1,2,3-triazole-substituted oleanolic acid derivatives: Synthesis and antiproliferative activity

Article abstract of DOI:10.3390/molecules18077661

Hybrid compounds are relevant products when searching for structure-activity relationships of natural products. Starting from the naturally occurring triterpene oleanolic acid, alkyl esters were prepared and treated with different aromatic azides using click chemistry to produce hybrid compounds. Some 18 new oleanolic acid derivatives were synthesized and the structures were confirmed by spectroscopic and spectrometric means. The antiproliferative activity of the new derivatives was evaluated towards normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), promyelocytic leukemia (HL-60), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells. The alkyne esters 1 and 3 showed activity on all cell lines but without selectivity (19.6-23.1 μM and 14.1-56.2 μM, respectively), their respective methyl esters were inactive. Compounds with a benzene and p-anisole attached to the triazole ring, showed no antiproliferative effect. Introduction of a chlorine atom into the benzene ring (compound 9) elicited a selective effect against AGS cells (IC50 value: 8.9 μM). The activity was lost when the COOH function at C-28 was methylated. Better antiproliferative effect was found for compounds 11 and 15 bearing a p-toluenesulphonyl group, with values in the range of 10.8-47.1 μM and 11.5-22.2 μM, respectively. The effect, however, was not associated with selectivity.

Full text of DOI:10.3390/molecules18077661

Molecules 2013, 18, 7661-7674; doi:10.3390/molecules18077661
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
1,2,3-Triazole-Substituted Oleanolic Acid Derivatives:
Synthesis and Antiproliferative Activity
Mariano Walter Pertino 1,*, Cecilia Lopez 1, Cristina Theoduloz 2 and
Guillermo Schmeda-Hirschmann 1
1
Instituto de Química de Recursos Naturales, Universidad de Talca, Casilla 747, Talca, Chile
2
Facultad de Ciencias de la Salud, Universidad de Talca, Talca, Chile
* Author to whom correspondence should be addressed; E-Mail: mwalter@utalca.cl;
Tel.: +56-71-2418-866; Fax: +56-71-2201-573.
Received: 30 May 2013; in revised form: 26 June 2013 / Accepted: 27 June 2013 /
Published: 1 July 2013
Abstract: Hybrid compounds are relevant products when searching for structure-activity
relationships of natural products. Starting from the naturally occurring triterpene oleanolic
acid, alkyl esters were prepared and treated with different aromatic azides using click
chemistry to produce hybrid compounds. Some 18 new oleanolic acid derivatives were
synthesized and the structures were confirmed by spectroscopic and spectrometric means.
The antiproliferative activity of the new derivatives was evaluated towards normal lung
fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), promyelocytic leukemia
(HL-60), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells. The alkyne esters 1
and 3 showed activity on all cell lines but without selectivity (19.6–23.1 μM and
14.1–56.2 μM, respectively), their respective methyl esters were inactive. Compounds with
a benzene and p-anisole attached to the triazole ring, showed no antiproliferative effect.
Introduction of a chlorine atom into the benzene ring (compound 9) elicited a selective
effect against AGS cells (IC50 value: 8.9 μM). The activity was lost when the COOH
function at C-28 was methylated. Better antiproliferative effect was found for compounds
11 and 15 bearing a p-toluenesulphonyl group, with values in the range of 10.8–47.1 μM
and 11.5–22.2 μM, respectively. The effect, however, was not associated with selectivity.
Keywords: oleanolic acid; click chemistry; antiproliferative activity

Molecules 2013, 18
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1. Introduction
Terpenes are compounds that present several biological activities. In the last decades, some studies
have shown the widespread promise of triterpenes as templates for selected bioactivities. Modifications
of oleanolic acid (OA) as well as some closely-related triterpenes such as betulinic acid and
dihydrobetulinic acid have led to anti-HIV agents [1], anti-human melanoma compounds [2],
anticancer prodrugs [3] and to molecules with antiproliferative and/or cytotoxic effects [4,5]. A review
on pentacyclic triterpenes as tools in cancer therapy that includes OA has recently been published [6].
Most of the work on bioactive OA derivatives has been carried out with naturally occurring
compounds isolated from plants. Esters or amides at C-3 and/or C-28 were prepared to disclose
structure-activity relationships of the products on selected biological targets [4,7–9]. However, little
has been done on the application of click chemistry techniques to obtain structural diversity starting
from OA. Recently, OA derivatives were prepared and assessed for cytotoxic effect using N-aryl-N'-
hydroxyguanidine to prepare C3-esters. Some of the compounds prepared showed strong and selective
cytotoxic activity against SMMC-7721 cells [10]. An additional article reported the synthesis and
cytotoxic activity of twelve OA derivatives on PC3, A549 and MCF-7 cells. Some of the semisynthetic
compounds showed similar or higher cytotoxicity than OA [9].
Click chemistry is a term which describes an efficient 1,3-dipolar cycloaddition reaction between
alkynes and azides to obtain 1,4-disubstituted triazoles. Triazoles have been shown to possess desirable
features in medicinal chemistry. The triazole are stable to acid and basic hydrolysis and reductive and
oxidative conditions, because of their high aromatic stabilization. In addition, this heterocycle has a
high dipole moment and might participate actively in hydrogen bond formation as well as in
dipole–dipole and π stacking interactions [11]. Last, this compound is relatively resistant to metabolic
degradation [12]. For many years, alkylating agents have been studied with regard to cancer
chemotherapy, and this has led to the development of many new and more selective alkylating agents
including molecules that are based on the triazole moiety [13–16]. Recently, 1,2,3-triazole have shown
antiproliferative properties [17,18]. Wang [19] proposed that planar heteroaromatic triazole derived
compounds might lead to a more facile interaction with DNA, proteins, or cells. They assessed abilities
for apoptotic induction, using a BGC target cell line, which is a common model for testing general
antitumor compound activities and for clarifying molecular mechanisms. Besides, this heterocycle also
possesses other activities like cytotoxic [20], anti-HIV [21], antibiotics [22] and bactericidal effects [23].
Click chemistry of natural products has acquired great importance in recent years. Some of the
molecules studied include different alkaloids [24,25], coumarins [26], saponins [27], steroids [28] and
triterpenes such as betulinic acid [29–31]. Derivatives of OA linked to glycosides using click
chemistry and esterification methods have recently been reported in the search for inhibitors of
glycogen phosphorylase [32]. The aim of the present work was to synthesize some new hybrid
compounds between OA alkynyl esters and aromatic azides using click chemistry, developing
methodologies that can be applied to other terpenes and natural products as well. The new compounds
were assessed for antiproliferative effect using several human tumor cell lines.

