The TAM (Axl, Mer, and Tyro3) family is implicated in the survival and chemoresistance of tumours and has emerged as a potential therapeutic target. A novel series of 7-aryl-2-anilino-pyrrolopyrimidines were identified as potent Axl/Mer tyrosine kinase inhibitors without significant inhibition of Tyro3. A representative compound 27 exhibited IC50 values of 2 nM and 16 nM for Mer and Axl, respectively, and considerable inhibition for Mer phosphorylation in cells. Docking studies suggested that the formation of a salt bridge between the nitrogen of the aniline moiety with ASP678 of the Mer kinase domain as well as an interaction with the hinge region that most kinase inhibitors have in common would be essential to retain activity. These results could provide useful information for finding promising inhibitors of Axl/Mer for the treatment of cancer.
The TAM (Axl, Mer, and Tyro3) family is implicated in the survival and chemoresistance of tumours and has emerged as a potential therapeutic target. A novel series of 7-aryl-2-anilino-pyrrolopyrimidines were identified as potent Axl/Mer tyrosine kinase inhibitors without significant inhibition of Tyro3. A representative compound 27 exhibited IC50 values of 2 nM and 16 nM for Mer and Axl, respectively, and considerable inhibition for Mer phosphorylation in cells. Docking studies suggested that the formation of a salt bridge between the nitrogen of the aniline moiety with ASP678 of the Mer kinase domain as well as an interaction with the hinge region that most kinase inhibitors have in common would be essential to retain activity. These results could provide useful information for finding promising inhibitors of Axl/Mer for the treatment of cancer.
Entities:
Keywords:
Axl; MER; TAM familty; kinase inhibitor; pyrrolopyrimidine
The TAM (TYRO3-AXL-MER) family consists of three receptor tyrosine kinases, Axl, Mer, and Tyro3. Several endogenous ligands have been identified for TAM receptors. GAS6 binds to all three receptors but has a higher affinity for Axlcompared to Mer and Tyro3. Protein S is known to be a specific ligand for Mer and Tyro3. TAM receptors are widely distributed in many tissues, including the nervous system, and they are involved in cell proliferation, survival, and migration as well as immune responses.OncogenicTAM receptor signalling is involved in tumour development. Particularly, ectopic expression of TAM receptors has been associated with a poor prognosis in a variety of cancers. Furthermore, it has been demonstrated that blockage of TAM signalling could improve the effectiveness of immunotherapy for cancer treatment. TAM receptors, mainly the Mer receptor, induce M2 polarisation of macrophages in tumour microenvironments, which promotes tumour progression.Recent studies have demonstrated that Axl and Mer are implicated in resistance to chemotherapy and targeted therapy,. Thus, Axl/Mer inhibitors could provide a significant benefit for the treatment of patients with acquired resistance. With regard to a role of Mer in tumour associated macrophages, radiation therapy induced the upregulation of Mer in macrophages without changing the expression of Axl and tyro3. Mertk knockout mice showed better overall survival than wild type mice after radiation therapy. Therefore, the Mer tyrosine kinase could be a target to prevent the resistance of tumours to radiation therapy.Recent studies revealed that Axl is a key molecule in hematological malignancies including multiple myeloma and metastaticbreast cancer. The combination of a pan-TAM kinase inhibitor, BMS-777607, with anti-PD1 resulted in a better anti-tumour effect than each monotherapy alone in a mouse model. Currently, many inhibitors for multiple TAM receptors are under clinical or preclinical investigation. Representative TAM kinase inhibitors are shown in Figure 1.
Figure 1.
Structures and IC50 values of TAM kinase inhibitors.
