Synthesis and identification of epoxy derivatives of 5-methylhexahydroisoindole-1,3-dione and biological evaluation

Article abstract of DOI:10.3390/molecules26071923

Cyclic imides belong to a well-known class of organic compounds with various biological activities, promoting a great interest in compounds with this functional group. Due to the structural complexity of some molecules and their spectra, it is necessary to use several spectrometric methods associated with auxiliary tools, such as the theoretical calculation for the structural elucidation of complex structures. In this work, the synthesis of epoxy derivatives of 5-methylhexahydroisoindole-1,3-diones was carried out in five steps. Diels–Alder reaction of isoprene and maleic anhydride followed by reaction with m-anisidine afforded the amide (2). Esterification of amide (2) with methanol in the presence of sulfuric acid provided the ester (3) that cyclized in situ to give imides 4 and 4-ent. Epoxidation of 4 and 4-ent with meta-chloroperbenzoic acid (MCPBA) afforded 5a and 5b. The diastereomers were separated by silica gel flash column chromatography, and their structures were determined by analyses of the spectrometric methods. Their structures were confirmed by matching the calculated¹ H and¹³ C NMR chemical shifts of (5a and 5b) with the experimental data of the diastereomers using MAE, CP3, and DP4 statistical analyses. Biological assays were carried out to evaluate the potential herbicide activity of the imides. Compounds 5a and 5b inhibited root growth of the weed Bidens pilosa by more than 70% at all the concentrations evaluated.

Full text of DOI:10.3390/molecules26071923

molecules
Article
Synthesis and Identification of Epoxy Derivatives of
5
-Methylhexahydroisoindole-1,3-dione and
Biological Evaluation
Kariny B. A. Torrent and Elson S. Alvarenga *
Department of Chemistry, Universidade Federal de Viçosa, Viçosa, MG 36570-900, Brazil;
karinybragatto@gmail.com
Correspondence: elson@ufv.br
*
Abstract: Cyclic imides belong to a well-known class of organic compounds with various biological
activities, promoting a great interest in compounds with this functional group. Due to the structural
complexity of some molecules and their spectra, it is necessary to use several spectrometric methods
associated with auxiliary tools, such as the theoretical calculation for the structural elucidation of
complex structures. In this work, the synthesis of epoxy derivatives of 5-methylhexahydroisoindole-
1
,3-diones was carried out in five steps. Diels–Alder reaction of isoprene and maleic anhydride
followed by reaction with m-anisidine afforded the amide ( ). Esterification of amide ( ) with
methanol in the presence of sulfuric acid provided the ester ( ) that cyclized in situ to give imides
and 4-ent. Epoxidation of and 4-ent with meta-chloroperbenzoic acid (MCPBA) afforded 5a and 5b
2
2
3
4
.
4
The diastereomers were separated by silica gel flash column chromatography, and their structures
were determined by analyses of the spectrometric methods. Their structures were confirmed by
matching the calculated H and 13C NMR chemical shifts of (5a and 5b) with the experimental data
of the diastereomers using MAE, CP3, and DP4 statistical analyses. Biological assays were carried
out to evaluate the potential herbicide activity of the imides. Compounds 5a and 5b inhibited root
growth of the weed Bidens pilosa by more than 70% at all the concentrations evaluated.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
1
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Torrent, K.B.A.; Alvarenga,
E.S. Synthesis and Identification of
Epoxy Derivatives of 5-
Methylhexahydroisoindole-1,3-dione
and Biological Evaluation. Molecules
Keywords: cyclic imide; theoretical calculations; DFT; CP3; DP4; herbicide; phytotoxic
2
021, 26, 1923. https://doi.org/
1
0.3390/molecules26071923
Academic Editor: Olav Schiemann
1. Introduction
In 2050, it is estimated that the global population will reach the mark of 9.1 billion
Received: 7 March 2021
Accepted: 25 March 2021
Published: 30 March 2021
people. As a result, world food production will need to increase by 70–100%. Weeds are
agricultural pests and cause the highest percentage of loss of income from food production.
They compete with the plantations for sunlight, water, and nutrients, harbor insects and
pathogens (fungi, bacteria, and viruses), promoting losses in production. In addition,
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
weeds destroy native habitats, threatening plants and animals in the local ecosystem [1,2].
Cyclic imides belong to an important class of organic compounds, being particularly
important in synthetic, pharmacological, and industrial chemistry. Imides are compounds
with high biological potential because they are electrically neutral and hydrophobic, being
able to cross biological membranes [3–5]. Among its biological activities, herbicidal activity
stands out and some commercial herbicides are presented in Figure 1 [6].
Spectroscopic and spectrometric techniques are tools used in the structural elucidation
of new substances, and nuclear magnetic resonance (NMR) spectroscopy is frequently
used. However, in some situations, the data obtained by these techniques are not sufficient
for structural elucidation of organic compounds with complex structures, or even due
to overlapping signals in the H NMR spectra, requiring the use of other tools, such as
computational chemistry [7–9].
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
1
4
.0/).
Molecules 2021, 26, 1923. https://doi.org/10.3390/molecules26071923
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1923
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Figure 1. Commercial herbicides containing cyclic imides in their structures (
a) chlorphthalim,
(
b) pentoxazone, (c) flumioxazin, (d) flumiclorac-pentyl, and (e) cinidon-ethyl.
Structural elucidation of molecules using computational chemistry, chemical shifts,
and spin–spin couplings is calculated using the functional density theory (DFT) and
considering solvation effects and relativistic effects. Calculated data are compared to
those obtained experimentally through statistical methods to establish the structure of the
molecule [10–12].
Statistical methods commonly used in the structure determination of organic com-
pounds are mean absolute error (MAE), CP3, and DP4 probabilities. DP4 tool is applied
when there are experimental data for one compound, which is compared to two or more
candidate structures, whereas CP3 is used when NMR chemical shifts are available for
two or more substances. The calculation of DP4 can be done at http://www.jmg.ch.cam.
ac.uk/tools/nmr/DP4 (accessed on 28 January 2021) for assigning one set of experimen-
tal data to one of many candidate structures. The calculation of CP3 can be done at
http://www-jmg.ch.cam.ac.uk/tools/nmr/CP3.html (accessed on 28 January 2021) for
assigning two or more set of experimental data to one of many potential structures. The
probability that the assignment combination is correct (5aexp
= 5acalc, 5bexp = 5bcalc) is
calculated by the Bayes’ theorem (Equation S1) [13–16].
Therefore, we decided to use the known Diels–Alder reaction to prepare the tetrahy-
drophthalimide ( ) in three steps, which was converted into the epoxy compounds (5a
4
and 5b). From this reaction, we obtained two novel pairs of diastereomers whose relative
configurations were determined by comparison of the experimental with the computed
NMR chemical shifts.
In this context, we prepared two novel epoxy compounds derived from 5-methylhexa
hydroisoindole-1,3-dione from the Diels–Alder reaction between maleic anhydride and
isoprene. The diastereoisomers had their structures elucidated by NMR, and their relative
configurations were confirmed by comparing the experimental with the calculated NMR
chemical shifts. The herbicidal activities of these compounds were evaluated against seeds
of Bidens pilosa L., Cucumis sativus L., Lactuca sativa L., and Sorghum bicolor L.

