Synthesis and Evaluation as Prodrugs of Hydrophilic Carbamate Ester Analogues of Resveratrol

Article abstract of DOI:10.1021/acs.molpharmaceut.5b00464

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is an unfulfilled promise for health care: its exploitation is hindered by rapid conjugative metabolism in enterocytes and hepatocytes; low water solubility is a serious practical problem. To advantageously modify the physicochemical properties of the compound we have developed prodrugs in which all or part of the hydroxyl groups are linked via an N-monosubstituted carbamate ester bond to promoieties derived from glycerol or galactose, conferring higher water solubility. Kinetic studies of hydrolysis in aqueous solutions and in blood indicated that regeneration of resveratrol takes place in an appropriate time frame for delivery via oral administration. Despite their hydrophilicity some of the synthesized compounds were absorbed in the gastrointestinal tract of rats. In these cases the species found in blood after administration of a bolus consisted mainly of partially deprotected resveratrol derivatives and of the products of their glucuronidation, thus providing proof-of-principle evidence of behavior as prodrugs. The soluble compounds largely reached the lower intestinal tract. Upon administration of resveratrol, the major species found in this region was dihydroresveratrol, produced by enzymes of the intestinal flora. In experiments with a fully protected (trisubstituted) deoxygalactose containing prodrug, the major species were the prodrug itself and partially deprotected derivatives, along with small amounts of dihydroresveratrol. We conclude that the N-monosubstituted carbamate moiety is suitable for use in prodrugs of polyphenols. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.molpharmaceut.5b00464

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Article
Synthesis and Evaluation as Prodrugs of
Hydrophilic Carbamate Ester Analogs of Resveratrol
Michele Azzolini, Andrea Mattarei, Martina La Spina, Ester
Marotta, Mario Zoratti, Cristina Paradisi, and Lucia Biasutto
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00464 • Publication Date (Web): 07 Aug 2015
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Synthesis and Evaluation as Prodrugs of Hydrophilic
Carbamate Ester Analogs of Resveratrol
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‖
,#
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Michele Azzolini§, ,#, Andrea Mattareiɸ , Martina La Spina , Ester Marottaɸ, Mario Zoratti , Cristina
Ƣ
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‖
,
Paradisiɸ, Lucia Biasutto§,
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§Dept. of Biomedical Sciences, University of Padova, viale G. Colombo 3, 35131 Padova, Italy
ƢNÓOS Srl, via Campello sul Clitunno 34, 00181 Roma, Italy
ɸDept of Chemical Sciences, University of Padova, via Marzolo 1, 35121 Padova, Italy
‖CNR Neuroscience Institute, viale G. Colombo 3, 35131 Padova, Italy
KEYWORDS. Resveratrol, bioavailability, prodrugs, carbamate ester, pharmacokinetics, galactose,
glycerol.
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ABSTRACT
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Resveratrol (3,5,4’ꢀtrihydroxyꢀtransꢀstilbene) is an unfulfilled promise for health care: its exploitation
is hindered by rapid conjugative metabolism in enterocytes and hepatocytes; low water solubility is a
serious practical problem. To advantageously modify the physicochemical properties of the compound
we have developed prodrugs in which all or part of the hydroxyl groups are linked via an Nꢀ
monosubstituted carbamate ester bond to promoieties derived from glycerol or galactose, conferring
higher water solubility. Kinetic studies of hydrolysis in aqueous solutions and in blood indicated that
regeneration of resveratrol takes place in an appropriate time frame for delivery via oral administration.
Despite their hydrophilicity some of the synthesized compounds were absorbed in the gastrointestinal
tract of rats. In these cases the species found in blood after administration of a bolus consisted mainly
of partially deprotected resveratrol derivatives and of the products of their glucuronidation, thus
providing proofꢀofꢀprinciple evidence of behavior as prodrugs. The soluble compounds largely reached
the lower intestinal tract. Upon administration of resveratrol, the major species found in this region was
dihydroresveratrol, produced by enzymes of the intestinal flora. In experiments with a fully protected
(trisubstituted) deoxygalactose containing prodrug, the major species were the prodrug itself and
partially deprotected derivatives, along with small amounts of dihydroresveratrol. We conclude that the
Nꢀmonosubstituted carbamate moiety is suitable for use in prodrugs of polyphenols.
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■ INTRODUCTION
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Resveratrol (3,5,4’ꢀtrihydroxyꢀtransꢀstilbene) is well known to the public as a “natural drug”,
potentially useful on major health battlefields: afflictions of the cardiovascular system,1ꢀ3
neurodegeneration,4ꢀ7 aging,8, 9 diabetes, obesity, metabolic syndrome,10ꢀ12 cancer.13, 14
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This multiplicity of potential biomedical effects stems from the impact resveratrol has on the activity
of apical or key proteins in interconnected cellular signaling networks. Among its major effects
observed in vitro, resveratrol inhibits phosphodiesterases, leading to an increase of cellular cAMP.15, 16
Signaling along the cAMPꢀAMPKꢀmTORC1 axis is thought to largely account for the antagonistic
activity of resveratrol vs metabolic syndrome.17, 18 Modulation of this pathway,19 but also of others, e.g.
the activity of MTA120 and of microRNA’s,21, 22 is important in cancer. After much debate, a consensus
now seems to have been reached that resveratrol also induces upregulation of SIRT1, an important
NADꢀdependent histone deacetylase.23, 24 Enhancement of SIRT1 activity has pleiotropic epigenetic
effects, reportedly culminating in life extension and health improvement in mice.25, 26 Another,
interwined27ꢀ29 major way resveratrol acts is by antagonizing inflammation.30, 31 Chronic inflammation
is well recognized to be a factor in carcinogenesis.32ꢀ34 A relevant example is provided by the frequent
insurgence of polyposis and colon cancer in humans35ꢀ37 and laboratory animals38, 39 affected by
inflammatory bowel disease. Resveratrol has been reported to be beneficial in rodent models of colitis,
polyposis and colon cancer induced by sodium dextran sulfate (DSS) or azoxymethane + DSS.40ꢀ44
Resveratrol is not, however, free of shortcomings. The major one is its builtꢀin propensity, common
to all polyphenols, to undergo Phase II metabolic conjugation to give sulfated and glucuronidated
derivatives.45ꢀ50 In addition resveratrol undergoes reduction of the double bond plus other
transformations by the colonic flora.51, 52 It has therefore often been pointed out53 that the results of
studies carried out in vitro with high concentrations of unmodified resveratrol may be of little
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pharmacological or nutritional relevance. Another unfavorable characteristic of resveratrol is its low
water solubility.
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A variety of formulations and nanoꢀstructured delivery systems are being tested with the goal of
overcoming these obstacles.54, 55 Another approach relies on the construction of prodrugs, i.e. reversible
derivatives with favorable physicochemical properties which can offer initial protection from
metabolism and regenerate the active compound with appropriate kinetics.55, 56 As the molecular
mechanisms of action are clarified, and polyphenols tend to be viewed more and more as drugs rather
than as nutrients, it seems well justified to apply this strategy and to develop a pharmacology of
bioactive natural compounds. Indeed, in view of the manyꢀfold activities and possible applications of
resveratrol there is a need for customized delivery vehicles and/or chemical elaborations to best achieve
the specific task at hand. Thus, returning to the example of the use of resveratrol against colitis and
associated pathologies, specific delivery to the colon has been obtained using a formulation (Ca
pectinate beads)57 or sophisticated prodrugs comprising a glucose moiety linked to resveratrol via a
glycosidic bond and an hydrocarbon chain linked to the sugar ring by an ester bond.58 In addition to the
acetal bond system,58, 59 other groups including carboxyester, sulfonate and carbonate have been tested
as protective, reversible capping groups for the hydroxyls of resveratrol. However, none of them has
proven satisfactory.55, 56
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The carbamoyl ester linkage is a promising alternative.60 A first family of resveratrol prodrugs was
produced and tested, in which the phenolic OH groups are masked as –OꢀC(=O)ꢀN(CH3)R where R is a
methoxypolyethylene glycolꢀ350 (mPEGꢀ350) or a butylꢀglucosyl group.61 These derivatives are highly
soluble in aqueous media but too stable to be used as prodrugs.61 Nꢀmonosubstituted carbamates are
expected to undergo faster hydrolysis.62 Here we report the synthesis and characterization of monoꢀ, diꢀ
and triꢀsubstituted resveratrol derivatives incorporating promoieties imparting water solubility
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(dihydroxypropyl and 6ꢀdeoxygalactosyl moieties) linked via Nꢀmonosubstituted carbamate ester
bonds to the phenolic oxygens of resveratrol. The kinetics and products of hydrolysis have been
studied, and the fate of the prodrugs after oral administration to rats has been investigated. The results
indicate that these new compounds may be useful for all applications in which water solubility and
protection from metabolism and oxidation are important.
