Biomimetic in vitro oxidation of lapachol: A model to predict and analyse the in vivo phase i metabolism of bioactive compounds

Article abstract of DOI:10.1016/j.ejmech.2012.06.042

The bioactive naphtoquinone lapachol was studied in vitro by a biomimetic model with Jacobsen catalyst (manganese(III) salen) and iodosylbenzene as oxidizing agent. Eleven oxidation derivatives were thus identified and two competitive oxidation pathways postulated. Similar to Mn(III) porphyrins, Jacobsen catalyst mainly induced the formation of para-naphtoquinone derivatives of lapachol, but also of two ortho-derivatives. The oxidation products were used to develop a GC-MS (SIM mode) method for the identification of potential phase I metabolites in vivo. Plasma analysis of Wistar rats orally administered with lapachol revealed two metabolites, α-lapachone and dehydro-α-lapachone. Hence, the biomimetic model with a manganese salen complex has evidenced its use as a valuable tool to predict and elucidate the in vivo phase I metabolism of lapachol and possibly also of other bioactive natural compounds.

Full text of DOI:10.1016/j.ejmech.2012.06.042

European Journal of Medicinal Chemistry 54 (2012) 804e812
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Biomimetic in vitro oxidation of lapachol: A model to predict and analyse
the in vivo phase I metabolism of bioactive compounds
Michael Niehues a, Valéria Priscila Barros a, Flávio da Silva Emery a, Marcelo Dias-Baruffi a,
,
Marilda das Dores Assis b, Norberto Peporine Lopes a
*
a Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Cafe S/N, 14040-903 Ribeirao Preto, SP, Brazil
b Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The bioactive naphtoquinone lapachol was studied in vitro by a biomimetic model with Jacobsen catalyst
(manganese(III) salen) and iodosylbenzene as oxidizing agent. Eleven oxidation derivatives were thus
identified and two competitive oxidation pathways postulated. Similar to Mn(III) porphyrins, Jacobsen
catalyst mainly induced the formation of para-naphtoquinone derivatives of lapachol, but also of two
ortho-derivatives. The oxidation products were used to develop a GCeMS (SIM mode) method for the
identification of potential phase I metabolites in vivo. Plasma analysis of Wistar rats orally administered
Received 19 January 2012
Received in revised form
20 June 2012
Accepted 21 June 2012
Available online 28 June 2012
with lapachol revealed two metabolites, a-lapachone and dehydro-a-lapachone. Hence, the biomimetic
Keywords:
Lapachol
model with a manganese salen complex has evidenced its use as a valuable tool to predict and elucidate
the in vivo phase I metabolism of lapachol and possibly also of other bioactive natural compounds.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Naphtoquinones
Biomimetic oxidation
Jacobsen catalyst
Drug metabolism
Phase I metabolism
1. Introduction
a manganese(III) porphyrin complex and H2O2 as oxidizing agent,
presented five oxidation products and a selectivity in the catalysis
The prediction and elucidation of drug metabolites is a relevant
step in drug development. One of the main elimination pathways
for drugs, especially non-polar compounds, in the human body
consists in the biotransformation of compounds by oxidation
reactions catalysed by enzymes of the cytochrome P450 (CYP)
superfamily, also known as phase I metabolism [1]. For that
scenario several biomimetic enzymatic and non-enzymatic models
have been described, although lastly none of these systems was
able to adequately mimic the totality of CYP-mediated reactions [2].
However, a widely used chemical model is based on synthetic
metalloporphyrins in presence of monooxygen donors, which
basically mimic the iron(III) peroxo and iron(IV) oxo intermediates
in the catalytic cycle of CYP 450 [3,4]. Several studies have reported
potential oxidative metabolites of bioactive compounds, revealing
that epoxidation, aliphatic and aromatic hydroxylation or oxidation
of the heteroatoms are the most common reactions [5e8]. With
regard to lapachol, a recent study on its biomimetic oxidation with
to generate para-naphtoquinones [9]. Other biomimetic models,
such as Fenton’s reagent or electrochemical induced oxidation were
also shown to be interesting tools for the prediction of phase I
metabolites [10,11]. In the past years metal salen complexes such as
the Jacobsen catalyst (Mn(salen)), have received increasing atten-
tion as biomimetic model compounds for the active site of CYP 450.
With respect to their electronic structure and catalytic activities,
metallosalen complexes exhibit similar features to metal-
loporphyrins, catalysing usually epoxidation reactions [12]. Until
now two species could be characterized by tandem mass spec-
trometry as the principal oxidation intermediates of the Mn(salen)
with PhIO: the oxomanganese(V) complex as the actual oxygen
transfer agent in epoxidation reactions, and the dimeric
m-oxo
bridged with two terminal iodosylbenzene ligands which acts as
reservoir species [13,14] Carbamazepine and primidone are two
clinically used pharmaceuticals, on which this system was tested
and successfully demonstrated as being a useful tool for the
prediction of the in vivo drug metabolism [7,15].
