D-amino acid position influences the anticancer activity of galaxamide analogs: An apoptotic mechanism study

Article abstract of DOI:10.3390/ijms18030544

Galaxamide, an extract from Galaxaura filamentosa, is a cyclic pentapeptide containing five L-leucines. Due to the particular cyclic structure and the excellent anticancer activity, synthesis of Galaxamide and its analogs and their subsequent bio-applications have attracted great attention. In the present work, we synthesized six Galaxamide analogs by replacing one of the L-leucines with phenylalanine and varying the D-amino acid position. The anticancer effect of the synthesized Galaxamide analogs was tested against four in vitro human cancer cell lines, human hepatocellular cells (HepG₂), human breast cancer cell (MCF-7), human breast adenocarcinoma cells (MDA-MB-435) and a human cervical carcinoma cell line (Hela). Results showed that Galaxamide analogs with different_D-amino acid positions displayed distinct anticancer potential. The Galaxamide analog containing D-amino acid at position 5 (Analog-6) presented the strongest anticancer activity. The mechanism study revealed that Analog-6 could cause the early apoptosis of HepG₂ cells by inhibiting their growth in the sub-G1 stage of the cell cycle and induce the chromatin condensation and fragmentation, which can be seen as 68% of HepG₂ cells inhibited in the sub-G1 stage. Moreover, a mitochondria-mediated pathway was found to be involved in the apoptotic process of Analog-6 on HepG₂ cells.

Full text of DOI:10.3390/ijms18030544

International Journal of
Molecular Sciences
Article
D-Amino Acid Position Influences the Anticancer
Activity of Galaxamide Analogs: An Apoptotic
Mechanism Study
1
2
3
3
3
3
Defa Bai , Siming Yu , Shenghui Zhong , Bingxin Zhao , Shaoling Qiu , Jianwei Chen ,
3
3,
1,3,
Jignesh Lunagariya , Xiaojian Liao * and Shihai Xu
*
1
College of Pharmacy, Jinan University, Guangzhou 510632, China; baidf@foxmail.com
Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical
Engineering, Jinan University, Guangzhou 510632, China; siming_yu@hotmail.com
Department of Chemistry, College of Chemistry and Materials Science, Jinan University, Guangzhou 510632,
China; zhongsh@stu2014.jnu.edu.cn (S.Z.); zbx840622@163.com (B.Z.); qslhappyrain@gmail.com (S.Q.);
2
3
1
8826237580@163.com (J.C.); jignesh.lunagariya@gmail.com (J.L.)
*
Correspondence: tliaoxj@jnu.edu.cn (X.L.); txush@jnu.edu.cn (S.X.); Tel.: +86-20-8522-1346 (S.X.)
Academic Editor: Anthony Lemarié
Received: 5 December 2016; Accepted: 27 February 2017; Published: 10 March 2017
Abstract: Galaxamide, an extract from Galaxaura filamentosa, is a cyclic pentapeptide containing five
L-leucines. Due to the particular cyclic structure and the excellent anticancer activity, synthesis of
Galaxamide and its analogs and their subsequent bio-applications have attracted great attention.
In the present work, we synthesized six Galaxamide analogs by replacing one of the L-leucines
with phenylalanine and varying the D-amino acid position. The anticancer effect of the synthesized
Galaxamide analogs was tested against four in vitro human cancer cell lines, human hepatocellular
cells (HepG ), human breast cancer cell (MCF-7), human breast adenocarcinoma cells (MDA-MB-435)
2
and a human cervical carcinoma cell line (Hela). Results showed that Galaxamide analogs
with different D-amino acid positions displayed distinct anticancer potential. The Galaxamide
analog containing D-amino acid at position 5 (Analog-6) presented the strongest anticancer activity.
The mechanism study revealed that Analog-6 could cause the early apoptosis of HepG cells by
2
inhibiting their growth in the sub-G1 stage of the cell cycle and induce the chromatin condensation
and fragmentation, which can be seen as 68% of HepG cells inhibited in the sub-G1 stage. Moreover,
2
a mitochondria-mediated pathway was found to be involved in the apoptotic process of Analog-6 on
HepG cells.
2
Keywords: Galaxamide analogs; cyclopeptide; D-amino acid; anticancer; mechanism
1
. Introduction
Cyclopeptides are a kind of peptide with a cyclic structure, which are widely distributed in nature.
Because of the particular cyclic structure, cyclopeptides present many advantages over their linear
counterparts, including: (1) cyclopeptides have a slower degradation behavior than the linear ones
due to the fact that proteases can hardly access the cycle hole and cleave the amide bonds [
cyclopeptides bind more strongly for protein targets thanks to their constrained bond rotation and
conformation [ ]. Moreover, many recent studies have demonstrated the potential use of cyclopeptides
as a clinical drug, such as antitumor, antiviral anti-inflammatory and antibiotic [ ]. Therefore, the
1]; (2) the
2
3,4
study of cyclopeptides has attracted enormous attention and becomes one of the most important parts
of the research and development of antitumor drugs.
Cyclopeptides are mainly extracted from marine species. Since the first bioactive cyclopeptide
ulithiacyclamide was isolated from the sea squirt by scientist of Chris Ireland in 1980 [5], more and more
Int. J. Mol. Sci. 2017, 18, 544; doi:10.3390/ijms18030544
www.mdpi.com/journal/ijms

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cyclopeptides have been isolated from various marine animals during the past three decades. Those
cyclopeptides have been used as important resources in designing and developing novel antitumor
drugs. Among those, cyclic-pentapeptide became the focus of our research group.
In our initial work, a novel cyclic-pentapeptide named Galaxamide was isolated from a marine
algae Galaxaura filamentosa collected from Xisha Island in the South China Sea [
cyclic-pentapeptide composed of five L-leucines, including two N-methylated ones. N-methylated
amino acid is an important component endowing the anticancer potential to the cyclopeptides [ ].
We found that Galaxamide exhibited strong in vitro antiproliferative activities against human renal cell
carcinoma GRC-1 and human hepatocellular carcinoma HepG cell lines, remarkably with half maximal
6]. Galaxamide is a
7
2
inhibitory concentration (IC ) values of 4.26 and 4.63
µ
g/mL, respectively [
presented excellent bioactivity in other cyclic pentapeptides [ ], we also incorporated phenylalanine
into our extracted Galaxamide by replacing one of the leucines with phenylalanine. The obtained
6,8]. Since phenylalanine
50
9
Galaxamide analog could inhibit the growth of HepG , U87 and MCF-7 human carcinoma cell lines
2
more efficiently than the natural Galaxamide [10]. Furthermore, we synthesized Galaxamide analogs
by changing L-leucine to D-leucine; Galaxamide analogs with D-leucine exhibited stronger inhibition
against human carcinoma cells than the natural Galaxamide. More recently, we demonstrated that
more D-leucine incorporated in Galaxamide resulted in better antitumor activity; the analog with
four D-leucines displayed the highest apoptosis effect against a wide range of human cancer cell lines,
such as HepG2, MCF-7, SW480 and U87 [8].
In the present work, we aimed to elucidate the relationship between the D-amino acid position
in Galaxamide analogs (Figure 1) and their anticancer activity. For this purpose, we first synthesized
the Galaxamide analog by replacing one leucine with phenylalanine and then gradually changed
the D-amino acid position from phenylalanine to the other leucines to test the changes in their
anticancer activity.
Figure 1. Chemical structure of Galaxamide and its analogs.

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2
. Results and Discussion
2
.1. Synthesis of Galaxamide Analogs
In the present work, all of the Galaxamide analogs were designed and synthesized using the classic
3 + 2” strategy; the schematic illustration of the synthetic route is detailed in Scheme 1. As shown
in Scheme 1, firstly, dipeptide was synthesized by the condensation reaction of N-Boc-Me-Leu-OH
and L(D)-Leu-OBn TosOH, where 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT)
“
·
was used as the condensation reagent. After removing the C-terminal Bn group, the deprotected
dipeptide was labeled as Fragment 1. Previous to the synthesis of tripeptide, the N-terminal Boc
group was removed from the synthesized dipeptide. Then, the deprotected dipeptide was reacted
with N-Boc-L(D)-phenylalanine to produce tripeptide. After removing the N-terminal Boc group, the
deprotected tripeptide was marked as Fragment 2. The linear pentapeptide was synthesized through
the condensation reaction of N-terminal protected dipeptide Fragment 1 and C-terminal protected
tripeptide Fragment 2. To synthesize cyclic pentapeptide, the N-terminal Boc and C-terminal Bn groups
were first removed from the obtained linear pentapeptide. Then, the deprotected linear pentapeptide
was dissolved in a mixed solvent of THF/CH CN/CH Cl (2:2:1) with a final concentration of 0.1 M,
3
2
2
and a mixture of condensation reagents of HATU/DEPBT/TBTU was added within the ice bath.
The whole process was traced by High Performance Liquid Chromatography (HPLC), and the product
was separated and purified by Reverse Phase-HPLC (RP-HPLC); finally, cyclic pentapeptides were
obtained. General chemical structure of the synthesized Galaxamide analogs is shown in Figure 2.
O
OH
O
OBn
NH2
BocN
O
a
H
b
BocN
O
H
+
N
N
OBn
N
OH
Boc
O
O
Fragment 1
c
NHBoc
NH2
d
O
N
H
O
O
N
O
H
N
N
OBn
OBn
O
O
Fragment 2
Boc
N
HN
O
O
O
O
Fragment 1
Fragment 2
NH
HN
a
e
NH
N
O
N
H
O
N
O
O
N
H
N
OBn
O
O
Cyclized Galaxamide
Analogs
Linear Pentapeptide
Scheme 1.
Synthesis of macrocycles.
Reagents and conditions: a, DEPBT, DIEA,
THF; b, H2, 10% Pd/C, EtOAc; c, (i) TFA, DCM; (ii) N-Boc- L(D)-Phe-OH, DEPBT,
DIEA, THF; d, TFA, DCM; e, (i) TFA, DCM; (ii) H2, 10% Pd/C, EtOAc; (iii) DEPBT,
HATU, TBTU. Note: DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), DIEA
(
N,N-Diisopropylethylamine), THF (tetrahydrofuran), EtOAc (ethyl acetate), TFA (trifluoroacetic
acid), DCM (dichloromethane), TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate), HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid
hexafluorophosphate).

