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Identification of Novel Matrix Metalloproteinase Inhibitors by Screening of Phenol Fragments Library.

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Arch. Pharm. Chem. Life Sci. 2011, 344, 557–563
557
Full Paper
Identification of Novel Matrix Metalloproteinase Inhibitors by
Screening of Phenol Fragments Library
Maria Teresa Rubino, Dariana Maggi, Antonio Laghezza, Fulvio Loiodice, and Paolo Tortorella
Dipartimento Farmaco-Chimico, Università degli Studi ‘‘Aldo Moro’’, Bari, Italy
In the last 20 years, a great variety of synthetic, low molecular weight MMP inhibitors (MMPIs) have
been synthesized and tested, although none has reached clinical utility. Exploration of novel ZBGs and
development of non-hydroxamate MMPI has become a focus in current research. It’s well-known that
polyphenols can produce beneficial effects on human health by their antioxidant properties as well as
they have the ability to block gelatinase activity. In this work we tested a series of selected phenols
as MMP inhibitors. The most interesting hit (B6) shows sub-micromolar activity against MMP-2
(IC50 0.59 0.05 mM, LE ¼ 1.07) and a fairly good selectivity spectrum.
Keywords: Inhibitors / Matrix metalloproteinases / Natural products screening / Phenols
Received: November 15, 2010; Revised: March 8, 2011; Accepted: March 9, 2011
DOI 10.1002/ardp.201000350
Introduction
Matrix metalloproteinases (MMPs) are a family of structurally
correlated zinc dependent endopeptidases involved in the
proteolysis of extracellular matrix proteins. Their proteolytic
role is crucial for the normal functions of the organisms
while their deregulated activity causes pathogenic conditions ranging from cardiovascular disease to cancer. In
general, an MMP inhibitor (MMPI) consists of a ‘‘zinc-binding
group’’ (ZBG) and a ‘‘backbone’’. The backbone is a classic
drug-like structure that interacts with the protein through
non-covalent interactions such as hydrogen bonding, hydrophobic packing, and electrostatic interactions. ZBG binds the
catalytic zinc (II) blocking substrate access to the active site
and rendering the metal incapable of peptide hydrolysis. In
addition, the ZBG acts as an anchor to lock the MMP inhibitors in the active site and direct its backbone into the target
substrate-binding pockets [1]. Classical ZBGs include carboxylates, hydroxamates, thiols, phosphonates or phosphinates.
The hydroxamic acid group is by far the most commonly used
ZBG in inhibitor design and has generally been found to be
the most effective [2]. The failure of hydroxamic acid-based
MMPIs in vivo, stemming from poor pharmacokinetics (low
oral bioavailability and short half-life), ability to bind other
Correspondence: Paolo Tortorella, Dipartimento Farmaco Chimico,
Università degli Studi ‘‘Aldo Moro’’, via Orabona 4, 70126 Bari, Italy.
E-mail: ptortorella@farmchim.uniba.it
Fax: þ39 080 5442231
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
metal ions, and lack of specificity due to very strong binding
to the catalytic zinc ion, have led to intensive efforts for
developing new MMPIs. As a consequence, the design of
new, even if weaker, ZBGs and their incorporation into a
non peptidic or peptidomimetic scaffold that contains
groups interacting specifically with the S1’ pocket, the region
with the lowest structural homology in the active site of
MMPs, could lead to inhibitors with better selectivity, bioavailability and pharmacokinetics of possible clinical success.
Recently, Cohen and co-workers identified new bidentate
ZBGs more potent than hydroxamic acid [3–5], some of which
have been developed as potent inhibitors of MMPs [5–7].
Considering that fragment-based drug design (FBDD) has
been successful in identifying clinical candidates with desirable chemical properties (such as low molecular weight) for
a wide range of targets, including metalloproteins [8, 9],
we employed this simple and effective approach to identify
new chemical hits for this specific role. Following the principles and advice suggested to realize fragment libraries [9],
we chose a group of phenols as a chemical starting point for a
preliminary screening.
Polyphenols are widespread in nature; the two most
important groups of dietary phenols are phenolic acids
and flavonoids, this latter being the largest and most studied
group [10]. Fruit and beverages such as tea and red wine
constitute the main sources of polyphenols. Some polyphenols such as quercetin are found in all plant products
(fruit, vegetables, cereals, leguminous plants, fruit juices,
tea, wine, infusions, etc.), whereas others are specific to
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M. T. Rubino et al.
particular foods (flavanones in citrus fruit, isoflavones in
soya, phloridzin in apples). In most cases, foods contain
complex mixtures of polyphenols, which are often poorly
characterized [11]. Phenols are also widely used as antioxidant food additives, dietary supplements or as drugs.
