Preparation of sesquiterpene lactone derivatives: Cytotoxic activity and selectivity of action

Article abstract of DOI:10.3390/molecules24061113

Cancer is one of the most important causes of death worldwide. Solid tumors represent the great majority of cancers (>90%) and the chemotherapeutic agents used for their treatment are still characterized by variable efficacy and toxicity. Sesquiterpene lactones are a group of naturally occurring compounds that have displayed a diverse range of biological activities including cytotoxic activity. A series of oxygenated and oxy-nitrogenated derivatives (4–15) from the sesquiterpene lactones cumanin (1), helenalin (2), and hymenin (3) were synthesized. The silylated derivatives of helenalin, compounds 13 and 14, were found to be the most active against tumor cell lines, with GI₅₀ values ranging from 0.15 to 0.59 μM. The ditriazolyl cumanin derivative (11) proved to be more active and selective than cumanin in the tested breast, cervix, lung, and colon tumor cell lines. This compound was the least toxic against splenocytes (CC₅₀ = 524.1 μM) and exhibited the greatest selectivity on tumor cell lines. This compound showed a GI₅₀ of 2.3 μM and a SI of 227.9 on WiDr human colon tumor cell lines. Thus, compound 11 can be considered for further studies and is a candidate for the development of new antitumor agents.

Full text of DOI:10.3390/molecules24061113

Article
Preparation of Sesquiterpene Lactone Derivatives:
Cytotoxic Activity and Selectivity of Action
María F. Beer 1,2, Augusto E. Bivona 3,4, Andrés Sánchez Alberti 3,4, Natacha Cerny 5,
Guillermo F. Reta 1, Víctor S. Martín 6, José M. Padrón 6, Emilio L. Malchiodi 3,4
Valeria P. Sülsen 2,7,*, † and Osvaldo J. Donadel 1,*,†
,
1
INTEQUI-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis,
Almirante Brown 1445, CP D5700HGC, San Luis, Argentina; mflorenciabeer@gmail.com (M.F.B.);
gfreta@gmail.com (G.F.R.)
2
3
CONICET-Universidad de Buenos Aires. Instituto de Química y Metabolismo del Fármaco (IQUIMEFA),
Junín 956 2°P (1113), Buenos Aires, Argentina
Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires,
Junín 956 2°P (1113), Buenos Aires, Argentina; Instituto de Estudios de la Inmunidad Humoral (IDEHU),
UBA-CONICET. Junín 956 4°P (1113), Buenos Aires, Argentina; augustobivona@gmail.com (A.E.B.);
andres.sanchez.alberti@gmail.com (A.S.A.); emalchio@ffyb.uba.ar (E.L.M.)
4
5
6
7
CONICET-Universidad de Buenos Aires. Instituto de Microbiología y Parasitología Médica (IMPaM),
Facultad de Medicina. Paraguay 2155. Piso 13, Buenos Aires, Argentina
CONICET-Universidad Nacional de Luján, Instituto de Ecología y Desarrollo Sustentable (INEDES),
Ruta 5 y Avenida Constitución-(6700), Luján, Argentina; natachacerny@gmail.com
Instituto Universitario de Bio-Orgánica Antonio González (IUBO-AG), Universidad de La Laguna, Avda.
Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain; vmartin@ull.es (V.S.M.); jmpadron@ull.es (J.M.P.)
Cátedra de Farmacognosia, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires,
Junín 956 2°P (1113), Buenos Aires, Argentina
* Correspondence: odonadel@gmail.com (O.J.D.); vsulsen@ffyb.uba.ar (V.P.S.);
Tel.: + 54-266-452-0300 (ext. 3151) (O.J.D.); + 54-11-5287-4272 (V.P.S.)
† These authors contributed equally to this work.
Received: 08 February 2019; Accepted: 15 March 2019; Published: 20 March 2019
Abstract: Cancer is one of the most important causes of death worldwide. Solid tumors represent
the great majority of cancers (>90%) and the chemotherapeutic agents used for their treatment are
still characterized by variable efficacy and toxicity. Sesquiterpene lactones are a group of naturally
occurring compounds that have displayed a diverse range of biological activities including
cytotoxic activity. A series of oxygenated and oxy-nitrogenated derivatives (4–15) from the
sesquiterpene lactones cumanin (1), helenalin (2), and hymenin (3) were synthesized. The silylated
derivatives of helenalin, compounds 13 and 14, were found to be the most active against tumor cell
lines, with GI50 values ranging from 0.15 to 0.59 μM. The ditriazolyl cumanin derivative (11) proved
to be more active and selective than cumanin in the tested breast, cervix, lung, and colon tumor cell
lines. This compound was the least toxic against splenocytes (CC50 = 524.1 μM) and exhibited the
greatest selectivity on tumor cell lines. This compound showed a GI50 of 2.3 μM and a SI of 227.9 on
WiDr human colon tumor cell lines. Thus, compound 11 can be considered for further studies and
is a candidate for the development of new antitumor agents.
Keywords: sesquiterpene lactones; antiproliferative activity; Asteraceae; cumanin; helenalin;
hymenin
Molecules 2019, 24, 1113; doi:10.3390/molecules24061113
www.mdpi.com/journal/molecules

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1. Introduction
According to recent studies, 60% of newly identified chemical entities are natural products,
semi-synthetic analogs, or synthetic compounds based on their pharmacophores [1]. This occurs
together with the increased incidence of life-threatening diseases such as AIDS, cancer, hepatitis, etc.
[2]. It is noteworthy that some semi-synthetic compounds derived from a natural product sometimes
show higher bioactivity than the original natural compound. Considering the wide range of
biological activities, such as antiparasitic, antiproliferative, anti-inflammatory, antiviral,
antibacterial, and antifungal activity, sesquiterpene lactones have attracted scientific interest [3–9].
Analyzing the structure of molecules used in cancer therapy, most of them show nitrogenated
functional groups like amide and carbamate moieties in their framework [10]. Recently, it a series of
disubstituted 1,2,3-triazoles has been reported exhibiting potent cytotoxicity in the nanomolar range
and tubulin inhibitory activity in the low micromolar range [11]. This kind of compound is
considered an interesting unit in the design of anticancer drugs. Such heterocycles may act
according to their dipolar character, rigidity, and ability to form hydrogen bridge bonds, or simply
as connectors [12].
Synthetic organic chemists have shown great interest in 1,2,3-triazoles for developing new
biologically active molecules. Triazole moieties do not occur naturally, but 1,2,3-triazole cores may
form the basis of small-molecule pharmaceutical leads. Molecules containing this heterocyclic
nucleus have being reported to have anti-HIV, antimicrobial, anti-allergic, antifungal, and antitumor
activity [13].
In a previous work, we reported the cytotoxicity enhancement of some sesquiterpenes and
iridoids when their lipophilicity was increased by adding alkyl and/or aryl-silyl functionalities on
the natural framework [12].
In consideration of the abovementioned factors, we herein describe our findings aimed at the
synthesis and cytotoxic evaluation of oxygenated and oxy-nitrogenated derivatives from
sesquiterpene lactones (STLs) cumanin (1), helenalin (2) and hymenin (3) (Figure 1). It should be
emphasized that these STLs can be isolated in significant quantities from the natural sources.
Furthermore, these plants are widely distributed in Argentina [14–16].
2. Results and Discussion
2.1. Chemistry
The STLs cumanin (1), helenalin (2), and hymemin (3) were isolated from Ambrosia tenuifolia
Spreng., Gaillardia megapotamica var. megapotamica Spreng., and Parthenium hysterophorus L.,
respectively (Figure 1).
Figure 1. Structures of natural sesquiterpene lactones cumanin (1), helenalin (2), and hymenin (3).
In preliminary bioactivity tests, these STLs showed significant cytotoxic activity, with helenalin
being the most active (2). Therefore, STLs have been used as starting materials for the preparation of
a series of oxygenated and oxo-nitrogenous products; these modifications led to an improved
activity of the obtained derivatives. In this way, derivatives 4 to 15 were prepared (Figure 2).

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Figure 2. Structures of sesquiterpene lactones derivatives obtained from cumanin (1), helenalin (2),
and hymenin (3).
The presence of a hydroxyl group in the structure of the natural compound allowed for the
preparation of acetylated and silylated derivatives from these STLs. In this regard, the diacetylated
derivative 4 was prepared under standard conditions from structure 1, and silylated derivatives 5 to
7 of were obtained under standard conditions and by varying the silylating agent (Scheme 1). The
silylation reaction of cumanine with TBDPSiCl led to the monosilylated derivative at C3. The
introduction of this voluminous group resulted in high steric hindrance. NMR, COSY, and HMBC
analysis allowed for the coupling of H4 (δ = 3.45, dd, J = 8 Hz) with the hydrogen of the hydroxyl
group of the position 3; HSQC analysis confirms the above since the signal corresponding to OH
does not show C‒H coupling. HRMS-ES (m/z) analysis: [M + Na]+: Calcd. for C31H40O4Si2Na:
527.2594; found 527.2586, determines the presence of monosilylated derivative 7.
Acetylated and silylated derivatives (12 to 15) were prepared from STLs 2 and 3 (Scheme 2).

