Synthesis of Amino Acid-comprising Sialyltransferase Inhibitors and Their Antimetastatic Activities against Human Breast Cancer Cells

Article abstract of DOI:10.1002/jccs.201500348

The amino acid-containing lithocholic acids (LCA) represent a new class of human sialyltransferase (ST) inhibitors. In this study, we have reported their design, synthesis, and inhibitory activity against human STs. Among these derivatives, D-Glu-LCA 7, L-Asp-L-Asp-LCA 13, and L-Asp-L-Asp-Gly-Gly-LCA 22 with specific amino acid sequence were the most active ones with IC₅₀ values of 2.3-5.6 and 4.2-6.2 μM toward α-2,3-ST and α-2,6-ST, respectively. The current study demonstrates that the new class of ST in-hibitors inhibit cell migration in breast cancer cells by preventing closure of the wound rather than involv-ing a direct antiproliferative effect.

Full text of DOI:10.1002/jccs.201500348

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis of Amino Acid-comprising Sialyltransferase Inhibitors and Their
Antimetastatic Activities against Human Breast Cancer Cells
Chih-Wei Fu,a,b Kai-Hsuan Chang,a Ya Ching Jen,a Tzu Ting Changa and Wen-Shan Lia,c*
aInstitute of Chemistry, Academia Sinica, Taipei 115, Taiwan
bDepartment of Chemistry, National Central University, Jhong-Li 320, Taiwan
cDoctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
(Received: Aug. 26, 2015; Accepted: Oct. 11, 2015; Published Online: Dec. 14, 2015; DOI: 10.1002/jccs.201500348)
The amino acid-containing lithocholic acids (LCA) represent a new class of human sialyltransferase (ST)
inhibitors. In this study, we have reported their design, synthesis, and inhibitory activity against human
STs. Among these derivatives, D-Glu-LCA 7, L-Asp-L-Asp-LCA 13, and L-Asp-L-Asp-Gly-Gly-LCA 22
with specific amino acid sequence were the most active ones with IC50 values of 2.3-5.6 and 4.2-6.2 mM
toward a-2,3-ST and a-2,6-ST, respectively. The current study demonstrates that the new class of ST in-
hibitors inhibit cell migration in breast cancer cells by preventing closure of the wound rather than involv-
ing a direct antiproliferative effect.
Keywords: Amino acid-containing lithocholic acids; Antiproliferative effect; Cell migration;
Human sialyltransferase inhibitors; Wound healing.
INTRODUCTION
Cancer is the major diseases that awfully threaten the
ages (Scheme 1).7 Recent studies have showed that hyper-
sialylation of b1 integrins through ST6Gal-I promotes can-
cer progression by up-regulating cell motility in vitro,8 then
the deficiency of hypersialylation promotes carcinoma dif-
ferentiation in vivo.9 ST3Gal-I is acting as a tumor pro-
moter in breast cancer and exerts its effect in tumor devel-
opment in transgenic mice model.10 ST6GalNAcV can me-
diate breast cancer metastasis to the brain.11 Accordingly,
there has been some development in using inhibition of ST
functions as biological tools and perhaps therapeutics.12
Therefore, the design of effective ST inhibitors would be
highly desirable.
quality of human life and metastasis is the leading cause of
cancer-related deaths.1 Metastasis is the biologically com-
plicated process including adhesion, degradation and mi-
gration of tumor cells from their initial site to the secondary
organ.2 Collapse at any of these steps could block the entire
metastatic course and, as a result, could serve as a therapeu-
tic strategy for the treatment of cancer metastasis.3 Devel-
opment of such therapeutic agents that inhibit tumor metas-
tasis is highly attractive. However, study about the anti-
metastatic agents is very rare and limited.4
Hypersialylation at the non-reducing end of glyco-
proteins or glycolipids plays an important role in cellular
interactions such as cell-cell adhesion, tumor cell metasta-
sis and invasions.5 Modifications of sialylation in vivo are
mediated and regulated by various glycoprotein- and
glycolipid-specific sialyltransferases (STs).6 STs belong to
a family of sialic acid-dependent glycosyltransferases and
participate in the biosynthesis of sialylated glycocon-
jugates by catalyzing the transfer of the N-acetylneur-
aminic acid (NeuAc) portion of cytidine monophos-
phate-N-acetylneuraminic acid (CMP-Neu5Ac) with in-
version of configuration to acceptor hydroxyl groups at or
near the nonreducing terminus of oligosaccharide chains of
glycoproteins and glycolipids via a2-3, a2-6, or a2-8 link-
Proposed mechanism of sialyltransferase-
mediated sialylation
Scheme 1
* Corresponding author. Tel: +886-2-27898662; Fax: +886-2-27831237; Email: wenshan@gate.sinica.edu.tw
Supporting information for this article is available on the www under http://dx.doi.org/10.1002/jccs.201500348
J. Chin. Chem. Soc. 2016, 63, 171-180
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Fu et al.
Structures of LCA, L-Asp-LCA (1), D-Asp-
LCA(2) and a series of amino acid conjugates
of LCA used in this study
Chart 2
Pioneering studies on ST inhibitors have provided es-
sential information regarding the development of core
skeleton based on mimetic of transition-state analogues,
bisubstrate analogues, donor analogues, and acceptor ana-
logues (Chart 1).13 However, the lack of membrane perme-
ability found in these inhibitors has hampered their biologi-
cal and clinical applications. Instead, our studies have re-
vealed the importance of ST inhibitors, lithocholic acid
(LCA) derivatives,14 with practical pharmaceutical appli-
cations. For example, we have shown that AL10, 7-nitro-
2,1,3-benzoxadiazole analogue of L-Glu-LCA, signifi-
cantly suppresses lung cancer metastasis in vivo without
affecting liver and kidney functions of experimental ani-
mals.15 Upon treatment of 4T1 cell animal model with ST
inhibitor, L-Asp-LCA (1), results demonstrated that ST in-
hibitor-treated mice effectively delayed lung tumor metas-
tasis/colonization.16 Furthermore, inhibition of chemokine
(C-C motif) receptor 7 sialylation appears to be the action
for suppressing CCL19-stimulated proliferation, invasion
and anti-anoikis.17
Interestingly, cell-permeable property appeared to be pre-
sented for LCA analogues, such as L-Asp-LCA (1) and
D-Asp-LCA (2). Despite current efforts, however, other
amino acid conjugates of LCA and their biological func-
tions toward ST activity and cancer metastasis have not
been explored. We reasoned that a hit library of this type
should first inactivate ST function. Therefore, enzyme ac-
tivity-based strategy and lead optimization approach were
employed by using the results of prior round of screening to
generate four amino acid sequence patterns of ST inhibitors
sequentially, monopeptide-LCAs (R1-LCAs, 3-8), dipep-
tide-LCAs (R2-R1-LCAs, 9-17), tripeptide-LCAs (R3-R2-
R1-LCAs, 18-21), and tetrapeptide-LCAs (R4-R3-R2-R1-
LCAs, 22-23).
