Inhibitors of the tissue factor/factor VIIa-induced coagulation: Synthesis and in vitro evaluation of novel 2-aryl substituted pyrido[3,4-d][1,3]-, pyrido[2,3-d][1,3]-, pyrazino[2,3-d][1,3]-, pyrimido[4,5-d][1,3]-, pyrazolo[3,4-d][1,3]-, thieno[3,2-d][1,3]- and thieno[2,3-d][1,3]-oxazin-4-ones

Article abstract of DOI:10.1016/S0968-0896(00)00207-8

The synthesis of a series of 2-aryl substituted hetero annulated 1,3-oxazin-4-ones and their evaluation as specific inhibitors of the tissue factor (TF)/factor VIIa (FVIIa)-induced pathway of coagulation is reported. Inhibitory activities (IC₅₀ values) in the range 0.64 to >40μM on the activation of factor X (FX) by the TF/FVIIa complex were found for compounds having one or two electronegative substituents such as F and NO₂ in the 2-aryl substituent. Some of the compounds showed a selectivity ratio towards FX and thrombin of >50, thus being similar in specificity to 2-aryl substituted 4H-3,1-benzoxazin-4-ones described as potential drugs for oral antithrombotic treatment without side effects, such as bleeding, which is observed especially with thrombin inhibitors. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00207-8

Bioorganic & Medicinal Chemistry 8 (2000) 2803±2812
Inhibitors of the Tissue Factor/Factor VIIa-Induced Coagulation:
Synthesis and In Vitro Evaluation of Novel 2-Aryl Substituted
Pyrido[3,4-d][1,3]-, Pyrido[2,3-d][1,3]-, Pyrazino[2,3-d][1,3]-,
Pyrimido[4,5-d][1,3]-, Pyrazolo[3,4-d][1,3]-, Thieno[3,2-d][1,3]-
and Thieno[2,3-d][1,3]-oxazin-4-ones
Palle Jakobsen, a,* Anne Marie Horneman a,y and Egon Persson b
aMedicinal Chemistry Research, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark
bProtein Chemistry 3, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark
Received 14 July 2000; accepted 2 August 2000
AbstractÐThe synthesis of a series of 2-aryl substituted hetero annulated 1,3-oxazin-4-ones and their evaluation as speci®c inhibi-
tors of the tissue factor (TF)/factor VIIa (FVIIa)-induced pathway of coagulation is reported. Inhibitory activities (IC50 values) in
the range 0.64 to >40 mM on the activation of factor X (FX) by the TF/FVIIa complex were found for compounds having one or
two electronegative substituents such as F and NO2 in the 2-aryl substituent. Some of the compounds showed a selectivity ratio
towards FX and thrombin of >50, thus being similar in speci®city to 2-aryl substituted 4H-3,1-benzoxazin-4-ones described as
potential drugs for oral antithrombotic treatment without side e€ects, such as bleeding, which is observed especially with thrombin
inhibitors. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
principal initiators of blood coagulation in vivo.2 It is
often desirable to inhibit the coagulation cascade in a
patient. Anticoagulants such as heparin, coumarin and
its derivatives, indandione derivatives, low-molecular-
weight thrombin or FXa inhibitors, or other agents may
be used. Treatment with heparin and other antico-
agulants may, however, have undesirable side e€ects, for
example bleedings. At the initial stage of blood coagula-
tion, i.e., of the FVIIa/TF activity, inhibition, results in
signi®cantly less bleeding.3 In addition, clinically avail-
able anticoagulants act throughout the body, rather
than acting speci®cally at the site of injury where the
coagulation cascade is active. Other known antico-
agulants comprise thrombin and factor Xa inhibitors
derived from bloodsucking organisms. Antithrombins,
hirudin, hirulog and hirugen are recombinant proteins
or peptides derived from the leach Hirudo medicinalis,
whereas the factor Xa inhibitor antistatin and the
recombinant derivative rTAP are tick-derived proteins.
Inhibitors of platelet aggregation such as monoclonal
antibodies or synthetic peptides, which interfere with
the platelet receptor gpIIb/IIIa, are also e€ective as
anticoagulants. These agents are, however, not suitable
for oral administration. Speci®c protein inhibitors of
the FVIIa/TF activity are the physiological inhibitor
Blood coagulation is a complex process involving var-
ious blood components or factors that eventually give
rise to a ®brin clot. Activated factor X (FXa) is required
to convert prothrombin to thrombin, which then con-
verts ®brinogen to ®brin as a ®nal stage in forming a
®brin clot.
2-Aryl substituted 4H-3,1-benzoxazin-4-ones1 have been
shown to act as speci®c inhibitors of the activation of
factor X (FX) through the `extrinsic pathway' in which
factor VIIa (FVIIa) and its cofactor, tissue factor (TF),
exert the proteolysis of FX to FXa.
TF is a membrane-bound protein and does not nor-
mally circulate in plasma. Upon vessel disruption, how-
ever, it is exposed and forms a complex with FVIIa to
catalyse FX or factor IX activation in the presence of
Ca2+ and phospholipid. FVIIa and TF appear to be the
*Corresponding author. Tel.: +45-4443-4800; fax: +45-4466-3939; e-
mail: paj@novo.dk
yPresent address: Leo Pharmaceutical Products, Industriparken 55,
DK-2750 Ballerup, Denmark.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00207-8

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P. Jakobsen et al. / Bioorg. Med. Chem. 8 (2000) 2803±2812
(tissue factor pathway inhibitor) and the inactivated
FVII (FVIIai), which binds to TF and competes with
FVIIa.
In connection with our investigations of 2-aryl sub-
stituted 4H-3,1-benzoxazin-4-ones,1 we found it of
interest to investigate the in¯uence of alterations of ring
size and the presence of heteroatoms in the `benzo'-part
of the molecule. Heteroatoms which change basicity as
well as charge distribution and spatial arrangement in
the ring system might in¯uence the activity of the active
ester (the lactone) and thereby cause change in activity
and speci®city.
Scheme 1. Reaction pathway for pyridines and pyrimidines. (Com-
pound: R): 2, 7: 2,6-F2Ph; 3, 6, 10: 2-FPh; 4, 8: 2-thienyl; 5, 9: 2-fur-
anyl; 11: 2-NO2Ph; 12: 2-MePh; 13: 2-ClPh.
directly connected to the ring system, and none of the
previously described compounds has been reported to
speci®cally inhibit the TF/FVIIa-induced coagulation
pathway.
We report here the synthesis of some hetero annulated
1,3-oxazin-4-one analogues of 2-aryl substituted 4H-3,1-
benzoxazin-4-ones in which the benzo ring is changed to
5-membered or 6-membered heterocycles such as thio-
phen, pyrazol, pyrimidine, pyridine and pyrazine, and
their SAR in in vitro systems of TF/FVIIa-catalysed
activation of FX, as well as their e€ects on the amidolytic
activity of FXa and thrombin and the prolongation of
clotting time caused by selected compounds.
