Antimalarial naphthoquinones. Synthesis via click chemistry, in vitro activity, docking to PfDHODH and SAR of lapachol-based compounds

Article abstract of DOI:10.1016/j.ejmech.2017.12.051

Lapachol is an abundant prenyl naphthoquinone occurring in Brazilian Bignoniaceae that was clinically used, in former times, as an antimalarial drug, despite its moderate effect. Aiming to search for potentially better antimalarials, a series of 1,2,3-triazole derivatives was synthesized by chemical modification of lapachol. Alkylation of the hydroxyl group gave its propargyl ether which, via copper-catalyzed cycloaddition (CuAAC) click chemistry with different organic azides, afforded 17 naphthoquinonolyl triazole derivatives. All the synthetic compounds were evaluated for their in vitro activity against chloroquine resistant Plasmodium falciparum (W2) and for cytotoxicity to HepG2 cells. Compounds containing the naphthoquinolyl triazole moieties showed higher antimalarial activity than lapachol (IC₅₀ 123.5 μM) and selectivity index (SI) values in the range of 4.5–197.7. Molecular docking simulations of lapachol, atovaquone and all the newly synthesized compounds were carried out for interactions with PfDHODH, a mitochondrial enzyme of the parasite respiratory chain that is essential for de novo pyrimidine biosynthesis. Docking of the naphthoquinonolyl triazole derivatives to PfDHODH yielded scores between ?9.375 and ?14.55 units, compared to ?9.137 for lapachol and ?12.95 for atovaquone and disclosed the derivative 17 as a lead compound. Therefore, the study results show the enhancement of DHODH binding affinity correlated with improvement of SI values and in vitro activities of the lapachol derivatives.

Full text of DOI:10.1016/j.ejmech.2017.12.051

Accepted Manuscript
Antimalarial naphthoquinones. Synthesis via click chemistry, in vitro activity, docking
to PfDHODH and SAR of lapachol-based compounds
Geraldo Célio Brandão, Franciele C. Rocha Missias, Lucas Miquéias Arantes,
Luciana Ferreira Soares, Kuldeep K. Roy, Robert J. Doerksen, Alaíde Braga de
Oliveira, Guilherme Rocha Pereira
PII:
S0223-5234(17)31067-X
10.1016/j.ejmech.2017.12.051
EJMECH 10029
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 September 2017
Revised Date: 11 December 2017
Accepted Date: 13 December 2017
Please cite this article as: Geraldo.Cé. Brandão, F.C. Rocha Missias, Lucas.Miqué. Arantes, L.F.
Soares, K.K. Roy, R.J. Doerksen, Alaí. Braga de Oliveira, G.R. Pereira, Antimalarial naphthoquinones.
Synthesis via click chemistry, in vitro activity, docking to PfDHODH and SAR of lapachol-based
compounds, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.051.
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ACCEPTED MANUSCRIPT
Antimalarial naphthoquinones. Synthesis via click
chemistry, in vitro activity, docking to PfDHODH
and SAR of lapachol-based compounds
a
b
c
Geraldo Célio Brandão , Franciele C. Rocha Missias , Lucas Miquéias Arantes , Luciana
c
d,e
d
c
Ferreira Soares , Kuldeep K. Roy , Robert J. Doerksen , Alaíde Braga de Oliveira , Guilherme
b,c
Rocha Pereira, *
a
Departamento de Farmácia, Escola de Farmácia, UFOP, Campus Morro do Cruzeiro, s/n,
Balxita, CEP 35400-000, Ouro Preto, MG, Brazil
1

ACCEPTED MANUSCRIPT
b
Pontifícia Universidade Católica de Minas Gerais, PUC Minas, Departamento de Física e
Química Instituto de Ciências Exatas e Informática ICEI. Av. Dom José Gaspar, 500 Prédio 34
Coração Eucarístico - CEP 30535.901, Belo Horizonte, MG Brazil
c
Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, UFMG, Av. Antônio Carlos,
6
627, Campus Pampulha, CEP 31270-901, Belo Horizonte, MG, Brazil.
d
Department of BioMolecular Sciences and Research Institute of Pharmaceutical Sciences,
School of Pharmacy, University of Mississippi, University, MS, USA.
e
National Institute of Pharmaceutical Education and Research, 4, Raja S. C. Mullick Road,
Jadavpur, Kolkata 700 032, WB, India.
KEYWORDS malaria, atovaquone, Plasmodium falciparum, copper-catalyzed cycloaddition,
triazole, chloroquine, dihydroorotate dehydrogenase (DHODH).
ABSTRACT
Lapachol is an abundant prenyl naphthoquinone occurring in Brazilian Bignoniaceae that was
clinically used, in former times, as an antimalarial drug, despite its moderate effect. Aiming to
search for potentially better antimalarials, a series of 1,2,3-triazole derivatives was synthesized
by chemical modification of lapachol. Alkylation of the hydroxyl group gave its propargyl ether
which, via copper-catalyzed cycloaddition (CuAAC) click chemistry with different organic
azides, afforded 17 naphthoquinonolyl triazole derivatives. All the synthetic compounds were
evaluated for their in vitro activity against chloroquine resistant Plasmodium falciparum (W2)
and for cytotoxicity to HepG2 cells. Compounds containing the naphthoquinolyl triazole
2

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moieties showed higher antimalarial activity than lapachol (IC50 123.5 ꢀM) and selectivity index
(SI) values in the range of 4.5 to 197.7. Molecular docking simulations of lapachol, atovaquone
and all the newly synthesized compounds were carried out for interactions with pf DHODH, a
mitochondrial enzyme of the parasite respiratory chain that is essential for de novo pyrimidine
biosynthesis. Docking of the naphthoquinonolyl triazole derivatives to pfDHODH yielded scores
between -9.14 and -14.55 units, compared to -9.137 for lapachol and -12.95 for atovaquone and
disclosed the derivative 17 as a lead compound. Therefore, the study results show the
enhancement of DHODH binding affinity correlated with improvement of SI values and in vitro
activities of the lapachol derivatives.
1
. Introduction
Malaria remains a serious parasitic disease in tropical areas due to its high morbidity and
mortality rates. It is estimated that 212 million cases occurred globally in 2015, leading to 429,
0
00 deaths, most of which were in children aged under 5 years in Africa [1, 2]. The protozoans
Plasmodium falciparum and P. vivax are the main parasites responsible for this disease; the
former, the most virulent, is responsible for 99% of the deaths. Resistance to antimalarial drugs
has been observed as a consequence of genetic mutations and is a serious setback to antimalarial
programs, since it limits the use of effective drugs like chloroquine [3] and artemisinin [4].
Therefore, discovery of new categories of antimalarial lead compounds is an important priority
[5].
3

