Synthesis and in vitro anti Leishmania amazonensis biological screening of Morita-Baylis-Hillman adducts prepared from eugenol, thymol and carvacrol

Article abstract of DOI:10.3390/molecules21111483

Leishmaniasis represents a series of severe neglected tropical diseases caused by protozoa of the genus Leishmania and is widely distributed around the world. Here, we present the syntheses of Morita-Baylis-Hillman adducts (MBHAs) prepared from eugenol, thymol and carvacrol, and their bioevaluation against promastigotes of Leishmania amazonensis. The new MBHAs are prepared in two steps from essential oils in moderate to good yields and present IC₅₀ values in the range of 22.30-4.71 μM. Moreover, the selectivity index to the most potent compound is very high (SIrb > 84.92), far better than that of Glucantime (SIrb 1.39) and amphotericin B (SIrb = 22.34). Conformational analysis were carried out at the M062X//6-31+G(d,p) level of theory to corroborate a hypothesis about the nitroaromatic bioreduction mechanism.

Full text of DOI:10.3390/molecules21111483

molecules
Article
Synthesis and In Vitro Anti Leishmania amazonensis
Biological Screening of Morita-Baylis-Hillman Adducts
Prepared from Eugenol, Thymol and Carvacrol
Francisco José Seixas Xavier 1, Klinger Antonio da Franca Rodrigues 2,
Ramon Guerra de Oliveira 1, Claudio Gabriel Lima Junior 1, Juliana da Câmara Rocha 3,
Tatjana Souza Lima Keesen 3, Marcia Rosa de Oliveira 2, Fábio Pedrosa Lins Silva 1,
*
and Mário Luiz Araújo de Almeida Vasconcellos 1,
*
1
Laboratório de Síntese Orgânica Medicinal da Paraíba (LASOM-PB), Departamento de Química,
Universidade Federal da Paraíba, Campus I, João Pessoa, Paraíba CEP 58059-900, Brazil;
seicxhas@hotmail.com (F.J.S.X.); ramonoliveirajp@gmail.com (R.G.d.O.); claudio@quimica.ufpb.br (C.G.L.J.)
Programa de Pós-graduação em Biologia Molecular and Departamento de Biologia Molecular,
Centro de Ciências Exatas and da Natureza, Universidade Federal da Paraíba, João Pessoa,
Paraíba CEP 58059-900, Brazil; klinger.antonio@gmail.com (K.A.d.F.R.); mrosajp@gmail.com (M.R.d.O.)
Departamento de Biotecnologia, Universidade Federal da Paraíba, Campus I, João Pessoa,
Paraíba CEP 58059-900, Brazil; ju_jurocha@hotmail.com (J.d.C.R.); tat.keesen@cbiotec.ufpb.br (T.S.L.K.)
Correspondence: pedrosalinssilva@gmail.com (F.P.L.S.); mlaav@quimica.ufpb.br (M.L.A.d.A.V.);
Tel.: +55-083-3216-7589 (F.P.L.S. & M.L.A.d.A.V.)
2
3
*
Academic Editor: Diego Muñoz-Torrero
Received: 11 October 2016; Accepted: 1 November 2016; Published: 8 November 2016
Abstract: Leishmaniasis represents a series of severe neglected tropical diseases caused by protozoa
of the genus Leishmania and is widely distributed around the world. Here, we present the syntheses
of Morita-Baylis-Hillman adducts (MBHAs) prepared from eugenol, thymol and carvacrol, and their
bioevaluation against promastigotes of Leishmania amazonensis. The new MBHAs are prepared in
two steps from essential oils in moderate to good yields and present IC50 values in the range of
22.30–4.71 µM. Moreover, the selectivity index to the most potent compound is very high (SIrb > 84.92),
far better than that of Glucantime® (SIrb 1.39) and amphotericin B (SIrb = 22.34). Conformational
analysis were carried out at the M062X//6-31+G(d,p) level of theory to corroborate a hypothesis
about the nitroaromatic bioreduction mechanism.
Keywords: Morita-Baylis-Hillman adducts; Leishmania amazonensis; eugenol; thymol; carvacrol
1. Introduction
Leishmaniasis is considered a set of neglected tropical diseases (NTD) caused by protozoa of the
genus Leishmania and is widely distributed around the world [1]. These diseases are classified into
three clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous
leishmaniasis (MCL). The most abundant species in the Americas, which causes general disfigurement
of the face (MCL) and leads to patients withdrawing from social life, are Leishmania braziliensis and
Leishmania amazonensis [2]. Approximately 500,000 new cases of VL are registered each year and are
responsible for the deaths of thousands of people. Six countries, one of which is Brazil, account for 90%
of deaths related to the disease. The World Health Organization (WHO) estimates that approximately
one billion people live in areas at risk, and the 0.9–1.3 million new cases are responsible for the
deaths of approximately 30,000 people annually [
3]. Leishmania spp. is distributed by sandflies, which
inject infective promastigotes into the skin of mammalian hosts during a blood meal. To date, there
is no leishmaniasis vaccine for humans [4]. Few drug treatment options are available, which are
Molecules 2016, 21, 1483; doi:10.3390/molecules21111483
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lengthy, toxic, and expensive. In some cases, hospitalization is required, leading to discontinuance of
treatment when some improvement is observed in the patient [ ]. In addition, Leishmania is developing
5
tolerance to the most effective drugs on the market; therefore, the drugs are losing efficiency. Thus,
the development of more efficient and cheaper drugs to improve the pharmacological action and the
discovery of novel therapeutic targets against trypanosomatids (e.g., Leishmania spp.) are major themes
of this field [6].
