Molecules 2014, 19, 10670-10697; doi:10.

3390/molecules190810670
OPEN ACCESS

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article

A QSAR, Pharmacokinetic and Toxicological Study of New

Artemisinin Compounds with Anticancer Activity
Josinete B. Vieira 1, Francinaldo S. Braga 1, Cleison C. Lobato 1,2, César F. Santos 1,
Josivan S. Costa 1, José Adolfo H. M. Bittencourt 3, Davi S. B. Brasil 1,4, Jocivânia O. Silva 2,3,
Lorane I. S. Hage-Melim 1,2, Williams Jorge C. Macêdo 1,5, José Carlos T. Carvalho 2,3 and
Cleydson Breno R. Santos 1,2,3,*
1
Laboratory of Modeling and Computational Chemistry, Federal University of Amapá, Rod JK
Km2, Macapá, Amapá 68902-280, Brazil; E-Mails: jnetbio.unifap2011.ap@gmail.com (J.B.V.);
fsbraga@unifap.br (F.S.B.); cleyson.cl@gmail.com (C.C.L.); cesar.fs@globomail.com (C.F.S.);
josivan.chemistry@gmail.com (J.S.C.); davibb@ufpa.br (D.S.B.B.); lorane@unifap.br (L.I.S.H.-M.);
williams.macedo@ufra.edu.br (W.J.C.M.)
2
Postgraduate Program in Pharmaceutical Sciences, Federal University of Amapá, Rod JK Km 2,
Macapá, Amapá 68902-280, Brazil; E-Mails: jocivania@unifap.br (J.O.S.);
farmacos@unifap.br (J.C.T.C.)
3
Laboratory of Drug Research, School of Pharmaceutical Sciences, Federal University of Amapá,
Macapá, Amapá 68902-280, Brazil; E-Mail: joseadolfo@unifap.br
4
Institute of Technology, Federal University of Pará, Av. Augusto Corrêa, 01, Belém,
Pará 66075-900, Brazil
5
Laboratory of Molecular Modeling and Simulation System, Federal Rural University of Amazônia,
Rua João Pessoa, 121, Campus Capanema-Centro, Capanema, Pará 68700-030, Brazil

* Author to whom correspondence should be addressed; E-Mail: breno@unifap.br;

Tel.: +55-96-4009-2920; Fax: +55-96-4009-2907.

Received: 5 June 2014; in revised form: 3 July 2014 / Accepted: 7 July 2014 /
Published: 24 July 2014

Abstract: The Density Functional Theory (DFT) method and the 6-31G** basis set were
employed to calculate the molecular properties of artemisinin and 20 derivatives with
different degrees of cytotoxicity against the human hepatocellular carcinoma HepG2 line.
Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were
employed to select the most important descriptors related to anticancer activity. The
significant molecular descriptors related to the compounds with anticancer activity were
the ALOGPS_log, Mor29m, IC5 and GAP energy. The Pearson correlation between
Molecules 2014, 19 10671

activity and most important descriptors were used for the regression partial least squares
(PLS) and principal component regression (PCR) models built. The regression PLS and
PCR were very close, with variation between PLS and PCR of R2 = ±0.0106, R2ajust = ±0.0125,
s = ±0.0234, F(4,11) = ±12.7802, Q2 = ±0.0088, SEV = ±0.0132, PRESS = ±0.4808 and
SPRESS = ±0.0057. These models were used to predict the anticancer activity of eight new
artemisinin compounds (test set) with unknown activity, and for these new compounds
were predicted pharmacokinetic properties: human intestinal absorption (HIA), cellular
permeability (PCaCO2), cell permeability Maden Darby Canine Kidney (PMDCK), skin
permeability (PSkin), plasma protein binding (PPB) and penetration of the blood-brain
barrier (CBrain/Blood), and toxicological: mutagenicity and carcinogenicity. The test set
showed for two new artemisinin compounds satisfactory results for anticancer activity and
pharmacokinetic and toxicological properties. Consequently, further studies need be done
to evaluate the different proposals as well as their actions, toxicity, and potential use for
treatment of cancers.

Keywords: artemisinin; anticancer activity; molecular modeling; B3LYP/6-31G**; QSAR

1. Introduction

Cancer, also called malignant neoplasm or malignant tumor, is a disease characterized by the
uncontrolled growth of abnormal cells in an organism [1]. While the origin of these is due to genetic
alterations may be by inactivation of tumor suppressor genes, activation of oncogenes, inactivation of
genes responsible for apoptosis and mutations produced by chemical, physical and biological agents,
and are characterized by loss of function coming from the absence of differentiation, uncontrolled
proliferation, invasiveness of adjacent tissues and metastasis [2,3].
On a global scale there was an increase to 14.1 million new cases of different types of cancer in
2012, causing 8.2 million deaths, in accordance with the online channel GLOBOCAN 2012 [4]. The
prevalence estimates for 2012 show that there were 32.6 million people (over the age of 15 years) who
have had a cancer diagnosed in the last five years. The types most commonly diagnosed around the
world were lung (1.8 million, 13.0% of the total), breast (1.7 million, 11.9%), and colon and rectum
(1.4 million, 9.7%). The most common determinants of death were lung cancers (1.6 million, 19.4% of
the total), liver (0.8 million, 9.1%) and stomach (0.7 million, 8.8%). Importantly, among the different
forms of cancer malignant tumors of the liver, hepatocellular carcinoma type, is the second most
common causing deaths around the world [5].
Nowadays a variety of factors has driven the search for new drugs of plant origin, particularly the
discovery of drugs that fight cancer effectively [6]. Chaturvedi [7] relates that nowadays the antitumor
action is the most widely studied biological activity of sesquiterpene lactones, where studies reveal
that these are capable of combating tumors via selective alkylation, thereby controlling and inhibiting
cell division. This set of factors and cellular functions leads the cells to lose action by apoptosis.
There are some drugs derived from sesquiterpene lactones such as artemisinin, that in clinical trials
showed activity to combat cancer [7–9]. Artemisia annua L. a plant species coming from temperate
Molecules 2014, 19 10672

regions such as China and Southeast Europe, contains the active principle artemisinin (qinghaosu), that
is widely used in traditional Chinese medicine for the treatment of malaria [10].
Recently artemisinin (Figure 1, compound 1) has been reported for its ability to exert a cytotoxic
effect on cancer cells [11]. Studies of the activity of artemisinin and its derivatives appear to indicate it
is mediated by its interaction through the endoperoxide function of the 1,2,13-trioxane ring [12].
Therefore, it becomes necessary to discover the mechanism of action of the compound to be studied in
order to determine how to carry out drug-receptor interactions, for this is necessary the utilization of
some tools such as the use of molecular modeling that enables one to determine cell sites or the
physiology involved in this process [13].
Molecular modeling is a tool that consists in the application of theoretical models to represent and
manipulate the structure of molecules, study chemical reactions and establish relationships between
structure and properties of matter [14,15]. In the theoretical chemistry area there are some strategies
that are promising in relation to the design of new drugs, such as rational design, which consists of
using information in different areas of human knowledge, especially those related to the electronic
levels of the drug, physical-chemical parameters (hydrophobic, steric and electronic) related with the
biological activity [16–19]. This type of strategy, unlike molecular modification, does not have high
time demands and is low in financial investment. Among the various techniques we can highlight
planning with the help of computer, which is a resource that increases considerably the possibilities of
scientific research in discovery of new drugs [20–23].
In this paper, a QSAR study of artemisinin and 20 derivatives with logarithm of relative activity,
logRA (see Figure 1) that showed different degrees of cytotoxicity against the human hepatocellular
carcinoma HepG2 line [24]. Initially, the structures were modeled, and many different molecular
descriptors were computed. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis
(HCA) were employed to choose the molecular descriptors that are most related to the anticancer
biological property investigated. Then, a QSAR model was elaborated through the Principal
Component Regression (PCR) and Partial Least Square (PLS) methods that were used to perform
predictions for eight new artemisinin compounds (test set) with unknown anticancer activity [25–28].
For these eight compounds the following pharmacokinetic properties: human intestinal absorption (HIA),
cellular permeability (PCaCO2), cell permeability Maden Darby Canine Kidney (PMDCK), skin permeability
(PSkin), plasma protein binding (PPB) and penetration of the blood-brain barrier (CBrain/Blood), and
toxicological ones, mutagenicity and carcinogenicity, were predicted. These predictions aid in the
interactions between micromolecules and their molecular targets, predicting, also, possible toxic
consequences of the drug candidate and to aid in future studies searching for other new anticancer drugs.

2. Results and Discussion

2.1. Determination of the Theoretical Geometrical Parameters for the 1,2,13-Trioxane Ring of
Artemisinin (Bond Length, Bond Angle, and Torsion Angle of Atoms in this Ring) in Different Methods
and Basis Sets

We determined the geometrical parameters for the 1,2,13-trioxane ring of artemisinin (bond length,
bond angle, and torsion angle of atoms in this ring), as shown in Table 1.
Molecules 2014, 19 10673

Figure 1. Structure and biological activity of artemisinin and derivatives with anticancer
activity against human hepatocellular carcinoma HepG2 line.

