Synthetic and Biological Studies of Sesquiterpene Polygodial: Activity of 9-Epipolygodial against Drug-Resistant Cancer Cells

Article abstract of DOI:10.1002/cmdc.201500360

Polygodial, a terpenoid dialdehyde isolated from Polygonum hydropiper L., is a known agonist of the transient receptor potential vanilloid 1 (TRPV1). In this investigation a series of polygodial analogues were prepared and investigated for TRPV1-agonist and anticancer activities. These experiments led to the identification of 9-epipolygodial, which has antiproliferative potency significantly exceeding that of polygodial. 9-Epipolygodial was found to maintain potency against apoptosis-resistant cancer cells as well as those displaying the multidrug-resistant (MDR) phenotype. In addition, the chemical feasibility for the previously proposed mechanism of action of polygodial, involving the formation of a Paal-Knorr pyrrole with a lysine residue on the target protein, was demonstrated by the synthesis of a stable polygodial pyrrole derivative. These studies reveal rich chemical and biological properties associated with polygodial and its direct derivatives. These compounds should inspire further work in this area aimed at the development of new pharmacological agents, or the exploration of novel mechanisms of covalent modification of biological molecules with natural products.

Full text of DOI:10.1002/cmdc.201500360

DOI: 10.1002/cmdc.201500360
Full Papers
Synthetic and Biological Studies of Sesquiterpene
Polygodial: Activity of 9-Epipolygodial against Drug-
Resistant Cancer Cells
Ramesh Dasari,[a] Annelise De Carvalho,[b] Derek C. Medellin,[a] Kelsey N. Middleton,[a]
FrØdØric Hague,[c] Marie N. M. Volmar,[d] Liliya V. Frolova,[e] Mateus F. Rossato,[f]
Jorge J. De La Chapa,[g] Nicholas F. Dybdal-Hargreaves,[h] Akshita Pillai,[i] VØronique Mathieu,[b]
Snezna Rogelj,[e] Cara B. Gonzales,[g] Jo¼o B. Calixto,[f] Antonio Evidente,[j] Mathieu Gautier,[c]
Gnanasekar Munirathinam,[i] Rainer Glass,[d] Patricia Burth,[k] Stephen C. Pelly,[l] Willem
A. L. van Otterlo,[l] Robert Kiss,[b] and Alexander Kornienko*[a]
Polygodial, a terpenoid dialdehyde isolated from Polygonum
hydropiper L., is a known agonist of the transient receptor po-
tential vanilloid 1 (TRPV1). In this investigation a series of poly-
godial analogues were prepared and investigated for TRPV1-
agonist and anticancer activities. These experiments led to the
identification of 9-epipolygodial, which has antiproliferative
potency significantly exceeding that of polygodial. 9-Epipoly-
godial was found to maintain potency against apoptosis-resist-
ant cancer cells as well as those displaying the multidrug-re-
sistant (MDR) phenotype. In addition, the chemical feasibility
for the previously proposed mechanism of action of polygo-
dial, involving the formation of a Paal–Knorr pyrrole with
a lysine residue on the target protein, was demonstrated by
the synthesis of a stable polygodial pyrrole derivative. These
studies reveal rich chemical and biological properties associat-
ed with polygodial and its direct derivatives. These compounds
should inspire further work in this area aimed at the develop-
ment of new pharmacological agents, or the exploration of
novel mechanisms of covalent modification of biological mole-
cules with natural products.
Introduction
Polygodial (1, Figure 1) is a bicyclic sesquiterpene first isolated
as a pungent component of the sprout of Polygonum hydropi-
per L. (Polygonaceae), a plant once used as a pepper substitute
in Europe and still a popular condiment for sashimi in Japan.[1]
It is a member of a family of more than 80 terpenoids contain-
ing an a,b-unsaturated 1,4-dialdehyde functionality; these
[a] Dr. R. Dasari, D. C. Medellin, K. N. Middleton, Prof. A. Kornienko
Department of Chemistry and Biochemistry
Texas State University, San Marcos, TX 78666 (USA)
E-mail: a_k76@txstate.edu
[g] J. J. De La Chapa, Prof. C. B. Gonzales
Department of Comprehensive Dentistry
Cancer Therapy and Research Center
University of Texas Health Science Center at San Antonio
San Antonio, TX 78229 (USA)
[b] A. De Carvalho, Prof. V. Mathieu, Prof. R. Kiss
Laboratoire de CancØrologie et de Toxicologie ExpØrimentale
FacultØ de Pharmacie, UniversitØ Libre de Bruxelles, 1050 Brussels (Belgium)
[h] N. F. Dybdal-Hargreaves
Department of Pharmacology
University of Texas Health Science Center at San Antonio
San Antonio, TX 78229 (USA)
[c] Dr. F. Hague, Dr. M. Gautier
Laboratoire de Physiologie Cellulaire et MolØculaire, FacultØ des Sciences
UniversitØ de Picardie Jules Verne, 80000 Amiens (France)
[i] A. Pillai, Dr. G. Munirathinam
Department of Biomedical Sciences, College of Medicine
University of Illinois, 1601 Parkview Ave., Rockford, IL 61107 (USA)
[d] M. N. M. Volmar, Prof. R. Glass
Neurosurgical Research, University Clinics Munich
Marchioninistr. 15, 81377 Munich (Germany)
[j] Prof. A. Evidente
Dipartimento di Scienze Chimiche, Università di Napoli Federico II
Complesso Universitario Monte Sant’Angelo
Via Cintia 4, 80126 Napoli (Italy)
[e] Prof. L. V. Frolova, Prof. S. Rogelj
Departments of Chemistry and Biology
New Mexico Institute of Mining and Technology
801 Leroy Place, Socorro, NM 87801 (USA)
[k] Prof. P. Burth
Departamento de Biologia Celular e Molecular
Instituto de Biologia, Universidade Federal Fluminense
Outeiro de S¼o Jo¼o Batista, s/n Campus do Valonguinho
Centro-Niterói, RJ 24020-140 (Brazil)
[f] Dr. M. F. Rossato, Prof. J. B. Calixto
Center of Innovation and Preclinical Studies
Av. Luiz Boiteux Piazza 1302, Cachoeira do Bom Jesus
Florianópolis, SC 88056-000 (Brazil)
and
[l] Dr. S. C. Pelly, Prof. W. A. L. van Otterlo
Department of Pharmacology
Federal University of Santa Catarina, Florianópolis, SC (Brazil)
Department of Chemistry and Polymer Science, Stellenbosch University
Stellenbosch, Private Bag X1, Matieland, 7602 (South Africa)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500360.
ChemMedChem 2015, 10, 2014 – 2026
2014
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
the discovery of polygodial’s vanilloid activity and its potential
use as an anti-nociceptive that has generated recent enthusi-
asm in the scientific literature.[13–18] In a manner similar to cap-
saicin, a pungent component of hot chili pepper used as
a spice in the culinary traditions of many cultures, polygodial
was found to inhibit the pain response invoked by formalin in-
jection and to block acetic acid induced writhing in mice.[19] To
produce their nociceptive activities, polygodial, capsaicin, and
the other vanilloids are believed to target the transient recep-
tor potential vanilloid 1 (TRPV1), a temperature-sensitive ion
channel with preference for Ca2+ ions.[20–24] Further research
has shown that in addition to its expression in sensory neurons
and involvement in different modalities of pain, TRPV1 is also
upregulated in various human cancer cells,[25–27] and its activa-
tion in human glioma cells leads to endoplasmic reticulum
stress followed by cell death.[28] TRPV1 thus appears to be
a promising target for cancer drug development, and there are
many reports of studies investigating TRPV1 ligands, such as
the TRPV1 agonists capsaicin[29–31] and resiniferatoxin,[27,32] as
well as the TRPV1 antagonists capsazepine[29,30] and
SB366791,[29] as potential anticancer agents. However, the re-
sults of mechanistic studies have complicated matters by ques-
tioning whether the anticancer effects of vanilloids are mediat-
ed by TRPV1, and have revealed that TRPV1 antagonists gener-
ally do not prevent vanilloid agonist-induced cell death.[27,29–32]
Despite several reports of cytotoxic activity associated with
polygodial,[33–39] to our knowledge, this TRPV1 agonist or relat-
ed bicyclic sesquiterpene dialdehydes have not been investi-
gated as potential anticancer agents. This report details our
synthetic study of polygodial (1), the generation of a series of
polygodial analogues, and the evaluation of the synthesized
compounds for TRPV1 and anticancer activities. This work led
to the discovery of 9-epipolygodial (2) as a promising agent
against drug-resistant cancer and polygo-11,12-diol as a non-
cytotoxic TRPV1 agonist. In addition, our study concludes that
the anticancer effects of this series of compounds are also
non-TRPV1-mediated, paralleling the previous findings with
other TRPV1 agonists.
Figure 1. Structures of selected a,b-unsaturated 1,4-dialdehyde terpenoids
and a proposed pyrrole adduct of 1 with methylamine (compound 4).
compounds have been isolated from a variety of natural sour-
ces including terrestrial plants, fungi, algae, liverworts, arthro-
pods, sponges, and mollusks.[2] Some additional members of
this family are shown in Figure 1 and are believed to protect
the producing organisms from predators.[2,3] Indeed, a signifi-
cant number of early biological investigations involving these
dialdehydes focused on their hot taste to the human tongue
and antifeedant activities, both of which appeared to depend
on the configuration of the aldehyde group at C9. Specifically,
these studies found that polygodial (1) possesses potent anti-
feedant activity against African armyworms (Spodoptera exemp-
ta)[4] and fish[5] and tastes hot to the human tongue.[6] In con-
trast, 9-epipolygodial (2) is tasteless to humans and devoid of
antifeedant activity toward insects[4] or fish.[5]
The antifeedant effects of polygodial and related bicyclic
sesquiterpenes with a b configuration at C9 have been theor-
ized to arise from their covalent interaction with receptors in-
volved in taste perception. Electrophysiological studies re-
vealed that when the maxillary palp (equivalent to taste buds)
of S. exempta larva is repeatedly brought into contact with
filter paper infused with warburganal (3, a related dialdehyde
shown in Figure 1) the sense of taste is irreversibly blocked. As
a consequence of this irreversible blockage, armyworms placed
on a warburganal-treated maize leaf and subsequently trans-
ferred to an untreated leaf starve to death.[7]
The formation of covalent adducts of polygodial with bio- Results and Discussion
logical molecules involved in taste perception has been pro-
posed to occur through either a reaction with thiol[6] or
Chemistry
amine[8] groups. Furthermore, based on NMR spectroscopic
monitoring of the reaction of 1 with methylamine in phos-
phate buffer at pH 9, the formation of pyrrole adduct 4
(Figure 1) was proposed.[8–10] However, to our knowledge, no
pyrrole adduct from the reaction of polygodial with primary
amines has been isolated and characterized; therefore, the fea-
sibility of such processes in chemical model systems remains
to be demonstrated.
