Amarisolide F, an Acylated Diterpenoid Glucoside and Related Terpenoids from Salvia amarissima

Article abstract of DOI:10.1021/acs.jnatprod.8b00565

Nine terpenoids were isolated from the leaves and flowers of Salvia amarissima, including a new acylated diterpenoid glucoside, amarisolide F (1), a new neo-clerodane diterpenoid, amarissinin D (2), which was isolated as an acetyl derivative (2a), and four known diterpenoids. The structure of amarisolide F (1) was elucidated by NMR and MS data analyses, as well as its methanolysis products 7 and 8, which also constituted new diterpenoids, named amarissinin E and 8-epi-amarissinin E, respectively. The absolute configuration of compound 7 was established by single-crystal X-ray diffraction. The cytotoxicity and anti-MDR effect of 1 in three phenotypes of the MCF-7 cell lines were assayed. Compound 1 was 2-3.6-fold more active than amarissinins A (3) and B (4), but several orders of magnitude less active than teotihuacanin (6) and reserpine.

Full text of DOI:10.1021/acs.jnatprod.8b00565

Note
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Amarisolide F, an Acylated Diterpenoid Glucoside and Related
Terpenoids from Salvia amarissima
†
‡
§,⊥
A. Toscano,∥
Mabel Fragoso-Serrano, Naytze
Angel G. Alpuche-Solís, Alfredo Ortega, and Elihu
́
Ortiz-Pastrana, Norma Luna-Cruz, Ruben
́
⊥
∥
,§
́
Bautista*
†
Departamento de Farmacia, Facultad de Química, Universidad Nacional Auton
, Ciudad de Mexico 04510, Mexico
Departamento de Química, Centro de Investigacion y de Estudios Avanzados del IPN, Avenida IPN 2508, Col. San Pedro
Zacatenco, Ciudad de Mexico 07360, Mexico
CONACYT-Consorcio de Investigacion, Innovacion
Investigacion Científica y Tecnologica A. C., Camino a la Presa San Jose
de Biología Molecular, Instituto Potosino de Investigacion Científica y Tecnolog
055, Lomas 4ta seccion, San Luis Potosí 78216, Mexico
Instituto de Química, Universidad Nacional Autonoma de Mex
Mexico 04510, Mexico
oma de Mexico, Circuito Exterior, Ciudad
́ ́
Universitaria, Coyoacan
́
́
́
‡
́
́
́
§
́
́
́
y Desarrollo para las Zonas Aridas (CIIDZA), Instituto Potosino de
2055, Lomas 4ta seccion, San Luis Potosí 78216, Mex
ica A. C., Camino a la Presa San Jose
́
́
́
́
́
́
ico
⊥
Division
́
́
́
2
∥
́
́
́
ico, Circuito Exterior, Ciudad Universitaria, Coyoacan, Ciudad de
́ ́
́
́
*
S Supporting Information
ABSTRACT: Nine terpenoids were isolated from the leaves
and flowers of Salvia amarissima, including a new acylated
diterpenoid glucoside, amarisolide F (1), a new neo-clerodane
diterpenoid, amarissinin D (2), which was isolated as an acetyl
derivative (2a), and four known diterpenoids. The structure of
amarisolide F (1) was elucidated by NMR and MS data
analyses, as well as its methanolysis products 7 and 8, which
also constituted new diterpenoids, named amarissinin E and
8
-epi-amarissinin E, respectively. The absolute configuration
of compound 7 was established by single-crystal X-ray
diffraction. The cytotoxicity and anti-MDR effect of 1 in
three phenotypes of the MCF-7 cell lines were assayed.
Compound 1 was 2−3.6-fold more active than amarissinins A
(
3) and B (4), but several orders of magnitude less active than teotihuacanin (6) and reserpine.
