Acetal derivatives as prodrugs of resveratrol

Article abstract of DOI:10.1021/mp400226p

The pharmacological exploitation of resveratrol is hindered by rapid phase-II conjugative metabolism in enterocytes and hepatocytes. One approach to the solution of this problem relies on prodrugs. We report the synthesis and characterization as well as t

Full text of DOI:10.1021/mp400226p

Article
pubs.acs.org/molecularpharmaceutics
Acetal Derivatives as Prodrugs of Resveratrol
Andrea Mattarei,†,‡ Michele Azzolini,§ Massimo Carraro,‡ Nicola Sassi,†,§ Mario Zoratti,†,§
Cristina Paradisi,*,‡ and Lucia Biasutto*,†,§
†CNR Institute of Neuroscience, viale G. Colombo 3, 35121 Padova, Italy
‡Department of Chemical Sciences, University of Padova, via F. Marzolo 1, 35131 Padova, Italy
§Department of Biomedical Sciences, University of Padova, viale G. Colombo 3, 35121 Padova, Italy
S
* Supporting Information
ABSTRACT: The pharmacological exploitation of resveratrol is
hindered by rapid phase-II conjugative metabolism in enterocytes
and hepatocytes. One approach to the solution of this problem
relies on prodrugs. We report the synthesis and characterization
as well as the assessment of in vivo absorption and metabolism of
a set of prodrugs of resveratrol in which the OH groups are
engaged in the formal (−OCH2OR) or the more labile acetal
(−OCH(CH3)OR) linkages. As carrier group (R) of the prodrug,
we have used short ethyleneglycol oligomers (OEG) capped by a
terminal methoxy group: −O−(CH2CH2O)n−CH3 (n = 0, 1, 2,
3, 4, 6). These moieties are expected to exhibit, to a degree, the
favorable properties of longer polyethyleneglycol (PEG) chains,
while their relatively small size makes for a more favorable drug loading capacity. After administration of formal-based prodrugs
to rats by oral gavage, significant concentrations of derivatives were measured in blood samples over several hours, in all cases
except for n = 0. Absorption was maximal for n = 4. Complete deprotection to give resveratrol and its metabolites was however
too slow to be of practical use. Administration of the acetal prodrug carrying tetrameric OEG chains resulted instead in the
protracted presence of resveratrol metabolites in blood, consistent with a progressive regeneration of the parent molecule from
the prodrug after its absorption. The results suggest that prodrugs of polyphenols based on the acetal bond and short
ethyleneglycol oligomers of homogeneous size may be a convenient tool for the systemic delivery of the unconjugated parent
compound.
KEYWORDS: resveratrol, oligoethyleneglycol, bioavailability, pharmacokinetic, prodrugs
hepatocytes.33−35 These conjugates can then be re-exported
INTRODUCTION
■
from cells by MDR pumps.36,37
Resveratrol (Figure 1), one of the most intensely studied plant
Part of the bioefficacy of resveratrol may well be due to
polyphenols, exhibits a range of activities1 of biomedical
hormesis, that is, a response to a low level of stress induced by
interest. It acts on several cellular signaling pathways,2−11
the bioactive compound, affording protection against subse-
overlapping to a considerable extent those mediating the effects
quent higher doses.38,39 Such a model implies that even a
of dietary restriction and exercise. Ensuing beneficial effects
modest, in absolute terms, increase of bioavailability may have
significant consequences, if hormesis induction is achieved. A
include lifespan extension in model systems,12 improvements of
functionality in aging,2,8,10,12,13 protection of the cardiovascular
very relevant aspect currently under investigation is the
system,4,14 anti-inflammatory activity,4 for example in arthri-
bioactivity of metabolites formed by phase II “detoxification”.
tis,15,16 cancer chemoprevention,17−19 and potentiation of
The available results suggest that, in general, these conjugates
chemotherapy,20,21 neuroprotection,22,23 antagonism of the
metabolic syndrome,24,25 improvement of glucose handling in
diabetes,26 and of fat mobilization.27,28 Despite the evidence
summarized in the cited reviews, the efficacy of resveratrol
administration in vivo remains in doubt.29,30 Meta-reviews
consider the still preliminary results of human clinical trials
inconclusive.31,32 This is because resveratrol, like other
polyphenols, has a relatively poor bioavailability due to a
built-in propensity to phase II metabolism, that is, “detox-
ification” via covalent modification of the hydroxyl groups by
the sulfo- and glucuronosyl-transferases of enterocytes and
are less bioactive than resveratrol itself,40−44 hence the dual
goal of improving its absorption and of slowing down
metabolism to hopefully enhance favorable effects.
Various approaches are being tested: formulations,45−47
nanovectors,48,49 and prodrugs50−53 are the major ones and
could represent a useful pharmacological tool to improve the in
Received: April 17, 2013
Revised: June 1, 2013
Accepted: June 4, 2013
© XXXX American Chemical Society
A
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Figure 1. Resveratrol (1) and new derivatives synthesized: formals (2a−g), acetals (3), and ketals (4).
vivo activity of resveratrol.54 We report here the synthesis,
properties, and pharmacokinetics in rats of a family of prodrugs
of resveratrol utilizing the acetal bond system to link
protective/solubilizing groups to resveratrol hydroxyls (“acetal”
is used here, as is customary, as a generic label including all
three bonding groups used in this work, see Figure 1). This
linkage, often used for the protection of hydroxyls in synthesis
procedures, offers the advantage of low polarity and steric
hindrance and is therefore expected to allow passage through
biomembranes. Furthermore it is acid-sensitive, a characteristic
which may lead to a requirement for protection by a suitable
formulation during gastric transit, but also offers in principle a
tool for targeted release of the active principle in acidic
environments. Its use in prodrugs has precedents,55 and
recently a variant has been used to produce derivatives of
quercetin, another remarkable polyphenol, with enhanced
stability and membrane permeability.56−58 Notably, the acetal
bond system links the polyphenolic scaffold to the glycosidic
moiety in polyphenol glycosides, a major class of natural
derivatives, and glucuronides, a major product of phase II
metabolism. These natural derivatives are characterized by high
hydrophilicity and solubility and poor adsorption in their native
form.
