Transformations of Some Sesquiterpene Lactones by Filamentous Fungi and Cytotoxic Evaluations

Article abstract of DOI:10.1002/cbdv.201700211

Biotransformation is an economically and ecologically viable technology which has been used to modify the structures of many classes of biologically active products. Some fungi may be useful for the biotransformation of sesquiterpene lactones (SLs), leading to unusual structural changes that modify their biological activities, and other transformations mimic their biosynthetic pathways, generating evidences for the proposed biogenesis. Eight filamentous fungi were screened for their ability to transform different SLs (1?–?9), and microbial reactions yielded compounds 10?–?17, which in turn have been isolated as natural products, thus mimicking their biosynthesis. Their structures were identified based on NMR and MS spectroscopic analyses. The cytotoxicities of SLs 1, 4, 6, 7 and 9, and their biotransformed produts (10, 14, 15 and 17) against human cancer cell lines U251 (glia), PC3 (prostate), K562 (leukemia), HCT-15 (colon), MCF7 (breast), and SKLU-1 (lung), were determined, confirming that the presence of Michael acceptor is an important feature for the bioactivity.

Full text of DOI:10.1002/cbdv.201700211

Article Type: Full Paper
Transformations of Some Sesquiterpene Lactones by
Filamentous Fungi. Cytotoxic Evaluations.
Arturo Cano-Flores,a and Guillermo Delgado*,b
a Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México. Av.
Guelatao No 66 (Eje 7 Oriente). Col Ejército de Oriente, Iztapalapa 09230. Ciudad de México,
México
b Instituto de Química, Universidad Nacional Autónoma de México. Ciudad Universitaria, Circuito
Exterior, Coyoacán 04510. Ciudad de México, México, Email: delgado@unam.mx
Biotransformation is an economically and ecologically viable technology which has been used to
modify the structures of many classes of biologically active products. Some fungi may be useful
for the biotransformation of sesquiterpene lactones (SLs), leading to unusual structural changes
that modify their biological activities, and other transformations mimic their biosynthetic
pathways, generating evidences for the proposed biogenesis. Eight filamentous fungi were
screened for their ability to transform different SLs (1-9), and microbial reactions yielded
compounds 11 -– 18, which in turn have been isolated as natural products, thus mimicking their
biosynthesis. Their structures were identified based on NMR and MS spectroscopic analyses.
The cytotoxicities of SLs 1, 4, 6, 7 and 9, and their biotransformed produts (10, 14, 15 and 17)
against human cancer cell lines U251 (glia), PC3 (prostate), K562 (leukemia), HCT-15 (colon),
MCF7 (breast), and SKLU-1 (lung), were determined, confirming that the presence of Michael
acceptor is an important feature for the bioactivity.
Keywords: biotransformations, sesquiterpene lactones, germacrolide, filamentous fungi,
cytotoxicities
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doi: 10.1002/cbdv.201700211
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Introduction
There is an increasing body of information about the use of biocatalysis for selective
transformations of synthetic and natural products. The principal advantages of the biocatalytic
processes are their mild reaction conditions, environmental safety, and high selectivity [1].
Special attention has been paid to filamentous fungi because they catalyze situ-, regio- and
stereo-selective reactions [2]. Sesquiterpene lactones (SLs) constitute a group of compounds
mainly isolated from species of the family Asteraceae. SLs possess a wide spectrum of
bioactivity [3] such as anti-inflammatory properties [4-7] and cytotoxicity against tumor cell
lines [7-10]. It is widely accepted that thiol groups such as cysteine residues in proteins, as well
as the free intracellular glutathione (GSH), serve as the major targets for SLs [11,12]. The
interaction between SLs and protein thiol groups or GSH leads to reduction of enzymatic
activity, or causes the disruption of GSH metabolism and the intracellular cell redox balance
[13,14]
The main types of reported biotransformations of SLs with various filamentous fungi include
hydrogenations, hydroxylations, acylations, methoxylations, and epoxidations, among others
[15-22]. Particularly, the biotransformations of the antimalarial sesquiterpene lactone
artemisinin or related compounds have been studied [23-27].
