Multivalent agents containing 1-substituted 2,3,4-trihydroxyphenyl moieties as novel synthetic polyphenols directed against HIV-1

Article abstract of DOI:10.1039/c4ob00445k

The synthesis and the assessment of the anti-HIV activity of a set of molecules inspired by the multivalent structures of some naturally-occurring polyphenols (tannins) are reported. Different multibranched scaffolds have been derived from pentaerythritol as the central core which distribute spatially synthetic polyphenolic subunits based on 1-substituted 2,3,4-trihydroxyphenyl moieties. A tetrapodal compound (13b) bearing four N-(2,3,4-trihydroxyphenyl) amide groups, exhibits remarkable selective activity against HIV-1 with EC ₅₀ values in the micromolar scale, in the same range as those reported for the most representative anti-HIV tannins. Preliminary SAR studies emphasize the importance of the 1-substituted 2,3,4-trihydroxyphenyl moiety, the presence of an amide as the linker and the multivalent architecture of these molecules, since the anti-HIV activity increases with the number of polyphenolic moieties. The data support the interest in synthetic polyphenols and represent a promising starting point for further design and development of selective HIV-1 inhibitors.

Full text of DOI:10.1039/c4ob00445k

Organic &
Biomolecular Chemistry
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PAPER
Multivalent agents containing 1-substituted 2,3,4-
trihydroxyphenyl moieties as novel synthetic
polyphenols directed against HIV-1†‡
Cite this: Org. Biomol. Chem., 2014,
12, 5278
Aida Flores,a María José Camarasa,a María Jesús Pérez-Pérez,a Ana San-Félix,a
Jan Balzarinib and Ernesto Quesada*a
The synthesis and the assessment of the anti-HIV activity of a set of molecules inspired by the multivalent
structures of some naturally-occurring polyphenols (tannins) are reported. Different multibranched
scaffolds have been derived from pentaerythritol as the central core which distribute spatially synthetic
polyphenolic subunits based on 1-substituted 2,3,4-trihydroxyphenyl moieties. A tetrapodal compound
(13b) bearing four N-(2,3,4-trihydroxyphenyl)amide groups, exhibits remarkable selective activity against
HIV-1 with EC50 values in the micromolar scale, in the same range as those reported for the most rep-
resentative anti-HIV tannins. Preliminary SAR studies emphasize the importance of the 1-substituted
2,3,4-trihydroxyphenyl moiety, the presence of an amide as the linker and the multivalent architecture of
these molecules, since the anti-HIV activity increases with the number of polyphenolic moieties. The data
support the interest in synthetic polyphenols and represent a promising starting point for further design
and development of selective HIV-1 inhibitors.
Received 26th February 2014,
Accepted 29th April 2014
DOI: 10.1039/c4ob00445k
www.rsc.org/obc
against the virus.5,6 Additionally, HAART does not eliminate
the persistence of latent long-term reservoirs of HIV.7 As a
Introduction
Acquired Immune Deficiency Syndrome (AIDS) remains a result, novel strategies and alternative therapeutic agents
major global health concern due to its worldwide extension directed against HIV are still required.8
and the high mortality rate still associated with this disease.1
Polyphenols are a heterogeneous class of naturally-occurring
Current antiretroviral therapy against Human Immunodefi- compounds, usually secondary metabolites isolated from higher
ciency Virus (HIV), the etiological agent of AIDS, consists of 26 plants, which have been structurally characterised by the pres-
clinically approved drugs,2 which are administrated in combi- ence of polyhydroxy-aromatic moieties on their molecular frame-
nation in the so-called Highly Active Antiretroviral Therapy works.9 Many polyphenols have been proposed as anti-HIV
(HAART).3 This chemotherapeutic arsenal targets different therapeutics, exerting different modes of action, ranging from
critical stages of the infectious cycle of the virus and has con- virus entry, reverse transcription, integration and virus matur-
verted AIDS from a lethal disease into a chronic, controlled ing.10 Some of the most noticeable polyphenols described so
condition.4
far as agents against HIV include catechins,11 theaflavins12
In spite of the positive effects of the HAART multidrug (considered lately as inexpensive and safe microbicide
regime, the emergence of resistance to all drugs currently in candidates for the prevention of HIV sexual transmission13), or
clinical use as well as the appearance of side effects, particu- quercetin,14 most of them found in various gallate forms.
larly after long-term treatment, are major hurdles in the fight
Among them, the family of hydrolysable tannins has
attracted particularly strong interest due to its remarkable
activity against HIV. This heterogeneous group of polyphenols
usually comprises a common architecture based on a multi-
branched molecular core that spatially distributes several
galloyl or galloyl-derived subunits, attached to the central
scaffold through ester linkages.15 Among these natural pro-
ducts must be noted some structurally simplified polyphenols
such as 1,3,4,5-tetragalloylapiitol. This secondary plant meta-
bolite contains an acyclic polyhydroxylated hydrocarbon core
(D-apiitol) functionalised with four gallate moieties. This
aInstituto de Química Médica CSIC, Juan de la Cierva 3, 28006 Madrid, Spain.
E-mail: EQ1@iqm.csic.es; Fax: +34 91 564 48 53; Tel: +34 91 258 76 20
bRega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000
Leuven, Belgium
†Paper dedicated to Professor Richard J. K. Taylor on the occasion of his 65th
birthday.
‡Electronic supplementary information (ESI) available: Detailed experimental
procedures for the synthesis of described compounds, as well as characterization
data and copies of 1H and 13C NMR spectra of key intermediates and final com-
pounds, are provided. See DOI: 10.1039/c4ob00445k
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tannin exemplifies how a structurally simple polyol can act as analogues in which changes were focused on the nature of the
an efficient distributing scaffold for polyphenolic subunits scaffolds that distribute spatially the most ubiquitous polyphe-
resulting in a molecule endowed with anti-HIV activity.16
nolic moiety, the galloyl residue.28 Thus, the promising anti-
The biological activities associated with polyphenols have viral profile of natural polyphenols and the lack of previous
been mainly attributed to the presence of differently functiona- work on structurally-modified analogues of these compounds
lised polyhydroxy-aromatic moieties on their structures. In par- prompted us to initiate a program pursuing the discovery of
ticular, galloyl subunits are structural motifs widely found in multivalent compounds containing novel synthetic polypheno-
natural polyphenols. The presence of these groups constitutes lic subunits.
