Synthesis and biological evaluation of 2,5-bis(alkylamino)-1,4- benzoquinones

Article abstract of DOI:10.3390/molecules15085629

A series of twelve 2,5-bis(alkylamino)-1,4-benzoquinones were prepared in yields ranging from 9-58% via the reaction between p-benzoquinone and various amines. The structures of the synthesized compounds were confirmed by IR, ¹H- and ¹³

Full text of DOI:10.3390/molecules15085629

Molecules 2010, 15, 5629-5643; doi:10.3390/molecules15085629
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of 2,5-Bis(alkylamino)-1,4-
benzoquinones
Luiz Cláudio Almeida Barbosa 1,*, Ulisses Alves Pereira 1, Célia Regina Alvares Maltha 1,
Róbson Ricardo Teixeira 1, Vânia Maria Moreira Valente 1, José Roberto Oliveira Ferreira 2,
Letícia Veras Costa-Lotufo 2, Manoel Odorico Moraes 2 and Cláudia Pessoa 2
1
Department of Chemistry, Federal University of Viçosa. Av. P.H. Rolfs, CEP 36570-000, Viçosa,
MG, Brazil
2
Department of Physiology and Pharmacology, Federal University of Ceará, St. Coronel Nunes de
Melo, 1127, CEP 60431-970, Fortaleza, CE, Brazil
* Author to whom correspondence should be addressed; E-Mail: lcab@ufv.br;
Tel.: +55-31-3899-3068.
Received: 14 June 2010; in revised form: 14 July 2010 / Accepted: 16 July 2010 /
Published: 13 August 2010
Abstract: A series of twelve 2,5-bis(alkylamino)-1,4-benzoquinones were prepared in
yields ranging from 9–58% via the reaction between p-benzoquinone and various amines.
The structures of the synthesized compounds were confirmed by IR, 1H- and 13C-NMR and
MS analyses. The phytotoxicity of the 2,5-bis(alkylamino)-1,4-benzoquinones was
evaluated against two crop species, Cucumis sativus and Sorgum bicolor, at 1.0 × 10-3
mol/L. In general, the quinones displayed inhibitory effects on the dicotyledonous species
C. sativus (7–74%). On the other hand stimulatory effects were observed on S. bicolor
(monocotyledonous). Similar results were observed in the biological assays carried out
with the weed species Ipomoea grandifolia (dicotyledonous) and Brachiaria decumbens
(monocotyledonous). In addition, the cytotoxicity of the 2,5-bis(alkylamino)-1,4-
benzoquinones was assayed against HL-60 (leukemia), MDA-MB-435 (melanoma), SF-
295 (brain) and HCT-8 (colon) human cancer cell lines and human peripheral blood
mononuclear cells (PBMC), as representatives of healthy cells, using a MTT and an
Alamar Blue assay. Compound 12 was the most active, displaying cytotoxicity against all
cancer cell lines tested.
Keywords: quinones; herbicide; cytotoxicity

Molecules 2010, 15
5630
1. Introduction
Plant roots perform a variety of functions such as mechanical support, water/nitrogen uptake, and
production of exudates. It is known that roots are capable of producing and secreting compounds
(exudates) into the rhizosphere. Root exudation includes release of ions, free oxygen and water,
enzymes, mucilage, and a range of organic compounds (primary and secondary metabolites) [1-4].
Various organic compounds present in root exudates are involved in chemical-mediated plant-plant
or allelopathy interactions. Allelopathy is concerned with the chemical interactions that occur among
plants and is mediated by the release of allelochemicals into the rhizosphere [5,6]. The study of such
interactions has led to the discovery of several phytotoxic substances that have potential use as
herbicides [7], or could be used as lead structures for the development of more active compounds [8].
Among such substances is a compound called sorgoleone (1, Figure 1), isolated from root exudates of
Sorghum bicolor [9] and characterized as 2-hydroxy-5-methoxy-3-[(8Z,11Z)-pentadeca-8,11,14-
trienyl]- cyclohexa-2,5-dien-1,4-dione.
Figure 1. Structures of several benzoquinones.
O
O
O
O
O
OH
O
OH
MeO
MeO
O
Maesaquinone (2)
Sorgoleone (1)
Me Me

3
Lapachone ( )
O
O
O
O
O
O
O
O
O
OMe
OR
H2N
H2N
OCONH2
OMe
Me
n
MeO
X
N
OHC
OMe
O
NH
O
(6): X = O; n=3
(7): X = CH2; n=4
Cordiaquinone J (10)
4
Mitomycin C ( )
5
Globiferin ( )
(8) R = tridecyl
(9) R = octadecyl
Since its discovery, it has been demonstrated that 1 is a potent inhibitor of chlorophyll formation in
Lemma minor L., and it also inhibits the growth of several grass and broadleaf weeds at concentrations
as low as 10 mol/L [10]. Further investigations revealed that 1 is an effective inhibitor of electron
-
transfer between QA to QB at the reducing side of photosystem II [11] and also of the mitochondrial
electron transport [12,13]. During in vitro assays, 1 has been shown to be more active than the
commercial herbicide atrazine in inhibiting photosystem II [14]. This quinone also causes disturbance
of plasma H+-ATPase activity in root cells [15].
It has also been demonstrated that several benzoquinones displayed cytotoxic effects. For example,
the naturally occurring maesaquinone (2, Figure 1) exhibited in vitro cytotoxicity against several solid
tumor cells [16]. -Lapachone (3, Figure 1), has a diversity of useful biological activities against
various cancer cell lines such as human ovarian and prostate tumors and, at lower doses, is a
radiosensitizer of several human cancer cell lines [17]. The quinone mitomycin C (4, Figure 1) has
been used in chemotherapy against certain solid tumors [18,19]. More recently, the biological profile
of globiferin (5, Figure 1) a terpenoid benzoquinone isolated from root extracts of Cordia globifera

