Design, synthesis and biological evaluation of 4-Aryl-5,7-dihydro- 6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives as a PI3Kα inhibitor

Article abstract of DOI:10.1248/bpb.b19-00080

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 K_ow values and molecular docking studies were performed to further investigate the drug-like properties of target compounds and interactions between 9c and PI3Kα.

Full text of DOI:10.1248/bpb.b19-00080

Vol. 42, No. 6
Biol. Pharm. Bull. 42, 1013–1018 (2019)
1013
Regular Article
Design, Synthesis and Biological Evaluation of 4-Aryl-5,7-dihydro-
H-pyrrolo[2,3-d]pyrimidin-6-one Derivatives as a PI3Kα Inhibitor
6
a
a
b
c
,a
a
Shengquan Hu, Zhichang Zhao, Yeming Ni, Hongxing Xin, Hong Yan,* and Xiuqing Song
a
Beijing Key Laboratory of Environmental and Viral Oncology, College of Life Science and Bio-engineering, Beijing
b
University of Technology; Beijing 100124, China: Fan Gongxiu Honors College, Beijing University of Technology;
Beijing 100124, China: and Jumpcan Pharmaceutical Group Company Limited; Taizhou, Jiangsu 225441, China.
c
Received January 30, 2019; accepted February 28, 2019
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
1
13
by H-NMR, C-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 pros-
tate 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 113ꢀ9nM
in the same order of magnitude as BEZ235. In addition, the LogKow values and molecular docking studies
were performed to further investigate the drug-like properties of target compounds and interactions between
9
c and PI3Kα.
Key words phosphoinositide 3-kinase α; pyrrolo[2,3-d]pyrimidin-6-one; scaffold hopping; Cell Counting
Kit-8
15)
INTRODUCTION
based on structure of Silenafil through scaffold hopping
Fig. 2).
Here, the scaffold hopping was utilized in designing a
(
Phosphoinositide 3-kinase (PI3K)-AKT-mammalian target
of rapamycin (mTOR) signal pathway has been paid attention novel series of 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
to as anti-tumor drug target by the medicinal community for derivatives as a PI3Kα inhibitor. Both of VS-5584 and
it regulating the growth, differentiation and apoptosis of cells CH-5132799 are centered on aromatic heterocyclic core, which
1
–4)
directly.
treat human malignancies by targeting key nodes in the PI3K exposed region substituent.
signaling pathway. PI3K inhibitors are considered as one of PI3K inhibitors substituted the 7-position as a solvent exposed
Its inhibitors are currently in various stages to were substituted by a morpholine, a headgroup and a solvent
9,16)
Most of the Chugai’s series
5
)
9)
the promising molecularly targeted cancer therapeutics. Co- region with an aromatic ring, sulfonyl, acyl or ureido. Only
panlisib (Fig. 1), a selectively PI3Kα/δ inhibitor, was approved a few substituted benzyl groups at the 7-position such as
17)
by the U.S. Food and Drug Administration (FDA) in 2017 for 1-B-49 reported by patent CN101501035. A purine deriva-
the treatment of adult patients experiencing relapsed follicular tive No. 8 in patent WO2009045174A1 reported by Nagaraj
lymphoma who have received at least two prior systemic ther- et al. employ 4-methoxybenzyl as solvent exposing region
6
)
18)
apies. Duvelisib (Fig. 1), dual inhibitor of PI3Kδ/γ, was more as well. According to the skeleton structure of 1-B-49 and
effective against 17p-deficient chronic lymphocytic leukemia compound No. 8 the aromatic heterocyclic core was changed
than Ofatumumab, that approved by FDA as a fully human to pyrrolo[2,3-d]pyrimidin-6-one by scaffold hopping (Fig. 3).
7
)
monoclonal antibody. VS-5584 and CH-5132799 (Fig. 1), A common morpholinyl group in the PI3K inhibitors caught
PI3K/mTOR inhibitors, are potential drugs for the treatment our attention from the early LY294002 (Fig. 1) to the phase
of solid tumors, lymphomas and malignant mesothelioma in II clinical trial stage GSK-2636771. Therefore, the vital mor-
8
–10)
phase I clinical stage.
GSK-2636771 (Fig. 1), a selective pholinyl group, immobilized at the 2-position of pyrrolo[2,3-
inhibitor of PI3Kβ in phase II clinical trial for the treatment d]pyrimidin-6-one, were retained. As target compound, the
1
1,12)
of advanced solid tumors.
In addition some potential PI3K 4-position substituent changed to various aromatic six-mem-
bered rings for seeking out a PI3K inhibitor with altered drug
inhibitor projects are also moving forward.
Based on the bioisosteric replacement of the core motif of properties and intellectual property rights.
a molecule, the scaffold hopping have been considered as one
of successful strategies for improving the physicochemical CHEMISTRY
properties and biological properties of compounds in me-
dicinal chemistry. It widely applied not only with the aim
General synthetic procedures are shown in Chart 1. The
of improving pharmacokinetics or pharmacodynamics but starting materials of the target compounds of diethyl malonate
also highly with commercial value such as intellectual prop- (1) and ethyl chloroacetate (2) reacted to offer the interme-
13,14)
erty aspects.
One of the instances was Vardenafil, a new diate 3 by nucleophilic substitution under basic conditions.
phosphodiesterase type 5 (PDE ) inhibitor for the treatment Intermediate 3 was reacted with urea in a solution of sodium
5
of erectile dysfunction, that has been designed successfully methoxide in methanol to form the corresponding barbitu-
*
To whom correspondence should be addressed. e-mail: hongyan@bjut.edu.cn
©
2019 The Pharmaceutical Society of Japan

