Synthesis and evaluation of antimigratory and antiproliferative activities of lipid-linked [13]-macro-dilactones

Article abstract of DOI:10.1016/j.bmcl.2010.07.083

The biological activities of a family of novel, lipid-linked 13-membered-ring macro-dilactones are reported. These [13]-macro-dilactones were synthesized by diacylation of functionalized diols, followed by ring-closing metathesis under conditions we had p

Full text of DOI:10.1016/j.bmcl.2010.07.083

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5472–5476
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of antimigratory and antiproliferative activities
of lipid-linked [13]-macro-dilactones
*
*
Anniefer N. Magpusao, Richard T. Desmond, Katelyn J. Billings, Gabriel Fenteany , Mark W. Peczuh
Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The biological activities of a family of novel, lipid-linked 13-membered-ring macro-dilactones are
reported. These [13]-macro-dilactones were synthesized by diacylation of functionalized diols, followed
by ring-closing metathesis under conditions we had previously reported. Antimigratory, cytostatic and
cytotoxic activities of the compounds against cancer cells were evaluated. Compound 13 was the most
potent in the series, while compound 10 had the broadest concentration range of subtoxic antiprolifer-
ative activity. These compounds share common structural components, namely the [13]-macro-dilactone
Received 9 June 2010
Revised 19 July 2010
Accepted 20 July 2010
Available online 25 July 2010
Keywords:
templated by an octyl a-glucoside 4,6-diol.
Macro-dilactones
Octyl glucoside
Migrastatin
Ó 2010 Elsevier Ltd. All rights reserved.
Inhibitors
Cell migration
Cell growth
A general objective of bioorganic chemistry and chemical biology
is to generate synthetic small molecules that mimic the robust bio-
logical activities of complex natural products. Macrolactones are an
enticing class of molecules in this regard because they possess a
variety of molecular architectures and activities. Consideration of
migrastatin (1)1 and iso-migrastatin (iso-1)2 (Fig. 1) illustrates some
key points. These macrolactones are products of a polyketide syn-
thase pathway.3 The macrocycles thus contain an even number of
atoms; 1 has a 14-membered ring and iso-1 has a 12-membered
ring. As their names imply, these and related molecules have been
shown to inhibit the migration of a variety of human and mouse
cancer cells1a,4a–h and tumor metastasis in mouse xenograft
models.4d,5 Cell migration is a fundamental biological phenomenon
involved in a range of normal and disease states, including embry-
onic development, wound healing, blood vessel formation (angio-
genesis), immune system function, and tumor metastasis. Cell
migration has become recognized as a process that could be targeted
for drug development, particularly for the treatment of cancer at the
level of angiogenesis (which is involved in the early development of
malignant solid tumors and depends on the growth and migration of
vascular endothelial cells) and cancer cell invasion and metastasis.6
The antimigratory activity of iso-1 exceeds that of 1 by several
orders of magnitude,4g and 1 is thought to be a shunt metabolite
of iso-1.3b A number of synthetic and semisynthetic analogs of
migrastatin have also been shown to possess considerable antimi-
gratory activity.4b–h Recently, the synthetic macroketone 2, which
potently inhibits cell migration,4b has been shown to bind and inhi-
bit fascin, a crosslinker of actin filaments involved in cell migration,
suggesting that fascin could be an antimetastatic drug target.5 In
addition, the natural product-like molecule 3, which contains a
macrocycle fused to a quinic acid scaffold, is also a potent inhibitor
of cell migration.4e The ability of 2 and 3 to recapitulate the biolog-
ical activity of the natural product itself suggests that other macro-
cycles of intermediate complexity may similarly inhibit the
migration of cells.
We recently reported on the synthesis of some macro-dilactones
that complement natural [12]- and [14]-macrocycles because they
contain 13 atoms in their ring (e.g., compound 4 in Fig. 1).7 A struc-
tural analysis of the new [13]-macro-dilactones demonstrated that
they possessed helical chirality that arose from the size of the ring,
the planar nature of their alkene and ester functional groups, and
the stereochemistry of individual backbone stereocenters. As a con-
sequence of the helical chirality, epoxidation of the double bond
embedded in the macrocyclic backbone was highly selective. The
unique molecular topology exhibited by the [13]-macro-dilactones
and the fact that their ring size was similar to the migrastatin and
iso-migrastatin isomers prompted us to consider their potential
ability to inhibit cell migration. We were additionally inspired by
the remarkable activity of the natural macrolactones despite their
relatively sparse functionality and stereochemistry. Reported here
are the synthesis and antimigratory and antiproliferative activities
* Corresponding authors. Tel.: +1 860 486 6645 (G.F.); tel.: +1 860 486 1605
(M.W.P.).
