Reaction of rosmarinic acid with nitrite ions in acidic conditions: Discovery of nitro- and dinitrorosmarinic acids as new anti-HIV-1 agents

Article abstract of DOI:10.1021/jm7011134

Rosmarinic acid was reacted with nitrite ions under acidic conditions to give 6′-nitro- and 6′,6″-dinitrorosmarinic acids according to the reaction time. Both compounds were active as HIV-1 integrase inhibitors at the submicromolar level. They also inhibi

Full text of DOI:10.1021/jm7011134

J. Med. Chem. 2008, 51, 2575–2579
2575
Reaction of Rosmarinic Acid with Nitrite Ions in Acidic Conditions: Discovery of Nitro- and
Dinitrorosmarinic Acids as New Anti-HIV-1 Agents
Mélanie Dubois,† Fabrice Bailly,† Gladys Mbemba,‡ Jean-François Mouscadet,‡ Zeger Debyser,§ Myriam Witvrouw,§ and
Philippe Cotelle*,†
Laboratoire de Chimie Organique et Macromoléculaire, UMR CNRS 8009, UniVersité de Lille 1, 59655 VilleneuVe d’Ascq, France,
Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée, UMR CNRS 8113, Ecole Normale Supérieure de Cachan, 61 AVenue
du Président Wilson, 94235 Cachan, France, and Molecular Medicine, K. U. LeuVen and IRC KULAK, KapucijnenVoer 33, B-3000 LeuVen,
Flanders, Belgium
ReceiVed September 7, 2007
Rosmarinic acid was reacted with nitrite ions under acidic conditions to give 6′-nitro- and 6′,6′′-
dinitrorosmarinic acids according to the reaction time. Both compounds were active as HIV-1 integrase
inhibitors at the submicromolar level. They also inhibited the viral replication in MT-4 cells with modest
and similar selectivity indexes. The nitration of rosmarinic acid strongly improves the anti-integrase inhibition
and the antiviral activity without increasing the cellular toxicity.
Introduction
scription (IC50 between 100 and 200 µM) and affected distinct
phases of early natural endogenous reverse transcription.15
One of the major exogenous sources of nitrosating species is
represented by nitrite ions, which are present in high levels in
human saliva (50–200 µM).16 In the acidic environment of the
stomach, nitrite ions are converted to nitrous acid that can react
with catechols to give nitro derivatives.17,18 The concentration
of rosmarinic acid in plant infusion can reach millimolar levels,19
and therefore, a reaction between rosmarinic acid and nitrite
ions under acidic conditions in the stomach is not unrealistic.
We therefore underwent to study such reaction and to isolate
and purify the products.
Our convergent interests for antioxidant (reactivity toward
reactive nitrogen species) and anti-integrase properties of
polyphenols led us to evaluate their antiviral properties.
Fortunately, we found a remarkable improvement of the anti-
IN and antiviral activities of these products versus rosmarinic
acid. In the present paper, we report the synthesis, the anti-
integrase activities, and the antiviral properties of 6′-nitro and
6′,6′′-dinitrorosmarinic acids. Finally magnesium-chelating prop-
erties of test compounds were investigated by UV–vis
spectroscopy.
Rosmarinic acid is an active component of many culinary
plants (sage, rosemary, mint, melissa, thyme, prunella, origan,
sweet basil, etc.) mostly responsible for antiinfective, anti-
inflammatory, and antioxidative activity of these herbs.1 It has
also been suggested that rosmarinic acid may have inhibitory
effects in Alzheimer amyloid-ꢀ peptide2,3 and Parkinson R-sy-
nuclein aggregation4,5 and interesting properties in the field of
cancer as Fyn kinase inhibitor6 and as apoptosis of Jurkat and
peripheral T-cells inducer.7 It is also known as an aldose
reductase inhibitor8 (an enzyme implicated in type II diabetes)
and possesses remarkable antioxidant properties as reactive
oxygen species scavenger and lipid peroxidation inhibitor.9
Among 51 samples from 46 herb species, 45 showed
significant inhibitory effects against HIV-1 induced cytopatho-
genicity in MT-4 cells. Particularly, Melissa officinalis, Mentha,
and Prunella Vulgaris extracts, where rosmarinic acid is the
major phenolic component, showed potent anti-HIV-1 activity
targeting reverse transcription step.10,11 Earlier, rosmarinic acid
was found to inhibit 3′-processing and strand transfer activities
of HIV-1 integrase (INa) in the presence of Mn2+ 12
By use of
.
