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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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Journal of Medicinal Chemistry

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 , 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 #03-10/11, Singapore 138667 (Singapore)

*Correspondence and requests for materials should be addressed to A.W.H. (email: [email protected])

Abstract: Protein Kinase C iota (PKC-ι) 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 azaindole-based scaffold was chosen for optimization. Driven by structureactivity relationship and supported by molecular modelling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.

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 PKC-ι being elevated in various cancers3 such as, lung,4-6 breast,7 ovarian,8-10 oesophageal,11 prostate,12 pancreatic,13 gastric,14 cholangiocarcinoma,15 colon,16 brain,17 cervical

18

and hepatocellular carcinoma

(HCC).19 More specifically, PKC-ι 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 Rac-Pak-Mek-Erk 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 PKC-ι activity would be an attractive route towards developing anticancer therapeutics.21-24 Herein, we describe the use of a fragment-based approach to discover a new class of potent and selective inhibitors against the atypical kinase PKC-ι.

Results and Discussion

A high concentration calliper-based biochemical screen of 1700 fragments (in-house curated) against PKC-ι identified 15 fragment hits (Table 1). Eleven of the 15 hits showed good ligand efficiencies (LE)

25

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 well-defined hinge binding anchor towards PKC-ι


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(IC50 = 452 – 1300 µM). The optimisation of the nicotinamide-based fragment 12 will be reported in a separate communication.

Table 1. Fragment screening against PKC-ιι yielded 15 fragment hits. From a fragment library of 1700 compounds, 23 fragments displayed point inhibition > 40 % (tested at 500 µM). Out of the 23 compounds, 15 displayed concentration-dependent inhibition with IC50 < 2.3 mM. The compounds have been ranked according to their ligand efficiencies.

Cmpd

Structure

IC50

LE

Cmpd

Structure

[µM]

IC50

LE

[µM]

1

615

0.44

9

927

0.34

2

452

0.41

10

1677

0.32

3

589

0.40

11

1423

0.32

4

37

0.40

12

424

0.29

5

1300

0.39

13

1556

0.27

1570

0.27

2216

0.26

OH

6

728

0.38

N

14 NH2

7

1312

0.36

15



229

8

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0.35

Preliminary SAR

In the absence of an X-ray 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 4-position of the azaindole core. As shown in Table 2, modification to an amide was not tolerated (compound 16), while substitutions with pyridine and phenyl-amide (compounds 17 & 18) resulted in a weak inhibition of PKC-ι (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

Cmpd

Structure

N H

IC50 [µM]

LE

Cmpd

16

> 384

-

17

374

0.31

Structure

IC50 [µM]

LE

23

95

0.29

24

221

0.28


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18

371

0.26

25

41

0.33

19

201

0.34

26

> 384

-

20

148

0.28

27

20

0.34

21

75

0.31

28

> 384

-

105

0.29

29

215

0.26

O NH

22

Interestingly, a bulky 4-bromo-pyrrole substituent (compound 19, IC50 = 201 µM) was also tolerated. Altogether, the initial SAR results suggested that the direct linking of the two aromatic groups to be favourable for inhibition of PKC-ι. More crucially, the retention of potency despite introduction of the larger aromatic groups in compounds 17 − 19 validated the hypothesis that the 4-position 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 bi-aryl 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 µM) which is ~15-fold more potent than the 4bromoazaindole fragment hit. Importantly, the installation of the benzimidazole in 25 has



widened the opportunities and vectors for subsequent fragment growing. Closer examination of the SAR in Table 2 also revealed the N-3 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 N-1 as a vector for further expansion.

Next, equipped with larger/more potent compounds and better understanding of the SAR, we attempted to dock compound 25 into an X-ray crystal structure of PKC-ι (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 Hbond donor-acceptor interactions with the backbone residue of Val-326 (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 Lys-274 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 N1 of benzimidazole as a potential vector to engage two distal aspartic acids (Asp-330 and Asp-373) 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 2-fold 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-ιι; a) Cross-sectional cropped view of the active site of PKC-ι (PDB code: 3A8W) with compound 25 docked. The expansion from the benzimidazole nitrogen with a base-containing moiety could potentially engage one of the two aspartic acids (Asp-330, Asp-373); b) ligand interaction diagram indicating the hydrogen bonding interactions with the backbone of Val-326 and the possible interaction between flexible Lys-274 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 N-1 position of benzimidazole (Table 3). Interestingly,

branching

out

with

an

azetidine-containing

compound

30

was

counterproductive and led to a ~9-fold loss in potency to 188 µM as compared to compound 27. The larger saturated 6-member rings, (compounds 31 − 32) were tolerated but only resulted in a modest 2-fold 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 N-1 benzimidazole was synthesized in an attempt to engage Asp-330 and Asp-373.

