Novel homo-bivalent and polyvalent compounds based on ligustrazine and heterocyclic ring as anticancer agents

Article abstract of DOI:10.3390/molecules24244505

Bivalent and polyvalent inhibitors can be used as antitumor agents. In this experiment, eight ligustrazine dimers and seven ligustrazine tetramers linked by alkane diamine with different lengths of carbon chain lengths were synthesized. After screening their antiproliferation activities against five cancer cell lines, most ligustrazine derivatives showed better cytotoxicity than the ligustrazine monomer. In particular, ligustrazine dimer 8e linked with decane-1,10-diamine exhibited the highest cytotoxicity in FaDu cells with an IC₅₀ (50% inhibiting concentration) value of 1.36 nM. Further mechanism studies suggested that 8e could induce apoptosis of FaDu cells through the depolarization of mitochondrial membrane potential and S-phase cell cycle arrest. Inspired by these results, twenty-seven additional small molecule heterocyclic dimers linked with decane-1,10-diamine and nine cinnamic acid dimers bearing ether chain were synthesized and screened. Most monocyclic and bicyclic aromatic systems showed highly selective anti-proliferation activity to FaDu cells and low toxicity to normal MCF 10A cells. The structure-activity relationship revealed that the two terminal amide bonds and the alkyl linker with a chain length of 8–12 carbon were two important factors to maintain its antitumor activity. In addition, the ADMET calculation predicted that most of the potent compounds had good oral bioavailability.

Full text of DOI:10.3390/molecules24244505

molecules
Article
Novel Homo-Bivalent and Polyvalent Compounds
Based on Ligustrazine and Heterocyclic Ring as
Anticancer Agents
Jiawen Wang 1,†, Ge Hong 2,3,†, Guoliang Li 2, Wenzhi Wang 2 and Tianjun Liu 1,2,3,
*
1
Graduate Institute, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China;
m13260192393@163.com
2
3
Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of
Medical Sciences & Peking Union Medical College, Tianjin 300192, China; hongge6688@aliyun.com (G.H.);
liguoliangsx@163.com (G.L.); wxwnx123@163.com (W.W.)
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Correspondence: Liutj@bme.org.cn
*
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Qiao-Hong Chen
Received: 7 November 2019; Accepted: 6 December 2019; Published: 9 December 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Bivalent and polyvalent inhibitors can be used as antitumor agents. In this experiment,
eight ligustrazine dimers and seven ligustrazine tetramers linked by alkane diamine with different
lengths of carbon chain lengths were synthesized. After screening their antiproliferation activities
against five cancer cell lines, most ligustrazine derivatives showed better cytotoxicity than the
ligustrazine monomer. In particular, ligustrazine dimer 8e linked with decane-1,10-diamine exhibited
the highest cytotoxicity in FaDu cells with an IC50 (50% inhibiting concentration) value of 1.36 nM.
Further mechanism studies suggested that 8e could induce apoptosis of FaDu cells through the
depolarization of mitochondrial membrane potential and S-phase cell cycle arrest. Inspired by these
results, twenty-seven additional small molecule heterocyclic dimers linked with decane-1,10-diamine
and nine cinnamic acid dimers bearing ether chain were synthesized and screened. Most monocyclic
and bicyclic aromatic systems showed highly selective anti-proliferation activity to FaDu cells and low
toxicity to normal MCF 10A cells. The structure-activity relationship revealed that the two terminal
amide bonds and the alkyl linker with a chain length of 8–12 carbon were two important factors to
maintain its antitumor activity. In addition, the ADMET calculation predicted that most of the potent
compounds had good oral bioavailability.
Keywords: bivalency; polyvalency; antitumor; apoptosis; cell cycle
1. Introduction
Natural products play a vital role in the development of the drug, especially anticancer drugs [1].
Ligustrazine (2,3,5,6-tetramethylpyrazine, TMP), an important component of the Chinese traditional
medicinal herb Chuanxiong (Ligusticum chuanxiong Hort), has been of wide clinical use for cardiovascular
and cerebrovascular diseases [2]. Recently, TMP has been reported to possess anticancer activity
in vitro and in vivo, inducing cancer cell apoptosis and cell cycle modulation [3,4
]. More importantly,
the introduction of different substituted groups into ligustrazine has helped to improve the antitumor
activity. Many ligustrazine derivatives with potent antitumor activity have been developed in the past
few years, like monocarbonyl ligustrazine-curcumin hybrids [5], ligustrazine-betulinic acid hybrids [6],
and ligustrazine-rhein derivatives [ ]. This advancement stimulated our interest in using TMP as the
7
scaffold to synthesize new antitumor agents.
Molecules 2019, 24, 4505; doi:10.3390/molecules24244505
www.mdpi.com/journal/molecules

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In the interaction between host and guest molecules, both bivalency and polyvalency could
enhance the affinity and activity of monovalent ligand [ ]. This phenomenon has attracted attention in
8,9
drug design. Many effective bivalent anti-cancer drugs have been found, such as artemisinin-derivative
dimers [10], jesterone dimer [11], indole-3-carbinol dimer [12], bis-daunorubicin [13]. Previous work
in this field has designed and synthesized several ligustrazine dimers linked by cyclohexanone and
oxime [14], curcumin [15], or triterpenes [16], which had a good cytotoxic effect on human cancer
cells. The result showed that the dimerization of ligustrazine could obviously improve the antitumor
potency of its monomer.
With an effort to developing new antitumor agents, we designed and synthesized a series of novel
TMP dimers and tetramers linked with different alkyl diamines and screened their anti-proliferation
potential on a panel of human cancer cell lines, including HeLa, Hep G2, MCF-7, FaDu, and A549.
Based on these experimental results, according to the importance of small molecular heterocyclic rings
in drug discovery [17], additional dimers series of aromatic rings instead of ligustrazine, a total of
51 new compounds were synthesized, screened, and their structure-activity relationship was briefly
discussed. Meanwhile, through morphological observation and flow cytometric analysis, the antitumor
mechanisms of the most potent one were preliminarily discussed in this study. ADMET properties of
all compounds were also evaluated to explore the drug-likeness.
2. Results and Discussion
2.1. Chemistry
In this study, 51 novel designed compounds were synthesized. Series 6a
obtained according to the synthetic method described in Schemes 1 and 2. The important intermediate
2-chloromethyl-3,5,6-trimethyl-pyrazine was prepared from ligustrazine by the tandem reaction:
Boekelheide reaction [18] and deprotection and the chlorination reaction [19]. The key intermediate
2-carboxylic acid-3,5,6-trimethyl-pyrazine was synthesized by the one-pot reaction, as previously
–g and 8a–f were
5
7
reported [20]. To explore the length effect, the TMP moieties, with 2–12 methylene alkyl chain as a
linker, were synthesized.
Scheme 1. Synthesis of the TMP complexes 6a–6i. Reagents and conditions: (a) 30% H2O2, CH3COOH,
95 ◦C, 2 h; (b) (CH3CO)2O, 140 ◦C, 2.5 h; (c) CH3ONa, CH3OH, r.t., 0.5 h; (d) SOCl2, CH2Cl2, r.t., 2.5 h;
(e) n-diaminoalkane (piperazine and monoethanolamine), K2CO3, CH3CN, 95 ◦C, 2 h.
Scheme 2. Synthesis of the Bis-TMP hybrids 8a
h; (g) n-diamino-alkane, EDCI/DMAP, r.t., 12 h.
–8f. Reagents and conditions:
(f) KMnO4, H2O, 37 ◦C, 24
Using heteroaromatic acids with decane-1,10-diamine as raw materials, the target
compounds 9e 35e (outlined in Scheme 3) linked by amide chain were synthesized
with EDCI (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and DMAP
–

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(4-Dimethylaminopyridine) in anhydrous CH2Cl2 at room temperature. Series 9d
,
9f
–
l
(shown in
Scheme 4) linked by ether chain or amide chain were synthesized by the above method. All the newly
synthesized compounds were characterized by 1H-NMR, 13C-NMR, and high-resolution mass spectra
(HRMS). Details were provided in the experimental part. The purity of the compounds was over 95%
measured by HPLC (Figure 1).
Scheme 3. Synthesis of compounds 9e–35e. Reagents and conditions: (h) decane-1,10-diamine,
EDCI/DMAP, r.t., 12 h.
Scheme 4. Synthesis of compounds 9d, 9f–9m. Reagents and conditions: (i) cinnamic acid, EDCI/DMAP,
r.t., 12 h.
Figure 1. The purity of compound 8e as a representative analyzed by HPLC (Kromasil C18 column,
eluted by methanol/water (80/20) containing 0.1% trifluoroacetic acid at a flow rate of 1 mL/min).
2.2. Anti-Proliferative Activity In Vitro
For the newly synthesized compounds, their anti-proliferative activity on human cancer cell
lines, HeLa (cervical carcinoma), Hep G2 (hepatoma carcinoma), MCF-7 (breast carcinoma), FaDu
(head and neck carcinoma), A549 (lung carcinoma), and normal mammary epithelial cell line MCF
10A, was screened by MTT assay [21] with doxorubicin (DOX) as the positive control. The IC50
values of these compounds are summarized in Table 1. Among the ligustrazine derivatives, 6a
–g
and 8a , the tetramers 6f and 6g had broad-spectrum cytotoxic activities in all cell lines (IC50
–
f

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6.57–20.83
The dimeric ligustrazine 8d
tumor cell lines with IC50 values between 0.00136 and 6.35
µ
M), and the cytotoxicity activities increased with the increase of carbon chain length.
8e 8f showed the most promising anticancer activity in most tested
M, a much better result than DOX and
,
,
µ
TMP. In particular, compound 8e, two ligustrazine rings linked with decane-1,10-diamine, exhibited
the best anti-proliferative ability in cells (except Hep G2), and compared with other linkers, its
IC50 values were 1.42
0.047 0.008 M against HeLa, MCF-7, FaDu, A549, and MCF 10A, respectively. In light of these
results, we replaced TMP with aromatic heterocycles similar to ligustrazine in electronic space.
The novel small aromatic molecule and heterocyclic dimers 9e 35e were synthesized, and their
anti-proliferative ability was evaluated. Compounds 9e 10e 11e 17e 18e 21e 22e, 23e, and 25e,
±
0.71
µM, 0.037
±
0.001
µM, 0.00136
±
0.00035
µM, 1.05
±
0.05 µM, and
±
µ
–
,
,
,
,
,
,
aromatic acid dimers (cinnamoyl, nicotinoyl, benzoyl, quinoline-3-carboxyl, quinoline-6-carboxyl,
benzofuran-2-carboxyl, benzothiophene-2-carboxyl, indol-3-carboxyl, chlorinated cinnamoyl), were
linked by decane-1,10-diamine, which had potent anti-proliferative ability in FaDu or A549 cell lines.
Their IC50 in FaDu cell lines were less than 1
and 697 nM (benzofuran-2-carboxyl). Also, they had nearly no toxicity in normal cells like MCF
10A. In series 9d , compounds 9d (IC50 < 1 M), two cinnamic acids linked by C8, C10, C12
chain with two terminal amide bonds, were more effective than compounds 9g (IC50 > 20 M), two
µM, in the range of 20 nM (benzothiophene-2-carboxyl)
–
n
–
f
µ
–
i
µ
cinnamic acids linked by the same alkyl chain with two terminal ester bonds instead of amide bonds.
This demonstrated the vital role of two-terminal amide bonds in antitumor activity. Compared with
compounds 9d
ether chain instead of alky chain, was weaker in tumor cells (IC50 > 20
alkyl chain played an important role in the structure. Then, we evaluated the position effect of the
substitution at the ortho, meta, or para positions of the same aromatic ring. Compounds 25 35e and
the unsubstituted compounds 9e and 11e were all derivatives of cinnamic acid or benzoic acid with
different substituents and electronic effects. The results of the compounds 26 27e or 28 29e confirmed
–
f, the inhibition of compounds 9j
–
m, two cinnamic acids linked by the similar length
µM), which confirmed that the
–
–
–
that the substituents had a general effect on the activity, -OH, -OCH3, -NH2, and other electron donor
groups could significantly reduce the activity, with the influence of -OH greater than that of -OCH3.
However, the electron attraction group like -Cl (25e) had no obvious effect. The compounds with
para-substituted analogs (31e, para position, IC50 = 5.594
activities in FaDu cells than ortho- and meta- substituted analogs (30e
IC50 > 20 M). It is worth noting that para -OCH3 substitution could enhance anticancer activity.
The IC50 value of compound 34e was 5.853 0.408 M, the IC50 value of para- OCH3 was half that of
the parent compound 33e, and the IC50 value of compound 33e was 10.393 0.949 M. Generally, there
µM) exhibited better cell growth inhibitory
,
32e, ortho and meta position,
µ
±
µ
±
µ
were three crucial elements in the structure-activity relationship of these systems: alkyl chain linker,
two-terminal amide bonds, and heteroaromatic substituents. This SAR (structure-activity relationship)
study would help to discover more effective compounds in the future.
To study the selective antiproliferative activities of bivalent and polyvalent inhibitors in normal
cells and cancer cell lines, the cytotoxicity of compounds in FaDu cells and normal mammary epithelial
MCF 10A cells was measured. The results listed in Table 1 showed that although the ligustrazine
dimers 8d and 8e had high cytotoxicity in MCF 10A, their IC50 values were 2.69
±
0.46 and 0.047
±
0.008 M, while the IC50 values in FaDu cells were 110 30 and 1.36 0.035 nM, respectively,
which were far lower than that in MCF 10A. The selective index (SI) between MCF10A and Fadu
µ
±
±
(IC50MCF 10A/IC50FaDu) was 24.45 and 34.56, respectively. In addition, the heteroaromatic acid dimers
9e–12e, 21e–23e, and 25e had the similar activity, which was stronger than doxorubicin in FaDu
cells, and had high selectivity in FaDu cells and MCF 10A cells with SI (IC50MCF 10A/IC50FaDu) values
over 28.69. In contrast, the SI (IC50MCF 10A/IC50FaDu) value of doxorubicin was 0.43. These results
encouraged us to further investigate the possible cellular mechanisms.

