Synthesis of Analogs of Trans-Fagaramide and Their Cytotoxic Activity

Article abstract of DOI:10.1007/s11094-018-1729-1

A series of 30 compounds were synthetized inspired by active trans-fagaramide structure skeleton. On this synthetic platform, 18 compounds were achieved via Knoevenagel condensation using maleic acid and piperonal, followed by peptide coupling with various amines, giving an average yield of 54%. Subsequently, nine compounds were obtained by palladium-mediated Heck coupling with an average yield of 79%. In addition, cytotoxic activity was evaluated against cardiomyoblast H9c2, breast adenocarcinoma MCF7, hepatocellular carcinoma HepG2, and glioblastoma U-87 cells. The results revealed two aryl halogen-substituted compounds moderately active against H9c2 and MCF7 with IC₅₀ values > 50 μM. One functionalized coumarin showed inhibitory activity against H9c2 (IC₅₀ > 50 μM). In contrast, p-aminophenyl-β-monosubstituted trans-fagaramide was found to inhibit MCF7 (IC₅₀ > 50 μM) without showing toxicity against H9c2 cells.

Full text of DOI:10.1007/s11094-018-1729-1

DOI 10.1007/s11094-018-1729-1
Pharmaceutical Chemistry Journal, Vol. 51, No. 11, February, 2018 (Russian Original Vol. 51, No. 11, November, 2017)
SYNTHESIS OF ANALOGS OF TRANS-FAGARAMIDE
AND THEIR CYTOTOXIC ACTIVITY
Melissa Barrera Tomas,1,2,3,* Tze Chieh Shiao,1 Phuong Trang Nguyen,1
Steve Bourgault,1 and René Roy1
Original article submitted May 28, 2017.
A series of 30 compounds were synthetized inspired by active trans-fagaramide structure skeleton. On this
synthetic platform, 18 compounds were achieved via Knoevenagel condensation using maleic acid and
piperonal, followed by peptide coupling with various amines, giving an average yield of 54%. Subsequently,
nine compounds were obtained by palladium-mediated Heck coupling with an average yield of 79%. In addi-
tion, cytotoxic activity was evaluated against cardiomyoblast H9c2, breast adenocarcinoma MCF7,
hepatocellular carcinoma HepG2, and glioblastoma U-87 cells. The results revealed two aryl halogen-substi-
tuted compounds moderately active against H9c2 and MCF7 with IC50 values > 50 mM. One functionalized
coumarin showed inhibitory activity against H9c2 (IC50 > 50 mM). In contrast, p-aminophenyl-b-
monosubstituted trans-fagaramide was found to inhibit MCF7 (IC50 > 50 mM) without showing toxicity
against H9c2 cells.
Keywords: trans-fagaramide; Knoevenagel condensation; peptide coupling; palladium-mediated Heck cou-
pling; cytotoxic activity.
1. INTRODUCTION
fication and characterization of FL118 molecule [10] showed
it to be an active compound capable of selectively inhibiting
multiple cancer survival and proliferation-associated anti-
apoptotic proteins (survivin, Mcl-1, XIAP, clAP2) (Fig. 1).
In this context, this paper describes the elaboration of a
synthetic platform of 30 analogs of trans-fagaramide gener-
ated via Knoevenagel condensation, peptide coupling,
Fischer esterification, and palladium-mediated Heck cou-
pling. Thus, some modifications in the trans-fagaramide
chemical structure may lead effective synthetic candidates
toward the determination of optimized and efficient drugs
against cancer cells.
Trans-fagaramide, a natural amide specifically found in
the Zanthoxylum genus [1, 2], is well known for its previ-
ously studied activity against cancer, cardiac affections,
rheumatism and as antifungal agent [3, 4]. Earlier studies of
trans-fagaramide biological effects revealed ovicidal activity
[5, 6] and moderate cytotoxicity against selected cancer cell
lines. Fundamentally, trans-fagaramide is formed by a me-
thylenedioxyphenyl group and an a,b-unsaturated amide [7],
which have inspired scientists to continue looking for active
molecules structurally related to trans-fagaramide skeleton.
Preceding reports showed considerable number of active
compounds against certain cancer cells, containing structural
units of trans-fagaramide [8]. In particular, some piperine
analogs having the skeleton structure of trans-fagaramide
were synthesized and evaluated for their activity against
MCF-7 breast cancer and Hela cervix, showing significant
activity against both human cancer cell types [9]. The identi-
2. RESULTS AND DISCUSSION
2.1 Synthesis of trans-Fagaramide and Its Analogs
by Knoevenagel Condensation, Peptide Coupling
and Fischer Esterification
1
The synthesis of trans-fagaramide (compound 2) and its
analogs were performed in two steps with an overall yield of
56%. First, Knoevenagel condensation between commer-
cially malonic acid and piperonal was used to afford the re-
quired precursor 1 (cinnamic acid) at 75% yield in the pres-
ence of excess basic solvents (pyridine and piperidine)
Department of Chemistry, Université du Québec à Montréal, CP 8888,
Succ. A, Montréal, Québec H3C 3P8, Canada.
2
Departamento de Química, Universidad Nacional Agraria La Molina,
Avenida La Molina, s/n La Molina, Lima, Perú.
3
Departamento de Química Orgánica, Universidad Nacional Mayor de San
Marcos, Avenida Universitaria, Calle Germán Amézaga 375, Lima, Perú.
*
e-mail: barreratomasmelissa@gmail.com.
995
0091-150X/17/5111-0995 © 2017 Springer Science+Business Media, LLC

996
Melissa Barrera Tomas et al.
Fig. 1. Examples of active analogs of trans-fagaramide [7 – 10].
[12, 13]. Compound 1 was further functionalized by its
activation using N,N¢-diisopropylcarbodiimide (DIC);
2.2. Synthesis of trans-Fagaramide Analogs
by Palladium-Mediated Heck Coupling
N,N,N¢,N¢-tetramethyl-O-(benzotriazol-1-yl)uranium tetra-
fluoroborate (TBTU), and hydroxybenzotriazole (HOBt) as
coupling agents [14, 15], which allowed the obtaining of
compounds 2 – 20 by following a peptide coupling reaction
employing various mono-substituted amines (Schemes 1a
and 1b). In order to obtain the optimum reaction conditions,
TBTU (well documented as a highly potent amide coupling
reagent [16]) and compound 1 were reacted in equal amounts
to avoid the formation of excess urea derivatives. These de-
rivatives are more or less soluble in organic solvents as well
as in aqueous phases, which makes them difficult to elimi-
nate [17]. However, this removal was easily achieved by the
work-up shown in the experimental part of this paper so as to
afford 18 analogs of trans-fagaramide without further purifi-
cation.
Subsequently, compounds 22 – 30 were achieved by pal-
ladium-mediated Heck coupling, another powerful tool in or-
ganic synthesis, which contributes in the formation of car-
bon – carbon and carbon – heteroatom bonds [18]. First,
compounds 2 and 21 were coupled successfully, generating
the corresponding eight products as mixtures of stere-
oisomers. The formation of aryl-substituted olefins were per-
formed by using various mono-substituted aryl iodides,
which reacted with the correspond olefin at elevated temper-
atures in DMF The coupling reaction was catalyzed by
.