Molecules 2013, 18
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2. Results and Discussion
Starting from the naturally occurring triterpene OA, eighteen derivatives including 14 new hybrid
compounds were prepared in moderate to good yields (47–93%) using click chemistry. The
compounds were 1,2,3-triazoles linked to the 3-O function of OA, either with a free or methylated
COOH function at C-28 (Scheme 1). Compounds 1–18 are described for the first time. All the products
were characterized by spectroscopic means.
Scheme 1. Preparation of oleanolic acid derivatives 1–18.
O
O
H
H
b
O
n
OR
N
N
O
n
OR
N
R1
O
O
1
2
3
4
n:2
n:2
n:3
n:3
R:H
5
6
7
8
9
n:2
n:2
n:2
n:2
n:2
R:H
R:CH3
R:H
R:CH3
R:H
R:CH3
R:H
R:CH3
R:H
R:CH3
R:H
R:CH3
R:H
R:CH3
R1: Phenyl
R1: Phenyl
R1: p-anisidyl
R1: p-anisidyl
R1: p-Chlorobenzyl
R1: p-Chlorobenzyl
R1: p-toluenesulfonyl
R1: p-toluenesulfonyl
R1: methyl phenyl sulfide
R1: methyl phenyl sulfide
R1: p-toluenesulfonyl
R:CH3
R:H
R:CH3
10 n:2
11 n:2
12 n:2
13 n:2
14 n:2
15 n:3
16 n:3
17 n:3
18 n:3
a
12
18
13
O
28
H
OH
R1: p-toluenesulfonyl
R1: methyl phenyl sulfide
R1: methyl phenyl sulfide
3
5
HO
Reagents and conditions: (a) appropriate alkyne acid, DCC, DMAP, CH2Cl2, 47–51%; for methyl esters
(R:CH3) CH2N2, Et2O, 92–93%; (b) appropriate aromatic azide, CuSO4·5H2O, sodium ascorbate,
CH2Cl2:H2O 1:1, 47–91%.
The new compounds were assessed for antiproliferative activity towards the following human cell
lines: normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), promyelocytic
leukemia (HL-60), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells. IC50 values > 100 µM were
considered inactive (Table 1). The alkyne esters 1 and 3 showed activity on all cell lines (19.6–23.1 μM
and 14.1–56.2 μM, respectively) but without selectivity. Their respective methyl esters 2 and 4 were
inactive. Compounds 5–8 with a benzene and p-anisole attached to the triazole ring, showed no
antiproliferative effects. However, introduction of a chlorine atom into the benzene ring (compound 9)
elicited a selective effect against AGS cells (IC50 value: 8.9 μM). The activity was lost when the
COOH function at C-28 was methylated (compound 10). Better antiproliferative effect was found for
compounds 11 and 15 bearing a p-toluenesulphonyl group, with values in the range of 10.8–47.1 μM
and 11.5–22.2 μM, respectively. The effect, however, was not associated with selectivity. Compounds
13 and 17 showed similar activity on all cell lines except on MRC-5 cells where compound 13 was
inactive and compound 17 showed an IC50 value of 46.7 μM. Almost all the methyl esters were

Molecules 2013, 18
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inactive with IC50 values > 100 µM. The only methyl ester that showed some activity was the
compound 12 on HL-60 cells (IC50 value: 22.4 μM). This fact shows the important role of the free
COOH function at C-28 in OA in the antiproliferative effect.
Table 1. Antiproliferative activity of oleanolic acid derivatives 1–18 against MRC-5
normal fibroblasts and selected tumor cell lines. a
(IC50 ± SD, µM) b
Compound
MRC-5
21.3 ± 0.9
>100
AGS
19.1 ± 1.0
>100
SK-MES-1
22.4 ± 1.9
>100
J82
23.1 ± 1.6
>100
HL-60
19.6 ± 0.1
>100
1
2
3
14.1 ± 1.2
>100
22.1 ± 1.5
>100
23.4 ± 1.8
>100
56.2 ± 4.3
>100
22.4 ± 0.1
>100
4
5
>100
>100
>100
>100
>100
6
>100
>100
>100
>100
>100
7
>100
95.1 ± 5.8
>100
>100
>100
>100
8
>100
>100
>100
>100
9
>100
8.9 ± 0.4
>100
50.4 ± 3.5
>100
35.4 ± 2.8
>100
35.8 ± 4.1
>100
10
>100
11
14.1 ± 0.8
76.0 ± 3.8
>100
10.8± 0.6
63.0 ± 3.8
31.6 ± 1.6
>100
21.4 ± 1.3
76.3 ± 5.1
65.5 ± 4.4
>100
47.1 ± 2.9
>100
15.9 ± 1.5
22.4 ± 1.8
18.1 ± 1.6
>100
12
13
>100
14
>100
>100
15
17.1 ± 1.0
>100
22.2 ± 1.6
>100
11.9 ± 0.6
>100
14.3 ± 0.6
>100
11.5 ± 1.0
84.2 ± 7.5
11.8 ± 0.9
>100
16
17
46.7 ± 2.4
>100
23.7 ± 1.4
>100
61.8 ± 3.1
>100
>100
18
>100
Etoposide c
0.33 ± 0.02
0.58 ± 0.02
1.83 ± 0.09
3.49 ± 0.16
2.23 ± 0.09
a
Cell lines: normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), promyelocytic
b
leukemia (HL-60), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells. Results are expressed as
mean values ± SD. Each concentration was tested in sextuplicate together with the control and repeated two
times in separate experiments. c Reference compound.
3. Experimental
3.1. General Procedures
Melting points were determined on a Koffler hot stage apparatus (Electrothermal 9100, Dubuque,
IA, USA) and were uncorrected. Optical rotations were measured on a Jasco DIP 370 (Jasco Analytical
Instruments, Easton, MD, USA) polarimeter in CHCl3 at 20 °C. IR spectra were recorded on a Nicolet
Nexus 470 FT-IR instrument (Thermo Electron Corporation, Waltham, MA, USA). The NMR spectra
were recorded on a Bruker Avance 400 (Bruker, Rheinstetten, Germany) spectrometer at 400 MHz for
1H and 100 MHz for 13C in CDCl3. Chemical shifts are given in ppm with TMS as the internal
standard. High-resolution mass spectra were measured on a VG Micromass ZAB-2F at 70 eV (Varian
Inc., Palo Alto, CA, USA). Merck silica gel (0.063–0.2) was used for column chromatography, pre-coated
Si gel plates (Merck, Kieselgel 60 F254, 0.25 mm) were used for TLC analysis. TLC spots were