Polyethyleneglycol (PEG) has been used in a number of
formulations and prodrugs to increase solubility, improve
absorption, and limit immune response.59,60 PEG is known to
be nontoxic, nonantigenic, and biocompatible, to be rapidly
eliminated from the body, to be soluble both in water and many
organic solvents, and to have pronounced solubilizing proper-
ties. Incorporation of PEG chains can increase resistance to
hydrolases and stability in the gastrointestinal tract. PEG has
been successfully used as a “carrier” of peptides and proteins as
well as of small bioactive molecules. In this latter case however
an intrinsic limitation is its low drug loading capacity, that is,
the high molecular weight of prodrugs incorporating polymeric
chains. We have previously reported the synthesis of a
resveratrol prodrug bearing PEG chains linked via carboxyester
moieties.51 In experiments assessing transport across rat
intestine, this construct succeeded in determining the trans-
location of unmodified resveratrol. When using resveratrol as
such, practically only conjugation metabolites appeared on the
basolateral side. To remedy the low loading capacity we have
now used as carrier groups short polyether chains of defined
size (oligoethyleneglycol: OEG) and performed a structure−
function study to determine the optimal chain size for
absorption in pharmacokinetics. To overcome the fragility of
the carboxyester moiety51,61 we have, as mentioned, turned to
the acetal linkage.
EXPERIMENTAL SECTION
■
Materials and General Methods. Resveratrol was
purchased from Waseta Int. Trading Co. (Shangai, P.R.
China). Other starting materials and reagents were purchased
from Aldrich, Fluka, Merck-Novabiochem, Riedel de Haen, J.T.
Baker, Cambridge Isotope Laboratories Inc., Acros Organics,
1
Carlo Erba and Prolabo, and were used as received. H NMR
spectra were recorded with a Bruker AC250F spectrometer
operating at 250 MHz and a Bruker AVII500 spectrometer
operating at 500 MHz. Chemical shifts (δ) are given in ppm
relative to the signal of the solvent. HPLC-UV analyses were
performed with an Agilent 1290 Infinity LC System (Agilent
Technologies), equipped with binary pump and a diode array
detector (190−500 nm). High-performance liquid chromatog-
raphy/electrospray ionization−mass spectrometry (HPLC/ESI-
MS) analyses and mass spectra were performed with a 1100
Series Agilent Technologies system, equipped with binary
pump (G1312A) and MSD SL Trap mass spectrometer
(G2445D SL) with an ESI source. ESI-MS positive spectra of
reaction intermediates and final purified products were
obtained from solutions in acetonitrile, eluting with a 1:1
water−acetonitrile mixture containing 0.1% formic acid. Thin-
layer chromatographies (TLCs) were run on silica gel
supported on plastic (Macherey-Nagel PolygramSIL G/UV254
,
silica thickness 0.2 mm) or on silica gel supported on glass
(Fluka) (silica thickness 0.25 mm, granulometry 60 Å, medium
porosity) and visualized by UV detection. Flash chromatog-
raphy was performed on silica gel (Macherey-Nagel 60, 230−
400 mesh granulometry (0.063−0.040 mm)) under air
pressure. The solvents were analytical or synthetic grade and
were used without further purification. All experiments
reported were performed in triplicate unless otherwise stated,
and means standard deviation values are reported.
General Procedure for the Preparation of the Formal
Derivatives of Resveratrol (2a−g). NaH (17.5 mmol, 4 eq
60% in mineral oil) was washed three times in 5 mL of n-
hexane. The suspension was decanted after each wash, and
hexane traces were removed under reduced pressure. A sample
of 10 mL of tetrahydrofuran (THF) was then added, and the
suspension was stirred for 5 min. A solution of resveratrol (1)
(4.4 mmol, 1 equiv) in 5 mL of anhydrous THF was then
added. After stirring for 30 min, a solution of the desired
chloromethyl ether (24.2 mmol, 5.5 equiv) in 5 mL of
anhydrous THF was added dropwise, and the mixture was
B
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vigorously stirred for 2 min at room temperature. Dimethyl-
formamide (DMF, 5 mL) was then added, and the reaction
progress was monitored by TLC. After the disappearance of
resveratrol, the reaction mixture was diluted in CH2Cl2 (150
mL) and washed with 0.3 N HCl (100 mL). The organic layer
was dried over MgSO4 and filtered. The solvent was evaporated
under reduced pressure, and the residue was purified by
chromatography to give the trisubstituted formal derivative of
resveratrol.
3.47−3.53 (m, 6H, 3 × −O−CH2−), 3.57−3.66 (m, 24H, 12 ×
−O−CH2−), 3.79−3.83 (m, 6H, 3 × −O−CH2−) 5.23−5.24
(m, 6H, 3 × −O−CH2−O−), 6.59 (t, 1H, 4JH,H = 2,0 Hz, H-4),
6.81 −7.03 (m, 6H, H-2, H-6, H-2′, H-6′, H-7, H-8), 7.39 (d,
3
2H, JH,H = 8,5 Hz, H-3′, H-5′) ppm. 13C NMR (250 MHz,
CDCl3, 25 °C): δ = 158.3, 156.9, 139.5, 130.8, 128.6, 127.6,
126.5, 116.3, 107.4, 103.9, 93.2, 93.2, 71.7, 70.4, 70.4, 70.2,
67.6, 58.9 ppm. Purity (HPLC-UV) ≥ 99%.
trans-3,4′,5-Tri(2,5,8,11,14-pentaoxapentadecyloxy)-
stilbene (2f). Purified by preparative HPLC (ACE 5 AQ
column 150 × 21.2 mm i.d.). Solvents A and B were H2O and
acetonitrile each containing 0.05% TFA. The gradient for B was
as follows: 37.5% for 2 min, from 37.5% to 65% in 10 min. The
flow rate was 17 mL/min. The eluate was monitored at 300 nm.
Yield: 2.59 g (66%) as a colorless oil. ESI-MS (ion trap): m/z
889.5 [M + H]+. 1H NMR (250 MHz, CDCl3, 25 °C): δ = 3.27
(s, 9H, 3 × −O−CH3), 3.43−3.46 (m, 6H, 3 × −O−CH2−),
3.52−3.61 (m, 36H, 18 × −O−CH2−), 3.74−3.77 (m, 6H, 3 ×
−O−CH2−) 5.17−5.18 (m, 6H, 3 × −O−CH2−O−), 6.54 (t,
trans-3,4′,5-Tri(methoxymethoxy)stilbene (2a). Purified by
flash-chromatography (CH2Cl2/AcOEt = 95:5). Yield: 1.15 g
(73% based on resveratrol) as a white powder. ESI-MS (ion
1
trap): m/z 361 [M + H]+. H NMR (250 MHz, CDCl3, 25
°C): δ = 3.49 (s, 3H, −O−CH3), 3.50 (s, 6H, 2 × −O−CH3),
4
5.19 (s, 6H, 3 × −O−CH2−O−), 6.64 (t, 1H, JH,H = 2.3 Hz,
4
H-4), 6.85 (d, 2H, JH,H = 2.0 Hz, H-2, H-6), 6.87−7.07 (m,
3
4H, H-2′, H-6′, H-7, H-8), 7.44 (d, 2H, JH,H = 8.8 Hz, H-3′,
H-5′) ppm. 13C NMR (250 MHz, CDCl3, 25 °C): δ = 158.5,
156.9, 139.8, 131.0, 128.8, 127.8, 126.7, 116.4, 107.6, 104.0,
94.5, 94.4, 56.1, 56.0 ppm. Purity (HPLC-UV) ≥ 95%.
