Exploring the antimalarial potential of the methoxy-thiazinoquinone scaffold: Identification of a new lead candidate

Article abstract of DOI:10.1016/j.bioorg.2018.12.031

A small library of antiplasmodial methoxy-thiazinoquinones, rationally designed on the model of the previously identified hit 1, has been prepared by a simple and inexpensive procedure. The synthetic derivatives have been subjected to in vitro pharmacolog

Full text of DOI:10.1016/j.bioorg.2018.12.031

BioorganicChemistry85(2019)240–252
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Exploring the antimalarial potential of the methoxy-thiazinoquinone
scaffold: Identification of a new lead candidate
Concetta Imperatorea,1, Marco Persicoa,1, Maria Senesea, Anna Aielloa, Marcello Casertanoa,
⁎
⁎
Paolo Lucianoa, Nicoletta Basilicob, , Silvia Parapinib, Antonella Paladinoc, Caterina Fattorussoa,
,
⁎
Marialuisa Mennaa,
a The NeaNat Group, Department of Pharmacy, University of Naples “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
b Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università di Milano, Via Pascal 36, 20133 Milan, Italy
c Istituto di Chimica del Riconoscimento Molecolare, CNR, Via M. Bianco 9, 20131 Milano, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
A small library of antiplasmodial methoxy-thiazinoquinones, rationally designed on the model of the previously
identified hit 1, has been prepared by a simple and inexpensive procedure. The synthetic derivatives have been
subjected to in vitro pharmacological screening, including antiplasmodial and toxicity assays. These studies af-
forded a new lead candidate, compound 9, endowed with higher antiplasmodial potency compared to 1, a good
selectivity index when tested against a panel of mammalian cells, no toxicity against RBCs, a synergistic anti-
plasmodial action in combination with dihydroartemisinin, and a promising inhibitory activity on stage V ga-
metocyte growth. Computational studies provided useful insights into the structural requirements needed for the
antiplasmodial activity of thiazinoquinone compounds and on their putative mechanism of action.
Organic synthesis
Thiazinoquinones
Antimalarial
DFT calculations
Reactive radical species
Redox-based mechanism of action
1. Introduction
disease, nevertheless, new therapeutic agents are always needed to
keep up with the evolution of parasite resistance [3]. In recent times we
According to the WHO World Malaria Report 2017 [1], in 2016 91
countries reported a total of 216 million cases of malaria, with an in-
crease of 5 million cases over the previous year: actually, nearly half of
the world's population is at risk of malaria [1,2]. Malaria deaths
worldwide were 445 000 in 2016, approximately the same number as
the previous year. Most of these cases were in the African Region
(90%), followed by the South-East Asia Region (7%) and the Eastern
Mediterranean Region (2%). Plasmodium falciparum (Pf) is the most
prevalent malaria parasite in sub-Saharan Africa, accounting for 99% of
estimated malaria cases in 2016 [1]. Great efforts have been made and
are still being done in an attempt to eliminate malaria; however, a more
attainable goal is controlling this disease.
are witnessing a significant increase of new chemotypes active against
Plasmodium but the challenge is now to assess their potential as anti-
malarial leads. In this contest, the marine environment has played an
important role in providing new chemical scaffolds [4]. In particular, a
group of molecules isolated from marine invertebrates and character-
ized by the presence of a thiazinoquinone bicyclic moiety, showed in-
teresting antiplasmodial properties [5,6]. This prompted us to explore
the antiplasmodial potential of this scaffold, which is also contained in
the structures of the aplidinones A and B (Fig. 1), two metabolites
isolated by us from the Mediterranean ascidian A. conicum [7]. Ac-
cordingly, we decided to build a chemical library designed on the
model of these simple natural thiazinoquinones [8,9]. A first series of
synthetic derivatives were prepared and tested in vitro against both
chloroquine (CQ)-sensitive (D10) and -resistant (W2) strains of Pf [8].
Many compounds showed interesting antiplasmodial activity, with IC50
A key role in the fight against malaria is played by the discovery of
new drugs. Indeed, although today different types of antimalarial drugs
are available and their use in combination is the best way to treat the
Abbreviations: Pf, Plasmodium falciparum; RBCs, red blood cells; SARs, structure-activity relationships; DFT, density functional theory; DHA, dihydroartemisinin;
NAC, N-acetyl-L-cysteine; CAN, cerium ammonium nitrate; HMEC-1, human microvascular endothelial cells line 1; SI, selectivity index; BMDM, bone marrow-derived
macrophages; THP-1, human monocytic leukaemia cells; MD, molecular dynamics; SA, Simulated annealing; MM, molecular mechanics; GM, global minimum; C-
PCM, conductor-like polarizable continuum model; RMSD, root mean square deviation; LUMO, lowest unoccupied molecular orbital; NBO, natural bond orbitals;
GSH, glutathione; iRBCs, infected red blood cells; ACT, artemisinin-based combination therapy
⁎ Corresponding authors.
E-mail addresses: nicoletta.basilico@unimi.it (N. Basilico), cfattoru@unina.it (C. Fattorusso), mlmenna@unina.it (M. Menna).
1 These authors contributed equally.
https://doi.org/10.1016/j.bioorg.2018.12.031
Received 13 August 2018; Received in revised form 19 December 2018; Accepted 22 December 2018
Availableonline03January2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

C. Imperatore et al.
BioorganicChemistry85(2019)240–252
Fig. 1. Structures of Aplidinones A and B, of the thiazinoquinone-based starting hit (1), and of the new derivatives 2–9.
values in the low micromolar range [8]. Some of them were also
strongly cytotoxic [9], as commonly found for thiazinoquinone com-
pounds [10–13], but others had a good selectivity index with respect to
normal human cells [8]. Structural requirements critical for both the
antiplasmodial and the cytotoxic activity already appeared from these
early studies: both the nature of the X group on the thiazinoquinone
ring of aplidinones (Fig. 1) and its position with respect to the nitrogen
or sulphur atom of the dioxothiazine ring, were shown to be crucial for
compound potency. On the other hand, the length of the alkyl chain
distinguished the antiplasmodial activity from the toxicity against
normal human cells [8]. Structure-activity relationships (SARs) were
investigated through computational and electrochemical studies and
the capacity to produce a toxic semiquinone species able to form a
stable adduct with Fe(III)–heme was found to be related to the anti-
plasmodial activity. As a whole, this first set of results confirmed the
thiazinoquinone as a new and promising chemotype active against Pf
and compound 1 (Fig. 1) was proposed as a new synthetic and low-cost
antimalarial hit candidate [8].
in combination with N-acetyl-L-cysteine (NAC) were also performed to
investigate 9 antiplasmodial mode of action. Both theoretical and ex-
perimental results support the hypothesis that the new optimized hit 9
is able to kill the parasite through the production of free radicals and
the consequent induction of cellular oxidative stress.
2. Results and discussion
2.1. Synthesis
The methoxy-derivatives 2–4 and 6–8 were prepared using a syn-
thetic approach similar to that adopted for the synthesis of compound 1
(Scheme 1) [8,9]. In the first step, the commercially available 1,2,4-
trimethoxybenzene (10) was treated with n-BuLi in THF and, succes-
sively, with three different alkyl bromides, affording the corresponding
1,2,4-trimethoxy-3-alkylbenzenes 11-R1, 11-R2, and 11-R3 with good
yields (80%, 76%, and 77%, respectively) [8].
The obtained methoxy alkyl benzenes 11-R1, 11-R2, and 11-R3
were oxidized with cerium ammonium nitrate (CAN) to give the qui-
nones 12-R1 (65% yield), 12-R2 (65% yield), and 12-R3 (74% yield),
respectively [14]. Subsequent reaction of each quinone with hypo-
taurine in the presence of catalytic amount of salcomine [15] allowed
the thiazinoquinone bicyclic scaffold to be constructed. As previously
observed for other unsymmetrical quinones [15,16], the biomimetic
addition of hypotaurine to benzoquinones 12 afforded both thiazino-
quinone regioisomers (RA and RB, Fig. 1) and was selective. Therefore,
a mixture of 2 and 6 was obtained from 12-R1, of 3 and 7 from 12-R2,
and of 4 and 8 from 12-R3, respectively. For all reactions, the outcome
was highly (9:1) in favour of RB.
In the present article, we report the optimization of the hit candi-
date 1, accomplished by the synthesis of a small and rationally designed
series of new methoxy-thiazinoquinone derivatives, compounds 2–9
(Fig. 1).
Starting from the previously obtained results [8], our current aim
was to investigate the role played on the antiplasmodial activity by both
the regiochemistry of the methoxy-thiazinoquinone system and the
nature of the side chain linked at the quinone ring. Thus, two set of
regioisomers (RA and RB, Fig. 1) were prepared, all characterized by
different R substituents (Fig. 1), rationally varied in shape and nature to
expand 1 SARs. Through this approach we identified a new hit candi-
date, compound 9, endowed by higher antiplasmodial potency com-
pared to 1 and a good (> 25) selectivity index when tested against a
panel of mammalian cells.
