Scaffold hopping of the SYK inhibitor entospletinib leads to broader targeting of the BCR signalosome
Radek Jorda1,2,#, Soňa Krajčovičová3,#, Petra Králová3, Miroslav Soural2,3, Vladimír Kryštof1*
1Laboratory of Growth Regulators, Palacký University & Institute of Experimental Botany, The Czech Academy of Sciences, Šlechtitelů 27, 78371 Olomouc, Czech Republic
2Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 5, 77900 Olomouc, Czech Republic
3Department of Organic Chemistry, Faculty of Science, Palacký University, 17. Listopadu 12, 77146 Olomouc, Czech Republic
# Equal contribution
* Corresponding author. Email: [email protected]
Abstract
Spleen tyrosine kinase (SYK) and Bruton’s tyrosine kinase (BTK) are attractive targets in human haematological malignancies with excessively activated B-cell receptor (BCR) signalling pathways. Entospletinib is a SYK inhibitor that has been evaluated as a clinical candidate. We designed and prepared five isosteres in which the imidazo[1,2-a]pyrazine scaffold of entospletinib was altered to pyrazolo[3,4-d]pyrimidine, pyrrolo[3,2- d]pyrimidine, imidazo[4,5-b]pyridine, imidazo[4,5-c]pyridine and purine. The last two isosteres were the most potent SYK inhibitors, with IC50 values in the mid-nanomolar range. Importantly, three compounds also inhibited BTK more effectively than did entospletinib. Further experiments then showed that BCR signalling was suppressed in Ramos cells by the potent compounds. Preliminary kinase inhibition screening also revealed LCK and SRC as additional targets. Our results further support the hypothesis that multikinase targeting compounds could produce more robust responses in the treatment of B lymphoid neoplasms.
Keywords
protein kinase, scaffold hopping, selectivity, Spleen tyrosine kinase, Bruton’s tyrosine kinase
Introduction
Chronic or recurrent stimulation of the B-cell receptor (BCR) signalling pathway plays a crucial role in promoting the cell proliferation and survival underlying the progression of many common B-cell malignancies. Spleen tyrosine kinase (SYK) together with Bruton’s tyrosine kinase (BTK) and phosphatidylinositol 3-kinases (PI3K), key mediators of BCR signalling, are attractive targets in human haematological malignancies, particularly in various lymphoid neoplasms.[1] SYK is a multiple-domain cytoplasmic tyrosine kinase, which cooperates with BTK and PI3K in a tightly regulated positive feedback loop to activate phospholipase PLC-γ2. Entospletinib (GS-9973) is an imidazo[1,2-a]pyrazine-based selective and orally available SYK inhibitor (IC50 = 7.7 nM, Figure 1A) undergoing clinical evaluation for autoimmune and oncology indications.[2] This drug is generally well tolerated with only mild to moderate adverse events,[3] but it has limited clinical activity in non- Hodgkin lymphoma, mantle cell lymphoma and diffuse large B-cell lymphoma.[4,5] Combination regimens based on entospletinib, however, are expected to have significantly higher efficacy in multiple malignancies.
Figure 1. Structure of entospletinib (A) and its interactions with SYK (B, PDB entry 4PUZ). Scaffolds isosteric to entospletinib investigated in this work (C). The nitrogens able to form a hydrogen bond with Ala451 are shown in blue (A and C).
Isosteric replacement of functional groups or alteration of the heterocyclic core (scaffold hopping) is a commonly used strategy in drug discovery and development.[6] In the case of [6+5] nitrogenous heterocycles, imidazo[4,5-c]pyridines and imidazo[4,5-b]pyridines have rarely been studied within the large family of different protein kinase inhibitors. One of the reasons is the relatively poor synthetic availability of polysubstituted imidazo[4,5-c]pyridines and imidazo[4,5-b]pyridines compared to their widely studied purine analogues. Nevertheless, we recently developed a synthetic approach that enables the preparation of these compounds in a multistep sequence starting from simple and readily available materials.[7,8] To further extend our understanding of the structure-activity relationships of imidazo[1,2-a]pyrazine SYK inhibitors, we applied
the scaffold hopping approach and altered the entospletinib heterocyclic core to the imidazopyridine moieties. The reported crystal structure of entospletinib bound to the SYK kinase domain reveals the critical interactions, including three crucial H-bonds between the protein and the inhibitor: N-1 of the imidazopyrazine system accepts a hydrogen bond from the backbone NH of Ala451, the aniline NH of 9 forms a bond with the carbonyl group of the same residue, and the benzimidazole NH donates a hydrogen to the carboxyl group of Asp512 (Figure 1B).[2] According to these findings, imidazo[4,5-b]pyridine and imidazo[4,5-c]pyridine analogues should be able to form the key interactions. Furthermore, we extended the family of compounds with purine, pyrazolo[3,4-d]pyrimidine and pyrrolo[3,2-d]pyrimidine isosteres of entospletinib (Figure 1C). The set of five isosteric derivatives was then used to analyse the impact of nitrogen/carbon replacement on SYK inhibition and the targeting of BCR signalling in cells.
Results and discussion
The syntheses of SYK isosteres
The target isosteres were synthesized in 2 – 8 steps depending on the type of the target compound. In the case of pyrazolo[3,4-d]pyrimidine 5 and pyrrolo[3,2-d]pyrimidine 6, the sequences started from commercially available dichloro derivatives 1 and 2. The first step was regioselective nucleophilic substitution with 4- morpholinoaniline, which yielded intermediates 3 and 4, respectively (Scheme 1). Interestingly, the subsequent Pd-catalysed Suzuki coupling was feasible directly, without the need for pyrazole/pyrrole protection, and yielded final compounds 5 and 6, respectively (Scheme 1).
Scheme 1. Synthesis of analogues 5 and 6. Reagents and conditions: (i) 4-morpholinoaniline, N,N- diisopropylethylamine (DIPEA), DMF, 100 °C, 16 h (84%, 3; 86%, 4); (ii) 1H-indazole-6-boronic acid pinacol ester, Pd(PPh3)4, NaHCO3, dioxane/H2O 4:1, 80 °C, 16 h (65%, 5); (iii) 1H-indazole-6-boronic acid pinacol ester, Pd(dppf)Cl2, Cs2CO3, dioxane/H2O 4:1, 80 °C, 16 h (70%, 6).
The synthesis of purine derivative 11 started from commercially available 2,6-dichloropurine 7, which, in contrast to the previous case, had to be protected at the N9 position with a tetrahydropyranyl (THP) group. Regioselective nucleophilic substitution of 8 at C6 provided intermediate 9, which, upon Pd-mediated coupling and THP cleavage using trifluoroacetic acid (TFA), yielded target product 11 (Scheme 2).
