Discovery of Novel Dual Poly(ADP-ribose)polymerase and Phosphoinositide 3-Kinase Inhibitors as a Promising Strategy for Cancer Therapy

Article abstract of DOI:10.1021/acs.jmedchem.9b00622

Concomitant inhibition of PARP and PI3K pathways has been recognized as a promising strategy for cancer therapy, which may expand the clinical utility of PARP inhibitors. Herein, we report the discovery of dual PARP/PI3K inhibitors that merge the pharmaco

Full text of DOI:10.1021/acs.jmedchem.9b00622

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Article
Discovery of Novel Dual Poly(ADP-ribose)polymerase
(PARP) and Phosphoinositide 3-kinase (PI3K)
Inhibitors as A Promising Strategy for Cancer Therapy
Junwei Wang, Hui Li, Guangchao He, Zhaoxing Chu, Kewen Peng, Yiran Ge, Qihua Zhu, and Yungen Xu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00622 • Publication Date (Web): 17 Dec 2019
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Discovery of Novel Dual Poly(ADP-ribose)polymerase (PARP) and
Phosphoinositide 3-kinase (PI3K) Inhibitors as A Promising Strategy
for Cancer Therapy
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Junwei Wanga, Hui Lia, Guangchao Heb, Zhaoxing Chub, Kewen Pengb, Yiran Geb,
Qihua Zhu*a,b and Yungen Xu*a,b
aState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, China
bJiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China
Pharmaceutical University, Nanjing 210009, China
Abstract: Concomitant inhibition of PARP and PI3K pathways has been recognized as a
promising strategy for cancer therapy, which may expand the clinical utility of PARP inhibitors.
Herein, we report the discovery of dual PARP/PI3K inhibitors that merge the pharmacophores of
PARP and PI3K inhibitors. Among them, compound 15 stands out as the most promising candidate
with potent inhibitory activities against both PARP-1/2 and PI3Kα/δ with pIC50 values greater than
8. Compound 15 displayed superior anti-proliferative profiles against both BRCA-deficient and
BRCA-proficient cancer cells in cellular assays. The prominent synergistic effects produced by the
concomitant inhibition of the two targets were elucidated by comprehensive biochemical and
cellular mechanistic studies. In vivo, 15 showed more efficacious anti-tumor activity than the
corresponding drug combination (Olaparib + BKM120) in MDA-MB-468 xenograft model with a
tumor growth inhibitory rate of 73.4% without causing observable toxic effects. All the results
indicate that 15, a first potent dual PARP/PI3K inhibitor, is a highly effective anticancer compound.
Keywords: PARP inhibitor; PI3K inhibitor; dual PARP/PI3K inhibitors; synergistic effects.
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INTRODUCTION
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In the context of cancer, single-agent/single-target therapeutics are poorly effective due to the
complexity of cancer pathways and the emergence of drug resistance.1 Consequently, combination
therapies targeting two or more pathways constitute the mainstays of modern cancer treatment.2
However, combination therapies are hampered by their complicated pharmacokinetics, intricate
toxicity profiles, undesirable drug-drug interactions, as well as poor patient compliance. Moreover,
expensive and time-consuming clinical research pertaining to drug combination presents yet another
great obstacle for the application of combination therapies against cancers.3-6 In an effort to
circumvent the limitations of combination regimens, we have focused on the development of dual
inhibitors capable of blocking two classes of enzymes simultaneously. Amid the numerous enzyme
targets, we found poly(ADP-ribose)polymerases (PARPs) and phosphoinositide 3-kinases (PI3Ks)
are two particularly suitable targets for our endeavor to develop dual inhibitors against BRCA-
proficient cancers.
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PARPs are a family of enzymes that catalyze the transfer of ADP-ribose from nicotinamide
adenine dinucleotide (NAD+) onto acceptor proteins.7,8 PARP-1 is the most abundant and well-
studied member of this family, which plays a crucial role in the repair of DNA single-strand breaks
(SSBs).9,10 The role of PARP-1 in DNA repair has made it a highly pursued therapeutic target for
cancer treatment, which prompted the development of numerous PARP-1 inhibitors.11-13 PARP
inhibitors were initially developed as chemo- or radio-sensitizing agents by inhibiting DNA damage
repair and promoting apoptosis of tumor cells.14 In 2005, two groups described the synthetic
lethality (SL) between PARP inhibition and BRCA1/2 mutations, suggesting a novel strategy for
treating patients with BRCA-mutant tumors.15,16 Therefore, beyond their use as chemosensitizers,
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PARP inhibitors are also promising monotherapy agents for patients with BRCA1/2 mutations.17,18
To date, four PARP inhibitors, Olaparib19, Rucaparib20, Niraparib21 and Talazoparib22 have been
approved by the FDA or EMA for the treatment of BRCA-mutant advanced ovarian and breast
cancers. In addition, a number of other PARP inhibitors, such as Veliparib23, are in clinical
evaluation for cancer therapies.24,25 However, clinical development of PAPR inhibitors has been
severely hampered by their narrow indication spectrum entailed by SL. Thus, only a small subgroup
of patients with BRCA1/2 mutations can benefit from this therapy, posing a great limitation on the
utility of PARP inhibitors.18
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The PI3K/AKT/mTOR pathway is a major signaling cascade implicated in cancer, and PI3K
is a well-established target for anticancer therapy.26,27 It was reported that PI3K plays a key
regulatory role in stabilizing and preserving DNA double-strand break (DSB) repair by interacting
with the homologous recombination (HR) complex.28,29 In 2012, Ibrahim and Juvekar groups
independently found that PI3K inhibition promotes HR deficiency by downregulating BRCA1/2
and sensitizes BRCA-proficient tumors to PARP inhibition, which provides a rationale for the
combined administration of PI3K and PARP inhibitors to expand the utility of PARP inhibitors
beyond BRCA1/2-mutant cancers.30,31 On the basis of these findings, a clinical trial of the
combination therapy of BKM120 and Olaparib has been initiated in patients with recurrent triple-
negative breast cancer (TNBC) and high-grade serous ovarian cancer.32
Such ground-breaking research encouraged us to develop small-molecule inhibitors
concomitantly targeting PARP and PI3K, given that no dual PARP/ PI3K inhibitors are currently
available in clinic or in the market. However, developing pharmacologically attractive dual
PARP/PI3K inhibitors is difficult, and the challenges include: retaining high potency and specificity
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simultaneously for the two targets within a single molecule; striking an appropriate balance in
targeting PARP and PI3K; and controlling the size and polarity of such hybrid compounds for a
favorable physiochemical and pharmacologic profile.