To prepare the benzyl-derivatives 5 and 9, the above synthetic
scheme was slightly modified to increase the yields in the first step
(Scheme 2). Therefore, after treatment of 1,2,4-trimethoxybenzene 10
with n-BuLi, benzaldehyde was added and the biaryl alcohol 13 was
obtained. Compound 13 was then easily reduced by triethylsilane –
trifluoroacetic acid in dichloromethane, and afforded compound 14
with very good yield [17,18]. Oxidation of 14 as described above
(Scheme 1) gave the quinone 15 which, in turn, was subjected to
condensation with hypotaurine in the presence of salcomine. Also in
this case, a mixture of the isomeric (RA and RB) thiazinoquinones 5 and
9 was thus obtained in the ratio of 9:1 in favour of 9.
Computational studies including density functional theory (DFT)
calculations were performed on 2–9 in order to rationalize the observed
SARs and shed light on the mechanism of action of this new promising
class of antiplasmodial compounds. The new optimized hit 9 was sub-
jected to further pharmacological characterization in order to explore
its antimalarial potential. No haemolysis was observed at anti-
plasmodial doses, a synergistic antiplasmodial action in combination
with dihydroartemisinin (DHA) was assessed, as well as, a promising
low (< 15) micromolar activity on late-stage gametocytes. Experiments
Each mixture of RA and RB (2/6, 3/7, 4/8, and 5/9) was easily
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C. Imperatore et al.
BioorganicChemistry85(2019)240–252
Scheme 1. Synthesis of compounds 2–4 and 6-
8a. (aReagents and conditions: (a) (1) n-BuLi,
THF, 0 °C, 1 h; (2) 11-R1: 1-bromo-2-methyl-
propane, 0 °C → rt, 24 h. 11-R2: 1-bromobutane,
0 °C → rt, 24 h. 11-R3: bromomethylcyclo-
hexane, 0 °C → rt, 24 h. (b) CAN, CH3CN, 0 °C,
30 min. (c) hypotaurine, EtOH/CH3CN, salco-
mine, rt, 24 h).
separated by HPLC into the individual components which were uni-
vocally distinguished and fully characterized by spectroscopic means
(Tables S1 and S2).
both strains, with IC50 values in the low micromolar range, and 9 re-
sulted active in the sub-micromolar range (Table 1). Thus, compound 9
resulted significantly more active than the identified starting hit 1.
Then, we evaluated the cytotoxicity of the active regioisomers 6–9
against human microvascular endothelial cells (HMEC-1; Table 2). Only
compound 8 showed cytotoxic activity in the low micromolar range.
Among the new series of thiazinoquinones, compound 9 exhibited not
only the highest antiplasmodial activity but also the higher selectivity
index (SI = IC50 on HMEC-1/IC50 on Pf strains; Table 2). The selective
toxicity of 9 was challenged also against other mammalian cell lines,
such as, immortalized mouse C57Bl/6 bone marrow macrophages
(BMDM) and human monocytic leukaemia cells (THP-1). Results
2.2. In vitro antimalarial activity and toxicity against human cells
Thiazinoquinones 2–9, as well as the quinone intermediate 15, were
tested for their in vitro antiplasmodial activity against both chloroquine
(CQ)-sensitive (D10) and CQ-resistant (W2) strains of Pf. Results are
reported in Table 1.
All the RA (2–5), as well as compound 15, showed an IC50 higher
than 10 µM. On the contrary, among the RB, 6–8 resulted active against
OCH3
OCH3
OCH3
OH
a
b
OCH3
OCH3
OCH3
10
13
14
OCH3
OCH3
OCH3
c
O
O
O
O
O
H
N
S
d
+
OCH3
S
OCH3
OCH3
N
H
5
9
15
O
O
O
O
O
Scheme 2. Synthesis of compounds 5 and 9. (aReagents and conditions: (a) (1) n-BuLi, THF, 0 °C, 1 h; (2) benzaldehyde, 0 °C → rt, 12 h. (b) (1) TFA, CH2Cl2, rt,
10 min; (2) TES, rt, 12 h. (c) CAN, CH3CN, 0 °C, 30 min. (d) hypotaurine, EtOH/CH3CN, salcomine, rt, 24 h).
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BioorganicChemistry85(2019)240–252
Table 1
clogD values and in vitro antiplasmodial activity of RA (2–5), RB (1 and 6–9), and compound 15 against CQ-sensitive (D10) and CQ-resistant (W2) P. falciparum
strains.a
Comp.
R
D10 IC50 (μM)b
W2 IC50 (μM)b
clogDc
Comp.
R
D10 IC50 (μM)b
W2 IC50 (μM)b
clogDc
RA-2
RA-3
RA-4
RA-5
15
CH2CH(CH3)3
(CH2)3CH3
CH2C6H11
CH2C6H5
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
−0.03
0.15
RB-6
RB-7
RB-8
RB-9
1
CH2CH(CH3)3
(CH2)3CH3
CH2C6H11
CH2C6H5
1.26
1.75
2.37
0.60
2.45
0.21
0.36
1.14
0.21
0.42
2.82
2.19
3.03
0.70
2.37
0.81
0.57
1.14
0.22
0.42
−0.03
0.15
1.15
1.15
0.41
0.41
CH2C6H5
2.67
CH2CH3
−0.91
a
b
c
Chloroquine (CQ) has been used as positive control (D10 IC50 = 0.04
Data are the mean SD of three different experiments in duplicate.
clogD calculated considering pH values: 7.4, 7.2 and 5.5.
0.01; W2 IC50 = 0.54
0.28; not cytotoxic).
alternative (Type II) NADH Dehydrogenases (complex I) [20]. In our case,
the size of the alkyl substituent strongly influenced human cell cytotoxicity
(1 vs. 7, 6 vs. 8; Table 2), while it has no significant effect on the anti-
plasmodial activity. This latter showed instead a significant improvement
when a benzyl substituent was attached as R (compound 9; Table 1); on the
contrary, HMEC-1 toxicity decreased when the cyclohexyl moiety of 8 was
replaced by the aromatic ring of 9 (Table 2). Taken together our results
suggest that, while the cytotoxic activity against human cells is possibly due
to the action on the mitochondrial electron transport chain, for the anti-
plasmodial activity a different, but still redox-based, mechanism of action
has to be evoked. Electrochemical studies previously performed on 1 as
well as on other active and inactive analogues, evidenced that the anti-
plasmodial activity is related to the ability to produce a one-electron re-
duced semi-quinone species able to form a stable adduct with Fe(III)-heme,
in which the metal is still redox-active [8]. On the other hand, previous
biochemical results on thiazinoquinone-based natural compounds active
against human cancer cells, demonstrated that their toxicity is due to the
collapse of the mitochondrial membrane potential, in agreement with the
currently observed SARs [10].
Table 2
IC50 against HMEC-1, Selectivity Index (SI) and clogD values of the active
compounds 1 and 6–9.
Comp.
a
IC50 (μM)
SIb
clogDc
D10
W2
1
6
7
8
9
> 100
> 40.8
21.2
22.9
1.6
> 42.2
9.4
−0.91
−0.03
0.15
26.8
40.2
3.9
6.4
7.6
0.1
1.4
18.3
1.3
1.15
17.6
27.7
25.2
0.41
a
b
c
Data are the mean
SD of three different experiments in duplicate.
SI = IC50 mammalian cells/IC50 P. falciparum strain.
logD calculated considering pH values: 7.4, 7.2 and 5.5.
confirmed a SI value > 25 (Table S3), as observed for the HMEC-1 cell
line.
Based on these results, the newly identified antiplasmodial hit 9 was
subjected to further in vitro pharmacological characterization (see the
dedicated paragraph).
To take into account the role played by molecular pharmacokinetics
on the observed SARs, distribution coefficient values of 1–9 were cal-
culated (clogD, Tables 1 and 2) (ACD/Percepta 2017). RA and RB
presented the same clogD values, indicating no influence of this para-
meter on the different antiplasmodial activity of the two sets of re-
gioisomers. In addition, also within the series of the active RB analo-
gues, no correlation was found between the clogD values and the
antiplasmodial activity (Table 1). On the contrary, it emerged that a
clogD value > 1 is required for cytotoxic activity against HMEC-1 cell
(Table 2), in agreement with Lipinski’s rules for drug absorption via
passive diffusion [21]. Interestingly, the above results are similar to
those previously obtained by us on a series of endoperoxide-based an-
timalarials [22], thus supporting the hypothesis that many anti-
plasmodial agents are actively transported into the infected erythrocyte
by using the large number of membrane transport proteins expressed by
the parasite on the host membrane [23].
2.3. Structure-activity relationships (SARs) of the new thiazinoquinone
derivatives
The whole of the antiplasmodial and cytotoxicity data allows
making some important considerations. First, the in vitro effects on the
parasite strains (Table 1) show that the antiplasmodial activity is not
merely due to the presence of the redox-active 1,4-benzoquinone
system. In fact, compound 15, analogue of the most active compound 9
but lacking the 1,1-dioxo-1,4-thiazine ring, resulted completely in-
active, thus indicating the thiazinoquinone moiety as crucial for the
antiplasmodial activity. In addition, among the tested thiazinoqui-
nones, only RB, featuring the amine group at the same side of the
methoxy group, showed significant antiplasmodial activity (Table 1).