Scheme 2. Synthesis of purine derivative 11. Reagents and conditions: (i) 3,4-dihydro-2H-pyran (DHP), p- toluenesulfonic acid (p-TSA), DMSO, 50 °C, 16 h (85%, 8); (ii) 4-morpholinoaniline, DIPEA, DMF, 100 °C, 16 h (84%, 9); (iii) 1H-indazole-6-boronic acid pinacol ester, XPhos-Pd-G2, K2CO3, dioxane/H2O 2:1, 80 °C, 16 h; (iv) neat TFA, r.t., 1 h (7% after 2 steps, 11).
The preparations of imidazo[4,5-b]pyridine 17 and imidazo[4,5-c]pyridine 18 were more difficult and were based on a different synthetic strategy inspired by previous reports.[7,8] Because the regioselective substitution of individual chlorines on the corresponding dichloro-imidazopyridines is impossible, the core heterocycle was built within the reaction sequence. For practical reasons, the synthesis was performed using polymer-supported amine 12. The polymer support served as a protecting group for 4-morhoplinoaniline, which enabled the regioselective formation of imidazopyridines 15/16 from pyridine-diamines 13/14. Additionally, the use of solid-phase synthesis was beneficial because it allowed the simple and fast preparation of the intermediates without the need for tedious purifications. Briefly, aminomethyl polystyrene resin was equipped with an acid-labile benzaldehyde linker, and resin-bound intermediate 12 was obtained upon reductive amination with 4-morhoplinoaniline (Scheme 3). Nucleophilic substitution and reduction of the nitro group using sodium dithionate provided intermediates 13 and 14, respectively. It is worth mentioning that their cyclization conditions using triethyl orthoformate depended on the position of the pyridine nitrogen atom (see Experimental part for details). After this step, we tried to continue the reaction sequence on the solid phase; however, Suzuki coupling with appropriate resin-bound intermediates was not feasible. For this reason, compounds 15 and 16 were cleaved from the resin, and the rest of the reaction sequence was completed using traditional solution-phase synthesis.
Scheme 3. Preparation of imidazo[4,5-b]pyridine 17 and imidazo[4,5-c]pyridine 18 analogues. Reagents and conditions: (i) a) DIPEA, DMF, r.t., 1 h; b) 4-(4-formyl-3-methoxyphenoxy)butanoic acid, 1-hydroxybenzotriazol (HOBt), N,N′-diisopropylcarbodiimide (DIC), DMF/CH2Cl2, r.t., 16 h; (ii) a) 4-morpholinoaniline, DMF/AcOH 10:1, r.t., 16 h, b) NaBH(OAc)3, DMF/AcOH 10:1, r.t., 4 h; (iii) 4,6-dichloro-3-nitropyridin-2-amine (yielded 13) or 2,6- dichloro-3-nitropyridin-4-amine (yielded 14), DIPEA, DMF, 80 °C, 16 h; (iv) Na2S2O4, K2CO3, DMF/H2O 2:1, 80 °C, 5 h; (v) CH(OEt)3/DMSO 1:1, 80 °C, 24 h (for 15) or 100 °C, 48 h (for 16); (vi) neat TFA, r.t., 2 h, (vii) DHP, p-TSA, EtOAc, 50 °C, 2 h; (viii) 1H-indazole-6-boronic acid pinacol ester, K2CO3, XPhos-Pd-G2, dioxane/H2O 2:1, 100 °C, 16 h; (ix) neat TFA, r.t., 17 h (overall yield 15%, 17; overall yield 15%, 18).
Hence, after THP protection, Suzuki coupling and deprotection using the same procedures as described earlier in this text, target imidazo[4,5-b]pyridine 17 and imidazo[4,5-c]pyridine 18 were obtained (Scheme 3).
Kinase inhibition by the synthesized isosteres
First, the inhibition constants of the synthesized isosteres for SYK and BTK were determined (Table 1). The most potent SYK inhibitors were imidazo[4,5-c]pyridine 18 and purine 11, with mid-nanomolar IC50 values, although the values were approximately threefold higher than that of entospletinib. The other three compounds did not display reasonable SYK inhibition (IC50 > 1 µM). These changes are undoubtedly linked to the two hydrogen bonds between entospletinib and Ala451, providing a basis for strong interactions. The interaction of exocyclic NH (H-bond donor) with the carboxyl of Ala451 is probably weaker in imidazo[4,5- b]pyridine 17 relative to those of entospletinib and imidazo[4,5-c]pyridine 18. This can be explained by the different proximity of the exocyclic NH group to the pyridine-like nitrogen (ortho vs. para position), which decreases the electron density on the NH in entospletinib and imidazo[4,5-c]pyridine 18, leading to an increased ability to serve as a H-bond donor. This explanation is also compatible with the high potency of purine 11. On the other hand, despite the presence of an o-pyridine-like nitrogen in both pyrazolo[3,4-
d]pyrimidine 5 and pyrrolo[3,2-d]pyrimidine 6, their lower potencies can be attributed to the amidic nitrogen of Ala451 not being able to interact due to the absence of a suitable H-bond acceptor (in 5) or to the presence of pyrrole-like nitrogen (in 6), as this part of the molecule serves as a H-bond donor.
The sensitivity of BTK to the prepared compounds did not follow the same pattern; 5, 11 and 18 inhibited BTK more effectively than did entospletinib. Purine 11 exhibited an 18-fold preference for BTK over entospletinib, which is in agreement with a previous report [9]; the ability of purine to bind to the active site is supported by interactions through a water network that forms a bridge to a conserved Lys430 (Supplementary Figure S1). Therefore, it is not surprising that nanomolar inhibition of BTK was also observed for derivatives 5 and 18, both of which have H-bond forming heterocyclic NH groups in their five-membered rings.
Table 1. Kinase inhibition data of entospletinib and its isosteres.