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Herein, we present the design, synthesis and biological evaluation of dual PARP/PI3K
inhibitors. Our data of the most promising compound 15 indicate that it can produce pronounced
synergistic anti-cancer effects and is more efficacious than the corresponding combination therapy
consisting of Olaparib and BKM120.
DESIGN OF DUAL INHIBITORS
Structures of Olaparib (PARP inhibitor) and BKM120 (PI3K inhibitor)33 served as the starting
point for our endeavor to design dual PARP/PI3K inhibitors. Analysis of the reported Olaparib-
PARP-1 complexes revealed that the phthalazine moiety is critical, since it binds to the catalytic
domain of PARP. However, the cyclopropanecarbonyl group (black section of Olaparib in Figure
1A) is not necessary for PARP inhibition since it extends out toward solvent, thus, structural
modifications can be tolerated in this region.34-37 On the other hand, scrutiny of the BKM120-PI3K
binding mode revealed that the morpholine group (black section of BKM120 in Figure 1) does not
interact with the ATP-binding region and thus we can replace it with a linker to incorporate the
pharmacophores of PARP inhibitors.33,38 Based on these observations, we designed a series of
potential dual PARP/PI3K inhibitors 1-18 by combining the red section of Olaparib with the blue
section of BKM120 through a linker, as shown in Figure 1.
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(A). Dual PARP/PI3K Inhibitors Design Strategy
4
5
O
NH
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8
9
PARP
solvent binding
channel
maintain critical PARP hinge
binding components in phthalazine
N
catalytic
domain
critical
binding
elements
O
F
N
O
N
N
O
explore interactions in
hydrogen bond receptor
pocket for improved PI3K potency
O
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merged key
pharmacophores
Olaparib
NH
N
N
N
PARP1/2 inhibitor
O
F
N
R1
N
O
ATP domain
binding group
Lin
ker
linker optimisation to search
for best balance of PARP
and PI3K inhibitory activities
N
N
N
CF3
N
N
Designed dual PARP/PI3K inhibitor
based on Olaparib and BKM120
O
NH2
BKM120
PI3K inhibitor
(B). Design of our dual PARP/PI3K inhibitors 1-18
O
O
O
N
O
O
O
N
N
NH
N
scaffold
hopping
ring
NH
N
NH
merging
N
N
CF3
N
N
N
O
F
N
N
O
F
O
F
N
N
R1
N
N
N
R1
N
N
NH2
14-18
1
2-13
1,3,5-triazin
5,6,7,8-tetrahydropyrido
[3,4-d]pyrimidine
Figure 1. Schematic showing design of a merged PARP-PI3K pharmacophore taking advantage of PARP catalytic
domain critical binding group and PI3K ATP domain binding group
Firstly, we chose the piperazine moiety of Olaparib as the linker to bridge the two structural
sections descried above, since this strategy appeared to be the most synthetically feasible one
(Figure 1B). To justify the rationale of this design strategy, a docking study was carried out to probe
the binding mode of 1 toward both PARP-1 (PDB code 5DS3)34 and PI3Kα (PDB code 4JPS)39.
The docked pose into the PARP-1 active site showed that compound 1 can form three key hydrogen
bonds with Ser904 and Gly863, and π-stacking interactions with Tyr896 and Tyr907. The docking
model in the PI3Kα active site showed that compound 1 can form a hydrogen bond with Val851
and two key hydrogen bonds with Asp810 and Asp933 (Figure 2). Thus, it can be concluded that
compound 1 fits in the active sites of PARP-1 and PI3Kα in a fashion similar to Olaparib and
BKM120, respectively, which indicates that compound 1 has the potential to inhibit the activities
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of both PARP-1 and PI3Kα.
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Figure 2. Docking studies of compound 1 with PARP-1 (5DS3) and PI3Kα (4JPS). Representation of the predicted
binding mode of compound 1 in the active site of PARP-1 (A, B) and PI3Kα (C, D). Hydrogen bonds are represented
as yellow dotted lines. For clarity, nonpolar H atoms are not represented.
RESULTS AND DISCUSSION
Structure Optimization and In Vitro PARP-1/PI3Kα Inhibition Assay.
Inspired by the docking results, we synthesized compound 1 and evaluated it for PARP-1 and
PI3Kα inhibitory activities in vitro. The results showed that compound 1 has good PARP-1 and
PI3Kα inhibitory activities with pIC50 values of 7.81 and 6.49, respectively. Although there is some
imbalance in its activities against the two enzymes, compound 1 is an appropriate lead compound
deserving further optimization.
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In our efforts to improve the PI3K inhibitory activity of 1, the pyrimidine scaffold was first
replaced by 1,3,5-triazine, leading to compound 2. Notably, the inhibitory activities of compound 2
against PARP-1 and PI3Kα both increased with pIC50 values of 8.02 and 6.77, respectively (Table
1). This result showed that the 1,3,5-triazine moiety is beneficial for both PARP and PI3K inhibition.
Consequently, we retained this 1,3,5-triazine moiety in further modifications. The reported
structure-activity relationships (SARs) of PI3K inhibitors suggest that modification of the R1
substituent serving as a hydrogen-bond donor may lead to an improved PI3K inhibitory
activity.33,40,41 Thus, we designed different aromatic moieties to replace the 2-amino-4-
(trifluoromethyl)-5-pyridinyl. Eleven compounds (3-13) were synthesized and evaluated for PARP-
1 and PI3Kα inhibitory activities. As expected, the change in R1 substituent brought about little
effect on PARP-1 inhibitory activity; compounds 3-13 all exhibited excellent PARP-1 inhibitory
activity in the low nanomolar range, as shown in Table 1. However, the inhibition of PI3Kα varied
considerably depending on the R1 substituent. The aromatic groups with a lower electron density
showed better PI3Kα inhibitory activity; for example, pyrimidine (pIC50: 7.04) and pyridine (pIC50:
6.88) rings were better than benzene (pIC50: 6.48), indazole (pIC50: 6.46) and indole (pIC50: 6.20)
rings. The position of the substituents on the ring also influences the activity; for example,
compound 2, bearing a trifluoromethyl at the para-position, showed stronger PI3Kα inhibitory
activity (pIC50: 6.77) than compound 3 (pIC50: 5.92) which bears a trifluoromethyl at the meta-
position. Nonetheless, the PI3Kα inhibitory activities of compounds 3-13 were much weaker than
BKM120, and only compound 4 displayed moderate PI3Kα inhibitory activity with pIC50 of 7.04.