Molecules 2010, 15
5631
was investigated [20]. It was found that this compound displays cytotoxic activity against NCI-H187
cell line. The biological profile of cordiaquinone J (10, Figure 1), a 1,4-naphthoquinone isolated from
the roots of Cordia leucocephala, has been shown to present antiproliferative effects related to reactive
oxygen species (ROS) generation [21].
In the last few years, we have targeted various natural products as lead structures towards the
development of plant growth regulators [22-29], including sorgolene (1) [30,31]. In this context,
several sorgoleone derivatives, such as compounds 6-9 (Figure 1), were prepared and subsequently
evaluated against crop and weed species. It has been found that the synthetic analogues 6 and 7 are
more effective than 1. Moreover, while compound 6 inhibited the root development of the weed
species Euphorbia heterophylla and Brachiaria decumbens, compound 7 was effective in inhibiting
the development of the aerial parts and roots of B. decumbens, an aggressive weed commonly found in
several crop plantations in Brazil. When tested against E. heterophylla, the aryl-p-benzoquinone 8
caused 34.2 and 76.5% inhibition of the aerial part and roots, respectively. The quinone 9 significantly
inhibited the aerial parts (5l.7%) and roots (85.2%) of the weed species Ipomoea grandifolia.
In continuation to our studies in this area we report in this paper the preparation and investigation of
the phytotoxic effects of twelve 2,5-bis(alkylamino)-1,4-benzoquinone analogues of sorgoleone (1).
Considering the cytotoxic effects reported in the literature for several benzoquinones, we also
evaluated the cytotoxicity of these analogues against several human cancer cell lines and peripheral
blood mononuclear cells (PMBC). The results of the cytotoxicity screening are also discussed.
2. Results and Discussion
2.1. Synthesis of 2,5-diamino-p-benzoquinones
Compounds 12-23 were synthesized by the reaction between p-benzoquinone 11 and various
amines [32-34]. A 3:2 molar ratio of benzoquinone/amine was required due to successive reductions
and oxidations involved in the formation of the products as shown in Scheme 1. The reaction was
carried out first in dry EtOH and later it was found that slightly better yields were obtained when wet
EtOH containing 2% (v/v) of H2O was used. Although no detailed investigation on the mechanism was
carried out, we believe that H2O might enhance the reactivity and polarizability of the quinone
carbonyl group facilitating the nucleophilic addition step [33].
After column chromatography purification, the 2,5-bis(alkylamino)-1,4-benzoquinones were obtained
as red compounds. Bayen and co-workers [32] reported the preparation of several 2,5-bis(alkylamino)-
1,4-benzoquinones in good yields employing similar conditions used in the present work. In our hands,
however, benzoquinones 12-23 were obtained in only low to fair yields (Scheme 1). This fact can
possibly be attributed to polymerization side reactions as previously reported [35]. These results are
also consistent with a great quantity of base line material observed during the column chromatographic
purification.
The IR spectra of compounds 12-23 exhibited strong absorptions in the 1,613 to 1,550 cm-1 range
due to carbonyl stretchings. The vinylic H-atoms in these quinones were characterized in the NMR
spectrum by signals observed at 5.3–5.4 ppm, while resonance signals for the C=O groups were
observed at 176-180 ppm. The expected molecular formulae were confirmed by high resolution ESI-MS.

Molecules 2010, 15
Scheme 1. Synthesis of 2,5-bis(alkylamino)-1,4-benzoquinones 12-23.
5632
O
OH
O
OH
O
OH
R1
EtOH
R1R2NH
N
+
R2
O
HO
O
O
OH
OH
11
O
O
R1
R2
OH
2
N
R1
R1
R2
6
5
1
4
R1
+
R1R2NH
N
N
R1
R2
N
3
R2
N
R2
O
O
HO
12-23
Compound
Amines
R1
R2
Isolated yield [%]
12
13
14
15
16
17
18
19
Ethylamine
CH3CH2
H
H
H
H
H
H
H
H
28
58
21
39
28
28
36
25
Isopropylamine
Butylamine
(CH3)2CH
CH3[CH2]2CH2
(CH3)2CHCH2
CH3[CH2]3CH2
Isobutylamine
Pentylamine
Isopentylamine
Hexylamine
Decylamine
(CH3)2CHCH2CH2
CH3 [CH2]4CH2
CH3 [CH2]8CH2
N
CH2CH2
20
21
2-(Piperidin-1-yl)ethylamine
2-Morpholinoethylamine
H
H
20
33
O
N CH2CH2
22
23
Diethylamine
CH3CH2
C6H11
CH3CH2
H
9
Cyclohexylamine
20
2.2. Phytotoxic activity
In a preliminary screen carried out on Petri dishes the effect of 2,5-bis(alkylamino)-1,4-
benzoquinones 12-23 on the radicle growth of S. bicolor and C. sativus was evaluated at a concentration
of 1.0 × 10-3 mol/L (Table 1).

Molecules 2010, 15
5633
Table 1. Effect of 2,5-bis(alkylamino)-1,4-benzoquinones on the radicle growth of C.
sativus and S. bicolor at 1.0 × 10-3 mol/L.
Cucumis sativus
Sorghum bicolor
24 h
48 h
24 h
48 h
Compound
Radicle
length
[cm]a
Radicle
length
[cm]a
Radicle
lenght
[cm]a
Radicle
Inhibition
Inhibition
Inhibition
Inhibition
[%]
length
[cm]a
[%]
[%]
[%]
12
13
14
15
16
17
18
19
20
21
1.40 def
1.75 cde
2.17 abc
2.02 abc
2.02 abc
1.89 bcd
1.25 ef
42
28
10
17
17
22
48
7
1.94 fg
3.91 bcd
4.87 a
59
18
-2
0.87 cd
1.90 ab
1.69 ab
1.14 bcd
1.52 abc
1.17 bcd
0.49 d
36
-40
-24
17
1.04 fg
30
-135
-148
-26
3.50 abc
3.70 ab
3.43 de
4.24 abcd
2.73 ef
1.25 g
28
11
43
74
22
51
7
1.87 defg
1.91 defg
2.25 bcdef
0.51 g
-12
14
-28
-51
64
66
2.26 abc
1.85 cd
2.46 a
3.73 ef
2.33 f
1.81 ab
1.23 bcd
2.09 a
-33
10
3.81 a
-156
-39
24
-2
2.07 cdef
3.21 abcd
4.41 abc
-54
-115
22
1.16 f
52
13
1.79 fg
62
7
0.85 cd
38
-8
0.92 fg
38
23
2.10 abc
2.42 ab
4.41 abc
4.76 ab
1.47 abc
1.36 abc
2.82 abcde
1.49 efg
-89
Control
a Means, in the same column, with the same letter are not significantly different at P = 0.05% by Tukey’s test.
This type of biological assay is commonly used as a general screening for identifying potential
phytotoxic substances [36]. With the exception of compound 14, all of the remaining substances
inhibited the radicle growth of the dicotyledonous species C. sativus after 48 h. Compound 18 displayed
the highest effectiveness against this species, causing 74% inhibition. A simple correlation could not be
found between the length of the side chain and the observed biological activity of compounds 12, 13, 14,
16, 18, and 19. Comparing quinones 14 (-2%) and 15 (28%) as well as 16 (11%) and 17 (43%) it can be
observed that the compounds with an unbranched side chain exhibited major inhibitory activity in
relation to the branched side chain. Considering compounds 20, 21, and 23, which present cyclic groups
in their side chain portions, the highest inhibitory effect was associated with compound 20. From the
results presented in Table 1 it is clear that while inhibitory effects were observed for C. sativus, after 48
hours radicle promotion was noticed with the compounds for the monocotyledonous species S. bicolor.
Exceptions to this generalization were the substances 12, 18, and 22.
The effect of the compounds 12-23 on the radicle growth of the weed species Ipomoea grandifolia
(dicotiledonous) and Brachiaria decumbens (monocotiledonous) was subsequently investigated. As
presented in Table 2, differential effects were observed between the two weed species. As a general
trend, inhibitory effects were observed for I. grandifolia while stimulation was noticed for
B. decumbens.

