Design, synthesis and biological evaluation of N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide derivatives as novel P-glycoprotein inhibitors reversing multidrug resistance

Article abstract of DOI:10.1016/j.bioorg.2019.01.039

The overexpression of P-glycoprotein plays an important role in the process of multidrug resistance (MDR). P-gp inhibitors are one of the effective strategies to reverse tumor MDR. Novel P-gp inhibitors with phthalazinone scaffolds were designed, synthesized and evaluated. Compound 26 was found to be the most promising for further study. Compound 26 possessed high potency (EC₅₀ = 46.2 ± 3.5 nM) and low cytotoxicity.26 possessed high MDR reversal activity towards doxorubicin-resistant K56/A02 cells. Reversal fold (RF) value reach to 44.26. 26 also increased accumulation of doxorubicin (DOX or ADM) or other MDR-related anticancer drugs with different structures. In conclusion, compound 26 deserves more research for its good features as P-gp inhibitor.

Full text of DOI:10.1016/j.bioorg.2019.01.039

Accepted Manuscript
Design, synthesis and Biological Evaluation of N-(4-(2-(6,7-dimethoxy-3,4-di-
hydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3,4-dihydrophthalazine-1-car-
boxamide derivatives as Novel P-Glycoprotein inhibitors Reversing Multidrug
Resistance
Qianqian Qiu, Jiaqi Zhou, Wei Shi, Mutta Kairuki, Wenglong Huang, Hai Qian
PII:
S0045-2068(18)31521-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.039
YBIOO 2743
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 December 2018
9 January 2019
21 January 2019
Please cite this article as: Q. Qiu, J. Zhou, W. Shi, M. Kairuki, W. Huang, H. Qian, Design, synthesis and Biological
Evaluation
of
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3,4-
dihydrophthalazine-1-carboxamide derivatives as Novel P-Glycoprotein inhibitors Reversing Multidrug Resistance,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.039
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Design,
synthesis
and
Biological
Evaluation
of
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3,4-dihydro
phthalazine-1-carboxamide derivatives as Novel P-Glycoprotein inhibitors Reversing
Multidrug Resistance
Qianqian Qiua,# , Jiaqi Zhoub,#, Wei Shib, Mutta Kairukib, Wenglong Huangb,c, , Hai Qianb,c,*
a School of Pharmacy, Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources
and Environmental Protection, Yancheng Teachers’ University, Yancheng 224007, PR China
b
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
c Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
Abstract: The overexpression of P-glycoprotein plays an important role in the process of
multidrug resistance (MDR). P-gp inhibitors are one of the effective strategies to reverse
tumor MDR. Novel P-gp inhibitors with phthalazinone scaffolds were designed, synthesized
and evaluated. Compound 26 was found to be the most promising for further study.
Compound 26 possessed high potency (EC50 = 46.2 ± 3.5 nM) and low cytotoxicity.26
possessed high MDR reversal activity towards doxorubicin-resistant K56/A02 cells. Reversal
fold (RF) value reach to 44.26. 26 also increased accumulation of doxorubicin (DOX or ADM)
or other MDR-related anticancer drugs with different structures. In conclusion, compound 26
deserves more research for its good features as P-gp inhibitor.
Keywords: Multidrug resistance, P-glycoprotein inhibitors, doxorubicin
1. Introduction
Drug resistance is one of the main causes of chemotherapy failure[1]. And Multidrug
 Corresponding authors : For Hai Qian: phone, +86-25-83271051; fax, +86-25-83271051;
E-mail, qianhai24@163.com; For Wenlong Huang: phone, +86-25-83271051; fax,
+86-25-83271051; E-mail, ydhuangwenlong@126.com
# These authors contributed equally to this work

resistance (MDR) accounts for a considerable proportion. Numerous studies have shown that
the development of MDR is closely related to the overexpression of P-glycoprotein (P-gp,
ABCB1), one of ATP-binding cassette (ABC) transport proteins[2, 3]. P-glycoprotein is a
cross-membrane homologous dimer composed of 1,280 amino acids, each monomer
consisting of a nucleotide-binding domain (NBD) and a transmembrane domain (TMD)[4, 5].
P-glycoprotein acts as a drug efflux pump, leading to chemotherapy drugs going into cancer
cells difficultly and decreasing the intra-cellular concentration of anticancer drugs[6, 7].
P-glycoprotein inhibitors are used to help anticancer drugs evade P-gp-mediated MDR.
Nowadays, three generations of P-gp inhibitors have been developed[8]. First-generation
inhibitors, such as Verapamil (1) and Cyclosporin A (2), are primarily those existing drugs
possessing inhibition against P-gp[9]. Such P-gp inhibition was found as side effects of drugs
during research[8]. Structural modification based on first-generation inhibitors develops
second-generation inhibitors that possess better selectivity and affinity but still obvious side
effects[10]. For example, Dexverapamil (3) and Valspodar (4)[11]. And in order to overcome
inherent defects of first-generation and second-generation inhibitors, third-generation
inhibitors represented by Tariquidar (5) and Zosuquidar (6) have been developed on basis of
QSAR and combinatorial chemistry methods[10]. However, no P-gp inhibitors have yet been
approved for marketing. Research on P-gp-mediated MDR reversal agents have been done for
almost four decades, no significant progress has been made. Hence, discovery of novel P-gp
inhibitors having high efficiency and safety is extremely urgent.
Figure 1. Structures of P-gp inhibitors

Studies have shown that several tyrosine kinase inhibitors (TKIs) having the structure of
quinazoline can effectively inhibit P-gp activity[12, 13]. Our group have designed,
synthesized and evaluated novel quinazoline P-gp inhibitors. Candidate compound LBM-11
was found possessing favorable activity, EC50 reaches to 57.9±3.5 nM[14]. In this article, we
choose the quinazoline P-gp inhibitor as lead compound, and design structure of
phthalazinone as isostere to replace quinazoline in order to obtain higher activity. Carbonyl
oxygen in designed phthalazinone structure could provide more interaction with P-gp protein.
And designed structure of amide bond is more similar to the third-generation P-gp inhibitor,
so activity is expected to reserve to the maximum extent.
Figure 2. Design of novel P-gp inhibitors
2.Results and discussion
2.1 Chemistry

