Synthesis and anti-migrative evaluation of moverastin derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.01.028

Cell migration of tumor cells is essential for invasion of the extracellular matrix and for cell dissemination. Inhibition of the cell migration involved in the invasion process represents a potential therapeutic approach to the treatment of tumor metastasis; therefore, a novel series of derivatives of moverastins (moverastins A and B), an inhibitor of tumor cell migration, was designed and chemically synthesized. Among these moverastin derivatives, several compounds showed stronger cell migration inhibitory activity than parental moverastins, and UTKO1 was found to have the most potent inhibitory activity against the migration of human esophageal tumor EC17 cells in a chemotaxis cell chamber assay. Interestingly, although moverastins are considered to inhibit tumor cell migration by inhibiting farnesyltransferase (FTase), UTKO1 did not inhibit FTase, indicating that UTKO1 inhibited tumor cell migration by a mechanism other than the inhibition of FTase.

Full text of DOI:10.1016/j.bmcl.2011.01.028

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1385–1389
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-migrative evaluation of moverastin derivatives
Masato Sawada a, , Shin-ichiro Kubo b, , Koji Matsumura b, Yasushi Takemoto a, Hiroki Kobayashi a,
a,
Etsu Tashiro a, Takeshi Kitahara b, Hidenori Watanabe b, , Masaya Imoto
⇑
⇑
a Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
b Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Cell migration of tumor cells is essential for invasion of the extracellular matrix and for cell dissemina-
tion. Inhibition of the cell migration involved in the invasion process represents a potential therapeutic
approach to the treatment of tumor metastasis; therefore, a novel series of derivatives of moverastins
(moverastins A and B), an inhibitor of tumor cell migration, was designed and chemically synthesized.
Among these moverastin derivatives, several compounds showed stronger cell migration inhibitory activ-
ity than parental moverastins, and UTKO1 was found to have the most potent inhibitory activity against
the migration of human esophageal tumor EC17 cells in a chemotaxis cell chamber assay. Interestingly,
although moverastins are considered to inhibit tumor cell migration by inhibiting farnesyltransferase
(FTase), UTKO1 did not inhibit FTase, indicating that UTKO1 inhibited tumor cell migration by a mecha-
nism other than the inhibition of FTase.
Received 6 December 2010
Revised 6 January 2011
Accepted 7 January 2011
Available online 11 January 2011
Keywords:
Cell migration
Chemical synthesis
UTKO1
Ó 2011 Elsevier Ltd. All rights reserved.
Despite significant advances in understanding the fundamental
aspects of cancer, the development of metastatic lesions remains
the predominant cause of death for most cancer patients.1,2 Cell
migration is a crucial event in the spread of cancer and, conse-
quently, the metastatic process.3,4 This prompted us to develop
inhibitors of tumor cell migration as novel anti-metastatic drugs.
Previously, we screened for inhibitors of cancer cell migration
derived from microbial origin, and obtained moverastin A and B
(1 and 2, respectively), new members of the cylindrol family, from
Aspergillus sp. F7720.5 Their structures including the absolute ste-
reochemistries were confirmed unambiguously by the synthesis
as outlined in Scheme 1. Furthermore, moverastin A and B were
found to inhibit FTase; therefore, moverastins were considered to
inhibit the migration of tumor cells by inhibiting the farnesylation
of H-Ras, and subsequent H-Ras-dependent activation of the
PI3K/Akt pathway. However, because the inhibitory activity of
moverastins for tumor cell migration was rather modest (IC50 value
Structures of moverastin derivatives (UTKO1–12) synthesized in
this study are shown in Figure 1. UTKO1–6 were synthesized by
employing the same approach as that for our previous synthesis of
moverastins.5 The enol triflates (7, 10, 12, 15, 18) were prepared
starting from readily available ketones or aldehydes (5, 9, 11, 13,
16, respectively) as shown in Scheme 2. Coupling reactions between
the enol triflates (or 2-iodopropene for UTKO4) and aldehyde 4 were
carried out successfully using the Nozaki–Hiyama–Kishi proce-
dure6,7 and UTKO1–6 were obtained after acid hydrolysis of MOM
ether. The dihydro analog (UTKO7) and etherified analogs (UTKO9
and 10) of UTKO1 were also synthesized by its hydrogenation, Mits-
unobu reaction or methylation (Scheme 3). The unsaturated ketone
analog of UTKO1 was also obtained by the PDC oxidation-deprotec-
tion of 19 which is the intermediate from 7 to UTKO1. UTKO11 and
12, deformylated analogs of moverastin and UTKO1, respectively,
were also synthesized from aldehyde 21 instead of 4 (Scheme 4).
Detailed synthetic procedure for UTKO compounds will be
published elsewhere.
of 7 lM), we considered it an attractive lead compound in the
search for other, more potent agents.
Next, the cell migration inhibitory activity of these moverastin
derivatives was examined by the chemotaxis cell chamber (BD Bio-
sciences) assay using conditioned medium of human esophageal
tumor EC17 cells as a source of chemoattractants as previously
reported with some modifications.8 In this assay, EC17-conditioned
medium was initially placed in the lower compartment. EC17 cells
were incubated in the upper chamber, where they were allowed to
migrate and penetrate the filter separating the chambers in order
to enter the lower chamber. After 24 h of incubation, the number
of cells attached to the lower side of the filter was counted. The
IC50 values obtained in this study are listed in Table 1. Among
In this study, we chemically synthesized a series of moverastin
derivatives and assessed their potential as tumor cell migration
inhibitors in several in vitro assays.
⇑
Corresponding authors. Tel./fax: +81 45 566 1557 (M.I.); tel.: +81 3 5841 5119;
fax: +81 3 5841 8019 (H.W.).
E-mail addresses: ashuten@mail.ecc.u-tokyo.ac.jp (H. Watanabe), imoto@bio.
keio.ac.jp (M. Imoto).
These authors contributed equally to the study.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.028

