3-Ylidenephthalides as a new class of transient receptor potential channel TRPA1 and TRPM8 modulators

Article abstract of DOI:10.1016/j.bmcl.2013.08.039

Following the recent identification of the naturally occurring 3-ylidene-4,5-dihydrophthalide ligustilide and its oxidation product dehydroligustilide as novel TRPA1 modulators, a series of seventeen 3-ylidenephthalides was synthesized and tested on TRPA1 and TRPM8 channels. Most of these compounds acted as strong modulators of the two channel types with EC₅₀ and/or IC₅₀ values distinctly lower than those of the reference compounds.

Full text of DOI:10.1016/j.bmcl.2013.08.039

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5614–5618
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Ylidenephthalides as a new class of transient receptor potential
channel TRPA1 and TRPM8 modulators
Giorgio Ortar a, , Aniello Schiano Moriello b,c, Enrico Morera a, Marianna Nalli a, Vincenzo Di Marzo c,
⇑
Luciano De Petrocellis b
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, piazzale Aldo Moro 5, 00185 Roma, Italy
b Endocannabinoid Research Group, Istituto di Cibernetica, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
c Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Following the recent identification of the naturally occurring 3-ylidene-4,5-dihydrophthalide ligustilide
and its oxidation product dehydroligustilide as novel TRPA1 modulators, a series of seventeen 3-ylide-
nephthalides was synthesized and tested on TRPA1 and TRPM8 channels. Most of these compounds acted
as strong modulators of the two channel types with EC50 and/or IC50 values distinctly lower than those of
the reference compounds.
Received 5 July 2013
Revised 5 August 2013
Accepted 7 August 2013
Available online 14 August 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Transient receptor potential channels
TRPA1
TRPM8
3-Ylidenephthalides
3-Ylidenephthalides [3-ylidene-1(3H)-isobenzofuranones]
represent an important group of both naturally occurring and syn-
thetic products endowed with remarkably wide-ranging biological
activities (Fig. 1).1 Most of them exist in the thermodynamically
more stable Z-configuration. In fact, (E)-3-ylidenephthalides have
appeared less frequently in the chemical literature, being isolated
with difficulty from E–Z mixtures in which the more stable
Z-isomers predominated.2 Typically, the content of (Z)-3-butylidene-
4,5-dihydrophthalide [(Z)-ligustilide] is about ten times higher
than that of the E-isomer in medicinal plants from the traditional
chinese medicine.3 The biological activity of ligustilide, which is
a major component of the aromas of celery (Apium graveolens)
and lovage (Levisticum officinale), has been extensively investigated
revealing an impressive pleiotropic pharmacological profile that
includes, inter alia, reduction of cerebral infarct volume and
improvement of neurobehavioral deficits, attenuation of lipop-
olysaccaride (LPS)-induced endotoxic shock, inhibition of vascular
smooth muscle cell proliferation, antioxidant, antiapoptotic, anti-
thrombotic, antinflammatory, and analgesic effects.3 The ability
of ligustilide to penetrate the brain when administered through
the nasal route deserves a special mention, because possibly rele-
vant to its neuroprotective actions.4
OH
O
O
O
O
O
O
Senkyunolide-B
Ligustilide
Senkyunolide-A
(both isomers)
OH
HO
O
O
O
O
O
O
Senkyunolide-C
Dehydroligustilide
Senkyunolide-E
(both isomers)
OH
H
O
O
O
O
O
O
Butylphthalide
Neocnidilide
Chuangxinol
(both isomers)
Recently, ligustilide has been reported to behave as a transient
Figure 1. Structures of some representative 3-ylidenephthalides and related
receptor potential ankyrin type-1 (TRPA1) agonist (EC50 = 44 lM),
compounds.
⇑
with only modest desensitizing properties on mustard oil
(MO)-induced activation of this channel.5 This profile of activity
Corresponding author. Tel.: +39 6 49913612; fax: +39 6 49913133.
E-mail address: giorgio.ortar@uniroma1.it (G. Ortar).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.039

G. Ortar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5614–5618
5615
O
compounds was able to activate significantly these channels
but, when TRPM8 was activated by menthol, a potent inhibition
was observed by application of either compound. These observa-
tions indicate that 3-ylidenephthalide could be a new and useful
template for the identification of TRPA1 and TRPM8 modulators.
