Introduction
Malaria is an infectious and life-threatening disease that affects nearly half the
population of the world, it is typically found in tropical and subtropical climates
where the parasite can grow. Africa is responsible for 90% of worldwide malaria morbidity
and death, particularly pediatric mortality.[1]
Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi are the most common plasmodium parasites that cause malaria.[2]
[3]
P. falciparum is the most dangerous human malaria parasite, and it is responsible for the majority
of malaria infections in Sub-Saharan Africa, such as Nigeria. It is also the parasite
most likely to develop drug resistance.[3]
Several antimalarial drugs have been used to reduce sensitivity of these parasites
to these antimalarial drugs. The most potent antimalarial agents are the artemisinin-based
combination therapies (ACTs); however, with reports of potential resistance to ACTs,
there are great concerns among researchers in the field of drug design for potent
antimalarial agents against the drug-resistant strains of Plasmodium. Although ACTs remain the first line of defense against malaria, backup or alternative
solutions are needed to deal with ACT-resistant strains produced by these parasites.[2]
One of the more recent areas of exploration in drug design is the fragment-based drug
design, where most active scaffolds or moieties of active drug compounds can be used
in designing drug compounds with synergistic-like effects in terms of activity and
physicochemical properties such as reduced or less toxic effects caused by a process
called molecular hybridization.[1]
[4] To combat this disease, new antimalarial agents with novel biological targets are
required.
Acetophenone is an organic compound that is used in the synthesis of many organic
pharmaceuticals, and it is also a precursor for many resins and fragrances. General
structure of acetophenone is shown in [Fig. 1]. Proteases form one of the most explored or studied antimalarial mediating targets.[5]
[6]
[7]
[8] And acetophenone is one of the important fragments with protease-inhibiting activity.
A condensation of acetophenone and aldehyde gives a very important class of organic
compounds known as chalcones. Chalcones offer a wide range of medicinal properties,
including antiplasmodial activity.[2] Acetophenones form part of the most active fragment of the chalcones and other classes
of organic compounds. Modifications on the acetophenone ring improve its activity.
Some acetophenones used in the synthesis of chalcones have been modified through the
addition of electron-withdrawing or -donating substituents such as methoxy, hydroxy,
chlorine, and alkyl to give different pharmacological activities. The presence of
electron-donating groups on the acetophenone ring, according to Li et al,[9] is advantageous to antimalarial activity. Structure–activity relationship (SAR)
evaluation shows that the size and hydrophobicity of the substituents attached on
the acetophenone are critical parameters for regulating the activity of the ring.[10]
Fig. 1 General structure of acetophenone.
In this work, two prenylated acetophenones, 3,5-diprenyl acetophenone (I) and 5-diprenyl acetophenone (II), were chosen to assess their antimalarial activity to correlate the theoretical
studies, e.g., rule of three (Ro3) for active fragment and rule of five (Ro5) for
orally bioavailable agents, and in silico pharmacokinetic (PK) studies with their experimental activity, and deduce further
insights into their SAR. This is the first report of the in vivo antimalarial activity of the two prenylated acetophenones.
Materials and Methods
Chemical Reagents and Instruments
All reagents and solvents utilized in this study were of analytical grade. The reagents
were 2,4-dihydroxyacetophenone, anhydrous 1,4-dioxane, boron trifluoride diethyl etherate,
3-methyl-2-buten-1-ol, 2-chloroquinolinyl-3-carbaldehyde, ethanol, sodium hydroxide,
hexane, and ethyl acetate. All reagents were procured from Sigma Aldrich, Germany.
The melting points of the compounds were measured without correction using the Gallenkamp
melting point instrument. Experiments of proton nuclear magnetic resonance (1H-NMR) and carbon-13 nuclear magnetic resonance (13C-NMR) were conducted in the Department of Chemistry, University of Kwazulu-Natal,
South Africa, and Multi-user Laboratory, Ahmadu Bello University, Zaria, using 400 MHz
Bruker and 400 MHz Agilent, respectively. Chemical shift (d) in ppm downfield from tetra-methyl silane as the internal standard was used to capture
the nuclear magnetic resonance data. Wavenumbers were captured as Fourier-transform
infrared spectroscopy (FTIR) using an Agilent spectrophotometer (cm−1).
