Design and synthesis of novel platelet fibrinogen receptor antagonists with 2H-1,4-benzoxazine-3(4H)-one scaffold. A systematic study

Article abstract of DOI:10.1016/j.ejmech.2004.09.004

New platelet glycoprotein IIb/IIIa (GP IIb/IIIa, integrin α_IIbβ₃) antagonists were prepared on a 2H-1,4-benzoxazine-3(4H)-one scaffold. Their anti-aggregatory activities in human platelet rich plasma and their affinity towards α

Full text of DOI:10.1016/j.ejmech.2004.09.004

European Journal of Medicinal Chemistry 40 (2005) 25–49
www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel platelet fibrinogen receptor antagonists
with 2H-1,4-benzoxazine-3(4H)-one scaffold. A systematic study
Marko Anderluh a, Jožko Cesar a, Petra Štefanicˇ a, Danijel Kikelj a, Damjan Janeš a,
Jernej Murn a, Kristina Nadrah a, Mojca Tominc a, Elisabeth Addicks b, Athanassios Giannis b,1
Mojca Stegnar c,2, Marija Sollner Dolenc a,*
,
a Faculty of Pharmacy, University of Ljubljana, Aškercˇeva 7, 1000 Ljubljana, Slovenia
b Institut für Organische Chemie, Universität Leipzig, Johannnisallee 29, 04103 Leipzig, Germany
c University Medical Centre, Department of Angiology, Zaloška 7, 1525 Ljubljana, Slovenia
Received 10 May 2004; received in revised form 1 September 2004; accepted 6 September 2004
Available online 08 December 2004
Abstract
New platelet glycoprotein IIb/IIIa (GP IIb/IIIa, integrin aIIbb3) antagonists were prepared on a 2H-1,4-benzoxazine-3(4H)-one scaffold.
Their anti-aggregatory activities in human platelet rich plasma and their affinity towards aIIbb3 and aVb3 integrins were assessed. Various
substitution positions and side chain variations were studied. In contrast to the generally accepted model, compounds containing ethyl esters
as aspartate mimetics were in general more active than the corresponding free acids. We suggest an explanation for the observed behaviour of
these new compounds.
© 2004 Elsevier SAS. All rights reserved.
Keywords: 2H-1,4-benzoxazine-3(4H)-one; Fibrinogen receptor; Fibrinogen receptor antagonists; Platelet aggregation
1. Introduction
carboxylic group and a basic functionality, in appropriate spa-
tial positions, are the key pharmacophore elements of the
aIIbb3 antagonists, mimicking the aspartate b-carboxylate and
the arginine guanidinium group in the RGD sequence. In addi-
tion, an alkyl- or aryl-sulphonamide or carbamate functional
group at the position a- to the carboxy terminus of the antago-
nist constitutes an additional, exosite-binding group [3]. This
approach has led to the discovery of tirofiban (Scheme 1),
one of three parenteral aIIbb3 antagonists currently being used
for the management of acute coronary syndrome and for
therapy adjunctive to coronary surgical procedures [4].
Platelet aggregation plays a vital role in primary haemo-
stasis, but under pathological conditions, such as those fol-
lowing an atherosclerotic plaque rupture, this process may
lead to an arterial thrombosis that can result in myocardial
infarction, ischaemic stroke, and peripheral artery disease [1].
The final common step in platelet aggregation is the binding
of fibrinogen to its glycoprotein IIb/IIIa (GPIIb/IIIa, integrin
aIIbb3) receptor, which is located on the surface of activated
platelets. Development of aIIbb3 antagonists has been one of
the main focuses in antithrombotic research over the last
decade [2]. They are based predominantly on the RGD (Arg-
Gly-Asp) sequence found in many natural ligands of the
receptor. According to the generally accepted model, a free
However, several orally active drug candidates targeted
against integrin aIIbb3 have so far proved to be ineffective in
clinical trials. This could be due to their poor pharmacoki-
netic properties; modest bioavailability and relatively short
half-life, resulting in high variation of the plasma drug con-
centration (peak to trough ratio) and thus preventing the sus-
tained inhibition required for a beneficial pharmacological
effect [5]. In addition, several results suggest that, at low
doses, aIIbb3 antagonists may activate the receptor, leading to
* Corresponding author. Tel.: +386 1 47 69 572; fax: +386 1 42 58 031.
E-mail addresses: giannis@chemie.uni-leipzig.de (A. Giannis),
mojca.stegnar@trnovo.kclj.si (M. Stegnar), marija.sollner@ffa.uni-lj.si
(M.S. Dolenc).
1 Tel.: +49 341 97 36 527; fax: +49 341 97 36 599.
2 Tel.: +386 1 52 28 052; fax: +386 1 52 28 070.
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.09.004

26
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
whole series of compounds. We attempt to resolve this para-
dox with a hypothesis that could contribute to an explanation
of the failure in clinical trials of several drug candidates.
2. Chemistry
2.1. Preparation of 2H-1,4-benzoxazine-3(4H)-one
scaffolds
Our strategy for the preparation of potential aIIbb3 antago-
nists was to attach various substituents with a pharmacoph-
ore group to the 2H-1,4-benzoxazine-3(4H)-one scaffold. In
order to achieve different substitution patterns on the scaf-
fold, four starting heterocycles were synthesized, as shown in
Scheme 3.
Initially, 2-amino-4-nitrophenol 1 was used to perform
one-step O-alkylation and, following cyclization with diethyl
2-bromo-2-methylmalonate, to give ethyl 2-methyl-6-nitro-
2H-1,4-benzoxazine-3(4H)-one-2-carboxylate (2) [10]. The
corresponding 7-nitro derivative 4 was prepared from
2-amino-5-nitrophenol (3). Alternatively, introduction of a
hydroxyl group at position 2 was achieved by N-acylation of
3 with 2,2-dichloroacetylchloride to give 5 and subsequent
cyclization under basic conditions to give 6 [11]. 2H-1,4-
benzoxazine-3(4H)-one derivative bearing no substituents at
position 2 (7) was obtained from 3 and ethyl 2-bromoacetate
[12].
Scheme 1. Structures of Tirofiban and Roxifiban.
increased aggregation [6]. Orally active, low molecular
weight aIIbb3 antagonists, like roxifiban (Scheme 1), with
higher affinity towards the receptor and consequently more
favourable pharmacokinetic properties, are therefore still of
interest [7].
We report the design and synthesis of a new series of
IIbb3 antagonists. Various 6,6-bicyclic heterocycles have
a
already been used as templates in developing RGD mimetics
[8,9]. In our work, we focused on 2H-1,4-benzoxazine-
3(4H)-one as a peptidomimetic building block. This parent
bicyclic heterocycle was chosen because of its ready syn-
thetic accessibility and, more importantly, the various substi-
tution options that allow different spatial positions of the
pharmacophore functional groups to be studied (Scheme 2).
The compounds synthesized were tested for their anti-
aggregatory activity againstADP-induced aggregation in hu-
man platelet rich plasma. Their in vitro affinities towards the
isolated aIIbb3 and the closely related aVb3 integrin were also
assessed. While antagonists with a free carboxylic group
expressed higher affinity towards the aIIbb3 in vitro, the
corresponding carboxylic esters showed higher anti-
aggregatory activity against ADP-induced aggregation.
These results are somehow paradoxical and have not been
reported before, but were consistent through almost the
2.2. 2,6- and 2,7-Di-substituted 2H-1,4-benzoxazine-
3(4H)-ones with an ethoxycarbonyl or carboxy substituent
at position 2
Scaffolds 2 and 4 were used as starting materials for the
synthesis of the target compounds 24–31 (Scheme 4). Ini-
tially, a variety of residues were attached to position 4 of the
benzoxazinone ring by NaH assisted alkylation with differ-
ent alkyl halides. The nitro group was then reduced by cata-
lytic hydrogenation and the resulting amines (12–17) were
acylated with p-cyanobenzoyl chloride to give 18–23. Ni-
triles 18–23 were subsequently subjected to Pinner reaction
Scheme 3. Synthetic route to 2H-1,4-benzoxazine-3(4H)-ones 2, 4, 6 and 7.
(a) EtOOCC(CH3)BrCOOEt, KF, DMF, 60 °C; (b) Cl2CHCOCl, Et3N,
Et2O; (c) NaHCO3, H2O, 100 °C; (d) BrCH2COOEt, KF, DMF, 60 °C.
Scheme 2
.
RGD tripeptide as the lead compound and strategy for the
synthesis of 2H-1,4-benzoxazine-3(4H)-one-based RGD mimetics.

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
27
Scheme 4
. (a) NaH, MeI (8), PhCH2Br (9), BrCH2COOEt (10), EtBr (11), toluene; (b) H2, Pd/C, EtOH; (c) 4-CNC6H4COCl, Et3N, CH2Cl2; (d) 1. HCl(g), EtOH;
2. CH3COO–NH4+, EtOH; (e) 1.NaOH(aq), dioxane; 2. HCl(aq)
.
[8]; treatment with dry gaseous HCl in absolute ethanol and
reaction of the resulting imidates with ammonium acetate
yielded amidines 24–29 in the form of their acetate salts.
Finally, saponification of ethyl esters 24 and 29 was per-
formed in order to obtain free carboxylic acids 30 and 31.
under the same conditions. The resulting nitro compound 33
was converted by catalytic hydrogenation to amine 34, which
was acylated with p-cyanobenzoyl chloride to give 35. The
amidine 36 was obtained by Pinner conversion of nitrile 35
and further saponification of 36 to give the free acid 37
(Scheme 5).
2.3. 2,7-Di-substituted 2H-1,4-benzoxazine-3(4H)-ones
with ethylcarbonylmethoxy or carboxymethoxy substituent
at position 2
The synthesis of target compound 44 is outlined in
Scheme 6. The initial benzoxazinone 4 was N-alkylated to
give 38 in which the ethyl ester group was cleaved to give 39
[13]. In order to introduce a methylene spacer between the
benzoxazine ring and the carboxyl moiety at position 2, a
modified Arndt-Eistert synthesis was conducted on carboxy-
lic acid 39 [14]. After mixed anhydride activation of the acid,
reaction with trimethylsilyldiazomethane (TMSCHN2) as a
substitute for diazomethane yielded diazoketone 40, which
was then subjected to a Wolff rearrangement. The resulting
homologated ester 41 was reduced to give amine 42. The
In our next series, alkylation of scaffold 6 bearing a hy-
droxyl group at position 2 of the benzoxazine ring gave a
mixture of N-alkylated and N,O-dialkylated products that
could be separated by flash chromatography (32; N-alkylated
product: N,O-dialkylated product 3:1). Therefore an ethyl
group was introduced to position 4 by KF-mediated alkyla-
tion followed by O-alkylation of 32 with ethyl bromoacetate
+
Scheme 5
.
(a) EtBr, KF, DMF; (b) BrCH2COOEt, KF, DMF; (c) H2, Pd/C, EtOH; (d) 4-CNC6H4COCl, Et3N, CH2Cl2; (e) 1. HCl (g), EtOH; 2. CH3COO–NH4
,
EtOH; (f) 1. NaOH(aq), EtOH, 2.CH3COOH.
Scheme 6
MeOH; (f) 4-CNC6H4COCl, Et3N, CH2Cl2; (g) 1. HCl(g), MeOH; 2. CH3COO–NH4+, MeOH.
.
(a) MeI, KF, DMF; (b) NaOH, dioxane; (c) 1. EtOCOCl, Et3N, THF; 2. TMSCHN2, MeCN; (d) PhCOO–Ag+, Et3N, MeOH, ultrasound; (e) H2, Pd/C,

28
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
+
Scheme 7
.
(a) GlyOEt, 58, EDC, HOBt, NMM, DMF; (b) H2, Pd/C, EtOH; (c) 4-CNC6H4COCl, Et3N, CH2Cl2, 0 °C; (d) 1. HCl(g), EtOH; 2. CH3COO–NH4
,
EtOH; (e) NH2OH × HCl, Et3N, EtOH, 50 °C; (f) 1. EtOCOCl, pyridine, –15 °C; 2. 120 °C; (g) NaOH(aq), EtOH; (h) H2, Pd/C, CH3COOH, DMF; (i) 59, EDC,
HOBt, NMM, DMF; (j) H2, Pd/C, CH3COOH, DMF; (k) 1. NaOH(aq), EtOH; 2.HCl.
amidine 44 was obtained by acylation of 42 with
p-cyanobenzoyl chloride and subsequent Pinner conversion
of the nitrile 43.
philic group neighbouring the carboxylic acid residue has
been proved to be beneficial for increasing aIIbb3 binding
affinity in numerous studies [3], we prepared L-2-amino-3-
(toluene-4-sulfonylamino)-propionic acid ethyl ester hydro-
chloride (58), starting from L-asparagine [16]. This was
coupled to 39 by the standard EDC/HOBt method to give 46.
The nitro group was then reduced to give amine 48. 4-(1,2,4-
oxadiazol-5(4H)-one)benzoic acid (59 [15]) was coupled to
the resulting amine 48 to give 55. Removal of the cyclic
carbamate protection group by catalytic hydrogenation
yielded amidine 56. Saponification of the ester group with
NaOH in ethanol yielded free acid 57.
The synthesis of target compounds 67, 68, 69 and 70 is
presented in Scheme 8. The initial acid 4 was coupled to
L-phenylalanine ethyl ester hydrochloride or L-3-amino-2-
(toluene-4-sulfonylamino)-propionic acid ethyl ester hydro-
chloride (71 [17]). Reduction of 61 and 62 by catalytic
hydrogenation furnished key intermediate amines 63 and 64,
which were acylated with p-cyanobenzoyl chloride. The re-
sulting nitriles 65 and 66 were converted to amidines 67 and
68 by Pinner’s procedure and ester groups were removed to
yield 69 and 70.
In the next series we decided to further lengthen the
substituent at position 2 of the benzoxazinone ring, as de-
picted in Scheme 7. For this purpose, glycine ethyl ester was
coupled to the acid 39 by the standard EDC/HOBt coupling
procedure to give 45. The nitro group was then reduced by
catalytic hydrogenation and the resulting amine 47 was acy-
lated with p-cyanobenzoyl chloride to give 49. Nitrile 49 was
subjected to Pinner reaction to yield amidine 50. Since the
1,2,4-oxadiazol-5(4H)-one ring has been described as an
easily accessible and versatile precursor of the amidino moi-
ety [15], we utilized this approach for preparing target com-
pound 55 from 49. Nitrile 49 was first converted to ami-
doxime 51 with hydroxylammonium chloride in basic
ethanolic media. This was then O-acylated with ethyl chlo-
roformate and subsequently cyclized under reflux in pyridine
to yield the 1,2,4–oxadiazol-5(4H)-one derivative (52). The
ester group was then saponified with NaOH in ethanol to give
53 and the cyclic carbamate protection group removed by
catalytic hydrogenation to give 54. As an additional lipo-

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
29
Scheme 8
. (a) NaOH(aq), dioxane; (b) L-PheOEt, 71, EDC, HOBt, NMM, DMF; (c) H2, Pd/C, EtOH; (d) 4-CNC6H4COCl, Et3N, CH2Cl2; (e) 1. HCl(g), EtOH;
2. CH3COO–NH4+, EtOH; (f) 1. NaOH(aq), EtOH; 2. CH3COOH.
Scheme 9. (a) BrCH2COOEt, KF, DMF, 60 °C; (b) NaOH(aq), dioxane; (c) 71, EDC, HOBt, NMM, DMF; (d) H2, Pd/C, EtOH; (e) 59, EDC, HOBt, NMM, DMF;
(f) 1. H2, Pd/C, CH3COOH, DMF; 2. CH3COO–NH4+, EtOH; (g) 1. NaOH(aq), EtOH; 2. CH3COOH; (h) Br(CH2)4COOEt, BnN+(Et)3Cl-, MeCN, 60 °C; (i) 59,
EDC, HOBt, NMM, DMF; (j) NaOH(aq), EtOH; (k) H2, Pd/C, CH3COOH, DMF.