Structures and IC50 values of TAM kinase inhibitors.Pyrazolopyrimidine UNC569 was derived from an analysis of the co-crystal structure of 1 with Mer tyrosine kinase and showed potent inhibitory activity against the TAM family. PyrrolopyrimidineUNC2025 showed more potent inhibitory activity against Mer than UNC569, but both exhibited strong activity against Tyro3. The MET tyrosine kinase inhibitor, BMS-777607, also showed activity as a pan- TAM inhibitor.Basically, the development of inhibitors specific to a single TAM receptor would be difficult because of structural similarities among the tree TAM receptors. However, Tyro3 is widely expressed in the adult central nervous system (CNS). Especially, Tyro3 is distributed in the nervous system at higher levels than Mer and Axl, indicating that inhibition of tyro3could potentially lead to a toxicity issue even though Tyro3could also be a therapeutic target for cancer. Mer is associated with resistance induced by Axl inhibition. Therefore, for the development of TAM kinase inhibitors, Axl/Mer inhibitors could provide an advantage over pan-TAM inhibitors. Moreover, the activation of Tyro3could suppress retinal degeneration associated with Mer inhibition. Therefore, it could be a plausible hypothesis that the discovery of Axl/Mer inhibitors that do not affect Tyro3could give a better toxicity profile. Herein, we describe the identification of novel small-molecule inhibitors for Mer and Axl, and an investigation of their structure-activity relationship.
Materials and methods
Chemistry
All commercially available reagents were purchased from Sigma Aldrich®, Alfa Aesar, Tokyo Chemical Industry, Combi Blocks, Ark Pharm, Inc., or AstaTech. USP-grade solvents were purchased from Samchun Pure Chemical. HPLC grade solvents were purchased from either Fisher Scientific or J.T. Baker®. Microwave irradiation was performed using an Anton Paar Monoave 300. All reactions were monitored by thin-layer chromatography (TLC), using silica gel 60F254 from Merck and UV light visualisation. Flash chromatography was performed by Combiflash Rf+ (Teledyne Isco, USA) using silica gel (ZEOprep 60, 4063 µm, Zeochem LLC, USA) manually, a prepacked flash column Welux™ Column ultra-pure silica gel 4063 µm 60 Å (Intertechnologies Co., Ltd., Republic of Korea), or a RediSep® Rf Gold (Teledyne Isco, USA). 1H and 13C-NMR spectra were obtained using Jeol Resonance ECZ 600 R (600 MHz) or Varian Gemini 2000 (300 Mhz). Chemical shifts were reported in parts per million (ppm, δ) using tetramethylsilane (TMS) as the internal standard. Coupling constants (J) were provided in Hertz (Hz). Splitting patterns were described as follows: s, singlet; d, doublet; t, triplet; q, quartette; p, pentet; dd, doublet of doublets; dt, doublet of triplets; td, triplet of doublets; m, multiplet; br, broad signal. High-resolution mass spectra (HRMS) were obtained using a Q Exative™ Hybrid Quadropole-Orbitrap Mass Spectrometer (Thermo Scientific) with the ESI method.
A mixture of 2-chloro-7H-pyrrolo[2,3-d]pyrimidine 6 (150 mg, 0.977 mmol), 2-bromothiazole (436.2 µL, 4.88 mmol), copper(I) iodide (1.9 mg, 0.01 mmol), tripotassium phosphate (414.6 mg, 1.95 mmol), and 1,2-trans-cyclohexanediamine (12.0 µL, 0.098 mmol) in toluene (1 ml) was stirred at 100 °C for 24 h, and then cooled down to room temperature. The reaction mixture was concentrated and diluted with dichloromethane (100 ml). The organic layer was washed with water and brine. After the mixture was dried over MgSO4, the organic layer was concentrated in vacuo and purified by MPLC with chloroform/acetonitrile to give the title compound 8 (51 mg, 22%).1H-NMR (300 MHz, CDCl3) δ 8.90 (s, 1H), 8.25 (d, J = 3.9 Hz, 1H), 7.63 (d, J = 3.3 Hz, 1H), 7.29 (d, J = 3.3 Hz, 1H), 6.77 (d, J = 3.9 Hz, 1H); 13C-NMR (150 MHz, CDCl3) δ 155.3, 154.6, 151.4, 150.7, 138.6, 127.6, 119.1, 116.8, 102.9.