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■ RESULTS AND DISCUSSION
Synthesis. The new derivatives of resveratrol (1) synthesized in this work, compounds 2ꢀ11, are
shown in Scheme 1.
Scheme 1. Molecular structure of resveratrol (1, trans 3,5,4’ꢀtrihydroxy stilbene) and of its carbamate
ester derivatives synthesized in this work (2ꢀ11).
R’
DHPꢀC
H
R’’
H
R’’’
2
3
H
DHPꢀC
DHPꢀC
DHPꢀC
DHPꢀC
H
H
4
DHPꢀC
H
H
DHPꢀC
DHPꢀC
H
5
6
DHPꢀC
DGALꢀC
H
7
8
DGALꢀC
H
9
DGALꢀC DGALꢀC
H
10
11
H
DGALꢀC DGALꢀC
DGALꢀC DGALꢀC DGALꢀC
The synthesis of 2ꢀ6 is outlined in Scheme 2. The starting material for the dihydroxypropyl
carbamate (DHPꢀC) moiety was commercially available 2,2ꢀdimethylꢀ1,3ꢀdioxolaneꢀ4ꢀmethanamine
(12) which was allowed to react with bisꢀ(4ꢀnitrophenyl) carbonate in the presence of 4ꢀ
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(dimethylamino)pyridine (DMAP) to give the corresponding activated urethane (13) in good yield.
Compound 13 was added in slight excess to resveratrol (1) in pyridine and in the presence of DMAP
and allowed to react at 70°C to obtain a mixture of all the possible monoꢀ, diꢀ and triꢀ substituted
resveratrol derivatives (14ꢀ18). The last step consisted in the removal of the isopropylidene protecting
groups, freeing the hydroxyl functions necessary to enhance the solubility in water of the final products
(2ꢀ6) which were finally isolated by preparative reverseꢀphase HPLC. Purity was in all cases > 95%, as
assessed by HPLCꢀUV analysis.
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Scheme 2. Synthesis of derivatives 2ꢀ6.a
X’
DDMꢀC
H
X’’
X’’’
H
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18
H
DDMꢀC
DDMꢀC
DDMꢀC
DDMꢀC
H
DDMꢀC
H
H
DDMꢀC
DDMꢀC
DDMꢀC
a Reagents and conditions: (a) bisꢀ(4ꢀnitrophenyl) carbonate, DMAP, THF, r.t., 3 h; (b) resveratrol
(1), pyridine, DMAP, 70°C, 16 h; (c) trifluoroacetic acid : water 9:1, r.t., 1.5 h.
The synthesis of derivatives 7ꢀ11 (Scheme 3) was somewhat more complex, because the starting
reagent, amine (22), is not commercially available and had to be synthesized. The starting material in
this case was 1,2:3,4ꢀdiꢀOꢀisopropylideneꢀαꢀDꢀgalactopyranose (19), possessing one free hydroxyl
group, which was esterified with tosyl chloride to obtain compound 20. The tosylate group of 20 was
easily displaced in the second step by sodium azide giving the azide (21) in nearly quantitative yield.
Azide 21 was then reduced under Staudinger reaction conditions to give the desired primary amine (22)
in excellent yield. The subsequent synthetic steps are equivalent to those reported above for derivatives
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(2ꢀ6), and consisted in the production of the active urethane (23) followed by transesterification with
resveratrol to give a mixture of all the possible monoꢀ, diꢀ and triꢀ substituted resveratrol derivatives
(24ꢀ28). Cleavage of the isopropylidene protecting groups afforded the desired resveratrolꢀ6ꢀdeoxyꢀ
galactosyl carbamate ester conjugates (7ꢀ11), which were isolated by preparative HPLC.
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Scheme 3. Synthesis of derivatives 7ꢀ11.a
Y’
Y’’
H
Y’’’
H
24 DIGꢀC
25
H
DIGꢀC
H
26 DIGꢀC DIGꢀC
27
H
H
DIGꢀC DIGꢀC
28 DIGꢀC DIGꢀC DIGꢀC
a Reagents and conditions: (i) tosyl chloride, pyridine, DMAP, from 0°C to r.t., 5 h; (ii) sodium azide,
DMSO, 160°C, 1.5 h; (iii) triphenylphosphine, THF, r.t., 4 h; (iv) water, reflux, 16 h; (a) bisꢀ(4ꢀ
nitrophenyl) carbonate, DMAP, THF, r.t., 3 h; (b) resveratrol (1), pyridine, DMAP, 70°C, 16 h; (c)
trifluoroacetic acid : water 9:1, r.t., 1.5 h.
Log Pow, Solubility and TPSA. The octanol/water partition coefficient, water solubility, and the
polar surface area were estimated for all new compounds synthesized (Table 1) (see Experimental
Section). The molecular polar surface area is the sum of the surfaces of polar atoms (primarily oxygen,
nitrogen and their hydrogens) in a molecule, as calculated from molecular models taking into account
atomic dimensions (modeled as spheres).63, 64 The ability to permeate cell membranes is considered to
be inversely related to this parameter, which is instead positively linked to water solubility.
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Table 1. Log Pow, water solubility and topological PSA values of resveratrol and of its carbamate ester
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derivatives.a
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Compound
Log Pow
2.69 ± 0.12 b, [59]
1.57
PSA (Å2)
60.68
Solubility (g/L)
Resveratrol (1)
0.039 ± 0.009 b, [59]
2
3
119.24
119.24
177.80
177.80
236.36
168.93
168.93
277.18
277.18
385.43
0.049
0.054
0.12
1.57
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0.15
5
0.15
0.13
6
ꢀ1.27
0.20
7
0.52
1.38
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0.52
1.51
9
ꢀ1.94
2.53
10
11
ꢀ1.94
2.80
ꢀ4.34
> 4.22 b
a
Algorithmꢀpredicted values unless otherwise indicated.
b Experimental value.
Hydrolysis studies. All the compounds proved to be stable over a 24 h period in acidic solution (0.1
N HCl) at 37°C. In contrast, they underwent hydrolysis of the carbamoyl bond in 0.1 M PBS buffer at
pH 6.8 and at 37°C. Kinetic results obtained with the dihydroxypropyl carbamate ester derivatives 2ꢀ6
are shown in Figure 1, which reports time profiles for reactants, intermediates and products. The
experimental data of reactant conversion are nicely fitted by a first order exponential decay equation,
from which rate constants (k) for hydrolysis of 2ꢀ6 were derived.
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3
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Time (h)
Figure 1. Hydrolysis of resveratrol dihydroxypropyl carbamate ester derivatives (2ꢀ6) in PBS 0.1 M,
pH 6.8, 37°C. Hydrolysis of the trisubstituted compound 6 forms disubstituted isomers 4 and 5. In turn,
hydrolysis of 4 leads to a mixture of monosubstituted isomers 2 and 3, while 5 can only give rise to 3.
Finally, hydrolysis of either 2 or 3 yields resveratrol, 1. Data are expressed as % of the initially loaded
compound. The fit is for pseudoꢀfirst order kinetics.