Lapachol (1) (Fig. 1), 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-
naphthoquinone, is a natural occurring naphtoquinone present in
several vegetal species, mainly in ones from the Bignoniaceae family
* Corresponding author. Tel.: þ55 16 36024168.
E-mail address: npelopes@fcfrp.usp.br (N.P. Lopes).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.042

M. Niehues et al. / European Journal of Medicinal Chemistry 54 (2012) 804e812
805
O
O
O
O
OH
O
OH
H
O
O
O
O
O
O
O
1
2*
5
3
4
O
O
O
O
O
OH
O
O
O
O
O
OH
O
O
O
O
O
6
8
9
10
7
O
O
O
O
O
O
OH
O
O
O
OH
12
11
13
Fig. 1. Chemical structures of lapachol (1) and derivatives (2e13). Compound 2 is labelled with *, since it has only been determined by mass spectrometry (EI). Compound 13 is
a synthetic derivative used to develop a GCeMS method in order to monitor putative in vivo metabolites of 1.
(Tabebuia sp.) [16]. Many biological activities are reported, among
which the cytotoxic effects against different cancer types and the
trypanocidal activity may be considered as the most prominent
ones [17e20]. Other interesting bioactivities of lapachol, such as
against malaria or antiviral and moluscicidal activity, are resumed in
the recent review by Lemos et al. [21]. Due to its anti-cancer activity
early clinical studies were performed, but drug development
stopped due to toxic side effects of the naphtoquinone within
administered active dosages and insufficient plasma concentration
levels [22]. Even so, lapachol was temporarily used in Brazil as
coadjuvant in the treatment of certain cancer types [23]. The main
biological interest of lapachol can therefore be centralized on its
cytotoxicity by mechanisms of oxidative stress induction, thereby
generating reactive oxygen species, and also of alkylation of cellular
nucleophiles [24,25]. For that reason and in order to reduce
unwanted toxicity as well as to improve activity, several synthetic
derivatives of lapachol have been reported [26e28].
Hence, the objective is to improve the proportion between lapa-
chol, Mn(salen) and iodosylbenzene (PhIO) as terminal oxygen
donor, in order to obtain high yields. Due to the reactivity of lapa-
chol, excess of PhIO showed a negative influence on product yields,
since most of the substrate was consumed and decomposed. Milder
conditions with proportions of 1:30:30 (catalyst/substrate/oxidant)
resulted in the formation of several products with acceptable yields
and showed that lapachol was consumed by approximately 90%
after 24 h.
The reaction was monitored by a GCeMS method, revealing
a series of peaks of which at least eleven (peaks 2e12) could be
assigned as oxidation products from lapachol (1) (Fig. 2). A first
analysis of the processed electron ionization (EI) mass spectra for
the respective peaks in conjunction with the NIST mass spectral
library, V.8.0 (National Institute of Standards and Technology, Gai-
thersburg, MD, USA), and Wiley Registry of Mass Spectral data, 7th
ed. (Wiley, Hoboken, NJ, USA), showed the presence of common
Even though lapachol has been intensively studied and already
passed by pre-clinical and clinical experiments, there still is no
published evidence for its metabolism in animals or humans.
Consequently, the identification of further in vitro oxidation
metabolites as well as the detailed elucidation of the in vivo
metabolism is of great importance for this naphtoquinone. In
addition, isolated oxidation derivatives enable the development of
quantitative hyphenated techniques to determine pharmacokinetic
and pharmacodynamic parameters of lapachol. Having said this,
the present study focuses on the identification, isolation of oxida-
tion derivatives by the biomimetic model with Mn(salen) and the
development of analytical methods for the elucidation of the phase
I metabolism.
cyclized pyranic derivatives in the reaction mixture, dehydro-
lapachone (7) and -lapachone (8) (Figs. 1 and 2A). Furthermore,
peak 9 showed a characteristic spectrum for the isomeric furanic
derivative dehydro- -isolapachone (9), with a base peak at m/z 197
a-
a
a
along with a prominent fragment at m/z 212 [29]. The mass spectra
of peaks numbered with 3e5 showed high probabilities (>90%) in
the spectra libraries for other previously reported compounds,
namely phtalic anhydride (3), naphtho[1,2-b]furan-4,5-dione (4)
and norlapachol (5) (Figs. 1 and 2A). The EI fragmentation (70 eV) of
9 for instance showed a low intensity ion at m/z 199 and a molec-
ular peak [M]þ at m/z 198, which is well in accordance with data
from literature and was confirmed by its exact mass experiment
(error < 20 ppm) [30]. Peak 2 is suggested to be 3,4-epoxy-4-
methylpentanal (2), as reported by comparison of the EI-MS frag-
mentation spectra (see supplementary material) to recent data
from another biomimetic study with lapachol [9]. However, the
isolation did not succeed and the determination of spectroscopic
data will be necessary to fully confirm the suggested structure of 2.
In despite of the value of EI mass spectra and related libraries,
no additional products could therewith be identified and apart
2. Results and discussion
2.1. In vitro oxidation of lapachol
The first step for the identification and isolation of oxidation
derivatives is the definition of the adequate catalysis conditions.