a multiplate integration procedure, aqueous and ethanolic
extracts of 50 Thai plants were screened for their inhibitory
activity against HIV-1 integrase.13 Rosmarinic acid, its methyl
ester, and its calcium and magnesium salts were isolated from
the ethanolic extract of Coleus parVifolius Benth. The calcium
and magnesium rosmarinates were found to be 5 times more
active than rosmarinic acid against IN activity. The same salts
isolated from water extract of Cordia spinescens were found to
be potent inhibitors of reverse transcriptase14 but inactive against
protease. Rosmarinic acid also directly inhibited reverse tran-
Results and Discussion
Chemistry. The reaction of caffeic acid derivatives with
nitrite ions under conditions simulating those within the stomach
is well-documented, and nitration of the aromatic ring is known
to occur only on caffeic esters.20,21 Applying the same conditions
to rosmarinic acid 1 afforded selectively mono or dinitro
derivatives depending on the reaction time. When the reaction
medium is saturated after 5 min by the addition of sodium
chloride and immediately extracted by ethyl acetate, the
6′-nitrorosmarinic acid 2 can be easily isolated in 15% yield
(Scheme 1). A reaction time of 10 min allowed us to isolate
the 6′,6′′-dinitrorosmarinic acid 3 in 25% yield. In both cases,
mono- and dinitrorosmarinic acids are the main products but
the yields dramatically decreased because of difficulty in
purification (crystallization in water). The positions of the
nitration on the aromatic ring can be easily determined from
* To whom correspondence should be addressed. Phone: +33 320 43
49 39. Fax: +33 320 33 63 09. E-mail: philippe.cotelle@univ-lille1.fr.
†
Université de Lille 1.
Ecole Normale Supérieure de Cachan.
K. U. Leuven and IRC KULAK.
‡
§
a Abbreviations: BSA, bovine serum albumin; CPE, cytopathic effect;
DMSO, dimethyl sulfoxide; DTT, dithiothreitol; HEPES, 4-(2-hydroxyethyl)-
piperazine-1-ethanesulfonic acid; HIV, human immunodeficiency virus; EI,
electronic impact; ESI, electrospray ionization; IN, integrase; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PEG, polyethylene
glycol.
1
the aromatic portion of the H NMR spectra. The presence of
two and four singlets for mono- and dinitrorosmarinic acid,
respectively, attested that the remaining hydrogen atoms were
10.1021/jm7011134 CCC: $40.75
2008 American Chemical Society
Published on Web 03/20/2008

2576 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Scheme 1. Synthesis of Compounds 2 and 3a
Brief Articles
determined using the Stern-Volmer equation to provide a
measure of the binding affinity between the compounds and
BSA (Figure 1). The binding affinities ranked in the following
order: 6′-nitrorosmarinic acid 2 (KSV ) 17460 × 103 M-1) >
6′,6′′-dinitrorosmarinic acid 3 (KSV ) 3300 × 103 M-1) >
rosmarinic acid 1 (KSV ) 20 × 103 M-1). The best binding
affinities were obtained for the nitrated species, when compared
to rosmarinic acid.
The biological studies also seemed to show an influence of
the nitro groups on the anti-HIV and particularly on the anti-
IN properties of the rosmarinic derivatives. This led us to
investigate their magnesium-chelating capacities. UV–vis spectra
of compounds were not affected by the addition of MgCl2. This
was not the case for Mg(OAc)2. Figure 2 shows a slight
modification of the UV–vis spectrum of rosmarinic acid in the
presence of magnesium acetate. In contrast, in the same
conditions, UV–vis spectra of the nitrated species were sharply
modified with large hypochromic effects at 248 and 334 nm
for 2 (Figure 3), a large hypochromic effect at 280 nm, and the
simultaneous apparition of a new peak at 322 nm for 3 (Figure
4). Since the solutions took a slight orange color upon addition
of Mg(OAc)2, the formation of phenolates was very probable.