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Table 1. Inhibitory effects of synthetic compounds on the growth of different types of human cancer
cells and normal mammary epithelial cells.
Antiproliferative Activity IC50 ± SEM (µM)
Comp.
HeLa[a]
Hep G2[b]
MCF-7[c]
FaDu[d]
A549[e]
MCF 10A[f]
SI[g]
NCh
6a
6b
6c
6d
6e
6f
6g
6h
6i
8a
8b
8c
8d
8e
8f
9e
10e
11e
12e
13e
14e
15e
16e
17e
18e
19e
20e
21e
22e
23e
24e
25e
26e
27e
28e
29e
30e
31e
32e
33e
34e
35e
9d
>100
>100
>100
>100
62.67 ± 4.19
10.16 ± 0.55
7.89 ± 0.86
>100
>100
>100
>100
>100
3.31 ± 0.14
1.42 ± 0.71
4.96 ± 1.62
>100
>100
>100
>100
>100
>100
37.74 ± 4.32
>100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>100
>100
>100
>100
50.77 ± 1.35
10.72 ± 0.22
9.62 ± 0.11
>100
>100
>100
>100
>100
>20
>100
>100
>100
>100
66.36 ± 0.05
20.83 ± 0.14
9.83 ± 0.17
>100
>100
>100
>100
>100
0.480 ± 0.003
0.037 ± 0.001
0.158 ± 0.009
>100
>100
>100
>100
>100
>100
21.48 ± 1.47
>100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>100
>100
>100
>100
49.50 ± 1.61
10.94 ± 0.12
6.80 ± 0.05
>100
>100
>100
>100
>100
>100
>100
90.29 ± 3.41
NCh
>100
NCh
>100
>100
85.44 ± 2.28
12.97 ± 0.55
6.57 ± 0.30
>100
>100
>100
>100
46.59 ± 0.65
2.69 ± 0.46
0.047 ± 0.008
0.051 ± 0.021
>100
38.60 ± 2.48
80.54 ± 7.60
>100
NCh
>100
1.73
1.19
20.05 ± 0.41
9.23 ± 0.16
>100
>100
>100
0.97
NCh
NCh
NCh
NCh
>100
>100
>100
NCh
80.41 ± 1.36
0.11 ± 0.03
0.00136 ± 0.00035
0.174 ± 0.010
0.054 ± 0.002
0.25 ± 0.02
0.50 ± 0.06
1.33 ± 0.07
48.31 ± 3.17
6.33 ± 0.65
8.85 ± 0.45
88.11 ± 1.37
0.236 ± 0.005
0.697 ± 0.021
8.74 ± 0.44
>20
6.35 ± 0.05
1.05 ± 0.05
2.46 ± 0.05
11.27 ± 0.81
2.94 ± 0.25
64.30 ± 3.46
8.77 ± 0.47
>100
44.37 ± 0.29
23.08 ± 0.12
>100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
24.45
34.56
0.29
>1851.85
154.40
161.08
>75.19
>2.07
3.13
>20
>20
>100
>100
>100
>100
>100
97.75 ± 1.55
26.34 ± 5.36
>100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>100
19.82 ± 1.41
54.65 ± 5.92
>100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
6.18
NCh
>84.75
>28.69
>2.29
NCh
>30.08
>1000
>31.35
>3.78
>357.14
NCh
0.665 ± 0.028
0.020 ± 0.002
0.638 ± 0.089
5.296 ± 0.366
0.056 ± 0.016
>20
4.621 ± 0.539
>4.33
NCh
>20
5.067 ± 0.461
>20
>3.95
NCh
5.594 ± 0.628
>3.58
NCh
>20
10.393 ± 0.949
5.853 ± 0.408
>20
1.054 ± 0.091
0.027 ± 0.002
>20
>20
>20
>20
>1.92
>3.42
NCh
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>18.98
>740.74
NCh
9f
9g
9h
9i
NCh
>20
>20
>20
NCh
NCh
9j
9k
9l
9m
>20
>20
>20
>20
>20
>20
>100
28.04 ± 1.09
>20
>20
>20
>100
6 ± 0.07
4.447 ± 0.208
>20
>20
>20
>100
>20
>20
>20
>100
>4.50
NCh
>20
NCh
>20
>100
1.27 ± 0.13
NCh
TMP >100
DOX 11.39 ± 0.48
>100
0.55 ± 0.03
0.43
[a] HeLa (cervical cancer cell line); [b] Hep G2 (hepatocarcinoma cell line); [c] MCF-7 (breast cancer cell line); [d]
FaDu (head and neck squamous carcinoma cell line); [e] A549 (epithelial cancer cell line); [f] MCF 10A (normal
mammary epithelial cell); [g] SI: selectivity index. It was calculated as: SI = IC50 (MCF 10A)/IC50 (FaDu); [h] NC:
not calculated.
2.3. Colony Formation Assay
As ligustrazine dimer 8e had the most potent antiproliferative activity in all synthesized
compounds, the colony formation assay in FaDu cells was conducted. As shown in Figure 2,
FaDu cells treated with compound 8e at concentrations 0.75 nM, 1.50 nM, 3 nM, 6 nM for 10 days
formed smaller and fewer colonies compared to untreated control group, indicating that compound
8e had a dose-dependent inhibition on the proliferation of FaDu cells, and the colony inhibition rate
reached around 85.4% at 6 nM.

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Figure 2. Effect of 8e on the colony formation in FaDu cells. (
after treatment at the indicated concentrations for 10 days; (
A
B
) Representative images of FaDu colonies
) The percentage of colonies compared
with control group, n = 3. **: p < 0.01; ***: p < 0.001 versus control.
2.4. Live/Dead Staining
Live-dead double staining experiment was conducted to visually evaluate the cell toxicity of
compound 8e, the most potent one obtained by MTT assay. Propidium iodide (PI)-stained dead cells
in red and calcein AM-stained living cells in green. The number of dead cells increased with the dose
of 8e while living cells decreased. Simultaneously, the densities of 8e-treated cells were much lower
than that of the control group. The concentration-dependent loss of FaDu cell viability was consistent
with the MTT results (Figure 3).
Figure 3. The confocal images of FaDu cells after incubation with compound 8e at concentrations of 0,
2, 5, and 10 nM for 24 h, respectively. Viable cells were stained in green with Calcein-AM, and dead
cells were stained in red with PI. The scale bar was 100 µm.

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2.5. Morphological Observation by Hoechst 33,342 Staining
To investigate whether the anti-proliferation behavior of 8e was related to the apoptosis pathway,
the interaction between 8e- and FaDu cells was analyzed through Hoechst 33,342 staining with a laser
scanning confocal microscope. As shown in Figure 4, cells without treatment as the control group
exhibited almost negligible apoptotic characteristics with homogenous and round blue-stained nuclei.
On the contrary, the typical apoptotic nuclei with irregular nuclear morphology and condensation
of chromatin (much brighter stained cells) were observed, and most FaDu cells were shrunken and
rounded up from the culture dish after exposure to 8e. The result demonstrated that inducing apoptosis
in FaDu cells might be one cause of cell growth inhibition by compound 8e.
Figure 4. Hoechst staining of FaDu cells treated with compound 8e,
2.6. Apoptosis Analysis by Annexin V-FITC/PI Staining
(A) Control group; (B) 20 nM group.
To substantiate whether the cell death induced by the ligustrazine dimer 8e was associated
with apoptosis, the interaction of FaDu cells with 8e was further analyzed by an annexin V-FITC/PI
staining, and the apoptosis ratios were quantitated by flow cytometry. The cells were divided into
four quadrants: Q1 represented necrotic cells (annexin V−/PI+), Q2 represented late apoptotic cells
(annexin V+/PI+), Q3 represented early apoptotic cells (annexin V+/PI−), Q4 represented living cells
(annexin V−/PI−). As shown in Figure 5, after being exposed to different concentrations of 8e (2, 5, 10
nM) for 24 h in FaDu cells, the apoptotic cells (including the early and late apoptosis ratios) increased
gradually from 8.23% of the control to 34.2%, 57.7%, 79.5%, respectively. The results revealed that
8e could induce FaDu cells’ apoptosis in a dose-dependent manner. This was consistent with other
reports showing TMP and its derivatives could induce apoptosis of cancer cells [22–24].

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Figure 5. Flow cytometry analysis of apoptosis using annexin V-FITC/PI double staining in FaDu cells
treated with compound 8e at the indicated concentrations for 24 h. The percentage of cells was given in
the respective quadrants.
2.7. Mitochondrial Membrane Potential (∆Ψm) Analysis
Mitochondria play an essential role in cell apoptotic progression by regulating mitochondrial
membrane potential (∆Ψm). Depolarization of ∆Ψm is considered an indicator of cell apoptosis [25]. To
better understand the mechanism of 8e-induced FaDu cells apoptosis, the depolarization of ∆Ψm was
quantitated by JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) dye. As a mitochondrial-specific
dual-fluorescence probe, JC-1 selectively entered the mitochondria. In healthy cells with high ∆Ψm, it
accumulated in the mitochondrial matrix as J-aggregates with red fluorescence, while in the reduced
∆Ψm cells, it remained in the cytoplasm as a monomer with green fluorescence. The change of ∆Ψ
m
could be easily detected by the change of fluorescence color. Flow cytometric analysis of FaDu cells
treated with different concentrations of 8e (2, 5, 10 nM) for 24 h revealed that the ratio of green to
red fluorescence was significantly higher than that of the control group in a concentration-dependent
manner, indicating that the number of lower ∆Ψm cells increased (Figure 6). These results suggested
that 8e could induce apoptosis of FaDu cells by decreasing ∆Ψm.

