 125.6 (d, J = 3.2 Hz), 122.9 (d, J = 4.8
Hz), 119.7 (d, J = 11.7 Hz), 116.8 (d, J = 22.0 Hz); 19F NMR (376
MHz, DMSO-d6) δ −112.35 to −112.50 (m, 1F). HRMS (HESI)
calcd for C14H9FN2O2S2 [M + H]+ 321.0167, found 321.0142.
2-((6-(3,4-Difluorophenyl)thieno[3,2-d]pyrimidin-4-yl)thio)-
acetic Acid (11x). Prepared using general procedure A, with 3,4-
difluorophenylboronic acid. After purification, a white solid was
2-((6-(6-Acetylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11q). Prepared using general procedure A, with
(6-acetylpyridin-3-yl)phenylboronic acid. After purification, a pale
1
orange solid was obtained (22 mg, 30%): mp 181.0−184.0 °C. H
NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 9.36 (d, J = 2.4 Hz,
1H), 9.01 (s, 1H), 8.55 (dd, J = 8.2, 2.4 Hz, 1H), 8.41 (s, 1H), 8.08
(d, J = 8.2 Hz, 1H), 4.29 (s, 2H), 2.69 (s, 3H); 13C NMR (176 MHz,
DMSO) δ 198.6, 169.2, 161.8, 158.9, 154.2, 153.2, 147.0, 146.8,
135.3, 131.2, 127.7, 123.1, 121.5, 31.6, 25.7. HRMS (HESI) calcd for
C15H11N3O3S2 [M + H]+ 346.0320, found 346.0310.
1
obtained (32 mg, 45%): mp 206.0−209.0 °C. H NMR (500 MHz,
DMSO-d6) δ 12.96 (s, 1H), 8.97 (s, 1H), 8.20−8.16 (m, 2H), 7.83
(dd, J = 8.2, 3.9 Hz, 1H), 7.66−7.60 (m, 1H), 4.27 (s, 2H); 13C NMR
(176 MHz, DMSO) δ 169.3, 161.3, 159.1, 154.1, 150.9 (dd, J = 120.7,
12.8 Hz), 149.5 (dd, J = 117.2, 12.8 Hz), 149.0, 129.6−129.5 (m),
127.1, 124.1−123.9 (m), 121.3, 118.6 (d, J = 17.7 Hz), 116.1 (d, J =
18.8 Hz), 31.5; 19F NMR (376 MHz, DMSO-d6) δ −135.61 to
−135.88 (m, 1F), −136.89 to −137.13 (m, 1F). HRMS (HESI) calcd
for C14H8F2N2O2S2 [M + H]+ 339.0073, found 339.0058.
2-((6-(Thiophen-2-yl)thieno[3,2-d]pyrimidin-4-yl)thio)acetic
Acid (11r). Prepared using general procedure A, with 2-thienylbor-
onic acid. After purification, a pale orange solid was obtained (29 mg,
45%): mp 179.0−182.0 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.95
(s, 1H), 8.93 (s, 1H), 7.85 (s, 1H), 7.83 (dd, J = 5.0, 1.2 Hz, 1H),
7.80 (dd, J = 3.7, 1.2 Hz, 1H), 7.26−7.23 (m, 1H), 4.25 (s, 2H); 13C
NMR (176 MHz, DMSO) δ 169.4, 160.9, 159.3, 154.3, 144.7, 134.7,
129.8, 129.0, 128.3, 126.3, 119.3, 31.5. HRMS (HESI) calcd for
C12H8N2O2S3 [M + H]+ 308.9826, found 308.9811.
2-((6-(3-Fluoro-4-(methylthio)phenyl)thieno[3,2-d]-
pyrimidin-4-yl)thio)acetic Acid (11y). Prepared using general
procedure A, with 3-fluoro-4-(methylthio)phenylboronic acid. After
purification, a pale yellow solid was obtained (32 mg, 42%): mp
1
203.0−206.0 °C. H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H),
2-((6-(Benzo[b]thiophen-2-yl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetic Acid (11s). Prepared using general procedure A, with
benzo[b]thien-2-ylboronic acid. After purification, a pale orange solid
8.95 (s, 1H), 8.17 (s, 1H), 7.92 (dd, J = 11.4, 2.0 Hz, 1H), 7.80 (dd, J
= 8.1, 2.0 Hz, 1H), 7.51−7.44 (m, 1H), 4.26 (s, 2H), 2.57 (s, 3H);
13C NMR (176 MHz, DMSO) δ 169.3, 161.1, 159.3, 158.6 (d, J =
1
was obtained (30 mg, 40%): mp >250 °C. H NMR (500 MHz,
242.1 Hz), 154.1, 149.8 (d, J = 2.5 Hz), 129.9 (d, J = 8.1 Hz), 128.7
(d, J = 17.1 Hz), 127.3 (d, J = 3.3 Hz), 126.8, 123.3 (d, J = 2.8 Hz),
120.6, 112.9 (d, J = 23.4 Hz), 31.5, 13.5; 19F NMR (376 MHz,
DMSO-d6) δ −112.48 (dd, J = 11.4, 8.1 Hz). HRMS (HESI) calcd for
C15H11FN2O2S3 [M + H]+ 367.0044, found 367.0027.
DMSO-d6) δ 12.97 (s, 1H), 8.98 (s, 1H), 8.16 (s, 1H), 8.08−8.06 (m,
1H), 7.98 (s, 1H), 7.96−7.93 (m, 1H), 7.48−7.46 (m, 2H), 4.27 (s,
2H); 13C NMR (176 MHz, DMSO) δ 169.3, 161.2, 159.0, 154.3,
144.4, 139.6, 134.4, 126.9, 126.3, 125.4, 124.8, 124.7, 122.7, 121.2,
31.6. HRMS (HESI) calcd for C16H10N2O2S3 [M + H]+ 358.9982,
found 358.9971.