The synthetic route to prepare phthalazinone derivatives 15-33 is shown in Scheme 1.
Intermediate 8 was prepared from Naphthalene oxidized by KMnO4. The synthesized 8 was
stirred in EtOH with substituted phenyl hydrazine at R.T. for 15h to afford intermediate
9a-s[15]. Under reflux condition, 9a-s reacted with SOCl2 to produce intermediate 10a-s.
3,4-dimethoxyphenylethylamine and paraformaldehyde were mixed in EtOH and stirred at
R.T. for 3h and then refluxed for 4h after acidification with concentrated hydrochloric acid to
afford intermediate 12. Under reflux conditions, intermediate 12 and 4-Nitrophenethyl
bromide were reacted for 17 h to produce compound 13. Compound 13 was reduced under
hydrogen atmosphere with Pd/C as catalyst to give intermediate 14. Add triethylamine to the
mixture of 10a-s and intermediate 14 in dichloromethane and the solution was stirred at R.T.
overnight to produce target compounds 15-33. Structures of all the target compounds are
presented in Table 1.
Scheme 1. Reagents and conditions: (i) 0.5 mol/L NaOH aq, KMnO4, H2O, reflux, 2 h; (ii)
substituted phenyl hydrazine, EtOH, rt, 15 h; (iii) SOCl2, reflux, 6 h; (iv) (CH2O)n, EtOH, r.t.,
3h; con. HCl, reflux, 4 h; (v) 1-(2-bromoethyl)-4-nitrobenzene, K2CO3, CH3CN, reflux, 17 h;
(vi) H2/Pd-C, DCM/EtOH, r.t., 24 h; (vii) 10a-s, Et3N, DCM, rt, overnight.
Table 1. Structures of target compounds

Compound
15
Ar
Compound
22
Ar
Compound
29
Ar
16
23
30
17
18
24
25
31
32
33
19
20
21
26
27
28
2.2 Biological Evaluation
2.2.1 Cytotoxicity assay in vitro
The effects of synthesized compounds on the viability of sensitive K562 cells and its
ADM-resistant K562/A02 cells with overexpressed P-gp were determined preliminarily by
the MTT assay. As present in Table 2, Most of the tested compounds show no cytotoxicity to
two strains of cells. IC50 value is greater than 50 μM. Compound 28 and 29 show weak
cytotoxicity to K562 cells. Positive control Verapamil (VRP) shows certain cytotoxicity to
both K562 and K562/A02 cells.
Table 2. Cytotoxicity of target compounds towards K562 and K562/A02 cells

IC50 (μM)
K562
IC50 (μM)
K562/A02
Compound
Compound
K562/A02
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
K562
>100
>100
>100
>100
83.42 ± 2.40
>100
15
16
17
18
19
20
21
22
23
24
25
26
27
72.19 ± 3.39
43.75 ± 1.35
38.23 ± 2.10
64.15 ± 1.77
>100
>100
28
>100
73.01 ± 3.29
84.81 ± 2.94
>100
29
>100
30
>100
31
>100
>100
>100
32
>100
69.86 ± 4.03
34.96 ± 2.24
4.23 ± 0.36
0.63 ± 0.09
>100
33
78.35 ± 4.42
62.64 ± 2.63
57.22 ± 3.04
VRP
LBM-11
ADM
31.71 ± 2.37
12.30 ± 0.73
67.28 ± 2.69
2.2.2 MDR reversal activity test on K562/A02 cells in vitro
According to the results of cytotoxicity test, all target compounds show no cytotoxicity at
the concentration of 2 μM. So target compounds are tested MDR reversal activity towards
ADM-resistant K562/A02 cells with overexpressed P-gp at such concentration. Results are
presented in Table 3. All target compounds show MDR reversal activity at 2 μM. Most of the
compounds are superior to the control Verapamil. Among them, compound 15, 20, 23, 25, 26
and 28 are far better than Verapamil. These six compounds are tested MDR reversal activity at
concentration of 100 nM. As shown in Table 4, they still possess potent MDR reversal
activity even at a low concentration. Especially for compound 26, Reversal fold (RF) value
reach 20.
Table 3. MDR reversal activity of target compounds on K56/A02 cells at concentration of 2
μM
Compound
IC50/ADM
RF
Compound
IC50/ADM
RF
(2.0 μM)
(μM)
(2.0 μM)
(μM)

2.22 ± 0.37
3.38 ± 0.14
3.06 ± 0.25
5.39 ± 0.96
2.83 ± 0.32
1.89 ± 0.21
3.93 ± 0.64
4.82 ± 0.59
2.10 ± 0.32
12.46 ± 2.78
2.48 ± 0.13
30.31
19.90
21.99
12.48
23.77
35.60
17.12
13.96
32.04
5.40
1.52 ± 0.26
2.43 ± 0.37
2.07 ± 0.12
6.16 ± 0.31
11.81 ± 0.28
13.54 ± 0.56
21.46 ± 2.09
4.23 ± 0.75
14.88 ± 1.50
2.27 ± 0.43
67.28 ± 4.19
44.26
27.69
32.50
10.92
5.70
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
4.97
3.14
32
15.91
4.52
33
VRP (5.0 μM)
LBM-11
0.1% DMSO
29.64
1.00
27.13
IC50 ( ±s, n=3) is determined by MTT assay testing cytotoxicity on K562/A02 cells when
2μM tested compound is combined on the cells with doxorubicin of different concentrations;
Reversal fold (RF) = IC50 of doxorubicin with no reversal agents existing / IC50 of doxorubicin
with reversal agents existing; 0.1% DMSO is blank control.
Table 4. MDR reversal activity of preferred compounds on K56/A02 cells at concentration of
100 nM
Compound
IC50/ADM
(μM)
RF
Compound
(100 nM)
28
IC50/ADM
(μM)
RF
(100 nM)
8.34 ± 0.91
4.39 ± 0.42
7.15 ± 0.24
9.93 ± 1.30
3.26 ± 0.13
7.75
14.72
9.04
5.36 ± 0.37
15.37 ± 0.84
4.20 ± 0.25
64.61 ± 3.41
12.05
4.20
15
20
23
25
26
VRP (5.0 μM)
LBM-11
15.39
1.00
6.51
0.1% DMSO
19.82
IC50 ( ±s, n=3) is determined by MTT assay testing cytotoxicity on K562/A02 cells when
2μM tested compound is combined on the cells with doxorubicin of different concentrations;
Reversal fold (RF) = IC50 of doxorubicin with no reversal agents existing / IC50 of doxorubicin
with reversal agents existing; 0.1% DMSO is blank control.

2.2.3 Dose-effect relationship research of MDR reversal activity of compound 26
As can be seen from the test results presented in the Figure 3, compound 26 still show
weak reversal activity at the concentration of 7.8 nM. EC50 refers to the
concentration of inhibitor required to reduce the IC50 of doxorubicin by half compared with a
control without a inhibitor[16]. According to reversal activity data at different concentration,
EC50 was calculated as 46.2 ± 4.7 nM.
Figure 3. EC50 value of 26 in reversing ADM-resistance in K562/A02 cells. The percent of
IC50 of ADM = [(IC50 of ADM at each modulator concentration / IC50 of ADM without
modulator) × 100%)]. EC50 refers to the concentration of modulator that can reduce the % of
IC50 of ADM by half. Each data point is presented as mean ± SD of three independent tests
2.2.4 Selective index of compound 26
As shown in the Figure 4, compound 26 shows no cytotoxicity to GES-1 cells (normal
cell). Even at the concentration of 100 μM, survival rate of GES-1 is still higher than 80%.
After calculation, selective index is greater than 2165, indicating that compound 26 is also
safe for normal cells when it plays the role of MDR reversing.