Chemistry
The target compounds were prepared from optionally
substituted ortho amino substituted heterocyclic car-
boxylic acids by means of reaction with an aryl carboxylic
acid chloride in toluene or DMF using triethylamine or
pyridine as base. The substitution pattern on the aroyl
chlorides was mainly chosen as electronegative groups
like F and NO2 in the 2- or 2,6-positions, based on the
knowledge from the benzoxazinone series.1 However,
few other substituents were also used. In some cases this
methodology gave very low yields, and we used the
method described by Wamho€ and Kroth14 in which
Hetero annulated 1,3-oxazin-4-ones have been reported
as anti-in¯ammatory agents,4,5 human leukocyte elastase
inhibitors,6,7 herpes proteases inhibitors,8,9 and anti-
allergia.10 Di€erent synthetic pathways to this group of
compounds have been reported;11 16 however, only few
of the reported compounds contain an aryl group
Scheme 2. Reaction pathway to pyrazino[2,3-d][1,3]oxazin-4-ones 14±16.

P. Jakobsen et al. / Bioorg. Med. Chem. 8 (2000) 2803±2812
2805
the ortho amino carboxylic acid esters were used, giving
a diaroylamino intermediate which was treated with
hydrazine followed by ring closure with Br2C2Cl4 and
PPh3. The reactions are depicted in Schemes 1±4.
The same methodology was used for the preparation of
pyrazolo[2,3-d][1,3]oxazin-4-one series. 2-Fluorobenzoyl
chloride reacted with ethyl 5-amino-1-methylpyrazole-4-
carboxylate giving a 1:1 mixture of the N-mono-N,N-di-
substituted intermediate which on ring closure gave
good yield of the hetero annulated oxazinone 17. For
2-methyl- and 2-nitrobenzoyl chloride only the N,N-
disubstituted intermediate was identi®ed, leading to
compounds 18 and 19 on hydrazine treatment and ring
closure (Scheme 3). The same reaction sequence was
used in the thienyl series (Scheme 4).
For the pyridine and pyrimidine systems the ring clo-
sure proceeded straightforwardly in pyridine solution
giving fair to good yields as reported for most benzo
analogues1 (Scheme 1).
In the pyrazine series this reaction also proceeded
smoothly when 2-¯uoro or 2,6-di¯uorobenzoyl chloride
was used as reagent; however, for the 2-methylbenzoyl
chloride this gave low yield and the N,N-disubstituted
intermediate was formed. This was subsequently treated
with hydrazine followed by ring closure by means of
Br2C2Cl4 and PPh3 (Scheme 2).
Biology
The compounds were evaluated for inhibitory activities
on the TF/FVIIa-induced activation of FX using a two-
Scheme 3. Synthetic pathway to pyrazolo[2,3-d][1,3]oxazin-4-ones 17±19.

2806
P. Jakobsen et al. / Bioorg. Med. Chem. 8 (2000) 2803±2812
Scheme 4. Synthetic pathway to thieno[3,2-d][1,3]- and thieno[2,3-d][1,3]-oxazin-4-ones 20 and 21.
step amidolytic assay. Compounds showing good inhi-
bitory activities in that assay were further tested for
activities on FXa and thrombin in order to sort out
structures with the best selectivity. Selected compounds
were further tested for their abilities to prolong clotting
time in a standard in vitro clotting assay. Selected data
are depicted in Table 1.
exerted no inhibitory e€ects on the FX activation. The
best selectivity for a heterocyclic structure taken as a
group was seen in the pyrimidine series (10±13); how-
ever, only 12 gave some prolongation in clotting ratio.
The strongest inhibitors were found in the pyridine ser-
ies 2, 3, 6 and 7, but only compound 2 had a reasonable
speci®city, the e€ect on FX and thrombin being
>200 mM, with a slight e€ect on the clotting ratio. The
strongest e€ect on the clotting time was observed for 18,
19 and 21 (1.84, 1.82, and 2.18, respectively); for 20 and
21 this might be due partly to the inhibitory e€ect on
thrombin resp. FX. It is of interest that compounds 12
and 18, which both have a 2-methylphenyl substituent,
exerted a good inhibition, selectivity as well as pro-
longation of clotting time; this was not seen in the benz-
oxazinone series.1
Discussion
In the pyrazine series 14±16, no inhibition of the FX
activation was observed even when the best 2-sub-
stituents were used. For the rest of the investigated com-
pounds all the heterocycles were active as inhibitors when
the optimal 2-aryl substituents were chosen. Thienyl and
furanyl groups in the 2-position (4, 5, 8 and 9) likewise
The expected mechanism of action for the serine pro-
tease inhibition by benzoxazinone-type compounds is
deactivation by means of reaction of the benzoxazinone
lactone with the active site serine, probably under the for-
mation of a drug-enzyme ester followed by its hydro-
lysis.17,18 This mechanism would expectedly be much
in¯uenced by the presence of electronegative groups
in¯uencing the polarity of the lactone carbonyl group and
thereby changing the reactivity. Also the presence of
bulky groups or rings would be expected to have in¯u-
ence on the reactivity. Obviously, the heterocyclic struc-
ture exhibiting the most negative e€ect on the activity as
a serine protease inhibitor was the pyrazine group, the
proximity of the nitrogen atom to the lactone diminish-
ing the polarity of the CˆO. The same e€ect was not
seen in the pyrimidine or pyridine series. This seems to be
in accordance with data for the benzoxazinone series in
which electronegative substituents in the 5 and 6 positions
gave good inhibitory e€ects in the FX activation assay,
while amino substitution at the same positions gave
compounds with less inhibitory e€ects in compounds car-
rying one of the best 2-substituents, namely 2,6-di¯uoro-
phenyl. The orientation of the thieno group in 20 and 21
seemed to in¯uence the activity on FX and thrombin,
while the e€ect on FVIIa/TF-induced FX activation
was almost the same for the two compounds.
Table 1. IC50 values (mM) for the inhibition of TF/FVIIa-catalysed
activation of FX, FXa and thrombin, and e€ects on TF/FVIIa-
induced clotting. The ratio of the clotting times with (330 mM) and
without test compound is given
Compound
no.
FX Activation
IC50 (mM)
FXa
IC50 (mM)
Thrombin
IC50 (mM)
Clotting
ratio
2
3
1.3
2.6
>200
90
>200
1.24
1.04
4
5
6
>40
>40
2.8
32
5.6
>200
>200
>200
139
14.4
126.5
36.9
1.37
1.1
1.2
1.34
1.06
0.96
1.23
0.95
7
8
9
0.64
30
25
15
5.6
1.1
46.5
10
11
12
13
14
15
16
17
18
19
20
21
>200
>200
125
78
182
12
>200
>200
>200
>200
>40
18
>200
>200
>200
42
129
35
16
141
1.84
1.48
1.82
2.18
7.7
5.3
3.5

P. Jakobsen et al. / Bioorg. Med. Chem. 8 (2000) 2803±2812
2807
Conclusion
Thrombin activity assay
The synthetic approach described easily yielded a series
of 2-aryl substituted hetero annulated 1,3-oxazin-4-ones
in fair to good yields by standard synthetic methods.