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Figure 2. Amino acids used in the synthesis of Galaxamide analogs. Red wavy lines show the chemical
bond where amino acid was replaced. Dash line boxes highlight leucine in Galaxamide has been
replaced by phenylalanine in our Analogs. Amino acids in solid line boxes has D/L configuration.
2
.2. Biological Activity
2
.2.1. Anticancer Activity of Galaxamide and Its Analogs on Different Human Cancer Cell Lines
To examine the anticancer potential of Galaxamide and its analogs, four different human cancer
cell lines, such as human hepatocellular cells (HepG ), human breast cancer cells (MCF-7), human
2
breast adenocarcinoma cells (MDA-MB-435) and a human cervical carcinoma cell line (Hela), were
selected for the in vitro cancerous cell models. IC50 values of Galaxamide and its analogs on the four cell
lines are shown in Table 1. Analog-1 and Analog-2 are Galaxamide analogs in which one of the leucines
was replaced with phenylalanine, but varied in phenylalanine configuration (L-phenylalanine and
D-phenylalanine for Analog-1, Analog-2, respectively). Generally, the incorporation of phenylalanine
obviously improved the anticancer activity of Galaxamide, but with an exception for Hela cells.
In particular, when L-phenylalanine was changed to D-phenylalanine, the inhibition activity of the
Galaxamide analog was further reinforced to some extent, especially for MCF-7 cells. In MCF-7 cells,
we found a 30% decrease in IC50 of Analog-2 (4.95
suggesting that D-amino acid can improve the anticancer effect of Galaxamide on cancer cells, which is
consistent with our previous work [8].
±
0.18) compared to Analog-1 (13.03
±
0.33),
On the basis of the obtained Galaxamide analog with one D-phenylalanine, we synthesized several
other Galaxamide analogs by gradually changing the D-amino acid position from phenylalanine to
other leucines. The anticancer activity of those analogs was also tested against the above-mentioned
cell lines. Remarkably, varying the D-amino acid position greatly affected the anticancer activity of the
analogs. Analog-6 with the D-amino acid at position 5 is the best among those analogs at inhibiting the
growth of all of the cancer cells; the IC50 values were 4.1
.42 0.25 (Hela) and 6.76 0.22 (MDA-MB-435), which are nearly half those of the natural
Galaxamide. Beside Analog-6, Analog-4 with the D-amino acid at position 3 also showed strong
inhibition effect against HepG , MCF-7, Hela and MDA-MB-435 with IC values of 7.2 0.15,
0.18, respectively. The anticancer ability of Analog-3 (D-amino acid at
±
0.20 (HepG ), 8.15
±
0.18 (MCF-7),
2
3
±
±
±
2
50
8
.62
±
0.26, 4.2
±
0.32, 7.67
±

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position 2) was only slightly changed compared to that of Galaxamide against HepG , MCF-7, Hela
2
and MDA-MB-435 with IC50 values of 7.71
while Analog-5 (D-amino acid at position 2) displayed a decreased inhibition effect with IC values of
±
0.11, 18.16
±
0.33, 4.39
±
0.26, 12.6
±
0.29, respectively,
50
1
0.72 ± 0.32, 15.21 ± 0.29, 7.4 ± 0.33, 20.43 ± 0.23, respectively.
Table 1. Cytotoxicity of Galaxamide and its analogs on different human cancer cell lines.
IC50 (µg/mL) in Different Cell Lines
Compound
HepG2
MCF-7
Hela
MDA-MB-435
Galaxamide
Analog-1
Analog-2
Analog-3
Analog-4
Analog-5
Analog-6
9.45 ± 0.25
5.85 ± 0.13
4.56 ± 0.22
7.71 ± 0.11
7.2 ± 0.15
14.08 ± 0.28
13.03 ± 0.33
9.64 ± 0.27
18.16 ± 0.33
8.62 ± 0.26
15.21 ± 0.29
8.15 ± 0.18
4.94 ± 0.19
6.85 ± 0.26
4.95 ± 0.18
4.39 ± 0.26
4.2 ± 0.32
7.4 ± 0.33
3.42 ± 0.25
11.08 ± 0.22
9.76 ± 0.18
9.5 ± 0.32
12.6 ± 0.29
7.67 ± 0.18
20.43 ± 0.23
6.76 ± 0.22
10.72 ± 0.32
4.1 ± 0.20
2
.2.2. Inhibition Effect of Galaxamide and Its Analogs on Cell Cycle
Since Analog-4 and -6 exhibited a better inhibition effect on the growth of cancer cells than
the others, further investigation regarding their apoptotic mechanism was performed on HepG2.
In particular, HepG cells were exposed to Galaxamide, Analog-4 and Analog-6 with concentrations
2
of 0, 5, 10 and 15
propidium iodide (PI) staining method, results are shown in Figure 3. Generally, treatment with
Galaxamide, Analog-4 and -6 all caused changes in the cell cycle of HepG cells, mainly in the
µg/mL for 48 h. Then cell cycle of HepG cells was analyzed using the
2
2
sub-G1 stage, where a subdiploid peak was detected, indicating their apoptotic effects on HepG cells.
2
Moreover, a concentration-dependent apoptotic effect was observed, an increase in the concentration
of Galaxamide, Analog-4 and -6 resulted in more apoptotic cells in the sub-G1 stage. Consistent with
the above cytotoxicity evaluation, Analog-6 displayed the strongest apoptotic effects on HepG cells,
2
where 68% of HepG cells were inhibited in the sub-G1 stage.
2
Figure 3. Cell cycle of HepG cells after 48 h of treatment with Galaxamide, Analog-4 and -6 with
2
concentrations of 0, 5, 10 and 15
peak) and G2/M (dark purple).
µg/mL. Sub-G1 (light blue peak), G0/G1 (light purple peak), S (pink

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2
.2.3. Effect of Galaxamide and Analog-6 on the Cell Nuclear Integrity
We then investigated the effect of Galaxamide and Analog-6 on the cell morphology and nuclear
integrity. HepG cells were treated with an increasing concentration of Analog-6 for 48 h and then
2
stained by Hoechst 33342 (a DNA fluorescent dye). The effect of Galaxamide on HepG cells was
2
used as a control. Noting that Hoechst 33342 is a DNA fluorescent dye, the fluorescently-stained DNA
presents a blue color under the fluorescence microscope.
In Figure 4, nuclei in the control cells show a regular round shape and are homogenously
distributed inside the cells. Increasing the concentration of Galaxamide and Analog-6 caused a
decrease in the number of nuclei and changes in the nuclei shape. For HepG cells exposed to
2
Galaxamide, slight shrinking of nuclei was observed. However, the nuclei morphology of HepG cells
2
was severely damaged at high concentrations of Analog-6, nuclei turned to being irregular and shrunk
greatly, and the number of regular nuclei was largely decreased. Quantification of Hoechst data also
shows that there is a significant difference in the percentage of cells with a regular nuclei shape between
those treated with Analog-6 and Galaxamide, where the percentage in the Analog-6-treated group
is much lower (Figure S1). The finding suggests that Analog-6 could cause obvious cell apoptosis,
leading to the severe chromatin condensation.
Figure 4. Fluorescent microscope images of HepG cells treated with different concentrations of
2
Galaxamide and Analog-6 after Hoechst 33342 staining. White arrows in the each images indicate the
cells with damaged nuclei (chromatin condensation and fragmentation). Scale bar is 50 µm.
2
.2.4. Apoptotic Mechanism Study of Galaxamide and Analog-6
To further study the apoptotic mechanism of Analog-6, the Annexin V-fluorescein isothiocyanate
(
FITC) and propidium iodide (PI) dual staining method was used. Briefly, HepG cells were treated
2
with 0, 5, 10 and 15
µg/mL Analog-6 for 48 h. Then, cells were washed several times with PBS
and stained by dual staining of two fluorescent dyes, Annexin V-FITC and PI, and analyzed by flow
cytometry. Results are shown in Figure 5, where HepG cells exposed to Galaxamide were used as
2
the control. Note that Q1, Q2, Q3 and Q4 in the figure represent four quadrants of necrotic cells,
late apoptotic cells, normal cells and early apoptotic cells, respectively.
Generally, exposure of Galaxamide and Analog-6 mainly caused the early apoptosis of HepG cells.
2
Moreover, a dose-dependent apoptotic affect was observed in both cases, an increase in concentration
led to a higher percentage of early apoptotic cells. In particular, when the Analog-6 concentration
reached 15 µg/mL, almost 50% of the cells were found to be apoptotic in the early stage. In comparison,
Galaxamide displayed a much weaker apoptotic effect with only 12.1% early apoptotic cells at a
concentration of 15 µg/mL, further confirming the stronger cancer inhibition activity of Analog-6.

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Galaxamide
0
μg/mL
5 μg/mL
10 μg/mL
15 μg/mL
a
0
.5%
1.8%
10.6%
12.1%
FITC
FITC
FITC
FITC
Analog-6
b
0
μg/mL
5 μg/mL
10 μg/mL
15 μg/mL
0
.8%
7.3%
16.9%
49.4%
FITC
FITC
FITC
FITC
Figure 5. Flow cytometry results of HepG cells stained by Annexin V-fluorescein isothiocyanate (FITC)
2
and propidium iodide (PI) after 48 h of exposure to 0, 5, 10 and 15
µ
g/mL Galaxamide (
); Comparison of the percentage of early apoptotic cells induced by Galaxamide ( ) and Analog-6 (
* p < 0.01.
a
) and Analog-6
(b
c
d);
*
2
.2.5. Western Blot Analysis
There are two major pathways involved in cells apoptosis: the mitochondria-mediated intrinsic
pathway and the death receptor-mediated extrinsic pathway [11]. To elucidate the possible mechanism
of the apoptotic effects of Analog-6, Western blot experiments were performed. HepG cells were
2
treated with 0, 5, 10 and 15 µg/mL Analog-6 for 48 h, and then expression of cleaved caspase-3,
caspase-9 and poly(ADP-ribose) polymerase (PARP) was quantified, results are shown in Figure 6.
As was shown, an increase in the Analog-6 concentration resulted in the increasing expression
of cleaved caspase-3, caspase-9 and PARP, suggesting that a mitochondria-mediated pathway was
involved in the apoptotic process. At the concentration of 15
µg/mL, an increased fold of 3.4, 3.3 and
6
.8 was found for cleaved caspase-3, caspase-9 and PARP, respectively. Therefore, Analog-6 exerted
the early apoptotic effects on HepG cells through a mitochondria-mediated pathway, which is similar
2
to the previous reports [9,12].