By consequence, not only the dioxothiazinoquinone ring system, but
also a specific relative orientation of the quinone ring substituents, are
required for the antiplasmodial activity.
2.4. Computational studies
Other interesting observations can be made based on the SARs of the
active regioisomers 6–9, particularly considering the structural features
differentiating the antiplasmodial from the cytotoxic activity. It has been
reported for substituted 2-hydroxy-naphthoquinones (atovaquone analo-
gues) that the inhibitory activity against the mitochondrial bc1 complex
increases by increasing the length of the alkyl substituent, with a nine
carbon atoms side-chain resulting the most effective one [19]. The same
has been demonstrated for quinone-based compounds acting on Pf
To rationalize the observed SARs and gain new insight into the
redox-based mechanism of action of our antiplasmodial thiazinoqui-
nones, the steric and electronic features of the new series of compounds
were investigated by mean of computational studies, including con-
formational analysis and DFT calculations.
Conformational analysis. The conformational space of compounds
2–9 and 15, was sampled by applying a calculation protocol including a
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C. Imperatore et al.
BioorganicChemistry85(2019)240–252
Table 3
By consequence, the torsion angle τ2 showed just two possible va-
ΔEGM values (kcal/mol) and torsion angle values (degrees) of the MM con-
lues for each family (τ2 mean value =
91°; standard deviation
formers of compound 9.
of
9°), giving rise to two subsets of conformers (named a and b), for
a total number of four sub-families: Ia (τ1+/τ2+), Ib (τ1+/τ2−), IIa
(τ1−/τ2+), and IIb (τ1−/τ2−).
The two families presented isoenergetic minima, which resulted to
be the mirror image of each other, namely, conformational enantiomers
(Fig. S2, Supporting information). On the other hand, conformers be-
longing to the same subfamily slightly differed in the conformation of
R, still presenting the same rotation of the first methylene group (i.e.,
the same value of τ2) and an overall similar volume occupation of the R
substituent (Fig. S1). Going into details, the isopropyl moiety of 2 and 6
resulted to shift among three conformations for each sub-family
Sub-family
ΔEGM (kcal/mol)a
Torsion Angles (°)b
(τ3 = ∼60°, ∼−60° and ∼180°; τ3GM
=
56°; Tables S4 and S5)
with a maximum contribution to the overall conformational energy of
about 1.4 kcal/mol. Similarly, each sub-family of compounds 3 and 7
presented three possible values for the τ3 and τ4 torsion angles of the R
c
τOMe
τ1d
τ2e
τ3f
Ia
0.00–0.30
0.11–0.36
0.11–0.36
0.00–0.30
−39.20
−40.43
40.43
58.44
95.56
−76.31
−74.61
74.52
IIa
Ib
−58.32
58.32
97.38
butyl chain (∼180°, ∼60°, and ∼−60°; τ3GM
=
179°;
−97.38
−95.60
τ4GM
=
180°; Tables S6 and S7), with a maximum contribution to
IIb
39.24
−58.44
76.26
the overall conformational energy of about 2.3 kcal/mol. On the other
hand, the cyclohexyl ring of 4 and 8 assumed three orientations with
respect to the thiazinoquinone ring (τ3 = ∼60°, ∼−60° and ∼180°;
a
The values reported refer to the lowest and the highest energy conformers
of the sub-family.
b
c
d
e
The values reported refer to the lowest energy conformer of the sub-family.
τ3GM
=
56°; Tables S8 and S9) and the two chair conformations (τ4
τOMe torsion angle is defined by j, k, l, and m atoms.
∼180° and ∼−80°; τ4GM
=
180°; Tables S8 and S9). However, in
τ1 torsion angle is defined by a, b, c, and d atoms.
this case, some conformers, such as, those with τ3 ∼180° and τ4 −80°,
are energetically disfavoured (ΔEGM = 4.7 kcal/mol) while all the
others are grouped within an energy range of 2.3 kcal/mol.
τ2 torsion angle is defined by e, f, g, and h atoms.
f
τ3 torsion angle is defined by f, g, h, and i atoms.
Finally, in the case of compounds 5 and 9, the phenyl ring assumed
just one orientation in all sub-families, corresponding to a τ3 value of
about −78° for family I and 78° for family II (standard devia-
molecular dynamics (MD; simulated annealing (SA)) procedure com-
bined with molecular mechanics (MM) energy minimization (see
Experimental section for details). Then, all resulting conformers within
5 kcal/mol from the global minimum energy (GM) value were classified
into families and sub-families according to the values of their torsion
angles. In Table 3 are reported the results obtained for 9, the most
active compound of the series, while the complete set of results for
compounds 2–8 and 15 is reported in Tables S4–S11 (Supporting
Information). In Fig. 2 are reported the lowest energy conformers of all
compounds superimposed by the quinone ring; the superimposition of
all minima of all compounds is reported in Fig. S1.
tion =
4°; Tables 3 and S10) with all the conformers within
0.34 kcal/mol from the GM.
In summary, our results evidenced that the new thiazinoquinone
derivatives 2–9 show a limited energetically accessible conformational
space (Figs. 2and S1), characterized by (i) the presence of two specular
sets of conformers having the opposite flip of the thiazinoquinone ring
and (ii) a fixed orientation of the hydrogen atoms of the first methylene
moiety of the R substituent (at 2.6 Å from either the closest quinone
oxygen atom and the methoxy oxygen atom; Fig. 2), which restrained
the conformational freedom of R, particularly in the derivatives 5 and 9
where the benzyl substituent presented just one conformation.
Importantly, the conformational behaviour resulted to be the same
between the active (B) and inactive (A) regioisomers (Table 3, Tables
S4–S11) evidencing that the different antiplasmodial activity is not due
to their conformational features.
Firstly, the conformers were grouped into two families, named I and
II, according to the two thiazinoquinone ring flips, that is, having po-
sitive or negative value of τ1 (torsion angles defined as in Table 3 and
Tables S4–S11), respectively (τ1 mean value =
63°; standard de-
viation =
5°).
group (τOMe mean value =
All conformers presented (i) the same orientation of the methoxyl
36°; standard deviation =
5°), (ii) the
rotation of the R substituent constrained by favourable electronic in-
teractions occurring between the hydrogen atoms of the first methylene
group and the oxygen atoms of, both, the quinone ring and the meth-
oxyl group (intramolecular distance = 2.6 Å; Fig. 2).
2.5. DFT calculations
In order to properly investigate the role of electronic properties on
the antiplasmodial activity, DFT studies were performed on a selected
Fig. 2. (A) Top view and (B) transversal view of
the lowest energy conformers of each sub-family
of compounds 2–9 and 15 superimposed by e, f,
k, and j atoms (for atom definition see Table 3).
Carbon atoms are colored according to family
classification
(Ia = green,
IIa = magenta,
Ib = pink, and IIb = light blue); heteroatoms are
colored by atom type (O = red, N = blue,
S = yellow). Hydrogens are omitted for sake of
clarity, with the exception of those of the first R
methylene group, whose intramolecular distance
from the oxygen atoms of the quinone and
methoxyl groups are reported.
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BioorganicChemistry85(2019)240–252
Table 4
Table 5
ΔEGM values (kcal/mol) and torsion angle values (degrees) of the DFT con-
Calculated energy values (kcal/mol) of the LUMO of compounds 4, 8, 5, 9, and
15.
formers of compound 9.
Comp.
4
8
5
9
15
ELUMO
−14.27
−19.74
−15.53
−20.49
−13.60
corresponding inactive regioisomers 4 and 5, and compound 15 (in-
active), which lacks the 1,1-dioxo-1,4-thiazine ring, showed the highest
LUMO energy value.
Then, in the active regioisomers 8 and 9 the LUMO is localized on
the quinone carbonyl function adjacent to the methoxyl substituent
while in the inactive regioisomers 4 and 5 it is placed on the quinone
carbonyl function adjacent to the R substituent (Fig. 3, B, D vs A, C).
Thus, the propensity of the compound to be firstly reduced on one
quinone carbonyl function rather than the other resulted a crucial issue
for antiplasmodial activity. Interestingly, in the studied compounds
such propensity is not dependent on the nature of the R substituent but
on the regiochemistry of the thiazinoquinone system, favouring the
carbonyl function close to the NH group.
Sub-family
ΔEGM (kcal/mol)
Torsion Angles (°)
a
τOMe
τ1b
τ2c
τ3d
Ia
0.00
0.16
0.16
0.00
−61.99
−62.43
62.43
60.22
93.27
−75.24
−67.94
67.94
IIa
Ib
−60.32
60.32
97.69
−97.69
−93.25
IIb
62.01
−60.23
75.21
a
b
c
τOMe torsion angle is calculated considering j, k, l, and m atoms.