Molecules 2010, 15
5634
Table 2. Effect of 2,5-bis(alkylamino)-1,4-benzoquinones on the radicle growth of I.
grandifolia and B. decumbens at 1.0 × 10-3 mol/L.
Ipomoea grandifolia
Brachiaria decumbens
24 h
48 h
24 h
48 h
Compound
Radicle
length
[cm]a
Radicle
length
[cm]a
Radicle
lenght
[cm]a
Radicle
Inhibition
Inhibition
Inhibition
[%]
Inhibition
[%]
length
[cm]a
[%]
[%]
12
13
0.91 de
1.19 cd
1.41 abc
1.43 abc
1.48 abc
1.23 cd
0.94 de
1.69 ab
1.22 cd
1.32 bcd
0.77 e
48
32
20
19
16
30
47
4
0.91 d
67
10
0
0.87 cd
1.82 a
47
-11
-3
1.06 bcd
2.90 a
43
-56
-65
-26
19
2.47 ab
2.74 a
14
1.69 ab
1.48 abc
1.10 bcd
1.59 abc
0.45 d
3.07 a
15
2.39 ab
2.41 ab
1.49 bcd
0.94 d
13
12
46
66
18
43
12
51
1
10
33
3
2.35 ab
1.50 bcd
2.21 ab
0.45 d
16
17
-19
76
18
73
-2
19
2.25 abc
1.57 bcd
2.40 ab
1.35 cd
2.72 a
1.67 ab
1.51 abc
1.80 ab
0.65 d
2.98 a
-60
-4
20
31
26
56
23
8
1.93 abc
2.82 a
21
-10
60
-5
-51
54
22
0.86 cd
3.02 a
23
1.35 bc
1.76 a
1.73 ab
1.64 ab
-62
Control
2.72 a
1.86 abc
a Means, in the same column, with the same letter are not significantly different at P = 0.05% by Tukey’s test
2.3. ATP assay
Considering a previous report on the literature concerning the influence of 2,5-diaminobenzo-
quinones on ATP synthesis [37], we assessed the ability of compounds 12, 13, and 23 to interfere with
this process. The choice of these substances was made based on the higher availability of them in our
laboratories. These three compounds displayed inhibitory activity on the ATP synthesis in isolated
spinach chloroplasts. The best inhibitory activity was observed for compound 13 which presented an
IC50 equal to 150 µM (data not shown).
2.4. Anti-tumor activity
The anti-tumor activity of the 2,5-bis(alkylamino)-1,4-benzoquinone sorgoleone analogues 12-14,
16-23 was evaluated against four human cancer cell lines: HL-60 (promyelocytic leukemia), HCT-8
(colon), SF-295 (central nervous systems) and MDA-MB-435 (melanoma), obtained from the National
Cancer Institute (Bethesda, MD, USA), using the MTT assay as previously described [38]. To
investigate the selectivity of different compounds toward normal proliferating cells, an Alamar Blue
assay was performed with PBMC after 72 h of drug exposure. The results of the IC50 data [g/mL] for
the antitumor activities are presented in Table 3. Doxorubicin was used as positive control. After 72 h,
compounds 12 and 20 exhibited cytotoxicity against all tumor cell lines tested. Compound 12 was a
more potent proliferation inhibitor (IC50 values in the range 2.0–6.0 µg/mL) than compound 20. The
other compounds investigated were not able to significantly inhibit cell growth under the assay
conditions, presenting IC50 values higher than 25 µg/mL.

Molecules 2010, 15
5635
The cytotoxic activity of the compounds 12 and 20 against normal cells (PBMC) was assessed by
Alamar Blue assay [39]. This assay was chosen because of its low toxicity to normal cells [40]. It was
found that compound 20 was cytotoxic, with an IC50 value 21.8 µg/mL. Compound 12 showed no
activity against PBMC at the highest concentration tested (>25 µg/mL). This compound exhibited the
most selective cytotoxicity against the tumor lines and is not toxic to normal cells. It is important to
emphasize that selectivity is one important feature towards the development of new antitumoral
drugs [41].
Table 3. Cytotoxic activity of 2,5-bis(alkylamino)-1,4-benzoquinone analogues to sorgoleone.
Cellsa
IC50b [µg/mL]; Confident interval
Compound
HL-60
2.3(1.3-3.9)
>25
SF-295
6.0 (4.6-7.9)
>25
HCT-8
5.2(2.4-11.2)
>25
MDA-MB-435
5.6(3.8-8.4)
>25
PBMC
>25
Nd
Nd
Nd
Nd
Nd
Nd
12
13
14
16
17
18
19
20
21
22
23
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
20.3(17.6-23.4) 11.3(8.6-14.9)
13.5(9.1-20.1) 21.5(18.7-24.8) 21,8 (15,6-30,1)
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
Nd
Nd
Nd
Doxorrubicin 0.02(0.01-0.02) 0.23(0.19-0.25) 0.01(0.01-0.02) 0.48(0.34-0.66) 0,96 (0,51-1,71)
a Cells were plated in 96-well plates incubated under a 5% CO2 atmosphere at 37 oC for 72 h in the
presence of pure compounds (0.39-25 g/mL). Each concentration was tested in triplicate and the
b
analyses were performed in duplicate; Data are presented as IC50 [g/mL] values and 95%
confidence interval (given in parentheses) obtained from at least three independent experiments.
Compound 15 was not evaluated due to limited amount. Nd: not determined.
The quinone structural motif is present in many anticancer drugs such as anthracyclines
(daunorubicin, doxorubicin), mitomycin and mitoxantrone, which are used clinically in the therapy of
solid tumors [42]. The mechanisms by which quinones cause these effects can be quite complex.
Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular
proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can, by
intermediacy of their semiquinone radicals, lead to formation of reactive oxygen species (ROS), which
can cause severe oxidative stress within cells through the formation of oxidized cellular
macromolecules, including lipids, proteins, and DNA. ROS can also activate a number of signaling
pathways. Additionally, quinones, including azaquinones, can work as DNA intercalators, inhibitors of
topoisomerases and of some enzymes of the mitochondrial electron transfer chain.
3. Experimental
3.1. General
Hydroquinone and amines were purchased from Aldrich (Milwaukee, WI, USA) Reagents and
solvents were purified, when necessary, according to procedures described by Perrin and Armarego