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Figure 1. Chemical structures of lapachol (1), atovaquone (2) and ubiquinone (3).
Quinones represent a large and important class of carbonyl-conjugated cyclic compounds with a
great number of therapeutic applications. Naturally-occurring naphthoquinones are commonly
found throughout different plant families, fungi and certain animals [6]. In this project, we
started with lapachol (1) because it is a well-known antimalarial compound. Lapachol is a
hydroxy-prenyl naphthoquinone occurring in woody trees of South American Bignoniaceae [5].
Lapachol has been studied as an interacting molecule with various protein targets such as
plasmodial HSP70, [7] though in many studies its detailed mechanism of action is unknown
and/or unreported [8]. It has been shown to interact with DHODH, but much more strongly with
human DHODH (1 nM) than with PfDHODH (16 ꢀM) [9]. Other modified lapachols, such as 3-
(3-methylbut-1-en-1-yl)-1,4-dioxo-1,4-dihydronaphthalen-2-yl acetate, have shown better
antimalarial activity than lapachol itself, [10-12] showing the potential to improve upon lapachol
in the search for new antimalarials.
Atovaquone (2) is a synthetic hydroxy-naphthoquinone, structurally related to lapachol, which is
very active against P. falciparum in both erythrocytic and liver stages of the protozoa life cycle
and was introduced in malaria therapeutics in 2000. However, resistance was observed and it is
presently used in association with other antimalarial drugs [13, 14]. Atovaquone is also active
against other protozoa parasites including Toxoplasma and Pneumocystis. It has been shown to
4

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be an inhibitor of coenzyme Q and ubiquinone (3), which play important roles in the parasitic
respiratory chain, an effect that has been related to their structural similarities. Atovaquone
blocks the mitochondrial electron transport chain, specifically targeting the cytochrome bc1
complex in the Plasmodium respirational system and further interfering in many processes
including protein synthesis and heme biogenesis that are important for its survival [15, 16]. A
similar mechanism of action could be produced by lapachol and analogues, since they also have
a naphthoquinone core [17].
Recent studies suggest a “cross-relation” between the respiratory chain and pyrimidine
biosynthesis [15]. Following the availability of the complete parasite genome, it was realized that
P. falciparum is dependent on de novo pyrimidine biosynthesis [18]. Thus, the parasite is also
susceptible to the inhibition of dihydroorotate dehydrogenase (DHODH), a flavin-dependent
mitochondrial enzyme that catalyzes the fourth and rate-limiting step in the de novo pyrimidine
biosynthetic pathway [19, 20]. Meanwhile, as demonstrated by reported X-ray co-crystal
structures of human and plasmodial DHODHs bound to selective inhibitors, significant
differences exist between the DHODHs from different species, in the whole sequence as well as
among the amino acids forming the inhibitor binding site [18, 21, 22].
Therefore, P. falciparum DHODH (PfDHODH) represents a target for the discovery and
development of parasite-specific inhibitors that could lead to promising antimalarial lead
compounds [18, 23]. Different chemical scaffolds have been identified as potent inhibitors of
PfDHODH that show strong selectivity for the plasmodial DHODH over that of the human host
[23, 24]. A number of co-crystal structures of the human and PfDHODH with bound inhibitors
are available in the RCSB Protein Data Bank (www.rcsb.org), and these have provided structural
insights into key protein-ligand interactions governing the selective inhibition of PfDHODH [18,
5

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2
1, 22]. Several computational studies have been done using various approaches with a goal of
finding optimized PfDHODH inhibitors [25, 26]. In this work, we applied molecular docking to
explore binding modes as well as to provide important information about the quantitative
structure-activity relationship (QSAR) for the new compounds’ interaction with this enzyme.
Additionally, the present results might support the proposal of more potent PfDHODH inhibitors
[20, 27] based on the lapachol pharmacophore.
Scheme 1. Naphthoquinonolyl-1,2,3-triazole hybrids from three different routes via click
reactions.
Recently, naphthoquinone derivatives have shown promising results as antiparasitic lead
compounds [28]. Conjugated hybrid compounds could be an effective path to discovery of new
drugs by associating two different pharmacophore groups with different mechanisms of action in
a single molecule, [29, 30] which could yield a drug that kills the plasmodial parasites in two
different ways. The chloroquine pharmacophoric group, responsible to inhibit the polymerization
6

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of the harmful heme group into the hemozoin non toxic pigment, was also planned to be
introduced in a final hybrid compound to have two possible mechanisms of action, as shown in
Scheme 1 [29]. A synthetic approach to produce a hybrid molecule is via click chemistry,
introduced by Sharpless, and currently widely used, [31] including for natural product activity
enhancement [32-39]. Hybrid molecules can be easily produced by combining a terminal alkyne
of a naphthoquinone intermediate (6) with different organic azides. Our strategy was to modify a
known active natural product lapachol (1), first by alkylating at the hydroxyl position. This ether
naphthoquinolyl intermediate was then reacted with different organic azides supporting
sulfonamide or 7-chloroquinoline moieties besides miscellaneous aryl groups using a copper
cycloaddition click reaction and to evaluate the antiplasmodial activity of the new compounds.
2
. Results and discussion
Lapachol is an alkyl naphthoquinone natural product occurring in Handronthus species (Syn.
Tabebuia) disclosing a large spectrum of biological activities. During the Second World War,
lapachol analogues including hydrolapachol and lapidone were studied in a search for alternative
antimalarial compounds. Later, a lapachol modified structure led to development of the
antimalarial atovaquone, a synthetic chloride naphthoquinone [40]. Structural modification of
lapachol, which is abundant in Brazilian Handroanthus spp. (Bignoniaceae), was the strategy
aimed to prepare potentially more potent antimalarial naphthoquinones. The possibility of
linking different moieties to the lapachol hydroxyl group was the inspiration for the present
work, once its easy alkylation by a Williamson reaction would afford the alkynyl moiety
necessary for the click reactions to be carried out. From the propargyl ether 2, a total of 17 new
naphthoquinonolyl-1,2,3-triazole hybrid compounds were synthesized and spectroscopically
characterized.
7

