Sidechain Diversification of Grandifloracin Allows Identification of Analogues with Enhanced Anti-Austerity Activity against Human PANC-1 Pancreatic Cancer Cells

Article abstract of DOI:10.1002/cmdc.201900549

The natural product (+)-grandifloracin is a potent “anti-austerity” agent, able to suppress the ability of various pancreatic cancer cell lines to tolerate conditions of nutrient deprivation. Such anti-austerity agents represent a promising approach to ca

Full text of DOI:10.1002/cmdc.201900549

Full Papers
DOI: 10.1002/cmdc.201900549
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Sidechain Diversification of Grandifloracin Allows
Identification of Analogues with Enhanced Anti-Austerity
Activity against Human PANC-1 Pancreatic Cancer Cells
Benjamin E. Alexander,[a] Sijia Sun,[b] Matthew J. Palframan,[a] Gabriele Kociok-Köhn,[c]
Dya Fita Dibwe,[b] Shiro Watanabe,[b] Lorenzo Caggiano,*[d] Suresh Awale,*[b] and
Simon E. Lewis*[a]
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The natural product (+)-grandifloracin is a potent “anti-
austerity” agent, able to suppress the ability of various
pancreatic cancer cell lines to tolerate conditions of nutrient
deprivation. Such anti-austerity agents represent a promising
approach to cancer chemotherapy. Here we report the synthesis
and biological evaluation of racemic analogues of grandifloracin
bearing diverse sidechains, of which two show enhanced
potency in comparison with the natural product. Additionally,
several unexpected by-products containing modifications of the
grandifloracin core were isolated, identified and similarly
evaluated for biological activity.
Introduction
combination therapies as the first line therapies of choice, even
these impart only modest survival benefits.[5] It is clear a major
breakthrough will require the use of entirely new therapeutic
strategies exploiting emerging targets.[6]
Pancreatic cancer is one of the most aggressive human
malignancies, and as it is often initially asymptomatic, most
patients already have metastases upon presentation. Therefore,
surgical resection is usually not appropriate.[1] Pancreatic tumours
show intrinsic resistance to chemotherapeutic agents that are
effective against other tumour types.[2] There are no truly effective
drugs for pancreatic cancer – It has a median survival of less than
6 months and the lowest 5-year survival rate (5.5%) of all cancers.
For example, �23,000 people in Japan[3] and �10,000 in the UK[4]
die of pancreatic cancer each year. Across the EU, pancreatic
cancer has now surpassed breast cancer to become the third
leading cause of cancer related death. Although use of gemcita-
bine as a single agent therapy has been superseded by
The “anti-austerity” therapeutic strategy was first described by
the Esumi group at the Japan National Cancer Center Research
Institute in 2000.[7] It targets the ability of certain cancer cell lines
to survive severe nutrient deprivation. Pancreatic tumours are
inherently hypovascular in nature, leading to limited supply of
essential nutrients to rapidly growing tumour cells.[7] Therefore
these cells are under constant metabolic stress, which may
contribute to genomic instability, impaired cellular repair, muta-
genesis and chemoresistance. In order to survive under such
extreme conditions, the cancer cells alter their metabolism and
acquire tolerance of nutrient starvation. Pancreatic cancer cell
lines, in contrast to other cell lines, demonstrate an extraordinary
capacity for survival in conditions of severe nutrient deprivation.
Thus, discovery of agents that eliminate the cancer cells’ tolerance
of nutrient starvation (anti-austerity agents, Figure 1) is a novel
strategy in anticancer drug discovery.[8,9]
Elucidation of how pancreatic cancer cells can survive and
proliferate in such “austere” conditions is ongoing; numerous
proteins and pathways have been implicated.[10] Nevertheless,
Esumi has described an assay to identify anti-austerity agents
without knowledge of their specific target.[11] Briefly, two cell
cultures are grown in different media: one in normal medium
(DMEM, Dulbecco’s modified Eagle’s medium) and one in
nutrient-deprived medium (NDM). The cultures are treated with
serial dilutions of the agent being evaluated and incubated for
24 h, and then cell survival is determined. Compounds which
are cytotoxic in NDM, without cytotoxicity in DMEM are deemed
anti-austerity agents. Quantitative data are expressed as PC50
values, denoting preferential cytotoxicity in NDM. By this
approach, several dozen anti-austerity agents have been
identified.[10,12–24] In several cases efficacy has been demon-
strated in vivo (xenograft PANC-1 tumours in nude mice).[8,9,25]
[a] Dr. B. E. Alexander, Dr. M. J. Palframan, Dr. S. E. Lewis
Department of Chemistry
University of Bath
Bath BA2 7AY (UK)
E-mail: s.e.lewis@bath.ac.uk
[b] S. Sun, Dr. D. F. Dibwe, Prof. S. Watanabe, Prof. S. Awale
Institute of Natural Medicine
University of Toyama
2630 Sugitani Toyama 930-0194 (Japan)
E-mail: suresh@inm.u-toyama.ac.jp
[c] Dr. G. Kociok-Köhn
Materials and Chemical Characterisation Facility (MC2)
University of Bath
Bath BA2 7AY (UK)
[d] Dr. L. Caggiano
Department of Pharmacy and Pharmacology
University of Bath
Bath BA2 7AY (UK)
E-mail: l.caggiano@bath.ac.uk
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.201900549
© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This
is an open access article under the terms of the Creative Commons Attri-
bution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
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Figure 2. The two enantiomers of grandifloracin 1.
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configuration of new natural products in this class, as it cannot
be inferred from the absolute configuration of co-isolates, nor
from biosynthetic speculation. The possible biosynthetic origins
of this enantiodivergence have previously been discussed.[32]
The stems of Uvaria dac were also found to contain several
further novel polyoxygenated cyclohexenes.[35,36] All the isolates
were evaluated in the anti-austerity assay, with (+)-1 proving
the most potent, exhibiting PC50 =14.5 μM against PANC-1 cells
in nutrient-deprived medium.
These results prompted us (S.A. and co-workers) to under-
take more extensive biological investigations of (+)-1, which
showed that it induces PANC-1 cell death under conditions of
nutrient deprivation through hyperactivation of autophagy.[37]
While suppression of autophagy is a more well-established
strategy in cancer chemotherapy,[38] hyperactivation of autoph-
agy is an alternative approach which has been clinically
validated in other tumour types – for example, the autophagy
inducer temozolomide has therapeutic benefits against apopto-
sis-resistant cancers.[39,40] In the case of (+)-1, we found no
change in Bcl-2 and caspase-3 expression (apoptosis marker
proteins), but instead observed pronounced upregulation of
Figure 1. The anti-austerity strategy in anticancer drug discovery.
The natural product arctigenin has been identified as a
potent anti-austerity agent, and has commenced clinical
evaluation in the form of “GBS-01”, an extract of the fruit of
Arctium lappa Linné (rich in arctigenin), which is registered in
the Japanese Pharmacopoeia as a traditional herbal medicine. A
successful phase I trial has established favourable clinical
responses and toxicity profile in patients with advanced
pancreatic cancer refractory to gemcitabine.[26] A Phase IIa trial
to evaluate efficacy and safety has been completed, and a
patent was recently granted on use of arctigenin in combina-
tion therapy with gemcitabine.[27] Thus, although no chemo-
therapeutic agent with known anti-austerity activity has yet
received approval, the approach is exceedingly promising.