Therefore, we decided, as a continuation of our ongoing interest
in the SAR exploration of natural product ligands of TRP chan-
nels,6 to prepare and test on TRPA1 and TRPM8 channels several
representative 3-ylidenephthalides, including dehydroligustilide
(compound 4j, Table 1).
TRPA1 and TRPM8 channels are involved in a wide array of
physiological functions and their dysfunction can cause various
pathological conditions, including pain, cancer and bladder, respi-
ratory and skeletal disorders.7 As a consequence, modulation of
TRPA1 and TRPM8 channels provides an attractive approach for
the treatment of the aforementioned ‘channelopathies’ and indeed
have been among the most actively investigated drug targets with-
in the TRP channel realm over the past few years.
R1
R2
R1
R2
R4
O
a
b
R4
O
OH
R3
R3
1
2
O
R4
R1
R2
R4
R1
R2
c
O
O
OTf
R3
R3
4
3
Scheme 1. Synthesis of compounds 4. Reagents and conditions: (a) AlCl3, 100 °C,
1 h; (b) (CF3SO2)2O, 2,6-di-tert-butyl-4-methylpyridine, CH2Cl2, rt, 16 h; (c) CO,
Pd(OAc)2 (dppp), Et3N (or K2CO3), DMF (or toluene), 60 °C (or 100 °C), 1–4 h.
is reversed by aromatization to dehydroligustilide, which acts as
The 3-ylidenephthalides 4 were synthesized by a palladium-
catalyzed cyclocarbonylation of 2-triflyloxyacetophenone deriva-
tives 3, an original approach to 3-ylidenephthalides disclosed by
us 20 years ago,8 which still offers distinct advantages over related
a potent blocker of MO-activated currents (IC50 = 23 lM). The ef-
fects of ligustilide and dehydroligustilide on mouse transient
receptor potential vanilloid type-1 (TRPV1) and melastatin
type-8 (TRPM8) channels have been tested as well. None of the
Table 1
Results of TRPA1 and TRPM8 assays of 3-ylidenephthalides 4a
Compound
TRPA1
TRPA1
(EC50
TRPA1
(IC50
TRPM8
TRPM8
TRPM8
(IC50
TRPM8
(IC50, l
M)f
(efficacy)b
,
l
M)
,
l
M)c
(efficacy)d
(EC50
,
l
M)
,
l
M)e
O
50.5 1.4
28.6 1.0
11.3 0.4
85.1 1.9
10.2 0.5
24.5 3.2
24.2 3.6
50.8 1.2
17.2 1.3
32.2 0.1
>100
>100
>100
<10
<10
<10
<10
<10
ND
>200
>200
>200
>200
>200
>200
O
4a
O
ND
ND
ND
ND
O
4b
O
O
4c
4d
4e
O
33.95 1.7
90.1 1.8
128.4 3.7
O
O
>100
>200
>200
F
O
O
MeO
10.1 0.2
42.8 0.1
>100
<10
ND
>200
>200
O
4f
(continued on next page)

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G. Ortar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5614–5618
Table 1 (continued)
Compound
TRPA1
TRPA1
(EC50
TRPA1
(IC50
TRPM8
TRPM8
(EC50
TRPM8
(IC50
TRPM8
(IC50, l
M)f
(efficacy)b
,
l
M)
,
l
M)c
(efficacy)d
,
lM)
,
l
M)e
Cl
O
20.7 0.5
4.7 0.5
46.7 7.2
<10
ND
74.2 1.6
135.5 14.1
O
4g
4h
4i
MeO
O
24.6 0.7
21.7 2.9
>100
<10
ND
ND
ND
ND
161.4 3.3
>200
O
O
43.7 1.5
15.8 2.2
36.3 4.3
44.5 3.8
3.3 0.3
<10
<10
<10
7.7 0.3
140.7 0.3
2.5 0.1
12.1 0.2
153.3 5.6
6.8 0.1
O
O
<10
ND
O
4j
O
94.3 1.9
2.8 0.2
O
4k
O
93.6 6.3
3.0 0.9
2.9 0.2
<10
ND
8.3 0.4
10.2 0.1
O
4l
O
117.1 2.9
1.1 0.1
0.80 0.03
<10
ND
1.0 0.1
1.9 0.1
O
4m
O
125.7 2.6
4.3 0.4
2.4 0.1
<10
ND
7.2 0.4
25.1 2.6
O
4n

G. Ortar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5614–5618
5617
Table 1 (continued)
Compound
TRPA1
TRPA1
(EC50
TRPA1
(IC50
TRPM8
TRPM8
(EC50
TRPM8
(IC50
TRPM8
(IC50, l
M)f
(efficacy)b
,
l
M)
,
l
M)c
(efficacy)d
,
lM)
,
l
M)e
Cl
O
111.6 1.3
2.0 0.1
1.5 0.1
<10
ND
14.9 1.7
48.1 0.3
O
4o
OMe
O
65.1 0.5
1.3 0.1
2.2 0.2
12.9 2.7
1.8 0.1
3.4 0.5
11.3 0.1
O
4p
O
O
103.0 1.5
3.1 0.2
3.2 0.2
<10
ND
2.4 0.1
4.4 0.5
4q
a
Data are means SEM of N = 3 determinations.
