Synthesis and structural studies of 4-thioxopyrimidines with antimicrobial activities

Article abstract of DOI:10.1007/s00706-006-0577-y

This work describes a two-step, one-pot synthetic method for the formal aza-[3 + 3] cycloaddition between N-alkyl substituted enaminones and benzoyl isothiocyanate, which afforded 4-thioxopyrimidines in reasonable yields. Reaction of acyclic enaminone wit

Full text of DOI:10.1007/s00706-006-0577-y

Monatshefte fu¨r Chemie 138, 111–119 (2007)
DOI 10.1007/s00706-006-0577-y
Printed in The Netherlands
Synthesis and Structural Studies of 4-Thioxopyrimidines
with Antimicrobial Activities
Silvio Cunha1;ꢀ, Rodrigo M. Bastos1, Priscila de O. Silva1, Giselle A. Nobre Costa1,
2;3
2
3
4
´
Ivo Vencato , Carlito Lariucci , Hamilton B. Napolitano , Cecılia M. A. de Oliveira ,
Lucılia Kato , Cleuza C. da Silva , Diego Menezes , and Marcos A. Vannier-Santos
4
5
6
6
´
1
´
Instituto de Quımica, Universidade Federal da Bahia, Campus de Ondina, Salvador-BA, Brazil
´ ´
Instituto de Fısica, Universidade Federal de Goias, Goiania-GO, Brazil
2
3
4
5
6
^
Ciencias Exatas e Tecnologicas, Universidade Estadual de Goias, Anapolis-GO, Brazil
´
´
´
^
Instituto de Quımica, Universidade Federal de Goias, Goiania-GO, Brazil
´
´
^
Departamento de Quımica, Universidade Estadual de Maringa, Maringa-PR, Brazil
´
´
´
´ ´
Laboratorio de Biomorfologia Parasitaria e Microscopia Eletronica, Centro de Pesquisas Gonc°
^
Fundaca˜o Oswaldo Cruz (CPqGM-FIOCRUZ), Candeal, Salvador-BA, Brazil
alo Moniz,
°
Received June 19, 2006; accepted (revised) July 13, 2006; published online January 19, 2007
# Springer-Verlag 2007
Summary. This work describes a two-step, one-pot synthetic
ic methodologies for this class of heterocycles [2].
Among them, the formal aza-[3þ 3] cycloaddition
involving enaminones as synthons for the preparation
of the bioactive heterocyclic nucleus [3] caught our
attention because enaminones are easily prepared in
good yields and have been used in the synthesis of a
broad spectrum of compounds [4]. However, while
thioxopyrimidine synthesis is well described with
N-aryl substituted enaminones and isothiocyanates
[2, 5], very little is known about the scope of this
aza-annulation with monosubstituted N-alkyl enamin-
ones, including sterically hindered ones, as the
nucleophilic component in the synthesis of thioxopyri-
midines via formal aza-[3 þ 3] cycloaddition.
method for the formal aza-[3þ 3] cycloaddition between
N-alkyl substituted enaminones and benzoyl isothiocyanate,
which afforded 4-thioxopyrimidines in reasonable yields.
Reaction of acyclic enaminone with a sterically hindered
group attached to the nitrogen atom afforded pyridine-2-
thione, yet in low yield. The antibacterial, antifungal, and
trypanocidal activities of the thioxopyrimidines were evalu-
ated and five compounds exhibited moderate activity against
Candida albicans, Micrococcus luteus, and Trypanosoma
cruzi. The solid state structures of a thioxopyrimidine, an
organic disulfide, and a 1,2,4-triazole were determined by
X-ray diffraction analysis.
Keywords. Cyclizations; Enaminones; Heterocycles; Pyrimi-
dine-4-thiones; X-ray structure determination.
According to Hsung et al., the formation of hetero-
cycle cores from an enaminone can be envisioned as a
formal aza-[3þ 3] cycloaddition between an enamin-
one and a functionalized ꢀ,ꢁ-unsaturated carbonyl
electrophile (or its equivalent), Fig. 1. The regio-
chemistry of this stepwise process can be classified
according to the orientation of carbonyl moieties of
enaminone and electrophile. The head-to-head re-
giochemistry (Hsung’s aza-annulation [3a]) results
from the orientation of both carbonyl carbons at the
Introduction
Thioxopyrimidine is an essential structural unit of
several heterocycles, which displays a wide range of
interesting biological and pharmacological properties,
such as anticancer and antimicrobial activities [1].
Despite these characteristics there are few synthet-
ꢀ
Corresponding author. E-mail: silviodc@ufba.br

112
S. Cunha et al.
Results and Discussion
We were unable to reproduce the yields of thioxopy-
rimidines 4a–4b using the reported protocol [5a, 5h].
Thus, a systematic investigation of the reaction con-
ditions for the formal aza-[3 þ 3] cycloaddition was
undertaken. Attempts to optimize the reaction con-
ditions revealed that the formation and yields of
heterocycles 4a–4b were dependent of solvent, tem-
perature, and the nature of enaminone employed. In
the best condition, 4a was obtained at room temper-
ature from ꢁ-enamino ketone 1a and benzoyl iso-
thiocyanate (2) using isopropyl ether as solvent in
30% yield. On the other hand, ꢁ-enamino ester 1b
afforded 4b in better yield (72%), but with a two-
step one-pot procedure (first, ethyl ether as solvent
and reaction at 0–5ꢁC; second, solvent exchange for
ethyl alcohol and reaction at room temperature)
according to Scheme 1. Formation of thioxopyrimi-
dines 4a–4b proceeded via acyclic intermediates
3a–3b as has been previously observed [5a, 5c].