806
M. Niehues et al. / European Journal of Medicinal Chemistry 54 (2012) 804e812
Fig. 2. Total ion chromatograms (TIC) of the GCeMS analysis of the in vitro oxidation reaction mixtures with the substrate (A) lapachol, (B)
a-lapachone (8) and (C) dehydro-a-
lapachone (7) oxidized by iodosylbenzene and Mn(salen). Numbers above the peaks indicate the positions of lapachol (1) and respective derivatives (2e12).
from that, the confirmation of compounds by other spectroscopic
techniques was necessary. Therefore an isolation of oxidation products
by high performance liquid chromatography coupled to a diode array
detector (HPLC-DAD) was aimed, allowing better insights into other
compounds generated during catalysis. The chromatographic separa-
tion of an upscaled reaction mixture incubated for 24 h led to the
isolation of 10 compounds. After purification, reinjection with the
GCeMS method and further spectroscopic analysis four additional
oxidation products (6, 10e12) were identified and previously sug-
gested structures confirmed (Fig.1). Peak 6 in the GCeMS analysis was
shown as an epoxidized derivative of lapachol, 10,11-epoxy-lapachol
(6), which was a predictable product since the main characteristic of
Mn(salen) is the epoxidation of olefinic double bonds as present in the
prenyl moiety lapachol (Figs. 1 and 2A). Compound 10 was identified
as 2-(1-hydroxy-1-methylethyl)-2,3-dihydronaphtho[2,3-b]furan-4,9-
dione (10), a hydroxylated homologue of the previously identified
rinacanthin A. Another oxidation product, compound 12, isolated in
a 5.2% yield from the reaction mixture (Table 1), is one of the few ortho-
derivatives identified in this type of catalysis with lapachol. Compound
12,
2-(1-hydroxy-1-methylethyl)-2,3-dihydronaphtho[1,2-b]furan-
4,5-dione (12), is basically the ortho-isomer to 10 and despite their
similarity they are easily distinguishable by their EI fragmentations.
However, possibly due to hydroxylegroup interactions with the
stationary phase or small residues and its lower volatility, 12 often
exhibited less intense and strongly enlarged signals in TICs of the
GCeMS analysis (Fig. 2A). The remaining isolated products were also
confirmed by their 1H NMR spectra as reported in literature
(compounds 4, 5, 7 and 8) or by comparison to commercial available
references (3).
Other noticeable peaks in the TIC of the reaction mixture with
lapachol (Fig. 2A), like ones at 13.1 min and 14.8 min, are artifacts of
the Mn(salen) and iodosylbenzene which could equally be
dehydro-a-isolapachone (9). Although peak 11 in the GCeMS chro-
matogram is not the most intense, the isolation of the respective
oxidation product by HPLC-DAD revealed that this is the main oxida-
tion product formed during the performed catalysis conditions.
Of the initial lapachol mass, 7.1% are yielded (Table 1) as the hydrox-
ylated pyranic derivative 3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-
naphtho[2,3-b]pyran-5,10-dione (11), also commonly known as
Table 1
Yields of lapachol oxidation products (2e12) isolated by HPLC-DAD. Percentage yield
values are relative to the initial mass of lapachol as substrate in the catalysis.
Compound
Yield [%]
2
3
4
5
6
7
8
9
10
11
12
e
4.1
<1
1.7
3.3
4.2
<1
<1
4.2
7.1
5.2

M. Niehues et al. / European Journal of Medicinal Chemistry 54 (2012) 804e812
807
observed in blank GCeMS analysis without the substrate (data not
shown). Signals at 18.1 min, 18.7 min and 19.9 min (Fig. 2A) pre-
sented inconclusive mass spectra, which nevertheless indicated
them as being potentially epoxidized or hydroxylated derivatives of
lapachol and/or of respective derivatives. However, possibly due to
their reactivity and/or instability, the isolation of these compounds
did not succeed.
In summary, the compounds identified in the reaction mixture
with lapachol indicate a selectivity of Mn(salen) to catalyse para-
naphtoquinone derivatives, such as compounds 7 or 10 (Fig. 1).
Mn(III) porphyrin catalysed oxidations have been shown for such
selectivity [9], equally identifying the oxidation products 2, 3, 7 and
10 in such reactions. However, ortho-naphtoquinone and other
types of products, such as norlapachol (5), were also detected,
suggesting therefore different oxidation mechanisms. Furthermore,
Mn(salen) catalysed reactions led in comparison to Mn(III)
porphyrin complexes [9] to a higher number of generated and
identified compounds, which is advantageous for the prediction
and elucidation of the in vivo phase I metabolism.
In pathway B lapachol epoxidation occurs in the prenyl side
chain, which is followed by a tautomeric equilibrium leading to
para-quinonoid (Pathways B1 and B2) and ortho-quinonoid
metabolites (pathway B3) (Fig. 3B). All cases therefore are a conse-
quence of intramolecular nucleophilic ring opening of the epoxide.
