JNJ-64619178

Rational Design, synthesis and biological evaluation of novel triazole derivatives as potent and selective PRMT5 inhibitors with antitumor activity

Kongkai Zhu1 · Jingwei Shao2 · Hongrui Tao1 · Xue Yan1 · Cheng Luo3 · Hua Zhang1 · Wenhu Duan2

Abstract

Protein arginine methyltransferase 5 (PRMT5) is responsible for the mono-methylation and symmetric dimethylation of arginine, and its expression level and methyl transferring activity have been demonstrated to have a close relationship with tumorigenesis, development and poor clinical outcomes of human cancers. Two PRMT5 small molecule inhibitors (GSK3326595 and JNJ-64619178) have been put forward into clinical trials. Here, we describe the design, synthesis and biological evaluation of a series of novel, potent and selective PRMT5 inhibitors with antiproliferative activity against Z-138 mantle cell lymphoma cell line. Among them, compound C_4 exhibited the highest potency with enzymatic and cellular level IC50 values of 0.72 and 2.6 μM, respectively, and displayed more than 270-fold selectivity toward PRMT5 over several other isoenzymes (PRMT1, PRMT4 and PRMT6). Besides, C_4 demonstrated obvious cell apoptotic effect while reduced the cellular symmetric arginine dimethylation levels of SmD3 protein. The potency, small size, and synthetic accessibility of this compound class provide promising hit scaffold for medicinal chemists to further explore this series of PRMT5 inhibitors.

Keywords PRMT5 inhibitor · Anti-proliferative · Cellular target validation · Design and synthesis

Introduction

Protein arginine methylation catalyzed by protein argi- nine methyltransferases (PRMTs) is an important type of post-translational modification that affects crucial cellular processes including cell growth and proliferation, apop- tosis, metastasis and angiogenesis via modulation of gene transcription and/or protein function [1–3]. The identified nine human PRMTs (PRMT1–9) are further subdivided into type I, type II, and type III enzymes according to the states of the methylated arginine. PRMT5 belongs to the predominant type II PRMTs which transfer methyl groups from S-adenosylmethionine (SAM) to a guanidine nitrogen of protein arginine residues, resulting in the reaction prod- ucts ω-NG-monomethylarginine (MMA), symmetric ω-NG, N′G-dimethylarginine (sDMA) and S-adenosylhomocysteine (SAH). Proteomic analysis of human tissues revealed that PRMT5, -1 and -4 exhibited more universal expression compared to PRMT2, -3, -6, -7, and -8 [4]. Through the symmetric dimethylation of diverse substrate proteins in the nucleus and cytoplasm, PRMT5 mediated various cel- lular processes, such as transcription [5], RNA metabolism [6], signal transduction [7], and cellular differentiation [8], etc. The symmetric dimethylation of histones (H4, H3, and H2A), Sm proteins (B/B′, D1, and D3) are the hallmarks associated with PRMT5 activity [9, 10].
Interestingly, accumulating studies implicated that PRMT5 was an attractive therapeutic target in cancer. It is overexpressed or upregulated in several human cancers, including lymphomas [11–13], breast cancer [14], lung can- cer [15], colorectal cancer [16], hepatocellular carcinoma [17], multiple myeloma [18], and glioblastoma [19]. It was reported that selective inhibition of PRMT5 could block ini- tiation and maintenance of B cell transformation [20]. The importance of PRMT5 in breast cancer stem cell (BCSC) maintenance was also highlighted [21]. Besides, PRMT5 was documented to control melanoma growth through the SKI/SOX10 regulatory axis [22]. PRMT5 has been proposed as an effective target in mantle cell lymphoma (MCL) [23], glioblastoma [19], multiple myeloma [18], and mixed line- age leukemias (MLL)-rearranged acute myeloid leukemia [24].
Tremendous efforts from a number of groups have been made to discover PRMT5 inhibitors [20, 23, 25–33], and all reported PRMT5 inhibitors are summarized in Fig. 1. Among them, LLY-283 [31], Sinefungin [34], and A9145C [10] are SAM binding site inhibitors, while EPZ015666 [23] together with its analogue GSK3326595 [35] are substrate binding site inhibitors. EPZ015666 is the first PRMT5 inhibitor to be described with potent activities in both in vitro and in vivo models of Mantle cell lymphoma. Besides, the phase I of clinical trial of GSK3326595 for the treatment of solid tumors and non- Hodgkin’s lymphoma is going on. And compound JNJ- 64619178 [36] was reported to bind into the SAM bind- ing pocket and meanwhile to reach the substrate binding pocket. Although more than 10 classes of PRMT5 inhibi- tors have been reported, most of them exhibit low potency or lack in vivo activity. Therefore, the search for more potent PRMT5 inhibitors remains a pressing task.
In the present study, we report the identification of a series of PRMT5 inhibitors with potent antiproliferative activity against Z-138 mantle cell lymphoma cell line. The most potent one (C_4) was employed to characterize its cell apoptotic effect and cellular target validation.