Salvia amarissima (Lamiaceae) is a herbaceous and perennial
plant that is endemic to Mexico. The distribution of this plant is
found in semiarid and wooded areas from the states of San Luis
the Salvia genus and search for new bioactive natural products,
scale-up of the isolation of teotihuacanin (6) to obtain
semisynthetic derivatives as potential MDR modulators was
performed. Herein, we describe the isolation and structural
elucidation of a new diterpenoid glucoside (1), its methanolysis
products (7/8), which also constituted new diterpenoids named
amarissinin E/8-epi-amarissinin E, and the crystal structure of
the O-acetyl derivative (2a) as a new diterpenoid, amarissinin D
1
,2
Potosi to Oaxaca. This plant species is popularly known as
“
hierba del cancer”, “insulina”, “hierba azul”, “chupona”,
3
,4
“
peludito”, and “nadri”. The common names for this plant
̃
species describe some of their medicinal uses to treat cancer and
3
,5
diabetes, as well as rheumatism. In addition, this plant
together with 17 other Salvia species constitute the group of
(
2).
Chemical reinvestigation of an acetone-soluble extract of the
6
́
medicinal plants called “chia”. On the other hand, it is
7
noteworthy that similar to other Salvia species, the metabolic
leaves and flowers of S. amarissima in order to scale-up the
isolation of teotihuacanin (6) led to the isolation of a new
diterpenoid glucoside (1), named amarisolide F, together with
the known diterpenoids amarissinin A (3), acetylamarissinin B
profile of S. amarissima is susceptible to geographic and climate
conditions. The phytochemical profiles of three populations of
this plant species have been determined; from the first two
populations, distributed in the states of Puebla and Oaxaca, the
glucoside diterpenoids amarisolides A−E (11−15) were
5
,8
Special Issue: Special Issue in Honor of Drs. Rachel Mata and
Barbara Timmermann
isolated. From a third population, distributed in the valley
of Teotihuacan, state of Mexico, the diterpenoids amarissinins
7
,9
A−C (3−5) and teotihuacanin (6) were isolated. Further-
Received: July 16, 2018
more, as a continuation of the studies of the phytochemistry of
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00565
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Table 1. H (500 MHz) and 13C (125 MHz) NMR Data of
1
Chart 1
Amarisolide F (1) and Amarissinin E/8-epi-Amarissinin E
(
7/8) in CDCl /DMSO-d , 3:1
3
6
b,c,d
1
7/8
δH mult. (J
in Hz)
position
δ
C
δ
H
mult. (J in Hz)
δ
C
a
a
1
6.01 d
129.4, CH 5.95 ddd (10.3, 4.6, 1.9)/ 130.2/128.7,
5.98 ddd (10.4, 4.8, CH
.8)
124.7, CH 5.80 dt (10.3, 4.6)/5.92 124.2/128.1,
ddd (10.4, 2.4, 1.0) CH
(10.4)
1
2
3
5.70 br d
10.4)
2.42 dd
13.1,
(
a
a
29.2, CH2 2.44 dd (4.6, 1.9) /2.56 30.0/30.4, CH2
ddd (18.8, 4.8, 1.0)
(
a
1
0.1)
2.42 dd
13.1,
a
3b
2.44 dd (4.6, 1.9) /2.37
a
(
1
ddd (18.8, 2.4, 1.8)
a
0.1)
4
5
6
75.4, C
47.7, C
19.1, CH
76.1/77.1, C
48.2/48.1, C
19.9/21.1, CH
a
a
2.01 d
6.8)
2
2
1.99 m /1.93 dd (13.4,
2
(
3.2)
a
a
a
6
7
b
1.92 m
1.99 m /1.72 m
α
1.31 br d
24.4, CH
1.39 ddd (14.9, 4.5, 3.4)/ 25.0/27.6, CH
2
a
a
(10.3)
1.22 m
7
β
1.92 br d
1.73 m/1.56 dt (13.7,
3.3)
a
(9.7)
8
9
1
1
3.42 br s
43.7, CH
43.1, C
2.96 t (3.4)/2.39 t (3.3) 48.4/50.0, CH
43.7/44.4, C
0
76.8, C
75.1/75.9, C
1a
3.74 d
(18.3)
42.3, CH
2
3.51 d (17.6)/3.27 d
(18.0)
45.7/47.6, CH
2
7
,9
(
4), and amarissinin C (5), as well as spathulenol (9) and 5,7-
O-diacetylacacetin (10) (Chart 1). Derivatives 2a and 5a
resulted from the acetylation of amarissinin D (2) and
amarissinin C (5), respectively. Compounds 7/8 were obtained
11b
3.21 d
3.37 d (17.6)/3.20 d
(18.0)
(18.3)
1
1
2
3
194.8, C
128.4, C
196.2/196.5, C
a
128.1 /
128.7,
as methanolysis products by the treatment of 1 with Na CO in
a
2
3
C
MeOH. The structures of these compounds also represent new
diterpenoids, which were named amarissinin E/8-epi-amarissi-
nin E.
