Coumarin Antifungal Lead Compounds from Millettia thonningii and Their Predicted Mechanism of Action

Article abstract of DOI:10.3390/molecules21101369

Fungal pathogens continue to pose challenges to humans and plants despite efforts to control them. Two coumarins, robustic acid and thonningine-C isolated from Millettia thonningii, show promising activity against the fungus Candida albicans with minimum

Full text of DOI:10.3390/molecules21101369

molecules
Article
Coumarin Antifungal Lead Compounds from
Millettia thonningii and Their Predicted Mechanism
of Action
Daniel M. Ayine-Tora 1,2, Robert Kingsford-Adaboh 2, William A. Asomaning 2,
Jerry J.E.K. Harrison 2, Felix C. Mills-Robertson 3, Yahaya Bukari 4, Patrick O. Sakyi 2,
Sylvester Kaminta 5 and Jóhannes Reynisson 1,
*
1
School of Chemical Sciences, University of Auckland, 23 Symonds Street, 1142 Auckland, New Zealand;
dayi479@aucklanduni.ac.nz
2
3
Department of Chemistry, University of Ghana, LG 56, Legon-Accra, Ghana;
kadabohs@yahoo.com (R.K.-A.); waasoman@ug.edu.gh (W.A.A.); harriebow@gmail.com (J.J.E.K.H.);
poskeydinho@yahoo.com (P.O.S.)
Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology,
Kumasi, Ghana; mirobfc2002@yahoo.com
Botany Department, University of Ghana, LG 55, Legon-Accra, Ghana; sparrowson@yahoo.com
Centre for Plant Medicine Research, 73, Mampong-Akuapem, Ghana; slykmt@yahoo.co.uk
Correspondence: j.reynisson@auckland.ac.nz; Tel.: +64-9-373-7599 (ext. 83746)
4
5
*
Academic Editor: Thomas J. Schmidt
Received: 14 July 2016; Accepted: 7 October 2016; Published: 15 October 2016
Abstract: Fungal pathogens continue to pose challenges to humans and plants despite efforts to
control them. Two coumarins, robustic acid and thonningine-C isolated from Millettia thonningii,
show promising activity against the fungus Candida albicans with minimum fungicidal concentration
of 1.0 and 0.5 mg/mL, respectively. Molecular modelling against the putative bio-molecular target,
lanosterol 14α-demethylase (CYP51), revealed a plausible binding mode for the active compounds,
in which the hydroxyl group binds with a methionine backbone carboxylic group blocking access to
the iron catalytic site. This binding disrupts the synthesis of several important sterols for the survival
of fungi.
Keywords: natural products; Candida albicans; molecular modelling; CYP51; Sclorotium; isoflavone
1. Introduction
Candida albicans is one of the most common fungal pathogens causing infection despite major
efforts to control it [
mortality in immunocompromised patients suffering from diseases such as cancer or AIDS [
Candidiasis infection can affect the skin, oral cavity, oesophagus, gastrointestinal tract, vagina and
vascular system of humans [ ]. There are a number of drugs such as flucoconazole, nystatin,
1,
2]. C. albicans has emerged as one of the main causes of morbidity and
3–6].
7–9
voriconazole, terbinafine and echinocandin to address fungal infections [10]. However, drug resistance,
restricted systemic usage due to dose-related toxicity and the emergence of new strains of fungal
infections all undermine the efficacy of these drugs [11–15]. Hence, it is necessary to develop new
antifungal treatments to address these emerging challenges.
Milletia thonningii (Schum-Thonn) Baker is a deciduous tree that grows in tropical climates.
It belongs to the family Papilionaceae and is indigenous to West and Central Africa [16–18].
Preparations from this tree are used in traditional medicine for alleviating several diseases and
disorders such as dysentery, intestinal pain, worms, tuberculoid leprosy, menstrual disorder, measles
and chicken pox [16,18]. In addition, the seeds show molluscicide activity [19] and the juice from the
leaves is lethal to Bulinus snails, the host for schistosomiasis [20].