Similar behavior was observed with the 6ꢀdeoxygalactosyl carbamate ester derivatives 7ꢀ11 (kinetic
plots not shown). The pseudo first order hydrolysis rate constants obtained in all these kinetic
experiments are collected in Table 2. As was reasonably expected, hydrolysis of multifunctional
derivatives proceeds in a stepwise manner. Thus, reaction of the trisubstituted derivative 6 leads to
disubstitued resveratrols 4 and 5, which in turn hydrolize to the monosubstituted compounds 2 and 3
(Figure 1). Interestingly, the data in Table 2 show that different isomers undergo hydrolysis at different
rates. Specifically, if one considers the disubstituted isomers (3,4’ꢀ vs 3,5ꢀ; i.e. 4 vs 5, 9 vs 10), it
appears that the presence of two carbamoyl moieties in the same ring (at positions 3 and 5) accelerates
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the hydrolysis process. This conclusion is consistent with the product distribution observed in the
hydrolysis of the trisubstituted derivatives 6 and 11. In both cases, the 3,4’ꢀ isomers, 4 and 9, are
present in larger amounts with respect to the more reactive 3,5ꢀ isomers, 5 and 10, respectively.
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Table 2. Rate constants (k) for hydrolysis of resveratrol derivatives 2ꢀ11 in aqueous PBS 0.1 M, at pH
6.8 and at 37°C.
102 ⋅ k (hꢀ1)
102 ⋅ k (hꢀ1)
Substitution pattern Compound
6
Compound
11
30.72 ± 0.08
32.9 ± 0.3
20.63 ± 0.09
23.8 ± 0.7
5
10
8.7 ± 0.1
3.62 ± 0.03
3.66 ± 0.08
8.8 ± 0.1
3.15 ± 0.03
3.69 ± 0.09
4
3
2
9
8
7
When only one carbamoyl function is present in the molecule, the rate of its hydrolysis is not
affected by the specific position occupied (see the similar reactivity of 4’ꢀ and 3ꢀ monosubstituted
derivatives, 2 vs 3 and 7 vs 8 in Table 2).
Our results are consistent with a base catalyzed mechanism of hydrolysis proceeding via
deprotonation and release of an isocyanate reactive intermediate, as sketched in Scheme 4.65
Scheme 4. Mechanism of base catalyzed hydrolysis of Nꢀmonosubstituted carbamate esters.
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O
O
C
R'
R
R'
R
C
RO-
+
O C NR'
N
H
O
N
O
B-
BH
9
This mechanism is not viable under acidic conditions, which accounts for the observation that
derivatives 2ꢀ11 are stable in acidic solutions. It is also obviously not viable for N,Nꢀdisubstituted
carbamate esters, which accounts for the observation that N,Nꢀdisubstituted analogs of 2ꢀ11 were found
to be stable also in neutral and basic solutions.61
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To verify whether these derivatives would withstand longꢀterm storage in aqueous formulations we
followed the hydrolysis of compound 11 in citrate buffer (5 mM each) at pH 5.0 and 6.0, at 4°C and at
room temperature, over several months. The results (Figure 2) indicate that indeed hydrolysis is very
slow under these conditions.
A
B
C
D
Time (days)
Figure 2. Long term stability of 11 against hydrolysis in aqueous citrate buffer. A: pH 6, R.T.; B: pH 6,
4 °C; C: pH 5, R.T.; D: pH 5, 4 °C.
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Hydrolysis in Blood. Hydrolysis in blood (Figure 3) was faster than in PBS pH 6.8. Reaction rates
were similar for dihydroxypropyl and 6ꢀdeoxygalactosyl carbamate ester derivatives. As found for
reaction in aqueous PBS solutions, hydrolysis in blood is favoured by the presence of two carbamoyl
groups on the same ring. Thus, reaction of 5 and 10 is faster than that of their isomers 4 and 9,
respectively. In the case of monosubstituted derivatives, the rate of hydrolysis depends on the
substitution position, the compounds substituted in 4’ꢀ reacting markedly more rapidly than their 3ꢀ
substituted isomers (see Figure 3 and Table 3). This indicates the involvement of a regioselective
(enzymatic) hydrolysis mechanism, since in aqueous buffered solutions the rates are similar for
isomeric monosubstituted pairs (2 and 3, 7 and 8).
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Time (h)
Figure 3. Hydrolysis of resveratrol dihydroxypropyl carbamates (2ꢀ6) in rat blood. Data are expressed
as % of the initially loaded compound.
Table 3. Rate constants (k) for hydrolysis of resveratrol derivatives 2ꢀ11 in rat blood, at 37°C.
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Pattern of
substitution
102 ⋅ k (hꢀ1)
102 ⋅ k (hꢀ1)
Compound
6
Compound
140 ± 10
390 ± 50
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136 ± 4
110 ± 20
5
36 ± 2
8.1 ± 0.3
35 ± 3
34 ± 2
11.4 ± 0.9
70 ± 6
4
3
2
9
8
7
Plasma albumin has carbamoyl esterase activity66, 67 and it has been reported to be the main
component of plasma catalyzing hydrolysis of phenolic carbamate esters.68
Pharmacokinetic Studies. In vitro stability studies thus indicated that the rate of hydrolysis of the
carbamoyl bond in our new resveratrol derivatives is of the correct order of magnitude for application
as prodrugs. We therefore proceeded to investigate the behavior of our compounds in pharmacokinetics
studies in vivo.
Dihydroxypropyl carbamate ester derivatives
Following administration of a single intragastric bolus, the monoꢀsubstituted derivatives were readily
absorbed, but also extensively metabolized. Low concentrations of the unmodified derivative were
detected in blood, together with considerably higher amounts of glucuronides(s). Remarkably, sulfated
metabolites were below detection limits in all cases. An example is shown in Figure 4, which shows
the HPLCꢀUV chromatogram of a blood sample withdrawn 2 hr after oral administration of 2. The
major metabolite was identified from its ESI mass spectra (Figure 4, panels B and C) and assigned to
the product of monoglucuronidation still bearing the protecting carbamoyl group (Scheme 5).
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Intens.
mAU
BiPh
A
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2
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Time [min]
UV Chromatogram, 298-302 nm
Intens.
+MS, 11.9min #420, Background Subtracted
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x10
522.1
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Intens.
-MS, 11.9min #419, Background Subtracted
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x10
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1.0
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0.2
0.0
656.0
785.9
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400
600
800
m/z
Figure 4. HPLCꢀUVꢀMS analysis of a blood sample taken 2h after administration of 2. A) UV
chromatogram, recorded at 300 nm; “BiPh” stands for 4,4’ꢀdihydroxybiphenyl, which was used as
internal standard. B) and C) are ESI mass spectra of the metabolite, acquired in the positive (B) and
negative (C) ion current mode, respectively.
HPLCꢀUVꢀESI/MS analysis showed that the other monosubstituted derivative 3 produced instead
two different isomeric monoglucuronidated metabolites, both still bearing the protective carbamoyl
group (Scheme 5).
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Scheme 5. Glucuronidated metabolites formed from 2 and 3.
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It was not possible to attribute the two peaks observed in HPLCꢀUV and HPLCꢀMS chromatograms
to the two isomeric glucuronides formed from 3. Therefore they are referred to by their
chromatographic retention times, which under our standard analysis protocol were 2.38 and 3.08 min,
respectively. The isomer with the retention time of 2.38 min, which is very similar to that (2.35 min) of
the monoglucuronidated metabolite formed from 2, probably also has a similar structure, i.e., it most
likely carries the glucuronic acid moiety in position 4’. It follows that in the metabolite with r.t. 3.08
the two substituents probably occupy positions 3 and 5.
In pharmacokinetic experiments, for both 2 and 3, the overall concentration of stilbenic compounds
was reached approximately 1 hour after administration (Figure 5). However, interestingly, the two
isomers have different kinetic profiles: thus, 2 and its metabolite persisted in the bloodstream for a
significantly longer time than 3 and its metabolites.
The triꢀ and diꢀsubstituted derivatives (4ꢀ6) were poorly absorbed. The relative amount of nonꢀ
conjugated vs conjugated (glucuronidated) derivatives in the bloodstream was inversely correlated to
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the number of protected hydroxyls in the original prodrug. This suggests that partially protected
resveratrol still functions as a substrate for glucuronosyl transferases.
7
8
9
When 6 was administered, the intact derivative rapidly reached the bloodstream, and underwent
hydrolysis to the 3,4’ꢀdisubstituted derivative (4). In addition, metabolites 2ꢀglucuronide and 3ꢀ
glucuronide with r.t. 3.08 min (Scheme 5) were also detected, and reached maximum concentration at
later times (tmax at 8 h and 2 h, respectively).
