Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine-based protein kinase D inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112638

The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitors with structural variety at position 1 were synthesized and evaluated for biological activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC₅₀ values in the range of 94–108 nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD (IC₅₀ = 17–35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.

Full text of DOI:10.1016/j.ejmech.2020.112638

Journal Pre-proof
Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine-based
protein kinase D inhibitors
Philippe Gilles, Rudra S. Kashyap, Maria João Freitas, Sam Ceusters, Koen Van
Asch, Anke Janssens, Steven De Jonghe, Leentje Persoons, Mathias Cobbaut, Dirk
Daelemans, Johan Van Lint, Arnout R.D. Voet, Wim M. De Borggraeve
PII:
S0223-5234(20)30610-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.112638
EJMECH 112638
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2020
Revised Date: 12 May 2020
Accepted Date: 1 July 2020
Please cite this article as: P. Gilles, R.S. Kashyap, M.J. Freitas, S. Ceusters, K. Van Asch, A. Janssens,
S. De Jonghe, L. Persoons, M. Cobbaut, D. Daelemans, J. Van Lint, A.R.D. Voet, W.M. De Borggraeve,
Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine-based protein kinase D
inhibitors, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112638.
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Research paper
Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine-
based protein kinase D inhibitors
Philippe Gilles[a], Rudra S. Kashyap[c], Maria João Freitas[c], Sam Ceusters[a], Koen Van Asch[b], Anke Janssens[b],
Steven De Jonghe[d], Leentje Persoons[d], Mathias Cobbaut[c][e], Dirk Daelemans[d], Johan Van Lint[c], Arnout R.D.
Voet[b] and Wim M. De Borggraeve*[a]
a KU Leuven, Department of Chemistry, Molecular Design and Synthesis, Celestijnenlaan 200F - box 2404, B-3001 Leuven, Belgium
b KU Leuven, Department of Chemistry, Biochemistry, Molecular and Structural Biology, Celestijnenlaan 200G - box 2403, B-3001 Leuven, Belgium
c KU Leuven, Department of Cellular and Molecular Medicine, Laboratory of Protein Phosphorylation and Proteomics, O&N I Herestraat 49 - box 901,
B-3000 Leuven, Belgium
d KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Laboratory of Virology and
Chemotherapy, Herestraat 49 - box 1043, B-3000 Leuven, Belgium
e The Francis Crick Institute, Protein Phosphorylation Laboratory, 1 Midland Road, London NW1 1AT, UK (present address)
Highlights
•
•
•
•
A novel series of pyrazolo[3,4-d]pyrimidine based PKD inhibitors with structural variety at position 1 was synthesized.
17m showed the most potent pan-PKD inhibitory activity with IC50 values ranging from 17 to 35 nM.
17m and 3-IN-PP1 potently blocked PKD-dependent cortactin phosphorylation in PANC-1 cells.
3-IN-PP1 exhibited low micromolar cytotoxic activity against various cancer cell lines.
A R T I C L E I N F O
A B S T R A C T
Keywords:
The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly
reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents
has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD
inhibitors with structural variety at position 1 were synthesized and evaluated for biological
activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC50 values in the range of 94-108
nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD
(IC50 = 17-35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited
PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-
proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines
demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.
Protein Kinase D inhibitor
anticancer agents
pyrazolo[3,4-d]pyrimidines
3-IN-PP1
1-NM-PP1
1. Introduction
PKD1 has tumor-suppressive roles in prostate, breast and
colorectal cancer and is able to reduce EMT via the
phosphorylation of Snail.[7-9] Furthermore, PKD1 expression is
significantly downregulated in invasive breast cancer cells due to
epigenetic hypermethylation of the PRKD1 gene promoter,
hence further strengthening its potential role in repressing tumor
growth.[10] In addition, global knockout (KO) of PKD1 in mice
results in embryonic lethality, while KO PKD2 mice are viable,
thereby signifying PKD1’s vital role.[11, 12] On the other hand,
PKD2 is markedly more linked to processes that mediate cancer
growth. For example, it is a crucial regulator of angiogenesis
through endothelial cell proliferation and migration.[13]
Furthermore, depletion of PKD2 by inhibition of HSP90 is
associated with increased cell death of several human cancer
cell lines.[14] Recent studies show that PKD2 is the major
isoform in human colon cancer cells and PKD2 silencing studies
reveal significant downregulation of AKT and ERK, kinases that
are both involved in cell proliferation.[15] In addition,
downregulation of PKD2 prevents glioblastoma tumor growth,
presumably via suppression of cyclin D1.[16]
Protein kinase D (PKD) is a serine/threonine kinase that belongs
to the Ca2+/calmodulin-dependent kinase group and comprises
three isoforms that share high sequence identity: PKD1, PKD2
and PKD3.[1] These isoforms all contain a conserved catalytic
domain, a regulatory pleckstrin homology (PH) domain and two
N-terminal cysteine-rich, zinc finger-like motifs. Additionally, PKD
1 and 2 have a PDZ-binding motif at the very end of their C-
terminus.[2, 3]
The PKD family is repeatedly associated with multiple cancers
such as prostate, breast, colorectal, pancreatic, gastric cancer
and glioblastoma.[4] Indeed, PKD activity is related to a plethora
of physiological conditions and dysregulated PKD function is
linked to multiple oncogenic processes including cell growth and
proliferation, cell survival, motility, apoptosis inhibition,
angiogenesis and epidermal to mesenchymal transition (EMT).[5,
6] The existence of PKD isozymes introduces an additional layer
of complexity in the cell signaling of these malignancies, as
these isoforms can either promote or reduce tumor formation.[3]

Figure 1. Reported PKD inhibitors
Remarkably, PKD2 silencing in pancreatic cancer cells
investigated as potential treatment of cardiac hypertrophy.[28,
orthotopically implanted in mice significantly impairs tumor
angiogenesis and growth, thus indicating its crucial role.[17] In
comparison with PKD1 and PKD2, PKD3 is studied to a much
lesser extent. Nevertheless, it has been categorized as tumor
promoting.[18, 19] Overall, it is becoming more and more clear
that the PKD family members have isozyme-specific roles within
the cell. However, a complete understanding of their exact roles
in both physiological and pathological conditions remains to be
uncovered.