Molecules 2016, 21, 1369; doi:10.3390/molecules21101369
www.mdpi.com/journal/molecules

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The many uses of M. thonningii as a traditional source of remedies have attracted research into its
constituents. For instance, some isoflavones isolated from this plant were reported to demonstrate
lethal toxicity to brine shrimp [21] as well as to inhibit activation of hypoxia-inducible factor-1 (HIF-1)
in human breast tumour T47D cells [22]. Interestingly, coumarin derivatives have been shown to
demonstrate antifungal activity [23–26].
The aim of this work was to investigate the activity of the constituent of M. thonningii against
C. albicans and investigate a mechanism against lanosterol 14α-demethylase (CYP51), a common
anti-fungal target. This enzyme converts lanosterol to ergosterol, the disruption of this enzyme results
in structural changes of plasma membranes, and causes a change in the concentrations of various
14α-methyl sterols, which has cytostatic and cytotoxic consequences [27–29]. It is hoped that the
findings will provide new lead compounds as well as useful insights into the rational design of the
active compounds of M. thonningii for antifungal treatment.
2. Results and Discussion
2.1. Isolation and Synthesis
In order to investigate the activity of the compounds from M. thonningii against C. albicans,
the constituents of the seeds of M. thonningii were isolated. The isolation yielded robustic
acid (
4’-O-methylalpinumisoflavone (
The yield of compound was low but more was successfully synthesised via demethylation
of for biological testing. Compound was also acetylated to form a new derivative,
acetyl-4-O-methylalpinumisoflavone ( ), to help understand the structure activity relationships of the
chemical series. The molecular structures are displayed in Figure 1.
1), thonningine-C (
2
), alpinumisoflavone (
3
), O,O-dimethylalpinumisoflavone (
4) and
5
). These compounds are known constituents of the plant [20
,30–32].
5
4
5
6
Figure 1. Structures of isolated coumarins (1, 2) and isoflavanones (3, 4, 5) from M. thonningii and a
synthesised derivative (6).
2.2. Investigation into the Selective Demethylation of Isoflavone 4
Previous studies focused on determination of reagents to give optimum yields for demethylation
of compounds such as visnagen and khellin. According to these investigations, a combination of
formic acid and KI gives the optimal yield. Replacing KI with NaBr, KBr, and NaI did not improve the
yield of this reaction. In addition, using glacial acetic acid instead of formic acid resulted in a lower

Molecules 2016, 21, 1369
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yield. Therefore, formic acid and KI were used for the demethylation of isoflavone
4
to synthesise
derivative 5 [33].
Interestingly, the demethylation of isoflavone
4 only occurred at the methoxy group on the
benzopyrone moiety (X) but not on the phenyl group (Y) (see Figure 2). This is in line with a previous
investigation, which showed that methoxy groups ortho to acyl moieties are highly reactive and acid
sensitive compared to aromatic methoxy groups [34]. In order to shed light on this phenomenon,
thermochemical density functional theory (DFT) calculations were performed to determine bond
dissociation energies (BDE) for the bonds X and Y, and proton affinities (PA) for derivative
4. It has
been shown that BDEs and PAs can be reliably calculated using the DFT method [35 38]. PAs of the
–
carbonyl and methoxy oxygen atoms were calculated to determine the preferred protonation site due
to the acidic nature of the reaction medium. The PA values show that the carbonyl oxygen is the
preferred protonation site (see Table S1 in the Supplemetary Information). The BDEs of neutral and
protonated forms of
4 were determined for both homolytic and heterolytic cleavages of the O-C bond
and the results are shown in Table 1.
Figure 2. Structure of O,O-dimethylalpinumisoflavone (4) and the two carbon-oxygen bonds under
investigation X and Y.
Table 1. Bond dissociation energies (BDEs) in kcal/mol.