2-((6-(3-Fluoro-4-methoxyphenyl)thieno[3,2-d]pyrimidin-4-
yl)thio)acetic Acid (11z). Prepared using general procedure A, with
3-fluoro-4-methoxyphenylboronic acid. After purification, a pale
2-((6-(4-Cyanophenyl)thieno[3,2-d]pyrimidin-4-yl)thio)-
acetic Acid (11t). Prepared using general procedure A, with 4-
cyanophenylboronic acid. After purification, a pale brown solid was
1
yellow solid was obtained (29 mg, 40%): mp 209.0−211.5 °C. H
1
obtained (34 mg, 50%): mp 226.0−230.0 °C. H NMR (500 MHz,
NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 8.93 (s, 1H), 8.06 (s,
N
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1H), 7.95 (dd, J = 12.5, 2.4 Hz, 1H), 7.77−7.74 (m, 1H), 7.35−7.28
(m, 1H), 4.25 (s, 2H), 3.93 (s, 3H); 13C NMR (176 MHz, DMSO) δ
169.4, 160.9, 159.5, 154.1, 151.6 (d, J = 245.4 Hz), 150.4 (d, J = 2.4
Hz), 148.9 (d, J = 10.6 Hz), 126.6, 124.8 (d, J = 7.1 Hz), 123.6 (d, J =
3.1 Hz), 119.6, 114.3, 114.3 (d, J = 17.8 Hz), 56.2, 31.5; 19F NMR
(376 MHz, DMSO-d6) δ −134.17 (dd, J = 12.5, 9.2 Hz). HRMS
(HESI) calcd for C15H11FN2O3S2 [M + H]+ 351.0273, found
351.0260.
Ethyl 2-((6-(4-chlorophenyl)thieno[3,2-d]pyrimidin-4-yl)-
oxy)acetate (14). Into a 20 mL microwave vial provided with a
magnetic stir bar, reagents were loaded in the following order: 7-
chloro-2-(4-chlorophenyl)benzo[b]thiophene 13a (1.0 equiv, 0.23
mmol, 65 mg), anhydrous DMF (3.0 mL), ethyl 2-hydroxyacetate
(2.0 equiv, 0.46 mmol, 48 mg), and NaH (2.6 equiv, 0.60 mmol, 25
mg, 60% dispersion in mineral oil). The vial was sealed, and the
reaction mixture was stirred at 100 °C for 30 min in a microwave
reactor. The crude was quenched over ice/water, solvents were
evaporated in vacuo and purified over silica gel with hexanes/EtOAc
in a gradient up to 50% of EtOAc. After purification, a white solid was
2-((6-(4-(Trifluoromethyl)phenyl)thieno[3,2-d]pyrimidin-4-
yl)thio)acetic Acid (11aa). Prepared using general procedure A,
with 4-(trifluoromethyl)phenylboronic acid. After purification, a pale
1
1
orange solid was obtained (39 mg, 50%): mp 173.0−176.0 °C. H
recovered (37 mg, 45%): mp 145.0−148.0 °C. H NMR (700 MHz,
DMSO-d6) δ 8.75 (s, 1H), 8.12 (s, 1H), 7.97 (dd, J = 8.6, 2.4 Hz,
2H), 7.63−7.59 (m, 2H), 5.21 (s, 2H), 4.18 (q, J = 8.2 Hz, 2H), 1.20
(t, J = 8.2 Hz, 3H). 13C NMR (176 MHz, DMSO) δ 167.6, 162.8,
162.3, 154.3, 150.5, 134.8, 131.0, 129.3, 128.3, 120.8, 115.9, 62.8,
60.8, 13.9. HRMS (HESI) calcd for C16H14N2O3SCl [M + H]+
349.0335, found 349.0400.
NMR (500 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.30 (s, 1H), 8.21 (d, J =
8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H), 4.28 (s, 2H); 13C NMR (176
MHz, DMSO) δ 169.2, 161.6, 159.1, 154.2, 149.4, 135.7, 130.0 (q, J =
32.0 Hz), 127.6, 127.4, 126.3 (q, J = 3.7 Hz), 123.9 (q, J = 272.0 Hz),
122.2, 31.6; 19F NMR (376 MHz, DMSO-d6) δ −61.35. HRMS
(HESI) calcd for C15H9F3N2O2S2 [M + H]+ 371.0135, found
371.0118.
2-((6-(4-Chlorophenyl)thieno[3,2-d]pyrimidin-4-yl)oxy)-
acetic Acid (15). In a 10 mL round-bottom flask provided with a
reflux condenser and magnetic stir bar, compound 14 (1.0 equiv, 0.06
mmol, 21 mg) was dissolved in THF (2 mL), followed by the addition
of LiOH (2.2 equiv, 0.12 mmol, 3 mg) predissolved in water. This
mixture was refluxed at 70 °C for 4−5 h. Solvents were removed
under reduced pressure, the white solid obtained was taken into ice/
water, and the pH adjusted to 4−5 with 2 M HCl(aq). The resulting
solid was filtrated, thoroughly rinsed with water, and air/vacuum
2-((6-(3-(Trifluoromethoxy)phenyl)thieno[3,2-d]pyrimidin-
4-yl)thio)acetic Acid (11ab). Prepared using general procedure A,
with 3-(trifluoromethoxy)phenylboronic acid. After purification, a
1
whitish solid was obtained (41 mg, 50%): mp 210.0−213.0 °C. H
NMR (500 MHz, DMSO-d6) δ 12.96 (s, 1H), 8.98 (s, 1H), 8.27 (s,
1H), 8.04−7.98 (m, 2H), 7.71−7.66 (m, 1H), 7.53 (d, J = 8.2 Hz,
1H), 4.27 (s, 2H); 13C NMR (176 MHz, DMSO) δ 169.3, 161.5,
159.1, 154.1, 149.4, 149.0 (d, J = 1.8 Hz), 134.1, 131.5, 127.2, 125.9,
122.5, 121.7, 120.0 (q, J = 258.4 Hz), 119.4, 31.5; 19F NMR (376
MHz, DMSO-d6) δ −56.74. HRMS (HESI) calcd for C15H9F3N2O3S2
[M + H]+ 387.0085, found 387.0071.