5-Bromo-2-chloro-N-phenylpyrimidin-4-amine (10a)
A mixture of 5-bromo-2,4-dichloropyrimidine 9 (683 mg, 3.0 mmol), aniline (273 µL, 3 mmol), and triethylamine (1254 µL, 9.0 mmol) in anhydrous isopropanol (10 ml) was stirred at room temperature for 12 h. The reaction mixture was diluted with dichloromethane (100 ml) and washed with saturated NaHCO3 solution (10 ml) and water. After drying over MgSO4, the organic layer was concentrated in vacuo and purified by MPLC with dichloromethane/methanol to give the title compound 10a (584 mg, 68%).1H-NMR (600 MHz, CDCl3) δ 8.28 (s, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.45–7.34 (m, 2H), 7.27 (d, J = 13.9 Hz, 1H), 7.23–7.16 (m, 1H); 13C-NMR (150 MHz, CDCl3) δ 159.24, 157.59, 157.16, 137.00, 129.27, 125.27, 121.30, 103.78.
5-Bromo-2-chloro-N-(4-(trifluoromethoxy)phenyl)pyrimidin-4-amine (10 b)
Following the procedure for 10a, 4-(trifluoromethoxy)aniline and 9 provided the title compound 10 b (89%).1H-NMR (600 MHz, CDCl3) δ 8.31 (s, 1H), 7.65 (d, J = 9.0 Hz, 2H), 7.31 (s, 1H), 7.26 (s, 1H), 7.24 (s, 1H); 13C-NMR (151 MHz, CDCl3) δ 159.20, 157.86, 157.04, 145.99, 135.63, 122.50, 121.91, 120.53(q, J = 257 Hz), 103.775.
A mixture of 10a (584 mg, 2.05 mmol), TMS acetylene (291 µL, 2.05 mmol), (1420 µL, 10.25 mmol) Pd(PPh3)Cl2 (28.7 mg, 0.041 mmol) and CuI (7.8 mg, 0.041 mmol) in anhydrous toluene (5 ml) was stirred at 80 °C for 4 h. After being cooled to room temperature, the reaction mixture was filtered through celite. The filtrate was concentrated in vacuo and purified by MPLC with dichloromethane/methanol to give 11a. Next, a mixture of 11a (607 mg, 2.01 mmol), TBAF 1 M in THF (4.02 ml, 4.02 mmol) in THF (25 ml) was stirred at 60 °C for 12 h. After the mixture was filtered through celite, the mixture was diluted with dichloromethane (100 ml) and washed with saturated NaHCO3 solution (10 ml) and brine. After drying over Na2SO4, the organic layer was concentrated in vacuo and purified by MPLC with dichloromethane/methanol to give the title compound 12a (236 mg, 52%).1H-NMR (600 MHz, CDCl3) δ 8.88 (s, 1H), 7.69 (dd, J = 8.5, 0.9 Hz, 2H), 7.51–7.55 (m, 3H), 7.40 (t, J = 7.5 Hz, 1H), 6.73 (d, J = 3.7 Hz, 1H); 13C-NMR (150 MHz, CDCl3) δ 154.34, 151.70, 151.57, 136.77, 129.78, 129.72, 127.58, 123.94, 118.66, 101.54.
A mixture of trans-4-((2-Chloro-5-((trimethylsilyl)ethynyl)pyrimidin-4-yl)amino)cyclohexanol11c (38.7 mg, 0.12 mmol), TBAF 1 M in THF (240 µL, 0.24 mmol). in THF (10 ml) was stirred at 60 °C for 24 h. After the mixture was filtered through celite, the mixture was diluted with dichloromethane (100 ml) and washed with saturated NaHCO3 solution (10 ml) and brine. After drying over Na2SO4, the organic layer was concentrated in vacuo and purified by MPLC with dichloromethane/methanol to give the title compound 12c (25 mg, 83%).1H-NMR (600 MHz, CDCl3) δ 8.78 (s, 1H), 7.27 (s, 1H), 6.57 (d, J = 3.7 Hz, 1H), 4.72–4.76 (m, 1H), 3.77–3.81 (m, 1H), 2.10–2.18 (m, 4H), 1.82–1.89 (m, 3H), 1.62 (ddd, J = 24.2, 13.1, 3.3 Hz, 2H); 13C-NMR (150 MHz, CDCl3) δ 153.30, 151.62, 150.96, 126.61, 117.96, 100.25, 69.71, 52.52, 34.41, 31.05.