Bond Dissociation
X
Y
Difference
Homolytic
Heterolytic
Homolytic-protonated
Heterolytic-protonated
51.2
216.0
42.5
54.0
227.8
62.7
2.8
11.8
20.2
56.2
111.4
167.6
The results in Table 1 show that demethylation of bond X is more thermochemically favourable
for both protonated and non-protonated forms of . Furthermore, the difference in BDE values for
non-protonated are small, which can result in a mixture of demethylated products, in particular for
4
4
the homolytic cleavage. Thus, it can be argued that protonation of the carbonyl oxygen is necessary for
the selective demethylation to proceed. After protonation, the reaction is kinetically driven by KI to
the desired product.
2.3. Biological Testing
The compounds were tested against the wild C. albicans strain and a reference strain
ATCC18804 [39–41]. The agar-well diffusion method was used to evaluate the zones of inhibition (ZOI).
This was followed by the determination of minimum inhibitory concentrations (MIC) and minimum
fungicidal concentrations (MFC) for the active compounds. The results are shown in Table 2.
As can be seen in Table 2, the ZOIs show that compounds
and the reference strain, but no activity was observed for derivatives
1
,
2
and
3
are active against C. albicans
and . The antifungal drug
4
,
5
6
clotrimazole was used as a reference, and it gave ~27% larger ZOI for the wild type strain and 16% for
the reference strain 18804. All the compounds were tested at 2 mg/mL concentration. Considering
that clotrimazole is an optimised drug in clinical use, it can be stated that compounds
1–3 are quite
active. According to the MIC results, compound is more active than and 3, and, finally, the MFC
2
1

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measurements revealed that coumarins
1
and
2
are fungicidal with 1.0 and 0.5 mg/mL, respectively.
Interestingly, the isoflavone
lead compounds for further development for antifungal treatment. Furthermore, compounds
also showed activity against the Sclerotium fungal strain qualitatively confirming their efficacy (data
not shown).
3
is only fungistatic. This activity of the coumarins makes them promising
1, 2
and
3
Table 2. Zones of inhibition (ZOI), minimum inhibitory concentrations (MIC) and minimum fungicidal
concentrations (MFC).
ZOI (mm)
MIC (mg/mL)
MFC (mg/mL)
Compound
WILD
18804
WILD
18804
WILD
18804
Robustic acid (1)
Thonningine-C (2)
Alpinumisoflavone (3)
10.3
10.3
10.7
0
0
0
14.7
14.7
15.0
0
0
0
0.25
0.13
0.25
X
X
X
1.00
0.25
0.50
X
X
X
1.0
0.5
F
X
X
X
X
X
1.0
0.5
F
X
X
X
X
X
O,O-dimethylalpinumisoflavone (4)
4-O-methylalpinumisoflavone (5)
Acetyl-4-O-methylalpinumisoflavone (
Clotrimazole
6)
14.4
0
17.6
0
X
X
X
X
DMSO (20%)
Zones of inhibition = ZOI; minimum inhibitory concentrations = MIC; minimum fungicidal concentrations =
MFC; WILD = wild C. albicans strain; 18804 = reference ATCC18804 strain; X = No data; F = Fungistatic.
2.4. Molecular Modelling
It is established that microorganisms produce a unique class of sterols such as ergosterol necessary
for their growth and survival [42]. The absence of these sterols in mammalian host cells makes
these pathways an excellent target for therapeutic antifungal design [43
clinically employed antifungals such as the azoles have been designed to disrupt CYP51, a cytochrome
P450 enzyme responsible for the synthesis of the sterols [28 45 46]. Coumarin antifungal derivatives
designed in silico against the CYP51 enzyme have also showed promising preliminary results [47 48].
from
,44]. In this light, some
,
,
,
It can therefore be hypothesised that CYP51 is the putative target of the active compounds 1–3
M. thonningii.
The coumarins and isoflavones were docked to the crystal structure of CYP51 (Protein data bank
ID: 4ZE1) [49]. The co-crystallised posaconazole was removed and redocked. The root-mean-square
deviation (RMSD) between the heavy atoms of the co-crystallised ligand and its docked counterparts
was 1.33 Å and 1.51 Å using ChemScore (CS) and GoldScore (GS) respectively, verifying the prediction
power of the method.