Thus, with the aim of exploring the bioreactivity and bioactivity of several SLs, we carried out
the transformations of arglanin (1) [28-30], 11,13- dehydroeriolin (2) [31, 32], budlein B (3)
[33,34], ludovicin A (4) [28], santamarin (5) [28], epoxysantamarin (6) [28], parthenolide (7)
[35], sphaerocephalin (8) [36] and deacetylconfertiflorin (9) [37] by eight filamentous fungi
previously used by our group [38]. The biotransformations afforded the products 10-17, which
were previously characterized as natural products. Therefore, these derivatizations imitate
their natural biosynthesis. The cytotoxicities of all the available compounds against human
cancer cell lines U251 (glia), PC-3 (prostate), K562 (leukemia), HCT-15 (colon), MCF7 (breast),
and SKLU-1 (lung) were also evaluated.
Results and Discussion
The biotransformation of arglanin (1) [28] with Rhizopus nigricans, Beauveria bassiana,
Curvularia lunata, Aspergillus niger, and Cunnighamella blakesleeana afforded vulgarin (10) [39]
as the major product (62, 72, 75, 78, 77% yied, respectively), while with Mucor miehei, Rhizopus
oligosporus and Fusarium moliniforme, the biotransformation proceeded in relative low yield (<
10% by NMR analysis of the mixture).
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1
The H NMR spectrum of vulgarin 10 did not show the two downfield doublets at H 6.15 (d)
and 5.48 (d) derived to the exocyclic vinylic hydrogens in 1. Instead, a new methyl signal at H
1.27 (d, J = 7 Hz) corresponding to CH3-13 (H 1.27) and a signal at H 2.35 (dq, J = 13.0, 7.0 Hz)
for H-11 appeared. The orientation of the methyl group at C-11 was established as α- by the
observed NOESY correlations between H-6 (H 4.17), H3-15β (H 1.57), H3-14β (H 1.22) with
H-11β (H 2.35). The HMBC and HSQC correlations confirmed the structure of vulgarin (10) [39]
for this compound.
The stereoselective hydrogenation of the olefin was the transformation observed for 11,13-
dehydroeriolin (2) with A. niger, C. blakesleeana, B. bassiana and F. moliniforme, affording eriolin
(11) [31, 40]. The configuration at C-11 was deduced from the observed NOESY correlation of
the -oriented H-8 (H 4.17) and H-6 (H 2.16) with H-11 (H 2.57), as well as the correlation
between H-7 (H 1.93) with CH3-13 (H 1.27), establishing the 11R- configuration [40].
Incubations of budlein B (3) [33, 34] with C. lunata and A. niger in medium B (see
Experimental), afforded allo-schkuhriolide (12) [34]. The formation of 13 can be rationalized
via an oxidation of the allylic alcohol of C-14, affording the aldehyde, followed by isomerization
of the C1-C10 double bond.
The biotransformation of ludovicin A (4) [28] with C. lunata in medium B produced tuberiferin
(13, 31%) [41] and ludovicin B (14, 52%) [30]. Compound 13 had the molecular formula
C15H18O3 (M+, m/z 248), as determined by EIMS. Its IR spectrum showed bands for a -lactone
(1768 cm-1) and for an ,-unsaturated ketone (1675 cm-1). The 1H NMR spectrum revealed the
presence of hydrogens representative of an exocyclic methylene conjugated with the -lactone
at H 6.12 and 5.43 (d, J = 3 Hz for H-13a y H-13b), and the vinylic hydrogens of an ,-
unsaturated ketone at H 6.71 and H 5.91 (d, J = 9 Hz each) corresponding to H-1 and H-2,
respectively. The signal at H 2.62 (dq, J = 7 Hz) was assigned to H-4, based on the COSY
correlation of H-4 with H-5 (H 2.1, dd, J = 7, 10 Hz) and H-15 (H 1.4, d, J= 7 Hz, 3H). The -
orientation of H-4 was determined by its NOESY correlation with H-6 and H3-14. The 1H and 13
NMR data and physical properties were in agreement for those of tuberiferin (13), previously
isolated from Sonchus tuberifer 41]. Compound 14 was previously characterized from A.
ludoviciana ssp mexicana [30]. The formation of 13 and 14 from 4 could be explained by the
regioselective opening of the oxirane, followed by removal of a proton to generate the allylic
C
alcohol 14; semipinacolic-type rearrangement (or a 1,3-isomerization of the allylic proton from
C-3 to C-15, followed by keto-enol tautomerization) would produce the ketone, and a
dehydration reaction would afford the ,-unsaturated ketone 13.
