Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00060

Protein kinase C iota (PKC-i) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-i.

Full text of DOI:10.1021/acs.jmedchem.8b00060

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Fragment-based drug discovery of potent Protein Kinase C-iota inhibitors
Jacek Kwiatkowski, Boping Liu, Doris Hui Ying Tee, Guoying Chen, Nur Huda Binte Ahmad, Yun
Xuan Wong, Zhi Ying Poh, Shi Hua Ang, Eldwin Sum Wai Tan, Esther HQ Ong, Nurul Dinie Binte
Rahad, Anders Poulsen, Vishal Pendharkar, Kanda Sangthongpitag, May Ann Lee, Sugunavathi D/
O Sepramaniam, Soo Yei Ho, Joseph Cherian, Jeffrey Hill, Thomas H. Keller, and Alvin W Hung
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00060 • Publication Date (Web): 24 Apr 2018
Downloaded from http://pubs.acs.org on April 24, 2018
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Fragment-based drug discovery of potent Protein Kinase C-iota
inhibitors
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Jacek Kwiatkowski, Boping Liu, Doris Hui Ying Tee, Guoying Chen, Nur Huda Binte
Ahmad, Yun Xuan Wong, Zhi Ying Poh, Shi Hua Ang, Eldwin Sum Wai Tan, Esther HQ
Ong, Nurul Dinie , Anders Poulsen, Vishal Pendharkar, Kanda Sangthongpitag, May Ann
Lee, Sugunavathi D/O Sepramaniam, Soo Yei Ho, Joseph Cherian, Jeffrey Hill, Thomas H.
Keller, Alvin W. Hung*.
Experimental Therapeutics Centre, Agency for Science, Technology and Research
(A*STAR), 11 Biopolis Way, Helios #03ꢀ10/11, Singapore 138667 (Singapore)
*Correspondence and requests for materials should be addressed to A.W.H. (email:
whung@etc.aꢀstar.edu.sg)
Abstract: Protein Kinase C iota (PKCꢀι) is an atypical kinase implicated in the promotion of
different cancer types. A biochemical screen of fragment library has identified several hits,
from which an azaindoleꢀbased scaffold was chosen for optimization. Driven by structureꢀ
activity relationship and supported by molecular modelling, a weakly bound fragment was
systematically grown into a potent and selective inhibitor against PKCꢀι.
Introduction
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Protein Kinase C (PKC) is an important class of enzymes involved in cell differentiation,
migration and proliferation processes.1 In total, the PKC family consists of ten distinct
enzymes grouped into three categories: classical (α, β−1,β−2, γ), novel (δ, θ, ε, η) and
atypical (ι, ζ).2 Studies have shown the levels of atypical kinase PKCꢀι being elevated in
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various cancers3 such as, lung,4ꢀ6 breast,7 ovarian,8ꢀ10 oesophageal,11 prostate,12 pancreatic,13
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gastric,14 cholangiocarcinoma,15 colon,16 brain,17 cervical
and hepatocellular carcinoma
(HCC).19 More specifically, PKCꢀι has been shown to phosphorylate the epithelial cell
transforming sequence 2 (ECT2) and bind partitioning defective 6 homolog (Par6).4 The
complex of the three enzymes is believed to be critical for triggering tumor growth and
invasion through a RacꢀPakꢀMekꢀErk signalling cascade. Consequently, high levels of ECT2
have been shown to correlate with poor prognosis for patients suffering from HCC.20 Taken
together, the data provide strong evidence that inhibition of PKCꢀι activity would be an
attractive route towards developing anticancer therapeutics.21ꢀ24 Herein, we describe the use
of a fragmentꢀbased approach to discover a new class of potent and selective inhibitors
against the atypical kinase PKCꢀι.
Results and Discussion
A high concentration calliperꢀbased biochemical screen of 1700 fragments (inꢀhouse curated)
against PKCꢀι identified 15 fragment hits (Table 1). Eleven of the 15 hits showed good
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ligand efficiencies (LE) of above 0.3 and presented good opportunities for optimization.
Particularly appealing were the three structurally related azaindole fragments 1, 2 and 5. The
three compounds showed high ligand efficiencies (LE = 0.39 – 0.44) and possessed an
azaindole core hypothesized to form a wellꢀdefined hinge binding anchor towards PKCꢀι
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(IC50 = 452 – 1300 ꢀM). The optimisation of the nicotinamideꢀbased fragment 12 will be
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reported in a separate communication.
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Table 1. Fragment screening against PKC-ι yielded 15 fragment hits. From a fragment
library of 1700 compounds, 23 fragments displayed point inhibition > 40 % (tested at 500
ꢀM). Out of the 23 compounds, 15 displayed concentrationꢀdependent inhibition with IC50 <
2.3 mM. The compounds have been ranked according to their ligand efficiencies.