In the last ten years, our research group has been continuously dedicated to the design,
synthesis and biological evaluation of Morita-Baylis-Hillman adducts (MBHAs) as antileishmanial
treatments and as treatments against other parasitic NTDs [
7]. The Morita-Baylis-Hillman reaction
(MBHR, Scheme 1) has been consolidated as an efficient methodology for C-C bond formation [7–9
].
Polyfunctional MBHA products are obtained in one step by the MBHR by using nucleophilic catalysis
(DABCO is the most common catalyst) under metal-free conditions and may be performed in
an aqueous solvent mixture or in a solvent-free medium. MBHR is now classified as an important
green reaction in organic syntheses [7].
XH
X
EAG
nucleophilic catalyst
i.e. NR3
+
EAG
N
N
R
R
H
DABCO
MBHA
R = alkyl, aryl, heteroaryl
X = O, NCO2R, NTs, NSO2Ph.
EAG = COR, CHO, CN, CO2R
Scheme 1. General Morita-Baylis-Hillman reaction. EAG = Electron attractor group.
In 2011, we reported that MBHA
hybridization [10] of the analgesic methyl salicylate (
1
(a chalcone-like compound), designed from the molecular
) and the MBHA (Scheme 2), presented a lower
2
3
IC50 in a congener series of compounds and was shown to be an efficient molecular hybrid and
a promising leishmanicidal drug.
Scheme 2. Molecular hybridization between MBHA 3 and methyl salicylate 2 for the design of hybrid 1.
In connection with our interest in discovering new compounds with efficient leishmanicidal
properties [ 11 15], we present here the design, syntheses, and in vitro leishmanicidal evaluations
against L. amazonensis of nine new o-, m- and p-nitro MBHA hybrids based on the synergistic biological
proprieties of the abundant and inexpensive essential oil ingredients eugenol ( ), thymol ( ) and
carvacrol ( ) (Figure 1). It is important to note that eugenol ( ) is an analgesic with leishmanicidal [16
and several others biological activities [17 18]. Thymol ( ) and carvacrol (
leishmanicidal activities [19]. In Figure 1, we also present MBHAs 16
7, –
4
5
6
4
]
,
5
6) present analgesic and
,
17 and
3, which were also
biologically evaluated to compare motifs [15]. A conformational DFT study is presented to corroborate
our proposed mechanism of the reduction of nitroaryl derivatives concerning these nitro-MBHAs [19].

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Figure 1. The MBHA hybrids synthetized, biologically evaluated and conformationally studied in
this paper and eugenol (
4
), thymol (
5
), and carvacrol (
6
). Compounds
7
–
9
–
are hybrids derived from
15 are hybrids of carvacrol.
eugenol; compounds 10 12 are hybrids made from thymol. Compounds 13
–
Compounds 16, 17 and 3 are MBHAs.
2. Results and Discussion
The new acrylates 18
Eugenol, thymol and carvacrol acrylate 18
and DMAP (catalytic) at 0 ◦C in dry dichloromethane (Scheme 3). For the synthesis of hybrids
–
20 and hybrids
7
–
–
15 (Figure 1) were synthesized according to Scheme 3.
20 were prepared by the respective reactions with DCC
15
7– ,
acetonitrile was used without drying, however, we observed hydrolysis reactions of the hybrids
leading to the products at low yields (<40% yields). To optimize the reaction yields, dry acetonitrile
was used as a solvent at room temperature to thus obtain hybrids
(Scheme 3).
7
–15 in moderate to good yields
R3
Acrylic Acid, DCC, DMAP,CH2Cl2, 48h, 0oC
R3
O
R2
R2
O
OH
R1
R1
18-20
4- 6
Aldehyde, CH3CN,
DABCO (100 mol %),
3h, 25oC
Eugenol
Thymol
4: R1 = OCH3, R2 = CH2CH=CH2, R3 = H
5: R1 = CH(CH3)2, R2 = H, R3 = CH3
Carvacrol 6: R1 =CH3 , R2 = H, R3 = CH(CH3)2
Hybrid
Hybrid
Hybrid
Hybrid
Hybrid
Hybrid
Hybrid
Hybrid
Hybrid
7: R1 = OCH3, R2 = CH2CH=CH2, R3 = H, R4=R5=H, R6 = NO2, 78%
1 = OCH3, R2 = CH2CH=CH2, R3 = H, R4=R6=H, R5 = NO2, 71%
8
R
9: R1 = OCH3, R2 = CH2CH=CH2, R3 = H, R5=R6=H, R4 = NO2, 75%
10:R1 = CH(CH3)2, R2 = H, R3 = CH3,R4=R5=H, R6 = NO2, 80%
11:R1 = CH(CH3)2, R2 = H, R3 = CH3,R4=R6=H, R5 = NO2, 73%
12:R1 = CH(CH3)2, R2 = H, R3 = CH3,R5=R6=H, R4 = NO2, 58%
13:R1 =CH3 , R2 = H, R3 = CH(CH3)2, R4=R5=H, R6 = NO2, 92%
R3
O
OH R4
R2
14 R1 =CH3 , R2 = H, R3 = CH(CH3)2, R4=R6=H, R5 = NO2, 63%
R5
R6
O
15:R1 =CH3 , R2 = H, R3 = CH(CH3)2, R5=R6=H, R4 = NO2, 72%
R1
Acrylate 18 = R1 = OCH3, R2 = CH2CH=CH2, R3 = H, 68%
Acrylate 19 = R1 = CH(CH3)2, R2 = H, R3 = CH3, 73%
Acrylate 20 = R1 =CH3 , R2 = H, R3 = CH(CH3)2, 76%
7-15
Scheme 3. Reagents, conditions, rates, and yields (%) of our preparations of new compounds
and 18–20.
7–15

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The antileishmanial activities against promastigotes of L. amazonensis of compounds
presented in Table 1. Some results in this Table should be highlighted here.