29]
We and others have been investigating derivatives of
pyrazolo[3,4-d]pyrimidine 1-NA-PP1 as PKD inhibitors.[30, 31]
1-NA-PP1 was originally designed as a specific inhibitor of
analog sensitive v-src protein kinases.[32, 33] These kinases
were generated via site-directed mutagenesis of their
gatekeeper residue, thereby creating a unique back pocket able
to accommodate the naphthalene moiety. However, 1-NA-PP1
and its close derivative 1-NM-PP1 do have off-targets against a
number of non-mutated protein kinases, such as wild-type
PKD.[34] Elaborating on these findings, we previously
discovered that replacement of the naphthalene moiety of 1-NA-
Despite the opposing roles of the PKD isozymes in cancer, pan-
PKD inhibitors do show antitumoral activity in various cancer
models,
both
in vitro and
in
vivo
(Figure
1).
Benzoxoloazepinolone CID755673 and its derivatives are potent, PP1 with an indole substituent (3-IN-PP1) further increased the
ATP non-competitive, PKD inhibitors that reduce cell
proliferation, cell migration and invasion in several prostate
cancer cell lines (such as LNCaP and PC3).[20, 21] The
selectivity of this compound, however, remains unclear as
PKD1-independent biological effects were observed and kinase
off-targets were identified.[22, 23] The same group also
discovered pteridine analogue SD-208 as a potent PKD inhibitor.
Treatment of PC3 xenograft mice with SD-208 reduced tumor
growth and enhanced apoptosis.[24] In addition, SD-208 also
reduced prostate cancer cell invasion, which might be related to
off-target inhibitory activity towards TGF-βRI. CRT0066101 is a
potent pan-PKD inhibitor exhibiting remarkable in vivo anti-
cancer potency. Treatment of both hetero- and orthotopic
PANC-1 xenograft mice with CRT0066101 leads to a significant
tumor growth reduction.[25] In addition, CRT0066101 shows
also promising activity in various colorectal cancer models
including the HCT-116 cell lines and HCT-116 mice
xenografts.[15] Moreover, CRT0066101 significantly reduces
tumor growth of both estrogen receptor negative as well as triple
negative breast cancer.[26, 27] Although the specificity of
CRT0066101 against PKD was confirmed in a panel of more
than 90 kinases,[25] doubts about the selectivity were raised.
inhibitory potency against PKD by tenfold.[31] Since no X-ray
crystallographic structures of the catalytic domain of PKD are
available, we rationalized our findings with an in-house
generated homology model of PKD. We speculated that the
increased PKD inhibition was due to an alternative binding mode
which has also been observed for other pyrazolo[3,4-
d]pyrimidine based transferase inhibitors. These novel insights
encouraged us to further explore the structure-activity
relationship (SAR) of the pyrazolo[3,4-d]pyrimidines as PKD
inhibitors, focusing on the largely ignored 1-position.
Furthermore, in the course of this work, off-target PKD activity
was observed in a chemoproteomic profiling study of BKI1369, a
pyrazolopyrimidine derived bumped kinase inhibitor (BKI)
designed to target parasitic kinase Toxoplasma gondii calcium-
dependent protein kinase 1 (TgCDPK1).[35] This pyrazolo[3,4-
d]pyrimidine contains an ethoxyquinolyl substituent in the 3-
position and a methylene piperidine substituent in the 1-position.
In the present study, we report the design and synthesis of novel
3-IN-PP1 analogues and their biochemical evaluation as PKD
inhibitors. The most promising PKD inhibitors were evaluated for
in cellulo inhibition of PKD and their effect on cell proliferation in
a pancreatic adenocarcinoma cell line (PANC-1). Finally, a
selection of compounds was screened for their cytotoxic activity
in a panel of eight different cancer cell lines.
Besides their anti-cancer applications, PKD inhibitors based on
other chemotypes have been profiled in other disease areas.
Examples
include
the
3,5-diarylisoxazoles
and
2,6-
naphthyridines that both potently inhibit PKD and have been
2

2. Results and Discussion
2.1 Chemistry
The pyrazolo[3,4-d]pyrimidines were synthesized according to
protocols reported by Todorovic and Bishop.[32, 36]
Conventional methods to functionalize the 3-position of this
pyrazolopyrimidine scaffold rely on either palladium-catalyzed
cross-coupling reactions or introduction of the side chain prior to
the heterocycle construction, if sp2-sp3 bond formation is
required. The synthesis of methylene bridged 3-IN-PP1 is shown
in Scheme 1. N-tosyl-protected 2-(indol-3-yl)acetic acid 4 was
prepared from commercially available indole-3-acetic acid 1.
Subsequent condensation with malononitrile, followed by O-
methylation with dimethyl sulfate afforded compound 6. A first
cyclization of 6 with tert-butyl hydrazine allowed to construct the
pyrazole moiety yielding intermediate 7 and a second ring
closure with formamide formed the pyrimidine ring furnishing
protected pyrazolo[3,4-d]pyrimidine 8. Finally, deprotection of
the indole moiety under basic conditions yielded methylene-
linked 3-IN-PP1 9.
As part of the SAR study at the 3-position, the synthesis of
functionalized indolyl derivatives was envisioned. Previously, 3-
IN-PP1 was synthesized via a Suzuki-coupling between 3-
bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine and 1-
(phenylsulfonyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1H-indole.[31] The latter was prepared from indole in three steps
including protection of the indole nitrogen atom and selective
bromination at position 3, followed by borylation. After the cross-
coupling reaction, a final deprotection step was performed. This
laborious route prompted us to explore alternative synthetic
procedures. Inspired by the Smith group’s work on selective
borylation, 3-borylated 1H-indole 11a was prepared in one step
Scheme 2. Synthesis of 3-functionalized 3-IN-PP1 derivatives. Reagents and
conditions: (a) i. HBpin, Et3N, THF, 80 °C, 20 min; ii. [Ir(OMe)(COD)] 2, 3,4,7,8-
tetramethyl-1,10-phenanthroline, 80 °C, 4 h; (b) [I r(OMe)(COD)]2, 3,4,7,8-
tetramethyl-1,10-phenanthroline, 80 °C, 4 h; (c) Bo ronic acid pinacol ester,
K2CO3, Pd(PPh3)4, dioxane/water (4/1), 150 ºC, Microwave (MW), 15 min.; (d)
MeI, KOH, DMF, rt.; (e) Pd(dba)2, PCy3, B2Pin2, KOAc, dioxane, 80 °C,
overnight.
