Understanding the cytotoxic effects of new isovanillin derivatives through phospholipid Langmuir monolayers

Article abstract of DOI:10.1016/j.bioorg.2018.10.029

Twenty-one isovanillin derivatives were prepared in order to evaluate their cytotoxic properties against the cancer cell lines B16F10-Nex2, HL-60, MCF-7, A2058 and HeLa. Among them, seven derivatives exhibited cytotoxic activity. We observed that for obtaining smaller IC₅₀ values and for increasing the index of selectivity, two structural features are very important when compared with isovanillin (1); a hydroxymethyl group at C-1 and the replacement of the hydroxyl group at C-3 by different alkyl groups. As the lipophilicity of the compounds was changed, we decided to investigate the interaction of the cytotoxic isovallinin derivatives on cell membrane models through Langmuir monolayers by employing the lipids DPPC (1,2-diplamitoyl-sn-glycero-3-phosphocoline) and DPPS (1,2-diplamitoyl-sn-glycero-3-phosphoserine). The structural changes on the scaffold of the compounds modulated the interaction with the phospholipids at the air-water interface. These results were very important to understand the biophysical aspects related to the interaction of the cytotoxic compounds with the cancer cell membranes.

Full text of DOI:10.1016/j.bioorg.2018.10.029

BioorganicChemistry83(2019)205–213
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Understanding the cytotoxic effects of new isovanillin derivatives through
phospholipid Langmuir monolayers
Ana C. de Carvalhoa, Natália Girolab, Carlos R. de Figueiredob, André C. Machadoa,
⁎
Lívia S. de Medeirosa, Rafael C. Guadagnina, Luciano Caselia, Thiago A.M. Veigaa,
a
Department of Chemistry, Federal University of São Paulo, Diadema, São Paulo, Brazil
b
Department of Microbiology, Immunology and Parasitology, Experimental Oncology Unit (UNONEX), Federal University of São Paulo, São Paulo, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Twenty-one isovanillin derivatives were prepared in order to evaluate their cytotoxic properties against the
cancer cell lines B16F10-Nex2, HL-60, MCF-7, A2058 and HeLa. Among them, seven derivatives exhibited cy-
totoxic activity. We observed that for obtaining smaller IC50 values and for increasing the index of selectivity,
two structural features are very important when compared with isovanillin (1); a hydroxymethyl group at C-1
and the replacement of the hydroxyl group at C-3 by different alkyl groups. As the lipophilicity of the compounds
was changed, we decided to investigate the interaction of the cytotoxic isovallinin derivatives on cell membrane
models through Langmuir monolayers by employing the lipids DPPC (1,2-diplamitoyl-sn-glycero-3-phosphoco-
line) and DPPS (1,2-diplamitoyl-sn-glycero-3-phosphoserine). The structural changes on the scaffold of the
compounds modulated the interaction with the phospholipids at the air-water interface. These results were very
important to understand the biophysical aspects related to the interaction of the cytotoxic compounds with the
cancer cell membranes.
Cytotoxic assay
Isovanillin derivatives
Langmuir monolayers
1. Introduction
Artemisinin was isolated from Artemisia annua L [4] and presents
activities against malaria [5], cancer [6] and cytomegalovirus [7].
The structural diversity of natural products represents an important
role for the development of drugs although there are still several aspects
associated with them. Restrictions for discovering new drugs from
specialized metabolites include metabolic instability, low solubility and
low absorption in the gut to the blood preventing oral formulation [1].
For this reason, promoting structural modifications on natural products
could be very useful in the search for an ideal molecule in order to
increase the activity and selectivity, improving its physicochemical
properties, such as solubility, distribution, ionization capacity, bio-
chemical and/or pharmacokinetic properties [2].
However, the treatment of some malaria patients can be difficult due to
the poor solubility in water or oil. In attempt to solve this problem some
analogues were prepared in order to improve its solubility and stability
[8]. One of these analogues, Artemisone, was promising and has been
submitted to phase III clinical trials [9,10]. Oridonin [11], isolated from
Isodon rubescens also had its structure modified. This compound de-
monstrated anticancer [12] and antibacterial activities [13]. However,
the low solubility did not allow its clinical use. Alternatively, the in-
troduction of water soluble groups increased its cytotoxicity against
some cancer cell lines [10].
Many natural compounds and their derivatives have been produced
for clinical application in the treatment of several human diseases. Among
these compounds, we highlight penicillin (antibacterial), morphine and
benzomorphanes (analgesics), artemisinin (antimalarial), camptothecin
and topotecan (anticancer). Despite the limitations presented by natural
products, structural modifications may lead to the discovery of new drugs
[3], and may increase the activity or efficiency against drug-resistant cells
or the introduction of water-soluble groups could improve the water so-
lubility of natural products to cross the cell membrane [3].
Plant bioactive components with amphiphilic or hydrophobic
structures are cytotoxic agents, and may interact with biological and
biomimetic membranes, changing their fluidity, microviscosity, elasti-
city and permeability [14]. Therefore, it is extremely important to in-
vestigate the interaction between compounds and the surface of cell
membranes by using models, as lipid Langmuir monolayers [15,16].
This approach is based on the spreading of amphiphilic molecules at the
air-liquid interface, forming a monomolecular film. When the mono-
layer is composed by membrane lipids spread on aqueous subphases, it
⁎
Corresponding author.
E-mail address: tveiga@unifesp.br (T.A.M. Veiga).
https://doi.org/10.1016/j.bioorg.2018.10.029
Received 30 May 2018; Received in revised form 10 October 2018; Accepted 15 October 2018
Availableonline19October2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

A.C. de Carvalho et al.
BioorganicChemistry83(2019)205–213
can represent a model for half-membrane, where the interface inter-
action between molecules can be investigated [15].
All NMR spectra were recorded on a Bruker spectrometer, Ultrashield
300 – Advance III operating at 300 MHz (1H) and 75 MHz (13C). HRMS
data were carried out on a Bruker Q-TOF mass spectrometer (Micro
TOF-QII) equipped with an electrospray (ESI) source, operated at a
resolution of 18.000 FWHM, in positive or negative mode, in the range
m/z 100–1200. Drying gas temperature and flow was set to 200 °C and
5 L min−1 respectively. Capillary voltage was set to 3000 V and nozzle
voltage to 500 V. The instrument was calibrated using sodium formate
automatically infused prior to each analytical run and providing a mass
accuracy better than 2 ppm. The samples were introduced into the MS
system at 10 µL min−1 via an infusion pump (kdScientific).