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Scheme 1. Synthesis of compounds 4–7.
Scheme 2. Synthesis of compounds 12–15.

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Synthetic organic chemistry has encouraged great interest in 1,2,3-triazoles in the development
of new biologically active molecules [12]. The triazole moiety does not occur in nature, but
1,2,3-triazole cores may form the basis of small-molecule pharmaceutical leads. Molecules
containing this heterocyclic nucleus have been reported to have anti-HIV, antimicrobial,
anti-allergic, antifungal, and antitumor activities [12]. One of the most popular reactions within the
click chemistry paradigm is the Cu (I)-catalyzed 1,3-dipolar Hüisgen cycloaddition of alkynes and
azides. This reaction proceeds with great efficiency and selectivity in aqueous media and yields
triazole moieties [17]. Our first objective was to obtain alkynes 8 and 9 using STL 1 as the starting
material (see Scheme 3). Compound 1 was treated with propargyl bromide and sodium hydride in
THF to obtain derivatives 8 and 9, and working conditions were optimized in order to increase the
yield of both products (for the characterization of the propargyl group at C3 see the Supplementary
Materials). Subsequently, under Hüisgen conditions, 8 and 9 were converted into the corresponding
mono and di-triazole derivatives 10 and 11, respectively (Scheme 3).
Scheme 3. Synthesis of compounds 8–11.
2.2. Biological Results
2.2.1. Antiproliferative Activity
In vitro antiproliferative activity was evaluated using the protocol of the National Cancer
Institute (NCI) after 48 h of drug exposure using the sulforhodamine B (SRB) assay. Results,
expressed as GI50 values, are shown in Table 1. Data revealed that oxygenated derivatives (5–7,
13–15) are more active than natural products (1–3) with GI50 values ranging from 0.15 to 6.8 μM, in
all cell lines. Helenalin silylated derivatives 13 and 14 were found to be the most active against all
tested cell lines, with GI50 values ranging from 0.15 to 0.59 μM. We cannot discard that increasing
lipophilicity may well result in an increase in cytotoxicity in vitro.
Most of the cumanin derivatives have shown higher cytotoxic activity than the natural parent
compound 1. The presence of two 1,2,3-triazole groups in derivative 11 has increased the activity
values by one order of magnitude compared to cumanin (1). Compounds 4, 8, 9, and 10 displayed
moderate activity and similar results to 1, with the exception of compounds 8 and 9, which were
more active than 1 against WiDr cells.
Hymenin derivative (15) has shown increased activity compared with natural compound 3.

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Table 1. Antiproliferative activity of the natural sesquiterpene lactones and its derivatives against
A549, HBL 100, HeLa, SW1573, T47-D, and WiDr cells. Results are expressed as 50% growth
inhibition (GI50) ± SD.
A549
(µM)
HBL100
(µM)
HeLa
(µM)
SW1573
(µM)
WiDr
(µM)
Compound
T47-D (µM)
1
2
19 (±1.4)
2.3 (±7.8)
9.7 (±0.4)
24 (±5.1) *
21 (±1.1)
2.8 (±1.1)
17 (±0.94)
23 (±5.4)
19 (±0.9)
1.7 (±2)
14 (±3.7)
2.2 (±4.8)
6.4 (±0.11)
11 (±2.1)
1.8 (±0.02)
1.2 (±0.02)
1.5 (±0.2)
28 (±8.6)
20 (±4.2)
32 (±19)
30 (±5.7)
2.9 (±4)
36 (±6.8)
2.7 (±5.1)
18 (±0.28)
27 (±5.7)
3
14 (±0.39)
16 (±2.5)
2.9 (±0.01) *
1.3 (±0.04) **
2.0 (±0.28) *
19 (±13)
20 (±0.58)
26 (±4.6)
4
5
2.4 (±0.08) **** 3.6 (±0.05) ****
1.9 (±0.04) *** 2.2 (±0.37) ****
2.2 (±0.52) *** 2.4 (±0.41) ****
3.4 (±0.15) *** 3.1 (±0.44) ****
6
1.2 (±0.2) ****
1.6 (±0.3) ****
18 (±1.1)
2.3(±0.04) ****
3.7 (±0.28) ****
17 (±1.2)
7
8
24 (±5.8)
24 (±3.7)
7.2 (±3.4) ****
2.1 (±0.2) ****
30 (±14)
9
17 (±1.5)
19 (±0.24)
24 (±0.37)
32 (±11)
10
11
12
13
14
15
18 (±1.5)
29 (±7.2) *
37 (±13)
3.7 (±0.9) ****
n.d.
6.4 (±1.1) ***
3.6 (±0.7)
5.9 (±0.53)
n.d.
4.7 (±0.45)
4.7 (±3.5)
0.28 (±0,02)
0.19 (±0.03)
9.7 (±8) ***
1.6 (±0.7)
0.29 (±0.01)
0.36 (±0.03)
2.3 (±0.58) ****
n.d.
0.59 (±0.06)
0.28 (±0.02)
3.4 (±0.8) ***
0.36 (±0.07) **
0.20 (±0.1) **
4.6 (±1.1) ***
0.19 (±0.04)
0.15 (±0.02)
0.56 (±0.08)
0.26 (±0.2)
6.8 (±1.7) ***
3.3 (±0.1) **** 2.4 (±0.67) *** 4.2 (±1.0) ****
The GI50 mean of each derivative was compared to the activity of its parent compound. For
compounds 1, 2, and their respective derivatives, a one-way Anova + Dunnett’s test was carried out.
T-test was used for compound 3 and its derivative. Asterisks indicate significant differences. * p <
0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
2.2.2. Cytotoxicity on Primary Cell Culture Activity
The cytotoxicity of the sesquiterpene lactones and its derivatives was evaluated using mouse
splenocytes. Table 2 shows the results of the cytotoxicity assay, as CC50 (the concentration of each
compound that causes 50% cell death) and the selectivity indexes. Cumanin (1) was the least toxic
natural compound against splenocytes (CC50 = 29.4 μM) compared with the other sesquiterpene
lactones (2 and 3). Cumanin derivatives (4–11) have shown CC50 values and selectivity indexes
higher than natural compound 1. Compounds 4–9 displayed moderate selectivity, while compound
10 showed low selectivity on the tested cell lines. The incorporation of two triazole groups in
cumanin (1) reduced cytotoxicity on normal cells and improved selectivity against tumor cell lines.
Compound 11 was the least toxic against splenocytes (CC50 = 524.1 μM) and presented the greatest
selectivity on the tested cell lines. This compound showed a GI50 of 2.3 and a SI of 227.9 on human
colon tumor cell line WiDr.
Compounds 2 and 3, as well as their corresponding derivatives, displayed a low selectivity of
action against tumor cell lines. Similar results have been obtained for compound 12 by Lee et al. [18].
Table 2. Cytotoxicity and selectivity indexes of natural sesquiterpene lactones and their derivatives.
Cytotoxicity results in splenocytes are expressed as CC50 (μM) ± SD.
Selectivity Indexes
Splenocytes
(µM)
Compound
A549 HBL100 HeLa SW1573 T47-D WiDr
1
2
3
4
5
6
7
8
9
29.4 (±0.2)
1.5
0.5
1.4
0.4
1.5
0.7
2.1
0.5
1.0
0.4
0.8
0.4
1.2 (±0.3)
4.4 (±0.8)
0.4
0.3
0.3
0.7
0.2
0.2
240.8 (±3.8) ****
66.4 (±0.4) ***
91.8 (±0.5) ****
142.7 (±1.5) ****
180.6 (±5.4) ****
93.5 (±8.9) ****
10.0
27.7
76.5
89.2
10.0
5.5
10.5
18.4
39.9
38.6
10.6
4.9
15.0
22.9
70.6
71.3
9.5
21.9
36.9
76.5
95.1
6.4
9.3
8.9
34.9
41.7
42.0
7.5
30.2
38.2
46.0
25.1
44.5
3.9
4.7
3.9

