Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma

Article abstract of DOI:10.1016/j.ejmech.2021.113481

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity fo

Full text of DOI:10.1016/j.ejmech.2021.113481

European Journal of Medicinal Chemistry 221 (2021) 113481
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Structure-activity relationship study of THZ531 derivatives enables
the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with
efficacy in Ewing sarcoma
,
b
,
,
b
,
,
,
e
Baishan Jiang a 1, Jie Jiang a 1, Ines H. Kaltheuner c 1, Amanda Balboni Iniguez d
,
Kanchan Anand c, Fleur M. Ferguson a b, Scott B. Ficarro a f, Bo Kyung Alex Seong d
,
,
,
,
e
Ann Katrin Greifenberg c, Sofia Dust c, Nicholas P. Kwiatkowski a b, Jarrod A. Marto a
,
,
,
f
,
Kimberly Stegmaier d e, Tinghu Zhang a **, Matthias Geyer c ***, Nathanael S. Gray a
,
,
b,
,
b, *
a Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02115, USA
b Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA
c Institute of Structural Biology, University of Bonn, Venusberg-Campus 1, 53127, Bonn, Germany
d Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02115, USA
e The Broad Institute, Cambridge, MA, 02142, USA
f Blais Proteomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02215, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers
as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described
THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity
relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused li-
brary of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent
inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer
cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of
Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic
properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived
xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken
together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy re-
ported to date.
Received 7 February 2021
Received in revised form
13 April 2021
Accepted 14 April 2021
Available online 20 April 2021
Keywords:
CDK12/13
Covalent inhibitor
Structure-activity relationship
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
and 20) [3,4]. Although cell cycle CDKs were the first to be
discovered and studied, tCDKs have been of increasing interest
The human genome encodes 21 cyclin-dependent kinases
(CDKs), serine/threonine kinases that require cyclin binding for
activity [1,2]. Based on the nature of cellular processes they regu-
late, CDKs are divided into two subfamilies: cell cycle CDKs (CDK1-
6, 11 and CDK14-18) and transcriptional CDKs (tCDKs; CDK7-13, 19
given their role as regulators of gene transcription, either as the
kinases of RNA polymerase (pol) II protein complex or as part of
transcriptional mediator [5,6]. CDK12 and its close homolog CDK13,
both activated through cyclin K binding, were recently identified as
kinases responsible for RNA pol II Ser2 phosphorylation which is
required to sustain transcriptional elongation [7e10]. Although
genetic deletion of either CDK12 or CDK13 leads to a down-
regulation of more than 1000 overlapping genes [10], genes that
regulate protein translation are more sensitive to CDK13 deletion,
while DNA-damage responsive (DDR) genes are more sensitive to
CDK12 deletion. This suggested an opportunity for combining
CDK12-targeting inhibitors with inhibitors of PARP, a single-strand
break DNA repair protein, to achieve an enhanced cancer cell killing
* Corresponding author.Department of Cancer Biology, Dana-Farber Cancer
Institute, Harvard Medical School, Boston, MA, 02115, USA.
** Corresponding author. Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.
*** Corresponding author.
E-mail address: nathanael_gray@dfci.harvard.edu (N.S. Gray).
These three authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113481
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
effect [11e15]. For example, a combination treatment of Olaparib, a
PARP inhibitor, and Dinaciclib, a clinical pan-CDK inhibitor, syner-
gistically suppress tumor growth in an ovarian patient-derived
xenograft (PDX) model [16]. A similar synergistic effect was
observed in a mouse model of Ewing sarcoma [17]. These initial
proof-of-concept results intensified our interest in developing
small molecule inhibitors for CDK12.
We have recently described an initial CDK12/13-selective co-
valent inhibitor THZ531, which was developed starting from THZ1,
a CDK7/CDK12/13 covalent inhibitor (Fig. 1) [18,19]. We also
established that CDK12/13 selectivity of THZ531 over CDK7 is
structurally derived from 3-aminopiperdine moiety, which dis-
criminates the targeted cysteine Cys1039 on CDK12 (or Cys1017 on
CDK13) from Cys312 on CDK7. To further understand its structural-
activity relationships (SAR), we designed and synthesized a focused
library of THZ531 analogs with 3-aminopiperidine scaffold or its
variation. Here, we present the results of these efforts, including a
structure-activity relationship (SAR) and a selectivity/potency
improvement for this series of compounds.
phosphorylation of His-c-Myc (aa 17e167) as a substrate of re-
combinant CDK12 and 13 proteins. The commercial kinase assays
from Invitrogen, ADAPTA or Z’LYTE, were chosen to measure the
biochemical inhibitory IC50 values for CDK2/CycA, CDK7/CycH and
CDK9/CycT1 using a fixed-time point format.
The initial THZ531 modifications included replacing the chloride
with bromide, cyclopropane and trifluoromethyl. As shown in
Table 1, 10b (bromide derivative) maintained nearly equal potency
as THZ531 on all CDKs tested, whereas 10c and 10d (cyclopropane
and trifluoromethyl derivatives, respectively) exhibited a dramatic
loss of activity, indicating the potential for a steric clash between
these bulky groups and Phe813 on CDK12 or Phe791 on CDK13,
respectively. Next, changing the size of the central saturated ring of
THZ531 with 3-aminopyrrolidine (10e) and 3-aminoazepan (10f)
led to a divergent activity shift. The smaller pyrrolidine group
enhanced the activities on CDK2/7/9/12/13, whereas bulkier 3-
aminoazepan abrogated the activities on CDK12/13. We therefore
fixed 3-aminopiperdine as the central ring and introduced a pyri-
dine ring to compound 10g, which displayed a slight increase of
IC50 relative to THZ531. Interestingly, removing methylene
dimethyl amine (DMA) from compound 10g restored the inhibitory
activity on CDK12/13 by compound 10h, likely due to less steric
interactions.
2. Results and discussion
2.1. Chemistry
Further modifications were focused on decreasing the size of the
compound by removing carbonyl group and conjugated piperidine
to the extended aromatic ring which contains the covalent
warhead. As shown in Table 2, compound 15a has 3e5-fold increase
of activity on CDK2, CDK7 and CDK12/13, while almost no change
on CDK9. However, this increased activity on CDK2 and CDK7 was
mitigated once the DMA group was removed, as shown by com-
pound 15b which retained the activity on CDK12/13 while
decreasing activity on CDK2, 7 and 9. The same trend was observed
with compounds 15c and 15d, which are pyridine versions of 15a
and 15b, respectively, suggesting that removal of the DMA group
improves selectivity for CDK12/13 over CDK2 and 7. Next, we
introduced small substitution groups, such as F, Me or OMe to
either 3- or 6-postion of the phenyl ring (compounds 15e-15h). The
mono-substitution with F or Me on either the 3 or 6 position was
well tolerated by CDK12/13 (15e and 15f). The bis-substitution with
F and Me resulted in a compound that unexpectedly gained some
activity on CDK2, although it still maintained activity for CDK12/13
(15g). Installing an OMe group at the 3-postion to replace F indeed
abrogates this gained activity on CDK2 (15h). Additionally, com-
pound 15h exhibited differential activity on CDK12 and CDK13,
with IC50 of 293.2 nM and 4385 nM, respectively. Therefore, 15h
provides a potential lead for developing selective inhibitors for
CDK12 over CDK13.
The synthesis of 10a-10h is illustrated in Scheme 1. The
commercially available (1-(phenylsulfonyl)-1H-indol-3-yl) boronic
acid was coupled with 2,4-pyrimidine using a Suzuki reaction,
followed by SN2 reaction with N-Boc protected 3-aminopyrrolidine
or its analogs: 3-aminopyrrolidine and 3-aminoazepan. The
removal of Boc group allowed intermediate 5 to react with 4-
nitrobenzoic acid or 5-nitropicolinic acid. Subsequent reduction
of nitro group (7) with SnCl2 in acidic conditions led to compound
8, which then reacted with either acryloyl chloride or (E)-4-
bromobut-2-enoyl chloride followed by dimethyl amine to
replace the bromide. The final product 10 was obtained after
deprotection of phenylsulfonyl group from 9.
Scheme 2 described the synthesis for compounds 15a-15h. The
common intermediate 5 was converted to a series of analogs 12 by a
nucleophilic substitution with 1-fluoro-4-nitrobenzene or 2-
fluoro-5-nitropyridine or related derivatives. The nitro group was
reduced to free amino group, which was then coupled with acryl-
amide or with (E)-4-bromobut-2-enoyl chloride. Compound 14 was
then heated under basic conditions to deprotect the phenylsulfonyl
group and afford the final product 15.
The synthesis of 22a-22e is depicted in Scheme 3. Substitution
of 1-fluoro-4-nitrobenzene with 3-aminocyclohexan-1-ol afforded
18, which was coupled with 3a at elevated temperatures to provide
19. Subsequent reduction resulted in free amine compound 20,
which was then coupled with acrylamide or (E)-4-bromobut-2-
enoyl chloride and afforded 21. The final product 22 was obtained
upon deprotection in the presence of 1 M NaOH solution.