CH3 CH3
CH3 2 3
1 H H
H
5 6
4 O O O O
H3C H3C
5a 7
H3C 3 O O
2 1 O O
12a 8 H H
H H
O 12 8a
13 O O
H CH3 CH3
H
O 9 H
11 10 NH CH3 NH C10H21
CH3
O O O
logRA = 0.00000 logRA = −0.0132 logRA = 1.5396
CH3 CH3 CH3
4 H 5 H
6 H

O O O O H3C O O
H3C H3C

O O O
H H H
H H H
O O O
CH3 CH3 CH3

NH C12H25 NH C18H37
NH C14H29

O
O O

logRA = 1.9075 logRA = 2.3240 logRA = 1.3635

CH3 CH3 CH3

7 8 H
9 H
H

O O H3C O O H3C O O
H3C

O O O
H H H
H H H
O O O
CH3 CH3 CH3

O C2H5 O C12H25 O C18H37

O O O

logRA= −0.0132 logRA = 2.1294 logRA= −0.0132

Molecules 2014, 19 10674

Figure 1. Cont.

CH3 CH3 CH3

10 11 H 12 H
H

O O H3C O O H 3C O O
H3C

O O O
H H H
H H H
O O O
CH3 CH3 CH3

C 2H 5 C 12 H 25
C2H5

OH OH
OH

logRA = −0.0132 logRA = −0.0132 logRA = 1.5396

CH3 CH3 CH3
13 H 14 H 15 H

O O H 3C O O O O
H3C H 3C

O O O
H H H
H H H
O O O
CH3 CH3 CH3

C12H25 C 18H 37 C 18H 37

OH OH OH

logRA = 1.3433 logRA = −0.0132 logRA = −0.0132

CH3 CH3 CH3
16 17 H 18
H H

O O H 3C O O H3C O O
H3C

O O O
H H H
H H H
O O O
CH3 CH3 CH3

C 18 H 37 CH 2(CH 2) 6CH 2OH

C2H 5

O O
O

logRA = −0.0132 logRA = −0.0132 logRA = 0.3604

CH3 CH3
19 CH3 20 H 21 H
H
H3C H3C
H 3C O O
O O O
O
O O
O
O O
O H H H
H H
O C 14 H 29 H
O C 12 H 25 O C12H25
NH O
NH
H3C H3C H3C

logRA = 1.8728 logRA = 2.1002 logRA = 1.4185

Molecules 2014, 19 10675

Table 1. Theoretical and experimental parameters for the 1,2,13-trioxane ring in artemisinin (compound 1).
Semiempirical Hartree-Fock/HF DFT/B3LYP
Parameters [a] EXP[29]
AM1 PM3 ZINDO 6-31G 6-31G* 6-31G** 3-21G 3-21G* 3-21G** 6-311G 6-31G 6-31G* 6-31G** [b] 3-21G
Bond length (Å)
O1O2 1.288 1.544 1.237 1.447 1.391 1.390 1.461 1.461 1.462 1.429 1.524 1.459 1.459 1.524 1.469
O2C3 1.447 1.403 1.400 1.435 1.393 1.396 1.440 1.440 1.439 1.432 1.452 1.413 1.414 1.455 1.416
C3O13 1.427 1.428 1.396 1.435 1.388 1.408 1.436 1.435 1.435 1.434 1.473 1.441 1.441 1.473 1.445
O13C12 1.416 1.403 1.392 1.403 1.400 1.376 1.407 1.407 1.407 1.401 1.425 1.395 1.396 1.430 1.379
C12C12a 1.537 1.555 1.513 1.533 1.533 1.532 1.529 1.529 1.530 1.530 1.438 1.539 1.539 1.535 1.523
C12aO1 1.468 1.427 1.416 1.469 1.429 1.429 1.477 1.477 1.477 1.438 1.499 1.455 1.455 1.504 1.461
Bond angle (°)
O1O2C3 112.530 110.340 114.310 108.800 106.100 109.460 107.100 107.080 107.060 109.210 107.300 108.280 108.280 105.590 108.100
O2C3O13 103.600 104.810 105.370 106.760 110.800 107.800 107.270 107.285 107.300 106.670 107.730 108.490 108.490 108.220 106.600
C3O13C12 115.480 116.010 115.843 117.300 112.800 115.300 115.670 115.680 115.710 116.960 114.990 114.080 114.060 113.200 114.200
O13C12C12a 113.510 115.200 113.270 112.280 108.700 112.300 112.080 112.080 112.030 112.360 113.640 113.250 113.240 113.300 114.500
C12C12aO1 111.070 113.180 107.290 110.910 110.500 110.545 111.570 111.600 111.600 110.760 111.740 111.290 111.280 112.410 110.700
C12aO1O2 113.740 112.290 118.380 113.240 112.700 112.700 111.290 111.290 111.290 113.360 111.400 111.600 111.590 109.620 111.200
Torsion angle (°)
O1O2C3O13 −77.800 −73.310 −70.403 −71.840 −73.369 −73.400 −74.670 −74.700 −74.690 −71.940 −73.460 −73.900 −73.910 −76.610 −75.500
O2C3O13C12 42.070 52.700 36.370 33.390 31.034 31.100 32.300 32.360 32.180 33.010 34.970 32.800 32.780 33.750 36.000
C3O13C12C12a 11.400 2.811 17.420 25.320 27.432 27.400 28.290 28.190 28.330 25.380 26.260 27.460 25.500 29.060 25.300
O13C12C12aO1 −41.770 −40.510 −46.610 −49.410 −50.100 −50.143 −50.860 −50.770 −50.700 −49.470 −51.200 −51.270 −51.340 −52.190 −51.300
C12C12aO1O2 12.050 19.940 18.110 12.510 10.900 10.924 9.989 9.940 9.750 12.480 12.740 11.730 11.780 9.060 12.700
C12aO1O2C3 47.050 35.630 40.130 46.700 48.700 48.674 50.330 50.350 50.530 46.870 46.900 47.850 47.830 51.060 47.800
Standard
4.776 8.388 4.372 1.663 2.484 1.762 1.722 1.714 1.797 1.658 0.843 1.227 1.103 1.915 ˗
Deviation
[a]
: The atoms are numbered according to compound 1 in Figure 1; [b]: Valence basis set separately validated for calculating the molecular properties.
Molecules 2014, 19 10676

Table 1 illustrates that for the DFT method, all four basis sets (B3LYP/6-31G, B3LYP/6-31G*,
B3LYP/6-31G**, and B3LYP/3-21G) can accurately describe all of the structural parameters with
respect to their magnitude and sign when compared with the experimental values.
Meanwhile, in the semiempirical (AM1, PM3, and ZINDO) and Hartree-Fock (HF/6-31G,
HF/6-31G*, HF/6-31G**, HF/3-21G, HF/3-21G*, HF/3-21G**, and HF/6-311G) methods there is not
good agreement between the experimental and theoretical values for the torsion angles, especially the
angle formed by atoms O2C3O13C12, with deviations −6.070° (AM1), −16.700° (PM3), −0.370°
(ZINDO), +2.610° (HF/6-31G), +4.966° (HF/6-31G*), +4.900° (HF/6-31G**), +3.700° (HF/3-21G),
+3.640° (HF/3-21G*), +3.820° (HF/3-21G**), +2.990° (HF/6-311G), +1.030° (B3LYP/6-31G),
+3.200° (B3LYP/6-31G*), +3.220° (B3LYP/6-31G**) and +2.250° (B3LYP/3-21G) and exhibited
standard deviations of 4.776 (AM1), 8.388 (PM3), 4.372 (ZINDO), 1.663 (HF/6-31G), 2.484
(HF/6-31G*), 1.762 (HF/6-31G**), 1.722 (HF/3-21G), 1.714 (HF/3-21G*), 1.797 (HF/3-21G**),
1.658 (HF/6-311G), 0.843 (B3LYP/6-31G), 1.227 (B3LYP/6-31G*), 1.103 (B3LYP/6-31G**) and
1.915 (B3LYP/3-21G), respectively.
Table 1 shows that for artemisinin (compound 1) the B3LYP/6-31G, B3LYP/6-31G*, B3LYP/6-31G**
basis sets show excellent results for bond length, bond angle and torsion angle compared to the
experimental data. The B3LYP/6-31G method described geometrical parameters well, with values
close to the experimental results. However, the minimum base 6-31G has several deficiencies; thus, a
polarization function was included to improve upon this base (i.e., p orbitals represented by *). Thus,
6-31G* refers to basis set 6-31G with a polarization function for heavy atoms (i.e., atoms other than
hydrogen), and 6-31G** refers to the inclusion of a polarization function for hydrogen and helium
atoms [29–35].
When basis sets with polarization functions are used in calculations involving anions, good results
are not obtained due to the electronic cloud of anionic systems, which tend to expand. Thus,
appropriate diffuse functions must be included because they allow for a greater orbital occupancy in a
given region of space. It then becomes necessary to include diffuse functions in the basis function
associated with the configuration of a neutral metal atom to obtain a better description of the metal
complex. The 6-31G** basis is particularly useful in the case of hydrogen bonds [30–35].
Cristino et al. [36] used the B3LYP/6-31G* method to model artemisinin and 19 10-substituted
deoxoartemisinin derivatives, with different degrees of activity against the Plasmodium falciparum
D-6 strains of Sierra Leone. Chemometric methods (PCA, HCA, KNN, SIMCA, and SDA) were
employed to reduce the dimensionality and to determine which subset of descriptors is responsible for
the classification between more and less active agents.
Figueiredo et al. [37] conducted studies using the B3LYP/6-31G* method for antimalarial
compounds against Plasmodium falciparum K1. These studies led to multivariate models for
artemisinin derivatives and series of dispiro-1,2,4-trioxolanes. The application of these models has
enabled the prediction of activity for compounds designed without known biological activity.
Moreover, a new series of antimalarial compounds is currently in the study phase.
Araújo et al. [38] used density functional theory (6-31G*) to verify the performance of a base set in
reproducing experimental data, particularly geometrical parameters, and to calculate the interaction
energies, electronic states, and geometrical arrangements for complexes composed of a heme group
and artemisinin. The results demonstrated that the interaction between artemisinin and the heme group
Molecules 2014, 19 10677