A mixture of 14a (25.1 mg, 0.1 mmol) and Pd/C (10 w/w%) in MeOH (10 ml) were sealed with a septum and substituted with H2 gas. The reaction mixture was stirred at room temperature for 4 h. The mixture was filtered off and the filtrate was concentrated in vacuo to give the corresponding amine 15a. Successively, a mixture of 15a, 7 (26.0 mg, 0.1 mmol), BINAP (9.3 mg, 0.0150 mmol), sodium tert-butoxide (19.2 mg, 0.2 mmol), and Pd2(dba)3 (4.6 mg, 0.005 mmol) in anhydrous toluene (4 ml) was stirred at 100 °C for 20 h. After being cooled to room temperature, the reaction mixture was concentrated and diluted with dichloromethane (100 ml). The organic layer was washed with water and brine. After the mixture was dried over MgSO4, the organic layer was concentrated in vacuo and purified by MPLC with chloroform/acetonitrile to give the title compound 16 (25 mg, 56%).1H-NMR (300 MHz, CDCl3) δ 8.68 (s, 1H), 7.56–7.61 (m, 3H), 7.24 (br, 1H), 7.14 (d, J = 3.9 Hz, 1H), 6.98–7.04 (m, 2H), 6.93 (dd, J = 2.4, 8.7 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H), 6.53 (d, J = 3.6 Hz, 1H), 3.87 (s, 3H), 3.66 (s, 3H), 3.07 (br, 4H), 2.68 (br, 4H), 2.39 (s, 3H); 13C-NMR (150 MHz, CDCl3) δ 158.3, 156.3, 152.5, 151.9, 136.3, 135.4, 130.9, 126.4, 125.7, 118.4, 114.4, 113.0, 110.2, 102.8, 101.1, 55.6, 55.3(2 C), 50.7, 45.9; IR(neat): 2935, 2798, 1599, 1562, 1530, 1510, 1417, 1249, 1212, 1151, 1033, 1012, 832, 733 cm−1. HRMS (ESI): m/z calcd for C25H28N6O2 [M + H]+ 445.2347 found 445.2344.
A mixture of 3-methoxy-5-nitrobenzonitirle 22 (359 mg, 2.0 mmol) and 1.0 M diisobutyl aluminium hydride in THF (5.0 ml, 5.0 mmol) in anhydrous toluene (20 ml) was stirred at 0 °C for 3 h. The reaction mixture was concentrated and diluted with dichloromethane (100 ml). The organic layer was washed with water and brine. After the mixture was dried over Na2SO4, the organic layer was concentrated in vacuo and purified by MPLC with hexanes/ethyl acetate to give the title compound 23 (84 mg, 23%).1H-NMR (600 MHz, CDCl3) δ 10.04 (s, 1H), 8.28 (s, 1H), 7.97 (t, J = 2.2 Hz, 1H), 7.71 (t, J = 1.2 Hz, 1H), 3.96 (s, 3H); 13C-NMR (150 MHz, CDCl3) δ 189.73, 160.94, 149.78, 138.30, 119.26, 117.17, 114.64, 56.46.
A mixture of 23 (83 mg, 0.458 mmol), 1-methylpiperazine (66 µL, 0.596 mmol), acetic acid (26 µL, 0.458 mmol), and NaBH(OAc)3 (291 mg, 1.375 mmol) in 1,2-dichloroethane (5 ml) was stirred at room temperature for 12 h. The mixture was diluted with dichloromethane (100 ml) and washed with saturated NaHCO3 solution (10 ml) and brine. After drying over Na2SO4, the organic layer was concentrated in vacuo and purified by MPLC with hexane/ethyl acetate to give the title compound 24 (40 mg, 33%).1H-NMR (600 MHz, CDCl3) δ 7.82 (s, 1H), 7.61 (t, J = 2.1 Hz, 1H), 7.23 (s, 1H), 3.89 (s, 3H), 3.55 (s, 2H), 2.41 (d, J = 113.6 Hz, 11H); 13 C-NMR (150 MHz, CDCl3) δ 160.08, 149.26, 141.81, 121.49, 116.13, 106.77, 77.24, 77.03, 76.82, 62.03, 55.88, 55.03, 53.00, 45.97.