Epidemiological studies have suggested an association
between the consumption of food or beverages containing
polyphenols and the prevention of some human diseases,
such as chronic obstructive pulmonary heart diseases,
chronic inflammation, as well as reduction of risk of many
type of cancer [12]. The antioxidant and free radical scavenging properties of plant polyphenols linked with their
ability to coordinate metal ions and to interact with different
enzymatic systems are the main causes for their beneficial
effects on human health [13].
Natural polyphenols have been evaluated for their ability
to block gelatinase activities [10, 13, 14], furthermore phenol
and some of its derivatives bind to carbonic anhydrase in a
very interesting manner [15]. Therefore, as part of our continuing efforts to explore new potential ZBGs [16–22], with
both improved affinity and selectivity for the Zn2þ active ion,
we evaluated the inhibition activity of a series of phenol
Arch. Pharm. Chem. Life Sci. 2011, 344, 557–563
fragments against MMP-2, -8, -9, and -14 (Fig. 1) with the
aim to discover a zinc binding motif that could be a potentially useful starting point in the design of new metalloprotease inhibitors.
Results and discussion
The phenol fragment library (PFL, Fig. 1) consists of 40 compounds with a molecular weight in the range 94–240 subdivided into four sets (A-D, 10 compounds for each set). Sets A
and B consist of simple para, metha, and ortho-substituted
phenols and some compounds in which the hydroxy group
is methylated, acetylated or substituted with an amino group
to explore whether the phenolic moiety is important for the
binding and if the pKa of the OH group is a crucial parameter.
Set C comprises tri- and tetra-substituted polyphenols, in
most case characterized by substitutions in positions 1, 2,
and 3 of the phenyl ring. Some of them were expressly
prepared (C4, C6-C8, and C10). Set D is constituted by 1,4benzoquinone (D10) and chemically various phenols that
were selected from commercially available compounds,
or expressly prepared (D8 and D9). Anthraflavic acid and
Figure 1. Components (40 fragments) of phenol fragment library (PFL).
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2011, 344, 557–563
Phenol Matrix Metalloproteinase Inhibitors
lawsone (D1 and D2) are more complex natural phenols; the
other phenols, instead, present structural similarities to P1’
substituents of known MMPIs.
Chemistry
Most of these phenol fragments were obtained from commercial supplier (Sigma Aldrich). C4 and C10 were prepared
according to literature procedure [23]. C6, C7, and D9 were
synthesized by dealkylation of the appropriate dimethoxy
derivatives with BBr3. D8 was prepared by Suzuki–Miyaura
cross-coupling of 1-bromo-2-methoxybenzene with the commercially available 4-methoxybenzeneboronic acid. C8 was
obtained by following the Curtius–Yamada rearrangement
on 2,3-bis(benzyloxy)benzoic acid with diphenylphosphoryl
azide and basic hydrolysis of the intermediate ethyl carbamate (Scheme 1). Final deprotection of the catecholic groups
led to the desired aniline (C8).
Biological activities
The screening of the whole fragments library was carried out
against the complete set of MMPs at a preliminary concentration of 100 mM in order to identify active fragments useful
as hits (Fig. 2).
Set A: The simple phenol A1 turned out to be a very weak
inhibitor of gelatinases, MMP-8, and MMP-14. The more acidic
4-bromo, 4-nitro, and 4-fluoro phenols (A3, A5, and A10,
respectively) resulted slightly more active compared to the
unsubstituted A1, but presented the same inhibitory activity
of the electrodonor 4-methoxy substituted A2. This could
represent an indirect proof that the protonation state of
the phenol moiety is not such a relevant parameter for the
binding of the inhibitor within the enzyme cavity. MMPs
inhibition was insensitive to the introduction of a carboxylic
group (A4) in the para position of A1, while the introduction
of an amino or a hydroxy group (A6 and A8, respectively) in
the same position led to an impressive increase of activity.
The acetylation of the p-amino group, as in paracetamol (A7),
or the substitution of one of the hydroxy groups of hydroquinone with a thiol moiety (A9), led to very weak inhibitors
OH
OBn
OH
although thiols are fairly good ZBGs. It is thus clear that quite
minor structural changes in the molecule of the phenol
result into drastic changes in its MMP inhibition properties.