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Figure 6. Western blot results display the expression of the cleaved caspase-3, caspase-9 and cleaved
PARP of HepG cells after being treated with 0, 5, 10 and 15 µg/mL Analog-6 for 48 h.
2
3
. Discussion
In our previous work, we synthesized several Galaxamide analogs and tested their cytotoxicity
on a normal cell line (human normal liver cell line L02) and a broad range of cancerous cell lines
in vitro. It was revealed that Galaxamide and its analogs exhibited very low cytotoxicity on normal
L02 (IC50 > 40 µg/mL) [8]. Moreover, the number of D-amino acids and the D-leucines greatly affected
the anticancer behavior of Galaxamide analogs against in vitro cancerous cell lines. In particular,
the analog containing four D-leucines presented the best anticancer potential. To explore a more potent
anticancer agent, in the present work, we synthesized another six Galaxamide analogs (Analog-1
to Analog-6) and studied extensively the structure-activity relationship. Generally, when leucine
in Galaxamide was replaced by phenylalanine (Analog-1 and Analog-2), the anticancer activity of
Galaxamide was improved, suggesting that phenylalanine can act as an active site against cancer cells
when incorporated in Galaxamide. Introduction of phenylalanine to sansalvamide A displayed similar
results [
activity of the Galaxamide analog. Analog-2 with D-phenylalanine showed a better inhibitory effect
than Analog-1 with L-phenylalanine; our previous work also revealed a similar trend [ ]. A possible
9]. Besides, we found that the configuration of phenylalanine also matters for the anticancer
8
mechanism is that the D-phenylalanine could more efficiently block the cyclic structure of Galaxamide,
forming a more constrained confirmation, which could facilitate the stronger binding of Galaxamide
analog to protein targets expressed by cancer cells [13]. Further change in the position of amino acid
with the D configuration from phenylalanine to other leucines led to changes in the anticancer potential.
Galaxamide analogs with D-amino acid in positions 3 and 5 (Analog-4 and Analog-6) displayed
enhanced anticancer activity compared to the Galaxamide analog (Analog-2) with D-phenylalanine.
In particular, Analog-6 demonstrated a two-fold elevated anticancer potential compared to Analog-2
on both HepG and MDA-MB-435 cell lines. This might be due to the more constrained and stable
2
cyclic structure of Galaxamide achieved when D-amino acid was situated in positions 3 (Analog-4)
and 5 (Analog-6), whereas Analog-6 presented the most stable cyclic configuration with the lowest
energy and exhibited the most active amino acid residues for targeting cancer cells. Analog-3 with the
D-amino acid in position 2 may have a similar structure-activity relationship as Analog-2, since their
anticancer behavior is similar. The constrained structure of the Galaxamide analog may be destroyed

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to some extent when the D-amino acid was placed in position 4 (Analog-5), since Analog-5 displayed a
decreased anticancer effect on the tested cancer cells.
Many cyclic-pentapeptides like RA-V (deoxybouvardin) and Sansalvamide A have presented
great anticancer potential in cancer cells in vitro, mainly by triggering cell apoptosis. However, the
exact apoptotic mechanism behind that is not clear yet. Sansalvamide A caused cell apoptosis through
inhibiting the cell growth in the G0/G1 stage, while our synthesized Galaxamide analogs (Analog-4
and Analog-6) could inhibit HepG cell growth in the sub-G1 stage, suggesting a different apoptotic
2
mechanism was involved. Further investigation of Analog-6 revealed that it caused the HepG cell
2
apoptosis through a mitochondria-mediated pathway. By performing Western blot assays, we found
a significant increase in the expression of cleaved caspase-3, caspase-9 and PARP, which is a sign
of the activation of the cascade of the caspase family. This may be caused by the decrease of the
mitochondrial membrane potential. A decrease in the potential is associated with changes in the
mitochondrial membrane permeability, which can promote the entrance of the apoptotic proteins
within the cytoplasm to the outer membrane and release of cytochrome C (Cyt-C) to cytoplasm [14].
The released Cyt-C in the cytoplasm then coupled with the apoptotic protease-activating factor
1
(Apaf-1), forming the Apaf-1/Cyt-C complex and initiating the activation of the cascade of the
caspase family [15]. The activated caspase-9 activates the downstream caspase-3, which then activates
endonuclease for degrading PARP, finally leading to cell apoptosis [16]. This suggests that Analog-6
exerted the early apoptotic effect through a mitochondria-mediated pathway.
4
. Experimental Section
4
.1. Synthesis of Galaxamide Analogs
Galaxamide analogues were synthesized following our reported protocols with some
modification [
whole process was performed in a liquid phase with N-Boc-L(D)-phenylalanine, N-Boc-L(D)-leucine
and L(D)-Leu-OBn TosOH as the starting materials. After sequential experimental steps of amine
6,8]. Briefly, a classic “3 + 2” synthetic strategy was used in the synthesis, and the
·
protection and deprotection, acid deprotection, methylation and condensation over amino acids,
cyclization was finished using a combination of condensation reagents DEPBT, HATU and TBTU,
leading to the product of cyclic pentapeptides.
4
.1.1. Synthesis of N-Boc-Leu-OH
In a 500-mL round-bottom flask under N atmosphere, 1 M NaOH solution (63 mL) in distilled
2
water and 3.93 g L-Leu-OH (30 mmol) were added in sequence. The system was then immerged into
the ice bath for cooling for 15 min. After the solution of L-Leu-OH become clear, 7.86 g Boc O dissolved
2
in THF (84 mL, 36 mmol) were added and stirred for 10 min; the pH was adjusted to around 10.5.
The reaction temperature was then slowly increased to room temperature, and the solution was stirred
overnight. After 24 h, the solvent was removed by vacuum distillation, and the rest was extracted
twice using 200 mL ethyl ether. The resultant aqueous phase was cooled in the ice bath, acidified
by adding an appropriate amount of 1 M HCl solution to increase the pH to 2. Then, the aqueous
phase was further extracted three times with ethyl acetate (150 mL), washed twice with saturated NaCl
solution (150 mL). Subsequently, the organic layer was dried by anhydrous sodium sulfate and filtered.
After complete distillation of the organic solvent, 6.94 g white powder were obtained with a yield
1
of 98.4%. H-NMR (500 MHz, CDCl ),
(
δ 4.87 (m, 1H), 4.31 (m, 1H), 1.75 (m, 2H), 1.54 (m, 1H), 1.45
H
3
+
+
+
s, 9H), 0.95 (d, 6H, J = 6.4 Hz); MS (ESI) m/z: 232.2 [M + H] , 249.2 [M + NH ] , 254.3 [M + Na] .
4
4
.1.2. Synthesis of N-Boc-D-Leu-OH
The experimental procedure for the synthesis of N-Boc- D-Leu-OH followed as per described in
the synthesis of N-Boc-Leu-OH. In this synthesis, 3.93 g D-Leu-OH were used instead of L-Leu-OH.
1
As a result, 6.74 g white product were obtained with a yield of 97.2%. H-NMR (500 MHz, CDCl ),
3