Pd(OAc) in the presence of tetrabutylammonium bromide
2
(TBABr) as a phase-transfer-catalyst (Scheme 2) [18]. In
contrast to other compounds, compound 26 contains a
coumarin unit in its chemical structure, which is formed via
the internal attack of the hydroxyl group from the benzene
ring to the carbonyl group.
Compound 21 was obtained by Fischer esterification be-
tween compound 1 (cinnamic acid) and methanol in the pres-
ence of H SO (acid catalyst) with an overall yield of 53%
2
4
Compound 26 (yield, 66%) was obtained as a white
solid. The rest of the compounds were obtained as mixtures
of stereoisomers at an average yield of 80% within an inter-
val of 68 – 96% (Fig. 3).
(Scheme 1c).
Analogs of trans-fagaramide were obtained by peptide
coupling at an average yield of 54% within 20 – 92% range
(Fig. 2).
Scheme 1. (a, b) Synthesis of trans-fagaramide (2) and its analogs (3 – 20) by peptide coupling; (c) synthesis of compound 21 by Fischer
esterification.

Synthesis of Analogs of Trans-Fagaramide
997
Fig. 2. Analogs of trans-fagaramide synthesized by peptide coupling.
2.3. Biological Testing
26 exhibited an inhibition activity against MCF7 and H9c2,
respectively, with IC above 50 mM. (Table 1). The rest of
50
The synthesized analogs of trans-fagaramide were tested
for their cytotoxic activity against breast adenocarcinoma
MCF7, hepatocellular carcinoma HepG2, glioblastoma U-87
cells as well as normal H9c2 cells. The results presented in
Table 1 are expressed as drug concentrations inhibiting fifty
compounds are not mentioned because of their lack of activ-
ity in these cells lines.
3. EXPERIMENTAL SECTION
percent of cell growth (IC ). Capsaicin was used as a posi-
50
3.1 General
tive control with IC ranging from 1 to 100 mM. The viabil-
50
ity of cells was measured using the resazurin assay [19, 20].
All solvents (Acros Organics or Aldrich) were used with-
out additional purification. The isolation and purification
were monitored by thin layer chromatography (TLC) on sil-
ica gel plate (Merck 60 F254) and by column chromatogra-
phy using silica gel (Silica Gel 60, particle size
Compounds 4 and 5 reduced the cellular viability of H9c2
and MCF7, with IC over 50 mM. This result showed that
50
the anticancer activity was related to the presence of
halo-substitued aryl structure. Likewise, compounds 25 and
Scheme 2. Synthesis of compounds 22 – 30 by palladium-mediated Heck coupling.

998
Melissa Barrera Tomas et al.
Fig. 3. Structures of trans-fagaramide analogs 22 – 30 synthesized by palladium-mediated Heck coupling.
0.063 – 0.200 mm). Compounds obtained by extraction were
analyzed and characterized in CDCl by 1H NMR at
30 mmol) and pyridine (5 mL, 61 mmol), piperidine (50 mL,
0.48 mmol) was slowly added drop by drop and the final re-
action mixture was stirred at room temperature for 45 min.
Then the reaction was heated at 100°C for 3 h. The mixture
was cooled and the excess of pyridine was neutralized by the
addition of 1 M HCl (20 mL), so that a white precipitate ap-
peared between pH 4 – 5. The precipitated acid was filtered,
washed with cold EtOAc, and recrystallized from saturated
hot ethanol. The filtrate was dried under vacuum in a desic-
cator for 48 h to afford 11.55 mmol (75%) of compound 1 as
3
300 MHz, 1H NMR at 600 MHz, and 13C NMR at 150 MHz
on a Varian Inova AS600 spectrometer. The molecular ions
were protonated as [M+H]+ for the confirmation of their em-
pirical formula. The notations used for the spectral analysis
are as follows: s (singlet), d (doublet), dd (doublet of dou-
blets), m (multiplet). Chemical shifts are reported in d (ppm)
using TMS as the internal standard; the coupling constants
(J) were measured in Hz. Infrared spectra were recorded on a
JASCO FT/IR-410 spectrophotometer.
white crystals; 1H-NMR ((CD ) SO, 600 MHz; d, ppm): 7.51
3 2
(1H, d, J = 16.0 Hz, benzene-CH), 7.36 (1H, d, J = 1.6 Hz,
Ha), 7.15 (1H, dd, J = 1.6 Hz, J = 8.2 Hz, Hb), 6.94 (1H, d,
J = 8.2 Hz, Hc), 6.40 (1H, d, J = 16.0 Hz, CHCO), 6.07 (2H,
s, OCH O); 13C-NMR ((CD ) SO, 150 MHz; d, ppm): 167.8
3.2. Synthesis and Characterization of Compound 1
and Synthetic trans-Fagaramide
2
3 2
(COOH), 149.1, 148.0, 128.7, 124.6, 108.5, 106.7 (arom),
(E)-3-(Benzo[d][1,3]dioxol-5-yl)acrylic acid (1). To a
143.9 (CHCH), 117.1 (CHCO), 101.6 (OCH O);
solution of piperonal (2.3 g, 15 mmol), malonic acid (3.1 g,
2
ESI+-HRMS (m/z): 193.0497 [M + H]+ (calcd. for C H O ,
10
9
4
193.0495).
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-isobutylacrylamide,
TABLE 1. Cytotoxic Activity* of trans-Fagaramide Analogs
against Cardiomyoblast H9c2, Breast Adenocarcinoma MCF7,
Hepatocellular Carcinoma HepG2, and Glioblastoma U-87
synthetic trans-fagaramide (2). Compound 1 (2.5 g,
13 mmol) in dry DCM (130 mL) was purged with N and
2
DIC (2.0 mL, 13 mmol) was added, followed sequentially by
HOBt (175.7 mg, 1.3 mmol) and isobutylamine (1.3 mL,
13 mmol). The mixture was stirred at room temperature for
24h (monitored by TLC) and then concentrated under vac-
uum to remove most of the CH Cl . The crude material was
IC50 (mM)
Compound
H9c2
>50
>50
>50
N/A
>50
MCF7
N/A
>50
HepG2
U-87
>50
Capsaicin
37 ± 10
N/A
2
2
4
N/A
N/A
N/A
N/A
diluted with EtOAc and washed successively with distilled
water in the presence of hexane (1/4 of the volume of
5
>50
N/A
EtOAc), then with saturated potassium bisulfate (KHSO ),
25
26
>50
N/A
4
and sodium bicarbonate (NaHCO ). The organic phase was
N/A
N/A
3
dried over anhydrous sodium sulfate (Na SO ), filtered, and
2
4
concentrated. The crude material was purified by column
chromatography on silica gel (eluent: hexane/EtOAc, 100:0
to 75:25) to afford 9.75 mmol (75%) of compound 2 as white
* Each value represents the mean ± SD from at least two individual
experiments performed in triplicate. Statistical significance was cal-
culated for p < 0.05; RM ANOVA on ranks and post hoc Stu-
dent–Newman–Keuls method; N/A = not assessed (compounds with
1
crystals; H-NMR (CDCl , 600 MHz; d, ppm): 7.53 (1H, d,
3
IC higher than 100 mM were considered inactive).