30
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
2.4. Preparation of 4,7-di-substituted
2H-1,4-benzoxazine-3(4H)-ones
negligible improvement in inhibition of platelet aggregation.
In the next step, the 2H-1,4-benzoxazine-3(4H)-one scaffold
was lengthened at position 2 with an oxymethylene moiety
(36 and 37) while retaining the same substituent at position 7.
Surprisingly, good affinity for aIIbb3, but poor anti-
aggregatory effect was observed. Because of the unfavour-
able effect of the oxymethylene group on activity, we decided
to replace it with a methylene spacer between the benzox-
azine ring and the carboxyl moiety. The resulting ester 44
was far more efficient in inhibiting aggregation but had
surprisingly low affinity for integrin aIIbb3. Further improve-
ment of anti-aggregatory activity was observed with deriva-
tives lengthened at position 2 with a carboxylglycine moiety
(50 and 54). As seen before with free acid derivatives, free
acid (54) had poor anti-aggregatory activity but strong affin-
ity for integrin aIIbb3.
Several groups have reported that introduction of an aryl-
or alkyl- sulphonamide group to the position a- to the car-
boxyl moiety resulted in higher activity of aIIbb3 antagonists
[3,17]. Importantly, additional binding groups should not
only enhance the affinity towards the receptor, but also pro-
long the drug’s plasma half-life [19]. For these reasons,
glycine in 54 was replaced by L-Phe, Na- and Nb-p-
toluenesulfonyl-2,3-diaminopropionates. In our series, intro-
duction of the p-toluensulfonamide group did not increase
the potency of the compounds. The b-sulfonamido deriva-
tives (56, 57) showed weaker anti-aggregatory activity and
the aIIbb3 affinity was comparable to that of the glycine
derivatives. The a-sulfonamido derivative 68 was a slightly
more potent anti-aggregatory agent than the b-sulfonamido
analogue 56 with free acid (70) which was totally insoluble
in aqueous media. L-Phe substituted derivatives (67, 69)
inhibited aggregation in a modestly improved manner, and
the ester and free acid derivatives had almost the same affin-
ity for aIIbb3, with IC50 reaching the nanomolar range. Com-
pounds 67 and 69 were selective versus aVb3; in fact, only
31, 69, 76 and 77 had any affinity for aVb3.
Of the 2,7-di-substituted 2H-1,4-benzoxazine-3(4H)-
ones, only a few were good inhibitors of platelet aggregation.
The probable explanation is that they fail to form the “cup-
shaped” conformation, which is presumably the one that fits
best the binding site of integrin aIIbb3 [2(a)]. Furthermore, a
series of 4,7-di-substituted 2H-1,4-benzoxazine-3(4H)-ones
were synthesized. Molecular modelling (HyperChem 7.5,
Hypercube, Inc.: conformations not included in the article)
revealed that 4,7-di-substituted analogues should have more
”cup-shaped” minimum conformations while retaining the
generally accepted distance requirement between carboxy-
late and amidine moieties (Table 1) [2(a)]. Compound 76 was
a fairly potent inhibitor of aggregation, but did not reach the
nanomolar range. Its free acid counterpart 77 had a high
affinity for aIIbb3, but was not tested for inhibition of aggre-
gation due to lack of solubility in aqueous media. Finally,
derivatives with more flexible conformations were synthe-
sized, 81 and 82. In contrast to the previously described
compounds, ester 81 inhibited aggregation only in the mid
4,7-Di-substituted 2H-1,4-benzoxazine-3(4H)-ones 76,
77, 81 and 82 were obtained from benzoxazine derivative 7
(Scheme 9). This was first N-alkylated with ethyl 2-bromo-
acetate, using KF as a base, to give 72. Saponification of 72
with NaOH in dioxane, and coupling of the resulting acid
with 71 gave 73. Subsequent catalytic hydrogenation of the
nitro group yielded amine 74, which was acylated with car-
boxylic acid 59 to give 75. Removal of the cyclic carbamate
protection group by catalytic hydrogenation gave 76. Saponi-
fication of 76 with NaOH in ethanol yielded 77.
Alternatively, 7 was N-alkylated with ethyl 5-bromo-
pentanoate, using the phase-transfer procedure with K2CO3
as base and benzyltriethylammonium chloride as the cata-
lyst, to give 78 [13]. Catalytic hydrogenation of the nitro
group and further coupling of 59 to the resulting amine 79
gave 80. 1,2,4-Oxadiazol-5(4H)-one group was converted to
amidinium 81 salt by catalytic hydrogenation in acetic
acid/DMF. Analogously, saponification of the ester group
and cleavage of the 1,2,4-oxadiazol-5(4H)-one group in 80
yielded 82.
3. Results and discussion
The ability of the synthesized compounds to inhibit plate-
let aggregation and the binding of fibrinogen to aIIbb3 and
aVb3 integrins was characterized in a platelet aggregation
assay and a solid-phase competitive displacement assay, re-
spectively. The results are presented in Table 1.
The first series of compounds were 2,6-di-substituted 2H-
1,4-benzoxazine-3(4H)-ones (24–27, 30) containing the
p-benzoylamidine moiety as an arginine mimetic, since it has
been proposed that a benzamidine moiety should interact
most favourably with the carboxylate moiety at the binding
site [18].A carboxylic acid (or ester) moiety at position 2 was
supposed to mimic the aspartate residue. Since 24 showed
modest inhibition of platelet aggregation, the free acid de-
rivative 30 was synthesized. To our surprise, it showed no
anti-agreggatory activity at all and this stimulated us to
optimise the ester 24. Different substituents were attached at
the ring nitrogen (position 4, compounds 25–27) to increase
non-polar interaction with the binding site, as proposed pre-
viously [8]. Only a methyl group was tolerated (compound
25), while bulkier substituents resulted in significant loss of
activity (compounds 26 and 27). All the compounds men-
tioned showed low affinity for both aIIbb3 and aVb3 integrins
(>100 µM), which is consistent with low anti-aggregatory
activity.
2,7-Di-substituted 2H-1,4-benzoxazine-3(4H)-ones were
more promising (28 compared to 24). Again, the correspond-
ing free acid 31 was practically inactive in inhibiting aggre-
gation while showing higher affinity for aIIbb3 (and, interest-
ingly, for aVb3). N-ethylation of 28 (29) resulted in

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
31
Table 1
Inhibition of platelet aggregation and inhibition of binding of fibrinogen to integrins aIIbb3 and aVb3 expressed as IC50 values (N.D. not determined because of
poor solubility in aqueous media)
Compound Structural formula
IC50 (µM)
(20 µM ADP)
IC50 (µM)
(aIIbb3 affinity)
IC50 (µM)
(aVb3 affinity)
Distance between
carboxylate and amidine
C-atoms (Å) a
24
25
26
27
29
200
145
790
591
40
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
14.296
14.370
14.111
14.120
13.322
30
>250
>100
>100
14.302
28
31
36
37
44
45
71.595
43.840
76.375
0.257
>100
63.585
>100
>100
>100
12.948
11.648
15.376
14.393
14.116
>250
78
650
5.0
>100
50
54
56
1.8
>25
21
10.242
0.128
>100
>100
>100
15.765
16.783
17.724
33.276
57
25
6.023
>100
15.580
67
69
68
0.6
6.9
5.6
7.721
7.202
12.685
>100
16.125
15.533
15.523
83.477
>100
70
N.D.
N.D.
N.D.
16.522
(continued on next page)

32
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
Table 1 (continued)
Compound Structural formula
IC50 (µM)
(20 µM ADP)
IC50 (µM)
(aIIbb3 affinity)
IC50 (µM)
(aVb3 affinity)
Distance between
carboxylate and amidine
C-atoms (Å) a
76
77
9.5
4.606
0.308
20.562
9.213
19.081
N.D.
19.026
81
18.9
2.5
0.123
0.0167
1.145
>100
18.074
17.732
12.729
82
>100
RGDS
260
3.296 × 10–3
a Distance between pharmacophore centres: conformations of potential energy minima were generated with HyperChem 7.5, Hypercube, Inc.
micromolar range, while the free acid 82 was able to bind to
IIbb3 with dramatically improved potency. The astonishing
pothesis [21]. Fibrinogen binding to aIIbb3 receptor triggers
outside-in signalling, which leads to receptor activation and
further platelet activation [22]. The ability of fibrinogen to
activate its receptor is intrinsic activity. The same effect can
be observed by a small RGD peptide and, presumably, all
small binding molecules [23]. This results in release of intra-
cellular Ca2+ (positive influence on platelet release reaction)
and exposure of ligand-induced binding sites (LIBS), which
are presumed to play an important role in receptor clustering
after fibrinogen binding in the last stage of platelet aggrega-
tion [23,24]. Alternatively, the aIIbb3 receptors might remain
in an activated conformation after partial agonist dissocia-
tion, thus leaving free the fibrinogen binding site [6]. Conse-
quently, aggregation of platelets could occur after fibrinogen
binding to free aIIbb3 receptors, even though the majority of
a
fact was the high affinity observed for the ester analogue.
In most cases, there is seen to be a reasonable correlation
between the anti-aggregatory potency of the ester analogues
and the affinity of free acids for aIIbb3. Compounds 37 (and
ester analogue 36) and 82 (and ester analogue 81) however,
showed high affinity for aIIbb3 but failed to adequately inhibit
platelet aggregation. These discrepancies are believed to be
the consequence of compounds 36, 37, 81 and 82 not being
full antagonists of the aIIbb3 receptor; despite their high
affinity for the receptor, they would not be able to inhibit
aggregation, due to remaining intrinsic activity (see below).
Notably, in almost all cases, esters were more potent than
the free acids in inhibiting platelet aggregation, in contrast
with the generally accepted model. Grumel et al. [20] re-
ported an equivocal observation, where ester analogues of
synthesized RGD mimetics showed similar or better anti-
aggregatory activity than the corresponding free acids. This
could be easily explained by the fact that free acid analogues
are zwitterions which are known to dissolve poorly in aque-
ous media. The activity of ester analogues could be the
consequence of in situ hydrolysis of the ester bond in the
assay, as numerous esterases are present in the blood (and
therefore in the assay plasma). However, cleavage of the ester
bond would form the same free acids that are not soluble
enough to allow measurement of their anti-aggregatory activ-
ity. Eventually, free acids formed after hydrolysis would
precipitate in the aqueous assay medium. But again, this does
not explain the anti-aggregatory activity of the esters.
a
IIbb3 receptors are occupied (Fig. 1). The full antagonists,
however, should be devoid of this activity, as inhibition of
fibrinogen binding should prevent, not only aggregation, but
also all pro-aggregatory processes. We believe that com-
pounds having small alkyl ester groups instead of carboxylic
ones could be regarded as full antagonists. In support of the
above hypothesis, Dickfeld et al. [25] reported that some
known fibrinogen receptor “antagonists” (tirofiban, integre-
lin, lamifiban; free acid compounds) do inhibitADP-induced
platelet aggregation but fail to inhibit TRAP-induced platelet
aggregation and platelet release (ATP and P-selectin secre-
tion), and even induce LIBS exposure. Therefore, these “an-
tagonists” act also in a pro-aggregatory manner, which is a
consequence of their intrinsic activity. Based on these results,
Dickfeld et al. proposed that development of aIIbb3 receptor
antagonists with less or zero LIBS activity and enhanced
release blocking might be advantageous. Nevertheless, cur-
rently used “antagonists” (tirofiban, integrelin) inhibit ADP-
mediated platelet aggregation potently by merely binding to
We propose that some or all of the free acid analogues
might be partial agonists of the aIIbb3 receptor, while esters
might be full antagonists of the fibrinogen receptor in terms
of receptor activation, even though both are inhibitors of
platelet aggregation. Cox et al. also reported a similar hy-
a
IIbb3 receptors with high affinity. The major therapeutic

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
33
Fig. 1. Proposed mechanism of fibrinogen receptor partial agonist/antagonist activity.
problem with these compounds is that at least 80% of all
IIbb3 receptors must be occupied to achieve therapeutically
inhibit platelet aggregation with less than 80% receptor occu-
pancy.
a
useful anti-aggregatory activity [21], so antagonists with
extremely high affinity are required. High receptor occu-
pancy, however, correlates with high bleeding risk, so plasma
levels of such agents must be kept within a relatively narrow
range. We assume that full antagonists would not provoke
bleeding risks to any great extent, since they are expected to
What can be said about the molecular mechanism of this
“ester-free acid paradox”? aIIbb3 receptors exist in active and
inactive conformations [25]. In the natural pathway, fibrino-
gen binds only to activated receptors, leading to platelet
aggregation. In contrast, it has been shown that RGD pep-
tides and their mimetics can bind to the inactive conforma-

34
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
tion of aIIbb3 [26]. Later, Xiong et al. [27,28] elucidated the
crystal structure of the extracellular segment of integrin aVb3
with the RGD-containing ligand. The active conformation of
aVb3 was shown to complex Mn2+ ions in the b3-subunit
carboxylate-binding site, while the inactive conformation
lacks Mn2+ ions in this site. These ions are supposed to form
an ionic bond with the carboxylate moiety of the binding
molecule, which would not be possible in the inactive con-
formation. Since integrins aVb3 and aIIbb3 share the same
b3-subunit, we propose that the same could be true of the
integrin aIIbb3. Namely, free acids bind to the active form of
integrin aIIbb3 by forming an ionic bond with the metal ion in
the binding site. This explains the high binding affinity of the
free acids. Since fibrinogen binds to the active conformation
of the receptor and triggers further signalling, the active
conformation must be responsible for the outside-in signal-
ling. The inactive conformation of aIIbb3 lacks the metal ion
and has more free space in the “carboxylate-binding hole”. It
could therefore accommodate an even larger group than the
carboxylate, i.e. the small alkyl ester group (see Fig. 2).
Since, in the inactive conformation of integrin aVb3 (and
presumably in the inactive conformation of aIIbb3), there is
no metal ion in the binding site, there is no requirement for a
binding group to form an ionic bond. Instead, hydrogen
bonds could be formed between ester oxygens and the metal-
ion chelating groups of the b3-subunit of the integrin aIIbb3
(for more details see [28]). Since formation of an ionic bond
releases more free energy than hydrogen bonding, a lower
affinity is observed for esters. It seems reasonable to predict
that the inactive conformation of aIIbb3 is incapable of
outside-in signalling and should therefore not contribute to
pro-aggregatory processes. Compounds which bind to the
inactive conformation of the receptor could diminish the
capacity of total fibrinogen-binding sites, not only by mere
occupation, but also by prolonging the lifetime of the inactive
conformation of the receptor. Being bound to the inactive
conformation of aIIbb3 receptor means that they should also
be incapable of triggering outside-in signalling. Compounds
with an ester moiety could, therefore, be envisaged as full
fibrinogen receptor antagonists.
The proposed hypothesis of fibrinogen partial
agonists/antagonists action could offer an answer to the pre-
viously described fact that low doses of fibrinogen receptor
“antagonists” having a carboxylic acid moiety do not inhibit
but, rather, promote platelet aggregation [29]. Considering
free acid analogues as partial agonists of the aIIbb3 receptor,
one might expect that, in lower doses, a free acid compound
would bind to the receptor to an insufficient extent to inhibit
aggregation, but sufficient enough to trigger platelet activa-
tion and LIBS exposure. In higher doses, compounds with
high affinity for the receptor would occupy almost all platelet
a
IIbb3 receptors, thus preventing the binding of fibrinogen.
The proposed hypothesis could offer some new insight into a
molecular explanation for previously reported phenomena
that some fibrinogen receptor “antagonists” failed to reach
the desired therapeutic effect after administration to patients
in clinical trials [5,30,31]. Though Scarborough and Gretler
[30], reported that poor pharmacokinetics have a major im-
pact on therapeutic insufficiency of some fibrinogen receptor
“antagonists” a combination of poor pharmacokinetics and
the proposed mechanism appears reasonable.
4. Conclusions
In conclusion, we have prepared novel 2H-1,4-
benzoxazine-3(4H)-one template-based
aIIbb3 receptor
ligands. Although aspartate mimetic moieties and substitu-
tion positions were varied, almost all the compounds synthe-
sized showed rather unusual binding behaviour, ethyl esters
being more active than free acids against ADP-induced hu-
man platelets aggregation. Although the impact of this phe-
nomenon on in vivo antagonist properties is yet to be clari-
fied, we propose a new hypothesis. Compounds bearing
small alkyl ester moieties may be full antagonists of aIIbb3
receptor, while the corresponding free acids could act as
partial agonists. The proposed hypothesis could offer a new
insight into the several aIIbb3 antagonists that fail to elicit
appropriate therapeutic activity. The ester compounds could
not be used as therapeutic agents, as hydrolysis under in vivo
conditions would form the corresponding free acids, but
could be useful as a starting points for the design of new,
therapeutically active, fibrinogen receptor antagonists.
Fig. 2. DThe aspartate binding site in the b3-subunit (vitronectin receptor,
presented as protein surface) with Mn2+ ions (violet spheres) and aspartate
residue (“ball and stick” rendering) [28]. For clarity, only the aspartate
residue of the c(RGDfV) ligand is shown. The absence of a metal ion (to the
right of the carboxyl group) would clearly leave enough space for a small
alkyl group.