Core Cmpd. IC50 (µM)*
SYK BTK
pyrazolo[3,4-d]pyrimidine 5 1.597 0.167
pyrrolo[3,2-d]pyrimidine 6 9.545 >20
purine 11 0.201 0.073
imidazo[4,5-b]pyridine 17 8.206 1.45
imidazo[4,5-c]pyridine 18 0.301 0.369
entospletinib 0.082 0.801
ibrutinib n.d. 0.001 *assayed in triplicate
The observed differences in potencies against SYK and BTK motivated us to investigate the compounds’ selectivity over a wider panel of protein kinases. Their selectivities were therefore measured at a single concentration of 1 μM on 50 kinases (Figure 2). Entospletinib was assayed as a control under the same conditions; the only publicly available screening results on 359 kinases revealed 58 kinases that are effectively targeted with efficacies of > 90%, but these results were obtained with 10 µM compound, which was not directly comparable to our compounds.[2] As shown in Figure 2, entospletinib was found to block SYK most strongly, confirming our results (Table 1). Furthermore, profiling data confirmed the potency of entospletinib isosteres 5, 11, 17 and 18 on BTK. Interestingly, not SYK but SRC kinase was the top hit of entospletinib and all its isosteres. The kinase inhibition screening also revealed that purine 11 and imidazo[4,5-c]pyridine 18 significantly reduced the activity of LCK, another SRC family member. While LCK was only weakly inhibited by
entospletinib (LCK ranked 14
th
; 71% residual activity), purine 11 and imidazo[4,5-c]pyridine 18 were
substantially more potent (2nd and 4th ranks; 14% and 28% residual activity, respectively). Because SRC kinases are responsible for propagating the BCR signal in cells, their parallel inhibition seems to be necessary for potent suppression of tumour growth.[10,11]
Figure 2. Kinase inhibition by entospletinib and its isosteres (expressed as % of residual activity). The profiling was performed in duplicate at a compound concentration of 1 μM. A complete list of profiled kinases is included in the Supporting Information.
Cellular activities of synthesized isosteres
Based on the ability of some entospletinib isosteres to simultaneously diminish the activities of both SYK and BTK, we further investigated their effects on BCR signalling in Ramos cells, a model of Burkitt lymphoma in which BCR signalling can be activated by immunoglobulin M. We therefore treated Ramos cells with various doses of compounds for 1 h before stimulation of BCR signalling and analysed them by Western blot. As shown in Figure 3, entospletinib caused effective dephosphorylation of SYK at Y323 (autophosphorylation) and its three downstream targets, namely, PLCγ2, Akt and ERK. Dephosphorylation of BTK at Y223 is probably caused by direct inhibition of BTK by entospletinib or by the inhibition of upstream SRC kinase(s) that are able to phosphorylate BTK on Y551 and prime it for further autophosphorylation at Y223.[12] Rapid suppression of SYK phosphorylation was also observed in IgM-activated cells treated with imidazo[4,5-c]pyridine 18 and purine 11, which, in our opinion, clearly corresponds with the measured inhibition of recombinant SYK (Table 1). While the purine derivative caused a greater decrease in SYK-downstream proteins compared to entospletinib, imidazo[4,5-c]pyridine 18 did not completely diminish ERK phosphorylation (Figure 3). Weaker pyrazolo[3,4- d]pyrimidine 5 did not cause dephosphorylation of ERK but partly blocked activation of BTK; the same outcome was also obtained with the reference BTK inhibitor, ibrutinib, which selectively targets the BTK pathway only (see Supplementary Figure S2). In agreement with kinase assays, neither imidazo[4,5-b]pyridine 17 nor pyrrolo[3,2-d]pyrimidine 6 changed the expression of the proteins of interest.
Figure 3. Effects of entospletinib and its isosteres on SYK signalling in Ramos cells. The cells were treated with the test compounds for 1 h and then stimulated with anti-IgM (5 μg/ml) for 10 min before harvest. Proteins were then detected by immunoblotting in cell lysates. PCNA was probed for equal protein loading.
Discussion
BCR signalosome members have been validated as rational drug targets effective in B-cell malignancies. The clinical efficacies of entospletinib, fostamanib and idelalisib are encouraging, but preclinical experiments suggest that simultaneous or combinatory targeting of signalosome members might be even more beneficial.[13] Identification of drugs targeting rationally selected enzymes could result in higher efficacies even for patients who have developed resistance to the drugs. For example, point mutations in BTK or PLCγ2 reduce their affinity for drugs and diminishes their ability to inhibit activities.[14]
Isosteric replacement of functional groups or alteration of the heterocyclic core is a commonly used strategy in drug discovery and development. Within the realm of protein kinases, this approach was successfully applied, for example, in the modification of the aminoindazole core of TYK2 inhibitors,[15] pyrazole core of DLK inhibitors[16] and the development of pyrazolo[4,3-d]pyrimidine inhibitors of CDK.[17,18] In a similar fashion, we attempted to modify the heterocyclic core of entospletinib, and the work yielded
compounds with significantly different potency on SYK and altered kinase selectivity. Imidazo[4,5-c]pyridine 18 and purine 11 are weaker SYK inhibitors but simultaneously target BTK and LCK, which are other members of the BCR signalosome family. Although the detailed role of LCK in BCR signalling has not been fully elucidated, there is experimental evidence that it potentiates the signalling pathway and that LCK is a targetable mediator of BCR signalling in chronic lymphocytic leukaemia.[11,19] In line with such observations, the recently developed BCR signalosome inhibitor IQS019, which shows strong affinity for BTK, SYK and LYN, showed improved efficacy when compared to the BTK inhibitor ibrutinib; moreover, it was also active in cells with acquired resistance to ibrutinib. [20] Another promising example with a similar profile is ArQule 531, a multikinase inhibitor of BTK, SRC and LYN. [21] This preclinical candidate showed activity in chronic lymphocytic leukaemia; treated cells showed decreased BTK-mediated functions, including BCR signalling.[22] SYK, BTK, LCK and LYN are highly related to SRC, which is another kinase highly sensitive not only to ArQule 531 but also to entospletinib and its isosteres (except for 6) described herein. It is becoming clearer that multikinase targeting compounds could be especially helpful and produce more robust responses in the treatment of B lymphoid neoplasms, which respond poorly to current BCR inhibitors.
Experimental section Chemistry
General information
All reagents were of reagent grade and were used without further purification. Solvents and chemicals were purchased from Sigma-Aldrich (Milwaukee, US), Acros Organics (Geel, Belgium) or Fluorochem (Derbyshire, UK). Dry solvents were dried over 4 Å molecular sieves or stored as received from commercial suppliers. Aminomethyl resin (100-200 mesh, 1% DVB, 0.98 mmol/g) was obtained from AAPPTec (Louisville, US).
The LC/MS analyses were carried out on a UHPLC-MS system consisting of an Acquity UHPLC chromatograph with a photodiode array detector and a single quadrupole mass spectrometer (Waters) using an X-Select C18 column at 30 °C and a flow rate of 600 μL/min. The mobile phase was (A) 0.01 M ammonium acetate in H2O and (B) CH3CN, and the gradient was linearly programmed from 10% A to 80% B over 2.5 min and then held for 1.5 min. The column was re-equilibrated with 10% solution B for 1 min. The ESI source operated at a discharge current of 5 μA, vaporizer temperature of 350 °C and capillary temperature of 200 °C.
Purification was carried out on a C18 reversed-phase column (YMC Pack ODS-A, 20× 100 mm, 5 μm particles), and a gradient of 10 mM aqueous ammonium acetate and CH3CN at a flow rate of 15 mL/min was used. For lyophilization of the residual solvents at -110 °C, a ScanVac Coolsafe 110-4 was used.