50
J = 15.9 Hz, benzene-CH), 7.0 (1H s, H ), 6.97 (1H, d,
a

Synthesis of Analogs of Trans-Fagaramide
999
J = 8.2 Hz, H ), 6.78 (1H, d, J = 8.2 Hz, H ), 6.22 (1H, d,
(4H, m, arom), 7.33 (1H, s, NH), 7.03 – 6.80 (3H, m, arom),
b
c
J = 15.9 Hz, CHCO), 5.98 (2H, s, OCH O), 5.63 (1H, s,
6.35 (1H, d, J = 15.7 Hz, CHCO), 6.01 (2H, s, OCH O);
2
2
13C-NMR (CDCl , 75 MHz; d, ppm): 165.5 (CONH), 148.5,
NH), 3.20 (2H, t, CH ), 1.13 (1H, m, CH), 0.94 (6H, d, CH );
2
3
3
13C-NMR (CDCl , 150 MHz; d, ppm): 166.0 (CONH),
149.0, 148.2, 129.3, 123.8, 108.5, 106.3 (arom), 140.6
148.0, 128.9, 122.9, 108.8, 106.5 (arom), 142.2 (CHCH),
3
132.2, 130.3, 124.6, 121.6 (arom), 118.9 (CHCO), 101.7
(OCH O); ESI+-HRMS (m/z): 346.0078 [M + H]+ (calcd. for
(CHCH), 118.8 (CHCO), 101.4 (OCH O), 47.0 (CH ), 28.6
2
2
2
(CH), 20.1 (CH ); ESI+-HRMS (m/z): 248.1281 [M + H]+
C H BrNO , 346.0073).
3
16 13
3
(calcd. for C H NO , 248.1281.
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-isopropylacrylami-
de (6). Compound 1 (100 mg, 0.52 mmol) was used as re-
agent for the peptide coupling with isopropylamine (64 mL,
0.78 mmol) to afford 0.31 mmol (60%) of compound 6 as
14 18
3
3.3 General Procedure of Peptide Coupling
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm): 7.51 (1H,
To a solution of compound 1 (1 eq.) in dry DMF (5 mL)
at room temperature, TBTU (167.0 mg, 0.52 mmol) and
HOBt (35.16 mg, 0.26 mmol) were added while the solution
was stirred for 30 min. Then, the corresponding amine (1.5
eq.) and N,N-diisopropylethylamine (DIPEA) (0.52 mL,
3.12 mmol) were added sequentially and the reaction mix-
ture was stirred at room temperature for 2.5 h and concen-
trated under reduced pressure. The crude product was dis-
solved in EtOAc and washed sequentially with distilled wa-
ter in the presence of hexane (1/4 of the volume of EtOAc)
and then with saturated KHSO and NaHCO . The organic
3
d, J = 16.2 Hz, benzene-CH), 6.98 – 6.77 (3H, m, arom),
6.18 (1H, d, J = 16.2 Hz, CHCO), 5.98 (2H, s, OCH O), 5.45
2
(1H, s, NH), 4.21 (1H, m, CH), 1.22 (6H, d, CH ); 13C-NMR
3
(CDCl , 75 MHz; d, ppm): 165.3 (CONH)), 140.6 (CHCH),
3
149.1, 148.3, 129.5, 123.9, 108.7, 106.4 (arom), 119.2
(CHCO), 101.5 (OCH O), 41.7 (NHCH), 23.0 (CH );
2
3
ESI+-HRMS (m/z): 234.1136 [M + H]+ (calcd. for
C H NO , 234.1125).
13 16
3
(E)-3-(Benzo[d][1,3]dioxol-5-yl)- N-propylacrylamide
(7). Compound 1 (100 mg, 0.52 mmol) was used as reagent
for the peptide coupling with propylamine (64 mL,
0.78 mmol) to afford 0.47 mmol (90%) of compound 7 as
4
3
phase was dried over anhydrous Na SO and the filtrate was
2
4
evaporated to dryness in vacuo.
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-phenylacrylamide
(3). Compound 1 (100 mg, 0.52 mmol) was used as reagent
for the peptide coupling with aniline (89 mL, 0.78 mmol) to
afford 0.31 mmol (60%) of compound 3 as white solid;
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm): 7.53 (1H,
3
d, J = 15.7 Hz, benzene-CH), 6.98 – 6.77 (3H, m, arom),
6.23 (1H, d, J = 15.7 Hz, CHCO), 5.98 (2H, s, OCH O), 5.70
2
(1H, s, NH), 3.33 (2H, m, NHCH ), 1.60 (2H, m,
1H-NMR (CDCl , 300 MHz; d, ppm): 7.93 (1H, d, J = 16 Hz,
2
NHCH CH ), 0.95 (3H, m, CH ); 13C-NMR (CDCl ,
3
2
2
3
3
benzene-CH), 7.67 – 7.58 (5H, m, arom), 7.53 – 7.06 (3H,
m, arom), 6.65 (1H, d, J = 16 Hz, CHCO), 6.27 (2H, s,
OCH O); 13C-NMR (CDCl , 75 MHz; d, ppm): 165.0
75 MHz; d, ppm): 166.2 (CONH), 149.1, 148.3, 129.5,
123.9, 108.6, 106.4 (arom), 140.7 (CHCH), 119.0 (CHCO),
101.5 (OCH O), 41.6 (NHCH ), 23.1 (NHCH CH ) 11.6
2
3
2
2
2
2
(CONH), 149.5, 148.4, 138.2, 120.1, 108.7, 106.6 (arom),
142.3 (CHCH), 118.2 (CH CO) 101.1 (OCH O), 137.8,
(CH ); ESI+-HRMS (m/z): 233.1266 [M + H]+ (calcd. for
3
2
2
C H NO , 233.1285).
129.2, 124.4, 120.1 (arom); ESI+-HRMS (m/z):
13 16
3
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-cyclopentylacrylami-
de (8). Compound 1 (100 mg, 0.52 mmol) was used as re-
agent for the peptide coupling with cyclopentylamine
(80 mL, 0.78 mmol) to afford 0.48 mmol (92%) of compound
268.096 [M + H]+ (calcd. for C H NO , 248.1281).