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M. Sawada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1385–1389
OH
OH
10
Cl
OTf
OH
HO
HO
O
O
O
CHO
O
CHO
3
Ascochlorin
10R : Moverastin A (1)
10S : Moverastin B (2)
CH2OH
OH
OHC
MOMO
HO
CHO
4
orcinol
OH
Scheme 1. Synthesis and structures of moverastin A and B.
OH
OH
cis
OH
HO
OH
HO
OH
HO
CHO
CHO
CHO
UTKO1
UTKO2
UTKO3
OH
OH
OH
OH
HO
OH
HO
OH
HO
CHO
CHO
CHO
UTKO4
UTKO5
UTKO6
OH
OH
O
CHO
cis
cis
cis
OH
HO
O
HO
HO
UTKO9
CHO
OCH3
CHO
CHO
UTKO7
UTKO8
OH
OH
cis
cis
OH
HO
OH
HO
OH
HO
O
UTKO10
UTKO11
UTKO12
Figure 1. Structures of moverastin derivatives.
moverastin derivatives, UTKO1 showed the most potent inhibitory
activity of EC17 cell migration with an IC50 of 1.98 M (Fig. 2).
UTKO7, UTKO9 and UTKO12 are also significantly more active
inhibitors of EC17 cell migration than the parental natural product,
moverastin A, with IC50 values of 2.12, 2.00 and 2.17 lM, respec-
tively (Table 1). These inhibitory effects are not due to the toxic ef-
fect of the drug because their 50% inhibitory concentration for
EC17 cell viability, as estimated by trypan blue dye exclusion assay,
was at least five-fold higher than that for cell migration.
the migration of tumor cells by inhibiting the farnesylation of
H-Ras;5 therefore, next we examined the effect of moverastin
derivatives on FTase in vitro. For this assay, FTase was partially
purified from EC17 cells and recombinant GST-H-Ras and [3H]-
farnesylpyrophosphate were used as the substrates as described
before.5 As shown in Table 1, all moverastin derivatives tested,
including UTKO1, UTKO7, UTKO9, and UTKO12, which showed
strong inhibition of cell migration, did not inhibit FTase in vitro
l
up to 100 lM. These results indicated that a mechanism other than
Previously, we found that moverastin A showed inhibitory
activity against FTase, and demonstrated that moverastin A inhibited
the inhibition of FTase is responsible for UTKO-induced inhibition
of EC17 cell migration. To examine this possibility, several cancer