Figure 4. The survival rate of different concentrations of 26 was determined in GES-1 cells.
Data were analyzed with GraphPad Prism 5.0 software and presented as mean ± SD for three
independent tests.
2.2.5 MDR reversal activity test on other P-gp-mediated anticancer-drug-resistant cells
As shown in the Figure 5, compound 26 can obviously enhance cytotoxicity of
Paclitaxel (PTX), Vinblastine (VLB), Daunorubicin (DNR) to drug-resistant cells. PTX, VLB
and DNR are easily pumped by P-gp out of cancer cells. When compound 26 was combined
with these anticancer drugs, it can increase their concentration in cancer cells so cytotoxicity
was significantly increased. However, Cyclophosphamide (CTX) is not substrate of P-gp,
combined compound 26 cannot influence its concentration in cells. These results indicate that
inhibition of P-gp function by compound 26 is the cause of drug resistance reversal.

Figure 5 MDR reversal activity of 26 on P-gp-mediated resistance to other anticancer agents.
DNR, PTX, and VLB are P-gp substrates, whereas CTX is not. Each data point is presented as
mean ± SD of three independent tests
2.2.6 Effect of compound 26 on Doxorubicin accumulation
Effect of compound 26 on doxorubicin accumulation in drug-resistant cells can been
seen in Figure 6. Doxorubicin accumulation is significantly more in doxorubicin-sensitive
K562 cells than in doxorubicin-resistant K562/A02 cells, it is about 5.9 folds. Compared with
blank control, in different experiment group with different concentration of reversal agents
added, doxorubicin accumulation in K562/A02 cells increased to different degrees.
Compound 26 can significantly increase doxorubicin accumulation in doxorubicin-resistant
K562/A02 cells even at the low concentration of 0.1 μM, while Verapamil increases
doxorubicin accumulation only at the high concentration of 2.0 μM. These results indicates
that compound 26 inhibits the transport function of P-gp, causing significant doxorubicin
accumulation in drug-resistant cells.

Figure 6. Effect of compound 26 on intracellular doxorubicin accumulation in K562/A02
cells. 0.1% DMSO was used as negative control. VRP was chosen as positive controls. N = 3
independent experiments. The results are presented as the mean ± standard error of mean: (**)
P < 0.01, (***) P < 0.001 relative to the negative control (K562/A02).
2.2.7 Structure-activity relationship research of the target compounds 15-33
Based on reversal activity results, structure-activity relationship is analyzed as below
(Figure 7): Structure of component A is common in third-generation P-gp inhibitors, which
plays a crucial role as important pharmacophore; When phthalazinone substitutes quinazoline
in component B, reversal activity of compounds is enhanced on the whole; When component
C is phenyl ring, number and position of substituents on the ring have a significant influence
on the reversal activity. When substituted at 3-positon of the phenyl ring, reversal activity is
kept or slightly reduced. Activity is significantly enhanced when substituted at 4-position, and
becomes strongest with 4-Cl substituted. Multiple substituents reduce activity and such
reduction is obvious when substituents are electron withdrawing group.

Figure 7 Structure-activity relationship of target compounds:(A) Crucial pharmacophore of
third-generation P-gp inhibitors; (B) Phthalazinone enhances reversal activity; (C) Different
substituents influence activity significantly.
2.2.8 Docking experiments for compound 26
Docking experiments have been conducted to study interaction between P-gp protein and
Compound 26 (Figure 8). Results show that Compound 26 occupied hydrophobic pocket of
P-gp protein. Compound 26 has H-bond interaction with residue GLN 721, and Pi-Pi stacking
interaction with TYR 303. More importantly, tertiary amine N atom of Compound 26 could
capture hydrogen ion, forming positive charge center, to interact with residue PHE 728, which
is essential to maintain activity.
A

B
Figure 8 Structure docking results with P-gp for Compound 26. (A) 3D diagram (B) 2D
diagram
3. Conclusion
In this study, a series of novel P-gp-mediated MDR modulators with phthalazinone
scaffolds were designed and synthesized. All the structures of the synthesized compound were
characterized by NMR, HRMS. Biological evaluation in vitro demonstrated that a few
compounds possess potent MDR reversal activity. In particular, compound 26 exhibited high
activity against doxorubicin-resistant K562/A02 cells with a low EC50 (46.2±4.7nM).
Compound 26 show no cytotoxicity towards normal cells (IC50>100μM). Calculated SI of
compound26 was very high (>2165). Compound 26 also exhibited reversal activity on other
anticancer drugs that may have different structures and mechanisms of action such as P-gp
substrates (PTX, VLB, DNR) but exhibited no activity on the non-P-gp substrate CTX.
Compound 26 increased doxorubicin accumulation in drug-resistant cells, which
demonstrates that it can inhibit the transfer function of P-gp.
4. Experimental section
4.1 Material and Methods
4.1.1 Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin
(DOX), daunorubicin (DNR), vinblastine (VLB), paclitaxel (PTX), cyclophosphamide (CTX),

verapamil (VRP) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St.
Louis, MO, USA). RPMI-1640s and FBS were from GIBCO, USA. Human leukemia
sensitive cell line K562 and its doxorubicin-selected P-gp overexpressing daughter cell line
K562/A02 were kindly provided by Professor Bao-An Chen (Department of Hematology, The
Affiliated Zhong-Da Hospital, Southeast University (Nanjing, China). The cell lines were
grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum
(FBS) in a humidified incubator at 37 °C with 5% CO2. To confirm drug resistance
characteristics, 1 mg/mL ADM was added to K562/A02 cultures and maintained in drug-free
medium for 2 weeks before usage. The cells in exponential growth can be used for
experiments.
4.1.2 Methods
4.1.2.1 Cytotoxicity assay
Cell viability was determined by MTT method with a minor modification[17-20]. K562
and K562/A02 cells during logarithmic growth phase were seeded in 96-well microtiter plates
at 3 × 104 cells per well. P-gp inhibitors were prepared as 5 mM DMSO stocks. In the MTT
assay for MDR reversal experiments, cells were incubated in the presence of P-gp inhibitors
for 48 h. MTT dye (10 μL of 2.5 mg/mL in PBS) was added to each well 4 h prior to
experiment termination in a 37 °C incubator containing 5% CO2. The plates were then
centrifuged at 1500 rpm for 15 min and the supernatant was discarded without disturbing the
formazan crystals and cells in the wells, while the MTT formazan crystals were dissolved in
150 μL of DMSO and the plates agitated on a plate shaker for 5 min. The optical density (OD)
was read on a microplate reader (Thermo, USA) with a wavelength of 490 nm. Cell inhibition
rate was calculated. The IC50 values of the compounds for cytotoxicity were calculated by
GraphPad Prism 5.0 software (GraphPad software, San Diego, CA, USA) from the
dose−response curves. Cell inhibition rate=1-(average OD of experimental group/average OD
of control group)*100%.