For example, the interaction of digitonin (a saponin) in lipid
monolayers altered the surface pressure, surface potential and surface
morphology by disturbing the lipid acyl chain orientation and the
membrane fluidity [17]. Betulinic acid, α-amyrin and ursolic acid were
incorporated in mitochondria lipids to understand the interaction of
these triterpenes with phospholipids and correlate them with the ob-
served anticancer activity [18]. In many cases, a biologically active
natural product is an inspiration for derivatives preparations, but the
main structure remains unaltered, like as: taxotere (prepared from pa-
clitaxel) and topotecan (camptothecin’s derivative) [19]. Based on this
idea, we report the preparation of several derivatives from Isovanillin
(1), which is a natural product found in many plant species like Pluchea
sagittalis [20], Pycnocycla spinose [21], Mondia whitei [22], Benincasa
hispida [23], Arum Palaestinum [24], Thalictrum przewalskii [25], Va-
leriana officinalis var. latifolia [26]. Noteworthy its constitutional isomer
vanillin, inhibited mutagenicity induced by chemical and physical
mutagens in several models, and has chemopreventive effect in che-
mical carcinogenesis models, being an agent for the treatment of sickle
cell anemia [27]. Moreover, it suppressed in vitro invasion and in vivo
metastasis on breast cancer cells [28] as well as presented activity on
human colorectal cancer cell line [29]. Vanillin derivatives also showed
anticancer activity [30] against pulmonary adenocarcinoma and human
leukemia cells [31]. Moreover, others Vanillin derivatives showed an-
ticancer activity [30] against pulmonary adenocarcinoma and human
leukemia cells [31].
2.1. Preparation of 3-hydroxy-4-methoxybenzenemethanol (2)
In a round-bottom flask equipped with magnetic stirrer was added
sodium borohydride (0.2 mmol; 0.01 g) to the solution of 1 (0.2 mmol;
0.03 g) prepared in ethanol (1 mL). The mixture was stirred at room
temperature for 30 min. The consumption of all starting material was
detected by thin layer chromatography (TLC); the solvent was evapo-
rated under reduced pressure. Subsequently, 10 mL of saturated NaCl
solution was added and then extracted with ethyl acetate (3 × 10 mL).
The organic phase was dried over anhydrous MgSO4, filtered and eva-
porated, providing 0.03 g of colorless oil, 90% yield of 2 [34].
Compound 2. 1H NMR (300 MHz, CDCl3): 7.23 (dd, J = 7.7 and
2.8 Hz, 1H); 7.07 (d, J = 7.7 Hz 1H); 7.05 (d, J = 2.8 Hz, 1H); 4.55 (s,
2H); 3.82 (s, 3H). 13C NMR (300 MHz, CDCl3): 150.6; 139.7; 133.6;
125.5; 121.8; 112.3; 64.6; 55.9.
HRMS. m/z 154.0615 [M+H]+ (calcd for C11H15O3, 154.0629, Δ
9.0 ppm).
Thus, in order to synthetize isovanillin bioactive congeners, our reac-
tions led to the compounds 2 – 4j (Fig. 1). Cytotoxic activities of the de-
rivatives were evaluated against the cancer cell lines B16F10-Nex2, HL-60,
MCF-7, A2058 and HeLa. Then, we investigated the interaction of iso-
vallinin in cell membrane models by using Langmuir monolayers. For that,
selected lipids have been chosen. The lipids 1,2-diplamitoyl-sn-glycero-3-
phosphocoline (DPPC) and 1,2-diplamitoyl-sn-glycero-3-phosphoserine
(DPPS) were chosen because they represent models for tumorigenic and
non-tumorigenic cells, respectively [32], since DPPS is overexpressed in
tumorigenic cells [33]. Thus, we investigated the interaction of Isovallinin
in cell membrane models by using lipid monolayers.
2.2. Preparation of (n)-Oxy-4-methoxybenzaldehydes (3a – 3j)
In a round-bottom flask equipped with magnetic stirrer and reflux
condenser was added sodium carbonate (1.97 mmol, 0.40 g) to the so-
lution of 1 (1 mmol, 0.15 g) and dimethylformamide (DMF, 3 mL). After
10 min, the alkyl bromides (1 mmol) were added. The solutions have
been kept under reflux by 5 h [35], and after determination of the
consumption of all starting material by TLC, 20 mL of ethyl acetate
were added, followed by the extraction with distilled water
(4 × 20 mL) and saturated NaCl solution (1 × 20 mL). The organic
phase was dried over MgSO4, filtered and evaporated under reduced
pressure to obtain compounds 3a – 3j [35].
2. Experimental section
The alkyl bromides and NaBH4 were purchased from Sigma Aldrich.
Fig. 1. Scheme for preparation of the Isovanillin derivatives.
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A.C. de Carvalho et al.
BioorganicChemistry83(2019)205–213
Compound 3a. 1H NMR (300 MHz, CDCl3): 9.84 (s, 1H); 7.42 (dd,
J = 8.1 and 1.9 Hz, 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.03 (t, 2H); 3.95 (s, 3H); 1.89 (sext, 2H); 1.04 (t, 3H). 13C NMR
(75 MHz, CDCl3): 191.0; 154.8; 149.1; 130.1; 126.6; 110.6; 110.3;
70.501; 56.2; 41.0; 22.3; 10.4.
22.8; 14.2. HRMS. m/z 405.3349 [M+H]+ (calcd for C26H45O3,
405.3369 Δ 4.9 ppm).
Compound 3j. 1H NMR (300 MHz, CDCl3): 9.84 (s, 1H); 7.42 (dd,
J = 8.1 and 1.9 Hz, 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.06 (t, 2H); 3.95 (s, 3H); 1.80 (sext, 2H); 1.26 (bs, 34H); 0.87 (t,
3H). 13C NMR (75 MHz, CDCl3):191.0; 154.8; 149.2; 130.1; 126.6;
110.5; 110.2; 69.1; 56.1; 31.9; 29.7; 29.4; 29.00; 25.9; 22.7; 14.1.
