Design, synthesis and biological evaluation of novel 1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolins as potential EZH2 inhibitors

Article abstract of DOI:10.1039/c5ra02365c

The histone lysine methyltransferase EZH2 has been implicated as a key component in cancer aggressiveness, metastasis and poor prognosis. This study discovered a new class of hexahydroisoquinolin derivatives as EZH2 inhibitors. A structure-activity relationship study showed that the steric hindrance was important to the activity for EZH2. A preliminary optimization study led to the discovery of several potent compounds with low nanomolar to sub-nanomolar potency for EZH2. Biological evaluation indicated that SKLB1049 was a highly potent with improved solubility compared to EPZ6438, SAM-competitive, and cell-active EZH2 inhibitor that decreased global H3K27me3 in SU-DHL-6 and Pfeiffer lymphoma cells in a concentration- and time-dependent manner. Further study indicated that SKLB1049 caused cell arrest in G₀/G₁ phase. These compounds would be useful as chemical tools to further explore the biology of EZH2 and provided us with a start point to develop new EZH2 inhibitors.

Full text of DOI:10.1039/c5ra02365c

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Design, synthesis and biological evaluation of novel
1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolins
as potential EZH2 inhibitors
Cite this: RSC Adv., 2015, 5, 25967
Lidan Zhang,†ab Xuejiao Song,†b Ningyu Wang,b Lifeng Zhao,b Qiang Feng,bc
Xinyu You,ab Cuiting Peng,ab Tiantao Gao,b Menghua Xiong,b Bing He,bc Chao Gao,b
b
b
a
b
*
*
Yong Luo, Ying Xu, Qiyi Zhang and Luoting Yu
The histone lysine methyltransferase EZH2 has been implicated as a key component in cancer
aggressiveness, metastasis and poor prognosis. This study discovered new class of
a
hexahydroisoquinolin derivatives as EZH2 inhibitors. A structure–activity relationship study showed that
the steric hindrance was important to the activity for EZH2. A preliminary optimization study led to the
discovery of several potent compounds with low nanomolar to sub-nanomolar potency for EZH2.
Biological evaluation indicated that SKLB1049 was a highly potent with improved solubility compared to
EPZ6438, SAM-competitive, and cell-active EZH2 inhibitor that decreased global H3K27me3 in SU-DHL-
6 and Pfeiffer lymphoma cells in a concentration- and time-dependent manner. Further study indicated
that SKLB1049 caused cell arrest in G0/G1 phase. These compounds would be useful as chemical tools
to further explore the biology of EZH2 and provided us with a start point to develop new EZH2 inhibitors.
Received 6th February 2015
Accepted 4th March 2015
DOI: 10.1039/c5ra02365c
www.rsc.org/advances
inhibition of EZH2 is considered as an attractive therapeutic
target for the treatment of cancer.
Introduction
Cancer is a major public health problem in the world.1 Target-
ing epigenetic regulators has provided us with a new strategy for
cancer therapy.2 Enhancer of Zeste Homolog 2 (EZH2) is a
member of the histone lysine methyltransferase family and the
catalytic component of the polycomb repressive complex 2
(PRC2) as well.3 PRC2 catalyzes the methylation of histone 3
lysine 27 (H3K27), leading to trimethylation of H3K27
(H3K27me3), which is an epigenetic mark of transcriptionally
repressive and silences target genes.4 The over expression of
EZH2 has been observed in numerous blood and solid tumors,
such as Hodgkin's disease,5 non Hodgkin's lymphoma,6 pros-
tate,7 breast,8 renal,9 melanoma10 and tongue11 cancers, and is
widely implicated in cancer progression, metastasis and poor
prognosis.8,12 Recently, somatic mutations of tyrosine Y641,
Alanine A677 and A687 of EZH2 were reported to be associated
with germinal center B-cell like diffuse large B-cell lymphoma
(GCB-DLBCL) and Follicular Lymphoma (FL).13–15 Thus,
Over the recent years, multiple groups, including Glax-
oSmithKline,16 Epizyme,17–19 Novartis,20 UNC Eshelman21 and
Constellation,22,23 have reported various structures of small
molecule inhibitors of EZH2 (Fig. 1). The most potent
compound EPZ6438 was reported to inhibit EZH2 with an IC50
of 2.5 nM, and exhibited signicant tumor growth inhibition in
several human DLBCL17 and malignant rhabdoid18 xenogra
models. In 2013, a phase I/II study of EPZ6438 for the treatment
of advanced solid tumors and B cell lymphomas was initiated.
However, the effective dose of EPZ6438, 600 mg kgꢀ1 once a day,
was very high in the WSU-DLCL2 xenogra model, which could
attribute to its inferior physicochemical properties, such as
solubility. Thus highly potent EZH2 inhibitor with improved
physicochemical properties is emergently needed.
Our research group designed and synthesized a series of
novel EZH2 inhibitors bearing hexahydroisoquinolins, and the
potency of these compounds for EZH2 was characterized. Bio-
logical evaluation indicated that SKLB1049 was highly potent
for EZH2 methyltransferase with improved solubility than
EPZ6438. In cell-based assays, SKLB1049 remarkably decreased
global H3K27me3 levels, and selectively killed two DLBCL cell
lines harboring the EZH2 mutant, SU-DHL-6 and Pfeiffer cells,
but no apparent impact on normal cell lines. Further study
indicated that SKLB1049 caused cell cycle arrest in G0/G1 phase.
This study may contribute to the further design of novel scaf-
folds against EZH2.
aCollege of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065,
China. E-mail: yuluot@scu.edu.cn; qyzhang@scu.edu.cn; Fax: +86-28-8550-3817;
Tel: +86-28-8550-3817
bState Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy
and Cancer Center, West China Hospital, West China Medical School, Sichuan
University, Chengdu 610041, China
cCollege of Chemistry and Life Science, Chengdu Normal University, Chengdu 611130,
China
† These authors contributed equally to this work.
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Results and discussion
Chemistry
The compounds described in this study were synthesized
according to Scheme 1. Briey, treatment of ketone 1 with ethyl
acetate under sodium hydride24 or acetyl chloride under LDA25
afforded 1,3-diones 2. Then the key building blocks pyridine-2-
(1H)-ones 3 were prepared by treating 1,3-diones with cyanoa-
cetamide in the presence of triethylenediamine (DABCO) in
reuxing ethanol.26 Subsequently, reduction of 3 by sodium
borohydride and iodine afforded amines 4,27 which were sub-
jected to coupling reaction with various acids 8 afforded 9.28
Next, the title compounds 10 were gained by Suzuki coupling
reaction with various boronic acids or boronic esters.
Compound 7 used for preparation of acid 8 was synthesized
under a two-step reductive amination reaction, starting from
methyl 3-amino-5-bromo-2-methylbenzoate 5. The structures of
Fig. 1 Disclosed EZH2 inhibitors.
1
all the tested compounds were fully characterized by H NMR,
13C NMR and ESI-MS analysis.
SAR analysis
Modications of 4- and 5-positions of pyridone moiety.
Tables
1 and 2 provided the biological data of these
compounds. All synthesize derivatives were evaluated for its
EZH2 inhibitory activity in AlphaLISA29 assay and the anti-
proliferation activities of SU-DHL-6 and Pfeiffer cells in MTT
assay. To investigate the structure–activity relationships of C4
and C5 position of pyridone moiety, we rst synthesized
compounds 10a–c wherein pyridone C4–C5 position together
formed an alkyl ring. Evaluation of the ring size of the fused
system demonstrated that the seven- and eight-membered rings
(10b and 10c) were equipotent and inferior to the six-membered
ring (10a, EZH2WT IC50 ¼ 14 nM; EZH2Y641 IC50 ¼ 27.2 nM;
EZH2A677G IC50 ¼ 7.0 nM), which was chosen for further SAR
study. Simple methyl substitution at the 7-position of the
terminal fused ring (10d) improved the potency on all three
EZH2 enzymes: EZH2WT, EZH2Y641F and EZH2A677G, while the
ethyl (10e), tert-butyl (10f) and dimethyl analogs (10g) were less
active. This revealed that a relative small substituent in this
position was more potent, and the volume of the substitutents
was a crucial factor for the activity. Introduction of a methyl to
C6 position of 1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin
resulted in a less potent compound (10h). Comparison of 10h
and 10d suggested that methyl substituted at the 7-position of
the hexahydroisoquinoline was preferred relative to the 6-
position. Interestingly, introduction of a phenyl ring fused to
cyclohexane moiety led to an inactive compound 10i, which
suggested that a bulky substituent in C5 and C6 of hexahy-
droisoquinoline might change its conformation and was
harmful to the anti-EZH2 activity. In this series of pyridone
analogs (10a–10i), 10d displayed the most active for EZH2
inhibition. However, it did not show remarkably enhanced
antiproliferation activities for SU-DHL-6 and Pfeiffer cells
compared with 10a in this 4 days assay.