τ1 torsion angle is defined by a, b, c, and d atoms.
Starting from the DFT conformers of the parent quinones, the next
step was to generate the corresponding semiquinone radicals and
submit them to full DFT optimization. After DFT calculations, the
semiquinone species of the active regioisomers 8 and 9 presented the
oxygen atom of the methoxyl group involved in a hydrogen bond with
the newly generated hydroxyl group (Fig. 4B and S4B).
τ2 torsion angle is calculated considering e, f, g, and h atoms.
τ3 torsion angle is calculated considering f, g, h, and i atoms.
d
subset of compounds. In particular, calculations were performed on: (i)
the most active compound 9 (D10 IC50 = 0.6 μM, W2 IC50 = 0.7 μM),
presenting a benzyl substituent as R, (ii) its inactive regioisomer 5, (iii)
The unpaired electron spin density of the semi-quinone species was
calculated by using the natural bond orbitals (NBO) population analysis
[25]. It resulted that the radical is differently delocalized on the two
regioisomers (Figs. 4 and S4, A vs B). Importantly, the DFT results also
allowed to hypothesise the possible redox-based mechanism of action of
the active thiazinoquinones. Indeed, in the active regioisomers 8 and 9,
a “through space” hydrogen radical could be shifted from a carbon
atom of the R substituent to the carbonyl oxygen atom (Fig. 5).
The putative hydrogen radical shift would lead to the reduction to
hydroquinone, accompanied by the formation of a carbon radical on the
R substituent. In support to this hypothesis, the above reported con-
formational analysis results showed that all low energy conformers
present a hydrogen of the first methylene group of R placed at 2.6 Å
from the quinone oxygen, a distance suitable for an intra-molecular
hydrogen radical shift. In the semi-quinone form of 9 this distance is
shortened to 2.45 Å (Fig. 4B), in this compound the hydrogen radical
shift would be favoured by the formation of a benzyl radical (10) and
this could account for its higher antiplasmodial activity with respect to
the other analogues in which R is an aliphatic substituent.
compound
4 (not active) and (iv) 8 (D10 IC50 = 2.4 μM, W2
IC50 = 3.0 μM), that is, the couple of regioisomers presenting a cyclo-
hexylmethyl substituent as R, and, finally, (v) compound 15 (not ac-
tive), lacking the dioxothiazine ring. In order to mimic an aqueous
environment, all DFT calculations were performed using the conductor-
like polarizable continuum model (C-PCM) [24] as solvent model.
Moreover, to characterize every structure as minimum, a vibrational
analysis was carried out (see the Experimental Section for details).
Fully optimized DFT conformers were classified into families and
sub-families as previously described for the MM conformers (Tables 4
and S12–S15).
As can be inferred by comparing the data in Table 3 with those in
Table 4, after DFT optimization, the structures of the neutral quinone
species conserved their overall conformational features (root mean
square deviation (RMSD) mean value on all atoms: 0.2830 Å; Fig. S3)
and energy classification (Tables S12–S15).
The mechanism of action of antimalarial quinones is ascribed to
their redox properties, and, in particular, the antiplasmodial activity of
quinone-based compounds has been related to their semi-quinone form
[8]. Accordingly, starting from the lowest energy conformer of each
subfamily, we considered the one-electron reduction pathway of the
thiazinoquinone scaffold in protic solvents (Scheme 3).
On these bases, we propose that thiazinoquinones, upon interaction
with the redox partner, give rise to an oxygen-centered radical. Then,
an intramolecular radical shift to a carbon atom of the side chain R
occurs that leads to the putative toxic species for the Plasmodium en-
vironment.
According to the frontier orbital theory, the energy of the lowest
unoccupied molecular orbital (LUMO) accounts for the propensity of
the molecule to acquire an electron while its location allows to identify
the most probable site to be reduced (i.e., to accept the electron).
Hence, we analysed the energy and the location of the LUMO in all the
DFT minima. The results evidenced a relationship between the observed
antiplasmodial activity and, both, electron affinity (Table 5) and the
LUMO position in the quinone species (Fig. 3).
Heme metabolism represents a well-known antimalarial target [26]
and the one-electron reduction of the natural endoperoxide drug arte-
misinin by free heme has been reported to be the key bio-activation step
leading to the toxic radical species [27]. As above mentioned, previous
cyclovoltammetry studies [8] evidenced that the thiazinoquinone de-
rivatives showing antiplasmodial activity towards D10 and W2 strains
(< 10µ), produced a redox-active adduct between their semi-quinone
form and free heme, and that the stability of such adduct was related to
their antiplasmodial potency. On the contrary, in the same conditions,
the inactive analogues underwent a two-electron reduction/oxidation
cycle without any interaction with the free heme present in solution.
Accordingly, to complete our investigation, we performed a fragment-
based search in the Cambridge Crystallographic Structural Database
(software Conquest 1.19; Cambridge Structural Database System 2016)
to investigate which functional group of the methoxy-thiazinoquinone
Firstly, the rank of the calculated LUMO energy values (Table 5)
reflected the antiplasmodial activity (Table 1). Indeed, compounds 8
and 9 presented significantly lower LUMO energy with respect to the
Scheme 3. Quinone one-electron reduction pathway in protic solvents.
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C. Imperatore et al.
BioorganicChemistry85(2019)240–252
Fig. 3. Localization of the lowest unoccupied molecular orbital (LUMO) of 4 (A), 8 (B), 5 (C), and 9 (D). The ligands are displayed in ball&stick and colored by atom
type (C = gray, O = red, N = blue, S = yellow and H = white). The orbitals were visualized using GaussView with an isosurface value of 0.02 e−/a.u.3.
system could be suitable for iron interaction (for details see experi-
mental section). In summary, the analysis of 192 identified hits evi-
denced that only the oxygen atoms of the quinone and methoxyl
functions were found to coordinate iron in X-ray determined complexes
(Fig. 6). On the contrary, the SO2 function alone or adjacent to a qui-
none oxygen were not found to complex iron.
the methoxyl group favours the interaction with the redox partner (i.e.,
heme) without interfering with the redox reaction.
3. In vitro pharmacological characterization of compound 9
3.1. Effect of N-acetyl-L-cysteine (NAC) on the antiplasmodial activity
Taken together, our results suggested that (i) the 1,1-dioxo-1,4-
thiazine ring fused to the quinone ring, as well as, the regiochemistry of
the resulting thiazinoquinone system, play a key role in the electron
affinity of the compounds, (ii) the quinone oxygen near the amine
function is likely the first to be reduced, being involved in the first step
of the putative bio-activation reaction (i.e., one-electron reduction re-
action), (iii) the positioning of the hydrogen atoms of the first methy-
lene group of the R substituent with respect to the semi-quinone oxygen
radical is critical for the observed antiplasmodial activity since it may
favour an hydrogen radical shift from the carbon to the oxygen atom
generating a toxic radical species able to impair the Pf antioxidant
defences, (iv) the nature of substituent R affects the antiplasmodial
potency according to the stability of the putative carbon radical, and (v)
To support the hypothesis that the mechanism of action of our an-
tiplasmodial thiazinoquinones was related to the generation of toxic
free radicals, compound 9 was tested against Pf infected red blood cells
(RBCs) pre-treated with increasing doses of the free radical scavenger
NAC. As shown in Table 6, the antiplasmodial activity of compound 9
was dose dependently decreased (i.e., the IC50 was increased) by dif-
ferent doses of NAC. NAC is a well-known antioxidant which stimulates
the synthesis of glutathione (GSH) [28]. Pre-treatment of infected RBC
with NAC might lead to an increase of reduced GSH preventing the
accumulation of free radicals. For example, the antiplasmodial activity
of artesunate, which is known to induce free radicals formation, is also
reduced by NAC [29].
Fig. 4. NBO spin density isosurface of QH• spe-
cies of 5 (A) and 9 (B). The spin density was
visualized using GaussView with an isosurface
value of 0.01 e−/a.u.3. The blue surface (positive
spin density) corresponds to an excess of α-
electron density. The intramolecular distance
between the methylene hydrogen of R and the
oxygen atom of the quinone is reported.
Hydrogen bonds are highlighted by a red dashed
line. The ligands are displayed in ball&stick and
colored by atom type (C = gray, O = red,
N = blue, S = yellow and H = white).
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C. Imperatore et al.
BioorganicChemistry85(2019)240–252
Fig. 5. Proposed mechanism for the formation of the putative toxic free radical (10) through generation of a semi-quinone species of 9 (red arrows indicate electron
delocalization) followed by intramolecular hydrogen radical shift.