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
35
5. Experimental protocols
product was established with TLC. The reaction mixture was
cooled to RT and washed with water (3 × 5 ml) and brine
(5 ml). The toluene phase was dried over Na2SO4, filtered
and evaporated under vacuum. Cold ethanol was poured onto
the oily residue and mixed vigorously until the crude product
precipitated. The precipitate was filtered off and dried under
vacuum.
5.1. General methods and materials
Chemicals from Aldrich Chemical Co., Fluka, and Acros
Organics were used without further purification. Anhydrous
solvents were prepared according to standard procedures
[32]. Analytical TLC was performed on Merck silica gel
(60 GF 254) plates (0.25 mm) and components were visual-
ized with ultraviolet light (254 nm wavelength). Column
chromatography was carried out on silica gel 60 (particle size
240–400 mesh). Melting points were determined on a Re-
5.2.1.3. General procedure for reduction of nitro
compounds to amines by catalytic hydrogenation (12–17,
34, 42, 47, 48, 63, 64, 74, 79). Argon was bubbled into a
solution of nitro compound (5 mmol) in anhydrous ethanol
(50 ml) and palladium (10% by weight, 10% on active char-
coal) was added stepwise. Hydrogen was bubbled into the
suspension and stirring continued under 1 atm of hydrogen at
RT until no nitro compound was detected with TLC. The
catalyst was filtered off and the solvent evaporated under
vacuum.
1
ichert hot stage microscope and are uncorrected. H-NMR
spectra were recorded on Bruker AVANCE DPX300 spec-
trometer in CDCl3 or DMSO-d6 solution with TMS as the
internal standard. IR spectra were obtained on a Perkin-
Elmer 1600 FT-IR spectrometer. Microanalyses were per-
formed on a Perkin-Elmer C, H, N analyzer 240 C. Analyses
indicated by the symbols of the elements or functions were
within 0.4% of the theoretical values. Mass spectra were
obtained using a VG-Analytical Autospec Q mass spectrom-
eter. All yields relate to purified products.
Human integrin aVb3 was obtained from Chemicon. Hu-
man integrin aIIbb3, purified human fibrinogen, biotin
N-hydroxysuccinimide ester and anti-biotin (goat) peroxi-
dase conjugate were obtained from Calbiochem. Tris(hy-
droxymethyl)aminomethane (Tris) buffer, Tween 20 CaCl2,
MgCl2 and MnCl2 were obtained from Acros Organics. Bo-
vine serum albumin (BSA) was purchased from Aldrich
Chemical Co. BM chemiluminescence ELISA Substrate
(POD) was obtained from Roche Diagnostics, Mannheim.
RGDS was purchased from Bachem.
5.2.1.4. General procedure for acylation with carboxylic
acid chlorides (18–23, 35, 43, 49, 65, 66). To a cooled
(–10 °C) solution of amine (1.0 mmol) and triethylamine
(1.1 mmol) in distilled dichloromethane (10 ml), carboxylic
acid chloride was added stepwise and the mixture stirred for
6 h. During stirring, the temperature was allowed to rise to
RT. If the product precipitated from the reaction mixture, it
was filtered off and washed with a small portion of dichlo-
romethane. Otherwise, the solvent was evaporated and the
residue dissolved in ethyl acetate (20 ml) and washed with
water (20 ml), 10% aqueous citric acid (2 × 20 ml), saturated
aqueous NaHCO3 (2 × 20 ml) and brine (20 ml). The organic
phase was dried over Na2SO4, filtered and evaporated under
vacuum.
5.2. Synthesis
5.2.1.5. General procedure for conversion of nitriles into
amidines (Pinner reaction) (24–29, 36, 44, 50, 67, 68)
[33]. Into an ice-cooled solution of nitrile (1.0 mmol) in
anhydrous ethanol (20 ml), gaseous HCl was bubbled until
complete saturation was achieved (~30 min). The mixture
was stirred for 24 h at RT and solvent then evaporated under
vacuum. The residue was dispersed in diethyl ether (20 ml),
filtered and washed with diethyl ether (2 × 20 ml). The crude
residue was further dried with a membrane pump and dis-
solved in anhydrous ethanol (10 ml). Ammonium acetate
(3.0 mmol) was added to the solution and stirred for another
24 h at RT. Diethyl ether was added dropwise into the reac-
tion mixture until all the solid product was precipitated. The
crude product was filtered off and dried under vacuum.
5.2.1. General procedures
5.2.1.1. General procedure for the alkylation with alkyl
halides in the presence of KF (2, 4, 7, 32, 33, 38, 72)
[10]. To a suspension of 100 mmol KF in 25 ml of anhydrous
N,N-dimethylformamide (DMF), 40 mmol of alkylhalide
and 40 mmol of starting compound were added. The mixture
was stirred for 30 min at room temperature (RT) after which
it was heated to 60 °C and stirred until complete conversion
to the final product was established with thin-layer chroma-
tography (TLC). The reaction mixture was cooled to RT and
poured on 100 g of ice. The ice was allowed to melt and the
precipitate was filtered off, washed with water and, unless
stated otherwise, recrystallized from ethanol.
5.2.1.6. General procedure for saponification of the ester
group with NaOH(aq)/dioxane (30, 31, 39, 60, 73). Into a
stirred solution of ester (20 mmol) in dioxane (70 ml), 2 M
NaOH(aq) (30 mmol) was added. The reaction was allowed to
continue for the next 24 h at RT and the solvent was then
evaporated. The crude residue was dissolved in water (60 ml)
and the solution washed with ethyl acetate (2 × 50 ml). The
5.2.1.2. General procedure for N-alkylation of amides with
NaH and alkylhalides (8, 9, 10, 11, 38) [13]. To a suspension
of 1.0 mmol of amide in 5 ml of anhydrous toluene NaH
(1.0 mmol) was added and the suspension stirred for 30 min
at RT. Alkyl halide (1.1 mmol) was added and the mixture
heated at 100 °C until complete conversion to the final

36
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
water phase was acidified with 4 M HCl to pH~3, extracted
with ethyl acetate (2 × 50 ml) and dried over Na2SO4. The
solvent was evaporated under vacuum to yield crude product.
5.2.3. Ethyl 2-methyl-7-nitro-2H-1,4-benzoxazine-3(4H)-
one-2-carboxylate (4) [10]
Prepared according to the general procedure as in Section
5.2.1.1 from 2-amino-5-nitrophenol (6.17 g, 40.0 mmol) and
diethyl 2-bromo-2-methylmalonate (7.64 ml, 40.0 mmol). m
= 9.30 g (83%). M.p. 191–193 °C. IR (KBr, cm–1): *3088,
1749, 1697, 1605, 1540, 1508, 1340, 1237, 1123, 1012, 826,
744. *Primary amines usually show two IR signals in the
3300–3400 cm–1 region differing by approximately
100 cm–1. This region is however covered with “low” base
5.2.1.7. General procedure for saponification of the ester
group with NaOH(aq)/ethanol (37, 53, 57, 69, 70, 77, 82). To
a dispersion of ethyl ester (0.29 mmol) in 96% ethanol
(10 ml), 2 M NaOH(aq) (0.3 ml, 0.6 mmol) was added and
stirred at RT until no starting compound was detected by
TLC. Unless otherwise stated, the mixture was treated with
acetic acid (1.0 ml) and cooled at 4 °C overnight. Precipitated
product was filtered off, washed with ice-cooled ethanol
(10 ml) and dried under vacuum.
1
line. H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.07 (t, 3H,
J = 7.2 Hz, CH2CH3), 1.75 (s, 3H, 2-CH3), 4.12 (m, 2H,
CH2), 7.10 (d, 1H, J = 8.9 Hz, Ar–H5), 7.84 (d, 1H,
J = 2.6 Hz, Ar–H8), 7.95 (dd, 1H, J = 8.9, 2.6 Hz, Ar–H6),
11.60 (s, 1H, –CONH–). MS (FAB) = 281 (MH+).
5.2.1.8. General procedure for coupling with EDC/HOBt/
NMM (45, 46, 55, 61, 62, 73, 75, 80). To a cooled (–10 °C)
solution of carboxylic acid (1.0 mmol) and amine (1.0 mmol)
in anhydrous N,N-dimethylformamide (5 ml), 1-hydroxy-
benzotriazole (HOBT) monohydrate and N-methyl-
morpholine (2.3 mmol) were added. The pH of the mixture
was checked with wet pH-indicator strip (~8) and N-ethyl-
N′-(dimethylaminopropyl)carbodiimide hydrochloride (EDC)
(1.3 mmol) was added. The temperature was allowed to rise
to RT and the mixture was stirred until complete conversion
to the final product was detected with TLC. The solvent was
evaporated and the residue was dissolved in ethyl acetate
(20 ml), washed with aqueous citric acid (10%, 2 × 20 ml),
saturated aqueous NaHCO3 (2 × 20 ml) and brine (20 ml) and
dried over Na2SO4. The solvent was evaporated under
vacuum to yield crude product.
5.2.4. 2,2-Dichloro-N-(2-hydroxy-4-nitrophenyl)acetamide
(5)
Dichloroacetyl chloride (4.91 ml, 50 mmol) was dissolved
in diethylether (20 ml) and added dropwise to a cooled
(–10 °C) suspension of 2-amino-5-nitrophenol (8.11 g,
50 mmol) and triethylamine (6.96 ml, 50 mmol) in diethyl-
ether (100 ml). After 1 h of stirring, the temperature was
raised to RT and stirring continued for another 4 h. Precipi-
tated triethylamonium chloride was filtered off and the fil-
trate washed with water (100 ml), 0.5 M HCl (2 × 100 ml),
brine (100 ml) and then dried over Na2SO4. The solvent was
evaporated under vacuum and the residue recrystallized from
chloroform. m = 9.45 g (71%). M.p. 149–151 °C. IR (KBr,
cm–1): 3178, 1681, 1592, 1557, 1511, 1430, 1340, 1267,
1079, 942. 1H-NMR (CDCl3, 300 MHz): d (ppm) 6.12 (s, 1H,
CHCl2), 6.95 (s, 1H, OH), 7.81 (d, 1H, J = 2.3 Hz, Ar–H6),
7.92 (dd, 1H, J = 9.1, 2.3 Hz,Ar–H4), 8.25 (d, 1H, J = 9.1 Hz,
Ar–H3), 8.91 (s, 1H, –CONH–). MS (EI) = 264 (M+). Anal.
CHN C8H6Cl2N2O4.
5.2.1.9. General procedure for conversion of 5-oxo-4,5-
dihydro-1,2,4-oxadiazoles to amidines by catalytic hydroge-
nation (54, 56, 76, 81, 82) [15]. Argon was bubbled into a
solution of 5-oxo-4,5-dihydro-1,2,4-oxadiazole derivative
(0.23 mmol) in anhydrous DMF (10 ml) and acetic acid
(5 ml). Palladium (10% in weight, 10% on active charcoal)
was then added stepwise. Hydrogen was bubbled into the
suspension and stirring continued under 1 atm of hydrogen at
RT until no initial compound was detected. The catalyst was
filtered off and the solvent evaporated under vacuum. The
oily residue was treated with diethyl ether and the crude
product dried under vacuum.
5.2.5. 2-Hydroxy-7-nitro-2H-1,4-benzoxazine-3(4H)-one
(6)
5 (5.30 g, 20 mmol) was suspended in an aqueous solution
(150 ml) of NaHCO3 (3.36 g, 40 mmol) and boiled for
30 min. The reaction mixture was cooled to RT, acidified
with 1 M HCl (pH ~3) and extracted with ethyl acetate (2 ×
80 ml). The organic phase was washed with 1 M HCl (2 ×
100 ml), saturated aqueous NaHCO3 (2 × 100 ml), brine
(100 ml) and dried over Na2SO4. The solvent was evapo-
rated, the residue suspended in a mixture of diethylether
(20 ml) and petroleum ether (10 ml) and filtered off. m
= 2.96 g (71%). M.p. 183–187 °C. IR (KBr, cm–1): 3088,
5.2.2. Ethyl 2-methyl-6-nitro-2H-1,4-benzoxazine-3(4H)-
one-2-carboxylate (2) [10]
This was prepared according to the general procedure as
in Section 5.2.1.1 from 2-amino-4-nitrophenol (6.17 g,
40.0 mmol) and diethyl 2-bromo-2-methylmalonate
(7.64 ml, 40.0 mmol). m = 8.15 g (73%). M.p. 194–198 °C.
IR (KBr, cm–1): 3462, 1736, 1696, 1528, 1490, 1384, 1348,
1261, 1132, 1082, 1018, 966. 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.08 (t, 3H, J = 7.2 Hz, CH2CH3), 1.76 (s,
3H, 2-CH3), 4.14 (m, 2H, CH2), 7.25 (d, 1H, J = 9.0 Hz,
Ar–H8), 7.76 (d, 1H, J = 2.6 Hz, Ar–H5), 7.89 (dd, 1H, J =
9.0, 2.6 Hz, Ar–H7), 11.36 (s, 1H, –CONH–). MS (EI) = 280
(M+).
1
1690, 1605, 1506, 1342, 1216, 1014. H-NMR (DMSO-d6,
300 MHz): d (ppm) 5.62 (d, 1H, J = 5.8 Hz, 2-H), 7.11 (d, 1H,
J = 8.9 Hz,Ar–H5), 7.82 (d, 1H, J = 2.5 Hz,Ar–H8), 7.95 (dd,
1H, J = 8.9, 2.5 Hz, Ar–H6), 8.32 (d, 1H, J = 5.8 Hz, 2-OH),
11.42 (s, 1H, –CONH–). MS (EI) = 210 (M+). Anal. CHN
C8H6N2O5.