Scheme 1 Reagents and conditions: (a) NaH, EA, PhMe, 0 ꢁC-RT or
LDA, acetyl chloride, THF, ꢀ78 ꢁC; (b) cyanothioacetamide, DABCO,
ethanol, reflux; (c) NaBH4, I2, THF,
0
ꢁC-reflux; (d) sodium
triacetoxyborohyride, acetic acid, trichloromethane, 0 ꢁC-RT; (e)
sodium triacetoxyborohyride, acetic acid, 1,2-dichloroethane, 0ꢁ C-
RT; (f) 3 M NaOH, ethanol, RT, 3 h, then adjust to pH 4–5 with 1 N HCl;
(g) EDC, HOAT, NMM, DMSO, RT, 12–48 h; (h) PdCl2(dppf)$CH2Cl2,
Na2CO3, 10 : 1 dioxane–H2O, 110 ꢁC.
Substitutions of R2 position of central phenyl. Subsequent
SAR study of R2 position of central phenyl was further explored
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Table 1 In vitro potencies of newly synthesized inhibitors 10a–10i
EZH2 IC50a (nM)
Cells IC50 (mM)
Compound
X
EZH2WT
EZH2Y641F
EZH2A677G
SU-DHL-6
Pfeiffer
10a
14
27.2
7.0
20
19.5
10b
10c
600
610
>500
>500
NTb
NT
>30
>30
27
21
10d
10e
0.66
1.3
24
23
2.9
4.4
13.57
18
19
9
10f
10g
10h
6.4
3.3
20
94
NT
63
20
NT
12
23.5
NT
28
20
NT
22
10i
>5000
NT
NT
>30
>30
EPZ6438
0.27
1.2
2.3
1.0
0.9
NT
NT
NT
NT
GSK126
0.56
a
The IC50 values were averaged from two independent dose–response curves. b Not tested.
with a series of aryl and heteroaryl (Table 2). For this purpose, EZH2. Among aryl substitutuents, compound 10q was the most
the fused cyclohexane group was held constant. The different R2 potent compound with an IC50 of 7.3 nM for EZH2WT, which was
substituents had considerable inuence on the activity for 2-fold improvement compared with 10a. On the other hand,
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Table 2 In vitro potencies of newly synthesized inhibitors 10j–10u
EZH2 IC50a (nM)
Cells IC50 (mM)
Compound
10j
R2
EZH2WT
19
EZH2Y641F
NTb
EZH2A677G
NT
SU-DHL-6
>30
Pfeiffer
>30
–Br
10k
75
NT
NT
>30
>30
10l
10m
10n
140
9.6
55
1266
455
NT
NT
NT
>30
>30
>30
>30
>30
>30
875
10o
10p
17
10
NT
NT
38
>30
>30
>30
>30
161
10q (SKLB1049)
7.3
14
39
5.9
7.0
16
20
12.5
19.5
10a
27.2
10r
25
NT
45
>30
>30
10s
10t
15.5
19.5
NT
NT
NT
>30
>30
>30
>30
165
10u
4.0
39
5.9
16
11
a
The IC50 values were averaged from two independent dose–response curves. b Not tested.
Table 3 The solubility of compound 10a, 10q (SKLB1049), 10u and EPZ6438
Solution (mg mLꢀ1
)
Compound
Water
Normal saline
PH ¼ 6.5
Methanol
Ethanol
Acetone
Dichloromethane
10a
SKLB1049
10u
29.7
185.3
41.3
3.0
41.1
87.6
3.5
29.9
243.0
0.6
36 926.3
>30 000
>30 000
10 252.3
265 779
>200 000
>200 000
2121.3
44 339.3
>30 000
>30 000
284.9
443 393
>400 000
>400 000
186 437
EPZ6438
6.8
4.6
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Fig. 2 SKLB1049 was a potent, SAM-competitive and selective inhibitor of EZH2. (a) Chemical structures of SKLB1049. (b) Determination of the
inhibitory potential of SKLB1049 on WT EZH2 (green), Y641F mutant EZH2 (red), and A677G EZH2 (blue). (c) Determination of the inhibitory
potential of SKLB1049 on WT EZH1. (d) Determination of the mechanism of action of SKLB1049. Data were represented as mean values ꢂ SEM
(n ¼ 2).
Table 4 The MTT data (6 days)a
IC50 (mM)
Compound
SU-DHL-6
Pfeiffer
HEK-293
LO2
VERO
10a
SKLB1049
10u
10.02
6.17
2.65
6.12
2.76
1.19
1.65
1.47
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
10.81
EPZ6438
a
IC50 value was average of three determinations and deviation from the
average was <5% of the average value.
nanomolar potency for EZH2. The 1-methyl-3-oxo-2,3,5,6,7,8-
hexahydroisoquinolin which replaced pyridone moiety also
led to improved solubility in some cases. The solubility of 10a,
10q (SKLB1049) and 10u was signicantly improved over that of
EPZ6438 (Table 3). In particular, SKLB1049 exhibited an
apparent 60-fold increase of solubility in water (185.3 mg mLꢀ1
vs. <3.0 mg mLꢀ1) compared with EPZ6438.
SKLB1049 was a SAM-competitive, potent, and selective
inhibitor of EZH2. On the basis of the high enzyme and cells
inhibitory activities and improved solubility, SKLB1049 was
selected for subsequent studies for mechanism of action (MOA),
selectivity and cellular assays. To elucidate the MOA of EZH2
inhibition by SKLB1049, the IC50 values were determined at two
different SAM concentrations of 1 mM and 10 mM. As shown in
Fig. 2d, the results demonstrated that the increase of SAM
concentration could cause an obvious shi of IC50 value of
SKLB1049 against EZH2WT, which indicated that SKLB1049 was
a SAM-competitive EZH2 inhibitor. The MOA for SKLB1049 was
consistent with that of structurally similar molecule EPZ6438.
As shown in Fig. 2b, SKLB1049 was highly potent for both
Y641F and A677G mutants, with IC50 values of 39 nM and 5.9 nM,
respectively. The potency of SKLB1049 for WT enzymes was
similar with A677G mutant and superior to Y641F mutant. To
determine the selectivity prole of SKLB1049, the potency for
EZH1 was also evaluated (Fig. 2c). SKLB1049 showed a consid-
erable potency for EZH1, with an IC50 value of 84 nM, approxi-
mately 12-fold less potent than EZH2.
Fig. 3 Compound SKLB1049 reduced H3K27me3 in Pfeiffer and
SU-DHL-6 cells in a dose- and time-dependent manner. (a) Pfeiffer
cells was treated with 0.625 mM, 1.25 mM, 2.5 mM, 5 mM or 10 mM of
SKLB1049 for 4 days and whole cell extracts were analyzed by western
blotting for levels of H3K27me3. H3 and b-actin levels served as
loading controls. (b) Similarly effect of H3K27me3 levels with 5 mM
SKLB1049 in Pfeiffer cells over 6 days were determined. (c) SU-DHL-6
cells was treated of SKLB1049 for 4 days and effect of H3K27me3,
H3K27me2 and EZH2 levels were determined. H3 and b-actin levels
served as loading controls.
replacement of the 4-(morpholinomethyl) phenyl moiety (10a)
with 1-methyl-1H-pyrazole-4-yl (10r), 2-uoropyridin-5-yl (10s)
or 2-chloropyridin-5-yl (10t) retained excellent potency for
EZH2, and introduction of 2-piperazinyl pyridin-5-yl (10u,
EZH2WT IC50 ¼ 4.0 nM) in this position led to a 4-fold
improvement of potency. In this series (10a, 10j–10u), most
compounds did not show obvious effect on tumor cells viability.
However, 10q and 10u displayed slightly improved anti-
proliferative activities for SU-DHL-6 and Pfeiffer cells compared
with 10a.
SKLB1049 potently reduced H3K27me3 in cells. The ability
of SKLB1049 to reduce H3K27me3 in intact cells was evaluated
in SU-DHL-6 and Pfeiffer cells using Western blotting analysis
(Fig. 3). Treatment with SKLB1049 at 5 mM for 4 days and 6 days
The solubility assay of hexahydroisoquinolin analogs.
Structure–activity relationship study led to the discovery of
several potent compounds with low nanomolar to sub-
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Fig. 4 Viability across SU-DHL-6 and Pfeiffer cell lines treated with
10a, SKLB1049 and 10u for 4 and 6 days was assessed. Data were
represented as mean values ꢂ SEM (n ¼ 3).
Fig. 6 Compound SKLB1049 induced G0/G1 arrest in EZH2 mutant
SU-DHL-6 cells. Cell cycle analysis (by flow cytometry) was assessed in
SU-DHL-6 cells during incubation with either vehicle or 0.625 mM, 1.25
mM, 2.5 mM, 5 mM, 10 mM SKLB1049 for 14 days. Data were represented
as mean values ꢂ SEM (n ¼ 2).
potently abolished H3K27me3 mark in EZH2 A677G mutant
Pfeiffer cells in a concentration- and time-dependent manner.
Similarly, SKLB1049 remarkably reduced H3K27me3 and
H3K27me2 mark in a concentration-dependent manner in
SU-DHL-6 cells, with no affection on EZH2 levels.
To further evaluate the safety of our EZH2 inhibitors, the
antiproliferation activities of SKLB1049 for 6 days against three
normal cell lines, HEK 293, Vero and LO2 were measured by
MTT assay (Table 4). All these compounds showed no obvious
impact on these normal cells, indicating its good in vitro safety
prole.
SKLB1049 could remarkably arrested G0/G1 phase. The effect
on cell cycle progression of SKLB1049 was also assessed (Fig. 6).