The results we obtained with derivatives featuring DMA group
in both the 10 and 15 series suggested that either an amide bond or
the direct connection between piperidine and phenyl resulted in a
rigidity of the molecule and thus restrained the acrylamide
warhead. To explore this issue further, we introduced a more
flexible ether linker as a strategy to mitigate DMA’s influence, and
thus compound BSJ-01-175 (22a) and its DMA-free version 22b
were synthesized. As expected, both compounds inhibited CDK12
with the same biochemical IC50 of 155 nM, a 3-fold decrease
2.2. SAR analysis leads to the identification of BSJ-01-175
All compounds were tested for their CDK12/13 inhibitory ac-
tivity using
a
radiometric kinase assay that measures
Fig. 1. Structures of THZ1 and THZ531.
2

B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
a
Scheme 1. Synthesis of 10a-10ha. Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, CH3CN, 80 ꢀC; (b) DIPEA, NMP, 140 ꢀC; (c) HATU, DIPEA, DMF; (d) SnCl2, MeOH, Ethyl Acetate,
80 ꢀC; (e) Acryloyl chloride, DIPEA, CH3CN, 0 ꢀC. Or (E)-4-bromobut-2-enoyl chloride, DIPEA, CH3CN, 0 ꢀC; Dimethylamine, CH3CN; (f) NaOH, dioxane.
compared to THZ531 (Table 3). In addition, we observed improved
selectivity against CDK9 and CDK2. Next, we explored whether the
stereochemistry of the ether connection impacts the activity and
we therefore synthesized compound 22c with an S configuration.
Although compound 22c retained the potency of BSJ-01-175 on
CDK12/13, it also exhibited increased activity on both CDK2 and
CDK9. Finally, the exploration of substitution on the phenyl ring
turned out to be much less tolerated compared to the 15 series as
exemplified by a loss of potency with compound 22d, in which a F
group was installed at 2-position. Collectively, our medicinal
chemistry efforts have resulted in a group of compounds with
improved potency and selectivity over our starting point (THZ531).
These compounds include 10h, 15b, 15f, 15g, 15h, 20b and BSJ-01-
175, which were then further evaluated in mouse liver micro-
somal stability (MLM) assay (Table 4). Out of this collection, BSJ-01-
175 showed the best microsomal stability. Therefore BSJ-01-175
was chosen for further biochemical and structural characteriza-
tion as well as cell-based assays and animal studies.
2.3. Characterization of BSJ-01-175
2.3.1. BSJ-01-175 covalently binds to CDK12 at cysteine 1039
To assess whether BSJ-01-175 reacts covalently with CDK12, we
incubated CDK12/Cyclin K complex with a 10-fold molar excess of
compound BSJ-01-175 and analyzed reaction products by LC-MS.
After the reaction, CDK12, but not Cyclin K, showed an increase in
mass consistent with covalent addition of a single molecule of BSJ-
01-175 (Fig. 2A). To identify the site of modification, the labeled
protein complex was digested with gluc protease, and proteolytic
fragments were analyzed by CE-MS, revealing exclusive
3

B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
Scheme 2. Synthesis of 15a-15ha. aReagents and conditions: (a) DIPEA, DMF, 80 ꢀC; (b) SnCl2, MeOH, Ethyl Acetate, 80 ꢀC; (c) Acryloyl chloride, DIPEA, CH3CN, 0 ꢀC. Or (E)-4-
bromobut-2-enoyl chloride, DIPEA, CH3CN, 0 ꢀC; Dimethylamine, CH3CN; (d) NaOH, dioxane.
Scheme 3. Synthesis of 22a-22ea. aReagents and conditions: (a) NaH, DMF, 0 ꢀCe25 ꢀC; (b) DIPEA, NMP, 140 ꢀC; (c) SnCl2, MeOH, Ethyl Acetate, 80 ꢀC; (d) Acryloyl chloride, DIPEA,
CH3CN, 0 ꢀC. Or (E)-4-bromobut-2-enoyl chloride, DIPEA, CH3CN, 0 ꢀC; Dimethylamine, CH3CN; (e) NaOH, dioxane.
modification of Cys1039 (Fig. 2B).
3.8 Å, twelve CDK12 residues were found to make direct contacts
with BSJ-01-175, with one additional water mediated contact be-
tween the amino group of Lys756, the backbone amide of Asp877,
and the indole ring amine of the compound (Fig. 3B). A small twist
of the cyclohexane group of BSJ-01-175 induces a rotamer shift in
the phenyl ring in the linker region of the compound, leading to a
different orientation of the acrylamide moiety in the two CDK-
cyclin complexes of the asymmetric unit compared to THZ531
2.3.2. Structure determination of CDK12/CycK with BSJ-01-175
The specific interaction of compound BSJ-01-175 with CDK12
and the formation of a covalent bond to Cys1039 was analyzed
structurally. We determined the crystal structure of CDK12eCyclin
K in complex with BSJ-01-175 to 3.0 Å resolution with excellent
stereochemistry (see Supplementary Table S2 for diffraction data
collection and refinement statistics). As observed before [18,20], we
found two CDK-cyclin complexes per asymmetric unit, with the
CDK12 molecule forming a covalent bond between Cys1039 and
BSJ-01-175 (Fig. 3A and Supplementary Fig. S1). Within a shell of
(see below). In both complexes, the C-terminal extension helix aK
of CDK12 is displaced from the kinase lobe structure allowing
Cys1039 to reorient toward the ATP-binding site for reacting with
BSJ-01-175 (Supplementary Fig. S2). The aminopyrimidine of BSJ-
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B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
Table 1
IC50 values for compounds 10a-10 g against CDK2,7,9,12 and 13.
Compound
n
X
R1
R2
IC50 (nM)
CDK2
CDK7
CDK9
CDK12
CDK13
10a (THZ531)
10b
10c
2
2
2
C
C
C
Cl
Br
CH2NMe2
CH2NMe2
CH2NMe2
1300
1000
>10,000
452
115
625
193
184
>10,000
482.6
225.6
9979
657.1
572.6
>10,000
10d
10e
10f
10 g
10h
2
1
3
2
2
C
C
C
N
N
CF3
Cl
Cl
Cl
Cl
CH2NMe2
CH2NMe2
CH2NMe2
CH2NMe2
H
10,000
49.3
339
3870
3390
>10,000
6.33
388
402
919
>10,000
14.2
1220
658
>10,000
68.7
1450
764.9
231.7
10,000
50.2
1831
1636
798.9
530
Table 2
IC50 values for compounds 15a-15h against CDK2,7,9,12 and 13.
Compound
X
R1
R2
R3
IC50 (nM)
CDK2
CDK7
CDK9
CDK12
CDK13
15a
15b
15c
15d
15e
15f
C
C
N
N
C
C
C
C
H
H
H
H
H
F
F
H
H
H
H
Me
H
Me
Me
CH2NMe2
H
CH2NMe2
H
H
H
H
H
623
3550
492
1940
1100
4020
266
69.5
162
74
433
197
318
118
403
155
171
87.7
121
165
436
230
522
166.7
93.6
35.4
67.6
142.3
87.6
177.6
123
186.1
120
238.8
126.6
203.8
4385
15g
15h
130.5
293.2
MeO
3360
Table 3
IC50 values for compounds 22a-22e against CDK2,7,9,12 and 13.
Compound
Chirality
R1
R2
IC50 (nM)
CDK2
CDK7
CDK9
CDK12
CDK13
22a (BSJ-01-175)
R
R
S
H
H
H
F
CH2NMe2
H
CH2NMe2
CH2NMe2
4510
>10,000
650
187
587
112
269
367
585
132
286
156
155.1
181
282.6
328.1
182
22b
22c
22d
R
3570
857.2
484.2
Table 4
Mouse liver microsomal half-life.
mediate further hydrophobic interactions, stacked between Leu866
of the antiparallel -strand preceding the kinase T-loop, and Ile733
of the N-lobe 1 strand, respectively (Fig. 3B). The electron density
b
b
Compound
MLM T1/2 (min)
Compound
MLM T1/2 (min)
map of the following exposed linker sites is poorly defined, except
for the side chain of Cys1039, which is again clearly visible with its
covalent bond to the acrylamide moiety of BSJ-01-175
(Supplementary Fig. S1A).
10h
15b
15f
3.5
7.2
7.6
3.9
15h
BSJ-01-175
20b
4.3
8
5.4
15g
To compare BSJ-01-175 and THZ531 binding modes, we deter-
mined the crystal structure of the latter bound to CDK13/Cyclin K at
2.36 Å resolution (Fig. 3C, see Table S2 for refinement statistics). As
seen before and described above, two cyclin-kinase complexes
01-175 forms two hydrogen bonds to the backbone of Met816 of
the kinase hinge region, while the cyclohexane and phenyl rings
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B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
Fig. 2. CDK12 is labeled by BSJ-01e175 at Cys1039. (A) Mass spectra (left) and zero-charge mass spectra (right) of CDK12/cyclin K complex treated with (top) DMSO or (bottom) a
10-fold excess of BSJ-01-175 for 1 h at room temperature. Mass spectral peaks are labeled with glyphs and correspond to cyclin K (red), unmodified CDK12 (green), and CDK12
covalently labeled with BSJ-01-175 (blue). Cyclin K is unaffected by compound treatment, while CDK12 protein exhibits an increase in mass consistent with modification by a single
molecule of BSJ-01-175. (B) MS/MS spectrum of CDK12 peptide1025LSKMAPPDLPHWQDCHE1041 modified with BSJ-01-175 indicates covalent labeling of Cys1039. Ions of types b and
y are indicated with blue and red glyphs, respectively. Inhibitor related ions [26] are marked with green glyphs.