Synthesis of Prenylated 2,4-Dihydroxyacetophenone
The synthesis of compound I, II, and III was described in [Fig. 2]. The two prenylated acetophenones (I and II) were synthesized through an aromatic electrophilic substitution reaction. Equimolar
quantities of 3-methyl-2-buten-1-ol and boron trifluoride diethyl etherate catalyst
(20 mmol) were dissolved in anhydrous 1,4-dioxane to acquire a pink-red solution,
to which 20 mmol of 2,4-dihydroxyacetophenone was subsequently added. The solution
was continuously stirred for 2 hours. This reaction was performed at room temperature
under inert conditions (nitrogen atmosphere). Thin-layer chromatography profile was
used to monitor the progress of the reaction. On completion of the reaction, the mixture
was diluted with diethyl ether (100 mL) and washed with water (50 mL × 3). The organic
phase was dried over anhydrous Na2SO4 and evaporated under reduced pressure to obtain a residue, which was purified by
a column chromatography using n-hexane and n-hexane/ethylacetate (7:3) to afford compounds I, II, and trace amount of compound III.
Fig. 2 Reaction scheme.
Animals
Forty locally bred adult Swiss Albino mice, weighing 18 to 22 g, were housed in conventional
laboratory settings with unlimited access to pellet feed and water ad libitum. The animals were housed in clean polypropylene cages at the Department of Pharmacology
and Therapeutics' Animal House. All animal experiments followed Ahmadu Bello University's
research policy and guidelines for the use and care of laboratory animals, which are
widely acknowledged globally. Ethical approval was sought and obtained from ABU Committee
on Animal Use and Care.
Malarial Parasite
Malaria parasites (Plasmodium berghei NK 65) were provided by the Department of Pharmacology
and Therapeutics, Ahmadu Bello University, Zaria. The mice were inoculated intraperitoneally
with 0.2 mL standard inoculum containing approximately 107 parasitized red blood cells.
Evaluation of Theoretical Oral Bioavailability, Pharmacokinetics, and Toxicity
Prior to synthesis, the oral bioavailability of the two prenylated acetophenones was
theoretically predicted using Lipinski's Ro5 on SWISS ADME web tools. (http://www.swissadme.ch). Ro3 was used to predict the propensity of their activity. In silico PK study and toxicity evaluation was performed on Admetlab2.0 and Protox-II.
Antimalarial Activity Evaluation In Vivo
Inoculation of Plasmodium berghei Parasite
Infected blood was taken from the tail vein of a donor mouse with a parasitemia level
of 20 to 25% using heparinized capillary tubes and then transferred to a sterile plain
beaker. Two milliliters of blood were diluted with 10 mL of normal saline, yielding
0.2 mL of infected red blood cells. The mice were then given 0.2 mL of blood suspension
intraperitoneally.
Treatment
To test the curative efficacy of the synthesized compounds against an established
plasmodium infection, Ryley and Peters' approach was used.[11] The mice were infected with the parasite as described by Ryley and Peters' method
and were left untreated for 72 hours, following which parasitemia levels were estimated
to be between 20 and 25%.[11] The mice with the required parasitemia levels were split into eight groups (n = 5 in each group). Group 1 was given the negative control intraperitoneally [1%
(w/v), acacia (Sigma Aldrich)]. Groups 2 to 7 were given 25, 50, and 100 mg/kg of
test compounds, while group 8 was given 5 mg/kg of the positive control [chloroquine
(Jiangsu Ruinian Quianjin Pharmaceutical Co. Ltd.)]. All treatments were given intraperitoneally
for 4 days consecutively. On the fifth day, blood was obtained from the tail vein
of the treated mice from all groups. A thin film was made by smearing the blood samples
on microscopic slides. The slides were fixed in absolute methanol and stained with
3% Giemsa solution at pH 7.2. Average levels of parasitemia were calculated from six
different fields. The average suppression percentage of the parasite was calculated
for each group using [Equation (1)]:
where A is average levels of parasitemia of negative control; B is average levels of parasitemia in each treated group.