Treatment with SKLB1049 in SU-DHL-6 cells at a concentration
of 0.625 mM showed no change in cell cycle compared with the
DMSO control, while a concentration of 10 mM treatment
resulted in an increase of the percentage of cells in G0/G1 phase
from 44.89% to 66.09%, and a concomitant decrease in S phase
from 34.43% to 12.68%. However there was no considerable
change in the percentage of cells in the G2/M phase through 14
days treatment. These results suggested that SKLB1049 could
remarkably arrested cell cycle in G0/G1 phase in a concentration-
dependent manner.
SKLB1049 inhibited proliferation of cancer cell line. To
further investigate the effect of EZH2 inhibition on cell prolif-
eration, the inhibitory activities of 10a, SKLB1049 and 10u
against SU-DHL-6 and Pfeiffer cell lines for 6 days were evalu-
ated (Table 4). As shown in Fig. 4, cell lines were effectively
killed by EZH2 inhibitors in a time-dependent manner.
SKLB1049 and 10u demonstrated slightly greater potency than
10a in both cell lines. This was consistent with the better activity
of SKLB1049 and 10u on EZH2 activity. Pfeiffer was more
sensitive to EZH2 inhibition compared with SU-DHL-6, with
IC50 values of 2.76 mM, 1.19 mM and 1.65 mM for 10a, SKLB1049
and 10u, respectively. The difference in sensitivity between
Pfeiffer and SU-DHL-6 was not due to different levels of target
engagement because the similar signicant suppression of
global H3K27me3 in both cell lines was observed.
Then, bright-eld microscopy images of SU-DHL-6 cancer
cells aer incubation with SKLB1049 for 6 days at varying
concentrations were performed to assess the activity visually. As
shown in Fig. 5, when treated with SKLB1049 for 6 days, the
proliferation of SU-DHL-6 cancer cell was remarkably inhibited,
and these phenomena were more signicant as the concentra-
tion increasing.
Conclusions
We designed and synthesized a series of novel compounds
bearing hexahydroisoquinolin and their biological activities of
targeting EZH2 were characterized. Structure–activity relation-
ship study showed the steric hindrace was important to the
Fig. 5 Bright-field microscopy images of SU-DHL-6 cancer cells, after incubation with SKLB1049 for 6 days at varying concentrations: 5 mM,
10 mM.
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activity for EZH2. Preliminary optimization led to the discovery 1 h, the cooling bath was removed and stirring was continued
of several potent compounds with good solubility, in which for 3 h. The reaction mixture was poured into a saturated
SKLB1049 was selected for subsequent studies for mechanism aqueous NH4Cl (25 mL) and then extracted with CH2Cl2 (3ꢃ).
of action (MOA), selectivity and cellular assays. Biological eval- The combined extracts were washed with brine, dried, and
uation indicated that SKLB1049 was a highly potent candidate concentrated. Purication by silica ash chromatography
for EZH2 wild-type, Y641 mutant and A677 mutant enzymes (hexanes/EtOAc) giving 2c, 2d. Yeild: 50–60%.
with improved solubility than EPZ6438, through a SAM-
competitive manner. In cell-based assays, SKLB1049 remark-
ably reduced global H3K27me3 and H3K27me2 levels in a
concentration- and time-dependent manner, with no affection
on EZH2 levels. SKLB1049 could selectively killed two DLBCL
cell lines harboring the EZH2 mutants, SU-DHL-6 and Pfeiffer
cells in a concentration- and time-dependent manner, but with
no apparent impact on normal cell lines. Further study indi-
cated that SKLB1049 caused cell arrest in G0/G1 phase.
Together, our study may contribute to the further design of
novel scaffolds against EZH2. Further studies of the biological
mechanisms and anti-tumor research in vivo are in progress.
General procedure for the synthesis of 3
1,3-Diketones (14 mmol, 1.0 equiv), cyanoacetamide (14 mmol,
1.0 equiv) and DABCO (14 mmol, 1.0 equiv) was added to a
solution of EtOH (25 mL), then the resulting mixture was stirred
at 90 ꢁC for 10 h. The reaction mixture was cooled to rt and the
solid precipitate was ltered, washed with EtOH and dried to
afford the desired compound. Yeild: 20–60%.
General procedure for the synthesis of 4
To an ice-bath cooled THF (100 mL) solution of 3 (10 mmol, 1.0
equiv) were added NaBH4 (11 mmol, 1.1 equiv) and I2 (10 mmol,
1.0 equiv) and the mixture stirred for 30 min. The reaction
mixture was then heated at reux for 10 h and then allowed to
cool to room temperature. Aer cooling to 0 ꢁC, the reaction
mixture was acidied by slow addition of 3 N HCl (l mL). The
reaction mixture was concentrated in vacuo and the crude
product 4 used directly in the next step without further
purication.
Experimental
Chemistry
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without purication. The 1H
and 13C NMR spectra were recorded on a Bruker AVANCE III 400
spectrometer using DMSO-d6 or CDCl3 as the solvent. Chemical
shis (d) were reported in ppm relative to Me4Si (internal
standard), and coupling constants (J) were reported in Hz. Mass
Spectra (MS) were performed on a Waters Q-TOF Premier mass
spectrometer. Thin layer chromatography (TLC) used Qingdao
Haiyang Silica gel F-254 plates, and column chromatography
were performed using Qingdao Haiyang Silica gel 60 (300–400
mesh). HPLC analysis was performed on an UltiMate 3000
HPLC system (Dionex, USA). All tested compounds were puri-
ed until the purity was S95%, detected by HPLC under UV 254
nm wavelength, NMR and ESI-MS.
General procedure for the synthesis of 6
The
starting
material
methyl
3-amino-5-bromo-2-
methylbenzoate (5) was synthesized based on a literature
method. To a stirred solution of 5 (10 mmol, 1.0 equiv) and
cyclic ketone (15 mmol, 1.5 equiv) in dichloroethane (50 mL)
was added acetic acid (60 mmol, 6.0 equiv) and the reaction
mixture stirred at room temperature for 15 minutes, then the
ꢁ
reaction mixture was cooled to 0 C and sodium triacetoxybor-
ohydride (30 mmol, 3.0 equiv) was added. The reaction mixture
was stirred overnight at room temperature. Upon completion of
the reaction as determined by TLC, aqueous sodium bicar-
bonate solution was added to the reaction mixture until a pH of
7–8 was obtained. The organic phase was separated and the
aqueous phase was extracted with ethyl acetate. The combined
organic layers were dried over anhydrous sodium sulfate,
ltered and concentrated under reduced pressure. The crude
compound was puried by column chromatography eluting
with ethyl acetate: hexane to afford the desired compound 6 as a
solid. Yeild: 70%.
General procedure for the preparation of 1,3-diketones 2a–2h
Method A for 2a–2b, 2e–2h. A mixture of cyclic ketone (14
mmol, 1.0 equiv) and toluene (0.5 mL) was added dropwise to a
suspension of 60% sodium hydride (28 mmol, 2.0 equiv) in
ethyl acetate (28 mmol, 2.0 equiv). Aer hydrogen evolution
ꢁ
ceased, the reaction mixture was heated at 40 C for 3 h. Addi-
tional stirring at room temperature was continued and excess
sodium hydride was quenched with methanol. The reaction
mixture was poured into water, neutralized with concentrated
HCl, and extracted with CH2Cl2. The combined extracts were
washed with brine, dried, and concentrated. Purication by
General procedure for the synthesis of 7
silica ash chromatography (hexanes/EtOAc) giving 2a, 2b, 2e, To a stirred solution of 6 (10 mmol, 1.0 equiv) and acetaldehyde
2e. Yeild: 30–60%. (15 mmol, 1.5 equiv) in dichloroethane (50 mL) was added
Method B for 2c–2d. A solution of cyclic ketone (1.0 equiv) in acetic acid (60 mmol, 6.0 equiv) and the reaction mixture stirred
dry THF (0.67 M) was added to a stirred solution of freshly at room temperature for 15 minutes, then the reaction mixture
prepared LDA (lithium diisopropylamide) (1.0 equiv) in THF was cooled to 0 ꢁC and sodium triacetoxyborohydride (30 mmol,
ꢁ
(1.0 M) at ꢀ78 C. The resulting solution was stirred for 2 h. A 3.0 equiv) was added. The reaction mixture was stirred over-
solution of acetyl chloride (1.0 equiv) in THF (1.2 M) was added night at room temperature. Upon completion of the reaction as
to the above solution. Aer stirring at ꢀ78 ꢁC was continued for determined by TLC, aqueous sodium bicarbonate solution was
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added to the reaction mixture until a pH of 7–8 was obtained. 30.23, 26.60, 24.09, 22.15, 21.89, 15.93, 14.55, 12.67. ESI-MS m/z
The organic phase was separated and the aqueous phase was 613.2 (M + H)+.
extracted with ethyl acetate. The combined organic layers were
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-N-((1-
dried over anhydrous sodium sulfate, ltered and concentrated methyl-3-oxo-3,5,6,7,8,9-hexahydro-2H-cyclohepta[c]pyridin-
under reduced pressure. The crude compound was puried by 4-yl)methyl)-40-(morpholinomethyl)biphenyl-3-carboxamide (10b).
column chromatography eluting with ethyl acetate: hexane to Yield: 74%; 1H NMR (400 MHz, DMSO) d 11.40 (s, 1H), 8.19 (s,
afford the desired compound 7 as a liquid. Yeild: 60%.