D peak corresponding to neutral loss of BSJ-01-175.
were found in the asymmetric unit [21]. The twist of the THZ531
piperidine ring in these two forms is more pronounced than what
we observed for the cyclohexane ring in BSJ-01-175 (Fig. 3D). This
results in a significantly different orientation of the acrylamide
linker region (Supplementary Fig. S3). These conformational dif-
ferences are possible due to the high flexibility in the kinase
(Fig. 4A). A superimposition with the crystal structure of CDK12
bound to ADP (4NST) [20] instead reveals that the cyclohexane
moiety of BSJ-01-175 adopts a similar position as the imidazole ring
of His1040 directly preceding the C-terminal extension helix
(Fig. 4B). The conformational differences of the linker regions to the
bound cysteines may thus give rise to the improved stability of BSJ-
01-175 compared to THZ531.
extension helix aK harboring the cysteine site that is targeted by
the covalent linkage (Supplementary Fig. S4). We observed that
while in one of the forms the side chain of Gln1015 participates in a
hydrogen-bond network between the amine backbone of Asp797
and the carbonyl group of the THZ531 acrylamide moiety, the other
form features THZ531 that uses its linker region to form hydro-
2.3.3. BSJ-01-175 is a selective CDK12/13 inhibitor
To assess the selectivity of BSJ-01-175, we used the KiNativ
approach [22] that reports on how well a compound blocks irre-
versible binding of a non-specific ADP(ATP)-biotin probe to cellular
phobic contacts to the N-lobe
b
1 strand (Supplementary Fig. S3B).
kinases. In these experiments, Kelly cells were pre-exposed to 1 mM
The variable linker region to the cysteine becomes also apparent
from an overlay of the two CDK12 crystal structures bound to
THZ531 (PDB accession code 5ACB) [18] or bound to BSJ-01-175
of BSJ-01-175, then lysed, treated with the irreversible ADP(ATP)-
biotin probe and pulled down via an affinity column. These re-
sults were compared to the DMSO treated negative control as a
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B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
Fig. 3. Crystal structures of CDK12/CycK bound to BSJ-01e175 and CDK13/CycK bound to THZ531. (A) Complex structure of CDK12/CycK bound to BSJ-01-175 (PDB accession code
7NXK). The phosphorylated threonine in the T-loop of the kinase and its coordinating arginine residues are indicated. (B) Close up of the interactions of BSJ-01-175 with the kinase
N-lobe of CDK12. Hydrogen bonds of the aminopyrimidine are highlighted. (C) Structure of CDK13/CycK bound to THZ531 (PDB accession code 7NXJ). (D) Overlay of the CDK12/CycK
structure with CDK13/CycK bound to BSJ-01-175 and THZ531, respectively. The variable conformations of the acrylamide moiety binding covalently to the conserved cysteine
residues are shown.
benchmark. As shown in Table 5, most of the protein kinases (total
270 proteins, Supplementary Dataset 1) were not affected (BSJ-01-
175 blocked less than 50% of ADP(ATP)-biotin probe binding),
whereas BSJ-01-175 was able to protect 75% of CDK12 from probe
binding. Although DNAPK, RSK2 and CDK7 displayed a degree of
protection higher than 50%, only CDK7 has a targetable cysteine
and biochemically, weak inhibitory IC50 values against both DNPAK
(>10,000 nM) and RSK2 (4500 nM) were detected, suggesting BSJ-
01-175 is a weak inhibitor for both proteins. Therefore, we carefully
examined the selectivity for CDK12/13 over CDK7, as discussed in
the next section.
500 nM. Compared to THZ531, which engages CDK7 at 1
01-175 exhibited mild engagement even at higher concentrations
(5 M). The irreversibility on CDK12/13’s binding by BSJ-01-175 in
mM, BSJ-
m
cellular context was well-demonstrated by sustained engagement
even at 24 h after a washout experiment (Fig. 5B). Next, to under-
stand how the target engagement affects CDK12/13 downstream
signaling, we examined phosphorylation levels on Ser2 of RNA pol
II. With 500 nM of BSJ-01-175, p-Ser2 was significantly reduced,
whereas Ser5 and Ser7, two non-CDK12/13 phosphorylation sites
remained unchanged. At higher concentrations, BSJ-01-175
reduced the protein levels of p-Ser2/5/7, as well as total RNA pol
II (Fig. 5C). As a transcriptional regulator, loss of CDK12 function by
BSJ-01-175 was expected to cause transcriptional defects on spe-
cific DDR genes regulated by this kinase. We therefore measured
BRCA1 and BRCA2 expression in Jurkat cells pretreated with BSJ-01-
175 for 4 h. As shown in Fig. 5D, both BRCA 1 and 2 genes were
downregulated to below 30% of DMSO control. Lastly, we exposed
previously generated Cys1039Phe mutant Kelly cells, resistant to
covalent CDK12/13 inhibitors that target Cys1039 [23], to BSJ-01-
175 and observed a 5-fold increase in cell viability compared to
the wild type (WT) (Fig. 6) indicating strong dependence on co-
valent bond formation with Cys1039.
2.3.4. BSJ-01-175 inhibited CDK12/13 activity and the downstream
signaling
We employed BSJ-01-175 in a series of cell-based assays to
examine its cellular activity, and establish whether the phenotype
is CDK12/13-inhibition dependent. First, we carried out a rescue
experiment using THZ1-biotin, a probe that binds CDK7/12/13
covalently, allowing their pulldown via streptavidin beads. Here,
Jurkat cells treated with increasing amounts of BSJ-01-175 for 6 h
were lysed and then exposed to 1 mM of THZ1-biotin. The pulldown
samples from streptavidin beads were immuno-blotted with the
antibodies for cyclin K and cyclin H (a CDK7 binding partner), as
surrogate readouts for CDK12/13 and CDK7, respectively. As shown
in Fig. 5A, BSJ-01-175 competed off THZ1-biotin binding with
CDK12/13, while leaving CDK7 untouched at concentration of
2.3.5. BSJ-01-175 suppresses tumor growth of Ewing sarcoma
Our previous study has shown that a pan CDK7/12/13 covalent
inhibitor THZ1 alone or combination with PARP inhibitor Olaparib
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B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
Fig. 4. Overlay of CDK12 crystal structures in complex with BSJ-01e175, THZ531 or ADPꢁAlF3ꢁ2Mg2þ. (A) Superimposition of the two CDK12 structures complexed with BSJ-01-
175 (7NXK; chain A, light blue/orange) or THZ531 (5ACB; chain C, magenta/green). Key residues and important secondary structure elements are indicated. (B) Superimposition of
the CDK12eBSJ-01-175 structure (7NXK; chain A, light blue/orange) and the CDK12eADPꢁAlF3ꢁ2Mg2þ nucleotide bound complex (4NST; chain A, teal/green). The imidazole ring of
H1040 from the DCHEL motif at the beginning of the C-terminal extension helix
aK adopts a similar position as the cyclohexane ring in BSJ-01-175 (right picture).
can suppress the growth of Ewing Sarcoma in vivo [17]. To under-
stand whether this efficacy can be recapitulated with a selective
CDK12/13 inhibitor, we first measured the antiproliferative activity
of BSJ-01-175 in TC71 Ewing sarcoma cells, and observed a slight
decrease of activity compared to THZ531 (Fig. 7A).
kinase inhibitors. For example, THZ1, the first covalent inhibitor of
the CDK family, inhibits three CDKs, CDK7, CDK12 and CDK13, by
reacting with cysteines that are located at similar positions by
sequence alignment but arranged differently in 3-dimensional
space. Our subsequent work produced THZ531, a selective CDK12/
13 inhibitor that features 3-amino-piperidine moiety that is
responsible for imparting selectivity for CDK12/13 relative to CDK7
[18]. Here, we describe results of our med-chem efforts and identify
several compounds of interest. Most significantly, we report and
characterize BSJ-01-175 that exhibited an exquisite selectivity
Next, the pharmacokinetic (PK) properties of BSJ-01-175 was
examined following two routes of administration (IV and PO), and
displayed a moderate stability with T1/2 of 2.25 h, a low clearance
(24.9 mL/min/kg), and 17% oral bioavailability (Table 6). Given these
promising results, we administrated BSJ-01-175 with IP dosing of
10 mg/kg three doses weekly to Ewing sarcoma PDX model derived
from TC71 cells. Compared to the vehicle control, BSJ-01-175 led to
a significant suppression of tumor growth throughout 3 weeks of
drug treatment period (Fig. 7B). However, we did observe weight
loss toxicity (Fig. 7C), requiring dosing protocol to be adjusted to
two three-day treatment holidays at Day 10 and Day 16. Whether
body weight loss is caused by CDK12/13 inhibition or a metabolic
liability of BSJ-01-175 remains to be addressed.
profile at
a concentration that completely inhibited CDK12-
regulated signaling. Compared to THZ531, BSJ-01-175 was devel-
oped to include DMA, a group known to improve solubility and
metabolic stability, resulting in a 3-fold activity improvement and
suitable stability for animal studies. Importantly, we show that BSJ-
01-175 demonstrates efficacy in Ewing sarcoma PDX model. This
study also led to compound 15h, a potential lead for development
of CDK12 specific inhibitor. Further modifications of BSJ-01-175 or
its analog 15h are needed to develop specific covalent CDK12 or 13
inhibitors, and/or optimize the current series into less toxic com-
pounds not resulting in the body weight loss induced by BSJ-01-
175. Taken together, our study confirms that the THZ1 scaffold
can be optimized both in terms of selectivity and pharmacological
3. Conclusion
Targeting a unique cysteine with a well-designed covalent in-
hibitor represents a powerful avenue to achieve highly selective
8

B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
Table 5
Intracellular KiNativ™ profiling assay identifies CDK12 as major target of BSJ-01-175.
