Evaluation of synthetic coumarins for antiausterity cytotoxicity against pancreatic cancers

Article abstract of DOI:10.1016/j.bmcl.2016.01.054

A series of functionalized coumarins were synthesized and evaluated for their capacity to inhibit the resistance to starvation of pancreatic cancer cells. This form of cytotoxicity, termed 'antiausterity' activity, was evaluated using a preferential cytotoxicity assay that compared cell survival in nutrient poor and nutrient rich conditions. Six of the seventeen compounds showed weak antiausterity activity against PANC-1. Compound 34 was active against PANC-1, MIA PaCa-2, and Capan-1 cancer cell lines. All of the compounds tested were simplified structural analogs of previously reported natural product leads. Six of the compounds, including 34, contain functionalized triazoles as novel potential bioisosteres of the side chain of the natural product angelmarin. Overall, the analogs were found to have low antiausterity activity relative to the corresponding natural products.

Full text of DOI:10.1016/j.bmcl.2016.01.054

Accepted Manuscript
Evaluation of Synthetic Coumarins for Antiausterity Cytotoxicity against Pan-
creatic Cancers
Conner M. Farley, Dya Fita Dibwe, Jun-ya Ueda, Eric A. Hall, Suresh Awale,
Jakob Magolan
PII:
S0960-894X(16)30054-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.01.054
BMCL 23519
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
2 November 2015
14 January 2016
19 January 2016
Please cite this article as: Farley, C.M., Dibwe, D.F., Ueda, J-y., Hall, E.A., Awale, S., Magolan, J., Evaluation of
Synthetic Coumarins for Antiausterity Cytotoxicity against Pancreatic Cancers, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.01.054
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Evaluation of Synthetic Coumarins for Antiausterity Cytotoxicity against Pancreatic Cancers
Conner M. Farley,a Dya Fita Dibwe,b Jun-ya Ueda,b Eric A. Hall,a Suresh Awale,b Jakob Magolana,*
a Department of Chemistry, University of Idaho, Moscow, ID 83844-2343, USA
b
Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630
Sugitani, Toyama 930-0194.
Abstract: A series of functionalized coumarins were synthesized and evaluated for their capacity to
inhibit the resistance to starvation of pancreatic cancer cells. This form of cytotoxicity, termed
‘antiausterity’ activity, was evaluated using a preferential cytotoxicity assay that compared cell survival in
nutrient poor and nutrient rich conditions. Six of the seventeen compounds showed weak antiausterity
activity against PANC-1. Compound 34 was active against PANC-1, MIA PaCa-2, and Capan-1 cancer
cell lines. All of the compounds tested were simplified structural analogs of previously reported natural
product leads. Six of the compounds, including 34, contain functionalized triazoles as novel potential
bioisosteres of the side chain of the natural product angelmarin. Overall, the analogs were found to have
low antiausterity activity relative to the corresponding natural products.
Keywords: antiausterity agents, pancreatic cancer, coumarins, apoptosis, starvation-resistance