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δ
4.87 (m, 1H), 4.31 (m, 1H), 1.75 (m, 2H), 1.54 (m, 1H), 1.45 (s, 9H), 0.95 (d, 6H, J = 6.4 Hz); MS (ESI)
H
+
+
+
m/z: 232.2 [M + H] , 249.2 [M + NH ] , 254.3 [M + Na] .
4
4
.1.3. Synthesis of N-Boc-Phe-OH
The experimental procedure for the synthesis of N-Boc-Phe-OH followed as per described in the
synthesis of N-Boc-Leu-OH, and 4.95 g L-Phe-OH were used instead of 3.93 g L-Leu-OH. As a result,
7
.72 g white product were obtained with a yield of 97%.
4
.1.4. Synthesis of N-Boc-D-Phe-OH
The experimental procedure for the synthesis of N-Boc-D-Phe-OH followed as per described in
the synthesis of N-Boc-Leu-OH, and 4.95 g L-Phe-OH (30 mmol) were used instead of 3.93 g L-Leu-OH.
As a result, 7.73 g white product were obtained with a yield of 97.2%.
4
.1.5. Synthesis of N-Boc-Me-Leu-OH
In a 250-mL round-bottom flask under N atmosphere, N-Boc-Leu-OH (4.62 g) and THF (60 mL)
2
were added with stirring within the ice bath. After the solution of N-Boc-Leu-OH in THF become
clear, CH I (6.3 mL, 100 mmol) was added slowly and allowed to react for 30 min. Then, a 60% NaH
3
dispersion in mineral oil (1.6 g, 40 mmol) was added slowly. The temperature of the system was
increased slowly, and the solution was stirred overnight. After 24 h, an additional amount of 60% NaH
in mineral oil (0.8 g, 20 mmol) was added after being cooled in the ice bath; again, the temperature
increased slowly to room temperature, and it was stirred for overnight. After completion of the reaction,
it was cooled in an ice bath, and distilled water (15 mL) was added slowly. Further, ethyl acetate
(100 mL) was added to the flask under cooling in the ice bath. Then, the organic solvent was removed
by vacuum distillation, and the residual was extracted twice using ethyl ether (60 mL). The combined
ether phase was washed with saturated NaCl solution (50 mL) and combined with the aqueous phase.
Under cooling in the ice bath, the solution was acidified by adding an appropriate amount of 1 M HCl
to increase the pH to 2 to 3, and it was extracted three times using ethyl acetate (100 mL). Combining
the organic phase together, it was washed by 150 mL 5% Na S O solution three times and saturated
2
2
3
NaCl solution (100 mL). Subsequently, the solution was dried by anhydrous sodium sulfate, filtered,
concentrated, dried under vacuum and purified using silica-gel column chromatography (eluant ratio
of Vpetroleum ether/Vacetone = 40:1); finally, 4.25 g white needle-like powder were obtained with a yield of
◦
26
◦
+ 30.7 (c = 0.5, EtOH), H-NMR (400 MHz, CDCl ), δ 4.82 (m, 1H),
3 H
1
8
4
2
6.3%. m.p.: 62.6~64.3 C, [α]D
.62 (m, 1H), 3.34 (s, 3H), 1.71 (m, 2H), 1.58 (m, 1H), 1.46 (s, 9H), 0.95 (d, 6H, J = 6.4 Hz); MS (ESI) m/z:
+
+
+
46.2 [M + H] , 263.2 [M + NH ] , 268.3 [M + Na] .
4
4
.1.6. Synthesis of N-Boc-Me-D-Leu-OH
The experimental procedure for the synthesis of N-Boc-Me-D-Leu-OH followed as per described in
the synthesis of N-Boc-Me-Leu-OH, and N-Boc-D-Leu-OH (4.62 g) was used instead of N-Boc-Leu-OH.
1
As a result, 4.62 g white powder product were obtained with a yield of 87.2%. H-NMR (500 MHz,
CDCl3),
δ 6.04 (m, 1H), 4.45 (m, 1H), 1.78 (m, 2H), 1.50 (m, 1H), 1.40 (s, 9H), 0.91 (d, 6H, J = 6.4 Hz);
H
+
+
+
MS (ESI) m/z: 232.2 [M + H] , 249.2 [M + NH ] , 254.3 [M + Na] .
4
4
.1.7. Synthesis of N-Boc-Me-Phe-OH
The synthesis followed the experimental ways for N-Boc-Me-Leu-OH as above. However, in this
synthesis, 5.3 g N-Boc-Phe-OH were used instead of N-Boc-Leu-OH. As a result, 4.7 g colorless oily
1
product were obtained with a yield of 85.0%. H-NMR (500 MHz, CDCl ),
δ 7.35 (m, 5H), 7.02 (s, 1H),
H
3
+
+
4
.90 (m, 1H), 3.57 (s, 3H), 3.12–3.43 (m, 1H), 1.42 (s, 9H); MS (ESI) m/z: 280.2 [M + H] , 297.2 [M + NH ] ,
4
+
3
02.3 [M + Na] .

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4
.1.8. Synthesis of N-Boc-Me-D-Phe-OH
The experimental procedure for the synthesis of N-Boc-Me-Phe-OH followed as per described
in the synthesis of N-Boc-Me-Leu-OH, and 5.3 g N-Boc-D-Phe-OH were used instead of 5.3 g
N-Boc-Leu-OH.
4
.1.9. Synthesis of Dipeptide N-Boc-Me-Leu-Leu-OBn(D1)
In a 250-mL round-bottom flask under N atmosphere, 2.46 g N-Boc-Me-Leu-OH and 10 mL THF
2
were added under stirring and cooled in ice bath condition. Then, DEPBT (4.5 g, 15 mmol) and DIEA
(
(
2.6 mL, 15 mmol) was added, and allowed to react for 10 min. After that 4.72 g L-Leu-OBn
12 mmol) were added and stirred for complete dissolution. Then, the pH of reaction solution was
·
TosOH
adjusted to 7 to 8 by adding DIEA slowly. Subsequently, the temperature was increased slowly to room
temperature, and the reaction was monitored using thin layer chromatography (TLC). After overnight
stirring, the solvent was removed by vacuum distillation, and the residual crude product was purified
directly using silica-gel column chromatography (eluant ratio of Vpetroleum ether/Vacetone = 40:1). Finally,
1
3
7
2
.76 g product were obtained with a yield of 83.8%. H-NMR (500 MHz, CDCl ),
δ
7.35 (m, 5H),
3
H
+
.02 (s, 1H), 4.90 (m, 1H), 3.57 (s, 3H), 3.12–3.43 (m, 1H), 1.42(s, 9H); MS (ESI) m/z: 280.2 [M + H] ,
+
+
97.2 [M + NH ] , 302.3 [M + Na] .
4
4
.1.10. Synthesis of Dipeptide N-Boc-Me-D-Leu-Leu-OBn(D2)
The experimental procedure for the synthesis of N-Boc-Me-D-Leu-Leu-OBn(D2) followed as per
described in the synthesis of N-Boc-Me-Leu-Leu-OBn(D1). In this synthesis, N-Boc-Me-D-Leu-OH
was used instead of N-Boc-Me-Leu-OH. As a result, 3.82 g product were obtained with a yield of
1
8
5.2%. H-NMR (300 MHz, CDCl ),
δ
7.45–7.31 (m, 5H), 6.39 (d, J = 78.1 Hz, 1H), 5.24–5.11 (m, 2H),
H
3
4
(
.65 (s, 2H), 2.76 (s, 3H), 1.69 (dd, J = 12.4, 6.4 Hz, 2H), 1.65–1.57 (m, 2H), 1.49 (s, 9H), 0.97–0.87
m, 12H).; 13C-NMR (75 MHz, CDCl3),
δ
172.5, 171.1, 167.4, 135.4, 128.6 (2C), 128.4, 128.2 (2C), 99.98,
C
+
67.0, 56.1, 50.7 (2C), 36.3, 29.8, 28.4 (3C), 24.8 (2C), 22.9 (2C), 22.1, 21.7; MS (ESI) m/z: 449.3 [M + H] ,
+
+
4
66.5 [M + NH ] , 471.6 [M + Na] .
4
4
.1.11. Synthesis of Dipeptide N-Boc-Me-Leu-D-Leu-OBn(D3)
The experimental procedure for the synthesis of N-Boc-Me-Leu-D-Leu-OBn(D3) followed as per
described in the synthesis of N-Boc-Me-Leu-Leu-OBn(D1). In this synthesis, D-Leu-OBn
·
TosOH was
TosOH. As a result, 3.92 g oily product were obtained with a yield of
7.42–7.29 (m, 5H), 6.64–6.31 (m, 1H), 5.14 (q, J = 12.3 Hz, 2H),
used instead of L-Leu-OBn
·
1
87.4%. H-NMR (300 MHz, CDCl ),
δ
3
H
4
.84–4.47 (m, 2H), 2.69 (s, 3H), 1.82–1.58 (m, 4H), 1.58–1.50 (m, 2H), 1.48 (s, 9H), 0.94 (t, J = 7.1 Hz,
1
5
4
2H); 13C-NMR (75 MHz, CDCl3),
δ
172.6, 171.4, 157.1, 135.5, 128.6 (2C), 128.4, 128.2 (2C), 80.5, 66.9,
C
+
5.9, 50.8, 41.1, 36.0, 29.8, 28.3 (3C), 24.9, 24.7, 23.2, 22.8 (2C), 21.8; MS (ESI) m/z: 449.3 [M + H] ,
66.5 [M + NH ] , 471.6 [M + Na] .
+
+
4
4
.1.12. Synthesis of Dipeptide N-Boc-Me-Phe-Leu-OBn(D4)
The experimental procedure for the synthesis of N-Boc-Me-Phe-Leu-OBn(D4) followed as per
described in the synthesis of N-Boc-Me-Leu-Leu-OBn(D1). In this synthesis, N-Boc-Me-Phe-OH
was used instead of N-Boc-Me-Leu-OH. As a result, oily 3.98 g product were obtained with a yield
1
of 82.6%. H-NMR (300 MHz, CDCl ),
δ
7.45–7.30 (m, 5H), 7.25–7.17 (m, 5H), 5.37 (dd, J = 10.1,
H
3
5
.3 Hz, 2H), 5.15 (q, J = 12.3 Hz, 2H), 4.85 (dd, J = 15.6, 6.7 Hz, 1H), 3.13–2.98 (m, 1H), 2.86 (s, 3H),
.84–2.77 (m, 1H), 1.87–1.60 (m, 2H), 1.47–1.39 (m, 9H), 1.38–1.24 (m, 2H), 0.92 (dd, J = 8.4, 6.7 Hz, 6H);
2
13
C-NMR (75 MHz, CDCl3),
δ 172.7, 171.3, 155.3, 136.3, 135.6, 129.5 (2C), 129.4, 128.6 (2C), 128.4 (2C),
C
1
28.3 (2C), 126.8, 79.6, 77.2, 66.9, 54.8, 51.7, 38.7, 37.1, 28.0 (3C), 24.7, 23.3, 21.5; MS (ESI) m/z: 483.7
+
+
+
[M + H] , 500.6 [M + NH ] , 505.6 [M + Na] .
4