Materials and methods

PRMT5, PRMT1, PRMT4 and PRMT6 enzymatic activity inhibition assays Using a radioactivity-based assay to monitor the transfer of the methyl group from 3H-SAM to peptide substrate, we tested the inhibitory activity of our compounds against PRMT5:MEP50. The detailed methods to test the inhibi- tion activity of compound C_4 against PRMT5, PRMT1, PRMT4 and PRMT6 were as we have previously described [29].

Molecular docking

Molecular docking was performed using Glide pro- gram inserted in Maestro 7.5. The coordinates of PRMT5:EPZ015666 (PDB code: 4X61) were used to construct docking model. First, the protein part was mini- mized by Protein Preparation Wizard Workflow. After that, docking grids were created by defining residues within 15 Å around EPZ015666. Then the prepared (using LigPrep panel) GSK3326595 was docked to the pre-defined dock- ing grids with standard precision mode.

In vitro anti‑proliferation assay

Long-term proliferation assays were performed on Z-138 cells using the method we previously reported. In brief, exponentially growing cells were seeded in 24-well cell plates in growth medium (RPMI1640 medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% peni- cillin/streptomycin) at a density of 1 × 105 cells per well. Cells were incubated in the presence of increasing con- centrations of the tested compounds up to 200 μM. Viable cell number was determined every 4 days for up to 8 days using the Cell Titer-Glo Luminescent Cell Viability kit (Promega). On days of cell counting, growth media and compounds were replaced and cells split back to a density of 1 × 105 cells per well. IC50 values were determined from at least three independent assays and calculated from the concentration-dependence curves.

Western blot analysis

Exponentially growing Z-138 cells were incubated with compound C_4 with different concentrations for 4 days. Then cells were harvested and lysed in 100 μL of total lysis buffer, and total cell lysates were separated on 4–12% SDS- PAGE and transferred to PVDF membrane (Millipore). Afterward, blocking buffer (5% nonfat milk in 0.1% Tween 20 PBS buffer) was used to block the Blots. Subsequently, primary antibodies [SDMA (CST, 13222s), GAPDH (CST, 5174) and SmD3 (Sigma, HPA001170-100UL), all prepared in a 1:3 dilution of blocking buffer] were added to Blots and incubated overnight at 4 °C. The next day, after being washed three times with PBST, the Blots were incubated with 1:10,000 dilution of donkey anti-rabbit secondary antibody (HRP conjugated) for 2 h. Finally, the signal was detected using ChemiScope3400 imaging system (Clinx Sci- ence Instruments).

Flow cytometric analysis

Exponentially growing Z-138 cells were plated in 6-well plates at a density of 5 × 105 and treated with compound C_4, EPZ015666 (positive control) or DMSO (control). Cells were harvested at 48 h and were then measured using AnnexinV-FITC Apoptosis Detection Kit (Vazyme Biotech). FlowJo V7.6.1 was used to detect samples and to analyze data.