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10
11
12
13
14
15
113.6 (±12.0) ****
6.3
3.9
81.9
0.1
3.5
88.8
n.d.
5.8
3.5
111.5
0.1
3.1
54.0
0.2
3.8
227.9
n.d.
2.0
524.1 (±4.0) **** 141.6
0.4 (±0.1)
1.1 (±0.3)
1.4 (±0.2)
2.3 (±0.7)
n.d.
1.9
5.0
0.7
3.0
3.9
3.8
7.0
9.3
7.4
3.9
5.4
0.5
0.7
0.9
0.5
0.3
The CC50 mean of each derivative was compared to the activity of its parent compound. For
compounds 1, 2, and their respective derivatives, a one-way Anova + Dunnett’s test was carried out.
T-test was used for compound 3 and its derivative. Asterisks indicate significant differences. * p <
0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
3. Materials and Methods
3.1. General
Unless otherwise stated, all solvents were purified by standard techniques. Reactions requiring
anhydrous conditions were performed under an argon atmosphere. Anhydrous magnesium sulfate
was used for drying solutions. Reactions were monitored by thin-layer chromatography (TLC) on
silica gel plates (60 F254) (Merck KGaA, Darmstadt, IN, USA), and visualized with UV light, 2.5%
phosphomolybdic acid in ethanol, or vanillin with acetic and sulfuric acid in ethanol with heating.
Purification was performed by column chromatography (CC) on silica gel (230–400 mesh) using
n-hexane and ethyl acetate gradient as solvent. 1H NMR spectra were recorded on a Bruker (Bruker
Biospin GmbH, Silberstreifen, Rheinstetten, Germany) 200, 500, or 600 MHz, 13C NMR spectra were
recorded at 50 and 125 M Hz, and chemical shifts are reported relative to internal Me4Si (δ = 0).
Melting points were determined by using an Electrothermal IA9000 melting point apparatus; results
are reported in degrees Celsius and are uncorrected. Optical rotations were recorded on a 343 Perkin
Elmer polarimeter (Waltham, MA, USA). High-resolution ESI mass spectra were obtained from a
Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, a RF-only hexapole ion
guide, and an external electrospray ion source. HRMS spectra were obtained on a Micromass
AutoSpec (Oakville, ON, Canada) mass spectrometer.
3.2. Plant Material
The species used in the present work were Ambrosia tenuifolia Spreng. (Asteraceae), Gaillardia
megapotamica var. megapotamica (Asteraceae), and Parthenium hysterophorus (Asteraceae).
The aerial parts of Ambrosia tenuifolia Spreng. (Asteraceae) were collected in May 2010 in Ibicuy,
Entre Ríos Province, Argentina. The material was identified by Dr. Gustavo Giberti and the
Herbarium specimen is deposited in the Museum of Pharmacobotany at the Faculty of Pharmacy
and Biochemistry, University of Buenos Aires (BAF 717).
Gaillardia megapotamica var. megapotamica Spreng. (Asteraceae) was collected in November 1989
in the city of La Arenilla, San Luis Province, and identified by Prof. Eng. Luis del Vitto. The
specimens are registered as Del Vito & Petenatti under No. 4633, and deposited at the Herbarium of
the National University of San Luis.
Parthenium hysterophorus (Asteraceae) was collected in March 1988 in the town of San Roque,
department of La Capital, Province of San Luis, and identified by Prof. Eng. Luis del Vitto. They are
registered under the numbers 1672-UNSL, and the Herbarium specimens are deposited in the
National University of San Luis.
In all cases the aerial parts were collected, dried at room temperature to a constant weight,
manually fragmented or ground, as appropriate, and placed in plastic bags from which the oxygen
was removed and replaced by argon inert gas for storage until the moment of processing.
3.3. Sesquiterpene Lactone Extraction and Isolation
3.3.1. Isolation of Cumanin from A. tenuifolia

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Extraction conditions: 1.25 kg of aerial parts dried at room temperature to constant weight were
processed. The plant material was extracted with acetone (3 × 48 h) at room temperature and the
organic extracts were concentrated in vacuo. The residue (30 g) was collected and chromatographed
in a 63-cm, 7 cm diameter column, using Sigel 60 G (70–230 mesh) (Merck KGaA, Darmstadt, IN,
USA) and eluting with n-hexane-EtOAc mixtures of increasing polarity [16].
Under these conditions, 6 g of cumanin (1) were obtained in the fractions eluted with hexane:
ethyl acetate 2:8. These values represent a yield of 4.8 g of cumanin per kg of dry plant.
3.3.2. Isolation of Helenalin from G. megapotámica var. megapotámica
The dried to constant weight aerial parts (1.50 kg) of G. megapotamica var megapotamica were
extracted (3 × 48 h) with MeOH at room temperature. The organic extracts were combined and
concentrated in a rotary evaporator. The dry extract was solubilized in 1 L of a mixture of MeOH:
H2O (8:2) and stored in a refrigerator overnight. After this cooling time, the organic phase was
separated and the aqueous phase was washed with MeOH (3 × 500 mL). The organic phases were
combined, and solvent was removed by rotary evaporator [18]. Thirty-five grams of dry extract were
recovered and chromatographed on a 65-cm length and 7 cm in diameter column, using as
stationary phase of Sigel 60 G (70–230 mesh) and eluting with n-hexane:EtOAc mixtures of
increasing polarity. After two separative processes in CC, 10.2 g of helenalin (2) were obtained,
representing a yield of 6.8 g per kg of dry plant.
3.3.3. Extraction and Purification of Hymenin from P. hysterophorus
For preparing the crude extract of the dried to constant weight aerial parts of P. hysterophorus,
1.10 kg of plant material was used and extracted by maceration at room temperature with CHCl3 for
24 h. The maceration was filtered and the extract was taken to dryness in a rotary evaporator.
Subsequently, the dried extract was solubilized in hot EtOH and left overnight at room temperature.
Then, it was filtered and the ethanolic solution was extracted with CHCl3 (4 × 150 mL). The organic
phase was dried over anhydrous Na2SO4 and filtered, and the solvent was removed under reduced
pressure at a temperature lower than 40 °C until 20 g of a gummy-like dry residue was obtained [18].
The fractionation was carried out by preparative CC (65 × 7 cm) using Sigel 60G (70–230 mesh) as
stationary phase and eluting with mixtures of n-hexane and EtOAc of increasing polarities. After the
chromatographic separation, 3.4 g of hymenin (3) were obtained, representing a yield of 3.1 g per kg
of dry plant.
3.4. Chemistry
Preparation of derivative 4: compound 1 (50 mg) (1 eq., 0.188 mmol) of 1 was dissolved in 1 mL
of pyridine (0.2 M) and 1 mL of acetic anhydride (50 eq., 9.4 mmol) was added. The reaction was
monitored by TLC after extraction with EtOAc in acid medium. After 6 h, the reaction was complete
and quenched by pouring in saturated copper sulfate solution and extracting with ethyl acetate (3 ×
50 mL each)., The organic phase was washed with distilled water (3 × 25 mL each), dried with
anhydrous sodium sulfate, and concentrated under reduced pressure. Under these conditions, 49
mg of the derivative 4 was recovered as a white solid, yield 75%.
Preparation of derivative 5: compound 1 (50 mg) (1 eq., 0.187 mmol) was dissolved in 1.5 mL of
Cl2CH2 (0.1 M), 45 mg (3.5 eq., 0.658 mmol) of imidazole, and 69 mg of TMSiCl (0.08 mL, 0.657 mmol)
were added; reaction was carried out at room temperature and confirmed by TLC to be complete
after 3 h. The reaction was quenched with a concentrated solution of (NH4)2SO4 (40 mL) and
extracted with Et2O (3 × 20 mL). The organic phase was washed with water (30 mL) and dried with
Na2SO4, filtered, concentrated, and purified by isocratic CC (30 × 0.5 cm) using Sigel 60 G (70–230
mesh), as stationary phase, and a mixture of n-hexane-EtOAc (60:40) as a mobile phase to give 48 mg
of compound 5 as a yellow solid, yield 63%.
Preparation of derivative 6: compound 1 (50 mg) (1 eq., 0.187 mmol) was dissolved in 1.5 mL of
Cl2CH2 (0.1 M), 45 mg (3.5 eq., 0.657 mmol) of imidazole and 87 mg of DMISOPSiCl (0.1 mL, 0.657