Molecules 2010, 15
5636
[43]. p-Benzoquinone 11 was synthesized from hydroquinone employing a previously described
procedure [44]. Analytical thin layer chromatography analyses were conducted on aluminum backed
pre-coated silica gel plates. Column chromatography was performed on silica gel (60–230 mesh) and
eluting with hexane:diethyl ether mixtures. Melting points are uncorrected and were obtained on a
MQAPF-301 apparatus (Microquimica, Brazil). IR spectra were recorded on a Perkin Elmer Paragon
1000 FTIR spectrophotometer using KBr discs (1% w/w) and scanning from 400 to 4,000 cm-1. The
1H- and 13C-NMR spectra were recorded on a Varian Mercury 300 instrument (at 300 MHz and
75 MHz, respectively), using deuterated CHCl3 as solvent and tetramethylsilane (TMS) as internal
standard ( = 0); coupling constants (J) in Hz. MS were recorded on a Shimadzu GCMS-QP5050A
instrument under electron impact (70 eV) conditions. HRMS data were recorded under ESI conditions
on a Bruker MicroToF spectrometer (resolution = 10,000 FWHM) using a lock-spray source. The
lock-mass used for calibration was tetraoctylammonium bromide in positive ion mode.
3.2. Synthesis of compounds 12-16, 18-23, exemplified by the synthesis of 2,5-bis(isopentylamino)-1,4-
benzoquinone (17)
To a round-bottomed flask (125 mL) were added p-benzoquinone 11 (500 mg, 4.63 mmol) and
EtOH 98% (v/v) (10 mL). After complete dissolution of 11, isopentylamine (268 mg, 3.08 mmol)
dissolved in EtOH 98% (v/v) (6 mL) was added slowly. The mixture was stirred at r.t. until complete
consumption of the starting material (TLC analysis). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography eluting with 1:3 (v/v)
hexane/CH2Cl2 to give compound 17 as red crystals in 28% yield (122 mg, 0.44 mmol).
Mp 167.3–169.7 ºC; IR: 3,264 (NH), 2,956, 2,870, 1,642, 1,549, 1,495, 1,459, 1,364, 1,283, 1,231,
1,071, and 812 cm-1; 1H-NMR δ: 6.59 (bs, NH); 5.30 [s, H-C(3), H-C(6)]; 3.16 [q, J = 6.9 Hz,
2 H-C(1′), 2 H-C(1′′)]; 1.61-1.72 [m, H-C(3′), H-C(3′′)]; 1.54 [q, J = 6.9 Hz, 2 H-C(2′), 2 H-C(2′′)];
0.93 [d, J = 6.9 Hz, 3 H-C(4′), 3 H-C(5′), 3 H-C(4′′), 3 H-C(5′′)]; 13C-NMR δ: 176.1 [C(1), C(4)];
149.4 [C(2), C(5)]; 90.6 [C(3), C(6)]; 38.9 [C(1′), C(1′′)]; 35.0 [C(2′), C(2′′)]; 23.9 [C(3′), C(3′′)]; 20.4
[C(4′), C(5′), C(4′′), C(5′′)]; MS, m/z (%): 278 (57) [M+], 235 (87), 222 (100), 221 (51), 208 (15), 194
(14), 179 (27), 163 (23), 152 (15), 138 (14), 125 (18), 82 (17), 68 (27), 55 (24); HRMS (ESI TOF-MS):
Calcd. for C16H27N2O2 279.2067; found: 279.2069.
The other compounds 12-16, 18-23 were prepared employing a procedure similar to that described
1
for 17, and yields are presented in Scheme 1. All the compounds were fully characterized by IR, H-
and 13C-NMR and mass spectrometry. The structural characterization of compounds 12, 13, 14, 18, 22,
and 23 has already been described [34,45,46]. Compounds 15, 16, 17, 20, and 21 have already been
synthesized, although their complete spectroscopic data were not given [47-51]. Structures for 15, 16,
17, 20, and 21 and the remaining compounds are supported by the following spectroscopic data:
2,5-Bis(isobutylamino)-1,4-benzoquinone (15). Red crystals. Purified by column chromatography,
eluent hexane/diethylether 1:1 (v/v). Mp 188.4–191.5 ºC; IR: 3,274 (NH), 2,953, 2,869, 1,645, 1,554,
1,491, 1,443, 1,366, 1,255, 1,065, 815, and 723 cm-1; 1H-NMR δ: 6.71 (br. s, NH); 5.37 [s, H-C(3), H-
C(6)]; 2.97 [t, J = 6.6 Hz, 2 H-C(1′), 2 H-C(1′′)]; 1.90-2.04 [m, H-C(2′), H-C(2′′)]; 0.98 [d, J = 6.6 Hz,
3 H-C(3′), 3 H-C(4′), 3 H-C(3′′), 3 H-C(4′′)]; 13C-NMR δ: 178.3 [C(1), C(4)]; 151.8 [C(2), C(5)] 93.0