It is predicted that the hydroxyl group of the coumarins bind to the backbone carboxylic moiety
of methionine (MET509), which enables them to adopt a plausible binding configuration with methyl
groups blocking the iron catalytic binding site as shown in Figure 3A,B. The isoflavones
have hydroxyl groups, also bind to the methionine but have their phenyl moieties inserted into the
binding site; whereas the isoflavones and without the hydroxyl groups have similar configurations
3 and 5, which
4
6
to the coumarins but do not form hydrogen bonds with the methionine, as shown in Figure 4A,B,
respectively. This is a possible explanation why the coumarins are active but not the isoflavones.
Robustic acid (1) had the highest predicted affinity by CS and thonningine-C (2) for the GS results.
Both of the coumarins gave better scores for both functions when compared to clotrimazole (see
Table S2 in the Supplementary Materials). These predictions point toward CYP51 as the bio-molecular
target of these compounds but with the very important caveat that experimental verification is needed.
Nevertheless, the molecular modelling work supports a plausible hypothesis and points the way in
which assays these compounds should be tested first.

Molecules 2016, 21, 1369
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A
B
Figure 3.
(A
) the docked configuration of robustic acid (
1) in the binding site of lanosterol
14
α
-demethylase (CYP51); and (B) the docked configuration of thonningine-C (2) in the binding
site. Hydrogen bonds are shown as green dotted lines.
A
B
Figure 4.
(A
) the docked configuration of alpinumisoflavone (
3) in the binding site of CYP51; and (
B)
the docked configuration of O,O-dimethylalpinumisoflavone (4) in the binding site. Hydrogen bonds
are shown as green dotted lines.
2.5. Chemical Space
The calculated molecular descriptors molecular weight (MW), log P, hydrogen bond donors (HD),
hydrogen bond acceptors (HA) polar surface area (PSA) and rotatable bonds (RB) given in Table S3 in
the Supplementary Materials. All of the molecules are within the drug-like chemical space (for definition
of lead-like, drug-like and Known Drug Space regions, see ref. [50] and Table S4). The molecular weight
of the ligands is between 336.3 and 438.4 g
mol−1 and the log P values lie in the range of 3.6 and
·
4.6. These values demonstrate that the compounds are in a favourable area of chemical space for
further development.
3. Materials and Methods
3.1. Isolation
3.1.1. Plant Material
The seeds of Milletia thonningii were collected from the University of Ghana campus (Legon-Accra,
Ghana). A voucher specimen was identified by Mr. D. K. Abbiw and then later Mr. J.Y. Amponsah at

Molecules 2016, 21, 1369
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the Ghana National Herbarium, Department of Botany, University of Ghana. The voucher specimen
(catalogue NO: 14832) was deposited at the herbarium. The seeds were shade dried for three weeks and
then pulverised. All solvents were analytical grade. Thin-layer chromatography (TLC) was performed
on aluminium foil slides pre-coated gel (thickness 0.2 mm, type Kiesegel 60 F254 Merck, Darmstadt,
Germany) using petrol/ethyl acetate (10%, 15%, 20%, 25%, 30% and 35%); detection was performed
using UV light and iodine vapour. Column chromatography was carried out on aluminium oxide
(Fluka, Seelze, Germany).
3.1.2. Extraction and Isolation
The pulverised plant material (4.5 kg) was extracted using cold percolation with petroleum ether
(2 L) for five days at 27 ◦C. The extract was filtered and concentrated using rotary evaporator to give
a brown solid (18 g) and stored in a refrigerator. In addition,◦15 g of this solid were dissolved
in chloroform, mixed with aluminium oxide and dried at 50 C. This was placed on a column
(
50 mm × 1200 mm) pre-packed with aluminium oxide. The column was first eluted with petrol
(500 mL), and then with a 5% increment of ethyl acetate in petrol, and 500 mL for each increment. At 45%
of ethyl acetate in petrol, TLC profiling showed two dominant fractions F1a and F1b. These fractions
were purified using column chromatography (CC) (20 mm
×
600 mm) with aluminium oxide and an
increasing amount ethyl acetate in petrol. Afterwards, the defatted plant material was also extracted
◦
with ethyl acetate (2 L) for a week at 27 C. A yellow mixture (20 g) was obtained after solvent
removal and also stored in a refrigerator. CC of 15 g of the yellow mixture was conducted as above.