Cmpd

Structure

IC50 [µM]

Cmpd

Structure

30

188

35

7.0

31

9.5

36

1.1

32

7.7

37

0.8

33

6.0

38

0.12

34

7.5

39

0.048

IC50 [µM]

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 µM. On the other hand, the larger and more flexible benzylamino- analogues 36 and 37 showed ~20-fold improvement in potency as compared to compound 27. Further elaboration by adding a methoxy- group at C2 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 8-fold


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improvement of IC50. Lastly, taking into account that both meta- and para-substituted benzylamines were equally potent, the cyclic analogue 39 was synthesized. The rigidification of the methylene amines proved to be beneficial and afforded ~3-folds increase in potency as compared to compound 38 (IC50 = 0.048 µM).

Characterization of PKC-ιι 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 non-mutant 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 µM, only CDK7 and PKC-ε were significantly inhibited (> 95%). Secondly, the IC50s of 39 were determined for PKC-α (classical), PKC-ε (novel) and PKC-ζ (atypical). As shown in Figure 2a, compound 39 exhibited 6-fold selectivity against PKC-α and > 10-fold selectivity against PKC-ε when compared to the inhibition of PKC-ι. However, no selectivity was observed within the atypical PKC sub-family and compound 39 inhibited PKC-ζ with IC50 = 0.010 µM. Finally, to investigate if the activity in biochemical assays would translate into cellular activity, compound 39 was tested in HUH-7 cells. As shown in Figure 2, compound 39 exhibited weak anti-proliferative activity against HUH-7 cells with GI50 = 9 µM. 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∙10-6 cm/s in a CACO-2 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 pH-dependent solubility of the compound. At pH = 4, compound 39 exhibited good solubility of 790 µg/ml and at pH of 7.4,



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39 showed poor solubility of 1.3 µg/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.

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-ιι inhibitor 39

We next exploited the use of a Group Efficiency (GE) analysis

27-28

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 hydrogen-binding potential of N-3 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).

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 ASP-330; 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



kcal/mol-1 is added to the initial fragment hit. Details of the GE calculations and the structure of compound 52 is also shown in SI).

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 with pyrrolopyridine (compound 45, IC50 = 0.0065 µM) resulted in a ~7-fold improvement in inhibitory activity compared to compound 39 and 3-fold improvement in antiproliferative activity against HUH-7 cells (GI50= 3 µM). More importantly, compound 45 now exhibited superior selectivity within the PKC family (> 16 folds selectivity against both PKC-α and PKC-ε). 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 4-chloroazaindole; 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 PKC-ι, 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 [µM]

PKC-α IC50 [µM]

PKC-ε IC50 [µM]

40

0.065

0.43

4.2

41

0.030

0.25

0.53

42

4.8

1.4

5.8

43

0.11

0.35

0.57

44

0.57

0.34

1.1

45

0.0065

0.11

0.23

Cmpd

Structure

With a potent single digit nanomolar inhibitor of PKC-ι 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 µM). Furthermore, it was found that the 5-member ring of isoindoline could be expanded to tetrahydroisoquinoline leading to a 2-fold improvement in potency (compound 49, IC50 = 0.0028 µM).



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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 PKC-ι has translated into cellular activity against Huh7 and HCCLM3 cells. Additionally, effects of inhibition of PKCι on the phosphorylation of substrate ECT2 was investigated in HCCLM3 cells (SI Figure 2).

PKC-ιι IC50 [µM]

PKC-α α IC50 [µM]

PKC-εε IC50 [µM]

Huh7 / HCCLM3 GI50 [µM]

46

> 384

2.5

3.4

-/-

47

38

0.38

3.3

-/-

48

0.005

0.14

0.52

- / 3.4

49

0.0028

0.071

0.35

1.4 / 3.0

0.0038

0.064

0.17

0.86 / 3.3

Cmpd

Structure

O

50 N H


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0.0027

51

0.045

0.045

3.3 / 6.6

Additionally, the tetrahydroisoquinoline could be methylated at the α-position to the nitrogen (compound 50) which translated into an unexpected 3-fold improvement of cellular activity (GI50 = 0.86 µM) 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 (CACO-2 = 23 · 10-6 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 PKC-ι 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 hit-to-lead 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 fragment-based drug discovery pursuits. Herein we have shown that our fragment optimization strategy has systematically led to the discovery of a series of potent and



selective PKC-ι inhibitors. These novel compounds can be used as high quality chemical probes or starting points for future development of atypical PKC inhibitors.