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mmol) were added; the reaction was carried out at room temperature and confirmed by TLC to be
complete after 1 h. The reaction was quenched with a concentrated solution of (NH4)2SO4 (40 mL)
and extracted with Et2O (3 × 20 mL). The organic phase was washed with water (30 mL) and dried
with Na2SO4, filtered, concentrated, and purified by isocratic CC (30 × 0.5 cm) using Sigel 60 G
(70–230 mesh), as stationary phase, and a mixture of n-hexane:EtOAc (50:50) as mobile phase to give
51 mg of compound 6 as a yellow solid, yield 58%.
Preparation of derivative 7: compound 1 (50 mg) (1 eq., 0.187 mmol) was dissolved in 1.5 mL of
Cl2CH2 (0.1 M), 45 mg (3.5 eq., 0.657 mmol) of imidazole and 211 mg TBDPSiCl (0.2 mL, 0.657 mmol)
were added; reaction was carried out at room temperature. The reaction was confirmed to be
complete by TLC after 4 h. The reaction was quenched with a concentrated solution of (NH4)2SO4 (40
mL) and extracted with Et2O (3 × 20 mL). The organic phase was washed with water (30 mL) and
dried with Na2SO4, filtered, concentrated, and purified by CC (30 × 0.5 cm) using Sigel 60 G (70–230
mesh), as stationary phase and 160 mL of a mixture of n-hexane:EtOAc (80:20) and 320 mL of a
mixture of n-hexane:EtOAc (70:30) as mobile phase to give 43 mg of compound 7 as a yellow solid,
yield 45%.
Preparation of derivative 8: compound 1 (100 mg) (1 eq., 0.188 mmol) was dissolved in 5 mL of
dry THF (0.04 M), and 144 mg (10 eq., 3,82 mmol) of sodium hydride (60% in mineral oil) was added.
The mixture was stirred for 60 min in Ar atmosphere. Subsequently, 277.6 mg (1 eq., 0.752 mmol) of
TBAI and 134 mg of propargyl bromide (3 eq., 0.10 mL; 1.128 mmol) were added. The reaction was
confirmed to be complete after 4 h, and was quenched with water and extracted with ethyl acetate (2
× 50 mL). The organic phases were combined, dried with Na2SO4, filtered, concentrated, and purified
by CC (30 × 0.5 cm) using Sigel 60G (70–230 mesh) as stationary phase and 120 mL of CH2Cl2 and 280
mL of CH2Cl2:EtOAc (95:5) as mobile phase to give 40 mg of 8 as a white solid, yield 35%.
Preparation of derivative 9: compound 1 (100 mg) (1 eq., 0.382 mmol) of 1 was dissolved in 5 mL
of dry THF (0.04 M), 144 mg (10 eq., 3.82 mmol) of sodium hydride (60% in mineral oil) was added.
The mixture was stirred for 60 min in Ar atmosphere. Subsequently, 277.6 mg (1 eq., 0.752 mmol) of
TBAI and 186 mg of propargyl bromide (6 eq., 0.20 mL, 2.292 mmol) were added. The reaction was
confirmed by TLC to be complete after 4 h. The reaction was quenched with water and extracted
with ethyl acetate (2 × 50 mL). The organic phases were combined, dried with Na2SO4, filtered,
concentrated and purified by CC (30 × 0.5cm) using Sigel 60G (70–230 mesh) as stationary phase and
120 mL of CH2Cl2 and 280 mL of CH2Cl2:EtOAc (95:5) as mobile phase to give 28 mg of 9 as a white
solid, yield 21%.
Preparation of derivative 10: compound 8 (31.7 mg) (1 eq., 0.104 mmol) of 8 was dissolved in 3
mL (0.2 M) of a 1:1 ethanol/water mixture at room temperature, 18.84 mg of benzyl azide (1 eq., 0.104
mmol),1.67 mg of CuSO4.5H2O (0.1 eq., 0.0104 mmol), and 6.18 mg sodium ascorbate (0.3 eq., 0.0312
mmol) were added. The reaction was confirmed by TLC to be complete after 96 h. The reaction was
quenched with 20 mL of water and extracted with ethyl ether (3 × 30 mL). The organic phase was
dried with Na2SO4, filtered, concentrated under reduced pressure, and purified by CC using Sigel
60G (70–230 mesh) as stationary phase and a mixture of n-hexane:EtOAc (50:50) as mobile phase to
give 21.6 mg of 10 as a white amorphous solid. Yield 47%.
Preparation of derivative 11: To obtain dithriazole 11, compound 9 (20 mg) (1eq., 0.03 mmol)
was dissolved in 1 mL (0.1 M) of a 1:1 ethanol/water mixture at room temperature, and 15 mg of
benzyl azide was added (1 eq., 0.03 mmol), then 1 mg of CuSO4.5H2O (0.1 eq., 0.003 mmol) and 3.29
mg of sodium ascorbate (0.3 eq., 0.009 mmol) were also added. The reaction was confirmed by TLC
to be complete after 96 h. The reaction was quenched with 20 mL of water and extracted with ethyl
ether (3 × 30 mL). The ether phase was dried with Na2SO4, filtered, concentrated under reduced
pressure. Then, it was purified by CC of Sigel 60G (70–230 mesh) using a mixture of
n-hexane:EtOAc: (40:60) as mobile phase to give 8.9 mg (yield 21.9%) of compound 11 as a white
amorphous solid.
Preparation of derivative 12: 50 mg (1 eq., 0.190 mmol) of helenalin (2), 1 mL (50 eq., 9.5 mmol)
of acetic anhydride, and 1 mL (0.2M) of pyridine were stirred at room temperature. The reaction was
confirmed to be complete by TLC after 6 h. The reaction was quenched with saturated copper sulfate

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solution and extracted with ethyl acetate (3 × 50 mL). The organic phase was washed with water (3 ×
25 mL), dried with Na2SO4, and concentrated under reduced pressure. Then, it was purified by CC of
Sigel 60G (70–230 mesh) using n-hexane:EtOAc (40:60) as mobile phase. Forty-two milligrams of 12
were obtained (yield 70%) as an amorphous solid.
Preparation of derivative 13: 50 mg (1 eq., 0.190 mmol) of helenalin (2), and 45 mg (3.5 eq., 0.667
mmol) of imidazole, were dissolved in 1.5 mL (0.1.M) of Cl2CH2, at room temperature. After 15 min
of stirring, 0.08 mL (3.5 eq.; 0.667 mmol) of TMSiCl was added. After 6 h, the reaction was complete.
The reaction was quenched with 40 mL of ammonium chloride solution and extracted with ethyl
ether (3 × 20 mL). The organic phase was washed with water (30 mL) and dried with Na2SO4,
filtered, concentrated, and purified in CC (30 × 0.5 cm) of Silicagel 60G (70–230 mesh) using a
mixture of n-hexane:EtOAc (50:50) as eluent. Thirty-five milligrams (yield 56%) of 13 were obtained
as an amorphous solid.
Preparation of derivative 14: 50 mg (1 eq., 0.190 mmol) of 2 and 45 mg (3.5 eq., 0.667 mmol) of
imidazole were dissolved in 1.5 mL (0.1M) of Cl2CH2 at room temperature. After 15 min of stirring,
0.1 mL (3.5 eq.; 0.667mmol) of DMISOPSiCl was added. After 10 h, the reaction was complete. The
reaction was quenched with 40 mL of an oversaturated ammonium chloride solution and
subsequently extracted with ethyl ether (3 × 20 mL). The organic phase was washed with water (30
mL) and dried with Na2SO4, filtered, concentrated, and purified in CC (30 × 0.5 cm) of Silicagel 60G
(70–230 mesh) using a mixture of n-hexane:EtOAc (50:50) as eluent. 34.5 mg (yield 50%) of 14 was
obtained as an amorphous solid.
Preparation of derivative 15: 100 mg of hymenin (3) (1 eq., 0.351 mmol) and 84 mg (3.5 eq., 1.23
mmol) of imidazole in 1.5 mL of dichloromethane were dissolved at room temperature. After 15 min
of stirring, 0.2 mL (3.5 eq., 1.23 mmol) TMSiCl was added. The reaction lasted 72 h. After this time,
the reaction was poured into 40 mL of ammonium chloride solution and extracted with ethyl ether (3
× 20 mL). The organic phase was washed with water (30 mL) and dried with Na2SO4, filtered,
concentrated, and purified by CC (30 × 0.5 cm) of silica gel 60G (70–230 mesh) using n-hexane:EtOAc
(30:70) as eluent; 44.6 mg of 18 (yield 35%) were obtained as an amorphous solid.
3.5. Spectroscopic and Physical Data
Compound 1: White solid, m.p. 122–124 °C. [α]2D0: +144.3 (c 11.73; CHCl3). IR (KBr; υmax: cm−1):
1
3412, 1745, 2908, 1275, 984. H-NMR (δ = ppm, 600M Hz): 1.41 (1H, ddd, J = 13 and 7 Hz, H-1);
1.66–1.73 (1H, td, J = 13 and 5 Hz, H-2a); 2.05–2.11 (1H, m, H-2b); 4.21 (1H, td, J = 8 and 5 Hz, H-3);
3.51 (1H, d, J = 8 Hz, H-4); 1.59 (1H, t, J = 15, H-6a); 1.95 (1H, dd, J = 4 and 15 Hz, H-6b); 3.13 (1H, m,
H-7); 4.68 (1H, dtd, J = 12 and 5 Hz, H-8); 1.77 (1H, c, J = 12 Hz, H-9a); 2.13–2.18 (1H, m,H-9b);
1.97–2.03 (1H, m, H-10); 5.61 (1H, d, J = 3 Hz, H-13); 6.26 (1H, d, J = 3 Hz, H-13′); 1.04 (3H, d, J = 7 Hz,
13
H-14); 0.98 (3H, s, H-15). C-NMR (δ = ppm, 150M Hz): 41.6 (C-1); 35.3 (C-2); 68.4 (C-3); 77.1 (C-4);
44.3 (C-5); 40.7 (C-6); 37.8 (C-7); 80.1 (C-8); 36.7 (C-9); 30.4 (C-10); 139.8 (C-11); 169.9 (C-12); 123.0
(C-13); 16.7 (C-14); 17.8 (C-14). HRMS-ES m/z: [M + Na]+: Calcd. for C15H22O4Na: 289.1416; found:
289.1422.
Compound 2: White solid, m.p. 159–161 °C. [α]2D0: −64.56 (c 12.33; CHCl3). IR (KBr; υmax: cm−1):
3342, 2927, 1699, 1757, 1111, 962. 1H-NMR (δ = ppm, 200M Hz): 7.52 (1H, d, J = 6 Hz, H-2); 6.19 (1H, d,
J = 6 Hz, H-3); 4.85 (1H, d, J = 9 Hz, H-6); 3.25 (1H, m, H-7); 2.00–2.10 (1H, m, H-8); 1.71–1.79 (1H, m,
H-9a); 1.71–1.79 (1H, m, H-9b); 1.71–1.79 (1H, m, H-10); 5.54 (1H, d, J = 3 Hz, H-13); 6.26 (1H, d, J = 4
13
Hz, H-13′); 1.13 (3H, d, J = 6 Hz, H-14); 1.06 (3H, s, H-15). C-NMR (δ = ppm, 50M Hz): 83.2 (C-1);
164.0 (C-2); 130.6 (C-3); 208.3 (C-4); 57.5 (C-5); 79.8 (C-6); 41.8 (C-7); 25.8 (C-8); 31.7 (C-9); 37.8 (C-10);
138.9 (C-11); 170.4 (C-12); 120.8 (C-13); 17.7 (C-14); 14.8 (C-15). HRMS-ES (m/z): [M + Na]+: Calcd. for
C15H18O4Na: 285.1103; found 285.1107.
Compound 3: White solid, m.p. 173–174 °C. [α]2D0: −78.69 (c 11.17; CHCl3). IR (KBr; υmax: cm−1):
3450, 2972, 1741, 1282, 976. 1H-NMR (δ = ppm, 200M Hz): 7.52 (1H, d, J = 6 Hz, H-2); 6.19 (1H, d, J = 6
Hz, H-3); 4.85 (1H, d, J = 9 Hz, H-6); 3.25 (1H, m, H-7); 2.00–2.10 (1H, m, H-8); 1.71–1.79 (1H, m,
H-9a); 1.71–1.79 (1H, m, H-9b); 1.71–1.79 (1H, m, H-10); 5.54 (1H, d, J = 3 Hz, H-13); 6.26 (1H, d, J = 4
13
Hz, H-13′); 1.13 (3H, d, J = 6 Hz, H-14); 1.06 (1H, s, H-15). C-NMR (δ = ppm, 50M Hz): 83.2 (C-1);