The compounds described comprise several compounds
with excellent activity and selectivity, thus supporting
the information obtained for similar benzo compounds.
Good inhibition of the FVIIa/TF induced activation of
FX seems to require the presence of at least one elec-
tron-attracting substituent in the ortho position of the
2-aryl substituent, while reasonable inhibitory e€ect
was also obtained with a methyl substituent when the
annulated heterocycle was a pyrazole or pyrimidine.
Annulated pyrazine, however, gave no inhibitory e€ect
when the same 2-aryl substituents were used. Simple
heterocycles like thienyl or furanyl as the 2-substituents
caused disappearance of inhibitory e€ect.
170 mL thrombin (Boehringer Mannheim, 3.5 nM) in the
bu€er described above, 10mL test compound in DMSO
(or DMSO alone in the control) and 20mL S-2238 (Chro-
mogenix, 10mM in water) were mixed. The hydrolysis of
S-2238 was monitored as described for the FX activation
assay.
FXa activity assay
170 mL FXa (Enzyme Research Laboratories, 1.2 nM in
bu€er) in the bu€er described above, 10 mL test compound
and 20mL S-2765 (Chromogenix, 10mM in water) were
mixed. The hydrolysis of S-2765 was monitored as
described for the FX activation assay.
FVIIa/TF-initiated clotting assay
The test compounds, 20 mM in DMSO, were diluted in
citrated normal human plasma just before the analysis
(1+19) and placed in the sample carousel of an ACL
300 Research coagulometer. A 55-mL sample (com-
pound in plasma) was mixed with 55 mL of thrombo-
plastin (Innovin, Dade; diluted 1:500 after dissolution)
and incubated for 5 min. The clotting reaction was
started by adding 55 mL of a 25-mM CaCl2 solution,
yielding a ®nal compound concentration of 0.33 mM.
The ratio between the clotting time in the presence and
absence of test compounds was used to quantify the
anticoagulant e€ect.
Experimental
General procedures
Melting points (uncorrected) were measured on a Buchi
535. NMR spectra were recorded on a Bruker Avance
DPX 200 or 300 MHz instrument operating at room
temperature. Mass spectra were obtained on a Finnigan
MAT TSQ 70 apparatus using a direct inlet system. The
HPLC-MS analyses were performed on a PE Sciex API
100 LC/MS system using a WatersTM 3 mm  150 mm,
3.5 m, C-18 Symmetry column and positive ion spray
with a ¯ow rate at 20 mL/min. The column was eluted
with a linear gradient of 5±90% A, 85±0% B and 10%
C for 15 min at a ¯ow rate of 1 mL/min (solvent
A=acetonitrile, solvent B=water, and solvent
C=0.1% tri¯uoroacetic acid in water). Microanalyses
were performed by Novo Nordisk Analytical Depart-
ment.
2-(2,6-Di¯uoro-phenyl)-pyrido[3,4-d][1,3]oxazin-4-one (2).
3-Aminopyridine-4-carboxylic acid (0.28 g, 2.0 mmol)
was dissolved in dry pyridine (8 mL), and 2,6-di¯uoro-
benzoyl chloride (0.56 mL, 4.5 mmol) was added drop-
wise under stirring and cooling. The reaction was stirred
at room temperature for 24 h, after which it was con-
centrated in vacuo. The crude product was puri®ed by
¯ash chromatography on silica-gel using EtOAc:hep-
tane 1:4 and EtOAc:MeOH 10:1 as the eluent. Two
compounds were isolated: impure 2, 0.39 g, Rf 0.50
EtOAc:heptane 1:1, and a yellow crystalline compound,
0.11 g, Rf 0.50 EtOAc:MeOH 10:1. Impure 2 was dis-
solved in CH2Cl2, washed with aq NaHCO3 (5Â10 mL),
dried (Na2SO4) and concentrated to give 2 as colourless
crystals, 0.22 g (44%): mp 137±140 ꢀC; EI/SP MS: M+
260; 1H NMR (CDCl3) 9.30 (1H, s), 9.00 (1H, d), 8.05 (1H,
d), 7.55 (1H, p), 7.10 (1H, t); 13C NMR (CDCl3) 163.8,
163.7, 158.7, 158.6, 157.8, 153.1, 150.9, 150.1, 140.8,
134.3, 134.1, 133.9, 123.2, 120.4, 113.0, 112.9, 112.5,
112.4, 110.7, 110.4, 110.0.
FX activation assay
The compounds were dissolved in DMSO and mixed
with FVIIa (produced in-house) in 50 mM Hepes, pH
7.4, containing 0.1 M NaCl, 5 mM CaCl2, and 1 mg/mL
bovine serum albumin (1+5). 30 mL of this mixture was
then mixed with 45 mL TF (American Diagnostica, reli-
pidated in PC/PS vesicles) and 25 mL of FX (Enzyme
Research Laboratories), all in a Ca2+-containing bu€er.
This gave ®nal concentrations of 100 pM FVIIa, 5
pM TF, 175 nM FX, and various concentrations of the
compounds. After a 5-min incubation, the FVIIa/TF-
catalysed activation of FX was terminated by an addi-
tion of 50 mL bu€er containing enough EDTA to give
an excess of the Ca2+ ions present. 50 mL of a 2-mM
solution of S-2765 (Chromogenix, 2 mM in water) was
then added and the FXa formed was allowed to hydro-
lyse the substrate for 10 min, during which the absor-
bance at 405 nm was continuously monitored in a
SPECTRAmaxTM 340 plate reader. The slope of the
absorbance curve was compared to that of a control
where only DMSO was added to FVIIa/TF/FX.
2-(2-Fluoro-phenyl)-pyrido[3,4-d][1,3]oxazin-4-one (3). 3-
Aminopyridine-4-carboxylic acid (0.28 g, 2.0 mmol) was
dissolved in dry pyridine (10 mL), cooled in an ice bath,
and 2-¯uorobenzoyl chloride (0.53 mL, 4.5 mmol) was
added dropwise. The solution was stirred at room tem-
perature for 16 h, and the solvent was evaporated in
vacuo. The solid was puri®ed by ¯ash chromatography
on silica-gel using EtOAc:heptane 1:1 as the eluent, to
give 3 as colourless crystals, 0.44g (91%): mp 130±135 ꢀC;
1
Rf 0.50 (EtOAc:heptane 1:1); H NMR (CDCl3) 9.18

2808
P. Jakobsen et al. / Bioorg. Med. Chem. 8 (2000) 2803±2812
(1H, s), 8.83 (1H, d), 8.11 (1H, dt), 8.03 (1H, dd), 7.6-7.5
(1H, m), 7.35±7.11 (2H, m); 13C NMR (CDCl3) 164,
159.3, 158.0, 157.2, 157.1, 150.8, 149.2, 141.4, 135.2, 135.0,
131.6, 124.9, 124.8, 123.0, 120.4, 118.8, 118.6, 118.0, 117.6.