occurs at long distances through a complex in which the iron atom of the heme group retains its
electronic characteristics, with the quintet state being the most stable. These results suggest that the
interaction between artemisinin and heme is thermodynamically favorable.
Pereira et al. [39] studied four structures of artemisinin by reductive decomposition A, B1, B2, and
B3 with 13 species (QHS, 1/2, 3, 4, 5, 5a, 6, 7, 18, 18a, 19, 20, and 21), and the structures of the
studied species were analyzed in terms of geometrical parameters, Löwdin bond orders, atomic partial
charges, spin densities, electronic energies, free energies, and entropy. These studies were carried out
at the B3LYP/6-31G** level.
Carvalho et al. [40] used the B3LYP/6-31G** method to study artemisinin and 31 analogues with
antileishmanicidal activity against Leishmania donovani. The authors proposed a set of 13 artemisinins,
seven of which are less active and six of which that have not been tested; of these six, one is expected
to be more active against L. donovani.
Barbosa et al. [41] performed molecular modeling and chemometric studies involving artemisinin
and 28 derivatives exhibiting anticancer activity and the calculations of the compounds studied were
performed at the B3LYP/6-31G** level.
By comparing these methods with the DFT method (see Table 1), we find that all of the basis sets
(B3LYP/6-31G, B3LYP/6-31G*, and B3LYP/6-31G**) have low standard deviations in relation to the
semiempirical and Hartree-Fock methods at 0.843 (B3LYP/6-31G), 1.227 (B3LYP/6-31G*), and 1.103
(B3LYP/6-31G**). The variation was ±0.384 between B3LYP/6-31G and B3LYP/6-31G*, ±0.260
between B3LYP/6-31G and B3LYP/6-31G**, and ±0.124 between B3LYP/6-31G* and B3LYP/6-31G**.
This study highlighted the B3LYP/6-31G** basis set, which is closer to the experimental results and
shows good performance in the description when comparing the O2C3 and C3O13 bond length, O1O2C3
and C3O13C12 bond angles. The torsion angles or dihedral angle also showed good agreement with
the experimental values reported in the literature, showing that with the 6-31G** basis set, the torsion
angles O13C12C12aO1 and C12aO1O2C3 are closer to the artemisinin crystallographic data.

2.2. Principal Component Analysis (PCA) Results

The PCA results showed that the most important descriptors were the following: ALOGPS_logs,
Mor29m, IC5 and GAP energy. They were chosen from the complete data set (1716 descriptors) and
other variables were not selected because either they had a poor linear correlation with activity or they
did not give a distinct separation between the more and less active.
The values of the important descriptors of each selected compound identified via PCA as well as the
values of logRA, relative activity (RA) and the IC50 is the 50% inhibitory concentration are shown in
Table 2. The Table 2 shows the Pearson correlation matrix between the descriptors and logRA, and the
correlation between pairs of descriptors is less than 0.2420, while the correlation between the
descriptors and logRA is less than 0.7459. The descriptors selected by PCA represent the
characteristics necessary to separate between the more and less active with anticancer activity of these
compounds against human hepatocellular carcinoma HepG2.
Molecules 2014, 19 10678

Table 2. Physicochemical properties selected by PCA, experimental logRA values, IC50

and the correlation matrix.
Compounds ALOGPS_log Mor29m IC5 Gap Energy logRA RA IC50/µΜ
1- −2.3500 −0.3050 4.8620 0.2616 0.0000 1.0000 97
-
2 −3.5200 −0.3070 5.2530 0.2525 −0.0132 0.9700 100
3+ −6.3500 −0.4550 5.6840 0.2521 1.5396 34.6417 2.8
+
4 −6.8400 −0.5250 5.6240 0.2524 1.9075 80.8164 1.2
+
5 −7.1600 −0.5140 5.5010 0.2527 2.3240 210.8628 0.46
+
6 −7.4900 −0.5010 5.2250 0.2525 1.3635 23.0940 4.2
7- −3.6400 −0.2360 5.2170 0.2467 −0.0132 0.9700 100
8+ −7.0300 −0.5260 5.5970 0.2462 2.1294 134.7100 0.72
-
9 −7.6800 −0.1790 5.1970 0.2462 −0.0132 0.9700 100
-
10 −3.6800 −0.3650 5.2530 0.2367 −0.0132 0.9700 100
11- −3.6800 −0.3050 5.2530 0.2359 −0.0132 0.9700 100
+
12 −6.9700 −0.3940 5.5080 0.2457 1.5396 34.6417 2.8
+
13 −6.9700 −0.2910 5.5080 0.2552 1.3433 22.0444 4.4
-
14 −7.4000 −0.2280 5.1590 0.2217 −0.0132 0.9700 100
15- −7.4000 −0.2280 5.1590 0.2287 −0.0132 0.9700 100
-
16 −3.7500 −0.4430 5.1800 0.2194 −0.0132 0.9700 100
-
17 −7.6100 −0.3330 5.1680 0.2177 −0.0132 0.9700 100
+
18 −5.4900 −0.3470 5.6380 0.2199 0.3604 2.2929 42.3
19+ −6.7200 −0.5520 5.5430 0.2491 1.8728 74.6105 1.3
+
20 −7.0600 −0.5520 5.4190 0.2492 2.1002 125.9505 0.77
+
21 −6.8400 −0.5150 5.5160 0.2449 1.4185 26.2119 3.7
ALOGPS_log 0.2420 −0.4260 0.0497 −0.5265 - -
Mor29m −0.5892 −0.2971 −0.8249 - -
IC5 0.1767 0.7459 - -
Gap energy 0.5238 - -

The results of the PCA model are presented in Table 3. The model was constructed with three main
components (3 PCs). The first principal component (PC1) describes 38.6537% of the total information,
the second principal component (PC2) describes 21.5859%, and the third (PC3) 12.3501%. PC1
contains 48.3171% of the original data, and the combination of the first two components (PC1 + PC2)
contains 75.2996%, and all three (PC1 + PC2 + PC3) explain 90.7373% of the total information, losing
only 9.2627% of the original information. The descriptors ALOGPS_logs (0.4232), Mor29m (0.5937)
and IC5 (−0.6223) contribute the most to PC1, while in PC2, the descriptor GAP energy (0.7746) is
the primary contributor. The main components can be written as a linear combination of the selected
descriptors. Mathematical expressions for PC1 (1) and PC2 (2) are shown below:
PC1 = 0.4232ALOGPS_log + 0.5937Mor29m − 0.6223IC5 − 0.2845Gap energy (1)
PC2 = 0.5936ALOGPS_log − 0.1803Mor29m − 0.1225IC5 + 0.7746Gap energy (2)
Molecules 2014, 19 10679

Table 3. PCA and contribution of selected descriptors based on step multivariate analysis.
Main Component
Parameters
PC1 PC2 PC3
Variance (%) 38.6537 21.5859 12.3501
Cumulative variance (%) 48.3171 75.2996 90.7373
Contribution
Molecular descriptors
PC1 PC2
ALOGPS_log 0.4232 0.5936
Mor29m 0.5937 −0.1803
IC5 −0.6223 −0.1225
Gap energy −0.2845 0.7746

Figure 2 shows the scores for the 21 compounds studied. Based on the graph, PC1 distinguishes
between compounds that are more potent and less potent. The most potent compounds are located at
the left (3, 4, 5, 6, 8, 12, 13, 18, 19, 20 and 21), while the less potent compounds are located in the
right side of the graph (1, 2, 7, 9, 10, 11, 14, 15, 16 and 17).

Figure 2. Plot of PC1–PC2 scores for artemisinin and derivatives with anticancer activity
against human hepatocellular carcinoma HepG2 line. Positive values indicate more potent
analogs (in blue), and negative values indicate less potent analogs (in red).

Figure 3 shows the loadings for the four (4) descriptors that are most important in the classification
of compounds. Less potent compounds have high contributions from the descriptors ALOGPS_logs
and Mor29m, while more potent compounds have a high contribution from the descriptor GAP energy
and IC5. Thus, the descriptors GAP energy and IC5 are responsible for the location of more potent
compounds at the left side of the graph. The descriptors ALOGPS_logs and Mor29m places less potent
compounds in the right part of the graph. Figure 3 also shows that the higher the contribution of the
descriptors ALOGPS_logs and Mor29m in the first principal component, i.e., the higher the value for a
certain compound, the higher the score value will be, indicating that the compound is less potent than
Molecules 2014, 19 10680

others. The other descriptors contribute to a lesser degree. For example, the descriptor GAP energy has
negative weight in PC1, demonstrating that the most potent compounds generally have lower values of
this descriptor.

Figure 3. Plot of the PC1–PC2 loadings with the four descriptors selected to build the PLS
and PCR models of artemisinin and derivatives with biological activity against human
hepatocellular carcinoma HepG2 line.