Molecules 2013, 18
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visualized by spraying the chromatograms with p-anisaldehyde-ethanol-acetic acid-H2SO4
(2:170:20:10 v/v) and heating at 110 °C for 3 min. Reagents: N,N-Dicyclohexylcarbodiimide (DCC)
and dimethylaminopyridine (DMAP) were from Merck (Schuchardt, Germany). 4-Pentynoic acid,
5-hexynoic acid and aromatic azides were from Aldrich (Schuchardt, Germany). Copper (II) sulphate
pentahydrate was from Aldrich (St. Louis, MO, USA) and sodium ascorbate was from Sigma
(St. Louis, MO, USA).
3.2. General Procedure for the Synthesis of Compounds 1–18
Oleanolic acid was isolated from the aerial parts of Fabiana imbricata as described previously [2]
and purified by successive silica gel column chromatography. The compounds 1–18 were prepared
treating OA with the appropriate alkyne acid/DCC/DMAP to obtain the esters. Treatment with the
appropriate azide yielded the corresponding triazole.
3.2.1. Preparation of Alkynyl Esters 1, 3
4-Pentynoic acid or 5-hexynoic acid (1 eq) was dissolved in dry CH2Cl2 at room temperature under
constant stirring. Then, DCC (1 eq) was added, followed after 10 minutes by a catalytic amount of
DMAP and OA (1 eq) dissolved in dry CH2Cl2. The reaction was stopped by adding H2O, extracted
with CH2Cl2, dried over Na2SO4, concentrated and purified.
3.2.2. General Procedure for the Synthesis of Triazoles 5, 7, 9, 11, 13, 15 and 17
The alkynyl esters 1 or 3 (1 eq) and the corresponding azide (1 eq) were dissolved in CH2Cl2/H2O
(1:1), followed by the addition of CuSO4.5H2O (2 mol%) and sodium ascorbate (10 mol%). The
mixture was stirred at room temperature for 24 h. The reaction was stopped by adding H2O, extracted
with CH2Cl2, dried over anhydrous Na2SO4, concentrated and purified by column chromatography on
silica gel.
3.2.3. Preparation of Methyl Esters 2, 4, 6, 8, 10, 12, 14, 16 and 18
Methylation was performed using diazomethane in diethyl ether (Et2O). Methylation of 1 and 3
yielded the compounds 2 and 4, respectively. Methylation of the compounds 5, 7, 9, 11, 13, 15 and 17
afforded the corresponding methyl esters 6, 8, 10, 12, 14, 16 and 18.
Compound (1). Oleanolic acid (OA) (170 mg, 0.373 mmol), DCC (77 mg, 0.373 mmol), a catalytic
amount of DMAP and 4-pentynoic acid (88 mg, 0.373 mmol), in dry CH2Cl2 (20 mL), were stirred at
room temperature for 2–4 h. The reaction mixture was worked-up as described in 3.2.1. The residue
was purified by silica gel column chromatography, eluting with hexane/EtOAc (8:2), yielding 1 (94 mg,
47%): white solid; mp 235 °C; [α]2D0 +65 (c 0.058, CHCl3); IR νmax (film) 3309, 2941, 2873, 1731,
1695, 1466, 1270, 760 cm−1; 1H-NMR (CDCl3): δ 5.25 (1H, brs, H-12), 4.52 (1H, t, J = 8.3 Hz, H-3α),
2.80 (1H, dd, J = 13.5; 3.6 Hz, H-18), 2.48–2.56 (4H, m, OCOCH2CH2), 1.96 (1H, brs, H-5'), 1.11
13
(3H, s), 0.92 (3H, s), 0.91 (3H, s), 0.89 (3H, s), 0.86 (3H, s), 0.84 (3H, s), 0.72 (3H, s); C-NMR
(CDCl3): δ 184.39 (C-28), 171.52 (C-1'), 143.62 (C-13), 122.49 (C-12), 82.62 (C-4'), 81.33 (C-3),

Molecules 2013, 18
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69.07 (C-5'), 55.27 (C-5), 47.53, 46.53, 45.82, 41.52, 40.87, 39.26, 38.01, 37.73, 36.97, 33.85, 33.78,
33.07, 32.48, 30.66, 29.70, 28.05, 27.66, 25.93, 23.58, 23.53, 23.38, 22.83, 18.15, 17.19, 16.70, 15.36,
14.56; HREIMS m/z 536.3768 [M]+• (calcd for C35H52O4, 536.3866).
Compound (2). Compound 1 (50 mg, 0.093 mmol), was methylated with a solution of CH2N2 in ethyl
ether, yielding 47 mg (92%) of 2: white solid; mp 173 °C; [α]2D0 +71 (c 0.046, CHCl3); IR νmax (film)
3306, 2945, 2873, 1731, 1461, 1262, 754 cm−1; 1H-NMR (CDCl3): δ 5.25 (1H, brs, H-12), 4.50 (1H, t,
J = 8.1 Hz, H-3α), 3.59 (3H, s, OMe), 2.82 (1H, dd, J = 13.7; 3.8 Hz, H-18), 2.46–2.54 (4H, m,
OCOCH2CH2), 1.94 (1H, brs, H-5'), 1.10 (3H, s), 0.90 (3H, s), 0.89 (3H, s), 0.87 (3H, s), 0.84 (3H, s),
13
0.83 (3H, s), 0.69 (3H, s); C-NMR (CDCl3): δ 178.23 (C-28), 171.43 (C-1'), 143.78 (C-13), 122.24
(C-12), 82.60 (C-4'), 81.30 (C-3), 69.06 (C-5'), 55.28 (C-5), 51.51 (OMe), 47.52, 46.69, 45.83, 41.62,
41.27, 39.26, 38.06, 37.71, 36.90, 33.83 (2C), 33.11, 32.58, 32.36, 30.68, 28.05, 27.68, 25.90, 23.64,
23.53, 23.39, 23.04, 18.20, 16.82, 16.73, 15.34, 14.51; HREIMS m/z 550.4324 [M]+• (calcd for
C36H54O4, 550.4022).
Compound (3). Compound 3 was synthesized as described for compound 1, using OA and 5-hexynoic
acid yielding 105 mg (51%) of 3: white solid; mp 212 °C; [α]2D0 +66 (c 0.023, CHCl3); IR νmax (film)
3308, 2941, 2874, 1729, 1694, 1462, 1276, 757 cm−1; 1H-NMR (CDCl3): δ 5.27 (1H, brs, H-12), 4.50
(1H, t, J = 8.3 Hz, H-3α), 2.80 (1H, dd, J = 13.4; 3.5 Hz, H-18), 2.45 (2H, t, J = 7.5 Hz, H-2'), 2.26
(2H, dt, J = 7.0; 2.5 Hz, H-4'), 1.94 (1H, brs, H-6'), 1.83–1.88 (2H, m, H-3'), 1.12 (3H, s), 0.93 (3H, s),
0.92 (3H, s), 0.90 (3H, s), 0.86 (3H, s), 0.85 (3H, s), 0.74 (3H, s); 13C-NMR (CDCl3): δ 184.01 (C-28),
172.84 (C-1'), 143.59 (C-13), 122.52 (C-12), 83.23 (C-4'), 80.90 (C-3), 69.06 (C-5'), 55.26 (C-5),
47.53, 46.52, 45.81, 41.53, 40.89, 39.25, 38.02, 37.73, 36.97, 33.76, 33.42, 33.05, 32.49, 30.66, 29.69,
28.07, 27.65, 25.91, 23.79, 23.57, 23.38, 22.86, 18.15, 17.91, 17.15, 16.71, 15.36, 14.10; HREIMS m/z
550.4410 [M]+• (calcd for C36H54O4, 550.4022).
Compound (4). Compound 3 (50 mg, 0.089 mmol), was methylated with a solution of CH2N2 in ethyl
ether, yielding 47 mg (93%) of 4: white solid; mp 176 °C; [α]2D0 +60 (c 0.042, CHCl3); IR νmax (film)
3297, 2942, 2868, 1726, 1460, 1226, 754 cm−1; 1H-NMR (CDCl3): δ 5.27 (1H, brs, H-12), 4.50 (1H, t,
J = 8.1 Hz, H-3α), 3.61 (3H, s, OMe), 2.84 (1H, dd, J = 13.8; 3.8 Hz, H-18), 2.44 (2H, t, J = 7.5 Hz,
H-2'), 2.25 (2H, dt, J = 7.0; 2.5 Hz, H-4'), 1.96 (1H, brs, H-6'), 1.81–1.88 (2H, m, H-3'), 1.12 (3H, s),
13
0.92 (6H, s), 0.89 (3H, s), 0.85 (6H, s), 0.71 (3H, s); C-NMR (CDCl3): δ 178.25 (C-28), 172.78
(C-1'), 143.76 (C-13), 122.22 (C-12), 83.28 (C-4'), 80.87 (C-3), 69.02 (C-5'), 55.24 (C-5), 51.48
(OMe), 47.50, 46.67, 45.80, 41.59, 41.24, 39.24, 38.04, 37.69, 36.88, 33.81, 33.38, 33.06, 32.55,
32.33, 30.65, 28.04, 27.64, 25.86, 23.76, 23.60, 23.51, 23.36, 23.02, 18.17, 17.87, 16.79, 16.71, 15.31;
HREIMS m/z 564.4164 [M]+• (calcd for C37H56O4, 564.4179).
Compound (5). Compound 1 (76 mg, 0.142 mmol) and azidobenzene (17 mg, 0.142 mmol), were
dissolved in CH2Cl2/H2O (3 mL/3 mL) followed by the addition of 4 mg CuSO4.5H2O (0.014 mmol,
dissolved in 200 μL of water) and 6 mg of sodium ascorbate (0.028 mmol, dissolved in 200 μL of
water). The solution was stirred at room temperature for 24 h. The reaction mixture was worked-up as
described in 3.2.2 and was purified by silica gel CC eluting with hexane/EtOAc (8:2), yielding 5
(72 mg, 77%). White solid; mp 240 °C; [α]D20 +53 (c 0.064, CHCl3); IR νmax (film) 3415, 2937, 2856,