With a five year survival rate of less than 5% and no effective chemotherapy options available, a pressing
need exists for medicines to combat pancreatic cancer.1, 2 In 2000, Esumi and co-workers showed that
pancreatic cancer cells demonstrate an atypical capacity to survive for up to 3 days when deprived of
serum, glucose, and amino acids; an incubation medium that kills human fibroblast cells and other
cancers much more rapidly.3 Given the fact that fast-growing pancreatic tumors are characterized by
nutrient-deficient microenvironments in which these cancer cells continue to thrive,4 the inhibition of
starvation-resistance became a potential therapeutic strategy against this disease. This was coined the
‘antiausterity’ approach with inhibitors described as having ‘preferential cytotoxicity’ (PC50 or PC100)
indicating that they induce cell death upon 24 incubation in a nutrient deprived medium with no
corresponding cytotoxicity in under standard, nutrient rich, incubation conditions.5, 6 Various potential
biochemical mechanisms for cellular resistance to starvation have been investigated.7-11 Meanwhile,
antiausterity bioassay guided-isolation of natural products has identified numerous promising inhibitors,12-
21
or antiausterity agents, several of which share the coumarin structural scaffold. The first coumarin-
based inhibitor was angelmarin (1), a phenolic furanocoumarin isolated from Angelica pubescens in
2006, which kills 100 % of PANC-1 cells under nutrient-deprived conditions at a concentration of 0.01
µg/mL (PC100 = 0.03 µM).13 In 2008, more than a dozen densely substituted hydroxycoumarins from
Kayea assimica were found to be active against PANC-1.16,17 The most potent of these were
kayeassamins A, B, D, E, and G (compounds 2-6, Figure 1) all with PC100 concentrations of 1 µM. The
geranylated coumarin ostruthin (7), from Rhizoma et Radix Notopterygii, was also found to be active with
a PC50 of 7.2 µM .21

Figure 1. Coumarin natural products with anti-austerity activity against PANC-1.
Anti-austerity active natural products continue to be isolated,22, 23 and the intriguing bioactivity of these
compounds has also inspired the efforts of synthetic chemists.24-32 In 2012, Coster and co-workers
synthesized a series of active analogs of angelmarin the most potent of which were two trifluoromethyl-
substituted derivatives 8 and 9 with PC50 values of 23 and 79 nM respectively (Figure 2).26 Carrico-
Moniz and co-workers have made a number of polyisoprenyl ethers including coumarins 10-13 (similar to
ostruthin, 7) the most active of which was the farnesyl ether 12 with a PC50 of 4 µM.31, 32
Figure 2. Anti-austerity active synthetic analogs of natural products.

Here we report our evaluation of the antiausterity activity of seventeen coumarins synthesized with the
aim of structural simplification of the natural products illustrated in Figure 1. First, we tested eleven
coumarins functionalized with simple n-alkyl ethers (14) intended to mimic the lipophlicity of
polyisoprenyl ether side chains (15) of the kayeassamis (Figure 3). Secondly we evaluated six coumarins
with readily accessible triazole-containing side chains (16) intended as alternatives to the more
synthetically challenging scaffold of angelmarin (17 and 1).
Figure 3. Naturally occurring coumarin substituents, 15 and 17, and their simplified mimics, 14 and 16,
that were synthesized and evaluated in this study.
Synthetic compounds 18-34 were tested for preferential cytotoxicity against PANC-1 human pancreatic
cancer using a previously reported antiausterity cytotoxicity assay (Table 1).12 Briefly, cell survival was
measured after incubation for 24 hours under nutrient deprived medium (NDM) as well as incubation in
Dulbecco’s modified Eagle medium (DMEM) in the presence of the test compounds (see Supporting
Information). Of the eleven simple alkyl ethers evaluated (18-28) only four compounds, 18, 23, 24, and
25, showed weak preferential cytotoxicity against PANC-1 under nutrient deprived conditions. These
included the three n-octyloxy coumarins, 18, 23 and 25, which were each more active than their
analogous compounds with longer n-alkyl chains (Table 1). Five of the triazoles tested, 28-33,
demonstrated no antiausterity activity against PANC-1 while a sixth, the meta-trifluoromethylphenyl
derivative 34, was active with a PC50 of 29 μM.