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164.0 (C-2); 130.6 (C-3); 208.3 (C-4); 57.5 (C-5); 79.8 (C-6); 41.8 (C-7); 25.8 (C-8); 31.7 (C-9); 37.8 (C-10);
138.9 (C-11); 170.4 (C-12); 120.8 (C-13); 17.7 (C-14); 14.8 (C-15). HRMS-ES (m/z): [M + Na]+: Calcd. for
C15H18O4Na: 285.1103; found 285.1107.
Compound 4: White solid, m.p.: 85–86 °C. [α]2D0: +57.7 (c 4.92; CHCl3). IR (KBr; υmax: cm−1): 2966,
1755, 1743, 1250, 1066, 823, 598. 1H-NMR (δ = ppm, 200M Hz): 1.50–1.59 (1H, m, H-1); 1.71–1.91 (1H,
m, H-2a); 2.10–2.27 (1H, m, H-2b); 5.25 (1H, ddd, J = 8 and 15 Hz, H-3); 4.78 (1H, d, J = 8 Hz, H-4); 1.60
(1H, s, H-6a); 1.67 (1H, m, H-6b); 3.2 (1H, m, H-7); 4.68 (1H, ddd, J = 8 and 16 Hz, H-8); 1.66–1.87 (1H,
m, H-9a); 2.15–2.27 (1H, m, H-9b); 2.01–2.07 (1H, m, H-10); 5.56 (1H, d, J = 2 Hz, H-13); 6.25 (1H, d, J =
2 Hz, H-13′); 1.05 (3H, d, J = 7 Hz, H-14); 1.07(1H, s, H-15); 2.04 (3H, s, H-2′); 2.07 (3H, s, H-2″).
13C-NMR (δ = ppm, 50M Hz): 41.3 (C-1); 32.3 (C-2); 69.2 (C-3); 75.7 (C-4); 43.9 (C-5); 39.9 (C-6); 37.6
(C-7); 79.4 (C-8); 36.2 (C-9); 29.8 (C-10); 139.4 (C-11); 169.5 (C-12); 123.2 (C-13); 16.6 (C-14); 18.5 (C-15);
170.2 (C-1′); 20.8 (C-2′); 170.6 (C-1″); 20.6 (C-2″). HRMS-ES (m/z): [M + Na]+: Calcd. for C19H26O6Na:
373.1627; found 373.1635.
Compound 5: Yellow solid, m.p.: 98–100 °C. [α]2D0: +121.5 (c 9.1; CHCl3). IR (KBr; υmax: cm-1):
2958, 2927, 1747, 1466, 1379, 1250, 1169, 1086. 1H-NMR (δ = ppm, 200M Hz): 1.31–1.41 (1H, m, H-1);
1.52–1.69 (1H, m, H-2a); 2.17 (1H, ddd, J = 4, 8 and 12 Hz, H-2b); 4.07 (1H, ddd, J = 4 and 7 Hz, H-3);
3.42 (1H, d, J = 7 Hz, H-4); 1.52–1.69 (1H, m, H-6a); 1.73–1.88 (1H, m, H-6b); 3.10 (1H, m, H-7); 4.67
(1H, dtd, J = 8 and 11 Hz, H-8); 1.73–1.88 (1H, m, H-9a); 1.90–2.08 (1H, m, H-9b); 1.90–2.08 (1H, m,
H-10); 5.53 (1H, d, J = 2 Hz, H-13); 6.26 (1H, d, J = 2 Hz, H-13′); 1.04 (3H, d, J = 7 Hz, H-14); 0.99 (3H, s,
13
H-15); 0.13* (9H, s, H-1′, H-2′ and H-3′); 0.17*(9H, s, H-1″, H-2″ and H-3″). C-NMR (δ = ppm, 50M
Hz): 41.1 (C-1); 36.8 (C-2); 69.7 (C-3); 78.1 (C-4); 44.6 (C-5); 40.4 (C-6); 37.7 (C-7); 79.9 (C-8); 36.5 (C-9);
30.3 (C-10); 140.2 (C-11); 169.8 (C-12); 122.3 (C-13); 16.6 (C-14); 18.5 (C-15); 0.40* (C-1′, C-2′ and C-3′);
0.50* (C-1″, C-2″ and C-3″). HRMS-ES (m/z): [M + Na]+: Calcd. for C21H38O4Si2Na: 433.2206; found
433.2209.
Compound 6: Yellow solid, m.p.: 50–52 °C. [α]2D0: +104.41 (c 9.97; CHCl3). IR (KBr; υmax: cm−1):
2960, 2899, 1747, 1464, 1385, 1248, 1173, 1088. 1H-NMR (δ = ppm, 200M Hz): 1.31–1.39 (1H, m, H-1);
1.63–1.77 (1H, m, H-2a); 1.88–2.06 (1H, m, H-2b); 4.06 (1H, cd, J = 7 Hz, H-3); 3.4 (1H, d, J = 7 Hz, H-4);
1.52–1.57 (m, H6a); 1.76–1.88 (m, H6b); 3.09 (m, H7); 4.65 (ddd, J = 8 and 11 Hz, H8); 1.76–1.88 (m,
H9a); 2.09–2.23(1H, m, H-9b); 1.88–2.06 (1H, m, H-10); 5.51 (1H, d, J = 3 Hz, H-13); 6.24 (1H, d, J = 3
Hz, H-13′); 1.02 (3H, d, J = 5 Hz, H-14); 0.98 (3H, s, H-15); -0,01(6H, s, H-1′ and H-2′); 0.61* (3H, d, J = 8
Hz, H-4′); 0.63* (3H, d, J = 8 Hz, H-5′); -0,01(6H, s, H-1″ and H-2″); 0.61* (3H, d, J = 8 Hz, H-4″); 0.63*
(3H, d, J = 8 Hz, H-5″). 13C-NMR (δ = ppm, 50M Hz): 41.2 (C-1); 36.8 (C-2); 69.7 (C-3); 78.2 (C-4); 44.8
(C-5); 40.4 (C-6); 37.8 (C-7); 80.0 (C-8); 36.5 (C-9); 30.3 (C-10); 140.3 (C-11); 169.8 (C-12); 122.3 (C-13);
16.5 (C-14); 18.5 (C-15); -0,01 (C-1′, 2′ and 3′); 17.1 (C-4′); 17.0 (C-5′); −0,01 (C-1″, 2″ and 3″); 17.1 (C-4″);
17.0 (C-5″). HRMS-ES (m/z): [M + Na]+: Calcd. for C25H46O4Si2Na: 489.2832; found 489.2825.
Compound 7: Yellow solid, m.p.: 70–72 °C. [α]2D0: +42.28 (c 10.36; CHCl3). IR (KBr; υmax: cm−1):
1
3537, 2962, 1763, 1427, 1267, 1113, 702. H-NMR (δ = ppm, 200M Hz): 1.23–1.29 (1H, m, H-1);
1.50–1.62 (1H, m, H-2a); 1.50–1.62 (1H, m, H-2b); 4.25 (1H, dd, J = 8 Hz, H-3); 3.45 (1H, dd, J = 8 Hz,
H-4); 1.50–1.62 (1H, m, H-6a); 1.93–2.04 (1H, m, H-6b); 3.09 (1H, m, H-7); 4.58 (1H, ddd, J = 8 and 11
Hz, H-8); 1.70–1.79 (1H, m, H-9a); 2.06–2.18 (1H, m, H-9b); 1.80–1.91 (1H, m, H-10); 5.58 (1H, d, J = 2
Hz, H-13); 6.24 (1H, d, J = 2 Hz, H-13′); 0.95 (3H, d, J = 7 Hz, H-14); 1.05 (3H, s, H-15); 1.11 (9H, s, H-2′,
H-3′ and H-4′); 7.63 (8H, m, H-2″ and H-3″); 7.43 (2H, m, H-4″). 13C-NMR (δ = ppm, 50M Hz): 41.1
(C-1); 35.8 (C-2); 70.3 (C-3); 77.0 (C-4); 44.3 (C-5); 40.6 (C-6); 37.6 (C-7); 79.7 (C-8); 36.4 (C-9); 30.0
(C-10); 139.8 (C-11); 169.9 (C-12); 122.8 (C-13); 16.6 (C-14); 17.8 (C-15); 0.2 (C-1′); 27.2 (C-2′); 133.4
(C-1″); 127.8 (C-2″); 135.9 (C-3″); 130.1 (C-4″). HRMS-ES (m/z): [M + Na]+: Calcd. for C31H40O4Si2Na:
527.2594; found 527.2586.
Compound 8: White solid, m.p.: 100–101 °C. [α]2D0 +30.3 (c 4.84; CHCl3). IR (KBr; υmax: cm−1): 3523,
1
3278, 2918, 2114, 1761, 1385, 1269, 1120, 1086, 997. H-NMR (δ = ppm, 600M Hz): 1.45 (1H, m, H-1);
1.75–1.82 (1H, m, H-2a); 2.05–2.12 (1H, m, H-2b); 4.26 (1H, m, H-3); 3.47 (1H, d, J = 7 Hz, H-4); 1.60
(1H, t, J = 1, 4 and 7 Hz, H-6a); 1.95–2.01 (1H, m, H-6b); 3.16 (1H, m, H-7); 4.68 (1H, ddd, J = 8 and 11
Hz, H-8); 1.72–1.81 (1H, m, H-9a); 2.15–2.20 (1H, m, H-9b); 1.95–2.01 (1H, m, H-10); 5.55 (1H, s, H-13);
6.26 (1H, d, J = 2 Hz, H-13′); 1.04 (3H, d, J = 7 Hz, H-14); 1.02 (3H, s, H-15); 4.36 (1H, d, J = 2 Hz, H-1′a);