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Figure 6. Flow cytometry analysis of the mitochondrial membrane potential using the JC-1
(tetraethylbenzimidazolylcarbocyanine iodide) dye in FaDu cells treated with compound 8e at the
indicated concentrations for 24 h. The percentage of cells was given in the respective quadrants.
2.8. Cell Cycle Analysis
In order to investigate whether 8e induced cell cycle disturbances in FaDu cells, the flow cytometric
analysis was performed with PI staining. The fluorescence intensity was proportional to the content of
double-stranded DNA. The separation of cells in G0/G1, S, and G2/M was based upon the distribution
of DNA content. Cytometric profiles of the PI-stained DNA showed that after being treated with 8e
at different concentrations (2, 5, 10 nM) for 24 h, the cell cycle of FaDu cells was arrested. As shown
in Figure 7, the percentage of cells in the S phase (33.7%, 44.3%, and 54.7%) was markedly improved
compared with the control group (28.1%), and this effect was concentration-dependent in FaDu cells.
The population of the G0/G1 phase decreased from 51.9% to 41.8%, 44.2%, and 32.4%, respectively.
These data indicated that 8e significantly arrested cell cycle at S phase. This was different from the
previously reported cell cycle arrest in the G0/G1 phase induced by TMP [4], which revealed that the
dimerization of TMP could change the modulation of the cell cycle of its monomer.

Molecules 2019, 24, 4505
10 of 24
Figure 7. Flow cytometry analysis for cell cycle distribution of FaDu cells. (A) Representative cell
cycle distribution of FaDu cells with compound 8e treatment (0, 2, 5, and 10 nM) for 24 h. The tall
peak (left) represents cells in the G0/G1 phase, the small peak (right) represents cells in the G2/M
phase, and the middle area represents cells in the S phase. (
cycle distribution.
B) Quantification of the percentage of cell
2.9. In Silico ADMET Prediction
ADMET prediction is becoming increasingly crucial as most drugs fail in clinical trials owing to
poor pharmacokinetic parameters [26 27]. Table 2 shows the ADMET calculated descriptors for our
,
synthesized compounds and two biplots shown in Figure 8. All compounds had 0–4 HBD (hydrogen
bond donor) and 4–10 HBA (hydrogen bond acceptor), which were under Lipinski’s rule of five (HBD
< 5, HBA < 10). PPB (plasma protein binding) was one of the key properties related to drug efficacy.
Except for compounds 10–15e, 17–24e, and 9d, most of the compounds had weak (<90%) binding with
plasma proteins in the blood, which were predicted to be non-inhibitors of cytochrome P450 2D6,
and participated in the good- metabolism of phase I metabolism. T1/2 of the target molecules was
in the range of 1.63–2.29 h, showing the characteristic of rapid metabolism. The HT (hepatotoxicity)
values estimated the hepatoxicity of the chemical compounds. HT values for series 6a
29e 31 35e 9f 9i 9k, and 9m were zero, and there was no hepatoxicity, while other compounds
might be hepatotoxic. Moreover, the LD50 (median lethal dose) predicted that series 8a 35e, and
9d were over 500 mg/kg, implying they were less toxic. In addition, an ADMET model formed
–i, 13–14e, 20e,
,
–
,
,
,
–f, 9–
–
m
with descriptors 2D polar surface area (PSA) and AlogP98 could predict human intestinal absorption
and the blood-brain barrier penetration (BBB) at 95% and 99% confidence level. In the absorption
plot, compounds 6a–f, 6h–i, 8a–f, 9–11e, 13–19e, 23e, 30–33e, 9d, 9g, and 9j–m were predicted to be
easily absorbed by the intestine at 99% confidence level. While in the BBB plot, the most potent
compounds 8e and 8f were fallen out of the 99% ellipse (undefined), suggesting its poor blood-brain
barrier penetration ability. Therefore, these molecules might possess low or no side effects of the
central nervous system (CNS). In conclusion, ADMET prediction of the compounds might be helpful
for further designing new drugs with favorable oral bioavailability.

Molecules 2019, 24, 4505
11 of 24
Table 2. The ADMET properties of all synthetic compounds.
Comp. HBDa
RBNc
logSe
HIAg
LD50(mg/kg)m
HBAb
logPd
PSAf
PPBh
BBBi CYP3A4j T1/2 (h)k
HTl
6a
6b
6c
6d
6e
6f
6g
6h
6i
8a
8b
8c
8d
8e
8f
9e
10e
11e
12e
13e
14e
15e
16e
17e
18e
19e
20e
21e
22e
23e
24e
25e
26e
27e
28e
29e
30e
31e
32e
33e
34e
35e
9d
0
0
0
0
0
0
0
0
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
2
2
2
2
4
2
4
2
4
2
4
4
4
2
2
2
2
2
0
0
0
2
2
0
0
6
0
10
10
10
10
10
10
10
6
6
6
6
6
6
6
6
2
4
2
2
4
2
4
6
4
4
4
6
2
2
2
2
2
6
6
8
8
4
4
4
6
8
8
2
2
4
4
4
4
5
6
7
12
2
11
12
13
15
17
19
21
4
6
6
7
9
1.84
1.90
2.48
3.39
4.31
5.22
6.13
0.97
0.47
−0.09
0.49
1.40
2.31
3.22
4.14
6.21
2.97
5.27
7.09
2.97
4.06
3.83
1.53
5.65
5.65
4.31
5.33
6.46
7.40
6.27
5.86
7.54
5.69
6.14
5.66
6.11
3.78
3.78
3.78
5.21
5.18
5.18
5.30
7.12
6.59
7.50
8.42
2.57
2.44
3.86
3.73
−0.04
0.66
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
2.23
1.93
2.05
2.02
1.90
1.67
1.35
2.49
1.52
1.53 105.27
1.87 105.27
2.30 105.27
2.67 105.27
2.97 105.27
3.21 105.27
4.45
2.60
4.27
6.11
2.60
3.66
3.35
1.68 105.27
5.10
5.10
4.38 112.85
4.57 112.68
6.02
6.99
5.55
5.15
5.78
96.79
96.79
96.79
96.79
96.79
96.79
96.79
51.75
69.21
0.81
0.80
0.80
0.80
0.80
0.80
0.80
0.79
0.78
0.69
0.72
0.72
0.72
0.72
0.72
0.69
0.70
0.68
0.69
0.72
0.41
0.70
0.68
0.70
0.72
0.69
0.43
0.47
0.69
0.75
0.72
0.67
0.45
0.54
0.46
0.54
0.59
0.64
0.64
0.53
0.54
0.53
0.69
0.69
0.55
0.55
0.55
0.37
0.37
0.50
0.50
0.02
0.93
0.68
0.67
0.68
0.65
0.65
0.66
0.67
0.67
0.70
0.78
0.79
0.80
0.83
0.85
0.85
0.89
0.91
0.91
0.92
0.91
0.90
0.91
0.85
0.95
0.96
0.90
0.93
0.94
0.91
0.95
0.95
0.89
0.87
0.82
0.77
0.73
0.90
0.90
0.90
0.81
0.76
0.76
0.91
0.87
0.88
0.86
0.85
0.86
0.85
0.88
0.86
0.78
0.49
0.94
0.91
0.91
0.91
0.91
0.91
0.91
0.98
0.75
0.98
0.98
0.98
0.98
0.98
0.98
0.99
0.99
1.00
0.99
0.99
1.00
0.99
0.99
0.98
0.95
0.94
0.98
0.99
0.99
0.99
0.96
0.99
0.85
0.98
0.94
0.96
0.99
0.97
0.98
0.99
0.98
0.97
0.99
0.99
0.98
0.98
0.98
0.99
0.99
0.98
0.98
0.02
0.99
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2.02
2.13
2.14
2.12
2.14
2.18
2.23
2.00
1.77
1.81
1.88
1.89
1.92
2.03
2.01
1.93
1.85
1.96
2.12
1.89
1.78
1.89
1.82
2.22
1.99
1.91
2.29
2.00
2.07
2.27
2.01
1.98
1.93
1.82
2.02
1.95
1.96
1.93
1.94
1.76
1.63
1.72
1.92
1.95
1.96
1.96
1.96
1.97
1.90
1.89
1.83
2.71
1.77
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
0
0
1
1
1
1
1
0
1
1
1
1
1
1
1
1
0
1
0
0
0
0
0
1
0
1
1
0
1
0
1
0
1
0
459.04
429.48
462.17
447.58
425.74
460.97
508.77
664.69
1011.08
1027.40
988.28
1033.87
1099.10
1278.93
1318.54
961.82
1075.58
1653.45
1818.99
1070.63
1145.12
1128.86
1461.77
1217.90
1254.91
1167.64
973.08
11
13
15
15
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
15
17
19
19
21
13
13
13
17
19
19
13
17
15
17
19
13
16
15
18
5
60.22
82.74
60.22
60.22
82.74
85.33
82.74
82.74
82.74
85.33
60.22
70.92
90.33
60.22
966.72
904.88
995.92
1072.61
1322.11
1249.90
1217.58
1163.97
927.46
3.28 119.71
3.64 95.94
3.13 137.57
3.08 113.80
2.74 113.30
2.69 113.30
2.72 113.30
3.97
3.62 113.80
3.67 113.80
4.11
4.73
5.34
5.69
5.96
2.06
1.41
2.96
2.14
849.92
951.43
853.84
95.94
1184.47
1073.30
1061.01
1523.97
1003.19
6181.36
4624.51
3127.76
1574.67
1693.04
4511.05
4493.16
324.50
60.22
60.22
52.46
52.46
52.46
78.08
87.01
70.32
79.25
9f
9g
9h
9i
9j
9k
9l
9m
DOX
TMP
4.80 209.31
1.51 22.52
0
1194.46
a
HBD (hydrogen bond donor), b HBA (hydrogen bond acceptor), c RBN (number of rotatable bonds), d logP (log
of the octanol/water partition coefficient), log S (log of the aqueous solubility), and f PSA (polar surface area)
e
were predicted using Discovery Studio 2.0 software. HIA (human intestinal absorption), h PPB (plasma protein
g
binding), I CYP3D4 (CYP3D4 inhibition), j T1/2 (half lifetime), k HT (human hepatoxicity), and l LD50 (median
lethal dose) were calculated via http://admet.scbdd.com/home/index/