an essential requisite for any bioactivity in most polyphenolic
In this sense, we recently described a tripodal receptor con-
natural products.17 Very noticeably, flavonols lacking galloyl taining a triethylbenzene scaffold attached by amide linkers to
moieties do not provide effective protection against HIV-1 2,3,4-trihydroxybenzoyl groups (a galloyl isomer). It was stated
infection and the number and position of free phenol (–OH) that this moiety allowed the formation of an intramolecular
groups determine their biological properties.18
hydrogen bond between the bridged-amide function and the
Galloyl residues provide polyphenols with efficient donor ortho-phenolic substituent, thus rigidifying the structures com-
and acceptor-functionalities for hydrogen bonding, which give pared to galloyl-containing compounds (3,4,5-trihydroxyphenyl
these compounds the potential to bind to proteins and nucleic isomer). This pre-organisation provided this compound with
acids.19,20 Moreover, galloyl-containing polyphenols are selective recognition properties for mannose-based polysac-
notable anti-oxidants. A large body of evidence indicates that charides, which opened attractive prospects for its potential in
the oxidative environment facilitates viral replication21 and anti-infective strategies, including enveloped viruses such as
that HIV infection is enhanced by free-radical damage.22 In HIV.29
this sense, it has been suggested that gallate groups attenuate
Based on these precedents, we describe herein the synthesis
AIDS progression due to their anti-oxidant properties23 which and biological evaluation against HIV of a series of com-
weaken the effects of the infection and the ability of the virus pounds inspired by the architecture of some hydrolysable
to infect new cells.24
tannins (such as 1,2,4,5-tetragalloylapiitol) in which the natu-
As previously mentioned, a remarkable feature of the hydro- rally-occurring galloyl group (3,4,5-trihydroxybenzoyl moiety)
lysable tannins is their multi-branched structure. Thus, several has been replaced by synthetic 2,3,4-trihydroxyphenyl
a
subunits of the polyhydroxy-aromatic moieties (i.e. gallic acid) nucleus functionalised at position 1 with different linkers that
are distributed profusely at the periphery of the scaffolds. This allow their attachment to suitable scaffolds (Fig. 1).
architecture provides these compounds with a dense outer
As scaffolds, a series of simple mono-, bi-, tri- and tetra-
array of repeated polyphenolic motifs (multivalency).25 Actu- podal molecules were used to explore the role of the number of
ally, the multivalent concept of molecular design is currently polyphenolic subunits grafted onto a given backbone (multi-
starting to be successfully employed in anti-HIV drug dis- valency) in the biological activity. Thus, pentaerythritol was
covery.26,27 The combined above-mentioned factors (hydrogen proposed as a versatile molecule for construction of the four-
bonding ability, anti-oxidative effect and multivalent architec- branched core.30 The gradual loss of branches of the pentaery-
ture) allow the biological effect of polyphenols at different thritol core allowed access to tripodal and bipodal scaffolds,
levels.
while simple linear hydrocarbon chains of different lengths
Albeit their intimate mechanisms of action have not been were used for the monopodal molecules. In addition, alkyl
characterized in full detail, polyphenols have been proposed as chains (2 or 3 methylene groups) have been introduced as
useful candidates, either alone or in combination with conven- spacers to separate the polyphenolic subunits from the central
tional therapeutics, for the treatment and prevention of HIV-1 core in order to provide to the scaffolds both flexibility and
infection.10a However, naturally-occurring polyphenols are elongated branches, avoiding steric hindrance. Amide bonds
obtained as mixtures of compounds extracted from their are ubiquitous in nature and chemically stable,31 so they were
natural sources. This is a significant drawback because in the first choice for the linking functionalities, but esters, carba-
many cases antiviral activities are due to complex mixtures of mates, ureas or triazoles (all good hydrogen bond donor and
substances (plant extracts) whose composition can only be acceptor functionalities) were also used in order to evaluate
estimated while further isolation of the active components the role of the linker on the biological activity. Thus far, we
from the extracts can turn out to be an elusive and difficult describe the synthesis and anti-HIV activity of this new family
task. As a consequence, the development of chemical pro- of compounds designed as synthetic polyphenols.
cedures able to allow access to pure, controlled samples of
polyphenols is of great interest. Furthermore, naturally-occur-
ring polyphenols can motivate and inspire researchers to look
for novel synthetic compounds based on related architectures
and/or new structurally-modified polyphenolic moieties. Sur-
Results and discussion
Synthesis
prisingly, and despite this interest, polyphenols have been We prepared a first series of compounds in which the selected
only rarely explored as inspirational motifs of new anti-HIV 2,3,4-trihydroxyphenyl moiety was linked to a polyacid scaffold
agents. Moreover, synthetic polyphenols described to date are by amide linkages. With this purpose, 2,3,4-trihydroxy aniline (8)
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Fig. 1 Architecture of the proposed synthetic tannins.
Scheme 1 Synthesis of amine 2,3,4-trihydroxybenzene nucleus 7 via Curtius rearrangement.
was prepared from the commercially available 2,3,4-trihydroxy- K2CO3, heating),32 followed by saponification of the benzyl
benzoic acid (1) according to the pathway depicted in ester, gave free acid 333 in high yield. Conversion of the
Scheme 1. Perbenzylation of the starting material 1 (acetone, benzoic acid 3 into its aniline analogue 7 was performed
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Scheme 2 Synthesis of multipodal polyacid scaffolds 10–12.
following a three-step synthetic sequence. The first transform- that involves firstly the conjugate addition of suitable polyols
ation involved a Curtius reaction34 using diphenylphosphoryl- A, B and C to olefins with electron-withdrawing substitutents
azide (DPPA) as a mild azide-transfer reagent.35 In this reaction, followed by the conversion of the terminal functionalities
the corresponding acyl-azide derivative of 3 is formed and (CO2tBu or CN) into the free acids carried out as specifically
rearranged in situ to give the intermediate isocyanate 4, which required (Scheme 2). Thus, tetraacid 10 was synthesised from
was not isolated from the reaction medium. A subsequent one- the inexpensive, commercially available pentaerythritol (A),
pot reaction of 4 with alcohols (BnOH, MeOH) produced the which reacts readily with acrylonitrile in aqueous media gener-
corresponding benzyl (5) and methyl (6a) carbamates respect- ating tetranitrile intermediate 9 (72% yield), which was finally
ively in high yield.36 Removal of the Cbz (BnOCO) group in 5 hydrolysed providing the desired tetraacid 10 in 86% yield.38
was successfully achieved using an excess of tetrabutyl- Tripodal scaffold 11 was synthesised from the commercially
ammonium fluoride (TBAF) to afford the free aniline derivative available triol B and tert-butyl acrylate in 73% yield.39 tert-
7 (Scheme 1, method A).37 However, despite the satisfactory Butyl protecting groups were then straightforwardly removed
overall yield of the conversion, the large excess of TBAF that by treatment with TFA, affording triacid 11 in 72% yield.40
has to be used to drive the deprotection to completion and the Finally, bidentate scaffold 12 was synthesised through a
toxicity of this reagent made it difficult to scale up this trans- similar two-step sequence involving conjugate addition of
formation. Alternatively, the methyl carbamate 6a was efficien- commercially available diol C to acrylonitrile in 98% yield41
tly deprotected by alkaline hydrolysis using KOH as reagent and subsequent acid hydrolysis of nitrile groups to the corres-
giving 7 in 98% yield. This procedure (Scheme 1, method B) ponding free acids in 64% yield.
performed on 6a proved simpler from a practical point of view,
Multivalent benzyl-protected amides 13a–15a were prepared
higher yielding overall, easily scalable and inexpensive com- by coupling of 8 and polyacid scaffolds 10–12 mediated by
pared to the TBAF procedure (which is carried out specifically PyBOP/Et3N in dichloromethane as depicted in Scheme 3.
on the CBz-protected aniline 5).