Set B: The metha substitution of A1 seems to have a negative
influence on the activity; resorcinol (B1) was much less active
than the para derivative A8 and 3-hydroxybenzoic acid (B2)
was a very weak inhibitor. On the contrary, the ortho substitution had different effects; in fact, pyrocatechol (B6) was the
most potent inhibitor of this series of compounds, whereas
aminothiophenol and aminophenol (B3 and B4) showed a 10fold decrease of the activity against MMP-2 and next to no
activity against the other MMPs. A definitive fall of activity
occurred in the case of the ortho diamino, dimethyl pyrocatechol, and acetylsalicylic acid derivatives (B8, B9, and B10,
respectively). Salicylic acid B5 resulted completely inactive
and a very weak inhibitor was B7 for the MMP-9 at 100 mM.
Data of the two series A and B (Fig. 2) showed very interesting inhibitors. In particular, three simple compounds (A6,
A8, and B6) presented a very effective inhibitory activity
against MMP-2 and -14.
Set C: Polysubstitution seems to have a positive influence
on the inhibitory activity of phenol, especially if the substituents are in positions 1, 2, and 3 of the phenyl ring and
even more if the three substituents are three hydroxyl groups
as for compounds C1 (pyrogallol) and C9 (gallic acid). The
introduction of an amino moiety on the aromatic ring of the
catechol brought to a very interesting percent inhibition
values against all the MMPs tested (C6 and C8), whereas
the benzylamino derivates C7 showed an intermediate
degree of inhibition for MMP-2, -8, and -14. The introduction
of metha-hydroxy groups (C2 and C3), the complete methylation of the hydroxy groups of gallic acid and its methyl ester
(C4 and C10) and the substitution of a hydroxy group of C1
with a carboxylic one (C5) resulted in a drastic decrease of
activity on the entire set of MMPs.
Set D: Most phenols constituting this group present a
phenyl ring, with or without a hydroxy substituent, either
directly linked in para position (D6, D7, and D9) or connected
through a short linker (D3-5). This structural enlargement led
OBn
OBn
OH
OBn
a, b, c
d, e
OH
OH
f
OH
NH2
O
559
O
NH2
C8
Scheme 1. Synthesis of C8. Reagents and conditions: (a) MeOH, H2SO4, reflux; (b) BnBr, K2CO3, DMF, 858C; (c) 8 N NaOH, dioxane/
MeOH, 858C; (d) (PhO)2P(O)N3, EtOH, Et3N, THF, 808C; (e) KOH/EtOH, reflux; (f) 10% Pd/C, H2, THF/MeOH.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. T. Rubino et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 557–563
Figure 2. Representation of percent inhibition values against MMP-2, -8, -9, and -14 by phenol fragments A-D 1-10 at 100 mM (standard
deviations from triplicate measurements for all values are 10%).
to no substantial increase of inhibitory activity on the whole
set of the MMPs evaluated; our results confirmed for compounds D4, D6, and D7 the values already obtained in a
previous FBDD by NMR screening on stromelysin from
Hajduk and co-workers [24]. The expressly prepared methylated analog of compound D9 (D8) confirmed the low activity
of the methylated derivatives. Anthraflavic acid, as well as
some other anthraquinones [25], and lawsone (D1 and D2)
showed moderate and weak MMPs inhibitory activity,
respectively, whereas 1,4-benzoquinone (D10) showed the
same good inhibition of hydroquinone (A8) against all four
MMPs evaluated at the concentration tested.
The PFL tested, therefore, generated 8 compounds (A6,
A8, B6, C1, C6, C8, C9, and D10) that showed an inhibition
activity of all or some of the tested metalloproteinases
between 77 and 100% at 100 mM. For these compounds
IC50 and ligand efficiency (LE, binding affinity per heavy
atom) values were determined (Table 1) to confirm the
Table 1. Inhibition data (IC50, mM) and LE of selected hits against MMPs.