1
dried, yielding a white solid (16 mg, 82%): mp 234.0−236.0 °C. H
NMR (400 MHz, DMSO-d6) δ 13.20 (s, 1H), 8.76 (s, 1H), 8.13 (s,
1H), 7.98 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 5.12 (s, 2H);
13C NMR (176 MHz, DMSO) δ 169.1, 162.8, 162.5, 154.4, 150.4,
2-((6-(4-(Methylthio)phenyl)thieno[3,2-d]pyrimidin-4-yl)-
thio)acetamide (12). Into a 20 mL vial provided with a magnetic
stir bar, reagents were loaded in the following order: compound (4)
(1.0 equiv, 0.06 mmol, 20 mg), DMF (1.0 mL), N,N-diisopropylethyl-
amine (2.2 equiv, 0.13 mmol, 20 mg), HATU (1.0 equiv, 0.06 mmol,
23 mg), and ammonia (excess, 0.1 mL of an 28% aqueous solution).
The vial was sealed, and the reaction mixture was stirred at room
temperature for 12 h. It was quenched with water, solid slowly
precipitated, filtered, thoroughly rinsed with water, and air and
vacuum dried. No further purification was needed, yielding a pale
brown solid (5 mg, 25%): mp 231.0−234.0 °C. 1H NMR (400 MHz,
DMSO-d6) δ 8.93 (s, 1H), 8.06 (s, 1H), 7.92−7.89 (m, 2H), 7.69 (s,
1H), 7.42−7.39 (m, 2H), 7.24 (s, 1H), 4.16 (s, 2H), 2.55 (s, 3H);
13C NMR (176 MHz, DMSO) δ 168.4, 161.4, 159.4, 154.0, 151.2,
141.7, 128.1, 127.0, 126.6, 126.0, 119.3, 32.9, 14.2. HRMS (HESI)
calcd for C15H13N3OS3 [M + H]+ 348.0299, found 348.0279.
General Procedure B: Synthesis of 4-Chloro-6-arylthieno-
[3,2-d]pyrimidines (13a, 13b). In a 50 mL round-bottom flask
provided with a reflux condenser, a rubber septum, a magnetic stir bar,
and under positive N2 pressure, the following reagents were added: 4-
chloro-6-iodothieno[3,2-d]pyrimidine 10 (1.0 equiv, 1.20 mmol, 355
mg), NaHCO3 (1.0 equiv, 1.20 mmol, 100 mg), CsF (1.0 equiv, 1.20
mmol, 185 mg), Pd(Ph3)4 (3 mol %, 40 mg), and the corresponding
arylboronic acid (1.0 equiv, 1.20 mmol). A previously N2-degassed
solution of 1,4-dioxane/water (3:1, 7.0 mL) was added, and the
reaction mixture was stirred under mild reflux for 3 h. After this
period, the solvent was removed under reduced pressure and the
resulting solid was purified over silica gel with hexanes/EtOAc in a
gradient up to 30% of the polar solvent.
134.8, 131.1, 129.4, 128.3, 120.8, 116.1, 62.8. HRMS (HESI) calcd
for C14H9ClN2O3S [M + H]+ 321.0100, found 321.0080.
Ethyl (6-(4-(methylthio)phenyl)thieno[3,2-d]pyrimidin-4-
yl)glycinate (16a). Into a 20 mL vial provided with a magnetic
stir bar, reagents were loaded in the following order: compound 13b
(1.0 equiv, 0.27 mmol, 80 mg), EtOH (5.0 mL), ethyl glycinate
hydrochloride (2.5 equiv, 0.68 mmol, 95 mg), and triethylamine (10
equiv, 2.7 mmol, 0.4 mL). The vial was sealed, and the reaction
mixture was stirred at 90 °C for 18 h. After this period, solvent was
evaporated in vacuo and purified over silica gel with hexanes/EtOAc
in a gradient up to 60% of EtOAc. After purification, a pale yellow
1
solid was recovered (85 mg, 78%): mp 160.5−164.0 °C. H NMR
(400 MHz, DMSO-d6) δ 8.43 (s, 1H), 8.33 (s, 1H), 7.82 (s, 1H),
7.80 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 4.21 (d, J = 5.9 Hz,
2H), 4.13 (q, J = 7.1 Hz, 2H), 2.54 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H).
HRMS (HESI) calcd for C17H18N3O2S2 [M + H]+ 360.0762, found
360.0844.
(6-(4-(Methylthio)phenyl)thieno[3,2-d]pyrimidin-4-yl)-
glycine (16b). In a 10 mL round-bottom flask provided with a reflux
condenser and magnetic stir bar, compound 16a (1.0 equiv, 0.06
mmol, 20 mg) was dissolved in THF (2 mL), followed by the addition
of LiOH (2.2 equiv, 0.12 mmol, 3 mg) predissolved in water. This
mixture was refluxed at 70 °C for 4−5 h. Solvents were removed
under reduced pressure, the white solid obtained was taken into ice/
water, and the pH adjusted to 4−5 with 2 M HCl(aq). The resulting
solid was filtrated, thoroughly rinsed with water, and air/vacuum
dried, yielding a pale orange solid (17 mg, 92%): mp, decomposition.
1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H), 8.84 (s, 1H), 8.58
7-Chloro-2-(4-chlorophenyl)benzo[b]thiophene (13a). Pre-
(s, 1H), 7.84−7.78 (m, 3H), 7.41 (d, J = 8.1 Hz, 2H), 4.20 (d, J = 5.9
pared using general procedure B, with (4-chlorophenyl)boronic acid.
Hz, 2H), 2.55 (s, 3H).
1
After purification, a white solid was obtained (185 mg, 55%): H
2-((6-Bromothieno[2,3-d]pyrimidin-4-yl)thio)acetic Acid
(18). In a 50 mL round-bottom flask provided with a rubber septum,
a magnetic stir bar, and under positive N2 pressure, 6-bromo-4-
chlorothieno[2,3-d]pyrimidine (1.0 equiv, 4.0 mmol, 1.0 g) 17 was
suspended in CH3CN or EtOH (15 mL) at 0 °C (ice bath), followed
by the dropwise addition of 2-mercaptoacetic acid (1.25 equiv, 5.0
mmol, 0.35 mL) and triethylamine (2.5 equiv, 10.0 mmol, 1.4 mL).
The ice bath was removed, and the reaction mixture was stirred
overnight at room temperature. After this period, solvent was
NMR (400 MHz, CDCl3) δ 8.97 (s, 1H), 7.74 (s, 1H), 7.73−7.69 (m,
2H), 7.53−7.47 (m, 2H).
