Synthesis and biological evaluation of 2,4-disubstituted phthalazinones as Aurora kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.04.048

A series of 2,4-disubstituted phthalazinones were synthesized and their biological activities, including antiproliferation, inhibition against Aurora kinases and cell cycle effects were evaluated. Among them, N-cyclohexyl-4-((4-(1-methyl-1H-pyrazol-4-yl)-1-oxophthalazin-2(1H)-yl) methyl) benzamide (12c) exhibited the most potent antiproliferation against five carcinoma cell lines (HeLa, A549, HepG2, LoVo and HCT116 cells) with IC₅₀ values in range of 2.2–4.6 μM, while the IC₅₀ value of reference compound VX-680 was 8.5–15.3 μM. Moreover, Aurora kinase assays exhibited that compound 12c was potent inhibitor of AurA and AurB kinase with the IC₅₀ values were 118 ± 8.1 and 80 ± 4.2 nM, respectively. Molecular docking studies indicated that compound 12c forms better interaction with both AurA and AurB. Furthermore, compound 12c induced G2/M cell cycle arrest in HeLa cells by regulating protein levels of cyclinB1 and cdc2. These results suggested that 12c is a promising pan-Aurora kinase inhibitor for the potential treatment of cancer.

Full text of DOI:10.1016/j.bmc.2018.04.048

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 2,4-disubstituted phthalazinones
as Aurora kinase inhibitors
b,c
Wei Wang a, Xiu Feng a, Huan-Xiang Liu a, Shi-Wu Chen a, , Ling Hui
⇑
a School of Pharmacy, Lanzhou University, Lanzhou 730000, China
b Experimental Center of Medicine, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
c Key Laboratory of Stem Cells and Gene Drug of Gansu Province, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,4-disubstituted phthalazinones were synthesized and their biological activities, including
antiproliferation, inhibition against Aurora kinases and cell cycle effects were evaluated. Among them,
N-cyclohexyl-4-((4-(1-methyl-1H-pyrazol-4-yl)-1-oxophthalazin-2(1H)-yl) methyl) benzamide (12c)
exhibited the most potent antiproliferation against five carcinoma cell lines (HeLa, A549, HepG2, LoVo
Received 27 March 2018
Revised 20 April 2018
Accepted 21 April 2018
Available online xxxx
and HCT116 cells) with IC50 values in range of 2.2–4.6
lM, while the IC50 value of reference compound
VX-680 was 8.5–15.3 M. Moreover, Aurora kinase assays exhibited that compound 12c was potent inhi-
l
Keywords:
Aurora kinase
Antitumor
Phthalazinone
Cell cycle
bitor of AurA and AurB kinase with the IC50 values were 118 8.1 and 80 4.2 nM, respectively.
Molecular docking studies indicated that compound 12c forms better interaction with both AurA and
AurB. Furthermore, compound 12c induced G2/M cell cycle arrest in HeLa cells by regulating protein
levels of cyclinB1 and cdc2. These results suggested that 12c is a promising pan-Aurora kinase inhibitor
for the potential treatment of cancer.
Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction
oped including VX-680, AT9283, AZD1152 and AMG900 (Fig. 1).14
VX-680 is the first generation of Aurora kinase inhibitor, which
Mitotic kinases play an essential role in mitosis, Aurora and
other mitotic kinases are often observed over-expression in human
solid and many hematologic cancers.1 As one of serine/threonine
kinases, Aurora kinase family is involved in centrosome matura-
tion, mitotic spindle formation, chromosome segregation and
cytokinesis during mitosis.2,3 The family includes three kinases
designated as Aurora A (AurA), B (AurB) and C (AurC), which pos-
sess a carboxy-terminal catalytic domain and an amino-terminal
regulatory domain4–7However, these kinases have quite different
and nonoverlapping functions in mitotic processes.8 AurA
increases during late G2 to M phase, and it is involved in centro-
some maturation, centrosome separation, bipolar-spindle assem-
bly and other mitotic events9–11 AurB is located at chromosome
17p13.1, and it is essential for chromosome segregation and execu-
tion of cytokinesis.12 AurC is a chromosomal passenger protein,
which appears to have overlapping functions with AurB during
mitotic cell division process.13
blocks cell-cycle progression and induces apoptosis in human
tumor. Crystal structure for VX-680 in complex with Aurora
kinases show that it occupies the ATP-binding site in Aurora
kinases to inhibit catalytic activity.15,16 AT9283 is a benzimidazole
derivative, which suppresses growth and survival in HCT116 cells
and produced the polyploid phenotype by inhibiting AurB kinase.17
AZD1152, a quinazoline derivative, is a selective inhibitor of AurB
kinase and displays striking activity in vivo.18 AMG900 is potent
pan-Aurora kinase inhibitor, which belongs to phthalazine deriva-
tive. It inhibits autophosphorylation of AurA and pHisH3 on Ser10
in vitro and suppresses the growth of human tumor xenografts
in vivo.19
Phthalazinones exhibited broad activities, such as antihyperten-
sive,20 antifungal effect,21 Histamine H1 receptor antagonist22 and
bradykinin B1 receptor antagonist.23 Prime et al. replaced phtha-
lazine with phthalazinone scaffolds and found phthalazinone is
an important pharmacology core for Aurora kinases.24 Compound
1 is a phthalazinone derivative and the crystal structure for 1 in
complex with Aurora kinases show that the amino-pyrazole of 1
can form hydrogen bounds with the hinge region of Aurora A,
and the benzyl of 1 may form hydrophobic interaction with Aurora
kinases.24 However, basis on crystal structure analysis, we consid-
ered that the benzyl of 1 was too small to completely occupy the
Aurora kinases have become a promising anticancer targets
since they are related to oncogenesis and tumor progression, and
many small molecule Aurora kinases inhibitors have been devel-
⇑
Corresponding author.
E-mail address: chenshw@lzu.edu.cn (S.-W. Chen).
https://doi.org/10.1016/j.bmc.2018.04.048
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
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W. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 1. Some Aurora kinase inhibitors in clinical trials.
were characterized by physicochemical and spectral means, and
both analytical and spectral data of all the compounds were in full
agreement with the proposed structures.
2.2. Biology
2.2.1. Antiproliferation activities of target compounds
Fig. 2. The design of target compounds derived from 1.
The antiproliferation activities of target compounds 11a-g, 12a-
g and 13a-c were screened against five human tumor cell lines
(HeLa, A549, HepG2, LoVo and HCT116) by MTT assay after treat-
ment for 72 h, with 1 and VX-680 as reference compounds.28 As
shown in Table 1, some target compounds showed better or equiv-
alent antiproliferation activity in five human tumor cell lines com-
pared with VX-680 and 1. Furthermore, compounds 12a-g with the
1-methyl-1H-pyrazole chain at the C-4 position showed more
potent antiproliferation activity than those of compounds 11a-g
with the 3,5-dimethylisoxazole chain in these five human tumor
cell lines. And compounds 13b-c displayed significantly reduce as
well as 13a showed the slightly decrease compared with 12c in
their vitro antiproliferation activities, suggesting that the 1-
methyl-1H-pyrazole group at the 4-positon of phthalazinone plays
a crucial role in maintaining the antiproliferative activities. In addi-
tion, the antiproliferation activities of compounds 12a-c were suc-
cessively increasing with the increasing of amine substituent size
of 4-methylbenzamide at 2-positon of phthalazinone. Among the
synthetic target compounds, compound 12c showed the strongest
growth-inhibitory activities in the five human tumor cell lines with
the IC50 values were 2.2 0.2, 3.3 0.5, 4.6 0.7, 2.6 0.3 and 3.8
hydrophobic pocket of Aurora kinases, which might accommodate
a larger group. Herein, we designed and synthesized a series of 2,4-
disubstited phthalazinones (Fig. 2) by optimizing N-2 and C-4 frag-
ments as Aurora kinase inhibitors, and evaluated their activities in
human tumor cell lines.
2. Results and discussion
2.1. Chemistry
Intermediates 3a-g were easily obtained through reaction of 4-
(chloromethyl) benzoic acid (2) with corresponding amine, under
1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride
(EDCI) in the presence of N-hydroxybenzotriazole (HOBT) in
dichloromethane at room temperature.25 Simultaneously, treat-
ment of 4-nitrobenzoyl chloride (4) with morpholine or 2-
chloroaniline afforded 5a-b. 5a-b were reduced to provide 4-
amino-benzamides 6a-b (Scheme 1).26
0.3 lM for the HeLa, A549, HepG2, LoVo and HCT116 tumor cell
lines, respectively.
