Synthesis and biological evaluation of novel 8-morpholinoimidazo[1,2-a]pyrazine derivatives bearing phenylpyridine/phenylpyrimidine-carboxamides

Article abstract of DOI:10.3390/molecules22020310

Herein we designed and synthesized three series of novel 8-morpholinoimidazo[1,2-a] pyrazine derivatives bearing phenylpyridine/phenylpyrimidine-carboxamides (compounds 12a-g, 13a-g and 14a-g). All the compounds were evaluated for their IC₅₀ values against three cancer cell lines (A549, PC-3 and MCF-7). Most of the target compounds exhibited moderate cytotoxicity against the three cancer cell lines. Two selected compounds 14b, 14c were further tested for their activity against PI3Kα kinase, and the results indicated that compound 14c showed inhibitory activity against PI3Kα kinase with an IC₅₀ value of 1.25 μM. Structure-activity relationships (SARs) and pharmacological results indicated that the replacement of the thiopyranopyrimidine with an imidazopyrazine was beneficial for the activity and the position of aryl group has a significant influence to the activity of these compounds. The compounds 13a-g in which an aryl group substituted at the C-4 position of the pyridine ring were more active than 12a-g substituted at the C-5 position. Moreover, the cytotoxicity of compounds 14a-g bearing phenylpyrimidine-carboxamides was better than that of the compounds 12a-g, 13a-g bearing phenylpyridine-carboxamides. Furthermore, the substituents on the benzene ring also had a significant impact on the cytotoxicity and the pharmacological results showed that electron donating groups were beneficial to the cytotoxicity.

Full text of DOI:10.3390/molecules22020310

molecules
Article
Synthesis and Biological Evaluation of Novel
-Morpholinoimidazo[1,2-a]pyrazine Derivatives
8
Bearing Phenylpyridine/Phenylpyrimidine-
Carboxamides
1
1,2,
1
1
2
2
Shan Xu , Chengyu Sun
*, Chen Chen , Pengwu Zheng , Yong Zhou , Hongying Zhou and
1
,
Wufu Zhu *
1
School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, China;
Shanxu9891@126.com (S.X.); chen5950677@163.com (C.C.); Zhengpw@126.com (P.Z.)
Department of Pharmacy, The Affiliated Hospital of Chongqing Three Gorges Medical College,
Chongqing 404000, China; zhouyong_0801@126.com (Y.Z.); 15223632072@163.com (H.Z.)
Correspondence: sunchengyu0902@126.com (C.S.); zhuwufu-1122@163.com (W.Z.);
Tel./Fax: +86-791-8380-2393 (W.Z.)
2
*
Academic Editor: Diego Muñoz-Torrero
Received: 29 December 2016; Accepted: 15 February 2017; Published: 17 February 2017
Abstract: Herein we designed and synthesized three series of novel 8-morpholinoimidazo[1,2-a]
pyrazine derivatives bearing phenylpyridine/phenylpyrimidine-carboxamides (compounds 12a
3a and 14a ). All the compounds were evaluated for their IC50 values against three cancer
cell lines (A549, PC-3 and MCF-7). Most of the target compounds exhibited moderate cytotoxicity
against the three cancer cell lines. Two selected compounds 14b 14c were further tested for their
activity against PI3K kinase, and the results indicated that compound 14c showed inhibitory
activity against PI3K kinase with an IC50 value of 1.25 M. Structure-activity relationships (SARs)
–g,
1
–
g
–g
,
α
α
µ
and pharmacological results indicated that the replacement of the thiopyranopyrimidine with
an imidazopyrazine was beneficial for the activity and the position of aryl group has a significant
influence to the activity of these compounds. The compounds 13a
substituted at the C-4 position of the pyridine ring were more active than 12a
position. Moreover, the cytotoxicity of compounds 14a bearing phenylpyrimidine-carboxamides
was better than that of the compounds 12a 13a bearing phenylpyridine-carboxamides.
–
g
in which an aryl group
–g
substituted at the C-5
–g
–
g,
–g
Furthermore, the substituents on the benzene ring also had a significant impact on the cytotoxicity and
the pharmacological results showed that electron donating groups were beneficial to the cytotoxicity.
Keywords:
imidazopyrazine; phenylpyridine-carboxamide; phenylpyrimidine-carboxamide;
PI3Kα; synthesis
1
. Introduction
At present, with the in-depth study of cancer pathogenesis at the molecular level, molecular
targeted therapy has become a research hotspot. Numerous molecular signaling pathways have been
shown to be associated with the development and progression of cancer. The PI3K-Akt-mTOR signaling
pathway is one of them, which was found frequently activated in different cancers. It can promote
the development of tumor by inhibiting cell apoptosis, promoting angiogenesis, cell proliferation and
invasion [
occurrence and development of cancer. Currently, a number of inhibitors acting on this pathway have
been reported, such as PI-103, GDC-0941, MLN0128, PKI-587 and ETP46992 [ ] (Figure 1). Among
them, PI-103 was the first PI3K/mTOR dual inhibitor with IC50 values of 8 and 20 nM against PI3K
1,2]. Therefore, the inhibition of the PI3K-Akt-mTOR pathway can effectively suppress the
3–7
α
Molecules 2017, 22, 310; doi:10.3390/molecules22020310
www.mdpi.com/journal/molecules