Grandifloracin is a dimeric natural product which is notable for
having been isolated from Nature in both enantiomeric forms. The
first reported isolation of (À )-grandifloracin, (À )-1 was in 1997,
from the tropical flowering plant Uvaria grandiflora,[28,29] followed
by later isolations from Uvaria rufa[30] and Uvaria calamistrata.[31]
Although the relative stereochemistry of (À )-1 was established,
the absolute configuration remained unknown until 2011. As part
of our ongoing interest in polyoxygenated cyclohexene natural
products,[32] we (S.E.L. and co-workers) carried out a total synthesis
of grandifloracin[33] employing a starting material of known
absolute configuration.[34] Our synthetic grandifloracin had a
positive optical rotation, from which we could therefore infer the
absolute configuration of (À )-grandifloracin, (À )-1, as shown in
Figure 2.
microtubule-associated protein
1 light chain 3 (LC3), an
autophagy marker. This was dose-dependent and time-depend-
ent following administration of (+)-1. Furthermore, LC3 activa-
tion mediated by (+)-1 was marginal in DMEM, but vastly more
pronounced in NDM. In addition to the above, it was found that
(+)-1 strongly inhibited the activation of Akt. Specifically, we
observed complete inhibition of Akt phosphorylation at Ser473
upon treatment with (+)-1. Complete inhibition of mTOR
phosphorylation at Ser2448 was similarly observed (mTOR is a
downstream effector of Akt). The serine/threonine kinase Akt/
mTOR pathway is constitutively activated in most pancreatic
cancer cell lines and is associated with tolerance of nutrient
deprivation,[41] tumour progression and metastasis.[42] Three
previously identified anti-austerity agents have been shown to
suppress Akt activation[43–45] and it is believed their activity
derives at least in part from this. However, our study explicitly
shows (+)-1 to be a dual inhibitor of both Akt and mTOR, both
clinically relevant prosurvival factors.
Motivated by these findings, we (S.E.L., L.C. and co-workers)
undertook an initial analogue study on (+)-1.[46] We prepared
three analogues of (+)-1 via modifications of our previous
synthesis,[33] all of which were slightly more active than (+)-1
(Figure 3). Two of these, (+)-2 and (+)-3, were variants bearing
different ester sidechains, whereas the third, (+)-4, was the
product of hydrogenation of (+)-1. All three of these showed
At that point, (+)-grandifloracin had not been isolated from
a natural source, so we described the synthetic (+)-1 as the
“non-natural enantiomer of grandifloracin”.[33] However, in 2012
we (S.A. and co-workers) reported the isolation of (+)-grandi-
floracin from Uvaria dac Pierre ex Finet & Gagnep,[35] thereby
showing that both enantiomers occur in Nature. The isolation
from Nature of several other polyoxygenated cyclohexene
natural products in both enantiomeric series has been reported
for different species in several genera, or even from the same
species. Care must therefore be taken when assigning absolute
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spirocyclic epoxide (�)-9. Here also a spontaneous Diels-Alder
dimerisation occurs to give bis(epoxide) (�)-10. This reaction
sequence was first reported by Adler,[49] and variants thereof
have been employed to access diverse synthetic targets.[50,51]
Hydrolytic opening of the epoxides in (�)-10 gave previously
unknown tetraol (�)-11, which underwent selective benzoyla-
tion at the primary alcohols in preference to the tertiary
alcohols, giving (�)-1 (Scheme 1b).
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Figure 3. Analogues of (+)-1 previously reported by us.
Both these racemic syntheses (as well as our earlier
synthesis) rely on the Diels–Alder dimerization as the key
complexity-forming event in what are overall very concise
synthetic routes. This cycloaddition is remarkably regio- and
stereoselective and the origins of this selectivity have been
studied.[52] The Stoltz route in particular is appropriate for
analogue synthesis, since sidechain diversity may be introduced
in the final step, by derivatisation of common building block
(�)-11. Therefore, for the present study we opted to adopt the
Stoltz approach. Whilst this synthetic route produces racemic
analogues, any that were found to be of particular interest
could then be subject to resolution and biological evaluation of
the individual enantiomers; conditions for preparative chiral
HPLC resolution of (�)-1 have been reported.[48]
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slightly enhanced potency with respect to (+)-1, and the
retention of activity in the saturated analogue (+)-4 indicated
that the enone motif is not required for anti-austerity activity.
From this we concluded that covalent modification through
Michael addition is not the mechanism of action by which (+)-1
interacts with its target(s). Encouraged by these findings, we
embarked on a programme of synthesis of further analogues of
(+)-1, the results of which are reported here.
Our previous syntheses of grandifloracin and analogues
employed a single-enantiomer starting material and hence gave
the final products as single enantiomers also. While ours
remains the only synthesis of enantiopure grandifloracin (+)-1,
two syntheses of racemic grandifloracin, (�)-1 have been
reported. In 2007, Quideau et al. published a synthesis of (�)-1
that employed an oxidative dearomatisation of an achiral Results and Discussion
starting material, mediated by SIBX, a stabilised formulation of
IBX (λ5-iodane 2-iodoxybenzoic acid).[47] Thus, salicyl benzoate 5
undergoes oxidation to form cyclohexadienone 6, which in turn
spontaneously dimerises by a Diels-Alder reaction to afford (�)-
1 (Scheme 1a). In 2015 Stoltz and co-workers reported a total
synthesis of (�)-1 whereby the oxidative dearomatisation/
dimerisation sequence occurs prior to introduction of the ester
sidechains.[48] Thus, salicyl alcohol undergoes oxidation by
sodium periodate to afford intermediate 8. The primary alcohol
then effects an intramolecular nucleophilic attack to give
Synthesis
Tetraol (�)-11 was synthesised and purified by chromatography
on silica, as per the reported procedure in Scheme 1b. From this
purified material we were able to grow crystals of (�)-11 as its
monohydrate, suitable for x-ray diffraction, and the structure
obtained is shown in Figure 4. In the solid state, (�)-11 is
engaged in a complex network of intermolecular hydrogen
bonding interactions (see ESI). For the purposes of synthesising
new ester analogues of (�)-1, we instead opted to take crude
(�)-11 directly into the acylation step with the relevant acid
chloride (Scheme 2).
As shown in Scheme 2, both aliphatic and aromatic ester
analogues were synthesised. Unoptimised yields over two steps
Figure 4. Solid-state structure of (�)-11·H2O, crystallised from ethanol/water.
Ellipsoids are represented at 50% probability. H atoms are shown as spheres
of arbitrary radius. CCDC #1952565.
Scheme 1. Reported syntheses of (�)-1.
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adjacent methylene AB protons which is observable in the HMBC
spectrum. These same methylene protons also exhibit a JCH
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coupling to a second carbonyl carbon, again observable in the
HMBC spectrum. The chemical shift of this carbonyl (δ=
198.1 ppm) identifies it as the conjugated ketone (and differ-
entiates it from the non-conjugated ketone carbonyl carbon at
δ=204.2 ppm), thereby confirming the connectivity present in
(�)-17 (see ESI for full details).
In addition to ester analogues (�)-12 to (�)-17, we also sought
to synthesise ether analogues of grandifloracin. We reasoned that
ether sidechains would be much more hydrolytically stable than
ester analogues, and might therefore exhibit appreciably different
PC50 values. Our first attempts to access analogues of grandiflor-
acin bearing ether sidechains involved treating bis(epoxide) (�)-10
with an oxygen nucleophile, in the hope of effecting epoxide ring
opening. Such an approach would provide the desired analogues
in a single step. Thus, as shown in Scheme 4, we treated (�)-10
with benzyl alcohol under basic conditions. However, this afforded
unexpected product (�)-19, with none of the bis(benzyl ether)
(�)-20 being produced.
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No incorporation of a benzyl group took place in the
formation of byproduct (�)-19. Instead, aromatisation of the
ring containing the enone occurred, with the remaining
epoxide being unchanged. The same product formed when
benzyl alcohol was omitted from the reaction. The structure of
(�)-19 was confirmed by x-ray crystallography (Figure 5).
In an attempt to favour the desired reaction pathway, we
employed a pre-formed alkoxide in place of an alcohol,[53]
Scheme 2. Telescoped synthesis of grandifloracin ester analogues from (�)-
10. Yields shown are isolated yield over two steps. a4 equivalents of pyridine
used as base/catalyst.
for these analogues were moderate, but this telescoped
procedure rapidly afforded pure material in sufficient quantity
for biological evaluation.