The general synthesis of the initial phthalazinone analogues
starts from phthalic anhydride and is outlined in Scheme 2. First,
hydrazine insertion into phthalic anhydride affords phthalazine-
1,4-dione (7) in quantitative yield. Subsequent dibromination fol-
lowed by monohydrolysis gives the desired bromophthalazinone
(9) in good yield.24 Subsequent treatment of 9 with 3a-g in
dimethylformamide (DMF) in the presence of NaH at room temper-
ature to get intermediates 10a-g. Finally, 10a-g were reacted with
3,5-dimethylisoxazole-4-boronic acid or 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole via palladium-
catalyzed Suzuki coupling reaction to produce target compounds
11a-g or 12a-g. Simultaneously, 10c were reacted with 1-
methyl-1H-pyrazol-4-amine or 6a-b via palladium-catalyzed
Buchwald-Hartwig coupling to produce target compounds 13a-
c.27 The newly synthesized compounds 11a-g, 12a-g and 13a-c
2.2.2. Aurora kinases inhibitory activities of the selected compounds
Next, compounds 12b, 12c, 12f, 12g and 13a were selected to
identify the inhibition activities against Aurora kinases by
Kinase-Glo luminescent kinase assay in vitro,29 with VX-680 as ref-
erence compound. As showed in Table 2, compound 12c exhibited
the most potent inhibition on AurA and AurB with the IC50 values
were 118 8.1 and 80 4.2 nM, respectively. The other com-
pounds showed slightly less inhibition activities against AurA
and AurB. Aurora kinases inhibition results of those compounds
were consistent with antiproliferative activity, suggesting that
those compounds inhibit cancer cell proliferation by targeting Aur-
ora kinases.
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W. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
Scheme 1. Reagents and conditions: (i) amine, EDCI, HOBt, DCM, rt; (ii) amine, triethylamine, CH2Cl2, 0 °C; (iii) NH4Cl, Fe, ethanol: H2O = 4:1, 80 °C.
shown in Fig. 4. For AurA (Fig. 4A), the amine group at C2 of 12c
can form hydrogen bond with Asp287. For AurB (Fig. 4B), com-
pound 12c can form hydrogen bond with Ala173. As shown in
Fig. 4C and D, the N-cyclohexyl-4-methylbenzamide moiety of
12c can better occupy the hydrophobic pocket of AurA and AurB
than that of compound 1. The model would also be helpful for us
to further understand the phthalazinone analogues were potent
Aurora kinases inhibitors.
2.2.5. Compound 12c induces cell cycle arrest in G2/M phase
The Aurora inhibitors including VX-680 often induce cancer
cells cycle arrest in the G2/M phase.36 Therefore, we also investi-
gated the effects of 12c on cell-cycle progression using fluores-
cence-activated cell sorting analysis of propidium iodide-stained
HeLa cells.25 As shown in Fig. 5, treatment with 12c resulted in a
dose-dependent accumulation of cells in the G2/M phase with a
concomitant decrease in the population of G1 phase cells. Exposure
Scheme 2. Reagents and conditions: (i) hydrazine, AcOH, 120 °C; (ii) POBr3, DCE,
reflux; (iii) AcOH, 120 °C; (iv) 3a-g, NaH, DMF, rt; (v) a) 3,5-Dimethylisoxazole-4-
boronic acid (for 11a-g), Pd(dppf)2Cl2, Na2CO3, H2O-dioxane, N2, reflux; b) 1-
Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)-1H-pyrazole (for 12a-g),
Pd(dppf)2Cl2, Na2CO3, H2O-dioxane, N2, reflux; c) 1-methyl-1H-pyrazol-4-amine or
6a-b (for 13a-c from 10c), Pd2dba3, 2-(di-tert-butylphosphino)biphenyl, t-BuOK,
toluene, N2, reflux.
to 0.5 and 5.0 lM of compound 12c for 24 h, the percentages of
cells in G2/M phase arrest were 34.66% and 87.17%, respectively,
compared with 9.63% in untreated cultures.
2.2.3. Compound 12c blocks Aurora kinases phosphorylation in HeLa
cells
AurA is activated by phosphorylation of Thr288, and AurB is
activated by phosphorylation of Thr23230–32 To further identifica-
tion the Aurora kinases were targets of compound 12c, the phos-
phorylation of AurA on Thr288 and AurB on Thr232 were
investigated in HeLa cells treated with various concentrations of
2.2.6. Compound 12c reduces expression of cyclinB1 and cdc2
In eukaryotic cells, progression of cells from the G2 phase to
M phase is triggered by activation of the cyclinB1-dependent
cdc2 kinase.37 As key regulators of the cell cycle, cyclinB1 and
cdc2 are regulated by several complex mechanisms. Therefore,
we used western blot to measure protein expression levels of
cyclinB1 and cdc2 in HeLa cells following treatment with com-
compound 12c (0, 0.5, 2.5 and 5.0 l
M) for 6 h by western blot.33
As shown in Fig. 3, compound 12c obviously decreased phosphory-
lation of AurA on Thr288, and reduced phosphorylation of AurB on
Thr232 in a dose-dependent manner. These results suggested that
compound 12c is a potential Aurora kinases inhibitor although its
potency was slightly weaker than that of VX-680.
pound 12c (0, 0.5, 2.0 and 5.0 lM) for 24 h, VX-680 as reference
compound.38 As shown in Fig. 6, with increasing concentration
of 12c, a marked decrease in cyclinB1 and cdc2 protein levels
compared with the control and VX-680 were observed, suggest-
ing that 12c influenced the protein expression levels of cyclinB1
and cdc2 and hence promote cell-cycle arrest in the G2/M
phase.
2.2.4. Aurora kinases binding model of compound 12c
To gain insight into the interaction of compound 12c with AurA
(PDB code: 3D14) and AurB (PDB code: 4C2V), docking simulation
was performed using Glide module (Glide, version 6.7, Schrödin-
ger, LLC, New York, NY, 2015) of Schrödinger software (Maestro,
version 10.1, Schrödinger, LLC, New York, NY, 2015).34 All the fig-
ures displaying the docking results were obtained using the scien-
tific software Pymol.35 The overview of the binding site of 12c is
3. Conclusions
In summary, we described a series of 2,4-disubstited phthalazi-
nones small molecule inhibitors that showed potent cytotoxicity
against the human tumor cell lines. Furthermore, we specifically
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W. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Table 1
The cytotoxicities of compounds 11a-g, 12a-g and 13a-c.
Compds
NR1R2
R’
(IC50, l
M)a
HeLab
A549b
HepG2b
LoVob
HCT116b
11a
＞100
＞100
＞100
＞100
＞100
11b
11c
11d
11e
11f
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
82.2 4.8
＞100
＞100
86.2 4.3
＞100
97.3 5.6
＞100
＞100
84.4 5.6
＞100
＞100
＞100
＞100
84.9 7.3
36.8 4.3
11g
83.5 4.9
84.3 8.4
85.1 6.4
41.2 7.3
12a
12b
12c
12d
12e
12f
40.0 2.6
16.7 3.7
2.2 0.2
65.5 3.2
＞100
51.2 3.4
32.3 6.2
3.3 0.5
＞100
48.2 5.1
23.7 4.2
2.6 0.3
20.1 5.3
14.3 2.4
3.8 0.3
73.9 5.7
＞100
84.3 6.8
4.6 0.7
73.1 4.5
＞100
67.4 2.7
73.3 5.4
54.3 5.9
37.3 3.6
77.1 7.3
52.3 6.3
83.3 6.4
50.1 6.1
28.5 4.3
27.7 2.2
47.3 3.8
27.4 2.9
69.5 5.3
50.5 4.3
12g
13a
3.2 0.2
6.8 0.6
8.3 0.5
5.3 0.2
5.4 0.3
13b
13c
23.2 2.4
87.3 4.6
64.3 2.5
40.4 5.2
82.4 3.4
54.5 7.3
79.5 4.5
48.2 4.3
＞100
＞100
1
–
–
–
–
6.3 0.6
8.5 2.1
42.6 2.3
15.3 3.2
11.3 0.9
13.7 2.4
22.2 2.2
12.4 1.5
22.7 1.1
10.1 1.3
VX680
a
IC50 values are presented as the means SD of triplicate experiments.
MTT method drug exposure for 72 h.
b
found that compound 12c inhibited Aurora kinases by decreasing
phosphorylation of AurA on Thr288 and AurB on Thr232. Addition-
ally, treatment with compound 12c also resulted G2/M accumula-
tion via the cell-cycle regulators cyclin B1 and cdc2 in HeLa cells.
Taken together, compound 12c is a potential anticancer agent by
targeting Aurora kinase.
