Synthesis, structure-activity relationship and in vitro pharmacodynamics of A-ring modified caged xanthones in a preclinical model of inflammatory breast cancer

Article abstract of DOI:10.1016/j.ejmech.2019.02.047

Inflammatory breast cancer (IBC) is a highly metastatic, lethal form of breast cancer that lacks targeted therapeutic strategies. Inspired by the promising cytotoxicity of gambogic acid and related caged xanthones in spheroids^MARY-X, an in vitr

Full text of DOI:10.1016/j.ejmech.2019.02.047

Accepted Manuscript
Synthesis, structure-activity relationship and in vitro pharmacodynamics of A-ring
modified caged xanthones in a preclinical model of inflammatory breast cancer
Oraphin Chantarasriwong, Andrew T. Milcarek, Theodore Habarth Morales, Aspen L.
Settle, Celso O. Rezende, Jr., Bashayer D. Althufairi, Maria A. Theodoraki, Mary L.
Alpaugh, Emmanuel A. Theodorakis
PII:
S0223-5234(19)30161-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.047
EJMECH 11139
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 January 2019
Revised Date: 14 February 2019
Accepted Date: 14 February 2019
Please cite this article as: O. Chantarasriwong, A.T. Milcarek, T.H. Morales, A.L. Settle, C.O. Rezende
Jr., B.D. Althufairi, M.A. Theodoraki, M.L. Alpaugh, E.A. Theodorakis, Synthesis, structure-activity
relationship and in vitro pharmacodynamics of A-ring modified caged xanthones in a preclinical
model of inflammatory breast cancer, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.02.047.
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Synthesis, structure-activity relationship and
in vitro pharmacodynamics of A-ring
modified caged xanthones in a preclinical
model of inflammatory breast cancer
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Oraphin Chantarasriwong,a,b,‡ Andrew T. Milcarek,c,‡
Theodore Habarth Morales,d Aspen L. Settle,d Celso
O. Rezende Jr.,a Bashayer D. Althufairi,a Maria A.
Theodoraki,d* Mary L. Alpaugh,c* and
Emmanuel A. Theodorakisa*
Corresponding authors:
* Emmanuel A. Theodorakis; email: etheodorakis@ucsd.edu
ORCID: https://orcid.org/0000-0001-9845-6919
* Mary L. Alpaugh; email: alpaugh@rowan.edu
ORCID: https://orcid.org/0000-0002-0008-2718
* Maria A. Theodoraki; email: theodorakim@arcadia.edu
ORCID: https://orcid.org/0000-0002-0775-8928
‡ The authors contributed equally.

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com/locate/ejmech
Research paper
Synthesis, structure-activity relationship and in vitro pharmacodynamics of A-
ring modified caged xanthones in a preclinical model of inflammatory breast
cancer
Oraphin Chantarasriwong,a,b,‡ Andrew T. Milcarek,c,‡ Theodore Habarth Morales,d Aspen L. Settle,d Celso
O. Rezende Jr.,a Bashayer D. Althufairi,a Maria A. Theodoraki,d* Mary L. Alpaugh,c* and Emmanuel A.
Theodorakisa*
a.
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.
Department of Chemistry, Faculty of Science, King Mongkut’s University of Technology Thonburi, 126 Pracha Uthit Rd., Bang Mod, Thung Khru, Bangkok
b.
10140, Thailand.
c.
Department of Molecular and Cellular Biosciences, Rowan University, Glassboro, NJ 08028
Department of Biology, Arcadia University, Glenside, PA, 19038
d.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Inflammatory breast cancer (IBC) is a highly metastatic, lethal form of breast cancer that lacks
targeted therapeutic strategies. Inspired by the promising cytotoxicity of gambogic acid and
related caged xanthones in spheroidsMARY-X, an in vitro preclinical IBC model, we constructed a
library of synthetic analogs and performed structure-activity relationship studies. The studies
revealed that functionalizing the A-ring of the caged xanthone framework can significantly
affect potency. Specifically, introduction of hydroxyl or fluorine groups at discrete positions of
the A-ring leads to enhanced cytotoxicity at submicromolar concentrations. These compounds
induce complete dissolution of spheroidsMARY-X with subsequent apoptosis of both the
peripherally- and centrally-located cells, proliferative and quiescent-prone (e.g. hypoxic),
respectively. These results highlight the structural flexibility and pharmacological potential of
the caged xanthone motif for the design of IBC-targeting therapeutics.
Keywords:
Keyword_1 spheroids
Keyword_2 natural product
Keyword_3 synthetic methods
Keyword_4 apoptosis
Keyword_5 breast cancer
2019 Elsevier Ltd. All rights reserved
.
