Preparation, antimicrobial activity, and toxicity of 2-amino-4-arylthiazole derivatives

Article abstract of DOI:10.1002/hc.20182

Seven 2-amino-4-aryl-1,3-thiazoles (1a-g) and their corresponding 2-aminoacetyl (2a-g) and 2-aminoacetyl-5-bromo (3a-g) derivatives were synthesized and tested in vitro against 11 reference strains, three Gram-positive and four Gram-negative bacteria, two yeasts, and two moulds. Toxicity of the compounds was also evaluated using the brine shrimp test. Compounds 1a, 1b, 1e-g, and 3b showed moderate antimicrobial activity at different concentrations. The results indicated that acetylation of the amino group and bromination at position 5 of the thiazole moiety cause lost of activity. Compounds 1a, 1e, and 1f showed toxicity to brine shrimp nauplii below 10 ppm. Most other compounds showed moderate toxicity, LD₅₀ above 100 ppm. Structures of all compounds were confirmed by NMR and MS data.

Full text of DOI:10.1002/hc.20182

Heteroatom Chemistry
Volume 17, Number 4, 2006
Preparation, Antimicrobial Activity, and
Toxicity of 2-Amino-4-arylthiazole Derivatives
Pedro Morales-Bonilla,1 Andrea Pe´rez-Carden˜a,1
Esther Quintero-Ma´rmol,1 Jose´ Luis Arias-Te´llez,2
and Gonzalo J. Mena-Rejo´n1
1Facultad de Quı´mica, Universidad Auto´noma de Yucata´n, 41 No. 421 Col. Industrial,
C.P. 97150, Me´rida, Yucata´n, Me´xico
2Facultad de Estudios Superiores-Cuautitla´n, Universidad Nacional Auto´noma de Me´xico,
Cuautitla´n Izcalli, C.P. 54700, Estado de Me´xico, Me´xico
Received 27 April 2005; revised 12 September 2005
INTRODUCTION
ABSTRACT: Seven 2-amino-4-aryl-1,3-thiazoles
(1a–g) and their corresponding 2-aminoacetyl (2a–g)
and 2-aminoacetyl-5-bromo (3a–g) derivatives were
synthesized and tested in vitro against 11 reference
strains, three Gram-positive and four Gram-negative
bacteria, two yeasts, and two moulds. Toxicity of
the compounds was also evaluated using the brine
shrimp test. Compounds 1a, 1b, 1e–g, and 3b
showed moderate antimicrobial activity at different
concentrations. The results indicated that acetylation
of the amino group and bromination at position 5 of
the thiazole moiety cause lost of activity. Compounds
1a, 1e, and 1f showed toxicity to brine shrimp nau-
plii below 10 ppm. Most other compounds showed
moderate toxicity, LD50 above 100 ppm. Structures
of all compounds were confirmed by NMR and MS
It is well known that the thiazole derivatives are
important compounds due to their broad range
of biological activities [1–3]. 2-Aminothiazoles
as well as 2-amino-4-arylthiazoles and their
derivatives have shown radioprotective [4,5],
bactericidal, antifungal [6–8], antiviral [9,10], in-
secticidal [8], and anesthetic [11–13] activities.
This type of compounds can be obtained using
a solid phase reaction, which involves the cy-
clocondensation of p-substituted acetophenones
and thiourea in the presence of iodine [14–16].
Other synthetic methods involve the use of ke-
tones, N-bromosuccinimide, thiourea, and benzoyl
peroxide [17], or bromination of ketones in the
presence of AlCl3 followed by cyclocondensa-
tion of the intermediate ꢀ-bromoketones with
thiourea. As part of our search for antimicrobial
heterocyclic compounds, we have undertaken
the preparation of seven 2-amino-4-(p-substituted-
phenyl)-1,3-thiazoles (1a–g) and their corresponding
2-aminoacetyl (2a–g) and 2-aminoacetyl-5-bromo
(3a–g) derivatives, and evaluated their in vitro
antimicrobial activity against bacteria, yeasts, and
filamentous fungi. The toxicity of all prepared
compounds was also tested using the Artemia salina
test.
