Synthesis of analogs of amathamide A and their preliminary antimicrobial activity

Article abstract of DOI:10.3390/10010295

Syntheses of three non-brominated analogs of amathamide A (1), a natural alkaloid isolated from the Tasmanian marine bryozoan Amathia wilsoni, are described. Antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudo

Full text of DOI:10.3390/10010295

Molecules 2005, 10, 295-301
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis of Analogs of Amathamide A and Their Preliminary
Antimicrobial Activity
Moisés Ramírez-Osuna 1, Daniel Chávez 1, Lourdes Hernández 2, Elias Molins 3, Ratnasamy
Somanathan 1 and Gerardo Aguirre 1,*
1
Centro de Graduados e Investigación, Instituto Tecnológico de Tijuana, Apartado Postal 1166,
22000, Tijuana, B.C., México
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México, D.F, México
Instituto de Ciencia de Materiales de Barcelona (CSIC), Campus de la UAB, 08193 Cerdanyola
2
3
(Barcelona), Spain
* Author to whom correspondence should be addressed. Tel.: (+52) 664 623-3772; Fax: (+52) 664
623-4043; E-mail: gaguirre@tectijuana.mx
Received: 20 August 2004; in revised form 6 January 2005 / Accepted: 7 January 2005 / Published:
31 January 2005
Abstract: Syntheses of three non-brominated analogs of amathamide A (1), a natural
alkaloid isolated from the Tasmanian marine bryozoan Amathia wilsoni, are described.
Antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli,
Pseudomona aeruginosa, and Candida albicans was tested. Test results for amathamide
A (1) showed a weak activity against C. albicans and E. coli. The three non-natural
analogs 2-4 proved to be inactive compounds.
Keywords: Organic synthesis, Amathamide A, Antimicrobial activity.
Introduction
Amathamide A (1) is a brominated alkaloid isolated from the Tasmanian bryozoan Amathia
wilsoni Kirkpatrick [1,2]. Biological activity of this type of enamide alkaloids has not been well
studied, and only nematocidal, antifungal, and antibacterial activitity has been described for
amathamides A (1), B, G, H, and I, isolated from A. wilsoni and A. convolute species [3]. Our previous
work relating to the synthesis of two natural amathamides (A and B) [4] prompted us to investigate the

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296
synthesis and biological evaluation of analogs of this type of alkaloid. Therefore, we selected
amathamide A (1) as a lead compound and we carried out the synthesis of three non-brominated
analogs 2-4, maintaining the N-methyl or substituting it by a single hydrogen or t-butylcarbamate
(Figure 1). Antimicrobial activity was then determined against two Gram-positive bacteria (B. subtilis
and S. aureus), two Gram-negative bacteria (E. coli and P. aeruginosa) and a yeast (C. albicans).
Figure 1.
R
H
N
N
R1
O
R
OMe
Compound
R
Br
H
H
H
R1
CH3
CH3
Boc
H
1
2
3
4
Results and Discussion
Amathamide A (1) was synthesized as previously reported [4]. For the three non-natural analogs,
the synthetic route was modified as shown in Scheme 1. m-Anisaldehyde was condensed with
nitromethane in 81 % yield in the presence of ammonium acetate and then thiophenol was
incorporated via Michael addition with a catalytic amount of N-isopropylcyclohexylamine to obtain
the product 6 in 90 % yield. Nitro group reduction was achieved in 45% yield in the conventional
manner [5] using Zn in AcOH/HCl. Acylation of the amine with (S)-N-t-butoxycarbonylproline in
presence of DCC/HOBt gave 8 in 20 % yield. Removal of the Boc (t-butoxycarbonyl) protective group
from compound 8 affords 9 in very good yield and acylation of the latter with formaldehyde and
reduction with NaBH3CN afford 10 (80 % yield). Elimination of thiophenol of 10 via oxidation with
NaIO4 to the sulfoxide and refluxing in toluene/K2CO3, gave the non-brominated analogue 2. On the
other hand, elimination of thiophenol from 8 led to analogue 3, and removal of Boc afforded 4. In this
manner, we obtained the three non-natural analogs of amathamide A (1).
The preliminary antimicrobial activity of amathamide A (1) and the three non-natural analogs 2-4
was determined against the Gram-positive bacteria S. aureus and B. subtuilis, the Gram-negative
bacteria E. coli and P. aeruginosa and the yeast C. albicans by using the agar dilution-streak assay
(Mitscher method) [6]. Only compound 1 was active in the bioassay at the final concentration of 200
µg/mL. Minimal inhibitory concentrations (MIC) for 1 were determined [7,8]. The MICs of 1 for C.
albicans and E. coli were weak, and less active (MICs greater than 128 µg/mL) for P. aeruginosa, S.
aureus, and B. subtilis (Table 1).
Scheme 1.