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4
.1.13. Synthesis of Dipeptide N-Boc-D-Phe-Me-Leu-OBn(D5)
The experimental procedure for the synthesis of N-Boc-D-Phe-Me-Leu-OBn(D5) followed as per
described in the synthesis of N-Boc-Me-Leu-Leu-OBn(D1). In this synthesis, N-Boc-Me-D-Phe-OH
was used instead of N-Boc-Me-Phe-OH. As a result, oily 4.05 g product were obtained with a yield of
1
8
4.1%. H-NMR (300 MHz, CDCl ),
δ
7.46–7.33 (m, 5H), 7.32–7.16 (m, 5H), 6.48 (dd, J = 95.7, 7.5 Hz,
H
3
1H), 5.17 (q, J = 12.3 Hz, 2H), 4.84 (ddd, J = 54.7, 17.6, 8.4 Hz, 2H), 3.54–3.27 (m, 1H), 3.01–2.84 (m, 1H),
2
.76 (s, 3H), 1.74–1.60 (m, 1H), 1.54 (dd, J = 14.7, 7.7 Hz, 2H), 1.34 (s, 9H), 0.90 (d, J = 5.4 Hz, 6H);
13
C-NMR (75 MHz, CDCl3),
δ
172.5, 170.6, 156.7, 137.5, 135.4, 129.1, 129.0, 128.6 (2C), 128.4 (2C), 128.2,
C
+
126.4, 80.5, 67.0, 59.4, 50.8, 41.2, 33.9, 28.2 (3C), 25.0, 24.8, 22.8, 21.8; MS (ESI) m/z: 483.7 [M + H] ,
+
+
5
00.6 [M + NH ] , 505.6 [M + Na] .
4
4
.1.14. Synthesis of Tripeptide N-Boc-Leu-Me-Leu-Leu-OBn(T1)
D (3.14g, 7 mmol) was first dissolved in 32 mL dichloromethane, and then, 1.47 mL anisole
1
were added under N protection. The solution was cooled in the ice bath and stirred continuously;
2
TFA (8 mL) was added slowly to the solution and allowed to react for 4 h. The solvent was removed by
vacuum distillation, then additional appropriate dichloromethane was added and vacuum distillated
again. This step was repeated three times for complete removal of TFA; finally, an oily product named
as D -A was obtained after drying, and it was used for further synthesis.
2
BOC-Leu-OH (1.85 g, 8 mmol) was dissolved in THF (5 mL) under stirring and cooled in an ice
bath in N atmosphere. Subsequently, DEPBT (3.00 g, 10 mmol) and DIEA (1.8 mL, 10 mmol) were
2
added into the solution and reacted for 10 min. Thereafter, an appropriate amount of the previously
synthesized D -A THF solution (10 mL) was added, and DIEA was used to adjust the pH of the solution
2
to 7 to 8. The temperature was slowly increased to room temperature, and the reaction was monitored
by TLC. After completion of the reaction, the solvent was removed by vacuum distillation, and the
residual crude product was purified directly using silica-gel column chromatography (eluant ratio of
V
/Vacetone = 40:1). Finally, 2.95 g colorless oily product were obtained with an overall yield of
petroleum ether
1
8
0.8%. H-NMR (500 MHz, CDCl ),
δ
7.32 (m, 5H), 6.32 (d, 1H, J = 4.0 Hz), 5.09 (m, 4H), 4.56 (m, 2H),
H
3
13
2
.94 (s, 3H), 1.63 (m, 3H), 1.46 (m, 3H), 1.41 (s, 9H), 1.23 (m, 3H), 0.90 (m, 18H); C-NMR (125 MHz,
CDCl3),
δ
174.1, 172.3, 170.2, 155.7, 135.3, 128.5, 128.4, 128.3, 128.2, 127.9, 79.6, 67.0, 54.3, 50.6, 49.0, 41.9,
C
+
4
5
0.9, 35.9, 30.1, 28.3 (3C), 24.7, 24.6, 23.4 (2C), 22.9, 22.8, 22.0 21.6,21.5; MS (ESI) m/z: 562.8 [M + H] ,
79.8 [M + NH ] , 584.9 [M + Na] .
+
+
4
4
.1.15. Synthesis of Tripeptide N-Boc-Leu-Me-Leu-D-Leu-OBn(T2)
The experimental procedure for the synthesis of N-Boc-Leu-Me-Leu-D-Leu-OBn(T2) followed
as per described in the synthesis of N-Boc-Leu-Me-Leu-Leu-Obn(T1). In this synthesis, D3 was used
1
instead of D1. H-NMR (300 MHz, CDCl ), δ 7.40–7.32 (m, 5H), 5.20–5.19 (m, 1H), 5.10 (dd, J = 12.6,
3
H
5
0
.5 Hz, 1H), 4.60–4.56 (m, 1H), 2.90 (s, 3H), 1.70–1.63 (m, 6H), 1.42 (s, 9H), 1.35 (d, J = 10.8 Hz, 3H),
.97–0.90 (m, 18H); 13C-NMR (75 MHz, CDCl3),
δ
174.9, 172.4, 170.6, 155.7, 135.5, 128.5 (2C), 128.5,
C
1
2
28.3 (2C), 128.2, 79.6, 66.9, 54.2, 50.7, 49.2, 41.6, 40.9, 35.6, 30.2, 28.3 (3C), 24.8, 24.6, 23.4, 23.1, 22.8, 22.0,
+
+
1.7, 21.6; MS (ESI) m/z: 562.8 [M + H]+, 579.8 [M + NH ] , 584.9 [M + Na] .
4
4
.1.16. Synthesis of Tripeptide N-Boc-Phe-Me-Leu-D-Leu-OBn(T3)
The experimental procedure for the synthesis of N-Boc-Phe-Me-Leu-D-Leu-OBn(T3) followed as
per described in the synthesis of N-Boc-Leu-Me-Leu-Leu-Obn(T1). In this synthesis, Boc-Phe-OH was
1
used instead of Boc-Leu-OH. H-NMR (300 MHz, CDCl ),
δ 7.37–7.33 (m, 5H), 7.32–7.30 (m, 1H),
H
3
7
4
1
.28–7.19 (m, 5H), 6.20 (d, J = 8.3 Hz, 1H), 5.15–5.03 (m, 2H), 4.81 (dt, J = 9.1, 6.6 Hz, 1H),
.65–4.52 (m, 2H), 3.09 (dd, J = 13.8, 6.6 Hz, 1H), 2.96–2.85 (m, 01H), 2.81 (s, 3H), 1.95–1.71 (m, 2H),
.58–1.46 (m, 2H), 1.39 (s, 9H), 1.30 (m, 2H), 0.95 (t, J = 5.4 Hz, 9H), 0.80 (dd, J = 6.6, 1.4 Hz, 3H); MS
+
+
+
(ESI) m/z: 595.7 [M + H] , 612.7 [M + NH ] , 617.9 [M + Na] .
4

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4
.1.17. Synthesis of Tripeptide N-Boc-Phe-Me-Leu-Leu-OBn(T4)
The experimental procedure for the synthesis of N-Boc-Phe-Me-Leu-Leu-OBn(T4) followed as
per described in the synthesis of N-Boc-Leu-Me-Leu-Leu-OBn(T1). In this synthesis, Boc-Phe-OH was
1
used instead of Boc-Leu-OH. H-NMR (300 MHz, CDCl ),
δ 7.93 (d, J = 7.9 Hz, 1H), 7.42–7.32 (m, 5H),
H
3
7
4
.32–7.18 (m, 5H), 6.18 (d, J = 8.0 Hz, 1H), 5.31–5.20 (m, 1H), 5.20–5.13 (m, 2H), 4.85–4.71 (m, 1H),
.71–4.55 (m, 1H), 3.18–2.99 (m, 1H), 2.90–2.81 (m, 1H), 2.79 (s, 3H), 1.88 (dd, J = 9.5, 3.6 Hz, 1H),
.76–1.66 (m, 1H), 1.66–1.57 (m, 2H), 1.57–1.44 (m, 2H), 1.40 (d, J = 1.6 Hz, 9H), 0.98–0.76 (m, 12H);
1
13
C-NMR (75 MHz, CDCl3),
δ 173.2, 172.3, 170.2, 155.2, 135.4, 129.3, 128.9, 128.6 (2C), 128.5, 128.4,
C
1
2
28.3 (2C), 128.2, 128.0, 127.0, 79.9, 67.1, 55.0, 50.8, 41.1, 39.0, 36.4, 30.6, 28.3 (3C), 24.9, 24.7 (2C) 23.1,
+
+
+
2.8, 22.0, 21.9; MS (ESI) m/z: 595.7 [M + H] , 612.7 [M + NH ] , 617.9 [M + Na] .
4
4
.1.18. Synthesis of Tripeptide N-Boc-Phe-Me-D-Leu-Leu-OBn(T5)
The experimental procedure for the synthesis of N-Boc-Phe-Me-D-Leu-Leu-OBn(T5) followed
as per described in the synthesis of N-Boc-Leu-Me-Leu-Leu-OBn(T1). In this synthesis, D2 was used
1
instead of D1. H-NMR (300 MHz, CDCl ),
δ 7.41–7.33 (m, 5H), 7.33–7.20 (m, 5H), 6.89 (d, J = 7.4 Hz,
H
3
1H), 5.25 (d, J = 6.8 Hz, 1H), 5.21–5.13 (m, 2H), 5.08 (d, J = 12.2 Hz, 1H), 4.71 (dd, J = 14.8, 6.8 Hz, 1H),
4.66–4.57 (m, 1H), 3.04–2.94 (m, 1H), 2.60 (s, 3H), 1.79–1.67 (m, 2H), 1.55 (dd, J = 12.9, 5.8 Hz, 2H),
1
.43 (s, 9H), 1.39–1.24 (m, 2H), 0.91 (dd, J = 6.0, 2.9 Hz, 6H), 0.86 (d, J = 6.5 Hz, 6H); MS (ESI) m/z: 95.7
+
+
+
[M + H] , 612.7 [M + NH ] , 617.9 [M + Na] .
4
4
.1.19. Synthesis of Linear Pentapeptide N-Boc-Me-Leu-Leu-Phe-Me-D-Leu-Leu-OBn(P1)
In a 50-mL round-bottom flask with N protection, D1 (1.68 g) was dissolved in ethyl acetate
2
(20 mL), and then 10% catalyst Pd/C catalyst (0.56 g) was added. The flask was sealed and flushed
with H , the solution was stirred at room temperature and monitored for the progress of the reaction
2
by TLC. After 4–6 h of reaction when the TLC point of the raw materials disappeared, H flow was
2
closed, and the reaction was stopped. The solution was first filtered to recycle the catalyst Pd/C, and
the rest of the solution was distillated in a vacuum to remove the solvent residues and was dried in the
oven. Finally, the colorless product of D1-B was obtained and used for the following step.
The previously-obtained product T5 (1.61 g) was dissolved in CH Cl (20 mL) under N
2
2
2
atmosphere, then anisole (0.76 mL, 7.2 mmol) was added under stirring and cooled in an ice bath.
TFA (8 mL) was added to the solution slowly and stirred for reaction 4 h. Thereafter, the solvent
was removed by vacuum distillation, then additional appropriate dichloromethane was added and
distillated under vacuum again to remove traces of TFA. This step was repeated three times for
complete removal of TFA. Finally, the oily product of T5-A was obtained for the next step.
The above obtained T5-A was dissolved in 5 mL THF under stirring and cooled in an ice bath.
Subsequently, DEPBT (2.15 g, 7.2 mmol) and DIEA (1.26 mL, 7.2 mmol) were added into the solution
and reacted for 10 min Thereafter, an appropriate amount of the above obtained D1-B THF solution
(5 mL) was added, and DIEA was used to adjust the pH of the solution to 7–8. The temperature was
increased slowly to room temperature and monitored for the progress of reaction by TLC. The solution
was stirred 24 h; the solvent was removed by vacuum distillation; and the residual crude product was
purified directly using silica-gel column chromatography (eluant ratio of Vpetroleum ether/Vacetone = 20:1).
1
H-NMR (300 MHz, CDCl3),
δ 7.31 (m, 7H), 7.23 (m, 3H), 6.76 (d, J = 8.4 Hz, 1H), 6.36 (d, J = 8.4 Hz,
H
1H), 5.30 (d, J = 8.4 Hz, 1H), 5.13 (s, 2H), 5.01 (m, 1H), 4.83 (m, 2H), 4.70 (m, 1H), 4.56 (m, 1H),
2
.96 (s, 3H), 2.73 (m, 2H), 2.67 (s, 3H), 1.64 (m, 2H), 1.59 (m, 6H), 1.49 (m, 4H), 1.41 (s, 9H), 0.91 (m, 24H);
1
3
C-NMR (75 MHz, CDCl3),
δ 172.62, 172.5, 171.8, 169.8, 169.5, 137.7, 128.9, 128.8, 128.2 (2C), 128.1,
C
1
3
28.1, 128.0, 128.0, 127.8 (2C), 127.5, 126.6, 76.8, 66.6, 66.2, 54.2, 54.1, 50.2, 47.2, 40.8, 40.7, 39.5, 39.3, 35.5,
0.0, 29.9, 27.9 (3C), 24.4, 24.3, 24.3, 24.2, 22.8, 22.6, 22.5, 22.4, 21.7, 21.6, 21.2, 21.1; MS (ESI) m/z: 836.9
+
+
+
[M + H] , 853.9 [M + NH ] , 858.9 [M + Na] .
4