was dissolved in dry pyridine (8 mL) and 2,6-di¯uoro-
benzoyl chloride (0.52 mL, 4.5 mmol) was added under
stirring and cooling. Stirring was maintained for 18 h at
room temperature, whereupon the solvent was evapo-
rated. The residue was dissolved in CH2Cl2 (40 mL), the
organic phase was washed with saturated NaHCO3
(5Â20 mL) and concentrated. The solid was puri®ed by
¯ash chromatography on silica-gel using EtOAc:hep-
tane 1:1±EtOAc:MeOH 10:1 as the eluent. This gave
two products. Compound 7 was isolated as a colourless
crystalline compound, 0.18 g (35%): Rf 0.30 (EtOAc:
heptane 1:1). Furthermore, a yellow crystalline compound
was isolated, 0.11g, Rf 0.25 (EtOAc:MeOH 10:1); this
compound was not fully characterized. Compound 7 had
mp 209±210 ꢀC; EI/SP MS: M+ 260; 1H NMR (CDCl3)
9.08 (1H, dd), 8.63 (1H, dd), 7.51±7.40 (2H, m), 7.08 (2H,
t); 13C NMR (CDCl3) 163.9, 163.8, 159.3, 158.8, 158.7,
158.0, 157.4, 154.9, 138.3, 134.4, 134.2, 133.9, 125.1, 113.6,
112.9, 112.8, 112.5, 112.4, 110.8, 110.4, 110.1.
2-Thiophen-2-yl-pyrido[3,4-d][1,3]oxazin-4-one (4). 3-
Aminopyridine-4-carboxylic acid (0.28 g, 2.0 mmol) was
dissolved in dry pyridine (8 mL), and 2-thiophene-
carbonyl chloride (0.47 mL, 4.5 mmol) was added drop-
wise under cooling and stirring. The solution was stirred
at room temperature for 30 h and subsequently con-
centrated in vacuo. Puri®cation by ¯ash chromato-
graphy on silica-gel using CH2Cl2:acetone 50:1 as the
eluent gave crude 3, 0.50 g. The crude product was dis-
solved in CH2Cl2 (40 mL) and washed with aqueous
NaHCO3 (5Â20 mL), dried (Na2SO4) and concentrated
to give 4, 0.35 g (76%). The compound was recrys-
tallized from EtOAc; mp 167±168 ꢀC; EI/SP MS: M+
1
230; H NMR (CDCl3) 9.09 (1H, d), 8.74 (1H, d), 8.00
(2H, dd), 7.68 (1H, dd), 7.21 (1H, dd); 13C NMR (CDCl3)
158.0, 155.8, 150.4, 148.5, 141.8, 133.9, 133.0, 128.9,
122.5, 120.4. Anal. calcd for: C, 57.38%; H, 2.63%; N,
12.17%; found: C, 57.38%; H, 2.59%; N, 12.00%.
2-Thiophen-2-yl-pyrido[2,3-d][1,3]oxazin-4-one (8). 2-
Aminopyridine-3-carboxylic acid (0.28 g, 2.0 mmol) was
dissolved in dry pyridine (8 mL) and 2-thiophenecarbo-
nyl chloride (0.47 mL, 4.5 mmol) was added under stir-
ring and cooling. Stirring was maintained for 20 h at
room temperature, whereupon the solvent was evapo-
rated. The residue was dissolved in CH2Cl2 (40 mL), the
organic phase was washed with saturated NaHCO3
(5Â20 mL) and concentrated. The solid was puri®ed by
¯ash chromatography on silica-gel using EtOAc:heptane
1:1 as the eluent. This gave compound 8 as colourless
crystals, 0.28 g (61%): Rf 0.25 (EtOAc:heptane 1:1); mp
199±200 ꢀC; EI/SP MS: M+ 230; 1H NMR (CDCl3) 8.98
(1H, dd), 8.54 (1H, dd), 8.10 (1H, dd), 7.72 (1H, dd), 7.47
(1H, dd), 7.24 (1H, dd); 13C NMR (CDCl3) 159.3, 158.2,
158.0, 157.3, 138.3, 134.7, 133.8, 133.7, 129.0, 123.6,
112.9. Anal. calcd for: C, 57.38%; H, 2.63%; N,
12.17%; found: C, 57.50%; H, 2.60%, N, 11.92%.
2-Furan-2-yl-pyrido[3,4-d][1,3]oxazin-4-one (5). 3-Amino-
pyridine-4-carboxylic acid (0.28 g, 2.0 mmol) was dis-
solved in dry pyridine (8 mL), and 2-furan-carbonyl
chloride (0.45 mL, 4.5 mmol) was added under stirring
and cooling. The solution was stirred at room tempera-
ture for 30 h, the solvent was evaporated and the residue
was dissolved in CH2Cl2 (40 mL). The organic phase
was washed with saturated NaHCO3 (5Â20 mL) and
concentrated in vacuo to give 5, 0.32 g (76%). The
compound was dissolved in EtOAc and ®ltered through
activated charcoal, followed by recrystallization from
EtOAc to give 5 as colourless crystals: mp 173±174 ꢀC;
1
EI/SP MS: M+ 214; H NMR (CDCl3) 9.15 (1H, d),
8.78 (1H, d), 7.98 (1H, dd), 7.76 (1H, dd), 7.43 (1H, dd),
6.67 (1H, dd). Anal. calcd for: C, 61.69%; H, 2.28%; N,
13.08%; found: C, 61.67%; H, 2.78%; N, 12.81%.
2-Furan-2-yl-pyrido[2,3-d][1,3]oxazin-4-one (9). 2-Amino-
pyridine-3-carboxylic acid (0.28 g, 2.0 mmol) was dis-
solved in dry pyridine (8 mL) and 2-furan-carbonyl
chloride (0.45 mL, 4.5 mmol) was added under stirring
and cooling. Stirring was maintained for 20 h at room
temperature, whereupon the solvent was evaporated.
The residue was dissolved in CH2Cl2 (40 mL), the
organic phase was washed with saturated NaHCO3
(10Â20 mL) and concentrated. The solid was puri®ed by
¯ash chromatography on silica-gel using EtOAc:hep-
tane 3:2 as the eluent. Compound 9 was isolated as col-
ourless crystals, 0.28 g (65%): Rf 0.28 (EtOAc:heptane
2-(2-Fluoro-phenyl)-pyrido[2,3-d][1,3]oxazin-4-one (6). 2-
Aminopyridine-3-carboxylic acid (0.28 g, 2.0 mmol) was
dissolved in dry pyridine (8 mL) and 2-¯uorobenzoyl
chloride (0.53 mL, 4.5 mmol) was added under stirring
and cooling. Stirring was maintained for 18 h at room
temperature, whereupon the solvent was evaporated.