Examples of some of the sialyltransferase in-
hibitors
Chart 1
All peptides attached to LCA were synthesized on so-
lution phase utilizing Fmoc- and Boc-amino acid building
blocks. The coupling and activating reagents, dicyclo-
hexylcarbodiimide (DCC), ethyl-(N’,N’-dimethylamino)
propylcarbodiimide hydrochloride (EDC) and N-hydro-
xybenzotriazole (HOBt), in dichloromethane (DCM) or
N,N-dimethylformamide (DMF) were used, respectively.
Scheme 2 illustrates the preparation of peptidyl LCA con-
jugates (3-8, 9-17, 18-21 and 22-23). The monopeptide-
LCAs 3, 5, 6 and 8 were obtained by condensing the corre-
sponding Fmoc- and Boc-amino acid, Fmoc-Gly-OH,
Fmoc-Pro-OH, Fmoc-L-Glu(OBut)-OH and Boc-L-Glu-
OBut, with 24 using DCC as coupling agent, followed by
alkaline hydrolysis of the Fmoc and acidolytic cleavage of
the remaining tBu or Boc groups. To prevent the side reac-
tions, hydroxyl group of Fmoc-Ser-OH was tert-butyl-
diphenylsilyl (TBDPS)-protected prior to coupling. Thus
reaction of Fmoc-Ser(OTBDPS)-OH with 24 followed by
treatment with tetrabutylammonium fluoride (n-Bu4NF)
In this study, we developed and synthesized a series
of amino acid conjugates of lithocholic acid (aa-O-LCA li-
brary; Chart 2), and studied their antimetastatic effects
against human breast cancer cell lines, MCF-7 and MDA-
MB231.
RESULTS AND DISCUSSION
Design and synthesis of amino acid conjugates of
lithocholic acid
LCA, which potentially mimics a pentacyclic ring of
Soyasaponin I,18 showed a promising inhibitory property
toward a-2,3-ST, indicating an acceptable pharmacophore.14
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Sialyltransferase Inhibitors and Their Antimetastatic Activities
afforded the amino alcohol which could be subsequently
converted to 4 using a similar deprotection procedure. To
prepare the D-form Glu-LCA 7, esterification of the sec-
ondary alcohol 24 using Boc-D-Glu(OAll)-OH was fol-
lowed by liberation of the d-carboxylic acid of D-Glu with
Pd(PPh3)4 in the presence of morpholine and subsequent
removal of tBu and Boc groups.
following the general peptide synthesis described in
Scheme 2, in which 24 was first reacted with Fmoc-L-
Asp(OBut)-OH using DCC/4-dimethylaminopyridine
(DMAP) as coupling agent, followed by alkaline-mediated
deprotection of Fmoc. The free amino group of protected
L-Asp-LCA was reacted with Boc-Glu-OBut in the pre-
sence of EDC/HOBt followed by removal of the remaining
Boc and tBu groups to yield 17. Similarly, the tripep-
tide-LCAs 18-21 and tetrapeptide-LCAs 22-23, developed
in this study, were accomplished by condensation of the
corresponding Fmoc-amino acids with 24 within yields
ranging from 7 to 26%. The peptide synthesis in this study
could have low yields, e.g., 4, 22 or others, and might suffer
from incorporation of lengthy protection/deprotection pro-
cedures as well as tedious HPLC separation of final prod-
ucts in some cases.
Preparation of peptidyl LCA analogues 3-
23. Reagents and conditions: a) Fmoc-amino
acid-OH, DCC, DMAP, DCM; b) DBU,
DCM; c) TFA, 2% H2O, RT; d) Fmoc-Ser
(OTBDPS)-OH, DCC, DMAP, DCM; e) n-
Bu4NF, HOAc, THF; f) Boc-D-Glu(OAll)-
OH, DCC, DMAP, DCM; g) Pd(PPh3)4,
morpholine, THF; h) Boc-Glu-OBut, DCC,
DMAP, DCM; i) Fmoc-amino acid-OH,
EDC, HOBt, DMF; j) Boc-Glu-OBut, EDC,
HOBt, DMF
Scheme 2
Inhibition of a-2,3-ST and a-2,6-ST by amino acid
conjugates of lithocholic acid
The inhibition assays of a-2,3-ST and a-2,6-ST were
performed following the general procedure of Schmidt et
al.,19 in which CMP-Neu5Ac was used as a substrate and
the modified disaccharide20 with a 4,5-dimethoxy-2-nitro-
benzyl group was employed as an UV-labelled acceptor
(Scheme 3). The resulting IC50 values of 1-23 are summa-
rized in Table 1. The presence of the specific amino acid
residue demonstrated to be essential for inhibitory potency.
Among the monopeptide-LCAs, L-Asp-LCA 1, D-Asp-
LCA 2, L-Glu-LCA 6 and D-Glu-LCA 7 exhibited a 1.2–
8-fold potency increase over 3-5 and 8, suggesting that
these four amino acids are important for promoting affinity
toward a-2,3-ST. Similarly, D-Glu-LCA 7 (IC50 = 6.2 mM)
exhibited a 2.5-fold potency increase over L-Asp-LCA 1,
indicating that the presence of a D-Glu substituent in LCA
leads to enhanced efficacy against a-2,6-ST.
a-2,3-ST and a-2,6-ST mediated sialyla-
Scheme 3
tions of UV-labelled acceptors
In addition, Scheme 2 illustrated the synthesis of di-
peptide-LCAs 9-16 from monopeptide-LCAs. As a repre-
sentative example, the assembly of dipeptide-LCA 10 was
performed by using Fmoc solution-phase peptide synthesis
strategy. Consecutively coupling of 24 with Fmoc-Gly-OH
and Fmoc-Asp(OBut)-OH to give protected Gly-Asp-
LCA. Dipeptide-LCA 10 was obtained in 35% yield after
the similar deprotection procedures as described for mono-
peptide-LCA. Conjugate dipeptide-LCA 17 was obtained
Of the 9 dipeptide-LCAs 9-17 designed for the
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LCAs 22-23. Incorporation of Gly or L-Asp at position R1
in tripeptide-LCAs 19 (IC50 = 20.4 mM) and 20 (IC50 = 31.6
mM), respectively, exhibited lower inhibitory potencies
against a-2,3-ST when compared to that of corresponding
dipeptide-LCA 13. The relative IC50 value (46.5 mM) of 20
show similar trend against a-2,6-ST, an observation that
indicates that the extra incorporation of Gly or L-Asp at po-
sition R1 is not useful for suppressing ST activity. Unfa-
vourable results were also observed for compounds 18
(L-Asp-Gly-Gly-LCA) and 21 (L-Glu-L-Glu-L-Glu-LCA).