Therefore, the first round of structural optimization culminated in compound 4 that displayed
acceptable PARP-1 and PI3Kα inhibitory activities with pIC50 values of 9.08 and 7.04, respectively.
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Although the PARP-1 inhibitory activity is comparable to Olaparib and the PI3Kα inhibitory
activity is only 3.5-fold less potent than BKM120, there is a 120-fold difference in the inhibitory
activity between PARP-1 and PI3Kα. Thus, further structural optimization efforts are needed to
strike an appropriate inhibitory balance between PARP-1 and PI3Kα.
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Table 1. In Vitro PARP-1 and PI3Kα Inhibitory Activities of Compounds 2-13
O
O
N
NH
N
N
N
O
F
N
N
R1
N
2-13
a
pIC50
Compd
R1
PARP-1
PI3Kα
CF3
N
2
8.02±0.06
6.77±0.07
NH2
CF3
3
4
8.95±0.09
9.08±0.06
8.97±0.07
8.93±0.06
9.08±0.08
9.14±0.05
8.96±0.05
9.36±0.09
9.17±0.13
9.19±0.16
9.16±0.07
5.92±0.12
7.04±0.09
6.88±0.12
6.48±0.09
6.25±0.13
6.15±0.06
5.64±0.09
6.46±0.08
< 5
N
NH2
N
N
NH2
5
N
NH2
6
NH2
F
7
NH2
F
8
NH2
NH2
9
F
10
11
12
13
NH
N
H
N
N
6.20±0.06
6.16±0.14
NH
N
H
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Olaparib
BKM120
9.08±0.06
NDb
NDb
7
7.61±0.05
8
9
apIC50 values for enzymatic inhibition of PARP-1 and PI3Kα; data are expressed as the mean ± SD from the dose-
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response curves of three independent experiments. bND, not detected.
It was observed that section A of compound 4 was crucial for the maintenance of PARP-1
inhibitory activity, while the morpholine and aromatic amine moieties were necessary for PI3Kα
inhibitory activity (Figure 3). Thus, only the piperazine linker can be modified. Via scaffold hopping,
we modified the linker of compound 4 by merging the pyrimidine and piperazine components into
a bicyclic system (Figure 3). Unexpectedly, the newly designed compound 14 not only exhibited
excellent PARP-1 and PI3Kα inhibitory activities with pIC50 values of 8.59 and 8.32, respectively,
but also struck an inhibitory balance between the two targets (Table 2).
O
O
N
O
A
O
N
N
NH
N
Scaffold Hopping
NH
N
N
N
O
F
N
O
F
N
N
N
N
N
N
B
N
NH2
N
NH2
4
14
Figure 3. Further compound design and structure optimization
Encouraged by this result, we kept the tetrahydropyrido[3,4-d]pyrimidine scaffold while
replacing the R1 substituent of 14, leading to compounds 15-18. The inhibitory activities of the
newly designed compounds are listed in Table 2. To our disappointment, although all the
compounds maintained high PARP-1 inhibitory activity in the low nanomolar range (pIC50: 8.10-
8.59), the PI3Kα inhibitory activities of compounds 16-18 reduced considerably. As a result, the
third round of structural optimization culminated in compounds 14 and 15, which remained potent
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and well-balanced dual PARP-1/PI3Kα inhibitory activities with pIC50 values of 8.59/8.22 and
8.32/8.25, respectively.
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Table 2. In Vitro PARP-1 and PI3Kα Inhibitory Activities of Compounds 14-18
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O
N
O
NH
N
O
F
N
N
N
R1
14-18
a
pIC50
Compd
R1
PARP-1
PI3Kα
N
14
15
16
8.59±0.15
8.32±0.16
N
NH2
CF3
8.22±0.06
8.10±0.11
8.21±0.07
8.25±0.09
7.52±0.08
7.35±0.09
N
N
NH2
NH2
17
18
NH
NH
N
8.31±0.08
7.36±0.08
apIC50 values for enzymatic inhibition of PARP-1 and PI3Kα; data are expressed as the mean ± SD from the dose-
response curves of three independent experiments.
In Vitro Anti-proliferation Assay.
Considering their prominent enzymatic inhibitory activities, we then preliminarily screened
compounds 14 and 15 in cellular assays using HCC1937 (BRCA1-deficient breast cancer cells),
HCT116 (BRCA2-deficient colorectal cancer cells), MDA-MB-231 and MDA-MB-468 cancer cell
lines (BRCA-proficient triple-negative breast cancer cells). As demonstrated in Table 3, Olaparib
showed strong anti-proliferative activity against BRCA-deficient cells, but weak inhibitory activity
against BRCA-proficient cells. In contrast, compounds 14 and 15 not only showed significant
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inhibitory activity against BRCA-deficient cells HCC1937 and HCT116, but also displayed potent
anti-proliferative activity against BRCA-proficient cells MDA-MB-231 and MDA-MB-468. In
addition, the anti-proliferative activities of compound 15 against these cancer cells are at least four
times more potent than BKM120.
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Table 3. In Vitro Growth-Inhibitory Activities of Compounds 14 and 15
pIC50
Compd
HCT116
HCC1937
5.46±0.16
5.98±0.40
5.34±0.03
5.20±0.06
5.77±0.05
MDA-MB-231
5.45±0.05
6.00±0.17
4.74±0.09
5.22±0.05
5.80±0.05
MDA-MB-468
5.59±0.01
6.01±0.15
4.86±0.11
5.40±0.04
5.89±0.23
14
15
5.80±0.06
6.06±0.08
5.52±0.08
5.44±0.07
5.91±0.10
Olaparib
BKM120
Olaparib+BKM120
To explore whether the applications of dual PARP/PI3K inhibitors could be further extended
to other BRCA-proficient cancer cell lines, we evaluated compound 15 for its anti-proliferative
activity against a panel of eight other BRCA-proficient cell lines that represent different tumor types
including breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, prostate cancer, non-
small cell lung cancer, kidney cancer, lymphoma and leukemia. It is noteworthy that, compared
with Olaparib and BKM120, compound 15 exhibited considerably more potent in vitro antitumor
activity against most of these BRCA-proficient cancer cells (Table 4). The potency of 15 against
various tumor cell lines demonstrates its therapeutic potential in BRCA-mutant cancers that
typically circumscribe the application of traditional PARP inhibitors.