Statistical Analysis
The data were analyzed with SPSS 20.0 software and displayed as mean ± standard error
of the mean. The mean difference between the results obtained was compared using a
one-way ANOVA (analysis of variance) followed by Dunnett's post-hoc test. Statistical
significance was defined as a p-value of less than 0.05.
Results
Synthesis and Characterization
Following the synthetic procedure, compounds I, II, and III were obtained and characterized by the following spectral data:
-
Compound I: chemical name “1-[2,4-dihydroxy-3,5-bis(3-methylbut-2-en-1-yl)-phenyl]-ethan-1-one,”
white crystals, yield: 6.25%, mp 110–112°C, FT-IR (ν cm−1): 3,384 (Ar-OH), 2,974 and 2,914 (sp2 C-H), 2,728 (sp3 C-H), 1,617 (C = O). 1H-NMR (MeOD, 400 MHz) δ 7.49 (d, 1H), 6.27 (d, 1H), 5.31 (t, 1H), 3.33 (d, J = 7.2 Hz, 2H), 3.23 (d, J = 8.0 Hz, 2H), 2.50 (s, 3H), 1.76 (s, 12H). 13C NMR (MeOD, 400 MHz) δ 203.91, 160.67, 159.84, 132.74, 131.01, 128.78, 121.89, 119.63,
115.05, 113.86, 27.62, 24.53, 21.19, 16.54.
-
Compound II: chemical name “1-[2,4-dihydroxy-5-(3-methylbut-2-en-1-yl)-phenyl] ethan-1-one,”
white crystals, yield: 10%, mp 145–146°C, FT-IR (ν cm−1): 3,272 (Ar-OH), 2,967 and 2,911 (sp2 C-H), 2,728 (sp3 C-H), 1,617 (C = O). 1H-NMR (CD3OD 400 MHz) δ 7.58 (s, 1H), 5.51 (s, 0.5H), 5.37 (s, 0.5H), 5.11 (s, 1H), 3.54 (s,
1H), 3.45 (s, 1H), 2.69 (s, 3H), 1.86 (s, 6H). 13C NMR (CD3OD, 400MHz) δ 202.65, 163.11, 132.03, 131.62, 122.25, 120.57, 112.64, 101.63, 27.18,
24.73, 24.53, 16.43.
-
Compound III: chemical name “1-[2,4-dihydroxy-3-(3-methylbut-2-en-1-yl)-phenyl] ethan-1-one,”
white crystals, yield: 0.07%, mp 154–156°C, FT-IR (ν cm−1): 3,276 (Ar-OH), 2,967 and 2,911 (sp2 C-H), 2,851 (sp3 C-H), 1,613 (C = O).
Drug-Likeness
Compound II efficiently passed Ro3 for active fragments having molecular weight ≤300 g/mol, MlogP
≤ 3, number of hydrogen bond acceptor ≤3, number of hydrogen bond donor ≤3, number
of rotatable bonds ≤3, polar surface area ≤60 Å, while compound I passed Ro3 on the average according to results in [Table 1]. Ro3 has been useful in ensuring that fragment libraries really do consist of compounds
with active fragment-like properties. Any compound that passes the Ro3 on average
could be useful when constructing fragment libraries for efficient lead discovery.
As shown in [Table 2], both compounds passed the Lipinski's Ro5 as indicated in the reported studies,[12]
[13] suggesting that they have high probability of being orally bioavailable.
Table 1
Rule of three evaluation of the fragments
|
Compound
|
MW ≤ 300 (g/mol)
|
MlogP ≤ 3
|
HBA ≤ 3
|
HBD ≤ 3
|
nRB ≤ 3
|
PSA ≤ 60 Å2
|
|
I
|
288.38
|
3.05
|
3
|
2
|
5
|
57.53
|
|
II
|
220.26
|
1.89
|
3
|
2
|
3
|
57.53
|
Abbreviations: MW, molecular weight; HBA, number of hydrogen bond acceptor; HBD, number
of hydrogen bond donor; MlogP, lipophilicity; nRB, number of rotatable bonds; PSA,
polar surface area.
