Synthesis and antimicrobial activity of new (4,4,4-trihalo-3-oxo-but-1- enyl)-carbamic acid ethyl esters, (4,4,4-trihalo-3-hydroxy-butyl)-carbamic acid ethyl esters, and 2-oxo-6-trihalomethyl-[1,3]oxazinane-3-carboxylic acid ethyl esters

Article abstract of DOI:10.1016/j.bmc.2005.12.031

This work presents a three-step synthesis of a new series of 4-substituted 2-oxo-6-trihalomethyl-[1,3]oxazinane-3-carboxylic acid ethyl esters, from β-alkoxyvinyl trihalomethyl ketones of general formula X ₃C-C(O)-CHC(R)-OR¹, where R

Full text of DOI:10.1016/j.bmc.2005.12.031

Bioorganic & Medicinal Chemistry 14 (2006) 3174–3184
Synthesis and antimicrobial activity of new
(4,4,4-trihalo-3-oxo-but-1-enyl)-carbamic acid ethyl esters,
(4,4,4-trihalo-3-hydroxy-butyl)-carbamic acid ethyl esters, and
2-oxo-6-trihalomethyl-[1,3]oxazinane-3-carboxylic acid ethyl esters
Nilo Zanatta,a,* Deise M. Borchhardt,a Sydney H. Alves,b Helena S. Coelho,a
Adriana M. C. Squizani,a Tiago M. Marchi,a Helio G. Bonacorsoa and
Marcos A. P. Martinsa
aNu´cleo de Quı´mica de Heterociclos (NUQUIMHE), Departamento de Quı´mica, Universidade Federal de Santa Maria,
97.015-900, Santa Maria, RS, Brazil
bLaborato´rio de Pesquisas Micolo´gicas (LAPEMI), Departamento de Microbiologia e Parasitologia,
Universidade Federal de Santa Maria, 97.015-900, Santa Maria, RS, Brazil
Received 14 October 2005; revised 14 December 2005; accepted 16 December 2005
Available online 18 January 2006
Abstract—This work presents a three-step synthesis of a new series of 4-substituted 2-oxo-6-trihalomethyl-[1,3]oxazinane-3-carbox-
ylic acid ethyl esters, from b-alkoxyvinyl trihalomethyl ketones of general formula X3C–C(O)–CH@C(R)–OR1, where R = H, Me,
Ph, and 4-Me-Ph; R1 = Me and Et; and X = F and Cl. The Michael addition–substitution of the ethyl carbamate on b-alkoxyvinyl
trihalomethyl ketones furnished the corresponding (4,4,4-trihalo-3-oxo-but-1-enyl)-carbamic acid ethyl esters. These compounds
underwent reduction with NaBH4 leading to the respective (4,4,4-trihalo-3-hydroxy-butyl)-carbamic acid ethyl esters. The 3-hy-
droxy-butyl carbamates were submitted to cyclization reaction with triphosgene to give a series of 4-substituted 2-oxo-6-trihalo-
methyl-[1,3]oxazinane-3-carboxylic acid ethyl esters. The in vitro antimicrobial activity, of some of the three new series of the title
compounds, was assessed against a panel of microorganisms including yeast like fungi, bacteria, and algae, and their minimal inhib-
itory concentration and minimal fungicidal, bactericidal, and algacidal concentrations were determined. Some of the analyzed
carbamates exhibited significant in vitro antimicrobial activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
of immunosuppressed patients. These patients now
survive longer and become highly susceptible to life-
threatening fungal infections.1 Concomitant with the in-
creased incidence of fungal infections there has been a
dramatic increase in the use of antifungals for the treat-
ment of both systemic and localized fungal infections. In
this context, the expanded use of antifungal agents has
accelerated the development of antifungal drug resis-
tance followed by frequent therapeutic failures and
increasing mortality rate.2 The most important and
dreadful microorganisms include fungi such as Candida
albicans, Candida glabrata, Candida dubliniensis, and
Cryptococcus neoformans, and bacteria such as Staphy-
lococcus aureus, Pseudomonas aeruginosa, and Esche-
richia coli. Prototheca zopfii, an alga of medical and
veterinary concern may, be added to this list.3 The anti-
mycotic drugs available for the treatment of systemic
fungal infections are limited (11 active compounds)
A higher incidence of microbial infections caused by
fungi, yeast, bacteria, and algae has been documented
since the 1980s with the parallel emergence of either
new pathogens or the resistance phenomenon. Fungi de-
serve attention because they are emerging as important
nosocomial pathogens causing severe morbidity and
mortality in immunocompromised patients. Modern
therapies and management such as bone marrow or sol-
id-organ transplants, new and more aggressive chemo-
therapy have resulted in a rapidly expanding number
Keywords: Carbamates; Carbamic acid ethyl ester; 1,3-Oxazinan-2-
ones; Antimicrobial activity; Minimal inhibitory concentration; MIC.
*
Corresponding author. Tel./fax: +55 55 3220 8756; e-mail:
zanatta@base.ufsm.br
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.031

N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
3175
which can be divided into four classes as follows: (i)
polyene macrolides; (ii) azole derivatives; (iii) DNA
and RNA inhibitors; and (iv) 1,3-b-glucan synthase
inhibitors (echinocandins).4 Hence, there is a great de-
mand for novel agents with a widened spectrum of
activity and reduced toxicity.
trichloromethyl containing heterocycles are more active
than the corresponding trifluoromethyl substituted ana-
logues13 as, for example, NTPDase inhibitory effect in
synaptosomes from rat cerebral cortex,13a antinocicep-
tive effect in mice,13b and hypothermic and antipyretic
effects.13c
Biological evaluation of carbamate function containing
compounds was reported recently.5,6 The introduction
of a carbamate group in organic molecules has shown
promising antimicrobial activity.7 Furthermore, carba-
mate groups are frequently used as prodrug linkers or
as versatile synthetic building blocks that allow one to
combine structure novelty with the potential for stream-
line synthesis.5,8 Carbamates are attractive due to the
easy preparation and chemical stability.9,10 Also, cyclic
carbamates are described in the literature as biologically
active compounds showing, for example, antitubercular
activity.11 On the other hand, it has been reported that
the introduction of a trifluoromethyl group in heterocy-
cles frequently results in much more potent activity than
that of the parent compounds, a fact that is probably
related to the high lipophilicity of the trifluoromethyl
group.12 Recently, it has been shown that, in some cases,
In the course of our research concerning the application
of b-alkoxyvinyl trihalomethyl ketones to obtain triha-
lomethylated compounds and considering the pharma-
cological and synthetic importance of carbamates,9,10
1,3-aminoalcohols,14 1,3-oxazinan-2-ones,15 and trihalo-
genated compounds,12,13 we are reporting the synthesis
of 4-substituted 2-oxo-6-trihalomethyl-[1,3]oxazinane-
3-carboxylic acid ethyl esters 7a–d and 8a–d, as well as
their intermediate compounds (4,4,4-trihalo-3-oxo-but-
1-enyl)-carbamic acid ethyl esters 3a–d and 4a–d and
the respective (4,4,4-trihalo-3-hydroxy-butyl)-carbamic
acid ethyl esters 5a–d and 6a–d, which are all new com-
pounds (Scheme 1). The obtained trihalomethylated car-
bamates were evaluated for their in vitro antimicrobial
activity against 18 microorganisms such as yeast like
fungi, bacteria, and alga considered important human
pathogens.
O
R
O
NHCO2Et
R
(i)
X3C
OR1
41-80%
X3C
3a-d, 4a-d
1a-d, 2a-d
(ii)
(ii)
31-70%
38-82%
R
R
R
HO
F3C
H
HO
H
HO
Cl3C
H
NHCO2Et
Cl3C
NHCO2Et
NHCO2Et
5a-d
6'a-c
6a-d
(iii)
(iii)
20-30%
25-54%
CCl3
CF3
R
O
O
HO
Cl3C
H
NHCO2Et
R
N
O
R
N
O
CO2Et
8a-d
CO2Et
6'a-c
7a-d
X
F
Compounds
1, 3, 5, 7
l 2, 4, 6, 8
Compound
R
H
Me
Ph
R1
Et
Me
Me
a
b
c
C
d
4-Me-Ph Me
Scheme 1. Reagents and conditions: (i) NH2COOEt, CH2Cl2/CHCl3, p-TsOH, 50/70 ꢁC, 24–48 h; (ii) NaBH4, EtOH, rt/70 ꢁC, 5–24 h;
(iii) (CCl3O)2CO, (CH2)2Cl2, Et3N, 85 ꢁC, 5–22 h.

3176
N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
2. Chemistry
ion current, registered on a GC–MS equipped with an
achiral capillary column. This is an indication that only
a pair of enantiomers was formed and that the reduction
reaction was stereoselective.