Fig. 5. BSJ-01e175 specifically targets CDK12/13 and suppresses the transcription of BRAC1 and BRAC2. (A) Competitive pulldown assay in Jurkat cells treated with BSJ-01-175 at
indicated concentrations for 6 h. Cell lysates were incubated with bio-THZ1 that competes for the binding pocket of the target kinase. Western blotting showing the pulldown (PD)
or input (total lysate) of Cyclin H and Cyclin K. (B) BSJ-01-175 binds intracellular CDK12-cyclin K complexes 24 h after washout in competitive pulldown assay. Jurkat cells were
treated with BSJ-01-175 for 3 h, followed by washout for indicated times. Cell lysates were incubated with bio-THZ1 that competes for the binding pocket of the target kinase.
Western blotting showing the pulldown (PD) or input (total lysate) of Cyclin H and Cyclin K. (C) BSJ-01-175 inhibits phosphorylation of RNA pol II CTD and downregulates total RNA
pol II CTD in Jurkat cells. Cells were treated with BSJ-01-175 or THZ531 for 6 h and cell lysates were subjected to immunoblotting analysis. THZ531 was used as a positive control. (D)
RT-qPCR of BRCA1/2 gene expression following BSJ-01-175 or THZ531 treatment (4 h) in Jurkat cells. Data are presented as the mean SEM (n ¼ 3). The figure is one representation
from three independent experiments.
9

B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
Fig. 6. Anti-proliferation activity of BSJ-01e175 on Kelly cells with WT CDK12 or C1039F mutant. Cell proliferation assay in WT and CDK12C1039F Kelly cells. Cells were treated
with the indicated compounds for 72 h and subjected to CellTiter-Glo assay. Data are presented as mean SEM(n ¼ 3). The figure is one representation from three independent
experiments.
properties, and suggest that further work in this area will likely lead
to clinically relevant chemical matter.
(MþH).
To a stirred solution of 5a (327 mg, 0.7 mmol) in 5 mL of pyridine
was added 4-nitrobenzoyl chloride 6a (126 mg, 0.7 mmol) and then
was heated to 80 ꢀC. The reaction mixture was stirred for 2 h and
then was cooled to room temperature and concentrated under
reduced pressure. The resulting crude was purified by silica gel
column with DCM/methanol (10/1) to give the desired compound
(R)-(3-((5-chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)pyrimidin-
2-yl)amino)piperidin-1-yl)(4- nitrophenyl)methanone 7a (315 mg,
73%). LC-MS: m/z 617 (MþH).
4. Experimental section
4.1. Chemistry
Unless otherwise noted, reagents and solvents were obtained
from commercial suppliers and were used without further purifi-
cation. 1H NMR spectra were recorded on 500 MHz (Bruker A500),
The nitro compound 7a (308 mg, 0.5 mmol) was suspended in
15 mL of ethyl acetate/methanol (5:1), followed by addition of SnCl2
(948 mg, 5 mmol). After stirring for 2 h at 80 ꢀC, the reaction
mixture was cooled down to room temperature and poured onto
saturated aqueous NaHCO3. The mixture was stirred for 10 min
followed by extraction with 100 mL of CHCl3 and 2-propanol (4:1).
The organic layer was washed with water and brine, dried over
sodium sulfate, filtered through a pad of Celite and concentrated
under reduced pressure. The resulting crude was purified by silica
gel column with DCM/methanol (10/1) to provide the desired
and chemical shifts are reported in parts per million (ppm, d)
downfield from tetramethylsilane (TMS). Coupling constants (J) are
reported in Hz. Spin multiplicities are described as s (singlet), br
(broad singlet), d (doublet), t (triplet), q (quartet), and m (multi-
plet). Mass spectra were obtained on a Waters Micromass ZQ in-
strument. Preparative HPLC was performed on a Waters Sunfire C18
column (19 ꢂ 50 mm, 5
mM) using a gradient of 5e95% methanol in
water or acetonitrile in water containing 0.05% trifluoroacetic acid
(TFA) over 22 min (28 min run time) or 35 min (45 min run time) at
a flow rate of 20 mL/min. All of the final compounds reported had
purities greater than 95% based on 1H nuclear magnetic resonance
(NMR) and liquid chromatographyꢃmass spectrometry (LCꢃMS).
compound
(R)-(4-aminophenyl)(3-((5-chloro-4-(1-(phenyl-
sulfonyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)piperidin-1-yl)
methanone 8a (161.4 mg, 55%). LC-MS: m/z 587 (MþH).
To the solution of the compound 8a (117.4 mg, 0.2 mmol) in 5 mL
4.1.1. General procedures for synthesis of compound 10a-10h
To a solution of 2,4,5-trichloropyrimidine 2a (378 mg, 2 mmol)
in 5 mL of CH3CN under N2 was added 1-(phenylsulfonyl)-1H-
indol-3-ylboronic acid 1 (602 mg, 2 mmol) and 2 mL of saturated
aqueous Na2CO3. The mixture was degassed 3 times before the
addition of Pd(PPh3)4 (115 mg, 0.1 mmol). After degassing a further
3 times, the mixture was heated to 80 ꢀC for 2 h. The resulting
precipitate was filtered and washed with water and ether to afford
the desired compound 3-(2,5-dichloropyrimidin-4-yl)-1-(phenyl-
sulfonyl)-1H-indole 3a as a light grey solid (687 mg, 85%). LC-MS:
m/z 404 (MþH).
of CH3CN was added DIPEA (70 mL, 0.4 mmol). The reaction mixture
was cooled down to 0 ꢀC and then treated with 4-bromobut-2-
enoyl chloride (36.4 mg, 0.2 mmol) or acryloyl chloride (18 mg,
0.2 mmol) in DCM. The reaction mixture was stirred for 10 min at
0
ꢀC to give the acrylamide 9h (LC-MS: m/z 641 (MþH)), and bro-
mide intermediate. Dimethylamine in THF (1 M, 2.0 mL) was then
added to the bromide intermediate solution. The resulting mixture
was warmed up to room temperature, stirred for 30min and then
concentrated under reduced pressure. The resulting crude was
purified by reverse phase HPLC (5e95% MeOH in H2O) to give the
desired compound 9a (91 mg, 65%). LC-MS: m/z 698 (MþH).
The compound 9a (70 mg, 0.1 mmol) was dissolved in 2 mL of
1,4-dioxane and 2 mL of 1 M aqueous NaOH. The solution was
allowed to stir at room temperature for 2 h followed by neutrali-
zation with 2 mL of 4 M HCl in Dioxane. The solution was then
extracted with 30 mL of CHCl3 and 2-propanol (4:1) and was
washed with water. The removal of solvent provided the crude
which was purified by reverse phase HPLC (5e95% MeOH in H2O) to
To a solution of 3a (403 mg, 1 mmol) in 5 mL of NMP was added
tert-butyl (R)-3-aminopiperidine-1-carboxylate 4a (200 mg,
1 mmol) and DIPEA (0.52 mL, 3 mmol). The solution was heated for
2 h at 130 ꢀC. The cooled solution was diluted with 100 mL of CHCl3
and 2-PrOH (4:1) and then washed with water. The organic phase
was then evaporated to give a brown crude which was dissolved in
3 mL of DCM, followed by a slow addition of 3 mL of TFA at 80 ꢀC.
After 30 min stirring at room temperature, the solvent was then
removed. The residue was purified by silica gel chromatography
column with DCM/methanol (10/1) to give the product (R)-5-
chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)-N-(piperidin-3-yl)
pyrimidin-2-amine 5a (350 mg, 76% in two steps). LC-MS: m/z 468
give
(R,E)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidine-1- carbonyl)phenyl)-4-(dimethylamino)but-2-
enamide 10a (25 mg, 43%). LC-MS: m/z 558 (Mþ1), 1H NMR
(500 MHz, DMSO‑d6) 11.83 (s,1H),10.94 (s,1H),10.41 (s,1H), 8.53 (s,
10

B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-
2-enamide (10b) LC-MS: m/z 602 (MþH). 1H NMR (500 MHz,
DMSO‑d6)
d 11.82 (s, 1H), 10.50 (s, 1H), 10.09 (s, 1H), 8.66e8.25 (m,
2H), 7.69 (br, 2H), 7.48 (d, J ¼ 8.1 Hz,1H), 7.38 (br, 2H), 7.25e7.06 (m,
2H), 6.83e6.72 (m, 1H), 6.54e6.41 (m, 1H), 4.44 (br, 1H), 3.95 (br,
4H), 3.13 (br, 2H), 2.80 (s, 6H), 2.06 (br, 1H), 1.95e1.78 (m, 1H), 1.66
(s, 1H), 1.54 (s, 1H).
(R,E)-N-(4-(3-((5-cyclopropyl-4-(1H-indol-3-yl)pyrimidin-2-
yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)
but-2-enamide (10c). LC-MS: m/z 564 (MþH). 1H NMR (500 MHz,
DMSO‑d6)
d 11.89 (s, 1H), 10.52 (s, 1H), 10.07 (s, 1H), 8.68 (br, 2H),
8.08 (s, 1H), 7.68 (br, 1H), 7.55 (d, J ¼ 8.0 Hz, 1H), 7.41 (s, 1H),
7.30e7.11 (m, 2H), 6.87e6.67 (m, 1H), 6.56e6.40 (m, 1H),3.96 (br,
4H), 3.54 (br, 3H), 2.82 (s, 6H), 2.18e2.08 (m, 1H), 2.03 (br, 1H), 1.87
(br, 1H), 1.75 (br, 1H), 1.60 (br, 1H), 1.13e0.93 (m, 2H), 0.74e0.52 (br,
2H).