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4
.1.20. Synthesis of Linear Pentapeptide N-Boc-Me-Leu-Leu-Phe-Me-Leu-D-Leu-OBn(P2)
The experimental procedure was followed as per described for the synthesis of P1. In this
synthesis, T3 and T3-A were used instead of T5 and T5-A in the corresponding places, respectively.
1
H-NMR (300 MHz, CDCl3),
δ 7.36–7.27 (m, 5H), 7.27–7.12 (m, 5H), 6.94 (d, J = 8.3 Hz, 1H), 6.55
H
(
4
1
8
d, J = 27.8 Hz, 2H), 5.14 (s, 1H), 5.15–5.09 (m, 2H), 5.04 (dd, J = 19.1, 6.7 Hz, 1H), 4.72–4.54 (m, 2H),
.45 (dd, J = 18.2, 10.1 Hz, 1H), 3.30–3.05 (m, 1H), 2.98–2.87 (m, 1H), 2.85 (s, 3H), 2.71 (s, 3H),
.92–1.52 (m, 8H), 1.48 (d, J = 4.5 Hz, 9H), 1.39–1.23 (m, 4H), 0.99–0.74 (m, 24H); MS (ESI) m/z:
+
+
+
36.7 [M + H] , 853.8 [M + NH ] , 858.7 [M + Na] .
4
4
.1.21. Synthesis of Linear Pentapeptide N-Boc-Me-D-Leu-Leu-Phe-Me-Leu-Leu-OBn(P3)
The experimental procedure was followed as per described for the synthesis of P1. In this
synthesis, D2 and D2-B were used instead of D1 and D1-B, and T4 and T4-A were used instead of T5
1
and T5-A in the corresponding places, respectively. H-NMR (300 MHz, CDCl ),
δ 7.46–7.33 (m, 5H),
H
3
7
5
2
.20 (dt, J = 12.2, 7.3 Hz, 5H), 7.05 (d, J = 8.1 Hz, 1H), 6.23 (d, J = 4.7 Hz, 1H), 5.70 (dd, J = 11.8, 5.2 Hz, 1H),
.27–5.16 (m, 2H), 5.16–5.09 (m, 1H), 4.77–4.56 (m, 2H), 4.31 (dd, J = 13.0, 5.3 Hz, 1H), 3.63–3.46 (m, 1H),
.92 (s, 3H), 2.84 (s, 3H), 2.82–2.71 (m, 1H), 2.10–1.61 (m, 8H), 1.44 (s, 9H), 1.26 (dd, J = 15.0, 5.5 Hz,
13
1
H), 1.02 (d, J = 6.4 Hz, 3H), 1.00–0.92 (m, 12H), 0.91 (d, J = 1.9 Hz, 6H), 0.74–0.58 (m, 6H); C-NMR
(
75 MHz, CDCl3), 174.9, 173.3 172.7, 171.6, 170.3, 155.7, 137.3, 135.6, 128.8, 128.6 (2C), 128.5 (2C),
δ
C
1
2
8
28.4 (2C), 128.3 (2C), 126.5, 79.7, 66.9, 56.9, 54.8, 51.0, 49.2, 48.3, 41.6, 40.4, 40.3, 35.3, 33.6, 30.9, 30.4,
+
8.3 (3C), 24.8 (2C), 24.6, 24.5, 24.1, 23.4, 23.3, 22.9 (2C), 22.1, 21.7, 21.4; MS (ESI) m/z: 836.7 [M + H] ,
+
+
53.8 [M + NH ] , 858.7 [M + Na] .
4
4
.1.22. Synthesis of Linear Pentapeptide N-Boc-Me-Leu-D-Leu-Phe-Me-Leu-Leu-OBn(P4)
The experimental procedure was followed as per described for the synthesis of P1. In this
synthesis, D3 and D3-A were used instead of D1and D1-A, and T4 and T4-B were used instead of T5
1
and T5-B in the corresponding places, respectively. H-NMR (300 MHz, CDCl ),
δ 7.36 (dt, J = 11.0,
H
3
3
1
3
.6 Hz, 5H), 7.30–7.20 (m, 5H), 6.48 (d, J = 8.6 Hz, 1H), 6.38 (d, J = 8.1 Hz, 1H), 5.30 (d, J = 9.1 Hz,
H), 5.24–5.15 (m, 2H), 5.14–5.03 (m, 2H), 5.00–4.89 (m, 1H), 4.84 (d, J = 9.0 Hz, 1H), 4.63–4.49 (m, 1H),
.11–2.95 (m, 1H), 2.93 (s, 3H), 2.86 (s, 3H), 2.82–2.67 (m, 1H), 1.81–1.50 (m, 8H), 1.46 (d, J = 7.8 Hz, 4H),
13
1.40 (s, 9H), 1.08–0.73 (m, 24H); C-NMR (75 MHz, CDCl3), δ 173.5, 173.1, 172.4, 170.5, 170.2, 155.2,
C
1
4
2
35.5, 134.0, 129.3, 128.7 (2C), 128.6 (3C), 128.4, 128.2 (2C), 127.0, 79.8, 66.9, 55.0, 54.3, 51.9, 50.7, 47.7,
1.4, 41.0, 39.0, 35.5, 30.6, 30.2, 28.4, 28.3 (3C), 24.9, 24.8, 24.6, 23.4, 23.1, 23.1, 22.9, 22.8, 22.0, 22.0, 21.7,
+
+
+
1.6; MS (ESI) m/z: 836.7 [M + H] , 853.8 [M + NH ] , 858.7 [M + Na] .
4
4
.1.23. Synthesis of Linear Pentapeptide N-Boc-Leu-Me-Leu-Leu-Me-Phe-Leu-OBn(P5)
The experimental procedure was followed as per described for the synthesis of P1. In
this synthesis, T4 and T4-A were used instead of T5 and T5-A in the corresponding places,
1
respectively. H-NMR (300 MHz, CDCl ),
δ 8.55–8.15 (m, 1H), 7.43–7.33 (m, 5H), 7.32–7.17 (m, 5H),
H
3
7
4
.15–7.09 (m, 1H), 5.19 (dd, J = 15.3, 2.9 Hz, 1H), 5.15–5.11 (m, 1H), 4.90–4.70 (m, 1H), 4.69–4.53 (m, 2H),
.42–4.19 (m, 1H), 3.35–3.20 (m, 1H), 3.07 (dd, J = 18.9, 8.5 Hz, 1H), 2.95 (s, 3H), 2.77 (s, 3H),
13
1.81–1.65 (m, 8H), 1.44 (s, 9H), 1.32–1.22 (m, 2H), 0.99–0.48 (m, 24H); C-NMR (75 MHz, CDCl3),
δ
174.1, 172.9, 172.8, 171.4, 169.4, 155.8, 137.9, 135.8, 129.6, 129.2 (2C), 128.9 (2C), 128.6 (2C), 128.2,
C
1
28.0, 127.0, 77.2, 67.0, 66.8, 62.7, 53.5, 51.5, 49.0, 47.0, 42.0, 39.9, 35.4, 34.0, 29.9, 29.5, 28.3 (3C), 24.8, 24.6
+
+
(2C), 24.3, 23.9, 23.5, 23.4, 23.1, 22.9, 21.7, 21.2, 19.9; MS (ESI) m/z: 836.7 [M + H] , 853.8 [M + NH ] ,
4
+
8
58.7 [M + Na] .
4
.1.24. Synthesis of Linear Pentapeptide N-Boc-Leu-Me-Leu-Leu-Me-D-Phe-Leu-OBn(P6)
The experimental procedure was followed as per described for the synthesis of P1. In this
synthesis, D1 and D1-A were used instead of T1 and T1-A, and T5 and T5-B were used instead of