Table 4. In Vitro Growth-Inhibitory Activities of 15 in Various Cancer Cell Lines
Cell lines
Cell type
pIC50
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5.29±0.18
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leukemia
5.97±0.06
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6.03±0.01
6.05±0.06
6.27±0.06
6.02±0.04
5.97±0.16
5.55±0.18
4.93±0.14
6.13±0.11
5.77±0.04
5.20±0.13
5.27±0.06
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5.40±0.13
4.99±0.12
5.61±0.07
6.46±0.08
5.89±0.06
5.93±0.05
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5.73±0.05
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DU145
A549
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Caki-1
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lymphoma
colorectal cancer
Cellular Mechanism of Action Studies.
1. Western Blot Analysis of Protein Expression in Vitro
Cellular mechanistic studies of 14 and 15 were carried out to explain their superior anti-
proliferative profiles against BRCA-proficient cancer cells. Firstly, western blot analysis was
conducted to explore whether compounds 14 and 15 affect PARP’s activity and disturb the PI3K
pathway in exerting their anti-proliferative effects. As shown in Figure 4, the autophosphorylation
levels of AKT and S6 reduced while the autophosphorylation level of ERK increased after treating
cells with compounds 14 and 15, indicating that they can inhibit the PI3K pathway and activate the
ERK pathway. In addition, cell apoptosis was pronounced given the significantly elevated cleaved
PARP level observed after the treatment of 14 or 15, suggesting these two compounds severely
jeopardized the DNA-repairing functions of PARP.
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Figure 4. Effects of BKM120, Olaparib, BKM120 (1 μM) + Olaparib (2 μM), compounds 14 and 15 on protein
expression of MDA-MB-468 cancer cells. Western blot analysis of proteins (pAKT, pS6S240, pERK and cleaved
PARP) as indicated in MDA-MB-468 cancer cells treated with the indicated compounds for 72 h. β-actin was used
as a loading control. The data are shown as the mean ± SD of three independent experiments. *p < 0.05 vs. control,
**p < 0.01, ***p < 0.001; #p < 0.05, ##p < 0.01, ###p < 0.001.
2. Quantitative Real-Time PCR
To verify whether BRCA1/2 was downregulated in MDA-MB-468 cancer cells treated with
compounds 14 and 15, the expression of BRCA1/2 at the mRNA level was measured by real-time
PCR. As shown in Figure 5, compounds 14 and 15 both displayed a much stronger capability to
downregulate the expression of BRCA1/2 at the mRNA level than Olaparib, BKM120 and their
combination, suggesting that compounds 14 and 15 probably induced HR deficiency through the
downregulation of BRCA1/2.
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Figure 5．Effects of BKM120, Olaparib, BKM120 (1 μM) + Olaparib (2 μM), compounds 14 and 15 on BRCA1
and BRCA2 expression in MDA-MB-468 cells. Real-time PCR analysis of BRCA1 and 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 independent experiments. ***p < 0.001 vs. control; #p < 0.05, ##p < 0.01,
###p < 0.001.
3. Immunofluorescence staining analysis of RAD51 and γH2AX
Next, immunofluorescence staining analysis of the RAD51 protein, a marker for the
competency of HR repair, was conducted to further confirm the above observations. As shown in
Figure 6A, compared with Olaparib, BKM120 and their combination, compounds 14 and 15
significantly reduced the formation of RAD51 foci, indicating impaired HR repair efficiency.
Furthermore, it was also observed that the nuclear foci of γH2AX, a biological marker for DSBs,
increased significantly after the treatment of compounds 14 and 15 (Figure 6B).
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Figure 6. (A) Effects of BKM120, Olaparib, BKM120 (1 μM) + Olaparib (2 μM), compounds 14 and 15 on RAD51
expression in MDA-MB-468 cells. (B) Effects of BKM120, Olaparib, BKM120 (1 μM) + Olaparib (2 μM),
compounds 14 and 15 on γH2AX expression in MDA-MB-468 cells. Representative images of immunofluorescence
staining of RAD51 and γH2AX in MDA-MB-468 cancer cells treated as indicated for 72 h. Cell nuclei were stained
with DAPI. The data are shown as the mean ± SD of three independent experiments. ***p < 0.001 vs. control; ##p <
0.01, ###p < 0.001.
4. Comet assay
To gain more insights into the therapeutic effect of the dual PARP/PI3K inhibitors, a comet
assay was conducted to evaluate the extent of DNA damage induced. As shown in Figure 7, both
compounds 14 and 15 induced large amounts of DNA damage. Notably, compound 15 generated a
much higher tail intensity compared with Olaparib, BKM120 or their combination, suggesting its
superiority in incurring DNA damage.
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Figure 7. Effects of BKM120, Olaparib, BKM120 (1 μM) + Olaparib (2 μM), compounds 14 and 15 on the DNA
damage response in MDA-MB-468 cells. (A) The MDA-MB-468 cancer cells were treated with the indicated
compound as single agents or in combination for 72 h. Comet images ×200 taken by fluorescence microscope are
shown. (B) The quantitative analysis was performed with the comet analysis software CASP, and the olive tail
moment was employed to evaluate DNA damage. The data are shown as the mean ± SD of three independent
experiments. ***p < 0.001 vs. control; ###p < 0.001.
5. Apoptosis Analysis
Finally, to determine the effects of compounds 14 and 15 on cell death, we conducted an
apoptosis assay using Annexin-V by FACS analysis in the MDA-MB-468 cell line. As shown in
Figure 8, compounds 14 and 15 led to a significant increase in cell apoptosis compared with both
single-agent treatments and the combination of Olaparib and BKM120. These results suggest that
the PI3K inhibition mediated by 14 and 15 can be exploited to induce HR deficiency and thus
enhance the sensitivity of BRCA-proficient triple-negative breast cancer cells to PARP inhibition.
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Figure 8. Effects of BKM120, Olaparib, BKM120 (1 μM) + Olaparib (2 μM), compounds 14 and 15 to induce
apoptosis of MDA-MB-468 cells. (A) MDA-MB-468 cells were treated with the 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; ###
p
< 0.001.
In Vivo Antitumor Effects Study.