The Paal–Knorr condensation of primary amines with 1,4-dicar-
bonyl compounds is a well-studied classical pyrrole synthe-
sis.[40] The biological significance of this reaction, however, ap-
pears to be severely underappreciated despite the wide occur-
rence of the 1,4-dicarbonyl functionality in natural products
that could potentially react with lysine residues on proteins.
The most well-studied example of the biological relevance of
the Paal–Knorr reaction is its involvement in n-hexane-induced
axonal atrophy in the central nervous system. It has been dem-
onstrated both in vitro and in vivo that 2,5-hexanedione, a neu-
rotoxic n-hexane metabolite, undergoes a selective Paal–Knorr
condensation with lysine residues of axonal cytoskeleton pro-
teins, forming 2,5-dimethylpyrrole adducts within specific re-
gions of neurofilaments (Scheme 1A).[41] To our knowledge, the
Later studies revealed anti-allergic and anti-inflammatory ac-
tivities[11] associated with polygodial. Moreover, the discovery
of its antifungal properties has led to its therapeutic use to
control localized candidiasis (Kolorex capsules).[12] Notably,
except for mild stomach discomfort and dizziness, polygodial
is well tolerated by the majority of patients.[13] However, it was
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2015
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Scheme 2. Reaction mechanism of the modified Paal–Knorr pyrrole conden-
sation of 1 with primary amines.
Chemical derivatization of 1 included two previously de-
scribed orthogonal reduction processes,[43,44] one with sodium
borohydride to give polygo-11,12-diol (6), the other with
Raney nickel to yield dihydro analogue 8, which was present
as an equilibrating mixture of dialdehyde 8a and lactol 8b
(Scheme 3). Next, it was found that the reaction with hydrazine
produced pyridazine 7, whereas acid-catalyzed enolization/epi-
merization gave the C9-epimeric mixture of 1 and 2, from
which 9-epipolygodial (2) was isolated in 40% yield. The C12-
Scheme 1. A) Paal–Knorr pyrrole formation implicated in the neurotoxicity of
hexane and B) proposed lysine pyrrolylation by 1 and chemical demonstra-
tion of its feasibility.
only other demonstrated exam-
ple of lysine pyrrolylation with
a 1,4-dicarbonyl compound in-
volves lysozyme modification
with spongian diterpenes, which
is responsible for the Golgi-mod-
ifying properties of these marine
natural products.[42]
To support the intriguing pro-
posal that a modified Paal–Knorr
condensation with
a
target
lysine residue is responsible for
the covalent interaction of poly-
godial with biological macromo-
lecules as described above, com-
pound
1
was reacted with
benzyl amine under mild acid
catalysis (Scheme 1B). Although
TLC and MS monitoring of the
Scheme 3. General derivatization of polygodial.
reaction mixture indicated the
possible formation of pyrrole 5a
(R=Bn), attempts to isolate it
were unsuccessful, likely due to the susceptibility of the vinyl
pyrrole functionality to oxidation. This problem was solved by
using aniline and p-nitroaniline to make the resulting pyrrole
ring system electron deficient, and in the latter case pyrrole 5c
(R=p-NO2-C6H5) was successfully isolated as a stable polygo-
dial derivative. Scheme 2 delineates a possible mechanism for
this modified Paal–Knorr condensation, which likely involves
attack by the primary amine at the more reactive C12-aldehyde
to give imine A, which tautomerizes to dienamine B, eventual-
ly leading to the irreversible formation of pyrrole 5.
aldehyde group in 2 proved to be more reactive and was se-
lectively converted into an unsaturated ester in 9 upon a reac-
tion with methyl (triphenylphosphoranylidene)acetate. Finally,
compound 2 was found to undergo the Paal–Knorr pyrrole for-
mation in a manner similar to epimeric 1 (Scheme 3).
Previous studies[34] also suggested that the reactive aldehyde
groups in polygodial (1) could be masked by the formation of
cyclic bis-acetals (Scheme 4). Indeed, when 1 was treated with
various alcohols in the presence of p-toluenesulfonic acid, ace-
tals 10–16 were produced as mixtures of two diastereomers
with configurations at the C11 and C12 positions as shown for
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2016
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
shows that common chemotherapeutic agents that work by in-
duction of apoptosis rapidly eliminate a certain population of
sensitive cells, thus generating low GI50 values. However, these
high potencies can be misleading, as the remaining cells can
resist the effects of chemotherapeutic agents even at concen-
trations 100- or 1000-fold higher than their GI50 values.[46,50] It
was thus instructive to learn that compound 2 eliminates all
cells in the cultures and generates no resistant populations
(close to 0% cell viability) at concentrations just slightly ex-
ceeding its GI50 values (Figure 2A). The contrasting effects on
cell viability between 2 and the chemotherapeutic agents pa-
clitaxel and podophyllotoxin are shown in Figure 2B and 2C.
As can be seen, paclitaxel and podophyllotoxin have no effect
on the proliferation of ~50% of cells in U87 GBM and A549
NSCLC cell cultures at concentrations up to 50 mm, whereas 2
exhibited potent growth inhibitory properties against most of
the cells in these cultures, and, with increasing concentration,
rapidly reached the antiproliferative levels of an indiscriminate
cytotoxic agent, phenyl arsine oxide (PAO). Compound 2 dem-
onstrated similar behavior in docetaxel-resistant SCC4 and cis-
platin-resistant SCC25 human oral cancer cell lines, as well as
docetaxel-resistant PC-3 human prostate cells (data not
shown).
Scheme 4. Synthesis of cyclic bis-acetals 10a,b–16a,b.
a and b (Scheme 4). The idea behind the synthesis of a series
of such analogues was to facilitate the selective pyrrole forma-
tion at polygodial’s target site. Although aldehyde-containing
compounds do not present any particular metabolic concerns
and are often present in clinical drugs (e.g., orally bioavailable
male contraceptive gossypol, a dialdehyde also currently stud-
ied in cancer clinical trials[45]), moderating the reactivity of the
dialdehyde functionality could be an attractive tool to improve
the pharmacokinetic properties of such compounds. Indeed,
with the right reactivity–stability balance of such cyclic bis-ace-
tals, the pyrrole formation at the target protein’s active site
would still be possible, yet the wasteful nonspecific reactions
with endogenous free amines and accessible lysine residues
on abundant proteins could be minimized.
In contrast to the intrinsic resistance described above,
tumors often initially respond to chemotherapy, but eventually
become refractory to continued treatment. In such an instance
of acquired resistance, cancer cells usually develop a multi-
drug-resistant (MDR) phenotype, affecting a broad spectrum of
structurally and mechanistically diverse antitumor agents.[51,52]
The phenomenon of MDR has plagued conventional therapy
with vinca alkaloids,[52] for example, or taxanes,[53] and it was
therefore of interest to evaluate 2 against MDR cells. The MDR
uterine sarcoma cell line MES-SA/Dx5 is resistant to multiple
functionally and structurally unrelated molecules,[54] and it was
established by growing the parent uterine sarcoma MES-SA
Antiproliferative activities
The synthesized compounds were evaluated for in vitro
growth inhibition using the colorimetric MTT assay against
a panel of five cancer cell lines including apoptosis-resistant
human glioblastoma (GBM) U373,[46] human A549 non-small-
cell lung cancer (NSCLC),[47] and human SKMEL-28
melanoma,[48] as well as apoptosis-sensitive human
Hs683 anaplastic oligodendroglioma[46] and human
Table 1. In vitro growth inhibitory effects of polygodial derivatives.
MCF7 breast cancer.[49] Analysis of these data reveals
that polygodial (1) and most of the synthesized deriv-
atives displayed little activity in this cancer cell line
panel, with the exception of 9-epipolygodial (2). Com-
pound 2 was at least 20-fold more potent than poly-
godial 1 and did not appear to discriminate between
apoptosis-resistant and apoptosis-sensitive cells by
displaying similar single-digit micromolar potencies in
both cell types (Table 1). Notably, the potent activity
of compound 2 was initially surprising given its recent
evaluation against a panel of cancer cell lines by Mon-
tenegro et al., who reported no activity associated
with this compound up to 200 mm.[39] However, we re-
tested this compound using multiple repeats with dif-
ferent assays across a large number of different cell
lines, as can be seen below, and thus we are confi-
dent in our conclusions.
Compd
GI50 [mm][a]
A549
SKMEL-28
MCF7
U373
Hs683
Mean
1
2
5
6
7
8
9
84Æ9
65Æ3
3Æ0.1
>100
>100
>100
72Æ1
70Æ1
77Æ2
30Æ1
56Æ2
>100
>100
>100
>100
11 Æ1
49Æ3
75Æ2
2Æ0.1
66Æ3
93Æ3
72Æ3
25Æ1
28Æ1
77Æ2
38Æ3
39Æ2
81Æ3
83Æ3
66Æ3
99Æ6
95Æ1
84Æ6
4Æ1
6Æ0.1
5Æ0.2
6Æ0.2
98Æ2
>100
>100
>100
>100
>100
82Æ2
>100
>89Æ7
>99Æ1
>93Æ5
50Æ12
40Æ8
93Æ2
33Æ1
33Æ0.5
88Æ3
56Æ2
30Æ0.3
83Æ2
78Æ4
97Æ4
35Æ1
86Æ3
29Æ1
96Æ3
64Æ2
28Æ1
39Æ1
84Æ2
29Æ1
42Æ1
10a
10b
11 a,b
12a
13a
13b
14b
15a,b
16a,b
84Æ4
43Æ7
39Æ5
>100
>100
>93Æ4
>92Æ5
>93Æ7
>100
>100
>100
>100
>100
>100
>100
>100
>100
69Æ4
33Æ1
22Æ3
35Æ2
34Æ10
41Æ1
34Æ1
41Æ1
28Æ1
38Æ3
[a] Concentration required to decrease cell viability by 50% relative to control after
72 h treatment with the indicated compounds as determined by MTT assay; values
are the meanÆSEM of sextuplicates of each experiment, performed once.