ꢀ
C
data.
2006 Wiley Periodicals, Inc. Heteroatom Chem
17:254–260, 2006; Published online in Wiley InterScience
(www.interscience.wiley.com). DOI 10.1002/hc.20182
Correspondence to: Gonzalo J. Mena-Rejo´n; e-mail: mrejon@
tunku.uady.mx.
Contract grant sponsor: Facultad de Qu´ımica de la Universidad
Auto´noma de Yucata´n.
Contract grant number: RP-01-2001.
2006 Wiley Periodicals, Inc.
c
ꢀ
254

Preparation, Antimicrobial Activity, and Toxicity of 2-Amino-4-arylthiazole Derivatives 255
SCHEME 1 Synthesis of 2-acetamide-5-bromo-4(4-substituted-phenyl)-1,3-thiazoles.
RESULTS AND DISCUSSION
The general tendency observed indicated that
the presence of electron-withdrawing groups or
electron-donating groups decreases the reaction
yield. Nevertheless, the chloro group was the ex-
ception giving the highest yield (Table 1). This
behavior could be due to a selective action of
the activating or deactivating groups over the
steps of the Hantzch’s reaction. It is possible that
electron-withdrawing substituents increases the
positive charge on carbonyl carbon by resonance
effects, avoiding an efficient protonation of the
carbonyl oxygen (step 1); while electron-donating
substituents decrease the positive charge on the
carbonyl carbon, thus making it less attractive
to nucleophilic attack of the thiourea nitrogen
(step 3). Acetylation of compounds 1a–g with acetic
2-Acetamido-5-bromo-4-phenyl-1,3-thiazole
(3a)
and six p-substituted-phenyl derivatives (3b–g)
were prepared from their corresponding 2-amino-
4-phenyl-1,3-thiazole and 2-amino-4-(p-substituted-
phenyl)-1,3-thiazoles (1a–g) (Scheme 1). Com-
pounds 1a–g were obtained by a Hantzch’s modified
method, a one-pot single-stage solid phase reaction
[16]. The compound formation can be explained
by a three-step mechanism. This process yields
the 2-amino-4-(p-substituted-phenyl)-1,3-thiazoles
derivatives as the hydroiodides, which are converted
into the free bases when treated with aqueous
alkaline solutions (Scheme 2). The reaction yields
were clearly affected by the p-phenyl substituents.
SCHEME 2 Mechanism of the Hantzch’s reaction.
Heteroatom Chemistry DOI 10.1002/hc

256 Morales-Bonilla et al.
TABLE 1 Characteristic Physical Data of 2-Amino-4(4-
compounds showed activity against some bacteria.
1b and 1f exhibited a selective activity; 1b was ac-
tive against K. pneumoniae, while 1f showed ac-
tivity against St. agalactiae. Compound 1g showed
nonselective antibacterial activity and was active
against all Gram-positive bacteria. Unfortunately,
the minimum inhibitory concentrations (MICs) were
very high in all cases, indicating a very slight an-
tibacterial activity. On the other hand, five com-
pounds (1a, 1b, 1e, 1f, 3b) showed moderate anti-
fungal activity, 1b and 1f were active against A. niger
and T. mentagrophytes below 500 ꢁg/mL. 2-Amino-
4-(p-chlorophenyl)-1,3-thiazole (1f) was the most
active compound that inhibited growth of T. men-
tagrophytes at 100 ꢁg/mL, followed by 2-amino-4-(p-
methylphenyl)-1,3-thiazole (1b), which inhibited the
fungus growth at 200 ꢁg/mL. This is an important
fact because T. mentagrophytes, a keratinophilic fil-
amentous fungus, is the causal agent inducing der-
matophytosis infections of hair, skin, and nails [18–
22]. In addition, Trichophyton species may cause
invasive infections in immune-compromised hosts
[23]. In spite of the moderate activity of compounds
1b and 1f, it is remarkable because of the lack of ef-
fective antifungal drugs and the pathogenicity of this
species.