For analogues (�)-12 to (�)-16, no under- or over-acylated
byproducts were isolated, implying a good selectivity for acylation
of the two primary hydroxyl groups in (�)-11 over the two tertiary
ones. In contrast, the use of nicotinoyl chloride as the acylating
agent resulted in the isolation only of monoacyl product (�)-17
instead of the expected bis(nicotinoyl) ester (�)-18 (Scheme 3).
The structure of (�)-17 was assigned based on 2D-NMR data.
Scheme 4. Unexpected synthesis of byproduct (�)-19.
3
Thus, the ester carbonyl carbon exhibits a JCH coupling to the
Figure 5. Solid-state structure of (�)-19, crystallised from ethyl acetate/petrol
°
(40-60 C). Ellipsoids are represented at 50% probability. H atoms are shown
Scheme 3. Unexpected synthesis of monoacyl product (�)-17.
as spheres of arbitrary radius. CCDC #1952563.
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treating (�)-10 with sodium methoxide in methanol. Here
again, however, aromatisation was favoured over incorporation
of the nucleophile, with byproduct (�)-21 being formed instead
of the desired bis(methoxy) analogue (�)-22 (Scheme 5).
It is interesting to note that while the reactions shown in
Schemes 4 and 5 both failed to provide the desired ether
analogues, they did not form the same byproduct. Rather, (�)-
21 is an analogue of (�)-19 from which a molecule of
formaldehyde has formally been lost. A mechanistic proposal
which would account for these outcomes is shown in Scheme 6.
Exposure of bis(epoxide) (�)-10 to base leads to an elimination
and concomitant epoxide opening to give cyclohexadienone
(�)-23. The alkoxide in (�)-23 is reprotonated to give (�)-24,
which can then undergo aromatisation via base-mediated
elimination of the doubly allylic proton to give phenoxide (�)-
25. This phenoxide would be in equilibrium with cyclohexadie-
none tautomer (�)-26, from which a retro-aldol process would
lead to extrusion of formaldehyde and formation of the
observed byproduct (�)-21.
selectivity between the two sets of reaction conditions is
seemingly high, with no (�)-19 being isolated in the synthesis
of (�)-21 and vice versa. The different solvents employed in the
reactions to form (�)-19 and (�)-21 may influence the reaction
outcome by altering the position of the tautomeric equilibrium
between (�)-25 and (�)-26. Thus, for retro-aldol product (�)-21
to form at an appreciable rate, tautomer (�)-26 must be
sufficiently populated. The hydrogen bond donor properties of
methanol might stabilise tautomer (�)-26 sufficiently for this to
be the case, whereas in THF if the concentration of (�)-26 is
negligible, (�)-19 will be formed in preference. Base strength
may also play a role, as (�)-25 and (�)-26 will also be in
equilibrium with their (neutral) conjugate acids. Since the pKa
of protonated DBU is lower than that of methanol,[54,55] (�)-26
will be more extensively protonated under the DBU/THF
reaction conditions, and the conjugate acid of (�)-26 may
undergo the retro-aldol process less readily. It is interesting to
note that Stoltz et al. reported isolating a byproduct with a
structure closely analogous to (�)-19 (specifically, with no loss
of formaldehyde) when performing acylations of tetraol (�)-11
in acetonitrile using cesium carbonate as base.
As we were unable to access grandifloracin analogues
through the direct opening of epoxides with nucleophiles, we
next explored the possibility of functional group interconver-
sion to replace the epoxides with alternative electrophilic
groups. Specifically, we attempted to derivatise the vicinal diol
motifs in tetraol (�)-11 as cyclic carbonates, which might then
undergo nucleophilic substitution at the methylene carbons,
giving the desired analogues with loss of carbon dioxide.
However, treatment of (�)-11 with carbonyldiimidazole[56,57] led
to unexpected byproduct (�)-27 instead of the desired bis
(carbonate) (�)-28 (Scheme 7).
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In the case of byproduct (�)-19, the mechanism shown in
Scheme 6 may proceed only as far as intermediate (�)-25,
before protonation in the workup to afford (�)-19. The
Formation of (�)-27 seemingly occurs by one vicinal diol
forming the cyclic carbonate as expected, and the primary
alcohol of the other vicinal diol participating in an intra-
molecular oxa-Michael reaction with the enone. Surprisingly,
attempts to induce this oxa-Michael reaction by treating (�)-11
with other reagents such as imidazole itself were unsuccessful.
This may imply that prior formation of the cyclic carbonate
from the other diol is necessary to induce a conformational
change that favours the oxa-Michael process. The structure of
(�)-27 was confirmed by x-ray crystallography (Figure 6).
Scheme 5. Unexpected synthesis of byproduct (�)-21.
Scheme 6. Proposed mechanism for formation of byproduct (�)-21.
Scheme 7. Unexpected synthesis of oxa-Michael product (�)-27.
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Figure 7. Preferential cytotoxic activity of A) (�)-12 and B) (�)-13 against the
PANC-1 human pancreatic cancer cell line in nutrient-deprived medium
(NDM) and Dulbecco’s modified Eagle’s medium (DMEM).
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a
Table 1. Measured PC50 values for compounds tested. See reference 35.
Figure 6. Solid-state structure of (�)-27, crystallised from MeOH/CH2Cl2.
Ellipsoids are represented at 50% probability. H atoms are shown as spheres
of arbitrary radius. CCDC #1952564.
Compound
PC50/μM
(+)-1
14.5a
4.8
7.0
27.4
>100
51.8
>100
96.6
36.6
>100
>100
(�)-12
(�)-13
(�)-14
(�)-15
(�)-16
(�)-17
(�)-19
(�)-21
(�)-27
(�)-30
However, the introduction of a methoxy substituent decreases
the activity. Replacement of the phenyl groups with other ring
systems or isobutyl groups led to dramatic loss of antiausterity
activity. Byproducts (�)-19 and (�)-21 were weakly active in
both nutrient-deprived and normal media, likely due to their
epoxides acting as electrophiles for non-selective alkylation.[59]
This evidence suggests that the grandifloracin core is essential
for the antiausterity activity, and that (�)-12 is an ideal
candidate for further investigation of biological activity.
PANC-1 cells, even when exposed to complete nutrient
deprivation for 24 hours, remain alive and show intact cellular
morphology. On the other hand, treatment with (�)-12
dramatically alters the cancer cells’ morphology, resulting in the
swelling and rounding of cells and leakage of the cytoplasmic
contents into the media. Such difference of live and dying cells
can be visualized by the ethidium bromide (EB) – acridine
orange (AO) double-staining fluorescence assay. AO is a cell-
membrane permeable dye and emits bright-green fluorescence
in living cells, while EB is only permeable through the
membrane of dead or dying cells and emits predominantly red
fluorescence. As shown in Figure 8, PANC-1 cells treated with
(�)-12 (10 μM) and the untreated control in NDM were
incubated for 24 h, and then stained with the EB/AO reagent.
The PANC-1 cells in the control group showed intact morphol-
ogy and emitted bright green fluorescence, suggesting 100%
cell survival even in the conditions complete nutrition starva-
tion. However, PANC-1 cells, when treated with (�)-12 emitted
predominantly red fluorescence due to EB, with rounded
morphology characteristic of dead cells.
Scheme 8. Synthesis of bis(azido) analogue (�)-30.
Finally, we briefly explored the synthesis of nitrogen-
containing analogues of grandifloracin. Since we had been
unable to effect nucleophilic addition of nucleophiles to bis
(epoxide) (�)-10 or a cyclic carbonate derivative, we adopted a
different synthetic approach, based on Quideau’s route (Sche-
me 1a), incorporating nitrogen into the salicyl substrate for
oxidative dearomatisation / dimerization. Thus, as shown in
Scheme 8, salicyl alcohol underwent a benzylic substitution to
give known azide 29.[58] This was treated with SIBX to give bis
(azido) analogue (�)-30.