13 14
3
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-(4-chlorophenyl)-
acrylamide (4). Compound 1 (100 mg, 0.52 mmol) was used
as reagent for the peptide coupling with 4-chloroaniline
(99.5 mg, 0.78 mmol) to afford 0.29 mmol (56%) of com-
1
8 as white solid; H-NMR (CDCl , 300 MHz; d, ppm): 7.52
3
pound 4 as white solid; 1H-NMR (CDCl , 300 MHz; d,
(1H, d, J = 15.3 Hz, benzene-CH), 6.98 – 6.77 (3H, m,
arom), 6.18 (1H, d, J = 15.3 Hz, CHCO), 5.98 (2H, s,
3
ppm): 7.66 (1H, d, J = 15.8 Hz, benzene-CH), 7.58 – 7.29
(4H, m, arom), 7.33 (1H, s, NH), 7.03 – 6.80 (3H, m, arom),
OCH O), 5.66 (1H, s, NH), 4.33 (1H, m, NHCH), 2.07 (2H,
2
m, CH CH CH ), 1.66 (4H, m, CH CH CH ), 1.44 (2H, m,
6.35 (1H, d, J = 15.8 Hz, CHCO), 6.01 (2H, s, OCH O);
2
2
2
2
2
2
2
CH CH CH ); 13C-NMR (CDCl , 75 MHz; d, ppm): 165.7
13C-NMR (CDCl , 75 MHz; d, ppm): 174.6 (CONH), 148.5,
148.0, 129.2, 121.3, 108.8, 106.5, 101 (arom), 142.8
2
2
2
3
3
(CONH), 149.1, 148.3, 129.5, 123.9, 108.7, 106.4 (arom),
140.6 (CHCH), 119.1 (CHCO), 101.5 (OCH O), 51.5
(CHCH), 136.8, 133.3, 129.0, 121.3 (arom), 117.8 (CHCO),
101.7 (OCH O); ESI+-HRMS (m/z): 302.0586 [M + H]+
2
(NHCH), 33.4 (CH CH CH CH ), 23.7 (CH CH CH CH );
2
2
2
2
2
2
2
2
2
ESI+-HRMS (m/z): 260.1293 [M + H]+ (calcd. for
(calcd. for C H ClNO , 302.0578).
16 13
3
C H NO , 260.1281).
(E)-3-(Benzo[d][1, 3]dioxol-5-yl)-N-(4-bromophenyl)-
acrylamide (5). Compound 1 (100 mg, 0.52 mmol) was used
as reagent for the peptide coupling with 4-bromoaniline
(134.2 mg, 0.78 mmol) to afford 0.23 mmol (45%) of com-
15 18
3
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-cyclohexylacrylami-
de (9). Compound 1 (100 mg, 0.52 mmol) was used as re-
agent for the peptide coupling with cyclohexylamine (90 mL,
0.78 mmol) to afford 0.36 mmol (70%) of compound 9 as
pound 5 as white solid; 1H-NMR (CDCl , 300 MHz; d,
ppm): 7.66 (1H, d, J = 15.7 Hz, benzene-CH), 7.53 – 7.43
3
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm): 7.51 (1H,
3

1000
Melissa Barrera Tomas et al.
d, J = 15.6 Hz, benzene-CH), 6.99 – 6.77 (3H, m, arom),
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-tert-butylacrylami-
de (13). Compound 1 (100 mg, 0.52 mmol) was used as re-
agent for the peptide coupling with t-butylamine (82 mL,
0.78 mmol, 1.5 equiv) to afford 0.27 mmol (52%) of com-
6.18 (1H, d, J = 15.6 Hz, CHCO), 5.98 (2H, s, OCH O), 5.46
2
(1H, s, NH), 3.90 (1H, m, NHCH), 2.01 – 1.15 (10H, m,
cyclohexyl); 13C-NMR (CDCl , 75 MHz; d, ppm): 165.1
(CONH), 149.1, 148.3, 129.5, 123.9, 108.7, 106.4 (arom),
3
1
pound 13 as white solid; H-NMR (CDCl , 300 MHz; d,
3
140.5 (CHCH), 119.3 (CHCO), 101.5 (OCH O), 48.4
ppm): 7.47 (1H, d, J = 15.8 Hz, benzene-CH), 6.97 – 6.76
(3H, m, arom), 6.15 (1H, d, J = 15.8 Hz, CHCO), 5.98 (2H,
s, OCH O), 5.44 (1H, s, NH), 1.42 pm (9H, s, CH );
2
(NHCH),
33.4
(CH CH CH CH CH ),
25.7
(CH CH CH CH CH );
2
2
2
2
2
(CH CH CH CH CH ),
25.0
2
2
2
2
2
2
2
2
2
2
2
3
ESI+-HRMS (m/z): 274.1438 [M + H]+ (calcd. for
13C-NMR (CDCl , 75 MHz; d, ppm): 165.5 (CONH), 149.0,
3
C H NO , 274.1438).
16 20
3
148.3, 129.5, 123.8, 108.7, 106.4 (arom), 140.1 (CHCH),
120.2 (CHCO), 101.5 (OCH O), 51.6 (C(CH ) ), 29.0
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-pentylacrylamide
(10). Compound 1 (100 mg, 0.52 mmol) was used as reagent
for the peptide coupling with n-amylamine (90 mL,
0.78 mmol) to afford 0.2 mmol (38%) of compound 10 as
2
3 3
(C(CH ) ); ESI+-HRMS (m/z): 248.1285 [M + H]+ (calcd.
3 3
for C H NO , 248.1281).
14 18
3
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-benzylacrylamide
(14). Compound 1 (100 mg, 0.52 mmol) was used as reagent
for the peptide coupling with benzylamine (90 mL,
0.78 mmol) to afford 0.18 mmol (38%) of compound 14 as
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm): 7.53 (1H,
d, J = 15.7 Hz, benzene-CH), 6.94 – 6.77 (3H, m, arom),
3
6.20 (1H, d, J = 15.7 Hz, CHCO), 5.98 (2H, s, OCH O), 5.63
2
(1H, s, NH), 3.37 (1H, m, NHCH ), 1.56 (2H, m,
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm): 7.58 (1H,
2
3
NHCH CH ), 1.34 (4H, m, NHCH CH CH CH ), 0.90 (3H,
2
2
2
2
2
2
d, J = 15.7 Hz, benzene-CH), 7.35 – 7.26 (5H, m, arom),
6.99 – 6.78 (3H, m, arom), 6.24 (1H, d, J = 15.7 Hz, CHCO),
m, CH ) 13C-NMR (CDCl , 75 MHz; d, ppm): 166.1
(CONH), 149.1, 148.3, 129.5, 123.9, 108.7, 106.5 (arom),
3
3
5.99 (2H, s, OCH O), 5.87 (1H, s, NH), 4.57 (2H, d,
2
140.7 (CHCH), 119.0 (CHCO), 101.6 (OCH O), 39.9
NHCH ); 13C-NMR (CDCl , 75 MHz; d, ppm): 166.0
2
2
3
(NHCH ), 29.5 (NHCH CH ), 29.3 (CH CH CH ), 22.5
2
2
2
2
2
3
(CONH), 149.2, 148.4, 129.3, 124.0, 108.7, 106.5 (arom),
138.4, 128.9, 128.1, 127.7 (arom), 141.3 (CHCH), 118.5
(CHCO), 101.6 (OCH O), 44.0 (NHCH ); ESI+-HRMS
(CH CH ),
14.1
(CH );
ESI+-HRMS
(m/z):
2
3
3
262.1426 [M + H]+ (calcd. for C H NO , 262.1438).
