Discovery of novel PARP/PI3K dual inhibitors with high efficiency against BRCA-proficient triple negative breast cancer

Article abstract of DOI:10.1016/j.ejmech.2020.113054

Co-targeting PARP and PI3K by PARP/PI3K dual inhibitors has been recognized as a promising chemotherapeutic strategy for the treatment of triple negative breast cancer (TNBC) in our previous work. To further explore novel and more potent PARP/PI3K dual in

Full text of DOI:10.1016/j.ejmech.2020.113054

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of novel PARP/PI3K dual inhibitors with high efficiency
against BRCA-proficient triple negative breast cancer
,
,
, b, **
Junwei Wang a 1, Guangchao He b 1, Hui Li a, Yiran Ge b, Shuping Wang b, Yungen Xu a
,
, b, *
Qihua Zhu a
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, China
b Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Co-targeting PARP and PI3K by PARP/PI3K dual inhibitors has been recognized as a promising chemo-
therapeutic strategy for the treatment of triple negative breast cancer (TNBC) in our previous work. To
further explore novel and more potent PARP/PI3K dual inhibitors, a series of compounds were designed,
synthesized and evaluated for their pharmacological properties, resulting in the candidate compound 12,
a potent and highly selective PARP/PI3K dual inhibitor. Compared to Olaparib, compound 12 exhibits a
superior antiproliferative profile against BRCA-proficient MDA-MB-468 cells. In MDA-MB-468 cell-
derived xenograft model, compound 12 displayed excellent antitumor efficacy at a dose of 50 mg/kg,
which is considerably more efficacious than the single administration of Olaparib or BKM120. Further-
more, compound 12 displayed good metabolic stability and high safety. Taken together, these results
suggest that compound 12 as a novel PARP/PI3K dual inhibitor is worthy for further study.
© 2020 Elsevier Masson SAS. All rights reserved.
Received 20 September 2020
Received in revised form
17 November 2020
Accepted 24 November 2020
Available online xxx
Keywords:
Triple negative breast cancer
PARP/PI3K dual Inhibitors
Antitumor activity
Structural modifications
Structure-activity relationships
1. Introduction
are recruited to damaged DNA for repairing of the double-strand
breaks (DSBs) [10,11]. The loss or mutation of BRCA1/2 in breast
Triple negative breast cancer (TNBC) which characterized by
deficient expression of estrogen, progesterone, and human
epidermal growth factor receptor 2 receptors, is a particularly
aggressive subtype of breast cancer with poorer prognosis and
higher recurrence [1,2]. Current treatment options for TNBC are
limited to chemotherapeutic regimens that are highly toxic and
cannot be cured. Despite advances in targeted drugs [3e7], the
treatment of TNBC still remains a major challenge in clinical prac-
tice. Cancer Genome Atlas revealed that the genomic alterations of
TNBC included extensive copy number alterations, p53 mutations,
phosphoinositide 3-kinase (PI3K) pathway activation, and de-
ficiencies in DNA damage repair, such as homologous recombina-
tion (HR), which provided a rationale for the design of TNBC
targeted therapy drugs [8,9].
epithelial cells disables DNA damage repair via HR, leading to
genomic instability and predisposing patients to hereditary breast
cancer [11]. Poly(ADP-ribose)polymerase (PARP) is a kind of nuclear
enzyme which plays important roles in the repair of DNA single-
strand breaks (SSBs) through the base excision repair (BER)
pathway and non-homologous end joining (NHEJ) pathway [12].
When PARP is inhibited, unrepaired SSBs accumulate in BRCA-
deficient cells and transform into DSBs that HR cannot repair,
leading to continuous and lethal DNA damage [13e15]. Therefore,
the loss of BRCA1/2 will result in HR deficiency and make cancer
cells sensitive to PARP inhibitors.
Based on the concept of synthetic lethality, four PARP inhibitors,
Olaparib [16,17], Rucaparib [18], Niraparib [19,20] and Talazoparib
[21] have been approved to treat BRCA-mutant advanced ovarian
cancer and metastatic breast cancer (Fig. 1). However, only a frac-
tion of TNBC patients harbor BRCA mutations are benefit to this
treatment, limiting the clinical application of PARP inhibitors.
Meanwhile, the resistance caused by reversion of BRCA mutations
and other mechanisms reduces the clinical efficacy of PARP in-
hibitors [22,23]. Therefore, development of advanced therapies for
TNBC without BRCA mutation remains in high demand and
challenging.
BRCA1 and BRCA2 proteins are essential components of HR that
* Corresponding author. State Key Laboratory of Natural Medicines, China Phar-
maceutical University, Nanjing, 210009, China.
** Corresponding author. State Key Laboratory of Natural Medicines, China Phar-
maceutical University, Nanjing, 210009, China.
E-mail addresses: xyg@cpu.edu.cn (Y. Xu), zhuqihua@vip.126.com (Q. Zhu).
Contributed equally.
1
https://doi.org/10.1016/j.ejmech.2020.113054
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
Please cite this article as: J. Wang, G. He, H. Li et al., Discovery of novel PARP/PI3K dual inhibitors with high efficiency against BRCA-proficient
triple negative breast cancer, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.113054

J. Wang, G. He, H. Li et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Gly863, and
p-stacking interactions with Tyr907. In addition,
compound 3 can form a hydrogen bond with Val851 and three key
hydrogen bonds with Asp810, Asp933 and Tyr836 (Fig. 3). The
docking results showed that compound 3 fitted well in the active
site pocket of PARP-1 and PI3K
teractions with the key residues in the corresponding active site,
indicating its potential to inhibit both PARP-1 and PI3K
a, and formed efficient binding in-
a
.
2.2. Structure optimization and in vitro PARP-1/PI3K
a inhibition
assay
To validate the feasibility of our design, compound 3 was syn-
thesized and evaluated for PARP-1 and PI3K inhibitory activities
in vitro. The results showed that compound 3 exhibited good PARP-
1 and PI3K inhibitory activity with the inhibition ratio more than
a
a
50% at 10 nM and 100 nM, respectively. Encouraged by this
promising result, compound 3 was chosen as new lead compound
for further optimization to find more potent dual PARP/PI3K in-
hibitors. Compounds (4-11) were designed and synthesized by
varying the R1 substituent and 1,3,5-triazine scaffold, and their
Fig. 1. Chemical structures of representative PARP inhibitors.
PARP-1 and PI3Ka inhibitory activities were summarized in Table 1.
PI3K/AKT/mTOR pathway is aberrant activated in TNBC, which
stabilizes and preserves DSB repair by interacting with the HR
complex [24,25]. Thus, co-targeting of PARP and PI3K may be a
promising chemotherapeutic strategy to treat BRCA-proficient
TNBC. In 2012, Ibrahim and Juvekar independently found that
PI3K inhibition impairs the expression of BRCA1/2 and sensitizes
BRCA-proficient TNBC to PARP inhibitors, providing a rationale to
combine PARP and PI3K inhibitors in the treatment of BRCA-
proficient TNBC [26,27]. Based on these findings, a clinical trial
with BKM120 and Olaparib is being initiated in patients with TNBC
[28]. Despite the highly significant therapeutic relevance of drug
combination therapy, it turned out to be considerably challenging
due to complicated pharmacokinetics, dissimilar toxicity profiles,
undesirable drug-drug interactions, as well as issues of patient
compliance [29e31]. Thus, the design of dual-target or multi-target
drugs becomes an effective strategy to overcome these drawbacks.