7-Chloro-2-(4-(methylthio)phenyl)benzo[b]thiophene
(13b). Prepared using general procedure B, with (4-(methylthio)-
phenyl)boronic acid. After purification, a pale yellow solid was
obtained (300 mg, 85%): 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s,
1H), 8.21 (s, 1H), 7.97−7.90 (m, 2H), 7.44−7.38 (m, 2H), 2.56 (s,
3H).
O
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removed under reduced pressure, solid taken into ice/water, and the
pH adjusted to 2−3 with 2 M aq. HCl. The resulting solid was
filtrated, thoroughly rinsed with water, and air and vacuum dried,
yielding a white solid. No further purification was needed (0.91 g,
75%). mp 194.5−198.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.90
(s, 1H), 8.83 (s, 1H), 7.83 (s, 1H), 4.18 (s, 2H); 13C NMR (176
MHz, DMSO) δ 169.3, 165.9, 161.6, 152.6, 128.0, 122.1, 116.2, 31.4.
HRMS (HESI) calcd for C8H6N2O2S2Br [M + H]+ 304.8976, found
304.9059.
General Procedure C: Synthesis of 6-Arylthieno[2,3-d]-
pyrimidines (19a−19g). In a 25 mL round-bottom flask provided
with a reflux condenser, a rubber septum, a magnetic stir bar, and
under positive N2 pressure, the following reagents were added: 2-((6-
bromothieno[2,3-d]pyrimidin-4-yl)thio)acetic acid (18) (1.0 equiv,
0.25 mmol, 75 mg), K2CO3 (4.0 equiv, 1.0 mmol, 140 mg), Pd(Ph3)4
(5 mol %, 15 mg), and the corresponding arylboronic acid (1.0 equiv,
0.25 mmol). A previously N2-degassed solution of 1,4-dioxane/water
(3:1, 2.5 mL) was added, and the reaction mixture was stirred under
mild reflux for 3 h. After this period, the solvent was removed under
reduced pressure, solids taken into ice/water, and the pH adjusted to
2−3 with 2 M HCl(aq). The resulting solid was filtrated, thoroughly
rinsed with water, air and vacuum dried, and purified over silica gel
using CH2Cl2/MeOH/HOAc (95/4/1) in a gradient up 5−10% of
the polar solvent.
155.8, 152.0, 139.3, 130.9, 128.8, 127.2, 121.2, 120.6, 115.3, 112.5,
55.9, 31.3. HRMS (HESI) calcd for C15H12N2O3S2 [M + H]+
333.0368, found 333.0370.
2-((6-(3,4-Dichlorophenyl)thieno[2,3-d]pyrimidin-4-yl)thio)-
acetic Acid (19f). Prepared using general procedure C, with 3,4-
dichlorophenylboronic acid. After purification, a white solid was
1
obtained (35 mg, 38%): mp >250 °C. H NMR (400 MHz, DMSO-
d6) δ 12.91 (s, 1H), 8.84 (s, 1H), 8.31 (d, J = 2.2 Hz, 1H), 8.17 (s,
1H), 7.86 (dd, J = 8.4, 2.2 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 4.22 (s,
2H); 13C NMR (176 MHz, DMSO) δ 169.5, 164.7, 162.9, 152.6,
140.4, 132.8, 132.2, 131.9, 131.3, 128.5, 128.0, 126.7, 116.5, 31.5.
HRMS (HESI) calcd for C14H8Cl2N2O2S2 [M + H]+ 370.9482, found
370.9459.
2-((6-(Benzo[b]thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl)-
thio)acetic Acid (19g). Prepared using general procedure C, with
benzo[b]thien-2-ylboronic acid. After purification, a pale orange solid
1
was obtained (36 mg, 40%): mp 225.0−228.0 °C. H NMR (400
MHz, DMSO-d6) δ 12.92 (s, 1H), 8.85 (s, 1H), 8.06−8.02 (m, 2H),
7.95−7.90 (m, 1H), 7.80 (s, 1H), 7.46−7.43 (m, 2H), 4.23 (s, 2H);
13C NMR (176 MHz, DMSO) δ 169.4, 164.7, 162.4, 152.7, 139.7,
139.1, 136.6, 134.7, 128.2, 125.9, 125.3, 124.4, 123.9, 122.6, 115.5,
31.5. HRMS (HESI) calcd for C16H10N2O2S3 [M + H]+ 358.9983,
found 358.9988.
Protein Purification and Crystallization. The kinase domain of
STK17B (aa 25−329) was expressed as an N-terminal His-tagged
fusion in E. coli. The recombinant protein was purified by Ni2+-affinity
chromatography and subsequently size-exclusion chromatography.
The protein in 25 mM HEPES, pH 7.5, 500 mM NaCl, and 0.5 mM
TCEP was mixed with 1 mM inhibitor, and the complex was
crystallized using a sitting drop vapor diffusion method at 4 °C with
the crystallization condition listed in SI Table 2. Crystals were cryo-
protected with mother liquor supplemented with 20% ethylene glycol.
Diffraction data collected at Diamond Light Source, BESSY II and
DESY were processed with iMosflm44 and scaled with a Scala or
Aimless from CCP4 suite.45 The structures were solved by molecular
replacement using a Phaser46 and the coordinates of DAPK1 (PDB:
3EHA). Manual model rebuilding was performed in COOT,47
alternated with refinement using REFMAC5.48 Data collection and
refinement statistics are summarized in SI Table 2.
Biological Assays. Kinase Selectivity. The KINOMEscan
scanMAX assay panel was performed at DiscoverX Corporation as
previously described49 across 403 wild-type human kinases and 65
mutant human kinases at a compound concentration of 1 μM. IC50
was determined on kinases where an inhibitor displayed >75%
inhibition at 1 μM. False positives in the scanMAX assay were
identified as those kinases where an inhibitor produced an IC50 > 10
μM.
KinaseSeeker Split Luciferase Assay. Stock solutions (10 mM) of
the compounds were serially diluted in DMSO to make assay stocks.