15 20
3
2
2
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-sec-butylacrylami-
de (11). Compound 1 (100 mg, 0.52 mmol) was used as re-
agent for the peptide coupling with sec-butylamine (80 mL,
0.78 mmol) to afford 0.17 mmol (32%) of compound 11 as
(m/z): 282.1141 [M + H]+ (calcd. for C H NO , 282.1125).
17 16
3
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-butylacrylamide
(15). Compound 1 (100 mg, 0.52 mmol) was used as reagent
for the peptide coupling with n-butylamine (80 mL,
0.78 mmol) to afford 0.18 mmol (35%) of compound 15 as
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm): 7.52 (1H,
d, J = 15.5 Hz, benzene-CH), 6.98 – 6.77 (3H, m, arom),
3
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm): 7.53 (1H,
3
6.20 (1H, d, J = 15.5 Hz, CHCO), 5.98 (2H, s, OCH O), 5.43
2
d, J = 15.8 Hz, benzene-CH), 6.99 – 6.77 (3H, m, arom),
(1H, s, NH), 4.05 (1H, m, NHCH), 1.51 (2H, m,
NHCHCH ), 1.18 (3H, m, NHCHCH ), 0.93 (3H, m,
6.21 (1H, d, J = 15.8 Hz, CHCO), 5.98 (2H, s, OCH O), 5.61
2
2
3
(1H, s, NH), 3.38 (2H, m, NHCH ), 1.52 (2H, m,
NHCHCH CH ); 13C-NMR (CDCl , 75 MHz; d, ppm):
165.5 (CONH), 149.0, 148.3, 129.5, 123.8, 108.6, 106.4
2
2
3
3
NHCH CH ), 1.35 (2H, m, CH CH ), 0.88 (3H, t, CH CH );
2
2
2
3
2
3
13C-NMR (CDCl , 75 MHz; d, ppm): 166.1 (CONH), 149.1,
(arom), 140.6 (CHCHCO), 119.3 (CHCO), 101.5 (OCH O),
3
2
148.3, 129.5, 123.9, 108.7, 106.4 (arom), 140.7 (CHCH),
119.0 (CHCO), 101.5 (OCH O), 39.6 (NHCH ), 31.9
45.9 (NHCH), 29.9 (NHCHCH ), 20.7 (NHCHCH ), 10.5
2
3
(CHCH CH ); ESI+-HRMS (m/z): 248.1273 [M + H]+;
2
2
2
3
(CH CH CH ), 20.3 (CH CH ), 13.9 (CH ); ESI+-HRMS
(calcd. for C H NO , 248.1281).
2
2
3
2
3
3
14 18
3
(m/z248.1269): [M + H]+ (calcd. for C H NO , 248.1281).
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-hexylacrylamide (12).
Compound 1 (100 mg, 0.52 mmol) was used as reagent for
the peptide coupling with hexylamine (110 mL, 0.78 mmol)
to afford 0.17 mmol (33%) of compound 12 as white solid;
14 18
3
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-phenethylacrylami-
de (16). Compound 1 (100 mg, 0.52 mmol) was used as re-
agent for the peptide coupling with phenetylamine (100 mL,
0.78 mmol) to afford 0.21 mmol (40%) of compound 16 as
1H-NMR (CDCl , 300 MHz; d, ppm): 7.53 (1H, d,
J = 15.3 Hz, benzene-CH), 6.99 – 6.77 (3H, m, arom), 6.20
3
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm): 7.53 (1H,
3
d, J = 15.7 Hz, benzene-CH), 7.36 – 7.21 (5H, m, arom),
6.97 – 6.77 (3H, m, arom), 6.14 (1H, d, J = 15.7 Hz, CHCO),
(1H, d, J = 15.3 Hz, CHCO), 5.98 (2H, s, OCH O), 5.62 (1H,
2
s, NH), 3.37 (2H, m, NHCH ), 1.52 (2H, m, NH CH CH ),
2
2
2
2
5.98 (2H, s, OCH O), 5.57 (1H, s, NH), 3.65 (2H, m,
1.31 (6H, m, NH CH CH CH CH CH ), 0.88 (3H, m,
2
2
2
2
2
2
2
NHCH ), 2.89 (2H, m, CH -benzene) 13C-NMR (CDCl ,
CH CH ); 13C-NMR (CDCl , 75 MHz; d, ppm): 166.1
(CONH), 149.1, 148.3, 129.5, 123.9, 108.7, 106.4 (arom),
2
2
3
2
3
3
75 MHz; d, ppm): 166.1 (CONH), 149.1, 148.4, 129.3,
124.0, 108.7, 106.5 (arom), 140.9 (CHCH), 139.0, 129.0,
140.7 (CHCH), 119.0 (CHCO), 101.6 (OCH O), 39.9
2
128.8, 126.7 (arom) 118.8 (CHCO), 101.6 (OCH O), 40.9
(NHCH ), 31.6 (NHCH CH ), 29.8 (NHCH CH CH ), 26.8
2
2
2
2
2
2
2
(NHCH ), 35.8 (CH - benzene); ESI+-HRMS (m/z):
(CH CH CH ), 22.7 (CH CH ), 14.2 (CH ); ESI+-HRMS
2
2
2
2
3
2
3
3
(m/z): 276.1602 [M + H]+ (calcd. for C H NO , 276.1594).
296.1294 [M + H]+ (calcd. for C H NO , 296.1281).
16 22
3
18 18 3

Synthesis of Analogs of Trans-Fagaramide
1001
(E)-N-allyl-3-(Benzo[d][1,3]dioxol-5-yl)acrylamide
(17). Compound 1 (100 mg, 0.52 mmol) was used as reagent
for the peptide coupling with allylamine (60 mL, 0.78 mmol)
to afford 0.39 mmol (75%) of compound 17 as white solid;
3.4. General Procedure of Fischer Esterification
(E)-Methyl 3-(benzo[d][1,3]dioxol-5-yl)acrylate (21).
To a solution of compound 1 (2.0 g, 10.4 mmol) in dry meth-
anol (25 mL) was added H SO dropwise and stirred for
1H-NMR (CDCl , 300 MHz; d, ppm): 7.55 (1H, d,
2
4(cc)
3
some minutes. The mixture was heated with reflux for 12 h,
cooled to room temperature, and concentrated under pres-
sure. The crude material was diluted with EtOAc, and
J = 15.5 Hz, benzene-CH), 6.99 – 6.77 (3H, m, arom), 6.25
(1H, d, J = 15.5 Hz, CHCO), 5.98 (2H, s, OCH O), 5.90 (1H,
2
m, CHCH ), 5.85 (1H, s, NH), 5.20 (2H, m, CHCH ), 4.01
2
2
(2H, m, NHCH ) 13C-NMR (CDCl , 75 MHz; d, ppm): 166.0
washed with aqueous NaHCO and brine. The organic phase
3
2
3
was dried over anhydrous Na SO , concentrated, and
(CONH), 149.2, 148.4, 129.3, 124.0, 108.6, 106.5 (arom),
2
4
recrystallized from saturated hot Hex/EtOAc (1/4) to afford
141.1 (CHCH), 134.3 (CHCH ), 118.6 (CHCO), 116.7
2
(CHCH ), 101.6 (OCH O), 42.3 (NHCH ); ESI+-HRMS
7.37 mmol (70%) of compound 21 as white crystals;
1H-NMR (CDCl , 300 MHz; d, ppm): 7.58 (d, 1H,
2
2
2
(m/z): 232.0974 [M + H]+ (calcd. for C H NO , 232.0968).