1. Introduction
R1
6
O
OH
Tropical trees of the genus Garcinia (Clusiaceae), grown
mostly in Southeast Asia, India, Africa and Brazil, are widely
known not only for their high value as sources of food but also
for their role in ethnomedicine.[1-3] For instance, gamboge, the
dried resin from Garcinia hanburyi, has been used in traditional
Eastern medicine for its anti-infective and anti-parasitic
properties.[4,5] Efforts to isolate the bioactive constituents of
gamboge led to the identification of gambogic acid (GBA, 1)
(Figure 1), a caged xanthonoid that displays potent anticancer
activities both in vitro and in vivo [6-10] and presents a promising
therapeutic window for clinical applications.[11-14] In fact, GBA
has entered clinical trials in China for the treatment of non-small
cell lung, colon and renal cancers.[15-17]
A
CO2H
R2
18
6
2
O
10
B
9
O
A
O18
O
C
10
O
B
9
O
O
C
O
2: cluvenone (CLV); R1, R2= H
3: MAD28; R1= OH, R2= H
4: MAD44; R1= H, R2= OH
1: gambogic acid (GBA)
Figure 1. Chemical structures of representative caged xanthones:
gambogic acid (GBA), cluvenone (CLV), MAD28 and MAD44
specific oxidations and functionalizations of this framework
contribute to the structural diversity of this family. The
pharmacological promise of CXs has inspired several structure-
function studies.[25-30] These efforts unveiled that cluvenone (2,
CLV) represents the minimum pharmacophore of the CX
family[31-33] and suggested that A-ring functionalization could
lead to analogs with improved bioactivity.[34-38] Along these lines,
MAD28 (3), the C6-hydroxylated cluvenone, was found to be
approximately 3 times more active than MAD44 (4), the C18-
By virtue of its unusual chemical architecture, GBA has
become the archetype of a family of bioactive natural products
that are commonly referred to as caged xanthones (CXs).[18-21]
Several members of this family exhibit potent activity against
leukemia, glioblastoma, lung cancer and colorectal cancer cell
lines.[22-24] Central to their structure is a xanthone framework in
which the C ring has been converted into a caged motif. Plant-

produce caged xanthone MAD67 (12) (69% yield over two
hydroxylated cluvenone (Figure 1), in inhibitiAngCgrCowEthPiTnEa DT- MANUSCRIPT
cell acute lymphoblastic leukemia (CEM) cell line.[39] More
recently, MAD28 (1.25 µM) was shown to exhibit selective
cytotoxicity against human epithelial breast cancer cells (MDA-
MB-231 or MCF7) with no apparent detrimental effect in normal
breast cells (MCF10A).[40]
steps).[57] Acid-catalyzed deprotection of the C18-MOM group
then gave rise to MAD6O9 (13).[37]
OH
6
OH
HO
O
(a) POCl3,
ZnCl2
6
HO 18
HO 18
+
HO
(b) Ph2CCl2
O
The promising activity of MAD28 against breast cancer led us
to evaluate its cytotoxicity in spheroidsMARY-X, a 3-dimensional
multicellular system formed from a patient-derived xenograft of
inflammatory breast cancer (IBC).[41] IBC is a highly aggressive
and often lethal subtype of breast cancer accounting for about 3-
10% of diagnosed breast carcinomas.[42-46] Its high mortality rate
is due to its metastatic nature and triple negative signature that
renders IBC resistant to chemotherapeutic regimens.[47-49]
Additionally, IBC lacks molecular markers making diagnosis and
design of targeted therapeutic approaches very challenging. It is
accepted that the lymphovascular embolus is a marker of cancer
aggressiveness, recurrence, metastatic progression and
therapeutic failure in many tumor types.[50-53] The spheroidsMARY-X
maintain the 3-dimensional and pathophysiological features of
the lymphovascular embolus.[54] As such, they provide a relevant
model to test efficacy of therapeutics to IBC and metastatic
disease in general. We found that both GBA and MAD28 induce
spheroid dissolution and cell death in the spheroidsMARY-X assay
with IC50 values of 0.42 and 0.66 µM, respectively.[41]
Importantly, this activity was 20-fold greater than that observed
with taxol (IC50= 7.8 µM) while other FDA-approved drugs
failed to induce any response.[41]
OH
HO
OH
13
12
%)
(42
O
O
5
6
7
Ph
(100%)
Ph
(c) MOMCl
(d) H2, Pd/C
(e) 10% Pd(PPh3)4
O
OH
O
OH
6
O
O
RO
RO
18
O
O
(9)
O
13
12
13
12
(89%)
O
O
OH
HO
8: R= MOM
10: R= MOM
(f) DMF, 120 °C
OH
OH
6
O
O
RO
RO
(69%)
18
O
13 O
12
O
O
13
12
In this study, we synthesized and evaluated a library of caged
xanthones in which the A-ring has been chemically modified and
found that such functionalizations substantially modulate the
observed cytotoxicity in the spheroidsMARY-X assay. Notably,
attachment of electron withdrawing groups at the A-ring as well
as introducing a free hydroxyl group at C6 enhances cytotoxicity
and results in complete dissolution with subsequent apoptosis of
O
O
12: MAD67; R= MOM
13: MAD69; R= H
(g)
11: R= MOM
Scheme 1. Reagents and conditions: (a) 27 equiv. POCl3, 10
equiv. ZnCl2, 4.5 h, 65 °C, 62%; (b) 3 equiv. Ph2CCl2,
acetonitrile, MW, 12 min, 150 °C, 67%; (c) 2 equiv. MOMCl, 3
equiv. DIPEA, CH2Cl2, 1 h, 25 °C, 100%; (d) H2, 10% Pd/C,
THF/MeOH (3:1), 18 h, 25 °C, 100%; (e) 10 equiv. t-butyl 2-
methylbut-3-en-2-yl carbonate (9), 10 mol% Pd(PPh3)4, THF, 2
h, 0 °C, 89%; (f) DMF, 4 h, 120 °C, 69%; (g) 15% HCl, acetone,
24 h, 25 °C, 32%.
the
spheroidsMARY-X at
submicromolar
concentrations.
Spatial/temporal analysis of the multicellular IBC spheroid
showed pyknotic nuclei throughout as well as activation of PARP
(i.e. cleaved PARP) at 3 hours, an indication of irreversible
apoptosis. Fluorescent overlay analysis of cPARP and the
proliferation marker Ki67 indicated that these compounds induce
apoptosis irrespective of proliferation status. Specifically, they
target both the peripherally-located proliferative cells as well as
the centrally-located quiescent-prone (e.g. hypoxic) cells of the
multicellular spheroid. The latter represents the often resistant
and chemo-refractory cell population of this disease. Thus, the
caged xanthone motif represents a promising platform for future
preclinical development of IBC-targeted therapeutics.
Treatment of 13 with tri(ethylene glycol) monomethyl ether
tosylate resulted in the formation of compounds 14 and 15 in
variable yields (Scheme 2).[58] Monoalkylated compound 14 was
isolated as the only product when the reaction was performed at
room temperature, likely due to the higher reactivity of the C18
phenol. On the other hand, at 80 °C the reaction gave rise to
dialkylated product 15 in 55% yield. Under similar conditions,
caged xanthones 3 and 4[39] were converted to derivatives 16 and
17, respectively.