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O
NO2
S
S
H
a
c
b
NO2
NH2
OCH3
OCH3
5
6
7
OCH3
OCH3
d
S
S
S
g
f
NH
NH
NH
N
N
N
O
O
O
Boc
CH3
H
10
9
8
OCH3
OCH3
OCH3
e
e
H
N
H
N
H
N
N
N
H
N
f
O
O
CH3
O
Boc
OCH3
OCH3
OCH3
2
4
3
(a) CH3NO2, AcOH, AcONH4; (b) PhSH; (c) Zn, HCl, AcOH; (d) DCC/HOBt,
DMAP, (S)-N-t-butoxycarbonylproline; (e) NaIO4, MeOH; Toluene, K2CO3, reflux.
(f) HF, reflux (g) H2CO, NaBH3CN.
Table 1. Minimal Inhibitory Concentration (MIC) of 1, Nystatin and Streptomycin
toward Pathogenic Bacteria and yeast.
Pathogen
C. albicans
E. coli
P. aeruginosa
S. aureus
B. subtilis
1a
142.8
71.4
71.4
142.8
142.8
Nystatina
Streptomycina
10.0
-
-
-
-
-
10.0
0.5
1.6
3.1
aResults are means of two determinations and are expressed as µg/mL

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Conclusions
The non-brominated analogues of amathamides obtained following a simpler synthetic route
proved to be inactive compounds. Thus, we conclude that the presence of bromine on the aryl ring
contributes to the antimicrobial activity of amathamide A (1). According to Narkowicz et al. [3], the E
orientation of the double bond of amathamide A (1) conferred the highest biological activity compared
with the less constrained saturated version, as in amathamide I, or the Z orientation of the double bond
as in amathamide B. This information leaves only the possibility of altering the acyl group and/or
substitution on the amide nitrogen in an effort to obtain analogues with enhanced antimicrobial activity
for further biological testing. The effect of other aryl substituents could also be explored.
Acknowledgments
We gratefully acknowledge support for this project by CONACYT grant E130,1517 (2001).
Experimental
General
Melting points were determined on a Fisher-Johns melting point apparatus and are uncorrected. (IR)
1
spectra were taken on a Perkin Elmer FT-IR 1600 spectrometer. H- (200 MHz) and 13C-NMR (75
MHz) spectra were recorded on a Bruker Avance DPX-300 MHz spectrometer. Spectra were run in
CDCl3 with tetramethylsilane (TMS) used as internal standard. The EIMS data was obtained on a
Finnigan MAT-90 instrument and all experiments were performed in the electron-impact mode (EI) at
70 eV using a direct insertion probe.
(E)-3-Methoxy-β-nitrostyrene (5): To a solution of m-anisaldehyde (73.4 mmol, 10.0 g) in glacial
AcOH (120 mL) was added AcONH4 (2.71 g) and nitromethane (13.1 mL). The solution was heated
under reflux for 1 h. The mixture was cooled and the precipitate was washed with water and
recrystalized from AcOH/H2O to give a yellowish solid (10.6 g, 81%); mp 88-89º C; IR (KBr): 3108,
1
1636, 1577, 1511 cm-1; H-NMR: δ 7.97 (d, J = 14.0 Hz), 7.56 (d, J = 14.0 Hz), 7.41-7.02 (m, 4H),
3.85 (s, 3H, OCH3).
2-(3-Methoxyphenyl)-2-(thiophenyl)-1-nitroethane (6): Compound 5 (22.3 mmol, 4.0 g) was dissolved
in CH2Cl2 (100 mL) and was added thiophenol (22.9 mmol, 2.6 mL) and 0.5 mL of N-isopropylcyclo-
hexylamine. The resulting solution was stirred for 1 h. at rt. The solution was concentrated and
subjected to flash chromatography using hexane/CH2Cl2 (80:20). Removal of the solvent gave a
1
brownish oil (5.8 g, 90%). IR (film): 3057, 2957, 1599, 1554, 1262 cm-1; H-NMR: δ 7.45-7.22 (m,
6H), 6.85-6.79 (m, 3H), 4.83-4.60 (m, 3H), 3.75 (s, 3H, OCH3).
2-(3-Methoxyphenyl)-2-(thiophenyl)-1-aminoethane (7): Compound 6 (17.3 mmol, 5.0 g) was
dissolved in AcOH (46 mL) and Zn powder (172.4 mmol, 11.3 g) was added. Then conc. HCl (37 mL)