Fig. 7. Percentage of methaemoglobin formation after 24 h treatment of RBC
with different doses of compounds 5 or compound 9. RBC were left in medium
or pretreated with increasing concentration of NAC for 3 h and then incubated
Fig. 6. QH• species of 9 (green) superimposed on the X-ray structure of 2-
methoxyhydroquinonate in complex with iron (orange; CSD code: EPIMIB). The
superimposition was made fitting the iron coordinating groups. vdW volume of
iron atom (cyan) is scaled (0.5) for clarity. Heteroatoms are colored: O, red; N,
blue; Fe, magenta; and Cl, light green.
with compound 5 or 9 for further 24 h. Methaemoglobin formation was then
determined. Data are from a representative experiment in triplicate.
out on compound 5, the inactive regioisomer of 9. As shown in Fig. 7,
the levels of methaemoglobin in control RBCs were less than 1%, as
reported for normal RBCs [31]. Compound 5 did not induced methae-
moglobin formation at any of the doses used, whereas 9 increased the %
of methaemoglobin formation at doses higher than 7.4 µM, but no more
than 30% methaemoglobin at the highest concentration. To verify the
ability of NAC to protect from methaemoglobin formation, the experi-
ments were conducted pretreating RBCs with NAC. Differently from
what observed for the activity of compound 9 on the parasite, NAC was
not able to protect from methaemoglobin formation induced by com-
pound 9. These results clearly indicate that only the active thiazino-
quinone is able to oxidize haemoglobin, however only at higher con-
centration and, contrarily to what observed for the antiplasmodial
activity, the presence of NAC did not affect methaemoglobin formation.
To further exclude a direct toxic effect of compounds 5 and 9 on
RBCs, haemolysis was measured in uninfected and infected RBCs
(iRBCs) treated with the compounds. No appreciable haemolytic ac-
tivity of either uninfected or infected RBCs was observed compared to
untreated controls with compounds 5 and 9 even at very high con-
centration (Fig. 8).
Table 6
In vitro antiplasmodial activity of compound 9 against P. falciparum, W2 (CQ-R)
strain, pretreated with increasing concentration of N-acetyl-L-cysteine (NAC).
Comp. W2 (CQ-R)
- IC50 (µM)a NAC 0.1 IC50
NAC 1 IC50
(µM)a
NAC 10 IC50
(µM)a
(µM)a
9
0.7
0.2
0.7
0.2
1.0
0.2
1.3
0.3
a
Data are the mean and SD of three experiments in duplicate.
3.2. Red blood cells (RBCs) toxicity
The formation of methaemoglobin, which can be generated by
oxidative stress, was evaluated in RBCs treated with different doses of
compound 9. The formation of methaemoglobin could be indicative of
possible toxicity on RBCs. However, methaemoglobin was formed only
at doses of compound 9 higher than the IC50 on asexual parasite, in-
dicating that the active doses were not toxic.
3.3. Combination with dihydroartemisinin (DHA)
The experiment was carried out using the algorithm of Winterbourn
[30] and was performed in the complete medium used for parasite
growth in the presence or not of NAC. Similar experiments were carried
Experiments were conducted to investigate a possible application of
compound 9 in an artemisinin-based combination therapy (ACT).
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BioorganicChemistry85(2019)240–252
the postulated redox-based mechanism of action of our thiazinoqui-
nones, it is not surprising that the in vitro antimalarial activity of DHA is
enhanced by addition of another free radical generating compound,
such as 9 [27,33,34].
3.4. Activity on late stage gametocytes
Eradicating malaria will require medicines that prevent transmis-
sion of the parasite between humans and mosquitoes. The goal to
prevent Pf transmission from humans to mosquitoes requires the iden-
tification of a molecular target in mature (IV–V) stage gametocytes, the
sexual stages circulating in the bloodstream. To investigate the trans-
mission blocking potential of the methoxy-thiazinoquinone scaffold,
compound 9 was tested against V stage Pf gametocytes. An IC50 of
13.8
5.6 µM was obtained, indicative of a lower activity against the
Fig. 8. Haemolytic effect of compounds 5 and 9, evaluated as release of hae-
moglobin in the supernatants (OD405). RBC or iRBC were incubated for 24 h
with different concentration of compound 5 or 9. The histogram represents the
ratio between the OD treated versus untreated RBC or iRBC. Data are from a
representative experiment in triplicate.
sexual blood parasite stage with respect to the asexual stage.
Nevertheless, this represented an encouraging result since, while some
antimalarial drugs are active against immature (I–III) stage gameto-
cytes, targeting mature (IV–V) stage gametocytes is currently proble-
matic as they are refractory to most antimalarial drugs, with the ex-
ception of primaquine. DHA itself induces a 40% of mature gametocytes
growth inhibition only at 10 μM [35]. Even a drug in clinical devel-
opment with activity against gametocytes, such as OZ277, has been
shown to have in vitro activity against stage V gametocytes with an IC50
of 6.4 μM [35,36].
Commonly, studies on drug combinations are performed in order to
identify synergistic interactions to reduce the doses of the drugs, and,
consequently, the side effects, maintaining the therapeutic efficacy. In
the case of antimalarial drugs, combination therapies are used both to
improve drug efficacy, and to delay the development of resistance to
the individual components of the combination by reducing the selection
of drug-resistant parasites.
4. Conclusions
The in vitro interaction of 9 with DHA was analysed by the use of
isobolograms. Compound 9 was applied alone or in combination with
DHA to W2 strain of Pf. In addition, a dose-response curve of DHA alone
was performed to obtain the DHA IC50. When tested in combination,
five fixed doses of DHA (from 0.002 to 0.8 nM) were added to the dose-
response curve of compound 9 (from 0.075 to 5 µM). The isobologram
was, then, constructed by plotting a pair of fractional IC50s (IC50 of the
drug in combination/IC50 of the drug alone) for each combination of
compound 9 with DHA (Fig. 9).
Starting from the previously identified hit candidate 1, a small and
rationally designed series of new antiplasmodial methoxy-thiazinoqui-
none derivatives has been developed. In particular, the role played on
the activity by both the regiochemistry of the methoxy-thiazinoquinone
system and the nature of the side chain linked at the quinone ring has
been explored. These studies afforded a new hit candidate, compound
9, endowed with higher antiplasmodial potency compared to 1, no
toxicity against RBCs, a good (> 25) selectivity index when tested
against a panel of mammalian cells, and a promising inhibitory activity
on stage V gametocyte growth. A synergistic antiplasmodial action in
combination with DHA was evidenced, too. At the same time, compu-
tational studies shed light on the putative mechanism of action of an-
tiplasmodial thiazinoquinones. In particular, it emerged that the re-
giochemistry of the thiazinoquinone system plays a key role both in the
electron affinity of the compounds and in determining the first site to be
reduced in the putative bio-activation step. Then, in the active RB, the
positioning of the hydrogen atoms of the first methylene of the R alkyl
group with respect to the semi-quinone oxygen radical represents a key
issue which could favour a hydrogen radical shift and form a toxic
carbon radical species able to impair the Pf antioxidant defences. In this
view, the higher antiplasmodial potency of 9 reflects the stability of the
benzyl radical formed after the putative radical shift.
An isobologram close to the diagonal indicates an additive effect,
curves above or below the diagonal indicate antagonistic or synergistic
effect, respectively. The isobologram obtained for compound 9 provides
a graphical representation of a synergistic interaction of this compound
with DHA against W2 strain. This underlies a synergistic mechanism of
action through which the combined drugs cooperate in killing the
parasite [32]. Our results can be explained taking into account that
artemisinin, as other endoperoxide-based antimalarials, are reported to
be bioactivated via the reduction of the dioxane function, likely by
heme or ferrous iron, thereby producing toxic free radicals which in-
duce oxidative stress within the parasite [27,33,34]. Thus, according to
5. Experimental section
5.1. General methods
Commercial reagents: Sigma–Aldrich. Solvents: Carlo Erba. TLC:
Silica Gel 60 F254 (plates 5 × 20, 0.25 mm) Merck. Preparative TLC:
Silica Gel 60 F254 plates (20 × 20, 2 mm). Spots revealed by UV lamp
then by spraying with 2 N sulfuric acid and heating at 120 °C.