2. Results and Discussion
2.1. Synthesis of A-ring modified caged xanthones
R1
14: CR170A; R1= OH, R2= O(CH2CH2O)3Me
15: CR170B; R1, R2= O(CH2CH2O)3Me
6
O
The synthesis of the C6,C18-dihydroxylated caged xanthones
12 and 13 is shown in Scheme 1. Condensation of phloroglucinol
(5) with 2,3,4-trihydroxybenzoic acid (6) under ZnCl2/POCl3
conditions[55] produced a tetrahydroxylated xanthone that was
subsequently protected with diphenyldichloromethane to form
compound 7 (42% yield over two steps).[56] Selective protection
of the more reactive C18-phenol with MOMCl/DIPEA followed
by deprotection of the diphenyl ketal (H2, Pd/C) gave rise to
xanthone 8 (100% yield over two steps). Pd-catalyzed reverse
prenylation of 8 with carbonate 9[35] produced 10 in 89% yield.
Heating of 10 in DMF at 120 °C resulted in a site-selective
Claisen rearrangement of the C12-allyl ether to form intermediate
11 that underwent an intramolecular Diels-Alder cyclization to
R2
18
O
O
16: CR136; R1= O(CH2CH2O)3Me, R2= H
17: CR140; R1= H, R2= O(CH2CH2O)3Me
O
Scheme 2. Reagents and conditions: 14: 1 equiv. 13, 2 equiv.
K2CO3, 2 equiv. TsO(CH2CH2O)3Me, DMF, 8 h, 25 °C, 75%; 15:
1 equiv. 13, 3 equiv. K2CO3, 3 equiv. TsO(CH2CH2O)3Me, DMF,
10 h, 80 °C, 55%; 16: 1 equiv. 3, 2 equiv. K2CO3, 3 equiv.
TsO(CH2CH2O)3Me, DMF, 8 h, 25 °C, 68%; 17: 1 equiv. 4, 2
equiv. K2CO3, 2 equiv. TsO(CH2CH2O)3Me, DMF, 8 h, 80 °C,
86%.

The synthesis of the caged xanthones 23 and 25 is shown in
prenylation of 29 with carbonate 30 gave xanthone 31. In the
final step, heat-induced Claisen/Diels-Alder reaction cascade
produced the regular caged xanthone 32 in 64% yield
accompanied by a small amount of the neo cage isomer 33 (3%
yield).
ACCEPTED MANUSCRIPT
Scheme 3. Treatment of commercially available 2-fluoro-1,4-
dimethoxybenzene (18) with n-BuLi and methyl chloroformate
afforded, after ester deprotection, the corresponding carboxylic
acid 19 (96% combined yield).[59] A two-step condensation of 19
with pyrogallol (20) produced xanthone 21 in 30% combined
yield. This compound was subsequently allylated with 9 under
Pd(0) catalysis and the resulting adduct 22 was rearranged to
caged xanthone 23 via a Claisen/Diels-Alder reaction cascade.
Various conditions, e.g. PhI(OCOCF3)2 or cerium ammonium
nitrate, were evaluated for the in situ oxidative demethylation of
23 to 25 but they proved to be unsuccessful.[60] To overcome this
issue, we sought to accomplish this conversion in two distinct
steps.[61,62] Along these lines, we converted 21 to 25 via: (a)
boron tribromide induced demethylation of the C6 methyl ether;
(b) Pd(0) mediated allylation of the resulting xanthone with 9; (c)
Claisen/Diels-Alder rearrangement; and (d) PhI(OCOCF3)2-
mediated oxidation of the resulting caged xanthone 24 (19%
combined yield).
F
F
6
O
X
O
(b) AlCl3
(81%)
F
F 18
+
HO
OH
F
HO
F
OH
OH
20
26: X= OH
27: X= Cl
28 HO
(a)
(c) Na2CO3
(48%)
F
(d) 10% Pd(PPh3)4
O
F
O
6
O
F
F
18
O
O
O
(30)
O
13
12
(100%)
O
O
OMe
R
OMe
HO
OH
(c) POCl3, ZnCl2
(d) DIPEA
6
O
31
29
HO
+
F
F
(30%)
(e) DMF
120 °C
O
O
O
MeO
(a, b)
HO
OH
MeO
O
F
F
F
18: R= H
+
HO
OH
O
21
12
13
20
19: R= CO2H
12
O
O
13 O
O
(e) 10% Pd(PPh3)4
O
(g, h,i)
O
(77%)
(28%)
32: RA25 (64%)
33: RA25N (3%)
OMe
O
OH
O
O
O
6
(9)
Scheme 4. Reagents and conditions: (a) 2 equiv. (COCl)2, DMF
(cat.), CH2Cl2, 3 h, 0 °C to 25 °C, 100%; (b) 1 equiv. 20, 3 equiv.
AlCl3, CHCl3/CH2Cl2 (1:2.5), 18 h, 25 °C; then 24 h, 60 °C, 81%;
(c) 1 equiv. Na2CO3, DMF, 3 h, 90 °C, 48%; (d) 10 equiv. bis(2-
methylbut-3-en-2-yl) carbonate (30), 10 mol% Pd(PPh3)4, THF,
40 min, 5 °C, 100%; (e) DMF, 1 h, 120 °C, 64% for 32, 3% for
33.
O
MeO
O
MeO
O
O
O
24
22
(68%)
(j) PhI(OCOCF3)2
O
(f) DMF, 120 °C (88%)
OMe
O
O
2.2.
