Biological and Pharmaceutical Bulletin
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Design, Synthesis and Biological Evaluation of 4-Aryl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one Derivatives as a PI3Kα Inhibitor
Shengquan HuZhichang ZhaoYeming NiHongxing XinHong YanXiuqing Song
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2019 年 42 巻 6 号 p. 1013-1018


A novel series of 4-aryl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives were designed as a phosphoinositide 3-kinase α (PI3Kα) inhibitor by scaffold hopping. The target compounds, characterized by 1H-NMR, 13C-NMR and high resolution (HR)-MS, were synthesized from diethyl malonate and ethyl chloroacetate by nucleophilic substitution, ring-closure, chlorination and Suzuki reaction, etc. The biological activities were evaluated with cytotoxic activity in vitro on Uppsala 87 Malignant Glioma (U87MG) and prostate cancer-3 (PC-3) by Cell Counting Kit-8 (CCK-8). The results showed that compound 9c displayed the higher inhibition than the positive control PI-103, and high PI3Kα inhibitory activity with IC50 of 113 ± 9 nM in the same order of magnitude as BEZ235. In addition, the Log Kow values and molecular docking studies were performed to further investigate the drug-like properties of target compounds and interactions between 9c and PI3Kα.


Phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signal pathway has been paid attention to as anti-tumor drug target by the medicinal community for it regulating the growth, differentiation and apoptosis of cells directly.14) Its inhibitors are currently in various stages to treat human malignancies by targeting key nodes in the PI3K signaling pathway. PI3K inhibitors are considered as one of the promising molecularly targeted cancer therapeutics.5) Copanlisib (Fig. 1), a selectively PI3Kα/δ inhibitor, was approved by the U.S. Food and Drug Administration (FDA) in 2017 for the treatment of adult patients experiencing relapsed follicular lymphoma who have received at least two prior systemic therapies.6) Duvelisib (Fig. 1), dual inhibitor of PI3Kδ/γ, was more effective against 17p-deficient chronic lymphocytic leukemia than Ofatumumab, that approved by FDA as a fully human monoclonal antibody.7) VS-5584 and CH-5132799 (Fig. 1), PI3K/mTOR inhibitors, are potential drugs for the treatment of solid tumors, lymphomas and malignant mesothelioma in phase I clinical stage.810) GSK-2636771 (Fig. 1), a selective inhibitor of PI3Kβ in phase II clinical trial for the treatment of advanced solid tumors.11,12) In addition some potential PI3K inhibitor projects are also moving forward.

Fig. 1. Structures of PI3K Inhibitors

Based on the bioisosteric replacement of the core motif of a molecule, the scaffold hopping have been considered as one of successful strategies for improving the physicochemical properties and biological properties of compounds in medicinal chemistry. It widely applied not only with the aim of improving pharmacokinetics or pharmacodynamics but also highly with commercial value such as intellectual property aspects.13,14) One of the instances was Vardenafil, a new phosphodiesterase type 5 (PDE5) inhibitor for the treatment of erectile dysfunction, that has been designed successfully based on structure of Silenafil through scaffold hopping15) (Fig. 2).

Fig. 2. Example of Scaffold Hopping

Here, the scaffold hopping was utilized in designing a novel series of 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives as a PI3Kα inhibitor. Both of VS-5584 and CH-5132799 are centered on aromatic heterocyclic core, which were substituted by a morpholine, a headgroup and a solvent exposed region substituent.9,16) Most of the Chugai’s series PI3K inhibitors substituted the 7-position as a solvent exposed region with an aromatic ring, sulfonyl, acyl or ureido.9) Only a few substituted benzyl groups at the 7-position such as 1-B-49 reported by patent CN101501035.17) A purine derivative No. 8 in patent WO2009045174A1 reported by Nagaraj et al. employ 4-methoxybenzyl as solvent exposing region as well.18) According to the skeleton structure of 1-B-49 and compound No. 8 the aromatic heterocyclic core was changed to pyrrolo[2,3-d]pyrimidin-6-one by scaffold hopping (Fig. 3). A common morpholinyl group in the PI3K inhibitors caught our attention from the early LY294002 (Fig. 1) to the phase II clinical trial stage GSK-2636771. Therefore, the vital morpholinyl group, immobilized at the 2-position of pyrrolo[2,3-d]pyrimidin-6-one, were retained. As target compound, the 4-position substituent changed to various aromatic six-membered rings for seeking out a PI3K inhibitor with altered drug properties and intellectual property rights.