Pathway B1 takes place when hydroxyl addition occurs in the most
hindered carbon atom of the epoxide, a pyran ring is formed,
resulting in derivative 11. This hydroxylated derivative is then
induced into a hypothetical cycle of dehydration, epoxidation and
hydrolysis, leading thereby to the derivative 7, a theoretic epoxi-
dized intermediate derivative and finally again in 11. Hydroxyl
addition to a less hindered carbon atom of the epoxide 6 (Pathway
B2), leads to the hydroxylated homologue 10 of dehydro-a-iso-
lapachone (9). Further dehydration leads to the formation of 9. The
same heterocyclization mechanism occurred in Pathway B3, leading
to the ortho-quinonoid nor-ß-lapachone type derivative 12, which
would lead by potential steps of dehydration-oxidation and loss of
a 2-propanon unit to the formation of 4. However, we were not able
to identify such precursors of 4, so that another mechanistic
pathway to 4 cannot be excluded. As known from literature [32],
2.1.1. In vitro oxidation of
Apart from lapachol itself, two derivatives, dehydro-
chone (7) and -lapachone (8), were also briefly studied by the
a
-lapachone and dehydro-
a
-lapachone
lapachol may decompose into a-lapachone in small amounts in
solution, by light influence or slightly acidic conditions, such as
during the isolation by preparative HPLC, the transformation
a-lapa-
a
biomimetic model with Mn(salen) and iodosylbenzene in order to
understand if those were precursors to oxidation products previ-
ously identified. Interestingly the GCeMS analysis of the reaction
of lapachol 1 to a-lapachone 8 is also observed during 24 h reactions.
2.3. In vivo analysis of lapachol metabolism
mixture with 8 revealed that practically none of the a-lapachone is
consumed and oxidized into other products (Fig. 2B). Conse-
quently, it can be assumed that the quinone ring double bond in
pyranic derivatives of lapachol exhibits an unreactive site for an
oxygen transfer by the active Mn(salen) complexes. In contrast, 7
presents a second double bond in the pyran ring, exposing an
alternative position for putative interactions with a reactive oxo-
manganese(V) complex. As observed in the chromatogram of the
catalysis of 7, several products are formed, of which one was also
observed during the prior described biomimetic transformation of
lapachol. The hydroxylated derivative 11 identified in both reac-
tions, exhibits the most intense signal among the compounds
monitored during the oxidation experiment with 7 (Fig. 2C). Since
7 is in this case the starting substrate we conclude that during the
biomimetic oxidation of lapachol, 7 also acts as the precursor to
the formation of 11, possibly via the formation of an epoxidized
intermediate.
A non-toxic dose of lapachol (25 mg/kg body weight) was
administered to female Wistar rats. At different time-points blood
samples were collected after animals’ treatment and plasma
samples extracted for the identification of lapachol and potential
phase I metabolites. The analysis of metabolites was performed by
developing a quantitative GCeMS (SIM mode) method with the
previously identified oxidation products (2e12), plus ß-lapachone
(13). During the analysis the respective characteristic m/z ions were
monitored (Table 2). External calibrations performed for 1, 7 and 8
presented respective detection limits at 50 ppb (0.05
mg/mL).
Repeatability and linearity was within acceptable ranges of detec-
ted concentrations. Because a residual component (fatty acid) of the
plasma matrix was interfering in analysis with the elution and
monitoring of 8 at 18.58 min (see Fig. 4D), a second temperature
gradient (see 4.6, extended SIM method) with an extended runtime
was performed in order to clearly assign the derivative 8 at
20.02 min (Fig. 4C).
2.2. Oxidation mechanisms
The analysis of plasma samples evidenced the presence of
three compounds, as observed in the respective overlay TIC’s for
the monitored m/z ions of 1, 7 and 8 (Fig. 4AeC). After 1.5 h a mean
Asalreadymentionedintheintroduction, metallosalencomplexes
suchasMn(salen), havetheabilitytocatalyseepoxidationreactionsin
presence of monooxygen donors such as iodosylbenzene. Two
oxidationsitesoflapacholarethereforeconsidered possibletoreceive
an oxygen by transfer of the oxomangenese(V) complex: the double
bond inthequinonering and thedoublebond intheprenyl sidechain.
Based on compounds (2e12) identified in the reaction mixture, two
competitive oxidation mechanisms were postulated (Fig. 3A and B).
Pathway A initiates with the oxygen transfer to the double bond
in the quinone ring of lapachol giving rise to the intermediate 14,
basically in accordance to the first step of the Hooker oxidation [31]
(Fig. 3A). In the process of the new ring formation the carbon atom
of the original ring attached to the hydroxyl is eliminated as carbon
dioxide. A carbon atom of the side chain takes its place and by
rearrangement 5 is formed. In accordance to the mechanism
postulated by Pires et al. [9], the epoxidation of the prenyl moiety in
14 leads in two steps to the formation of 3 and in one step to
compound 2, which by further oxidation generates a lactone
derivative.