Molecules 2017, 22, 310
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and mTOR, respectively [
3
]. GDC-0941 was the first PI3K inhibitor entering clinical studies and was
developed by Genentech based on the modification and optimization of PI-103 [4].
Figure 1. Structures of some reported PI3K pathway inhibitors and design strategy of the target compounds.
In our previous research [
and synthesized as PI3K-Akt-mTOR pathway inhibitors, such as compound
8
–
11], several series of thiopyranopyrimidine derivatives were designed
and compound II
I
.
In order to screen for compounds with excellent anti-tumor activity, further studies on these compounds
were carried out in this research. According to the reported PI3K pathway inhibitors, we found that
most of them contained (fused-)pyrimidine/pyrazine skeleton structure and the SARs results indicated
that this skeleton was beneficial to the activity. Therefore, we replaced the thiopyranopyrimidine
with an imidazopyrazine inspired by ETP46992 which is a PI3K
α inhibitor with an IC50 value of
0.9 nM. In addition, we maintained the amide scaffold based on the SARs results of our previous
research. Then, we changed the position of aryl group on the pyridine/pyrimidine to investigate
the effect on activity. Lastly, we introduced various substituents on the benzene ring to investigate
their effect on anti-tumor activity. As a result, three series of 8-morpholinoimidazo[1,2-a]pyrazine
derivatives bearing phenylpyridine-carboxamides or phenylpyrimidine-carboxamides were designed
and synthesized. The design strategy for all target compounds is shown in Figure 1.
Herein, we report the design, synthesis and the cytotoxicity of all target compounds against
three human cancer cell lines A549 (lung cancer), PC-3 (prostate cancer), and MCF-7 (breast cancer),
as well as the activity of two selected compounds 14b, 14c against PI3Kα kinase. In addition,
a molecular modeling docking study of the most promising compound 14c with PI3K
α kinase
was performed.
2
. Results and Discussion
The preparations of target compounds 12a–g, 13a–g and 14a–g were described in Scheme 1.
The key intermediate
was brominated to yield 3,5-dibromopyrazin-2-amine (
substituted with morpholine to give compound . Subsequently, compound
5
was synthesized from 2-aminopyrazine in five steps. The 2-aminopyrazine
) and the 3-bromine atom was then
was cyclized with
1
2
2

Molecules 2017, 22, 310
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chloroacetaldehyde to give compound
3. Compound
3
was coupled with p-bromonitrobenzene
by Suzuki coupling and then reduced with hydrazine hydrate to obtain the key intermediate
compound . The 5-(substituted phenyl)picolinic acids 6a , 4-(substituted phenyl)picolinic acids
and 6-(substituted phenyl)pyrimidine-4-carboxylic acids 8a were synthesized through the
procedures reported in our previous research [11 12]. Carboxylic acids 6a 7a and 8a were
converted into the corresponding acyl chlorides and then immediately amidated with compound
to obtain the target compounds 12a 13a and 14a
target compound are listed in Table 1.
5
–g
7
a
–
g
–g
,
–
g,
–
g
–g
5
–g
,
–g
–g, respectively (Scheme 1). The yields for each
Scheme 1. Synthetic routes of target compounds 12a
–
g
,
13a
–
g
and 14a
–
g
. Reagents and conditions:
◦
◦
(
a) Br , pyridine, CH Cl 40 C, 1.5 h; (b) morpholine, 80 C, reflux, 1 h; (c) chloroacetaldehyde,
2
2
◦
2,
◦
isopropanol, 45 C~65 C, 2 h; (d) (1) bis(pinacolato)diboron, KAc, Pd(PPh )Cl , p-bromonitrobenzene,
3
2
1
,2-dimethoxyethane, reflux, 2–3 h; (2) H O, Na CO , Pd(PPh ) Cl , reflux, 6–8 h; (e) hydrazine
2 2 3 3 2 2
hydrate, FeCl , Activated carbon, ethanol, reflux, 1–3 h; (f) thionyl chloride, reflux, 1 h; (g) DIPEA,
3
CH Cl , 0.5 h.
2
2
2
.1. Biological Evaluation
The target compounds 12a
–
g,
13a
–
g
and 14a
–
g
were evaluated for their in vitro cytotoxicity
against three cancer cell lines (A549, PC-3 and MCF-7) by the standard MTT assay, with compound II
and GDC-0941 as positive controls. Two selected compounds (14b
,
14c) were further tested for PI3K
α
®
kinase inhibitory activity together with reference compound II, GDC-0941 and PI-103 by a Kinase-Glo
Luminescent Kinase Assay. The results expressed as inhibition rates or IC50 values are summarized in
Tables 1 and 2, where the values are the average of at least two independent experiments.

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Table 1. Structures and cytotoxicity of target compounds 12a–g, 13a–g and 14a–g.
IC50 (µM) a
Compd.
R
Yield (%)
A549
PC-3
MCF-7
1
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
4g
Compound II
H
3-fluoro
96.1
79.4
90.2
83.5
93.3
72.6
89.1
97.0
88.0
91.2
81.8
93.1
91.5
98.0
82.7
79.5
77.8
90.9
93.2
80.8
77.6
-
58.73 ± 1.99
50.28 ± 2.04
ND
NA
NA
ND
NA
ND
ND
NA
NA
1
1
1
1
1
1
1
1
NA
NA
2,4-difluoro
4-trifluoromethyl
4-methoxy
3-methyl
4-methyl
H
NA
NA
15.27 ± 0.88
69.70 ± 1.13
NA
NA
19.11 ± 1.10
68.12 ± 0.89
NA
26.78 ± 1.47
42.32 ± 0.86
NA
NA
NA
55.64 ± 1.32
59.68 ± 2.22
65.61 ± 0.52
NA
3-fluoro
NA
NA
1
1
1
1
1
1
1
1
2,4-difluoro
4-trifluoromethyl
4-methoxy
3-methyl
4-methyl
H
4-methyl
4-methoxy
4-bromo
4-chloro
4-fluoro
NA
41.99 ± 1.49
23.95 ± 0.81
10.53 ± 0.82
11.23 ± 1.08
10.75 ± 1.62
8.88 ± 0.97
6.39 ± 1.04
14.36 ± 1.01
17.38 ± 1.01
58.17 ± 4.67
NA
62.48 ± 1.99
74.92 ± 1.14
74.19 ± 1.86
7.89 ± 0.81
NA
6.69 ± 2.04
10.23 ± 1.62
NA
NA
25.04 ± 1.19
12.65 ± 0.95
37.14 ± 0.97
NA
1
1
1
1
NA
47.06
ND
NA
4-trifluoromethyl
96.63 ± 2.58
11.34 ± 0.11
0.20 ± 0.08
b,c
-
-
8.37 ± 0.10
9.26 ± 0.82
b
GDC-0941
-
6.99 ± 0.21
0.07 ± 0.03
a
The values are an average of two separate determinations; b Used as a positive control; c Compounds reported in
our previous research; NA: No Activity; ND: Not Determine.
Table 2. PI3Kα kinase activity of selected compounds and positive controls.
PI3Kα
Compound No.
IC50 (µM) a or 10 µM Inhibitory % a
∼
1
1
4b
4c
Compound II
58.0 ± 4.4 =10 µM
1.25 ± 0.13
7.39 ± 0.19 [11]
0.003 [4]
b,c
b
GDC-0941
PI-103 b
0.075 ± 0.018
a
The values are an average of two separate determinations; b Used as a positive control; c Compounds reported in
our previous research.
From Table 1, we can see that most of the target compounds showed moderate cytotoxicity against
the three cancer cell lines, with IC50 values ranging from 6.39 to 74.9 µM and most of them showed
more activity against the A549 cancer cell line than the other two cancer cell lines (PC-3, MCF-7).
The IC50 values of the most promising compound 14c against the A549 cancer cell lines was 6.39
µM, which was superior to the previously prepared compound II (IC50 values 8.37 µM). This means
that the replacement of the thiopyranopyrimidine with an imidazopyrazine was beneficial to the
cytotoxicity. Comparing the activity of the three series of target compounds, we found that an aryl
group at the pyridine C-4 position (compounds 13a
–g) was more active than that at the C-5 position
(
compounds 12a ). Besides, the data of Table 1 show that the cytotoxicity of compounds 14a–g
–
g