2.3. Hierarchical Cluster Analysis (HCA) Results

The HCA method classified the compounds into two classes (more active and less active) and was
based on the Euclidean distance and the incremental method [42]. In the incremental linkage, the
distance between two clusters is the maximum distance between a variable in one cluster and a
variable in the other cluster. The descriptors employed to perform HCA were the same as those used
for PCA, i.e., ALOGPS_logs, Mor29m, IC5 and GAP energy.
In the HCA technique, the distances between pairs of samples are computed and compared. Small
distances imply that compounds are similar, while dissimilar samples will be separated by relatively
large distances. The dendrogram in Figure 4 shows the HCA graphic as well as the compounds
separated into two main classes. The scale of similarity varies from 0 for samples with no similarity to 1
for samples with identical similarity. By analyzing the dendrogram, some conclusions can be drawn
even though the compounds present some structural diversity.
HCA showed results similar to those obtained with PCA. The compounds are grouped according
to their biological activities. The most potent compounds are 3, 4, 5, 6, 8, 12, 13, 18, 19, 20 and 21.
The less potent compounds are 1, 2, 7, 9, 10, 11, 14, 15, 16 and 17. Compound 18 has the lowest value
of logRA = 0.3604, among the compounds classified as most potent of the series studied. Whereas, the
compound 5 has the highest value of logRA = 2.3240, whereas the variation between the activities of
the compounds 5 and 18 is ±1.9636 between them.
Molecules 2014, 19 10681

Figure 4. HCA dendrogram for artemisinin and derivatives with anticancer activity against
human hepatocellular carcinoma HepG2. Positive values indicate more potent analogs, and
negative values indicate less active compounds.

2.4. Partial Least Squares (PLS) and Principal Component Regression (PCR) Results

The statistical quality [43] of the PLS and PCR models was gauged by parameters such as
correlation coefficient or squared correlation coefficient (R2), explained variance (R2ajust, i.e., adjusted
R2), standard deviation (s), variance ratio (F—a statistic of assessing the overall significance),
cross-validated correlation coefficient (Q2), standard error of validation (SEV), predicted residual error
sum of squares (PRESS) and standard deviation of cross-validation (SPRESS) [44–46]. The best
regression models were selected based on high values of R2, R2ajust, Q2 and F and low values of s, SEV,
PRESS and Spress.
The calculated properties and the experimental activity values for the compounds studied were used
to build the PLS and PCR regression models (see Table 4). The models built using the PLS and PCR
were based on three latent variables and 21 compounds.
The regression equations obtained for PLS (Equation (3)) and PCR (Equation (4)) models that
relate the descriptors and anticancer activity are the following:
logRA = −0.2748ALOGPS_log − 0.4307Mor29m + 0.3894IC5 + 0.2734 Gap energy (3)
n = 21, R2 = 0.9473, R2ajust = 0.9381, s = 0.2280, F(4,17) = 71.9013, Q2 = 0.9151, SEV = 0.2620,
PRESS = 0.8937, SPRESS = 0.0590.
logRA = −02904ALOGPS_log − 0.4074Mor29m + 0.4270IC5 + 0.1953 Gap energy (4)
n = 21, R2 = 0.9367, R2ajust = 0.9256, s = 0.2514, F(4,17) = 59.1211, Q2 = 0.9063, SEV = 0.2752,
PRESS = 1.0745, SPRESS = 0.0647.
Molecules 2014, 19 10682

The results obtained with the PLS and PCR models were very close, with variation between
PLS and PCR of R2 = ±0.0106, R2ajust = ±0.0125, s = ±0.0234, F(4,11) = ±12.7802, Q2 = ±0.0088,
SEV = ±0.0132, PRESS = ±0.4808 and SPRESS = ±0.0057. The quality of the PLS and PCR models can
be demonstrated by comparing the measured and the predicted activities. The validation errors
obtained by the leave-one-out cross-validation method are shown in Table 4. For the PLS model, only
six compounds (1, 3, 5, 18, 20 and 21) had high validation errors, and the PCR model yielded seven
compounds (1, 3, 4, 5, 17, 18 and 20) with high residual values.

Table 4. Predicted PLS and PCR results and validation errors for logRA (experimental).
Predicted Validation Error Experimental
Compounds
PLS PCR PLS PCR logRA
1− −0.4002 −0.3420 −0.4002 −0.3420 0.0000
2− 0.3129 0.2298 0.3161 0.2166 −0.0132
3+ 1.9110 1.8824 0.3714 0.3428 1.5396
4+ 2.0905 2.0404 0.1830 1.1329 1.9075
5+ 1.8148 1.7574 −0.5092 −0.5666 2.3240
6+ 1.4038 1.3075 0.0403 −0.0560 1.3635
7− −0.1312 −0.1548 −0.1444 −0.1680 −0.0132
8+ 1.9071 1.9093 −0.2223 −0.2201 2.1294
9− 0.2824 0.2716 0.2692 0.2584 −0.0132
10− 0.1883 0.1772 0.1751 0.1640 −0.0132
11− −0.0429 −0.0270 −0.0561 −0.0402 −0.0132
12+ 1.3212 1.3357 −0.2184 −0.2039 1.5396
13+ 1.1437 1.1276 −0.1996 −0.2157 1.3433
14− −0.1448 0.0796 −0.1580 0.0664 −0.0132
15− 0.0023 0.1410 −0.0109 0.1278 −0.0132
16− 0.0131 0.1077 0.0001 0.0945 −0.0132
17− 0.1968 0.3439 0.1836 0.3307 −0.0132
18+ 0.7639 0.8522 0.4035 0.4918 0.3604
19+ 1.9530 1.9139 0.0802 0.0411 1.8728
20+ 1.7459 1.6991 −0.3543 −0.4011 2.1002
21+ 1.7443 1.7392 0.3258 0.3207 1.4185

The measured versus predicted values using our PLS and PCR models are presented in Figure 5a,b,
respectively. The PLS and PCR plots identify compounds with higher activity (blue) and compounds
with lower activity (red). According to the PLS and PCR models, the four variables present different
magnitudes of regression coefficients (in absolute value). The models reveal that compounds with high
biological potency against human hepatocellular carcinoma HepG2 have a combination of higher
values of IC5 and GAP energy and lower values of ALOGPS_logs and Mor29m for the PLS and
PCR models.
Molecules 2014, 19 10683

Figure 5. Plot of experimental versus predicted values for logRA modeled by (a) PLS and (b) PCR.

The eight compounds of the test set (22–29) were molded from the most stable structure of
artemisinin, compound 1 of Figure 1, and constructed using GaussView 5.0 program, carrying the
complete optimization of the geometry of each compound with the basis set of separated valence
B3LYP/6-31G** using the DFT method as implemented in Gaussian 03 program. After obtain the
most stable geometry of each compound was determined only selected descriptors in PCA and used in
the construction of the QSAR models, namely ALOGPS_logs, Mor29m, IC5 and GAP energy, shown
in Table 5.
Molecules 2014, 19 10684

Table 5. Molecular properties selected by analysis of main components of test set with
anticancer activity unknown.
Test Set ALOGPS_log Mor29m IC5 Gap energy, a.u.
22 −5.030000 −0.412000 5.514000 0.252200
23 −5.760000 −0.443000 5.628000 0.252200
24 −7.390000 −0.515000 5.364000 0.252400
25 −7.140100 −0.305100 5.571100 0.219700
26 −6.030000 −0.311000 5.572000 0.252400
27 −4.820000 −0.518000 5.856000 0.251700
28 −7.350000 −0.601000 5.280000 0.227600
29 −7.010000 −0.543000 5.488000 0.232300

The QSAR models (PLS and PCR) were built used to predict the unknown anticancer activity of
eight new artemisinin derivatives shown in Figure 6, compounds 22–29. Table 6 shows the results of
the logRA by PCR and PLS models. According to Table 6 the PLS and PCR models showed that all
the compounds of the test set are predicted to be more active, they had values of logRA greater than
zero (logRA > 0) in both models (PLS and PCR) with residues of prediction ranging from 0.0650
to −0.0560, suggesting that these new compounds in the two models (PLS and PCR) are more potent
than artemisinin may be synthesized and tested for anticancer activity.

Figure 6. Compounds of the test set artemisinin derivatives with unknown anticancer
activity against human hepatocellular carcinoma HepG2.

22 23 24
CH3 CH3 CH3
H H H

O O H3C O O H3C O O
H3C

O O O
H H H
H H H
O O O
CH3 CH3 CH3

NH C6H13 NH C8H17 NH C16H33

O O O

25 26 27
CH3 CH3 CH3
H H H

H3 C O O O O H3C O O
H3C

O O O
H H H
H H H
O O O
CH3 CH3 OH
CH3
OH
C 12H25 N
C8H17 C8H17

O O
O
Molecules 2014, 19 10685

Figure 6. Cont.

28 29
CH3 CH3
H H

H3C H3C
O O O O
O O
O O
H H
H H
O O
NH C 16H 33 C 12H25
H3C H3C

Table 6. Anticancer activity predicted (logRA) by PCR and PLS models for the test set
compounds and residues of prediction between models.
Test Set Predicted (logRA) Residues of Prediction
Compounds PLS PCR (PLS-PCR)
22 1.2458 1.2048 0.0410
23 1.6431 1.6210 0.0221
24 1.6804 1.6154 0.0650
25 0.6841 0.8649 −0.1808
26 1.1631 1.1564 0.0067
27 2.1201 2.1163 0.0038
28 1.3444 1.3850 −0.0406
29 1.5410 1.5970 −0.0560

2.5. Pharmacokinetic and Toxicological Results

The prediction of Absorption, Distribution, Metabolism and Excretion (ADME) proprieties for
artemisinin and its derivatives of the test set (compounds 22–29) classified by PLS and PCR models as
more potent are shown in Tables 7 and 8. In Table 7, one can observe the absorption values (HIA,
PCaCO2 and PMDCK) predicted for the compounds. The prediction of human intestinal absorption is
a major objective in the optimization and selection of candidates for the development of oral
medications. The focus on the discovery of modern drugs is not simply in the pharmacological activity,
but also in search of more favorable pharmacokinetic properties [47]. The results of human intestinal
absorption are the sum of absorption and bioavailability, evaluated from the proportion of excretion or
cumulative excretion in urine, bile and feces [48,49].
The test compounds showed good human intestinal absorption, having values of HIA > 90%, being
close to that of artemisinin (compound 1). Compound 27 showed the lowest absorption equal to 94.2039%,
whereas compound 26 showed the highest value of HIA equal to 98.1189%, as shown in Table 7.
The PCaco2 (nm/s) and PMDCK (nm/s) cell models have been used as a reliable in vitro model for the
prediction of oral drug absorption, being the Caco-2 cells derived from human colon adenocarcinoma
and have various routes of drug transport through the intestinal epithelium [49]. The results of
the compounds shown in Table 7 showed an average permeability of 45.4351, as proposed by
Yazdanian [50]. The values obtained of PCaCO2 (nm/s) were higher than 30.3276 nm/s (compound 1,
Molecules 2014, 19 10686

artemisinin). The compounds 25 and 26 showed higher values of cell permeability of 51.2476 and
51.5452 nm/s, respectively.