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Natural product lapachol (1) was isolated by basic/acid work up and further purified by silica
column chromatography and the synthesized compounds were evaluated for their in vitro
antimalarial activity against P. falciparum W2 that is a chloroquine resistant and mefloquine-
sensitive strain. Table 1 shows the yields of the click reactions, the IC values for the in vitro
5
0
antimalarial assay (pLDH method), the CC values for the cytotoxicity to HepG2 cells (MTT
5
0
method) and the SI for each compound. In general, low cytotoxicity (CC > 100 µM) was
50
observed except for the hybrid lapachol-AZT derivative (18) (CC50 < 100 µM). All the
compounds disclosed IC50 < 100 µM and were thus more active than lapachol (IC50 = 123.5 ±
1
1.5 µM), except possibly for 21, for which the exact IC50 was not determined (IC50 > 104.6
µM). Eleven compounds showed IC < 10 µM, with the most active being 13, 14 and 25 with
5
0
IC of 4.0 ± 1.6, 4.6 ± 1.9, and 4.1 ± 1.0 µM, respectively. The most promising compounds are
5
0
1
7 and 25 that disclosed the highest SI values (197.7 and 171.0, respectively). Compound 7 also
has a favorable SI (128.8). Its effect might be because of a second mechanism of action, that is,
the inhibition of heme polymerization, similarly to chloroquine, due to the presence of a
chloroquinolyl pharmacophore group.
Table 1. Yields of click reactions affording lapachol derivatives 1, 7, 11, 13-28, in vitro
antimalarial activity (IC µM) against P. falciparum (W2 strain), cytotoxicity (CC µM, HepG2
5
0
50
cells) and selectivity index (SI).
a
b
c
R
IC50 (µM)
CC50 (µM)
SI
R
IC50 (µM)
CC50 (µM)
SI
8

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1
7
lapachol
123.5 ± 11.5
15.1 ± 3.0
> 4130.7
> 33.4
128.8
20
21
8.4 ± 4.1
264.6 ± 40.3
31.5
1944.9 ± 55.3
>
104.6
482.8 ± 42.7
1154.0± 37.8
< 4.6
117.7
1
1
3
112.7
34.0
22
23
9.8 ± 3.2
8.4 ± 1.3
8
.2 ± 0.5
924.2 ± 65.2
136.0 ± 23.8
1
1
4.0 ± 1.6
707.2 ± 30.1
539.8 ± 106.6
84.2
46.1
4
4.6 ± 1.9
7.8 ± 1.1
570.3 ± 62.6
413.5 ± 37.9
657.5 ± 29.2
124.0
24
11.7 ± 0.6
4.1 ± 1.0
1
1
5
6
25
26
701.1 ± 55.9
531.5 ± 79.9
171.0
64.8
7
5.6
8
.2 ± 5.5
75.6
8
5
.7 ± 3.4
.2 ± 1.8
1
1
7
8
197.7
4.5
27
28
14.2 ± 1.4
18.8 ± 3.3
200.1 ± 29.3
256.0 ± 33.1
14.1
13.6
1028.1± 29.2
60.7 ± 5.3
1
3.6 ± 3.4
1
9
19.1 ± 1.0
100.3±
5.2
2
5.1
d
-3
4
Chloroquine
0.4± 0.03
393.0 ± 27.2
982.5
2
Atovaquone
1.22 x 10
±
> 40.0
> 327.9
0
.0009
a
IC50: concentration that inhibits 50% of the parasite growth in relation to control cultures with
no drugs.
b
CC : concentration that kills 50% of HepG2 cells, 24 h after incubation with the compounds
50
determined by the MTT method.
9

ACCEPTED MANUSCRIPT
c
SI: Selectivity Index = CC50/ IC50
Molecular modeling can provide important information about the interaction of the obtained
compounds and potential parasite targets, such as DHODH. The inhibitor-binding site of
PfDHODH, located in proximity to the cofactor-binding site, is characterized by the presence of
two regions: the H-bond pocket, comprising H185, Y528 and R265, and the hydrophobic pocket.
It has been crystallographically proven that the 1,4-naphthoquinone substructure of atovaquone
binds into the H-bond pocket, while its large hydrophobic substituent resides in the hydrophobic
pocket. Interestingly, the compounds reported in this paper also possess a 1,4-naphthoquinone
moiety, similarly to atovaquone, as well as a large hydrophobic substituent attached at position 3.
On the basis of this information, molecular modeling studies were designed aiming to explore
the putative binding modes of these new compounds at the inhibitor-binding site of PfDHODH.
In general, several available structures of the DHODH enzyme with bound inhibitors of various
sizes show that there is an overlap of the portions of the structures that fit into the H-bond pocket
and exhibit polar interactions with H185, Y528 and R265. On the other hand, the size of the
hydrophobic pocket is variable, depending on the conformations of the side chains of F171 and
F188. In addition, we observed that M536 and Y168 are two additional residues with a high
degree of conformational flexibility in the same hydrophobic pocket. Therefore, in order to study
the binding mode of the new ligands of variable size, we used the Induced Fit docking protocol
(Schrӧdinger package), in which the ligand-binding site flexibility was taken into account to
explain the binding modes of ligands varying in size. A summary of docking scores predicted for
the interaction of the obtained compounds with PfDHODH (PDB-Id: 1TV5) is given in Table 2,
together with their IC50 values.
10