Int. J. Mol. Sci. 2017, 18, 544
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1
D5 and D5-B in the corresponding places, respectively. H-NMR (500 MHz, CDCl ),
δ
7.31 (m, 7H),
H
3
7
5
2
.26–7.23 (m, 3H), 6.76 (d, 1H, J = 10.0 Hz), 6.36 (d, 1H, J = 10.0 Hz), 5.30 (d, 1H, J = 10.0 Hz), 5.13 (s, 2H),
.05–5.01 (m, 1H), 4.90–4.83 (m, 2H), 4.73–4.70 (m, 1H), 4.59–4.56 (m, 1H), 2.96 (s, 3H), 2.77–2.73 (m, 2H),
.67 (s, 3H), 1.67–1.64(m, 2H), 1.62–1.67 (m, 6H), 1.50–1.46 (m, 4H), 1.41 (s, 9H), 0.98–088 (m, 24H), MS
+
+
+
(ESI) m/z: 837.0 [M + H] , 854.0 [M + NH ] , 859.0 [M + Na] .
4
4
.1.25. Synthesis of Cyclo(Me-Leu-Leu-Phe-Me-Leu-Leu) (Analog-1)
P5 (0.12 g, 0.15 mmol) was first dissolved in dichloromethane (20 mL) in a 50-mL round-bottom
flask, and then, TFA (5 mL) was added under stirring and cooled in an ice bath. After the reaction
was stirred for 4 h, the solvent was removed by vacuum distillation, then additional appropriate
dichloromethane was added and distillated under vacuum again. This step was repeated three times
for complete removal of TFA; finally, an oily product was obtained after drying. Then, the obtained oily
product was dissolved in ethyl acetate (20 mL) under N atmosphere, and 10% Pd/C (0.2 g) catalyst
2
was added. The flask was sealed and flushed with H ; the solution was stirred under room temperature
2
and monitored for the progress of reaction by TLC. After 4–6 h of reaction when the TLC point of
the raw materials disappeared, H flow was closed, and the reaction was stopped. The solution was
2
first filtered to recycle the catalyst Pd/C, and the rest of the solution was distillated under vacuum to
remove the solvent residues and was dried in the oven. Finally, a colorless product was obtained. The
as-obtained product was then dissolved in 50 mL THF/CH2Cl2/CH3CN (2:1:2) mixed solvent under
N atmosphere; 0.067 g HATU, 0.065 g DEPBT and 0.07 g TBTU were added in sequence and allowed to
2
react for 4 days. The solvent was removed by vacuum distillation, and the solid product was dissolved
in CH Cl and washed by saturated NaHCO and H O separately. The resultant organic layer was
2
2
3
2
dried by anhydrous Na SO and concentrated, then purified by RP-HPLC (Agilent Eclipse ZORBAX
2
4
SB-C18, 5
H-NMR (300 MHz, CDCl ),
µ
m, 9.4
×
250 nm). Finally, 11.2 mg white powder with a yield of 40.2% were obtained.
8.15 (d, J = 9.7 Hz, 1H), 8.15 (d, J = 9.7 Hz, 1H), 7.53 (d, J = 8.7 Hz, 1H),
1
δ
H
3
7.53 (d, J = 8.7 Hz, 1H), 7.36–7.25 (m, 3H), 7.18–7.11 (m, 2H), 5.80 (d, J = 7.3 Hz, 1H), 4.86–4.72 (m, 2H),
4.27 (ddd, J = 11.1, 7.3, 3.6 Hz, 1H), 3.67 (dd, J = 10.3, 5.8 Hz, 1H), 3.67 (dd, J = 10.3, 5.8 Hz, 1H),
3.45 (d, J = 7.8 Hz, 1H), 3.39 (dd, J = 8.7, 6.4 Hz, 1H), 3.12 (dd, J = 12.9, 5.8 Hz, 1H), 2.95 (s, 3H),
2.81 (s, 3H), 2.32 (ddd, J = 14.2, 8.7, 5.7 Hz, 1H), 2.02–1.90 (m, 1H), 1.90–1.73 (m, 4H), 1.60–1.55 (m, 1H),
1
(
.50–1.32 (m, 4H), 1.26–1.18 (m, 1H), 0.95–0.91 (m, 9H), 0.89 (d, J = 4.7 Hz, 9H), 0.86 (s, 3H), 0.83
d, J = 6.5 Hz, 3H); 13C-NMR (75 MHz, CDCl3),
δ
C
174.5, 173.0, 172.6, 171.2, 169.7, 136.5 (2C), 129.2 (2C),
1
2
29.1, 127.6, 71.1, 68.0, 53.3, 49.3, 48.1, 41.5, 41.1, 40.7, 40.0, 39.6, 38.8, 34.0, 26.1, 25.2, 25.1, 25.0, 23.6,
+
+
+
3.4, 23.2, 23.1, 22.6, 22.1, 21.8, 21.3; MS (ESI) m/z: 628.8 [M + H] , 645.8 [M + NH ] , 650.8 [M + Na] .
4
1
For the detailed characterization of mass spectra, mass-mass spectra, H-NMR and HPLC
regarding Analog-1, refer to Figures S2A, S3A, S4A and S5A.
4
.1.26. Synthesis of Cyclo(Me-Leu-Leu-D-Phe-Me-Leu-Leu) (Analog-2)
The experimental procedure was followed as described in the synthesis of Analog-1. In this
synthesis, P6 was used instead of P5. Finally, 31.8 mg white powder with a yield of 42.3% were
obtained. 1H-NMR (500 MHz, CDCl3),
8.13 (s, 1H), 7.74–7.71 (m, 1H), 7.29–7.19 (m, 5H), 6.91
δ
H
(d, J = 10.0 Hz, 1H), 5.18 (t, J = 10.0Hz, 1H), 4.91–4.87 (m, 1H), 4.79–4.74 (m, 3H), 3.09–3.05 (m, 2H),
2
.94–2.91 (m, 1H), 2.84 (s, 1H), 2.80 (s, 2H), 2.62 (s, 3H), 1.93–1.88 (m, 1H), 1.82–1.77 (m, 1H),
13
1.61–1.50 (m, 6H), 1.43–1.37 (m, 1H), 1.12–1.06 (m, 1H), 0.97–0.89 (m, 18H), 0.73–0.66 (m, 6H); C-NMR
125 MHz, CDCl3), 175.9, 171.7, 171.5, 170.1, 170.0, 136.2, 129.1 (2C), 128.8 (2C), 127.2, 57.8, 53.7 (2C),
(
δ
C
4
2
8.7, 47.8, 41.5, 41.1, 37.7, 37.5, 34.5, 31.4, 29.5, 24.9 (2C), 24.8, 24.5, 23.2, 23.1, 22.9 (2C), 22.2, 21.9, 21.8,
+
+
+
1.6; MS (ESI) m/z: 628.8 [M + H] , 645.9 [M + NH ] , 650.8 [M + Na] .
4
1
For the detailed characterization of mass spectra, mass-mass spectra, H-NMR and HPLC of
Analog-2, refer to Figures S2B, S3B, S4B and S5B.