E-mail addresses: gabriel.fenteany@uconn.edu (G. Fenteany), mark.peczuh@
uconn.edu (M.W. Peczuh).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.083

A. N. Magpusao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5472–5476
5473
O
O
NH
H3CO
O
O
O
HO
O
HN
O
O
O
O
OH
O
iso-1
OCH3
1
OH
OCH3
O
2
H
N
O
O
HO
O
HO
O
O
O
OH
3
4
Figure 1. Migrastatin (1), iso-migrastatin (iso-1), ‘macroketone’ 2, quinic acid-based macrolide 3, and [13]-macro-dilactone 4.
of a family of [13]-macro-dilactones ( Fig. 2). A unifying feature
among the active [13]-macro-dilactones was the presence of an oc-
tyl (C8) glycoside. It is worth noting that two natural products con-
taining a [13]-macrolactone, spongidepsin,8 and amphidinolide P,9
both have modest antiproliferative activity.
to contain up to three components that might impart biological
activity. The primary feature was the [13]-macro-dilactone itself,
which is common among 4–13. The nature of the 1,3-diol used
to template the macro-dilactone was also varied. Macrocycles 4
and 7, for example, utilized acyclic 1,3-diols from 1,3-butane diol
and an acylated threoninol,10 respectively. The 1R,2S and 1S,2S iso-
mers of 2-hydroxycyclohexyl methanol11 were used to template 5
[13]-Macro-dilactones and related molecules evaluated in this
investigation are shown in Figure 2. The molecules were designed
O
O
O
O
O
O
O
O
O
O
H
N
O
C9
OCH3
OBn
O
O
O
O
O
O
OBn
O
O
O
O
O
8a C1 = α
8b C1 = β
5
4
6
7
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
C8
OR
C8
C8
C8
OBn
H
O
H
OH
OH
O
O
O
O
O
OR
OH
OH
OBn
O
O
O
10 R = Bn
12
9
13
11 R = CH3
O
O
O
OH
O
O
O
O
O
O
O
O
C8
OBn
O
C8
OH
O
C8
OBn
C8
OR
Ph
Ph
O
O
HO
OBn
OH
OBn
OR
O
15 C1 = α R = Bn
16 C1 = α R = H
17 C1 = β R = Bn
18 C1 = β R = H
19a C1 = α
19b C1 = β
14
20
Figure 2. [13]-Macro-dilactones and related molecules synthesized and evaluated in this study.

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A. N. Magpusao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5472–5476
PhCH(OCH3)2
pTSA
ment of a lipophilic alkyl chain to the molecule represented the fi-
nal structural feature.
O
O
O
O
O
C8
OR
HO
HO
C8
OH
The [13]-macro-dilactones in Figure 2 were synthesized by a
strategy we have previously described.7 Scheme 1 depicts the syn-
thesis of [13]-macro-dilactone 10. It is illustrative of the approach
taken for all the [13]-macro-dilactones. Preparation of 10 com-
menced with the protection of octyl glucoside 2112 as its 4,6-O-
Ph
O
DMF
20%
OR
OH
19a R = H
20 R = Bn
NaH
BnBr
21
92%
benzylidene. The
a-anomer of the benzylidene, 19a, was isolated
1) pTsOH, MeOH
in 20% yield, as was the b-anomer, from an initial ꢀ1:1 mixture
of C1-anomers. The C2 and C3 hydroxyl groups were then
efficiently converted to their corresponding benzyl ethers to
deliver 20 (92% yield). The 4,6-O-benzylidene of 20 was then
removed, followed by acylation of the exposed hydroxyl groups
with 4-pentenoic acid to give 14 in 60% yield over two steps.
