Structure-activity relationship of 7-aryl-2-anilino-pyrrolopyrimidines as Mer and Axl tyrosine kinase inhibitors

Abstract 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.


Introduction
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 1 . GAS6 binds to all three receptors but has a higher affinity for Axl compared 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.
Oncogenic TAM receptor signalling is involved in tumour development 1 . Particularly, ectopic expression of TAM receptors has been associated with a poor prognosis in a variety of cancers 1 . Furthermore, it has been demonstrated that blockage of TAM signalling could improve the effectiveness of immunotherapy for cancer treatment 2 . TAM receptors, mainly the Mer receptor, induce M2 polarisation of macrophages in tumour microenvironments, which promotes tumour progression 3 .
Recent studies have demonstrated that Axl and Mer are implicated in resistance to chemotherapy and targeted therapy 4,5 . 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 6 . 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 7 and metastatic breast cancer 8 . 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 9 . Currently, many inhibitors for multiple TAM receptors are under clinical or preclinical investigation 10 . Representative TAM kinase inhibitors are shown in Figure 1.
Pyrazolopyrimidine UNC569 was derived from an analysis of the co-crystal structure of 1 with Mer tyrosine kinase 11 and showed potent inhibitory activity against the TAM family. Pyrrolopyrimidine UNC2025 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) 12 . Especially, Tyro3 is distributed in the nervous system at higher levels than Mer and Axl, indicating that inhibition of tyro3 could potentially lead to a toxicity issue even though Tyro3 could 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 Tyro3 could suppress retinal degeneration associated with Mer inhibition 13 . Therefore, it could be a plausible hypothesis that the discovery of Axl/Mer inhibitors that do not affect Tyro3 could give a better toxicity profile. Herein, we describe the identification of novel smallmolecule inhibitors for Mer and Axl, and an investigation of their structure-activity relationship.

Chemistry
All commercially available reagents were purchased from Sigma AldrichV R , 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 V R . Microwave irradiation was performed using an Anton Paar Monoave 300. All reactions were monitored by thin-layer chromatography (TLC), using silica gel 60F 254 from Merck and UV light visualisation. Flash chromatography was performed by Combiflash Rfþ (Teledyne Isco, USA) using silica gel (ZEOprep 60, 4063 mm, Zeochem LLC, USA) manually, a prepacked flash column Welux TM Column ultra-pure silica gel 4063 mm 60 Å (Intertechnologies Co., Ltd., Republic of Korea), or a RediSep V R Rf Gold (Teledyne Isco, USA). 1 H and 13 C -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, d) 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 TM Hybrid Quadropole-Orbitrap Mass Spectrometer (Thermo Scientific) with the ESI method.
1H 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 H 2 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 mL, 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 MgSO 4 , the organic layer was concentrated in vacuo and purified by MPLC with chloroform/ acetonitrile to give the title compound 25 (13 mg, 29%).  160.9, 158.2, 156.2, 152.8, 151.7, 151.0, 142.0, 130.9, 126.5, 125.7,

Kinase assay
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% CO 2 under a humidified atmosphere.

Western blot
MKN 28 cells (500,000 cells/2 ml) were seeded in each well of 6well plates and incubated for 24 h. Then, the cells were treated with 20 mL 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 5Â SDS-PAGE

Results and discussion
To find the novel Mer and Axl inhibitors, we screened our inhouse chemical library for the TAM family. As shown in Table 1, 7aryl pyrrolopyrimidines 2 and 3 were found to be hits, with IC 50 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. considerable inhibition of Tyro3. Dimethylaminopyrrolidine 18 showed a slight decrease in activity against Mer and improved activity against Axl. Next, the effects of meta-substitution were explored. Interestingly, substitution of N-methyl piperazine (25) at the metaposition (R 3 ) led to great potency for Mer with an IC 50 of 2 nM. The one-carbon extended N-methyl piperazine derivative 26 was also highly potent, but it showed relatively high activity for Tyro3 compared 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.  Figure 2, the docking model showed that N 1 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.

As shown in
Next, a brief structure-activity relationship investigation was carried out for R 4 , as shown in Table 3. According to the predicted docking model for Mer, it was postulated that R 4 as a 4methoxyphenyl group is positioned in the hydrophobic pocket and is involved in a p-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 N 7 -substituents with an aromatic group may be suitable to bind Mer or Axl.
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 human gastric cancer cell 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.
In summary, we report here the discovery of 7-aryl-2-anilinopyrrolopyrimidine derivatives as potent inhibitors of Axl and Mer kinases without considerable inhibition of Tyro3. The most potent compound 27 had IC 50 values of 2 nM and 16 nM for Mer and Axl, respectively, but just 40% inhibition of Tyro3 at 1 mM. In addition, compound 27 exhibited considerable inhibition for Mer phosphorylation in a cancer cell line. Structure-activity relationship and docking studies showed that forming a salt bridge and an aromatic group at the N 7 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.

Disclosure statement
No potential conflict of interest was reported by the authors.