Anhydrous solvents: Sigma–Aldrich or prepared by distillation ac-
cording to standard procedures. High-resolution ESI-MS analyses were
performed on a Thermo LTQ Orbitrap XL mass spectrometer (Thermo-
Fisher, San Josè, CA, USA). The spectra were recorded by infusion into
the ESI (Thermo-Fisher, San Josè, CA, USA) source dissolving the
sample in MeOH. 1H (700 MHz) and 13C (175 MHz) NMR spectra were
Fig. 9. Isobologram of the interaction between different doses of compound 9
and DHA on W2 strain of P. falciparum. The data are the mean of three in-
dependent experiments.
recorded on
a Agilent INOVA spectrometer (Agilent Technology,
248

C. Imperatore et al.
BioorganicChemistry85(2019)240–252
Cernusco sul Naviglio, Italy) equipped with a 13C enhanced HCN Cold
Probe; chemical shifts were referenced to the residual solvent signal
(CDCl3: δH = 7.26, δC = 77.0; DMSO‑d6 δH = 2.39, δC = 39.0). For an
accurate measurement of the coupling constants, the one-dimensional
1H NMR spectra were transformed at 64 K points (digital resolution:
0.09 Hz). Homonuclear (1H-1H) and heteronuclear (1H-13C) con-
nectivities were determined by COSY and HSQC experiments, respec-
tively. Two and three bond 1H-13C connectivities were determined by
C11H14O3Na+: 217.0835, found: 217.0841. (12-R2): 1H NMR (CDCl3):
6.66 (1H, d, J = 10.0 Hz, H-6); 6.51 (1H, d, J = 10.0 Hz, H-5); 4.01 (3H,
s, -OCH3); 2.42 (2H, t, J = 7.4 Hz, CH2CH2CH2CH3); 1.47 (2H, m,
CH2CH2CH2CH3); 1.39 (2H, m, CH2CH2CH2CH3); 0.92 (3H, d,
J = 6.7 Hz, CH3); HRMS (ESI): m/z [M+Na+
]
+calcd. for
C11H14O3Na+: 217.0835, found: 217.0843. (12-R3): 1H NMR (CDCl3):
6.69 (1H, d, J = 8.8 Hz, H-6); 6.52 (1H, d, J = 8.8 Hz, H-5); 4.01 (3H, s,
-OCH3); 2.35 (2H, d, J = 7.4 Hz, CH2-cyclohexyl); 1.55 (1H, m, CH);
1.53–0.95 (4H, m); 1.15–1.66 (4H, m), 1.60 (2H, m); HRMS (ESI): m/z
gradient 2D HMBC experiments optimized for a 2,3J of 8 Hz. JH-H va-
3
lues were extracted from 1D 1H NMR. High performance liquid chro-
matography (HPLC) separations were achieved on a Shimadzu LC-10AT
(Shimadzu, Milan, Italy) apparatus equipped with a Knauer K-2301
(LabService Analytica s.r.l., Anzola dell'Emilia, Italy) refractive index.
[M+Na+
]
+calcd. for C14H18O3Na+: 257.1148, found: 257.1139.
5.2.3. Condensation of quinones 12R1- R3 with hypotaurine
1.5 mmol of each quinone 12R1–R3 (290 mg of 12-R1, 290 mg of
12-R2, 350 mg of 12-R3) were dissolved in a mixture of EtOH/
CH3CN = 1: 1 and heated in a water bath under stirring; then, a solu-
tion of hypotaurine (163.7 mg, 1.5 mmol) in 8 mL of water and a cat-
alytic amount of salcomine were added in portions. The mixture was
stirred for 24 h at room temperature and the yellow solution became
orange/red. Most of the ethanol was removed in vacuo and the residue
was poured into water. The mixture was extracted with diethyl ether
(three times) and the organic phase was washed with brine, dried over
sodium sulfate, and filtered. Solvent removal gave residues containing
mixtures of compounds 2/6 (from 12-R1, 170 mg, 38% yield), 3/7
(from 12-R2, 173 mg, 39% yield) as well as of compounds 4/8 (from
12-R3, 167 mg, 33% yield); they were separated as reported below.
5.2. Syntheses
5.2.1. 2-isobutyl-1,3,4-trimethoxybenzene
(11-R1),
2-butyl-1,3,4-
trimethoxybenzene (11-R2) and 2-(cyclohexylmethyl)-1,3,4-trimethoxy-
benzene (11-R3)
1.68 g (10 mmol) of 1,2,4-trimethoxybenzene (10) were dissolved in
25 mL of anhydrous THF and 12.5 mL of a n-BuLi 1.6 M solution
(20 mmol) were added, under argon atmosphere at 0 °C; the mixture
was stirred for 1 h. Then, 20 mmol of 1-bromo-2-methylpropane
(2.2 mL), 1-bromobutane (2.1 mL), or bromomethylcyclohexane
(2.8 mL) were added and the mixture was left under stirring for 24 h
(the end of the reaction was controlled with TLC, eluent: chloroform/
hexane = 7:3). After this time, the mixture was poured into cold water
(150 mL) and extracted three times with diethyl ether. The ethereal
solution was washed with brine several times, dried over sodium sul-
fate, filtered and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (hexane/EtOAc = 9:1)
to afford 11-R1 (1.8 g, 80.3% yield), 11-R2 (1.7 g, 76.0% yield) and 11-
R3 (2.0 g, 76.5% yield) as colorless oil. 11-R1: 1H NMR (CDCl3): 6.68
(1H, d, J = 8.8 Hz, H-6); 6.51 (1H, d, J = 8.8 Hz, H-5); 3.79 (3H, s,
-OCH3); 3.78 (3H, s, -OCH3); 3.73 (3H, s, -OCH3); 2.49 (2H, d,
5.2.4. Separation of crude mixtures of 2/6, 3/7 and 4/8 isomers
Separation of isomers 2 and 6 mixture (170 mg) was achieved by
HPLC on a SiO2 column (Luna 5 µm, 250 × 4.60 mm) eluting with
EtOAc/hexane 6:4 (v/v) and afforded pure compounds 2 (8 mg) and 6
(70 mg). Mixture of isomers 3 and 7 (170 mg), as well as that of 4 and 8
(167 mg), were separated in the same conditions and yielded pure
compounds 3 (7 mg), 7 (67 mg), 4 (5 mg), and 8 (49 mg).
J = 7.3 Hz, CH2); 1.89 (1H, m, J = 7.3 Hz-6.7 Hz, CH); 0.86 (6H, d,
5.2.5. 6-isobutyl-7-methoxy-3,4-dihydro-2H-benzo[b][1,4]thiazine-5,8-
dione 1,1-dioxide (2)
J = 6.7 Hz, CH3); HRMS (ESI): m/z [M+Na+
]
calcd. for
+
13H20O3Na+: 247.1305, found: 247.1314. 11-R2: 1H NMR (CDCl3):
HPLC EtOAc/hexane 6:4 (v/v), (tR): 13.5 min (single peak). 1H and
13C NMR data are reported in Tables S1 and S2. HRMS (ESI): m/z [M
C
6.69 (1H, d, J = 8.8 Hz, H-6); 6.52 (1H, d, J = 8.8 Hz, H-5); 3.77 (3H, s,
-OCH3); 3.74 (3H, s, -OCH3); 3.72 (3H, s, -OCH3); 2.63 (2H, t,
J = 7.4 Hz, CH2CH2CH2CH3); 1.49 (2H, m, CH2CH2CH2CH3); 1.38 (2H,
m, CH2CH2CH2CH3); 0.92 (3H, d, J = 6.7 Hz, CH3); HRMS (ESI): m/z
+Na+
]
+calcd. for C13H17NO5SNa+: 322.0720, found: 322.0727.
[M+Na+
]
+calcd. for C13H20O3Na+: 247.1305, found: 247.1315. 11-
5.2.6. 7-isobutyl-6-methoxy-3,4-dihydro-2H-benzo[b][1,4]thiazine-5,8-
dione 1,1-dioxide (6)
R3: 1H NMR (CDCl3): 6.69 (1H, d, J = 8.8 Hz, H-6); 6.52 (1H, d,
J = 8.8 Hz, H-5); 3.80 (3H, s, -OCH3); 3.78 (3H, s, -OCH3); 3.74 (3H, s,
-OCH3); 2.50 (2H, d, J = 7.4 Hz, CH2-cyclohexyl); 1.55 (1H, m, CH);
1.53–0.95 (4H, m); 1.15–1.66 (4H, m), 1.60(2H, m); HRMS (ESI): m/z
HPLC EtOAc/hexane 6:4 (v/v), (tR): 24 min (single peak). 1H and
13C NMR data are reported in Tables S1 and S2. HRMS (ESI): m/z [M
+Na+
]
+calcd. for C13H17NO5SNa+: 322.0720, found: 322.0729.
[M+Na+
]
+calcd. for C16H24O3Na+: 287.1618, found: 287.1626.
5.2.7. 6-butyl-7-methoxy-3,4-dihydro-2H-benzo[b][1,4]thiazine-5,8-dione
1,1-dioxide (3)
5.2.2. 2-isobutyl-3-methoxycyclohexa-2,5-diene-1,4-dione (12-R1), 2-butyl-3-
methoxycyclohexa-2,5-diene-1,4-dione (12-R2), 2-(cyclohexylmethyl)-3-
methoxycyclohexa-2,5-diene-1,4-dione (12-R3)
HPLC EtOAc/hexane 6:4 (v/v), (tR): 11.5 min (single peak). 1H and
13C NMR data are reported in Tables S1 and S2. HRMS (ESI): m/z [M
3.5 mmol of each compound 11R1–R3 (780 mg of 11-R1, 780 mg of
11-R2, 925 mg of 11-R3) dissolved in 90 mL of acetonitrile were added
dropwise to a solution of CAN (9.6 g, 17.5 mmol) in water (100 mL) at
0 °C. The mixture was stirred for 30 min at room temperature (the end
of the reaction was checked by TLC, eluent: chloroform/EtOAc = 7:3).