1
4
6.77 br s
7.44 d
108.4, CH 6.78 dd (1.6, 1.0)/6.76
dd (1.6, 1.2)
108.7, CH
15
143.6, CH 7.47 t (1.6)/7.49 t (1.6) 144.6/144.9, CH
(5.5)
Amarisolide F (1) was isolated as a beige, amorphous powder.
Its molecular formula of C H O was deduced from its
1
1
1
1
6
8.15 br s
147.2, CH 8.11 t (1.0)/8.08 t (1.2) 147.9/148.7, CH
28
35 14
7
172.0, C
174.9, C
173.3/173.4, C
174.5, 174.3, C
HRESIMS (m/z 595.2046, calcd 595.2021), indicating 12
indices of hydrogen deficiency. The ESIMS/MS analysis showed
8
9α
4.94 d
71.7, a
CH2
4.98 dd (8.4, 2.2)/5.01
dd (8.8, 2.3)
71.2/70.6, CH2
+
potassium and sodium adduct ions at m/z 633 [M + K] and 617
(8.2)
+
[
M + Na] and ions produced by the cleavage of a glycosidic
19β
4.37 d
(8.2)
4.35 d (8.4)/4.26 d (8.8)
fragment containing an acetyl group at m/z 429 [(M − 162
+
(
hexose unit) − 42 (ketene)) + K] and 413 [(M − 162 (hexose
20
1.32 s
21.9, CH
93.7, CH
3
1.19 s/1.13 s
22.7/15.7, CH
3
+
10
1
′
5.46 d
(8.2)
unit) − 42 (acetate)) + Na] . The IR spectrum showed
−
1
absorption bands of hydroxy groups (3408 cm ), a γ-lactone
a
−
1
−1
2′
2.93 dd
71.7, CH
76.4, CH
69.4, CH
74.4, CH
carbonyl (1742 cm ), a ketocarbonyl (1710 cm ), an ester
carbonyl (1672 cm ), and a furan ring (872 cm ). These
assignments were supported by analysis of the H and C NMR
data (Table 1), which indicated the presence of a mono-
substituted furan ring (δ 128.4, C-13; δ 8.15, br s, H-16; 7.44,
(8.7, 8.2)
−1
−1
3
′
3.39 dd
(9.0, 8.7)
1
13
4′
3.16 dd
(9.3, 9.0)
C
H
5
′
3.52 dd
d, J = 5.5 Hz, H-15; 6.77, br s, H-14) and an 18,19-γ-lactone (δC
(9.3, 3.9)
1
8
74.9, C-18, 71.7, C-19; δ 4.94, d, J = 8.2 Hz, H-19α; 4.37, d, J =
.2 Hz, H-19β). Additional signals in the H and C NMR
H
6′a
6′b
4.30 d a
(3.9)
63.3, CH2
1
13
spectra of 1 at δ 6.01 (d, J = 10.4 Hz) and 5.70 (br d, J = 10.4
4.30 d
H
a
(3.9)
Hz) and δ 76.8 (C) and 75.4 (C) were assigned to H-1 (δC
C
1
″
170.8, C
1
29.4), H-2 (δC 124.7), C-10, and C-4, respectively, and
1(2)
2″
2.10 s
20.7, CH3
suggested the presence of a Δ
two tertiary hydroxy groups at C-4 and C-10, as has been
described for amarissinin C (5) and infuscatin. The signals for
double bond, together with
a
b
c
Overlapped signal. Determined in CDCl . OCH signals appeared
at δH 3.61 (s)/3.75 (s), respectively. OCH signals appeared at δ
1.6/51.9.