The residue was dissolved in CH2Cl2 (40 mL), the
organic phase was washed with saturated NaHCO3
(5Â20 mL) and concentrated. The crude product was
puri®ed by ¯ash chromatography on silica-gel using
EtOAc:heptane 1:1 as the eluent. This gave 6 as a col-
ourless solid, 0.40g (83%): Rf 0.20 (EtOAc:heptane 1:1);
ꢀ
1
3:2); mp 147±148 C; EI/ SP MS: M+ 214; H NMR
(CDCl3) 8.98 (1H, dd), 8.52 (1H. dd), 7.74 (1H, dd),
7.52 (1H, dd), 7.46 (1H, dd), 6.65 (1H, dd); 13C NMR
(CDCl3) 159.0, 158.0 (d), 152.9, 148.3, 144.3, 138.2,
123.8, 119.4, 113.4, 113.0.
mp 146±147 ꢀC; EI/SP MS: M+ 242; H NMR (CDCl3)
1
9.04 (1H, dd), 8.60 (1H, dd), 8.28 (1H, dd), 7.68±7.50 (2H,
m), 7.38±7.18 (2H, m); 13C NMR (CDCl3) 164.7, 159.5,
159.4, 159.0, 158.0, 157.8, 138.2, 135.5, 135.3, 132.1,
124.8, 124.7, 124.4, 118.7, 118.5, 118.0, 117.6, 113.2.
Anal. calcd for: C, 64.47%; H, 2.91%; N, 11.57%;
found: C, 64.33%; H, 2.91%; N, 11.44%.
7-Ethylthio-2-(2-¯uoro-phenyl)-pyrimido[4,5-d][1,3]oxa-
zin-4-one (10). 4-Amino-5-carboxy-2-ethyl mercapto-
pyrimidine (100 mg, 0.50 mmol) was dissolved in dry
DMF (5 mL) and Et3N (0.14 mL, 1.0 mmol), and sub-
sequently 2-¯uorobenzoyl chloride (0.12 mL, 1.0 mmol)
was added dropwise. The solution was stirred at room
2-(2,6-Di¯uoro-phenyl)-pyrido[2,3-d][1,3]oxazin-4-one (7).
2-Aminopyridine-3-carboxylic acid (0.28 g, 2.0 mmol)

P. Jakobsen et al. / Bioorg. Med. Chem. 8 (2000) 2803±2812
2809
temperature for 17 h and the solvent was evaporated.
The solid was stirred with 0.2 M HCl (5 mL) followed
by addition of saturated NaHCO3 (10 mL). The aqu-
eous phase was extracted with CH2Cl2 (3Â10 mL), and
the organic phase was washed with saturated NaHCO3
(10 mL) and concentrated. Puri®cation by ¯ash chro-
matography on silica-gel using EtOAc:heptane 1:1 as
the eluent gave 10, crystalline compound, 80 mg (53%).
The compound was recrystallized from EtOAc. 1H NMR
(CDCl3) showed traces of impurities: 9.27 (1H, s), 8.28
(1H, dt), 7.72±7.58 (1H, m), 7.39±7.20 (2H, m), 3.30 (2H,
q), 1.47 (3H, t); 13C NMR (CDCl3) 181.0, 163,5, 162.9,
161.7, 158.3, 156.1, 135.0, 134.8, 131.0, 123.4, 123.3, 116.8,
116.7, 116.2, 104.1, 24.8, 12.8; mp 155±156 ꢀC; EI/SP
MS: M+ 303.
DMF (5 mL), and Et3N (0.17 mL, 1.2 mmol) and sub-
sequently 2-chlorobenzoyl chloride (0.15 mL, 1.2 mmol)
were added dropwise. The solution was stirred at room
temperature for 24 h and the solvent was evaporated.
The solid was stirred with 0.2 M HCl (5 mL) followed
by addition of saturated NaHCO3 (10 mL). The aqu-
eous phase was extracted with CH2Cl2 (3Â10 mL), and
the organic phase was washed with saturated NaHCO3
(3Â10 mL) and concentrated. Puri®cation by ¯ash
chromatography on silica-gel using EtOAc:heptane 1:3
as the eluent gave impure 13 (100 mg). The solid was
dissolved in CH2Cl2 (10 mL), washed with saturated
NaHCO3 (3Â5 mL), dried (Na2SO4) and concentrated
to give compound 13 as colourless crystals, 80 mg
(42%). The compound was recrystallized from EtOAc:
1
heptane. H NMR (CDCl3) 9.24 (1H, s), 8.02 (1H, dt),
7-Ethylthio-(2-nitro-phenyl)-pyrimido[4,5-d][1,3]oxazin-4-
one (11). 4-Amino-5-carboxy-2-ethyl mercaptopyrimidine
(120 mg, 0.60 mmol) was dissolved in dry DMF (5 mL),
and Et3N (0.17 mL, 1.2 mmol) and subsequently 2-nitro-
benzoyl chloride (0.16 mL, 1.2 mmol) were added drop-
wise. The solution was stirred at room temperature for
18h and the solvent was evaporated. The solid was stirred
with 0.2 M HCl (5 mL) followed by addition of saturated
NaHCO3 (10 mL). The aqueous phase was extracted with
CH2Cl2 (3Â10mL), and the organic phase was washed
with saturated NaHCO3 (3Â10 mL) and concentrated.
Puri®cation by ¯ash chromatography on silica-gel using
EtOAc:heptane 1:2 as the eluent gave 11 as a crystalline
compound, 120 mg (60%). The compound was recrys-
tallized from EtOAc:heptane. 1H NMR (CDCl3) 9.28 (1H,
s), 8.20±8.12 (1H, m), 8.03±7.93 (1H, m), 7.86±7.72 (2H,
m), 3.31 (2H, q), 1.46 (3H, t). 13C NMR (CDCl3) 183.3,
165.8, 163.1, 160.4, 157.3, 148.7, 134.2, 133.8, 131.9, 126.7,
125.6, 106.0, 26.8, 14.6. Rf 0.45 (EtOAc:heptane 1:1); mp
113±114 ꢀC; EI/SP MS: M+ 330.
7.56 (2H, m), 7.49±7.38 (1H, m), 3.30 (2H, q), 1.44 (3H,
t); 13C NMR (CDCl3) 181.9, 165.3, 162.3, 159.1, 156.9,
133.7, 133.4, 131.9, 131.1, 128.7, 126.6, 104.9, 25.6, 13.6.
Rf 0.20 (EtOAc:heptane 1:3); mp 154±155 ꢀC; EI/SP
MS: M+ 319. Anal. calcd for: C, 52.59%; H, 3.15%; N,
13.14%; found: C, 52.71%; H, 3.14%; N, 12.97%.
2-(2-Fluoro-phenyl)-pyrazino[2,3-d][1,3]oxazin-4-one (14).