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
37
5.2.6. 2-Methyl-7-nitro-2H-1,4-benzoxazine-3(4H)-one (7)
[10]
ethyl bromide. m = 2.01 g (91%). M.p. 75–79 °C. IR (KBr,
cm–1): 2983, 1747, 1715, 1602, 1522, 1391, 1338, 1263,
1
1140, 1022, 884, 793, 745. H-NMR (CDCl3, 300 MHz):
Prepared according to the general procedure as in Section
5.2.1.1 from 2-amino-5-nitrophenol (10.0 g, 60 mmol) and
ethyl bromoacetate (6.85 ml, 60 mmol). m = 7.37 g (63%).
M.p. 235–239 °C. IR (KBr, cm–1): 3080, 1698, 1598, 1509,
d (ppm) 1.16 (t, 3H, J = 7.2 Hz, NCH2CH3), 1.32 (t, 3H,
J = 7.2 Hz, OCH2CH3), 1.92 (s, 3H, 2-CH3), 4.17 (m, 4H,
2 × CH2), 7.06 (d, 1H, J = 8.6 Hz, Ar–H5), 7.98 (m, 2H,
Ar–H6, Ar–H8). MS (EI) = 308 (M+). Anal. CHN
C14H16N2O6.
1
1392, 1341, 1222, 1044, 887, 742. H-NMR (DMSO-d6,
300 MHz): d (ppm) 4.72 (s, 2H, CH2), 7.06 (d, 1H, J
= 8.7 Hz, Ar–H5), 7.75 (d, 1H, J = 2.3 Hz, Ar–H8), 7.89 (dd,
1H, J = 8.7, 2.3 Hz, Ar–H6), 11.30 (s, 1H, –CONH–). MS
(FAB) = 195 (MH+).
5.2.11. Ethyl 6-amino-2-methyl-2H-1,4-benzoxazin-3(4H)-
one-2-carboxylate (12) [10]
Prepared according to the general procedure as in Section
5.2.1.3 from 2.50 g (8.93 mmol) of 2. m = 2.07 g (93%). M.p.
132–133 °C. IR (KBr, cm–1): 3383, 1753, 1703, 1617, 1521,
1494, 1421, 1351, 1228, 1125, 1015, 867. 1H-NMR (DMSO-
d6, 300 MHz): d (ppm) 1.06 (t, 3H, J = 7.2 Hz, CH2CH3),
1.60 (s, 3H, 2-CH3), 4.06 (m, 2H, CH2), 4.86 (s, 2H, NH2),
6.11–6.15 (m, 2H, Ar–H5, Ar–H7), 6.67 (d, 1H, J = 9.4 Hz,
Ar–H8), 10.62 (s, 1H, –CONH–). MS (EI) = 250 (M+).
5.2.7. Ethyl 2,4-dimethyl-6-nitro-2H-1,4-benzoxazin-
3(4H)-one-2-carboxylate (8)
Prepared according to the general procedure as in Section
5.2.1.2 from 2.10 g (7.5 mmol) of 2 and 0.69 ml (11.0 mmol)
methyl iodide. m = 0.77 g (36%). M.p. 101–103 °C. IR (KBr,
cm–1): 3472, 1745, 1636, 1522, 1474, 1381, 1348, 1277,
1152, 1019, 895. 1H-NMR (DMSO-d6, 300 MHz): d (ppm)
1.06 (t, 3H, J = 7.2 Hz, CH2CH3), 1.79 (s, 3H, 2-CH3), 3.41
(s, 3H, N–CH3), 4.09 (m, 2H, CH2); 7.32 (d, 1H, J = 9.0 Hz,
Ar–H8), 7.99 (m, 2H, Ar–H7, Ar–H5). MS (EI) = 294 (M+).
Anal. CHN C13H14N2O6.
5.2.12. Ethyl 6-amino-2,4-dimethyl-2H-1,4-benzoxazin-
3(4H)-one -2-carboxylate (13)
Prepared according to the general procedure as in Section
5.2.1.3 from 0.82 g (2.8 mmol) of 8. m = 0.53 g (72%). M.p.
116–119 °C. IR (KBr, cm–1): 3447, 3364, 2981, 1724, 1675,
5.2.8. Ethyl 4-benzyl-2-methyl-6-nitro-2H-1,4-benzoxazin-
3(4H)-one-2-carboxylate (9)
1
1613, 1516, 1383, 1278, 1234, 1134, 1014, 858. H-NMR
Prepared according to the general procedure as in Section
5.2.1.2 from 2.38 g (8.5 mmol) of 2 and 1.19 ml (10.0 mmol)
benzyl bromide. m = 2.48 g (79%). M.p. 87–90 °C. IR (KBr,
cm–1): 3484, 1749, 1636, 1525, 1456, 1385, 1342, 1172,
(DMSO-d6, 300 MHz): d (ppm) 1.03 (t, 3H, J = 7.2 Hz,
CH2CH3), 1.63 (s, 3H, 2-CH3), 3.23 (s, 3H, N–CH3), 4.05
(m, 2H, CH2), 4.93 (s, 2H, NH2), 6.23 (dd, 1H, J = 8.7,
2.3 Hz, Ar–H7), 6.37 (d, 1H, J = 2.3 Hz, Ar–H5), 6.73 (d, 1H,
J = 8.7 Hz, Ar–H8). MS (EI) = 264 (M+). Anal. CHN
C13H16N2O4.
1
1128, 1002, 878, 727. H-NMR (DMSO-d6, 300 MHz):
d (ppm) 1.08 (t, 3H, J = 7.2 Hz, CH2CH3), 1.88 (s, 3H,
2-CH3), 4.15 (m, 2H, –CH2–CH3), 5.05 (d, 1H, J = 16.6 Hz,
–CH2–Ph), 5.50 (d, 1H, J = 16.6 Hz, –CH2–Ph), 7.27–7.41
(m, 6H, Ph, 5H,Ar–H8), 7.82 (d, 1H, J = 2.5 Hz,Ar–H5), 7.95
(dd, 1H, J = 8.6, 2.5 Hz, Ar–H7). MS (EI) = 370 (M+). Anal.
CHN C19H18N2O6.
5.2.13. Ethyl 6-amino-4-benzyl-2-methyl-2H-1,4-
benzoxazin-3(4H)-one -2-carboxylate (14)
Prepared according to the general procedure as in Section
5.2.1.3 from 1.46 g (3.95 mmol) of 9. The crude product was
recrystallized from ethanol. m = 1.10 g (82%). M.p. 153–
156 °C. IR (KBr, cm–1): 3458 (wide signal), 1723, 1674,
1609, 1513, 1446, 1398, 1340, 1234, 1125, 1006, 838.
1H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.06 (t, 3H,
J = 7.2 Hz, CH2CH3), 1.71 (s, 3H, 2-CH3), 4.10 (q, 2H,
J = 7.2 Hz, –CH2–CH3), 4.79 (d, 1H, J = 16.4 Hz, –CH2–Ph),
4.94 (s, 2H, NH2), 5.27 (d, 1H, J = 16.4 Hz, –CH2–Ph), 6.20
(m, 2H, Ar–H5, Ar–H7), 6.76 (d, 1H, J = 9.0 Hz, Ar–H8),
7.27–7.38 (m, 5H, Ph). MS (EI) = 340 (M+). Anal. CHN
C19H20N2O4.
5.2.9. Ethyl 4-(2-ethoxy-2-oxoethyl)-2-methyl-6-nitro-2H-
1,4-benzoxazin-3(4H)-one-2-carboxylate (10)
Prepared according to the general procedure as in Section
5.2.1.2 from 1.68 g (6.0 mmol) of 2 and 0.95 ml (8.5 mmol)
ethyl bromoacetate. m = 1.65 g (75%). M.p. 108–110 °C. IR
(KBr, cm–1): 3414, 2997, 1749, 1707, 1617, 1522, 1379,
1344, 1210, 1018, 885, 749. 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.07 (t, 3H, J = 7.2 Hz, 2-COOCH2CH3),
1.20 (t, 3H, J = 7.2 Hz, NCH2COOCH2CH3), 1.80 (s, 3H,
2-CH3), 4.05–4.20 (m, 4H, 2 × CH2–CH3), 4.81 (d, 1H, J
= 17.7 Hz, N–CH2–), 4.97 (d, 1H, J = 17.7 Hz, N–CH2–),
7.37 (d, 1H, J = 9.0 Hz,Ar–H8), 8.00 (m, 2H,Ar–H7,Ar–H5).
MS (FAB) = 367 (MH+). Anal. CHN C16H18N2O8.
5.2.14. Ethyl 6-amino-4-(2-ethoxy-2-oxoethyl)-2-methyl-
2H-1,4-benzoxazin-3(4H)-one -2-carboxylate (15)
Prepared according to the general procedure as in Section
5.2.1.3 from 1.83 g (5.0 mmol) of 10. The crude product was
recrystallized from ethanol. m = 1.36 g (80%). M.p. 155–
160 °C. IR (KBr, cm–1): 3448 (wide signal), 1734, 1684,
5.2.10. Ethyl 4-ethyl-2-methyl-7-nitro-2H-1,4-benzoxazin-
3(4H)-one-2-carboxylate (11)
Prepared according to the general procedure as in Section
5.2.1.2 from 2.00 g (7.1 mmol) of 4 and 0.99 ml (8.6 mmol)
1
1625, 1512, 1387, 1236, 1127, 1016, 940, 849. H-NMR
(DMSO-d6, 300 MHz): d (ppm) 1.06 (t, 3H, J = 7.2 Hz,

38
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
2-COOCH2CH3), 1.21 (t, 3H,
J
=
7.2 Hz,
N–CH3), 4.08 (m, 2H, CH2), 7.09 (d, 1H, J = 8.7 Hz, Ar–H8),
7.44 (dd, 1H, J = 8.7, 2.3 Hz,Ar–H7), 7.66 (d, 1H, J = 2.3 Hz,
Ar–H5), 7.99–8.12 (m, 4H,Ar–4H), 10.50 (s, 1H, –CONH–).
MS (EI) = 393 (M+). Anal. CHN C21H19N3O5.
NCH2COOCH2CH3), 1.64 (s, 3H, 2-CH3), 4.02–4.19 (m,
4H, 2 × CH2–CH3), 4.54 (d, 1H, J = 17.5 Hz, N–CH2–), 4.65
(d, 1H, J = 17.5 Hz, N–CH2–), 4.90 (s, 2H, NH2), 6.21–6.26
(m, 2H, Ar–H5, Ar–H7), 6.76 (d, 1H, J = 8.3 Hz, Ar–H8). MS
(EI) = 336 (M+). Anal. CHN C16H20N2O6.
5.2.19. Ethyl 4-benzyl-6-[(4-cyanobenzoyl)amino]-2-
methyl-2H-1,4-benzoxazin-3(4H)-one-2-carboxylate (20)
Prepared according to the general procedure as in Section
5.2.1.4 from 1.02 g (3.0 mmol) of amine 14 and 540 mg
(3.15 mmol) 4-cyanobenzoyl chloride. The crude product
was suspended in ethanol and filtered off. m = 1.20 g (85%).
M.p. 168–171 °C. IR (KBr, cm–1): 3451, 2231, 1732, 1697,
1654, 1620, 1559, 1513, 1439, 1374, 1340, 1263, 1125,
5.2.15. Ethyl 7-amino-2-methyl-2H-1,4-benzoxazin-3(4H)-
one-2-carboxylate (16)
Prepared according to the general procedure as in Section
5.2.1.3 from 3.00 g (10.7 mmol) of 4. m = 2.30 g (86%). M.p.
139–141 °C. IR (KBr, cm–1): 3401, 3228, 2986, 1746, 1698,
1658, 1518, 1426, 1386, 1316, 1242, 1126, 1009, 843.
1H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.08 (t, 3H,
J = 7.2 Hz, CH2CH3), 1.62 (s, 3H, 2-CH3), 4.07 (q,
J = 7.2 Hz, 2H, CH2), 4.92 (s, 2H, NH2), 6.17–6.58 (m, 3H,
Ar–3H), 10.43 (s, 1H, –CONH–). MS (EI) = 250 (M+).
EI-HRMS calcd. for C12H14N2O4: 250.096030. Found:
250.095357.
1
1017, 862, 734. H-NMR (DMSO-d6, 300 MHz): d (ppm)
1.07 (t, 3H, J = 7.2 Hz, CH2CH3), 1.79 (s, 3H, 2-CH3), 4.12
(q, 2H, J = 7.2 Hz, –CH2–CH3), 4.87 (d, 1H, J = 16.4 Hz,
–CH2–Ph), 5.34 (d, 1H, J = 16.4 Hz, –CH2–Ph), 7.12 (d, 1H,
J = 8.9 Hz, Ar–H8), 7.27–7.39 (m, 5H, Ph), 7.43 (dd, 1H,
J = 8.9, 2.3 Hz, Ar–H7), 7.58 (d, 1H, J = 2.3 Hz, Ar–H5),
7.96–8.04 (m, 4H,Ar–4H), 10.43 (s, 1H, –CONH–). MS (EI)
= 469 (M+). Anal. CHN C27H23N3O5.
5.2.16. Ethyl 7-amino-4-ethyl-2-methyl-2H-1,4-benzo-
xazin-3(4H)-one-2-carboxylate (17)
Prepared according to the general procedure as in Section
5.2.1.3 from 1.80 g (5.84 mmol) of 11. m = 1.57 g (97%).
M.p. 105–112 °C. IR (KBr, cm–1): 3467, 3371, 2988, 1727,
5.2.20. Ethyl 6-[(4-cyanobenzoyl)amino]-4-(2-ethoxy-2-
oxoethyl)-2-methyl-2H-1,4-benzoxazin-3(4H)-one-2-
carboxylate (21)
Prepared according to the general procedure as in Section
5.2.1.4 from 1.34 g (4.0 mmol) of amine 15 and 710 mg
(4.2 mmol) 4-cyanobenzoyl chloride. The crude product was
suspended in ethanol and filtered off. m = 1.61 g (87%).
M.p. 130–131 °C. IR (KBr, cm–1): 3426, 2230, 1734, 1646,
1
1671, 1630, 1513, 1416, 1266, 1124, 1007, 834. H-NMR
(CDCl3, 300 MHz): d (ppm) 1.18 (t, 3H, J = 7.2 Hz,
NCH2CH3), 1.26 (s, 3H, 2-CH3), 1.32 (t, 3H, J = 7.2 Hz,
OCH2CH3), 3.62 (s, 2H, NH2), 4.02 (m, 4H, 2 × CH2),
6.35–6.78 (m, 3H, Ar–3H). MS (FAB) = 279 (MH+). Anal.
CHN C14H18N2O4.
1
1558, 1386, 1253, 1014, 860, 758. H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.08 (t, 3H, J = 7.2 Hz, 2-COOCH2CH3),
1.22 (t, 3H, J = 7.2 Hz, NCH2COOCH2CH3), 1.73 (s, 3H,
2-CH3), 4.02–4.21 (m, 4H, 2 × CH2–CH3), 4.61 (d, 1H, J
= 17.7 Hz, N–CH2–), 4.77 (d, 1H, J = 17.7 Hz, N–CH2–),
7.13 (d, 1H, J = 8.7 Hz,Ar–H8), 7.46 (dd, 1H, J = 8.7, 2.3 Hz,
Ar–H7), 7.50 (d, 1H, J = 2.3 Hz, Ar–H5), 8.01–8.10 (m, 4H,
Ar–4H), 10.46 (s, 1H, –CONH–). MS (EI) = 465 (M+). Anal.
CHN C24H23N3O7.
5.2.17. Ethyl 6-[(4-cyanobenzoyl)amino]-2-methyl-2H-1,4-
benzoxazin-3(4H)-one-2-carboxylate (18)
Prepared according to the general procedure as in Section
5.2.1.4 from 1.00 g (4.0 mmol) of amine 12 and 715 mg
(4.2 mmol) 4-cyanobenzoyl chloride. m = 1.20 g (80%). M.p.
198–199 °C. IR (KBr, cm–1): 3303, 2992, 2230, 1741, 1686,
1621, 1520, 1380, 1253, 1124, 1016, 857. 1H-NMR (DMSO-
d6, 300 MHz): d (ppm) 1.08 (t, 3H, J = 7.2 Hz, CH2CH3),
1.69 (s, 3H, 2-CH3), 4.11 (m, 2H, CH2), 7.02 (d, 1H,
J = 8.7 Hz, Ar–H8), 7.29 (dd, 1H, J = 8.7, 2.5 Hz, Ar–H7),
7.54 (d, 1H, J = 2.5 Hz, Ar–H5), 8.00–8.10 (m, 4H, Ar–4H),
10.44 (s, 1H, 6-NH), 11.01 (s, 1H, –CONH–). MS
(FAB) = 380 (MH+). Anal. CHN C20H17N3O5.
5.2.21. Ethyl 7-[(4-cyanobenzoyl)amino]-2-methyl-2H-1,4-
benzoxazin-3(4H)-one-2-carboxylate (22)
Prepared according to the general procedure as in Section
5.2.1.4 from 1.00 g (4.0 mmol) of amine 16 and 664 mg
(4.0 mmol) 4-cyanobenzoyl chloride. m = 1.27 g (83%). M.p.
212–214 °C. IR (KBr, cm–1): 3391, 2231, 1692, 1557, 1518,
1
1413, 1325, 1131, 1014, 854, 753. H-NMR (DMSO-d6,
5.2.18. Ethyl 6-[(4-cyanobenzoyl)amino]-2,4-dimethyl-2H-
1,4-benzoxazin-3(4H)-one-2-carboxylate (19)
300 MHz): d (ppm) 1.08 (t, 3H, J = 7.2 Hz, CH2CH3), 1.70 (s,
3H, 2-CH3), 4.10 (q, 2H, J = 7.2 Hz, CH2), 6.87–7.56 (m, 3H,
Ar–3H), 8.00–8.10 (m, 4H, Ar–4H), 10.44 (s, 1H, 7-NH),
10.92 (s, 1H, –CONH–). MS (FAB) = 380 (MH+). Anal.
CHN C20H17N3O5.
Prepared according to the general procedure as in Section
5.2.1.4 from 460 mg (1.74 mmol) of amine 13 and 312 mg
(1.83 mmol) 4-cyanobenzoyl chloride. The crude product
was suspended in ethanol and filtered off. m = 570 mg (83%).
M.p. 201–204 °C. IR (KBr, cm–1): 3450, 2229, 1732, 1695,
1663, 1618, 1542, 1437, 1365, 1244, 1155, 1129, 1014, 859,
5.2.22. Ethyl 7-[(4-cyanobenzoyl)amino]-4-ethyl-2-methyl-
2H-1,4-benzoxazin-3(4H)-one-2-carboxylate (23)
Prepared according to the general procedure as in Section
5.2.1.4 from 1.20 g (4.31 mmol) of amine 17 and 716 mg
1
758. H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.05 (t, 3H,
J = 7.2 Hz, CH2CH3), 1.72 (s, 3H, 2-CH3), 3.32 (s, 3H,