The reaction progress was easily followed because
the solution turned red, the typical color of the in-
termediates 3a and 3b. Besides, formation of the
C-adduct 3a and not the regioisomeric N-adduct
has been corroborated by absence of olefinic C–H
Fig. 1. Regiochemistry of formal aza-[3 þ 3] cycloaddi-
tion of enaminones and a generic ꢀ,ꢁ-unsaturated carbonyl
eletrophile
same side, whereas the head-to-tail regiochemistry
(Hicmott-Stille’s aza-annulation [3b, 3c]) arises from
carbonyl carbons oriented at opposite sides [3d, 3e].
Herein we disclose our results concerning the for-
mal aza-[3þ 3] cycloaddition of acyclic N-alkyl sub-
stituted enaminones with benzoyl isothiocyanate for a
direct 4-thioxopyrimidine synthesis with emphasis on
synthetic, mechanistic, and structural implications.
1
in both H and 13C NMR spectra [6].
Scheme 1

4-Thioxopyrimidines with Antimicrobial Activities
113
Because the two-step one-pot procedure affords
the best yields, it was extended to the ꢁ-enamino
ketone 1a, but a slow reaction took place. After con-
sumption of 3a [7] the disulfide 5a was the sole
product, Scheme 1. Additionally, attempts to opti-
mize the yield of 4b by refluxing in the second step
failed. Only the disulfide 5b was isolated in low
yield, Scheme 1. Formation of disulfides 5a–5b pro-
ceeded via 4a–4b [5a, 5c]. Noteworthy, a previous
study reported that the synthesis of 5a–5b via
4a–4b took place only under basic conditions [5c].
Herein, the spontaneous conversion under base-free
conditions suggests that 4a–4b undergo air oxidation
to 5a–5b, probably via reversible transformation be-
cause, when disulfide 5a was left in CDCl3 in the
NMR tube it was slowly converted into thioxopyri-
midine 4a, and the ratio of 5a:4a after two days was
1.5:1, according to the integral of methyl groups in
Fig. 2. ORTEP drawing of disulfide 5b with atom-number-
ing scheme; the displacement ellipsoids are drawn at 30%
probability levels
structures of 5b is shown in Fig. 2 as an example (for
4b vide infra). The only structural difference be-
tween 5a and 5b is the C5 substituent of pyrimidine
rings, but they presented significant different fea-
tures. Thus, disulfide 5a is a planar molecule in the
solid state with pyrimidine rings antiperiplanar at
the S–S bond (torsional angle C4-S-Si-C4i ¼ 180ꢁ,
symmetry code: (i)¼ ꢂx þ 1; ꢂy þ 1; ꢂz). Curiously,
this conformation is not observed in 5b, where the
1
the H NMR spectrum.
Although thioxopyrimidines 4a–4b and disulfides
5a–5b are known compounds [5a, 5c] we wanted to
corroborate their structure unambiguously. Thus we
undertook the structural characterization by an X-ray
study of 4b and 5a–5b, which afforded monocrys-
tals. The ORTEP [8] representation of the solid state
Scheme 2

114
S. Cunha et al.
corresponding torsional angle C4a-S1a-S1b-C4b is
75.99(7)ꢁ, with the pyrimidines positioned synclinal.
In order to investigate the scope and limitations of
formal aza-[3 þ 3] cycloaddition a series of structur-
ally different enaminones was subjected to reaction
with benzoyl isothiocyanate (2). Acyclic N-alkyl
substituted ꢁ-enamino ketones 1c–1f and ꢁ-enamino
esters 1g–1i were submitted to the two-step one-pot
condition, Scheme 2. Thus, a series of new 4-thiox-
opyrimidines 4c–4i could be obtained in reasonable
yields from enaminones owing to a primary alkyl
group connected to the nitrogen atom, Table 1.
Mechanistically, the formation of N-alkyl 4-thiox-
opyrimidines 4c–4i can be envisioned as an ionic
stepwise process (Scheme 2) initiated by attack of
the nucleophilic ꢀ carbon of the enaminone to the
electrophilic sp hybridized carbon of 2, which rep-
resents a soft–soft interaction, yielding C-adducts
3c–3i. In sequence, 3c–3i suffer an intramolecular
N-acylation and elimination of water. The regiose-
lectivity of this formal aza-[3þ 3] cycloaddition cor-
responds to the head-to-tail regiochemistry (Hicmott-
Stille’s aza-annulation, Fig. 1) [3d, 3e]. The struc-
tures of compounds 4b, 4e, 4h, and 4i were also
investigated by means of an X-ray analysis and an
Table 1. Isolated yields and IC50 of anti-Trypanosoma cruzi
activity of 4-thioxopyrimidines
IC50
(mM)
Compound R1
R2
Yield=% Time=d
4c
4d
4e
4f
4g
4h
4i
CH3 CH2Ph
CH3 Bu
CH3 CH2CH2OH
CH3 CH2CH2OCH3
OEt CH2Ph
72
70
65
60
74
73
66
5
4
5
2
2
2
5
159.9
95.4
148.2
nta
nta
Fig. 3. ORTEP drawing of 4b with atom-numbering scheme;
the C12 atom is disordered and its H-atoms were not found;
the H-atoms of C13-methyl are also disordered; the displace-
ment ellipsoids are drawn at 30% probability levels
OEt Bu
OEt CH2CH2OH
99.0
153.3
ant Not tested
Scheme 3

4-Thioxopyrimidines with Antimicrobial Activities
115
ORTEP representation of the solid state structure of
4e is shown in Fig. 3 as an example.
tion step that is necessary to form the expected
thioxopyrimidine. Moreover, formation of 6 involves
the incorporation of a methyl group and the nucleo-
philic ꢀ carbon of enaminone 1j into the heterocy-
clic ring. To the best of our knowledge, there is only
one previous example of pyridin-2-thione synthesis
through an enaminone annulation [5f].
