Bioactivity of a family of chiral nonracemic aminobenzylnaphthols towards candida albicans

Article abstract of DOI:10.3390/molecules19045219

Chiral nonracemic aminobenzylnaphthols were obtained by a Betti multicomponent reaction between 2-naphthol, aryl aldehydes and enantiopure arylethylamine. Moreover, some new aminobenzylnaphthols were synthesized by a similar reaction between 2-naphthol, aryl aldehydes and prolinol. These aminobenzylnaphthols, synthesized from different components and thus having different structural features, were tested as anti-yeast agents inhibiting Candida albicans. The effect towards the test strain was studied with a microdilution approach and three different concentrations (150, 300 and 450 μg/mL) were tested. The best results were found for the aminobenzylnaphthols obtained from 1-naphthylethylamine and from natural prolinol. The use of the two-way ANOVA highlighted the better performances of the prolinol derivative among the differently structured aminobenzylnaphthols that were screened. The activity towards C. albicans of this prolinol derivative resulted to be interesting and could represent a promising alternative to overcome the problem of the strains resistant to the traditional antifungals.

Full text of DOI:10.3390/molecules19045219

Molecules 2014, 19, 5219-5230; doi:10.3390/molecules19045219
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Bioactivity of a Family of Chiral Nonracemic
Aminobenzylnaphthols towards Candida albicans
Maria Annunziata M. Capozzi 1, Cosimo Cardellicchio 2, Angela Magaletti 1,
Antonio Bevilacqua 3,*, Marianne Perricone 3 and Maria Rosaria Corbo 3
1
Department of Chemistry, University of Bari, Via Orabona 4, 70125 Bari, Italy;
E-Mails: maria.capozzi@uniba.it (M.A.M.C.); angelamagaletti@gmail.com (A.M.)
2
CNR-ICCOM, at the Department of Chemistry, University of Bari, Via Orabona 4, 70125 Bari,
Italy; E-Mail: cardellicchio@ba.iccom.cnr.it
3
Department of the Science of Agriculture, Food and Environment, University of Foggia,
Via Napoli 25, 71122 Foggia, Italy; E-Mails: marianne.perricone@unifg.it (M.P.);
mariarosaria.corbo@unifg.it (M.R.C.)
* Author to whom correspondence should be addressed; E-Mail: antonio.bevilacqua@unifg.it;
Tel.: +39-0881-589231.
Received: 20 February 2014; in revised form: 8 April 2014 / Accepted: 16 April 2014 /
Published: 23 April 2014
Abstract: Chiral nonracemic aminobenzylnaphthols were obtained by a Betti multi-
component reaction between 2-naphthol, aryl aldehydes and enantiopure arylethylamine.
Moreover, some new aminobenzylnaphthols were synthesized by a similar reaction
between 2-naphthol, aryl aldehydes and prolinol. These aminobenzylnaphthols, synthesized
from different components and thus having different structural features, were tested as
anti-yeast agents inhibiting Candida albicans. The effect towards the test strain was
studied with a microdilution approach and three different concentrations (150, 300 and
450 µg/mL) were tested. The best results were found for the aminobenzylnaphthols obtained
from 1-naphthylethylamine and from natural prolinol. The use of the two-way ANOVA
highlighted the better performances of the prolinol derivative among the differently
structured aminobenzylnaphthols that were screened. The activity towards C. albicans of
this prolinol derivative resulted to be interesting and could represent a promising
alternative to overcome the problem of the strains resistant to the traditional antifungals.
Keywords: Candida albicans; Betti reaction; prolinol; anti-yeast activity; microdilution
approach; interactive effects