At 80% of ethyl acetate in petrol, TLC profiling showed four dominant fractions—F2a, F2b, F2c and
F2d. These fractions were also purified on CC. The compounds were characterised by TLC, mass
spectrometry, IR, 1H and 13C-NMR spectra and compared to published data. F1a and F2b were found
to be the same compound and identified as isoflavone (
isoflavones ( ) (1.2 g) and ( ) (23 mg), respectively. F2a and F2d were also identified as coumarin (
(1.4 g) and (2) (642 mg), respectively.
4) (4.8 g). F1b and F2c were identified as
3
5
1)
◦
Robustic Acid ( ): Colourless block crystal, melting point (mp.) 207–209 C. IR
1612 cm−1; Gas chromatography tandem Mass spectrometry (GC-MS) m/z 380, 365; 1H-NMR
(400 MHz, CDCl3) : 9.91 (1H, s), 7.49 (2H, d, J = 8 Hz), 6.98 (2H, d, J = 8 Hz), 6.64 (1H, s), 6.53 (1H,
d, J = 10 Hz), 5.80 (1H, d, J = 10 Hz), 3.98 (3H, s), 3.83 (3H, s), 1.49 (6H, s); 13C-NMR (100 MHz, CDCl3)
: 162.2, 159.7, 158.6, 156.8, 153.5, 151.8, 131.1, 131.4, 123.0, 113.2, 114.7, 110.3, 103.6, 101.5, 101.4, 98.0,
64.0, 54.9, and 27.6. This is in agreement with published data [30,31].
1
νmax: 3381, 1701,
δ
δ
◦
Thonningine-C (
m/z 437; H-NMR (400 MHz, CDCl3)
2
): Yellow granules; mp. 200–203 C; IR
ν
max: 3288, 3093, 1716, 1658, 1620 cm−1; GC-MS
1
δ
: 10.23 (1H, s), 7.48 (1H, s), 7.45 (1H, d, J = 8 Hz), 7.00 (1H,
s), 6.99 (1H, d, J = 8 Hz), 6.90 (1H, s), 6.89 (1H, m), 3.94 (1H, s), 3.91 (1H, s), 3.90 (1H, s), 3.85 (1H, s),
2.23 (1H, s); 13C-NMR (100 MHz, CDCl3)
: 165.0, 160.1, 158.8, 158.5, 158.3, 143.8, 131.1, 122.1, 113.0,
110.4, 103.9, 102.1, 95.8, 64.7, 54.7, and 19.9. This is in agreement with published data [32].
δ
Alpinumisoflavone (
3
): Yellow needle-like crystal; mp. 204–207 ◦C; IR
ν
max: 3454, 1654, 1614, and 1573
1
cm−1; GC-MS m/z: 336, 321; H-NMR (400 MHz, CDCl3)
δ
: 13.11 (1H, s), 7.82 (1H, s), 7.39 (2H, d,
J = 8 Hz), 6.88 (2H, d, J = 8 Hz) 6.71 (1H, d, J = 10 Hz), 6.33 (1H, s), 5.64 (1H, d, J = 10 Hz), 5.25 (1H, s),
4.25 (1H, s), 3.93 (1H, s), 1.47 (6H, s); 13C-NMR (100 MHz, CDCl3)
δ: 180.4, 159.0, 156.8, 156.3, 155.4,
152.0, 129.8, 127.6, 123.0, 122.5, 115.1, 114.9, 105.6, 105.1, 94.3, and 27.8. This is in agreement with
published data [30,51].