substituted-phenyl)-1,3-thiazoles and Derivatives
Compound
Substituent
MP ( ◦C)
Yield (%)
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
H
Me
MeO
HO
Br
Cl
NO2
H
Me
MeO
HO
Br
Cl
NO2
H
Me
MeO
OH
Br
Cl
NO2
148–150
130–132
200–203
175–177
227–230
203–205
283–285
208–210
204–206
188–190
189–191
264–266
233–235
–
238–240
218–220
242–244
210–212
228–230
236–238
236–238
69.4
66.1
36.1
40.0
55.0
85.8
43.7
88.8
81.0
78.8
71.5
45.5
52.4
75.0
82.0
75.0
83.6
88.0
60.5
67.4
89.0
2g
3a
3b
3c
3d
3e
3f
3g
anhydride/sodium acetate gave the corresponding
aminoacetyl derivatives 2a–g in moderate to good
yields depending of the p-phenyl substituent, with
an opposite tendency compared with compounds
1a–g (Table 1). The 5-bromo derivatives 3a–g were
easily obtained from the aminoacetyl derivatives
2a–g, using bromine in acetic acid. The results
indicate that the presence of the N-acetyl group is
required for the bromination at 5-position.
The structures of the synthesized compounds
and the general method are shown in Scheme 1. All
compounds were identified by 1H and 13C NMR spec-
troscopy and mass spectrometry. The NMR assign-
ments are based on HSQC and HMBC experiments
(Tables 2 and 3). All data were in full agreement with
the assigned structures. The chemical shifts of pro-
tons and carbons at 5-position are clearly affected by
p-substituents in the phenyl group. The nitro group
deshields the protons at positions 7 and 11, while the
carbons at the same positions were not affected. The
EIMS and IR data are shown in Tables 4 and 5.
All the compounds were tested against Staphy-
lococcus aureus, Bacillus subtilis, Streptococcus.
agalactiae (Gram-positive bacteria), Escherichia coli,
Pseudomonas aeruginosa, Klebsiella pneumoniae,
Shigella flexneri serotype 4 (Gram-negative bacteria),
Candida albicans, Saccharomyces cerevisae (yeasts),
Aspergillus niger, and Trichophyton mentagrophytes
(filamentous fungi). The 86% of the tested com-
pounds were not active against bacteria; only three
All active compounds were 2-amino-4-(p-
substituted-phenyl)-1,3-thiazoles, except 3b. This
result suggests that acetylation of the amino group
or the bromination at 5-position has a negative
effect on the antimicrobial activity.
Toxicity of the prepared compounds was evalu-
ated using Artemia salina nauplii [24], and cytotox-
icity was inferred based on the same brine shrimp
lethality assay, which has been found to have a
good relationship with antitumoral activity [25]. The
results indicate that 71% of the synthesized thia-
zoles can be considered moderate-low toxic, since
they show toxicity at concentrations above 100 ppm
(Table 6). However, compounds 1a, 1e, and 1f
showed toxicity below 10 ppm. Correlation of these
results and those obtained in the antifungal test indi-
cated that 1b was the better antifungal compound of
the synthesized thiazoles due to its moderate toxicity.
Although the brine shrimp lethality bioassay is an ex-
cellent choice to elementary toxicity investigations,
it is necessary the use of human line cells to evaluate
the cytotoxicity of the synthesized compounds.