M. Sawada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1385–1389
1387
a
b
O
OTf
OTf
O
cis
cis
97%
55%
5
6
7
CHO
c
a, b
O
96%
62%
8
9
10
b
O
OTf
OTf
61%
11
12
CHO
c
a, b
O
85%
30%
13
16
14
15
c
a, b
CHO
O
OTf
67%
63%
17
18
UTKO1 (R=
UTKO2 (R=
UTKO3 (R=
, 59%)
7
or
, 68%)
OH
10
or
R
, 32%)
12
d, e
4
or
+
OH
HO
2-iodopropene
UTKO4 (R= H, 72%)
UTKO5 (R=
or
15
or
CHO
, 38%)
18
UTKO6 (R=
, 52%)
Scheme 2. Synthesis of UTKO1–6. Reagents and conditions: (a) H2, Pd-C, EtOAc (for 6) or EtOH (for 10, 15 and 18); (b) KHMDS, Comin’s reagent, THF, À78 °C; (c)
Ph3P@CHCOMe, CH2Cl2 (for 9 and 14) or xylene (for 17), reflux; (d) CrCl2, NiCl2, DMF, rt; (e) concd HCl, THF, rt.
cell lines were investigated with respect to the inhibitory potential
of moverastin A and the most potent moverastin derivative,
UTKO1. The inhibitory effect of moverastin A depends on cell type,
and there is a significant negative correlation between the sensitiv-
ity of each cell to moverastin A and the expression level of H-Ras, a
substrate of FTase (r = À0.86, p = 0.0013) (Fig. 3). This result
OH
OMOM
a
cis
UTKO7
OH
cis
93%
HO
HO
OH
MOMO
CHO
O
19
CHO
OH
CHO
d, e
b
UTKO1
cis
cis
UTKO9
27%
63%
OCH3
CHO
cis
c
O
HO
UTKO10
OH
HO
12%
CHO
UTKO8
Scheme 3. Synthesis of UTKO7–10. Reagents and conditions: (a) Rh-Al2O3, EtOH; (b) DEAD, PPh3, THF; (c) MeI, K2CO3, (n-Bu)4NÁHSO4, EtOAc–toluene, reflux; (d) PDC, CH2Cl2;
(e) concd HCl, THF, rt.