NMR spectra were recorded on a JEOL ECX500 spectrometer at a magnetic field strength of 11.75 T (with operating frequencies of 500.16 MHz for 1H and 125.77 MHz for 13C). Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J) are reported in Hertz (Hz). The 1H and 13C NMR chemical shifts (δ in ppm) were referenced to the residual signals of DMSO-d6 [2.50 (1H) and 39.52 (13C)]. Abbreviations in NMR spectra: br s – broad singlet, d – doublet, dd – doublet of doublets, m – multiplet, s – singlet.
HRMS analysis was performed using an LC chromatograph (Dionex Ultimate 3000, Thermo, MA, US) and an Exactive Plus Orbitrap high-resolution mass spectrometer (Thermo Fischer Scientific, MA, USA) operating in positive full scan mode (120 000 FWMH) in the range of 100-1000 m/z. The settings for electrospray ionization were as follows: oven temperature of 150 °C and source voltage of 3.6 kV. The acquired data were internally calibrated with phthalate as a contaminant in CH3OH (m/z 297.15909). Samples were diluted to a final concentration of 0.1 mg/mL in H2O and CH3OH (50:50, v/v). Before HPLC separation (Phenomenex Gemini column, 50 × 2.00 mm, 3 µm particles, C18), the samples were injected by direct infusion into the mass spectrometer using an autosampler. The mobile phase was isocratic 80% ACN and 20% buffer (0.01 M ammonium acetate) or 95% MeOH + 5% water + 0.1% HCOOH at a flow rate of 0.3 mL/min.
Solid-supported reactions were carried out in plastic reaction vessels (syringes, each equipped with a porous disk) using a manually operated synthesizer (Torviq, Niles, US, www.torviq.com) or in dried glassware, unless stated otherwise. The volume of wash solvent was 10 mL per 1 g of resin. For washing, the resin slurry was shaken with fresh solvent for at least 1 min before changing the solvent. Resin-bound intermediates were dried under a stream of nitrogen for prolonged storage and/or quantitative analysis. For the LC/MS analysis, a sample of resin (~5 mg) was treated with CH2Cl2/TFA (1:1, 1 mL, v/v), the cleavage cocktail was evaporated under a stream of nitrogen, and the cleaved compounds were extracted into CH3CN (1 mL). The yields of the final compounds prepared by solid-phase synthesis were calculated from the loading of compound 12 (0.56 mmol/g) according to a published procedure. [23]
Experimental procedures
General procedure A for substitution with 4-morpholinoaniline
To a stirred solution of starting material (1 eq) in DMF (2 mL/0.5 mmol of SM) were added 4-morpholinoaniline (1.1 eq) and DIPEA (1.1 eq). The reaction mixture was stirred at 100 °C for 16 h, diluted with saturated NH4Cl (50 mL/0.5 mmol of SM) and extracted with EtOAc (5 × 50 mL/0.5 mmol). The organic extracts were combined, dried over MgSO4, filtered and concentrated under reduced pressure. The crude products were purified by flash chromatography (CH2Cl2/CH3OH 15:1 to 7:1 v/v) to yield the desired products.
General procedure B-1 for the Suzuki coupling
A flask with starting material (0.2 mmol) was properly flushed with nitrogen. 1H-Indazole-6-boronic acid pinacol ester (1.3 eq), Pd(PPh3)4 (0.1 eq) and NaHCO3 (2 eq) in a properly degassed solution of dioxane/H2O (4:1, 5 mL, v/v) were added to the flask with starting material. The reaction mixture was stirred at 80 °C for 40 h. When it reached full conversion to the desired intermediate, the mixture was diluted with saturated NH4Cl (40 mL) and brine (20 mL) and extracted with EtOAc (5 × 60 mL). The organic extracts were combined, dried over MgSO4, filtered and concentrated under reduced pressure. The crude products were purified by flash chromatography (CH2Cl2/CH3OH 10:1 v/v) to yield the desired products.
General procedure B-2 for the Suzuki coupling
A flask with starting material (0.1 mmol) was properly flushed with nitrogen. 1H-Indazole-6-boronic acid pinacol ester (1.5 eq), Pd(dppf)Cl2 (0.2 eq) and Cs2CO3 (3 eq) in a properly degassed solution of dioxane/H2O (4:1, 5 mL, v/v) were added to the flask with starting material. The reaction mixture was stirred at 80 °C for 24 h. When it reached full conversion to the desired intermediate, the mixture was diluted with saturated NH4Cl (50 mL) and extracted with EtOAc (5 × 50 mL). The organic extracts were combined, dried over MgSO4, filtered and concentrated under reduced pressure. The crude products were purified by flash chromatography (CH2Cl2/CH3OH/AcOH 5:1:0.1 v/v) to yield the desired products.
General procedure B-3 for the Suzuki coupling
A pressure ampule with starting material (0.5 mmol) was properly flushed with nitrogen. 1H-Indazole-6-boronic acid pinacol ester (1.3 eq), XPhos-Pd-G2 (0.1 eq) and K2CO3 (2 eq) in a properly degassed solution of dioxane/H2O (2:1, 12 mL, v/v) were added to the pressure ampule with starting material. The reaction mixture was stirred at 125 °C for 5 – 10 h. When it reached full conversion to the desired intermediates, the mixture was diluted with water (100 mL) and extracted with EtOAc (3 × 100 mL). The organic extracts were combined, dried over MgSO4, filtered and concentrated under reduced pressure. The crude products were lyophilized overnight and used in the next step without further purification.
General procedure C for DHP protection
To a stirred solution of starting material (1 eq) in EtOAc (2 mL) were added 3,4-dihydro-2H-pyran (4 eq) and pTSA (6 mol%). The reaction mixture was stirred at 50 °C for 90 min. When it reached full conversion to the desired intermediates, it was diluted with saturated Na2CO3 (50 mL) and extracted with EtOAc (3 × 50 mL). The organic extracts were combined, dried over MgSO4, filtered and concentrated under reduced pressure. The crude products used in the next step without further purification.
General procedure D for THP deprotection
The corresponding intermediates (0.5 mmol) were dissolved in neat TFA (5 mL) and stirred at ambient temperature for 16 h, during which it reached full conversion to the desired final compounds. The residual acid was concentrated under a stream of nitrogen to dryness, and the crude products were purified by semi- preparative HPLC.
