Hormonemate Derivatives from Dothiora sp., an Endophytic Fungus

Article abstract of DOI:10.1021/acs.jnatprod.6b00680

A search for cytotoxic agents from cultures of the endophytic fungus Dothiora sp., isolated from the endemic plant Launaea arborescens, led to the isolation of six new compounds structurally related to hormonemate, with moderate cytotoxic activity against different cancer cell lines. By using a bioassay-guided fractionation approach, hormonemates A-D (1-4), hormonemate (5), and hormonemates E (6) and F (7) were obtained from the acetone extract of this fungus. Their structures were determined using a combination of HRMS, ESI-qTOF-MS/MS, 1D and 2D NMR experiments, and chemical degradation. The cytotoxic activities of these compounds were evaluated by microdilution colorimetric assays against human breast adenocarcinoma (MCF-7), human liver cancer cells (HepG2), and pancreatic cancer cells (MiaPaca_2). Most of the compounds displayed cytotoxic activity against this panel.

Full text of DOI:10.1021/acs.jnatprod.6b00680

Article
pubs.acs.org/jnp
Hormonemate Derivatives from Dothiora sp., an Endophytic Fungus
,
†
†
†
†
Mercedes Per
́
ez-Bonilla,* Víctor Gonzal
́
ez-Menen
́
dez, Ignacio Per
́
ez-Victoria, Nuria de Pedro,
†
‡
‡
ez-Tejero,‡
Jesus
́
Martín, Joaquín Molero-Mesa, Manuel Casares-Porcel, María Reyes Gonzal
Francisca Vicente, Olga Genilloud, Jose
́
†
†
†
,†
́
R. Tormo, and Fernando Reyes*
†
́ ́ ́
Fundacion MEDINA, Centro de Excelencia en Investigacion de Medicamentos Innovadores en Andalucía, Parque Tecnologico
Ciencias de la Salud, Avda. del Conocimiento 34, 18016 Armilla, Granada, Spain
‡
Departamento de Botan
́
ica, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain
*
S Supporting Information
ABSTRACT: A search for cytotoxic agents from cultures of
the endophytic fungus Dothiora sp., isolated from the endemic
plant Launaea arborescens, led to the isolation of six new
compounds structurally related to hormonemate, with
moderate cytotoxic activity against different cancer cell lines.
By using a bioassay-guided fractionation approach, hormone-
mates A−D (1−4), hormonemate (5), and hormonemates E
(
6) and F (7) were obtained from the acetone extract of this
fungus. Their structures were determined using a combination
of HRMS, ESI-qTOF-MS/MS, 1D and 2D NMR experiments,
and chemical degradation. The cytotoxic activities of these
compounds were evaluated by microdilution colorimetric assays against human breast adenocarcinoma (MCF-7), human liver
cancer cells (HepG2), and pancreatic cancer cells (MiaPaca_2). Most of the compounds displayed cytotoxic activity against this
panel.
ancer is one of the top ten leading causes of death
globally, and the second after cardiovascular diseases in
Endophytes are relatively underexplored as potential sources
of novel natural products for exploitation in medicine,
agriculture, or food. Of the approximately 300,000 higher
plant species that exist on the earth, each individual plant is
C
middle income countries according to the World Health
Organization, and a big effort has been directed to identify new
pathways, biomarkers, and agents that are likely to be effective
6
,7
host to one or more endophytes.
Consequently, the
1
,2
against the treatment of this disease. The search for novel
drugs is a priority in cancer chemotherapy due to the rapid
development of resistance to multiple chemotherapeutic drugs,
the high toxicity usually associated with this type of treatment,
and their undesirable side effects. The discovery of novel
natural drugs with reduced side-effects, high selectivity for
cancer cell lines, low toxicity to normal cells, and efficient
killing of cancer cells is desirable.
Arid zones in Andalucıa have special edaphological and
́
climatic conditions, such as low rainfall, high sunshine levels,
opportunity to find new and interesting endophytic micro-
organisms among myriads of plants in different settings and
ecosystems is very wide. In addition, if endophytes can produce
the same rare and important bioactive compounds as their host
plant, this would not only reduce the need to harvest slow-
growing and possibly rare plants but also help to preserve the
world’s ever diminishing biodiversity. Thus, more background
information on a given plant species and its microorganism
biology would be exceedingly helpful in directing the search for
3
8
bioactive products.
During the period 1990−2010, one hundred anticancer
compounds belonging to 19 different chemical classes, with
activity against 45 different cell lines, were isolated from over 50
different fungal species belonging to six different endophytic
fungal groups. Of the total compounds isolated from
endophytic fungi, 57% were novel compounds or analogues
and specific lithology, where loamy materials and evaporites
́
abound. Among them, the Tabernas desert in Almerıa is
designated as one of the Mediterranean’s biodiversity hot spots4,
with one of the richest steppic to subdesertic floras in Europe.
Native plants from these ecosystems possess distinctive survival
characteristics for these conditions, which has led to the
existence of a high degree of endemic plants with highly
adapted endophyte ecosystems, previously poorly studied. It is
precisely this singularity which turns them into a valuable
potential source for the isolation of new and unique host-
9
of known compounds. The identification and isolation of
important plant-derived anticancer drugs, such as Taxol,
camptothecin, podophyllotoxin, vinblastine, and vincristine,
from endophytic fungi isolated from the producing plants and
4
,5
specific endophytes. Investigation of the secondary metabo-
lites of microorganisms from unusual or specialized niches may
increase the chances of finding novel compounds.
Received: July 21, 2016
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00680
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
the identification of the gene/gene products controlling the
production of these drugs could provide alternative sources for
10
the production of key bioactive natural products. In fact, it is
becoming increasingly clear that host specificity is a bona fide
phenomenon in endophyte relationships with higher plants.
Knowledge of such interactions can provide guidance as to
which endophytes might be selected in the search for novel
11
medicinal natural products.
In our search for bioactive compounds from endophytes of
endemic plants of the Tabernas desert, we observed
cytotoxicity against the human cell lines MCF-7 and HepG2
in an acetone extract of the fungus Dothiora sp., isolated from
Launaea arborescens. Additionally, the LC-HRMS profile of the
extract revealed the presence of diverse secondary metabolites,
which prompted a more detailed investigation of the extract.
Extraction of the culture broth followed by a bioassay-guided
fractionation of the extract led to the isolation of six new
compounds structurally related to hormonemate. Despite the
fact that the isolation of hormonemate had been reported
1
2
previously, no spectroscopic data have been so far disclosed
for this molecule. Structure elucidation of the new compounds
and hormonemate itself is reported herein for the first time.
RESULTS AND DISCUSSION
■
Cultures of Dothiora sp. in YES medium were extracted with
acetone and proved to be active against the MCF-7 and HepG2
human cancer cell lines. These extracts were fractionated using
a bioassay-guided approach, by column chromatography and
semipreparative reversed phase HPLC, to yield compounds 1−
7.
Compound 1 was obtained as a colorless syrup. A molecular
formula of C H O was assigned after analysis by ESI-TOF.