from
indole
using
pinacolborane
(HBpin)
and
a
I
I
R2
NH2
NH2
NH2
phenantroline/iridium catalytic system.[37] Subsequently, this
unprotected borylated indole 11a was successfully coupled to 3-
bromo-pyrazolo[3,4-d]pyrimidin-4-amine 10 (synthesized from
a or b
c
N
N
N
N
N
N
N
N
N
N
N
N
H
R1
R1
(ethoxymethylene)malononitrile according to
a
literature
2 = 3ꢀindolyl
2 = 1ꢀnaphthyl
14
16
17,
18,
R
R
procedure), yielding compound 12a (3-IN-PP1). Using a similar
methodology, a set of functionalized indoles, a pyrrole and a
pyrazole were borylated furnishing intermediates 11b-11h
(Scheme 2). Subsequent coupling with 3-bromo-pyrazolo[3,4-
d]pyrimidine 10 yielded compounds 12b-h.
R1 substituents
O
O
O
3-IN-PP1
16a,
17a,
16b,
17b,
16c,
17c,
16d,
17d,
56%
69%
40%
49%
21%
65%
28%
55%
OH
OH
CN
a, b, c
d, e
NC
N
H
1
N
Ts
N
Ts
4
6
Ph
f
R
Ts
16e,
17e,
16f,
17f,
16g,
17g,
16h,
17h,
16i,
17i,
25%
84%
47%
44%
54%
72%
48%
34%
35%
58%
N
N
NH2
g
NC
N
N
N
N
N
N
H2N
N
Ac
N
CBz
O
HN
HO
8
9
7
, R = Ts
, R = H
h
16j,
17j,
16k,
17k,
16l,
17l,
18l,
-
16n,
17n,
18n,
28%
47%
15%
71%
44%
68%
79%
50%
31%
43%
d
17m,
18m,
83%
72%
Scheme 1. Synthesis of the methylene bridged 3-IN-PP1 derivative 9.
Reagents and conditions: (a) MeOH, PTSA, reflux, overnight; (b) TsCl, Et3N,
DCM, rt; (c) LiOH, MeOH, rt, overnight; (d) i. SOCl2, reflux; ii. malononitril, NaH,
THF,
0 °C; (e) Me 2SO2, NaHCO3, dioxane/H2O; (f) tert-butyl hydrazine
Scheme 3. Synthesis of 1-functionalized pyrazolo[3,4-d]pyrimidines. Reagents
and conditions: (a) Alkyl mesylate or halide, Cs2CO3, DMF, 80 ºC, 4 h; (b)
Alcohol, DIAD, PPh3, THF, 0 ºC, 1 h; (c) Boronic acid pinacol ester, K2CO3,
Pd(PPh3)4, dioxane/water (4/1), 150 ºC, MW, 15 min.; (d) Pd/C, THF, 50 ºC, 8
h.
hydrochloride, Et3N, EtOH, 2 h; (g) formamide, reflux; (h) NaOH, EtOH, reflux,
1 h.
3

Quinoline derivative 11i was synthesized using a palladium-
catalyzed borylation of 5-bromoquinoline and was subsequently
coupled similarly to 3-bromo-pyrazolo[3,4-d]pyrimidine 10 to
afford 12i. Finally, using iodomethane under alkaline conditions,
the nitrogen of the indolyl moiety of 3-IN-PP1 (12a) was
methylated affording compound 13.
pyrazolo[3,4-d]pyrimidines with bulkier substituents at position 1
was evaluated for PKD2 inhibition (Table 2). The introduction of
a single methylene linker between the pyrazolo[3,4-d]pyrimidine
scaffold and the tert-butyl substituent gave the neopentyl
derivative 17a that was slightly less potent than 3-IN-PP1 (25%
versus 15% residual PKD2 activity). The insertion of an ethylene
linker (affording neohexyl analogue 17b) led to a complete loss
of PKD2 inhibition. The presence of larger cycloaliphatic
substituents (17c-e) or aromatic moieties (17f-i) afforded
compounds with strongly reduced PKD2 inhibitory potency
(between 43-100% residual PKD2 activity) in comparison to 3-
IN-PP1. As the incorporation of these bulky hydrophobic
substituents was detrimental for PKD inhibition, the influence of
polar groups was assessed. Notably, the pyranyl analogue 17j
and the N-acetylpiperidinyl derivative 17k are more potent PKD2
inhibitors when compared to the corresponding cyclohexyl
analogue 17d, indicating that more polar substituents are better
accommodated. Based on the potent off-target PKD inhibition of
For the synthesis of pyrazolo[3,4-d]pyrimidine analogues with
structural variation at position 1, we were inspired by the work of
Todorovic.[36] Iodination of 1H-pyrazolo[3,4-d]pyrimidin-4-amine
using NIS is followed by alkylation and cross-coupling. The
alkylation is carried out either via SN2 reactions, using an alkyl
mesylate or alkyl halide, or a Mitsunobu reaction. A diverse set
of hydrophobic and hydrophilic substituents was introduced at
the 1-position (16a-l, 16n) followed by a Suzuki coupling with 3-
boryl-1H-indole affording the desired 3-IN-PP1 analogues 17a-l
and 17n (Scheme 3). In order to have a methylene piperidinyl
substituent at position 1, -Boc was initially selected as protecting
group for the nitrogen atom of the piperidine moiety.
Unfortunately, multiple efforts to cleave the Boc-group under
acidic conditions failed to yield pure 17m. Therefore, we opted
for a -Cbz protecting group and its reductive deprotection
yielded the corresponding free amine 17m in excellent yield. In
addition, two analogues containing a naphthyl substituent
instead of the indolyl moiety (18m and 18n) were prepared. BKI-
1369 was synthesized following a slightly modified version of the
original procedure reported by Vidadala et al. (see Supporting
Information).[38]
BKI1369 (vide supra),
a methyl piperidinyl and a 2,2-
dimethylbutan-1-ol group at position 1 were both evaluated,
while keeping the indole moiety at position 3. Remarkably, both
17m and 17n displayed excellent PKD2 inhibition with almost no
remaining kinase activity when tested at a concentration of 1 µM.