Procedure for preparation of BAL resin
Aminomethyl polystyrene resin (1 g, loading 0.98 mmol/g) was swollen in CH2Cl2 (10 mL) for 30 min, washed with DMF (3 × 10 mL), shaken with DMF/TEA (5:1, 10 mL) for an additional 30 min and then washed again with DMF (5 × 10 mL). Backbone amide linker (BAL) (700 mg, 2.94 mmol) and HOBt (450 mg, 2.94 mmol) were dissolved in DMF/CH2Cl2 (1:1, 10 mL, v/v), and DIC (460 µL, 2.94 mmol) was added. The resulting solution was added to a polypropylene fritted syringe with the aminomethyl resin. The reaction slurry was shaken at ambient temperature for 16 h and then washed with DMF (3 × 10 mL) and CH2Cl2 (3 × 10 mL). The negative bromophenol blue test confirmed the quantitative acylation of the amino groups.
Procedure for reductive amination with 4-morpholinoaniline
BAL resin (1 g, loading 0.98 mmol/g) was swollen in CH2Cl2 (10 mL) for 30 min and then washed with dry THF (3 × 10 mL) and dry DMF (3 × 10 mL). A solution of 4-morpholinoaniline (872 mg, 4.9 mmol) in DMF/AcOH (10:1, 10 mL, v/v) was added to a polypropylene fritted syringe with BAL resin and shaken at ambient temperature for 16 h. Then, NaBH(OAc)3 (210 mg, 2.94 mmol) in DMF/AcOH (20:1, 5 mL, v/v) was added portion-wise to the reaction mixture over a period of 4 h. Then the mixture was washed with DMF (5 × 10 mL) and CH2Cl2 (3 × 10 mL) and neutralized with DMF/TEA (10:1, 10 mL, v/v) for an additional 30 min to obtain resin 12.
Procedure for arylation with substituted halopyridines
Resin 12 (2 × 1 g) was swollen in CH2Cl2 (10 mL) for 30 min and then washed with CH2Cl2 (10 mL). 4,6-Dichloro- 3-nitropyridine-2-amine (420 mg, 2 mmol) or 2,6-dichloro-3-nitropyridine-4-amine (420 mg, 2 mmol) and DIPEA (520 µL, 3 mmol) were dissolved in anhydrous DMF (10 mL) and added to vials containing resins 12. The reaction slurry was stirred at 65 °C for 20 h and then washed with DMF (3 × 10 mL), DMSO (3 × 10 mL), DMF (3 × 10 mL) and CH2Cl2 (5 × 10 mL). The analytical sample (~ 5 mg) of the resins was reacted with Fmoc-Cl (68 mg, 0.22 mmol) and DIPEA (45 µL, 0.26 mmol) in CH2Cl2 at ambient temperature for 1 h. Subsequent cleavage from the resin and UHPLC-MS analysis confirmed the full conversion to the arylated products.
Procedure for the reduction of the nitro group
The corresponding resins (1 g) were swollen in CH2Cl2 (10 mL) for 30 min and then washed with CH2Cl2 (3 × 10 mL). A solution of Na2S2O4 (1.74 g, 10 mmol), K2CO3 (1.38 g, 10 mmol) and TBAHS (610 mg, 1.8 mmol) in DMF/H2O (2:1, 15 mL, v/v) was added to the vials with the resins. The reaction slurry was stirred at 80 °C for 5 h and then washed with DMF (5 × 10 mL), H2O (5 × 10 mL), DMF (5 × 10 mL) and CH2Cl2 (5 × 10 mL). Subsequent cleavage from the resin and UHPLC-MS analysis confirmed the full conversion to reduced products 13 and 14.
Procedure for cyclization to the imidazopyridine scaffold
Corresponding resins 13 and 14 (2 × 1 g) were swollen in CH2Cl2 (10 mL) for 30 min and then washed with CH2Cl2 (3x 10 mL). A solution of triethylorthoformate/DMSO (1:1, 10 mL, v/v) was added to the vials with the resins. The reaction slurry was stirred at 80 °C for 24 h and then washed with DMSO (5 × 10 mL), DMF (5 × 10 mL) and CH2Cl2 (5 × 10 mL). Subsequent cleavage from the resin and UHPLC-MS analysis confirmed the full conversion to cyclized products.
General procedure E for cleavage from the resin
The cleavage of the intermediates from the resin on an analytical scale (~ 5 mg) prior to analysis was carried out in CH2Cl2/TFA (1:1, 1 mL, v/v) for 30 min according to the General Information. The cleavage of the intermediates from the resin after cyclization on a preparative scale (1 g) was conducted using the following procedure: The corresponding resin was swollen in CH2Cl2 (10 mL) for 30 min and then washed with CH2Cl2 (5 × 10 mL). A solution of CH2Cl2/TFA (1:1, 10 mL, v/v) was added to each polypropylene fritted syringe with resin.
The reaction slurry was shaken at ambient temperature for 3 h and then washed with CH2Cl2/TFA (1:1, 3x 5 mL, v/v) and CH2Cl2 (3x 5 mL). The cleavage cocktail and the combined washes were concentrated under a stream of nitrogen, and the residual solvents were lyophilized overnight. Crude intermediates 15 and 16 were used without further purification.
Analytical data of intermediates and final compounds
6-chloro-N-(4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 3
Compound 3 was prepared by General procedure A from starting material 1 as pale brown solid (262 mg, 84% yield). 1H NMR (500 MHz, DMSO-d6): δ 13.65 (br s, 1H), 10.22 (s, 1H), 8.30 (br s, 1H), 7.75 – 7.25 (m, 2H), 7.00 (d, J = 8.5 Hz, 2H), 3.82 – 3.66 (m, 4H), 3.14 – 3.03 (m, 4H) ppm. 13C NMR (126 MHz, DMSO-d6): δ 156.8, 155.6, 154.4, 147.9, 132.8, 129.9, 122.6, 115.3, 99.3, 66.1, 48.5 ppm. HRMS (ESI): m/z calcd for C15H16ClN6O [M+H]+ = 331.1069, found [M+H]+ = 331.1069.
2-chloro-N-(4-morpholinophenyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine 4
Compound 4 was prepared by General procedure A from starting material 2 as dark brown solid (100 mg, 86% yield). 1H NMR (500 MHz, DMSO-d6): δ 11.19 – 11.06 (m, 1H), 9.33 (s, 1H), 7.66 – 7.64 (m, 1H), 7.60 – 7.58 (m, 2H), 7.00 – 6.98 (m, 2H), 6.41 – 6.40 (m, 1H), 3.77 – 3.73 (m, 4H), 3.11 – 3.06 (m, 4H) ppm. 13C NMR (126 MHz, DMSO- d6): δ 150.2, 148.9, 147.5, 147.2, 131.0, 129.7, 121.2, 115.6, 112.5, 101.3, 66.1, 48.8 ppm. HRMS (ESI): m/z calcd for C16H17ClN5O [M+H]+ = 330.1116, found [M+H]+ = 330.1116.