4
1H, JH,H = 2,0 Hz, H-4), 6.76−6.98 (m, 6H, H-2, H-6, H-2′,
trans-3,4′,5-Tri(ethoxymethoxy)stilbene (2b). Purified by
flash-chromatography (hexane/AcOEt = 50:50). Yield: 1.34 g
(76%) as a yellow oil. ESI-MS (ion trap): m/z 403 [M + H]+.
1H NMR (250 MHz, CDCl3, 25 °C): δ = 1.11−1.17 (m, 9H, 3
× CH2−CH3), 3.62−3.70 (m, 6H, 3 × −O−CH2-), 5.24 (s, 6H,
3 × −O−CH2−O−), 6.57 (t, 1H, 4JH,H = 2,0 Hz, H-4), 6.87 (d,
H-6′, H-7, H-8), 7.34 (d, 2H, 3JH,H = 8,5 Hz, H-3′, H-5′) ppm.
13C NMR (250 MHz, CDCl3, 25 °C): δ = 158.1, 156.7, 139.3,
130.5, 128.4, 127.4, 126.3, 116.1, 107.2, 103.7, 93.0, 92.9, 71.5,
70.1, 70.0, 69.9, 67.4, 58.5 ppm. Purity (HPLC-UV) ≥ 98%.
trans-3,4′,5-Tri(2,5,8,11,14,17,20-heptaoxahenicosyloxy)-
methylenoxy)stilbene (2g). Purified by preparative HPLC
(ACE 5 AQ column 150 × 21.2 mm i.d.). Solvents A and B
were H2O and acetonitrile each containing 0.05% TFA. The
gradient for B was as follows: from 27.5% to 65% in 13 min.
The flow rate was 17 mL/min. The eluate was monitored at
300 nm. Yield: 3.25 g (64%) as a colorless oil. ESI-MS (ion
4
2H, JH,H = 2.0 Hz, H-2, H-6), 7.00−7.21 (m, 4H, H-2′, H-6′,
3
H-7, H-8), 7.54 (d, 2H, JH,H = 8,5 Hz, H-3′, H-5′) ppm. 13C
NMR (250 MHz, CDCl3, 25 °C): δ = 158.0, 156.6, 139.3,
130.3, 128.5, 127.8, 126.3, 116.2, 107.1, 104.5, 92.5, 92.4, 63.7,
63.6, 15.0 ppm. Purity (HPLC-UV) ≥ 95%.
1
trans-3,4′,5-Tri-((2-methoxyethoxy)methoxy)stilbene (2c).
Purified by flash-chromatography (hexane/AcOEt = 50:50).
Yield: 1.44 g (67%) as a yellow oil. ESI-MS (ion trap): m/z 493
trap): m/z 1153.6 [M + H]+. H NMR (250 MHz, CDCl3, 25
°C): δ = 3.21 (s, 9H, 3 × −O−CH3), 3.47−3.51 (m, 6H, 3 ×
−O−CH2−), 3.57−3.64 (m, 60H, 30 × −O−CH2−), 3.77−
3.81 (m, 6H, 3 × −O−CH2−) 5.22−5.23 (m, 6H, 3 × −O−
1
[M + H]+. H NMR (250 MHz, CDCl3, 25 °C): δ = 3.38 (s,
4
3H, −O−CH3), 3.39 (s, 6H, 2 × −O−CH3), 3.56−3.60 (m,
CH2−O−), 6.57 (t, 1H, JH,H = 2,0 Hz, H-4), 6.79 −7.01 (m,
3
6H, 3 × −O−CH2−), 3.82−3.86 (m, 6H, 3 × −O−CH2−),
6H, H-2, H-6, H-2′, H-6′, H-7, H-8), 7.38 (d, 2H, JH,H = 8,5
4
5.28−5.29 (m, 6H, 3 × −O−CH2−O−), 6.64 (t, 1H, JH,H
=
Hz, H-3′, H-5′) ppm. 13C NMR (250 MHz, CDCl3, 25 °C): δ
= 158.2, 156.8, 139.5, 130.7, 128.5, 127.5, 126.5, 116.2, 107.3,
103.9, 93.2, 93.1, 71.6, 70.3, 70.3, 70.2, 70.1, 67.5, 58.8 ppm.
Purity (HPLC-UV) ≥ 95%.
2,0 Hz, H-4), 6.87 (d, 2H, 4JH,H = 2,0 Hz, H-2, H-6), 6.93−7.06
(m, 4H, H-2′, H-6′, H-7, H-8), 7.43 (d, 2H, 3JH,H = 8,5 Hz, H-
3′, H-5′) ppm. 13C NMR (250 MHz, CDCl3, 25 °C): δ =
158.3, 156.9, 139.6, 130.8, 128.7, 127.6, 126.6, 116.3, 107.5,
104.0, 93.3, 93.2, 71.5, 67.5, 58.9 ppm. Purity (HPLC-UV) ≥
95%.
trans-3,4′,5-Tri(2-methoxypropan-2-yloxy)stilbene (3). 2-
Methoxypropene (1.58 g, 21.9 mmol, 10.0 equiv) was added to
a mixture of resveratrol (0.5 g, 2.19 mmol, 1 equiv) and
pyridinium p-toluenesulphonate (55 mg, 0.22 mmol, 0.1 equiv)
under nitrogen pressure and strictly anhydrous conditions. The
neat reaction mixture was stirred at room temperature for 72 h.