The naphthyl derivatives 18m and 18n are both less potent
PKD2 inhibitors when compared to their indole-substituted
congeners 17m and 17n, respectively. This is in agreement with
what previously has been observed for 1-NA-PP1 and 3-IN-
PP1.[31]
2.2 PKD inhibition
Table 1. PKD2 inhibition of 3-substituted pyrazolo[3,4-d]pyrimidines
All compounds were evaluated for PKD inhibition against full-
length PKD using Promega’s ADP-Glo kinase activity assay and
syntide-2 as a substrate. The known PKD inhibitors 1-NM-PP1
and 3-IN-PP1 were included as reference compounds. In a first
round of screening, all compounds were evaluated at a single
concentration of 1 µM and the data were expressed as the
percentage of residual PKD2 activity. The first series
encompassed a focused library with structural variation at
position 3 of the pyrazolo[3,4-d]pyrimidine scaffold (Table 1).
Previously, we demonstrated that 1-NM-PP1 was a more potent
PKD2 inhibitor compared to 1-NA-PP1. Introducing a similar
methylene bridge in 3-IN-PP1 (12a) yielded compound 9 that
unfortunately was less potent than 3-IN-PP1 against PKD2 (48%
versus 15% residual activity). The methylated 3-IN-PP1
analogue 13 showed significantly reduced PKD2 inhibition (53%
residual activity), when compared to 3-IN-PP1 (15% residual
activity), indicating the involvement of this nitrogen atom in
hydrogen bonding. The introduction of subtle decorations on the
indole moiety (e.g. chlorine, fluorine and methoxy substituents)
afforded compounds 12b-12e that were completely devoid of
PKD2 inhibition (72-97% residual activity). In addition, pyrrole
12f, pyrazole 12g and quinoline 12i lacked activity against PKD2.
Furthermore, replacing the indole substituent of 3-IN-PP1 for an
azaindole group resulted in a compound (12h) that is only
slightly less active than 3-IN-PP1. Together, these observations
further demonstrate the very narrow space for improvements at
the 3-position which is in complete agreement with Wipf’s
findings and our previous work.[30, 31]
CPD
R1
RA (%)a
CPD
12d
R1
RA (%)a
1-NM-PP1
20 ± 1
72 ± 5
3-IN-PP1
15 ± 1
48 ± 2
53 ± 10
12e
12f
97 ± 7
68 ± 26
94 ± 9
9
13
12g
12b
12c
96 ± 7
82 ± 2
12h
12i
25 ± 3
82 ± 1
a
Determined using ADP-Glo™ kinase assay; CPD = Compound; RA
residual activity at 1 µM.
=
In contrast, the 1-position of the pyrazolo[3,4-d]pyrimidine
scaffold is significantly understudied since only methyl and tert-
butyl substituents have been extensively evaluated. A library of
4

Table 2. PKD2 inhibition of 1-substituted pyrazolo[3,4-d]pyrimidines
CPD
17a
R1
R2
RA (%)a
CPD
17f
R1
R2
RA (%)a
CPD
17k
R1
R2
RA (%)a
25 ± 1
68 ± 4
44 ± 1
17b
17c
17d
17e
93 ± 1
43 ± 3
92 ± 8
100
17g
17h
17i
69 ± 7
100
17m
18m
17n
18n
5 ± 2
19 ± 3
4 ± 1
96 ± 4
46 ± 2
17j
27 ± 5
a Determined using ADP-Glo™ kinase assay; CPD = Compound; RA = residual activity at 1 µM.
The most promising PKD2 inhibitors were subjected to dose-
response curves in order to determine the half maximal
inhibitory concentration (IC50) against PKD1, 2 and 3 (Table 3
and Figure S1 in Supporting Information). To have a direct
comparison with known PKD inhibitors, CRT0066101 and 1-NM-
PP1 were included as a benchmark in this study. The observed
trends between 3-IN-PP1 and these references is in agreement
with literature data.[25, 31] Furthermore, the off-target PKD
activity of BKI1369 relative to the current state-of-the-art was
also investigated. BKI-1369 is slightly less active against PKD1
in comparison to the other two isoforms. This difference was
also reported in the above-mentioned chemoproteomic profiling
study.[35]
The replacement of the tert-butyl group of 3-IN-PP1 by a 4-
methylpiperidine moiety afforded compound 17m, which is 3- to
5-fold more potent against PKD than 3-IN-PP1. With IC50 values
of 35, 35 and 17 nM for PKD1, 2 and 3 respectively, compound
17m is the most potent pyrazolo[3,4-d]pyrimidine based PKD
inhibitor reported to date. Compound 17n, bearing a 2,2-
dimethylpropan-3-ol moiety, showed only slightly higher IC50
values when compared to compound 17m, which further
confirms the crucial role of a hydrogen bond donor moiety in the
substituent at position 1. As expected, the naphthalene
analogue 18m was less potent; fully correlating with the
observed trends between 3-IN-PP1 and 1-NA-PP1.[31] As the
presence of an indolyl group at position 3 was optimal for PKD
inhibition, we also investigated subtle structural modifications of
this moiety. Interestingly, the N-methylated 3-IN-PP1 analogue
(13) is a 30- to 40-fold less potent PKD inhibitor when compared
to 3-IN-PP1, whereas aza-indole analogue 12h displays only a
3- to 4-fold drop in PKD inhibitory activity.
Table 3. IC50 values expressed in nM for a subset of the synthesized
pyrazolo[3,4-d]pyrimidine analogues, CRT0066101, 1-NM-PP1 and BKI1369
IC50 PKD1
(nM) a
IC50 PKD2
(nM) a
IC50 PKD3
(nM) a
Compound
2.3 Molecular docking studies.
CRT0066101
1-NM-PP1
BKI1369
3-IN-PP1
17m
16 ± 2
9 ± 2
6 ± 2
We further investigated the binding mode of this class of
compounds in the ATP binding site of PKD. According to our
previously developed model, an alternative binding mode was
hypothesized that better correlated with 3-IN-PP1’s increased
activity.[31] This alternate binding mode is also observed in X-
ray crystallographic studies of APH(3')-Ia, an aminoglycoside
phosphotransferase of Acinetobacter baumannii with an active
site fold similar to protein kinases (PDB ID: 4GKI, 4GKH). With
our expanded activity dataset at hand, including the library of 1-
functionalized 3-IN-PP1 derivatives, we further evaluated this
binding mode hypothesis. Since no X-ray structures of PKD’s
catalytic domain are publicly available, a homology model was
constructed.