Based on the excellent enzymatic and antiproliferative activities of compounds 14 and 15 in
vitro, we then evaluated their antitumor activities in vivo in MDA-MB-468 xenograft mouse model.
Compounds 14 and 15, Olaparib, BKM120, and the combination of Olaparib and BKM120 were
administered by intraperitoneal injection twice daily (BID) for 34 consecutive days. As shown in
Figure 9, compounds 14 and 15 significantly suppressed the tumor growth at a dose of 50 mg·kg⁻¹
and they were both well-tolerated with no mortality. The tumor suppression effects of 14 (TGI:
52.7%) and 15 (TGI: 73.4%) were both 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 inhibitors are superior to the single-target inhibitors in the
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antitumor efficacy.
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Figure 9. Effects of Olaparib, BKM120, Olaparib (50 mg·kg⁻¹) + BKM120 (27.5 mg·kg⁻¹), compounds 14 and 15
treatment on the tumorigenicity of MDA-MB-468 cells in vivo. The combination dose was referred to the literature.30
(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 ± SD of 6 mice. *p < 0.05 vs. control, ***p < 0.001; #p <
0.05. (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.
In Vivo Western Blot Analysis.
Since compound 15 displayed more promising antitumor activity in vivo, it was submitted to
mechanistic study to bolster our conclusion that the prominent antitumor effect of compound 15 is
indeed engendered by its PARP/PI3K dual-targeting capability. Western blot analysis of the excised
tumor tissue from MDA-MB-468 tumor bearing mice was carried out. In good agreement with the
in vitro western blot analysis, compound 15 strongly inhibited the expression of pAKT and pS6,
while it stimulated the expression of pERK and pETS1 (Figure 10). Moreover, 15 significantly
downregulated the expression of BRCA1/2 and increased the level of cleaved PARP. In conclusion,
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our medicinal chemistry endeavor led to the discovery of compound 15, demonstrating that the dual-
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inhibition of PARP and PI3K with a single chemical entity is feasible.
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Figure 10. Western blot analysis of the excised tissue from MDA-MB-468 tumor bearing mice after treating with
compound 15. (A) Effects of compound 15 on AKT and S6 phosphorylation levels. (B) Effects of compound 15 on
ERK and ETS1 phosphorylation levels. (C) Effects of compound 15 on BRCA1/2 expressions. (D) Effects of
compound 15 on cleaved PARP expressions.
Kinase Selectivity Study.
In order to profile compound 15’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 Table 5
indicate that compound 15 possesses strong inhibitory effect on PARP-1/2 and PI3Kα/δ.
Table 5. PARP and PI3K Isoform Selectivity Profile of Compound 15
a
pIC50
Compd
PARP-1
PARP-2
PI3Kα
PI3Kβ
PI3Kδ
PI3Kγ
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15
8.22±0.06
8.44±0.06
8.25±0.09
6.54±0.10
8.13±0.07
6.08±0.05
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BKM12033
8.30
9.00
7.28
6.78
6.94
6.58
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apIC50 values of compound 15 are expressed as the mean ± SD from the dose-response curves of three independent
experiments.
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To further elucidate its kinase selectivity, compound 15 was tested at a single concentration of
1 μM against 374 kinases in the Reaction Biology Corporation (RBC) kinase panel (see Supporting
Information). The results showed that compound 15 displayed weak inhibitory activities against 374
kinases at 1 μM concentration (Figure 11 and Table S1). It can be thus concludes that compound 15
is a highly selective dual PARP/PI3K inhibitor.
Figure 11. Kinase selectivity profile of compound 15 as shown by an RBC kinase panel screen against 374 kinases
assayed at 1 μM in duplicate. Compound selectivity is represented in a dendrogram view of the human kinome
phylogenetic tree. The color code for inhibition is indicated.
CHEMISTRY
The synthetic routes of the target compounds are summarized in Schemes 1-3. The synthesis
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of compound 1 is depicted in Scheme 1. The starting material 2,4,6-trichloropyrimidine (20) was
mono-substituted by morpholine to give intermediate 21 which was further substituted by N-Boc-
piperazine and followed by N-deprotection to give intermediate 22. Condensation of 22 with 23
afforded intermediate 24. Finally, target compound 1 was obtained by Suzuki coupling of 24 with
boric acid ester 25a.
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Scheme 1. Synthetic Route of Compound 1.
O
O
N
O
N
O
N
NH
N
O
O
F
Cl
NH
N
OH
a
N
N
b
N
N
O
F
N
N
N
N
23
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Cl
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N
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Cl
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HCl
c
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CF3
N
O
NH
N
N
NH2
O
F
N
N
CF3
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N
d
N
NH2
1
Reagents and conditions: (a) morpholine, DIPEA, DCM, -78 °C, 0.5 h; (b) i) N-Boc-piperazine, K2CO3, DMF, 25°C,
5 h; ii) HCl/EtOAc, 25 °C, 2 h; (c) PyBOP, DIPEA, DMF, 25 °C, 6 h; (d) Pd(PPh3)4, K2CO3, dioxane/H2O, reflux,
N2, 8 h.
Compounds 2-13 were synthesized via a similar route with compound 1 (Scheme 2). The
nucleophilic substitution of starting material 2,4,6-trichloro-1,3,5-triazine (26) with morpholine
produced intermediate 27, which underwent substitution with N-Boc-piperazine and N-deprotection
giving intermediate 28. Condensation of 28 with 23 in the presence of PyBOP and DIPEA furnished
intermediate 29. Target compounds 2-13 were finally obtained by Suzuki coupling of 29 with
corresponding boric acid esters 25a-l.
Scheme 2. Synthetic Route of Compounds 2-13.
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O
N
O
O
N
O
N
NH
N
Cl
O
F
N
N
a
N
N
23
b
N
N
N
N
N
N
N
Cl
Cl
N
Cl
Cl
N
Cl
Cl
N
N
NH
HCl
c
26
27
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O
O
O
NH
N
N
N
B
R1
O
O
N
N
25a-l
N
N
R1
d
F
2-13
CF3
N
CF3
NH2
N
3, 25b R1
=
2, 25a R1
=
4, 25c R1
8, 25g R1
=
=
5, 25d R1
=
NH2
N
NH2
NH2
N
N
NH2
NH2
F
F
6, 25e R1
10, 25i R1
=
9, 25g R1
13, 25l R1
=
=
7, 25f R1
=
F
NH2
NH2
=
11, 25j R1
=
12, 25k R1
=
N
H
HN
NH
N
NH
N
Reagents and conditions: (a) morpholine, DIPEA, DCM, -78°C, 0.5 h; (b) i) N-Boc-piperazine, K2CO3, DMF, 25°C,
5 h; ii) HCl/EtOAc, 25 °C, 2 h; (c) PyBOP, DIPEA, DMF, 25 °C, 6 h; (d) Pd(PPh3)4, K2CO3, dioxane/H2O, reflux,
N2, 6~8 h.