The excess of 2-methoxypropene was removed under reduced
pressure, and the crude product was purified by flash
chromatography (hexane/acetone = 98:2 + 2% of triethyl-
amine). Yield: 0.20 g (21%) as pale yellow oil. ESI-MS (ion
trap): m/z 445 [M + H]+. 1H NMR (300 MHz, acetone-d6, 25
°C): δ = 1.45 (s, 6H, −O−C(CH3)2−O−), 1.48 (s, 12H, 2 ×
−O−C(CH3)2−O−), 3.38 (s, 3H, −O−CH3), 3.40 (s, 6H, 2 ×
−O−CH3), 6.91 (t, 1H, 4JH,H = 2.1 Hz, H-4), 6.98 (d, 2H, 4JH,H
= 2.1 Hz, H-2, H-6), 7.01−7.16 (m, 4H, H-2′, H-6′, H-7, H-8),
trans-3,4′,5-Tri((2-(2-methoxyethoxy)ethoxy)methoxy)-
stilbene (2d). Purified by flash-chromatography (in two steps,
using, respectively, CH2Cl2/acetone = 80:20 and hexane/
AcOEt = 20:85). Yield: 1.84 g (67%) as a yellow oil. ESI-MS
(ion trap): m/z 625 [M + H]+. 1H NMR (250 MHz, CDCl3, 25
°C): δ = 3.33−3.34 (m, 9H, 3 × −O−CH3), 3.48−3.52 (m, 6H,
3 × −O−CH2−), 3.59−3.67 (m, 12H, 6 × −O−CH2−), 3.82−
3.86 (m, 6H, 3 × −O−CH2−) 5.24−5.25 (m, 6H, 3 × −O−
4
CH2−O−), 6.61 (t, 1H, JH,H = 2,0 Hz, H-4), 6.83 −7.04 (m,
3
6H, H-2, H-6, H-2′, H-6′, H-7, H-8), 7.40 (d, 2H, JH,H = 8,5
Hz, H-3′, H-5′) ppm. 13C NMR (250 MHz, CDCl3, 25 °C): δ
= 158.3, 156.8, 139.5, 130.8, 128.6, 127.6, 126.5, 116.3, 107.4,
103.9, 93.2, 93.2, 71.7, 70.4, 70.3, 70.2, 67.6, 58.9 ppm. Purity
(HPLC-UV) ≥ 99%.
3
7.50 (d, 2H, JH,H = 8.7 Hz, H-3′, H-5′) ppm. 13C NMR (300
MHz, acetone-d6, 25 °C): δ = 157.0, 156.0, 139.7, 132.6, 129.2,
128.2, 127.9, 121.9, 114.2, 113.8, 104.2, 49.2, 25.4, 25.1 ppm.
Purity (HPLC-UV) ≥ 95%.
trans-3,4′,5-Tri(2,5,8,11-tetraoxadodecyloxy)stilbene (2e).
Purified by flash-chromatography (hexane/AcOEt/diethyl
ether/acetone = 10:30:30:30). Yield: 2.15 g (65%) as a yellow
trans-3,4′,5-Tri(2,5,8,11,14-pentaoxahexadecan-15-
yloxy)stilbene (4). Tetraethylene glycol methyl vinyl ether (0.8
g, 3.4 mmol, 6.0 equiv) was added to a mixture of resveratrol
1
oil. ESI-MS (ion trap): m/z 757 [M + H]+. H NMR (250
MHz, CDCl3, 25 °C): δ = 3.33−3.34 (m, 9H, 3 × −O−CH3),
C
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(0.13 g, 0.57 mmol, 1 equiv) and pyridinium p-toluenesulph-
onate (50 mg, 0.19 mmol, 0.3 equiv) under nitrogen pressure
and strictly anhydrous conditions. The neat reaction mixture
was stirred at room temperature for 24 h. The crude product
was purified by flash chromatography (hexane/acetone = 5:3 +
2% of triethylamine). Yield: 0.23 g (43%) as a pale yellow oil.
UV. Each measurement was repeated in triplicate and also with
a mixing time of 48 and 72 h to verify the constancy of the
solubility values. The overall average is presented.
Octanol/Water Partition Coefficient (log Pow). Octanol
and bidistilled water were reciprocally saturated by shaking
them together for 24 h before use. A known amount
(approximately 5 × 10−7 mol) of the compound was dissolved
in 5 mL of octanol and placed in a sealed vial at 25 °C with 5
mL of water under stirring. After 24 h the two phases were
separated and centrifuged (12 000 g, 20 min). A small aliquot of
each phase was then diluted with acetonitrile and subjected to
HPLC analysis. Each measurement was repeated in triplicate
and also with a mixing time of 48 and 72 h to verify the
constancy of the log Pow values. The overall average is
presented.
Hydrolysis Reactions. The chemical stability of all new
compounds was tested in aqueous media mimicking gastric (0.1
N HCl, NormaFix) and intestinal (0.1 M PBS buffer, pH 6.8)
pH values. A 50 μM solution of the compound was made from
a 5 mM stock solution in DMSO and incubated at 37 °C for 24
h; samples withdrawn at different times were analyzed by
HPLC-UV. Hydrolysis products were identified by HPLC/ESI-
MS analysis of selected samples. Nonlinear curve fitting was
performed using Origin 8.0 data analysis software, using the
equations described in the Supporting Information.
Stability in Blood. Rats were anesthetised and blood was
withdrawn from the jugular vein, heparinised and transferred
into tubes containing EDTA. Blood samples (1 mL) were
spiked with 5 μM of compound (dilution from a 5 mM stock
solution in DMSO) and incubated at 37 °C for 4 h (the
maximum period allowed by blood stability). Aliquots were
taken after 10 min, 30 min, 1 h, 2 h, and 4 h and treated as
described below (blood sample treatment and analysis).
Cleared blood samples were finally subjected to HPLC-UV
analysis. Ad hoc tests demonstrated that compound 4
withstood the extraction procedure without undergoing
detectable hydrolysis.
Blood Sample Treatment and Analysis. Before starting
the treatment, 4,4′-dihydroxybiphenyl was added as internal
standard to a carefully measured blood volume (25 μM final
concentration). Blood was then stabilized with a freshly
prepared 10 mM solution of ascorbic acid (0.1 vol) and
acidified with 0.6 M acetic acid (0.1 vol); after mixing, an excess
of acetone (4 vol) was added, followed by sonication (2 min)
and centrifugation (12 000 g, 7 min, 4 °C). The supernatant
was finally collected and stored at −20 °C. Before analysis,
acetone was allowed to evaporate at room temperature using a
Univapo 150H (UniEquip) vacuum concentrator centrifuge,
and up to 40 μL of CH3CN was added to precipitate residual
proteins. After centrifugation (12 000 g, 5 min, 4 °C), cleared
samples were directly subjected to HPLC-UV analysis.
Metabolites and hydrolysis products were identified by
HPLC/ESI-MS analysis and/or comparison of chromato-
graphic retention time with true samples.
1
ESI-MS (ion trap): m/z 932 [M + H]+. H NMR (500 MHz,
acetone-d6, 25 °C): δ = 1.45−1.47 (m, 9H, 3 × −O−
CH(CH3)−O−, 3.27 (s, 6H, 2 × −O−CH3), 3.28 (s, 3H, −O−
CH3), 3.44−3.47 (m, 6H, 3 × −O−CH2−), 3.51−3.62 (m,
36H, 18 × −O−CH2−), 3.64−3.85 (m, 6H, 3 × −O−CH2−)
5.52−5.57 (m, 3H, 3 × −O−CH(CH3)−O−), 6.65 (t, 1H,
4
4JH,H = 2.0 Hz, H-4), 6.91 (d, 2H, JH,H = 1.7 Hz, H-2, H-6),
7.03−7.21 (m, 4H, H-2′, H-6′, H-7, H-8), 7.53 (d, 2H, 3JH,H
=
8.6 Hz, H-3′, H-5′) ppm. 13C NMR (500 MHz, acetone-d6, 25
°C): δ = 159.3, 157.8, 140.6, 131.9, 129.5, 128.6, 127.5, 118.4,
109.6, 106.6, 100.4, 100.4, 72.6, 71.2, 71.2, 71.2, 71.0, 66.0,
66.0, 65.9, 58.8, 20.6, 20.5 ppm. Purity (HPLC-UV) ≥ 95%.
