Synthesis of an Orthogonally Protected Polyhydroxylated Cyclopentene from l-Sorbose

Article abstract of DOI:10.1002/asia.201600736

The use of l-sorbose in the synthesis of functionalized cyclopentene derivatives was accomplished. These cyclopentene derivatives are related to those found in naturally occurring jatrophane frameworks and in other bioactive compounds. The formation of allyl α-l-sorbopyranoside was a key synthetic step. Regioselective introduction of protecting groups was followed by the hydrolysis of the allyl glycoside to furnish a fully protected acyclic l-sorbose derivative. This acyclic intermediate was subsequently used to give an orthogonally protected polyhydroxylated cyclopentene, which has potential for further synthesis of bioactive compounds. The protected cyclopentene itself showed a clear cytotoxic activity when tested against a panel of human cancer cell lines (HT29, LS174T, SW620, A549, and HeLa cells).

Full text of DOI:10.1002/asia.201600736

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Synthesis of an orthogonally protected polyhydroxylated cyclopentene from L-
sorbose
Authors: Daniele Lo Re; Leigh Jones; Ernest Giralt; Paul Vincent Murphy
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Chemistry - An Asian Journal
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COMMUNICATION
Synthesis of an orthogonally protected polyhydroxylated
cyclopentene from L-sorbose
Daniele Lo Rea*, Leigh Jonesa, Ernest Giraltb,c and Paul Murphya*
Dedicated to Ma Teresa Plaza López-Espinosa on the occasion of her retirement ((optional))
Abstract: The use of L-sorbose in synthesis of functionalized
cyclopentene derivatives was accomplished. These cyclopentene
derivatives are related to those found in naturally occurring
jatrophone frameworks and in other bioactive compounds. The
formation of allyl α-L-sorbopyranoside was a key synthetic step.
Regioselective introduction of protecting groups was followed by the
hydrolysis of the allyl glycoside to furnish a fully protected acyclic L-
sorbose derivative. This acyclic intermediate was subsequently
used to give an orthogonally protected polyhydroxylated
cyclopentene, which has potential in further bioactive compound
synthesis. The protected cyclopentene itself showed a clear
cytotoxic activity when tested against a panel of human cancer cell
lines (HT29, LS174T, SW620, A549, HeLa cells).
at C-3 enhanced the ABCB1 inhibitory activity (figure 1). They
also show that there is a correlation between overall lipophilicity
and ABCB1 inhibition.[9]
The occurrence of resistance to anticancer agents is a major
obstacle for successful cancer chemotherapy. The emergence
of resistance to anticancer drugs, in particular multidrug
resistance (MDR) has made many of the available anticancer
drugs ineffective.[1] A glycoprotein, ABCB1, also known as P-
glycoprotein, is a membrane protein member of the ABC
transporters superfamily. These membrane-embedded transport
proteins decrease the intracellular drug accumulation, by and
ATP-dependent efflux. This reduces the cytotoxicity of the
anticancer agent and enables the tumor cells to survive.
Jatrophanes, such as 1-3, are natural compounds extracted
from plants of the genus of Euphorbia. A broad range of
biological properties have been reported for constituents of the
plant extract.[2] In particular, jatrophanes are potent and specific
P-glycoprotein modulators.[3] In addition, a variety of other
biological activities have been reported: inhibitory activity on the
mammalian mitochondrial respiratory chain,[4] antiviral activity,[5]
microtubole interaction,[6] antiplasmodial activity,[7] cytotoxicity
against various human cancer cell lines[8].
Figure 1. a) Structure of selected jatrophane natural products; b) synthetic
jatrophane analogues and their activity against ABCB1; c) orthogonally
protected pyranose 18 and open-chain sorbose derivatives 20 and the
orthogonal protected cyclopentene 7 prepared herein.