EXPERIMENTAL
Melting points were determined on an Electrother-
mal apparatus model IA9100 in open capillar-
ies and are uncorrected. IR spectra (KBr disks)
were recorded using a Nicolet Magna-550 FT
Heteroatom Chemistry DOI 10.1002/hc

Preparation, Antimicrobial Activity, and Toxicity of 2-Amino-4-arylthiazole Derivatives 257
TABLE 2 1H NMR Data (400 MHz) in DMSO-d6 of 2-Amino-4(4-substituted-phenyl)-1,3-thiazoles and Derivates
H5
H7, 11
H8, 10
H9
NH2
NH
Me
MeO
OH
Ac
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
6.99
6.90
6.85
6.71
7.07
7.05
7.36
7.54
7.53
7.00
7.29
7.68
7.64
7.95
7.79 (d, 7.6)
7.67 (dd, 6.8, 1.2)
7.71 (dd, 7.2, 1.3)
7.59 (dd, 6.4, 2)
7.74 (dd, 6.8, 2)
7.79 (d, 8.4)
8.21 (d, 8.4)
7.81 (d, 8.0)
7.80 (d, 7.8.0)
7.73 (d, 8.0)
7.67 (d, 8.2)
7.86 (d, 8.2)
7.89 (d, 8.0)
8.27 (d, 8.1)
7.83 (d, 8.0)
7.72 (d, 4)
7.36 (t, 7.2)
7.16 (d, 7.2)
6.92 (dd, 7.5, 1.3)
6.74 (dd, 6.4, 2.0)
7.54 (dd, 6.8, 2.0)
7.40 (d, 8.4)
8.01 (d, 8.4)
7.42 (t, 8.0)
7.25 (d, 7.8)
6.94 (d, 8.0)
6.78 (d, 8.2)
7.65 (d, 8.2)
7.48 (d, 8.0)
8.14 (d, 8.1)
7.46 (t, 8.0)
7.26 (d, 8.0)
7.01 (d, 8.0)
7.28
7.25 (t, 7.0)
7.04
7.01
6.98
6.95
7.07
7.08
7.18
2.35
3.76
9.50
7.34 (t, 8.0)
7.39 (t, 8.0)
12.19
12.26
11.73
12.15
12.29
12.26
12.36
12.48
12.46
12.44
12.55
12.56
12.55
12.66
1.75
2.19
1.75
2.13
2.19
2.16
2.16
2.17
2.32
2.19
2.19
2.16
2.16
2.21
2.34
2.28
3.83
3.81
9.62
2g
3a
3b
3c
3d
3e
3f
7.79 (d, 8.0)
7.92
7.81 (d, 8.0)
7.87 (d, 8.3)
8.38 (d, 8.0)
10.50
7.69 (d, 8.0)
7.54 (d, 8.3)
8.18 (d, 8.0)
3g
TABLE 3 13C NMR Data (100 MHz) in DMSO-d6 of 2-Amino-4(4-substituted-phenyl)-1,3-thiazoles and Derivatives
C2
C4
C5
C6
C7, 11
C8, 10
C9
Me
MeO
Ac
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
169.1
168.2
169.1
168.4
168.4
168.4
168.9
159.8
158.8
159.8
158.0
158.7
159.0
158.8
157.2
157.1
159.4
158.1
158.0
158.1
158.0
150.8
149.9
150.7
150.3
148.7
148.6
148.0
149.6
149.8
149.4
149.3
148.1
148.4
146.8
146.0
146.0
146.0
146.4
145.4
145.4
147.3
102.4
100.6
100.3
99.0
135.8
132.3
128.8
126.6
134.1
131.6
141.0
134.4
132.7
127.4
126.0
134.1
133.1
140.5
134.2
138.2
126.9
122.5
133.3
133.0
139.9
126.5
125.5
127.8
127.2
127.6
127.3
124.3
126.4
126.6
127.3
127.4
128.2
128.2
124.5
128.2
128.1
129.6
129.9
130.7
129.5
124.4
129.4
129.0
114.8
115.5
131.4
128.5
126.5
129.1
130.3
114.4
115.7
132.3
129.5
126.7
128.7
129.2
114.1
115.6
132.2
130.6
129.5
128.1
136.4
159.5
157.0
120.2
133.7
146.2
128.5
138.1
158.1
157.5
121.4
134.1
146.6
128.7
130.6
157.0
157.8
122.5
134.0
144.2