HRMS. m/z 455.3490 [M+Na]+ (calcd for C28H48O3Na, 455.3501
Δ 2.4 ppm).
HRMS. m/z 195.1028 [M+H]+ (calcd for C11H15O3, 195.1021, Δ
3.5 ppm).
Compound 3b. 1H NMR (300 MHz, CDCl3): 9.84 (s, 1H); 7.43 (dd,
J = 8.1 and 1.9 Hz, 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.03 (t, 2H); 3.95 (s 3H); 1.85 (qui, 2H); 1.50 (sext, 2H); 0.99 (t,
3H). 13C NMR (75 MHz, CDCl3): 191.0; 154.8; 149.2; 130.1; 126.6;
110.6; 11.2; 68.7; 56.2; 31.0; 19.2; 13.9.
2.3. Preparation of Oxy-4-methoxybenzenemethanols (4a – 4j)
HRMS. m/z 209.1176 [M+H]+ (calcd for C12H17O3, 209.1178, Δ
0.9 ppm).
In a round-bottom flask with magnetic stirrer was added sodium
borohydride (NaBH4) and the alkylated derivatives (3a – 3j) in di-
chloromethane (DCM, 1 mL) or ethanol (EtOH, 1 mL) solution. The
solutions were stirred at room temperature for 30 min [34]. After de-
pletion of the starting material, the solvent was removed under reduced
pressure. Thereafter, saturated NaCl solution (10 mL) was added and
the extraction with ethyl acetate (3 × 10 mL) has been done. The or-
ganic phase was dried over anhydrous MgSO4, filtered and evaporated
under reduced pressure producing yellowish solids with on average of
98% yield [34].
Compound 3c. 1H NMR (300 MHz, CDCl3): 9.84 (s, 1H); 7.42 (dd,
J = 8.1 and 1.9 Hz, 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.06 (t, 2H); 3.97 (s, 3H); 1.70 (qui, 2H) 1.50 (sext, 2H); 0.95 (t,
3H). 13C NMR (75 MHz, CDCl3): 191.0; 154.8; 149.2; 130.1; 126.6;
110.6; 110.2; 56.2; 28.7; 28.0; 22.4; 14.0.
HRMS. m/z 223.1327 [M+H]+ (calcd for C13H19O3, 223.1334, Δ
3.1 ppm).
Compound 3d. 1H NMR (300 MHz, CDCl3): 9.84 (s, 1H); 7.42 (dd,
J = 8.1 and 1.9 Hz, 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.06 (t, 2H); 3.95 (s, 3H); 1.75 (qui, 2H); 1.40 (sext, 2H); 1.28 (s,
8H); 0.88 (t, 3H). 13C NMR (75 MHz, CDCl3): 191.0; 154.8; 149.2;
130.1; 126.6; 110.6; 110.2; 69.1; 56.2; 41.0; 31.8; 29.3; 29.0; 26.0;
22.6; 14.1.
Compound 4a. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 10.9
and 1.7 Hz, 1H); 6.87 (d, J = 1.7 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.55
(bs, 2H); 3.95 (t, 2H); 3.83 (s, 3H); 1.84 (sext, 2H); 1.02 (t, 3H). 13C
NMR (75 MHz, CDCl3): δ 148.6; 133.7; 119.37; 112.14; 111.6; 70.3;
65.1; 56.1; 22.5; 10.4.
HRMS.: m/z 265.1799 [M+H]+ (calcd for C16H25O3, 265.1804, Δ
1.9 ppm).
HRMS. m/z 219.1003 [M+Na]+ (calcd for C11H16O3Na, 219.0997
Δ 2.7 ppm).
Compound 3e. 1H NMR (300 MHz, CDCl3): 9.84 (s, 1H); 7.42 (dd,
J = 8.1 and 1.9 Hz, 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.07 (t, 2H); 3.95 (s, 3H); 1.87 (sext, 2H); 1.28 (bs, 15H); 0.88 (t,
3H). 13C NMR (75 MHz, CDCl3): 191.0; 154.9; 149.2; 130.1; 126.6;
110.6; 110.3; 69.1; 56.2; 32.9; 32.00; 29.6; 29.4; 29.4; 29.1; 26.00;
22.7; 14.2.
Compound 4b. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 8.8
and 1.5 Hz 1H); 6.87 (d, J = 1.8 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.61
(bs, 2H); 4.03 (t, 2H); 3.49 (s, 3H); 1.84 (sext, 2H); 0.98 (t, 3H). 13C
NMR: δ 149.0; 148.7; 130.8; 120.3; 112.8; 111.4; 71.7; 68.7; 56.1;
31.3; 19.2; 13.9
HRMS. 233.1157 m/z 233.1003 [M+Na]+ (calcd for C12H18O3Na,
233.1154 Δ 1.8 ppm).
HRMS. m/z 293.2121 [M+H]+ (calcd for C18H29O3, 293.2117 Δ
1.4 ppm).
Compound 4c. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 10.8
and 1.4 Hz 1H); 6.87 (d, J = 1.7 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.59
(bs, 2H); 4.00 (t, 2H); 3.84 (s, 3H); 2.03 (s, OH); 1.84 (qui, 2H); 1.34
(sext, 2H); 0.92 (t, 3H). 13C NMR (75 MHz, CDCl3): δ 149.0; 148.6,
130.8, 120.4, 112.8; 111.4; 71.7; 69.0; 56.1; 28.9; 28.1; 22.5 14.0
HRMS. m/z 247.1296 1324 [M+Na]+ (calcd for C12H18O3Na,
247.1310 Δ 5.6 ppm).
Compound 3f. 1H NMR (300 MHz, CDCl3): 9.84 (s, 1H); 7.42 (dd,
J = 8.1 and 1.9 Hz, 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.07 (t, 2H); 3.95 (s, 3H); 1.85 (sext, 2H); 1.28 (bs, 15H); 0.88 (t,
3H). 13C NMR (75 MHz, CDCl3): 191.0; 154.9; 149.2; 130.1; 126.6;
110.6; 110.3; 69.1; 56.2; 32.0; 29.7; 29.7; 29.6; 29.4; 29.4; 29.1; 26.0;
22.7; 14.2.