Prior to initiating IC50 determinations, the test compounds were
evaluated for false positives against split-luciferase. Each test
compound was screened in duplicate at 1 μM against STK17A and
STK17B. Cfluc-kinase was translated along with Fos-Nfluc using a
cell-free system (rabbit reticulocyte lysate) at 30 °C for 90 min. A 24
μL aliquot of this lysate containing either 1 μL of DMSO (for no-
inhibitor control) or compound solution in DMSO was incubated for
30 min at room temperature followed by 1 h in presence of a kinase
specific probe. An amount 80 μL of the luciferin assay reagent was
added to each solution, and luminescence was immediately measured
on a luminometer. The % inhibition was calculated using the
following equation: % inhibition = [ALU (control) − ALU (sample)]
/ ALU (control) × 100. For IC50 determinations, each compound was
tested at eight different concentrations. The % inhibition was plotted
against compound concentration and the IC50 was determined for
each compound using an 8-point curve.
2-((6-(4-(Methylthio)phenyl)thieno[2,3-d]pyrimidin-4-yl)-
thio)acetic Acid (19a). Prepared using general procedure C, with 4-
(methylthio)phenylboronic acid. After purification, a pale yellow solid
1
was obtained (45 mg, 53%): mp 194.0−196.5 °C. H NMR (400
MHz, DMSO-d6) δ 12.89 (s, 1H), 8.81 (s, 1H), 7.91 (s, 1H), 7.85 (d,
J = 8.6 Hz, 2H), 7.38 (d, J = 8.6 Hz, 2H), 4.20 (s, 2H), 2.54 (s, 3H);
13C NMR (176 MHz, DMSO) δ 169.5, 164.3, 161.9, 152.2, 143.1,
140.5, 128.8, 128.5, 126.9, 126.1, 113.7, 31.4, 14.3. HRMS (HESI)
calcd for C15H12N2O2S3 [M + H]+ 349.0139, found 349.0112.
2-((6-(4-Methoxyphenyl)thieno[2,3-d]pyrimidin-4-yl)thio)-
acetic Acid (19b). Prepared using general procedure C, with 4-
methoxyphenylboronic acid. After purification, a pale yellow solid was
1
obtained (39 mg, 48%): mp 175.0−178.0 °C. H NMR (400 MHz,
DMSO-d6) δ 12.88 (s, 1H), 8.79 (s, 1H), 7.87−7.83 (m, 2H), 7.79 (s,
1H), 7.09−7.05 (m, 2H), 4.20 (s, 2H), 3.83 (s, 3H); 13C NMR (176
MHz, DMSO) δ 169.5, 164.2, 161.5, 160.3, 151.9, 143.6, 128.9,
128.0, 124.7, 114.7, 112.6, 55.4, 31.4. HRMS (HESI) calcd for
C15H12N2O3S2 [M + H]+ 333.0367, found 333.0345.
2-((6-(3-(Methylthio)phenyl)thieno[2,3-d]pyrimidin-4-yl)-
thio)acetic Acid (19c). Prepared using general procedure C, with 3-
(methylthio)phenylboronic acid. After purification, a pale yellow solid
1
was obtained (57 mg, 65%): mp 160.0−163.0 °C. H NMR (400
MHz, DMSO-d6) δ 12.90 (s, 1H), 8.83 (s, 1H), 8.04 (s, 1H), 8.05−
8.03 (m, 1H), 7.66−7.59 (m, 1H), 7.45 (dd, J = 8.7, 7.8 Hz, 1H),
7.37−7.33 (m, 1H), 4.21 (s, 2H), 2.58 (s, 3H); 13C NMR (176 MHz,
DMSO) δ 169.5, 164.5, 162.4, 152.41, 142.8, 139.9, 132.9, 129.8,
128.6, 126.7, 123.3, 123.2, 115.1, 31.4, 14.5. HRMS (HESI) calcd for
C15H12N2O2S3 [M + H]+ 349.0139, found 349.0122.
2-((6-(3-Methoxyphenyl)thieno[2,3-d]pyrimidin-4-yl)thio)-
acetic Acid (19d). Prepared using general procedure C, with 3-
methoxyphenylboronic acid. After purification, a pale yellow solid was
1
obtained (42 mg, 50%): mp 185.0−188.0 °C. H NMR (400 MHz,
DMSO-d6) δ 12.89 (s, 1H), 8.82 (s, 1H), 8.01 (s, 1H), 7.50−7.47 (m,
1H), 7.45−7.40 (m, 2H), 7.09−6.99 (m, 1H), 4.22 (s, 2H), 3.87 (s,
3H); 13C NMR (176 MHz, DMSO) δ 169.5, 164.4, 162.3, 159.8,
152.3, 143.3, 133.5, 130.5, 128.6, 119.0, 115.6, 114.8, 111.5, 55.4,
31.4. HRMS (HESI) calcd for C15H12N2O3S2 [M + H]+ 333.0367,
found 333.0370.
2-((6-(2-Methoxyphenyl)thieno[2,3-d]pyrimidin-4-yl)thio)-
acetic Acid (19e). Prepared using general procedure C, with 2-
methoxyphenylboronic acid. After purification, a pale yellow solid was
1
obtained (46 mg, 55%): mp 176.5−180.0 °C. H NMR (400 MHz,
NanoBRET Assays. NanoBRET assays were performed as
previously described.24 For STK17B, the C-terminal NanoLuc fusion
(STK17B-NL) was encoded in pFN32K expression vector, including
flexible Gly-Ser-Ser-Gly linkers between NanoLuc and STK17B. A 10
μg/mL solution of DNA in Opti-MEM without serum was made
DMSO-d6) δ 12.89 (s, 1H), 8.81 (s, 1H), 7.99 (dd, J = 7.8, 1.7 Hz,
1H), 7.91 (s, 1H), 7.46 (ddd, J = 8.7, 7.3, 1.7 Hz, 1H), 7.24 (dd, J =
8.7, 1.1 Hz, 1H), 7.12 (ddd, J = 7.8, 7.3, 1.1 Hz, 1H), 4.21 (s, 2H),
3.98 (s, 3H); 13C NMR (176 MHz, DMSO) δ 169.6, 164.9, 161.6,
P
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containing 9 μg/mL of carrier DNA (Promega) and 1 μg/mL of
STK17B-NL for a total volume of 1.05 mL. To this solution was then
added 31.5 μL of FuGENE HD (Promega) to form a lipid:DNA
complex. The solution was then mixed by inversion eight times and
incubated at room temperature for 20 min. The resulting transfection
complex (1.082 mL) was then gently mixed with 21 mL of HEK-293
cells (ATCC) suspended at a density of 2 × 105 cells/mL in DMEM
(Gibco) + 10% FBS (Corning). An amount of 100 μL of this solution
was dispensed into 96-well tissue culture-treated plates (Corning
#3917) followed by incubation (37 °C/5% CO2) for 24 h. The media
was removed and replaced with 85 μL of room temperature Opti-
MEM without phenol red. NanoBRET Tracer K9 (Promega) was
used at a final concentration of 0.66 μM. A total of 5 μL per well (20×
working stock of NanoBRET Tracer K9 [13.2 μM] in Tracer Dilution
Buffer (Promega N291B)) was added to all wells, except the “no
tracer” control wells. All test compounds were prepared initially as
concentrated (10 mM) stock solutions in 100% DMSO (Sigma), then
diluted in Opti-MEM media (99%) to prepare 1% DMSO working
stock solutions. A total of 10 μL per well of the 10-fold test compound
stock solutions (final assay concentration 0.1% DMSO) was added.