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Absorption of disubstituted derivatives was rapid, with the unmodified prodrug peaking in blood
about 30 minutes after gavage. Administration of the 3,4’ꢀ(dihydroxypropyl) derivative (4) resulted in
detectable levels of 2ꢀglucuronide and of 3ꢀglucuronide with retention time 2.38 min; no other
intermediate products were detected. On the other hand, administration of the 3,5ꢀ(dihydroxypropyl)
isomer (5) resulted in appearance of 3 and of its glucuronide with r.t. 2.38 min.
6
5
4
3
2
Time (h)
Figure 5. Blood pharmacokinetic profiles after oral administration of resveratrol derivatives (2ꢀ6). Data
represent average values + standard deviation. N = 3 in all cases.
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6-Deoxygalactosyl carbamate ester derivatives
4
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Pharmacokinetic determinations provided no evidence of absorption for 3,4’,5ꢀ and 3,5ꢀderivatives
(11 and 10) after oral administration.
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Figure 6 shows the pharmacokinetic profiles of 3,4’ꢀ, 3ꢀ and 4’ꢀ substituted derivatives (9, 8 and 7
respectively). Compounds were generally poorly absorbed. Absorption of 9 was rapid, reaching 10
minutes after administration a low peak blood concentration, which then remained approximately
constant for up to 2 hours. Derivative 8 reached an early peak in blood, with the concentration decaying
in an approximately exponential manner to undetectable levels after about 4 hours. The 4’ꢀsubstituted
derivative (7) again differed significantly from its isomer: it showed a delayed absorption with the
maximum concentration of total species in blood reached at approximately 2 hours after administration.
Metabolites or resveratrol could not be reliably identified in any pharmacokinetic run with
deoxygalactosyl carbamate ester derivatives.
9
8
7
Time (h)
Figure 6. Blood pharmacokinetic profiles after oral administration of resveratrol 6ꢀdeoxygalactosyl
derivatives 7ꢀ9. Data represent average values + standard deviation. N = 3 in all cases.
Intestinal Levels. The highly soluble derivative 11 did not enter systemic circulation after oral
administration. Poor intestinal absorption may however be expected to result in high levels in the
lumen of the lower intestinal tract, suggesting possible applications for the treatment of intestinal
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disorders. After chronic administration, a striking difference was observed in the intestinal fate of
resveratrol and of 11: in the colon and caecum the former was present mainly as its bacterial metabolite
dihydroresveratrol (Figure 7), while the latter was mainly present as partially hydrolyzed derivatives
(Figure 8). Low amounts of resveratrol and dihydroresveratrol were also detected.
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A
B
Intens.
x10
+MS, 22.0min #1541
Dihydroꢀ1
[M.H]+
5
Intens.
mAU
231.1
3
2
1
0
200
100
0
200
400
600
m/z
0
5
10
15
Time [min]
Figure 7. Resveratrol and its metabolites in the lower intestine of rats after chronic administration. A)
Amounts of resveratrol (1), dihydroresveratrol (dihydroꢀ1) and resveratrol sulfates (1ꢀsulfates) in
caecum and colon after 48h of chronic treatment with approx. 220 µmol/kg×day of resveratrol. B)
HPLCꢀUV chromatogram (286 nm) and ESI mass spectrum of dihydroresveratrol (insert).
Intens.
x10
Intens.
x10
Intens.
x10
-MS, 7.1min #492
878.0
-MS, 9.4min #663
A
B
5
5
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11
9 + 10 -MS, 8.2min #575
7 + 8
673.0
467.9 [M.Cl]ꢀ
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0.6
0.4
0.2
0.0
3
2
1
[M.Cl]ꢀ
[M.Cl]ꢀ
639.1
226.9
850.0
510.0
226.8
200
Intens.
mAU
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600
800 m/z
200
400
600
800 m/z
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800 m/z
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Time [min]
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Figure 8. Compound 11 and derived species in the lower intestine of rats after chronic administration.
A) Amounts of resveratrol derivatives in caecum and colon after 48h of chronic treatment with approx.
220 µmol/kg×day of 11. B) HPLCꢀUV chromatogram (286 nm) and ESI mass spectra of 11 and its
hydrolysis products (inserts).
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■ CONCLUSIONS
The new resveratrol derivatives we developed have properties and reactivity that match expectations
for their use as prodrugs. In particular, they are stable in acid solution, and can therefore survive the
gastric stage. In nearꢀneutral solution and blood the rate of hydrolysis is conveniently slow.
The behavior of these prodrugs visꢀàꢀvis absorption from the intestine is bound to depend on the
properties of the chosen promoieties. While water solubility is desirable for obvious practical reasons,
high hydrophilicity is known to hinder biomembrane permeation due to the hydrophobic barrier formed
by the hydrocarbon chains of phospholipids. Accordingly, while in in vivo experiments
dihydroxypropyl derivatives were absorbed, and monosubstituted ones (2, 3) satisfactorily so,
derivatives comprising the more hydrophilic 6ꢀdeoxygalactose moiety (7ꢀ11) performed poorly in this
respect. Solute carriers might intervene to aid translocation, but apparently 7ꢀ11 are not recognized
and/or transported by intestinal glucose carriers.
The trisubstituted compound 11 is the most soluble, and was not absorbed in the gastrointestinal
tract; however, interestingly, chronic administration of this compound resulted in its accumulation in
the lower intestine. The presence in colon and caecum of partially hydrolyzed derivatives of 11 points
to protracted release of resveratrol, which is rapidly reduced to dihydroresveratrol by bacterial
enzymes. These findings suggest a possible use of glycosylꢀresveratrol derivatives for the treatment of
colitis and prevention of colon cancer.
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Unmodified resveratrol was below detection limits in the bloodstream in all cases, undoubtedly
reflecting the powerful phase II metabolism of the liver. Blood however is well suited to accommodate
hydrophilic compounds such as glucuronides (and, to an extent, our prodrugs), and the composition of
prodrugꢀderived species in other organs may well be different. Important interꢀorgan variations have
been reported in the case of pterostilbene and its metabolites.69, 70
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■ EXPERIMENTAL SECTION
Materials. Resveratrol was purchased from Waseta Int. Trading Co. (Shangai, P.R.China). Other
starting materials and reagents were purchased from SigmaꢀAldrich, Fluka, MerckꢀNovabiochem,
Riedel de Haen, J.T. Baker, Cambridge Isotope Laboratories Inc., Acros Organics, Carlo Erba and
1
13
Prolabo, and were used as received. H and C NMR spectra were recorded with a Bruker AC250F
spectrometer and a Bruker Avance DMX 600. Chemical shifts (δ) are given in ppm relative to the
signal of the solvent. TLCs were run on silica gel supported on plastic (MachereyꢀNagel Polygram®SIL
G/UV254, silica thickness 0.2 mm) and visualized by UV detection, ninhydrin reaction or KMnO4
oxidation. Flash chromatography was performed on silica gel (MachereyꢀNagel 60, 230ꢀ400 mesh
granulometry (0.063ꢀ0.040 mm)) under air pressure. The solvents were analytical or synthetic grade
and were used without further purification. Preparative HPLC was performed using a Shimadzu LCꢀ8A
equipped with a UV/VIS detector SPDꢀ20A Prominence® and a reverse phase column (ACE 5AQ (150
× 21.2 mm)).
Animals. Adult male Wistar rats (approximately 400 g body weight) from the facility of the
Department of Biomedical Sciences were used for pharmacokinetic experiments. All experiments
involving animals were performed after approval by the University of Padova Ethical Committee for
Experimentation on Animals (CEASA) (Permit Number: 80/2011) and of the Italian Ministry of
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Health, and with the supervision of the Central Veterinary Service of the University of Padova, in
compliance with Italian Law DL 116/92, embodying UE Directive 86/609.
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Synthetic procedures
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Please refer to the Results sections for an outline of the general strategy. Synthetic targets
(compounds 2ꢀ11) as well as some intermediates were purified by preparative HPLC. For all new
compounds purity was > 95%, as assessed by HPLCꢀUV analysis.