HRMS. m/z 307.2272 [M+H]+ (calcd for C19H31O3, 307.2273 Δ
0.3 ppm).
Compound 4d. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 10.5
and 1.3 Hz, 1H); 6.87 (d, J = 1.6 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.55
(bs, 2H); 3.97 (t, 2H); 3.81 (s, 3H); 1.80 (sext, 2H); 1.25 (bs, 10H) 0.85
(t, 3H). 13C NMR (75 MHz, CDCl3): δ 148.4; 148.2; 133.8; 119.1;
112.0; 111.4; 68.8; 55.7; 31.4; 29.1; 25.7; 22.4; 13.7.
Compound 3g. 1H NMR (300 MHz, CDCl3): 9.84 (s, 1H); 7.42 (dd,
J = 8.1 and 1.9 Hz, 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.07 (t, 2H); 3.95 (s, 3H); 1.86 (qui, 2H); 1.26 (bs, 18H); 0.88 (t,
3H). 13C NMR (75 MHz, CDCl3): 191.0; 154.8; 149.1; 130.0; 126.6;
110.5; 110.2; 69.0; 56.1; 31.9; 29.6; 29.6; 29.5; 29.5; 29.3; 29.3; 28.9;
25.9; 22.6; 14.1.
HRMS. m/z 289.1782 [M+Na]+ (calcd for C16H26O3Na, 289.1779
Δ 1.0 ppm).
Compound 4e. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 9.7
and 1.3 Hz, 1H); 6.87 (d, J = 1.6 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.58
(bs, 2H); 3.99 (t, 2H); 3.84 (s, 3H); 1.83 (sext, 2H); 1.26 (bs, 7H) 0.87 (t,
3H). 13C NMR (75 MHz, CDCl3): δ 149.1; 148.8; 133.7; 119.4; 112.2;
111.6; 69.1; 65.4; 56.2; 32.0; 29.7; 29.5; 29.4; 29.3; 26.1; 22.8; 14.2.
HRMS. m/z 317.2079 [M+Na]+ (calcd for C18H30O3Na, 317.2092
Δ 4.0 ppm).
HRMS. m/z 321.2435 [M+H]+ (calcd for C20H33O3, 321.2430 Δ
1.5 ppm).
Compound 3h. 1H NMR (300 MHz, CDCl3): 9.84 (s, 1H); 7.45 (dd,
J = 8.1 and 1.9 Hz 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.07 (t, 2H); 3.95 (s, 3H); 1.86 (sext, 2H); 1.25 (bs, 26H); 0.88 (t,
3H). 13C NMR (75 MHz, CDCl3): 190.9; 154.8; 149.2; 130.1; 126.6;
110.6; 110.3; 69.1; 56.2; 31.9; 29.7; 29.6; 29.5; 29.4; 29.0; 25.9; 22.7;
14.1.
Compound 4f. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 9.6 and
1.4 Hz, 1H); 6.87 (d, J = 1.7 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.55 (bs,
2H); 3.97 (t, 2H); 3.82 (s, 3H); 1.82 (sext, 2H); 1.26 (bs, 10H); 0.87 (t,
3H). 13C NMR (75 MHz, CDCl3): δ 148.9; 133.8; 119.3; 112.1; 111.5;
69.1; 65.1; 56.0; 31.9; 29.5; 29.4; 26.0; 22.7; 14.1.
HRMS. m/z 377.3075 [M+H]+ (calcd for C24H41O3, 377.3057 Δ
4.7 ppm).
Compound 3i. 1H NMR (300 MHz, CDCl3): 9.83 (s, 1H); 7.45 (dd,
J = 8.1 and 1.9 Hz, 1H); 7.39 (d, J = 1.9 Hz, 1H); 6.97 (d, J = 8.1 Hz,
1H); 4.05 (t, 2H); 3.93 (s, 3H); 1.84 (sext, 2H); 1.24 (bs, 30H); 0.86 (t,
3H). 13C NMR (75 MHz, CDCl3): 191.0; 154.9; 149.3; 130.2; 126.7;
110.6; 110.3; 69.2; 56.2; 32.0; 29.8; 29.8; 29.7; 29.7; 29.5; 29.1; 26.0;
HRMS. m/z 331.2247 [M+Na]+ (calcd for C19H32O3Na, 331.2249
Δ 0.6 ppm).
Compound 4g. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 7.2
and 1.6 Hz, 1H); 6.87 (d, J = 1.6 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.54
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(bs, 2H); 3.97 (t, 2H); 3.82 (s, 3H); 1.82 (sext, 2H); 1.26 (bs, 10H) 0.87
(t, 3H). 13C NMR (75 MHz, CDCl3): δ 149.1; 148.9; 133.7; 119.5;
112.2; 111.7; 69.2; 65.5; 56.2; 32.1; 29.7; 29.5; 29.3; 26.1; 22.8; 14.2.
HRMS. m/z 345.2400 [M+Na]+ (calcd for C20H34O3Na, 345.2406
Δ 1.7 ppm)
lipid solutions on the air-water interface of water contained in a
Langmuir trough (KSV – model mini) made of Teflon® and equipped
with surface pressure sensor and a polarization modulation infrared
reflection-absorption spectrometer (PM-IRRAS) obtained from KSV
(PMI 550 instrument).
Compound 4h. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 7.2
and 1.6 Hz, 1H); 6.87 (d, J = 1.7 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.60
(d, J = 5.8 Hz, 2H); 4.00 (t, 2H); 3.85 (s, 3H); 1.84 (sext, 2H); 1.25 (bs,
26H); 0.88 (t, 3H). 13C NMR (75 MHz, CDCl3): δ 149.0; 133.5; 119.3;
120.0; 111.3; 69.0; 65.3; 56.1; 31.9; 29.7; 29.4; 29.2; 26.0; 22.7; 14.1.
