Synthesis of Migrastatin Analogues as Inhibitors of Tumour Cell Migration: Exploring Structural Change in and on the Macrocyclic Ring

Article abstract of DOI:10.1002/chem.201502861

Migrastatin and isomigrastatin analogues have been synthesised in order to contribute to structure-activity studies on tumour cell migration inhibitors. These include macrocycles varying in ring size, functionality and alkene stereochemistry, as well as glucuronides. The synthesis work included application of the Saegusa-Ito reaction for regio- and stereoselective unsaturated macroketone formation, diastereoselective Brown allylation to generate 9-methylmigrastatin analogues and chelation-induced anomerisation to vary glucuronide configuration. Compounds were tested in vitro against both breast and pancreatic cancer cell lines and inhibition of tumour cell migration was observed in both wound-healing (scratch) and Boyden chamber assays. One unsaturated macroketone showed low affinity for a range of secondary drug targets, indicating it is at low risk of displaying adverse side effects.

Full text of DOI:10.1002/chem.201502861

DOI: 10.1002/chem.201502861
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Natural Products |Hot Paper|
Synthesis of Migrastatin Analogues as Inhibitors of Tumour Cell
Migration: Exploring Structural Change in and on the Macrocyclic
Ring
Daniele Lo Re,[a] Ying Zhou,[a] Joanna Mucha,[c] Leigh F. Jones,[a] Lorraine Leahy,[b]
Corrado Santocanale,[b] Magdalena Krol,[c] and Paul V. Murphy*[a]
Abstract: Migrastatin and isomigrastatin analogues have
been synthesised in order to contribute to structure–activity
studies on tumour cell migration inhibitors. These include
macrocycles varying in ring size, functionality and alkene ste-
reochemistry, as well as glucuronides. The synthesis work in-
cluded application of the Saegusa–Ito reaction for regio-
and stereoselective unsaturated macroketone formation, dia-
stereoselective Brown allylation to generate 9-methylmigras-
tatin analogues and chelation-induced anomerisation to vary
glucuronide configuration. Compounds were tested in vitro
against both breast and pancreatic cancer cell lines and in-
hibition of tumour cell migration was observed in both
wound-healing (scratch) and Boyden chamber assays. One
unsaturated macroketone showed low affinity for a range of
secondary drug targets, indicating it is at low risk of display-
ing adverse side effects.
Introduction
Tumour metastasis is a process in which cancer cells detach
from a primary tumour, migrate and then colonize a distant
organ and the process leads to mortality for cancer patients.[1]
Small molecules that interfere with tumour cell migration[2]
have significant potential as anti-metastatic drugs[3] or as
tools[4] for the study of tumour cell migration. Migrastatin (1),
is a natural product that inhibits tumour cell migration (mm
range) in vitro. Truncated[5] or simpler analogues of migrastatin,
such as 2, 3, MGSTA-4, and MGSTA-5 (Figure 1), prepared in
Danishefsky’s laboratory,[6] have been reported to show orders
of magnitude higher activity than migrastatin itself in vitro and
both 3 and MGSTA-5[7] and other compounds[8] have demon-
strated inhibitory activity in vivo. We have been engaged in
the synthesis of migrastatin analogues[9] and recently through
collaboration with Anderson and Nobis have shown that mac-
roketone MGSTA-5 inhibited E-cadherin dynamics in vivo, in
a manner consistent with increased cell adhesion and reduced
invasive potential.[10] It has been reported that the target for
Figure 1. a) Migrastatin (1) and truncated analogues 2–3 IC50 values (in pa-
rentheses) in Boyden chamber assay against 4T1 mouse mammary cancer
cells.[6] b) Modification of migrastatin core in this paper.
MGSTA-5 is fascin, which is recognized as being involved in
cell motility and migration processes and is upregulated in
many human tumours as well as in several cancer cell lines in-
cluding breast,[11] pancreatic[12] and colon cancer cells.[13] Impor-
tantly, fascin is also involved in the chemotherapeutic resist-
ance of breast[14] and colon[15] cancer cells. However, whether
fascin is the target for MGSTA-5 is still debated[16] and there is
a belief that there may be other unidentified target(s) of mi-
grastatin and its analogues.[16]
[a] Dr. D. Lo Re, Dr. Y. Zhou, Dr. L. F. Jones, Prof. P. V. Murphy
School of Chemistry, National University of Ireland, Galway (Ireland)
E-mail: paul.v.murphy@nuigalway.ie
[b] L. Leahy, Prof. C. Santocanale
Centre for Chromosome Biology
School of Natural SciencesNational University of Ireland
Galway(Ireland)
For these reasons the synthesis of analogues of migrastatin,
including new analogues, continues to be of interest.[6–8,16–19]
Here we report the preparation and migrastatin analogues
shown in Figure 2. The modifications have included variation
in macrocycle ring size and functionality within the ring as well
as varying alkene geometry and glucuronidation. Included are
[c] Dr. J. Mucha, Prof. M. Krol
Department of Physiological Sciences, Faculty of Veterinary Medicine
Warsaw University of Life Science, Warsaw (Poland)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502861.
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the allylic bromide 9 and subse-
quently reacting 9 with b-keto-
sulfones 10a–c as previously de-
scribed[6] to give 11 a–c. Ring-
closing metathesis followed by
TBS removal afforded MGSTA-5[6]
and new analogues MGSTA-2
and MGSTA-9. Macroketone 12b
was reacted with LHMDS and
TMSCl to give an enolate which
was then oxidized by Saegusa–
Ito conditions to afford the a,b-
unsaturated macroketone 13
which after desilylation gave
MGSTA-6.
Macrothiolactones
were prepared by the Mitsunobu
reaction of thioacids 15a–b with
the allylic alcohol 5. Thioacids
15a and 15b were firstly readily
prepared by treating the corre-
sponding carboxylic acid with
Lawesson’s reagent under micro-
wave irradiation.[20] The thioacids
15a–b were not stable to silica
gel and hence were instead puri-
fied by distillation under re-
duced pressure (Kugelrohr, P=
50”10À3 mbar, T=458C, 2 h) or
were used without distillation in
Figure 2. Migrastatin analogues prepared in this manuscript.
the next step. Coupling of 15a–
b to 5 using Mitsunobu condi-
the syntheses of related isomigrastatin analogues. The synthe-
sis of these various analogues will facilitate establishment of
structure–activity relationships and in helping to try to identify
more potent analogues. The activity of selected compounds in
relevant bioassays was subsequently investigated and results
of these are described. The project has also provided a good
testing ground for synthetic methodology such as Brown ally-
lation, the Saeguso–Ito reaction and Lewis acid promoted
anomerisation.
tions, and subsequent RCM and TBS group removal afforded
macrothiolactones MGSTA-3 and 7. The compounds MGSTA-
1 to 9, were purified using flash chromatography (representa-
tive mobile phase: petroleum ether/ethyl acetate, 1:1). The
purity of these macrocyclic agents could be increased by their
distillation under reduced pressure (Kugelrohr, P=0.05 mbar,
T=908C, distillation time from 2 h to 24 h).
Synthesis of isomigrastatin analogues
Isomigrastatin 18, a natural product isolated from Streptomy-
ces platensis[21] and a precursor to migrastatin, is a potent in-
hibitor of tumour cell migration.[17d] In view of this, we have
used monoprotected diol 19, readily available in four steps
from 4,[10] for the preparation of truncated isomigrastatins.
Firstly the direct exchange of the OH in 19 for Br was attempt-
ed, but when 19 was treated with polymer-bound Ph3P and
CBr4 in CH2Cl2, the methoxy group at C-4 migrated to the C-
1 position giving allylic bromide 20 (68%), with no trace of the
desired compound being found. Attempts to form the tosylate
or chloride from 19 led to complex reaction mixtures. On the
other hand, when 19 was coupled with carboxylic acid 6b
under Mitsunobu conditions, the desired ester 22 was isolated
in a 57% yield together with a small amount of rearranged
compound 21 (14%). With 22 in hand, RCM was next attempt-
ed. Unfortunately, reaction of 22 with the Grubbs II in toluene
Results and Discussion
Modification of the macrocycle structure and ring size
The synthetic work began from intermediate 5, which was pre-
pared in ~two gram quantity using the route recently devel-
oped in our laboratory.[10] This intermediate was first used to
prepare 13–15-membered ring structures based on macrolac-
tones, macroketones and macrothiolactones. Firstly, the allylic
alcohol 5 was coupled to carboxylic acids 6a–c (Scheme 1)
under Mitsunobu conditions and the esters formed were then
treated with the Grubbs II metathesis catalyst to afford the
macrolactones 8a–c by ring-closing metathesis (RCM). Removal
of the TBS group using HF.pyridine gave MGSTA-4[6] and its
new 13- and 15-membered analogues MGSTA-1 and MGSTA-8.
Macroketones, were prepared by conversion of alcohol 6 into
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relative configuration was determined by obtaining
an X-ray crystal structure determination for 26. Diol
26 was then protected as its di-O-TBS ether and sub-
sequent treatment with 10% TsOH in MeOH removed
the TBS group from the primary position to give the
alcohol 27. This alcohol 27 was oxidized with the
Dess–Martin periodinane and the aldehyde obtained
was then treated with the Ando phosphonate[17b,k,24]
35 to give 28. Reduction of the ester group in 28
using DIBAL-H gave a primary alcohol and its subse-
quent Mitsunobu reaction gave 29. However, RCM of
29, under several conditions and reaction times, only
gave complex reaction mixtures (TLC) with no trace
of the desired product. Alcohol 32 was prepared
with a view to resolving this problem and for applica-
tion in isomigrastatin analogue synthesis. Since the
presence of the TBS group was problematic in the at-
tempted RCM with compound 22, the secondary al-
cohol was protected as its MOM ether. Regioselective
protection of the primary alcohol in 26 with a TBDPS
group gave 30 and subsequent protection of its sec-
ondary alcohol as MOM ether afforded the orthogo-
nally protected alkene 31, which gave 32 after TBDPS
removal.
The Mitsunobu reaction of 32 gave MGSTA-10.
Ring-closing metathesis of MGSTA-10 afforded the
cis- and trans-alkene-containing macrocycles MGSTA-
11 and MGSTA-12 in a 1:0.85 ratio. The MOM pro-
tecting group was removed from both intermediates
by using TMSBr to give truncated isomigrastatin ana-
logues MGSTA-13 and MGSTA-14. For the synthesis
of migrastatin-core analogue MGSTA-15 bearing the
methyl group at C-9, the primary alcohol 32 was oxi-
dized using the Dess–Martin periodinane, and then
treated with the Ando phosphonate[17b,k,24] 35 giving
the trisubstituted alkene 33, with the Z configuration.