b
c
d
e
f
As percent of the effect of allyl isothiocyanate (100 lM).
Determined against the effect of allyl isothiocyanate (100
As percent of the effect of ionomycin (4 M).
Determined against the effect of icilin (0.25
Determined against the effect of menthol (50
lM).
l
lM).
lM). ND, not determined when efficacy is lower than 10%.
transition metal-catalyzed heteroannulation reactions in terms of
starting materials availability (Scheme 1).9 The cyclocarbonylation
of triflates 3a–k,q was carried out at 60 °C under a CO atmosphere
in DMF as solvent, using Pd(OAc)2 as catalyst, 1,3-bis(diphenylph-
oshino)propane (dppp) as ligand, and Et3N as base. The reaction of
triflates 3l–p required somewhat different conditions (toluene as
solvent, K2CO3 as base, 100 °C) because, under standard conditions,
3l was completely rearranged to the corresponding triflone 5
through a base-catalyzed process (Scheme 2).8,10 Assignment of
(Z)-configuration to phthalides 4h–q was based on 1H NMR data,
in particular the chemical shift of the C-8 olefinic proton.1h,2b,9,11
In addition, phthalides 4j and 4l exhibited physical properties well
in agreement with those reported in literature for known
Z-isomers.11f,12
Triflates 3 were in turn obtained by triflation of the correspond-
ing phenols 2. Non-commercially available 1-(2-hydroxy-
aryl)alkan-1-ones 2d, h–q were prepared by a AlCl3-promoted
Fries rearrangement of aryl esters 1d, h–q.
The 17 phthalides synthesized were tested for their ability to in-
duce Ca2+ elevation in HEK293 cells stably transfected with either
the rat TRPA1 or the rat TRPM8 cDNAs (Table 1).13 Control exper-
iments were carried out using non-transfected HEK293 cells. The
antagonist or desensitizing activity was evaluated by adding the
test compounds 5 min before stimulation of cells with reference
agonists.
3-Methylenephthalides 4a–g produced a moderate activation of
TRPA1 with efficiencies between 10.1% and 85.1% and EC50 values
between 4.7 and 50.8 lM and behaved, with the exception of 4d, g,
as weak TRPA1 desensitizers. They were, again with the exception
of 4d, g, essentially inactive in TRPM8 activity assays. The best
result in terms of activation and subsequent desensitization
properties towards TRPA1 was observed with the 5,7-dimethyl-
substituted phthalide 4d, followed by 5-chloro-6-methyl-3-meth-
ylenephtahilde 4g, a result that supports the hypothesis that
dehydroligustilide might bind to a hydrophobic pocket via a
non-covalent mechanism.5
Homologation of the methylene moiety, while maintaining the
5,7-dimethyl substitution (compounds 4i, k), greatly increased the
ability to elicit a ‘true’ antagonistic response on TRPM8 channels
(that is, inhibition without agonism per se, and hence not due to
desensitization) with IC50 values 610 lM. Phthalide 4k also dis-
played single-digit micromolar EC50 and IC50 values (2.8 and 3.3,
respectively), on TRPA1 channels.