Molecules 2013, 18
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1
1727, 1693, 1462, 1277, 761 cm−1; H-NMR (CDCl3): δ 7.79 (1H, s, H-5'), 7.68 (2H, d, J = 7.8 Hz,
H-2'' and H-6''), 7.49 (2H, t, J = 7.8 Hz, H-3'' and H-5''), 7.40 (1H, t, J = 7.3 Hz, H-4''), 5.25 (1H, brs,
H-12), 4.50 (1H, t, J = 8.3 Hz, H-3α), 3.12 (2H, t, J = 7.2 Hz, H-3'), 2.81 (1H, dd, J = 13.4; 3.5 Hz,
H-18), 2.79 (2H, t, J = 7.0 Hz, H-2'), 1.10 (3H, s), 0.90 (6H, s), 0.88 (3H, s), 0.81 (3H, s), 0.79 (3H, s),
0.72 (3H, s); 13C-NMR (CDCl3): δ 183.97 (C-28), 172.58 (C-1'), 147.20 (C-4'), 143.68 (C-13), 137.20
(C-1''), 129.72 (2C, C-3'' and C-5''), 128.59 (C-4''), 122.43 (C-12), 120.44 (2C, C-2'' and C-6''), 119.55
(C-5'), 81.26 (C-3), 55.28 (C-5), 47.53, 46.50, 45.86, 41.53, 40.90, 39.25, 38.03, 37.72, 36.95, 34.02,
33.75, 33.08, 32.50, 32.46, 30.68, 27.99, 27.67, 25.92, 23.58, 23.59, 23.38, 22.85, 21.17, 18.15, 17.14,
16.70, 15.36; HREIMS m/z 656.4529 [M+H]+ (calcd for C41H58N3O4, 656.4427).
Compound (6). Compound 5 (50 mg, 0.076 mmol), was methylated with a solution of CH2N2 in ethyl
ether, yielding 44 mg (86%) of 6: white solid; mp 168 °C; [α]2D0 +44 (c 0.050, CHCl3); IR νmax (film)
2944, 2876, 1730, 1463, 1260, 756 cm−1; 1H-NMR (CDCl3): δ 7.79 (1H, s, H-5'), 7.69 (2H, d, J = 7.7
Hz, H-2'' and H-6''), 7.51 (2H, t, J = 7.7 Hz, H-3'' and H-5''), 7.42 (1H, t, J = 7.3 Hz, H-4''), 5.27 (1H,
brs, H-12), 4.51 (1H, t, J = 8.3 Hz, H-3α), 3.62 (3H, s, OMe), 3.13 (2H, t, J = 7.2 Hz, H-3'), 2.85 (1H,
dd, J = 13.4; 3.5 Hz, H-18), 2.80 (2H, t, J = 7.0 Hz, H-2'), 1.12 (3H, s), 0.92 (3H, s), 0.91 (3H, s), 0.89
13
(3H, s), 0.83 (3H, s), 0.80 (3H, s), 0.71 (3H, s); C-NMR (CDCl3): δ 178.35 (C-28), 172.56 (C-1'),
147.24 (C-4'), 143.53 (C-13), 137.56 (C-1''), 129.73 (2C, C-3'' and C-5''), 128.58 (C-4''), 122.26
(C-12), 120.46 (2C, C-2'' and C-6''), 119.50 (C-5'), 81.26 (C-3), 55.30 (C-5), 51.56 (OMe), 47.54,
46.72, 45.84, 41.63, 41.28, 39.27, 38.08, 37.73, 36.91, 34.03, 33.84, 33.11, 32.56, 32.37, 30.70, 27.99,
27.67, 25.90, 23.64, 23.55, 23.40, 23.05, 21.20, 18.19, 16.82, 16.71, 15.34; HREIMS m/z 670.5248
[M+H]+ (calcd for C42H60N3O4, 670.5286).
Compound (7). Compound 7 was synthesized as described for compound 5, using compound 1 (100 mg,
0.187 mmol) and 4-azidoanisole (28 mg, 0.187 mmol) yielding 68 mg (53%) of 3: white solid; mp 210 °C;
[α]2D0 +35 (c 0.045, CHCl3); IR νmax (film) 3416, 2946, 2877, 1732, 1695, 1460, 1255, 747 cm−1;
1H-NMR (CDCl3): δ 7.70 (1H, s, H-5'), 7.57 (2H, d, J = 8.7 Hz, H-2'' and H-6''), 6.98 (2H, d,
J = 8.7 Hz, H-3'' and H-5''), 5.25 (1H, brs, H-12), 4.50 (1H, t, J = 8.3 Hz, H-3α), 3.85 (3H, s, PhOMe),
3.11 (2H, t, J = 7.2 Hz, H-3'), 2.80 (1H, dd, J = 13.4; 3.5 Hz, H-18), 2.78 (2H, t, J = 7.0 Hz, H-2'),
13
1.11 (3H, s), 0.91 (6H, s), 0.87 (3H, s), 0.82 (3H, s), 0.79 (3H, s), 0.73 (3H, s); C-NMR (CDCl3): δ
183.70 (C-28), 172.60 (C-1'), 159.67 (C-4''), 146.99 (C-4'), 143.68 (C-13), 130.97 (C-1''), 122.45
(C-12), 122.10 (2C, C-2'' and C-6''), 119.75 (C-5'), 114.64 (2C, C-3'' and C-5''), 81.22 (C-3), 55.33
(OMe), 55.25 (C-5), 47.52, 46.50, 45.84, 41.54, 40.92, 39.25, 38.05, 37.73, 36.95, 34.07, 33.77, 33.01,
32.47, 32.30, 30.67, 27.93, 27.69, 25.83, 23.65, 23.53, 23.38, 23.20, 21.18, 18.13, 16.76, 16.64, 15.29;
HREIMS m/z 686.4309 [M+H]+ (calcd for C42H60N3O5, 686.4533).
Compound (8). Compound 7 (40 mg, 0.058 mmol), was methylated with a solution of CH2N2 in ethyl
ether, yielding 36 mg (89%) of 8: white solid; mp 151 °C; [α]2D0 +47 (c 0.015, CHCl3); IR νmax (film)
2941, 2872, 1722, 1460, 1252, 755 cm−1; 1H-NMR (CDCl3): δ 7.70 (1H, s, H-5'), 7.58 (2H, d, J = 8.9 Hz,
H-2'' and H-6''), 6.99 (2H, d, J = 8.9 Hz, H-3'' and H-5''), 5.27 (1H, brs, H-12), 4.50 (1H, t, J = 8.3 Hz,
H-3α), 3.86 (3H, s, PhOMe), 3.61 (3H, s, OMe), 3.11 (2H, t, J = 7.2 Hz, H-3'), 2.84 (1H, dd, J = 13.4;
3.5 Hz, H-18), 2.78 (2H, t, J = 7.0 Hz, H-2'), 1.11 (3H, s), 0.91 (6H, s), 0.89 (3H, s), 0.82 (3H, s), 0.79