Sterically hindered alkyl substituents, like isopro-
pyl and cyclohexyl groups, were not tolerated in the
formal aza-[3 þ 3] cycloaddition, being a limiting
factor in the N-alkyl 4-thioxopyrimidines synthesis.
Although a complex mixture of products was formed
with N-cyclohexyl substituted ꢁ-enamino ester 1j,
pyridin-2-thione 6 could be isolated and its structure
and regiochemistry of cyclization was investigated
In the reaction of enaminones with phenyl isothio-
cyanate, it has been demonstrated that the obtained
C-adduct is a versatile intermediate for the synthesis
of pyrazoles in good yields [9]. These results in-
spired us to try a similar reaction with the unstable
intermediate 3. We reasoned that this would be a
good strategy to form complex heterocycles because
intermediate 3 possesses an additional electrophilic
carbonyl when compared with the analogous C-
adduct [9]. Additionally, the second step of the
aza-annulation here studied is a slow reaction (see
Scheme 2 and Table 1), which suggests the possibil-
ity of trapping the C-adduct 3 with a nitrogen nu-
cleophile. Therefore, were reacted enaminones 1a
and 1b with benzoyl isothiocyanate (2) and tenta-
tively trapped intermediate 3 with diverse bisnu-
cleophiles. Disappointingly, this reactions afforded
complex mixtures. Probably, the additional electro-
philic carbonyl center of 3 confers a great reactiv-
1
by H,13C long range correlation, as indicated in
Scheme 3. Thus, the ortho hydrogens of the phenyl
ring and the hydrogen at C5 of the pyridin-2-thione
nucleus present a common correlation with the C-6
carbon near to the endocyclic nitrogen of 6, and
this spectral feature is in accordance with the indi-
cated structure. A mechanistic rationalization is also
shown in Scheme 3, whereby formation of C-adduct
3j is followed by a sequence of tautomeric equilibria
that first result in 3j9 and by the involvement of the
methyl group form transient enamine 3j0, which
reacts with the benzoyl moiety to yield the hetero-
cyclic core 69. After this, elimination of water
affords 60 and proton migration results in pyridin-
2-thione 6. The steric hindrance of the cyclohexyl
group obviously inhibits the intramolecular N-acyla-
Scheme 4

116
S. Cunha et al.
In conclusion, we developed a simple two-step one-
pot synthesis procedure for the formal aza-[3 þ 3] cy-
cloaddition between N-alkyl substituted enaminones
and benzoyl isothiocyanate which afforded 4-thioxo-
pyrimidines in good yields and under mild conditions.
This work also suggests the potential of intermediate 3
in the formation of complex N-heterocycles. Efforts
are underway to optimize yields and elucidate the
mechanistic details of the reaction of intermediate 3
with bisnucleophiles and define the scope, limitations,
and synthesis applications. This will be reported in
due course.
Fig. 4. ORTEP drawing of 7 with atom-numbering scheme;
the displacement ellipsoids are drawn at 30% probability
levels
Experimental
´
Melting points were determined on a Microquımica MQAPF
301 hot plate apparatus. Infrared spectra were recorded with
KBr discs on a FT-IR BOMEM MB100 instrument. NMR
spectra were obtained for 1H at 300 MHz and for 13C at
75MHz using a Varian Gemini 300 spectrometer. Chemical
shifts are reported in ppm units downfield from reference
(internal TMS). Elemental analyses were performed on a 2400
CHN Perkin Elmer instrument. Their results agreed favoura-
bly with the calculated values. Enaminones 1a–1k were pre-
pared according to known procedures [10]. The physical
properties (mp, spectra) of known compounds 4a, 4b, 5a
and 5b are in agreement with the literature [5a, 5c, 5h].
ity to this intermediate under these conditions and
no selective reaction occurred. When hydrazine was
employed only disulfides 5a and 5b were isolated,
Scheme 4. Meanwhile, the reaction of the phenylhy-
drazine and C-adduct 3k from enaminone 1k pro-
vided 7, albeit in very low yield. Despite this
finding, formation of heterocycle 7 is noteworthy
because it combines a pyrazole linked to a 1,2,4-
triazole ring. Scheme 4 summarizes theses results
and also shows a mechanistic proposal for the for-
mation of 7, whose structure was unambiguously
assigned by X-ray analysis, as shown in Fig. 4.
The thioxopyrimidines were individually tested for
antibacterial activity against B. subtilis ATCC 6633,
S. aureus ATCC 6638, M. luteus ATCC 10240,
S. mutans ATCC 24175, Salmonella choleraesuis
ATCC 14028, E. coli ATCC 94863, P. aeruginosa,
C. albicans ATCC 18804, A. niger ATCC 16404,
and C. cladosporioides IMI 178517 by broth micro-
dilution method. For the purpose of antimicrobial
evaluation, 4e and 4i were acetylated affording deriv-
atives 4j and 4k in excellent yields, Scheme 2.