Scheme 1 outlines the synthetic strategy developed to
obtain the [1,3]oxazinan-2-ones 7a–d and 8a–d. This
strategy starts with the reaction of the readily available
4-alkoxy-1,1,1-trifluoro[chloro]-alk-3-en-2-ones (1 and
2)16 with ethyl carbamate to give a series of (4,4,4-tri-
halo-3-oxo-but-1-enyl)-carbamic acid ethyl esters 3a–d
and 4a–d.
The structures of all synthesized compound were ana-
lyzed by GC–MS, 1H, and 13C NMR spectroscopy
and the data are reported in Section 6. Figure 1 shows
the atom numbering used for the NMR assignment of
compounds 3–8.
Although the Michael addition reaction of amines to 4-
alkoxy-1,1,1-trihalo-alk-3-en-2-ones (enones), followed
by the elimination of the 4-alkoxy group, leading to
the corresponding enamino ketones has been widely
reported,17 the reaction of weak nitrogen nucleophiles
such as amides and carbamates with enones is not very
well developed.18 In this work, the reaction of enones
1 and 2 with ethyl carbamate was carried out in dichlo-
romethane or chloroform in the presence of p-toluene-
sulfonic acid under reflux for 24–48 h. It was observed
that the substitution on the b-carbon makes the attack
of the ethyl carbamate on the b-alkoxyvinyl ketones
more difficult. For the 1-substituted 4,4,4-trihalo-3-
oxo-but-1-enyl carbamates (e.g., 3b–d and 4b–d) it was
necessary to use an excess of ethyl carbamate and longer
reflux time in order to obtain the desired products.
Compounds 3 and 4 are in the Z form, which is evi-
denced by the deshielding nature of the N–H chemical
shift in the range of 10.5–11.5 ppm.17
The major evidence for the reduction of the 4,4,4-tri-
halo-3-oxo-but-1-enyl carbamates 3a–d and 4a–d to
the respective 3-hydroxy-butyl carbamates 5a–d and
6a–d comes from the chemical shift changes observed
for the C-2, C-3, and C-4 (see Fig. 1). For example,
the chemical shift of the C-2 (C@O) shifted from the
range of 179–182 ppm, for the 3-oxo-but-1-enyl carba-
mates 3a–d and 4a–d, to 66–84 ppm for the 3-hydroxy-
butyl carbamates 5a–d and 6a–d. The chemical shift of
the carbons of the carbon–carbon double bond (C-3
and C-4) shifted from the range of 94–99 and 146–
165 ppm, respectively, for the 3-oxo-but-1-enyl carba-
mates, to 29–38 and 36–53 ppm, respectively, for the
3-hydroxy-butyl carbamates. Also the reduction can be
1
confirmed from the H NMR spectra by observing the
disappearance of the doublet and the doublet of dou-
blets of the vinylic hydrogens H-3 and H-4 in the range
of 5.7–6.5 and 7.6–8.0 ppm, respectively, for the 4,4,4-
trihalo-3-oxo-but-1-enyl carbamate to appear as com-
plex multiplets in the range of 1.6–2.6 and 3.5–
5.3 ppm, respectively, for the 3-hydroxy-butyl carba-
mates. The 3-hydroxy-butyl carbamates also showed
a multiplet at about 3.3–4.3 ppm relative to the H-2,
absent in the enaminones.
The reduction of compounds 3 and 4 with sodium boro-
hydride in ethanol at room temperature furnished the
corresponding (4,4,4-trihalo-3-hydroxy-butyl)-carbamic
acid ethyl esters 5 and 6, in moderate to good yields.
The 4,4,4-trihalo-3-oxo-but-1-enyl carbamates 3a–d
and 4d underwent reduction of both the carbon–carbon
double bond and the carbonyl giving only the corre-
sponding trihalomethylated 3-hydroxy-butyl carba-
mates 5a–d and 6d, while the 4,4,4-trichloro-3-oxo-but-
1-enyl carbamates 4a–c furnished a mixture of the
desired 6a–c and the partially reduced 4,4,4-trichloro-
3-hydroxy-alk-1-enyl carbamates 60a–c in approximate-
ly 1:1 ratio. In our hands, the separation of compounds
6a–c from 60a–c was not possible by column chromato-
graphy, so the cyclization step was carried out with the
mixture of both 6 and 60. It is interesting to note that the
reduced compounds, such as 5b–d and 6b–d, which have
more than one stereogenic center, presented only a sin-
The [1,3]oxazinan-2-ones 7 and 8 were obtained by the
cyclization of 3-hydroxy-butyl carbamates 5 and 6 with
triphosgene in dichloroethane in the presence of triethyl-
amine, under argon atmosphere, for 5 or 22 h at reflux.
The literature reports that the cyclization of 1,3-amino
alcohols to construct 1,3-oxazin-2-ones has been done
using phosgene14,19 or by an intramolecular cyclization
of a 1,3-hydroxy carbamate in the presence of a strong
base.20,21 In this work, the inconvenience of handling
the highly toxic phosgene gas was avoided by using a
crystalline stable solid triphosgene22 to successfully per-
form the cyclocarbonylation of the 3-hydroxy-butyl car-
bamates 5 and 6 to the respective [1,3]oxazinan-2-ones 7
and 8. We also tried to perform the intramolecular cycli-
1
gle set of signals in both H and 13C NMR spectra. In
addition, a single peak was observed in the total
CX3
5
6
7
8
O
2
NHCO2CH2CH3
OH
2
R
6
O
5
4
1
2
4
X3C
1
R
3
N
X3C
1
NHCO2CH2CH3
4
3
R
O
3
5
6
7
8
3, 4
5, 6
CO2CH2CH3
7
8
9
7, 8
Figure 1. Atom numbering used for the NMR assignment of compounds 3–8.

N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
3177
zation of 5 and 6 in the presence of strong bases, accord-
ing to the literature,20,21 but these procedures did not
furnish the corresponding [1,3]oxazinan-2-ones 7 and 8.
carbamates precursors 5b–d and 6b–d they present only
one set of signals in both the 1H and the 13C NMR spec-
tra. In addition, a single peak was observed in the mass
chromatogram registered on a GC–MS equipped with
an achiral capillary column. This indicates that only a
pair of enantiomers has been obtained for the
compounds 7b–d and 8b–d. To confirm this hypothesis,
1H and 13C NMR spectra were registered in the presence
of tris-[3-trifluoromethyl-hydroxymethylen-d-camphor-
ate]-europium, a chiral lanthanide shift reagent, in a mo-
lar ratio of 1:0.3 of the 7d and the lanthanide,
respectively. The NMR spectra registered under this
The cyclization of the mixtures of 6a–c and 60a–c carried
out with triphosgene underwent cyclization only the
double reduced compounds 6a–c furnishing the respec-
tive [1,3]oxazinan-2-ones 8a–c. Thus, the product
mixture of 60a–c and 8a–c was purified by column chro-
matography obtaining 8a–c as pure compounds and
60a–c were not recovered. Table 1 reports the yields,
melting points, and elemental analysis of all compounds
synthesized in this work.
1
condition show that some signals in both the H and
the 13C spectra were duplicated and exhibited the same
intensity, suggesting that the [1,3]oxazinan-2-ones 7b–d
and 8b–d were obtained as a racemic mixture rather than
mixtures of diastereoisomers.
The [1,3]oxazinan-2-ones 7a–d and 8b–d display similar
1H and 13C NMR spectra as the corresponding amino
alcohol precursors. The main evidence of the formation
of compounds 7 and 8 is the appearance of a second car-
bonyl carbamate in the range of 146–148 ppm. Further-
more, it was observed that the resonance of H-6 of 7 and
8 moved downfield, in average, 0.40 ppm in relation to
the respective (H-2) of the 3-hydroxy-butyl carbamate
3 and 4 (see Fig. 1). In addition, the molecular ion in
the mass spectra showed that the oxazinanes 7 and 8
have 26 mass units more than the corresponding
3-hydroxy-butyl carbamates 5 and 6.