Biological Evaluation
Grandifloracin analogues (�)-12 to (�)-17 and (�)-30, as well as
byproducts (�)-19, (�)-21 and (�)-27, were evaluated in the
anti-austerity assay against PANC-1 cells as previously de-
scribed. (Figure 7, Table 1). Their activity is represented by a
PC50 value, the concentration at which 50% of cancer cells were
killed preferentially in nutrient-deprived medium (NDM) without
apparent toxicity in the normal nutrient-rich medium (DMEM).
Among the tested compounds, (�)-12 and (�)-13 displayed the
most potent activity with PC50 values of 4.8 μM and 7.0 μM
respectively, more potent than the natural product (+)-grandi-
floracin (1, PC50 14.5 μM). Interestingly, introducing a p-fluoro or
p-trifluoromethyl-substituent onto the phenyl groups increased
the activity by 3-fold and 2-fold respectively. (12>13>1).
The effect of (�)-12 against PANC-1 cells in NDM was
further investigated in a parallel real-time time-lapse micro-
scopy experiment. The control (NDM only) and treated (25 μM
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Figure 8. Morphological changes of PANC-1 cells induced by (�)-12 (bottom row), in comparison to untreated cells (i.e., the control, top row) in nutrient-
deprived medium (NDM). PANC-1 tumour cells were treated with 10 μM of (�)-12 in NDM and incubated for 24 h. Cells were stained with ethidium bromide
(EB) and acridine orange (AO) and photographed under fluorescence (red and green) and phase contrast modes using an EVOS FL digital microscope.
of (�)-12 in NDM) PANC-1 cells were incubated within a CO2
incubator equipped with real-time microscopy system. The
images were captured every 15 min on the digital cell imaging
system for 24 h. Treatment of PANC-1 cells with (�)-12 inhibited
cell mobility within 30 minutes and induced cell death within
4 h, leading to cell death after 8 h (Figure 9, Movie 1). PANC-1
cells cultured under the same conditions in NDM (control)
continued to survive until the end of the experiment (24 h).
Thus, this study further demonstrated the first live evidence of
the anticancer therapeutic potential of (�)-12.
Pancreatic cancer is known for its high metastatic potential.
Most patients at the point of diagnosis show colonies of
migrated pancreatic tumours in the duodenum, liver, and other
organs where the tumour cells have sufficient nutrition to grow
into large tumours. Compounds having the ability to inhibit
such colony formation have therapeutic benefit against cancer
‘colonization’. Therefore, the effect of (�)-12 was investigated
for its effect against PANC-1 cell colony formation in normal
nutrient-rich medium. Compound (�)-12 was exposed to PANC-
1 cells at the concentration of 25, 50 and 100 μM in nutrient-
rich DMEM medium for 24 hours. The medium was then
replaced by fresh DMEM and incubated for a further 10 days to
allow colony formation. As shown in Figure 10, (�)-12 inhibited
the colony formation ability of PANC-1 cells, even when
exposed for only 24 hours at the non-cytotoxic concentration.
The average colony area of each well within 12 well plates was
found to be 60% for the control, which was significantly
decreased to 55%, 23% and 3% for those treated with (�)-12
at 25, 50 and 100 μM concentrations. (See ESI for statistical
analysis).
Conclusions
We have prepared several analogues of grandifloracin and
determined their PC50 values in the anti-austerity assay. Of the
compounds tested, (�)-12 exhibited the best PC50 value and
hence was subjected to additional biological evaluation. The
fluorescence microscopy, live imaging and colony formation
experiments all provide additional evidence of the anti-austerity
Figure 9. Captures of the live imaging of the effect of (�)-12 (10 μM) on
PANC-1 cells at different intervals of time (hour: minute)
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point apparatus, and are uncorrected. High resolution mass
spectrometry (HRMS) was carried out using a micrOTOF ESI-TOF
spectrometer coupled to an Agilent 1200 LC system for autosam-
pling. X-ray crystallography was carried out on a Nonius Kappa CCD
diffractometer with MoÀ Kα radiation (λ=0.71073 Å).
1
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7
Synthetic Procedures
(1R*,4 S*,4aS*,8R*,8aR*,10R*)-8,10-Dihydroxy-8,10-bis(hydroxymethyl)-
8
9
4,4a,8,8a-tetrahydro-1,4-ethanonaphthalene-7,9(1H)-dione
(�)-11.
This was synthesised according to a literature procedure.[48] Bis
(epoxide) (�)-10[49] (240 mg, 1.00 mmol) was dissolved in water
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°
(10 mL) and stirred at 60 C for 48 h. Toluene (2 mL) was added and
the combined solvents were removed in vacuo. No further
purification was conducted.
General procedure for synthesis of analogues (�)-12 to (�)-17. To
crude (�)-11 (280 mg, 1.00 mmol, 1 eq.) in dichloromethane at
room temperature was added the relevant acyl chloride, as well as
DMAP, trimethylamine, and/or pyridine. The resulting solution was
stirred, then the solvent was removed in vacuo and the residue
partitioned between ethyl acetate (25 mL) and water (25 mL). The
organic layer was washed further, then dried over MgSO4, filtered
and the solvent removed in vacuo. The crude mixture was then
purified by column chromatography (SiO2), and then recrystallized
if necessary to give the desired product.
Figure 10. Effect of (�)-12 on colony formation by PANC-1 cells. (A) PANC-
1 cell colonies treated with different concentrations of (�)-12. (B) Graph
showing mean values of the area occupied by PANC-1 cell colonies (three
replicates). ****p<0.0001, *p<0.05 when compared with the untreated
control group.
((1R*,4 S*,4aS*,8R*,8aR*,10R*)-8,10-Dihydroxy-7,9-dioxo-1,4,4a,7,8,8a-
hexahydro-1,4-ethanonaphthalene-8,10-diyl)bis(methylene) bis(4-fluo-
robenzoate) (�)-12. The general procedure was employed, using
10 mL of CH2Cl2, para-fluorobenzoyl chloride (0.26 mL, 2.20 mmol,
2.2 eq.), DMAP (10 mg, 0.05 mmol, 0.05 eq.) and the dropwise
addition of triethylamine (0.56 mL, 4.00 mmol, 4 eq.). Reaction time
was 24 h at room temperature. Further washes employed HCl(aq)
(0.1 M, 25 mL), water (25 mL), brine (25 mL). Chromatography
eluent was petrol:EtOAc (4:1!1:1), and subsequent recrystallization
from chloroform and petrol gave the desired product as a white
activity of (�)-12. The measured PC50 value for (�)-12 indicates
a 3-fold increase in potency compared to (+)-1. However, it
should be noted that (�)-12 was prepared and evaluated as a
racemate. If (�)-12 were resolved, the single enantiomer (+)-12
would be expected to show a further increase in potency. Taken
as a whole, these results are strongly encouraging for drug
discovery activities related to the grandifloracin core; further
efforts in this regard are ongoing in our laboratories and will be
reported in due course.