Herein, we present the design, synthesis and biological evaluation
of novel dual PARP/PI3K inhibitors based on the structural opti-
mization of compound 1 [32]. And our medicinal chemistry efforts
finally led to the discovery of a promising candidate 12, which
displayed good physicochemical properties, and potent in vitro and
in vivo activities against BRCA-proficient TNBC (Fig. 2).
As expected, R1 substituent had no significant effect on PARP-1
inhibitory activity, and most compounds exhibited potent PARP-1
inhibitory activity with the inhibition ratio more than 50% at
10 nM. However, PI3K
depending on the R1 substituent. The aromatic groups with a lower
electron density showed better PI3K inhibitory activity. For
example, compounds 6e8 with a pyrimidine or pyridine ring
showed stronger PI3K inhibitory activity than compound 9 with a
a inhibitory activity was considerably
a
a
benzene ring, compound 10 with an indole ring and compound 11
with a benzimidazole ring (Table 1). In addition, we found that
1,3,5-triazine scaffold is more beneficial for PI3Ka inhibition than
pyrimidine scaffold. Overall consideration of PARP-1 and PI3K
a
inhibition ratio, compounds 6e8 were selected to evaluate the IC50
values (Table 2). As a result, the structural optimization culminated
in compound 8, which remained potent and balanced PARP-1 and
PI3Ka inhibitory activities with the IC50 values of 5.8 nM and
6.3 nM, respectively.
2.3. Physical and chemical property optimization
Although compound 8 displayed excellent in vitro inhibitory
activity against PARP-1 and PI3Ka, poor solubility and pharmaco-
kinetic parameters profile hindered its further investigation. To
improve its solubility, we tried to synthesis hydrochloride, tartrate
and phosphate salts of 8. However, there was little improvement in
the solubility of these salts. We hypothesized that the addition of a
hydrophilic group might be an effective way to increase its solu-
bility. As a result, compound 13, which incorporated a basic, solu-
bilizing N-acetyl morpholine group on the amino of pyrimidine was
designed. However, we failed to obtained this compound after
several attempts of synthesis. To our delight, when introducing a
hydrophilic group to compound 6, the corresponding product 12
was successfully obtained (Fig. 4). Although its enzymes inhibitory
activities were weaker than its parent compound 6, the solubility of
its hydrochloride was significantly increased (Table 3).
2. Results and discussion
2.1. Design of novel dual PARP/PI3K inhibitors
In our previous work, we have discovered a highly potent dual
PARP/PI3K inhibitor through pharmacophore merging and scaffold
hopping strategies, demonstrating that co-targeting PARP and PI3K
with a single chemical entity is feasible [32]. As part of our ongoing
program aiming at discovery of dual PARP/PI3K inhibitor with new
structural type and improved druggable, we hypothesized that
replacing the phthalazine moiety with a structurally smaller
pharmacophore of PARP inhibitor could retain the potency and
reduce the size of such hybrid compound for a favorable physi-
ochemical and pharmacologic profile. Thus, compound 3 was
designed by replacing the phthalazine moiety with benzofuran-7-
carboxamide moiety which is a structurally smaller pharmaco-
phore of PARP inhibitor (Fig. 1). A docking study was subsequently
performed to verify the rationality of the design by analyzing the
binding mode of compound 3 in the active sites of PARP-1 (PDB
2.4. In vitro antiproliferative activity study
Considering their good inhibitory activities against PARP-1 and
PI3Ka, compounds 6 and 12 were selected to evaluate their anti-
proliferative activity against BRCA-proficient TNBC cells (MDA-MB-
468). As shown in Table 4, compounds 6 and 12 both showed potent
antiproliferative activity against MDA-MB-468 cells with the IC50
code 5DS3) [33] and PI3K
a (PDB code 4JPS) [34]. As shown in Fig. 2,
compound 3 can form three key hydrogen bonds with Ser904 and
values of 4.06
mM and 1.40 mM, respectively. In particular,
2

J. Wang, G. He, H. Li et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 2. Schematic illustration of the discovery of compound 12.
Fig. 3. Docking studies of compound 3 with PARP-1 (5DS3) and PI3K
PI3K
a
(4JPS). Representation of the predicted binding mode of compound 3 in the active site of PARP-1 (A) and
a
(B). Hydrogen bonds are represented as yellow dotted lines. For clarity, nonpolar H atoms are not represented. (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article.)
compound 12 showed much stronger antiproliferative activity than
Olaparib against BRCA-proficient TNBC cells.
analysis in the MDA-MB-468 cell line. As shown in Fig. 6, compound
12 led to a significant increase in cell apoptosis compared to both
single agent treatments and the combination of Olaparib and
BKM120. The result suggested the superiority of PARP-1/PI3K dual
inhibitors in inducing apoptosis of BRCA-proficient TNBC cells.
2.5. Effect on the expression of BRCA1/2
To verify whether BRCA1/2 was downregulated in MDA-MB-468
cancer cells after treating with PARP/PI3K dual inhibitor, the
expression of BRCA1/2 at the mRNA level was measured by real-
time PCR. As shown in Fig. 5, compound 12 displayed a stronger
capability to downregulate the expression of BRCA1/2 at the mRNA
level than Olaparib and BKM120, suggesting that compound 12
probably induced HR deficiency through the downregulation of
BRCA1/2.
2.7. Metabolic stability study of compound 12
To evaluate the metabolic stability, compound 12 was incubated
with liver microsomes of different species (mouse, rat, dog, monkey
and human) for 1 h (Table 6). The results showed that compound 12
possessed good metabolic stability in all species, especially more
stable in human liver microsomes (HLM).
2.6. Apoptosis analysis
2.8. Permeability study of compound 12
Further, to determine the effect of compound 12 on cell death,
With good metabolic stability, the permeability and drug
an apoptosis assay was conducted using Annexin-V by FACS
transport characteristics of compound 12 was investigated in a
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J. Wang, G. He, H. Li et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 1
In vitro PARP-1 and PI3K
2.9. Toxicity study of compound 12
a
inhibitory activities of compounds 3e11.
To further evaluate the safety of compound 12 in vivo, additional
maximum tolerated dose (MTD) determination tests was con-
ducted. As shown in Table 8, no rats died and no significant weight
loss was observed after single-dose treatment of 1000 mg/kg.
Therefore, compound 12 exhibited an MTD values over 1000 mg/kg.
Meanwhile, compound 12 exhibited very weak hERG inhibition
with an IC50 of 29.4 mM (IC50 > 10 mM: very weak inhibition or no
inhibition). All above results indicated that compound 12 exhibited
high safety.
Compd
X
C
R1
Inhibition Ratio
PARP-1 (10 nM)
51%
PI3Ka (100 nM)
2.10. In vivo antitumor effects study
3
80%
Based on the excellent enzymatic and antiproliferative activities
of compound 12 in vitro, we then evaluated its antitumor activity
in vivo in MDA-MB-468 xenograft mouse model. Compounds 12,
Olaparib, BKM120, and the combination of Olaparib and BKM120
were administered by intraperitoneal injection twice daily (BID) for
34 consecutive days. As shown in Fig. 7, compound 12 significantly
suppressed the tumor growth at a dose of 50 mg/kg and it was well-
tolerated with no mortality. The tumor suppression effects of
compound 12 (TGI: 54.6%) was more effective than Olaparib (TGI:
28.5%) and BKM120 (TGI: 33.4%), and even the combination of
Olaparib and BKM120 (TGI: 48.4%). It is also noteworthy that no
significant weight fluctuations were observed during the whole
process. The results suggest that dual PARP/PI3K inhibitor is su-
perior to the single-target inhibitors in the antitumor efficacy
against BRCA-proficient TNBC.