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
39
(4.31 mmol) 4-cyanobenzoyl chloride. m = 1.00 g (57%).
M.p. 143–147 °C. IR (KBr, cm–1): 3293, 2982, 2228, 1756,
1696, 1655, 1514, 1395, 1326, 1188, 1130, 1017, 862, 763.
1H-NMR (CDCl3, 300 MHz): d (ppm) 1.17 (t, 3H, J = 7.2 Hz,
NCH2CH3), 1.29 (t, 3H, J = 7.2 Hz, OCH2CH3), 1.86 (s, 3H,
2-CH3), 4.07 (m, 4H, 2xCH2), 7.00–7.46 (m, 3H, Ar–3H),
7.82–7.97 (m, 4H, Ar–4H), 10.46 (s, 1H, –CONH–). MS
(FAB) = 408 (MH+). Anal. CHN C22H21N3O5.
= 8.7, 2.3 Hz, Ar–H7), 7.70 (d, 1H, J = 2.3 Hz, Ar–H5), 7.94
(d, 2H, J = 8.5 Hz, Ar–2H), 8.10 (d, 2H, J = 8.5 Hz, Ar–2H),
10.56 (s, 1H, Ar–NHCO). MS (FAB) = 487 (MH+-free base).
Compound was too hygroscopic for CHN analysis.
5.2.26. Ethyl 6-({4-[amino(imino)methyl]benzoyl}amino)-
4-(2-ethoxy-2-oxoethyl)-2-methyl-2H-1,4-benzoxazin-
3(4H)-one-2-carboxylate in the form of acetate (27)
Prepared according to the general procedure as in Section
5.2.1.5 from 1.16 g (2.50 mmol) of nitrile 21. m = 1.13 g
(83%). M.p. 137–139 °C. IR (KBr, cm–1): 3366, 2986, 1748,
5.2.23. Ethyl 6-({4-[amino(imino)methyl]benzoyl}amino)-
2-methyl-2H-1,4-benzoxazin-3(4H)-one-2-carboxylate in
the form of acetate (24)
1
1677, 1512, 1378, 1262, 1126, 1017, 862, 708. H-NMR
(DMSO-d6, 300 MHz): d (ppm) 1.08 (t, 3H, J = 7.1 Hz,
Prepared according to the general procedure as in Section
5.2.1.5 from 1.00 g (2.6 mmol) of nitrile 18. m = 965 mg
(80%). M.p. 191–195 °C. IR (KBr, cm–1): 3316, 2987, 1713,
1654, 1621, 1558, 1493, 1407, 1291, 1233, 1124, 1017, 861,
2-COOCH2CH3), 1.22 (t, 3H,
J
=
7.1 Hz,
NCH2COOCH2CH3), 1.73 (s, 3H, 2-CH3), 1.78 (s, 3H,
CH3COOH), 4.04–4.22 (m, 4H, 2 × CH2–CH3), 4.61 (d, 1H,
J = 17.8 Hz, N–CH2–), 4.77 (d, 1H, J = 17.8 Hz, N–CH2–),
7.12 (d, 1H, J = 8.7 Hz, Ar–H8), 7.46–7.54 (m, 2H, Ar–H7,
Ar–H5), 7.90–8.09 (m, 4H, Ar–4H), 10.42 (broad, 4H,
CONH, NH=C–NH2). MS (FAB) = 483 (MH+-free base).
Compound was too hygroscopic for CHN analysis.
1
700. H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.08 (t, 3H,
J = 7.2 Hz, CH2CH3), 1.69 (s, 3H, 2-CH3), 1.74 (s, 3H,
CH3COOH), 4.10 (m, 2H, CH2), 7.01 (d, 1H, J = 8.7 Hz,
Ar–H8), 7.30 (dd, 1H, J = 8.7, 2.5 Hz, Ar–H7), 7.55 (d, 1H,
J = 2.5 Hz, Ar–H5), 7.92 (d, 2H, J = 8.7 Hz, Ar–2H), 8.08 (d,
2H, J = 8.7 Hz, Ar–2H), 10.40 (broad, 3H, –CONH–,
HN=C–NH2). MS (EI) = 396 (M+-free base). EI-HRMS
calcd. for C20H20N4O5: 396.1434. Found: 396.1445.
5.2.27. Ethyl 7-({4-[amino(imino)methyl]benzoyl}amino)-
2-methyl-2H-1,4-benzoxazin-3(4H)-one-2-carboxylate in
the form of acetate (28)
Prepared according to the general procedure as in Section
5.2.1.5 from 500 mg (1.21 mmol) of nitrile 22. m = 477 mg
(78%). M.p. 193–196 °C. IR (KBr, cm–1): 3454, 1637, 1560,
5.2.24. Ethyl 6-({4-[amino(imino)methyl]benzoyl}amino)-
2,4-dimethyl-2H-1,4-benzoxazin-3(4H)-one-2-carboxylate
in the form of hydrochloride (25)
1
1522, 1406, 1118, 863. H-NMR (DMSO-d6, 300 MHz):
Prepared according to the general procedure as in Section
5.2.1.5 from 490 mg (1.25 mmol) of nitrile 19. The crude
product was dissolved in a saturated ethanolic solution of
HCl and precipitated by the addition of diethyl ether.
m = 90 mg (16%). M.p. 188–190 °C. IR (KBr, cm–1): 3424,
1731, 1662, 1618, 1555, 1516, 1472, 1425, 1302, 1249,
1120, 861. 1H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.05 (t,
3H, J = 7.2 Hz, CH2CH3), 1.72 (s, 3H, 2-CH3), 3.32 (s, 3H,
N–CH3), 4.08 (m, 2H, CH2), 7.09 (d, 1H, J = 8.9 Hz, Ar–H8),
7.54 (dd, 1H, J = 8.9, 2.3 Hz,Ar–H7), 7.76 (d, 1H, J = 2.3 Hz,
Ar–H5), 7.99 (d, 2H, J = 8.5 Hz, Ar–2H), 8.20 (d, 2H,
d (ppm) 1.08 (t, 3H, J = 7.2 Hz, CH2CH3), 1.70 (s, 3H,
2-CH3), 1.80 (s, 3H, CH3COOH), 4.11 (q, 2H, J = 7.2 Hz,
CH2), 6.87–7.58 (m, 3H, Ar–3H), 8.09–8.11 (m, 4H,
Ar–4H), 10.43 (s, 1H, 7-NH). MS (FAB) = 397 (MH+-free
base). Anal. CHN C20H20N4O5 × CH3COOH × 2 H2O.
5.2.28. Ethyl 7-({4-[amino(imino)methyl]benzoyl}amino)-
4-ethyl-2-methyl-2H-1,4-benzoxazin-3(4H)-one-2-
carboxylate in the form of acetate (29)
Prepared according to the general procedure as in Section
5.2.1.5 from 300 mg (0.74 mmol) of nitrile 23. m = 151 mg
(48%). M.p. 146–158 °C. IR (KBr, cm–1): 3136, 3046, 1737,
1679, 1606, 1548, 1514, 1407, 1317, 1280, 1123, 1011, 861,
+
J = 8.5 Hz, Ar–2H), 9.35 (broad, 2H, H2N=C–NH2), 9.57
+
(broad, 2H, H2N=C–NH2), 10.64 (s, 1H, –CONH–). MS
(FAB) = 411 (MH+-free base). Compound was too hygro-
scopic for CHN analysis.
1
705. H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.05 (t, 3H,
J = 7.2 Hz, OCH2CH3), 1.15 (t, 3H, J = 7.2 Hz, NCH2CH3),
1.73 (s, 3H, 2-CH3), 1.79 (s, 3H, CH3COOH), 4.08 (m, 4H, 2
× CH2), 7.22–7.67 (m, 3H, Ar–3H), 7.94–8.13 (m, 4H,
Ar–4H), 10.54 (s, 1H, NH). MS (FAB) = 425 (MH+-free
base). Compound was too hygroscopic for CHN analysis.
5.2.25. Ethyl 6-({4-[amino(imino)methyl]benzoyl}amino)-
4-benzyl-2-methyl-2H-1,4-benzoxazin-3(4H)-one-2-
carboxylate in the form of acetate (26)
Prepared according to the general procedure as in Section
5.2.1.5 from 705 mg (1.50 mmol) of nitrile 20. m = 625 mg
(76%). M.p. 163–166 °C. IR (KBr, cm–1): 3456, 1748, 1639,
5.2.29. Sodium 6-({4-[Amino(imino)methyl]benzoyl}-
amino)-2-methyl-2H-1,4-benzoxazin-3(4H)-one-2-
carboxylate (30)
Prepared according to the general procedure as in Section
5.2.1.6 from 24 (770 mg, 1.69 mmol) without acidification.
The compound precipitated from the reaction mixture.
m = 0.11 g (14%). M.p. 216–222 °C. IR (KBr, cm–1): 3354,
1
1559, 1551, 1402, 1334, 1265, 1131, 1012, 936, 862. H-
NMR (DMSO-d6, 300 MHz): d (ppm) 1.07 (t, 3H, J = 7.2 Hz,
CH2CH3), 1.79 (s, 3H, 2-CH3), 1.80 (s, 3H, CH3COOH),
4.12 (m, 2H, –CH2–CH3), 4.89 (d, 1H, J = 16.2 Hz, –CH2–
Ph), 5.33 (d, 1H, J = 16.2 Hz, –CH2–Ph), 7.11 (d, 1H, J
= 8.7 Hz, Ar–H8), 7.25–7.39 (m, 5H, Ph), 7.49 (dd, 1H, J

40
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
1
1697, 1618, 1518, 1398, 1235, 1132, 866, 780. H-NMR
(DMSO-d6 + D2O, 300 MHz): d (ppm) 1.49 (s, 3H, 2-CH3),
6.78 (d, 1H, J = 8.7 Hz,Ar–H8), 7.11 (dd, 1H, J = 8.7, 2.3 Hz,
Ar–H7), 7.31 (d, 1H, J = 2.3 Hz, Ar–H5), 7.87–7.99 (m, 4H,
4 ×Ar–H). MS (EI) = 368 (M+-free acid). Compound was too
hygroscopic for CHN analysis.
5.2.33. Ethyl 2-[(7-amino-4-ethyl -2H-1,4-benzoxazin-
3(4H)-one-2-yl -2-yl)oxy]acetate (34)
Prepared according to the general procedure as in Section
5.2.1.3 from 465 mg (1.4 mmol) of 33. The oily residue was
immediately used for the next step without further character-
ization. m = 390 mg (95%).
5.2.34. Ethyl 2-({7-[(4-cyanobenzoyl)amino]-4-ethyl-2H-
1,4-benzoxazin-3(4H)-one-2-yl}oxy)acetate (35)
5.2.30. 7-({4-[Amino(imino)methyl]benzoyl}amino)-2-
methyl-2H-1,4-benzoxazin-3(4H)-one-2-carboxylate in
the form of hydrochloride(31)
Prepared according to the general procedure as in Section
5.2.1.4 from 380 mg (1.3 mmol) of amine 34 and 233 mg
(1.4 mmol) 4-cyanobenzoyl chloride. m = 530 mg (96%).
M.p. 57–61 °C. IR (KBr, cm–1): 3565, 2230, 1748, 1683,
Prepared according to the general procedure as in Section
5.2.1.6 from 28 (400 mg, 0.92 mmol). m = 0.089 g (14%).
M.p. 185–189 °C. IR (KBr, cm–1): 3447, 1653, 1557, 1514,
1406, 1350, 1178, 1026, 904, 756, 670.1H-NMR (DMSO-d6
+ D2SO4, 300 MHz): d (ppm) 1.24 (s, 3H, 2-CH3), 6.87–6.91
(dd, 1H, J = 8.7, 2.6 Hz, Ar–H7), 7.35 (d, 1H, J = 8.7 Hz,
Ar–H8), 6.87–6.91 (d, 1H, J = 2.6 Hz, Ar–H5), 7.96 (s, 1H,
–CONH–), 7.90–8.15 (dd, 4H, J = 8.3 Hz, Ar–4H) 9.12–9.45
(2 s, broad, 4H, NH2=C+–NH2), 10.40 (s, 1H, –CONH–). MS
(FAB) = 369 (MH+-free acid). Compound was too hygro-
scopic for CHN analysis.
1
1607, 1514, 1439, 1274, 1108, 1046, 856, 790. H-NMR
(DMSO-d6, 300 MHz): d (ppm) 1.16 (t, 6H, J = 7.2 Hz,
2CH3), 3.97 (m, 2H, N–CH2CH3), 4.11 (m, 2H,
O–CH2CH3), 4.30 (d, 1H, J = 16.2 Hz, O–CH2–CO), 4.44 (d,
1H, J = 16.2 Hz, O–CH2–CO), 7.31 (d, 1H, J = 9.0 Hz,
Ar–H5), 7.52 (dd, 1H, J = 9.0, 2.3 Hz, Ar–H6), 7.67 (d, 1H,
J = 2.3 Hz, Ar–H8), 8.04 (d, 2H, J = 8.3 Hz, Ar–2H), 8.11 (d,
2H, J = 8.3 Hz, Ar–2H), 10.53 (s, 1H, –CONH–). MS (FAB)
= 424 (MH+). Anal. CHN C22H21N3O6 × 0.5 H2O.
5.2.35. Ethyl 2-{[7-({4-[amino(imino)methyl]benzoyl}-
amino)-4-ethyl-2H-1,4-benzoxazin-3(4H)-one-2-yl]oxy}-
acetate in the form of acetate (36)
5.2.31. 4-Ethyl-2-hydroxy-7-nitro-2H-1,4-benzoxazin-
3(4H)-one (32)
Prepared according to the general procedure as in Section
5.2.1.1 from 2.10 g (10.0 mmol) of 6 and 0.84 ml
(11.0 mmol) ethyl bromide. The final product was first sepa-
rated from the starting compound by column chromatogra-
phy using ethyl acetate/hexane = 3/2 as an eluent and after-
wards from N,O-dialkylated byproduct by the same method
using ether/petrolether = 2/1 as an eluent. m = 0.69 g (29%).
M.p. 133–136 °C. IR (KBr, cm–1): 3296, 3064, 2968, 1774,
1667, 1601, 1527, 1449, 1346, 1275, 1092, 1033, 918, 810,
Prepared according to the general procedure as in Section
5.2.1.5 from 444 mg (1.05 mmol) of nitrile 35. m = 450 mg
(86%). M.p. 202–206 °C. IR (KBr, cm–1): 3384, 2982, 1742,
1687, 1608, 1514, 1438, 1351, 1223, 1108, 1031, 800, 705.
1H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.17 (m, 6H,
2CH3), 1.76 (s, 3H, CH3–COOH), 3.97 (q, 2H, J = 7.2 Hz,
N–CH2CH3), 4.11 (m, 2H, O–CH2CH3), 4.30 (d, 1H,
J = 16.0 Hz, O–CH2–CO), 4.45 (d, 1H, J = 16.0 Hz, O–CH2–
CO), 7.31 (d, 1H, J = 8.9 Hz, Ar–H5), 7.55 (dd, 1H, J = 8.9,
2.3 Hz, Ar–H6), 7.70 (d, 1H, J = 2.3 Hz, Ar–H8), 7.94 (d, 2H,
J = 8.5 Hz, Ar–2H), 8.11 (d, 2H, J = 8.5 Hz, Ar–2H),
9.60–10.85 (broad, 3H, –CONH–, NH2). MS (FAB) = 441
(MH+-free base). Anal. CHN C22H25N4O6 × CH3COOH ×
0.5 H2O.
1
746. H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.16 (t, 3H,
J = 6.8 Hz, CH3), 3.85–4.17 (m, 2H, CH2), 5.71 (s, 1H, Hz,
2-H), 7.50 (d, 1H, J = 9.0 Hz,Ar–H5), 7.85 (d, 1H, J = 2.6 Hz,
Ar–H8), 8.00 (dd, 1H, J = 9.0, 2.6 Hz, Ar–H6), 8.38 (s, 1H,
2-OH). Coupling between the signals at 5.71 in 8.38 ppm was
determined by COSY NMR. MS (EI) = 238 (M+). Anal.
CHN C10H10N2O5.
5.2.36. 2-{[7-({4-[amino(imino)methyl]benzoyl}amino)-4-
ethyl-2H-1,4-benzoxazin-3(4H)-one-2-yl]oxy}acetic acid
(37)
5.2.32. Ethyl 2-[(4-ethyl-7-nitro-2H-1,4-benzoxazin-3(4H)-
one-2-yl 2-yl)oxy]acetate (33)
Prepared according to the general procedure as n Section
5.2.1.7 from 100 mg (0.20 mmol) of ester 36. The crude
product was purified by column chromatography utilizing
methanol/water/acetonitrile/acetic acid = 2/2/6/1 as an elu-
ent. m = 45 mg (48%). M.p. 235–240 °C. IR (KBr, cm–1):
3394, 1666, 1598, 1513, 1412, 1337, 1274, 1100, 1043, 863,
Prepared according to the general procedure as in Section
5.2.1.1 from 524 mg (2.2 mmol) of 32 and 0.23 ml
(2.3 mmol) ethyl bromide. m = 595 mg (83%). M.p. 112–
113 °C. IR (KBr, cm–1): 2990, 1744, 1701, 1603, 1519, 1400,
1
1341, 1215, 1105, 1025, 883, 807, 745. H-NMR (DMSO-
1
d6, 300 MHz): d (ppm) 1.17 (m, 6H, 2 × CH3), 3.97–4.16 (m,
4H, 2 × CH2–CH3), 4.39 (d, 1H, J = 16.2 Hz, OCH2CO), 4.51
(d, 1H, J = 16.2 Hz, OCH2CO), 5.81 (s, 1H, Hz, 2-H), 7.56
(d, 1H, J = 9.0 Hz, Ar–H5), 7.98 (d, 1H, J = 2.6 Hz, Ar–H8),
8.05 (dd, 1H, J = 9.0, 2.6 Hz, Ar–H6). MS (EI) = 324 (M+).
EI-HRMS calcd. for C14H16N2O7: 324.0958. Found:
324.0963.
704. H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.12 (t, 3H,
J = 7.2 Hz, CH3), 3.93 (q, 2H, J = 7.2 Hz, N–CH2–), 4.17 (d,
1H, J = 16.2 Hz, O–CH2–CO), 4.31 (d, 1H, J = 16.2 Hz,
O–CH2–CO), 7.28 (d, 1H, J = 8.9 Hz, Ar–H5), 7.53 (dd, 1H,
J = 8.9, 2.5 Hz, Ar–H6), 7.69 (d, 1H, J = 2.5 Hz, Ar–H8), 7.95
(d, 2H, J = 8.5 Hz, Ar–2H), 8.13 (d, 2H, J = 8.5 Hz, Ar–2H).
MS (FAB) = 413 (MH+). Anal. CHN C20H20N4O6 × 2 H2O.