However, tetrapeptide-LCA 22, in which extra Gly is
at position R1 of corresponding tripeptide-LCA 19, inhib-
ited a-2,3-ST at the low micromolar level (IC50 = 2.3 mM).
Compound 22 was at least 9-fold more potent than 19,
2-fold more potent than 13, 5-fold more potent than 1, and
15-fold more potent than 23. The inhibitory activity of
compound 22 was also evaluated against a-2,6-ST with an
IC50 value of 6.1 mM (Table 1), representing 2.6-7.6-fold
improvement over L-Asp-LCA 1 and tripeptide L-Asp-L-
Asp-L-Asp-LCA 20 and similar activity compared to
monopeptide D-Glu-LCA 7 and dipeptide L-Asp-L-Asp-
LCA 13. Collectively, the results show that the presence of
specific amino acid sequence in LCA such as D-Glu-LCA
7, L-Asp-L-Asp-LCA 13, and L-Asp-L-Asp-Gly-Gly-LCA
22, leads to significantly enhanced efficacy against a-2,3-
ST and a-2,6-ST.
Table 1. Inhibition of a-2,3-ST and a-2,6-ST by selected
peptidyl LCA analogues 1-23
Compound [a] a-2,3-ST (IC50, mM)[a] a-2,6-ST (IC50, mM)[a]
1
12.2 ± 2.1[b]
6.8 ± 1.0[b]
14.3 ± 1.5
19.9 ± 2.0
42.1 ± 3.9
7.0 ± 0.9
15.8 ± 0.2[e]
2
-
3
-
4
-
5
-
6
13.2 ± 2.1
7
5.4 ± 0.7
6.2 ± 1.3
8
15.8 ± 1.5
~100
-
9
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
16.8 ± 2.6
> 100 (16)[c]
15.5 ± 1.8
5.6 ± 0.7
> 15 (8)[d]
21.1 ± 1.9
22.0 ± 1.7
34.0 ± 3.8
19.9 ± 3.7
20.4 ± 2.7
31.6 ± 4.7
> 15 (2)[d]
2.3 ± 0.3
-
-
-
4.2 ± 1.2
-
-
-
-
-
-
46.5 ± 5.3
-
6.1 ± 1.2
34.7 ± 3.8
-
[a] Inhibitor concentration at which half-maximal enzyme
activity is obtained (IC50, mean ± S.E.M., n = 3). [b] Value is
from Ref. 14. [c] The percent (%) inhibition, in parentheses, at
100 mM is expressed as the percent (%) inhibition of enzyme
activity. [d] The percent (%) inhibition, in parentheses, at 15 mM
is expressed as the percent (%) inhibition of enzyme activity. [e]
Value is from Ref. 16.
In Vitro cell viability effects of amino acid conjugates
of lithocholic acid
The antiproliferative effect21 of compounds 1-23 was
tested on two breast cancer cell lines, MCF-7 (weakly
metastatic human breast cancer cell) and MDA-MB-231
(strongly metastatic human breast cancer cell). 1-23 did
not cause promising inhibitory effect on cell growth (>
90% survival) in both cell lines at the tested concentrations
(20 and 50 mM). These results indicate that compounds
1-23 exhibit the inhibitory efficacy of ST by blocking the
catalytic process of ST-mediated sialylation rather than in-
volving a direct antiproliferative effect. The low anti-
proliferative effect of compounds 1-23 may also benefit the
study of inhibition of migration of breast cancer cells in
next section.
second round of screening, compound 13 showed the best
inhibition constant with IC50 values of 5.6 and 4.2 mM
against a-2,3-ST and a-2,6-ST, respectively. Dipeptide-
LCAs containing two glycines (compound 9) or two pro-
lines (compound 11), which are relatively hydrophobic,
were shown to have low ability to suppress a-2,3-ST activ-
ity (at least 18-fold less potent than 13), suggesting that the
hydrophobic amino acid reduce ST activity. Surprisingly,
replacement of the L-Asp-L-Asp with L-Glu-L-Glu, as in
14, reduced activity dramatically when compared to that of
13. Similarly, compounds 15-17, where dipeptide is con-
structed by using different combination of two single
amino acids (L-Asp, L-Glu and g-Glu), displayed an ap-
proximately 4–6-fold lower inhibitory potency than 13.
These results uncover the importance of specific
amino acid sequence for improvement of inhibitory po-
tency and provide us a clue to tackle the difficulty in the
next design of tripeptide-LCAs 18-21 and tetrapeptide-
Inhibition of migration of metastatic breast cancer
cells by ST inhibitors
Increased cell migration is one of the important fac-
tors associated with tumor metastasis, the most universal
cause of death in cancer patients. Therefore, the ability of
ST inhibitors, representative compounds 2, 5, 6, 13, 20 and
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CHEMICAL SOCIETY
Sialyltransferase Inhibitors and Their Antimetastatic Activities
22, to inhibit breast cancer cell migration was investigated
in vitro using a qualitative wound healing assay.22 For the
control experiments, a sterile pipet tip was used to scratch
the confluent MCF-7 cell monolayers and generated a clear
wound strip at 0 h. The wound was almost closed at 24 h
(Figure 1a). Addition of 20 mM of ST inhibitors inhibited
MCF-7 cell migration and avoided closure of the wound at
24 h after scratching (Figure 1b). A greatly slower rate of
wound closure was observed in the presence of compounds
5, 6, and, while compounds 13 and 22 were considered
more effective. This result is in line with the previous ob-
servations, the similar trend of ST inhibition (Table 1). In
contrast, compounds 2, 5, 6, 13, 20, and 22 completely in-
hibited the migration of strongly metastatic human breast
cancer cell, MDA-MB-231 (Figure 2), indicating that most
of the LCA derivatives are selective for MDA-MB-231
cells rather than MCF-7 cells. Collectively, these findings
suggest that migration of MDA-MB-231 rather than
MCF-7 is relatively sensitive to these compounds. The cel-
lular studies further demonstrate that potent ST inhibitor
reduces a-2,3-ST/a-2,6-ST activities, prevents closure of
the wound, and sequentially inhibits breast cancer cell mi-
gration.