A mixture of 20a (25.1 mg, 0.1 mmol) and Pd/C (10 w/w%) in MeOH (10 ml) was sealed with a septum and substituted with H2 gas. The reaction mixture was stirred at room temperature for 4 h. The mixture was filtered off and the filtrate was concentrated in vacuo to give compound 21a. Successively, a mixture of 21a, 7 (26.0 mg, 0.1 mmol), 4 M HCl in dioxane (50 µL, 0.2 mmol) in anhydrous isopropanol (2 ml) was heated at 160 °C for 1 h in a microwave reactor. After being cooled to room temperature, the reaction mixture was concentrated and diluted with dichloromethane (100 ml). The organic layer was washed with water and brine. After the mixture was dried over MgSO4, the organic layer was concentrated in vacuo and purified by MPLC with chloroform/acetonitrile to give the title compound 25 (13 mg, 29%).1H-NMR (300 MHz, CDCl3) δ 8.69 (s, 1H), 7.59 (d, J = 9 Hz, 2H), 7.25 (s, 1H), 7.14 (d, J = 3.6 Hz, 1H), 7. 02 (d, J = 9 Hz, 2H), 6.99 (t, J = 2.1 Hz, 1H), 6.80 (t, J = 2.1 Hz, 1H), 6.54 (d, J = 3.6 Hz, 1H), 6.11 (t, J = 2.1 Hz, 1H), 3.88 (s, 3H), 3.70 (s, 3H), 3.10 (t, J = 5.1 Hz, 4H) 2.49 (t, J = 5.1 Hz, 4H), 2.34 (s, 3H); 13C-NMR (150 MHz, CDCl3) δ 160.9, 158.2, 156.2, 152.8, 151.7, 151.0, 142.0, 130.9, 126.5, 125.7, 114.5, 113.2, 101.1, 98.7, 96.2, 95.2, 55.5, 55.2, 55.1, 48.9, 46.1; IR(neat): 2934, 2837, 1593, 1568, 1536, 1518, 1483, 1452, 1418, 1248, 1209, 1161, 1032, 1003, 831, 734, 699 cm−1; HRMS (ESI): m/z calcd for C25H28N6O2 [M + H]+ 445.2347 found 445.2345.
All kinase assays were carried out at Km ATP by Eurofins Discovery's Kinase Screening and Profiling services (France).
Cell culture
MKN28 cells were obtained from the Korea Institute of Science and Technology (KIST). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO2 under a humidified atmosphere.
Western blot
MKN 28 cells (500,000 cells/2 ml) were seeded in each well of 6-well plates and incubated for 24 h. Then, the cells were treated with 20 µL of DMSO stock solution of the corresponding compounds and incubated for a further 1.5 h. After that, the cells were washed with cold Dulbecco's Phosphate-Buffered Saline (DPBS) twice and lysed with RIPA buffer supplemented with protease inhibitor and phosphatase inhibitor cocktails on ice. Equal amounts of protein samples were boiled with 5× SDS-PAGE loading buffer and separated by 10% SDS-PAGE gels, then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% BSA in 1× TBST (1× TBS with 0.1% Tween-20) for 1 h and incubated at 4 °C overnight with a 1:1000 dilution of the following primary antibodies in blocking buffer: pMer (Tyr749 + 753 + 754, ab14921), Mer (CST, # 4319), and ß-actin (SC, # SC-47778). After three washes with 1× TBST, the membranes were incubated with a 1:2000 dilution of the following secondary antibodies at RT for 1 h followed by extensive washing three times. Secondary antibodies: anti-rabbit IgG, HRP-linked antibody (CST, # 7074S); anti-mouse IgG, HRP-linked antibody (CST, # 7076S). Antibody binding was visualised by an enhanced chemiluminescent (ECL) system (Bio-Rad, Clarity Western ECL Substrate, # 1705061) and VILBER FUSION SOLO X. Antibodies were purchased from Abcam, Cell Signalling Technology (CST), and Santa Cruz Biotechnology (SC). ß-actin was used as a loading control.