35
62 15
1
The H NMR data of 1 indicated the presence of five doublets
at 4.20, 5.09, 5.10, 5.11, and 5.15 ppm, corresponding to five
oxygenated methines. In addition, three hydroxylated methines
at 3.52−3.90 ppm, together with two oxygenated methylenes at
3.63 (2H), and 4.22 and 4.47 ppm were observed, suggesting
the presence of a glycol moiety. Five methines at 1.88−2.10
ppm appeared in the aliphatic region, along with ten overlapped
methylene protons at 1.35 and 1.53 ppm and ten overlapped
aliphatic methyl groups at 0.93−1.02 ppm (Table 1).
1
3
Although the C NMR spectrum displayed several over-
alditol moiety is attached to C-1 via an ester linkage (Figure 1).
Methine protons at 5.09−5.15 ppm exhibited long-range
correlations to the carbonyl carbons at 170.6−175.7 ppm (C-
1), suggesting that the five 2-hydroxy-3-methylpentanoyl
moieties are connected to each other through ester linkages
between the 1- and 2- positions (Figure 1), similar to that in
lapped signals, 35 carbons were identified based on multiplicity
1
3
edited HSQC and HMBC experiments. The C NMR data
exhibited different sets of signals corresponding to five ester
carbonyl carbons appearing at 170.6 (×2), 170.8, 171.2, and
1
7
75.7 ppm; eight oxygenated methine carbons at 70.5, 71.6,
2.0, 74.2, 76.1, 76.3, 76.5, and 76.7 ppm; two oxygenated
13,14
liamocins and the antibiotic substance exophilin A.
methylene carbons at 64.8 and 68.7 ppm; five aliphatic methine
carbons at 37.8, 38.0, 38.1 (×2), and 40.1 ppm; five aliphatic
methylene carbons at 26.9 (×2) and 27.0 (×3); and ten methyl
carbons at 12.0 (×3), 12.1, 12.3, 14.0, 14.7 (×2), and 14.8
As further evidence, the saponification reaction of compound
1 with NaOH led to the cleavage of the ester bond and released
the alditol and the acid salt. Subsequent acidification of the
alkoxide ion with hydrochloric acid produced the carboxylic
acid. NMR analysis of both units and their comparison with
standards verified that the sugar alcohol was arabitol and the
carboxylic acid was 2-hydroxy-3-methylpentanoic acid or
isoleucic acid. As a result, the structure of compound 1 consists
of a single arabitol headgroup 1-O-acylated with a chain of five
2-hydroxy-3-methylpentanoic ester units that was named
hormonemate A.
(
×2). The 1D and 2D NMR data established five identical
partial substructures of 2-hydroxy-3-methylpentanoyl moieties
and one alditol of five carbons. The C-2 position of all but one
of the 2-hydroxy-3-methylpentanoyl units was acylated, as
confirmed by the downfield chemical shift of proton H-2 (δH
5
.09−5.15). The remaining unit showed an upfielded chemical
shift for H-2 (δH 4.20 ppm), indicating its terminal, non-
acylated, position. HMBC correlations were used to establish
the points of attachment between the different moieties. Long-
range correlation of the methylene protons at 4.22 and 4.47
ppm to the carbonyl carbon at 171.2 (C-1) revealed that the
Compound 2 was also obtained as a colorless syrup. Its ESI-
TOF spectrum had an ammonium adduct at m/z 710.4331
corresponding to a molecular formula of C H O . The
34
60 14
proton and carbon chemical shifts of compound 2 were similar
B
DOI: 10.1021/acs.jnatprod.6b00680
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Journal of Natural Products
Article
Table 1. NMR Spectroscopic Data (500 Mz, CD OD) for Hormonemate A (1) and Hormonemate B (2)
3
hormonemate A (1)
δH (J in Hz)
hormonemate B (2)
δH (J in Hz)
position
δC, type
HMBC
δC, type
erythritol
HMBC
L-arabitol
1
1
2
3
4
4
5
′a
′b
′
68.7, CH2
4.22, dd (11.4, 6.6)
4.47, dd (11.4, 2.6)
3.90, m
2′, 3′, 1A
3′, 1A
3′, 5′
1′, 2′, 5′
2′, 3′, 5′
68.2, CH2
4.21, dd (11.6, 6.6)
4.41, dd (11.6, 3.0)
3.75, m
2′, 3′, 1A
3′, 1A
4′
1′
2′, 3′
2′, 3′
70.5, CH
72.0, CH
71.6, CH
71.3, CH
73.5, CH
64.6, CH2
′
3.52, dd (8.8, 1.7)
3.90, m
3.56, m
′a
′b
′
3.61, dd (11.2, 5.9)
3.75, m
64.8, CH2
3.63, d (6.3)
3′
D-allo-isoleucic acid A
1
2
3
4
4
5
6
171.2, C
76.7, CH
37.8, CH
27.0, CH2
171.0, C
76.6, CH
37.9, CH
27.1, CH2
5.15, d (3.2)
2.10, m
1, 3, 4, 6, 1B
4, 5, 6
5.14, d (3.2)
2.10, m
1, 3, 4, 6, 1B
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
a
1.35, m
2, 3, 5, 6
2, 3, 5, 6
4
1.35, m
b
1.53, m
1.53, m
12.0, CH3
14.8, CH3
0.96, m
12.0, CH3
14.9, CH3
0.96, m
1.02, m
2, 3, 4
1.01, m
2, 3, 4
D-allo-isoleucic acid B
1
2
3
4
4
5
6
170.6, C
76.5, CH
38.1, CH
27.0, CH2
170.6, C
76.3, CH
38.1, CH
27.0, CH2
a
b
5.11 , d (3.6)
1, 3, 4, 6, 1C
4, 5, 6
5.11 , d (3.2)
1, 3, 4, 6, 1C
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
2.10, m
1.35, m
1.53, m
0.96, m
1.02, m
2.10, m
1.35, m
1.53, m
0.96, m
1.01, m
a
2, 3, 5, 6
2, 3, 5, 6
4
b
12.1, CH3
14.8, CH3
12.1, CH3
14.8, CH3
2, 3, 4
2, 3, 4
D-allo-isoleucic acid C
1
2
3
4
4
5
6
170.6, C
76.3, CH
38.1, CH
27.0, CH2
170.6, C
76.6, CH
38.1, CH
27.0, CH2
a
b
5.10 , d (3.8)
1, 3, 4, 6, 1D
4, 5, 6
5.10 , d (3.6)
1, 3, 4, 6, 1D
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
2.10, m
1.35, m
1.53, m
0.96, m
1.02, m
2.10, m
1.35, m
1.53, m
0.96, m
1.01, m
a
2, 3, 5, 6
2, 3, 5, 6
4
b
12.0, CH3
14.7, CH3
12.1, CH3
14.7, CH3
2, 3, 4
2, 3, 4
D-allo-isoleucic acid D
1
2
3
4
4
5
6
170.8, C
76.1, CH
38.0, CH
26.9, CH2
170.8, C
76.1, CH
38.1, CH
27.0, CH2
5.09, d (3.3)
2.10, m
1, 3, 4, 6, 1E
4, 5, 6
5.09, d (3.2)
2.10, m
3, 4, 6, 1E
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
a
1.35, m
2, 3, 5, 6
2, 3, 5, 6
4
1.35, m
b
1.53, m
1.53, m
12.0, CH3
14.7, CH3
0.96, m
12.1, CH3
14.7, CH3
0.96, m
1.02, m
2, 3, 4
1.01, m
2, 3, 4
D-allo-isoleucic acid E
1
2
3
4
4
5
6
175.7, C
74.2, CH
40.1, CH
26.9, CH2
175.7, C
74.2, CH
40.1, CH
26.9, CH2
4.20, d (3.5)
1.88, m
3, 4, 6
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
4.20, d (3.2)
1.88, m
3, 4, 6
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
a
1.35, m
1.35, m
b
1.53, m
1.53, m
12.3, CH3
14.0, CH3
0.96, m
12.3, CH3
14.1, CH3
0.96, m
0.93, m
2, 3, 4
1.01, m
2, 3, 4
a,b
Interchangeable signals.