[Nꢀ(2,2ꢀdimethylꢀ1,3ꢀdioxolaneꢀ4ꢀmethan)ꢀcarbamoyl]ꢀ4ꢀnitrophenol (13): Bisꢀ4ꢀ(nitrophenyl)
carbonate (3.47 g, 11.4 mmol, 1 eq.) in 20 mL of anhydrous THF and DMAP (2.79 g, 22.8 mmol, 2
eq.) were mixed in a 100 mL roundꢀbottom flask with a magnetic stirrer under N2. 2,2ꢀdimethylꢀ1,3ꢀ
dioxolaneꢀ4ꢀmethanamine (12, 1.5 g, 11.4 mmol, 1 eq.) in THF (20 mL) was then added dropwise at
RT. After three hours the mixture was taken up in EtOAc (150 mL), transferred to a 500 mL separation
funnel and washed with 0.5 M HCl (3 × 150 mL). The organic phase was then dried over MgSO4 and
filtered. The solvent was evaporated under reduced pressure and the residue was purified by flash
chromatography on silica gel (eluent: CH2Cl2/EtOAc, 9.5:0.5) to afford 13 (75% yield). 1HꢀNMR (250
MHz, CDCl3) δ (ppm): 1.38 (s, 3H, ꢀCH3), 1.48 (s, 3H, ꢀCH3), 3.28ꢀ3.61 (m, 2H, ꢀNHꢀCH2), 3.70ꢀ4.14
(m, 2H, ꢀCH2ꢀ), 4.28ꢀ4.37 (m, 1H, ꢀCHꢀ), 7.32 (d, 2H, ArꢀH, 3JHꢀH: 9.25 Hz), 8.25 (d, 2H, ArꢀH, 3JHꢀH
:
13
9.25 Hz); CꢀNMR (62.9 MHz, CDCl3) δ (ppm): 155.7, 153.3, 144.7, 125.1, 121.9, 109.6, 74.2, 66.5,
43.6, 26.7, 25.0; ESIꢀMS (ion trap): m/z 297 [M+H]+.
(4’ꢀmonoꢀ, 3ꢀmonoꢀ, 3,4’ꢀdiꢀ, 3,5ꢀdiꢀ, 3,4’,5ꢀtriꢀ) [Nꢀ(2,2ꢀdimethylꢀ1,3ꢀdioxolaneꢀ4ꢀmethan)ꢀ
carbamoyl]ꢀresveratrol (14ꢀ18): 13 (1.24 g, 4.2 mmol, 2.6 eq.) and pyridine (10 mL) were placed in a
100 mL roundꢀbottom flask with magnetic stirrer and under N2. Resveratrol (1, 0.37 g, 1.6 mmol, 1.0
eq.) and DMAP (0.79 g, 6.4 mmol, 4.0 eq.) in pyridine (10 mL) was added and the reaction was
allowed to proceed at 70°C overnight. The mixture was then taken up with 150 mL of EtOAc,
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transferred to a separation funnel and washed with 0.5 M HCl (5 × 150 mL). The organic phase was
then dried over MgSO4 and filtered. The solvent was evaporated under reduced pressure. The resulting
solid was purified by flash chromatography (eluent: CH2Cl2/Acetone, 7.5:2.5) to afford 0.57 g of a
mixture of 14ꢀ18 used as such for the subsequent synthetic step. ESIꢀMS (ion trap): 14, 15: m/z 386
[M+H]+; 16, 17: m/z 543 [M+H]+; 18: m/z 700 [M+H]+.
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(4’ꢀmonoꢀ, 3ꢀmonoꢀ, 3,4’ꢀdiꢀ, 3,5ꢀdiꢀ, 3,4’,5ꢀtriꢀ)[Nꢀ(2,3ꢀdihydroxypropyl)ꢀcarbamoyl]ꢀ
resveratrol conjugates (2ꢀ6): The mixture (14ꢀ18, 0.57 g) obtained in the previous step was added to
a solution of TFA/water (90:10, 5 mL). The resulting mixture was vigorously stirred for 1.5 h at RT
and then dried under vacuum. The residue was then separated by preparative HPLC (from 0% to 40%
ACN in 17 minutes) and lyophilized to afford derivatives: 2 (0.129 g, cumulative yield of the final two
steps: 23%), 3 (0.068 g, cumulative yield of the final two steps: 12%), 4 (0.135 g, cumulative yield of
the final two steps: 18%), 5 (0.038 g, cumulative yield of the final two steps: 5%) and 6 (0.085 g,
cumulative yield of the final two steps: 9%).
2: 1HꢀNMR (250 MHz, DMSOꢀd6) δ (ppm): 2.95ꢀ3.25 (m, 2H, ꢀCH2ꢀ), 3.35 (d, 2H, 3JHꢀH = 5.25 Hz, ꢀ
4
CH2ꢀ), 3.51ꢀ3.66 (m, 1H, ꢀCHꢀ), 6.17 (t, 1H, 4JH,H = 2.0 Hz, Hꢀ4), 6.45 (d, 2H, JH,H = 2.0 Hz, Hꢀ2, Hꢀ
3
6), 7.03ꢀ7.10 (m, 4H, Hꢀ3’, Hꢀ5’, Hꢀ7, Hꢀ8), 7.57 (d, 2H, 3JHꢀH = 8.5 Hz, Hꢀ2’, Hꢀ6’), 7.65 (t, 1H, JHꢀH
= 5.75 Hz, ꢀNꢀH); 13CꢀNMR (62.9 MHz, DMSOꢀd6) δ (ppm): 158.5, 154.5, 150.5, 138.8, 133.8, 128.6,
127.2, 127.1, 121.9, 104.7, 102.3, 70.3, 63.8, 44.1; ESIꢀMS (ion trap): m/z 346 [M+ H]+.
3: 1HꢀNMR (250 MHz, DMSOꢀd6) δ (ppm): 2.93ꢀ3.23 (m, 2H, ꢀCH2ꢀ), 3.34 (d, 2H, 3JHꢀH = 5.25 Hz, ꢀ
CH2ꢀ), 3.52ꢀ3.61 (m, 1H, ꢀCHꢀ), 6.38 (t, 1H, 4JH,H = 2.0 Hz, Hꢀ4), 6.73ꢀ6.78 (m, 4H, Hꢀ2, Hꢀ6, Hꢀ3’, Hꢀ
3
3
3
5’), 6.90 (d, 1H, JHꢀH = 16.5, Hꢀ7), 7.07 (d, 1H, JHꢀH = 16.5, Hꢀ8), 7.42 (d, 2H, JHꢀH = 8.75, Hꢀ2’, Hꢀ
3
13
6’), 7.58 (t, 2H, JHꢀH = 5.75 Hz, 2 × ꢀNꢀH); CꢀNMR (62.9 MHz, DMSOꢀd6) δ (ppm): 158.0, 157.4,
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154.5, 152.2, 139.3, 128.9, 128.0, 127.8, 124.6, 115.5, 110.0, 109.7, 107.8, 70.3, 63.8, 44.0; ESIꢀMS
(ion trap): m/z 346 [M+ H]+.
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4: 1HꢀNMR (250 MHz, DMSOꢀd6) δ (ppm): 1HꢀNMR (250 MHz, DMSOꢀd6) δ (ppm): 2.95ꢀ3.25 (m,
3
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4H, 2 × ꢀCH2ꢀ), 3.36 (d, 4H, JHꢀH = 5.25 Hz, 2 × ꢀCH2ꢀ), 3.53ꢀ3.62 (m, 2H, ꢀCHꢀ), 6.41ꢀ6.43 (m, 1H,
Hꢀ4), 6.77ꢀ6.80 (m, 2H, Hꢀ2, Hꢀ6), 7.02ꢀ7.19 (m, 4H, Hꢀ3’, Hꢀ5’, Hꢀ7, Hꢀ8), 7.52ꢀ7.61 (m, 4H, 2 × ꢀ
NHꢀ, Hꢀ2’, Hꢀ6’); 13CꢀNMR (62.9 MHz, DMSOꢀd6) δ (ppm): 158.1, 154.5, 154.4, 152.2, 150.6, 138.7,
133.6, 128.0, 127.6, 127.3, 121.9, 110.3, 110.1, 108.4, 70.2, 70.3, 63.7, 44.0; ESIꢀMS (ion trap): m/z
463 [M+H]+.
