Synthesis and antifungal activity against fusarium oxysporum of some brassinin analogs derived from L-Tryptophan: A DFT/B3LYP study on the reaction mechanism

Article abstract of DOI:10.3390/molecules21101349

An efficient methodology to obtain novel antifungal analogs of brassinin 1 is described. Starting from L-Tryptophan 2, N,N0-dialkylthiourea 4, 4-[(1H-indol-3-yl)methylene]-2-sulfanylidene- 1,3-Thiazolidin-5-one 5 and alkyl (2S)-3-(1H-indol-3-yl)-2-{[(alkylsulfanyl)carbonothioyl]amino} propanoate 6 type compounds were obtained as main products in different ratios depending on the reaction conditions via a tandem dithiocarbamate formation and Michael addition reaction. In order to understand the dependence of the reaction conditions on the mechanism pathway, a DFT/B3LYP study was performed. The results suggested the existence of competitive mechanistic routes which involve the presence of an ionic dithiocarbamate intermediate 9. Antifungal activities of all products were then evaluated against Fusarium oxysporum through mycelial growth inhibition using a microscale amended-medium assay. IC⁵⁰ values were thus determined for each compound. These results showed that 6-related compounds can be considered as promissory antifungal agents.

Full text of DOI:10.3390/molecules21101349

molecules
Article
Synthesis and Antifungal Activity against
Fusarium oxysporum of Some Brassinin Analogs
Derived from L-tryptophan: A DFT/B3LYP Study on
the Reaction Mechanism
Diego Quiroga *, Lili Dahiana Becerra, John Sadat-Bernal, Nathalia Vargas and
Ericsson Coy-Barrera
Bioorganic Chemistry Laboratory, Facultad de Ciencias Básicas y Aplicadas,
Universidad Militar Nueva Granada, Campus Nueva Granada, Cajicá 49300, Colombia;
ldbecerrag@unal.edu.co (L.D.B.); john.bernal@unimilitar.edu.co (J.S.-B.); u0500887@unimilitar.edu.co (N.V.);
ericsson.coy@unimilitar.edu.co (E.C.-B.)
*
Correspondence: diego.quiroga@unimilitar.edu.co; Tel.: +57-1-6500-000 (ext. 3270)
Academic Editor: Derek J. McPhee
Received: 14 September 2016; Accepted: 8 October 2016; Published: 11 October 2016
Abstract: An efficient methodology to obtain novel antifungal analogs of brassinin
1 is described.
0
Starting from L-tryptophan
,3-thiazolidin-5-one
propanoate type compounds were obtained as main products in different ratios depending on
2, N,N -dialkylthiourea
4, 4-[(1H-indol-3-yl)methylene]-2-sulfanylidene-
1
5 and alkyl (2S)-3-(1H-indol-3-yl)-2-{[(alkylsulfanyl)carbonothioyl]amino}
6
the reaction conditions via a tandem dithiocarbamate formation and Michael addition reaction.
In order to understand the dependence of the reaction conditions on the mechanism pathway,
a DFT/B3LYP study was performed. The results suggested the existence of competitive mechanistic
routes which involve the presence of an ionic dithiocarbamate intermediate 9. Antifungal activities of
all products were then evaluated against Fusarium oxysporum through mycelial growth inhibition
using a microscale amended-medium assay. IC50 values were thus determined for each compound.
These results showed that 6-related compounds can be considered as promissory antifungal agents.
Keywords: Brassinin; phytoalexin; L-tryptophan derivatives; dithiocarbamate; Fusarium oxysporum
1
. Introduction
Phytoalexins are secondary metabolites produced by plants for protecting against microbial
pathogens, heavy metals, and UV irradiation. They are crucial components of the defense mechanisms
in plants. Among the cruciferous (Brassicaceae) phytoalexins, brassinin has a fundamental role in
the biosynthetic pathway of phytoalexins derived from L-tryptophan . Brassinin is an important
precursor of several phytoalexins such as cyclobrassinin, brassilexin, spirobrassinin, dioxybrassinin,
brassicanate A, and rutalexin produced by Brassica spp. and other cruciferous species [ ].