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IC50
IC50
Cmpd
1
Structure
LE
Cmpd
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Structure
LE
[ꢀM]
[ꢀM]
615
452
589
0.44
927
0.34
0.41
0.40
0.40
1677 0.32
1423 0.32
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37
424
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1300
728
0.39
0.38
0.36
1556 0.27
1570 0.27
2216 0.26
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OH
N
NH2
1312
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0.35
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Preliminary SAR
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In the absence of an Xꢀray crystal structure to guide fragment optimization, our first priority
was to explore potential vectors for fragment expansion. From the preliminary structure
activity relationship (SAR) of the hits, one obvious fragment growing strategy was directed at
replacement of the Br and Cl groups at the 4ꢀposition of the azaindole core. As shown in
Table 2, modification to an amide was not tolerated (compound 16), while substitutions with
pyridine and phenylꢀamide (compounds 17 & 18) resulted in a weak inhibition of PKCꢀι
(compound with a phenyl substituent was insoluble and the IC50 is therefore not presented).
Table 2. Extension of the 4-position of the azaindole hits. Combinatorial replacement of
chlorine/bromine on an azaindole template led to significant improvement of potency.
N
N
H
Cmpd
16
Structure
IC50 [ꢀM]
LE
Cmpd Structure IC50 [ꢀM]
LE
> 384
ꢀ
95
0.29
23
24
374
0.31
221
0.28
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371
0.26
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201
148
0.34
0.28
> 384
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ꢀ
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27
0.34
75
0.31
0.29
> 384
215
ꢀ
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22
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29
O
NH
105
0.26
Interestingly, a bulky 4ꢀbromoꢀpyrrole substituent (compound 19, IC50 = 201 ꢀM) was also
tolerated. Altogether, the initial SAR results suggested that the direct linking of the two
aromatic groups to be favourable for inhibition of PKCꢀι. More crucially, the retention of
potency despite introduction of the larger aromatic groups in compounds 17 − 19 validated
the hypothesis that the 4ꢀposition of the azaindole would be ideal for fragment growing. In an
attempt to further explore the binding pocket, larger bicyclic rings were attached to the
original azaindole moiety in a combinatorial fashion. Gratifyingly, most of these biꢀaryl
compounds were accommodated and displayed marked improvement in potency as compared
to the original fragment hits (Table 2, compounds 20 ꢀ 29). Consequently, this effort led to
the discovery of benzimidazole 25 (IC50 = 41 ꢀM) which is ~15ꢀfold more potent than the 4ꢀ
bromoazaindole fragment hit. Importantly, the installation of the benzimidazole in 25 has
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widened the opportunities and vectors for subsequent fragment growing. Closer examination
of the SAR in Table 2 also revealed the Nꢀ3 nitrogen on the benzimidazole of 25 to be key
for activity (compare with compound 26) and a further methyl scan (compounds 27 − 29)
around the benzimidazole indicated Nꢀ1 as a vector for further expansion.
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Next, equipped with larger/more potent compounds and better understanding of the SAR, we
attempted to dock compound 25 into an Xꢀray crystal structure of PKCꢀι (Figure 1; PDB:
3A8W, details on generating the model are described in the supporting information). As
shown in the docking pose in Figure 1a and 1b, the azaindole group in 25 engages in two Hꢀ
bond donorꢀacceptor interactions with the backbone residue of Valꢀ326 (hinge binding
region). The benzimidazole portion of compound 25 is then predicted to occupy the rest of
the adenosine binding site of ATP. More significantly, the benzimidazole nitrogen is
hypothesized to form a hydrogen bonding interaction with a flexible Lysꢀ274 residue,
corroborating previous SAR results where a sharp drop in activity was observed for
compounds 26 and 28. Docking study further indicated the possibility of branching out of Nꢀ
1 of benzimidazole as a potential vector to engage two distal aspartic acids (Aspꢀ330 and
Aspꢀ373) for further hydrogen binding interactions. Importantly, this prediction was in
agreement with SAR results from a methyl scans around the imidazole part of compound 25
where compound 27 was shown to be 2ꢀfold more potent than 25, (compound 27, Table 2).
On the contrary, methylated compounds 28 and 29 showed marked reduction in potency,
indicative of a lack of space for further fragment growing.
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Figure 1. Molecular modelling of azaindole fragment into PKC-ι; a) Crossꢀsectional
cropped view of the active site of PKCꢀι (PDB code: 3A8W) with compound 25 docked. The
expansion from the benzimidazole nitrogen with a baseꢀcontaining moiety could potentially
engage one of the two aspartic acids (Aspꢀ330, Aspꢀ373); b) ligand interaction diagram
indicating the hydrogen bonding interactions with the backbone of Valꢀ326 and the possible
interaction between flexible Lysꢀ274 and the benzimidazole nitrogen.
Guided by both the molecular modelling and SAR, a series of compounds was designed and
synthesized with the aim of extending out of Nꢀ1 position of benzimidazole (Table 3).
Interestingly, branching out with an azetidineꢀcontaining compound 30 was
counterproductive and led to a ~9ꢀfold loss in potency to 188 ꢀM as compared to compound
27. The larger saturated 6ꢀmember rings, (compounds 31 − 32) were tolerated but only
resulted in a modest 2ꢀfold improvement in inhibitory activity when compared to compound
27.