5: 1HꢀNMR (250 MHz, CDCl3) δ (ppm): 1HꢀNMR (250 MHz, DMSOꢀd6) δ (ppm): 2.96ꢀ3.25 (m, 4H,
2 × ꢀCH2ꢀ), 3.36 (d, 4H, 3JHꢀH = 5.25 Hz, 2 × ꢀCH2ꢀ), 3.54ꢀ3.63 (m, 2H, 2 × ꢀCHꢀ), 6.64ꢀ6.80 (m, 3H, Hꢀ
4, Hꢀ2, Hꢀ6), 6.97ꢀ7.24 (m, 4H, Hꢀ3’, Hꢀ5’, Hꢀ7, Hꢀ8), 7.40 (d, 2H, 3JHꢀH = 8.5 Hz, Hꢀ2’, Hꢀ6’), 7.70 (t,
2H, 3JHꢀH = 5.75 Hz, 2 × ꢀNꢀH); 13CꢀNMR (62.9 MHz, DMSOꢀd6) δ (ppm): 157.6, 154.3, 151.8, 139.4,
130.0, 128.2, 127.7, 123.7, 115.8, 115.6, 114.0, 70.3, 63.8, 44.2; ESIꢀMS (ion trap): m/z 463 [M+H]+.
6: 1HꢀNMR (250 MHz, CDCl3) δ (ppm): 1HꢀNMR (250 MHz DMSOꢀd6) δ (ppm): 2.94ꢀ3.25 (m, 6H,
3 × ꢀCH2ꢀ), 3.35 (d, 6H, JHꢀH = 5.25 Hz, 3 × ꢀCH2ꢀ), 3.53ꢀ3.62 (m, 3H, 3 × ꢀCHꢀ), 6.78 (t, 1H, 4JH,H
=
3
2.0 Hz, Hꢀ4), 7.10ꢀ7.36 (m, 6H, Hꢀ2, Hꢀ6, Hꢀ3’, Hꢀ5’, Hꢀ7, Hꢀ8), 7.59ꢀ7.74 (m, 5H, Hꢀ2’, Hꢀ6’, 3 × ꢀNꢀ
13
H); CꢀNMR (62.9 MHz, DMSOꢀd6) δ (ppm): 154.4, 154.2, 151.7, 150.8, 138.8, 133.5, 129.1, 127.5,
126.7, 122.0, 116.2, 114.6, 70.3, 63.8, 44.1; ESIꢀMS (ion trap): m/z 580 [M+H]+.
(1,2:3,4ꢀdiꢀOꢀisopropylideneꢀ6ꢀOꢀ(pꢀtoluensulfonate))ꢀαꢀDꢀgalactopiranose (20): Pyridine (3.1
mL, 38.4 mmol, 2.0 eq.) and DMAP (3.51 g, 28.7 mmol, 1.5 eq.) were added to a solution of 1,2:3,4ꢀ
diꢀOꢀisopropylideneꢀαꢀDꢀgalactopiranose (19, 5.0 g, 19.2 mmol, 1.0 eq.) in CH2Cl2 (20 mL), and the
mixture was stirred at 0°C for 15 min. A solution of tosyl chloride (5.49 g, 28.7 mmol, 1.5 eq.) in
CH2Cl2 (20 mL) was then added dropwise and the mixture was stirred at room temperature for 5 hours.
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After adding H2O (30 mL), the mixture was diluted in CH2Cl2 (150 mL) and washed with 0.5 N HCl (5
× 100 mL). The organic layer was dried over MgSO4 and filtered. The solvent was evaporated under
reduced pressure and the residue was purified by flash chromatography (eluent: CH2Cl2/EtOAc, 9:1) to
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afford 7.33 g of 20 (92 % yield). HꢀNMR (250 MHz, CDCl3) δ (ppm): 1.26 (s, 3H, ꢀCꢀCH3), 1.30 (s,
3H, ꢀCꢀCH3), 1.33 (s, 3H, ꢀCꢀCH3), 1.49 (s, 3H, ꢀCꢀCH3), 2.43 (s, 3H, ArꢀCH3), 4.00ꢀ4.20 (m, 4H, Hꢀ4,
Hꢀ5, Hꢀ6), 4.28 (dd, 1H, 3J3ꢀ2 = 2.5 Hz, 3J3ꢀ4 = 2.5 Hz, Hꢀ3), 4.57 (dd, 1H, 3J2ꢀ1 = 2.5 Hz, Hꢀ2), 5.44 (d,
1H, 3J1ꢀ2 = 5 Hz, Hꢀ1), 7.32 (d, 2H, ArꢀH, 3JHꢀH = 10 Hz), 7.80 (d, 2H, 3JHꢀH = 7.5 Hz); 13CꢀNMR (62.9
MHz, CDCl3) δ (ppm): 144.7, 132.7, 129.7, 128.0, 109.5, 108.9, 96.0, 70.4, 70.3, 70.3, 68.1, 65.8, 25.9,
25.7, 24.9, 24.3, 21.6; ESIꢀMS (ion trap): m/z 415 [M+H]+.
(1,2:3,4ꢀdiꢀOꢀisopropylideneꢀ6ꢀdeoxyꢀazido)ꢀαꢀDꢀgalactopiranose (21): Sodium azide (5.84 g,
89.8 mmol, 5.0 eq.) was added to a solution of 20 (7.22 g, 17.4 mmol, 1.0 eq.) in DMSO (40 mL), and
the mixture was stirred at 160 °C for 1.5 hours under nitrogen. The resulting mixture was poured into
ice water (500 mL), and extracted with EtOAc (2 × 250 mL). The organic layer was dried over MgSO4
and filtered. The solvent was evaporated under reduced pressure and the residue was purified by flash
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chromatography (eluent: CH2Cl2/EtOAc, 97:3) to afford 4.60 g of 11 (93 % yield). HꢀNMR (250
MHz, CDCl3) δ (ppm): 1.33 (s, 6H, 2 × ꢀCꢀCH3), 1.44 (s, 3H, ꢀCꢀCH3), 1.53 (s, 3H, ꢀCꢀCH3), 3.34 (dd,
1H, 2J6*ꢀ6 = 12.5 Hz, 3J6*ꢀ5 = 5 Hz, Hꢀ6*), 3.50 (dd, 1H, 2J6ꢀ6* = 12.5 Hz, 3J6ꢀ5 = 7.5 Hz, Hꢀ6), 3.87ꢀ3.93
(m, 1H, Hꢀ5), 4.18 (dd, 1H, J4ꢀ3 = 2.5 Hz, 3J4ꢀ5 = 7.5 Hz, Hꢀ4), 4.31 (dd, 1H, J3ꢀ2 = 2.5 Hz, 3J3ꢀ4 = 2.5
3
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Hz, Hꢀ3), 4.61 (dd, 1H, J2ꢀ1 = 7.5 Hz, J2ꢀ3 = 2.5 Hz, Hꢀ2), 5.53 (d, 1H, J1ꢀ2 = 5 Hz, Hꢀ1); CꢀNMR
(62.9 MHz, CDCl3) δ (ppm): 109.5, 108.7, 96.3, 71.0, 70.7, 70.3, 66.9, 50.6, 25.9, 25.9, 24.8, 24.3;
ESIꢀMS (ion trap): m/z 286 [M+H]+.
