Elongation of the Hydrophobic Chain as a Molecular Switch: Discovery of Capsaicin Derivatives and Endogenous Lipids as Potent Transient Receptor Potential Vanilloid Channel 2 Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.8b00734

The transient receptor potential vanilloid type-2 (TRPV2) protein is a nonselective Ca²⁺ permeable channel member of the TRPV subfamily, still considered an orphan TRP channel due to the scarcity of available selective and potent pharmacological tools and endogenous modulators. Here we describe the discovery of novel synthetic long-chain capsaicin derivatives as potent TRPV2 antagonists in comparison to the totally inactive capsaicin, the role of their hydrophobic chain, and how the structure-activity relationships of such derivatives led, through a ligand-based approach, to the identification of endogenous long-chain fatty acid ethanolamides or primary amides acting as TRPV2 antagonists. Both synthetic and endogenous antagonists exhibited differential inhibition against known TRPV2 agonists characterized by distinct kinetic profiles. These findings represent the first example of both synthetic and naturally occurring TRPV2 modulators with efficacy in the submicromolar/low-micromolar range, which will be useful for clarifying the physiopathological roles of this receptor, its regulation, and its targeting in pathological conditions.

Full text of DOI:10.1021/acs.jmedchem.8b00734

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Article
Elongation of the hydrophobic chain as a molecular switch:
discovery of capsaicin derivatives and endogenous lipids as potent
Transient Receptor Potential Vanilloid Channel 2 antagonists
Aniello Schiano Moriello, Silvia Lopez Chinarro, Olalla Novo Fernández, Jordi Eras, Pietro Amodeo,
Ramon Canela-Garayoa, Rosa Maria Vitale, Vincenzo Di Marzo, and Luciano De Petrocellis
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00734 • Publication Date (Web): 03 Sep 2018
Downloaded from http://pubs.acs.org on September 4, 2018
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Elongation of the hydrophobic chain as a molecular switch: discovery of capsaicin
derivatives and endogenous lipids as potent Transient Receptor Potential Vanilloid
Channel 2 antagonists
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‡⁋ ⁋
⁋†
⁋†
†
Aniello Schiano Moriello , Silvia Lopez Chinarro , Olalla Novo Fernández , Jordi Eras , Pietro
§
†
§*
‡§#
Amodeo , Ramon CanelaꢀGarayoa , Rosa Maria Vitale , Vincenzo Di Marzo
& Luciano De
‡
§*
Petrocellis
‡
Endocannabinoid Research Group ꢀ Institute of Biomolecular Chemistry (ICB)ꢀNational Research
Council (CNR), Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy
Institute of Biomolecular Chemistry (ICB)ꢀNational Research Council (CNR), Via Campi Flegrei 34,
§
8
0078 Pozzuoli (NA), Italy
⁋
†
Epitech Group SpA, Saccolongo, Padova, Italy
Departament de Química, Universitat de LleidaꢀAgrotecnio Center, Avda. Alcalde Rovira Roure, ⁋9⁋,
Eꢀ25⁋98, Lleida, Spain
MicrobiomeꢀEndocannabinoidome Axis in Metabolic Health (CERCꢀMEND)
Université Laval, Quebec City, Canada
These authors contributed equally to the work
#
⁋
*
Corresponding authors: rmvitale@icb.cnr.it, ldepetrocellis@icb.cnr.it
Abstract
2+
The transient receptor potential vanilloid 2 (TRPV2) is a nonꢀselective Ca permeable channel
member of the TRPV subfamily, still considered an orphan TRP channel due to the scarcity of available
selective and potent pharmacological tools and endogenous modulators. Here we describe the
discovery of novel synthetic longꢀchain capsaicinꢀderivatives as potent TRPV2 antagonists in
comparison to the totally inactive capsaicin, the role of their hydrophobic chain, and how the structureꢀ
activity relationships of such derivatives led, through a ligandꢀbased approach, to the identification of
endogenous longꢀchain fatty acid ethanolamides or primary amides acting as TRPV2 antagonists. Both
synthetic and endogenous antagonists exhibited differential inhibition against known TRPV2 agonists
characterized by distinct kinetic profiles. These findings represent the first example of both synthetic
and naturallyꢀoccurring TRPV2 modulators with efficacy in the sub/lowꢀmicromolar range, which will
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be useful to clarify the physioꢀpathological roles of this receptor, its regulation, and its targeting in
pathological conditions.
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1
. Introduction
TRPV2 belongs to the polymodal transient receptor potential (TRP) superfamily of calciumꢀpermeable
nonꢀselective cation channels, activated by a wide variety of physical and chemical stimuli. Due to its
mechanosensor property, TRPV2 is considered a stretchꢀmodulated channel and a regulator of calcium
homeostasis in different tissues and organs, in particular the heart, where it is ⁋0ꢀfold more abundant
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⁋
than in skeletal muscle . Different lines of evidence suggest for TRPV2 a key role in physiological
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–4
cardiac function as well as in cardiomyopathies and dystrophic diseases . Besides the heart, TRPV2 is
also found in the brain, vascular smooth muscle cells, the gastrointestinal tract, macrophages and the
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7–9
urothelial tract , and it is involved in a number of physioꢀpathological processes , including cancer ,
⁋
0–⁋3
particularly of the urinary tract
.
Despite its biological and pharmacological relevance, TRPV2 is still considered an orphan TRP
channel due to the scarcity of selective drugs and known endogenous ligands. The 2ꢀ
aminoethoxydiphenyl borate (2APB) is one of the first nonꢀselective activators identified for rat
⁋4
TRPV2 (EC = ⁋29 ꢁM), although inactive at the human orthologue, suggesting a strong species
50
⁋
5,⁋6
9ꢀ
9
specificity
. Cannabis sativa derivatives such as ꢂ tetrahydrocannabinol (ꢂ ꢀTHC), cannabidiol
9
ꢀ
9
⁋7,⁋8
(CBD) and ꢂ tetrahydrocannabivarin (ꢂ ꢀTHCV) are TRPV2 activators
, and so is pꢀ(diꢀnꢀ
⁋
9
propylsulfamyl)ꢀbenzoic acid (Probenecid) . However, all these agonists are known to modulate other
20
TRP channels. Most TRPV channels are proposed to be modulated also by phosphoinositide lipids .