1013 ± 200
183 ± 24
108 ± 20
35 ± 10
786 ± 40
48 ± 2
616 ± 110
43 ± 6
94 ± 4
108 ± 33
17 ± 8
35 ± 5
18m
64 ± 9
55 ± 9
78 ± 2
17n
50 ± 12
43 ± 5
76 ± 4
12h
343 ± 98
3319 ± 260
282 ± 25
3286 ± 270
456 ± 27
4055 ± 680
13
a Determined using ADP-Glo™ kinase assay
5

dramatically eroded to 0.56 when evaluating the alternative
binding mode (red curve). Furthermore, the highest docking
score only moderately correlated with the activity data (AUC =
0.69), presumably due to docking power related limitations. As a
control, eliminating the bias of the ligand on the homology model,
we also created
a
structural model relying on the
aforementioned aminoglycoside phosphotransferase APH(3')-Ia
(PDB ID: 4GKI). This transferase shares a similar fold and has
1-NM-PP1 bound in the alternative, inverted binding mode.
Interestingly, also the classic binding mode was preferred over
the alternative in this second homology model, thus ruling out
binding mode preferences imposed during the homology
modelling (Figure S3 in Supporting Information). Taken together,
these results indicate that the activity profiles of our dataset
generally correlate better with the classical binding mode rather
than the earlier hypothesized alternative mode. Despite this
observed preference, it should be noted that binding modes can
be highly compound dependent and only co-crystals can reveal
more case-specific details. Figure 2B displays the postulated
binding mode for compound 17m, which is similar to the one
observed for inhibitor RM-1-95 in the crystal structure of
TgCDPK1.[39] The increased potency of 17m can be explained
by two hydrogen bonds formed by the indole and piperidine
nitrogen atoms. Similar interactions are observed for the nearly
equally active compound 17n, also containing this dual
hydrogen bond pattern. In our SAR study, both of these
interactions were found to be crucial for potent PKD inhibition.
The methylated analogue 13 was significantly less active while
the importance of the hydrogen bond donor on the substituent at
position 1 was also demonstrated. Furthermore, the orientation
of the indole moiety resembles that of a recently reported
pyrazolo[3,4-d]pyrimidine-based covalent EGFR kinase inhibitor
(PDB ID: 5J9Z).[40]
Figure 2. (A) ROC Analysis of the binding mode of pyrazolo[3,4-d]pyrimidine
based PKD inhibitors. Activity cut-off 50% inhibition. Blue: ROC based on
score of the classical binding mode. Green: ROC based on the highest scored
retrieved. Red: ROC obtained for the alternative binding mode. (B) Predicted
binding mode of compound 17m after docking into the homology model of
PKD (PDB ID 3MWU as template).
As discussed above, some bumped kinase inhibitors of
TgCDPK1 display cross-reactivity against PKD, thus suggesting
that both kinases might share a similar environment in the active
site. Therefore, the crystallographic X-ray structure of TgCDPK1
in complex with RM-1-95, in classic binding mode, was chosen
as a template (PDB ID: 3MWU). Our dataset containing 66
compounds (Table S1 in Supporting Information) was docked
into the ATP-pocket using GOLD. Among these 66 docked
ligands, 33 retrieved the classical binding mode as the primarily
scored pose. To assess the fitness of each binding mode to the
dataset, receiver operating characteristic (ROC) curves were
constructed for three scores: the highest retrieved score, the
classical binding mode score and the alternative binding mode
score (Figure 2A). The curve designated by the black dashed
line (X=Y) displays random behavior (AUC = 0.50). When
considering the classic binding mode an AUC of 0.83 is obtained,
thus showing good fitness. On the other hand, the AUC
Figure 3. The effect of 1-NM-PP1, 3-IN-PP1 and 17m on PKD mediated phosphorylation of cortactin at S298. PANC-1 cells co-transfected with FLAG-PRKD1 or
FLAG-PRKD2 and myc-CTTN were pretreated with the indicated compounds (20 µM) for 1 hour prior to stimulation with 250 nM phorbol 12,13-dibutyrate (PDBu)
for 15 min. Phosphorylation of cortactin was followed via western blot. Quantification of pS298/Myc-Cort signals of three independent experiments is shown in the
graph. Graph bars and error bars represent the normalized mean and calculated standard error of the mean (SEM), respectively. A one-way ANOVA was
performed to determine the statistical significance with the control conditions (DMSO).
6

2.4 PKD inhibition in PANC-1 cells reduces cortactin
phosphorylation
2.6 antitumoral screening reveals 3-IN-PP1 as
spectrum anticancer agent.
a broad-
Cellular PKD activity was monitored by analyzing the levels of
phosphorylation at S298 of cortactin (CTTN), a known substrate
of PKD.[41] Both PKD 1 and 2 were investigated for PKD-
dependent cortactin phosphorylation in PANC-1 cells and
CRT0066101 was used as a positive control. After stimulating
the cells with phorbol 12,13-dibutyrate (PDBu), all tested
inhibitors significantly decreased the levels of pS298 CTTN in
comparison to the control (Figure 3). Hence, all compounds are
clearly inhibiting PKD in this cellular environment. Although 17m
is a more potent PKD inhibitor than 3-IN-PP1 in the biochemical
assay, it appears that it is less capable of inhibiting PKD-
mediated CTTN phosphorylation in a cellular environment.
When PDBu stimulated pancreatic cancer cells (PANC-1) were
treated with 17m, residual CTTN phosphorylation was noticed.
This was also observed for the less potent inhibitor 1-NM-PP1.
In contrast, treatment with 3-IN-PP1 resulted in minimal residual
PKD activity. As expected for pan-PKD inhibitors, no differences
were observed between inhibition of PKD 1 and 2. Furthermore,
the biochemical inhibitory activity of 3-IN-PP1 against CAMKIIα
and PKCδ, two related kinases, was investigated. No notable
inhibition was observed at compound concentrations of 1 and 10
µM (Figure S2 in Supporting Information).