Spectra of the fully deprotonated compounds in the presence
of a large excess of NaOH were investigated. They were
completely different (Figure 2 and Supporting Information) and
exhibited a large peak around 385 nm (1, 2) and 430 nm (3). In
the presence of NaOAc, the spectra of all compounds were
slightly modified (Supporting Information). All these results
undoubtedly attest that the weak base acetate partially depro-
tonates the nitrated compounds, which leads to the formation
of a complex with the magnesium ion. This situation was not
encountered for rosmarinic acid. Thus, the nitration favorably
affects the chelating properties of the rosmarinic derivatives,
and this supports the best anti-IN activity obtained for the
dinitrated species 3.
a Reagents and conditions: (i) NaNO2, acetate buffer (0.2 M AcOH/0.2
M AcONa), room temperature, 5 min; (ii) NaNO2, acetate buffer (0.2 M
AcOH/0.2 M AcONa), room temperature, 10 min.
in the para position. The catechol where the nitration took place
in 2 was determined by comparison of the 13C NMR spectra of
mononitrorosmarinic acid and rosmarinic acid. The 13C NMR
signals of the caffeic acid moiety are not modified by the
mononitration, whereas a high-field shift is observed for CR
and Cꢀ signals after dinitration. Expectedly the catechol ring
substituted by an alkyl group (3,4-dihydroxyphenyllactic acid
part of rosmarinic acid) is the most reactive toward the aromatic
electrophilic substitution, allowing us to control the mono- and
the dinitration with the reaction time.
Biology. 6′-Nitro and 6′,6′′-dinitrorosmarinic acids 2 and 3
were tested in IN inhibition assays, which have been recently
reviewed.22,23 The overall IN enzymatic activity has been
evaluated using two different protocols in the presence (protocol
1, Table 1 column 2) and in the absence (protocols 1 and 2,
Table 1, columns 3 and 4) of bovine serum albumin (BSA) with
magnesium ions as cofactor. The 3′-processing and strand
transfer reaction inhibition were evaluated using protocol 1
(Table 1, columns 5 and 6). Antiviral properties and cytotoxicity
were evaluated in the viral replication in MT-4 cells (Table 1,
columns 8 and 9, respectively). Comparison of the overall
integrase inhibitions in the absence of BSA using two different
protocols of purification of the enzyme and two reaction media
shows that our compounds are very sensitive to the conditions
of the assays. But the most dramatic differences are obtained
in the presence of BSA. This is probably due to the formation
of complexes between BSA and the polyphenolic drugs.24 In
all the pharmacological assays, 6′,6′′-dinitrorosmarinic acid 3
is always more active than 6′-nitrorosmarinic acid 2 and
rosmarinic acid 1, indicating that the nitration of the caffeic
acid moiety allows for better anti-IN and anti-HIV activities. 3
presents a pronounced selectivity toward the strand transfer
reaction (see column 7 of Table 1) and therefore was found to
have the most powerful antiviral activity. Whereas 1 had weak
antiviral properties limited by an important cytotoxicity, 2 and
3 were found to present noticeable antiviral activities in the
MT-4 cells assays with modest therapeutic indexes of about
∼4. Nevertheless one must recognize that these antiviral
activities remain modest in comparison to those of the naph-
tyridine reference compound, L-870,810. The IN assays on the
overall reaction in the presence of BSA seem to be the most
relevant ones compared to MT-4 cell assays.
Conclusion
In the present paper, we have found that rosmarinic acid may
readily react with nitrite ions under acidic conditions mimicking
acidity in gastric juice. Two new nitrorosmarinic acids were
isolated and revealed remarkable activities against HIV-1
integrase and antiviral properties. The anti-IN activities of 2
and 3 are expected to be due to their Mg2+-chelating properties.
The antiviral potency of infusion of rosmarinic acid-rich plants
(such as sage) has to be evaluated in order to propose an
alternative (or a complementation) to the classical antiretroviral
therapy.
Since the concentrations of nitrite ions in stomach may reach
millimolar levels, nitrorosmarinic acids may be considered as
possible metabolites of rosmarinic acid. The great number of
biological properties of rosmarinic acid indicates that the
properties of its nitro derivatives have to be checked, particularly
in the field of diabetes, cancer, and neurodegenerative diseases.
The nitration of rosmarinic acid strongly affects the binding
properties to BSA. Compounds 2 and 3 will be therefore tested
in the inhibition of Aꢀ, R-synuclein, and τ filament formation.