2
+
TRPV2ꢀmediated Ca
influx has been reported following stimulation by endogenous
2
⁋
lysophospholipids such as lysophosphatidylcoline (LPC) and lysophosphatidylinositol (LPI) , LPC
22
being a relatively potent activator (EC = 3.4 ꢁM) . To date, the nature of endogenous regulators of
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0
2
3
TRPV2 activity still remains elusive .
Also synthetic inhibitors of TRPV2 are either not specific or endowed with low potency, as exemplified
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by: Ruthenium red (IC = 0.6 ꢁM) a pore blocker that inhibits other twelve ion channels ; La and
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3+ 26
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⁋6
Gd ; citral; the alkylated imidazole SKF96365; tetraethylammonium and 4ꢀaminopyridine, two
2
+
potassium channel blockers; ⁋ꢀ(2ꢀ(trifluoromethyl) phenyl) imidazole, an inhibitor of capacitative Ca
⁋
6
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29–34
entry; and Tranilast , which has been used in several studies
, even though it has never been
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validated as TRPV2 antagonist.
TRPV2 shares high sequence identity (>50%) with TRPV⁋ but its threshold of activation by
2
4
temperature is higher (> 52 °C) , and, unlike TRPV⁋, is not sensitive to capsaicin. The recently solved
3
5,36
cryoꢀEM structures of both TRPV⁋ and TRPV2
, along with mutagenesis and computational studies,
showed that the TRPV⁋ binding site of capsaicin is not conserved in TRPV2. Furthermore, the
replacement of critical residues leads to a mutant (TRPV2ꢀQuad) against which capsaicin behaves as an
3
7
antagonist, rather than an agonist as in TRPV⁋ . These intriguing results prompted us to investigate a
series of capsaicinꢀderivatives, in which the vanillylamide polar head of capsaicin bears a longer alkyl
chain, featuring different length, unsaturation degree and type of polar substituents. The structureꢀ
activity relationship (SAR) of these synthetic compounds then suggested the screening of structurallyꢀ
related endogenous lipids sharing at least one functional group with the capsaicinꢀderivatives, with the
aim of finding new endogenous modulators.
2. Results
2
.1
Synthesis
Commercial fatty acids such as ricinoleic acid, oleic acid and palmitic acid were used as starting
material to synthesize the 23 compounds tested. Scheme 1 shows the synthesis of the α,βꢀunsaturated
ketone 5 by the rutheniumꢀcatalyzed oxidation in anhydrous toluene of the homoallylic alcohol of the
3
8
methyl ricinoleate 4. Shvo’s catalyst and acrolein were used as catalyst and hydrogen scavenger,
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respectively. The addition of bis(pinacolato)diboron (Bpin) to the enone 5 in presence of triꢀnꢀbutyl
2
n
40
4⁋
phosphine (P( Bu) ) yielded the βꢀboronketone 6 in 46% yield. Enzymatically controlled hydrolysis
3
®
of the methyl ester 6 with Novozym 435 lipase led to the carboxylic acid 7 quantitatively. This acid 7
was coupled, without any further purification, with 4ꢀhydroxyꢀ3ꢀmethoxybenzylamine hydrochloride 3
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by HATU and DIPEA in DMFanh. achieving the amide 8. The oxidative hydrolysis of the boron
substituent of the compound 8 led to the βꢀhydroxyketone 9 in a 76% yield (Scheme 1).
Scheme 1. Synthesis of compound 9.
4
3
The irradiation of alcohol 4 with diphenyl sulphide in isooctane in a photochemical reactor for 3 h led
to the isomer 10 in 37% yield after several recrystallizations at ꢀ30 °C. This compound was used to
synthesize two new longꢀchain Nꢀvanillylamides (12, 15). The hydrolysis of the methyl ester of 10 led
to the corresponding carboxylic acid 11. The subsequent coupling of 11 with the 4ꢀhydroxyꢀ3ꢀ
methoxybenzylamine hydrochloride 3 using the same conditions described above yielded compound 12
4
4
in a 34% yield. Compound 10 was also oxidized with CrO in pyridine to prepare the trans ketone 13
3
(
49% yield), which was enzymatically hydrolysed to synthesise the corresponding acid 14 in a 78%
yield. Subsequently, 14 was coupled with the vanillyl amine 3 to yield the (E)ꢀNꢀ(4ꢀhydroxyꢀ3ꢀ
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methoxybenzyl)ꢀ⁋2ꢀoxooctadecꢀ9ꢀenamide 15 after purification by liquid column chromatography
(⁋7% yield) (Scheme 2).
Scheme 2. Synthesis of compounds 12 and 15.
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Scheme 3 shows the synthesis of the sulphurꢀ and selenoꢀderivatives of 3. Mercaptopropionic acid 16
was coupled with 4ꢀhydroxyꢀ3ꢀmethoxybenzylamine hydrochloride 3 using HATU and DIPEA in DMF
anh achieving the amide 17 (74% yield). The synthesis of the selenoꢀderivatives started with
bromopropionic acid 18, which was treated with KSeCN in water: The neutralization with Na CO ,
2
3
yielded the selenocyanatopropionic acid 19 in 80% without purification. Finally, compound 19 was
coupled with the 4ꢀhydroxyꢀ3ꢀmethoxybenzylamine hydrochloride 3 to obtain compound 20 after
purification by liquid column chromatography (60% yield).
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Scheme 3. Synthesis of sulphur- and seleno-intermediates.
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Amide 17 was Sꢀalkylated with the previously synthesized alkylating derivatives 30a-c, 32 and 35 (see
supporting information) in DMF and triethylamine obtaining the longꢀchain Nꢀvanillylamides 39-43
and 45 in 4⁋ꢀ68% yield. NꢀVanillylamide 44 was successfully achieved after removing the TBDMS
protecting group with acetic acid at room temperature (8⁋% yield). New longꢀchain Nꢀvanillylamides
were obtained from compound 20, which was firstly treated with NaBH in ethanol at room
4
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5
temperature to remove the cyano protection and regenerate the selenol group. Subsequent Seꢀ
alkylation was carried out in oneꢀpot with the addition of diverse set of alkylating reagents (30a-c, 35
and 38). NꢀVanillylamides 46-49 and 51 were synthesized in 7⁋ꢀ87% yields. Compound 50 was
successfully prepared after removing the TBDMS protecting group with acetic acid at room
temperature (79% yield) (Scheme 4).