4
5
C
C
85%
38%
60%
26%
6
N
60%
77%
7
8
9
N
N
N
61%
70%
51%
79%
86%
5%
2.11. Kinase selectivity study
To demonstrate whether the compound has potential off-target
effect, compound 12 was tested at a single concentration of 1 mM
against 374 kinases in the Reaction Biology Corporation (RBC) ki-
nase panel (see Supporting Information). The results showed that
compound 12 displayed weak inhibitory activities against 374 ki-
10
11
N
N
62%
36%
8%
nases at 1 mM concentration (Fig. 8A and Table S1), demonstrating
that compound 12 is a highly selective dual PARP/PI3K inhibitor. To
further profile compound 12’s selectivity against PARP and PI3K
isoform, we tested its inhibitory activities against PARP-1/2 and
four class I isoforms of PI3K. The results in Fig. 2B indicated that
compound 12 possesses strong inhibitory effect on PARP-1/2 and
29%
Olaparib
BKM120
91%
ND
ND
82%
PI3Ka (Fig. 8B).
2.12. Chemistry
The starting material 13 underwent a replacement with a cyano,
then hydrolysis of the cyano and ester gave intermediate 15. The
following condensation of 15 with 16 or 17 in the presence of PyBOP
and DIPEA furnished intermediate 18 or 19. Target compounds 3, 4,
6e10 and 12 were finally obtained by Suzuki coupling of 18 or 19
with the corresponding boric acid esters 20a-f (Scheme 1).
The reaction of starting material 21 with 22 or 23 in the pres-
ence of K2CO3 gave intermediate 24 or 25, which underwent sub-
stitution with N-Boc-piperazine and N-deprotection to give
intermediate 28 or 29. Compounds 5 and 11 were finally obtained
by the condensation of 15 with 28 or 29 in the presence of PyBOP
and DIPEA (Scheme 2).
Table 2
IC50 values of representative compounds against PARP-1 and PI3K
a
.
Compd
IC50 (nM)a
PARP-1
PI3K
a
6
7
8
7.3 0.2
10.8 0.1
5.8 0.6
39.6 1.3
118.5 2.5
6.3 0.9
a
The data are expressed as the mean SD from the dose-response curves of three
independent experiments.
Caco-2 monolayer cell model (Table 7). The results demonstrated
that compound 12 showed good membrane permeability with
Papp (A-B) values more than 1 ꢀ 10ꢁ6 cm/s.
3. Conclusions
In conclusion, to develop novel PARP/PI3K dual inhibitors, lead
compound 3 was designed by replacing the phthalazine moiety of
previous discovered PARP/PI3K dual inhibitor 1 with a benzofuran-
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J. Wang, G. He, H. Li et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 4. Strategy to improve water solubility.
Table 3
MB-468 cell-derived xenograft model, compound 12 displayed
excellent antitumor efficacy at a dose of 50 mg/kg, which is
considerably more efficacious than the single administration of
Olaparib or BKM120. Compound 12 also possessed good metabolic
stability and high safety. In view of its encouraging in vitro and
in vivo properties, compound 12 as a novel PARP/PI3K dual inhib-
itor is worthy of further profiling and is currently under preclinical
study.
Solubility and enzymes inhibitory activities of compounds 6 and 12.
Compd
Solubility pH ¼ 7
IC50 SD (nM)
PARP-1
PI3K
a
6$ HCl
12$ HCl
<100
mg/mL
7.3 0.2
13.8 1.9
39.6 1.3
64.0 2.5
>10 mg/mL
Table 4
In vitro antiproliferative activity of compounds 6 and 12.
4. Experimental sections
Compd
IC50 SD (m
M)a
4.1. Materials and physical measurements
MDA-MB-468
6
12
Olaparib
BKM120
4.06 0.49
1.40 0.13
13.72 2.52
3.97 0.28
All cell culture reagents were purchased from Gibco (Invitrogen,
USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT), STZ, wortmannin, SB216763 and chloroquine were
purchased from Sigma-Aldrich company (USA). All chemical re-
agents were purchased from Sinopharm Chemical Reagent Co., Ltd.
(China), Alfa Aesar, Adamas-beta, J&K and TCI. All solvents were
used without further purifications. Reaction progress was moni-
tored using analytical thin layer chromatography (TLC) on pre-
coated silica gel GF254 (Yantai, China) plates and the spots were
detected under UV light (254 nm). Column chromatography was
performed on silica gel (200e300 mesh) from Yantai (China).
Melting point were recorded on a WRS-1B melting point apparatus
and uncorrected. 1H NMR and 13C NMR spectra were measured in
a
The data are shown as the mean SD from the dose-
response curves of three independent experiments.
7-carboxamide moiety. Subsequent structural optimization leading
to the candidate compound 12, which showed potent and highly
selective inhibitory activities against PARP and PI3K with IC50
values of 13.8/6.2 nM (PARP1/2) and 64 nM (PI3Ka), respectively.
Moreover, compound 12 showed much stronger antiproliferative
activity than Olaparib against BRCA-proficient TNBC cells. In MDA-
Fig. 5. Effects of BKM120, Olaparib, BKM120 (1
BRCA2 expression in MDA-MB-468 cancer cells treated with the indicated compounds. Gene expression was normalized to GAPDH. The data are shown as the mean SD of three
m
M) þ Olaparib (2
mM), compound 12 on BRCA1 and BRCA2 expression in MDA-MB-468 cells. Real-time PCR analysis of BRCA1 and
independent experiments. ***p < 0.001 vs. control.
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J. Wang, G. He, H. Li et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 6. Effects of BKM120, Olaparib, BKM120 (1
indicated concentrations of compounds for 72 h. The apoptosis rate was measured using flow cytometry. (B) The percentage of cells in each population. The data are shown as the
mean SD of three independent experiments. ***p < 0.001 vs. control.
m
M) þ Olaparib (2
m
M) and compound 12 to induce apoptosis of MDA-MB-468 cells. (A) MDA-MB-468 cells were treated with the
Table 8
Table 6
Weight changes of ratsa treated with a single doseb of 1000 mg/kg.
Evaluation of the metabolic stability of different species liver microsomes.
weight (g)c
a
Species
k (minꢁ1
)
T1/2 (min)b
CLint (m
L/min/mg)c
Compd
adaptation period
Day 2
Day 4
Day 7
Human
Monkey
Dog
Rat
Mouse
0.00734
0.0153
0.0147
0.0165
0.0398
107
14.7
30.7
29.3
32.9
79.7
59.1
47.3
42.2
17.4
Control
12
258 13
256 11
273 15
267 14
280 12
272 10
298 14
281 12*
*p < 0.05 vs. control, **p < 0.01.
a
SpragueꢁDawley rat; n ¼ 6 animals, half male and half female.
a
k is the drug elimination rate constant
T1/2 is the elimination half-life.