Screening of caged xanthones using the
O
O
spheroidsMARY-X assay
X
O
O
MeO
O
O
O
The activity of the synthetic caged xanthones was determined
using the spheroidsMARY-X assay. This system allows quick
assessment of the drug response via brightfield microscopy and
ImageJ circularity analysis (see Fig. S1, Supplemental
Information), where spheroid dissolution is displayed as a
deviation from the well-circumscribed spheroid edge which
correlates with induction of apoptosis (i.e. drug response).[41,63]
Briefly, 30-50 spheroidsMARY-X were seeded in a multi-well plate
and treated with increasing doses of each compound or DMSO
(vehicle) for 24 hours. All the compounds were tested at five
different concentrations, ranging from 0.5 µM to 2.5 µM. CX
analogs that showed no response (NR), low response (LR) and
moderate response (MR) at the highest concentration (2.5 µM)
are presented in Figure 2. MAD69 and CR240C showed no
response (NR) as there was no deviation of the well-
circumscribed edge of the spheroidsMARY-X comparable to the
vehicle only (DMSO, control) treated spheroidsMARY-X (Fig. S1).
Analogs MAD44 and P67 showed low response (LR) while
CR140, CR170B showed low to moderate response (LR-MR);
both pairs of drugs resulted in only minor deviations from the
well-circumscribed spheroid edge. In contrast, the extent of the
deviation was increased for spheroidsMARY-X treated with CR170A
and CR136 leading to a moderate response (MR).
25: CR240C
23: P67
Scheme 3. Reagents and conditions: (a) 1.2 equiv. 1.6 M n-
BuLi, THF, 1 h, –78 °C; then 1.2 equiv. ClCO2CH3, THF, 4 h, –
78 °C, 96%; (b) 5 equiv. LiOH, MeOH/H2O (3:1), 1.5 h, 70 °C,
100%; (c) 1 equiv. pyrogallol (20), 27 equiv. POCl3, 10 equiv.
ZnCl2, 2.5 h, 65 °C, 40%; (d) 5 equiv. DIPEA, ACN, MW, 22
min, 180 °C, 74%; (e) 10 equiv. t-butyl 2-methylbut-3-en-2-yl
carbonate (9), 10 mol% Pd(PPh3)4, THF, 24 h, 0 °C, 77%; (f)
DMF, 4 h, 120 °C, 88%; (g) 1 equiv. BBr3 (1 M in CH2Cl2),
CH2Cl2, 2 h, 0 °C to 25 °C, 62%; (h) 10 equiv. t-butyl 2-
methylbut-3-en-2-yl carbonate (9), 10 mol% Pd(PPh3)4, THF, 18
h, 0 °C, 63%; (i) DMF, 4 h, 120 °C, 72%; (j) 1.2 equiv.
PhI(OCOCF3)2, CH2Cl2, 1 h, –78 °C to –30 °C, 68%.
The synthesis of A-ring fluorinated caged xanthones is
described in Scheme 4. Xanthone 29 containing fluorine at C6
and C18 was prepared via: (a) conversion of 2,4,6-trifluoro
benzoic acid (26) to its acyl chloride 27; (b) Friedel-Crafts
acylation of 27 with pyrogallol (20); and (c) intramolecular
nucleophilic aromatic substitution of the resulting benzophenone
28 (39% combined yield, Scheme 4). Pd(0)-catalyzed reverse

Out of the thirteen caged xanthones tested, five exhibited
ACCEPTED MANUSCRIPT
complete response (CR) as evidenced by a total dissolution of the
spheroidsMARY-X after 24 hours treatment at 2.5 µM. Specifically,
MAD28, MAD67, CLV, RA25 and RA25N induced complete
response (CR) leading to a single cell suspension of the formerly
intact, well-circumscribed spheroidsMARY-X. These analogs were
subjected to a separate, more detailed screening, aiming to
quantify their effect on spheroidsMARY-X and determine their IC50
values. During this second screening, nine concentrations from
0.25 µM to 2.25 µM at 0.25 µM dose intervals of each compound
were generated by serial dilutions and were used to treat the
spheroidsMARY-X for 24 hours. The effect of these compounds in
four representative concentrations (0.5, 0.75, 1.0 and 2.0 µM) is
shown in Figure 3 (the entire 9-concentration panel is shown in
Supplemental Information; Fig. S2). The brightfield images
acquired from this screening were further analyzed using ImageJ
software[64] in order to quantify the circularity/response of the
spheroidsMARY-X and construct a dose-response graph that allows
for calculation of IC50 values (Figure 4).
Figure 2. Screening of A-ring modified caged xanthones in
spheroidsMARY-X Brightfield images of spheroidsMARY-X after 24
.
hours of treatment with 2.5 µM show a gradation of drug
response from no deviation (black arrowheads) from well-
circumscribed spheroid edge to an increase in deviation (blue
arrowheads) and loss of well-circumscribed spheroid edge, as
follows: MAD69, CR240C: no response (NR); MAD44, P67:
low response (LR); CR140, CR170B: low response to moderate
response (LR-MR); and CR170A, CR136: moderate response
(MR).
Figure 3. Dose-dependent response of spheroidsMARY-X upon treatment with MAD28, CLV, MAD67, RA25 and RA25N. Representative
brightfield images of spheroidsMARY-X 24 hours post drug treatment at 0.5, 0.75, 1.0 and 2.0 µM concentrations (bar 100 µm).

Table 1
ACCEPTED MANUSCRIPT
CX analogs
R1
6
O
R2
Respon
se
IC50
18
O
17
R1
R2
O
O
(µM)
CLV
H
H
CR
0.63
0.51
MAD28
MAD44
MAD67
MAD69
CR170A
CR170B
CR136
CR140
P67
OH
H
CR
H
OH
LR
OH
OMOM
CR
0.38
Figure 4. Dose-response curves and IC50 values of CX analogs
inducing complete response. Circularity values obtained via
ImageJ analysis were used to calculate the IC50 values for
MAD28, MAD67, CLV, RA25 and RA25N. The non-linear fit
curves and calculation of IC50 value for each analog were
performed using GraphPad Prism.
OH
OH
NR
OH
O(CH2CH2O)3Me
MR
LR-MR
MR
LR-MR
LR
O(CH2CH2O)3Me
O(CH2CH2O)3Me
O(CH2CH2O)3Me
H
H
O(CH2CH2O)3Me
OMe
C=O
F
OMea
C=Oa
F
2.3.