Since the discovery of diketoacids,26 nitrorosmarinic acids
constitute the first new family of selective strand transfer
inhibitors with antiviral properties. Two issues have to be
addressed: (i) the caffeic and lactic parts of 3 must be
synthesized separately and tested in order to define the contribu-
tion of these potential pharmacophores to the anti-IN and
antiviral activities; (ii) the therapeutic index and therefore the
antiviral activities must be increased.
Physicochemical Studies. In the overall integrase inhibition
experiments, IC50 values obtained in presence of BSA were
considerably higher than those in absence of BSA, suggesting
the possible formation of complexes between BSA and the
polyphenolic drugs,24 which could drastically decrease the
catechol availability and therefore the IN inhibitory activities
of the compounds. This was confirmed by a study of the
interaction of rosmarinic derivatives with BSA by a fluorescence-
based method.25 All the compounds quenched significantly BSA
tryptophan fluorescence, and quenching constants (KSV) were

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2577
Table 1. Inhibition of HIV-1 IN Catalytic Activities, Antiviral Activity, and Cytotoxicity of Rosmarinic Acid 1, 6′-Nitrorosmarinic Acid 2,
6′,6′′-Dinitrorosmarinic Acid 3, and Reference Compound L-870,810
IC50 (µM)
overalla
BSAf
+
c
d
compd
overalla
overallb
NTh
0.026 ( 0.005 >247
0.030 ( 0.005 52.2
3′-Pa
NTh
STa
3′P/STg
EC50 (µM)
CC50 (µM)
SIe
1
2
3
63.5 ( 15
NTh
NTh
>55
55
54.7 ( 17.1 1.4 ( 1.5
3.7 ( 1.9
0.07 ( 0.02
>67
746
48
39 ( 9
160 ( 18.5
45.5 ( 4.5
2.2 ( 0.2
4.0
3.8
457
20.8 ( 14.9 0.15 ( 0.09
12 ( 3
L-870,810 NTh
0.0005 ( 0.0003 NTh
0.12 ( 0.03 0.0025 ( 0.0007
0.0047 ( 0.0007
a Concentration required to inhibit by 50% the in vitro integrase activity assays using protocol 1. b Concentration required to inhibit by 50% the in vitro
integrase activity assays using protocol 2. c Effective concentration required to reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells. d Cytotoxic
concentration to reduce MT-4 cell viability by 50%. e Selectivity index: CC50/EC50 ratio. f Bovine serum albumin was added in the medium (0.1 mg/mL).
g IC50(3′-P)/IC50(ST) ratio. h NT: not tested.
Figure 3. UV–visible spectra of 6′-nitrorosmarinic acid 2 (50 µM in
Figure 1. Stern-Volmer plots describing BSA tryptophan quenching
at pH 7.4 caused by rosmarinic acid derivatives association: (9) 6′-
nitrorosmarinic acid 2, y ) 17.4x + 1, R2 ) 0.99; (2) 6′,6′′-
dinitrorosmarinic acid 3, y ) 3.3x + 1, R2 ) 0.96; ([) rosmarinic
acid 1, y ) 0.02x + 1, R2 ) 0.96.
ethanol, 1) in presence of Mg(OAc)2 (16.67 µM 2, 33.33 µM 3, 50.00
µM 4, 66.66 µM 5, 83.33 µM 6, 100.00 µM 7, 116.67 µM 8, 133.33
µM 9).
Figure 4. UV–visible spectra of 6′,6′′-dinitrorosmarinic acid 3 (50
µM in ethanol, 1) in presence of Mg(OAc)2 (16.67 µM 2, 33.33 µM 3,
50.00 µM 4, 66.66 µM 5, 83.33 µM 6, 100.00 µM 7).
Figure 2. UV–visible spectra of rosmarinic acid 1 (50 µM in ethanol,
1) in presence of 50 µM Mg(OAc)2 (2), 100 µM Mg(OAc)2 (3), 333
µM NaOH (4), and 666 µM NaOH (5).
spectrometer in the appropriate solvent with TMS as internal
reference. Melting points were obtained on a Reichert Thermopan
melting point apparatus equipped with a microscope and are
uncorrected. Mass spectra were recorded on a Thermo-Finnigan
PolarisQ mass spectrometer (70 eV, electron impact). HRMS were
obtained on an Apex Qe 9.4 T Bruker Daltonics spectrometer.
Elemental analyses were performed by CNRS Laboratories (Ver-
naison).