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4.33 (1H, d, J = 2 Hz, H-1′b); 2.5 (1H, s, H-3′). 13C-NMR (δ = ppm, 150M Hz): 41.6 (C-1); 35.1 (C-2); 66.5
(C-3); 83.9 (C-4); 44.2 (C-5); 40.7 (C-6); 38.0 (C-7); 79.9 (C-8); 36.4 (C-9); 30.2 (C-10); 140.2 (C-11); 169.8
(C-12); 122.7 (C-13); 16.6 (C-14); 18.8 (C-15); 58.5 (C-1′); 75.2 (C-2′); 80.0 (C-3′). HRMS-ES (m/z): [M +
Na]+: Calcd. for C18H24O4Na: 327.1572; found 327.1574.
Compound 9: White solid, m.p.: 125–126 °C. [α]2D0: +59.0 (c 5.73; CHCl3). IR (KBr; υmax: cm−1): 3431,
1
2927, 2355, 2119, 1759, 1387, 1273, 1088, 997. H-NMR (δ = ppm, 600M Hz): 1.48 (1H, m, H-1);
1.74–1.85 (1H, m, H-2a); 2.055–2.12 (1H, m, H-2b); 4.25 (1H, m, H-3); 3.62 (1H, d, J = 7 Hz, H-4); 1.60
(1H, t, J = 15 Hz, H-6a); 1.97–2.04 (1H, m, H-6b); 3.18 (1H, m, H-7); 4.69 (1H, ddd, J = 8 and 11 Hz,
H-8); 1.74–1.85 (1H, m, H-9a); 2.16–2.22 (1H, m, H-9b); 1.97–2.04 (1H, m, H-10); 5.58 (1H, s, H-13);
6.26 (1H, s, H-13′); 1.05 (3H, d, J = 7 Hz, H-14); 1.04 (3H, s, H-15); 4.43 (1H, d, J = 16 Hz, H-1′a); 4.30
(1H, d, J = 1 6 Hz, H-1′b); 2.46 (1H, brs, H-3′); 4.22 (1H, d, J = 16 Hz, H-1″a); 4.16 (1H, d, J = 16 Hz,
H-1″b); 2.42 (1H, brs, H-3″). 13C-NMR (δ = ppm, 150M Hz): 41.4 (C-1); 32.9 (C-2); 72.2 (C-3); 81.7 (C-4);
44.0 (C-5); 40.1 (C-6); 37.8 (C-7); 79.8 (C-8); 36.4 (C-9); 30.2 (C-10); 140.0 (C-11); 169.7 (C-12); 122.6
(C-13); 16.6 (C-14); 18.4 (C-15); (57.0C-1′); 80.2 (C-2′); 74.5 (C-3′); 56.4 (C-1″); 79.7 (C-2″); 74.3 (C-3″)
HRMS-ES (m/z): [M + Na]+: Calcd. for C21H26O4Na: 365.1729; found 365.1729.
Compound 10: White amorphous solid, m.p.: 74–75 °C. [α]2D0: +10.5 (c 3.80; CHCl3). IR (KBr; υmax:
cm−1): 3433, 2924, 2360, 1757, 1637, 1456, 1205, 1076, 725. 1H-NMR (δ = ppm, 600M Hz): 1.41–1.45 (1H,
m, H-1); 1.72–1.79 (1H, m, H-2a); 2.07–2.11 (1H, m, H-2b); 4.15 (1H, m, H-3); 3.52 (1H, d, J = 7 Hz,
H-4); 1.51 (1H, t, J = 15 Hz, H-6a); 1.68 (1H, dd, J = 15 Hz, H-6b); 3.07 (1H, m, H-7); 4.64 (1H, ddd, J = 3
and 8- Hz, H8); 1.72–1.79 (1H, m, H-9a); 2.13–2.19 (1H, m, H-9b); 1.94–1.99 (1H, m, H-10); 5.43 (1H, d,
J = 2 Hz, H-13); 6.2 (1H, d, J = 2 Hz, H-13′); 1.03 (3H, d, J = 7 Hz, H-14); 1.00 (3H, s, H-15); 4.75 (2H, d, J
= 12, H-1′); 7.53 (1H, s, H-3′); 5.56 (2H, d, J = 3, H-1″); 7.40 (2H, m, H-3″ and H-5″); 7.30 (1H, m, H-4″).
13C-NMR (δ = ppm, 150M Hz): 41.3 (C-1); 34.9 (C-2); 66.3 (C-3); 84.4 (C-4); 44.2 (C-5); 40.7 (C-6); 37.8
(C-7); 79.8 (C-8); 36.3 (C-9); 30.0 (C-10); 139.8 (C-11); 169.7 (C-12); 122.7 (C-13); 16.6 (C-14); 18.7 (C-15);
63.8 (C-1′); 145.1 (C-2′); 122.7 (C-3′); 54.4 (C-1″); 134.2 (C-2″); 129.3 (C-3″); 128.2 (C-4″); 129.0 (C-5″).
HRMS-ES (m/z): [M + Na]+: Calcd. for C25H31N3O4Na: 460.2212; found 460.2217.
Compound 11: White amorphous solid, m.p.: 80–81 °C. [α]2D0: +7.3 (c 1.79; CHCl3). IR (KBr; υmax:
cm−1): 3433, 2962, 2924, 1757, 1456, 1261, 1092, 1120, 800. 1H-NMR (δ = ppm, 600M Hz): 1.37–1.42 (1H,
m, H-1); 1.71–1.77 (1H, m, H-2a); 2.01–2.04 (1H, m, H-2b); 3.96–4.00 (1H, m, H-3); 3.45 (1H, d, J = 7 Hz,
H-4); 1.68 (1H, dd, J = 15 Hz, H-6a); 1.71–1.77 (1H, m, H-6b); 3.03 (1H, m, H-7); 4.61–4.65 (1H, m, H-8);
1.71–1.77 (1H, m, H-9a); 2.13–2.18 (1H, m, H-9b); 1.95–1.98 (1H, m, H-10); 5.43 (1H, d, J = 2 Hz, H-13);
6.19 (1H, d, J = 2 Hz, H-13′); 1.01 (3H, d, J = 7 Hz, H-14); 0.99 (3H, s, H-15); 4.64–4.67 (2H, m, H-1′a) /
4.65–4.71 (2H, m, H-1′a); 4.47 (2H, d, J = 12, H-1′b) / 4.54 (2H, d, J = 12, H-1′b); 7.61 (2H, s, H-3′a); 7.58
(1H, s, H-3′b); 5.58 (d, J = 10, H-1″a); 5.54 (d, J = 10, H-1″b); 7.29–7.40 (10H, m, H-3″, H-4″ and H-5″).
13C-NMR (δ = ppm, 150M Hz): 41.0 (C-1); 33.2 (C-2); 73.9 (C-3); 83.6 (C-4); 44.3 (C-5); 40.3 (C-6); 37.8
(C-7); 79.8 (C-8); 36.3 (C-9); 30.1 (C-10); 139.7 (C-11); 169.8 (C-12); 122.9 (C-13); 16.7 (C-14); 18.5 (C-15);
62,3 (C-1′a); 63.2 (C-1′b); 145.2 (C-2′a); 145.3 (C-2′b); 123.1 (C-3′a); 123.4 (C-3′b); 54.5 (C-1″); 134.4
(C-2″); 128.3 (C-3″); 129.2 (C-4″); 129.0 (C-5″). HRMS-ES (m/z): [M + Na]+: Calcd. for C35H40N6O4Na:
631.3009; found 631.3015.
Compound 12: Amorphous solid, m.p.: 164–165 °C. [α]2D0: −76.3 (c 9.3; CHCl3). IR (KBr; υmax:
cm−1): 2927, 2854, 1767, 1734, 1709, 1657, 1466, 1238, 949. 1H-NMR (δ = ppm, 200M Hz): 3.02 (1H, dt, J
= 3 and 2 Hz, H-1); 7.65 (1H, dd, J = 2 and 6 Hz, H-2); 6.07 (1H, dd, J = 3 and 6 Hz, H-3); 5.38 (1H, br s,
H-6); 3.51 (1H, ddd, J = 8 and 12, H-7); 4.88 (1H, ddd, J = 2 and 7 Hz, H-8); 1.75 (1H, cd, J = 19 Hz,
H-9a); 2.43 (1H, m, H-9b); 2.07–2.18 (1H, m, H-10); 6.14 (1H, d, J = 3 Hz, H-13); 6.45 (1H, d, J = 3 Hz,
13
H-13′); 1.27 (3H, d, J = 7 Hz, H-14); 1.01 (3H, s, H-15); 2.00 (3H, brs, H-2′). C-NMR (δ = ppm, 50M
Hz): 53.2 (C-1); 162.1 (C-2); 129.7 (C-3); 208.9 (C-4); 55.4 (C-5); 77.6 (C-6); 47.7 (C-7); 78.1 (C-8); 40.2
(C-9); 26.1 (C-10); 137.4 (C-11); 169.7 (C-12); 125.0 (C-13); 20.0 (C-14); 18.4 (C-15); 169.5 (C-1′); 21.0
(C-2′). HRMS-ES (m/z): [M + Na]+: Calcd. for C17H20O5Na: 327.1208; found 327.1215.
Compound 13: Amorphous solid. m.p.: 82–84 °C. [α]2D0: −41.6 (c 11.2; CHCl3). IR (KBr; υmax: cm−1):
2960, 2929, 1761, 1714, 1464, 1246, 1109, 845. 1H-NMR (δ = ppm, 200M Hz): 3.20 (1H, dt, J = 6 and 12
Hz, H-1); 7.64 (1H, dd, J = 2 and 6 Hz, H-2); 5.99 (1H, dd, J = 3 and 6 Hz, H-3); 4.42 (1H, d, J = 2 Hz,
H-6); 3.30 (1H, m, H-7); 4.95 (1H, td, J = 3 and 8 Hz, H-8); 1.78 (1H, cd, J = 8 Hz, H-9a); 2.30 (1H, m,