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Table 3. Substitutions on benzimidazole nitrogen targeting aspartic acids. A series of
compounds with basic groups extending out of Nꢀ1 benzimidazole was synthesized in an
attempt to engage Aspꢀ330 and Aspꢀ373.
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Cmpd
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Structure
Cmpd
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Structure
IC50 [ꢀM]
IC50 [ꢀM]
188
7.0
9.5
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0.12
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0.048
Similarly, compounds substituted with aromatic pyridines and phenyls 33 − 35 were tested
and found to be slightly improving potency to IC50 = 6.0 – 7.5 ꢀM. On the other hand, the
larger and more flexible benzylaminoꢀ analogues 36 and 37 showed ~20ꢀfold improvement in
potency as compared to compound 27. Further elaboration by adding a methoxyꢀ group at Cꢀ
2 of the benzylamine to force an increase in the dihedral angle between the benzimidazole
and benzylamine (compound 38)26 was also beneficial and resulted in an additional 8ꢀfold
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improvement of IC50. Lastly, taking into account that both metaꢀ and paraꢀsubstituted
benzylamines were equally potent, the cyclic analogue 39 was synthesized. The rigidification
of the methylene amines proved to be beneficial and afforded ~3ꢀfolds increase in potency as
compared to compound 38 (IC50 = 0.048 ꢀM).
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Characterization of PKC-ι inhibitor 39
At this stage, the most potent lead compound 39 was profiled to guide the subsequent
optimizations. Firstly, the selectivity of 39 within a panel of 101 nonꢀmutant kinases was
assessed. Satisfyingly, compound 39 was found to exhibit good overall selectivity within a
representative subset of the kinome (Figure 2) and at a screening concentration of 1 ꢀM,
only CDK7 and PKCꢀε were significantly inhibited (> 95%). Secondly, the IC50s of 39 were
determined for PKCꢀα (classical), PKCꢀε (novel) and PKCꢀζ (atypical). As shown in Figure
2a, compound 39 exhibited 6ꢀfold selectivity against PKCꢀα and > 10ꢀfold selectivity against
PKCꢀε when compared to the inhibition of PKCꢀι. However, no selectivity was observed
within the atypical PKC subꢀfamily and compound 39 inhibited PKCꢀζ with IC50 = 0.010
ꢀM. Finally, to investigate if the activity in biochemical assays would translate into cellular
activity, compound 39 was tested in HUHꢀ7 cells. As shown in Figure 2, compound 39
exhibited weak antiꢀproliferative activity against HUHꢀ7 cells with GI50 = 9 ꢀM.
Next the compound 39 was characterized in a series of in vitro pharmacokinetic experiments.
As shown in Figure 2, the compound exhibited low permeation rates of 0.57∙
10ꢀ6 cm/s in a
CACOꢀ2 assay, indicative of its suboptimal penetration capabilities. The observed low influx
rate of 39 could likely be attributed to the prescence of the protonated base in the scaffold.
The effect of the basic amine is also reflected in the pHꢀdependent solubility of the
compound. At pH = 4, compound 39 exhibited good solubility of 790 ꢀg/ml and at pH of 7.4,
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39 showed poor solubility of 1.3 ꢀg/ml. Gratifyingly, compound 39 exhibited no clearance
issues against mouse and human liver microsomes (MLM and HLM) and displayed only
weak inhibition of CYP 3A4 and CYP 2D6. Clearly, the goal forward was to generate a more
potent compound with improved permeability and solubility in neutral pH for greater cellular
potency.
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Figure 2. Characterization of compound 39; a) selectivity within PKC family, cellular
activity and in-vitro pharmacokinetic properties; b) kinase selectivity of compound 39;
KINOMEscanTM was performed by DiscoverX. The results are visualized using
TREEspotTM. Kinases are sorted according to the phylogenetic tree and the degree of
inhibition is proportional to the size of the maroon sphere. S scores are: S(35) = 0.085, S(10)
= 0.032.
Optimization of PKC-ι inhibitor 39
27ꢀ28
We next exploited the use of a Group Efficiency (GE) analysis
to further aid the
optimization of the scaffold. As indicated in Figure 3, despite having an overall efficient
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compound (LE = 0.35), our GE analysis quickly revealed inefficient parts in the molecule. In
particular, the central benzimidazole moiety with GE = 0.29 was not contributing as much to
activity as the azaindole, isoindoline and methoxy groups. With this information in hand, we
then set out to replace the central core of compound 39 with the hope of improving the
potency of the compounds without further increasing the size of the molecule. To support the
replacement of the benzimidazole, the hydrogenꢀbinding potential of Nꢀ3 was calculated in a
series of compounds with alternative placement of nitrogen atoms around the molecular
framework (SI Figure 3). This computational assessment indicated possibilities to improve
overall inhibitory activity of the scaffold through the modifications of the central
benzimidazole core and warranted the synthesis of compounds 40 – 45 (Table 4).