to those of compound 1 for the polyester chain of five isoleucic
acids. The major difference between the H NMR data of both
carbons at 64.6 and 68.2 ppm (Table 1). A cross peak between
the methylene protons H-1′ at 4.21 and 4.41 ppm and the
carbonyl carbon at 171.0 (C-1) indicated that the alditol moiety
is attached to the polyester chain of isoleucic acids via an ester
linkage.
Saponification followed by acidification and spectroscopic
analysis of the products obtained, secured that the sugar alcohol
was erythritol. As a consequence, the structure of compound 2
consists of a single erythritol headgroup 1-O-acylated with a
1
compounds was the presence of only two hydroxylated
methines at 3.56−3.75 ppm, together with two oxygenated
methylenes (3.61 and 3.75, and 4.21 and 4.41 ppm), suggesting
the presence of a glycol moiety having four carbon atoms
1
3
instead of five. The C NMR spectrum showed 34 carbons
with only seven oxygenated methine carbons at 71.3, 73.5, 74.2,
76.1, 76.3, and 76.6 (×2) ppm and two oxygenated methylene
C
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and 4.23 and 4.50 ppm, suggesting that the alditol was
connected to the chain of isoleucic acids by a terminal position.
The downfield shift of the methylene carbon at 68.9 ppm in the
1
3
C NMR spectrum (C-1′) and the presence of a cross-peak in
the HMBC spectrum between the methylene protons H-1′ at
.23 and 4.50 ppm and the carbon at 171.1 ppm (C-1)
4
confirmed the evidence of an ester linkage between position 1′
of the alditol and position 1 of the polyester chain of isoleucic
acids, confirming the structure previously described as
12
hormonemate.
The HRMS spectrum of compound 6 presented an
ammonium adduct at m/z 740.4437 corresponding to a
molecular formula of C H O . The proton and carbon
Figure 1. Key HMBC correlations observed for compound 1.
35
62 15
chain of five isoleucic ester groups which was named
hormonemate B.
chemical shifts of compound 6 were similar to those of
1
13
compound 1. The main difference between the H and
C
Compound 3, obtained as a colorless syrup, was assigned a
molecular formula of C H O based on the ESI-TOF MS of
the ammonium adduct. The NMR data of this compound were
very similar to those of compounds 1 and 2, with the major
difference being the presence of four oxygenated methines at
NMR spectra of both compounds was the chemical shift of the
oxygenated protons of the alditol. An oxygenated methine at
3
6
64 16
1
5.03 ppm appeared in the H NMR spectrum together with a
downfield shifted signal at 76.8 ppm in the carbon spectrum. In
addition, an upfield shifted resonance was observed for the
methylene protons at 3.80 and 3.90 ppm along with the
corresponding upfield shift of their carbon at 61.6 ppm. The
long-range correlation in the HMBC spectrum between the
methine proton at 5.03 ppm and the carbon at 170.5 ppm (C-
1) supported the evidence of an ester linkage between position
2′ of the alditol and position 1 of the polyester chain, to give
the structure that we named hormonemate E.
3
3
.40, 3.92, 3.96, and 5.22, and two oxygenated methylenes at
.50 and 3.66 ppm and 3.62 and 3.79 ppm, suggesting the
13
presence of an alditol with six carbon atoms. The C NMR
data displayed 36 carbons, accounting for the presence of five 2-
hydroxy-3-methylpentanoyl units, and four oxygenated methine
carbons at 70.8, 72.0, 72.4, and 74.8 ppm, and two oxygenated
methylene carbons at 64.7 and 65.2 ppm corresponding to the
alditol moiety (Table 2). The downfield shifted resonance of
Compound 7 was assigned a molecular formula of
1
proton H-3′ at 5.22 ppm in the H NMR data along with the
C H O , based on its HRMS analysis. The chemical shifts
29
52 13
downfield shift of the corresponding carbon C-3′ at 74.8 ppm
in the 13C NMR data, and the long-range HMBC correlation
between this proton and the carbon at 170.9 ppm (C-1) were
consistent with the existence of an ester linkage between the 3′-
position of the alditol and position 1 of the chain of five
isoleucic acids. COSY and HMBC correlations (Table 2) to
other protons and carbons of the alditol confirmed this
structural proposal, and compound 3 was named hormonemate
C.
of the protons and carbons of compound 7 were similar to
1
13
those of compound 1. The difference between the H and
C
NMR data of both compounds was the absence of signals
corresponding to one unit of D-allo-isoleucic acid. Compound 7
consisted therefore in a single arabitol headgroup O-acylated
with polyester tails containing four isoleucic ester groups, and
was called hormonemate F.
Figure 2 displays the overlapped HSQCs spectrum of
hormonemate A (1) together with the standard L-arabitol.
The zoom shot of the alditol region shows the shift of the
methylene protons (H-1a and H-1b) from 3.66/3.83 ppm to
4.22/4.47 ppm due to the esterification of this position. As
expected, the methine proton H-2 is also affected, with a slight
shift downfield. These findings indicate that the O-linkage
between the backbone of the arabitol to the 2-hydroxy-3-
methylpentanoyl polyester tail is through position 1 of the
arabitol moiety. Similarly, Figure S1 (Supporting Information)
points out the 1-O-linkage between erythritol and the 2-
hydroxy-3-methylpentanoyl polyester chain, due to the
remarkable chemical downshift of the methylene protons H-
1a/H-1b, as well as the downshift of the methine proton H-2.
The aforementioned evidence was also applicable to esterified
mannitol as shown in Figure S2 (Supporting Information).
Protons H-1′ and carbon C-1′ of the mannitol unit are highly
affected by the esterification with the polyester chain (3.69/
3.84 ppm in the mannitol to 4.23/4.50 in the esterified
mannitol). The methine proton at position C-2′ is likewise
being affected with a slight downfield shift from 3.74 ppm to
3.90. At the same time, the remaining protons and carbons
were assigned from the multiplicity edited HSQC and HMBC
data and were slightly affected.