1H), 7.58 (d, J ¼ 7.7 Hz, 2H), 7.39 (d, J ¼ 4.0 Hz, 2H), 7.36 (s, 1H),
7.22 (s, 1H), 4.38 (d, J ¼ 4.0 Hz, 2H), 3.83 (d, J ¼ 10.6 Hz, 2H),
3.58 (s, 4H), 3.48 (s, 2H), 3.25 (t, J ¼ 11.2 Hz, 2H), 3.16 (m, 2H),
3.02 (m, 1H), 2.65 (d, J ¼ 9.4 Hz, 2H), 2.36 (s, 4H), 2.26 (s, 3H),
2.19 (s, 3H), 1.74 (s, 2H), 1.67 (m, 2H), 1.53 (m, 4H), 1.45 (s, 2H),
1.24 (s, 2H), 0.83 (t, J ¼ 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 170.16, 163.55, 150.05, 149.57, 147.42, 139.30, 137.89, 133.62,
131.73, 131.34, 131.24, 130.16, 128.37, 128.25, 126.84, 123.98,
121.93, 121.15, 120.73, 67.33, 66.23, 62.44, 58.41, 53.06, 41.54,
37.07, 33.29, 31.89, 30.47, 29.70, 29.37, 27.45, 26.49,
25.63, 22.70, 20.79, 16.94, 14.78, 14.15, 12.84. ESI-MS m/z 627.2
(M + H)+.
General procedure for the synthesis of 8
Ester 7 (10 mmol, 1.0 equiv) in ethanol (25 mL) was added
aqueous NaOH (15 mmol, 1.5 equiv in 5 mL water) and the
resulting mixture was stirred at 60 ꢁC for 1 h. Upon completion
of the reaction as determined by TLC, the solvent was removed
under reduced pressure and the residue obtained was acidied
with 1 N HCl until a pH 4 was obtained. The precipitate was
ltered off, washed with water and dried to afford the desired
acid 8. Yeild: 80%.
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-N-((1-
methyl-3-oxo-2,3,5,6,7,8,9,10-octahydrocycloocta[c]pyridin-4-yl)-
methyl)-40-(morpholinomethyl)biphenyl-3-carboxamide (10c).
General procedure for the synthesis of 9
The above acid 8 (1.0 mmol, 1.0 equiv) and amino 4 (2 mmol, 2.0
equiv) were dissolved in DMSO (10 mL), then HOAT (1.5 mmol,
1.5 equiv), EDCI (1.5 mmol, 1.5 equiv) and N-methylmorpholine
(3 mmol, 3 equiv) were added to it. The reaction mixture was
stirred at room temperature for 20 h. Upon completion of the
reaction as determined by TLC, the reaction mixture was poured
onto ice-cold water (100 mL), stirred for 30 minutes and the
precipitated solid was collected by ltration, washed with water
(50 mL) and air dried. The crude compound was puried by
column chromatography eluting to afford the desired
compound 9 as a solid. Yeild: 65–78%.
1
Yield: 84%; H NMR (400 MHz, DMSO) d 11.35 (s, 1H), 8.18 (t,
J ¼ 4.8 Hz, 1H), 7.57 (d, J ¼ 8.0 Hz, 2H), 7.39 (d, J ¼ 4.4 Hz, 2H),
7.36 (s, 1H), 7.21 (s, 1H), 4.37 (d, J ¼ 4.6 Hz, 2H), 3.83 (d, J ¼ 10.4
Hz, 2H), 3.57 (s, 4H), 3.48 (s, 2H), 3.25 (t, J ¼ 11.3 Hz, 1H), 3.08
(m, 2H), 3.02 (m, 1H), 2.60 (m, 2H), 2.36 (s, 4H), 2.24 (s, 3H),
2.21 (s, 3H), 1.66 (m, 2H), 1.58 (s, 2H), 1.54 (d, J ¼ 8.0 Hz, 2H),
1.39 (s, 2H), 1.27 (s, 2H), 0.83 (t, J ¼ 6.8 Hz, 3H). 13C NMR (100
MHz, CDCl3) d 170.16, 164.15, 149.94, 149.52, 144.99, 144.42,
139.41, 137.81, 133.72, 130.18, 126.94, 123.96, 122.71, 120.71,
117.96, 67.34, 66.19, 62.48, 58.40, 53.09, 41.51, 37.07, 31.93,
30.80, 30.47, 29.80, 29.71, 29.57, 26.18, 25.65, 25.52, 16.18,
14.75, 14.15, 12.83. ESI-MS m/z 641.2 (M + H)+.
General procedure for the synthesis of 10
To a stirred solution of 9 (1.0 mmol, 1.0 equiv) in dioxane–water
N-((1,7-Dimethyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-
mixture (20 mL/2 mL) was added boronic ester derivatives (1.08 4-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-
mmol, 1.08 equiv) followed by addition of Na2CO3 (3.6 mmol, 40-(morpholinomethyl)biphenyl-3-carboxamide (10d). Yield:
3.6 equiv). The solution was purged with argon for 15 minutes 79%; 1H NMR (400 MHz, DMSO) d 11.49 (s, 1H), 8.16 (t, J ¼ 4.4
and then PdCl2(dppf)$CH2Cl2 (0.1 mmol, 0.1 equiv) was added Hz, 1H), 7.57 (d, J ¼ 8.0 Hz, 2H), 7.45–7.26 (m, 1H), 7.21 (s, 1H),
and the solution was again purged with argon for a further 10 4.31 (t, J ¼ 4.0 Hz, 2H), 3.83 (d, J ¼ 10.2 Hz, 2H), 3.64–3.53 (m,
ꢁ
min. The reaction mixture was heated at 100 C for 4 h. Aer 4H), 3.48 (s, 2H), 3.25 (t, J ¼ 11.2 Hz, 2H), 3.14–3.04 (m, 2H),
completion (monitored by TLC), the reaction mixture was 3.04–2.87 (m, 1H), 2.61 (m, 2H), 2.36 (s, 4H), 2.25 (s, 3H), 2.10 (s,
diluted with water and extracted with 10% MeOH/DCM. The 3H), 1.90 (m, 1H), 1.80 (m, 1H), 1.66 (m, 4H), 1.52 (m, 2H), 1.19
combined organic layers were dried over anhydrous sodium (m, 1H), 1.01 (d, J ¼ 6.4 Hz, 3H), 0.83 (t, J ¼ 6.8 Hz, 3H). 13C NMR
sulphate, ltered and concentrated under reduced pressure. (100 MHz, CDCl3) d 170.17, 163.60, 150.66, 149.63, 140.58,
The crude compound was puried by column chromatography 139.36, 137.71, 133.75, 130.28, 126.81, 123.92, 121.10, 120.76,
eluting with methanol: DCM to the title compound as a solid.
114.88, 67.33, 66.01, 58.39, 52.88, 41.50, 36.12, 33.48, 30.47,
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-N-((1- 30.39, 28.52, 27.37, 21.91, 16.58, 14.78, 12.84. ESI-MS m/z 627.2
methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)- (M + H)+.
40-(morpholinomethyl)biphenyl-3-carboxamide (10a). Yield:
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-N-((7-ethyl-1-methyl-
1
85%; H NMR (400 MHz, DMSO) d 11.50 (s, 1H), 8.17 (s, 1H), 3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)-4-methyl-40-
7.58 (d, J ¼ 6.8 Hz, 2H), 7.38 (m, 3H), 7.22 (s, 1H), 4.32 (s, 2H), (morpholinomethyl)biphenyl-3-carboxamide (10e). Yield: 74%.
3.83 (d, J ¼ 9.6 Hz, 2H), 3.58 (s, 4H), 3.49 (s, 2H), 3.25 (t, J ¼ 10.9 1H NMR (400 MHz, DMSO) d 11.49 (s, 1H), 8.16 (s, 1H), 7.57 (s,
Hz, 2H), 3.08 (m, 2H), 3.02 (m, 1H), 2.75 (s, 2H), 2.37 (s, 6H), 1H), 7.38 (s, 2H), 7.21 (s, 1H), 4.31 (s, 2H), 3.83 (s, 2H), 3.57 (s,
2.25 (s, 3H), 2.10 (s, 3H), 1.65 (s, 6H), 1.53 (m, 2H), 0.84 (s, 3H). 4H), 3.48 (s, 4H), 3.25 (s, 2H), 3.08 (s, 2H), 2.93 (m, 1H), 2.56 (s,
13C NMR (100 MHz, DMSO) d 169.03, 161.53, 150.04, 148.84, 4H), 2.36 (s, 3H), 2.25 (s, 3H), 2.11 (s, 2H), 1.88 (m, 2H), 1.65 (m,
140.62, 139.60, 138.56, 136.95, 132.56, 129.48, 126.37, 122.78, 2H), 1.53 (m, 2H), 1.34 (m, 2H), 1.14 (m, 1H), 0.93 (s, 3H), 0.83
120.76, 111.76, 66.29, 66.16, 62.04, 57.81, 53.14, 41.05, 34.57, (s, 3H). 13C NMR (100 MHz, CDCl3) d 170.17, 163.60, 150.97,
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149.64, 140.66, 140.56, 139.39, 133.79, 130.30, 126.80, 123.91, 139.64, 139.26, 138.20, 137.74, 135.98, 133.10, 129.81, 129.50,
121.05, 120.75, 114.85, 67.33, 58.39, 52.80, 41.48, 36.13, 35.16, 128.26, 128.01, 126.86, 124.58, 123.97, 123.19, 120.79, 116.36,
31.33, 30.48, 29.71, 29.38, 29.00, 28.00, 27.32, 22.71, 16.59, 67.29, 66.70, 62.88, 58.38, 53.41, 41.45, 36.79, 30.44, 28.17, 22.81,
14.78, 14.15, 12.84, 11.44. ESI-MS m/z 641.2 (M + H)+.