Entry
MMP-2
A6
A8
B6
C1
C6
C8
C9
D10
a
LEa (kcal/mol)
IC50 (mM)
10.8
3.1
0.59
2.0
4.4
20.0
2.4
3.1
1.7
0.7
0.05
1.6
1.6
2.1
1.4
0.6
MMP-8
7.4
4.4
10.0
9.0
9.1
26.0
37.0
5.1
2.8
2.8
3.1
3.1
0.9
8.1
19.1
1.2
MMP-9
21.2
6.4
57.0
5.0
7.4
16.0
23.0
4.9
10.4
1.8
7.2
3.1
1.2
1.4
9.1
0.2
MMP-14
2.5
2.5
6.2
9.7
5.6
16.3
50.0
3.8
0.2
1.5
2.7
0.4
0.3
0.9
9.2
0.7
0.85
0.94
1.07
0.87
0.82
0.72
0.64
0.94
Calculated for fragments against MMP-2.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 557–563
quality of initial screening hits and for selecting the most
promising ligands to be optimized as MMPIs. The ligand
efficiency is a precious concept in lead evaluation that
allows to compare compounds across different series
considering their potency [26, 27] but also the molecular
weight and the size of the fragment as well as its physicochemical properties [9].
The p-amino phenol A6 showed comparable activity
against all tested MMPs with a good LE and a slight selectivity
for MMP-14. Hydroquinone A8 and 1,4-benzoquinone D10
resulted slightly more active than A6 against MMP-2, -8,
and -9. On the other hand, the ortho substitutions led to a
great variation in the biological activity. Catechol (B6)
resulted to have sub-micromolar potency and the best LE
against MMP-2 (IC50 0.59 0.05 mM, LE ¼ 1.07). It was also
a MMP-2 selective inhibitor with 17, 97, and 10-fold decrease
of activity against MMP-8, -9, and -14, respectively. This
compound resulted more than 2000-fold more potent
against MMP-2 than acetohydroxamic acid (IC50 15 mM)
[28] thus suggesting that inhibitors containing this chemical
structure could have the potential for even greater binding
affinity than those containing a simple alkyl hydroxamic
acid. In the meantime, while we were completing our
biological tests, an article appeared [29], where Cohen and
co-workers described the screening of a chelator fragment
library against matrix metalloproteinases. Among the
96 components of the library, pyrocatechol B6 was also
included, but surprisingly with a very different activity
against MMP-2 (0–25% inhibition at 1 mM). This unexpected
result prompted us to repeat the biological assay also
at higher concentration but our preliminary data was
confirmed. A third OH group in the other ortho-position
(pyrogallol C1) decreased the activity against MMP-2 in
comparison with the catechol producing about 40-fold
reduction of the selectivity towards MMP-9, but leaving
unchanged the activities against MMP-8 and -14. It’s
interesting to note the change of inhibitory activity against
MMP-2 from A6, B6, and C6. The introduction of a hydroxyl
group into the structure of A6 to give the catechol C6
increased the activity which was further enhanced by
elimination of the amino group (see C6 vs. B6). This last
modification, moreover, led to an increase of selectivity
towards the MMPs tested. From these data, it appears evident
the importance of the catecholic group to obtain a more
potent MMPI, even though the simple movement of the
amino group from 4 to 3 position of the catechol system
significantly reduced selectivity and inhibitory activity (see
C8 vs. C6). Finally, gallic acid (C9), which maintained the
pyrogallol moiety with incorporation of a carboxy moiety,
showed the same inhibitory activity of C1 against MMP-2
leading to about 15, 10, and 21-fold increase of selectivity
towards MMP-8, -9, and -14, respectively.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Phenol Matrix Metalloproteinase Inhibitors
561
Conclusion
In conclusion, we investigated the inhibition profiles of some
phenols against MMP-2, -8, -9, and -14. Phenols incorporating
hydroxy and amino moieties in ortho- or para-position were
effective MMP inhibitors. Catechol (B6) showed a very interesting activity on the whole series of tested MMPs with a fairly
good selectivity towards MMP-2. The result herein reported
makes this class of derivatives of great interest for the design
of inhibitors for some of the medicinal targets belonging to
this enzyme family.
Experimental
Biological methods
The PFL was screened against four targets: MMP-2 (proMMP-2,
Calbiochem), MMP-9 and MMP-14 (catalytic domain purchased
from Calbiochem), and MMP-8 (catalytic domain acquired from
Biomol).
Proenzyme was activated immediately prior to use with
p-aminophenylmercuric acetate (APMA 1 mM) for 1 h at 378C.