Table 7. Absorption properties for artemisinin (compound 1) and compounds of the test set.
Absorption
Compounds [a]
HIA(%) PCaCO2(nm/s) [b] PMDCK(nm/s) [c] Pskin [d]
1 96.3143 30.3276 72.4627 −3.00248
22 95.9522 48.074 0.2820 −2.78573
23 96.0180 49.0102 2.7481 −2.38535
24 96.1170 50.8969 64.4258 −1.10239
25 97.6636 51.2473 54.1962 −1.00477
26 98.1189 51.5452 13.6801 −1.4846
27 94.2039 35.0362 0.0437 −2.66011
28 96.1170 46.0453 64.766 −0.792156
29 97.6636 46.7337 55.4025 −0.768943
[a]
: percentage of human intestinal absorption; [b]: cell permeability (Caco-2 in nm/s); [c]
: cell permeability
Maden Darby Canine Kidney in nm/s; [d]: skin permeability.

Table 8. Distribution properties in percentages of PPB and penetration of the blood brain
barrier for artemisinin (compound 1) and compounds of the test set.
Distribution
Compounds [a]
PPB(%) CBrain/CBlood [b]
1 93.368123 1.30488
22 90.481620 3.1575
23 91.279366 5.35648
24 93.306402 11.0801
25 96.696312 8.39023
26 95.399268 2.65831
27 90.056670 1.91129
28 93.838777 10.9862
29 97.347576 8.08563
[a]
: percentage of plasma protein binding; [b] penetration of the blood brain barrier.

In accordance with Irvine et al. [51], PMDCK (nm/s) system cells can be used as tool for rapid
screening permeability. The test compounds (22, 23, 26 and 27) were those that presented low
permeability in the PMDCK (<25) cell system. In the studied set, compounds 22 and 27 showed the
lowest permeability values PMDCK equal to 0.2820 and 0.0437 nm/s, respectively. Compounds 24, 25,
28 and 29 showed the highest permeability values varying in the range from 54.1962 to 64.7660 nm/s,
close to the permeability value of artemisinin.
In the pharmaceutical, cosmetic and agrochemical industries, predicting the rate of skin
permeability is a crucial parameter for transdermal administration of medications and for the risk
assessment of chemical products that come into contact with the skin accidentally [52]. The test set
compounds showed negative values of skin permeability, i.e, it is not important to be administered for
transdermal use, and also not present any risk accordance results described in Table 7.
Molecules 2014, 19 10687

The distribution of a drug depends on its plasma protein binding (PPB) and partition in adipose
tissue and other tissues. In plasma the drug may be in unbound or bound form, which depends on the
affinity that the drug presents by the plasmatic protein (drug target). If the protein binding is reversible,
then a chemical equilibrium will exist between bound and unbound states. The proteins binding can
influence in the biological half-life in the body. The bound portion may act as a reservoir or deposit to
which the drug is slowly released in the unbound form. As the non-bound form being metabolized
and/or excreted from the body, fraction bound to will be released in order to that maintain
balance [53,54]. In Table 8 shows the results of the distribution properties (PPB% and CBrain/CBlood) for
artemisinin and classified as most potent compounds of test set. Compounds 22–29 showed strong
plasma protein binding with PPB > 90.0566%, being close to the value of PPB of artemisinin which
was equal to 93.3681%. Compounds 25, 26 and 29 showed higher strength in plasma protein binding
equal to 96.6963%, 95.3992% and 97.3475%, respectively.
The penetration of the blood brain barrier is critical in the pharmaceutical field, because compounds
that act on the central nervous system (CNS) should go through it, and inactive compounds in CNS
should not go in order to avoid collateral effects of CNS [55]. In the test set, all compounds showed
absorption values to the CNS higher than 1, and in accordance with the classification proposed by
Ma et al. [56], compounds that have values greater than 1 (CBrain/CBlood > 1) are classified as active in
the CNS may cause collateral effects, and compounds that have values below 1 (CBrain/CBlood < 1) are
classified as inactive in the CNS. Therefore, compounds 22–29 had a variation of CBrain/CBlood in
relation to the artemisinin of 1.8526, 4.0516, 9.7752, 7.0853, 1.3534, 0.6064 and 9.6813, respectively.
Since the compound 27 showed the value of penetration of the blood brain barrier (CBrain/CBlood) closest
to of artemisinin (CBrain/CBlood = 1.304) having the smallest variation between test compounds studied
(CBrain/CBlood[compound 27] − CBrain/CBlood[artemisinin]), showing value equal to 0.6064.
Table 9 shows the results of the toxicological properties of mutagenicity (Ames Test) and
carcinogenicity (Mouse and rat) for artemisinin and its derivatives of the test set (22–29) classified by
PLS and PCR models as more potent with anticancer activity against human hepatocellular carcinoma
HepG2. One of the important reasons for the discovery of new drugs is the evaluation of the toxicity of
drug candidates. This means that the conception of drugs with consideration of its toxicity is very
important, as well as predicts the mutagenicity and carcinogenicity of new compounds that may be toxic.

Table 9. Toxicological properties of mutagenicity (Ames Test) and carcinogenicity (mouse

and rat) for artemisinin and its derivatives of the test set (22–29).
Ames Test Carcinogenicity
Compounds
Mutagenicity Mouse Rat
1 Mutagenic Negative Positive
22 Non-mutagenic Negative Positive
23 Non-mutagenic Negative Positive
24 Non-mutagenic Negative Positive
25 Non-mutagenic Positive Positive
26 Non-mutagenic Positive Positive
27 Non-mutagenic Negative Negative
28 Non-mutagenic Negative Positive
29 Non-mutagenic Negative Positive
Molecules 2014, 19 10688

The Ames test is a simple method to test mutagenicity of a compound, suggested by Ames, where
various strains of Salmonella typhimurium bacterium with mutations in the genes involved in histidine
synthesis, so they require histidine for growth, are used. The variable being tested is the ability of the
mutagenic agent to provoke the reversal of the growth in histidine-exempt medium [57]. In this
method, compound 1 (artemisinin) presented positive prediction, which means that this compound was
predicted as a mutagen. The other compounds (22–29) showed a negative prediction, ie, were
predicted as non-mutagenic, as shown in Table 9.
Carcinogenicity is the ability that a substance has to induce alterations that lead to cancer. The
carcinogenicity assays require a long time (>2 years). The principal methodologies use “in vivo”
assays, using mice or rats by exposing them to a chemical compound, where the observed variable is
the existence of cancer. In this study, PreADMET server was used to predict the result which is
constructed from the data of the NTP (National Toxicology Program) and the USA/FDA, which are
the results of in vivo tests for carcinogenicity in mice and rats for 2 years.
In the prediction of carcinogenicity in mouse, compounds 25 and 26 showed positive prediction, ie,
no evidence of carcinogenic activity. The others compounds were predicted as negative, which means
that there is evidence of carcinogenic activities in mouse, for such compounds (1, 22–24 and 27–29).
In the prediction of carcinogenicity in rat, the following compounds 1, 22–26, 28 and 29 had positive
prediction, demonstrating that show no carcinogenic activity. Whereas compound 27 showed negative
prediction, meaning that this compound may exhibit carcinogenic activity.

3. Experimental Section

3.1. Anticancer Compounds Studied

Initially, 21 artemisinins (artemisinin and its derivatives) with different degrees of cytotoxicities
against human hepatocellular carcinoma HepG2 were selected from the literature (Figure 1) [24]. The
employed strategy was based on the knowledge that the endoperoxide group presented in artemisinin
and its derivatives is responsible for their antimalarial, antileishmanicidal and anticancer activities. The
compounds, the subjects of this study, consisted of artemisinin, amides, esters, alcohols, ketones,
derivatives with polar hydroxyl and carboxylic acid groups and five-membered ring derivatives. All
compounds have been associated with in vitro bioactivity against a human hepatocellular carcinoma
cell line, HepG2.
The numbering of the atoms used in this study is shown in Figure 1 (compound 1—artemisinin).
The logarithm of the IC50 value of artemisinin over the IC50 value of the compounds (logarithm of
relative activity, logRA) was used to reduce inconsistencies caused by individual experimental
environments:
logRA = log(IC50 of artemisinin/IC50 of analog) (5)
where IC50 is the 50% inhibitory concentration. In this study, the following classification based on the
anticancer responses was adopted: compounds with logRA > 0.00, ranging from 0.3604 to 2.324,
were assumed to be more potent analogs (3, 4, 5, 6, 8, 12, 13, 18, 19, 20 and 21), and those with
logRA ≤ 0.00, ranging from 0.0000 to −0.0132, were considered to be less potent analogs (2, 7, 9, 10,
11 and 14–17). The compound 5 (logRA = 2.324) is the most potent compound in the series studied.
Molecules 2014, 19 10689