Hormonemate D (4) was obtained as a colorless syrup. The
HRESIMS data showed an ammonium adduct with the
molecular formula C H O . Its NMR data were similar to
3
6
64 16
those of compound 3, with the major difference being in the
chemical shift of the four oxygenated methines at 3.68, 3.46,
4
.11, and 5.03, and two oxygenated methylenes at 3.60 and 3.79
ppm and 3.81 and 3.91 ppm, suggesting that the alditol was
connected to the chain of isoleucic acids at a different position.
The 13C NMR data also displayed 36 carbons with nine
oxygenated methine carbons at 69.1, 71.6, 72.8, 74.2, 76.0, 76.3,
76.5, 76.6, and 76.8 ppm, together with two oxygenated
methylene carbons at 61.8 and 65.2 ppm (Table 2). In this case,
1
the deshielded proton at 5.03 ppm in the H NMR spectrum
corresponded to H-2′ according to COSY and HMBC
measurements. Its downshifted carbon at 76.8 ppm in the
1
3
C NMR data and the cross-peak in the HMBC spectrum
between this proton and the carbon at 170.5 ppm (C-1)
provided further evidence of an ester linkage between position
C-2′ of the alditol and position C-1 of the chain of five isoleucic
acids.
Compound 5 was assigned a molecular formula of C H O
36
64 16
based on its HRESIMS analysis. Its NMR data were similar to
those of compounds 3 and 4, with the major difference being in
the chemical shift of the four oxygenated methines at 3.68−3.90
ppm, and the two oxygenated methylenes at 3.64 and 3.81 ppm
Further evidence of the structures proposed was also
obtained through analysis of compounds 1, 2, and 5 by
quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS/
D
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Table 2. NMR Spectroscopic Data (500 Mz, CD OD) for Hormonemate C (3), Hormonemate D (4), and Hormonemate (5)
3
hormonemate C (3)
δH (J in Hz)
hormonemate D (4)
δH (J in Hz)
hormonemate (5)
δH (J in Hz)
position
D-mannitol
′a
′b
δC, type
HMBC
δC
HMBC
δC
HMBC
1
64.7, CH2
3.50, dd (11.7, 6.9) 2′, 3′
3.66, dd (11.7, 3.5) 3′
61.8
3.81, dd (12.3, 3.4) 2′, 3′
3.91, dd (12.3, 3.4) 3′
68.9
4.23, dd (11.4, 6.7) 2′, 1A
4.50, dd (11.4, 2.4) 3′, 1A
1
2
3
4
5
6
6
′
′
′
′
′a
′b
72.0, CH
74.8, CH
70.8, CH
72.4, CH
65.2, CH2
3.92, m
3′
76.8
69.1
71.6
72.8
65.2
5.03, m
3′, 4′, 1A
1′, 2′
5′, 6′
3′, 4′, 6′
70.4
71.1
71.1
73.0
65.2
3.90, m
3.78, m
3.78, m
3.68, m
3.64, m
3.81, m
4′
5.22, dd (7.8, 1.4)
3.96, dd (9.4, 1.4)
3.40, m
1′, 2′, 1A
3′, 5′, 6′
4′
4.11, d (8.3)
1′, 5′
2′, 6′
3′
5′
5′
d
d
f
3.46 , d (8.3)
f
3.68 , m
3.62, dd (11.4, 5.6) 4′, 5′
3.79, dd (11.4, 2.7) 4′
3.60, dd (11.1, 6.0) 4′, 5′
3.79, dd (11.1, 4.6) 4′, 5′
D-allo-isoleucic acid A
1
2
3
4
4
5
6
170.9, C
76.7, CH
37.7, CH
170.5
76.6
37.7
27.2
171.1
76.6
37.8
27.1
a
e
5.13, d (2.8)
2.12, m
1, 3, 4, 6, 1B
5.20, d (3.0)
2.10, m
1, 3, 4, 6, 1B
5.16, d (3.2)
2.10, m
1, 3, 4, 6, 1B
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
a
27.5, CH2
1.36, m
1.36, m
1.36, m
b
1.53, m
1.54, m
1.53, m
12.0, CH3
14.8, CH3
0.96, m
12.0
14.9
0.96, m
12.0
14.9
0.95, m
1.02, m
2, 3, 4
1.02, m
2, 3, 4
1.01, m
2, 3, 4
D-allo-isoleucic acid B
b
h
i
1
2
3
4
4
5
6
170. 8, C
76.3, CH
38.1, CH
170. 5
170.6
76.5
38.1
27.0
a
c
e
g
j
5.12 , d (3.3)
1, 3, 4, 6, 1C
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
76.5
5.12 , d (3.5)
1, 3, 4, 6, 1C
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
5.11 , d (3.2)
1, 3, 4, 6, 1C
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
2.12, m
1.36, m
1.53, m
0.96, m
1.02, m
38.1
26.9
2.10, m
1.36, m
1.54, m
0.96, m
1.02, m
2.10, m
1.36, m
1.53, m
0.95, m
1.01, m
a
27.0, CH2
b
12.1, CH3
14.7, CH3
12.1
14.8
12.1
14.8
2, 3, 4
2, 3, 4
2, 3, 4
D-allo-isoleucic acid C
b
h
i
1
2
170.6, C
76.6, CH
170.6
170.5
76.3
a
c
e
g
j
5.10 , d (3.4)
1, 3, 4, 6,
76.3
5.11 , d (3.7)
1, 3, 4, 6, 1D
5.10 , d (3.4)
1, 3, 4, 6,
1D
1
D
3
4
4
5
6
38.1, CH
2.12, m
1.36, m
1.53, m
0.96, m
1.02, m
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
38.1
27.0
2.10, m
1.36, m
1.54, m
0.96, m
1.02, m
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
38.1
27.0
2.10, m
1.36, m
1.53, m
0.95, m
1.01, m
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
a
26.9, CH2
b
12.1, CH3
14.7, CH3
12.1
14.7
12.1
14.7
2, 3, 4
2, 3, 4
2, 3, 4
D-allo-isoleucic acid D
1
2
3
4
4
5
6
170.7, C
76.1, CH
38.1, CH
170.7
76.0
38.0
27.0
170.7
76.0
38.0
26.9
a
5.09, (3.3)
2.12, m
1.36, m
1.53, m
0.96, m
1.02, m
1, 3, 4, 6, 1E
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
5.09, d (3.4)
2.10, m
1, 3, 4, 6, 1E
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
5.09, d (3.2)
2.10, m
1, 3, 4, 6, 1E
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
a
27.1, CH2
1.36, m
1.36, m
b
1.54, m
1.53, m
12.1, CH3
14.7, CH3
12.0
14.7
0.96, m
12.1
14.7
0.95, m
2, 3, 4
1.02, m
2, 3, 4
1.01, m
2, 3, 4
D-allo-isoleucic acid E
1
2
3
4
4
5
6
175.7, C
74.2, CH
40.1, CH
27.1, CH2
175.7
74.2
40.1
27.0
175.6
74.2
40.1
26.9
4.20, d (3.2)
1.88, m
1, 3, 4, 6
4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
4.20, d (3.4)
1.88, m
1, 3, 4, 6
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
4.20, d (3.4)
1.88, m
1, 3, 4, 6
1, 4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
a
1.36, m
1.36, m
1.36, m
b
1.53, m
1.54, m
1.53, m
12.3, CH3
14.1, CH3
0.96, m
12.3
14.0
0.96, m
12.3
14.0
0.95, m
0.92, m
2, 3, 4
0.93, m
2, 3, 4
0.92, m
2, 3, 4
a−jInterchangeable signals.