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
41
5.2.37. Ethyl 2,4-dimethyl-7-nitro-2H-1,4-benzoxazine-
3(4H)-one-2-carboxylate (38) [10]
sound bath. After 30 min the solvent was evaporated and the
residue dissolved in 60 ml of ethyl acetate and extracted with
10% aqueous citric acid (2 × 20 ml), saturated aqueous
NaHCO3 (2 × 20 ml) and brine (30 ml). The organic phase
was dried over Na2SO4, filtered and evaporated under
vacuum. The crude product was purified by column chroma-
tography utilizing ethyl acetate/hexane = 3/2 as the eluent.
m = 1.32 g (84%). IR (NaCl, cm–1): 2953, 1741, 1693, 1600,
1524, 1322, 1209, 1074, 881, 744. 1H-NMR (CDCl3,
300 MHz): d (ppm) 1.54 (s, 3H, 2-CH3), 2.86 (d, 1H,
J = 16.6 Hz, CH2), 3.34 (d, 1H, J = 16.6 Hz, CH2), 3.46 (s,
3H, N–CH3), 3.68 (s, 3H, O–CH3), 7.03 (d, 1H, J = 9.0 Hz,
Ar–H5), 7.81 (d, 1H, J = 2.6 Hz, Ar–H8), 7.94–7.98 (dd, 1H,
J = 9.0, 2.6 Hz, Ar–H6). MS (EI) = 294 (M+). EI-HRMS
calcd. for C13H14N2O6: 294.0852. Found: 294.0862.
Prepared according to the general procedure as in Section
5.2.1.1 from 5.60 g (20.0 mmol) of 4 and 1.32 ml
(21.0 mmol) methyl iodide. m = 5.86 g (96%). M.p. 118–
120 °C. IR (KBr, cm–1): 3069, 2984, 1751, 1698, 1602, 1522,
1506, 1377, 1341, 1245, 1126, 1073, 1024, 910, 817, 748,
550, 505. 1H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.05 (t,
3H, J = 7.2 Hz, CH2CH3), 1.78 (s, 3H, 2-CH3), 3.40 (s, 3H,
N–CH3), 4.10 (m, 2H, CH2); 7.43 (d, 1H, J = 8.9 Hz, Ar–H5),
7.87 (d, 1H, J = 2.5 Hz,Ar–H8), 8.02 (dd, 1H, J = 8.9, 2.5 Hz,
Ar–H6). MS (FAB) = 295 (MH+).
5.2.38. 2,4-Dimethyl-7-nitro-2H-1,4-benzoxazine-3(4H)-
one-2-carboxylic acid (39)
Prepared according to the general procedure as in Section
5.2.1.6 from 5.88 g (20.0 mmol) of ester 38. m = 5.00 g
(94%). M.p. 158–160 °C. IR (KBr, cm–1): 3540, 3381, 1760,
5.2.41. Methyl 2-(7-amino-2,4-dimethyl-2H-1,4-
benzoxazin-3(4H)-one-2-yl)acetate (42)
Prepared according to the general procedure as in Section
5.2.1.3 from 382 mg (1.3 mmol) of 41 with methanol as
solvent. The oily residue was immediately used for the next
step without further characterization. m = 324 mg (95%).
1
1678, 1599, 1527, 1341, 1250, 1114, 1026, 871, 745. H-
NMR (DMSO-d6, 300 MHz): d (ppm) 1.76 (s, 3H, 2-CH3),
3.39 (s, 3H, N–CH3), 7.41 (d, 1H, J = 9.1 Hz, Ar–H5), 7.84
(d, 1H, J = 2.6 Hz, Ar–H8), 7.98–8.02 (dd, 1H, J = 9.1,
2.6 Hz, Ar–H6). MS (EI) = 266 (M+). EI-HRMS calcd. for
C8H6Cl2N2O4: 266.0539. Found: 266.0549.
5.2.42. Methyl 2-{7-[(4-cyanobenzoyl)amino]-2,4-
dimethyl-2H-1,4-benzoxazin-3(4H)-one-2-yl} acetate (43)
Prepared according to the general procedure as in Section
5.2.1.4 from 320 mg (1.21 mmol) of amine 42 and 228 mg
(1.31 mmol) 4-cyanobenzoyl chloride. m = 352 mg (74%).
M.p. 138–142 °C. IR (KBr, cm–1): 3372, 2953, 2228, 1732,
5.2.39. 2-(2-Diazoacetyl)-2,4-dimethyl-7-nitro-2H-1,4-
benzoxazin-3(4H)-one (40)
A 1.6 g (6.0 mmol) of acid 39 was dissolved in 30 ml of
freshly distilled THF and 0.86 ml (6.2 mmol) triethylamine
was added. The mixture was flushed with argon and cooled to
–15 °C. A solution of ethyl chloroformate (0.59 ml,
6.2 mmol) in 5 ml of THF was added and the resulting
mixture stirred for 30 min at –5 °C. The precipitated triethy-
lammonium chloride was filtered off. Acetonitrile (20 ml)
was added to the filtrate, followed by 6.0 ml (12.0 mmol) of
trimethylsilyldiazomethane (2.0 M solution in hexane, Ald-
rich). The mixture was stirred for 24 h at 4 °C. Afterwards
80 ml of diethyl ether was added and extracted with 10%
aqueous citric acid (2 × 30 ml), saturated aqueous NaHCO3
(2 × 30 ml) and brine (30 ml). The organic phase was dried
over Na2SO4, filtered and evaporated under vacuum. The
crude product was used for the next step. m = 1.59 g (92%).
M.p. 128–130 °C. IR (KBr, cm–1): 3452, 3085, 2126, 1691,
1
1673, 1517, 1394, 1215, 1153, 1000, 866, 635. H-NMR
(DMSO-d6, 300 MHz): d (ppm) 1.41 (s, 3H, 2-CH3), 2.83 (d,
1H, J = 16.2 Hz, CH2), 3.15 (d, 1H, J = 16.2 Hz, CH2), 3.29
(s, 3H, N–CH3), 3.57 (s, 3H, O–CH3), 7.14 (d, 1H,
J = 8.7 Hz, Ar–H5), 7.41–7.45 (dd, 1H, J = 8.7, 2.3 Hz,
Ar–H6), 7.49 (d, 1H, J = 2.3 Hz, Ar–H8), 8.02 (d, 2H,
J = 8.7 Hz,Ar–2H), 8.09 (d, 2H, J = 8.7 Hz,Ar–2H), 10.45 (s,
1H, –CONH–). MS (FAB) = 394 (MH+). Anal. CHN
C21H19N3O5 × 0.5 H2O.
5.2.43. Methyl 2-[7-({4-[amino(imino)methyl]
benzoyl}amino)-2,4-dimethyl-2H-1,4-benzoxazin-3(4H)-
one-2-yl]acetate in the form of acetate (44)
1
Prepared according to the general procedure as in Section
5.2.1.5 from 330 mg (0.84 mmol) of nitrile 43 utilizing
anhydrous methanol as the solvent. m = 47 mg (12%). M.p.
180–184 °C. IR (KBr, cm–1): 3391, 2946, 1713, 1670, 1606,
1636, 1600, 1525, 1341, 1108, 1022, 876, 744, 531. H-
NMR (DMSO-d6, 300 MHz): d (ppm) 1.73 (s, 3H, 2-CH3),
3.38 (s, 3H, N–CH3), 6.48 (s, 1H, CH–N2), 7.40 (d, 1H,
J = 8.7 Hz, Ar–H5), 7.89–8.02 (m, 2H, Ar–H8,H6). MS (EI)
= 290 (M+). EI-HRMS calcd. for C12H10N4O5: 290.0651.
Found: 290.0662.
1
1516, 1413, 1314, 1238, 1149, 1009, 860, 705. H-NMR
(DMSO-d6, 300 MHz): d (ppm) 1.41 (s, 3H, 2-CH3), 1.80 (s,
3H, CH3COOH), 2.83 (d, 1H, J = 16.2 Hz, CH2), 3.15 (d, 1H,
J = 16.2 Hz, CH2), 3.30 (s, 3H, N–CH3), 3.57 (s, 3H,
O–CH3), 7.14 (d, 1H, J = 8.9 Hz, Ar–H5), 7.44–7.48 (dd, 1H,
J = 8.9, 2.1 Hz, Ar–H6), 7.51 (d, 1H, J = 2.1 Hz, Ar–H8), 7.93
(d, 2H, J = 8.3 Hz, Ar–2H), 8.10 (d, 2H, J = 8.3 Hz, Ar–2H),
10.43 (broad, 1H, –CONH–). MS (FAB) = 411 (MH+-free
base). Compound was too hygroscopic for CHN analysis.
5.2.40. Methyl 2-(2,4-dimethyl-7-nitro-2H-1,4-benzoxazin-
3(4H)-one-2-yl)acetate (41)
A 1.45 g (5.0 mmol) of diazoketone 40 was suspended in
50 ml of anhydrous methanol and a solution of 0.23 g
(1.0 mmol) of silver benzoate in 2.8 ml of triethylamine was
gradually added while the mixture was sonicated in an ultra-

42
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
5.2.44. Ethyl 2-{[(2,4-dimethyl-7-nitro-2H-1,4-
1.14 (m, 6H, 2 × CH2CH3), 1.62 (s, 3H, 2-CH3), 1.63 (s, 3H,
2-CH3), 2.39 (s, 6H, 2 × Ph–CH3), 3.00 (m, 4H, 2 × NH–
CH2–CH), 3.19 (s, 3H, N–CH3), 3.20 (s, 3H, N–CH3), 3.88–
4.04 (m, 4H, 2 × O–CH2–CH3), 4.19 (m, 1H, CH), 4.30 (m,
1H, CH), 5.01 (s, 2H, NH2), 5.02 (s, 2H, NH2), 6.28 (m, 2H,
2 × Ar–H6), 6.31 (d, 1H, J = 2.5 Hz, Ar–H8), 6.37 (d, 1H,
J = 2.5 Hz, Ar–H8), 6.75–6.82 (m, 2H, 2 × Ar–H5), 7.40 (d,
4H, J = 8.3 Hz, 2 × SO2–Ph–CH3, 2H), 7.59–7.75 (m, 6H, 2
× SO2–Ph–CH3, 2H, 2 × SO2NH), 8.07 (d, 1H, J = 7.9 Hz,
–CONH–), 8.22 (d, 1H, J = 7.5 Hz, –CONH–). MS (EI)
= 504 (M+). EI-HRMS calcd. for C23H28N4O7S: 504,1679.
Found: 504,1695.
benzoxazine-3(4H)-one-2-yl)carbonyl]amino}acetate (45)
Prepared according to the general procedure as in Section
5.2.1.8 from 39 (0.96 g, 3.6 mmol) and glycine ethyl ester
(0.92 g, 3.8 mmol). m = 0.92 g (73%). M.p. 147–150 °C. IR
(KBr, cm–1): 3340, 2993, 1746, 1697, 1602, 1531, 1342,
1207, 1128, 1070, 1027, 898, 816, 746, 589. 1H-NMR
(DMSO-d6, 300 MHz): d (ppm) 1.07 (t, 3H, J = 7.2 Hz,
CH2CH3), 1.78 (s, 3H, 2-CH3), 3.37 (s, 3H, N–CH3), 3.72 (d,
2H, J = 6.0 Hz, NH–CH2–), 3.96 (q, 2H, J = 7.2 Hz,
–CH2CH3), 7.39 (d, 1H, J = 8.7 Hz, Ar–H5), 7.98–8.02 (m,
2H, Ar–H6, H8), 8.73 (t, 1H, J = 6.0 Hz, NH). MS (FAB)
= 352 (MH+). Anal. CHN C15H17N3O7 × 0.25 H2O.
5.2.48. Ethyl 2-{[(7-{[(4-cyanophenyl)carbonyl]amino}-
2,4-dimehyl-2H-1,4-benzoxazine-3(4H)-one-2-yl)carbonyl]-
amino}acetate (49)
5.2.45. Ethyl (2S)-2-{[(2,4-dimethyl-7-nitro-2H-1,4-
benzoxazine-3(4H)-one-2-yl)carbonyl]amino}-3-{[(4-
methylphenyl)sulphonyl]amino}propanoate (46)
Prepared according to the general procedure as in Section
5.2.1.8 from 39 (0.64 g, 2.4 mmol) and 58 (0.76 g, 2.4 mmol).
m = 1.18 g (92%). M.p. 75–80 °C. IR (KBr, cm–1): 3472,
Prepared according to the general procedure as in Section
5.2.1.4 from 47 (0.65 g, 2.0 mmol) and 4-cyanobenzoyl
chloride (0.36 g, 2.1 mmol). m = 0.68 g (76%). M.p. 171–
174 °C. IR (KBr, cm–1): 3376, 2986, 2228, 1734, 1697, 1514,
1
1700, 1602, 1526, 1457, 1382, 1342, 1159, 1093, 814, 663.
1431, 1389, 1221, 1151, 1018, 865, 761, 634. H-NMR
20
D
␣
͓ ͔
= –8.6° (c 0.81; MeOH). 1H-NMR (DMSO-d6,
(DMSO-d6, 300 MHz): d (ppm) 1.11 (t, 3H, J = 7.2 Hz,
CH2CH3), 1.73 (s, 3H, 2-CH3), 3.30 (s, 3H, N–CH3), 3.62–
3.80 (m, 2H, NH–CH2–), 4.01 (m, 2H, –CH2CH3), 7.15 (d,
1H, J = 8.9 Hz, Ar–H5), 7.39–7.42 (dd, 1H, J = 8.9, 2.3 Hz,
Ar–H6), 7.74 (d, 1H, J = 2.3 Hz, Ar–H8), 8.02 (d, 2H,
J = 8.5 Hz, Ar–2H), 8.10 (d, 2H, J = 8.5 Hz, Ar–2H), 8.54 (t,
1H, J = 6.0 Hz, CO–NH–CH2), 10.51 (s, 1H, –CONH–). MS
(FAB) = 451 (MH+). Anal. CHN C23H22N4O6.
300 MHz): d (ppm) 0.93 (t, 3H, J = 7.2 Hz, CH2CH3), 1.07 (t,
3H, J = 7.2 Hz, CH2CH3), 1.78 (s, 3H, 2-CH3), 1.79 (s, 3H,
2-CH3), 2.39 (s, 6H, 2 × Ph–CH3), 2.92 (m, 2H, NH–CH2–
CH), 3.02 (m, 2H, NH–CH2–CH), 3.37 (s, 3H, N–CH3), 3.38
(s, 3H, N–CH3), 3.80 (q, 2H, J = 7.2 Hz, O–CH2–CH3), 3.96
(q, 2H, J = 7.2 Hz, O–CH2–CH3), 4.18 (m, 1H, CH), 4.26 (m,
1H, CH), 7.34–7.42 (m, 6H, 2 × SO2–Ph–CH3, 2H, 2 ×
Ar–H5), 7.53–7.66 (m, 5H, 2 × SO2–Ph–CH3, 2H, SO2NH),
7.70 (t, 1H, J = 6.0 Hz, SO2NH), 7.97–8.04 (m, 4H, 2 ×
Ar–H6, 2 ×Ar–H8), 8.53 (d, 1H, J = 8.3 Hz, CONH), 8.59 (d,
1H, J = 8.3 Hz, –CONH–). MS (FAB) = 535 (MH+). Anal.
CHN C23H26N4O9S.
5.2.49. Ethyl 2-({[7-({4-[amino(imino)methyl]benzoyl}-
amino)-2,4-dimehyl-2H-1,4-benzoxazine-3(4H)-one-2-yl)-
carbonyl]amino}acetate in the form of acetate (50)
Prepared according to the general procedure as in Section
5.2.1.5 from 49 (0.60 g, 1.33 mmol). m = 0.33 g (47%). M.p.
146–149 °C. IR (KBr, cm–1): 3318, 2988, 1688, 1512, 1384,
1260, 1153, 1018, 863, 649. 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.11 (t, 3H, J = 7.2 Hz, CH2CH3), 1.73 (s,
3H, 2-CH3), 1.76 (s, 3H, CH3COOH), 3.30 (s, 3H, N–CH3),
3.63–3.79 (m, 2H, NH–CH2–), 4.00 (q, 2H, J = 7.2 Hz,
–CH2CH3), 7.15 (d, 1H, J = 8.9 Hz, Ar–H5), 7.41–7.45 (dd,
1H, J = 8.9, 2.1 Hz, Ar–H6), 7.76 (d, 1H, J = 2.1 Hz, Ar–H8),
7.94 (d, 2H, J = 8.3 Hz, Ar–2H), 8.11 (d, 2H, J = 8.3 Hz,
Ar–2H), 8.54 (s, 1H, CO–NH–CH2), 10.48 (s, 1H,
–CONH–). MS (FAB) = 468 (MH+-free base). Anal. CHN
C23H25N5O6 × 1.2 CH3COOH × 1 H2O.
5.2.46. Ethyl 2-{[(7-amino-2,4-dimethyl-2H-1,4-
benzoxazine-3(4H)-one-2-yl)carbonyl]amino}acetate (47)
Prepared according to the general procedure as in Section
5.2.1.3 from 45 (0.76 g, 2.15 mmol). m = 0.68 g (98%). M.p.
146–148 °C. IR (KBr, cm–1): 3380, 2989, 1743, 1681, 1639,
1518, 1394, 1312, 1187, 1018, 836, 615. 1H-NMR (DMSO-
d6, 300 MHz): d (ppm) 1.13 (t, 3H, J = 7.2 Hz, CH2CH3),
1.66 (s, 3H, 2-CH3), 3.21 (s, 3H, N–CH3), 3.66–3.74 (m, 2H,
NH–CH2–), 4.02 (m, 2H, –CH2CH3), 5.56 (s, 2H, NH2),
6.31–6.35 (dd, 1H, J = 8.5, 2.3 Hz, Ar–H6), 6.39 (d, 1H,
J = 2.3 Hz, Ar–H8), 6.82 (d, 1H, J = 8.5 Hz, Ar–H5), 8.37 (t,
1H, J = 6.0 Hz, NH). MS (EI) = 321 (M+).
5.2.50. Ethyl 2-({[7-({4-[amino(hydroxyimino)methyl]-
benzoyl}amino)-2,4-dimehyl-2H-1,4-benzoxazine-3(4H)-
one-2-yl)carbonyl]amino}acetate (51)
To a suspension of hydroxylammonium chloride (0.22 g,
3.0 mmol) in anhydrous ethanol (20 ml), triethylamine
(0.42 ml, 3.00 mmol) was added and the resulting solution
stirred for 20 min, after which a solution of 49 (0.45 g,
1.0 mmol) in anhydrous ethanol (10 ml) was added and the
mixture heated at 50 °C overnight. The product was precipi-
5.2.47. Ethyl (2S)-2-{[(7-amino-2,4-dimethyl-2H-1,4-
benzoxazine-3(4H)-one-2-yl)carbonyl]-amino}-3-{[(4-
methylphenyl)sulphonyl]amino}propanoate (48)
Prepared according to the general procedure as in Section
5.2.1.3 from 46 (1.07 g, 2.0 mmol). m = 0.96 g (95%). M.p.
82–88 °C. IR (KBr, cm–1): 3366, 2981, 1734, 1685, 1519,
1397, 1309, 1159, 1093, 1029.
MeOH). H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.05–
␣
2D0= + 5.9° (c 0.63;
͓ ͔
1