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Scheme 4. Synthesis of no-branched sulphur- and seleno-derivatives.
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Scheme 5 shows the synthesis of aminoꢀbranched analogues. The first step consisted in the treatment
of Lꢀcystine 21 or Lꢀselenocystine 25 with Boc O in presence of triethylamine to afford the protected
2
⁋
2
46,47
derivatives 22 and 26 (quantitative and 65% yield, respectively).
These compounds were coupled
with 4ꢀhydroxyꢀ3ꢀmethoxybenzylamine hydrochloride 3 using EDCI, HOBt and triethylamine (TEA)
in anhydrous DMF achieving the amides 23 and 27 (74% and 88% yield). The reduction of compound
n
2
3 with P( Bu) in wet dichloromethane afforded compound 24 in a 73% yield after purification by
3
liquid column chromatography. New longꢀchain Nꢀvanillylamides were afforded from compound 24,
which was Sꢀalkylated with the previously synthesized alkylating derivatives 30a-c and 32 in presence
of triethylamine obtaining the longꢀchain Nꢀvanillylamides 52, 53 and 54 in moderate yields (50ꢀ79%
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yield). The NꢀBoc deprotection was carried out using trifluoroacetic acid in dichloromethane yielding
Nꢀvanillylamides 55, 56 and 57 as trifluoroacetic salts in quantitative yields. Compound 27 was
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reduced with NaBH in ethanol at room temperature to cleave the diselenium bond. The Seꢀalkylation
4
was carried out with the addition of the alkylating derivatives 30a-b to afford the Nꢀvanillylamides 58
and 59 in 74ꢀ88% yields. Finally, The NꢀBoc deprotection was carried out using the same conditions
described above to afford the Nꢀvanillylamides 60 and 61 as trifluoroacetic salts.
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Scheme 5. Synthesis of amino-branched analogues.
O
O
O
O
R1ꢀ3
n
P( Bu)
NH
SH 30a-b or 32
NHBoc
NH
S
3
23
NHBoc
TEA
OH
OH
2
4, 73%
R (52, 71%), R (53, 50%), R (54, 79%)
1
2
3
R =
1
14
6
O
TFA
O
R1ꢀ3
R =
NH
NH
S
2
6
NH —CF COOH
2
3
O
OH
R =
3
R (55, quant.)
1
15
R (56, quant.)
R (57, quant.)
3
O
2
O
O
O
R1ꢀ2
O
R1ꢀ2
3
0a-b
NH
Se
TFA
Se
NH2—CF3COOH
27
NaBH4
NHBoc
OH
OH
R (60, quant.)
R (61, quant.)
1
R (58, 71%), R (59, 50%)
1
2
2
Acids 63a-b, which were previously obtained from the hydrolysis of their respective methyl esters 62a-
b (see supporting information), were coupled with the 4ꢀhydroxyꢀ3ꢀmethoxybenzylamine
hydrochloride 3 using HATU and DIPEA in anhydrous DMF achieving the amides 64 and 65 after
purification by liquid column chromatography (64 and 63% yield) (Scheme 6).
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Scheme 6. Synthesis of compounds 64 and 65.
O
O
NH
11
7
OH
R =
R =
(a)
64, 64%
9
9
6
3
RꢀCOOH
(b)
0
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9
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9
0
1
2
3
4
5
6
7
8
9
0
3
HATU
O
6
3a-b
O
NH
11
4
OH
65, 63%
Methyl palmitate 28a was treated with an excess of hydrazine hydrate in ethanol to synthesize the
palmitic acid hydrazide 66 (80% yield). The addition of the aromatic aldehyde vanillin 1 to compound
5
0
6
6 in presence of acetic acid in reflux conditions gave the Schiff's base compound 67 in 58% yield. A
similar compound was synthesized starting from oleaic acid 70, which was coupled to tertꢀbutyl
hydrazinecarboxylate 69 using HATU and DIPEA in DMF to yield the oleylhydrazide 71 in a 94%
yield. The NꢀBoc deprotection of oleylhydrazide 71 with TFA in DCM for 2 h led to oleylhydrazide 72
in 92% yield. Compound 72 refluxed with vanillin 1 in the presence of acetic acid in methanol
produced the Schiff base 73 in 22% yield (Scheme 7).
Scheme 7. Synthesis of compounds 67 and 73.
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2.2
Biological evaluation
2.2.1 Capsaicin-derivatives activate TRPV1 channel
5
⁋
The capsaicin scaffold (Figure 1)
can be ideally divided into three regions: head, neck and tail,
0
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9
0
formed by the vanillyl moiety, the amidic group and the lipophilic alkyl chain, respectively. Structural
variations, including incorporation of sulphur atom, into the head and the neckꢀregions have been
52–55
described in the literature
.
Figure 1. Chemical structure of capsaicin. The vanillyl head, the amide neck and hydrophobic tail
are shaded in yellow, cyan and grey, respectively.