Results and discussion
To find the novel Mer and Axl inhibitors, we screened our in-house chemical library for the TAM family. As shown in Table 1, 7-aryl pyrrolopyrimidines 2 and 3 were found to be hits, with IC50 of 39 nM and 95 nM against Mer, respectively, and little activity for Tyro3. 3-phenyl aniline 4 and piperazine 5 did not have activity against the TAM family. Thus, derivatives of compound 2 were synthesised to explore the structure-activity relationship.
Table 1.
Activity for TAM kinases of methoxy phenyl pyrrolopyrimidines.
Compound
R1
IC50 (nM)
% Inhibition at 1 µM
MERTK
AXL
TYRO3
2
39
1240
38
3
95
751
44
4
>3000
>3000
12
5
>1000
>1000
0
UNC569
3
420
NDa
Data are mean values. aND: not determined.
Activity for TAM kinases of methoxy phenyl pyrrolopyrimidines.Data are mean values. aND: not determined.The synthetic routes for the derivatives are illustrated in Schemes 1 and 2. As shown in Scheme 1, 2-chloro-pyrrolo[2,3-d]pyrimidine intermediates with various substituents were prepared by reacting commercially available 2-chloro-7H-pyrrolo[2,3-d]pyrimidine 6 with boronic acids using the Chan–Lamcoupling reaction. Thiazole 8 was synthesised by the Ullmanncoupling reaction. For the synthesis of further derivatives, intermediates 10a–c were synthesised by nucleophilic substitution of amine with 9. Sonogashira coupling of 10a–c with TMS acetylene followed by intramolecular cyclisation gave the desired intermediates 12a–c.
Scheme 1.
Reagents and conditions: (a) (OH)2B-Ar, Cu(OAc)2, pyridine, 4 Å MS, CH2Cl2, rt, 6–24 h; (b) 2-bromothiazole, CuI, K3PO4, 1,2-trans-cyclohexanediamine, THF, 110 °C, 24 h; (c) BINAP, Pd2(dba)3, Cs2CO3, dioxane, 100 °C, 8 h; (d) HCl, i-PrOH, MW 160 °C, 1 h; (e) 4-(trifluoromethoxy)aniline, TEA, i-PrOH; (f) ethynyltrimethylsilane, Pd(PPh3)2, CuI, TEA, toluene, 80 °C, 4 h; (g) TBAF, THF, 60 °C, 4 h.
Reagents and conditions: (a) (OH)2B-Ar, Cu(OAc)2, pyridine, 4 Å MS, CH2Cl2, rt, 6–24 h; (b) 2-bromothiazole, CuI, K3PO4, 1,2-trans-cyclohexanediamine, THF, 110 °C, 24 h; (c) BINAP, Pd2(dba)3, Cs2CO3, dioxane, 100 °C, 8 h; (d) HCl, i-PrOH, MW 160 °C, 1 h; (e) 4-(trifluoromethoxy)aniline, TEA, i-PrOH; (f) ethynyltrimethylsilane, Pd(PPh3)2, CuI, TEA, toluene, 80 °C, 4 h; (g) TBAF, THF, 60 °C, 4 h.Commercially available 1-bromo-2-methoxy-4-nitrobenzene 13 was coupled with amines by the Buchwald–Hartwig coupling reaction. The resulting alkylamino compound 14 was hydrogenated to yield aniline 15, followed by coupling with 2-chloropyrrolopyrimidines (7, 8, and 12a-c) to give the desired compounds 16–18. Synthesis of meta-substituted derivatives started from 3-bromo-5-methoxy nitrobenzene. Compounds 22–32 were obtained using the same synthetic methods as those used to prepare 16–18. Derivative 23 was synthesised from 3-cyano-5-methoxy nitrobenzene, as outlined in Scheme 2(c).
Scheme 2.