HRMS. m/z 401.3001 [M+Na]+ (calcd for C24H42O3Na, 401. 3032
Δ 7.7 ppm)
For preliminary tests, derivatives 3e, 3f, 3g, 4e, 4f, 4g and 4h,
solubilized in chloroform to a final concentration of 0.5 mg mL−1, were
spread individually on the air-water interface in order to test their
surface activity. For mixed Isovanillin-lipid monolayers, both com-
pounds were co-spread at the air-water interface with selected pro-
portions. After 30 min for chloroform evaporation and homogenization,
surface pressure-area (π−A) isotherms were obtained by compressing
the monolayer at a rate of 5 Å2 molecule−1 min−1. All experiments
were carried out at a controlled room temperature (300 K) and con-
ducted at least three times to test the reproducibility.
Compound 4i. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 9.7 and
1.6 Hz 1H); 6.87 (d, J = 1.6 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.58 (d,
J = 5.8 Hz, 2H); 4.00 (t, 2H); 3.84 (s, 3H); 1.82 (sext, 2H); 1.25 (s, 14);
0.87 (t, 3H). 13C NMR (75 MHz, CDCl3): δ 149.1; 148.8; 133.7; 119.5;
112.2; 111.7; 69.1; 65.4; 56.2; 32.1; 29.8; 29.7; 29.7; 29.5; 29.5; 29.3;
26.1; 22.8; 14.2.
3. Results and discussion
HRMS. m/z 429.3358 [M+Na]+ (calcd for C20H34O3Na, 429.3345
Δ 3 ppm)
Isovanillin structural modifications have been performed in order to
change some functional groups, but keeping the main scaffold to verify
if the variations could increase the cytotoxic activity that was not ob-
served for compound 1. Firstly the aldehyde carbonyl was replaced by
an oxymethylene group to obtain compound 2. This compound also did
not present activity against tested cancer cell lines, B16F10-Nex2, HL-
60, MCF-7, A2058 and HeLa. Due to this fact, we have decided to ob-
serve if the replacement of the hydroxyl group (position C-3) by an
alkyl chain with a variable number of methylene groups could turn the
compound more lipophilic, and consequently more active.
Compound 4j. 1H NMR (300 MHz, CDCl3): δ 6.92 (dd, J = 9.5 and
1.5 Hz, 1H); 6.87 (d, J = 1.6 Hz, 1H); 6.85 (d, J = 8.1 Hz, 1H); 4.61 (d,
J = 5.8 Hz, 2H); 4.02 (t, 2H); 3.86 (s, 3H); 1.83 (sext, 2H); 1.25 (bs,
34H); 0.88 (t, 3H). 13C NMR (75 MHz, CDCl3): δ 119.2; 118.9; 133.7;
119.5; 112.2; 111.7; 69.2; 65.5; 56.2; 32.1; 26.1; 22.8; 14.3.
HRMS. 457.3637 m/z 457.358 [M+Na]+ (calcd for C20H34O3Na,
457.3658 Δ 4.5 ppm).
2.4. Tumor cells and culture conditions
In this way, in order to investigate the relationship between the
length of the alkyl chain and the cytotoxic effects, derivatives 3a – 3j
were prepared with alkyl chains of 3, 4, 5, 8, 10, 11, 12, 16, 18 and 20
carbons at C-3 along with carbonyl group at C-1. Compounds 3e and 3f
presented activity against B16F10-Nex2, HL-60, MCF-7, A2058 and
HeLa. In its turn, derivative 3g also presented activity against all of
them, except for MCF-7 (Table 1).
The murine melanoma cells B16F10 were originally obtained from
the Ludwig Institute for Cancer Research (LICR), São Paulo Branch. The
lineage B16F10-Nex2 was isolated at the Experimental Oncology Unit
(UNONEX) from the Federal University of São Paulo (UNIFESP) and
registered in the Banco de Células do Rio de Janeiro (BCRJ), N° 0342.
Human melanoma cell lines (A2058), breast adenocarcinoma (MCF-7),
uterine cervix adenocarcinoma (HeLa) and promyelocytic leukemia
(HL-60) have been provided by the LICR. Both cell lines were cultivated
in culture bottles, using RPMI 1640 (Gibco, Grand Island, NY) supple-
mented with 10 mM of N-(2-hydroxyethyl)-piperazine-N′-2-ethane-
sulfonic acid (HEPES; Sigma-Aldrich, St. Louis, MO), 24 mM of sodium
bicarbonate, 40 mg L−1 of Gentamicin (Hipolabor, Minas Gerais,
Brazil) at pH 7.2, and 10% of fetal bovine serum (Invitrogen). Cells
were cultivated at 37 °C in a humidified atmosphere and 5% of CO2.
Due to the activity of the compounds 3e, 3f, 3g we have decided to
prepare their derivatives originating compounds 4e, 4f and 4g. Thus,
they were obtained with a hydroxymethylene group at C-1 obtained by
using NaBH4 as reducing agent. Compounds 4e, 4f and 4g presented
smaller IC50 values (Table 1) than their related aldehydes. Thus, to
investigate if the merger between both structural features is responsible
for maintaining the activity, we also prepared the derivatives 4a, 4b,
4c, 4d, 4h, 4i and 4j from compounds 3a, 3b, 3c, 3d, 3h, 3i and 3j,
respectively.
Initially, the analysis of the IC50 values (Table 1) suggested the
importance of the structural features for increasing the cytotoxic ac-
tivity: the alkyl chain from 10 to 17 carbons and the hydroxymethylene
group at C-1. Derivatives 3e, 3f, 3g, 4e, 4f, 4g and 4h presented cy-
totoxicity against cell lines B16F10-Nex2, HL-60, MCF-7, A2058, HeLa,
except 3g and 4g that did not present effect against MCF-7 (Table 1).
Compound 4g showed higher cytotoxicity and index of selectivity (I.S.)
2.5. In vitro cytotoxic assay
To estimate the IC50 values, 1 × 104 of tumor cell lines were seeded
in 96-well plates and treated with different concentrations of each
compound ranging from 0 to 100 µg ml−1 for 24 h. Viable cells were
quantified by colorimetric assay, using MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO).