Molecules 2017, 22, 310
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bearing a phenylpyrimidine-carboxamides was better than that of compounds 12a
–
g,
13a
–g
bearing
a phenylpyridine-carboxamide. In addition, due to the different substituents on the benzene ring,
the cytotoxicity of the same class of compounds varied too. The results showed that the compounds
containing electron donating groups (i.e., 12e
the compounds containing electron-withdrawing ones (i.e., 12b–d, 13b–d and 14d–g).
The activity of the selected compounds 14b 14c as well as the positive control compounds against
the PI3K kinase is shown in Table 2. The activity of compound 14b against PI3K kinase was
moderate, with an inhibitory value of 58.0% at 10 M. The most promising compound 14c showed
an IC50 value of 1.25 M against PI3K kinase, which was 6-fold more active than the lead compound II
IC50 values 7.39 M). This indicated that the replacement of the thiopyranopyrimidine structure with
–g, 13e–g and 14a–c) exhibited higher cytotoxicity than
,
α
α
µ
µ
α
(
µ
the imidazopyrazine moiety enhanced the activity against PI3Kα kinase.
As described above, the replacement of the thiopyranopyrimidine with an imidazopyrazine
was beneficial to the activity of these derivatives. In addition, the series of compounds bearing
phenylpyrimidine-carboxamide structures showed the best activity among all three series of target
compounds. Moreover, electron donating substituents on the benzene ring were beneficial to the
cytotoxicity. Comparing Tables 1 and 2, we found that the differences in cytotoxicity between
compounds 14b and 14c were not particularly significant, but they were significant for PI3Kα activity.
This suggested that compound 14b may have potential activity on other targets, which may be
an interesting direction in our future studies.
2
.2. Molecular Docking Study
To explore the binding modes of the target compounds with the active site of PI3Kα, molecular
docking simulation studies were carried out by using the SURFLEX-DOCK module of the SYBYL
software package. Based on the results of the in vitro activity tests, we selected compound 14c as
a ligand example, and the structure of PI3K
model [9,10].
α (PDB ID code: 4L23) was selected as the docking
The binding mode of compound 14c with PI3K
the imidazopyrazine scaffold and morpholine group of compound 14c were tightly packed into the
channel formed by the amino acid residues of PI3K . We found that the compound 14c formed
two hydrogen bonds with residues of the PI3K molecule: the oxygen atom of morpholine with the
α is shown in Figure 2a,b. As depicted in Figure 2a,
α
α
Val851 residue and the carbonyl of the amide scaffold with the SER773 residue.
This result indicates that the morpholine and the amide scaffold are very important to maintain
the activity against PI3Kα kinase of the target compounds. In addition, according to the comparison of
docking results of the compound 14c and PI-103 (Figure 2b), we see that the main skeleton structures
of compound 14c and the PI-103 molecule scaffold were almost completely overlapped, especially
the morpholine group. This result suggested that the replacement of the thiopyranopyrimidine with
an imidazopyrazine was advantageous in improving the activity. What’s more, the docking results
also suggest that the benzene ring of phenylpyridine/phenylpyrimidine cannot bind well to PI3Kα,
which may be one reason why the anti-tumor activity of these compounds is not particularly good.
For this reason, we may be able to improve the anti-tumor activity of them by modify this part in the
future work.

Molecules 2017, 22, 310
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Figure 2. Binding models: The proteins were displayed by cyan ribbon. Hydrogen bonds were showed
in dashed lines (yellow). ( ) Binding models of compound 14c (shown in Capped Sticks) with PI3K
b) Binding models of compound 14c and native ligand PI 103 (shown in red Sticks) with PI3Kα.
a
α;
(
3
. Experimental Section
3
.1. General Information
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik,
Flawil, Switzerland). NMR spectra were recorded using a Bruker 400 MHz spectrometer (Bruker
Bioscience, Billerica, MA, USA) with TMS as an internal standard. Mass spectra (MS) were taken in
ESI mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA). TLC analysis was carried out on
silica gel plates GF254 (Qingdao Haiyang Chemical, Qingdao, China). All the reagents were obtained
from commercial suppliers and used without purification, unless otherwise specified.