Structure-activity relationship study
Comparison of CLV (IC50= 0.63 µM) and MAD28 (IC50=
0.51 µM) indicates enhancement of cytotoxicity when there is a
free OH group at the C6 center of the A-ring of the CX scaffold
(Table 1). Alkylation of this group decreases the cytotoxicity as
shown with CR136 (MR) and also with P67 (LR). These results
may be due to an intramolecular hydrogen bonding between the
C6-OH and the near carbonyl group that, in turn, increases the
electrophilicity of the C10 enone. On the other hand, installation
of a free OH group at C18 substantially reduces the cytotoxicity
as shown in both MAD44 and MAD69 (LR and NR,
respectively). In the latter structure, even the presence of an OH
group at C6 is not sufficient to induce cytotoxicity. Alkylating
the free C18 phenol group partially enhances the cytotoxicity as
shown by comparing CR170B (LR-MR) to MAD69 (NR) as
well as MAD44 (LR-MR) to CR140 (LR). Moreover, when the
C18 phenol is alkylated, the presence of a free OH group at the
C6 center improves activity as shown by comparing CR170A
(MR) to CR170B (LR-MR). In fact, MAD67 that contains a
MOM-protected C18 phenol and a free C6 hydroxyl group
induces complete response (CR; i.e. leads to a single cell
suspension of the formerly intact, well-circumscribed
spheroidsMARY-X) and has the highest cytotoxicity (IC50= 0.38
µM).
CR240C
RA25
NR
CR
1.12
0.89
RA25Nb
F
F
CR
a
R2 is located at the C17 center
RA25N contains a neo caged motif
b
2.4.
Evaluation of the spatial and temporal induction of
apoptosis in spheroidsMARY-X by selected caged xanthones
The spheroidsMARY-X is a spontaneously-forming 3-dimensional
model system that accurately recapitulates the growth, behavior
and microenvironment of solid tumors.[67-72] To investigate
whether the lead compounds can target any cell of the
multicellular spheroid or are restricted to targeting a specific cell
subpopulation, we evaluated the status of proliferation and
apoptosis within the spheroids using immunofluorescence. For
this experiment, the spheroidsMARY-X were suspended in an
extracellular matrix (ECM) plug, which provides a scaffold that
prevents dispersion of the cells during drug treatment. The ECM
plugs containing spheroidsMARY-X were then exposed to 2.5 µM of
MAD28, MAD67 and RA25, or DMSO (control) and monitored
using brightfield microscopy for loss of the well-circumscribed
spheroid edge. Treatment was terminated after 3 hours, when
deviations from the well-circumscribed spheroid edge of all
compound-treated spheroids were detected (Figure 5; brightfield
column, white arrowheads). The ECM plugs of vehicle only
(DMSO) and compound-treated samples were then fixed and
stained with the nuclear stain, 4’,6-diamidine-2’-phenylindole
dihydrochloride (DAPI), and immunostained with the apoptotic
marker, cleaved fragment of poly (ADP-ribose) polymerase
(cPARP) and the proliferative marker Ki67 (Figure 5). Spheroids
treated with vehicle only (DMSO) were found to have intact
nuclei (i.e. viable cells) as shown with DAPI staining in sections
corresponding to the middle of the spheroids. However, treatment
for 3 hours with 2.5 µM of MAD28, MAD67, and RA25 was
sufficient to induce apoptosis, as shown by the appearance of
pyknotic nuclei (Figure 5; DAPI column, white arrowheads).
These pyknotic nuclei correlate to the loss of well-circumscribed
edges in all three compound-treated samples (Figure 5;
brightfield column, white arrowhead). Pyknotic nuclei were
Conversion of the A-ring phenol to a quinone leads to
complete loss of activity as shown with CR240C (NR) indicating
that the A-ring should maintain its aromatic character. On the
other hand, replacement of hydrogens with fluorines at the A-ring
maintains the CR phenotype but leads to a slight decrease of the
cytotoxicity as shown by comparing the difluorinated analogs
RA25 (IC50= 1.12 µM) and RA25N (IC50= 0.89 µM) to CLV
(IC50= 0.63 µM). The finding that both RA25 and RA25N induce
complete response (CR) and have similar cytotoxicity (IC50= 1.12
µM and 0.89 µM, respectively) indicates that both isomeric
forms of the caged motif have comparable activity. Similar
observations have been reported when comparing other caged
xanthones that contain regular versus neo caged structures.[65,66]

Diels-Alder reaction cascade. The overall strategy proceeds in 5-
detected both in cells on the periphery and AthCe cCenEtePr TofEtDhe MANUSCRIPT
spheroid. This indicates that not only can the compounds
effectively target the peripherally-located proliferative cells but
also the centrally-located, quiescent-prone cells of the spheroids.
Detection of cPARP after treatment with all three compounds,
suggests that three hours of treatment at 2.5 µM is adequate to
activate caspases and irreversible initiation of apoptosis
throughout the spheroid (Figure 5; cPARP column). Staining
with Ki-67, a commonly used proliferation marker, and
fluorescent overlay analysis (i.e. induction of apoptosis vs.
proliferation status) shows that MAD28, MAD67 and RA25
target all cells of the multicellular spheroid and thus are
efficacious irrespective of proliferation status (Figure 5; Ki67
column). This impartial cancer cell targeting is clearly
advantageous over the effect of various FDA-approved
anticancer drugs that mainly target proliferating cells.[40,41,73,74].
6 steps and delivers A-ring modified analogs in good yields.