(1,2:3,4ꢀdiꢀOꢀisopropylideneꢀ6ꢀdeoxyꢀamino)ꢀαꢀDꢀgalactopiranose
(22):
A
solution
of
triphenylphosphine (5.49 g, 20.9 mmol, 1.3 eq.) in THF (35 mL) was added dropwise to a solution of
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21 (4.60 g, 16.1 mmol, 1.0 eq.) in THF (60 mL), and the mixture was stirred at RT for 4 hours under
nitrogen. Distilled water (25 mL) was then added and the resulting mixture was heated to reflux and
vigorously stirred for 15 hours. Solvents were removed under vacuum and the residue was purified by
flash chromatography (eluent: CH2Cl2/MeOH, 95:5 + 1% of triethylamine) to afford 4.05 g of 22 (97 %
yield). 1HꢀNMR (250 MHz, CDCl3) δ (ppm): 1.32 (s, 6H, 2 × ꢀCꢀCH3), 1.34 (s, 2H, ꢀNH2), 1.43 (s, 3H,
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ꢀCꢀCH3), 1.51 (s, 3H, ꢀCꢀCH3), 2.82 (dd, 1H, J6*6 = 15 Hz, J6*ꢀ5 = 5 Hz, Hꢀ6*), 2.95 (dd, 1H, J6ꢀ6*
=
12.5 Hz, 3J6ꢀ5 = 7.5 Hz, Hꢀ6), 3.65ꢀ3.71 (m, 1H, Hꢀ5), 4.21 (dd, 1H, 3J4ꢀ3 = 7.5 Hz, 3J4ꢀ5 = 2.5 Hz, Hꢀ4),
4.30 (dd, 1H, 3J3ꢀ2 = 5 Hz 3J3ꢀ4 = 2.5Hz, Hꢀ3), 4.58 (dd, 1H, 3J2ꢀ1 = 10 Hz, 3J2ꢀ3 = 2.5 Hz, Hꢀ2), 5.53 (d,
3
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1H, J1ꢀ2 = 2.5Hz, Hꢀ1); CꢀNMR (62.9 MHz, CDCl3) δ (ppm): 109.1, 108.4, 96.3, 71.7, 70.7, 70.5,
69.4, 42.3, 26.0, 25.9, 24.9, 24.3; ESIꢀMS (ion trap): m/z 260 [M+H]+.
[Nꢀ(1,2:3,4ꢀdiꢀOꢀisopropylideneꢀ6ꢀdeoxyꢀαꢀDꢀgalactopyranosyl)ꢀcarbamoyl]ꢀ4ꢀnitrophenol (23):
A solution of 22 (1.0 g, 3.9 mmol, 1.0 eq.) in THF (15 mL) was added dropwise to a solution of bis (4ꢀ
nitrophenyl) carbonate (1.17 g, 3.9 mmol, 1.0 eq.) and DMAP (1.01 g, 7.8 mmol, 2.0 eq.) in THF (20
mL) and the mixture was stirred at RT for 5 hours. The resulting mixture was diluted in EtOAc (150
mL) and washed with 0.5 N HCl (3 × 100 mL). The organic layer was dried over MgSO4 and filtered.
The solvent was evaporated under reduced pressure and the residue was purified by flash
1
chromatography (eluent: CH2Cl2/Hexane, 75:25) to afford 1.27 g of 23 (78 % yield). HꢀNMR (250
MHz, CDCl3) δ (ppm): 1.34 (s, 3H, ꢀCꢀCH3), 1.35 (s, 3H, ꢀCꢀCH3), 1.46 (s, 3H, ꢀCꢀCH3), 1.51 (s, 3H, ꢀ
CꢀCH3), 3.34ꢀ3.44 (m, 1H, Hꢀ6*), 3.55ꢀ3.65 (m, 1H, Hꢀ6), 3.95ꢀ4.01 (m, 1H, Hꢀ5), 4.23 (dd, 1H, 3J4ꢀ3
=
10 Hz, 3J4ꢀ5 = 2.5 Hz, Hꢀ4), 4.34 (dd, 1H, 3J3ꢀ2 = 2.5 Hz, 3J3ꢀ4 = 5 Hz, Hꢀ3), 4.63 (dd, 1H, 3J2ꢀ1 = 7.5 Hz,
3J2ꢀ3 = 2.5 Hz, Hꢀ2), 5.55 (d, 1H, 3J1ꢀ2 = 5 Hz, Hꢀ1), 5.60 (dd, 1H, 3JNHꢀ6* = 7.5 Hz, JNHꢀ6 = 5 Hz, ꢀNH),
13
7.28ꢀ7.32 (m, 2H, Hꢀ2’, Hꢀ6’), 8.21ꢀ8ꢀ25 (m, 2H, Hꢀ3’, Hꢀ5’); CꢀNMR (250 MHz, CDCl3) δ (ppm):
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155.9, 153.3, 144.7, 125.0, 121.9, 109.5, 108.8, 96.3, 71.5, 70.7, 70.4, 66.0, 41.8, 26.0, 25.9, 24.9, 24.3;
ESIꢀMS (ion trap): m/z 425 [M+H]+.
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(4’ꢀmonoꢀ, 3ꢀmonoꢀ, 3,4’ꢀdiꢀ, 3,5ꢀdiꢀ, 3,4’,5ꢀtriꢀ)ꢀ[Nꢀ(1,2:3,4ꢀdiꢀOꢀisopropylideneꢀ6ꢀdeoxyꢀαꢀDꢀ
galactopyranosyl)ꢀcarbamoyl]ꢀresveratrol (24ꢀ28): A solution of resveratrol (0.15 g, 0.66 mmol, 1.0
eq.) and DMAP (0.32 g, 2.64 mmol, 4.0 eq.) in pyridine (10 mL) was added to a solution of 23 (1.13 g,
2.7 mmol, 4.0 eq.) in pyridine (10 mL) and the resulting mixture was allowed to react under vigorous
stirring at 70°C for 16 hours. The reaction mixture was then diluted in EtOAc (200 mL) and washed
with 0.5 N HCl (5 × 150 mL). The organic layer was dried over MgSO4 and filtered. The solvent was
evaporated under reduced pressure and the residue was purified by flash chromatography using
EtOAc:Hexane 5.5:4.5 as eluent to afford 0.39 g of a mixture of 24ꢀ28 used as such for the subsequent
synthetic step. ESIꢀMS (ion trap): 24, 25: m/z 514 [M+H]+; 26, 27: m/z 799 [M+H]+; 28: m/z 1084
[M+H]+.
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(4’ꢀmonoꢀ, 3ꢀmonoꢀ, 3,4’ꢀdiꢀ, 3,5ꢀdiꢀ, 3,4’,5ꢀtriꢀ)ꢀ[Nꢀ(6ꢀdeoxyꢀgalactosyl)ꢀcarbamoyl]ꢀresveratrol
(7ꢀ11): The mixture (24ꢀ28, 0.39 g) obtained in the previous step was added to a solution of TFA/water
(90:10, 3 mL) and vigorously stirred at RT. After 1.5 hours the resulting mixture was precipitated with
40 mL of diethyl ether, the material was centrifuged and the solvent was decanted. The white powder
obtained was then washed with diethyl ether three more times to eliminate traces of TFA. The residue
was then dried under vacuum, separated by preparative HPLC (from 0% to 38% ACN in 17 minutes)
and lyophilized to afford derivatives: 7 (0.040 g, cumulative yield of the final two steps: 14%), 8 (0.023
g, cumulative yield of the final two steps: 8%), 9 (0.093 g, cumulative yield of the final two steps:
22%), 10 (0.025 g, cumulative yield of the final two steps: 6%) and 11 (0.094 g, cumulative yield of the
final two steps: 17%).
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7: 1HꢀNMR (600 MHz, DMSOꢀd6) δ(ppm): 8.00 (t, 1H, JNHꢀ6*= JNHꢀ6 = 6 Hz, ꢀNHꢀ), 7.56 (d, 2H, J2’’ꢀ
3’’ = 12 Hz, Hꢀ2’’, 6’’), 7.07 (d, 2H, J3’’ꢀ2’’ = 6 Hz, Hꢀ3’’, 5’’ ), 7.10ꢀ7.31 (m, 2H, Hꢀ8, 9), 6.43 (s, 2H,
Hꢀ2’, 6’), 6.15 (s, 1H, Hꢀ4’), 4.89ꢀ5.00 (m, 1H, Hꢀ1), 4.24ꢀ3.17 (m, 12H, Hꢀ2, 3, 4, 5, 6, 6*, 6 OH);
13CꢀNMR (151 MHz, DMSOꢀd6) δ (ppm): 158.6, 154.5, 150.5, 138.8, 133.9, 128.7, 127.3, 127.1,
121.9, 104.7, 102.4, 97.6, 92.7, 73.3, 72.4, 71.9, 69.5, 69.2, 68.9, 68.6, 68.0, 41.8, 41.6; ESIꢀMS (ion
trap): m/z 434 [M+H]+.