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Figure 3. Optimization of compound 39; a) docking of compound 39; b) ligand interaction
diagram indicating the additional hydrogen bond and salt bridge formed between isoindoline
nitrogen and aspartic acid ASPꢀ330; c) group efficiency analysis of compound 39 with pIC50s
approximated to pKds. HA is defined as the heavy atom count. From the GE analysis,
benzimidazole was identified as the most inefficient group in compound 39. *ꢁGrigid = 4.2
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kcal/molꢀ1 is added to the initial fragment hit. Details of the GE calculations and the structure
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of compound 52 is also shown in SI).
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As shown in Table 4, the introduction of nitrogen atoms into the phenyl part of the
benzimidazole was tolerated and did not result in significant changes in inhibitory activity
(compounds 40 and 41). In contrast, alterations to the imidazole moiety resulted in 10 ꢀ 100
folds decrease in potency (compounds 42 − 44). Interestingly, replacement of benzimidazole
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with pyrrolopyridine (compound 45, IC50 = 0.0065 ꢀM) resulted in a ~7ꢀfold improvement in
inhibitory activity compared to compound 39 and 3ꢀfold improvement in antiproliferative
activity against HUHꢀ7 cells (GI50= 3 ꢀM). More importantly, compound 45 now exhibited
superior selectivity within the PKC family (> 16 folds selectivity against both PKCꢀα and
PKCꢀε). It is also noteworthy that compound 45 now possess a high ligand efficiency of 0.39,
which corresponds closely to the LE of the starting fragment hit 4ꢀchloroazaindole; this is
indicative of a successful fragment growing approach. Compound 45 was also identified as
the most active molecule in our calculations (see SI for details); however comparing the
docking poses of compound 39 and 45 against PKCꢀι, it is structurally unclear why the
pyrrole would confer a 7 folds improvement in potency over the imidazole.
Table 4. Scaffold hopping with replacements of the benzimidazole. Profound effects on
activity were observed upon the replacement of imidazole with pyrazole or pyrrole.
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PKC-ι
IC50 [ꢀM]
PKC-α
IC50 [ꢀM]
PKC-ε
IC50 [ꢀM]
Cmpd
40
Structure
0.065
0.43
4.2
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0.030
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0.25
1.4
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5.8
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0.11
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0.34
0.11
0.57
1.1
0.57
0.0065
0.23
With a potent single digit nanomolar inhibitor of PKCꢀι in hand, the next immediate goal was
to improve the cellular potency of the inhibitor. To this end, isoindoline was modified in an
attempt to modulate the pharmacokinetic properties of the compounds. As summarized in
Table 5, the basic amine of the isoindoline was found crucial for activity and conversion to
amides was not tolerated, as shown in compounds 46 and 47. On the other hand, alkylation of
the amine did not affect the potency (compound 48, IC50 = 0.005 ꢀM). Furthermore, it was
found that the 5ꢀmember ring of isoindoline could be expanded to tetrahydroisoquinoline
leading to a 2ꢀfold improvement in potency (compound 49, IC50 = 0.0028 ꢀM).
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Table 5. Compounds 48 – 51 were found to be the most potent pyrazole azaindole
compounds in the series. The biochemical inhibition against PKCꢀι has translated into
cellular activity against Huh7 and HCCLM3 cells. Additionally, effects of inhibition of PKCꢀ
ι on the phosphorylation of substrate ECT2 was investigated in HCCLM3 cells (SI Figure 2).
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Huh7 /
HCCLM3
GI50 [ꢀM]
PKC-ι
PKC-α
PKC-ε
Cmpd
46
Structure
IC50 [ꢀM] IC50 [ꢀM] IC50 [ꢀM]
> 384
38
2.5
3.4
3.3
ꢀ / ꢀ
ꢀ / ꢀ
0.38
47
0.005
0.14
0.52
0.35
ꢀ / 3.4
48
49
0.0028
0.071
1.4 / 3.0
O
0.0038
0.064
0.17
0.86 / 3.3
50
N
H
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0.0027
0.045
0.045
3.3 / 6.6
51
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Additionally, the tetrahydroisoquinoline could be methylated at the αꢀposition to the nitrogen
(compound 50) which translated into an unexpected 3ꢀfold improvement of cellular activity
(GI50 = 0.86 ꢀM) and improvement in solubility. In an attempt to modulate basicity of the
amine, fluorine was introduced and the resulting compound 51 showed a substantial
improvement in permeability (CACOꢀ2 = 23 · 10ꢀ6 cm/s). The effect is likely caused by the
electronegativity of the fluorine, which caused a decrease in the basicity of the
tetrahydroisoquinoline by 1 log unit and increased the fraction of neutral species to ~25 %, as
compared to compound 48 (pKa measurements are shown in SI).
