First total synthesis and phytotoxic activity of Streptomyces sp. metabolites abenquines

Article abstract of DOI:10.1016/j.tetlet.2016.03.038

The first total synthesis of abenquines A, B2, C and D has been achieved in three steps starting from commercially available 2,5-dimethoxyaniline, with overall yields of 41-61%. Four analogues bearing the amino acids d-valine (17), l-methionine (18), and glycine (19), and benzylamine (20), were also prepared in 45-72% yield. The inhibitory properties of these compounds were evaluated against the photoautotrophic growth of a model Synechococcus sp. strain. Abenquine C and its enantiomer were substantially ineffective, whereas all other abenquines significantly inhibited cell proliferation, with concentrations causing 50%-inhibition of algal growth ranging from 10^-5 to 10^-6 M.

Full text of DOI:10.1016/j.tetlet.2016.03.038

Tetrahedron Letters 57 (2016) 1811–1814
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
First total synthesis and phytotoxic activity of Streptomyces sp.
metabolites abenquines
b
c
Amalyn Nain-Perez a, Luiz C. A. Barbosa a,b, , Celia R. A. Maltha , Giuseppe Forlani
⇑
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 Life Science and Biotechnology, University of Ferrara, via L. Borsari 46, I-44121 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The first total synthesis of abenquines A, B2, C and D has been achieved in three steps starting from com-
mercially available 2,5-dimethoxyaniline, with overall yields of 41–61%. Four analogues bearing the
amino acids D-valine (17), L-methionine (18), and glycine (19), and benzylamine (20), were also prepared
in 45–72% yield. The inhibitory properties of these compounds were evaluated against the photoau-
totrophic growth of a model Synechococcus sp. strain. Abenquine C and its enantiomer were substantially
ineffective, whereas all other abenquines significantly inhibited cell proliferation, with concentrations
causing 50%-inhibition of algal growth ranging from 10ꢀ5 to 10ꢀ6 M.
Received 17 February 2016
Revised 8 March 2016
Accepted 11 March 2016
Available online 12 March 2016
Keywords:
Abenquines
Aminoquinones
Herbicidal activity
Phytotoxin
Ó 2016 Elsevier Ltd. All rights reserved.
Introduction
followed by the isolation8 and synthesis9 of several new naki-
jiquinones and analogues bearing amino acid residues. One amino-
Among the plethora of natural products, such as polyketides,
terpenoids, steroids, alkaloids, amino acids, and carbohydrates,1
the quinones represent an important class of substances endowed
with a vast array of biological activities.2 These compounds are
found in plants, microorganisms, and marine organisms.2 Despite
the large structural variation among the natural quinones, those
bearing an amino group ortho to the carbonyl group are less com-
mon. One of the earliest natural aminoquinones identified is strep-
tonigrin (1) (Fig. 1), a highly functionalized antibiotic isolated from
Streptomyces flocculus in 1959.3 This compound entered phase II
clinical trials as an anticancer agent in the 1970s, and has attracted
the attention of many research groups.4 In the early 1980s laven-
damycin (2), structurally related to streptonigrin, was isolated
from Streptomyces lavendulae, and was shown to exert cytotoxic
and antimicrobial activities.5 Lavendamycin was also the object
of synthetic investigations, including the preparation of analogues
for biological studies.4b,6
quinone structurally related to nakijiquinones was then isolated
from the marine sponge Dactylospongia elegans and named
smenospongine.10 This compound and other natural analogues iso-
lated from marine sources presented antimicrobial and cytotoxic
activities.11 Other natural aminoquinones endowed with biological
activities include metachromins,12 geldanamycin,13 mytomicin,14
and dysifragilones.15
Recently, a new group of benzoquinones bearing natural amino
acid residues (abenquines A–D, 6–10) (Fig. 1) were isolated from a
strain of Streptomyces sp. collected from the Atacama desert in
Chile. These abenquines showed cytotoxic activity against bacteria
and dermatophytic fungi, and were inhibitory of phosphodi-
esterase type 4b activity.16 Several groups,17 including ours,18 have
described aminobenzoquinone phytotoxicity, but little work has
been done to address their development as new herbicides. In view
of our interest in this area and considering that abenquines are
available in short supply, we report here the first total synthesis
of such compounds and a preliminary assessment of their phyto-
toxic effects.
In 1994 two new terpenoid aminoquinones, nakijiquinones A
(3) and B (4), were isolated from a marine sponge (family Spongi-
dae). Besides representing the first sesquiterpenoid quinones with
amino acid residues of natural origin, they also exhibited cytotoxic
activity against some cancer cell lines.7 This discovery was
Results and discussion
Initially we envisaged that all abenquines could be obtained
from quinone 11, which in turn could easily be prepared from
the commercially available 2,5-dimethoxyaniline 13 (Scheme 1).
⇑
Corresponding author. Tel.: +55 31 34093396; fax: +55 31 3899 3065.
E-mail addresses: lcab@ufmg.br, lcab@ufv.br (L.C.A. Barbosa).
http://dx.doi.org/10.1016/j.tetlet.2016.03.038
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