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was added dropwise and the solution was stirred overnight at rt. The solution was neutralized with 2 N
NH4OH. The residue was extracted into ethyl acetate (2 × 100 mL) and the solvent was removed under
reduced pressure to give a yellowish green oil (2.0 g, 45%); IR (film): 3372, 3057, 2936, 1587, 1483,
1261, 1044, 694 cm-1; 1H-NMR: δ 7.4-7.18 (m, 6H), 6.96-6.70 (m, 3H), 4.12 (t, J = 8 Hz), 3.77 (s, 3H,
OCH3), 3.20-3.00 (m, 2H), 2.00-1.40 (bs, 2H, NH2).
2(S)-N-[2(3-Methoxyphenyl)-2-(thiophenyl)ethanyl]-1-t-butoxycarbonylproline (8): To a solution of
(S)-N-t-butoxycarbonylproline (19.5 mmol, 4.1 g) in dry THF (100 mL) was added dicyclohexyl-
carbodiimide (19.5 mmol, 4.0 g), 1-hydroxybenzotriazole (19.5 mmol, 2.6 g) and 4-N,N-dimethyl-
aminopyridine in catalytic amounts. A solution of 7 (19.5 mmol, 5.05 g) in dry THF (20 mL) was
added and the suspension was stirred for 24 h, filtered and the solvent was removed in vacuo. The
residue was purified by flash chromatography using CH2Cl2/MeOH (95:5). Removal of the solvent
1
gave a brownish oil (1.8 g, 20%); IR (film): 3313, 3056, 2974, 1697 cm-1; H-NMR: δ 7.50-7.20 (m,
6H), 6.90-6.76 (m, 3H), 4.50-4.30 (m, 1H), 4.30-4.10 (m, 1H), 3.75 (s, 3H, OCH3), 3.90-3.50 (m, 2H),
13
3.50-3.20 (m, 2H), 2.40-1.60 (m, 4H), 1.39 (s, 9H, boc); C-NMR: δ 159.6, 140.7, 133.7, 132.2,
129.5, 128.8, 127.3, 127.2, 120.0, 113.3, 113.2, 113.1, 80.3, 55.1, 52.3, 46.9, 43.8, 43.6, 28.3: IEMS,
m/z: M+ 456 (nd), [M-PhSH]+ 346 (23), 198 (3), 149 (62), 132 (2), 70 (100), 57 (33).
2(S)-N-[2(3-Methoxyphenyl)-2-(thiophenyl)ethanyl]-2-pyrrolinecarboxamide (9): Compound 8 (4.38
mmol, 2.0 g) was dissolved in acetonitrile (30 mL) and HF (50%, 0.7 mL) was added and the mixture
heated overnight under reflux with magnetic stirring. The mixture was cooled and the solvent was
removed in vacuo. The residue was treated with K2CO3 (50%, 20 mL) and extracted with
dichloromethane (2 × 20 mL). The organic layer was dried over anhydrous sodium sulfate and
concentrated to give 9 (1.51 g, 97%). Compound 9 was used in the next step without further
purification. IR (film): 3322, 3054, 2960, 1665 cm-1.
2(S)-N-[2(3-Methoxyphenyl)-2-(thiophenyl)ethanyl]-1-methyl-2-pyrrolinecarboxamide (10): 9 (4.24
mmol, 1.5 g) was dissolved in acetonitrile (14 mL) and formaldehyde (37%, 2.3 mL) and sodium
cyanoborohydride (9.19 mmol, 0.6 g) were added and the solution was stirred for 2 h. at rt. The
solution was neutralized with AcOH and stirring was continued for 2 h. The solvent was removed
under vacuum and the residue was washed with water and extracted with CH2Cl2 (2 × 15 mL). The
organic layer was dried over anhydrous sodium sulfate and concentrated to give a brownish oil of the
title compound as a mixture of diastereomers (1.2 g, 80%). IR (film): 3328, 3056, 2942, 1663, 1261
1
cm-1; H-NMR: δ 7.56 (bs, 2H, N-H), 7.38-7.160 (m, 12H), 6.88-6.75 (m, 6H), 4.42-4.32 (m, 2H),
3.87-3.55 (m, 4H), 3.76 (s, 6H, OCH3), 3.00-2.93 (m, 2H), 2.80 (dd, J =10.0, 4.0 Hz, 2H), 2.32-2.00
(m, 4H), 2.22 (s, 3H, NCH3), 2.15 (s, 3H, NCH3), 1.50-1.80 (m, 6H); 13C-NMR: δ 174.9, 174.8, 159.8,
141.0, 141.0, 134.1, 132.1, 132.1, 129.9, 129.6, 129.1, 128.9, 127.5, 127.4, 127.3, 120.4, 120.3, 113.4,
113.4, 113.4, 113.3, 68.8, 56.6, 56.5, 55.2, 52.2, 43.6, 43.5, 41.6, 41.5, 31.0, 30.9, 24.22.
2(S)-N-[(E)-2(3-Methoxyphenyl)ethanyl]-1-methyl-2-pyrrolinecarboxamide (2): To a solution of 10
(1.7 mmol, 0.6 g) in methanol (10 mL) was added a solution of sodium periodate (1.9 mmol, 0.4 g) in
water (10 mL) and the resulting mixture heated under reflux for 1.5 h. The methanol was removed in