3.2. Molecular Modeling and Calculations of Descriptors or Properties Molecular

Molecular modeling started with the construction of the structure of artemisinin using the GaussView
3.0 program [58], which was then optimized with different methods and basis sets—semiempirical
(AM1, PM3, and ZINDO), Hartree-Fock (HF/6-31G, HF/6-31G*, HF/6-31G**, HF/3-21G, HF/3-21G*,
HF/3-21G**, and HF/6-311G), and DFT (B3LYP/6-31G, B3LYP/6-31G*, B3LYP/6-31G**, and
B3LYP/3-21G).
These calculations were executed to find the method and basis sets with the best fit between the
computational time and accuracy of the information compared to the experimental data [59]. After initial
determination and structural optimization, the theoretical geometrical parameters of artemisinin in the
region of the 1,2,13-trioxane ring (bond length, bond angle and torsion angle) were determined with the
aim of evaluating the quality of the molecular wave function and standard deviation of method studied
comparing the theoretical geometrical parameters with the experimental data (see Table 1).
The experimental structure of artemisinin was taken from the Cambridge Structural Database CSD,
with REFCODES: QNGHSU10, crystallographic R factor 3.6 [60]. All the other structures (see Figure 1)
were built with the optimized structure of artemisinin using the Gaussian 03 program [61] with the
DFT method and B3LYP/6-31G** basis set. After the structures were determined in 3D, various
descriptors for each molecule of the set studied were calculated.
The descriptors are important for the quantitative description of molecular structure and to finding
appropriate predictive models [62]. The computation of the descriptors was performed employing the
following software: Gaussian 03 program [61], e-Dragon [63,64], Molekel [65] and HyperChem 6.02 [66].
The e-Dragon program calculated 1666 descriptors that were divided into the following 20 classes:
48 constitutional descriptors; 47 descriptors of quantity and trajectory; 47 information indexes;
107 adjacency indexes; 21 topological charge indexes; 41 molecular Radic profiles; 150 RDF descriptors;
154 functional groups; 14 charge descriptors; 33 connectivity indexes; 96 2-D autocorrelations,
64 Burden eigenvalues; 44 indexes based on eigenvalues; 74 geometric descriptors; 160 MORSE-3D;
120 fragments centered in the atom; 31 molecular property descriptors; 119 topological indexes; 99 WHIM
descriptors; and 197 Getaway descriptors. Other descriptors such as the following were obtained:
(a) QUANTUM CHEMICAL descriptors: In our study, we calculated the following 25 quantum-
chemical descriptors: total energy (TE), energy of the highest occupied molecular orbital (HOMO), a
level below the energy of the highest occupied molecular orbital (HOMO − 1), lowest unoccupied
molecular orbital energy (LUMO), a level above the energy of the lowest unoccupied molecular orbital
(LUMO + 1), difference in energy between HOMO and LUMO (GAP = HOMO − LUMO), Mulliken
electronegativity (χ), molecular hardness (η), molecular softness (1/η), and charge on the atom n
(where n = 1, 2, 3, 4, 5, 5a, 6, 7, 8, 8a, 9, 10, 11, 12, 12a, 13). The atomic charges used in this study
were obtained with the key word POP = CHELPG using the electrostatic potential [67], with this
strategy, it was possible to obtain the best potential molecular series of points defined around the
molecule, and atomic charges offer the general advantage of being physically more satisfactory than
Mulliken charges [68].
(b) Descriptors related to quantitative properties of chemical structure and biological activity: In our
data matrix, QSAR descriptors were included, i.e., total surface area (TSA), molecular volume (MV),
molar refractivity (MR), molar polarizability (MP), coefficient of lipophilicity (logP), molecular mass
Molecules 2014, 19 10690

(MM) and hydration energy (HE) according to the HyperChem 6.02 program. The molecular
descriptors were selected to provide valuable information about the influence of electronic, steric,
hydrophilic and hydrophobic features on the anticancer activity of artemisinins.

3.3. Variable Selection and Model Building QSAR (PLS and PCR)

After the determination of all molecular descriptors, it was possible to construct a data matrix to
develop step multivariate analysis. The step multivariate analysis was necessary to make the autoscale or
standardizing data matrix X = (n, m) consisting of twenty-one (21) lines (the anticancer compounds
studied) and one thousand seven hundred sixteen (1,716) columns (in this case, the calculated descriptors
for each molecule), where n is the number of compounds studied and m is the number of variables.
The aim of using the standardizing matrix is to give each variable equal weight in mathematical
terms, so each variable was centered on the mean and scaled to unit variance. To reduce the data set,
variables were selected based on the analysis of the correlation matrix between variables (descriptors)
and the logarithm of the relative activity (logRA).
The descriptors with small or no correlation (under the 0.20 correlation value cutoff) were
discarded, resulting in only two hundred and thirteen (213) descriptors remaining from the initial set of
one thousand seven hundred sixteen (1,716) descriptors. After this data compression, two
complementary methods for exploratory data analysis were employed (PCA and HCA) to study
intersample and intervariable relationships and to select the properties that contribute the most to the
classification of the compounds into two groups [27,28]. One group contained more potent analogs and
the other less potent analogs. PCA was employed to reduce the dimensionality of the data, find
descriptors that could be useful in characterizing the behavior of the compounds acting against a
human hepatocellular carcinoma cell line (HepG2) and look for natural clustering in the data and
outlier samples.
While performing PCA, several attempts to obtain a good classification of the compounds were
made. At each attempt, the score and loading plots were analyzed based on the variables employed in
the analysis. The score plot gives information about the compounds (similarity and differences). The
loading plot gives information about the variables (how they are connected to each other and which
best describe the variance in the original data) [27,28]. The descriptors selected by PCA were used to
perform HCA, PLS and PCR.
The objective of HCA was to present the compounds distributed in natural groups and the results
confirm the PCA results. Thus, several approaches were attempted to establish links between
samples/cluster. All of them were of an agglomerative type because each sample was first defined as
its own cluster, and then others were grouped together to form new clusters until all the samples were
part of a single cluster [28].
The QSAR models for the new artemisinin compounds with anticancer activity were constructed by
the PCR and PLS methods based on the autoscaled data and the leave-one-out crossvalidation
procedure [25–28]. The final purpose of the multivariate analysis (PLS and PCR) was the construction
of a mathematical model that can be used to predict anticancer activity of the compounds studied. The
statistical parameters used to assess the quality of the models were the Prediction Residual Error Sum
of Squares (PRESS), Equation (6), the Standard Error of Validation (SEV), Equation (7), the total
Molecules 2014, 19 10691

variance explained, R2 (correlation between the estimated values predicted by the model built with the
full data set and actual values of y), Q2 (the cross-validated correlation coefficient) and SPRESS
(standard deviation of cross-validation) given by Equations (8)–(10), respectively [27,28,69–71]:
n
PRESS =  (y i −ŷ i ) 2 (6)
i =1

PRESS
SEV = (7)
n
 PRESS 
2
R = 1−  cal
 (8)
 i =1 (yi − y) 
n 2

 PRESS 
2
Q =1−  val
 (9)
 n (y i − y) 2 
 i =1 

PRESS
S PRESS = (10)
n − k −1
In Equations (6) and (7), n is the number of compounds used for the calibration or validation model,
yi is the experimental value of the physicochemical property for the sample and ŷi is the value
predicted by a calibration or validation model. In Equations (8) and (9), PRESScal is the Calibration
Prediction Error Sum of Squares and PRESSval is the Validation Prediction Error Sum of Squares.
Both PRESScal and PRESSval are evaluated from Equation (6) by changing ŷi for a calibration or
validation model. The values of explained variance (R2ajust, i.e., adjusted R2), standard deviation (s) and
F (Fisher test) were determined. The multivariate data analyses (PCA, HCA, PLS and PCR) were
performed by employing Pirouette 3.01 software [42].

3.4. Pharmacokinetic and Toxicological Properties of Test Compounds

At a molecular level, a system is coordinated by transporters, channels, receptors and enzymes; this
system affects the absorption, distribution, metabolism, excretion and toxicity (ADME/Tox) of a
molecule in humans. Understanding the interactions between small molecules and their molecular
targets should improve the ability to predict the toxic consequences that are responsible for the
removal of many commercialized drugs and failures in the final stage drug development [35,72–74].
Traditional ADME/Tox studies provide a detailed understanding of individual proteins, in which it
is possible to examine if the molecule also binds to receptors that affect the regulation of other
proteins, and if it interferes with endogenous metabolic, regulatory proteins and transport.
Alternatively the main metabolic via may be mediated by a polymorphic enzyme and likely affect the
therapeutic dose [73,75,76].
The properties ADME/Tox for artemisinin and its derivatives of the test set (22–29) were calculated
using the server PreADMET [49]. This server calculates pharmacokinetic properties as: human
intestinal absorption, cellular permeability Caco-2 in vitro, cell permeability Maden Darby Canine
Molecules 2014, 19 10692

Kidney (MDCK), skin permeability, plasma protein binding and penetration of the blood-brain barrier,
and toxicological properties as mutagenicity and carcinogenicity.