plasma concentration for lapachol (1) of 37
which successively diminished until a plasma concentration of
g/mL after 12 h (Fig. 5). The derivatives dehydro- -lapachone
(7) and -lapachone (8) were also detected, but with clearly lower
intensities in TIC’s (Fig. 4B and C). Both were detected after 1.5 h
with plasma concentrations of 0.6 g/mL and 1.4 g/mL, respec-
tively. While 7 was not more detectable after 6 h, compound 8
showed its concentration maximum at 3 h (6.0 g/mL), which
mg/mL was observed,
2
m
a
a
m
m
m
after 12 h decreased to a plasma concentration of 0.7
(Fig. 5).
mg/mL
None of the remaining monitored derivatives were detected in
the rat plasma samples. In search of other putative metabolites
samples were also analysed by GCeMS in Scan mode. However, no
other metabolite was identified, either because there simply was no
additional derivative of lapachol (1) present in samples or because
the sensitivity of the GCeMS system in SCAN mode did not allow
detecting those (data not shown). However, the results determined
that the majority of administered lapachol remains unmetabolized

808
M. Niehues et al. / European Journal of Medicinal Chemistry 54 (2012) 804e812
A
O
O
O
O
OH
OH
OH
COOH
OH
[O]
COOH
OH
OH
OH
O
O
O
14
1
[O]
H
CO2
O
OH
O
O
O
CO2
OH
H
OH
O
H
O
O
O
OH
O
O
O
2
5
3
B
O
O
O
O
O
O
[O]
O
O
O
7
-H2O
O
O
O
8
B1
OH
..
O
H
O
H+
11
OH
O
H
O
O
O
O
[O]
[O]
B2
O
CH2
..
O
O
O
O
-H2O
OH
OH
6
O
10
9
1
O
O
O
B3
O
O
O
?
OH
O
O
OH
4
12
Fig. 3. Hypothetical mechanisms for the in vitro oxidation of lapachol (1) and respective derivatives (2e12). Pathway A shows the mechanism starting with the oxidation of the
double bond in the quinone ring of (1). Pathway B shows the mechanism starting with the oxidation of the double bond in the side chain of (1).
after oral administration in rats. In addition, it seems that most of
lapachol circulating in the blood stream is eliminated or in another
way processed after 12 h, since it is barely detectable in plasma 12 h
after administration (Fig. 5). According to the mass spectrometric
data, orally administered lapachol is in rats partially converted into
the metabolites 7 and 8. However, lapachol could easily be trans-
formed by cyclization in the acidic environment of the gastric
product of lapachol and may therefore also be considered as an
in vivo oxidation metabolite, probably generated by CYP 450
enzymes mediated catalysis of lapachol. Even though the detected
concentration is comparatively low, both compounds should be
regarded with interest since different biological activities have
been reported for them. a-Lapachone (8) has for instance shown to
inhibit cellular DNA topoisomerase II by a novel mechanism [33]
and also demonstrated interesting trypanocidal activity [20].
passage to
human stomach may induce such a reaction [32], so that 8 would
not be a genuine phase I metabolite. Dehydro- -lapachone (7) on
a-lapachone (8). Hydrochloric acid existent in the
Dehydro-a-lapachone was, for instance, recently reported to
exhibit antivascular activity in orthotopic mammary tumors in
mice by targeting the cell adhesion [34].
a
the other hand, has been shown in this study as a genuine oxidation
Table 2
Compounds and respective ions monitored in the GCeMS (SIM mode) analysis of plasma samples.
Compound
1
2
3
4
5
6
7
8
9
10
11
12
14
m/z 1
m/z 2
m/z 3
m/z 4
199
227
242
e
59
97
115
76
114
142
170
198
77
197
212
225
240
197
225
240
e
159
181
227
242
197
212
225
240
144
172
200
225
159
188
243
258
159
197
230
258
159
214
227
242
104
148
e
172
213
228
e

M. Niehues et al. / European Journal of Medicinal Chemistry 54 (2012) 804e812
809
Fig. 4. Overlayed TICs from the GCeMS (SIM mode) plasma analyses for the monitored compounds: (A) lapachol (1), (B) dehydro-
a
-lapachone (7) and (C)
a-lapachone (8). D
presents in summary the TIC’s of all signals (see Table 2) monitored in the analysis by GCeMS in SIM mode (total runtime was 37 min).
2.4. Experimental data
2.4.2. Naphtho[1,2-b]furan-4,5-dione 4 (C12H6O3)
1H NMR (lit. [30]) (CDCl3, 500 MHz)
(ppm): 6.83 (d, 1H,
d
2.4.1. Phtalic anhydride 3 (C8H4O3)
J ¼ 2.1 Hz, eCH]) 7.45 (m,1H, AreH) 7.50 (d,1H, J ¼ 2.1 Hz, eCH]),
7.67 (m, 1H, AreH), 7.72 (m, 1H, AreH), 8.08 (m, 1H, AreH). HRESI-
MS m/z 199.0437 [M þ H]þ. MS (EI) m/z (rel. int., %): 199 (14), 198
(100), 170 (5), 142 (33), 114 (73), 113 (32), 88 (16), 76 (23), 63 (15).