Molecules 2021, 26, 1923
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2. Results and Discussions
2.1. Synthesis
Diels–Alder reaction of isoprene and maleic anhydride followed by nucleophilic
addition of m-anisidine afforded the cis-carbamoyl-carboxylic acid ( ). The cis geometry of
compound (2) was directly influenced by the stereoselectivity of the Diels–Alder reaction.
The cis-carbamoyl-carboxylic acid ( ) was identified as a mixture of constitutional isomers
by spectrometric methods. Treating compound ( ) with sulfuric acid in methanol afforded
methyl cis-carbamoyl carboxylate ( ), which was identified in the presence of compound
). The relative quantities of compounds and in the mixture were determined by gas
chromatography with flame ionization detection (GC-FID, Figure S1 in the Supporting
Information (SI)). Sample peak areas were integrated to furnish 63% of imide ( ) and
7% of methyl cis-carbamoyl carboxylate ( ). Compounds and were not separated,
before reaction with meta-chloroperbenzoic acid (MCPBA) to afford (5a 5a-ent) and (5b
b-ent). Hereafter, we refer to the enantiomeric mixtures (5a 5a-ent) and (5b 5b-ent
2
2
2
3
(
4
3
4
4
3
3
3
4
+
+
5
+
+
)
as (5a) and (5b), respectively. Compounds (5a) and (5b) were obtained in 85% yield after
silica-gel column chromatography (Scheme 1).
Scheme 1. Synthesis of epoxy hexahydroisoindole-1,3-diones.
2.1.1. Structure Identification of Compound (5b)
Compound (5b) was isolated as the major product in 66% yield by silica-gel column
1
chromatography. The singlets (s) at
δ
= 1.32 and 3.80 ppm observed in the H NMR were
assigned to the methyl and methoxy groups, respectively. The triplet (t) at
with J = 2 Hz was assigned to H2 due to coupling with H4 and H6 . The doublet of
δ
= 6.84 ppm
0
0
0

Molecules 2021, 26, 1923
4 of 15
doublets of doublets (ddd) at
δ
= 6.84 and 6.92 ppm (J = 8.0, 2.0, and 1.0 Hz) were assigned
0
0
to H6 and H4 , respectively. The most deshielded signal, at
δ
= 7.36 ppm (t, J = 8.0 Hz), was
0
assigned to H5 (Figure 2). The doublet at
δ
= 3.04 ppm (J = 4.0 Hz) was assigned to H6a due
◦
to coupling with H6x (dihedral angle of 42 ). The absence of coupling of H6a with H6y can
◦
be explained by their dihedral angle of 75 . According to the Karplus equation, a dihedral
◦
angle of 75 should provide coupling constants of 0–1 Hz. The chemically equivalent
and magnetically different hydrogens H2a and H5a were assigned to the non-first order
multiplet at
was coupled to the geminal H6x (J = 15.0 Hz) and the vicinal H5a (J = 7.0 Hz). The signal
at = 2.18 ppm (dd) was assigned to H2y, which was coupled to H2x (J = 15.0 Hz) and
δ = 2.89 ppm. The signal at δ = 2.22 ppm (dd) was assigned to H6y, which
δ
H2a (J = 7.0 Hz). COSY (Correlation Spectroscopy) contour map (Figure S7) was of great
value to assist in these assignments. The nuclear Overhauser effect (NOE) of H2x/H6x
observed in the NOESY (Nuclear Overhauser Effect Spectroscopy, Figure S8) was used
to confirm the distance separating these cross-relaxing hydrogens and, by extension, the
relative stereochemistry of H2y/H6y.
2
x
H
2y
H
H
O
1
6
2
'
'
5'
2
6
3
5
2
a
1
a
1
'
O
4 N
4'
6
a
5a
3'
H
6y
O
O
H
x
H
6
5
a
7
'
Figure 2. 13C (100 MHz; δTMS = 0 ppm) and H (400 MHz; δ
1
= 0 ppm) NMR spectra of compounds 5a and 5b.
TMS
1
After the assignment of the hydrogens by interpretation of the H NMR, COSY, and
NOESY spectra, the corresponding carbons were assigned using HMQC (Heteronuclear