Instead, the effect of a sulphur atom in the alkyl chain has been less investigated. The recent
5
6
57
availability of the 3D structure of TRPV⁋ along with mutagenesis studies allowed the identification
of the capsaicin binding site, where the alkyl chain is hosted in a phenylalanineꢀrich hydrophobic
region close to Thr550, a residue involved in Hꢀbond interaction with the ligand amide group. The
presence of a sulphur atom near the neck region should in principle lead to an increment of activity due
to favourable dipoleꢀdipole and aromaticꢀsulphur interactions. Since sulphur can be substituted with
selenium via isosteric replacement, we also synthetized the corresponding seleniumꢀanalogs. Selenium
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5
5
6
is an essential trace element whose role in medicine and biology is just starting to be elucidated. Some
seleniumꢀcontaining compounds have provided protection against many degenerative conditions,
including cancer. Thus, a series of novel capsaicinꢀderivatives, i.e. 9, 12, 15, 39, 46, 55, 60, 42, 57, 44,
56, 40, 45, 65, 41, 48, 64, 47, 61, 51, 50, 67, 73, whose structures are reported in Tables 1 and 2,
featuring the same “head” and “neck” as capsaicin but differing in length and nature of the hydrocarbon
tail, were tested on human TRPV⁋ heterologously expressed in human embryonic kidney (HEK)ꢀ293
cells by fluorometric assay (see Tables S1 and S2 in SI). The predicted activities as TRPV⁋ agonists
were confirmed for many compounds within the series, exhibiting EC50 values from highꢀ to subꢀ
nanomolar range. A SAR analysis of the results also disclosed the critical role of the region flanking the
amide group in modulating the activity. In fact, the insertion of a positive charge next to the amide
group was detrimental for activity (compounds 55-57 and 60), and the introduction of an imido group
between the aromatic moiety and the amido group led to totally inactive compounds (compounds 67
and 73). Conversely, the introduction of a single polar substituent (hydroxyl, ester or ketone) was wellꢀ
tolerated, and the introduction of a sulphur or selenium atom in the hydrophobic tail even improved the
0
1
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9
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0
1
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4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7
activity. However, on the basis of the antagonist activity exhibited by capsaicin on TRPV2 Quad , the
new compounds were also tested on TRPV2 to determine if the elongation and the functionalization of
the alkyl chain could elicit a functional response at this receptor.
2
.2.2 Capsaicin-derivatives inhibit TRPV2 channels activated by LPC
The activity of the synthesized capsaicinꢀderivatives on TRPV2 was evaluated in vitro. The assays
were conducted using a fluorometric assay with rat TRPV2 heterologously expressed in HEKꢀ293
2
+
cells. The tested compounds did not significantly activate TRPV2ꢀmediated Ca elevation in
transfected HEKꢀ293 cells. Instead, preincubation (5 min) of TRPV2ꢀHEKꢀ293 cells with different
concentrations of the tested compounds, followed by incubation with LPC (3 µM), caused inhibition of
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intracellular Ca elevation due to TRPV2 response to LPC. The corresponding IC values are reported
50
in Table 1.
The structureꢀactivity relationships (SARs) of these compounds suggested a critical influence on the
capability to exert TRPV2 antagonism of the alkyl chain and, in particular, of its hydrophobicity, length
and degree of unsaturation. Hydrophobicity is important since, as shown in Table 1, the activity
dramatically dropped after introduction in the chain of polar substituents such as hydroxyl, keto or ester
groups (these latter arising from esterification of the hydroxyl group), or their combinations (42, 44, 50,
0
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9
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9
0
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8
9
0
4
5, 51, 9, 12, 15). However, the presence of an amino group next to the amide (55, 60, 56, 61), which
had marginal effects for alreadyꢀactive compounds, by only slightly increasing their potency (60 vs 46),
was instead dramatic for those inactive compounds bearing a hydroxyl or an ester moiety in the alkyl
chain, whose activity was completely rescued (see 42 vs 57). The complete recovery of activity after
introduction of an amino group next to the amide in derivatives bearing a polar substituent in the alkyl
chain suggests that reinforcement of the polar interactions of the “head” avoids the competition with
the polarꢀsubstituted alkyl chain for interaction with receptor polar residues in a region where the polar
head, but not the alkyl chain, should be hosted to elicit a measurable effect. The chain is fairly more
tolerant to changes not substantially affecting the hydrophobicity of the alkyl group: replacement of
sulphur with selenium in the alkyl chain did not affect significantly ligand activity (39 vs 46); its
replacement with a carbon atom determined an increase in potency (64 vs 40/47). While polar
functionalization of the alkyl chain caused a dramatic drop of activity, amino or imino groups (67, 73)
were well tolerated in the region close to the amide moiety of capsaicin. In particular, the imino
derivatives were among the most active compounds within the series (IC50 = 0.28 and 0.⁋2 ꢁM;
respectively). Also length and unsaturation degree of the alkyl chain significantly affected the activities
of the tested compounds. The C⁋6:0 and C⁋8:0 saturated analogs were inactive, whereas the C20:0
derivative showed an IC50 =3.⁋ꢁM. The insertion of a single double bond in C⁋8 chain (Olvanil)
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dramatically increased the antagonism, with an IC = 0.⁋6 ꢁM.
5
0
Thus, the screening led to the identification of several very potent TRPV2 antagonists, exhibiting IC50
values in the subꢀ to lowꢀmicromolar range. This result is quite remarkable since, despite its close
homology to TRPV⁋, TRPV2 is insensitive to capsaicin, being the residues responsible for capsacin
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0
5
8
binding and receptor activation in TRPV⁋ not conserved in TRPV2.
The most striking result from the SAR of capsaicinꢀderivatives against LPC is that the elongation of the
alkyl chain of capsaicin causes a switch of such scaffold from inactivity towards potent antagonism at
rat recombinant TRPV2. Intriguingly, the dependence of TRPV2 modulation on the length of the ligand
alkyl chain has already been observed for lysophospholipids, which require a carbon chain longer than
2
⁋
C⁋2 to stimulate the receptor.
2.2.3 Capsaicin-derivatives inhibit TRPV2 channels activated by CBD
Due to different latency in the activation profile between LPC and cannabidiol (CBD) (see Figure 2),
we also investigated the effect of a representative panel of capsaicin derivatives against CBD, to
ascertain whether the inhibitory activity/potency would vary against agonists exhibiting different
kinetics of action. Also in this case, the assays were conducted using a fluorometric assay with
recombinant rat TRPV2 heterologously expressed HEKꢀ293 cells. The preincubation (5 min) of
TRPV2ꢀHEKꢀ293 cells with different concentrations of the tested compounds, followed by incubation
2
+
with CBD (2 µM), caused an inhibition of the Ca elevation due to the TRPV2 response to CBD. The
corresponding IC values of the tested compounds are reported in Table 1. While the trend identified
5
0
in LPC antagonism for capsaicin derivatives bearing all carbon atoms, selenium or sulphur was
substantially conserved, a different behavior was observed with those derivatives featuring polar
substituents (i.e. 50/51), since their activity against CBD was not negatively affected by these
functional groups, as instead observed against LPC. The iminoꢀderivatives 67 and 73 (see Table 2), i.e.