Whereas 4g, 4j, and 4i exhibited antimicrobial activ-
ity with MIC values of 100 mg=cm3 for C. albicans,
compounds 4b and 4c were active against M. luteus
with MIC values of 100 and 50 mg=cm3.
1-(4-Methyl-2-phenyl-6-thioxo-1,6-dihydro-5-pyrimidinyl)-
1-ethanone (4a, C13H12N2OS)
To a solution of 1.0 mmol enaminone 1a in 5 cm3 isopropyl
ether was added dropwise a solution of 1.1 mmol benzoyl
isothiocyanate in 5 cm3 dry ethyl ether under ice-bath cooling
and magnetic stirring. After 15min the ice-bath was removed
and the reaction mixture was left at room temperature for 5
days, while the progress of the reaction was monitored by
TLC, after which time the solvent was evaporated. The resi-
due was recrystallized from ethyl acetate=petroleum ether to
1
give 30% 4a as yellow solid, mp 120.0–121.0ꢁC. H NMR
(300MHz, CDCl3): ꢂ ¼ 2.66 (3H, s), 2.75 (3H, s), 7.32–7.43
(3H, m), 8.25 (2H, d, J ¼ 6.9 Hz) ppm; 13C NMR (75 MHz,
CDCl3): ꢂ ¼ 23.84 (CH3), 32.02 (CH3), 128.63 (CH), 128.91
(CH), 129.75 (CH), 131.65 (C), 136.51 (C), 162.83 (C), 163.59
(C), 165.58 (C), 201.21 (C) ppm; IR (KBr): ꢃꢀ¼ 3309, 1682,
1537 cmꢂ1
.
General Synthesis Procedure for 4b, 5a, 4c–4i, and 6
To a solution of 1.0mmol enaminone 1a–1j in 5 cm3 dry ethyl
ether was added dropwise a solution of 1.2 mmol benzoyl
isothiocyanate in 5 cm3 dry ethyl ether under ice-bath cooling
and magnetic stirring. After 15min the ice-bath was removed
and the reaction mixture was left for 30 min at room tempera-
ture, after which time the solvent was evaporated. 10 cm3
ethyl alcohol were added and the solution was reacted at room
temperature for the time indicated in each case (or in Table 1
for 4c–4i), while the progress of the reaction was monitored
Additionally, thioxopyrimidines 4c, 4d, 4e, 4h, and
4i were also evaluated in vitro against epimastigotes
of Trypanosoma cruzi and their IC50 values were de-
termined, Table 1. All tested derivatives showed try-
panocidal activity, albeit modest. This result suggests
that the structural modification in the 4-thioxopyrimi-
dine scaffold to improve the anti-Trypanosoma cruzi
property deserves attention.

4-Thioxopyrimidines with Antimicrobial Activities
117
by TLC. The solvent was evaporated and the crude residue
was treated as indicated in each case.
144.99 (C), 157.29 (C), 192.40 (C), 201.79 (C) ppm; IR (KBr):
ꢃꢀ¼ 3327, 1703, 1593, 1514, 1479, 1288, 1190, 1055 cmꢂ1
.
Ethyl 4-methyl-2-phenyl-6-thioxo-1,6-dihydro-
1-[1-(2-Methoxyethyl)-6-methyl-2-phenyl-4-thioxo-1,4-
5-pyrimidinecarboxylate (4b, C14H16N2O3S)
dihydro-5-pyrimidinyl]-1-ethanone (4f, C16H18N2O2S)
Reaction time: 3 days. Recrystallized from ethyl acetate=
petroleum ether, mp 150.0–151.1ꢁC. 1H NMR (300 MHz,
CDCl3 þ (CD3)2CO): ꢂ ¼ 1.42 (3H, t, J ¼ 6.9 Hz), 2.44 (3H,
s), 4.46 (2H, q, J ¼ 6.9 Hz), 7.51–7.79 (3H, m), 8.10–8.14
(2H, m) ppm; 13C NMR (75 MHz, CDCl3 þ (CD3)2CO):
ꢂ ¼ 14.09 (CH3), 22.40 (CH3), 61.89 (CH2), 128.30 (CH),
128.44 (C), 128.95 (CH), 131.33 (C), 132.55 (C), 156.78 (C),
158.41 (C), 166.44 (C), 182.28 (C) ppm.
Recrystallized from CH2Cl2=petroleum ether, mp 162.4–
164.0ꢁC. 1H NMR (300MHz, CDCl3): ꢂ ¼ 2.32 (3H, s),
2.64 (3H, s), 3.33 (2H, t, J ¼ 5.1Hz), 4.20 (2H, t, J ¼ 5.1 Hz),
7.48–7.51 (5H, m) ppm; 13C NMR (75 MHz, CDCl3):
ꢂ ¼ 17.14 (CH3), 30.72 (CH3), 49.42 (CH2), 59.49 (CH3),
70.78 (CH2), 128.73 (CH), 129.08 (CH), 130.84 (CH),
137.36 (C), 142.90 (C), 156.56 (C), 164.10 (C), 201.83 (C)
ppm; IR (KBr): ꢃꢀ¼ 1702, 1595 cmꢂ1
.