Ring-closing metathesis of the alkene moieties in 14 provided 10
(50% yield).
2) 4-pentenoic acid,
DCC, DMAP, DCM
60% (2 steps)
O
O
O
O
O
O
O
O
C8
OBn
Grubbs' 2nd Gen.
CH3Ph, 90 °C
C8
OR
With the [13]-macro-dilactones in hand, the next objective was
to assay their antimigratory activity. In addition, intermediates
14–20 from the synthesis of the macrocycles were also tested.
We employed a scratch-wound assay, where a small wound—
mechanically scratched in a cell monolayer—triggers cell migration
and closure of the wound. The progress of wound closure was fol-
lowed over time,13 essentially as previously described.14 The activ-
ity of these compounds was evaluated in BT-20 human breast
carcinoma cells, T47D human breast carcinoma cells, MDA-MB-
231 human breast carcinoma cells, MDA-MB-435 human mela-
noma cells, 4T1 mouse breast carcinoma cells, and Madin–Darby
canine kidney (MDCK) epithelial cells. Compounds 10, 11, 12, 13,
19a, and 19b displayed weak antimigratory activity in BT-20,
MDA-MB-435, and MDCK cells but not in T47D cells, MDA-MB-
231, or 4T1 cells. Of these, 11–13 were the most bioactive, and they
appeared to have the greatest activity in BT-20 cells of all the cell
lines tested. The activity of these compounds was thus further
examined in BT-20 cells. The rate of wound closure over a range
of compound concentrations was determined from digital micro-
scope images.14,15 Potential cytotoxicity was determined at the
end of each experiment. The concentration–response profiles
revealed a very narrow range of subtoxic antimigratory activity be-
tween the minimum inhibitory concentration (MIC) and the mini-
mum lethal concentration (MLC), as shown in Table 1. This
precluded determination of meaningful half-maximal inhibitory
O
O
OBn
OR
50%
O
O
14 R = Bn
10
Scheme 1. Synthesis of octyl glucoside-fused [13]-macro-dilactone 10.
Table 1
Antimigratory activity of compounds 11, 12, and 13 in BT-20 breast cancer cells
Compound
MICa
(
lM)
MLCb
(lM)
11
12
13
75
50
50
125
125
75
a
Minimum inhibitory concentration defined as the lowest concentration at
which there was a statistically significant decrease by Student’s t-test in the mean
rate of migration in the wound closure assay compared to parallel controls from
three independent experiments for each compound.
Minimum lethal concentration defined as the lowest concentration at which
there was evidence of cytotoxicity based on the trypan blue dye exclusion assay
conducted at the end of each wound closure experiment.
b
and 6. The remainder of the macrocycles were templated via the C4
and C6 hydroxyls of a protected glucoside, as in 8–13. The attach-
Figure 3. Antiproliferative activity at 100 lM in BT-20 breast cancer cells. Cells were plated at uniform density onto 96-well tissue culture plates and allowed to attach and
grow for 24 h. At that point, the mean cell number (‘initial’) was determined in a tetrazolium salt-based assay. Compounds were added to experimental cultures, which were
grown for another 48 h, along with parallel controls that had been treated with 1% DMSO alone. Values represent means with error bars signifying standard errors of the mean
for three independent experiments for each treatment (with four replicate wells for each experiment for 12 total), normalized to the mean initial cell number. Black bars
represent compounds that had no statistically significant effect on cell growth. Blue bars represent compounds that appeared cytostatic at 100
numbers that were significantly lower by Student’s t-test than the mean control cell number at 48 h but not significantly reduced from the mean initial cell number. Red bars
represent compounds that appeared cytotoxic at 100 M, defined as cell numbers that were significantly lower by Student’s t-test than the mean initial cell number.