4. Experiment
4.1. Chemistry
All starting materials and regents were purchased commercially
and used without further purified, unless otherwise stated. All
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W. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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Table 2
The kinase inhibitions of selected compounds in vitro.
Compds
(IC50, nM)a
Aurora A
Compds
(IC50, nM)a
Aurora A
Aurora B
Aurora B
12b
12c
12f
583 15.3
118 8.1
183 6.1
265 11.5
80 4.2
105 7.3
12g
13a
VX-680
168 12.4
145 7.4
134 13.2
132 6.1
15 1.4
1
0.2
a
The IC50 values are the means of at least two experiments.
Fig. 3. Western blot for inhibition of p-T288, AurA, p-T232 and AurB with various concentrations of compound 12c in HeLa cells for 6 h. The results are expressed as the
mean SD; *P < 0.05, **P < 0.001 versus control.
Fig. 4. The binding mode of 12c with AurA (PDB code: 3D14) and AurB (PDB code: 4C2V). (A) The interaction between 12c and AurA. (B) The interaction between 12c and
AurB. (C) The binding pocket of AurA was shown in surface. (D) The binding pocket of AurB was shown in surface. The surface is colored by gray.
reactions were monitored by thin layer chromatograph (TLC) on
silica gel GF254 (0.25 mm thick). Column chromatography (CC)
was performed on Silica Gel 60 (230–400 mesh, Qingdao Ocean
Chemical Ltd., China). 1H NMR and 13C NMR spectra were recorded
with a Agilent NMR inova 600 spectrometer with TMS as an inter-
nal standard, all chemical shift values are reported as ppm. Mass
spectra were recorded on a Bruker Dalton APEXII49e and Esquire
6000 (ESI-ION TRAP) spectrometer with ESI source as ionization,
respectively. Melting points were determined in Kofler apparatus
and were uncorrected. EPR signals were obtained on a Bruker
A300-9.5/12 spectrometer.
4.1.1. Procedures for synthesis of 3a-g
4.1.1.1. 4-(Chloromethyl)-N-cyclopropylbenzamide (3a). A mixture
of 4-(chloromethyl)benzoic acid (2) (1.0 g, 5.9 mmol), cyclo-
propanamine (0.824 g/mL, 5.9 mmol), EDCI (7.1 mmol) and HOBt
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W. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 5. Effect of 12c on cell cycle progression. (A) Control HeLa cells; (B) HeLa cells treated with 0.5
lM 12c for 24 h; (C) HeLa cells treated with 2.0 lM 12c for 24 h; (D) HeLa
cells treated with 5.0 M 12c for 24 h; (E) HeLa cells treated with 5.0 M VX680 for 24 h; (F) Bar graphs showing compound 12c on cell cycle progression.
l
l
Fig. 6. Western blot analysis of expression of cyclinB1 and cdc2 in HeLa cells treated with 12c (0, 0.5, 2.0 and 5.0 lM) or VX-680 (5.0 lM) for 24 h. The results are expressed
as the mean SD; *P < 0.05, **P < 0.001 versus control.
(7.1 mmol) was stirred in anhydrous DCM (20 mL) for 6 h at room
temperature. After the reaction was finished, the mixture was fil-
tered and filtrate was evaporated in vacuo, the crude product
was further purified by column chromatography on silica gel to
yield compound 3a (92%) as a white solid. 1H NMR (600 MHz,
CDCl3) d 7.73 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 6.24 (s,
1H), 4.60 (s, 2H), 2.90 (q, J = 3.6 Hz, 1H), 0.90–0.86 (m, 2H), 0.64–
0.60 (m, 2H).
(q, J = 4.2 Hz, 1H), 2.04–2.00 (m, 2H), 1.77–1.72 (m, 2H), 1.68–
1.63 (m, 1H), 1.47–1.38 (m, 2H), 1.28–1.18 (m, 3H).
4.1.1.4. (4-(Chloromethyl)phenyl)(piperidin-1-yl)methanone (3d).
With piperidine, a similar procedure as that described for 3a gave
pure 3d (88%) as a white solid. 1H NMR (600 MHz, CDCl3) d 7.42 (d,
J = 7.8 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 4.60 (s, 2H), 3.71 (brs, 2H),
3.33 (brs, 2H), 1.68 (brs, 4H), 1.52 (brs, 2H).
4.1.1.5. (4-(Chloromethyl)phenyl)(morpholino)methanone (3e). With
morpholine, a similar procedure as that described for 3a gave pure
3e (89%) as a white solid. 1H NMR (600 MHz, CDCl3) d 7.44 (d, J =
8.4 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 4.60 (s, 2H), 3.84–3.40 (m, 8H).
4.1.1.2.
4-(Chloromethyl)-N-cyclopentylbenzamide
(3b). With
cyclopentanamine, a similar procedure as that described for 3a
gave pure 3b (90%) as a white solid. 1H NMR (600 MHz, CDCl3) d
7.74 (d, J = 7.8 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 6.24 (t, J = 1.8 Hz,
1H), 4.61 (s, 2H), 2.90 (q, J = 7.2 Hz, 1H), 2.12–2.07 (m, 2H), 1.75–
1.65 (m, 4H), 1.49 (q, J = 6.6 Hz, 2H).
4.1.1.6. 4-(Chloromethyl)-N-(1-methylpiperidin-4-yl)benzamide (3f).
With 1-methylpiperidin-4-amine, a similar procedure as that
described for 3a gave pure 3f (70%) as a white solid. 1H NMR
(600 MHz, DMSO d6) d 8.26 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 7.2 Hz,
2H), 7.50 (d, J = 8.4 Hz, 2H), 4.81 (s, 2H), 3.72 (q, J = 3.6 Hz, 1H),
2.77 (d, J = 10.8 Hz, 2H), 2.17 (s, 3H), 1.96 (t, J = 10.8 Hz, 2H), 1.75
(d, J = 11.4 Hz, 2H), 1.61–1.55 (m, 2H).
4.1.1.3. 4-(Chloromethyl)-N-cyclohexylbenzamide (3c). With cyclo-
hexanamine, a similar procedure as that described for 3a gave pure
3c (93%) as a white solid. 1H NMR (600 MHz, CDCl3) d 7.74 (d, J =
8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 5.96 (s, 1H), 4.60 (s, 2H), 2.90
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4.1.1.7. 4-(Chloromethyl)-N-[(2,2,6,6-tetramethyl-1-oxyl)piperidin-4-
yl]benzamide (3g). With 4-NH2-TEMPO, a similar procedure as that
described for 3a gave pure 3g (90%) as a pink solid. 1H NMR (600
MHz, CDCl3) (3g is a free-radical, some signals appear broadened
and other signals are missing) d 7.77 (brs, 2H), 7.50 (s, 2H), 4.62
(s, 2H). MS (ESI) m/z 325.1 ([M+2H]+).
described for 10a gave pure 10c (68%) as a white solid. 1H NMR
(600 MHz, CDCl3) d 8.42 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 7.8 Hz,
1H), 7.88 (t, J = 7.2 Hz, 1H), 7.82 (t, J = 7.2 Hz, 1H), 7.71 (d, J = 7.8
Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 5.90 (d, J = 7.8 Hz, 1H, NH), 5.40
(s, 2H), 3.95 (t, J = 4.2 Hz, 1H), 2.00 (d, J = 10.2 Hz, 2H), 1.73
(d, J = 13.2 Hz, 2H), 1.64 (d, J = 12.6 Hz, 1H), 1.45–1.37 (m, 2H),
1.26–1.17 (m, 3H).
4.1.2. Procedures for synthesis of 5a-b and 6a-b
4.1.2.1. Morpholino(4-nitrophenyl)methanone (5a). To
a
stirred
4.1.3.4.
4-Bromo-2-(4-(piperidine-1-carbonyl)benzyl)phthalazin-1
solution of 4-nitrobenzoyl chloride (4) (11.8 mmol) and triethy-
lamine (11.8 mmol) in 15 mL dry CH2Cl2 at 0 °C was dropwise
added dichloromethane solution of morpholine (14.2 mmol), and
then the the temperature of the mixture warmed to room temper-
ature naturally. After stirring for 24 h, the reaction mixture was
concentrated under reduced pressure gave the crude product 5a
(91%) as a yellow solid. 1H NMR (600 MHz, CDCl3) d 8.30 (d, J =
7.8 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 3.83–3.38 (m, 8H).
(2H)-one (10d). With 3d, a similar procedure as that described
for 10a gave pure 10d (63%) as a white solid. 1H NMR (600 MHz,
CDCl3) d 8.43 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 7.2 Hz, 1H), 7.88
(t, J = 7.8 Hz, 1H), 7.82 (t, J = 8.4 Hz, 1H), 7.51 (d, J = 7.8 Hz, 2H),
7.36 (d, J = 7.8 Hz, 2H), 5.38 (s, 2H), 3.68 (brs, 2H), 3.31 (brs, 2H),
1.66–1.45 (m, 6H).