6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5
Compound 5 was prepared by General procedure B-1 from starting material 3 as dark brown solid (50 mg, 65% yield). 1H NMR (500 MHz, DMSO-d6): δ 13.55 (br s, 1H), 13.26 (s, 1H), 9.91 (s, 1H), 8.60 – 8.59 (m, 1H), 8.20 (dd, J = 8.6, 1.3 Hz, 1H), 8.12 (s, 1H), 7.83 (dd, J = 8.6, 0.8 Hz, 1H), 7.81 – 7.59 (m, 2H), 7.08 – 7.06 (m, 2H), 3.80 – 3.76 (m, 4H), 3.17 – 3.13 (m, 4H) ppm. 13C NMR (126 MHz, DMSO-d6): δ 160.9, 155.9, 140.1, 139.4, 136.2, 133.4, 132.3, 131.1, 125.3, 123.9, 122.4, 120.2, 120.0, 115.4, 109.9, 66.1, 48.8 ppm. HRMS (ESI): m/z calcd for C22H21N8O [M+H]+ = 413.1834, found [M+H]+ = 413.1833.
2-chloro-N-(4-morpholinophenyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine 6
Compound 6 was prepared by General procedure B-2 from starting material 4 as dark brown solid (40 mg, 70% yield). 1H NMR (500 MHz, DMSO-d6): δ 13.32 (br s, 1H), 12.29 (br s, 1H), 10.36 (br s, 1H), 8.56 – 8.55 (m, 1H), 8.17 (d, J = 8.5 Hz, 1H), 8.12 (s, 1H), 7.97 – 7.93 (m, 2H), 7.85 (d, J = 8.6 Hz, 1H), 7.71 – 7.70 (m, 1H), 7.07 – 7.05 (m, 2H), 6.57 – 6.54 (m, 1H), 3.79 – 3.75 (m, 4H), 3.14 – 3.10 (m, 4H) ppm. 13C NMR (126 MHz, DMSO- d6): δ 154.5, 146.9, 140.2, 133.3, 131.9, 129.3, 123.6, 121.0, 120.1, 120.1, 115.6, 113.1, 109.5, 100.6, 66.1, 49.0 ppm. HRMS (ESI): m/z calcd for C23H22N7O [M+H]+ = 412.1879, found [M+H]+ = 412.1880.
2,6-dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine 8
Compound 8 was prepared by General procedure C from starting material 7 as pale yellow solid (338 mg, 85% yield). The analytical data corresponded with literature.[24]
2-chloro-N-(4-morpholinophenyl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine 9
Compound 9 was prepared by General procedure A from starting material 8 as pale amorphous solid (433 mg, 84% yield). 1H NMR (500 MHz, DMSO- d6): δ 10.11 (s, 1H), 8.48 (s, 1H), 7.63 (d, J = 8.7 Hz, 2H), 6.97 – 6.92 (m, 2H), 5.61 (dd, J = 10.9, 2.0 Hz, 1H), 4.03 – 3.99 (m, 1H), 3.78 – 3.72 (m, 4H), 3.72 – 3.67 (m, 1H), 3.11 – 3.05 (m, 4H), 2.28 – 2.18 (m, 1H), 2.00 – 1.93 (m, 2H), 1.79 – 1.70 (m, 1H), 1.63 – 1.55 (m, 2H) ppm. 13C NMR (126 MHz, DMSO- d6): δ 152.7, 152.5, 149.9, 147.6, 139.8, 130.5, 122.7, 118.4, 115.1, 80.9, 67.6, 66.1, 48.8, 29.9, 24.4, 22.2 ppm. HRMS (ESI): m/z calcd for C20H24ClN6O2 [M+H]+ = 415.1643, found [M+H]+ = 415.1644 .
2-(1H-indazol-6-yl)-N-(4-morpholinophenyl)-9H-purin-6-amine 11
Compound 11 was prepared from starting material 9 by General procedure B-3, followed by General procedure C, as yellow amorphous solid (32 mg, 7% yield). 1H NMR (500 MHz, DMSO- d6): δ 13.21 (br s, 1H), 13.13 (br s, 1H), 9.65 (br s, 1H), 8.56 (s, 1H), 8.25 (s, 1H), 8.19 (d, J = 8.5 Hz, 1H), 8.10 (s, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.83 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 8.7 Hz, 2H), 3.78 – 3.76 (m, 4H), 3.12 – 3.10 (m, 4H) ppm. 13C NMR (126 MHz, DMSO- d6): δ 157.6, 151.5, 151.2, 146.7, 140.3, 139.9, 136.7, 133.4, 132.1, 123.5, 121.8, 120.1, 120.0, 118.3, 115.3, 109.3, 66.1, 49.1 ppm. HRMS (ESI): m/z calcd C22H21N8O for [M+H]+ = 413.1835, found [M+H]+ = 413.1833 .
5-chloro-N-(4-morpholinophenyl)-3H-imidazo[4,5-b]pyridin-7-amine 15
Compound 15 was prepared from the starting material 13 by cyclization, followed by General procedure E, as a dark brown solid. Crude compound was used without further purification. MS (ESI): m/z 330 [M+H]+.
6-chloro-N-(4-morpholinophenyl)-1H-imidazo[4,5-c]pyridin-4-amine 16
Compound 16 was prepared from starting material 14 by cyclization, followed by General procedure E, as a dark brown solid. Crude compound was used without further purification. MS (ESI): m/z 330 [M+H]+.
5-(1H-indazol-6-yl)-N-(4-morpholinophenyl)-3H-imidazo[4,5-b]pyridin-7-amine 17
Compound 17 was obtained from starting material 15 by General procedure C, General procedure B-3 and General procedure D, as a pale yellow solid (40 mg, 15% yield, calculated from loading of the resin 12). 1H NMR (500 MHz, DMSO- d6): δ 13.06 (br s, 1H), 12.81 (s, 1H), 8.71 (s, 1H), 8.19 (s, 1H), 8.07 (s, 1H), 8.02 (s, 1H), 7.80 – 7.79 (m, 1H), 7.69 – 7.68 (m, 1H), 7.32 (d, J = 8.5 Hz, 2H), 7.20 (s, 1H), 7.00 (d, J = 8.6 Hz, 2H), 3.78 – 3.71 (m, 4H), 3.11 – 3.09 (m, 4H) ppm. 13C NMR (126 MHz, DMSO- d6): δ 152.7, 148.4, 147.5, 143.8, 140.4, 139.7, 138.3, 133.2, 132.1, 125.8, 123.5, 122.6, 120.3, 119.6, 115.9, 107.7, 102.60, 66.1, 48.8 ppm. HRMS (ESI): m/z calcd C23H22N7O for [M+H]+ = 412.1878, found [M+H]+ = 412.1880.
