Design and synthesis of analogues of marine natural product galaxamide, an N-methylated cyclic pentapeptide, as potential anti-tumor agent in vitro

Article abstract of DOI:10.3390/md14090161

Herein, we report design and synthesis of novel 26 galaxamide analogues with N-methylated cyclo-pentapeptide, and their in vitro anti-tumor activity towards the panel of human tumor cell line, such as, A549, A549/DPP, HepG2 and SMMC-7721 using MTT assay. We have also investigated the effect of galaxamide and its representative analogues on growth, cell-cycle phases, and induction of apoptosis in SMMC-7721 cells in vitro. Reckon with the significance of conformational space and N-Me aminoacid (aa) comprising this compound template, we designed the analogues with modification in N-Me-aa position, change in aa configuration from L to D aa and substitute one Leu-aa to D/L Phe-aa residue with respective to the parent structure. The efficient solid phase parallel synthesis approach is employed for the linear pentapeptide residue containing N-Me aa, followed by solution phase macrocyclisation to afford target cyclo pentapeptide compounds. In the present study, all galaxamide analogues exhibited growth inhibition in A549, A549/DPP, SMMC-7721 and HepG2 cell lines. Compounds 6, 18, and 22 exhibited interesting activities towards all cell line tested, while Compounds 1, 4, 15, and 22 showed strong activity towards SMMC-7221 cell line in the range of 1-2 μg/mL IC₅₀. Flow cytometry experiment revealed that galaxamide analogues namely Compounds 6, 18, and 22 induced concentration dependent SMMC-7721 cell apoptosis after 48 h. These compounds induced G0/G1 phase cell-cycle arrest and morphological changes indicating induction of apoptosis. Thus, findings of our study suggest that the galaxamide and its analogues 6, 18 and 22 exerted growth inhibitory effect on SMMC-7721 cells by arresting the cell cycle in the G0/G1 phase and inducing apoptosis. Compound 1 showed promising anti-tumor activity towards SMMC-7721 cancer cell line, which is 9 and 10 fold higher than galaxamide and reference DPP (cisplatin), respectively.

Full text of DOI:10.3390/md14090161

marine drugs
Article
Design and Synthesis of Analogues of Marine
Natural Product Galaxamide, an N-methylated Cyclic
Pentapeptide, as Potential Anti-Tumor Agent in Vitro
Jignesh Lunagariya 1,†, Shenghui Zhong 1,†, Jianwei Chen 1, Defa Bai 2, Poonam Bhadja 3,
Weili Long 1, Xiaojian Liao 1,*, Xiaoli Tang 4,* and Shihai Xu 1,
*
1
Department of Chemistry, Life Science School, Jinan University, Guangzhou 510632, China;
jignesh.lunagariya@gmail.com (J.L.); shenghuizhong@foxmail.com (S.Z.); 18826237580@163.com (J.C.);
gallopfeel@gmail.com (W.L.)
College of Pharmacy, Jinan University, Guangzhou 510632, China; tbaidf@jnu.edu.cn
Institute of Biomineralization and Lithiasis Research, Department of Chemistry, Life Science School,
Jinan University, Guangzhou 510632, China; poonambhadja@gmail.com
Alpert Medical School, Brown University, 55 Claverick St. 4th Floor, Providence, RI 02903, USA
Correspondence: tliaoxj@jnu.edu.cn (X.L.); xiaoli_tang@brown.edu (X.T.); txush@jnu.edu.cn (S.X.);
Tel.: +86-20-85-221-346 (S.X.)
2
3
4
*
†
These authors contributed equally to this work.
Academic Editor: Paul Long
Received: 2 August 2016; Accepted: 26 August 2016; Published: 3 September 2016
Abstract: Herein, we report design and synthesis of novel 26 galaxamide analogues with
N-methylated cyclo-pentapeptide, and their in vitro anti-tumor activity towards the panel of human
tumor cell line, such as, A549, A549/DPP, HepG2 and SMMC-7721 using MTT assay. We have
also investigated the effect of galaxamide and its representative analogues on growth, cell-cycle
phases, and induction of apoptosis in SMMC-7721 cells in vitro. Reckon with the significance of
conformational space and N-Me aminoacid (aa) comprising this compound template, we designed
the analogues with modification in N-Me-aa position, change in aa configuration from L to D aa and
substitute one Leu-aa to D/L Phe-aa residue with respective to the parent structure. The efficient solid
phase parallel synthesis approach is employed for the linear pentapeptide residue containing N-Me
aa, followed by solution phase macrocyclisation to afford target cyclo pentapeptide compounds.
In the present study, all galaxamide analogues exhibited growth inhibition in A549, A549/DPP,
SMMC-7721 and HepG2 cell lines. Compounds
all cell line tested, while Compounds 15, and 22 showed strong activity towards SMMC-7221 cell
line in the range of 1–2 g/mL IC50. Flow cytometry experiment revealed that galaxamide analogues
namely Compounds 18, and 22 induced concentration dependent SMMC-7721 cell apoptosis after
6, 18, and 22 exhibited interesting activities towards
1, 4,
µ
6,
48 h. These compounds induced G0/G1 phase cell-cycle arrest and morphological changes indicating
induction of apoptosis. Thus, findings of our study suggest that the galaxamide and its analogues
6, 18 and 22 exerted growth inhibitory effect on SMMC-7721 cells by arresting the cell cycle in the
G0/G1 phase and inducing apoptosis. Compound
1 showed promising anti-tumor activity towards
SMMC-7721 cancer cell line, which is 9 and 10 fold higher than galaxamide and reference DPP
(cisplatin), respectively.
Keywords: anti-tumor; apoptosis; cyclic pentapeptide; galaxamide analogues; macrocyclisation
1. Introduction
The marine environment is the vital source of living organisms [
1,2], which provides diversity of
natural products that pave the way for medicinal scientists to discover well-suited bioactive compounds
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www.mdpi.com/journal/marinedrugs

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for the biological systems [
3]. Among them, peptides are very interesting target following their
important role as hormones [
4
], neurotransmitters [
5
], growth factors [
6
], ion channel ligands [
7
],
],
or anti-infectives [ ]. Nearly, more than 7000 naturally occurring peptides have been identified [
8
9
including several bioactive peptides. Peptides and their homologous compounds (proteins and
antibodies) can be used in multiple pathologies, including allergy and asthma [10], arthritis [11],
cancer [12], cardiovascular diseases [13], diabetes [14], gastrointestinal dysfunction [15], HIV [16],
infective diseases [17], inflammation [18], pain [19], and so on.
At present, around 100 peptide medicines on the market, and which is expected to grow
considerably [20], with about 140 currently in clinical trials and more than 500 therapeutic peptides in
preclinical development [9].
In recent time, approve drug and candidates from small molecules in clinical trials are decreasing,
which led scientists to pay more attention towards peptides and other alternatives [21]. The probability
of regulatory approval is 20% for the peptide drugs with respect to 10% for the small molecule [22,23],
as the small molecules are not selective and can accumulate in specific organs such as the kidney and
liver, resulting in severe toxic side effects [24].
In peptide drug portfolio, around half of the peptides in clinical trial have target indication in
oncology, metabolic, cardiovascular and infectious diseases [24
have been demonstrated as anti-tumor agent, and shown cytotoxicity towards several types of tumors,
including pancreatic [26 28], colon [29 32], breast, prostate, and melanoma cancers [26 33 34], which
,25]. In particular, cyclic pentapeptides
–
–
, ,
proved its high potential in targeting the cancer. Following discovery of natural product sansalvamide,
a cyclic depsipeptide, several reports have been demonstrated anti-tumor activity of Sansalvamide A
analogues, including N-methylsanslavamide A analogues as a variety of anti-tumor agent by Silverman
group [26]. Accordingly, McAlpine group also published numerous articles on San A, such as,
analogues as treatment of MSS colon cancer [30], synthesis of analogues as potential anti-tumor [34],
and potent derivative against pancreatic cancer cell lines [27]. Along the same line, galaxamide 1,
an N-methylated cyclic pentapeptide, has been discovered from marine algae Galaxaura filamentosa with
structural determination and first total synthesis by our group [35]. Galaxamide 1and general structure
of its analogues have shown in Figure 1. Galaxamide and its analogues have also shown potential anti
tumor activity [36]. All of these analogues, including sansalvamide analogues, have clearly suggested
that the incorporation of N-Me and D aa alter the conformational space and hydrogen bonding,
which consequently play a crucial role in the cytotoxicity and other pharmacokinetic property of the
compound. Hence, in continuation of our interest to find more potent lead compound as anti-tumor
agent on the basis of conformational space and hydrogen bonding, we report the solid phase synthesis
of 26 galaxamide analogues with modification in N-Me-aa position, change in aa configuration from L
to D aa and substitute one Leu-aa to D/L Phe-aa residue with respective to the parent structure, and
their in vitro anti-tumor activity against A549, A549/DPP, SMMC-7721 and HepG2 cell lines using
MTT assay. We further investigated its mechanism of action by detecting cell morphology, cell-cycle
progression, and apoptosis using SMMC-7721 as representative cell line model.
Figure 1. Structure of galaxamide and general structure of its derivative.