4.1.3.5. 4-Bromo-2-(4-(morpholine-4-carbonyl)benzyl)phthalazin-1
(2H)-one (10e). With 3e, a similar procedure as that described
for 10a gave pure 10e (65%) as a white solid. 1H NMR (600 MHz,
CDCl3) d 8.43 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 7.2 Hz, 1H), 7.88
(t, J = 7.2 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 7.8 Hz, 2H),
7.38 (d, J = 7.2 Hz, 2H), 5.39 (s, 2H), 3.76–3.40 (m, 8H).
4.1.2.2.
N-(2-Chlorophenyl)-4-nitrobenzamide
(5b). With
2-
chloroaniline, a similar procedure as that described for 5a gave pure
5b (90%) as a yellow solid. 1H NMR (600 MHz, CDCl3) d 8.52 (d, J = 8.4
Hz, 1H), 8.44 (s, 1H, NH), 8.38 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 8.4 Hz,
2H), 7.45 (d, J = 7.2 Hz, 1H), 7.39–7.35 (m, 1H), 7.17–7.13 (m, 1H).
4.1.3.6.
4-((4-Bromo-1-oxophthalazin-2(1H)-yl)methyl)-N-(1-
4.1.2.3. (4-Aminophenyl)(morpholino)methanone (6a). A solution of
morpholino(4-nitrophenyl)methanone (5a) (6.3 mmol) with NH4Cl
(6.3 mmol) and Fe (31.5 mmol) in 4:1 ethanol:H2O (100 mL) was
heated at 80 °C for 3 h. After the mixture was cooled, the solvent
was evaporated and the residue was purified by column chro-
matography on silica gel to yield compound 6a (82%) as a white
solid. 1H NMR (600 MHz, CDCl3) d 7.26 (d, J = 8.4 Hz, 2H), 6.65 (d,
J = 8.4 Hz, 2H), 3.89 (s, 2H, NH2), 3.70–3.64 (m, 8H).
methylpiperidin-4-yl) benzamide (10f). With 3f, a similar procedure
as that described for 10a gave pure 10f (54%) as a white solid. 1H
NMR (600 MHz, DMSO d6) d 8.33 (d, J = 7.8 Hz, 1H), 8.25 (d,
J = 7.2 Hz, 1H), 8.07 (t, J = 7.2 Hz, 1H), 8.00–7.96 (m, 2H), 7.80 (d,
J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 5.37 (s, 2H), 3.78 (d,
J = 6.0 Hz, 1H), 2.90 (d, J = 6.0 Hz, 2H), 2.29 (s, 3H), 2.20 (brs, 2H),
1.79 (d, J = 11.4 Hz, 2H), 1.66–1.60 (m, 2H).
4.1.3.7. 4-((4-Bromo-1-oxophthalazin-2(1H)-yl)methyl)-N-[(2,2,6,6-
tetramethyl-1-ox yl)piperidin-4-yl]benzamide (10g). With 3g, a sim-
ilar procedure as that described for 10a gave pure 10g (58%) as a
pink solid. 1H NMR (600 MHz, CDCl3) (10g is a free-radical, some
signals appear broadened and other signals are missing) d 8.43
(d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.89 (t, J = 7.8 Hz, 1H),
7.84 (d, J = 7.8 Hz, 1H), 7.74 (brs, 2H), 7.57 (s, 2H), 5.42 (s, 2H).
MS (ESI) m/z 512.2 ([M+H]+).
4.1.2.4. 4-Amino-N-(2-chlorophenyl)benzamide (6b). With com-
pound 5b, a similar procedure as that described for 6a gave pure
6b (80%) as a white solid. 1H NMR (600 MHz, CDCl3) d 8.56
(d, J = 9.0 Hz, 1H), 8.35 (s, 1H, NH), 7.76 (d, J = 8.4 Hz, 2H), 7.40
(d, J = 8.4 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H),
6.73 (d, J = 7.8 Hz, 2H), 4.11 (s, 2H, NH2).
4.1.3. Procedures for synthesis of 10a-g
4.1.3.1.
4-((4-Bromo-1-oxophthalazin-2(1H)-yl)methyl)-N-cyclo-
4.1.4. Procedures for synthesis of 11a-g and 12a-g
propylbenzamide (10a). 4-Bromophthalazin-1(2H)-one (9) (1.3
mmol) and NaH (1.3 mmol) were dissolved in anhydrous DMF
and stirred for 30 min at room temperature, then added 4-(chloro-
methyl)-N-cyclopropylbenzamide (3a) (1.0 mmol) into reaction
mixture. After stirring for 48 h, concentration of the reaction mix-
ture under reduced pressure gave the crude product was further
purified by column chromatography on silica gel to yield com-
pound 10a (62%) as a white solid. 1H NMR (600 MHz, CDCl3) d
8.42 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.88 (t, J = 7.2 Hz,
1H), 7.82 (t, J = 7.2 Hz, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 7.8
Hz, 2H), 6.19 (s, 1H, NH), 5.39 (s, 2H), 2.89–2.87 (m, 1H), 0.87–
0.84 (m, 2H), 0.62–0.57 (m, 2H).
4.1.4.1. N-Cyclopropyl-4-((4-(3,5-dimethylisoxazol-4-yl)-1-oxophtha-
lazin-2(1H)-yl) methyl)benzamide (11a). To stirred solution of 10a
(0.25 mmol) in 1,4-dioxane:H2O (4:1, 10 mL) was added the 3,5-
dimethylisoxazole-4-boronic acid (0.50 mmol), Pd(dppf)2Cl2
(0.005 mmol), Na2CO3 (4 mmol) under nitrogen, followed the mix-
ture was refluxed for 36 h before being cooled to the room temper-
ature. The reaction mixture was filtered and filtrate was
evaporated in vacuo, the crude product was further purified by col-
umn chromatography on silica gel to yield compound 11a (84%) as
a white solid. m.p.: 184–186 °C. 1H NMR (600 MHz, CDCl3) d 8.52
(d, J = 6.6 Hz, 1H), 7.84–7.80 (m, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.51
(d, J = 8.4 Hz, 2H),7.43 (d, J = 6.6 Hz, 1H), 6.20 (s, 1H, NH), 5.47 (s,
2H), 2.89–2.87 (m, 1H), 2.31 (s, 3H), 2.15 (s, 3H), 0.88–0.84 (m,
2H), 0.62–0.57 (m, 2H). 13C NMR (150 MHz, CDCl3) d 168.3 (2C),
159.3, 158.9, 140.2, 138.1, 134.0, 133.4, 132.0, 129.5, 128.8 (2C),
128.3, 127.6, 127.1 (2C), 125.7, 111.1, 54.4, 23.1, 11.9, 10.6, 6.8
(2C). MS (ESI) 415.2 for [M+H]+.
4.1.3.2.
4-((4-Bromo-1-oxophthalazin-2(1H)-yl)methyl)-N-
cyclopentylbenzamide (10b). With 3b, a similar procedure as that
described for 10a gave pure 10b (60%) as a white solid. 1H NMR
(600 MHz, CDCl3) d 8.43 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 7.2 Hz,
1H), 7.88 (t, J = 7.2 Hz, 1H), 7.82 (t, J = 7.2 Hz, 1H), 7.70 (d, J = 7.8
Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 5.98 (d, J = 7.8 Hz, 1H, NH), 5.40
(s, 2H), 4.38 (q, J = 7.2 Hz, 1H), 2.10–2.04 (m, 2H), 1.73–1.63 (m,
4H), 1.60–1.43 (m, 2H).
4.1.4.2. N-Cyclopentyl-4-((4-(3,5-dimethylisoxazol-4-yl)-1-oxophtha-
lazin-2(1H)-yl) methyl)benzamide(11b). With compound 10b,
a
similar procedure as that described for 11a gave pure 11b (80%)
as a white solid. m.p.: 194–196 °C. 1H NMR (600 MHz, CDCl3) d
8.52 (d, J = 7.8 Hz, 1H), 7.84–7.78 (m, 2H), 7.71 (d, J = 7.8 Hz, 2H),
7.52 (d, J = 7.8 Hz, 2H), 7.43 (d, J = 7.8 Hz, 1H), 6.03 (d, J = 7.2 Hz,
4.1.3.3.