Our experience in working with cells that are in-
trinsically resistant to various pro-apoptotic stimuli
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2017
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
sphere conditions known to pro-
mote the growth of stem-like
cells from human glioma tissue.
Neurospheres have been shown
to recapitulate human gliomas
on both histological and genetic
levels more faithfully than
serum-cultured glioma cell lines
when injected into the brains of
mice,[55–58] and they are generally
resistant to radiation and che-
motherapy.[59–62] The small neuro-
sphere cell culture panel chosen
for this study included cells car-
rying a tumor-suppressor cyclin-
dependent kinase inhibitor 2A
(cdkn2a) deletion[63] as well as
epidermal growth factor recep-
tor variant III (EGFRvIII)[64] and
platelet-derived growth factor
subunit (PDGFB)[65] amplifica-
tions, representing frequent mu-
tations in high-grade astrocytic
tumors. The data shown in
Figure 3 indicate that compound
2 used at 4 mm (average GI50 in
Table 1) is as toxic to these cells
as cannabidiol (CBD) at 10 mm,
an orphan drug advanced to
phase II clinical trials for the
treatment of GBM.
Vanilloid activities
Figure 2. A) Elimination of all cells in all five cultures tested with analogue 2, and contrasting effects on the viabili-
ty of all cells between 2 and standard chemotherapeutic agents paclitaxel and podophyllotoxin (PODO) in B) U87
GBM and C) A549 NSCLC cell cultures.
To evaluate the affinities of the
synthesized polygodial deriva-
tives for the vanilloid site of
cell line in the presence of increasing concentrations of doxo-
rubicin. Paclitaxel (Table 2) and vinblastine lost their potency
by 1000-fold when tested for antiproliferative activity against
the MDR cell line relative to the parent line. In contrast, there
was little variation in the sensitivities of the two cell lines
toward 2 (Table 2).
TRPV1, the compounds were assessed for their inhibition of
specific binding of [3H]resiniferatoxin (RTX) in rat spinal cord
membranes.[16] The results (Figure 4) demonstrate that at
10 mm, polygodial (1) displayed 76% inhibition, whereas
polygo-11,12-diol (6), 7,8-dihydropolygodial (8), and unsaturat-
ed ester 9 turned out to be more potent in this assay and
showed 94, 97, and 93% inhibition, respectively. The lack of ac-
tivity of 9-epipolygodial (2), the most promising analogue
active against drug-resistant cancer, was initially surprising.
However, this result is consistent with earlier findings that this
natural product is tasteless to humans and devoid of antifee-
dant activity,[3,4] strongly suggesting that the biological effects
of 2 are not mediated by TRPV1.
The ability of 2 to overcome drug resistance was further
evaluated using glioma cell cultures maintained under neuro-
Table 2. Antiproliferative effects of 2 against MDR cells.
Compd
GI50 [mm][a]
MES-SA
MES-SA/Dx5
In a complementary assessment of TRPV1 activities, meas-
urements of Ca2+ entry into MDA-MB-231 breast cancer cells
abundantly expressing TRPV1 receptors[66] were performed
(Figure 5). In a manner similar to capsaicin (Figure 5A), polygo-
dial (1) at its average GI50 concentration of 80 mm (from
Table 1) caused [Ca2+]i increase in these assays (Figure 5B).
This activity was completely inhibited with the selective TRPV1
paclitaxel
vinblastine
2
0.007Æ0.001
0.006Æ1
9.8Æ0.3
5.0Æ1.4
6.8Æ0.4
10.7Æ0.7
[a] Concentration required to decrease cell viability by 50% relative to
DMSO control after 48 h treatment with the indicated compounds as de-
termined by MTT assay; values are the meanÆSD of two independent ex-
periments, each performed in four replicates.
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2018
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
The results of the measure-
ments of [Ca2+]i into MDA-MB-
231 breast cancer cells induced
by 9-epipolygodial (2) and
polygo-11,12-diol (6) were also
consistent with the findings in
the above-described [3H]RTX dis-
placement assay. Compound 2
had no effect on [Ca2+
] (Fig-
i
ure 6A), while killing approxi-
mately half the cells at the same
concentration of 4 mm (Fig-
ure 6B), clearly through a TRPV1-
independent mechanism. In con-
trast, compound 6, while lacking
any toxicity (Figure 6D), led to
a rapid increase in [Ca2+]i (Fig-
ure 6C) in a TRPV1-dependent
manner, as confirmed by the in-
hibition of this process with the
TRPV1-specific antagonist capsa-
zepine (Figure 6C).
Figure 3. Activity of 2 against neurosphere glioma cell cultures with clinically relevant mutations. Transgenic
mouse gliomas of defined molecular subtypes were generated by forced expression of EGFRvIII (classical GBM
subtype, reporter: green fluorescent protein) or PDGFB (proneural GBM subtype, reporter: dsRed) in cdkn2a-defi-
cient sub-ventricular neural precursors (NPC). These two different mouse gliomas and cdkn2a-deficient NPC were
treated for 24 h either with 10 mm CBD versus a corresponding vehicle control (containing 0.01% DMSO) or com-
pound 2 at 4 mm versus vehicle control (0.004% DMSO). Cytotoxicity was measured 24 h after incubation, and
baseline cytotoxicity levels in the controls were arbitrarily defined as 1. Readouts from treated cells were normal-
ized to their respective vehicle controls, and the fold change in relative cytotoxicity was calculated. Each bar rep-
resents the mean ÆSD; statistical significance, as determined by unpaired t-tests, is indicated: ***p<0.001.
Previous studies of the anticancer effects associated with
TRPV1-targeting agents question whether these are genuinely
mediated by TRPV1. Indeed, it is puzzling why both TRPV1 ag-
onists[29–32] and antagonists[29,30] administered independently
would exert antiproliferative effects against cancer cells
through similar cell-death mechanisms.[29,67] It remains to be
established whether there are additional functionally different
intracellular targets that possess structural binding require-
ments similar to those at the vanilloid site on TRPV1. For exam-
ple, vanilloids have indeed been shown to serve as ligands for
cannabinoid receptors,[68] and both TRPV1 agonists and antag-
onists were found to inhibit mitochondrial function by concen-
tration-dependent decreases in oxygen consumption and mi-
tochondrial membrane potential.[29] Thus, structural modifica-
tions of polygodial may have dissimilar effects on TRPV1
versus this alternative hypothetical target genuinely responsi-
ble for cancer cell death. This is supported by the results of
the present work with the identification of compound 2,
which, compared with polygodial, has significantly improved
antiproliferative properties and yet lacks any effects on TRPV1,
and compound 6, which appears to have TRPV1-agonistic
properties superior to those of polygodial while lacking any
antiproliferative effects.
Figure 4. Evaluation of selected polygodial analogues in a [3H]RTX TRPV1
displacement assay. Effect of a single concentration (10 mm) of the selected
analogues in the specific binding of [3H]RTX to the vanilloid site of TRPV1 re-
ceptor from rat spinal cord membranes. Results are expressed as the mean
ÆSEM from three independent experiments, analyzed by one-way analysis
of variance (ANOVA), followed by Dunnett’s multiple comparison test
(***p<0.05, ****p<0.001).
antagonist capsazepine (Figure 5D). Furthermore, TRPV1-like
currents were recorded using whole-cell patch-clamp in MDA-
MB-231 cells following external perfusion with polygodial at
80 mm (Figure 5C). Taken together, these results showed that
polygodial activated TRPV1 in the plasma membrane of MDA-
MB-231 cells. Moreover, an MTT assay using a 24 h nontoxic
treatment with capsazepine (5 mm) was performed to inhibit
the activity of TRPV1 in the presence of polygodial (80 mm).
The results indicate that capsazepine alone does not affect the
viability of MDA-MB-231 cells, whereas polygodial strongly in-
hibits MDA-MB-231 viability. Interestingly, capsazepine did not
counterbalance the effect of polygodial, suggesting that the
anticancer effects of polygodial are also independent of TRPV1
activity (Figure 5E).
Computer modeling
In a search for a theoretical explanation of the experimental re-
sults obtained in this investigation, the likely binding modes of
polygodial (1) and polygo-11,12-diol (6) were examined by
using the cryo-EM derived structure of TRPV1 (PDB ID: 3J5R).[69]
These cryo-EM studies clearly identified the binding pocket of
capsaicin and RTX, thereby allowing for molecular modeling
studies to probe this region for the likely binding conforma-
tions of 1 and 6. Visual inspection of the binding pocket re-
vealed a polar “southern region” providing possible hydrogen
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2019
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
the binding pocket) from the
original structure. Docking of
1 and 6 into the refined TRPV1
structure resulted in more sensi-
ble poses given the nature of
the binding pocket, and provid-
ed an explanation for the mea-
sured binding affinities for the
two structures. In the case of
1 (Figure 7B), the compound is
well accommodated in the cap-
saicin pocket, with the aldehyde
functionalities orientated toward
the southern more polar region
of the pocket. Hydrogen bond-
ing is observed for the aldehyde
functionalities to the side chains
of Tyr511 and Thr550, with the
latter serving as hydrogen bond
donors. In addition, the hydro-
phobic portion of 1 is accommo-
dated in the northern apolar
region of the pocket. Of interest
is the fact that located at the
southernmost point of the bind-
ing pocket is a lysine residue
Lys571 (Figure 7A). Although the
side chain of this residue is not
oriented toward the binding
pocket, its close proximity to the
aldehyde functionalities of
1
(ꢀ10 Š) may explain the ob-
served irreversible binding. The
predicted binding pose for diol
6 differs from that of 1 in that
the two hydroxy functionalities
can behave as either hydrogen
Figure 5. Effect of polygodial on TRPV1 activity in MDA-MB-231 breast cancer cells. A) Effect of capsaicin on MDA-
MB-231 [Ca2+]i. B) Effect of polygodial on MDA-MB-231 [Ca2+]i. C) TRPV1-like current activation. D) Effect of capsa-
zepine on polygodial-mediated [Ca2+]i response. E) Effect of co-treatment with polygodial and capsazepine on
24 h cell viability (***p<0.001; two-way ANOVA followed by Holm–Sidak tests).