Molecules 2010, 15
5637
[C(3), C(6)]; 50.3 [C(1′), C(1′′)]; 28.0 [C(2′), C(2′′)]; 20.5 [C(3′), C(4′), C(3′′), C(4′′)]; MS, m/z (%):
250 (84) [M+], 235 (13), 207 (100), 193 (40), 165 (28), 164 (31), 151 (60), 138 (23), 123 (20), 67 (21),
53 (46); HRMS (ESI TOF-MS): Calcd. for C14H23N2O2 251.1754; found: 251.1756.
2,5-Bis-(pentylamino)-1,4-benzoquinone (16). Red crystals. Purified by column chromatography,
eluent hexane/dichloromethane 1:2 (v/v). Mp 134.6–137.1 ºC; IR: 3,263 (NH), 2,957, 2,927, 2,857,
1,644, 1,553, 1,497, 1,463, 1,369, 1271, and 812 cm-1; 1H-NMR (* indicates assignments that could be
reversed) δ: 6.62 (br. s, NH), 5.28 [s, H-C(3), H-C(6)]; 3.12 [q, J = 6.8 Hz, 2 H-C(1′); 2 H-C(1′′)];
1.59-1.68 [m, 2 H-C(2′), 2 H-C(2′′)]; 1.21-1.38 [m, 2 H-C(3′), 2 H-C(4′), 2 H-C(3′′), 2 H-C(4′′)];
0.86-0.91 [m, 3 H-C(5′), 3 H-C(5′′)]; 13C-NMR δ: 177.1 [C(1), C(4)]; 150.4 [C(2), C(5)]; 91.6 [C(3),
C(6)]; 41.6 [C(1′), C(1′′)]; 28.1 [C(3′), C(3′′)]*; 27.0 [C(2′), C(2′′)]; 21.3 [C(4′), C(4′′)]*; 13.0
[C(5′), C(5′′)]; MS, m/z (%): 278 (100) [M+], 235 (73), 223 (30), 207 (40), 193 (46), 179 (28), 165
(62), 164 (19), 151 (45), 138 (23), 137 (23), 110 (23), 67 (54), 54 (56); HRMS (ESI TOF-MS): Calcd.
for C16H27N2O2 279.2067; found: 279.2070.
2,5-Bis-(decylamino)-1,4-benzoquinone (19): Red crystals. Purified by column chromatography, eluent
hexane/diethylether 2:1 (v/v). Mp 126.4–127.3 ºC; IR: 3,256 (NH), 2,954, 2,917, 2,848, 1,644, 1,554,
1,503, 1,456, 1,365, 1,291, and 683 cm-1; 1H-NMR δ: 6.62 (br. s, NH); 5.30 [s, H-C(3), H-C(6)]; 3.13
[q, J = 6.6 Hz, 2 H-C(1′), 2 H-C(1′′)]; 1.50-1.70 [m, 2 H-C(2′), 2 H-C(2′′)]; 1.15-1.40 [m, 14 H-C(3′-
13
9′), 14 H-C(3′′-9′′)]; 0.88 [t, J = 6.6 Hz, 3 H-C(10′), 3 H-C(10′′)]; C-NMR (* indicates assignments
that could be reversed) δ: 178.3 [C(1), C(4)]; 151.6 (C(2), C(5)]; 92.8 [C(3), C(6)]; 42.8 (C(1′), C(1′′)];
32.1 [C(2′), C(2′′)]*; 29.7 [C(8′), C(8′′)]*; 29.7 [C(3′), C(3′′)]*; 29.5 [C(4′), C(4′′)]*; 29.4 [C(5′),
C(5′′)]*; 28.5 [C(6′), C(6′′)]*; 27.2 [C(7′), C(7′′)]*; 22.9 [C(9′), C(9′′)]*; 14.4 [C(10′), C(10′′)]; MS, m/z
(%): 418 (100) [M+], 362 (35), 333 (11), 305 (31), 165 (25), 162 (27), 151 (17), 138 (17), 68 (20), 54
(30). HRMS (ESI TOF-MS): Calcd. for C26H47N2O2 419.3632; found: 419.3637.
2,5-Bis-(1-(2-aminoethyl)piperidino)-1,4-benzoquinone (20). Red crystals. Purified by column
chromatography, eluent hexane/methanol 1:3 (v/v). Mp 164.2–165.5 ºC; IR: 3,291 (NH), 3,251 (NH),
1
2,935, 2,849, 1,641, 1,551, 1,493, 1,462, 1,364, 1,289, 1,223, 1,126, 993, and 693 cm-1; H-NMR δ:
7.04 (br. s, NH); 5.30 [s, H-C(3), H-C(6)]; 3.19 [q, J = 6.0 Hz, 2 H-C(1′), 2 H-C(1′′)]; 2.58 [t,
J = 6.0 Hz, 2 H-C(2′), 2 H-C(2′′)]; 2.34-2.42 [m, 2 H-C(3′), 2 H-C(7′), 2 H-C(3′′), 2 H-C(7′′)]; 1.59
[quint., J = 5.5 Hz, 2 H-C(4′), 2 H-C(6′), 2 H-C(4′′), 2 H-C(6′′)]; 1.40-1.44 [m, 2 H-C(5′), 2 H-C(5′′)];
13C-NMR δ: 178.5 [C(1), C(4)]; 151.6 [C(2), C(5)]; 93.3 [C(3), C(6)]; 56.11 [C(2′), C(2′′)]; 54.5
[C(3′), C(7′), C(3′′), C(7′′)]; 39.3 (C(1′), C(1′′)]; 26.1 [C(4′), C(6′), C(4′′), C(6′′)]; 24.5 [C(5′), C(5′′)];
MS, m/z (%): 360 (2) [M+], 99 (11), 98 (100), 70 (6), 55 (14). HRMS (ESI TOF-MS): Calcd. for
C20H33N4O2 361.2598; found: 361.2599.
2,5-Bis-(4-(2-aminoethyl)morfoline)-1,4-benzoquinone (21). Red crystals. Purified by column
chromatography, eluent hexane/ethanol 1:4 (v/v). Mp 186.8–188.0 ºC; IR: 3,354 (NH), 2,869, 2,811,
1,645, 1,613, 1,495, 1,455, 1,354, 1,293, 1,115, 1,026, and 809 cm-1; 1H-NMR δ: 7.00 (br. s, NH); 5.28
[s, H-C(3), H-C(6)]; 3.72 [t, J = 4.5 Hz, 2 H-C(4′), 2 H-C(5′), 2 H-C(4′′), 2 H-C(5′′)]; 3.20 [q,
J = 6.0 Hz, 2 H-C(1′), 2 H-C(1′′)]; 2.64 [t, J = 6.0 Hz, 2 H-C(2′), 2 H-C(2′′)]; 2.47 [t, J = 4.5 Hz, 2 H-

Molecules 2010, 15
5638
13
C(3′), 2 H-C(6′), 2 H-C(3′′), 2 H-(6′′)]; C-NMR (CDCl3) δ: 178.3 [C(1), C(4)]; 151.0 [C(2), C(5)];
93.1 [C(3), C(6)]; 66.8 [C(4′), C(5′), C(4′′), C(5′′)]; 55.5 [C(2′), C(2′′)]; 53.2 [C(3′), C(6′), C(3′′),
C(6′′)]; 38.5 [C(1′), C(1′′)]; MS, m/z (%): 364 (3) [M+], 101 (10), 100 (100), 70 (8), 56 (24); HRMS
(ESI TOF-MS): Calcd. for C18H29N4O4 365.2183; found: 365.2186.
3.3. Biological assays
3.3.1. Phytotoxic activity
The phytotoxic activity of the 2,5-bis(alkylamino)-1,4-benzoquinones 12-23 was preliminarily
evaluated as the ability of these compounds to interfere with the radicle growth of the cultivars
Sorghum bicolor and Cucumis sativus. For these biological assays, stock solutions at 1.0 × 10-3 mol/L
were prepared as follows: each compound 12-23 was dissolved in xylene (84 L), Tween 80 surfactant
(127 L) and pentan-3-one (42 L). The resultant suspension was shaken for 1 min and then
transferred to a volumetric flask and the volume supplemented with water to 88 mL. The resultant
suspension was sonicated for 5 min.
3.3.2. Assay of radicle elongation on Petri dishes
This biological assay was carried out as previously described [52,53]. Groups of seven
pregerminated plants of S. bicolor (purchased from Geneze Company, Paracatu, Minas Gerais State,
Brazil) were placed in Petri dishes (i.d. = 9 cm) containing washed sand (660 g) and the solution
(88 mL) containing the compound to be evaluated. The Petri dishes were sealed with Parafilm,
o
incubated at 28 C, and inclinated at 75o. After 24 h and 48 h, the radicle length was measured to the
nearest millimeter. All treatments were replicated four times in a completely randomized design. The
percentage of radicle growth inhibition was calculated in relation to the root length of the control.
Positive values represent inhibition and negative values correspond to stimulation. The data were
analyzed using Tukey′s test at 0.05 probability level [54]. This biological assay was also conducted
with C. sativus L. (purchased from ISLA Company, Porto Alegre, Rio Grande do Sul State, Brazil), B.
decumbens (Marangatú Company, Ribeirão Preto, São Paulo State) and I. grandifolia (Agro Cosmos,
Engenheiro Coelho, São Paulo State).
3.3.3. Measurement of the ATP synthesis
Intact chloroplasts were isolated from spinach leaves (Spinacea oleracea L.) obtained from local
market as previously described [55]. Chloroplasts were suspended in the following medium:
400 mmol/L sucrose, 5 mmol/L MgCl2, 10 mmol/L KCl, and buffered with 0.03 mol/L Na+-tricine at
pH 8.0. They were stored as a concentrated suspension in the dark for 1 h at 0 oC. Intact chloroplasts were
efficiently lysed to yield free thylakoids prior to each experiment by incubating them in the following
electron transport medium: 100 mmol/l sorbitol, 10 mmol/L KCl, 5 mmol/L KCN, and 30 mmol/L
tricine buffer (pH 8 with the addition of KOH). Chlorophyll concentration was measured
spectrophotometrically as reported [56].
ATP synthesis was measured as the pH rise from 8.000 to 8.100 using a microelectrode connected
to a Corning potentiometer with expanded scale. The pH change was recorded with a Gilson recorder.