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
43
tated by cooling in the refrigerator and filtered off. m = 0.34 g
(70%). M.p. 150–153 °C. IR (KBr, cm–1): 3346, 2986, 2936,
1732, 1697, 1608, 1516, 1430, 1389, 1317, 1253, 1161,
1088, 1015, 928, 859, 814, 705. 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.11 (t, 3H, J = 7.2 Hz, CH2CH3), 1.73 (s,
3H, 2-CH3), 3.32 (s, 3H, N–CH3), 3.63–3.80 (m, 2H, NH–
CH2–), 4.00 (q, 2H, J = 7.2 Hz, –CH2CH3), 5.93 (s, 2H,
NH2), 7.13 (d, 1H, J = 8.7 Hz, Ar–H5), 7.40–7.44 (dd, 1H,
J = 8.7, 2.3 Hz, Ar–H6), 7.76 (d, 1H, J = 2.3 Hz, Ar–H8), 7.83
(d, 2H, J = 8.5 Hz, Ar–2H), 7.95 (d, 2H, J = 8.5 Hz, Ar–2H),
8.53 (t, 1H, J = 6.0 Hz, CO–NH–CH2), 9.83 (s, 1H, OH),
10.29 (s, 1H, –CONH–). MS (FAB) = 484 (MH+). Anal.
CHN C23H25N5O7 × 0.5 H2O.
5.2.53. 2-({[7-({4-[Amino(imino)methyl]benzoyl}amino)-
2,4-dimethyl-2H-1,4-benzoxazine-3(4H)-one-2-yl)carbonyl]-
amino}acetic acid in the form of acetate (54)
Prepared according to the general procedure as in Section
5.2.1.9 from 53 (0.11 g, 0.23 mmol). m = 0.08 g (72%). M.p.
242–245 °C. IR (KBr, cm–1): 3338, 1659, 1514, 1389, 1314,
1150, 1032, 863, 703. 1H-NMR (DMSO-d6 + D2O,
300 MHz)*: d (ppm) 1.74 (s, 3H, 2-CH3), 7.2 (d, 1H,
J = 9.1 Hz, Ar–H5), 7.32–7.37 (dd, 1H, J = 9.1, 1.9 Hz,
Ar–H6), 7.49 (t, 1H, J = 4.9 Hz, CO–NH–CH2), 7.68 (d, 2H,
J = 8.7 Hz, Ar–2H), 7.86 (d, 1H, J = 1.9 Hz, Ar–H8), 8.01 (d,
2H, J = 8.7 Hz, Ar–2H), 10.42 (s, 1H, –CONH–). Signals
between 3.1 in 3.9 ppm (N–CH3, NH–CH2–) are covered
with the signal for H2O. MS (FAB) = 440 (MH+-free base).
Anal. CHN C21H21N5O6 × CH3COOH × 2.25 H2O.
5.2.51. Ethyl 2-{[(2,4-dimethyl-7-{[4-(5-oxo-4,5-dihydro-
1,2,4-oxadiazol-3-yl)benzoyl]amino}- 2H-1,4-benzoxazine-
3(4H)-one-2-yl)carbonyl]amino}acetate (52)
5.2.54. Ethyl (2S)-2-{[(2,4-dimethyl-7-{[4-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)benzoyl]amino}-2H-1,4-
benzoxazine-3(4H)-one-2-yl)carbonyl]amino}-3-{[(4-
methylphenyl)sulphonyl]amino}propanoate (55)
Prepared according to the general procedure as in Section
5.2.1.8 from 48 (0.91 g, 1.8 mmol) and 59 (0.39 g, 1.9 mmol).
m = 0.84 g (67%). The crystalline product was immediately
used for the next step without further characterization.
Into an ice-cooled solution of 51 (0.3 g, 0.62 mmol) in
freshly distilled pyridine (15 ml) argon was bubbled for
5 min. Afterwards, ethyl chloroformate (0.06 ml, 0.66 mmol)
was added dropwise, stirred for 45 min in an ice-bath and
then 30 min at RT. Again, the argon was bubbled in and the
mixture heated under reflux (~120 °C) for a further 6 h. The
solvent was evaporated under vacuum and the crude residue
recrystallized from methanol. m = 0.18 g (57%). M.p. 221–
225 °C. IR (KBr, cm–1): 3366, 2987, 1760, 1693, 1513, 1430,
1250, 1150, 1018, 862, 758. 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.11 (t, 3H, J = 7.2 Hz, CH2CH3), 1.73 (s,
3H, 2-CH3), 3.34 (s, 3H, N–CH3), 3.62–3.80 (m, 2H, NH–
CH2–), 4.00 (q, 2H, J = 7.2 Hz, –CH2CH3), 7.15 (d, 1H,
J = 8.9 Hz, Ar–H5), 7.40–7.44 (dd, 1H, J = 8.9, 2.3 Hz,
Ar–H6), 7.76 (d, 1H, J = 2.3 Hz, Ar–H8), 7.96 (d, 2H,
J = 8.7 Hz, Ar–2H), 8.12 (d, 2H, J = 8.7 Hz, Ar–2H), 8.54 (t,
1H, J = 6.0 Hz, CO–NH–CH2), 10.45 (s, 1H, –CONH–),
13.11 (s, 1H, –CONH–). MS (FAB) = 509 (MH+). Anal.
CHN C24H23N5O8 × 0.25 H2O.
5.2.55. Ethyl (2S)-2-{[(2,4-dimethyl-7-{[4-(amino(imino)-
methyl)benzoyl]amino}-2H-1,4-benzoxazine-3(4H)-one-2-
yl)carbonyl]amino}-3-{[(4-methylphenyl)sulphonyl]-
amino}propanoate in the form of acetate (56)
Prepared according to the general procedure as in Section
5.2.1.9 from 55 (0.80 g, 1.16 mmol). m = 0.53 g (65%). M.p.
151–155 °C. IR (KBr, cm–1): 3333, 1676, 1515, 1390, 1316,
1157, 1092, 1030, 816, 661. ␣ 2D0= –3.1° (c 0.66; MeOH).
͓ ͔
1H-NMR (DMSO-d6, 300 MHz): d (ppm) 0.97 (t, 3H,
J = 7.2 Hz, CH2CH3), 1.10 (t, 3H, J = 7.2 Hz, CH2CH3), 1.71
(s, 3H, 2-CH3), 1.72 (s, 3H, 2-CH3), 1.75 (s, 6H, 2 × CH3–
COOH), 2.30 (s, 3H, Ph–CH3), 2.38 (s, 3H, Ph–CH3), 3.01
(m, 4H, 2 × NH–CH2–CH), 3.30 (s, 6H, 2 × N–CH3), 3.85
(m, 2H, O–CH2–CH3), 3.99 (q, 2H, J = 7.2 Hz, O–CH2–
CH3), 4.16 (m, 1H, CH), 4.29 (m, 1H, CH), 7.15 (m, 2H, 2 ×
Ar–H5), 7.30 (d, 2H, J = 8.29 Hz, SO2–Ph–CH3), 7.39 (d, 2H,
J = 7.9 Hz, SO2–Ph–CH3), 7.45 (m, 2H, 2 × Ar–H6), 7.58 (d,
2H, J = 7.9 Hz, SO2–Ph–CH3), 7.64 (d, 2H, J = 8.3 Hz,
SO2–Ph–CH3), 7.79 (m, 2H, 2 ×Ar–H8), 7.87–8.01 (m, 4H, 2
× CO–Ph–, 2H), 8.04–8.14 (m, 4H, 2 × CO–Ph–, 2H), 8.33
(s, 1H, CONH–CH), 8.45 (s, 1H, CONH–CH), 10.50 (s, 2H,
2 × –CONH–. MS (FAB) = 651 (MH+-free base). Anal. CHN
C31H34N6O8S × CH3COOH × 2 H2O.
5.2.52. 2-{[(2,4-Dimethyl-7-{[4-(5-oxo-4,5-dihydro-1,2,4-
oxadiazol-3-yl)benzoyl]amino}-2H-1,4-benzoxazine-3(4H)-
one-2-yl)carbonyl]amino}acetic acid (53)
Prepared according to the general procedure as in Section
5.2.1.7 from 52 (0.15 g, 0.29 mmol). m = 0.14 g (99%). M.p.
206–209 °C. IR (KBr, cm–1): 3447, 1779, 1680, 1552, 1513,
1436, 1290, 1205, 1032, 946, 816, 756, 670. 1H-NMR
(DMSO-d6, 300 MHz): d (ppm) 1.72 (s, 3H, 2-CH3), 3.30 (s,
3H, N–CH3), 3.52–3.75 (m, 2H, NH–CH2–), 7.13 (d, 1H,
J = 8.9 Hz, Ar–H5), 7.40–7.44 (dd, 1H, J = 8.9, 2.3 Hz,
Ar–H6), 7.72 (d, 1H, J = 2.3 Hz, Ar–H8), 7.96 (d, 2H,
J = 8.5 Hz, Ar–2H), 8.11 (d, 2H, J = 8.5 Hz, Ar–2H), 8.38 (t,
1H, J = 6.0 Hz, CO–NH–CH2), 10.42 (s, 1H, –CONH–),
12.66 (s, 1H, –CONH–). MS (FAB) = 482 (MH+). Anal.
CHN C22H19N5O8 × 1.75 H2O.
5.2.56. (2S)-2-{[(2,4-Dimethyl-7-{[4-(amino(imino)methyl)-
benzoyl]amino}-2H-1,4-benzoxazine-3(4H)-one-2-yl)-
carbonyl]amino}-3-{[(4-methylphenyl)sulphonyl]-
amino}propanoic acid in the form of acetate (57)
Prepared according to the general procedure as in Section
5.2.1.7 from 56 (0.29 g, 0.4 mmol). The compound was
treated with 4 M HCl, washed with diethyl ether and dried in