Conclusions
A fragment screen against PKCꢀι afforded 15 fragment hits. Using a combination of parallel
synthesis and guided by SAR, we were able to rapidly identify vectors and space for initial
fragment expansion. With the identification of a larger and more potent compounds, the next
phase of optimization was driven by molecular modelling. Finally, a GE analysis was
employed to further optimize an advanced hitꢀtoꢀlead stage compound and improve
selectivity within the PKC family. It is noteworthy that while fragment screening has
become more established over the years, fragment optimization remains a challenging feat in
most fragmentꢀbased drug discovery pursuits. Herein we have shown that our fragment
optimization strategy has systematically led to the discovery of a series of potent and
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selective PKCꢀι inhibitors. These novel compounds can be used as high quality chemical
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probes or starting points for future development of atypical PKC inhibitors.
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Experimental
Chemistry
The detailed synthesis of the key compound 49 is described below. All other compounds (17
– 51) were prepared as described in the SI. Solvents and reagents were purchased from
1
commercial source and used without further purification. H NMR spectra were obtained for
all compounds using a Bruker Ultrashield 400 PLUS/R system, operating at 400 MHz.
Chemical shifts are reported as parts per million relative to the solvent peak. The compounds’
purities were ≥ 95% as determined by a VARIAN ProStar HPLC instrument using
Acetonitrile/water (with 0.1% formic acid) as eluent. Mass spectrometric data was recorded
on an Agilent 1290 Infinity series, single quad spectrometer with an ESI ion source.
Scheme 1. Synthesis of compound 49.a
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a
Regents and conditions: (a) toluene, reflux, 6 h; (b) ClCO2Et (1 equiv.), P(OEt)3 (1.2
o
equiv.), rt, 18 h; then TiCl4 (1.2 equiv.), CHCl3; 10 C ꢀ reflux, 48 h; (c) LiBH(OEt)3 (10
equiv), THF, rt, overnight (d) Boc2O (1.2 equiv.), Na2CO3 aq, CH2Cl2, rt, 2 h; (e) pinacolatoꢀ
o
diboron (1.2 equiv.), KOAc(2.3equiv.), PdCl2(dppf)·CH2Cl2 (5 mol%), dioxane, 110 C, 16
o
h; (f) NIS (1.5 equiv.), 0 C – rt, 16 h; (g) PdCl2(dppf)·CH2Cl2, K3PO4, dioxane/water (4/1),
100 oC, 0.5 h; (h) TFA/CH2Cl2 (10 vol.%), rt, 1 h.
Procedure
Step 1: 3ꢀ bromoꢀ4ꢀmethoxy benzaldehyde (10 g, 46.50 mmol, 1 equiv.) was dissolved in
toluene (100 mL) and to this solution aminoacetaldehyde dimethyl acetal (7.65 mL, 69.75
mmol, 1.5 equiv.) was added and the reaction mixture was refluxed for 6 h using DeanꢀStark
apparatus. Reaction mixture was then concentrated and the crude product was used for the
next step ꢀ (3‐bromo‐4‐methoxyphenyl)ꢀmethylidene(2,2‐dimethoxyethyl)amine (14.5 g).
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1H NMR (CDCl3, 400 MHz): δ 8.18 (s, 1H), 8.02 (d, 1H), 7.64ꢀ7.64 (dd,
J
=2.4 Hz, 1H)
4
5
6
7
8
6.93ꢀ6.91 (d,
Hz, 2H), 3.43 (s, 6H).
Step 2: the crude (3 bromo
J
= 8.4 Hz, 1H), 4.68ꢀ4.67(t,
J
= 5.2 Hz, 1H) 3.94 (s, 3H), 3.77ꢀ3.76(t, J = 1.6
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‐
‐4
‐methoxyphenyl)methylidene(2,2‐dimethoxyethyl)amine (14.04
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g, 46.49 mmol, 1 equiv.) was dissolved in THF (100 ml) and the solution was cooled to 0 oC.
Ethylchloroformate (4.46 ml, 46.49 mmol, 1 equiv.) was added dropwise and the mixture was
stirred for 5 min. After this time, triethylphosphite (9.65 ml, 55.78 mmol, 1.2 equiv.) was
added dropwise and the mixture was stirred at RT for 18 hrs. Reaction was then concentrated
and coꢀdistilled (2 x 50 mL) with toluene. To this crude residue (15 g) chloroform (100 ml)
o
was added, followed by TiCl4 (1.2 equiv.) dropwise at 15ꢀ20 C. The reaction mixture was
then refluxed for 48 hrs and then poured onto crushed ice and basified to pHꢀ9 with aqueous
ammonia. The layers were separated. The aqueous layer was extracted with chloroform (2 x
200 mL). Organic layer was dried and concentrated. Compound was purified on column
chromatography using 230ꢀ400 mesh silica gel and 10ꢀ20% ethyl acetate in petroleum ether
as solvent system to afford 7
‐bromo‐6‐methoxyisoquinoline as an offꢀwhite solid (2.5 g).