20.8
56.0
102.5
102.4
106.9
108.0
108.0
106.3
105.3
109.3
109.4
112.6
97.2
96.5
95.6
95.2
98.3
98.5
100.3
22.8
23.5
22.7
22.8
23.1
23.3
22.7
22.5
22.8
22.8
22.9
23.0
23.2
22.8
21.8
21.1
55.5
55.5
2g
3a
3b
3c
3d
3e
3f
3g
spectrophotometer. A Bruker Avance 400 Ultrashield
spectrometer, operating at 400 MHz for 1H and
100 MHz for 13C with XWINMR software pack-
age, was used for NMR experiments measured in
DMSO-d6 with TMS as internal reference, chem-
ical shifts are recorded in δ values. EIMS were
obtained on a Hewlett–Packard 5892 mass spectrom-
eter using a Hewlett–Packard 5970 series II gas chro-
matograph as injection system, equipped with an
Ultra II (5% diphenyl–95% dimethylsiloxane) fused-
silica column (25 m × 0.2 mm i.d.; 0.33 ꢁm film).
2-Amino-4-phenyl-1,3-thiazole (1a) and
Derivatives (1b–g) (General Procedure)
Thiourea (0.98 g, 12.9 mmol) and I2 (3.28 g,
12.9 mmol) were triturated and mixed with ace-
tophenone (1.03 g, 8.6 mmol). The mixture was
Heteroatom Chemistry DOI 10.1002/hc

258 Morales-Bonilla et al.
TABLE 4 Mass Spectrometric Data of 2-Amino-4(4-substituted-phenyl)-1,3-thiazoles and Derivatives
EIMS (70 eV) m/z
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
3e
3f
176 [M]+; 134, 104, 89, 77, 63, 51
190 [M]+; 147, 118, 91, 77, 63, 51
206 [M]+; 191, 164, 149, 134, 121, 77, 63
192 [M]+; 150, 121, 105, 93, 77
264 [M]+; 212, 182, 175, 148, 133, 120, 102, 89, 75, 63
210 [M]+; 168, 133, 102, 89, 63
221 [M]+; 191, 175, 149, 121, 89, 63
218 [M]+; 176, 134, 104, 89, 77, 43
232 [M]+; 190, 148, 118, 91, 65, 43
248 [M]+; 206, 191, 164, 149, 121, 77, 43
234 [M]+; 192, 150, 121, 93, 77, 65, 43
306[M]+; 254, 212, 182, 174, 133, 120, 89, 75, 43
252 [M]+; 210 (PB), 168, 133, 89, 43
263[M]+; 221, 191, 175, 146, 121, 103, 89, 43
306[M]+; 254, 212, 174, 146, 133, 103, 89, 77, 43
320 [M]+; 268, 226, 188, 147, 103, 91, 77, 43
336 [M]+; 284, 269, 227, 204, 175, 163, 134, 120, 89, 63, 43
322 [M]+; 192, 150, 121, 93, 77, 65, 43
394 [M]+; 332, 252, 211, 182, 174, 146, 132, 102, 93, 75, 43
340 [M]+; 288, 252, 208, 167, 137, 123, 102, 71, 43
351 [M]+; 299, 269, 253, 219, 178, 146, 132, 120, 102, 43
3g
TABLE 5 Infrared Spectroscopic Data of 2-Amino-4(4-substituted-phenyl)-1,3-thiazoles and Derivatives
(cm−1
IR (KBr) ν
)
max
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
3436, 3254, 3157, 3157, 3115, 1599, 1518, 1331, 715
3401, 3291, 3180, 3120, 1626, 1518, 1335, 821
3441, 3274, 3168, 3118, 1626, 1538, 1494, 1331, 1250, 1177, 1033, 836