4.1.2.2 MDR reversal activity test
Method is similar to cytotoxicity assay. K562 and K562/A02 cells during logarithmic
growth phase were seeded in 96-well microtiter plates at 3 × 104 cells per well. P-gp
inhibitors were prepared as 5 mM DMSO stocks. In the MTT assay for MDR reversal
experiments, cells were incubated in the presence of anticancer agents (DOX, DNR, VLB,
PTX, or CTX) with P-gp inhibitors for 48 h. MTT dye (10 μL of 2.5 mg/mL in PBS) was
added to each well 4 h prior to experiment termination in a 37 °C incubator containing 5%
CO2. The plates were then centrifuged at 1500 rpm for 15 min and the supernatant was
discarded without disturbing the formazan crystals and cells in the wells, while the MTT
formazan crystals were dissolved in 150 μL of DMSO and the plates agitated on a plate
shaker for 5 min. The optical density (OD) was read on a microplate reader (Thermo, USA)
with a wavelength of 490 nm. Cell inhibition rate was calculated. The IC50 values of the
compounds for cytotoxicity were calculated by GraphPad Prism 5.0 software (GraphPad
software, San Diego, CA, USA) from the dose−response curves. Cell inhibition
rate=1-(average OD of experimental group/average OD of control group)*100%. Reversal
fold (RF)=IC50A/IC50B. IC50A refers to IC50 of doxorubicin towards K562/A02 cells with no
reversal agents existing. IC50B refers to IC50 of doxorubicin towards K562/A02 cells with
reversal agents existing.
4.1.2.3 Dose-effect relationship research of MDR reversal activity
The test compound was diluted with serum-containing medium to a final concentration
of 5.0 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.31 μM, 0.156 μM, 0.078 μM, 0.04 μM, 0.02 μM
and 0.01 μM. MDR reversal activity of the compound is tested at different concentration of
test compound. Detailed method is same as section 4.1.2.2.
4.1.2.4 Selective index research
IC50 value of preferred compound towards GES-1 cells is determined by MTT method.
Detailed method is same as section 4.1.2.1. Selective index (IS) = IC50A/EC50B. IC50A refers
to IC50 of preferred compound towards GES-1 cells. EC50B refers to the concentration of
preferred compound that can reduce the % of IC50 of ADM by half

4.1.2.5 Doxorubicin accumulation assay in vitro
Reported procedure was employed with minor modification to detect concentration of
doxorubicin in cells[16, 21]. 2 × 105 cells of K562 and K562/A02 were incubated with 20.0
μM DOX and different concentrations of tested compound and VRP for 150 min at 37 °C,
with 0.1% DMSO as a negative control. After incubation, the cells were washed with cold
PBS and lysed with lysis buffer (0.75 M HCl, 0.2% Triton-X100 in 2- propanol). The
fluorescence level of DOX in the lysate was measured by fluorescence spectrophotometer
(RF-5301 PC, Shimadzu) using an excitation and an emission wavelength pair of 460 and 587
nm.
4.1.2.6 Docking experiments
Receptor protein (PDB ID:3G60) was prepared and optimized using relevant module by
Schrodinger software. Receptor Grid Generation Module was used to generate docking area.
Structure of ligands were drawn by ChemOffice2014 software and prepared using LigPrep
module. In the end, molecular docking between ligands and receptor was conducted with
extra precision.
4.2 Apparatus and chemistry
Melting points were taken on a RY-1 melting-point apparatus and were uncorrected;
NMR s1pectra were recorded on a Bruker ACF300/500MHz instrument, tetramethylsilane
(TMS) as internal standard; Purification by column chromatography were carried out over
silica gel (100-200 or 200-300 mesh); Reactions were monitored by thin layer
chromatography on GF/UV254 plates and were visualized using UV light at 254 or 365nm;
ESI-MS data was recorded on Waters ACQUITY UPLC systems with mass(Waters, Milford,
MA). All chemical reagents were obtained from commercial sources and used without
purification unless otherwise indicated.
4.3 Synthesis
4.3.1 Synthesis of 2-(carboxycarbonyl)benzoic acid (8)

Naphthalene (16.0g, 125.0 mmol) was dissolved in 0.5 N NaOH (250 mL) and refluxed.
Potassium permanganate (106 g, 670.0 mmol) was dissolved in 750 mL of boiling water and
added dropwise to the refluxing solution in 1.5 h. after completion of the dropwise addition,
the reaction was continued for 30 minutes to complete oxidation, and the reaction was
quenched by adding 100 mL of ethanol, and the mixture was cooled to room temperature and
suction filtered. The filter cake was acidified with 30% hydrochloric acid, and extracted with
ethyl acetate (75 mL×3). The organic layers were combined, dried with anhydrous sodium
sulfate. Solvent was evaporated under reduced pressure to give compound 8. Yield 35.0%,
white solid, m.p. 142-144°C
4.3.2 Preparation of intermediates 9a-s and 10a-s
Prepare a mixed solution of 30 mL of ethanol and water (1:2), dissolve the corresponding
aromatic hydrazine hydrate (10.30 mmol) in 20ml of the mixed solution and drop into the 10
mL mixed solution of compound 8. The mixture was stirred at room temperature for 15 h.
Precipitation was filtered and washed with dichloromethane twice, then vacuum dried to give
intermediates 9a-s. To the corresponding intermediate 9a-s (10.0 mmol), thionyl chloride (15
mL) was added and the mixture was heated to reflux for 5-7 h. Extra liquid was evaporated
under reduced pressure to give compound 10a-s
4.3.3 Synthesis of 4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]phenylamine
(14)
4.3.3.1 Synthesis of 6,7-Dimethoxy-1,2,3,4-tetrahydro-isoquinoline hydrochloride (12)
To a 250 mL single-necked flask was added 3,4-dimethoxyphenethylamine (11) (18.1 g,
100 mmol), paraformaldehyde (3.6 g, 120 mmol), and anhydrous ethanol (30 mL). After
stirring at room temperature for 3 h, the mixture was adjusted to pH=2 with concentrated
hydrochloric acid, and then refluxed for 4 h. Cooling and filtering to give white crystalline
hydrochloride solid 14.2 g. Yield 61.8%.
4.3.3.2 Synthesis of 6,7-dimethoxy-2-(4-nitrophenethyl)-1,2,3,4-tetrahydroisoquinoline (13)
Mixture of 4-nitrophenethyl bromide (2.42 g, 10 mmol), compound 12 (2.41g, 10.5