Molecules 2010, 15
5639
The reaction medium contained 100 mmol/L sorbitol, 5 mmol/L MgCl2, 10 mmol/L KCl and 1 mol/L
K+-tricine at pH 8.0 in the presence of 1 mmol/L ADP and 3 mmol/L KH2PO4. Methylviologen
(0.05 mol/L) was added as electron acceptor for the Hill reaction. The effect of compounds 12, 13 and
23 on the ATP synthesis was evaluated at 100 mol/L, 200 mol/L, 300 mol/L, 400 mol/L, and
500 mol/L. All mixtures were illuminated with actinic light of a projector lamp (GAP 2660) passed
through a 5 cm aqueous solution of 2% CuSO4 as filter [57].
3.4. Cytotoxicity screening
3.4.1. Cell lines and cell cultures
The cytotoxicity of compounds 12-14 and 16-23 was tested against HL-60 (human leukemia),
MDA-MB-435 (human breast cancer), HCT-8 (human colon) and SF-295 (human central nervous
system) cell lines obtained from the National Cancer Institute (Bethesda, MD, USA). Cells cultured in
RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 U/mL
penicillin, 100 μg/mL streptomycin at 37 °C under a 5% CO2 atmosphere.
Heparinized blood (from healthy, non-smoker donors who had not taken any drug at least 15 days
prior to sampling) was collected and Peripheral Blood Mononuclear Cells (PBMC) were isolated by a
standard method of density-gradient centrifugation over Histopaque-1077. PBMC were washed and
resuspended in RPMI 1640 medium supplemented with 20% fetal bovine serum, 2 mmol/L glutamine,
100 U/mL penicillin, 100 μg/mL streptomycin at 37 °C under a 5% CO2 atmosphere.
Phytohemaglutinin (2 %) was added at the beginning of culture. After 24 h of culture, cells were
treated with the test compounds 12-14 and 16-23.
3.4.2. MTT assay
Tumor cell growth was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-
dimethyl-2-thiozolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product [38].
For the experiments, cells were plated in 96-well plates (0.7 × 105 cells/mL for MDA-MB-435, HCT-8
and SF-295 cell lines, and 0.3 × 106 cells/mL for leukemia cells). After 24 h, the test compounds (0.39
to 25 μg/mL) dissolved in DMSO (0.1%), were added to each well and incubated for 72 h. DMSO
(0.1%) and doxorubicin were used as negative and positive controls, respectively. Thereafter, the
plates were centrifuged and then the medium was replaced by fresh medium (150 μL) containing
0.5 mg/mL MTT. Three hours later, the MTT formazan product was dissolved in 150 µL DMSO, and
absorbance was measured using a multiplate reader (Spectra Count, Packard, Ontario, Canada). Drug
effect was quantified as the percentage of control absorbance of the reduced dye at 595 nm in relation
to control wells.
3.4.3. Alamar Blue assay
In order to investigate selectivity of compounds toward a normal proliferating cell, the Alamar Blue
assay was performed with PBMC after 72 h drug exposure [39]. Briefly, PBMC were plated in 96-well
plates (3 × 105 cells/well in 100 µL of medium). After 24 h, the compounds (0.09–25 µg/mL)
dissolved in DMSO were added to each well (using the HTS - high-throughput screening - Biomek

Molecules 2010, 15
5640
3000 - Beckman Coulter, Inc., Fullerton, California, USA) and incubated for 72 h. Doxorubicin was
used as positive control. Twenty four hours before the end of the incubation, 10 µL of stock solution
(0.312 mg/mL) of the Alamar Blue (resazurin - Sigma Aldrich Co. - St. Louis, MO, USA) was added
to each well. The absorbance was measured using a multiplate reader (DTX 880 Multimode Detector,
Beckman Coulter, Inc). The drug effect was quantified as the percentage of control absorbance at
570 nm and 595 nm.
4. Conclusions
A series of 2,5-bis(alkylamino)-1,4-benzoquinone analogues of sorgoleone (1) were synthesized and
biologically evaluated in terms of their phytotoxicity and cytotoxicity. The assessment of phytotoxicity
revealed that the compounds can act as plant growth regulators with various patterns of activity.
Moreover, differences were noticed concerning the effects of the compounds on monocotyledonous
and dicotyledonous species. Further investigation on the phytotoxicity of the 2,5-bis(alkylamino)-1,4-
benzoquinones showed that they are capable of inhibiting the ATP synthesis in isolated chloroplasts.
The cytotoxicity assays revealed that the compounds 2,5-bis(ethylamino)-1,4-benzoquinone 12 and
2,5-bis(1-(2-aminoethyl)piperidino)-1,4-benzoquinone 20 exhibited activity against all of the human
cell lines used in the biological evaluation. The 2,5-bis(ethylamino) derivative 12 exhibited the most
selective cytotoxicity against the tumor cell lines tested and is not toxic to normal cells.
Acknowledgements
We are grateful to the following Brazilian Agencies: Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) for research fellowships (to L.C.A.B. and U.A.P.) and financial
support; and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) and FINEP for financial
support. We are also grateful to Dr. Blas Lotina-Hennsen for the ATP synthesis inhibition
biological assays.
References and notes
1. Oliveros-Bastidas, A.J.; Macías, F.A.; Fernández, C.C.; Marín, D.; Molinillo, J.M.G. Exudados
dela raiz y su relevancia actual en las interacciones alelopaticas. Quim. Nova. 2009, 32, 198-213.
2. Bais, H.P.; Weir, T.L.; Perry, L.G.; Gilroy, S.; Vivanco, J.M. The role of root exudates in
rhizosphere interactions with plants and other organisms. Annu. Rev. Plant Biol. 2006, 57, 233-266.
3. Walker, T.S.; Bais, H.P.; Grotewold, E.; Vivanco, J.M. Root exudation and rhizosphere biology.
Plant Physiol. 2003, 132, 44-51.
4. Bertin C.; Yang, X.; Weston L.A. The role of root exudates and allelochemicals in the
rhizosphere. Plant Soil. 2003, 256, 67-83.
5. Macías, F.A.; Molinillo, J.M.G.; Varela, R.M.; Galindo, J.C.G. Allelopathy - A natural alternative
for weed Control. Pest Manag. Sci. 2007, 63, 327-348.
6. Macías, F.A.; Galindo, J.C.G.; Molinillo, J.M.G.; Cutler, H.G. Allelopathy – Chemistry and Mode
of Action of Allelochemicals; CRC Press: Boca Raton, FL, USA, 2004.
7. Copping, L.G.; Duke, S.O. Natural products that have been used commercially as crop protection
agents - A review. Pest Manag. Sci. 2007, 63, 524-554.