Santamarin (6) [28] was transformed by the different fungi tested to 3α-hydroxyreynosin (15)
in relatively low yield, but epoxysantamarin (6) was relatively efficiently transformed by C.
lunata and A. niger (see Experimental).
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Screening-scale experiments showed that eight microorganisms used in this study were capable
of converting parthenolide (7) into compound 16 [35]. However, with B. bassiana (medium A)
and C. lunata (medium B), 1,10-epoxy-11H,13-dihydroparthenolide (17) [42] was obtained.
The HRFABMS established a molecular formula of C15H23O4 [M+H]+, 267.1595, calcd. 267.1597,
and the 1H NMR spectrum revealed a signal at H 1.28 (d, J = 7.0 Hz, 3H), which corresponded to
CH3-13, indicative of the hydrogenation of the exocyclic olefin. The presence of an epoxide at C-1
and C-10 in 17 was confirmed on the basis of 13C NMR, HMQC and HMBC experiments.
Particularly, H-5 (H 2.82, d, J = 10 Hz) and H-1 (H 2.82, dd; J = 10, 2 Hz) appeared at the same
chemical shift, and the corresponding carbons appeared at C 84.3 and C 63.8, respectively.
Furthermore, the HMBC correlation of C-5 with H-3 (H 2.16), H-7 (H 2.28) and H3-15 (H
1.47), C-1 (C 63.8) with H-9 (H 2.25) H-2, H-3 and H3-14, confirmed structure 17.
Sphaerocephalin (8) [36] and deacetylconfertiflorin (9) [37] were not biotransformed by any of
the eight fungi in the tested conditions (using medium A).
The cytotoxic activities of the natural compounds used as starting material (1, 4, 6, 7 and 9),
and the biotransformation products (10, 14, 15, and 17) were evaluated against selected
human cancer cell lines: U251 (glia), PC-3 (prostate), K562 (leukemia), HCT-15 (colon), MCF7
(breast), SKLU-1 (lung), following standard procedures, and the results are summarized in
Table 1 (some bioactivities were not determined due to the paucity of available material). All
the compounds displayed activity (as determined by IC50 values below 100 M), with lactones
10 and 17 being less active. It was confirmed that the cytotoxicities of sesquiterpene lactones
reside chiefly on the presence of Michael acceptor groups, although the presence of additional
polar groups and stereoelectronic considerations are also important. Parthenolide (7) was the
most active compound, but displayed limited selectivity.
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Scheme 1. Biotransformations of sesquiterpene lactones 1 – 6 with filamentous fungi. a: R.
nigricans, b: B. bassiana, c: C. lunata, d: A. niger, e: C. blakesleeana, f: M. miehei, g: R. oligosporus,
h: F. moliniforme.
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Table. Table. Cytotoxicity IC50 values (M) of sesquiterpene lactones against selected cancer cell lines.
Compound
U251
PC-3
K562
HCT-15
(colon)
MCF7
SKLU-1
(lung)
nd
(glía)
(prostate)
10.980.8
39.760.6
32.80.5
6.40.2
(leukemia)
(breast)
1
10.250.15
35.730.35
26.470.5
5.340.12
26.570.28
65.700.52
nd
12.180.64
36.430.4
nd
10.980.54
10.050.37
38.660.83
nd
4
nd
nd
6
nd
nd
7
nd
nd
1.320.45
20.11.9
nd
7.090.9
19.890.9
nd
9
24.780.9
75.640.37
50.090.6
27.981.49
78.141.1
nd
22.270.87
73.290.12
36.580.3
21.280.12
76.973.7
nd
22.140.76
10
84.410.27
14
nd
nd
nd
nd
nd
nd
15
nd
nd
23.120.4
72.770.81
0.320.02
17
nd
nd
Helenalin
0.190.03
0.210.02
The sesquiterpene lactone helenalin was taken as reference [43]. Nd: not determined, due to the paucity of available material.