Molecules 2021, 26, 1923
5 of 15
Multiple Quantum Coherence, Figure S9) and HMBC (Heteronuclear Multiple Bond Coher-
ence, Figure S10) cross-correlations. The experimental NMR chemical shifts and a complete
assignment of the signals for compound 5b are shown in Table 1.
Table 1. Experimental H and 13C NMR chemical shifts, COSY, and NOESY correlations for com-
1
pound 5b *.
Assign.
δ
(ppm)
Assign.
δ
(ppm) m, J (Hz)
H
COSY
NOESY
C
C1
C1A
C2
-
C2a
C5a
C6
-
21.9
56.7
28.0
-
35.3
36.7
23.6
-
H1
-
1.32, s
-
-
-
-
-
H2x
H2y
H2a
H5a
H6x
H6y
H6a
2.59, d, J = 15.0 Hz
2.18, dd, J = 15.0 and 7.0 Hz
2.89, nfom
H2y
H2a, H2x
H2y
H2y
H2x, H2a
H2y
H6y
H6y
H6x, H5a
H6x
2.89, nfom
H6y
2.77, dd, J = 15.0 and 4.0 Hz,
2.22, dd, J = 15.0 and 7.0 Hz
3.04, d, J = 4.0 Hz
H6y, H6a
H6x, H5a
H6x
H2
H2 , H5 ,
H6
H4 , H6
H2 , H4 ,
C6a
57.6
0
0
0
C2
112.4
114.6
129.8
119.0
H2
6.84, t, J = 2.0 Hz
-
0
0
6
.92, ddd, J = 8.0, 2.0, and
.0 Hz
0
0
C4
H4
-
-
-
0
1
0
0
0
0
0
0
C5
H5
7.36, t, J = 8.0 Hz
.89, ddd, J = 8.0, 2.0, and
6
0
0
C6
H6
0
1.0 Hz
H5
0
0
C7
C1
C3
55.4
133.8
160.1
179.6
179.7
H7
3.80, s
-
-
-
-
-
-
-
-
-
-
0
-
-
-
-
-
-
-
-
0
C3
C5
*
abbreviations: s = singlet; d = doublet; dd = doublet of doublets; t = triplet; ddd = doublet of doublet of doublets;
J = coupling constant; m = multiplicity; Assign. = assignment. COSY= Correlation Spectroscopy; NOESY= Nuclear
Overhauser Effect Spectroscopy.
2.1.2. Structure Identification of Compound (5a)
Compound (5a) was isolated as the minor product in 19% yield by silica-gel column
1
chromatography. The singlets (s) at
δ
= 1.40 and 3.80 ppm observed in the H NMR were
0
assigned to the methyl and methoxy groups, respectively. The aromatic hydrogens H2 ,
0
0
0
H4 , H5 , and H6 were located at the deshielded region
assignments were done according to their chemical shifts and couplings. The signals of
H2a, H5a, and H6a were superimposed as a multiplet at = 2.98–3.20 ppm (Figure 2). The
δ = 6.79–7.36 ppm, and their
δ
cross-correlation with C2a, C5a, and C6a, observed in the HMQC (Figure S18), confirmed
1
the assignments of H2a, H5a, and H6a to the multiplet at
NMR. The doublet of doublets at
δ
= 2.98–3.20 ppm in the H
δ
= 2.08 and 2.68 ppm were assigned to H6y and H6x,
respectively. The multiplicity of H6y was due to geminal coupling with H6x (J = 15.0 Hz)
and vicinal coupling to H5a (J = 10.0 Hz). The hydrogens H2y and H2x were assigned to the
doublet of doublets at
δ = 2.01 and 2.50 ppm, respectively. The relative stereochemistry of
5a
was confirmed by the cross-correlations of H2a/H2x, H5a/H6x, and H6y/H6a observed
in the NOESY (Figure S17).
Table 2 presents nuclei dihedral angles and coupling constants predicted by Karplus
equation [17] for compounds 5a and 5b. The dihedral angles were visualized in GaussView
6.0 [18] for the most stable conformer after structure optimization using Gaussian 16 [18].
NOESY and coupling constants data were used to define the relative stereochemistries of
compounds 5a and 5b.