1H NMR (CDCl3, 500 MHz)
d (ppm): 2.17 (s, 1H), 7.97 (s, 3H, 1
CH3), 7.85 (dd, 2H, J ¼ 7.8 Hz and, J ¼ 17.4 Hz, AreH). HRESI-MS m/z
149.0233 [M þ H]þ. MS (EI) m/z (rel. int., %): 148 (17), 105 (8), 104
(100), 76 (91), 74 (20), 50 (50).
2.4.3. Norlapachol 5 (C14H12O3)
1H-NMR (lit. [38] (CDCl3, 300 MHz))
d (ppm): 1.68 (s, 3H, 1 CH3),
40.0
1.99 (s, 3H, 1 CH3), 2.17 (s, 1H), 6.00 (s, 1H), 7.69 (m, 1H, AreH),
7.76(m, 1H, AreH), 8.12 (dd, 2H, J ¼ 7.8 Hz and, J ¼ 17.4 Hz,
AreH). HRESI-MS m/z 229.0878 [M þ H]þ. MS (EI) m/z (rel. int., %):
228 (77), 213 (100), 200 (9), 185 (21), 172 (16),157 (32),129 (32), 115
(15), 77 (29).
lapachol (1)
dehydro-α-lapachone (7)
α-lapachone (8)
35.0
30.0
25.0
20.0
15.0
10.0
5.0
2.4.4. 10,11-Epoxy-lapachol 6 (C15H14O4)
1H NMR (lit. [39]) (CDCl3, 400 MHz)
d (ppm): 1.23 (s, 3H, 1 CH3),
1.38 (s, 3H, 1 CH3), 1.72 (dd, 1H, J ¼ 4.8 Hz and J ¼ 5.8 Hz, eCH2e),
2.14 (s,1H, J ¼ 4.8 Hz and J ¼ 5.8 Hz, eCH2e), 2.29 (dd,1H, J ¼ 5.8 Hz
and J ¼ 8.3 Hz, -CH-O), 7.82 (m, 2H, AreH), 8.17 (m, 1H, AreH), 8.23
(m, 1H, AreH). HRESI-MS m/z 259.0941 [M þ H]þ. MS (EI) m/z (rel.
int., %): 258 (2), 240 (76), 225 (24), 212 (57), 197 (100), 172 (25), 149
(32), 133 (38), 115 (40), 105 (60), 77 (57).
0.0
0
5
10
15
time [h]
2.4.5. Dehydro-
a-lapachone 7 (C15H12O3)
1H NMR (lit. [40]) (CDCl3, 500 MHz)
d
(ppm): 1.64 (s, 6H, 2 CH3),
Fig. 5. Time-dependent profiles of plasma concentrations for lapachol (1), dehydro-
lapachone (7), and -lapachone (8). Quantitative analysis performed by GCeMS (SIM
mode). Values are relative to the respective vehicle controls and given as mean (n ¼ 3).
a-
5.81 (d, 1H, J ¼ 10.0 Hz, eCH]), 6.74 (d, 1H, J ¼ 10.0 Hz, eCH]),
a
7.74e7.81 (m, 2H, ArH), 8.17(d, 2H, J ¼ 7.6 Hz, ArH). HRESI-MS m/z

810
M. Niehues et al. / European Journal of Medicinal Chemistry 54 (2012) 804e812
241.0859 [M þ H]þ. MS (EI) m/z (rel. int., %): 240 (11), 225 (100), 212
presented with an oxomanganese(V) complex as oxygen transfer
agent, are an important tool to mimic certain reactions of CYP 450
and therefore also for the prediction and elucidation of the in vivo
phase I metabolism. The outstanding advantage of such models is
the possibility to generate and isolate oxidation derivatives in
higher yields at an acceptable cost, such as the ones shown in this
study, and by that means have valuable compounds for further
toxicological studies as well as for the development and validation
of methods for the analysis of in vivo metabolites. As reported in
previous articles [7,15], this model is also applicable to different
classes of bioactive compounds. Furthermore, these systems can
help minimizing the use of large animal quantities for in vivo
metabolism experiments. Although we were able to determine first
insights into the oxidative metabolism of lapachol, future and more
detailed in vivo studies are necessary to fully evidence the metab-
olism and pharmacokinetic parameters of lapachol and its
metabolites.
(5), 197 (30), 133 (8), 115 (14), 105 (16), 76 (18).
2.4.6.
a-Lapachone 8 (C15H14O3)
1H NMR (lit. [38]) (CDCl3, 500 MHz)
d
(ppm): 1.37 (s, 6H, 2 CH3),
1.75 (t, 1H, J ¼ 6.6 Hz, eCH2e), 2.55 (t, 2H, J ¼ 6.6 Hz, eCH2e), 7.61
(t, 2H, J ¼ 7.7 Hz, ArH), 8.01 (m, 2H, ArH). HRESI-MS m/z 243.1015
[M þ H]þ. MS (EI) m/z (rel. int., %): 242 (36), 227 (100), 214 (11), 199
(14), 181 (14), 159 (42), 115 (8), 102 (21).