Conclusions
11(13)
The hydrogenation of the 
double bond was the common process in microbial
transformation of sesquiterpene lactones 1, 2, 4 and 7 by the filamentous fungi used. The
11(13)
reduction of the 
olefin appears to be dependent on the compound involved, the
concentration used, and the fungi assayed. The fungal transformation of the tested lactones are
in accordance to the commonly accepted biogenetic routes, since the obtained products have
been found also as natural products. The eudesmanolides (1, 4, 5, 6) appear to be more reactive
toward biotransformations in comparison with germacrolides (2, 3 and 7). This is the first
report on the biotransformations of compounds 1 – 6, and it is interesting to note that two of
the nine assayed compounds (sphaerocephalin 8 and deacetylconfertiflorin 9) were not
transformed by the tested fungi. The presence of the -methylene--lactone is responsible in
most cases for the cytotoxicities of these substances, and they displayed limited bioselectivities.
Experimental Section
General
Melting points were determined on a Fischer-Jones apparatus. Optical Rotation were measured
on a Perkin-Elmer 341 polarimeter. IR: Nicolet Magna FT-IR 750 spectrometer. 1H and 13C NMR
spectra were taken on a Varian Unity-plus 500 (at 500/125 MHz) instrument. EI-MS: Jeol JMS-
AX505HA mass spectrometer and Jeol JMS-SX 102 A) for HREIMS. TLC spots were revealed by
spraying with ceric ammonium sulfate, followed by heating. Vaccum Column Chromatographies
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(VCC) were done following the reported procedures [44]. Column Chromatography (CC) were
performed using silica gel 70-230 and TLC using silica gel 60F254 (Merck) plates.
Fungi, Media and Culture Conditions
Rhizopus nigricans (ATCC 6227b), Beauveria bassiana (ATCC 13144), Curvularia lunata (ATCC
13432), Aspergillus niger (ATCC 16404), Cunninghamella blakesleeana (ATCC 8688a), Mucor
miehei (ATCC 16457), Rhizopus oligosporus (ATCC 22959), and Fusarium moliniforme (ATCC
102091) were obtained from Instituto de Biotecnología, UNAM, Mexico, maintained on potato
dextrose agar (PDA), and stored at 4 °C. The medium A for R. nigricans, B. bassiana, A. niger, C.
blakesleeana, M. miehei, R. oligosporus and F. moliniforme were prepared by mixing the following
ingredients in distilled H2O (1 L): peptone (1 g), yeast extract (1 g), beef extract (1 g) and
glucose (5 g). The pH was adjusted to pH 7 before autoclaving. The medium B for A. niger, C.
blakesleeana and C. lunata was prepared by mixing glucose (10 g), glycerol (10 g), peptone (5 g),
yeast extract (5 g), KH2PO4 (5 g) and NaCl (5 g). For, the pH was adjusted to 7 before
autoclaving.
Incubation Experiments, Recovery and Purification of Products
Eight fungi, R. nigricans, B. bassiana, C. lunata, A. niger, C. blakesleeana, M. miehei, R. oligosporus,
F. moliniforme, were screened for their capabilities to metabolize compounds 1 – 9 at analytical
scale (10-15 mg), and according with these results, experiments were carried out on a
preparative scale (150-220 mg), following the same experimental processes [38]. Erlenmeyer
cell culture flasks (250 mL) containing 125 mL of medium A or medium B were inoculated with
a dense suspension (2 mL) of the corresponding fungi. Incubation was maintained at 25°C with
gyratory shaking (125 rpm) for 14 days after which the substrates (2-4 mL) in acetone were
added. These biotransformation experiments were monitored by TLC, including two controls,
“culture control” and “substrate control”, for determining that the isolated products are not
secondary metabolites from the fungi, and that the culture media have not done any
transformation on the substrate.
Cultures were filtered and pooled, and cells were washed thoroughly with water, the liquid was
saturated with NaCl, and extracted with CH2Cl2. The organic extracts were dried with Na2SO4
and concentrated under reduced pressure. The obtained residue was chromatographed by VCC
or CC using gradient elution system of n-hexane-EtOAc. Representative experimental
procedures are described below.