3–
17 are
Table 1. The antileishmanial activities against promastigotes of L. amazonensis. Values represent the
average IC50 calculated in three independent experiments standard error of the mean. For this
calculation, we used the probabilistic regression models with SPSS 13.0 software. SIrb (selectivity index
on red blood cell) = HC50/IC50
±
.
Entry
Compounds
IC50 (µM)
HC50 (µM)
SIrb
miLogP
1
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
4
7
8
9
5
10
11
12
6
13
14
15
16
17
3
77.15 ± 1.77
8.75 ± 0.44
>400
>400
>400
>400
-
-
-
-
>5.18
>45.71
>38.13
>84.92
2.10
3.05
3.02
3.00
3.34
4.05
4.03
4.00
3.81
4.52
4.50
4.47
1.53
1.50
1.48
−2.68
−2.49
10.49 ± 0.52
4.71 ± 0.24
115.12 ± 5.77
11.40 ± 0.57
10.56 ± 0.53
5.91 ± 0.30
-
-
-
-
-
-
-
138.11 ± 6.91
18.08 ± 0.90
22.30 ± 1.12
13.60 ± 0.68
15.77 ± 0.79
22.38 ± 1.12
12.06 ± 0.60
1633.49 ± 437.29
0.52 ± 0.03
-
-
-
-
-
>400
>400
>400
1175.02
11.61
>25.36
>17.87
>33.16
1.39
22.34
Glucantime®
AmphotericinB
We can observe that eugenol (
essential oils thymol ( , Entry 5) and carvacrol (
(compounds , Entries 2–4), thymol (10 12, Entries 6–8) and carvacrol (13
higher antileishmanial activity than the corresponding essential oils ingredients. The hybrids of
eugenol (compounds , Entries 2–4) showed lower IC50 values than the other series. Another very
important dataset shown in Table 1, which is in agreement with previously obtained data by our
research group [ ], indicates that the nitrated MBHAs presenting an ortho nitro group on the aromatic
4
, Entry 1) exhibits greater leishmanicidal activity than the other
, Entry 9). All hybrids derived from eugenol
15, Entries 11–13) exhibit
5
6
7
–9
–
–
7–9
7
ring are more bioactive against promastigotes of L. amazonensis than the corresponding meta and para
nitroaryl isomers. Several nitro aromatics are used as anti-infective agents, e.g., drugs to treat parasitic
infections. The biological activity of the nitro compounds is connected to a nitro group reduction that
generates RNO−• or more reduced intermediates (Scheme 4) [19].
−
Scheme 4. General scheme of the 2e nitro aromatic reduction (Ar = aromatic group).

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Currently, the exact mechanism of action of MBHAs against Leishmania targets is not known.
To propose a biological mechanism of action for these new nitroaryl hybrids and to elucidate the
reasons behind the higher bioactivity of the ortho nitro regioisomer compared to the meta and para
regioisomers, the calculation of the more stable conformation of
medium were performed. These geometries are shown in Figure 2.
7 and 9 in a simulated aqueous
7
9
and
Figure 2. Conformational minima of hybrids
7
9 obtained by M062X//6-31+G(d,p) in implicit
water simulation as the level of the calculations.
In previous electrochemical studies by cyclic voltammetry of the corresponding adducts 16
,
17 and
3
(Figure 1), it was observed that ortho aryl
3
is more easily reduced to the corresponding anion radical
−•
(ArNO2 ) of the isomers 16 and 17
deviation of the NO2 moiety in ortho aryl
[
20,
21]. It was proposed by QTAIM calculations that the dihedral
with the aromatic ring is caused by the intramolecular
9
hydrogen bonding (IHB) of the seven-member grouping (HO—O-N-O, IHB = 2.104 Å) [22], which is
responsible for the highest electron affinity of this nitrogen. These data are accepted as the reason
behind the ease of the biological reduction the ortho nitro compounds related to their greater
leishmanicidal activity [21]. We can note from Figure 2 that, in the hybrid
nearly coplanar with an aromatic ring (dihedral angle NO2
Ar = 1.7◦), which is very different from
the observation of hybrid (dihedral angle NO2
Ar = 31.0◦). Thus, the radical intermediates from
7, the nitro group is
−
9
−
ortho MBHA could present higher lifetimes than those of the para nitro regioisomer in cytoplasmic
medium, increasing the cleavage of the nuclear membrane of protozoa [21].
3. Materials and Methods
3.1. Experimental Chemistry
General
All commercially available reagents and solvents were obtained from the provider Sigma-Aldrich®
(St. Louis, MO, USA) and used without further purification. Reactions were monitored by TLC using
Silica gel 60 UV254 pre-coated silica gel plates (Macherey-Nagel, Bethlehem, PA, USA) and detection
was performed using a UV lamp. Flash column chromatography was performed on 300–400 mesh
silica gels. Organic layers were dried over anhydrous MgSO4 or Na2SO4 prior to evaporation using
1
a rotary evaporator. H-NMR and 13C-NMR spectra were recorded using a Mercury Spectra AC 20

Molecules 2016, 21, 1483
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spectrometer (200 MHz for 1H, 50 MHz for 13C, Varian, (Varian, Palo Alto, CA, USA). Chemical shifts
were reported relative to internal tetramethylsilane (
0.00 ppm) for 1H, using CDCl3 as the solvent.
FTIR spectra were recorded using a model IRPrestige-21 spectrophotometer (Shimadzu, Kyoto, Japan)
in KBr pellets. The high-resolution mass spectrometry (HRMS) of new compounds 15 (Figure 1) was
δ
7–
performed using a Q-Tof quadrupole/orthogonal instrument (Waters, Milford, MA, USA) in positive
and negative mode.