Transformations proceeded at room temperature in 48 h, but
Unfortunately, free aniline 7 proved to be unstable and gentle heating at 60 °C led the reaction to completion in
decomposition was observed even when it was stored under shortened reaction times (overnight) and satisfactory yields.
controlled conditions (low temperature and inert atmosphere).
Hydrogenolysis of all the benzyl-protected compounds
To solve this limitation, 7 was converted immediately after iso- 13a–15a (H2, 3.1 bar, 25 °C, catalytic Pd/C) afforded the free-
lation into its HCl salt 8. Hence, hydrochloride 8 was stable phenols 13b–15b in good yields. The choice of benzyl as pro-
and could be stored at room temperature for months without tecting group for the phenol functionalities proved successful
apparent degradation.
as easy instrumental procedures and minimal work-up were
Next, scaffolds 10–12 with 4, 3 and 2 branched carboxylic required to achieve the fully deprotected compounds at high
acids, respectively, were synthesised following a two-step route purity (≥95% HPLC).
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Scheme 3 Synthesis of amide-linked tetra-, tri- and bipodal compounds 13–15.
In order to evaluate the influence on the activity of the posi- able tetraamine 22 (prepared as will be discussed later) with
tion of the phenol groups on the aromatic ring, the 3,4,5- the corresponding acid 3 afforded 23a, whose further deprotec-
isomer derivative of 13b, namely compound 17b, was obtained tion yielded 23b in 69% yield.
(Scheme 4). This compound was synthesized similarly to 13b
We were encouraged to extend the study to new molecules
in 47% overall yield after two steps. The first transformation with modified linkers between the scaffold and the 2,3,4-tri-
involves a PyBOP-mediated coupling of scaffold 10 and the hydroxyphenyl moieties. Thus, isocyanate 4 emerged as a key
corresponding 3,4,5-benzyloxy aniline 16 followed by hydro- intermediate that allowed its direct conversion into carbamate
genolysis of the benzyl groups in 17a to afford 17b. Additionally, (OCONH) and urea-linked (NHCONH) derivatives, obtained
with the aim of analysing the role of the free amide proton on straightforwardly by reaction of 4 with alcohols and amine-
the activity, the N-methyl derivative 18b was prepared in 41% containing cores, respectively (Schemes 9 and 10). For the syn-
overall yield by reaction of 13a with methyl iodide followed by thesis of these compounds, scaffolds 22 and 24–26 were pre-
removal of the benzyl groups in 18a by hydrogenation.
pared as shown in Schemes 7 and 8.
Next, to complete the amide series of compounds with
Treatment of tetraacid 10 with HCl gas in methanol42 fol-
monopodal structures and to properly evaluate the effect of lowed by reduction of the in situ-generated methyl ester inter-
branching on the scaffolds, compounds 19b–21b were pre- mediate with LiAlH4 afforded tetraol 24. This sequential
pared. Thus, 8 was straightforwardly functionalised with acetic method proved more reliable than the direct reduction of the
anhydride in pyridine to afford acetate 19a. Ethyl (20a; short- tetraacid 10 with borane–THF complex as described.43 Tri- and
chained derivative) and palmitoyl (21a; long-chained deriva- bipodal alcohols 2544 and 2645 were obtained similarly by
tive) monopodal amides were synthesized by reaction of 8 with reduction of their corresponding methyl ester intermediates D
the corresponding acyl chlorides in order to additionally evalu- and E (Scheme 7).
ate the role of the amphiphilic character of these molecules.
Conversion of the polyol 24 into the tetraamine 22
Finally, hydrogenolysis of the benzyl groups of 19a–21a (Scheme 8) was performed firstly via tetraazide intermediate
afforded the monopodal free-phenols 19b–21b in satisfactory 27, which was obtained by a tandem procedure involving a
yields (Scheme 5).
Mitsunobu activation of the primary alcohols of 24 (DIAD,
Preliminary biological evaluation of compounds 13b–21b PPh3) followed by an in situ reaction with diphenylphosphoryl-
indicated that the tetrapodal compound 13b showed promis- azide (DPPA), acting as an azide-transfer reagent. This trans-
ing anti-HIV-1 activity (see biological evaluation section). We formation allows a simple and straightforward synthesis of
then considered preparing the tetrapodal amide 23b in which tetraazide 27 alternatively to the described procedure,46 which
the linker is reversed (NHCO instead of CONH), compared to involves previous activation of the alcohols as mesylates, iso-
13b, in order to evaluate the influence of this structural para- lation and subsequent conversion into their azide derivatives
meter on the activity (Scheme 6). Thus, coupling of the suit- by reaction with sodium azide. Finally, 27 was reduced to the
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Scheme 4 Synthesis of amide-linked tetrapodal compounds 17–18.
Scheme 5 Synthesis of monopodal amide-linked compounds 19–21.
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Scheme 6 Synthesis of tetrapodal amide 23b.
Scheme 7 Synthesis of tetrapodal, tripodal and bipodal polyol scaffolds 24–26.
tetraamine 22 in 63% yield by catalytic hydrogenation under pounds were completed with the monodentate methyl deriva-
standard conditions. Alternatively, compound 22 could be tive 6b (the synthesis of this benzyl-protected derivative was
obtained directly from the tetranitrile 9 by hydrogenation at previously described) and the octyl derivative 31b in order to
high pressure (70 bar, 70 °C; Scheme 8). This reaction was per- evaluate the effect of the amphiphilic nature of the carbamate.
formed in
a
continuous-flow reactor using immobilized
Regarding urea-linked derivatives, tetradentate amine 22
Co-RANEY® as the catalyst, providing the tetraamine in a single (Scheme 10) reacted very poorly with isocyanate 4. Thus, only
step and quantitative yield. This was finally the method of by forcing reaction conditions under microwave irradiation
choice to reach the tetravalent amine-containing scaffold 22.
(100 °C for 90 min) was it possible to isolate tetrapodal urea
Reaction of polyols 24–26 with 4, generated in situ by the 32a, albeit with a poor 17% yield. Additionally, reaction of 4
treatment of 3 with DPPA as previously mentioned, afforded with propylamine afforded monodentate compound 33a in
carbamates 28a–30a in reasonable yields (Scheme 9). Debenzy- 52% yield. Monodentate urea 33b containing free phenol
lation by hydrogenation of 28a–30a proceeded satisfactorily groups was successfully isolated in 99% yield after hydrogen-
affording free polyphenols 28b–30b. Carbamate-linked com- ation. However, tetradentate urea 32b was not obtained pure
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Scheme 8 Synthesis of tetrapodal polyamine (22) and polyazide (27) scaffolds.