1812
A. Nain-Perez et al. / Tetrahedron Letters 57 (2016) 1811–1814
O
O
With this plan in mind, compound 13 was treated with acetyl
O
O
H3CO
H2N
chloride and quantitatively converted into amide 12 on a 2 g scale
(Scheme 2). We then investigated the conversion of this amide into
abenquine B2 (8) via three routes, as shown in Scheme 2. Initially
compound 12 was demethylated by treatment with BBr3 using a
methodology previously applied for the synthesis of alternariol
analogues.19 Although the required hydroquinone 14 was obtained
in low yield (50%), no attempt was made to optimize this reaction
step since we focused on the investigation of a direct conversion of
14 into abenquine B2 using the methodology proposed by Zhong-
Lu et al.20 Thus, we reacted 14 with isoleucine in ethanol for 24 h,
but the required product 8 was isolated in only 11% yield. This low
yield was not surprising, since we have recently shown that a
direct amination followed by spontaneous oxidation of hydro-
quinone is a low efficiency process.18b
N
COOH
N
COOH
CH3
N
H2N
N
H2N
CH3
OH
HN
OCH3
1 ( )-Streptonigrin
2 Lavendamycin
OCH3
R
HN
CO2H
O
NH2
OH
O
O
O
OH
H
H
Based on our recent success in obtaining 2,5-bis-(alkyl/
arylamino)-1,4-benzoquinones directly from benzoquinones,18b
we then converted compound 12 into benzoquinone 11. As
reported by Whiteley,21 the oxidation of 12 with a large excess
of cerium ammonium nitrate (CAN) in acetonitrile and water can
afford the target product 11. When we attempted the reaction
using only two CAN equivalents, the reaction gave a complex mix-
ture of polar compounds. This result is in agreement with previous
data showing that 1,4-dimethoxybenzene with one substituent
gives rise to a dimerization reaction.22 An alternative treatment
of 12 with Dess–Martin periodinane (DMP) resulted in the required
quinone 11 in a better yield of 50%. To improve it further, a hyper-
valent iodine reagent, namely phenyliodine diacetate (PIDA),23 was
used and afforded quinone 11 in a 75% yield. Despite repeating this
reaction several times, the yield could not be increased anymore,
as other side products always formed.
3 Nakijiquinone A (R = H)
5 Smenospongine
4
Nakijiquinone B (R = isopropyl)
O
H
N
O
H
N
HO
R
O
HO
O
O
N
H
O
N
H
O
O
6 Abenquine A (R = benzyl)
N
7
8
Abenquine B1 (R = isobutyl)
H
Abenquine B2 (R = sec-butyl)
10 Abenquine D
9 Abenquine C (R = isopropyl)
Figure 1. Structures of some natural aminoquinones.
Compound 11 was subsequently reacted with isoleucine in the
presence of sodium bicarbonate in ethanol. After 2 h at reflux, the
required abenquine B2 was isolated in 81% yield. Therefore, start-
ing from the commercially available aniline 13, abenquine B2 was
obtained in just 3 steps and 60.8% yield. The structure of this com-
pound was proven by extensive spectroscopic analysis. The molec-
ular formula C14H18N2O5 was established by HRMS (ESI), in
accordance with the signal at m/z = 295.1290 ([M+H]+). The IR
spectra of abenquine B2 showed absorptions typical of amino acid
groups (broad band at 3404–2736 cmꢀ1), and another band at
O
O
OMe
OMe
H
N
H
N
H
N
HO
R
O
NH2
O
O
O
N
H
O
O
OMe
OMe
11
12
13
Abenquines A-D
Scheme 1. Retrosynthetic analysis of abenquines A–D.
OH
H
N
BBr3, DCM
L-isoleucine
O
-78 ºC to rt,
5
NaHCO3
Route 1
OMe
overnigth, 50%
EtOH, reflux, 24 h
11%
OH
14
Route 2
OMe
O
O
O
H
N
H
N
H
N
L-isoleucine
CH3COCl
NH2
HO
O
PhI(OAc)2
NaHCO3
Et3N, DCM
O
O
O
N
H
H2O, MeOH
rt, 1 h, 75%
EtOH, reflux,
2 h
81%
0 ºC to rt, 2 h
100%
OMe
OMe
O
13
8 Abenquine B2
12
11
Route 3 Br2, DCM, rt
100%
OMe
O
O
H
N
H
N
(NH4)2Ce(NO3)6
L-isoleucine
O
O
ACN/H2O
rt, 30 min, 70%
Br
Br
NaHCO3
EtOH, reflux, 2 h
40%
OMe
16
15
Scheme 2. Investigated routes for the synthesis of abenquine B2.