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6
the two most active compounds against LPC (0.28 and 0.⁋2 ꢁM, respectively), were less potent against
CBD (IC50 = 6.0 and 3.0 ꢁM, respectively). The trend of activity of C⁋6:0, C⁋8:0 and C⁋8:⁋
derivatives was similar to that observed for LPC, although C⁋8:⁋ (Olvanil) was less potent as an
antagonist (IC50 =⁋.7 ꢁM), whereas, differently from what observed with LPC, C20:0 was totally
inactive. These results demonstrate a dependence of the antagonist activity on the type of agonist
against which antagonism is tested.
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. TRPV2 is activated by LPC (3 µM) and CBD (2 µM). The graph shows the representative
2
+
traces of [Ca ] increase evoked by the two agonists in HEK293 cells overexpressing TRPV2.
i
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4
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5
5
6
Table 1. Antagonist potency of Capsaicinꢀlike compounds at TRPV2 against LPC (3 µM) and CBD
(
2 µM), reported as IC (µM).
50
Caps-like
Palvanil
Structure
LPC
CBD
>⁋0
>⁋0
>⁋0
a
(C⁋6:0)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Stevanil
(C⁋8:0)
>⁋0
Olvanil
(C⁋8:⁋)
0.⁋6±0.02
2.6±0.2
>⁋0
⁋.7±0.⁋
2.⁋±0.⁋
>⁋0
Livanil
(C⁋8:2)
9
(C⁋8:0)
1
2
>⁋0
7.5 ± ⁋.3
4.4 ± 0.3
>⁋0
(C⁋8:⁋)
15
>⁋0
(C⁋8:⁋)
Eicosavan
illamide
(C20:0)
3.⁋ ± 0.2
3.8 ± 0.8
b
39
nd
(C⁋9/S)
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Caps-like
46
Structure
LPC
CBD
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
O
4.3 ± 0.9
nd
O
(C⁋9/Se)
N
Se
H
HO
55
⁋.4 ± 0.2
nd
nd
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(C⁋9/S)
60
⁋.2 ± 0.03
(C⁋9/Se)
42
>⁋0
nd
nd
(C2⁋/S/O)
57
⁋.4 ± 0.⁋
(C2⁋/S/O)
44
>⁋0
nd
nd
nd
nd
(C2⁋/S:⁋)
56
⁋.9 ± 0.⁋
2.5 ± 0.⁋
>⁋0
(C2⁋/S:⁋)
40
(C2⁋/S:⁋)
45
O
(C2⁋/S:⁋)
O
O
O
N
S
H
HO
65
0.82 ± 0.⁋2 ⁋.8 ± 0.3
(C22:2)
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Caps-like
Structure
LPC
CBD
41
(C22:2)
⁋.4 ± 0.07
2.8 ± 0.4
48
⁋.4 ± 0.06
2.3 ± 0.⁋
(C22:2)
0
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8
9
0
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9
0
1
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6
7
8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
64
0.49 ± 0.07 ⁋.5 ± 0.2
(C22:⁋)
47
⁋.8 ± 0.0⁋
⁋.7 ± 0.0⁋
>⁋0
3.2 ± 0.2
0.98 ± 0.⁋4
2.3 ± 0.3
(C2⁋/Se:⁋)
61
(
C2⁋/Se:⁋)
51
O
O
(
C2⁋/Se:⁋)
O
O
N
Se
H
HO
50
>⁋0
⁋.4 ± 0.⁋
(C2⁋/Se:⁋)
a
In parenthesis, number of C atoms in the alkyl chain: number of unsaturations. When heteroatom X
b
occurs within alkyl chain, it is indicated as “/X”; nd: Not Determined
Table 2. Antagonist potency of Capsaicinꢀimino compounds at TRPV2 against LPC (3 µM) and
CBD (2 µM), reported as IC (µM).
5
0
Imino-caps
Structure
LPC
CBD
67
0.28 ± 0.04 6.0 ± ⁋.0
(⁋6:0)
73
0.⁋2 ± 0.0⁋ 3.0 ± 0.4
(⁋8:⁋)
a
In parenthesis, number of C atoms in the alkyl chain: number of unsaturations.
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2.2.4 Evaluation of endogenous lipids as potential TRPV2 antagonists
Since the activity of the tested compounds appears to critically depend on the nature of alkyl chain, but
is less affected by changes in the polar head, we decided to ascertain the role of the head group of
capsaicin, i.e. the vanillyl moiety, by testing a series of naturallyꢀoccurring lipids bearing different
polar heads and differing in length and unsaturation of the alkyl chain, in order to determine the
structural and functional requisites for TRPV2 modulation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.2.5 Long-chain ethanolamides exhibit differential inhibition of TRPV2 upon activation by
LPC or CBD
To evaluate the contribution of the aromatic moiety to the overall activity, a panel of natural occurring
ethanolamides differing in length and unsaturation degree was tested for both agonism and antagonism
at TRPV2, using both LPC and CBD as reference activators. Ethanolamides share with the tested
capsaicinꢀderivatives the nature of both the alkyl chain and the hydrophilic groups (amide and hydroxyl
moieties) in the polar head. The IC values (against CBD 2 ꢁM and LPC 3 ꢁM) are reported in Table
5
0
3. Ethanolamides featuring saturated alkyl chains, regardless of their lengths, were inactive against both
agonists, whereas the introduction of a single double bond was sufficient to switch from inactivity to
activity against both agonists (see PEA vs POEA, or SEA vs OEA), similarly to what already observed
for capsaicinꢀderivatives. However, while the C20:0 capsaicinꢀderivative was active against LPC, the
homolog ethanolamide was inactive. Moreover, while OEA was less active than the counterpart
Olvanil, LEA was more potent than Livanil against both reference agonists. Increasing the number of
double bonds increased the potency against CBD, but not LPC.
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Potency of Fatty Ethanolamides as functional antagonists at TRPV2 against LPC (3 µM)
and CBD (2 µM), reported as IC (µM).