3
3
d
11
3
C
5
the glycosidic moiety were resolved and assigned by the
identification of the anomeric carbon at δ 93.7 (CH, C-1′), its
C
12
HSQC correlation with the anomeric proton (δ 5.46, d, J = 8.2
spectra of the resulting spin system (Table 1). The coupling
H
Hz, H-1′), and the subsequent analysis of the COSY and HSQC
constants (8.2−9.3 Hz) for this sugar moiety (Table 1) suggest
B
DOI: 10.1021/acs.jnatprod.8b00565
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
trans-diaxial dispositions of all the involved protons in the spin
8
system, as is common for a β-glucopyranoside. In addition, the
NMR data of the sugar moiety and furan ring of 1 were similar to
those of O-acetylglucoside diterpenoids from Salvia chamae-
1
0
dryoides. The C-6 location of the acetoxy group in the sugar
moiety was established by the HMBC correlations between H2-
6
′ (δ 4.30, d, J = 3.9 Hz) and C-1″ (δ 170.8). The glycosidic
H
C
linkage position of the aglycone was established at C-17 (δC
1
1
72.0) based on the HMBC correlations of this carbon with H-
′ and H-8 (δ 3.42, br s). The establishment of the relative
H
configuration of 1 was carried out considering an α-disposition
of H -20, which is common in neo-clerodane diterpenoids,
11
3
followed by the analysis of its NOESY spectrum. NOE
correlations of H -20 with H-8 and H-19β determined their α-
3
dispositions (Figure 1). However, the NOESY spectrum did not
1
Figure 3. H NMR spin analysis of compounds 7/8 (simulated of 7 in
blue, simulation of 8 in red, experimental in black).
with H-8 and H-19β determined their α-dispositions (Figure
4
a) in compound 7. These correlations were the same as those
Figure 1. Key NOESY correlations of compound 1.
Figure 4. Key NOESY correlations of compound 7 (a) and 8 (b).
permit definition of the orientation of the tertiary hydroxy
groups and prompted formation of the aglycone using the
13
observed for compound 1 and indicated that the absolute
configuration of the aglycone in 1 is the same as the one shown
in 7. The NOESY correlations observed for compound 8 of H-
published methodology. Thus, treatment of 1 with Na CO in
2
3
MeOH gave two fractions, a nonpolar fraction containing the
aglycone and a polar fraction containing mainly glucose, which
was directly analyzed by HPLC and coeluted with a reference of
D-glucose. In addition, the specific rotation of the polar fraction
1
1b (δ 3.20, d, J = 18.0 Hz) with H -20 (δ 1.13, s) and H-8
H 3 H
(
δH 2.39, t, J = 3.3 Hz) (Figure 4b) confirmed that H-8 is β-
was closely similar ([α]20 +26) to the reference value. The
5
oriented.
D
Compound 2 was isolated as the O-acetyl derivative (2a) from
nonpolar fraction yielded an epimeric mixture of the
methanolysis products 7/8 in a 55:45 relation. Several attempts
to separate this mixture by chromatography were unsuccessful.
Crystallization from acetone/hexanes gave crystalline 7, which
was used to perform a single-crystal X-ray diffraction study. The
1
a previously analyzed mixture by H NMR (Figure S11,
Supporting Information), which showed the presence of
diagnostic signals (δH 7.71, 7.43, 6.55, 4.86, and 4.36) of a
7
,8
neo-clerodane diterpenoid and was resolved by acetylation
1
4
with Ac O in pyridine at room temperature to give crystalline
2
Flack parameter of 0.03(8) permitted definition of the
configuration as (4R,5S,8S,9R,10S) (Figure 2). Spectroscopic
and spectrometric data of the remaining epimeric mixture of
compound 2a. The structure of this compound was directly
determined by a single-crystal X-ray diffraction analysis (Figure
5).
Compound 1 did not show cytotoxicity (IC50 > 42 μM)
against five human cancer cell lines: HeLa, HCT-15, HCT-116,
1
13
compounds 7/8 were acquired. The correct H and C NMR
assignment (Table 1) for each compound was made by using the
1
Mnova Spin Simulation software. A comparison of the H NMR
experimental spectrum of the mixture 7/8 and the simulated
spectra are shown in Figure 3. The NOE correlations of H -20
3
Figure 2. ORTEP drawing of compound 7.
Figure 5. ORTEP drawing of compound 2a.