Table 1. Preferential cytotoxicity of synthetic coumarins 18-35 against PANC-1.
Entry Structure
R
Compound #
PC50 (μM)a
48
1
2
3
5
n-C8H17
n-C12H25
n-C14H29
18
19
20
21
> 100
> 100
> 100
6
> 100
22
7
8
n-C8H17
n-C12H25
29
51
23
24
9
n-C8H17
n-C16H33
n-C12H25
63
25
26
27
10
11
> 100
> 100
12
n-C16H33
n-C8H17
> 100
28
13
14
> 100
> 100
29
30
15
16
17
> 100
> 100
> 100
29
31
32
33
18
34
a.
PC50 describes cytotoxicity against PANC-1 (i.e. LC50 estimated using the graphical method) after 24
hour incubation in nutrient deprived medium (NDM) with no corresponding cytotoxicity at 100 μM in
Dulbecco’s modified Eagle medium (DMEM) (See Supporting Information.).
The difference in antiausterity activity between the para- and meta-trifluoromethylphenyl derivatives 33
and 34 was surprising given the opposite trend evident in angelmarin analogs 8 and 9 (see Figure 2)
reported by Coster and co-workers.26 This prompted us to evaluate both compounds 33 and 34 against
two additional pancreatic cancer cell lines, Capan-1 and MIA PaCa-2 (Figure 4). Compound 34 showed
preferential cytotoxicity against both Capan-1 and MIA PaCa-2 cells with PC50 concentrations of 8.5 and

18 μM respectively. In contrast, compound 33 was selectively cytotoxic only to MIA PaCa-2 cells (PC50
= 9.6 μM) without antiausterity activity against PANC-1 or Capan-1. The mechanism of cell death was
determined to be apoptotic (PANC-1, compound 34) based on a modified ethidium bromide and acridine
orange (EB/AO) staining assay (Figure 5).33
Figure 4. Preferential cytotoxicity of compounds 35 and 36 against three cancer cell lines after
incubation for 24 h in nutrient deprived medium (NDM) and Dulbecco’s modified Eagle medium
(DMEM).
Figure 5. Dual ethidium bromide (EB)/acridine orange (AO) stained fluorescent images of PANC-1 cells
at 24 h incubation in nutrient deprived medium (NDM); control, live cells emitted bright green
fluorescence; treatment with compound 35 (50 μM) resulted in orange fluorescence concentrated on one
side of cells indicative of apoptotic cell death (see Supporting Information).

All of the compounds evaluated above were readily accessible in one or two synthetic operations from
inexpensive substrates. Compounds 18-28 were products of the alkylation of the corresponding 7-
hydroxycoumarins with alkyl halides or alkyl sulfonates under typical conditions of K2CO3 in refluxing
acetone. Compounds 29-34 were prepared by assembly of the triazole rings via copper mediated
cycloaddition between 7-propargyloxycoumarin (37) and a series of alkyl azides (Scheme 1).
Scheme 1. Syntheses of Compounds 18-34.
In conclusion, of the seventeen synthetic coumarins evaluated for antiausterity activity against PANC-1
cancer cells, six were found to have weak activity. Evaluation of two trifluoromethylphenyl compounds
33 and 34 against three cancer cell lines showed that position of the trifluoromethyl substituent on the
phenyl ring of these compounds (meta vs para) was correlated to selectivity for activity against MIA
PaCa-2 cell line. This relatively small change in structure had a substantial effect on activity. Overall,
this work leads to the conclusion that these structural simplifications of naturally occurring coumarin
functionalities (Figure 3) result in decreased antiausterity activity relative to the natural products.

Acknowledgements
This work was supported by the University of Idaho. E.A.H. received research support from an IDeA
award from the NIH, NIGMS (Grant #P20GM103408). This work was also supported by a Grant from
Toyama Support Center for Young Principal Investigators in Advanced Life Sciences, Japan, and a Grant
in Aid for Scientific Research (No. 24510314) from the Japan Society for the Promotion of Science
(JSPS) to S.A. We thank Dr. Alex Blumenfeld and Dr. Lee Deobald for NMR and HRMS analyses
respectively.
Conflict of Interest
The authors have declared that there is no conflict of interest.
Supplementary data
Supplementary data (cytotoxicity assay, staining assay, synthetic protocols, characterization data and
NMR spectra for compounds 18–34) associated with this article can be found at….
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Graphical abstract