lM, defined as mean cell
l

A. N. Magpusao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5472–5476
5475
Table 2
Activities of antiproliferative compounds in BT-20 breast cancer cells
b
Compound
MICa
(l
M)
IC50
(
lM)
95% confidence intervalc
(lM)
MLCd
(l
M)
Therapeutic index (MLC/MIC)
10
11
12
13
15
17
19a
19b
10
50
50
10
25
25
10
50
85.1
82.8–87.4
89.2–93.6
102.6–111.5
37.3–38.8
51.2–53.8
60.1–62.1
43.8–50.1
69.4–74.8
300
300
>300
50
75
100
75
30
6
>6
5
3
4
91.4
107.0
38.1
52.9
61.1
47.1
72.1
7.5
6
300
a
Minimum inhibitory concentration, defined as the lowest concentration at which there was a statistically significant reduction by Student’s t-test in mean cell number
from the mean control cell number at 48 h from three independent experiments (with four replicates for each treatment per experiment for 12 replicates total) in a
tetrazolium-salt-based assay.
b
Half-maximal concentration for inhibition of cell growth, calculated from mean cell numbers at 48 h for a range of concentrations of each compound.
c
95% confidence interval for the IC50
.
d
Minimum lethal concentration, defined as the lowest concentration at which there was a statistically significant reduction by Student’s t-test in mean cell number at 48 h
from the mean initial cell number.
concentration (IC50) values for the compounds’ antimigratory
activity. We concluded that the effects on wound closure observed
at subtoxic concentrations were likely due to incipient toxicity.
We next evaluated the effects of the compounds on the viability
and growth of BT-20 cells.16 We first tested these compounds at
100 lM and found that compounds 9, 10, 11, 12, 13, 15, 17, 19a,
and 19b displayed either subtoxic antiproliferative (cytostatic) or
cytotoxic activity at this concentration (Fig. 3). Compounds that
Acknowledgments
This work was supported by National Institutes of Health Grant
GM077622 (to G. Fenteany) and National Science Foundation
Career Award CHE-0546311 (to M.W. Peczuh). K.J. Billings was
supported in part by National Science Foundation Research
Experience for Undergraduates Grant CHE-0754580. W.S. Fyvie
(University of Connecticut) prepared 4–6 and 8a, 8b.
were considered cytostatic at 100 lM (9, 10, 11, 12, and 19b) re-
duced the rate of cell proliferation over 48 h compared to controls
treated with dimethyl sulfoxide (DMSO) alone but did not reduce
cell numbers below the initial values. Compounds that were con-
sidered cytotoxic at this concentration (13, 15, 17, and 19a) not
only inhibited cell growth but also caused a reduction in cell num-
bers from the initial values due to cell death.
Supplementary data
Supplementary data (synthetic schemes and spectral data)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.07.083.
Compounds 10, 11, 12, 13, 15, 17, 19a, and 19b exhibited either
pronounced cytostatic or cytotoxic activity in the initial assay at
References and notes
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Danishefsky, S. J. J. Am. Chem. Soc. 2010, 132, 3224.
5. Chen, L.; Yang, S.; Jakoncic, J.; Zhang, J. J.; Huang, X. Y. Nature 2010, 464, 1062.
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9. Ishibashi, M.; Takahashi, M.; Kobayashi, J. J. Org. Chem. 1995, 60, 6062.
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12. Porzelle, A.; Williams, C. M.; Schwartz, B. D.; Gentle, I. R. Synlett 2005, 2923.
13. BT-20, T47D, MDA-MB-231, MDA-MB-435, 4T1, and MDCK cells were obtained
from the American Tissue Culture Collection or Kam C. Yeung (University of
Toledo). Cells were grown in a tissue culture incubator at 37 °C with 5% CO2 in
minimum essential medium with 10% fetal bovine serum (FBS) for BT-20 cells
or 10% newborn calf serum for MDCK cells, RPMI 1640 with 10% FBS (T47D and
100 lM and were tested over a range of concentration to establish
concentration–response profiles for each compound. Compound 9
only very weakly inhibited cell proliferation, and, when tested over
a range of concentrations, the concentration–response profile was
virtually flat.17 Compounds 10, 11, 12, 13, 15, 17, 19a, and 19b, on
the other hand, displayed cytostatic activity that in most cases ap-
peared separable from the cytotoxicity observed at higher concen-
trations (Table 2). The criterion for this conclusion was based on
the MLC/MIC ratio (‘therapeutic index,’ often also defined as half-
maximal lethal concentration divided by the IC50). By this measure,
therefore, these compounds have subtoxic cytostatic activity, par-
ticularly in the case of 10, with a therapeutic index of 30.