mmol) and anhydrous potassium carbonate (3.48 g, 25.2 mmol) were heated and refluxed in
acetonitrile (50 ml) for 18 h. The mixture was cooled, filtered, and the filter cake was washed
with dichloromethane. The filtrate was evaporated under reduced pressure to obtain the
yellow solid residue, recrystallized by ethanol to obtain the yellow acicular solid 2.76 g. Yield
81.2 %
4.3.3.3 Preparation of 14
Compound 13 (11.02g, 32.2mmol) was dissolved in ethanol/dichloromethane (1:1, 60ml)
mixture, and hydrogen reduction reaction was catalyzed by Pd/C(0.58g) at room temperature
for 48 h. Dichloromethane washed the filter cake. The filtrate was evaporated under reduced
pressure to obtain a light yellow solid. Recrystallization by dichloromethane/petroleum ether
resulted in a pale white solid of 7.9 g. Yield 79.0%.
4.3.4 Preparation of target compounds 15-33
Compound 14 (0.31g, 1 mmol), triethylamine (1.2mmol) was dissolved in
dichloromethane (10mL). Under the condition of ice bath, it was added by drops into
dichloromethane solution of corresponding compound 10a-s (1.1 mmol). After addition was
completed, mixture was moved to room temperature and continued to react for 10 h. Reaction
solution was washed with 10% potassium carbonate (20 mL ×3), saturated saline (20 mL ×3),
The organic layer was dried with anhydrous sodium sulfate, then the solvent was removed by
vacuum evaporation to obtain the crude product. Then the column chromatography
CH2Cl2/MeOH (80:1) was used to obtain compound 15-33
4.3.4.1
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3-phenyl-3,4-di
hydrophthalazine-1-carboxamide (15)
Yield 65.9%, light yellow solid, m.p. 170-172°C. 1H NMR (300 MHz, DMSO-d6) δ: 10.61 (s,
1H), 8.52 – 8.25 (m, 2H), 8.13 – 7.89 (m, 2H), 7.77 (d, J = 7.4 Hz, 2H), 7.68 (d, J = 8.1 Hz,
2H), 7.55 (t, J = 7.4 Hz, 2H), 7.47 (d, J = 7.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 2H), 6.64 (d, J =
6.5 Hz, 2H), 3.70 (s, 6H), 3.54 (s, 2H), 2.82 (s, 2H), 2.69 (s, 6H) ; 13C NMR (75 MHz,

DMSO-d6) δ: 161.45, 141.26, 140.42, 136.56, 136.10, 134.16, 132.61, 128.94, 128.57, 127.87,
126.84, 126.31, 126.08, 125.88, 120.23, 111.76, 109.96, 59.46, 55.43, 55.05, 50.53, 32.42,
28.28; ESI-MS m/z: 561.6 [M+H]+. Anal. Calcd for C34H32N4O4: C, 72.84; H, 5.75; N, 9.99.
Found: C, 72.82; H, 5.77; N,9.98
4.3.4.2
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3-(m-tolyl)-3,4-
dihydrophthalazine-1-carboxamide (16)
Yield 70.3%, yellow solid, m.p. 112-114°C. 1H NMR (300 MHz, DMSO-d6) δ: 10.64 (s, 1H),
8.51 – 8.28 (m, 2H), 8.07 – 7.89 (m, 2H), 7.68 (d, J = 7.6 Hz, 2H), 7.55 (d, J = 9.0 Hz, 2H),
7.43 (s, 2H), 7.27 (d, J = 7.5 Hz, 2H), 6.64 (d, J = 5.8 Hz, 2H), 3.70 (s, 6H), 3.54 (s, 2H), 2.78
(d, J = 19.8 Hz, 2H), 2.69 (s, 6H), 2.40 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ: 161.48,
158.06, 147.10, 146.71, 141.21, 140.39, 138.10, 136.55, 136.13, 134.11, 132.57, 128.93,
128.51, 128.39, 127.91, 126.79, 126.42, 125.89, 123.26, 120.23, 111.75, 109.96, 59.47, 55.43,
55.04, 50.52, 32.43, 28.28, 20.86; ESI-MS m/z: 575.6 [M+H]+. Anal. Calcd for C35H34N4O4:
C, 73.15; H, 5.96; N, 9.75. Found: C, 73.16; H, 5.98; N,9.73
4.3.4.3
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3-(p-tolyl)-3,4-d
ihydrophthalazine-1-carboxamide（17）
Yield 54.8%, yellow solid, m.p. 145-147°C.1H NMR (300 MHz, DMSO-d6) δ: δ 10.60 (s,
1H), 8.41 (t, J = 6.9 Hz, 2H), 8.08 – 7.92 (m, 2H), 7.66 (dd, J = 8.3, 7.6 Hz, 4H), 7.36 -7.26
(m, 4H), 6.64 (d, J = 4.0 Hz, 2H), 3.70 (s, 6H), 3.55 (s, 2H), 2.80 (s, 2H), 2.70 (s, 6H), 2.40 (s,
3H)；13C NMR (75 MHz, DMSO-d6) δ:161.49, 158.13, 147.05, 146.81, 141.18, 140.37,
138.11, 136.56, 136.09, 134.10, 132.57, 128.93, 128.53, 128.39, 127.88, 126.85, 126.76,
126.55, 125.87, 123.25, 120.22, 111.68, 109.90, 59.46, 55.40, 55.02, 50.50, 32.39, 28.25,
20.85; ESI-MS m/z: 575.6 [M+H]+. Anal. Calcd for C35H34N4O4: C, 73.15; H, 5.96; N, 9.75.
Found: C, 73.17; H, 5.97; N,9.77
4.3.4.4