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1. Introduction
Candida spp., namely C. albicans, are one the most important causes of hospital acquired systemic
infections, with crude mortality rate of up 50% [1,2]. These yeasts can cause two types of pathologies:
superficial (oral and vaginal candidiasis) and life-threatening systemic infections [3]. C. albicans, and
to a lesser extent the other species of the genus, can be easily found in the oral cavity of up to 75% of
the population [3]. In healthy individuals this colonization remains benign; however, C. albicans could
pose a strong threat in immunocompromised patients, who can suffer recalcitrant infections on the skin
and in the oral cavity [4]. The rise in the number of immunocompromised patients has caused a
dramatic increase of the incidence of fungal infections due to C. albicans and other related human
opportunistic pathogens [5,6] and this issue is a great challenge for human health due also to the fact
that some strains of these yeasts are resistant to some traditional antifungals, like the azoles [7].
Some alternatives to the azoles are extracts from plants (like the oils from Iamium album, Cuminum
cyminum, Salvadora persica) [8,9], the combination of terpenes and fluconazole [10], bioremediation [11],
as well as other agents [12,13].
Previously, some papers [14–19] reported the Betti reaction as a route for the asymmetric synthesis
of chiral nonracemic amino benzylnaphthols. This procedure is a straightforward condensation of
2-naphthol, aryl aldehydes and suitable chiral nonracemic amines (Schemes 1 and 2) to yield more
complex molecular structures. When an enantiopure (S)-amine was used, a (S,S)-aminobenzylnaphthol
was easily isolated [14,20].
The experimental procedure that was applied [14] to the synthesis of aminobenzylnaphthols 1–6
(Scheme 1) was extended in the present paper to the reaction of 2-naphthol, aryl aldehyde and (S)- or
(R)-prolinol (Scheme 2), to yield the corresponding aminobenzylnaphthols 7–9.
Scheme 1. Synthesis of aminobenzylnaphthols 1–6.
X
Ar
Me
NH
OH
CHO
X
OH
Ar
60 °C
+
+
H2N
Me
1-6
Compound
(S, S)-1
(S, S)-2
(S, S)-3
(S, S)-4
(S, S)-5
(S, S)-6
X
Ar
Ph
H
F
Ph
Cl
H
F
Ph
1-Np
1-Np
1-Np
Br

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Scheme 2. Synthesis of aminobenzylnaphthols 7–9.
X
CHO
H
N
OH
N (S)
(S)
(S)
60 °C
OH
+
+
OH
OH
X
7, 9
X = H, Cl
CHO
H
N
N
OH
(R)
(R)
60 °C
OH
+
+
OH
OH
(R)
8
Compound
(S, S)-7
X
Amine
H
H
(S)-Prolinol
(R)-Prolinol
(S)-Prolinol
(R, R)-8
(S, S)-9
Cl
At this stage, taking into account that C. albicans is a great threat for human health and the
production of novel compounds with a significant bioactivity towards this yeast is an important issue
for both organic chemistry and medicine, we decided to test the bioactivity of aminobenzylnaphthols
1–9 against a collection strain of C. albicans to correlate the results of the screening to the structural
features of the investigated molecules.
2. Results and Discussion
The experiments were performed on aminobenzylnaphthols divided into three main groups,
depending on the enantiopure amine that was employed in the Betti reaction (Schemes 1 and 2). In
particular, compounds 1–3 were synthesized having (S)-phenylethylamine as a precursor; compounds
4–6 were obtained starting from (S)-1-(1-naphthyl)ethylamine; finally, compounds 7–9 were the new
aminobenzylnaphthols related to the prolinol structure.
When testing the bioactivity of a chemical, the choice of the type of approach is a critical step;
namely, the evaluation of the antimicrobial activity relies upon two main approaches: the agar
diffusion and the microdilution test. In this paper, we utilized the microdilution method, due to its
suitability to assess the Minimal Inhibitory Concentration and to establish an eventual time-kill response.
Moreover, due to a possible interference of DMSO in the evaluation of the anti-yeast activity [21], two
types of controls were prepared and respectively, analyzed (without and with the solvent, samples C1
and C2, respectively). In all the experiments the differences in cell counts with and without this solvent
were generally not significant (P > 0.05, one-way ANOVA, data not shown), with some exceptions to
this generalized statement.
Table 1 shows the anti-yeast effect of the compounds 1–3 belonging to the first group. An anti-yeast
effect was observed only after 6 h, with a decrease in yeast count of 0.5 log cfu/mL (cfu, colony