The configuration of the newly formed double bond
was supported by NOE spectroscopic experiments,
for which NOEs were observed for 33 as indicated in
Scheme 1. Synthesis of migrastatin core analogues: a) 6a–c, Ph3P, DIAD, PhCH3, RT; 7a
(69%), 7c (76%); b) Grubbs II catalyst, PhCH3, reflux, 8a (68%), 12a (99%), 17a (88%),
8c (73%), 12c (60%); c) HF·Py, THF, RT; MGSTA-1 (61%), MGSTA-2 (84%), MGSTA-3
(85%), MGSTA-6 (90%), MGSTA-7 (63%), MGSTA-8 (54%), MGSTA-9 (82%); d) CBr4, Ph3P
polymer-bound, CH2Cl2; e) 10a–c, DBU, PhCH3 then 9, RT, 11 a (51%) 11 c (51%), from 9;
f) Na/Hg, MeOH, RT; g) TMSCl, LHMDS, THF, 08C, 2 h; h) Pd(OAc)2, CH3CN, RT, 2 h, 78%
from 12b; i) Lawesson’s reagent, CH2Cl2, MW, 1008C, 10 min; j) 15a,c, Ph3P, DIAD, PhCH3,
RT; 16a (42%), 16b (39%); k) Grubbs II catalyst, CH2Cl2, MW, 1008C, 30 min, 17b (49%).
DBU=1,8-diazabicyclo[5.4.0]undec-7-ene; DIAD=diisopropyl azodicarboxylate;
LHMDS=lithium bis(trimethylsilyl)amide; TMSCl = chlorotrimethylsilane.
only gave a complex mixture (TLC evidence) and the desired
Scheme 3. The reduction of 33 with DIBAL-H and subsequent
esterification using Mitsunobu conditions gave the RCM pre-
cursor to S6 (see Supporting Information for structure of S6).
As for 29, the RCM reaction of this precursor was unsuccessful
and macrocyclic product could not be detected under several
reaction conditions. Fortunately, addition of 1,4-benzoqui-
none[25] to the reaction mixture led to formation of desired 34
in modest yield, which after deprotection afforded the 9-
methyl-macrolactone MGSTA-15.
1
product was not detected (MS and H NMR spectroscopic evi-
dence). The TBS group was removed from 22 and the secon-
dary alcohol generated then protected as the MOM ether 23.
In this case, 23 readily cyclized using Grubbs II to afford macro-
cycle 24 (Scheme 2) as previously described.[22] A mechanistic
proposal for formation of 20 and 21 is suggested in
Scheme 2B.
Synthesis of 9-methylmacrolactone analogues of migrastatin
and isomigrastatin
Synthesis of migrastatin glucuronides
Brown allylation was used as the key step to generate an ana-
logue bearing a methyl group instead of methoxy group at C-
9. Thus Brown allylation[23] of 25 (Scheme 3) with cis-butene
and b-methoxydiisopinocampheylborane installed two contig-
uous stereocenters with subsequent removal of the benzoate
group enabling diol 26 to be isolated in moderate yield; the
Glucuronides are an important class of phase 2 metabolites
and glucuronidation in vivo of small molecules usually leads to
compounds with higher water solubility, which are more easily
excreted through the kidney.[26] Glucuronide metabolites are
often biologically inactive but in some cases can display inter-
esting bioactivity.[27,28] The 6-O-glucuronide of morphine (M6G)
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Scheme 2. A) Synthesis of 24: a) CBr4, Ph3P polymer-bound, CH2Cl2, RT; 1 h,
68%; b) 6b, Ph3P, DIAD, PhCH3, RT; 57%; c) TBAF, THF, RT, 3 days, 60%;
d) MOMCl, CH2Cl2, DIPEA, 08C to RT, 18 h, 60%; e) Grubbs II catalyst, PhCH3,
1
reflux; 31% IC50 values using 4T1 mouse mammary adenocarcinoma cells.
2IC50 values using MDA-MB-231 human breast adenocarcinoma cells.[17d]
B) Proposed mechanism for formation of 20 and 21 under Mitsunobu and
Appel conditions. TBAF=tetrabutylammonium fluoride; MOMCl=methoxy-
methyl chloride; DIPEA = N,N-diisopropylethylamine.
displays full agonist properties at the m1-opioid receptor and
appears to be more potent than morphine itself with fewer
side effects.[26,29] In addition, the glucuronide of ezetimibe, a se-
lective inhibitor of cholesterol absorption, is more active than
ezetimibe itself.[26,30] The biological activity of thiocolchicoside,
a myorelaxant used for painful muscle contraction, has been
attributed to the 3-O-glucuronidated metabolite.[26] A glucuro-
nide can be hydrolyzed through an enzymatic or non-enzymat-
ic reaction to give the active aglycon[27] In this latter context
glucuronidation has been used as for the preparation of SN-38
and taxol prodrugs.[27,31] The strategy relies on the fact that b-
glucuronidase is generally overexpressed in tumour tissues. In
view of these interesting properties of glucuronides and of the
possibility that in vivo generation of glucuronides can occur
we decided to prepare the glucuronide of MGSTA-5. Thus,
MGSTA-5, which had been prepared on a 100 mg scale, was
coupled with trichloroacetimidate 36[32] using TMSOTf afford-
ing exclusively the protected b-glucuronide 37 (Scheme 4). Si-
multaneous removal of benzoate protective groups and hy-
drolysis of the methyl ester using aqueous NaOH gave the
macroketone b-glucuronide MGSTA-16. In order to obtain the
a-anomer of this glucuronide, we applied the chelation-in-
duced anomerization,[33] which has been of interest in our lab-
oratory[33d] for the synthesis of the a-glucuronide. Thus, glucur-
onide 37 was treated with TiCl4 in CDCl3 at 48C giving the a-
glucuronide 38 as the only product. Deprotection and ester
hydrolysis gave MGSTA-17.
Scheme 3. Synthesis of methyl migrastatin core analogues: a) i) cis-Butene,
nBuLi, THF, 45 min, À208C; ii) (+)-Ipc2BOMe, 30 min, À788C; iii) BF3·Et2O,
then 25, 28 h; iv) NaOH 1n, H2O2 30%, 16 h; b) K2CO3, MeOH, RT, 24 h, 32%
from 25; c) TBSOTf, DCM, DIPEA, 08C, 5 h; d) p-TSA, MeOH, 3 h 08C, 62%
from 26; e) DMP, CH2Cl2, RT, 18 h, 88%; f) 35, THF, NaH, 08C, 1 h, then alde-
hyde obtained from oxidation of 27, À788C to RT, 18 h, 85%; g) DIBAL,
CH2Cl2, À788C, 10 min 63%; h) Ph3P, DIAD, 6-heptenoic acid, RT, 2 h, 81%;
i) Grubbs II catalyst, PhCH3, reflux; j) TBDPSCl, imidazole, CH2Cl2, RT, 62%;
k) MOMCl, DIPEA, CH2Cl2, RT, 48 h, 89%; l) TBAF 1m, THF, RT, 18 h, 98%;
m) Ph3P, DIAD, 6-heptenoic acid, RT, 4 h, 88%; n) Grubbs II catalyst, PhCH3,
1 h, heat at reflux, 33% MGSTA-12 and 31% MGSTA-11; o) TMSBr, CH2Cl2,
À208C, 2 h, 77% (MGSTA-13), 74% (MGSTA-14); p) DMP, CH2Cl2, pyridine,
RT, 4 h, 70%; q) 35, THF, NaH, 08C, 1 h, then aldehyde obtained from the oxi-
dation of 32, À788C to RT, 15 h, 73%; r) DIBAL, CH2Cl2, À788C, 15 min. 76%;
s) Ph3P, DIAD, 6-heptenoic acid, RT, 3 h, 78%; t) Grubbs II catalyst, PhCH3,
benzoquinone, 808C, 5 h, 13%; u) TMSBr, CH2Cl2, À208C, 3 h, 60%. DIBAL=
diisobutylaluminium hydride; DMP=Dess–Martin periodinane; p-TSA=p-tol-
uenesulfonic acid; Ipc2BOMe = (+)-B-methoxydiisopinocampheylborane;
TBSOTf = tert-butyldimethylsilyl trifluoromethanesulfonate; TBDPSCl = tert-
butyl(chloro)diphenylsilane; TMSBr = bromotrimethylsilane.
Synthesis of glutarimide-containing analogues of migrasta-
tin for which alkene geometry is varied
The preparation of MGSTA-18 and MGSTA-19, containing the
glutarimide side chain, for which olefin geometry is modified
in the macrocyclic core was included in the synthetic study.
Thus, diene precursors were prepared from freshly made alde-
hyde 39 (Scheme 5) by using Wittig reagent 40 to give the E-
alkene 41 with high stereoselectivity (E/Z=25:1); this reaction
provided the isomer, which would enable preparation of mi-
grastatin analogues with an E-alkene at C-3 in the macrocycle.
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Scheme 4. Synthesis of MGSTA-17 and MGSTA-16: a) 36 TMSOTf, MS
(AW 300), CH2Cl2, À788C, 5 h, 60%; b) TiCl4, CDCl3, 48C, 69%; c) NaOH aq.,
MeOH, RT, 18 h, MGSTA-17 (61%), MGSTA-16 (73%). TMSOTf = Trimethyl-
silyl trifluoromethanesulfonate.
Scheme 6. Synthesis of 53a,b: a) DMP, CH2Cl2, RT, 1.5 h; b) dimethyl methyl-
phosphonate, BuLi, À78 to 08C, 15 min; c) DMP, CH2Cl2, RT, 1 h, 69% over
three steps; d) 47, LiCl, DBU, CH3CN, 1 h, RT, 94%; e) 50, PhCH3, RT, 89%;
f) AcOH, THF, H2O, RT, 3 h, 97%; g) (E)-2,6-heptadienoic acid, 2,4,6-thrichloro-
benzoyl chloride, DIPEA, pyridine, PhCH3, 88%; h) 6-heptenoyl chloride,
DMAP, CH2Cl2, RT, 75%; i) Grubbs II catalyst, PhCH3, 1 h, reflux, 70%; j) HF·Py,
THF, RT, 75%. DMAP = 4-Dimethylaminopyridine
an alcohol intermediate. Oxidation of this alcohol afforded
phosphonate 48. Then glutarimide aldehyde 47 was treated
with 48 using the Masamune–Roush variant of the Horner–
Wadsworth–Emmons reaction, giving the corresponding E-
enone which was reduced with the Stryker reagent 50 and
subsequent selective cleavage of TES protecting group gave al-
cohol 49 (Scheme 6). The synthesis of stereoisomers of migras-
tatin was possible with 49 in hand, as it had a trisubstituted
alkene with the E-configuration (Scheme 6). Thus the modified
Yamaguchi acylation from 2,6-heptadienoic acid gave 51. How-
ever, the subsequent RCM reaction, attempted using both the
Grubbs II and Hoveyda–Grubbs II catalysts, did not give rise to
a ring-closed product. The coupling of 49 with 6-heptenoyl
chloride, which gave 52 was carried out. Substrate 52 does not
contain the unsaturated ester and in this case the RCM reac-
tion succeeded and gave the ring-closed product as a mixture
of E- and Z-stereoisomers (2:1), which were difficult to sepa-
rate. Removal of the TBS group from the mixture was effective-
ly achieved using HF/pyridine to give both 53a and 53b. The
presence of both Z/E isomers in the mixture was deduced by
Scheme 5. Synthesis of 47: a) 40, PhCH3, reflux, 22 h, 96%; b) DIBAL, CH2Cl2,
À788C, 2 h, 84%; c) DMP, CH2Cl2, pyridine, 18 h, 92%; d) 42, MgCl2, Et3N,
TMSCl, RT, 48 h then TFA, MeOH; e) TESCl, CH2Cl2, imidazole, RT, 12 h, 88%
over two steps; f) LiBH4, MeOH, THF, RT, 1 h, 86%; g) N-methylimidazole,
TsCl, MeCN then 1-dodecanthiol, 70 min 08C, 80%; h) Pd/C 10%, Et3Si, ace-
tone, RT, 1 h, 91%. TFA = Trifluoroacetic acid; TESCl = chlorotriethylsilane;
TsCl = 4-toluenesulfonyl chloride.