1
2
1
1–7
Although these plants can produce mixtures of phytoalexins having different biological effects on
several pathogens, some phytopathogenic fungi transform indole phytoalexins in biologically inactive
products [1–7]. Paldoxins, phytoalexin detoxification inhibitors, are an environmentally friendly
alternative via inhibiting specific metabolic reactions in fungal phytopathogens. Some of the synthetic
strategies based on heterocyclic systems, such as indole-3-substituted derivatives, have been proposed
as promissory paldoxins (Figure 1) [2]. In this context, the importance of sulfur functionalized organic
groups (i.e., dithiocarbamate moieties) and the presence of indole-based skeleton in this kind of
compounds have been evidenced [8].
To obtain these compounds, a suitable synthetic strategy involves the reaction between amines
and carbon disulfide [9–11]. Wang et al. [12] studied the reaction between o-phenylenediamine and
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carbon disulfide to obtain 2-mercaptobenzimidazole, using triethylamine as catalyst in THF, observing
the formation of an ionic dithiocarbamate-type intermediate [12]. Therefore, the reaction between
dithiocarbamic acid salts with Michael acceptors offers an ecofriendly alternative to obtaining alkyl
dithiocarbamates via the formation of the above-mentioned ionic intermediate. Bardajee et al. [13
]
achieved the synthesis of alkyl dithiocarbamates using a solvent-free multicomponent reaction between
alkylamines, carbon disulfide, and Michael acceptors in the presence of the KF and aluminum oxide
mixture. Azizi et al. [14] reported a method for the synthesis of alkyl dithiocarbamates using mild
reaction conditions starting from similar synthetic precursors.
Figure 1. Chemical structures for compounds 1–7.
Recently, our research has been directed towards the synthesis of Brassinin(
1)-like analogs
using conventional -aminoacids as synthetic precursors to generate antifungal agents against
α
Fusarium oxysporum.
Here, we describe the functionalization of L-tryptophan
, 4-[(1H-indol-3-yl)methylene]-2-sulfanylidene-1,3-thiazolidin-5-one
2
5
towards
and alkyl
0
N,N -dialkylthiourea
4
(
2S)-3-(1H-indol-3-yl)-2-{[(alkylsulfanyl)carbonothioyl]amino}propanoate 6 type compounds. In order
to understand the influence of the reaction conditions in the ratios of the mentioned products,
a DFT study at the B3LYP/6-31G(d,p) level of theory was then performed. An in vitro assay against
Fusarium oxysporum was performed to evaluate the antifungal activity of the synthesized products.
2
. Results and Discussion
The first step of the present synthetic methodology involved the esterification of 2, as previously
reported by Li and Sha [15]. To evaluate the esterification method, the reactions were performed using
four different aliphatic alcohols (R = Me, Et, 2-Pr, and n-Bu). We synthesized alkyl (2S)-2-amino-3-(1H-
indol-3-yl)propanoate compounds 7a
–
d
with yields of 85%–96%. Reactions between 7a
–b, carbon
disulfide, and Michael acceptors 8a
–
d were studied under different conditions such as acid-basic
medium character, temperature and solvents (Scheme 1).
The reaction mixtures were maintained for 24 h in constant stirring carrying out each trial at
◦
different temperatures (
and acetonitrile , while
−
10, 0, 10, and 40 C). We identified 4a
–b
as main products in soft acid catalysis
was
5
was obtained in a soft basic medium and methanol. The formation of 6a
–
f
detected in low yields (<5%) in both acid and basic catalysis. When reactions were carried out in strong
◦
acid or basic media at 40 C, the number of detectable products increased. These results suggest that
reaction pathways were dependent on the solvent and the medium’s acid-basic character, implying
competitive mechanistic routes. The first route involves the formation of
of the ionic dithiocarbamate intermediate and a second molecule of
and elimination releasing sulfide hydrogen. The second route involves the 5-exo-trig intramolecular
cyclization of to produce favored by the basic medium within a kinetic control. A third route
4
through condensation
9
7, a consecutive addition
9
5
involves the reaction of intermediate
effect of polar solvents (Scheme 2).