CLint is the intrinsic clearance.
b
c
Oral administration once a day.
b
c
Values presented are the mean of six independent mice.
4.2. Chemistry
Table 7
Measurement of bidirectional Papp and efflux ratio of compound 12a..
4.2.1. General procedure for the synthesis of compounds 3, 4, 6e10,
12
Direction
Recovery (%)
Papp (10ꢁ6 cm/s)b
Efflux Ratio
To a solution of compound 13 (9.30 g, 36.46 mmol) in DMF
(100 mL) was added Zn(CN)2 (8.56 g, 72.92 mmol) and Pd(PPh3)4
(2.10 g, 1.82 mmol). The suspension was heated with stirring under
a nitrogen atmosphere at 80 ꢂC for 6e8 h. The reaction was checked
by TLC. After completion of the reaction, the mixture was cooled to
room temperature and filtered. The filtrate was poured into water
(300 mL) and stirred for 10 min. The precipitate was filtered and
washed with water, then dried under vacuum. The crude product
was purified by silica gel column chromatography (PE/EtOAc ¼ 50:1
to 20:1) to give intermediate 14 as white solid (4.70 g, 64.0% yield).
A to B
B to A
67.3
99.9
8.42
22.1
2.6
a
Test compound concentration: 5
Values are reported as the mean of two experiments.
m
M; incubation time: 120 min.
b
DMSO‑d6 or CDCl3 solutions using an AVANCE III 300 or 600
spectrometer. Chemical shifts are reported in scale (ppm) relative
to internal TMS, J values are given in Hertz, and spin multiplicities
are expressed as s (singlet), d (doublet), t (triplet), or m (multiplet).
Low and high-resolution mass spectra were obtained in the ESI
mode.
d
m.p.: 130e132 ꢂC. 1H NMR (300 MHz, CDCl3)
J ¼ 8.0, 1.0 Hz, ArH), 7.79 (1H, dd, J ¼ 7.6, 1.0 Hz, ArH), 7.62 (1H, s,
d (ppm): 7.97 (1H, dd,
6

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European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 7. Effects of Olaparib, BKM120, Olaparib (50 mg/kg) þ BKM120 (27.5 mg/kg), and compound 12 treatment on the tumorigenicity of MDA-MB-468 cells in vivo. (A) The resulting
tumors excised from the animals after treatment. (B) The tumor masses for six groups of animals were compared, and each histogram represents the Mean S.D. of 6 mice.
*p < 0.05 vs. control; ***p < 0.001. (C) The nude mouse body weight was measured every 2 days. (D) The tumor volumes of the nude mice were measured and calculated once every
2 days.
Fig. 8. (A) Kinase selectivity profile of compound 12 as shown by an RBC kinase panel screen against 374 kinases assayed at 1 mM in duplicate. Compound selectivity is represented
in a dendrogram view of the human kinome phylogenetic tree. The color code for inhibition is indicated. (B) Inhibitory activity of compound 12 against PARP-1/2 and four isoforms
of PI3K. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
ArH), 7.43 (1H, t, J ¼ 7.8 Hz, ArH), 4.02 (3H, s, CH3).
PyBOP (4.12 g, 7.92 mmol) were dissolved in DMF (50 mL), followed
by the addition of DIPEA (3.82 mL, 23.10 mmol). The mixture was
stirred at 25 ꢂC for 6 h. After completion, the solution was poured
into water (150 mL) and stirred for 10 min. The precipitate was
filtered and washed with water, then dried under vacuum. The
crude product was purified by silica gel column chromatography
(DCM/MeOH ¼ 80:1 to 30:1) to give compound 19 as white solid
(2.50 g, 80.4% yield). m.p.: 142e144 ꢂC. 1H NMR (300 MHz,
To a solution of intermediate 14 (4.70 g, 23.36 mmol) in MeOH
(50 mL) was added 30%H2O2 (16 mL), 1 mol/L NaOH (47 mL). The
mixture was heated with stirring at 40 ꢂC for 1 h. After completion
of the reaction, the mixture was cooled to room temperature and
added 2 mol/L HCl to adjust the pH to 2. The precipitate was
filtered, washed with water and dried under vacuum to give 15 as
white solid (4.50 g, 93.9% yield). m.p.: 178e180 ꢂC. 1H NMR
(300 MHz, DMSO‑d6)
d
(ppm): 13.77 (1H, s, COOH), 7.92 (1H, d,
DMSO‑d6)
d
(ppm): 7.94 (1H, d, J ¼ 7.8 Hz, ArH), 7.86 (1H, d,
J ¼ 7.5 Hz, ArH), 7.85 (1H, s, 1/2CONH2), 7.83 (1H, d, J ¼ 7.6 Hz, ArH),
7.74 (1H, s, ArH), 7.66 (1H, s, 1/2CONH2), 7.42 (1H, t, J ¼ 7.4 Hz, ArH).
Intermediate 15 (1.35 g, 6.58 mmol), 17 (2.12 g, 6.60 mmol) and
J ¼ 7.1 Hz, ArH), 7.84 (1H, s, 1/2CONH2), 7.77 (1H, s, 1/2CONH2), 7.58
(1H, s, ArH), 7.46 (1H, t, J ¼ 7.7 Hz, ArH), 3.90e3.66 (16H, m, 6CH2N,
2CH2O). The synthesis methods of intermediate 18 refer to that of
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European Journal of Medicinal Chemistry xxx (xxxx) xxx
Scheme 1. Reagents and conditions: (a) Zn(CN)2, Pd(PPh3)4, DMF, N2, 80 ꢂC, 6 h; (b) 1 mol/L NaOH, H2O, MeOH, 40 ꢂC, 1 h; (c) PyBOP, DIPEA, DMF, 25 ꢂC, 6 h; (d) Pd(PPh3)4, K2CO3,
dioxane/H2O, reflux, N2, 8 h.
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, 25 ꢂC, 4 h; (b) N-Boc-piperazine, K2CO3, aceton, 30 ꢂC, 10 h; (c) HCl/EtOAc, 25 ꢂC, 4 h; (d) PyBOP, DIEA, DMF, 25 ꢂC, 8 h.
intermediate 19.
ArH), 7.91 (1H, d, J ¼ 8.3 Hz, ArH), 7.82 (1H, d, J ¼ 7.1 Hz, ArH), 7.79
(1H, s, 1/2CONH2), 7.72 (1H, s, 1/2CONH2), 7.53 (1H, s, ArH), 7.42
(1H, t, J ¼ 7.6 Hz, ArH), 6.82 (1H, s, ArH), 6.73 (2H, s, NH2), 6.25 (1H,
s, ArH), 4.03e3.59 (16H, m, 6CH2N, 2CH2O). 13C NMR (75 MHz,
To a solution of intermediate 18 or 19 (0.64 mmol) in dioxane
(20 mL) was added the corresponding boric acid esters
(0.70 mmol), Pd(PPh3)4 (0.06 mmol) and K2CO3 (2.56 mmol) which
was dissolved in water (2 mL). The suspension was heated with
stirring under a nitrogen atmosphere at 100 ꢂC for 6e8 h. The re-
action was checked by TLC. After completion, the mixture was
cooled to room temperature and evaporated under vacuum. The
residue was partitioned between EtOAc (20 mL) and water (20 mL),
and the water phase was extracted two times with the same vol-
ume of EtOAc. The combined organic phase was washed with water
and brine, dried over Na2SO4 overnight, and the solvent was
evaporated under vacuum. The crude products were purified by
silica gel column chromatography (DCM/MeOH ¼ 100:1 to 20:1) to
yield the target compounds 3, 4, 6e10, 12.