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2. Results
2.1. Chemistry
Recently, in most cases, synthesis of cyclo pentapeptide was employed on segment based solution
phase synthetic strategy [27 30 34 36]. Synthesis of galaxamide and its analogues have been reported
,
,
–
by our group with a similar strategy [35,36].
In present study, we synthesized linear pentapeptide using Fmoc peptide synthetic strategy with
2-chloro trityl resin solid phase (Scheme 1), which is very proficient in a way to efficient, speed, easy
to handle and high purity of crude product. Synthesis of D/L N-Me Leu aa residue were performed
as per reported method by Zhang et al. [37] (Scheme 2). Briefly, N-Fmoc protected aa was converted
into 5-oxazolidinones derivative using para formaldehyde in the presence of catalytic amount of
p-toluenesulfonic acid (PTSA) in toluene under azeotropic water removal condition, followed by
reduction with triethylsilane in the presence of lewis acid to provide N-Me aa. This method is more
convenient over N-Me with sodium hydride/methyl iodide according to Benoiton’s protocol [38].
Scheme 1. Reaction scheme for linear peptide on solid phase. Reagents and conditions: a,
(i) dichloro methane (DCM), N,N-Diisopropylethylamine (DIPEA) 4 eq., room temperature (rt);
(ii) DCM/MeOH/DIPEA (4/5/1), 30 min, rt; b, (i) 20% piperidine in N,N-Dimethylformamide (DMF)
(2
×
10 min); (ii) DIPEA 2 eq., DEPBT 2 eq., DCM/DMF (1/1), 3 h, rt; c, 1% TFA in DCM for
(2 × 30 min), rt.
a
Fmoc
b
N
Fmoc
Fmoc
N
COOH
N
COOH
O
H
O
Scheme 2. Freidinger synthesis of N-methylated Leucine. Reagents and conditions: a, (CH2O)n,
PTSA (cat.), toluene, azeotropic removal of water; b, Et3SiH, CF3COOH, CHCl3.
First aa residue was immobilized on resin using 4.0 eq. N,N-Diisopropylethylamine (DIPEA) in
dichloro methane (DCM) for 2 h at room temperature (rt). Subsequent aa coupling including N-me
aa was performed via 3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) mediated
activation in DCM with DIPEA as base. DEPBT was chosen as more convenient for N-Me aa coupling

Mar. Drugs 2016, 14, 161
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and negligible racemization [39]. Solid-phase reactions were monitored by use of a qualitative Kaiser
test [40] for the detection of primary amines and the chloranil test [41] for detection of secondary
amines. Coupling reaction was completed within 2 h except for coupling with N-Me aa, which
generally took 8–10 h. Upon cleavage from solid support with 1% TFA in DCM linear pentapeptide
was obtained in 50%–60% overall yield with 90%–95% purity which was verified by RP-HPLC.
The linear pentapeptide was isolated by precipitation from diethylether, collected by centrifugation,
which can be used for macrocyclisation without further purification. Key challenge for macrocyclisation
is lower yield due to many intermolecular reactions, resulting into various complex oligomeric
products. Recently, some advancement on macrocyclisation reaction of pentapeptide have been
suggested that (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) is
better coupling reagent for obtaining higher yield with short reaction time [42]. After screening
range of dilution of solvent DCM and base equivalent, employing PyBOP as a coupling reagent for
macrocyclisation, promising reaction condition with 0.0007 M dilution of solvent DCM in the presence
of 2 eq. of base DIPEA and 2 eq. PyBOP have been established (Scheme 3). After 24 h reaction, targeted
cyclo pentapeptides were obtained in 44%–66% yield after preparative RP-HPLC purification.
Scheme 3. Macrocylclisation of linear pentapeptide.
Structure of Macrocyclic Analogues
We designed and synthesized three subsets of galaxamide derivatives. These were mainly
differentiated through N-Me position, addition to that multi changes have been incorporated with
position and number of D configuration aa residues, which was common in all three subset (Figure 2).
Figure 2. Amino acid used at various position for galaxamide derivative.
Some compounds also include two N-Me aa residue product (Compound 19, and 26). The first
5,
subset of compounds contains N-Me aa residue at third position (Compound 1–5, Figure 3). The second

Mar. Drugs 2016, 14, 161
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subset contain N-Me aa residue at fourth position (Compounds
6–
19, Figure 4). The third subset contain
N-Me aa residue at fifth position (Compounds 20–26, Figure 5).
O
O
O
O
O
N
N
O
NH
NH
NH
N
NH
NH
O
O
O
HN
NH
HN
HN
H
H
H
N
N
N
O
O
O
O
O
O
Compound 3
Compound 1
Compound 2
O
O
O
N
NH
NH
O
N
NH
NH
O
O
HN
HN
H
N
N
O
O
O
O
Compound 5
Compound 4
Figure 3. Subset 1: N-Me at third position galaxamide derivative.
Figure 4. Subset 2: N-Me at fourth position galaxamide derivative.

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Figure 5. Subset 3: N-Me at fourth position galaxamide derivative.
2.2. Biological Activity
2.2.1. Inhibitory Activity of Galaxamide and Its Analogues
Galaxamide and its analogues were evaluated for their cytotoxicity against panel of tumor cell
lines, such as, Lung cancer cell line A549, drug-resistant lung cancer cell line A549/DPP, human
hepatocellular carcinoma HepG2, SMMC-7721 and also against human normal liver cell line L02
using MTT assay. These compounds selectively differently interact with cancer cell over normal
liver cell line and exhibited less cytotoxicity to normal cell with >40
Compounds 14 and 15 which showed 34.63 and 36.87 g/mL IC50 value and was also seven fold higher
than galaxamide against HepG2. Compounds 18 and 22 exhibited interesting activities towards all
cell line tested, while Compounds 15 and 22 showed strong activity towards SMMC-7221 cell line
in the range of 1–2 g/mL IC50. Additionally, we have further studied three Compounds 18 and 22
µg/mL IC50 value, except for the
µ
6,
1, 4,
µ
6,
as representative of all these analogues to understand the pathway. We have chosen these compounds
as they have expressed interesting activities towards all cell line and had moderate differences with
Compounds 1, 4 and 15 which showed strongest activity in SMMC-7721 cell line. The results are
summarized in Figure 6.
2.2.2. Galaxamide and Its Analogues Caused G0/G1 Arrest
The cell cycle of cancer cells was assessed to continue the investigation into the toxicity of
galaxamide and its analogues. Figure 7 illustrates the representative experimental results of propidium
iodide (PI) fluorescence intensity, which was proportional to the DNA content and indicated the
cell cycle phases. To investigate the mechanism of galaxamide and its analogues on cell growth
inhibition, SMMC-7721 cells were exposed to representative compounds with the concentrations of 4,
8 and 16 µg/mL for 48 h before submitted to cell-cycle analysis. Result of this experiment showed
that treatment of SMMC-7721 cells with these compounds promoted a considerable increase in the
percentage of cells in the G0/G1 phase, which suggested that compounds caused cell-cycle arrest
at G0/G1 phase in which the cell grows and prepares to synthesize DNA. Thus, these analogues
compounds directly interact with the nucleus of cancer cells, and did not induce DNA fragmentation
in cells, thereby altered the cell-cycle progression and inhibited growth of cancer cells.

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30
25
20
15
10
5
Subset 1
0
Galaxamid
e
1
2
3
4
5
DPP
A549
8.74
27.2
5.27
15.01
7.76
5.17
11.41
18.82
2.77
8.89
19.71
16.58
1.53
6.89
23.61
5.23
10.34
17.42
4.63
12.34
16.72
8.43
A549-DPP
HepG2
12.15
1.25
SMMC-7721
35
5.48
2.74
11.21
12.56
Subset 2
30
25
20
15
10
5
0
Galax
amide
6
7
8
9
10
11
12
13
14
15
16
17
18
19
DPP
A549
1.73 6.05 10.62 9.24 16.06 4.47 7.54 13.81 6.75 3.63 5.71 7.15 2.76 5.21 10.34 12.34
5.13 14 17.34 10.08 10.88 13.24 29.34 27.46 15.39 8.24 22.43 14.53 6.91 17.73 17.42 16.72
A549-DPP
HepG2
7.23 10.41 22.61 23.87 23.02 7.41 13.93 10.22 5.97 16.81 13.49 10.48 6.96 4.25 4.63 8.43
2.34 14.97 16.29 3.72 6.54 7.54 1.84 7.57 6.05 1.84 7.89 2.9 5.43 2.7 11.21 12.56
SMMC-7721
30
Subset 3
25
20
15
10
5
0
Galaxam
ide
20
21
22
23
24
25
26
DPP
A549
7.41
16.15
9.68
6.47
18.6
15.3
8.21
9.4
12.22
6.41
1.44
5.85
9.13
3.45
9.26
7.92
7.04
5.18
20.43
22.88
6.85
4.27
11.33
7.12
10.34
17.42
4.63
12.34
16.72
8.43
A549-DPP
HepG2
SMMC-7721
26.62
4.32
3.35
6.65
11.21
12.56
Figure 6. Cytotoxicity of compounds galaxamide and its three subset analogues against A549,
A549/DPP, HepG2 and SMMC-7721 (IC50, µg/mL) using MTT assay. The IC50 values are reported as
the means
±
SD from three independent experiments. Lung cancer cell line A549, drug-resistant lung
cancer cell line A549/DPP, human hepatocellular carcinoma HepG2 and SMMC-7721.

Mar. Drugs 2016, 14, 161
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(a)
(b)
(c)
(d)
Figure 7. Galaxamide and its analogues caused G0/G1 phase arrest in SMMC-7721 cells. SMMC-7721
cells were incubated with or without 4, 8 and 16 g/mL galaxamide ( ) and compounds 6 ( ), 18 (c)
µ
a
b
and 22 (d) for 48 h. Cell cycle was determined by PI staining using flow cytometry.
2.2.3. Galaxamide and Its Analogues Induced Apoptosis of SMMC-7721 Cells
SMMC-7721 cell apoptosis was examined by flow cytometric analysis using annexin V/PI
double staining after 48 h treatment of galaxamide and its analogues with different concentrations
(4, 8 and 16
µg/mL). Apoptosis plays an important role in the treatment of cancer, as it is a
popular target of cancer treatment strategies. The abundance of literature suggests that targeting
apoptosis in cancer is feasible. As shown in Figure 8, the lower right quadrants accounted the early
apoptotic rate, and column statistics of apoptotic rate presented in Figure 9. We found that the
representative Compounds 6, 18 and 22 induced SMMC-7721 cell apoptosis in dose-dependent manner.
Hence, these analogues caused apoptosis in cancer cells through growth arrest during cell-cycle
progression pathway.

Mar. Drugs 2016, 14, 161
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(a)
(b)
(c)
(d)
Figure 8. Galaxamide and its analogues induced apoptosis in SMMC-7721 cells. SMMC-7721 cells were
incubated with or without 4, 8 and 16 g/mL galaxamide ( ) and compounds 6 ( ), 18 ( ) and 22 (d)
µ
a
b
c
for 48 h. The apoptosis of SMMC-7721 cells was determined by annexin V/PI staining. B1 (the upper
left) quadrant represents necrotic cells, B2 (the upper right) quadrant represents late apoptotic and
dead cells, B3 (the bottom left) represents normal cells, B4 (the bottom right) quadrant represents early
apoptotic cells.