Molecules 2017, 22, 310
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3
3
.2. Chemistry
,5-Dibromo-2-aminopyrazine (1)
A solution of dichloromethane (200 mL) and pyridine (25.3 mL, 0.315 mol) was added to
a three-necked flask containing 2-aminopyrazine (14.27 g, 0.15 mol) and stirred well. In the dark and
while refluxing a solution of bromine (16.2 mL, 0.315 mol) in dichloromethane (100 mL) was slowly
added dropwise to the three-necked flask. About 1 h later the addition finished and the mixture
◦
was refluxed at 40 C for 30 min more. After TLC monitoring indicated the reaction was complete,
the reaction mixture was cooled to room temperature and distilled water (50 mL) was added and the
mixture was stirred vigorously for 10 min. Then the organic layer was collected and washed twice
with distilled water. Silica gel (10 g) and activated carbon (1 g) were added to the organic layer and the
mixture was decolorized under reflux for 30 min. After hot filtration, the filtrate was collected and
vacuum distilled. The residue was refluxed with n-hexane (45 mL) for 2 h, filtered while hot again and
1
the solid product was dried and weighed to give 18.15 g of a pale yellow solid (47.8% yield). H-NMR
(
DMSO-d ) δ 8.14 (s, 1H), 7.01 (s, 2H).
6
3
-Morpholino-5-bromo-2-aminopyrazine (2)
A mixture of morpholine (50 mL) and 3,5-dibromo-2-aminopyrazine (12.50 g) was refluxed for
h until the reaction was complete by TLC analysis. The solution was cooled to room temperature and
1
added to a beaker containing ice water (300 mL) with continuous stirring. The solid was precipitated
and filtered. After drying, 12.30 g of a yellow solid with a metallic luster was obtained (a yield of
1
9
6.1%). H-NMR (DMSO-d ) δ 7.70 (s, 1H), 6.28 (s, 2H), 3.85–3.62 (m, 4H), 3.04 (d, J = 4.0 Hz, 4H).
6
4
-(6-Bromoimidazo[1,2-a]pyrazin-8-yl)morpholine (3)
Chloroacetaldehyde (4.4 g) was mixed with isopropanol (15 mL) and a portion of the mixture
(
10 mL) was added to a round bottom flask containing compound 2 (1.7 g). Then the temperature
◦
was raised to 45 C and stirring was continued. After 1 h, the remaining solution was added and the
temperature raised to 65 C with continuous stirring. After TLC indicated the reaction was finished,
◦
the reaction was stopped and cooled to room temperature. Then the solution was added into a beaker
containing ice water (300 mL) with continuous stirring. The brown-yellow solid that precipitated
1
was filtered and dried to give the product in a yield of 26.8%. H-NMR (DMSO-d )
δ 7.93 (s, 1H),
6
7
.25 (d, J = 7.3 Hz, 1H), 7.01 (d, J = 7.3 Hz, 1H), 3.45 (s, 4H), 3.77 (d, J = 4.3 Hz, 4H).
4
-(6-(4-Nitrophenyl)imidazo[1,2-a]pyrazin-8-yl)morpholine (4)
Compound
4 was prepared using the synthetic method described in our previous article [11].
Briefly, a solution of 1-bromo-4-nitrobenzene (2 g, 0.01 mol), bis(pinacolato) diboron (3.8 g, 0.015 mol),
potassiumacetate (2.9 g, 0.03 mol) and bistriphenylphosphine palladium dichloride (0.18 g, 0.25 mmol)
◦
was added to 1,4-dioxane (50 mL) and heated to 80 C. The reaction was continued for 3 h under N
2
and monitored by TLC. H O (20 mL) was added to the solution and continued for 5 min. Subsequently,
2
compound
3 (1.7 g, 0.006 mol), Na CO (2.1 g, 0.02 mol) and bistriphenylphosphine dichloride (0.18 g,
2 3
◦
0
.25 mmol) were added successively, then the mixture was heated to 100 C. The reaction was continued
for about 8 h. The mixture was cooled and concentrated under vacuum. H O (100 mL) was added,
2
stirred for 30 min and then filtered. The filter cake was decolorized with activated carbon (5 g) and
silica gel (2 g) in a mixed solvent (CH Cl :CH OH = 5:1, 100 mL), filtered while hot and concentrated
2
2
3
+
1
under vacuum to obtain
.78 (d, J = 8.2 Hz 2H), 8.33 (d, J = 8.2 Hz 2H), 8.01 (s, 1H), 7.20 (d, J = 8.7 Hz, 1H), 6.95 (d, J = 8.7 Hz,
H), 3.91 (s, 4H), 3.77 (s, 4H).
4 with a yield of 63.0%. ESI-MS m/z: [M + H] 326.1. H-NMR (DMSO-d ) δ
6
8
1