6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)-1H-imidazo[4,5-c]pyridin-4-amine 18
Compound 18 was obtained from starting material 16 by General procedure C, General procedure B-3 and General procedure D, as a pale brown solid (40 mg, 15% yield, calculated from loading of the resin 12). 1H NMR (500 MHz, DMSO- d6): δ 13.14 (br s, 1H), 8.84 (br s, 1H), 8.28 (s, 1H), 8.25 (s, 1H), 8.08 (s, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.87 – 7.80 (m, 2H), 7.60 – 7.55 (m, 1H), 7.02 – 6.91 (m, 2H), 3.76 – 3.74 (m, 4H), 3.10 – 3.04 (m, 4H) ppm. 13C NMR (126 MHz, DMSO- d6): δ 147.2, 146.4, 145.6, 140.7, 140.6, 139.8, 138.2, 133.9, 133.0, 125.7, 122.3, 120.1, 119.9, 119.2, 115.6, 107.4, 96.9, 66.1, 49.4 ppm. HRMS (ESI): m/z calcd C23H22N7O for [M+H]+ = 412.1879, found [M+H]+ = 412.1880.
Cell Culture
The Ramos cell line was obtained from DSMZ and was cultivated according to the provider’s instructions. The cells were grown in Iscove’s modified Dulbecco’s medium supplemented with 10% foetal bovine serum, 4 mM glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin and were cultivated in a humidified CO2 incubator at 37 °C.
Chemicals
Entospletinib and ibrutinib were purchased from MedChemExpress. Goat F(ab’)2 anti-human IgM was purchased from Southern Biotech.
Western blotting
Cell lysates were prepared, and then the proteins were separated on SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes. After blocking, overnight incubation with specific primary antibodies and incubation with peroxidase-conjugated secondary antibodies, the peroxidase activity was detected with SuperSignal West Pico reagents (Thermo Scientific) using a CCD camera LAS-4000 (Fujifilm). The following specific antibodies were purchased from Cell Signaling Technology (anti-phospho SYK Y323; anti- SYK, clone D3Z1E; anti-phospho PLCγ2 Y1217; anti- PLCγ2; anti-phospho BTK Y223; anti-BTK, clone C82B8; anti- phospho ERK1/2 T202/Y204; anti- ERK1/2; anti-phospho Akt S473, clone D9E; anti-Akt, clone C67E7). The anti- PCNA (clone PC-10) antibody was generously gifted by Dr. B. Vojtěšek (Masaryk Memorial Cancer Institute, Brno, Czech Republic). All antibodies were diluted in TBS containing 3% BSA and 0.1% Tween 20.
Kinase reaction
The BTK and SYK (ProQinase, GST-tagged) kinase reactions were assayed with the peptide substrate poly(Glu,Tyr) (4:1, 1 mg/mL, Merck) in the presence of 15/1.5 μM ATP for BTK/SYK, 0.05 μCi [γ-33P]ATP, and the test compound in a final volume of 10 μL, all in a reaction buffer (60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 2.5 μg/50 μL PEG20.000). The reactions were stopped by adding 5 μL of 3% aq. H3PO4. Aliquots were spotted onto P-81 phosphocellulose (Whatman), washed 3× with 0.5% aq. H3PO4, and finally air-dried. Kinase inhibition was quantified using a FLA-7000 digital image analyser. The concentration of the test compound required to reduce the kinase activity by 50% was determined from the dose-response curves and is reported as the IC50 value.
Acknowledgements
The authors wish to acknowledge the support from Palacký University (IGA_LF_2020_012), the Ministry of Health of the Czech Republic (17-31834A) and the European Regional Development Fund (Project ENOCH, No. CZ.02.1.01/0.0/0.0/16_019/0000868).
References
[1]J. Sharman, P.J. Di, Targeting B-cell receptor signaling kinases in chronic lymphocytic leukemia: the promise of entospletinib, Ther. Adv. Hematol. 7 (2016) 157-170.
[2]K.S. Currie, J.E. Kropf, T. Lee, P. Blomgren, J. Xu, Z. Zhao, S. Gallion, J.A. Whitney, D. Maclin, E.B. Lansdon, P. Maciejewski, A.M. Rossi, H. Rong, J. Macaluso, J. Barbosa, J.A. Di Paolo, S.A. Mitchell, Discovery of GS- 9973, a selective and orally efficacious inhibitor of spleen tyrosine kinase, J. Med. Chem. 57 (2014) 3856- 3873.
[3]S. Ramanathan, J.A. Di Paolo, F. Jin, L. Shao, S. Sharma, M. Robeson, B.P. Kearney, Pharmacokinetics, Pharmacodynamics, and Safety of Entospletinib, a Novel pSYK Inhibitor, Following Single and Multiple Oral Dosing in Healthy Volunteers, Clin. Drug Investig. 37 (2017) 195-205.
[4]D.J. Andorsky, K.S. Kolibaba, S. Assouline, A. Forero-Torres, V. Jones, L.M. Klein, D. Patel-Donnelly, M. Smith, W. Ye, W. Shi, C.A. Yasenchak, J.P. Sharman, An open-label phase 2 trial of entospletinib in indolent non-Hodgkin lymphoma and mantle cell lymphoma, Br. J. Haematol. 184 (2019) 215-222.
[5]F.T. Awan, M.J. Thirman, D. Patel-Donnelly, S. Assouline, A.V. Rao, W. Ye, B. Hill, J.P. Sharman, Entospletinib monotherapy in patients with relapsed or refractory chronic lymphocytic leukemia previously treated with B-cell receptor inhibitors: results of a phase 2 study, Leuk. Lymphoma. 60 (2019) 1972-1977.
[6]H. Sun, G. Tawa, A. Wallqvist, Classification of scaffold-hopping approaches, Drug Discov. Today. 17 (2012) 310-324.
[7]B. Lemrova, P. Smyslova, I. Popa, T. Ozdian, P. Zajdel, M. Soural, Directed solid-phase synthesis of trisubstituted imidazo[4,5-c]pyridines and imidazo[4,5-b]pyridines, ACS Comb. Sci. 16 (2014) 558-565.
[8]D. Vanda, M. Soural, V. Canale, S. Chaumont-Dubel, G. Satala, T. Kos, P. Funk, V. Fulopova, B. Lemrova, P. Koczurkiewicz, E. Pekala, A.J. Bojarski, P. Popik, P. Marin, P. Zajdel, Novel non-sulfonamide 5-HT6 receptor partial inverse agonist in a group of imidazo[4,5-b]pyridines with cognition enhancing properties, Eur. J. Med. Chem. 144 (2018) 716-729.
[9]Q. Shi, A. Tebben, A.J. Dyckman, H. Li, C. Liu, J. Lin, S. Spergel, J.R. Burke, K.W. McIntyre, G.C. Olini, J. Strnad, N. Surti, J.K. Muckelbauer, C. Chang, Y. An, L. Cheng, Q. Ruan, K. Leftheris, P.H. Carter, J. Tino, G.V. De Lucca, Purine derivatives as potent Bruton’s tyrosine kinase (BTK) inhibitors for autoimmune diseases, Bioorg. Med. Chem. Lett. 24 (2014) 2206-2211.