3.2. General Procedure for Esterification of Eugenol (4), Thymol (5) and Carvacrol (6) with Acrylic Acid;
Preparation of Compounds 18–20
Eugenol (3, 1.640 g, 10.0 mmol), thymol (4, 1.500 g, 10.0 mmol) or carvacrol (5, 1.500 g, 10.0 mmol)
and dry dichloromethane (10.0 mL) were added in a bottle flask. After homogenization in an ice-bath
and magnetic stirring, DCC (2.060 g, 10.0 mmol) and DMAP (0.122 g, 1 mmol) were added. The reaction
was monitored by TL chromatography for 48 h. Filtration on a separating funnel was performed. Water
was added and the reaction mixture was extracted with dichloromethane (30.0 mL
×
2). The organic
phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure using a rotary
evaporator. Purification was performed using a chromatographic column containing 30.0 g of flash
silica gel, using 100.0 mL of hexane as eluent followed by a mixture of 300 mL ethyl acetate/hexane
(30%). The fractions were collected, and the solvent was evaporated from the obtained product
using a rotary evaporator. The expected products were colorless, somewhat viscous oils and solid at
temperatures below 0 ◦C.
3.2.1. 4-Allyl-2-methoxyphenyl acrylate (18)
68% yield; FTIR KBr: 1745 cm−1 (C=O); 1637 cm−1 (C=C); 1602 (C=C); 1035 cm−1 (C-O-C). 1H-NMR
δ
(ppm): 6.98 (d, J = 7.8 Hz, 1H, H-Ar); 6.84–6.73 (m, 2H, H-Ar); 6.60 (dd, J = 17.3, 1.6 Hz, 1H, CH2=CH);
6.33 (dd, J = 17.3, 10.3 Hz, 1H, CH2=CH); 6.06–5.84 (m, 2H, CH=CH2); 5.17–5.03 (m, 2H, CH2=CH);
3.79 (s, 3H, CH3OPh); 3.37 (d, J = 6.7 Hz, 2H, CH2Ph). 13C-NMR
(ppm): 164.28 (C3); 150.91 (C7);
δ
139.07 (C9); 137.67 (C4); 137.04 (C1); 132.46 (C10); 127.64 (C2); 122.50 (C5); 120.68 (C6); 116.18 (C12);
112.75 (C8); 55.81 (C13); 40.10 (C11).
3.2.2. 2-Isopropyl-5-methylphenyl acrylate (19)
73% yield; FTIR KBr: 1743 cm−1 (C=O); 1622 cm−1 (C=C). 1H-NMR
δ (ppm): 7.19 (d, J = 7.9 Hz, 1H,
H-Ar); 7.00 (d, J = 8,9 Hz, 1H, H-Ar);)6.69 (s, 1H, H-Ar); 6.57 (dd, J = 17.2, 1.4 Hz, 1H, CH2=CH); 6.33
(dd, J = 17.3, 10.3 Hz, 1H, CH=CH2); 5.97 (d, J = 10.3 Hz, 1H, CH2=CH); 2.98–2.75 (m, 1H, HC-(CH3)2);
2.13 (s, 3H, H3C-Ph), 1.23 (d, J = 6.9 Hz, 6H, (CH3)2-CH). 13C-NMR
δ
(ppm): 164.41 (C3); 149.12 (C6);
148.10 (C4); 132.43 (C1); 130.93 (C8); 127.86 (C2); 127.23 (C9); 124.24 (C7); 119.73 (C5); 33.60, (C11); 23.95
(C12/120 ); 15.80 (C ).
10
3.2.3. 5-Isopropyl-2-methylphenyl acrylate (20)
76% yield; FTIR KBr: 1743 cm−1 (C=O); 1635 cm−1 (C=C); 1622 cm−1 (C=C). 1H-NMR
δ (ppm): 7.08
(d, J = 7.8 Hz, 1H, H-Ar); 6.97 (d, J = 7.8 Hz, 1H, H-Ar);) 6.69 (s, 1H, H-Ar); 6.57 (dd, J = 17.2, 1.4 Hz,
1H, CH2=CH); 6.33 (dd, J = 17.3, 10.3 Hz, 1H, CH=CH2); 5.97 (d, J = 10.3 Hz, 1H, CH2=CH); 2.98–2.75
(m, 1H, HC-(CH3)2); 2.13 (s, 3H, H3C-Ph), 1.23 (d, J = 6.9 Hz, 6H, (CH3)2-CH). 13C-NMR
δ
(ppm):
164.41 (C3); 149.12 (C6); 148.10 (C4); 132.43 (C1); 130.93 (C8); 127.86 (C2); 127.23 (C9); 124.24 (C7); 119.73
(C5); 33.60, (C11); 23.95 (C12e120 ); 15.80 (C ).
10
3.3. General Synthesis of the MBHAs 16, 17 and 3
The compounds 16, 17 and 3 synthesized in this work are not new and were characterized using
1H-NMR and 13C-NMR for comparison with the compounds described in the literature [14]. Reactions
were carried out using the corresponding aldehydes (1 mmol), methyl acrylate (0.5 mL) and of DABCO

Molecules 2016, 21, 1483
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(1 mmol) at 0 ◦C. After some time, the reaction media was directly filtered through silica gel using
EtOAc-hexane (2:8) as the solvent, and the reaction products were concentrated under reduced pressure.
The products were then ready for biological evaluations without the need of further purification.
3.3.1. 2-[Hydroxy(4-nitrophenyl)methyl]propanoate (16)
85% yield; 1H-NMR
5.60 (d, J = 5.8 Hz, 1H), 3.71 (s, 3H), 3.41 (d, J = 5.8 Hz, 1H). 13C-NMR
δ
(ppm): d, 8.16 (d, 2H, J = 8.8 Hz), 7.54 (d, 2H, J = 8.4 Hz), 6.36 (s, 1H), 5.86 (s, 1H),
(ppm): 52.2 (1C), 72.6 (1C),
δ
123.5 (1C), 137.2 (1C), 127.3 (1C), 140.8 (1C), 147.3 (1C), 148.5 (1C), 166.3 (1C).