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Table 2. In vitro antimalarial activity (IC50 in µM) and predicted docking scores (kcal/mol) of
lapachol and the synthetic naphthoquinonolyl compounds with PfDHODH by the Induced Fit
docking protocol.
Compound
IC50
Docking score
-9.137
Compound
IC50
Docking score
-10.994
1
Lapachol
123.5 ± 11.5
19
19.1 ± 1.0
-3
2
Atovaquone
1.22 x 10 ±
-12.945
20
8.4 ± 4.1
-12.914
0
.0009
7
15.1 ± 3.0
8.2 ± 0.5
4.0 ± 1.6
4.6 ± 1.9
7.8 ± 1.1
8.7 ± 3.4
5.2 ± 1.8
13.6 ± 3.4
-11.498
-9.375
-12.504
-12.587
-13.449
-13.449
-14.55
*
21
22
23
24
25
26
27
28
> 104.6
9.8 ± 3.2
8.4 ± 1.3
11.7 ± 0.6
4.1 ± 1.0
8.2 ± 5.5
14.2 ± 1.4
19.4 ± 3.3
-10.12
-12.709
-12.608
-12.014
-12.96
1
1
1
1
1
1
1
1
3
4
5
6
7
8
-12.296
-12.08
-13.621
*Failed to dock into the inhibitor-binding site
The molecular docking data supported the potential interaction of lapachol and the synthetic
naphthoquinonolyl compounds with the PfDHODH enzyme as the putative mechanism of action
and the inhibition of parasite growth in vitro. In general, compounds fit well into the binding
pocket of PfDHODH, the tightness of binding varying with the size of the ligand, which is
expressed by the docking scores. In order to explain the binding of the studied compounds, the
ligand-binding site is labeled as three pockets based on the binding of atovaquone: pocket 1 (P1)
occupied by the 1,4-naphthoquinone moiety, pocket 2 (P2) accommodating cyclohexane, and
pocket 3 (P3) occupied by the 4-chlorobenzene ring (Figure 2). The 1,4-naphthoquinone, a
11

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common substructure, displayed direct interactions with R265 and Y528 in pocket P1, and its
binding pose was stabilized by hydrophobic interactions with I172, I263 and V532 amino acids.
The unsaturated isoprenyl moiety at position 2 of the 1,4-napthoquinone moiety occupied the
proximal hydrophobic pocket P2 comprising interactions with the amino acids F188, V223, F227
and Y528. It has been shown experimentally that the side chain of F188 attains various rotameric
states in P2 in order to allow the protein to accommodate ligands of variable sizes as well as to
provide access to P3. Interestingly, since the binding site was kept flexible during docking
studies, F188 attained a down-pointing conformation in most of the inhibitor-docked PfDHODH
structures, so as to accommodate the large substituents present at position 3 of 1,4-
naphthoquinone. Additionally, F188 exhibited aromatic interactions with the 1,2,3-triazole
moiety in an edge-to-face manner. In addition, the 1,2,3-triazole moiety was oriented parallel to
the disulfide bridge between C175 and C184, which connects the two helices α1 and α2 of
PfDHODH, and exhibited hydrophobic interactions with C175, L176, C184, and other proximal
amino acids. Among the studied novel molecules (1, 7, 11, 13-28), major differences were
observed in the relative binding of the various substituents linked to the nitrogen N1 of the 1,2,3-
triazole moiety into P3 of PfDHODH. To accommodate these substituents, F171 in the α1 helix
attained distinct conformations and was found to exhibit aromatic π–π stacking or other types of
hydrophobic interactions. The better activity observed for compound atovaquone (2) over
lapachol (1) is apparently due to the presence of the hydrophobic propargyl group which fit
better into the hydrophobic pocket. The piperidine NH moiety of 14 formed H-bonds with the
OH of Y168 and the backbone C=O of F171. The piperidine ring in 13 (docking score = -12.504)
exhibited bidentate H-bonding with the backbone C=O of F171 and with Y168 in pocket P3. The
substitution of the acetyl group for the piperidyl nitrogen (compound 19, docking score = -
12

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1
0.994) reduced the binding score, due to loss of H-bonding with F171 and Y168, which could
be one of the reason behind its lesser potency against P. falciparum. In comparison to 24
docking score = -12.014), compound 25, which contains an o-methoxy group in addition to a p-
(
nitro, showed better binding (docking score = -12.960), due to the hydrophobic contribution in
pocket P3. The sulfonamide analogue 21 displayed poorer binding affinity (docking score = -
1
0.120), which agreed with its experimentally-demonstrated lower activity (IC ). The docking
50
study suggested 17 as the compound with the highest affinity for PfDHODH. As shown in
Figure 2, the 3-pyridyl substituent (17) exhibited aromatic π–π stacking interactions with F171
(
in the α1-helix) as well as H-bonding with the backbone NH of M536 along with hydrophobic
interactions. In addition, it favored the CH – π interaction between the 1,2,3-triazole and F188
Figure 2). Attempts of docking with 18 failed, probably because of its overall large size.
(
Possibly the pocket P3 failed to accommodate the bulky substituent on N1 of its 1,2,3-triazole
core. Therefore, compound 18 is predicted not to act through the inhibition of PfDHODH
enzyme for its antimalarial activity (IC50 = 13.6 ± 3.4 µM). The quinoline analogue 7 also
displayed comparatively poorer binding affinity that was apparently due to the bulkiness of its
quinoline ring, leading to impaired interaction in pocket P3. Overall, the docking study suggested
compound 17 as the lead against PfDHODH (score = -14.550) (IC50 = 5.2 ± 1.8 µM; SI = 197.7).
The structure-activity relationships could be further explored by incorporating small substitutions
on the 3-pyridyl ring. Such structural modifications are expected to enhance enzyme binding
affinity and thus lead to compounds with higher potency against the parasite.
13

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Figure 2. (a) Superposed view of the X-ray structure of rat DHODH with bound atovaquone
(cyan carbon) (PDB-ID: 1UUM) and two structures of PfDHODH with bound DSM267 (dark
green carbon) (PDB-ID: 3SFK) and A26 (orange carbon) (PDB-ID: 1TV5). The inhibitor and
proximal co-factor binding sites are encircled with black dashed lines. The inhibitor binding site
is categorized into three sub-pockets as Pocket 1 (P1), Pocket 2 (P2) and Pocket 3 (P3) (roughly
14