Chemistry

All purchased chemicals and solvents were used without further purification unless otherwise noted. Flash chroma- tography was performed using silica gel (300–400 mesh). All reactions were monitored by thin-layer chromatography (TLC), using silica gel plates with fluorescence F254 and visualized under UV light. 1H NMR data were recorded on Varian Mercury 300 or 400 NMR spectrometers. Chemical shifts (δ) were reported in parts per million (ppm), coupling constant (J) values were in hertz, and the splitting patterns were abbreviated as follows: s for singlet; d for doublet; t for triplet; q for quartet and m for multiplet. High resolution ESI–MS spectra were recorded on a Finnigan/MAT95 spec- trometer. All the final compounds are racemates. Compound 5 was synthesized according to the reported procedures [37]. All the compounds were synthesized as illustrated in Schemes 1, 2, and 3. Compound 1 was reacted with 2-(chlo- romethyl)oxirane in the presence of base to afford the inter- mediate epoxide 2. Benzenesulfonyl chloride derivatives were treated with ammonium hydroxide to afford benze- nesulfonamides. Compounds A_1–A_10 were prepared through ring-opening reaction of epoxide (compound 2) under solid–liquid phase-transfer catalytic conditions.
The synthesis of compounds B_1 and B_2 was shown in Scheme 2. Compound 5 was treated with tetrahydro- 2H-pyran-4-amine or 11b, affording 6 or 7. Reduction of 6 yielded the amine intermediate which was converted to compound B_1 by reacting with 2. Compound B_2 was obtained by a two-step procedure: reduction of 7 and reduc- tive amination reaction.
The synthesis of compounds C_1–C_6 was started from 3- or 4-bromonitrobenzene, which was first subjected to a Sonogashira cross-coupling with TMS-acetylene and then deprotected with K2CO3 to yield 10. Compound 2 was treated with NaN3 or NH3 in MeOH to furnish 11. Compounds C_1 and 12 were prepared via click chemistry from intermediates 10 and 11a. Reduction of C_1 and 12 with hydrogen afforded C_2 or C_5 that were reacted with ketones to afford the target compounds.

Results

Rational design, synthesis, and structure–activity relationship analysis

As the best two previously reported substrate binding site inhibitors are GSK3326595 and EPZ015666, we thus choose them as template compounds for further structure optimization. Fortunately, the complex crystal structure of PRMT5 with EPZ015666 was previously determined, which provided many clues for structure- based molecular design. Therefore, the detailed inter- action between EPZ015666 and PRMT5 was analyzed and shown in Fig. 2a. As can be seen from Fig. 2a, the N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypro- pyl) amino fragment of EPZ015666 forms crucial inter- action with many residues (L319, T323, F327, E435, L437, E444 and W579) contributing to its high inhibi- tory potency against PRMT5. It is worth to note that the cation-π interaction between tetrahydroisoquinoline ring and the cofactor SAM was first proposed and believed to contribute to the good selectivity of EPZ015666. Most importantly, the hydroxyl group in EPZ015666 forms stable H-bond interactions with L437, E444 and W579, which indicates that it is the key pharmacophore. So we retained this hydroxyl group in the subsequent molecular design. Giving that the chirality of the hydroxyl-bonding carbon was different in EPZ015666 and GSK3326595, and the co-crystal structure of PRMT5 complexed with GSK3326595 was not determined, molecular docking was thus used to probe the binding mode of GSK3326595. As can be seen from Fig. 2b, c, the N-(3-(3,4-dihydroisoquin- olin-yl)-2-hydroxypropyl) group in GSK3326595 could be well overlapped with that in EPZ015666, which suggested that it was the privileged fragment and the chirality of the hydroxyl-bonding carbon had little effect on the PRMT5 inhibitory activity. As shown in Fig. 2a, apart from the N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypro- pyl) amino fragment, the remaining fragment (left part) of EPZ015666 established well-defined hydrophobic the free amino group with extra rings significantly improved the activity by 14- to 17-fold (C_3, C_4 vs. C_2) or sixfold (C_6 vs. C_5), and the best inhibition against PRMT5 was found for compound C_4 with a cyclobutyl substitution on the amino moiety, showing a IC50 value of 0.72 ± 0.20 µM.
Selectivity testing indicated that C_4 is a selective PRMT5 inhibitor ate the selectivity over other PRMTs (PRMT1, PRMT4, and PRMT6). As shown in Table 2, the IC50 values of compound C_4 against PRMT1, PRMT4, and PRMT6 were all above 200 μM, which indicated that it was a selective PRMT5 inhibitor.