Fig. 3. Design of Target Compounds


General synthetic procedures are shown in Chart 1. The starting materials of the target compounds of diethyl malonate (1) and ethyl chloroacetate (2) reacted to offer the intermediate 3 by nucleophilic substitution under basic conditions. Intermediate 3 was reacted with urea in a solution of sodium methoxide in methanol to form the corresponding barbituric acid (4). Intermediate 4 was chlorinated by phosphorus oxychloride and nucleophilic aromatic substitution reactions to obtain intermediate 7, which was ring-closed under acidic conditions to form the corresponding ring-closing product (8). Intermediate 8 reacted with the corresponding boronic acid or boric acid ester under the palladium catalysis to give the target compounds 9aq, respectively. All the intermediates and target compounds (shown in Table 1) were structurally characterized by 1H-NMR and 13C-NMR spectra. The target compounds were also structurally characterized by high resolution (HR)-MS.

Chart 1

Reagents and conditions: (a) EtONa, EtOH; (b) EtONa, MeOH; (c) DIPEA, POCl3; (d) DIPEA, DMF; (e) 4-DMAP, DMF; (f) TsOH, Toluene; (g) (dppf)2PdCl2, Na2CO3, dioxane/H2O.

In order to verify which chlorine of intermediate 6 was replaced during the synthesis of intermediate 7, another synthesis route was designed (Chart 2). Two chlorines at the 4-position and 6-position on intermediate 10 are symmetrical. Therefore, intermediate 10 react with 4-methoxybenzylamine to yield the unique product intermediate 7. By comparing the NMR spectra, it was found that the spectra of intermediate 7 synthesis by Charts 1 and 2 can completely overlap. This indicates that the chlorine at the 2-position of intermediate 6 was substituted by morpholine to form intermediate 7.

Chart 2

Reagents and conditions: (d) DIPEA, DMF; (e) 4-DMAP, DMF.


Cytotoxicity Assay

Cytotoxic activity of compounds in vitro was detected by Cell Counting Kit-8 (CCK-8).1921) Human malignant glioblastoma cell line Uppsala 87 Malignant Glioma (U87MG) and human prostate cancer cell line prostate cancer-3 (PC-3) were selected as test cell lines.22) The inhibition rates of 17 compounds (50 µM) and PI-103 (50 µM) which served as the positive control were presented in Table 1.

Table 1. Structures, Log Kow Predicted and Inhibition Rate of Target Compounds

a) The value predicted by KOWWIN. b) The mean value of five times measurements.

As can be seen from the Table 1, compounds 9a, 9b, 9c and 9f showed a certain inhibitory effect against cell line PC-3, and the inhibition rate was 13 to 67%. The compound 9c, with 6-aminopyridin-3-yl at 4-position of pyrrolo[2,3-d]pyrimidin-6-one, showed strong cytostatic effect against cell line PC-3 and was stronger than that of the positive control PI-103. Compound 9c showed intense cytostatic effect against cell line U87MG over the positive control as well. Further results of IC50 test of 9c against cell line PC-3 was 50.42 µM and against cell line U87MG was 28.39 µM (Table 2).

Table 2. IC50 of 9c against U87MG, PC-3, PI3Kα and mTOR

a) NT: not tested. b) The mean value of twice measurements.

Kinase Enzyme Assay

For further target verification, the enzyme inhibitory activity was carried out as described previously.23) As shown in Table 2, that 9c has strong PI3Kα inhibitory activity with IC50 of 113 ± 9 nM. Under the same test conditions, BEZ235, a PI3K/mTOR inhibitor (reported PI3Kα inhibitory activity with IC50 of 4 nM),24) only shown PI3Kα inhibitory activity with IC50 of 45 ± 13 nM, which was on the same order of magnitude as 9c. 9c also showed a certain mTOR inhibitory activity with IC50 of 1094 ± 71 nM.


For study the physicochemical properties of the target compounds and better understanding the possible binding modes of 9c in the PI3Kα active site, the computational studies were performed. The Log Kow values of the target compounds were calculated using KOWWIN, a part of the Estimation Programs Interface (EPI) suite environmental modeling program, which was developed and was maintained by the United States Environmental Protection Agency (EPA) and the Syracuse Research Corporation (SRC).25) The molecular docking analysis was carried out using the AutoDock software package as implemented through the graphical user interface AutoDockTools (ADT 1.5.2). The protein crystal structures of PI3Kα (PDB: 4L23)26) was chosen as receptor protein.