1H NMR (CDCl3, 400 MHz): δ 9.01 (s, 1H), 8.48ꢀ8.47 (d, 1H), 8.25 (s, 1H) 7.65ꢀ7.64 (d,
J
=
4.4 Hz, 1H), 7.26 (s, 1H) and 4.07 (s, 3H); LCꢀMS RT = 2.04 min., m/z 237.9 [M+H]+, 99.3
%.
Step 3: 7‐bromo‐6‐methoxyisoquinoline (1 g, 4.22 mmol, 1 equiv.) was dissolved in dry THF
(20 mL) and lithium triethylborohydride (42 mL, 42.2 mmol, 10 equiv.) was added dropwise
at RT and the reaction was left at RT stirring overnight. After that time, TLC showed the
completion of the reaction, which was then quenched with 5 mL of methanol added dropwise,
followed by acidification with aqueous HCl to pH = 2. Reaction mixture was then washed
with ethyl acetate to remove impurities and the pH was then adjusted to pH 9 using aqueous
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Na2CO3. The crude 7
the next step without purification.
Step 4: to the crude 7 bromo methoxy‐1,2,3,4‐tetrahydroisoquinoline (1.02 g, 4.2 mmol, 1
‐bromo‐6‐methoxy‐1,2,3,4‐tetrahydroisoquinoline was used directly to
‐
‐6
‐
9
equiv.) in aqueous Na2CO3 (20 mL) was added DCM (20 mL) followed by (Boc)2O (1.16
mL, 5.06 mmol, 1.2 equiv.) and the reaction mixture was stirred at RT for 2 hrs. The reaction
was complete as indicated by TLC and worked up. Layers were separated and the aqueous
layer extracted with CHCl3. Combined organic layers were then dried over Na2SO4 and
concentrated. Compound was purified on column chromatography using 230ꢀ400 mesh silica
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gel and 0ꢀ5 % ethyl acetate in petroleum ether as solvent to yield tert
methoxy 1,2,3,4
‐butyl 7‐bromo‐6‐
‐
‐
tetrahydroisoquinoline
‐2‐carboxylate as an offꢀwhite solid (850 mg).
1H NMR (CDCl3, 400 MHz): δ 7.27ꢀ7.26 (d,
J
= 6.8 Hz, 1H), 6.64 (s, 1H), 4.47 (s, 2H) 3.86
= 8 Hz, 2H), and 1.48 (s, 9H); LCꢀMS
retention time = 4.42 min., m/z 285.9 [Mꢀ55], 98.4 %.
(s, 3H), 3.63ꢀ3.62 (t,
J = 12 Hz, 2H), 2.78ꢀ2.76 (t, J
Step 5: tertꢀbutyl 7ꢀbromoꢀ6ꢀmethoxyꢀ3,4ꢀdihydroisoquinolineꢀ2(1H)ꢀcarboxylate (400 mg,
1.16 mmol) in anhydrous 1,4ꢀ dioxane (7.0 mL) was treated with 4,4,4',4',5,5,5',5'ꢀ
octamethylꢀ2,2'ꢀbi(1,3,2ꢀdioxaborolane) (326 mg, 1.2 mmol) and potassium acetate (229 mg,
2.3 mmol). The mixture was purged with nitrogen gas for 5 minutes prior to the addition of
[1,1′ꢀBis(diphenylphosphino)ferrocene]dichloropalladium(II) (47.7 mg, 0.058 mmol). The
o
mixture was purged with nitrogen gas for another 5 minutes before heating at 110 C for 16
hours. The reaction mixture was then filtered through a pad of cellite, washing with ethyl
acetate. The filtrate was poured into water, and the layers were separated. The aqueous layer
was extracted with ethyl acetate twice, and the combined organic layer was dried with
sodium sulfate, filtered and concentrated under reduced pressure. The crude product was
purified by flash column chromatography (Redisep Silica Gel, Hexane:Ethyl acetate, eluting
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at 22 % ethyl acetate, monitoring using 230 nm) to afford tertꢀbutyl 6ꢀmethoxyꢀ7ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ3,4ꢀdihydroisoquinolineꢀ2(1H)ꢀcarboxylate (59a, 392
mg, 69 %).
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1H NMR (400 MHz, DMSOꢀ
d6) δ (ppm) 7.28 (s, 1H), 6.74 (s, 1H), 4.40 (s, 2H), 3.70 (s, 3H),
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3.52ꢀ3.49 (m, 2H), 2.78ꢀ2.75 (m, 2H), 1.42 (s, 9H), 1.25 (s, 12H); APCIꢀMS, m/z 290.1
[C21H32BNO5ꢀ100]+; HPLC purity: 53.0 %; HPLC retention time: 7.03 mins.
Step 6: to 5ꢀbromopyrazolo[1,5ꢀa]pyridine (55d, 4.0 g, 20.3 mmol) in acetonitrile (20.0 mL)
o
at 0 C was added NꢀIodosuccinimide (4.56 g, 20.3 mmol) and stirred at room temperature
for 16 hours. The reaction mixture was poured into saturated sodium thiosulfate, and
extracted with ethyl acetate twice. The combined organic layer was washed with brine, dried
with sodium sulfate, filtered and concentrated under reduced pressure to afford 5ꢀbromoꢀ3ꢀ
iodopyrazolo[1,5ꢀa]pyridine (56d, 6.3 g, 96 %).