°
solid, 90 mg (17% yield): Rf 0.33 (petrol:EtOAc 1:1); m.p. 185–189 C
(crystallised from petrol:EtOAc); νmax/cmÀ 1 (ATR) 3461 (bs), 1724 (s),
1
1687 (s); H NMR (CDCl3, 500 MHz) δ ppm 8.09-8.05 (m, 2H), 7.96–
7.93 (m, 2H), 7.15–7.12 (m, 2H), 7.11-7.08 (m, 2H), 6.59 (dd, J=10.1,
4.3 Hz, 1H), 6.43 (ddd, J=8.0, 6.6, 1.3 Hz, 1H), 6.21 (dd, J=10.1,
1.6 Hz, 1H), 6.02 (ddd, J=7.9, 6.1, 1.6 Hz, 1H), 4.45 (d, J=11.9 Hz,
1H), 4.40 (d, J=11.2 Hz, 1H), 4.36 (d, J=11.2 Hz, 1H) 4.31 (s, 1H),
4.22 (d, J=11.9 Hz, 1H), 3.66 (dt, J=6.7, 1.9 Hz, 1H), 3.44 (ddt, J=
8.4, 4.2, 2.0 Hz, 1H), 3.39 (ddd, J=6.2, 2.3, 1.3 Hz, 1H), 3.35 (dd, J=
8.5, 2.2 Hz, 1H), 3.21 (br s, 1H); 13C NMR (CDCl3, 126 MHz) δ ppm
208.2, 198.2, 166.23 (d, 1JCF =254 Hz), 166.15 (d, 1JCF =254 Hz), 165.9,
Experimental Section
General Conditions
3
3
All reactions were carried out under an atmosphere of N2 or argon,
through the use of a Schlenk line, unless otherwise stated.
Anhydrous solvents were either purchased from Fisher Scientific or
Sigma-Aldrich, or purified through anhydrous alumina columns
using an Innovative Technology Inc. PS-400-7 solvent purification
system. Solvents were deoxygenated either by channelling a stream
of N2 through the liquid (sparging), or by the freeze-thaw-pump
method. Thin layer chromatography (TLC) was carried out on
aluminium plates coated with silica gel (Alugram®SIL G/UV 254 nm),
and visualisation was achieved with UV light or KMnO4, ceric
ammonium molybdate and iodine dips, followed by gentle heating.
Solvents were removed using Büchi rotary evaporators and with
high vacuum on a Schlenk line. Flash column chromatography was
carried out using Davisil LC 60 Å silica gel (35–70 micron) purchased
from Sigma-Aldrich. NMR spectra were run in CDCl3 unless
otherwise stated, on Bruker Avance 250, Bruker Avance 300, Bruker
Avance 400, Bruker Avance 500 II+ or Agilent A500a instruments.
IR spectra were recorded on a Perkin-Elmer 1600 FT-IR instrument.
Capillary melting points were recorded on a Büchi 535 melting
165.0, 146.8, 135.2, 132.63 (d, JCF =9.5 Hz), 132.45 (d, JCF =9.4 Hz),
4
4
128.7, 128.2, 125.70 (d, JCF =3.2 Hz), 125.67 (d, JCF =3.1 Hz), 115.90
(d, 2JCF =22.2 Hz), 115.78 (d, 2JCF =22.1 Hz), 75.5, 74.6, 68.5, 52.3,
41.2, 40.1, 37.4; 19F NMR (CDCl3, 471 MHz) δ ppm À 104.6 (s, 1F),
À 104.8 (s, 1F); ESI-MS [M+Na]+ m/z C28H22F2NaO8 requires
547.1180, found 547.1155.
((1R*,4 S*,4aS*,8R*,8aR*,10R*)-8,10-Dihydroxy-7,9-dioxo-1,4,4a,7,8,8a-
hexahydro-1,4-ethanonaphthalene-8,10-diyl)bis(methylene)
bis(4-
(trifluoromethyl)benzoate) (�)-13. The general procedure was
employed, using 20 mL of CH2Cl2, para-(trifluoromethyl)benzoyl
chloride (0.30 mL, 2.00 mmol, 2.0 eq.), and pyridine (0.32 mL,
°
4.00 mmol, 4 eq.). Reaction time was 1 h at 0 C and then 4 h at
room temperature. Further washes employed NaHCO3(aq) (saturated,
2×20 mL), water (20 mL), brine (20 mL). Chromatography eluent
was petrol:EtOAc (6:1!1:1), and subsequent recrystallization from
chloroform gave the desired product as a white solid, 60 mg (10%
°
yield): Rf 0.72 (1:1 petrol:ethyl acetate); m.p. 186–188 C (crystallised
1
from petrol:EtOAc); νmax/cmÀ 1 (ATR) 3422 (bs), 1712 (s), 1710 (s); H
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NMR (CDCl3, 500 MHz) δ ppm 8.17 (br d, J=8.1 Hz, 2H), 8.04 (br d,
J=8.2 Hz, 2H), 7.74 (br d, J=8.2 Hz, 2H), 7.69 (br d, J=8.5 Hz, 2H),
6.61 (dd, J=10.1, 4.3 Hz, 1H), 6.44 (ddd, J=8.1, 6.6, 1.3 Hz, 1H), 6.22
(dd, J=10.1, 1.6 Hz, 1H), 6.04 (ddd, J=7.8, 6.1, 1.6 Hz, 1H), 4.50 (d,
J=11.9 Hz, 1H), 4.43 (d, J=11.2 Hz), 4.40 (d, J=11.2 Hz), 4.33 (s,
1H), 4.26 (d, J=11.9 Hz, 1H), 3.68 (dt, J=6.6, 1.9 Hz, 1H), 3.45 (ddt,
J=8.3, 4.1, 1.9 Hz, 1H), 3.41 (ddd, J=6.1, 2.3, 1.3 Hz, 1H), 3.36 (dt,
J=8.5, 2.2 Hz, 1H), 3.15 (s, 1H); 13C NMR (CDCl3, 126 MHz) δ ppm
using 10 mL of CH2Cl2, 3,4,5- pivaloyl chloride (0.27 mL, 2.20 mmol,
2.2 eq.), DMAP (10 mg, 0.05 mmol, 0.05 eq.) and triethylamine
(0.56 mL, 4.00 mmol, 4 eq.). Reaction time was 20 h at room
temperature. Further washes employed HCl(aq) (0.1 M, 25 mL), water
(25 mL), brine (25 mL). Chromatography eluent was CH2Cl2:MeOH
(99:1), giving the desired product as a white solid, 80 mg (18%
1
2
3
4
5
6
7
8
9
°
yield): Rf 0.23 (CH2Cl2:MeOH 99:1); m.p. 158–161 C (crystallised
1
from CH2Cl2:MeOH); νmax/cmÀ 1 (ATR) 3451 (bs), 1719 (s), 1695 (s); H
208.2, 198.0, 165.6, 164.7, 146.9, 135.11, 135.10 (q, 2JCF =32.7 Hz),
NMR (CDCl3, 500 MHz) δ ppm 6.54 (dd, J=10.1, 4.3 Hz, 1H), 6.34
(ddd, J=8.2, 6.6, 1.3 Hz, 1H), 6.14 (dd, J=10.1, 1.7 Hz, 1H), 5.96
(ddd, J=7.9, 6.1, 1.6 Hz, 1H), 4.25 (d, J=11.9 Hz, 1H), 4.19 (d, J=
11.2 Hz, 1H), 4.17 (s, 1H), 4.07 (d, J=11.2 Hz, 1H), 3.92 (d, J=
11.9 Hz, 1H), 3.50 (dt, J=6.6, 2.0 Hz, 1H), 3.36 (ddt, J=8.4, 4.0,
1.8 Hz, 1H), 3.31 (ddd, J=6.1, 2.3, 1.3 Hz, 1H), 3.22 (dd, J=8.5,
2.2 Hz, 1H), 3.15 (s, 1H), 1.22 (s, 9H), 1.13 (s, 9H); 13C NMR (CDCl3,
126 MHz) δ ppm 208.2, 198.1, 179.0, 178.0, 146.5, 135.1, 128.6,
128.1, 75.6, 74.6, 71.3, 67.9, 52.3, 41.1, 40.1, 39.1, 38.9, 37.3, 27.3,
27.2; ESI-MS [MÀ H]À m/z C24H31O8 requires 447.2024, found
447.2043.