Molecules 2013, 18
7668
(3H, s), 0.71 (3H, s); 13C-NMR (CDCl3): δ 178.26 (C-28), 172.51 (C-1'), 159.63 (C-4''), 146.97 (C-4'),
143.77 (C-13), 130.62 (C-1''), 122.21 (C-12), 122.05 (2C, C-2'' and C-6''), 119.63 (C-5'), 114.68 (2C,
C-3'' and C-5''), 81.19 (C-3), 55.58 (PhOMe), 55.27 (C-5), 51.50 (OMe), 47.51, 46.67, 45.81, 41.59,
41.25, 39.24, 38.05, 37.69, 36.89, 34.03, 33.81, 33.07, 32.54, 32.33, 30.66, 27.95, 27.64, 25.86, 23.60,
23.52, 23.36, 23.02, 21.17, 18.16, 16.78, 16.67, 15.31; HREIMS m/z 700.4257 [M+H]+ (calcd for
C43H62N3O5, 700.4689).
Compound (9). Compound 9 was synthesized as described for compound 5, using compound 1 (120
mg, 0.224 mmol) and 1-azido-4-chlorobenzene (34 mg, 0.224 mmol) yielding 84 mg (54%) of 9: white
solid; mp 184 °C; [α]2D0 +46 (c 0.052, CHCl3); IR νmax (film) 3432, 2942, 2880, 1720, 1688, 1460,
1271, 756 cm−1; 1H-NMR (CDCl3): δ 7.78 (1H, s, H-5'), 7.64 (2H, d, J = 8.6 Hz, H-2'' and H-6''), 7.46
(2H, d, J = 8.6 Hz, H-3'' and H-5''), 5.25 (1H, brs, H-12), 4.49 (1H, t, J = 8.3 Hz, H-3α), 3.13 (2H, t,
J = 7.0 Hz, H-3'), 2.80 (1H, dd, J = 13.4; 3.5 Hz, H-18), 2.78 (2H, t, J = 6.9 Hz, H-2'), 1.10 (3H, s),
0.90 (6H, s), 0.88 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.72 (3H, s); 13C-NMR (CDCl3): δ 184.16 (C-28),
172.53 (C-1'), 147.51 (C-4'), 143.65 (C-13), 135.60 (C-1''), 134.28 (C-4''), 129.89 (2C, C-3'' and C-5''),
122.46 (C-12), 121.54 (2C, C-2'' and C-6''), 119.49 (C-5'), 81.29 (C-3), 55.28 (C-5), 47.53, 46.51,
45.85, 41.51, 40.88, 39.24, 38.02, 37.72, 36.96, 33.92, 33.78, 33.08, 32.47, 32.34, 30.68, 27.99, 27.65,
25.92, 23.60, 23.58, 23.39, 22.87, 21.13, 18.21, 17.17, 16.70, 15.36; HREIMS m/z 690.4133 [M+H]+
(calcd for C41H57ClN3O4, 690.4037).
Compound (10). Compound 9 (50 mg, 0.073 mmol), was methylated with a solution of CH2N2 in ethyl
ether, yielding 45 mg (88%) of 10: white solid; mp 206 °C; [α]2D0 +53 (c 0.015, CHCl3); IR νmax (film)
2949, 2873, 1725, 1461, 1257, 760 cm−1; 1H-NMR (CDCl3): δ 7.77 (1H, s, H-5'), 7.62 (2H, d, J = 8.7 Hz,
H-2'' and H-6''), 7.43 (2H, d, J = 8.7 Hz, H-3'' and H-5''), 5.23 (1H, brs, H-12), 4.46 (1H, t, J = 8.1 Hz,
H-3α), 3.58 (3H, s, OMe), 3.08 (2H, t, J = 7.2 Hz, H-3'), 2.81 (1H, dd, J = 13.6; 3.4 Hz, H-18), 2.75
(2H, t, J = 7.2 Hz, H-2'), 1.08 (3H, s), 0.88 (3H, s), 0.87 (3H, s), 0.85 (3H, s), 0.78 (3H, s), 0.75 (3H,
13
s), 0.67 (3H, s); C-NMR (CDCl3): δ 178.21 (C-28), 172.39 (C-1'), 147.42 (C-4'), 143.76 (C-13),
135.59 (C-1''), 134.16 (C-4''), 129.83 (2C, C-3'' and C-5''), 122.20 (C-12), 121.46 (2C, C-2'' and C-6''),
119.40 (C-5'), 81.20 (C-3), 55.26 (C-5), 51.50 (OMe), 47.50, 46.65, 45.80, 41.57, 41.24, 39.22, 38.04,
37.68, 36.87, 33.87, 33.81, 33.11, 32.53, 32.33, 30.66, 27.96, 27.64, 25.88, 23.63, 23.53, 23.36, 23.00,
21.13, 18.16, 16.79, 16.70, 15.32; HREIMS m/z 704.4243 [M+H]+ (calcd for C42H59ClN3O4,
704.4194).
Compound (11). Compound 11 was synthesized as described for compound 5, using compound 1 (120
mg, 0.224 mmol) and p-toluenesulfonyl azide (48 mg, 0.224 mmol) yielding 91 mg (55%) of 11: white
solid; mp 163 °C; [α]2D0 +39 (c 0.054, CHCl3); IR νmax (film) 3420, 2946, 2871, 1730, 1693, 1460,
1272, 759 cm−1; 1H-NMR (CDCl3): δ 7.96 (2H, d, J = 8.3 Hz, H-2'' and H-6''), 7.91 (1H, s, H-5'), 7.36
(2H, d, J = 8.3 Hz, H-3'' and H-5''), 5.26 (1H, brs, H-12), 4.47 (1H, t, J = 8.3 Hz, H-3α), 3.03 (2H, t,
J = 7.2 Hz, H-3'), 2.81 (1H, dd, J = 13.5; 3.1 Hz, H-18), 2.70 (2H, t, J = 7.2 Hz, H-2'), 2.43 (3H, s,
13
PhMe), 1.11 (3H, s), 0.91 (3H, s), 0.90 (3H, s), 0.89 (3H, s), 0.77 (3H, s), 0.73 (6H, s); C-NMR
(CDCl3): δ 184.33 (C-28), 172.05 (C-1'), 147.19 (C-4'), 146.37 (C-4''), 143.64 (C-13), 133.17 (C-1''),
130.43 (2C, C-2'' and C-6''), 128.62 (2C, C-3'' and C-5''), 122.47 (C-12), 121.02 (C-5'), 81.36 (C-3),