In summary, in an effort to explore cell-permeable
and low cytotoxic ST inhibitors for alteration of sialic acids
on cancer cell surfaces, we disclose herein the rapid discov-
ery and preparation of new amino acid conjugates of
lithocholic acid based on the systematically rational de-
sign. Development of amino acid sequence-dependent ST
inhibitors was achieved and some of these inhibitors, 7, 13
and 22, were found to have significant effect in not only
suppression of a-2,3-ST and a-2,6-ST activities but also
inhibition of breast cancer cell migration. Given the effec-
tive results that amino acid conjugate of LCA-based mole-
cules inhibit cancer cell migration, application of using
these cell-permeable ST inhibitors against tumor metasta-
sis in vivo is of great promise. Meanwhile, further optimi-
zation of this class of potent ST inhibitors should pave the
way for the development of new type of antimetastatic
agents that are of clinical value.
EXPERIMENTAL
Materials: Amino acids were purchased from Advanced
Fig. 2. Effect of ST inhibitors on wound healing of
MDA-MB-231 cells. Confluent cells were
scraped with a pipette tip to produce a ~1 mm-
wide wound area and the cells were treated with
medium containing vehicle at 0 h and 24 h a),
and ST inhibitors, compounds 2, 5, 6, 13, 20,
and 22, were added to the medium at a final
concentration of 20 mM, respectively. Individ-
ual imagines of the wound healing process were
photographed after incubation of cells at 37 °C
for 24 h b).
Fig. 1. Effect of ST inhibitors on wound healing of
MCF-7 cells. Confluent cells were scraped with
a pipette tip to produce a ~1 mm-wide wound
area and the cells were treated with medium
containing vehicle at 0 h and 24 h a), and ST in-
hibitors, compounds 2, 5, 6, 13, 20, and 22,
were added to the medium at a final concentra-
tion of 20 mM, respectively. Individual imag-
ines of the wound healing process were photo-
graphed after incubation of cells at 37 °C for 24
h b).
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Chemtech. Chemicals and buffers were purchased from Sigma,
Aldrich, or Acros Organics. CMP-Neu5Ac was synthesized start-
ing with sialic acid as described by Kajihara and co-workers.23
Synthesis of lactose acceptor was prepared from lactose via the
procedures similar to those of Halcomb.20 Preparation of
LacNAc-based acceptor was accomplished by modifying the pro-
cedure described in previous report14 and the results will be pub-
lished elsewhere. Rat liver a-2,3-ST and a-2,6-ST were obtained
from CalBiochem at a concentration of 560 mU/mL and 0.76
mg/mL, stored at –80 °C and consumed within one week. 1H and
13C NMR spectra were recorded with Bruker AMX400 or 500
MHz instruments. Proton chemical shifts (d) are reported in parts
per million (ppm) relative to the methine singlet at 7.24 ppm for
the residual CHCl3 in the deuteriochloroform, or the methyl
pentet at 3.30 ppm for the residual CHD2OD in the methanol-d4.
Carbon chemical shifts are reported in parts per million relative to
the internal 13C signals in CDCl3 (77.0 ppm) and CD3OD-d4 (49.0
ppm). Mass spectra were obtained with a FAB JMS-700 double
focusing mass spectrometer (JEOL, Tokyo, Japan) and ESI
Finnigan LCQ mass spectrometer (Thermo Finnigan, San Jose,
CA, United States) in negative mode. The final compounds were
purified by reversed phase HPLC (Waters 1525 Binary Pump
System with a Waters 2487 Dual l Absorbance Detector), using
Vydac 201SP510 C18 (1 cm ´ 25 cm) column to purity greater
than 95% as judged by analytical HPLC. The purity of com-
pounds was determined by reversed phase analytical HPLC
(Waters 2695 System with a 996 PDA detector), using Vydac
201SP54 C18 column (250 ´ 4.6 mm, 5 mm) and Sephadex LH-20
(170 ´ 10 mm) column. The separation procedure was performed
using H2O/0.1% trifluoroacetic acid (TFA) and CH3CN/0.05%
TFA as eluents.
the enzyme reaction, the solution mixture was quenched by heat
at 100 °C for 5 min. The activity was followed by measuring the
quantity of sialylated disaccharide product by RP-HPLC (Waters
2695 System with a 996 PDA detector; Supelco DiscoveryÒ HS
C18, 5 mm, 4.6 mm ´ 25 cm). Assays were performed in triplicate.
In Vitro Quantification of Cell Proliferation: The cells
were seeded with 3 ~ 5 ´ 104 cells/well in 200 ml DMEM or L-15
containing 10% FBS and incubated at 37 °C in 5% CO2 for 24 h.
The cells were exposed to each of the ST inhibitors, at the re-
quired concentration (a final concentration at 20 mM or ranging
from 0.2 to 100 mM) and allowed to incubate for 48 h. The MTT
solution (100 ml, 2 mg/ml) was added to each well for 3 h. Then,
100 ml DMSO/well was added and optical absorbance was de-
tected at 540 nm by a Molecular Devices Spectramax Plus 384
microplate reader. Survival ratio is expressed in a percent of a
value with respect to untreated cells. The IC50 values were deter-
mined as the ST inhibitor concentrations that inhibit cell growth
by 50% compared with growth of vehicle-treated cells. All mea-
surements were determined from replicates of 5 wells, and each
experiment was repeated two times.
Wound Healing Migration Assay: Breast cancer cells (1 ´
106 cells/mL) in DMEM or L-15 containing 10% FBS were
seeded into 6-well culture plates for 24 h. After they grew to con-
fluence, wounds were created with sterile pipette tips to obtain a
clean ~1-mm-wide wound area followed by addition of ST inhibi-
tors (20 mM) in the culture medium at 5% CO2. The migration and
cell movement was examined and photographed (Zeiss Axiovert
40CFL microscope) after 24 h.
General Procedure for Esterification of the 3-Hydroxy
Lithocholic Acid (24) and Amino Acid: To a solution of the pro-
tected lithocholic acid 24 (0.35 mmol), amino acid (0.45 mmol)
and 4-(dimethylamino)pyridine (0.10 mmol) in dichloromethane
(8 cm3) was added a solution of dicyclohexylcarbodiimide in di-
chloromethane (2 cm3). The reaction was stirred at room tempera-
ture for 30 min and the solvent was removed by rotary evapora-
tion. The resulting residue was purified by column chromatogra-
phy on silica gel eluting with ethyl acetate and hexane to afford
the pure product.
Cell Culture: Human breast carcinoma cell line MCF-7
and human adenocarcinoma cells MDA-MB-231 (BCRC-60436;
BCRC-60425, Bioresource Collection and Research Center, Food
Industry Research and Development Institute; Hsinchu, Taiwan)
were grown in Dulbecco’s modified Eagle’s medium (DMEM) or
L-15 medium with 10% fetal bovine serum (FBS) at 37 °C in a hu-
midified atmosphere containing 5% CO2 in air.