Molecules 2010, 15
5641
8. Barbosa, L.C.A.; Teixeira, R.R.; Montanari, R.M. Phytotoxic Natural Products as Models for the
development of crop protection agents. In Current Trends in Phytochemistry; Epifano, F., Ed.;
Research Signpost: Kerala, India, 2008; pp. 21-59.
9. Chang, M.; Netzly, D.H.; Butler, L.G.; Lynn, D.G. Chemical regulation of distance.
Characterization of the first natural host germination stimulant for Striga asiatica. J. Am. Chem.
Soc. 1986, 108, 7858-7860.
10. Einhellig, F.A.; Souza, I.F. Phytotoxicity of sorgoleone found in grain Sorghum root exudates. J.
Chem. Ecol. 1992, 18, 1-11.
11. Gonzales, V.M.; Kazimir, J.; Nimbal, C.; Weston, L.A.; Cheniae, G.M. Inhibition of a
photosystem II electron transfer reaction by the natural product sorgoleone. J. Agric. Food Chem.
1997, 45, 1415-1421.
12. Rasmussen, J.A.; Hejl, A.M.; Einhellig, F.A.; Thomas, J.A. Sorgoleone from root exudate inhibits
mitochondrial functions. J. Chem. Ecol. 1992, 18, 197-207.
13. Czarnota, M.A.; Paul, R.N., Dayan, F.E.; Nimbal, C.I.; Weston, L.A. Mode of action, localization,
of production, chemical nature, and activity of sorgolene: A potent PSII inhibitor in Sorghum spp.
root exudates. Weed Technol. 2001, 15, 813-825.
14. Streibig, J.C.; Dayan, F.E.; Rimando, A.M.; Duke, S.O. Joint action of natural and synthetic
photosystem II inhibitors. Pest. Sci. 1999, 55, 137-146.
15. Hejl, A.M.; Koster, K.L. The allelochemical sorgoleone inhibits root H+-ATPase and water
uptake. J. Chem. Ecol. 2004, 30, 2181-2191.
16. Kubo, I.; Chaudhuri, S.K. Structure of maesaquinone. Bioorg. Med. Chem. Lett. 1994, 4, 1131-1134.
17. Silva, M.N. da; Ferreira, V.F.; de Souza, M.C.B.V. Um panorama atual da química e da
farmacologia de naftoquinonas, com ênfase na β-lapachona e derivados. Quim. Nova 2003, 26,
407-416.
18. Pan, S.S.; Andrews, P.A.; Glover, C.J. Reductive activation of mitomycin C and mitomycin C
metabolites catalyzed by NADPH-cytochrome P-450 reductase and xanthine oxidase. J. Biol.
Chem. 1984, 259, 959-966.
19. Tomasz, M.; Palom, Y. The mitomycin bioreductive antitumor agents: cross-linking and
alkylation of DNA as the molecular basis of their activity. Pharmacol. Ther. 1997, 76, 73-87.
20. Dettrakul, S.; Surerum, S.; Rajviroongit, S.; Kittakoop, P. Biomimetic transformation and
biological activities of globiferin, a terpenoid benzoquinone from Cordia globifera. J. Nat. Prod.
2009, 72, 861-865.
21. Marinho-Filho, J.D.; Bezerra, D.P.; Araújo, A.J.; Montenegro, R.C.; Pessoa, C.; Diniz, J.C.;
Viana, F.A.; Pessoa, O.D.; Silveira, E.R.; de Moraes, M.O.; Costa-Lotufo, L.V. Oxidative stress
induction by (+)-cordiaquinone J triggers both mitochondria-dependent apoptosis and necrosis in
leukemia cells. Chem. Biol. Interact. 2010, 183, 369-379.
22. Barbosa, L.C.A.; Costa, A.V.; Veloso, D.P.; Lopes, J.L.C.; Hernandez-Terrones, M.G.; King-
Diaz, B.; Lotina-Hennsen, B. Phytogrowth-inhibitory lactones derivatives of glaucolide B. Z
Naturforsch. 2004, 59c, 803-810.
23. Chaves, F.C.; Barbosa, L.C.A.; Demuner, A.J.; Silva, A.A. New helminthosporal analogues with
plant-growth regulatory properties synthesized via oxyallyl cation. Z. Naturforsch. 2006, 61b,
1287-1294.