The orange liquid was then poured into 100 mL of cold water and ex-
tracted three times with diethyl ether. The combined organic layers
were washed with brine, dried over sodium sulfate, and filtered. The
solvent removal under reduced pressure afforded 12-R1 (445 mg, 65%),
12-R2 (445 mg, 65%), and 12-R3 (610 mg, 74%) sufficiently pure for
the following reaction. 12-R1: 1H NMR (CDCl3): 6.66 (1H, d,
J = 10.0 Hz, H-6); 6.51 (1H, d, J = 10.0 Hz, H-5); 3.99 (3H, s, -OCH3);
2.34 (2H, d, J = 7.3 Hz, CH2); 1.81 (1H, m, J = 7.3 Hz-6.7 Hz, CH);
+Na+
]
+calcd. for C13H17NO5SNa+: 322.0720, found: 322.0733.
5.2.8. 7-butyl-6-methoxy-3,4-dihydro-2H-benzo[b][1,4]thiazine-5,8-dione
1,1-dioxide (7)
HPLC EtOAc/hexane 6:4 (v/v), (tR): 22 min (single peak). 1H and
13C NMR data are reported in Tables S1 and S2. HRMS (ESI): m/z [M
+Na+
]
+calcd. for C13H17NO5SNa+: 322.0720, found: 322.0735.
5.2.9. 6-(cyclohexylmethyl)-7-methoxy-3,4-dihydro-2H-benzo[b][1,4]
thiazine-5,8-dione 1,1-dioxide (4)
HPLC EtOAc/hexane 6:4 (v/v), (tR): 11.8 min (single peak). 1H and
13C NMR data are reported in Tables S1 and S2. HRMS (ESI): m/z [M
0.89 (6H, d, J = 6.7 Hz, CH3); HRMS (ESI): m/z [M+Na+
]
+calcd. for
+Na+
]
+calcd. for C16H21NO5SNa+: 362.1033, found: 362.1042.
249

C. Imperatore et al.
BioorganicChemistry85(2019)240–252
5.2.10. 7-(cyclohexylmethyl)-6-methoxy-3,4-dihydro-2H-benzo[b][1,4]
thiazine-5,8-dione 1,1-dioxide (8)
5.2.15. Separation of crude mixtures of 5 and 9 isomers
Separation of isomers 5 and 9 mixtures (175 mg) was achieved by
HPLC on a SiO2 column (Luna 5 µm, 250 × 4.60 mm) eluting with
EtOAc/hexane = 6:4 (v/v) and afforded pure compounds 5 (7 mg) and
9 (63 mg).
HPLC EtOAc/hexane 6:4 (v/v), (tR): 21.4 min (single peak). 1H and
13C NMR data are reported in Tables S1 and S2. HRMS (ESI): m/z [M
+Na+
]
+calcd. for C16H21NO5SNa+: 362.1033, found: 362.1038.
5.2.11. phenyl(2,3,6-trimethoxyphenyl)methanol (13)
5.2.16. 6-benzyl-7-methoxy-3,4-dihydro-2H-benzo[b][1,4]thiazine-5,8-
dione 1,1-dioxide (5)
To a solution of 1,2,4-trimethoxybenzene (1.0 mL, 6.7 mmol) in THF
(25 mL) was added 6 mL n-BuLi 1.6 M solution (10 mmol) under argon
atmosphere at 0 °C; the mixture was stirred for 1 h. Then, the benzal-
dehyde (1.0 mL, 10 mmol) was added and the mixture was left under
stirring at room temperature overnight. and quenched with saturated
NH4Cl solution (30 mL). The two layers were separated and the aqueous
layer was extracted with ether (three times). The combined organic
layers were washed with brine, dried over anhydrous sodium sulfate,
and concentrated in vacuo. The crude material was purified by column
chromatography on silica gel (hexane/EtOAc = 8:2) to afford the cor-
responding biaryl alcohol 13 (1.3 g, 70% yield): 1H NMR (CDCl3): 6.82
(1H, d, J = 8.8 Hz, H-6); 6.62 (1H, d, J = 8.8 Hz, H-5); 3.82 (3H, s,
-OCH3); 3.74 (3H, s, -OCH3); 3.61 (3H, s, -OCH3); 6.25 (1H, d,
J = 11.7 Hz, CH-OH); 7.36 (2H, d, J = 7.0 Hz); 7.28 (2H, t, J = 7.6 Hz);
7.19 (1H, t, J = 7.6 Hz); 4.29 (1H, d, J = 11.7 Hz, OH). HRMS (ESI): m/
HPLC EtOAc/hexane 6:4 (v/v), (tR): 14 min (single peak). 1H and
13C NMR data are reported in Tables S1 and S2. HRMS (ESI): m/z [M
+Na+
]
+calcd. for C16H15NO5SNa+: 356.0563, found: 356.0565.
5.2.17. 7-benzyl-6-methoxy-3,4-dihydro-2H-benzo[b][1,4]thiazine-5,8-
dione 1,1-dioxide (9)
HPLC EtOAc/hexane 6:4 (v/v), (tR): 27 min (single peak). 1H and
13C NMR data are reported in Tables S1 and S2. HRMS (ESI): m/z [M
+Na+
]
+calcd. for C16H15NO5SNa+: 356.0563, found: 356.0565.
5.3. Parasite growth and drug susceptibility assay
Unless stated otherwise all reagents were from Sigma Italia, Milan,
Italy.
z [M+Na+
]
+calcd. for C16H18O4Na+: 297.1097, found: 297.1109.
The CQ sensitive (D10) and the CQ resistant (W2) strains of Pf were
maintained in vitro as described [37] at 5% hematocrit (human type A-
positive red blood cells) in RPMI 1640 (EuroClone, Celbio) medium
with the addition of 1% AlbuMax (Invitrogen, Milan, Italy), 0.01%
hypoxanthine, 20 mM HEPES, and 2 mM glutamine. Cultures were
maintained at 37 °C in a standard gas mixture of 1% O2, 5% CO2, and
94% N2. Compounds were dissolved in DMSO and then diluted with
medium to achieve the required concentrations (final DMSO con-
centration < 1%, which is nontoxic to the parasite). Drugs were placed
in 96 well flat-bottom microplates and serial dilutions made. Asyn-
chronous cultures (parasitemia of 1–1.5%; 1% final hematocrit) were
aliquoted into the plates and incubated for 72 h at 37 °C. In some ex-
periments, the W2 strain culture was pretreated with NAC (0.1, 1,
10 µM) for 3 h at 37 °C. Parasite culture was then washed with PBS
before the chemosensitivity assay. Parasite growth was determined
spectrophotometrically (OD650) by measuring the activity of the para-
site lactate dehydrogenase (pLDH), according to a modified version of
Makler's method in control and drug-treated cultures [38]. Anti-
plasmodial activity is expressed as the 50% inhibitory concentrations
(IC50) which is the dose of compound necessary to inhibit cell growth
by 50%.
5.2.12. 2-benzyl-1,3,4-trimethoxybenzene (14)
Trifluoroacetic acid (0.5 mL, 7.2 mmol) was slowly added to a so-
lution of phenyl(2,3,6-trimethoxyphenyl)methanol (13) (650 mg,
2.4 mmol) and triethylsilane (1.1 mL, 7.2 mmol) in dichloromethane
(30 mL). The mixture was stirred at room temperature overnight. The
organic phase was washed with a sodium carbonate solution (30 mL)
and dried. The solvent removal afforded 14 (570 mg, 93%) sufficiently
pure for the following reaction. 1H NMR (CDCl3): 6.73 (1H, d,
J = 8.8 Hz, H-6); 6.56 (1H, d, J = 8.8 Hz, H-5); 3.80 (3H, s, -OCH3);
3.72 (3H, s, -OCH3); 3.68 (3H, s, -OCH3); 4.01 (1H, s, CH2-benzyl); 7.22
(2H, d, J = 7.0 Hz); 7.18 (2H, t, J = 7.6 Hz); 7.09 (1H, t, J = 7.6 Hz).
HRMS (ESI): m/z [M+Na+
]
+calcd. for C16H18O3Na+
: 281.1148,
found: 281.1154.
5.2.13. 2-benzyl-3-methoxycyclohexa-2,5-diene-1,4-dione (15)
502 mg (2.2 mmol) of 14 dissolved in 90 mL of acetonitrile were
added dropwise to a solution of CAN (6.0 g, 11.0 mmol) in water
(110 mL) at 0 °C. The mixture was stirred for 30 min at 0 °C (the end of
the reaction was checked by TLC, eluent: chloroform/EtOAc = 7:3).
The orange liquid was then poured into 100 mL of cold water and ex-
tracted three times with diethyl ether. The combined organic layers
were washed with brine, dried over sodium sulfate, and filtered. The
solvent removal under reduced pressure afforded 324 mg of 15 (65%),
sufficiently pure for the following reaction. 1H NMR (CDCl3): 6.66 (1H,
d, J = 10.0 Hz, H-6); 6.57 (1H, d, J = 10.0 Hz, H-5); 4.02 (3H, s,
-OCH3); 3.77 (1H, s, CH2-benzyl); 7.25 (2H, d, J = 7.0 Hz); 7.23 (2H, t,
J = 7.6 Hz); 7.16 (1H, t, J = 7.6 Hz). HRMS (ESI): m/z [M
5.3.1. Combination of compound 9 with DHA
The in vitro interactions of compound 9 with DHA were determined
by the analysis of the isobolgrams. The drugs were applied alone or in
combination against W2 strain of Pf. Five fixed doses of DHA (from 0.8
to 0.002 nM) were added to the dose-response curves of compound 9
(from the dose of 5 µM to 0.075 µM). In addition, a dose-response curve
of DHA alone was performed to obtain the IC50 of DHA alone.