For “no compound” and “no tracer” control wells, a total of 10 μL per
well of Opti-MEM plus DMSO (9 μL Opti-MEM with 1 μL DMSO)
was added (final concentration 1% DMSO). 96-well plates containing
cells with NanoBRET Tracer K9 and test compounds at eleven
concentrations (100 μL total volume per well) were equilibrated (37
°C/5% CO2) for 2 h. The plates were cooled to room temperature for
15 min. To measure NanoBRET signal, a NanoGlo substrate
(Promega) at a ratio of 1:166 to Opti-MEM media in combination
with extracellular NanoLuc Inhibitor (Promega) diluted 1:500 (10 μL
[30 mM stock] per 5 mL Opti-MEM plus substrate) was combined to
create a 3X stock solution. A total of 50 μL of the 3× substrate/
extracellular NanoLuc inhibitor was added to each well. The plates
were read within 10 min on a GloMax Discover luminometer
(Promega, Madison, WI, USA) equipped with 450 nm BP filter
(donor) and 600 nm LP filter (acceptor), using 0.3 s integration time
utilizing the “NanoBRET 618” protocol. Raw milliBRET (mBRET)
values were obtained by dividing the acceptor emission values (600
nm) by the donor emission values (450 nm) and then multiplying by
1000. Averaged control values were used to represent complete
inhibition (no tracer control: Opti-MEM + DMSO only), and no
inhibition (tracer only control: no compound, Opti-MEM + DMSO +
Tracer K9 only) and was plotted alongside the raw mBRET values.
Biological replicates were normalized and fit using a Sigmoidal, 4PL
binding curve in Prism Software (version 8, GraphPad, La Jolla, CA,
USA). The experiment was performed n = 3. Average IC50 and
standard error (SE) were calculated in Prism Software.
1.3 mM EDTA) to an empty well of a 96-well plate and maintained at
37 °C. The diluted 0.1 mM compound (8.75 μL) was added to the
microsomal solution in the reaction plate and mixed thoroughly by
repeated pipetting. The resulting solutions were preincubated for 5
min at 37 °C before preparing the T = 0 plates. An aliquot (160 μL)
of each reaction solution was added to an empty well of a 96-well
plate, as an exact replicate of the Reaction Plate. Cold (4 °C) MeOH
(400 μL) was added to each well and mixed thoroughly by repeated
pipetting. NADPH regeneration solution (40 μL) was added to each
well and mixed thoroughly by repeated pipetting. For the T = 30 min
incubation plates (−NADPH), an aliquot (160 μL) of each reaction
solution was added to an empty well of a 96-well plate, as an exact
replicate of the reaction plate. LC−MS-grade H2O (40 μL) was added
to each well and mixed thoroughly by repeated pipetting. The plate
was sealed and incubated at 37 °C for the incubation period. After
incubation, cold (4 °C) MeOH (400 μL) was added to each well and
mixed thoroughly by repeated pipetting. For the T = 30 min
incubation plate (+NADPH), NADPH (95 μL) was added to the
remaining solution (microsomes + test compound) in each well in the
previously prepared reaction plate to initiate the reaction. The plate
was sealed and incubated at 37 °C for the incubation period. An
aliquot (100 μL) was removed from each well at the desired time
point and dispensed into a well of a 96-well plate. An amount of 200
μL of cold (4 °C) MeOH was added to quench the reaction. All plates
were sealed, vortexed, and centrifuged at 3000 RPM, 4 °C for 15 min,
and the supernatants are transferred for analysis by LC-TOFMS. The
supernatant (20 μL) was injected onto an AQUASIL C18 column and
eluted using fast-generic gradient program. TOFMS data was acquired
using Agilent 6538 ultrahigh-accuracy TOF MS in extended dynamic
range (m/z 100−1000) using generic MS conditions in positive
mode. Following data acquisition, exact mass extraction and peak
integration were performed using MassHunter Software (Agilent
Technologies). The stability of the compound is calculated as the
percent remaining of the unchanged parent compound at each time
point (T = 30 min) relative to the peak area at T = 0 min.
Lipophilicity. LogD at pH 7.0 was calculated using the ChemAxon
web interface https://disco.chemaxon.com/calculators/demo/
plugins/logd/.
Enzyme Assays. Assays were performed at Eurofins discovery
services as previously described.50 Compounds were provided as 10
mM stock solutions and screened in 10 point dose−response at the
Km of ATP using the full-length recombinant protein. For STK17B,
the enzyme was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA,
0.05% BSA, 10 mM sodium chloride, 250 μM KKRPQRRYSNVF, 10
mM Magnesium acetate, and [γ-33P-ATP]. The reaction was initiated
by the addition of the Mg/ATP mix. After incubation for 2 h at room
temperature, the reaction was stopped by the addition of phosphoric
acid to a concentration of 0.5%. An amount of 10 μL of the stopped
reaction was spotted onto a P30 filtermat and washed four times for 4
min in 0.425% phosphoric acid and once in methanol prior to drying
and scintillation counting.