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8: HꢀNMR (600 MHz, DMSOꢀd6) δ (ppm): 7.68 (t, 1H, JNHꢀ6*= JNHꢀ6 = 6 Hz, ꢀNHꢀ ), 7.41 (d, 2H,
J2’’ꢀ3’’= 6 Hz, Hꢀ2’’, 6’’), 6.16ꢀ7.08 (m, 7H, Hꢀ3’’,5’’, 2’, 4’, 6’, 8, 9), 3.17ꢀ4.95(m, 13H, Hꢀ1, 2, 3, 4, 5,
6, 6*,6 OH); 13CꢀNMR (151 MHz, DMSOꢀd6) δ (ppm): 158.5, 157.9, 154.9, 152.6, 139.8, 129.4, 128.4,
128.3, 125.0, 115.9, 115.5, 115.1, 114.8, 110.4, 110.2, 108.2, 97.9, 93.1, 73.7, 72.8, 72.3, 69.9, 69.6,
69.3, 69.0, 68.5, 42.1, 42.0; ESIꢀMS (ion trap): m/z 434 [M+H]+.
1
9: HꢀNMR (600 MHz, DMSOꢀd6) δ (ppm): 7.71 (m, 2H, ꢀNHꢀ), 7.58 (d, 2H, J2’’ꢀ3’’= 6 Hz, Hꢀ2’’,
6’’), 7.08ꢀ7.18 (m, 4H, Hꢀ3’’5’’, 8, 9), 6.81 (s, 1H, Hꢀ2’), 6.79 (s, 1H, Hꢀ4’), 6.41 (s, 1H, Hꢀ6’), 4.94ꢀ
13
5.01 (m, 2H, 2 × Hꢀ1), 3.18ꢀ4.26 (m, 22H, 2 × Hꢀ1, 2, 3, 4, 5, 6, 6*, 8 OH); CꢀNMR (151 MHz,
DMSOꢀd6) δ (ppm): 158.2, 154.5, 154.5, 152.3, 150.6, 138.9, 133.7, 128.2, 127.7, 127.4, 121.9, 110.4,
110.2, 108.4, 101.7, 97.5, 97.5, 92.7, 82.6, 82.2, 76.2, 73.3, 73.3, 72.4, 72.4, 72.0, 71.9, 69.5, 69.2,
68.9, 68.6, 68.1, 68.0, 41.7, 41.6; ESIꢀMS (ion trap): m/z 639 [M+H]+.
1
10: HꢀNMR (600 MHz, DMSOꢀd6) δ (ppm): 7.74 (t, 2H, JNHꢀ6*= JNHꢀ6 = 6 Hz, ꢀNHꢀ), 7.42 (d, 2H,
J2’’ꢀ3’’= 6 Hz, Hꢀ2’’, 6’’), 6.97ꢀ7.18 (m, 4H, Hꢀ3’’5’’, 8, 9), 6.79 (s, 1H, Hꢀ2’), 6.76(s, 1H, Hꢀ4’), 3.07ꢀ
4.24 (m, 23H, Hꢀ6’, 2 × Hꢀ1, 2, 3, 4, 5, 6, 6*, 8 OH); 13CꢀNMR (151 MHz, DMSOꢀd6) δ (ppm): 157.6,
154.3, 154.2, 151.7, 151.7, 130.1, 130.0, 128.2, 127.8, 115.6, 115.3, 97.5, 97.5, 92.7, 73.3, 73.3, 72.4,
71.9, 71.9, 69.4, 69.3, 68.9, 68.8, 68.6, 68.0, 41.8, 41.7, 41.6. ESIꢀMS (ion trap): m/z 639 [M+H]+.
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11: 1HꢀNMR (600 MHz, DMSOꢀd6) δ (ppm): 7.68ꢀ7.81 (m, 3H, ꢀNHꢀ), 7.60 (d, 2H, J2’’ꢀ3’’= 6 Hz, Hꢀ
2’’, 6’’), 7.11ꢀ7.33 (m, 6H, Hꢀ3’’5’’, 8, 9, 2’, 6’), 6.78 (s, 1H, Hꢀ4’), 2.98ꢀ4.95 (m, 33H, 3 × Hꢀ1, 2, 3,
4, 5, 6, 6*, 12 OH); 13CꢀNMR (151 MHz, DMSOꢀd6) δ (ppm): 154.4, 154.2, 151.7, 150.8, 138.9, 134.5,
133.5, 129.2, 127.5, 126.7, 122.0, 116.3, 101.7, 97.5, 92.7, 82.6, 82.1, 76.1, 73.3, 72.4, 71.9, 69.4, 69.2,
68.9, 68.6, 68.0, 68.0, 41.8, 41.7. ESIꢀMS (ion trap): m/z 845 [M+H]+.
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Log Pow, Solubility and TPSA. Unless otherwise indicated, the Log Pow and solubility values
reported are predictions obtained using the software ALOGPS 2.1 (available at Virtual Computational
Chemistry Laboratory, http://www.vcclab.org). “Topological” Polar Surface Area63 values were
predicted using the Molinspiration Property Calculator (www.molinspiration.com).
HPLCꢀUV Analysis. Samples (2 µl) were analyzed by HPLC/UV (1290 Infinity LC System, Agilent
Technologies) using a reverse phase column (Zorbax RRHD Eclipse Plus C18, 1.8 µm, 50 x 2.1 mm
i.d.; Agilent Technologies) and a UV diode array detector (190ꢀ500 nm). Solvents A and B were water
containing 0.1% trifluoroacetic acid (TFA) and acetonitrile, respectively. The gradient for B was as
follows: 2% (0.5 min), from 2% to 10% in 0.3 min, then from 10% to 18% in 1.7 min, then from 18%
to 28% in 0.5 min, 28% for 0.5 min then from 28% to100% in 1.8 min; the flow rate was 0.6 mL/min.
The eluate was preferentially monitored at 286, 300 and 320 nm (corresponding to absorbance maxima
of the internal standard, derivatives and resveratrol, respectively). The column compartment was
thermostated at 35°C.
HPLC/ESIꢀMS Analysis. HPLC/ESIꢀMS 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 ESI source operating in fullꢀscan positive or negative ion mode, with
nebulizer pressure 70 psi, dry gas flow 12 L/min, dry gas temperature 350°C. ESIꢀMS positive mass
spectra of reaction intermediates and final purified products were obtained by flow injection analysis of
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solutions in acetonitrile, eluting with a water:acetonitrile, 1:1 mixture containing 0.1% formic acid.
HPLC/ESIꢀMS was also performed on selected samples from hydrolysis studies and pharmacokinetics.
Samples (20 µl) were analyzed using a reversed phase column (SynergiꢀMAX, 4 µm, 150 x 4.6 mm
i.d.; Phenomenex, Castel Maggiore (BO), Italy). Solvents A and B were water containing 0.1% TFA or
5 mM NH4OAc and acetonitrile, respectively. The gradient for B was as follows: 10% for 2 min, from
10% to 35% in 20 min, from 35% to 100% in 20 min, then 100% for 2 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 fullꢀscan positive or negative ion mode.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hydrolysis Reactions. The chemical stability of all new compounds was tested in aqueous media
approximating gastric (0.1 N HCl, NormaFix) and intestinal (0.1 M PBS buffer, pH 6.8) pH values. A 5
µM solution of the compound was prepared from a 5 mM stock solution in DMSO, and incubated at
37°C for 24 hours; samples withdrawn at different times were analysed by HPLCꢀUV. Hydrolysis
products were identified by comparison of chromatographic retention time with authentic standards.
Nonꢀlinear curve fitting was performed using Origin 8.0 data analysis software; the hydrolysis reaction
rate constants (k) of the starting compounds were calculated through interpolation of data with the
equation for pseudoꢀfirst order reactions: [C] = [C]0·eꢀkt, where:
[C] : concentration of the compound
[C]0 : concentration of the compound at the initial time t0
t: time.
To evaluate its the longꢀterm stability under mildly acidic conditions, we stored compound 11
dissolved in citrate buffer (5 mM) / sucrose (5 mM) solution, pH 5.0, at 4°C or R.T., following the
disappearance of 11 by HPLC analysis of samples taken at intervals over a period of several months.
29
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