(R,E)-N-(4-(3-((4-(1H-indol-3-yl)-5-(trifluoromethyl)pyr-
imidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethy-
lamino)but-2-enamide (10d). LC-MS: m/z 592 (MþH). 1H NMR
(500 MHz, DMSO‑d6) d 11.88 (s, 1H), 10.50 (br, 1H), 9.83 (s, 1H), 8.50
(s, 1H), 8.45e8.22 (m, 2H), 7.74 (br, 2H), 7.53e7.34 (m, 2H),
7.23e7.08 (m, 2H), 6.95 (br, 1H), 6.83e6.63 (m, 1H), 6.44 (br, 1H),
4.53 (br, 2H), 3.96 (br, 4H), 3.57 (br, 1H), 2.80 (s, 6H), 1.99e1.88 (m,
1H), 1.82e1.70 (m, 1H), 1.69e1.56 (br, 1H).
(R,E)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)pyrrolidine-1-carbonyl)phenyl)-4-(dimethylamino)but-
2-enamide (10e). LC-MS: m/z 544 (MþH). 1H NMR (500 MHz,
DMSO‑d6) d 11.87 (s, 1H), 10.49 (s, 1H), 9.88 (s, 1H), 8.47 (s, 1H), 8.63
(br, 1H), 8.29 (s, 1H), 7.80e7.42 (m, 3H), 7.29e7.15 (s, 2H), 7.07 (s,
1H), 6.82e6.69 (m, 1H), 6.63e6.30 (m, 1H), 4.65e4.30 (br, 2H), 3.94
(br, 2H), 3.47 (br, 2H), 2.80 (s, 6H), 2.23 (s, 1H), 2.15e1.93 (m, 1H),
1.31e1.04 (m, 1H).
(R,E)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)azepane-1-carbonyl)phenyl)-4-(dimethylamino)but-2-
enamide (10f). LC-MS: m/z 572 (MþH). 1H NMR (500 MHz,
DMSO‑d6) d 11.86 (s, 1H), 10.44 (s, 1H), 9.82 (s, 1H), 8.60 (s, 1H), 8.45
(s, 1H), 8.20 (br, 1H), 7.74 (d, J ¼ 8.2 Hz, 1H), 7.62 (s, 1H), 7.50 (d,
J ¼ 8.1 Hz, 1H), 7.45e7.28 (m, 2H), 7.25e7.08 (m, 2H), 6.83e6.65 (m,
1H), 6.56e6.30 (m, 1H), 4.40 (s, 1H), 4.21e4.01 (m, 1H), 3.95 (s, 2H),
3.47e3.19 (m, 2H), 2.80 (s, 6H), 2.13e1.94 (m, 1H), 1.94e1.76 (m,
1H), 1.76e1.41 (m, 2H).
(R,E)-N-(6-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidine-1-carbonyl)pyridin-3-yl)-4-(dimethylamino)
but-2-enamide (10g). LC-MS: m/z 559 (MþH). 1H NMR (500 MHz,
Fig. 7. BSJ-01e175 suppresses tumor growth of Ewing sarcoma. (A) Antiproliferation
of BSJ-01-175 on TC71 cells, data are presented as the mean SEM (n ¼ 3). The figure is
one representation from two independent experiments. (B) Tumor volume measure-
ments of a subcutaneous TC71 xenograft mouse model of Ewing sarcoma treated with
vehicle or 10 mg/kg BSJ-01-175 IP qD. Treatment was stopped on day 21. Data are
plotted as mean values SD. *p value < 0.05, 2-way ANOVA with Tukey post hoc test.
(C) Relative weight measurements of mice in B. Data are plotted as mean values SD.
***p value < 0.001. 2-way ANOVA with Tukey post hoc test. Mice used in (B) and (C)
are 5 in vehicle group and 5 in BSJ-01-175 group.
DMSO‑d6)
d 11.99e11.79 (m, 1H), 10.81e10.54 (M, 1H), 9.88 (s, 1H),
8.92e8.57 (m, 1H), 8.39e8.22 (m, 1H), 8.02 (d, J ¼ 8.6 Hz, 1H), 7.63
(d, J ¼ 8.5 Hz, 1H), 7.55e7.29 (m, 2H), 7.25e7.17 (m, 1H), 7.16e7.05
(m, 2H), 6.91e6.68 (m, 1H), 6.56e6.39 (m, 1H), 3.96 (d, J ¼ 9.2 Hz,
2H), 3.85e3.61 (m, 3H), 3.48 (s, 1H), 3.07 (s, 1H), 2.81 (s, 6H),
2.17e1.94 (m, 2H), 1.84e1.70 (m, 1H), 1.70e1.45 (m, 1H).
(R)-N-(6-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidine-1-carbonyl)pyridin-3-yl)acrylamide (10h). LC-
1H), 8.44 (s, 1H), 8.25 (s, 1H), 7.67 (d, J ¼ 7.0 Hz, 1H), 7.52 (d,
J ¼ 8.0 Hz, 1H), 7.36 (d, J ¼ 8.5 Hz, 2H), 7.22 (t, J ¼ 8.0 Hz,1H), 7.16 (d,
J ¼ 8.0 Hz, 1H), 6.83 (m, 1H), 6.52 (d, J ¼ 15.0 Hz, 1H), 4.02 (br, 2H),
3.93e3.80 (m, 3H), 3.20 (br, 2H), 2.80 (s, 6H), 2.10 (m, 1H), 1.86 (m,
1H), 1.71 (m, 1H), 1.61 (m, 1H).
MS: m/z 502 (MþH). 1H NMR (500 MHz, DMSO‑d6)
d 11.80 (s, 1H),
10.66 (s,1H), 9.78 (s,1H), 8.98e8.67 (m,1H), 8.39e8.22 (m,1H), 8.10
(d, J ¼ 8.6 Hz, 1H), 7.68 (d, J ¼ 8.5 Hz, 1H), 7.55e7.29 (m, 2H),
7.25e7.17 (m, 1H), 7.16e7.05 (m, 2H), 6.85e6.61 (m, 1H), 6.56e6.39
(m, 1H), 4.55 (br, 1H), 3.86 (d, J ¼ 9.0 Hz, 2H), 3.85e3.61 (m, 1H),
(R,E)-N-(4-(3-((5-bromo-4-(1H-indol-3-yl)pyrimidin-2-yl)
Table 6
Mouse Pharmacokinetics of compound BSJ-01-175.
Route
Dose (mg/kg)
Tmax (h)
Cmax (ng/mL)
AUClast (h$ng/mL)
T1/2 (h)
2.2
CL (mL/min/kg)
24.9
Vss (L/kg)
3.9
F (%)
17
IV
PO
3
10
1511
272
1832
1043
2
11

B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
3.48 (s, 1H), 3.07 (s, 1H), 2.27e1.99 (m, 2H), 1.88e1.73 (m, 1H),
1.71e1.45 (m, 1H).
(15g). LC-MS: m/z 505 (MþH). 1H NMR (500 MHz, DMSO‑d6)
d 11.87
(s, 1H), 9.38 (s, 1H), 8.90e8.57 (m, 1H), 8.49 (d, J ¼ 3.1 Hz, 1H), 8.28
(s, 1H), 7.47 (d, J ¼ 8.1 Hz, 1H), 7.41e7.23 (m, 2H), 7.23e7.07 (m, 1H),
6.92 (d, J ¼ 9.4 Hz, 1H), 6.57e6.47 (m, 1H), 6.26e6.17 (m, 1H),
5.77e5.69 (m, 1H), 3.52 (s, 3H), 3.36e3.27 (m, 1H), 2.76e2.58 (m,
2H), 2.19e1.94 (m, 4H), 1.86 (br, 1H), 1.74 (br, 1H), 1.55e1.49 (m, 1H).
(R)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
4.1.2. General procedures for synthesis of compound 15a-15h
Substituted
1-fluoro-4-nitrobenzenes
or
2-fluoro-5-
nitropyridine 11 (1 mmol, 1 equiv) were added to a solution of 5a
(327 mg, 0.7 mmol, 1 equiv) in 3 mL of DMF, followed by addition of
0.1 mL of DIPEA. The resulting mixture was heated to 80 ꢀC and kept
stirring for 8 h. The reaction mixture was then cooled to room
temperature and the solvent was evaporated to give a brown res-
idue, which was purified by silica gel chromatography column with
DCM/methanol (10/1) to afford the desired nitro products 12. The
final compounds 15a-15h were synthesized with similar proced-
ures as 10a-10h.
amino)piperidin-1-yl)-5-methoxy-2-methylphenyl)acrylamide
(15h). LC-MS: m/z 517 (MþH). 1H NMR (500 MHz, DMSO‑d6)
d 11.86
(s, 1H), 9.41 (s, 1H), 8.69 (br, 1H), 8.48 (d, J ¼ 3.1 Hz, 1H), 8.29 (s, 1H),
7.48 (d, J ¼ 8.2 Hz, 1H), 7.31e6.89 (m, 4H), 6.56e6.48 (m, 1H),
6.25e6.18 (m, 1H), 5.76e5.69 (m, 1H), 4.17 (br, 2H), 3.61 (br, 3H),
3.35 (s, 2H), 2.82 (br, 2H), 2.11 (s, 3H), 2.07e1.94 (m, 1H), 1.88 (br,
1H), 1.81 (br, 1H), 1.61 (br, 1H).