O,O-dimethylalpinumisoflavone (
1512, 1389, 1362, 1244, 1067 cm−1, GC-MS m/z: 364, 349; 1H-NMR (400 MHz, CDCl3)
7.47 (2H, d, J = 8 Hz), 6.97 (2H, d, J = 8 Hz), 6.76 (1H, d, J = 10 Hz), 6.60 (1H, s), 5.74 (1H, d, J = 10 Hz),
3.83 (3H, s), 3.90 (3H, s),1.47 (6H, s); 13C-NMR (100 MHz, CDCl3)
: 173.3, 157.8, 157.0, 156.3, 154.1,
4
): Colourless granules (8.9 g); mp. 137–139 ◦C; IR
ν
max: 1636, 1605,
δ: 7.76 (1H, s),
δ

Molecules 2016, 21, 1369
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148.6, 129.0, 128.6, 123.9, 122.6, 114.4, 112.2, 111.6, 111.5, 98.9, 61.0, 53.6, and 26.6. This is in agreement
with published data [30,31].
3.2. Synthesis
3.2.1. Synthesis of 40-O-methylalpinumisoflavone (5)
To a solution of O,O-dimethylalpinumisoflavone (4) (1.00 g, 2.75 mmol) in 30 mL of ethyl acetate,
KI (5.10 g, 30.73 mmol) was added, followed by formic acid (30 mL) as described by Mustafa et al. for
the demethylation of khellin and visnagen [33]. This mixture was refluxed for 110 min. After the reflux,
50 mL of water was added and the product precipitated. The product was purified by chromatography
0
(ethyl acetate/petroleum ether, 1:5) to yield 780 mg of 4 -O-methylalpinumisoflavone (
5
) as pale yellow
needle-like crystals. mp.: 136–138 ◦C; IR
1H-NMR(400 MHz, CDCl3)
: 13.16 (1H, s), 7.82 (1H, s), 7.47 (2H, d, J = 8 Hz), 7.00 (2H, d, J = 8 Hz),
6.75 (1H, d, J = 10 Hz), 6.34 (1H, s), 5.64 (1H, d, J = 10 Hz), 3.85 (3H, s), 1.48(6H, s); 13C-NMR (100 MHz,
CDCl3) : 179.5, 158.4, 158.1, 155.9, 155.5, 151.0, 128.7, 126.7, 122.1, 121.6, 114.1, 112.7, 104.7, 104.2, 93.4,
ν
max: 3423, 1651, 1614, 1512 cm−1; GC-MS m/z: 350, 335;
δ
δ
53.9, and 26.9. This is in agreement with published data [30].
3.2.2. Synthesis of Acetyl-40-methylalpinumisoflavone (6)
0
A solution of 4 -O-methylalpinumisoflavone (
5) (253 mg, 0.72 mmol) in 20 mL of acetic anhydride
was refluxed for 120 min. After the reflux, a solution of sodium bicarbonate (10 g, 119 mmol) in
100 mL of water was added to the mixture to neutralise the acetic acid produced, which resulted
in the precipitation of the product. The product was filtered and purified by chromatography
(ethyl acetate/petroleum ether, 1:5) to afford 146 mg of acetyl-40-methylalpinumisoflavone (
6
) as
max: 1766, 1635, 1608, 1514 cm−1; GC-MS m/z: 393,
: 7.75 (1H, s), 7.41 (2H, d, J = 8 Hz), 6.96 (2H, d, J = 8 Hz), 6.71 (1H, s),
6.51 (1H, d, J = 10 Hz), 5.78 (1H, d, J = 10 Hz), 2.45 (3H, s), 3.83 (3H, s),1.49 (6H, s); 13C-NMR (100 MHz,
CDCl3) : 175.0, 169.3, 159.6, 158.1, 157.6, 150.8, 145.0, 132.0, 130.4, 125.7, 124.0, 115.2, 114.0, 113.0, 102.2,
a colourless block crystal. mp.: 204–206 ◦C; IR
335; 1H-NMR(400 MHz, CDCl3)
ν
δ
δ
94.0, 55.3, 28.4, and 21.1.
3.3. Bioactivity Studies
C. albicans were isolated and identified at the Komfo Anokye Teaching Hospital at Kumasi, Ghana.
The organisms were brought to the Microbiology Department of the Centre for Plant Medicine Research
(CPMR) at Mampong-Akuapem, Ghana.