The synthesis of compounds 14-18 is outlined in Scheme 3. The first step is pinner pyrimidine
synthesis, commercially available ethyl 1-benzyl-3-oxopiperidine-4-carboxylate (30) was reacted
with urea in the presence of NaOMe to produce intermediate 31. The chlorodehydroxylation of 31
with POCl3 gave intermediate 32 which was further substituted by morpholine and followed by
benzyl deprotection to give intermediate 34. Condensation of 34 with 23 in the presence of PyBOP
and DIPEA furnished intermediate 35. Finally, compounds 14-18 were obtained through Suzuki
coupling of 35 with the corresponding boric acid esters.
Scheme 3. Synthetic Route of Compounds 14-18.
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O
N
O
Cl
N
CO2Et
O
d
N
c
NH
a
b
N
N
N
N
Bn
N
H
O
Bn
Cl
Bn
N
Bn
O
N
Cl
30
31
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33
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O
O
O
N
O
N
NH
NH
N
N
N
23
25a,c,d,i,k
O
F
O
N
N
N
e
f
HN
N
N
N
R1
N
Cl
N
Cl
F
34
35
14-18
CF3
N
N
14 R1
=
15 R1
=
16 R1
=
18 R1
=
17 R1
=
N
NH2
N
NH2
NH2
NH
NH
N
Reaction conditions: (a) urea, NaOMe, MeOH, 0 °C to reflux, N2, 24 h; (b) POCl3, 0 °C to reflux, N2, 6 h; (c)
morpholine, DIPEA, (CH3)2CHOH/CH2Cl2, 50 °C, 3 h; (d) 1-chloroethyl chloroformate, CH2Cl2, 0 to 25 °C, 8 h,
MeOH，reflux, 1 h; (e) PyBOP, DIPEA, DMF, 25 °C, 6h; (f) Pd(PPh3)4, K2CO3, dioxane/H2O, reflux, N2, 4~6 h.
CONCLUSIONS
PARP inhibitors have clinical effectiveness restricted to a small subgroup of patients with
BRCA mutations. Recently, it was reported that PI3K inhibition could promote HR deficiency and
sensitize BRCA-proficient tumors to PARP inhibition. Therefore, co-targeting of PARP and PI3K
has been recognized as a promising chemotherapeutic strategy to expand the utility of PARP
inhibitors. In our efforts to obtain dual PARP/PI3K inhibitors, lead compound 1 was designed by
combining the pharmacophores of PARP and PI3K inhibitors. Subsequent structural optimization
was conducted, focusing on increasing the inhibitory activities and improving inhibitory balance,
and led to the candidate compounds 14 and 15. They both showed potent and well-balanced
inhibitory activities against PARP-1 and PI3Kα with pIC50 values greater than 8.22. Compound 15
displayed more potent anti-proliferative activity against a panel of BRCA-proficient cancer cells
than Olaparib. Cellular mechanistic studies showed that compounds 14 and 15 strongly inhibited
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the growth of MDA-MB-468 cells through suppressing the PI3K signaling pathway,
downregulating BRCA1/2 expression and inducing DNA damage and apoptosis. In MDA-MB-468
cell-derived xenograft model, compounds 14 and 15 displayed excellent antitumor efficacy at a dose
of 50 mg·kg⁻¹, which is considerably more efficacious than the single administration of Olaparib or
BKM120 and even their combined administration. In view of the structure together with its
encouraging in vitro and in vivo properties, compound 15 as a first dual PARP/PI3K inhibitor is
worthy of further profiling. Our data demonstrate that dual PARP/PI3K inhibitors have good
synergistic effect and should be extensively evaluated as a new class of targeted therapy against a
wide range of oncologic diseases.
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EXPERMENTAL SECTION
General Procedures. Unless otherwise specified, reagents were purchased from commercial
suppliers and used without further purification. Melting points were determined by an X-4 digital-
display micromelting-point apparatus (Beijing Tech Instrument Company, Ltd., Beijing, China).
NMR spectra were recorded on a Bruker AVANCE AV-600 spectrometer (300 MHz and 500 MHz
for 1H and 75 MHz for 13C) or a Bruker AVANCE AV-300 spectrometer (300 MHz for 1H and 75
MHz for 13C; Bruker, Billerica, MA). Mass spectra were obtained on an Agilent 1100 LC/MSD
mass spectrometer (Agilent, Santa Clara, CA) and Micromass Q-tofmicro MS (Waters, Milford,
MA). All reactions were monitored by TLC (Silica gel GF254, Merck, Kenilworth, NJ), and spots
were visualized with UV light or iodine. Flash column chromatography on silica gel (200-300 mesh)
was used for the routine purification of reaction products. The purities of the biologically evaluated
compounds were >95% as determined by HPLC.
General Procedure A for the Synthesis of Compound 1. 2,4,6-trichloropyrimidine 20 (10.00
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g, 54.52 mmol) and DIPEA (9.10 mL, 55.06 mmol) were dissolved in dichloromethane (DCM) (100
mL). After cooling to -78 °C, a solution of morpholine (4.75 mL, 54.52 mmol) in DCM (10 mL)
was added dropwise. The mixture was stirred at -78 °C for 0.5 h, then the precipitate was filtered
and washed with water. The white powder was purified with mixed solvent (n-hexane: ethyl acetate
= 50:1, v/v), then filtered and dried in a vacuum desiccator to give intermediate 21 (9.40 g, yield:
74%). m.p.: 122-124 oC. 1H NMR (300 MHz, CDCl3) δ (ppm): 6.56 (1H, s, ArH), 3.83-3.80 (4H,
m, 2CH2O), 3.76-3.72 (4H, m, 2CH2N).