9 with a Michael acceptor, which is not favored by the solvation

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Scheme 1. Synthesis of compounds 4, 5, and 6.
Scheme 2. Proposed mechanism for forming 4, 5, and 6.
Computational calculations were carried out using the DFT method at the B3LYP/6-31G(d,p) level
of theory [16]. The formation of is proposed as the first step in the mechanistic route. Using Fukui
9
index values, we established both electrophilic and nucleophilic characters of the carbonyl carbon
−
(
in
C=O, f+ between 0.050 and 0.100) and the sulfur atoms (R-(C=S)-S , f
−
between 0.110 and 0.400)
9
, respectively. These results suggest strong intramolecular both electrophilic and nucleophilic
0
characters and the intramolecular cyclization of
9
towards 5 (∆G calculated = −5.77 kcal/mol) in
3
a basic medium. The HOMO in
while LUMO is partially located in the
of the C=S, and the * orbital of the aromatic rings (Figure 2). In the second route,
on the carbon atom of the thiocarbonyl moiety to yield hydrogen sulfide and
9 is located in sp and non-bonding orbitals in the sulfide fragment,
π
* orbital of the C=O bond in the ester group, the
π* orbital
π
9
undergoes
nucleophilic attack by
9
7
0
(∆G calculated = +33.80 kcal/mol) in an acid medium. The solvation effect of intermediate
evaluated using IEFPCM and IPCM models. The results suggest that the interaction between
methanol tends to reduce its stability, which can be verified according to the total energy of
9
9
was
and
9
in each

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model (there is an energy difference of +56.8 kcal/mol and +72.9 kcal/mol, respectively). Moreover,
the methanol solvation effect tends to enhance the polarizability of the C=O bond in the ester fragment
according to the Mulliken charge values of the carbon atom (+0.189 in gas phase, +0.200 for each
solvation model). These results support the hypothesis that the presence of a polar protic solvent in
the reaction medium exerts an influence in the reaction pathway.
Figure 2. HOMO and LUMO for intermediate
9 (Isovalue of 0.02). White balls represent
hydrogen atoms, gray balls represent carbon atoms, blue balls represent nitrogen atoms, red balls
represents oxygen atoms, yellow balls represent sulfur atoms, dark red and green regions represent
HOMO-LUMO orbitals.
In order to enhance the formation of compounds 6, some one-pot reactions of L-tryptophan 2
with carbon disulphide, Michael acceptors, trimethylsilane chloride, and the respective alcohol were
carried out in dry THF employing triethylamine (TEA) as base. TMSCl and the alcohol were used
to protect the carboxylic acid group in situ. The tandem dithiocarbamate formation and Michael
addition reaction produced compounds
a Michael acceptor yielded only polymeric resins, which were therefore not characterized. The Michael
addition occurred as a competitive step (Scheme 2), where intermediate suffers a weak solvent effect,
which lead to its stabilization as we proposed for polar solvents. Therefore, intermediate attacks the
electrophilic carbon atom in the Michael acceptor promoting the C–S coupling with . These results
6 in higher yields (75%–95%). However, using acrolein as
9
9
6
demonstrate that the third route is preferred using a one-pot reaction and mild conditions.