General Procedure for the Preparation of the Non-
commercially Available Chloromethyl Ethers. Paraformal-
dehyde (10.5 mmol, 1.05 equiv) was added to a solution of the
desired alcohol (10.0 mmol, 1 equiv) in trimethylsilyl chloride
(5 mL), and the reaction mixture was stirred for 2.5 h at room
temperature. The solvent was evaporated under reduced
pressure, and the chloromethyl ether obtained was used
without further purification.
HPLC-UV Analysis. Samples (2 μL) were analyzed using a
reversed phase column (Zorbax RRHD Eclipse Plus C18, 1.8
μm, 50 × 2.1 mm i.d.). Solvents A and B were H2O containing
0.1% TFA and CH3CN, respectively. The gradient for B was as
follows: 10% for 2 min, from 10% to 20% in 3 min, then from
20% to 30% in 1 min, then from 30% to 100% in 1 min; the
flow rate was 0.6 mL/min. The eluate was preferentially
monitored at 286, 300, and 320 nm. These analytical conditions
were used to determine the purity of the synthesized
compounds. Formal derivatives 2a−g proved to be stable
under these analytical conditions; on the contrary, 0.1% TFA in
the eluting aqueous phase (A) caused partial hydrolysis of
derivative 4. For this reason, HPLC analysis of 4 was performed
in the absence of TFA. Samples from pharmacokinetic studies
of 4 were analyzed both in the absence and in the presence of
TFA, to optimally quantify acetal derivatives or resveratrol and
phase II metabolites, respectively. Optimal resolution for di-
and monosubstituted isomers was achieved using another
reversed phase column (Zorbax RRHD Eclipse Plus Phenyl-
Hexyl, 1.8 μm, 50 × 2.1 mm i.d.), with a gradient for B as
follows: 28% for 3 min, from 28% to 65% in 2 min, then from
65% to 100% in 0.7 min, 100% for 0.5 min.
HPLC/ESI-MS Analysis. Samples (20 μL) were analyzed
using a reversed phase column (Synergi-MAX, 4 μm, 150 × 4.6
mm i.d.; Phenomenex). Solvents A and B were H2O containing
0.1% TFA and CH3CN, respectively. The gradient for B was as
follows: 10% for 2 min, from 10% to 35% in 20 min, then from
35% to 100% in 20 min; the flow rate was 1 mL/min. The
eluate was preferentially monitored at 286, 300, and 320 nm.
MS analysis was performed with an ESI source operating in full-
scan mode in positive ion mode.
The recovery yields of resveratrol and its metabolites have
been reported previously.62,63 Internal standard recovery was
68.7 6.3% (N = 7). For the new prodrugs the corresponding
recoveries, expressed as the ratio to the recovery of internal
standard, were as follows: 2a: 1.19 0.07; 2b: 1.18 0.08.; 2c:
1.21 0.07; 2d: 0.62 0.06; 2e: 0.93 0.04; 2f: 0.77 0.02;
2g: 0.94 0.05; 4: 0.69 0.04 (N = 5). Recoveries of partially
protected (disubstituted) derivatives were assumed to be the
same as those of the corresponding fully substituted prodrug.
Solubility in Water. A known amount (approximately 2 ×
10−5 mol) of the compound was mixed with 2 mL of bidistilled
water and kept under stirring in a sealed vial at 25 °C. After 24
h the solution was filtered using a 0.45 μm PTFE filter and
centrifuged (10 000 g, 10 min, 25 °C). A small aliquot was
withdrawn, diluted with acetonitrile, and analyzed by HPLC-
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Scheme 1. Synthesis of Formal Derivatives (2a−g) from Resveratrol (1) and Alkoxymethylene Chlorides
Knowledge of these ratios allowed us to determine the
unknown amount of analyte in a blood sample by measuring
the recovery of the internal standard.
resveratrol (1) on the appropriate alkoxymethylene chloride
as sketched in Scheme 1. Alkoxymethylene chlorides are well-
known and useful reagents for protection of phenolic hydroxyls
with formal groups.64 Despite its apparent simplicity the key
step of coupling resveratrol with the alkoxymethylene chloride
proved rather tricky. Attempts to perform the reaction in
tetrahydrofuran (THF) were not successful: when sodium
hydride was added to a THF resveratrol solution, a precipitate
was formed, and it did not react appreciably upon addition of
alkoxymethylene chloride at room temperature. The desired
product was instead obtained in high yield by adding a small
amount of dimethylformamide (DMF), which resulted in a very
fast change of the suspension color from pale yellow to a dark
orange, followed by color fading within a few minutes. It is
interesting to note that, when pure DMF was used as solvent, a
very fast reaction occurred leading to a complex product
mixture, the precipitation of a gummy colored material, and low
yields in the desired resveratrol derivative.
Permeation of the Rat Intestinal Wall (Ex Vivo).
Intestine was excised from 18 h fasted rats and transferred
into a saline solution (154 mM NaCl in water) at 37 °C. The
jejunum was cut into 1 cm long strips, opened longitudinally,
rinsed free of luminal content, and mounted in Ussing-type
chambers. Apical and basolateral compartments were filled with
1 mL of oxygenated HEPES buffer each (248 mM NaCl, 55.3
mM glucose, 50 mM NaHCO3, 9.9 mM KCl, 1.9 mM MgSO4,
40 mM HEPES, pH 6.8) and incubated in a water bath at 37 °C
until all chambers (normally six per experimental run) were
assembled. The buffer was then removed and substituted with 1
mL of a 20 μM solution of the compound to be tested in the
same buffer on the apical side. During the experiment, oxygen
was continuously bubbled in each basolateral compartment. An
aliquot of the initial apical solutions was incubated separately at
37 °C for the period of the experiment, to verify the stability of
each compound in the absence of jejunum. At the end of the
experiment (2.5 h) 800 μL of chamber contents on both apical
and basolateral sides were collected and mixed with 8 μL of 100
mM ascorbic acid in water, 8 μL of 6 M acetic acid, and 80 μL
of 250 μM 4,4′-dihydroxybiphenyl (internal standard) in
CH3CN. The samples were then centrifuged (12 000 g, 7
min, 4 °C), and supernatants were frozen and maintained at
−20 °C until HPLC-UV analysis.