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4
-(8-Morpholinoimidazo[1,2-a]pyridin-6-yl)aniline (5)
The synthesis of compound is similar to the synthetic method reported in our previous
article [11]. Compound (3.3 g, 0.01 mol) was refluxed with hydrazine hydrate (5 g, 0.1 mol),
5
4
ferric chloride (0.5 g, 0.002 mol) and an appropriate amount of activated carbon in ethanol (50 mL)
for 30 min–1 h and monitored by TLC. After filtering while hot and concentrating under vacuum
+
+
1
compound
5
was obtain in a yield of 60.2%. ESI-MS m/z: [M + H] 296.2. [M + H] 326.1. H-NMR
(DMSO-d6) δ 8.12 (d, J = 8.5 Hz 2H), 7.89 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H),
6
.60 (d, J =8.5 Hz 2H), 5.56 (s, 2H), 3.75 (d, J = 3.5 Hz, 4H), 3.32 (s, 4H).
General Procedure for the Preparation of Compounds 6a–g, 7a–g, 8a–g, 9a–g, 10a–g and 11a–g
Compounds 6a 7a and 8a were synthesized according to the procedures reported by our
research group [11 12]. Then compounds 6a 7a and 8a (0.02 mmol) were dissolved in thionyl
chloride (8 mL) and refluxed for 1 h. The reaction mixture was evaporated to yield the corresponding
–g
,
–g
–g
,
–g
,
–g
–g
chloride which was dissolved in dichloromethane (10 mL). The solution of 9a–g, 10a–g or 11a–g was
used for the next step without further purification.
General Procedure for the Preparation of the Target Compounds 12a–g, 13a–g and 14a–g
A solution of phenylpyrimidine chloride 9a 10a or 11a in dichloromethane (10 mL)
was added dropwise to a solution of (0.12 mol) and N,N-diisopropylethylamine (0.048 mol) in
–
g,
–
g
–g
5
dichloromethane (10 mL) in an ice bath. Upon completion of the addition, the reaction mixture
was removed from the ice bath, held at room temperature for 15 min and monitored by thin-layer
chromatography (TLC). The mixture was washed with 10% K CO (50 mL
×
3) followed by
1), and the organic layer was separated, dried over anhydrous sodium sulfate,
and evaporated to yield the target compounds 12a 13a and 14a which were recrystallized from
isopropanol. In addition, all the target compounds were purified by silica gel column chromatography
CH Cl :CH OH = 100:1).
2
3
brine (50 mL
×
–g
,
–
g
–g
(
2
2
3
N-(4-Morpholinoimidazo[1,2-a]pyridin-6-yl) phenyl)-4-phenylpyridine amide (12a). A yellow solid; yield:
◦
6.1%; m.p. 268–270 C; ESI-MS [M + H] m/z: 477.2; H-NMR (CDCl ) δ 10.46 (s, 1H), 9.06 (s, 1H),
3
.86 (s, 2H), 8.39 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.13–8.04 (m, 4H),
.96 (s, 3H), 7.83 (s, 2H), 4.21 (s, 4H), 4.01 (s, 4H).
+
1
9
8
7
5
-(3-Fluorophenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)picolinamide (12b). A yellow solid;
◦
+
1
yield: 79.4%; m.p. 261–264 C; ESI-MS [M + H] m/z: 495.2; H-NMR (400 MHz, CDCl )
δ
10.18 (s, 1H),
.85 (s, 2H), 8.40 (d, J = 8.1 Hz, 2H), 8.29 (d, J = 8.1 Hz, 1H), 8.09 (s, 3H), 7.88 (s, 1H), 7.75 (s, 1H), 7.52 (d,
J = 5.1 Hz, 1H), 7.45 (s, 1H), 7.36 (d, J = 9.2 Hz, 1H), 7.20 (d, J = 9.0 Hz, 1H), 4.17 (s, 4H), 4.03 (s, 4H).
3
8
5
-(2,4-Difluorophenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)picolinamide (12c). A pale
◦
+
1
yellow solid; yield: 90.2%; m.p. 255–257 C; ESI-MS [M + H] m/z: 513.2; H-NMR (DMSO-d )
δ
6
1
7
3
0.97–10.71 (m, 1H), 8.86 (s, 1H), 8.35 (d, J = 9.2 Hz, 1H), 8.22 (s, 1H), 8.04 (s, 1H), 7.84–7.71 (m, 1H),
.60 (d, J = 12.2 Hz, 2H), 7.51–7.37 (m, 1H), 7.32–7.17 (m, 1H), 6.51 (s, 2H), 6.30 (s, 2H), 3.92 (s, 4H),
.74 (s, 4H).
N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-5-(4-(trifluoromethyl)phenyl)picolinamide
(
12d).
10.46
s, 1H), 8.88 (s, 2H), 8.33 (d, J = 8.0 Hz, 2H), 8.23 (d, J = 14.9 Hz, 1H), 8.12 (s, 2H), 7.99 (d, J = 22.6 Hz
◦
+
1
A gray solid; yield: 83.5%; m.p. 233–235 C; ESI-MS [M + H] m/z: 545.2; H-NMR (CDCl )
δ
3
(
5
,
13
H), 7.56 (s, 1H), 7.48 (s, 1H), 4.08 (d, J = 18.4 Hz, 8H). C-NMR (DMSO-d ) δ 162.66, 157.43, 149.99,
6
1
1
47.87, 147.28, 138.75, 137.36, 136.99, 136.53, 135.92, 132.97, 132.96, 132.52, 132.29, 128.69 (2C),
28.68 (2C), 128.52, 126.28 (2C), 123.14, 120.76 (2C), 116.11, 66.73 (2C),46.71 (2C).
5
-(4-Methoxyphenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)picolinamide (12e). A pale
◦
+
1
yellow solid; yield: 93.3%; m.p. 255–257 C; ESI-MS [M + H] m/z: 507.2; H-NMR (DMSO-d )
6