(R,E)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidin-1-yl)phenyl)-4-(dimethylamino)but-2-
enamide (15a). LC-MS: m/z 530 (MþH). 1H NMR (500 MHz,
4.1.3. General procedures for synthesis of compound 22a-22d
To a suspension of NaH (0.8 g, 8.25 mmol, 2.5 equiv) in 3.0 mL of
anhydrous DMF was added 3-aminocyclohexanol HCl salt (0.5 g,
3.3 mmol, 1.0 equiv) slowly at 0 ꢀC and kept stirring for 0.5 h, then
substituted 1-fluoro-4-nitrobenzene (3.3 mmol, 1.0 equiv) was
added. The reaction mixture was kept stirring at 0 ꢀC for another
30min, then warm to room temperature and kept stirring for 2 h
1 mL of H2O was added dropwise to quench the reaction. The
mixture was extracted with DCM (100 mL), washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by silica gel chroma-
tography column to give the desired products 18 as a brown solid.
The final compounds 22a-22d were synthesized with similar pro-
cedures as 10a-10h.
DMSO‑d6) d 11.88 (s, 1H), 10.15 (s, 1H), 9.82 (s, 1H), 8.68 (s, 1H), 8.49
(d, J ¼ 3.1 Hz, 1H), 8.30 (s, 1H), 7.52 (d, J ¼ 8.4 Hz, 2H), 7.48 (d,
J ¼ 8.2 Hz, 1H), 7.36 (d, J ¼ 7.7 Hz, 1H), 7.24e6.86 (m, 4H), 6.74e6.62
(m, 1H), 6.45e6.32 (m, 1H), 3.92 (br, 2H), 3.59 (br, 2H), 2.79 (s, 6H),
2.73e2.62 (m, 1H), 2.04 (br, 1H), 1.88 (br, 1H), 1.72 (br, 1H), 1.55 (br,
1H).
(R)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidin-1-yl)phenyl)acrylamide (15b). LC-MS: m/z 473
(MþH). 1H NMR (500 MHz, DMSO‑d6)
d 11.88 (s, 1H), 10.05 (s, 1H),
8.49 (d, J ¼ 3.1 Hz, 1H), 8.30 (s, 1H), 7.57 (s, 2H), 7.48 (d, J ¼ 8.2 Hz,
1H), 7.40 (s, 1H), 7.27e6.87 (m, 4H), 6.44e6.32 (m, 1H), 6.27e6.13
(m, 1H), 5.71 (dd, J ¼ 10.1, 2.1 Hz, 1H), 4.10 (br, 2H), 3.81 (s, 2H), 2.85
(br, 2H), 2.08 (s, 1H), 1.96e1.85 (m, 1H), 1.77 (br, 1H), 1.58 (br, 1H).
(R,E)-N-(6-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidin-1-yl)pyridin-3-yl)-4-(dimethylamino)but-2-
enamide (15c). LC-MS: m/z 531 (MþH). 1H NMR (500 MHz,
(E)-N-(4-(((1R,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-
2-yl)amino)cyclohexyl)oxy)phenyl)-4-(dimethylamino)but-2-
enamide (22a). LC-MS: m/z 545 (MþH). 1H NMR (500 MHz,
DMSO‑d6) d 12.12 (br, 1H), 10.92 (s, 1H), 10.38 (s, 1H), 8.71e8.51 (m,
DMSO‑d6)
d
11.87 (s, 1H), 10.35 (s, 1H), 9.88 (s, 1H), 8.79e8.57 (br,
2H), 8.36 (s, 1H), 7.59 (d, J ¼ 8.7 Hz, 2H), 7.54 (d, J ¼ 8.1 Hz, 1H), 7.34
(d, J ¼ 50.9 Hz,1H), 7.25 (t, J ¼ 7.6 Hz,1H), 7.21e7.13 (m, 2H), 6.95 (d,
J ¼ 9.0 Hz, 2H), 6.79e6.48 (m, 1H), 6.48 (d, J ¼ 15.3 Hz, 1H), 4.81 (br,
1H), 3.89 (t, J ¼ 6.1 Hz, 2H), 3.53e3.41 (m, 2H), 2.74 (s, 3H), 2.73 (s,
3H), 2.22e2.10 (m, 1H), 2.02 (br, 1H), 1.88e1.70 (m, 3H), 1.69e1.50
(m, 2H), 1.48e1.33 (m, 1H).
1H), 8.47 (d, J ¼ 3.0 Hz, 1H), 8.38 (s, 1H), 8.31 (s, 1H), 7.83 (s, 1H),
7.54e7.35 (m, 2H), 7.09 (br, 2H), 6.82e6.61 (m, 1H), 6.47e6.32 (m,
1H), 4.32 (br, 1H), 4.10 (br, 1H), 3.95 (dd, J ¼ 7.4, 3.3 Hz, 2H),
3.10e2.89 (m, 2H), 2.80 (s, 6H), 2.25e1.95 (m, 1H), 1.87 (s, 1H),
1.72e1.51 (m, 2H).
(R)-N-(6-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidin-1-yl)pyridin-3-yl)acrylamide (15d). LC-MS: m/z
N-(4-(((1R,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)cyclohexyl)oxy)phenyl)acrylamide (22b). LC-MS: m/z 488
474 (MþH). 1H NMR (500 MHz, DMSO‑d6)
d
11.87 (s, 1H), 10.25 (s,
(MþH). 1H NMR (500 MHz, DMSO‑d6)
d 11.90 (s, 1H), 10.99 (br, 1H),
1H), 8.48 (d, J ¼ 3.0 Hz, 1H), 8.39 (br, 2H), 8.32 (s, 1H), 8.04e7.66 (m,
1H), 7.54e7.40 (m, 2H), 7.19 (br, 2H), 6.43e6.32 (m, 1H), 6.30e6.22
(m, 1H), 5.82e5.74 (m, 1H), 4.27 (s, 1H), 4.06 (br, 3H), 3.15e2.99 (m,
2H), 2.05 (br, 1H), 1.91 (br, 1H), 1.74e1.57 (m, 2H).
(R)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidin-1-yl)-2-methylphenyl)acrylamide (15e). LC-
10.36 (s, 1H), 8.71 (br, 1H), 8.60 (d, J ¼ 8.4 Hz, 2H), 8.36 (br, 1H), 7.66
(d, J ¼ 8.7 Hz, 2H), 7.54 (d, J ¼ 8.1 Hz, 1H), 7.49e7.31 (m, 1H), 7.25 (t,
J ¼ 7.6 Hz, 1H), 7.21e7.13 (m, 3H), 7.09e6.96 (m, 2H), 6.62e6.45 (m,
1H), 6.40 (d, J ¼ 15.3 Hz, 1H), 5.88 (s, 1H), 4.85 (br, 1H), 3.90 (br, 2H),
3.57e3.45 (m, 2H), 2.22e1.94 (m, 2H), 1.88e1.69 (m, 3H), 1.65e1.52
(m, 2H), 1.46 (br, 1H).
MS: m/z 487 (MþH). 1H NMR (500 MHz, DMSO‑d6)
d
11.87 (s, 1H),
(E)-N-(4-(((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-
2-yl)amino)cyclohexyl)oxy)phenyl)-4-(dimethylamino)but-2-
enamide (22c). LC-MS: m/z 545 (MþH). 1H NMR (500 MHz,
9.34 (s, 1H), 8.81e8.61 (br, 1H), 8.49 (d, J ¼ 3.1 Hz, 1H), 8.30 (s, 1H),
7.48 (d, J ¼ 8.2 Hz, 1H), 7.44e7.07 (m, 3H), 6.92 (br, 2H), 6.55e6.38
(m, 1H), 6.25e6.08 (m, 1H), 5.70 (dd, J ¼ 10.1, 2.1 Hz, 1H), 3.86 (br,
2H), 3.55 (br, 1H), 2.97e2.58 (m, 2H), 2.27e1.94 (m, 5H), 1.87 (br,
1H), 1.73 (br, 1H), 1.63e1.36 (m, 1H).
DMSO‑d6) d 11.88 (s, 1H), 10.16 (s, 1H), 9.79 (s, 1H), 8.62 (s, 1H), 8.49
(s, 1H), 8.25 (d, J ¼ 4.6 Hz, 1H), 7.50 (br, 2H), 7.39e7.26 (m, 1H),
7.25e7.10 (m, 2H), 6.98e6.90 (m, 1H), 6.75e6.62 (m, 1H), 6.41 (d,
J ¼ 15.3 Hz, 1H), 4.39 (br, 2H), 3.92 (br, 3H), 2.80 (s, 3H), 2.79 (s, 3H),
2.47e2.28 (m, 1H), 2.20e1.94 (m, 2H), 1.79 (br, 1H), 1.55e1.37 (m,
1H), 1.35e1.17 (m, 2H).