The 4-substituted 6-trichloromethyl-[1,3]oxazinan-2-
ones are good probes to obtain information about the
three-dimensional structure of these compounds by ana-
lyzing the coupling constants between the hydrogens of
the backbone structure (H-4, H-5/H-50, and H-6). The
trichloromethylated oxazinanes 8b–d are better probes
than the trifluoromethylated analogues 7b–d because
the splitting pattern of the H-6 signal is more simple
to analyze due to the lack of further coupling with the
fluorine nuclei. For example, compound 8c shows cou-
pling constants between the H-6 and H-5 axial and
The oxazinanes 7b–d and 8b–d have more than one
stereogenic center but as for the 3-hydroxy-butyl
Table 1. Melting points, yields, and elemental analysis of compounds 3–8
Compound
Mp (ꢁC)
Yielda (%)
Molecular formula
Elemental analysis
Calculated
H
Found
H
C
N
C
N
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
60a
60b
60c
7a
7b
7c
7d
8a
8b
8c
8d
69–72
Oil
Oil
80
70
64
47
80
48
68
41
78
58
38
82
40
40
36
31
35
30
30
51
25
54
28
C7H8NO3F3
C8H10NO3F3
C13H12NO3F3
39.82
42.67
54.36
55.82
32.27
35.00
46.39
47.96
39.07
41.92
53.61
55.08
31.78
34.49
45.84
47.41
32.03
34.75
46.11
39.84
42.36
53.00
54.38
33.07
35.49
45.86
47.33
3.82
4.48
4.21
4.68
3.10
3.67
3.59
4.02
5.62
6.16
5.54
5.94
4.57
5.07
4.73
5.12
3.84
4.37
4.17
4.18
4.74
4.45
4.87
3.47
3.97
3.85
4.24
6.63
6.22
4.88
4.65
5.38
5.10
4.16
3.99
6.51
6.11
4.81
4.59
5.29
5.03
4.11
3.95
5.34
5.06
4.14
5.81
5.49
4.41
4.23
4.82
4.60
3.82
3.68
39.81
42.90
54.63
57.13
32.54
35.13
47.09
48.31
38.88
41.85
3.81
4.49
4.23
4.93
2.95
3.91
3.29
3.97
5.56
6.44
6.09
4.61
4.71
5.27
4.86
3.80
3.64
6.95
5.97
Oil
C14H14NO3F3
118–123
Oil
Oil
C7H8NO3Cl3
C8H10NO3Cl3
C13H12NO3Cl3
Oil
Oil
C14H14NO3Cl3
C7H12NO3F3
C8H14NO3F3
C13H16F3NO3
C14H18NO3F3
C7H12Cl3NO3
C8H14Cl3NO3
C13H6Cl3NO3
C14H18NO3Cl3
C7H10Cl3NO3
C8H12Cl3NO3
C13H14Cl3NO3
C8H10NO4F3
C9H12NO4F3
C14H14NO4F3
C15H15NO4F3
C8H10Cl3NO4
C9H12Cl3NO4
Oil
Oil
5.95
b
108–110
Oil
Oil
55.51
5.88
b
4.61
b
b
Oil
Oil
47.27
5.51
b
4.06
Oil
Oil
b
b
Oil
80–85
Oil
39.77
42.69
52.98
54.64
4.16
4.74
4.45
5.65
5.57
4.48
3.98
95–100
60–70
4.86
b
79–83
153–156
Oil
22
20
20
35.97
46.08
3.95
3.91
4.47
3.54
C
C
14H14Cl3NO4
15H16Cl3NO4
b
a Yields after purification.
b Not registered.

3178
N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
H-5 equatorial of 11.2 and 1.8 Hz, respectively. Also,
the observed coupling constant between H-4 and H-5
axial and H-5 equatorial is 10.9 and 7.6 Hz, respectively.
These values of coupling constants are consistent with a
chair conformation and that the CCl3 occupies an equa-
torial position and the 4-substituent group is in a pseu-
do-equatorial position. All the other synthesized 4-
100 lL of the inoculum. The plates were incubated at
35 ꢁC for 24 h for bacteria and Candida species; 72 h
for S. cerevisae, C. neoformans, and P. zopfii. Growth
or a lack thereof in the antimicrobial agent containing
wells was determined by comparison with the growth
control, indicated by turbidity. All tests were carried
out in duplicate and the lowest concentration that com-
pletely inhibited visible growth of the organism was
recorded as the MIC.
substituted
6-trihalomethyl-[1,3]oxazinan-2-ones
showed similar coupling constants and, therefore, they
should have the same three-dimensional structure. This
observation is in agreement with the conformational
assignment study of some 3-acyloxy-1,3-oxazinanes
done by Ali and co-workers.23 According to these obser-
vations the compounds 7b–d and 8b–d, which have two
stereogenic centers, but presented only a single set of
signals in both the 1H and the 13C NMR spectra,
should be composed by a pair of enantiomers with
configuration 4-(R)/6-(R) and 4-(S)/6-(S).
The minimal fungicidal, bactericidal, and algacidal con-
centrations were determined by subculture of 20 lL of
the content of each well that remained clear. The media
employed were Sabouraud’s dextrose agar for fungi and
P. zopfii, and Mueller–Hinton agar for bacteria. The
plates were incubated at 35 ꢁC for the same duration
as for the MIC determinations and the lowest concen-
tration required to demonstrate complete absence of
growth was named cidal.
3. Biological activity
The interpretation of the results was based on fluconaz-
ole breakpoints for the fungi, amphotericin B for the
Prototheca, and based on imipenem for bacterial patho-
gens, according to M27-A224 and M7-A425 techniques,
respectively.
The in vitro antimicrobial activity of the carbamate
compounds was assessed against a panel of microorgan-
isms including yeast like fungi such as C. albicans ATCC
44373, C. dubliniensis CBS 7987, C. glabrata ATCC
10231, C. neoformans var. gattii (sorotype D) ATCC
28952, and Saccharomyces cerevisiae ATCC 2601, bacte-
ria such as E. coli ATCC 25922, P. aeruginosa ATCC
27850, S. aureus ATCC 25923 and Micrococcus luteus
ATCC 9341, Staphylococcus haemolyticus (clinically
isolated), Staphylococcus warneri (clinically isolated),
Staphylococcus saprophyticus (clinically isolated),
Enterococcus faecalis ATCC 29212, and the alga P. zop-
fii (clinically isolated). All microorganisms were com-
pared to the control drugs: fluconazole, imipenem, and
amphotericin B for fungi, bacteria, and alga, respective-
ly. The minimal inhibitory concentration (MIC) and
minimal fungicidal, bactericidal, and algacidal concen-
trations were determined by broth microdilution meth-
ods according to NCCLS standards.24 Compounds
were dissolved in DMSO (1 mL) and the solutions were
diluted with medium. By further progressive dilutions
with test medium the required concentrations (64, 32,
16, 8, 4, 2, 1, 0.5 and 0.25 lg/mL) were obtained. The
antimicrobial activities were evaluated based on mini-
mal inhibitory concentration (MIC) according to the
procedures NCCLS M27-A2 for fungi and algae and,
NCCLS M7-A4 for bacteria that grow aerobically. Bac-
teria were inoculated into Mueller–Hinton agar and
after overnight growth, four or five colonies were direct-
ly suspended in saline solution so that the turbidity
matches the turbidity of the McFarland standard
(ꢀ108 cfu/mL). The suspension was diluted 1:100 in
saline followed by new dilution 1:20 in Mueller–Hinton
broth, resulting in a final inoculum concentration of
5· 104 cfu for well.
4. Results and discussion
Thirteen of the new synthesized compounds 3a,d, 4a–d,
5a,b,d, 7a,c,d, and 8c were evaluated for their in vitro
antimicrobial activity against a panel of microorganisms
including yeast like fungi, bacteria, and alga by deter-
mining their minimal inhibitory concentration (MIC)
and minimal fungicidal, bactericidal, and algacidal con-
centrations by broth microdilution methods according
to NCCLS standards.24 Some of the evaluated com-
pounds exhibited significant in vitro activity against
the tested microorganism strains with a MIC range of
0.5–8 lg/mL; a moderate antifungal activity was also
found with the MIC range varying from 16 to 32 lg/
mL (Tables 2 and 3). When we consider the susceptibil-
ity of the microorganisms, another point deserves atten-
tion. In general, C. albicans was similar or more resistant
than C. dubliniensis which is in agreement with the liter-
ature for antifungal agents and compounds like NaCl,
alcohols, and others.26,27 In general C. glabrata, C. trop-
icalis, and C. krusei showed susceptibility patterns simi-
lar to C. albicans. Based on breakpoints established for
fluconazole (M27-A2), the three varieties of C. neofor-
mans studied were sensitive to three compounds such
as 4b,c and 7d.