A. Nain-Perez et al. / Tetrahedron Letters 57 (2016) 1811–1814
1813
1626 cmꢀ1 due to stretch vibration of the amide carbonyl group.24
The 1H NMR spectrum showed a singlet at 2.22 ppm (MeCO group)
and another singlet at 5.43 ppm typical of the vinylic H-20 and the
H-2 of amino acid as doublet at 3.99 ppm. The 13C NMR spectrum
analysis confirmed the carbonyl group of the amide at 172.7 ppm
and the signals at 11.7, 15.6, 26.7, 38.2, 64.4 ppm associated with
the amino acid residues. A detailed assignment of the NMR spectra
(1H and 13C) and other physical data for this compound are pre-
sented as Supplementary data. Although this compound has been
described previously, no spectroscopic data were reported in the
literature,16 and a comparison was not possible.
Having produced for the first time four natural abenquines, we
applied the developed methodology to the synthesis of analogues
in order to further study their biological activities. In view of that,
starting from intermediate 11, an enantiomer of abenquine C (17)
was prepared in 68% yield. Other abenquine analogues (18 and 19)
were obtained in 45% and 60% yield. A simple derivative of benzy-
lamine (20) was also obtained in 72% yield (Fig. 2).
The phytotoxicity of abenquines and their analogues was
assessed as their ability to inhibit the photoautotrophic growth
of
a
model cyanobacterial strain, Synechococcus elongatus
PCC6301, using a methodology previously described for some
2,5-diaminobenzoquinones.18b The addition to the culture
medium of increasing levels of a given compound in the range
A further attempt (route 3) to improve the overall yield of this
synthesis involved the initial bromination of amide 13 to afford 15
in quantitative yield. The rationale for this transformation was
from 1 to 100 lM in all cases determined a significant reduction
that, according to Hayashi et al.,22c the oxidation of
a
of the growth rate constant. A mild effect was evident for
abenquine C and its isomer 17, with a concentration inhibiting
growth by 50% (IC50) higher than 10ꢀ4 M (Table 1). For the other
disubstituted 1,4-dimethoxybenzene derivative proceeds in
better yield and produces less dimer than compound 13. We
then oxidized compound 15 employing the same reagents used
for the production of 11. It was found that the use of CAN and
PIDA afforded the bromoquinone 16 in 70% yield, while the
treatment with DMP did not produce the required compound.
Based on the efficient substitution of a bromine for an amino
group in case of 2,3-dibromofuran-2(5H)-one,25 we envisaged that
the same reaction would take place with compound 16. Conse-
quently, we reacted 16 with isoleucine under the conditions
reported above, but abenquine B2 was formed in a disappointing
40% yield. In summary, the shortest and most efficient approach
for the preparation of abenquine B2 in a satisfactory yield con-
sisted of route 2. This procedure afforded abenquines A (6), C (9)
and D (10) in 68%, 70% and 55% yield, respectively (Scheme 3).
The structures of abenquines A, C, and D were confirmed
by spectroscopic analysis. For all compounds (6, 9, 10) the
infrared spectra presented a strong absorption in the range
3554–2794 cmꢀ1 corresponding to the amino acid group. In the
1H NMR spectra, the methyl group of amide was observed for all
compounds as a singlet at 2.21–2.18 ppm. In addition, the vinylic
hydrogen-20 is located at 5.24–5.39 ppm and the hydrogen-2
corresponding to amino acid at 3.70–4.12 ppm. The 13C NMR
spectra confirmed the carbonyl group of the amide (signals at
172.6–172.7 ppm) and the carboxylic carbon at 176.4–177.1 ppm.
The high resolution mass spectra for all three compounds were
in agreement with the corresponding molecular formulas. A
detailed assignment of the NMR spectra (1H and 13C) and physical
data for each compound are presented in the Supplementary data
(Experimental section). All spectroscopic data were in agreement
with previously reported data.16
O
H
N
O
H
N
HO
O
HO
O
O
O
N
H
S
N
H
O
O
17
18
(68%)
(45%)
O
H
N
O
H
N
HO
O
O
O
N
H
N
H
O
O
19
20
(60%)
(72%)
Figure 2. Abenquine analogues prepared.
Table 1
Abenquine concentrations causing 50%-inhibition of cyanobacte-
rial growth
Compound
IC50
6
8
9
9.6 ⁄ 10ꢀ6
M
M
1.4 ⁄ 10ꢀ5
>10ꢀ4
M
10
17
18
19
20
1.2 ⁄ 10ꢀ5
2.3 ⁄ 10ꢀ4
5.5 ⁄ 10ꢀ6
7.7 ⁄ 10ꢀ6
2.0 ⁄ 10ꢀ6
M
M
M
M
M
H
O
O
O
H
N
OH
O
H
N
O2
H2O
O
R
O
HO
R
O
O
R
O
N
NaHCO3
EtOH, rt
O
R
O
O
NH3
N
H
N
H
HCl(aq)
O
11
CH2
6 68% (Abenquine A)
H3C
H3C
9
70% (Abenquine C)
10
55% (Abenquine D)
N
H
Scheme 3. Synthesis of abenquines A, C and D.