3.3.2. 2-[Hydroxy(3-nitrophenyl)methyl]propanoate (17)
1
98% yield; H-NMR
δ (ppm): d, 8.22 (t, J = 1.8 Hz, 1H); 8.08–8.13 (m, 1H); 7.69–7.73 (m, 1H); 7.50
(t, J = 8 Hz, 1H); 6.38 (s, 1H); 5.89 (s, 1H); 5.60 (d, J = 4.4 Hz, 1H); 3.70 (s, 3H); 3.46 (d, J = 5.4 Hz, 1H).
13C-NMR
δ
(ppm): 52.2 (1C), 72.5 (1C), 121.5 (1C), 122.7 (1C), 127.2 (1C), 129.3 (1C), 132.7 (1C), 140.9
(1C), 143.5 (1C), 148.2 (1C), 166.3 (1C).
3.3.3. 2-[Hydroxy(2-nitrophenyl)methyl]propanoate (3)
92% yield; 1H-NMR
J = 7.8/1.2/1.0 Hz, 1H); 7.43 (ddd, J = 8.0/1.6/1.6 Hz, 1H); 6.38 (s, 1H); 5.89 (d, 1H); 5.60 (d, J = 4.4 Hz
1 Hz); 3.70 (s, 3H); 3.60 (d, J = 5.4 Hz, 1H). 13C-NMR
(ppm): 52.20 (1C), 67.56 (1C), 124.55 (1C), 126.48
δ (ppm): d, 7.92 (dd, J = 8.2/1.2 Hz, 1H); 7.73 (dd, J = 7.8/1.8 Hz, 1H); 7.62 (ddd,
,
δ
(1C), 128.67 (C), 128.67 (C) 133.47 (1C), 136.05 (1C), 140.65 (1C), 148.32 (1C), 166.39 (1C).
3.4. General Synthesis of the Hybrids 7–15
To a 25.0 mL flask, acrylate 18, 19 or 20 (0.5 mmol), the corresponding nitro-substituted aldehyde
(0.6 mmol), and dry acetonitrile (1.0 mL) were added. To the resulting solution DABCO (0.5 mmol)
was added. The reaction mixture was stirred at room temperature and monitored by TLC for 3 h.
The purified product was obtained by using 6.0 g of silica and 10.0 mL of acetonitrile to form
a dispersion and added to a chromatographic column containing 30.0 g of flash silica gel. A mixture
of 300 mL ethyl acetate/hexane 5% followed by 200 mL 15% acetate/hexane was used as an eluent.
The fractions were separated and transferred to a rotary evaporator. The respective products were
yellowish viscous oils at ambient temperature. All spectra are available in the Supplementary information.
3.4.1. 4-Allyl-2-methoxyphenyl 2-(hydroxy(4-nitrophenyl)methyl)acrylate (7)
78% yield; FTIR: 3450 cm−1 (O-H); 1735 cm−1 (C=O); 1637 cm−1 (C=C); 1602 cm−1 (C=C); 1531 and
1
1350 cm−1 (R-NO2), 1035 cm−1 (C-O-C). H-NMR
δ (ppm): 8.21 (d, J = 8.8 Hz, 2H, H-Ar); 7.63 (d,
J = 8.8 Hz, 2H, H-Ar); 6.95–6.89 (m, 1H, H-Ar); 6.81–6.73 (m, 2H, H-Ar); 6.64 (s, 1H, H-C-OH); 6.02 (d,
J = 7.9 Hz, 1H, CH2=C); 5.96–5.70 (m, 2H, CH=CH2 and CH2=C); 5.16–5.03 (m, 2H, CH2=CH); 3.72 (s,
3H, CH3OPh); 3.40 (t, J = 13.3 Hz, 3H, CH2Ph and HO-C). 13C-NMR
δ (ppm): 168.81 (C8); 155.16 (C14);
153.19 (C4); 152.16 (C3); 145.44 (C6); 144.22 (C12); 141.95 (C9); 141.56 (C16); 133.51 (C7); 132.24 (C1e10);
128.29 (C2e20); 127.01 (C10); 125.46 (C11); 121.03 (C17); 117.37 (C13); 77.33 (C5); 60.45 (C18); 44.78 (C15).
HRMS (TOF MS ES−) calculated for C20H19NO6: 369.3700 [M + H]+; found: 370.1298.
3.4.2. 4-Allyl-2-methoxyphenyl-2-(hydroxy(3-nitrophenyl)methyl)acrylate (8)
71% yield; FTIR: 3450 cm−1 (O-H); 1735 cm−1 (C=O); 1637 cm−1 (C=C); 1602 cm−1 (C=C); 1531 and
1350 cm−1 (R-NO2), 1035 cm−1 (C-O-C). 1H-NMR
δ (ppm): 8.33 (s, 1H, H-Ar); 8.17 (d, J = 7,0 Hz, 1H,
H-Ar); 7.80 (d, J = 8.2 Hz, 1H, H-Ar); 7.54 (t, J = 7.9 Hz, 1H, H-Ar); 6.93 (d, J = 8.6 Hz, 1H, H-Ar); 6.75
(d, J = 7.3 Hz, 2H, H-Ar); 6.66 (s, 1H, H-C-OH); 6.03 (s, 1H, CH2=C); 5.75–5.95 (m, 2H, CH=CH2 and
CH2=C); 5.14–5.04 (m, 2H, CH2=CH); 3.72 (s, 3H, CH3OPh); 3.36 (d, J = 6.7 Hz, 3H, CH2Ph and HO-C).