The compounds possessing antiproliferative activity, 10, 11, 12,
13, 15, 17, 19a, and 19b, share common structural components
that had been included in their design. To our surprise, the [13]-
macro-dilactone unit was not essential for activity. All of the bioac-
tive compounds contain a glucosyl unit and an octyl (C8) chain.
Furthermore, the majority of these molecules contain an a-linkage
between the glucose unit and the alkyl chain. However, the most
promising compounds, as measured by either therapeutic index
(compound 10) or IC50 (compound 13), contained the [13]-
macro-dilactone. In the case of 10 in particular, the cytostatic
and cytotoxic activities were clearly separable, with a wide con-
centration range for its subtoxic antiproliferative activity. Based
on this and its micromolar IC50, it is likely that 10 does not act
by merely having a non-specific effect on cells, such as disrupting
the integrity of cellular membranes. Instead, this compound may
target some factor(s) involved in cell cycle progression. In sum-
mary, we have demonstrated the application of our published syn-
thesis to access a novel class of antiproliferative agents.

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4T1 cells), Dulbecco’s modified Eagle medium with 10% FBS (MDA-MB-231 and
cells were wounded 30 min after treatment with different concentrations of
these compounds or DMSO alone, and digital images of the wounds were
captured at 3, 6, 9, 12, 24, 36, 48, 60, and 72 h post-wounding. The trypan blue
dye exclusion assay was performed at the end of each experiment. The area of
the wound at each time point was subsequently calculated from the digital
images with NIH ImageJ software (http://rsbweb.nih.gov/ij/). Statistically
significant differences were determined by unpaired, two-tailed Student’s
t-test (p >0.05).
MDA-MB-435 cells). The medium was changed every 2 days. Cells were plated
on 24-well tissue culture-treated plates. When the cells reached confluence,
the medium was changed again one more time, and the experiment was
started the next day. Cells were treated with 100 lM of each compound or 1%
DMSO alone, corresponding to the concentration of DMSO carrier solvent in the
experimental treatments. (We found in preparatory experiments that 1%
DMSO had no detectable effect on the migration or growth of any of the cells
compared to medium alone.) The compounds or DMSO alone, mixed with fresh
medium before addition to the cell cultures, were delivered to the cells as the
medium was changed. After 30 min, the cell monolayers were scraped with a
micropipet tip to produce oval-shaped wounds of 0.5–1.0 mm2 in area.
Progress of wound closure was followed by observation on an inverted
microscope every 12 h for 72 h. At each time point, wounds were scored as
either closed or opened. Compounds that resulted in wounds that were still
open after the wounds for the control DMSO treatments were closed were
considered antimigratory. The trypan blue dye exclusion assay was performed
at the end of each experiment to gauge potential cytotoxicity of the
compounds.
16. BT-20 cells were plated onto 96-well tissue culture-treated plates at 5000 cells
per well in 100
incubator at 37 °C with 5% CO2. Initial cell numbers were determined for cells
on control plate by adding the tetrazolium salt WST-8 as per the
lL of growth medium and incubated for 24 h in a tissue culture
a
manufacturer’s instructions (Cell Counting Kit-8, Dojindo Molecular
Technologies), incubating for 3 h at 37 °C and then measuring absorbance at
450 nm in
a UV–visible absorbance plate reader (Molecular Devices
Spectramax Plus 384). The experimental plates were treated with
compounds or DMSO alone and incubated in the tissue culture incubator for
another 48 h. Cell numbers were then measured as above. Statistically
significant differences were determined by unpaired, two-tailed Student’s
t-test (p >0.05). IC50 values were calculated with GraphPad Prism software
from concentration–response data.
14. Mc Henry, K. T.; Ankala, S. V.; Ghosh, A. K.; Fenteany, G. ChemBioChem 2002, 3,
1105.
15. Concentration–response profiles for compounds 11, 12, and 13, which
appeared most active in the initial wound closure assay, were obtained in a
quantitative and kinetics wound closure assay. Confluent monolayers of BT-20
17. At concentrations above 500 lM, 9 by itself exhibited light scattering at the
wavelength used in the spectrophotometric assay, preventing evaluation of
this compound at high concentrations.