The assays were performed in triplicate in a total volume of
100 mL per well in 96-well microtitre plates (Corning, white,
NBS). For assay measurements, the inhibitor stock solutions
(DMSO, 10 mM) were further diluted, at 6 different concentrations for each MMP in the fluorometric assay buffer (FAB:
50 mM Tris, pH ¼ 7.5, 200 mM NaCl, 1 mM CaCl2, 1 mM
ZnCl2, 0.05% NaN3, 0.05% Brij 35, and 1% DMSO). Activated
enzyme and inhibitor solutions were incubated in the assay
buffer for 30 min at 258C before the addition of the fluorogenic substrate solution (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2,
Calbiochem) in DMSO (final concentration 2.5 mM). After further
incubation at 378C for 3 h, the hydrolysis was stopped by the
addition of a 3% acetic acid solution and the fluorescence was
measured (lex ¼ 340 nm, lem ¼ 405 nm) using a PERKIN-ELMER
Victor V3 plate reader. Control wells lacked inhibitor. The MMP
inhibition activity was expressed in relative fluorescent units
(RFU). Percent of inhibition was calculated from control reactions without the inhibitor. IC50 was determined using GraphPad
Prism5 software.
Chemical methods
Melting points were determined in open capillaries on a
Gallenkamp electrothermal apparatus and are uncorrected.
Mass spectra were recorded on a HP MS 6890-5973 MSD
spectrometer, electron impact 70 eV, equipped with HP
chemstation or with Agilent LC-MS 1100 Series LC-MSD
Trap System VL spectrometer, electrospray ionization (ESI).
1
H-NMR spectra were recorded using the suitable deuterated
solvent on a Varian Mercury 300 NMR Spectrometer. Chemical
shifts are expressed as parts per million (d). Flash column
chromatography was performed using Merck silica gel 40–
63 mm mesh. All chemicals were purchased from commercial
supplier (Sigma Aldrich) and were used without any further
purification.
Phenol fragment library (PFL) was comprised of 40 fragments
(Fig. 1). Most of these fragments were obtained from commercial
supplier (Sigma Aldrich). The details for fragments that were
synthesized are presented below.
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562
M. T. Rubino et al.
Methyl 3,4,5-trimethoxy benzoate (C4) and 3,4,5-trimethoxy
benzoic acid (C10) were prepared according to literature [23].
Arch. Pharm. Chem. Life Sci. 2011, 344, 557–563
White solid. Yield: 96%. M. p.: 171–1738C; 1H-NMR (CDCl3): d 3.92
(s, 6H, OCH3), 3.93 (s, 3H, OCH3), 7.40 (s, 2H, aromatics). MS (ESI)
m/z: 211 [M–H], MS2 m/z (%): 153 ([C8H9O3þ], 100). Anal.
calcd. for C10H12O5: C 56.60%, H 5.70%, found: C 56.77%,
H 5.71%.
aqueous layer. The organic solution was washed with saturated
solutions of NaHCO3 and brine, dried over Na2SO4, and evaporated to dryness to give a residue that was purified by column
chromatography on silica gel (eluent EP/CH2Cl2 9.8:0.2) affording
the title compound as a white solid. Yield: 72%. M. p.: 96–998C;
1
H-NMR (CDCl3): d 3.80 (s, 6H, CH3), 6.93–7.04, (m, 4H, aromatics)
7.27–7.32, (m, 2H, aromatics), 7.45–7.49, (m, 2H, aromatics). GC–
MS m/z (%): 214 ([M]þ, 100). Anal. calcd. for C14H14O2: C 78.48%,
H 6.59%, found: C 78.35%, H 6.64%.
Methyl 3,4,5-trimethoxy benzoate C10
1,2-Dihydroxy-3-aminobenzene C8
Yellowish solid. Yield: 98%. M. p.: 83–848C; 1H-NMR (CDCl3): d 3.90
(s, 12H, CH3), 7.29 (s, 2H, aromatics). GC–MS m/z (%): 226 ([M]þ,
100). Anal. calcd. for C11H14O5: C 58.40%, H 6.24%, found:
C 58.77%, H 6.27%.