16.40, 14.79, 12.86. ESI-MS m/z 661.2 (M + H)+.
N-((7-tert-Butyl-1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquino-
5-Bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-
lin-4-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4- N-((1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)-
methyl-40-(morpholinomethyl)biphenyl-3-carboxamide
(10f). benzamide (10j). Yield: 74%. 1H NMR (400 MHz, DMSO) d 11.38
1
Yield: 67%. H NMR (400 MHz, DMSO) d 11.49 (s, 1H), 8.16 (t, (s, 1H), 8.24 (s, 1H), 7.31 (s, 1H), 7.09 (s, 1H), 4.33 (d, J ¼ 4.6 Hz,
J ¼ 4.4 Hz, 1H), 7.57 (d, J ¼ 8.0 Hz, 2H), 7.45–7.26 (m, 1H), 7.21 2H), 3.83 (d, J ¼ 10.0 Hz, 2H), 3.24 (t, J ¼ 10.8 Hz, 2H), 3.09–2.97
(s, 1H), 4.31 (t, J ¼ 4.0 Hz, 2H), 3.83 (d, J ¼ 10.2 Hz, 2H), 3.64– (m, 2H), 2.97–2.83 (m, 1H), 2.72 (s, 2H), 2.38 (s, 2H), 2.15 (s, 3H),
3.53 (m, 4H), 3.48 (s, 2H), 3.25 (t, J ¼ 11.2 Hz, 2H), 3.14–3.04 (m, 2.09 (s, 3H), 1.59 (m, 6H), 1.51 (m, 2H), 0.79 (t, J ¼ 6.8 Hz, 2H).
2H), 3.04–2.87 (m, 1H), 2.61 (m, 2H), 2.36 (s, 4H), 2.25 (s, 3H), 13C NMR (100 MHz, DMSO) d 167.74, 161.55, 150.38, 141.24,
2.10 (s, 3H), 1.90 (m, 1H), 1.80 (m, 1H), 1.66 (m, 4H), 1.52 (m, 140.33, 132.96, 127.06, 124.75, 120.87, 117.38, 111.76, 66.09,
2H), 1.19 (m, 1H), 1.01 (s, 9H), 0.83 (t, J ¼ 6.8 Hz, 3H). 13C NMR 58.15, 40.75, 34.55, 29.83, 26.61, 24.20, 22.17, 16.00, 14.82,
(100 MHz, DMSO) d 170.04, 159.80, 155.87, 142.77, 141.48, 12.42. ESI-MS m/z 516.1 (M + H)+.
140.30, 139.71, 138.87, 136.80, 129.17, 128.90, 128.52, 128.20,
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-N-((1-
127.99, 116.49, 114.00, 67.12, 67.08, 63.42, 54.66, 51.93, 46.90, methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)-
1
46.37, 36.13, 32.67, 29.95, 29.31, 28.02, 26.85, 24.74, 16.01, 15.61, biphenyl-3-carboxamide (10k). Yield: 77%. H NMR (400 MHz,
14.39, 12.58. ESI-MS m/z 669.2 (M + H)+.
CDCl3) d 13.30 (s, 1H), 7.46 (d, J ¼ 6.6 Hz, 2H), 7.36–7.21 (m,
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-40-(mor- 5H), 4.57 (s, 2H), 3.94 (d, J ¼ 10.2 Hz, 2H), 3.31 (t, J ¼ 9.6 Hz,
pholinomethyl)-N-((1,7,7-trimethyl-3-oxo-2,3,5,6,7,8-hexahydro- 2H), 3.10 (d, J ¼ 6.4 Hz, 2H), 3.02 (m, 1H), 2.95 (s, 2H), 2.30 (s,
isoquinolin-4-yl)methyl)biphenyl-3-carboxamide (10g). Yield: 2H), 1.97 (s, 3H), 1.71 (m, 7H), 1.26 (s, 4H), 0.89 (t, J ¼ 6.4 Hz,
1
69%. H NMR (400 MHz, DMSO) d 11.51 (s, 1H), 8.21 (s, 1H), 3H). 13C NMR (100 MHz, CDCl3) d 169.72, 163.52, 151.73,
7.57 (s, 2H), 7.45–7.26 (m, 1H), 7.21 (s, 1H), 4.31 (t, J ¼ 4.0 Hz, 140.90, 140.21, 139.50, 138.74, 128.80, 127.51, 126.88, 123.88,
2H), 3.83 (d, J ¼ 10.2 Hz, 2H), 3.64–3.53 (m, 4H), 3.48 (s, 2H), 120.97, 72.79, 67.23, 42.50, 31.94, 30.19, 29.71, 29.37, 27.43,
3.25 (t, J ¼ 11.2 Hz, 2H), 3.14–3.04 (m, 2H), 3.04–2.87 (m, 1H), 24.85, 22.71, 22.19, 16.49, 15.07, 14.15, 12.60. ESI-MS m/z 514.2
2.61 (m, 2H), 2.36 (s, 4H), 2.25 (s, 3H), 2.10 (s, 3H), 1.90 (m, 1H), (M + H)+.
1.80 (m, 1H), 1.66 (m, 4H), 1.52 (m, 2H), 1.19 (m, 1H), 1.1 (s, 6H),
40-Chloro-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-N-
0.83 (t, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 170.18, ((1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)-
1
150.25, 149.62, 141.12, 140.84, 139.39, 133.80, 130.37, 126.85, biphenyl-3-carboxamide (10l). Yield: 80%. H NMR (400 MHz,
123.89, 120.75, 114.48, 67.33, 65.87, 58.37, 52.81, 41.47, 38.56, DMSO) d 11.59 (s, 1H), 8.19 (s, 1H), 7.66 (s, 2H), 7.49 (d, J ¼ 5.3
36.25, 34.73, 30.46, 28.75, 28.01, 24.61, 16.65, 14.76, 12.84. ESI- Hz, 2H), 7.42 (s, 1H), 7.25 (s, 1H), 4.33 (s, 2H), 3.82 (s, 2H), 3.25
MS m/z 641.2 (M + H)+.
(s, 2H), 3.05 (m, 3H), 2.75 (s, 2H), 2.37 (s, 2H), 2.27 (s, 3H), 2.10
N-((1,6-Dimethyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)- (s, 3H), 1.64 (s, 6H), 1.53 (m, 2H), 0.83 (s, 3H).
methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-40-
13C NMR (100 MHz, CDCl3) d 169.79, 163.47, 151.54, 140.86,
(morpholinomethyl)biphenyl-3-carboxamide (10h). Yield: 80%. 139.64, 138.67, 137.35, 133.81, 133.52, 128.90, 128.09, 123.66,
1H NMR (400 MHz, DMSO) d 11.49 (s, 1H), 8.15 (t, J ¼ 4.8 Hz, 121.38, 120.95, 115.38, 72.83, 67.24, 59.30, 42.19, 35.95, 31.93,
1H), 7.54 (m, 2H), 7.48–7.30 (m, 3H), 7.22 (s, 1H), 4.33 (d, J ¼ 4.8 30.23, 29.70, 29.37, 27.43, 24.87, 22.70, 22.17, 16.51, 15.05,
Hz, 2H), 3.83 (d, J ¼ 10.0 Hz, 2H), 3.70–3.53 (m, 4H), 3.48 (s, 2H), 14.15, 12.71. ESI-MS m/z 548.2 (M + H)+.
3.25 (t, J ¼ 11.2 Hz, 2H), 3.09 (m, 2H), 3.04–2.90 (m, 2H), 2.36 (s,
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-N-((1-
6H), 2.25 (s, 3H), 2.10 (s, 3H), 1.81 (m, 1H), 1.66 (m, 3H), 1.52 methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)-
(m, 2H), 1.21 (m, 2H), 1.01 (d, J ¼ 6.4 Hz, 3H), 0.83 (t, J ¼ 6.8 Hz, 40-(triuoromethyl)biphenyl-3-carboxamide (10m). Yield: 64%.