The alkoxymethylene chloride agents used for the synthesis
of 2a−c are commercially available, whereas those for 2d−g
had to be synthesized in situ from the corresponding alcohol via
reaction with paraformaldehyde as shown in Scheme 2. The
Scheme 2. Synthesis of Alkoxymethylene Chloride from
Alcohol and Paraformaldehyde
Pharmacokinetics. Derivatives 2a−g and 4 were adminis-
tered to overnight-fasted male Wistar rats from the stabulary of
the Department of Biomedical Sciences as a single intragastric
dose (88 μmol/kg, dissolved in 250 μL DMSO). Blood samples
were obtained by the tail bleeding technique: before drug
administration, rats were anesthetised with isoflurane, and the
tip of the tail was cut off; blood samples (80−100 μL each)
were then taken from the tail tip at different time points after
drug administration. Blood was collected in heparinised tubes,
kept in ice, and treated as described below within 10 min. The
AUC values were calculated using the trapezoidal rule. All
experiments involving animals were performed with the
permission and supervision of the University of Padova Ethical
Committee for Experimentation on Animals (CEASA) and
Central Veterinary Service, in compliance with Italian Law DL
116/92, embodying UE Directive 86/609.
high reactivity of alkoxymethylene chlorides does not allow
their purification via column chromatography on silica. Thus,
after removal of excess trimethylchlorosilane under vacuum,
they were used immediately in the next step without any further
purification.
The reactivity of the acetal bond can be modulated by the
introduction of suitable substituents onto the methylenic
carbon. In particular, alkyl groups are known to favor acid-
catalyzed hydrolysis.64 To assess the stability of these bonds in
the context of our compounds and conditions we therefore
synthesized the ketal 3 and the acetal 4 by reaction of
resveratrol with the appropriate isopropenyl and vinyl ethers,
respectively, in the presence of catalytic amounts of pyridinium
p-toluenesulfonate (Scheme 3).
Determination of Solubility and of log Pow. We
determined the effect of varying the length of the OEG chain
on the solubility in water and on the octanol/water partition
coefficient (log Pow) (Table 1) of the formal derivatives. Log
Pow is indeed considered to be an important predictive
parameter for absorption of passively absorbed drugs.65
Statistics. Significance in comparisons was assessed using
the Wilcoxon Rank Test.
RESULTS
■
Synthesis. Resveratrol formal derivatives 2a−g were
synthesized via nucleophilic substitution by deprotonated
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Scheme 3. Synthesis of Ketal 3 and Acetal 4
Table 1. Water Solubility and log Pow of Resveratrol and of Its Formal Derivatives (N = 3)
2a
2b
2c
2d
2e
>6000
2.34 0.01
2f
>6000
1.70 0.01
2g
>6000
0.43 0.02
1
solubility (μM)
log Pow
a
a
90 10
230 50
170 40
3.41 0.03
3.88 0.09
3.67 0.06
2.95 0.06
2.69 0.12
a
Below detection limit.
Compounds 2a−c are less soluble than resveratrol itself; the
solubility markedly increased with the presence of additional
ethoxy groups in the chain. The octanol−water partition
coefficients of formal OEG derivatives show an opposite trend.
Compounds 2e and 2f possess a remarkable solubility in water
and have, at the same time, a log Pow value in the optimum
range for oral administration and absorption.
Stability Studies. Hydrolysis of 2a and 2b was not assayed
due to the very low solubility of these compounds in water-
based media. The other formals proved stable under near-
neutral pH conditions (no reaction over 24 h at 37 °C in 0.1 M
phosphate buffer, pH 6.8) as well as in blood (no reaction over
4 h). In 0.1 N HCl they all hydrolyzed at similar rates. For each
derivative, suitable HPLC elution conditions were found to
separate all components of the hydrolysis reaction mixture
comprising, besides the reagent and the final product
(resveratrol), four reaction intermediates, that is, the two
isomeric disubstituted (3,4′- and 3,5-) and the two isomeric
monosubstituted (3- and 4′-) derivatives. As an example, the
concentration vs time profiles of all six species involved in the
hydrolysis if 2f are shown in Figure 2a. Kinetic analysis of the
data was then performed by assuming that hydrolysis to
resveratrol occurs via consecutive losses of the three protecting
groups in pseudofirst order processes and by considering each
pair of isomeric intermediates as a single species, that is, the
two monosubstituted and the two disubstituted intermediates
are handled as species B and C, respectively (Scheme 4).
Figure 2b shows the time course of the four species (A, B, C,
and D) involved in the case of 2f and the fit of the experimental
data obtained using a set of equations analogous to those
utilized by Kozerski et al.66 and presented as Supporting
Information. The excellent fit observed allows to conclude that
the assumptions made are justified and that the rate of
hydrolysis of each individual formal bond does not depend
significantly on the position occupied on the resveratrol scaffold
or on the presence of other analogous linkages in the molecule.
In fact, at any given time the concentration of one disubstituted
resveratrol (the 3,4′-disubstituted) was close to twice that of
the other disubstituted isomer (the 3,5-disubstituted), indicat-
ing that the formal bonds at the various positions react at about
the same rate both in the trisubstituted compound and in the
disubstituted ones. Consistently, the amount of one mono-
substituted (the 3-substituted) resveratrol was always close to
Figure 2. Kinetics of the hydrolysis of 2f in 0.1 N HCl. Data from a
single representative experiment. (a) Time profiles of reagent, product,
and four reaction intermediates as obtained by HPLC analysis; (b)
kinetic fit of the experimental data shown in panel a according to
Scheme 4 (see text and Supporting Information).
twice that of the other isomer (the 4′-substituted molecule)
(Figure 2a).
Analogous analyses and procedures were used to obtain
kinetic data for the hydrolysis of the other formal derivatives
(2c−e and 2g). The obtained rate constant values are reported
in Table S1 (Supporting Information).
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derivatives described above. It is indeed apparent that the rate
of hydrolysis of this class of protecting groups of phenolic
hydroxyls is not significantly influenced by the specific site of
substitution or by the presence of additional similar groups in
the molecule.
Scheme 4. Kinetic Scheme for the Hydrolysis of 2c−g
Transport across Explanted Rat Intestinal Segments.
Using Ussing chambers we also assayed the transport of formals
2a−g across explanted rat jejunum segments to verify what
effect the length of the OEG chain might have on this process.
In these experiments, the same molar amount of resveratrol or
prodrug was placed in the apical-side chamber, and the solution
on the basolateral side was collected and analyzed after 2.5 h
(see Materials and Methods for experimental details). Figure 4
The ketal derivative 3 proved too labile, with about 50%
complete hydrolysis to resveratrol in 10 min at pH 7.4 and
room temperature (not shown). This compound therefore was
not studied further. In contrast, acetal 4 was stable at pH 6.8
and in blood but underwent hydrolysis under acidic conditions.