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Figure 9. Galaxamide and its analogues induced apoptosis in SMMC-7721 cells. Annexin V positive
cells of three independent experiments were shown in column statistics. Data expressed as mean SD.
* p < 0.05; ** p < 0.01 vs. control
±
2.2.4. Morphological Changes of SMMC-7721 Cells Induced by the Treatment of Galaxamide
and Its Analogues
Hoechst33342 staining was performed for the morphological alteration analysis of SMMC-7721
cells using fluorescence microscopy. As shown in Figure 10, treatment of SMMC-7721 cells with 4, 8 and
16 µg/mL galaxamide and representative Compounds 6, 18, 22 analogues resulted in cell detachment
from cell culture plates. This action shows dose-dependent toxicity of evaluated compounds towards
cancer cells. Herein, control cells exhibited normal morphology. However, galaxamide and its
analogues-treated cells lost substrate attachment, became rounded, shrank, condensed their chromatin
and displayed membrane blebbing. These morphological features shows that these compounds share
cell growth inhibition by inducing apoptosis and cell growth arrest, eventually lead to cell death of
cancerous cells.
(a)
(b)
(c)
Figure 10. Cont.

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(d)
Figure 10. Morphological observation of galaxamide and its analogues-induced apoptosis in
SMMC-7721 cells as indicated by Hoechst33342 staining. SMMC-7721 cells were incubated with
or without 4, 8, 16
morphology was determined by Hoechst33342 staining with to visualize the nucleus (blue) under a
fluorescent microscope at a magnification of 200 ; scale bar, 50 m. Arrows indicate apoptotic features
(condensed chromatin and nuclear fragmentation).
µg/mL galaxamide (a) and compounds 6 (b); 18 (c) and 22 (d) for 48 h, and then cell
×
µ
3. Discussion
3.1. Structure-Activity Relationship
In our previous article, we have reported in vitro anti-tumor activity of few analogues of
galaxamide with inclusion of D Phe and variation in number of D Leu aa residue, from which,
one analogues showed promising anti-tumor activity against HepG2 with the three fold enhancement
than galaxamide [36]. In continuation to find more potent compound, we have demonstrated
26 galaxamide analogues with inclusion of one D/L Phe aa residue, only one N-Me Leu residue
at three, four and five position of macrocycle called subset 1, subset 2 and subset 3 (except Compounds 5,
19 and 26 where two N-Me were introduced), respectively including variation in position and number
of D Leu aa residue. In general, particular trend was unobserved in the structure-activity relationship
as many investigations have recently suggested that the major factor between potency and structure
involves conformational space which lock the macrocycle into its low energy conformation and
thus binding to its biological target in that position with appropriately present side chain [27].
Furthermore, compounds mostly showed dissimilar trend in activity against all cancer cell line except
A549 and A549/DPP where similar trend was observed in increase and decrease of cytotoxicity. All the
compounds showed less activity compared to galaxamide in HepG2 cancer cell line, though showed
comparable activity with DPP. This might be reason that two N-Me aa is favorable for HepG2 cancer
cell as the Compounds
Compound which contain N-me at 4 and D Leu at 5 position showed strongest activity (1.73 and
5.73 g/mL IC50) against A549 and A549/DPP, respectively followed by Compound 18, which include
D Leu at 2 and D N-Me Leu at 4 position showed excellent activity (2.76 and 6.91 g/mL IC50,
respectively). Compounds 12 15, and 22 showed strongest activity in the range of 1–2 g/mL IC50
value followed by Compounds 17, and 19 which showed activity in the range of 2–3 g/mL
5, 19, and 26 with two N-Me showed comparable activity with galaxamide.
6
µ
µ
1
,
4,
,
µ
3,
5
,
6,
µ
IC50 value against SMMC-7221 cell line. There was no particular structure-activity relationship
(SAR) observed, though all subset 1 compounds with N-Me at 3 position showed excellent activity.
While strongest activity shown by Compound 1 from Subset 1 had 1.25 µg/mL IC50 value which is 9 and
10 fold higher than parent galaxamide and current marketed drug DPP, respectively. In particular, from
the SAR it is observed that the N-Me at 3 position is very critical which might change the conformation
that boost the activity with enhancement of binding to the biological target.
3.2. Mechanism of Apoptosis Pathway
It is well known that apoptosis is a programmed process influence the density of a cell in
multicellular organisms in order to regulate tissue homeostasis. It becomes a matter of life and death
and the survival of the organism lies in the balance. Deregulation of cell proliferation and/or apoptosis
leads to pathological conditions of cancer. The life spans of both normal and cancer cells within a

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living system are regarded to be substantially affected by the rate of apoptosis. The understanding of
apoptosis events and its pathways has provided basic novel targets in the management and therapy
for cancer. Two major pathways are involved in mammalian cells apoptosis—mitochondria-mediated
intrinsic pathway and death receptor-mediated extrinsic pathway [43]. Thus, the chemopreventive
agents that can modulate apoptosis, are often useful in cancer therapy. Recently, the anti-tumor
activity of cyclic peptides has attracted much attention [44]. Many natural and synthetic cyclopeptides
have been proven to be clinically effective for treating cancer could inhibit tumors by triggering
cancer cell apoptosis. RA-V (deoxybouvardin), an unique natural cyclopeptide which was derived
from the medical plant Rubia yunnanensis, can induce mitochondria-mediated apoptosis, exerted
anti-tumor activity against human breast cancer [45]. Sansalvamide A, isolated from a marine fungus
of the genus Fusarium, is a cyclic tetrapeptide exhibited better anti-tumor activity [33]. In this
study, we have shown that galaxamide and its analogues resulted in an induction of apoptosis in
dose-dependent manner in SMMC-7721 cells, confirmed by annexin V/PI staining assay. Therefore,
inducing apoptosis might be the primary mechanism for the anti-cancer activity of galaxamide
analogues. Moreover, higher concentration of representative galaxamide derivatives induced better
apoptotic effect than galaxamide.
Cell cycle and apoptosis are considered to be two major regulatory mechanisms for cell growth.
When specific checkpoints during the cell cycle are arrested, apoptotic cell death occurs [46].
These checkpoints maintain the fidelity of DNA replication, repair, and division. Moreover, most
cancer therapeutics cause cell cycle arrest, and have been confirmed to be valuable agent for cancer
treatment [47]. It has been reported that a cyclic-peptide sansalvamide analogue inhibits pancreatic
cancer cell growth through G0/G1 cell-cycle arrest and apoptosis [28]. Herein, flow cytometric
examination showed that the growth inhibition of SMMC-7721 cells induced by galaxamide and its
analogues occurs through the cell cycle arrest in G0/G1 phase. Therefore, suggests that these agents
may slow down the growth of cancer cells by artificially imposing the cell cycle checkpoint. G0/G1
phase arrest of cell cycle regulation provides an opportunity for cells to follow the apoptotic pathway.
The G1 to S cell cycle progression is controlled by several cyclin-dependent kinase (CDK) complexes,
the activities of which are dependent on the balance of cyclins and cyclin-dependent kinase inhibitors
(CKIs). p53, the most extensively studied tumor suppressor, mediates a variety of anti-proliferative
processes through cell cycle checkpoints, DNA repair and apoptosis [48]. Previous reports have found
that p21, the target of p53, is one of the major CKIs, which directly inhibit the activity of CDKs, thereby
leading to cell cycle arrest in the G1 phase [49]. The exact mechanism of apoptosis and cell cycle
deregulation by galaxamide and its analogues needs a further exploration of signal transduction
pathways. It is, however, tempting to speculate that cyclin kinase inhibitors, cyclin dependent kinases
and their regulatory cyclins proteins operating in G1 phase of the cell cycle may be involved in the
galaxamide-mediated apoptosis and cell cycle arrest. Thus, present investigation revealed that the
galaxamide and its analogues exerted growth inhibitory effect on SMMC-7721 cells by arresting the
cell cycle in the G0/G1 phase and inducing apoptosis.
4. Experimental Section
4.1. General
NMR spectra were recorded on a Bruker Avance 300 spectrometer (300 MHz for 1H and 75 MHz
for 13C) in CDCl3. Chemical shifts are reported as
δ values in parts per million (ppm) relative to
tetramethylsilane (TMS) and J values are expressed in Hertz. The ESI mass spectra were obtained on
a LCQ DECA XP LC-MS mass spectrometer. Silica gel (200–300 mesh) for column chromatography
and silica GF254 for TLC were produced by Qingdao Marine Chemical Company (Qingdao, China).
All air- or moisture-sensitive reactions were conducted under nitrogen atmosphere. Starting materials
and reagents used in reactions were obtained commercially from Acros, Aldrich, GL Biochem and
were used without purification, unless otherwise indicated.