3
13 14
3
J = 16.3 Hz, benzene-CH), 7.01 (s, 1H, Ha), 6.99 (d, 1H,
J = 8.0 Hz, Hb), 6.80 (d, 1H, J = 8.0 Hz, Hc), 6.26 (d, 1H,
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-(4-methylbenzyl)-
acrylamide (18). Compound 1 (100 mg, 0.52 mmol) was
used as reagent for the peptide coupling with methylben-
zylamine (100 mL, 0.78 mmol) to afford 0.37 mmol (72%) of
J = 16.3 Hz, CHCO), 5.99 (s, 2H, OCH O), 3.78 (s, 3H,
2
CH ); 13C-NMR (CDCl , 75 MHz; d, ppm): 167.7 (CONH),
3
3
compound 18 as white solid; 1H-NMR (CDCl , 300 MHz; d,
149.7, 148.5, 128.9, 124.5, 108.7, 106.6 (arom), 144.7
(CHCH), 115.8 (CHCO), 101.4 (OCH O), 51.7 (CH );
3
ppm): 7.58 (1H, d, J = 15.8 Hz, benzene-CH), 7.23 – 7.14
2
3
ESI+-HRMS (m/z): 208.0675 [M + H]+ (calcd. for C H O ,
(4H, m, arom), 6.99 – 6.78 (3H, m, arom), 6.21 (1H, d,
11 11
4
208.0686).
J = 15.8 Hz, CHCO), 5.99 (2H, s, OCH O), 5.79 (H, s, NH),
2
4.52 (2H, d, NHCH ), 2.34 (3H, s, CH ); 13C-NMR (CDCl ,
2
3
3
75 MHz; d, ppm): 165.9 (CONH), 149.2, 148.4, 129.6,
124.0, 108.7, 106.5 (arom), 141.2 (CHCH), 137.5, 135.4,
3.5. General Procedure of Platinum-Mediated Heck
Reaction
129.3, 128.1 (arom), 118.6 (CHCO), 101.6 (OCH O), 43.8
ESI+-HRMS
2
(NHCH ),
21.2
(CH );
(m/z):
To a solution of compound 2 or 21 (100 mg, 1 eq.) dis-
2
3
296.1281 [M + H]+ (calcd. for C H NO , 296.1281).
solved in anhydrous DMF (3 mL) under nitrogen atmosphere
18 18
3
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-(naphthalen-2-yl)-
acrylamide (19). Compound 1 (100 mg, 0.52 mmol) was
used as reagent for the peptide coupling with 2-naphtylamine
(112 mg, 0.78 mmol) to afford 0.18 mmol (35%) of com-
were sequentially added Pd(AcO) (catalytic amount), the
2
appropriate aryl iodide (0.97 mmol, 2 eq.), tetrabutylammo-
nium bromide (TBABr) (161.2 mg, 1 eq.), and NaHCO
3
(122.2 mg, 3 eq.). The reaction mixture was stirred at room
temperature for 15 min under nitrogen atmosphere and then
heated at 105 °C overnight (monitored by TLC). After cool-
ing to room temperature, the reaction mixture was concen-
trated under reduced pressure and the crude product was dis-
solved in EtOAc and washed with distilled water in the pres-
ence of hexane (1/4 of the volume of EtOAc). The organic
1
pound 19 as white solid; H-NMR (CDCl , 300 MHz; d,
3
ppm): 8.32 (1H, s, NH), 7.79 – 7.39 (7H, m, arom), 7.70 (1H,
d, J = 15.3 Hz, benzene-CH), 7.01 – 6.76 (3H, m, arom),
6.45 (1H, d, J = 15.3 Hz, CHCO), 5.99 (2H, s, OCH O);
2
13C-NMR (CDCl , 75 MHz; d, ppm): 164.5 (CONH), 149.5,
3
148.8, 129.1, 124.4, 108.7, 106.6 (arom), 142.4 (CHCH),
layer was washed sequentially with saturated KHSO
135.7, 134.0, 130.8, 128.9, 127.9, 127.7, 126.6, 125.2, 120.3,
4
(10 mL), NaHCO (10 mL), and brine (10 mL), dried over
117.3 (napthalene), 119.0 (CHCO), 101.6 (OCH O);
3
2
ESI+-HRMS (m/z): 318.1114 [M + H]+ (calcd. for
Na SO , filtered, and concentrated to dryness under reduced
2
4
C H NO , 318.1125).
pressure.
Benzo[d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-N-isobu-
20 17
3
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-(prop-2-yn-1-yl)ac-
rylamide (20). Compound 1 (100 mg, 0.52 mmol) was used
as reagent for the peptide coupling with propargylamine
(50 mg, 0.78 mmol) to afford 0.41 mmol (80%) of compound
tylacrylamide (22). Compound 2 was used as reagent with
4-fluoro iodobenzene. The crude material was purified by
column chromatography on silica gel using the eluting mix-
21 as white solid; 1H-NMR (CDCl , 300 MHz; d, ppm): 7.57
ture hex/Et O/EtOAc (10:0:0 to 6:1:3), to afford 0.31 mmol
2
3
(76%) of a mixture of stereoisomers cis/trans (3:1) of com-
(1H, d, J = 15.2 Hz, benzene-CH), 6.99 – 6.78 (3H, m,
arom), 6.23 (1H, d, J = 15.2 Hz, CHCO), 5.99 (2H, s,
OCH O), 5.85 (1H, s, NH), 4.18 (2H, m, NHCH ), 2.26 (1H,
1
pound 22 as a white solid; H-NMR (CDCl , 300 MHz; d,
3
ppm): 7.28 – 6.88 (7H, m, arom), 6.54 (1H, s, CCH), 6.08
(2H, s, OCH O), 4.22 (1H, s, NH), 3.02 (2H, m, CH ), 2.08
2
2
m, CH ); 13C-NMR (CDCl , 75 MHz; d, ppm): 165.8
2
2
3
3
(1H, m, CH), 0.87 (6H, d, CH (2x)); 13C-NMR (CDCl ,
(CONH), 149.4, 148.4, 129.1, 124.2, 108.7, 106.5 (arom),
3
3
75 MHz; d, ppm): 166.7 (COO), 162.1, 148.5, 148.1, 138.7,
131.3, 129.6, 121.9, 121.1, 115.8, (arom), 152.2 (CCHCO)
108.3 (CCHCO), 101.2 (OCH O), 48,1 (CH ), 29.2 (CH)
141.8 (CHCH), 117.8 (CHCO), 101.6 (OCH O), 79.7
2
(CCH), 71.9 (CCH) 29.6 (NHCH ); ESI+-HRMS (m/z):
2
230.0808 [M + H]+ (calcd. for C H NO , 230.0812).