3490, 3381, 3128, 1600, 1511, 1331, 1273, 1178, 841
3446, 3361, 3118, 1622, 1532, 1348, 827, 650
3441, 3363, 3122, 1622, 1532, 1348, 827, 732
3403, 3309, 3156, 1642, 1597, 1534, 1507, 1328, 847
3158, 3071, 1639, 1580, 1540, 1301, 711
3172, 3065, 1643, 1580, 1309, 812
3164, 3063, 1642, 1575, 1304, 825
3188, 3068, 1671, 1475, 1300, 752
3175, 3070, 1644, 1557, 1305, 831
3175, 3070, 1644, 1557, 1305, 831
3164, 3071, 1642, 1576, 1526, 1340, 855
3172, 3065, 1648, 1560, 1298, 771, 693
3171, 3067, 2993, 1645, 1550, 1300, 995, 803
3407, 3149, 2963, 1639, 1555, 1302, 831, 756
3189, 1671, 1551, 1455, 1295, 1238, 752
3163, 3063, 2988, 1643, 1550, 1467, 1308, 990, 819
3173, 3072, 2996, 1644, 1552, 1460, 981, 825
3179, 3078, 3005, 2925, 1658, 1552, 1506, 1336, 1283, 704
2g
3a
3b
3c
3d
3e
3f
3g
heated at 150◦C on a sand bath for 2 h. The obtained
solid was triturated with Et2O, filtered, and washed
with the same solvent to remove the unreacted ace-
tophenone. The crude product was dissolved in hot
water, and the thiazole was precipitated by the addi-
tion of NH4OH and was separated by filtration. The
obtained solid was purified by crystallization from a
mixture of EtOH:H2O (1:4).
2-Acetamide-4-phenyl-1,3-thiazole (2a) and
Derivatives (2b–g) (General Procedure)
Equimolar amounts of 2-amino-4-phenyl-1,3-
thiazole (1 g, 5.68 mmol) and sodium acetate were
dissolved in acetic anhydride (7 mL). The reaction
mixture was refluxed for 1 h. Then, 20 mL of cold
water was slowly added with vigorous stirring. The
Heteroatom Chemistry DOI 10.1002/hc

Preparation, Antimicrobial Activity, and Toxicity of 2-Amino-4-arylthiazole Derivatives 259
TABLE 6 Minimum Inhibitory Concentration (mg/mL) Against Microorganisms and Toxicity Against Artemia salina of 2-Amino-
4(4-substituted-phenyl)-1,3-thiazoles Synthesized
Sa
Bs
Sta
Ec
Pa
Kp
Sf
Ca
Sc
An
Tm
LD50
1a
1b
1e
1f
1g
3b
6.25
3.13
6.25
1.56
3.13
0.20
0.79
0.10
3
223
7
3
36
215
1.56
6.25
0.78
0.40
0.40
3.13
6.25
6.25
3.13
6.25
12.5
Sa, Staphylococcus aureus; Bs, Bacillus subtilis; Sta, Streptococcus agalactiae; Ec, Escherichia coli; Kp, Pseudomonas aeruginosa; Kp,
Klebsiella pneumoniae; Sf, Shigella flexneri serotype 4; Ca, Candida albicans; Sc, Saccharomyces cerevisae; An, Aspergillus niger; Tm,
Trichophyton mentagrophytes; LD50, lethal dose in ppm for 50% of the brine shrimp.
product was precipitated by addition of NH4OH and
separated by filtration. The crude product was dried
and crystallized from a mixture of EtOH:H2O (1:4).
