Synthesis of guggulsterone derivatives as potential anti-austerity agents against PANC-1 human pancreatic cancer cells

Article abstract of DOI:10.1016/j.bmcl.2020.126964

E- and Z-guggulsterones and nine guggulsterone derivatives (GSDs) were synthesized and evaluated for their preferential cytotoxicity against human PANC-1 cell in nutrient deprived medium utilizing antiausterity strategy. Among the synthesized compounds, GSD-1 and GSD-7 showed potent cytotoxicity against PANC-1 cells under nutrient-deprived conditions in a concentration dependent manner, with a PC₅₀ value of 1.6 μM and 3.2 μM, respectively. The effect of GSD-1 and GSD-7 was further evaluated in a real time using live cell imaging. Both of these compounds altered PANC-1 cell morphology, leading to cell death at sub micromolar concentration range. GSD-1 and GSD-7 also inhibited PANC-1 cell colony formation in a concentration-dependent manner. GSD-1 and GSD-7 are lead structure for the anti-austerity drug development.

Full text of DOI:10.1016/j.bmcl.2020.126964

Journal Pre-proofs
Synthesis of guggulsterone derivatives as potential anti-austerity agents against
PANC-1 human pancreatic cancer cells
Aki Kohyama, Rei Yokoyama, Dya Fita Dibwe, Sahar El-Mekkawy, Meselhy
R. Meselhy, Suresh Awale, Yuji Matsuya
PII:
S0960-894X(20)30017-2
DOI:
https://doi.org/10.1016/j.bmcl.2020.126964
BMCL 126964
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
2 December 2019
8 January 2020
9 January 2020
Please cite this article as: Kohyama, A., Yokoyama, R., Dibwe, D.F., El-Mekkawy, S., Meselhy, M.R., Awale, S.,
Matsuya, Y., Synthesis of guggulsterone derivatives as potential anti-austerity agents against PANC-1 human
pancreatic cancer cells, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.126964
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Synthesis of guggulsterone derivatives as potential anti-austerity
agents against PANC-1 human pancreatic cancer cells
Aki Kohyamaa, Rei Yokoyamaa, Dya Fita Dibweb,
,b
,a
Meselhy R. Meselhyd, Suresh Awale , Yuji Matsuya
aFaculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-
0194, Japan
bDivision of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama,
2630 Sugitani, Toyama 930-0194, Japan
cDepartment of Chemistry of Natural Compounds, National Research Centre, Elbohouth St.,
Giza, Egypt
dDepartment of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt
* Corresponding Authors:
suresh@inm.u-toyama.ac.jp (Suresh Awale)
matsuya@pha.u-toyama.ac.jp (Yuji Matsuya)

ABSTRACT
E-and Z- guggulsterones and nine guggulsterone derivatives (GSDs) were synthesized and
evaluated for their preferential cytotoxicity against human PANC-1 cell in nutrient deprived
medium utilizing antiausterity strategy. Among the synthesized compounds, GSD-1 and GSD-
7 showed potent cytotoxicity against PANC-1 cells under nutrient-deprived conditions in a
concentration dependent manner, with a PC50 value of 1.6 M and 3.2 M, respectively. The
effect of GSD-1 and GSD-7 was further evaluated in a real time using live cell imaging. Both
of these compounds altered PANC-1 cell morphology, leading to cell death at sub micromolar
concentration range. GSD-1 and GSD-7 also inhibited PANC-1 cell colony formation in a
concentration-dependent manner. GSD-1 and GSD-7 are lead structure for the anti-austerity
drug development.
Keywords: Guggulsterone; Pancreatic cancer; PANC-1; antiausterity agent; preferential
cytotoxicity; drug discovery; steroid

(Graphical Abstract)
natural
synthetic
O
H
O
H
SAR study
H
H
H
H
O
O
E-guggulsterone
PC50 = 42 µM
GSD-1
PC50 = 1.6 µM