(R)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidin-1-yl)-3-fluorophenyl)acrylamide (15f). LC-MS:
m/z 491 (MþH). 1H NMR (500 MHz, DMSO‑d6)
d 11.88 (s, 1H),
10.15 (s, 1H), 8.72 (br, 1H), 8.49 (t, J ¼ 8.4 Hz, 1H), 8.29 (s, 1H),
7.66e7.57 (m, 1H), 7.47 (d, J ¼ 8.1 Hz, 1H), 7.33 (br, 1H), 7.23 (s, 1H),
7.17 (s, 1H), 7.05 (t, J ¼ 9.3 Hz, 1H), 6.93 (s, 1H), 6.43e6.31 (m, 1H),
6.27e6.17 (m, 1H), 5.74 (dd, J ¼ 10.1, 2.1 Hz, 1H), 3.51 (br, 2H),
3.32e3.21 (m, 1H), 2.75e2.58 (m, 2H), 2.04 (br, 1H), 1.91e1.80 (m,
1H), 1.74 (br, 1H), 1.60e1.47 (m, 1H).
(E)-N-(4-(((1R,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-
2-yl)amino)cyclohexyl)oxy)-3-fluorophenyl)-4-(dimethyla-
mino)but-2-enamide (22d). LC-MS: m/z 563 (MþH). 1H NMR
(500 MHz, DMSO‑d6)
d 11.86 (s, 1H), 10.37 (s, 1H), 9.80 (s, 1H), 8.66
(d, J ¼ 8.0 Hz, 1H), 8.50 (s, 1H), 8.25 (d, J ¼ 3.6 Hz, 1H), 7.69 (dd,
J ¼ 13.3, 2.5 Hz, 1H), 7.48 (d, J ¼ 8.1 Hz, 1H), 7.33e7.09 (m, 4H),
6.82e6.66 (m, 1H), 6.49e6.33 (m, 1H), 4.79 (s, 1H), 3.97e3.91 (m,
4H), 2.80 (s, 3H), 2.78 (s, 3H), 2.16 (br, 1H), 1.81 (br, 3H), 1.68e1.48
(R)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)
amino)piperidin-1-yl)-5-fluoro-2-methylphenyl)acrylamide
12

B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
(m, 2H), 1.48e1.18 (m, 1H).
immunoblotting as described above.
4.2. Cell lines
4.6. Antiproliferation assay
TC71 (male) cells were cultured in RPMI þ 10% FBS þ1% PSQ. STR
genotyping was previously performed to validate cell line identity
(Crompton et al., 2014). Jurkat (male), Kelly (male) and Kelly
CDK12C1039F were cultured in were cultured in RPMI media
(Gibco, #11875119) containing 10% fetal bovine serum (Gibco,
#10437028) and 1% Penicillin/Streptomycin (Thermo Fisher Sci-
entific, #10378016) at 37 ꢀC in 5% CO2 humidified air. Cell lines
were tested negative for mycoplasma contamination (Lonza,
#LT07-318).
Kelly wild type or CDK12C1039F cells were seeded at the density
of 5000 cells/well in 96-well plates and cultured overnight. Com-
pound 22a or THZ531 were then added to cells and incubated for
72 h. Cell viability was determined by using CellTiter-Glo (Promega,
#G7571) according to the manufacturer’s instructions. IC50 values
(defined as the concentration of compound needed to reduce cell
viability to 50% of the vehicle DMSO control) were estimated by
using GraphPad Prism 8.2.0 (GraphPad Software).
4.7. qRT-PCR
4.3. Immunoblotting and antibodies
1 mg of total RNA from cells was reverse transcribed with oli-
Cells were lysed in RIPA buffer (150 mM NaCl, 1.0% IGEPAL® CA-
630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0)
(Sigma, #R0278) with protease/phosphatase inhibitor cocktail
(Roche). The protein concentrations were measured by BCA anal-
ysis (Pierce). Equal amounts of protein were resolved by 4e12%
Tris-Base gels (Invitrogen), and then transferred to the Immuno-
Blot PVDF membrane (BioRad). Proteins were probed with appro-
priate primary antibodies and IRDye®800-labeled goat anti-rabbit
IgG and IRDye®800-labeled goat anti-mouse IgG (LI-COR) sec-
ondary antibodies. The membranes were detected on Odyssey CLx
system.
go(dT) and SuperScript IV Reverse Transcriptase (Life Technologies,
#18090050). RT-qPCR was performed using a SYBR Green (Life
Technologies, #4367659) and the 7500 Fast Real-Time PCR system
(Appiled Biosystems). All runs were accompanied by the internal
control Gapdh gene. The samples were run in triplicate and
normalized to GAPDH using a DD cycle threshold-based algorithm
to provide arbitrary units representing relative expression levels.
The primer sequences for specific genes are shown in Table S1.
4.8. Mass spectrometric analysis of recombinant CDK12 protein
Antibodies used in this study include anti-following proteins:
Cyclin H (Cell Signaling Technology, #2927S, 1:1000), Cyclin K
(Bethyl Laboratories, #A301-939A, 1:1000), p-RNA polII Ser2
(Sigma Aldrich, #04-1571-I, 1:1000), p-RNA polII Ser5 (Sigma
Aldrich, #04-1572-I, 1:1000), p-RNA polII Ser7 (Sigma Aldrich, #04-
1570-I, 1:1000), RNA PollI (Cell Signaling Technology, #14958S,
CDK12/Cyclin K complex (5 mg) was treated with DMSO or a 10-
fold molar excess of BSJ-01-175 for 1 h at room temperature and
analyzed by LC-MS using an HPLC system (Shimadzu, Marlborough,
MA) interfaced to an LTQ ion trap mass spectrometer (Thermo-
Fisher Scientific, San Jose, CA). After injection, proteins were
desalted for 4 min on column with 100% A, and then eluted with an
HPLC gradient (0e100% B in 1 min; A ¼ 0.2 M acetic acid in water;
B ¼ 0.2 M acetic acid in acetonitrile). The mass spectrometer was
programmed to acquire full scan mass spectra (m/z 300e2000) in
profile mode (spray voltage ¼ 4.5 kV). Mass spectra were decon-
voluted using MagTran software version 1.03b2 [24].
1:1000), and b-Actin (Cell Signaling Technology, #3700, 1:1000).
4.4. In vitro kinase assays
The commercial kinase assays from Invitrogen, ADAPTA or
Z’LYTE, were chosen to measure the biochemical inhibitory IC50
values for CDK2/CycA, CDK7/CycH, CDK9/CycT1, DNA-PK, RSK2,
JNK1, JNK2 and JNK3.
To identify the site of covalent modification, BSJ-01-175 treated
CDK12/Cyclin
K complex was reduced with 10 mM tris(2-
CDK12/13 kinase assay: Radioactive kinase activity measure-
carboxyethyl)phosphine for 10 min at room temperature, alky-
lated with 20 mM methyl methanethiosulfonate for 10 min at room
temperature, and digested with Glu-C protease (Promega, Madison,
WI) overnight at 37 ꢀC. Peptides were desalted using C18 (SOLA,
ThermoFisher Scientific, Madison, WI), dried by vacuum centrifu-
gation, reconstituted in 50% acetonitrile, 1% formic acid, 100 mM
ammonium acetate, and analyzed by CE-MS using a ZipChip CE-MS
instrument and autosampler (908 devices, Boston, MA) interfaced
to a QExactive HF mass spectrometer (ThermoFisher Scientific).
Peptides were resolved at 500 V/cm using an HR chip with a
background electrolyte consisting of 50% acetonitrile with 1% for-
mic acid. The mass spectrometer was operated in data dependent
mode, and subjected the 5 most abundant ions in each MS scan (m/
z 300e2000, 60 K resolution, 3E6 target, 100 ms max fill time) to
MS/MS (15 K resolution, 1E5 target, 100 ms max fill time). Dynamic
exclusion was enabled with a repeat count of 1 and an exclusion
duration of 5 s. Raw mass spectrometry data files were converted
to.mgf using multiplierz software [25] and searched against a
forward-reverse human refseq database using Mascot version 2.6.2.
Search parameters specified fixed methylthio modification of
cysteine, variable methionine oxidation, and variable BSJ-01-175
modification of cysteine. BSJ-01-175 modified spectra were exam-
ined, and figures prepared using mzStudio software. BSJ-01-175
specific product ions were assigned as described [26].
ments were performed using 0.2 mM [32P]-
g
-ATP containing
0.45 mCi [32P]/mL (PerkinElmer). CDK12/CycK or CDK13/CycK
complexes were pre-incubated at 0.2 M with varying concentra-
m
tions of compound for 5 min at 30 ꢀC, followed by addition of
substrate and an additional incubation for 15 min at 30 ꢀC. Re-
actions were stopped by EDTA, added to a final concentration of
50 mM. Mixtures were spotted onto filter sheets of Amersham
Protran nitrocellulose membrane (GE Healthcare) and washed
three times for 5 min with 0.75% (v/v) phosphoric acid. Radioac-
tivity was counted in a Beckman Liquid Scintillation Counter
(Beckman-Coulter) for 1 min.
