Phytotoxicity, structural and computational analysis of 2-methyl-1,5-diarylpentadienones

Article abstract of DOI:10.1016/j.molstruc.2017.04.028

In our studies aimed to produce new chemicals used in weed control, 2-methyl-1,5-diarylpentadienones were synthesized by the reaction of p-methoxybenzaldehyde, p-nitrobenzaldehyde and p-N,N-dimethylbenzaldehyde, respectively, with 2-butanone, resulting in four model compounds. The phytotoxicity of these compounds against wheat coleoptiles and Sesame seedling was observed at μM concentrations, indicating good potential for their usage in weed management in the field. Spectroscopic and computational studies were performed in order to gain understanding on their mechanisms of action and to clarify some structural complexities due existence of conformers and substituent effects. These compounds probably act as hydroxyphenylpyruvate dioxygenase inhibitors. The tested compounds were characterized by spectroscopic and single crystal X-ray diffraction analyses. Solid crystalline state of the compound A (2-Methyl-1-(p-methophyphenyl)-5-(phenyl)-diarylpentadienone) is observed in the monoclinic space group P2₁/_c with unit cell dimensions a = 14.3366(4) ?, b = 11.3788(4) ?, c = 9.6319(3) ?, β = 96.596, V = 1560.88(9) ?³ and Z = 4. Compound C (2-Methyl-1-(p-methophyphenyl)-5-(p-nitrophenyl)-diarylpentadienone) crystallizes in the monoclinic space group P2₁/_c with unit cell dimensions a = 17.8276(9) ?, b = 7.3627(4) ?, c = 12.9740(6) ?, β = 107.6230(10), V = 1623.04(14) ?³ and Z = 4. LC-UV-MS analysis furnished important data helpful for their characterization. The spectroscopic data and computational (DFT) analysis revealed the fact that each of the compounds A-D occurs in solution as four conformers.

Full text of DOI:10.1016/j.molstruc.2017.04.028

Accepted Manuscript
Phytotoxicity, structural and computational analysis of 2-Methyl-1,5-
diarylpentadienones
Zia Ud Din, Edson Rodrigues-Filho, Viviane de Cassia Pereira, Sonia Cristina Juliano
Gualtieri, Victor Marcelo Deflon, Pedro Ivo da Silva Maia, Aleksey E. Kuznetsov
PII:
S0022-2860(17)30470-2
10.1016/j.molstruc.2017.04.028
MOLSTR 23650
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 4 November 2016
Revised Date: 10 April 2017
Accepted Date: 10 April 2017
Please cite this article as: Z.U. Din, E. Rodrigues-Filho, V. de Cassia Pereira, S.C.J. Gualtieri,
V.M. Deflon, P.I. da Silva Maia, A.E. Kuznetsov, Phytotoxicity, structural and computational
analysis of 2-Methyl-1,5-diarylpentadienones, Journal of Molecular Structure (2017), doi: 10.1016/
j.molstruc.2017.04.028.
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ACCEPTED MANUSCRIPT