The subsequent steps were similar to those used by Danishef-
sky and co-workers[6,17h] for their preparation of migrastatin.
Thus, the reduction of the ester with DIBAL-H gave the corre-
sponding allylic alcohol which was subsequently oxidized
using the Dess–Martin reagent and the aldehyde generated
was reacted with propionyl oxazolidinone 42 in the presence
of MgCl2, triethylamine and TMSCl to afford, after treatment
with TFA, the desired anti-aldol product 43. Protection of the
hydroxy group as a TES ether, and reductive removal of the
auxiliary gave primary alcohol 44 in good yield (Scheme 5).
The glutarimide aldehyde 47 needed for the introduction of
the glutarimide chain was prepared from 45 via thioester 46
(Scheme 5).[34]
1
alkene proton signals in the H NMR spectrum of the mixture
of 53a and 53b with coupling constants J=10.7 (Z) and
15.2 Hz (E).
Encouraged that the presence of the E-alkene in 52 did not
totally preclude macrocycle formation by RCM, we revised the
approach to MGSTA-18 and MGSTA-19. Danishefsky and his
co-workers, in their total synthesis of isomigrastatin,[35] report-
ed that RCM gave the 12-membered ring only when the
alkene group adjacent to the carbonyl group was absent. They
used an a-phenylselenide derivative and introduced the alkene
adjacent to the carbonyl group after RCM. In order to obtain
With 44 in hand the preparation of 51 was next completed.
Hence Dess–Martin oxidation of 44 gave the corresponding al-
dehyde, which was reacted with the anion generated by reac-
tion of dimethyl methylphosphonate with butyl lithium to give
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MGSTA-18 and MGSTA-19, the selenium containing acid 55
was, therefore, first prepared from commercially available 54 in
two steps. Yamaguchi acylation of 49 with racemic 55 and sub-
sequent removal of the TBS group provided a mixture of dia-
stereoisomers 56. The RCM from 56 using the Grubbs II re-
agent gave a 1:2 mixture of E-isomer 57a (not shown) and the
Z-isomer 57b. The polarity of 57a and 57b was similar on TLC,
but it was possible to separate small amounts of 57a and 57b
through repeated chromatographic purification steps. Howev-
er, despite repeated chromatography it was not possible to
obtain these compounds with high purity at the scale at which
the reaction was carried out. Thus the oxidative deselenation
of the Z-isomer 57b was carried out with the material ob-
tained and this gave the E-isomer MGSTA-19 in 42% yield,
along with 23% of the Z-isomer MGSTA-18 (Scheme 7).
tion of cell proliferation or toxicity, rather than an interaction
with the cell motility machinery, the effect of newly synthe-
sized migrastatin-core analogues on three breast (MCF7,
MCF7-Dox, MDA-MB361) and one pancreatic cancer (HPAC) cell
lines were evaluated using the MTT assay. The newly synthe-
sized analogues did not cause any toxicity or arrest of prolifer-
ation at the tested concentrations (1, 10 and 100 mm) (see the
Supporting Information provided). Therefore, wound-healing
assays were next carried out to address the effect on cell mi-
gration following exposure to compounds. The migrastatin an-
alogues when given at the concentration of 10 mm strongly in-
hibited migration of the low-invasive MCF7[36] and doxorubicin
resistant MCF7-Dox breast cancer cells, with average scratch
closure of 14.6 and 14.9%, respectively compared to 100% of
scratch closure in the controls.
Interestingly, treatment of MDA-MB361 breast cancer cells
with migrastatin analogues resulted in the inhibition of cell mi-
gration with an average scratch closure of 42.0% compared to
100% scratch closure in the controls. MDA-MB361 are known
to have poor cell–cell adhesion.[36] As a result, some migrasta-
tin analogues were tested on the MDA-MB-231 cell line that
displays a more invasive phenotype,[36] and with this in vitro
model, the average scratch closure was 63.3% compared to
100% scratch closure in the controls. When the migrastatin an-
alogues where tested on the pancreatic cancer cell line, HPAC,
we noted an average of scratch closure of 33.5% (see the Sup-
porting Information) compared to 100% scratch closure in the
controls.
Next a selected number of the compounds tumour cell mi-
gration inhibitory activity was evaluated using the Boyden
chamber assay as this is considered a superior model with
which to conduct cell migration studies.[1] Eight of the com-
pounds were selected based on considering macrocycle ring
size and other structural features: compounds MGSTA-13 and
MGSTA-14 were selected because of their relationship to the
isomigrastatin macrocyclic core; MGSTA-9 contains an extra
carbon in the macrocyclic ring compared to migrastatin;
MGSTA-6 possesses an a,b-unsaturated carbonyl group that is
present in migrastatin; MGSTA-15 is the direct analogue of
MGSTA-4 with a methyl group instead of a methoxy at the 9
position; MGSTA-16 and 17 are the glucuronides and MGSTA-
5 (Danishesky’s macroketone), was selected for use as the in-
ternal control. As shown in Figures 3 and 4, the selected com-
pounds inhibit cell migration in the less-invasive MCF7 cells in
the nanomolar range. The only exception was MGSTA-6, which
inhibited cell migration with considerably lower potency
(IC50 >1 mm) when compared with the macroketone MGSTA-5
(IC50 =13 nm). The glucuronides were found to be less potent
than the macroketone against this cell type with the b-glucur-
onide more potent than its a-anomer. MGSTA-5,6 and 14 were
tested using the more invasive MDA-MB361 cell line. Interest-
ingly, MGSTA-6 (IC50 =410 nm) was 2.4-fold more potent than
MGSTA-5 (IC50 =974 nm) in this particular cell line, contrasting
with the result for the MCF7 cells. In addition, the isomigrasta-
tin-core analogue MGSTA-14 strongly inhibited cell migration
of MDA-MB361 cells (IC50 =301 nm). MGSTA-5,6,9,15,16 and 17
all strongly inhibited cell migration in HPAC cells; however, the
Scheme 7. Synthesis of MGSTA-18 and MGSTA-19: a) BuLi, iPr2NH, Ph2Se2,
THF; b) LiOH, THF/H2O/MeOH, 86%; c) 55, 2,4,6-trichlorobenzoyl chloride,
DIPEA, PhCH3, RT, 3 h then 49, toluene, pyridine, RT, 4 days, 92%; d) HF·pyri-
dine, pyridine, RT, 24 h, 61%; e) Grubbs II catalyst, PhCH3, reflux; 5 min, 78%;
f) mCPBA, CH2Cl2, À788C, 30 min then DIPEA, RT, 2 h, MGSTA-18 (23%),
MGSTA-19 (42%). mCPBA=meta-chloroperoxybenzoic acid.
The structural assignment for MGSTA-19 was supported by
a typical a,b-unsaturated ester proton signal (d=6.83) with J
value typical of a E-alkene (16.0 Hz), whereas MGSTA-18 had
a signal at d=6.23 and J value typical of an Z-alkene (11.1 Hz).
Biological evaluation of migrastatin-core analogues
With a variety of congeners now prepared, biological studies
were next carried out with the capacity of compounds to in-
hibit tumor cell migration investigated using both wound heal-
ing and trans-well assays. Firstly, in order to eliminate the pos-
sibility that any decrease of migration was caused by an inhibi-
Chem. Eur. J. 2015, 21, 18109 – 18121
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ent molecular changes occurring
during the complex event of
metastasis in invasive and non-
invasive cells. In
a previous
paper, we showed that MGSTA-6
inhibited the formation and
elongation of filopodia by inter-
fering with fascin1-dependent
cross-linking of actin filaments
and subsequently leads to inhib-
ition of stress fibre formation.[9]
The importance of these cytos-
keleton rearangements in cancer
invasion indicates a correlation
between high fascin expression
and poor prognosis in human
carcinomas.[37] Stress fibre forma-
tion is the last and crucial step
of complex changes occurring in
cytoskeleton of invasive cancer
cell before its movement and it
could tentatively explain the
more pronounced biological ac-
tivity of MGSTA-6 in MDA-
MB361 cells.
In vitro pharmacological profile
of MGSTA-6
Figure 3. The effect of selected migrastatin analogues on human breast (MCF7, MDA-MB361) and pancreatic
(HPAC) cancer cell lines. a) Quantification of migration of examined cell lines is presented as Relative Fluorescent
Units (RFU). The treatment of cancer cells with selected migrastatin analogues at different concentrations, de-
creased cell migration through the membrane. b) Micrograph of the migrated MCF7, MDA-MB361 and HPAC cells
taken with Olympus microsopy BX60 at 4” magnification.
The pharmacological profile of
a biologically active small mole-
cule can be classified into pri-
mary (interaction with the in-
tendent target) and secondary
(interactions with different tar-
gets). Interaction with secondary
targets can lead to adverse drug
reaction (ADRs). From the drug
discovery point of view, it is ex-
tremely important to identify the
potential side effects as early as
possible. Recently,[38] it has been
suggested that drug candidates
should be screened against a va-
riety of targets (receptors, ion
channels, enzymes and trans-
porters). Compounds that inter-
act with several of the above
mentioned targets, are more
prone to induce ADRs. Unsatu-
rated macroketone MGSTA-6
Figure 4. The effect of selected migrastatin analogues on human breast cancer cell lines MCF7 and MDA-MB36 as-
sessed with Boyden chamber assay.
curve dose response was very wide and the IC50 could not be
extrapolated.
strongly inhibits cell migration in MDA-MB361 and HPAC cells
(see above) and was earlier shown to interfere with the
fascin1-dependent cross-linking of actin filaments leading to
inhibition of stress fibre formation in canine mammary cancer
cells.[9] This agent was, therefore, selected from the panel of
agents for screening against 55 targets (receptors, ion channels
Thus, MGSTA-6 showed good cell-migration inhibition in
highly metastatic MDA–MB361 but not in less invasive MCF7
cells. This apparent paradox could be related with cytoskeleton
proteins targeted by migrastatin analogues[9,19] and with differ-
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Full Paper
30 min and then the solvent was removed under reduced pressure
and the residue was purified by flash chromatography (FC).
and transporters) known to be linked to ADRs. At the concen-
tration of 10 mm, MGSTA-6 only showed weak inhibition of
two targets: adenosine receptor A2A (27%) and prostanoid EP4
receptor (39%). It has been suggested[38] that compounds with
a promiscuity index (percentage of targets giving more than
50% inhibition at 10 mm in a set of at least 50 targets) of more
than 20% should be considered promiscuous and this has
been linked to market withdrawal and clinical trial failure. Un-
saturated macroketone MGSTA-6, with a promiscuity index be-
tween 0–5%, is a possible candidate as a tumour cell migra-
tion inhibitor with potentially low side effects. However, the
pharmacological panel in this study does not include enzymes
and further investigations would be necessary in order to con-
firm the selectivity of MGSTA-6 (Table S1, Supporting Informa-
tion).