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4.2. Procedure for D/L Fmoc-N-Me Leucine
4.2.1. Step-1 Synthesis of Oxazolidinones from Fmoc Leu
The Fmoc Leu (5 mmol), paraformaldehyde (6.66 eq., 1 g) and p-toluenesulphonic acid (0.12 eq.,
100 mg) were suspended in toluene (100 mL) in 250 mL three neck RBF. The mixture was refluxed for
30 min in a Dean-Stark setup and reaction was monitored by TLC (97.5:2:0.5 CHCl3:MeOH:AcOH).
The solution was cooled, washed with saturated NaHCO3 and dried over anhydrous NaSO4.
Concentration in vacuo gave the crude product which in most cases solidified upon standing. In some
cases, purification via flash chromatography was required (silica gel, 3:7 ethylacetate:pentane).
4.2.2. Step-2 Synthesis of N-methylated Fmoc Leu from Oxazolidinones
To a solution of the oxazolidinone (1 eq.) and Lewis acid (2 eq.) in dry DCM (20 mL/1 mmol
oxazolidinone) was added triethylsilane (2 eq.). The reaction was stirred at ambient temperature until
TLC (1:3 ethylacetate:hexane) showed the absence of starting material. An additional amount of DCM
was added (twice the reaction volume) and the organic phase was washed with 1 M HCl. The organic
phase was dried over anhydrous sodium sulphate and concentrated in vacuo. The crude product was
purified via column chromatography on silica gel (hexane and ethylacetate).
4.3. General Procedure for the Synthesis of Pentapeptides-COOH
Synthesis of linear peptide was carried out using 2-chlorotrityl chloride (CTC) resin (1.05 mmol/g)
following the standard Fmoc strategy. All reactions were performed on 0.250 g of resin scale.
First N-Fmoc protected aa (2 eq.) residue was coupled with CTC resin (0.250 g) using DIPEA
(4 eq.) in DCM (5 mL) for 2 h at rt. After filtration, the remaining unreacted sites were capped
by a solution of DCM/MeOH/DIPEA (4:5:1, 10 mL) for 30 min. Resin was washed with DCM
(3
group was carried out using 20% piperidine in DMF for (2
measured by concentration of Fmoc group by ultraviolet standard curve method. The first aa bound
resin was thoroughly washed with DCM (3 5 mL), DMF (3 5 mL) and MeOH (3 5 mL). All other
×
5 mL), N,N-Dimethylformamide (DMF) (3
×
5 mL) MeOH (3
×
5 mL). Deprotection of Fmoc
×
10 min) at rt. Loading of resin was
×
×
×
N-Fmoc aa (2.0 eq.) coupling, including N-Me were carried out using standard solid phase peptide
synthesis using DEPBT (2.0 eq.) as coupling reagent in the presence of DIPEA (2 eq.) in DMF/DCM
(1:1, 5 mL) at rt for 3 h. Reaction was monitored using Kaiser test for the primary amine and chloranil
test for the secondary amine. Generally, coupling with N-Me aa took 5–6 h further time to complete the
reaction. Cleavage was performed using 1% TFA in DCM for (2
×
30 min) at rt. After cleavage, filtrate
was immediately neutralized with 1% pyridine in MeOH and concentrated under reduced pressure.
Crude peptide was isolated via precipitation from diethylether (20 mL), collected through centrifuge
as a white solid, which can be directly used for macrocyclisation reaction without further purification.
4.4. General Procedure for Macrocyclisation Reaction
All the macrocyclisation reaction was performed at 80 mg scale. The linear pentapeptide (80 mg,
0.126 mmol) was dissolved in dry DCM (0.0007 M, 181 mL) in 250 mL round bottom flask under
nitrogen atmosphere. Coupling reagent PyBOP (2 eq., 0.253 mmol, 131 mg) and DIPEA (2 eq.,
0.253 mmol, 44 µL) was added at rt. Stirred the reaction for one day and monitored the progress
of reaction on TLC as well as on reverse phase HPLC using a gradient of acetonitrile and DI water
with 0.1% TFA. Upon completion of reaction, work up with saturated sodium bicarbonate solution
(16 mL) and ammonium chloride (16 mL) solution was carried out. Organic layer collected, dried over
anhydrous sodium sulphate and concentrated under reduced pressure. Crude cyclic product was
purified on preparative reverse phase column chromatography using MeOH and DI water gradient at
210 nM.

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4.5. Spectral Data
L Fmoc-N-Me Leucine. Yield: 88%; 1H NMR (300 MHz, Chloroform-d)
δ
ppm: 12.74 (s, 1 H), 7.85 (t,
J = 7.4 Hz, 2 H), 7.60 (td, J = 7.6, 3.6 Hz, 2 H), 7.43–7.23 (m, 4 H), 4.64–4.17 (m, 4 H), 2.68 (s, 3 H),
1.74–1.19 (m, 3 H), 0.89–0.63 (m, 6 H); 13C NMR (75 MHz, Chloroform-d)
δ
ppm:178.31, 156.24,
143.70(2C), 141.35(2C), 127.76(2C), 127.11(2C), 125.10(2C), 120.03(2C), 67.12, 52.42, 47.21, 41.44, 24.81,
22.91, 21.73(2C); MS (ESI) m/z: 368.4 [M + H]+, 385.5 [M + NH4]+, 390.6 [M + Na]+.
D Fmoc-N-Me Leucine. Yield: 89%; 1H NMR (300 MHz, Chloroform-d)
J = 7.4 Hz, 2 H), 7.61 (td, J = 7.6, 3.6 Hz, 2 H), 7.42–7.22 (m, 4 H), 4.63–4.16 (m, 4 H), 2.67 (s, 3 H),
1.75–1.20 (m, 3 H), 0.90–0.64 (m, 6 H); 13C NMR (75 MHz, Chloroform-d)
ppm:178.31, 156.24,
δ ppm: 12.71 (s, 1 H), 7.83 (t,
δ
143.70(2C), 141.35(2C), 127.76(2C), 127.11(2C), 125.10(2C), 120.03(2C), 67.12, 52.42, 47.21, 41.44, 24.81,
22.91, 21.73(2C); MS (ESI) m/z: 368.5 [M + H]+, 385.5 [M + NH4]+, 390.6 [M + Na]+.
Compound
NMR (300 MHz, Chloroform-d)
1
cyclo(Phe-D-Leu-N-Me-Leu-D-Leu-D-Leu). Yield: 57.3%, Wt: 44.5 mg, white powder; 1H
ppm: 7.90 (dd, J = 24.9, 8.1 Hz, 2 H), 7.76–7.67 (m, 1 H), 7.24 (d,
δ
J = 13.6 Hz, 5 H), 6.94 (d, J = 6.9 Hz, 1 H), 5.04 (d, J = 8.7 Hz, 1 H), 4.79 (d, J = 7.7 Hz, 1 H), 4.56 (q,
J = 10.1, 9.6 Hz, 2 H), 3.71 (dq, J = 14.2, 7.6, 7.2 Hz, 2 H), 3.22 (s, 3 H), 3.13 (s, 1 H), 1.58 (d, J = 18.4 Hz,
4 H), 1.43 (d, J = 8.0 Hz, 8 H), 1.06-0.71 (m, 24 H); 13C NMR (75 MHz, Chloroform-d)
δ ppm: 174.67,
173.89, 173.14, 172.33, 170.78, 137.12, 129.39(2C), 128.31(2C), 126.55, 58.11, 56.75, 54.05, 51.75, 48.41,
41.63, 40.29, 38.40, 37.00, 36.80, 31.04, 25.16, 25.06, 25.00, 24.68, 23.15, 22.94, 22.83, 22.74, 22.53, 22.28,
21.42, 21.31; MS (ESI) m/z: 614.7 [M + H]+, 631.7 [M + NH4]+, 636.6 [M + Na]+.
Compound
NMR (300 MHz, Chloroform-d)
2
cyclo(D-Phe-Leu-N-Me-Leu-D-Leu-D-Leu). Yield: 61.5%, Wt: 47.8 mg, white powder; 1H
8.50 (d, J = 7.9 Hz, 1 H), 7.58 (d, J = 8.7 Hz, 1 H), 7.40–7.13 (m, 6 H),
δ
6.96 (d, J = 8.5 Hz, 1 H), 5.04 (t, J = 7.5 Hz, 1 H), 4.85 (q, J = 7.7 Hz, 1 H), 4.49 (dd, J = 11.0, 6.6 Hz, 2 H),
3.81 (q, J = 8.5, 6.9 Hz, 1 H), 3.29 (dd, J = 14.2, 7.1 Hz, 1 H), 3.15 (dd, J = 14.1, 4.9 Hz, 1 H), 2.70 (s, 3 H),
1.85 (dd, J = 13.4, 6.6 Hz, 2 H), 1.55 (dtq, J = 40.8, 13.0, 6.9, 6.4 Hz, 10 H), 1.04–0.72 (m, 24 H); 13C NMR
(75 MHz, CDCl3)
δ ppm: 174.02, 171.76, 170.58, 170.03, 170.01, 135.98, 129.19(2C), 129.0(2C), 127.54,
55.05, 53.88(2C), 48.61(2C), 41.16, 40.58, 37.38, 37.25, 34.75, 29.36, 24.88, 24.80(3C), 23.05, 22.81, 22.75,
22.67, 22.51, 22.31, 21.94, 21.66; MS (ESI) m/z: 614.6 [M + H]+, 631.6 [M + NH4]+, 636.5 [M + Na]+.
Compound
NMR (300 MHz, Chloroform-d)
3
(cyclo(Phe-Leu-D-N-Me-Leu-D-Leu-D-Leu). Yield: 51.7% , Wt: 40.2 mg, white powder; 1H
ppm: 8.07 (d, J = 6.8 Hz, 1 H), 7.93 (d, J = 8.3 Hz, 1 H), 7.33–7.11
δ
(m, 6 H), 6.75 (d, J = 9.1 Hz, 1 H), 5.02 (t, J = 7.7 Hz, 1 H), 4.91 (t, J = 7.0 Hz, 1 H), 4.49 (ddd, J = 11.3,
6.9, 4.3 Hz, 1 H), 4.36-4.10 (m, 1 H), 3.67 (q, J = 7.8 Hz, 1 H), 3.30 (dd, J = 14.1, 4.3 Hz, 1 H), 3.03 (d,
J = 2.0 Hz, 3 H), 2.82 (dd, J = 14.1, 11.5 Hz, 1 H), 1.84–1.31 (m, 12 H), 1.00–0.67 (m, 24 H); 13C NMR
(75 MHz, CDCl3) δ ppm: 173.39, 172.82, 172.20, 172.18, 171.19, 136.59, 128.86(2C), 128.74(2C), 127.07,
58.88, 56.74, 54.87, 52.07, 48.23, 41.54, 41.28, 38.36, 37.11, 36.65, 30.68, 25.57, 25.20, 25.15, 24.72, 22.86,
22.71, 22.63, 22.57(2C), 22.44, 22.24, 22.14; MS (ESI) m/z: 614.4 [M + H]+, 631.6 [M + NH4]+, 636.3
[M + Na]+.
Compound
NMR (300 MHz, Chloroform-d)
4
cyclo(D-Phe-Leu-D-N-Me-Leu-Leu-D-Leu). Yield: 53.8%, Wt: 41.8 mg, white powder; 1H
ppm: 7.30 (d, J = 7.5 Hz, 4 H), 7.22–7.12 (m, 3 H), 6.98 (d, J = 9.9 Hz,
δ
1 H), 6.25 (d, J = 7.8 Hz, 1 H), 5.05 (t, J = 7.6 Hz, 1 H), 4.93–4.79 (m, 1 H), 4.63 (td, J = 8.8, 4.5 Hz, 1 H),
4.51-4.36 (m, 1 H), 3.85 (dt, J = 9.6, 6.1 Hz, 1 H), 3.31 (dd, J = 14.3, 4.5 Hz, 1 H), 3.04–2.91 (m, 1 H), 2.72
(s, 3 H), 1.85 (dt, J = 13.6, 6.9 Hz, 1 H), 1.73–1.40 (m, 10 H), 1.34 (ddd, J = 13.6, 8.8, 6.0 Hz, 1 H), 0.93
(ddd, J = 17.6, 11.4, 6.3 Hz, 18 H), 0.75 (d, J = 6.5 Hz, 3 H), 0.69 (d, J = 6.5 Hz, 3 H); 13C NMR (75 MHz,
CDCl3)
δ ppm: 174.38, 172.30, 171.71, 170.16, 169.99, 136.24, 128.93(2C), 128.89(2C), 127.28, 54.96, 53.90,
53.75, 51.05, 48.41, 41.30, 40.59, 39.08, 37.37, 34.61, 29.42, 24.92, 24.82, 24.77, 24.49, 22.85, 22.80, 22.78,
22.69, 22.54, 22.15, 22.04, 21.83; MS (ESI) m/z: 614.7 [M + H]+, 631.7 [M + NH4]+, 636.6 [M + Na]+.
Compound
5
cyclo(D-Phe-D-Leu-N-Me-Leu-D-Leu-N-Me-Leu). Yield: 53.9%, Wt: 41.9 mg, white powder;
ppm: 7.33–7.09 (m, 6 H), 7.04 (d, J = 9.6 Hz, 1 H), 5.97 (d,
1H NMR (300 MHz, Chloroform-d)
δ