2-Aminopyrazine-3-carboxylic acid (0.14 g, 1.0 mmol)
was dissolved in dry DMF (5 mL), Et3N (0.28 mL,
2.0 mmol) was added, followed by addition of 2-¯uoro-
benzoyl chloride (0.25 mL, 2.1 mmol). The suspension
was stirred at room temperature for 17 h, after which
the solvent was evaporated. The residue was suspended
and stirred with 0.2 M HCl (5 mL). Saturated NaHCO3
(10 mL) was added, and the aqueous phase was extrac-
ted with CH2Cl2 (3Â10 mL). The organic phase was
washed with saturated NaHCO3 (2Â10 mL) followed by
concentration. The solid was puri®ed by ¯ash chroma-
tography on silica-gel using EtOAc:heptane 1:3±1:0 as
the eluent. The crude product was isolated, 50 mg
(21%). The product was dissolved in CH2Cl2 (10 mL),
washed with saturated NaHCO3 (5Â5 mL), and the sol-
vent was evaporated to give a solid (45 mg) which was
recrystallized from EtOAc to give compound 14 as col-
ourless crystals: mp 218±220 ꢀC (decomposition); EI/SP
MS: M+ 243; 1H NMR (CDCl3) 9.05 (1H, d), 8.89 (1H,
d), 8.29 (1H, dt), 7.71±7.51 (1H, m), 7.39±7.11 (2H, m);
13C NMR (CDCl3) 169.0, 165.2, 159.9, 159,4, 159.3,
136.5, 136.3, 136.3, 132.9, 124.5, 117.6, 117.2. Anal.
calcd for: C, 59.27%; H, 2.48%; N, 17.28%; found: C,
59.32%; H, 2.46%, N, 16.94%.
7-Ethylthio-2-O-tolyl-pyrimido[4,5-d][1,3]oxazin-4-one (12).
4-Amino-5-carboxy-2-ethyl mercaptopyrimidine (120mg,
0.60 mmol) was dissolved in dry DMF (5 mL), and Et3N
(0.17 mL, 1.2 mmol) and subsequently 2-methylbenzoyl
chloride (0.16 mL, 1.2 mmol) were added dropwise. The
solution was stirred at room temperature for 24 h and
the solvent was evaporated. The solid was stirred with
0.2 M HCl (5 mL) followed by addition of saturated
NaHCO3 (10 mL). The aqueous phase was extracted with
CH2Cl2 (3Â10mL), and the organic phase was washed
with saturated NaHCO3 (3Â10mL) and concentrated.
Puri®cation by ¯ash chromatography on silica-gel using
EtOAc: heptane 1:6±1:4 as the eluent gave 12 as a crystal-
line compound, 60mg (33%). The compound was
2-(2,6-Di¯uoro-phenyl)-pyrazino[2,3-d][1,3]oxazin-4-one-
(15). 2-Aminopyrazine-3-carboxylic acid (0.14 g, 1.0
mmol) was dissolved in dry DMF (5 mL), Et3N
(0.27 mL, 2.0 mmol) was added, followed by addition of
2,6-di¯uorobenzoyl chloride (0.25 mL, 2.0 mmol). The
suspension was stirred at room temperature for 16 h,
after which the solvent was evaporated. The residue was
suspended and stirred with 0.2 M HCl (5 mL). Saturated
NaHCO3 (10 mL) was added, and the aqueous phase
was extracted with CH2Cl2 (3Â10 mL). The organic
phase was washed with saturated NaHCO3 (10Â10 mL)
followed by concentration. The solid was puri®ed by
¯ash chromatography on silica-gel using EtOAc:hep-
tane 1:2±1:0 as the eluent. Impure 15 was isolated,
1
recrystallized from EtOAc:heptane. H NMR (CDCl3)
9.23 (1H, s), 8.16 (1H, dd, b), 7.52 (1H, dt, b), 7.36 (2H,
t, b), 3.30 (2H, q), 2.78 (3H, s), 1.47 (3H, t); 13C NMR
(CDCl3) 180.2, 16.5, 161.2, 157.7, 156.1, 138.9, 131.7,
130.6, 129.6, 126.9, 124.6, 103.4, 24.2, 20.9, 12.3. Rf 0.45
(EtOAc:heptane 1:3); mp 127±128 ꢀC; EI/SP MS: M+
299. Anal. calcd for: C, 60.19%; H, 4.38%; N, 14.04%;
found: C, 60.44%; H, 4.33%; N, 13.90%.
2-(2-Chloro-phenyl)-7-ethylthio-pyrimido[4,5-d][1,3]oxa-
zin-4-one (13). 4-Amino-5-carboxy-2-ethyl mercapto-
pyrimidine (120 mg, 0.60 mmol) was dissolved in dry

2810
P. Jakobsen et al. / Bioorg. Med. Chem. 8 (2000) 2803±2812
70 mg (21%). The product was dissolved in CH2Cl2
(10 mL), washed with saturated NaHCO3 (5Â5 mL),
and the solvent was evaporated to give 15, 50 mg (19%),
as a solid. The compound was recrystallized from
EtOAc to give compound 10 as colourless crystals: H
NMR (CDCl3) 9.05 (1H, d), 8.95 (1H, d), 7.65±7.48
(1H, m), 7.10 (2H, dd); EI/SP MS: M+ 261.
15.0 mmol) was added dropwise. The suspension was
stirred at 40 ꢀC for 17 h. The suspension was subse-
quently stirred with NaHCO3 (1.26 g, 15.0 mmol), and
the solvent was evaporated. The solid was suspended in
CH2Cl2 (40 mL) and washed with H2O (20 mL) and aq
NaHCO3 (3Â15 mL). The organic phase was dried
(Na2SO4), ®ltered and concentrated, and the crude pro-
duct was crystallized from EtOAc:heptane to give 5-
[bis-(2-methyl-benzoyl)-amino]-1-methyl-1H-pyrazole-4-
carboxylic acid ethyl ester as slightly coloured crystals,
1.60 g (79%): mp 170±171 ꢀC; Rf 0.51 (EtOAc:heptane
1:1); EI/SP MS: M+ 405.
1
2-O-Tolyl-pyrazino[2,3-d][1,3]oxazin-4-one (16). Com-
pound 16 was prepared in three steps starting from
3-aminopyrazine-2-carboxylic acid methyl ester as
described by Wamho€.14 1H NMR (CDCl3) 9.80 (1H,
d), 8.87 (1H, d), 8.19 (1H, dd), 7.58±7.45 (1H, m), 7.5
(2H, dt, b), 2.80 (3H, t); 13C NMR (CDCl3) 162.7,
157.8, 155.0, 151.9, 146.2, 141.1, 133.5, 132.8, 131.5,
139.5, 128.5, 126.8, 23.2. Rf 0.70 (EtOAc); mp 174±
175 ꢀC (lit. 166 ꢀC); EI/SP MS: M+ 271. Anal. calcd for:
C, 65.27%; H, 3.79%; N, 17.56%; found: C, 65.26%;
H, 3.77%; N, 17.39%.
5-[Bis-(2-methyl-benzoyl)-amino]-1-methyl-1H-pyrazole-4-
carboxylic acid ethyl ester (1.49 g, 3.7 mmol) was dis-
solved in dioxane (10 mL) and 2-propanol (6 mL), and
hydrazine hydrate (0.18 mL, 3.7 mmol) was added
dropwise. The solution was re¯uxed for 1.5 h and the
solvent was evaporated. Puri®cation by ¯ash chroma-
tography on silica-gel using EtOAc:heptane 2:3±1:1 as
the eluent gave 1-methyl-5-(2-methyl-benzoylamino)-
1H-pyrazole-4-carboxylic acid ethyl ester as colourless
crystals, 1.0 g (94%): Rf 0.40 (EtOAc:heptane 1:1); mp
119±120 ꢀC; EI/SP MS: M+ 287. Anal. calcd for: C,
62.71%; H, 5.96%; N, 14.62%; found: C, 62.84%; H,
6.00%; N, 14.58%.