Molecular Modeling. Homology Model of STK17A. A homology
model for the kinase domain of human STK17A (Uniprot: Q9UEE5)
was generated using MODELLER 9.24.31 The crystal structure of
human STK17B bound to 11s (PDB: 6Y6H) with R41 in folded
conformation A was used as the template. 11s was docked into the
STK17A model using AutoDock Vina 1.1.2.51
Molecular Dynamic Simulation of STK17B in Apo Form. The co-
crystal structure of human STK17B with 11s (PDB ID: 6Y6H) was
downloaded. All the hetero atoms including 11s and water molecules
were removed. PDB files corresponding to the P-loop (residues 37−
46) with R41 in conformations A and B were generated.
Approximately 1.25 μS unbiased full-atom MD simulation was
performed using GROMACS 2018.152 starting from the two
conformations. The hydrogen atoms were converted to virtual sites
to remove the fastest vibrational freedom. The protein was
parameterized with the amber99sb-ildn and then solvated with
TIP3P water model in a dodecahedron box. To neutralize the charge
on the protein, sodium and chloride ions were added to the system.
Verlet cutoff was used to define the neighbor list for nonbonded
Kinetic Solubility. Assays were performed at Analiza, Inc. 50-fold
dilutions of each DMSO stock solution were prepared in singleton by
combining 6 μL of DMSO stock with 294 μL of the appropriate
media in a Millipore solubility filter plate with 0.45 μM polycarbonate
filter membrane using Hamilton Starlet liquid handling. The final
DMSO concentration was 2.0%, and the maximum theoretical
compound concentration was 200 μM (assuming stock concentration
of 10 mM). The filter plate was heat sealed for the duration of the 24
h incubation period. Phosphate-buffered saline solution 10×, PBS
(Fisher Bioreagent part number BP399-500), 50 mL, was added to
approximately 450 mL of HPLC-grade H2O. The volume of the
solution was then adjusted to 500 mL for a total dilution factor of
1:10 and a final PBS concentration of 1×. The samples were place on
a rotary shaker (200 RPM) at ambient temperature (22.4−24.0 °C)
and then vacuum filtered. All filtrates were injected into the nitrogen
detector for quantification on Analiza’s Automated Discovery
Workstation. The solubility was calculated as μg/mL and corrected
for background nitrogen.
Microsomal Stability. Assays were performed at Analiza, Inc.
DMSO stock solutions (10 mM) were diluted to 0.5 mM with DMSO
and again to 0.1 mM with acetonitrile. The resulting solution
contained a 0.1 mM test article in 20% DMSO/80% acetonitrile. A
reaction plate was prepared by adding 691.25 μL prewarmed (37 °C)
microsomal solution (0.63 mg/mL protein in 100 mM K3PO4 with
Q
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interactions.53 Particle mesh Ewald (PME) was used to calculate long-
range interactions. Energy minimization was performed with the
steepest-descent algorithm and then with conjugate descent with
Fmax <500 kJmol−1 nm−1. The canonical ensemble was carried out by
heating the system from 0 to 310 K, using velocity rescaling for 100
ps.54 The isothermal-isobaric ensemble (P = 1 bar, T = 310 K) was
carried out using the Berendsen barostat for 100 ps.55 The
unrestrained MD productions were collected using a time step of 5
fs after the isothermal-isobaric ensemble. The trajectories were
processed and analyzed using the built-in tools of Gromacs and then
visualized with PyMOL 2.3.2.
Molecular Dynamic Simulation of STK17B in Inhibitor Bound
Form. RosettaLoop56 was used to model the missing residues (187−
295) in STK17B bound to 4 (PDB ID: 6Y6F). Unbiased full atom
MD simulation was performed using GROMACS 2018.1.52 4 and 11s
were parameterized with GAFF (General AMBER Force Field)57
using ACPYPE (AnteChamber PYthon Parser interfacE).58 The
protein was parameterized with the amber99sb-ildn and then solvated
with a TIP3P water model in a dodecahedron box. The charge was
neutralized by adding sodium and chloride ions to the system. The
Verlet cutoff was used to define the neighbor list for nonbonded
interactions.53 Protein parameterization and simulation protocols
were identical to those used in the STK18B apo simulation described
above. VMD59 was used to calculate RMSD and H-bond frequency.
Xmgrace was used to plot the data. Trajectory was visualized with
PyMOL 2.3.2.
MD of STK17B/11s conformation A (SI Movie 4)
(MP4)
MD of STK17B/11s conformation B (SI Movie 5)
(MP4)
AUTHOR INFORMATION
Corresponding Author
■
Timothy M. Willson − Structural Genomics Consortium,
UNC Eshelman School of Pharmacy, University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina
27599-7264, United States; orcid.org/0000-0003-4181-
8223; Email: tim.willson@unc.edu
Authors
Alfredo Picado − Structural Genomics Consortium, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599-7264,
United States
Apirat Chaikuad − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, Frankfurt 60438, Germany;
Structural Genomics Consortium, Buchmann Institute for
Molecular Life Sciences (BMLS), Frankfurt 60438,
Germany; orcid.org/0000-0003-1120-2209
Sequence Alignment of STK17A and STK17B Orthologs. Kinase
domains of STK17A and STK17B orthologs were identified using
previously curated hierarchical eukaryotic protein kinase (ePK)
profiles.60 The sequences were then aligned with MAFFT version
7.450.61 The alignment was then visualized using BOXSHADE
version 3.2.1.
Basicity Calculation. pKa was obtained from density functional
theory (DFT) calculations using the molecular electrostatic potential
as the descriptor. All DFT calculations were performed with the
Gaussian 16 package version A0362 with tight self-consistent field
convergence criterion and ultrafine integration grids. A full-structure
optimization was first carried out at the B3LYP/6-311+G(d,p) level of
theory. After the structure optimization, single-point calculations were
performed to obtain the molecular electrostatic potential using the
pop=chelpg keyword63 for each of the nuclei followed by a full NBO
analysis.64
Carrow I. Wells − Structural Genomics Consortium, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599-7264,
United States; orcid.org/0000-0003-4799-6792
Safal Shrestha − Institute of Bioinformatics, University of
Georgia, Athens, Georgia 30602, United States
William J. Zuercher − Structural Genomics Consortium, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599-7264,
United States; orcid.org/0000-0002-9836-0068
Julie E. Pickett − Structural Genomics Consortium, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599-7264,
United States; orcid.org/0000-0002-9535-8528
Frank E. Kwarcinski − Luceome Biotechnologies, Tucson,
Arizona 85719, United States
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01174.