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separated by gray long-dashed lines). (b-i) Depiction of predicted binding modes of (b) 1, (c) 11,
d) 14, (e) 13, (f) 24, (g) 21, (h) 17, and (i) 7, all with PfDHODH (1TV5). The protein is shown as
gray-colored cartoon, while ligands are shown as ball-and-stick form. The inhibitor binding sites
in images b-i are depicted as surface view, colored according to the electrostatic potential (blue:
electropositive, red: electronegative). Some π-π interactions are shown with blue dashed lines
and some hydrogen bonds or other electrostatic interactions are shown with black dotted lines.
3
. Conclusion
The present work describes the synthesis of 18 naphthoquinonolyl-1,2,3-triazole compounds
based on natural product modification with antiplasmodial activity. Lapachol derivatives
disclosed higher activity than the natural product. Six of these final modified lapachol 1,2,3-
triazole compounds showed SI > 100. The most promising compounds such as compounds 17
,
2
2
and 25 have some structural similarities such as a substituted aryl side chain in the triazole
ring. The triazole naphthoquinolyl compounds with only phenyl (compound 19 with SI = 5.3) or
benzyl (compound 15 with SI = 53.0) moieties had moderate activity. However, the presence of
heteroatoms such as a chlorine substituent or a nitrogen on the aromatic ring played an important
role (SI = 51.0, 75.6, 128.8 and 197.7 for compounds 22
promising compounds such as 25 SI of 171.0 demonstrate that nitro and methoxy groups also
can play an important role, depending upon their site of attachment. Atovaquone and compound
with terminal alkyne and pyrimidine moieties, with SI of 112.7 and 124.0, respectively, also
, 16, 7 and 17, respectively). Other
(
)
1
1
provide important information for future studies. Docking studies support the possibility that
these compounds do target PfDHODH. Based on the best docking scores, 16 was identified as
the best lead compound against PfDHODH. Structure-activity relationship studies could further
15

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be explored by incorporating small substitutions on the 3-pyridyl ring of 17. Doing so is
expected to enhance enzyme binding affinity and thus lead to compounds with higher potency
against the Pf parasite.
4
4
4
. Materials and Methods
.1. Biological activity
.1.1. Continuous cultures of Plasmodium falciparum
P. falciparum W2 clone, which is chloroquine-resistant and mefloquine-sensitive [41], was kept
in a continuous culture at 37 °C in human erythrocytes using the candle jar method [41, 42]. The
antimalarial effect of the compounds was measured by the pLDH assay [43, 44]. Parasites were
kept in complete culture medium (RPMI) containing sodium bicarbonate (21 mM), D-glucose
(11 mM), HEPES (25 mM), hypoxanthine (300 µM) and gentamicin (40 µg/ml) that was
supplemented with 10% human plasma on culture dishes, the culture medium being changed
daily. All experiments were performed in triplicate. The compounds were tested in triplicate at
each concentration. The cultures with predominantly ring-stage parasites were concentrated by
sorbitol-synchronization [45]. A suspension of red blood cells with 0.05% parasitemia and 1.5%
hematocrit was distributed in a 96-well micro titer plate (180 µl/well). The parasite growth was
evaluated by the pLDH assay, as summarized below.
4
.1.2. Evaluation of the in vitro antimalarial activity by the pLDH assay
The antimalarial effects of the compounds and of the controls were measured by the lactate
dehydrogenase of Plasmodium falciparum (pLDH) assay as previously described [20], with
16

ACCEPTED MANUSCRIPT
minor modifications. Briefly, ring-stage parasites in sorbitol-synchronized blood cultures were
added to 96-well culture plates at 2% parasitemia and 1% hematocrit and then incubated with the
-1
test drugs that were diluted in complete medium, from 50 mg mL stock solutions in DMSO, at
a final concentration of 0.002% (v/v) and stored at –20 ºC. After a 48 h incubation period, the
plates were frozen (–20 ºC for 24 h) and thawed for the pLDH assay. The hemolyzed cultures
were transferred to another 96-well culture plate, and Malstat® and nitro blue tetrazolium salt
and phenazine ethosulphate (NBT/PES) reagents were added. After 1 h of incubation at 37 ºC in
®
the dark, the absorbance was read at 570 nm in a spectrophotometer (Infinite 200 PRO, Tecan).
The results were evaluated with the software Microcal Origin 8.5 for determination of the dose-
response curves plotted with sigmoidal fit [46]. The IC was determined by comparison with
5
0
controls with standard drug and without drugs.
4
.1.3. Cytotoxicity evaluation in human hepatoma cell cultures – HepG2 cell
2
Hepatoma cells HepG2 cell were maintained at 37 ºC in 5% CO
2
in 75 cm sterile culture flasks
®
(
Corning ) with RPMI 1640 culture medium supplemented with 5% FBS, penicillin (10 U/ml),
and streptomycin (100 g/ml), with changes of medium twice a week. The cells were maintained
in weekly passages (at 1:3 dilutions in sterile culture flasks) and grown to 80% [47]. They were
used for experiments after being trypsinized (0.05% trypsin/0.5 mM EDTA) and plated on 96
well microplates [48]. The test samples and controls (chloroquine and atovaquone) were diluted
to a final concentration of 0.02% DMSO in culture medium to yield four concentrations in serial
dilutions starting at 1,000 mg/ml. After 24 h incubation at 37 °C, 18 µl of MTT solution (5
mg/ml in PBS) were added to each well, followed by another 90 min incubation at 37 °C. The
supernatant was then removed, and 180 µl of DMSO was added to each well. The culture plates
were read in a spectrophotometer with a 570 nm filter [47]. The minimum cytotoxicity
17

ACCEPTED MANUSCRIPT
concentration was determined as described previously, with slight modifications (DMSO was
used instead of ethanol for solubilizations and the positive control used was chloroquine instead
of primaquine). Each test was performed in duplicate, and the concentration that killed 50% of
the cells (CC ) was determined [49]. The selectivity index (SI) for the antimalarial activity was
50
then calculated based on the ratio between CC50 and IC50 for the in vitro activity against P.
falciparum as described [50]. Most of the compounds showed SI > 10 and could be considered
non-toxic [51].
4
4
.2. Extraction
.2.1. Plant material
Trunkwood of Handroanthus serratifolius (Vahl) S.O. Grose were collected from specimens
identified by Dr. Prof. João Renato Stehmann, Instituto de Ciências Biológicas, Universidade
Federal de Minas Gerais. A voucher specimen (BHCB 32995) is deposited in the herbarium of
Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais, Belo Horizonte, MG,
Brazil.
4
.2.2. Extraction and isolation of lapachol
The pulverized wood material (3000 g) was macerated with 10 L of a 2.5% sodium carbonate
solution for 12 h. The suspension was filtered and the filtrate was acidified with HCl(aq) conc.,
resulting in precipitation of a dark viscous material (180.0 g) that was filtered under vacuum,
dried in an oven at 45 ºC and further extracted with dichloromethane in a Soxhlet apparatus for
1
2 h. Further purification was performed by chromatography through a silica gel column. The
product was eluted with dichloromethane/hexane (1:1 v/v) as a bright yellow solid (5.3 g) and
1
13
was characterized as lapachol (1) by classic methods including H NMR and C NMR.
18