1-[4-(5-Acetyl-6-methyl-2-phenyl-4-
Ethyl 1-benzyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydro-
5-pyrimidinecarboxylate (4g, C21H20N2O2S)
pyrimidinyldisulfanyl)-6-methyl-2-phenyl-5-
Recrystallized from ethyl acetate=petroleum ether, mp 119.1–
120.1ꢁC. 1H NMR (300MHz, CDCl3): ꢂ ¼ 1.39 (3H, t,
J ¼ 7.2 Hz), 2.20 (3H, s), 4.41 (2H, q, J ¼ 7.2 Hz), 5.17 (2H,
s), 6.97 (2H, d, J ¼ 6.3Hz) 7.32–7.50 (8H, m) ppm; 13C NMR
(75 MHz, CDCl3): ꢂ ¼ 13.99 (CH3), 17.19 (CH3), 53.63 (CH2),
62.24 (CH2), 125.20 (CH), 125.31 (CH), 128.18 (CH), 128.53
(CH),128.69(CH),129.56(CH),130.78(C),131.93(C),133.34
(C), 134.27 (C), 137.99 (C), 143.28 (C), 156.69 (C), 166.00 (C),
pyrimidinyl]-1-ethanone (5a, C26H22N4O2S2)
Reaction time: 6 days. Recrystallized from ethyl ether=
petroleum ether, mp 189.0–190.1ꢁC. 1H NMR (300 MHz,
CDCl3): ꢂ ¼ 2.35 (3H, s), 2.65 (3H, s), 7.54–7.63 (3H, m),
8.01 (2H, d, J ¼ 7.5 Hz) ppm; 13C NMR (75 MHz, CDCl3):
ꢂ ¼ 22.25 (CH3), 30.62 (CH3), 127.39 (CH), 129.45 (CH),
130.52 (C), 133.05 (CH), 136.52 (C), 155.51 (C), 158.15 (C),
179.28 (C), 201.95 (C) ppm; IR (KBr): ꢃꢀ¼ 1694, 1558, 1218,
193.96 (C) ppm; IR (KBr): ꢃꢀ¼ 1730, 1599, 1238 cmꢂ1
.
1199 cmꢂ1
.
Ethyl 1-butyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydro-
5-pyrimidinecarboxylate (4h, C18H22N2O2S)
1-(1-Benzyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydro-
5-pyrimidinyl)-1-ethanone (4c, C20H18N2OS)
Recrystallized from ethanol, mp 174.6–175.6ꢁC. 1H NMR
(300MHz, CDCl3): ꢂ ¼ 0.72 (3H, t, J ¼ 7.2 Hz), 1.11 (2H,
sextet, J ¼ 7.2 Hz) 1.41 (3H, t, J ¼ 7.2 Hz), 1.49 (2H, quintet,
J ¼ 7.8Hz), 2.34 (3H, s), 3.90 (2H, d, J ¼ 7.8 Hz), 4.32 (2H, q,
J ¼ 7.2Hz), 7.48–7.50 (5H, m) ppm; 13C NMR (75 MHz,
CDCl3): ꢂ ¼ 13.34 (CH3), 14.22 (CH3), 17.11 (CH3), 19.72
(CH2), 32.25 (CH2), 50.03 (CH2), 62.46 (CH2), 128.53 (CH),
129.00 (CH), 130.90 (CH), 132.16 (C), 133.60 (C), 142.55
(C), 156.45 (C), 166.26 (C), 193.34 (C) ppm; IR (KBr):
Trituration with n-hexane, mp 163.8–164.2ꢁC. 1H NMR
(300 MHz, CDCl3): ꢂ ¼ 2.14 (3H, s), 2.64 (3H, s), 5.20 (2H,
s), 6.97 (2H, d, J ¼ 6.9 Hz), 7.53 (8H, m) ppm; 13C NMR
(75 MHz, CDCl3): ꢂ ¼ 16.67 (CH3), 30.67 (CH3), 53.87 (CH),
125.50 (CH), 128.51 (CH), 128.82 (CH), 128.98 (CH), 129.82
(CH), 131.13 (CH), 134.46 (C), 137.99 (C), 142.47 (C), 157.06
(C), 194.06 (C), 201.43 (C) ppm; IR (KBr): ꢃꢀ¼ 1708, 1596,
1289, 1127 cmꢂ1
.
ꢃꢀ¼ 3052, 1721, 1603cmꢂ1
.
1-(1-Butyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydro-
5-pyrimidinyl)-1-ethanone (4d, C17H20N2OS)
Ethyl 1-(2-hydroxyethyl)-6-methyl-2-phenyl-4-thioxo-
Recrystallized from CH2Cl2=petroleum ether, mp 222.3–
223.2ꢁC. 1H NMR (300MHz, CDCl3): ꢂ ¼ 0.72 (3H, t,
J ¼ 7.5 Hz), 1.10 (2H, sextet, J ¼ 7.5 Hz), 1.50 (2H, quintet,
J ¼ 7.5 Hz), 2.29 (3H, s), 2.63 (3H, s), 3.91 (2H, m), 7.49–
7.53 (5H, m) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.11
(CH3), 16.15 (CH3), 19.18 (CH3), 30.34 (CH2), 32.00 (CH2),
49.82 (CH2), 128.35 (CH), 128.78 (CH), 130.74 (CH), 133.32
(C), 137.68 (C), 141.53 (C), 156.35 (C), 193.08 (C), 201.36
1,4-dihydro-5-pyrimidinecarboxylate (4i, C16H18N2O3S)
Recrystallized from ethanol, 1H NMR (300 MHz, DMSO-d6):
ꢂ ¼ 1.29 (3H, t, J ¼ 6.9 Hz), 2.35 (3H, s), 3.38 (1H, br) 3.40
(2H, t, J ¼ 5.4Hz), 4.04 (2H, t, J ¼ 5.4Hz), 4.26 (2H, q,
J ¼ 6.9 Hz), 7.53-7.61 (5H, m) ppm; 13C NMR (75 MHz,
DMSO-d6): ꢂ ¼ 13.69 (CH3), 17.27 (CH3), 51.59 (CH2), 58.86
(CH2), 61.15 (CH2), 128.25 (CH), 128.38 (CH), 129.96 (CH),
130.53 (C), 134.53 (C), 145.29 (C), 156.36 (C), 165.59 (C),
(C) ppm; IR (KBr): ꢃꢀ¼ 1699, 1590, 1287 cmꢂ1
.