Scheme 9 Synthesis of carbamate-linked series from key isocyanate 4.
after removal of the benzyl protecting groups in 32a. In this biologically against HIV with a purity of 82% (as determined
case, the material isolated after hydrogenolysis contained by HPLC).
several minor by-products. Attempts to purify this crude
As will be seen in the biological section, the data obtained
product (reverse-phase chromatography, recrystallization, from the evaluation against HIV of the amide (13b–15b,
exchange resins, etc.) failed to increase purity of this tetraden- 17b–21b) and carbamate (6b, 28b–31b) series clearly indicated
tate urea. Thus, exceptionally, compound 32b was evaluated that the more 2,3,4-trihydroxyphenyl moieties on the periphery
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Scheme 10 Synthesis of tetrapodal and monopodal urea-linked compounds from isocyanate 4.
of the molecules, the better the anti-HIV activity. Thus, our acetic anhydride in pyridine. Finally, removal of the benzyl
next series of structural modifications were performed based ethers in 37a and 38a was carried out by hydrogenolysis using
on these findings and we decided to focus only on the tetra- the standard protocol. Deprotected compound 38b undergoes
podal compounds, while the monopodal derivatives were a phenomenon of intramolecular acyl migration between
obtained for comparative purposes.
vicinal phenols, producing spontaneously in the hydrogen-
Next, compounds with an ester as the linker were prepared ation medium a (2 : 1) mixture of 1-acetyl/2-acetyl isomers 38b/
1
(Scheme 11). In this case, derivative 36 was the key intermedi- 38c as determined by H-NMR. Tetraester 37b did not exhibit
ate. This compound was obtained in a three-step sequence by this spontaneous trend of transesterification between the
reduction of 2 with lithium aluminium hydride under stan- phenol groups, although all compounds 37b and 38b/38c
dard conditions (LiAlH4, THF) to afford benzyl alcohol 34 in proved labile in solution, probably due to partial hydrolysis of
80% yield.47 Subsequent conversion of this alcohol into the ester moieties.
its aldehyde derivative 35 was initially performed by using
Finally, in order to explore new structural modifications on
pyridinium chlorochromate (PCC) as the oxidizing agent the linker, we considered it of interest to replace the amide
(Scheme 11, method A) as described in the literature for the bridge of the most active tetrapodal compound 13b by a non-
isomeric 3,4,5-trimethoxy analogue of 34.48 However, this hydrolysable surrogate. The triazole group has been proposed
transformation afforded, after chromatography, only moderate as a non-classical bioisostere of the amide moiety retaining
yields (45%) of the desired aldehyde 35. Alternatively, a MnO2- the main parameters of the original group (electronic pro-
mediated oxidation (Scheme 11, method B) was assayed giving perties, lipophilicity, size and geometry) while introducing
aldehyde 35 in better yield (69%) and avoiding tedious chro- structural diversity. Additionally, triazole groups have the
matographic purifications. Finally,
(H2O2–HCO2H) converted the aromatic aldehyde 35 into its ration (click chemistry).50 Thus, two non-hydrolysable triazole-
phenol derivative 36 as described.49
linked tetrapodal agents 40b and 43b were synthesized as
a Dakin-type reaction advantages of a remarkable chemical stability and easy gene-
Coupling of compound 36 with the tetra-acid scaffold 10 shown in Scheme 12. Two different azide–alkyne pairs were
was achieved readily by Steglich reaction mediated by DCC used. For the synthesis of 40b, acetylene 39 was required. Com-
and a catalytic amount of DMAP to give tetradentate ester 37a pound 39 was obtained from the benzyl alcohol 34 through a
in 62% yield. A simple monodentate ester 38a was also sequential one-pot oxidation–propargylation protocol.51 Thus,
obtained for comparative purposes by treatment of 36 with 34 was oxidised by activated MnO2 (THF, reflux) with no
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Scheme 11 Synthesis of monopodal and tetrapodal esters 37b and 38b/38c.
Scheme 12 Synthesis of tetrapodal triazole-linked polyphenols.
isolation of intermediate aldehyde 35, which was transformed reagent) in the presence of K2CO3 at room temperature.52
in situ into the corresponding acetylene 39 by treatment with Subsequently, Huisgen reaction (Cu(I)-catalysed [1,3]-dipolar
dimethyl-(1-diazo-2-oxopropyl)phosphonate (Bestmann–Ohira cycloaddition)53 between 39 and the previously mentioned
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tetraazide 27 in the presence of CuSO4 and sodium ascorbate
afforded tetra-triazole 40a in good yield (81%).
Whereas some of the described compounds inhibited
HIV-1 replication in the lower micromolar concentration
On the other hand, tetrapodal derivative 43a was obtained range, none of these derivatives proved active against HIV-2 at
by coupling of aryl-azide 41 and its acetylenic pentaerythritol sub-toxic concentrations (Table 1). Therefore, the active com-
counterpart 42. Compound 41 was obtained from the hydro- pounds should be considered as specific inhibitors of HIV-1
chloride 8, which was converted into its diazo derivative (not replication in CD4+ T-lymphocyte cells.
isolated) by treatment with sodium nitrite and subsequently
With respect to the architecture of the tested compounds, it
substituted in situ by treatment with sodium azide to afford was shown that the number of polyhydroxyphenyl moieties is
41, although in low yield (23%; non-optimised procedure). important for the activity. Thus, monopodal (6b, 19b–21b,
Huisgen coupling of 41 with 42 (synthesized by O-propargyl- 31b, 33b and 38b,c) and bipodal (15b and 30b) compounds
ation of pentaerythritol54) provided 43a in satisfactory yield were all inactive against HIV replication at subtoxic concen-
(60%). Finally, removal of benzyl protecting groups in both 40a trations. In the monopodal series of amides (19b–21b) and car-
and 43a was carried out under standard conditions affording bamates (6b and 31b), the length of the hydrocarbon side
deprotected polyphenols 40b and 43b in 60% and 85% yield, chain (Me, Et, octyl and palmitoyl) linked to the phenolic
respectively.
moiety did not affect their antiviral behaviour, indicating that
the hydrophobic character of the molecule has no significant
effect on activity. It should be noted additionally that com-
pound 21b, bearing a long palmitoyl hydrocarbon chain, has a
Biological assays. Antiviral activity
The new synthetic polyphenols were evaluated for their inhibi-
tory activity against HIV-1(IIIB) and HIV-2 (ROD) in a cell- noticeably amphiphilic nature, which precluded its biological
+
testing at a higher concentration of 2 µM due to its limited
aqueous solubility.
Among the tripodal derivatives, only compound 14b exhibi-
ted a pronounced anti-HIV-1 activity (EC50 = 4.2 µM) although
its activity is 5-fold lower than its toxicity threshold (selectivity
based assay (CD4 T-lymphocyte cell culture) where virus
infected cells were incubated in the presence of the selected
compounds.
It must be noted firstly that none of the benzyl-protected
polyphenols exhibited activity against HIV at subtoxic concen-
trations (data not shown), which clearly indicates that free index ∼6).
Regarding the tetrapodal series, compounds 40b and 43b,
phenol functions are crucial for any antiviral activity in this
with non-hydrolysable triazole linkers, and 37b and 32b, con-
family of compounds. The results of the biological evaluation
of the deprotected compounds against HIV are summarized in nected to the scaffold with ester and urea linkers respectively,
were all inactive. In contrast, the tetrapodal amide-linked
Table 1.