B. subtilis (465), St. agalactiae (4768), E. coli (128),
P. aeruginosa (260), Klebsiella pneumoniae (4209),
Shigella flexneri serotype 4 (9748), Candida albicans
(752), Saccharomyces cerevisae (287), Aspergillus
niger (16888), and Trichophyton mentagrophytes
(4807); all of them were purchased from the
American Type Culture Collection. Diluted bacte-
rial culture (1.5 × 108 UFC/mL) was spread on a
sterile Muller–Hinton agar plates. 6-mm Diameter
disks were impregnated with 5 ꢁL of 2% (w/v) of a
chloroform solution of each synthesized compound,
after that the disks were placed on the plates. The
plates were incubated for 24 h at 37◦C under aero-
bic conditions. The inhibition zone diameter around
each disk was measured and recorded. Amikacin
(0.03 mg/mL), nystatin (50 IU/mL), and itraconazole
(0.025 mg/mL) were used as positive control for bac-
teria, yeast, and fungi, respectively; chloroform was
used as negative control. All determinations were
performed in triplicate.
2-Acetamide-5-bromo-4-phenyl-1,3-thiazole (3a)
and Derivatives (3b–g) (General Procedure)
Bromine (2 mL, 4.0 mmol) dissolved in acetic acid
(10 mL) was slowly dropped to a cold solution
(10◦C) of 2-acetamide-4-phenyl-1,3-thiazole (8.72 g,
4.0 mmol) in acetic acid (10 mL). The mixture re-
action was kept on stirring until white solid precipi-
tated. The product was separated by filtration, dried,
and crystallized from a mixture of EtOH:H2O (1:3).
Brine Shrimp Lethality Assay
Dry cysts, purchased from San Francisco Bay Brand
Co., were washed for 30 min with an aqueous bleach
solution (50%), rinsed thoroughly with water, and
then were incubated in a hatcher at 28–30◦C with
strong aeration, under a continuous light regime. Ap-
proximately 12 h after hatching, the phototropic nau-
plii were collected with a pipette from the lighted side
and concentrated in a 20 mL vial. Each test consisted
of exposing groups of 10 nauplii aged 12 h (in instar
II/III) to each synthesized compound. Each test was
performed in triplicate. Each compound was tested
at 10, 100, and 1000 ppm. The concentrations were
obtained by transferring 5, 50, and 500 ꢁL from a
stock solution (1% in DMSO) to 10 mL vials and
filtered, and sterilized seawater was added to get a
final volume of 5 mL. The number of survivors was
counted, and the LD50 was calculated. Larvae were
considered dead if they did not exhibit any internal
or external movement during 10 s of observation un-
der stereoscopic microscope.
Determination of Minimum Inhibitory
Concentration (MIC)
MIC values were determined by a microdilution tech-
nique in 96-well cell culture plates [26]. Each ac-
tive compound was dissolved in DMSO to give a
concentration of 400 mg/mL. Twofold serial dilu-
tions were made in broth over a range to give fi-
nal concentrations of 200 mg–100 ꢁg/mL. In each
microplate well, 50 ꢁL of a bacterial suspension
(1.5 × 108 UFC/mL) was added to 50 ꢁL of se-
rial dilutions. The plates were incubated at 37◦C
for 24 h after to indicate bacterial growth, 50 ꢁL
p-iodonitrotetrazolium violet dissolved in water was
added to the microplate wells and incubated at
37◦C for 10–30 min. The MIC was defined as the
lowest concentration at which no bacterial growth
was observed. All determinations were performed in
triplicate.
Antimicrobial Assays
Synthesized compounds were screened, using the
disk-diffusion method, against S. aureus (4012),
Heteroatom Chemistry DOI 10.1002/hc

260 Morales-Bonilla et al.
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ACKNOWLEDGMENTS
The authors wish to thank Durcy Ruiz-Ciau and
Gumersindo Miro´n-Lo´pez for GC–MS and IR techni-
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to Dr. Leovigildo Quijano for the critical review and
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