191.85 (C) ppm; IR (KBr): ꢃꢀ¼ 3408, 1727, 1606 cmꢂ1
.
1-[1-(2-Hydroxyethyl)-6-methyl-2-phenyl-4-thioxo-1,4-
dihydro-5-pyrimidinyl]-1-ethanone (4e, C15H16N2O2S)
Recrystallized from CH2Cl2=petroleum ether, mp 235.7–
236.6ꢁC. 1H NMR (300 MHz, DMSO-d6): ꢂ ¼ 2.29 (3H, s),
2.52 (3H, s), 3.42 (2H, t, J ¼ 5.4Hz), 4.01 (2H, t, J ¼ 6.0 Hz),
7.55–7.62 (5H, m) ppm; 13C NMR (75 MHz, DMSO-d6): ꢂ ¼
17.37 (CH3), 31.13 (CH3), 52.38 (CH2), 59.70 (CH2), 129.13
(CH), 129.21 (CH), 130.81 (CH), 135.06 (C), 137.29 (C),
Ethyl 4-cyclohexylamino-6-phenyl-2-thioxonicotinate
(6, C20H24N2O2S)
Recrystallized from ethanol, 1H NMR (300MHz, CDCl3):
ꢂ ¼ 1.40 (3H, t, J ¼ 7.2 Hz), 1.30–1.88 (10H, m), 3.56 (1H,
m), 4.36 (2H, q, J ¼ 7.2Hz), 6.41 (1H, s), 7.46–7.52 (3H, m),
7.85 (2H, d, J ¼ 5.7 Hz), 10.16 (1H, br) ppm; 13C NMR
(75 MHz, DMSO-d6): ꢂ ¼ 14.43 (CH3), 24.20 (CH2), 25.24

118
S. Cunha et al.
(CH2), 33.05 (CH2), 51.78 (CH), 60.50 (CH), 91.79 (CH),
126.27 (C), 128.84 (C), 131.36(C), 131.47 (CH), 159.71 (C),
160.41 (C), 162.04 (C), 170.12 (C) ppm.
21.02 (CH3), 30.70 (CH3), 48.42 (CH2), 61.82(CH2), 129.11
(CH), 129.47 (CH), 161.59 (CH), 133.27 (C), 138.00 (C),
142.14 (C), 156.85 (C), 170.49 (C), 193.66 (C), 201.59 (C)
ppm; IR (KBr): ꢃꢀ¼ 1741, 1697 cmꢂ1
.
Ethyl 4-(5-ethyloxycarbonyl-6-methyl-2-phenyl-
2-(5-Ethyloxycarbonyl-6-methyl-2-phenyl-4-thioxo-1,4-
4-pyrimidinyldisulfanyl)-6-methyl-2-phenyl-
dihydro-1-pyrimidinyl)ethyl acetate (4k, C18H20N2O4S)
1
5-pyrimidinecarboxylate (5b, C28H26N4O4S2)
Yield 85% mp 190.2–192.0ꢁC; H NMR (300MHz, CDCl3):
To a solution of 1.0 mmol enaminone 1a in 5 cm3 dry ethyl
ether was added dropwise a solution of 1.2mmol benzoyl
isothiocyanate in 5 cm3 dry ethyl ether under ice-bath cooling
and magnetic stirring. After 15 min the ice-bath was removed
and the reaction mixture was left for 30 min at room tempera-
ture, after which time the solvent was evaporated. 10cm3
ethyl alcohol were added and the solution was reacted at
reflux, while the progress of the reaction was monitored by
TLC. After 1 day, the solvent was evaporated and the residue
was recrystallized from ethyl acetate=petroleum ether to give
75% 5b, mp 129.7–130.4ꢁC. 1H NMR (300MHz, CDCl3):
ꢂ ¼ 1.42 (3H, t, J ¼ 7.0 Hz), 2.44 (3H, s), 4.45 (2H, q, J ¼
7.0 Hz), 7.51–7.63 (3H, m), 8.05–8.08 (2H, m) ppm;
13C NMR (75 MHz, CDCl3): ꢂ ¼ 14.03 (CH3), 22.73 (CH3),
62.05 (CH2), 127.67 (CH), 129.21 (CH), 131.20 (C), 132.76
(C), 156.61 (C), 159.99 (C), 166.07 (C), 178.51 (C) ppm; IR
ꢂ ¼ 1.40 (3H, t, J ¼ 7.2 Hz), 1.99 (3H, s), 2.37 (3H, s), 4.02 (2H,
t, J ¼ 5.7 Hz), 4.27 (2H, t, J ¼ 5.7 Hz), 4.43 (2H, q, J ¼ 7.2 Hz),
7.50–7.59 (5H, m) ppm; IR (KBr): ꢃꢀ¼ 1744, 1729cmꢂ1
.