1H NMR (400 MHz, DMSOꢀ
d6) δ (ppm) 8.72ꢀ8.70 (m, 1H), 8.15 (s, 1H), 7.74 (s, 1H), 7.09
(dd, 1H, J = 2.1, 7.3 Hz); APCIꢀMS, m/z 322.8 and 324.8, Br isotope (1:1) [C7H4BrIN2+H]+;
HPLC purity: 97.6 %; HPLC retention time: 6.16 mins
Step 7: 6ꢀbromoꢀ3ꢀiodoimidazo[1,2ꢀb]pyridazine (216 mg, 0.669 mmol) in anhydrous 1,4ꢀ
dioxane (2.0 mL) was treated with tertꢀbutyl 6ꢀmethoxyꢀ7ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
dioxaborolanꢀ2ꢀyl)ꢀ3,4ꢀdihydroisoquinolineꢀ2(1H)ꢀcarboxylate (390 mg, 1.00 mmol) and
tripotassium phosphate (283 mg, 1.34 mmol) in water (0.5 ml). The mixture was purged with
nitrogen gas for 15 minutes before addition of [1,1′ꢀBis(diphenylphosphino)ferrocene]ꢀ
o
dichloropalladium (II) (54.6 mg, 0.0670 mmol). The mixture was then heated to 110 C for
30 minutes. The reaction mixture was filtered through a pad of celite and washed with ethyl
acetate. The filtrate was concentrated under reduced pressure and purified by flash column
chromatography (Redisep silica gel, hexane : ethyl acetate, eluting at 22% ethyl acetate) to
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afford tertꢀbutyl 7ꢀ(5ꢀbromopyrazolo[1,5ꢀa]pyridinꢀ3ꢀyl)ꢀ6ꢀmethoxyꢀ3,4ꢀdihydroisoquinolineꢀ
2(1H)ꢀcarboxylate (60, 201 mg, 65%) as a yellow solid.
ESIꢀMS, m/z 458.1 and 460.1, Br Isotope (1:1) [C22H24BrN3O3+H]+; HPLC purity: 88.78%;
HPLC retention time: 7.31 mins.
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Step
8:
tertꢀbutyl
7ꢀ(5ꢀbromopyrazolo[1,5ꢀa]pyridinꢀ3ꢀyl)ꢀ6ꢀmethoxyꢀ3,4ꢀdihydroꢀ
isoquinolineꢀ2(1H)ꢀcarboxylate (200 mg, 0.436 mmol) in anhydrous 1,4ꢀdioxane (2.0 mL)
was treated with 4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀpyrrolo[2,3ꢀb]pyridine
(159 mg, 0.655 mmol) and tripotassium phosphate (185 mg, 0.870 mmol) in water (0.5 mL).
The mixture was purged with nitrogen gas for 15 minutes before addition of [1,1′ꢀ
Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (35.6 mg, 0.0440 mmol). The
mixture was heated to 110 oC for 30 minutes. Upon completion of reaction, the reaction
mixture was filtered over celite (washing with ethyl acetate). The filtrate was concentrated
under reduced pressure and purified by flash column chromatography (Redisep silica gel,
hexane: ethyl acetate, eluting at 72% ethyl acetate) to afford tertꢀbutyl 7ꢀ(5ꢀ(1Hꢀpyrrolo[2,3ꢀ
b]pyridinꢀ4ꢀyl)pyrazolo[1,5ꢀa]pyridinꢀ3ꢀyl)ꢀ6ꢀmethoxyꢀ3,4ꢀdihydroisoquinolineꢀ2(1H)ꢀ
carboxylate (150 mg, 69 %).
1H NMR (400 MHz, DMSOꢀd6) δ (ppm) 11.8 (s, 1H), 8.84 (d, 1H, J = 7.2 Hz), 8.32 (d, 1H, J
= 4.8 Hz), 8.22 (s, 1H), 7.96 (d, 1H, J = 1.2 Hz), 7.59 (d, 1H, J = 2.8), 7.32ꢀ7.30 (m, 1H),
6.95 (s, 1H), 6.67 (d, 1H, J = 2.4 Hz), 4.49 (s, 2H), 3.81 (s, 3H), 3.57 (t, 2H, J = 6.0 Hz), 2.82
(t, 2H, J = 5.2 Hz), 1.42 (s, 9H); APCIꢀMS, m/z 496.2 [C29H29N5O3+H]+; HPLC purity: 99.73
%; HPLC retention time: 6.40 mins.
