Research paperDesign, synthesis, biological evaluation and docking studies of novel 2-substituted-4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives as dual PI3Kα/mTOR inhibitors
Graphical abstract
Four series of 2-substituted-4-morpholino- 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives were synthesized and evaluated for their activity against PI3Kα and mTOR kinase and cancer cell lines. The most promising compound 11 showed good antitumor potency for A549, PC-3 and MCF-7 cell lines with IC50 values of 0.52 ± 0.10 μM, 1.41 ± 0.10 μM, and 4.82 ± 0.24 μM, and strong antitumor activities against PI3Kα/mTOR with IC50 values of 6.72 ± 0.30 μM and 0.94 ± 0.10 μM.
Introduction
The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/the mammalian target of rapamycin (mTOR) pathway is an intracellular signaling pathway important in regulating various cellular processes, including growth regulation, apoptosis, and survival in many cancers [1], [2], [3]. In recent years, many (fused-)pyrimidine/triazine derivatives were reported as PI3K-Akt-mTOR signal pathway inhibitors (Fig. 1) [4], [5], [6], [7], [8], [9]. Among them, GDC-0941, which is a potent, orally bioavailable inhibitor of PI3K, exerted antitumor activity against an array of human tumor cell lines and is currently undergoing Phase II clinical trials [10]. PF-05139962 [6], compoundⅠ [7], PKI-402 [11] and PKI-587 [12] (Fig. 1) are several aryl urea derivatives which exhibit potent anti-tumor activity. Structure-activity relationships (SARs) showed that aryl urea moieties were essential for the anti-tumor activity.
In our previous research, we reported several series of analogous of PF-05139962, 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine hydrazone (Fig. 1) which demonstrated excellent antitumor activity. Biological evaluation results showed that thiopyrano[4,3-d]pyrimidines were important for the activity of the target compounds.
In continuation of our previous research, and in order to screen compounds which possess excellent in vitro/in vivo anti-tumor activity as well as improved pharmacokinetic properties, further studies on analogous of PF-05139962 were carried out in this research. The design strategy for all target compounds is shown in Fig. 2 and is described as follow. Firstly, the urea group was kept unchanged, and a series of phenyl pyridine urea compounds 9–12 was designed by replacing the ethyl group of PF-05139962 with aryl group and replacing the phenyl group with pyridine group. What's more, sulfur atom was oxidized to study the influence to the target compounds. This series of compounds showed excellent cytotoxicity and moderate PI3Kα/mTOR kinases inhibitory activity. Furthermore, investigations were carried out in details to study the effect of urea to the activity and therefore urea was replaced by sulfonylurea group to yield compounds 13–18 and 19–22. However, this two series of compounds showed poor cytotoxicity and moderate mTOR kinase inhibitory activity. Thus, we shifted our focus back to the phenyl pyridine urea series. To investigate the influences of pyridine urea to the activity and inspired by previous compounds 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine hydrazone (Compound Ⅱ in Fig. 2), phenyl pyridine urea was replaced by piperazine phenylurea to afford compounds 23–28. The activity of 23–28 was similar to that of 9–12 and was much better than that of compounds 13–18 and 19–22.
The studies of Wentao Yue showed that PI3K, Akt and mTOR gene of the PI3K-Akt-mTOR signaling pathway expression in the non-small cell lung cancer were (61 ± 23)%, (77 ± 32)%, (43 ± 21)% respectively [13]. And the level of this pathway in breast cancer also up to 70% [14]. In the prostate cancer cells, the missing of tumor suppressor gene (PTEN) resulted in the high expression of PI3K-Akt-mTOR signaling pathway. In addition, the studies of GDC-0941 showed it has strong inhibitory activities against A549 (lung cancer cell lines), PC-3 (prostate cancer cell lines) and MCF-7(breast cancer cell lines), suggesting that these three cells have a high sensitivity for PI3K-Akt-mTOR signaling pathway inhibitors [15], [16], [17]. Therefore, A549, PC-3 and MCF-7 cell lines were chose as the test cell line for in vitro antitumor experiments in this study.
Herein we disclosed the design, synthesis and antitumor activity of four series of compounds against A549, PC-3, MCF-7 and PI3Kα, mTOR and c-Met kinases. Moreover, docking studies were presented in this paper as well.
Section snippets
Chemistry
The preparation of target compounds 9–28 was described in Scheme 1, Scheme 2.
Compounds 7a–d and 8a–e were synthesized according to the procedures reported previously by our group [18], [19]. The different substituent of phenylamine reacted with triphosgene to get compound compounds 7a–d. 8a–e was synthesized from the different substituent of chlorobenzene through three steps.
The key intermediates 4-(2-chloro-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine (4a) and
Biological evaluation
The target compounds (9–28) were evaluated for the cytotoxicity against three cancer cell lines A549, PC-3 and MCF-7 by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation assay, together with reference compounds sorafenib and GDC-0941. In addition, four selected compounds 10, 11, 24 and 27 were further evaluated for their IC50 values against mTOR, PI3Kα, c-Met kinases by LANCE® Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assay,
Conclusions
In summary, four series of 4-(2-chloro-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-yl)morpholine derivatives were designed, synthesized and evaluated for antitumor activity against three cancer cell lines and mTOR, PI3Kα and c-Met kinases in vitro. The pharmacological results indicated that seven of the synthesized compounds displayed moderate antitumor activity and four selected compounds showed equal to more potency than lead compound sorafenib and GDC-0941. The most promising compound 11
Chemistry
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. NMR spectra were performed using Bruker 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal standard. Mass spectra (MS) were taken in ESI mode on Agilent 1100 LCMS (Agilent, Palo Alto, CA, USA). All the materials were obtained from commercial suppliers and used without purification, unless otherwise specified. Yields were not
Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science Funds (No. 81460527), Science and Technology Project Founded by the Education Department of Jiangxi Province (GJJ150796), Top-notch talent project of Jiangxi Science & Technology Normal University (2015QNBJRC001), Program of Key Laboratory of Drug Design and Optimization, Jiangxi Science & Technology Normal University (300098010306), College Students' Science and Technology Innovation Project (20140802034)
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These authors contribute equally to this work.