Molecules 2019, 24, 4505
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Figure 8. The plot of PSA (polar surface area) versus AlogP98 for the newly synthesized compounds
showing the 95% and 99% confidence limit ellipses corresponding to the intestinal absorption and
blood-brain barrier models.
3. Conclusions
Using ligustrazine, aromatic, or heteroaromatic acids as the main raw materials and linked by alkyl
or ether chain, bivalent antitumor agents were synthesized and screened from five different human
cancer cell lines and one normal mammary epithelial cell line. SAR revealed that the two terminal amide
bonds and the alky chain were the dominant factors with 10 C atom chain showing the most potent
antitumor effect. Among different aromatic moieties, the ligustrazine dimer 8e was the best candidate;
its IC50 was 1.36 nM in FaDu cell, it showed a broad-spectrum antitumor activity with IC50 in the range
of 0.00136–1.42 µM, and the selective ratio MCF 10A/FaDu was 34.56. The subsequent fluorescence
staining and flow cytometry analysis indicated that 8e could induce apoptosis through depolarization
of the mitochondrial membrane potential in FaDu cells. Further mechanism investigation showed that
8e could arrest cell cycle at the S phase in FaDu cells. Furthermore, ADMET predicted that most of
the potent compounds followed Lipinski’s law and became ‘drug-like’ molecules, further confirming
that the dimerization of ligustrazine could improve the antitumor activity of its monomer. The highly
selective inhibition of these dimers on the proliferation of FaDu cells might be related to the effect on
a specific target in FaDu cells. Based on the characteristics of FaDu cells, further targeted screening
would give insight into the selectivity of such potent compounds.
4. Materials and Methods
4.1. Chemistry
1H-NMR and 13C-NMR spectra were recorded on a Mercury Vx-300 (300 MHz) or AVANCE III
(400 MHz). Chemical shifts were reported in ppm (δ) using the residue solvent line as the internal
standard (for TMS: 0 ppm, 1H and 13C; for CHCl3-d: 7.26 ppm, 1H and 77.16 ppm, 13C). Coupling
constants were given in Hertz (J). High-resolution MS (HRMS) were recorded on an Agilent 6520 Q-TOF
LC/MS. Melting points were uncorrected and were determined on a digital melting point apparatus
(Shenguang WRS-1B, shanghai, China). Flash column chromatography was carried out by using
silica gel (200–300 mesh). Reactions were monitored by thin-layer chromatography (TLC) on silica gel
GF254 plates (Qingdao Haiyang Chemical Plant, Qingdao, China). Reagents and solvents were used
as purchased from commercial sources without further purification. The purity of the synthesized
compounds was over 95% analyzed by HPLC (Waters 2695 Alliance system, Waters Corp., Milford, MA,
USA), with the Kromasil C18 column eluted by methanol/water (80/20) containing 0.1% trifluoroacetic
acid at a flow rate of 1 mL/min. Compounds
4
and
5
were prepared, as reported previously [19].
9m can be seen in supplementary materials.
1H-NMR, 13C-NMR and HR-MS spectra of compounds 6a
–

Molecules 2019, 24, 4505
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4.2. General Synthetic Procedure for 6a–g
A mixture of alkane-diamine (10 mmol), TMP-Cl (50 mmol), KOH (60 mmol), and
dimethylformamide (35 mL) was heated to reflux. The reaction progress was monitored via TLC. After
2 h, the reaction mixture was cooled to room temperature. Subsequently, the solid was filtered off, and
the filtrate was added with H2O (100 mL) and extracted with ethyl acetic acid three times. The organic
layers were combined and washed with H2O and brine, and then separated and dried over anhydrous
Na2SO4 for 8 h. The solvent was removed under reduced pressure; the resulting residue was separated
on flash column chromatography eluted by a mixture of petroleum ether/acetone in a volume ratio of
7:1–3:1, and further recrystallized from acetone to give pale white solid.
4.2.1. N1,N1,N2,N2-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Ethane-1,2-Diamine (6a)
◦
1
Pale white solid, yield 28%, m.p. 207.6–208.3 C; H-NMR (300 MHz, CDCl3)
δ
: 3.60 (s, 8H),
2.67 (s, 4H), 2.43 (s, 12H), 2.41 (s, 12H), 2.23 (s, 12H); 13C-NMR (101 MHz, DMSO)
δ: 149.91, 149.63,
148.37, 147.85, 58.75, 52.14, 50.83, 21.61, 21.42, 20.65; HRMS (ESI) m/z: 597.4134 [M + H]+, calcd. for
[C34H49N10]+ 597.4142.
4.2.2. N1,N1,N3,N3-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Propane-1,3-Diamine (6b)
Pale white solid, yield 27%, m.p. 113–114.3 ◦C; 1H-NMR (300 MHz, CDCl3)
δ: 3.65 (s, 8H), 2.46 (s,
12H), 2.43 (s, 12H), 2.41–2.38 (m, 4H), 2.32 (s, 12H), 1.81–1.71 (m, 2H). 13C-NMR (101 MHz, DMSO)
δ:
149.93, 149.53, 148.51, 147.82, 58.63, 53.36, 21.61, 21.44, 20.70; HRMS (ESI) m/z: 611.4286 [M + H]+, calcd.
for [C35H51N10]+ 611.4298.
4.2.3. N1,N1,N4,N4-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Butane-1,4-Diamine (6c)
◦
1
Pale white solid, yield 37%, m.p. 129.2–129.5 C; H-NMR (300 MHz, CDCl3) δ: 3.62 (s, 8H),
2.44 (s, 24H), 2.40–2.37 (m, 4H), 2.30 (s, 12H), 1.37 (s, 4H); 13C-NMR (101 MHz, DMSO) δ: 150, 149.53,
148.60, 147.79, 58.54, 54.85, 24.39, 21.64, 21.47, 20.71; HRMS (ESI) m/z: 625.4451 [M + H]+, calcd. for
[C36H53N10]+ 625.4455.
4.2.4. N1,N1,N6,N6-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Hexane-1,6-Diamine (6d)
◦
1
Pale white solid, yield 43%, m.p. 146.7–147.9 C; H-NMR (300 MHz, CDCl3)
δ: 3.67 (s, 8H),
2.46–2.45 (m, 24H), 2.43–2.40 (m, 4H), 2.34 (s, 12H), 1.43 (br, 4H), 1.04 (br, 4H); 13C-NMR (101 MHz,
DMSO) δ: 150.05, 149.48, 148.71, 147.77, 58.64, 54.93, 27.34, 26.56, 21.65, 21.48, 20.74; HRMS (ESI) m/z:
653.4765 [M + H]+, calcd. for [C38H57N10]+ 653.4768.
4.2.5. N1,N1,N8,N8-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Octane-1,8-Diamine (6e)
Pale white solid, yield 48%, m.p. 107.8–108.6 ◦C; 1H-NMR (300 MHz, CDCl3)
(br, 24H), 2.42–2.38 (m, 4H), 2.34 (s, 12H), 1.48–1.40 (m, 4H), 1.05 (br, 8H); 13C-NMR (101 MHz, CDCl3)
δ: 3.67 (s, 8H), 2.45
δ
: 150.09, 149.47, 148.75, 147.76, 58.69, 54.90, 29.52, 27.46, 26.55, 21.65, 21.47, 20.73; HRMS (ESI) m/z:
681.5076 [M + H]+, calcd. for [C40H61N10]+ 681.5081.
4.2.6. N1,N1,N10,N10-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Decane-1,10-Diamine (6f)
Pale white solid, yield 76%, m.p. 67.4–68 ◦C; 1H-NMR (300 MHz, CDCl3)
(br, 24H), 2.41–2.39 (m, 4H), 2.35 (s, 12H), 1.47 (br, 4H), 1.11 (br, 12H); 13C-NMR (101 MHz, DMSO)
δ: 3.69 (s, 8H), 2.46
δ:
150.10, 149.45, 148.78, 147.76, 58.71, 54.92, 29.73, 29.52, 27.48, 26.57, 21.64, 21.47, 20.74; HRMS (ESI) m/z:
709.5378 [M + H]+, calcd. for [C42H65N10]+ 709.5394.
4.2.7. N1,N1,N12,N12-Tetrakis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Dodecane-1,12-Diamine (6g)
Pale white solid, yield 76%, m.p. 84.7–85.3 ◦C; 1H-NMR (300 MHz, CDCl3)
(br, 24H), 2.44 (br, 4H), 2.35 (s, 12H), 1.47 (br, 4H), 1.15–1.12 (br, 16H); 13C-NMR (101 MHz, DMSO)
δ: 3.69 (s, 8H), 2.46
δ:

Molecules 2019, 24, 4505
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150.10, 149.44, 148.78, 147.74, 58.72, 54.89, 29.75, 29.53, 27.46, 26.56, 21.64, 21.47, 20.73; HRMS (ESI) m/z:
737.5689 [M + H]+, calcd. for [C44H69N10]+ 737.5707.
4.3. 1,4-Bis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Piperazine (6h)
A mixture of piperazine (0.800 g, 9.29 mmol), TMP-Cl (3.80 g, 22.29 mmol), KOH (1.56 g, 27.66
mmol), and dimethylformamide (20 mL) was heated under reflux for 2 h. The reaction was monitored
by TLC. The reaction was quenched, filtered, and the filtrate was added with 100 mL H2O and extracted
with ethyl acetate three times. The organic layers were combined, washed with H2O and brine, and
then dried over anhydrous Na2SO4 for 8 h. The solvent was evaporated in vacuo. The resulting
residue was separated on flash column chromatography with a mixture of petroleum ether/acetone
7:1–3:1(volume ratio) as eluent, and then further recrystallized from acetone to afford compound 6h.
Pale white solid, yield 62%, m.p. 155.8–156.3 ◦C; 1H-NMR (300 MHz, CDCl3)
δ: 150.01, 149.57, 148.10, 147.98, 61.88, 53.26, 50.70,
21.61, 21.51, 21.04; HRMS (ESI) m/z: 355.2610 [M + H]+, calcd. for [C20H31N6]+ 355.2610.
δ
: 3.58 (s, 4H), 2.55
(s, 8H), 2.47 (br, 18H); 13C-NMR (101 MHz, DMSO)
4.4. 2-(Bis((3,5,6-Trimethylpyrazin-2-yl)Methyl)Amino)Ethanol (6i)
A mixture of ethanolamine (0.500 g, 8.19 mmol), TMP-Cl (3.35 g, 19.65 mmol), K2CO3 (3.39 g,
24.56 mmol), and acetonitrile (20 mL) was heated under reflux for 3 h. The product formation was
monitored via TLC. After cooling, the mixture was filtered, and the filtrate was evaporated in vacuo.
The residue was purified by flash column chromatography with an eluent of petroleum ether/acetone
= 10:1–3:1 to afford compound 6i.
Light yellow oil, yield 72%; 1H-NMR (300 MHz, CDCl3)
δ: 3.82 (13s, 4H), 3.66 (t, J = 5 Hz, 2H),
2.87 (t, J = 5.1 Hz, 2H), 2.41 (s, 6H), 2.37 (s, 6H), 2.35 (s, 6H); 13C-NMR (101 MHz, DMSO)
δ: 149.54,
148.94, 148.27, 147.87, 59.67, 58.15, 57.36, 21.41, 21.21, 20.52; HRMS (ESI) m/z: 330.2290 [M + H]+, calcd.
for [C18H28N5O]+ 330.2294.
4.5. General Synthetic Procedure for 8a–f
Compound
7 was gained according to the method described by Wu [20]. A mixture of
tetramethylpyrazine acid (4.40 mmol), EDCI (4.72 mmol), triethylamine (12 mmol), DMAP (2.40
mmol), and anhydrous dichloromethane (10 mL) was stirred to dissolve, then diamino-alkane (2
mmol) was added and stirred at room temperature until the reaction was finished (monitored by TLC).
The reaction mixture was washed with brine and water, extracted with dichloromethane (20 mL),
dried over anhydrous sodium sulfate for 8 h, filtered, and the solvent was evaporated. The resulting
residue was purified by flash column chromatography with an eluent of petroleum ether/acetone =
15:1–3:1(volume ratio) to give pale white solid.
4.5.1. N,N0-(Propane-1,3-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (8a)
Pale white solid, yield 16%, m.p. 125.4–126.4 ◦C; 1H-NMR (300 MHz, CDCl3)
Hz, 2H), 3.58–3.52 (m, 4H), 2.92 (s, 6H), 2.56 (s, 6H), 2.50 (s, 6H), 1.99–1.90 (m, 2H); 13C-NMR (101
δ: 8.30 (t, J = 5.9
MHz, CDCl3) δ: 165.57, 154.09, 151.29, 147.80, 139.09, 36.89, 29.87, 22.94, 22.07, 21.47; HRMS (ESI) m/z:
371.2189 [M + H]+, calcd. for [C19H27N6O2]+ 371.2195.
4.5.2. N,N0-(Butane-1,4-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (8b)
Pale white solid, yield 30%, m.p. 168.4–168.6 ◦C; 1H-NMR (300 MHz, CDCl3)
δ: 8.07 (t, J = 5.5 Hz,
2H), 3.50–3.48 (m, 4H), 2.91 (s, 6H), 2.56 (s, 6H), 2.52 (s, 6H), 1.75–1.73 (m, 4H); 13C-NMR (101 MHz,
CDCl3)
δ: 165.25, 154.15, 151.38, 147.75, 139.07, 50.90, 39.13, 27.49, 22.96, 22.06, 21.48; HRMS (ESI) m/z:
385.2352 [M + H]+, calcd. for [C20H29N6O2]+ 385.2352.