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 commercial source and used without further purification. 1H 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

equiv.), rt, 18 h; then TiCl4 (1.2 equiv.), CHCl3; 10 oC - reflux, 48 h; (c) LiBH(OEt)3 (10 equiv), THF, rt, overnight (d) Boc2O (1.2 equiv.), Na2CO3 aq, CH2Cl2, rt, 2 h; (e) pinacolatodiboron (1.2 equiv.), KOAc(2.3equiv.), PdCl2(dppf)·CH2Cl2 (5 mol%), dioxane, 110 oC, 16 h; (f) NIS (1.5 equiv.), 0 oC – 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: 3- bromo-4-methoxy 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 Dean-Stark 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).



1

H NMR (CDCl3, 400 MHz): δ 8.18 (s, 1H), 8.02 (d, 1H), 7.64-7.64 (dd, J =2.4 Hz, 1H)

6.93-6.91 (d, J = 8.4 Hz, 1H), 4.68-4.67(t, J = 5.2 Hz, 1H) 3.94 (s, 3H), 3.77-3.76(t, J = 1.6 Hz, 2H), 3.43 (s, 6H). Step 2: the crude (3‐bromo‐4‐methoxyphenyl)methylidene(2,2‐dimethoxyethyl)amine (14.04 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 co-distilled (2 x 50 mL) with toluene. To this crude residue (15 g) chloroform (100 ml) was added, followed by TiCl4 (1.2 equiv.) dropwise at 15-20 oC. The reaction mixture was then refluxed for 48 hrs and then poured onto crushed ice and basified to pH-9 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 230-400 mesh silica gel and 10-20% ethyl acetate in petroleum ether as solvent system to afford 7‐bromo‐6‐methoxyisoquinoline as an off-white solid (2.5 g). 1

H NMR (CDCl3, 400 MHz): δ 9.01 (s, 1H), 8.48-8.47 (d, 1H), 8.25 (s, 1H) 7.65-7.64 (d, J =

4.4 Hz, 1H), 7.26 (s, 1H) and 4.07 (s, 3H); LC-MS 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‐bromo‐6‐methoxy‐1,2,3,4‐tetrahydroisoquinoline was used directly to the next step without purification. Step 4: to the crude 7‐bromo‐6‐methoxy‐1,2,3,4‐tetrahydroisoquinoline (1.02 g, 4.2 mmol, 1 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 230-400 mesh silica gel and 0-5 % ethyl acetate in petroleum ether as solvent to yield tert‐butyl 7‐bromo‐6‐ methoxy‐1,2,3,4‐ tetrahydroisoquinoline‐2‐carboxylate as an off-white solid (850 mg). 1

H NMR (CDCl3, 400 MHz): δ 7.27-7.26 (d, J = 6.8 Hz, 1H), 6.64 (s, 1H), 4.47 (s, 2H) 3.86

(s, 3H), 3.63-3.62 (t, J = 12 Hz, 2H), 2.78-2.76 (t, J = 8 Hz, 2H), and 1.48 (s, 9H); LC-MS retention time = 4.42 min., m/z 285.9 [M-55], 98.4 %. Step 5: tert-butyl 7-bromo-6-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate (400 mg, 1.16 mmol) in anhydrous 1,4- dioxane (7.0 mL) was treated with 4,4,4',4',5,5,5',5'octamethyl-2,2'-bi(1,3,2-dioxaborolane) (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 mixture was purged with nitrogen gas for another 5 minutes before heating at 110 oC 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



at 22 % ethyl acetate, monitoring using 230 nm) to afford tert-butyl 6-methoxy-7-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (59a, 392 mg, 69 %). 1

H NMR (400 MHz, DMSO-d6) δ (ppm) 7.28 (s, 1H), 6.74 (s, 1H), 4.40 (s, 2H), 3.70 (s, 3H),