5
0
Ethanolamides
Structure
LPC
>⁋0
CBD
>⁋0
a
b
PEA (C⁋6:0)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
POEA (C⁋6:⁋)
3.5 ± 0.0⁋ ⁋.7 ± 0.⁋
O
SEA (C⁋8:0)
OEA (C⁋8:⁋)
>⁋0
>⁋0
HO
N
H
⁋.8 ± 0.⁋
⁋.4 ± 0.⁋
5.4 ± 0.2
0.65 ± 0.07
LEA (C⁋8:2)
ArachidoylꢀEA
>⁋0
>⁋0
(C20:0)
AEA (C20:4)
6.6 ± 0.⁋
0.96 ± 0.09
EPEA (C20:5)
>⁋0
2.3 ± 0.2
DocosaenoylꢀEA
(C22:⁋)
0.74 ± 0.02 >⁋0
DHEA(C22:6)
>⁋0
⁋.6±0.⁋
a
Abbreviations: Ethanolamide (EA), Palmitoyl Ethanolamide (PEA),Palmitoleoyl Ethanolamide
(POEA),Oleoyl Ethanolamide (OEA), Lynoleoyl Ethanolamide (LEA), Arachidonoylethanolamide
b
(AEA), Eicosapentaenoyl Ethanolamide (EPEA), Docosahexaenoyl Ethanolamide (DHEA); In
parenthesis, number of C atoms in the alkyl chain: number of unsaturations.
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2.2.6 Long-chain primary amides exhibit differential inhibition of TRPV2 channels upon
activation by LPC or CBD
To also evaluate the role of the hydroxyl group, we tested a series of amide derivatives. As for
capsaicinꢀ and ethanolamineꢀderivatives, also for the amides the activity strongly depended upon the
presence of at least one double bond. In particular, Erucamide is active as TRPV2 antagonist with a
potency comparable to that of its capsaicinꢀderivative (0.67 vs 0.49 ꢁM) against LPC, but it is less
potent than the capsaicin counterpart against CBD (7.⁋ vs ⁋.5 ꢁM). As observed with the
ethanolamides, also the C20:0 amideꢀderivative was inactive against both activators (Table 4).
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9
0
Table 4. Antagonist potency of Fatty Amides at TRPV2 against LPC (3 µM) and CBD (2 µM),
reported as IC (µM).
5
0
Amides
a
Structure
LPC
>⁋0
CBD
>⁋0
b
PA (C⁋6:0)
SA(C⁋8:0)
OA (C⁋8:⁋)
LA (C⁋8:2)
>⁋0
>⁋0
2.⁋ ± 0.⁋
2.2 ± 0.⁋
2.⁋ ± 0.2
⁋.2 ± 0.⁋
ErA (C22:⁋)
0.67 ± 0.⁋3 7.⁋ ± 0.7
Eicosanamide
>⁋0
>⁋0
(C20:0)
a
Abbreviations: Palmitamide (PA), Stearamide (SA), Oleamide (OA), Linoleamide (LA), Erucamide
b
(ErA); In parenthesis, number of C atoms in the alkyl chain: number of unsaturations.
2
.2.7 Free fatty acids are poor inhibitors of TRPV2 channels
Finally, to investigate the role of the amide group, we tested against both LPC and CBD a panel of
longꢀchain fatty acids, featuring alkyl chains comparable with those occurring in the alreadyꢀtested
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compounds. The results are reported in Table 5. Fatty acids with alkyl chains from C⁋6 up to C22 are
by far less potent antagonists against both reference agonists than the other classes of compounds
bearing similar alkyl chains, thus suggesting that the amide group is mandatory for potent antagonism.
0
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Table 5. Lack of strong antagonist activity of Fatty acids at TRPV2 against LPC (3 µM) and CBD (2
µM), reported as IC values (µM).
5
0
Acids
Structure
LPC
>⁋0
CBD
>⁋0
a
Palmitic acid (C⁋6:0)
Oleic acid (C⁋8:⁋)
>⁋0
>⁋0
>⁋0
>⁋0
Arachidic acid (C20:0)
Arachidonic acid
>⁋0
>⁋0
(C20:4)
Erucic acid (C22:⁋)
>⁋0
>⁋0
>⁋0
>⁋0
Docosadienoic acid
(C22:2)
a
In parenthesis, number of C atoms in the alkyl chain: number of unsaturations.
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2.2.8 Schild Analysis on selected TRPV2 antagonists
The effect of increasing concentrations of antagonist 61, Olvanil and DocosaenoylꢀEA vs LPC and 61,
Olvanil and 50 vs CBD were tested against concentration–response curves of LPC and CBD (where the
ꢀ
4
effects of each concentration of LPC and CBD were expressed as percent of their effect of 2x⁋0 M in
the absence of the antagonist) to calculate Schild’s plots. These compounds have been selected as
representative of antagonists active either against both activators (61, Olvanil), or selectively towards
LPC (DocosaenoylꢀEA)/CBD (50) alone. In all cases, the plots analyzed by linear regression gave
slope values significantly less than unity, as reported in Table 6, indicative of a nonꢀcompetitive
behavior. However, this result may be also indicative of a nonꢀequilibrium condition and we do not
definitely rule out a competitive behavior.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
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Table 6. Slope values from linear regression of Schild analysis and tꢀtest statistics
LPC
CBD
N
a
b
c
a
b
c
Compounds
61
Slope
N
P
Slope
P
ꢀ0.58±0.087
ꢀ0.77±0.049
ꢀ0.54±0.046
ꢀ
4
6
6
ꢀ
<0.0024
<0.00⁋
<0.00⁋
ꢀ
ꢀ0.74±0.048
ꢀ0.55±0.068
ꢀ
4
6
ꢀ
<0.002
<0.00⁋
ꢀ
Olvanil
DocosaenoylꢀEA
5
0
ꢀ0.63±0.039
5
<0.00⁋
a
b
mean value ± standard deviation; number of experiments (each one performed at least in triplicate)
c
used for Schild regression; P values calculated from t-test values for the “slope=1 hypothesis”.
3
. Discussion
Novel capsaicinꢀderivatives, initially designed as TRPV⁋ agonists, behave as potent TRPV2
antagonists. The different types of modifications introduced in this compounds determine different
agonist/antagonist profiles and, in particular, opposite behaviors in terms of relative potency/efficacy
within a derivative series on the two channels. In fact, the insertion of a positive charge or an imido
group close the amido group, detrimental for TRPV⁋ agonism, is wellꢀtolerated for TRPV2
antagonism, and even leads in some cases to an increment or a rescue of activity. Conversely, the
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insertion of a sulfur/selenium atom and/or the presence of a polar group, which increase TRPV⁋
agonism, leave unaffected, or even decrease, TRPV2 antagonism.