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4
.1.27. Synthesis of Cyclo(Me-Leu-Leu-Phe-Me-D-Leu-Leu) (Analog-3)
The experimental procedure was followed as described in the synthesis of Analog-1. In this
synthesis, P1 was used instead of P5. Finally, 17.0 mg white powder with a yield of 38.5% were
1
obtained. H-NMR (300 MHz, CDCl ),
δ 7.97 (d, J = 9.1 Hz, 1H), 7.73 (dd, J = 5.7, 3.3 Hz, 1H), 7.55
H
3
(dd, J = 5.7, 3.3 Hz, 1H), 7.25 (dd, J = 7.6, 3.4 Hz, 4H), 6.20 (d, J = 9.4 Hz, 1H), 4.24 (dd, J = 5.9, 3.7 Hz,
1
1
H), 3.59 (dt, J = 9.6, 6.7 Hz, 1H), 3.26 (dd, J = 8.1, 5.2 Hz, 1H), 3.10 (s, 2H), 2.96 (s, 2H), 1.59–1.40 (m, 8H),
13
.32 (dd, J = 19.1, 8.3 Hz, 6H), 0.94 (dd, J = 8.7, 4.4 Hz, 20H), 0.83 (dd, J = 8.1, 6.7 Hz, 6H); C-NMR
172.9, 172.8, 171.3, 171.1, 168.8, 136.1, 130.9, 129.5, 128.8 (2C), 128.6 (2C), 127.0,
(75 MHz, CDCl3),
δ
C
6
2
8.2, 64.0, 59.0, 55.1, 49.7, 47.3, 41.7, 39.1, 38.7, 37.1, 30.6, 30.4, 28.9, 25.3, 25.0, 24.8, 23.8, 23.3, 23.0, 22.9,
+
+
+
2.7, 22.6, 22.5, 21.8; MS (ESI) m/z: 628.8 [M + H] , 645.8 [M + NH ] , 650.8 [M + Na] .
4
1
For the detailed characterization of mass spectra, mass-mass spectra, H-NMR and HPLC of
Analog-3, refer to Figures S2C, S3C, S4C and S5C.
4
.1.28. Synthesis of Cyclo(Me-Leu-Leu-Phe-Me-Leu-D-Leu) (Analog-4)
The experimental procedure was followed as described in the synthesis of Analog-1. In this
synthesis, P2 was used instead of P5. Finally, 17.6 mg white powder with a yield of 34.2% were
1
obtained. H-NMR (300 MHz, CDCl ),
δ 7.28–7.17 (m, 5H), 6.95 (d, J = 9.8 Hz, 1H), 6.69 (d, J = 9.9 Hz,
H
3
1
7
3
H), 5.28 (dd, J = 17.0, 8.1 Hz, 1H), 5.12 (t, J = 7.7 Hz, 1H), 4.90 (dd, J = 16.4, 8.6 Hz, 1H), 4.76 (dd, J = 15.4,
.1 Hz, 1H), 4.59 (dd, J = 8.7, 6.1 Hz, 1H), 4.37 (dd, J = 17.4, 7.7 Hz, 1H), 3.22 (dd, J = 13.1, 8.3 Hz, 1H),
.05–2.96 (m, 1H), 2.81 (s, 3H), 2.61 (s, 3H), 1.66–1.41 (m, 8H), 1.39–1.21 (m, 4H), 1.00–0.79 (m, 24H);
1
3
C-NMR (75 MHz, CDCl3),
δ 173.4, 172.1, 170.6, 169.6, 167.8, 136.2, 129.2 (2C), 128.8, 128.7, 127.1,
C
5
2
8.5, 53.8, 51.7, 49.2, 48.4, 40.9, 40.6, 38.7, 38.0, 34.2, 29.1 (2C), 25.0, 24.9, 24.8, 24.7, 24.6, 23.0, 22.9, 22.7,
+
+
+
2.6, 22.5, 22.2, 22.1; MS (ESI) m/z: 628.8 [M + H] , 645.8 [M + NH ] , 650.8 [M + Na] .
4
1
For the detailed characterization of mass spectra, mass-mass spectra, H-NMR and HPLC of
Analog-4, refer to Figures S2D, S3D, S4D and S5D.
4
.1.29. Synthesis of Cyclo(Me-D-Leu-Leu-Phe-Me-Leu-Leu (Analog-5)
The experimental procedure was followed as described in the synthesis of Analog-1. In this
synthesis, P3 was used instead of P5. Finally, 39 mg white powder with a yield of 36.5% were obtained.
1
H-NMR (300 MHz, CDCl3),
δ 7.66 (d, J = 8.7 Hz, 1H), 7.28–7.17 (m, 5H), 6.74 (d, J = 9.8 Hz, 1H),
H
5.26–5.15 (m, 1H), 5.11 (dd, J = 9.4, 6.2 Hz, 1H), 5.00 (td, J = 8.4, 6.4 Hz, 1H), 4.85–4.79 (m, 1H),
4.63–4.50 (m, 1H), 4.46 (dd, J = 10.4, 6.6 Hz, 1H), 3.30–3.17 (m, 1H), 2.96 (t, J = 5.7 Hz, 1H), 2.84 (s, 3H),
2
.54 (s, 3H), 2.12–1.76 (m, 4H), 1.62–1.48 (m, 8H), 1.01–0.75 (m, 24H); 13C-NMR (75 MHz, CDCl3),
δ
173.5, 171.8, 171.5, 169.6, 167.9, 137.3, 129.6, 129.4, 128.3 (2C), 126.5, 58.3, 53.8, 51.7, 51.1, 45.8, 41.0,
C
3
8.6, 37.8, 37.2, 34.0, 29.7, 29.1, 25.4 24.9, 24.8, 24.7, 24.4, 23.3, 23.0, 22.9, 22.6, 22.4, 22.2, 21.9; MS (ESI)
+
+
+
m/z: 628.8 [M + H] , 645.8 [M + NH ] , 650.8 [M + Na] .
For the detailed characterization of mass spectra, mass-mass spectra, H-NMR and HPLC of
Analog-5, refer to Figures S2E, S3E, S4E and S5E.
4
1
4
.1.30. Synthesis of Cyclo(Me-Leu-D-Leu-Phe-Me-Leu-Leu) (Analog-6)
The experimental procedure was followed as described in the synthesis of Analog-1. In this
1
synthesis,P4 was used instead of P5. Yield: 42.5%. H-NMR (300 MHz, CDCl ), δ 7.28–7.17 (m, 5H),
3
H
7.03–6.87 (m, 1H), 6.71 (d, J = 9.6 Hz, 1H), 5.25 (dd, J = 16.3, 8.2 Hz, 1H), 5.11 (t, J = 7.6 Hz, 1H), 4.91
(t, J = 7.6 Hz, 1H), 4.76 (dd, J = 15.5, 7.0 Hz, 1H), 4.57 (dd, J = 8.8, 5.9 Hz, 1H), 4.34 (dd, J = 17.1,
7
8
.6 Hz, 1H), 3.21 (dd, J = 13.0, 8.6 Hz, 2H), 2.79 (s, 3H), 2.60 (s, 3H), 1.53 (ddd, J = 17.6, 12.8, 7.0 Hz,
H), 1.42–1.22 (m, 4H), 1.00–0.79 (m, 24H); 13C-NMR (75 MHz, CDCl3),
173.3, 172.0, 170.9, 169.9,
δ
C
1
2
67.7, 136.1, 129.2 (2C), 128.8 (2C), 127.2, 58.5, 53.8, 51.8, 50.2, 49.3, 48.4, 40.9, 40.4, 37.9, 34.2, 29.1, 25.1,
+
4.9, 24.8, 24.6, 23.0, 22.9, 22.7, 22.6, 22.4, 22.4, 22.2, 22.1, 21.7; MS (ESI) m/z: 628.8 [M + H] , 645.8
+
+
[M + NH ] , 650.8 [M + Na] .
4

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1
For the detailed characterization of mass spectra, mass-mass spectra, H-NMR and HPLC of
Analog-6, refer to Figures S2F, S3F, S4F and S5F.
4
.2. In Vitro Cell Experiments
4
.2.1. Cell Culture
Human hepatocellular cells (HepG ), human breast cancer cells (MCF-7), human breast
2
adenocarcinoma cells (MDA-MB-435) and a human cervical carcinoma cell line (Hela) were selected as
our in vitro cancerous cell models. The cells were all cultured in DMEM medium containing 10% fetal
◦
bovine serum, 1% penicillin-streptomycin and antifungal agent, incubated at 37 C with 5% CO and
2
9
0% humidity.
4
.2.2. MTT Measurement
The cytotoxicity of Galaxamide and its analogs on the four cancerous cells was evaluated using
the MTT method. Briefly, cells were seeded in 96-well tissue culture plates at a concentration of
3
2.5
×
10 cells/well. When cells’ confluence reached 70%, Galaxamide and its analogs with a
concentration of 0, 5, 10 and 15
µg/mL were added to the cell cultures and incubated for 48 h.
After that, 20 L MTT reagents were added to each well and incubated between 3–5 h according
µ
to the nature of cells; the color intensity of the cell culture at 570 nm was measured using a
microplate spectrophotometer. Finally, the cell viability was calculated accordingly. Note: MTT is
3
-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide.
4
.2.3. Flow Cytometry Experiments
5
HepG cells were seeded in 6-well tissue culture plates at a concentration of 1
Galaxamide and its analogs with a concentration of 0, 5, 10 and 15
×
10 cells/well.
2
µg/mL were added to the cell
cultures and incubated for 48 h. After that, 5 L Annexin V-fluorescein isothiocyanate (FITC) and 5 µL
µ
of PI were added for dual staining of the cells and analyzed by FACScan flow cytometry. Cell cycle
and apoptotic cells were determined by counting the FITC-positive and PI-negative fractions.
4
.2.4. Hoechst 33342 Staining
5
HepG cells were seeded in 6-well tissue culture plates at a concentration of 1
×
10 cells/well.
2
Galaxamide and its analogs with a concentration of 0, 5, 10 and 15 µg/mL were added to the cell
cultures and incubated for 48 h. After that, cells were fixed with 3.7% of paraformaldehyde for 10 min
and washed three times with PBS. Then, Hoechst 33342 was added for cell staining, then visualized
using a fluorescent microscope.
4
.2.5. Western Blot Experiments
Target proteins were first extracted using Radio-Immunoprecipitation Assay (RIPA) buffer and
quantified by the Bradford method. Then, proteins were transferred to a polyvinylidene difluoride
PVDF) membrane, and the membrane was blocked with 5% non-fat milk for 1 h and incubated with a
(
primary antibody (Cell Signaling Technology, Danvers, MA, USA, rabbit monoclonal antibody, 1:1000)
◦
at 4 C overnight. After that, the membrane was washed two times with TBST buffer (Tris-buffered
saline with 0.1% Tween-20) and incubated with a secondary antibody (goat-anti-rabbit horseradish
peroxidase (HRP)-conjugated 1:15,000) for 1 h under room temperature. Finally, the membrane was
washed three times with TBST, and the intensity of the specific immunoreactive bands was detected by
enhanced chemiluminescence.

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4
.2.6. Statistical Analysis
All data in the manuscript were expressed in the form of the mean
The statistical analysis were performed using Student’s t-test, and differences between groups were
considered significant when p < 0.05.
±
the standard deviation.
5
. Conclusions
In the present work, we synthesized six Galaxamide analogs using a classic “3 + 2” synthetic
strategy, where five of the analogs were designed with the varied position of the D-amino acid.
Their anticancer activity was tested against four human cancer cell lines, such as HepG , MCF-7,
2
MDA-MB-435 and Hela cells. We found that changes in the D-amino acid position resulted in the
alteration of the anticancer potential. Analog-2 (with a D-phenylalanine at position 1), Analog-4 (with
a D-leucine at position 3) and Analog-6 (with a D-leucine at position 5) showed improved anticancer
activity compared to natural product Galaxamide. Among those analogs, Analog-6 exhibited the best
anticancer effects. Analog-6 could cause the early apoptosis of HepG cells by inhibiting their growth
2
in the sub-G1 stage of the cell cycle and induced chromatin condensation. Moreover, Analog-6 exerted
the early apoptotic effect through a mitochondria-mediated pathway. The findings in our work will
provide a database for the rational design of Galaxamide analogs with improved anticancer activity
that are promising for the development of anticancer drugs.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/3/544/s1.
Acknowledgments: This research work was financially supported by the general projects of the National Natural
Science Foundation (Nos. 21672084, 41376155, 21372100, 31201028), the Chinese Fundamental Research Funds
for the Central Universities (21616113, 21616306) and the major special project of collaborative innovation of
industry-university-research of Guangzhou (201605131220049).
Author Contributions: Shihai Xu, Xiaojian Liao and Defa Bai conceived of and designed the experiments. Defa Bai,
Shenghui Zhong, Bingxin Zhao, Shaoling Qiu and Jianwei Chen performed the experiments. Defa Bai, Siming Yu,
Jignesh Lunagariya and Shihai Xu analyzed the data. Defa Bai, Siming Yu, Jignesh Lunagariya, Xiaojian Liao and
Shihai Xu wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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