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Biotransformation of Arglanin (1) with C. blakesleeana
To 72 h of growth of C. blakesleeana in medium A, arglanine (1) (210.9 mg) was dissolved in
acetone (10 mL) and distributed in 13 Erlenmeyer cell culture flasks. After 14 days of
incubation and monitoring the reaction by TLC, the crude brownish residue (193.4 mg) was
purified by VCC and preparative TLC. This procedure afforded 162.2 mg (77%) of vulgarin (10):
mp. 175-177 ºC. D 25 = +31.58 (c = 9.5 x 10-3, CHCl3). IR max (CHCl3) 3571, 2977, 2937, 1789,
1
1683, cm-1. H NMR (500 MHz, CDCl3): 6.61 (1H, d, J = 11.0 Hz, H-3), 5.90 (1H, d, J = 11.0 Hz, H-
2), 4.17 (1H, dd, J = 11.5, 10.5 Hz, H-6), 2.44 (1H, d, J = 11.5 Hz, H-5), 2.35 (1H, dq, J = 12.0, 7.0
Hz, H-11), 2.05 (1H, ddd, J = 12.0, 3.0, 3.0 Hz, H-9β), 2.01(1H, dddd, J = 13.0, 12.0, 3.0, 3.0 Hz, H-
8), 1.73 (1H, m, H-7), 1.58 (1H, m, H-7), 1.56 (3H, s, CH3-15), 1.51 (1H, m, H-9), 1.27 (3H, d, J =
7 Hz, CH3-13), 1.22 (3H, s, CH3-14). 13C NMR (125 MHz, CDCl3): 205.6 (C-1), 151.7 (C-2), 170.1
(C-3), 70.1 (C-4), 54.6 (C-5), 79.6 (C-6), 52.4 (C-7), 22-7 (C-8), 34.2 (C-9), 46.2 (C-10), 40.5 (C-
11), 179.9 (C-12), 12.5 (C-13), 23.8 (C-14), 19.8 (C-15). EM (IE) m/z: 264 [M+] (15.8), 231 (12.3),
203 (19.2), 132 (36.3), 133 (100), 121 (10.9), 98 (31.5), 93 (28.1), 55 (15.0).
Biotransformation of 11,13-Dehydroeriolin (2) with A. niger
11,13-Dehydroeriolin (2, 200 mg), dissolved in 10 mL de Me2CO, was distributed among 20
flask containing 125 mL of medium A. The crude product obtained in each case was purified by
VCC using n-hexane and increasing quantities of EtOAc (5-100%), this afforded 122.2 mg
(61.12%) of eriolin (11) [30], mp 234-236°C [CH2Cl2/i-Pr2O]. Similar conditions and procedures
were followed for the biotransformation of 2 (200 mg) with F. moliniforme, to obtain 11 (137.2
mg, 68.6%), while with B. bassiana (70%) and C. blakesleeana (64%), 11 was obtained in ca. 5%
yield.
Biotransformation of Budlein B (3) with C. lunata
To 72 h of growth of C. lunata in medium B, budlein B (3, 189.7 mg) was dissolved in acetone
(10 mL) and distributed in 15 Erlenmeyer cell culture flasks. After of 14 days of incubation, the
obtained organic residue (150.4 mg) was purified by VCC and preparative TLC, obtaining 3
(142.3 mg, 75%) and 12 (41.7 mg, 22%). Similar conditions and procedures were followed for
the biotransformation of 3 (150 mg) with A. niger, to obtain 12 (87.2 mg, 58.1%) [34].