A solution of 2,3-dihydroxybenzoic acid (12.99 mmol) in MeOH
(17 mL) and H2SO4 (0.7 mL) was held at reflux for 6 h. The solvent
was removed under reduced pressure, the residue was dissolved
in EtOAc (70 mL) and washed with saturated solution of NaHCO3,
brine, and dried over Na2SO4. The organic phase was evaporated
to dryness to afford the corresponding methyl ester as yellowish
solid in quantitative yield. The compound was immediately used
in the next step without further purification. M.p.: 77–808C;
1
H-NMR (CDCl3): d 3.95 (s, 3H, CH3), 5.67 (s, 1H, OH), 6.79
(m, 1H, aromatic), 7.09–7.12 (m, 1H, aromatic), 7.35–7.38
(m, 1H, aromatic), 10.99 (s, 1H, OH). GC–MS m/z (%): 168 ([M]þ,
36), 136 ([C7H4O3]þ, 100).
To a stirred solution of methyl 2,3-dihydroxybenzoate
(12.17 mmol) and K2CO3 (24.34 mmol) in anhydrous DMF
(16 mL), benzyl bromide (24.34 mmol) was added and the reaction mixture was stirred at 858C under Ar atmosphere. After 6 h
the suspension was filtered and the resulted solution was evaporated affording a yellowish oil. The unreacted benzyl bromide
was removed by distillation and the solid obtained was crystallized from hexane to afford the methyl 2,3-bis(benzyloxy) benzoate as a white solid. Yield: 71%. M. p.: 64–678C; 1H-NMR (CDCl3):
d 3.85 (s, 3H, CH3), 5.11 (s, 2H, CH2Ph), 5.14 (s, 2H, CH2Ph), 7.10–
7.16 (m, 2H, aromatics), 7.30–7.46 (m, 11H, aromatics).
To a stirred solution of methyl 2,3-bis(benzyloxy) benzoate
(8.64 mmol) in dioxane/MeOH (2:1, 33 mL), 8 N NaOH
(173 mmol) was added. The reaction mixture was heated for
2.5 h at 858C, then evaporated. 2 N HCl was added to the residue
stirring for 10 min. This aqueous phase was extracted with
CHCl3 (100 mL). The organic phase was washed with brine, dried
over Na2SO4, filtered, and evaporated to dryness to obtain the 2,3bis(benzyloxy) benzoic acid as a white solid. Yield: 97%. M. p.:
122–1248C; 1H-NMR (CDCl3): d 5.19 (s, 2H, CH2Ph), 5.27 (s, 2H,
CH2Ph), 7.16–7.50 (m, 12H, aromatics), 7.73–7.76 (m, 1H,
aromatic).
To a solution of 2,3-bis(benzyloxy) benzoic acid (4.19 mmol)
in anhydrous THF (15.5 mL) were added diphenylphosphoryl
azide (4.40 mmol), absolute ethanol (2.5 mL), and anhydrous
Et3N (5.03 mmol). After 6 h at 808C, the mixture was concentrated under reduced pressure in order to remove most ethanol,
and then it was diluted with EtOAc (50 mL); the organic phase
was washed with saturated aqueous NaHCO3 solution, 1 N HCl,
brine, dried over Na2SO4, and concentrated under reduced pressure. The crude solid was purified by column chromatography
on silica gel (eluent EP/EtOAc 9.5:0.5) affording the ethyl 2,3bis(benzyloxy)phenylcarbamate as a white solid. Yield: 84%.
M. p.: 67.5–708C; 1H-NMR (CDCl3): d 1.28 (s, 3H, CH3), 1.28
(s, 2H, CH3CH2), 5.05 (s, 2H, CH2Ph), 5.15 (s, 2H, CH2Ph), 6.69–
6.73, (m, 1H, aromatic), 6.98–7.04, (m, 1H, aromatic), 7.13
(br s, 1H, NH) 7.31–7.44, (m, 8H, aromatics), 7.45–7.48, (m, 2H,
aromatics), 7.68–7.71, (m, 1H, aromatic).
3,4,5-Trimethoxy benzoic acid C4
1,2-Dihydroxy-4-aminobenzene C6, 3,4-Dihydroxy-benzylamine C7, and 2,40 -Dihydroxybiphenyl D9
BBr3 (1 M CH2Cl2 solution, 5.9 mmol) was carefully added dropwise under N2 atmosphere to a cooled (708C) red solution of the
suitable dimethoxy derivative (1.96 mmol) in anhydrous toluene
(21 mL). After 5 h at r.t. an excess of frozen MeOH (10 mL) was
added and stirring was continued for additional 30 min. The
organic solvent was removed under reduced pressure, and the
crude solids were crystallized or triturated with hexane/MeOH or
hexane affording the final compounds (C6, C7, D9) as colored
solids in 44–64% yields.