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J = 9.3 Hz, 1 H), 5.11 (t, J = 7.7 Hz, 1 H), 4.92 (dd, J = 9.9, 4.6 Hz, 1 H), 4.80 (tt, J = 9.1, 4.1 Hz, 2 H), 4.16
(dd, J = 10.5, 5.4 Hz, 1 H), 3.22 (dd, J = 14.0, 4.9 Hz, 1 H), 3.13 (s, 3 H), 3.10–2.96 (m, 1 H), 2.70 (s, 3 H),
1.78–1.21 (m, 12 H), 1.00–0.69 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 175.88, 171.73, 171.51,
170.12, 170.06, 136.18, 129.17(2C), 128.79(2C), 127.25, 57.86, 53.74(2C), 48.71, 47.83, 41.45, 41.13, 37.70,
37.47, 34.46, 31.40, 29.47, 24.96(2C), 24.83, 24.55, 23.27, 23.11, 22.95(2C), 22.24, 21.97, 21.82, 21.65; MS
(ESI) m/z: 628.8 [M + H]+, 645.8 [M + NH4]+, 650.9 [M + Na]+.
Compound
(300 MHz, Chloroform-d)
6
cyclo(Phe-Leu-Leu-N-Me-Leu-D-Leu). Yield: 64.5%, Wt: 50.1 mg, white powder; 1H NMR
7.36-7.19 (m, 5 H), 7.15 (d, J = 9.2 Hz, 1 H), 7.02 (d, J = 9.2 Hz, 1 H), 6.72 (t,
δ
J = 7.4 Hz, 2 H), 5.07 (t, J = 7.6 Hz, 1 H), 4.89 (dt, J = 9.1, 6.8 Hz, 1 H), 4.37 (dddd, J = 20.4, 10.0, 8.7,
5.3 Hz, 3 H), 3.24 (dd, J = 14.0, 6.1 Hz, 1 H), 3.01–2.91 (m, 1 H), 2.89 (s, 3 H), 1.88 (ddd, J = 14.1, 9.5,
4.7 Hz, 1 H), 1.73 (dt, J = 13.7, 7.8 Hz, 2 H), 1.63–1.40 (m, 7 H), 1.38–1.24 (m, 2 H), 1.00–0.85 (m, 18 H),
0.79 (t, J = 6.4 Hz, 6 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 173.39, 172.88, 171.03, 170.55, 170.40, 135.72,
128.98(2C), 128.88(2C), 127.42, 56.74, 54.34, 51.83, 51.13, 48.10, 41.17, 40.83, 39.57, 37.12, 35.24, 30.18,
25.08, 24.72(3C), 23.26, 22.77, 22.74, 22.60, 22.39, 22.30(2C), 21.39; MS (ESI) m/z: 614.6 [M + H]+, 631.6
[M + NH4]+, 636.5 [M + Na]+.
Compound
(300 MHz, Chloroform-d)
7
cyclo(Phe-Leu-D-Leu-N-Me-Leu-Leu). Yield: 52.2%, Wt: 40.6 mg, white powder; 1H NMR
ppm: 7.85 (d, J = 8.2 Hz, 1 H), 7.68 (d, J = 7.4 Hz, 2 H), 7.30–7.16 (m, 5 H),
δ
6.95 (d, J = 7.3 Hz, 1 H), 5.04–4.85 (m, 2 H), 4.31 (ddt, J = 32.9, 9.1, 6.4 Hz, 2 H), 4.11 (q, J = 7.5 Hz, 1 H),
3.41 (dd, J = 13.8, 9.2 Hz, 1 H), 3.19 (s, 3 H), 2.76–2.68 (m, 1 H), 1.87–1.49 (m, 9 H), 1.41 (dp, J = 20.1,
6.5 Hz, 3 H), 1.01–0.77 (m, 24 H); 13C NMR (75 MHz, Chloroform-d)
δ ppm: 174.24, 173.83, 172.80,
171.67, 171.25, 136.83, 129.14(2C), 128.46(2C), 126.81, 59.24, 56.38, 54.26, 52.19, 48.18, 40.73, 40.37, 39.80,
36.88, 36.41, 30.99, 25.23, 25.05, 24.94, 24.86, 23.05, 22.89, 22.72, 22.61, 22.50, 22.28, 22.21, 21.48; MS (ESI)
m/z: 614.7 [M + H]+, 631.7 [M + NH4]+, 636.6 [M + Na]+.
Compound
(300 MHz, Chloroform-d)
8
cyclo(Phe-D-Leu-Leu-N-Me-Leu-Leu). Yield: 44.8%, Wt: 34.8 mg, white powder; 1H NMR
ppm: 7.81 (d, J = 8.3 Hz, 1 H), 7.62 (dd, J = 11.7, 7.7 Hz, 2 H), 7.23 (h,
δ
J = 5.0 Hz, 5 H), 6.96 (d, J = 7.4 Hz, 1 H), 4.98 (dd, J = 10.1, 5.9 Hz, 1 H), 4.90 (d, J = 7.6 Hz, 1 H),
4.39–4.29 (m, 1 H), 4.22 (d, J = 9.2 Hz, 1 H), 4.11 (d, J = 7.5 Hz, 1 H), 3.42 (dd, J = 13.7, 9.3 Hz, 1 H),
3.30-3.20 (m, 1 H), 3.19 (s, 3 H), 1.73–1.52 (m, 9H), 1.42 (dp, J = 13.6, 6.7 Hz, 3 H), 1.03–0.79 (m, 24 H);
13C NMR (75 MHz, Chloroform-d)
δ ppm: 174.25, 173.77, 172.93, 171.69, 171.20, 136.85, 129.12(2C),
128.48(2C), 126.82, 59.35, 56.34, 54.22, 52.16, 48.24, 40.64, 40.23, 39.71, 36.81, 36.36, 30.97, 25.23, 25.02,
24.95, 24.84, 23.06, 22.90, 22.73, 22.61, 22.51, 22.29, 22.16, 21.47; MS (ESI) m/z: 614.5 [M + H]+, 631.5
[M + NH4]+, 636.4 [M + Na]+.
Compound
(300 MHz, Chloroform-d)
6.52 (s, 2 H), 5.07–4.87 (m, 1 H), 4.71 (d, J = 7.7 Hz, 1 H), 4.46 (qd, J = 10.7, 9.9, 4.4 Hz, 3 H), 3.13 (d,
J = 8.2 Hz, 2 H), 3.09 (s, 3 H), 1.79–1.26 (m, 12 H), 1.04–0.65 (m, 24 H); 13C NMR (75 MHz, CDCl3)
9
cyclo(D-Phe-Leu-Leu-N-Me-Leu-D-Leu). Yield: 60.2%, Wt: 46.8 mg, white powder; 1H NMR
ppm: 7.34 (d, J = 9.2 Hz, 1 H), 7.30–7.17 (m, 5 H), 7.06 (d, J = 7.4 Hz, 1 H),
δ
δ
ppm: 174.81, 172.13, 171.18, 170.98, 170.67, 136.97, 129.31(2C), 128.44(2C), 126.68, 56.13, 55.93, 52.57,
50.69, 47.97, 41.87, 41.37, 37.59, 37.11, 36.78, 30.94, 25.11, 25.06, 24.85, 24.40, 23.05, 22.99, 22.91, 22.57,
22.52, 22.15, 21.80, 21.49; MS (ESI) m/z: 614.3 [M + H]+, 631.3 [M + NH4]+, 636.4 [M + Na]+.
Compound 10 cyclo(D-Phe-Leu-D-Leu-N-Me-Leu-Leu). Yield: 53.1%, Wt: 41.3 mg, white powder; 1H NMR
(300 MHz, Chloroform-d) δ ppm: 7.49 (d, J = 8.6 Hz, 1 H), 7.33–7.13 (m, 5 H), 6.96 (dd, J = 8.8, 2.4 Hz,
1 H), 6.76 (t, J = 9.5 Hz, 1 H), 6.57 (dd, J = 9.7, 5.1 Hz, 1 H), 5.17 (dd, J = 11.8, 4.3 Hz, 1 H), 4.76 (q,
J = 7.9 Hz, 1 H), 4.64 (q, J = 6.9 Hz, 1 H), 4.60–4.38 (m, 2 H), 3.21 (dd, J = 14.2, 7.4 Hz, 1 H), 3.14 (s, 3 H),
2.97–2.84 (m, 1 H), 2.13–1.36 (m, 12 H), 1.05–0.78 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 175.07,
174.57, 172.58, 172.02, 170.54, 137.03, 129.13(2C), 128.44(2C), 126.58, 56.79, 53.61, 51.78, 51.04, 49.14,
40.09, 40.04, 39.88, 36.58, 34.95, 30.97, 25.35, 24.91(2C), 24.77, 23.38, 22.94, 22.90, 22.56, 22.33, 22.23,
22.15, 20.84; MS (ESI) m/z: 614.6 [M + H]+, 631.5 [M + NH4]+, 636.6 [M + Na]+.