A selection of the newly synthesized PKD inhibitors (compounds
13, 17n and 17m), as well as a number of known PKD inhibitors
(CRT0066101, 1-NM-PP1, 3-IN-PP1 and BKI1369) were
evaluated for their antitumoral activity (Table 4) in a panel of
eight different cancer cell lines: LN-229 (glioblastoma), Capan-1
(pancreatic adenocarcinoma), HCT-116 (colorectal carcinoma),
NCI-H460 (lung carcinoma), DND-41 (acute lymphoblastic
leukemia), HL-60 (acute myeloid leukemia), K-562 (chronic
myeloid leukemia) and Z-138 (non-Hodgkin lymphoma).
Inhibition of cell proliferation was monitored in real-time over a
period of three days using an IncuCyte live-cell automated
imager determining cellular confluence as a measure of cellular
viability. Overall, CRT0066101 was the most potent
antiproliferative agent with IC50 values in the range of 0.6-2.2 µM
against the various tumor cell lines. Among the pyrazolo[3,4-
d]pyrimidine based PKD inhibitors, 3-IN-PP1 was the most
potent cytotoxic agent with IC50 values in the low µM range for
most cancer cell lines, with the exception for the glioblastoma
cell line for which the activity was clearly less (IC50 = 39.2 µM).
Overall, in most tumor cell lines (except for LN-229), 3-IN-PP1
was only slightly less active than the reference PKD inhibitor
CRT0066101. On the other hand, 3-IN-PP1 is much more potent
as antitumoral agent than the reference pyrazolo[3,4-
d]pyrimidine based PKD inhibitor 1-NM-PP1. The N-methyl-
indole analogue of 3-IN-PP1 (compound 13) was endowed with
dramatically reduced anti-proliferative properties. This was
completely in line with the weak PKD inhibition for compound 13.
Despite their potent activity against PKD, compound 17n and
especially compound 17m, displayed only weak antiproliferative
activity. This was in agreement with the studies on cellular
cortactin phosphorylation and PANC-1 proliferation in which
compound 17m displayed overall weaker cellular activity. In
addition, pyrazolo[3,4-d]pyrimidine BKI1369, also carrying a
piperidinyl group, lacked antitumoral activity. These findings
indicate that, although a piperidinyl group is optimal for PKD
inhibition, it is not tolerated for cellular activity. Different
mechanisms including metabolism, permeability and off-target
effects could be responsible for these observations.
2.5 3-IN-PP1 reduces PANC-1 cell proliferation.
The effect of PKD inhibitors 17m, 3-IN-PP1 and 1-NM-PP1 on
the proliferation of PANC-1 cells was determined (Figure 4). 3-
IN-PP1 (at a concentration of 5 µM) significantly inhibited PANC-
1 cell proliferation after incubation for 96 (p=0.041) and
144 hours (p<0.001), thus performing better than the same
concentration of 1-NM-PP1. Despite its more potent PKD
inhibition, 17m does not significantly affect PANC-1 cell
proliferation at a concentration of 5 µM. This correlates very well
with the data from the cellular cortactin phosphorylation study in
which 17m was also less potent. A reduced cellular permeability
or increased metabolism in comparison to 3-IN-PP1 may
account for these effects.
3. Conclusion
Based on our previously identified PKD inhibitor 3-IN-PP1, a
novel series of pyrazolo[3,4-d]pyrimidines was synthesized.
Systematic structural variation at position 1 of this scaffold led to
the discovery of 17m, which is the most potent pyrazolo[3,4-
d]pyrimidine based pan-PKD inhibitor reported to date with IC50
values of 35, 35 and 17 nM for PKD1, 2 and 3, respectively.
Despite the fact that compound 17m was 3- to 5-fold more
potent in a biochemical PKD assay, when compared to 3-IN-PP1,
compound 17m was less potent in cellular environments. On the
other hand, 3-IN-PP1 very potently inhibited PKD activity in cells
and blocked the proliferation of PANC-1 pancreatic cancer cells.
Finally, a broad antitumoral screening revealed that 3-IN-PP1
endowed with low-micromolar antiproliferative activity against 7
out of 8 cancer cell lines. Altogether, these data demonstrate
that within the pyrazolo[3,4-d]pyrimidine based PKD-inhibitors
developed to date, 3-IN-PP1 clearly is the most potent
antiproliferative agent.
Figure 4. Cell proliferation of PANC-1 cells in presence of 1-NM-PP1, 3-IN-
PP1 and 17m at a concentration of 5 µM. 3-IN-PP1 significantly reduces
PANC-1 cell proliferation at 96 hours (p = 0.041) and 144 hours (p < 0.001). A
two-way ANOVA was performed using Dunnett’s multiple comparisons test.
7

Table 4. Inhibition of tumor cell growth of selected PKD inhibitors, data expressed as compound concentration required to inhibit tumor cell viability by 50% (IC50).
Compound
IC50 (µM)a
LN-229
Capan-1
HCT-116
NCI-H460
DND-41
HL-60
K-562
Z-138
CRT0066101
1-NM-PP1
BKI1369
3-IN-PP1
13
0.6 ± 0.1
>100
1.8 ± 0.1
23 ± 0.3
>100
1.7 ± 0.5
>100
1.9 ± 0.2
>100
1.0 ± 0.0
>26.1
1.0 ± 0.4
24.1 ± 7
>100
2.2 ± 0.2
>36.3
0.7 ± 0.3
>23.7
45.2 ± 6.8
39.2 ± 4.9
>78.3
>55.2
>100
35.5 ± 19.7
9.8 ± 0.2
>41.4
>100
30.4 ± 1.7
9.7 ± 3.5
>38.2
1.6 ± 0.4
21.3 ± 9
4.7 ± 0.1
40.7 ± 14.5
2.5 ± 0.2
63 ± 7.9
13.4 ± 0.7
>100
4 ± 0.8
73.9 ± 22.7
14 ± 2.6
>95.8
7.8 ± 0.7
>25.6
3.7 ± 0.9
>33.7
17n
52.6 ± 0.4
80.6 ± 7
13.1 ± 0.6
42.7 ± 4.9
27.6 ± 4.6
17.9 ± 4.1
12 ± 0.9
>100
19.6 ± 4.3
35.3 ± 10.4
17m
a Data are expressed as the mean ± SEM of n=2 independent experiments.