Pancreatic cancer is deadly among various cancers and ranks 6th as the most frequent
cause of cancer death.1 The 5-year survival rate of the patients is the lowest among different
cancer types because the patients are diagnosed usually at the advanced metastatic stage.2 In
addition, therapeutic protocols for the treatment of pancreatic cancer often fail to be effective
due to drug resistance and impaired drug delivery pathways.2c,2d,3 Therefore, novel
chemotherapeutic agents to target pancreatic cancer are urgently required. An “anti-austerity
strategy” has been developed4,5 as a promising approach for drug discovery against pancreatic
cancer. Human Pancreatic tumors are hypovascular in nature with limited supply of nutrients,
the cancer cells are in starvation state within the tumor microenvironment. Therefore, tolerance
to nutrition starvation is one of the critical factors for the pancreatic cancer cells to survive in
tumor microenvironment. 4,5 It has been found that Human PANC-1 pancreatic cancer survives
for over 3 days even in the complete absence of essential nutrients such as glucose, amino
acids, and serum. Discovery of agents inhibiting such tolerance to starvation leading to cancer
cell survival is the promising anti-austerity strategy in anti-cancer drug discovery.6,7 In this
approach, the compounds or extracts are exposed to pancreatic cancer cell line under the
nutrient deprived medium (NDM) and the normal medium (DMEM). Compounds that show
preferential cytotoxicity under NDM is selected as anti-austerity agents. The activity is
represented as a PC50 value, which is the concentration at which 50% of the tumor cells were
preferentially killed in NDM without exhibiting cytotoxicity in DMEM. This strategy has been
successfully employed in the screening of thousands of medicinal plant extracts and the
compounds for the discovery of unique anti-cancer agents from the natural source.6,7 A natural
lignan “arctigenin” has been discovered as one of the promising antiausterity agent, which has
successfully passed early Phase II human clinical trial, with significant survival benefits among
the pancreatic patients without toxic side effect.8 It encouraged us to explore on natural product
candidates to discover potent antiausterity agents through medicinal chemistry approach.

Guggulsterone (GS) is a steroidal natural product isolated from the gum resin of
Commiphora mukul, a plant widely used in the Indian traditional medicine to treat various
diseases.9 It has been reported for anti-proliferative activity against various cancer cells,10,11
therefore is an attractive candidate for cancer chemotherapy drug development. Meanwhile,
there have been remarkable efforts in synthesizing potential anti-austerity agents.12-14 In this
project, we initiated a medicinal chemistry project and synthesized new GS derivatives (GSD)
in an attempt to discover potential anti-austerity agents.
Figure 1. Molecular design of guggulsterone derivatives.
Scheme 1. Synthesis of guggulsterone derivatives
a. R-CH2PPh3Br (4 eq), t-BuOK (4 eq), THF, reflux, 1−24 h; b. SeO2 (1 eq), TBHP (2.4 eq),
CH2Cl2, 0 °C, 3−3.5 h; c. cyclohexanone (10 eq), Al(OiPr)3 (2.5 eq), benzene, 80 °C, 2−3 h
16−63% (3 steps); d. p-TsOH (0.1 eq), benzene, 80 °C, 1 h, 13−26%; e. Pd/C, H2, EtOH, 2.5−6
h, 93−94%

First, we designed the following guggulsterone derivatives; alkyl-substituted derivatives on D-
ring (E-type), these (Z)-isomers (Z-type), and enone-deficient derivatives (Fig. 1). Generally,
intrinsic electrophiles are reactive toward various intracellular molecules to play essential roles
in cellular signals. In this context, two enone moieties on GS are expected to play crucial roles
for the biological activity. Thus, these enone-deficient derivatives, as well as the side chain-
modulated derivatives, were designed for the structure-activity-relationship (SAR) study.
For the concise and flexible synthesis of our designed GS derivatives in a short time, Kang’s
synthetic method was utilized.15a,16 It is composed of Wittig reaction, allylic oxidation,17 and
Oppenauer reaction by using dehydroepiandrosterone (1) as a starting material. The use of
various Wittig reagents enabled us to introduce different alkyl groups on D-ring (Scheme 1).
The higher stereoselectivities were observed to give E-alkenes 2a-e with trace amounts of Z-
isomers. The resulting alkenes 2a-e were oxidized to dienones via allylic oxidation and the
subsequent Oppenauer reaction to provide all E-type derivatives, including natural E-GS.
Furthermore, E-GS, GSD-2, and GSD-4 were partially converted to Z-isomers under Kang’s
isomerization condition.18 For the synthesis of enone-deficient derivatives, GSD-7 was
obtained as an inseparable mixture of E/Z isomer via Wittig reaction followed by Oppenauer
reaction. Synthesis of GSD-8 was achieved from the known compound 4 through the three-step
sequence. Hydrogenation of GSD-8 proceeded in a stereoselective manner to give GSD-9 as a
single diastereomer. Considering previous reports,19 the reaction would proceed from sterically
less hindered -face, providing the 17-ethyl derivative with a -configuration.
6