Determination of Inhibitory Activity (IC50) of ST Inhib-
itor: Each reaction solution containing 200 mM MES buffer, 100
mM sodium chloride, 0.5 mM disodium-EDTA, 0.01 % Triton
X-100, 20 mM modified disaccharide, 1.4 mU a-2,3-ST (or a-
2,6-ST) and inhibitor (in dimethyl sulfoxide) was adjusted to a to-
tal volume of 55 mL. The solution was incubated at 37 °C for 10
min, then 1 mM CMP-Neu5Ac (final concentration, 3 mL) was
added to initiate the reaction. The solution was incubated at 37 °C
until a detectable amount of product was produced. To terminate
General Procedure for Peptide Bond Formation: To a
solution of peptidyl lithocholic acid (0.17 mmol) and amino acid
(0.18 mmol) in dimethylformamide (DMF) (3 cm3) was added
1-hydroxybenzotriazole (HOBT) (0.18 mmol) and ethyl-(N’,N’-
dimethylamino)propylcarbodiimide hydrochloride (EDC) (0.18
mmol). The reaction was stirred at room temperature for 1 hour
and the solvent was removed by vacuum system. The mixture was
dissolved in dichloromethane (30 cm3) and washed with water (30
cm3) twice. The extracts were evaporated and dried to afford the
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Sialyltransferase Inhibitors and Their Antimetastatic Activities
crude product as the sticky oil, which was purified by column
chromatography on silica gel eluting with ethyl acetate and
hexane to afford the pure product.
found, 464.3380.
Monopeptide-LCA 5. 1H NMR (400 MHz, CD3OD): d 4.87
(m, 1H), 4.38 (m, 1H), 3.35 (m, 2H), 2.33 (m, 1H), 2.20 (m, 1H),
2.11-1.71 (m, 11H), 1.63-1.07 (m, 19H), 0.98 (s, 3H), 0.96 (d, J =
6.5 Hz, 3H), 0.70 (s, 3H); 13C NMR (125 MHz, CD3OD): d
178.29, 169.83, 78.81, 61.03, 58.02, 57.65, 47.31, 44.05, 43.44,
41.98, 41.62, 37.32, 36.85, 36.03, 35.83, 33.25, 32.45, 32.14,
29.63, 29.35, 28.25, 27.67, 27.57, 25.38, 24.65, 23.85, 22.08,
18.88, 12.60; HRMS (ESI) calculated for C29H48NO4 (M + H)+,
474.3583; found, 474.3583.
Monopeptide-LCA 6. 1H NMR (400 MHz, CD3OD): d 5.48
(d, J = 6.6 Hz, 1H ), 4.07 (t, J = 6.7 Hz, 1H), 2.53 (m, 2H),
2.28-2.15 (m, 4H), 2.03-1.89 (m, 6H), 1.75-1.69 (m, 2H), 1.62-
1.58 (m, 3H), 1.45-1.35 (m, 8H), 1.30-1.26 (m, 6H), 1.13-1.09 (m,
6H), 0.98 (s, 3H), 0.96 (d, J = 6.5 Hz, 3H), 0.69 (s, 3H); 13C NMR
(125 MHz, CD3OD): d 178.29, 174.55, 169.83, 77.21, 56.53,
56.19, 52.23, 42.60, 42.01, 40.52, 40.15, 35,88, 35.40, 34.63,
34.39, 31.86, 31.01, 30.73, 27.90, 26.82, 26.21, 23.93, 22.42,
20.64, 17.45, 11.16; HRMS (FAB) calculated for C29H48NO6 (M
+ H)+, 506.3482; found, 506.3484.
Monopeptide-LCA 7. 1H NMR (400 MHz, CD3OD): d 4.07
(t, J = 6.7 Hz, 1H), 2.55 (m, 2H), 2.28-2.15 (m, 4H), 2.03-1.89 (m,
6H), 1.75-1.69 (m, 2H), 1.62-1.58 (m, 2H), 1.45-1.35 (m, 7H),
1.30-1.26 (m, 5H), 1.13-1.09 (m, 6H), 0.98 (s, 3H), 0.96 (d, J =
6.5 Hz, 3H), 0.70 (s, 3H); 13C NMR (125 MHz, CD3OD): d
176.70, 175.55, 168.29, 77.11, 56.37, 56.03, 51.93, 42.44, 41.86,
40.37, 40.00, 35.72, 35.24, 34.46, 34.23, 31.67, 30.85, 30.54,
28.88, 27.75, 26.63, 26.03, 25.29, 23.77, 22.26, 20.48, 17.28,
10.99; HRMS(FAB) calculated for C29H48NO6 (M + H)+,
506.3482; found, 506.3489.
General Procedure for Deprotection of Allyl Group: To
a solution of the Allyl-protected steroidal compound (0.36 mmol)
in tetrahydrofuran (5 cm3) was added tetrakis(triphenylphos-
phine)palladium(0) (0.03 mmol) and morpholine (1.5 cm3) drop-
wise. The reaction was stirred at room temperature for 2 hours.
Solvent was removed by rotary evaporation. The mixture was dis-
solved in dichloromethane (30 cm3) and washed with water (30
cm3) twice. The extracts were dried and evaporated to afford the
crude product as the sticky oil, which was purified by flash
column eluting with ethyl acetate and hexane to give the pure
product.
General Procedure for Deprotection of Fmoc Group: To
a solution of the Fmoc-protected steroidal compound (0.15
mmol) in dichloromethane (5 cm3) was added neat 7,11-diaza-
bicyclo[5.4.0]undec-11-ene (0.15 mmol) dropwise. The reaction
was stirred at room temperature for 30 min. Solvent was removed
by rotary evaporation to yield the crude product as oil, which was
purified by flash column eluting with ethyl acetate and hexane to
give the pure product.
General Procedure for Deprotection of Boc and tBu
Groups: A solution of protected compound (0.15 mmol) in TFA
(2 cm3) was treated at room temperature with 2% water (0.04
cm3). After 30 min, solvent was removed by rotary evaporation.
The resulting mixture was neutralized with saturated sodium bi-
carbonate and purified by reverse phase HPLC to afford the pure
product.
Monopeptide-LCA 3. 1H NMR (400 MHz, CD3OD): d 4.90
(m, 1H), 3.79 (s, 2H), 2.31 (m, 1H), 2.21 (m, 1H), 2.04 (m, 1H),
1.96-1.78 (m, 6H), 1.62-1.42 (m, 9H), 1.34-1.07 (m, 10H), 0.98
(s, 3H), 0.96 (d, J = 6.5 Hz, 3H), 0.70 (s, 3H); 13C NMR (125
MHz, CD3OD): d 178.29, 168.15, 78.30, 58.03, 57.65, 44.06,
43.44, 42.00, 41.63, 41.35, 37.32, 36.85, 36.07, 35.83, 33.33,
32.46, 32.15, 29.35, 28.26, 27.66, 25.38, 23.88, 22.08, 18.89,
12.61; HRMS (ESI) calculated for C26H44NO4 (M + H)+,
434.3270; found, 434.3260.