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Compound 11 cyclo(D-Phe-D-Leu-Leu-N-Me-Leu-D-Leu). Yield: 50.4%, Wt: 39.2 mg, white powder; 1H
NMR (300 MHz, Chloroform-d) ppm: 8.35–8.13 (m, 1 H), 7.48 (d, J = 8.8 Hz, 1 H), 7.31–7.13 (m, 5 H),
δ
6.90 (d, J = 9.8 Hz, 1 H), 6.24 (d, J = 6.8 Hz, 1 H), 4.98 (t, J = 7.2 Hz, 1 H), 4.91–4.75 (m, 1 H), 4.48 (td,
J = 9.3, 6.2 Hz, 1 H), 4.31–4.08 (m, 2 H), 3.27 (d, J = 10.0 Hz, 1 H), 3.18 (dd, J = 13.6, 7.0 Hz, 1 H), 2.60 (d,
J = 1.5 Hz, 3 H), 1.90–1.74 (m, 2 H), 1.60–1.27 (m, 10 H), 1.00–0.79 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ
ppm: 174.32, 171.97, 171.24, 170.97, 170.20, 136.61, 128.83(2C), 128.76(2C), 126.98, 56.23, 53.89(2C),
51.46, 48.17, 41.41, 40.69, 40.63, 34.75, 34.62, 29.44, 24.86, 24.78(3C), 23.22, 22.77, 22.75, 22.70, 22.63,
22.29, 21.95, 21.04; MS (ESI) m/z: 614.6 [M + H]+, 631.6 [M + NH4]+, 636.4 [M + Na]+.
Compound 12 cyclo(D-Phe-D-Leu-D-Leu-N-Me-Leu-Leu). Yield: 46.1%, Wt: 35.8 mg, white powder; 1H
NMR (300 MHz, Chloroform-d)
δ ppm: 7.36–7.17 (m, 6 H), 7.10 (s, 1 H), 6.96 (d, J = 10.3 Hz, 2 H), 5.06
(dd, J = 10.6, 5.3 Hz, 1 H), 4.87 (q, J = 7.5, 7.1 Hz, 1H), 4.72 (dd, J = 17.3, 9.2 Hz, 1 H), 4.40 (q, J = 7.8 Hz,
1 H), 3.84 (q, J = 8.3, 7.7 Hz, 1 H), 3.22–3.14 (m, 1H), 3.03 (s, 3 H), 3.00–2.95 (m, 1 H), 1.86–1.40 (m, 12 H),
1.02–0.73 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 174.15, 172.69, 172.31, 171.75, 171.41, 136.65,
129.19(2C), 128.50(2C), 126.79, 56.98, 55.94, 55.11, 51.92, 48.67, 41.08, 39.72, 38.69, 36.65, 36.44, 30.74,
25.33, 24.84(2C), 24.74, 23.20, 23.05, 22.97, 22.89, 22.38, 22.24, 21.59, 21.27; MS (ESI) m/z: 614.5 [M + H]+,
631.5 [M + NH4]+, 636.6 [M + Na]+.
Compound 13 cyclo(D-Phe-D-Leu-D-Leu-N-Me-Leu-Leu). Yield: 50.3%, Wt: 39.1 mg, white powder; 1H
NMR (300 MHz, Chloroform-d) δ ppm: 7.49 (d, J = 8.6 Hz, 1 H), 7.23 (q, J = 7.2, 6.7 Hz, 5 H), 6.96 (dd,
J = 8.8, 2.4 Hz, 1 H), 6.76 (t, J = 9.5 Hz, 1 H), 6.57 (dd, J = 9.7, 5.1 Hz, 1 H), 5.17 (dd, J = 11.8, 4.3 Hz, 1 H),
4.76 (q, J = 7.9 Hz, 1 H), 4.64 (q, J = 6.9 Hz, 1 H), 4.57–4.43 (m, 2 H), 3.28–3.19 (m, 1 H), 3.14 (s, 3 H),
2.96–2.85 (m, 1 H), 1.94 (dtt, J = 34.9, 9.8, 5.1 Hz, 2 H), 1.76–1.46 (m, 10 H), 1.03–0.78 (m, 24 H); 13C NMR
(75 MHz, Chloroform-d) δ ppm: 173.48, 172.91, 171.04, 170.58, 170.42, 135.73, 129.00(2C), 128.87(2C),
127.44, 56.90, 54.39, 51.83, 51.27, 48.20, 41.22, 40.82, 39.68, 37.04, 35.27, 30.20, 25.09, 24.76(2C), 24.72,
23.23, 22.77, 22.73, 22.59, 22.41, 22.33, 22.30, 21.42; MS (ESI) m/z: 614.4 [M + H]+, 631.4 [M + NH4]+,
636.6 [M + Na]+.
Compound 14 cyclo(D-Phe-D-Leu-D-Leu-N-Me-Leu-D-Leu). Yield: 55.5%, Wt: 43.1 mg, white powder; 1H
NMR (300 MHz, Chloroform-d)
δ ppm; 8.28 (q, J = 9.0, 8.3 Hz, 1 H), 7.48 (d, J = 8.7 Hz, 1 H), 7.31–7.16
(m, 5 H), 6.89 (d, J = 9.8 Hz, 1 H), 6.23 (d, J = 6.9 Hz, 1 H), 4.98 (t, J = 7.1 Hz, 1 H), 4.85 (q, J = 7.4 Hz,
1 H), 4.55–4.40 (m, 1 H), 4.32–4.08 (m, 2 H), 3.32–3.12 (m, 2H), 2.59 (s, 3 H), 1.83 (ddq, J = 18.3, 9.8, 5.1,
4.0 Hz, 2 H), 1.64–1.31 (m, 10 H), 1.00–0.78 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 174.33, 171.97,
171.23, 170.98, 170.18, 136.63, 128.84(2C), 128.74(2C), 126.96, 56.20, 53.87(2C), 51.45, 48.17, 41.41, 40.70,
40.63, 34.74, 34.62, 29.43, 24.85(2C), 24.78(2C), 23.22, 22.77, 22.74, 22.70, 22.62, 22.29, 21.95, 21.04; MS
(ESI) m/z: 614.5 [M + H]+, 631.4 [M + NH4]+, 636.3 [M + Na]+.
Compound 15 cyclo(Phe-Leu-Leu-D-N-Me-Leu-Leu). Yield: 49.2%, Wt: 38.2 mg, white powder; 1H NMR
(300 MHz, Chloroform-d) δ ppm: 7.98 (t, J = 7.6 Hz, 2 H), 7.77 (d, J = 6.2 Hz, 1 H), 7.25 (q, J = 8.6 Hz,
5 H), 6.83 (d, J = 6.6 Hz, 1 H), 5.06 (dd, J = 10.9, 5.2 Hz, 1 H), 4.81 (q, J = 7.4 Hz, 1 H), 4.59 (q, J = 7.6 Hz,
1 H), 4.48 (q, J = 7.1, 6.7 Hz, 1 H), 3.72 (dt, J = 11.4, 6.3 Hz, 1 H), 3.31 (d, J = 6.0 Hz, 1 H), 3.23 (s, 3 H),
3.05 (dd, J = 13.1, 9.5 Hz, 1 H), 2.13 (td, J = 12.3, 10.4, 4.9 Hz, 1 H), 2.00–1.85 (m, 1 H), 1.78–1.32 (m,
10 H), 1.06–0.81 (m, 21 H), 0.68 (d, J = 6.5 Hz, 3 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 174.28, 172.06,
171.23, 170.90, 170.23, 136.56, 128.82(2C), 128.78(2C), 127.02, 56.44, 53.98, 51.46, 50.86, 48.15, 41.39, 40.66,
40.59, 34.64, 34.78, 29.49, 24.87, 24.80, 24.78(2C), 23.22, 22.77(2C), 22.69, 22.63, 22.29, 21.95, 21.06; MS
(ESI) m/z: 614.5 [M + H]+, 631.5 [M + NH4]+, 636.4 [M + Na]+.
Compound 16 cyclo(Phe-D-Leu-Leu-D-N-Me-Leu-Leu). Yield: 53.3%, Wt: 41.4 mg, white powder; 1H NMR
(300 MHz, Chloroform-d) δ ppm: 7.32 (d, J = 9.4 Hz, 1 H), 7.26–7.20 (m, 5 H), 7.11 (d, J = 7.2 Hz, 1 H),
6.66–6.53 (m, 2 H), 4.96 (td, J = 8.7, 5.7 Hz, 1 H), 4.71 (t, J = 7.8 Hz, 1 H), 4.45 (dtt, J = 13.7, 9.3, 4.5 Hz,
3 H), 3.20–3.10 (m, 2 H), 3.08 (s, 3 H), 1.77–1.30 (m, 12 H), 1.02–0.89 (m, 18 H), 0.82 (d, J = 6.5 Hz, 3 H),
0.77 (d, J = 6.4 Hz, 3 H); 13C NMR (75 MHz, CDCl3)
δ
ppm: 174.74, 172.19, 171.22, 171.01, 170.72,
136.96, 129.33(2C), 128.42(2C), 126.67, 55.95, 55.76, 52.63, 50.72, 47.93, 41.79, 41.33, 37.60, 37.10, 36.77,