Molecules 2010, 15
5642
24. Barbosa, L.C.A.; Demuner, A.J.; Maltha, C.R.A.; da Silva, P.S.; Silva, A.A. Sintese e avaliação
da ativadade fitotóxica de novos análogos oxigenados do ácido helmintospórico. Quim. Nova
2003, 26, 655-660.
25. Demuner, A.J.; Barbosa, L.C.A.; Veloso, D.P.; Howarth, O.W. Synthesis and plant growth
regulatory activity of 6α,7β-dihydroxyvouacapan-17β-oic acid derivatives. Aust. J. Chem. 1998,
51, 61-66.
26. Barbosa, L.C.A.; Demuner, A.J.; Alvarenga, E.S.; Oliveira, A.; King-Diaz, B.; Lotina-Hennsen,
B. Phytogrowth- and photosynthesis-inhibiting properties of nostoclide analogues. Pest Manag.
Sci. 2006, 62, 214-222.
27. Barbosa, L.C.A.; Demuner, A.J.; Maltha, C.R.A.; Teixeira, R.R.; Souza, K.A.P.; Bicalho, K.U.
Phytogrowth activity of 3-(3-chlorobenzyl)-5-arylidenefuran-2(5H)-ones. Z. Naturforsch. 2009,
64b, 245-251.
28. Barbosa, L.C.A.; Rocha, M.E.; Teixeira, R.R.; Maltha, C.R.A.; Forlani, G. Synthesis of 3-(4-
bromobenzyl)-5-(aryl methylene)-5H-furan-2-ones and their activity as inhibitors of the
photosynthetic electron transport chain. J. Agric. Food Chem. 2007, 55, 8562-8569.
29. Teixeira, R.R.; Barbosa, L.C.A.; Forlani, G.; Piló-Veloso, D.; Carneiro, J.W.M. Synthesis of
photosynthesis-inhibiting nostoclide analogues. J. Agric. Food Chem. 2008, 56, 2321-2329.
30. Lima, L.S.; Barbosa, L.C.A.; Alvarenga, E.S.; Demuner, A.J.; Silva, A.A. Synthesis and
phytotoxicity evaluation of substituted para-benzoquinones. Aust. J. Chem. 2003, 56, 625-630.
31. Barbosa, L.C.A.; Alvarenga, E.S.; Demuner, A.J.; Virtuoso, L.S.; Silva, A.A. Synthesis of new
phytogrowth-inhibitory substituted aryl-p-benzoquinones. Chem. Biod. 2006, 3, 553-567.
32. Bayen, S.; Barooah, N.; Sarma, R.J.; Sen, T.K.; Karmakar, A.; Baruah, J.B. Synthesis, structure
and electrochemical properties of 2,5-bis(alkyl/arylamino)1,4-benzoquinones and 2-arylamino-
1,4-naphthoquinones. Dye Pigment. 2007, 75, 770-775.
33. Hassan, S.S.M.; Iskander, M.L.; Nashed, N.E. Spectrophotometric determination of aliphatic
primary and secondary amines by reaction with p-benzoquinone. Talanta 1985, 32, 301-305.
34. Tindale, C.R. Reactions of biogenic amines with quinones. Aust. J. Chem. 1984, 37, 611-617.
35. Nithianandam, V.S.; Erhan, S. Quinone-amine polymers: 18. A novel method for the synthesis of
poly(alkyl aminoquinone)s. Polymer 1998, 39, 4095-4098.
36. Einhellig, F.A.; Schon, M.K.; Rasmussen, J.A. Synergistic effects of four cinnamic acid
compounds on grain sorghum. J. Plant Growth Regul. 1982, 1, 251-258.
37. Hennsen, B.L.; Achine, L.; Ruvalcaba, N.M.; Ortiz, A.; Hernández, J.; Farbán, N.; Martínez, M.
A. 2,5-Diamino-p-benzoquinone derivatives as photosystem I electron acceptors: Synthesis and
electrochemical and physicochemical properties. J. Agric. Food Chem. 1998, 46, 724-730.
38. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays. J. Immunol. Method. 1983, 16, 55-63.
39. Ahmed, S.A.; Gogal, R.M.J.; Walsh, J.E. A new rapid and simple non-radioactive assay to
3
monitor and determine the proliferation of lymphocytes: An alternative to H-thymidine
incorporation assay. J. Immunol. Method. 1994, 170, 211-224.
40. Zhi-Jun, Y.; Sriranganathan, N.; Vaught, T.; Arastu, S.K.; Ahmed, S.A. A dye-based lymphocyte
proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis,
and immunophenotyping studies. J. Immunol. Method. 1997, 210, 25-39.

Molecules 2010, 15
5643
41. Chari, R.V.J. Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc. Chem. Res.
2008, 41, 98-107.
42. O′Brien, P.J. Molecular mechanisms of quinone cytotoxicity. Chem.-Biol. Interact. 1991, 80,
1-41.
43. Perrin, D.D.; Armarego, W.L.F. Purification of Laboratory Chemicals, 3rd ed.; Pergamon:
Oxford, UK, 1988.
44. Brondani, D.J.; Bieber, L.W. Regioselective lithiation and alkylation of 2-methoxy-hydroquinone.
Quim. Nova 1995, 18, 144-146.
45. Zhou, Q.; Swager, T.M. Probing delocalization across alkyne-containing linkages: Synthesis and
cyclic voltammetry of bridged phenylenediamines. J. Org. Chem. 1995, 60, 7096-7100.
46. Wellington, K.W.; Steenkamp, P.; Brady, D. Diamination by N-coupling using a commercial
laccase. Bioorg. Med. Chem. 2010, 18, 1406-1414.
47. Garreau, Y. Oxidation products of gentisic acid in the presene of primary amines. Bull. Soc. Chim.
Fr. 1950, 501-502.
48. Garreau, Y. [Interaction of] phenols and sulfites. Ann. Chim. Applicata 1938, 10, 485-558.
49. Torigoe, M. Cancerocidal substances. XIII. Effect of some quinones and related compounds upon
the Yoshida sarcoma. Pharm. Bull. 1955, 3, 337-342.
50. Makarova, A.N.; Berlin, A.Y. Reaction of ethylenimino-1,4-benzoquinones with amines. I.
Reaction between ethylenimino-1,4-benzoquinones and secondary amines. Zh. Org. Khim. 1959,
29, 666-672.
51. Cavalitto, C.J.; Soria, A.E.; Hoppe, J.O. Amino- and ammonium-alkylaminobenzoquinones as
curarimimetic agents. J. Am. Chem. Soc. 1950, 72, 2661-2665.
52. Barbosa, L.C.A.; Pereira, U.A.; Teixeira, R.R.; Maltha, C.R.A.; Fernandes, S.A.; Forlani, G.
Synthesis and phytotoxic activity of ozonides. J. Agric. Food Chem. 2008, 56, 9434-9440.
53. Barbosa, L.C.A.; Maltha, C.R.A.; Cusati, R.C.; Teixeira, R.R.; Rodrigues, F.F.; Silva, A.A.;
Drew, M.G.B.; Ismail, F.M.D. Synthesis and biological evaluation of new ozonides with
improved plant growth regulatory activity. J. Agric. Food Chem. 2009, 57, 10107-10115.
54. Gomes, F.P. Curso de Estatística Experimental, 13th ed.; Nobel: Piracicaba, Brasil, 1990.
55. Macías, M.L.; Rojas, I.S.; Matar, R.; Lotina-Hennsen, B. Effect of selected coumarins on spinach
chloroplast photosynthesis. J. Agric. Food Chem. 1999, 47, 2137-2140.
56. Strain, H.H.; Coppe, B.T.; Svec, W.A. Analytical procedures for the isolation, identification,
estimation and investigation of the chlorophylls. In Methods of Enzymology; San Pietro, A., Ed.;
Academic Press: New York, NY, USA, 1971, p. 452.
57. Demuner, A.J.; Barbosa, L.C.A.; Veiga, T.A.M.; Barreto, R.W.; King-Diaz , B.; Lotina-Hennsen,
B. Phytotoxic constituents from Nimbya alternantherae. Bioch. Syst. Ecol. 2006, 34, 790-795.
Sample Availability: Contact the authors.
© 2010 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 license
(http://creativecommons. org/licenses/by/3.0/).