MS). Data shown in the Supporting Information (Figures S53,
S54, and S55) confirmed the connectivity of the monomers,
and were also consistent with the structures proposed.
ing this was the D-allo (2R,3S) form of the acid based on the
25
negative value of its specific optical rotation [α] = −30.4° (c
D
16
0.01, MeOH). NMR and optical rotation data from the alditol
residues were compared with those obtained for the authentic
L-arabitol, erythritol, and D-mannitol (Figures S45−S50). In the
To determine the absolute configuration of the 2-hydroxy-3-
methylpentanoic acid moiety present in all the compounds of
the series, its NMR and optical rotation data after alkaline
2
5
case of arabitol, its negative specific optical rotation [α]D
=
15
hydrolysis were compared with those published, demonstrat-
−17.8° (c 0.06, MeOH) was consistent with that of standard L-
E
DOI: 10.1021/acs.jnatprod.6b00680
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 2. Overlapped multiplicity edited HSQC spectra of hormonemate A (1) and L-arabitol, showing the “alditol” region. Red and blue signals
correspond to hormonemate A whereas green and pink signals are those from L-arabitol.
arabitol, [α]25 = −9.2° (c 0.08, MeOH). Erythritol is the meso
Table 3. Cytotoxic Activity of the Isolated Compounds
Expressed as Inhibitory Concentration (IC50
D
form of an alditol with four carbon atoms, so the specific optical
rotation measured was zero. Measurement of the optical
rotation of mannitol in the presence of 10% ammonium
)
HepG2
MCF-7
MiaPaca_2
compounds
μg/mL
μM
μg/mL
μM
μg/mL
μM
2
5
molybdate and 1 N sulfuric acid gave a positive value of [α] =
D
hormonemate A
20.9
29.0
11.3
15.6
>40.0
>40.0
>40.0
>40.0
>40.0
27.4
>55.4
+
3
103.1°, confirming the presence of D-mannitol in compounds
−5.
Recently, a family of similar structures has been isolated from
(1)
hormonemate B
2)
19.9
28.7
>53.2
36.2
7.7
11.1
27.8
18.3
24.3
19.0
>65.7
1.4
>57.8
>53.2
>53.2
>53.2
36.4
(
hormonemate C
3)
>40.0
27.3
20.9
13.8
18.2
13.9
>40.0
0.8
the polymorphic fungus Aureobasidium pullulans, a micro-
organism isolated from tiger lily flowers. A. pullulans, often
considered to be a black yeast because cultures produce
melanin, is an ascomycetous yeast belonging to the
Dothideomycetes class, order Dothideales: the same as
Dothiora sp. Apart from polysaccharide pullulans, A. pullulans
produces extracellular heavy oils; these oils are 3,5-dihydrox-
ydecanoyl and 5-hydroxy-2-decenoyl esters of mannitol,
(
hormonemate D
(4)
hormonemate
>40.0
18.7
>53.2
26.2
(5)
hormonemate E
(6)
hormonemate F
>40.0
0.1
>65.7
0.2
>40.0
1.0
>65.7
1.9
(7)
doxorubicin
13,14
arabitol, or glycerol, named as liamocins.
Interestingly, these alditol lipids exerted an antiproliferative
EXPERIMENTAL SECTION
14,17
■
effect on the human lung cancer cell line A549
as well as in
General Experimental Procedures. Optical rotations were
measured using a Jasco P-2000 polarimeter. UV spectra were obtained
with an Agilent 1100 DAD. IR spectra were recorded on a JASCO FT/
IR-4100 spectrometer equipped with a PIKE MIRacle single reflection
ATR accessory. NMR spectra were recorded on a Bruker Avance III
spectrometer (500 and 125 MHz for H and C NMR, respectively)
equipped with a 1.7 mm TCI MicroCryoProbe (Bruker Biospin).
Chemical shifts were reported in ppm using the signals of the residual
two human breast cancer cell lines (T47D and SK-BR3) and a
18
human cervical cell line (HeLa). In addition, liamocins also
19
showed antibacterial activity and act as biosurfactants with
important biotechnological applications in the food industry,
medicine, and cosmetics due to their specific modes of action
1
13
20
and low toxicity. Similarly, hormonemates could also act as
antibacterial and/or biosurfactants, expanding their potential
solvent as internal reference (δ
3.31 and δ 49.15 for CD OD, δ
H
C 3 H
1
9,20
4.75 for deuterium oxide). LC-MS and LC-HRMS analysis were
applications.
2
1
performed as described previously.
The cytotoxic activity of the hormonemates was tested
against three tumor cell lines. Table 3 shows the IC50 values
obtained for the pure compounds. No clear structure−activity
relationship could be established from the values obtained.
Overall, human breast cancer cells (MCF-7) exhibited the
greatest sensitivity to hormonemates, mainly to hormonemate
B (IC50 = 11.1 μM) and hormonemate A (IC50 = 15.6 μM).
Additionally, an IC around 30 μM was measured for most of
Experimental Biological Material. Cell culture medium, fetal
bovine serum, L-glutamine, sodium pyruvate, MEM nonessential
amino acids, penicillin-streptomycin, triple express, PBS, and bovine
insulin were purchased from Life Technologies (Gigco, Invitrogen
Corporation, CA, USA). Actinomycin D, doxorubicin, MTT (3-(4,5-
dimethylthiazol-2-yl-)-2,5-diphenyl tetrazolium bromide), and MMS
(
methylmethanesulfonate) were purchased from Sigma Chemical Co.
(St. Louis MO. USA). Cells were maintained and plated with a cell
culture robotic system, SelecT (TAP Biosystems). All cell lines were
obtained from the American Type Culture Collection (ATCC,
Manassas, VA, USA). Medium, serum, and complements were
purchased from Invitrogen. HepG2 cells (human liver carcinoma,
CCL-8065) were grown in ATCC-formulated Eagle’s M essential
medium (MEM) with 10% qualified FBS, 2 mM L-glutamine, 1 mM
5
0
the compounds against human hepatic cancer cells (HepG2).
Except hormonemate E (IC50 = 36.4 μM), none of them were
active against the pancreatic cell line (MiaPaca_2) at the
highest concentration tested.