(6E,8S,9S,10R,11Z)-9-Hydroxy-8-methoxy-10,12-dimethyloxacy-
clotrideca-6,11-dien-2-one (MGSTA-1): Purification by FC (PE/
EtOAc 10:1) afforded MGSTA-1 (15.1 mg, 61%) as a white solid;
1
[a]D À13.08 (c=0.03 in CHCl3); H NMR (CDCl3, 500 MHz): d=5.65–
5.61 (m, 1H, H-11) 5.62–5.57 (dd, 1H, J6,7trans =15.5, J5,6 =7.5 Hz, H-
6), 5.26 (ddt, 1H, J7,8 =6.2, J=1.4 Hz, H-7), 5.13 (dd, 1H, J13,13’ =12.1,
J
Me,13 =1.2 Hz, H-13), 3.82 (d, 1H, H-13’), 3.41–3.34 (m, 1H, H-8),
3.31–3.27 (m, 4H, OCH3, H-9), 2.42–2.28 (m, 3H, CH2, H-10), 2.27–
2.12 (m, 2H, CH2), 2.05–1.94 (m, 1H, CH2), 1.82 (d, 3H, CH3 at C-12),
1.77–1.68 (m, 1H, CH2), 0.95 ppm (d, 3H, J10,10Me =6.8 Hz, CH3 at C-
10); 13C NMR (CDCl3, 125 MHz): d=173.4 (C-2), 136.4 (C-11), 132.9
(C-6), 129.3 (C-7), 128.5 (C-12), 82.7 (C-8), 77.6 (C-9), 63.3 (C-13), 56.8
(OCH3), 32.9 (C-10), 32.0 (CH2), 31.7 (CH2), 24.0 (CH3 at C-12), 21.8
(CH2), 13.0 ppm (CH3 at C-10); HRMS-ESI: m/z: calcd for C15H24O4Na:
291.1572; found: 291.1584.
(4Z,6R,7S,8S,9E)-7-Hydroxy-8-methoxy-4,6-dimethylcyclotrideca-
4,9-dienone (MGSTA-2): Purification by FC (PE/EtOAc 10:1) afford-
ed MGSTA-2 (11.8 mg, 84%) as a yellow syrup; [a]D 87.4 (c=1.64
Conclusion
in CHCl3); 1H NMR (CDCl3, 500 MHz): d=5.62 (ddd, 1H, J9,10trans
=
The stereoselective synthesis of intermediates based on Brown
allylation and alkoxyallylation has enabled the preparation of
a series of migrastatin and isomigrastatin analogues. Chelation-
induced anomerisation is compatible with the various func-
tionalities found in the migrastatin macroketone skeleton and
could be exploited to give macroketone glycosides for biologi-
cal study. The Saeguso–Ito reaction enabled synthesis of
MGSTA-6 in a regio- and stereoselective fashion. Compounds
showed inhibitory activity when tested against breast and pan-
creatic cancer cell lines (MCF-7, MCF7-Dox, MDA-MB631, MDA-
MB261, HPAC) using the wound healing assay and Boyden
chamber assay (nm range). One inhibitor (MGSTA-6) has low
activity against a variety of receptors, ion channels and trans-
porters, which indicates that it, and possibly other migrastatin
analogues, may have potential to inhibit tumour metastasis
without adverse drug reactions.
15.0, J10,11 =8.5, J10,11’ =5.9 Hz, H-10), 5.30 (d, 1H, J5,6 =10 Hz, H-5),
5.22 (ddt, 1H, J8,9 =7.6, J=1.3 Hz, H-9), 3.37–3.34 (m, 1H, H-8),
3.31–3.29 (m, 4H, H-7 and OCH3), 2.82 (s, 1H, OH), 2.64–2.59 (m,
1H, CH2), 2.51–2.45 (m, 2H, CH2), 2.43–2.25 (m, 4H, H-6, CH2), 2.10–
2.03 (m, 1H, CH2), 1.91–1.81 (m, 2H, CH2), 1.76–1.70 (m, 1H, CH2),
1.70 (d, 3H, J=1.3 Hz, CH3 at C-4), 0.95 ppm (d, 3H, J6,Me =6.9 Hz,
CH3 at C-6); 13C NMR (125 MHz, CDCl3): d=210.7 (C-1), 135.7 (C-10),
133.9 (Cq), 130.7 (C-5), 129.5 (C-9), 83.9 (C-8), 77.8 (C-7), 56.4
(OCH3), 42.5 (CH2), 40.2 (CH2), 32.9 (C-6), 31.9 (CH2), 26.3 (CH2), 23.6
(CH3), 21.5 (CH2), 13.9 ppm (CH3); HRMS-ESI: m/z: calcd for
C16H26O3Na: 289.1780; found: 289.1772.
(6E,8S,9S,10R,11Z)-9-Hydroxy-8-methoxy-10,12-dimethylthiacy-
clotrideca-6,11-dien-2-one (MGSTA-3): Purification by FC (PE/
EtOAc 15:1) afforded MGSTA-3 (5.0 mg, 85%) as a yellow syrup;
[a]D =1.6 (c=0.46 in CHCl3); 1H NMR (CDCl3, 500 MHz): d=5.65–
5.48 (m, 2H, H-6,10), 5.15–5.05 (dd, 1H, J6,7trans =15.4, J7,9 =7.7 Hz,
H-7), 4.21 (d, 1H, J=13.4 Hz, H-13), 3.33–3.27 (m, 2H, H-7,8), 3.26
(s, 3H, OCH3), 2.84 (s, 1H, OH), 2.78 (d, 1H, H-13’), 2.66 (ddd, 1H,
J=16.7, 7.0, 2.0 Hz, H-3), 2.57–2.51 (m, 1H, H-3’), 2.51–2.42 (m, 1H,
H-5), 2.22 (m, 1H, H-4), 2.12 (m, 1H, H-10), 2.01–1.92 (m, 1H, H-5’),
1.76–1.74 (s, 3H, Me at C-12), 1.69–1.62 (m, 1H, H-4’), 0.97 ppm (d,
3H, J10,Me =6.8 Hz, Me at C-10);13C NMR (125 MHz, CDCl3): d=198.0
(C-2), 136.8 (C-6), 133.9 (C-10), 130.1 (C-11), 128.8 (C-7), 83.1 (C-8 or
C-9), 75.0 (C-8 or C-9), 56.3 (OCH3), 43.7 (C-3), 34.3 (C-5), 32.5 (C-
10), 28.9 (C-13), 25.3 (CH3 at C-12), 22.7 (C-4), 12.6 ppm (CH3 at C-
10); HRMS-ESI: calcd for C15H23O3S: 283.1368; found: 283.1375.
Experimental Section
General
NMR spectra were recorded on a 500 MHz spectrometer at 308C.
Chemical shifts are reported relative to internal Me4Si inCDCl3 (d=
1
0.0 ppm) for H and CDCl3 (d=77.16 ppm) for 13C at 308C, unless
otherwise stated. 13C signals were assigned with the aid of HSQC.
Coupling constants are reported in Hertz. High-resolution mass
spectra were measured by using an LC time-of-flight mass spec-
trometer and were measured in positive and/or negative mode as
indicated. TLC was performed on aluminium sheets pre-coated
with Silica Gel 60 (HF254, Merck Millipore) and spots were visual-
ised by charring with vanillin solutions. Chromatography was car-
ried out by using silica gel 60 (0.040–0.630 mm, E. Merck). Di-
chloromethane, tetrahydrofuran, MeOH and toluene were used as
obtained from a PureSolv solvent purification system. Petroleum
ether is the fraction with b.p.=40–608C.
(2E,6E,8S,9S,10R,11Z)-9-[(tert-Butyldimethylsilyl)oxy]-8-methoxy-
10,12-dimethylcyclotetradeca-2,6,11-trien-1-one (13): To stirred
12b (26.7 mg; 0.068 mmol) in THF (0.5 mL) at 08C, TMSCl (26 mL;
0.203 mmol) and LHMDS (203 mL; 0.203 mmol, 1m in THF) were
added dropwise. The resulting solution was stirred for 2 h at 08C,
quenched with NH4Cl and extracted with EtOAc. The organic layers
were dried over Na2SO4, filtered and the solvent was removed
under reduced pressure. The residue was dissolved in CH3CN
(0.5 mL) and Pd(OAc)2 (18 mg; 0.082 mmol) was added. The mix-
ture was stirred for 3 h and then diluted with NaHCO and extract-
ed with CH2Cl2. The organic layers were dried over Na2SO4, filtered
and the solvent was removed under reduced pressure. Purification
by FC (PE/EtOAc 20:1) afforded 14 (17 mg: 64%) as a yellow syrup;
General procedure for TBS deprotection
To a stirred solution of TBS-protected macrocycle in THF (24 mm),
HF·pyridine 70% (250 equiv) was added at RT. The resulting solu-
tion was stirred at RT for 18 h. After the careful addition of
MeOTMS (350 equiv), the mixture was stirred for an additional
1
[a]D 42.98 (c=1.2 in CHCl3); H NMR (CDCl3, 500 MHz): d=6.74 (dt,
1H, J2,3 =16.2, J3,4 =7.0 Hz, H-3), 5.93 (d, 1H H-2), 5.64 (ddd, 1H,
J
6,7 =15.4, J=8.9, J=4.7 Hz, H-6), 5.37–5.14 (m, 2H, H-7,11), 3.45–
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3.27 (m, 2H, H-8,9), 3.21 (s, 3H, OCH3), 2.70–2.31 (m, 7H, H-10,
CH2), 2.31–2.20 (m, 1H, CH2), 2.05 (m, 1H, CH2), 1.69 (d, 3H, J=
1.5 Hz, Me at C-12), 0.90 (s, 9H, C(CH3)3), 0.87 (d, 3H, J=6.6 Hz, Me
at C-10), 0.04 (s, 3H, SiCH3), 0.00 ppm (s, 3H, SiCH3); 13C NMR
(125 MHz, CDCl3): d=201.5 (C-1), 148.2 (C-3), 132.8 (C-6), 132.0,
132.0, 131.7, 131.4, 85.2 (C-8 or C-9), 79.0 (C-8 or C-9), 56.4 (OCH3),
38.4 (CH2), 34.5 (C-10), 32.4 (CH2), 31.6 (CH2), 30.7 (CH2), 26.3
(C(CH3)3), 23.0 (Me at C-12), 18.8 (C(CH3)3), 12.7 (Me at C-10), À3.7
(SiCH3), À4.9 ppm (SiCH3); HRMS-ESI: m/z: calcd for C23H39O3Si:
391.2668; found: 391.2678.