Monopeptide-LCA 4. 1H NMR (400 MHz, CD3OD): d 4.04
(m, 1H), 3.98 (dd, J = 11.6, 4.7 Hz, 1H), 3.91 (dd, J = 11.6, 3.4 Hz,
1H), 3.69 (m, 1H), 2.30 (m, 1H), 2.20 (m, 1H), 2.04-1.74 (m, 7H),
1.63-1.42 (m, 9H), 1.36-1.04 (m, 11H), 0.98 (s, 3H), 0.96 (d, J =
6.5 Hz, 3H), 0.70 (s, 3H); 13C NMR (125 MHz, CD3OD): d
178.42, 168.71, 78.55, 60.99, 58.05, 57.68, 56.36, 44.05, 43.45,
41.99, 41.63, 37.31, 36.86, 36.07, 35.84, 33.32, 32.49, 32.24,
29.36, 28.27, 27.68, 27.58, 25.38, 23.87, 22.08, 18.89, 12.60;
HRMS (ESI) calculated for C27H46NO5 (M + H)+, 464.3376;
1
Monopeptide-LCA 8. H NMR (400 MHz, CD3OD): d
4.76-4.70 (m, 1H), 4.00 (t, J = 6.6 Hz, 1H), 2.56-2.54 (m, 2H),
2.20-2.21 (m, 4H), 1.92-1.86 (m, 6H), 1.65-1.48 (m, 4H), 1.46-
1.42 (m, 7H), 1.32-1.28 (m, 4H), 1.16-1.09 (m, 5H), 0.97 (s, 3H),
0.96 (d, J = 6.5 Hz, 3H), 0.69 (s, 3H); 13C NMR (125 MHz,
CD3OD): d 178.27, 173.48, 171.75, 76.50, 58.03, 57.62, 53.50,
44.06, 43.49, 42.00, 41.64, 37.33, 36.83, 36.21, 35.85, 33.50,
32.45, 32.14, 31.14, 29.35, 28.30, 27.78, 27.70, 26.90, 25.40,
23.97, 22.09, 18.92, 12.64; HRMS(FAB) calculated for C29H48NO6
(M + H)+, 506.3482; found, 506.3489.
Dipeptide-LCA9. 1H NMR (400 MHz, CD3OD): d 4.76 (m,
1H), 4.00 (s, 2H), 3.74 (s, 2H), 2.31 (m, 1H), 2.21(m, 1H),
2.04-1.57 (m, 9H), 1.50-1.05 (m, 18H), 0.96 (s, 3H), 0.94 (d, J =
6.5 Hz, 3H), 0.70 (s, 3H); 13C NMR (125 MHz, CD3OD): d
178.29, 170.82, 167.94, 77.25, 58.03, 57.64, 44.06, 43.48, 42.39,
42.00, 41.64, 41.54, 37.33, 36.86, 36.16, 35.85, 33.45, 32.46,
32.14, 29.36, 28.30, 27.73, 27.69, 25.39, 23.91, 22.09, 18.89,
J. Chin. Chem. Soc. 2016, 63, 171-180
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Article
Fu et al.
12.60; HRMS (ESI) calculated for C28H47N2O5 (M + H)+,
491.3485; found, 491.3484.
1.07-1.02 (m, 5H), 0.97 (s, 3H), 0.96 (d, J = 6.5 Hz, 3H), 0.70 (s,
3H); 13C NMR (125 MHz, CD3OD): d 176.75, 174.62, 174.27,
170.71, 168.41, 75.86, 70.09, 56.30, 55.96, 52.14, 42.43, 41.89,
40.32, 39.98, 35.74, 35.26, 34.56, 34.25, 31.82, 30.86, 30.54,
29.51, 28.60, 26.68, 26.29, 26.15, 26.05, 23.78, 22.29, 20.47,
17.28, 10.99; HRMS (ESI) calculated for C34H55N2O9 (M + H)+,
635.3908; found, 635.3907.
Dipeptide-LCA 15. 1H NMR (400 MHz, CD3OD): d 4.75-
4.72 (m, 2H), 3.90 (t, J = 6.4 Hz, 1H), 2.80-2.77 (m, 2H),
2.53-2.47 (m, 2H), 2.25-2.22 (m, 1H), 2.10-2.07 (m, 3H), 1.93-
1.80 (m, 5H), 1.81-1.66 (m, 4H), 1.42-1.36 (m, 7H), 1.25-1.21 (m,
5H), 1.07-1.02 (m, 6H), 0.89 (s, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.62
(s, 3H); 13C NMR (125 MHz, CD3OD): d 176.71, 174.31, 172.13,
169.84, 168.18, 76.09, 56.29, 56.25, 52.13, 49.14, 42.43, 41.89,
40.32, 39.98, 35.74, 35.26, 34.56, 34.25, 31.82, 30.86, 30.54,
29.51, 28.60, 26.68, 26.29, 26.15, 26.05, 23.78, 22.29, 20.47,
17.28, 10.99; HRMS (FAB) calculated for C33H53N2O9 (M + H)+,
621.3751; found, 621.3755.
Dipeptide-LCA 16. 1H NMR (400 MHz, CD3OD): d 4.75-
4.73 (m, 1H), 4.46-4.45 (m, 1H), 2.97-2.95 (m, 1H), 2.83-2.80 (m,
1H), 2.43 (t, J = 7.3 Hz, 2H), 2.40-2.38 (m, 1H), 2.23-2.20 (m,
2H), 2.01-1.89 (m, 7H), 1.75-1.67 (m, 3H), 1.48-1.41 (m, 8H),
1.31-1.24 (m, 6H), 1.16-1.09 (m, 6H), 0.97 (s, 3H), 0.95 (d, J =
6.5 Hz, 3H), 0.69 (s, 3H); 13C NMR (125 MHz, CD3OD): d
176.88, 174.79, 171.97, 170.75, 168.29, 75.79, 56.29, 55.96,
52.21, 49.73, 42.44, 41.87, 40.35, 39.97, 35.73, 35.26, 35.09,
34.53, 34.24, 31.82, 30.86, 30.54, 29.51, 28.60, 26.68, 26.29,
26.15, 26.05, 23.78, 22.29, 20.47, 17.31, 11.02; HRMS (FAB)
calculated for C33H53N2O9 (M + H)+, 621.3731; found, 621.3729.