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Phytotoxicity, Structural and Computational Analysis of 2-Methyl-1,5-diarylpentadienones
Zia Ud Dina), Edson Rodrigues-Filhoa), Viviane de Cassia Pereirab), Sonia Cristina Juliano Gualtierib),
Victor Marcelo Deflonc), Pedro Ivo da Silva Maiac) and Aleksey E. Kuznetsov*d)
a) LaBioMMi, Departamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905,
São Carlos, SP, Brazil.
b) Laboratório de Ecofisiologia de Sementes, Universidade Federal de São Carlos, CP 676, 13565-
905, São Carlos, SP, Brazil.
c) GQIEB, Instituto de Química de São Carlos, Universidade de São Paulo,
13566-590 São Carlos, SP, Brazil.
d) Instituto de Química, Universidade de São Paulo – SP, Av. Prof. Lineu Prestes, 748 – Butantã,
05508-000, São Paulo - SP – Brasil;
phone: +55(16)98109-6002; e-mail: aleksey73kuznets@gmail.com
Abstract: In our studies aimed to produce new chemicals used in weed control, 2-methyl-1,5-
diarylpentadienones were synthesized by the reaction of p-methoxybenzaldehyde, p-nitrobenzaldehyde
and p-N,N-dimethylbenzaldehyde, respectively, with 2-butanone, resulting in four model compounds.
The phytotoxicity of these compounds against wheat coleoptiles and Sesame seedling was observed at
µM concentrations, indicating good potential for their usage in weed management in the field.
Spectroscopic and computational studies were performed in order to gain understanding on their
mechanisms of action and to clarify some structural complexities due existence of conformers and
substituent effects. These compounds probably act as hydroxyphenylpyruvate dioxygenase inhibitors.
The tested compounds were characterized by spectroscopic and single crystal X-ray diffraction
analyses. Solid crystalline state of the compound A (2-Methyl-1-(p-methophyphenyl)-5-(phenyl)-
diarylpentadienone) is observed in the monoclinic space group P21/c with unit cell dimensions a =
14.3366(4) Å, b = 11.3788(4) Å, c = 9.6319(3) Å, β = 96.596, V = 1560.88(9) Å3 and Z = 4.
Compound C (2-Methyl-1-(p-methophyphenyl)-5-(p-nitrophenyl)-diarylpentadienone) crystallizes in
the monoclinic space group P21/c with unit cell dimensions a = 17.8276(9) Å, b = 7.3627(4) Å, c =
12.9740(6) Å, β = 107.6230(10), V = 1623.04(14) Å3 and Z = 4. LC-UV-MS analysis furnished
important data helpful for their characterization. The spectroscopic data and computational (DFT)
analysis revealed the fact that each of the compounds A-D occurs in solution as four conformers.
Keywords: Diarylpentadienone • Phytotoxic activity • XRD • Coleoptile

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1. Introduction
The yield of agriculture products is dependent on control of the undesired plants, pests and weeds,
which contribute negatively for crop yields. Approximately 7,000 weed species have been recognized
until now; perhaps a few hundred of them are presenting troubles to the world's agriculturalists [1].
Although control of undesired plants using natural compounds have long being considered as a good
and ecologically friendly approach, during the last 50 years researches have been focused almost
exclusively on synthetic herbicides [2]. These compounds can be synthesized and optimized in
laboratory for large scale production [3].
Different synthetic herbicides present at the market target the same biological process in weeds so their
enormous use induces resistance in treated weeds. Due to this, the development of novel phytotoxic
agents with different target processes has recently become a primary goal of the weed control research
[4]. Potential phytotoxic agents are inspired by the vast variety of biologically active secondary
metabolites of plants [5]. Phytological investigations have characterized some natural substances as
good inhibitory agents [6], which disrupt mitochondrial function and cause apoptosis in Arabidopsis
roots at concentrations as low as 35 ꢀM [7].
Natural products chalcones and their precursors have exhibited good phytotoxic potential [8,9] causing
programmed cell death in Arabidopsis thaliana roots [6,7]. The reduction of mitochondrial trans
membrane potential was detected after chalcone treatment. Unsymmetrical 1,5-diarylpentadienones
structurally resemble natural products chalcones possessing broad range of pharmacological
properties. However, unsymmetrical 1,5-diarylpentadienones (Figure 1) have not been studied for their
herbicide activity before. Their structure is composed of two aryl groups and a carbonyl group in
conjugation with two C=C bonds. Diarylpentadienones and their congeners exhibited various
biological activities such as antioxidant [10,11], cytotoxicity [12], anti-leishmanial activity [13], anti-
tuberculous activity [14], antimalarial [15], antifungal [16], antifertility [17], antimitotic [18],
antibacterial [19], and chemoprotective activity [20].

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Figure 1. Comparison of the structure of 2-methyl-1,5-diarylpentadienones with chalcone.
The aim of this study is to discuss some interesting structural aspects of a series of diarylpentadienones
recently produced and characterized by our research group, and also to evaluate their phytotoxic
activity in order to gain understanding on their structural requirement to be potential herbicides. These
compounds have different electron-donating and withdrawing groups affecting the dienone system,
and analysis of the effects of these groups may help to understand the mechanism of inhibition.
Docking studies were also carried out to predict the probability of binding of the compounds A-D with
hydroxyphenylpyruvate dioxygenase (HPPD), a well-recognized important molecular target for
inhibition of plant development. Moreover, theoretical calculations were performed to obtain detailed
information about the possible conformations of the compounds A-D, which were revealed by LC-UV-
MS analysis.
Compounds A-D were produced in two steps according to the scheme shown in Figure 2, by the
reaction of anisaldehyde and p-N,N-dimethylbenzaldehyde with 2-butanone at first step to produce
intermediate 1 and 2 [21]. After purification intermediate 1 was treated with benzaldehyde, p-N,N-
dimethylbenzaldehyde, and p-nitrobenzaldehyde to yield compounds A, B and C, respectively. The
intermediate 2 yields compound D by reacting with p-N,N-dimethylbenzaldehyde.
Figure 2. Synthetic scheme for 2-methyl-5-phenylpentadienones.
2. Material and Methods
2.1. Instrumentation
All chemicals were purchased from Organics, Sigma-Aldrich, Acros Chemicals and Fisher Scientific
Ltda and used without further purification. The deuterated solvents of Apolo were used for the NMR