F
DOI: 10.1021/acs.jnatprod.6b00680
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 4. NMR Spectroscopic Data (500 Mz, CD OD) for Hormonemate E (6) and Hormonemate F (7)
3
hormonemate E (6)
δH (J in Hz)
hormonemate F (7)
δH (J in Hz)
position
δC, type
HMBC
δC, type
HMBC
L-arabitol
1
1
2
3
4
5
′a
′b
′
′
′
61.6, CH2
3.80, dd (12.3, 3.3)
3.90, dd (12.3, 4.5)
5.03, m
3′
68.7, CH2
4.23, dd (11.4, 6.5)
4.47, dd (11.4, 2.7)
3.90, m
2′, 3′, 1A
3′, 1A
5′
1′, 2′, 5′
3′
2′, 3′
3′, 4′, 1A
1′, 2′
5′
76.8, CH
70.0, CH
71.8, CH
64.5, CH2
70.5, CH
72.1, CH
71.7, CH
64.8, CH2
3.87, d (8.4)
3.61, m
3.53, dd (8.7, 1.8)
3.89, dd (6.2, 1.8)
3.63, d (6.2)
′
3.61, m
3′, 4′
3′, 4′
D-allo-isoleucic acid A
1
2
3
4
4
5
6
170.5, C
171.2, C
76.7, CH
37.9, CH
27.1, CH2
a
76.5, CH
37.9, CH
5.21, d (3.1)
2.10, m
1, 3, 4, 6, 1B
4, 5, 6
5.15, d (3.3)
2.09, m
1, 3, 4, 6, 1B
4, 5, 6
a
27.3, CH2
1.35, m
2, 3, 5, 6
2, 3, 5, 6
4
1.36, m
2, 3, 5, 6
2, 3, 5, 6
4
b
1.53, m
1.53, m
12.0, CH3
14.9, CH3
0.98, m
12.0, CH3
14.9, CH3
0.97, m
1.02, m
2, 3, 4
1.02, m
2, 3, 4
D-allo-isoleucic acid B
b
d
1
2
3
4
4
5
6
170. 6, C
170.9, C
a
c
76.4, CH
38.2, CH
26.9, CH2
5.12 , d (3.2)
1, 3, 4, 6, 1C
4, 5, 6
76.5, CH
37.1, CH
27.0, CH2
5.10, d (3.4)
2.09, m
1, 3, 4, 6, 1C
4, 5, 6
2.10, m
1.35, m
1.53, m
0.98, m
1.02, m
a
2, 3, 5, 6
2, 3, 5, 6
4
1.36, m
2, 3, 5, 6
2, 3, 5, 6
4
b
1.53, m
12.1, CH3
14.8, CH3
12.1, CH3
14.8, CH3
0.97, m
2, 3, 4
1.02, m
2, 3, 4
D-allo-isoleucic acid C
b
d
1
2
3
4
4
5
6
170.7, C
170.7, C
a
c
76.7, CH
38.1, CH
5.10 , d (3.4)
1, 3, 4, 6, 1D
4, 5, 6
76.1, CH
38.2, CH
27.0, CH2
5.09, d (3.4)
2.09, m
1, 3, 4, 6, 1D
4, 5, 6
2.10, m
1.35, m
1.53, m
0.98, m
1.02, m
a
27.0, CH2
2, 3, 5, 6
2, 3, 5, 6
4
1.36, m
2, 3, 5, 6
2, 3, 5, 6
4
b
1.53, m
12.1, CH3
14.8, CH3
12.1, CH3
14.7, CH3
0.97, m
2, 3, 4
1.02, m
2, 3, 4
D-allo-isoleucic acid D
1
2
3
4
4
5
6
170.8, C
170.7, C
74.2, CH
40.1, CH
27.0, CH2
a
76.1, CH
38.1, CH
5.09, d (3.4)
2.10, m
1, 3, 4, 6, 1E
4, 5, 6
4.20, d (3.5)
1.88, m
1, 3, 4, 6
4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
a
27.0, CH2
1.35, m
2, 3, 5, 6
2, 3, 5, 6
4
1.36, m
b
1.53, m
1.53, m
12.0, CH3
14.7, CH3
0.98, m
12.3, CH3
14.1, CH3
0.97, m
1.02, m
2, 3, 4
0.93, m
2, 3, 4
D-allo-isoleucic acid E
1
2
3
4
4
5
6
175.7, C
74.2, CH
40.1, CH
27.0, CH2
4.20, d (3.4)
1.88, m
1, 3, 4, 6
4, 5, 6
2, 3, 5, 6
2, 3, 5, 6
4
a
1.35, m
b
1.53, m
12.3, CH3
14.1, CH3
0.98, m
0.93, m
2, 3, 4
a−dInterchangeable signals.
sodium pyruvate, and 100 μM MEM nonessential amino acids. MCF-7
cells (human breast HTB-22) were maintained in the previous
medium supplemented with 0.01 mg/mL bovine insulin. MiaPaca_2
cell line (ATCC CRL-1420), a fibroblast primary pancreatic
adenocarcinoma cell line which has mutated K-RAS, P16 y P53
genes, was grown in Dulbecco’s modified Eagle’s medium,
supplemented with 10% fetal bovine serum, 2.5% fetal horse serum,
identification was made by one of us, J. Molero-Mesa from the
Deparment of Botany, Faculty of Pharmacy, University of Granada,
where a voucher specimen has been deposited.
Producing Fungus, Fungal Dimorphic Growth, and Culti-
vation. The producing organism (CF-285353) was isolated from
aerial parts of the plant by using a method for isolating fungal
5
endophytes described previously. Its ITS1-5.8S-ITS228S region or
1% L-glutamine, and 1% penicillin/streptomycin. Cell cultures were
independent ITS and 28S rDNA was completely sequenced and
compared with GenBank (KU295575) and the NITE Biological
Resource Center (http://www.nbrc.nite.go.jp/) databases by using the
BLAST application. Frozen stock cultures in 10% glycerol (−80 °C)
maintained in a humidified incubator at 37 °C with 5% CO2.
Plant Material. Aerial parts of L. arborescens were collected from
́
Tabernas desert (Almerıa, Spain), in May 2014. The taxonomical
G
DOI: 10.1021/acs.jnatprod.6b00680
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
3
C NMR data see Table 1; (+)-HRESIMS m/z 740.4437 [M+NH ]+
were maintained in the collection of Fundacion
medinadiscovery.com).
Different morphologies of this strain have recently been reported as
dependent on the number of subcultures performed. Changes in the
fermentation morphology (pigmentation and conidia/hyphae con-
version) during the growth of the fungus affected the production of
the secondary metabolites, and hormonemates were only present
when Dothiora sp. (Dothideales) was grown as a yeast, featuring a
black thick broth with high density of conidia. However, when a
subculturing step was added, it resulted in a yellowish broth containing
hyphae and a few conidia. Similar findings were observed for
Hormonema dematioides (Dothideales), producing hormonemate at
́
MEDINA (www.
4
(calcd for C H NO , 740.4427).