Table 2
Theoretical oral bioavailability of compounds I and II based on Lipinski's rule of
five
|
Lipinski's rule of five[a]
|
|
Compound
|
Mol. Wt[b]
|
HBA
|
HBD
|
nRB
|
MlogP
|
Remarks
|
|
I
|
288.38
|
3
|
2
|
5
|
3.05
|
Pass
|
|
II
|
220.26
|
3
|
2
|
3
|
1.89
|
Pass
|
Abbreviations: HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; nRB, number
of rotatable bonds; MlogP, lipophilicity.
a Lipinski rule (Mol. Wt ≤ 500, MlogP ≤ 4.15, N or O ≤ 10, NH or OH ≤ 5 and nRB ≤ 10).[4]
[5]
b Molecular weight in g/mol.
In Silico PK (ADME)
Absorption
As shown in [Table 3], compounds I and II had optimal and medium permeability in both in-vivo and in-vitro predictive models, respectively. The in-vivo permeability of compound II in the predictive human colon adenocarcinoma cell lines (Caco-2) was slightly higher
when compared with compound I, with values of being −4.64 and −4.70 cm/s, respectively. However, permeability co-efficient
(Papp) values of both compounds were 15 × 10−6 cm/s, which is greater than 2 × 10−6 cm/s but less than 20 × 10−6 cm/s in Madin − Darby canine kidney cells (MDCKs), suggesting that they had medium
permeability in MDCK model.
Table 3
Absorption
|
Compound
|
Caco-2 permeability
|
MDCK permeability
|
Pgp-inhib.
|
Pgp-subs.
|
HIA
|
F30%
|
F20%
|
F10%
|
|
I
|
−4.70
|
15 × 10−6
|
0.82
|
0.05
|
0.02
|
0.36
|
0.96
|
0.55
|
|
II
|
−4.64
|
15 × 10−6
|
0.07
|
0.02
|
0.01
|
0.29
|
0.92
|
0.55
|
Note: Empirical decision for P-gp, HIA, and F: 0–0.3, excellent; 0.3–0.7, good; 0.7–1.0,
poor.
Compound I had strong substrate affinity, while poor inhibitory affinity for P-gp enzyme with
scores of 0.05 and 0.82, respectively. On the other hand, compound II had comparable substrate affinity for P-gp with scores of 0.02 and stronger inhibitory
affinity for P-gp with scores of 0.07, respectively. The observed disparities between
Caco-2 permeability and MDCK permeability for both compounds can be explained by P-gp
inhibitory and substrate affinities. Drugs that are P-gp substrates usually have disparities
in their Caco-2 and MDCK permeability. An instance is quinidine, a P-gp substrate
that had high permeability in the Caco-2 model but medium permeability in the MDCK
model. This explained why both compounds being P-gp substrates with comparable affinities
indicated high permeability in the Caco-2 model and medium permeability in the MDCK
model.[14]
Compounds I and II had high human intestinal absorption with scores of 0.02 and 0.01, suggesting that
more than 30% of the compounds is absorbed in human intestine. Also, the result showed
that compound II is more readily absorbed in the human intestine in comparison to compound I.
Oral bioavailability prediction suggested that the two compounds are orally bioavailable
complementing the results of Lipinski's rule's results. Compounds I and II had excellent 30% oral bioavailability with F30% scores of 0.36 and 0.29, good 10%
oral bioavailability with F10% scores of 0.55 and 0.55 (SwissADME), yet, poor 20%
oral bioavailability with F20% scores of 0.96 and 0.92, respectively.
Distribution
As shown in [Table 4], compound I was predicted to have protein plasma binding (PPB) of 89.96%, a value considered
as optimal PPB, and compound II predicted a higher PPB value of 97.49%. However, statistics have shown that lump-sums
of United States Food and Drug Administration (FDA)-approved drugs have PPB greater
than 99%.[14] The volume distribution (VD) values of both compounds fall between 0.04 and 20 L/kg
for optimal distribution as indicated by the results in [Table 4]. As a consequence of their PPB values, compound I demonstrated medium fraction unbound (F
u) of 7.638% while compound II indicated a low F
u of 3.974%. In spite of their low F
u, both compounds had excellent blood–brain barrier penetration with scores less than
0.1.