Step 9: tertꢀbutyl 7ꢀ(5ꢀ(1Hꢀpyrrolo[2,3ꢀb]pyridinꢀ4ꢀyl)pyrazolo[1,5ꢀa]pyridinꢀ3ꢀyl)ꢀ6ꢀ
methoxyꢀ3,4ꢀdihydroisoquinolineꢀ2(1H)ꢀcarboxylate (150.0 mg, 0.303 mmol) in
dichloromethane (2.0 mL) was treated with trifluoroacetic acid (0.50 mL, 38.2 mmol). The
mixture was stirred at room temperature for 1 hour. Upon completion, the reaction mixture
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was concentrated under reduced pressure and the crude product was purified by preparative
HPLC (acetonitrile: water; 0.1% formic acid; gradient from 15% to 35% acetonitrile). The
pure fractions were combined and concentrated under reduced pressure. The product was
then passed through Agilent PLꢀHCO3 column to remove the TFA counterion; the filtrate
was concentrated in vacuo and freezeꢀdried to afford 7ꢀ(5ꢀ(1Hꢀpyrrolo[2,3ꢀb]pyridinꢀ4ꢀ
yl)pyrazolo[1,5ꢀa]pyridinꢀ3ꢀyl)ꢀ6ꢀmethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline as a pale yellow
solid (49, 87.5 mg, 73%).
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1H NMR (400 MHz, DMSOꢀ
d6) δ (ppm) 11.9 (s, 1H), 8.82 (d, 1H, J = 7.2 Hz), 8.31 (d, 1H, J
= 4.9 Hz), 8.18 (s, 1H), 7.94 (d, 1H, J = 1.2 Hz), 7.61ꢀ7.60 (m, 1H), 7.32ꢀ7.29 (m, 2H), 7.15
(s, 1H), 6.84 (s, 1H), 6.68ꢀ6.66 (m, 1H), 3.83 (s, 2H), 3.78 (s, 3H), 2.95 (t, 2H, J = 5.7 Hz),
2.73 (t, 2H, J = 5.5 Hz); APCIꢀMS, m/z 396.1 [C24H21N5O+H]+; HPLC purity: 99.4 %;
HPLC retention time: 6.65 mins; mp 161.9ꢀ164.3 oC
Molecular Modelling
The Xꢀray structure of PKCꢀι in complex with ATP was downloaded from the protein data
bank (PDB code: 3A8W). The PKCꢀι structure was prepared using the protein preparation
wizard in Maestro release 2017ꢀ3 (www.schrodinger.com). Settings included the addition of
hydrogen atoms, bond assignments, removal of all water molecules, protonation state
assignment and optimization of the hydrogen bond network. The PKCꢀι apo structure was
generated with the removal of ATP ligand from the prepared structure and the inhibitors were
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then manually docked into the ATPꢀsite using Maestro. Figures of the crossꢀsectional docked
structures were prepared with PyMOL software.
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Enzymatic Assay
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PKC enzymes (Carna Biosciences, Chuoꢀku, Kobe, Japan) were assayed using Microfluidics
Lab Chip® technology (PerkinElmer, Waltham, MA, USA). The Caliper assay was
performed in buffer comprising of 100 mM Hepes, pH 7.5, 10 mM MgCl2, 1 mM DTT
(Dithiothreitol), 0.003 % of Brij35 and 0.004 % of Tween 20. The PKCꢀι biochemical assays
were performed using 36 ꢀM of ATP, 1.5 ꢀM peptide (5FAM) RFARKGSLRQKNV
(obtained from GenScript) and 0.15 nM of PKCꢀι. Concertationꢀdependent IC50 curves of the
inhibitors were generated and fitted using GraphPad Prism software. The mean IC50s of
compounds are shown from three separate experiments. The experimental for the biochemical
assays of other isoenzymes of PKC are shown in the SI.
Cellular assays
Cell viability assay was performed using CellTiterꢀGlo Luminescent cell viability assay from
Promega. The Huh7/HCCLM3 cells were treated with PKCꢀι compounds that were serially
diluted in growth medium. Plates were incubated for 72 h at 37 oC in 5 % CO2. After 72 h, an
equal volume of CellTiterꢀGlo reagent was added. Plates were rocked on a rotator for 2 h.
Luminescence emitted was measured with the Tecan Safire II (Perkin Elmer). GI50s were
generated and fitted using using GraphPad Prism software.
Author Information
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Corresponding Author: whung@etc.aꢀstar.edu.sg
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The authors declare no competing financial interest.
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Acknowledgements
This work was in part supported by the Agency for Science, Technology and Research
(A*STAR) Joint Council grant 1231B105.
Supporting information
The Supporting Information is available free of charge on the ACS Publications website at
DOI:
Provided in the SI are: biochemical assay procedures, protein and substrate production;
cellular assays procedures; details on generation of molecular model and calculations of
hydrogenꢀbonding potential; group efficiency analysis calculations; solubility and pKa
measurements; synthesis and analytical data of new compounds.
Abbreviations used
ECT2, epithelial cell transforming sequence 2; Par6, bind partitioning defective 6 homolog;
HCC, hepatocellular carcinoma; HLM, human liver microsomes; MLM, mouse liver
microsomes; GE, group efficiency; HA, heavy atom; NIS, Nꢀiodosuccinimide
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