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30.93, 25.10, 25.05, 24.84, 24.42, 23.02, 22.97, 22.90, 22.58, 22.49, 22.18, 21.82, 21.50; MS (ESI) m/z: 614.6
[M + H]+, 631.6 [M + NH4]+, 636.5 [M + Na]+.
Compound 17 cyclo(D-Phe-Leu-Leu-D-N-Me-Leu-Leu). Yield: 56.4%, Wt: 43.8 mg, white powder; 1H NMR
(300 MHz, Chloroform-d)
δ ppm: 8.02 (d, J = 7.8 Hz, 1 H), 7.33–7.14 (m, 6 H), 7.08 (dd, J = 14.4, 9.8 Hz,
2 H), 5.04 (t, J = 7.6 Hz, 1 H), 4.83 (dt, J = 9.3, 6.8 Hz, 1 H), 4.61–4.46 (m, 1 H), 4.26 (dt, J = 10.5, 6.2 Hz,
1 H), 4.06 (ddd, J = 11.1, 7.1, 3.6 Hz, 1 H), 3.18–3.12 (m, 1 H), 3.02 (d, J = 6.0 Hz, 1 H), 2.75 (s, 3 H),
1.90–1.20 (m, 12 H), 0.90 (dt, J = 11.6, 6.4 Hz, 18 H), 0.74 (d, J = 6.5 Hz, 3 H), 0.64 (d, J = 6.2 Hz, 3 H); 13
C
NMR (75 MHz, CDCl3)
δ ppm: 173.77, 172.03, 171.55, 171.21, 170.21, 135.91, 129.03(2C), 128.69(2C),
127.04, 56.94, 53.95, 53.19, 51.00, 47.90, 41.47, 40.53, 40.24, 36.97, 34.87, 29.53, 24.93, 24.82, 24.69, 24.32,
23.10, 22.81, 22.74, 22.73, 22.61, 22.20, 22.10, 21.08; MS (ESI) m/z: 614.8 [M + H]+, 631.7 [M + NH4]+,
636.6 [M + Na]+.
Compound 18 cyclo(D-Phe-D-Leu-Leu-D-N-Me-Leu-Leu). Yield: 56.2%, Wt: 43.7 mg, white powder; 1H
NMR (300 MHz, Chloroform-d)
δ ppm: 7.49 (d, J = 8.9 Hz, 1 H), 7.24 (q, J = 6.9 Hz, 6 H), 7.12 (d,
J = 7.0 Hz, 1 H), 6.81–6.65 (m, 1 H), 4.96 (t, J = 8.0 Hz, 1 H), 4.81 (t, J = 8.0 Hz, 2 H), 4.51 (td, J = 9.3,
4.1 Hz, 1 H), 4.26 (q, J = 7.5 Hz, 1 H), 3.23 (dd, J = 14.3, 6.2 Hz, 1 H), 3.06 (s, 3 H), 2.95 (dd, J = 14.3,
9.1 Hz, 1 H), 1.74–1.34 (m, 12 H), 1.01–0.73 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 173.50, 173.47,
172.91, 171.04, 170.42, 135.72, 129.00(2C), 128.87(2C), 127.44, 56.93, 54.38, 51.81, 51.30, 48.21, 41.22, 40.80,
39.68, 37.02, 35.25, 30.20, 25.08, 24.75(2C), 24.72, 23.24, 22.78, 22.73, 22.59, 22.41, 22.33, 22.29, 21.41; MS
(ESI) m/z: 614.6 [M + H]+, 631.6 [M + NH4]+, 636.5 [M + Na]+.
Compound 19 cyclo(D-Phe-D-N-Me-Leu-Leu-N-Me-Leu-Leu). Yield: 45.5%, Wt: 35.4 mg, white powder; 1H
NMR (300 MHz, Chloroform-d)
δ ppm: 7.28–7.14 (m, 5 H), 6.88 (d, J = 8.6 Hz, 1 H), 6.82 (d, J = 7.3 Hz,
1 H), 6.54 (d, J = 9.6 Hz, 1 H), 5.30–5.02 (m, 2 H), 4.67 (q, J = 7.8, 7.3 Hz, 1 H), 4.45 (dt, J = 8.0, 3.8 Hz,
1 H), 3.43 (dd, J = 8.9, 4.5 Hz, 1 H), 3.32 (dt, J = 10.7, 5.3 Hz, 1 H), 3.08 (s, 3H), 2.93 (s, 3 H), 2.88 (d,
J = 6.4 Hz, 1 H), 1.74–1.38 (m, 12 H), 0.99-0.74 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 173.91,
173.12, 172.52, 171.48, 170.89, 136.81, 129.32(2C), 128.36(2C), 126.69, 66.77, 56.19, 51.69, 51.28, 48.48,
40.36, 40.19, 40.02, 38.14, 37.46, 36.86, 30.86, 25.30, 25.15, 24.87, 24.83, 23.40, 23.31, 23.00, 22.90, 22.22,
22.12, 21.47, 21.17; MS (ESI) m/z: 628.7 [M + H]+, 645.9 [M + NH4]+, 650.9 [M + Na]+.
Compound 20 cyclo(Phe-Leu-Leu-D-Leu-N-Me-Leu). Yield: 46.1%, Wt: 35.8 mg, white powder; 1H NMR
(300 MHz, Chloroform-d) δ ppm: 7.95 (t, J = 7.9 Hz, 2 H), 7.73 (d, J = 6.3 Hz, 1 H), 7.33–7.17 (m, 5 H),
6.88 (d, J = 6.8 Hz, 1 H), 5.05 (dd, J = 10.8, 5.2 Hz, 1 H), 4.80 (d, J = 7.3 Hz, 1 H), 4.59 (s, 1 H), 3.71 (s,
1 H), 3.35–3.25 (m, 1 H), 3.23 (s, 3 H), 3.08 (dd, J = 13.2, 9.2 Hz, 1 H), 2.12 (ddd, J = 13.7, 10.2, 5.2 Hz,
1 H), 1.97–1.84 (m, 1 H), 1.58 (dt, J = 15.4, 4.4 Hz, 7 H), 1.21–1.05 (m, 3 H), 1.08–0.66 (m, 24 H); 13C NMR
(75 MHz, Chloroform-d) δ ppm: 174.72, 173.98, 173.17, 172.32, 170.71, 137.12, 129.43(2C), 128.28(2C),
126.52, 58.33, 56.93, 56.41, 51.84, 48.37, 41.77, 40.25, 38.27, 37.05, 36.83, 31.06, 25.18, 25.09, 25.02, 24.66,
23.17, 22.91, 22.84, 22.74, 22.54, 22.37, 21.39, 21.30; MS (ESI) m/z: 614.7 [M + H]+, 631.7 [M + NH4]+,
636.6 [M + Na]+.
Compound 21 cyclo(Phe-D-Leu-D-Leu-D-Leu-N-Me-Leu). Yield: 55.1%, Wt: 42.8 mg, white powder; 1H
NMR (300 MHz, Chloroform-d)
δ ppm: 7.43 (d, J = 8.8 Hz, 1 H), 7.30–7.19 (m, 6 H), 7.06 (s, 1 H), 6.89 (d,
J = 8.5 Hz, 1 H), 4.90–4.79 (m, 1 H), 4.71 (t, J = 7.7 Hz, 1 H), 4.35 (d, J = 7.7 Hz, 2 H), 4.11 (d, J = 9.8 Hz,
1 H), 3.32–3.18 (m, 2 H), 3.06 (s, 3 H), 1.65–1.43 (m, 12 H), 1.00–0.86 (m, 24 H); 13C NMR (75 MHz,
CDCl3)
δ ppm: 174.50, 172.67, 172.53, 171.60, 171.40, 137.01, 129.40(2C), 128.36(2C), 126.71, 57.63, 55.68,
54.29, 52.91, 48.48, 41.39, 39.36, 39.17, 37.22, 36.89, 30.94, 25.26, 25.24, 24.87, 24.77, 23.02, 22.97, 22.94,
22.80, 22.44, 21.99, 21.85, 21.31; MS (ESI) m/z: 614.7 [M + H]+, 631.6 [M + NH4]+, 636.6 [M + Na]+.
Compound 22 cyclo(D-Phe-Leu-Leu-D-Leu-N-Me-Leu). Yield: 65.4%, Wt: 50.8 mg, white powder; 1H NMR
(300 MHz, Chloroform-d) δ ppm: 7.65–7.45 (m, 1 H), 7.40 (d, J = 8.7 Hz, 1 H), 7.26 (dt, J = 15.9, 6.0 Hz,
5H), 6.96–6.81 (m, 1 H), 6.74 (s, 1 H), 5.18 (t, J = 7.6 Hz, 1 H), 4.81 (q, J = 7.4 Hz, 1 H), 4.71 (d, J = 7.3 Hz,
1 H), 4.51 (td, J = 9.2, 5.2 Hz, 1 H), 3.88 (dt, J = 9.9, 5.0 Hz, 1 H), 3.13 (dd, J = 13.3, 5.8 Hz, 1 H), 2.99 (s,
1 H), 2.93 (s, 3 H), 1.89–1.12 (m, 12 H), 1.06–0.68 (m, 24 H); 13C NMR (75 MHz, CDCl3) δ ppm: 173.38,