4.5. Cellular target engagement assays
Cells were lysed in IP lysis buffer (25 mM Tris-HCl pH 7.4,
150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) (Thermo
Fisher Scientific, Cat#87788) containing protease/phosphatase in-
hibitor cocktail (Roche). The protein concentrations were measured
by BCA analysis (Pierce). Cell lysates were incubated with 1 mM
biotinylated THZ1 at 4 ꢀC overnight and incubated for 3 more hours
at room temperature. Lysates with probe were then incubated with
streptavidin beads (Thermo Fisher, #20349) for 2 h at 4 ꢀC. The
protein-probe complexes on the beads were then subjected to
13

B. Jiang, J. Jiang, I.H. Kaltheuner et al.
European Journal of Medicinal Chemistry 221 (2021) 113481
4.9. KiNativ profiling
Villigen, at 1.000 Å wavelength and 100 K temperature using the
PILATUS 6 M detector. The XDS package was used to process,
integrate and scale the data [28]. The structure was solved by
molecular replacement using the program PHASER [29] and the
coordinates of CDK12/CycK (4NST) as search model. The model was
refined by alternate cycles of refinement using PHENIX [30]. The
manual rebuilding was made using the graphical program COOT
[31]. Molecular diagrams were drawn using PyMOL (http://www.
pymol.org/).
Kelly cells were plated in fresh media in 15 cm plates and treated
for 6 h with DMSO or 1 mM BSJ-01-175. To harvest cells, plates were
washed 3 times with cold PBS, then collected by scraping into 4 mL
of cold PBS containing protease and phosphatase inhibitors. Cells
were pelleted by centrifugation at 1350g at 4 ꢀC for 5 min and
immediately frozen in liquid nitrogen. The frozen samples were
sent to ActivX Biosciences (La Jolla, CA) for the remainder of the
KiNativ profiling experiment.
The purified human CDK13/CycK complex was mixed with
THZ531 in molar ratios of 1:5 and incubated on ice for 30 min.
Initial micro-crystals were obtained using the hanging drop vapor
4.10. Recombinant proteins
diffusion technique at 293 K by mixing 1
ml protein-complex so-
Recombinant human kinases GST-CDK12 (714e1063)/GST-
lution with 1
m
l of the reservoir solution of JCSGþ screen containing
Cyclin
K
(1e267) and GST-CDK13 (694e1039)/GST-Cyclin
K
0.1 M Bis-Tris (pH 5.5), 25% PEG 3350 and 0.2 M MgCl2. These
conditions were used as template for further refinement to obtain
optimal crystals. Final crystallization conditions were set up using
the hanging drop vapor diffusion technique at 293 K using 24-well
plates (Molecular Dimension). Best crystals grew in about 10 days
(1e267) were co-expressed with CDK-Activating Kinase (CAK) from
S. cerevisiae in Sf9 insect cells using the MultiBacTurbo system [27].
Cells expressing the respective kinase complexes were harvested
by centrifugation and resuspended in lysis buffer (50 mM Hepes pH
7.4, 300 mM NaCl, 5% glycerol and 5 mM
bMe) and disrupted by
to a size of about approximately 200 ꢂ 40 ꢂ 30
m
m3 using 0.1 M
sonication. The lysate was cleared by centrifugation in a Beckman
Avanti J-26S XP centrifuge with a JA-25.50 rotor (20,000 r.p.m. for
45 min at 4 ꢀC) and applied to GSTrap FF columns (GE Healthcare)
MES (pH 6.8), 24% PEG 3350 and 0.2 M MgCl2. For cryo-protection,
crystals were transferred to a solution that contained 15% ethylene
glycol in mother-liquor. After 5e10 s soaking, crystals were flash-
frozen in liquid nitrogen.
€
equilibrated with Lysis buffer using an AKTA prime-plus chroma-
tography system (GE Healthcare). Following extensive washes with
10 column volumes (CV) of lysis buffer, the protein was eluted in
elution buffer (50 mM Hepes pH 7.4, 300 mM NaCl, 5% glycerol,
Diffraction data were collected at beamline ID23-1 at the Eu-
ropean Synchrotron Radiation Facility (ESRF), Grenoble, France, at
0.97242 Å wavelength and 100 K temperature using the PILATUS
6 M detector. Data processing, integration, scaling, molecular
replacement and model building was performed as described
above. The coordinates of CDK13/CycK (5EFQ) were used as search
model.
5 mM bMe and 10 mM Glutathione).
Proteins were further purified by size exclusion chromatography
(SEC) on a Superdex 200 PG column (GE Healthcare) equilibrated in
SEC buffer (20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol and
1 mM TCEP). Fractions of the main peak containing stoichiometric
kinase complexes as determined by SDSePAGE were pooled and
concentrated using Amicon filters (Millipore). Protein was ali-
quoted, snap frozen in liquid nitrogen and stored at ꢃ80 ꢀC.
4.12. Mouse liver microsomal stability
The MLM assays were previously reported and are commercially
available from Scripps Florida [32].
4.11. Crystallization and structure determination
4.13. In vivo pharmacokinetic studies
For crystallization of kinase complexes bound to small molecule
inhibitors, proteins were purified as described above. After SEC,
compounds were diluted in DMSO, added in a 1:1.5 ratio to the
pooled kinase complexes and incubated overnight at 4 ꢀC. Excess
free compound was removed by buffer exchange using PD10
desalting column (GE Healthcare). Proteins were concentrated,
aliquoted, snap frozen and stored at ꢃ80 ꢀC.
Male Swiss albino mice were dosed with BSJ-01-175 solution
formulation (intravenous, 5% NMP, 5% Solutol HS-15, 90% Normal
saline, dose 3 mg/kg) or (oral, 5% NMP, 5% Solutol HS-15, 90%
Normal saline, dose 10 mg/kg). Blood samples were collected at
Pre-dose, 0.08, 0.25, 0.5, 1, 2, 4, 8 and 24 h (IV and IP) and Pre-dose,
0.25, 0.5, 1, 2, 4, 6, 8 and 24 h (PO). The blood samples were
collected from sets of three mice at each time point in labeled
microcentrifuge tubes containing K2EDTA as an anticoagulant.
Plasma samples were separated by centrifugation and stored
below ꢃ70 ꢀC until bioanalysis. All samples were processed for
analysis by precipitation using acetonitrile and analyzed with a
partially validated LC/MS/MS method (LLOQ - 1.22 ng/mL for IV and
PO, LLOQ e 5.02 ng/mL for IP). Pharmacokinetic parameters were
calculated using the noncompartmental analysis tool of WinNonlin
Enterprise software (Version 6.3).
The CDK12/CycK protein complex, was stored in a buffer of
20 mM Hepes, pH 7.5, 150 mM NaCl and 1 mM TCEP. Initial needle
like crystals were obtained using the hanging drop vapor diffusion
technique at 293 K by using the JCSGþ screen (Molecular Dimen-
sion). These crystals grew as clusters that showed high mosaicity
while testing on the diffractometer and diffracted to about 7 Ang
resolution. To optimize crystal growth, several techniques were
tried, e.g., micro-seeding, streak-seeding and macro-seeding, out of
which micro-seeding helped in growing big single crystals by
mixing 1
ml protein solution with 1 ml of the reservoir solution
containing 0.1 M MES, pH 6.0, 30% PEG-mixture (medium weight
pegs), 0.3 M NDSB and 0.2 M MgCl2. Crystallization drops were set
up by mixing protein sample and seed stock dilutions in a 1:1 ratio
and crystals were grown using the hanging drop vapor diffusion
technique at 293 K. Best crystals grew within one week to a size of
4.14. In vivo efficacy studies
In vivo studies were performed at the Dana-Farber Cancer
Institute, and all animal protocols were approved by the Dana-
Farber Cancer Institute Animal Care and Use Committee. Nude
mice were maintained according to institutional guidelines.
Three million TC71 Ewing sarcoma cells were implanted sub-
cutaneously into the right flank of 7e8 weeks old female nude
mice. Following engraftment, mice with palpable tumors were
divided into two groups and treated with 10 mg/kg BSJ-01-175
about approximately 150 ꢂ 40 ꢂ 30
m
m [3] using a 10ꢃ2 dilution of
the seed stock. We harvested many rod-shaped crystals by trans-
ferring them into 15% (v/v) ethylene glycol in mother-liquor and
flash freezing in liquid nitrogen.
Diffraction data were collected at the Swiss Light Source,
14

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European Journal of Medicinal Chemistry 221 (2021) 113481
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Author contributions
Conceptualization: N.G., T.Z. M.G.; Chemistry: B.J., F.F.; Biology:
J.J., I. K., A. I., K.A., B. S. N.K., K.S. S.D., A.G.; Mass spectrum labeling:
S.F., J.M.; Writing: T.Z., M.G., B.J. J.J, N.G.; The manuscript was
written through contributions of all authors. All authors have given
approval to the final version of the manuscript. zThese authors
contributed equally.
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Declaration of competing interest
The authors declare the following financial interests/personal
relationships which may be considered as potential competing
interests: N.S.G. is a founder, science advisory board member (SAB)
and equity holder in Gatekeeper, Syros, Petra, C4, B2S, Aduro and
Soltego. The Gray lab receives or has received research funding
from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi,
Her2llc, Deerfield. Ephiphanes and Sanofi. N.S.G., T. Z. and B. J. are
named inventors on patent applications covering compounds
described in this manuscript.
Acknowledgment
We thank Jim Sun at NMR facility of Dana-Farber cancer insti-
tute for his assistance on NMR data collection and Milka Kostic for
editorial suggestions on the manuscript. We would like to thank the
beamline scientists at Swiss Light Source (Villigen, Switzerland)
and the European Synchrotron Radiation Facility (Grenoble, France)
for access and support. Milka Kostic is greatly acknowledged for the
editing and proof reading. M.G. is supported by the Deutsche For-
schungsgemeinschaft (GE 976/9-2) under Germany’s Excellence
Strategy e EXC2151e390873048.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113481.
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