6-(2-Fluoro-phenyl)-1-methyl-1H-pyrazolo[3,4-d][1,3]oxa-
zin-4-one (17). Ethyl 5-amino-1-methylpyrazol-4-
carboxylate (0.51g, 3.0 mmol) was dissolved in dry pyri-
dine (15 mL) and 2-¯uorobenzoyl chloride (0.44 mL, 3.6
mmol) was added dropwise. The solution was stirred at
50 ꢀC for 5 h, and the solvent was evaporated. The resi-
due was dissolved in CH2Cl2 (20 mL) and washed with
saturated NaHCO3 (3Â10 mL). The organic phase was
dried (Na2SO4) and concentrated, and the crude pro-
duct was puri®ed by ¯ash chromatography on silica-gel
using EtOAc:heptane 1:3 as the eluent. This gave two
products: the bis-acylated compound, 0.3 g, Rf 0.52
(EtOAc:heptane 1:1), and 5-(2-¯uoro-benzoylamino)-1-
methyl-1H-pyrazole-4-carboxylic acid ethyl ester, 0.22 g
(25%) as colourless crystals: Rf 0.38 (EtOAc:heptane
1:1); mp 106±108 ꢀC; EI/ SP MS: M+ 291. Anal. calcd
for: C, 57.73%; H, 4.84%; N, 14.43%; found: C,
58.16%; H, 4.87%; N, 14.07%.
1-Methyl-5-(2-methyl-benzoylamino)-1H-pyrazole-4-
carboxylic acid ethyl ester (0.52 g, 1.88 mmol) was dis-
solved in dry toluene (15 mL) at 80 ꢀC. Triphenylphos-
phine (0.54 g, 2.07 mmol) was added, followed by
addition of Et3N (0.78 mL, 5.64 mmol). Br2C2Cl4
(0.67 g, 2.07 mmol) was dissolved in dry toluene (3 mL)
and added dropwise. The reaction was stirred at 80 ꢀC
for 18 h, after which the suspension was ®ltered and the
®ltrate was concentrated. Puri®cation by ¯ash chroma-
tography on silica-gel using EtOAc:heptane 1:3 as the
eluent gave 18 as colourless crystals, 0.40 g (89%): Rf
1
0.25 (EtOAc:heptane 1:3); H NMR (CDCl3) 8.08 (1H,
5-(2-Fluoro-benzoylamino)-1-methyl-1H-pyrazole-4-
carboxylic acid ethyl ester (0.16 g, 0.55 mmol) was dis-
solved in dry toluene (6 mL). Triphenylphosphine
(0.16 g, 0.60 mmol) was added, followed by addition of
Et3N (0.23 mL, 1.65 mmol). Br2C2Cl4 (0.20 g, 0.60 mmol)
was dissolved in dry toluene (1 mL) and added drop-
wise. The reaction was stirred at 80 ꢀC for 24 h, after
which the solvent was evaporated. Puri®cation by ¯ash
chromatography on silica-gel using EtOAc:heptane 1:3
as the eluent gave 17 as colourless crystals, 95 mg
dd), 8.04 (1H, s), 7.50±7.28 (3H, m), 4.03 (3H, s), 2.72
(3H, s); 13C NMR (CDCl3) 163.5, 155.2, 151.4, 140.1,
137.0, 132.6, 131.0, 129.3, 126.6, 99.3, 35.0, 23.0. The
compound was recrystallized from EtOAc:heptane; mp
130±131 ꢀC; EI/SP MS: M+ 241. Anal. calcd for: C,
64.72%; H, 4.60%; N, 17.42%; found: C, 64.60%; H,
4.63%; N, 17.38%.
1-Methyl-6-(2-nitro-phenyl)-1H-pyrazolo[3,4-d][1,3]oxa-
zin-4-one (19). Ethyl 5-amino-1-methylpyrazol-4-car-
boxylate (0.85 g, 5.0 mmol) was dissolved in dry
pyridine (25 mL), and 2-nitrobenzoyl chloride (2.0 mL,
15.0 mmol) was added dropwise. The suspension was
stirred at 40 ꢀC for 17 h. The suspension was subse-
quently stirred with NaHCO3 (1.26 g, 15.0 mmol) and
the solvent was evaporated. The solid was suspended in
CH2Cl2 (40 mL) and washed with H2O (20 mL) and aq
NaHCO3 (3Â15 mL). The organic phase was dried
(Na2SO4), ®ltered and concentrated, and the crude pro-
duct was crystallized from EtOAc:heptane to give 5-
[bis-(2-nitro-benzoyl)-amino]-1-methyl-1H-pyrazole-4-
carboxylic acid ethyl ester as slightly coloured crystals,
1.83 g (78%): mp 167±168 ꢀC; Rf 0.32 (EtOAc:heptane
1:1); EI/SP MS: M+467.
1
(73%). Rf 0.30 (EtOAc:heptane 1:2); H NMR (CDCl3)
8.16 (1H, dt), 8.09 (1H, s), 7.66±7.52 (1H, m), 7.36±7.18
(2H, m), 4.07 (3H, s); 13C NMR (CDCl3) 164.6, 160.0,
159.9, 159.4, 154.8, 151.2, 137.2, 135.2, 135.0, 131.7,
124.8, 124.7, 118.9, 118.7, 118.0, 117.6, 99.6, 35.1. The
compound was recrystallized from EtOAc:heptane; mp
134±135 ꢀC; EI/SP MS: M+ 245. Anal. calcd for: C,
58.78%; H, 3.29%; N, 17.14%; found: C, 58.85%; H,
3.22%; N, 16.89%.
1-Methyl-6-(2-methyl-phenyl)-1H-pyrazolo[3,4-d][1,3]ox-
azin-4-one (18). Ethyl 5-amino-1-methylpyrazol-4-car-
boxylate (0.85 g, 5.0 mmol) was dissolved in dry pyridine
(25 mL), and 2-methylbenzoyl chloride (1.96 mL,

P. Jakobsen et al. / Bioorg. Med. Chem. 8 (2000) 2803±2812
2811
5-[Bis-(2-nitro-benzoyl)-amino]-1-methyl-1H-pyrazole-4-
carboxylic acid ethyl ester (1.71 g, 3.7 mmol) was dis-
solved in dioxane (10 mL) and 2-propanol (6 mL), and
hydrazine hydrate (0.18 mL, 3.7 mmol) was added
dropwise. The solution was re¯uxed for 1.5 h and the
solvent was evaporated. Puri®cation by ¯ash chroma-
tography on silica-gel using EtOAc:heptane 1:1 as the
eluent gave 1-methyl-5-(2-nitro-benzoylamino)-1H-pyr-
azole-4-carboxylic acid ethyl ester as colourless crystals,
0.95 g (81%): Rf 0.20 (EtOAc:heptane 1:1); mp 204±
205 ꢀC; EI/SP MS: M+ 318. Anal. calcd for: C, 52.83%;
H, 4.43%; N, 17.60%; found: C, 52.89%; H, 4.44%; N,
17.54%.