Whereas 6-nitrocaffeic acid can be easily obtained by
demethylation of the commercially available 6-nitro-3,4-
dimethoxycinnamic acid using boron tribromide according to a
previously reported method,27 attempts to obtain and isolate the
6-nitro-3,4-dihydroxyphenyllactic acid were unsuccessful be-
cause of an instability of the formed nitrated species occurring
during column chromatography.
Synthesis of 2 and 3. To a solution of 544 mg (7.9 mmol) of
sodium nitrite in 50 mL of acetate buffer (0.2 M AcOH/0.2 M
AcONa) was added 360 mg (1.0 mmol) of rosmarinic acid. After
5 min (2) or 10 min (3) the solution was saturated with sodium
chloride and extracted with AcOEt (3 × 50 mL). The combined
organic layers were dried over Na2SO4 and concentrated in vacuo.
The crude product was crystallized in water (two times for 2) to
give 3 as an orange powder and 2 as a yellow powder.
Experimental Section
Chemistry. General Details. All reagents and solvents were
purchased from Aldrich-Chimie (Saint-Quentin-Fallavier, France)
of ACS reagent grade and were used as provided. TLC analyses
were performed on plastic sheets precoated with silica gel 60F254
(Merck). SiO2, 200–400 mesh (Merck), was used for column
chromatography. NMR spectra were obtained on an AC 200 Bruker
2: RD (MeOH) +61.9 ° (c 0.17 g/L); mp 168 °C; 1H NMR
2
3
(acetone-d6) 3.40 (dd, 1H, J ) 14.1 Hz, J ) 9.4 Hz, H3), 3.71

2578 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Brief Articles
2
3
3
(dd, 1H, J ) 14.1 Hz, J ) 4.2 Hz, H3), 5.38 (dd, 1H, J ) 9.4
Acknowledgment. This work was financially supported by
grants from le Centre National de la Recherche Scientifique
(CNRS), l’Agence Nationale de la Recherche contre le Sida
(ANRS), and the European Commission (Grant LSHB-CT-2003-
503480). The European TRIoH Consortium is gratefully ac-
knowledged. The mass spectrometry facility used in this study
was funded by the European Community (FEDER), the Région
Nord-Pas de Calais (France), the CNRS, and the Université des
Sciences et Technologies de Lille. We are grateful to Martine
Michiels, Nam Joo Vanderveken, and Barbara Van Remoortel
for excellent technical assistance.
3
3
Hz, J ) 4.2 Hz, H2), 6.26 (d, 1H, J ) 15.8 Hz, HR), 6.89 (d,
1H, J ) 8.2 Hz, H5′′), 7.01 (s, 1H, H2′), 7.06 (dd, 1H, J ) 8.2
3
3
4
4
Hz, J ) 2.0 Hz, H6′′), 7.18 (d, 1H, J ) 2.0 Hz, H2′′), 7.55 (d,
1H, 3J ) 15.8 Hz, Hꢀ), 7.61 (s, 1H, H5′); 13C NMR 171.2 (COOH),
166.7 (CdO), 151.2 (C3′), 149.1 (C4′′), 146.8 (C3′′), 146.4 (Cꢀ),
145.2 (C4′), 142.3 (C6′), 127.5 (C1′′), 126.0 (C1′), 122.8 (C6′′),
120.0 (C2′), 116.5 (C5′′), 115.3 (CR), 114.7 (C2′′), 113.3 (C5′),
72.7 (C2), 35.1 (C3); MS (EI) 404 (M+0 - 1, 57%), 242 (lactic
fragment, 100%), 180 (caffeic fragment, 50%); HRMS (nano-ESI-
FT-ICR)(negative mode) calcd for C18H15NO10 404.062 32, found
404.062 33. Anal. (C18H15NO10) C, H, N.
1
3: RD (MeOH) +25.3 ° (c 0.21 g/L); mp 45–46 °C; H NMR
Supporting Information Available: Additional chemical and
biological information. This material is available free of charge
via the Internet at http://pubs.acs.org.