7.3 Hz, H-9), 3.44–3.33 (m, 2H, H-7,8), 3.28 (s, 3H, OMe), 2.78 (d,
1H, J7,OH =1.3 Hz, OH), 2.51–2.31 (m, 5H), 2.29–2.17 (m, 2H), 2.14–
2.01 (m, 2H), 1.73–1.56 (m, 5H), 1.52–1.34 (m, 2H), 1.29–1.08 (m,
2H), 0.93 ppm (d, 3H,
J
6,Me =6.8 Hz, C6Me); 13C NMR (CDCl3,
125 MHz): d=213.0 (C-1), 136.5 (C-10), 133.4 (C-4), 129.8 (C-5),
128.1 (C-9), 84.2 (C-7 or C-8), 76.6 (C-7 or C-8), 56.3 (OMe), 43.1
(CH2), 41.8 (CH2), 32.7 (C-6), 31.8 (CH2), 27.9 (CH2), 27.8 (CH2), 26.4
(CH2), 25.7 (CH2), 23.4 (Me at C-4), 13.0 ppm (Me at C-6). HRMS-ESI:
m/z: calcd for C18H30O3Na: 317.2093; found: 317.2087.
(2R,3R,4S)-2,4-Dimethylhex-5-ene-1,3-diol (26): To stirred tBuOK
(1.63 g; 14.57 mmol) in THF (4 mL) at À458C n-butyllithium in hep-
tane (9.1 mL, 14.57 mmol, 1.6m) was added. The mixture was
cooled to À788C and b-methoxydiisopinocampheylborane (5.5 g,
17.4 mmol) in THF (17 mL) was added via cannula. The mixture
was stirred at À788C for an additional 1 h and boron trifluoride
etherate (2.4 mL, 19.45 mmol) was added dropwise at À788C. Im-
mediately, a solution of aldehyde 25 (3.94 g, 20.52 mmol) in THF
(7 mL) was added, and the mixture was stirred at À788C for 18 h.
Then aqueous NaOH (10.66 mL; 32 mmol, 3m) was added followed
by H2O2 (4.5 mL, 30%), and the biphasic mixture was allowed to
warm to room temperature slowly and refluxed for 1 h. The solu-
tion was diluted with CH2Cl2 and washed with water. The organic
layer was separated, and then dried over Na2SO4 and the solvent
was removed under reduced pressure. The residue was subjected
to silica gel chromatography (PE/EtOAc, 1:6) affording a mixture of
the title compound and pinenol during the workup (9.87 g) which
can be used directly in the next step. The mixture (9.87 g) was dis-
solved in dry methanol (200 mL) and K2CO3 (2.32 g) was added.
After stirring at room temperature for 24 h AcOH was added until
pH 7 was reached. The solvent was removed under reduced pres-
sure and the residue was purified by chromatography (PE/EtOAc
1:10!Et2O) to afford 26 (940 mg; 32% from 25) as white crystal
(needles). M.p.=175–1818C; 1H NMR (CDCl3, 500 MHz): d=5.62
(ddd, 1H, J5,6trans =17.1, J5,6cis =10.3, J4,5 =8.7 Hz, H-5), 5.08–5.04 (m,
(2E,6E,8S,9S,10R,11Z)-9-Hydroxy-8-methoxy-10,12-dimethylcyclo-
tetradeca-2,6,11-trienone (MGSTA-6): Purification by FC (PE/EtOAc
5:1) afforded MGSTA-6 (7.6 mg, 90%) as a yellow syrup; [a]D 92.28
1
(c=0.6 in CHCl3); H NMR (CDCl3, 500 MHz): d=6.73 (dt, 1H, J2,3
=
16.4, J3,4 =7.1 Hz, H-3), 5.93 (d, 1H, H-2), 5.69 (ddd, 1H, J6,7 =15.4,
J=9.4, J=4.4 Hz, H-6), 5.37 (d, 1H, J10,.11 =9.9 Hz, H-11), 5.24 (ddd,
1H, J7,8 =8.4, J=1.4 Hz, H-7), 3.44 (appt, 1H, J7,8 =J8,9 =8.9 Hz, H-8),
3.34 (dd, 1H, J=1.3 Hz, H-9), 3.31 (s, 3H, OCH3), 2.70 (s, 1H, OH),
2.64 (m, 1H, CH2), 2.57–2.46 (m, 4H, H-10, CH2), 2.42–2.22 (m, 3H,
CH2), 2.14 (m, 1H, CH2), 1.72 (d, 3H, J=1.4 Hz, Me at C-12),
0.92 ppm (d, 3H, J=6.8 Hz, Me at C-10); 13C NMR (125 MHz, CDCl3):
d=201.3 (C-1), 147.8 (C-3), 134.9 (C-6), 132.9 (C-12), 131.7 (C-2),
130.9 (C-11), 130.5 (C-7), 84.1 (C-8), 77.2 (C-9), 56.6 (OCH3), 38.4
(CH2), 32.7 (C-10), 32.3 (CH2), 31.3 (CH2), 30.6 (CH2), 22.9 (Me at C-
12), 12.6 ppm (Me at C-10); HRMS-ESI: m/z: calcd for C17H25O3:
277.1804; found: 277.1801.
(7E,9S,10S,11R,12Z)-10-Hydroxy-9-methoxy-11,13-dimethylthia-
cyclotetradeca-7,12-dien-2-one (MGSTA-7): Purification by FC
(hexane/EtOAc 15:1) afforded MGSTA-7 (12.8 mg, 63%) as a yellow
syrup; [a]D 93.2 (c=0.07 in CHCl3);1H NMR (CDCl3, 500 MHz): d=
5.80–5.70 (m, 1H, H-7), 5.58 (d, 1H, J11,12 =9.6 Hz, H-12), 5.28–5.20
(ddt, 1H, J7,8 =15.8, J8,9 =7.3, J=1.4 Hz, H-8), 3.72 (d, 1H, J14,14’
12.4 Hz, H-14), 3.43 (dd, 1H, J9,10 =9.0 Hz, H-9), 3.35 (dd, 1H, J10,OH
=
=
1.6 Hz, H-10), 3.30 (s, 3H, OMe), 3.18 (d, 1H, H-14’), 2.80 (brs, 1H,
OH), 2.61 (m, 1H), 2.49–2.37 (m, 2H, H-11, CH2), 2.22 (m, 1H), 2.02
(m, 1H), 1.77 (d, 3H, J=1.4 Hz, Me at C-13), 1.75–1.59 (m, 3H),
1.45–1.35 (m, 1H), 0.95 ppm (d, 3H, J=6.9 Hz, Me at C-11);
13C NMR (CDCl3, 125 MHz): d=200.9 (C-2), 135.0 (C-7), 134.2 (C-12),
128.4 (C-13), 127.8 (C-8), 83.8 (C-9), 76.6 (C-10), 56.6 (OMe), 43.5
(CH2), 32.9 (C-11), 31.1 (C-14), 29.6 (CH2), 27.4 (CH2), 25.6 (CH2), 25.1
(Me at C-13), 12.8 ppm (Me at C-11); HRMS-ESI: m/z: calcd for
C16H26O3NaS: 321.1500; found: 321.1496.
1H, H-6trans), 4.98 (dd, 1H, Jgem =1.8 Hz, H-6cis), 3.71 (dd, 1H, J1,1’
=
10.5, J1,2 =4.1 Hz, H-1), 3.66 (dd, 1H, J1’,2 =5.5 Hz, H-1’), 3.56 (dd,
1H, J3,4 =9.1, J2,3 =2.4 Hz, H-3), 2.67 (brs, 2H, OH), 2.33–2.26 (m,
1H, H-4), 1.82 (dddd, 1H, J2,Me =6.8 Hz, H-2), 1.09 (d, 3H, J4,Me
=
6.7 Hz, CH3 at C-4), 0.93 ppm (d, 3H, CH3 at C-2); 13C NMR (CDCl3,
125 MHz): d=141.0 (C-5), 114.9 (C-6), 77.5 (C-3), 67.9 (C-1), 42.4 (C-
4), 36.7 (C-2), 17.3 (CH3 at C-4), 9.1 ppm (CH3 at C-2); HRMS-ESI:
m/z: calcd for C8H15O2Na: 143.1072; found: 143.1071.
(8E,10S,11S,12R,13Z)-11-Hydroxy-10-methoxy-12,14-dimethylox-
acyclopentadeca-8,13-dien-2-one (MGSTA-8): Purification by FC
(PE/EtOAc 10:1!5:1) afforded MGSTA-8 (28.7 mg, 54%) as a white
Ring-closing metathesis on MGSTA-10
1
syrup; [a]D 728 (c=1.7 in CHCl3); H NMR (CDCl3, 500 MHz): d=5.79
To a stirred solution of MGSTA-10 (100 mg; 0.33 mmol), in de-
gassed PhCH3 (650 mL) the Grubbs II catalyst (57 mg, 0.067 mmol)
was added by cannula. The mixture was stirred at reflux for
60 min. The PhCH3 was removed under reduced pressure. Purifica-
tion of the residue by FC (EtOAc/Cy 1:30) afforded MGSTA-11
(25 mg, 31%) and MGSTA-12 (23.1 mg; 33%) as yellow syrups.
(dq, 1H, J12,13 =9.4, J13,Me =1.6 Hz, H-13), 5.64 (ddd, 1H, J8,9 =15.3,
J
8,7 =8.9, J8,7’ =5.2 Hz, H-8), 5.16 (dd, 1H, J9,10 =8.0 Hz, H-9), 4.52 (d,
1H, J15a,15b =11.8 Hz, H-15a), 4.27 (d, 1H, H-15b), 3.46–3.34 (m, 2H,
H-10, H-12), 3.29 (s, 3H, OMe), 2.82 (t, 1H, JOH,12 =1.3 Hz, OH), 2.51
(q, 1H, J=7.4 Hz), 2.39–2.26 (m, 2H), 2.28–2.18 (m, 1H, H-7), 2.11–
2.00 (m, 1H, H-7’), 1.81 (d, 3H, C-14Me), 1.74–1.55 (m, 2H), 1.50–
1.33 (m, 2H, H-6,6’), 1.30–1.10 (m, 2H), 0.96 ppm (d, 3H, C-12Me);
13C NMR(CDCl3, 125 MHz): d=175.0 (C-2), 136.7 (C-8), 136.3 (C-13),
128.2 (C-14), 128.0 (C-9), 84.0 (C-11 or C-10), 76.0 (C-11 or C-10),
64.0 (C-15), 56.2 (OMe), 35.1 (CH2), 32.9 (C-12), 32.0 (C-7), 27.6
(CH2), 27.4 (C-6), 25.8 (CH2), 23.5 (Me), 13.2 ppm (Me); HRMS-ESI:
m/z: calcd for C17H28O4Na: 319.1885; found: 319.1882.