■
Parvathi Sinha − Luceome Biotechnologies, Tucson, Arizona
85719, United States
Chandi S. de Silva − Luceome Biotechnologies, Tucson,
Arizona 85719, United States
sı
Experimental and calculated pKa values for thienopyr-
idine (SI Table 1), X-ray data collection and refinement
statistics (SI Table 2), SMARTCyp prediction of most
liable points to metabolism in 4 (SI Figure 1), X-ray
structure of the catalytic domain of STK17B/11h
complex (SI Figure 2), multiple sequence alignment of
the kinase domain of STK17A and STK17B orthologs
(SI Figure 3), and binding pocket comparison of
STK17A/11s with STK17B/11s (SI Figure 4) (PDF)
DiscoverX KINOMEscan data (SI File 1) (XLSX)
NMR spectra (SI File 2) (PDF)
Purity traces for lead compounds (SI File 3) (PDF)
Molecular formula strings (SI File 4) (CVS)
Coordinates of human STK17A homology model (SI
File 5) (PDB)
MD of apo-STK17B conformation A (SI Movie 1)
(MP4)
Reena Zutshi − Luceome Biotechnologies, Tucson, Arizona
85719, United States
Shubin Liu − Research Computing Center, University of North
Carolina, Chapel Hill, North Carolina 27599-3420, United
States; orcid.org/0000-0001-9331-0427
Natarajan Kannan − Institute of Bioinformatics and
Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, Georgia 30602, United States
Stefan Knapp − Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, Frankfurt 60438, Germany; Structural
Genomics Consortium, Buchmann Institute for Molecular Life
Sciences (BMLS), Frankfurt 60438, Germany; German
Translational Cancer Network (DKTK) site Frankfurt/
Mainz, Frankfurt am Main 60596, Germany; Frankfurt
Cancer Institute (FCI), Frankfurt am Main 60596,
Germany; orcid.org/0000-0001-5995-6494
David H. Drewry − Structural Genomics Consortium, UNC
Eshelman School of Pharmacy, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599-7264,
United States; orcid.org/0000-0001-5973-5798
MD of apo-STK17B conformation B (SI Movie 2)
(MP4)
MD of STK17B/4 (SI Movie 3) (MP4)
R
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01174
Corporation; FuGene HD, transfections reagent trademark of
Promega Corporation; DMEM, Dulbecco’s modified Eagle
medium; FBS, fetal bovine serum; LC-TOFMS, liquid
chromatography-time-of-flight-mass spectrometry; MOPS, 3-
(N-morpholino)propanesulfonic acid
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
REFERENCES
■
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Innovation, Genome Canada, Innovative Medicines Initiative
EUbOPEN (no 875510), Janssen, Merck KGaA Darmstadt
Germany, MSD, Novartis Pharma AG, Ontario Ministry of
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Research Foundation-FAPESP, Takeda, and Wellcome were
acknowledged. Funding for this project was provided by
National Institutes of Health (1R44TR001916-02) to
Luceome Biotechnologies and the SGC-UNC. Additional
funding for the SGC-UNC was provided by UNC Lineberger
Comprehensive Cancer Center, PharmAlliance, and National
Institutes of Health (1R01CA218442-01 and 1U24DK116204-
01). S.K. is grateful for support by the German cancer network
(DKTK) and the Frankfurt cancer center (FCI). Funding for
N.K. from the National Institutes of Health (1U01CA239106-
01) is acknowledged. The authors thank staff at Diamond
Light Source, BESSY II, and DESY for their assistance during
data collection, and the UNC Department of Chemistry Mass
Spectrometry Core Laboratory for their assistance with
compound characterization. NanoBRET assays were aided by
the provision of a CAMKK2-NLuc clone by Carol C. Preta and
Priscila Zonzini (SGC-UNICAMP, Brazil). Other NanoBRET
constructs and tracer K9 were kindly provided by Promega
Corporation (Madison WI). The authors thank Alexander
Tropsha (UNC Chapel Hill) for insights and discussion of
computational models and electrostatics calculations.
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ABBREVIATIONS
■
NanoBRET, bioluminescense resonance energy-transfer assay,
trademark of Promega Corporation; STK17A, serine/threo-
nine-protein kinase 17A; STK17B, serine/threonine-protein
kinase 17B; DRAK1, DAP kinase-related apoptosis-inducing
protein kinase (DRAK)1; DRAK2, DAP kinase-related
apoptosis-inducing protein kinase (DRAK)2; TGFβ1, trans-
forming growth factor Beta 1; DFG, “Asp-Phe-Gly”; MAP4K4,
mitogen-activated protein kinase kinase kinase kinase 4;
PKIS2, published kinase inhibitor set 2; TPL2, tumor
progression locus 2 kinase; MAPK3K8, mitogen-activated
protein kinase 3K8; COT, cancer Osaka thyroid kinase; KD,
dissociation constant; SNAr, nucleophilic aromatic substitution;
Log, logarithm of distribution coefficient; CAMKK1, calcium/
calmodulin-dependent protein kinase kinase 1; CAMKK2,
calcium/calmodulin-dependent protein kinase kinase 2;
AURKB, Aurora kinase B; PIM2, serine/threonine-protein
kinase PIM-2; MET, mesenchymal epithelial transition kinase;
NEK6, serine/threonine-protein kinase Nek6; WEE1, Wee1-
like protein kinase; STK38L, serine/threonine-protein kinase
38-like; HIPK4, Homeodomain-interacting protein kinase 4;
pKa, -logarithm of acid dissociation constant Ka; CSNK2,
casein kinase II; CDCl3, deuterated chloroform; EtOAc, ethyl
acetate; EtOH, ethanol; HEPES, 4-(2-hydroxyethyl)-1-piper-
azineethanesulfonic acid; TCEP, tris(2-carboxyethyl)-
phosphine; Fluc, split-firefly luciferase pairs; ALU, Arithmetic
logic unit; NanoLuc, luciferase trademark of Promega
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