Hydrolysis was too rapid for determination of kinetic
parameters at pH 1 (HCl 0.1N). The pH of the rat stomach
is however 3−4, higher than in humans.67 We therefore
determined the kinetics of hydrolysis of 4 as a function of pH in
a higher pH range (20 mM acetate buffer, pH 3.6−5.6). Figure
3 presents the k values, determined with reference to Scheme 4
as described above, for the consecutive hydrolysis steps of 4 in
the pH range 4.0−5.6 (a reliable fit could not be obtained for
the reaction at pH 3.6).
Figure 4. Transport across the explanted rat intestinal segments.
Plotted is the amount found in the basolateral chamber (mean
standard deviation; N = 3), expressed as percentage of the total
amount of stilbenoid compounds recovered at the end of the 2.5-h
incubation. See text and Materials and Methods for details. *:
significantly different from basolateral translocation of 1 (p ≤ 0.1).
The results shown in Figure 3 at any given pH value support
and extend the conclusions reached for reaction of the formal
summarizes the results, showing the amounts of stilbene
derivatives found in the basolateral compartment at the end of
the experiment expressed as percentage of the total species
recovered in the apical and basolateral sides. When the
compound provided was resveratrol, no resveratrol could be
detected in the basolateral chamber, which contained instead
metabolites adding up to 0.6
0.3% of the total recovered
amount. No compounds ascribable to a stilbenoid skeleton
could be detected in the basolateral chamber when compounds
2a or 2b were loaded. The other formal derivatives appeared
instead on the basolateral side without alteration. In the case of
compounds 2d and 2e the fraction translocated exceeded that
of resveratrol (metabolites) (1.4 0.4% and 1.5 0.3% for 2d
and 2e, respectively; N = 3). A substituent chain containing 3
or 4 monomeric units seems therefore optimal for permeation
of the intestinal wall.
Pharmacokinetics and Absorption. All synthesized
derivatives, except 3, were then used in pharmacokinetic tests
in rats. The administration of 88 μmol/kg of compounds 2a−b
did not result in the appearance of resveratrol, derivatives, or
any metabolites in blood samples. Compound 2c was absorbed
to a small extent, but circulating species were below the
quantification limit (0.12 μM).62 In contrast, compounds 2d−f
appeared in blood in unmodified form within 10 min of
administration, with a concentration peak between 30 and 60
min. The products of partial hydrolysis, including disubstituted
formal derivatives and coeluting trisubstituted species carrying
shortened OEG chains, were also found with a similar kinetic
profile but in lower concentration. As an example, the results of
HPLC/ESI-MS analysis of a blood sample from a pharmaco-
Figure 3. Kinetics of the hydrolysis of 4 at different pH values. (a)
Observed rate constants ( standard error) for the hydrolysis of 4, as a
function of pH; (b) kinetic fit of the experimental data at pH 4.0,
according to Scheme 4 (see text and Supporting Information).
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kinetic experiment with 2f are shown in Figure 5. Besides a
major peak due to 2f, a few minor and coeluting chromato-
graphic peaks are detected which are assigned to products due
to hydrolysis of one formal linkage, to give a disubstituted
derivative, and to progressive loss of the terminal methyl groups
(Δ m/z = −14) and single ethyleneglycol units (Δ m/z = −44)
(Figure 5). Interestingly, this type of degradation of the
substituent groups does not take place upon incubation of the
original precursors (2f in this case) in solution. We presume
these species derive from the action of bacterial enzymes in the
gut. Degradation of PEGs by intestinal microbes is known to
occur.68−70 No resveratrol or resveratrol conjugates (glucur-
onides, sulfates) could be detected in the blood samples with
any of the formal-based prodrugs.
The results of pharmacokinetic tests with compound 2f are
summarized in Figure 6, which also shows the pharmacoki-
netics of resveratrol when administered as such for comparison.
Analogous graphs for compounds 2d, 2e, and 2g are provided
as Supporting Information (Figure S1).
The outcome of pharmacokinetic experiments with the acetal
derivative 4 (Figure 7) was clearly different from those of the
tests with the formals. The major species found in blood was
resveratrol glucuronide(s) as identified in HPLC/ESI-MS
analyses by comparison with an authentic sample. Its
concentration peaked at approximately 8 h after administration.
The area under the curve (AUC) parameter for the formal
derivatives increases with the length of the OEG chain reaching
a maximum for derivative 2f (Figure 8). Interestingly, in the
case of this compound the AUC vastly exceeds that measured
for resveratrol as suchand is comparable to the combined
value for resveratrol and its metabolitesafter administration
of the same dose of resveratrol. In the case of 4 the overall
AUC was somewhat lower than for 2f, which carries the same
substituent group.
DISCUSSION AND CONCLUSIONS
■
The prodrugs we synthesized and tested possess desirable
properties. In the case of acetal derivatives, the protective
groups are rapidly hydrolyzed in a strongly acidic environment.
In contrast, acid hydrolysis of formals is sufficiently slow, even
at pH ∼ 1, similar to that of the human stomach, not to
constitute a deterrent: in our pharmacokinetic experiments
hydrolysis of formal bond-linked protective groups was indeed
very limited. A well-developed formulation technology is
available in any case to permit the safe transit of acid-sensitive
drugs through the gastric compartment.71−73 Importantly, short
OEG chains appear to be a good choice as carrier group in
these resveratrol prodrugs: specifically, for chains comprising
three or more monomeric units the derivatives are much more
soluble in water than resveratrol. The method we used to
determine water solubility led to an estimated value of about
170 μM for resveratrol. It is to note that accurate solubility data
are notoriously hard to obtain for compounds which can form
colloids, and literature values for resveratrol range from less
than 1 μM74 to about 10 μM75 and all the way up to 300 μM.76
Solubility is relevant: essentially water-insoluble 2a and 2b do
not cross the intestinal wall in Ussing chamber experiments
even if kept in solution by the presence of DMSO as a
cosolvent and are not absorbed from the gastrointestinal tract
after oral administration. OEG-decorated, more soluble
derivatives can permeate explanted rat intestinal segments
(Figure 4) and are absorbed after oral administration (Figures
6, 7, and 8). The AUC of stilbenoid compounds in blood was
Figure 5. HPLC/ESI-MS analysis of a blood sample taken 1 h after
administration of 2f in a pharmacokinetic experiment. (a) UV
chromatogram (320 nm) showing the presence of 2f and of its partial
hydrolysis products (inserts reproduce the corresponding mass
spectra). Disubstituted resveratrol derivatives are not well-resolved
from the products of partial loss of ethyleneglycol units. (b) Extracted
ion chromatograms (EIC) for the m/z values corresponding to intact
2f (m/z 906 [M + NH4]+), disubtituted derivatives (m/z 686 [M +
NH4]+) and products of partial loss of ethyleneglycol units (m/z 892,
848, 804, 760, all due to [M + NH4]+).