Isobolograms were constructed by plotting a pair of fractional IC50s
for each combination of compound 9 with DHA. The fractional IC50 for
compound 9 was calculated by dividing the IC50 of compound 9 in
combination with DHA by the IC50 of compound 9 alone and this data
were plotted on the horizontal axis. The corresponding DHA fractional
IC50 was calculated by dividing each fixed concentration by the IC50 of
the DHA alone and was plotted on the vertical axis. An isobologram
close to the diagonal indicates an additive effect. Curves above or below
the diagonal indicate antagonistic or synergistic effect, respectively.
+Na+
]
+calcd. for C14H12O3Na+: 251.0679, found: 251.0689.
5.2.14. Condensation of quinone 15 with hypotaurine
324 mg (1.42 mmol) of quinone 15 was dissolved in 26 mL of a
mixture of EtOH/CH3CN 1:1 and heated in a water bath under stirring;
then, a solution of hypotaurine (155 mg, 1.42 mmol) in 2 mL of water
and a catalytic amount of salcomine were added in portions. The
mixture was stirred for 24 h at room temperature (the end of the re-
action was checked by TLC, eluent: chloroform: EtOAc = 7:3). The
yellow solution became orange/red. Most of the ethanol was removed
in vacuo and the residue was poured into water. The mixture was ex-
tracted with diethyl ether (three times) and the organic phase was
washed with brine, dried over sodium sulfate, and filtered. Solvent
removal gave residues containing a mixture of the isomers 5 and 9
(175 mg, 37%) which was separated as reported below.
5.4. Cytotoxicity assay
The long-term human microvascular endothelial cell line (HMEC-1)
was maintained in MCDB 131 medium (Invitrogen, Milan, Italy) sup-
plemented with 10% heat-inactivated fetal calf serum (HyClone, Celbio,
250

C. Imperatore et al.
BioorganicChemistry85(2019)240–252
Milan, Italy), 10 ng/ml of epidermal growth factor (Chemicon), 1 µg/ml
of hydrocortisone, 2 mM glutamine, 100 U/ml of penicillin, 100 µg/ml
of streptomycin, and 20 mM Hepes buffer (EuroClone). The human
THP-1 monocytic leukaemia cells were maintained in RPMI 1640
(EuroClone) supplemented with 100 U/ml of penicillin, 100 μg/ml
streptomycin, 20 mM HEPES, 1 mM Sodium Pyruvate, 0.05 mM β-
mercaptoethanol, and 10% heat-inactivated fetal calf serum
(EuroClone). The immortalized mouse C57Bl/6 bone marrow macro-
phages (BMDM) were maintained in DMEM, supplemented with 10%
fetal calf serum (EuroClone), 25 mM HEPES, 4 mM L-glutamine, and
10 μg/ml ciprofloxacin.
5.6.2. Redox property analysis
The lowest energy conformer of each sub-family of 4–5, 8–9 and 15
have been then subjected to DFT calculations. The calculations were
carried out using the Gaussian 09 package [42]. All structures were
fully optimized at the B3LYP/6–31+G(d,p) level [43,44] using the
conductor-like polarizable continuum model (C-PCM) [24]. The C-PCM
method allows the calculation of the energy in the presence of a solvent.
In this case all structures were optimized as a solute in an aqueous
solution. In order to characterize every structure as minimum a vibra-
tional analysis was carried out at the same level of theory using the
keyword freq. The RMS force criterion was set to 3 × 10−4 a.u. Mole-
cular orbitals and spin density have been calculated using the natural
bond orbital (NBO) method [25]. Starting from the structure of the DFT
Q (i.e., the starting quinone species) GM conformers, the redox states
Q•− and QH• were generated. In particular, following the reduction
pathway of quinones showed in Scheme 3, each species was generated
starting from the DFT optimized species of the previous step and sub-
mitted to full DFT optimization.
For the cytotoxicity assays, cells were treated with serial dilutions of
test compounds for 72 h and cell proliferation was evaluated using the
MTT [39]. The results are expressed as IC50
.
5.5. Haemolysis and formation of methaemoglobin
Infected RBC, (iRBC, at 1–2% parasitemia) or fresh RBCs were di-
luted to 1% hematocrit in medium and incubated for 24 h with different
doses of compounds 5 and 9 (from 59.5 to 0.9 µM). Haemolysis was
evaluated by measuring spectrophotometrically the release of hae-
moglobin in the supernatants (absorbance at 405 nm, Soret band) and
calculating the ratio of untreated to compounds-treated samples. The
percentage of haemolysis for RBCs was determined by referring to a
standard curve prepared with serially diluted RBCs lysed with saponin
(1%).
Finally, a fragment-based search in the Cambridge Crystallographic
Structural Database (CSD) using the CSDS software Conquest 1.19
(Cambridge Structural Database System 2016) was performed in order
to investigate which functional group of the methoxy-thiazinoquinone
system could be suitable for iron interaction. In particular, the fol-
lowing fragments in complex with iron were used as probes: 1-meth-
oxypropan-2-ol, 1-methoxypropan-2-one, 1-methoxyprop-1-en-2-ol, 3-
methoxyprop-1-en-2-ol, 1-(methylamino)propan-2-ol, 1-(methylamino)
propan-2-one, 1-(methylamino)prop-1-en-2-ol, 3-(methylamino)prop-
1-en-2-ol, 1-(methylsulfonyl)propan-2-ol, 1-(methylsulfonyl)propan-2-
one, 1-(methylsulfonyl)prop-1-en-2-ol, 3-(methylsulfonyl)prop-1-en-2-
ol, and (methylsulfonyl)methane). The identified hits were analysed
using the softwares Conquest 1.19 and Insight 2005 (Accelrys, San
Diego, CA).
To evaluate the formation of methaemoglobin, RBC were incubated
in medium or in the presence of NAC (1–10 µM) for 3 h. RBC were then
washed with PBS and incubated for 24 h at 37 °C with different doses of
compound 5 and 9 (from 59.5 to 0.9 µM) At the end of the incubation,
RBC were lysed and the percentage of oxyhaemoglobin or methae-
moglobin was determined using the algorithm of Winterbourn based on
the measurement of optical densities at 560, 577 and 630 nm [30].
Author contributions
5.6. Molecular modeling studies
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
Molecular modeling calculations were performed on E4 Server Twin
2 × Dual Xeon-5520, equipped with two nodes. Each node: 2 × Intel®
Xeon® QuadCore E5520-2.26Ghz, 36 GB RAM. The molecular modeling
graphics were carried out on a personal computer equipped with Intel
(R) Core(TM) i7-4790 processor and SGI Octane 2 workstations.
Acknowledgment
This work was also supported by Ministero dell’Istruzione,
dell’Università
e della Ricerca (PRIN) Projects 2010C2LKKJ_006;
20154JRJPP_004. The authors acknowledge AVIS-Milano for providing
fresh human blood for parasite cultures.
5.6.1. Conformational property analysis
The apparent pKa and clogD values of new thiazinoquinone com-
pounds were calculated by using the ACD/Percepta software (ACD/
Percepta, Advanced Chemistry Development, Inc., Toronto, ON,
Canada, 2017, http://www.acdlabs.com.). All compounds were con-
sidered neutral in all calculations performed as a consequence of the
estimation of percentage of neutral/ionized forms computed at pH 7.4
(blood pH value), pH 7.2 (cytoplasm pH value), and 5.5 (Pf food va-
cuole pH), using the Handerson–Hasselbalch equation. Atomic poten-
tials and charges were assigned using the CFF91 force field [40]. The
conformational space of the compounds was sampled through 200 cy-
cles of Simulated Annealing (ε = 80 * r). The following protocol was
applied: the system was heated up to 1000 K over 2000 fs (time
step = 3.0 fs); the temperature of 1000 K was applied to the system for
2000 fs (time step = 3.0 fs) with the aim of surmounting torsional
barriers; successively, temperature was linearly reduced to 300 K in
1000 fs (time step = 1.0 fs). Resulting conformations were then sub-
jected to molecular mechanic (MM) energy minimization within Insight
2005 Discover module (CFF91 force field; ε = 80 * r) until the max-
imum RMS derivative was less than 0.001 kcal/Å, using Conjugate
Gradient [41] as minimization algorithm. Finally, the resulting con-
formers were ranked by their potential energy values (i.e., ΔE from the
global energy minimum) and grouped into conformational sub-families
on the basis of dihedral angle values (i.e., τ1 and τ2).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2018.12.031.
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