Biotransformation of Ludovicin A (4) with C. lunata
Ludovicin A (4, 202.4 mg), dissolved en 10 mL of Me2CO, was distributed among 20 Erlenmeyer
cell culture flasks containing 125 mL of medium B (pH 7). The biotransformation was stopped
after 14 days. The crude residue (188.4 mg) was purified by VCC using n-hexane and increasing
quantities of EtOAc, yielding recovered 4 (24.3 mg, 12%), 13 (62.7 mg, 31%) and 14 (105.3 mg,
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52%) [30]. Tuberiferin (13): white solid (62.7 mg, 31%), mp. 145-147 ºC. IR max (CHCl3): 1768,
1675, 1457 cm-1; 1H NMR (300 MHz, CDCl3),  6.71 (1H, d, J = 9 Hz, H-1), 6.12 (1H, d, J = 3 Hz, H-
13a), 5.91 (1H, d, J = 9 Hz, H-2), 5.43 (1H, d, J = 3 Hz, H-13b), 2.62 (1H, dc, J = 7, 4 Hz, H-4), 2.1
(1H, dd, J = 10, 7 Hz, H-5), 3.98 (1H, t, J = 10 Hz, H-6), 2.58 (1H, m, H-7), 2.13 (1H, m, H-8), 1.79
(1H, ddd, J = 10, 3 Hz, H-8), 1.75 (1H, m, H-9), 1.68 (1H, dd, J = 10, 3 Hz, H-9), 1.40 (3H, d, J =
4 Hz, H-15), 1.17 (3H, s, H-14). 13C NMR (75 MHz, CDCl3 ),  157.8 (C-1), 126.8 (C-2), 200.4 (C-3),
42.1 (C-4), 52.3 (C-5), 82,0 (C-6), 50.2 (C-7), 21.2 (C-8), 37.2 (C-9), 38.5 (C-10), 138.3 (C-11),
170.1 (C-12), 117.5 (C-13), 19.3 8 (C-14), 14.8 (C-15). EM (IE) m/z: 248 M+, 1.2, 232 (2.1), 218
(2.1), 189 (48.0), 148 (32.5), 108 (18.0) 93 (31.0), 76 (76.0), 51 (26.0), 40 (15.0).
Biotransformation of Epoxysantamarin (6) with C. lunata
Compound 6 (100.2 mg) was dissolved in acetone (10 mL) and distributed in 15 Erlenmeyer
cell culture flasks containing 2 mL of A. niger (48 h of growth) in medium A. After 6 days of
incubation, the starting material was consumed and the crude residue was purified by CCV and
preparative TLC, recovering 6 (17.8 mg, 17.8 %) and 15 (53.4 mg, 53.3%) [28].
Biotransformation of parthenolide (7) with B. bassiana
Substrate 7 (200 mg) was dissolved in Me2CO (10 mL) and distributed among 20 Erlenmeyer
cell culture flasks and incubated for 14 days. After this time, the cultures were filtered and
pooled; the cells were washed thoroughly with water and the liquid was saturated with NaCl
and extracted twice with CH2Cl2. Extracts were pooled, dried with anhydrous Na2SO4, the
organic solvent was removed, and the residue was chromatographed on a silica gel column,
affording 7 (24.1 mg, 12.1%), 16 (83.9 mg, 42%) and 17 (19.8, mg, 9.9%). 11H,13-
Dihydroparthenolide (16): mp. 134-36 °C. IR max (CHCl3): 2980, 2932, 1774 cm-1. 1H NMR (300
MHz, CDCl3): 5.15 (1H, dd, J = 12.0, 2.3, H-1), 3.80 (1H, t, J = 10.0, 8.4 Hz, H-6), 2.69 (1H, d, J =
10.0, H-5), 2.37 (1H, dddd, J = 13.0, 13.0, 12.0, 5.0 Hz, H-2), 2.28 (1H, m, H-7), 2.28 (1H, m, H-
8), 2.27 (1H, dq, J = 10.3, 6.8, H-11), 2.25 (1H, m, H-9), 2.11 (1H, dddd, J = 13.0, 13.0, 6.0, 2.3
Hz, H-2), 2.16 (1H, m, H-3), 1.80 (1H, m, H-8), 1.80 (1H, m, H-9), 1.68 (3H, s, CH3-14), 1.27
(3H, s, CH3-15), 1.25 (1H, d, J = 6.8, CH3-13), 1.21 (1H, dt, J = 13.0, 5.6 Hz, H-3). 13C NMR (75
MHz, CDCl3): 125.2 (C-1), 24.7 (C-2), 36.7 (C-3), 61.4 (C-4), 66.4 (C-5), 82.2 (C-6), 51.9 (C-7),
29.8 (C-8), 41.2 (C-9), 134.4 (C-10), 42.5 (C-11), 177.3 (C-12), 13.3 (C-13), 16.9 (C-14), 17.1 (C-
15).
1,10-Epoxy-11H,13-dihydroparthenolide (17): mp. 146-148°C. IR  3686, 2972, 2932, 1780,
1602 cm-1. The HRFABMS established a molecular formula of C15H23O4 [M+H]+, 267.1595, Calcd.