Structure-activity relationship studies reveal that the cytotoxicity
in the spheroidsMARY-X assay can be regulated by selective
functionalization of the A-ring. Notably, introduction of a free
hydroxyl group at the C6 position leads to compounds that
exhibit complete response in the spheroid assay at low
micromolar concentrations. Specifically, MAD28 and MAD67
were found to induce complete dissolution of the spheroids with
IC50 values of 0.51 and 0.38 µM, respectively. Conversely,
introduction of a free hydroxyl group at C18 as well as further
oxidation of the A-ring leads to a decrease in cytotoxicity as
evidenced with MAD44, MAD69, CR240C and related
structures. Exchange of hydrogen atoms with fluorines at the A-
ring leads to compounds RA25 and RA25N that maintain the
complete response phenotype albeit at a slight increase of the
effective concentration (IC50 values 1.12 and 0.89 µM,
respectively). Additional studies in ECM-immobilized spheroids
showed that MAD28, MAD67 and RA25 effectively target both
proliferative (peripherally located) and quiescent-prone (centrally
located) cells of the multicellular spheroid. Therefore, these
compounds hold promise for the treatment of resistant and
chemo-refractory quiescent cancer cells.[75] Overall, these
findings can be used to accelerate and streamline the
development of IBC-based therapeutics based on the caged
xanthone pharmacophore.
3.
Conclusions
We report here the synthesis of a library of caged xanthones
and their in vitro evaluation in spheroidsMARY-X
, a preclinical
model of inflammatory breast cancer. The synthetic strategy
starts with acid-induced condensation of two appropriately
functionalized aromatic rings to form the central xanthone motif
that is then converted to the caged structure using the Claisen
Figure 5. Analysis of induction of apoptosis in spheroidsMARY-X after 3 hours of treatment with MAD28, MAD67 and RA25 at 2.5
µM. Brightfield microscopy panels show the loss of well-circumscribed spheroid edge upon treatment in comparison to the control
(DMSO). The immunofluorescence microscopy panels show optical slices from the middle of the spheroidsMARY-X and present DAPI
stained nuclei (pyknotic nuclei indicated by arrowheads), localization of c-PARP, Ki-67 and the merged pictures. (bar 20 µm)

4.
Experimental section
Et2O–hexane); 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 9.1 Hz,
ACCEPTED MANUSCRIPT
1H), 7.13 (d, J = 9.0 Hz, 1H), 6.97 (d, JH,F = 9.1 Hz, 1H), 6.78
(t, JH,F = 10.9 Hz, 1H), 6.27 – 6.13 (m, 2H), 5.23 – 5.16 (m,
3H), 5.04 (d, J = 10.8 Hz, 1H), 1.57 (s, 6H), 1.56 (s, 6H); 13C
NMR (100 MHz, CDCl3) δ 174.3, 166.8 (d, JC,F = 15.4 Hz),
164.3 (dd, JC,F = 15.0, 8.1 Hz), 161.7 (d, JC,F = 15.6 Hz), 158.0
(d, JC,F = 15.9 Hz), 157.4, 151.8, 143.7, 143.5, 135.4, 121.3,
117.2, 114.5, 113.4, 101.1 (d, JC,F = 25.1 Hz), 101.0 (t, JC,F =
24.2 Hz), 84.0, 82.6, 27.4, 27.2; HRMS calc. for C23H22F2O4Na
(M + Na)+ 423.1378, found 423.1377.
General information for chemical purification and
compounds characterization. Reactions were monitored by
thin-layer chromatography (TLC) carried out on 0.25 mm E.
Merck silica gel plates (60F-254) and visualized under UV light
and/or by treatment with a solution of CAM or KMnO4 stain
followed by heating. Preparative thin-layer chromatography
separations were carried out on 0.25 or 0.50 mm E. Merck silica
gel plates (60F-254). Flash column chromatography was
performed on silica gel (Merck Kieselgel 60, 230-400 mesh)
using hexane/ethyl acetate or hexane/ethyl ether as standard
eluents. 1H NMR and 13C NMR spectra were recorded on a 400
or 500 MHz Varian or a 500 JEOL instrument. Chemical shifts
(δ) are quoted in parts per million (ppm) referenced to the
appropriate residual undeuterated solvent peak, with the
abbreviations s, bs, d, t, q, dd, m, denoting singlet, broad singlet,
doublet, triplet, quartet, doublet of doublets, multiplet,
respectively. J is a coupling constant given in Hertz (Hz). High
resolution mass spectra (HRMS) were recorded on a VG7070HS
mass spectrometer under chemical ionization (CI) conditions, on
General procedure for Claisen/Diels-Alder reaction. A
solution of allylated compound (1 equiv) in DMF was heated at
120 °C. The onset of a yellow color indicated the formation of
the caged xanthone. The reaction progress was monitored by
TLC. Upon completion, the yellow reaction mixture was cooled
to room temperature and the solvent was removed under vacuum
by rotary evaporation. The crude material was purified by flash
column chromatography (silica, EtOAc or Et2O in hexane) to
yield the caged xanthone.
8-hydroxy-10-(methoxymethoxy)-2,2-dimethyl-3a-(3-
methylbut-2-en-1-yl)-1,2-dihydro-1,5-methanofuro[2,3-
d]xanthene-4,7(3aH,5H)-dione (12): 69%, yellow solid. Rf =
0.24 (25% EtOAc–hexane); 1H NMR (400 MHz, CDCl3) δ 12.38
(s, 1H), 7.43 (d, J = 7.1 Hz, 1H), 6.20 (d, J = 2.1 Hz, 1H), 6.17
(d, J = 2.1 Hz, 1H), 5.19 (s, 2H), 4.45 – 4.41 (m, 1H), 3.51 – 3.48
(m, 1H), 3.47 (s, 3H), 2.61 (d, J = 8.8 Hz, 2H), 2.43 (d, J = 9.6
Hz, 1H), 2.33 (dd, J = 13.6, 4.7 Hz, 1H), 1.68 (s, 3H), 1.38 (s,
3H), 1.32 – 1.31 (m, 1H), 1.29 (s, 3H), 1.09 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 203.2, 179.7, 166.1, 164.9, 161.1, 135.4,
134.2, 133.9, 118.5, 101.8, 97.2, 95.7, 94.1, 90.4, 84.6, 83.7,
56.7, 49.0, 47.0, 30.6, 29.9, 29.3, 25.8, 25.3, 17.0; HRMS calc.
for C25H28O7Na (M + Na)+ 463.1727, found 463.1729.
a
VG ZAB-ZSE mass spectrometer under fast atom
bombardment (FAB) conditions, or on a Bruker microTOF mass
spectrometer under electrospray ionization (ESI) conditions.