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M. Sawada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1385–1389
OH
OH
OMOM
OMOM
OH
HO
a
b, c
OHC
MOMO
6%
UTKO11
O
3 or 7
76%
MOMO
20
21
cis
OH
HO
32%
UTKO12
Scheme 4. Synthesis of UTKO11 and UTKO12. (a) O3, CH2Cl2, À78%, then PPh3; (b) CrCl2, NiCl2, DMF, rt; (c) concd HCl, THF, rt.
140
120
100
80
(a)
H-ras
60
tubulin
%
41
14
100
80
19
10
36
40
(b)
20
0
0
1
10
moverastin A
r = -0.86
UTKO1 ( M)
UTKO1 ( M)
IC50 ( M)
0
3
0.3
10
1
UTKO1
30
r = -0.38
Figure 2. Effect of UTKO1 on migration of EC17 cells. EC17 cells were incubated
with various concentrations of UTKO1 in the top chamber; the lower chamber
contained EC17-conditioned medium, obtained from 24-hour cultures of EC17 cells
maintained in RPMI1640 supplemented with 1% FBS. After 24 h, the number of cells
that migrated through the filter to the lower surface was counted. The results are
the mean SD of five different fields.
IC50 ( M)
supported our previous conclusion that moverastins inhibited
tumor cell migration due to the inhibition of FTase. On the other
hand, the cell migration inhibitory activity of UTKO1 also depends
on cell type, but there is no correlation with the expression levels
of H-Ras (r = À0.38, p = 0.40) (Fig. 3). These results suggested that
the inhibitory mechanism of cell migration by UTKO1 is different
from that of moverastin A. To understand the molecular basis by
which UTKO1 inhibits tumor cell migration, biochemical identifica-
tion of the protein target for UTKO1 is now under investigation.
Figure 3. Correlation between expression levels of H-Ras and inhibition of
migration by moverastin and UTKO1. (a) Western blot analysis of H-Ras
A
expression levels in seven tumor cell lines. Cell lysates were separated by
SDS–PAGE and then subjected to immunoblotting using anti-H-Ras antibody.
Quantitation of expression levels of H-Ras was analyzed by Image gauge and
normalized with the level of tubulin. Percentages represent the relative expression
level of H-Ras in tumor cells compared to the level in EC17 cells. (b) Simple linear
correlations between two parameters were calculated. Correlation coefficients (r)
are shown in this figure.

M. Sawada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1385–1389
1389
Table 1
Although UTKO1 was initially synthesized as an analogous com-
pound of moverastins, it possesses a different biological function
from cylindrol family, and therefore, UTKO1 is expected to be a
new lead compound in the search for more potent anti-metastatic
and anti-cancer agents.
Effects of UTKO compounds on cell migration, cell viability and in vitro FTase activity
in EC17 cells
IC50 (lM)
Cell viabilitya
Cell migration
FTaseb
Moverastin A
UTKO1
UTKO2
UTKO3
UTKO4
UTKO5
UTKO6
UTKO7
UTKO8
7.22
1.98
8.43
7.53
30.0
10.6
6.52
2.12
4.41
2.00
4.57
21.4
2.17
>77
46
87
>80
>130
45
55
16
18
18
14.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Acknowledgement
This work was supported by a grant from the Ministry of
Education, Culture, Sports, Science, and Technology.
Supplementary data
UTKO9
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.028. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
UTKO10
UTKO11
UTKO12
18
>28
17
a
For cell viability assay,
a trypan blue dye (15250-061, Gibco, Invitrogen)
exclusion assay were used to examine cell viability and performed according to
previously reported protocols.9
References and notes
b
For in vitro FTase assay, partially purified enzymes from EC17 cells were
incubated with [3H]-FPP plus recombinant GST-H-Ras in the presence or absence of
test compound. The reaction was terminated by the addition of TCA. The radioac-
tivity of the TCA insoluble fraction was measured.
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Symp. Quant. Biol. 2005, 70, 291.
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4. Friedl, P.; Wolf, K. Nat. Rev. Cancer 2003, 3, 362.
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Shindo, K.; Kitahara, T.; Imoto, M. Chem. Biol. 2005, 12, 1337.
6. Takai, K.; Kimura, K.; Kuroda, T.; Hiyama, T.; Nozaki, H. Tetrahedron Lett. 1983,
24, 5281.
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Chem. Soc. 1986, 108, 6048.
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867.
Our preliminary structure–activity relationship study revealed
that UTKO12 retained the same level of inhibitory activity toward
EC17 cell migration as that of UTKO1, indicating that formyl group
on benzene ring is not required for the inhibitory activity toward
EC17 cell migration. On the other hand, the formyl groups of
moverastins are essential for the FTase inhibition, because the
inhibitory activity of UTKO11 toward FTase has been lost.
9. Ormerod, M. G.; Collins, M. K.; Rodriguez-Tarduchy, G.; Robertson, D. J. Immunol.
Methods 1992, 153, 57.