N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(2-ethylphenyl)-4-ox
o-3,4-dihydrophthalazine-1-carboxamide (18)
Yield 56.8%, yellow solid, m.p. 119-121°C.1H NMR (300 MHz, DMSO-d6) δ: 10.50 (s, 1H),
8.39 (s, 2H), 8.04 (d, J = 7.3 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.46 (s, 4H), 7.27 (s, 2H), 6.64
(d, J = 6.5 Hz, 2H), 3.70 (s, 6H), 3.54 (s, 2H), 2.75 (d, J = 32.8 Hz, 8H), 1.23 (s, 3H), 1.11 (d,
13
J = 7.3 Hz, 2H); C NMR (75 MHz, DMSO-d6) δ: 161.11, 158.90, 157.67, 150.55, 147.11,
146.87, 137.21 , 135.66, 132.98, 130.87, 129.50, 128.58, 127.85, 126.66, 125.92, 125.31,
122.93, 122.00, 113.69, 111.74, 109.97, 59.53, 55.45, 55.09, 50.52, 32.24, 28.16, 13.67;
ESI-MS m/z: 589.7 [M+H]+. Anal. Calcd for C36H36N4O4: C, 73.45; H, 6.16; N, 9.52 Found:
C, 73.44; H, 6.15; N,9.53
4.3.4.5
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(3-methoxyphenyl)-4
-oxo-3,4-dihydrophthalazine-1-carboxamide (19)
Yield 54.8%, yellow solid, m.p. 117-119°C.1H NMR (300 MHz, DMSO-d6) δ: 10.65 (s, 1H),
8.40 (s, 2H), 8.02 (d, J = 9.2 Hz, 2H), 7.68 (d, J = 7.9 Hz, 2H), 7.44 (d, J = 8.0 Hz, 1H), 7.41
– 7.17 (m, 4H), 7.04 (d, J = 7.7 Hz, 1H), 6.64 (d, J = 6.1 Hz, 2H), 3.82 (s, 3H), 3.69 (s, 6H),
3.54 (s, 2H), 2.82 (s, 2H), 2.69 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ: 161.45, 159.34,
158.29, 158.12, 146.99, 144.66, 146.13, 142.30, 140.10, 136.54, 136.10, 134.04, 132.57,
129.29, 128.94, 127.95, 126.84, 126.63, 126.29, 125.89, 120.21, 118.39, 114.44, 113.52,
112.17, 111.75, 109.96, 59.51, 55.44, 55.07, 50.53, 32.43, 28.29, 20.64; ESI-MS m/z: 591.6
[M+H]+. Anal. Calcd for C35H34N4O5: C, 71.17; H, 5.80; N, 9.49. Found: C, 71.16; H,5.83;
N,9.52
4.3.4.6
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(4-methoxyphenyl)-4
-oxo-3,4-dihydrophthalazine-1-carboxamide (20)
Yield 61.7%, light brown solid, m.p. 128-130°C.1H NMR (300 MHz, DMSO-d6) δ: 10.59 (s,
1H), 8.40 (t, J = 7.7 Hz, 2H), 8.07 – 7.90 (m, 2H), 7.68 (d, J = 8.8 Hz, 4H), 7.27 (d, J = 8.2
Hz, 2H), 7.08 (d, J = 8.9 Hz, 2H), 6.64 (d, J = 6.6 Hz, 2H), 3.83 (s, 3H), 3.70 (s, 6H), 3.54 (s,

2H), 2.80 (d, J = 7.4 Hz, 2H), 2.69 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ: 161.53, 158.59,
158.18, 147.16, 146.90, 140.11, 136.41, 136.15, 134.27, 133.99, 132.47, 128.92, 127.89,
127.34, 126.89, 126.76, 126.41, 126.22, 125.83, 120.22, 113.68, 111.78, 109.99, 59.30, 55.42,
54.90, 50.43, 32.29, 28.12; ESI-MS m/z: 591.3 [M+H]+. Anal. Calcd for C35H34N4O5: C,
71.17; H, 5.80; N, 9.49. Found: C, 71.17; H,5.81; N,9.51
4.3.4.7
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(2-fluorophenyl)-4-o
xo-3,4-dihydrophthalazine-1-carboxamide (21)
Yield 69.2%, light yellow solid, m.p. 187-189°C.1H NMR (300 MHz, DMSO-d6) δ: 10.65 (s,
1H), 8.49 – 8.31 (m, 2H), 8.12 – 7.96 (m, 2H), 7.77 (s, 1H), 7.67 (d, J = 7.1 Hz, 2H), 7.58 (s,
1H), 7.52 – 7.36 (m, 2H), 7.27 (d, J = 7.5 Hz, 2H), 6.65 (d, J = 6.0 Hz, 2H), 3.70 (s, 6H), 3.54
(s, 2H), 2.81 (s, 2H), 2.69 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ: 161.04, 159.68, 146.77,
146.36, 140.81, 136.47, 136.03, 134.47, 130.70, 129.55, 128.93, 127.34, 126.82, 126.63,
125.89, 125.02, 120.24, 116.20, 115.87, 111.76, 109.97, 109.79, 108.13, 59.45, 58.65, 55.44,
55.05, 50.52, 32.42, 28.06; ESI-MS m/z: 579.6 [M+H]+. Anal. Calcd for C34H31FN4O4: C,
70.57; H, 5.40; N, 9.68. Found: C, 70.56; H,5.42; N,9.69
4.3.4.8
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(3-fluorophenyl)-4-o
xo-3,4-dihydrophthalazine-1-carboxamide（22）
Yield 55.8%, yellow solid, m.p. 108-110°C.1H NMR (300 MHz, DMSO-d6) δ: 10.68 (s, 1H),
8.42 (t, J = 6.3 Hz, 2H), 8.07 – 7.97 (m, 2H), 7.77 – 7.68 (m, 4H), 7.31 (d, J = 7.9 Hz, 4H),
13
6.72 (d, J = 9.0 Hz, 2H), 3.93 (s, 2H), 3.72 (s, 6H), 2.99 (s, 6H), 2.87 (s, 2H); C NMR (75
MHz, DMSO-d6) δ: 161.46, 159.96,147.46, 147.10, 139.97, 137.10, 135.01, 133.63, 133.29,
129.93, 128.56, 128.12, 126.37, 125.15, 124.54, 123.14, 122.46, 122.25, 114.18, 111.68,
109.87, 58.5, 55.44, 54.05, 50.07, 32.43, 28.19; ESI-MS m/z: 579.6 [M+H]+. Anal. Calcd for
C34H31FN4O4: C, 70.57; H, 5.40; N, 9.68. Found: C, 70.56; H,5.39; N,9.67
4.3.4.9
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(4-fluorophenyl)-4-o