(9S,10R,11R,Z)-10-(Methoxymethoxy)-9,11-dimethyloxacyclodo-
dec-7-en-2-one (MGSTA-11): 1H NMR (CDCl3, 500 MHz): d=5.48–
5.40 (m, 1H, H-8), 5.34 (td, 1H, J7,8 =10.6, J6,7 =5.0 Hz, H-7), 4.67 (d,
1H, J=6.6 Hz, OCH2O), 4.62 (d, 1H, J=6.6 Hz, OCH2O), 4.38 (dd,
1H, J12,12’ =11.4, J12,11 =3.1 Hz, H-12), 3.89 (dd, 1H, J11,12’ =5.2 Hz, H-
12’), 3.43–3.38 (m, 4H, H-10, OCH3), 2.71 (dqd, 1H, J8,9 =9.9, J9,Me
=
6.8, J9,10 =2.9 Hz, H-9), 2.44 (ddd, 1H, J=12.7, 8.5, 3.9 Hz, CH2), 2.33
(ddd, 1H, J=13.3, 8.9, 3.7 Hz, CH2), 2.26–2.14 (m, 1H, CH2), 2.14–
2.02 (m, 1H, H-11), 1.91–1.75 (m, 2H, CH2), 1.64 (dddt, 2H, J=20.6,
8.7, 5.8, 3.2 Hz, CH2), 1.42–1.29 (m, 1H, CH2), 1.11 (d, 3H, J=7.3 Hz,
CH3 at C-11), 0.98 ppm (d, 3H, J=6.8 Hz, H- CH3 at C-9); 13C NMR
(CDCl3, 125 MHz): d=174.5 (C-2), 135.8 (C-8), 128.6 (C-7), 98.3
(4Z,6R,7S,8S,9E)-7-Hydroxy-8-methoxy-4,6-dimethylcyclopenta-
deca-4,9-dienone (MGSTA-9): Purification by FC (PE/EtOAc 10:1)
afforded MGSTA-9 (11.8 mg, 82%) as a white syrup; [a]D 878 (c=
1
0.9 in CHCl3); H NMR (CDCl3, 500 MHz): d=5.66 (ddd, 1H, J 14.7,
8.8, 5.2 Hz, H-10), 5.41 (d, 1H, J5,6 =9.6 Hz, H-5), 5.17 (dd, 1H, J8,9
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(OCH2O), 85.6 (C-10), 67.4 (C-12), 56.3 (OCH3), 36.2 (C-11), 34.6
(CH2), 34.5 (C-9), 28.8 (CH2), 25.9 (CH2), 23.6 (CH2), 15.1 (CH3 at C-11),
14.6 ppm (CH3 at C-9); HRMS-ESI: m/z: calcd for C15H26O4Na:
293.1729; found: 293.1731.
(3”10 mL). The combined organic layers were dried and the sol-
vent was removed under reduced pressure. Purification by FC
(EtOAc/Cy 1:7) gave MGSTA-15 (4.5 mg, 60%) as a colourless oil.
1
[a]D 37.68 (c=0.19 in CHCl3); H NMR (CDCl3, 500 MHz): d=5.60 (dt,
1H, J11,12 =10.2, J12,Me =1.4 Hz, H-12), 5.46 (ddd, 1H, J7,8 =15.3, J=
7.8, J=5.8 Hz, H-7), 5.30 (ddt, 1H, J=8.6, J=1.3 Hz, H-8), 4.56 (dd,
1H, J14,14’ =13.1, J=1.0 Hz, H-14), 4.34 (d, 1H, H-14’), 3.24 (d, 1H,
J=9.3 Hz, H-10), 3.02–2.95 (m, 1H, H-11), 2.46–2.39 (m, 1H, CH2),
2.30–2.18 (m, 2H, H-9, CH2), 2.17–2.10 (m, 1H, CH2), 1.98–1.90 (m,
1H, CH2), 1.82–1.77 (m, 1H, CH2), 1.76 (d, 3H, CH3 at C-13), 1.64–
1.57 (m, 1H, CH2), 1.53–1.47 (m, 1H, CH2), 1.41–1.32 (m, 1H CH2),
(9S,10R,11R,E)-10-(Methoxymethoxy)-9,11-dimethyloxacyclodo-
dec-7-en-2-one (MGSTA-12): 1H NMR (CDCl3, 500 MHz): d=5.42
(ddd, 1H, J7,8 =14.9, 8.3, 6.3 Hz, H-7), 5.25 (dd, 1H, J8,9 =8.2 Hz, H-
8), 4.70–4.66 (m, 2H, OCH2O), 4.47 (dd, 1H, J12,12’ =10.6, J11,12
=
2.9 Hz, H-12), 3.54 (appt, 1H, J12,12’ =J11,12’ =10.7 Hz, H-12’), 3.40 (s,
3H, OCH3), 3.32 (d, 1H, J=10.1 Hz, H-10), 2.46–2.38 (m,1H, CH2),
2.17–2.06 (m, 3H, H-9, CH2), 2.02–1.93 (m, 1H, H-11), 1.74–1.61 (m,
4H, CH2), 1.20–1.12 (m, 1H, CH2), 1.06 (d, 3H, J=6.8 Hz, CH3 at C-
9), 0.89 ppm (d, 3H, J=7.3 Hz, CH3 at C-11); 13C NMR (CDCl3,
125 MHz): d=173.4 (C-2), 135.5 (C-7), 131.4 (C-8), 98.7 (OCH2O),
83.9 (C-10), 66.5 (C-12), 56.0 (OCH3), 40.4 (C-9), 35.7 (C-11), 34.7
(CH2), 30.4 (CH2), 26.9 (CH2), 22.6 (CH2), 17.2 (CH3 at C-9), 10.6 ppm
(CH3 at C-11); HRMS-ESI: m/z: calcd for C15H26O4Na: 293.1729;
found: 293.1732.
1.09 (d, 3H, J9,Me =6.7 Hz, CH3 at C-9), 0.92 ppm (d, 3H, J11,Me
=
7.0 Hz, CH3 at C-11); 13C NMR (CDCl3, 125 MHz): d=174.0 (C-2),
134.7 (C-12), 133.9 (C-8), 129.9 (C-7), 127.5 (C-13), 78.9 (C-10), 65.0
(C-14), 41.4 (C-9), 34.6 (C-11), 34.0 (CH2), 30.0 (CH2), 27.6 (CH2), 23.5
(CH2), 23.4 (CH3 at C-13), 18.1 (CH3 at C-9), 12.9 ppm (CH3 at C-11);
HRMS-ESI: m/z: calcd for C16H26O3Na: 289.1780; found: 289.1775.
(4Z,6R,7S,8S,9E)-7-O-(Methyl-2’,3’,4’-tri-O-benzoyl-b-d-glucopyra-
nosyluronate)-4,6-dimethylcyclotetradeca-8-methoxy-4,9-dien-
one (37): To stirred macroketone MGSTA-5 (5 mg, 18 mmol), the tri-
chloroacetimidate 36 (24 mg, 36 mmol), and powdered molecular
sieves 4 A (25 mg) in dry CH2Cl2 (0.5 mL) was cooled to À408C
under nitrogen atmosphere. To this mixture was added 10%
TMSOTf in CH2Cl2 (11 mL, 7.2 mmol) dropwise, and the mixture was
stirred at À408C for 5 h, and then EtOAc was added (1 mL,
7.2 mmol). The mixture was filtered and concentrated. FC of the
residue (PE/EtOAc 4:1) gave 37 (8 mg, 57%).[a]D À9.48 (c=0.58 in
(9S,10R,11R,Z)-10-Hydroxy-9,11-dimethyloxacyclododec-7-en-2-
one (MGSTA-13): To stirred MGSTA-11 (28.1 mg; 0.104 mmol) in
CH2Cl2 (5 mL), TMSBr (20.6 mL; 0.0156 mmol) was added at À208C.
The mixture was stirred for 1 h. Satd NaHCO3 was then added and
the solution was extracted with CH2Cl2 (3”10 mL). The combined
organic layers were dried and the solvent was removed under re-
duced pressure. Purification by FC (EtOAc/Cy 1:8) gave MGSTA-13
(18.2 mg, 77%) as a colourless oil. [a]D À208 (c=1.5 in CHCl3);
1H NMR (CDCl3, 500 MHz): d=5.54–5.47 (m, 1H, H-8), 5.39 (td, 1H,
1
J
7,8 =10.6, J6,7 =5.3 Hz, H-7), 4.47 (dd, 1H, J12,12’ =11.5, J12,11 =3.4 Hz,
H-12), 3.82 (dd, 1H, J11,12’ =4.5 Hz, H-12’), 3.54 (ddd, 1H, J10,11 =7.6,
10,OH =4.9, J9,10 =2.2 Hz, H-10), 2.73 (dqd, 1H, J8,9 =9.2, J9,Me
CHCl3); H NMR (CDCl3, 500 MHz): d=8.00–8.00 (m, 2H, ArH), 7.93–
7.91 (m, 2H, ArH), 7.88–7.81 (m, 2H, ArH), 7.53–7.36 (m, 7H, ArH),
7.31–7.28 (m, 2H, ArH), 5.94 (appt, 1H, J2’,3’ =J3’,4’ =9.6 Hz, H-3’),
5.69 (dd, 1H, J3’,4’ =J4’,5’ =9.7 Hz, H-4’), 5.62–5.52 (m, 3H, H-2’, H-
5,10), 5.23 (dd, 1H, J9,10trans =15.7, J8,9 =7.5 Hz, H-9), 5.21 (d, 1H,
J
=
6.8 Hz, H-9), 2.42 (ddd, 1H, J=12.9, 8.8, 4.0 Hz, CH2), 2.33 (ddd, 1H,
J=13.1, 8.8, 3.7 Hz, CH2), 2.24–2.14 (m, 1H, CH2), 2.01–1.93 (m, 1H,
H-11), 1.91–1.84 (m, 1H, CH2), 1.83–1.74 (m, 1H, CH2), 1.70–1.63 (m,
2H, CH2), 1.43 (d, 1H, OH), 1.37–1.28 (m, 1H, CH2), 1.14 (d, 3H,
J
1’,2’ =7.9 Hz, H-1’), 4.27 (d, 1H, H-5’), 3.69 (s, 3H, OMe), 3.48–3.38
(m, 2H, H-7,8), 2.87 (s, 3H, OMe), 2.56–2.41 (m, 2H, H-6, CH2), 2.40–
2.30 (m 2H, CH2), 2.28–2.06 (m, 5H, CH2), 1.70 (d, 3H, J=1.5 Hz, Me
at C-4), 1.68–1.57 (m, 2H, CH2), 1.52–1.45 (m, 2H, CH2), 0.91 ppm
(d, 3H, J6,Me =6.8 Hz, Me at C-6); 13C NMR (125 MHz, CDCl3): d=
212.3 (C-1), 167.6 (C=O), 165.9 (C=O), 165.4 (C=O), 165.0 (C=O),
134.5 (C=C), 133.5 (ArCH), 133.4 (ArCH), 133.2 (ArCH), 132.0 (Cq),
130.5 (C=C), 130.0 (ArCH), 130.0 (ArCH), 129.9 (ArCH), 129.8 (Cq),
129.4 (C-9), 129.1 (Cq), 129.1 (Cq), 128.6 (ArCH), 128.5 (ArCH), 128.4
(ArCH), 101.5 (C-1’), 84.9 (C-7 or 8), 84.5 (C-7 or 8), 72.9 (C-5), 72.7
(C-3’), 72.1 (C-2’), 70.8 (C-4’), 56.5 (OCH3), 52.9 (OCH3), 42.3 (CH2),
41.0 (CH2), 33.2 (C-6), 30.5 (CH2), 28.8 (CH2), 27.2 (CH2), 23.4 (CH2),
23.2 (CH3 at C-4), 13.5 ppm (CH3 at C-6); HRMS-ESI: m/z: calcd for
C45H50O12Na: 805.3200; found: 805.3202.