4-((4-Bromo-1-oxophthalazin-2(1H)-yl)methyl)-N-cyclo-
similar procedure as that
hexylbenzamide (10c). With 3c,
a
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8
W. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
1H, NH), 5.47 (s, 2H), 4.38 (q, J = 6.6 Hz, 1H), 2.31 (s, 3H), 2.15 (s,
3H), 2.11–2.04 (m, 2H), 1.73–1.62 (m, 4H), 1.50–1.43 (m, 2H). 13C
NMR (150 MHz, CDCl3) d 168.3, 166.6, 159.3, 158.8, 139.9, 138.0,
134.4, 133.4, 132.0, 129.4, 128.8 (2C), 128.2, 127.5, 127.1 (2C),
125.7, 111.0, 54.4, 51.7, 33.2 (2C), 23.8 (2C), 12.0, 10.7. MS (ESI)
443.2 for [M+H]+.
128.9 (2C), 127.8, 127.5, 127.3, 125.5 (2C), 110.7, 54.2, 29.4, 11.9,
10.5. ESR (DMSO): g = 2.007. MS (ESI) 529.3 for [M+H]+.
4.1.4.8.
thalazin-2(1H)-yl)m
N-Cyclopropyl-4-((4-(1-methyl-1H-pyrazol-4-yl)-1-oxoph-
ethyl)benzamide(12a). With 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, a simi-
lar procedure as that described for 11a gave pure 12a (86%) as a
white solid. m.p.: 175–177 °C. 1H NMR (600 MHz, CDCl3) d 8.50
(d, J = 7.2 Hz, 1H), 7.97 (d, J = 7.2 Hz, 1H), 7.83 (s, 1H), 7.82–7.77
(m, 2H), 7.72 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H),7.51 (d, J = 8.4 Hz,
1H), 6.25 (s, 1H, NH), 5.45 (s, 2H), 4.01 (s, 3H), 2.87 (q, J = 3.6 Hz,
1H), 0.87–0.81 (m, 2H), 0.61–0.57 (m, 2H). 13C NMR (150 MHz,
CDCl3) d 168.5, 158.9, 140.5, 140.3, 139.2, 133.8, 133.2, 131.5,
130.3, 129.3, 128.7 (2C), 128.2, 127.4, 127.1 (2C), 126.0, 117.0,
54.4, 39.2, 23.1, 6.8 (2C). MS (ESI) 400.2 for [M+H]+.
4.1.4.3. N-Cyclohexyl-4-((4-(3,5-dimethylisoxazol-4-yl)-1-oxophtha-
lazin-2(1H)-yl)me thyl)benzamide (11c). With compound 10c, a
similar procedure as that described for 11a gave pure 11c (86%)
as a white solid. m.p.: 198–200 °C. 1H NMR (600 MHz, CDCl3) d
8.54–8.50 (m, 1H), 7.84–7.79 (m, 2H), 7.71 (d, J = 7.2 Hz, 2H),
7.52 (d, J = 8.4 Hz, 2H), 7.44–7.42 (m, 1H), 5.93 (d, J = 7.8 Hz, 1H,
NH), 5.47 (s, 2H), 3.95 (t, J = 3.6 Hz, 1H), 2.32 (s, 3H), 2.16 (s, 3H),
2.00 (d, J = 9.6 Hz, 2H), 1.75–1.72 (m, 2H), 1.66–1.63 (m, 1H),
1.45–1.37 (m, 2H), 1.25–1.17 (m, 3H). 13C NMR (150 MHz, CDCl3)
d 168.3, 166.1, 159.3, 158.8, 139.8, 138.0, 134.5, 133.4, 132.0,
129.4, 128.8 (2C), 128.1, 127.5, 127.1 (2C), 125.7, 111.0, 54.5,
48.7, 33.2 (2C), 25.5, 24.8 (2C), 12.0, 10.7. MS (ESI) 457.2 for
[M+H]+.
4.1.4.9.
N-Cyclopentyl-4-((4-(1-methyl-1H-pyrazol-4-yl)-1-oxoph-
thalazin-2(1H)-yl) methyl)benzamide (12b). With compound 10b,
a similar procedure as that described for 12a gave pure 12b
(88%) as a white solid. m.p.: 184–186 °C. 1H NMR (600 MHz, CDCl3)
d 8.51 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 7.2 Hz, 1H), 7.83 (s, 1H),7.82–
7.77 (m, 2H), 7.73 (s, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz,
2H), 6.04 (d, J = 6.6 Hz, 1H, NH), 5.46 (s, 2H), 4.37 (q, J = 7.2 Hz, 1H),
4.01 (s, 3H), 2.09–2.03 (m, 2H), 1.72–1.61 (m, 4H), 1.49–1.43 (m,
2H). 13C NMR (150 MHz, CDCl3) d 166.8, 158.9, 140.3, 140.2,
139.2, 134.2, 133.2, 131.5, 130.3, 129.2, 128.7 (2C), 128.1, 127.4,
127.0 (2C), 125.9, 117.0, 54.4, 51.6, 39.2, 33.2 (2C), 23.7 (2C). MS
(ESI) 428.2 for [M+H]+.
4.1.4.4. 4-(3,5-Dimethylisoxazol-4-yl)-2-(4-(piperidine-1-carbonyl)
benzyl)phthalazin -1(2H)-one(11d). With compound 10d, a similar
procedure as that described for 11a gave pure 11d (78%) as a white
solid. m.p.: 188–190 °C. 1H NMR (600 MHz, CDCl3) d 8.53 (d, J = 7.8
Hz, 1H), 7.84–7.79 (m, 2H), 7.50 (d, J = 7.2 Hz, 2H), 7.43 (t, J = 7.8
Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 5.46 (s, 2H), 3.68 (brs, 2H), 3.30
(brs, 2H), 2.32 (s, 3H), 2.16 (s, 3H), 1.66 (s, 4H), 1.48 (s, 2H). 13C
NMR (150 MHz, CDCl3) d 169.9, 168.3, 159.4, 158.9, 137.9, 137.8,
136.1, 133.4, 132.0, 129.4, 128.8 (2C), 128.2, 127.6, 127.1 (2C),
125.7, 111.1, 54.5, 48.7, 43.1, 26.5, 25.6, 24.5, 12.0, 10.7. MS (ESI)
443.3 for [M+H]+.
4.1.4.10.
N-Cyclohexyl-4-((4-(1-methyl-1H-pyrazol-4-yl)-1-oxoph-
thalazin-2(1H)-yl)m ethyl)benzamide (12c). With compound 10c, a
similar procedure as that described for 12a gave pure 12c (90%)
as a white solid. m.p.: 192–194 °C. 1H NMR (600 MHz, CDCl3) d
8.51 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.83 (s, 1H), 7.81–
7.77 (m, 2H), 7.73 (s, 1H), 7.70 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.8
Hz, 2H), 5.91 (d, J = 6.6 Hz, 1H, NH), 5.46 (s, 2H), 4.01 (s, 3H),
3.96–3.94 (m, 1H), 1.99 (brs, 2H), 1.75–1.71 (m, 2H), 1.66–1.62
(m, 2H), 1.45–1.37 (m, 2H), 1.27–1.18 (m, 2H). 13C NMR (150
MHz, CDCl3) d 166.3, 158.9, 140.3, 140.2, 140.1, 139.2, 134.4,
133.1, 130.3, 129.2, 128.7 (2C), 128.1, 127.4, 127.0 (2C), 125.9,
117.0, 54.5, 48.6, 39.2, 33.1, 25.5 (2C), 24.9, 24.8. MS (ESI) 442.2
for [M+H]+.
4.1.4.5. 4-(3,5-Dimethylisoxazol-4-yl)-2-(4-(morpholine-4-carbonyl)
benzyl)phthalazin -1(2H)-one(11e). With compound 10e, a similar
procedure as that described for 11a gave pure 11e (79%) as a white
solid. Yield 79%. m.p.: 137–139 °C. 1H NMR (600 MHz, CDCl3) d
8.51 (d, J = 6.0 Hz, 1H), 7.83–7.78 (m, 2H), 7.52 (d, J = 8.4 Hz, 2H),
7.43 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.2 Hz, 2H), 5.45 (s, 2H), 3.76–
3.40 (m, 8H), 2.32 (s, 3H), 2.15 (s, 3H). 13C NMR (150 MHz, CDCl3)
d 169.7, 168.3, 159.3, 158.9, 138.5, 138.0, 134.8, 133.4, 132.0,
129.4, 128.9 (2C), 128.2, 127.5, 127.4 (2C), 125.7, 111.0, 66.8
(2C), 54.5, 48.6, 42.6, 12.0, 10.7. MS (ESI) 445.2 for [M+H]+.