DMSO‑d6)
d (ppm): 165.3, 162.8, 162.7, 160.3, 159.9, 159.0, 150.8,
150.3,148.2,136.0,127.6,126.8,125.9,124.8,123.7,121.3,119.1,110.9,
104.6, 92.0, 65.8, 49.8, 43.8, 43.3. HRMS (ESI): m/z [MþH]þ. Calcd
for C28H27F3N8O4: 597.2180; Found: 597.2177. IR (cmꢁ1): 3481.4,
3423.6, 3323.0, 3212.3, 2858.5, 1683.7, 1627.1, 1571.9, 1554.7, 1535.1,
1513.8, 1434.4, 1408.7, 1373.3, 1261.1, 1241.4, 1195.2, 1175.1, 1153.0,
1135.5, 1116.9, 1008.5, 994.4, 975.0, 962.6, 938.8, 872.1, 838.7, 816.8,
742.9, 670.0, 500.9.
4.2.3. 2-(4-(20-amino-2-morpholino-[4,50-bipyrimidin]-6-yl)
piperazine-1-carbonyl)benzofuran-7-carboxamide (4)
The product was light yellow solid, 47.2% yield, m.p.: >250 ꢂC. 1H
4.2.2. 2-(4-(6-(6-amino-4-(trifluoromethyl)pyridin-3-yl)-2-
morpholinopyrimidin-4-yl)piperazine-1-carbonyl)benzofuran-7-
carboxamide (3)
NMR (300 MHz, DMSO‑d6) d (ppm): 8.99 (2H, s, ArH), 7.94 (1H, dd,
J ¼ 7.8, 1.3 Hz, ArH), 7.89e7.82 (2H, m, ArH, 1/2CONH2), 7.76 (1H, s,
1/2CONH2), 7.57 (1H, s, ArH), 7.46 (1H, t, J ¼ 7.7 Hz, ArH), 7.09 (2H, s,
NH2), 6.66 (1H, s, ArH), 3.95e3.88 (8H, m, 4CH2N), 3.72e3.64 (8H,
The product was light yellow solid, 45.8% yield, m.p.:
208e210 ꢂC. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm): 8.17 (1H, s,
m, 2CH2N, 2CH2O). 13C NMR (150 MHz, DMSO‑d6)
d (ppm): 170.3,
8

J. Wang, G. He, H. Li et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
169.3, 168.5, 165.9, 164.6, 163.9, 162.2, 162.0, 156.0, 154.0, 132.9,
131.8, 130.6, 128.8, 125.1, 125.0, 116.1, 91.8, 71.1, 49.2, 45.2. HRMS
(ESI): m/z [MþH]þ. Calcd for C26H27N9O4: 530.2259; Found:
530.2250. IR (cmꢁ1): 3444.4, 3320.9, 3165.5, 2955.1, 2849.1, 2359.9,
1644.8, 1584.2, 1562.0, 1477.7, 1433.3, 1418.5, 1389.7, 1361.7, 1267.5,
1251.5, 1228.2, 1212.8, 1192.8, 1180.1, 1110.0, 1000.8, 973.9, 934.8,
880.9, 807.8, 734.3, 556.3, 532.6.
159.0, 150.8, 148.1, 139.6, 127.6, 126.8, 125.97, 125.1, 124.3, 123.8,
121.6, 119.1, 114.9, 114.5, 114.3, 111.1, 72.8, 65.9, 43.1, 42.4. HRMS
(ESI): m/z [MþH]þ. Calcd for C27H27FN8O4: 547.2212; Found:
547.2211. IR (cmꢁ1): 3480.8, 3398.5, 3340.8, 3237.9, 2958.7, 2856.8,
1667.2, 1624.5, 1548.0, 1517.6, 1435.9, 1385.1, 1305.2, 1280.0, 1264.0,
1233.3, 1219.47, 1168.9, 1111.0, 1002.7, 983.4, 944.6, 913.0, 809.2,
770.7, 737.1.
4.2.4. 2-(4-(4-(6-amino-4-(trifluoromethyl)pyridin-3-yl)-6-
morpholino-1,3,5-triazin-2-yl)-piperazine-1-carbonyl)benzofuran-
7-carboxamide (6)
4.2.8. 2-(4-(4-(1H-indol-5-yl)-6-morpholino-1,3,5-triazin-2-yl)
piperazine-1-carbonyl)benzofuran-7-carboxamide (10)
The product was light yellow solid, 45.2% yield. m.p.:
The product was off-white solid, 48.4% yield, m.p.: >250 ꢂC.
164e166 ꢂC. HPLC: 99.53%. 1H NMR (300 MHz, DMSO‑d6)
d (ppm):
HPLC: 98.06%. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm): 8.62 (1H, s,
11.35 (1H, s, NH), 8.70 (1H, s, ArH), 8.22 (1H, d, J ¼ 8.8 Hz, ArH), 7.95
(1H, d, J ¼ 7.8 Hz, ArH), 7.90e7.84 (2H, m, ArH, 1/2CONH2), 7.79 (1H,
s, 1/2CONH2), 7.60 (1H, s, ArH), 7.51e7.42 (3H, m, ArH), 6.60 (1H, s,
ArH), 4.12e3.84 (12H, m, 4CH2N, 2CH2O), 3.72e3.63 (4H, m,
ArH), 7.92 (1H, d, J ¼ 7.8 Hz, ArH), 7.83 (1H, d, J ¼ 7.4 Hz, ArH), 7.77
(1H, s, 1/2CONH2), 7.71 (1H, s, 1/2CONH2), 7.55 (1H, s, ArH), 7.43
(1H, t, J ¼ 7.7 Hz, ArH), 6.94 (2H, s, NH2), 6.83 (1H, s, ArH), 3.92e3.65
(16H, m, 6CH2N, 2CH2O). 13C NMR (75 MHz, DMSO‑d6)
d
(ppm):
2CH2N). 13C NMR (75 MHz, DMSO‑d6)
d (ppm): 171.1, 165.6, 165.1,
174.2, 169.8, 168.8, 165.9, 163.4, 157.2, 155.6, 153.4, 140.6, 132.4,
131.4, 130.1, 129.6, 128.3, 125.9, 124.6, 123.4, 115.8, 109.4, 70.6, 59.6,
47.9, 47.2. HRMS (ESI): m/z [MþH]þ. Calcd for C27H26F3N9O4:
598.2133; Found: 598.2136. IR (cmꢁ1): 3422.6, 3207.6, 2854.9,
2361.1, 1682.4, 1649.3, 1622.2, 1560.6, 1525.2, 1484.9, 1437.5, 1420.0,
1389.8, 1365.9, 1274.2,1259.8,1227.1, 1166.4,1128.41, 1056.3, 1001.8,
981.3, 963.4, 938.6, 924.0, 861.7, 813.6, 746.9, 677.7, 545.3.
165.0,159.1,151.4,149.2,138.6,128.1,128.0,127.8,127.2,126.8,125.9,
124.1, 122.0, 121.6, 120.4, 111.6, 111.3, 102.6, 74.0, 66.5, 55.3, 43.7.