Table 4
Distribution
|
Compound
|
PPB (%)
|
Volume distribution (L/kg)
|
BBB (%)
|
F
u (%)
|
|
I
|
89.96
|
3.452
|
0.049
|
7.638
|
|
II
|
97.49
|
0.800
|
0.082
|
3.974
|
Abbreviations: PPB, plasma protein binding; BBB, blood–brain barrier; F
u, fraction unbound.
Note: Empirical decision for BBB: 0–0.3, excellent; 0.3–0.7, good; 0.7–1.0, poor.
Metabolism
Compound I is a strong inhibitor and substrate of CYP2D6 and CYP3A4, a weak inhibitor of CYP2C9,
and a noninhibitor of CYP1A2 and CYP2C19. Also, the compound has high substrate affinity
for CYP1A2 and CYP2C19, and was nonsubstrate of CYP2C9 enzyme ([Table 5]
). On the other hand, compound II was a strong inhibitor and substrate of CYP3A4, a weak inhibitor of CYP2C9, and a
noninhibitor of CYP2D6, CYP1A2, and CYPC19 enzymes. Compound II is also a strong substrate of CYP2C19, a weak substrate of CYP1A2 and CYP2D6, and
a nonsubstrate of CYP2C19 ([Table 5]).
Table 5
Metabolism
|
Compound
|
CYP1A2
|
CYP2C19
|
CYP2C9
|
CYP2D6
|
CYP3A4
|
|
Inhib.
|
Subs.
|
Inhib.
|
Subs.
|
Inhib.
|
Subs.
|
Inhib.
|
Subs.
|
Inhib.
|
Subs.
|
|
I
|
0.85
|
0.33
|
0.74
|
0.16
|
0.69
|
0.82
|
0.50
|
0.32
|
0.13
|
0.14
|
|
II
|
0.97
|
0.64
|
0.81
|
0.08
|
0.59
|
0.81
|
0.88
|
0.53
|
0.28
|
0.20
|
Abbreviations: Inhib., inhibitors; Subs., substrate.
Note: Empirical decision: 0–0.3, excellent; 0.3–0.7, good; 0.7–1.0, poor.
Excretion
Clearance of compound I (15.726 mL/min/kg) is slightly higher than that of compound II (15.045 mL/min/kg), suggesting a high clearance rate of both of the compounds. The
half-life (T
1/2) score of compound II was 0.85 ([Table 6]), suggesting a short half-life of compound II within 3 hours. However, T
1/2 of compound I was 0.57, suggesting a moderate half-life of compound I of ≤3 hours.
Table 6
Excretion
|
Compound
|
Clearance (mL/min/kg)
|
Half-life (T
1/2)
|
|
I
|
15.726
|
0.568
|
|
II
|
15.045
|
0.850
|
Note: Empirical decision for T
1/2: 0–0.3, excellent; 0.3–0.7, good; 0.7–1.0, poor.
Toxicity
[Tables 7]
[8]
[9]
[10]
[11] to [12] illustrate the different toxicity endpoints of compounds I and II. The percentage of predicted accuracy and percentage of average similarity of each
compound, compared with the datasets of the models used on Protox-II, were 68.07 and
69.12% for compounds I and II ([Table 9]).
Table 7
Organ toxicity
|
Compound
|
LD50 (mg/kg)
|
Toxic class
|
H-HT/DILI
|
Carcinogenic
|
Immunotoxic
|
Mutagenic
|
Cytotoxic
|
|
I
|
2,830
|
5
|
0.68 (I)
|
0.68 (I)
|
0.99 (I)
|
0.75 (I)
|
0.76 (I)
|
|
II
|
2,830
|
5
|
0.57 (I)
|
0.70 (I)
|
0.62 (I)
|
0.75 (I)
|
0.71 (I)
|
Abbreviation: I, inactive.
Note: Empirical decision for H-HT/DILI: 0–0.3, excellent; 0.3–0.7, good; 0.7–1.0,
poor.
Table 8
Organ toxicity continued
|
Compound
|
hERG blocker
|
OAT
|
FDAMDD
|
Skin sensitivity
|
Respiratory toxicity
|
|
I
|
0.01
|
0.21
|
0.04
|
0.84
|
0.16
|
|
II
|
0.01
|
0.07
|
0.19
|
0.53
|
0.40
|
Abbreviations: FDAMDD, FDA maximum recommended daily dose; hERG, human ether-a-go-go-related
gene; OAT, oral acute toxicity.