4.1.4.11. 4-(1-Methyl-1H-pyrazol-4-yl)-2-(4-(piperidine-1-carbonyl)
benzyl)phthalazin -1(2H)-one(12d). With compound 10d, a similar
procedure as that described for 12a gave pure 12d (82%) as a white
solid. m.p.: 178–180 °C. 1H NMR (600 MHz, CDCl3) d 8.52 (d, J = 6.6
Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.84 (s, 1H), 7.83–7.79 (m, 2H), 7.75
(s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 2H), 5.45 (s, 2H),
4.02 (s, 3H), 3.68 (brs, 2H), 3.31 (brs, 2H), 1.65 (brs, 2H), 1.62 (brs,
2H), 1.47 (brs, 2H). 13C NMR (150 MHz, CDCl3) d 170.0, 158.9,
140.2, 139.2, 138.2, 135.8, 133.1, 131.5, 130.3, 129.3, 128.6 (2C),
128.2, 127.4, 127.1 (2C), 125.9, 117.1, 54.5, 48.7, 43.1, 39.2, 26.5,
25.6, 24.6. MS (ESI) 428.3 for [M+H]+.
4.1.4.6. 4-((4-(3,5-Dimethylisoxazol-4-yl)-1-oxophthalazin-2(1H)-yl)
methyl)-N-(1-met hylpiperidin-4-yl)benzamide (11f). With com-
pound 10f, a similar procedure as that described for 11a gave pure
11f (75%) as a white solid. m.p.: 207–209 °C. 1H NMR (600 MHz,
DMSO d6) d 8.46 (brs, 1H, NH), 8.37–8.35 (m, 1H), 7.93–7.90 (m,
2H), 7.82 (d, J = 7.8 Hz, 2H), 7.58–7.56 (m, 1H), 7.40 (d, J = 7.8 Hz,
2H), 5.42 (s, 2H), 3.96 (brs, 1H), 2.97 (brs, 2H), 2.64 (s, 3H), 2.30
(s, 3H), 2.07 (s, 3H), 1.94–1.84 (m, 4H), 1.22–1.18 (m, 2H). 13C
NMR (150 MHz, DMSO d6) d 168.3, 165.6, 159.0, 158.1, 140.3,
137.4, 134.0, 133.5, 132.4, 128.9, 127.6 (2C), 127.5, 127.4, 126.6
(2C), 126.3, 110.6, 53.6, 52.5, 44.1, 42.4, 29.0, 28.6, 26.5, 11.6,
10.1. MS (ESI) 472.3 for [M+H]+.
4.1.4.12.
4-(1-Methyl-1H-pyrazol-4-yl)-2-(4-(morpholine-4-car-
4.1.4.7. 4-((4-(3,5-Dimethylisoxazol-4-yl)-1-oxophthalazin-2(1H)-yl)
bonyl)benzyl)phthalazi n-1(2H)-one (12e). With compound 10e, a
similar procedure as that described for 12a gave pure 12e (81%)
as a white solid. m.p.: 170–172 °C. 1H NMR (600 MHz, CDCl3) d
8.51 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.85 (s, 1H), 7.80–
7.78 (m, 2H), 7.75 (s, 1H), 7.53 (d, J = 7.8 Hz, 2H), 7.36 (d, J = 8.4
Hz, 2H), 5.45 (s, 2H), 4.02 (s, 3H), 3.74–3.40 (m, 8H). 13C NMR
(150 MHz, CDCl3) d 170.2, 158.9, 140.2, 139.2, 138.8, 134.6,
133.2, 131.5, 130.3, 129.3, 128.7 (2C), 128.2, 127.4 (3C), 125.9,
117.0, 66.8 (2C), 54.6, 48.2, 42.7, 39.3. MS (ESI) 430.2 for [M+H]+.
methyl)-N-[(2,2,
6,6-tetramethyl-1-oxyl)piperidin-4-yl]benzamide
(11g). With compound 10g, a similar procedure as that described
for 11a gave pure 11g (78%) as a pink solid. m.p.: 164–166 °C. 1H
NMR (600 MHz, CDCl3) (compound 11g is a free-radical, some sig-
nals appear broadened and other signals are missing) d 8.53 (brs,
1H), 7.84–7.74 (m, 3H), 7.59 (brs, 2H), 7.47 (brs, 1H), 7.18 (s, 1H),
5.49 (s, 2H), 2.33 (s, 3H), 2.16 (s, 3H). 13C NMR (150 MHz, CDCl3)
d 168.0, 166.5, 159.0, 158.6, 140.2, 137.8, 133.3, 131.9, 129.1,
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W. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
4.1.4.13. 4-((4-(1-Methyl-1H-pyrazol-4-yl)-1-oxophthalazin-2(1H)-
yl)methyl)-N-(1-m ethylpiperidin-4-yl)benzamide (12f). With com-
pound 10f, a similar procedure as that described for 12a gave pure
12f (80%) as a white solid. m.p.: 200–202 °C. 1H NMR (600 MHz,
DMSO d6) d 8.37 (d, J = 6.6 Hz, 1H), 8.25 (d, J = 7.2 Hz, 1H), 8.24
(s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.99–7.90 (m, 2H), 7.83 (s, 1H),
7.80 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 5.40 (s, 2H), 3.94
(s, 3H), 3.78–3.74 (m, 1H), 2.86 (d, J = 11.4 Hz, 2H), 2.25 (s, 3H),
2.14 (brs, 2H), 1.77 (d, J = 10.8 Hz, 2H), 1.66–1.58 (m, 2H). 13C
NMR (150 MHz, DMSO d6) d 172.4, 165.5, 157.9, 140.2, 139.8,
138.5, 133.8, 132.0, 131.2, 128.5 (3C), 127.5, 127.4, 126.6 (2C),
126.3, 115.8, 53.9. 53.6, 45.9, 45.2, 38.7, 30.8 (2C), 21.2. MS (ESI)
457.3 for [M+H]+.
MHz, DMSO d6) d 9.77 (s, 1H), 9.09 (s, 1H), 8.39 (d, J = 8.4 Hz,
1H), 8.36 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 7.8 Hz, 1H), 8.02 (t, J = 7.2
Hz, 1H), 7.92 (t, J = 7.2 Hz, 1H), 7.88 (d, J = 9.0 Hz, 2H), 7.81 (d, J
= 8.4 Hz, 2H), 7.67 (d, J = 9.0 Hz, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.53
(dd, J = 7.8, 1.2 Hz, 1H), 7.45 (d, J = 7.8 Hz, 2H), 7.36 (td, J = 7.8,
1.2 Hz, 1H), 7.26 (td, J = 7.8, 1.2 Hz, 1H), 5.32 (s, 2H), 3.70 (t, J =
3.6 Hz, 1H), 1.77 (d, J = 7.2 Hz, 2H), 1.67 (d, J = 4.8 Hz, 2H), 1.55
(d, J = 11.4 Hz, 1H), 1.29–1.20 (m, 4H), 1.08–1.05 (m, 1H). 13C
NMR (150 MHz, DMSO d6) d 165.1, 164.8, 156.9, 144.6, 140.9,
140.4, 135.3, 134.1, 133.2, 132.1, 129.4, 129.1, 128.5, 128.1, 127.9
(2C), 127.5 (2C), 127.4 (2C), 127.0 (2C), 126.9, 125.8, 124.9,
123.6, 117.6 (2C), 52.7, 48.3, 32.3 (2C), 25.2, 24.9 (2C). MS (ESI)
606.3 for [M+H]+.
4.1.4.14. 4-((4-(1-Methyl-1H-pyrazol-4-yl)-1-oxophthalazin-2(1H)-
yl)methyl)-N-[(2,2, 6,6-tetramethyl-1-oxyl)piperidin-4-yl]benzamide
(12g). With compound 10g, a similar procedure as that described
for 12a gave pure 12g (84%) as a pink solid. m.p.: 150–152 °C. 1H
NMR (600 MHz, DMSO d6) (compound 12g is a free-radical, some
signals appear broadened and other signals are missing) d 8.52
(brs, 1H), 7.99 (s, 1H), 7.85–7.74 (m, 5H), 7.58 (s, 2H), 7.19 (s,
1H), 5.48 (s, 2H), 4.03 (s, 3H), 2.16 (s, 3H). 13C NMR (150 MHz,
CDCl3) d 165.6, 157.5, 139.4, 138.9, 137.9, 131.9, 130.3, 129.1,
127.9, 127.6 (2C), 126.8, 126.2, 126.1, 124.6 (2C), 115.6, 53.2,
38.1, 23.6. ESR (DMSO): g = 2.007. MS (ESI) 514.3 for [M+H]+.