In vitro antifungal activity testing against Fusarium oxysporum was performed using a microscale
amended-medium assay (resistant strain G1 obtained from Cape gooseberry, provided by the collection
of Phytopathology Laboratory at UMNG). Compounds
4, 5, 6, and 7 were evaluated at five different
concentrations in the 10–300 g/mL range. The results were expressed as half-maximal inhibitory
µ
concentration (IC50 in mM) for each compound, using a non-linear regression in the program GraphPad
Prism version 5.00 for Windows. All the results are summarized in Table 1.
Table 1. Antifungal activity of the compounds 4–7 against Fusarium oxysporum.
Compound
IC50 a (mM) ± SD
4
a
0.49 ± 0.09
0.76 ± 0.23
1.5 ± 0.5
1.1 ± 0.2
1.8 ± 0.9
2.5 ± 0.7
0.16 ± 0.05
2.1 ± 0.8
1.1 ± 0.7
0.59 ± 0.21
1.7 ± 0.5
3.1 ± 1.7
>50
4b
4
c
4
d
5
6
a
6
b
6
c
6
d
6
6
7
e
f
a
7
b
7
c
7.9 ± 3.8
7
d
0.76 ± 0.14
a
Data expressed as mean values ± standard deviation (SD) of three replicates.

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Results for compounds 7a
–
d
suggest that the size of the substituent has an effect on antifungal
activity. However, these results are unsatisfactory considering that 7a
protection of the carboxylic group does not enhance the antifungal activity of these compounds.
Results for showed lower IC50 values to that of their precursors, suggesting that the addition of
–d are indole derivatives. Thus,
6
a dithiocarbamate group increases the inhibitory effect on the fungus. The effect of the alkyl substituent
at the ester group and the electron withdrawing group at the dithiocarbamate fragment were evident
in this kind of compound. These structural moieties tend to enhance the antifungal activity, specifically
using ethyl and nitrile groups. However, compounds 6e and 6f showed the opposite. This behavior
may indicate that bulky substituents in the dithiocarbamate fragment can promote the formation of
hydrophobic interactions avoiding the correct binding within specific fungal targets.
3
. Materials and Methods
3
.1. General Information
All chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and Merck KGaA
(
Darmstadt, Germany) and used without further purification. Dry solvents were purchased in sufficient
purity. Thin layer chromatography (TLC) was done on TLC silica gel 60 F254 (Merck KGaA), and
compounds were detected at 254 nm. Column chromatography was conducted manually on silica gel
6
0 (0.040–0.063 mm) from Merck KGaA. Nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker Avance AV-400 MHz spectrometer (Billerica, MA, USA). All shifts are given below in (ppm)
δ
using the signal of tetramethylsilane (TMS) as a reference. All coupling constants (J) are given in Hz.
Splitting patterns are typically described as follows: s: singlet, d: doublet, t: triplet, and m: multiplet.
The HPLC analyses were performed on a Shimadzu Prominence HPLC instrument (Kyoto, Japan)
using a Synergi HydroRP-C18 column (150 mm
×
4.6 mm
×
4 µm) (Torrance, CA, USA). The mobile
phases consisting of A (formic acid 1% in acetonitrile) and B (formic acid 0.1% in water) were used
with the gradient mode at a flow rate of 1.5 mL/min. The UV detection at 270 nm was performed with
a diode array detector (Shimadzu, Kyoto, Japan). The mass spectrometry experiments were performed
on a LC/MS 2020 spectrometer (Shimadzu) with electrospray ionization in positive ion mode. The sign
of the optical specific rotations for all the compounds was determined with a Jasco P-2000 Polarimeter
(JASCO Co., Ltd., Mary’s Court, PA, USA) in a quartz cell (1.0 cm), and the value is an average of
ten measures.
3
.2. General Procedure for the Synthesis of Alkyl Esters (7a–d)
Compounds 7a were prepared as described in the literature [15] with some modifications:
trimethylsilane chloride (2 mmol) was added to a solution of L-tryptophan (1 mmol) in the respective
–
d
2
alcohol (5.0 mL). The reaction mixture was stirred at room temperature for 24 h, and the solvent was
slowly evaporated at room temperature over a period of about 1 week, and the solid residue was
recrystallized from ethanol. The crude reaction product in its hydrochloride form was treated with
a 10% aqueous solution of sodium bicarbonate and extracted with ethyl acetate (5
compounds 7a–d.
×
10 mL) to yield
+
Methyl (2S)-2-amino-3-(1H-indol-3-yl)propanoate (7a): ESI-MS in positive mode m/z: [M
−
−
−
−
H] : 219.00;
25
[
α] = + 5.80 ± 0.200 (c 0.1, H O).
2
D
+
Ethyl (2S)-2-amino-3-(1H-indol-3-yl)propanoate (7b): ESI-MS in positive mode m/z: [M
H] : 233.05;
2
5
[
α] = −7.73 ± 0.306 (c 0.1, H O).
2
D
+
2
-Propyl-(2S)-2-amino-3-(1H-indol-3-yl)propanoate (7c): ESI-MS in positive mode m/z: [M
H] : 247.00;
25
[
α] = −21.5 ± 0.306 (c 0.1, H O).
2
D
+
n-Butyl-(2S)-2-amino-3-(1H-indol-3-yl)propanoate (7d): ESI-MS in positive mode m/z: [M
H] : 261.00;
2
5
[
α] = −15.1 ± 0.231 (c 0.1, H O).
2
D

Molecules 2016, 21, 1349
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0
3
.3. General Procedure for the Synthesis of N,N -Dialkylthioureas (4a–b)
A 10% aqueous solution of hydrochloric acid (1.0 mL) and carbon disulfide (2 mmol) was added
to a solution of the respective ester (7a , 1.0 mmol) in acetonitrile. The reaction mixture was stirred at
room temperature for 24 h, the reaction was then neutralized with 10% aqueous solution of sodium
bicarbonate, and the product was extracted with CHCl (4 5 mL). The extract was concentrated
–
d
×
3
under reduced pressure and the residue was purified via column chromatography on Silica gel and
eluted with a hexane–ethyl acetate (7:3) mixture, to afford compounds 4a–b.
0
0
0
Dimethyl (2S,2 S)-2,2 -(carbonothioyldiazanediyl)-3,3 -bis(1H-indol-3-yl)dipropanoate (4a): Yellow oil.
+
ESI-MS in positive mode m/z: [M − H] : 479.05.
Diethyl (2S,2 S)-2,2 -(carbonothioyldiazanediyl)-3,3 -bis(1H-indol-3-yl)dipropanoate (4b): Yellow oil. ESI-MS
in positive mode m/z: [M − H] : 507.05.
0
0
0
+
3
.4. Synthesis of 4-[(1H-Indol-3-yl)methylene]-2-sulfanylidene-1,3-thiazolidin-5-one (5)
A 10% aqueous solution of potassium hydroxide (1.0 mL) and carbon disulfide (2 mmol) was
added to a solution of the respective ester (7a–d, 1.0 mmol) in methanol. The reaction mixture
was stirred at room temperature for 24 h. The product was extracted with ethyl acetate (4 × 5 mL).
The extract was concentrated under reduced pressure and the residue was purified via column
chromatography on Silica gel and eluted with a hexane-ethyl acetate (7:3) mixture to afford compound 5:
+
25
ESI-MS in positive mode m/z: [M − H] : 261.15; [α]D = −2.60 ± 0.200 (c 0.1, MeOH).
3
.5. General Procedure for the Synthesis of Alkyl (2S)-3-(1H-indol-3-yl)-2-{[(alkylsulfanyl)carbonothioyl]amino}
propanoate (6a–f)
Method A: Triethylamine (2 mmol), carbon disulfide (1 mmol) and the respective Michael acceptor
were added to a solution of the respective ester (7a , 1 mmol) in THF. The reaction mixture was
8
a
–
d
–b
stirred at room temperature for 24 h. It was then concentrated under reduced pressure and the residue
was purified via column chromatography on Silica gel and eluted with a hexane-ethyl acetate (7:3)
mixture to yield compounds 6a–f.
Method B via tandem dithiocarbamate formation/Michael addition reaction: The respective
alcohol (R = Me, Et; 1 mmol), trimethylsilane chloride and triethylamine (2 mmol) were added to
a solution of L-tryptophan
respective Michael acceptor 8a
2
(1 mmol) in THF. Then, a solution of carbon disulfide (1 mmol) and the
in THF was added dropwise. The reaction mixture was stirred at
–
d
room temperature for 24 h and it was then concentrated under reduced pressure and the residue was
purified via column chromatography on Silica gel and eluted with a hexane–ethyl acetate (7:3) mixture
to yield compounds 6a–f.
Methyl 2-({[(2-cyanoethyl)sulfanyl]carbonothioyl}amino)-3-(1H-indol-3-yl)propanoate (6a): 1H-NMR
(
400.1 MHz, CDCl ): 8.20 (s, 1H), 7.26–7.13 (m, 4H), 6.84 (s, 1H), 5.56–5.74 (m, 1H), 3.80 (s, 3H),
3
3
.47–3.66 (m, 1H), 3.09–3.20 (m, 1H), 2.9–83.05 (m, 2H), 2.87–2.98 (m, 2H). ESI-MS in positive mode
+
25
m/z: [M − H] : 347.80. [α]D = +7.00 ± 0.200 (c 0.1, MeOH).
Ethyl 2-({[(2-cyanoethyl)sulfanyl]carbonothioyl}amino)-3-(1H-indol-3-yl)propanoate (6b):
1H-NMR
(
400.1 MHz, CDCl ): 8.22 (s, 1H), 7.26–7.13 (m, 4H), 6.84 (s, 1H), 5.54–5.75 (m, 1H), 4.20 (q, 2H),
3
3
.47–3.66 (m, 1H), 3.10–3.20 (m, 1H), 2.98–3.05 (m, 2H), 2.89–3.00 (m, 2H), 1.40 (t, 3H). ESI-MS in
+
25
positive mode m/z: [M − H] : 362.15. [α] = +4.33 ± 0.306 (c 0.1, MeOH).
D
Methyl 3-(1H-indol-3-yl)-2-({[(3-methoxy-3-oxopropyl)sulfanyl]carbonothioyl}amino)propanoate (6c): 1H-NMR
(
(
400.1 MHz, CDCl ): 8.15 (s, 1H), 7.55–7.10 (m, 4H), 6.97 (s, 1H), 5.49–5.56 (m, 1H), 3.60 (s, 3H), 3.58
3
s, 3H), 3.51–3.55 (m, 1H), 3.46–3.50 (m, 2H), 3.38–3.44 (m, 1H), 2.65–2.87 (m, 2H). ESI-MS in positive
+
25
mode m/z: [M − H] : 380.80. [α]D = −2.80 ± 0.917 (c 0.1, MeOH).

Molecules 2016, 21, 1349
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Ethyl 3-(1H-indol-3-yl)-2-({[(3-methoxy-3-oxopropyl)sulfanyl]carbonothioyl}amino)propanoate (6d): 1H-NMR
400.1 MHz, CDCl ): 8.18 (s, 1H), 7.52–7.09 (m, 4H), 6.97 (s, 1H), 5.47–5.54 (m, 1H), 4.10 (q, 2H), 3.70 (s,
(
3
3
H), 3.50–3.54 (m, 1H), 3.38–3.45 (m, 2H), 3.21–3.34 (m, 1H), 2.63–2.85 (m, 2H), 1.11 (t, 3H). ESI-MS in
+
25
positive mode m/z: [M − H] : 395.05. [α] = +13.4 ± 1.06 (c 0.1, MeOH).
D
Methyl 3-(1H-indol-3-yl)-2-({[(2-methyl-4-oxopentan-3-yl)sulfanyl]carbonothioyl}amino)propanoate (6e):
1
H-NMR (400.1 MHz, CDCl ): 8.19 (s, 1H), 7.60–7.15 (m, 4H), 7.13 (s, 1H), 4.4–4.60 (m, 1H), 3.75
3
(
s, 3H), 3.36–3.49 (m, 1H), 3.34–3.40 (m, 1H), 2.17 (s, 2H), 1.63 (s, 2H), 1.25 (s, 3H), 1.11 (s, 3H).
+
25
ESI-MS in positive mode m/z: [M − H] : 393.35. [α]D = +22.9 ± 0.611 (c 0.1, MeOH).
Ethyl 3-(1H-indol-3-yl)-2-({[(2-methyl-4-oxopentan-3-yl)sulfanyl]carbonothioyl}amino)propanoate (6f): 1H-NMR
(
400.1 MHz, CDCl ): 8.21 (s, 1H), 7.60–7.20 (m, 4H), 7.15 (s, 1H), 4.48–4.56 (m, 1H), 4.21 (q, 2H), 3.50–3.54
3
(
m, 1H), 3.34–3.40 (m, 1H), 2.15 (s, 2H), 2.01 (s, 3H), 1.37 (s, 3H), 1.28 (t, 3H), 1.11 (s, 3H). ESI-MS in
+
25
positive mode m/z: [M − H] : 407.15. [α] = −87.5 ± 0.643 (c 0.1, MeOH).
D
3
.6. Antifungal Assay
Compounds 7d were in vitro evaluated for their antifungal activity against F. oxysporum using
an amended-medium protocol to assess the mycelial growth inhibition. Potato dextrose agar (PDA)
medium was then prepared in flasks and sterilized. Compounds 7d were vigorously mixed with
PDA in order to get a final concentration of the test compounds in the medium in a 10–300 g/mL
4–
4–
µ
range. Amended-medium was then poured into sterilized 24-well plates. An isolate of F. oxysporum
◦
(
2
G1) was incubated in PDA at (20
±
1) C for 8 days to get new mycelium for the antifungal assays.
-mm-diameter mycelial plugs were thus inoculated in the center of each PDA-containing well.
The inoculated 24-well plates were incubated at (20
◦
±
1) C for 5 days. PDA (neat) was used
as control. Three replicates were performed for each treatment. The radial mycelial growth of
the fungal colonies was measured in comparison to that of the control, using the ImageJ software.
The mycelial growth inhibition of the test compounds was calculated via the formula: inhibition
growth (%) = (C − T) × (100/C), where C represents the diameter of fungal growth on untreated PDA,
and T represents the diameter of fungal growth on treated PDA. Inhibition growth (%) data were
then used to build the corresponding dose-response curves in order to calculate the half-maximal
inhibitory concentrations (IC50 expressed in mM) for each compound, using a non-linear regression in
the program GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA) for Windows.
4
. Conclusions
In summary, we have synthesized novel indole analogs derived from L-tryptophan, identifying
the main reaction conditions for the different mechanistic routes towards formation of or 6.
4,
5,
In addition, an in vitro antifungal testing against Fusarium oxysporum was performed for all
compounds, demonstrating the promissory behavior of the synthesized compounds as templates for
the development of a novel kind of antifungal agents.
Acknowledgments: Product derived from the project INV-CIAS-1787 funded by Vicerrectoría de Investigaciones
at UMNG-Validity 2015.
Author Contributions: D.Q., J.S.-B., and E.C.-B. designed research and revised the manuscript; L.D.B. and N.V.
performed the synthesis and characterization of the products; E.C.-B. coordinated the HPLC-ESI-MS analyses.
D.Q. performed DTF/B3LYP calculations, L.D.B. and N.V. performed the in vitro antifungal testing. All authors
analyzed the data, and read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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