Molecules 2017, 22, 310
9 of 13
δ
7
4
11.05 (s, 1H), 8.74 (d, J = 5.1 Hz, 1H), 8.57 (s, 1H), 8.47 (s, 1H), 8.03 (d, J = 8.6 Hz, 2H), 8.02 (s, 2H),
.87 (d, J = 9.8 Hz, 2H), 7.86 (d, J = 8.6 Hz, 2H), 7.51 (s, 1H), 7.14 (d, J = 8.6 Hz, 2H), 4.32 (s, 4H),
.19 (s, 2H), 3.85 (d, J = 3.9 Hz, 3H).
N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-5-(m-tolyl)picolinamide (12f). A pale yellow solid;
◦
+
1
yield: 72.6%; m.p. 241–243 C; ESI-MS [M + H] m/z: 491.2; H-NMR (CDCl )
δ
10.17 (s, 1H),
3
8
8
2
.84 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.30–8.22 (m, 1H), 8.20 (d, J = 7.5 Hz, 1H), 8.10 (d, J = 9.8 Hz, 2H),
.06 (s, 2H), 7.93 (d, J = 8.2 Hz, 2H), 7.74 (s, 1H), 7.56 (d, J = 7.3 Hz, 3H), 4.25 (s, 4H), 3.98 (s, 4H),
.44 (s, 3H).
N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-5-(p-tolyl)picolinamide (12g). A pale yellow solid;
◦
+
1
yield: 89.1%; m.p. 258–260 C; ESI-MS [M + H] m/z: 491.2; H-NMR (DMSO-d )
9
2
δ
10.79 (s, 1H),
.02 (d, J = 9.4 Hz, 2H), 8.59 (s, 1H), 8.35 (d, J = 8.1 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H), 8.05 (d, J = 8.7 Hz,
H), 7.77 (s, 2H), 7.59 (s, 2H), 7.37 (t, J = 8.5 Hz, 3H), 4.30 (s, 4H), 3.80 (s, 4H), 2.40 (s, 3H).
6
N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-4-phenylpicolinamide (13a). A yellow solid; yield:
7.0%; m.p. 261–262 C; ESI-MS [M + H] m/z: 477.2; 1H-NMR (DMSO-d6)
◦
+
δ 9.55 (s, 1H),
9
7
.59 (d, J = 5.0 Hz, 1H), 7.34 (s, 1H), 7.20 (s, 1H), 6.79 (d, J = 7.8 Hz, 5H), 6.74–6.65 (m, 3H), 6.34
(dd, J = 8.4, 4.3 Hz, 4H), 3.07 (s, 4H), 2.61–2.51 (m, 4H).
4
-(3-Fluorophenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)picolinamide (13b). A white solid;
◦
+
1
yield: 88.0%; m.p. 237–239 C; ESI-MS [M + H] m/z: 495.2; H-NMR (DMSO-d )
δ
10.85 (s, 1H),
6
8
3
3
.84 (d, J = 5.0 Hz, 1H), 8.80 (d, J = 5.0 Hz, 1H), 8.60 (s, 1H), 8.45 (s, 1H), 8.33 (s, 1H), 8.06 (dd, J = 5.7,
.5 Hz, 3H), 7.96 (s, 1H), 7.85–7.79 (m, 2H), 7.63 (d, J = 7.7 Hz, 1H), 7.45–7.33 (m, 2H), 4.30 (s, 4H),
.80 (d, J = 4.2 Hz, 4H).
4
-(2,4-Difluorophenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)picolinamide (13c). A pale
◦
+
1
yellow solid; yield: 91.2%; m.p. 266–267 C; ESI-MS [M + H] m/z: 513.2; H-NMR (DMSO-d )
δ
10.76
s, 1H), 8.79 (d, J = 5.1 Hz, 1H), 8.52 (s, 1H), 8.26 (s, 1H), 7.96 (d, J = 7.3 Hz, 4H), 7.89 (s, 1H), 7.85–7.74
m, 2H), 7.52 (s, 1H), 7.43 (d, J = 9.6 Hz, 1H), 7.26 (t, J = 8.3 Hz, 1H), 4.23 (s, 4H), 3.74 (d, J = 4.3 Hz, 4H).
6
(
(
N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-4-(4-(trifluoromethyl)phenyl)picolinamide
A pale yellow solid; yield: 81.8%; m.p. 269–272 C; ESI-MS [M + H] m/z: 545.2; H-NMR (DMSO-d )
(
13d).
◦
+
1
6
δ
10.86 (s, 1H), 8.88 (d, J = 5.0 Hz, 1H), 8.59 (s, 1H), 8.49 (s, 1H), 8.15 (d, J = 8.0 Hz, 2H), 8.13–8.07
(m, 2H), 8.03 (d, J = 11.5 Hz, 3H), 8.00 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.59 (s, 1H), 4.30 (s, 4H),
3
.80 (d, J = 4.0 Hz, 4H).
4
-(4-Methoxyphenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)picolinamide (13e). A pale
◦
+
1
yellow solid; yield: 93.1%; m.p. 257–260 C; ESI-MS [M + H] m/z: 507.2; H-NMR (DMSO-d )
6
δ
10.82 (s, 1H), 8.76 (d, J = 5.1 Hz, 1H), 8.60 (s, 1H), 8.40 (s, 1H), 8.05 (d, J = 8.6 Hz, 2H), 8.02 (s, 2H), 7.97
d, J = 9.8 Hz, 2H), 7.91 (d, J = 8.6 Hz, 2H), 7.59 (s, 1H), 7.14 (d, J = 8.6 Hz, 2H), 4.30 (s, 4H), 3.85 (s, 2H)
.80 (d, J = 3.9 Hz, 3H).
(
3
,
N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-4-(m-tolyl)picolinamide (13f). A pale red solid;
◦
+
1
yield: 91.5%; m.p. 233–234 C; ESI-MS [M + H] m/z: 491.2; H-NMR (DMSO-d )
8
3
δ
10.82 (s, 1H),
.79 (d, J = 4.9 Hz, 1H), 8.59 (s, 1H), 8.42 (s, 1H), 8.30 (s, 1H), 8.05 (d, J = 8.4 Hz, 3H), 7.83 (d, J = 8.1 Hz,
H), 7.59 (s, 1H), 7.43–7.36 (m, 3H), 4.30 (s, 4H), 3.80 (d, J = 3.7 Hz, 4H), 2.40 (s, 3H).
6
N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-4-(p-tolyl)picolinamide (13g). A brown solid; yield:
◦
1
9
7
6
8.0%; m.p. 271–273 C; ESI-MS [M + H]+ m/z: 491.2; H-NMR (DMSO-d6)
δ 9.87 (s, 1H),
.90 (d, J = 5.2 Hz, 1H), 7.56 (s, 1H), 7.44 (s, 1H), 7.22–7.15 (m, 3H), 6.95 (d, J = 8.5 Hz, 3H), 6.73 (s, 1H),
.54 (s, 4H), 3.45 (s, 4H), 3.15–2.68 (m, 4H), 2.33 (s, 3H). C-NMR (DMSO-d ) δ 162.98, 151.15, 149.90,
13
6
1
1
49.64, 149.31, 147.87, 140.08, 138.78, 135.91, 134.03, 132.94, 132.30, 130.48 (2C), 127.35 (2C), 127.31,
26.29 (2C), 120.70, 119.64 (2C), 116.13, 116.11, 66.73 (2C), 46.70 (2C), 21.29.