3.3.1. Preparation of Solutions and Media
20 mg of all the compounds were dissolved in DMSO (dimethylsulphoxide, 10 mL of 20%,
(Sigma-Aldrich, D5879, Münich, Germany)), and 100 µg/mL solution of clotrimazole was also prepared
using DMSO (20%). Nutrient agar, bacteriological peptone (Sigma-Aldrich, P0556) and Sabouraud 4%
glucose agar (Fluka Biocheika 84088, Sigma-Aldrich, Bangalore, India) were prepared according to the
manufacturer’s instructions. The stock cultures of C. albicans were subcultured onto fresh Nutrient
agar plates and then incubated for 24 hours and stored in a refrigerator overnight.
3.3.2. Determination of the Potency of the Compounds
The agar-well diffusion method was used to investigate the antimicrobial properties of the
isoflavones and the coumarins [52,53].
About three to five colonies of the same morphological type of the microbes were◦suspended
in test tubes containing 5 mL of sterilised bacteriological peptone and incubated at 37 C for 16 h.
After the 16 hours, the microorganisms were subcultured in sterilised bacteriological peptone water and
incubated again for another two hours. The turbidity was adjusted to a 0.5 McFarland standard [52].
The dried Sabouraud 4% glucose agar plates were then flooded with the fungi and the surface was

Molecules 2016, 21, 1369
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allowed to dry. A sterilised cock borer of an internal diameter of 6 mm was used to punch wells in the
medium. The compounds were then dispensed into their respective labelled wells with concentrations
of 2 mg/mL for all the compounds tested; and DMSO (20%) was dispensed into its labelled well as
a negative control whilst clotrimazole (2 mg/mL) was dispensed as a positive control. Triplicates of
each plate with the same labelled compounds were made for each fungus. The plates were kept in
the refrigerator overnight for complete diffusion. The plates were then incubated at 35 ◦C for 48 h.
After the incubation period, the diameter of each inhibition zone was measured in millimeters (mm)
with a metre rule.
3.3.3. Determination of Minimum Inhibitory Concentration and the Fungicidal or Fungistatic Effect of
the Active Compounds
The MICs of the active compounds were determined using the microplate dilution method as
described by Eloff [54]. A hundred microliters (100
was placed in the first well of the second row of the 96-well microplate. About 100
active compound was added to 100 L of sterilised bacteriological peptone water in the next well of the
second column. This was mixed with a micropipette and 100 L of this dilution was added to the well
in the next column, which contained 100 L of sterilised bacteriological peptone water. This process
was repeated to produce two-fold serial dilutions in the original concentration. About 100 L of DMSO
(20%) was placed in the last column. This process was repeated for three rows on the 96-well microplate.
In addition, 100 L of clotrimazole was placed in the last row. Furthermore, 100 L of subcultured
µL) of 2 mg/mL solution of an active compound
µL of the same
µ
µ
µ
µ
µ
µ
fungi were placed in all the wells except the first row, which contains only peptone water. The plates
◦
were then covered and incubated at 35 C for 48 h, after which 40
µL of iodonitrotetrazolium chloride
(INT, 0.2 mg/mL) was added to each well and examined after 120 min of incubation. Fungi growth
was indicated by a red colour of formazan, which is the reduced form of the INT. The minimum
concentration at which there was no colour was taken as the minimum inhibitory concentration.
Samples from the wells with no colour, and, therefore, wells with no observable fungi growth
were incubated at 35 ◦C for 48 h on fresh Sabouraud 4% glucose agar plates using inoculation pins.
The minimum concentration at which no growth of microbes was seen was taken as the minimum
fungicidal concentration.
3.4. Thermochemical Calculations
The calculations were done using Gaussian 09 software suite [55] (Gaussian Inc., Wallingford,
CT, USA) employing a restricted non-local B3LYP functional hybrid method [56
–58]. The standard
6-31+G (d, p) [59 60] basis set was used for the geometry optimisation and frequency analysis while
,
the larger 6-311+G (2df, p) basis set was employed to perform the energy calculations. The zero-point
vibrational energies (ZPE) were scaled using Wong’s value of 0.9804 [61]. In all cases, the normal modes
revealed no imaginary frequencies, which indicate that they represent the minima on the potential
energy surface. The PAs and BDEs were calculated as described by Foresman and Frisch [62].