3
5
66
15
25
Hormonemate B (2). colorless syrup; [α]D +27.4° (c 0.25,
5
MeOH); UV (DAD) end absorption; IR (ATR) νmax 3374, 2965,
−1 1
2937, 2879, 1749, 1462, 1385, 1185, 1127, 1105, 1046, 1023 cm ; H
and 13C NMR data see Table 1; (+)-HRESIMS m/z 710.4331 [M
+
+NH ] (calcd for C H NO , 710.4321).
4
34 64
14
2
5
Hormonemate C (3). colorless syrup; [α]
+5.25° (c 0.08,
D
MeOH); UV (DAD) end absorption; IR (ATR) νmax 3363, 2965,
−
1 1
2937, 2879, 1750, 1642, 1385, 1185, 1127, 1105, 1021 cm ; H and
13
+
C NMR data see Table 2; (+)-HRESIMS m/z 770.4547 [M+NH
(calcd for C36 68NO16, 770.4533).
Hormonemate D (4). colorless syrup; [α]
MeOH); UV (DAD) end absorption; IR (ATR) νmax 3363, 2965,
]
4
H
1
2
25
its mature state, in a melanized culture with a dark-green appearance
and the polymorphic fungus Aureobasidium pullulans (Dothideales),
+11.3° (c 0.17,
D
13
−1 1
producing liamocins. Hence, hormonemates could be identified as
biomarkers for the conidia morphology of the fungus Dothiora sp.
To scale up the fermentation to 600 mL, 10 mycelial discs of fungal
strain grown on YM agar plates at 22 °C for 7 days were used to
inoculate 50 mL of SMYA (Difco neopeptone 10 g, maltose 40 g,
2937, 2879, 1750, 1642, 1385, 1185, 1127, 1105, 1021 cm ; H and
13
+
C NMR data see Table 2; (+)-HRESIMS m/z 770.4546 [M+NH
]
4
(calcd for C H NO , 770.4533).
Hormonemate (5). colorless syrup; [α]
UV (DAD) end absorption; IR (ATR) νmax 3363, 2965, 2937, 2879,
36
68
16
2
5
+39.2° (c 1.11, MeOH);
D
−1
1
13
Difco yeast extract 10 g, agar 4 g, distilled H O 1 L). After 7 days
1750, 1642, 1385, 1185, 1127, 1105, 1021 cm ; H and C NMR
2
+
incubation at 22 °C and 220 rpm, 3 mL aliquots of this culture were
used to inoculate YES medium (Difco yeast extract 20 g, sucrose 150
g, MgSO ·7H O 0.5 g, trace elements 1 mL (ZnSO ·7H O 1 g/100
data see Table 2; (+)-HRESIMS m/z 770.4547 [M+NH
68NO16, 770.4533).
Hormonemate E (6). colorless syrup; [α]
MeOH); UV (DAD) end absorption; IR (ATR) ν
4
] (calcd for
C H
36
2
5
+32.5° (c 0.27,
3387, 2965,
4
2
4
2
D
mL and CuSO ·5H O 0.5 g/100 mL) and distilled H O 1 L)
max
4
2
2
−
1
1
13
2
937, 2879, 1751, 1462, 1185, 1126, 1046, 1023 cm ; H and
C
distributed among 6 × 100 mL in 500 mL Erlenmeyer flasks. The
flasks were incubated statically at 22 °C, 70% relative humidity for 14
days.
Isolation and Identification of Metabolites. The conidia
culture and fermentation broth (600 mL) were extracted by adding
acetone (600 mL) and shaking at 220 rpm for 2 h. After filtration, the
acetone extract was concentrated under reduced pressure to a final
volume of 600 mL (100% water). The aqueous residue was loaded
onto a SP207ss resin column (65 g, 32 × 100 mm) and eluted with an
+
NMR data see Table 4; (+)-HRESIMS m/z 740.4437 [M+NH4]
(
calcd for C H NO , 740.4427).
35 66 15
25
Hormonemate F (7). colorless syrup; [α]D + 0.6° (c 0.03, MeOH);
UV (DAD) end absorption; IR (ATR) νmax 3387, 2965, 2937, 2879,
1
data see Table 4; (+)-HRESIMS m/z 626.3750 [M+NH ] (calcd for
C H NO , 626.3746).
Alkaline Hydrolysis of 1, 2, and 5. To solutions in THF (0.4
mL) of compounds 1 (3.1 mg), 2 (3.1 mg), and 5 (2.6 mg) was added
NaOH 2 N (0.2 mL), and the resulting solution was stirred for 15 min
at room temperature. After hydrolysis was completed, reaction
mixtures were neutralized with HCl 1 N and evaporated to dryness.
The residues were subjected to semipreparative reversed-phase HPLC
−1
1
13
751, 1462, 1385, 1185, 1126, 1046, 1023 cm ; H and C NMR
+
4
29 56
13
acetone−H O stepped gradient (10/90 for 6 min, 20/80 for 6 min,
2
40/60 for 6 min, 60/40 for 6 min, 80/20 for 6 min, and 100/0 for 12
min, 10 mL/min, 20 mL/fraction) to give 19 fractions. Bioactive
fractions 15 to 18 were pooled and subjected to preparative reversed-
phase HPLC (Zorbax SB-C18 PrepHT, 21.2 × 250 mm, 7 μm, 16 mL/
(
Zorbax SB-C , 9.4 × 250 mm, 5 μm, 3.6 mL/min, detection at 210
18
min, UV detection at 210 and 280 nm, 10 mL/fraction) using H O +
2
nm) using a linear gradient of CH CN in water (5−10% CH CN over
3
3
0
.1% TFA (solvent A) and CH CN + 0.1% TFA (solvent B). Elution
3
2
0 min after a 10 min period at 5% CH CN) for elution. This afforded
3
was carried out using isocratic conditions of 5% B for 5 min and then a
linear gradient from 5% to 100% B in 43 min, yielding 79 fractions that
were tested against MCF-7 and HepG2 cells. Subfractions 53−57 and
the alditols, L-arabitol (0.4 mg, 4.0 min), erythritol (0.3 mg, 4.0 min),
and D-mannitol (0.4 mg, 4.0 min), together with D-allo-isoleucic acid
(
1.9, 2.1, and 1.6 mg respectively, 6.0 min).
60 were active against MCF-7 and fractions 53 and 60 against HepG2
25
D-allo-Isoleucic acid. white amorphous powder; [α]D −30.4° (c
cells. Subfractions 57 and 60 of this chromatography yielded
compounds 1 (tR 40 min, 54.0 mg) and 2 (tR 42 min, 18.3 mg),
respectively.
Subfraction 53 was further purified by semipreparative reversed-
phase HPLC (Zorbax SB-C18 SemiPrep column, 9.4 × 250 mm, 5 μm,
1
0
.01, MeOH); H NMR (500 MHz, D O) δ 4.00 (1H, d, J = 2.80 Hz,
2
H-2), 1.79−1.74 (1H, dddq, J = 2.80, 4.20, 7.04, 9.88 Hz, H-3), 1.44−
1
.35 (1H, m, H-4b), 1.31−1.22 (1H, m, H-4a), 0.91 (3H, t, J = 7.41
13
Hz, H-5), 0.77 (3H, d, J = 7.04 Hz, H-6); C NMR (D O) (obtained
2
from HSQC) δ 75.32 (C-2), 38.76 (C-3), 26.63 (C-4), 13.67 (C-6)
and 11.99 (C-5).