Molecules 2021, 26, 1923
6 of 15
Table 2. Dihedral angles for compounds 5a and 5b and corresponding coupling constants according
to Karplus equation [17].
5
a
5b
Nuclei
Dihedral Angle (φ)
J (Hz) *
Dihedral Angle (φ)
J (Hz) *
2y-2a
2x-2a
6y-5a
6x-5a
6y-6a
6x-6a
160
42
160
41
75
42
13
7
13
7
0.7
7
44
70
44
73
75
44
6.5
1
6
0.9
0.7
6
*
J = coupling constant.
The carbon atoms of compound 5a were fully assigned using DEPT (Distortion-
less Enhancement by Polarization transfer, Figure S15), HMQC (Figure S18), and HMBC
1
13
(
Figure S19) spectra. H and C NMR spectra of compounds 5a and 5b are presented in
Figure 2.
Table 3 summarizes the experimental NMR data for compound 5a, coupling constants,
and cross-correlations observed in the COSY and NOESY spectra.
Table 3. Experimental H and 13C NMR chemical shifts, COSY, and NOESY correlations for com-
1
pound 5a *.
Assign.
δ
(ppm)
Assign.
δ
(ppm) m, J (Hz)
H
COSY
NOESY
C
H2y, H2x,
H6a
C1
21.8
H1
1.40, s
-
-
C1A
C2
-
C2a
C5a
55.0
28.8
-
35.3
36.9
-
-
-
H2x
H2y
H2a
H5a
2.50, dd, J = 15.0 and 8.0 Hz
2.01, dd, J = 15.0 and 10.0 Hz
2.98–3.20, m
2.98–3.20, m
.68, ddd, J = 15.0, 8.0, and
H2y, H2a
H2x, H2a
H2x, H2y
H6x, H6y
H6y, H5a,
H6a
H2y, H6x
H2x
H2x
H6x
H5a, H2x,
H6y
2
C6
23.8
H6x
4
.0 Hz
-
-
H6y
H6a
H2
2.08, dd, J = 15.0 and 10.0 Hz
H6x, H5a
H6x, H6y
H4 , H6
H6x, H6a
H1, H6y
H5 , H7
C6a
C2
56.1
2.98–3.20, m
0
0
0
0
0
0
0
0
0
0
0
112.2
114.5
129.9
118.6
6.79, t, J = 2.0 Hz
6
6
.93, ddd, J = 8.0, 2.0, and
.0 Hz
0
0
0
0
C4
H4
H2 , H5
H5 , H7
1
0
0
0
0
C5
H5
7.36, t, J = 8.0 Hz
.84, ddd, J = 8.0, 2.0, and
H4 , H6
H4 , H6
0
0
0
0
C6
H6
H2 , H5
H5
1.0 Hz
0
0
0
0
C7
C1
C3
C3
C5
55.4
132.7
160.1
178.5
178.6
H7
3.80, s
-
-
-
-
-
H2 , H4
0
-
-
-
-
-
-
-
-
-
-
-
-
0
*
abbreviations: s = singlet; dd = doublet of doublets; t = triplet; ddd = doublet of doublet of doublets; J = coupling
constant; m = multiplicity; Assign. = assignment.
2
.2. Computational Analysis
All H and 13C NMR signals were assigned for candidate structures 5a and 5b by
1
interpretation of the NMR spectra. The diastereomers were further analyzed by comparing
the calculated chemical shifts for each candidate structure with their experimental data.
The geometries of the conformers, identified by an initial molecular mechanics con-
formational search [19], for each of 5a and 5b were optimized at the M06-2X/6-31+G(d,p)
level of theory and then subjected to NMR chemical shift calculation using the B3LYP
(Becke, 3-parameter, Lee–Yang–Parr) functional and 6-311+G(2d,p) basis set. The shifts for
each diastereomer were Boltzmann averaged according to the M06-2X energies of the full

Molecules 2021, 26, 1923
7 of 15
set of conformers. The experimental H and 13C NMR chemical shifts for 5a and 5b and
1
the linearly corrected computed shifts are shown in Tables S5–S8.
The corrected mean absolute error (CMAE) and the statistical analyses CP3 and DP4
were then used to identify the better match between the experimental and computed
data sets.
Linear regressions of the H and 13C NMR chemical shifts of (5aexp
1
+
5bexp
)
vs.
(5acalc
+
5bcalc) and (5aexp
+
5bexp) vs. (5bcalc
+
5acalc) were carried out, as shown in Tables S5–S8.
The chemical shifts after linear correction were compared with the experimental data to
calculate the CMAE.
1
Incorrect matching 5bexp 5acalc for the H NMR chemical shifts presented larger
6
=
CMAE (0.10 ppm) than the correct matchings 5aexp
=
5acalc and 5bexp
=
5bcalc (0.04 ppm).
5bcalc
The correct matching for the 13C NMR chemical shifts 5aexp
=
5acalc and 5bexp
=
presented a smaller CMAE value than the incorrect matching (1.66 vs. 1.91). The correct
matchings are highlighted in green in Table 4.
Table 4. Methods used for comparing the experimental and calculated chemical shifts data a,b
.
Pairwise
Comparisons
CMAEproton d
CMAEcarbon e
CP3 all Data c
DP4 all Data f
CP3proton
CP3carbon
5
aexpt vs. 5acalc
0.04 ppm
0.10 ppm
0.04 ppm
0.10 ppm
1.66 ppm
1.91 ppm
1.66 ppm
1.91 ppm
88.1%
11.9%
88.1%
11.9%
100%
0%
100%
0%
100%
0
100%
0
100%
0%
93.9%
6.1%
5
aexpt vs. 5bcalc
5
bexpt vs. 5bcalc
5
bexpt vs. 5acalc
a
The chemical shifts used in the CP3 analyses were not corrected by linear regression. b Calculations were carried out using the B3LYP/6–
3
11+G(2d,p)//M06-2X/6–31+G(d,p) level of theory. The CP3 analyses were performed before and after the assignment of the signals.
c
The probabilities were identical in both situations (print screens of the CP3 analyses are shown in the SI). CP3 analysis was performed
with proton and carbon data. d After linear correction (ALC) of the calculated H NMR chemical shifts (Tables S7 and S8). After linear
1
e
correction (ALC) of the calculated C NMR chemical shifts (Tables S5 and S6). f DP4 analysis was performed with proton and carbon data.
1
3
The statistical approach CP3 [14
signals provided 100% probability for 5aexp
,
20] without linear correction and assignment of the
5acalc and 5bexp 5bcalc matching using
C NMR chemical shifts. The results reported by CP3 were obtained by comparing
=
=
13
differences in calculated data with differences in experimental chemical shifts for equivalent
nuclei. CP3 calculations were performed by transferring the NMR data to the URL located
at http://www-jmg.ch.cam.ac.uk/tools/nmr/CP3.html (accessed on 28 January 2021).
Structure assignment was done using chemical shifts obtained in single-point calculations
on geometries optimized at M06-2X/6-31+G(d,p) level of theory. The advantage of CP3
over CMAE is that it does not need linear regression and correction of the chemical shifts.
Another major advantage of CP3 is that it can be carried out without assignment of the
NMR signals, unlike CMAE, which needs NMR interpretation by experts for the assignment
of the signals.
DP4 [13,15,20] is applied when only one set of experimental chemical shifts is available,
unlike CP3, which applies to the situation of assigning a pair of diastereoisomers when
more than one experimental data sets are available. Therefore, DP4 complements the
probabilities obtained by CP3.
The calculated chemical shifts for candidate structures 5a and 5b were compared with
the experimental data for compound named 5a using the DP4 probability method. These
values were transferred to the web at http://www-jmg.ch.cam.ac.uk/tools/nmr/DP4/
(accessed on 28 January 2021), and the DP4 probability value was calculated automatically.
DP4 analysis without linear correction and without assignment of the signals provided
1
13
100% probability for 5aexp
=
5acalc and 93.9% matching for 5bexp
=
5bcalc using H and
C
NMR chemical shifts. The DP4 and CP3 statistical probabilities are shown in Table 4.

Molecules 2021, 26, 1923
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2.3. Biological Assay
The biological tests were carried out with seeds of three dicotyledonous plants (lettuce,
cucumber, and beggartick) and one monocotyledonous plant (sorghum).
The results of the bioassays were presented in bar graphs with their respective stan-
dard deviation values. The values of inhibition and stimulation of the substances were
evaluated according to the growth (positive values) or inhibition (negative values) of
®
the root and stem of the tested seeds. Dual (Syngenta Company, São Paulo, Brazil; S-
metolachlor, Figure 3) was used as positive control and an aqueous solution of 0.3% DMSO
(v/v) as a negative control.
O
O
Cl
(S)
N
Figure 3. Commercial herbicide metolachlor (Dual Gold®).
Assessing the development of lettuce seeds (Figure 4), all substances interfered with
the growth of the aerial and root parts of the seeds; however, the results observed for the
root were more satisfactory. In general, substance 5a presented better inhibition results
than substance 5b. At the concentration of 500
µM, substance 5a presented inhibition of
4
3% of the aerial and 52% of the root parts. At the concentration of 100
µM, substance 5a
was the most active, even compared with the positive control, with 69% roots inhibition.
Lactuca sativa
5
a-stalk
5b-stalk
Dual-stalk
5a-root
5b-root
Dual-root
2
0
0
0
1
-
-
-
-
-
-
-
-
10
20
30
40
50
60
70
80
5
00
300
150
100
50
Figure 4. Development of Lactuca sativa (Dicotyledonae) seeds in relation to the positive control Dual.
Assessing the development of the aerial and root parts of cucumber seeds (Figure 5),
all substances interfered with their development; however, none of the tested substances
presented satisfactory results compared with the commercial herbicide.

Molecules 2021, 26, 1923
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Cucumis sativus
5a-stalk
5b-stalk
Dual-stalk
5a-root
5b-root
Dual-root
30
20
10
0
-
-
-
-
-
-
-
10
20
30
40
50
60
70
5
00
300
150
100
50
Figure 5. Development of Cucumis sativus (Dicotyledonae) seeds in relation to the positive control Dual.
Evaluating the development of sorghum seeds (Figure 6), all substances interfered
with the growth of the aerial and root parts of the seeds; however, substance 5a presented
better inhibition results, in general, compared with substance 5b. At the concentration of
5
00
µM, substance 5a presented close to 50% inhibition of stem and root parts of sorghum.
At 150
µM, substance 5a presented more than 55% inhibition of the roots and stems—results
superior to that presented by the commercial herbicide at the same concentration.
Sorghum bicolor
5
a-stalk
5b-stalk
Dual-stalk
5a-root
5b-root
Dual-root
0
10
20
30
40
50
60
70
80
90
-
-
-
-
-
-
-
-
-
5
00
300
150
100
50
Figure 6. Development of Sorghum bicolor (Monocotyledonae) seeds in relation to the positive control Dual.
Assessing the development of the weed Bidens pilosa (beggartick) seeds, both epoxides
affected their development (Figure 7). Epoxide 5a presented better inhibition of the stalk
and root parts of beggartick than 5b at the concentration of 500 µM. At 500 µM, 5a and

Molecules 2021, 26, 1923
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5b
presented 86% and 81% inhibition of the root parts of beggartick, respectively. The
herbicide activity, displayed by 5a and 5b, was comparable with those presented by Dual.
Bidens pilosa
5a-stalk
5b-stalk
Dual-stalk
5a-root
5b-root
Dual-root
4
0
0
0
2
-
-
-
-
20
40
60
80
-100
5
00
300
150
100
50
Figure 7. Development of Bidens pilosa (Dicotyledonae) seeds in relation to the positive control Dual.
The optimized structures of the most stable conformers of epoxides 5a and 5b are
shown in Figure 8. Epoxide 5b has a folded structure, while 5a has a flatter structure.
Despite the diastereomeric relationship, 5a and 5b have completely different conforma-
tions. That is, 5b looks like a folded envelope, while 5a is more like an open envelope.
Considering that 5a and 5b are diastereomers, the difference in the biological activity of
these compounds may be associated with their shape in space, which differentiates them
in the biological target.
5
a
5b
Figure 8. The most stable conformers of epoxides 5a and 5b optimized at M06-2X+G(d,p).

Molecules 2021, 26, 1923
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3. Materials and Methods
3.1. General
All compounds were purified by silica-gel column chromatography and recrystal-
lization from diethyl ether. Reaction progress was followed by visualizing thin layer
chromatography (TLC) plates with the spotted reaction mixture and starting material
solution in an ultraviolet (UV) chamber [21]. The plates were then chemically stained with
vanillin spray reagent.
Melting points were obtained on an MQAPF-301 (Microquímica Equipamentos Ltda,
Palhoça, Brazil) melting point apparatus and were not corrected. Infrared (IR) spectra were
acquired using a Varian 660-IR spectrophotometer (equipped with GLADI-ATR, Agilent,
Santa Clara, CA, USA) with the attenuated total reflectance (ATR) method. NMR spectra
were performed on a Brucker Avance DRX 400 MHz equipment (Billerica, MA, USA),
using CDCl (Sigma-Aldrich, São Paulo, Brazil) as solvent. Chemical shifts were reported
3
using tetramethylsilane (TMS) signals (δ = 0.0 ppm) as reference. The mass spectra were
obtained on a Shimadzu equipment (Kyoto, Japan) GC-MS-QP5050A and GC-MS-QP2010
Ultra, after electron impact ionization (EI) at 70 eV. The NMR, IR, and mass spectra of the
synthesized compounds can be found in the Supplementary Material.
3.2. Synthesis
3.2.1. Synthesis of Amide (2)
Maleic anhydride (9.8 g, 0.1 mol) and isoprene (6.8 g, 0.1 mol) were irradiated in
a microwave reactor for 5 min. After this period, the sealed tube was cooled to room
temperature, providing a white solid that was washed with hexane to obtain anhydride
(1) in 99% yield. Then, 3.0 mmol (500.0 mg) of the anhydride (1) was transferred to a
round-bottomed flask and suspended in 3.0 mL of anhydrous dichloromethane (DCM).
The mixture was stirred until complete solubilization. m-Anisidine (3.0 mmol) was added
to the DCM solution, and the reaction mixture was stirred for 30 min. The solvent was
removed under reduced pressure, and the residue was recrystallized using a mixture of
hexane and DCM, providing amide (2) in 65% yield.
3
3
.2.2. Synthesis of 2-(3-methoxyphenyl)-5-methyl-3a,4,7,7a-tetrahydro-1H-isoindole-1,
(2H)-dione (4)
The amide (2, 0.500 mg) was dissolved in methanol (30 mL), and concentrated sulfuric
acid (2.0 mL) was added dropwise to the solution. The mixture was stirred at room
temperature for 1 h and transferred to a separation funnel. The solution was shaken
with a mixture of DCM and saturated aqueous sodium bicarbonate (3
The organic phase was dried with anhydrous magnesium sulfate, filtered, and the solvent
evaporated under reduced pressure. The residue was analyzed by GC/FID, and integration
×
30 mL, 1:1 v/v).
of the peak areas afforded a mixture containing 37% of the ester (
4). The crude reaction yield was 95%.
3) and 63% of the imide
(
3
.2.3. Synthesis of Epoxides 5a and 5b
meta-Chloroperbenzoic acid (MCPBA, 0.43 g) was added to the crude mixture of
compounds ( and , 0.500 g) dissolved in DCM (10 mL). The mixture was stirred for 1 h and
transferred to a separating funnel. The mixture was diluted with DCM (30 mL) and washed
with saturated potassium bicarbonate solution (3 25 mL). The organic phase was dried
3
4
×
with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure.
The residue was purified by silica-gel column chromatography eluting with hexane/ethyl
acetate (1:1) to afford 5a and 5b, which were further purified by recrystallization from
diethyl ether. Compounds 5a and 5b were obtained in 19% and 66% yields, respectively.

Molecules 2021, 26, 1923
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(
1aS*,2aS*,5aR*,6aR*)-4-(3-methoxyphenyl)-1a-methylhexahydro-3H-oxireno[2,3-
f]isoindole-3,5(4H)-dione (5a)
Yield 19%, white crystalline solid, TLC: Rf 0.5 (hexane/ethyl acetate 1:1), m.p. 124.7–
◦
1
1
25.7 C. H NMR (400 MHz, CDCl )
δ
: 1.40 (3H, s, H1), 2.01 (1H, dd, J = 15 e 10 Hz, H2y),
3
2
8
.08 (1H, dd, J = 15 e 10 Hz, H6y) 2.50 (1H, dd, J = 15 e 8 Hz, H2x), 2.68 (1H, ddd, J = 15,
0
e 4 Hz, H6x), 2.98–3.20 (3H, m, H2a, H5a e H6a), 3.80 (3H, s, H7 ), 6.79 (1H, t, J = 2 Hz,
0
0
0
H2 ), 6.84 (1H, ddd, J = 8, 2 e 1 Hz, H6 ), 6.93 (1H, ddd, J = 8, 2 e 1 Hz, H4 ), 7.36 (1H, t,
J = 8 Hz, H5 ). C NMR (100 MHz, CDCl )
0
13
δ
: 21.8 (C1), 23.8 (C6), 28.8 (C2), 35.3 (C2a), 36.9
3
0
0
0
0
0
(C5a), 55.0 (C1a), 55.4 (C7 ), 56.1 (C6a), 112.2 (C2 ), 114.5 (C4 ), 118.6 (C6 ), 129.9 (C5 ), 132.7
0
0
(C1 ), 160.1 (C3 ), 178.5 (C3), 178.6 (C5). IR (solid) 3088, 2931, 1776, 1700, 1599, 1258, 790.
+
MS (EI) m/z (%): 288 ([M + 1], 16), 287 ([M .], 90), 244 (40), 149 (44), 123 (32), 81 (38), 53 (41),
43 (100).
(
1aS*,2aR*,5aS*,6aR*)-4-(3-methoxyphenyl)-1a-methylhexahydro-3H-oxireno[2,3-
f]isoindole-3,5(4H)-dione (5b)
Yield 66%, white crystalline solid, TLC: Rf 0.2 (hexane/ethyl acetate 1:1), m.p. 126.2–
◦
1
1
27.4 C. H NMR (400 MHz, CDCl )
δ
: 1.32 (3H, s, H1), 2.18 (1H, dd, J = 15 e 7 Hz, H2y),
3
2
.22 (1H, dd, J = 15 e 7 Hz, H6y), 2.59 (1H, d, J = 15 Hz, H2x), 2.77 (1H, dd, J = 15 e 4 Hz,
0
H6x), 2.89 (2H, m, H2a e H5a), 3.04 (1H, d, J = 4 Hz, H6a), 3.80 (3H, s, H7 ), 6.84 (1H, t,
J = 2 Hz, H2 ), 6.89 (1H, ddd, J = 8, 2 e 1 Hz, H6 ), 6.92 (1H, ddd, J = 8, 2 e 1 Hz, H4 ), 7.36
0
0
0
0
13
(
(
(
1H, t, J = 8 Hz, H5 ). C NMR (100 MHz, CDCl )
C2a), 36.7 (C5a), 55.4 (C7 ), 56.7 (C1a), 57.6 (C6a), 112.4 (C2 ), 114.6 (C4 ), 119.0 (C6 ), 129.8
C5 ), 133.8 (C1 ), 160.1 (C3 ), 179.6 (C3), 179.7 (C5). IR (solid) 3077, 2990, 2927, 1780, 1705,
δ
: 21.9 (C1), 23.6 (C6), 28.0 (C2), 35.3
3
0
0
0
0
0
0
0
+
1
591, 1258, 849. MS (EI) m/z (%): 288 ([M + 1], 18), 287 ([M .], 100), 244 (22), 204 (50), 149
(
50), 93 (56), 43 (78).
3.3. Computation
The conformational searches were performed for candidate structures 5a and 5b
(Figure 2) using Maestro 2018-1 (Maestro version 11.5.011) [22]. Eight conformers were
found for structure (5a) and four conformers for structure (5b). The conformers were
submitted to geometry optimization and frequency calculation using DFT at M06-2X/6-
3
1+G(d,p) level of theory. Chemical shifts were obtained from the NMR shielding tensor
values, which were computed for each candidate structure at B3LYP/6-311+G(2d,p) level in
Gaussian 16 [18]. The shielding tensor of all nuclei was converted into referenced chemical
shifts by subtracting the computed shielding tensors of TMS calculated at the same level of
1
13
theory ( H = 31.8360; C = 183.8702). These operations were repeated for each candidate
structure 5a and 5b. Solvation was addressed with the universal solvation model (smd) [23
]
during optimizations/frequencies and shielding tensors calculations.
Linear regression of the NMR chemical shifts of 5aexp
5bexp vs. 5bcalc 5acalc was performed in MS Excel and transferred to the SI
Tables S5–S8). With the values of the intercept and slope, the for each candidate
+ 5bexp vs. 5acalc + 5bcalc and
5
(
aexp
+
+
δ
scaled
structure was obtained. The experimental data set was compared with the calculated data
after linear correction to determine the CMAE values, which are summarized in Table 3
(
cf. green highlighted values) [20]. The CMAE analyses for each candidate structure were
performed for the plot of the calculated ( calc) chemical shifts against experimental (δexp
chemical shifts, using the H and C NMR data.
Finally, the CMAE was calculated by the summation of the absolute value of ∆δ
δ
)
1
13
(
∆δ
=
δexp − δ ) of all N-nucleus and divided by the total number of N-nucleus. The
calc
1
13
calculated H and C chemical shifts using B3LYP/6-311+G(2d,p)//M06-2X/6-31+G(d,p)
level of theory are presented in the Supporting Information.
A goodness-of-fit probability was determined using the DP4 and CP3 methods de-
scribed by Goodman [14,24]. DP4 analysis was accomplished by inputting computed and

Molecules 2021, 26, 1923
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experimental chemical shifts into the DP4 analysis tool (located at http://www-jmg.ch.
cam.ac.uk/tools/nmr/DP4/ (accessed on 28 January 2021)).
3.4. Biological Assay
The bioassays were performed with the synthesized substances 5a and 5b.
Lettuce seeds (Lactuca sativa) were placed on germination paper in a Petri dish (5.5 cm
diameter). The solution containing the dissolved compound (2.5 mL) was added to the Petri
dish, which was identified, sealed with plastic film, and transferred to the germination
◦
chamber (Bio-Oxygen Demand B.O.D.). The chamber was maintained at 25 C in the
absence of light for 120 h (5 days). The experiments were performed in triplicate with
15 seeds of each species. The experiments were conducted also with cucumber (Cucumis
sativus), sorghum (Sorghum bicolor), and beggartick (Bidens pilosa) seeds.
Solutions of each tested substance were prepared in five distinct concentrations
(
500
sulfoxide (DMSO) and diluted with distilled water to prepare the most concentrated solu-
tion (500 M) containing 0.3% DMSO. The remaining solutions were prepared from the
00 µM solution by the corresponding dilution with 0.3% aqueous DMSO [25–27].
The percentage of growth of shoots and roots was calculated according to the following
µM, 300 µM, 150 µM, 100 µM, and 50 µM). The compounds were dissolved in dimethyl-
µ
5
formula:
(
S − C)
G(%) =
100
(1)
C
where S is the average length of the germinating shoots or roots of the plants, and C
corresponds to the average growth of the negative control.
◦
After the period of 5 days, the plates were frozen at 0 C for 24 h to facilitate handling
of the seedlings in the next step. They were then placed on a dark board and photographed.
The aerial and root lengths of the seedlings were measured digitally.
The commercial herbicide Dual Gold (metolachlor) was used as a positive control,
using 0.3% aqueous DMSO solution with the corresponding concentration (500
µM, 300 µM,
1
50 M, 100 M, and 50 M). The seeds of the plants were obtained commercially, except
µ
µ
µ
beggartick seeds, which were collected at the campus of Universidade Federal de Viçosa,
Viçosa, MG, Brazil.
4
. Conclusions
The synthesis of a novel pair of epoxy derivatives from 5-methylhexahydrophtalimides
5a and 5b) was carried out in five steps. The first step was a Diels–Alder reaction, followed
(
by nucleophilic addition, esterification, cyclization, and finally epoxidation with MCPBA.
The diastereomers obtained were isolated and characterized by the spectrometric methods,
and 5b was the major product. Epoxide 5b was formed via attack of the MCPBA on the
most hindered face of 4a and 4a-ent, which suggests that the steric effects are smaller than
electronic effects in determining the outcome of this reaction.
Computational calculations followed by CMAE, CP3, and DP4 analyses were per-
formed. The structures established by theoretical calculations were consistent with the
structures found by exhaustive NMR interpretation.
Finally, biological tests were carried out with epoxides 5a and 5b, using lettuce,
cucumber, sorghum, and beggartick seeds. The two tested substances interfered in the
development of the seeds, and greater inhibition of growth was observed for the root part of
the seeds. Substance 5a, in general, presented better inhibition results in the development
of both the root and aerial parts of the seeds than substance 5b. Therefore, 5a and 5b
proved to be suitable lead compounds for further investigation in the development of novel
herbicides.
Supplementary Materials: The following are available online: Figure S1. Chromatogram of the mix-
ture of tetrahydrophthalimide (4 + 4-ent) and ester (3). Figure S2. IR spectrum of compound
1
5
b
.
Figure S3. Mass spectrum of compound 5b
.
Figure S4. H NMR spectrum (400 MHz,
1
3
CDCl3
δ
= 7.27 ppm) of compound 5b. Figure S5. C NMR spectrum (100 MHz, CDCl3
CHCl3

Molecules 2021, 26, 1923
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δ
= 77.0 ppm) of compound 5b. Figure S6. DEPT spectrum (100 MHz, CDCl3
δ
= 77.0 ppm)
CDCl3
CDCl3
of compound 5b. Figure S7. COSY contour map of compound 5b. Figure S8. NOESY contour map
of compound 5b. Figure S9. HMQC contour map of compound 5b. Figure S10. HMBC contour
map of compound 5b. Figure S11. IR spectrum of the compound 5a. Figure S12. Mass spectrum of
1
compound 5a. Figure S13. H NMR spectrum (400 MHz, CDCl
δ
= 7.27 ppm) of compound 5a.
3
CDCl3
Figure S14. 1 C NMR spectrum (101 MHz, CDCl3
3
δ
= 77.0 ppm) of compound 5a. Figure S15.
CDCl3
DEPT spectrum (100 MHz, CDCl3
δ
= 77.0 ppm) of compound 5a. Figure S16. COSY contour
CDCl3
map of compound 5a. Figure S17. NOESY contour map of compound 5a. Figure S18. HMQC contour
1
map of compound 5a. Figure S19. HMBC contour map of compound 5a. Figure S20. H NMR
spectrum (300 MHz, DMSO,
δ
= 2.50 ppm) of compound
2
. Figure S21. 13C NMR spectrum (75
DMSO
1
MHz, DMSO,
δ
= 40.0 ppm) of compound
2. Figure S22. H NMR spectrum (300 MHz, CDCl
DMSO
3
1
3
δ
= 7.27 ppm) of the mixture of compounds
δ
3
and
= 77.0 ppm) of the mixture of compounds
CDCl3
4
. Figure S23. C NMR spectrum (75 MHz,
CDCl3
1
CDCl
3
and
4
. Figure S24. CP3 analysis of H
3
and 13C NMR data of compound 5a ( C NMR Expt A, H NMR Expt A) and 5b ( C NMR Expt B,
13
1
13
1
1
13
H NMR Expt B) before assignment of the signals and without linear regression. H and C NMR
1
13
calc A refer to 5a candidate structure while H and C NMR calc B refer to 5b candidate structure.
Figure S25. CP3 analysis of H and 13C NMR data of compound 5a
1
(
13
C NMR Expt A, H NMR Expt
1
13
1
A) and 5b ( C NMR Expt B, H NMR Expt B) before assignment of the signals and without linear
1
13
1
13
regression. H and C NMR calc A refer to 5a candidate structure while H and C NMR calc B
refer to 5b candidate structure. Equation S1: Bayes’ theorem for calculating conditional probability.
P(A1|B) is the probability, without previous NMR interpretation, that the proposed assignment
combination (5aexp = 5acalc, 5bexp = 5bcalc) is correct. Table S1. Development of lettuce seeds in
relation to the control. Table S2. Development of cucumber seeds in relation to the control. Table S3.
Development of sorghum seeds in relation to control. Table S4. Development of beggartick seeds in
relation to control. Table S5. CMAE analyses calculated after linear correction of the calculated 13
C
NMR chemical shifts. Table S6. CMAE analyses calculated after linear correction of the calculated
1
1
1
3
C NMR chemical shifts. Table S7. CMAE analyses calculated after linear correction of the calculated
H NMR chemical shifts. Table S8. CMAE analyses calculated after linear correction of the calculated
H NMR chemical shifts.
Author Contributions: Conceptualization, K.B.A.T. and E.S.A.; methodology, K.B.A.T. and E.S.A.;
software, K.B.A.T. and E.S.A.; validation, K.B.A.T. and E.S.A.; resources, E.S.A.; writing—original
draft preparation, K.B.A.T.; writing—review and editing, E.S.A.; supervision, E.S.A.; project adminis-
tration, E.S.A.; funding acquisition, E.S.A. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by FAPEMIG, CAPES, CNPq, and RQ-MG.
Institutional Review Board Statement: Not applicant.
Informed Consent Statement: Not applicant.
Data Availability Statement: The data presented in this study are available in this article and
Supplementary Material.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 5a and 5b are available from the authors.
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