4. Conclusions

The DFT method and the B3LYP/6-31G** basis set revealed themselves to be adequate to optimize
the structures of artemisinin and derivatives for subsequent study. The predictive classification models
for artemisinin derivatives were obtained with a set of molecular descriptors selected by chemometric
approaches. PCA and HCA methods classified the compounds studied into groups according to their
degree of anticancer activity against a human hepatocellular carcinoma cell line (HepG2). The
descriptors ALOGPS_logs, Mor29m, IC5 and GAP energy were responsible for distinguishing
compounds with higher and lower anticancer activity. The molecular features represented by these
descriptors are in good agreement with previous SAR analysis performed on artemisinin derivatives.
The combination of these structural attributes is believed to govern the anticancer effects of the
compounds studied in this work. The PLS and PCR models obtained here showed not only statistical
significance but also predictive ability. The test set showed for two new artemisinin compounds
satisfactory results for anticancer activity and pharmacokinetic and toxicological properties. Through
this strategy and our findings, useful information was obtained that could be of use in experimental
syntheses and biological evaluation to understand the molecular and structural requirements for designing
new ligands to be used as anticancer agents. Consequently, further studies need be done to evaluate the
different proposals as well as their actions, toxicity, and potential use for treatment of cancers.

Acknowledgments

We gratefully acknowledge the support provided by the Brazilian Agency National Council of
Scientific and Technological Development (CNPq Proc. 306676/2010-9) and Institute of Exact and
Natural Sciences of Federal University of Pará for use of the GaussView and Gaussian software. The
authors would like to thank the Postgraduate Program in Pharmaceutical Sciences, Laboratory of Modeling
and Computational Chemistry (LMCC) of Federal University of Amapá for computational support.

Author Contributions

Authors J.B.V., F.S.B., C.C.L. and C.B.R.S. designed the study, involved in writing the first draft
and data collection. Authors C.F.S., J.S.C., D.S.B.B. and W.J.C.M. managed the literature search,
analyses of the study and manuscript preparation. Author C.B.R.S. and J.B.V. performed the
statistical analysis and also aided in data interpretation and was actively involved in reading the
manuscript. J.A.H.M.B., J.O.S., L.I.S.H.M. and J.C.T.C. developed and predicted pharmacological and
toxicological properties of compounds. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

Molecules 2014, 19 10693

References

1. Rosenberg, S.A. Progress in human tumour immunology and immunotherapy. Nature 2001, 411, 380.
2. Cairns, J. The origin of human cancers. Nature 1981, 289, 353–357.
3. Brentani, R.R.; Chammas, R.; Coelho, F.R.G. Mecanismos de invasão e metástases. In Bases da
Oncologia, 1st ed.; Brentani, M.N., Coelho, F.R.G., Iyeyasu, H., Kowalski, L.P., Eds.; Livraria e
Editora Marina: São Paulo, Brazil, 1998; pp. 91–98.
4. Ferlay, J.; Soerjomataram, I.; Ervik, M.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.;
Forman, D.; Bray, F. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC
Cancer Base No. 11. Available online: http://globocan.iarc.fr (accessed on 15 February 2014).
5. International Agency for Research on Cancer. Latest World Cancer Statistics Global Cancer
Burden Rises to 14.1 Million New Cases in 2012: Marked Increase in Breast Cancers must be
Addressed; World Health Organization 2013; N° 223, pp. 1–3. Available online:
http://www.iarc.fr/en/media-centre/pr/2013/pdfs/pr223_E.pdf (accessed on 10 January 2014).
6. Carvalho, J.E. Fitoterápicos: Alimento ou medicamento? In Ciência de Alimentos: Avanços e
Perspectivas; Mercadante, A.Z., Bobbio, F.O., Bobbio, P.A., Pereira, J.L., Pastore, G.M., Eds.;
Faculdade de Engenharia de Alimentos da Unicamp: Campinas, Brazil, 2001; Volume 2,
pp. 196–202.
7. Chaturvedi, D. Sesquiterpene lactones : Structural diversity and their biological activities.
In Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry; Research
Signpost: Kerala, India, 2011; pp. 313–334.
8. Zhang, S.; Won, Y.; Ong, C.; Shen, H. Anti-cancer potential of sesquiterpene lactones:
Bioactivity and molecular mechanisms. Curr. Med. Chem. Anticancer Agents 2005, 5, 239–249.
9. Ghantous, A.; Gali-Muhtasib, H.; Vuorela, H.; Saliba, N.; Darwiche, N. What made sesquiterpene
lactones reach cancer clinical trials? Drug Discov. Today 2010, 15, 668–78.
10. Santos, C.B.R. Desenvolvimento Racional de Fármacos Antimaláricos Derivados da Artemisinina
usando Métodos Computacionais SAR e QSAR; Universidade Federal do Amazonas: Amazonas,
Brazil, 28 February 2014.
11. Yusuke, O.; Catherine, A.; Elstad, E.C.; Donald, Y.S.; Raymond, M.Q.; Henry, C.L. Effect of
hyperbaric oxygen on the anticancer effect of artemisinin on molt-4 human leukemia cells.
Anticancer Res. 2010, 30, 4467–4470.
12. Pinheiro, J.C.; Kiralj, R.; Ferreira, M.M.C.; Romero, O.A.S. Artemisinin derivatives with
antimalarial activity against plasmodium falciparum designed with the aid of quantum chemical
and partial least squares methods. QSAR Comb. Sci. 2003, 22, 830–842.
13. Balunas, M.J.; Kinghorn, A.D. Drug discovery from medicinal plants. Life Sci. 2005, 78, 431–441.
14. Dos Santos, H.F. O conceito de modelagem molecular. Cadernos Temáticos de Química Nova na
Escola 2001, 4, 4–5.
15. Santos, C.B.R.; Lobato, C.C.; Sousa, M.A.C.; Macêdo, W.J.C.; Carvalho, J.C.T. Molecular
modeling: Origin, fundamental concepts and applications using structure-activity relationship and
quantitative structure-activity relationship. Rev. Theor. Sci. 2014, 2, 91–115.
16. Cohen, N.C. Guidebook on Molecular Modeling in Drug Design; Academic Press: San Diego, CA,
USA, 1996.
Molecules 2014, 19 10694

17. Sant’Anna, C.M.R. Glossário de termos usados no planejamento de fármacos (recomendações da

IUPAC para 1997). Quim. Nova 2002, 25, 505–512.
18. Carvalho, I.; Borges, A.D.L.; Bernardes, L.S.C. Medicinal chemistry and molecular modeling: An
Integration to teach drug structure-activity relationship and the molecular basis of drug action.
J. Chem. Educ. 2005, 82, 588–596.
19. Wermuth, C.G. The Practice of Medicinal Chemistry, 3rd ed.; Academic Press: Burlington, MA,
USA, 2009.
20. Ribeiro, F.A.L.; Ferreira, M.M.C. QSPR models of boiling point, octanol-water partition
coefficient and retention time index of polycyclic aromatic hydrocarbons. J. Mol. Struct.
(Theochem) 2003, 663, 109–126.
21. Cohen, N.C; Blaney, J.M.; Humblet, C.; Gund, P.; Bany, D.C. Molecular modeling software and
methods for medicinal chemistry. J. Med. Chem. 1990, 33, 883–894.
22. Bernadinelli, G.; Jefford, C.W.; Maric, D.; Thomson, C.; Weber, J. Computational studies of the
structures and properties of potential anti-malarial compounds based on the 1,2,4-trioxane ring
structure: I. Artemisinin-like molecules. Int. J. Quantum. Chem. 1994, 52, 117–131.
23. Kokpol, S.K.; Hannongbua, S.V.; Thongrit, N.; Polman, S.; Rode, B.M.; Schwendinger, M.G.
Analysis of structure-activity relation for primaquine antimalarial drugs by a quantum
pharmacological approach. Anal. Sci. 1988, 4, 565–568.
24. Liu, Y.; Wong, V.K.-W.; Ho, B.C.-B.; Woang, M.-K.; Che, C.-M. Synthesis and cytotoxicity
studies of artemisinin derivatives containing lipophilic alkyl carbon chains. Org. Lett. 2005, 12,
1561–1564.
25. Gramatica, P. Principles of QSAR models validation: Internal and external. QSAR Comb. Sci.
2007, 26, 694–701.
26. Geladi, P.; Kowalski, B.R. Partial least-squares regression: A tutorial. Anal. Chim. Acta 1986,
185, 1–17.
27. Geladi, P. Notes on the history and nature of partial least squares (PLS) modeling. J. Chemom.
1988, 2, 231–246.
28. Ferreira, M.M.C. Multivariate QSAR. J. Braz. Chem. Soc. 2002, 13, 742–753.
29. Santos, C.B.R.; Lobato, C.C.; Vieira, J.B.; Brasil, D.S.B.; Brito, A.U.; Macêdo, W.J.C.;
Carvalho, J.C.T.; Pinheiro, J.C. Evaluation of quantum chemical methods and basis sets applied in
the molecular modeling of artemisinin. Comp. Mol. Biosc. 2013, 3, 66–79.
30. Hehre, W.J.A. Guide to Molecular Mechanics and Quantum Chemical Calculations;
Wavefunction Inc.: Irvine, CA, USA, 2003.
31. Mulliken, R.S.; Liu, B. Self-consistent-field wave functions of P2 and PO, and the role of d
functions in chemical bonding and of s-p hybridization in N2 and P2. J. Am. Chem. Soc. 1971, 93,
6738–6744.
32. Levine, I.N. Quantum Chemistry, 4th ed.; Prentice-Hall: New York, NY, USA, 1991.
33. Leach, A. Molecular Modelling-Principles and Applications, 2nd ed.; Pearson Education Limited:
Upper Saddle River, NJ, USA, 2001.
34. Santos, C.B.R.; Vieira, J.B.; Lobato, C.C.; Hage-Melim, L.I.S.; Souto, R.N.P.; Lima, C.S.;
Costa, E.V.M.; Brasil, D.S.B.; Macêdo, W.J.C.; Carvalho, J.C.T. A SAR and QSAR study of new
artemisinin compounds with antimalarial activity. Molecules 2014, 19, 367–399.
Molecules 2014, 19 10695