When we compared the MICs with the MCM (minimal
cidal concentrations) in 160 (68.3%) of the cases, the
MICs were similar to MCMs and in 74 (31.7%) of the
cases the MCMs were one or more dilution more elevat-
ed. When the MICs or MCMs were P64 lg/mL, the
comparison was not established. The compounds with
a trichloromethyl group showed higher activity in the
three types of tested microorganisms. Compounds 4c
and b, where R = Ph and CH3, respectively, exhibited
higher activity. Considering the compounds bearing
Fungi and P. zopfii were inoculated into potato dextrose
agar and the procedures of inoculum standardization
were similar among them; however, the medium test
was RPMI 1640 broth. Each well of the microdilution
tray was filled with 100 lL of compound diluted in

N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
Table 2. Antibacterial activity in vitro of carbamates (MIC/MBC, lg/mL)
3179
Compound
MICa/MBCb
Bacteria Gram-positive
Bacteria Gram-negative
S. a.c
S. h.d
S. w.e
S. s.f
M. l.g
E. f.h
E. c.i
P. a.j
3a
3d
4a
4b
4c
4d
5a
5b
5d
7a
7c
7d
8c
Ik
16/64
64/64
32/64
1/1
16/32
32/64
32/64
1/2
16/64
64/64
32/64
1/1
32/64
64/>64
32/64
2/2
16/64
64/64
>64/>64
16/64
64/64
32/64
1/1
16/64
64/64
64/64
16/32
4/4
32/64
64/64
64/64
64/64
2/2
1/1
2/2
2/2
1/2
2/2
2/2
2/2
32/>64
16/16
64/64
64/>64
32/64
>64
32/>64
16/32
64/>64
64/>64
32/64
>64
32/64
16/16
64/64
64/64
32/32
64/>64
64/64
64/>64
0.06
32/>64
16/16
64/64
64/64
32/64
>64
8/16
8/64
32/>64
16/64
64/64
64/>64
32/64
>64
32/32
64/>64
64/64
64/>64
64/>64
64/>64
64/>64
64/>64
0.06
32/32
32/>64
64/64
32/>64
64/64
64/>64
32/>64
64/>64
2.0
64/>64
64/64
64/64
>64
64/64
>64
0.06
64/>64
>64
0.06
64/64
>64
0.06
64/>64
>64
0.03
64/64
>64
0.06
a Minimal inhibitory concentration.
b Minimal bactericidal concentration microorganisms.
c Staphylococcus aureus ATCC 25923.
d Staphylococcus haemolyticus (clinical isolate).
e Staphylococcus warneri (clinical isolate).
f Staphylococcus saprophyticus (clinical isolate).
g Micrococcus luteus ATCC 9341.
h Enterococcus faecalis ATCC 29212.
i Escherichia coli ATCC 25922.
j Pseudomonas aeruginosa ATCC 27850.
k Control: imipenem.
Table 3. Antifungal activity in vitro of carbamates (MIC/MFC, lg/mL)
Compound
MICa/MFCb
Fungi
Alga
P. z.l
C. a.c
C. d.d
C. g.e
C. t.f
C. k.g
C. n.h
C. n.i
C. n.j
S. c.k
3a
3d
4a
4b
4c
4d
5a
5b
5d
7a
7c
7d
8c
Fm
An
64/64
16/64
32/32
16/64
32/64
32/64
32/64
64/64
64/64
64/64
64/64
32/32
64/64
4.0
64/64
8/32
64/>64
16/32
32/32
16/32
32/32
64/>64
64/64
64/64
64/64
64/>64
64/>64
64/64
64/64
8.0
64/64
16/32
32/32
16/64
32/64
32/64
32/32
64/64
64/64
64/64
64/64
32/64
64/64
4.0
64/64
16/64
32/32
16/64
32/64
32/64
32/64
64/64
64/64
64/64
64/>64
32/32
64/64
64
16/32
>64
16/64
>64
32/32
>64
64/64
>64
64/64
>64
64/64
8/16
32/32
2/2
4/4
32/64
2/4
4/4
32/32
2/2
2/4
16/16
4/8
4/4
64/64
4/4
16/16
64/64
64/64
64/64
64/64
64/64
64/>64
32/32
64/64
2.0
0.5/0.5
8/8
32/32
16/64
64/64
16/16
16/32
32/32
8/32
32/64
16/32
64/>64
16/16
16/64
32/32
8/32
32/32
32/64
64/64
16/32
16/32
32/64
8/16
32/64
64/64
32/64
32/64
64/64
64/>64
32/64
64/64
1.0
64/64
32/32
64/64
64/64
64/64
64/64
64/64
32/64
2.0
32/32
2.0
32/32
2.0
0.5
a Minimal inhibitory concentration.
b Minimal fungicidal concentration microorganisms.
c Candida albicans ATCC 44373.
d Candida dubliniensis CBS 7987.
e Candida glabrata ATCC 10231.
f Candida tropicalis ATCC 750.
g Candida krusei ATCC 6258.
h Cryptococcus neoformans var. gattii (sorotype B) ATCC 56990.
i Cryptococcus neoformans var. gattii (sorotype C) ATCC 24066.
j Cryptococcus neoformans var. grubii (sorotype A) ATCC 90012.
k Saccharomyces cerevisiae ATCC 2601.
l Prototheca zopfii (clinical isolate).
m Control: fluconazole.
n Control: amphotericin B.

3180
N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
the trichloromethyl group, compound 4b (R = CH3)
showed the best bacteriostatic activity from the series
of compounds tested to Gram-positive cocci (S. aureus,
S. haemolyticus, S. warneri, S. saprophyticus, E. faecalis,
and M. luteus). Compound 4b also showed better bacte-
riostatic activity against other microorganisms if com-
pared with compound 4a (R = H). The 4a showed
moderate antifungal activity against S. cerevisiae. Com-
pound 4c (R = Ph) was the most active compound
against the alga P. zopfii with MIC similar to that ob-
tained for amphotericin B. The activity of 4c was also
significant against P. aeruginosa as well as for Gram-
negative rods and Gram-positive cocci. The antifungal
activity of 4c was more significant against C. neoformans
and S. cerevisiae, and showed moderate activity against
Candida species. In general, 4c showed a large spectrum
of antimicrobial activity.
to their anti-Cryptococcus and anti-Candida specificity.
It was observed that the antimicrobial activity is very
sensitive to the R-substituent and that the trichlorome-
thylated compounds, in general, showed higher activity
than the trifluoromethylated analogues.
6. Experimental
The solvents were purified and dried before used and the
4-alkoxy-1,1,1-trifluoro[chloro]alk-3-en-2-ones
were
prepared according to the literature procedures.16 The
CHN elemental analysis was performed on a Perkin-El-
mer 2400 CHN elemental analyzer (University of Sa˜o
Paulo, SP, Brazil). Melting points were determined with
a Reichert Thermovar apparatus and are uncorrected.
1H and 13C NMR spectra were acquired on a Bruker
DPX 200 spectrometer (1H at 200.13 MHz and 13C at
One interesting result was demonstrated for the com-
pound 4d (R = 4-Me-Ph). The antimicrobial activity of
4d was restricted to P. zopfii and M. luteus, both with
moderate susceptibility to this compound. Although,
4d has only a methyl group attached to the phenyl resi-
due more than 4c, the former exhibited much lower
activity against all studied microorganisms if compared
with 4c. These results show that small changes in the R
group induced significant differences in antimicrobial
activity. In addition, the cyclic compound 8c also exhib-
ited much lower activity than its precursor 4c.
50.32 MHz) or on a
Bruker DPX 400 (1H at
400.13 MHz and 13C at 100.62 MHz) in CDCl3 or
DMSO-d6 and using TMS as the internal reference.
The mass spectrums were registered in a HP 5973
MSD connected to a HP 6890 GC and interfaced by a
HP Pentium PC. The GC was equipped with a split–
splitless injector, autosampler, cross-linked HP 5 capil-
lary column (30 m, 0.32 mm, of internal diameter), and
helium was used as the carrier gas.
6.1. General procedure for the synthesis of (4,4,4-trihalo-3-
oxo-but-1-enyl)-carbamic acid ethyl esters (3a–d and 4a–d)
Compound 3a showed a weak fungistatic activity
against C. neoformans which was absent in Candida spe-
cies. Bacteriostatic activity to Gram-positive cocci and
Gram-negative rod as E. coli was also observed. In con-
trast, the 3d exhibited a specific anti-Candida activity
and the activity against C. dubliniensis must be empha-
sized. The 3-hydroxy-butyl carbamates 5a and d showed
very similar results to their precursors 3a and d. The 1,3-
oxazinan-2-one 7a exhibited activity similar to its pre-
cursor 5a against C. neoformans, but it was inactive
against other micoorganisms. Compound 7d was similar
to its precursor 5d but its anticryptococcal activity was
more pronounced.
To a solution of ethyl carbamate (1.33 g, 15 mmol for 1a
and 2a and 2.00 g, 22.5 mmol for 1b–d and 2b–d) in
chloroform or dichloromethane (10 mL), a solution of
1 or 2 (15 mmol) in chloroform or dichloromethane
(10 mL) was slowly added under stirring at room tem-
perature. 4-Toluenesulfonic acid (catalytic amount)
was added to the reaction vessel and the stirring was
continued for 24 h at 50 ꢁC (for 3a and 4a), 24 h at
70 ꢁC (for 3c,d and 4c,d), and 48 h at 70 ꢁC (for 3b
and 4b). The isolation of compounds was carried out
by neutralization of the reaction mixture with sodium
carbonate solution (1 M) and water (10 mL) followed
by extraction with chloroform or dichloromethane
(3· 10 mL). The organic layers were combined, dried
with anhydrous magnesium sulfate, and the solvent
was removed with rotatory evaporator. Compounds
3b,c and 4b,c were purified by florisil column chroma-
tography (Aldrich 100–200 mesh) using hexane as the
eluant. The compound 3d and 4d were purified by silica
gel column chromatography (Aldrich 60A, 230–400
Mesh) using hexane/dichloromethane, 2:1 as the eluant.