1814
A. Nain-Perez et al. / Tetrahedron Letters 57 (2016) 1811–1814
Donohoe, T. J.; Jones, C. R.; Barbosa, L. C. A. J. Am. Chem. Soc. 2011, 133, 16418;
(e) Donohoe, T. J.; Jones, C. R.; Kornahrens, A. F.; Barbosa, L. C. A.; Walport, L. J.;
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compounds the treatment was much more effective, and at the
highest doses tested algal growth was completely abolished.
Natural abenquines were almost equipotent, with IC50 values
around 10ꢀ5 M. The other synthetic analogues were significantly
more effective, and in the case of compound 20 an IC50 value of
2.0 ⁄ 10ꢀ6 M was found. The occurrence of this variability in a
limited number of analogues seems very promising for the future
tailoring of their scaffold aimed at increasing their effectiveness
(Supplementary information).
In conclusion, we have developed a short and efficient method
for the synthesis of natural abenquines that can also been applied
to the preparation of analogues. These compounds were shown to
exert phytotoxic activity against the cyanobacterium Synechococ-
cus elongatus. Further work is underway in our laboratories to elu-
cidate their mechanism(s) of action and to produce more active
analogues with potential use as herbicides.
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Acknowledgments
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We thank the Brazilian agencies Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and Fundação
de Amparo à Pesquisa de Minas Gerais (FAPEMIG), and the Univer-
sity of Ferrara (FAR 2014) for financial support. We are grateful to
Dr. Carlos Zani and Dr. Markus Kohlhoff for obtaining the HRMS
data.
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Supplementary data
Supplementary data (full experimental procedures and charac-
terization data for all compounds) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/j.
tetlet.2016.03.038.
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