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ric acid (4). Intermediate 4 was chlorinated by phosphorus and target compounds (shown in Table 1) were structurally
1
13
oxychloride and nucleophilic aromatic substitution reactions characterized by H-NMR and C-NMR spectra. The target
to obtain intermediate 7, which was ring-closed under acidic compounds were also structurally characterized by high reso-
conditions to form the corresponding ring-closing product (8). lution (HR)-MS.
Intermediate 8 reacted with the corresponding boronic acid
In order to verify which chlorine of intermediate 6 was
or boric acid ester under the palladium catalysis to give the replaced during the synthesis of intermediate 7, another
target compounds 9a–q, respectively. All the intermediates 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 indi-
cates that the chlorine at the 2-position of intermediate 6 was
substituted by morpholine to form intermediate 7.
BIOLOGICAL ASSAY
Cytotoxicity Assay Cytotoxic activity of compounds
19–21)
in vitro was detected by Cell Counting Kit-8 (CCK-8).
Human malignant glioblastoma cell line Uppsala 87 Malignant
Glioma (U87MG) and human prostate cancer cell line prostate
2
2)
cancer-3 (PC-3) were selected as test cell lines. The inhibi-
tion rates of 17 compounds (50µM) and PI-103 (50µM) which
served as the positive control were presented in Table 1.
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
Fig. 1. Structures of PI3K Inhibitors
9
c, 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 re-
sults of IC50 test of 9c against cell line PC-3 was 50.42µM and
Fig. 2. Example of Scaffold Hopping
Fig. 3. Design of Target Compounds

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1015
Reagents and conditions: (a) EtONa, EtOH; (b) EtONa, MeOH; (c) DIPEA, POCl ; (d) DIPEA, DMF; (e) 4-DMAP, DMF; (f) TsOH, Toluene; (g) (dppf) PdCl , Na CO ,
3
2
2
2
3
dioxane/H O.
2
Chart 1
Reagents and conditions: (d) DIPEA, DMF; (e) 4-DMAP, DMF.
Chart 2
against cell line U87MG was 28.39µM (Table 2).
Kinase Enzyme Assay For further target verification, the the Syracuse Research Corporation (SRC). The molecular
United States Environmental Protection Agency (EPA) and
2
5)
enzyme inhibitory activity was carried out as described previ- docking analysis was carried out using the AutoDock software
2
3)
ously. As shown in Table 2, that 9c has strong PI3Kα in- package as implemented through the graphical user interface
hibitory activity with IC50 of 113± 9nM. Under the same test AutoDockTools (ADT 1.5.2). The protein crystal structures of
2
6)
conditions, BEZ235, a PI3K/mTOR inhibitor (reported PI3Kα PI3Kα (PDB: 4L23) was chosen as receptor protein.
2
4)
inhibitory activity with IC50 of 4nM), only shown PI3Kα
The range of predicted LogKow value of the target com-
inhibitory activity with IC50 of 45± 13nM, which was on pounds was between 2.19–6.21 (shown in Table 1). All
the same order of magnitude as 9c. 9c also showed a certain the target compounds have a molecular weight between
mTOR inhibitory activity with IC50 of 1094± 71 nM.
416–500g/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
THEORETICAL CALCULATION
For study the physicochemical properties of the target com- rule of five, which is a rule of thumb to evaluate druglike-
2
7)
pounds and better understanding the possible binding modes ness. These results indicate that the synthesized target com-
of 9c in the PI3Kα active site, the computational studies pound has good drug-like properties.
were performed. The LogKow values of the target compounds
Initially, the reliability of the docking method and pa-
were calculated using KOWWIN, a part of the Estimation rameters being verified. (see Supplementary Materials for
Programs Interface (EPI) suite environmental modeling details) Docking analysis shown, two conventional hydrogen
program, which was developed and was maintained by the bond interactions between 9c and PI3Kα were observed. The