+
Table 1 Anti HIV-1(IIIB) and anti-HIV-2(ROD) activity and cytostatic properties of the test compounds in human CD4 T-lymphocyte (CEM) cell
cultures
EC50 a (µM)
Linker
Multiplicity
Compound
HIV-1
HIV-2
CC50 b (µM)
Carbamate
Amide
Amide
Monopodal
Tetrapodal
Tripodal
6b
>10
>10
≥10
>10
>10
>10
>10
>50
>50
>2c
>10
>10
>10
>50
>10
>10
>50
>50
>50
>10
>10
171 85
25 11
24 8.5
60 54
21 5.7
63 37
112 0.0
108 2.1
>2c
21 2.8
20 4.2
24 4.9
86 17
124 11
22 0.0
>250
13b
14b
15b
17b
18b
19b
20b
21b
23b
28b
29b
30b
31b
32bd
33b
37b
38b/38c
40b
43b
1.2 0.6
4.2 0.85
>10
5.6 1.6
>10
Amide
Bipodal
Amide (3,4,5-isomer)
Amide (N-Me derivative)
Amide
Amide
Amide
Amide (NHCOAr)
Carbamate
Carbamate
Carbamate
Carbamate
Urea
Tetrapodal
Tetrapodal
Monopodal
Monopodal
Monopodal
Tetrapodal
Tetrapodal
Tripodal
>50
>50
>2c
10 0.0
8.4 2.3
>10
>50
>10
>10
>50
>50
>50
Bipodal
Monopodal
Tetrapodal
Monopodal
Tetrapodal
Monopodal
Tetrapodal
Tetrapodal
Urea
Ester
Ester
Triazole
102 21
>250
27 2.8
48 30
>10
>10
Triazole
a 50% effective concentration or compound concentration required to protect 50% of the cells against the cytopathic effect of the virus. b 50%
cytostatic concentration or compound concentration required to inhibit CEM cell proliferation by 50%. All data are mean values (standard
deviation for at least three independent experiments). c Compound precipitation was detected at higher concentrations. d Product evaluated with
82% purity.
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derivative 13b was endowed with the highest antiviral activity
The tetrapodal amide-linked compound 13b is the most
against HIV-1 (EC50 = 1.2 µM), a concentration markedly below active member of this series, exhibiting low micromolar inhibi-
the toxicity threshold, resulting in a selectivity index of ∼20. tory activity against HIV-1, in the same range or even improved
Very noticeably, reversion in the sequence of the amide linker compared to those values that have been reported for related
(NH–CO in 17b instead of CO–NH in 13b) caused a 4- to 5-fold naturally-occurring polyphenols.10a The 2,3,4-trihydroxyphenyl
loss of antiviral activity (17b, EC50 = 5.6 µM) keeping similar moiety present in these compounds proved superior to
levels of cytostatic activity (CC50: 21 µM). Also, the tetrapodal the naturally-occurring gallate group (3,4,5-trihydroxybenzoyl
derivative 28b attached to the scaffold through a carbamate moiety). It is noteworthy that any structural changes per-
group (NH–CO–O) was 7-fold less active than 13b (EC50
=
formed on the linker of the prototype amide-bridged com-
8.4 µM), linked by an amide (NHCO) moiety, but again, kept a pound 13b led to a decrease in antiviral activity. The specific
similar cytostatic potential as 13b and 17b. Overall, it can be molecular target of prototype compound 13b as well as its
concluded that the best architecture is the tetrapodal configur- exact mechanism of action are both still under investigation.
ation and that, despite a common tetrapodal scaffold, the
linker group clearly affects the activity.
Furthermore, it must be noted that naturally-occurring poly-
phenols are extracts from natural sources whose composition
The amide series (compounds 13b–21b) can help to esta- is variable, while the synthesis of the compounds described
blish interesting structure–activity relationships. On the one herein has been carried out through a set of simple and
hand, the distribution of the free phenolic OH groups around reliable transformations that allowed the isolation of pure,
the aromatic ring is important for the biological activity. Thus, controlled samples of the substances ready for biological
compound 17b, bearing polyphenolic subunits with a galloyl- evaluation.
type distribution (3,4,5-trihydroxyphenyl moieties), was 7-fold
For all the above reasons, this contribution can be con-
less active (EC50 = 5.6 µM) than compound 13b, substituted by sidered as an encouraging starting point for further develop-
the 2,3,4-trihydroxyphenyl isomer, showing that this moiety is ment of synthetic polyphenols as selective HIV-1 inhibitors.
superior for this series of compounds. Moreover, compound
18b, in which amide NH groups have been eliminated by the
introduction of methyl groups, was inactive compared to 13b.
This indicates the importance of the free NH groups probably
to keep the capacity to form H-bridges with the molecular
Experimental
Chemical synthesis
target. Finally, compared to its less branched tripodal (14b),
General methods. Reactions were carried out with magnetic
bipodal (15b) and monopodal (19b–21b) analogs, the antiviral stirring in round-bottomed flasks unless otherwise noted. Air
profile of 13b indicates that at least four 2,3,4-trihydroxyphenyl or moisture-sensitive reactions were conducted in oven-dried
moieties located at the periphery of the molecule are required glassware under a positive pressure of dry argon. Microwave-
for antiviral activity. These data support the importance of a mediated reactions were performed in a Biotage Initiator™ 2.0
multi-branched architecture in the design of these families of reactor. Debenzylation of benzyl-protected phenols was per-
compounds. Moreover, it is interesting to notice that the cyto- formed in a Parr-hydrogenator working at 3.1 bar and thermo-
static activity of the antivirally active test compounds were stated at 25 °C. High-pressure hydrogenation of tetranitrile 9
quite comparable, which opens promising future prospects of was performed on an H-Cube® Continuous-flow Hydrogen-
further improvement of the selectivity index by optimizing ation reactor using Co-RANEY® as catalyst immobilized in a
(increasing) the antiviral potency.
CatCart®-Catalyst Cartridge System (both from Thales Nano).
All solvents were pre-dried, freshly distilled prior to use as
specifically described.55 Analytical thin layer chromatography
(TLC) was performed on pre-coated aluminum silica gel plates
Conclusions
Herein we have described the synthesis and anti-HIV pro- 60 (F254, 0.25 mm). Products were visualized from the TLC
perties of a series of synthetic polyphenols containing a under UV lamps (254 or 260 nm) or by heating after treatment
central pentaerythritol-based core functionalised with 2,3,4-tri- with developer solution (cerium ammonium molybdate (CAM),
hydroxyphenyl subunits. Amide, carbamate, urea, ester and phosphomolybdic acid (PMA) or vanillin). Separations were
triazole groups have been used as linkers to attach the poly- performed on silica gel by preparative flash column chromato-
phenolic moieties to the scaffolds. The design of these com- graphy56 or by Centrifugal Circular Thin-Layer Chromato-
pounds has been inspired by the multivalent architecture of graphy (CCTLC). Large scale purifications were conducted on
some representative naturally-occurring tannins. The effect of an automated flash purification system apparatus (Biotage
the number of polyphenolic moieties has been studied by the Isolera One). Oils or waxy solids were converted into foams by
synthesis of a tetra-, tri- and bipodal series of compounds repeated freeze-drying (lyophilization) from acetonitrile or
while monopodal derivatives were obtained for comparative DMSO and traces of water for better handling of the products.