Reagents and conditions: (a) amines, Pd2(dba)3, BINAP, Toluene, Cs2CO3, 100 °C, 6 − 24 h; (b) H2, Pd/C, MeOH, rt, 3 − 9 h; (c) 7, 8, or 12, HCl, i-PrOH, MW 160 °C, 1 h; (d) DIBAL, toluene, 0 °C, 3 h; (e) 1-methylpiperazine, NaBH(OAc)3, AcOH, DCE, rt, 12 h.
Reagents and conditions: (a) amines, Pd2(dba)3, BINAP, Toluene, Cs2CO3, 100 °C, 6 − 24 h; (b) H2, Pd/C, MeOH, rt, 3 − 9 h; (c) 7, 8, or 12, HCl, i-PrOH, MW 160 °C, 1 h; (d) DIBAL, toluene, 0 °C, 3 h; (e) 1-methylpiperazine, NaBH(OAc)3, AcOH, DCE, rt, 12 h.Introduction of an alkyl amino group at the para-position resulted in an improvement of activity over compound 2 with weak activity against Tyro3 (Table 2). N-Methyl piperazinyl derivative 16 showed 2.6-fold and 12-fold decreased IC50 for Mer and Axl, respectively, compared to 2. Compound 17 also had IC50 of 10 nM for Mer and approximately 200 nM for Axl without considerable inhibition of Tyro3. Dimethylaminopyrrolidine 18 showed a slight decrease in activity against Mer and improved activity against Axl.
Table 3.
Inhibitory activity of R4 derivatives.
Compound
R4
IC50 (nM)
%Inhibition at 1 µM
MER
AXL
TYRO3
33
9
57
23
34
17
48
35
35
64
247
16
36
484
2018
55
UNC569
3
420
NDa
aND: not determined.
Next, the effects of meta-substitution were explored. Interestingly, substitution of N-methyl piperazine (25) at the meta-position (R3) led to great potency for Mer with an IC50 of 2 nM. The one-carbon extended N-methyl piperazine derivative 26 was also highly potent, but it showed relatively high activity for Tyro3compared to the other derivatives. 4-Pyrrolidinyl piperidine derivative 27 was also equipotent to the known compound (UNC569) for Mer and Axl but still showed weak activity against Tyro3. Methylated derivative 28 displayed 2–3-fold weaker activity than 27. 3-Pyrrolidinyl piperidine 29 had moderate activity for Mer and Axl whilst 4-oxetanyl piperazine 27 showed decreased activity for Axl. Interestingly, the introduction of an azaspirodecanyl group (31) led to complete loss of activity for all TAMs. However, the insertion of oxygen (32) resulted in the recovery of activity similar to 30. These data suggest that a heteroatom at an appropriate distance from the aniline ring might be necessary to achieve good activity for Mer and Axl. A docking study was carried out to further understand the binding mode of the described compounds.As shown in Figure 2, the docking model showed that N1 and NH of 16 and 25 interact with MET674 of the hinge region through hydrogen bonding, and the nitrogen atom in the piperazine moiety forms a salt bridge with ASP678. The distances between ASP678 and the nitrogen atom in the piperazine moiety of para-derivative 16 and meta-derivative 25 were calculated as 4.64 Å and 1.77 Å, respectively. This suggests that the piperazine moiety at the meta-position could be placed closer to ASP678 than when it is at the para-position, which may induce stronger binding of 25 than that of 16. In addition, this docking model provided a reasonable explanation for the complete loss of activity of 31, which cannot interact with ASP678 owing to the absence of a nitrogen atom. The addition of an oxygen atom at the spiro-ring in 32 led to recovered activity, which also supported the docking model. These data indicate that the formation of a salt bridge with ASP678 is important for retaining activity against TAM family kinases.
Figure 2.
Predicted docking orientation of 16 and 25 with the Mer kinase domain (PDB ID: 3TCP). Docking mode of (a) 16 and (b) 25 with Mer. 2 D-interaction diagram of the binding model of (c) 16 and (d) 25. Estimated binding energies were −7.52 kcal/mol and −8.26 kcal/mol for 16 and 25, respectively. Hydrogen bonds and a salt bridge between the ligand and the backbone are shown in dashed lines. The docking study was performed by AutoDock Vina.