Note: Empirical decision: 0–0.3, excellent; 0.3–0.7, good; 0.7–1.0, poor.
Table 9
Organ toxicity continued
|
Compound
|
Eye corrosion
|
Eye irritation
|
Prediction-accuracy (%)
|
Average similarity (%)
|
|
I
|
0.004
|
0.468
|
68.07
|
69.12
|
|
II
|
0.055
|
0.935
|
68.07
|
69.12
|
Note: Empirical decision for eye corrosion/irritation: 0–0.3, excellent; 0.3–0.7,
good; 0.7–1.0, poor.
Table 10
Nuclear receptor pathway toxicity
|
Compound
|
NR-AR
|
NR-AR-LBD
|
NR-AhR
|
NR-Ar
|
NR-ER
|
NR-ER-LBD
|
NR-PPAR-γ
|
|
I
|
0.99 (I)
|
0.99 (I)
|
0.91 (I)
|
0.98 (I)
|
0.85 (I)
|
0.93 (I)
|
0.96 (I)
|
|
II
|
0.98 (I)
|
0.99 (I)
|
0.76 (I)
|
0.94 (I)
|
0.77 (I)
|
0.86 (I)
|
0.96 (I)
|
Abbreviations: I, inactive.
Table 11
Stress response pathway toxicity
|
Compound
|
SR-ARE
|
SR-ATAD5
|
SR-HSE
|
SR-MMP
|
SR-p53
|
|
I
|
0.87 (I)
|
0.97 (I)
|
0.87 (I)
|
0.76 (I)
|
0.84 (I)
|
|
II
|
0.80 (I)
|
0.97 (I)
|
0.80 (I)
|
0.61 (A)
|
0.77 (I)
|
Abbreviations: A, active; I, inactive.
Table 12
Environmental toxicity
|
Compound
|
BCF [log10(L/kg)]
|
IGC50
|
LC50FM
|
LC50DM
|
|
I
|
1.09
|
3.77
|
5.12
|
6.13
|
|
II
|
0.85
|
3.77
|
4.44
|
5.31
|
Abbreviations: BCF, bioconcentration factor; IGC50, concentration of a substance in water in mg/L that could cause 50% growth inhibition
to Tetrahymena pyriformis after 48 hours; LC50FM, concentration of a substance in water in mg/L that could cause 50% of fathead
minnow to die after 96 hours; LC50DM, the concentration of the designed hydrazones in water in mg/L that could cause
50% of Daphnia magna to die after 48 hours.
Note: Unit for IGC50, LC50FM, and LC50DM is −log10[(mg/L)/(1,000 × MW)]
Organ Toxicity
Lethality dose (LD50) of compounds I and II were 2,830 mg/kg ([Table 7]), which were categorized as Class V: (2,000 mg/kg < LD50 ≤ 5,000 kg/kg), suggesting that they may be harmful if swallowed according to the
toxic class of the globally harmonized system of classification of labeling of chemicals.
Predicted rat or mice oral acute toxicity (OAT) scores of compounds I and II were 0.21 and 0.07, which fall in the category of OAT >500 mg/kg for low toxicity
translating that the compounds are safe. Our data also showed that compounds I and II are predicted as noncauser of human hepatotoxicity or drug-induced liver injury with
inactivity probabilities of 0.68 and 0.57, noncarcinogenic with probabilities of 0.68
and 0.70, nonimmunotoxic with probabilities of 0.99 and 0.62, nonmutagenic with probabilities
of 0.75 and 0.75, and noncytotoxic with probabilities of 0.76 and 0.71, respectively
([Table 7]).
Furthermore, compounds I and II are also nonblocker of the human ether-a-go-go related gene (hERG) as indicated by
their scores of 0.01 ([Table 8]), which translates to an excellent safety profile. Therefore, they may not cause
hERG toxicities which include long QT syndrome, arrhythmia, and Torsades de Pointes
that were associated with palpitations, fainting, or even sudden death.[14] Compound I could not cause respiratory toxicity with excellent safety score of 0.16, while compound
II has a score of 0.40 ([Table 8]), suggesting a lower safety profile when compared with compound I. Also, both compounds are nontoxic, with FDA maximum recommended daily dose below
0.2 mmol/kg-bw/d ([Table 8]). However, compound I was predicted to be skin sensitive with a score of 0.84, therefore may not be formulated
for topical application. While compound II was relatively non-skin sensitive with a score of 0.53, it may be formulated for
topical application.