purposes. Multibranched architecture seems to play a crucial
Mass spectra (low and high-resolution) were registered
role in the design of these compounds, since the increase in with a quadrupole mass spectrometer Hewlett-Packard 1100
the number of peripheral polyphenolic subunits is associated equipped with an electrospray source (ES) in negative or posi-
with an increased activity.
tive mode of detection. NMR spectra were obtained on Varian
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INOVA-300, -400, UNITY-500, MERCURY-400 or Bruker from cold diethyl ether, dried and isolated as a yellowish solid,
AVANCE-300 NMR spectrometers using CDCl3 or methanol-d4 which was used without further purification (736 mg,
1
as solvents. H and 13C NMR spectra are reported in parts per 1.79 mmol, 98%). Isolated compound 7 by this method B is
million (ppm) referred to protonated residual peaks or specific identical to that obtained by the method A.
signals due to deuterated solvents as internal references
Preparation of aniline hydrochlorides 8 and 16. General
procedure
in CDCl3 or CD3OD. The following abbreviations are used to
describe peaks: s (singlet), d (doublet), t (triplet), qt (quartet),
qn (quintet), m (multiplet) and br (broad).
The free amine was re-dissolved in Et2O and a 1 N solution of
HPLC analyses were carried out using either a Waters Alli- HCl in Et2O was added dropwise until an abundant precipitate
ance HPLC/MS system (quoted as HPLC/MS) or on an Agilent was formed. The precipitate was filtered off, gently washed
1120 apparatus (quoted as HPLC) eluting with acetonitrile– with additional amounts of Et2O and then efficiently dried to
water. All retention times are quoted in minutes. Elemental give a solid that did not need further purification. Hydrochlo-
analyses were performed with a Carlo Erba CHN-1108 or a rides 8 and 16 can be stored without noticeable degradation
Heraeus CHN-O-RAPID microanalyzers. Melting points were for months if kept in a cool and dry place.
measured on a Mettler Toledo model MP70 melting point
apparatus and are uncorrected. Lyophilised compounds melt general procedure, aniline 7 was converted into its hydrochlo-
at a wide range of temperatures. ride 8, isolated as a pale brownish solid. Mp 174.9–176.2 °C;
2,3,4-Tris(benzyloxy)aniline hydrochloride (8). Following the
A representative set of compounds are described in this 1H NMR (400 MHz, CDCl3) δ: 10.0 (br s, 3 H), 7.5 (d, J = 4.2 Hz,
Experimental section. A detailed full description of all the 2 H), 7.4–7.2 (m, 11 H), 7.2 (br d, J = 9.4 Hz, 1 H), 7.1 (dd, J =
analytical methods, experimental procedures, spectroscopic 5.1, 1.6 Hz, 2 H), 6.6 (d, J = 9.0 Hz, 1 H), 5.3 (s, 2 H), 5.1 (s,
characterization and selected copies of 1H and 13C-NMR 2 H), 5.0 (s, 2 H); 13C NMR (100 MHz, CDCl3) δ: 152.8, 146.0,
spectra of intermediates and final compounds are provided 142.2, 136.8, 136.6, 136.3, 128.7, 128.6, 128.3, 128.2, 127.9,
separately in the ESI‡ available.
127.5, 118.9, 118.3, 109.3, 75.9, 75.6, 71.2; m/z (ESI+): 823 ([2M −
HCl + H]+, 30%), 413 ([M − HCl + 2H]+, 38%), 412 ([M − HCl +
H]+, 100%).
Preparation of 2,3,4-tris(benzyloxy)aniline (7) from carbamate
precursors
PyBOP-mediated synthesis of multivalent amides 13a–15a and
17a. General procedure
Method A. Reaction with TBAF. Specific method for benzyl-
carbamate deprotection. Cbz-protected aniline
5 (1.1 g,
2 mmol) was dissolved in dry, freshly distilled THF (5 mL) To a suspension of the suitable polyacid (1 mmol) in dry
under argon atmosphere at room temperature. A solution of CH2Cl2 (20 mL mmol−1) at 0 °C (ice-bath) was added sequen-
TBAF in THF (1 M solution, 30 mL, 15 equiv.) was added and tially PyBOP (1.2 equiv. per acid group) and Et3N (3.75 equiv.
the reaction mixture was heated at 60 °C overnight. TLC moni- per acid group) in a pressure tube. When the acid is solubil-
toring indicated complete deprotection of carbamate. Volatiles ized (approx. 5 min), aniline hydrochloride 8 (1.2 equiv. per
were removed under vacuum and the residue embedded in a acid group) was added and the pressure tube sealed. The reac-
pad of celite and directly purified by column chromatography tion mixture was warmed to 60 °C and stirred overnight until
on SiO2 (hexane–EtOAc (2 : 1) v/v as eluent). Free amine 7 was consumption of the starting material (TLC monitoring). Then,
isolated as a waxy solid, converted into a white foam after reaction was cooled to room temperature, washed with a 10%
repeated freeze-drying from CH3CN–H2O (627 mg, 1.5 mmol, citric acid aqueous solution and then with a 10% NaHCO3
1
76%). TLC (Rf = 0.32; hexane–EtOAc (3 : 2) v/v; CAM); H NMR aqueous solution, dried over anhydrous Na2SO4, filtered and
(400 MHz, CDCl3) δ: 7.45–7.3 (m, 15 H), 6.6 (d, J = 8.8 Hz, 1H), concentrated under vacuum. The crude product was purified
6.49 (d, J = 8.8 Hz, 1H), 5.1 (s, 2H), 5.07 (s, 2H), 5.04 (s, 2H); as determined.
13C NMR (100 MHz, CDCl3) δ: 145.5, 143.4, 140.8, 137.9, 137.8,
Tetrakis[5-oxo-5-[(2,3,4-tribenzyloxyphenyl)amino]-2-oxapentyl]-
137.7, 135.5, 128.6, 128.5, 128.4, 128.2, 128.0, 127.9, 127.8, methane (13a). A mixture of tetra-acid 1038 (21 mg,
111.7, 110.1, 75.7, 75.2, 72.5; m/z (ESI+) 434 ([M + Na]+, 26%), 0.050 mmol), hydrochloride 8 (108 mg, 0.240 mmol), PyBOP
412 ([M + H]+, 100%); HPLC/MS: tR = 2.55 min. polar gradient (126 mg, 0.240 mmol) and Et3N (104 µL, 0.725 mmol) in
CH3CN–H2O from (70 : 30) up to (100 : 0) in 5 min (98% CH2Cl2 (1 mL) was treated as described in the general pro-
purity).