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4.5.3. N,N0-(Hexane-1,6-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (8c)
Pale white solid, yield 47%, m.p. 114.2–114.7 ◦C; 1H-NMR (300 MHz, CDCl3)
δ
: 8.02 (t, J = 5.4
Hz, 2H), 3.45–3.38 (m, 4H), 2.90 (s, 6H), 2.55 (s, 6H), 2.51 (s, 6H), 1.67–1.63 (m, 4H), 1.47–1.43 (m, 4H);
13C-NMR (101 MHz, CDCl3)
δ: 165.12, 154.03, 151.35, 147.63, 139.11, 39.33, 29.74, 26.82, 22.98, 22.06,
21.48; HRMS (ESI) m/z: 413.2662 [M + H]+, calcd. for [C22H33N6O2]+ 413.2665.
4.5.4. N,N0-(Octane-1,8-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (8d)
Pale white solid, yield 68%, m.p. 113.6–113.8 ◦C; 1H-NMR (300 MHz, CDCl3)
δ: 8 (t, J = 5.3 Hz,
2H), 3.45–3.38 (m, 4H), 2.91 (s, 6H), 2.56 (s, 6H), 2.52 (s, 6H), 1.65–1.60 (m, 4H), 1.37–1.37 (m, 8H);
13C-NMR (101 MHz, CDCl3)
δ: 165.10, 154.02, 151.40, 147.65, 139.20, 39.45, 29.81, 29.34, 27.11, 23.01,
22.08, 21.53; HRMS (ESI) m/z: 441.2975 [M + H]+, calcd. for [C24H37N6O2]+ 441.2978.
4.5.5. N,N0-(Decane-1,10-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (8e)
Pale white solid, yield 66%, m.p. 110.3–110.6 ◦C; 1H-NMR (300 MHz, CDCl3)
2H), 3.44–3.37 (m, 4H), 2.91 (s, 6H), 2.55 (s, 6H), 2.51 (s, 6H), 1.64–1.57 (m, 4H), 1.34–1.30 (m, 12H);
δ: 8 (t, J = 5.2 Hz,
13C-NMR (101 MHz, CDCl3)
δ: 165.07, 153.97, 151.33, 147.66, 139.21, 39.47, 29.80, 29.56, 29.39, 27.14,
22.94, 22.02, 21.49; HRMS (ESI) m/z: 469.3287 [M + H]+, calcd. for [C26H41N6O2]+ 469.3291.
4.5.6. N,N0-(Dodecane-1,12-Diyl)Bis(3,5,6-Trimethylpyrazine-2-Carboxamide) (8f)
Pale white solid, yield 72%, m.p. 98.3–99.1 ◦C; 1H-NMR (300 MHz, CDCl3)
3.45–3.38 (m, 4H), 2.91 (s, 6H), 2.56 (s, 6H), 2.52 (s, 6H), 1.67–1.57 (m, 4H), 1.34–1.27 (m, 16H); 13C-NMR
δ: 8 (t, J = 4.9 Hz, 2H),
(101 MHz, CDCl3) δ: 165.07, 153.98, 151.37, 147.62, 139.22, 39.49, 29.83, 29.66, 29.44, 27.18, 22.99, 22.07,
21.51; HRMS (ESI) m/z: 497.3602 [M + H]+, calcd. for [C28H45N6O2]+ 497.3604.
4.6. General Synthetic Procedure for 9e–35e
Compounds 9e–35e were obtained by using one-pot reaction. A mixture of aromatic acid (6.30
mmol), EDCI (7.50 mmol), DMAP (0.60 mmol), and anhydrous dichloromethane (20 mL) was stirred to
dissolve, then decane-diamine (3 mmol) was added and stirred at room temperature for 12 h. The
mixture solution was filtered under reduced pressure. After that, the residue was washed with little
amount of CH2Cl2 and water successively, and dried to give the solid. Then, the residue was purified
on preparative TLC eluted with chloroform/methanol = 40:1–7:1 to yield compounds 26e
and 31e.
, 28e, 30e,
4.6.1. (2E,20E)-N,N0-(Decane-1,10-Diyl)Bis(3-Phenylacrylamide) (9e)
Pale white solid, yield 62%, m.p. 169.8–170.5 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD)
(d, J = 15.7 Hz, 2H), 7.49–7.46 (m, 4H), 7.36–7.31 (m, 6H), 6.47 (d, J = 15.6 Hz, 2H), 3.33–3.28 (m, 4H),
δ: 7.54
1.57–1.54 (m, 4H), 1.52–1.28 (m, 12H); 13C-NMR (101 MHz, CDCl3+CD3OD)
δ: 150.10, 149.45, 148.78,
147.76, 58.71, 54.92, 29.73, 29.52, 27.48, 26.57, 21.64, 21.47, 20.74; HRMS (ESI) m/z: 433.2851 [M + H]+,
calcd. for [C28H45N6O2]+ 433.2855
4.6.2. N,N0-(Decane-1,10-Diyl)Dinicotinamide (10e)
Pale white solid, yield 67%, m.p. 167.4–167.8 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD)
2H), 8.55 (d, J = 3.9 Hz, 2H), 8.11 (dt, J = 8, 1.7 Hz, 2H), 7.35 (dd, J = 7.9, 4.9 Hz, 2H), 3.34 (t, J = 7.2 Hz,
δ: 8.84 (s,
4H), 1.58–1.51 (m, 4H), 1.27–1.24 (m, 12H); 13C-NMR (101 MHz, CDCl3+CD3OD)
δ: 165.85, 151.29,
147.53, 135.93, 130.87, 123.79, 40.16, 29.28, 29.12, 26.86; HRMS (ESI) m/z: 383.2441 [M + H]+, calcd. for
[C28H45N6O2]+ 383.2447.

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4.6.3. N,N0-(Decane-1,10-Diyl)Dibenzamide (11e)
Pale white solid, yield 70%, m.p. 154.3–154.6 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD)
δ
: 7.77–7.73
(m, 4H), 7.51–7.33 (m, 6H), 3.42–3.35 (m, 4H), 1.63–1.56 (m, 4H), 1.34–1.30 (m, 12H); 13C-NMR (101
MHz, CDCl3+CD3OD) δ: 168.39, 134.55, 131.36, 128.45, 126.86, 40.02, 29.36, 29.27, 29.13, 26.84. HRMS
(ESI) m/z: 381.2702 [M + H]+, calcd. for [C24H33N2O2]+ 381.2542.
4.6.4. N,N0-(Decane-1,10-Diyl)Bis(2-Naphthamide) (12e)
Pale white solid, yield 64%, m.p. 163.9–164 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD)
δ: 8.22–8.17
(m, 2H), 7.89–7.82 (m, 4H), 7.54–7.39 (m, 8H), 3.49–3.44 (m, 4H), 1.67–1.60 (m, 4H), 1.38–1.34 (m, 12H);
13C-NMR (101 MHz, CDCl3 + CD3OD)
: 170.38, 134.50, 133.56, 130.33, 129.96, 128.22, 126.89, 126.29,
δ
125.10, 124.77, 124.65, 39.94, 29.36, 29.30, 29.11, 26.85. HRMS (ESI) m/z: 481.2853 [M + H]+, calcd. for
[C32H37N2O2]+ 481.2855.
4.6.5. N,N0-(Decane-1,10-Diyl)Diisonicotinamide (13e)
Pale white solid, yield 60%, m.p. 175.6–176.3 ◦C; 1H-NMR (300 MHz, CDCl3+CD3OD)
J = 5.6 Hz, 4H), 7.71 (dd, J = 6.1, 2.8 Hz, 4H), 3.41–3.33 (m, 4H), 1.64–1.57 (m, 4H), 1.33–1.31 (m, 12H);
δ: 8.62 (d,
13C-NMR (101 MHz, CDCl3+CD3OD)
δ: 165.88, 149.48, 142.45, 121.46, 40.04, 29.16, 28.99, 26.71. HRMS
(ESI) m/z: 383.2445 [M + H]+, calcd. for [C22H31N4O2]+ 383.2447.
4.6.6. N,N0-(Decane-1,10-Diyl)Bis(Furan-2-Carboxamide) (14e)
Pale white solid, yield 62%, m.p. 132.6–133.3 ◦C; 1H-NMR (300 MHz, CDCl3)
δ: 7.41 (dd, J =
1.7, 0.8 Hz, 2H), 7.09 (dd, J = 3.5, 0.7 Hz, 2H), 6.48 (dd, J = 3.5, 1.7 Hz, 2H), 6.37 (br, 2H), 3.45–3.38
(m, 4H), 1.65–1.55 (m, 4H), 1.36–1.30 (m, 12H); 13C-NMR (101 MHz, CDCl3)
δ: 158.53, 148.38, 143.77,
114.02, 112.22, 39.29, 29.80, 29.48, 29.33, 26.98. HRMS (ESI) m/z: 383.1944 [M + Na]+, calcd. for
[C20H28N2NaO4]+ 383.1947.
4.6.7. N,N0-(Decane-1,10-Diyl)Dipicolinamide (15e)
Pale white solid, yield 68%, m.p. 156.6–156.9 ◦C; 1H-NMR (300 MHz, CDCl3)
Hz, 2H), 8.69 (d, J = 2.5 Hz, 2H),.8.47–8.46 (m, 2H), 7.77 (br, 2H), 3.47–3.40 (m, 4H), 1.65–1.55 (m, 4H),
δ: 9.35 (d, J = 1.4
1.34–1.26 (m, 12H); 13C-NMR (101 MHz, CDCl3)
δ: 163.01, 147.26, 144.76, 144.55, 142.58, 39.61, 29.68,
29.51, 29.34, 27.03. HRMS (ESI) m/z: 383.2441 [M + H]+, calcd. for [C22H31N4O2]+ 383.2447
4.6.8. N,N0-(Decane-1,10-Diyl)Bis(Pyrazine-2-Carboxamide) (16e)
Pale white solid, yield 70%, m.p. 77.6–80.1 ◦C; 1H-NMR (300 MHz, CDCl3)
8.20–8.17 (m, 2H), 8.05 (br, 2H), 7.85–7.80 (m, 2H), 7.42–7.38 (m, 2H), 3.49–3.43 (m, 4H), 1.68–1.58 (m,
δ: 8.54–8.51 (m, 2H),
4H), 1.38–1.26 (m, 12H); 13C-NMR (101 MHz, CDCl3)
δ: 164.31, 150.25, 148.09, 137.49, 126.13, 122.34,
39.61, 29.77, 29.56, 29.41, 27.11. HRMS (ESI) m/z: 385.2350 [M + H]+, calcd. for [C20H29N6O2]+ 385.2352.
4.6.9. N,N0-(Decane-1,10-Diyl)Bis(Quinoline-3-Carboxamide) (17e)
Pale white solid, yield 59%, m.p. 206.7–207.1 ◦C; 1H-NMR (300 MHz, DMSO-d6 + CD3OD)
(d, J = 1.8 Hz, 2H), 8.09 (s, 2H), 7.39 (dd, J = 8.3, 3.1 Hz, 4H), 7.17 (t, J = 7.6 Hz, 2H), 7 (t, J = 7.4 Hz, 2H),
2.68 (t, J = 7 Hz, 4H), 0.99–0.88 (m, 4H), 0.76–0.62 (m, 12H). 13C-NMR (101 MHz, CDCl3)
: 165.92,
δ: 8.57
δ
148.01, 136.52, 131.47, 128.81, 127.93, 127.56, 127.35, 127.06, 40.11, 29.20, 29.15, 29.04, 26.78. HRMS (ESI)
m/z: 483.2756 [M + H]+, calcd. for [C30H35N4O2]+ 483.2760.
4.6.10. N,N0-(Decane-1,10-Diyl)Bis(Quinoline-6-Carboxamide) (18e)
Pale yellow solid, yield 73%, m.p. 197.7–198.3 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD)
δ: 8.71
(dd, J = 4.3, 1.6 Hz, 2H), 8.19 (d, J = 1.6 Hz, 2H), 8.16 (dd, J = 8.3, 1.3 Hz, 2H), 7.95 (dd, J = 8.8, 1.9
Hz, 2H), 7.91 (s, 2H), 7.35 (dd, J = 8.3, 4.3 Hz, 2H), 3.28 (t, J = 7.2 Hz, 4H), 1.53–1.45 (m, 5H), 1.22–1.13