2
3
(acetone-d6) 3.36 (dd, 1H, J ) 13.9 Hz, J ) 9.0 Hz, H3), 3.69
2
3
3
(dd, 1H, J ) 13.9 Hz, J ) 4.3 Hz, H3), 5.40 (dd, 1H, J ) 9.0
3
3
Hz, J ) 4.3 Hz, H2), 6.33 (d, 1H, J ) 15.9 Hz, HR), 6.98 (s,
1H, H2′), 7.23 (s, 1H, H2′′), 7.58 (s, 1H, H5′), 7.61 (s, 1H, H5′′),
8.12 (d, 1H, J ) 15.9 Hz, Hꢀ); 13C NMR 170.9 (COOH), 166.0
3
References
(1) Petersen, M.; Simmonds, M. S. Rosmarinic acid. Phytochemistry 2003,
62, 121–125.
(2) Ono, K.; Hasegawa, K.; Naiki, H.; Yamada, M. Curcumin has potent
anti-amyloidogenic effects for Alzheimer’s beta-amyloid fibrils in vitro.
J. Neurosci. Res. 2004, 75, 742–750.
(3) Alkam, T.; Nitta, A.; Mizoguchi, H.; Itoh, A.; Nabeshima, T. A natural
scavenger of peroxynitrites, rosmarinic acid, protects against impair-
ment of memory induced by Aꢀ25–35. BehaV. Brain Res. 2007, 180,
139–145.
(CdO), 151.5 (C3′′), 151.1 (C3′), 147.8 (C4′′), 145.1 (C4′), 142.8
(Cꢀ), 142.6 (C6′′) 142.2 (C6′), 125.8 (C1′), 124.4 (C1′′), 120.3
(CR), 120.0 (C2′), 115.4 (C2′′), 113.3 (C5′), 113.1 (C5′′), 73.0 (C2),
35.1 (C3); MS (EI) 449 (M+0 - 1, 45%), 242 (lactic fragment,
100%), 226 (caffeic fragment, 52%); HRMS (nano-ESI-FT-ICR)
(negative mode) calcd for C18H14N2O12 449.047 40, found 449.043 22.
Anal. (C18H14N2O12) C, H, N.
Integrase Inhibition (Protocol 1). To determine the susceptibil-
ity of the HIV-1 integrase enzyme toward different compounds,
we used an enzyme-linked immunosorbent assay. This assay uses
an oligonucleotide substrate of which one oligo (5′-ACTGCTA-
GAGATTTTCCACACTGACTAAAAGGGTC-3′) is labeled with
biotin on the 3′ end and the other oligo is labeled with digoxigenin
at the 5′ end. For the overall integration assay the second
5′-digoxigenin labeled oligo is 5′-GACCCTTTTAGTCAGTGTG-
GAAAATCTCTAGCAGT-3′. The integrase was diluted in 750
mM NaCl, 10 mM Tris, pH 7.6, 10% glycerol, and 1 mM
ꢀ-mercaptoethanol. To perform the reaction, 4 µL of diluted
integrase (corresponds to 1.6 µM WT integrase22) and 4 µL of
annealed oligos (7 nM) were added to a final reaction volume of
40 µL containing 10 mM MgCl2, 5 mM DTT, 20 mM HEPES, pH
7.5, 5% PEG, and 15% DMSO. The reaction was carried out for
1 h at 37 °C. These reactions were followed by an immunosorbent
assay on avidin coated plates.28
Integrase Inhibition (Protocol 2). Oligonucleotides were pur-
chased from Eurogentec and further purified on 18% acrylamide/
urea denaturing gel: U5B, GTGTGGAAAATCTCTAGCA; U5A,
5′-ACTGCTAGAGATTTTCCACAC. Wild-type HIV-1 integrase
was purified as described previously.29 The assay was performed
in a reaction volume of 20 µL containing 0.025 pmol of labeled
U5A/U5B double-stranded DNA substrate and 1 pmol of integrase
in buffer [20 mM HEPES (pH 7.2), 10 mM MgCl2, 25 mM NaCl,
and 1 mM DTT]. Products were separated on an 18% acrylamide/
urea denaturing gel and quantified on a phosphoimager using
ImageQuant software (Amersham Pharmacia Biotech).