Molecules 2013, 18
7669
55.23 (C-5), 47.53, 46.53, 45.82, 41.51, 40.88, 39.25, 38.03, 37.67, 36.95, 33.78, 33.49, 33.10, 32.46,
32.30, 30.68, 27.95, 27.65, 25.94, 23.60, 23.49, 23.37, 23.14, 21.87, 20.91, 18.13, 17.17, 16.65, 15.37;
HREIMS m/z 734.4814 [M+H]+ (calcd for C42H60N3O6S, 734.4203).
Compound (12). Compound 11 (60 mg, 0.082 mmol), was methylated with a solution of CH2N2 in
ethyl ether, yielding 53 mg (87%) of 12: white solid; mp 154 °C; [α]2D0 +35 (c 0.053, CHCl3); IR νmax
(film) 2946, 2876, 1726, 1467, 1260, 757 cm−1; 1H-NMR (CDCl3): δ 7.94 (2H, d, J = 8.3 Hz, H-2'' and
H-6''), 7.90 (1H, s, H-5'), 7.34 (2H, d, J = 8.3 Hz, H-3'' and H-5''), 5.25 (1H, brs, H-12), 4.44 (1H, t,
J = 8.2 Hz, H-3α), 3.60 (3H, s, OMe), 3.01 (2H, t, J = 7.2 Hz, H-3'), 2.83 (1H, dd, J = 13.4; 3.4 Hz,
H-18), 2.68 (2H, t, J = 7.0 Hz, H-2'), 2.41 (3H, s, PhMe), 1.10 (3H, s), 0.90 (3H, s), 0.87 (6H, s), 0.74
13
(3H, s), 0.70 (3H, s), 0.69 (3H, s); C-NMR (CDCl3): δ 178.26 (C-28), 171.97 (C-1'), 147.14 (C-4'),
146.36 (C-4''), 143.81 (C-13), 133.19 (C-1''), 130.40 (2C, C-2'' and C-6''), 128.60 (2C, C-3'' and C-5''),
122.21 (C-12), 121.00 (C-5'), 81.31 (C-3), 55.23 (C-5), 51.54 (OMe), 47.51, 46.68, 45.81, 41.60,
41.26, 39.25, 38.03, 37.63, 36.87, 33.83, 33.46, 33.11, 32.54, 32.35, 30.69, 27.91, 27.66, 25.90, 23.64,
23.46, 23.39, 23.03, 21.84, 20.91, 18.17, 16.80, 16.66, 15.33; HREIMS m/z 748.4274 [M+H]+ (calcd
for C43H62N3O6S, 748.4359).
Compound (13). Compound 13 was synthesized as described for compound 5, using compound 1 (120 mg,
0.224 mmol) and azidomethyl phenyl sulfide (37 mg, 0.224 mmol) yielding 98 mg (62%) of 13: white
solid; mp 170 °C; [α]2D0 +13 (c 0.055, CHCl3); IR νmax (film) 3426, 2939, 2871, 1723, 1691, 1461,
1276, 756 cm−1; 1H-NMR (CDCl3): δ 7.37 (1H, s, H-5'), 7.29 (5H, s, Ph), 5.57 (2H, s CH2S), 5.25 (1H,
brs, H-12), 4.47 (1H, t, J = 8.3 Hz, H-3α), 3.00 (2H, t, J = 7.3 Hz, H-3'), 2.81 (1H, dd, J = 13.5; 3.5 Hz,
H-18), 2.68 (2H, t, J = 7.5 Hz, H-2'), 1.11 (3H, s), 0.91 (3H, s), 0.90 (3H, s), 0.89 (3H, s), 0.80 (3H, s),
13
0.78 (3H, s), 0.73 (3H, s); C-NMR (CDCl3): δ 184.07 (C-28), 172.42 (C-1'), 147.12 (C-4'), 143.66
(C-13), 132.09 (C-1''), 132.02 (2C, C-2'' and C-6''), 129.46 (2C, C-3'' and C-5''), 128.57 (C-4''), 122.47
(C-12), 120.70 (C-5'), 81.17 (C-3), 55.28 (C-5), 53.65 (CH2S), 47.54, 46.52, 45.85, 41.53, 40.89,
39.26, 38.02, 37.71, 36.96, 33.98, 33.79, 33.07, 32.49, 32.44, 30.67, 28.02, 27.66, 25.91, 23.59, 23.52,
23.38, 22.86, 21.19, 18.15, 17.17, 16.70, 15.35; HREIMS m/z 702.3102 [M+H]+ (calcd for
C42H60N3O4S, 702.4304).
Compound (14). Compound 13 (60 mg, 0.086 mmol), was methylated with a solution of CH2N2 in
ethyl ether, yielding 55 mg (90%) of 14: white solid; mp 195 °C; [α]2D0 +17 (c 0.102, CHCl3); IR νmax
1
(film) 2947, 2875, 1731, 1460, 1257, 763 cm−1; H-NMR (CDCl3): δ 7.37 (1H, s, H-5'), 7.28 (5H, s,
Ph), 5.56 (2H, s CH2S), 5.26 (1H, brs, H-12), 4.46 (1H, t, J = 8.1 Hz, H-3α), 3.60 (3H, s, OMe), 2.99
(2H, t, J = 7.3 Hz, H-3'), 2.84 (1H, dd, J = 13.7; 3.9 Hz, H-18), 2.68 (2H, t, J = 7.6 Hz, H-2'), 1.10
13
(3H, s), 0.90 (3H, s), 0.89 (3H, s), 0.88 (3H, s), 0.80 (3H, s), 0.76 (3H, s), 0.70 (3H, s); C-NMR
(CDCl3): δ 178.30 (C-28), 172.39 (C-1'), 147.12 (C-4'), 143.80 (C-13), 132.08 (C-1''), 132.00 (2C,
C-2'' and C-6''), 129.45 (2C, C-3'' and C-5''), 128.56 (C-4''), 122.34 (C-12), 120.69 (C-5'), 81.18 (C-3),
55.29 (C-5), 53.63 (CH2S), 51.54 (OMe), 47.53, 46.70, 45.83, 41.61, 40.27, 39.25, 38.06, 37.70, 36.90,
33.96, 33.83, 33.11, 32.56, 32.36, 30.69, 28.01, 27.66, 25.90, 23.64, 23.52, 23.39, 23.04, 21.19, 18.18,
16.81, 16.71, 15.33; HREIMS m/z 716.4378 [M+H]+ (calcd for C43H62N3O4S, 716.4461).