3.5. Molecular Modelling
Compounds
resolution 2.05 Å) [49], which was obtained from Protein Data Bank (PDB) [63
FJ 2.6 program [65] (Fijitsu Limited, Kanagawa, Japan was used to prepare the crystal structure for
1
–
6
were docked into the crystal structure of CYP51 (Protein Data Bank ID: 4ZE1
,
64]. The Scigress version
)
docking, hydrogen atoms were added, the co-crystallised ligand as well as crystallographic water
molecules were removed. The Scigress software suite was also used to build the inhibitors and the
MM2 [66] force field was used for structural optimisation. The centre of the binding was defined
as the position the carbonyl oxygen of methionine 509 (x = 4.024, y = 15.088, z = 99.736) with a
10 Å radius. Hundred docking runs were allowed for each ligand with default search efficiency
(100%). The GoldScore [67], and ChemScore [68,69], scoring functions were implemented using
the parameter file for cytochromes P450 and heme-containing proteins [70] developed to predict

Molecules 2016, 21, 1369
9 of 13
binding modes and relative binding energies of the ligands using the GOLD v5.4 software suite
(Cambridge Crystallographic Data Centre, Cambridge, UK). This heme-containing proteins parameter
file was developed using data from the Cambridge Structural Database (CSD) and Protein Data
Bank. The root square mean deviation (RSMD) of the co-crystallised ligand for CSD-ChemScore,
CSD-GoldScore, PDB-ChemScore, and PDB-GoldScore were 1.333 Å, 1.710 Å, 1.595 Å and 1.509 Å,
respectively. The CSD-Chemscore, which had the best RSMD value, was used for further analysis.
The docking scores of the compounds are given in Table S1 in the supplementary materials.
The QikProp 3.2 [71] software package (Schrödinger LLC, New York, NY, USA) was used to
calculate the molecular descriptors of the molecules. The reliability has been established for the
calculated descriptors [72].
4. Conclusions
Previous studies have focused on azoles, polyenes, griseofulvin, flucytosine and allylamine
molecular scaffolds for antifungal design, e.g., the clinically used drugs voriconazole, ketoconazole
and fluconazole are derived from azole containing molecules and terbinafine from allylamine [10].
However, not much attention has been paid to the development of coumarins as antifungals. This study
demonstrates their potential for antifungal drug development.
Robustic acid (1) and thonnigine-C (2) were active against the fungal strains tested. The predicted
mechanism of action showed that the hydroxyl group binds to the methionine backbone of CYP51
helping the coumarins to adopt a pose that blocks access to the catalytic site of the enzyme.
Interestingly, the coumarins under study appear to be non-toxic in mammalian systems due to the
numerous traditional medicinal use of the M. thonningii. This non-toxic nature presents these coumarins
as good antifungal leads, which might be developed further as antifungal drugs and food preservatives.
This study extends the number of coumarins with antifungal properties reported [23
to providing direction for rational design of coumarins as antifungal agents.
–26] in addition
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
10/1369/s1. Table S1: Bond dissociation energies, Table S2: Results of the scoring function for the ligands, Table S3:
The calculated molecular descriptors for the ligands, Table S4: Definition of lead-like, drug-like and known drug
space (KDS) in terms of molecular descriptors.
Acknowledgments: Government of Ghana Graduate Studies Allowance and Department of Chemistry, UG
Graduate Assistantship to DMA-T is duly acknowledged.
Author Contributions: Robert Kingsford-Adaboh, William A. Asomaning and Jerry J.E.K. Harrison designed the
research; Daniel M. Ayine-Tora conducted the research with support of Jerry J.E.K. Harrison and Patrick O. Sakyi;
Felix C. Mills-Robertson, Sylvester Kaminta and Yahaya Bukari conducted biological studies; Jóhannes Reynisson
and Daniel M. Ayine-Tora designed and conducted the modelling and computational studies and wrote the paper.
All the authors read the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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2016 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
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).