HRMS (ESI): m/z [MþH]þ. Calcd for C29H28N8O4: 553.2306; Found:
553.2306. IR (cmꢁ1): 3396.6, 2852.8, 2355.1, 1676.0, 1618.6, 1542.7,
1519.9, 1485.1, 1434.5, 1384.8, 1364.1, 1346.5, 1275.3, 1260.1, 1228.2,
1170.1, 1111.0, 1067.7, 1000.1, 979.46, 806.6, 770.7, 739.6, 716.7,
542.1, 429.0.
4.2.5. 2-(4-(4-(6-aminopyridin-3-yl)-6-morpholino-1,3,5-triazin-
2-yl)piperazine-1-carbonyl)-benzofuran-7-carboxamide (7)
The product was light yellow solid, 46.0% yield. m.p.: >250 ꢂC. 1H
4.2.9. 2-(4-(4-morpholino-6-(6-(2-morpholinoacetamido)-4-
(trifluoromethyl)pyridin-3-yl)-1,3,5-triazin-2-yl)piperazine-1-
carbonyl)benzofuran-7-carboxamide (12)
NMR (300 MHz, DMSO‑d6)
d
(ppm): 8.88 (1H, s, ArH), 8.34 (1H, d,
The product was light yellow solid, 64.7% yield. m.p.:
J ¼ 7.7 Hz, ArH), 7.90 (1H, d, J ¼ 7.8 Hz, ArH), 7.82 (1H, s, ArH), 7.80
(1H, s, 1/2CONH2), 7.75 (1H, s, 1/2CONH2), 7.55 (1H, s, ArH), 7.42
(1H, t, J ¼ 7.6 Hz, ArH), 7.06 (2H, s, NH2), 6.62 (1H, d, J ¼ 8.6 Hz, ArH),
4.00e3.57 (16H, m, 6CH2N, 2CH2O). 13C NMR (75 MHz, DMSO‑d6)
163e165 ꢂC. HPLC: 99.40%. 1H NMR (300 MHz, DMSO‑d6)
d (ppm):
10.78 (1H, s, NH), 8.93 (1H, s, ArH), 8.58 (1H, s, ArH), 7.94 (1H, dd,
J ¼ 7.8, 1.3 Hz, ArH), 7.90e7.77 (3H, m, ArH, CONH2), 7.59 (1H, s,
ArH), 7.45 (1H, t, J ¼ 7.7 Hz, ArH), 3.99e3.77 (12H, m, 2CH2N,
4CH2O), 3.71e3.61 (8H, m, 4CH2N), 3.32 (2H, s, COCH2N), 2.58 (4H,
d
(ppm): 168.6, 165.0, 164.2, 161.9, 158.6, 150.8, 149.6, 148.7, 136.6,
131.4, 127.6, 126.6, 125.4, 123.6, 120.1, 119.9, 111.0, 106.9, 66.0, 43.2,
28.9, 26.5. HRMS (ESI): m/z [MþH]þ. Calcd for C26H27N9O4:
530.2259; Found: 530.2252. IR (cmꢁ1): 3458.8, 3376.6, 3183.6,
2920.8, 2850.6, 2361.0,1683.0,1628.4, 1569.8, 1532.6, 1519.9,1487.1,
1436.2, 1419.3, 1388.4, 1364.3, 1261.9, 1225.7, 1178.6, 1154.2, 1118.2,
1017.1, 1001.1, 976.4, 940.8, 854.0, 814.2, 742.3, 543.0, 516.8, 490.7,
458.9, 409.1.
t, J ¼ 4.5 Hz, 2CH2N). 13C NMR (150 MHz, DMSO‑d6)
d (ppm): 175.0,
174.0,170.3,169.1,163.9,158.2,156.4,156.1,153.8,141.9, 132.8,132.3,
131.9, 131.8, 130.67, 128.8, 126.9, 125.1, 116.3, 114.5, 71.4, 71.1, 66.5,
58.2, 48.5, 48.3, 45.2. HRMS (ESI): m/z [MþH]þ. Calcd for
C
33H36F3N10O6: 725.2766; Found: 725.2760. IR (cmꢁ1): 3446.2,
2928.5, 2862.3, 2360.8, 1708.2, 1629.3, 1585.4, 1510.5, 1438.8,
1383.9, 1351.58, 1259.9, 1227.9, 1170.9, 1123.1, 1067.4, 1000.5, 979.1,
854.9, 759.7, 669.6.
4.2.6. 2-(4-(4-(2-aminopyrimidin-5-yl)-6-morpholino-1,3,5-
triazin-2-yl)piperazine-1-carbonyl)-benzofuran-7-carboxamide (8)
The product was white solid, 59.4% yield. m.p.: >250 ꢂC. 1H NMR
4.2.10. General procedure for the synthesis of compounds 5 and 11
Intermediate 21 (500 mg, 2.97 mmol), 22 (700 mg, 2.99 mmol)
and K2CO3 (411 mg, 2.97 mmol) were dissolved in DMF (25 mL). The
mixture was stirred at 100 ꢂC for 8 h. The reaction was checked by
TLC. After completion of the reaction, the mixture was cooled to
room temperature and then poured into water (80 mL). The pre-
cipitate was filtered and washed with water, then dried under
vacuum. The crude product was purified by silica gel column
chromatography (PE/EtOAc ¼ 50:1 to 20:1) to give intermediate 24
as white solid (1.00 g, 91.7% yield). m.p.: 197e200 ꢂC. 1H NMR
(300 MHz, DMSO‑d6)
d (ppm): 9.06 (2H, s, ArH), 7.90 (1H, d,
J ¼ 7.3 Hz, ArH), 7.82 (1H, s, 1/2CONH2), 7.81 (1H, d, J ¼ 5.4 Hz, ArH),
7.74 (1H, s, 1/2CONH2), 7.55 (1H, s, ArH), 7.42 (1H, t, J ¼ 7.1 Hz, ArH),
7.30 (2H, s, NH2), 4.00e3.64 (16H, m, 6CH2N, 2CH2O). 13C NMR
(75 MHz, DMSO‑d6) d (ppm): 170.8, 169.8, 168.6, 165.8, 163.4, 162.4,
155.6, 153.4, 147.6, 132.4, 131.4, 130.1, 128.3, 124.6, 123.3, 115.8, 70.7,
51.1, 48.0, 47.4. HRMS (ESI): m/z [MþH]þ. Calcd for C25H26N10O4:
531.2211; Found: 531.2209. IR (cmꢁ1): 3478.7, 2860.0, 2359.8,
1660.8,1631.6,1563.0,1509.0,1440.3,1420.6,1362.3,1329.8,1265.2,
1233.4, 1113.8, 1068.6, 1001.0, 977.5, 873.1, 856.3, 811.61, 783.6,
755.0, 658.7, 624.5, 543.1, 511.5, 469.0.
(300 MHz, DMSO‑d6)
d, J ¼ 7.7 Hz, ArH), 7.69 (1H, t, J ¼ 53.6 Hz, CHF2), 7.55e7.42 (2H, m,
d
(ppm): 8.31 (1H, d, J ¼ 8.1 Hz, ArH), 7.86 (1H,
ArH), 7.09 (1H, s, ArH), 3.74 (8H, brs, 2CH2O, 2CH2N).