[10]E. Battistello, N. Katanayeva, E. Dheilly, D. Tavernari, M.C. Donaldson, L. Bonsignore, M. Thome, A.L. Christie, M.A. Murakami, O. Michielin, G. Ciriello, V. Zoete, E. Oricchio, Pan-SRC kinase inhibition blocks B-cell receptor oncogenic signaling in non-Hodgkin lymphoma, Blood. 131 (2018) 2345-2356.
[11]F. Talab, J.C. Allen, V. Thompson, K. Lin, J.R. Slupsky, LCK is an important mediator of B-cell receptor signaling in chronic lymphocytic leukemia cells, Mol. Cancer Res. 11 (2013) 541-554.
[12]D.J. Rawlings, A.M. Scharenberg, H. Park, M.I. Wahl, S. Lin, R.M. Kato, A.C. Fluckiger, O.N. Witte, J.P. Kinet, Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases, Science. 271 (1996) 822-825.
[13]S.L. Tan, C. Liao, M.C. Lucas, C. Stevenson, J.A. DeMartino, Targeting the SYK-BTK axis for the treatment of immunological and hematological disorders: recent progress and therapeutic perspectives, Pharmacol. Ther. 138 (2013) 294-309.
[14]S. Cheng, A. Guo, P. Lu, J. Ma, M. Coleman, Y.L. Wang, Functional characterization of BTK(C481S) mutation that confers ibrutinib resistance: exploration of alternative kinase inhibitors, Leukemia. 29 (2015) 895-900.
[15]T. Yogo, H. Nagamiya, M. Seto, S. Sasaki, H. Shih-Chung, Y. Ohba, N. Tokunaga, G.N. Lee, C.Y. Rhim, C.H. Yoon, S.Y. Cho, R. Skene, S. Yamamoto, Y. Satou, M. Kuno, T. Miyazaki, H. Nakagawa, A. Okabe, S. Marui, K. Aso, M. Yoshida, Structure-Based Design and Synthesis of 3-Amino-1,5-dihydro-4H-pyrazolopyridin-4- one Derivatives as Tyrosine Kinase 2 Inhibitors, J. Med. Chem. 59 (2016) 733-749.
[16]S. Patel, S.F. Harris, P. Gibbons, G. Deshmukh, A. Gustafson, T. Kellar, H. Lin, X. Liu, Y. Liu, Y. Liu, C. Ma, K. Scearce-Levie, A.S. Ghosh, Y.G. Shin, H. Solanoy, J. Wang, B. Wang, J. Yin, M. Siu, J.W. Lewcock, Scaffold- Hopping and Structure-Based Discovery of Potent, Selective, And Brain Penetrant N-(1H-Pyrazol-3- yl)pyridin-2-amine Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12), J. Med. Chem. 58 (2015) 8182-8199.
[17]R. Jorda, L. Havlicek, I.W. McNae, M.D. Walkinshaw, J. Voller, A. Sturc, J. Navratilova, M. Kuzma, M. Mistrik, J. Bartek, M. Strnad, V. Krystof, Pyrazolo[4,3-d]pyrimidine bioisostere of roscovitine: evaluation
of a novel selective inhibitor of cyclin-dependent kinases with antiproliferative activity, J. Med. Chem. 54 (2011) 2980-2993.
[18]R. Jorda, K. Paruch, V. Krystof, Cyclin-dependent kinase inhibitors inspired by roscovitine: purine bioisosteres, Curr. Pharm. Des. 18 (2012) 2974-2980.
[19]K.J. Till, J.C. Allen, F. Talab, K. Lin, D. Allsup, L. Cawkwell, A. Bentley, I. Ringshausen, A.D. Duckworth, A.R. Pettitt, N. Kalakonda, J.R. Slupsky, Lck is a relevant target in chronic lymphocytic leukaemia cells whose expression variance is unrelated to disease outcome, Sci. Rep. 7 (2017) 16784.
[20]P. Balsas, A. Esteve-Arenys, J. Roldan, L. Jimenez, V. Rodriguez, J.G. Valero, A. Chamorro-Jorganes, R.P. de la Bellacasa, J. Teixido, A. Matas-Cespedes, A. Moros, A. Martinez, E. Campo, A. Saez-Borderias, J.I. Borrell, P. Perez-Galan, D. Colomer, G. Roue, Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma, J. Hematol. Oncol. 10 (2017) 80.
[21]O.A. Elgamal, A. Mehmood, J.Y. Jeon, B. Carmichael, A. Lehman, S.J. Orwick, J. Truxall, V.M. Goettl, R. Wasmuth, M. Tran, S. Mitchell, R. Lapalombella, S. Eathiraj, B. Schwartz, K. Stegmaier, S.D. Baker, E. Hertlein, J.C. Byrd, Preclinical efficacy for a novel tyrosine kinase inhibitor, ArQule 531 against acute myeloid leukemia, J. Hematol. Oncol. 13 (2020) 8.
[22]S.D. Reiff, R. Mantel, L.L. Smith, J.T. Greene, E.M. Muhowski, C.A. Fabian, V.M. Goettl, M. Tran, B.K. Harrington, K.A. Rogers, F.T. Awan, K. Maddocks, L. Andritsos, A.M. Lehman, D. Sampath, R. Lapalombella, S. Eathiraj, G. Abbadessa, B. Schwartz, A.J. Johnson, J.C. Byrd, J.A. Woyach, The BTK Inhibitor ARQ 531 Targets Ibrutinib-Resistant CLL and Richter Transformation, Cancer Discov. 8 (2018) 1300-1315.
[23]P. Kralova, V. Fulopova, M. Malon, T. Volna, I. Popa, M. Soural, Stereoselective Polymer-Supported Synthesis of Morpholine- and Thiomorpholine-3-carboxylic Acid Derivatives, ACS Comb. Sci. 19 (2017) 173-180.
[24]H.M. Guo, Ch. Xia, H.Y. Niu, X.T. Zhang, S.T. Kong, D.Ch. Wang, G.R. Qu, Intermolecular hydrogen abstraction reaction between nitrogen radicals in purine rings and alkyl ethers: a highly selective method for the synthesis of N-9 alkylated purine nucleoside derivatives, Adv. Synth. Catal. 353 (2011) 53-56.
Highlights
Five isosteres of entospletinib with altered central heterocyclic scaffolds were prepared Two isosteres exhibited SYK inhibition in the mid-nanomolar range
Three isosteres inhibited BTK more effectively than did entospletinib The cellular effect on the BCR signalosome was verified
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: