Synthesis of novel glycerol-derived 1,2,3-triazoles and evaluation of their fungicide, phytotoxic and cytotoxic activities

Article abstract of DOI:10.3390/molecules22101666

The synthesis of a series of 1,2,3-triazoles using glycerol as starting material is described. The key step in the preparation of these triazolic derivatives is the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), also known as click reaction, between 4-(azidomethyl)-2,2-dimethyl-1,3- dioxolane (3) and different terminal alkynes. The eight prepared derivatives were evaluated with regard to their fungicide, phytotoxic and cytotoxic activities. The fungicidal activity was assessed in vitro against Colletotrichum gloeosporioides, the causative agent of papaya anthracnose. It was found that the compounds 1-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)- cyclo-hexanol (4g) and 2-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)propan-2-ol (4h) demonstrated high efficiency in controlling C. gloeosporioides when compared to the commercial fungicide tebuconazole. The triazoles did not present any phytotoxic effect when evaluated against Lactuca sativa. However, five derivatives were mitodepressive, inducing cell death detected by the presence of condensed nuclei and acted as aneugenic agents in the cell cycle of L. sativa. It is believed that glycerol derivatives bearing 1,2,3-triazole functionalities may represent a promising scaffold to be explored for the development of new agents to control C. gloeosporioides.

Full text of DOI:10.3390/molecules22101666

molecules
Article
Synthesis of Novel Glycerol-Derived 1,2,3-Triazoles
and Evaluation of Their Fungicide, Phytotoxic and
Cytotoxic Activities
Adilson Vidal Costa 1,*, Marcos Vinicius Lacerda de Oliveira 1, Roberta Tristão Pinto 1,
Luiza Carvalheira Moreira 1, Ediellen Mayara Corrêa Gomes 2, Thammyres de Assis Alves 3
,
,
ID
Patrícia Fontes Pinheiro 1 ID , Vagner Tebaldi de Queiroz 1, Larissa Fonseca Andrade Vieira 4
ID
Robson Ricardo Teixeira 5
and Waldir Cintra de Jesus Júnior 6
ID
1
Graduate Program in Agrochemistry, Universidade Federal do Espírito Santo, Alto Universitário, S/N,
Guararema, Alegre ES 29500-000, Brazil; marcao_lacerda@hotmail.com (M.V.L.O.);
roberta_tristao@hotmail.com (R.T.P.); luiza.carvalheira@yahoo.com.br (L.C.M.);
patriciafontespinheiro@yahoo.com.br (P.F.P.); vagnertq@gmail.com (V.T.Q.)
2
3
4
5
Graduate Program in Plant Production, Universidade Federal do Espírito Santo, Alto Universitário, S/N,
Guararema, Alegre ES 29500-000, Brazil; ediellengomes@hotmail.com
Graduate Program in Biotechnology, Universidade Federal do Espírito Santo, Alto Universitário, S/N,
Guararema, Alegre ES 29500-000, Brazil; thammyresalves@gmail.com
Department of Biology, Universidade Federal de Lavras, Lavras MG 37200-000, Brazil;
lfandrade.vieira@gmail.com
Departament of Chemistry, Universidade Federal de Viçosa, Av. P.H. Rolfs, S/N, Viçosa MG 36570-900,
Brazil; robsonr.teixeira@ufv.br
Universidade Federal de São Carlos, Campus Lagoa do Sino, Buri SP 18290-000, Brazil; wcintra@yahoo.com
Correspondence: avcosta@hotmail.com; Tel.: +55-28-3552-8659
6
*
Received: 1 September 2017; Accepted: 3 October 2017; Published: 7 October 2017
Abstract: The synthesis of a series of 1,2,3-triazoles using glycerol as starting material is described.
The key step in the preparation of these triazolic derivatives is the copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC), also known as click reaction, between 4-(azidomethyl)-2,2-dimethyl-1,3-
dioxolane (3) and different terminal alkynes. The eight prepared derivatives were evaluated
with regard to their fungicide, phytotoxic and cytotoxic activities. The fungicidal activity was
assessed in vitro against Colletotrichum gloeosporioides, the causative agent of papaya anthracnose.
It was found that the compounds 1-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)-
cyclo-hexanol (4g) and 2-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)propan-2-ol (4h
)
demonstrated high efficiency in controlling C. gloeosporioides when compared to the commercial
fungicide tebuconazole. The triazoles did not present any phytotoxic effect when evaluated against
Lactuca sativa. However, five derivatives were mitodepressive, inducing cell death detected by the
presence of condensed nuclei and acted as aneugenic agents in the cell cycle of L. sativa. It is believed
that glycerol derivatives bearing 1,2,3-triazole functionalities may represent a promising scaffold to
be explored for the development of new agents to control C. gloeosporioides.
Keywords: glycerol; 1,2,3-triazoles; fungicide; cytotoxic activity; click chemistry
1. Introduction
The 1,2,3-triazoles are five-membered nitrogenated aromatic heterocyclic molecules of exclusively
synthetic origin, remarkably stable and essentially inert to oxidation, reduction and hydrolysis [
1].
This class of compounds has attracted the attention of researchers because of its high effectiveness,
low toxicity and vast range of biological activities, including antibacterial [2], cytotoxicity [3], antitumor [4
],
Molecules 2017, 22, 1666; doi:10.3390/molecules22101666
www.mdpi.com/journal/molecules

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antiprotozoal [
among others.
5], antifungal [
6,
7], antimalarial [
8
], tripanossomicide [
9
], and phytotoxicity effects [10],
Among the available methodologies to prepare triazoles, the copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC), also known as click reaction, proposed by Sharpless and Meldal, stands out
as a versatile strategy affording products in high yields and being extremely useful in the preparation
of 1,4-disubstituted 1,2,3-triazoles [11].
Glycerol is a natural polyol discovered in 1779 by the Swedish scientist Carl Wilhelm Scheele [12].
Its first industrial application is related to the transformation of glycerol to nitroglycerin as reported
in 1860 by Alfred Nobel [13]. Since then, chemists started to find applications for glycerol, which it
is widely used in the pharmaceutical and cosmetic industries. Other applications include its uses as
a plasticizer in the polymer industry and as a moistening agent or solvent in the food industry [14].
This compound has also found several applications as a building block for the construction of useful
chemicals [15–17] and as a medium for chemical reactions [18,19].
Within this context, in the present investigation we describe the synthesis of a series of novel
1,2,3-triazoles using glycerol as the starting material. The key step involved in their preparation was
the CuAAC reaction between 4-(azidomethyl)-2,2-dimethyl-1,3-dioxolane (3) and different terminal
alkynes. The fungicide activity of the obtained 1,2,3-triazoles was assessed in vitro on the fungus
Colletotrichum gloeosporioides, which is an important papaya crop pest. In addition, the phytotoxicity
and cytotoxicity of the glycerol triazolic derivatives were evaluated on Lactuca sativa L. (lettuce),
which is an efficient model for the investigation of toxic effects of chemical compounds [20], and the
results are also discussed.
2. Results and Discussion
2.1. Synthesis
The new triazoles were synthesized from glycerol following the synthetic route shown in
Scheme 2. The acetal
by p-toluenesulfonic acid. The treatment of compound
the ester p-toluenesulfonate in 75% yield. The bimolecular nucleophilic substitution reaction
between ester sulfonate and sodium azide afforded compound in 93% yield. The CuAAc reaction
(click reaction) between organic azide and different commercially available terminal alkynes gave
rise to eight novel triazoles 4a 84%, Figure 1). IR and NMR
1
was obtained in 63% yield via the reaction of glycerol with acetone, catalyzed
1
with p-toluenesulfonyl chloride afforded
2
2
3
3
–
4h in synthetically useful yields (65%
−
(1H and 13C) spectroscopies as well as mass spectrometry analyses confirmed the structures of the
synthesized compounds.
Scheme 1. Cont.

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Compound
R
Yield (%)
4a
4b
4c
76
82
68
4d
4e
4f
75
70
84
65
4g
4h
73
Scheme 2. Synthesis of novel 1,2,3-triazole derivatives 4a
to produce triazole derivatives in four steps. Intermediates (
–
4h. Glycerol was utilized as starting material
), ( ) and ( ) were obtained in 63%, 75%
1
2
3
and 93% yield, respectively. Compounds 4a–4h were obtained in 68%–84% yield.
Figure 1. Examples of chromosomal and nuclear alterations observed in the meristematic cells of
Lactuca sativa (lettuce) exposed to the synthesized triazoles 4a
–
4h. (A
) Condensed nuclei, (
B) sticky
chromosomes and micronuclei (arrow), (
C
) micronuclei (arrow), ( ) c-metaphases, (E
D
) non-oriented
chromosomes in metaphase (arrow).
2.2. Biological Evaluation
The mycelial growth results (in cm) of C. gloeosporioides treated with the triazoles 4a–4h are
presented in Table 1. The triazoles presented inhibitory effects in all treatments, compared to the
control. The best results were associated with compounds 4f and 4g which, at the highest concentration,
presented 0.46 and 0.85 cm mean radial diameters, respectively.
The synthesized triazoles 4a
C. gloeosporioides (Table 2). The most effective compounds were 4d and 4g at the concentration of
1000
g mL−1, presenting 2.49 and 2.99 spores mL−1, respectively. These compounds significantly
reduced the sporulation more than 98%.
–4h also presented antisporulating activity in vitro against
µ

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Table 1. Mean values for mycelial growth (cm) of Colletotrichum gloeosporioides treated with triazoles (4a–4h).
−1
Concentrations (µg mL
)
Compounds
1
10
100
500
1000
4a
4b
4c
4d
4e
4f
5.39 e *
5.72 d
5.75 d
5.93 d
6.15 c
6.17 c
6.57 b
6.06 c
5.36 d
5.53 d
5.73 c
5.90 b
6.15 b
6.10 b
6.19 b
6.18 b
5.01 e
5.29 d
5.65 c
4.92 e
5.27 d
5.97 b
5.63 c
5.64 c
4.02 c
3.86 d
4.88 b
3.72 d
4.07 c
1.50 g
2.86 f
3.27 e
1.74 d
2.64 b
2.33 c
1.66 d
1.05 e
0.46 g
0.85 f
1.22 e
4g
4h
Tebuconazole
Control
1.83 f
7.14 a
0.30 e
7.14 a
0.00 f
7.14 a
0.00 h
7.14 a
0.00 h
7.14 a
* Means followed by the same letter in the column do not differ at 5% probability by the Scott-Knott test.
−1
Table 2. Mean values for spores mL of Colletotrichum gloeosporioides treated with Triazoles 4a–4h.
−1
Concentrations (µg mL
)
Compounds
1
10
100
500
1000
4a
4b
4c
4d
4e
4f
148.28 b *
145.15 b
128.30 c
102.16 e
114.46 d
112.30 d
86.72 f
145.33 b
125.40 c
99.06 d
85.48 e
74.59 g
79.54 f
46.18 h
74.41 i
78.78 b
71.61 c
45.69 f
66.66 d
58.13 e
49.54 f
30.28 h
41.60 g
37.40 b
37.66 b
37.04 b
13.79 e
28.10 c
19.34 d
12.26 e
10.20 e
18.61 b
17.51 b
19.83 b
2.49 d
19.00 b
4.68 c
4g
4h
2.99 d
3.45 c
83.04 f
Tebuconazole
Control
9.35 g
3.28 m
0.00 i
0.00 f
0.00 e
206.70 a
206.70 a
206.70 a
206.70 a
206.70 a
* Means followed by the same letter in the column do not differ at 5% probability by the Scott- Knott test.
The significant antisporulating performance demonstrated by the triazoles can be explained by
rapid penetration and translocation as well as toxic action on sporulation and spore germination [21].
The efficiency of 1,2,3-triazoles in reducing sporulation was demonstrated by Chen and co-workers [22].
These authors synthesized eighteen triazolic compounds and verified that they showed fungicide
activity against C. lagenarium, inhibiting the reproduction of the phytopathogen by 61% at
a concentration of 200 µ
g mL−1. In another study, Silva and colleagues [23] synthesized fifteen new
1,2,3-triazoles observed that the compounds displayed fungicide activity against Aspergillus niger and
low cytotoxicity.
The regression models of the data obtained from the fungicide activity evaluation of triazoles
4a–4h on C. gloeosporioides are listed in Table 3. The lowest values of ED50 and ED100 were obtained for
the compounds 4f and 4g in relation to mycelial growth. Compounds 4g and 4h were the most active
in terms of sporulation.
As can be seem from Table 3, the triazoles 4a–4h presented lower ED50 and ED100 values for
sporulation in relation to mycelial growth of C. gloeosporioides, meaning that such compounds have
greater inhibitory action on the reproductive structures than on the vegetative growth structures.
Reducing the sporulation of a phytopathogen is of great importance, since inhibition or death of
these structures is directly linked to the reproduction of the species, thus affecting its propagation,
reproductive cycle and resistance [24].

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Table 3. Regression equation models obtained from fungicide evaluation of triazoles (4a–4h) and tebuconazole against Colletotrichum gloeosporioides.
−1
SP
21.48
−1
)
SP
Regression Equations
ED50 (µg mL
MG
)
ED100 (µg mL
MG
Solutions
4a
MG
SP
Y = 23,742 + 0.049x **
Y = 4145 + 0.6421 logx **
529.50
715.72
682.63
529.94
502.20
394.80
496.02
519.76
0.26
1537.77
1798.08
1.72194
1396.40
1213.07
1035.83
1091.26
1248.41
35.32
1439.60
1384.10
1.42583
1048.40
1425.00
1112.17
1069.37
1098.15
13.71
R2 = 0.98
R2 = 0.91
4b
Y = 16,937 + 0.046x **
R2 = 0.92
Y = 4295 + 0.608 logx *
R2 = 0.99
14.45
4.97
0.98
2.41
10.70
0.44
0.83
<1
4c
Y = 17,159 + 0.048x **
R2 = 0.90
Y = 4642 + 0.515 logx **
R2 = 0.91
4d
Y = 19,419 + 0.057x **
R2 = 0.97
Y = 4033 + 0.494 logx *
R2 = 0.81
4e
Y = 14,677 + 0.070x **
R 2= 0.99
Y = 4823 + 0.463 logx **
R2 = 0.99
4f
Y = 19,205 + 0.078x *
R2 = 0.88
Y = 25.6 + 20.7x
−
1.2x2 **
R2 = 0.99
4g
4h
Y = 8334 + 0.084x **
R2 = 0.97
Y = 5183 + 0.517 logx **
R2 = 0.99
Y = 14,334 + 0.069x **
R2 = 0.98
Y = 5043 + 0.514 logx2 **
R2 = 0.95
Tebuconazole
Y = 5627 + 1.097 logxns
R2 = 0.55
*
Y = 6598 + 0.640 logx2 **
R2 = 0.99
MG = Mycelial growth; SP = Sporulation; ED50 and ED100 correspond to, respectively, minimum concentration necessary to inhibit 50% and 100% of mycelial growth and pathogen
sporulation. Regression coefficient followed by * or ** are significant at the 5 or 1% probability, respectively.

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2.3. Cytotoxic and Phytotoxic Effect
The investigation of the phytotoxicity and cytotoxicy effects of triazoles 4a–4h on L. sativa revealed
that the percentage of germinated seeds in all treatments was higher than 98%, including the control
treatments (distilled water and DCM) (Table 4). In addition, the germination speed index (GSI) of all
treatments was statistically similar, not differing from the applied controls. With regard to root length
(RL), variation was observed, ranging from 6.81 mm for compound 4a to 9.91 mm for compound 4c
,
at 250
µ
g mL−1 concentration, with this being the only treatment that significantly increased RL−in1
relation to the controls. On the other hand, compounds 4a and 4e at 250 µ µg mL
g mL−1 and 4b at 100
significantly reduced RL by about 10%.
Table 4. Macroscopic and microscopic parameters evaluated in Lactuca sativa seeds treated with the
triazoles 4a–4h at three concentrations and negative controls (distilled water and dichloromethane).
−1
Compounds Concentrations (µg mL
)
G%
GSI
RG
MI%
CA%
NA%
50
100
250
98.40 a *
100.00 a
98.40 a
11.13 ab
11.12 ab
11.70 ab
7.76 ab
8.08 ab
6.81 ab
6.88 c
6.70 d
6.78 d
0.88 a
0.78 a
1.00 b
0.36 c
0.40 c
0.36 c
4a
4b
4c
4d
4e
50
100
250
98.40 a
98.40 a
100.00 a
11.43 ab
10.97 ab
11.35 a
7.22 ab
7.13 ab
9.05 a
6.76 d
6.26 d
6.90 c
0.72 a
0.96 b
0.80 a
0.38 c
0.38 c
0.34 c
50
100
250
100.00 a
98.40 a
100.00 a
11.27 a
11.01 a
10.43 a
8.18 a
8.54 a
9.91 a
6.94 c
6.26 d
7.36 c
0.80 a
0.68 a
0.82 a
0.30 c
0.40 c
0.52 d
50
100
250
100.00 a
98.40 a
99.20 a
11.28 a
10.98 a
10.57 a
8.20 a
8.77 a
8.80 a
7.10 c
7.16 c
7.20 c
0.62 a
0.64 a
0.68 a
0.20 a
0.28 c
0.30 c
50
100
250
100.00 a
99.20 a
99.20 a
11.37 a
10.73 a
11.46 a
7.37 a
7.88 a
6.92 a
7.3 c
6.88 c
7.20 c
0.66 a
0.48 a
0.56 a
0.34 c
0.36 c
0.16 a
50
100
250
100.00 a
97.60 a
99.20 a
11.51 a
10.77 a
11.31 a
8.13 a
8.17 a
7.84 a
7.88 a
7.76 a
7.26 c
0.44 a
0.82 a
0.48 a
0.06 b
0.20 a
0.24 a
4f
50
100
250
100.00 a
100.00 a
96.80 a
11.05 a
11.53 a
10.35 a
7.66 a
7.87 a
7.95 a
7.9 a
8.14 b
7.96 a
0.54 a
0.50 a
0.44 a
0.20 a
0.08 b
0.06 b
4g
50
100
250
100.00 a
99.20 a
100.00 a
11.66 a
11.47 a
11.27 a
8.07 a
7.95 a
9.26 a
8.00 a
8.04 a
8.12 b
0.36 c
0.50 a
0.56 a
0.02 b
0.08 b
0.12 a
4h
water
dichloromethane
0
0
98.40 a
100.00 a
10.90 a
11.31 b
7.52 a
7.48 b
7.88 a
8.10 b
0.48 a
0.48 a
0.16 a
0.14 a
G% = Germination; GSI = Germination speed index; RG = Root growth; MI% = Mitotic index; CA% = Chromosome
aberrations; NA% = Nuclear aberrations. * Means followed by the same letter do not differ statistically by the Tukey
test (p < 0.05).
Among the macroscopic parameters evaluated in the assays to investigate the effects of chemical
substances, germination appears to be the least sensitive [25]. On the other hand, root growth (RG) is
directly affected by the germination delay, which is reflected in the GSI reduction as a consequence of
the toxic effect of the tested compound. Hence, significant decreases in RG reflect the toxicity of the
substance [26].
Evaluating the phytotoxicity of the triazolic active principles difenoconazole and tebuconazole,
found in commercial fungicides, Bernardes and co-workers [27] found that the first did not affect the
percentage of germinated seeds, similarly to what was observed in the present work. The triazoles

Molecules 2017, 22, 1666
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tested here did not affect the GSI of lettuce seeds, whereas significant reductions in GSI were observed
for difenoconazole and tebuconazole.
Root growth and initial development of the lettuce plantle−t1were significan−tl1y reduced only in
treatments with three of the triazoles evaluated here (4a, 250
µg mL
;
4b, 100
µg mL
; 4e, ,
250 µg mL−1
Table 4), whereas tebuconazole and difenoconazole, in the work of Bernardes and co-workers,
had significant effects on the reduction of GSI and RG at all evaluated concentrations. It is worth
noting, however, that the concentrations tested in the present work are one thousand times more
diluted than those utilized by Bernardes and co-workers [27], which varied from 7 to 200 g L−1
.
The herbicide potential of the triazoles 4a–4h did not become evident in the applied tests,
since they did not compromise plantlet development, but the mechanism of action of triazoles
presenting herbicide activity is related to photosynthesis. According to Rodrigues and Almeida [28],
the commercial triazolic herbicide amicarbazone acts by inhibiting photosystem II and photosynthesis,
thus preventing the fixation of CO2 and NADPH2 and affecting weed growth. Sulfentrazone is another
herbicide from the triazole class and acts via inhibition of the enzyme protoporphyrinogen oxidase.
This enzyme is important in the chlorophyll synthesis chain [29] which also impacts the inhibition of
photosynthesis and causes death of the plant due to lack of nutrients.
Nevertheless, the mechanism of action of the triazoles 4a–4h is in accordance with the
observations of Borgati and co-workers [10]. In evaluating the effect of 13 triazolic derivatives
containing benzyl-halogenated groups, the authors observed an RG reduction in Allium cepa (onion),
Cucumis sativus (cucumber) and L. sativa (lettuce), comparable to 2,4-dichlorophenoxyacetic acid (2,4-D),
a commercial herbicide. Furthermore, the phytotoxic action of a compound is directly related to its
antiproliferative capacity and aneugenic action, hindering microtubule proliferation. In the present
work, the aneugenic action was observed as discussed below.
The microscopic evaluation (Table 4) showed that, overall, the treatments with triazoles 4a to
4e reduced MI compared to the control treatments, whereas triazoles 4f, 4g and 4h presented MI
statistically similar to the control. Comparable effects were observed with regard to nuclear alterations,
which comprises condensed nuclei (Figure 1A), and micronuclei (Figure 1B), where condensed nucleus
was the most frequent. In this case, an increase in nuclear alterations was noticed in the treatments
with triazoles 4a to 4e, while NA frequencies significantly identical to the controls were registered for
the applied concentrations of triazoles 4f–4h (Table 4).
The relationship between MI reduction and an increase in condensed nuclei, observed as the most
frequent nuclear alteration in the present work, has also been reported by Palmieri and colleagues [30].
This observation is related to the fact that condensed nuclei represent cytological evidence for the
occurrence of cell death [31]. Hence, an increase in the frequency of cells with condensed nucleus
reflects the increase in the number of cells in death process, which will no longer undergo division,
thus reducing MI.
Regarding the frequency of chromosome alterations, which may lead the cell to activate its death
−1
mechanism, a variation was observed between 0.36 for the triazole 4h at the concentration of 50
and 1.0 for the triazole 4a at 250
µg L
µ
g L−1 (Table 4). Significant differences were established in the
treatments where the increase in the frequency of total chromosome alterations was higher than 50%
that observed in the controls (Table 4).
Distribution of the frequency of each detected alteration (bridges, c-metaphases, sticky chromosomes,
non-oriented chromosomes and polyploid cells) is presented in Figure 2.
The presence of sticky chromosomes (Figure 1C) was the most frequent alteration in all evaluated
treatments, followed by c-metaphases (Figures 1D and 2). Among the tested treatments, only triazole 4c
at 250 µg L−1 presented all detected alterations simultaneously (Figure 2).
The observed chromosome alterations allow drawing conclusions concerning the mechanism
of action of the tested triazoles, which is related to malfunctioning or non-polymerization of the
mitotic spindle. According to Leme and Marin-Morales [32], chromosome alterations that bring about
the loss or gain of chromosomes reflect aneugenic mechanisms of action of the tested compound.

Molecules 2017, 22, 1666
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In addition, an aneugenic mechanism of action is attributed to the presence of c-metaphases, polyploid
cells and non-oriented chromosomes in metaphase (Figure 1E) or lost in anaphase/telophase [33].
Together, these changes comprise more than 50% of all chromosome alterations observed in each
treatment (Figure 2).
Figure 2. Distribution of the frequency of bridge, c-metaphase, stick, loss and polyploid observed in
meristematic cells of Lactuca sativa using three different concentrations (50, 100 and 250
the triazoles 4a–4h.
µ
g mL−1) of
The presence of c-metaphases with polyploid cells is the most concrete evidence for the
predominance of an aneugenic mechanism of action. Chromosomes spread across the cell and
showing well-defined centromeres, which characterizes the c-metaphase (Figure 1D), arise from
the absence or stabilization of the mitotic spindle [34]. In the absence of the spindle, without the
possibility of segregation of the chromosomes, the cell cannot finalize the division process. Commonly,
with prolongation of the compound
chromatid segregation; as a consequence, in the subsequent cycle this cell will display twice the number
of chromosomes, characterizing a polyploid cell [35 36]. The presence of non-oriented chromosomes
's effect, the cell restores interphase without occurrence of
,
together with the set of chromosomes on the equatorial plaque (Figure 1E), also observed in the
present study as an effect of exposure to the triazoles, reinforces the aneugenic mechanism of
action. These delayed chromosomes may lead to chromosome loss during the segregation process in
anaphase/telophase, generating a chromosome imbalance in the daughter nuclei due to irregularities
in the spindle, giving rise to daughter cells with different shapes and nuclear sizes [37].
In addition to these aneugenic alterations, the occurrence of metaphases with sticky chromosomes
(Figure 1C) is also attributed to the mechanism of aneugenic action, since one of the factors responsible
for their formation is related to proteins of the chromosomal protein scaffold [35]. The consequence
of sticky chromosomes is abnormal chromosome segregation, which may compromise the balancing
of their number in the daughter nuclei and may lead to cell death in more severe cases when the
interchromatid bond [35] is very intense [33].
3. Materials and Methods
3.1. General Information
The terminal alkynes used in the click chemistry reactions were purchased from Sigma-Aldrich
(St. Louis, MO, USA) and used as received. Other reagents and solvents were procured from Vetec
(Rio de Janeiro, Brazil). IR spectra were obtained using a Tensor 27 spectrometer (Bruker, Karlsruhe,
Germany). The samples were analyzed by attenuated total reflectance (ATR) scanning from 4000 to
500 cm−1. Mass spectra were recorded on a GCMS-QPPlus 2010 device (Shimadzu, Kyoto, Japan) under

Molecules 2017, 22, 1666
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1
electron impact (70 eV) conditions of positive ion mode. H- and 13C-NMR spectra were recorded
on a Mercury 300 instrument (Varian, Palo Alto, CA, USA) at 300 MHz and 75 MHz, respectively,
using CDCl3 and TMS as internal standards. NMR data are presented as follows: chemical shift (δ)
in ppm, multiplicity, the number of protons, J values in Hertz (Hz). Multiplicities are shown as the
following abbreviations: s (singlet), brs (broad singlet), d (doublet), dd (double of a doublet), t (triplet),
quart (quartet), quint (quintet), sept (septet). Melting points were obtained with PFMII equipment
(Tecnopon, São Paulo, Brazil) and are not corrected. Analytical thin layer chromatography analysis was
conducted on aluminum backed precoated silica gel plates using different solvent systems. TLC plates
were visualized using potassium permanganate solution, phosphomolybdic acid solution and/or UV
light. Flash column chromatography was performed using silica gel 60 (60−230 mesh).
3.2. Synthesis of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (1)
Glycerol (100 mL, 1.36 mol), acetone (100 mL, 1.36 mol), p-toluenesulfonic acid (0.0800 g,
0.460 mmol) and copper sulfate pentahydrate (10.0 g, 62.65 mmol) were added to a round-bottom flask.
The resulting reaction mixture was stirred at room temperature for two days. After that, the mixture
was filtered, yielding a bluish viscous liquid. This liquid (20 g) was purified by silica gel column
chromatography eluted with hexane-ethyl acetate (3/1 v:v). Compound
1
was obtained in 63% yield as
: 1.27 (s, 3H, C 3),
1
a colorless liquid. IR (cm−1
,
υmax): 3385, 2937, 1372, 1213, 1156. H-NMR (CDCl3)
δ
H
1.33 (s, 3H, CH3), 2.99 (brs, 1H, O
J = 4.2 Hz), 3.66 (dd, 1H, J = 8.2 Hz, J = 6.6 Hz), 3.94 (dd, 1H, J = 8.2, J = 6.6 Hz), 4.09–4.16 (m, 1H).
13C-NMR (CDCl3) H3), 62.8, 65.6, 76.0, 109.1. MS (m/z, %): 117 ([M-15]+, 38),
: 25.0 ( H3), 26.4 (
101 (22), 72 (10), 57 (25), 43 (100), 31 (12).
H), 3.49 (dd, 1H, J = 11.5 Hz, J = 5.2 Hz), 3.58 (dd, 1H, J = 11.5 Hz,
δ
C
C
3.3. Synthesis of (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate (2)
Compound (12.52 g, 98.48 mmol) and pyridine (50 mL, 640.0 mmol) were added into
1
a round-bottom flask. The mixture was cooled in ice bath and stirred for 20 min. Next, 4-toluenesulfonyl
chloride (27.00 g, 142.2 mmol) dissolved in dry dichoromethane (10 mL) was added. The resulting
yellowish solution was magnetically stirred in ice bath for 2 h. After this time, water (10 mL) was added
to the reaction mixture and the phases were separated. The organic phase was washed several times
with HCl (1 mol L−1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced
pressure. Compound
(3/1 v:v). This procedure afforded compound
2995, 2985, 1600, 1365, 1265, 1176, 978. 1H-NMR (CDCl3)
2.45 (s, 3H, tosyl-CH3), 3.76 (dd, 1H, J = 8.8 Hz, J = 5.2 Hz), 3.93–4.04 (m, 3H), 4.23–4.31 (m, 1H), 7.34
(d, 2H, J = 8.2 Hz), 7.79 (d, 2H, J = 8.2 Hz). 13C-NMR (CDCl3)
: 21.6 ( H3-tosyl), 25.1 ( H3), 26.6
H3), 66.1, 69.4, 72.8, 110.0, 127.9, 129.8, 132.4, 145.0. MS (m/z, %): 271 ([M-15]+, 90), 173 (10), 155 (76),
2
was purified by column chromatography eluted with hexane-ethyl acetate
as a colorless liquid in 75% yield. IR (cm−1
υmax):
: 1.30 (s, 3H, CH3), 1.33 (s, 3H, CH3),
2
,
δ
δ
C
C
(C
101 (86), 91 (89), 65 (24), 59 (10), 43 (100), 31(4).
3.4. Synthesis of 4-(azidomethyl)-2,2-dimethyl-1,3-dioxolane (3)
A round-bottom flask was charged with compound
2 (0.900 g, 3.15 mmol), NaN3 (1.00 g,
15.73 mmol) and DMF (3.00 mL). The resulting reaction mixture was stirred and refluxed for 8 h.
After that, the mixture was filtered yielding a yellowish liquid. The solution was extracted with
ethyl acetate (3
reduced pressure. Compound
any further purification procedure. IR (cm−1
×
30 mL). The organic phase was dried over Na2SO4, filtered and concentrated under
3 was obtained as a yellow liquid in 93% yield and not submitted to
,
υmax): 3497, 2932, 2102, 1736, 1662, 1439, 1386, 1244,
1
1091, 1046, 659. H-NMR (CDCl3)
δ
: 1.32 (s, 3H, CH3), 1.42 (s, 3H, CH3), 3.24 (dd, 1H, J = 12.6 Hz,
J = 5.4 Hz), 3.35 (dd, 1H, J = 12.6 Hz, J = 4.7 Hz), 3.72 (dd, 1H, J = 8.5 Hz, J = 5.7 Hz), 4.00 (dd, 1H,
J = 8.5 Hz, J = 6.3 Hz), 4.19–4.26 (m, 1H). 13C-NMR (CDCl3)
δ: 25.4 (CH3), 26.6 (CH3), 53.0, 66.7, 74.7,
110.2. MS (m/z, %): 142 ([M-15]+, 39), 101 (61), 83 (4), 72 (10), 59 (12), 43 (100), 31 (5). Elemental analysis:
calc. for C6H11N2O2 (MW 143.16): 50.34% C, 7.74% H, 19.57% N; found 50.42% C, 7.62% H, 19.20% N.

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3.5. General Procedure for Copper(I)-Catalyzed Azide-Alkyne Cycloaddition Reactions for the Preparation of
Triazoles 4a–4h
The azide
3
(1.00 g, 6.40 mmol), the terminal alkyne (9.60 mmol), an aqueous solution of
5H2O aqueous solution
tert-butyl alcohol (6 mL of water/6 mL of tert-butyl alcohol), CuSO4
·
(0.100 mol L−1, 1.00 mL, 0.0960 mmol), and sodium ascorba◦te (0.0600g, 0.288 mmol) were added
to a round-bottom flask. The reaction mixture was stirred at 50 C for 8 h. After the end of the reaction
as verified by TLC analysis, distilled water (10.0 mL) was added and the aqueous layer was extracted
with dichloromethane (3
×
20 mL). The organic extracts were combined and the resulting organic
phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography eluted with ethyl acetate-methanol
(9:1 v/v). The described procedure afforded triazoles 4a–4h in 65%–84% yield. The structures of
compounds 4a–4h are supported by the following data:
−1
(1-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methanol (4a). Yellow liquid. IR (cm
3355, 2972, 1472, 1365, 1202, 1040, 910, 833; 1H-NMR (CDCl3)
3.44 (s, 1H, O ), 3.72 (dd, 1H, J = 8.8 Hz, J = 5.5 Hz), 4.10 (dd, 1H, J = 8.8 Hz, J = 6.0 Hz), 4.38 (dd, 1H,
J = 12.9 Hz, J = 6.0 Hz), 4.41–4.48 (m, 1H), 4.53 (dd, 1H, J = 12.9 Hz, J = 3.3 Hz), 4.75 (s, 2H), 7.68 (s, 1H).
, υmax):
δ
: 1.32 (s, 3H, CH3), 1.37 (s, 3H, CH3),
H
13C-NMR (CDCl3)
δ: 25.3 (CH3), 26.9 (CH3), 52.5, 56.3, 66.6, 74.4, 110.6, 123.6, 148.0. MS (m/z, %): 213
([M+], 1), 198 ([M-15]+, 42), 155 (49), 138 (18), 113 (18), 101 (44), 83 (10), 73 (23), 57 (23), 43 (100), 31 (12).
Elemental analysis: calc. for C9H15N3O3 (MW 213.23): 50.69% C, 7.01% H, 19.71% N; found 50.76% C,
6.92% H, 19.63% N.
−1
2-(1-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)ethanol (4b). Yellow liquid. IR (cm
, υmax)
υmax: 3384, 2928, 1648, 1554, 1457, 1373, 1217, 1151, 1116, 1051, 968, 880, 831. 1H-NMR (CDCl3)
1.30 (s, 3H, CH3), 1.35 (s, 3H, CH3), 2.91 (t, 2H, J = 6.3 Hz), 3.40 (s, 1H, O ), 3.70 (dd, 1H, J = 8.8 Hz,
J = 5.5 Hz), 3.88 (t, 2H, J = 6.3 Hz), 4.08 (dd, 1H, J = 8.8 Hz, J = 6.0 Hz), 4.35 (dd, 1H, J = 12.6 Hz,
J = 5.4 Hz), 4.39–4.45 (m, 1H), 4.48 (dd, 1H, J = 12.6 Hz, J = 3.3 Hz), 7.52 (s, 1H). 13C-NMR (CDCl3)
δ:
H
δ
:
25.4 (CH3), 26.9 (C
H3), 28.9, 52.5, 61.6, 66.6, 74.3, 110.4, 123.3, 145.4. MS (m/z, %): 228 ([M.+], 2), 227
(M+, 2), 212 ([M-15]+, 35), 197 (8), 169 (65), 152 (16), 127 (15), 110 (20), 101 (25), 68 (37), 57 (62), 43 (100),
32 (50). Elemental analysis: calc. for C10H17N3O3 (MW 227.26): 52.85% C, 7.54% H, 18.49% N; found
52.76% C, 7.43% H, 18.37% N.
3-(1-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)propan-1-ol
IR (cm−1
(CDCl3)
J = 7.4 Hz), 3.41 (s, 1H, O
J = 8.7 Hz, J = 5.7 Hz), 4.35 (dd, 1H, J = 12.6 Hz, J = 5.2 Hz), 4.38-4.45 (m, 1H), 4.48 (dd, 1H, J = 12.6 Hz,
(
4c). Yellow liquid.
1
,
υmax): 3380, 2938, 1648, 1552, 1456, 1373, 1257, 1216, 1151, 1058, 969, 880, 831. H-NMR
δ
: 1.30 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.88 (quint, 2H, J1=7.4 Hz, J2=6.3 Hz), 2.78 (t, 2H,
), 3.69 (t, 2H, J = 6.3 Hz), 3.70 (dd, 1H, J = 8.7 Hz, J = 5.7 Hz), 4.07 (dd, 1H,
H
J = 3.2 Hz), 7.44 (s, 1H). 13C- NMR (CDCl3)
δ: 22.1, 25.4 (CH3), 26.7 (CH3), 32.2, 52.4, 61.7, 66.6, 74.3,
110.4, 122.6, 147.8. MS (m/z, %): 242 ([M+], 2), 241 ([M.+], 4), 226 ([M-15]+, 31), 211 (12), 197 (9), 183 (39),
166 (11), 112 (46), 101 (28), 83 (223), 68 (25), 57 (61), 43 (100), 41 (41), 31 (18). Elemental analysis: calc.
for C11H19N3O3 (MW 241.29): 54.76% C, 7.94% H, 17.41% N; found 54.78% C, 7.93% H, 17.38% N.
−1
1-(1-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)ethanol (4d). Yellow liquid. IR (cm
, υmax):
1
3385, 2985, 1373, 1217, 1149, 1066, 892, 830. H-NMR (CDCl3)
δ
: 1.33 (s, 3H, CH3), 1.37 (s, 3H, CH3),
1.58 (d, CH3, J = 6.6 Hz), 3.50 (s, 1H, OH), 3.73 (dd, 1H, J = 8.8 Hz, J = 5.4 Hz), 4.10 (dd, 1H, J = 8.8 Hz,
J = 5.7 Hz), 4.39 (dd, 1H, J = 12.9 Hz, J = 5.5 Hz), 4.42–4.49 (m, 1H), 4.52 (dd, 1H, J = 12.9 Hz, J = 3.0 Hz),
5.07 (quart, 1H, J = 6.6 Hz), 7.61 (s, 1H). 13C-NMR (CDCl3)
δ: 23.0, 25.1 (CH3), 26.6 (CH3), 52.2, 62.9,
66.3, 74.0, 110.1, 121.4, 152.7. MS (m/z, %): 228 ([M+], 1), 227 ([M.+], 1), 212 ([M-15]+, 36), 169 (38),
152 (16), 127 (12), 101 (36), 73 (23), 57 (29), 43 (100), 31 (8). Elemental analysis: calc. for C10H17N3O3
(MW 227.26): 52.85% C, 7.54% H, 18.49% N; found 52.81% C, 7.49% H, 18.43% N.
−1
1-(1-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)propan-2-ol (4e). Yellow liquid. IR (cm
, υmax):
1
3384, 2985, 1648, 1552, 1457, 1373, 1216, 1151, 1117, 1065, 1045, 968, 941, 880, 831. H-NMR (CDCl3)
δ
:

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1.26 (d, 3H, CH3, J = 6.3 Hz), 1.33 (s, 3H, CH3), 1.37 (s, 3H, CH3), 2.72–2.92 (m, 1H), 3.46 (s, 1H, OH),
3.74 (dd, 1H, J = 8.8 Hz, J = 5.7 Hz), 4.10 (dd, 1H, J = 8.8 Hz, J = 5.8 Hz), 4.15–4.17 (m, 2H), 4.39 (dd, 1H,
J = 12.6 Hz, J = 5.7 Hz), 4.42–4.49 (m, 1H), 4.52 (dd, 1H, J = 12.6 Hz, J = 3.3 Hz), 7.52 (s, 1H). 13C- NMR
(CDCl3) δ: 23.1 (CH3), 25.4 (CH3), 26.9 (CH3), 35.0, 52.5, 66.6, 67.3, 74.3, 110.4, 123.4, 145.5. MS (m/z, %):
242 ([M+], 1), 226 ([M-15]+, 31), 197 (94), 183 (12), 166 (11), 139 (22), 115 (26), 101 (22), 83 (26), 68 (50),
57 (69), 43 (100). Elemental analysis: calc. for C11H19N3O3 (MW 241.29): 54.76% C, 7.94% H, 17.41% N;
found 54.78% C, 7.91% H, 17.43% N.
−1
2-(1-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)butan-2-ol (4f). Yellow liquid. IR (cm
, υmax):
1
3407, 2979, 1457, 1373, 1217, 1151, 1046, 993, 919, 831. H-NMR (CDCl3)
δ
: 0.84 (t, 3H, J = 7.4 Hz, CH3),
1.33 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.56 (d, 3H, J = 3.6 Hz, CH3), 1.89 (sept, 2H, J = 7.4 Hz), 3.46 (s, 1H,
), 3.71 (dd, 1H, J = 8.7 Hz, J = 5.5 Hz), 4.09 (dd, 1H, J = 8.7 Hz, J = 6.0 Hz), 4.39 (dd, 1H, J = 12.9 Hz,
J = 5.2 Hz), 4.42–4.49 (m, 1H), 4.51 (dd, 1H, J = 12.9 Hz, J = 4.4 Hz), 7.55 (s, 1H). 13C-NMR (CDCl3)
O
H
δ
:
8.5 (CH3), 25.4 (CH3), 26.7 (CH3), 28.2 (CH3), 36.1, 52.3, 66.6, 71.4, 74.4, 110.4, 121.5, 154.7. MS (m/z, %):
256 ([M+], 1), 240 ([M-15]+, 25), 226 (95), 180 (9), 125 (40), 101 (20), 84 (22), 57 (45), 43 (100), 31 (9).
Elemental analysis: calc. for C12H21N3O3 (MW 255.31): 56.45% C, 8.29% H, 16.46% N; found 56.38% C,
8.25% H, 16.42% N.
1-(1-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)cyclohexanol (4g). White solid. Melting
−1
1
point (m.p.): 85
(CDCl3) : 1.34 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.52–1.99 (m, 10H), 3.48 (s, 1H, O
J = 8.5 Hz, J = 5.4 Hz), 4.11 (dd, 1H, J = 8.5 Hz, J = 6.0 Hz), 4.38–4.55 (m, 3H), 7.58 (s, 1H). 13C-NMR
−
89 oC. IR (cm
,
υmax): 3290, 2933, 1448, 1371, 1223, 1162, 1071, 968, 898, 828. H-NMR
δ
H
), 3.73 (dd, 1H,
(CDCl3) δ: 21.9, 23.1, 25.3 (CH3), 26.6 (CH3), 38.1, 39.7, 52.1, 66.4, 69.5, 72.0, 74.0, 110.1, 121.0, 152.0.
MS (m/z, %): 281 ([M+], 33), 263 (32), 248 (34), 238 (18), 210 (19), 176 (12), 152 (19), 134 (39), 121 (18),
101 (27), 79 (35), 68 (33), 57 (65), 43 (100), 31 (12). Elemental analysis: calc. for C14H23N3O3 (MW 281.35):
59.77% C, 8.24% H, 14.94% N; found 59.72% C, 8.19% H, 14.89% N.
−1
2-(1-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)propan-2-ol (4h). Yellow liquid. IR (cm
, υmax):
1
3388, 2983, 1457, 1374, 1212, 1150, 1052, 960, 830. H-NMR (CDCl3)
δ
: 1.32 (s, 3H, CH3), 1.34 (s, 3H,
), 3.72 (dd, 1H, J = 8.8 Hz, J = 5.2 Hz),
4.09 (dd, 1H, J = 8.8 Hz, J = 5.7 Hz), 4.38 (dd, 1H, J = 12.6 Hz, J = 5.2 Hz), 4.41–4.47 (m, 1H), 4.51 (dd, 1H,
C
H3), 1.61 (s, 3H, CH3), 1.62 (s, 3H, CH3), 2.98 (s, 1H, OH
J = 12.6 Hz, J = 3.3 Hz), 7.57 (s, 1H). 13C-NMR (CDCl3)
δ: 25.1 (CH3), 26.6 (CH3), 30.2 (CH3), 30.4 (CH3),
52.1, 66.3, 68.3, 73.9, 110.1, 120.6, 155.7. MS (m/z, %): 241 ([M+], 1), 226 ([M-15]+, 49), 208 (11), 183 (24),
166 (10), 101 (35), 94 (24), 73 (21), 57 (38), 43 (100), 31 (12). Elemental analysis: calc. for C11H19N3O3
(MW 241.29): 54.76% C, 7.94% H, 17.41% N; found 54.73% C, 7.96% H, 17.39% N.
3.6. Evaluation of Fungicidal Activity
To evaluate the fungicidal effect of the triazoles 4a
gloeosporioides, aqueous solutions were prepared at 1, 10, 100, 500 and 1,000
–
4h on the fungal species Colletotrichum
g mL−1 containing
µ
3.5% (v/v) of dimethyl sulfoxide (DMSO). The fungicide tebuconazole was used as positive control
and a 3.5% (v/v) DMSO solution as negative control. The C. gloeosporioides isolate was obtained from
wounded papaya fruit tissues. Pure cultures were incubated in PDA (potato-dextrose-agar) culture
medium at 25 ◦C for 10 days.
Fungitoxic activity was determined based on the sensitivity of C. gloeosporioides mycelial growth
to the applied treatments, according to methodology described by Edgington, Khew and Barron [38
and Rampersad and Teelucksingh [39], with modifications [40].
]
For sporulation analysis, a spore suspension was prepared for each treatment by adding distilled
water (10 mL) to the Petri dishes. With the help of a Drigalsky spatula, light friction was applied
on the fungal colony to release the reproductive structures of the fungus from the culture medium.
The obtained mixture was filtered with the help of a glass funnel and gauze layer. The obtained
suspension was homogenized and the number of conidia was determined in a Neubauer chamber
(hemocytometer).

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3.7. Evaluation of Phytotoxicity and Cytotoxicity
The phytoxic and cytotoxic effects of compounds 4a–4h were evaluated in order on the plant
model Lactuca sativa L. (2n = 2x = 18) [41]. The assays were performed using three different
concentrations (50, 100 and 250
were used as negative controls.
µ
g mL−1) of each compound in dichloromethane and distilled water
Twenty-five lettuce seeds were placed in a 9 cm diameter Petri dish containing filter paper
moistened with solution from each treatment (2 mL). The experiments followed a completely
randomized design (CRD), with five repetitions per treatment. The dishes were sealed with transparent
◦
plastic film to prevent evaporation and kept moist in BOD incubator at 24
±
2 C without light
throughout the experiment period. The number of germinated seeds was evaluated from 8 to 48 h,
at 8 h intervals. The macroscopic parameters assessed were germination speed index (GSI), percentage
of germinated seeds (GR), root length after 48 h (RL) and aerial growth (AG) after 120 h, as previously
described [42].
For microscopic analysis, ten roots from each Petri dish were collected after 48 h of exposure,
fixed in an ethanol-acetic acid solution (3:1 v/v), and stored at -4 ◦C for at least 24 h. The slides were
prepared using the squash technique and stained with 2% acetic orcein. Slides were evaluated and the
parameters mitotic index (MI), chromosomal alterations (CA) and nuclear alterations (NA) frequencies
were determined.
3.8. Statistical Analyses
For the fungicidal assays, a completely randomized design (CRD) was adopted using a 9 × 5 + 1
factorial scheme, with nine treatments (triazoles 4a–4h and tebuconazole), five concentrations
(1, 50, 100, 500 and 1,000 µ
g mL−1), and one additional treatment (negative control). The data on
mycelial growth and sporulation sensitivity were subjected to analysis of variance. When the ANOVA
was significant at a 5% probability level, the quantitative factor (concentrations) were evaluated by
means of regression analysis, and the qualitative factor (triazole compounds) by the means clustering
test of Scott-Knott. The values of the percentage of fungal growth inhibition (GIP) and sporulation
inhibition (SIP) [21] were used to determine ED50 and ED100 (concentration of the fungicide active
ingredient necessary to inhibit by 50% and 100% the mycelial growth and sporulation of the pathogen)
by means of adjustments to the regression equations [39,40].
To assess phytotoxicity and cytotoxicity, the experiments were done in a CRD using a 8 × 4 + 2
factorial scheme, with eight treatments (triazoles 4a–4h), three concentrations (50, 100 and
250
µ
g mL−1), and two additional treatments (water and dichoromethane). When significant,
the obtained phytotoxicity and cytotoxicity data were subjected to analysis of variance and the means
were compared by the Tukey test at a 5% significance level.
4. Conclusions
The four-step methodology presented in this work demonstrates that chemical transformations
using glycerol as a starting material were effective for obtaining new triazoles in a quick and efficient
way. The compounds 1-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)cyclo-hexanol
(4g) and 2-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-1H-1,2,3-triazol-4-yl)propan-2-ol (4h) were
highly efficient when compared with the commercial fungicide tebuconazole in inhibiting the
sporulation of C. gloeosporioides, with ED50 values of 0.44 and 0.83 mg L−1. In addition, compounds 4g
and 4h do not exert either phytotoxicity or cytotoxicity against the plant model Lactuca sativa L.
Compounds 4g and 4h thus stand out among the other triazoles synthesized here as potential
candidates for developing novel antifungal agents against C. gloeosporioides. Glycerol-derived
1,2,3-triazoles may thus represent a novel scaffold to be exploited aiming at the development of new
active ingredients for fungus control. Work is currently in progress in our laboratories to synthesize
new derivatives with increased effectiveness.

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Acknowledgments: The authors acknowledge the Research and Innovation Support Foundation of the State
of Espírito Santo (FAPES), the National Council for Science and Technology (CNPq), the Graduate Program in
Agrochemistry, Universidade Federal do Espírito Santo, and the research group on Natural Products and Organic
Synthesis (GEAPS-UFES).
Author Contributions: A.V.C., P.F.P., R.R.T., M.V.L.O., R.T.P. and L.C.M. were responsible for the synthesis and
characterization of the novel 1,2,3-triazoles; W.C.J.J., L.F.A.V., E.M.C.G. and T.A.A. performed the biological assays
and provided the experimental procedures and results; V.T.Q. carried out the statistical analyzes and conducted
the paper writing; all authors read, commented and approved the manuscript.
Conflicts of Interest: The authors declare that there is no conflict of interests regarding the publication of
this paper.
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Sample Availability: Samples of the compounds all are available from the authors.
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