Intermediate 24 (1.00 g, 2.73 mmol), N-Boc-piperazine (0.54 g,
2.87 mmol) and K2CO3 (0.57 g, 4.09 mmol) were dissolved in DMF
(25 mL). The mixture was stirred at 80 ꢂC for 10 h. The reaction was
checked by TLC. After completion of the reaction, the mixture was
cooled to room temperature and then poured into water (80 mL).
The precipitate was filtered and washed with water, then dried
under vacuum. The crude product was purified by silica gel column
chromatography (PE/EtOAc ¼ 30:1 to 5:1) to give intermediate 26
as white solid (1.25 g, 88.7% yield). m.p.: 195e198 ꢂC. 1H NMR
4.2.7. 2-(4-(4-(4-amino-3-fluorophenyl)-6-morpholino-1,3,5-
triazin-2-yl)piperazine-1-carbonyl)-benzofuran-7-carboxamide (9)
The product was light yellow solid, 48.6% yield. m.p.: >250 ꢂC. 1H
NMR (300 MHz, DMSO‑d6)
d (ppm): 7.96e7.91 (3H, m, ArH), 7.84
(1H, d, J ¼ 7.2 Hz, ArH), 7.80 (1H, s, 1/2CONH2), 7.73 (1H, s, 1/
2CONH2), 7.56 (1H, s, ArH), 7.44 (1H, t, J ¼ 7.7 Hz, ArH), 6.80 (1H, t,
J ¼ 8.9 Hz, ArH), 5.78 (2H, s, NH2), 4.02e3.62 (16H, m, 6CH2N,
2CH2O). 13C NMR (75 MHz, DMSO‑d6)
d (ppm): 168.7, 165.3, 164.3,
9

J. Wang, G. He, H. Li et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
(300 MHz, CDCl3)
d
(ppm): 8.22 (1H, d, J ¼ 7.1 Hz, ArH), 7.90 (1H, d,
165.0, 163.8, 163.3, 162.4, 158.7, 153.7, 150.9, 148.6, 141.2, 127.6,
126.6, 125.4, 123.8, 121.8, 120.5, 119.9, 114.9, 111.0, 108.6, 79.5, 65.7,
45.8, 44.5, 25.8. HRMS (ESI): m/z [MþH]þ. Calcd for C30H28F2N8O4:
603.2274; Found: 603.2278. IR (cmꢁ1): 3362.8, 3195.9, 2967.3,
2866.9, 1685.0, 1635.7, 1618.1, 1604.2, 1524.3, 1449.5, 1425.25,
1392.5, 1347.1, 1283.9, 1235.4, 1227.8, 1216.2, 1200.4, 1188.1, 1114.8,
1094.6, 1041.2, 1011.7, 1002.6, 959.9, 768.7, 747.8, 736.1, 585.2.
J ¼ 7.1 Hz, ArH), 7.51 (1H, t, J ¼ 53.6 Hz, CHF2), 7.44e7.35 (2H, m,
ArH), 5.52 (1H, s, ArH), 3.84e3.81 (4H, m, 2CH2O), 3.65e3.57 (12H,
m, 6CH2N).
To a solution of compound 26 (1.20 g, 2.33 mmol) in EtOAc
(10 mL) was added the EtOAc solution of HCl (5 mL). The mixture
was stirred at 25 ꢂC for 5 h. The reaction was checked by TLC. After
completion of the reaction, the precipitate was filtered and washed
with EtOAc, then dried under vacuum to give intermediate 28 as
white solid (0.89 g, 84.8% yield). m.p.: 196e198 ꢂC. 1H NMR
4.2.12. 2-(4-(4-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6-
morpholino-1,3,5-triazin-2-yl)piperazine-1-carbonyl)benzofuran-
7-carboxamide (11)
(300 MHz, DMSO‑d6)
d (ppm): 9.10 (2H, brs, NH, HCl), 8.20 (1H, d,
The product was white solid, 41.4% yield, m.p.: 189e191 ꢂC. 1H
J ¼ 8.1 Hz, ArH), 7.82 (1H, d, J ¼ 7.7 Hz, ArH), 7.68 (1H, t, J ¼ 53.1 Hz,
CHF2), 7.49e7.39 (2H, m, ArH), 6.08 (1H, s, ArH), 3.88e3.65 (12H, m,
2CH2O, 4CH2N), 3.18 (4H, brs, 2CH2N).
NMR (300 MHz, CDCl3)
d
(ppm): 8.33 (1H, d, J ¼ 7.7 Hz, ArH), 8.22
(1H, d, J ¼ 7.3 Hz, ArH), 7.92 (1H, d, J ¼ 7.1 Hz, ArH), 7.86 (1H, d,
J ¼ 8.0 Hz, ArH), 7.56 (1H, t, J ¼ 53.6 Hz, CHF2), 7.45 (3H, m, ArH),
7.35 (1H, s, ArH), 7.22 (1H, s, 1/2CONH2), 6.02 (1H, s, 1/2CONH2),
4.03e3.81 (16H, m, 6CH2N, 2CH2O). 13C NMR (75 MHz, DMSO‑d6)
Intermediate 21 (1.43 g, 8.50 mmol), 23 (2.00 g, 8.51 mmol) and
K2CO3 (1.18 g, 8.51 mmol) were dissolved in DMF (25 mL). The
mixture was stirred at 25 ꢂC for 4 h. The reaction was checked by
TLC. After completion of the reaction, the mixture was cooled to
room temperature and then poured into water (80 mL). The pre-
cipitate was filtered and washed with water, then dried under
vacuum to give intermediate 25 as white solid (2.70 g, 86.5% yield).
d
(ppm): 165.0, 164.3, 161.2, 158.8, 150.9, 148.6, 145.8, 141.3, 132.8,
127.6, 126.6, 125.9, 125.4, 124.3, 123.6, 120.6, 119.9, 115.8, 111.0,
108.5, 87.0, 65.8, 45.9, 43.6, 43.0. HRMS (ESI): m/z [MþH]þ. Calcd for
C
29H27F2N9O4: 604.2227; Found: 604.2233. IR (cmꢁ1): 3453.1,
m.p.: 244e247 ꢂC. 1H NMR (300 MHz, CDCl3)
J ¼ 7.7 Hz, ArH), 7.91 (1H, d, J ¼ 7.5 Hz, ArH), 7.59 (1H, t, J ¼ 53.4 Hz,
CHF2), 7.53e7.43 (2H, m, ArH), 4.02e3.95 (4H, m, CH2), 3.87e3.81
(4H, m, CH2).
d (ppm): 8.44 (1H, d,
3169.5, 2958.1, 2861.4, 1675.3, 1638.9, 1578.0, 1516.6, 1446.19,
1413.8, 1369.2, 1308.5, 1271.3, 1249.3, 1228.9, 1210.8, 1148.25,
1108.8, 1048.8, 1003.2, 983.3, 961.4, 849.6, 804.1, 765.5, 554.3,
539.9, 513.9.