bonding interactions with the side chains of Tyr511 and
Thr550, while the “northern region” of the pocket is predomi-
nantly apolar (Figure 7A). Initial docking studies of 1 and 6
suggested that the preferred orientation of the ligands was
indeed to facilitate hydrogen bonding to the side chains of
Tyr511 and Thr550, via the carbonyl and hydroxy groups of
1 and 6, respectively. Moreover, the hydrophobic regions of
1 and 6 were orientated toward the northern hydrophobic
region of the pocket. However, visual critique of the binding
mode suggested that for both 1 and 6, the binding poses are
suboptimal, with large parts of the molecules’ hydrophobic
portions not well accommodated within the capsaicin binding
pocket. Given the poor resolution of the TRPV1 structure, opti-
mization of the binding pocket was warranted, and to this end
1 was manually manipulated to a better fit within the pocket
(by visual inspection), and the TRPV1–polygodial structure was
subjected to minimization by using fixed-atom constraints of
the protein backbone (not including residues within 7 Š of 1)
and a generalized Born implicit solvation model. The resulting
TRPV1 structure exhibited an RMSD of 0.66 Š (residues within
bond donors or acceptors (Figure 7C). For the hydrogen bond-
ing interaction with the side chain of Tyr511 this is of no con-
sequence, but regarding the hydrogen bonding to Thr550, it is
here where the significantly stronger measured binding of 6
(relative to 1) to TRPV1 might be explained. In the case of 1, in
order to facilitate double hydrogen bonding interactions, the
side chains of both Tyr511 and Thr550 must act as donors.
However, in the case of Thr550, in the apo form of the protein,
this hydroxy side chain forms a hydrogen bond to the amide
carbonyl group of Ala546. To form a hydrogen bond to the car-
bonyl group of 1, the hydrogen bond to Ala546 must be sacri-
ficed. However, in the case of diol 6, the hydrogen bond be-
tween Thr550 and Ala546 need not be undone, as 6 may form
a hydrogen bond with the hydroxy group of Thr550, as a hy-
drogen bond donor. Docking of 9-epipolygodial (2) interesting-
ly revealed that inversion of stereochemistry at C9 results in
a pose incapable of facilitating both hydrogen bonding inter-
actions. Hydrogen bonding only to the less preferred Thr550
(as donor) was observed, and this may explain the substantially
decreased TRPV1 activity.
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2020
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 6. A) Lack of an effect of 9-epipolygodial (2) on [Ca2+]i despite B) a pronounced effect on the viability of MDA-MB-231 breast cancer cells at 4 mm.
C) Effect of polygo-11,12-diol (6) on [Ca2+]i into TRPV1-expressing MDA-MB-231 breast cancer cells, which is blocked with capsazepine. D) Lack of an effect of
polygo-11,12-diol (6) on cell viability at these concentrations.
Figure 7. Molecular modeling showing the capsaicin binding region of TRPV1 with a solvent-interpolated charge surface containing A) docked polygodial (1)
and the close proximity of Lys571. B) Hydrogen bonding interactions to Tyr511 and Thr550 are observed for docked 1. C) Polygo-11,12-diol (6) docked within
the pocket assumes a similar pose; however, the ability to act as a hydrogen bond donor to Thr550 means that this residue can maintain its structural hydro-
gen bonding interaction with Ala546.
Conclusions
cal applications and have had a major impact on human
health.[70–72] For example, an estimated 80 billion tablets of as-
pirin are consumed annually in the United States, and
a number of blockbuster drugs such as clopidogrel, lansopra-
zole, and esomeprazole are covalent inhibitors.[72] Furthermore,
Despite a safety concern involving potential tissue damage or
haptenization of proteins eliciting an immune response, cova-
lent drugs have been approved as treatments for diverse clini-
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2021
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
dehyde stains. 1H and 13C NMR spectra were recorded on
a Bruker 400 spectrometer. Chemical shifts (d) are reported in ppm
relative to the TMS internal standard. Abbreviations are as follows:
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). The syn-
thesized compounds are at least 95% pure according to an HPLC–
MS analysis. Polygodial (1) was purchased from VWR.
covalent inhibitors are of key importance in the treatment of
cancer and may be more effective in eradicating drug-resistant
tumor cells.[73] Thus, mutations in the binding site of a drug
target represent an important cancer cell resistance mecha-
nism, and it has been noted that irreversible inhibitors main-
tain activity against such mutations caused by treatment with
reversible inhibitors.[73] In one experimental confirmation of
this proposal, treatment of NSCLC patients with reversible epi-
dermal growth factor receptor (EGFR) inhibitors produced
a good initial response but then led to relapse in ~50% of pa-
tients due to the development of resistance involving the ex-
pression of EGFR with mutations at T790M and/or L858R in the
ATP binding site.[73,74] The NSCLC cell line harboring the
T790M-L858R double-mutant form of EGFR was not affected
by a panel of reversible inhibitors, but was, by contrast, signifi-
cantly affected by irreversible inhibitors.[73,75] The effectiveness
of irreversible inhibitors against resistant mutants may lie in
the fact that mutations generally only affect the rate of cova-
lent complex formation, and given sufficient exposure time,
even mutated protein targets that react considerably more
slowly will become irreversibly inactivated.[72]
Compound 2: To a solution of 1 (3 mg, 0.0128 mmol) in dry tolu-
ene (1.5 mL) were added 4 Š molecular sieves and p-toluenesulfon-
ic acid (cat.). The mixture was stirred at room temperature for 40 h.
Solid NaHCO3 was added to the reaction mixture and stirred for
10 min, filtered, and the filtrate was concentrated under reduced
pressure. The crude product was purified by preparative TLC (8:92
EtOAc/hexanes) to obtain 1.2 mg of 2 as a colorless oil (40% yield);
1H NMR (400 MHz, C6D6): d=9.69 (d, J=2.4 Hz, 1H), 9.19 (s, 1H),
6.20 (dd, J=4.9, 2.4 Hz, 1H), 1.96–1.88 (m, 1H), 1.62–1.51 (m, 1H),
1.45–1.39 (m, 2H), 1.34–1.25 (m, 2H), 1.20–1.12 (m, 2H), 0.96–0.87
(m, 2H), 0.65 (s, 3H), 0.63 (s, 3H), 0.57 ppm (d, J=0.7 Hz, 3H);
13C NMR (100 MHz, CDCl3): d=202.2, 192.8, 153.4, 137.4, 58.5, 44.2,
42.0, 37.7, 37.1, 32.9, 32.7, 25.5, 21.9, 21.5, 18.4 ppm; HRMS (ESI)
calcd for C15H22NaO2 [M+Na] 257.1517, found 257.1519.
Compound 5c: To a solution of 1 (3 mg, 0.0128 mmol) and 4-nitro-
aniline (1.9 mg, 0.014 mmol) in THF (2 mL) was added AcOH
(4.3 mL, 0.077 mmol). The mixture was stirred at room temperature
for 20 h. After completion of the reaction, as monitored by TLC,
the reaction mixture was concentrated under reduced pressure
and co-distilled with toluene. The crude product was purified by
preparative TLC (15:85 EtOAc/hexanes) to obtain 2.4 mg of 5c as
The present work has led to the identification of 9-epipoly-
godial, an analogue of the widely studied polygodial, which is
more potent than the latter by a factor of 20 against all cancer
cells in the panel used in this work. Encouragingly, this com-
pound retained activity against a significant number of drug-
resistant cell lines with which it was challenged. Furthermore,
the experimental results in our chemical system point to the
fact that this compound likely works by covalent inhibition of
its anticancer target through formation of a pyrrole adduct
with a lysine residue. Such mode of reactivity in covalent
target inhibition is greatly understudied. It is likely, however,
that this type of reactivity toward a lysine residue over the
common imine formation, may lead to greater selectivity for
the target site, and thus decreased off-target effects. Indeed,
a more complex chemical mechanism of the Paal–Knorr pyrrole
condensation over the simple imine formation may be possible
only at select protein binding sites capable of catalyzing this
multistep transformation. In support of this reasoning, polygo-
dial’s therapeutic application to control Candida albicans infec-
tions is well tolerated by most patients.[13]
1
a yellow viscous liquid (56% yield); H NMR (400 MHz, CDCl3): d=
8.27 (d, J=9.2 Hz, 2H), 7.43 (d, J=9.2 Hz, 2H), 6.98 (d, J=2.1 Hz,
1H), 6.79 (dd, J=2.1, 0.7 Hz, 1H), 6.55 (dd, J=9.4, 2.9 Hz, 1H), 5.89
(dd, J=9.4, 2.9 Hz, 1H), 2.12 (t, J=2.9 Hz, 1H), 2.08–2.01 (m, 1H),
1.81–1.72 (m, 1H), 1.69–1.59 (m, 4H), 1.09 (s, 3H), 1.05 (s, 3H),
0.99 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=145.4, 139.2, 128.7
(2C), 125.6 (2C), 123.5, 120.4, 118.2, 113.3, 110.5, 100.0, 53.1, 41.3,
36.4, 33.0, 29.7, 22.3, 21.6, 18.8, 14.1 ppm; HRMS (ESI) calcd for
C21H25N2O2 [M+H] 337.1916, found 337.1916.
Compound 6: To a solution of 1 (3 mg, 0.0128 mmol) in MeOH
(2 mL) was added NaBH4 (1.0 mg, 0.027 mmol). The mixture was
stirred at room temperature for 2 h. After completion of the reac-
tion, as monitored by TLC, H2O was added to the reaction mixture
and MeOH was evaporated. The reaction mixture was diluted with
EtOAc, and the organic phase was washed with 1n HCl and H2O,
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The crude product was purified by preparative TLC
(10:90 EtOAc/hexanes) to obtain 2.4 mg of 6 as a colorless oil
Finally, this work also resulted in the discovery of nontoxic
TRPV1 agonists such as polygo-11,12-diol. Given the considera-
ble interest in the medicinal chemistry community toward the
exploitation of TRPV1-targeting agents as anti-nocicep-
tives[14–18,21–24] and the cytotoxicity associated with some of the
studied compounds,[27–32] these findings could be significant.