3.52-3.49 (m, 2H), 2.78-2.75 (m, 2H), 1.42 (s, 9H), 1.25 (s, 12H); APCI-MS, m/z 290.1 [C21H32BNO5-100]+; HPLC purity: 53.0 %; HPLC retention time: 7.03 mins. Step 6: to 5-bromopyrazolo[1,5-a]pyridine (55d, 4.0 g, 20.3 mmol) in acetonitrile (20.0 mL) at 0 oC was added N-Iodosuccinimide (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 5-bromo-3iodopyrazolo[1,5-a]pyridine (56d, 6.3 g, 96 %). 1

H NMR (400 MHz, DMSO-d6) δ (ppm) 8.72-8.70 (m, 1H), 8.15 (s, 1H), 7.74 (s, 1H), 7.09

(dd, 1H, J = 2.1, 7.3 Hz); APCI-MS, 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: 6-bromo-3-iodoimidazo[1,2-b]pyridazine (216 mg, 0.669 mmol) in anhydrous 1,4dioxane (2.0 mL) was treated with tert-butyl 6-methoxy-7-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-3,4-dihydroisoquinoline-2(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]dichloropalladium (II) (54.6 mg, 0.0670 mmol). The mixture was then heated to 110 oC 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 tert-butyl 7-(5-bromopyrazolo[1,5-a]pyridin-3-yl)-6-methoxy-3,4-dihydroisoquinoline2(1H)-carboxylate (60, 201 mg, 65%) as a yellow solid. ESI-MS, m/z 458.1 and 460.1, Br Isotope (1:1) [C22H24BrN3O3+H]+; HPLC purity: 88.78%; HPLC retention time: 7.31 mins. Step

8:

tert-butyl

7-(5-bromopyrazolo[1,5-a]pyridin-3-yl)-6-methoxy-3,4-dihydro-

isoquinoline-2(1H)-carboxylate (200 mg, 0.436 mmol) in anhydrous 1,4-dioxane (2.0 mL) was treated with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]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 tert-butyl 7-(5-(1H-pyrrolo[2,3b]pyridin-4-yl)pyrazolo[1,5-a]pyridin-3-yl)-6-methoxy-3,4-dihydroisoquinoline-2(1H)carboxylate (150 mg, 69 %). 1

H NMR (400 MHz, DMSO-d6) 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), 7.32-7.30 (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); APCI-MS, m/z 496.2 [C29H29N5O3+H]+; HPLC purity: 99.73 %; HPLC retention time: 6.40 mins. Step

9:

tert-butyl

7-(5-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrazolo[1,5-a]pyridin-3-yl)-6-

methoxy-3,4-dihydroisoquinoline-2(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



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 PL-HCO3 column to remove the TFA counterion; the filtrate was concentrated in vacuo and freeze-dried to afford 7-(5-(1H-pyrrolo[2,3-b]pyridin-4yl)pyrazolo[1,5-a]pyridin-3-yl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline as a pale yellow solid (49, 87.5 mg, 73%). 1

H NMR (400 MHz, DMSO-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), 7.61-7.60 (m, 1H), 7.32-7.29 (m, 2H), 7.15 (s, 1H), 6.84 (s, 1H), 6.68-6.66 (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); APCI-MS, m/z 396.1 [C24H21N5O+H]+; HPLC purity: 99.4 %; HPLC retention time: 6.65 mins; mp 161.9-164.3 oC

Molecular Modelling

The X-ray structure of PKC-ι in complex with ATP was downloaded from the protein data bank (PDB code: 3A8W). The PKC-ι structure was prepared using the protein preparation wizard in Maestro release 2017-3 (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 PKC-ι 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 ATP-site using Maestro. Figures of the cross-sectional docked structures were prepared with PyMOL software.

Enzymatic Assay

PKC enzymes (Carna Biosciences, Chuo-ku, 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 PKC-ι biochemical assays were performed using 36 µM of ATP, 1.5 µM peptide (5FAM) RFARKGSLRQKNV (obtained from GenScript) and 0.15 nM of PKC-ι. Concertation-dependent 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 CellTiter-Glo Luminescent cell viability assay from Promega. The Huh7/HCCLM3 cells were treated with PKC-ι 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 CellTiter-Glo 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



Corresponding Author: [email protected] The authors declare no competing financial interest.

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 hydrogen-bonding 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, N-iodosuccinimide

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