In Vitro Anti-HIV and Drug Susceptibility Assays. The
inhibitory effect of antiviral drugs on the HIV-1-induced cytopathic
effect (CPE) in human lymphocyte MT-4 cell culture was deter-
mined by the MT-4/MTT-assay.30 This assay is based on the
reduction of the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) by mitochondrial dehydrogenase of
metabolically active cells to a blue formazan derivative, which can
be measured spectrophotometrically. The 50% cell culture infective
dose (CCID50) of the HIV-1 (IIIB) strain was determined by titration
of the virus stock using MT-4 cells. For the drug susceptibility
assays MT-4 cells were infected with 100–300 CCID50 of the virus
stock in the presence of 5-fold serial dilutions of the antiviral drugs.
The concentrations of various compounds achieving 50% protection
against the CPE of the different HIV strains, which are defined as
EC50, were determined. In parallel the 50% cytotoxic concentration
(CC50) was determined.
(4) Ono, K.; Yamada, M. Antioxidant compounds have potent anti-
fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils
in vitro. J. Neurochem. 2006, 97, 105–115.
(5) Masuda, M.; Suzuki, N.; Taniguchi, S.; Oikawa, T.; Nonaka, T.;
Iwatsubo, T.; Hisanaga, S.; Goebert, M.; Hasegawa, M. Small molecule
inhibitors of R-synuclein assembly. Biochemistry 2006, 45, 6085–6094.
(6) Jelie, D.; Mildner, B.; Kootrun, S.; Nujie, K.; Verbanac, D.; Culie,
O.; Antolovie, R.; Brandt, W. Homology modeling of human fyn
kinase structure: discovery of rosmarinic acid as a new fyn kinase
inhibitor and in silico study of its possible binding modes. J. Med.
Chem. 2007, 50, 1090–1100.
(7) Hur, Y.-G.; Yun, Y.; Won, J. Rosmarinic acid induces p56lck-
dependent apoptosis in Jurkat and peripheral T cells via mitochondrial
pathway independent from Fas/Fas ligand interaction. J. Immunol.
2004, 172, 79–87.
(8) Koukoulitsa, C.; Zika, C.; Geromichalos, G. D.; Demopoulos, V. J.;
Skaltsa, H. Evaluation of aldose reductase inhibition and docking
studies of some secondary metabolites, isolated from Origanum
Vulgare L. ssp. hirtum. Bioorg. Med. Chem. 2006, 14, 1653–1659.
(9) Qiao, S.; Li, W.; Tsubouchi, R.; Haneda, M.; Murakami, K.; Takeuchi,
F.; Nisimoto, Y.; Yoshino, M. Rosmarinic acid inhibits the formation
of reactive oxygen and nitrogen species in RAW264.7 macrophages.
Free Radical Res. 2005, 39, 995–1003.
(10) Yamasaki, K.; Nakano, M.; Kawahata, T.; Mori, H.; Otake, T.; Ueba,
N.; Oishi, I.; Inami, R.; Yamane, M.; Nakamura, M.; Murata, H.;
Nakanishi, T. Anti-HIV-1 activity of herbs in Labiatae. Biol. Pharm.
Bull. 1998, 21, 829–833.
(11) Kageyama, S.; Kurokawa, M.; Shiraki, K. Extract of Prunella Vulgaris
spikes inhibits HIV replication at reverse transcription in vitro and
can be absorbed from intestine in vivo. AntiViral Chem. Chemother.
2000, 11, 157–164.
(12) Tewtrakul, S.; Miyashiro, H.; Nakamura, N.; Hattori, M.; Kawahata,
T.; Otake, T.; Yoshinaga, T.; Fujiwara, T.; Supavita, T.; Yuenyong-
sawad, S.; Rattanasuwon, P.; Dej-Adisai, S. HIV-1 integrase inhibitory
substances from Coleus parVifolius. Phytother. Res. 2003, 17, 232–
239.
(13) Mazumder, A.; Neamati, N.; Sunder, S.; Schulz, J.; Pertz, H.; Eich,
E.; Pommier, Y. Curcumin analogs with altered potencies against
HIV-1 integrase as probes for biochemical mechanisms of drug action.
J. Med. Chem. 1997, 40, 3057–3063.
(14) Lim, Y. A.; Kojima, S.; Nakamura, N.; Miyashiro, H.; Fushimi, H.;
Komatsu, K.; Hattori, M.; Shimotohno, K.; Gupta, M. P.; Correa, M.
Inhibitory effects of Cordia spinescens extracts and their constituents
on reverse transcriptase and protease from human immunodeficiency
virus. Phytother. Res. 1997, 11, 490–495.