1
Dipeptide-LCA 10. H NMR (400 MHz, CD3OD): d 4.76
(m, 1H), 4.26 (q, J = 4.3 Hz, 1H), 3.98 (dd, J = 38.5, 17.6 Hz, 2H),
3.02 (dd, J = 17.9, 5.9 Hz, 1H), 2.85 (dd, J = 17.9, 8.7 Hz, 1H),
2.31 (m, 1H), 2.22 (m, 1H), 2.04-1.35 (m, 16H), 1.34-1.05 (m,
10H), 0.96 (s, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.70 (s, 3H); 13C
NMR (125 MHz, CD3OD): d 178.27, 172.99, 170.54, 170.02,
77.20, 57.96, 57.59, 51.12, 44.05, 43.45, 42.54, 41.97, 41.62,
37.32, 36.84, 36.31, 36.15, 35.83, 33.44, 32.45, 32.14, 28.29,
27.74, 27.66, 25.39, 23.93, 22.09, 18.91, 12.63; HRMS (ESI) cal-
culated for C30H49N2O7 (M + H)+, 549.3540; found, 549.3545.
1
Dipeptide-LCA 11. H NMR (400 MHz, CD3OD): d 4.71
(m, 1H), 4.56 (dd, J = 8.6, 5.6 Hz, 1H), 4.48 (dd, J = 8.7, 5.2 Hz,
1H), 3.69 (m, 1H), 3.60 (m, 1H), 3.44 (m, 1H), 3.33 (m, 1H), 2.56
(m, 1H), 2.35-2.12 (m, 4H), 2.10-1.76 (m, 14H), 1.70-1.52 (m,
2H), 1.47-1.05 (m, 15H), 0.97 (s, 3H), 0.96 (d, J = 6.5 Hz, 3H),
0.70 (s, 3H); 13C NMR (125 MHz, CD3OD): d 178.27, 172.72,
168.38, 77.06, 61.08, 60.45, 58.10, 57.67, 48.25, 47.81, 44.06,
43.43, 42.04, 41.68, 37.32, 36.86, 36.11, 35.85, 33.40, 32.45,
32.14, 30.18, 29.64, 29.36, 28.28, 27.70, 26.04, 25.38, 25.22,
23.91, 22.08, 18.89, 12.61; HRMS (ESI) calculated for C34H55N2O5
(M + H)+, 571.4111; found, 571.4111.
1
Dipeptide-LCA 12. H NMR (500 MHz, CD3OD): d 4.72
(m, 1H), 4.50 (m, 2H), 3.36 (m, 2H), 3.02 (dd, J = 18.0, 3.1 Hz,
1H), 2.74 (dd, J = 18.0, 9.9 Hz, 1H), 2.32 (m, 2H), 2.20 (m, 1H),
2.08-1.80 (m, 8H), 1.62-1.04 (m, 20H), 0.97 (s, 3H), 0.96 (d, J =
6.5 Hz, 3H), 0.69 (s, 3H); 13C NMR (125 MHz, CD3OD): d
178.32, 172.68, 172.64, 168.08, 77.08, 57.89, 57.58, 50.17,
48.35, 44.05, 43.45, 41.99, 41.60, 37.33, 36.87, 36.11, 35.84,
33.25, 32.46, 32.15, 30.18, 29.37, 28.28, 27.70, 27.63, 26.11,
25.38, 23.90, 22.09, 18.89, 12.60; HRMS (ESI) calculated for
C33H53N2O7 (M + H)+, 589.3853; found, 589.3850.
1
Dipeptide-LCA 17. H NMR (400 MHz, CD3OD): d 5.48
(s, 1H), 4.10-4.08 (m, 1H), 3.97-3.95 (m, 1H), 2.80-2.77 (m, 2H),
2.53-2.47 (m, 2H), 2.25-2.22 (m, 1H), 2.10-2.07 (m, 3H), 1.93-
1.80 (m, 5H), 1.81-1.66 (m, 4H), 1.42-1.36 (m, 7H), 1.25-1.21 (m,
5H), 1.07-1.02 (m, 6H), 0.95-0.90 (m, 6H), 0.69 (s, 3H); 13C NMR
(125 MHz, CD3OD): d 177.18, 173.32, 172.78, 170.86, 170.76,
76.35, 56.81, 56.47, 49.69, 42.90, 42.37, 40.81, 40.46, 36.19,
35.71, 35.01, 34.72, 31.74, 31.34, 31.31, 31.00, 28.22, 27.16,
26.54, 26.49, 24.24, 22.78, 22.69, 20.94, 17.75, 13.42, 11.45;
HRMS (FAB) calculated for C33H53N2O9 (M + H)+, 621.3751;
found, 621.3767.
Tripeptide-LCA 18. 1H NMR (400 MHz, CD3OD): d 4.74
(m, 1H), 4.00 (dd, J = 8.5, 4.6 Hz, 1H), 3.98 (m, 4H), 3.03 (dd, J =
17.9, 4.6 Hz, 1H), 2.87 (dd, J = 17.9, 8.4 Hz, 1H), 2.31 (m, 1H),
2.21(m, 1H), 2.04-1.53 (m, 10H), 1.50-1.05 (m, 18H), 0.96 (s,
3H), 0.95 (d, J = 6.5 Hz, 3H), 0.69 (s, 3H); 13C NMR (125 MHz,
CD3OD): d 178.28, 172.99, 171.61, 170.90, 169.92, 77.10, 58.00,
57.61, 51.24, 44.06, 43.48, 43.31, 42.41, 41.98, 41.63, 37.34,
1
Dipeptide-LCA 13. H NMR (500 MHz, CD3OD): d 4.77
(m, 2H), 4.25 (dd, J = 9.2, 4.0 Hz, 1H), 3.04 (dd, J = 18.1, 4.0 Hz,
1H), 2.85 (m, 3H), 2.31 (m, 1H), 2.21 (m, 1H), 2.03-1.85 (m, 8H),
1.70-1.41 (m, 12H), 1.34-1.05 (m, 13H), 0.95 (s, 3H), 0.93 (d, J =
6.5 Hz, 3H), 0.69 (s, 3H); 13C NMR (125 MHz, CD3OD): d
176.31, 171.73, 170.96, 169.40, 167.49, 75.58, 55.90, 55.62,
49.00, 48.82, 42.05, 41.49, 39.96, 39.60, 35.34, 34.87, 34.64,
34.40, 34.13, 33.85, 31.39, 30.47, 30.15, 27.38, 26.29, 25.64,
23.39, 21.90, 20.09, 16.89, 10.60; HRMS (ESI) calculated for
C32H51N2O9 (M + H)+, 607.3589; found, 607.3595.