ACCEPTED MANUSCRIPT
4
4
.3. Chemistry
.3.1. General
Chemicals and reagents were purchased from commercial suppliers and used as received unless
noted otherwise. Reactions were monitored by thin layer chromatography (TLC) on precoated
0
.2 mm silica gel 60 F254 (Merck) plates and visualized in several ways with an ultraviolet light
source at 254 nm, by spraying with Hanissam reagent (Ceric ammonium molibidate-CAM),
anisaldehyde sulfuric acid, Dragendorff reagent or iodine. All reactions were performed in
standard dry glassware without inert atmosphere. Evaporation and concentration were done in
standard rotavapors (Büchi and IKA) under vacuum. TLC was carried out with silica gel 60 with
fluorescent indicator (e.g., Silica Gel F-254 or IB-F, Merck) after previous activation with
heating at 100 °C overnight and visualization by UV light or Dragendorff reagent. Melting points
1
13
(
mps) were measured in a Fisher-Jones melting point apparatus and are uncorrected. H and C
NMR spectra were measured on a Bruker Advance DPX 200, Fourier 300HD and DRX400 with
FT analysis. Chemical shifts are reported (ppm) with SiMe as internal standard. Coupling
constants (J) are given in Hertz. The deuterated solvents used were CD OD, CDCl or DMSO-
δ
4
3
3
d
6
. All 2D NMR data were recorded at 400 MHz (Bruker DRX400), heteronuclear single
quantum coherence (HSQC) using J 145 Hz, and heteronuclear multiple-bond correlation
HMBC) using J 8 Hz. Infrared spectra were recorded on a FT-IR, Spectrum One (Perkin-Elmer)
(
-1
with ATR system and are reported in wave numbers (cm ). Samples were diluted with
methanol-formic acid 0.1% solution. High resolution mass spectrometry (HRMS) data were
recorded on a Shimadzu liquid chromatography-mass spectrometry ion trap and time-of-flight
®
(
LCMS-IT-TOF) spectrometer using electrospray ionization (ESI) and Waters ACQUITY
tandem quadrupole detector (TQD).
19

ACCEPTED MANUSCRIPT
4
.3.2. Materials
4
,7-Dichloroquinoline,
benzyl
bromide,
aniline,
3-(bromomethyl)pyridine,
3-
(bromomethyl)pyridine hydrobromide, 2-bromopyridine, 2-azidotolueno solution, sulfonamide,
2
-methoxy-4-nitroaniline, 4-fluoroaniline, 4-methoxy-2-nitroaniline, 4-aminobenzonitrile, 4-
hydroxypiperidine, 1-Boc-4-hydroxypiperidine, molecular sieves A4, molecular sieves A3,
triethylamine, 3-bromoprop-1-yne, Ziduvudine, Hydrogen Chloride 1M solution, RPMI 1640
medium, sodium bicarbonate, D-glucose, HEPES, hypoxanthine, gentamicin, D-sorbitol, PBS,
BSA, TMB, FBS, penicillin, streptomycin, tripsin/EDTA, and DMSO were obtained from
®
®
Sigma-Aldrich USA, Ltd. Copper sulphate pentahydrate was obtained from Reagen , ascorbic
®
acid was obtained from Synth , MPFG-55P and MPFM-55A antibodies were purchased from
®
ICLLABS , sulfuric acid, diethyl ether, dichloromethane, hexane, ethyl acetate, chloroform,
®
sodium bicarbonate and sodium sulphate were obtained from FMaia , and were used without
further purification. Glassware was from Hialoquímica, Ltd.
4
.3.3 Synthetic Approach
Lapachol (
naphthoquinonolyl intermediate (11) in good yield, after optimizing conditions to provide this
regioisomer rather than 12. Using parallel synthesis, 4,7-dichloroquinoline ( ) was
functionalized in sodium azide, via a nucleophilic substitution reaction (S AR), to provide
compound [52]. Next, these two intermediates were combined by Copper-Catalyzed-Azide-
Alkyne Cycloaddition (CuAAC) to produce the lapachol-1,2,3-triazole hybrid as shown in
Scheme 2
1) was O-alkylated using propargyl bromide propyne in acetone, providing the alkyne
8
N
9
7
.
20

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of the naphthoquinonolyl hybrid 7 containing a 7-chloroquinoline moiety
obtained via click reaction between the terminal alkyne 11 and the organic azide 9 in a
convergent route.
N
Different azides could also by prepared by bimolecular nucleophilic substitution (S 2) or
aromatic nucleophilic substitution (S Ar) as shown in Scheme 3 [53]. Commercially available
N
alkyl and aryl halides and Zidovudine® (AZT) were used as shown in Scheme 3. Compounds
1
3-17 were prepared in high yield reactions (60 to 95%). Compound 14 was obtained after Boc
deprotection of 13 using anhydrous acid conditions.
21

ACCEPTED MANUSCRIPT
O
O
R
N
N
1
1
O
X
N3
O
O
NaN , DMF
3
click reaction
r.t.
O
R
R
N
X=Ms, Br.
R
HO
N
N
O
O
N
N
Boc
H
N
NH
O
13
14
15
16
17
18
Scheme 3. Naphthoquinonolyl hybrid synthetic route for click reactions between 11 and organic
azides 13-18.
N
Another explored route to azides was provided by aromatic nucleophilic substitution (S Ar) via
diazotization reaction of commercially available anilines (Scheme 4).
22

ACCEPTED MANUSCRIPT
Scheme 4. Aromatic nucleophilic substitution (S
N
Ar) forming diazonium salts “in situ”
providing different organic azides as starting materials for click reactions with alkyne 11.
The structures of the products were characterized by spectroscopic data including HRMS-ESI-
1
13
IT-TOF, IR, H and C NMR (Cf. Supporting Information).
4
.3.4. Isolation, Synthesis and characterization
Lapachol
-1
Yield: 0.5% from bark, orange solid, IR (λmax, cm ): 3348, 2972, 2907, 1659, 1638, 1591, 1456,
1
1
367, 1351, 1336, 1237, 1209, 1182, 1153, 1046, 1028, 936, 846, 675. H NMR (200 MHz,
CDCl3):
m, 1H, H12), 3.30 (s, 3H, J = 6.6 Hz, H11), 1.79 (s, 3H, H14), 1.68 (s, 3H, H15). C NMR (50
MHz, CDCl ): 184.46, 181.71, 152.72, 134.77, 133.73, 133.04, 132.78, 129.54, 126.78,
δ 8.13-8.05 (m, 2H, H and H ), 7.78-7.63 (m, 2H, H and H ), 7.34 (s, 1H, OH), 5.21
7 8 6 9
1
3
(
3
δ
23