4 °C. Next, cell lysate was incubated with FLAG ant ibody beads (M2
Sigma Aldrich, St. Louis, USA) for 2 hours at 4 °C. The beads were
washed 2 times with the aforementioned buffer but using 500 mM of
NaCl instead of 150 mM. The proteins were eluted in 50 mM Tris pH 7.4,
50 mM NaCl, supplemented with 100 ꢁg/mL FLAG peptide and stored in
buffer containing 25% glycerol. Protein concentration was determined via
the Bradford assay using BSA as a standard and purity was determined
by Coomassie stained SDS-polyacrylamide gel.
4. Materials and methods
4.1 Chemistry
Synthetic procedures, experimental details, spectral information (NMR
spectra) are found in the supporting information of this article.
4.3.2 PKD screening assay
4.2 Biomolecular modelling
An in vitro luminescence based assay
(ADP-Glo™ from Promega
All homology modelling was performed using MOE2018 (Chemical
Computing Group, Canada) with the Amber10-EHT force field. During the
homology modelling process the crystallographically determined position
of the ATP competitive inhibitors were retained for induced fit modelling.
GOLD 5.7.2 was used for docking using the GoldScore and 100% search
efficiency parameters.[42] The template ligands were used to determine
the binding site with a radius of 6 Å. In order to prioritize the alternative
binding mode, the nitrogens forming hydrogen bonds with the hinge
served as pharmacophore features. Following docking, the binding
modes and corresponding scores were sorted as classical, inverted and
best energy. To evaluate the predictive value of the models, the Scikit-
Learn Python library was used to calculate the ROC curves and AUC.[43]
The residual activity cut-offs were chosen at 40, 50 or 60% to separate
the active from inactive inhibitors.
Corporation) was used to determine the PKD phosphorylation activity.
Syntide-2 was used as a substrate and assays were performed in the
presence of various compounds at a concentration of 1 µM for initial
screening, and concentrations ranging from 10 µM to 1 nM for IC50
determinations. Reactions were carried out in white 96-well flat bottom
plates (NUNC) with a 10 µL reaction volume containing 50 mM Tris-HCl
pH 7.4, 10 mM MgCl2, 8 nM of PDBu stimulated FLAG PKD, 250 µM
Syntide-2 (Sigma Aldrich, St. Louis, USA) and 10 µM ATP. After
incubation of 15 minutes at 30 °C, the reaction was stopped and
incubated with ADP-Glo reagent I and II (ratio 1:1:2, i.e. reaction volume:
reagent 1: reagent 2, respectively) according to the manufacturer’s
protocol. Subsequently, the luminescence was measured using a Perkin-
Elmer Victor X4 Multiple Plate Reader in counts per second (CPI).
Percentage of PKD inhibition was determined relative to a DMSO control
(0.1% DMSO). Graphs were plotted and IC50 values were determined
using the GraphPad Prism 7 software package.
4.3 Biological studies
4.3.3 Cell culture
4.3.1 Production and purification of recombinant FLAG tagged PKD1/2/3
in HEK293T cells.
PANC-1 cells were grown in Dulbecco’s modified eagle medium (DMEM)
(Sigma, St. Louis, MO, USA) supplemented with 10% (v/v) fetal bovine
serum (GE Healthcare, Little Chalfont, UK), 2 mM L-glutamine (Sigma, St.
Louis, MO, USA), 100 U/mL penicillin and 100 ꢁg/mL streptomycin
(Sigma, St. Louis, MO, USA).
HEK293T cells were cultured in DMEM (Sigma Aldrich, St. Louis, USA)
supplemented with 1% Glutamax (Gibco, Life Technologies, California,
USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma Aldrich,
St. Louis, USA) and 10% Fetal bovine serum (Hyclone, GE Healthcare,
Wisconsin, USA). Cells were plated at a density of 5 x 106 cells per 10
cm plate and transiently transfected with 10 µg FLAG tagged pdcDNA-
FLAG-PKD2 using polyethyleneimine (PEI) (Polysciences Inc.,
Washington, USA) in a 5:1 (w/w) ratio. 48 hours after transfection cells
were stimulated with 500 nM phorbol 12,13-dibutyrate (PDBu) (Sigma
Aldrich, St. Louis, USA) for 15 minutes and subsequently lysed in a
buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM
Na3VO4; 50 mM NaF; 5 mM NaPPi (sodium pyrophosphate); 0.2 µM
microcystine; 1 mM AEBSF (4-(2-aminoethyl)-benzene sulfonyl fluoride
hydrochloride); 10 µg/mL leupeptin; and 1 % triton X-100. Insoluble
material was removed by centrifugation at 10000 × g for 15 minutes at
4.3.4 Cellular screening assay using phospho-cortactin as a readout of
PKD inhibition
PANC-1 cells were seeded at 0.3 x 106 per condition in a 6-well plate and
allowed to attach. Twenty-four hours later, 0.5 µg of pcDNA-FLAG-
PRKD1 or pcDNA-FLAG-PRKD2 and 0.5 µg of pcDNA3-Myc-CTTN were
transfected using a PEI at a 1:3 (m/m) plasmid/ PEI ratio. After 24 hours,
the cells were treated with the indicated compounds (Figure 3) at a final
concentration of 20 µM or DMSO control for 1 hour, followed by either
stimulation or no stimulation with 250 nM of phorbol-12,13-dibutyrate
8

(PDBu) for 15 minutes. Lysis was done in 50 mM Tris, pH 7.4, 150 mM
NaCl, 15 mM EDTA, 1% NP-40 supplemented with phosphatase
inhibitors (Phosphostop, Roche, Germany), and protease inhibitors
(cOmplete, Roche, Germany). Protein quantification was determined
using the BCA protein assay (Pierce™ BCA Protein Assay Kit, Thermo
Fisher Scientific, Waltham, MA, USA). Next, equal amounts of lysate (10
µg) were resolved in an SDS-PAGE and blotted into a nitrocellulose
membrane. Antibody unspecific binding was blocked with either 5% BSA
(Sigma, St. Louis, MO, USA) or 5% low-fat milk. Blots were probed with
anti-Cortactin pSer-298 antibody (homemade),[31] anti-Myc 9E10 (Sigma,
St. Louis, Missouri) and anti-FLAG M2 (Sigma, St. Louis, Missouri).