Method B. Alkaline hydrolysis of carbamates. General pro- product was purified by CCTLC (EtOAc–hexane 10–80%) to
cedure. A 40% solution of KOH in methanol (5 mL mmol−1
afford 13a (58 mg, 0.029 mmol, 58%) as a yellowish waxy foam
cedure (reaction proceeded at room temperature). The crude
)
was added to the methyl carbamate 6a (1 g, 1.83 mmol). The after freeze-drying. TLC (Rf = 0.26 hexane–EtOAc (2 : 3) v/v;
1
slurry was stirred under reflux overnight (monitored by TLC). CAM); H NMR (500 MHz, CDCl3) δ: 7.80 (br s, 4 H), 7.75 (d,
Then, it was cooled to room temperature and the solvent was J = 9.3 Hz, 4 H), 7.41–7.27 (m, 60 H), 6.57 (d, J = 9.4 Hz, 4 H),
halved, diluted with H2O (10 mL mmol−1) and extracted with 5.04 (s, 16 H), 5.01 (s, 8 H), 3.44 (t, J = 5.7 Hz, 8 H), 3.18 (s,
EtOAc (3 × 10 mL mmol−1). Combined organic layers were 8 H), 2.26 (t, J = 5.7 Hz, 8 H); 13C NMR (125 MHz, CDCl3)
dried over anhydrous Na2SO4, filtered and concentrated under δ: 169.2, 149.2, 142.6, 141.5, 137.4, 137.0, 136.8, 128.7, 128.6,
vacuum to yield the desired free amine 7, which was triturated 128.5, 128.3, 128.0, 127.9, 127.5, 126.1, 115.7, 109.4, 76.1, 75.5,
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71.2, 69.2, 67.0, 45.0, 37.7; m/z (ESI+) 1000 ([1/2M + 2H]+, δ: 7.66 (br s, 4 H), 7.42–7.29 (m, 60 H), 6.91 (br s, 4 H), 6.71
100%); HRMS (ESI+) [M H]+ C125H121N4O20 requires (d, J = 9.3 Hz, 4 H), 5.06 (s, 8 H), 5.05 (s, 8 H), 5.04 (s, 8 H),
+
1997.8574, found: 1997.8568; Anal. calcd for C125H120N4O20 4.16 (t, J = 6.4 Hz, 8 H), 3.47 (t, J = 6.3 Hz, 8 H), 3.42 (s, 8 H),
(1996.3): C, 75.13; H, 6.05; N, 2.80; found: C, 74.93; H, 6.13; 1.87 (qn, J = 6.3 Hz, 8 H); 13C NMR (100 MHz, CDCl3) δ: 153.5,
N, 2.95%.
The synthesis of compounds 14a–15a and 17a was per- 128.3, 128.0, 127.9, 127.5, 126.3, 113.6, 109.8, 75.9, 75.5, 71.4,
formed similarly following the general procedure. For full 69.7, 67.6, 62.4, 45.3, 29.2; HRMS (ESI+) [M Na]+
148.3, 141.8, 141.7, 137.4, 137.0, 136.9, 128.6, 128.5, 128.4,
+
details, see ESI‡ available.
C129H128N4O24Na requires 2139.8816, found: 2139.8811.
The synthesis of carbamates 29a–31a and ureas 32a–33a
was performed similarly following the general procedure. For
full experimental details, see ESI‡ available.
Preparation of carbamates 28a–31a and ureas 32a–33a by
Curtius rearrangement. General procedure
To a solution of the appropriate benzoic acid (1 equiv.) in
Preparation of 1,2,3-triazole-bridged compounds 40a and 43a
by copper-catalysed 1,3-dipolar azide–alkyne cycloaddition
(Huisgen reaction). General procedure
toluene (5 mL mmol−1
) under argon was added DPPA
(1.1 equiv.) and Et3N (1.1 equiv.) at room temperature. The
mixture was heated at 75 °C until complete disappearance of
the starting material (as monitored by TLC), yielding the iso- To a suspension of the corresponding azide (1 equiv. per
cyanate intermediate 4 (not isolated). Then, the corresponding alkyne) and alkyne (1 equiv. per azide) in tBuOH–H2O (2 : 1)
alcohols or amines were added (1.2 equiv. per isocyanate v/v (64 mL mmol−1
) at room temperature was added
group to be functionalised). The reaction was stirred addition- (+)-sodium L-ascorbate (0.4 equiv.) and CuSO4 (0.04 equiv.).
ally at 75 °C overnight (monitored by TLC), cooled to room The heterogeneous mixture was stirred at room temperature
temperature and subsequently washed with a 1 N aqueous overnight and then treated with H2O. The resulting solid was
solution of HCl and then with a saturated aqueous solution of filtered, washed with H2O and purified as determined.
NaHCO3, dried over Na2SO4, filtered and concentrated
under reduced pressure. The crude product was purified as 2-oxapentyl]methane (40a). A suspension of tetra-azide 27
determined. (20 mg, 0.04 mmol), alkyne 39 (72 mg, 0.17 mmol), (+)-sodium
Tetrakis[5-(4-(2,3,4-tribenzyloxyphenyl)-1H-1,2,3-triazol-1-yl)-
Methyl (2,3,4-tris(benzyloxy)phenyl)carbamate (6a). Accord- L-ascorbate (3 mg, 0.017 mmol) and CuSO4 (1 mg,
ing to the general procedure, acid 333 (1.55 g, 3.5 mmol), DPPA 0.002 mmol) in tBuOH–H2O (2.8 mL) was treated as described
(830 µL, 4 mmol) and Et3N (540 µL, 4 mmol) in toluene in the general procedure. The crude solid was purified by
(18 mL) were reacted to form isocyanate intermediate 4, which CCTLC on silica gel (polar gradient on elution, EtOAc–hexane
was treated with methanol (170 μL, 0.4 mmol). After work-up, from (40 : 60) up to (100 : 0) v/v) affording 40a (75 mg,
the crude mixture was purified by CCTLC (polar gradient on 0.04 mmol, 81%) as a yellowish oil. TLC (Rf = 0.32 hexane–
1
elution using EtOAc–hexane from (5 : 95) up to (20 : 80)) and EtOAc (3 : 7) v/v; PMA); H NMR (500 MHz, CDCl3) δ: 7.89 (d,
further recrystallized from MeOH to yield 6a (1.22 g, 2.6 mmol, J = 8.8 Hz, 4 H), 7.74 (s, 4 H), 7.44–7.28 (m, 60 H), 6.84 (d, J =
74%) as a white solid. Mp 89.1–90.7; TLC (Rf = 0.34 hexane– 8.9 Hz, 4 H), 5.10 (s, 8 H), 5.08 (s, 8 H), 5.03 (s, 8 H), 4.31 (t,
1
EtOAc (4 : 1) v/v; CAM); H NMR (400 MHz, CDCl3) δ: 7.7 (br d, J = 7.0 Hz, 8 H), 3.35 (t, J = 5.7 Hz, 8 H), 3.33 (s, 8 H), 1.97 (qn,
1 H), 7.4–7.3 (m, 15 H), 6.89 (br s, 1 H), 6.76 (d, J = 9.2 Hz, J = 6.4 Hz, 8 H); 13C NMR (125 MHz, CDCl3) δ: 152.8, 149.9,
1 H), 5.09 (s, 4 H), 5.08 (s, 2 H), 3.71 (s, 3 H); 13C NMR 143.1, 141.8, 137.5, 137.4, 136.7, 128.6, 128.5, 128.3, 128.1,
(100 MHz, CDCl3) δ: 154.0, 148.4, 141.7, 137.3, 137.0, 128.0, 127.5, 122.5, 122.0, 118.4, 109.9, 75.6, 75.5, 70.9, 69.5,
136.8, 128.6, 128.5, 128.4, 128.3, 128.1, 127.9, 127.5, 67.6, 47.2, 45.4, 30.3; HRMS (ESI+) [M + H]+ C133H129N12O16
126.2, 113.4, 109.8, 75.8, 75.6, 71.4, 52.2; m/z (ESI+) 492 MH requires 2149.9650, found: 21.9644.