Given the scarcity of known endogenous ligands for TRPV2, the discovery of such longꢀchain
capsaicinꢀderivatives as potent TRPV2 antagonists prompted us to investigate the following classes of
longꢀchain fatty acid derivatives with at least one functional group in common with capsaicin
derivatives as potential TRPV2 modulators: i) ethanolamides, ii) primary amides and iii) free fatty
acids, to evaluate the role of the amide group itself. Antagonists were found both in the ethanolamide
and primary amide, but not in fatty acid, series.
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Activities for both synthetic and endogenous ligands were tested against either LPC or CBD as
activators, since, on the basis of their different kinetics of activation, CBD can be defined as a direct
TRPV2 agonist, whereas LPC induces TRPV2 activation indirectly, via its Gꢀproteinꢀcoupled receptors
2
⁋
and PI3,4 Kinase mediated pathways. We found that this different mode of activation is differentially
counteracted by the investigated compounds, which can be classified as follows: a) compounds
endowed with similar antagonist efficacy against both agonists, b) compounds selectively active
against LPC, c) compounds selectively active against CBD. To determine the nature of antagonism, a
Schild regression was carried out for the representative members of each class, i.e. Olvanil,
DocoesanoylꢀEA and compound 50 and in all 3 cases the antagonists behaved as nonꢀcompetitive
ligands, suggesting that these compounds may act as allosteric antagonists. However, we cannot
completely rule out a competitive behavior since a Schild plot slope <⁋ may also suggest nonꢀ
equilibrium conditions. Moreover, since the hydrophobicity of the alkyl chain of the investigated
compounds is a critical requisite for LPC but not for CBD inhibition, it is reasonable to speculate that a
different binding site is involved in LPC antagonism, with structural/functional requisites different
from those of CBD. This site might be either on TRPV2 or on other targets activated by LPC in its
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signaling cascade and would be the target of those compounds selectively antagonizing activation by
LPC. A common critical requisite for activity of both ethanolamides and amides as TRPV2 antagonists
is the occurrence of at least one double bond in the alkyl chain, since saturated lipids, regardless of the
length of their acyl chains, are totally inactive. This suggests that a bent conformation of the alkyl chain
is required for a better accommodation into the active site, as previously reported for other TRPV⁋
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59
agonists . Also C⁋6:0 and C⁋8:0 derivatives of capsaicin result inactive against both CBD and LPC,
whereas the C20:0 derivative is selectively active against LPC. Instead, a different behavior is observed
with iminoꢀcapsaicin derivatives since they are active also when bearing saturated alkyl chain. The
aromatic moiety contributes to the overall activity at TRPV2 of the compounds characterized in the
present work, since it occurs in the most active antagonists.
4
. Conclusions
In summary, the search for structurallyꢀrelated synthetic or endogenous lipids with structural similarity
to capsaicinꢀderivatives led to identification of Olvanil and 73 as potent TRPV2 antagonists against
LPC (0.⁋6 and 0.⁋2 ꢁM, respectively) and of LEA (linoleoylꢀethanolamide) as potent TRPV2
antagonist against CBD (0.65 ꢁM). This finding is both surprising ꢀ since all other synthetic and
endogenous compounds tested here on TRPV2 behave as antagonists and capsaicin is inactive at this
channel ꢀ and of great physiological importance, since novel potent endogenous antagonists were been
identified following this study.
In conclusion, starting from the testing of a series of synthetic capsaicinoids as modulators of rat
TRPV2, we discovered not only new tools for the pharmacological manipulation of the latter, but also
that previously described endogenous lipids, i.e. long chain fatty acid ethanolamides and primary
amides, behave as negative modulators of this channel. These data are of great potential importance
given the increasingly important role assigned to TRPV2 in temperature sensing, pain, insulin
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secretion, immune response, muscle and heart function and cancer.
5
. Experimental Section
4
.1
Compounds
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0
Stevanil, Livanil, ethanolamides, amides and fatty acids when not described in the synthetic section
have been purchased from CaymanꢀVinci Biochem. Palvanil and PEA are a kind gift from Epitech
Group SpA, Saccolongo, Padova, Italy whereas Olvanil is a precious gift from dr. Alberto Minassi,
Dipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Novara, Italy.
5
.2
Synthetic Procedures.
Reactions requiring anhydrous conditions were performed in blazed or ovenꢀdried glassware using
anhydrous solvents and under inert atmosphere (argon). The solvents and reagents were purchase from
Acros Organics, Sigma Aldrich, Fluka, Merk, Panreac, Strem Chemicals or TCI Chemicals. Petroleum
ether, EtOAc, DCM and MeOH were used without further purification. In case of anhydrous reactions,
solvent and reagents were properly dried. Acrolein was distilled at atmospheric pressure and used
immediately. The reactions were monitored until completion by TLC on silica gel 60Fꢀ254 precoated
plates (Merck). Visualization of the compounds was performed by UV light (254 nm) and stained was
performed either by immersion in a 5% solution of concentrated H SO in methanol or 5% w/v
2
4
phosphomolibdic acid in ethanol followed by heating. Flash column chromatography was performed
using silica gel (technical grade, 60 Å, 40ꢀ63 µm) (Sigma Aldrich) under air pressure. NMR spectra
were recorded on a MERCURYplus AS400 MHz Varian spectrometer. Chemical shifts are reported in
parts per million (ppm, δ units). Coupling constants (J) are reported and expressed in hertz (Hz),
splitting patterns are designated as: br (broad), s (singlet), d (doublet), dd (double doublet), t (triplet), q
(
quartet), dt (double triplet), td (triple doublet), ddd (double double doublet), p (pentuplet) and m
⁋
3
(multiplet). All C NMR spectra were proton decoupled. High resolution mass spectra (HRꢀMS) were
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recorded on at the Serveis Cientificotècnics of Universitat de Lleida (SCTꢀUdL) and Servei de
Recursos Científics i Tècnics of Universitat Rovira i Virgili (URV) with an Agilent G65⁋0AA QꢀTOF
+
MS spectrometer in positive electrospray ionization (ESI ) and Agilent LC⁋200 Series coupled to
+
MS62⁋0 TOF spectrometer in electrospray ionization (ESI ) respectively. Mobile phase was composed
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of ACN/MeOH 50:50. Flow rate: 0.6 mL/min. Infrared spectra were recorded on Jasco FTꢀIR 6300
using a diamond ATR crystal cell. Melting points were measured using Gallenkamp capillary apparatus
and are uncorrected. Optical rotations were measured at 20 °C with a Perkin Elmer 24⁋ nc polarimeter
(λ=589 Na, path length ⁋ dm). Some recorded values were within the error limit of the polarimeter and
therefore were not possible to determine them. It has been indicated as <⁋º. Analytical UPLCꢀMS
was performed on a binary Acquity UPLC with a Acquity PDA UPLC eLambda 800 nm triple
quadrupole mass spectrometer (Xevo TQꢀS) using a Acquity UPLC® BEH C⁋8 50 x 2.⁋ mm, ⁋.7 µm
C⁋8 column. UV detection = 2⁋0 – 500 nm, mass spectrometry= ESI+ (scan ⁋00ꢀ850 m/z). Flow rate
was 0.3 mL/min using a solvent gradient of B ⁋00% over 6 min (total runtime with equilibration back
to starting conditions = 2 min) where A = MeOH and B = : 85/⁋5/0.2 MeOH/H2O/AcOH. Purities were
measured by UV absorption at 254 nm or TIC and are ≥95% unless otherwise stated. Purity of final
compounds was assessed by reversedꢀphase UHPLC with UV diode array detection; all tested
compounds were >95% purity.