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analysis. Thin layer chromatography was performed with precoated silica gel G-25-UV254 plates and
detection was carried out at 254 nm under UV, and by vaniline in H2SO4 solution. Mass spectra (ESI-
HRMS) were measured using a Thermo Scientific LTQ Orbitrap XL spectrometer operated at a
resolution of 60,000 FWHM, in positive mode, m/z 90-800. HRMS/MS data were obtained by CID. 1H
13
NMR and C NMR analyses were performed on a Brüker AVANCE 400 spectrometer operating at
400.15 MHz and 100.62 MHz, respectively. CDCl3 was used as a solvent and tetramethylsilane (TMS)
as internal reference. Compounds A-D were dissolved in organic solvents at concentrations about 10
mg/mL each and placed into a 5-mm NMR tube. Chemical shifts (δ ppm) were measured with
accuracy of 0.01 (1H) and 0.1 ppm (13C). The UV-vis spectra were recorded using a Perkin Elmer
Lambda 25 spectrophotometer using 1 cm optical length quartz cuvettes at 25 °C and methanol as
solvent. X-ray diffraction intensities were measured at room temperature (296 K) using Mo-Kα
radiation (λ = 71.073 pm) on a Bruker APEX II Duo diffractometer.
2.2. Synthesis of the compounds A-D
Chalcone analogues can be formed by the direct reaction of aldehyde and ketone using basic or acid
catalysis. Acid catalysts used for cross-aldol condensation reaction include sulfuric, hydrochloric [23]
and Lewis acids. Compounds 1 and 2 were synthesized by treating anisaldehyde, and p-N,N-
dimethylbenzaldehyde with 2-butanone in a 50 mL double-necked round bottom flask. Dry HCl gas
was passed via the content of the flask until it was saturated and coloration appeared. The reaction
mixture was stirred for 8 hrs. The crude product was diluted with toluene and washed with NaHSO3
solution. The organic layer was separated, dried with anhydrous Na2SO4 and evaporated in vacuum.
The residue was distilled under reduced pressure to give pure compound 1 and 2, which were
solidified by keeping in a refrigerator for 24 hrs. Intermediate compound 1 was further treated with
benzaldehyde, N,N-dimethylbenzaldehyde and p-nitrobenzaldehyde, and yielded compounds A-C,
while intermediate compound 2 was treated with benzaldehyde to yield compound D. All compounds
were produced in a good yield and were characterized by different spectroscopic and spectrometric
analyses (see Electronic Supporting Information).
A solution of 1 and 2 with aldehyde in ethanol (5 mL) was stirred for 5 minutes at room temperature
and sodium hydroxide solution in ethanol (4 mL, 50 mmole) was added and the stirring was continued
for 7 hrs. The solvent was evaporated in vacuum. Residue was dissolved in ethyl acetate, extracted
with NaHSO3 solution, dried with Na2SO4, and concentrated in vacuum yielding the crude product
which was collected as precipitate and further purified by column chromatography and recrystallized
from ethanol to give pure compounds A-D.