In comparison with 4k, and in agreement with literature data,5
dehydroligustilide (4j) which lacks the 5,7-dimethyl substitution,
inhibited MO-induced activation of TRPA1 with a IC50 value of
44.5 lM, with no appreciable activating capacity towards both
TRPA1 and TRPM8 channels. Furthermore, preincubation of
TRPM8-HEK293 cells with dehydroligustilide and then continued
incubation with either icilin or menthol caused only a rather
modest inhibition of the TRPM8 response to these agonists, with
O
O
O
IC50 values ꢀ150
l
M. Compared to literature data,5 4j was thus
SO2CF3
OH
SO2CF3
found to be a weak TRPM8 inhibitor and this discrepancy could
be due to the use of TRPM8 from a different species. The large
activity gain observed following the introduction of two methyl
groups in dehydroligustilide is noteworthy and may be tenta-
5
3l
Scheme 2. Base-catalyzed rearrangement of triflate 3l to triflone 5.

5618
G. Ortar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5614–5618
with brine and extracted with AcOEt, washed twice with brine, dried (Na2SO4),
and evaporated under vacuum. The residue was purified by silica gel column
chromatography eluting with light petroleum/AcOEt mixtures. Method B (for
triflates 3l–p): a mixture of triflate 3 (1 mmol), K2CO3 (138 mg, 1 mmol),
Pd(OAc)2 (22 mg, 0.1 mmol), and 1,3-bis(diphenylphosphino)propane (41 mg,
0.1 mmol) in toluene (6 mL) was purged with carbon monoxide for 5 min and
then stirred under a CO balloon at 100 °C for 2–4 h. The reaction mixture was
then worked-up as in method A. Data for selected compounds: compound 4k:
tively ascribed to an improvement of the fitting into a lipophilic
region of the channel.
A profile similar to that of 4k was exhibited by the 3-(2-phen-
ylethylidene)phthalide 4q and by most of the 3-benzylidenephtha-
lides (compounds 4l–p), which acted as robust TRPA1 activators
and desensitizers and TRPM8 antagonists. Again, the 5,7-dimethyl
substitution proved to be beneficial for both TRPA1 and TRPM8
modulating properties (compare compounds 4l and 4m), while
para-substituents on the benzylidene phenyl ring appeared to ex-
ert only little influence on activity, suggesting again a non-covalent
binding.
In conclusion, in this Letter we have presented a series of 3-
ylidenephtahlides that act as strong modulators of TRPA1 and/or
TRPM8 channels with EC50 and/or IC50 values distinctly lower than
those of ligustilide and dehydroligustilide. The 3-ylidenephthalide
structure qualifies as a versatile new template for the development
of novel TRP channel modulators.
yield 58%; mp 35–37 °C; IR (CHCl3) 1760, 1677, 1457, 1378 cmÀ1 1H NMR
;
(300 MHz, CDCl3) d 0.98 (3H, t, J = 7.5 Hz), 1.53 (2H, sextet, J = 7.5 Hz), 2.41 (2H,
q, J = 7.5 Hz), 2.43 (3H, s), 2.63 (3H, s), 5.54 (1H, t, J = 7.5 Hz), 7.05 (1H, s), 7.23
(1H, s); 13C NMR (75 MHz, CDCl3) d 13.80, 17.37, 21.96, 22.61, 27.72, 108.15,
117.24, 119.92, 132.16, 138.86, 140.50, 145.02, 145.60, 167.41. Compound 4l:
yield 82%; mp 92–93 °C; IR (CHCl3) 1769, 1661, 1610, 1474, 1352 cmÀ1 1H
,
NMR (300 MHz, CDCl3) d 6.43 (1H, s), 7.32–7.95 (9H, m); 13C NMR (75 MHz,
CDCl3) 107.07, 119.81, 123.40, 125.57, 128.42, 128.77, 129.77, 130.12,
133.08, 134.48, 140.60, 144.57, 167.06. Compound 4m: yield 15%; mp 140–
d
142 °C; IR (KBr) 1772, 1654, 1601 cmÀ1 1H NMR (300 MHz, CDCl3) d 2.46 (3H,
;
s), 2.65 (3H, s), 6.33 (1H, s), 7.09–7.84 (7H, m); 13C NMR (75 MHz, CDCl3) d
17.42, 22.03, 105.83, 117.47, 128.08, 128.71, 129.96, 132.67, 133.42, 139.24,
141.74, 144.62, 145.32, 167.20. Compound 4q: Yield 34%; mp 88–90 °C; IR
(neat) 1768, 1686, 1278, 1045 cmÀ1 1H NMR (300 MHz, CDCl3) d 3.81 (2H, d,
;
J = 7.8 Hz), 5.78 (1H, t, J = 7.8 Hz), 7.22–7.90 (9H, m); 13C NMR (75 MHz, CDCl3)
d 32.06, 107.73, 119.85, 124.57, 125.30, 126.50, 128.58, 128.69, 129.67, 134.34,
140.70, 145.72, 167.01.