The range of predicted Log Kow value of the target compounds was between 2.19–6.21 (shown in Table 1). All the target compounds have a molecular weight between 416–500 g/mol. The number of hydrogen bond donors (the total number of nitrogen–hydrogen and oxygen-hydrogen bonds) or hydrogen bond acceptors (all nitrogen or oxygen atoms) of all target compounds are also satisfied Lipinski’s rule of five, which is a rule of thumb to evaluate druglikeness.27) These results indicate that the synthesized target compound has good drug-like properties.

Initially, the reliability of the docking method and parameters being verified. (see Supplementary Materials for details) Docking analysis shown, two conventional hydrogen bond interactions between 9c and PI3Kα were observed. The oxygen of morpholine formed a hydrogen bond with Lys802, while amino group on the pyridine formed an additional hydrogen bond interaction with Glu849. The 6-aminopyridin-3-yl on 4-position of pyrrolo[2,3-d]pyrimidin-6-one formed hydrophobic interactions deeply with the hydrophobic pocket formed by Ile848, Ser854, Val850, Val851, Glu849, Trp780, Tyr836, Met922, Phe930 and Ile932. There are also some Pi-sigma and Pi-sulfur interaction between the pocket and 9c. There are three carbon hydrogen bond interactions between Asn920, Asp933 and methoxy on benzyl, morpholinyl (Fig. 4). These effects have obvious advantages over the result of the docking of 1-B-49 or No. 8 in Fig. 3 (see Supplementary Materials for details).

Fig. 4. Dominant Conformation of Compound 9c in the PI3Kα Active Site

The superimpositions of dominant conformation of 9c in binding site of PI3Kα, compared with 9a, 9b, 9d and 9e, have shown that there were two kinds of binding mode (Fig. 5). The 6-aminopyridin-3-yl on 9c occupied the hydrophobic pocket and form hydrogen bonds with Glu849 in the pocket. In addition, compound 9a, 9b, 9d and 9e perform the same binding mode, which the methoxybenzyl stretched into the hydrophobic pocket. Combined with biological activity and docking patterns, it can be concluded that the 6-aminopyridin-3-yl occupy the hydrophobic pocket and formed hydrogen bond with the residues of Glu849 may contribute to improved PI3Kα inhibition activity.

Fig. 5. Dominant Conformation of 9c Superimposed with That of Compound 9a, 9b, 9d, and 9e in Binding Site of PI3Kα


In this paper, a series of 4-aryl-pyrrolo[2,3-d]pyrimidin-6-one derivatives were designed as PI3Kα inhibitor by scaffold hopping of the Chugai’s series PI3K inhibitor 1-B-49. The target compounds were synthesized via nucleophilic substitution, ring-closure, chlorination, Suzuki reaction and verified by the 1H-NMR, 13C-NMR, HR-MS. Human malignant glioblastoma cell line U87MG and human prostate cancer cell line PC-3 were selected as test cell lines to detect the inhibitory activities of target compounds by CCK-8. The results shown that compound 9c displayed the highest inhibition and was better than that of positive control PI-103. The result of PI3Kα kinase enzyme assays also show that 9c has strong PI3Kα inhibitory activity with IC50 of 113 ± 9 nM. The Log Kow values of the target compounds were calculated using KOWWIN and the results indicate that the synthesized target compounds have good drug-like properties. An in silico molecular docking study indicated that the 6-aminopyridin-3-yl on the dominant conformation of 9c contribute to improved PI3Kα inhibition activity by extending to the hydrophobic pocket and formed hydrogen bond with the residues of Glu849. With the value as novel PI3Kα inhibitor, the compound 9c could be as a lead compound for further optimization in the research and development of new PI3Kα inhibitors.


This study was supported by 2015 Beijing Natural Science Foundation (No. KZ201510005007). Thanks to Xiuqing Song for the NMR test. The services of cytotoxic activity were kindly provided by Nanjing Ogpharmaceutical Co., Ltd. (Nanjing, China). The service of enzyme IC50 was kindly provided by Huawei Pharmaceutical Co., Ltd. (Shandong, China).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials. Full experimental detail, characterization data for intermediates and target compounds and 1H-, 13C-NMR spectra and HR-MS with this article can be found in the online version.

© 2019 The Pharmaceutical Society of Japan