Molecules 2013, 18
7670
Compound (15). Compound 15 was synthesized as described for compound 5, using compound 3 (120 mg,
0.218 mmol) and p-toluenesulfonyl azide (32 mg, 0.218 mmol) yielding 76 mg (47%) of 15: white
solid; mp 186 °C; [α]2D0 +51 (c 0.048, CHCl3); IR νmax (film) 3430, 2943, 2873, 1729, 1689, 1461,
1272, 757 cm−1; 1H-NMR (CDCl3): δ 7.94 (2H, d, J = 8.2 Hz, H-2'' and H-6''), 7.87 (1H, s, H-5'), 7.34
(2H, d, J = 8.1 Hz, H-3'' and H-5''), 5.23 (1H, brs, H-12), 4.47 (1H, t, J = 8.1 Hz, H-3α), 2.79 (1H, dd,
J = 13.4; 3.0 Hz, H-18), 2.73 (2H, t, J = 7.5 Hz, H-4'), 2.40 (3H, s, PhMe), 2.32 (2H, t, J = 7.3 Hz,
H-2'), 1.96 (2H, m, H-3'), 1.09 (3H, s), 0.90 (3H, s), 0.89 (3H, s), 0.87 (3H, s), 0.81 (6H, s), 0.71 (3H,
13
s); C-NMR (CDCl3): δ 184.46 (C-28), 172.82 (C-1'), 147.19 (C-4'), 147.12 (C-4''), 143.63 (C-13),
133.14 (C-1''), 130.44 (2C, C-2'' and C-6''), 128.57 (2C, C-3'' and C-5''), 122.47 (C-12), 120.71 (C-5'),
80.98 (C-3), 55.24 (C-5), 47.51, 46.50, 45.82, 41.48, 40.84, 39.23, 38.03, 37.70, 36.96, 33.81, 33.08,
32.47, 32.33, 31.60, 30.65, 28.10, 27.66, 25.94, 24.65, 24.26, 23.66, 23.59, 23.38, 22.82, 21.84, 18.15,
17.19, 16.75, 15.37; HREIMS m/z 748.4327 [M+H]+ (calcd for C43H62N3O6S, 748.4359).
Compound (16). Compound 15 (50 mg, 0.067 mmol), was methylated with a solution of CH2N2 in
ethyl ether, yielding 44 mg (86%) of 16: white solid; mp 165 °C; [α]2D0 +32 (c 0.079, CHCl3); IR νmax
(film) 2943, 2871, 1728, 1459, 1254, 757 cm−1; 1H-NMR (CDCl3): δ 7.96 (2H, d, J = 8.1 Hz, H-2'' and
H-6''), 7.87 (1H, s, H-5'), 7.36 (2H, d, J = 8.1 Hz, H-3'' and H-5''), 5.26 (1H, brs, H-12), 4.48 (1H, t,
J = 8.3 Hz, H-3α), 3.60 (3H, s, OMe), 2.84 (1H, dd, J = 13.5; 3.2 Hz, H-18), 2.74 (2H, t, J = 7.5 Hz,
H-4'), 2.43 (3H, s, PhMe), 2.33 (2H, t, J = 7.3 Hz, H-2'), 1.98 (2H, m, H-3'), 1.11 (3H, s), 0.90 (6H, s),
13
0.88 (3H, s), 0.82 (6H, s), 0.70 (3H, s); C-NMR (CDCl3): δ 178.27 (C-28), 172.74 (C-1'), 147.16
(C-4'), 147.11 (C-4''), 143.80 (C-13), 133.19 (C-1''), 130.42 (2C, C-2'' and C-6''), 128.60 (2C, C-3'' and
C-5''), 122.34 (C-12), 120.62 (C-5'), 80.99 (C-3), 55.27 (C-5), 51.55 (OMe), 47.53, 46.69, 45.83,
41.61, 41.27, 39.26, 38.06, 37.70, 36.91, 33.81, 33.11, 32.57, 32.37, 31.58, 30.69, 28.10, 27.66, 25.91,
24.68, 24.27, 23.64, 23.56, 23.39, 23.04, 21.84, 18.20, 16.82, 16.77, 15.35; HREIMS m/z 762.4427
[M+H]+ (calcd for C44H64N3O6S, 762.4516).
Compound (17). Compound 17 was synthesized as described for compound 5, using compound 3 (120 mg,
0.218 mmol) and azidomethyl phenyl sulfide (36 mg, 0.218 mmol) yielding 87 mg (56%) of 17: white
solid; mp 198 °C; [α]2D0 +43 (c 0.011, CHCl3); IR νmax (film) 3426, 2940, 2876, 1723, 1692, 1461,
1270, 754 cm−1; 1H-NMR (CDCl3): δ 7.30 (1H, s, H-5'), 7.29 (5H, s, Ph), 5.57 (2H, s CH2S), 5.25 (1H,
brs, H-12), 4.49 (1H, t, J = 8.1 Hz, H-3α), 2.80 (1H, dd, J = 13.4; 3.2 Hz, H-18), 2.71 (2H, t, J = 7.5 Hz,
H-4'), 2.31 (2H, t, J = 7.3 Hz, H-2'), 1.95 (2H, m, H-3'), 1.11 (3H, s), 0.91 (6H, s), 0.88 (3H, s), 0.84
13
(3H, s), 0.82 (3H, s), 0.73 (3H, s); C-NMR (CDCl3): δ 184.15 (C-28), 173.06 (C-1'), 147.75 (C-4'),
143.69 (C-13), 132.28 (2C, C-2'' and C-6''), 131.95 (C-1''), 129.47 (2C, C-3'' and C-5''), 128.66 (C-4''),
122.44 (C-12), 120.55 (C-5'), 80.88 (C-3), 55.27 (C-5), 53.72 (CH2S), 47.55, 46.52, 45.86, 41.53,
40.89, 39.26, 38.03, 37.73, 36.98, 33.92, 33.76, 33.10, 32.52, 32.41, 30.68, 28.12, 27.67, 25.95, 24.92,
24.73, 23.62, 23.57, 23.41, 22.89, 18.22, 17.18, 16.79, 15.39; HREIMS m/z 716.4423 [M+H]+ (calcd
for C43H62N3O4S, 716.4461).
Compound (18). Compound 17 (50 mg, 0.068 mmol), was methylated with a solution of CH2N2 in
ethyl ether, yielding 45 mg (91%) of 18: white solid; mp 166 °C; [α]2D0 +39 (c 0.016, CHCl3); IR νmax
1
(film) 2946, 2870, 1723, 1461, 1259, 755 cm−1; H-NMR (CDCl3): δ 7.28 (1H, s, H-5'), 7.26 (5H, s,