3H). 13C NMR (100 MHz, CDCl3) d 170.17, 163.53, 151.06, 1H NMR (400 MHz, DMSO) d 11.48 (s, 2H), 8.19 (s, 1H), 7.86 (m,
149.56, 140.62, 139.90, 139.40, 137.90, 133.59, 130.05, 126.82, 2H), 7.80 (m, 2H), 7.48 (s, 1H), 7.30 (s, 1H), 4.32 (s, 2H), 3.84 (d, J
123.92, 120.93, 120.78, 114.46, 77.39, 77.07, 76.75, 67.33, 66.32, ¼ 7.5 Hz, 2H), 3.26 (m, 2H), 3.11 (m, 2H), 3.03 (m, 1H), 2.75 (s,
62.53, 58.41, 53.14, 41.51, 36.07, 35.78, 30.47, 29.71, 28.51, 2H), 2.38 (s, 2H), 2.27 (s, 3H), 2.10 (s, 3H), 1.65 (s, 6H), 1.53 (m,
24.77, 21.91, 16.62, 14.78, 12.85. ESI-MS m/z 627.2 (M + H)+.
2H), 0.84 (s, 3H). 13C NMR (100 MHz, CDCl3) d 169.57, 165.86,
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-40-(mor- 165.24, 163.10, 151.87, 143.76, 140.96, 139.83, 137.26, 129.70,
pholinomethyl)-N-((2-oxo-2,3,5,6-tetrahydrobenzo[f]isoquinolin-1- 129.46, 128.31, 127.40, 127.21, 125.73, 125.50, 123.93, 122.80,
yl)methyl)biphenyl-3-carboxamide (10i). Yield: 74%. 1H NMR 120.86, 115.87, 67.18, 35.91, 31.94, 30.12, 29.71, 29.37, 27.45,
(400 MHz, DMSO) d 11.65 (s, 1H), 8.33 (s, 1H), 7.92 (s, 1H), 7.58 (d, 24.89, 22.71, 22.13, 22.09, 16.59, 15.17, 14.14, 12.58. ESI-MS m/z
J ¼ 7.6 Hz, 3H), 7.46 (t, J ¼ 8.0 Hz, 3H), 7.30 (s, 3H), 7.25 (s, 1H), 582.2 (M + H)+.
4.47 (d, J ¼ 4.0 Hz, 2H), 3.83 (d, J ¼ 10.0 Hz, 2H), 3.57 (s, 4H), 3.48
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-40-methoxy-4-
(s, 2H), 3.25 (t, J ¼ 11.3 Hz, 2H), 3.09 (d, J ¼ 6.7 Hz, 2H), 3.02 (m, methyl-N-((1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-
1H), 2.80 (m, 2H), 2.64 (m, 2H), 2.36 (s, 4H), 2.28 (s, 6H), 1.66 (m, 4-yl)methyl)biphenyl-3-carboxamide (10n). Yield: 77%. 1H NMR
2H), 1.53 (m, 2H), 1.09 (t, J ¼ 7.0 Hz, 3H), 0.83 (t, J ¼ 6.6 Hz, 3H). (400 MHz, DMSO) d 11.55 (s, 1H), 8.15 (t, J ¼ 4.8 Hz, 1H), 7.55 (d,
13C NMR (100 MHz, CDCl3) d 170.32, 164.21, 149.58, 149.47, J ¼ 8.8 Hz, 2H), 7.35 (s, 1H), 7.18 (s, 1H), 7.00 (d, J ¼ 8.8 Hz, 2H),
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4.32 (d, J ¼ 4.4 Hz, 2H), 3.82 (d, J ¼ 10.8 Hz, 2H), 3.78 (s, 3H), 129.64, 127.78, 121.54, 121.24, 120.71, 119.02, 111.54, 66.31,
3.24 (t, J ¼ 11.2 Hz, 2H), 3.07 (m, 2H), 3.00 (m, 1H), 2.74 (s, 2H), 57.80, 41.08, 38.54, 34.55, 30.28, 26.60, 24.09, 22.14, 21.89,
2.37 (s, 2H), 2.23 (s, 3H), 2.10 (s, 3H), 1.64 (s, 6H), 1.53 (m, 2H), 15.93, 14.46, 12.71. ESI-MS m/z 518.1 (M + H)+.
0.83 (t, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO) d 169.11,
3-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-5-(6-uoropyridin-
161.52, 158.78, 150.38, 148.77, 139.54, 136.82, 132.20, 127.64, 3-yl)-2-methyl-N-((1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquino-
122.36, 120.29, 114.29, 66.30, 57.80, 55.15, 35.10, 30.26, 26.60, lin-4-yl)methyl)benzamide (10s). Yield: 85%; 1H NMR (400
24.08, 22.15, 21.89, 15.93, 14.49, 12.66. ESI-MS m/z 555.2 (M + MHz, DMSO) d 11.51 (s, 1H), 8.53 (s, 1H), 8.27 (t, J ¼ 7.2 Hz, 1H),
H)+.
8.18 (s, 1H), 7.48 (s, 1H), 7.28 (s, 1H), 7.26 (s, 1H), 4.32 (d, J ¼ 4.0
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-N-((1- Hz, 2H), 3.83 (d, J ¼ 10.5 Hz, 2H), 3.25 (t, J ¼ 11.2 Hz, 2H), 3.10
methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)- (d, J ¼ 6.4 Hz, 2H), 3.03 (m, 1H), 2.75 (s, 2H), 2.37 (s, 2H), 2.21 (s,
40-morpholinobiphenyl-3-carboxamide (10o). Yield: 87%. 1H 3H), 2.10 (s, 3H), 1.64 (s, 6H), 1.52 (m, 2H), 0.82 (t, J ¼ 6.4 Hz,
NMR (400 MHz, DMSO) d 11.49 (s, 1H), 8.12 (t, J ¼ 4.6 Hz, 1H), 3H). 13C NMR (100 MHz, CDCl3) d 169.60, 164.35, 163.12,
7.49 (d, J ¼ 8.0 Hz, 2H), 7.34 (s, 1H), 7.16 (s, 1H), 7.01 (d, J ¼ 8.4 161.88, 158.63, 151.99, 148.49, 145.63, 145.45, 140.94, 139.95,
Hz, 2H), 4.31 (d, J ¼ 4.4 Hz, 2H), 3.83 (d, J ¼ 10.8 Hz, 2H), 3.79– 139.62, 134.18, 133.96, 123.72, 120.96, 115.86, 109.66, 109.39,
3.68 (m, 4H), 3.25 (t, J ¼ 11.2 Hz, 2H), 3.13 (m, 4H), 3.08 (m, 2H), 67.20, 59.49, 42.25, 35.85, 30.20, 27.46, 24.92, 22.41, 16.50,
3.05 (s, 1H), 2.74 (s, 2H), 2.38 (s, 2H), 2.22 (s, 3H), 2.10 (s, 3H), 15.12, 12.91. ESI-MS m/z 533.0 (M + H)+.
1.64 (m, 6H), 1.52 (m, 2H), 0.83 (t, J ¼ 6.8 Hz, 3H). 13C NMR (100
5-(6-Chloropyridin-3-yl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-
MHz, CDCl3) d 170.07, 163.26, 151.46, 150.59, 140.83, 138.17, methyl-N-((1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-
127.53, 120.83, 115.70, 115.21, 77.39, 77.07, 76.75, 67.17, 66.76, yl)-methyl)benzamide (10t). Yield: 72%; 1H NMR (400 MHz,
49.10, 36.24, 30.11, 29.71, 27.41, 24.89, 22.25, 22.19, 16.57, DMSO) d 11.48 (s, 1H), 8.71 (d, J ¼ 2.4 Hz, 1H), 8.34–7.91 (m,
15.04, 12.65. ESI-MS m/z 599.0 (M + H)+.
2H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.50 (s, 1H), 7.30 (s, 1H), 4.32 (d, J ¼
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-N-((1- 4.8 Hz, 2H), 3.83 (d, J ¼ 9.6 Hz, 2H), 3.25 (t, J ¼ 11.2 Hz, 2H),
methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)- 3.15–3.06 (m, 2H), 3.03 (m, 1H), 2.75 (s, 2H), 2.38 (s, 2H), 2.27 (s,
30-morpholinobiphenyl-3-carboxamide (10p). Yield: 70%; 1H 3H), 2.10 (s, 3H), 1.66 (m, 6H), 1.57–1.44 (m, 2H), 0.82 (t, J ¼ 6.8
NMR (400 MHz, DMSO) d 11.47 (s, 1H), 8.13 (t, 1H), 7.36 (s, 1H), Hz, 3H). 13C NMR (100 MHz, DMSO) d 168.71, 161.53, 150.10,
7.30 (t, J ¼ 8.0 Hz, 1H), 7.20 (s, 1H), 7.09 (s, 1H), 7.02 (d, J ¼ 8.0 149.31, 149.06, 147.70, 140.37, 139.81, 137.75, 134.67, 133.92,
Hz, 1H), 6.94 (d, J ¼ 8.0 Hz, 1H), 4.32 (d, J ¼ 4.0 Hz, 2H), 3.83 (d, 132.61, 124.25, 123.11, 120.96, 120.67, 111.52, 66.31, 57.78,
J ¼ 12.0 Hz, 2H), 3.76 (s, 4H), 3.25 (t, J ¼ 12.0 Hz, 2H), 3.17 (m, 40.95, 34.58, 30.24, 26.61, 24.08, 22.13, 21.89, 15.93, 14.68,
4H), 3.12–3.05 (m, 1H), 3.02 (m, 1H), 2.75 (s, 2H), 2.38 (s, 2H), 12.70. ESI-MS m/z 549.2 (M + H)+.