13C-NMR
δ
(ppm): 164.84 (C10); 151.22 (C16); 149.09 (C4); 144.23 (C6); 141.43 (C9); 140.21 (C14); 138.02
(C11); 137.63 (C18); 133.62 (C1); 130.09 (C2); 129.58 (C8); 123.55 (C5); 123.06 (C3); 122.45 (C12); 121.48

Molecules 2016, 21, 1483
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(C13); 117.02 (C19); 113.40 (C15); 73.35 (C7); 56.53 (C20); 40.83 (C17). HRMS (TOF MS ES
for C20H19NO6: 369.3700 [M + H]+; found: 370.1390.
−
) calculated
3.4.3. 4-Allyl-2-methoxyphenyl 2-(hydroxy(2-nitrophenyl)methyl)acrylate (9)
75% yie−ld1; FTIR: 3458 cm−1 (O-H); 1737 cm−1 (C=O); 1637 cm−1 (C=C); 1604 cm−1 (C=C); 1527 and
−1
1348 cm (R-NO2), 1033 cm (C-O-C). 1H-NMR
δ (ppm): 7.96–8.06 (m, 1H, H-Ar); 7.83 (d, J = 7.8 Hz,
1H, H-Ar); 7.67 (dd, J = 8.3, 4.6 Hz, 1H, H-Ar); 7.50 (d, J = 6.7 Hz, 1H, H-Ar); 6.93 (d, J = 8.6 Hz, 1H,
H-Ar); 6.79–6.70 (m, 2H, H-Ar); 6.61 (s, 1H, H-C-OH); 6.31 (s, 1H, CH2=C); 6.05–5.81 (m, 2H, CH=CH2
and CH2=C); 5.16–5.01 (m, 2H, CH2=CH); 3.73 (s, 4H, CH3OPh and HO-C); 3.36 (d, J = 6.7 Hz, 2H,
CH2Ph). 13C-NMR
δ
(ppm): 164.12 (C10); 150.52 (C16); 148.32 (C5); 140.25 (C9); 139.21 (C14); 137.43
(C6); 136.92 (C18); 135.99 (C11); 133.50 (C2); 129.09 (C3); 128.72 (C1); 128.06 (C8); 124.67 (C4); 122.34
(C12); 120.65 (C13); 116.18 (C19); 112.61 (C15); 67.88 (C7); 55.72 (C20); 40.04 (C17). HRMS (TOF MS ES
calculated for C20H19NO6: 369.3700 [M + H]+; found: 370.1298.
−
)
3.4.4. 2-Isopropyl-5-methylphenyl 2-(hydroxy(4-nitrophenyl)methyl)acrylate (10)
80% yield; FTIR: 3485 cm−1 (O-H); 1732 cm−1 (C=O); 1606 cm−1 (C=C); 1523 and 1348 cm−1 (R-NO2).
1H-NMR
(ppm): 8.20 (d, J = 8.8 Hz, 2H, H-Ar); 7.63 (d, J = 8.6 Hz, 2H, H-Ar); 7.06 (d, J = 7.8 Hz, 2H,
δ
H-Ar); 6.78 (d, J = 7.3 Hz, 1H, H-Ar); 6.67 (s, 1H, H-C-OH); 6.12 (s, 1H, CH2=C); 5.75 (d, J = 4.8 Hz, 1H,
CH2=C); 3.50 (d, J = 4.9 Hz, 1H, HO-C); 2.85 (hept, J = 6.8 Hz, 1H, HC-(CH3)2); 1.95 (s, 3H, H3C-Ph);
1.20 (d, J = 6.9 Hz, 6H, (CH3)2-CH). 13C-NMR
δ
(ppm): 164.24 (C8); 148.63 (C4); 148.27 (C9); 147.48
(C3); 140.93 (C11); 131.01 (C13); 128.23 (C6); 128.16 (C7); 127.56 (C2/20 ); 126.95 (C14); 124.49 (C12); 123.63
(C1/10 ); 119.46 (C10); 72.36 (C5); 33.50 (C16); 23.85 (C17/170 ); 15.57 (C15). HRMS (TOF MS ES ) calculated
−
for C20H21NO5: 355.1400 [M − H]−; found: 354.1426.
3.4.5. 2-Isopropyl-5-methylphenyl 2-(hydroxy(3-nitrophenyl)methyl)acrylate (11)
73% yield; FTIR: 3493 cm−1 (O-H); 1730 cm−1 (C=O); 1637 cm−1 (C=C); 1622 cm−1 (C=C); 1531 and
1350 cm−1 (R-NO2).1H-NMR
(ppm): 8.30 (s, 1H, H-Ar); 8.15 (dd, J = 8.2, 1.1 Hz, 1H, H-Ar); 7.79
δ
(d, J = 7.7 Hz, 1H, H-Ar); 7.53 (t, J = 7.9 Hz, 1H, H-Ar); 7.06 (dd, J = 7.8 Hz, 2H, H-Ar); 6.79 (s, 1H,
H-Ar); 6.68 (s, 1H, H-C-OH); 6.13 (s, 1H, CH2=C); 5.74 (d, J = 5.2 Hz, 1H, CH2=C); 3.36 (d, J = 5.6 Hz,
1H, HO-C), 2.76–2.60 (m, 1H, HC-(CH3)2); 1.95 (s, 3H, H3C-Ph); 1.20 (d, J = 6.9 Hz, 6H, (CH3)2-CH).
13C-NMR
δ
(ppm): 164.29 (C10); 148.69 (C11); 148.29 (C4); 143.56 (C13); 140.81 (C6); 132.90 (C1); 131.01
(C2); 129.44 (C15); 128.36 (C8); 128.28 (C9); 126.99 (C16); 124.51 (C5); 122.89 (C3); 121.65 (C14); 119.49 (C12);
72.17 (C7); 33.51 (C17); 23.87 (C18/180 ); 15.60 (C19). HRMS (TOF MS ES ) calculated for C20H21NO5:
−
355.1400 [M − H]−; found: 354.1154.