3-(3,5-Dimethyl-1-phenyl-1H-4-pyrazolyl)-1,5-diphenyl-
1H-1,2,4-triazole (7, C25H21N5)
To a solution of 788.7 mg (5.1mmol) enaminone 1k in 15cm3
n-hexane were added 849.8 mg (5.2mmol) benzoyl isothio-
cyanate. After 30min at room temperature the solvent was
evaporated and 10cm3 ethanol and 1.0 cm3 (10.2 mmol) phe-
nylhydrazine were added. After 91h the solvent was evapo-
rated and the residue was recrystallized from ethyl alcohol=
1
petroleum ether to give 5% 7, mp 188.2–189.0ꢁC. H NMR
(300MHz, CDCl3): ꢂ ¼ 2.69 (6H, s, 2CH3), 7.34–7.59 (15H,
m) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 12.79 (CH3), 14.19
(CH3), 110.94 (C), 125.56 (CH), 125.61 (CH), 127.92 (CH),
128.53 (C), 128.75 (CH), 128.78 (CH), 129.23 (CH), 129.29
(CH), 129.52 (CH), 130.09 (C), 138.73 (C), 139.72 (C),
139.89 (C), 149.14 (C), 153.86 (C), 158.40 (C) ppm.
(KBr): ꢃꢀ¼ 1731, 1581, 1567, 1233, 1086cmꢂ1
.
Synthesis of the Intermediate Benzamides 3
To a solution of 1.0mmol enaminone 1a–1j in 5 cm3 n-hexane
was added dropwise a solution of 1.1 mmol benzoyl isothio-
cyanate in 5 cm3 n-hexane under ice-bath cooling and mag-
netic stirring. After 15min the ice-bath was removed and the
reaction mixture was left for 30 min at room temperature, after
which time the solvent was evaporated. The residue was
recrystallized from ethyl acetate=petroleum ether.
Antibacterial and Antifungal Assay. Determination
of Minimal Inhibitory Concentration (MIC)
Values are means of three experiments. The bacteria cultures
used were grown for 24 h at 35ꢁC on nutrient agar. The fungi
and yeasts were cultivated for 72h at 26ꢁC on malt extract
agar and yeast malt agar. The inocula for the assays were
prepared by cell suspensions according to McFarland scale
0.5, except of filamentous fungi for which a modified method
was used [11]. Broth microdilution method was carried out to
determine the MIC of the compounds against the microrgan-
isms in sterile 96-well microplates. The 20% DMSO aqueous
stock solutions of the compounds were transferred into the
first well from which serial dilutions were performed so that
concentrations ranged from 100 to 0.78 mg=cm3. Chloramphen-
icol and olamine ciclopirox were used as the reference drugs
against bacteria and fungi. Aqueous DMSO (20%) was used as
negative control. The inoculum was added to all wells and the
plates were incubated under appropriate conditions. After
incubation, microrganisms’ growth was observed by the pres-
ence of turbidity on the well. MIC was defined as the lowest
concentration of the substances that inhibited visible growth.
N-[(E)-2-Acetyl-3-amino-1-thioxo-2-butenyl]benzamide
(3a, C13H14N2O2S)
Yield 75%, mp 130.0–131.0ꢁC; 1H NMR (300 MHz, CDCl3):
ꢂ ¼ 2.16 (3H, s), 2.21 (3H, s), 7.53 (2H, m), 7.63 (1H, m), 7.91
(2H, m) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 22.46 (CH3),
28.12 (CH3), 114.52 (C), 127.88 (CH), 129.20 (CH), 132.34
(C), 133.55 (CH), 160.82 (C), 162.31 (C), 193.01 (C), 207.12
(C) ppm.
Acetylation of 4-Thioxopyrimidines 4e and 4i
To a solution of 1.0 mmol 4e, 4i in 10cm3 DMF were added 5
drops of pyridine and 10 cm3 acetic anhydride. After 30min,
30cm3 CH2Cl2 were added and the mixture was extracted
with brine (5ꢃ30 cm3). The organic phase was dried over
MgSO4, filtered, and the solvent was evaporated, affording
pure 4j and 4k.
Anti-Trypanosoma cruzi Assay
Parasites epimastigotes of Trypanosoma cruzi Y-strain were
cultivated at 28ꢁC in plastic flasks containing 5 cm3 liver infu-
sion trypticase medium inoculated with 5ꢃ106 cells=cm3 and
supplemented with 10% fetal calf serum. Cells from the mid-
log phase were harvested by centrifugation at 2.500 rpm and
fixed in formaldehyde and axenic prolifareation was assessed
by counting in Neubauer chambers under light microscopy.