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vacuo and the aqueous residue was extracted with dichloromethane (2 × 20 mL). The organic layer
was dried over anhydrous sodium sulfate and concentrated to give sulfoxide as a brownish oil (0.57 g,
70%). The crude product was then used in the next step without purification. The sulfoxide (1.20
mmol, 0.465 g) was dissolved in toluene (30 mL), sodium carbonate was added (166 mg) and the
mixture heated under reflux for 2 h. The solvent was removed under vacuum and the residue was
purified by circular chromatography using CH2Cl2/MeOH (95:5). Removal of the solvent gave a
brownish oil (0.16 g, 35%). IR (film): 3286, 2942, 1681, 1650, 1504, 1262 cm-1; 1H-NMR: δ 9.21 (d, J
= 10.8 Hz, 1H, N-H), 7.40 (dd, J = 14.0, 12.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.94-6.85 (m, 2H),
6.73 (ddd, J = 8.2, 2.6, 0.8 Hz, 1H), 6.17 (d, J = 14.0 Hz, 1H), 3.81 (s, 3H, OCH3), 3.24-3.17 (m, 1H),
3.07 (dd, J = 9.8, 4.8 Hz, 1H), 2.42 (s, 3H, NCH3), 2.50-2.20 (m, 2H), 2.00-1.7 0 (m, 3H); 13C-NMR: δ
172.2, 159.9, 137.7, 129.6, 122.5, 118.4, 113.3, 112.5, 110.6, 68.7, 56.7, 55.2, 41.9, 31.0, 24.5; IEMS,
m/z: M+ 260 (26), 132 (15), 112 (18), 70 (100).
2(S)-N-[(E)-2(3-Methoxyphenyl)ethanyl]-1-t-butoxycarbonyl-2-pyrrolinecarboxamide (3): Compound
3 was synthesized from 8 (2.2 mmol, 1.0 g) using the same procedure described for amide 2.
Compound 3 was obtained as a white solid (0.30 g, 30%); IR (KBr): 3283, 2974, 1695, 1653, 1400,
1257, 1161, 774 cm-1; 1H-NMR: δ 9.28 (bs, 1H, N-H), 7.46 (dd, J = 14.6, 10.8 Hz, 1H), 7.19 (t, J = 7.8
Hz, 1H), 6.90 (d, J = 7.8, 1H), 6.85 (d, J = 2.4, 1H), 6.73 (dd, J = 8.2, 2.4 Hz, 1H), 6.08 (d, J = 14.6
Hz, 1H), 4.42-4.22 (m, 1H), 3.77 (s, 3H, OCH3), 3.56-3.2 (m, 2H), 2.60-1.80 (m, 4H), 1.49 (s, 9H);
IEMS, m/z M+ 346 (23), 245 (1), 198 (2), 149 (42), 132 (6), 70 (100), 57 (33).
2(S)-N-[(E)-2(3-Methoxyphenyl)ethanyl]-2-pyrrolinecarboxamide (4): Compound 4 was obtained
from 3 (0.6 mmol, 0.2 g) as a brownish oil (0.13 g, 90%) using the same procedure described for
amide 9. IR (film) 3272, 3062, 2956, 1679, 1649, 1528, 1260, 1043, 692 cm-1; 1H-NMR: δ 9.58 (d, J =
10.6 Hz, 1H, N-H), 7.44 (dd, J = 15.0, 11.3 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.91 (d, J = 7.8, 1H),
6.87 (d, J = 2.0 Hz, 1H), 6.72 (ddd, J = 8.0, 2.4, 0.8 Hz, 1H), 6.16 (d, J = 15 Hz, 1H), 3.91-3.75 (m,