Intermediate 25 (2.70 g, 7.36 mmol), N-Boc-piperazine (1.44 g,
7.73 mmol) and K2CO3 (1.53 g, 11.0 4 mmol) were dissolved in
acetone (50 mL). The mixture was stirred at 30 ꢂC for 8 h. The re-
action was checked by TLC. After completion of the reaction, the
mixture was evaporated under vacuum. The residue was dissolved
in DCM (50 mL) and washed with water (50 mL). The organic phase
was washed with brine and dried over Na2SO4 overnight. The sol-
vent was evaporated under vacuum to give intermediate 27 (3.50 g,
4.3. Biological activity assay
4.3.1. In vitro PARP and PI3K inhibition assay
PARP-1/2 and PI3Ka/b/g/d inhibition assays were carried out
according to previously reported procedures [27]. The IC50 values
were calculated using nonlinear regression with normalized dose-
response fit using Prism GraphPad software.
92.1% yield). m.p.: 184e187 ꢂC. 1H NMR (300 MHz, CDCl3)
d (ppm):
8.34 (1H, d, J ¼ 7.6 Hz, ArH), 7.90 (1H, d, J ¼ 6.9 Hz, ArH), 7.57 (1H, t,
J ¼ 53.6 Hz, CHF2), 7.48e7.39 (2H, m, ArH), 3.92e3.85 (8H, m, CH2),
3.80 (4H, s, CH2), 3.55 (4H, s, CH2), 1.50 (9H, s, CH3).
To a solution of compound 27 (3.50 g, 6.78 mmol) in EtOAc
(20 mL) was added the EtOAc solution of HCl (10 mL). The mixture
was stirred at 25 ꢂC for 5 h. The reaction was checked by TLC. After
completion of the reaction, the precipitate was filtered and washed
with EtOAc, then dried under vacuum to give intermediate 29 as
white solid (2.72 g, 88.6% yield). m.p.: 230e232 ꢂC. 1H NMR
4.3.2. In vitro antiproliferative activity assay
MDA-MB-468 cells were maintained in RPMI1640 medium
containing 10% (v/v) FBS at 37 ꢂC in a 5% (v/v) CO2 humidified
incubator. Cell proliferation assay was determined by the Cell Titer-
Glo cell viability assay. Briefly, cells were passaged the day before
dosing into a 96-well plate, allowed to grow for 12 h, and then
treated with different concentrations of compound for 7 days at 5%
CO2, 37 ꢂC. After incubation, 100
mL of Cell Titer-Glo reagent was
added to the assay plate, which was then incubated at room tem-
perature for 10 min to stabilize luminescence signal and read by
Envision plate reader. The inhibition rate (%) ¼ (1-(RLU compound-
RLU blank)/(RLU DMSO-RLU blank)) ꢀ 100%. The IC50 values were
calculated using nonlinear regression with normalized dose-
response fit using XLFit software.
(300 MHz, CDCl3)
d
(ppm): 8.36 (1H, d, J ¼ 7.3 Hz, ArH), 7.91 (1H, d,
J ¼ 7.1 Hz, ArH), 7.59 (1H, t, J ¼ 53.7 Hz, CHF2), 7.49e7.39 (2H, m,
ArH), 3.98e3.87 (8H, m, CH2), 3.81 (4H, s, CH2), 3.02 (4H, s, CH2).
Intermediate 15 (180 mg, 0.88 mmol), 28 or 29 (0.88 mmol) and
PyBOP (550 mg, 1.06 mmol) were dissolved in DMF (20 mL), fol-
lowed by the addition of DIPEA (0.44 mL, 2.64 mmol). The mixture
was stirred at 25 ꢂC for 8e10 h. The reaction was checked by TLC.
After completion of the reaction, the solution was poured into
water (60 mL) and stirred for 5 min. The precipitate was filtered and
washed with water, then dried under vacuum. The crude product
was purified by silica gel column chromatography (DCM/
MeOH ¼ 100:1 to 60:1) to yield the target compounds 5 and 11.
4.3.3. Real-time PCR
The relative expression of BRCA1 or BRCA2 mRNA was detected
using Real-time PCR. Briefly, total RNA was extracted from cultured
cells with TRIzol reagent (Life Technologies) according to the
manufacturers’ instructions. Reverse transcription reactions were
performed using PrimeScript™ RT reagent Kit with gDNA Eraser
(Takara). For transcript quantification, SYBR Green based qPCR was
performed with PrimeScript™ RT Master Mix (Takara) using Real
Time PCR System (Stratagene Mx3000p). The human BRCA1 for-
ward primer was 50-GTCCCATCTGTCTGGAGTTGA-30, the reverse
primer was 50-AAAGGACACTGTGAAGGCCC-3’. The human BRCA2
forward primer was 50-AAAGGACACTGTGAAGGCCC-30, and the
reverse primer was 50-TTCTTCCTCTCTTTCATTGCG-3’. GAPDH was
used as an internal control. The results were represented as fold
changes relative to the internal control.
4.2.11. 2-(4-(6-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-2-
morpholinopyrimidin-4-yl)piperazine-1-carbonyl)benzofuran-7-
carboxamide (5)
The product was off-white solid, 45.3% yield, m.p.: 126e128 ꢂC.
HPLC: 98.03%. 1H NMR (300 MHz, DMSO‑d6)
d (ppm): 8.23 (1H, d,
J ¼ 8.1 Hz, ArH), 7.91 (1H, d, J ¼ 7.8 Hz, ArH), 7.84e7.80 (2H, m, ArH),
7.75 (1H, s, 1/2CONH2) 7.70 (1H, t, J ¼ 52.7 Hz, CHF2), 7.56 (1H, s,
ArH), 7.49e7.36 (3H, m, ArH), 6.04 (1H, s, ArH), 3.96e3.64 (16H, m,
6CH2N, 2CH2O). 13C NMR (75 MHz, DMSO‑d6)
d (ppm): 229.5, 197.7,
10

J. Wang, G. He, H. Li et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
L. Ouyang, B. Liu, Discovery of a small molecule targeting ULK1- modulated
cell death of triple negative breast cancer in vitro and in vivo, Chem. Sci. 8
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4.3.4. In vivo antitumor activity study
Six-week-old male BALB/c nude mice were purchased from the
Beijing Vital River Laboratory Animal Technology Co., Ltd. All ani-
mals were housed in a specific pathogen-free facility and used ac-
cording to the animal-care regulations of Nanjing Biorn Life Science
Co., Ltd (Nanjing, Jiangsu, China). Prior to implantation, MDA-MB-
468 cells were harvested during exponential growth. 2 ꢀ 106 cells
were inoculated subcutaneously on the right flank of each BALB/c
nude mouse. Mice were randomly divided into four treatment
groups and control group when tumor size reached an approximate
volume of 100 mm3. Olaparib (50 mg/kg), BKM120 (27.5 mg/kg),
Olaparib þ BKM120 (50 mg/kg þ 27.5 mg/kg), and 12 (50 mg/kg)
were administered every 2 days for 34 days (6 mice per group) by
intraperitoneal administration, equal volume of PBS (5% DMSO, v/v)
was used as the negative control. Tumor size and body weight were
measured every 2 days. Tumor volume (V) was calculated using
equation V ¼ ab2/2, where a and b stand for the longest and
shortest diameter measured by vernier caliper, respectively.
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgment
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(BMN 673, talazoparib), a novel, highly potent, and orally efficacious poly
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This work was supported by the National Natural Science
Foundation of China (Grant No. 81502928) and Six Talent Peaks
Project in Jiangsu Province, China (2017, YY-059).
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113054.
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