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(m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
δ
: 167.43, 151.15, 148.21, 137.99, 132.82, 128.38,
127.79, 127.71, 127.64, 121.81, 40.14, 29.19, 29.04, 26.78. HRMS (ESI) m/z: 483.2758 [M + H]+, calcd. for
[C30H35N4O2]+ 483.2760.
4.6.11. N,N0-(Decane-1,10-Diyl)Bis(1H-Benzo[d]Imidazole-6-Carboxamide) (19e)
Brick red solid, yield 75%, m.p. 229.7–230.3 ◦C; 1H-NMR (300 MHz, CDCl3+ CD3OD)
(s, 2H), 8.07 (s, 2H), 7.73 (dd, J = 8.5, 1.6 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 3.42 (t, J = 7.1 Hz, 4H),
δ: 8.11
1.70–1.56 (m, 4H), 1.42–1.25 (m, 12H). 13C-NMR (101 MHz, CDCl3+ CD3OD)
δ: 168.61, 142.46, 128.70,
121.15, 39.32, 28.66, 28.62, 28.50, 26.19. HRMS (ESI) m/z: 461.2662 [M + H]+, calcd. for [C26H33N6O2]+
461.2665.
4.6.12. N,N0-(Decane-1,10-Diyl)Bis(2-Oxo-2H-Chromene-3-Carboxamide) (20e)
Pale white solid, yield 60%, m.p. 208.9–209.3 ◦C; 1H-NMR (300 MHz, CDCl3)
(s, 2H), 7.71–7.61 (m, 4H), 7.43–7.32 (m, 4H), 3.45 (dd, J = 13, 7 Hz, 4H), 1.63 (dt, J = 14.4, 7.2 Hz, 4H),
1.44–1.26 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
: 161.74, 161.44, 154.23, 148.38, 134.16,
δ: 8.90 (s, 2H), 8.80
δ
129.80, 125.30, 118.46, 117.97, 116.38, 39.69, 29.20, 29.02, 29.01, 26.77. HRMS (ESI) m/z: 517.2338 [M +
H]+, calcd. for [C30H33N2O6]+ 517.2339.
4.6.13. N,N0-(Decane-1,10-Diyl)Bis(Benzofuran-2-Carboxamide) (21e)
Pale white solid, yield 43%, m.p. 135.8–136.3 ◦C; 1H-NMR (300 MHz, DMSO-d6)
δ: 8.64 (s, 2H),
7.74 (d, J = 7.7 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.49 (s, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.4
Hz, 2H), 3.25 (dd, J = 13.1, 6.6 Hz, 4H), 1.58–1.43 (m, 4H), 1.37–1.14 (m, 12H). 13C-NMR (101 MHz,
DMSO-d6) δ: 157.94, 154.13, 149.37, 127.17, 126.61, 123.59, 122.63, 111.70, 109.02, 38.68, 29.01, 28.91,
28.71, 26.39. HRMS (ESI) m/z: 461.2438 [M + H]+, calcd. for [C28H33N2O4]+ 461.2430.
4.6.14. N,N0-(Decane-1,10-Diyl)Bis(Benzo[b]Thiophene-2-Carboxamide) (22e)
Pale yellow solid, yield 46%, m.p. 175–175.7 ◦C; 1H-NMR (300 MHz, DMSO-d6)
δ: 8.68 (s, 2H),
8.04 (s, 2H), 8.01–7.95 (m, 2H), 7.93–7.87 (m, 2H), 7.41 (p, J = 7 Hz, 4H), 3.25 (dd, J = 12.8, 6.6 Hz, 4H),
1.58–1.44 (m, 4H), 1.37–1.21 (m, 12H). 13C-NMR (101 MHz, DMSO-d6)
δ: 161.28, 140.28, 140.07, 139.16,
126, 125.02, 124.80, 124.33, 122.72, 28.99, 28.92, 28.72, 26.43. HRMS (ESI) m/z: 493.1976 [M + H]+, calcd.
for [C28H33N2O2S2]+ 493.1983.
4.6.15. N,N0-(Decane-1,10-Diyl)Bis(1-Methyl-1H-Indole-3-Carboxamide) (23e)
Pale white solid, yield 43%, m.p. 192.8–193.2 ◦C; 1H-NMR (300 MHz, DMSO-d6 + CD3OD)
δ: 8.08
(d, J = 7.7 Hz, 2H), 7.86 (s, 2H), 7.41 (d, J = 8 Hz, 2H), 7.20–7.13 (m, 2H), 7.13–7.06 (m, 2H), 3.78 (s, 6H),
3.22 (t, J = 7.1 Hz, 4H), 1.55–1.43 (m, 4H), 1.32–1.22 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
δ
: 166.26, 137.01, 131.75, 125.56, 122.27, 121.03, 120.13, 109.63, 39.26, 32.62, 29.33, 29.07, 28.94, 26.66.
HRMS (ESI) m/z: 487.3064 [M + H]+, calcd. for [C30H39N4O2]+ 487.3073.
4.6.16. N,N0-(Decane-1,10-Diyl)Bis(1H-Indole-6-Carboxamide) (24e)
Pale white solid, yield 47%, m.p. 168.4–169.2 ◦C; 1H-NMR (300 MHz, DMSO-d6)
δ: 11.32 (s, 2H),
8.30 (s, 2H), 7.92 (s, 2H), 7.59–7.41 (m, 6H), 6.46 (s, 2H), 3.25 (dd, J = 12.7, 6.4 Hz, 4H), 1.61–1.43 (m,
4H), 1.38–1.21 (m, 12H). 13C-NMR (101 MHz, DMSO)
δ: 167.04, 135.24, 129.61, 127.74, 127.66, 119.27,
117.90, 111.07, 101.14, 39.21, 29.27, 29, 28.83, 26.55. HRMS (ESI) m/z: 459.2760 [M + H]+, calcd. for
[C28H35N4O2]+ 459.2760.
4.6.17. (2E,20E)-N,N0-(Decane-1,10-Diyl)Bis(3-(4-Chlorophenyl)Acrylamide) (25e)
Pale white solid, yield 64%, m.p. 230.3–230.9 ◦C; 1H-NMR (300 MHz, CDCl3 +CF3COOD)
(d, J = 13 Hz, 2H), 7.54–7.38 (m, 8H), 6.51 (d, J = 11.9 Hz, 2H), 3.62–3.37 (m, 4H), 1.73–1.54 (m, 4H),
δ: 7.64

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1.46–1.18 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CF3COOD) δ: 129.71, 118.78, 115.96, 113.13, 110.30,
29.22, 28.99, 26.64. HRMS (ESI) m/z: 501.2075 [M + H]+, calcd. for [C28H35Cl2N2O2]+ 501.2076.
4.6.18. (2E,20E)-N,N0-(Decane-1,10-Diyl)Bis(3-(3-Hydroxy-4-Methoxyphenyl)Acrylamide) (26e)
Pale yellow solid, yield 35%, m.p. 192.9–195.3 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD)
δ: 7.42
(d, J = 15.3 Hz, 2H), 7.05 (s, 2H), 6.98 (d, J = 8.1 Hz, 2H), 6.83 (d, J = 8.3 Hz, 2H), 6.33 (d, J = 15.6 Hz,
2H), 3.89 (s, 6H), 3.29 (t, J = 7 Hz, 4H), 1.55–1.50 (m, 4H), 1.31–1.27 (m, 12H). 13C-NMR (101 MHz,
CDCl3 + CD3OD) δ: 167.37, 148.94, 146.05, 140.42, 128.11, 121.06, 118.25, 113.07, 111, 55.57, 39.50, 29.13,
28.98, 26.69. HRMS (ESI) m/z: 525.2951 [M + H]+, calcd. for [C30H41N2O6]+ 525.2965.
4.6.19. (2E,20E)-N,N0-(Decane-1,10-Diyl)Bis(3-(3,4-Dimethoxyphenyl)Acrylamide) (27e)
Pale white solid, yield 59%, m.p. 168.9–169.9 ◦C; 1H-NMR (300 MHz, DMSO-d6)
7.30 (br, 2H), 7.11 (br, 4H), 6.97 (br, 2H), 6.50 (br, 2H), 3.78 (br, 12H), 3.14 (br, 4H), 1.42 (br, 4H), 1.27 (br,
δ: 7.95 (br, 2H),
12H). 13C-NMR (101 MHz, CDCl3+ CD3OD)
δ: 167.15, 150.31, 148.90, 140.25, 127.89, 121.77, 118.56,
111.04, 109.74, 55.66, 55.58, 39.47, 29.16, 29.14, 28.99, 26.69. HRMS (ESI) m/z: 553.3272 [M + H]+, calcd.
for [C32H45N2O6]+ 553.3278.
4.6.20. (2E,20E)-N,N0-(Decane-1,10-Diyl)Bis(3-(4-Hydroxy-3,5-Dimethoxyphenyl)Acrylamide) (28e)
Pale yellow solid, yield 33%, m.p. 179.8–180.5 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD)
(d, J = 15.5 Hz, 3H), 6.76 (s, 4H), 6.37 (d, J = 15.6 Hz, 2H), 3.88 (s, 12H), 3.30 (t, J = 7.1 Hz, 4H), 1.61–1.49
δ: 7.44
(m, 4H), 1.38–1.24 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
δ: 166.94, 147.48, 141.03, 136.87,
126.16, 118.46, 104.99, 56.23, 39.63, 29.39, 29.21, 29.08, 26.79. HRMS (ESI) m/z: 585.3164 [M+H]+, calcd.
for [C32H45N2O8]+ 585.3176
4.6.21. (2E,20E)-N,N0-(Decane-1,10-Diyl)Bis(3-(3,4,5-Trimethoxyphenyl)Acrylamide) (29e)
Pale white solid, yield 67%, m.p. 193.4–194.3 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD)
J = 15.4 Hz, 2H), 6.76 (s, 4H), 6.44 (d, J = 15.6 Hz, 2H), 3.88 (s, 12H), 3.85 (s, 6H), 3.31 (t, J = 7.1 Hz, 4H),
1.63–1.47 (m, 4H), 1.40–1.23 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
: 166.73, 153.26, 140.53,
δ: 7.45 (d,
δ
139.33, 130.65, 120.18, 104.99, 60.81, 56, 39.63, 29.28, 29.21, 29.05, 26.76. HRMS (ESI) m/z:613.3472 [M +
H]+, calcd. for [C34H49N2O8]+ 613.3489.
4.6.22. N,N0-(Decane-1,10-Diyl)Bis(2-Aminobenzamide) (30e)
◦
1
Pale white solid, yield 67%, m.p. 90.2–91 C; H-NMR (300 MHz, CDCl3 + CD3OD)
δ: 7.36
(dd, J = 7.8, 1.2 Hz, 2H), 7.22–7.15 (m, 2H), 6.79–6.60 (m, 4H), 3.35 (dt, J = 9.7, 5.5 Hz, 4H), 1.66–1.49
(m, 4H), 1.41–1.24 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
δ: 173.92, 151.77, 135.96, 131.40,
121.48, 121.28, 120.96, 43.54, 33.31, 33.26, 33.11, 30.83. HRMS (ESI) m/z:411.2759 [M + H]+, calcd. for
[C24H35N4O2]+ 411.2760.
4.6.23. N,N0-(Decane-1,10-Diyl)Bis(4-Aminobenzamide) (31e)
Pale yellow solid, yield 31%, m.p. 186.7–187.7 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD)
(d, J = 8.6 Hz, 4H), 6.66 (d, J = 8.6 Hz, 4H), 3.34 (t, J = 7.2 Hz, 4H), 1.63–1.53 (m, 4H), 1.31–1.26 (m,
δ: 7.57
12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
δ: 168.44, 150.01, 128.49, 123.39, 114.04, 39.77, 29.33, 29.18,
29.06, 26.76. HRMS (ESI) m/z: 411.2755 [M + H]+, calcd. for [C24H35N4O2]+ 411.2760.
4.6.24. N,N0-(Decane-1,10-Diyl)bis(3-Aminobenzamide) (32e)
◦
1
Pale white solid, yield 57%, m.p. 135.7–136.3 C; H-NMR (300 MHz, CDCl3 + CD3OD)
7.30–6.97 (m, 6H), 6.83 (dd, J = 20.8, 7.2 Hz, 2H), 3.36 (t, J = 6.8 Hz, 4H), 1.67–1.47 (m, 4H), 1.46–1.06 (m,
12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
: 168.75, 146.84, 135.56, 129.27, 118.09, 116.55, 113.73,
δ:
δ