2.4.7. Dehydro-iso-
a
-lapachone 9 (C15H14O3)
(ppm): 1.77 (s, 3H, CH3),
1H NMR (lit. [40]) (CDCl3, 400 MHz)
d
3.00 (dd, 1H, J ¼ 9.5 and Hz, J ¼ 17.6 Hz, eCH2e), 3.35 (dd, 1H,
J ¼ 11.8 Hz and J ¼ 17.6 Hz, eCH2e) 4.98 (s, 1H, J ¼ 9.5 Hz,]CH2),
5.11 (s, 1H, ]CH2), 5.36 (s, 1H, J ¼ 9.5 and J ¼ 11.9 Hz, eCH2e),
7.72(m, 2H, AreH), 8.08 (m, 2H, AreH). HRESI-MS m/z 241.0849
[M þ Hþ]. MS (EI) m/z (rel. int., %): 240 (24), 225 (46), 212 (98), 197
(100), 183 (19), 169 (22), 141 (32), 133 (29), 115 (36), 105 (58), 76
(81).
4. Experimental protocols
2.4.8. 2-(1-Hydroxy-1-methylethyl)-2,3-dihydronaphtho[2,3-b]
4.1. Chemicals and materials
furan-4,9-dione 10 (C15H14O4)
1H NMR (lit. [38]) (CDCl3, 500 MHz)
d
(ppm): 1.34 (s, 3H, CH3),
The Jacobsen catalyst (Mn(salen)), (S,S)-(þ)-N,N0-Bis(3,5-di-tert-
butylsalicylidene)-1,2-cyclohexanediaminomanganese(III) chloride
was purchased from Sigma Aldrich, as well as iodosylbenzene
diacetate which was used to generate the oxidizing agent iodo-
sylbenzene (PhIO). The synthesis of PhIO was performed according
to [35], reaching a purity of 85e90% determined by iodometric
titration. Solvents used for in vitro, in vivo experiments and for the
isolation of compounds were of chromatographic grade (Malinck-
rodt Baker, MXN). Lapachol (1) and ß-lapachone (13) used for
1.49 (s, 3H, CH3), 3.25 (d, 2H, J ¼ 10.0 Hz, eCH2e) 4.93 (t, 1H,
J ¼ 10.0 Hz, eOeCHe) 7.74e7.83 (m, 2H, AreH), 8.16 (t, 2H,
J ¼ 6.9 Hz, AreH). HRESI-MS m/z 259.0965 [M þ H]þ. MS (EI) m/z
(rel. int., %): 260 (2), 243 (5), 225 (6), 200 (100), 172 (63), 144 (33),
115 (41), 105 (14), 76 (20), 59 (74).
2.4.9. 3,4-Dihydro-3-hydroxy-2,2-dimethyl-2H-naphtho[2,3-b]
pyran-5,10-dione 11 (C15H14O4)
1H NMR (lit. [40]) (CDCl3, 500 MHz)
d
(ppm): 1.44 (s, 3H, CH3),
experiments were synthesized and purified according to [36,37].
a-
1.49 (s, 3H, CH3), 2.70 (dd, 1H, J ¼ 5.1 Hz and 18.8 Hz, eCH2e), 2.89
(dd, 1H, J ¼ 5.1 Hz and 18.8 Hz, eCH2e), 3.90 (t, 1H, J ¼ 5.3 Hz,
eOeCHe) 7.66-7.75 (m, 2H, AreH), 8.11 (dd, 2H, J ¼ 7.5 Hz and
J ¼ 13.0 Hz, AreH). HRESI-MS m/z 259.0965 [M þ H]þ. MS (EI) m/z
(rel. int., %): 258 (100), 243 (17), 230 (5), 217 (6), 200 (10), 188 (24),
172 (18), 159 (25), 115 (27), 105 (19), 72 (57).
Lapachone (8) and dehydro- -lapachone (7) used for biomimetic
oxidation reactions were isolated from the correspondent biomi-
metic catalysis of lapachol.
a
4.2. Biomimetic oxidation with iodosylbenzene and Mn(salen)
In a typical experiment, reactions were carried out in 5 mL vials
containing screw caps and under exclusion of light. Mn(salen)
2.4.10. 2-(1-Hydroxy-1-methylethyl)-2,3-dihydronaphtho[1,2-b]
furan-4,5-dione 12 (C15H14O4)
(0.5
lapachone) (15
volume of 1.5 mL. Thereafter the oxidizing agent PhIO (15
was added and suspended in acetonitrile. Reaction mixtures were
stirred under light exclusion, at constant room temperature over
m
mol) and substrate (lapachol,
mol) were dissolved in acetonitrile to a total
mol)
a-lapachone and dehydro-a-
1H NMR (lit. [38]) (CDCl3, 500 MHz)
d
(ppm): 1.27 (s, 3H, CH3),
m
1.39 (s, 3H, CH3), 3.06 (d, 2H, J ¼ 9.5 Hz, eCH2e) 4.93 (t, 1H,
J ¼ 9.0 Hz, eOeCHe) 7.64e7.69 (m, 1H, AreH), 7.73-7.79 (m, 2H,
J ¼ 6.9 Hz, AreH), 8.11 (d, 2H, J ¼ 7.5 Hz). HRESI-MS m/z 259.0965
[M þ H]þ. MS (EI) m/z (rel. int., %): 260 (8), 258 (22), 240 (4), 230
(29), 212 (14), 197 (23), 172 (26), 160 (48), 159 (90), 144 (15), 115
(41), 105 (30), 72 (87).