4
135.01 (q, 2JCF =32.7 Hz), 132.69 (q, 4JCF =1.1 Hz), 132.61 (q, JCF
=
1.0 Hz), 130.4, 130.3, 128.8, 128.2, 125.74 (q, 3JCF =3.8 Hz), 125.69 (q,
3JCF =3.5 Hz), 123.67 (q, 1JCF =272 Hz, 2 C), 75.5, 74.5, 72.2, 68.8,
52.3, 41.1, 40.1, 37.4; 19F NMR (CDCl3, 471 MHz) δ ppm À 63.23 (s,
3F), À 63.25 (s, 3F); ESI-MS [M+Na]+ m/z C30H22F6NaO8 requires
647.1117, found 647.1100.
10
11
12
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((1R*,4 S*,4aS*,8R*,8aR*,10R*)-8,10-Dihydroxy-7,9-dioxo-1,4,4a,7,8,8a-
hexahydro-1,4-ethanonaphthalene-8,10-diyl)bis(methylene)
bis(4-
(trifluoromethyl)benzoate) (�)-14. The general procedure was
employed, using 10 mL of CH2Cl2, 3,4,5-trimethoxybenzoyl chloride
(0.51 g, 2.20 mmol, 2.2 eq.), DMAP (10 mg, 0.05 mmol, 0.05 eq.) and
triethylamine (0.56 mL, 4.00 mmol, 4 eq.). Reaction time was 22 h at
room temperature. Further washes employed HCl(aq) (0.1 M, 25 mL),
water (25 mL), brine (25 mL). Chromatography eluent was petrol:
EtOAc (2:1!1:1), and subsequent recrystallization from dichloro-
methane gave the desired product as a white solid, 70 mg (10%
(1R*,4 S*,4aS*,8R*,8aR*,10R*)-8,10-Dihydroxy-10-(hydroxymethyl)-7,9-
dioxo-1,4,4a,7,8,8a-hexahydro-1,4-ethanonaphthalen-8-yl)methyl nico-
tinate (�)-17. The general procedure was employed, using 20 mL of
CH2Cl2, 3,4,5- nicotinoyl chloride (0.39 mL, 2.20 mmol, 2.2 eq.),
DMAP (10 mg, 0.05 mmol, 0.05 eq.) and triethylamine (0.56 mL,
4.00 mmol, 4 eq.). Reaction time was 22 h at room temperature.
Further washes employed HCl(aq) (0.1 M, 25 mL), water (25 mL),
brine (25 mL). Chromatography eluent was CH2Cl2:MeOH (99:1),
giving the desired product as a cream-coloured solid, 110 mg (29%
°
yield): Rf 0.16 (petrol:EtOAc 1:1); m.p. 171–173 C (dec., crystallised
from dichloromethane); νmax/cmÀ 1 (ATR) 3446 (bs), 1716 (s), 1588 (s);
1H NMR (CDCl3, 500 MHz) δ ppm 7.30 (s, 2H), 7.19 (s, 2H), 6.60 (dd,
J=10.1, 4.2 Hz, 1H), 6.43 (ddd, J=8.0, 6.5, 1.3 Hz, 1H), 6.21 (dd, J=
10.1, 1.6 Hz, 1H), 6.02 (ddd, J=7.9, 6.0, 1.5 Hz, 1H), 4.43 (d, J=
11.9 Hz, 1H), 4.38 (d, J=11.1 Hz, 1H), 4.32 (d, J=11.2 Hz, 1H), 4.30
(s, 1H), 4.25 (d, J=11.9 Hz, 1H), 3.92 (s, 6H), 3.91 (s, 3H), 3.89 (s, 3H),
3.89 (s, 6H), 3.65 (dt, J=6.6, 1.9 Hz, 1H), 3.45–3.43 (m, 1H), 3.39
(ddd, J=6.2, 2.3, 1.4 Hz, 1H), 3.36 (dd, J=8.5, 2.1 Hz), 3.34 (s, 1H);
13C NMR (CDCl3, 126 MHz) δ ppm 208.2, 198.3, 166.6, 165.5, 153.2,
153.1, 146.8, 142.9, 142.7, 135.2, 128.6, 128.2, 124.3, 107.3, 107.1,
75.6, 74.7, 72.0, 68.7, 61.10, 61.07, 56.5, 56.45, 56.36, 52.4, 41.2, 40.2,
37.3; ESI-MS [M+Na]+ m/z C34H36NaO14 requires 691.2003, found
691.1962.
°
yield): Rf 0.56 (CH2Cl2:MeOH 99:1) m.p. 220–224 C (crystallised from
CH2Cl2:methanol); νmax/cmÀ 1 (ATR) 3460 (s), 1731 (s), 1688 (s); 1H
NMR (CD2Cl2, 500 MHz) δ ppm 9.07 (dd, J=2.2, 0.9 Hz, 1H), 8.76 (dd,
J=4.9, 1.7 Hz, 1H), 8.18 (dd, J=7.9, 2.2, 1.7 Hz, 1H), 7.39 (ddd, J=
8.0, 4.8, 0.9 Hz, 1H), 6.66 (dd, J=10.2, 4.2 Hz, 1H), 6.43 (dddd, J=
8.1, 6.6, 1.5, 0.6 Hz, 1H), 6.22 (dd, J=10.1, 1.8 Hz, 1H), 6.03 (dddd,
J=8.1, 6.3, 1.7, 0.6 Hz, 1H), 4.44 (d, J=11.2 Hz, 1H), 4.40 (d, J=
11.2 Hz, 1H), 4.32 (s, 1H), 3.53 (ddddd, J=8.5, 4.3, 2.4, 1.8, 0.6 Hz,
1H), 3.48 (ddd, J=6.3, 2.5, 1.4 Hz, 1H), 3.14 (dt, J=6.6, 2.0 Hz, 1H),
3.10 (d, J=6.2 Hz, 1H), 3.06 (dd, J=8.5, 2.2 Hz, 1H), 2.92 (d, J=
6.1 Hz, 1H). 13C NMR (CD2Cl2, 126 MHz) δ ppm 204.2, 198.1, 165.0,
154.4, 151.4, 147.8, 137.5, 134.8, 130.0, 128.5, 125.9, 123.9, 76.0,
72.3, 58.6, 54.4, 53.7, 41.5, 40.2, 39.5; ESI-MS [MÀ H]À m/z C20H18NO6
requires 368.1129, found 368.1144.
((1R*,4 S*,4aS*,8R*,8aR*,10R*)-8,10-Dihydroxy-7,9-dioxo-1,4,4a,7,8,8a-
hexahydro-1,4-ethanonaphthalene-8,10-diyl)bis(methylene)
dicyclo-
pentanecarboxylate (�)-15. The general procedure was employed,
using 10 mL of CH2Cl2, 3,4,5- cyclopentane carbonyl chloride
(0.27 mL, 2.20 mmol, 2.2 eq.), DMAP (10 mg, 0.05 mmol, 0.05 eq.)