([M + Na]+, 59%), 470 ([M + H]+, 100%); Anal. calcd for
C29H27NO5 (469.5): C, 74.18; H, 5.80; N, 2.98; found: C, 74.27; 2-oxapropyl]methane (43a). A suspension of tetra-alkyne 4254
H, 6.02; N, 3.20%. (14 mg, 0.048 mmol), azide 41 (83 mg, 0.190 mmol),
Tetrakis[3-(1-(2,3,4-tribenzyloxyphenyl)-1H-1,2,3-triazol-4-yl)-
Tetrakis[5-((2,3,4-tribenzyloxyphenyl)carbamoyl)oxy-2-oxapentyl]- (+)-sodium L-ascorbate (4 mg, 0.019 mmol) and CuSO4 (1 mg,
methane (28a). Following the general procedure, a solution of 0.002 mmol) in tBuOH–H2O (3.0 mL) was treated as described
acid 333 (478 mg, 1.1 mmol), DPPA (0.26 mL, 1.2 mmol) and in the general procedure. Additional (+)-sodium L-ascorbate
Et3N (0.17 mL, 1.2 mmol) in toluene (5.4 mL) was reacted to (0.4 equiv.) and CuSO4 (0.04 equiv.) were added to ensure full
form the isocyanate intermediate 4, which was treated with conversion of the reaction. The crude solid was purified by
tetraol 24 (100 mg, 0.27 mmol) (stirred overnight at 75 °C after CCTLC (EtOAc–hexane 20–100%) to afford 43a (57 mg,
addition of 24). Additional acid 3 (239 mg, 0.54 mmol), DPPA 0.028 mmol, 60%) as a yellowish solid. Mp 50.2–51.1 °C; TLC
1
(0.13 mL, 0.6 mmol) and Et3N (83 mL, 0.6 mmol) were needed (Rf = 0.30 hexane–EtOAc (3 : 7) v/v; PMA); H NMR (400 MHz,
for the reaction to proceed to completion. After the usual CDCl3) δ: 7.78 (d, J = 2.7 Hz, 4 H), 7.43–7.30 (m, 32 H,)
work-up, the crude material was purified by CCTLC (polar gra- 7.30–7.25 (m, 10 H), 7.21 (dd, J = 9.0, 4.6 Hz, 4 H), 7.18–7.14
dient EtOAc–hexane from (10 : 90) up to (50 : 50)) to afford 28a (m, 10 H), 7.05–6.98 (m, 8 H), 6.76 (d, J = 9.1 Hz, 4 H), 5.08 (s,
(100 mg, 0.05 mmol, 21%) as a yellowish oil. TLC (Rf
=
8 H), 5.06 (s, 8 H), 4.87 (s, 8 H), 4.54 (s, 8 H), 3.52 (s, 8 H);
1
0.4 hexane–EtOAc (2 : 3) v/v; CAM); H NMR (400 MHz, CDCl3) 13C NMR (125 MHz, CDCl3) δ: 153.8, 146.1, 145.0, 142.7, 137.2,
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136.4, 136.3, 128.8, 128.7, 128.5, 128.4, 128.3, 127.7, 125.6,
124.8, 120.5, 109.4, 76.4, 75.9, 71.3, 69.5, 65.2, 45.7; HRMS
Acknowledgements
(ESI+) [M + H]+ C125H113N12O16 requires 2037.8398, found: This work has been supported by the Spanish MICINN/
2037.8392; Anal. calcd for C125H112N12O16 (2038.30): C, 73.66; MINECO (projects SAF2009-13914-C02-01 and SAF2012-39760-
H, 5.54; N, 8.25; found: C, 73.58; H, 5.51; N, 8.19%.
C02-01), Comunidad de Madrid (BIPEDD-2 project CM-S2010/
BMD-2457) and the KU Leuven (GOA no. 10/014; EF 05/15;
PF 10/018). Dr A. Flores acknowledges financial support from a
contract JAE-Doc (Programa Junta para la Ampliación de Estu-
dios) co-funded by CSIC and UE (FSE, Fondo Social Europeo).
Sonsoles Velázquez is acknowledged for critical reading and
valuable comments on this article and Leen Ingels for dedi-
Preparation of free polyphenols 6b, 13b–15b, 17b–21b, 23b,
28b–33b, 37–38b/38c, 40b and 43b. Deprotection of benzylated
phenols by catalytic hydrogenation. General procedure
A solution of the corresponding benzyl-protected precursors cated technical assistance with the antiviral assays.
(1 mmol) in a binary mixture of solvents THF–methanol (1 : 1)
v/v (100 mL) containing catalytic amounts of Pd (on charcoal;
10 wt%) was hydrogenated overnight in a Parr Hydrogenator at
3.1 bar in a thermostated vessel at 25 °C. The Pd catalyst was
References
removed by filtration through a Whatman® filter paper 42 and
the solvent was removed under reduced pressure to give the
corresponding deprotected free-phenolic derivatives as single
products which were treated as specified. All debenzylated
compounds should be kept at −20 °C and protected from
direct exposure to light.
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Tetrakis[5-oxo-5-((2,3,4-trihydroxyphenyl)amino)-2-oxapentyl]-
methane (13b). A solution of 13a (50 mg, 0.025 mmol) in
THF–MeOH (2.5 mL) was treated as described in the general
procedure to afford 13b (24 mg, 0.026 mmol, quantitative
yield) as a yellowish foam after freeze-drying without further
1
purification. H NMR (400 MHz, CD3OD) δ: 6.70 (d, J = 8.6 Hz,
4 H), 6.32 (d, J = 9.0 Hz, 4 H), 3.59 (t, J = 5.7 Hz, 8 H), 3.42 (s,
8 H), 2.50 (t, J = 5.7 Hz, 8 H); 13C NMR (100 MHz, CD3OD)
δ: 173.7, 145.2, 140.0, 136.0, 120.4, 114.6, 108.1, 71.0, 68.7,
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Na]+ C41H48N4NaO20 requires
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(916.8): C, 53.71; H, 5.28; N, 6.11; found: C, 53.42; H, 5.58;
N, 6.31%; HPLC: tR = 2.86 min. polar gradient CH3CN–H2O
from (10 : 90) up to (100 : 0) in 5 min (96% purity).
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