m
a period of 24 h. After filtration by a 0.45 mm pore size membrane
filter, an aliquot was taken for further chromatographic analysis
and reaction mixtures then dried under nitrogen flow for storage
at ꢀ20 ꢁC. The isolation of potential oxidation products was per-
formed with upscaled reactions including up to 150 mg lapachol
and applying the previously described proportions.
3. Conclusion
The Mn(salen) catalysed oxidation of lapachol resulted in the
formation of at least eleven oxidation products, which may be
explained bytwo distinct oxidation mechanisms: one starting with
the epoxidation of double bond in the quinone ring and the other
initiated by oxidation of prenyl side chain of lapachol. The eleven
in vitro derivatives were used to monitor by GCeMS (SIM mode)
metabolites in rat plasma samples. Animals which were orally
administered with a non-toxic dosage of lapachol exhibited two
4.3. Oxidation product analysis, isolation and structure elucidation
Generated oxidation products were monitored and analysed by
GCeMS (GC-2010, GCMS-QP2010, Shimadzu). A RtxÒ-5 MS column
(30 ꢂ 0.25 mm, 0.25
mm film) (Restek, USA) was used, applying
a temperature gradient, 120 ꢁC (4min) / 8 ꢁC/min / 300 ꢁC
(12 min), with a total runtime of 37 min. Split injection (1:20) was
performed and electron ionization (EI) mass spectra acquired
at 70eV.
metabolites in plasma,
(7). Whereas -lapachone is probably already formed during the
acidic gastric passage and thereupon absorbed, dehydro- -lapa-
a-lapachone (8) and dehydro-a-lapachone
a
a
The isolation of oxidation derivatives from lapachol (1) was
achieved by a preparative HPLC-DAD method. An RP-C18 column
(250 mm ꢂ 21.20 mm, 100A, Luna 5 Micron, C18(2), Phenomenex,
USA) was employed and chromatographic conditions set for
chone is a genuine oxidation metabolite presumably originated by
CYP 450 enzymes mediated catalysis. In conclusion, these results
show that biomimetic oxidation models, such as the one here

M. Niehues et al. / European Journal of Medicinal Chemistry 54 (2012) 804e812
811
a 100 min runtime. An appropriate separation of products was
achieved by an elution gradient with acetonitrile (A) and purified
water (B), both acidified with 1% glacial acetic acid. The elution
gradient was as followed: 0.01 min (15% A) / 20 min (30% A) /
30 min (45% A) / 70 (55% A) / 90 min (80%) /100 min (100% A).
Isolated compounds were analysed by high resolution ESI-MS
(ultrOTOFQ, Bruker Daltonics) and 1H NMR (300 MHz, 400 MHz
and 500 MHz, Bruker Daltonics) in order to elucidate and confirm
structures.
120 ꢁC (4min) / 8 ꢁC/min / 210 ꢁC (12 min) / 8 ꢁC/min /
300 ꢁC (12 min), with a total runtime of 50 min. Splitless injection
of 1 mL and other parameters were as described before.
Acknowledgements
The authors thank FAPESP, CNPq, INCT_if (CNPq process nꢁ
573663/2008-4) and CAPES (Brazil) for financial support and
scholarship.
4.4. Lapachol in vivo metabolism
Appendix A. Supplementary material
Experiments were carried out on female Wistar rats (Rattus
norvegicus), weighing approximately 180 g. The animals were
acquired from the animal facilities of the Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil,
and kept in polyethylene boxes (n ¼ 3), in a climate-controlled
environment with a 12-h, light/dark cycle with free access to
water and food. All experiments were conducted in accordance
with the guidelines set by the Animal Care Committee (CEUA) of
the Universidade de São Paulo, Brazil (Campus of Ribeirão Preto).
The seventeen rats were divided into 6 groups. Lapachol, dis-
solved in an ethanolic solution (ethanol/water, 1:1) was adminis-
tered to the rats by oral gavage. In clinical studies, orally
administered lapachol was shown to be toxic at doses greater than
2.0 g/day (28.6 mg/kg for a patient of 70-kg body weight) [22].
Therefore, a single administration dose of 0.5 mL with a non-toxic
lapachol dose of 25 mg/kg body weight was given and at selected
times blood collected by cardiac punction into heparinized tubes.
For the treated animals blood was collected after 1.5 h, 3 h, 6 h and
12 h (4 groups, each time point n ¼ 3). Animals of the vehicle
control group (n ¼ 4) were treated with 0.5 mL of the ethanolic
solution and for every time period blood of one animal collected. A
single Wistar rat was taken for the blank plasma control.
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2012.06.042.
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