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77.48, 77.16, 76.84, 49.31, 49.09, 48.88, 48.67, 48.45, 39.93, 29.29, 29.22, 29.09, 26.79. HRMS (ESI) m/z:
411.2758 [M + H]+, calcd. for [C24H35N4O2]+ 411.2760
4.6.25. N,N0-(Decane-1,10-Diyl)Bis(3,5-Dimethoxybenzamide) (33e)
Pale white solid, yield 56%, m.p. 143.4–144.2 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD)
J = 2.1 Hz, 4H), 6.43 (t, J = 1.9 Hz, 2H), 3.69 (s, 12H), 3.25 (t, J = 7.2 Hz, 4H), 1.50–1.45 (m, 4H), 1.25–1.15
δ: 6.79 (d,
(m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
δ: 168.01, 160.73, 136.74, 104.94, 103.34, 55.40, 40.03,
29.28, 29.26, 29.11, 26.82. HRMS (ESI) m/z: 501.2959 [M + H]+, calcd. for [C28H41N2O6]+ 501.2965.
4.6.26. N,N0-(Decane-1,10-Diyl)Bis(3,4,5-Trimethoxybenzamide) (34e)
Pale white solid, yield 63%, m.p. 194–194.9 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD)
4H), 3.72 (s, 12H), 3.67 (s, 6H), 3.22–3.16 (m, 4H), 1.50–1.36 (m, 4H), 1.22–1.08 (m, 12H). 1H-NMR (300
MHz, CDCl3 + CD3OD). : 6.91 (s, 4H), 3.72 (s, 12H), 3.67 (s, 6H), 3.22–3.16 (m, 4H), 1.50–1.36 (m, 4H),
1.22–1.08 (m, 12H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
: 167.81, 152.77, 129.84, 104.42, 60.53, 55.85,
δ: 6.91 (s,
δ
δ
40.01, 29.17, 29.03, 26.73. HRMS (ESI) m/z: 561.3169 [M + H]+, calcd. for [C30H45N2O8]+ 561.3176.
4.6.27. N,N0-(Decane-1,10-Diyl)Bis(2,3,4-Trimethoxybenzamide) (35e)
Pale white solid, yield 26%, m.p. 112.3–112.5 ◦C; 1H-NMR (300 MHz, CDCl3)
(d, J = 8.9 Hz, 2H), 6.76 (d, J = 9 Hz, 2H), 3.96 (s, 6H), 3.90 (s, 6H), 3.87 (s, 6H), 3.44 (dd, J = 12.7, 6.9 Hz,
4H), 1.62 (dt, J = 14.3, 7.1 Hz, 4H), 1.41–1.29 (m, 12H). 13C-NMR (101 MHz, CDCl3)
: 164.90, 156.35,
δ: 7.94 (s, 2H), 7.89
δ
152.42, 141.83, 126.76, 119.31, 107.67, 61.71, 61.08, 56.15, 39.71, 29.78, 29.65, 29.43, 27.24. HRMS (ESI)
m/z: 561.3172 [M + H]+, calcd. for [C30H45N2O8]+ 561.3176.
4.7. General Synthetic Procedure for 9d, 9f
A mixture of cinnamic acid (6.30 mmol), EDCI (7.50 mmol), DMAP (0.60 mmol), and anhydrous
dichloromethane (20 mL) was stirred to dissolve, then diamino-alkane (3 mmol) was added and stirred
at room temperature for 12 h. The mixture solution was filtered under reduced pressure. After that,
the residue was washed with dichloromethane and water successively, and then dried to give a pale
white solid. TLC indicated that it was a single point.
4.7.1. (2E,20E)-N,N0-(Octane-1,8-Diyl)Bis(3-Phenylacrylamide) (9d)
Pale white solid, yield 63%, m.p. 186–186.4 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD)
(m, 12H), 6.53 (d, J = 15.7 Hz, 2H), 3.31 (t, J = 7.1 Hz, 4H), 1.59–1.55 (m, 4H), 1.35 (s, 8H). 13C-NMR (101
δ: 7.58–7.29
MHz, CDCl3 + CD3OD) δ: 166.96, 140.39, 134.78, 129.42, 128.59, 127.54, 120.60, 39.43, 29.04, 28.85, 26.57.
HRMS (ESI) m/z: 405.2537 [M + H]+, calcd. for [C26H33N2O2]+ 405.2542.
4.7.2. (2E,20E)-N,N0-(Dodecane-1,12-Diyl)Bis(3-Phenylacrylamide) (9f)
Pale white solid, yield 49%, m.p. 164.7–165 ◦C; 1H-NMR (300 MHz, CDCl3 + CD3OD)
δ: 7.56–7.49
(m, 6H), 7.40–7.28 (m, 6H), 6.53 (d, J = 15.7 Hz, 2H), 3.30 (t, J = 7.1 Hz, 4H), 1.62–1.51 (m, 4H), 1.38–1.23
(m, 16H). 13C-NMR (101 MHz, CDCl3 + CD3OD)
δ: 166.94, 140.22, 134.73, 129.29, 128.47, 127.42, 120.54,
39.42, 29.19, 29.03, 28.99, 26.66. HRMS (ESI) m/z: 461.3166 [M + H]+, calcd. for [C30H41N2O2]+ 461.3168.
4.8. General Synthetic Procedure for 9g–9i
A mixture of cinnamic acid (6.30 mmol), EDCI (7.50 mmol), DMAP (0.60 mmol), and anhydrous
dichloromethane (20 mL) was stirred to dissolve, then alkane-diol (3 mmol) was added and stirred
at room temperature for 12 h. The mixture solution was filtered under reduced pressure. After that,
the residue was washed with dichloromethane and water successively, subsequently, purified by
preparative TLC eluted with petroleum ether/ethyl acetate = 5:1 to give pale white solid.

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4.8.1. Octane-1,8-Diyl (2E,20E)-Bis(3-Phenylacrylate) (9g)
Pale white solid, yield 35%, m.p. 61.1–62.5 ◦C; 1H-NMR (300 MHz, CDCl3)
δ: 7.68 (d, J = 16 Hz,
2H), 7.58–7.46 (m, 4H), 7.43–7.30 (m, 6H), 6.45 (d, J = 16 Hz, 2H), 4.21 (t, J = 6.7 Hz, 4H), 1.77–1.68 (m,
4H), 1.46–1.40 (m, 8H). 13C-NMR (101 MHz, CDCl3)
δ: 167.22, 144.72, 134.62, 130.35, 129.01, 128.19,
118.43, 64.79, 29.31, 28.85, 26.05. HRMS (ESI) m/z: 407.2223 [M + H]+, calcd. for [C26H31O4]+ 407.2222.
4.8.2. Decane-1,10-Diyl (2E,20E)-Bis(3-Phenylacrylate) (9h)
Pale white solid, yield 33%, m.p. 118.6–119.2 ◦C; 1H-NMR (300 MHz, CDCl3)
2H), 7.57–7.48 (m, 4H), 7.38 (dd, J = 6.6, 3.6 Hz, 6H), 6.45 (d, J = 16 Hz, 2H), 4.21 (t, J = 6.7 Hz, 4H),
δ: 7.68 (d, J = 16 Hz,
1.77–1.62 (m, 4H), 1.48–1.25 (m, 12H). 13C-NMR (101 MHz, CDCl3)
δ: 167.23, 144.69, 134.64, 130.35,
129.01, 128.19, 118.46, 64.85, 29.58, 29.40, 28.88, 26.12. HRMS (ESI) m/z: 435.2534 [M + H]+, calcd. for
[C28H35O4]+ 435.2535.
4.8.3. Dodecane-1,12-Diyl (2E,20E)-Bis(3-Phenylacrylate) (9i)
Pale white solid, yield 28%, m.p. 63.1–64 ◦C; 1H-NMR (300 MHz, CDCl3)
2H), 7.52 (dt, J = 4.6, 3.2 Hz, 4H), 7.42–7.33 (m, 6H), 6.45 (d, J = 16 Hz, 2H), 4.21 (t, J = 6.7 Hz, 4H),
δ: 7.68 (d, J = 16 Hz,
1.77–1.65 (m, 4H), 1.43–1.25 (m, 16H). 13C-NMR (101 MHz, CDCl3)
δ: 167.22, 144.68, 134.64, 130.34,
129.01, 128.18, 118.47, 64.87, 29.68, 29.66, 29.42, 28.87, 26.12. HRMS (ESI) m/z: 463.2851 [M + H]+, calcd.
for [C30H39O4]+ 463.2828.
4.9. General Synthetic Procedure for 9j–9m
A mixture of cinnamic acid (6.30 mmol), EDCI (7.50 mmol), DMAP (0.60 mmol), and anhydrous
dichloromethane (20 mL) was stirred to dissolve, then diamino-ether (3 mmol) was added and stirred
at room temperature for 12 h. Then, the solid was filtered off, the filtrate was added with H2O (50 mL)
and extracted by dichloromethane three times. The combined organic layers were washed with H2O
and brine, dried with anhydrous sodium sulfate for 8 h, filtered, and evaporated. The resulting residue
was purified by preparative TLC with petroleum ether/ethyl acetate = 2:1–1:1 to give pale white solid
or light yellow oil.
4.9.1. (2E,20E)-N,N0-((Ethane-1,2-Diylbis(Oxy))Bis(Ethane-2,1-Diyl))Bis(3-Phenylacrylamide) (9j)
Pale white solid, yield 78%, m.p. 117.9–118.5 ◦C; 1H-NMR (300 MHz, CDCl3)
Hz, 2H), 7.51–7.41 (m, 4H), 7.37–7.27 (m, 6H), 6.48 (d, J = 15.6 Hz, 2H), 3.65–3.60 (m, 12H). 13C-NMR
δ: 7.62 (d, J = 15.6
(101 MHz, CDCl3) δ: 166.26, 141.19, 134.95, 129.77, 128.92, 127.92, 120.82, 70.50, 70.05, 39.63. HRMS
(ESI) m/z: 409.2121 [M + H]+, calcd. for [C24H29N2O4]+ 409.2127.
4.9.2.
(2E,20E)-N,N0-(((Oxybis(Ethane-2,1-Diyl))Bis(Oxy))Bis(Ethane-2,1-Diyl))Bis(3-Phenylacrylamide) (9k
)
Light yellow oil, yield 28%; 1H-NMR (300 MHz, CDCl3)
= 6.5, 3.1 Hz, 4H), 7.32–7.30 (m, 4H), 6.71 (t, J = 5.7 Hz, 2H), 6.47 (d, J = 15.6 Hz, 2H), 3.65 (s, 8H),
δ: 7.60 (d, J = 15.6 Hz, 2H), 7.45 (dd, J
3.63–3.54 (m, 8H). 13C-NMR (101 MHz, CDCl3)
δ: 166.17, 140.96, 134.98, 129.70, 128.89, 127.86, 120.93,
70.49, 70.27, 70, 39.59. HRMS (ESI) m/z: 453.2387 [M + H]+, calcd. for [C26H33N2O5]+ 453.2389.
4.9.3. (Ethane-1,2-Diylbis(Oxy))Bis(Ethane-2,1-Diyl) (2E,20E)-Bis(3-Phenylacrylate) (9l)
1
Light yellow oil, yield 65%; H-NMR (300 MHz, CDCl3)
δ
: 7.70 (d, J = 16 Hz, 2H), 7.54–7.48
(m, 4H), 7.40–7.34 (m, 6H), 6.48 (d, J = 16 Hz, 2H), 4.42–4.36 (m, 4H), 3.84–3.77 (m, 4H), 3.73 (s, 4H).
13C-NMR (101 MHz, CDCl3)
: 167.03, 145.21, 134.50, 130.44, 129.01, 128.23, 118, 70.79, 69.48, 63.78.
HRMS (ESI) m/z: 411.1805 [M + H]+, calcd. for [C24H27O6]+ 411.1808.
δ