C
DOI: 10.1021/acs.jnatprod.8b00565
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
MCF-7, and MDA-MB-231. Therefore, its anti-MDR effect was
tested in two phenotypes of the MCF-7 cell line, which was at
27, hexanes/EtOAc, 60:40). Fraction A2 (0.9 g) showed a complex
15
1
mixture by TLC and H NMR, which was acetylated using Ac O (3
2
4
2.1 μM (RFMCF‑7/Vin− 2.1 and RFMCF‑7/Vin 12.0), 2−3.6-fold
+
mL) in pyridine (1.5 mL) to give 1.1 g of reaction product. A mixture
(
150 mg) containing 9 as the major constituent precipitated, which was
separated by filtering and directly analyzed by GC-MS. The supernatant
0.85 g) was subjected to silica gel CC (2.0 × 12.0 cm, 25 mL) eluted
−
more active than amarissinin A (3, RF
3.1 and
MCF‑7/Vin
+
−
RFMCF‑7/Vin 5.8) and amarissinin B (4, RFMCF‑7/Vin 54.0 and
RFMCF‑7/Vin 2.6) in phenotype MCF-7/Vin+, respectively, but
(
+
with hexanes/EtOAc of increasing polarity. Fractions eluted with
hexanes/EtOAc, 70:30, gave 5.9 mg of 10. Fractions eluted with
hexanes-/EtOAc, 65:35, yielded 0.5 mg of 2a. From fractions eluted
with hexanes/EtOAc, 60:40, was obtained 11.6 mg of 4a. Fraction A3
afforded compound 5 (101.6 mg) after crystallization from acetone/
hexanes. From fraction B a solid was obtained and recrystallized from
acetone/hexanes to give 2.45 g of 5. Mother liquors (11.7 g) were
subjected to silica gel CC (4.0 × 8.0, 100 mL) eluted with mixtures of
hexanes/EtOAc, 65:35 (fr. B1, 0.4 g), hexanes/EtOAc, 60:40 (fr. B1,
4.12 g), and hexanes/EtOAc, 50:50 (fr. B3, 1.44 g). Fraction B1 yielded
350 mg of 5 by crystallization from EtOAc/hexanes. Fraction B2 was
exhibited a weak MDR modulatory effect in comparison to the
most active diterpenoid from the plant (teotihuacanin, 6;
−
+
RFMCF‑7/Vin 8437.5 and RF
10703) and reserpine, used
7,9
+
MCF‑7/Vin
−
as positive control (RFMCF‑7/Vin 5.0 and RFMCF‑7/Vin 29.2).
EXPERIMENTAL SECTION
■
General Experimental Procedures. The (uncorrected) melting
points were measured on a Fisher-Johns apparatus. Optical rotations
were measured on a PerkinElmer 343 polarimeter. The UV spectra
were recorded on an Agilent Cary Series UV−vis−NIR spectropho-
tometer. IR spectra were obtained on a Shimadzu IRTracer-100
spectrometer. NMR experiments were performed on a Bruker Avance
III 400 MHz or on a Varian Unity Plus 500 MHz. Chemical shifts were
relative to tetramethylsilane, and J values are given in Hz. X-ray
crystallographic data were obtained on a Bruker D8 Venture κ-
geometry diffractometer with Cu Kα radiation (λ = 1.541 78 Å).
HRESIMS data were recorded on a MALDI SYNAPT G2-Si mass
spectrometer. Waters HPLC equipment was composed of a 600E
multisolvent delivery system with a refractive index detector (Waters
1
shown by TLC and H NMR as a complex mixture, which was
acetylated with Ac O (12.4 mL) in pyridine (6.2 mL) to give 4.4 g of
2
reaction mixture. It was subjected to silica gel CC (4.0 × 8.0, 50 mL)
developed with hexanes/EtOAc, 60:40, to give 61 mg of 4a and with
hexanes/EtOAc, 50:50, to yield 87 mg of 5a. From fraction B3 43.0 mg
of 3 was isolated by crystallization with acetone/hexanes. From fraction
D (3.9 g), compounds 3 (365 mg) and 6 (214 mg) were obtained by
successive silica gel CC eluted with mixtures of CHCl /MeOH of
3
increasing polarity and finally crystallized from acetone/hexanes.
Fraction E (7.52 g) was submitted to silica gel CC (4.0 × 8.0, 50
mL) eluted with hexanes/EtOAc, 1:1, to obtain 1.15 g of 3. Fraction G
(10.32 g) was subjected to silica gel CC (4.5 × 8.0, 100 mL) eluted with
mixtures of hexanes/EtOAc and EtOAc/MeOH. The fractions
obtained from this column were analyzed by TLC and pooled as
follows: G1 (frs. 1−22, hexanes/EtOAc, 2:3), G2 (frs. 23−32, hexanes/
EtOAc, 3:7), G3 (frs. 33−53, hexanes/EtOAc, 1:4), G4 (frs. 54−67,
EtOAc/MeOH, 99:1), G5 (frs. 68−77, EtOAc/MeOH, 95:5), G6 (frs.