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Figure 8. AUC parameter, as calculated from plots analogous to those
of Figures 6 and 7. For compounds 2d−g the values reported are the
sum of the AUC parameters measured for the prodrug itself and its
partial hydrolysis products, which were in all cases a minor
component. Conjugation products of resveratrol were detectable
only after administration of resveratrol itself (1) and of 4. In these two
cases AUC values are shown for both the compound as administered
and for total blood stilbenoids.
molecules, bypassing the xenobiotics-inactivating defense
systems of the gut and liver.
When resveratrol is administered, the vast majority of the
circulating species consists of its phase II metabolites; in the
case of our derivatives, still fully or partially protected molecules
were found in the bloodstream. This highlights the short-
coming of the formal bond: it is probably too stable at
physiological pH values to regenerate the parent compound at
useful rates.
To destabilize the bond, we substituted one (4) or two (3) of
the methylene hydrogens with methyl groups. These
substitutions had indeed marked effects on the rate of
hydrolysis. Compound 3 proved to be too reactive to be of
practical use. Compound 4 was also deprotected at an
appreciable rate in the pH range of the rat stomach (Figure
3), but deprotection was clearly not complete in vivo. The
original prodrug and the species resulting from loss of one of
the protecting groups were present in blood samples taken over
the first hour or so after administration, along with resveratrol
glucuronide (Figure 8). At later times, the glucuronide was
essentially the only stilbenoid found in blood. Interestingly, its
concentration profile in time was different from that observed
after administration of resveratrol (Figure 6a): the peak
concentration was reached at about 8 h vs about 1 h in the
case of resveratrol.
Figure 6. Pharmacokinetic profiles for: (a) resveratrol (1) and (b)
compound 2f. “Hydrolysis products” stands for disubstituted
derivatives and coeluting species carrying shortened OEG chains as
described in the text. Reported are mean values standard deviation
(N = 5 and 4 for 1 and 2f, respectively).
The bond linkage in 4 bears a close resemblance to that
present in glycoside and glucuronide derivatives of polyphenols.
In the case of glycosides, studies conducted mostly with
quercetin derivatives have led to a model envisioning both the
cleavage of the acetal bond by lactase phlorizin hydrolase
located on the luminal intestinal surface, followed by diffusion
of the aglycone into enterocytes, and the transport of the
glycoside by glucose transporter SGLT1 followed by
deglycosylation inside the cell.77−80 Similar mechanisms may
apply to resveratrol glycosides as well.81 Considering their log
Pow values and what is known about PEG chains-containing
prodrugs, our OEG decorated compounds presumably can
enter enterocytes and reach the blood by diffusing through
biomembranes, independently of transporters. Given that it
contains an acetal bond, 4 might be a substrate for lactase
phloridzin hydrolase. The results of pharmacokinetics with this
prodrug could therefore be explained by slow hydrolysis in the
intestine generating resveratrol which would be promptly
Figure 7. Pharmacokinetics of 4. Reported are mean values standard
deviation (N = 4).
similar after administration of resveratrol or of 2f, the derivative
with tetraethyleneglycol chains. This is remarkable when
considering the much higher molecular weight and water
solubility of the compound and is presumably related to a
partition coefficient still favoring permeation of membranes
(Table 1). This result, in keeping with the remarkable
properties of PEG, offers the perspective of achieving high
level systemic or targeted delivery of resveratrol-generating
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imported by enterocytes and appear in blood as the
glucuronide. This model however does not explain why the
fully or partially protected species disappear from blood after
about one hour: if resveratrol is produced by their hydrolysis in
the intestine over the subsequent at least 7 h, they should
obviously still be present and continue to transit from the
intestinal lumen to blood through the intestinal wall. Of note,
we were unable to detect any species containing both OEG
chain(s) and glucuronide (or sulfate) moieties in blood. The
hypothesis that 4 is completely hydrolyzed in the stomach to
produce resveratrol which then moves to the intestine to be
absorbed is also unlikely, given the different pharmacokinetics
observed upon administration of resveratrol and of 4.
An alternative model envisions the rapid absorption of the
prodrug (as well as of some resveratrol formed in the stomach;
Figure 3b), which then disappears from blood because it
becomes associated with other, less polar, environments
(tissues, membranes) due to its physicochemical properties.
Slow hydrolysis, possibly mediated by ubiquitous glucuroni-
dase/glycosylase activities, would gradually release resveratrol
which would then be glucuronidated and thus converted to a
hydrophilic species, returning therefore to the blood.
Whether this model is correct remains to be investigated.
Regardless, this study establishes that linkage to short OEG
chains may be a convenient stratagem to protect polyphenols
and possibly other drugs from metabolism while still allowing
sustained absorption. Coupling such a substituent to the core
molecule through a chemical bond with appropriate stability
characteristics in physiological environments may well result in
a functional prodrug for oral delivery. The acetal bond system,
adopted by nature and exploited in this study, appears to be a
viable candidate.
ABBREVIATIONS
■
AcOEt, ethyl acetate; AUC, area under the curve; CEASA,
Ethical Committee for Experimentation on Animals; EIC,
extracted ion chromatograms; ESI, electrospray ionization; log
Pow, octanol/water partition coefficient; MDR, multi-drug
resistance; OEG, oligoethyleneglycol; PBS, phosphate-buffered
saline; PEG, polyethyleneglycol; PTFE, polytetrafluoroethy-
lene; SGLT1, sodium/glucose cotransporter 1; TFA, trifluoro-
acetic acid; TLC, thin-layer chromatography
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ASSOCIATED CONTENT
■
S
* Supporting Information
Kinetics of hydrolysis of compounds 2c−g in 0.1 M HCl;
pharmacokinetic profiles of compounds 2d, 2e, 2g. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*C.P.: e-mail: cristina.paradisi@unipd.it; phone: +39 049
8275661. L.B.: e-mail: lucia.biasutto@cnr.it; phone: +39 049
8276483.
Author Contributions
A.M. and M.A. contributed equally to this work.
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2007, 7, 344−351.
Notes
The authors declare no competing financial interest.
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■
We thank Prof. S. Garbisa for discussions and support, Dr. E.
Marotta for useful discussions, Dr. E. Bradaschia for performing
some of the pharmacokinetics, and Mr. M. Ghidotti for
technical help. This work was supported by grants from the
Fondazione Cassa di Risparmio di Padova e Rovigo
(CARIPARO) (“Developing a Pharmacology of Polyphenols”),
the University of Padova (postdoctoral fellowships to L.B. and
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