xo-3,4-dihydrophthalazine-1-carboxamide (23)
Yield 69.2%, light yellow solid, m.p. 166-168°C.1H NMR (300 MHz, DMSO-d6) δ: 10.60 (s,
1H), 8.41 (s, 2H), 8.09 – 7.94 (m, 2H), 7.83 (s, 2H), 7.68 (d, J = 6.9 Hz, 2H), 7.39 (t, J = 7.1
Hz, 2H), 7.28 (d, J = 6.9 Hz, 2H), 6.65 (d, J = 5.3 Hz, 2H), 3.70 (d, J = 1.2 Hz, 6H), 3.54 (s,
2H), 2.82 (s, 2H), 2.70 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ: 162.55, 161.37, 159.32,
158.16, 147.11, 146.87, 140.35, 137.52, 136.59, 136.07, 134.17, 132.62, 128.94, 128.32,
128.20, 127.88, 126.92, 126.82, 126.64, 126.37, 125.90, 120.24, 115.51, 115.20, 111.76,
109.96, 59.46, 55.43, 55.06, 50.52, 32.43, 28.29; ESI-MS m/z: 579.7 [M-H]+. Anal. Calcd for
C34H31FN4O4: C, 70.57; H, 5.40; N, 9.68. Found: C, 70.55; H,5.41; N,9.67
4.3.4.10
3-(2-bromophenyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-
oxo-3,4-dihydrophthalazine-1-carboxamide (24)
Yield 61.6%, yellow solid, m.p. 140-142°C.1H NMR (300 MHz, DMSO-d6 δ:10.60 (s, 1H),
8.24 (d, J = 8.1 Hz, 2H), 7.98-7.92 (m, 2H), 7.71 (d, J = 6.8 Hz, 2H), 7.69-7.62 (m, 4H), 7.21
(d, J = 8.0 Hz, 2H), 6.67 (d, J = 9.0 Hz, 2H), 3.70 (s, 6H), 3.54 (s, 2H), 2.81 (s, 2H), 2.69 (s,
6H); 13C NMR (75 MHz, DMSO-d6) δ: 161.70, 158.67,147.12, 146.87, 137.23, 137.02,
135.81, 133.09, 130.38, 129.52, 128.58, 128.35, 128.01, 126.58, 125.89, 123.03, 122.15,
121.11, 114.00, 111.69, 109.94, 59.44, 55.44, 55.06 , 50.45, 32.46, 28.27; ESI-MS m/z: 639.6
[M+H]+. Anal. Calcd for C34H31BrN4O4: C, 63.85; H, 4.89; N, 8.76. Found: C, 63.84; H,4.88;
N,8.76
4.3.4.11
3-(3-bromophenyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-
oxo-3,4-dihydrophthalazine-1-carboxamide (25)
Yield 51.0%, yellow solid, m.p. 105-107°C.1H NMR (300 MHz, DMSO-d6) δ:10.62 (s, 1H),
8.41 (t, J = 7.1 Hz, 3H), 8.12 – 7.96 (m, 4H), 7.67 (d, J = 8.4 Hz, 3H), 7.28 (d, J = 8.4 Hz,
2H), 6.65 (d, J = 6.5 Hz, 2H), 3.70 (d, J = 0.9 Hz, 6H), 3.54 (s, 2H), 2.81 (d, J = 7.1 Hz, 2H),
2.70 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ: 161.72, 159.46, 147.13, 146.89, 144.33,
136.83, 136.03, 133.31, 128.79, 128.64, 128.26, 126.63, 125.91, 123.49, 123.03, 122.23,

114.12, 111.75, 109.98, 59.49, 55.46, 55.09, 50.53, 32.54, 28.31; ESI-MS m/z: 639.5 [M+H]+.
Anal. Calcd for C34H31BrN4O4: C, 63.85; H, 4.89; N, 8.76. Found: C, 63.83; H,4.88; N,8.77
4.3.4.12
3-(4-chlorophenyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-
oxo-3,4-dihydrophthalazine-1-carboxamide (26)
Yield 62.8%, yellow solid, m.p.162-164°C .1H NMR (300 MHz, DMSO-d6) δ: 10.63 (s, 1H),
8.41 (t, J = 6.4 Hz, 2H), 8.19 – 7.93 (m, 2H), 7.85 (d, J = 8.2 Hz, 2H), 7.65 (dd, J = 18.7, 8.0
Hz, 4H), 7.27 (d, J = 7.6 Hz, 2H), 6.64 (d, J = 6.4 Hz, 2H), 3.70 (s, 6H), 3.54 (s, 2H), 2.82 (s,
2H), 2.69 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ: 161.33, 158.09, 147.10, 146.86, 140.56,
140.02, 136.60, 136.08, 134.24, 132.67, 132.19, 128.94, 128.53, 127.79, 126.86, 126.63,
126.39, 125.90, 120.24, 111.76, 109.96, 59.47, 55.46, 55.06, 50.52, 32.44, 28.29; ESI-MS m/z:
595.6 [M+H]+. Anal. Calcd for C34H31ClN4O4: C, 68.62; H, 5.25; N, 9.41. Found: C, 68.63;
H,5.24; N,9.41
4.3.4.13
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(4-nitrophenyl)-4-ox
o-3,4-dihydrophthalazine-1-carboxamide (27)
Yield 53.9%, yellow solid, m.p.183-185°C .1H NMR (300 MHz, DMSO-d6) δ: 10.69 (s, 1H),
8.54 – 8.26 (m, 4H), 8.18 (d, J = 8.7 Hz, 2H), 8.07 – 7.82 (m, 2H), 7.69 (d, J = 8.1 Hz, 2H),
7.29 (d, J = 8.0 Hz, 2H), 6.65 (d, J = 6.4 Hz, 2H), 3.70 (s, 6H), 3.54 (s, 2H), 2.80 (s, 2H), 2.70
(s, 6H); 13C NMR (75 MHz, DMSO-d6) δ: 161.17, 158.21, 146.89, 146.22, 141.21, 136.71,
135.99, 134.50, 132.88, 128.94, 127.85, 126.83, 125.93, 123.92, 120.29, 111.81, 110.02,
59.37, 55.50, 55.04, 50.49, 32.41, 28.25; ESI-MS m/z: 606.6 [M+H]+. Anal. Calcd for
C34H31N5O6: C, 67.43; H, 5.16; N, 11.56. Found: C, 67.44; H,5.16; N,11.55
4.3.4.14
3-(4-cyanophenyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-o
xo-3,4-dihydrophthalazine-1-carboxamide (28)
Yield 68.3%, yellow solid, m.p.165-167°C . 1H NMR (300 MHz, DMSO-d6) δ: 10.64 (s, 1H),