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4.9.4. ((Oxybis(Ethane-2,1-Diyl))Bis(Oxy))Bis(Ethane-2,1-Diyl) (2E,20E)-bis(3-Phenylacrylate) (9m)
Light yellow oil, yield 87%; 1H-NMR (300 MHz, CDCl3)
: 7.70 (d, J = 16 Hz, 2H), 7.55–7.48 (m,
4H), 7.38 (dd, J = 3.8, 2.7 Hz, 6H), 6.48 (d, J = 16 Hz, 2H), 4.37 (dd, J = 5.5, 4.1 Hz, 4H), 3.81–3.74 (m,
δ
4H), 3.70 (s, 8H). 13C-NMR (101 MHz, CDCl3)
δ: 167.03, 145.19, 134.52, 130.45, 129.02, 128.23, 118.04,
77.48, 77.36, 77.16, 76.84, 70.82, 70.79, 69.42, 63.80. HRMS (ESI) m/z: 472.2337 [M + NH4]+, calcd. for
[C26H34NO7]+ 472.2335.
4.10. Biological Assays
4.10.1. Cell Culture
The tested cells were obtained from Shanghai Institute of Biochemistry and Cell Biology of the
Chinese Academy of Sciences (Shanghai, China). HeLa and Hep G2 cell lines were cultured in Dulbecco’s
modified Eagle’s medium (DMEM; gibco, Life Technologies, Carlsbad, CA, USA) supplemented with
10% fetal bovine serum (FBS; Biological Industries), 100 U/mL penicillin-streptomycin (PS; gibco, Life
Technologies, Carlsbad, CA, USA). MCF-7 and A549 cell lines were maintained in RPMI-1640 media
(gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS and 100 U/mL PS. FaDu
cell lines were cultured in minimum essential medium (MEM; Gibco, Life Technologies, Carlsbad, CA,
USA) supplemented with 10% FBS and 100 U/mL PS. MCF 10A cell lines were maintained in Mammary
MEGM kit (Lonza/Clonetics) supplemented with 100 ng/mL cholera toxin (Sigma, Shanghai, China).
Cell lines were grown at 37 ◦C in a humidified atmosphere of 5% CO2.
4.10.2. Cell Viability Assay
The growth-inhibitory effects against HeLa, Hep G2, MCF-7, FaDu, and A549 cancer cells and
normal cell lines MCF 10A were determined by MTT assay [21]. All of the synthesized compounds
and the positive control drug doxorubicin were dissolved in 0.1% DMSO. Cells were seeded in 96 well
plates at a density of 1
×
104 cells per well and incubated for 24 h at 37 ◦C in a humidified atmosphere
of 5% CO2. The cells were then incubated with various concentrations of drugs for 48 h in a 5% CO2
incubator at 37 ◦C. After treatment with 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) solution for 4 h, the formazan crystals were dissolved in 100 µL DMSO in each well.
Plates were read on a microplate reader (Thermo Varioskan Flash 3001) at 490 nm. The experiment
was performed in triplicate. The inhibitory concentration at 50% (IC50) was calculated based on
concentration-inhibition relationships via regression using SPSS software (Version 17.0).
4.10.3. Colony Formation Assay
FaDu cells were seeded in a 6-well plate with 500 cells per well and incubated for 24 h. Then, the
cells were treated with compound 8e at various final concentrations of 0, 0.75, 1.50, 3 nM. Following
treatment for 10 days, the cells were washed with PBS twice and fixed with 4% paraformaldehyde
for 10 min at room temperature. Then, the cell colonies were visualized by staining with 0.1% crystal
violet for 10 min. The image was photographed with Handy camera, and the numbers of cell colonies
were analyzed using the open Image J software (Developed by National Institutes of Health). A group
of >50 cells was defined as one colony. Triplicate wells were set up for each concentration.
4.10.4. Live/Dead Staining
The Calcein AM/propidium iodide (PI) double staining was performed following the
manufacturer’s instructions (Dojindo Laboratory, Kumamoto, Japan). Briefly, FaDu cells were
seeded in 6-well plates overnight. Then, the 8e solutions at final concentrations of 2.5 nM, 5 nM, and 10
nM were added. After incubation for 24 h, the cells were collected, washed with PBS, and stained by a
mixture of 2
µM Calcein-AM (live cells, green) and 4.5 µM PI (dead cells, red) solution in the dark for
30 min at 37 ◦C (excitation 490 nm). Then, the cells were rinsed with PBS twice and re-suspended in the

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cell medium. Confocal fluorescence images were acquired using a laser scanning confocal microscope
(LSCM, Carl-Zeiss LSM 710).
4.10.5. Hoechst 33,342 Staining
For Hoechst staining, FaDu cells were seeded onto a glass-bottom of cell culture dish at a density
of 1
×
105 cells. After incubation for 24 h, the media containing 20 nM of the compound 8e was used to
replace the culture medium. Then, the cells were stained with Hoechst 33,342 solution (10
µg/mL in
the culture medium, Beyotime Institute of Biotechnology, Shanghai, China) at 37 ◦C in the dark for
20 min after treatment with 8e. Then, the cells were washed with the serum-free medium to remove
excess dye. LSCM (Carl-Zeiss LSM 710) was used to capture the images of nuclear morphological
changes to find apoptotic cells.
4.10.6. Flow Cytometric Analysis of Apoptosis by Annexin V-FITC/PI Staining
Apoptosis in FaDu cells was evaluated by an annexin V-FITC/PI apoptosis detection kit (Beyotime
Institute of Biotechnology, Shanghai, China). Briefly, FaDu cells were treated with various concentrations
of compound 8e (2, 5, 10 nM) or DMSO as vehicle control for 24 h at 37 ◦C; then, the cells were washed
twice with cold 1
×
PBS and centrifuged at 1000 rpm for 5 min. The harvested cells were resuspended
L annexin V-FITC and 10 L PI. After incubating for 15
gently in 400 L binding buffer, containing 5
µ
µ
µ
min in the dark at room temperature, the cells were analyzed with flow cytometry (CyFlow® Cube 6,
Sysmex). Data were shown as pseudo color graphs and analyzed using FlowJo Software (Tree Star Inc,
Ashland, OR).
4.10.7. Mitochondrial Membrane Potential (∆Ψm) Analysis
The mitochondrial membrane potential (∆Ψm) was detected by a mitochondrial membrane
potential assay kit with JC-1 (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, after
treatment with various concentrations of compound 8e (2, 5, 10 nM) for 24 h, FaDu cells were harvested
and stained with 500
µ
L 1
JC-1 dye solution at 37 ◦C for 20 min in the dark. Then, the treated cells
×
were washed twice and resuspended by 1 JC-1 staining buffer. The fluorescence of approximately
1 × 104 cells was analyzed using flow cytometry (CyFlow® Cube 6, Sysmex).
×
4.10.8. Cell Cycle Analysis
The cell cycle analysis was carried out by a cell cycle and apoptosis analysis kit with a propidium
iodide (PI) staining method (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, FaDu cells
were incubated with the above-mentioned doses of compound 8e for 24 h; all samples were collected,
washed once with cold 1
were washed once with cold 1
RNase A) at 37 ◦C for 30 min in the dark. The red fluorescence was detected using a flow cytometer
(CyFlow® Cube 6, Sysmex). The data were analyzed using FlowJo Software (Tree Star Inc, Ashland,
OR, USA).
×
PBS, and fixed by 70% ice-cold ethanol at 4 ◦C for 12 h. Then, the fixed cells
PBS and stained with 500 L 1 PI dye solution (containing 10 µL
×
µ
×
4.10.9. In Silico ADMET Prediction
A computational study of all synthesized compounds was carried out for the prediction of ADMET
(absorption, distribution, metabolism, excretion, and toxicity) properties. In this module, thirteen
mathematical models, such as HBD (hydrogen bond donor), HBA (hydrogen bond acceptor), RBN
(number of rotatable bonds), logP (log of the octanol/water partition coefficient), log S (log of the
aqueous solubility), and PSA (polar surface area) were predicted via Discovery Studio 2.0 Software
(Studio 2.5, Accelrys, Co. Ltd., San Diego, CA, USA) and HIA (human intestinal absorption), PPB
(plasma protein binding), CYP3D4 (CYP3D4 inhibition), T1/2 (half lifetime), HT (human hepatotoxicity),
and LD50 (median lethal dose) were calculated using web-based applications and then analyzed to

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predict the drug likeliness profile (Xiangya School of Pharmaceutical Sciences and Central South
University, http://admet.scbdd.com/home/index/) [28]. We also screened our novel derivatives through
the prediction model by Egan et al. [29,30]. Two molecular descriptors, AlogP98 and PSA, were
computed and plotted in a 2D plane to comprehensively evaluate the absorption and blood-brain
penetration of the compounds.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/24/4505/s1,
1H-NMR, 13C-NMR and HR-MS spectra of compounds.
Author Contributions: Ideas and experiment design: J.W., G.H., and T.L.; Chemistry and Biology: J.W., G.H., G.L.,
and W.W.; Analysis and interpretation of data: J.W., G.H., G.L., and W.W.; Writing and review of the manuscript:
J.W., G.H., G.L., W.W., and T.L.; Study supervision: T.L. and G.H.
Funding: This research was funded by the national major science and technology special project for “significant
new drugs development” (2018ZX09711001-005-018), “the medical and health science and technology innovation
project of Chinese Academy of Medical Science” (2017-I2M-3-021/2019-I2M-1-005), “the Tianjin special support
program for talent development” (TJTZJH-GCCCXCYTD-1-30).
Acknowledgments: The authors gratefully acknowledge the Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College and information technology center for affording the Discovery
Studio 2.0 Software.
Conflicts of Interest: The authors declare that they have no competing interests.
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Sample Availability: Samples of all compounds are available from the authors.
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