1
(80% yield); H NMR (400 MHz, CDCl3): d=5.83–5.78 (m, 1H), 4.38–
4.32 (m, 1H), 3.99 (d, J=12.1 Hz, 1H), 3.91 (dd, J=10.9, 2.2 Hz,
1H), 3.69 (dd, J=10.9, 8.2 Hz, 1H), 2.86 (brs, 1H), 2.14 (brs, 1H),
2.13–2.04 (m, 1H), 2.01–1.83 (m, 2H), 1.59–1.39 (m, 3H), 1.28–1.11
(m, 4H), 0.88 (s, 3H), 0.87 (s, 3H), 0.76 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=136.9, 127.6, 67.5, 61.5, 54.5, 49.4, 42.0, 39.3,
35.6, 33.2, 33.0, 23.6, 21.9, 18.8, 14.5 ppm; HRMS (ESI) calcd for
C15H26NaO2 [M+Na] 261.1830, found 261.1829.
Experimental Section
Compound 7: To a solution of 1 (3 mg, 0.0128 mmol) and hydra-
zine hydrate (0.7 mL, 0.014 mmol) in MeOH (1 mL) were added 4 Š
molecular sieves. The mixture was stirred at room temperature for
20 h. After completion of the reaction, as monitored by TLC, the re-
action mixture was filtered and the filtrate was concentrated under
reduced pressure. The crude product was purified by preparative
TLC (4:96 MeOH/CHCl3) to obtain 2.8 mg of 7 as a brown viscous
General: All reagents, solvents, and catalysts were purchased from
commercial sources (Acros Organics and Sigma–Aldrich) and used
without purification. All reactions were performed in oven-dried
flasks open to the atmosphere or under nitrogen or argon and
monitored by thin-layer chromatography (TLC) on TLC pre-coated
(250 mm) silica gel 60 F254 glass-backed plates (EMD Chemicals Inc.).
Visualization was accomplished with UV light, iodine, and p-anisal-
1
liquid (95% yield); H NMR (400 MHz, CDCl3): d=9.02 (s, 1H), 8.84
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2022
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
(s, 1H), 2.99–2.90 (m, 1H), 2.82 (ddd, J=18.7, 11.1, 7.7 Hz, 1H),
2.37–2.31 (m, 1H), 2.06–1.98 (m, 1H), 1.86–1.65 (m, 3H), 1.58–1.51
(m, 1H), 1.42 (td, J=12.7, 3.9 Hz, 1H), 1.31–1.23 (m, 2H), 1.23 (d,
J=0.7 Hz, 3H), 0.98 (s, 3H), 0.95 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=152.2, 148.3, 145.8, 135.8, 49.5, 41.2, 36.95, 36.4, 33.4,
33.0, 26.9, 24.2, 21.5, 18.6, 17.7 ppm; HRMS (ESI) calcd for C15H23N2
[M+H] 231.1861, found 231.1861.
(m, 5H), 2.78–2.71 (m, 1.5H), 2.50–2.43 (m, 1H), 2.05–1.79 (m, 5H),
1.71–1.58 (m, 12.5H), 1.54–1.39 (m, 12H), 1.39–1.22 (m, 11 H), 1.19–
1.13 (m, 2H), 0.95–0.85 (m, 18H), 0.81 (s, 3H), 0.80 (s, 4.5H), 0.78 (s,
4.5H), 0.75 (s, 3H), 0.71 ppm (s, 4.5H), total number of protons=
100H [40H (40H”1.0) of 11 a, 60H (40H”1.5) of 11 b]; HRMS (ESI)
calcd for C23H40NaO3 [M+Na] 387.2875, found 387.2879.
1
Compound 12a: 40% yield, colorless oil; H NMR (400 MHz, C6D6):
d=5.88–5.84 (m, 1H), 5.73–5.70 (m, 1H), 5.27 (d, J=6.2 Hz, 1H),
4.03–3.89 (m, 2H), 2.47–2.41 (m, 1H), 2.05–1.96 (m, 1H), 1.89–1.73
(m, 2H), 1.51 (dt, J=13.3, 2.9 Hz, 1H), 1.40–1.29 (m, 11 H), 1.15–
1.09 (m, 6H), 0.90 (s, 3H), 0.81 (s, 3H), 0.75 ppm (s, 3H); HRMS (ESI)
calcd for C21H36NaO3 [M+Na] 359.2562, found 359.2560.
Compound 8: To a suspension of Raney nickel (21.0 mg) in H2O
was added 1 (3 mg, 0.0128 mmol) in THF (2 mL). The resultant mix-
ture was stirred at room temperature for 20 h under hydrogen at-
mosphere using a balloon. After completion of the reaction, as
monitored by TLC, the reaction mixture was filtered through silica
pad. The bed was washed several times with Et2O and the filtrate
was concentrated under reduced pressure to obtain 2.9 mg of 8 as
1
Compound 13a: 35% yield, colorless oil; H NMR (400 MHz, C6D6):
d=5.74–5.70 (m, 1H), 5.34 (s, 1H), 4.90 (d, J=5.9 Hz, 1H), 3.86–
3.71 (m, 2H), 3.57–3.42 (m, 2H), 2.25–2.19 (m, 1H), 1.89–1.80 (m,
1H), 1.68–1.60 (m, 2H), 1.32–1.28 (m, 2H), 1.27–1.23 (m, 2H), 1.14–
1.02 (m, 2H), 0.73 (s, 3H), 0.70 (s, 3H), 0.67 ppm (s, 3H); HRMS (ESI)
calcd for C19H26F6KO3 [M+K] 455.1423, found 455.1423.
1
a colorless oil (95% yield); H NMR (400 MHz, C6D6): d=5.97 (t, J=
2.1 Hz, 1H), 5.38–5.31 (m, 1H), 2.41–2.35 (m, 1H), 2.25–2.18 (m,
1H), 2.06 (s, 1H), 1.86–1.74 (m, 1H), 1.52–1.40 (m, 3H), 1.35–1.31
(m, 1H), 1.31–1.27 (m, 1H), 1.12–1.00 (m, 4H), 0.78 (s, 3H), 0.74 (s,
3H), 0.72 ppm (s, 3H); HRMS (ESI) calcd for C15H24NaO2 [M+Na]
259.1674, found 259.1674.
1
Compound 13b: 55% yield, colorless oil; H NMR (400 MHz, C6D6):
d=5.43 (dd, J=6.8, 3.1 Hz, 1H), 5.09 (s, 1H), 4.90 (d, J=4.0 Hz,
1H), 3.80–3.65 (m, 2H), 3.63–3.44 (m, 2H), 2.52–2.46 (m, 1H), 1.83–
1.74 (m, 1H), 1.60–1.46 (m, 2H), 1.30–1.21 (m, 2H), 1.09–0.89 (m,
4H), 0.72 (s, 3H), 0.68 (s, 3H), 0.57 ppm (s, 3H); 13C NMR (100 MHz,
C6D6): d=136.0, 127.4, 125.7, 122.8, 106.5, 104.0, 65.3 (d), 63.9 (d),
57.7, 49.2, 42.2, 39.3, 32.9, 32.4, 29.8, 23.6, 21.4, 18.6, 14.1 ppm;
HRMS (ESI) calcd for C19H26F6KO3 [M+K] 455.1423, found 455.1423.
Compound 9: To a solution of 2 (1 mg, 0.0043 mmol) in CH2Cl2
(2 mL) was added methyl (triphenylphosphoranylidene)acetate
(7.1 mg, 0.0021 mmol). The mixture was stirred at room tempera-
ture for 20 h. After completion of the reaction, as monitored by
TLC, the reaction mixture was filtered and the filtrate was concen-
trated under reduced pressure. The crude product was purified by
preparative TLC (9:91 EtOAc/hexanes) to obtain 1.1 mg of 9 as
1
1
Compound 14b: 60% yield, colorless oil; H NMR (400 MHz, C6D6):
a colorless oil (90% yield); H NMR (400 MHz, C6D6): d=9.33 (d, J=
d=7.29–7.24 (m, 4H), 7.06–6.97 (m, 4H), 5.57–5.52 (m, 1H), 5.34 (s,
1H), 5.15 (d, J=4.0 Hz, 1H), 4.65 (dd, J=24.7, 12.3 Hz, 2H), 4.35 (d,
J=12.2 Hz, 2H), 2.78–2.72 (m, 1H), 1.98–1.86 (m, 1H), 1.72–1.61
(m, 1H), 1.46–1.38 (m, 2H), 1.22–1.10 (m, 3H), 1.10–0.96 (m, 2H),
0.78 (s, 3H), 0.73 (s, 3H), 0.69 ppm (s, 3H); HRMS (ESI) calcd for
C29H34Br2NaO3 [M+Na] 611.0772, found 611.0791.
4.8 Hz, 1H), 7.46 (d, J=15.8 Hz, 1H), 6.16 (d, J=15.8 Hz, 1H), 5.84–
5.79 (m, 1H), 3.44 (s, 3H), 2.63–2.59 (m, 1H), 1.91–1.82 (m, 1H),
1.66–1.58 (m, 1H), 1.44–1.39 (m, 2H), 1.25–1.13 (m, 5H), 0.65 (s,
6H), 0.59 ppm (d, J=0.8 Hz, 3H); 13C NMR (100 MHz, C6D6): d=
200.3, 168.0, 146.4, 141.4, 132.6, 117.1, 62.5, 51.1, 44.4, 42.2, 37.6,
36.4, 32.9, 32.7, 25.4, 21.8, 18.7, 14.2 ppm; HRMS (ESI) calcd for
C18H26NaO3 [M+Na] 313.1780, found 313.1779.
Compounds 15a+b (1.0:1.7 mixture): 80% yield, colorless oil;
1H NMR (400 MHz, C6D6): d=5.93–5.87 (m, 1H), 5.74–5.70 (m, 1H),
5.58–5.53 (m, 1.7H), 5.40 (s, 1.7H), 5.24 (d, J=6.1 Hz, 1H), 5.19 (d,
J=4.2 Hz, 1.7H), 4.04–3.94 (m, 5H), 3.79–3.67 (m, 5H), 3.61–3.33
(m, 11 H), 3.20 (s, 5H), 3.18 (s, 5H), 3.15 (s, 3H), 3.14 (s, 3H), 2.76–
2.70 (m, 1.7H), 2.47–2.41 (m, 1H), 1.95–1.83 (m, 4H), 1.75–1.62 (m,
2H), 1.53–1.42 (m, 2H), 1.42–1.25 (m, 8H), 1.27–1.19 (m, 4H), 1.15–
1.01 (m, 6H), 0.87 (s, 3H), 0.79 (s, 3H), 0.77 (s, 5H), 0.73 (s, 5H),
0.73 (s, 3H), 0.70 ppm (s, 5H), total number of protons=97H [36H
(36H”1.0) of 15a, 61H (36H”1.7) of 15b]; HRMS (ESI) calcd for
C21H36NaO5 [M+Na] 391.2460, found 391.2460.