8.43 (t, J = 6.2 Hz, 3H), 8.06 (d, J = 7.6 Hz, 4H), 7.67 (d, J = 7.0 Hz, 2H), 7.28 (d, J = 7.6 Hz,
2H), 6.65 (d, J = 6.3 Hz, 2H), 3.70 (s, 6H), 3.56 (s, 2H), 2.83-2.71 (m, 8H); 13 C NMR (75
MHz, DMSO-d6) δ:161.87, 158.10, 147.13, 146.87, 137.11, 135.69, 134.85, 132.98, 130.03,
128.54, 127.93, 126.50, 125.85, 125.68, 123.06, 122.15, 121.08, 115.24, 114.95, 113.91,
111.66, 109.90, 59.36, 55.41, 54.99, 50.36, 32.40, 28.21; ESI-MS m/z: 586.6 [M+H]+. Anal.
Calcd for C35H31N5O4: C, 71.78; H, 5.34; N, 11.96. Found: C, 71.77; H,5.33; N,11.96
4.3.4.15
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-3-(3,4-dimethylphenyl
)-4-oxo-3,4-dihydrophthalazine-1-carboxamide (29)
Yield 60.7%, yellow solid, m.p.133-135°C .1H NMR (300 MHz, DMSO-d6) δ: 10.56 (s, 1H),
8.39 (s, 2H), 7.99 (s, 2H), 7.67 (d, J = 7.4 Hz, 2H), 7.55 – 7.40 (m, 2H), 7.28 (s, 3H), 6.64 (d,
J = 6.3 Hz, 2H), 3.69 (s, 6H), 3.54 (s, 2H), 2.79-2.69 (m, 8H), 2.29 (s, 6H); 13 C NMR (75
MHz, DMSO-d6) δ: 161.54, 158.38, 147.13, 146.89, 139.85, 137.20, 135.70, 132.99, 128.96,
128.58, 127.88, 126.67, 125.93, 125.55, 122.96, 122.00, 113.94, 111.76, 109.99, 59.49, 55.43,
55.10, 50.50, 32.48, 28.31, 20.96, 19.91; ESI-MS m/z: 589.6 [M+H]+. Anal. Calcd for
C36H36N4O4: C, 73.45; H, 6.16; N, 9.52. Found: C, 73.44; H,6.16; N,9.54
4.3.4.16
3-(2,6-difluorophenyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)
-4-oxo-3,4-dihydrophthalazine-1-carboxamide (30)
Yield 62.7%, yellow solid, m.p. 166-168°C.1H NMR (300 MHz, DMSO-d6) δ: 10.62 (s, 1H),
8.41 (d, J = 7.4 Hz, 2H), 8.06 (d, J = 9.0 Hz, 2H), 7.78-7.50 (m, 5H), 7.29 (s, 2H), 6.64 (d, J =
6.3 Hz, 2H), 3.70 (s, 6H), 3.55 (s, 2H), 2.81-2.70 (m, 8H); 13 C NMR (75 MHz, DMSO-d6) δ:
162.18, 159.64, 147.11, 146.86, 137.98 (s), 137.68, 136.84, 136.01, 130.48, 128.60,128.33,
128.02, 126.70, 126.63, 125.98, 125.90, 125.23, 122.15, 112.67, 111.72, 109.95, 59.45, 55.41,
55.08, 50.47, 32.47, 28.30; ESI-MS m/z: 597.6 [M+H]+. Anal. Calcd for C34H30F2N4O4: C,
68.45; H, 5.07; N, 9.39. Found: C,68.44; H,5.06; N,9.39
4.3.4.17

3-(3,4-dichlorophenyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)
-4-oxo-3,4-dihydrophthalazine-1-carboxamide (31)
Yield 46.7%, yellow solid, m.p. 147-149°C.1H NMR (300 MHz, DMSO-d6) δ: 10.77 (s, 1H),
8.42 (d, J = 7.6 Hz, 2H), 8.11 – 7.93 (m, 3H), 7.89 – 7.79 (m, 2H), 7.69 (d, J = 8.3 Hz, 2H),
7.28 (d, J = 8.2 Hz, 2H), 6.65 (d, J = 6.3 Hz, 2H), 3.70 (s, 6H), 3.54 (s, 2H), 2.81 (d, J = 7.4
Hz, 2H), 2.70 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ: 161.23, 158.10, 147.11, 146.86,
140.79, 140.71, 136.62, 136.08, 134.32, 132.73, 130.88, 130.38, 128.92, 127.85, 127.80,
126.85, 126.64, 126.48, 126.25, 125.90, 120.33, 111.76, 109.97, 59.43, 55.47, 55.05, 50.50,
32.43, 28.27; ESI-MS m/z: 629.6 [M+H]+. Anal. Calcd for C34H30Cl2N4O4: C, 64.87; H, 4.80;
N, 8.90. Found: C,64.88; H,4.81; N,8.92
4.3.4.18
3-(3,5-bis(trifluoromethyl)phenyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)et
hyl)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide（32）
Yield 58.1%, yellow solid, m.p. 118-120°C .1 H NMR (300 MHz, DMSO-d6) δ: 10.73 (s, 1H),
8.63 (s, 2H), 8.44 (d, J = 5.1 Hz, 2H), 8.23 (s, 1H), 8.13 – 7.89 (m, 2H), 7.69 (d, J = 7.8 Hz,
2H), 7.29 (d, J = 7.4 Hz, 2H), 6.64 (d, J = 5.5 Hz, 2H), 3.70 (s, 6H), 3.54 (s, 2H), 2.82 (d, J =
7.4 Hz, 2H), 2.70 (s, 6H); 13 C NMR (75 MHz, DMSO-d6) δ: 161.95, 158.77, 147.12, 146.88,
137.18, 135.80, 132.99, 130.94, 128.47, 127.87, 126.64, 125.91, 125.32, 122.94, 122.27,
120.93, 113.71, 111.76, 111.28, 110.98, 109.97, 59.64, 55.46, 55.1, 50.56, 32.47, 28.29;
ESI-MS m/z: 697.6 [M+H]+. Anal. Calcd for C36H30F6N4O4: C, 62.07; H, 4.34; N, 8.04.
Found: C,62.06; H,4.33; N,8.03
4.3.4.19
N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3-(pyridin-2-yl)
-3,4-dihydrophthalazine-1-carboxamide (33)
Yield 58.1%, yellow solid, m.p. 147-149°C .1 H NMR (300 MHz, DMSO-d6) δ: 10.68 (s, 1H),
8.67 (s, 1H), 8.37 (dd, J = 7.2, 3.0 Hz 2H), 8.06-8.02 (m, 4 H), 7.78-7.58 (m, 3 H), 7.27 (d, J
= 6.9 Hz, 2 H), 6.67 (d, J = 7.0 Hz, 2H), 3.71 (s, 6 H), 3.59 (s, 2 H), 2.88 (d, J = 8.8 Hz, 2 H),
2.71 (s, 6 H),; 13 C NMR (75 MHz, DMSO-d6) δ:161.86, 158.08, 147.12, 146.88, 145.55,

136.83, 136.11, 133.35, 128.65, 128.29, 126.66, 125.92, 123.08, 122.29, 121.73, 114.42,
111.77, 109.99, 59.48, 55.47, 55.09, 50.52, 32.49, 28.31; ESI-MS m/z: 562.6 [M+H]+. Anal.
Calcd for C33H31N5O4: C, 70.57; H, 5.56; N, 12.47. Found: C,70.56; H,5.55; N,12.47
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
This study was supported by National Natural Science Foundation of China (No.
81872733 and 81803353)
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Graphical abstract