3.4.6. 2-Isopropyl-5-methylphenyl 2-(hydroxy(2-nitrophenyl)methyl)acrylate (12)
58% yield; FTIR: 3450 cm−1 (O-H); 1734 cm−1 (C=O); 1620 cm−1 (C=C); 1527 and 1348 cm−1 (R-NO2).
1H- NMR
δ (ppm): 7.87 (d, J = 7.5 Hz, 2H, H-Ar); 7.62 (t, J = 7.5 Hz, 1H, H-Ar); 7.42 (t, J = 7.7 Hz, 1H,
H-Ar); 7.03 (d, J = 7.8 Hz, 2H, H-Ar); 6.81 (s, 1H, H-Ar); 6.58 (s, 1H, H-C-OH); 6.30 (s, 1H, CH2=C);
5.86 (s, 1H, CH2=C); 3.82 (s, 1H, HO-C); 2.95–2.74 (m, 1H, HC-(CH3)2); 1.96 (s, 3H, H3C-Ph); 1.19 (d,
J = 6.9 Hz, 6H, (CH3)2-CH). 13C-NMR
δ (ppm): 164.36 (C10); 148.92 (C11); 148.12 (C5); 140.83 (C13);
136.39 (C8); 133.61 (C2); 130.93 (C15); 128.96 (C3); 128.79 (C1); 127.70 (C6); 127.66 (C9); 127.18 (C16);
124.78 (C4); 124.32 (C14); 119.61 (C12); 67.54 (C7); 33.53 (C17); 23.89 (C18/180 ); 15.55 (C19). HRMS (TOF
MS ES−) calculated for C20H21NO5: 355.1400 [M − H]−; found: 354.1335.
3.4.7. 5-Isopropyl-2-methylphenyl 2-(hydroxy(4-nitrophenyl)methyl)acrylate (13)
92% yield; FTIR: 3510 cm−1 (O-H); 1728 cm−1 (C=O); 1624 cm−1 (C=C); 1606 cm−1 (C=C); 1521 and
1
1348 cm−1 (R-NO2). H-NMR
δ
(ppm): 8.20 (d, J = 8.8 Hz, 2H, H-Ar); 7.63 (d, J = 8.6 Hz, 2H, H-Ar);
7.06 (dd, J = 21.4, 7.8 Hz, 2H, H-Ar); 6.78 (d, J = 1.2 Hz, 1H, H-Ar); 6.67 (s, 1H, H-C-OH); 6.12 (s, 1H,

Molecules 2016, 21, 1483
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CH2=C); 5.75 (d, J = 4.8 Hz, 1H, CH2=C); 3.50 (d, J = 4.9 Hz, 1H, HO-C); 2.85 (hept, J = 6.8 Hz, 1H,
HC-(CH3)2); 1.95 (s, 3H, H3C-Ph); 1.20 (d, J = 6.9 Hz, 6H, (CH3)2-CH). 13C-NMR
(ppm): 164.24 (C8);
δ
148.63 (C4); 148.27 (C9); 147.48 (C3); 140.93 (C11); 131.01 (C13); 128.23 (C6); 128.16 (C7); 127.56 (C2e20 );
126.95 (C14); 124.49 (C12); 123.63 (C1e10 ); 119.46 (C10); 72.36 (C5); 33.50 (C16); 23.85 (C17e170 ); 15.57 (C15).
HRMS (TOF MS ES−) calculated for C20H21NO5: 355.1400 [M − H]−; found: 354.1335.
3.4.8. 5-Isopropyl-2-methylphenyl 2-(hydroxy(3-nitrophenyl)methyl)acrylate (14)
63% yield; FTIR: 3450 cm−1 (O-H); 1735 cm−1 (C=O); 1637 cm−1 (C=C); 1602 cm−1 (C=C); 1531
1
and 1350 cm−1 (R-NO2). H-NMR
δ
(ppm): 8.30 (s, 1H, H-Ar); 8.14 (d, J = 8.2 Hz, 1H, H-Ar); 7.79 (d,
J = 7.7 Hz, 1H, H-Ar); 7.53 (t, J = 7.9 Hz, 1H, H-Ar); 7.08 (d, J =7.8 Hz, 2H, H-Ar); 6.79 (s, 1H, H-Ar); 6.68
(s, 1H, H-C-OH); 6.13 (s, 1H, CH2=C); 5.74 (d, J = 5.2 Hz, 1H, CH2=C); 3.36 (d, J = 5.6 Hz, 1H, HO-C),
2.97–2.71 (m, 1H, HC-(CH3)2); 1.95 (s, 3H, H3C-Ph); 1.20 (d, J = 6.9 Hz, 6H, (CH3)2-CH). 13C-NMR
δ
(ppm): 164.29 (C10); 148.69 (C11); 148.29 (C4); 143.56 (C13); 140.81 (C6); 132.90 (C1); 131.01 (C2); 129.44
(C15); 128.36 (C8); 128.28 (C9); 126.99 (C16); 124.51 (C5); 122.89 (C3); 121.65 (C14); 119.49 (C12); 72.17
(C7); 33.51 (C17); 23.87 (C18/180 ); 15.60 (C19). HRMS (TOF MS ES ) calculated for C20H21NO5: 355.1400
−
[M − H]−; found: 354.1154.
3.4.9. 5-Isopropyl-2-methylphenyl 2-(hydroxy(2-nitrophenyl)methyl)acrylate (15)
72% yield; FTIR: 3489 cm−1 (O-H); 1734 cm−1 (C=O); 1624 cm−1 (C=C); 1527 and 1348 cm−1 (R-NO2).