Molecules 2013, 18
7671
Ph), 5.54 (2H, s CH2S), 5.24 (1H, brs, H-12), 4.46 (1H, t, J = 8.1 Hz, H-3α), 3.58 (3H, s, OMe), 2.82
(1H, dd, J = 13.4; 3.2 Hz, H-18), 2.69 (2H, t, J = 7.5 Hz, H-4'), 2.29 (2H, t, J = 7.3 Hz, H-2'), 1.93
(2H, m, H-3'), 1.09 (3H, s), 0.88 (6H, s), 0.86 (3H, s), 0.81 (3H, s), 0.80 (3H, s), 0.68 (3H, s);
13C-NMR (CDCl3): δ 178.23 (C-28), 172.95 (C-1'), 147.71 (C-4'), 143.77 (C-13), 132.24 (2C, C-2''
and C-6''), 131.98 (C-1''), 129.42 (2C, C-3'' and C-5''), 128.61 (C-4''), 122.23 (C-12), 120.48 (C-5'),
80.82 (C-3), 55.26 (C-5), 53.64 (CH2S), 51.51 (OMe), 47.51, 46.67, 45.81, 41.59, 41.25, 39.24, 38.06,
37.70, 36.90, 33.89, 33.83, 33.12, 32.56, 32.35, 30.68, 28.10, 27.65, 25.90, 24.93, 24.71, 23.65, 23.57,
23.38, 23.03, 18.19, 16.81 (2C), 15.36; HREIMS m/z 730.4572 [M+H]+ (calcd for C44H64N3O4S,
730.4617).
3.3. Antiproliferative Assay
All human cell lines used in this work were purchased from the American Type Culture Collection
(ATCC, Manasas, VA, USA). Normal lung MRC-5 fibroblasts (CCL-171), SK-MES-1 lung cancer
cells (HTB-58) and J82 bladder carcinoma cells (HTB-1) were grown as monolayers in minimum
essential Eagle medium (MEM) with Earles’s salts, 2 mM L-glutamine and 1.5 g/L sodium
bicarbonate. Gastric adenocarcinoma AGS cells (CRL-1739) were grown as monolayers in Ham F-12
medium containing 1 mM L-glutamine and 1.5 g/L sodium bicarbonate. Promyelocytic leukemia HL-60
cells (CCL-240) were grown in suspension in RPM1 medium containing 1 mM sodium pyruvate and
2.0 g/L sodium bicarbonate. All media were supplemented with 10% heat-inactivated FBS, 100 IU/mL
penicillin and 100 µg/mL streptomycin. Cells were grown in a humidified incubator with 5% CO2 in
air at 37 °C. For the antiproliferative assay, adherent cells were plated at a density of 5 × 104 cells/mL
and HL-60 cells at 30 × 104 cells/mL. Cells were seeded in 96-well plates (100 µL/well). One day after
seeding, cells were treated with medium containing the compounds at concentrations ranging from
0 up to 100 µM during 3 days. The compounds were dissolved in DMSO (1% final concentration) and
complete medium. Untreated cells (medium containing 1% DMSO) were used as 100% viability
controls. Etoposide (98% purity, Sigma-Aldrich, St. Louis, MO, USA) was used as reference
compound. Each concentration was tested in sextuplicate and experiments were repeated 2 times. Cell
viability was determined by means of the MTT reduction assay at the end of the incubation with the
products. The results were transformed to percentage of controls and the IC50 values were graphically
obtained from the dose-response curves.
4. Conclusions
Click chemistry techniques were applied to obtain new heterocycles-terpene hybrid compounds
starting from the naturally occurring triterpene OA. The methods presented in this work allowed the
synthesis of several new compounds in good to reasonable yields. The compounds were assessed as
antiproliferative agents in four human tumor cell lines and on normal fibroblasts. The presence of the
free COOH function was relevant for the antiproliferative effect, regardless of the heterocycle moiety.
Some of the new products presented better effect than the starting compounds with relative selectivity
towards AGS (compound 9) or HL-60 cancer lines (compound 17). Additional studies and other assays
using different targets are necessary to disclose the potential of the new compounds as bioactive
agents. The new hybrid compounds are much less active than the reference compound etoposide and

Molecules 2013, 18
7672
cannot be considered as promising antiproliferative agents. However, other biological activities and
mechanisms of action remain to be investigated.
Acknowledgments
Financial support of FONDECYT Project 11100046 is gratefully acknowledged.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–18 are available from the authors.
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