4.2. Biology
4.2.1. Cell lines and culture conditions
HeLa (human cervical carcinoma cell line), A549 (human lung
cancer cell line), HepG2 (human hepatoma cell line), LoVo (human
colonic carcinoma cell line) and HCT-116 (human colonic carci-
noma cell line) were provided by General Hospital of Lanzhou Mil-
itary Command. The cell was routinely cultured in RPMI-1640
medium, supplemented with 10% fetal bovine serum (FBS). The
culture was maintained at 37 °C with a gas mixture of 5%
CO2/95% air. All media were supplement with 5 mg/mL penicillin
and 5 mg/mL streptomycin.
4.1.5. Procedures for synthesis of 13a-c
4.1.5.1.
N-Cyclohexyl-4-((4-((1-methyl-1H-pyrazol-4-yl)amino)-1-
4.2.2. Antiproliferation activities assays
oxophthalazin- 2(1H)-yl)methyl)benzamide (13a). To stirred solu-
tion of 10c (0.2 mmol) in dry toluene (8mL) was added the 1-
methyl-1H-pyrazol-4-amine (0.28 mmol), t-BuOK (0.28 mmol), 2-
(di-tert-butylphosphino)biphenyl (0.02 mmol) and Pd2dba3 (0.01
mmol) under nitrogen, followed the mixture was refluxed for 36
h before being cooled to the room temperature. The reaction mix-
ture was filtered and filtrate was evaporated in vacuo, the crude
product was further purified by column chromatography on silica
gel to yield compound 13a (67%) as a white solid. m.p.: 234–236
°C. 1H NMR (600 MHz, DMSO d6) d 8.98 (s, 1H), 8.30 (t, J = 6.6 Hz,
2H), 8.11 (d, J = 7.8 Hz, 1H), 7.95 (t, J = 7.2 Hz, 1H), 7.85 (t, J = 7.2
Hz, 1H), 7.79 (d, J = 7.8 Hz, 2H), 7.71 (s, 1H), 7.50–7.44 (m, 3H),
5.30 (s, 2H), 3.77–3.75 (m, 4H), 1.76 (brs, 2H), 1.68 (brs, 2H),
1.57 (d, J = 12.0 Hz, 1H), 1.30–1.20 (m, 4H), 1.09 (brs, 1H). 13C
NMR (150 MHz, DMSO d6) d 165.1, 156.5, 140.8, 140.7, 133.9,
133.0, 131.6, 129.7, 128.0 (2C), 127.9, 127.4 (2C), 127.0, 124.3,
123.4, 122.8, 120.4, 52.6, 48.2, 38.6, 32.3 (2C), 25.2, 24.9 (2C). MS
(ESI) 457.3 for [M+H]+.
Cells were incubated at 37 °C in a 5% CO2 atmosphere. The MTT
assay were used to determined growth inhibition. The HeLa, A-549,
HepG2, LoVo and HCT-116 cells were seeded in 96-well plates at a
lL of the medium with 10%
heat-inactivated NBS for 24 h. The synthetic compounds 11a–g,
density of 5 Â 103 cells/well in 150
12a–g, 13a–c and reference compound 1 and VX-680 were dis-
solved to five concentrations (0.01, 0.1, 1, 10 and 100
mediam and then added them to the well and incubated at 37 °C
for 72 h, then the media was aspirated, and 10 L of 5 mg/mL
lM) in the
l
MTT solution (dilute in sterile PBS) diluted in serum-free media
was added to each well. 4 h later, the supernatant was discarded
and 100 lL of DMSO was added to each well. The mixture was sha-
ken on an oscillator and measured at 490 nm using universal
microplate reader (Infinite M200 Pro, Tecan Inc.). IC50 values were
determined from
concentration.
a log plot of percent of control versus
4.2.3. Aurora kinase assays
The synthetic compounds and reference compound were
diluted to five concentrations (0.1, 1, 10, 100 and 1000 nM) in
the PBS and then added 5 mL to the 50 mL reaction mixture (40
mM Tris, pH = 7.4, 10 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, 10
mM ATP, 0.2 mg/ml Kinase and 100 mM Kemptide acetate salt), and
then the kinase reactions were incubated for 30 min at 37 °C.
Finally, we used Kinase-Glo luminescence kinase assay kit tested
luminescent signal of the reaction mixture, gave IC50 values by
GraphPad Prism software.
4.1.5.2.
N-Cyclohexyl-4-((4-((4-(morpholine-4-carbonyl)phenyl)
amino)-1-oxophthala zin-2(1H)-yl)methyl)benzamide (13b). With
compound 6a, a similar procedure as that described for 13a gave
pure 13b (81%) as a white solid. m.p.: 152–154 °C. 1H NMR (600
MHz, CDCl3) d 8.51–8.48 (m, 1H), 7.84 (t, J = 4.2 Hz, 1H), 7.76 (t,
J = 4.2 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 7.8 Hz, 2H),
7.35 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 6.10
(d, J = 8.4 Hz, 1H), 5.33 (s, 2H), 3.93 (t, J = 4.2 Hz, 1H), 3.80–3.50
(m, 8H), 1.98 (d, J = 10.2 Hz, 2H), 1.74–1.71 (m, 3H), 1.62 (d, J =
13.2 Hz, 1H), 1.42–1.34 (m, 2H), 1.25–1.15 (m, 4H). 13C NMR
(150 MHz, CDCl3) d 170.3, 166.3, 157.9, 142.4, 140.9, 140.4,
134.5, 132.9, 131.7, 128.8 (2C), 128.5 (2C), 128.1, 128.0, 127.1
(2C), 125.2, 121.9, 118.2 (2C), 66.9 (2C), 53.7, 48.7, 33.1 (2C),
29.7 (2C), 25.5, 24.9 (2C). MS (ESI) 566.3 for [M+H]+.
4.2.4. Western blotting analysis
HeLa cells were treated with 12c (0, 0.5, 2.0 and 5.0
680 (5.0 M) for 6 or 24 h. For total cell protein extracts, cells were
washed and lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 1% NP-
40, 2 mM EDTA, 10 mM NaCl, 10 g/ml Aprotinin, 10 g/ml
lM) or VX-
l
l
l
Leupeptin, 1 mM DTT, 0.1% SDS and 1 mM phenyl methyl sulfonyl
fluoride). Total proteins were achieved by centrifuging (12,000g for
20 min at 4 °C). The protein concentrations were determined
by using BCA protein assay kit (Beyotime, Jiangsu, China). For
4.1.5.3. N-(2-Chlorophenyl)-4-((3-(4-(cyclohexylcarbamoyl)benzyl)-
4-oxo-3,4-dihy drophthalazin-1-yl)amino)benzamide (13c). With
compound 6c, a similar procedure as that described for 13a gave
pure 13c (87%) as a white solid. m.p.: 277–279 °C. 1H NMR (600
western blot analysis, equal amounts of proteins (20–30 lg) were
Please cite this article in press as: Wang W., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.048

10
W. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7. Fu J, Bian M, Jiang Q, Zhang C. Roles of Aurora kinases in mitosis and
tumorigenesis. Mol Cancer Res. 2007;5:1–10.
8. Vader G, Lens SM. The Aurora kinase family in cell division and cancer. Bioch
Biophy Acta. 2008;1786:60–72.
9. Linardopoulos S, Blagg J. Aurora kinase inhibition: a new light in the sky? J Med
Chem. 2015;58:5186–5188.
10. Barr AR, Gergely F. Aurora-A: the maker and breaker of spindle poles. Cell Sci.
2007;120:2987–2996.
11. Saeki T, Ouchi M, Ouchi T. Physiological and oncogenic Aurora – a pathway. Int J
Biol Sci. 2009;5:758–762.
12. Vader G, Lens MSMA. The chromosomal passenger complex: guiding Aurora-B
through mitosis. J Cell Biol. 2006;173:833–837.
separated on 12% SDS-PAGE gels and transferred to polyvinylidine
difluoride (PVDF) membranes (Millipore Corporation, USA). The
blot was blocked in blocking buffer (5% non-fat dry milk in TBST)
for 2 h at room temperature, and then incubated with dilute solu-
tion (1:500–1:1000) of the antibody against AurA and AurB
(Abcam), the antibody against phospho-AurA (Thr288) and phos-
pho-AurB (Thr232) (Cell Signaling Technology), the antibodies
against cyclinB1 and cdc2 (BioLegend) and the antibody against
b-actin (ZSGB-BIO) in blocking buffer overnight at 4 °C. The blot
was then incubated with appropriate secondary antibody
(1:5000–1:10000 dilution), b-Actin was used as a loading control.
The protein bands were visualized using the Gel Imaging System
(ChemDoc-It610, UVP, USA).