1H), 3.80 (s, 3H, OCH3), 3.10-2.90 (m, 2H), 2.65-2.3 (bs, 1H, NH), 2.26-2.15 (m, 1H), 2.06-1.90 (m,
1H), 1.81-1.71 (m, 2H); 13C-NMR: δ 172.9, 160.0, 138.0, 129.8, 122.6, 118.5, 113.5, 112.6, 110.7,
60.5, 55.4, 47.5, 30.9, 26.4; IEMS m/z M+ 246 (25), 149 (16), 132 (3), 70 (100).
Bioassay Evaluation Procedures.
Antimicrobial activity against Gram-positive bacteria Staphylococcus aureus ATCC 6538 and
Bacillus subtilis ATCC 6633, Gram-negative bacteria Escherichia coli ATCC 8739 and Pseudomona
aeruginosa ATCC 9027, and yeast Candida albicans ATCC 10231 was determined by the agar
Mitscher method as previously described [5]. Test samples were dissolved (2 mg/mL) in 10 mL
nutrient agar medium No. 1 (Merck) for bacterial cultures and Sabouraud agar (Merck) supplemented
with glucose (4 %) for yeast, and added aseptically to each Petri dish and swirled carefully until the
agar began to set. After 24 h. of incubation (sterility test) the bacteria or yeast in a concentration of
approximately 0.5 McFarland were streaked in radial patterns on the agar plates containing the
samples, incubated at 37oC for 24 h, except for C. albicans which was incubated at 25oC. Complete
inhibition of bacterial growth was expected for a sample to be declared active. Inhibition was present

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at 200 µg/mL only for amathamide A (1). Minimal inhibitory concentrations (MICs) values were
determined by the dilution method previously described [6,7]. Two-fold serial dilutions were tested in
nutrient broth (Merck) for B. subtilis, S. aureus, E. coli and P. aeruginosa, and Sabouraud-glucose (2
%) nutrient broth (Merck) for C. albicans. An initial concentration of 1 mg/mL of sample was
prepared by dissolving in dimethyl sulphoxide. Serial dilutions (100-0.1 µg/mL) of sample were
prepared, and the liquid medium was inoculated by cultures in stationary phase at concentration of 105
CFU/mL. After overnight incubation, the MIC was determined as the lowest concentration of
compound preventing any visible growth. Streptomycin sulfate (Sigma) (1 µg/mL) and nystatin
(Sigma) (3 µg/mL) were used as positive controls.
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Sample availability: Contact authors
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