Finally, blots were incubated with the appropriate secondary HRP-linked
antibody: Goat anti-Rabbit or Horse anti-Mouse (Cell Signaling
Technologies Beverly, MA, USA). Band intensities were quantified with
Image Studio Lite (LI-COR Biosciences). Ponceau staining was used as
a loading control. The sharpness of the ponceau images was improved
by using the default settings of the sharpen tool in Image Studio Lite
software. Experiments were performed in triplicate (n=3). The statistical
measures used were the mean and SEM. A one-way ANOVA was
performed to determine the statistical significance with the control
conditions (DMSO). Statistical analysis was conducted using the
GraphPad Prism 7 software package.
4.3.7 Antitumor assays
All tumour cell lines were acquired from the American Type Culture
Collection (ATCC, Manassas, VA, USA), except for the DND-41 cell line,
which was purchased from the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ Leibniz-Institut, Germany). All
cell lines were cultured as recommended by the suppliers. Culture media
were purchased from Gibco Life Technologies and supplemented with
10% fetal bovine serum (HyClone, GE Healthcare Life Sciences).
Adherent cell lines LN-229, HCT-116, NCI-H460, and Capan-1 cells were
seeded at a density between 400 and 1250 cells per well, in 384-well,
black walled, clear-bottomed tissue culture plates (Greiner). After
overnight incubation, cells were treated with the test compounds at seven
different concentrations ranging from 100 to 6.4x10-3 µM. Suspension cell
lines HL-60, K-562, Z-138, and DND-41 were seeded at densities
ranging from 2500 to 5000 cells per well in 384-well, black walled, clear-
bottomed tissue culture plates containing the test compounds at the
same seven concentration points. The plates were incubated and
monitored at 37 °C for 72 hours in an IncuCyte (Ess en BioScience Inc.,
Ann Arbor, MI, USA) for real-time imaging. Images were taken every 3
hours, with one field imaged per well under 10× magnification. All
compounds were tested with duplicate data points and averaged.
4.3.5 CAMKIIα and PKCδ assay
Declaration of competing interests
An in vitro radiometric assay was used to determine the inhibition of
CAMKIIα and PKCδ in presence of various compounds at the indicated
concentrations. Reactions were carried out in 40 ꢁL of a mixture
containing 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 2 mM CaCl2, 30 µg/µL
Calmodulin, 50 ng of CAMKIIα (Carna biosciences, Kobe, JP), 2 ꢁg
Syntide-2 (Sigma Aldrich, St. Louis, USA), 25 ꢁM ATP and 2 ꢁCi [γ-32P]
ATP (Perkin-Elmer, Massachusetts, USA) for the CAMKIIα assay. For
the PKCδ assay, reactions were carried out in 40 ꢁL of a mixture
The authors declare no competing financial interest
Acknowledgements
PG thanks FWO (PhD fellowship of the Research Foundation -
Flanders) for the received fellowship (PhD fellowship 1S09017N).
The Hercules Foundation of the Flemish Government is
acknowledged for making mass spectrometry possible (grant
20100225-7) and supporting the purchase of a 400 MHz NMR
spectrometer through project AKUL1311. AV thanks FWO for
funding through Odysseus project G0F9316N. JVL thanks FWO
(Research Foundation - Flanders) for funding via project
G0C4118N, and KU Leuven via C2-project C24/17/074 (Interne
Fondsen KU Leuven/Internal Funds KU Leuven). PANC-1 cells
were a kind gift of Prof. Thomas Seufferlein (Department of
Internal Medicine I, University of Ulm, Ulm, Germany). We are
grateful to prof. dr. Jef Rozenski for HR-MS measurements.
containing 50 mM Tris-HCl pH 7.4, 10 mM MgCl2,
4 µL of
phosphatidylserine (PS)/PDBu vesicles stock, 50 ng of PKCδ (Carna
biosciences, Kobe, JP), 5 ꢁg MARCKS peptide (in-house synthesis), 25
ꢁM ATP and 2 ꢁCi [γ-32P] ATP (Perkin-Elmer, Massachusetts, USA).
After 10 minutes, the kinase reaction was stopped by spotting 30 ꢁL of
the reaction on Whatman P81 filter paper. The filter papers were washed
three times with an aqueous solution of 0.5% phosphoric acid, followed
by one wash with 100% acetone. Subsequently, the papers were air-
dried and counted using a Tri-Carb 2810 TR scintillation counter (Perkin-
Elmer). Percentage of inhibition was determined relative to a condition
where no inhibitor was added (0.1% DMSO). Graphs were plotted using
the GraphPad Prism 7 software package. PS/PDBu vesicles were
prepared as follows: PDBu and PS were mixed to final concentrations of
100 µg/mL PS and 250 nM PDBu and dried using a Savant Speedvac
concentrator (Thermo Fisher Scientific, Waltham, MA, USA). Dried lipids
were resuspended in 50 mM Tris pH 7.4 and sonicated three times for 10
minutes until a clear solution was achieved.
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10

CHEMISTRY DEPARTMENT
CELESTIJNENLAAN 200F BOX 2404
3001 HEVERLEE, BELGIUM
OUR REFERENCE
YOUR REFERENCE
HEVERLEE
Revision of EJMECH-D-20-00543R1, European Journal of Medicinal Chemistry
May 11th 2020
Highlights
•
A novel series of pyrazolo[3,4-d]pyrimidine based PKD inhibitors with structural variety at position 1 was
synthesized.
•
•
•
17m showed the most potent pan-PKD inhibitory activity with IC50 values ranging from 17 to 35 nM.
17m and 3-IN-PP1 potently blocked PKD-dependent cortactin phosphorylation in PANC-1 cells.
3-IN-PP1 exhibited low micromolar cytotoxic activity against various cancer cell lines.
PROF. DR. WIM DE BORGGRAEVE
TEL. + 32 16 32 76 93
wim.deborggraeve@chem.kuleuven.be
chem.kuleuven.be/moldesigns