Molecules 2017, 22, 310
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N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-6-phenylpyrimidine-4-carboxamide (14a). A pale
◦
+
1
yellow solid; yield: 82.7%; m.p. 272–275 C; ESI-MS [M + H] m/z: 478.1; H-NMR (DMSO-d )
δ
10.79
6
(s, 1H), 8.75 (d, J = 5.1 Hz, 1H), 8.58 (s, 1H), 8.39 (s, 1H), 8.04 (d, J = 8.7 Hz, 2H), 8.01 (s, 1H), 7.98 (d, J =
6
.3 Hz, 2H), 7.95 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.58 (s, 1H), 7.13 (d, J = 8.5 Hz, 2H), 4.29 (s, 4H), 3.80
(d, J = 4.3 Hz, 4H).
N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-6-(p-tolyl)pyrimidine-4-carboxamide (14b). A pale
◦
+
1
yellow solid; yield: 79.5%; m.p. 222–225 C; ESI-MS [M + H] m/z: 492.2; H-NMR (DMSO-d )
δ
6
10.82 (s, 1H), 8.77 (dd, J = 22.5, 5.1 Hz, 2H), 8.58 (s, 1H), 8.41 (s, 1H), 8.27 (s, 1H), 7.94–7.87 (m, 2H),
7
.58 (s, 2H), 7.41 (t, J = 8.3 Hz, 4H), 4.29 (s, 4H), 3.79 (s, 4H), 1.25 (d, J = 6.5 Hz, 3H).
6
-(4-Methoxyphenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)pyrimidine-4-carboxamide (14c).
◦
+
1
A pale brown solid; yield: 77.8%; m.p. 269–271 C; ESI-MS [M + H] m/z: 508.2; H-NMR (DMSO-d )
6
δ
10.96 (s, 1H), 9.38 (s, 1H), 8.57 (s, 1H), 8.32 (d, J = 8.0 Hz, 2H), 8.03 (dd, J = 14.7, 8.4 Hz, 4H),
7
.96 (s, 1H), 7.08 (d, J = 1.7 Hz, 2H), 7.05 (d, J = 1.6 Hz, 2H), 4.29 (s, 4H), 3.87 (s, 3H), 3.80 (s, 4H).
1
3
C-NMR (DMSO-d6)
δ 162.80, 162.53, 162.06, 158.73, 158.48, 157.98, 157.00, 138.37, 133.36, 132.96,
132.29, 129.59 (2C), 128.25, 128.20, 126.30 (2C), 120.98 (2C), 116.12, 114.98 (3C), 66.73 (2C), 55.95,
4
6.70 (2C).
6
-(4-Bromophenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)pyrimidine-4-carboxamide (14d).
◦
+
1
A white solid; yield: 90.9%; m.p. 221–223 C; ESI-MS [M + H] m/z: 557.1; H-NMR (DMSO-d )
δ
10.91
6
(s, 1H), 9.49 (s, 1H), 8.65 (s, 1H), 8.59 (d, J = 11.9 Hz, 2H), 8.38 (d, J = 8.3 Hz, 2H), 8.00–7.94 (m, 3H)
,
7
.85 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 2.7 Hz, 2H), 4.30 (s, 4H), 3.79 (d, J = 4.0 Hz, 4H).
6
-(4-Chlorophenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)pyrimidine-4-carboxamide (14e).
◦
+
1
A pale yellow solid; yield: 93.2%; m.p. 235–237 C; ESI-MS [M + H] m/z: 513. 1; H-NMR
DMSO-d6) 10.81 (s, 1H), 8.78 (d, J = 5.1 Hz, 1H), 8.58 (s, 1H), 8.41 (s, 1H), 8.05 (d, J = 8.8 Hz,
H), 8.00 (d, J = 7.5 Hz, 2H), 7.95 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.58 (s, 1H), 7.41 (d, J = 8.0 Hz, 2H),
(
δ
2
4
.29 (s, 4H), 3.88–3.67 (m, 4H).
6
-(4-Fluorophenyl)-N-(4-(8-morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)pyrimidine-4-carboxamide (14f).
◦
+
1
A gray solid; yield: 80.8%; m.p. 259–260 C; ESI-MS [M + H] m/z: 496.2; H-NMR (CDCl )
δ
3
10.05 (s, 1H), 9.33 (s, 1H), 8.97 (s, 1H), 8.63 (s, 1H), 8.17 (dd, J = 18.2, 8.6 Hz, 2H), 8.10–7.83 (m, 5H),
7
.75 (d, J = 5.2 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 4.32 (s, 4H), 3.96 (s, 4H).
N-(4-(8-Morpholinoimidazo[1,2-a]pyrazin-6-yl)phenyl)-6-(4-(trifluoromethyl)phenyl)pyrimidine-4-carboxamide
◦
14g). A white solid; yield: 77.6%; m.p. 278–281 C; ESI-MS [M + H] m/z: 546.2; H-NMR (DMSO-d )
6
+
1
(
δ
10.86 (s, 1H), 9.86 (d, J = 5.0 Hz, 1H), 8.49 (s, 1H), 8.15 (d, J = 8.0 Hz, 2H), 8.10 (d, J = 4.5 Hz, 1H),
8
.03 (dd, J = 20.1, 8.6 Hz, 4H), 7.95 (d, J = 8.4 Hz, 3H), 7.59 (s, 1H), 4.30 (s, 4H), 3.80 (s, 4H).
3
.3. In Vitro Cytotoxicity Assays
3
.3.1. The Selection of Cancer Cell Lines
The studies of Yue showed that the expression of the VEGF, PI3K, Akt and mTOR genes of the
PI3K-Akt-mTOR signaling pathway in non-small cell lung cancer (A549) were (40
±
59)%, (61
±
23)%,
(
77 32)%, (43 21)% respectively [13]. The level of this pathway in breast cancer (MCF-7) was also
±
±
up to 70% [14]. In prostate cancer cells (PC-3), the missing tumor suppressor gene (PTEN) resulted in
a high expression of the PI3K-Akt-mTOR signaling pathway. In addition, the studies of GDC-0941
showed it have strong inhibitory activity against A549, PC-3 and MCF-7 suggesting that these cells
have a high sensitivity for PI3K-Akt—mTOR signaling pathway inhibitors [
these cell lines as the test cell lines for our in vitro anti-tumor experiments.
4]. Therefore, we chose