(15) Hooker, C. W.; Lott, W. B.; Harrich, D. Inhibitors of human
immunodeficiency virus type 1 reverse transcriptase target distinct
phases of early reverse transcription. J. Virol. 2001, 75, 3095–3104.
(16) Duncan, C.; Dougall, H.; Johnston, P.; Green, S.; Brogan, R.; Leifert,
C.; Smith, L.; Golden, M.; Benjamin, N. Chemical generation of nitric
oxide in the mouth from the enterosalivary circulation of dietary nitrate.
Nat. Med. 1995, 1, 546–551.

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2579
(17) De la Bretèche, M. L.; Servy, C.; Lenfant, M.; Ducrocq, C. Nitration
of catecholamines with nitrogen oxides in mild conditions. Tetrahedron
Lett. 1994, 35, 7231–7232.
(18) D’Ischia, M.; Costantini, C. Nitric oxide-induced nitration of cat-
echolamine neurotransmitters: a key to neuronal degeneration. Bioorg.
Med. Chem. 1995, 3, 923–927.
(19) Lima, C. F.; Andrade, P. B.; Seabra, R. M.; Fernandes-Ferreira, M.;
Pereira-Wilson, C. The drinking of a SalVia officinalis infusion
improves liver antioxidant status in mice and rats. J. Ethnopharmacol.
2005, 97, 383–389.
from gel electrophoresis. Biosci. Biotechnol. Biochem. 2006, 70, 152–
160.
(25) Papadopoulou, A.; Green, R. J.; Frazier, R. A. Interaction of flavonoids
with bovine serum albumin: a fluorescence quenching study. J. Agric.
Food Chem. 2005, 53, 158–163.
(26) Hazuda, D. J.; Felock, P.; Witmer, M.; Wolfe, A.; Stillmock, K.;
Grobler, J. A.; Espeseth, A.; Gabryelski, L.; Schleif, W.; Blau, C.;
Miller, M. D. Inhibitors of strand transfer that prevent integration and
inhibit HIV-1 replication in cells. Science 2000, 287, 646–650.
(27) Grenier, J. L.; Cotelle, N.; Catteau, J. P.; Cotelle, P. Synthesis and
physico-chemical properties of nitrocaffeic acids. J. Phys. Org. Chem.
2000, 13, 511–517.
(28) Hwang, Y.; Rhodes, D.; Bushman, F. Rapid microtiter assays for
poxvirus topoisomerase, mammalian type IB topoisomerase and HIV-1
integrase: application to inhibitor isolation. Nucleic Acids Res. 2000,
28, 4884–4892.
(29) Leh, H.; Brodin, P.; Bischerour, J.; Deprez, E.; Tauc, P.; Brochon,
J. C.; LeCam, E.; Coulaud, D.; Auclair, C.; Mouscadet, J. F.
Determinants of Mg2+-dependent activities of recombinant human
immunodeficiency virus type 1 integrase. Biochemistry 2000, 39, 9285–
9294.
(30) Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn,
P.; Desmyter, J.; De Clercq, E. Rapid and automated tetrazolium-
based colorimetric assay for the detection of anti-HIV compounds.
J. Virol. Methods 1988, 20, 309–321.
(20) Cotelle, P.; Vezin, H. Reaction of caffeic acid derivatives with acidic
nitrite. Tetrahedron Lett. 2001, 42, 3303–3305.
(21) Napolitano, A.; d’Ischia, M. New insights into the acid-promoted
reaction of caffeic acid and its esters with nitrite: decarboxylation
drives chain nitrosation pathways toward novel oxime derivatives and
oxidation/fragmentation products thereof. J. Org. Chem. 2002, 67, 803–
810.
(22) Debyser, Z.; Cherepanov, P.; Pluymers, W.; De Clercq, E. Assays for
the evaluation of HIV-1 integrase inhibitors. Methods Mol. Biol. 2001,
160, 139–155.
(23) Witvrouw, M.; Van Maele, B.; Vercammen, J.; Hantson, A.; Engel-
borghs, Y.; De Clercq, E.; Pannecouque, C.; Debyser, Z. Novel
inhibitors of HIV-1 integration. Curr. Drug Metab. 2004, 5, 291–
294.
(24) Kusuda, M.; Hatano, T.; Yoshida, T. Water-soluble complexes
formed by natural polyphenols and bovine serum albumin: evidence
JM7011134