and triethylamine (0.56 mL, 4.00 mmol, 4 eq.). Reaction time was
(1’R*,2R*,4’S*)-7’-Hydroxy-8’-(hydroxymethyl)-1’,4’-dihydrospiro[oxir-
ane-2,10’-[1,4]ethanonaphthalen]-9’-one (�)-19. Bis(epoxide) (�)-1035
(240 mg, 1.00 mmol, 1 eq.) was dissolved in anhydrous THF (12 mL)
and DBU (0.30 mL, 2.00 mmol, 2 eq.) was added dropwise. The
resulting mixture was stirred at room temperature for 24 h, then
HCl(aq) (1.0 M, 10 mL) was added and then extracted with EtOAc (2×
20 mL). The combined organic layers were washed with brine
(10 mL), then dried over MgSO4, filtered and the solvent removed in
vacuo. The crude product was purified by column chromatography
(SiO2) eluting with petrol:EtOAc (1:1) to give (�)-19 as a white solid,
°
16 h at 40 C. Further washes employed HCl(aq) (0.1 M, 25 mL), water
(25 mL), brine (25 mL). Chromatography eluent was CH2Cl2:EtOAc
(9:1), giving the desired product as a white solid, 90 mg (19%
°
yield): Rf 0.38 (9:1 CH2Cl2:EtOAc) m.p. 149–153 C (crystallised from
1
CH2Cl2:EtOAc); νmax/ cmÀ 1 (ATR) 3435 (bs), 1726 (s), 1687 (s); H NMR
(CDCl3, 500 MHz) δ ppm 6.53 (dd, J=10.1, 4.3 Hz, 1H), 6.33 (ddd, J=
8.0, 6.6, 1.3 Hz, 1H), 6.14 (dd, J=10.1, 1.6 Hz, 1H), 5.95 (ddd, J=7.9,
6.0, 1.6 Hz, 1H), 4.25 (d, J=11.9 Hz, 1H), 4.20 (d, J=11.2 Hz, 1H),
4.17 (s, 1H), 4.08 (d, J=11.2 Hz, 1H), 3.92 (d, J=11.9 Hz, 1H), 3.50
(dt, J=6.8, 1.9 Hz, 1H), 3.36 (ddt, J=8.5, 4.2, 2.0 Hz, 1H), 3.30 (ddd,
J=6.1, 2.3, 1.3 Hz, 1H), 3.23–3.20 (m, 2H), 2.77 (p, J=8.4 Hz, 1H),
2.66 (tt, J=8.5, 6.8 Hz, 1H), 1.90 (dddd, J=14.5, 8.7, 7.5, 3.8 Hz, 2H),
1.84–1.77 (m, 4H), 1.74–1.65 (m, 6H), 1.60–1.55 (m, 4H); 13C NMR
(CDCl3, 126 MHz) δ ppm 208.2, 198.2, 177.2, 176.2, 146.5, 135.1,
128.6, 128.2, 75.6, 74.6, 71.2, 67.7, 52.3, 43.79, 43.74, 41.1, 40.1, 37.3,
30.20, 30.16, 30.15, 30.02, 25.91 (2 C), 25.85, 25.84; ESI-MS [M+Na]+
m/z C26H32NaO8 requires 495.1989, found 495.2023.
°
120 mg (51% yield): Rf 0.23 (Petrol:EtOAc 1:1); m.p. 186–191 C
(crystallised from petrol:EtOAc); νmax/cmÀ 1 (ATR) 3445 (bs), 3199 (bs),
1729; 1H NMR (CD3CN, 500 MHz) δ ppm 7.83 (s, 1H), 7.16 (d, J=
8.0 Hz, 1H), 6.79–6.75 (m, 2H), 6.71 (d, J=8.1 Hz, 1H), 4.82 (dd, J=
12.6, 5.2 Hz, 1H), 4.75 (dd, J=12.6, 5.6 Hz, 1H), 4.43 (dd, J=5.4,
2.2 Hz, 1H), 4.09 (dd, J=5.9, 2.1 Hz, 1H), 3.39 (t, J=5.4 Hz, 1H), 3.03
(d, J=5.9 Hz, 1H), 2.97 (d, J=5.9 Hz, 1H); 13C NMR (CD3CN, 126 MHz)
δ ppm 199.3, 156.6, 140.5, 136.0, 133.4, 128.3, 126.4, 124.0, 115.0,
57.9, 57.5, 57.1, 53.5, 43.6; ESI-MS [M+Na]+ m/z C14H12NaO4 requires
267.0633, found 267.0617.
((1R*,4 S*,4aS*,8R*,8aR*,10R*)-8,10-Dihydroxy-7,9-dioxo-1,4,4a,7,8,8a-
(1’R*,2R*,4’S*)-7’-Hydroxy-1’,4’-dihydrospiro[oxirane-2,10’-[1,4]ethano-
naphthalen]-9’-one (�)-21. Bis(epoxide) (�)-1035 (240 mg,
1.00 mmol, 1 eq.) was dissolved in methanol (10 mL) and stirred at
hexahydro-1,4-ethanonaphthalene-8,10-diyl)bis(methylene)
dicyclo-
pentanecarboxylate (�)-16. The general procedure was employed,
ChemMedChem 2019, 14, 1–12
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°
0 C. Sodium methoxide (0.5 M in MeOH, 4.40 mL, 2.20 mmol,
39.9, 38.4; ESI-MS [M+Na]+ m/z C14H14N6NaO4 requires 353.0969,
found 353.0993.
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2.2 eq.) was then added dropwise and the reaction mixture stirred
for 16 h at room temperature. The reaction mixture was extracted
with dichloromethane (3×10 mL), washed with NaHCO3(aq) (satu-
rated, 10 mL), water (10 mL), and brine (10 mL), then dried over
MgSO4, filtered and the solvent removed in vacuo. The crude
product was purified by column chromatography (SiO2), eluting
with petrol:EtOAc (1:1!1:2) to give (�)-21 as a white solid, 80 mg
Biological Evaluation
Preferential Cytotoxicity Assay Against PANC-1 Cells
The human pancreatic cancer cell line PANC-1 (RBRC-RCB2095,
Tsukuba, Japan) was purchased from the Riken BRC cell bank and
maintained in standard Dulbecco’s modified Eagle’s medium
°
(41% yield). Rf 0.47 (petrol:EtOAc 1:1); m.p. 170–174 C (crystallised
from petrol:EtOAc); νmax/cmÀ 1 (ATR) 3345 (bs), 1731 (s); 1H NMR
(CD3OD, 500 MHz) δ ppm 7.16 (d, J=8.1 Hz, 1H), 6.82 (d, J=2.4 Hz,
1H), 6.79 (ddd, J=7.8, 6.2, 1.6 Hz, 1H), 6.72 (ddd, J=7.7, 6.0, 1.8 Hz,
1H), 6.67 (dd, J=8.1, 2.4 Hz, 1H), 4.40 (ddd, J=6.0, 1.6, 0.5 Hz, 1H),
3.65 (dd, J=6.2, 1.8 Hz, 1H), 2.99 (d, J=6.0 Hz, 1H), 2.98 (d, J=
6.0 Hz, 1H); 13C NMR (CD3OD, 126 MHz) δ ppm 200.0, 158.5, 142.5,
136.3, 133.3, 127.6, 127.0, 114.6, 113.8, 57.6, 57.5, 53.4, 48.0; ESI-MS
[M+Na]+ m/z C13H10NaO3 requires 237.0522, found 237.0521.