Specific information on the synthetic/analytical protocols as well
as copies of the spectroscopic data for all compounds are shown
in the Supporting Information.
General
procedure
for
Pd(0)-catalyzed
reverse
prenylation. A solution of starting xanthone (1 equiv) in
anhydrous THF (approximate 0.2 mM) was degassed by argon
and was placed in an ice bath. To this homogeneous solution was
added tert-butyl 2-methylbut-3-en-2-yl carbonate (9) or bis(2-
methylbut-3-en-2-yl) carbonate (30) (10 equiv) via syringe,
followed by 10% mol Pd(PPh3)4. The mixture was stirred under
argon at 0 to 5 °C and the reaction progress was monitored by
TLC. Upon completion the reaction mixture was quenched with
water and extracted with EtOAc. The combined organic layers
were dried over MgSO4, filtered and concentrated by rotary
evaporation. The crude material was purified through flash
column chromatography (silica, EtOAc in hexane) to yield di-
allylated product.
8,11-dimethoxy-2,2-dimethyl-3a-(3-methylbut-2-en-1-yl)-
1,2-dihydro-1,5-methanofuro[2,3-d]xanthene-4,7(3aH,5H)-
dione (23): 88%, yellow solid. Rf = 0.40 (50% EtOAc–hexane);
1H NMR (400 MHz, CDCl3) δ 7.31 (d, J = 6.9 Hz, 1H), 7.04 (d, J
= 9.0 Hz, 1H), 6.47 (d, J = 9.0 Hz, 1H), 4.52 (t, J = 7.9 Hz, 1H),
3.88 (s, 3H), 3.85 (s, 3H), 3.44 (dd, J = 7.0, 4.2 Hz 1H), 2.61 (d, J
= 9.0 Hz, 2H), 2.40 (d, J = 9.6 Hz, 1H), 2.32 (dd, J = 13.5, 4.6
Hz, 1H), 1.78 (s, 3H), 1.39 (s, 3H), 1.30 (s, 3H), 1.30 – 1.27 (m,
1H), 1.09 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 203.3, 176.1,
154.3, 151.2, 143.0, 136.4, 132.5, 118.7, 118.5, 110.9, 102.8,
90.0, 84.5, 83.6, 57.0, 56.4, 48.5, 46.7, 29.5, 29.1, 29.0, 25.6,
25.4, 16.9; HRMS calc. for C25H28O6Na (M + Na)+, 447.1778
found 447.1776.
1-hydroxy-3-(methoxymethoxy)-5,6-bis((2-methylbut-3-en-
2-yl)oxy)-9H-xanthen-9-one (10): 89%, a colorless oil. Rf =
0.57 (25% EtOAc–hexane); H NMR (400 MHz, CDCl3) δ 7.82
1
(d, J = 9.0 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 6.56 (d, J = 2.2 Hz,
1H), 6.46 (d, J = 2.2 Hz, 1H), 6.29 – 6.14 (m, 2H), 5.25 (s, 2H),
5.21 – 5.15 (m, 3H), 5.03 (d, J = 10.9 Hz, 1H), 3.51 (s, 3H), 1.57
(s, 9H), 1.57 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 180.9,
164.0, 163.5, 157.5, 152.5, 143.7, 143.5, 120.4, 116.8, 114.4,
113.4, 98.8, 94.9, 94.4, 83.9, 82.5, 56.7, 27.4, 27.1; HRMS calc.
for C25H29O7 (M + H)+ 441.1988, found 441.1907.
8-hydroxy-11-methoxy-2,2-dimethyl-3a-(3-methylbut-2-en-
1-yl)-1,2-dihydro-1,5-methanofuro[2,3-d]xanthene-
4,7(3aH,5H)-dione (24): 72%, yellow solid. Rf = 0.50 (50%
1
EtOAc–hexane); H NMR (400 MHz, CDCl3) δ 11.55 (s, 1H),
7.49 (d, J = 7.0 Hz, 1H), 7.12 (dd, J = 8.9, 1.8 Hz, 1H), 6.47 (dd,
J = 8.9, 1.8 Hz, 1H), 4.43 – 4.39 (m, 1H), 3.84 (s, 3H), 3.55 –
3.52 (m, 1H), 2.64 (d, J = 9.5 Hz, 2H), 2.47 (d, J = 9.6 Hz, 1H),
2.36 (dd, J = 13.6, 4.2 Hz, 1H), 1.78 (s, 3H), 1.36 (s, 3H), 1.31 (s,
4H), 1.00 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 202.8, 181.6,
156.2, 149.5, 140.7, 135.4, 135.0, 134.0, 124.1, 119.0, 107.6,
106.5, 90.4, 84.7, 84.0, 57.9, 49.0, 47.1, 29.8, 29.4, 29.3, 25.7,
24.9, 16.8; HRMS calcd for [C24H26O6Na]+ (M + Na)+ 433.1622,
found 433.1623.
1,4-dimethoxy-5,6-bis((2-methylbut-3-en-2-yl)oxy)-9H-
xanthen-9-one (22): 77%, a colorless oil. Rf = 0.43 (50%
1
EtOAc–hexane); H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 9.1
Hz, 1H), 7.15 (d, J = 8.9 Hz, 1H), 7.08 (d, J = 9.0 Hz, 1H), 6.69
(d, J = 9.0 Hz, 1H), 6.41 (dd, J = 10.7, 17.6 Hz, 1H), 6.19 (dd, J =
10.8, 17.6 Hz, 1H), 5.21 – 5.13 (m, 3H), 4.97 (d, J = 10.9 Hz,
1H), 3.96 (s, 3H), 3.95 (s, 3H), 1.61 (s, 6H), 1.55 (s, 6H); 13C
NMR (100 MHz, CDCl3) δ 176.4, 156.2, 153.6, 151.2, 148.0,
143.8, 143.7, 142.6, 135.8, 120.8, 117.2, 116.2, 113.9, 113.0,
104.2, 83.9, 82.0, 56.7, 56.5, 27.1, 26.7; HRMS calc. for
C25H28O6Na (M + Na)+, 447.1778 found 447.1777.