1,2-Dihydroxy-4-aminobenzene C6
Grey solid. Yield: 64%. M. p.: 256–2598C; 1H-NMR (DMSO-d6):
d 6.58–6.61 (m, 1H, aromatic), 6.75–6.79 (m, 2H, aromatics),
9.26 (br s, 1H, OH), 9.52 (br s, 1H, OH), 9.64 (br s, 2H, NH2).
GC–MS m/z (%): 125 ([M]þ, 100). Anal. calcd. for C6H7NO2 HBr:
C 34.98%, H 3.91%, N 6.80%, found: C 35.18%, H 4.06%, N 6.78%.
3,4-Dihydroxy-benzyl-amine C7
Brownish solid. Yield: 44%. M. p.: 186–1888C; 1H-NMR (DMSO-d6):
d 3.80 (s, 2H, CH2), 6.65–6.74 (m, 2H, aromatics), 6.79–6.80
(m, 1H, aromatic), 7.80–9.20 (br, 4H, OH, NH2). Anal. calcd.
for C7H9NO2 HBr: C 38.20%, H 4.58%, N 6.63%, found:
C 38.21%, H 4.58%, N 6.37%.
2,40 -Dihydroxybiphenyl D9
Yellowish solid. Yield: 63%. M. p.: 165–1668C; 1H-NMR (DMSO-d6):
d 6.73–6.88, (m, 4H, aromatics) 7.03–7.09, (m, 1H, aromatic), 7.14–
7.17, (m, 1H, aromatic), 7.31–7.36 (m, 2H, aromatics), 9.33 (br s,
2H, OH). GC–MS m/z (%): 186 ([M]þ, 100). Anal. calcd. for C12H10O2:
C 77.40%, H 5.41%, found: C 77.02%, H 5.42%.
2,40 -Dimethoxy-biphenyl D8
A solution of 1-bromo-2-methoxybenzene (2.14 mmol), 4-methoxybenzeneboronic acid (4.28 mmol), and Cs2CO3 (3.21 mmol) in
anhydrous toluene (22 mL) was stirred at room temperature
under N2 atmosphere for 20 min. [Pd(PPh3)4] was added
(0.054 mmol) and the resulting mixture was heated at 908C.
After stirring overnight, the reaction mixture was cooled to
room temperature, diluted with 1 N HCl and EtOAc (1:1,
7.5 mL), stirred for 15 min and filtered through a pad of
Celite, followed by separation of the organic phase from the
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2011, 344, 557–563
To a solution of the previous ethyl carbamate (1.14 mmol)
in ethanol (8.1 mL), was added a freshly prepared solution of
potassium hydroxide (12.54 mmol) in ethanol (4.1 mL). After
18 h at reflux, the ethanol was removed under reduced pressure
and the resulting yellowish solid was diluted with EtOAc (40 mL),
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure affording 2,3-bis(benzyloxy)benzenamine as a
brownish-yellow oil. Yield: 96%. 1H-NMR (CDCl3): d 3.79 (br s, 1H,
NH), 5.02 (s, 2H, CH2Ph), 5.12 (s, 2H, CH2Ph), 6.37–6.44, (m, 2H,
aromatics), 6.81–6.86, (m, 1H, aromatic), 7.30–7.48, (m, 10H,
aromatics).
To a stirred suspension of 2,3-bis(benzyloxy)benzenamine
(1.05 mmol) in THF/MeOH (36 mL, 3:1), 10% Pd/C (66 mg) was
added. After stirring overnight under H2 atmosphere (6 atm), the
reaction mixture was filtered through a pad of Celite and concentrated under reduced pressure affording the desired 1,2-dihydroxy-3-aminobenzene (C8) as a brownish-yellow solid. Yield:
98%. M. p.: 1628C (dec.); 1H-NMR (DMSO-d6): d 4.39 (br s, 2H,
NH2), 6.02–6.09 (m, 2H, aromatics), 6.30–6.35 (m, 1H, aromatic),
7.79 (br s, 1H, OH), 8.66 (br s, 1H, OH); MS (ESI) m/z: 124 [M H],
MS2 m/z (%): 124 ([C6H6NO2þ], 100). Anal. calcd. for
C6H7NO2 HCl: C 44.60%, H 4.99%, N 8.67%, found: C 44.28%,
H 5.03%, N 8.40%.
The authors have declared no conflict of interest.
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