4
.2.1 Procedure I. Amine bond formation
To a 0.35 M solution of starting material in anhydrous DMF were added the amine 3 (⁋.⁋ eq.), HATU
⁋.5 eq.) and DIPEA (3 eq.). The mixture was stirred at room temperature for 20 h. To the mixture was
(
added EtOAc and brine, and the aqueous phase was extracted with EtOAc. The combined organic
phases were washed with ⁋ M HCl, saturated solution of NaHCO and brine. The organic phase was
3
dried over anhydrous Na SO , filtered and the solvent was removed under reduced pressure. The crude
2
4
residue was purified by silica gel column chromatography.
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4.2.2 Procedure II. Ester hydrolysis
To a 0.2 M solution of starting material in THF/H O (⁋:⁋) LiOH·H O (3 eq.) was added. The mixture
2
2
was stirred at room temperature until completion of the reaction. The reaction mixture was acidified
with ⁋ M HCl until pH ⁋ and extracted with EtOAc. The organic phase was dried over anhydrous
Na SO , filtered and the solvent was removed under reduced pressure to afford the corresponding
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compound.
.2.3 Procedure III. Boc protection
4
Et N (⁋.5 eq.) was added to a 0.3 M aqueous solution of starting material, cooled in an ice bath. Then
3
Boc O (⁋.5 eq.) was added dropwise and stirred overnight. After completion of the reaction, the solvent
2
was evaporated under reduced pressure. The residue was dissolved in EtOAc, washed with ⁋ M HCl
and brine, dried over anhydrous Na SO , filtered and evaporated under reduced pressure. The crude
2
4
residue was thoroughly washed with hexane for several times.
.2.4 Procedure IV. SS/SeSe bond cleavage
4
SS bond cleavage: To a 0.⁋5 M solution of starting material in wet THF was added triꢀnꢀbutyl
n
phosphine (P( Bu) ) (⁋.05 eq.). The reaction mixture was stirred at room temperature for 2 h. After
3
completion of the reaction, the solvent was removed under reduced pressure to afford the crude
product, which was purified by silica gel column chromatography.
SeSe bond cleavage and Se-alkylation: To a 0.⁋3 M solution of starting material in ethanol was added
NaBH (2.5 eq) at 0 ºC. The reaction mixture was stirred for 20 min, followed by addition of the
4
respective iodinated compound. The reaction mixture was stirred at room temperature for ⁋6 h. Then,
the reaction was quenched with ⁋ M HCl and extracted with EtOAc. The organic phase was dried over
anhydrous Na SO , filtered and the solvent was removed under reduced pressure. The crude residue
2
4
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4.2.5 Procedure V. Reduction of methyl ester
To a 0.2 M solution of starting material in anhydrous THF LiAlH (2 eq.) was added at 0 ºC. The
4
reaction mixture was stirred at room temperature for 24 h. Then, the reaction was quenched with ⁋ M
HCl, followed by extraction with DCM. The combined organic phases were dried over anhydrous
Na SO , filtered and the solvent was removed under reduced pressure. The solid residue was purified
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by silica gel column chromatography.
.2.6 Procedure VI. Iodination
4
To a 0.25 M solution of starting material in toluene iodine (⁋.2 eq.), imidazole (3 eq.) and PPh (⁋.2
3
eq.) were added. The mixture was stirred at 90 ºC for 2 h. The solvent was evaporated under reduced
pressure. The residue was dissolved in EtOAc, washed with saturated aqueous solution of KMnO4,
water and brine, dried over anhydrous Na SO , filtered and evaporated under reduced pressure. The
2
4
solid residue was purified by silica gel column chromatography.
.2.7 Procedure VII. S-Alkylation
4
To a 0.2 M solution of starting material in DMF, TEA (⁋.5 eq.) and the corresponding iodinated
compound (⁋.⁋2 eq.) were added. The reaction mixture was stirred at 90 ºC overnight. To the mixture
was added EtOAc and brine, and the aqueous phase was extracted with EtOAc. The combined organic
phases were washed with ⁋ M HCl, saturated solution of NaHCO and brine. The organic phase was
3
dried over anhydrous Na SO , filtered and the solvent was removed under reduced pressure. The crude
2
4
residue was purified by silica gel column chromatography.
.2.8 Procedure VIII. TBDMS deprotection
4
A 0.25 M solution of the starting material in a mixture of AcOH/THF/H O was stirred at room
2
temperature until deprotection was complete. The solvent was evaporated under reduced pressure to
obtain the reaction crude, which was purified by silica gel column chromatography.
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