After incubation, 10 μL of MTT solution (5 mg ml−1) was added to the
cells, followed by incubation for 3 h at 37 °C. Then, a solution of 10% of
sodium dodecyl sulfate (Sigma-Aldrich, St. Louis, MO) was added to
each well. The absorbance was measured with a spectrophotometric
automated plate reader (SpectraMax-M2, Molecular Devices,
Sunnyvale, CA) at 570 nm. IC50 was measured using GraFit 5 data
analysis software (Version 5.0.13).
than others against cell lines B16F10-Nex2 (IC50 = 30.4
4.6;
A2058
1.1;
I.S = 3.04),
(IC50 = 19.8
HL-60
(IC50 = 26.4
1.3;
I.S. = 3.78),
3.0; I.S. = 5.04) and HeLa (IC50 = 18.8
I.S. = 5.30) (Table 1). In comparison, its precursor 3g which presented
IC50 values bigger than 4g. However, in this specific case, we observed
that the hydroxyl group caused a decrease on IC50 and an increase of
the I.S., that is a very important aspect in cytotoxic studies. All com-
pounds, except 3g and 4g presented activity against MCF-7. This cell
line is deficient in caspase-3 [36]. Caspase-3 is a protein which is di-
rectly related to a cell death by apoptosis [37]. If the compound pre-
sented the activity against MCF-7, there are indications that the me-
chanism of action is not by induction of apoptosis via caspase-3 [38].
To understand and suggest how the active compounds interact with
membrane models, pure isovanillin derivatives were spread on the air-
water interface, and compressed in order to obtain surface pressure-
2.6. Langmuir monolayers
The lipids DPPC and DPPS were purchased from Sigma-Aldrich and
solubilized in chloroform (Synth) to prepare a 0.5 mg mL−1 solution.
Water was purified from a Milli-Q system (resistivity 18.2 MΩ⋅cm). The
Langmuir monolayers were then obtained by spreading an aliquot of
208

A.C. de Carvalho et al.
BioorganicChemistry83(2019)205–213
Table 1
Calculated IC50 (µg mL−1) values and index of selectivity (I.S.) for the assayed isovanillin derivatives.
Compounds
Cell lines
B16F10-Nex2
HL-60
MCF-7
A2058
HeLa
I.S. = 1.06
IC50 = 53.9
I.S. = 2.24
IC50 = 25.4
I.S. = 1.71
IC50 = 32.8
I.S. = 1.14
IC50 = 49.9
I.S. = 0.67
2.4
1.3
0.4
1.3
1.7
0.3
1.7
0.9
IC50 = 85
3.1
I.S. = 1.51
IC50 = 30.8
I.S. = 2.02
IC50 = 23.1
I.S. = 2.60
IC50 = 17.9
I.S. = 1.65
IC50 = 28.2
I.S. = 1.9
IC50 = 24.4
0.7
I.S. = 0.70
IC50 = 66.0
I.S. = 0.75
IC50 = 52
I.S. = 0.71
I.S. = 1.17
IC50 = 68
I.S. = 0.72
IC50 = 93
1.7
1.5
IC50 > 100
0.01
2.4
I.S. = 1.43
IC50 = 24.9
I.S. = 2.68
IC50 = 13.3
I.S. = 2.24
IC50 = 15.9
I.S. = 1.64
IC50 = 21.8
I.S. = 1.56
IC50 = 22.8
0.5
0.6
4.6
1.0
0.8
1.3
0.1
0.4
1.6
0.5
3.0
1.4
I.S. = 1.33
IC50 = 28.1
I.S. = 2.29
IC50 = 16.3
I.S. = 1.62
IC50 = 23.0
I.S. = 1.57
IC50 = 23.8
I.S. = 1.46
IC50 = 25.5
1.3
1.1
I.S. = 3.04
IC50 = 30.4
I.S. = 3.78
IC50 = 26.4
I.S. = 0.99
I.S. = 5.04
IC50 = 19.8
I.S. = 5.30
IC50 = 18.8
IC50 > 100
I.S. = 1.07
IC50 = 73.0
I.S. = 1.36
IC50 = 72.0
I.S. = 1.14
IC50 = 47.0
I.S. = 1.04
IC50 = 46.0
I.S. = 0.76
IC50 = 54.0
0.9
1.3
2
0.9
7.2
area isotherms (Fig. 2). The isotherms have showed that they are sur-
face active and, except for the compounds 3e and 4e, which are smaller
in size of alkyl chain, are able to increase the surface pressure to values
above zero. This high increase of surface pressure, even in relative large
molecular areas, can indicate an aggregation behavior. The minimum
areas attained before collapse for the major of the case were below
30 Å2, which is compatible with their approximate sizes. Particularly,
for the compounds 3f – 3h, which contain carbonyl at C-1, the general
profile of the curve is a smooth increase of the surface pressure upon
compression until a plateau is attained at 10, 13 and 30 mN/m, re-
spectively. At the plateau is probably that the collapse has been at-
tained, although for the compound 3f a sequential increase of surface
pressure is observed after this plateau up to 20 mN/m. The relative
values of surface pressure are compatible with their size of chain, i.e.,
longer chain leads to higher surface activities.
For the compounds 4e to 4h, which contain a hydroxymethylene
group at C-1, the profile of the isotherm for the compound 4e does not
change considerably in comparison with the previous group in 3e.
However, for the compounds 4f, 4g, and 4h the initial surface pressure
is initially higher and further compression leads to progressive increase
of the surface pressure with an indefinite collapse. It is probable
therefore that the variation of the polar group may change the rheo-
logical properties of the monolayer and also the dissipation of energy
when the film is mechanically compressed.
Although we have performed mixtures of DPPC with a larger
number of compounds (as shown later in Fig. 4), we decided to focus in
the effect of three of them to easy the physical chemical interpretation
as shown in Fig. 3. Panel A shows the surface pressure-area isotherms
for DPPC monolayers. The pure DPPC monolayer presents a typical
curve, collapsing at 50 Å2/molecule and a typical plateau around 12
209

A.C. de Carvalho et al.
BioorganicChemistry83(2019)205–213
3e
3f
3g
4e
4f
and values of surface pressure. The mixtures of DPPC with all com-
pounds presented maximum high values E (about 200 mN/m), fea-
turing the LC state. This value may represent mainly the effect of DPPC,
which alone also attains high values of E relatively (250 mN/m).