Table 1
compound
E/Z
R
PC50 (M)
IC50 (M)
(NDM)
42
(DMEM)
E-guggulsterone
GSD-1
E
Me
> 100
H
Et
1.6
50
11
> 100
> 100
> 100
42
GSD-2
E
E
E
Z
Z
Z
GSD-4
n-Pr
14
GSD-6
(CH2)3OMe
Me
52
Z-guggulsterone
GSD-3
37
Et
17
> 100
> 100
GSD-5
n-Pr
18
arctigenina
0.80
apositive control
Table 2
compound
PC50 (M)
(NDM)
42
IC50 (M)
(DMEM)
> 100
E-guggulsterone
GSD-7
3.2
> 100
GSD-8
> 100
18
> 100
GSD-9
> 100
7

A)
GSD-1
B)
GSD-7
PANC-1
125
PANC-1
125
NDM
NDM
100
75
DMEM
100
75
DMEM
50
50
PC50
3.16
=
PC50
1.6
=
25
0
25
0
mM
mM
0
1
2
3
0
1
2
3
Log concentration [µM]
Log concentration [µM]
Figure 2. Preferential cytotoxic activity of A) GSD-1 and B) GSD-7 against the PANC-1 human
pancreatic cancer cell line in nutrient-deprived medium (NDM) and Dulbecco’s modified Eagle’s
medium (DMEM).
Following the antiausterity strategy, the synthesized E-GS, Z-GS, and nine GSDs were
investigated for its anti-austerity activity against the PANC-1 tumor cell line (Table 1, 2, Figure
2). Among these, GSD-1 and GSD-7 showed the most potent preferential cytotoxicity against
PANC-1 cells in NDM, exhibiting a PC50 value of 1.6 M and 3.2 M, respectively. In
particular, the PC50 value of GSD-1 is comparable to that of arctigenin (PC50 0.8 M), a positive
control used in this study.8 The selectivity index of GSD-1 and GSD-7 in NDM was found to
be 6.5 times and 34 times compared to DMEM. Based on the observed activity, a clear structure
and activity can be deduced. In particular, table 1 shows that unsubstituted exocyclic double
bond within enone moiety in D-ring of GSDs seems to be essential for the potent activity.
(GSD-1 > E and Z-guggulsterone and GSD-2−6). All the GSD derivatives with substituents at
exocyclic double bond displayed weak activity. Table 2 shows cytotoxicities of enone-deficient
derivatives. GSD-8 and GSD-9 exhibited inactive or weak cytotoxicity. On the other hand,
GSD-7 showed potent preferential cytotoxicity with PC50 value of 3.2 M, implying that one
enone moiety on A-ring is important for cytotoxicity of GSD-7.
The promising antiausterity activity of GSD-1 and GSD-7 encouraged us to investigate
the effect of these compounds on PANC-1 cells in a real-time frame. Therefore, PANC-1 cells
8

were treated with GSD-1 (3 µM) and GSD-7 (5 µM) in NDM, and incubated in a stage-top
incubator at 37 °C in an atmosphere of 5% CO2. The image of cellular events was captured in
every 10 min in the phase-contrast mode on an EVOS FL digital cell imaging system for 24 h.
As seen in Movie 1, GSD-1 and GSD-7 inhibited the cell mobility within short time after
exposure leading to shrinkage of PANC-1 cells followed by swelling of cells and leakage of
cytoplasmic contents into the media causing cell death within 12 h (Figure 3, movie 1).
Figure 3. Captures of the live imaging of the effect of (A) GSD-1 (3 M), and (B) GSD-7 (5
M) on PANC-1 cells at different intervals of time (hour: minute) in nutrient deprived medium.
[Please insert movie 1 here in the web version]
[Please insert this movie clip in the HTML version of the paper]
9