Molecules 2017, 22, 310
11 of 13
3
.3.2. MTT Assay In Vitro
The in vitro cytotoxic activity of all the compounds 12a
–g
,
13a
–g
and 14a
–g
was evaluated with
the A549, PC-3 and MCF-7 cell lines by the standard MTT assay, with compounds II and GDC-0941
as positive controls. The cancer cell lines were cultured in minimum essential medium (MEM)
3
supplement with 10% fetal bovine serum (FBS). Approximately 4
×
10 cells, suspended in MEM
◦
medium, were plated onto each well of a 96-well plate and incubated in 5% CO at 37 C for 24 h.
2
The test compounds at indicated final concentrations were added to the culture medium and the cell
cultures were continued for 72 h. Fresh MTT was added to each well at a terminal concentration of
◦
5
µ
g/mL and incubated with cells at 37 C for 4 h. The formazan crystals were dissolved in 100
µL
of DMSO in each well, and the absorbance at 492 nm (for the MTT formazan) and 630 nm (for the
reference wavelength) was measured with an ELISA reader. All of the compounds were tested three
times in each of the cell lines. The results expressed as inhibition rates or IC50 (half-maximal inhibitory
concentration) were the averages of two determinations and calculated by using the Bacus Laboratories
Incorporated Slide Scanner (Bliss) software (the Bacus Laboratories Inc. Slide Scanner (BLISS) system,
Lombard, IL, USA).
3
.4. PI3Kα Kinase Assay
The selected compounds 14b, 14c were tested for their activity against PI3Kα
using a Kinase-Glo®
Luminescent Kinase Assay, with Compound II, GDC-0941 and PI103 as positive controls. The kinase
reaction is done in 384-well black plate. Each well is loaded with 50 L of test items (in 90% DMSO)
and 5 L reaction buffer containing 10 g/mL PI substrate (l- -phosphatidylinositol, Avanti Polar
Lipids, (Avanti Polar Lipids, Inc., Alabaster, AL, USA), prepared in 3% octylglucoside) and the PI3K
protein 10 nM is then added to it. The reaction is started by the addition of 5 L of 1 M ATP prepared
in the reaction buffer and is incubated for 60 min for p110 . It is terminated by the addition of 10
µ
µ
µ
α
α
µ
µ
α
µL
Kinase-Glo buffer. The plates are then read in a Synergy 2 reader (BioTek Instruments, Inc., Winooski,
VT, USA) for luminescence detection. All of the compounds were tested two times [9,10].
3
.5. Docking Studies
For docking purposes, we prepared the receptor protein PDB ID code: 4L23 (PI3Kα).
The three-dimensional structure of the PI3Kα were obtained from the RCSB Protein Data Bank [9,10].
We built a small organic molecule set (compound 14c) and used the Gasteiger-Hückel method to
optimize the molecular force field and structure. Hydrogen atoms were added to the structure allowing
for appropriate ionization at physiological pH. First of all, extract ligand substructure, then remove
water and excess structure, finally, add hydrogens and fix sidechain amides. The protonated state
of several important residues, such as Val851, Lys802, Ser773 and Asp933, were adjusted by using
SYBYL 6.9.1 (Tripos, St. Louis, MO, USA) in favor of forming reasonable hydrogen bond with the
ligand. Molecular docking analysis was carried out by the SURFLEX-DOCK module of SYBYL 6.9.1
package (Tripos) to explore the binding model for the active site of PI3Kα with its ligand. All atoms
located within the range of 5.0 Å from any atom of the cofactor were selected into the active site, and
the corresponding amino acid residue was, therefore, involved into the active site if only one of its
atoms was selected. Other default parameters were adopted in the SURFLEX-DOCK calculations.
All calculations were performed on a Silicon Graphics workstation (package version 6.9.1 on silicon
graphics origin300 workstation with IRIX 6.5 operating system, San Francisco, CA, USA). Lastly,
docking results and the optimized molecular docking model with the receptor proteins was obtained.
4
. Conclusions
Three new classes of 8-morpholino imidazo[1,2-a]pyrazine derivatives bearing phenylpyridine-
carboxamides or phenylpyrimidine-carboxamides were designed, synthesized and evaluated for
their cytotoxicity against three cancer cell lines and/or PI3K kinase. The pharmacological results
α

Molecules 2017, 22, 310
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indicated that most of the synthesized compounds displayed moderate cytotoxicity against the
three cell lines, with IC50 values ranging from 6.39 to 74.9 M. The most promising compound
4c showed a good inhibitory activity against PI3K kinase with an IC50 value of 1.25 0.13 M,
which was more potent than the lead compound II (IC50 values 7.39 M). Among them, the series of
µ
1
α
±
µ
µ
compounds containing phenylpyrimidine-carboxamide structures exhibited the best activity. For the
other two series, the compounds with the aryl unit substituted at the pyridine C-4 position were more
active than those substituted at the C-5 position. Moreover, the substituents on the benzene ring also
have an important impact on the cytotoxicity. The results demonstrated that electron donating groups
on the benzene ring were beneficial to the cytotoxicity. The initial SARs and docking studies with
the PI3Kα molecule showed that the morpholine group, imidazopyrazine and amide scaffold were
beneficial for these compounds to maintain a good activity.
Acknowledgments: We gratefully acknowledge the generous support provided by the National Natural Science
Funds of China (No. 81460527), Science and Technology Project of Jiangxi Science & Technology Normal University
(
2016XJZD007), the National Natural Science Funds of Jiangxi Province, China (20161BAB215216), Program of Key
Laboratory of Drug Design and Optimization, Jiangxi Science & Technology Normal University (300098010306),
College Students' Science and Technology Innovation Project of Jiangxi Province.
Author Contributions: S.X., C.C. and C.S. synthesized all of novel compounds, P.Z., Y.Z. and H.Z. run the
bioassay evaluation and statistics analysis, W.Z. started the project and designed the molecules, S.X. and C.S.
wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 12a–g, 13a–g and 14a–g are available from the authors.
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