General procedure for acetals 10–16: To a solution of 1 (3 mg,
0.0128 mmol) in dry toluene (2 mL) were added a selected alcohol
(0.5 mL), 4 Š molecular sieves and p-toluenesulfonic acid (cat.). The
resulting mixture was stirred at room temperature for 20 h. Solid
NaHCO3 was added and the reaction mixture was stirred for
10 min, filtered, and the filtrate was concentrated under reduced
pressure. The crude product was purified by preparative TLC (8:92
EtOAc/hexanes) to obtain acetal products 10–16.
1
Compound 10a: 35% yield, colorless oil; H NMR (400 MHz, C6D6):
d=5.90–5.86 (m, 1H), 5.68–5.65 (m, 1H), 5.18 (d, J=6.1 Hz, 1H),
3.99–3.86 (m, 2H), 3.55–3.45 (m, 2H), 2.48–2.42 (m, 1H), 2.03–1.93
(m, 1H), 1.84–1.71 (m, 2H), 1.52–1.42 (m, 1H), 1.35–1.25 (m, 5H),
1.19 (t, J=7.2 Hz, 3H), 1.17 (t, J=7.2 Hz, 3H), 0.87 (s, 3H), 0.80 (s,
3H), 0.74 ppm (s, 3H); HRMS (ESI) calcd for C19H32NaO3 [M+Na]
331.2249, found 331.2248.
Compounds 16a+b (1:1 mixture): 75% yield, colorless oil; 1H NMR
(400 MHz, C6D6): d=5.90–5.82 (m, 1H), 5.63–5.53 (m, 2H), 5.30 (s,
1H), 5.14–5.05 (m, 2H), 3.97–3.75 (m, 4H), 3.54–3.32 (m, 4H), 2.72–
2.62 (m, 1H), 2.45–2.36 (m, 1H), 2.10–1.86 (m, 8H), 1.82–1.74 (m,
4H), 1.73–1.50 (m, 16H), 1.49–1.26 (m, 8H), 1.26–0.92 (m, 10H),
0.88–0.68 ppm (m, 18H), total number of protons=80H [40H of
16a, 40H of 16b]; HRMS (ESI) calcd for C27H40NaO3 [M+Na]
435.2875, found 435.2878.
1
Compound 10b: 45% yield, colorless oil; H NMR (400 MHz, C6D6):
d=5.59–5.56 (m, 1H), 5.36 (s, 1H), 5.14 (d, J=4.3 Hz, 1H), 3.98–
3.83 (m, 2H), 3.57–3.44 (m, 2H), 2.76–2.70 (m, 1H), 1.95–1.86 (m,
1H), 1.74–1.62 (m, 2H), 1.50–1.43 (m, 1H), 1.35–1.27 (m, 4H), 1.26–
1.17 (m, 7H), 0.79 (s, 3H), 0.76 (s, 3H), 0.70 ppm (s, 3H); HRMS (ESI)
calcd for C19H32NaO3 [M+Na] 331.2249, found 331.2248.
Cell culture: Human cancer cell lines were obtained from the Amer-
ican Type Culture Collection (ATCC; Manassas, VA, USA), the Euro-
pean Collection of Cell Culture (ECACC; Salisbury, UK), and the
Deutsche Sammlung von Mikroorganismen und Zellkulturen
(DSMZ; Braunschweig, Germany). Human mammary carcinoma
MCF7 cells were cultured in RPMI-1640 medium supplemented
with 10% FBS. U87 cells (ATCC HTB-14) were cultured in DMEM,
whereas A549 cells (DSMZ ACC107) were cultured in RPMI-1640
Compounds 11 a +b (1:1.5 mixture): 80% yield, colorless oil;
1H NMR (400 MHz, C6D6): d=5.92–5.87 (m, 1H), 5.71–5.67 (m, 1H),
5.59 (dd, J=6.8, 3.1 Hz, 1.5H), 5.39–5.37 (m, 1.5H), 5.20 (d, J=
6.1 Hz, 1H), 5.15 (d, J=4.3 Hz, 1.5H), 4.03–3.86 (m, 5H), 3.56–3.43
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2023
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
medium supplemented with 10% heat-inactivated FBS. Hs683
(ATCC HTB-138) GBM cells were cultivated in RPMI-1640 medium
supplemented with 10% FBS. Human uterine sarcoma MES-SA and
MES-SA/Dx5 cells were cultured in RPMI-1640 medium supple-
mented with 10% FBS, with MES-SA/Dx5 maintained in the pres-
ence of 500 nm doxorubicin (Sigma). SKMEL-28 cells (ATCC HTB72)
and U373 GBM cells (ECACC 08061901) were cultured in RPMI-1640
medium supplemented with 10% heat-inactivated FBS. Cell culture
media were supplemented with 4 mm glutamine (Lonza BE17-
605E), 100 mgmLÀ1 gentamicin (Lonza 17-5182), and penicillin–
streptomycin (200 UmLÀ1 and 200 mgmLÀ1, respectively; Lonza 17-
602E). MDA-MB-231 (ATCC HTB-26) epithelial mammary adenocarci-
noma cells were cultured in Eagle’s minimum essential medium
(EMEM, Invitrogen) containing 5% FCS (Cambrex), 2 mm l-gluta-
mine (Invitrogen), 0.06% HEPES (Invitrogen), and penicillin
(50 IUmLÀ1)–streptomycin (50 mgmLÀ1, Invitrogen) at 378C in a hu-
midified atmosphere of 5% CO2 in air. Transformed mouse NPCs
were cultured in suspension under neurosphere conditions at 378C
in a humidified atmosphere of 95% O2 and 5% CO2 in DMEM F-12
(Invitrogen 11320-074) supplemented with 1” B27 supplement (In-
vitrogen 17504-044), 5% penicillin–streptomycin (Biochrom 10378-
017), 10 ngmLÀ1 EGF (R&D systems 236-EG), and 10 ngmLÀ1 FGF
(PeproTech 100-18B).
were detected and omitted, if any, using the Grubbs test. Graphs
were generated with GraphPad Prism 5 software (version 5.01).
[3H]Resiniferatoxin binding assays: To evaluate the possible affinity
of different analogues to the vanilloid site of TRPV1,
a [3H]resiniferatoxin ([3H]RTX) binding assay was performed as pre-
viously described.[79,80] Briefly, rat spinal cords were homogenized
in buffer A (10 mm HEPES pH 7.4, 5 mm KCl, 5.8 mm NaCl, 2 mm
MgCl2, 0.75 mm CaCl2, 137 mm sucrose) and centrifuged for 10 min
at 1000 g at 48C, and the supernatant was further centrifuged for
30 min at 35000 g at 48C. The resulting pellets were than resus-
pended in buffer A and frozen until assayed. The binding reaction
was performed in a final volume of 500 mL, containing buffer A
(plus 0.25 mgmLÀ1 BSA), membranes (0.5 mgmLÀ1), and 2 nm
[3H]RTX in the presence or absence of polygodial analogues
(10 mm). For measurements of nonspecific binding, nonradioactive
RTX (100 mm) was included. The reaction was started by incubating
tubes at 378C for 60 min and then stopped by transferring the
tubes to an ice bath and adding 100 mg bovine a1-acid glycopro-
tein (to decrease nonspecific binding). Finally, the bound and free
membranes [3H]RTX were separated by centrifugation for 30 min at
35000 g at 48C. The pellet was used to quantify the scintillation
counting. Specific binding was calculated as the difference be-
tween total and nonspecific binding, and the results are reported
as a percentage of specific binding.
Antiproliferative properties: The antiproliferative properties of the
synthesized compounds were evaluated by MTT assay.[76–78] All
compounds were dissolved in DMSO at a concentration of either
100 or 50 mm prior to cell treatment. The cells were trypsinized
and seeded at various concentrations depending on cell type. Cells
were grown for 24–72 h, treated with compounds at concentra-
tions ranging from 0.001 to 100 mm and incubated for 24, 48, or
72 h in 100 or 200 mL media depending on the cell line used. The
number of experiments and replicates varied depending on the
cell line. Cells treated with 0.1% DMSO were used as a negative
control; 1 mm PAO was used as a positive control.
Intracellular Ca2+ measurements: Cells were grown on glass cover-
slips for fluorescence imaging. The cytosolic calcium levels were
measured using Fura-2-loaded cells. Cells were loaded for 45 min
at 378C in a humidified atmosphere of 5% CO2 in air with 3.3 mm
Fura-2/AM prepared in saline solution. Fluorescence was excited at
l 350 and 380 nm alternately using a monochromator (Polychro-
me IV; TILL Photonics, Planegg, Germany), and captured by a Cool
SNAP HQ camera (Princeton Instruments, France) after filtration
through a long-pass filter (l 510 nm). Metafluor software 7.0 (Mo-
lecular Devices) was used for acquisition and analysis. All record-
ings were carried out at room temperature. Cells were perfused
with saline solutions consisting of 140 mm NaCl, 5 mm KCl, 2 mm
CaCl2, 2 mm MgCl2, 10 mm HEPES, and 5 mm glucose (adjusted to
pH 7.4 with NaOH).
Selection of doxorubicin-resistant cells: Selection of the MES-SA/Dx5
cell line was done according to Harker and Sikic.[54] The cells were
split and allowed to adhere overnight. The next day cells were ini-
tially exposed to doxorubicin (DOX) at a concentration of 100 nm,
which represents the GI50 concentration. The cells were maintained
at this DOX concentration until their growth rate reached that of
the untreated cells. The DOX concentration was then increased in
two-fold increments following the same growth criteria at each
concentration to a final DOX concentration of 500 nm. Each new
DOX concentration required approximately two passages to reach
the growth rate of the untreated cells.