35. Santos, C.B.R.; Vieira, J.B.; Formigosa, A.S.; Costa, E.V.M.; Pinheiro, M.T.; Silva, J.O.;
Macêdo, W.J.C.; Carvalho, J.C.T. Validation of computational methods applied in molecular
modeling of artemisinin with antimalarial activity. J. Comput. Theor. Nanosci. 2014, 11, 553–561.
36. Cristino, M.G.G.; Meneses, C.C.F.; Soeiro, M.M.; Ferreira, J.E.V.; Figueiredo, A.F.; Barbosa, J.P.;
Almeida, R.C.O.; Pinheiro, J.C.; Pinheiro, A.L.R. Computational modeling of antimalarial
10-substituted deoxoartemisinins. J. Theor. Comput. Chem. 2012, 11, 241–263.
37. Figueiredo, A.F.; Ferreira, J.E.V.; Barbosa, J.P.; Macêdo, W.J.C.; Cristino, M.G.G.; Lobato, M.S.;
Pinheiro, J.C.; Serra, R.T.A.A Computational study on antimalarial dispiro-1,2,4-trioxolanes.
J. Comput. Theor. Nanosci. 2011. 8, 1847–1856.
38. Araújo, J.Q.; Carneiro, J.W.M.; Araújo, M.T.; Leite, F.H.A.; Taranto, A.G. Interaction between
artemisinin and heme. A density functional theory study of structures and interaction energies.
Bioorg. Med. Chem. 2008, 16, 5021–5029.
39. Pereira, M.S.C.; Kiralj, R.; Ferreira, M.M.C. Theoretical study of radical and neutral intermediates
of artemisinin decomposition. J. Chem. Inf. Mod. 2008, 48, 85–98.
40. Carvalho, J.R.C.; Ferreira, J.E.V.; Barbosa, J.P.; Lobato, M.S.; Meneses, C.C.F.; Soeiro, M.M.;
Farias, M.S.; Almeida, R.C.O.; Ventura, K.C.; Pinheiro, J.C.; et al. Computational modeling of
artemisinins with antileishmanial activity. J. Comput. Theor. Nanosci. 2011, 8, 2193–2203.
41. Barbosa, J.P.; Ferreira, J.E.V.; Figueiredo, A.F.; Almeida, R.C.O.; Silva, O.P.P.; Carvalho, J.R.C.;
Cristino, M.G.G.; Pinheiro, J.C.; Vieira, J.L.F.; Serra, R.T.A. Molecular modeling and chemometric
study of anticancer derivatives of artemisinin. J. Serb. Chem. Soc. 2011, 76, 1263–1282.
42. Pirouette 3.01; Infometrix Inc.: Seattle, WA, USA, 2001.
43. Snedecor, G.W.; Cochran, W.G. Statistical Methods; Oxford and IBH: New Delhi, India, 1967;
p. 381.
44. Chatterjee, S.; Hadi, A.S.; Price, B. Regression Analysis by Examples, 3rd ed.; Wiley VCH:
New York, NY, USA, 2000.
45. Diudea, M.V. QSPR/QSAR Studies for Molecular Descriptors; Nova Science: Huntingdon, PA,
USA; New York, NY, USA, 2000.
46. Bikash, D.; Shovanlal, G.; Subrata, B.; Soma, S.; Tarun, J. QSAR study on some pyridoacridine
ascididemin analogues as anti-tumor agents. Bioorg. Med. Chem. 2003, 11, 5493–5499.
47. Yee, S. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal)
absorption in man-fact or myth. Pharm. Res. 1997, 14, 763.
48. Zhao, Y.H.; Le, J.; Abraham, M.H.; Hersey, A.; Eddershaw, P.J.; Luscombe, C.N.; Butina, D.;
Beck, G.; Sherborne, B.; Cooper, I.; et al. Evaluation of human intestinal absorption data and
subsequent derivation of a quantitative structure-activity relationship (QSAR) with the Abraham
descriptors. J. Pharm. Sci. 2001, 90, 749–784.
49. Yamashita, S.; Furubayashi, T.; Kataoka, M.; Sakane, T.; Sezaki, H.; Tokuda, H. Optimized
conditions for prediction of intestinal drug permeability using Caco-2 cells. Eur. J. Pharm. 2000,
10, 195–204.
50. Yazdanian, M.; Glynn, S.L.; Wright, J.L.; Hawi, A. Correlating partitioning and Caco-2 cell
permeability of structurally diverse small molecular weight compounds. Pharm. Res. 1998, 15,
1490–1494.
Molecules 2014, 19 10696

51. Irvine, J.D.; Takahashi, L.; Lockhart, K.; Cheong, J.; Tolan, J.W.; Selick, H.E.; Grove, J.R.
MDCK (Madin-Darby canine kidney) cells: A tool for membrane permeability screening.
J. Pharm. Sci. 1999, 88, 28–33.
52. Singh, S.; Singh, J. Transdermal drug delivery by passive diffusion and iontophoresis: A review.
Med. Res. Rev. 1993, 13, 569–621.
53. Godin, D.V. Pharmacokinetics: Disposition and metabolism of drugs. In Principles of
Pharmacology; Munson, P.L., Ed.; Chapman& Hall: New York, NY, USA, 1995.
54. Pratt, W.B., Taylor, P., Eds. Principles of Drug Action: The Basis of Pharmacology, 3rd ed.;
Churchill Livingstone: New York, NY, USA, 1990.
55. Ajay; Bemis, G.W.; Murcko, M.A. Designing libraries with CNS activity. J. Med. Chem. 1999,
42, 4942–4951.
56. Ma, X.; Chen, C.; Yang, J. Predictive model of blood-brain barrier penetration of organic
compounds. Acta Pharm. Sin. 2005, 26, 500–512.
57. Ames, B.N.; Gurney, E.G.; Miller, A.J.; Bartsch, H. Carcinogens as frameshift mutagens:
Metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens.
Proc. Nat. Acad. Sci. USA 1972, 69, 3128–3132.
58. GaussView 3.07; Gaussian Inc.: Pittsburgh, PA, USA, 1997.
59. Lisgarten, J.N.; Potter, B.S.; Bantuzeko, C.; Palmer, R.A. Structure, absolute configuration, and
conformation of the antimalarial compound, Artemisinin. J. Chem. Crystallogr. 1998, 28, 539–543.
60. Allen, F.H. The cambridge structural database: A quarter of a million crystal structures and rising.
Acta Crystallogr. B 2002, 58, 380–388.
61. Frisch, M.J. Gaussian 98 Revision A.11; Gaussian, Inc.: Pittsburgh, PA, USA, 2001.
62. Estrada, E.; Molina, E. Novel local (fragment-based) topological molecular descriptors for
QSPR/QSAR and molecular design. J. Mol. Graph. Model. 2001, 20, 54–64.
63. Virtual Computational Laboratory, VCCLAB 2005. Available online: http://www.vcclab.org
(accessed on 18 January 2013).
64. Tetko, I.V.; Gasteiber, J.; Todeschini, R.; Mauri, A.; Livingstone, D.; Ertl, P.; Palyulin, V.A.;
Radchenko, E.V.; Zefirov, N.S.; Makarenko, A.S.; et al. Virtual computational chemistry
laboratory-design and description. J. Comput. Aided Mol. Des. 2005, 19, 453–463.
65. Molekel 4.3; Swiss Center for Scientific Computing: Manno, Switzerland, 2000.
66. ChemPlus, Modular Extensions to HyperChem, Release 6.02; Molecular Modeling for Windows,
Hyper, Inc.: Gainesville, FL, USA, 2000.
67. Breneman, C.M.; Winberg, K.B. Determining atom-centered monopoles from molecular
electrostatic potentials. The need for high sampling density in formamide conformational analysis.
J. Comput. Chem. 1990, 11, 361–373.
68. Singh, U.C.; Kollman, P.A. An approach to computing electrostatic charges for molecules.
J. Comput. Chem. 1984, 5, 129–145.
69. Custódio, R.; Andrade, J.C.; Augusto, F. O ajuste de funções matemáticas a dados experimentais.
Quim. Nova 1997, 20, 219–225.
70. Pimentel, M.; Neto, B.B. Calibração: Uma revisão para químicos analíticos. Quim. Nova 1996,
19, 268–277.
Molecules 2014, 19 10697

71. Gaudio, A.C.; Zandonade, E. Proposição, validação e análise dos modelos que correlacionam
estrutura química e atividade biológica. Quim. Nova 2001, 24, 658–671.
72. Thou, T.; Wang, J. Structure-ADME relationship: Still a long way to go? Expert Opin. Drug
Metab. Toxicol. 2008, 4, 759–770.
73. Bittencourt, J.A.H.M.; Oliveira, N.K.S.; Cabral, M.S.; Ribeiro, J.R.; Henriques, S.V.C.;
Picanço, L.C.S.; Santos, C.B.R.; Stien, D.; Carvalho, J.C.T.; Silva, J.O. Antiophidian activity of
brosimum guianense (aubl) huber. Am. J. Pharmacol. Toxicol. 2014, 9, 148–156.
74. Tonmunphean, S.; Parasuk, V.; Kokpol, S. Automated calculation of docking of artemisinin to
heme. J. Mol. Model. 2001, 7, 26–33.
75. Van De Waterbeemd, H.; Gifford, E. ADMET in silico modelling: Towards prediction paradise?
Nat. Rev. Drug Discov. 2003, 2, 192–204.
76. Costa, M.S.; Kiralj, R.; Ferreira, M.M.C. Estudo teórico da interação existente entre a
artemisinina e o heme. Quim. Nova 2007, 30, 25–31.

Sample Availability: Not available.

© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).