As shown in [Table 9], compounds I and II are non-eye corrosive with excellent safety scores of 0.004 and 0.06, respectively.
However, compound II is an eye irritant with a score of 0.94, while compound I is relatively non-eye irritant with a score of 0.47.
Tox21 Pathway
Nuclear receptor pathway toxicity. Compounds I and II was predicted not to interact with any of the nuclear receptors with probabilities
between 0.85 for estrogen receptor (NR-ER) and 0.99 for androgen receptor (NR-AR)
according to results in [Table 10]. This suggests that the compounds may not cause nuclear receptor pathway toxicity.
Stress response pathway toxicity. Compound I was predicted as nontoxic to stress response pathways indicating noninteraction with
any of the stress response receptors. The noninteraction probabilities of the compound
ranged between 0.76 for mitochondrial membrane potential (SR-MMP) to 0.97 for ATPase
family AAA domain-containing proteins 5 (SR-ATAD5) ([Table 11]). While compound II was also inactive against nearly all of the stress response receptors with noninteraction
probabilities between 0.77 for phosphoprotein (tumor suppressor) p53 SR-p53 and 0.97
for SR-ATAD5 ([Table 11]). It is found to interact with SR-MMP with 0.61 activity probability.
Environmental toxicity. Compounds I and II had bioconcentration factors (BCFs) <3.000 log10(L/kg) according to the results in
[Table 12], which corresponds to BCF <1,000 L/kg categorized as nonbioaccumulative by the United
States Environmental Protection Agency under the Toxic Substances Control Ac. These
values are also below the 3.700 earmarked by Registration, Evaluation, Authorization
and Restriction of Chemicals (REACH) threshold for very bioaccumulative chemicals.
While compounds I and II had equal tetrahymena pyriformis (IGC50) values (3.77). Compound I demonstrated safer fathead minnow LC50 (LC50FM, 5.12) and daphnia magna LC50 (LC50DM, 6.13) when compared with compound II (4.44 and 5.31, respectively).
Antimalarial Activity
Mice with plasmodium infection were treated with compound I (25, 50, 100 mg/kg) and compound II (25, 50, 100 mg/kg). Chloroquine was used as a control drug. Then, parasitemia levels
were evaluated, and the results are shown in [Table 13]. Our data showed that parasitemia levels in mice treated with compounds I and II were significantly lowered than those treated with distilled water, suggesting the
antimalarial activity of the two compounds; however, parasitemia levels of compound
I and II-treated group were much higher than the control drug (chloroquine), suggesting a
much weaker activity of the two compounds when compared with the reference drug.
Table 13
Effect of compounds I and II on curative activity in Plasmodium berghei-infected mice
|
Treatment
|
Dosage (mg/kg)
|
Six fields (% inhibition of parasite growth)
|
Parasitemia levels
|
|
DW
|
10
|
–
|
27.90 ± 2.75
|
|
Compound I
|
25
|
68.03
|
8.92 ± 0.41
|
|
50
|
65.16
|
9.72 ± 0.30
|
|
100
|
69.75
|
8.44 ± 0.91
|
|
Compound II
|
25
|
33.33
|
18.60 ± 2.13
|
|
50
|
39.43
|
16.90 ± 1.30
|
|
100
|
72.16
|
7.77 ± 0.83
|
|
CQ
|
5
|
95.69
|
1.20 ± 0.10
|
Abbreviations: CQ, chloroquine; DW, distilled water.
Note: Values are presented as mean ± standard error of the mean; data were analyzed
by one-way ANOVA followed by Dunnett's post-hoc test, n = 5.