ACCEPTED MANUSCRIPT
1
15 3
26.02, 123.57, 119.73, 25.67, 22.65, 17.85. HRMS-ESI-IT-TOF: m/z calculated C15H O
(M+H) 243.1021, found 243.1152.
Azides
4
-Azido-7-chloro-quinoline (
9) [54]
To a solution of 4,7-dichloroquinoline (
8) (2.0 g, 10 mmol) in 5 mL anhydrous DMF and
molecular sieves A4, sodium azide (1.3 g, 20 mmol) was added in one portion at room
o
temperature, and the resulting mixture stirred at 85 C for 8 h, when TLC indicated reaction
completion. The reaction mixture was allowed to cool to room temperature and then it was
2 2 2 4
diluted with 100 mL CH Cl , washed with water (3 x 40 mL), dried over anhydrous Na SO , and
evaporated to dryness. The resulting product residue was purified by small column
chromatography eluted with CH Cl /Hexane mixture 1:1 to yield the final pure product as
2
2
colorless, needle-like crystals (1.8 g, 91%), mp 115 °C (from CH
.29.
2 2
Cl /Hex), Rf (EtOAc/Hex 3:7)
0
-1
IR (λmax, cm ): 3079, 3036, 2983, 2118, 1670, 1608, 1578, 1564, 1557, 1489, 1440, 1417, 1373,
1
351, 1300, 1278, 1199, 1146, 1090, 1071, 1011, 963, 896, 880, 840, 818, 777, 768, 671, 663.
1
H NMR (200 MHz, CDCl
3
):
δ
8.76 (1H, d, J = 5.0 Hz, H
2
), 8.00 (1H, d, J = 2.0 Hz, H
8
), 7.88
(
1H, d, J = 8.8 Hz, H ), 7.49 (1H, dd, J = 2.0 Hz, J = 8.8 Hz, H ) 7.13 (1H, d, J = 5.0 Hz, H ).
5
6
3
1
3
C NMR (50 MHz, CDCl3):
08.73.
δ 151.33, 149.59, 146.30, 136.57, 128.23, 127.52, 123.77, 119.91,
1
General methodology to produce azides by nucleophilic aromatic substitution
Sodium azide (1.5 equivalent) ( ) and a commercially-available alkyl halide (1.0 equivalent)
9
were mixed in anhydrous DMF (2 mL) and stirred at room temperature. The reaction was left
running overnight and was stopped when it was completed as shown by TLC. Work-up of the
24

ACCEPTED MANUSCRIPT
reaction mixture with CH
2
Cl
2
2 4
, brine and water (2 x 10 mL), was followed by drying over Na SO
and the product was used without further purification by column chromatography.
Tert-butyl 4-azidopiperidine-1-carboxylate (30).
-1
IR (λmax, cm ): 2976, 2932, 2863, 2090, 1687, 1468, 1451, 1416, 1392, 1364, 1129, 1087, 1019,
1
9
38, 863, 816, 769, 725. H NMR (200 MHz, CDCl
3
):
δ
4.82 (m, 1H, H
4
), 3.73-3.20 (m, 4H,
1
3
H ), 3.00 (s, 3H, H ), 1.99-1.81 (m, 5H, H and H ), 1.42 (s, 9H, H ). C NMR (50 MHz,
2
5
3
4
7
CDCl3): δ 154.40, 79.86, 40.45, 38.76, 31.59, 28.29, 8.55.
(Azidomethyl)benzene (31).
-1
Yield: 98%, viscous colorless oil, IR (λmax, cm ): 3127, 3047, 2924, 2849, 1610, 1593, 1560,
1
1
504, 1449, 1438, 1348, 1310, 1244, 1115, 1046, 1018, 922, 875, 834, 822, 813, 767, 672. H
1
3
NMR (200 MHz, CDCl
3
):
δ
7.49-7.44 (m, 5H), 4.39 (M, 2H). C NMR (50 MHz, CDCl
3
):
δ
1
35.38, 128.73, 128.50, 128.14, 128.13, 54.60 HRMS-ESI-IT-TOF: m/z calculated C H N
7
8
3
(M+H) 134.0718, found 134.0758.
25

ACCEPTED MANUSCRIPT
2
-(Azidomethyl)pyridine (32).
-1
IR (λmax, cm ): 3034, 2089, 1571, 1508, 1474, 1420, 1285, 1237, 1189, 1140, 1124, 1100, 1038,
1
1
019, 978, 935, 909, 857, 797, 700, 687. H NMR (200 MHz, CDCl
3
):
δ
8.60 (d, 1H, J = 4.2
Hz), 7.73 (t, 1H, J = 7.6 Hz), 7.34 (d, 1H, J = 7.6 Hz), 7.30 (t, 1H, J = 4.2 Hz), 4.39 (M, 2H).
1
3
C NMR (50 MHz, CDCl3):
δ 155.57, 149.55, 136.99, 122.91, 121.99, 53.49. HRMS-ESI-IT-
TOF: m/z calculated C H N (M+H) 135.0671, found 135.0744.
6
7
4
3
-Azidopyridine (33).
-1
IR (λmax, cm ): 3064, 3036, 2090, 1593, 1491, 1470, 1454, 1341, 1293, 1278, 1174, 1128, 1075,
1
1
025, 1002, 895, 810, 745, 685. H NMR (200 MHz, CDCl ):
δ
8.02 (m, 2H), 6.97-6.93 (m, 2H).
3
1
3
C NMR (50 MHz, CDCl3):
δ 145.34, 140.63, 136.36, 125.16, 123.45, HRMS-ESI-IT-TOF: m/z
calculated C (M+H) 121.0514, found 121.0596.
5
5 4
H N
Azidobenzene (34).
-1
IR (λmax, cm ): 3247, 3064, 3036, 2122, 2090, 1593, 1490, 1471, 1454, 1293, 1279, 1173, 1128,
1
1
075, 895, 820, 810, 685. H NMR (200 MHz, CDCl
3
):
δ
3
7.32 (t, 2H, J = 7.6 Hz, H ), 7.11 (t, 1H,
1
3
J = 7.6 Hz, H ), 7.00 (d, 2H, J = 7.6 Hz, H ). C NMR (50 MHz, CDCl ):
δ
139.96, 129.65,
4
2
3
1
24.77, 118.94.
26