78−83, EtOAc/MeOH, 80:20). Fraction G5 (1.48 g) was subjected to
2410). Control of the equipment, data acquisition, and processing of
the chromatographic information were performed with the Empower 2
software (Waters). GC-MS was performed on an Agilent 7890B
instrument coupled to an Agilent 5977A spectrometer. GC conditions:
DB-5ht (5% phenyl)methylpolysiloxane column (30 m × 0.32 mm, film
thickness 0.10 μm); He, 1.37 mL/min; 100 °C isothermal for 1 min,
linear gradient to 350 °C at 9 °C/min; final temperature hold, 3 min.
MS conditions: ionization energy, 70 eV; ion source temperature, 250
−
1
°
C; interface temperature, 270 °C; scan speed, 2 scans s ; mass range,
silica gel CC (3.0 × 8.0, 50 mL) and eluted with mixtures of CHCl /
3
1
4−600 amu. The Mass Hunter Wiley10 Nist11 G1035 (W10N11)
MeOH of increasing polarity. The fractions eluted with CHCl3/
was used for the identification of volatile compounds. Column
chromatography (CC) was performed on silica gel 230−400 mesh
MeOH, 9:1, yielded 413.4 mg of 1.
Amarisolide F (1): beige, amorphous powder; [α]
MeOH); UV (MeOH) λmax (log ε) 213 (3.85), 250 (3.54); IR (ATR)
νmax 3408, 1742, 1710, 1672, 872 cm ; H and C NMR (CDCl /
2
5
D
−6 (c 0.3,
(
Macherey−Nagel). TLC was carried out on precoated Macherey−
−
1
1
13
Nagel Sil G/UV254 plates of 0.25 mm thickness, and spots were
3
+
visualized by UV light at 254 nm and sprayed with 3% CeSO in 2 N
DMSO-d ) see Table 1; positive ESIMS m/z 633 [M + K] , 617 [M +
4
6
+
+
H SO , followed by heating.
Na] ; HRESIMS m/z 595.2046 [M + H] (calcd for C H O ,
2
4
28 35 14
Chemicals, Cell Lines, and Cell Culture. The resistant MCF-7/
595.2021).
Vin was developed through continuous exposure to vinblastine during
five consecutive years as previously described.
X-ray Single-Crystal Structure Determination of 4-O-
15
acetylamarissinin D (2a). Formula: C22H O , MW = 398.39,
22 7
Plant Material. The leaves and flowers of S. amarissima were
collected in the mountains that surround the Valley of Teotihuacan, in
the state of Mexico, in August 2015. A voucher specimen was deposited
monoclinic, space group P2
, unit cell dimensions a = 18.8711(11) Å, b
1
3
= 9.2292(5) Å, c = 22.5216(13) Å, β = 105.731(2)°, V = 3775.6(4) Å ,
3
Z = 8, D = 1.402 g/cm , F(000) = 1680. A total of 15 144 unique
c
́
reflections were collected, with 14 448 reflections greater than I ≥ 2σ(I)
(
MEXU-1407290) at the National Herbarium, Instituto de Biologia,
Universidad Nacional Autonoma de Mexico.
́
́
(Rint = 0.0401). The structure was solved by direct methods and refined
2
Extraction and Isolation. The dried and powdered leaves and
flowers from S. amarissima (3.5 kg) were extracted by percolation with
acetone (20 L). The extract was concentrated at reduced pressure to
obtain 190 g of gummy residue, which was defatted by partition with
by full-matrix least-squares on F , with anisotropic displacement
parameters for non-hydrogen atoms at final R indices [I > 2σ(I)], R
=
1
0.0319, wR = 0.0814; R indices (all data), R = 0.0339, wR = 0.0833.