Molecules 2019, 24, 1113
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H-9b); 2.22–2.33 (1H, m, H-10); 5.76 (1H, d, J = 3 Hz, H-13); 6.36 (1H, d, J = 3 Hz, H1–3′); 1.24 (3H, d, J
13
= 7 Hz, H-14); 0.9 (3H, s, H-15); 0.11 (9H, s, H-1′, H-2′ and H-3′). C-NMR (δ = ppm, 50M Hz): 51.5
(C-1); 163.4 (C-2); 129.2 (C-3); 210.8 (C-4); 57.6 (C-5); 76.0 (C-6); 51.7 (C-7); 78.7 (C-8); 40.1 (C-9); 26.0
(C-10); 138.1 (C-11); 170.0 (C-12); 122.9 (C-13); 20.1 (C-14); 18.4 (C-15); 0.4 (C-1′, 2′ and 3′). HRMS-ES
(m/z): [M + Na]+: Calcd. for C18H26O4SiNa: 357.1498; found 357.1492.
Compound 14: Amorphous solid, m.p.: 50–52 °C. [α]2D0: −33.9 (c 9.85; CHCl3). IR (KBr; υmax: cm−1):
1
2958, 2922, 2364, 1770, 1718, 1464, 1269, 1090, 858. H-NMR (δ = ppm, 200M Hz): 3.21 (1H, dt, J = 3
and 2 Hz, H-1); 7.64 (1H, dd, J = 2 and 6 Hz, H-2); 5.98 (1H, dd, J = 3 and 6 Hz, H-3); 4.42 (1H, d, J = 2
Hz, H-6); 3.39 (1H, m, H-7); 4.92 (1H, td, J = 3 and 8 Hz, H-8); 1.76 (1H, cd, J = 8 Hz, H-9a); 2.33 (1H,
m, H-9b); 2.1 (1H, m, H-10); 5.74 (1H, d, J = 3 Hz, H-13); 6.36 (1H, d, J = 3 Hz, H-13′); 1.23 (3H, d, J = 7
Hz, H-14); 0.88 (3H, s, H-15); 0.12*(3H, s, H-1′); 0.44*(3H, s, H-2′); 0.94 (1H, s, H-3′); 0.84*(3H, s, H-4′);
0.90*(3H, s, H-5′). 13C-NMR (δ = ppm, 50M Hz): 51.8 (C-1); 163.2 (C-2; 129.2 (C-3); 210.7 (C-4); 57.8
(C-5); 76.1 (C-6); 51.5 (C-7); 78.7 (C-8); 40.3 (C-9); 26.0 (C-10); 138.1 (C-11); 170.0 (C-12); 123.0 (C-13);
20.1 (C-14); 18.5 (C-15); −3.6* (C-1′); -3.8* (C-2′); 29.7 (C-3′); 16.8 (C-4′ and 5′). HRMS-ES (m/z): [M +
Na]+: Calcd. for C20H30O4SiNa: 385.1811; found 385.1819.
Compound 15: Amorphous solid, m.p.: 150–151 °C. [α]2D0: −94.3 (c 5.23; CHCl3). IR (KBr; υmax:
cm−1): 2960, 2927, 1763, 1714, 1468, 1379, 1053, 847. 1H-NMR (δ = ppm, 200M Hz): 7.52 (1H, d, J = 6 Hz,
H-2); 6.19 (1H, d, J = 6 Hz, H-3); 4.83 (1H, d, J = 9 Hz, H-6); 3.23 (1H, m, H-7); 1.92–2.05 (1H, m, H-8);
1.62–1.69 (1H, m, H-9); 1.62–1.69 (1H, m, H-10); 5.50 (1H, d, J = 3 Hz, H-13); 6.26 (1H, d, J = 4 Hz,
H-13′); 1.05 (3H, d, J = 5 Hz, H-14); 1.03 (3H, s, H-15); 0.15 (9H, brs, H-1′, H-2′ and H-3′). 13C- NMR (δ
= ppm, 50M Hz): 86.5 (C-1); 163.0 (C-2); 131.0 (C-3); 207.6 (C-4); 58.2 (C-5); 79.7 (C-6); 41.8 (C-7); 25.6
(C-8); 32.2 (C-9); 38.9 (C-10); 170.0 (C-11); 139.1 (C-12); 120.3 (C-13); 17.7 (C-14); 15.4 (C-15); 2.6 (C-1′,
2′ and 3′). HRMS-ES (m/z): [M + Na]+: Calcd. for C18H26O4SiNa: 357.1498; found 357.1502.
3.6. Cells, Culture, and Plating
The following human solid tumor cell lines were used in this study: HBL-100 (breast), HeLa
(cervix), SW1573 (non-small cell lung), T-47D (breast), A549 (lung), and WiDr (colon). Cells were
maintained in 25 cm2 culture flasks in RPMI 1640 supplemented with 5% heat-inactivated fetal calf
serum and 2 mM L-glutamine in a 37 °C, 5% CO2, 95% humidified air incubator. Exponentially
growing cells were trypsinized and resuspended in antibiotic containing medium (100 units
penicillin G and 0.1 mg of streptomycin per mL). Single-cell suspensions displaying >97% viability
by trypan blue dye exclusion test were subsequently counted. After counting, dilutions were made
to give the appropriate cell densities for inoculation onto 96-well microtiter plates. Cells were
inoculated in a volume of 100 μL per well at densities of 20,000 (WiDr), 15,000 (T-47D and A549), and
10,000 (HeLa, SW1573, and HBL-100) cells per well, based on their doubling times.
3.7. Antiproliferative Tests
Chemosensitivity tests were performed using the SRB assay of the NCI with slight
modifications. Briefly, pure compounds were initially dissolved in DMSO at 400 times the desired
final maximum test concentration. Control cells were exposed to an equivalent concentration of
DMSO (0.25% v/v, negative control). Each agent was tested in triplicate at different dilutions in the
range 1–100 μM. Drug treatment started on day 1 after plating. Drug incubation periods were 48 h,
after which cells were precipitated with 25 μL of ice-cold 50% (w/v) trichloroacetic acid and fixed for
60 min at 4 °C. Then, the SRB assay was performed. Optical density (OD) of each well was measured
at 492 nm, using BioTek’s PowerWave XS Absorbance Microplate Reader (Highland Park, Winooski,
VT, USA). Values were corrected for background OD from wells containing only culture medium.
The percentage growth (PG) was calculated with respect to untreated control cells (C) at each level of
drug concentrations based on the difference in OD at the start time (T0) and at the end of drug
exposure (T), according to NCI formulas. Therefore, if T is greater than or equal to T0, the calculation
is 100 × [(T–T0)/(C–T0)]. If T is lower than T0, denoting cell death, the calculation is 100 ×
[(T–T0)/(T0)]. The effect is defined as the growth percentage, where 50% growth inhibition (GI50)
represents the concentration at which PG is +50. Based on these calculations, a PG value of 0