Predicted docking orientation of 16 and 25 with the Mer kinase domain (PDB ID: 3TCP). Docking mode of (a) 16 and (b) 25 with Mer. 2 D-interaction diagram of the binding model of (c) 16 and (d) 25. Estimated binding energies were −7.52 kcal/mol and −8.26 kcal/mol for 16 and 25, respectively. Hydrogen bonds and a salt bridge between the ligand and the backbone are shown in dashed lines. The docking study was performed by AutoDock Vina.Next, a brief structure-activity relationship investigation was carried out for R4, as shown in Table 3. According to the predicted docking model for Mer, it was postulated that R4 as a 4-methoxyphenyl group is positioned in the hydrophobic pocket and is involved in a π–alkyl interaction with Val601. As expected, trifluoromethyl phenyl compound 33 and phenyl compound 34 showed excellent activity for Mer and Axl. Thiazole derivative 35 also retained activity, although its activity was less than those of 33 and 34. However, the introduction of a cycloalkyl group, a trans-4-hydroxycyclohexyl group, in 36 significantly decreased the activity for Mer and Axl. This suggests that the described compounds may interact with Mer in a different manner than UNC569. The data indicate that N7-substituents with an aromatic group may be suitable to bind Mer or Axl.
Table 2.
Activities of 3-methoxy aniline derivatives of compound 3.
Compound
R2
R3
IC50 (nM)
% Inhibition at 1 µM
MER
AXL
TYRO3
16
H
15
104
27
17
H
10
199
37
18
H
46
175
30
25
H
2
17
86
26
H
4
18
92
27
H
2
16
40
28
H
7
32
72
29
H
31
31
49
30
H
36
212
39
31
H
>3000
>3000
4
32
H
40
288
0
UNC569
3
420
NDa
aND: not determined.
Activities of 3-methoxy aniline derivatives of compound 3.aND: not determined.Inhibitory activity of R4 derivatives.aND: not determined.To determine the inhibitory activity of compounds on Mer phosphorylation in cells, western blot analysis was carried out. A representative compound 27 was used to treat a Mer-overexpressed humangastric cancercell line, MKN28. A potent Mer inhibitor, UNC2025, was used as a positive control (Figure 3). Compound 27 showed a better effect on blocking phosphorylation than UNC2025 at the indicated concentrations.
Figure 3.
Inhibitory effect of 27 on Mer phosphorylation in a Mer-overexpressed human gastric cancer cell line, MKN28. Cells were treated with the indicated compounds at 0.5 and 0.1 μM for 1.5 h. UNC2025 and β-actin were used as a positive control and a loading control, respectively. Western blot analysis for phosphorylated and total Mer from a representative experiment is shown. Bar graph represents the relative intensities of the total and phosphorylated Mer as determined by band densitometry using image analysis software.
Inhibitory effect of 27 on Mer phosphorylation in a Mer-overexpressed humangastric cancercell line, MKN28. Cells were treated with the indicated compounds at 0.5 and 0.1 μM for 1.5 h. UNC2025 and β-actin were used as a positive control and a loading control, respectively. Western blot analysis for phosphorylated and total Mer from a representative experiment is shown. Bar graph represents the relative intensities of the total and phosphorylated Mer as determined by band densitometry using image analysis software.In summary, we report here the discovery of 7-aryl-2-anilino-pyrrolopyrimidine derivatives as potent inhibitors of Axl and Mer kinases without considerable inhibition of Tyro3. The most potent compound 27 had IC50 values of 2 nM and 16 nM for Mer and Axl, respectively, but just 40% inhibition of Tyro3 at 1 µM. In addition, compound 27 exhibited considerable inhibition for Mer phosphorylation in a cancercell line. Structure-activity relationship and docking studies showed that forming a salt bridge and an aromatic group at the N7 position are essential for its Axl and Mer kinase inhibition activity. This work could provide useful information for the molecular design of Axl/Mer kinase inhibitors.
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Figure 1.
Scheme 1.
Scheme 2.
Figure 2.
Figure 3.