2
1
2
Flack parameter = 0.04(3). Crystallographic data reported in this paper
have been deposited in the Cambridge Crystallographic Data Centre
(deposition number: CCDC 1482296). The data can be obtained free
of charge via http://www.ccdc.ac.uk./data_request/cif.
hexanes (1 L) and MeOH/H O, 4:1 (3 × 1 L). The MeOH was
2
evaporated from the aqueous alcohol fraction, water (1 L) was added,
and the resulting mixture was partitioned with EtOAc (3 × 1 L) to give
4
6.3 g of residue. The EtOAc fraction was subjected to silica gel 60 G
4-O-Acetylamarissinin C (5a): colorless crystals, mp 198−201 °C;
1
CC (4.5 × 15.0 cm, 500 mL) eluted with mixtures of hexanes/EtOAc
and EtOAc/acetone as eluents. The fractions obtained from this
column were analyzed by TLC and pooled as follows: fraction A (frs.
H NMR (400 MHz, CDCl ) δ 7.44 (1H, t, J = 2.0 Hz, H-16), 7.37
3 H
(1H, dd, J = 2.0, 1.5 Hz, H-15), 6.40 (1H, t, J = 1.5 Hz, H-14), 6.12 (1H,
ddd, J = 10.4, 2.4, 1.2 Hz, H-1), 5.86 (1H, ddd, J = 10.4, 5.0, 2.1 Hz, H-
2), 5.67 (1H, dt, J = 7.3, 1.3 Hz, H-12), 4.81 (1H, dd, J = 8.6, 1.8 Hz, H-
19β), 4.30 (1H, d, J = 8.6 Hz, H-19α), 3.44 (1H, s, OH-10), 3.12 (1H,
dd, J = 16.0, 8.5 Hz, H-11α), 3.08 (1H, ddd, J = 19.7, 5.0, 1.2 Hz, H-3α),
2.54 (1H, ddd, J = 19.7, 5.4, 2.8 Hz, H-3β), 2.09 (1H, overlapped, H-
1
−11, 3.1 g, hexanes/EtOAc, 4:1), fraction B (frs. 12−18, 14.5 g,
hexanes/EtOAc, 7:3), fraction C (frs. 19−22, 2.6 g hexanes/EtOAc,
:3), fraction D (frs. 23−26, 3.89 g, hexanes/EtOAc, 3:2), fraction E
frs. 27−37, 7.52 g, hexanes/EtOAc, 2:3), fraction F (frs. 38−49, 4.3 g,
7
(
hexanes/EtOAc, 1:4), and fraction G (frs. 50−53, 10.3 g, EtOAc/
acetone, 80:20). Constituents from fraction A were separated by silica
gel CC (4.0 × 8.0 cm, 100 mL) as follows: A1 (frs. 1−12, hexanes/
EtOAc, 70:30), A2 (frs. 13−20, hexanes/EtOAc, 65:35), A3 (frs. 21−
7a), 2.08 (3H, s, H -2′), 1.94 (1H, dd, J = 16.3, 1.2 Hz, H-11β), 1.69
3
(2H, overlapped, H-7b, H-6a), 1.40 (1H, dd, J = 9.8, 2.7 Hz, H-6b),
1
3
1.29 (3H, s, H -20); C NMR (100 MHz, CDCl ) δ 172.2 (C, C-17),
3
3
C
169.3 (C, C-18), 166.9 (C, C-1′), 144.4 (CH, C-16), 138.7 (CH,C-15),
D
DOI: 10.1021/acs.jnatprod.8b00565
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
1
1
4
29.8 (CH, C-1), 127.2 (C, C-13), 126.3 (CH, C-2), 108.7 (CH, C-
AUTHOR INFORMATION
Corresponding Author
Tel: +52 444 834 2000. Fax: +52 444 834 2010. E-mail:
francisco.bautista@ipicyt.edu.mx (E. Bautista).
ORCID
́
Elihu Bautista: 0000-0002-7050-9182
Notes
The authors declare no competing financial interest.
■
*
4), 84.5 (C, C-4), 75.4 (C, C-5), 71.0 (CH, C-12), 70.2 (CH , C-19),
2
9.4 (C, C-5), 42.9 (CH, C-8), 41.5 (C, C-9), 34.0 (CH , C-11), 27.4
2
(
1
CH , C-20), 25.9 (CH , C-3), 24.0 (CH , C-6), 21.6 (CH , C-2′),
3
2
2
3
6.4 (CH , C-7).