Molecules 2014, 19
5222
forming units); however, this activity was not related to the amount of the compounds in the broth, as
it was found for the lowest concentrations (150 and 300 μg/mL) of the molecules 2 and 3 but not for
the highest one (450 μg/mL). This result could be due to the low solubility of the investigated
compounds 1–3 in aqueous systems. Thus, we could assume that the lower bioactivity at the highest
concentration was probably due a partial dissolution of the compounds in the broth, resulting in a
lower active concentration.
Table 1. Anti-yeact activity ([count in the control C2] − [count in the samples containing
the chemicals]) (log cfu/mL; cfu, colony forming units) of the compounds 1–3 towards
C. albicans in YPG broth. The letters indicate the significant differences (one-way
ANOVA and Tukey’s test, P < 0.05). The symbol “/” indicate a not significant effect.
Time (h)
Samples/
concentrations
(S,S)-1
6
24
/
/
/
/
/
/
150 μg/mL
300 μg/mL
450 μg/mL
(S,S)-2
/
/
/
150 μg/mL
300 μg/mL
450 μg/mL
(S,S)-3
0.50 a
0.55 a
/
/
/
/
150 μg/mL
300 μg/mL
450 μg/mL
0.53 a
0.55 a
/
A significant bioactivity was found for the compounds of the 2nd (4–6) and 3rd series (7–9), as
reported in Figures 1 and 2. Concerning the group of molecules 4–6, their bioactivity was strongly
affected by the structure, the concentration and the time. In particular, compound 4 reduced the count
of C. albicans by ca. 0.5 log cfu/mL and the effect of the concentration was not significant. On the
other hand, the concentration played a significant role for the compound 5, as it reduced the yeast by
0.68 ± 0.2 log cfu/mL at 150 µg/mL and 1.33–1.44 log cfu/mL at 300–450 µg/mL. Finally, the
strongest bioactivity was observed for the molecule 6, as it caused a decrease of the yeast by 2.7 log
cfu/mL. After 48 h the compounds of this group did not exert any type of anti-yeast effect.
As regards to the bioactivity of the molecules of the third group 7–9, after 24 h the effect of the
compound 7 was strong and relied upon the concentration, as it reduced C. albicans by 2.7–3.0 log cfu/mL
at 150/300 µg/mL and 4.34 ± 0.40 log cfu/mL at 450 µg/mL. Its stereoisomer 8, synthesized starting
from the unnatural (R)-prolinol, did not exert any effect towards C. albicans. An interesting and
significant bioactivity was found for the compound 9, which decreased the yeast by 1.20 ± 0.10 log cfu/mL
at 150 µg/mL and ca. 2.4 log cfu/mL at 300/450 µg/mL. After 48 h, a significant effect was recovered
only for the compound 7, which reduced the level of C. albicans by 1–3.68 log cfu/mL (Figure 2).

Molecules 2014, 19
Figure 1. Anti-yeast effect of the compounds 4–6 after 24 and 48 h. The results were
5223
reported as difference between the yeast count in the control C2 and the level of the target
in the samples containing the antimicrobial compounds. Mean values ± standard deviation.
The letters indicate significant differences (one-way ANOVA and Tukey’s test, P < 0.05). The
number after the compounds indicate its concentration in the broth (150, 300 or 450 µg/mL).
3
2.5
2
c
c
c
b
b
1.5
1
24 h
48 h
a
a
a
a
0.5
0
‐0.5
‐1
Figure 2. Anti-yeast effect of the compounds 7–9 after 24 and 48 h. The results were reported
as difference between the yeast count in the control C2 and the level of the target in the
samples containing the antimicrobial compounds. Mean values ± standard deviation. The
letters indicate significant differences (one-way ANOVA and Tukey’s test, P < 0.05) (Small
letters: significant differences after 24 h; Capital letters; significant differences after 48 h). The
number after the compounds indicate its concentration in the broth (150, 300 or 450 µg/mL).
5
4
e
C
d
c,
3
c
c
d
B
2
1
24 h
48 h
b
B
A
A
a
a
a
A
A
A
0
‐1
As a final step of this research, the effects of the molecules tested in the first run and able to reduce
the level of C. albicans (i.e., 5, 6, 7, and 9) were used as input values for a two-way ANOVA to
highlight the most promising compound. Two way ANOVA could be described as the most simple
factorial analysis; it is based on the analysis of variance, with an important difference: the results can
be classified by two factors (in this paper the kind of the compound and the concentration) and the
analysis can highlight the differences due to each factor and the variability linked to their interaction.

Molecules 2014, 19
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The main output of this analysis are the table of the statistical effects and the hypothesis
decomposition; the table of the effects reports a qualitative output, i.e., if a factor is significant or not,
whilst the graph of hypothesis decomposition shows the quantitative effect. Table 2 shows the
statistical effect of the analysis, whereas Figure 3 reports the hypothesis decomposition.
Table 2. Two-way ANOVA for the effects of the kind of chemicals and their concentration
on the anti-yeast effect after 24 h: comparison of the most promising compounds from the
second group (4–6) and the third groups (7–9). SS, sum of squares; df, degrees of freedom;
MS, mean square; F, Fisher test value.
Effects
Compound
Amount
Interaction
Within
SS
df
3
MS
F
p-value
1.49E-14
2.55E-06
1.03E-05
24.6148
3.17882
4.30922
1.65102
33.7539
8.20494
1.58941
0.7182
0.06879
119.3
23.1
10.44
2
6
24
35
Total
Figure 3. Decomposition of two-way ANOVA for the effects of the kind of the chemical
and the concentration (comparison of the most promising compounds from the second and
the third groups); the bars denote 95%-confidence intervals.
The statistical analysis pointed out a significant effect of the two factors (compound and
concentration); moreover, the ANOVA highlighted a significant interaction compound/concentration.
The hypothesis decomposition graph shows this type of interaction for compounds 9 and 7; namely the
effects of compound 9 were strengthened at 300 µg/mL, whilst molecule 7 underwent to a significant
increase of its activity at 450 µg/mL. Concerning the most promising compound, the highest bioactivity
was found for compound 7 at 450 µg/mL; moreover, this molecule also retained its effect after 48 h.

Molecules 2014, 19
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3. Experimental Section
3.1. Strain
Candida albicans DSM 70014 (DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen,
Braunschweig, Germany) was used as the test strain; the yeast was stored at 4 °C on YPG slants (yeast
extract, 10 g/L; bacteriological peptone, 20 g/L; glucose, 20 g/L; agar, 15 g/L) and grown before each
assay in YPG broth, incubated at 25 °C for 48–72 h. Thereafter, yeast cultures were centrifuged at
1,000 g for 10 min, the supernatant was discarded and the pellet suspended in sterile saline solution
(0.9% NaCl); yeast suspension was diluted to ca. 5 log cfu/mL.
3.2. Tested Molecules
Starting materials for the Betti reaction were purchased from Sigma–Aldrich (Milan, Italy) and
were used as received. NMR spectra were recorded on a Bruker AM500 spectrometer. HPLC analyses
were performed with an Agilent 1100 chromatograph, equipped with a DAD detector. Schemes 1 and
2 show the different aminobenzylnaphthols used in this research.
3.2.1. Synthesis of Aminobenzylnaphthols
(S,S)-Aminobenzylnaphthols 1–6 were synthesized [14] according to the previously reported
procedure by reacting 2-naphthol, aryl aldehydes and (S)-1-arylethylamine (Scheme 1). Physical and
spectral properties of the compounds 1–3 and 5–6 were already reported in the same paper [14].
Physical and spectral properties of aminobenzylnaphthol 4 were reported in a different work [22].
Aminobenzylnaphthols 7–8 (Scheme 2) were synthesized by reacting 2-naphthol, aryl aldehydes
and (S)- or (R)-prolinol for two days at 60 °C without any solvent. After this time, the crude reaction
mixture was cooled to room temperature and purified by column chromatography (eluent
n-hexane/ethyl acetate 7:3), followed by crystallization. When (S)-prolinol was used, two compounds,
whose NMR spectra are compatible with the (S,S) and (R,S)-aminobenzylnaphthol 7, were recovered.
Among them, the most abundant isomer was fully characterized and later used for the biological tests.
3.2.2. 1-[(2-Hydroxymethyl-pyrrolidin-1-yl)-benzyl]-naphth-2-ols 7 and 8—Predominant Stereoisomer
Isolated yield: 37%. Mp 138–140 °C (ethanol). []D25 (c = 0.7, chloroform) = +199 for 7 and −192
(c = 0.5, chloroform) for 8. 1H-NMR (CDCl3, 500 MHz) δ 14.03–13.86 (broad, 1 H), 7.82–7.78 (m, 1 H),
7.72–7.67 (m, 2 H), 7.64–7.60 (m, 2 H), 7.37–7.33 (m, 1 H), 7.30–7.26 (m, 2 H), 7.25–7.20 (m, 2 H),
7.17 (d, J = 8.9 Hz, 1 H), 5.44 (s, 1 H), 3.30–3.22 (m, 1 H), 3.21–3.11 (m, 2 H), 3.06–2.98 (m, 1 H),
13
2.69–2.62 (m, 1 H), 2.10–2.01 (m, 1 H), 1.93–1.83 (m, 3 H), 1.72–1.41 (broad m, 1 H). C-NMR
(CDCl3, 125 MHz) δ 155.6, 140.2, 131.8, 129.6, 129.4, 129.0, 128.9, 128.8, 128.6, 128.3, 126.4,
122.4, 120.9, 120.0, 70.2 (broad), 64.8, 61.8, 55.9 (broad), 28.5, 23.8. In the absence of a crystal
suitable for the X-ray analysis, the configuration of the newly formed stereogenic centre of the
aminobenzylnaphthols 7 and 8 was attributed by the following combined NMR and computational
1
analysis. The H-NMR spectra of the couple of diastereomeric aminobenzylnaphthols 7 (predominant

Molecules 2014, 19
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and less abundant stereoisomer) isolated from the reaction mixture were compared. The overlap of a
section of the two NMR spectra is illustrated in Figure 4.
Figure 4. NMR spectra of aminobenzylnaphthols 7/8.
As can be seen, the signals of the methylene hydrogen atoms of the hydroxymethyl moiety in the
case of less abundant stereoisomer of 7 are centered at 3.52 ppm (Figure 4, top). On the other hand, in
the case of the predominant stereoisomer of 7, the same signals move upfield to coalesce in the
3.21–3.11 ppm multiplet (Figure 4, bottom).
The structures of the two different stereoisomers (R,S)-7 and (S,S)-7 were optimized with an
HF-calculation (Spartan program), with the further constrain of a hydrogen bond between the naphthol
hydrogen atom and the nitrogen atom, fixed at the average value of 1.85 Å, as occurred in every other
aminobenzylnaphthols [14,17]. Within this constrain, the calculation shows that the exocyclic
methylene group points towards the phenyl moiety in the case of the (S,S)-aminobenzylnaphthol 7,
whereas the same group points towards free space in the case of the (R,S)-counterpart (Figure 5).
Figure 5. Spatial representation of aminobenzylnaphthols 7/8.
In a constrained structure, if some hydrogen atoms points towards an aryl group, an upfield shift of
their 1H-NMR signals is observed. Since this upfield shift was predicted for the (S,S)-configuration and
observed for the predominant stereoisomer, then the (S,S)-configuration can be attributed to the most
abundant stereoisomer. In summary, when natural (S)-prolinol was used in the Betti reaction,

Molecules 2014, 19
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(S,S)-aminobenzylnaphthol 7 was mainly obtained, in complete agreement with the observed
stereochemical course of the Betti reactions reported to date. Conversely, when unnatural (R)-prolinol
was used in the Betti reaction, (R,R)-aminobenzylnaphthol 8 was synthesized.
A chiral HPLC analysis (column Chiralcel OD-H, eluent n-hexane/i-propanol 9:1) was performed
also on the (S,S)-aminobenzylnaphthols 7 and (R,R)-8 to check that no racemization at the stereogenic
centre deriving from the (S)-prolinol occurred during the reaction. The separation conditions had been
preliminarily optimized with a simulated racemic mixture. The chiral HPLC analysis confirmed that no
racemization was observed.
3.2.3. 1-[(4-Chlorophenyl)-(2-hydroxymethylpyrrolidin-1-yl)-methyl]-naphth-2-ol (9)
This compound was obtained using the experimental conditions outlined for compounds 7 and 8.
25
1
Isolated yield: 36%. Mp 75–77 °C (ethanol). []D (c = 0.25, chloroform) = +83. H-NMR (CDCl3,
500 MHz) δ 13.86–13.74 (broad, 1 H), 7.76–7.64 (m, 3 H), 7.57–7.52 (m, 2 H), 7.37–7.31 (m, 1 H),
7.26–7.19 (m, 3 H), 7.13 (d, J = 9.2 Hz, 1 H), 5.41 (s, 1 H), 3.27–3.01 (m, 4 H), 2.66–2.57 (m, 1 H),
2.08–1.98 (m, 1 H), 1.91–1.80 (m, 3 H), 1.54–1.34 (broad, 1 H). 13C-NMR (CDCl3, 125 MHz) δ 155.5,
138.8, 134.1, 131.6, 130.7, 129.9, 129.0, 128.6, 126.6, 122.5, 120.6, 120.0, 116.4, 69.4 (broad), 64.7,
61.6, 55.7 (broad), 28.3, 23.8. The (S,S)-configuration of aminobenzylnaphthol 9, prepared starting
from (S)-prolinol, was attributed by analogy to the previously cited combined NMR and computational
analysis. Also in this case, the chiral HPLC analysis (Chiralcel OD-H column; eluent: n-hexane/
i-propanol 9:1) confirmed that no racemization occurred during the Betti reaction.
3.3. Evaluation of the Anti-Yeast Activity
The bioactivity of the aminobenzylnaphthols was assessed through a microdilution approach,
modified as follows. Aliquots of YPG broth (9.3 mL) were inoculated with C. albicans to 3 log cfu/mL
and added with variable amounts of DMSO and/or of the stock solutions; the preparation of the
samples is shown in Table 3. Two different kinds of control were prepared: C1, without the chemicals
and DMSO, and C2, containing DMSO but not the molecules. A stock solution of each chemical was
prepared by solving 7.5 g of the compound per liter of DMSO (dimethyl sulfoxide).
Table 3. Preparation of the sample to assess the bioactivity of the molecules through the
microdilution approach.
Stock solution
Saline solution
Samples
DMSO (mL)
Inoculum (mL)
(mL)
0.2
0.4
0.6
-
(mL)
150 μg/mL
300 μg/mL
0.4
0.2
-
-
-
0.1
0.1
0.1
0.1
0.1
450 μg/mL
-
Control 1 (C1)
Control 2 (C2)
-
0.6
-
-
0.6
The samples were stored at 25 °C and the viable count of C. albicans was assessed periodically
(after 6, 24 and 48 h) through the spread plating on YPG plates, incubated at 25 °C for 48–72 h.

Molecules 2014, 19
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The microbiological analyses were performed in duplicate over two different batches; the results
were analyzed through the one-way and two-way Analysis of Variance and Tukey’s test as the
post-hoc comparison test (P < 0.05) using the software Statistica for Windows ver. 10.0 (Statsoft,
Tulsa, OK, USA).
4. Conclusions
The increase of the incidence of the strains of C. albicans resistant to some traditional antifungals is
a great threat for human health; however, the multicomponent Betti reaction could be a promising and
simple way to design novel compounds to inhibit this yeast. The results of this paper suggest that the
bioactivity of aminobenzylnaphthols synthesized by this reaction could be affected by different factors,
connected to the different components of the Betti procedure:
1. The structure of the compounds, since biological activity was detected for aminobenzyl-
naphthols synthesized starting from 1-(1-naphthylethylamine) or from prolinol (groups 2 and
3), but not observed for molecules derived from 2-phenyethylamine.
2. The presence on the tested molecules of the halogen atom in the para-position of the phenyl
group deriving from the aryl aldehyde employed in the Betti reaction, since chlorine or bromine
atom can affect the bioactivity.
3. The stereochemistry of the amine used in the Betti reaction, since the aminobenzylnaphthols
deriving from the natural (S)-prolinol showed a strong effect towards C. albicans, whilst the
compound prepared by the unnatural (R)- prolinol does not.
As a final step of this research, a promising compound was selected, i.e., (S,S)-1-[(2-hydroxymethyl-
pyrrolidin-1-yl)-benzyl]-naphth-2-ol or (S,S)-7, that is able to reduce by 4.34 log cfu/mL the count of
C. albicans at 450 µg/mL after 24 h and to retain a significant effect also after 48 h. A future trend of
this research could be the application of this compound as a tool to disrupt or control biofilms formed
by Candida spp.
Author Contributions
The authors Capozzi, Cardellicchio and Magaletti were involved in the synthesis and the chemical
characterization of the compounds, whilst the authors Bevilacqua, Perricone and Corbo performed the
microbiological analysis and the statistical treatment of the data.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds tested in this research are available from the authors.
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