13 12
3
2
2

1002
Melissa Barrera Tomas et al.
and 20.1 (CH ); ESI+-HRMS (m/z): 299.2999 [M + H]+
(2H, s, OCH O); 13C-NMR (CDCl , 75 MHz; d, ppm): 160.9
3
2
3
(calcd. for C H O , 299.3001).
(COO), 154.3, 149.1, 148.2, 132.0, 129.0, 127.1, 124.3,
122.8, 119.1, 117.5, 109.0, 108.9 (arom), 155.3 (CCHCO)
115.0 (CCHCO), 101.8 (OCH O);
ESI+-HRMS
(m/z267.0658): [M + H]+ (calcd. for C H O , 267.0652).
17 15
5
Benzo[d][1,3]dioxol-5-yl)-N-isobutyl-3-(4-methoxyphe-
nyl)acrylamide (23). Compound 2 was used as reagent with
4-iodoanisol. The crude material was purified by column
chromatography on silica gel using the eluting mixture
hex/EtOAc (gradient: 5:1 to 1:1), to afford 0.24 mmol (68%)
of a mixture of stereoisomers cis/trans (1:1) of compound 23
2
16 11
4
Methyl 3-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphe-
nyl)acrylate (27). Compound 21 was used as reagent with
4-iodoanisol. The crude material was purified by column
chromatography on silica gel using the eluting mixture hex-
ane/Tol/EtOAc (50:0:0 to 40:5:5), to afford 142 mg (95%) of
a mixture of stereoisomers cis/trans (3:1) 27 as white solid;
as a white solid; 1H-NMR (CDCl , 300 MHz; d, ppm):
3
7.19 – 6.73 (7H, m, arom), 6.25 (1H, s, CCH), 5.99 (2H, s,
OCH O), 5.35 (1H, s, NH), 2.97 (2H, m, CH ), 2.04 (1H, m,
2
2
1H-NMR (CDCl , 300 MHz; d, ppm): 7.16 – 6.69 (7H, m,
arom), 6.24 (1H, s, CCH), 6.00 (2H, s, OCH O), 3.86 (3H, s,
CH), 0.85 (6H, d, CH (2x)); 13C-NMR (CDCl , 75 MHz; d,
3
3
3
ppm): 164.2 (COO), 160.1, 148.6, 148.0, 135.2, 131.3,
129.6, 121.1, 115.8, 108.5 (arom), 153.1 (CCHCO) 108.4
(CCHCO), 101.2 (OCH O), 55,6 (OCH ), 47.9 (CH ), 28.9
2
OCH ), 3,63 (3H, s, OCH ); 13C-NMR (CDCl , 75 MHz; d,
3
3
3
ppm): 163.9 (COO), 158.3, 148.2, 148.1, 133.6, 129.6,
124.7, 121.9, 120.1, 116.5, 109.0, (arom), 153.1 (CCHCO)
111.3 (CCHCO), 101.4 (OCH O), 55, 2 (OCH ), 52,9
2
3
2
(CH) and 20.1 (CH ); ESI+-HRMS (m/z): 354.17 [M + H]+
3
(calcd. for C H O , 354.17)
2
3
21 24
4
(OCH ); ESI+-HRMS (m/z): 313.1083 [M + H]+ (calcd. for
Benzo[d][1,3]dioxol-5-yl)-3-(4-hydroxyphenyl)-N-iso-
butylacrylamide (24). Compound 2 was used as reagent
with 4-iodophenol. The crude material was purified by col-
umn chromatography on silica gel using the eluting mixture
hex/Tol/EtOAc (5:1:1 to 5:1:4), to afford 0.28 mmol (70%)
of a mixture of stereoisomers cis/trans of compound 24 as a
3
C H O , 313.1071).
18 17
4
Methyl 3-(benzo[d][1,3]dioxol-5-yl)-3-(4-hydroxyphe-
nyl)acrylate (28). Compound 21 was used as reagent with
4-iodophenol. The crude material was purified by column
chromatography on silica gel using the eluting mixture hex-
ane/EtOAc (50:0 to 1:1), to afford 0.36 mmol (75%) of a
mixture of stereoisomers cis/trans (2:1) of compound 28 as
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm):
3
7.26 – 6.71 (7H, m, arom), 6.23 (1H, s, CCH), 5.97 (2H, s,
OCH O), 5.48 (1H, s, NH), 2.98 (2H, m, CH ), 1.58 (1H, m,
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm):
3
2
2
7.22 – 6,68 (7H, m, arom), 6.22 (1H, s, CCH), 5.98 (2H, s,
OCH O), 5.17 (1H, s, OH), 3.64 (3H, s, OCH ); 13C-NMR
CH), 0.78 (6H, d, CH (2x); 13C-NMR (CDCl , 75 MHz; d,
3
3
2
3
ppm): 166.7 (COO), 157.0, 148.2, 134.1, 132.2, 131.3,
124.6, 121.1, 115.8, 109.8 (arom), 153.1 (CCHCO) 108.4
(CCHCO), 101.2 (OCH O), 55.6 (OCH ), 47.9 (CH ), 28.9
(CDCl , 75 MHz; d, ppm): 166.9 (COO), 157.2, 147.8,
133.7, 132.7, 130.3, 123.3, 115.4, 114.7, 100.1 (arom), 147.8
3
2
3
2
(CCHCO) 108.1 (CCHCO), 101.3 (OCH O), 54.4 (OCH );
ESI+-HRMS
(m/z):
2
3
(CH)
and
20.1
(CH );
ESI+-HRMS (m/z): 299.2901 [M + H]+ (calcd. for C H O ,
3
340.1541 [M + H]+ (calcd. for C H NO , 340.1543)
17 17
4
20 22
4
299.2903).