2-(5-Acetyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydro-
1-pyrimidinyl)ethyl acetate (4j, C17H18N2O3S)
Yield 90%, yellow solid, mp 207.0–207.8ꢁC; 1H NMR
(300 MHz, CDCl3): ꢂ ¼ 2.00 (3H, s), 2.33 (3H, s), 2.63 (3H,
s), 4.04 (2H, t, J ¼ 5.7 Hz), 4.31 (2H, t, J ¼ 5.7 Hz), 7.50–7.59
(5H, m) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 17.15 (CH3),

4-Thioxopyrimidines with Antimicrobial Activities
119
Parasite’s growth in the absence or presence of increasing
concentrations of the different drugs was assessed by the
absorbance determined at 610 nm. The data are representative
of a minimum of three independent experiments performed
in triplicate, which yielded analogous results. Significant
[3] a) Hsung RP, Wei L-L, Sklenicka HM, Douglas CJ,
McLaughlin MJ, Mulder JA, Yao LJ (1999) Org Lett 1:
509; b) Hickmott PW, Sheppard G (1971) J Chem
Soc C 2112; c) Palvannan K, Stille JR (1992) J Org
Chem 57: 5319; For reviews see: d) Harrity JPA,
Provoost O (2005) Org Biomol Chem 3: 1349;
e) Hsung RP, Kurdyumov AV, Sydorenko N (2005)
Eur J Org Chem, 23
ꢀ
ꢀꢀ
differences in control relation ( P<0.05,
ꢀꢀ
P<0.01 and
P<0.001) were statistically analyzed using ANOVA.
[4] For reviews see: a) Ferrar HMC, Pereira FLC (2004)
Quim Nova 27: 89; b) Kascheres C (2003) J Braz Chem
Soc 14: 945; c) Negri G, Kascheres C, Kascheres AJ
(2004) J Heterocyclic Chem 41: 461
Crystallographic Data Collection and Structure
Determination of Compounds 4b, 5b, and 7
Single crystals X-ray diffraction data were collected at room
temperature using a Nonius CAD-4 diffratometer [12] with
[5] a) Goerdeler J, Pohland WH (1963) Chem Ber 96: 526;
b) Goerdeler J, Gnad J (1965) Chem Ber 98: 1531;
c) deStevens G, Smolisnky B, Dorfman L (1964) J
Org Chem 29: 1115; d) Erian AW (1999) J Prakt Chem,
341; e) Greenhill JV, Hanaee J, Steel PJ (1990) J Chem
Soc Perkin Trans 1, 1869; f) Carney RWJ, Wojtkunski J,
Fechtig B, Puckett RT, Biffar B, deStevens G (1971)
˚
CuKa radiation (ꢄ ¼ 1.54180 A). The structures were solved
by direct methods and refined anisotropically with full-matrix
least-squares on F2 using SHELXL97 [13]. The hydrogen
atoms were placed at calculated positions, except those in-
volved in H-bonds and weak interactions found on difference
maps, and refined with riding constraints. The crystallographic
data were deposited at the Cambridge Crystallographic
Data Center under the numbers CCDC 601662, 601657, and
601658. Copies of the data can be obtained, free of charge
via www.ccdc.cam.ac.uk=conts=retrieving.html (or from the
Cambrigde Crystallographic Data Centre, CCDC, 12 Union
Road, Cambridge, CB2 1EZ, UK (fax þ44 1223 336033, or
e-mail: deposit@ccdc.cam.ac.uk).
´
´
J Org Chem 36: 2602; g) Uher M, Ilavsky D, Foltın J,
ꢁ
´
Skvareniva K (1981) Collect Czech Chem Commun 46:
3128; h) El-Dean AMK, Abdel-Monea ME (2002) J Chin
Chem Soc 49: 1057
1
ꢁ
´
ꢁ
[6] For H NMR data of 3a at 50 C see: Machacev V,
ꢁ
˘
El-Bahaie S, Sterba V (1981) Collect Czech Chem
Commun 46: 256
[7] Consumption of 3 is easily followed because the solution
changes its color from red to yellow
Acknowledgements
[8] Farrugia LJ (1997) J Appl Cryst 30: 565
[9] Missio L, Braibante HS, Braibante MEF (1996)
J Heterocyclic Chem 33: 1243
[10] a) Braibante MEF, Braibante HS, Missio L, Andricopulo
A (1994) Synthesis, 898; b) Braibante MEF, Braibante,
HS, Salvatore SJSA (1990) Quim Nova 13: 67; c)
Braibante HS, Braibante MEF, Rosso GB, Oriques
DA (2003) J Braz Chem Soc 14: 994; d) Braibante
MEF, Braibante HS, Morel AF, Costa CC, Lima MG
(2006) J Braz Chem Soc 17: 184
The authors gratefully acknowledge the financial support of
´
`
gico – CNPq and Fundac°a˜o de Amparo a Pesquisa do Estado
da Bahia – FAPESB. We also thank FEPESB for a fellowship
to G.A.N.C. and CNPq for scholarships to R.M.B. and P.O.S.
(PIBIC-UFBA-CNPq), and for research fellowships to I.V.
and S.C.
´
Conselho Nacional de Desenvolvimento Cientıfico e Tecnolo-
References
[11] Kusucu C, Rapino B, McDermott L, Hadley S (2004)
J Clin Microbiol 42: 1224
[12] Enraf-Nonius (1993) CAD-4=PC 1.2 Enraf-Nonius,
Delft, The Netherlands
[1] a) Cocco MT, Congiu C, Onnis V, Piras R (2001)
Farmaco 56: 741; b) Cocco MT, Congiu C, Onnis V
(1995) Farmaco 50: 73
[2] For representative papers see: a) Aggarwal V, Ila H,
Junjappa H (1982) Synthesis, 65; b) Carney RWJ,
Wojtkunski J, deStevens G (1964) J Org Chem 29: 2887
[13] Sheldrick GM (1997) SHELXS97 and SHELXL9
Program for Crystal Structure Refinement; University
¨
of Gottingen, Germany