Compounds 3a and 4a were purified by recrystallization
from hexane.
5. Conclusion
In conclusion, this work presented a convenient three-
step synthesis of a new series of 4-substituted 6-triha-
lomethyl-1,3-oxazinan-2-ones 7a–d and 8a–d from the
readily available enones 1a–d and 2a–d. An important
step of this strategy was the preparation of the new ser-
ies of trihalomethylated 3-oxo-but-1-enyl carbamates
3a–d and 4a–d, by an unprecedented reaction of a very
weak carbamate nucleophile with b-alkoxyvinyl triha-
lomethyl ketones 1 and 2. Another important reaction
was the double reduction of compounds 3 and 4 that
was achieved in a single reaction step to furnish a new
series of trihalomethylated 3-hydroxy carbamates 5a–d
and 6a–d. Some compounds of the three studied series
exhibited significant antimicrobial activity against path-
ogenic microorganisms including fungi, bacteria, and
alga. Some compounds deserve additional studies due
6.1.1. (4,4,4-Trifluoro-3-oxo-1-but-1-enyl)-carbamic acid
ethyl ester (3a). MS EI (70 ev): m/z (%) = 211 (M+, 52),
166 (31), 142 (100), 114 (62), 70 (96); 1H NMR (CDCl3,
200 MHz) d 1.30 (t, 3H, JH8–H7 = 7.0 Hz, H-8), 4.32 (q,
2H, JH7–H8 = 7.0 Hz, H-7), 5.77 (d, 1H, JH3–H4 = 8.4 Hz,
H-3), 7.67 (dd, 1H, JH4–H3 = 8.4 Hz, JH4–H5 = 12.0 Hz,
H-4), 10.5 (br s, 1H, NH); 13C NMR (CDCl3,
100 MHz) d 13.6 (C-8), 62.7 (C-7), 94.0 (C-3), 115.5

N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
3181
(q, JC–F = 287.4 Hz, CF3), 146.7 (C-4), 152.3 (C-6),
181.2 (q, JC–F = 35.3 Hz, C-2).
(CCl3), 98.0 (C-3), 127.6, 128.1, 130.6, 134.9 (Ph), 151.9
(C-6), 161.9 (C-4), 182.2 (C-2).
6.1.2.
(4,4,4-Trifluoro-1-methyl-3-oxo-but-1-enyl)-car-
6.1.8. [4,4,4-Trichloro-1-(4-methyl-phenyl)-3-oxo-but-1-
enyl]-carbamic acid ethyl ester (4d). MS EI (70 ev): m/z
(%) = 349 (M+, 3), 304 (3), 232 (100), 204 (78), 118 (72),
91 (40); 1H NMR (CDCl3, 200 MHz) d 1.25 (t, 3H,
JH8–H7 = 7.2 Hz, H-8), 2.40 (s, 3H, CH3), 4.14 (q, 2H,
JH7–H8 = 7.2 Hz, H-7), 6.13 (s, 1H, H-3), 7.23 (d, 2H,
JH10–H11 = 8.0 Hz, Ph), 7.35 (d, 2H, JH11–H10 = 8.0 Hz,
Ph), 10.80 (br s, 1H, NH); 13C NMR (CDCl3,100 MHz)
d 14.1 (C-8), 21.5 (C-13), 62.5 (C-7), 96.3 (CCl3), 97.8
(C-3), 127.7, 128.9, 132.0, 141.3 (Ph), 152.1 (C-6), 162.2
(C-4), 182.2 (C-2).
bamic acid ethyl ester (3b). MS EI (70 ev): m/z (%) = 225
(M+, 60), 196 (5), 180 (38), 156 (85), 128 (63), 110 (87),
68 (36); 1H NMR (CDCl3, 200 MHz) d 1.21 (t, 3H,
JH8–H7 = 7.0 Hz, H-8), 2.42 (s, 3H, CH3), 4.16 (q, 2H,
JH7–H8 = 7.2 Hz, H-7), 5.55 (s, 1H, H-3), 11.47 (br
s, 1H, NH); 13C NMR (CDCl3, 100 MHz) d 13.9
(C-8), 21.7 (CH3), 62.5 (C-7), 96.4 (C-3), 116.3 (q,
JC–F = 287.2 Hz, CF3), 152.1 (C-6), 165.0 (C-4), 179.6
(q, JC–F = 34.3 Hz, C-2).
6.1.3. (4,4,4-Trifluoro-3-oxo-1-phenyl-1-but-1-enyl)-car-
bamic acid ethyl ester (3c). MS EI (70 ev): m/z (%) = 287
(M+, 31), 242 (10), 218 (100), 190 (60), 77 (55);
6.2. General procedure for the synthesis of (4,4,4-trihalo-3-
hydroxy-butyl)-carbamic acid ethyl esters (5a–d and 6a-d)
1H NMR (CDCl3, 200 MHz)
d
1.24 (t, 3H,
JH8–H7 = 7.2 Hz, H-8), 4.14 (q, 2H, JH7–H8 = 7.2 Hz, H-
7), 5.81 (s, 1H, H-3), 7.43 (m, 5H, Ph), 11.18 (br s, 1H,
NH); 13C NMR (CDCl3, 100 MHz) d 13.9 (C-8), 62.7
(C-7), 98.6 (C-3), 116.3 (q, JC–F = 287.8 Hz, CF3),
127.5, 128.1, 130.9, 134.3 (Ph), 151.7 (C-6), 163.4 (C-4),
180.1 (q, JC–F = 35.3 Hz, C-2).
To a solution of 3-oxo-but-1-enyl carbamates 3 and 4
(10 mmol) in anhydrous ethanol (10 mL) was slowly
added sodium borohydride (0.45 g, 12 mmol) under vig-
orous stirring at room temperature. The stirring was
continued for 5 h at room temperature (for 3a–d) or
24 h at 70 ꢁC (for 4a–d). The solvent was removed by
rotatory evaporator, dichloromethane (15 mL) and
water (15 mL) were added, and the mixture was stirred
at room temperature for 15 min. The organic layer
was separated and the water phase was extracted with
dichloromethane (2· 15 mL). The organic layers were
combined, dried under magnesium sulfate, and the sol-
vent was removed by rotatory evaporator. Compounds
5a and b were purified by florisil column chromatogra-
phy (Aldrich 100–200 mesh) with hexane as the eluant.
Compound 5c was purified by silica gel column chroma-
tography (Aldrich 60A, 230–400 Mesh), with dichloro-
methane as the eluant. Compound 5d was purified by
recrystallization from hexane. The compound 6d was
purified by dissolution in a mixture of 5% of dichloro-
methane in hexane. In this condition, a solid (impurity)
precipitated and was filtered off. The product 6d was ob-
tained as oil and was purified by column chromatogra-
phy as the compound 5c. Our effort to separate the
mixture of 3-hydroxy-butyl carbamates 6a–c from
3-hydroxyvinyl carbamates 60a–c (partially reduced
compounds) was unsuccessful. The mixture of 6 and
60 was obtained in a ratio of approximately 1:1.
6.1.4. [4,4,4-Trifluoro-1-(4-methyl-phenyl)-3-oxo-but-1-
enyl]-carbamic acid ethyl ester (3d). MS EI (70 ev): m/z
(%) = 301 (M+, 42), 272 (4), 256 (10), 232 (92), 204 (50),
91 (51); 1H NMR (CDCl3, 200 MHz) d 1.26 (t, 3H,
JH8–H7 = 7.0 Hz, H-8), 2.40 (s, 3H, CH3), 4.15 (q, 2H,
JH7–H8 = 7.0 Hz, H-7), 5.81 (s, 1H, H-3), 7.23 (d, 2H,
JH10–H11 = 8.0 Hz, Ph), 7.34 (d, 2H, JH11–H10 = 8.0 Hz,
Ph), 11.17 (br s, 1H, NH); 13C NMR (CDCl3, 100 MHz)
d 14.0 (C-8), 21.4 (CH3), 62.6 (C-7), 98.4 (C-3), 116.3 (q,
JC–F = 287.8 Hz, CF3), 127.7, 128.9, 131.3, 141.7 (Ph),
151.9 (C-6), 163.6 (C-4), 180.0 (q, JC–F = 34.6 Hz, C-2).