°
(DMEM) supplemented with 10% foetal bovine serum (FBS) at 37 C
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under
a
humidified atmosphere of 5% CO2 incubator. For
preferential cytotoxicity assay, PANC-1 cells were seeded in 96-well
plates (1.5×104/well) in DMEM and incubated overnight CO2
incubator for the cell attachment. The cells were then washed twice
with phosphate-buffered saline (PBS), and then added serially
diluted test samples in both nutrient-rich medium (DMEM) and
nutrient-deprived medium (NDM) with a control and blank in each
test plate. The treated plates were incubated for 24 h. The medium
in each well was then replaced by 100 μL of DMEM containing 10%
WST-8 cell counting kit solution, followed by 3 h of incubation, and
the absorbance at 450 nm was measured on an EnSpire Multimode
plate reader (PerkinElmer, Inc., Waltham, MA, USA). Cell viability
was calculated from the mean values from three wells using the
following equation:
(1’R*,4R*,4’R*,4a’R*,5’R*,8’S*,8a’S*)-4’-Hydroxy-1’,4’,4a’,5’,8’,8a’-hexa-
hydrospiro[[1,3]dioxolane-4,10’-[1,4](epoxymethano)[5,8]ethanonaph-
thalene]-2,3’,9’(2’H)-trione (�)-27. Crude tetraol (�)-11 (280 mg,
1.00 mmol, 1 eq.) was stirred in anhydrous dichloromethane
(10 mL) and carbonyl diimidazole (370 mg, 2.3 mmol, 2.3 eq.) was
added. The reaction mixture was stirred for 18 h at room temper-
ature, water (10 mL) was added and then extracted with dichloro-
methane (2×10 mL), washed with brine (10 mL), dried over MgSO4,
filtered and the solvent removed in vacuo. The crude product was
purified by column chromatography (SiO2), eluting with (CH2Cl2:
MeOH 9:1) to give (�)-27 as a white crystalline solid, 130 mg (45%
Cell viability ð%Þ ¼
½Absðtest sampleÞÀ AbsðblankÞ=AbsðcontrolÞÀ AbsðblankÞ� � 100 %
°
yield). Rf 0.35 (CH2Cl2:MeOH 9:1); m.p. 200–206 C (crystallised from
CH2Cl2:MeOH); νmax/cmÀ 1 (ATR) 2927 (bs), 1805 (s), 1726 (s); H NMR
1
(CD3CN, 500 MHz) δ ppm 6.26 (dddd, J=7.0, 6.2, 1.7, 0.8 Hz, 1H),
6.05 (tt, J=6.9, 1.0 Hz, 1H), 4.21 (app q, J=3.4 Hz, 1H), 4.18 (d, J=
8.8 Hz, 1H), 4.12 (d, J=8.8 Hz, 1H), 3.91 (s, 1H), 3.80 (dd, J=8.8,
1.0 Hz, 1H), 3.56 (d, J=8.9 Hz, 1 H), 3.55 (dt, J=6.9, 2.0 Hz, 1H), 3.43
(dt, J=6.3, 1.6 Hz, 1H), 3.05-2.99 (m, 2H), 2.71 (ddd, J=19.7, 3.2,
1.2 Hz, 1H), 2.50 (ddd, J=19.7, 2.1, 1.0 Hz, 1H); 13C NMR (CD3CN,
126 MHz) δ ppm 210.7, 202.4, 154.9, 132.1, 131.8, 80.4, 75.1, 72.3,
71.5, 70.7, 48.9, 43.9, 42.3, 41.8, 40.1; ESI-MS [MÀ HÀ H2O]À m/z
C15H11O6 requires 287.0561, found 287.0564.
Morphological Assessment of Cancer Cells
PANC-1 cells were seeded at a density of 2×105 cells/well in DMEM
in a 12-well plate dish and incubated overnight in a CO2 incubator
for the cell attachment. The cells were then washed twice with PBS,
followed by treatment with (�)-12 (10 mM) in NDM, or, untreated
control cells in NDM. The cells were then incubated for 24 h, and
then treated with EB/AO reagent, and cell morphology was
captured using Evos FL digital microscope (20× objective) with
phase-contrast and fluorescence modes.
(1R*,4 S*,4aS*,8R*,8aR*,10R*)-8,10-Bis(azidomethyl)-8,10-dihydroxy-
4,4a,8,8a-tetrahydro-1,4-ethanonaphthalene-7,9(1H)-dione (�)-30. To
a stirred solution of 2-(azidomethyl)phenol 29[44] (0.22 g, 1.49 mmol,
1 eq.) in anhydrous THF (10 mL) was added stabilised 2-iodoxyben-
zoic acid (SIBX) (1.85 g, 2.98 mmol of IBX, 2 eq.) as a solid in one
portion. The resulting suspension was stirred at room temperature
for 16 h, after which trifluoroacetic acid (0.68 mL, 8.94 mmol, 1 eq.)
was added and stirred for a further 4 h. The reaction mixture was
diluted with CH2Cl2 (100 mL) and H2O (50 mL). NaHCO3(aq) was
added slowly, with shaking, until all the acid had been neutralised.
The aqueous phase was extracted with CH2Cl2 (2×20 mL). The
combined organic phases were then washed with NaHCO3(aq)
(20 mL), H2O (20 mL), brine (20 mL) and then vigorously shaken
with Na2S2O3(aq) (1.0 M, 20 mL), washed again with brine (20 mL)
and dried over MgSO4, filtered and the solvent evaporated in vacuo
to give a dark brown oil. The crude mixture was then purified by
column chromatography (SiO2), eluting with CH2Cl2/MeOH 99:1 to
give the desired product as a light brown oil, 30 mg (12% yield): Rf
0.22 (petrol:EtOAc 1:1); νmax/cmÀ 1 (ATR) 3447 (bs), 2100 (s), 1727 (s),
Colony Formation Assay
PANC-1 cells were seeded at a density of 2 × 103 cells/well in DMEM
(2 mL/well) in a 12-well plate dish and incubated for 24 h for the
cell attachment. The medium was then changed to DMEM
containing (�)-12 at 100, 50, 25, or untreated control in DMEM,
with three replicates in each group, and incubated for 24 h. The
cells were then washed twice with PBS and the medium was
replaced by fresh DMEM (2 mL) without any test compound. Then
the cells were allowed to grow for 10 days. On the last day, the cells
were washed with PBS, fixed with 4% formaldehyde, and stained
with crystal violet for 10 min. Finally, the colony area was measured
using ImageJ plugin “Colony Area”,[60] and the data were analysed
by the GraphPad software Prism 6.
1
1690 (s); H NMR (CDCl3, 500 MHz) δ ppm 6.55 (dd, J=10.1, 4.0 Hz,
Time-Lapse Imaging
1H), 6.33 (ddd, J=8.1, 6.6, 1.2 Hz, 1H), 6.20 (dd, J=10.1, 1.5 Hz, 1H),
5.95 (ddd, J=8.2, 6.6, 1.6 Hz, 1H), 4.31 (s, 1H), 3.60 (dt, J=6.7,
1.9 Hz, 1H), 3.43 (d, J=12.5 Hz, 1H), 3.37 (d, J=13.0 Hz, 1H), 3.36-
3.32 (m, 2H), 3.29-3.27 (m, 1H), 3.27 (d, J=12.4 Hz, 1H), 3.20 (d, J=
12.9 Hz, 1H), 2.87 (br s, 1H); 13C NMR (CDCl3, 126 MHz) δ ppm 208.7,
198.4, 146.8, 135.3, 128.8, 128.1, 77.4, 75.2, 61.4, 57.1, 52.2, 41.6,
PANC-1 cells (1×105) were plated in 35-mm cell culture dishes and
allowed to attach overnight in DMEM. The cells were then washed
with PBS, followed by treatment with 10 μM (�)-12 in nutrient-
deprived medium (NDM), and placed immediately inside
a
°
humidified CO2 incubator at 37 C, equipped real-time digital time-
ChemMedChem 2019, 14, 1–12
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Acknowledgements
We thank EPSRC (DTP studentship to B.E.A.) and Cancer Research
at Bath (CR@B) for funding. The biological investigation was
supported by a grant from the Japanese Society for the Promotion
of Science (JSPS Kakenhi #16 K08319) and the Kobayashi Interna-
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Manuscript received: September 27, 2019
Revised manuscript received: November 11, 2019
Version of record online: ■■■, ■■■■
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FULL PAPERS
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Dr. B. E. Alexander, S. Sun, Dr. M. J.
Palframan, Dr. G. Kociok-Köhn,
Dr. D. F. Dibwe, Prof. S. Watanabe,
Dr. L. Caggiano*, Prof. S. Awale*,
Dr. S. E. Lewis*
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Austerity measures: Grandifloracin is
a natural product that is known to
exhibit anti-austerity activity, i.e. it is
able to abolish the ability of pancreat-
ic cancer cells to survive under
“austere” conditions of nutrient depri-
vation. Herein we report the synthesis
and biological evaluation of
analogues of grandifloracin, as well as
our findings on rearrangement and
fragmentation reactions of the
dimeric grandifloracin core. Live
imaging, fluorescence microscopy and
colony formation experiments with
the most active analogue are
described.
Sidechain Diversification of Gran-
difloracin Allows Identification of
Analogues with Enhanced Anti-
Austerity Activity against Human
PANC-1 Pancreatic Cancer Cells