Dipeptide-LCA 14. 1H NMR (400 MHz, CD3OD): d 4.79-
4.76 (m, 1H), 4.50-4.47 (m, 1H), 3.96 (t, J = 6.4 Hz, 1H), 3.64 (s,
1H), 2.57-2.51 (m, 2H), 2.44-2.42 (m, 2H), 2.18-2.03 (m, 4H),
1.81-1.66 (m, 10H), 1.54-1.48 (m, 3H), 1.38-1.20 (m, 10H),
178
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J. Chin. Chem. Soc. 2016, 63, 171-180

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Sialyltransferase Inhibitors and Their Antimetastatic Activities
36.85, 36.18, 36.05, 35.85, 33.46, 32.46, 32.14, 29.36, 28.31,
27.74, 27.69, 25.39, 23.91, 22.09, 18.90, 12.61; HRMS (ESI) cal-
culated for C32H52N3O8 (M + H)+, 606.3754; found, 606.3745.
Tripeptide-LCA 19. 1H NMR (500 MHz, CD3OD): d 4.82
(dd, J = 8.1, 5.2 Hz, 1H), 4.74 (m, 1H), 4.20 (dd, J = 8.6, 4.5 Hz,
1H), 3.91 (dd, J = 23.4, 17.5 Hz, 2H), 3.06 (dd, J = 17.9, 4.4 Hz,
1H), 2.89 (m, 2H), 2.75 (dd, J = 17.0, 8.2 Hz, 1H), 2.31 (m, 1H),
2.21(m, 1H), 2.03-1.54 (m, 8H), 1.50-1.04 (m, 15H), 0.96 (s, 3H),
0.95 (d, J = 6.5 Hz, 3H), 0.69 (s, 3H); 13C NMR (125 MHz,
CD3OD): d 178.30, 173.87, 173.26, 173.12, 170.71, 169.50,
77.06, 57.95, 57.58, 51.56, 51.21, 44.05, 43.47, 42.55, 41.96,
41.61, 37.34, 36.86, 36.78, 36.21, 36.17, 33.45, 32.46, 32.14,
29.37, 28.30, 27.74, 27.67, 25.39, 23.92, 22.09, 18.90, 12.61;
HRMS (ESI) calculated for C34H54N3O10 (M + H)+, 664.3809;
found, 664.3799.
Tripeptide-LCA 20. 1H NMR (400 MHz, CD3OD): d 4.80
(m, 1H), 4.72 (m, 2H), 4.20 (dd, J = 9.3, 4.3 Hz, 1H), 3.08-2.71
(m, 6H), 2.33 (m, 1H), 2.20 (m, 1H), 2.03-1.76 (m, 6H), 1.69-1.44
(m, 10H), 1.34-1.01 (m, 10H), 0.96 (m, 6H), 0.69 (s, 3H); 13C
NMR (125 MHz, CD3OD): d 178.31, 174.11, 173.84, 173.36,
172.45, 171.67, 169.58, 77.46, 57.87, 57.58, 51.55, 51.21, 50.78,
44.04, 43.50, 41.91, 41.58, 37.34, 36.86, 36.82, 36.35, 36.16,
35.85, 33.34, 32.46, 32.15, 29.37, 28.29, 27.64, 25.39, 23.91,
22.09, 18.89, 12.60; HRMS (ESI) calculated for C36H56N3O12 (M
+ H)+, 722.3864; found, 722.3856.
4.78-4.68 (m, 4H), 4.18 (dd, J = 8.4, 4.4 Hz, 1H), 3.03-2.74 (m,
8H), 2.31 (m, 1H), 2.21 (m, 1H), 2.03-1.78 (m, 6H), 1.65-1.42 (m,
10H), 1.34-1.00 (m, 10H), 0.96 (m, 6H), 0.69 (s, 3H); 13C NMR
(100 MHz, CD3OD): d 178.35, 174.35, 174.29, 174.10, 173.76,
172.73, 172.35, 171.67,169.99,77.42, 57.85, 57.58, 52.11, 51.44,
50.91, 44.05, 43.53, 41.82, 41.56, 37.37, 37.00, 36.87, 36.68,
36.46, 36.20, 35.86, 33.30, 32.47, 32.15, 29.39, 28.32, 27.67,
27.59, 25.40, 23.91, 22.10, 18.90, 12.60; HRMS (ESI) calculated
for C40H61N4O15 (M + H)+, 837.4133; found, 837.4146.
ACKNOWLEDGEMENTS
We are grateful for financial support of this work pro-
vided by the Academia Sinica and the National Science
Council (NSC 102-2325-B-001-025-). Instrumentation
support was provided by the NMR and Mass Spectrometry
facilities of the Institute of Chemistry at Academia Sinica,
Taiwan.
REFERENCES
1. Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Thun, M. J.
CA Cancer J. Clin. 2009, 59, 225-249.
2. a) Minn, A. J.; Gupta, G. P.; Siegel, P. M.; Bos, P. D.; Shu, W.;
Giri, D. D.; Viale, A.; Olshen, A. B.; Gerald, W. L.;
Massagué, J. Nature 2005, 436, 518-524; b) Shaikhibrahim,
Z.; Lindstrot, A.; Langer, B.; Buettner, R.; Wernert, N. Int. J.
Mol. Med. 2011, 27, 811-819.
Tripeptide-LCA 21. 1H NMR (400 MHz, CD3OD): d 4.84-
4.79 (m, 1H), 4.45-4.42 (m, 2H), 3.95 (t, J = 6.4 Hz, 1H), 2.51-
2.42 (m, 6H), 2.44-2.42 (m, 1H), 2.25-2.19 (m, 5H), 1.81-1.66 (m,
7H), 1.38-1.20 (m, 5H), 1.07-1.02 (m, 5H), 0.96 (s, 3H), 0.94 (d, J
= 6.5 Hz, 3H), 0.70 (s, 3H); 13C NMR (125 MHz, CD3OD): d
176.75, 175.05, 174.98, 171.86, 171.04, 168.38, 75.60, 56.28,
55.95, 52.77, 52.28, 52.06, 42.43, 41.89, 40.31, 39.99, 35.73,
35.26, 34.57, 34.25, 31.82, 30.86, 30.55, 29.70, 27.77, 26.68,
26.41, 26.14, 26.05, 26.00, 23.79, 22.30, 20.47, 17.28, 10.99;
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Tertapeptide-LCA22. 1H NMR (400 MHz, CD3OD): d 4.72
(m, 1H), 4.46 (m, 1H), 4.31 (m, 1H), 3.91 (m, 4H), 2.87 (m, 4H),
2.31 (m, 1H), 2.21 (m, 1H), 2.03-1.52 (m, 10H), 1.44-1.04 (m,
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