6.1.5. (4,4,4-Trichloro-3-oxo-but-1-enyl)-carbamic acid
ethyl ester (4a). MS EI (70 ev): m/z (%) = 259 (M+, 2),
142 (83), 114 (100); 1H NMR (CDCl3, 200 MHz) d
1.26 (t, 3H, JH8–H7 = 7.0 Hz, H-8), 4.22 (q, 2H,
JH7–H8 = 7.0 Hz, H-7), 6.38 (d, 1H, JH3–H4 = 13.5 Hz,
H-3), 7.99 (dd, 1H, JH4–H3 = 13.3 Hz, JH4–H5 = 1.8 Hz,
H-4), 10.50 (br s, 1H, NH); 13C NMR (CDCl3,
100 MHz) d 14.0 (C-8), 62.2 (C-7), 96.1 (C-3), 96.5
(CCl3), 146.6 (C-4), 152.9 (C-6), 180.4 (C-2).
6.1.6. (4,4,4-Trichloro-1-methyl-3-oxo-but-1-enyl)-car-
bamic acid ethyl ester (4b). MS EI (70 ev): m/z (%) = 273
6.2.1. (4,4,4-Trifluoro-3-hydroxy-butyl)-carbamic acid
ethyl ester (5a). MS EI (70 ev): m/z (%) = 215 (M+, 3),
186 (14), 170 (18), 142 (12), 102 (100); 1H NMR (CDCl3,
200 MHz) d 1.27 (t, 3H, JH8–H7 = 7.2 Hz, H-8), 1.66-1.94
(m, 2H, H-3), 3.27–3.40 (m, 1H, H-4), 3.53 (1H, OH),
3.69–3.73 (m, 1H, H-4), 4.11 (q, 2H, JH7–H8 = 7.0 Hz,
H-7), 4.87–4.90 (m, 1H, H-2), 5.23–5.25 (m, 1H, NH);
13C NMR (CDCl3, 100 MHz) d 14.2 (C-8), 29.7 (C-4),
36.5 (C-3), 61.1 (C-7), 67.6 (q, JC–F = 31.1 Hz, C-2),
125.0 (q, JC–F = 279.9 Hz, CF3), 157.8 (C-6).
1
(M+, 4), 228 (4), 156 (59), 128 (100); H NMR (CDCl3,
200 MHz) d 1.31 (t, 3H, JH8–H7 = 7.2 Hz, H-8), 2.52 (s,
3H, CH3), 4.22 (q, 2H, JH7–H8 = 7.0 Hz, H-7), 5.91 (s,
1H, H-3), 11.22 (br s, 1H, NH); 13C NMR (CDCl3,
50 MHz) d 14.2 (C-8), 22.2 (CH3), 62.3 (C-7), 95.1
(C-3), 96.3 (CCl3), 152.3 (C-6), 163.3 (C-4), 182.2 (C-2).
6.1.7. (4,4,4-Trichloro-3-oxo-1-phenyl-but-1-enyl)-car-
bamic acid ethyl ester (4c). MS EI (70 ev): m/z (%) = 335
(M+, 2), 218 (82), 190 (65), 172 (100), 146 (13), 77 (41);
6.2.2.
(4,4,4-Trifluoro-3-hydroxy-1-methyl-butyl)-car-
1H NMR (CDCl3, 200 MHz) d 1.17 (t, 3H, JH8–H7
=
bamic acid ethyl ether (5b). MS EI (70 ev): m/z (%) = 229
(M+, 3), 214 (39), 200 (9), 184 (16), 160 (3), 116 (100), 88
(24); 1H NMR (CDCl3, 200 MHz) d 1.16 (t, 3H,
JH8–H7 = 6.8 Hz, H-8), 1.16 (d, 3H, JH9–H4 = 6.4 Hz,
7.0 Hz, H-8), 4.06 (q, 2H, JH7–H8 = 7.0 Hz, H-7), 6.05 (s,
1H, H-3), 7.39 (m, 5H, Ph), 10.73 (br s, 1H, NH); 13C
NMR (CDCl3, 50 MHz) d 14.0 (C-8), 62.5 (C-7), 96.2

3182
N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
CH3), 1.66–1.79 (m, 2H, H-3), 3.49 (m, 1H, OH), 3.80–
4.00 (m, 2H, H-4, H-2), 4.03 (q, 2H, JH7–H8 = 7.2 Hz,
H-7), 4.83 (1H, NH); 13C NMR (CDCl3, 100 MHz)
d 14.4 (C-8), 21.4 (CH3), 36.9 (C-4), 44.6 (C-3), 61.1
(C-7), 68.7 (q, JC–F = 31.0 Hz, C-2), 125.0 (q,
JC–F = 280.2 Hz, CF3), 156.6 (C-6).
NMR (CDCl3, 200 MHz) d 1.17 (t, 3H, JH8–H7 = 7.2 Hz,
H-8), 1.87 (s, 3H, CH3), 1.68–1.90 (s, 1H, OH), 4.04
(q, 2H, JH7–H8 = 7.2 Hz, H-7), 4.62–4.69 (dd, 1H,
H2–H3 = 11.4 Hz, JH2–OH = 2.6 Hz, H-2), 4.74–4.91 (m,
1H, H-3), 6.42 (br s, 1H, NH); 13C NMR (CDCl3,
100 MHz) d 14.3 (C-8), 20.4 (CH3), 60.6 (C-7), 80.6 (C-2),
95.0 (CCl3), 103.9 (C-3), 152.7 (C-4), 156.2 (C-6).
6.2.3. (4,4,4-Trifluoro-3-hydroxy-1-phenyl-butyl)-carbam-
ic acid ethyl ester (5c). MS EI (70 ev): m/z (%) = 291 (M+,
3), 218 (4), 178 (100); 1H NMR (CDCl3, 200 MHz) d 1.18
(t, 3H, JH8–H7 = 7.0 Hz, H-8), 2.22–2.30 (m, 2H, H-3),
6.2.9. (4,4,4-Trichloro-3-hydroxy-1-phenyl-butyl)-carbamic
acid ethyl ester (6c). MS EI (70 ev): m/z (%) = 339 (M+, 1),
266 (16), 178 (100), 77 (33); 1H NMR (CDCl3, 200 MHz)
d 1.08–1.27 (m, 3H, H-8,), 2.13–2.25 (m, 2H, H-3, OH),
2.38–2.42 (m, 1H, H-3), 3.99 (q, 2H, JH7–H8 = 7.0 Hz, H-
7), 4.12–4.23 (m, 1H, H-4), 4.82–4.88 (m, 1H, H-2), 5.21
(br s, 1H, NH), 7.27–7.30 (m, 5H, Ph); 13C NMR (CDCl3,
100 MHz) d 14.4 (C-8), 38.3 (C-3), 53.4 (C-4), 61.1 (C-7),
79.5 (C-2), 97.5 (CCl3), 126.1, 127.2, 128.9, 133.3, (Ph),
156.0 (C-6).
3.61–3.77 (m, 2H, H-4, OH), 4.06 (q, 2H, JH7–H8
=
7.2 Hz, H-7), 4.86–4.89 (m, 1H, H-2), 5.32 (1H, NH),
7.32–7.35 (m, 5H, Ph); 13C NMR (CDCl3, 100 MHz)
d 14.4 (C-8), 36.4 (C-3), 52.9 (C-4), 61.3 (C-7), 69.0 (q,
JC–F = 31.5 Hz, C-2), 124.9 (q, JC–F = 280.2 Hz, CF3),
126.6, 128.1, 129.0, 140.4 (Ph), 156.2 (C-6).
6.2.4. (4,4,4-Trifluoro-3-hydroxy-1-(4-methyl-phenyl)-bu-
tyl)-carbamic acid ethyl ester (5d). MS EI (70 ev): m/z
(%) = 305 (M+, 7), 232 (43), 214 (7), 192 (100), 91 (52);
6.2.10. (4,4,4-Trichloro-3-hydroxy-1-phenyl-but-1-enyl)-
carbamic acid ethyl ester (60c). MS EI (70 ev): m/z
1
1H NMR (CDCl3, 200 MHz) d 1.12 (t, 3H, JH8–H7
=
(%) = 292 (M+ꢁ45, 11), 176 (9), 104 (100), 77 (72); H
7.2 Hz, H-8), 2.00–2.09 (m, 2H, H-3), 2.26 (s, 3H, CH3),
3.62–3.69 (m, 2H, H-4, OH), 3.99 (q, 2H,
JH7–H8 = 7.2 Hz, H-7), 4.74–4.77 (m, 1H, H-2), 5.14 (s,
1H, NH), 7.10–7.20 (m, 4H, Ph); 13C NMR (CDCl3,
100 MHz) d 14.3 (C-8), 20.9 (CH3), 36.4 (C-3), 52.5 (C-
4), 61.2 (C-7), 68.2 (q, JC–F = 31.15 Hz, C-2), 125.0 (q,
JC–F = 280.2 Hz, CF3), 126.5, 129.6, 137.5, 137.7 (Ph),
156.3 (C-6).