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corresponds to the number of cells present at the beginning of drug exposure, while negative PG
values denote net cell death.
3.8. Cytotoxicity on Primary Cell Culture
In a 96-well plate, spleen cells from Balb/c mouse (1.5 × 105) were incubated with different drug
dilutions (200, 100, 50, 10, and 5 μg/mL) in RPMI medium containing 10% fetal calf serum. After 48 h
of incubation at 37 °C (5% CO2), cells were harvested, washed once with PBS, and stained with 2.5
μg/mL propidium iodide (PI) for 5 min at room temperature. Subsequently, cell death was assessed
by flow cytometry using a BD FACSaria II cytometer. Cells incubated only with a drug vehicle were
used as a 100% viability control and death percentage was calculated according to the following
formula:
ꢇ
(%ꢅꢆ ꢈꢉꢊꢊꢋ)
ꢇ
ꢌꢍꢎꢏꢇꢐꢍꢑꢒꢐꢑꢌ
( )
ꢀꢁꢂꢃℎ % = ꢄ1 −
ꢝ × 100.
(%ꢅꢆ ꢈꢉꢊꢊꢋ)
ꢓꢔꢔ% ꢕꢖꢒꢗꢖꢘꢖꢐꢙ ꢚꢛꢜꢐꢍꢛꢘ
Then the concentration capable of causing cell death in 50% of splenocytes (CC50) was
determined using a non-linear regression approach.
3.9. Statistical Analysis
Results are presented as means ± SD. GraphPad Prism 5.0 software (GraphPad Software Inc.,
San Diego, CA, USA) was employed to carry out calculations. The results account for three to four
independent experiments.
4. Conclusions
Given these results, using natural compounds can be a viable strategy to prepare new active
molecules. Sesquiterpene lactones were the starting material for their transformation into several
oxygenated and oxo-nitrogenated derivatives by chemical reactions aiming at the hydroxylated
positions. Our strategy was to obtain new derivatives, including functionalities such as acetates, silyl
ethers, and 1,2,3-triazoles. Although sesquiterpene lactones showed interesting antiproliferative
activity values, a significant number of these synthetic derivatives showed greater activity than the
naturally occurring parent product. Many of the synthesized analogs were more selective toward
tumor cell lines than normal cells. Compound 11, the ditriazolyl cumanin derivative, proved to be
more active and selective than cumanin in the tested breast, cervix, lung, and colon tumor cell lines.
Thus, this compound can be considered for further studies and is a possible candidate for
developing new antitumor agents.
Finally, this work aims at illustrating the possibility of obtaining new naturally-occurring
anti-tumor leads from molecular frameworks, some of which exhibit significantly improved
bioactivity just by common chemical transformations.
Supplementary Materials: The following are available online. Figure S1: 1H-NMR of 1; Figure S2: 13C-NMR of 1;
Figure S3: 1H-NMR of 2; Figure S4: 13C-NMR of 2; Figure S5: 1H-NMR of 3; Figure S6: 13C-NMR of 3; Figure S7:
1H-NMR of 4; Figure S8: 13C-NMR of 4; Figure S9: 1H-NMR of 5; Figure S10: 13C-NMR of 5; Figure S11: 1H-NMR
1
of 6; Figure S12: 13C-NMR of 6; Figure S13: H-NMR of 7; Figure S14: 13C-NMR of 7; Figure S15: COSY of 7;
Figure S16: HSQC of 7; Figure S17: HMBC of 7; Figure S18: 1H-NMR of 8; Figure S19: 13C-NMR of 8; Figure S20:
COSY of 8; Figure S21: HSQC of 8; Figure S22: HMBC of 8; Figure S23: 1H-NMR of 9; Figure S24: 13C-NMR of 9;
1
1
Figure S25: H-NMR of 10; Figure S26: 13C-NMR of 10; Figure S27: H-NMR of 11; Figure S28: 13C-NMR of 11;
1
1
Figure S29: H-NMR of 12; Figure S30: 13C-NMR of 12; Figure S31: H-NMR of 13; Figure S32: 13C-NMR of 13;
1
1
Figure S33: H-NMR of 14; Figure S34: 13C-NMR of 14; Figure S35: H-NMR of 15; Figure S36: 13C-NMR of 15;
Figure S37: HRMS-ES of 1; Figure 38: HRMS-ES of 2; Figure S39: HRMS-ES of 3; Figure S40: HRMS-ES of 4;
Figure S41: HRMS-ES of 5; Figure S42: HRMS-ES of 6; Figure S43: HRMS-ES of 7; Figure S44: HRMS-ES of 8;
Figure S45: HRMS-ES of 9; Figure S46: HRMS-ES of 10; Figure S47: HRMS-ES of 11; Figure S48: HRMS-ES of 12;
Figure S49: HRMS-ES of 13; Figure S50: HRMS-ES of 14; Figure S51: HRMS-ES of 15.
Author Contributions: Conceptualization, E.L.M., V.P.S. and O.J.D.; Methodology, M.F.B., A.E.B., A.S.A., N.C.,
G.F.R., E.L.M., V.P.S. and O.J.D.; Investigation, M.F.B., V.P.S. and O.J.D.; Funding acquisition: E.L.M., V.P.S.

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and O.J.D.; Writing—Original Draft, M.F.B., V.P.S. and O.J.D.; Writing—Review, E.L.M., V.P.S. and O.J.D.;
Supervision, V.S.M., J.M.P., E.L.M., V.P.S., and O.J.D.
Funding: This research was supported by CONICET (PIP 090 and PIP 11220150100158CO), UNSL (Project
PROICO 2-2516), Tricontinental Talent Program of the Canary Islands CEI: Tricontinental Atlantic Campus, The
National Agency for Science and Technology Promotion (PICT 2015-3531), and the University of Buenos Aires
(UBACYT 20020130200270). This investigation is part of the activities within the “Research Network Natural
Products against Neglected Diseases” (ResNet NPND): http://www.resnetnpnd.org/.
Acknowledgments: M.F.B. thanks Monica Ferrari and Walter Stege for technical support. O.J.D. thanks the
Tricontinental Talent Program of the Canary Islands CEI: Tricontinental Atlantic Campus. This work is part of
the doctoral thesis of M.F.B.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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