2.24 (s, 3H), 2.10 (s, 3H), 1.64 (m, 6H), 1.59–1.43 (m, 2H), 0.83 (t,
3-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-N-((1-
J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO) d 169.10, 161.52, methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)-5-
151.60, 150.09, 148.71, 140.79, 139.53, 137.84, 132.50, 129.45, (6-(4-methylpiperazin-1-yl)pyridin-3-yl)benzamide (10u). Yield:
123.00, 121.03, 120.70, 117.74, 114.25, 113.23, 111.51, 66.28, 82%; 1H NMR (400 MHz, DMSO) d 11.48 (s, 1H), 8.39 (d, J ¼ 2.4
66.11, 57.83, 48.45, 41.14, 34.56, 30.27, 26.63, 24.08, 22.16, Hz, 1H), 8.12 (t, J ¼ 4.8 Hz, 1H), 7.75 (m, 1H), 7.36 (s, 1H), 7.17
21.90, 15.92, 14.54, 12.70. ESI-MS m/z 599.0 (M + H)+.
(s, 1H), 6.89 (d, J ¼ 8.8 Hz, 1H), 4.31 (d, J ¼ 4.8 Hz, 2H), 3.83 (d, J
5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-N-((1- ¼ 10.2 Hz, 2H), 3.52 (s, 4H), 3.25 (t, J ¼ 11.2 Hz, 2H), 3.08 (m,
methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)- 2H), 3.00 (m, 1H), 2.71 (s, 2H), 2.41 (m, 8H), 2.23 (s, 3H), 2.11 (s,
40-((4-methylpiperazin-1-yl)methyl)biphenyl-3-carboxamide
3H), 1.64 (s, 7H), 1.51 (m, 2H), 0.82 (t, J ¼ 6.8 Hz, 3H). 13C NMR
1
(10q). Yield: 72%; H NMR (400 MHz, DMSO) d 11.49 (s, 1H), (100 MHz, CDCl3) d 170.10, 163.57, 158.36, 151.14, 149.59,
8.15 (s, 1H), 7.56 (d, J ¼ 8.0 Hz, 2H), 7.48–7.28 (m, 3H), 7.22 (s, 145.90, 140.75, 139.50, 135.86, 135.42, 132.93, 125.77, 123.08,
1H), 4.32 (s, 2H), 3.83 (d, J ¼ 8.4 Hz, 2H), 3.47 (s, 2H), 3.27 (m, 121.04, 119.94, 114.94, 106.84, 67.32, 58.38, 54.53, 45.80, 44.79,
6H), 3.09 (m, 3H), 2.75 (s, 2H), 2.37 (s, 6H), 2.25 (s, 3H), 2.17 (s, 41.52, 35.88, 30.45, 27.39, 24.85, 22.27, 22.19, 16.52, 14.74,
3H), 2.10 (s, 3H), 1.65 (m, 6H), 1.53 (m, 2H), 0.83 (s, 3H). 13C 12.76. ESI-MS m/z 613.2 (M + H)+.
NMR (100 MHz, DMSO) d 169.04, 161.52, 150.04, 148.84, 139.59,
138.49, 136.96, 132.53, 129.38, 126.35, 122.77, 120.75, 111.51,
66.29, 61.59, 57.81, 54.53, 52.28, 45.45, 34.57, 30.24,
26.60, 24.09, 22.15, 21.89, 15.93, 14.54, 12.67. ESI-MS m/z 626.2
(M + H)+.
Enzyme inhibitory assays
The enzyme inhibitory assays were performed according to the
AlphaLISA assay protocols (PerkinElmer Inc.).
3-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-5-(1-methyl-
1H-pyrazol-4-yl)-N-((1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquino-
Western blot analysis
lin-4-yl)methyl)benzamide (10r). Yield: 60%; 1H NMR (400 MHz, Aer treatment with a series of concentrations of SKLB1049 for
ꢁ
CDCl3) d 12.53 (s, 1H), 7.65 (s, 1H), 7.51 (s, 1H), 7.36 (s, 1H), 7.20 various days at 37 C, cells were harvested, washed in ice-cold
(s, 1H), 7.17 (s, 1H), 4.58 (d, J ¼ 5.2 Hz, 2H), 3.94 (d, J ¼ 10.4 Hz, PBS, and lysed with RIPA buffer (Beyotime, China). And the
2H), 3.86 (s, 3H), 3.31 (t, J ¼ 10.0 Hz, 2H), 3.07 (d, J ¼ 6.8 Hz, protein concentrations were determined by the Bradford
2H), 2.95 (m, 3H), 2.38 (s, 2H), 2.32 (s, 3H), 2.10 (s, 3H), 1.73 (m, method. Proteins were separated by gel electrophoresis on 5–
4H), 0.87 (t, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO) d 10% SDS-PAGE gels and probed with specic antibodies (Cell
169.19, 161.55, 150.03, 148.73, 140.38, 139.69, 135.89, 130.76, Signaling Technology, USA) including anti-H3K27me3, anti-
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H3K27me2, anti-EZH2, anti-H3 and anti-b-actin. All of the
antibodies were used at a 1 : 1000 dilution, and the horseradish
peroxidase-coupled secondary antibodies (Zhong Shan Golden
Bridge Bio-technology, China) were used at 1 : 5000.
7 O. Tamgue, C.-S. Chai, L. Hao, J.-C. D. Zambe, W.-W. Huang,
B. Zhang, M. Lei and Y.-M. Wei, Asian Pac. J. Cancer Prev.,
2013, 14, 5663–5669.
8 C. G. Kleer, Q. Cao, S. Varambally, R. Shen, I. Ota,
S. A. Tomlins, D. Ghosh, R. G. Sewalt, A. P. Otte,
D. F. Hayes, M. S. Sabel, D. Livant, S. J. Weiss, M. A. Rubin
and A. M. Chinnaiyan, Proc. Natl. Acad. Sci. U. S. A., 2003,
100, 11606–11611.
9 N. Wagener, S. Macher-Goeppinger, M. Pritsch, J. Husing,
K. Hoppe-Seyler, P. Schirmacher, J. Ptzenmaier,
A. Haferkamp, F. Hoppe-Seyler and M. Hohenfellner, BMC
Cancer, 2010, 10, 524.
10 E. Hodis, I. R. Watson, G. V. Kryukov, S. T. Arold,
M. Imielinski, J. P. Theurillat, E. Nickerson, D. Auclair,
L. Li, C. Place, D. Dicara, A. H. Ramos, M. S. Lawrence,
K. Cibulskis, A. Sivachenko, D. Voet, G. Saksena,
N. Stransky, R. C. Onofrio, W. Winckler, K. Ardlie,
N. Wagle, J. Wargo, K. Chong, D. L. Morton, K. Stemke-
Hale, G. Chen, M. Noble, M. Meyerson, J. E. Ladbury,
M. A. Davies, J. E. Gershenwald, S. N. Wagner, D. S. Hoon,
D. Schadendorf, E. S. Lander, S. B. Gabriel, G. Getz,
L. A. Garraway and L. Chin, Cell, 2012, 150, 251–263.
11 Z. Li, Y. Wang, J. Qiu, Q. Li, C. Yuan, W. Zhang, D. Wang,
J. Ye, H. Jiang, J. Yang and J. Cheng, OncoTargets Ther.,
2013, 4, 2532–2549.
12 S. Varambally, S. M. Dhanasekaran, M. Zhou, T. R. Barrette,
C. Kumar-Sinha, M. G. Sanda, D. Ghosh, K. J. Pienta,
R. G. Sewalt, A. P. Otte, M. A. Rubin and A. M. Chinnaiyan,
Nature, 2002, 419, 624–629.
13 S. Guo, J. K. Chan, J. Iqbal, T. McKeithan, K. Fu, B. Meng,
Y. Pan, W. Cheuk, D. Luo, R. Wang, W. Zhang,
T. C. Greiner and W. C. Chan, Clin. Cancer Res., 2014, 20,
3078–3086.
Cell viability assay
Human cancer cell lines used in this investigation were
obtained from American Type Culture Collection (ATCC,
Rockville, MD). The MTT assay was used to measure the cell
viability aer compounds treatment. Cells were seeded in 96-
well microplates at a density of 2–5 ꢃ 103 cells per well, and
then, cultured for 24 h. Aer treatment with various concen-
trations of compound for 4 or 6 days, 20 mL of MTT solution (5
mg mLꢀ1) was added to each well and incubated 2–4 h at 37 ꢁC,
the formazan crystals were dissolved with 50 mL of acidied SDS
(20%, w/v). The absorbance of each well was measured with
Spectra MAX M5 microplate spectrophotometer at 570 nm
wavelength, and the median inhibitory concentration (IC50) of
each cell line was calculated.
Flow cytometry for cell cycle analysis
The cell cycle was measured by FCM assay. Approximately 1 ꢃ
105 cells were collected aer treatment with vehicle or
SKLB1049 in various concentrations for 14 days and xed with
70% ethanol overnight. The cells were then washed with cold
PBS and stained with 50 mg mLꢀ1 propidium iodide containing
100 mg mLꢀ1 RNase, and 0.1% Triton X-100. The cell cycle
proles were determined on a FACS Calibur ow cytometer
(Becton Dicknson, USA) and analyzed using the ModFit LT 3.2
soware (Verity Soware House, USA).