7.58±7.41 (1H, m), 7.08 (2H, t), 2.44 (3H, s); 13C NMR
(CDCl3) 163.8, 163.7, 158.7, 158.6, 155.6, 155.3, 153.7,
135.3, 133.7, 133.5, 133.3, 118.0, 112.8, 112.7, 112.4,
112.3, 110.8, 13.1. The compound was recrystallized
from EtOAc; mp 149±151 ꢀC; EI/SP MS: M+ 279.
Anal. calcd for: C, 55.91; H, 2.53%; N, 5.02%; found:
C, 55.95%; H,2.49%; N, 4.98%.
5-Methyl-2-(2-nitro-phenyl)-thieno[2,3-d][1,3]oxazin-4-
one (21). Ethyl 2-amino-4-methylthiophene-3-carboxy-
late (0.74 g, 4.0 mmol) was dissolved in dry CH2Cl2
(10 mL). 2-Nitrobenzoyl chloride (0.58 mL, 4.4 mmol)
was added followed by the addition of Et3N (0.61 mL,
4.4 mmol). The suspension was stirred at room tempera-
ture for 72 h, after which the organic phase was washed
with saturated NaHCO3 (3Â30 mL), dried (Na2SO4), ®l-
tered and concentrated. This gave 4-methyl-2-(2-nitro-
benzoylamino)-thiophene-3-carboxylic acid ethyl ester
as slightly coloured crystals (1.23 g, 92%): Rf 0.25
(EtOAc:heptane 1:2). The compound was recrystallized
from EtOAc; mp 135±137 ꢀC; EI/SP MS: M+ 334.
1-Methyl-5-(2-nitro-benzoylamino)-1H-pyrazole-4-car-
boxylic acid ethyl ester (0.37 g, 1.16 mmol) was dissolved
in dry toluene (75 mL) at 80 ꢀC. Triphenylphosphine
(0.34 g, 1.28 mmol) was added, followed by addition of
Et3N (0.48 mL, 3.49 mmol). Br2C2Cl4 (0.42 g, 1.28 mmol)
was dissolved in dry toluene (2 mL) and added drop-
wise. The reaction was stirred at 80 ꢀC for 18 h, after
which the suspension was ®ltered and the ®ltrate was
concentrated. Puri®cation by ¯ash chromatography on
silica-gel using EtOAc:heptane 2:3 as the eluent gave 19
as colourless crystals, 0.24 g (75%): Rf 0.37 (EtOAc:
4-Methyl-2-(2-nitro-benzoylamino)-thiophene-3-car-
boxylic acid ethyl ester (0.38 g, 1.1 mmol) was dissolved
in dry toluene (25 mL) at 80 ꢀC. PPh3 (0.33 g, 1.3 mmol)
was added followed by the addition of Et3N (0.47 mL,
3.4 mmol). C2Br2Cl4 (0.41 g, 1.3 mmol) was dissolved in
dry toluene (2 mL) and added to the reaction mixture.
The suspension was stirred at 80 ꢀC for 24 h, after which
it was ®ltered and the ®ltrate was concentrated. The
crude product was puri®ed by ¯ash chromatography on
silica-gel using EtOAc:heptane 1:4 as the eluent. This
gave 21 as a slightly yellow crystalline compound
1
heptane 1:1); H NMR (CDCl3) 8.12 (1H, s), 8.04±7.92
(2H, m), 7.80±7.70 (2H, m), 4.00 (3H, s); 13C NMR
(CDCl3) 160.2, 154.2, 150.6, 149.3, 137.4, 133.2, 131.4,
125.6, 124.9, 99.6, 35.2. The compound was recrys-
tallized from EtOAc:heptane; mp 175±176 ꢀC; EI/SP
MS: M+ 272. Anal. calcd for: C, 52.95%; H, 2.96%; N,
20.58%; found: C, 52.94%; H, 2.95%; N, 20.48%.
1
2-(2,6-Di¯uoro-phenyl)-7-methyl-thieno[3,2-d][1,3]oxa-
zin-4-one (20). Methyl 3-amino-4-methylthiophene-2-
carboxylate (0.68 g, 4.0 mmol) was dissolved in dry pyri-
dine (10 mL) and 2,6-di¯uorobenzoyl chloride (0.53 mL,
4.2 mmol) was added. The suspension was stirred at
room temperature for 7 days, after which the solvent
was evaporated. The solid was dissolved in CH2Cl2
(50 mL) and the organic phase was washed with satu-
rated NaHCO3 (3Â30 mL), dried (Na2SO4), ®ltered and
concentrated. This gave 3-(2,6-di¯uoro-benzoylamino)-
4-methyl-thiophene-2-carboxylic acid methyl ester as a
colourless crystalline compound (1.17 g, 94%): Rf 0.33
(EtOAc:heptane 1:2). The compound was recrystallized
from EtOAc; mp 159±160 ꢀC; EI/SP MS: M+ 311.
Anal. calcd for: C, 54.02%; H, 3.56%; N, 4.50%;
found: C, 53.97%; H, 3.53%; N,4.45%.
(0.29 g, 91%): Rf 0.31 (EtOAc:heptane 1:2); H NMR
(CDCl3) 8.02±7.92 (2H, m), 7.68±7.55 (2H, m), 6.97
(1H, m), 2.57 (3H, s); 13C NMR (CDCl3) 163.4, 157.2,
155.0, 149.1, 136.2, 133.3, 132.7, 131.4, 125.9, 125.0,
120.7, 117.7, 16.4. The compound was recrystallized
from EtOAc to give slightly coloured crystals; mp 155±
165 ꢀC; EI/SP MS: M+ 288. Anal. calcd for: C, 54.16%;
H, 2.80%; N, 9.72%; found: C, 54.11%; H, 2.75%; N,
9.67%.
Acknowledgement
The skilful technical assistance of Bo R. Pedersen is
gratefully acknowledged.
3-(2,6-Di¯uoro-benzoylamino)-4-methyl-thiophene-2-
carboxylic acid methyl ester (0.45 g, 1.45 mmol) was dis-
solved in dry toluene (25 mL) at 80 ꢀC. Then PPh3
(0.42 g, 1.59 mmol) was added followed by addition of
Et3N (0.60 mL, 4.34 mmol). C2Br2Cl4 (0.52 g, 1.59 mmol)
was dissolved in dry toluene (2 mL) and added to the
reaction mixture. The suspension was stirred at 80 ꢀC
for 7 h and at 50 ꢀC for 90 h, after which it was ®ltered
and the ®ltrate was concentrated. The crude product
was puri®ed by ¯ash chromatography on silica-gel using
EtOAc:heptane 1:4 as the eluent. This gave 20 as a col-
ourless crystalline compound (0.32 g, 80%): Rf 0.50
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