3.6 mL/min, UV detection at 210 and 280 nm, 1.8 mL/fraction) using
H O + 0.1% TFA (solvent A) and CH CN + 0.1% TFA (solvent B).
25
2
3
L-Arabitol. white amorphous powder; [α]D −17.8° (c 0.06,
Elution was carried out using isocratic conditions of 50% B for 65 min
1
MeOH); H NMR (500 MHz, D O) δ 3.95 (1H, td, J = 2.06, 7.18
2
to yield compounds 3 (t 38 min, 1.0 mg), 4 (t 40 min, 4.8 mg), and
R
R
Hz, H-4), 3.84 (1H, dd, J = 2.96, 11.71 Hz, H-1b), 3.77 (1H, td, J =
2.96, 8.10 Hz, H-2), 3.67−3.69 (3H, m, H-1a, H-5) and 3.60 (1H, dd, J
=
5
(t 49 min, 5.4 mg).
R
Subfraction 55 was further purified by preparative reversed-phase
2.06, 8.10 Hz, H-3).
Erythritol. white amorphous powder; [α]D 0° (c 0.95, MeOH); H
HPLC (Zorbax SB-C18 PrepHT, 21.2 × 250 mm, 7 μm, 16 mL/min,
UV detection at 210 and 280 nm, 10 mL/fraction) eluting with
isocratic conditions of 50% CH CN in H O for 75 min to yield
25
1
NMR (500 MHz, D O) δ 3.76 (2H, d, J = 11.46 Hz, H-1b, H-4b),
3.69−3.70 (2H, m, H-2, H-3) and 3.62 (2H, dd, J = 6.18, 11.46 Hz, H-
1a, H-4a).
2
3
2
compounds 5 (t 39 min, 3.2 mg), 6 (t 46 min, 7.4 mg), and 1 (t 52
R
R
R
D-Mannitol. white amorphous powder [α]2 0° (c 1.15, MeOH);
5
min, 15.8 mg).
D
[α]2 + 103.1° (c 0.11, 10% (NH ) MoO , 1 N H SO ); H NMR
(500 MHz, D O) δ 3.86 (2H, dd, J = 2.69, 11.76 Hz, H-1b, H-6b),
3.81 (2H, d, J = 8.47 Hz, H-3, H-4), 3.74−3.78 (1H, dd, J = 2.69, 8.47
Hz, H-2), 3.74−3.78 (1H, dd, J = 2.69, 6.03 Hz, H-5) and 3.67−3.69
(2H, dd, J = 6.03, 11.76 Hz, H-1a, H-6a).
5
D
1
Subfraction 56 was further purified by preparative reversed-phase
4
2
4
2
4
HPLC (Waters XBridge Prep C , 10 × 250 mm, 5 μm, 16 mL/min,
18
2
UV detection at 210 and 280 nm, 10 mL/fraction) eluting with
isocratic conditions of 55% CH CN in H O for 75 min to yield
3
2
compounds 7 (t 16 min, 1.5 mg), 6 (t 44 min, 27.1 mg), and 1 (t
R
R
R
4
8 min, 75.6 mg).
MTT Cytotoxicity Assay. The MTT reduction rate is an indicator
Hormonemate A (1). colorless syrup; [α] D2 5 +26.2° (c 1.18,
of the functional integrity of the mitochondria and, hence, of cellular
viability.
assay time: 20000 cells/well concentration for HepG2 and MiaPaca_2,
2
2,23
MeOH); UV (DAD) end absorption; IR (ATR) νmax 3387, 2965,
Two different cell concentrations were dependent on the
−
1 1
2937, 2879, 1751, 1462, 1385, 1185, 1126, 1046, 1023 cm ; H and
H
DOI: 10.1021/acs.jnatprod.6b00680
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
and 30000 cells/well for MCF-7 in the 24 h assay, and 10000 cell/well
for HepG2 and MiaPaca_2 and 15000 cells/well for MCF-7 in the 72
h assay. Extract and fractions were tested at a dilution 1/40 (5 μL of
extract/fractions into 195 μL of fresh medium), whereas pure
compounds were analyzed at 1/200 dilution (3 μL of pure compound
into 597 μL of fresh medium). MMS was used as positive control, and
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.5% DMSO was used as negative control. The maximum
concentration of DMSO was 0.5% to minimize any solvent toxicity
background. Pure compounds were tested in triplicate at 10
concentrations using 2-fold serial dilutions starting at a maximum
concentration of 40 μg/mL following the method previously
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described.
(
ASSOCIATED CONTENT
Supporting Information
■
(
*
S
M.; Tayeh, M.; Teeraworawit, S.; Kriwong, S.; Leathers, T. D. World J.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00680.
Microbiol. Biotechnol. 2014, 30, 2199−2204.
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D and 2D NMR spectra of hormonemates and
(
21) Martín, J.; Crespo, G.; Gonzal
́
ez-Menen
́
dez, V.; Per
́
ez-Moreno,
degradation products, and MS/MS fragmentation data
of selected compounds (PDF)
G.; San
Gonzal
Reyes, F. J. Nat. Prod. 2014, 77, 2118−2123.
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ez-Pacanowska, D.; Vicente, F.; Genilloud, O.; Bills, G. F.;
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ez, L. M.;
́
(
AUTHOR INFORMATION
■
Roeck-Holtzhauer, Y. J. Pharmacol. Toxicol. Methods 1998, 39, 143−
Corresponding Authors
E- mail: mercedes.perez@medinaandalucia.es (M.P.-B.). Tel.:
34 958993965.
E-mail: fernando.reyes@medinaandalucia.es (F.R.). Tel.: +34
58993965, ext. 7006.
ORCID
1
46.
*
(23) Liu, Y.; Nair, M. G. J. Nat. Prod. 2010, 73, 1193−1195.
(24) Audoin, C.; Bonhomme, D.; Ivanisevic, J.; de la Cruz, M.;
+
*
9
Cautain, B.; Monteiro, M. C.; Reyes, F.; Rios, L.; Per
O. P. Mar. Drugs 2013, 11, 1477−1489.
́
ez, T.; Thomas,
́
Ignacio Perez-Victoria: 0000-0002-4556-688X
Fernando Reyes: 0000-0003-1607-5106
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the Junta de Andalucıa
́
through Project
RNM-7987 is gratefully acknowledged. The polarimeter;
HPLC, IR, and NMR equipment; and plate reader used in
this work were purchased via grants for scientific and
technological infrastructures from the Ministerio de Ciencia e
Innovacion
́
[Grants No. PCT-010000-2010-4 (NMR), INP-
2
011-0016-PCT-010000 ACT6 (polarimeter, HPLC, and IR),
and PCT-01000-ACT7, 2011-13 (plate reader)].
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J. Nat. Prod. XXXX, XXX, XXX−XXX