Discussion
Synthesis and Characterization
Two prenylated acetophenones were synthesized using 3-methyl-2-buten-1-ol under nitrogen
conditions to minimize oxidative side-product(s) and efficient prenylation on positions
3 and 5 of the dihydroxy acetophenone. The synthesis was achieved in low yields of
6.25, 10.0, and 0.07% for compounds I, II, and III, respectively. Oral bioavailability is an important parameter in drug design as it
reduces drug failure resulting from poor PK profile. The two compounds passed Lipinski's
rule as revealed in [Table 2]. This showed that the compounds would be orally bioavailable.
Compounds I, II, and III were found to be white crystalline solids according to a reported study.[15] Melting points were within the range of 110 to 112°C for compound I, 145 to 146°C for compound II, and 154 to 156°C for compound III. The FTIR spectra showed the presence of prominent bands at 3,384–3,272 cm−1 (OH stretching vibration), 1,613–1,617 cm−1 (C = O stretching vibration), 2,851–2,728 cm−1 (sp3 C-H stretching vibration), 2,914–2,911 cm−1 and 2,974–2,967 cm−1 (sp2 C-H stretching vibration) ([Figs. 3]
[4]
[5]). The sp2 and sp3 stretches indicated the presence of the prenylated groups on compounds I, II, and III. The 1H NMR and 13C NMR spectra were used to elucidate the structures of compounds I and II. Also as shown by 1H and 13C NMR spectra, there were appearances of distinct peaks indicating prenylation had
occurred on the substituted acetophenone ring. The 1H-NMR of compound I showed distinct peaks at 5.31 and 6.27 ppm, indicating the presence of phenolic protons
on the aromatic group, peaks at 3.33 and 3.23 ppm corresponded to allylic protons
in the prenylated groups. Peaks at 7.49 ppm indicated the aromatic proton. Peaks at
1.76 ppm corresponded to signals for terminal methyl groups of the prenyl groups,
while the peak at 2.50 ppm corresponded to α-methyl of the ketone. For compound II, the proton peaks at 7.58 and 5.51 ppm corresponded to aromatic protons, the peaks
at 5.37 and 5.11 ppm indicated for phenolic protons, the peak at 3.54 ppm indicated
for allylic proton, the peak at 3.45 ppm corresponded to methylene protons of the
prenyl group, the peak at 2.69 ppm corresponded to α-methyl of the ketone, and the
peak at 1.751 ppm indicated for the terminal methyl groups on compound II accordingly.
Fig. 3 FTIR spectrum of compound I. FTIR, Fourier-transform infrared spectroscopy.
Fig. 4 FTIR spectrum of compound II. FTIR, Fourier-transform infrared spectroscopy.
Fig. 5 FTIR spectrum of compound III. FTIR, Fourier-transform infrared spectroscopy.
Antimalarial Activity
This study is the first report of antimalarial activity of prenylated acetophenone.
As shown in [Table 13], at doses of 25 and 50 mg/kg, compound I showed promising activity with percentage inhibition of 68.03 and 65.16% respectively,
demonstrating superior activities when compared with compound II (33.33 and 39.43%). This suggests that prenylation at position 3 of the acetophenone
is important for antimalarial activity. Furthermore, the superior activities of compound
I may be due to its better PK profiles in respect to their PPB, VD, and F
u as displayed in [Table 4].
However, at a higher dose of 100 mg/kg, compound II demonstrates supper-activity with the inhibition rate of parasitemia level being
72.16% when compared with compound I (69.75%). Furthermore, the antimalarial activity of compound II was dose-dependent. A closer analysis also indicated 100% increase in activity of
compound II when the dose was increased from 50 to 100 mg/kg.
To evaluate the possible prodrug effect of the compounds, the curative model was chosen
for the study. Krettli et al suggested that a compound should be considered active
when its parasitemia reduction is ≥30%.[16] In another study, antimalarial agents are classified into three categories as moderate,
good, and very good if the compound showed parasitemia suppression percentage equal
or greater than 50%.[17]
[18] Consequently, based on these criteria, compounds I and II were presumed to have very good antimalarial activity. Furthermore, compounds I and II demonstrated a superior antimalarial activity compared with nerolidylcatechol and
its derivatives in a similar study,[19] suggesting that the resorcinol moiety of compounds I and II is important for antimalarial activity and therefore confers more antimalarial activity
compared with the catechol moiety. Early communication of these findings has been
reported in a preprint.[20]