However, the decrease from 250 mN/m (pure DPPC) to 200 mN/m
(mixed monolayers) could be attributed to the effect of the isovanillin
derivatives disturbing the rheological properties of the film. Although
at this stage, the derivatives may be expelled from the interface, they
must be located nearby of the film since they are lipophilic and aqueous
insoluble. Therefore, such interaction may still affect the values of
surface elasticity of the film because of viscoelastic effects.
40
35
30
25
20
15
10
5
4g
4h
In Fig. 4, we showed once more the effect of the compounds 3g, 4g
and 4h; beyond that we observed the effect of all other compounds
investigated. We believe in this way we can better correlate the simi-
larity between the structures with the similarity between the isotherm
profiles. Broadly speaking, the compound 3e presented a similar iso-
therm profile to the isotherm generated by 3g. However, the collapse
was achieved at lower values of surface pressure. Furthermore, the
compounds 4e and 4f presented similar profile than 4g. Compound 3f,
however, present a very specific isotherm when mixed with DPPC,
presenting a high surface activity, even at high film areas, and the curve
presents an expanded monolayer in relation to the pure lipid mono-
layer, indicating that the compound has not been expelled from the
film. These features, in general, are also observed in the elasticity
curves (Fig. 4B).
0
10
20
30
40
50
60
2
70
80
Area per molecule (Å )
Fig. 2. Surface pressure-area isotherms for the isovanillin derivatives.
mN/m, representing the transition between the liquid-expanded (LE)
and liquid-condensed (LC) phases. The introduction of these com-
pounds in the monolayer shifts the isotherm to higher molecular areas
of DPPC at low surface pressure values, indicating the penetration of
the compound in the lipid monolayer. However at high surface pres-
sures, attained when the film is compressed to lower areas, the com-
pounds seem to be expelled from the monolayer since the areas for the
mixed films are lower than from pure DPPC. It is important to highlight
that the molecular areas shown in the x-axis is attributed only for DPPC
and not for the other treatments (DPPC + isovanillin derivatives). This
was done in order to compare the effect of small amounts of isovanillin
derivatives onto the DPPC monolayer.
The compounds 3e and 3g present a difference of only 2 methylene
groups in the alkyl chain at C-3, but 3f, even present a alkyl chain with
an intermediate size between 3e and 3g, it presents a more expanded
monolayer, indicating that a single addition of a methylene group in the
alkyl chain of the compound affected the surface activity properties on
the DPPC monolayer. However the profile of the isotherm is similar
even comparing 3f with 3e and 3g, and the main difference is on the
occupancy area. It is probably that 3f may be arranged molecularly
with DPPC at the air-water interface at such a way that either provides
repulsion among the chains, or the mixed monolayer component may
suffer geometrical restrictions to accommodate at the interface.
Furthermore, the compounds 4e, 4f and 4g also present differences only
in the size of the alkyl group (with 9, 10, and 11 methylene groups,
respectively). Compound 4h is also similar to these compounds, but this
distinctive isotherm can be explained by the higher number of methy-
lene groups (15). Curiously, it is the compound with size more com-
parable to that for DPPC, and it presents the isotherm with a profile
more similar to that for pure DPPC. This point leads us to an important
finding: that compounds with similar alkyl chain sizes may adapt better
at the air-water interface, causing less effect regarding 2D-transitions,
expelling from the interface, and collapse process. It likely, therefore,
that the compound 4h and DPPC may be molecularly arranged as a
mixed Langmuir monolayer that can be packed regularly when laterally
compressed.
For compound 3g, interestingly, we observe a second plateau
around 30 mN/m, which may represents the phase where the com-
pound is being expelled from the interface. For compound 4g, parti-
cularly, the effect is more evident as it causes an instantly raise in the
surface pressure even at high molecular areas. Further compression
leads the monolayer to a plateau at 40 mN/m and a sequential raise of
the surface pressure at very low areas (30 Å2). This plateau may present
not only the mechanism for expelling the compounds from the mono-
layer, but also the disruption of the film monomolecular structure.
Possibly 4g destroys the film, taking together some DPPC molecules to
the collapse structure since the area of 30 Å2 is too low considering the
structure of the lipid. This may be related to the cytotoxic effect ob-
served for 4g.
Fig. 3B shows the surface elasticity (E) of the monolayers, defined as
−A(∂π/∂A)T (A: molecular area; π: surface pressure; T: temperature).
At about 12 mN/m, the decrease of the E values is related to the LE-LC
transition. The plateaus for 3g and 4g observed in their surface pres-
sure-area isotherm at 30 and 40 mN/m, respectively, can be observed in
the elasticity curves by the low values of E in the correspondent curves
DPPC
DPPC + 4h
DPPC + 3g
DPPC + 4g
DPPC
DPPC + 4h
DPPC + 3g
DPPC + 4g
300
60
A
B
250
200
150
100
50
50
40
30
20
10
0
0
-50
0
10
20
30
40
50
60
20 30 40 50 60 70 80 90 100 110 120
Surface pressure (mN/m)
2
Area per molecule (Å )
Fig. 3. Surface pressure-area (A) and compressional modulus-surface pressure (B) isotherms for DPPC, pure or mixed with the compounds 3g, 4g and 4h (5% in mol).
210

A.C. de Carvalho et al.
BioorganicChemistry83(2019)205–213
DPPC
300
250
200
150
100
50
DPPC + 3e
DPPC + 3f
DPPC + 4e
DPPC + 4f
DPPC
60
50
40
30
20
10
0
DPPC + 4h
DPPC + 3g
DPPC + 4g
DPPC + 3e
DPPC + 3f
DPPC + 4e
DPPC + 4f
B
A
0
-50
40 50 60 70 80 90 100 110 120
0
10
20
30
40
50
60
2
Area per molecule (Å )
Surface pressure (mN/m)
Fig. 4. Surface pressure-area (A) and compressional modulus-surface pressure (B) isotherms for DPPC, pure or mixed with the isovanillin derivatives (5% in mol) and
indicated in the inserts.