Electrophysiological recordings: TRP currents were recorded using
the conventional patch-clamp technique in whole-cell configura-
tion. Briefly, the holding membrane potential was held at À40 mV,
and currents were elicited by a ramp depolarization from À100 to
+100 mV for 350 ms. The interval between each ramp depolariza-
tion was 10 s. The patch pipettes (3–5 MW) were made from hema-
tocrit glass using a vertical puller (P30 vertical micropipette puller;
Sutter Instruments). The following extracellular solution was used:
140 mm sodium gluconate, 5 mm potassium gluconate, 2 mm mag-
nesium gluconate, 2 mm calcium gluconate, 10 mm HEPES, 5 mm
glucose, and 5 mm TEA-Cl (adjusted to pH 7.4 with NaOH). The fol-
lowing intrapipette solution was used: 145 mm cesium gluconate,
8 mm sodium gluconate, 10 mm EGTA, 3 mm magnesium gluco-
nate, and 10 mm HEPES (adjusted to pH 7.2 with CsOH). Signals
were filtered at 1 kHz and digitized at 5 kHz using an Axopatch
200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA,
USA) combined with a Digidata 1322A instrument (Molecular Devi-
ces). Electrophysiological protocols and analyses were made using
pClamp 10 and Clampfit software (both by Molecular Devices), as
well as Origin 6.0 (Microcal Software, Northampton, MA, USA). All
experiments were performed at room temperature.
CytoTox-Fluor cytotoxicity assays: The CytoTox-Fluor cytotoxicity
assay (Promega) was used according to manufacturer’s instruc-
tions. In brief, 1.5”104 cells per well (five replicates per condition)
were plated in 24-well plates in 450 mL of culture medium (DMEM
F-12 without phenol red). They then received 50 mL of culture
medium supplemented with test compounds or the respective ve-
hicle control. After 24 h incubation at 378C, cell suspension (20 mL)
was transferred to a black 384-well plate and mixed with bis-AAF-
R110 substrate dilution (20 mL). After 2 h incubation at 378C, the
fluorescence intensity was measured using a Tecan Infinite F200
fluorescence plate reader (lex 485 nm, lem 520 nm). Blank values
were subtracted from all wells, and the fluorescence readout for
untreated cells (vehicle control) was normalized to 1. Readouts
from cells receiving various treatment conditions were normalized
to those of untreated cells, and the fold change of relative cytotox-
icity to untreated controls was calculated for each well. Outliers
Computer modeling: Molecular modelling was performed using Dis-
covery Studio 4.5 (DS). The receptor template was obtained from
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2024
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[23] J. B. Davis, J. Gray, M. J. Gunthorpe, J. P. Hatcher, P. T. Davey, P. Overend,
M. H. Harries, J. Latcham, C. Clapham, K. Atkinson, S. A. Hughes, K.
Rance, E. Grau, A. J. Harper, P. L. Pugh, D. C. Rogers, S. Bingham, A. Ran-
dall, S. A. Sheardown, Nature 2000, 405, 183–187.
[24] M. J. Caterina, D. Julius, Annu. Rev. Neurosci. 2001, 24, 487–517.
[25] L. S. Premkumar, M. Bishnoi, Curr. Top. Med. Chem. 2011, 11, 2192–2209.
[26] D. Gkika, N. Prevarskaya, Biochim. Biophys. Acta. 2009, 1793, 953–958.
[27] M. Hartel, F. F. di Mola, F. Selvaggi, G. Mascetta, M. N. Wente, K. Felix,
N. A. Giese, U. Hinz, P. Di Sebastiano, M. W. Buchler, H. Friess, Gut 2006,
55, 519–528.
the RCSB Protein Data Bank (PDB ID: 3J5R) and chains B and D
were retained for the simulations. Protein preparation was carried
out using the Prepare Protein protocol launched from within DS.
All docking simulations were carried out using a modified CDocker
protocol with pre-generation of ligand conformations to adequate-
ly sample conformational space. Minimizations were carried out
within DS, employing the CHARMm force field (version 39.1).
Acknowledgements
[28] K. Stock, J. Kumar, M. Synowitz, S. Petrosino, R. Imperatore, E. S. Smith,
P. Wend, B. Purfürst, U. A. Nuber, U. Gurok, V. Matyash, J. H. Wälzlein,
S. R. Chirasani, G. Dittmar, B. F. Cravatt, S. Momma, G. R. Lewin, A. Ligres-
ti, L. De Petrocellis, L. Cristino, V. Di Marzo, H. Kettenmann, R. Glass, Nat.
Med. 2012, 18, 1232–1238.
[29] A. Athanasiou, P. A. Smith, S. Vakilpour, N. M. Kumaran, A. E. Turner, D.
Bagiokou, R. Layfield, D. E. Ray, A. D. Westwell, S. P. H. Alexander, D. E.
Kendall, D. N. Lobo, S. A. Watson, A. Lophatanon, K. A. Muir, D. A. Guo,
T. E. Bates, Biochem. Biophys. Res. Commun. 2007, 354, 50–55.
[30] C. B. Gonzales, N. B. Kirma, J. J. De La Chapa, R. Chen, M. A. Henry, S.
Luo, K. M. Hargreaves, Oral Oncol. 2014, 50, 437–447.
[31] M. Skrzypski, M. Sassek, S. Abdelmessih, S. Mergler, C. Grotzinger, D.
Metzke, T. Wojciechowicz, K. W. Nowak, M. Z. Strowski, Cell. Signalling
2014, 26, 41–48.
[32] V. Farfariello, S. Liberati, M. B. Morelli, D. Tomassoni, M. Santoni, M. Na-
bissi, A. Giannantoni, G. Santoni, C. Amantini, Chem.-Biol. Interact. 2014,
224, 128–135.
This project was supported by grants from the National Institute
of General Medical Sciences (P20M103451), the National Cancer
Institute (CA186046-01A1), the Welch Foundation (AI-0045), and
the National Science Foundation (NSF award 0946998). S.R. and
L.F. acknowledge their NMT Presidential Research Support. L.F. ac-
knowledges the National Science Foundation (NSF award IIA-
1301346). R.K. is a director of research with the Fonds National
de la Recherche Scientifique (FRS-FNRS, Belgium). S.C.P. and
W.v.O. gratefully acknowledge support from the National Re-
search Foundation (NRF) of South Africa, as well as Stellenbosch
University. The authors are grateful to Anntherese Kornienko for
her help with creating Figure 2.
[33] N. Allouche, C. Apel, M. T. Martin, V. Dumontet, F. GuØritte, M. Litaudon,
Phytochemistry 2009, 70, 546–553.
Keywords: capsaicin
resiniferatoxin · vanilloid
·
capsazepine
·
ion channels
·
[34] T. Tozyo, F. Yasuda, H. Nakai, H. Tada, J. Chem. Soc. Perkin Trans. 1 1992,
1859–1866.
[35] A. F. Barrero, M. CortØs, E. A. Manzaneda, E. Cabrera, R. Chahboun, M.
Lara, A. R. Rivas, J. Nat. Prod. 1999, 62, 1488–1491.
[36] H. Anke, O. Sterner, Planta Med. 1991, 57, 344–346.
[37] A. Forsby, M. Andersson, L. Lewan, O. Sterner, Toxicol. In Vitro 1991, 5,
9–14.
[38] M. Andersson, F. Bocchio, O. Sterner, A. Forsby, L. Lewan, Toxicol. In Vitro
1993, 7, 1–6.
[39] I. Montenegro, G. Tomasoni, C. Bosio, N. Quinones, A. Madrid, H. Carra-
sco, A. Olea, R. Martinez, M. Cuellar, J. Villena, Molecules 2014, 19,
18993–19006.
[40] V. Amarnath, D. C. Anthony, K. Amarnath, W. M. Valentine, L. A. Wetter-
au, D. G. Graham, J. Org. Chem. 1991, 56, 6924–6931.
[41] L. Zhang, T. Gavin, A. P. DeCaprio, R. M. LoPachin, Toxicol. Sci. 2010, 117,
180–189.
[42] M. J. Schnermann, C. M. Beaudry, N. E. Genung, S. M. Canham, N. L. Un-
tiedt, B. D. W. Karanikolas, C. Sutterlin, L. E. Overman, J. Am. Chem. Soc.
2011, 133, 17494–17503.
[1] A. Ohsuka, Nippon Kagaku Zasshi 1963, 84, 748–752.
[2] M. Jonassohn, O. Sterner, Trends Org. Chem. 1997, 6, 23–43.
[3] V. Caprioli, G. Cimino, R. Colle, M. Gavagnin, G. Sodano, A. Spinella, J.
Nat. Prod. 1987, 50, 146–151.
[4] K. Nakanishi, I. Kubo, Isr. J. Chem. 1977, 16, 28–31.
[5] G. Cimino, S. De Rosa, S. De Stefano, G. Sodano, Comp. Biochem. Physiol.
1982, 73B, 471–474.
[6] I. Kubo, I. Ganjian, Experientia 1981, 37, 1063–1064.
[7] I. Kubo, K. Nakanishi in Advances in Pesticide Science, Part 2 (Ed.: H.
Geissbuhler), Pergamon Press, New York, 1979, p. 284.
[8] G. Cimino, A. Spinella, G. Sodano, Tetrahedron Lett. 1984, 25, 4151–
4152.
[9] M. D’Ischia, G. Prota, G. Sodano, Tetrahedron Lett. 1982, 23, 3295–3298.
[10] G. Cimino, G. Sodano, A. Spinella, Tetrahedron 1987, 43, 5401–5410.
[11] F. M. da Cunha, T. S. Frode, G. L. Mendes, A. Malheiros, V. C. Filho, R. A.
Yunes, J. B. Calixto, Life Sci. 2001, 70, 159–169.
[12] S. H. Lee, J. R. Lee, C. S. Lunde, I. Kubo, Planta Med. 1999, 65, 204–208.
[13] O. Sterner, A. Szallasi, Trends Pharmacol. Sci. 1999, 20, 459–465.
[14] E. Andre, J. Ferreira, A. Malheiros, R. A. Yunes, J. B. Calixto, Neurophar-
macology 2004, 46, 590–597.
[15] C. Della Monica, L. De Petrocellis, V. Di Marzo, R. Landi, I. Izzo, A. Spine-
lla, Bioorg. Med. Chem. Lett. 2007, 17, 6444–6447.