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To a solution of compound 21 (2.00 g, 8.54 mmol) in acetone (50 mL) was added N-Boc-
piperazine (3.98 g, 21.35 mmol) and Et3N (4.00 mL, 28.86 mmol). The mixture was stirred under
reflux for 10 h. After completion, the precipitate was filtered and washed with acetone, then dried
under vacuum to give a white solid (2.84 g). The solid was dissolved in ethyl acetate (EtOAc) (15
mL), and an HCl-saturated EtOAc solution was added slowly to pH 2. The mixture was stirred at
room temperature until completion of the reaction as evidenced by TLC. The precipitate was filtered,
washed with EtOAc and dried under vacuum to give 22 as a white solid (2.36 g, yield: 86%).
m.p.: >250 oC. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 9.53 (1H, s, HCl), 6.92 (1H, s, NH), 6.30
(1H, s, ArH), 3.90 (4H, t, J = 4.8 Hz, 2CH2O), 3.67-3.58 (8H, m, 4CH2N), 3.12 (4H, s, 2CH2N).
To a solution of intermediate 22 (468 mg, 1.46 mmol) in DMF (10 mL) was added compound
23 (436 mg, 1.46 mmol), PyBOP (912 mg, 1.75 mmol) and DIPEA (0.76 mL, 4.38 mmol) dropwise.
The mixture was stirred at room temperature for 4 h. After completion, the mixture was poured into
water (30 mL) and stirred for 10 min. The precipitate was filtered and washed with water, then dried
under vacuum to give intermediate 24 as a yellow solid (650 mg, yield: 79%). m.p.: 108-110 oC. 1H
NMR (300 MHz, DMSO-d6) δ (ppm): 12.57 (1H, s, CONH), 8.25 (1H, d, J = 7.6 Hz, ArH), 7.97-
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7.79 (3H, m, ArH), 7.46-7.41 (1H, m, ArH), 7.37-7.34 (1H, m, ArH), 7.22 (1H, t, J = 8.7 Hz, ArH),
6.19 (1H, s, ArH), 4.32 (2H, s, ArCH2), 3.72-3.53 (14H, m, 7CH2), 3.24-3.17 (2H, m, CH2).
4-(3-(4-(6-(6-amino-4-(trifluoromethyl)pyridin-3-yl)-2-morpholinopyrimidin-4-
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yl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (1). To
a
solution of
intermediate 24 (0.53 mmol) in dioxane (20 mL) was added boric acid ester 25a (0.58 mmol),
Pd(PPh3)4 (0.05 mmol) and K2CO3 (2.12 mmol) dissolved in water (2 mL). The suspension was
heated with stirring under a nitrogen atmosphere at 100 °C for 4-6 h. The reaction was monitored
by TLC. After completion of the reaction, the mixture was cooled to room temperature and
evaporated under reduced pressure. The residue was partitioned between EtOAc (20 mL) and water
(20 mL), and the water phase was extracted two times with the same volume of EtOAc. The
combined organic phases were washed with water and brine and evaporated under reduced pressure.
The crude product was purified by silica gel column chromatography (DCM/MeOH = 80:1 to 30:1,
v/v) to yield title compound 1 as a light yellow solid (148 mg, yield: 41%). m.p.: 142-144 oC. HPLC:
97.7%. 1H NMR (300 MHz, DMSO-d6+D2O) δ (ppm): 8.23 (d, J = 7.6 Hz, 1H, ArH), 8.11 (s, 1H,
ArH), 7.97-7.89 (m, 1H, ArH), 7.89-7.73 (m, 2H, ArH), 7.44-7.36 (m, 1H, ArH), 7.35-7.27 (m, 1H,
ArH), 7.20 (t, J = 9.0 Hz, 1H, ArH), 6.81 (s, 1H, ArH), 6.17 (s, 1H, ArH), 4.30 (s, 2H, ArNH2),
3.80-3.72 (m, 2H, CH2O), 3.68-3.41 (m, 12H, CH2O, 5CH2N), 3.23-3.08 (m, 2H, CH2N). 13C-NMR
(75 MHz, DMSO-d6) δ (ppm): 164.2, 162.9, 160.3, 160.0, 159.5, 157.9, 154.7, 152.6, 150.4, 145.2,
140.1, 134.7, 133.6, 131.6, 129.0, 128.7, 127.7, 126.0, 125.4, 123.7, 121.2, 120.4, 116.1, 104.5, 92.0,
65.8, 46.4, 43.8, 36.4, 28.8. HRMS (ESI): m/z [M+H]+. Calcd for C34H32F4N9O3: 690.2559; Found:
690.2571. IR (cm-1): 3339.43, 3209.64, 2923.01, 2852.32, 1633.70, 1573.89, 1540.13, 1497.33,
1466.83, 1436.56, 1364.45, 1282.53, 1243.12, 1192.04, 1166.02, 1136.28, 1119.34, 1000.95,
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974.15, 806.36, 770.70, 673.73, 483.04.
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General Procedure B for the Synthesis of Compounds 2-13. 2,4,6-trichloro-1,3,5-triazine
26 (10.00 g, 54.23 mmol) and DIPEA (9.92 mL, 56.94 mmol) were dissolved in dichloromethane
(DCM) (100 mL). After cooling to -78 °C, a solution of morpholine (4.73 mL, 54.23 mmol) in DCM
(10 mL) was added dropwise. The mixture was stirred at -78 °C for 0.5 h, then the precipitate was
filtered and washed with water. The white powder was dried in a vacuum desiccator to give
intermediate 27 (7.18 g, yield: 56%). m.p.: >250 oC. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 3.80-
3.76 (4H, m, CH2O), 3.69 (4H, t, J = 4.6 Hz, CH2N).
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To a solution of intermediate 27 (7.00 g, 29.78 mmol) in DMF was added N-Boc-piperazine
(5.55 g, 29.80 mmol) and K2CO3 (4.12 g, 29.81 mmol). The mixture was stirred at 25 °C for 5 h.
After completion, the mixture was poured into water (300 mL) and stirred for 10 min. The
precipitate was filtered and washed with water, then dried under vacuum to give a white solid (10.50
g). Without purification, the white solid was dissolved in ethyl acetate (EtOAc) (50 mL), and then
an HCl-saturated EtOAc solution was added slowly to pH 2. The mixture was stirred at room
temperature until completion of the reaction as evidenced by TLC. The precipitate was filtered,
washed with EtOAc and dried under vacuum to give intermediate 28 as a white solid (7.65 g, yield:
80%). m.p.: >250 oC. 1H NMR (300 MHz, MeOD) δ (ppm): 4.06 (4H, t, J = 5.2 Hz, 2CH2O), 3.81-
3.77 (4H, m, 2CH2N), 3.70-3.67 (4H, m, 2CH2N), 3.27 (4H, t, J = 5.4 Hz, 2CH2N).