267.1597]. 1H NMR (500 MHz,  CDCl3): 3.80 (1H, t, J = 10.0, 8.4 Hz, H-6), 2.83 (1H, d, J = 10.0, H-
5), 2.37 (1H, dd, J = 11.9 y 2.3, H-1), 2.37 (1H, dddd, J= 13.0, 13.0, 12.0, 5.0 Hz, H-2), 2.28 (1H,
m, H-7), 2.28 (1H, m, H-8), 2.27 (1H, dq, J = 10.3, 6.8, H-11), 2.25 (1H, m, H-9), 2.16 (1H, m,
H-3), 2.11 (1H, dddd, J = 13.0, 13.0, 6.0, 2.3 Hz, H-2), 1.80 (1H, m, H-8), 1.80 (1H, m, H-9),
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1.68 (3H, s, CH3-14), 1.27 (3H, s, CH3-15)1.25 (1H, d, J = 6.8, CH3-13), 1.21 (1H, dt, J= 13.0, 5.6
Hz,H-3). 13C NMR (CDCl3, 125 MHz): 63.8 (C-1), 23.8 (C-2), 35.2 (C-3), 60.5 (C-4), 64.5 (C-5),
81.4 (C-6), 51.8 (C-7), 25.5 (C-8), 40.1 (C-9), 60.4 (C-10), 42.7 (C-11), 176.8 (C-12), 12.9 (C-13),
16.9 (C-14), 17.4 (C-15).
Cytotoxicity bioassays
Human tumor cell lines were supplied by National Cancer Institute (NCI), USA. The cytotoxicity
of the tumors cells with the test compounds was determined using the protein-binding dye
sulforhodamine B (SRB) in microculture assay to measure cell growth. The cell lines were
cultured in RPM-1640 (Sigma Chemical Co Ltd. St. Louis, MO, USA), supplemented with 10%
fetal bovine serum, 2 M L-glutamine, 100 IU/mL penicillin G, 100 g/mL streptomycin sulfate
and 0.25 g/mL amphotericin B (Gibco). They were maintained at 37 °C in a 5% CO2
atmosphere with 95% humidity. For the assay, 5 x 104 cell/mL and 100 L/ well of these cells
suspensions were seeded in 96-well microtiter plates, and incubated to allow the cell
attachment. After 24 h, 100 L the test compounds and positive substances were added to each
well, and after 48 h adherent cell cultures were fixed in situ by adding 50 L of cold 50%
(wt/vol) trichloroacetic acid (TCA) and incubated for 60 min at 4°C. The supernatant was
discarded and the plates were washed three times with water and air-dried. Cultured fixed with
TCA were stained for 30 min with 100 L of 4% SRB solution. Protein bound dye was extracted
with 10 M un buffer tris base and the optical densities were read on an Ultra Microplate
Reader (Elx 808, BIO-TBK Instruments, Inc.) with a test wavelength of 515 nm. Results were
expressed as inhibitory concentration 50 (CI50) values, they were calculated according to the
protocol of Monks [45], where a dose-response curve was plotted for each compound, and the
concentration giving 50% inhibition (CI50) was estimated from linear regression equations.
Helenanin (IC50= 0.32 ± 0.02 M against U251) was used as positive control.
Supplementary Material
Supporting information for this article is available on the www under
http://dx.doi.org/10.1002/MS-number.
Acknowledgements
This work was taken in part from the PhD thesis of Arturo Cano Flores. submitted to Programa
de Maestría y Doctorado en Ciencias Química de la Universidad Nacional Autónoma de México.
The autors thank Rocío Patiño, Ángeles Peña, María Isabel Chávez, Beatriz Quiroz, María Teresa
Ramírez-Apan, Luis Velasco, Javier Pérez, and Antonio Nieto (Instituto de Química, UNAM), for
technical assistance. A. C.-F. thanks Programa de Apoyo para la Superación del Personal
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Académico, Dirección General de Asuntos del Personal Académico, UNAM. Financial support
from, UNAM, DGAPA PAPIIT IN216012, and Carrera de Biología, FES-Zaragoza, UNAM, is
gratefully acknowledged. This paper is dedicated to the memory of Dr. Jesús Romo (1922-1977)
and Dr, Xorge A. Domínguez (1926-1992).
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