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Table 1. Structures, LogKow Predicted and Inhibition Rate of Target Compounds
a) The value predicted by KOWWIN. b) The mean value of five times measurements.
Table 2. IC50 of 9c against U87MG, PC-3, PI3Kα and mTOR
a) NT: not tested. b) The mean value of twice measurements.
oxygen of morpholine formed a hydrogen bond with Lys802, 3-yl on 4-position of pyrrolo[2,3-d]pyrimidin-6-one formed
while amino group on the pyridine formed an additional hydrophobic interactions deeply with the hydrophobic pocket
hydrogen bond interaction with Glu849. The 6-aminopyridin- formed by Ile848, Ser854, Val850, Val851, Glu849, Trp780,

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1017
Fig. 4. Dominant Conformation of Compound 9c in the PI3Kα Active Site
CONCLUSION
In this paper,
a
series of 4-aryl-pyrrolo[2,3-d]-
pyrimidin-6-one derivatives were designed as PI3Kα inhibi-
tor by scaffold hopping of the Chugai’s series PI3K inhibitor
1-B-49. The target compounds were synthesized via nucleo-
philic substitution, ring-closure, chlorination, Suzuki reaction
1
13
and verified by the H-NMR, C-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 inhibi-
tion 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± 9nM. The
LogKow 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 mo-
lecular 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 hydropho-
bic pocket and formed hydrogen bond with the residues of
Glu849. With the value as novel PI3Kα inhibitor, the com-
Fig. 5. Dominant Conformation of 9c Superimposed with That of Com-
pound 9a, 9b, 9d, and 9e in Binding Site of PI3Kα
Tyr836, Met922, Phe930 and Ile932. There are also some Pi- pound 9c could be as a lead compound for further optimiza-
sigma and Pi-sulfur interaction between the pocket and 9c. tion in the research and development of new PI3Kα inhibitors.
There are three carbon hydrogen bond interactions between
Asn920, Asp933 and methoxy on benzyl, morpholinyl (Fig.
). These effects have obvious advantages over the result of Beijing Natural Science Foundation (No. KZ201510005007).
the docking of 1-B-49 or No. 8 in Fig. 3 (see Supplementary Thanks to Xiuqing Song for the NMR test. The services of
Materials for details).
cytotoxic activity were kindly provided by Nanjing Ogphar-
Acknowledgments This study was supported by 2015
4
The superimpositions of dominant conformation of 9c in maceutical Co., Ltd. (Nanjing, China). The service of enzyme
binding site of PI3Kα, compared with 9a, 9b, 9d and 9e, IC50 was kindly provided by Huawei Pharmaceutical Co., Ltd.
have shown that there were two kinds of binding mode (Fig. (Shandong, China).
5
). The 6-aminopyridin-3-yl on 9c occupied the hydrophobic
pocket and form hydrogen bonds with Glu849 in the pocket.
Conflict of Interest The authors declare no conflict of
In addition, compound 9a, 9b, 9d and 9e perform the same interest.
binding mode, which the methoxybenzyl stretched into the
hydrophobic pocket. Combined with biological activity and
Supplementary Materials The online version of this
docking patterns, it can be concluded that the 6-aminopyridin- article contains supplementary materials. Full experimen-
3
-yl occupy the hydrophobic pocket and formed hydrogen tal detail, characterization data for intermediates and target
bond with the residues of Glu849 may contribute to improved compounds and H-, C-NMR spectra and HR-MS with this
PI3Kα inhibition activity.
article can be found in the online version.
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