DPPS
DPPS
DPPS
DPPS + 3g
DPPS + 3e
DPPS + 3f
DPPS + 4e
DPPS + 4f
DPPS + 4g
DPPS + 4h
DPPS + 3g
DPPS + 3e
DPPS + 3f
DPPS + 4e
DPPS + 4f
DPPS + 4g
DPPS + 4h
70
60
50
40
30
20
10
0
500
400
300
200
100
0
A
B
C
250
200
150
100
50
0
0
10 20 30 40 50 60 70
0
20
40
60
0
50
100
150
200
2
250
Surface pressure (mN/m)
Surface pressure (mN/m)
Area per molecule (Å )
Fig. 5. Surface pressure-area (A) and compressional modulus-surface pressure (B and C) isotherms for DPPS, pure or mixed with the isovanillin derivatives (5% in
mol) and indicated in the inserts.
Fig. 5 shows the effect of the same compounds with DPPS, a lipid
encountered in high quantities in tumorigenic cells [33]. Curiously,
except for the compound 3g, all other tested compounds expanded the
lipid monolayer, which contrast with the effect on DPPC monolayers,
once the condensation of the monolayer presented the behavior more
commonly encountered. The profiles of the isotherms are almost all
similar, generally speaking. There is an initial increase of the surface
pressure at high molecular areas, with compression it is attained a
plateau at high surface pressures (30 or 40 mN/m), and after sequential
compression, the pressure raises again and the monolayer collapses.
Interestingly the compounds 3e, 3f and 3g present all a plateau at 30
mN/m, and they differs only in the number of methylene groups on the
alkyl chain. In its turn, compounds 4e, 4f and 4g presented a plateau at
40 mN/m and also only differ on the size of the alkyl chain, but with an
hydroximethylene group at C-1. It can be conclude, therefore, that the
chemical differential of the two set of compounds (the presence of a
carbonyl or a hydroxyl group) is the factor that determines the surface
pressure of this plateau. Consistently, the compound 4h was the only
one that did not present such plateau, showing a isotherm only more
expanded (shifted to higher areas) and more compressible than DPPS
(presented lower values of elasticity as shown in Panel 5C, and also
clear observing the inclination of the surface pressure-area isotherms).
In this case, the explanation is similar to that for DPPC. The similarity
between the size of the derivative and the phospholipid may determine
a better molecular accommodation at the air-water interface when the
lipid-derivative is packed due to the lateral compression.
cells, the incorporation of small amounts of the compound basically
expanded the monolayers, whereas for DPPC, a zwitterionic charged
lipid and model for non-tumorigenic cells, it condenses the monolayers.
Such distinctive interaction may be resulted from different electrostatic
interactions between the compounds and the lipid concerning not only
interactions between net charges, but also between polar and apolar
regions.
For the myriad of seven lipids employed in this work, compound 4h,
particularly, presented a less characteristic effect on the monolayer
isotherms, and we believe that it must be related to the similar size of
this compound to lipids employed. For the other derivatives, we can
separate them in two groups since those with hydroxyl and those with
carbonyl groups presented distinctive behavior in terms of 2D phase
transition concerning the surface pressure-area isotherms.
By employing lipid monolayers was possible to understand the cy-
totoxic activities of isovanilin derivatives. This approach is becaming an
interesting tool to go further on chemistry of interfaces. In the analyses
of natural products, some studies have used similar methods for dif-
ferent biological targets. Broniatowski et al. [39], evaluated the anti-
bacterial activity of some pentacyclic triterpenes. As the model mem-
brane, were applied mixtures of the representative Escherichia coli
phosphatidylethanolamine (POPE), with the cardiolipin (ECCL) or
phosphatidylglycerol (ECPG) extracted from the E. coli inner mem-
brane. Violacein, a bis-indole pigment, was investigated on cell mem-
brane models represented by selected lipids, dipalmitoylpho-
sphatidylcholine/DPPC
(healthy
cellular
membranes),
and
Although the correlation of the surface properties of each compound
with their cytotoxic properties presented in Table 1 is not straightfor-
ward, we can steadfastly conclude that the small changes in the che-
mical structure of the compounds not only alter the cytotoxicity ac-
tivities on several selected cell lines, but also they modulate the
interaction with the phospholipid at the air-water interface. This in-
teraction is highly dependent on the chemical structure of the lipid,
since for DPPS, a negative charged lipid and a model for tumorigenic
dipalmitoylphosphatidylserine/DPPS (tumorigenic cellular mem-
branes). Morphological analysis also suggests that violacein at the air-
water interface is homogenized when incorporated in both lipids [32].
These results therefore may have important implications in under-
standing how violacein acts on specific sites of the cellular membrane,
and evidence the fact that the lipid composition of the monolayer
modulates the interaction with the lipophilic drug.
Similar study was performed for Quercetin. The flavonoid
211

A.C. de Carvalho et al.
BioorganicChemistry83(2019)205–213
incorporation diminishes the monolayer surface elasticity for DPPC and
causes a relative decrease of the intensity for CeH stretch bands,
pointing to a disruption of the packed order of DPPC. These results can
be associated to the interaction between Quercetin and cell membrane
surfaces during biochemical processes, which may influence its phar-
maceutical properties [15]. A higher monolayer expansion was ob-
served for DPPS and DODAB in the presence of Eugenol [40]. These
results pointed to the fact that the interaction of Eugenol with lipid
monolayers at the air–water interface is modulated by the lipid com-
position, which may be important to comprehend at the molecular level
the interaction of this drug with biological surfaces.
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Acknowledgements
The authors thank the financial support from Fundação de Amparo a
Pesquisa do Estado de São Paulo (FAPESP, process 2010/51313-0) and
CAPES - Finance Code 001 (Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior) for the scholarship granted to ACC.
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A.C. de Carvalho et al.
BioorganicChemistry83(2019)205–213
HeLa: human epitheloid cervix carcinoma cell line
Glossary
DPPC: 1,2-diplamitoyl-sn-glycero-3-phosphocoline
DPPS: 1,2-diplamitoyl-sn-glycero-3-phosphoserine
MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
LE: liquid-expanded phase
B16F10-Nex2: murine melanoma cell line
HL-60: human leukemia cell line
MCF-7: human breast adenocarcinoma cell line
A2058: human melanoma cell line
LC: liquid-condensed phase
213