4-Aminophenyl)-3-(benzo[d][1,3]dioxol-5-yl)-N-isobu-
tylacrylamide (25). Compound 2 was used as reagent with
4-iodoaniline. The crude material was purified by column
chromatography on silica gel using the eluting mixture
hex/EtOAc (50:0 to 1:1), to afford 0.35 mmol (88%) of a
mixture of stereoisomers cis/trans of compound 25 as a
Methyl 3-(4-aminophenyl)-3-(benzo[d][1,3]dioxol-5-
yl)acrylate (29). Compound 21 was used as reagent with
4-iodoaniline. The crude material was purified by column
chromatography on silica gel using the eluting mixture hex-
ane/EtOAc (50:0 to 35:15), to afford 0.36 mmol (75%) of a
mixture of stereoisomers cis/trans (2:1) of compound 29 as
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm):
white solid; 1H-NMR (CDCl , 300 MHz; d, ppm):
3
3
7.08 – 6,59 (7H, m, arom), 6.20 (1H, s, CCH), 5.97 (2H, s,
OCH O), 5.36 (1H, s, NH), 2.96 (2H, m, CH ), 1.52 (1H, m,
7.26 – 6.58 (7H, m, arom), 6.21 (1H, s, CCH), 5.99 (2H, s,
OCH O), 3.86 (2H, s, NH), 3.63 (3H, s, OCH ); 13C-NMR
2
2
2
3
CH), 0.76 (6H, d, CH (2x)); 13C-NMR (CDCl , 75 MHz; d,
(CDCl , 75 MHz; d, ppm): 166.5 (COO), 148.7, 147.8,
133.5, 132.7, 128.9, 123.3, 121.3, 114.3, (arom), 152.8
3
3
3
ppm): 166.7 (COO), 149.1, 148.2, 134.2, 132.2, 131.3,
124.6, 121.1, 115.8, 109.8 (arom), 153.1 (CCHCO) 108.4
(CCHCO), 101.2 (OCH O), 55.6 (OCH ), 47.9 (CH ), 28.9
(CCHCO) 108.3 (CCHCO), 101.3 (OCH O), 54.4 (OCH );
2
3
ESI+-HRMS (m/z): 298.1086 [M + H]+ (calcd. for
2
3
2
(CH)
and
20.1
(CH );
ESI+-HRMS
(m/z):
C H NO , 298.1074).
3
17 16
4
170.0887 [M + H]+ (calcd. for C H N O , 170.0888)
Methyl 3-(benzo[d][1,3]dioxol-5-yl)-3-(4-fluorophe-
nyl)acrylate (30). Compound 21 was used as reagent with
4-fluoro iodobenzene. The crude material was purified by
column chromatography on silica gel using the eluting mix-
20 24
2
3
Benzo[d][1,3]dioxol-5-yl)-2H-chromen-2-one
(26).
Compound 21 was used as reagent with 2-iodophenol. The
crude material was purified by column chromatography on
silica gel using the eluting mixture hexane /Et O/EtOAc
ture hexane/Et O/EtOAc (40:0:0 to 40:5:2), to afford
2
2
(100:0:0 to 70:10:5) to afford 0.19 mmol (35%) of com-
0.46 mmol (96%) of a mixture of stereoisomers cis/trans of
compound 30 as white solid; 1H-NMR (CDCl , 300 MHz; d,
ppm): 7.26 – 6.75 (7H, m, arom), 6.26 (1H, s, CCH), 5.99
1
pound 26 as white solid; H-NMR (CDCl , 300 MHz; d,
3
3
ppm): 7.57 – 7.21 (7H, m, arom), 6.34 (1H, s, CCH), 6.03

Synthesis of Analogs of Trans-Fagaramide
1003
(2H, s, OCH O), 3.61 (3H, s, OCH ); 13C-NMR (CDCl ,
esterification, palladium-mediated Heck couplings, and pep-
tide coupling. Compounds 3 – 20 (18 compounds) were syn-
thesized by peptide coupling at an average yield of 54%.
Compound 21 was obtained by Fischer esterification with a
yield of 70%. The synthesis of compounds 22 – 30 was af-
forded by palladium-mediated Heck coupling: compound 26
was obtained with 66% yield as a white solid, and the rest of
compounds were obtained as mixtures of stereoisomers with
an average yield of 80%. The synthesized compounds were
elucidated using 1D spectroscopy (1H-NMR and 13C-NMR),
2D spectroscopy (COSY and HMQC), and mass spectrome-
try. Furthermore, all compounds were screened on cancer
cells and in toxicity experiments against breast
adenocarcinoma MCF7, hepatocellular carcinoma HepG2,
glioblastoma U-87 as well as normal cells H9c2. Compounds
2
3
3
75 MHz; d, ppm): 166.5 (COO), 162.1, 148.0, 148.1, 139.0,
131.5, 129.8 137.3, 129.8, 121.9, 115.8 (arom), 130.2
(CCHCO) 108.6 (CCHCO), 101.2 (OCH O), 52.3 (OCH );
2
3
ESI+-HRMS (m/z): 301.0878 [M + H]+ (calcd. for
C H FO , 301.0871).
17 14
4
3.6. Cytotoxic Activity Evaluation on MCF7, U-87, HepG2
and H9c2 Cells
3.6.1. Cell Culture. The human primary glioblastoma
cell line U-87 was grown in Eagle’s minimal essential me-
dium (EMEM) supplemented with 10% (v/v) fetal bovine se-
rum (FBS) and 1 mM sodium pyruvate. The human breast
cancer cell line MCF7 was grown in EMEM supplemented
with 10% (v/v) FBS, 100 units/mL penicillin/streptomycin,
1 mM sodium pyruvate, 0.01 mg/mL insulin and 10 nM
estradiol. The human perpetual cell line HepG2 was grown
in EMEM supplemented with 10% (v/v) FBS, 100 units/mL
penicillin/streptomycin and 1 mM sodium pyruvate. The rat
cardiomyoblast cell line H9c2 was grown in Dulbecco’s
Modified Eagle’s Medium (DMEM) high glucose supple-
mented with 10% (v/v) FBS and 100 units/mL penicil-
lin/streptomycin. All cell lines were cultured at 37°C with
4 and 5 showed IC values above 50 ìM against H9c2 and
50
MCF7. Likewise, compounds 25 and 26 exhibited inhibition
activity at IC values above 50 ìM against MCF7 and H9c2,
50
respectively. The rest of compounds showed no activity
against these cell lines. The lack of activity in most com-
pounds makes proper structure – activity assessment not pos-
sible.
5% CO and split when reached 70 – 80% confluences.
AUTHOR CONTRIBUTIONS
2
3.6.2. Cell Viability Assay. U87, MCF7, HepG2 and
H9c2 cells were seeded in black wall clear bottom 96-well
plates (treated with tissue culture) at a density of 5,000
cells/well (100 mL/cell). After 24 h incubation at 37°C in a
M. Barrera did all the synthesis as a part of her Master
Thesis and wrote the paper. T. C. Shiao contributed to the in-
terpretation of spectra. P. T. Nguyen and S. Bourgault per-
formed evaluation of the cytotoxic activity. R. Roy designed
thematics and supervised all experiments presented in the pa-
per.
5% CO incubator, the cell medium was removed and re-
2
placed with serum-free medium in the absence or presence of
test compounds. The compounds were dissolved in DMSO
and the final concentration of DMSO in the culture media
was maintained below 1% (v/v). Cells were incubated an-
CONFLICT OF INTEREST
other 48h at 37°C in 5% CO and the cell viability was mea-
2
sured using the resazurin reduction assay [21]. The cell via-
bility was expressed as percentage (%) of that in the control
calculated using the ratio of the fluorescence intensity of
treated sample to the vehicle control. The fluorescence was
measured on an automated 96-well Fluoroskan Ascent FL
plate reader (Labsystems) using an excitation wavelength of
530 nm and an emission wavelength of 590 nm. the
cytotoxicity was expressed as the drug concentration inhibit-
The authors declare no conflict of interest.
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