NMR (CDCl3, 200 MHz) d 1.08–1.27 (m, 3H, H-8),
2.13–2.25 (s, 1H, OH), 3.74–3.79 (m, 1H, H-2), 3.99
(q, 2H, JH7–H8 = 7.0 Hz, H-7), 4.84–4.88 (m, 1H, H-3),
5.21 (br s, 1H, NH), 7.27–7.30 (m, 5H, Ph); 13C NMR
(CDCl3, 100 MHz) d 14.1 (C-8), 60.1 (C-7), 72.4 (C-2),
99.5 (CCl3), 125.5 (C-4), 126.6, 127.5 (Ph), 128.4 (C-3),
129.1, 139.1 (Ph), 154.8 (C-6).
6.2.11. [4,4,4-Trichloro-3-hydroxy-1-(4-methyl-phenyl)-
butyl]-carbamic acid ethyl ester (6d). MS EI (70 ev):
m/z (%) = 353 (M+, 2), 280 (11), 192 (100), 148 (8),
6.2.5. (4,4,4-Trichloro-3-hydroxy-butyl)-carbamic acid eth-
yl ester (6a). MS EI (70 ev): m/z (%) = 263 (M+, 1), 234 (3),
190 (4), 117 (32), 102 (100); 1H NMR (CDCl3, 200 MHz)
d 1.22–1.29 (m, 3H, H-8), 1.77 (m, 1H, H-3), 2.25 (s, 1H,
OH), 2.57–2.61 (m, 1H, H-3), 3.40–3.71 (m, 2H, H-4),
4.07-4.20 (m, 2H, H-7), 4.27 (m, 1H, H-2); 13C NMR
(CDCl3, 100 MHz) d 14.4 (C-8), 32.0 (C-3), 37.7 (C-4),
61.1 (C-7), 80.6 (C-2), 103.2 (CCl3), 156.4 (C-6).
1
118 (40), 91 (42); H NMR (CDCl3, 200 MHz) d 1.20
(t, 3H, JH8–H7 = 7.20 Hz, H-8), 2.26 (s, 3H, CH3), 2.29
(m, 2H, H-3), 3.72–3.76 (m, 1H, H-4), 4.00 (q, 2H,
JH7–H8 = 7.20 Hz, H-7), 4.77–4.81 (m, 1H, H-2), 5.11
(s, 1H, NH), 7.07–7.19 (m, 4H, Ph); 13C NMR (CDCl3,
100 MHz) d 14.5 (C-8), 21.1 (CH3), 38.4 (C-3), 53.3 (C-
4), 61.1 (C-7), 80.8 (q, JC–F = 31.2 Hz, C-2), 103.6
(CCl3), 126.1, 126.6, 129.6, 137.7 (Ph), 156.0 (C-6).
6.2.6. (4,4,4-Trichloro-3-hydroxy-1-but-1-enyl)-carbamic
acid ethyl ester (60a). MS EI (70 ev): m/z (%) = 217
(M+ꢁ45, 2), 147 (3), 91 (100), 73 (90); 1H NMR (CDCl3,
200 MHz) d 1.22–1.29 (m, 3H, H-8), 2.25 (s, 1H, OH),
4.07–4.20 (m, 2H, H-7), 4.53 (m, 1H, H-2), 5.10 (m,
1H, H-3), 6.91 (m, 2H, H-4, NH); 13C NMR (CDCl3,
100 MHz) d 14.9 (C-8), 61.6 (C-7), 79.3 (C-2), 103.5
(CCl3), 128.3 (C-3), 156.2 (C-6), 157.3 (C-4).
6.3. General procedure for the synthesis of 2-oxo-6-triha-
lomethyl-[1,3]oxazinane-3-carboxylic acid ethyl esters
(7a–d and 8a–d)
To a solution of 2-hydroxy carbamates 5 and 6 (5 mmol)
in 1,2-dichloroethane (10 mL), triethylamine (0.70 mL,
5 mmol) and triphosgene (1.483 g, 5 mmol) were added
under stirring at room temperature. The stirring was
continued for 5 h (for 5a–d) or 22 h (for 6a–d) at
80 ꢁC. The mixture was allowed to cool and the triethy-
lammonium salt was filtered off. The solution was
washed with water (4· 15 mL). The aqueous layer was
extracted with dichloromethane (3· 10 mL). The organ-
ic layers were combined, dried under magnesium sulfate,
and the solvent was removed by rotatory evaporator.
Compounds 7d and 8b–d were purified by silica gel col-
umn chromatography (Aldrich 60A, 230–400 Mesh),
with hexane/dichloromethane, 1:1 as the eluant. Com-
pound 7b was purified by silica gel column chromatog-
raphy (Aldrich 60A, 230–400 Mesh), with hexane/
dichloromethane, 2:1 as the eluant. Compounds 7a
6.2.7. (4,4,4-Trichloro-3-hydroxy-1-methyl-butyl)-carbam-
ic acid ethyl ester (6b). MS EI (70 ev): m/z (%) = 262 (M+–
15, 4), 190 (4), 116 (100); H NMR (CDCl3, 200 MHz)
1
d 1.17 (t, 3H, JH8–H7 = 7.2 Hz, H-8), 1.26 (d, 3H,
JCH –H4 ¼ 6:0 Hz, CH3), 1.68–1.90 (m, 2H, H-3, OH),
3
2.04–2.25 (m, 1H, H-3), 3.56–3.67 (m, 1H, H-4), 3.76–
3.90 (m, 1H, H-2), 4.04 (q, 2H, JH7–H8 = 7.2 Hz, H-7),
4.93 (br s, 1H, NH); 13C NMR (CDCl3, 100 MHz) d 14.3
(C-8), 21.0 (CH3), 31.1 (C-3), 44.9 (C-4), 60.7 (C-7), 84.3
(C-2), 97.7 (CCl3), 157.6 (C-6).
6.2.8. (4,4,4-Trichloro-3-hydroxy-1-methyl-but-1-enyl)-car-
bamic acid ethyl ester (60b). MS EI (70 ev): m/z (%) = 230
1
(M+ꢁ45, 4), 216 (67), 136 (86), 114 (86), 71 (100); H

N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
3183
and c were purified by recrystallization from a mixture
of 5% of chloroform in hexane. Compound 8a could
not be purified and it was detected by GC–MS.
JC–F = 277.50 Hz, CF3), 125.2, 129.6, 137.9, 138.0
(Ph), 147.9 (C-2), 152.2 (C-7).
6.3.5. 2-Oxo-6-trichloromethyl-[1,3]oxazinane-3-carbox-
ylic acid ethyl ester (8a). This compound could not be
purified. It was only detected by GC–MS. MS EI
(70 ev): m/z (%) = 289 (M, 3), 262 (29), 217 (17), 157
(12), 119 (35), 100 (100).
6.3.1. 2-Oxo-6-trifluoromethyl-[1,3]oxazinane-3-carbox-
ylic acid ethyl ester (7a). MS EI (70 ev): m/z (%) = 241
(M+, 5), 214 (81), 196 (5), 169 (92), 152 (12), 69 (33);
1H NMR (CDCl3, 200 MHz)
d
1.36 (t, 3H,
JH9–H8 = 7.0 Hz, H-9), 2.05–2.42 (m, 2H, H-5), 3.62–
3.77 (m, 1H, H-4), 4.04–4.15 (m, 1H, H-4), 4.34 (q,
2H, JH8–H9 = 7.0 Hz, H-8), 4.64–4.77 (m, 1H, H-6);
13C NMR (CDCl3, 100 MHz) d 14.0 (C-9), 21.1 (C-5),
42.0 (C-4), 64.0 (C-8), 74.0 (q, JC–F = 34.5 Hz, C-6),
122.2 (q, JC–F = 278.2 Hz, CF3), 146.4 (C-2), 152.8
(C-7).
6.3.6. 4-Methyl-2-oxo-6-trichloromethyl-[1,3]oxazinane-
3-carboxylic acid ethyl ester (8b). MS EI (70 ev): m/z
(%) = 303 (M+, 1), 186 (12), 116 (96), 72 (100); 1H NMR
(CDCl3, 200 MHz) d 1.37 (t, 3H, JH9–H8 = 7.2 Hz, H-9),
1.44 (d, 3H, JCH –H4 ¼ 6:4 Hz, CH3), 2.01 (ddd,
3
1H,
JH5(ax)–H4 = 8.7 Hz, H-5), 2.86 (ddd, 1H, JH5(eq)–H5(ax)
JH5(ax)–H5(eq) = 13.8 Hz,
JH5(ax)–H6 = 11.2 Hz,
=
6.3.2. 4-Methyl-2-oxo-6-trifluoromethyl-[1,3]oxazinane-
3-caboxylic acid ethyl ester (7b). MS EI (70 ev): m/z
(%) = 255 (M+, 5), 228 (6), 168 (100), 124 (22), 70 (77);
13.8 Hz, JH5(eq)–H4 = 8.6 Hz, JH5(eq)–H6 = 2.8 Hz, H-5),
4.34 (q, 2H, JH8–H9 = 7.2 Hz, H-8), 4.42–4.50 (m, 1H,
H-4), 4.64 (dd, 1H, JH6–H5(ax) = 11.2 Hz, JH6–H5(eq)
=
1H NMR (CDCl3, 200 MHz)
d
1.35 (t, 3H,
2.8 Hz, H-6); 13C NMR (CDCl3, 100 MHz) d 14.1 (C-9),
22.7 (CH3), 32.9 (C-5), 49.7 (C-4), 63.8 (C-8), 83.1 (C-6),
96.4 (CCl3), 148.4 (C-2), 152.9 (C-7).