J
11,Me =7.0 Hz, CH3 at C-11), 0.96 ppm (d, 3H, CH3 at C-9);13C NMR
(CDCl3, 125 MHz): d=174.6 (C-2), 135.5 (C-8), 128.9 (C-7), 78.4 (C-
10), 67.4 (C-12), 36.3 (C-11), 34.8 (CH2), 33.8 (C-9), 28.8 (CH2), 26.1
(CH2), 23.7 (CH2), 15.0 (CH3 at C-11), 12.8 ppm (CH3 at C-9); HRMS-
ESI: m/z: calcd for C15H25NO3Na: 290.1732; found: 290.1726.
(9S,10R,11R,E)-10-Hydroxy-9,11-dimethyloxacyclododec-7-en-2-
one (MGSTA-14): To stirred MGSTA-12 (23.4 mg; 0.087 mmol) in
CH2Cl2 (6 mL), TMSBr (23 mL; 0.174 mmol) was added at À208C.
The mixture was stirred for 2 h. Saturated NaHCO3 was added and
the solution was extracted with CH2Cl2 (3”10 mL) and the com-
bined organic layers were dried and the solvent was removed
under reduced pressure. Purification by FC (EtOAc/Cy 1:10) gave
MGSTA-14 (14.5 mg, 74%) as a colourless oil. [a]D =6.498 (c=0.99
in CHCl3); 1H NMR (CDCl3, 500 MHz): d=5.44 (ddd, 1H, J7,8 =14.6,
(4Z,6R,7S,8S,9E)-7-O-(b-d-Glucopyranosyluronic acid)-4,6-dime-
thylcyclotetradeca-8-methoxy-4,9-dienone (MGSTA-16): To stirred
37 (12.7 mg; 16 mmol) in MeOH (1.2 mL), NaOH aq. 5% (48 mL,
59 mmol) was added. The resulting solution was stirred at RT for
20 h. A solution of 1m AcOH was added until the pH was 7. The
solvent was removed under reduced pressure and the residue was
passed through silica gel 100 C18-reversed-phase column (H2O to
H2O/MeOH 4:1 to H2O/MeOH 2:1 to H2O/MeOH 1:1 to H2O/MeOH
1:2 to MeOH). The residue was subjected to vacuum distillation to
remove the aromatic impurities and then passed again through
silica gel 100 C18-reverse-phase silica (eluant, H2O to H2O/MeOH
4:1 to H2O/MeOH 2:1 to H2O/MeOH 1:1 to H2O/MeOH 1:2 to
MeOH) to give MGSTA-16 (5.3 mg; 73%). [a]D +5.328 (c=0.36 in
MeOH); 1H NMR (CDCl3, 500 MHz): 5.85 (ddd, 1H, J9,10 =15.0, 9.6,
J
7,6 =8.0, J7,6’ =6.4 Hz, H-7), 5.28 (dd, 1H, J8,9 =8.1 Hz, H-8), 4.41 (dd,
1H, J12,12’ =10.7, 2.8 Hz, H-12), 3.56 (t, 1H, H-12’), 3.43 (dd, 1H, J=
9.7, J10,OH =6.4 Hz, H-10), 2.44–2.36 (m, 1H, CH2), 2.16–2.06 (m, 3H,
H-9, CH2), 2.03–1.96 (m, 1H, H-11), 1.74–1.62 (m, 4H, CH2), 1.45 (d,
1H, OH), 1.20–1.12 (m, 1H, CH2), 1.09 (d, 3H, J9,Me =6.8 Hz, CH3 at
C-9), 0.88 ppm (d, 3H, J11,Me =7.3 Hz, CH3 at C-11); 13C NMR (CDCl3,
125 MHz): d=173.4 (C-2), 135.3 (C-8), 131.2 (C-7), 76.3 (C-10), 66.6
(C-12), 39.9 (C-9), 35.9 (C-11), 34.9 (CH2), 30.2 (CH2), 27.4 (CH2), 22.5
(CH2), 16.8 (CH3 at C-9), 10.0 ppm (CH3 at C-11); HRMS-ESI: m/z:
calcd for C13H22O3Na: 249.1467; found: 249.1459.
(7E,9S,10R,11R,12Z)-10-Hydroxy-9,11,13-trimethyloxacyclotetra-
deca-7,12-dien-2-one (MGSTA-15): To a stirred solution of 34
(8.7 mg; 0.028 mmol) in CH2Cl2 (5 mL), TMSBr (5.5 mL; 0.042 mmol)
was added at À158C. The mixture was stirred for 3 h. Saturated
NaHCO3 was added and the solution was extracted with CH2Cl2
4.8 Hz, H-10), 5.44 (dd, 1H, J8,9 =9.0 Hz, H-9), 5.37 (d, 1H, J5,6
=
9.2 Hz, H-5), 4.44 (d, 1H, J1’,2’ =7.9 Hz, H-1’), 3.85 (appt, 1H, H-8),
3.60 (d, 1H, J4’,5’ =9.3 Hz, H-5’), 3.56–3.46 (m, 3H, H-7,3’,4’), 3.37
Chem. Eur. J. 2015, 21, 18109 – 18121
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(dd, 1H, J2’,3’ =7.8 Hz, H-2’), 3.32 (s, 3H, OCH3), 2.96 (m, 1H, CH2),
2.69 (m, 1H, CH2), 2.42 (m, 1H, CH2), 2.35–2.16 (m, 6H, CH2, H-6),
1.71–1.60 (m, 5H, Me at C-4, CH2), 1.50 (m, 1H, CH2), 1.36–1.25 (m,
1H, CH2), 0.87 ppm (d, 3H, JMe,6 =6.7 Hz, Me at C-6); 13C NMR
(125 MHz, CDCl3): d=218.8 (C-1), 175.4 (C-6’), 137.2 (C-10), 133.1
(C-4), 130.5 (C-5), 127.0 (C-9), 104.3 (C-1’), 87.7 (CH), 84.4 (C-8), 76.9
(C-5’), 75.4 (CH), 73.8 (C-2’), 71.9 (CH), 55.5 (OCH3), 40.4 (CH2), 38.7
(CH2), 32.9 (C-6), 29.1 (CH2), 28.6 (CH2), 24.7 (CH2), 22.1 (Me at C-4),
20.5 (CH2), 12.2 ppm (Me at C-6); HRMS-ESI: m/z: calcd for
C23H36O9Na: 479.2257; found: 479.2263.
MGSTA-18 and MGSTA-19
To 57b (21 mg, not fully pure, <32.5 mmol,) at À788C in 2.5 mL
CH2Cl2, mCPBA (ꢀ77%, 7.6 mg, 33.9 mmol) in CH2Cl2 (2.5 mL) was
added. After 30 min at À788C, TLC (PE/EtOAc 1:1.5) showed only
baseline material. iPr2NEt (6.8 mL, 39 mmol) was then added and the
mixture was allowed to warm to room temperature over 10 min.
After 2 h at room temperature, TLC showed there still to have
a baseline material, so more iPr2NEt (7 mL) was added. After 1 h the
mixture was concentrated under reduced pressure and purified by
chromatography on silica eluting with PE/EtOAc (1:1.5) to give
MGSTA-19 (4.7 mg) and MGSTA-18 (8.5 mg) in 65% yield for
2 steps.
(4Z,6R,7S,8S,9E)-7-O-(Methyl 2’,3’,4’-tri-O-benzoyl-a-d-glucopyra-
nosyluronate)-4,6-dimethylcyclotetradeca-8-methoxy-4,9-dien-
one (38): To stirred 37 (20.8 mg; 27 mmol), in CDCl3 (0.75 mL), TiCl4
(66 mL; 54 mmol; 0.82m in CDCl3) was added. After storage for 12 h
at 48C, the solution was diluted with CH2Cl2 and quenched with
NaHCO3. The organic layers were collected, dried over Na2SO4, fil-
tered and the solvent was removed under reduced pressure. FC of
the residue (PE/EtOAc 4:1) gave 38 (14.5 mg, 69%); [a]D +33.48
4-{(S)-5-[(2R,3E,5R,6S,7S,8Z,12Z)-6-Hydroxy-7-methoxy-3,5-di-
methyl-14-oxooxacyclotetradeca-3,8,12-trien-2-yl]-4-oxohexyl}pi-
peridine-2,6-dione (MGSTA-18): 1H NMR (500 MHz): d=7.70 (brs,
1H), 6.23 (ddd, 1H, J=11, 11, 6.5 Hz, CH=CHC=O, H-3), 5.78 (ddd,
1H, J=11, 11, 5.3 Hz, H-8), 5.72 (d, 1H, J=11.4 Hz, H-4), 5.55 (aptt,
1H, J=10.5 Hz, H-7), 5.51 (d, 1H, J=10 Hz, H-11), 5.39 (d, 1H, J=
11.0 Hz, CHOC=O), 3.79 (d, 1H, J=10.1 Hz), 3.19 (s, 3H), 3.04 (t, 1H,
J=10.2 Hz, 10.2 Hz), 3.00 (dd, 1H, J=11.1 Hz, 7.1 Hz), 2.80–2.68 (m,
4H), 2.52 (dt, 2H, J=6.9, 6.8, 3.3 Hz), 2.30–2.19 (m, 2H), 2.17–2.06
(m, 3H), 1.96 (m, 1H), 1.63 (d, 3H, J=1.0 Hz), 1.65–1.60 (m, 2H),
1.40–1.33 (m, 2H), 1.08 (d, 3H, J=6.6 Hz), 0.97 ppm (d, 3H, J=
7.1 Hz); 13C NMR (125 MHz): d=210.9 (C), 171.7 (C), 165.5 (C), 145.1
(CH), 139.6 (CH), 133.7 (CH), 129.5 (C), 127.0 (CH), 121.4 (CH), 81.7
(CH), 77.2 (CH), 73.1 (CH), 54.9 (CH3), 46.6 (CH), 41.3 (CH2), 37.7
(CH2), 35.9 (CH), 34.2 (CH2), 30.4 (CH), 29.8 (CH2), 28.3 (CH2), 20.1
(CH2), 17.2 (CH3), 13.4 (CH3), 10.5 ppm (CH3); ESI/MSÀ m/z: 512.26
[M+Na+], 488.27 [MÀHÀ]; HRMS-ESI: m/z: calcd for C27H39NO7Na:
512.2624; found: 512.2598.