1H- NMR
δ (ppm): 7.86 (d, J = 7.5 Hz, 2H, H-Ar); 7.62 (t, J = 7.5 Hz, 1H, H-Ar); 7.42 (t, J = 7.7 Hz, 1H,
H-Ar); 7.03 (d, J = 7.9 Hz, 2H, H-Ar); 6.81 (s, 1H, H-Ar); 6.58 (s, 1H, H-C-OH); 6.30 (s, 1H, CH2=C);
5.86 (s, 1H, CH2=C); 3.82 (s, 1H, HO-C); 2.99–2.67 (m, 1H, HC-(CH3)2); 1.96 (s, 3H, H3C-Ph); 1.19
(d, J = 6.9 Hz, 6H, (CH3)2-CH). 13C-NMR δ (ppm): 164.36 (C10); 148.92 (C11); 148.12 (C5); 140.83 (C13);
136.39 (C8); 133.61 (C2); 130.93 (C15); 128.96 (C3); 128.79 (C1); 127.70 (C6); 127.66 (C9); 127.18 (C16);
124.78 (C4); 124.32 (C14); 119.61 (C12); 67.54 (C7); 33.53 (C17); 23.89 (C18/180 ); 15.55 (C19). HRMS (TOF
MS ES) calculated for C20H21NO5: 355.1400 [M − H]−; found: 354.1516.
3.5. Biology
The promastigote viabilities were determined by the ability of living cells to reduce the
yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) to purple
formazan [23]. Promastigote forms of Leishmania amazonensis (IFLA/BR/67/PH8) in the log phase
of growth were incubated at 25 ◦C in 96-well cellular culture plates (TPP, Trasadingen, Switzerland)
with 1
×
105 cells/well in 100
µL Schneider’s Drosophila medium supplemented with 20% of FBS in
the presence or absence of different concentrations of compounds 4–15. The growth of promastigotes
forms was evaluated simultaneously in the presence of Glucantime®, as reference drug. After 72 h
of incubation, 10 mL of a 5 mg
the formed product formazan was dissolved in 50 mL of sodium dodecyl sulfate (SDS) at 10% for 24 h,
and the absorbance was measured by spectrophotometry at a 545 nm wavelength. The percentage
viability was calculated from the ratio of OD readings in the wells with compounds versus the wells
◦
·
mL−1 MTT solution was added. After 4 h of incubation at 25 C,
without compounds
×
100. The concentration that inhibited 50% of growth (IC50) was determined
by regression analysis using the SPSS 8.0 software for Windows (IBM, New York, NY, USA). All
experiments were performed at least three times, and each experiment was performed in triplicate [16].
Haemolytic assays (HC50 > 400 µM) showed that the 4, 7, 8, 9, 16, 17 and 3 exhibit low toxicity towards
human red blood cells, unlike amphotericin B (HC50 = 11.61 µM Table 1) [24].
3.6. Conformational Analysis
The relaxed potential energy surface scan (RPESS) was performed by taking particular care of the
dihedral angles θ1–θ10, which are illustrated in Figure 3. These calculations were executed by using the
semi-empiric PM6 level with angle variations from 0◦ to 360◦ in intervals of 10◦. Next, the minimum
conformation was fully optimized by M062x//6-31+G(d,p) as the level of the calculations without any

Molecules 2016, 21, 1483
10 of 12
symmetry constraint and with all geometries modeled at a minimum of potential energy because no
imaginary frequencies were found. The minima of hybrids and were optimized in water implicit
simulations using the CPCM methodology by GAUSSIAN09L [25].
7
9
Figure 3. Computational methodology used to minimize the new hybrids
7 and 9. Optimized
geometries (Cartesian coordinates) to 7 and 9 are available in the Supplementary information.
4. Conclusions
The nine new molecular hybrids designed in this study were synthesized in good yields in two
steps from eugenol, thymol and carvacrol and presented IC50 values in the range of 22.30–4.71 M to
promastigotes of Leishmania amazonensis. Besides, the most active hybrid does not exhibit significant
µ
9
toxicity in red blood cell studies (SIrb > 84.92) which suggests the possibility of an advancement
in CL and MCL treatment. The experimental studies of bioactivity and conformational DFT
calculations presented here are in full agreement with our first mechanistic proposal whereby the
biological activity of nitro compounds is connected to nitro group reduction that generates RNO−•
.
Separation of the enantiomers of the new compound
9 by kinetic resolution catalyzed by lipase B from
Candida antarctica [26] and performing in vivo studies will continue this present work.
Supplementary Materials: Supplementary information related to this article is available at www.mdpi.com/
1420-3049/21/11/1483/s1.
Acknowledgments: The authors gratefully acknowledge CNPq and CAPES for financial support;
Felipe T. Martins (UFG) for the HRMS data; and PRPG-UFPB by financial support of the highly qualified native
English speaking editors at American Journal Experts and the payment of publication fees of this article.
Author Contributions: Francisco José da Seixas Xavier was responsible for the chemical experiments and
analyzed the data; Klinger Antonio da Franca Rodrigues was responsible for antipromastigotes experiments,
Ramon Guerra de Oliveira and Claudio Gabriel Lima Junior participate in discussions during the work.
Juliana da Câmara Rocha developed the study of cytotoxicity and selectivity index of compounds.
Tatjana Keesen de Souza Lima directed the study of cytotoxicity and selectivity index of compounds.
Marcia Rosa de Oliveira directed the study of antipromastigotes experiments; Fábio Pedrosa Lins Silva and
Mário Luiz Araújo de Almeida Vasconcellos coordinate the chemical experiments and analyzed the data and
had all conception of this work. They also wrote this article.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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