14 R. D. Morin, N. A. Johnson, T. M. Severson, A. J. Mungall,
J. An, R. Goya, J. E. Paul, M. Boyle, B. W. Woolcock,
F. Kuchenbauer, D. Yap, R. K. Humphries, O. L. Griffith,
S. Shah, H. Zhu, M. Kimbara, P. Shashkin, J. F. Charlot,
M. Tcherpakov, R. Corbett, A. Tam, R. Varhol, D. Smailus,
M. Moksa, Y. Zhao, A. Delaney, H. Qian, I. Birol, J. Schein,
R. Moore, R. Holt, D. E. Horsman, J. M. Connors, S. Jones,
S. Aparicio, M. Hirst, R. D. Gascoyne and M. A. Marra, Nat.
Genet., 2010, 42, 181–185.
Acknowledgements
We thank Zhejiang Apeloa Medical Technology Co., Ltd, for
their technical and nancial support. We also thank Lei Li of
State Key Laboratory of Biotherapy (Sichuan University) for
NMR measurements.
Notes and references
15 R. D. Morin, M. Mendez-Lago, A. J. Mungall, R. Goya,
K. L. Mungall, R. D. Corbett, N. A. Johnson,
T. M. Severson, R. Chiu, M. Field, S. Jackman,
M. Krzywinski, D. W. Scott, D. L. Trinh, J. Tamura-Wells,
S. Li, M. R. Firme, S. Rogic, M. Griffith, S. Chan,
O. Yakovenko, I. M. Meyer, E. Y. Zhao, D. Smailus,
M. Moksa, S. Chittaranjan, L. Rimsza, A. Brooks-Wilson,
J. J. Spinelli, S. Ben-Neriah, B. Meissner, B. Woolcock,
M. Boyle, H. McDonald, A. Tam, Y. Zhao, A. Delaney,
T. Zeng, K. Tse, Y. Buttereld, I. Birol, R. Holt, J. Schein,
D. E. Horsman, R. Moore, S. J. Jones, J. M. Connors,
M. Hirst, R. D. Gascoyne and M. A. Marra, Nature, 2011,
476, 298–303.
1 R. Siegel, J. Ma, Z. Zou and A. Jemal, Ca-Cancer J. Clin., 2014,
64, 9–29.
2 S. Wee, D. Dhanak, H. Li, S. A. Armstrong, R. A. Copeland,
R. Sims, S. B. Baylin, X. S. Liu and L. Schweizer, Ann. N. Y.
Acad. Sci., 2014, 1309, 30–36.
3 R. Margueron and D. Reinberg, Nature, 2011, 469, 343–349.
4 J. A. Simon and C. A. Lange, Mutat. Res., 2008, 647, 21–29.
5 F. M. Raaphorst, F. J. van Kemenade, T. Blokzijl, E. Fieret,
K. M. Hamer, D. P. E. Satijn, A. P. Otte and
C. J. L. M. Meijer, Am. J. Pathol., 2000, 157, 709–715.
6 H. P. J. Visser, M. J. Gunster, H. C. Kluin-Nelemans,
E. M. M. Manders, F. M. Raaphorst, C. J. L. M. Meijer,
R. Willemze and A. P. Otte, Br. J. Haematol., 2001, 112, 16 M. T. McCabe, H. M. Ott, G. Ganji, S. Korenchuk,
950–958.
C. Thompson, G. S. Van Aller, Y. Liu, A. P. Graves,
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 25967–25978 | 25977

View Article Online
RSC Advances
Paper
A. D. P. Iii, E. Diaz, L. V. LaFrance, M. Mellinger, 22 S. Garapaty-Rao, C. Nasveschuk, A. Gagnon, E. Y. Chan,
C. Duquenne, X. Tian, R. G. Kruger, C. F. McHugh,
M. Brandt, W. H. Miller, D. Dhanak, S. K. Verma,
P. J. Tummino and C. L. Creasy, Nature, 2012, 492, 108–112.
17 S. K. Knutson, S. Kawano, Y. Minoshima, N. M. Warholic,
P. Sandy, J. Busby, S. Balasubramanian, R. Campbell,
F. Zhao, L. Bergeron, J. E. Audia, B. K. Albrecht,
J.-C. Harmange, R. Cummings and P. Trojer, Chem. Biol.,
2013, 20, 1329–1339.
K. C. Huang, Y. Xiao, T. Kadowaki, M. Uesugi, 23 W. D. Bradley, S. Arora, J. Busby, S. Balasubramanian,
G. Kuznetsov, N. Kumar, T. J. Wigle, C. R. Klaus,
C. J. Allain, A. Raimondi, N. J. Waters, J. J. Smith,
M. Porter-Scott, R. Chesworth, M. P. Moyer,
R. A. Copeland, V. M. Richon, T. Uenaka, R. M. Pollock,
K. W. Kuntz, A. Yokoi and H. Keilhack, Mol. Cancer Ther.,
2014, 13, 842–854.
V. S. Gehling, C. G. Nasveschuk, R. G. Vaswani, C. C. Yuan,
C. Hatton, F. Zhao, K. E. Williamson, P. Iyer, J. Mendez,
R. Campbell, N. Cantone, S. Garapaty-Rao, J. E. Audia,
A. S. Cook, L. A. Dakin, B. K. Albrecht, J. C. Harmange,
D. L. Daniels, R. T. Cummings, B. M. Bryant, E. Normant
and P. Trojer, Chem. Biol., 2014, 21, 1463–1475.
18 S. K. Knutson, N. M. Warholic, T. J. Wigle, C. R. Klaus, 24 T. Kaiho, K. San-Nohe, S. Kajiya, T. Suzuki, K. Otsuka, T. Ito,
C. J. Allain, A. Raimondi, M. Porter Scott, R. Chesworth,
M. P. Moyer, R. A. Copeland, V. M. Richon, R. M. Pollock,
J. Kamiya and M. Maruyama, J. Med. Chem., 1989, 32, 351–
357.
K. W. Kuntz and H. Keilhack, Proc. Natl. Acad. Sci. U. S. A., 25 F. Kopp, C. F. Stratton, L. B. Akella and D. S. Tan, Nat. Chem.
2013, 110, 7922–7927. Biol., 2012, 8, 358–365.
19 S. K. Knutson, T. J. Wigle, N. M. Warholic, C. J. Sneeringer, 26 N. Y. Wang, W. Q. Zuo, Y. Xu, C. Gao, X. X. Zeng, L. D. Zhang,
C. J. Allain, C. R. Klaus, J. D. Sacks, A. Raimondi,
C. R. Majer, J. Song, M. P. Scott, L. Jin, J. J. Smith,
X. Y. You, C. T. Peng, Y. Shen, S. Y. Yang, Y. Q. Wei and
L. T. Yu, Bioorg. Med. Chem. Lett., 2014, 24, 1581–1588.
E. J. Olhava, R. Chesworth, M. P. Moyer, V. M. Richon, 27 J. L. Burgess, N. Johnson, S. D. Knight, L. Lafrance,
R. A. Copeland, H. Keilhack, R. M. Pollock and
K. W. Kuntz, Nat. Chem. Biol., 2012, 8, 890–896.
20 W. Qi, H. Chan, L. Teng, L. Li, S. Chuai, R. Zhang, J. Zeng,
W. H. Miller, K. Newlander, S. Romeril, M. B. Rouse,
X. Tian, S. K. Verma and D. Suarez, PCT Int. Appl., WO
2012005805, 2012.
M. Li, H. Fan, Y. Lin, J. Gu, O. Ardayo, J. H. Zhang, 28 S. K. Verma, X. Tian, L. V. LaFrance, C. Duquenne,
X. Yan, J. Fang, Y. Mi, M. Zhang, T. Zhou, G. Feng,
Z. Chen, G. Li, T. Yang, K. Zhao, X. Liu, Z. Yu, C. X. Lu,
P. Atadja and E. Li, Proc. Natl. Acad. Sci. U. S. A., 2012, 109,
21360–21365.
D. P. Suarez, K. A. Newlander, S. P. Romeril, J. L. Burgess,
S. W. Grant, J. A. Brackley, A. P. Graves, D. A. Scherzer,
A. Shu, C. Thompson, H. M. Ott, G. S. Aller,
C. A. Machutta, E. Diaz, Y. Jiang, N. W. Johnson,
S. D. Knight, R. G. Kruger, M. T. McCabe, D. Dhanak,
P. J. Tummino, C. L. Creasy and W. H. Miller, ACS Med.
Chem. Lett., 2012, 3, 1091–1096.
21 K. D. Konze, A. Ma, F. Li, D. Barsyte-Lovejoy, T. Parton,
C. J. MacNevin, F. Liu, C. Gao, X.-P. Huang, E. Kuznetsova,
M. Rougie, A. Jiang, S. G. Pattenden, J. L. Norris,
L. I. James, B. L. Roth, P. J. Brown, S. V. Frye, 29 J. R. Simard, M. Plant, R. Emkey and V. Yu, Assay Drug Dev.
C. H. Arrowsmith, K. M. Hahn, G. G. Wang, M. Vedadi and
Technol., 2013, 11, 152–162.
J. Jin, ACS Chem. Biol., 2013, 8, 1324–1334.
25978 | RSC Adv., 2015, 5, 25967–25978
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