References and notes
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stably over-expressing recombinant rat TRPA1 or rat TRPM8 were grown on
100 mm diameter Petri dishes as mono-layers in minimum essential medium
(EMEM) supplemented with non-essential amino acids, 10% foetal bovine
serum, and 2 mM glutamine, and maintained at 5% CO2 at 37 °C. Stable
expression of each channel was checked by quantitative PCR (data not shown).
The effect of the substances on intracellular Ca2+ concentration ([Ca2+]i) was
determined by using Fluo-4, a selective intracellular fluorescent probe for Ca2+
On the day of the experiment, cells were loaded for 1 h at room temperature
with the methyl ester Fluo-4-AM (4 M in dimethyl sulfoxide containing 0.02%
.
l
Pluronic F-127, Invitrogen) in EMEM without foetal bovine serum, then were
washed twice in Tyrode’s buffer (145 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl2,
1.2 mM MgCl2, 10 mM D-Glucose, and 10 mM HEPES, pH 7.4), resuspended in
the same buffer, and transferred (about 100,000 cells) to the quartz cuvette of
the spectrofluorimeter (Perkin-Elmer LS50B equipped with PTP-1 Fluorescence
Peltier System; PerkinElmer Life and Analytical Sciences, Waltham, MA, USA)
under continuous stirring. The changes in [Ca2+]i were determined before and
after the addition of various concentrations of test compounds by measuring
cell fluorescence (kEX = 488 nm, kEM = 516 nm) at 25 °C. Curve fitting
(sigmoidal dose–response variable slope) and parameter estimation were
performed with GraphPad PrismÒ (GraphPad Software Inc., San Diego, CA).
Potency was expressed as the concentration of test substances exerting a half-
maximal agonist effect (i.e., half-maximal increases in [Ca2+]i) (EC50). The
effects of TRPA1 agonists are expressed as a percentage of the effect obtained
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with 100
lM allyl isothiocyanate (AITC). In the case of TRPM8 assays, the
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maximum Ca2+ influx effect on [Ca2+
] observed with application of 4 lM
i
ionomycin (Alexis). When significant, the values of the effect on [Ca2+]i in wild-
type (i.e., not transfected with any construct) HEK293 cells were taken as
baseline and subtracted from the values obtained from transfected cells.
Antagonist/desensitizing behaviour was evaluated against AITC (100
lM) for
8. Ciattini, P. G.; Mastropietro, G.; Morera, E.; Ortar, G. Tetrahedron Lett. 1993, 34,
3763.
TRPA1, icilin (0.25 M) and menthol (50 M) for TRPM8, by adding the test
l
l
compounds in the quartz cuvette 5 min before stimulation of cells with
agonists. Data are expressed as the concentration exerting a half-maximal
9. Rambabu, D.; Pavan Kumar, G.; Deepak Kumar, B.; Kapavarapu, R.;
Basaveswara Rao, M. V; Pal, M. Tetrahedron Lett. 2013, 54, 2989. and
references therein.
10. General procedure for the synthesis of compounds 4. Method A (for triflates 3a–k, q):
a mixture of triflate 3 (1 mmol), Et3N (0.28 mL, 2 mmol), Pd(OAc)2 (7 mg,
0.03 mmol), and 1,3-bis(diphenylphosphino)propane (12 mg, 0.03 mmol) in
DMF (3 mL) was purged with carbon monoxide for 5 min and then stirred
under a CO balloon at 60 °C for 1–4 h. The reaction mixture was then diluted
inhibition of agonist-induced [Ca2+
] elevation (IC50), which was calculated
i
again using GraphPad PrismÒ software. The effect on [Ca2+]i exerted by agonist
alone was taken as 100%. Dose–response curves were fitted by a sigmoidal
regression with variable slope. All determinations were performed at least in
triplicate. Statistical analysis of the data was performed by analysis of variance
at each point using ANOVA followed by the Bonferroni’s test.