Nevertheless, the potential to prepare cyclopentene derivatives
related to those found in 4 and which would ultimately be used
to give new jatrophone analogues has formed the motivation for
the work described herein. We envisaged that the preparation
of orthogonally protected polyhydroxylated cyclopentanol 7,
similar to the ring A of the jatrophane framework, is relevant to
the preparation of jatrophane analogues with increased
biological activity and for structure-activity relationships. In
addition, advanced intermediate 7 can be also used for the
It has been reported that modifications of the cyclopentene (ring
A) of the jatrophane framework, resulted in increased biological
preparation
of
biologically
active
cyclopentitols
or
cyclopentenols. These are polyhydroxylated cyclopentanes,[10]
[a]
Dr. D. Lo Re, Prof. P.V. Murphy, Dr. L. Jones
School of Chemistry
that display a plethora of biological activity such as found for the
[11]
glycosidase inhibitors trehazolin and mannostatin,[10a]
the
National University of Ireland, Galway
University Road, Galway, Ireland
E-mail: daniele.lore@irbbarcelona.org
E-Mail: paul.v.murphy@nuigalway.ie
Prof. Ernest Giralt
Institute for Research in Biomedicine (IRB Barcelona), The
Barcelona Institute of Science and Technology
C/Baldiri Reixac 10, Barcelona, E-08028, Spain
Prof. Ernest Giralt
anticancer and antibiotic pactamycin,[12] and the insecticide
ryanodine.[13] In addition, cyclopentenols, are also important
because of their presence in numerous biologically active
targets such as the antibiotic pentenomycin,[14] the anti-
inflammatory monotropein,[15] and the anticancer compound (-)-
neplanocin A.[16]
[b]
[c]
Department of Organic Chemistry
University of Barcelona
Marti i Franques 1-11, Barcelona, E-08028, Spain
It was envisaged that the construction of the polyhydroxylated
cyclopentene could be achieved through
a
ring-closing
metathesis[10c, 17] (RCM) reaction of fully protected diene such as
that in 11. Since RCM could be challenging in such a densely
substituted diene,[18] we first prepared 11 in order to validate the
synthetic strategy.
Supporting information for this article is given via a link at the
activity (figure 1).[9] For instance, Hierseman and co-workers[9]
demonstrated that introduction of aromatic lipophilic substituent
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Chemistry - An Asian Journal
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COMMUNICATION
chromatography. We next attempt the protection of the
secondary alcohol of 15 as the TBS ether. However, the
introduction of the TBS group failed under several conditions
(TBSOTf-TEA-THF, TBSOTf-NaH-THF, TBSOTf-DBU, TBSCl-
NaH-DMF). Fortunately, treatment of 15 with benzoyl chloride in
pyridine gave 18 in excellent yield (93%). The analysis of NMR
data for 19 confirmed that the hydroxyl group at C-4 was
protected as the MOM ether while the hydroxyl group at C-5 was
protected as the benzoate ester. In addition, the mixture of 15
and 17 obtained during regioselective MOM protection of 14 was
reacted with BzCl in pyridine giving both 18 and 21 that can be
easily separated using chromatography. Treatment of the 3,6-O-
p-methoxybenzylidene acetal 18 with NaCNBH3 and TMSCl[25]
gave the corresponding 4-O-PMB ether 19 in 59% yield.
Scheme 1. Preliminary validation of the synthetic strategy from L-sorbose:
synthesis of per-O-benzylated cyclopentane 12.
Compound 9 has been prepared from L-sorbose previously.[19]
However, in our hands, attempts to simultaneously install olefin
at C6 and C2 through directly oxidation of 9 followed by a
concomitant double Wittig reaction were unsuccessful. Oxidation
of the latent alcohol of 9 under Swern conditions followed by
Wittig reaction only gave a complex mixture while treatment of 9
with the Dess-Martin reagent gave no reaction. For these
reasons, aldehyde 10 was first prepared as reported[19] and then
treated with Ph3PCH3Br and tBuOK to give the desired diene 11.
The RCM of 11 using the Grubbs 2nd generation catalyst at room
temperature only gave only recovered starting material.
However, when the RCM was carried out by heating in refluxing
toluene, the polyhydroxylated cyclopentene 12 was isolated in
good yield (76%, Scheme 1).
Next, the protection of the secondary hydroxyl group of 19 was
attempted. Unfortunately, protection of 19 as a TIPS ether was
unsuccessful after several attempts (using TIPSCl or TIPSOTf,
DMAP, DMF, 130 oC or TIPSOTf, NaH, THF, RT), with 19 being
recovered unreacted. Surprisingly, even benzylation of 19 failed
under several conditions (NaH and BnBr or Dudley´s reagent[26]).
Instead, removal of the allyl group, followed by installation of the
protecting group at C-3 were studied. However, attempted
removal of the allyl protecting group using PdCl2 at several
different conditions gave the desired product in low yields (28-
41%). Fortunately, treatment of 19 with OsO4/NaIO4[23b] followed
by the simultaneous protection of both the C-3 and C-6 OH
groups as TBS ethers gave 20 in good yield (58% over two
steps). With 20 in hand, chain branching at C-2 using the Wittig
reaction was investigated. Treatment of 20 with Ph3PCH3Br and
tBuOK gave the desired alkene 22 in 61% yield. Regioselective
removal of the TBS group at the primary position of 22 using 10
% TsOH in MeOH gave a primary alcohol, which was oxidized
using Ley-Griffith conditions and gave aldehyde 23 in good yield.
Again, compound 23 was reacted with Ph3PCH3Br and tBuOK to
give diene 24 (Scheme 4). However, the reaction of 24 with the
Grubbs 2nd generation catalyst in toluene only gave a complex
mixture at different temperatures (from 80 oC to reflux).
Fortunately, RCM of 24 in dichloromethane under microwave
irradiation gave the orthogonally protected cyclopentene 25 in
good yield (71%). Removal of PMB group in 25 was successful
using DDQ-H2O leading to 26 (66%). Treatment of 26 with MnO2
gave the α,β-unsaturated aldehyde 27 in 74% yield. Aldehyde 27
was then reacted with isopropenylmagnesium bromide to give
secondary alcohol 28 (72%) that was finally oxidized with MnO2
giving the orthogonally protected advanced intermediate 7 in
93% yield (Scheme 3).
With these encouraging observations in hand, it was next
decided to exploit this strategy for the preparation of an
orthogonally protected L-sorbose derivative.
manipulation of L-sorbose is often carried out via its methyl L-
sorbopyranoside[19]
or its 2,3:4:6-di-O-isopropyliden-α-L-
Chemical
sorbofuranose.[18, 20] However, these synthetic approaches suffer
drawbacks. In fact, hydrolysis of the dioxolane ring (for the L-
sorbofuranose) or hydrolysis of the methyl glycoside (for the L-
sorbopyranoside) requires strong acidic conditions, which are
not compatible with several functional or protecting groups. On
other hand, the allyl group[21] can be removed in the presence of
different protecting groups[22] under relatively mild conditions.[23]
Since the synthesis of the L-sorbopyranoside 8 is achieved
using MeOH in the presence of acetyl chloride (AcCl), we
hypothesized that similar conditions could be used for the
preparation of the allyl glycoside 13. In fact, treatment of L-
sorbose with allyl alcohol and AcCl gave 13 in 64% yield.
However, at this stage we could not establish the
1
stereochemical configuration of 13. However, H-NMR analysis
Importantly, advanced intermediate 7 has potential to be used
for the preparation of analogues of MDR modulators such as
jatrophanes and can be also used for the preparation of
biologically active cyclopentitols. In addition, intermediate 7 was
screened against a panel of human cancer cell lines derived
from colon (HT29, Ls174t, SW620), lung (A549) and cervical
cancer (HeLa). We took advantage that 7 has a silyl ether, a
feature that could increase cellular uptake and enhance anti-
proliferative effects. In addition, It has also been demonstrated
that no “element-specific” toxicity is associated with
organosilicon compounds.[27] Padron and co-workers have
demonstrated that the presence of TBS ether enhance the anti-
proliferative effects of tetrahydropyrans derivatives in HL60
(human promyelocytic leukemia) and MCF7 (human breast
cancer) cell lines.[28] Furthermore, trialkylsilyl campothecine
suggested the formation of the pyranose ring (J3,4 = 9.5 Hz; J5,6
10.4; J4,5 = 8.7; J5,6’ = 5.4) had occurred. Fortunately,
=
regioselective protection of hydroxyl groups at C-1 and C-3
using anisaldehyde dimethyl acetal in presence of
camphorsulphonic acid gave 14 in good yield (scheme 2) which
crystallized from a mixture of MeOH-AcOEt 3:1. X-Ray crystal
structure[24] determination of the resultant crystals confirmed the
formation of the allyl α-L-sorbopyranoside 14 as the main
product (see SI). The regioselective protection of diol 14 was
next attempted. Unfortunately, the attempted reaction of 14 with
TIPSCl in pyridine was not successful. Similar results were
obtained when 14 was treated with MOMCl in pyridine.
Fortunately, regioselective protection of 14 was achieved using
MOMCl and DIPEA in dichloromethane, giving alcohol 15 in
moderate yield (55%), together with small amounts of 16 (14%)
derivatives such as cositecan[29] [30] and DB-67[31] are promising
,
and
a mixture of regioisomers 15 and 17 (16%), after
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Chemistry - An Asian Journal
10.1002/asia.201600736
COMMUNICATION
compounds for cancer treatment and are currently in phase III
and II clinical trials respectively.
Scheme 3. Synthesis of polyhydroxylated cyclopentene 7.
In conclusions, we have developed a synthetic approach for the
converstion of L-sorbose to allyl α-L-sorbopyranoside (13). This
intermediate was turned into an orthogonally protected L-
sorbopyranose derivative (18).
The cleavage of the allyl
glycoside in 19 is compatible with different protecting group
(acidic, basic and single electron oxidation sensitive).
Subsequently, we also prepared the orthogonal acyclic L-
sorbose derivative (20): this intermediate can be used for the
preparation of more complex chiral products. We demonstrated
its use in preparation of the orthogonally protected
polyhydroxylated cyclopentene 7 which could have potential as
an intermediate for the preparation of jatrophane analogues or
for the preparation of cyclopentitols which are of particular
significance because of their presence in a variety of medicinally
relevant natural products. Compound, 7 was tested in a panel of
human cancer cell lines (HT29, Ls174t, SW620, HeLa, A549)
and displayed good cytotoxic activity. In addition, 7 weakly
inhibit ABCB1 ABCC2 and ABCC3 demonstrating that it could
be used as a starting point for the development of truncated
jatrophane analogues as multi drug resistance (MDR)
modulators.
Scheme 2. a) Synthesis of open-chain intermediate 20; b) resolution of 15-17
mixture; c) crystal structure of 14.
Compound 7 showed good toxicity in the XTT assay with IC50
range of 6.1-35.1 µM. Compound 7 is generally less potent than
widely used chemotherapeutics cisplatin or oxaliplatin while in
HT29 colon cancer cell line, displayed an activity similar to
cisplatin. Previous studies have demonstrated the ability of
jathropane diterpenes to reduce the activity of ABC proteins
linked with multi-drug resistance (MDR).[32] Compound 7 can be
considered as an analogue of ring A of jatrophane (figure 1). In
light of that, we decided to test 7 against ABCC2 (MRP2),
ABCC3 (MRP3) and ABCB1 (MDR1 or P-gp). At the
concentration of 20 µM, compound 7 show weakly inhibit
ABCC2 (10.5 ± 0.25%), ABCC3 (6.1± 0.35%) and ABCB1 (7.1±
0.65%) indicating that truncated[33] jatrophane analogues might
have potential to be developed into MDR modulators.
Figure 2. Antiproliferative activity of 7: IC50 was determinated by XTT assay.
a: see ref [34]
Experimental Section
General methods and materials. All NMR spectra were recorded using
a 500 MHz spectrometer at 30 oC. Chemical shifts are reported relative
to internal Me4Si in CDCl3 (δ 0.0 ppm) for 1H and CDCl3 (δ 77.16) for 13
C
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Chemistry - An Asian Journal
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COMMUNICATION
at 30 ºC, unless otherwise stated. The 13C signals were assigned with the
(1.229 mL; 5.36 mmol) were added and the resulting solution was stirred
at 80 oC for 18h. TLC (petroleum ether-EtOAc 1:1) showed a new spot
with higher Rf and the absence of starting material. The solution was
diluted with dichloromethane and washed with H2O. The organic phase
was dried and the solvent was removed. Flash chromatography
(cyclohexane-EtOAc, 30:1 to 10:1 gradient elution gave 20 as a yellow oil
(525 mg; 58 % over two steps); [α]D , - 19.6° (c 0.41, CHCl ); 1H NMR
(CDCl3, 500 MHz): δ 8.03–7.97 (m, 2H, ArCH), 7.55–7.50 (m,31H, ArCH),
7.41 (t, 2H, J = 7.8 Hz, ArCH), 7.09 (d, 2H, J = 8.6 Hz, ArCH), 6.79 (d,
2H, J = 8.6 Hz, ArCH), 5.39 (td, 1H, J = 5.8, 4.3 Hz, H-5), 4.75 (d, 2H, J =
1.6 Hz, CH2), 4.50 (d, 1H, J=5.1 Hz, H-3), 4.34 – 4.24 (m, 2H, CH2), 4.22
– 4.13 (m, 3H, H-4 and CH2), 3.85 (dd, 2H, J = 5.9, 1.4 Hz, H-6), 3.78 (s,
3H, ArOCH3), 3.39 (s, 3H, OCH2OCH3), 0.90 (s, 9H, C(CH3)3), 0.86 (s,
9H, C(CH3)3), 0.08 (s, 3H, SiCH3), 0.03 (m, 9H, 3SiCH3); 13C NMR
(CDCl3, 125 MHz): δ 206.3 (C=O), 165.8 (C=O), 159.4 (ArC), 133.2
(ArCH), 130.0 (ArCH), 129.9 (ArC), 129.6 (ArCH), 129.5 (ArC), 128.5
(ArCH), 113.8 (ArCH), 98.2 (CH2), 77.5 (C-4), 76.3 (C-3), 73.1 (CH2),
72.9 (CH2), 60.5 (C-5), 56.4 (CH2), 55.4 (ArOCH3), 25.9 (C(CH3)3), 18.2
(C(CH3)3), -4.8 (SiCH3), -4.8 (SiCH3), -5.2 (SiCH3), -5.4 (SiCH3); HRMS-
ESI: calcd for C35H56O9Si2Na: 699.3361; Found: 699.3354 (M+Na+).
1
aid of HSQC. The H-NMR signals were assigned with the aid of COSY.
Coupling constants are reported in Hertz. High resolution mass spectra
were measured using an LC Time-of-flight mass spectrometer and were
measured in positive and/or negative mode as indicated. TLC were
performed on aluminium sheets precoated with Silica Gel 60 (HF254, E.
Merck) and spots visualized charring with vanillin solutions. Flash column
chromatography was carried out using silica gel 60 (0.040-0.630 mm, E.
Merck). Dichloromethane, tetrahydrofuran, MeOH, and toluene were
used as obtained from
a
PureSolvTM solvent purification system.
Petroleum ether is the fraction with bp 40-60 ºC.
Allyl 1,3-O-(4-methoxybenzylidine)-α-L-sorbopyranoside (14). To a
stirred solution of 13 (2.364 g; 10.7 mmol) in DMF (23 mL),
camphorsulphonic acid (248 mg; 1.07 mmol) and anisaldehyde dimethyl
acetal (1.82 mL; 10.7 mmol) were added. The solution was stirred at
room temperature for 5 h. The mixture was diluted with Et2O (30 mL) and
washed several times with water. The solvent was evaporated and flash
chromatography (EtOAc-petroleum ether 3:1) gave 14 as a white solid
o
o
(2.91 g; 80%); m.p. = 104 C-109 C; [α] - 41 (c 0.69, CHCl3); 1H NMR
(CDCl3, 500 MHz): δ 7.48 – 7.36 (d, J = D8.8 Hz, 2H, ArCH), 6.91 – 6.82
(d, J = 8.7 Hz, 2H, ArCH 5.99 (ddt, Jtrans = 16.9, Jcis = 10.5, 5.3 Hz, 1H,
OCH2CH=CH2), 5.51 (s, 1H, OCHO), 5.37 (dd, 1H, J = 17.3, Jgem = 1.7
Hz, OCH2CH=CH2), 5.18 (dd, 1H, Jcis = 10.6, OCH2CH=CH2), 4.26 (dd,
1H, J = 12.1, 2.0 Hz, CH2), 4.18 – 4.11 (m, 1H, OCH2CH=CH2), 4.11 –
3.98 (m, 2H, OCH2CH=CH2, CH), 3.79 (s, 3H, J = 1.4 Hz, OCH3), 3.75 –
3.62 (m, 2H, CH2, CH), 3.58 – 3.49 (m, 2H, CH2), 3.44 (dd, 1H, J = 9.8,
2.7 Hz, CH); 13C NMR (CDCl3, 125 MHz): 160.4 (ArC), 134.4
(OCH2CH=CH2), 129.7 (ArC), 128.0, (ArCH), 116.7 (OCH2CH=CH2),
113.8 (ArCH), 102.7 (OCHO), 92.8 (C-2), 82.0 (CH), 71.2 (CH), 70.7
(CH), 68.2 (CH2), 63.3 (CH2), 62.0 (CH2), 55.5 (OCH3); HRMS-ESI: calcd
for C17H22O7Na: 361.1263; Found: 361.1266 (M+Na+).
(1S,4R,5R)-4-(Tert-butyldimethylsilyl)oxy)-3-methacryloyl-5-
(methoxymethoxy)cyclopent-2-en-1-yl benzoate (7). To
a stirred
solution of 28 (20.0 mg; 0.045 mmol) in dichloromethane (1.0 mL), MnO2
(39 mg; 0.45 mmol) was added. The resulting solution was stirred at
room temperature for 48 h. The mixture was filtered through celite with
dichloromethane (50 mL) and the solvent was removed. The crude was
re-dissolved in dichloromethane (1.0 mL), MnO2 (20 mg; 0.23 mmol) was
added and the resulting mixture was stirred at room temperature for
additional
6
h. The mixture was filtered through celite with
Flash
dichloromethane (50 mL) and the solvent was removed.
chromatography (EtOAc-cyclohexane 1:10) gave 7 as a yellow oil (18.7
mg; 93%); 1H NMR (CDCl3, 500 MHz): δ 8.12 – 8.06 (m, 2H, ArCH), 7.61
– 7.56 (m, 1H, ArCH), 7.46 (t, 2H, J = 7.8 Hz, ArCH), 6.16 (d, 1H, J1,2
=
Allyl
5-O-benzoyl-1,3-O-(4-methoxybenzylidine)-4-O-
1.9 Hz, H-2), 6.00 (s, 1H, C(Me)=CH2), 5.90 (s, 1H, C(Me)=CH2), 5.71
(dd, 1H, J1,5 = 4.4 Hz, H-1), 5.02 (d, 1H, J4,5 = 4.0 Hz, H-4), 4.77 (q, 2H, J
= 6.8 Hz, OCH2O), 4.29 (appt, 1H, J1,4 = J1,5 =4.2 Hz, H-5), 3.33 (s, 3H,
OCH3), 1.93 (s, 3H, C(Me)=CH2), 0.85 (s, 9H, C(CH3)3), 0.15 (s, 3H,
SiCH3 ), 0.07 (s, 3H, SiCH3); 13C NMR (CDCl3, 125 MHz): δ 194.5 (C=O),
166.1 (C=O), 146.4 (C), 144.5 (C), 133.4 (ArCH), 133.2 (C-2), 130.0 (C),
129.9 (ArCH), 128.8 (C(Me)=CH2), 128.6 (ArCH), 97.2 (OCH2O), 90.6 (C-
5), 79.9 (C-1), 78.7 (C-4), 56.1 (OCH3), 25.8 (C(CH3)3), 18.1
(C(Me)=CH2), 17.4 (C(CH3)3), -4.6 (SiCH3), -5.0 (SiCH3); HRMS-ESI:
calcd for C24H34O6SiNa: 469.2022; Found: 469.2015.
(methoxymethoxy)-α-L-sorbopyranoside (18). To a stirred solution of
15 (430 mg; 1.12 mmol) in pyridine (10 mL) at 0 oC, BzCl (195 µL; 1.68
mmol) was added. The mixture was stirred at room temperature for 18 h.
TLC (petroleum ether-EtOAc, 2:1) showed a new spot with higher Rf and
the absence of 15. The solution was diluted with dichloromethane (20
mL), and washed with 1% HCl (20 mL), dried over Na2SO4, filtered and
the solvent was removed. Flash chromatography) (cyclohexane-EtOAc,
5:1) gave 18 as a yellow oil (508 mg; 93 %); [α] : -17.16 (c 0.85, CHCl3);
1H NMR (CDCl3, 500 MHz): 8.09 (m, 2H, ArDCH), 7.60–7.55 (m, 1H,
ArCH), 7.47–7.43 (m, 4H, ArCH), 6.89 (d, 2H, J -= 8.8 Hz, ArCH), 6.03
(ddt, 1H, Jtrans = 17.3, Jcis =10.6, 5.4 Hz, OCH2CH=CH2), 5.59 (s, 1H,
OCHO), 5.40 (dd, 1H, Jtrans = 17.2, J = 1.7 Hz, OCH2CH=CH2), 5.27–5.16
(m, 2H, OCH2CH=CH2, H-5), 4.84 (d, 1H, J = 6.8 Hz, OCH2O), 4.67 (d,
Cell Culture. A549 (non-small cell lung cancer, human), HT29
(colorectal adenocarcinoma, human), SW620 (colorectal, Dukes’ type C,
human) and Ls174t (colorectal, Dukes’ type B, human), HeLa (cervix
adenocarcinoma, human) cells were grown in 75 cm2 culture flasks
(Corning® Flask) as adherent monolayer cultures in Dulbecco´s Modified
Eagle Medium (DMEM) (GIBCO, ref. 41966-029). Culture mediums were
supplemented with 10% heat-inactivated fetal bovine serum, and with
Penicillin/Streptomycin (Gibco, 15140-122). Cultures were maintained at
37 °C in a humidified atmosphere containing 5% CO2 and 95% air.
1H, OCH2O), 4.47 (dd, 1H, J3,4 = J4,5 = 9.4 Hz, H-4), 4.32 (d, 1H, J1,1’
=
12.1 Hz, H-1), 4.21 (ddt, 1H, J = 12.7, 5.1, 1.6 Hz, OCH2CH=CH2), 4.08
(ddt, 1H, J = 12.7, 5.1, 1.6 Hz, OCH2CH=CH2), 4.01 (dd, 1H, J6,6’ = 10.6,
J6,5 = 6.4 Hz, H-6), 3.81 (s, 3H, ArOCH3), 3.74 (d, 1H, H-3), 3.68 – 3.61
(m, 2H, H-1’,6’), 3.18 (s, 3H, OCH2OCH3); 13C NMR (CDCl3, 125 MHz): δ
165.8 (CO), 160.3 (ArC), 134.4(OCH2CH=CH2), 133.5 (ArCH), 129.9
(ArC), 129.9 (ArCH), 129.6 (ArC), 128.6 (ArCH), 127.8 (ArCH), 116.8
(OCH2CH=CH2), 113.7 (ArCH), 102.4 (OCHO), 97.3 (OCH2O), 93.0 (C-
2), 82.1 (C-3), 73.0 (C-4), 71.7 (C-5), 68.1 (C-1), 62.3 (OCH2CH=CH2),
60.9 (C-6), 55.8 (OCH2OCH3), 55.4 (ArOCH3); HRMS-ESI: calcd for
C26H30O9Na: 509.1788; Found: 509.1788 (M+Na+).
Inhibition of Cell Viability Assay. Drug effects on exponentially growing
tumour cells were determined using XTT assay as described
previously.35 A549, HT29, SW620, Ls174t, HeLa cells were seeded at a
density of 3000 cells/well in 96-well plates and incubated for 24h.
Thereafter, cancer cells were exposed to drugs at different
concentrations during 72h. At 72h, 50 μL of XTT/ECR 1:50 solution and
incubated for a further 4 h at 37 oC. .Absorbance measured at 475 nm
was converted to percentages. UV-vis absorbance was measured at 475
nM using a microplate reader. Experiments were performed in triplicated
for each drug concentration and carried out independently at least three
times. The interpolation analysis was done using dose-dependent
inhibition pattern (log3d vs. normalized response {Variable slope}) with
Prism version 5.00 software (GraphPad Software, USA).
5-O-benzoyl-3,6-di-O-(Tert-butyldimethylsilyl)-1-O-(4-
methoxybenzyl)-4-O-(methoxymethoxy)-L-sorbose (20). To a stirred
solution of 19 (657 mg; 1.34 mmol) in dioxane (6.5 mL) and H2O (0.7
mL), NMO (473 mg; 4.039 mmol), OsO4 (1.419 mL; ~ 0.134 mmol, 2.5%
in t-BuOH), NaIO4 (1.146 g; 5.36 mmol) and H2O (2.6 mL) were added.
The resulting solution was stirred at 60 oC for 24 h. TLC (petroleum
ether-EtOAc 1:1) showed a new spot with lower Rf and the absence of
19. The mixture was diluted with dichloromethane and washed with H2O.
The organic layers were washed with aq satd Na2S2O3. The organic
phase was dried and the solvent was removed. The crude mixture was
dissolved in DMF (6.5 mL), DIPEA (1.87 mL; 10.72 mmol) and TBSOTf
For internal use, please do not delete. Submitted_Manuscript

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10.1002/asia.201600736
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Drug transporter inhibition assay. Cells are seeded in a 96-well culture
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at 37 oC for 15 min. Subsequently substrate is added to the plate
followed by 20-min incubation at 37 oC. The plate is then washed with
cold assay buffer followed by fluorescence reading for assays with
fluorogenic substrates. Cell lines: MDR1-MDCKII (Transporter: P-gp,
substrate: Calcein AM, positive control Verapamil IC50 = 29 µM); MRP2-
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=
Acknowledgements ((optional))
This work was supported by Science Foundation Ireland (grant Nos.
07/IN.1/B966 and 11/TIDA/B2047). The research leading to these results
also received funding from the People Programme (Marie Curie Actions)
of the European Union’s Seventh Framework Programme (FP7/2007-
2013) under grant agreement number PIEF-GA-2011-299042 and from
the European Union’s Seventh Framework Programme for research,
technological development and demonstration under grant agreement no
600404 and from MINECO-FEDER (Bio2013-40716-R). IRB Barcelona is
the recipient of a Severo Ochoa Award of Excellence from MINECO
(Government of Spain).
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