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2.3. Growth of wheat coleoptile (Triticuma estivum) and Sesamum indicum
Wheat seeds (Triticuma estivum) were sown in Petri dishes (15 cm diameter) having one sheet of filter
paper soaked with distilled water and grown in the dark at 22 °C for 3 days. Roots and caryopses were
detached from the shoots. Shoots were placed in a Van der Weij guillotine, the apical 2 mm were cut
off and discarded and the next 4 mm of the coleoptiles were recovered and used for the bioassay. This
bioassay was performed to evaluate the initial growth of wheat coleoptile in the presence of
compounds in concentrations 10-3 M; 3×10-4 M; 1×10-4 M, 3×10-5 M; and 10-5 M adopted from
literature [24][25][26]. As a positive control, the herbicide Oxyfluorfen (240g/L) was used at the same
concentrations. Seedling growth of sesame (Sesamum indicum) was carried by following methodology
described by Grisi et al. [27]. The concentrations 10-3 M; 3×10-4 M; 1×10-4 M; 3×10-5 M and 10-5 M
were used, and positive control herbicide Oxyfluorfen (240 g/L) was used at the same concentrations.
All results were tested for normality and homoscedasticity with the Shapiro-Wilk and Levene tests,
respectively. Normal and homoscedastic data were analysed with ANOVA followed by Tukey's test.
The heteroscedastic data were analysed with the non-parametric Kruskal-Wallis test, in the software
Past 2.14 [28].
2.4. X-ray characterization
Single crystals were grown by dissolving the compounds in 5 mL of hot ethanol and allowing them to
grow at room temperature for 24 hrs. Yellow prisms suitable for single crystal diffraction were
obtained and used for data collection. X-ray diffraction intensities were measured at room temperature
(296 K) using Mo-Kα radiation (λ = 71.073 pm) on a Bruker APEX II Duo diffractometer. Standard
procedures were applied for data reduction and absorption correction. The crystal structures were
solved by direct methods using the SHELX97 program. All non-hydrogen atoms were refined with
anisotropic displacement parameters with SHELXL97 [29]. The hydrogen atoms were calculated at
idealized positions using the riding model option of SHELXL97.
2.5. Docking study
Molecular docking was carried out using Auto Dock Tool from Scripts Vina Research Institute [30].
Auto Dock is a suite of automated docking tools, designed to predict how small molecules such as
substrates or drug candidates bind to a receptor of known 3D structure. Docking was used to predict

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both ligand orientation and binding affinity. For docking analysis, first the ligands were optimized on
the basis of energy by software Avogadro 1.1.1 [31]. Calculations were performed by removing water
molecules first and adding polar hydrogen atoms using the option available in autodock tool. Gasteiger
charges were also assigned for ligands and then the protein ligand were processed for obtaining file
.pdbqt, which are input files for autodock 4.2. Enzyme 1TFZ was optimized by considering only active
site that occupied by inhibitors. The preferred orientation of ligands and receptor by the formation of
stable complex was predicted in three dimensions. Binding energy interaction results were elucidated
using Discovery Studio Visualiser 4.1 [32].
2.6. Computational methods
Computational studies of the compounds A-D were performed using the Gaussian09 package [33].
Geometries of all the species were optimized in the gas phase without any symmetry constraints. The
resulting structures were assessed using vibrational frequency analysis to check whether the structures
represent true minimum-energy geometries. If any imaginary frequency was detected, we performed
further optimizations along the normal coordinates corresponding to the imaginary frequency(ies)
(without symmetry constraints). For all the species under investigation, we studied four possible
conformers, marked as 1-4 (see the manuscript text). All the calculations were done using the split-
valence 6-311++G(d,p) basis set [34] containing the sets of polarization and diffuse functions on both
heavier and hydrogen atoms, and the hybrid Becke three parameter hybrid functional with the non-
local correlation provided by Perdew/Wang 91 B3PW91 [35,36]. This approach is subsequently
referred to as B3PW91/6-311++G(d,p). For all the structures under investigation, we studied the
singlet states and for some of them we checked triplet states as well (see discussion in the text and
Electronic Supporting Information). To further refine the energies of the optimized conformers, we
performed single-point calculations on the gas-phase optimized geometries using the B3PW91
functional and the Dunning’s correlation consistent basis set cc-pVTZ [37]. In the paper, we discuss
the results obtained in the gas phase without zero-point correction ZPE (∆E0). The charge analysis was
performed using the Natural Bond Orbital (NBO) scheme with the ‘pop=nbo’ command as
implemented in the Gaussian09 package [38,39]. Molecular structures and frontier orbitals were
visualized using the OpenGL version of Molden 5.0 visualization software [40].
3. Results and Discussion

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Although the 2D-draw of these diarylpentadienones seems to indicate simple molecular structures,
there are some structural elements that render them some complexity due the possibility of existence of
conformers, and this may impact their activities. Thus, during former works we have seen that the α-
carbonylic and vinylic methyl group disturb the planarity of the cross-conjugated 1,5-
diarylpentadienyl-3-one system [21], which open possibility for different contributions of substituents
at the aromatic rings. Also, during some analysis of these compounds, the injection of dissolved pure
and crystalline diarylpentadienones in the HPLC-UV-MS, with the column temperature set at 30 oC, at
least four peaks are detected (data not shown here), being two major and two far minor peaks, with
exactly same MS data, but differing in UV-absorption (e.g. A: major peaks with longer λmax at c.a.
315 and 335; minor peaks with shorter λmax at c.a. 280 and 290). The UV data of some of these
compounds will be presented and discussed elsewhere. Here we presented the room temperature NMR
data, where only the more stable conformers are seen; the MS/MS data at relatively low energy
showing interesting fragmentation paths; their X-ray analysis; and their herbs inhibition activities
along with a suggestion for their mechanism of action. Also a gas-phase conformational study is
discussed.
3.1. NMR spectroscopic characterization
1H NMR spectrum of compound 1 showed two characteristic signals of two methyl groups in the
shielded region at δH 2.07 and 2.45, one methoxy singlet at δH 3.85, whereas one =CH was displayed at
δH 7.39 as singlet; additionally, two aromatic signals having integration for four protons were detected
at δH 6.93 and 7.41. 13C NMR spectra of 1 showed signals for two methyl groups at δC 12.9 and 25.8,
one OCH3 at δC 55.3 and six peaks from δC 113–159 for eight carbons, with two of these peaks having
double intensity for aromatic carbons. The characteristic peak at δC 200.3 is due to carbonyl carbon.
1
The H and 13C NMR spectra of 1 and 2 are similar except the presence of the N,N-dimethyl
substituent on benzene ring in 2 showed by signals at δH 3.02 and δC 40.15.
The reaction of the intermediate compound 1 with benzaldehyde, N,N-dimethylaminobenzaldehyde
and p-nitrobenzaldehyde yields compounds A-C, respectively, while the reaction of N,N-
dimethylaminobenzaldehyde with the compound 2 yields compound D. Also, extra signals in the
1
aromatic region were noted in the H spectra and the signal for methyl ketone group disappeared,
showing that an aromatic ring was added and the methyl group was modified. The appearance of a pair
of doublets at δH 7.53 and 7.69, with a coupling constant of c.a. 16 Hz, typical for two hydrogens in an
E-double bound, clearly confirms that the aldol condensation with the second aldehyde occur. Further
1
complete characterizations were performed by H NMR, 13C NMR, UV/Vis spectroscopy, mass
spectrometry and X-ray analysis (for details, see Electronic Supporting Information).

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Compounds A-D were produced in good yields after re-crystallization from ethanol: 51% for A, 93%
for B, 69.3% for C, and 67% for D. The reaction progress was monitored by TLC. The reaction time
depends on the nature of the aldehyde and substitution on it. Thus, for crossed aldol reaction, electron
donors (N,N-dimethylamine) at the p-position in the benzene rings decrease the reaction time, while
nitrobenzaldehyde reacted very fast.
3.2. Mass-spectral characterization
The mass-spectra profile of all four compounds revealed important and interesting fragmentation
paths. The pseudomolecular ion peak ([M+H]+) of the compound A (C19H18O2, 278 Da) appeared at
m/z 279.4 in its ESI(+)-MS/MS spectrum. Although these molecules demonstrate a high degree of
unsaturation, which contributes for their stability, the MS/MS spectra, obtained when precursor ions
were accelerated using relatively low energy (30 eV) for collisional induced dissociation (CID) against
argon, contain interesting ion fragments which correlates well with their structures (see Electronic
Supporting Information). Thus, the ions detected at m/z 121 and 91 were found related to the rings A
and B of the compound A, which are formed from anisaldehyde and benzaldehyde, respectively. The
peak at m/z 103 (100%) is due to production of styrene cation (path b). The cleavage that eliminates
neutral phenyl ring results in the production of m/z 171.08 (path d). Well expected fragment ions were
detected at m/z 77 (path i), 131 (path k) and 143 (path f). The acylium ions of m/z 131 and 143 were
probably formed after ESI-protonation at C(2) or C(4) respectively, followed by cleavage of C(2) –
C(3) or C(3) – C(4) bonds. The peaks that appeared at m/z 135 (path h) and 105 (path g) matches the
formulas for methoxybenzoyl and benzoyl acyllium ions, although compound A does not have an
oxygen atom directly attached to C(1) or C(5), respectively. These two apparently unexpected ions
could be formed after ESI-protonation at C(2) or C(4), cyclization assisted by π-electrons from C=O
forming an oxetane ring. Cleavages at this oxetane ring after H-rearrangement led to m/z 135 and 105
(Figure 3). The compounds B, C and D follow the same pattern. Their mass spectra are provided in the
Electronic Supporting Information.

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Figure 3. Fragmentation scheme followed for the compound A under collisional induced dissociation
(CID) after ESI(+) ionization.
3.3. Crystallographic characterization
ORTEP drawings of the compounds A and C with numbering scheme are presented in Figure 4 and
crystal packing diagram is shown in Figure S1. Selected bond distances and angles for these
compounds are listed in Table S1 for understanding the effect of substitution upon the compound
geometries The electron-donating and withdrawing groups significantly affect the bond distances and
angles in compounds. The bond lengths along the carbon chain between the two aromatic rings are
within the values for alternate single and double bonds. This alternation of single and double bonds
makes the structure more flexible and less planar. The C(10)−O(1) bond is a typical double bond,
which is in accord with the IR data. The bond distances N(1)−O(3) and N(1)−O(4) in the nitro group
of C are exactly the same due to resonance inside this group. As expected, only weak interactions are
observed in the crystal packing of these compounds. Detailed information about the structure
determination is given in Table S2 (see Electronic Supporting Information).

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Figure 4. ORTEP structures and atomic numbering scheme for the compounds A (left) and C (right).
Displacement ellipsoids for non-H atoms drawn at the 50% probability level.
3.4. Phytotoxic potential
The compounds studied induced inhibition of wheat coleoptiles growth in percentage compared to
positive control Oxyfluorfen (Figure S2). All compounds had inhibitory effect at all concentrations
tested on the growth of coleoptiles. However, the largest percentage of inhibition was caused by the
compound D at the concentration of 10-3 M, while the compound A was the best inhibitor at lower
concentration. Maybe A, being not very sensitive to pH variation compared to the compounds B-D,
does not disturb molecular target by changing interacting site geometry when its concentration
enhances.
Table S3 in Electronic Supporting Information shows comparison of the average of each treatment
with the average obtained for the herbicide Oxyfluorfen treatment at different concentrations. It was
noted that at all concentrations the effect of the herbicide is statistically similar to the effect caused by
the compounds studied, except for the compound B at 3.10-5 M. Thus, we can say that these
compounds have properties that resemble the herbicide. At the concentration of 10-4 M all the
compounds A, B, C, and D were found to be more potent than the reference herbicide (Table S3).
Sesame seedlings grown in contact with the compounds A and D exhibited lower values of shoot and
root length. The Figure S3 shows the average growth of the seedlings grown under the effect of these
compounds. It was observed that only the concentration of 3.10-5 M did not inhibit the growth of
shoots, since no significant difference was shown when compared with control treatment. The root
seedling growth was compromised by the action of the compounds A and D at all concentrations
tested, showing that these compounds have phytotoxic properties. Seedlings subjected to treatment
with the herbicide Oxyfluorfen exhibited the lowest average growth of shoot and root at all
concentrations tested.
3.5. Growth of cells of Sesamum indicum

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The seedlings of metaxylem cells were exposed to the compounds A and D at the concentrations of
3.10-4 M and 10-5 M. The seedlings showed significant reduction in length by comparing with negative
control treatment (Figure S4). The results of each treatment are also statistically compared in Table S4
(see Electronic Supporting Information). The cell lengths in the presence of the compounds A and D
were measured to be 71.85 µm and 63.42 µm respectively for the first concentration and 95.47 µm and
79.40 µm for the second concentration, respectively. The average value of the control was 166.5 µm.
3.6. Herbicide compounds - HPPD enzyme docking studies
4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme present in plants that breaks down
the amino acid tyrosine into components that plants utilize to create other molecules they need. Small
molecular entities can act as HPPD inhibitors [22]. All the compounds A-D were analyzed for their
binding capability to the enzyme HPPD Pdb (code 1TFZ) and the results obtained are tabulated in
Table S7. The active site calculation was carried out on the basis of the co-crystallized ligand in its
crystal structure. Centroid of the active site was measured and then all compounds were evaluated for
their in silico inhibition activity. The amino acids present in active sites and contributing actively are
His-205, Pro-259, Asn-261, His-287, Met-314, Phe-360, Glu-373, Phe-398, Gly-399, Lys-400, Asn-
402, and Phe-403. It is clear from the results obtained for the compounds A-D that they can have
strong interactions with amino acids residues, like H-bonding, steric interactions, π-interactions and
hydrophobic interactions. Compound B docked within the binding groove of HPPD with active
residues. H bonding, π-interactions and hydrophobic interactions between the aromatic ring of B and
that of amino acids residue in active pocket were observed (Figure 5). The results indicate that our
curcuminoid analogues could be useful inhibitors of HPPD.
Compound B interacts with binding sites of amino acids with good fitting interactions. Hydrogen
bonding was observed between the ligand with Asn-402 having distance 2.75 Å. The binding structure
is given in Figure 5.

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Figure 5. Docking diagram of the compound B with 1TFZ.
3.7. Computational studies
The numbering system used in these discussions appears in Figure S5. Gas-phase optimized structures
of the four conformers for each of the compounds studied are shown in Figure 6 as example, and in
Figures S6-S8. The main energetic and structural features of the conformers of the compounds A-D are
summarized below as follows:
(i) For all the four compounds, the singlet structures were found to be much lower in energy than the
triplet structures. The triplet structure was calculated for the check purposes in the gas phase for the
conformer 1 of the compound A (see Electronic Supporting Information, Table S5) and was found to
be higher in energy by ca. 44 kcal/mol. Thus, in this study our focus was only on the singlet structures.
(ii) For the compound A, the conformer 1 (Figure 6) was found to be the most stable structure, whereas
for the compounds B-D the conformer 3 (Figures S6-S8) was found to be the most stable species (see
Electronic Supporting Information, Table S5). The conformer 1 has the both double bonds, C(7) – C(8)
and C(11) – C(12) (see Figure 4 and Figure S5 for labeling), in trans-configuration and the atoms C(9)
and O(1) in trans-position relative to each other. The conformer 3 also has the both double bonds in
trans-configuration but the atoms C(9) and O(1) in cis-position relative to each other. Thus, it is
possible to convert conformer 1 to conformer 3 simply by the rotation around the single bond C(8) –
C(10). This is supported by the very small energy barriers between the conformers 1 and 3 for all the
compounds A-D, ranging from 0.05 to 1.3 kcal/mol at the B3PW91/6-311++G(d,p) level (0.2-1.5
kcal/mol at the B3PW91/cc-pVTZ level of theory, see Table S5). Also, the almost negligible energy

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difference between the conformers 1 and 3 of the compound A (0.05 kcal/mol) explains why the
computed lowest-lying structure (1) differs from the experimentally found structure (3), see Figure 4.
(iii) For all the four compounds we found two or three very close lying in energy structures for the
conformer 3 (see Figures S3-S6 and Table S5 in Electronic Supporting Information). The energy
differences between these structures generally vary from 0.002 to 1.4 kcal/mol at the B3PW91/6-
311++G(d,p) level and from 0.1 to 1.8 kcal/mol at the B3PW91/cc-pVTZ level of theory (see Table
S5).
(iv) Interestingly, all the four calculated conformers for all the four compounds are quite close to each
other in energy, the maximum energy differences (between the global minimum isomer, 1 or 3, and
isomer 4) being 5.9-8.0 kcal/mol at the B3PW91/6-311++G(d,p) level and 6.7-8.9 kcal/mol at the
B3PW91/cc-pVTZ level of theory (see Table S5).
(v) Geometry optimizations led to the noticeable structural distortions of the conformers 2-4 of all the
four compounds (see Figures S6-S8), the deviations from planarity are especially noticeable for the
conformers 2 and 4 of the compounds A-D. The conformer 2 has the double bond C(7) – C(8) in cis-
configuration and the double bond C(11) – C(12) in trans-configuration and the atoms C(9) and O(1)
in trans-position. The conformer 4 has the double bond C(7) – C(8) in trans-configuration and the
double bond C(11) – C(12) in cis-configuration and the atoms C(9) and O(1) in trans-position.
(vi) The energy differences between the lowest-lying conformer 3 (1 for the compound A) and the
conformers 2 and 4 are quite noticeable, ranging from ca. 4 to 8 kcal/mol at the B3PW91/6-
311++G(d,p) level (ca. 4-9 kcal/mol at the B3PW91/cc-pVTZ level), see Figures S6-S8 and Table S5
in Electronic Supporting Information. This is explained by the necessity to change the C=C bond(s)
conformation to convert from the conformer 3 to 2/4, thus the interchanging energy barriers are quite
high in this case, compared to the interchange between 1 and 3, see above.

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Figure 6. B3PW91/6-311++G(d,p) optimized gas phase structures of the isomers of compound A: side
views (bottom rows) and top views.
Analysis of the electron density distribution in the lowest-energy isomers of the compounds A-D, as
illustrated by the plots of the HOMOs (Figure 7), shows the following.
(i) The HOMOs of the compounds A, C, and D have dominating contributions from the double bond
C(7) – C(8) and the benzene ring attached to it (including noticeable contributions from the electron-
donating group CH3O in the compounds A and C and electron-withdrawing group NO2 in the
compound D), along with some contributions from the O(1) and CH3-group. The HOMO of the
compound B has dominating contributions from the double bond C(11) – C(12) and the benzene ring
attached to it, along with noticeable contributions from the electron-donating N(CH3)2 group and small
contributions from the O(1) and the double bond C(7) – C(8).
(ii) Thus, the both double bonds in all the four compounds A-D bear quite significant amounts of the
electron density, which is also supported by the results of the NBO analysis showing quite noticeable
negative charges on the atoms C(7)-C(8) and C(11)-C(12) of the double bonds (see Table S6,
Electronic Supporting Information). Also, large negative charges are accumulated on the carbonyl
oxygen O(1) and the methyl group (C(9)). Therefore, the parts of the compounds A-D containing the
double bonds should have significant reactivity towards various electrophiles, for example, towards
positively charged amino acids residues or protons. The same should be true for the O- and N-

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containing functional groups of these compounds. In total, the whole molecules of the compounds A-D
should have strong interactions with amino acids residues, like H-bonding, steric interactions, π-
interactions, and hydrophobic interactions, as described above in the section devoted to the docking
studies.
Figure 7. HOMOs and LUMOs of the lowest-energy conformers of the compounds A (a), B (b), C (c),
and D (d).
4. Conclusions
The investigated compounds A, B, C and D were shown to exhibit impressive phytotoxic activity.
These compounds are easy to synthesize and can be produced in desired amounts. Synthetic
chalconoids can replace the natural herbicides due to difficult and time-consuming purification
methods used to obtain those. The compounds A-D also show binding affinity to enzyme 1TFZ, which
may be the probable target for them. The DFT calculations provided valuable information on the
structural and electronic features of A-D. Based on the computational results, we were able to
determine the most stable conformers for each of the four compounds, make analysis of the electronic
structures of the compounds studied, and thus provide explanations of their reactivity features. It seems
that, at physiological conditions, the conformation mobility of these substances does not affect
significantly their bioactivity.
Electronic Supporting Information

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Electronic Supporting Information is free of charge. It contains the following data: NMR spectra of the
compounds 1, 2 and A-D; UV-vis spectra of the compounds 1, 2 and A-D; mass spectra of the
compounds 1, 2 and A-D; crystallographic information of the compounds A - C; phytotoxic properties
of the compounds A-D; synthetic details of the compounds 1, 2 and A-D; details of computational
studies of the compounds A-D; docking details of the compounds A-D.
Acknowledgements
The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP),
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de
Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) for financial su
pport and Third World Academy of Science (TWAS) for Ph.D. fellowship of ZUD. The computational
resources of the Centro Nacional de Processamento de Alto Desempenho do CENAPAD-UFC in
Fortaleza are highly apprecaited.
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HIGHLIGHTS
•
•
•
•
Phyto-toxic activity of compounds A-D was evaluated.
Docking was performed against possible target.
Spectroscopic data are presented and discussed.
DFT approach was applied for computational analysis.