Movie 1. Time-lapse video showing the detailed events upon exposure to GSD-1 (3 µM) and
GSD-7 (5 µM) against PANC-1 cells in nutrient-deprived medium (NDM) with one frame
per 10 min for 24 h on the EVOS FL digital imaging system (10 objective)
Phase contrast
Merged
AO
EB
Treated
(GSD-1)
Treated
(GSD-7)
Control
Figure 4. Morphological changes of PANC-1 cells induced by GSD-1 ((3 µM) and GSD-7 (5
µM), in comparison to untreated cells (i.e., the control) in nutrient-deprived medium (NDM).
PANC-1 tumor cells were treated with GSD-1 and GSD-7 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.
The effect of GSD-1 and GSD-7 on the alteration of PANC-1 morphology and cell death
was further visualized by the ethidium bromide (EB) – acridine orange (AO) double-staining
fluorescence assay. In this assay, the live cell emits bright-green fluorescence due to AO,
while dead or dying cells and emits predominantly red fluorescence due to EB. PANC-1 cells
10

in the control show an intact cellular morphology and emitted exclusive green fluorescence
of live cells even after exposed to nutrient deprivation for 24 hours. Contrary to this, PANC-
1 cells treated with GSD-1 and GSD-7 showed rounded cell morphology of PANC-1 cells’
and emitted exclusive red fluorescence, suggestive of total cell death.
Figure 5. Effect of GSD-1 and GSD-7 on colony formation by PANC-1 cells. (A)
Representative PANC-1 cell colonies treated with different concentrations of GSD-1 and
GSD-7. (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.
All these promising biological responses of GSD-1 and GSD-7 encouraged us to further
evaluate these compounds against colony formation potential of PANC-1 cells. Pancreatic
cancer is highly metastatic, and the invading cancer cells form metastatic colonies in the distant
organs such as stomach, liver and lungs. Therefore, antiausterity agents with inhibitory activity
against colony formation are therapeutically beneficial against pancreatic cancer. Hence, GSD-
1 and GSD-7 was investigated against PANC-1 cell colony formation following the protocol
described previously. PANC-1 cells (500 cells) were exposed to GSD-1 (1.25, 2.5 and 5.0 M)
and GSD-7 (12.5, 25 and 50 M) at non-cytotoxic concentration in DMEM and incubated for
11

24 h. After that, the cells were washed twice with PBS followed by addition of fresh DMEM
(without any test compound) and placed in a CO2 incubator to allow colony formation for 10
days. At the end of experiment, cells were stained with crystal violet, and the area occupied by
colony is evaluated. As shown in the Figure 5, the control PANC-1 cells grew and occupied
14%-18% of the total well area. Contrary to that, PANC-1 cells exposed with GSD-1 showed
complete inhibition of colony formation even at the lowest tested dose of 1.25 M. GSD-7 on
the other hand inhibited colony formation only at the maximum tested concentration of 50 M.
From these results, GSD-1 was concluded as the most rewarding candidate for the drug
development against pancreatic cancer. During nutrition starvation, Akt is overexpressed in
pancreatic tumor cells and therefore regarded as austerity marker. Antiausterity agents, such
as arctigenin, grandifloracin, kigamycin, ancistrolikokine E3 have been found to inhibit Akt
activation.4a,5,7a Therefore, it is possible that GSD derivatives might inhibit the activation of the
Akt pathway leading to preferential cytotoxicity during the starvation condition. Further works
on this area is currently in progress and will report in due course.
■ ACKNOWLEDGMENTS
This work was supported by a grant from the Japanese Society for the Promotion of Science
(JSPS Kakenhi #16K08319) and Kobayashi International Scholarship Foundation to S.A.,
JSPS Core-to-Core Program (B. Asia-Africa Science Platforms) to Y.M., and Tamura Science
and Technology Foundation to A.K.
Supplementary data
Supplementary data containing analytical data and NMR spectral data of the synthesized
compounds and experimental procedures.
12

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Declaration of interests
☒The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
17

natural
synthetic
H
O
H
SAR study
H
H
H
H
O
O
E-guggulsterone
GSD-1
µ
µ
PC50 = 42 M
PC50 = 1.6 M
18