[16] E. AndrØ, B. Campi, M. Trevisani, J. Ferreira, A. Malheiros, R. A. Yunes,
J. B. Calixto, P. Geppetti, Biochem. Pharmacol. 2006, 71, 1248–1254.
[17] M. D’Acunto, C. D. Monica, I. Izzo, L. De Petrocellis, V. di Marzo, A. Spine-
lla, Tetrahedron 2010, 66, 9785–9789.
[18] Y. Iwasaki, M. Tanabe, Y. Kayama, M. Abe, M. Kashio, K. Koizumi, Y. Oku-
mura, Y. Morimitsu, M. Tominaga, Y. Ozawa, T. Watanabe, Life Sci. 2009,
85, 60–69.
[43] A. F. Barrero, M. CortØs, E. A. Manzaneda, E. Cabrera, R. Chanboun, M.
Lara, A. R. Rivas, J. Nat. Prod. 1999, 62, 1488–1491.
[44] M. A. Cuellar, L. E. Moreno, M. D. Preite, ARKIVOC 2003, 10, 169–177.
[45] H. Keshmiri-Neghab, B. Goliaei, Pharm. Biol. 2014, 52, 124–128.
[46] F. Lefranc, G. Nuzzo, N. A. Hamdy, I. Fakhr, L. Moreno, Y. Banuls, G. Van
Goietsenoven, G. Villani, V. Mathieu, R. van Soest, R. Kiss, M. L. Ciavatta,
J. Nat. Prod. 2013, 76, 1541–1547.
[47] A. Mathieum, M. Remmelink, N. D’Haene, S. Penant, J. F. Gaussin, R. Van
Ginckel, F. Darro, R. Kiss, I. Salmon, Cancer 2004, 101, 1908–1918.
[48] V. Mathieu, C. Pirker, E. Martin de Lasalle, M. Vernier, T. Mijatovic, N.
De Neve, J. F. Gaussin, M. Dehoux, F. Lefranc, W. Berger, R. Kiss, J. Cell.
Mol. Med. 2009, 13, 3960–3972.
[49] L. V. Frolova, I. V. Magedov, A. E. Romero, M. Karki, I. Otero, K. Hayden,
N. M. Evdokimov, L. M. Y. Banuls, S. K. Rastogi, W. R. Smith, S. L. Lu, R.
Kiss, C. B. Shuster, E. Hamel, T. Betancourt, S. Rogelj, A. Kornienko, J.
Med. Chem. 2013, 56, 6886–6900.
[50] A. V. Aksenov, A. N. Smirnov, I. V. Magedov, M. R. Reisenaur, N. A. Akse-
nov, I. V. Aksenova, G. Nguyen, R. K. Johnston, M. Rubin, R. Kiss, V. Ma-
thieu, F. Lefranc, J. Correa, D. A. Cavazos, A. J. Brenner, S. Rogelj, A. Kor-
nienko, L. V. Frolova, J. Med. Chem. 2015, 58, 2206–2220.
[51] M. Saraswathy, S. Q. Gong, Biotechnol. Adv. 2013, 31, 1397–1407.
[52] G. K. Chen, G. E. Duran, A. Mangili, Br. J. Cancer 2000, 83, 892–898.
[19] G. L. Mendes, A. R. Santos, M. M. Campos, K. S. Tratsk, R. A. Yunes, V. Ce-
chinel Filho, J. B. Calixto, Life Sci. 1998, 63, 369–381.
[20] M. J. Caterina, M. A. Schumacher, M. Tominaga, T. A. Rosen, J. D. Levine,
D. Julius, Nature 1997, 389, 816–824.
[21] D. Julius, A. I. Basbaum, Nature 2001, 413, 203–210.
[22] M. Trevisani, D. Smart, M. J. Gunthorpe, M. Tognetto, M. Barbieri, B.
Campi, S. Amadesi, J. Gray, J. C. Jerman, S. J. Brough, D. G. D. Owen
Smith, A. D. Randall, S. Harrison, A. Bianchi, J. B. Davis, P. Geppetti, Nat.
Neurosci. 2002, 5, 546–551.
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2025
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[53] R. Geney, M. Ungureanu, D. Li, I. Ojima, Clin. Chem. Lab. Med. 2002, 40,
918–925.
[70] M. H. Potashman, M. E. Duggan, J. Med. Chem. 2009, 52, 1231–1246.
[71] J. G. Robertson, Biochemistry 2005, 44, 5561–5571.
[54] W. G. Harker, B. I. Sikic, Cancer Res. 1985, 45, 4091–4096.
[55] S. K. Singh, C. Hawkins, I. D. Clarke, J. A. Squire, J. Bayani, T. Hide, R. M.
Henkelman, M. D. Cusimano, B. P. Dirks, Nature 2004, 432, 396–401.
[56] X. Yuan, J. Curtin, Y. Xiong, G. Liu, S. Waschsmann-Hogiu, K. L. Black, J. S.
Yu, Oncogene 2004, 23, 9392–9400.
[57] R. Galli, E. Binda, U. Orfanelli, B. Cipelletti, A. Gritti, S. De Vitis, R. Fiocco,
C. Foroni, F. Dimeco, A. Vescovi, Cancer Res. 2004, 64, 7011–7021.
[58] J. Lee, S. Kotliarova, Y. Kotliarov, A. Li, Q. Su, N. M. Donin, S. Pastorino,
B. W. Purow, N. Christopher, W. Zhang, J. K. Park, H. A. Fine, Cancer Cell
2006, 9, 391–403.
[59] S. Bao, Q. Wu, R. E. McLendon, Y. Hao, Q. Shi, A. B. Hjelmeland, M. W.
Dewhirst, D. D. Bigner, J. M. Rich, Nature 2006, 444, 756–760.
[60] G. Liu, X. Yuan, Z. Zeng, P. Tunici, H. Ng, I. R. Abdulkadir, L. Lu, D. Irvin,
K. L. Black, J. S. Yu, Mol. Cancer 2006, 5, 67.
[61] T. C. Johannessen, R. Bjerkvig, B. B. Tysnes, Cancer Treat. Rev. 2008, 34,
558–567.
[72] J. Singh, R. C. Petter, T. A. Baillie, A. Whitty, Nat. Rev. Drug Discovery
2011, 10, 307–317.
[73] E. L. Kwak, R. Sordella, D. W. Bell, N. Godin-Heymann, R. A. Okimoto,
B. W. Brannigan, P. L. Harris, D. R. Driscoll, P. Fidias, T. J. Lynch, S. K. Ra-
bindran, J. P. McGinnis, A. Wissner, S. V. Sharma, K. J. Isselbacher, J. Set-
tleman, D. A. Haber, Proc. Natl. Acad. Sci. USA 2005, 102, 7665–7670.
[74] W. Pao, V. A. Miller, K. A. Politi, G. J. Riely, R. Somwar, M. F. Zakowski,
M. J. Kris, H. Varmus, PLoS Med. 2005, 2, 225–235.
[75] T. A. Carter, L. M. Wodicka, N. P. Shah, A. M. Velasco, M. A. Fabian, D. K.
Treiber, Z. V. Milanov, C. E. Atteridge, W. H. Biggs, P. T. Edeen, M. Floyd,
J. M. Ford, R. M. Grotzfeld, S. Herrgard, D. E. Insko, S. A. Mehta, H. K.
Patel, W. Pao, C. L. Sawyers, H. Varmus, P. P. Zarrinkar, D. J. Lockhart,
Proc. Natl. Acad. Sci. USA 2005, 102, 11011–11016.
[76] R. Dasari, L. M. Y. Banuls, M. Masi, S. C. Pelly, V. Mathieu, I. R. Green,
W. A. L. van Otterlo, A. Evidente, R. Kiss, A. Kornienko, Bioorg. Med.
Chem. Lett. 2014, 24, 923–927.
[62] S. Ma, T. K. Lee, B. J. Zheng, K. W. Chan, X. Y. Guan, Oncogene 2008, 27,
1749–1758.
[63] S. Purkait, P. Jha, M. C. Sharma, V. Suri, M. Sharma, S. S. Kale, C. Sarkar,
Neuropathology 2013, 33, 405–412.
[77] R. Dasari, A. Kornienko, Chem. Heterocycl. Compd. 2014, 50, 139–144.
[78] R. Scott, M. Karki, M. R. Reisenauer, R. Rodrigues, R. Dasari, W. R. Smith,
S. C. Pelly, W. A. L. van Otterlo, C. B. Shuster, S. Rogelj, I. V. Magedov, L. V.
Frolova, A. Kornienko, ChemMedChem 2014, 9, 1428–1435.
[64] H. K. Gan, A. H. Kaye, R. B. Luwor, J. Clin. Neurosci. 2009, 16, 748–754.
[65] P. Guo, B. Hu, W. Gu, L. Xu, D. Wang, H. J. S. Huang, W. K. Cavenee, S. C.
Cheng, Am. J. Pathol. 2003, 162, 1083–1093.
[79] A. Szallasi, T. Biro, S. Modarres, L. Garlaschelli, M. Petersen, A. Klusch, G.
Vidari, M. Jonassohn, S. De Rosa, O. Sterner, P. M. Blumberg, J. E. Krause,
Eur. J. Pharmacol. 1998, 356, 81–89.
[66] A. Ligresti, A. S. Moriello, K. Starowicz, I. Matias, S. Pisanti, L. De Petrocel-
lis, C. Laezza, G. Portella, M. Bifulco, V. Di Marzo, J. Pharmacol. Exp. Ther.
2006, 318, 1375–1387.
[80] M. F. Rossato, G. Trevisan, C. I. Walker, J. Z. Klafke, A. P. de Oliveira, J. G.
Villarinho, R. B. Zanon, L. F. Royes, M. L. Athayde, M. V. Gomez, J. Ferre-
ira, Biochem Pharmacol. 2011, 81, 544–551.
[67] C. A. Reilly, J. L. Taylor, D. L. Lanza, B. A. Carr, D. J. Crouch, G. S. Yost, Toxi-
col. Sci. 2003, 73, 170–181.
[68] R. Sancho, L. de La Vega, G. Appendino, V. Di Marzo, A. Macho, E.
Munoz, Br. J. Pharmacol. 2003, 140, 1035–1044.
Received: August 9, 2015
[69] E. Cao, M. Liao, D. Cheng, D. Julius, Nature 2013, 504, 113–118.
Published online on October 5, 2015
ChemMedChem 2015, 10, 2014 – 2026
www.chemmedchem.org
2026
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim