Amino-substituted para-Benzoquinones as Potential Herbicides

Received Date: 03-Nov-2015

Accepted Date: 05-Jan-2016

Article Type: Full Paper

Amino-substituted para-Benzoquinones as Potential Herbicides

by Amalyn Nain-Perez^a)^b), Luiz C. A. Barbosa*^a)^b), Marcelo C. Picanço^c), Samuele

Giberti^d), and Giuseppe Forlani*^d)

^a) Department of Chemistry, Universidade Federal de Minas Gerais, Av. Pres. Antônio Carlos,

6627, Campus Pampulha, CEP 31270-901, Belo Horizonte, MG, Brazil

^b) Department of Chemistry, Federal University of Viçosa, Viçosa, MG, Brazil

^c) Department of Entomology, Federal University of Viçosa, Viçosa, MG, Brazil

^d) Department of Life Science and Biotechnology, University of Ferrara, via L. Borsari 46, I-

44121 Ferrara, Italy

*Corresponding authors: Tel.: +55 3899 3065 and +39 0532 455311. E-mail address:

lcab@ufmg.br (Luiz C. A. Barbosa) and flg@unife.it (Giuseppe Forlani)

This article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process, which may

lead to differences between this version and the Version of Record. Please cite this article as

doi: 10.1111/cbdv.201500340

Although quinones present a large array of biological activities, a few studies on the

herbicidal potential of 2,5-bis(alkyl/arylamino)-1,4-benzoquinones have been reported to

date. In this work, starting from benzoquinone, thirteen 2,5-bis(alkyl/arylamino)-1,4-

benzoquinones were prepared in 46-93% yields. The products were fully characterized by

spectroscopic analyses and their phytotoxicity against Cucumis sativus and Sorgum bicolor

seedlings was investigated. At 100 ppm, compounds caused 10-88% growth inhibition of the

dicotyledonous species, whereas the monocotyledon was less affected. Most compounds

exerted little inhibitory effect on a cyanobacterial model strain. However, at 100 μM

compounds 8-10 caused about 50% inhibition of algal growth, and compounds 1 and 2

reduced cell viability in the 1-10 μM range. Ability of benzoquinone derivatives to interfere

with the light-driven ferricyanide reduction by isolated spinach chloroplasts was evaluated.

Some substances showed a moderate effect as uncouplers, but no relationship was found

between this property and their biological activity, indicating that the herbicidal effect is not

associated with the inhibition of the photosynthetic electron transport chain. Phytotoxic

compounds were not toxic to insects, strengthening the possibility that they may serve as lead

for the development of eco-friendly herbicides.

Keywords: Herbicidal activity, Algicidal activity, Insecticidal activity, Photosynthetic

inhibitors, 2,5-bis(alky/arylamino)-1,4-benzoquinones.

Introduction. - Modern agriculture is highly dependent on the use of synthetic

chemicals such as fertilizers and pesticides, including insecticides, herbicides and fungicides,

which are used worldwide to increase crop yield [1-2]. However, the repeated use of

substances that act by the same mechanism of action continuously leads to the development

of resistance in several organisms, such as insects, fungi and weeds, representing a major

issue in crop protection [3-5]. Thus, there is a continue need for the development of new

agrochemicals to provide farmers with effective tools to control pests [6].

New pesticides should be endowed with ‘ecological selectivity’, i.e., be effective in

pest control without affecting non-target organisms, such as pest natural enemies and

pollinators [7-8]. Among the formers are the predatory ants, as Solenopsis saevissima

(SMITH) (Hymenoptera: Formicidae) [9]; among the latter bees (Hymenoptera: Apidae)

constitute an essential group of beneficial insects, accounting for 90% of the reproductive

success of flowering plants [10]. In this perspective, the stingless bee Tetragonisca angustula

LATREILLE (Meliponinae) is of great importance in the Neotropics [11].

Biologically active quinones have recently attracted great interest due to their broad-

spectrum effects, such as antibacterial, antifungal [12], herbicidal [13–17], antifeedant [18],

urease inhibitory [16] and insecticidal [19-20] activities. Some naturally occurring quinones

are redox active molecules that play a main role as electron carriers in photosynthesis,

respiration, and information-transfer processes [21]. With this background, we set our study

directed to the synthesis and characterization of a series of 2,5-bis(alky/arylamino)-1,4-

benzoquinones. Even though there are several reports on the preparation of this type of

compounds [14, 16, 22-23] and their substituted derivatives [18], to date their potential use as

herbicides has been poorly investigated [24- 25].

Here we report the synthesis of a set of aminobenzoquinone derivatives 1-13 and the

assessment of their inhibitory effects on plant and algal growth, as well as their ability to

interfere with the photosynthetic electron transport chain in isolated spinach chloroplasts. In

view of the great concern about the impact of new herbicides on the environment, their

toxicity against some beneficial or pest insects that are widespread in Brazil was also

investigated.

Results and Discussion.- Synthesis. The initial attempt to prepare 2,5-

diaminobenzoquinones involved addition of an amine directly to hydroquinone solution as

reported in [16]. Using this protocol, pentylamine was reacted for 1 h with 1 equiv. of

hydroquinone and the required product 4 was obtained in only 24% yield (Table 1, Entry 1).

The same procedure was used employing several aliphatic and aromatic amines and the

yields obtained were consistently in the range of 13-35%, despite the claim of Zhong-Lu et al.

[16] that such methodology produces the required products in yields higher than 87% (see

Table 1, Entry 6 as an example; other data are not shown). Having demonstrated that this

methodology [16] did not work in our hands, the same reaction mixture (pentylamine and

hydroquinone, 1:1 ratio) was allowed to stir for 18 and 24 hours, yielding the required

compound 4 in 45% and 48%, respectively, and compound 10 in 45% (Table 1, Entries 2, 3,

and 7). Employing 2 equiv. of amine for 1 equiv. of hydroquinone, even after 36 hours only

24% and 10% of products 4 and 10, respectively, were isolated (Table 1, Entries 4 and 8). All

reactions were carried out in the presence of light or in the dark producing the same results.

Also, the reactions were carried out under normal atmosphere, under O₂, or under N₂. Under

O₂atmosphere, slightly better yields were observed, which did not represent a great

improvement (data not shown).

Despite a recent report of a new catalyst for the efficient conversion of hydroquinone

into the corresponding 2,5-diamino-p-benzoquinone [26], we turned our attention to the use

of p-benzoquinone. In a previous work on this area, we had prepared a series of 2,5-diamino-

p-benzoquinones in low yields (9-58%) employing a 2:3 ratio of amine/quinone [14]. In the

present work, the use of excess amine resulted in compound 10 in only 25% (Table 1, Entry

9). We then found that using a 1:3 ratio of amine/quinone the required products were obtained

in high yields (Table 1, Entries 5 and 10).

Having established a reliable protocol to produce 2,5-dialkylamino- and 2,5-

diarylaminobenzoquinones in high yield, a series of analogues 1-13 was prepared (Scheme 1).

In general, the procedure involves a reaction between 1 equiv. of amine and 3 equiv. of

benzoquinone, dissolved in MeOH, at room temperature and for approximately 1-16 hours,

under dark or light and normal atmosphere. For the synthesis of compounds 10-13, it was

necessary to add NaHCO₃to the EtOH solution of the amino acids to ensure the full

deprotonation of the zwitterion [22].

In general, the aliphatic amines were obtained in higher yields (74-93%), while for the

less nucleophilic aromatic amines the yields were 58-87%. For the amino acids, the yields of

the products varied from 46 to 68%. Although these yields are lower, the results are good

since the only report of reaction between benzoquinone and amino acids in good yields

involves the use of the enzyme laccase as catalyst [27].

The structure of each compound was confirmed by extensive spectroscopic analysis.

All compounds showed in their respective IR spectra typical absorptions expected for the

major functional groups present. For example, a broad N–H absorption band centered around

3200 cm^–1was observed. In addition, the stretch vibration of the C=O group of quinone was

observed between 1640 and 1630 cm^–1[28]. Through the ¹H-NMR spectrum, the vinylic H-

atom was confirmed at δ(H) 5.1 – 6.4 as a singlet, whereas ¹³C-NMR analysis confirmed the

C=O group at δ(C) 177 – 181 and the C=C bond of quinone at δ(C) 90 – 150. A detailed

assignment of the NMR spectra (¹H and ¹³C), ESI-MS and physical data for all new

compounds are presented in the experimental section.

Phytotoxic Activity. In a preliminary screening, the effect of 2,5-bis(alky/arylamino)-

1,4-benzoquinones 1-13 on the early growth of Sorghum bicolor and Cucumis sativus

seedlings was evaluated at 100 ppm (corresponding to 200 to 600 μM) by means of a Petri

dish assay [29-30]. The commercial herbicide Dual^®was used as a positive control (Fig. 1).

In the case of the monocotyledonous species, S. bicolor, compounds 1-4 and 9-10 inhibited

seedling growth by 50% or more. The amino acid derivatives 11-13 promoted root growth of

S. bicolor, a behavior already reported for some other quinones [14]. Against the

dicotyledonous species, C. sativus, a generally lower activity was evident, and only

compounds 1 and 7 inhibited growth by more than 50%, although compounds 5 and 12

strongly reduced root development. In both cases, compound 1 showed the highest

effectiveness, causing 60% and 85% inhibition of S. bicolor and C. sativus growth,

respectively. In previous reports, we and others have described the herbicidal activity (at

higher concentration, compared with the tests reported here) of a different series of

aminobenzoquinone derivatives [14; 25]. In those cases also it was observed that such class

of compounds is more phytotoxic toward dicotyledonous than monocotyledonous species.

Since compound 1 was the most active, the effect of increasing concentrations of this

substance on the growth of C. sativus was investigated, and compared with the commercial

herbicide Dual^®as reference. Results allowed to calculate the concentrations causing 50%

inhibition (IC₅₀) (Fig. 2), which in the case of the aminoquinone were 263 33 μM and 250

33 μM for roots and shoots, respectively, while for Dual^®they were one order of magnitude

lower, equal to 72 32 μM and 40 22 μM, respectively.

Further experiments were carried out to evaluate the ability of aminoquinones to

inhibit also the photoautotrophic growth of prokaryotes, using a model strain of the blue-

green alga Synechococcus. The results showed that compounds 8-10 were able to

significantly reduce cell proliferation at micromolar concentrations (Fig. 3). Once again, the

highest inhibitory potential was shown by compound 1, which at 100 μM completely

suppressed algal viability. In this case, similar effects were exerted also by compound 2.

To better define the inhibitory range, the effect of increasing levels of the two most

effective compounds was then investigated. The data obtained (Fig. 4) were used to calculate

IC₅₀values, which were 1.6 0.1 μM and 2.6 0.2 μM for compounds 1 and 2, respectively.

Interestingly, these values are two orders of magnitude lower than those previously found

against seedling growth (Fig. 3). In the absence of any information concerning the molecular

target(s) of these compounds, no plain explanation may be provided for this difference.

However, it is quite likely that the accessibility to the target molecules may play a role.

Indeed, in planta a drastic reduction of activity compared to that in vitro is frequently

observed, which may depend on poor penetration through the epidermal cuticle, poor

xylematic/phloematic translocation to the target tissue, or subcellular or tissue

compartmentalization. On the contrary, this is unlikely to occur in unicellular algae, where an

active compound needs to diffuse only a short distance to reach its target. Whatever the

reason, the results obtained with the algal strain clearly show that some aminoquinones are

able to exert phytotoxic activity in the low micromolar range.

Mode of action. At this stage of the research, a structure–activity relationship analysis

is unfeasible. However, an attempt to understand the effect of structural modifications of the

quinone scaffold on its biological activity was carried out using a relatively simple set of

rules that have been developed to help assess the bioavailability of agrochemicals on the basis

of key molecular structure descriptors and lipophilicity [29], a development of the Lipinsky

‘rule of five’ used in drug design [31-32]. In short, the most bioactive compounds against

plants and animals have been shown to be characterized by low molecular weight (≤ 500 Da),

moderate octan-1-ol/H₂O partition coefficient (log P ≤ 5), as well as a limited number of O

and N hydrogen donors or H-bonding sites. These rules have been applied successfully to

evaluate the herbicidal potential of a given substance [33]. The above parameters were thus

calculated for compounds 1–13 and Dual^®(Table 2). For all aminoquinones, comparable

values for log P and number of proton acceptor sites were obtained, all falling within the

range suggested by Tice et al. [31,34]. With the exception of compounds 5 and 7, log P

ranged in accordance with values proposed by Tice. In terms of the parameter Log S,

compounds 1-4, 9 and 11-12 have values within the expected range [35-37]. In summary,

only compounds 1-4 obey all the parameters proposed by Tice, and compound 1, the most

phytotoxic, has larger number of H donor sites and higher water solubility. Although

phytotoxicity cannot be justified only by these parameters, low log P and therefore high

hydrophylicity (log S) values of 1 and 2 should facilitate their absorption or permeation in the

plant cell [35-36].

Since some naturally occurring quinones are redox active molecules, they are

susceptible to interfere with electron transfer chains. In photosynthetic organisms,

plastoquinones play a central role in carrying electrons from photosystem II to the

cytochrome b6f complex. To verify whether the phytotoxic activity of aminoquinones may

depend on their ability to inhibit the chloroplastic electron transport, the light-driven

reduction of ferricyanide by isolated thylakoid membranes, the so-called Hill reaction, was

measured in the absence or in the presence of a given 2,5-bis(alkyamino)-1,4-benzoquinone,

as previously described [38] (Fig. 5). Contrary to what expected, no inhibition was observed.

Conversely, in the case of compounds 6 and 8-13, a slight stimulation was found, similar to

that due to a partial dissipation of the proton gradient across the membrane, which may be

caused by the presence of uncouplers [39]. Interestingly, the compounds exerting this effect

contain aromatic substituents, as well as amino acids fragments. However, benzoquinones are

well-known to undergo reversible oxido-reductive reactions [24]. This suggests that reduced

compounds 6 and 8-13 can react with the O₂in the medium producing the superoxide anion

[40], which in turn may damage the thylakoid membranes and partially dissipate the proton

gradient. On the whole, data seem therefore to point at the occurrence of other molecular

targets inside the cell, being aminoquinone phytotoxicity unrelated to the photosynthetic

electron transport chain.

Insecticidal and feeding deterrent activities. To verify whether aminoquinones may be

endowed with biological activity also against insects, the toxicity of a selected group of

compounds (1-7) bearing N-alkyl or N-aryl groups was evaluated on the beetle Rhyzopertha

dominica, the ant Solenopsis saevissima and the moth Tuta absoluta. These species were

selected because R. dominica causes massive loss of stored grains worldwide, S. saevissima is

a common household pests, and T. absoluta is a main pest affecting tomato fields in South

America, Europe and Africa. No significant mortality was observed for S. saevissima and R.

dominica, and similar results were obtained also with T. absoluta (Table 3).

As observed, the data suggest a low impact, if any, of these substances toward insects,

since a mortality rate of 30% is the limit used by the International Organization for Biological

Control for a compound to be considered as causing low or no impact on non-target

organisms [41]. An important group of beneficial insects are bees (Hymenoptera: Apidae),

and the disappearance of bee populations in several places on the planet is causing great

concern. No significant increase in mortality of the predatory ant S. saevissima (F_{5, 30}= 2.38,

P = 0.062) and the bee Tetragonisca angustula (F_{5, 30}= 2.24, P = 0.076) was caused by

substances (1 and 2). Moreover, when the feeding deterrent activity of compounds 1-7 was

evaluated against the butterfly Ascia monuste, once again no significant effects were observed

(Fig. 6).

Herbicides with ecological selectivity are ideal products for use on crops, since they

preserve populations of non-target organisms as natural pest enemies and pollinators [7]. The

decline of bee populations worldwide [11] is most likely due to the use of some pesticides

[42, 43]. Thus, a low toxicity to bees by aminoquinones would be of great importance in view

of their possible utilization for weed control.

Conclusions.- A group of new 2,5-bis(alkyl/arylamino)-1,4-benzoquinones were

synthesized by a simple methodology in satisfactory yield. These compounds showed a

moderate biological activity against the growth of both eukaryotic and prokaryotic

photosynthetic organisms. A significantly higher effectiveness in the latter case suggests that

the transport across plant tissues may limit their efficacy. The most active 2,5-

bis(methylamino)-1,4-benzoquinone (1) completely suppressed cyanobacterial growth at

concentrations as low as 10 μM. Despite the similarity with plastoquinone, the electron carrier

associated with the photosystem II, their phytotoxicity was found unrelated to interference

with the photosynthetic electron transport chain. Even at highest rate, benzoquinones were on

the contrary found to affect neither viability, nor feeding activity of various insects, a

desirable feature in the case of active ingredients for weed control. 2,5-Bis(alkyl/arylamino)-

1,4-benzoquinones may therefore represent a lead for the development of new herbicides.

Work is currently in progress in our laboratory to tailor their scaffold and increase their

effectiveness.

We thank the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq) and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), as

well as the University of Ferrara (FAR 2014) for financial support. We also thank Dr. Carlos

L. Zani and Dr. Markus Kohlhoff for HR-MS analysis.

Experimental Part

General Experimental Procedures. Hydroquinone and amines were purchased from

Aldrich. M.p.: MQAPF-301 apparatus (Microquimica, Brazil); uncorrected. IR Spectra:

PerkinElmer Paragon 1000 FT-IR spectrophotometer using KBr discs (1% w/w) and

scanning from 400 to 4,000 cm^–1; ෤ in cm^–1. ¹H- and ¹³C-NMR spectra: Varian Mercury 300

instrument (at 300 MHz and 75 MHz, resp.); δ in ppm rel. to Me₄Si as internal standard, J in

Hz. HR-MS: Bruker MicroToF spectrometer (resolution = 10,000 FWHM) using a lock-spray

source under ESI conditions; in m/z.

Synthesis

General Procedure for the Synthesis of Compounds 1 – 10, Exemplified by the

Synthesis of 2,5-Bis(methylamino)-1,4-benzoquinone (1). To a round-bottomed flask (25

ml) were added p-benzoquinone (0.162 g, 1.5 mmol), methylamine (0.015 g, 0.5 mmol) and

MeOH (5 ml). The mixture was stirred at r.t. for 1 h under normal atmosphere. The soln.

changed from colorless to red. MeOH was evaporated and the residue was purified by

column chromatography on silica gel eluting with hexane/AcOEt 2:1 to give compound 1 as

red crystals in 74% yield (0.37 mmol, 0.061 g). M.p.: 246 – 248°. IR: 3284, 3002, 2929,

1642, 1568, 1500, 1410, 1360, 1290, 1234, 1062, 706. ¹H-NMR ((D₆)DMSO): 2.75 (d, J =

5.37, H–C(1')); 5.15 (s, H–C(3,6)); 7.60 (s, NH). ¹³C-NMR ((D₆)DMSO): 28.61 (C(1'));

91.76 (C(3,6)); 152.25 (C(2,5)); 177.12 (C(1,4)). HR-ESI-MS: 167.0816 ([M + H]⁺,

+

C₈H₁₁N₂O₂; calc. 167.0776).

The other compounds 2 – 10 were prepared employing a procedure similar to that

described for 1. All the compounds were fully characterized by means of m.p., IR, ¹H- and

¹³C-NMR, and MS spectral data.

2,5-Bis(propylamino)-1,4-benzoquinone (2). Red crystals. Purified by column

chromatography, eluent hexane/AcOEt 3:1. Yield: 86%. The spectroscopy data matched with

those reported in [44].

2,5-Bis(isobutylamino)-1,4-benzoquinone (3). Red crystals. Purified by column

chromatography, eluent hexane/AcOEt 3:1. Yield: 83%. The spectral data matched with those

reported in [14].

2,5-Bis(pentylamino)-1,4-benzoquinone (4). Red crystals. Purified by column

chromatography, eluent hexane/AcOEt 3:1. Yield: 93%. The spectral data matched with those

reported in [14].

2,5-Bis(hexylamino)-1,4-benzoquinone (5). Red crystals. Purified by column

chromatography, eluent hexane/AcOEt 3:1. Yield: 78%. The spectral data matched with those

reported in [14].

2,5-Bis(benzylamino)-1,4-benzoquinone (6). Red crystals. Purified by column

chromatography, eluent CHCl₃/AcOEt 1:1. Yield: 87%. The spectral data matched with those

reported in [44].

2,5-Bis(4-methylanilino)-1,4-benzoquinone (7). Brown crystals. Purified by column

chromatography, eluent CHCl₃/hexane 1:2. Yield: 61%. M.p.: 299 – 300°. IR: 3236, 2916,

1634, 1556, 1480, 1288, 828. ¹H-NMR (CDCl₃): 2.36 (s, H–C(1'')); 6.01 (s, H–C(3,6)); 7.13

(d, J = 8.5, H–C(3',5')); 7.20 (d, J = 8.5, H–C(2',6')); 8.08 (s, NH). HR-ESI-MS: 341.1263

+

([M + Na]⁺, C₂₀H₁₈N₂NaO₂; calc. 341.1266).

2,5-Bis[(2-acetylphenyl)amino]-1,4-benzoquinone (8). Brown crystals. Yield: 65%.

M.p.: 267 – 268°. IR: 3206, 2922, 2980, 1652, 1642, 1604, 1566, 1454, 129, 770. ¹H-NMR

(CDCl₃): 2.67 (s, H–C(2'')); 6.42 (s, H–C(3,6)); 7.16 – 7.95 (m, H–C(3' – 6')); 11.21 (s, NH).

¹³C-NMR (CDCl₃): 28.65 (C(2'')); 100.11 (C(3,6)); 121.33; 123.48; 126.32; 134.20; 139.89

(C(1' – 6')); 144.77 (C(2,5)); 181.89 (C(1,4)); 201.22 (C(1'')). HR-ESI-MS: 375.1238 [M +

+

H]⁺, C₂₂H₁₉N₂O₄; calc. 375.1402).

2,5-Bis{[2-(hydroxymethyl)phenyl]amino}-1,4-benzoquinone (9). Brown crystals.

Purified by column chromatography, eluent AcOEt/Et₂O 1:1. Yield: 58%. M.p.: 211 – 212°.

IR: 3383, 3180, 2872, 1632, 1576, 1484, 1286, 904, 822, 738. ¹H-NMR ((D₆)DMSO): 4.47

(d, J = 5.0, H–C(1')); 5.49 (t, J = 5.2, OH); 5.53 (s, H–C(3,6)); 7.24 – 7.48 (m, H–C(2' – 6'));

9.36 (s, NH). ¹³C-NMR ((D₆)DMSO): 60.58 (C(1')); 95.02 (C(3,6)); 124.18; 126.17; 128.07;

128.70; 135.82; 136.25 (C(1' – 6')); 147.92 (C(2,5)); 179.38 (C(1,4)). HR-ESI-MS: 373.1149

+

([M + Na]⁺, C₂₀H₁₈N₂NaO₄; calc. 373.1164).

2,5-Bis{[3-(hydroxymethyl)phenyl]amino}-1,4-benzoquinone (10). Brown crystals.

Purified by column chromatography, eluent AcOEt/Et₂O 3:2. Yield: 77%. M.p.: 244 – 245°.

IR: 3426, 3240, 2908, 1638, 1568, 1428, 1288, 934, 876, 690. ¹H-NMR ((D₆)DMSO): 4.51

(d, J = 5.2, H–C(1')); 5.32 (t, J = 5.2, OH); 5.81 (s, H–C(3,6)); 7.16 – 7.41 (m, H–

C(2',4',5',6')); 9.34 (s, NH). ¹³C-NMR ((D₆)DMSO): 63.19 (C(1')); 95.95 (C(3,6)); 121.98;

122.85; 124.28; 129.68; 138.29; 144.60 (C(1' – 6')); 148.03 (C(2,5)); 180.39 (C(1,4)). HR-

+

ESI-MS: 373.1158 ([M + Na]⁺, C₂₀H₁₈N₂NaO₄; calc. 373.1164).

General Procedure for the Synthesis of (11 – 13), Exemplified by the Synthesis of 2,5-

Bis[(1-carboxyethyl)amino]-1,4-benzoquinone (= 2,2'-[(3,6-Dioxocyclohexa-1,4-diene-

1,4-diyl)diamino]dipropanoic Acid; 11). To a round bottom flask (25 ml) was added p-

benzoquinone (0.162 g, 1.5 mmol) in EtOH (10 ml). After complete dissolution, L-alanine

(0.045 g, 0.5 mmol) and NaHCO₃(0.084 g, 1.0 mmol) were added. The mixture was stirred

at r.t. under aerobic conditions. The reaction was monitored by TLC and stopped after 24 h.

The mixture was filtered to remove the NaHCO₃salt and unreacted amino acid, and washed

with EtOH. The resulting solution was concentrated under reduced pressure. The residue

obtained was acidified with 2.0 ml of concentrated HCl and dissolved with 5.0 ml of distilled

H₂O. The mixture was extracted with AcOEt (3 × 25 ml). The org. extracts were combined,

and the resulting org. phase was dried (MgSO₄), filtered, and concentrated under reduced

pressure. The obtained residue was purified by column chromatography on silica gel eluting

with acetone/MeOH 2.5:1.5 to give compound 11 as red crystals in 68% yield (0.048 g, 0.17

mmol). M.p.: 268 – 269°. IR: 3444, 3318, 2992, 2934, 1636, 1594, 1308, 780. ¹H-NMR

((D₆)DMSO/D₂O): 1.19 (d, J = 6.6, H–C(3')); 3.56 – 3.61 (m, H–C(1')); 5.13 (s, H–C(3,6));

7.75 (d, J = 6.6, NH). ¹³C-NMR ((D₆)DMSO/D₂O): 17.91 (C(3')); 52.08 (C(1')); 92.13

(C(3,6)); 150.03 (C(2,5)); 174.08 (C(1,4)); 177.18 (C(2')). HR-ESI-MS: 303.0597 ([M – 2 H

+

+ Na]^–, C₂₀H₁₈N₂NaO₄; calc. 303.0593).

2,5-Bis{[(1-carboxy-2,3-dimethyl)butyl]amino}-1,4-benzoquinone (= 2,2'-[(3,6-

Dioxocyclohexa-1,4-diene-1,4-diyl)diamino]bis(3,4-dimethylpentanoic Acid); 12). Red

crystals. Purified by column chromatography, eluent acetone/MeOH 2.5:1.5. Yield: 51%.

M.p. > 300°. IR: 3418, 3320, 2966, 2934, 2876, 1574, 1494, 1404, 1352, 816, 708. ¹H-NMR

((D₆)DMSO/D₂O): 0.78 (s, H–C(5',6')); 1.05 – 1.76 (m, H–C(3',4')); 3.63 (s, H–C(1')); 5.21

(s, H–C(3,6)). ¹³C-NMR ((D₆)DMSO/D₂O): 12.68 (C(5',6')); 16.68 (C(4')); 26.23 (C(3'));

62.52 (C(1')); 92.88 (C(3,6)); 152.12 (C(2,5)); 174.27 (C(1,4)); 177.85 (C(2')).

2,5-Bis[(1-carboxy-2-phenylethyl)amino]-1,4-benzoquinone (= 2,2'-[(3,6-

Dioxocyclohexa-1,4-diene-1,4-diyl)diamino]bis(3-phenylpropanoic Acid); 13). Red

crystals. Purified by column chromatography, eluent acetone/MeOH 2.5:1.5. Yield: 46%. The

spectral data matched with those reported in [27].

Chemical and Physicochemical Parameter Calculations. Molecular attributes

analyzed (cLog P (P = octan-1-ol/H₂O partition coefficient), the molecular weight (MW), the

amount of H-bond donors (HBD), the amount of H-bond acceptor (HBA), and the number of

rotable bonds (nRotb)) were calculated using the software provided by Molinspirations

Cheminformatics (Bratislava, Slovak Republic, www.molinspiration.com), whereas Log S

(solubility in H₂O, g/l) was calculated using the OSIRIS software [38].

Biological Assays. Phytotoxic Activity. The phytotoxic activity of 2,5-

bis(alkyl/arylamino)-1,4-benzoquinones 1 – 13 was evaluated as the ability to interfere with

the growth of Sorghum bicolor and Cucumis sativus seedlings. Stock solutions (100 ppm)

were prepared as follows: each compound (1.5 mg) was dissolved in DMSO (150 µl) and

Tween 80 surfactant (5 µl). The resultant suspension was shaken for 1 min, transferred into a

volumetric flask and the volume was brought with H₂O to 15 ml. The suspension was further

sonicated for 5 min. Growth in Petri dishes was carried out as previously described [29,30].

Aliquots (5 ml) of stock solutions were used to imbibe a sheet of filter paper (Whatman

number 1) placed in a Petri dish (100 × 15 mm). Seeds of Sorghum bicolor (L.) MOENCH

(Geneze Company, Paracatu, Minas Gerais State, Brazil) and Cucumis sativus L. (purchased

from a local market) were uniformly sown, 20 seeds for dish that was sealed with paraffin

film and placed in a growth chamber at 25° in the dark for 7 days. Seedlings were killed by

freezing for 24 h to facilitate handling, and the length of shoots and roots was determined for

each plantlet. Treatments were carried out in a completely randomized design with three

replications. The results were expressed as percentage growth with respect to untreated

controls. Data were analyzed by cluster analysis employing Statistica V 5.0 (Statistica^®,

Statsoft Inc., Tulsa, OK, USA, 1984 – 1995). Correlation parameters and curve fitting were

calculated using Graphpad Prism 4.0 (GraphPad Software Inc., San Diego CA, USA).

Measurement of the Photosynthetic Electron Transport by the Hill Reaction.

Photosynthetically active thylakoid membranes were isolated from market spinach (Spinacia

oleracea L.) leaves, as previously described [45]. Briefly, 20 g of deveined plant material

were resuspended in 100 ml of ice-cold 20 mM Tricine/NaOH buffer (pH 8.0), containing 10

mM NaCl, 5 mM MgCl₂and 0.4M sucrose, and homogenized for 30 s in a blender at maximal

speed. The homogenate was filtered through surgical gauze, and the filtrate was centrifuged

at 4° for 1 min at 500g; the supernatant was further centrifuged for 10 min at 1500g. Pelleted

chloroplasts were osmotically swollen by resuspension in sucrose-lacking buffer,

immediately diluted 1:1 with sucrose-containing buffer, and kept on ice in the dark until used.

Following dilution with 80% (v/v) acetone, the chlorophyll content was calculated on the

basis of Arnon’s formula. The basal rate of photosynthetic electron transport was measured

following light-driven ferricyanide reduction. Aliquots of membrane preparations

corresponding to 15 μg of chlorophyll were incubated at 24° in 1 ml cuvettes containing 20

mM Tricine/NaOH buffer (pH 8.0), 10 mM NaCl, 5 mM MgCl₂, 0.2M sucrose and 1 mM

K₃[Fe(CN)₆]. The assay was initiated by exposure to saturating light (800 μmol m⁻²s⁻¹), and

the rate of ferricyanide reduction was measured at 1 min intervals for 20 min in a GE

Healthcare Novaspec Plus spectrophotometer at 420 nm against an exact blank. Activity was

−1

calculated over the linear portion of the curve from a molar extinction coefficient of 1000 M

cm⁻¹. Compounds 1 – 13 were dissolved in DMSO so as to obtain 20 mM solns. that were

then diluted with H₂O, as appropriate. Their effect upon the photosynthetic electron transport

was evaluated in parallel assays in which the compounds were added to the reaction mixture

to a final concentration ranging from 0.1 to 200 μM. For each dose, the assay was carried out

at least in triplicate, and results were expressed as percentage of untreated controls. Reported

values are mean SE over replicates.

Cyanobacterial Growth. Synechococcus elongatus, strain PCC 6301, was grown at 24

1° under 14 h days (150 μmol m⁻²s⁻¹PAR) and 10 h nights in 250 ml Erlenmeyer flasks

containing 60 ml of ATCC minimal medium 616. Subcultures were made every 4 weeks by

transferring 10 ml of aliquots to 50 ml of fresh medium. Growth was followed by measuring

chlorophyll concentration. Culture aliquots (0.5 – 1.0 ml) were withdrawn, and cells were

sedimented by centrifugation for 3 min at 14000g. Pellets were resuspended with 1.0 ml of

MeOH, and solubilization was allowed to proceed for 30 min in the dark, with occasional

mixing. Samples were then centrifuged again, and total chlorophyll content in the supernatant

was estimated spectrophotometrically, as described [46]. Following logarithmic

transformation of data, growth constants and generation times were calculated from the linear

portion of each curve. Late log-grown cells were sedimented by centrifugation 5 min at

4000g and used to inoculate 25-well squared Petri dishes, 4.0 ml per well, to an initial

density of 2.0 mg l⁻¹chlorophyll. Suitable dilutions in ATCC minimal medium 616 of a 20

mM stock solution of a given compound in DMSO were added so as to obtain concentrations

ranging from 5 to 100 μM. A complete randomized design with four replications was adopted.

Cell growth in each well was followed for 1 week by daily destructive harvest, as indicated.

Following logarithmic transformation of data, growth constants were calculated and

expressed as percent of the mean value for untreated controls. Means SE over replicates are

reported.

Insecticidal Activity. For all species used (Rhyzopertha dominica (FABR.) (Coleoptera:

Bostrychidae), Solenopsis saevissima SMITH (Hymenoptera: Formicidae), Tuta absoluta

(MEYRICK) (Lepidoptera: Gelechiidae) and Tetragonisca angustula (Hymenoptera: Apidae),

bioassays were performed over 48 h. Compounds (5.0 mg) were dissolved in acetone (150 µl)

and a 0.5 µl of aliquot was applied to each insect through a microsyringe. Controls were

treated topically with the same volume of pure acetone. The experimental design was

completely randomized with three replications. The experiments were conducted in

polystyrene plastic pots (120 × 90 mm) containing 10 insects each. The pots were covered

with screen to prevent escape of insects, but allowing the entry of oxygen. After the

treatment, insects were fed (leaflets of tomato, cultivar Santa Clara, for T. absoluta; wheat

grain for R. dominica; candy (85% sugar and 15% honey) for S. saevissima). For the latter

species, H₂O was also provided by means of a moistened piece of cotton. Plastic jars with

insects were wrapped and kept at a temperature of 25 0.5°, at a relative humidity of 75

5% under a photoperiod of 12 h, except for the ant, which was maintained in the dark. Insect

viability was assessed 24 and 48 h after the treatment. The mortality of T. angustula was

subjected to analysis of variance (P < 0.05).

Feeding Deterrence. The compounds were dissolved in acetone at 50 mg ml^–1and

immediately sprayed using a Potter tower calibrated to 69 kPa onto cabbage (Brassica

oleracea var. acephala) leaf discs in Petri dishes (10 cm diameter; 78.54 cm²area), placed so

as that the discs covered the entire surface of the plate. Controls were treated with the same

volume (1 ml) of acetone. After evaporation of the solvent, 10 fourth instar caterpillars (Ascia

monustes (L.) (Lepidoptera: Pieridae)) were carefully transferred into each plate using a soft

bristle brush. Three replicates were set up per treatment. The Petri dishes were photographed

(Canon EOSDigital Rebel XS) 1, 4, 8, 10, 17, 24 and 32 h thereafter. Photos were edited

using the Paint program NET 3.5.11 (dot NDP, 2013), and the leaf area consumed was

measured using Image Tools 3.6 (Union D, 2013).

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Received November 3, 2015

Accepted January 5, 2016

Table 1. Optimization of the reaction conditions^a).

Entry

Quinone or Hydroquinone

([equiv.])

Hq (1)

Amine

([equiv.])

A (1)

A (2)

A (1)

B (1)

B (2)

B (1)

Time

[h]

1

Product

Yield

[%]

24

1

2

4

Hq (1)

18

24

36

1

45

4

3

Hq (1)

48

4

Hq (1)

24

4

5

Bq (3)

93

4

6

Hq (1)

18

36

24

36

10

7

Hq (1)

45

8

Hq (1)

10

9

Bq (1)

25

10

Bq (3)

77

^a) A = Amylamine; B = 3-aminobenzylacohol; Hq = Hydroquinone; Bq = p-

Benzoquinone.

Table 2. Optimal molecular descriptors proposed for herbicides as applied to compounds 1 –

13 and the commercial herbicide Dual^®.

Mol. weight

cLog P

H-Donors H-Acceptors

nRotb

Log S

≥ –4

Tice values

≥ 150 and ≤ 500 ≤ 5

≤ 3

≥ 2 and ≤ 12

≤ 12

(Herbicides)

Compound

166.18

0.127

2

4

0

4

6

8

3

2

–1.68

–2.5

1

2

222.288

250.342

278.396

306.45

1.884

2.371

4.013

5.024

2.924

5.293

4.097

2.975

3.023

–2.299

2.262

0.623

3.548

6

–3.11

–3.94

–4.35

–5.05

–5.27

–5.07

–3.99

–4.06

–2.48

–3.87

–4.75

–3.43

3

10

12

6

4

5

318.376

374.396

350.374

282.252

394.468

434.448

283.799

6

4

7

6

8

6

9

6

10

11

12

13

Dual

6

10

6

Table 3. Insecticidal activity of compounds 1 – 7 at the concentration of 30 mg (g of insect)^–1

against Rhyzopertha dominica, Solenopsis saevissima, and Tuta absoluta.

Compound

Mortality [%]

R. dominica

S. saevissima

24 h

T. absoluta

24 h

48 h

0.0

3.3 0.3

6.6 0.3

6.6 0.6

0.0

18.3 0.8

8.4 0.3

25.0 0.5

38.5 1.2

20.8 0.8

23.7 1.0

23.3 0.6

12.5 0.5

22.5 0.5

8.4 0.3

29.1 0.6

38.5 1.2

20.8 0.8

23.7 1.0

23.3 0.6

12.5 0.5

1

3.3 0.3

0.0

6.6 0.6

0.0

3.3 0.3

6.6 0.6

0.0

2

3

0.0

4

6.6 0.3

3.3 0.3

0.0

6.6 0.3

3.3 0.3

0.0

10.0 1.0

3.3 0.3

5

0.0

6

0.0

7

Control

0.0

3.3 0.0

Article Doi

Amino-substituted para-Benzoquinones as Potential Herbicides

DOI: 10.1002/cbdv.201500340

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