8,10-difluoro-2,2-dimethyl-3a-(3-methylbut-2-en-1-yl)-1,2-
dihydro-1,5-methanofuro[2,3-d]xanthene-4,7(3aH,5H)-dione
1
(32): 64% yellow solid; Rf = 0.38 (50% Et2O–hexane); H NMR
1,3-difluoro-5,6-bis((2-methylbut-3-en-2-yl)oxy)-9H-
(400 MHz, CDCl3) δ 7.42 (d, J = 6.9 Hz, 1H), 6.60 (d, JH,F = 9.5
xanthen-9-one (31): 100%, a colorless oil. Rf = 0.56 (33 %
Hz, 1H), 6.53 (t, JH,F = 10.9 Hz, 1H), 4.46 (t, J = 6.9 Hz, 1H),

3.52 – 3.49 (m, 1H), 2.60 (d, J = 8.3 Hz, 2H), 2.44 (d, J = 10.0
conjugated anti-Ki67 (BD Pharmingen, #558616) and cPARP
(Promega, #G7341) at 1:10 and 1:100, respectively. Incubation
with Cy5 conjugated goat anti-rabbit (Invitrogen, A10523)
secondary at 4 µg/ml followed at 4°C for 24 hours. After three
washes in 1x PBS, a drop of Vectashield mounting medium with
DAPI (Vector Laboratories, H-1200) was applied on top of the
Matrigel plugs with the treated cells and then the coverslips were
flipped and sealed on slides. The slides were visualized and
subjected to z-stacking using a Zeiss AxioObserver Z1 with
ApoTome2 inverted microscope.
ACCEPTED MANUSCRIPT
Hz, 1H), 2.34 (dd, J = 13.6, 4.6 Hz, 1H), 1.67 (s, 3H), 1.40 (s,
3H), 1.35 – 1.31 (m, 1H), 1.30 (s, 3H), 1.09 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 202.7, 173.5, 168.2 (d, JC,F = 16.0 Hz),
165.3 (dd, JC,F = 77.3, 15.9 Hz), 162.2 (d, JC,F = 16.0 Hz),
161.8 (dd, JC,F = 15.6, 5.5 Hz), 135.2, 134.7, 134.5, 118.9, 101.5
(dd, JC,F = 24.6, 4.4 Hz), 99.4 (t, JC,F = 25.5 Hz), 91.4, 84.6,
83.7, 49.0, 47.0, 29.9, 29.3, 29.2, 25.7, 25.5, 17.2; HRMS calc.
for C23H22F2O4Na (M + Na)+ 423.1378, found 423.1373.
6,8-difluoro-2,2-dimethyl-11a-(3-methylbut-2-en-1-yl)-
2,3,3a,11a-tetrahydro-3,10a-methanofuro[3,2-b]xanthene-
5,11-dione (33): 3%, yellow solid; Rf = 0.48 (50% Et2O–
hexane); 1H NMR (400 MHz, CDCl3) δ 7.22 (d, J = 7.0 Hz, 1H),
6.74 (d, JH,F = 9.6 Hz, 1H), 6.52 (t, JH,F = 11.0 Hz, 1H), 5.01 (t,
J = 6.9 Hz, 1H), 3.76 (dd, J = 6.9, 4.7 Hz,1H), 2.50 – 2.46 (m,
2H), 2.19 (dd, J = 9.9, 4.5 Hz, 1H), 2.12 – 2.04 (m, 1H), 1.87 (dd,
J = 13.2, 10.0 Hz, 1H), 1.72 (s, 3H), 1.60 (s, 3H), 1.38 (s, 3H),
1.35 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 199.6, 172.8, 168.5
(d, JC,F = 15.8 Hz), 165.9 (d, JC,F = 16.1 Hz), 162.7 (m), 162.2
(m), 136.7, 135.6, 131.8, 117.4, 102.0 (dd, JC,F = 24.9, 4.2 Hz),
99.7 (t, JC,F = 20.0 Hz), 85.0, 84.1, 79.2, 44.9, 42.5, 32.9, 30.3,
29.8, 26.9, 26.1, 18.4; HRMS calc. for C23H22F2O4Na (M + Na)+
423.1378, found 423.1379.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgment
We thank the California Breast Cancer Research Program
(IDEA Award No: 22IB-0024). We thank the Thailand Research
Fund for a New Researcher Grant (TRG5780085) to Dr. O.
Chantarasriwong and Kuwait University for a Fellowship to B.D.
Althufairi. This work was also supported by the Ellington
Beavers Award from Arcadia University. We also thank Teresa
Abendroth for editorial assistance.
Culture of spheroidsMARY-X
.
The spheroidsMARY-X were
cultured in Minimal Essential Medium (MEM) supplemented
with 10% heat-inactivated fetal bovine serum and antibiotics
(100 U/ml penicillin and 100 µg/ml streptomycin) at 37°C in an
air -5% CO2 atmosphere at constant humidity.
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Supplementary Material
The Supplementary Material contains detailed experimental
procedures, spectroscopic and analytical data as well as copies of
1H and 13C NMR of all synthetic compounds. It also contains
information on the drug response measurements in spheroidsMARY-
X
(Fig. S1) and a complete panel of dose response analysis of
spheroidsMARY-X (Fig. S2).

ACCEPTED MANUSCRIPT
•
Inflammatory breast cancer (IBC) is a highly metastatic and lethal subtype of breast
cancer with no known pharmacological treatment.
•
•
A synthetic library of caged xanthones allows structure-activity studies in an IBC model.
A-ring modified caged xanthones are potent against spheroidsMARY-X an in vitro model of
IBC.
•
Synthetic caged xanthones induce complete dissolution of spheroidsMARY-X and cell
apoptosis.
•
The caged xanthone motif represents a potent drug lead for the design of IBC-targeting
therapeutics.