ACCEPTED MANUSCRIPT
1
-Azido-3-fluorobenzene (35).
-1
IR (λmax, cm ): 3075, 2107, 1589, 1484, 1446, 1446, 1294, 1207, 1162, 1153, 1108, 1099, 944,
1
9
6
1
22, 858, 843, 767, 673, 660. H NMR (200 MHz, CDCl
3
):
δ
7.23 (t, 1H, J = 6.8 Hz, H
5
), 6.83-
1
3
.66 (m, 3H, H2,
H
4
and H
6
). C NMR (50 MHz, CDCl
3
):
δ 164.89, 160.97, 141.97, 141.77,
30.82, 130.63, 114.58, 111.92, 111.49, 106.79, 106.30.
Azido-benzenesulfonamide (36).
-1
IR (λmax, cm ): 3398, 3076, 2102, 1638, 1510, 1488, 1464, 1260, 1017, 951, 916, 849, 806, 822,
1
8
13, 764, 703. H NMR (200 MHz, DMSO-d
6
):
δ
3
7.84 (d, 2H, J = 7.0 Hz, H ), 7.37 (sl, 2H,
1
3
NH
2
), 7.28 (d, 2H, J = 7.0 Hz, H
2
). C NMR (50 MHz, DMSO-d
6
): 142.88, 140.45, 127.58,
δ
1
19.40. HRMS-ESI-IT-TOF: m/z calculated C H N O SNa (M+Na) 221.0109, found 221.0100.
6 6 4 2
1
-azido-4-chlorobenzene (37).
-1
IR (λmax, cm ): 2924, 2849, 2120, 2089, 1592, 1484, 1292, 1269, 1174, 1128, 1090, 1011, 821,
1
7
44, 707. H NMR (200 MHz, CDCl ):
δ
7.25 (d, 2H, J = 8.6 Hz, H ), 6.88 (d, 2H, J = 8.6 Hz,
3
3
1
3
H ). C NMR (50 MHz, CDCl ): δ 138.53, 130.08, 129.66, 120.09.
2
3
27

ACCEPTED MANUSCRIPT
4
-Azidobenzonitrile (38).
-1
IR (λmax, cm ): 3400, 3222, 3094, 3043, 2974, 2142, 2105, 1703, 1658, 1598, 1503, 1448, 1416,
1
1
δ
1
377, 1303, 1278, 1176, 1126, 1110, 965, 942, 919, 818, 747, 704. H NMR (200 MHz, CDCl ):
3
1
3
7.64 (d, 2H, J = 8.8 Hz, H ), 7.11 (d, 2H, J = 8.8 Hz, H ). C NMR (50 MHz, CDCl ):
δ
3
2
3
44.85, 133.71, 129.88, 128.15, 119.65, 118.17, 108.32. HRMS-ESI-IT-TOF: m/z calculated
Na (M+Na) 167.0334, found 167.0898.
7 4 4
C H N
1
-Azido-2-methoxy-4-nitrobenzene (39).
-1
Yield: 80%, orange solid, IR (λmax, cm ): 3116, 3086, 2924, 2849, 2112, 1607, 1588, 1509,
1
7
1
1
2
498, 1441, 1418, 1340, 1311, 1264, 1250, 1186, 1135, 1075, 1009, 916, 903, 818, 812, 743,
1
15, 698. H NMR (200 MHz, CDCl
3
):
δ
7.45 (d, 1H, J = 4.8 Hz, H
5
), 7.23 (s, 1H, H
156.55, 141.25, 127.09,
21.99, 120.97, 110.22, 56.06. HRMS-ESI-IT-TOF: m/z calculated C H N O Na (M+Na)
3
), 7.20 (d,
1
3
H, J = 5.4 Hz, H ), 3.86 (s, 3H, H ). C NMR (50 MHz, CDCl ):
δ
6
7
3
7
6
4
3
17.0338, found 217.0248.
1
-Azido-4-methoxy-2-nitrobenzene (40).
28

ACCEPTED MANUSCRIPT
-1
IR (λmax, cm ): 3116, 3086, 2924, 2849, 2112, 1607, 1588, 1509, 1498, 1441, 1418, 1340, 1311,
1
1
264, 1250, 1186, 1135, 1075, 1009, 916, 903, 818, 812, 743, 715, 698. H NMR (200 MHz,
CDCl3):
δ
7.45 (d, 1H, J = 4.8 Hz, H ), 7.23 (s, 1H, H ), 7.20 (d, 1H, J = 5.4 Hz, H ), 3.86 (s,
5 3 6
1
3
3
H, H ). C NMR (50 MHz, CDCl ):
δ
156.55, 141.25, 127.09, 121.99, 120.97, 110.22, 56.06.
7
3
HRMS-ESI-IT-TOF: m/z calculated C H N O Na (M+Na) 217.0338, found 217.0248.
7
6
4
3
1
-Azido-4-nitrobenzene (41).
-1
IR (λmax, cm ): 3225, 3003, 2954, 2910, 2836, 2099, 1712, 1501, 1284, 1240, 1180, 1172, 1108,
1
1
031, 822, 754. H NMR (200 MHz, CDCl
3
):
3
δ 8.23 (d, 2H, J = 9.0 Hz, H ), 7.13 (d, 2H, J = 9.0
1
3
Hz, H ). C NMR (50 MHz, CDCl ):
δ 146.81, 144.64, 125.50, 119.33.
2
3
Tert-butyl 4-(methylsulfonyl)piperidine-1-carboxylate (13).
1
H NMR (200 MHz, CDCl3):
δ
4.82 (m, 1H, H ), 3.73-3.20 (m, 4H, H ), 3.00 (s, 3H, H ), 1.99-
4
2
5
1
3
1
3
.81 (m, 5H, H and H ), 1.42 (s, 9H, H ). C NMR (50 MHz, CDCl ):
δ
154.40, 79.86, 40.45,
3
4
7
3
4
8.76, 31.59, 28.29, 8.55. HRMS-ESI-IT-TOF: m/z calculated C11H22NO S (M+H) 264.1270,
found 264.1242.
29