Anti‑proliferative activity on Z‑138 cells

Z-138 cells were used to assess the anti-proliferative effects upon treatment with our synthesized compounds. A long- term proliferation assay was developed to measure the cell interactions with PRMT5, which indicated that the rela- tive pocket was a hydrophobic pocket. Thus, the design strategy was to keep the N-(3-(3,4-dihydroisoquinolin- yl)-2-hydroxypropyl) amino moiety and to replace the left side part of EPZ015666 with groups that could form hydrophobic interactions with PRMT5. A total of 18 com- pounds (Fig. 3) were synthesized according to our design strategy by utilizing the traditional structure optimization methods, such as scaffold hopping, bioisosteric replace- ment and aromatic ring replacement (see Supplementary Material for synthetic details).
The inhibitory activities against PRMT5 at enzymatic level (Table 1) of the 18 compounds and EPZ015666 were evaluated using the method we previously reported [29, 38]. From Table 1, it could be seen that the activity of EPZ015666 (47 nM) remained the same level as reported (22 nM) in the literature, demonstrating the reliability of our assay, whereas replacing the amide moiety with the sul- fonamide group (A_1–A_10), benzo[d]thiazole ring (B_1 and B_2) or triazole-derived fragment (C_1–C_6) caused a distinct loss of activity. Compounds A_2–A_4 with an elec- tron-donating substituent on the benzene ring showed much better inhibitory activity than compounds (A_5–A_10) with corresponding electron-withdrawing groups, which was dif- ferent from the C series of compounds. Compound C_1 with an electron-withdrawing nitro substituent on the benzene ring showed much better inhibitory activity than C_2 with a corresponding electron-giving amino group, while the sub- stituting position of the amino group did not seem to impact the result (C_2 vs. C_5). Further structural modification of growth over 8 days. B and C series of compounds dem- onstrated potent concentration-dependent anti-proliferative effects, with IC50 values ranging from 2.60 to 57.57 μM (Table 3). As indicated by the assay results shown in Table 3, the cell level activities of these compounds were consist- ent with those of the enzyme level, and compound C_4 also proved to be the most potent one with an IC50 value of 2.60 ± 0.81 μM. The dose response curve for C_4 against Z-138 cell lines was shown in Fig. 4a.

Cellular symmetric dimethylation effects and inducement of cell apoptosis of C_4

The effect of C_4 on cellular symmetric arginine dimeth- ylation in Z-138 cells was tested by immunoblot using the pan-dimethyl symmetric arginine antibody symmetric dime- thyl arginine (SDMA). As shown in Fig. 4b, concentration- dependent decrease in the intensity of SmD3me2s was observed. SmD3 is one of the PRMT5 substrate proteins and can be used to track the cellular biochemical activity of PRMT5, so from the western blot result, we can conclude that compound C_4 could target PRMT5 in cellular context. Since PRMT5 inhibitors usually induce cancer cell apop- tosis [19, 33], the effect of C_4 on cell apoptosis was stud- ied in Z-138 cells for 48 h by flow cytometry. As shown in Fig. 5, concentration-dependent increase of apoptosis upon treatment with compound C_4 was observed. This result further confirmed the cellular target engagement of C_4 on PRMT5.

Discussion

In the present study, new PRMT5 inhibitors were suc- cessfully discovered by combination of structure-based drug design, synthesis and biological evaluation. Among our synthesized 18 compounds, compound C_4 showed the best PRMT5 inhibitory activity and profound cellular anti-proliferative activity with IC50 values of 0.72 ± 0.21 and 2.60 ± 0.81 μM, respectively. Besides, flow cytomet- ric analysis demonstrated that C_4 exhibited an obvious apoptotic effect. Compound C_4 also reduced the cellu- lar symmetric arginine dimethylation level of SmD3, a protein that can be used to track the biochemical activ- ity of PRMT5 in cells. The compounds identified in this work provide a new scaffold for future PRMT5 inhibitor development.

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