2
Amarissinin E/-8-epi-Amarissinin E (7/8). To a solution of 1 (50.4
mg) in MeOH (1.5 mL) was added Na CO (54 mg). The resulting
mixture was stirred at room temperature for 12 h. The supernatant was
filtered and concentrated under reduced pressure to afford a residue,
which was successfully extracted with 10 mL of EtOAc and 10 mL of
2
3
CHCl to give a nonpolar fraction (29.8 mg), which was extracted with
3
ACKNOWLEDGMENTS
We acknowledge the technical assistance of R. Gavin
■
1
0 mL of MeOH to give a polar fraction (22.1 mg). An epimeric mixture
̃
o, H. Rios,
quez, E. Garcia,
of 7/8 was obtained directly from the nonpolar fraction. Crystallization
of this mixture from acetone/hexanes yielded 7 as a pink solid, mp
́
I. Chav
́
ez, E. Huerta, A. Pen
̃
a, B. Quiroz, C. Mar
́
2
5
́
2
(
1
03−205 °C; [α] −2 (c 0.2, Me CO); UV (MeOH) λ (log ε) 204
L. Martinez, and M. P. Orta. IR and UV spectra were acquired at
Laboratorio de Materiales Nanoestructurados y Catalisis
Heterogenea, Instituto Potosino de Investigacion Cientifica y
Tecnologica. HRESIMS spectra were recorded by Dr. Francisco
Perez at Centro de Biociencias, Universidad Autonoma de San
D
2
max
4.02), 234 (3.29), 250 (3.45); IR (KBr) ν 3310, 2921, 1776, 1709,
́
max
−
1
1
13
671, 875 cm ; H and C NMR (CDCl /DMSO-d ) see Table 1;
́
́
́
3
6
+
positive HRESIMS m/z 427.1382 [M + Na] (calcd for C H O Na,
2
1
24
8
́
4
27.1363).
X-ray Single-Crystal Structure Determination of Amarissinin
E (7). Formula: C H O , MW = 404.40, orthorhombic, space group
́
́
́
Luis Potosi. E.B. is grateful to CONACYT for the Research
Fellowship. This research received partial financial support from
CONACYT (CB220535 and FORDECYT-CIIDZA296354)
21
24
8
P2 2 2 , unit cell dimensions a = 9.7873(3) Å, b = 10.4684(3) Å, c =
1
1 1
3
3
1
8.7272(6) Å, V = 1918.74(10) Å , Z = 4, D = 1.400 g/cm , F(000) =
c
and Direccion General de Asuntos del Personal Academico
́ ́
UNAM, IN215016). We also thank BA in TEFL Lucia Aldana
Navarro for the grammar and spelling review.
856. A total of 4082 unique reflections were collected, with 3739
́
(
reflections greater than I ≥ 2σ(I) (R = 0.0675). The structure was
int
2
solved by direct methods and refined by full-matrix least-squares on F ,
with anisotropic displacement parameters for non-hydrogen atoms at
final R indices [I > 2σ(I)], R = 0.0374, wR = 0.0956; R indices (all
DEDICATION
1
2
■
data), R1 = 0.0417, wR2 = 0.0995. Flack parameter = 0.03(8).
Crystallographic data reported in this paper have been deposited in the
Cambridge Crystallographic Data Centre (deposition number: CCDC
Dedicated to Dr. Rachel Mata, National Autonomous University
of Mexico, Mexico City, Mexico, for her pioneering work on
bioactive natural products.
1843755). The data can be obtained free of charge via http://www.
ccdc.ac.uk./data_request/cif.
REFERENCES
■
(
Sugar Analysis. The polar fraction (22.1 mg) obtained by the
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́
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(
́
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́
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(
́
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were seeded into 96-well plates and treated with various concentrations
of vinblastine (0.7 nM−11 μM) in the presence or absence of
compound 1 (42.1 μM) for 72 h. The ability of compound 1 to
potentiate vinblastine cytotoxicity was measured by calculating the
(
̃
a, M.
(
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ASSOCIATED CONTENT
Supporting Information
■
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*
S
(
The Supporting Information is available free of charge on the
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2
(
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NMR spectra for compounds 1 and 7/8 (PDF)
E
DOI: 10.1021/acs.jnatprod.8b00565
J. Nat. Prod. XXXX, XXX, XXX−XXX