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172.16, 172.01, 171.98, 170.05, 136.36, 129.22(2C), 128.68(2C), 127.03, 55.08, 54.31, 54.14, 50.99, 48.26,
40.57, 40.43, 40.05, 39.80, 35.11, 30.12, 24.98, 24.97, 24.74, 24.48, 23.17, 22.87, 22.80(2C), 22.50, 22.35,
21.70, 21.57; MS (ESI) m/z: 614.6 [M + H]+, 631.6 [M + NH4]+, 636.5 [M + Na]+.
Compound 23 cyclo(D-Phe-Leu-D-Leu-Leu-N-Me-Leu). Yield: 48.5%, Wt: 37.7 mg, white powder; 1H NMR
(300 MHz, Chloroform-d) δ ppm: 7.98 (t, J = 7.6 Hz, 1 H), 7.77 (d, J = 6.2 Hz, 1 H), 7.25 (q, J = 8.5 Hz,
6 H), 6.83 (d, J = 6.6 Hz, 1 H), 5.06 (dd, J = 10.9, 5.2 Hz, 1 H), 4.81 (q, J = 7.4 Hz, 1H), 4.60 (t, J = 7.8 Hz,
1 H), 4.48 (q, J = 7.1, 6.7 Hz, 1 H), 3.72 (dt, J = 11.4, 6.3 Hz, 1 H), 3.31 (d, J = 6.0 Hz, 1 H), 3.27 (s, 3H),
3.05 (dd, J = 13.1, 9.5 Hz, 1 H), 2.21–1.75 (m, 3 H), 1.73–1.36 (m, 9 H), 1.01–0.63 (m, 24 H); 13C NMR
(75 MHz, CDCl3) δ ppm: 174.59, 173.88, 173.14, 172.31, 170.65, 137.14, 129.43(2C), 128.26(2C), 126.49,
58.39, 57.10, 56.35, 51.83, 48.26, 41.98, 40.31, 38.23, 37.11, 36.87, 31.02, 25.18, 25.09, 25.03, 24.60, 23.19,
22.93, 22.87, 22.73, 22.55, 22.40, 21.34(2C), ; MS (ESI) m/z: 614.7 [M + H]+, 631.7 [M + NH4]+, 636.6
[M + Na]+.
Compound 24 cyclo(Phe-Leu-Leu-Leu-D-N-Me-Leu). Yield: 66.4%, Wt: 51.6 mg, white powder; 1H NMR
(300 MHz, Chloroform-d) δ ppm: 8.55 (d, J = 7.2 Hz, 1 H), 7.54 (d, J = 8.9 Hz, 1 H), 7.35–7.12 (m, 7 H),
5.07 (t, J = 7.6 Hz, 1 H), 4.85 (td, J = 11.7, 10.3, 7.1 Hz, 2 H), 4.17 (p, J = 5.5, 4.6 Hz, 1 H), 3.68 (td, J = 9.5,
8.0, 4.2 Hz, 1 H), 2.98 (qd, J = 13.7, 8.0 Hz, 2 H), 2.72 (s, 3 H), 1.84 (tt, J = 11.5, 5.8 Hz, 3 H), 1.53 (dtd,
J = 35.9, 13.2, 5.7 Hz, 9 H), 0.97–0.71 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 172.73, 172.14,
171.84, 171.24, 170.36, 136.19, 129.17(2C), 128.44(2C), 126.80, 53.96, 53.94, 53.86, 53.69, 48.13, 41.37, 40.92,
38.15, 37.26, 34.65, 29.39, 25.18, 24.75, 24.68, 24.21, 23.58, 23.13, 22.84, 22.73, 22.66, 22.19, 21.34, 20.96;
MS (ESI) m/z: 614.6 [M + H]+, 631.6 [M + NH4]+, 636.6 [M + Na]+.
Compound 25 cyclo(D-Phe-Leu-D-Leu-D-Leu-D-N-Me-Leu). Yield: 50.0%, Wt: 38.8 mg, white powder; 1H
NMR (300 MHz, Chloroform-d)
δ ppm: 7.60 (d, J = 6.2 Hz, 1 H), 7.32–7.18 (m, 6 H), 7.06 (d, J = 8.7 Hz,
1 H), 6.52 (d, J = 9.4 Hz, 1 H), 6.32 (d, J = 9.1 Hz, 1H), 5.08 (dd, J = 11.8, 4.3 Hz, 1 H), 4.85 (q, J = 7.9 Hz,
1 H), 4.55 (p, J = 8.5, 7.5 Hz, 3 H), 3.40 (dd, J = 13.8, 7.1 Hz, 1 H), 3.02 (s, 3 H), 2.97 (d, J = 7.9 Hz, 1 H),
1.94–1.26 (m, 12 H), 0.91 (ddt, J = 29.8, 10.5, 5.4 Hz, 24 H); 13C NMR (75 MHz, CDCl3)
δ ppm: 175.43,
174.81, 172.61, 171.77, 170.37, 137.61, 129.59(2C), 128.18(2C), 126.46, 56.65, 53.77, 51.76, 50.67, 49.26,
40.43, 39.64, 37.24, 37.06, 36.29, 31.04, 25.22, 24.88(2C), 24.55, 23.33, 22.86, 22.76, 22.69, 22.33, 22.20,
22.09, 20.75; MS (ESI) m/z: 614.6 [M + H]+, 631.6 [M + NH4]+, 636.5 [M + Na]+.
Compound 26 cyclo(D-Phe-N-Me-Leu-Leu-Leu-D-N-Me-Leu) Yield: 45.1%, Wt: 35.1 mg, white powder; 1H
NMR (300 MHz, Chloroform-d)
δ ppm: 7.29–7.17 (m, 6 H), 6.88 (d, J = 8.4 Hz, 1 H), 6.66 (d, J = 9.5 Hz,
1 H), 5.30 (dd, J = 11.5, 4.4 Hz, 1 H), 5.13 (td, J = 10.3, 5.0 Hz, 1 H), 4.57 (dq, J = 22.5, 7.4, 7.0 Hz, 2 H),
3.35 (dd, J = 10.9, 4.5 Hz, 1 H), 3.09 (s, 3 H), 3.05–3.00 (m, 1 H), 2.98 (s, 3 H), 2.91–2.87 (m, 1 H), 2.33–2.19
(m, 1 H), 2.07–1.86 (m, 2 H), 1.79–1.37 (m, 9 H), 0.99–0.78 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ ppm:
173.84, 172.88, 171.50, 170.82, 170.31, 137.09, 129.69(2C), 128.29(2C), 126.67, 56.15, 55.53, 55.12, 51.89,
48.53, 40.24, 37.86, 37.77, 36.17, 35.43, 30.92, 25.18, 25.10, 24.97, 24.85, 23.38, 23.02, 22.79, 22.75, 22.62,
22.13, 21.58, 20.81; MS (ESI) m/z: 628.7 [M + H]+, 645.8 [M + NH4]+, 650.7 [M + Na]+.
4.6. In Vitro Anti-Tumor Activity
4.6.1. Maintenance of Cell Culture
Lung cancer A549, drug-resistant lung cancer A549-DPP, human hepatocellular carcinoma HepG2
and hepatocellular carcinoma SMMC-7721 cells obtained from the China cell bank of the Institute of
Biochemistry and were cultured in DMEM culture medium (DMEM, Corning, NY, USA) containing
10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 1% penicillin-streptomycin and an antifungal
agent in a 5% CO2 humidified atmosphere at 37 ◦C. The culture medium was replaced once in a day.
Trypsin digestion method was adopted for cell propagation. Upon reaching 80%–90% confluence,
the cells were rinsed twice with PBS. A certain amount of 0.25% trypsin digestion solution was then
added and maintained for 3–5 min at 37 ◦C. Afterward, DMEM culture medium containing 10% fetal

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bovine serum was added to terminate the digestion. The cells were then blown well to form single
cell suspensions.
4.6.2. MTT Assay
The cell viability was determined by measuring the ability of cells to transform MTT (Genview,
Houston, TX, USA) to a purple formazan dye. The cells were seeded in 96-well tissue culture plates
at 2.5
for 48 h. After incubation, 20
added and incubated for 5 h. The medium was aspirated and replaced with 100
to dissolve the formazan salt. The color intensity of the formazan solution, which reflects the cell
×
103 cells/well for 24 h. The cells were then incubated with galaxamide and its analogues
µL/well of MTT solution (5 mg/mL phosphate buffered saline) were
µL/well of DMSO
growth condition, was measured at 570 nm using a microplate spectrophotometer (SpectroAma
Winooski, VT, USA).
™ 250,
4.6.3. Cell Apoptotic Analysis
The apoptotic cells were quantified using an Annexin V-FITC (Sigma-Aldrich, St. Louis, MO,
USA) cell apoptosis assay kit according to instruction provided with kit. In brief, about 1.5
105 cells
were plated in 6-well plates and treated with galaxamide and representative analogues (4, 8, 16 g/mL)
×
µ
for 48 h. The cells were resuspended in 200 mL binding buffer. Afterward, 5 mL annexin V-fluorescein
isothiocyanate (FITC) was added and then incubated in darkness at room temperature for 10 min.
The cells were again resuspended in 200 mL binding buffer and stained with 5 mL PI. The prepared
cells were then analyzed using a flow cytometry (Coulter Epics Elite, Miami, FL, USA). The cells in the
FITC-positive and PI-negative fraction were regarded as apoptotic cells.
4.6.4. Cell Morphological Observation
Exponentially growing SMMC-7721 cells (1
µg/mL of galaxamide and representative analogues. Apoptotic nuclear morphology was visualized
×
105 cells/mL) were incubated for 48 h with 4, 8,
16
by the Hoechsst33342 staining technique. Cells were fixed with 3.7% of paraformaldehyde for 10 min
at room temperature and washed three times with PBS. After fixation, cells were stained using
Hoechsst33342 (10 µg/mL) and incubated in the darkness for 10 min. After washing three times with
PBS, cells were visualized using fluorescence microscope.
4.6.5. Cell Cycle Analysis
SMMC-7721 cells with the density of about 1.5
×
105 were incubated with galaxamide and
representative analogues (4, 8, 16 g/mL) for 48 h. Afterwards, collected cells were washed twice with
µ
PBS and centrifugation (1000 rpm, 5 min), then fixed using 70% ethanol for 12 h at 4 ◦C. Ethanol was
removed by centrifugation (2000 rpm, 5 min), and the cells were washed twice with PBS. Cells were
then resuspended in 200 mL PI and kept at 37 ◦C for 15 min. The cell cycle was analyzed by flow
cytometry measuring the amount of PI-labeled DNA in fixed cells.
4.7. Statistical Analysis
Experimental data were expressed as the mean
±
SD from three independent experiments.
The experimental results were analyzed statistically using SPSS software. The statistically significant
differences were analyzed using Tukey’s test. p values < 0.05 were considered statistically significant,
and p < 0.01 considered extremely significant.
5. Conclusions
In conclusion, we have demonstrated robust parallel solid phase strategy for the synthesis
of galaxamide analogues, and synthesized 26 novel compounds with change of L aminoacid to D
enantiomeric aminoacid, changing the position of N-Me aminoacid residue and substitute one Leu to

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D/L Phe aminoacid residue with respect to parent structure. All compounds were evaluated in vitro
anti-tumor activity using MTT assay. The thorough SAR exploration provides insight of structural
motif and cytotoxicity correlation, and importance of N-Me at 3 position with this template compound.
In particular, Compound
1 exhibited promising activity to be a potential lead compound against
SMMC-7721 cancer cell line which was 9 and 10 fold higher than galaxamide and current cancer drug
DPP, respectively. Galaxamide analogues arrest the cell cycle in the G0/G1 phase and induce apoptosis,
which confirm with representative Compound 6, 8 and 22. Synthesis of next generation compound
utilizing information described here will be of great help to understand the clear SAR and find future
lead for the cancer.
Acknowledgments: This research work was financially supported by the general projects of the National Natural
Science Foundation (No. 21672084, 21172094, 41376155, 21372100, 31201028) and Cultivation Foundation of
Jinan University (21616113).
Author Contributions: Shihai Xu, Jignesh Lunagariya and Xiaojian Liao conceived and designed the experiments;
Shenghui Zhong, Jianwei Chen and Weili Long performed the synthesis experiments; Poonam Bhadja and Defa Bai
performed biological experiments; Jignesh Lunagariya, Shihai Xu and Xiaoli Tang wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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