13. Sasai K, Katayama H, Stenoien DL, et al. Aurora-C kinase is
a novel
chromosomal passenger protein that can complement Aurora-B kinase
function in mitotic cells. Cell Motil Cytoskelet. 2004;59:249–263.
14. Borisa AC, Bhatt HG. A comprehensive review on Aurora kinase: small molecule
inhibitors and clinical trial studies. Eur J Med Chem. 2017;140:1–19.
15. Harrington EA, Bebbington D, Moore J, et al. VX-680, a potent and selective
small-molecule inhibitor of the Aurora kinases, suppresses tumor growth
in vivo. Nat Med. 2004;10:262–267.
4.2.5. Analysis of cell cycle by flow cytometry
16. Pollard JR, Mortimore M. Discovery and development of aurora kinase
inhibitors as anticancer agents. J Med Chem. 2009;52:2629–2651.
17. Howard S, Berdini V, Boulstridge JA, et al. Fragment-Based discovery of the
The cell cycle was analyzed by flow cytometry. Firstly, HeLa
cells were treated with different concentrations of compound 12c
(0, 0.5, 2.0 and 5.0 lM) or VX-680 (5.0 lM) for 24 h. After incuba-
pyrazol-4-yl urea (AT9283),
a multitargeted kinase inhibitor with potent
tion, a total of (1–5)⁄105 cells were harvested from the treated and
normal samples. The cells were washed twice with PBS and fixed in
75% ice-cold ethanol for at least overnight. The sample was con-
centrated by removing the ethanol and then cells were then
washed three times with PBS, staining the cellular DNA with fluo-
rescent solution (1% (v/v) Triton X-100, 0.01% RNase, 0.05% PI) for
15 min in darkness. The cell cycle distribution was then detected
by flow cytometry (COULTER EPICS XL, USA). All experiments were
performed three times.
Aurora kinase activity. J Med Chem. 2009;52:379–388.
18. Yang J, Ikezoe T, Nishioka C, et al. AZD1152, a novel and selective aurora B
kinase inhibitor, induces growth arrest, apoptosis, and sensitization for tubulin
depolymerizing agent or topoisomerase II inhibitor in human acute leukemia
cells in vitro and in vivo. Blood. 2007;110:2034–2040.
19. Payton M, Bush TL, Chung G, et al. Preclinical evaluation of AMG 900, a novel
potent and highly selective pan-aurora kinase inhibitor with activity in taxane-
resistant tumor cell lines. Cancer Res. 2010;70:9846–9854.
20. Demirayak S, Karaburun AC, Beis R. Some pyrrole substituted aryl pyridazinone
and phthalazinone derivatives and their antihypertensive activities. Eur J Med
Chem. 2004;39:1089–1095.
21. Mood AD, Premachandre IDUA, Hiew S, et al. Potent antifungal synergy of
phthalazinone and isoquinolones with azoles against Candida albicans. ACS Med
Chem Lett. 2017;8:168–173.
22. Procopiou PA, Ford AJ, Gore PM, et al. Design of phthalazinone amide Histamine
H1 receptor antagonists for use in rhinitis. ACS Med Chem Lett. 2017;8:577–581.
23. Biswas K, Peterkin TAN, Bryan MC, et al. Discovery of potent, orally bioavailable
phthalazinone bradykinin B1 receptor antagonists.
2011;54:7232–7246.
24. Prime ME, Courtney SM, Brookfield FA, et al. Phthalazinone pyrazoles as potent,
selective, and orally bioavailable inhibitors of Aurora-A kinase. J Med Chem.
2011;54:312–319.
4.3. Molecular docking
The crystal structures of AurA (PDB code: 3D14) and AurB (PDB
code: 4C2V) were derived from the PDB database, and then AurA
(or AurB) and the compound 12c were performed docking using
the Glide module of Schrödinger software. Firstly, AurA (or AurB)
and the compound 12c were adopted optimization. Next, the bind-
ing sites of the protein and the compound 12c were defined.
Finally, molecular docking was performed. In the docking process,
we used a semi-flexible docking, the compound 12c was docked to
the appropriate combination pockets of protein, and finally we
chose the best combination model.
J
Med Chem.
25. Qin WW, Sang CY, Zhang LL, et al. Synthesis and biological evaluation of 2,4-
diaminopyrimidines as selective Aurora A kinase inhibitors. Eur J Med Chem.
2015;95:174–184.
26. Zhao J, Guan XW, Chen SW, Hui L. Synthesis and biological evaluation of
norcantharidin derivatives as protein phosphatase-1 inhibitors. Bioorg Med
Chem Lett. 2015;25:363–366.
27. Christensen H, Søren Kiil A, Damjohansen K, et al. Effect of solvents on the
product distribution and reaction rate of
reaction. Org Process Res Dev. 2006;10:762–769.
a
BuchwaldÀHartwig amination
Acknowledgments
28. Huang WT, Liu J, Liu JF, et al. Synthesis and biological evaluation of conjugates
of deoxypodophyllotoxin and 5-FU as inducer of caspase-3 and -7. Eur J Med
Chem. 2012;49:48–54.
29. Kashem MA, Nelson RM, Yingling JD, et al. Three mechanistically distinct kinase
assays compared: measurement of intrinsic ATPase activity identified the most
comprehensive set of ITK inhibitors. J Biomol Screen. 2007;12:70–83.
30. Bayliss R, Sardon T, Vernos I, Conti E. Structural basis of Aurora-A activation by
TPX2 at the mitotic spindle. Mol Cell. 2003;12:851–862.
We gratefully acknowledge the financial support from the
National Natural Science Foundation of China (No. 21672093 and
21372110).
A. Supplementary data
31. Li Y, Zhou WQ, Wei LL, et al. The effect of Aurora kinases on cell proliferation,
cell cycle regulation and metastasis in renal cell carcinoma. Int
J Oncol.
2012;41:2139–2149.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2018.04.048.
32. Giet R, Prigent C. The non-catalytic domain of the Xenopus laevis auroraA
kinase localises the protein to the centrosome. J Cell Sci. 2011;114:2095–2104.
33. Guan XW, Xu XH, Feng SL, et al. Synthesis of hybrid 4-deoxypodophyllotoxin-
5-fluorouracil compounds that inhibit cellular migration and induce cell cycle
arrest. Bioorg Med Chem Lett. 2016;26:1561–1566.
References
34. Hailt MM, Ebrahim HY, Mohyeldin MM, et al. The tobacco cembranoid
1. Li JJ, Li SA. Mitotic kinases: the key to duplication, segregation, and cytokinesis
errors, chromosomal instability, and oncogenesis. Pharmacol Ther.
2006;111:974–984.
2. Carmena M, Earnshaw WC. The cellular geography of aurora kinases. Nat Rev
Mol Cell Biol. 2003;4:842–854.
(1S,2E,4S,7E,11E)-2,7,11-cembratriene-4,6-diol as
a
novel angiogenesis
inhibitory lead for the control of breast malignancies. Bioorg Med Chem.
2017;25:3911–3921.
35. DeLano L. The PyMOL molecular graphics system. Palo Alto, CA, USA: DeLano
Scientific; 2002.
3. Katayama H, Brinkley WR, Sen S. The aurora kinases: role in cell transformation
and tumorigenesis. Cancer Metastasis Rev. 2003;22:451–464.
4. Glover DM, Leibowitz MH, Mclean DA, Parry H. Mutations in Aurora prevent
centrosome separation leading to the formation of monopolar spindles. Cell.
1995;81:95–105.
5. Nigg EA. Mitotic kinases as regulators of cell division and its checkpoints. Nat
Rev Mol Cell Biol. 2001;2:21–32.
6. Kilchmann F, Marcaida MJ, Kotak S, et al. Discovery of a selective aurora A
kinase inhibitor by virtual screening. J Med Chem. 2016;59:7188–7211.
36. Cheng C, Liu ZG, Zhang H, et al. Enhancing chemosensitivity in ABCB1- and
ABCG2-overexpressing cells and cancer stem-like cells by an Aurora kinase
inhibitor CCT129202. Mol Pharm. 2012;9:1971–1982.
37. Nar A, Ozen O, Tutuncu NB, Demirhan B. Cyclin A and cyclin B1 overexpression
in differentiated thyroid carcinoma. Med Oncol. 2012;29:294–300.
38. Liu JF, Sang CY, Qin WW, et al. Synthesis and evaluation of the cell cycle arrest
and CT DNA interaction properties of 4b-amino-40-O-demethyl-4-
deoxypodophyllotoxins. Bioorg Med Chem. 2013;21:6948–6955.
Please cite this article in press as: Wang W., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.048