1
(c=0.75 in CHCl3); H NMR (CDCl3, 500 MHz): d=8.05–7.99 (m, 2H,
ArH), 8.00–7.94 (m, 2H, ArH), 7.92–7.85 (m, 2H, ArH), 7.56–7.48 (m,
2H, ArH), 7.47–7.35 (m, 5H, ArH), 7.30 (m, 2H, ArH), 6.27 (appt, 1H,
J
2’,3’ =J3’,4’ =9.9 Hz, H-3’), 5.71 (ddd, 1H, J9,10 =14.9, 8.4, 5.9 Hz, H-
10), 5.58 (t, 1H, J4’,5’ =10.0 Hz, H-4’), 5.45 (d, 1H, J1’,2’ =3.7 Hz, H-1’),
5.37 (dd, 1H, H-2’), 5.31 (dd, 1H, J8,9 =8.1 Hz, H-9), 5.21 (d, 1H, H-
5’), 4.80 (d, 1H, J5,6 =9.6 Hz, H-5), 3.70 (appt, 1H, H-8), 3.67 (s, 3H,
OCH3), 3.56 (d, 1H, J7,8 =9.1 Hz, H-7), 3.27 (s, 3H, OCH3), 2.63–2.57
(m, 1H, CH2), 2.38–2.07 (m, 7H, H-6, CH2), 1.98–1.93 (m, 1H, CH2),
1.67–1.57 (m, 2H, CH2), 1.48–1.38 (m, 2H, CH2), 1.42 (d, 3H, JMe,4
=
5.8 Hz, Me at C-4), 0.85 ppm (d, 3H, JMe,6 =6.6 Hz, Me at C-6);
13C NMR (125 MHz, CDCl3): d=211.1 (C-1), 169.1 (C=O), 166.0 (C=O),
165.7 (C=O), 165.6 (C=O), 134.9 (C-10), 134.0 (Cq), 133.7 (ArCH),
133.4 (ArCH), 133.4 (ArCH), 130.1 (ArCH), 129.9 (ArCH), 129.9 (ArCH),
129.6 (C-5), 129.3 (Cq), 129.3 (Cq), 129.1 (C-9), 129.0 (Cq), 128.7
(ArCH), 128.6 (ArCH), 128.5 (ArCH), 97.3 (C-1’), 86.2 (C-7), 82.6 (C-8),
71.8 (C-2’), 70.9 (C-4’), 70.0 (C-3’), 68.7 (C-5’), 56.1 (OCH3), 52.8
(OCH3), 40.8 (CH2), 40.3 (CH2), 33.9 (C-6), 30.2 (CH2), 29.0 (CH2), 26.3
(CH2), 22.8 (Me at C-4), 22.2 (CH2), 12.8 ppm (Me at C-6); HRMS-ESI:
m/z: calcd for C45H50O12Na: 805.3200; found: 805.3212.
4-{(S)-5-[(2R,3E,5R,6S,7S,8Z,12E)-6-Hydroxy-7-methoxy-3,5-di-
methyl-14-oxooxacyclotetradeca-3,8,12-trien-2-yl]-4-oxohexyl}pi-
peridine-2,6-dione (MGSTA-19): 1H NMR (500 MHz): d=7.75 (brs,
1H, NH), 6.84 (ddd, 1H, J=15.5, 6.0, 7.0 Hz, CH=CHC=O, H-3), 5.85
(d, 1H, J=15.5 Hz, CH=CHC=O, H-2), 5.69 (ddd, 1H, J=11, 11,
5.5 Hz, H-6), 5.59 (dd, 1H, J=11, 9.5 Hz, H-7), 5.34 (d, 1H, J=
10.0 Hz, CH3C=CH, H-11), 5.24 ppm (d, 1H, J=10.4 Hz, CHÀOÀC=O,
H-13); 13C NMR (125 MHz): d=211.1 (C), 171.8 (C), 167.0 (C), 150.7
(CH), 136.2 (CH), 133.1 (CH), 131.6 (C), 127.8 (CH), 123.0 (CH), 82.7
(CH), 77.5 (CH), 74.8 (CH), 55.5 (CH3), 47.2 (CH), 41.1 (CH2), 37.73
(CH2), 37.72 (CH2), 35.6 (CH), 34.0 (CH2), 30.4 (CH2), 30.3 (CH), 29.3
(CH2), 20.0 (CH2), 17.6 (CH3), 13.5 (CH3), 11.4 ppm (CH3); ESI/MS m/z:
512.26 [M+Na+], 488.27 [MÀHÀ]; HRMS-ESI: m/z: calcd for
C27H39NO7Na: 512.2624; found: 512.2621.
(4Z,6R,7S,8S,9E)-7-O-(a-d-Glucopyranosyluronic acid)-4,6-dime-
thylcyclotetradeca-8-methoxy-4,9-dienone (MGSTA-17): To stirred
38 (9 mg; 11.5 mmol) in MeOH (0.8 mL), NaOH aq. 5% (26.2 mL,
32.2 mmol) was added. The resulting solution was stirred at RT for
48 h. A solution of 1m AcOH was added until pH 7 was reached.
The solvent was removed under reduced pressure and the residue
was passed through a silica gel 100 C18-reversed phase column
(eluant, H2O to H2O/MeOH 4:1 to H2O/MeOH 2:1 to H2O/MeOH 1:1
to H2O/MeOH 1:2 to MeOH) to give MGSTA-17 (3.2 mg; 61%). [a]D
+63.2 (c=0.032 in MeOH); 1H NMR (CDCl3, 500 MHz): d=5.81
Cell viability assay (MTT-assay)
(ddd, 1H, J9,10 =15.0, 10.1, 4.5 Hz, H-10), 5.46–5.39 (dd, 1H, J8,9
9.4 Hz, H-9), 5.34 (dd, 1H, J=9.9, 1.6 Hz, H-5), 5.01 (d, 1H, J1’,2’
3.9 Hz, H-1’), 4.39 (d, 1H, J4’,5’ =10.2 Hz, H-5’), 3.81 (dd, 1H, J2’,3’
=
=
=
Cell viability (metabolic activity of viable cells) was quantified by
MTT assay. Cells were seeded onto 96-well plate (Nunc Inc., Den-
mark) at the concentration of 1”104 cells per well. When cells
reached 60–70% confluence, the medium (DMEM) was replaced
with medium containing 10% FBS and six various migrastatin ana-
logues at the concentrations of: 1, 10, or 100 mm. The cultures
were incubated for 24 h. Then, cells were incubated with
0.5 mgmLÀ1 tetrazolium salt MTT diluted in phenol red-free DMEM
medium (Sigma Aldrich) for 4 h at 378C. To complete solubilization
of the formazan crystals, 100 mL of DMSO (dimethyl sulfoxide,
Sigma Aldrich) were added to each well. Cell viability was quanti-
fied by measuring photometric absorbance at 570 nm in a multi-
well plate reader (Infinite 200 PRO Tecanꢁ, TECAN, Switzerland). All
the samples were examined in triplicate, each experiment was con-
ducted three times (n=9).
9.9, J3’,4’ =8.9 Hz, H-3’) 3.74–3.71 (appt, 1H, H-8’), 3.56 (m, 2H, H-
7,2’), 3.48 (dd, 1H, J3’,4’ =9.0 Hz, H-4’), 3.29 (s, 3H, OCH3), 3.12–3.05
(m, 1H, CH2), 2.78–2.73 (m, 1H, CH2), 2.58–2.52 (m, 1H, CH2), 2.36–
2.10 (m, 6H, H-6, CH2), 1.72–1.62 (m, 2H, CH2), 1.70 (d, 3H, J=
1.4 Hz, Me at C-4), 1.53–1.44 (m, 1H, CH2), 1.27–1.13 (m, 1H, CH2),
0.89 ppm (d, 3H, J6,Me =6.7 Hz, Me at C-6); 13C NMR (125 MHz,
CDCl3): d=218.4 (C-1), 175.7 (C-6’), 136.8 (C-10), 133.4 (C-4), 130.6
(C-5), 127.3 (C-9), 99.2 (C-1’), 84.4 (C-7), 83.2 (C-8), 72.6 (C-3’), 71.9
(C-4’), 71.5 (C-2’), 71.3 (C-5’), 55.3 (OCH3), 40.3 (CH2), 37.8 (CH2), 33.8
(C-6), 29.0 (CH2), 28.7 (CH2), 24.2 (CH2), 22.0 (Me at C-4), 19.9 (CH2),
12.0 ppm (Me at C-6); HRMS-ESI: calcd for C23H36O9Na: 479.2257;
found: 479.2256.
Chem. Eur. J. 2015, 21, 18109 – 18121
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Full Paper
In vitro wound healing assay (scratch test)
Acknowledgements
The scratch assay was conducted to assess the influence of migras-
tatin analogues on MCF7, MCF7-Dox, MDA-MB361, MDA-MB231,
HPAC cancer cell motility. The cells were seeded in a high density
at 600 mm Petri dishes (Corning-Costar, Cambridge, USA). After
24 h, the medium was removed and replaced with medium
(DMEM) containing 10% FBS and one migrastatin analogues or
DMSO (control). The monolayer was wounded by scratching the
surface: as uniformly and straight as possible with a pipette tip
(1000 mL) at least three times. The images of cells invading the
scratch were captured at indicated time points (0, 6 and 24 or 48 h
where indicated) using phase-contrast microscopy (IX 70 Olympus
Optical Co., Germany). The pictures were analyzed using a comput-
er-assisted image analyzer (Olympus Microimageꢁ Image Analysis,
software version 4.0 for Windows, USA). The migration rate was ex-
pressed as a percentage of scratch closure and was calculated as
follows: % of scratch closure=aÀb/a, in which (a) is a distance be-
tween edges of the wound, and (b) is the distance which remained
cell-free during cell migration to close the wound.[39] The values
are presented as means of three independent wound fields from
three independent experiments (n=9).
This publication has emanated from research supported in
part by a research grant from Science Foundation Ireland (SFI)
and is co-funded under the European Regional Development
Fund under Grant Number 14/SP/2710. The research leading
to these results has also received funding in part from the
People Programme (Marie Curie Actions) of the European
Union’s Seventh Framework Programme FP7/2007-2013/ under
REA grant agreement No. PIEF-GA-2011-299042 and the Poland
National Science Council. We acknowledge networking support
and a short term scientific mission funded by the COST Action
CM1106. We thank Dr. A. Calon and Dr. E. Lonardo (Institute for
Research in Biomedicine, IRB Barcelona) for helpful discussion.
Keywords: anomerization · glycosidation · natural products ·
stereoselective synthesis · tumour cell migration
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Received: July 21, 2015
Published online on November 4, 2015
Chem. Eur. J. 2015, 21, 18109 – 18121
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