To a solution of intermediate 28 (8.10 g, 25.22 mmol) in DMF (100 mL) was added compound
23 (7.53 g, 25.25 mmol), PyBOP (14.40 g, 27.67 mmol) and DIPEA (12.50 mL, 75.63 mmol)
dropwise. The mixture was stirred at room temperature for 6 h. After completion, the mixture was
poured into water (300 mL) and stirred for 10 min. The precipitate was filtered and washed with
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water, then dried under vacuum. The crude product was purified by silica gel column
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chromatography (DCM/MeOH = 100:1 to 40:1, v/v) to give intermediate 29 as a light yellow solid
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(12.50 g, yield: 88%). m.p.: 148-150 oC. H-NMR (300 MHz, DMSO-d6) δ (ppm): 12.57 (1H, s,
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CONH), 8.27 (1H, d, J = 7.8 Hz, ArH), 7.99-7.81 (3H, m, ArH), 7.48-7.43 (1H, m, ArH), 7.38 (1H,
d, J = 6.5 Hz, ArH), 7.25 (1H, t, J = 9.0 Hz, ArH), 4.35 (2H, s, ArCH2), 3.80-3.63 (14H, m, 2CH2O,
5CH2N), 3.27-3.20 (2H, m, CH2N).
To a solution of intermediate 29 (0.53 mmol) in dioxane (20 mL) was added boric acid esters
25a-l (0.58 mmol), Pd(PPh3)4 (0.05 mmol) and K2CO3 (2.12 mmol), which was dissolved in water
(2 mL). The suspension was heated with stirring under a nitrogen atmosphere at 100 °C for 4-6 h.
The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to
room temperature and evaporated under reduced pressure. The residue was partitioned between
EtOAc (20 mL) and water (20 mL), and the water phase was extracted two times with the same
volume of EtOAc. The combined organic phases were washed with water and brine and evaporated
under reduced pressure. The crude products were purified by silica gel column chromatography
(DCM/MeOH = 80:1 to 30:1) to yield title compounds 2-13.
4-(3-(4-(4-(6-amino-4-(trifluoromethyl)pyridin-3-yl)-6-morpholino-1,3,5-triazin-2-yl)-
piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (2). Off-white solid, 51% yield,
m.p.: 159-161 oC. HPLC: 99.5%. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 12.61 (1H, s, CONH),
8.61 (1H, s, ArH), 8.27 (1H, d, J = 7.4 Hz, ArH), 7.97 (1H, d, J = 7.4 Hz, ArH), 7.91-7.80 (2H, m,
ArH), 7.47-7.43 (1H, m, ArH), 7.39 (1H, dd, J = 6.4, 1.9 Hz, ArH), 7.25 (1H, t, J = 9.0 Hz, ArH),
6.97 (2H, s, NH2), 6.82 (1H, s, ArH), 4.34 (2H, s, ArCH2), 3.85-3.63 (14H, m, 2CH2O, 5CH2N),
3.24-3.21 (2H, m, CH2N). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 169.5, 164.0, 161.2, 159.3,
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158.0, 154.8, 152.6, 144.8, 135.9, 134.8, 133.4, 131.5, 129.1, 127.9, 126.0, 125.4, 124.9, 123.7,
123.5, 121.2, 118.6, 116.1, 115.8, 104.6, 65.9, 46.2, 43.2, 36.5, 28.9. HRMS (ESI): m/z [M+H]+.
Calcd for C33H30F4N10O3: 691.2511; Found: 691.2518. IR (cm-1): 3338.61, 3210.89, 2921.32,
2854.73, 1636.67, 1560.36, 1540.11, 1518.99, 1436.52, 1391.19, 1365.02, 1259.55, 1226.52,
1162.84, 1115.67, 1013.32, 1000.47, 978.32.
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4-(3-(4-(4-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-6-morpholino-1,3,5-triazin-2-yl)-
piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (3). Light yellow solid, 55% yield,
1
m.p.: 244-246 oC. H NMR (300 MHz, DMSO-d6) δ (ppm): 12.62 (1H, s, CONH), 9.12 (1H, s,
ArH), 8.48 (1H, s, ArH), 8.26 (1H, d, J = 7.3 Hz, ArH), 7.99-7.80 (3H, m, ArH), 7.49-7.35 (2H, m,
ArH), 7.26 (1H, t, J = 8.9 Hz, ArH), 7.14 (1H, s, 1/2NH2), 6.42 (1H, s, 1/2NH2) 4.35 (2H, s, ArCH2),
3.94-3.51(14H, m, 2CH2O, 5CH2N), 3.22 (2H, m, CH2N). 13C NMR (75 MHz, DMSO-d6) δ (ppm):
167.4, 164.2, 164.0, 161.0, 159.3, 158.0, 157.3, 154.8, 153.2, 151.3, 144.8, 138.8, 134.8, 133.4,
131.6, 129.1, 128.9, 127.9, 126.0, 125.5, 123.8, 119.8, 116.1, 115.8, 66.0, 45.8, 43.1, 36.4, 25.8.
HRMS (ESI): m/z [M+H]+. Calcd for C33H30F4N10O3: 691.2511; Found: 691.2517. IR (cm-1):
3518.20, 3178.15, 2897.79, 2858.70, 1680.81, 1645.80, 1583.50, 1544.76, 1522.47, 1497.79,
1441.61, 1396.66, 1363.34, 1308.66, 1253.94, 1225.60, 1148.85, 1114.30, 1003.38, 983.10, 814.27,
681.51.
4-(3-(4-(4-(2-aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)piperazine-1-
carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (4). Light yellow solid, 58% yield, m.p.: 182-
183 oC. HPLC: 98.4%. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 12.61 (1H, s, CONH), 9.06 (2H,
s, ArH), 8.26 (1H, d, J = 6.8 Hz, ArH), 7.99-7.81 (3H, m, ArH), 7.49-7.43 (1H, m, ArH), 7.38 (1H,
d, J = 6.6, 1.9 Hz, ArH), 7.30 (2H, s, NH2), 7.25-7.22 (1H, m, ArH), 4.34 (2H, s, ArCH2), 3.94-3.57
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