JH9–H8 = 7.2 Hz, H-9), 1.42 (d, 3H, JCH3–H4 = 6.2 Hz,
CH3), 1.91 (ddd, 1H, JH5(ax)–H5(eq) = 13.7 Hz,
JH5(ax)–H6 = 11.6 Hz, JH5(ax)–H4 = 8.8 Hz, H-5), 2.57
(ddd, 1H, JH5(eq)–H5(ax) = 13.7 Hz, JH5(eq)–H4 = 8.7 Hz,
JH5(eq)–H6 = 3.1 Hz, H-5), 4.33 (q, 2H, JH8–H9 = 7.0 Hz,
H-8), 4.41–4.59 (m, 2H, H-6, H-4); 13C NMR (CDCl3,
100 MHz) d 14.0 (C-9), 22.6 (CH3), 29.9 (C-5),
49.5 (C-4), 63.8 (C-8), 72.3 (q, JC–F = 34.6 Hz, C-6),
122.0 (q, JC–F = 277.3 Hz, CF3), 148.2 (C-2), 152.8
(C-7).
6.3.7. 2-Oxo-4-phenyl-6-trichloromethyl-[1,3]oxazinane-
3-carboxylic acid ethyl ester (8c). MS EI (70 ev):
m/z (%) = 365 (M+, 9), 292 (53), 250 (13), 177 (41),
104 (100), 77 (95); 1H NMR (CDCl3, 200 MHz)
d
1.07 (t, 3H, JH9–H8 = 7.0 Hz, H-9), 2.21
(ddd, 1H, JH5(ax)–H5(eq) = 14.0 Hz, JH5(ax)–H6 = 11.2 Hz,
JH5(ax)–H4 = 10.9 Hz, H-5), 2.93 (ddd, 1H,
JH5(eq)–H5(ax) = 14.0 Hz, JH5(eq)–H4 = 7.6 Hz, JH5(eq)–H6
=
6.3.3. 2-Oxo-4-phenyl-6-trifluoromethyl-[1,3]oxazinane-3-
carboxylic acid ethyl ester (7c). MS EI (70 ev): m/z
(%) = 317 (M+, 15), 272 (2), 244 (100), 132 (14), 77 (71);
1.8 Hz, H-5), 4.08 (q, 2H, JH8–H9 = 7.2 Hz, H-8), 4.71
(dd, 1H, JH6–H5(ax) = 11.2 Hz, JH6–H5(eq) = 1.8 Hz H-6),
5.18 (dd, 1H, JH4–H5(ax) = 10.9 Hz, JH4–H5(eq) = 7.6 Hz,
H-4), 7.19–7.31 (m, 5H, Ph); 13C NMR (CDCl3,
100 MHz) d 13.8 (C-9), 34.8 (C-5), 58.1 (C-4), 63.8 (C-8),
83.0 (C-6), 96.3 (CCl3), 125.3, 128.3, 129.1, 141.2 (6C,
Ph), 148.1 (C-2), 152.3 (C-7).
1H NMR (CDCl3, 200 MHz) d 1.12 (t, 3H, JH9–H8
=
7.0 Hz, H-9), 2.18 (ddd, 1H, JH5(ax)–H5(eq) = 14.2 Hz,
JH5(ax)–H6 = 11.0 Hz, JH5(ax)–H4 = 10.8 Hz, H-5), 2.70
(ddd, 1H, JH5(eq)–H5(ax) = 14.2 Hz, JH5(eq)–H4 = 7.8 Hz,
JH5(eq)–H6 = 2.0 Hz, H-5), 4.13 (q, 2H, JH8–H9 = 7.2 Hz,
H-8), 4.71 (m, 1H, JH6–H5(ax) = 11.0 Hz, JH6–H5(eq)
=
6.3.8. 2-Oxo-4-(4-methyl-phenyl)-6-trichloromethyl-[1,3]oxaz-
inane-3-carboxylic acid ethyl ester (8d). MS EI (70 ev): m/z
(%) = 379 (M+, 11), 191 (46), 118 (100), 91 (98); 1H NMR
(CDCl3, 200 MHz) d 1.16 (t, 3H, JH9–H8 = 7.0 Hz,
H-9), 2.18-2.30 (ddd, 1H, JH5(ax)–H5(eq) = 14.2 Hz,
JH5(ax)–H6 = 11.4 Hz, JH5(ax)–H4 = 10.8 Hz, H-5), 2.34 (s,
3H, CH3), 2.97 (ddd, 1H, JH5(eq)–H5(ax) = 14.2 Hz,
JH5(eq)–H4 = 7.8 Hz, JH5(eq)–H6 = 1.8 Hz, H-5), 4.16 (q,
2.0 Hz, H-6), 5.25 (dd, 1H, JH4–H5(ax) = 10.8 Hz,
JH4–H5(eq) = 7.8 Hz, H-4), 7.23–7.38 (m, 5H, Ph); 13C
NMR (CDCl3, 100 MHz) d 13.7 (C-9), 31.8 (C-5), 58.0
(C-4), 63.8 (C-8), 72.3 (q, JC–F = 35.0 Hz, C-6), 121.9 (q,
JC–F = 277.30 Hz, CF3), 125.2, 128.3, 129.1, 140.9 (6C,
Ph), 147.9 (C-2), 152.3 (C-7).
6.3.4. 2-Oxo-4-(4-methyl-phenyl)-6-trifluoromethyl-[1,3]oxaz-
inane-3-carboxylic acid ethyl ester (7d). MS EI (70 ev):
m/z (%) = 331 (M+, 22), 258 (100), 91 (51), 118 (68);
2H, JH8–H9 = 7.0 Hz, H-8), 4.78 (dd, 1H, JH6–H5(ax) =
11.4 Hz, JH6–H5(eq) = 1.6 Hz, H-6), 5.21 (dd, 1H,
JH4–H5(ax) = 10.8 Hz, JH4–H5(eq) = 7.8 Hz, H-4), 7.17 (m,
4H, Ph); 13C NMR (CDCl3, 100 MHz) d 13.9 (C-9), 21.1
(CH3), 34.8 (C-5), 57.9 (C-4), 63.8 (C-8), 83.0 (C-6), 96.4
(CCl3), 125.3, 129.8, 138.2 (Ph), 148.2 (C-2), 152.4 (C-7).
1H NMR (CDCl3, 200 MHz) d 1.15 (t, 3H, JH9–H8
=
7.1 Hz, H-9), 2.18 (ddd, 1H, JH5(ax)–H5(eq) = 14.4 Hz,
JH5(ax)–H6 = 11.4 Hz, JH5(ax)–H4 = 10.5 Hz, H-5), 2.33
(s, 3H, CH3), 2.67 (ddd, 1H, JH5(eq)–H5(ax) = 14.4 Hz,
JH5(eq)–H4 = 7.8 Hz, JH5(eq)–H6 = 1.6 Hz, H-5), 4.15 (q,
2H, JH8–H9 = 7.0 Hz, H-8), 4.59–4.74 (dd, 1H,
JH6–H5(ax) = 11.4 Hz, JH6–H5(eq) = 1.6 Hz, H-6), 5.21
(dd, 1H, JH4–H5(ax) = 10.5 Hz, JH4–H5(eq) = 7.8 Hz, H-
4), 7.16 (m, 4H, Ph); 13C NMR (CDCl3, 100 MHz)
d 13.6 (C-9), 20.9 (CH3), 31.8 (C-5), 57.7 (C-4),
63.6 (C-8), 72.2 (q, JC–F = 34.3 Hz, C-6), 121.9 (q,
Acknowledgments
The authors thank the financial support from the Conse-
´
´
lho Nacional de Desenvolvimento Cientıfico e Tecnolog-
ico (Universal Grant No. 477682/01-4) and fellowships
(D. M. Borchhardt, H. S. Coelho, and T. M. Marchi).

3184
N. Zanatta et al. / Bioorg. Med. Chem. 14 (2006) 3174–3184
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14. Csomos, P.; Bernath, G.; Sohar, P.; Csampai, A.; Kimpe,
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