44
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
vacuo. m = 0.20 g (73%). M.p. 204–216 °C. IR (KBr, cm–1):
CH2CH3), 1.65 (d, 3H, J = 6.0 Hz, 2-CH3), 2.37 (s, 3H,
Ph–CH3), 3.06 (m, 1H, NH–CH2–CH), 3.30 (m, 1H, NH–
CH2–CH), 3.66 (m, 2H, O–CH2–CH3), 4.18 (m, 1H, NH–
CH2–CH), 7.03 (d, 1H, J = 8.7, Ar–H5), 7.35–7.60 (dd, 4H,
J = 8.7, 7.91 Hz, SO2–Ph–CH3), 7.89 (m, 1H, Ar–H6), 7.94
(d, 1H, Ar–H8), 8.19 (m, 1H, SO2NH), 8.38 (s, 1H,
–CONH–), 11.39 (dd, 1H, J = 11.30, 9.0 Hz, –CONH–). MS
(FAB) = 521 (MH+). Anal. CHN C22H24N4O9S.
3333, 1676, 1515, 1390, 1316, 1157, 1092, 1030, 816, 661.
␣
͓ ͔
2D0= –3.1° (c 0.66; MeOH). 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.70 (s, 6H, 2 × 2-CH3), 2.30 (s, 3H,
Ph–CH3), 2.38 (s, 3H, Ph–CH3), 3.00 (m, 4H, 2 × NH–CH2–
CH), 3.30 (s, 6H, 2 × N–CH3), 4.13 (m, 1H, CH), 4.22 (m,
1H, CH), 7.14 (m, 2H, 2 × Ar–H5), 7.29 (d, 2H, J = 8.3 Hz,
SO2–Ph–CH3), 7.38 (d, 2H, J = 7.9 Hz, SO2–Ph–CH3),
7.48–7.55 (m, 3H, 2 × Ar–H6, NHSO2), 7.58 (d, 2H,
J = 8.3 Hz, SO2–Ph–CH3), 7.64 (d, 2H, J = 8.3 Hz, SO2–Ph–
CH3), 7.70 (t, 1H, J = 6.0 Hz, NHSO2), 7.77 (d, 1H,
J = 2.3 Hz, Ar–H8), 7.82 (d, 1H, J = 2.3 Hz, Ar–H8), 8.00 (d,
4H, J = 7.9 Hz, 2 × CO–Ph–, 2H), 8.05 (d, 1H, J = 7.9 Hz,
CONH–CH), 8.20 (m, 4H, 2 × CO–Ph–, 2H), 8.28 (d, 1H,
J = 7.9 Hz, CONH–CH), 9.39 (s, 4H, 2 × NH2), 9.59 (s, 4H,
5.2.60. Ethyl 2-{[(7-amino-2-methyl-2H-1,4-benzoxazine-
3(4H)-one-2-yl)carbonyl]amino}-3-phenylpropanoate (63)
Prepared according to the general procedure as in Section
5.2.1.3 from 61 (1.49 g, 3.50 mmol). m = 1.37 g (99%). M.p.
69–71 °C. IR (KBr, cm–1): 3364, 2980, 1699, 1636, 1520,
20
D
1375, 1180, 1027, 810, 702.
␣
͓ ͔
= –14.8° (c = 0.50;
+
1
2 × NH2 ), 10.60 (s, 1H, –CONH–), 10.65 (s, 1H, –CONH–),
MeOH). H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.05 (t,
3H, J = 7.2 Hz, CH2CH3), 1.12 (t, 3H, J = 7.2 Hz, CH2CH3),
1.46 (s, 3H, 2-CH3), 1.60 (s, 3H, 2-CH3), 3.00 (m, 4H,
Ph–CH2–CH), 4.00 (m, 4H, O–CH2–CH3), 4.27 (m, 1H,
CH2–CH), 4.41 (m, 1H, CH2–CH), 4.88 (s, 1H, 7-NH2), 4.94
(s, 1H, 7-NH2), 6.16–6.22 (m, 2H, 2 × Ar–H6), 6.28–6.36
(2d, 2H, J = 2.3 Hz, 2 × Ar–H8), 6.50–6.53 (2d, 2H,
J = 6.0 Hz, 2 ×Ar–H5), 7.11–7.29 (m, 10H, 2 × 5Ar–H), 8.10
(d, 1H, J = 7.9 Hz, CONH), 8.24 (d, 1H, J = 7.9 Hz, CONH),
11.24 (s, 1H, CONH), 11.27 (s, 1H, CONH). MS
(FAB) = 398 (MH+). Anal. CHN C21H23N3O5.
12.81 (s, 2H, 2 × CH–COOH). MS (FAB) = 623 (MH+-free
base). Anal. CHN C29H30N6O8S × CF3COOH × 3 H2O.
5.2.57. 2-Methyl-7-nitro-2H-1,4-benzoxazine-3(4H)-one-2-
carboxylic acid (60) [10]
Prepared according to the general procedure as in Section
5.2.1.6 from 4 (6.72 g, 24.0 mmol). m = 5.62 g (93%). M.p.
163–165 °C. IR (KBr, cm–1): 3492, 3098, 2605, 1694, 1607,
1
1539, 1505, 1384, 1343, 1262, 1126, 975, 894, 745. H-
NMR (DMSO-d6, 300 MHz): d (ppm) 1.73 (s, 3H, 2-CH3),
7.10 (d, 1H, J = 8.7 Hz, Ar–H5), 7.81 (d, 1H, J = 2.5 Hz,
Ar–H8), 7.93 (dd, 1H, J = 8.7, 2.5, Ar–H6). MS (FAB) = 253
(MH+).
5.2.61. Ethyl 2-{[(7-amino-2-methyl-2H-1,4-benzoxazine-
3(4H)-one-2-yl)carbonyl]amino}-2-{[(4-
methylphenyl)sulphonyl]amino}propanoate (64)
5.2.58. Ethyl 2-{[(2-methyl-7-nitro-2H-1,4-benzoxazine-
3(4H)-one-2-yl)carbonyl]amino}-3-phenylpropanoate (61)
Prepared according to the general procedure as in Section
5.2.1.8 from 60 (1.26 g, 5.0 mmol) and L-phenylalanine ethyl
ester hydrochloride (1.27 g, 5.5 mmol). m = 1.85 g (86%).
M.p. 165–167 °C. IR (KBr, cm–1): 3346, 3248, 1732, 1662,
Prepared according to the general procedure as in Section
5.2.1.3 from 62 (3.02 g, 5.8 mmol). m = 2.89 g (100%). M.p.
103–106 °C. IR (KBr, cm–1): 3367, 2983, 1697, 1520, 1427,
1
1320, 1162, 1092, 1020, 814, 666, 551. H-NMR (DMSO-
d6, 300 MHz): d (ppm) 1.00 (m, 3H, CH2CH3), 1.44 (d, 3H,
J = 12.40 Hz, 2-CH3), 2.37 (s, 3H, Ph–CH3), 2.89 (m, 1H,
NH–CH2–CH), 3.41 (m, 1H, NH–CH2–CH), 3.76 (m, 2H,
CH2–CH3), 3.92 (m, 1H, NH–CH2–CH), 4.90 (s, 2H,
7-NH2), 6.16 (dd, 1H, J = 8.3, 2.3 Hz, Ar–H6), 6.26 (d, 1H,
J = 2.3 Hz, Ar–H8), 6.52 (d, 1H, J = 8.3 Hz, Ar–H5),
7.37–7.64 (dd, 4H, J = 8.3, 7.5 Hz, SO2–Ph–CH3), 8.04 (m,
1H, SO2NH), 8.23 (s, 1H, –CONH–), 11.26 (m, 1H,
1608, 1532, 1324, 1125, 1019, 881, 744, 491. ␣ 2D0= –25.8°
͓ ͔
(c = 0.40; MeOH). 1H-NMR (DMSO-d6, 300 MHz): d (ppm)
0.95 (t, 3H, J = 7.0 Hz, CH2CH3), 1.14 (t, 3H, J = 7.0 Hz,
CH2CH3), 1.53 (s, 3H, 2-CH3), 1.71 (s, 3H, 2-CH3), 2.93 (m,
2H, Ph–CH2–CH), 3.08 (m, 2H, Ph–CH2–CH), 3.87 (m, 2H,
O–CH2–CH3), 4.07 (m, 2H, O–CH2–CH3), 4.41 (m, 2H, 2 ×
1H, CH2–CH), 6.91–7.22 (m, 12H, 2 × Ar–H5, 2 × 5 Ar–H),
7.85–7.96 (m, 4H, 2 × Ar–H6, 2 × Ar–H8), 8.62 (t, 1H,
J = 8.3 Hz, CONH), 8.69 (t, 1H, J = 8.3 Hz, CONH), 11.32 (s,
2H, CONH). MS (FAB) = 428 (MH+). Anal. CHN
C21H21N3O7.
–CONH–). MS (FAB)
C22H26N4O7S × H20.
=
491 (MH+). Anal. CHN
5.2.62. Ethyl 2-{[(2-methyl-7-(4-cyanobenzoylamino)-2H-
1,4-benzoxazine-3(4H)-one-2-yl)carbonyl]amino}-3-
phenylpropanoate (65)
5.2.59. Ethyl 2-{[(2-methyl-7-nitro-2H-1,4-benzoxazine-
3(4H)-one-2-yl)carbonyl]amino}-2-{[(4-
methylphenyl)sulphonyl]amino}propanoate (62)
Prepared according to the general procedure as in Section
5.2.1.8 from 60 (2.02 g, 8.0 mmol) and 71 (2.84 g, 8.8 mmol).
m = 3.49 g (84%). M.p. 96–100 °C. IR (KBr, cm–1): 3271,
2984, 1722, 1608, 1532, 1327, 1162, 1092, 816, 663, 550.
1H-NMR (DMSO-d6, 300 MHz): d (ppm) 0.94 (m, 3H,
Prepared according to the general procedure as in Section
5.2.1.4 from 63 (0.569 g, 1.50 mmol) and 0.27 g (1.58 mmol)
of 4-cyanobenzoyl chloride. m = 0.655 g (83%). M.p. 225–
228 °C. IR (KBr, cm–1): 3344, 2230, 1736, 1687, 1655, 1516,
20
D
1394, 1247, 1186, 1117, 1016, 853, 756.
␣
͓ ͔
= –11.6°
(c = 0.40; MeOH). 1H-NMR (DMSO-d6, 300 MHz): d (ppm)
0.98 (t, 3H, J = 7.2 Hz, CH2CH3), 1.12 (t, 3H, J = 7.2 Hz,

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
45
CH2CH3), 1.51 (s, 3H, 2-CH3), 1.67 (s, 3H, 2-CH3), 3.01 (m,
5.2.65. Ethyl 2-{[(2-methyl-7-{[4-(amino(imino)methyl)-
benzoyl]amino}-2H-1,4-benzoxazine-3(4H)-one-2-yl)-
carbonyl]amino}-2-{[(4-methylphenyl)sulphonyl]amino}-
propanoate in the form of acetate (68)
4H, 2 × 2H, Ph–CH2–CH), 3.91 (q, 2H, J = 7.2 Hz, O–CH2–
CH3), 4.04 (q, 2H, J = 7.2 Hz, O–CH2–CH3), 4.29 (m, 1H,
CH2–CH), 4.40 (m, 1H, CH2–CH), 6.80 (d, 1H, J = 8.3 Hz,
Ar–H5), 6.83 (d, 1H, J = 8.3 Hz, Ar–H5), 6.99–7.31 (m, 12H,
2 × Ar–H6, 2 × 5 Ar–H), 7.69 (m, 2H, 2 × Ar–H8), 8.02–8.13
(m, 8H, Ar–4H), 8.39 (d, 1H, J = 8.3 Hz, –CONH–), 8.47 (d,
1H, J = 8.3 Hz, –CONH–), 10.46 (s, 1H, –CONH–), 10.47 (s,
1H, –CONH–), 10.69 (s, 1H, –CONH–), 10.71 (s, 1H,
Prepared according to the general procedure as in Section
5.2.1.5 from 0.75 g 66 (1.2 mmol). m = 0.17 g (20%). M.p.
232–235 °C. IR (KBr, cm–1): 3425, 1700, 1558, 1520, 1417,
1338, 1161, 1020, 815, 644. 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 0.95 (m, 3H, CH2CH3), 1.58 (s, 3H,
2-CH3), 1.80 (s, 6H, CH3COO–), 2.34 (s, 3H, Ph–CH3), 3.05
(m, 1H, NH–CH2–CH), 3.30 (m, 1H, NH–CH2–CH), 3.77
(m, 2H, CH2–CH3), 3.97 (m, 1H, NH–CH2–CH), 6.84 (d,
1H, J = 8.6 Hz, Ar–H5), 7.33 (m, 3H, Ar–H6, SO2–Ph–CH3)
7.63 (dd, 2H, J = 8.3 Hz, SO2–Ph–CH3), 7.68 (d, 1H,
J = 1.9 Hz, Ar–H8), 7.93–8.11 (dd, 4H, J = 8.26, 8.26 Hz,
Ar–H), 8.27 (m, 1H, –CONH–), 10.47 (s, 1H, –CONH–),
signals for amidine protons and the lactam proton were not
observed. MS (FAB) = 637 (MH+-free base). Compound was
too hygroscopic for CHN analysis.
–CONH–). MS (FAB)
C26H29N4O6 × 0.5 H2O.
=
527 (MH+). Anal. CHN
5.2.63. Ethyl 2-{[(2-methyl-7-(4-cyanobenzoylamino)-2H-
1,4-benzoxazine-3(4H)-one-2-yl)carbonyl]amino}-2-
{[(4-methylphenyl)sulphonyl]amino}propanoate (66)
Prepared according to the general procedure as in Section
5.2.1.4 from 64 (1.24 g, 2.5 mmol), 0.40 ml (2.6 mmol) Et3N
and 0.45 g (2.6 mmol) of 4-cyanobenzoyl chloride.
m = 1.07 g (69%). M.p. 233–237 °C. IR (KBr, cm–1): 3280,
2232, 1711, 1674, 1519, 1419, 1328, 1166, 1058, 855, 669,
562. 1H-NMR (DMSO-d6, 300 MHz): d (ppm) 0.90 (m, 3H,
CH2CH3), 1.58 (d, 3H, J = 10.9 Hz, 2-CH3), 2.36 (s, 3H,
Ph–CH3), 3.02 (m, 1H, NH–CH2–CH), 3.34 (m, 1H, NH–
CH2–CH), 3.73 (m, 2H, CH2–CH3), 3.94 (m, 1H, NH–CH2–
CH), 6.83 (d, 1H, J = 8.7 Hz, Ar–H5), 7.28 (dd, 1H, J = 8.7,
1.9 Hz, Ar–H6), 7.35 (d, 2H, J = 7.9 Hz, SO2–Ph–CH3) 7.64
(m, 3H′, SO2–Ph–CH3, Ar–H8), 8.02–8.09 (dd, 4H, J = 8.7,
7.9 Hz, Ar–H), 8.21 (m, 2H, SO2NH,–CONH–), 10.47 (s,
1H, –CONH–), 10.74 (m, 1H, –CONH–). MS (FAB) = 620
(MH+). Anal. CHN C30H29N5O8S.
5.2.66. 2-{[(2-Methyl-7-{[4-(amino(imino)methyl)benzoyl]-
amino}-2H-1,4-benzoxazine-3(4H)-one-2-yl)carbonyl]-
amino}-3-phenylpropanoic acid in the form of acetate (69
)
Prepared according to the general procedure as in Section
5.2.1.7 from 67 (0.147 g, 0.24 mmol). m = 0.103 g (73%).
M.p. >230 °C (decomposes without melting). IR (KBr,
cm–1): 3394, 1694, 1617, 1518, 1401, 1328, 1133, 864, 702.
1H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.59 (s, 3H,
2-CH3), 1.61 (s, 3H, 2-CH3), 1.78 (s, 1.5 H, CH3COO–), 2.97
(m, 4H, 2 × 2H, Ph–CH2–CH), 3.82 (m, 1H, CH2–CH), 3.93
(m, 1H, CH2–CH), 6.85 (m, 2H, Ar–H5), 6.95–7.25 (m, 10H,
2 × 5Ar–H), 7.42 (m, 1H, Ar–H6), 7.48–7.53 (m, 2H, Ar–H6,
Ar–H8), 7.63 (d, 1H, J = 1.9 Hz, Ar–H8), 7.74–7.91 (dd, 4H,
J = 8.3 Hz, Ar–H), 8.01–8.12 (dd, 4H, J = 8.3 Hz, Ar–H),
10.58 (s, 2H, 2 × CONH), signals for amidine protons,
carboxylic proton and for the lactam proton were not ob-
served; only half an equivalent of acetic acid was observed.
MS (FAB) = 516 (MH+-free base). Anal. CHN C27H25N5O6
× 0.5 CH3COOH × 5 H2O.
5.2.64. Ethyl 2-{[(2-methyl-7-{[4-(amino(imino)methyl)-
benzoyl]amino}-2H-1,4-benzoxazine-3(4H)-one-2-yl)-
carbonyl]amino}-3-phenylpropanoate in the form
of acetate (67)
Prepared according to the general procedure as in Section
5.2.1.5 from 65 (0.526 g, 1.00 mmol). m = 0.287 g (48%).
M.p. >220 °C (decomposes without melting). IR (KBr,
cm–1): 3352, 1701, 1578, 1518, 1412, 1180, 1014, 864, 702.
1H-NMR (DMSO-d6, 300 MHz): d (ppm) 0.99 (t, 3H,
J = 7.2 Hz, CH2CH3), 1.10 (t, 3H, J = 7.2 Hz, CH2CH3), 1.51
(s, 3H, 2-CH3), 1.66 (s, 3H, 2-CH3), 1.73 (s, 6H, CH3COO–),
3.01 (m, 4H, 2 × 2H, Ph–CH2–CH), 3.91 (q, 2H, J = 7.2 Hz,
O–CH2–CH3), 4.04 (q, 2H, J = 7.2 Hz, O–CH2–CH3), 4.29
(m, 1H, CH2–CH), 4.39 (m, 1H, CH2–CH), 6.81 (d, 1H,
J = 8.7 Hz, Ar–H5), 6.85 (d, 1H, J = 8.3 Hz, Ar–H5),
7.00–7.26 (m, 10H, 2 × 5Ar–H), 7.32 (s, 1H,Ar–H6), 7.36 (s,
1H, Ar–H6), 7.73 (m, 2H, 2 × Ar–H8), 7.95–8.11 (m, 8H, 2 ×
Ar–4H), 8.39 (s, 1H, –CONH–), 8.46 (s, 1H, –CONH–),
10.56 (s, 2H, 2 × –CONH–), signals for amidine protons and
for the lactam proton were not observed. MS (FAB) = 544
(MH+-free base). Compound was too hygroscopic for CHN
analysis.
5.2.67. 2-{[(2-Methyl-7-{[4-(amino(imino)methyl)benzoyl]-
amino}-2H-1,4-benzoxazine-3(4H)-one-2-yl)carbonyl]-
amino}-2-{[(4-methylphenyl)sulphonyl]amino}propanoic
acid in the form of acetate (70)
Prepared according to the general procedure as in Section
5.2.1.7 from 68 0.11 g, (0.2 mmol). m = 0.04 g (35%). M.p.
245–248 °C. IR (KBr, cm–1): 3425, 1700, 1558, 1520, 1417,
1338, 1161, 1020, 815, 644. 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.68 (s, 3H, 2-CH3), 1.78 (s, 3H,
CH3COO–), 2.28 (s, 3H, Ph–CH3), 2.97 (m, 2H, NH–CH2–
CH), 3.97 (m, 1H, NH–CH2–CH), 6.84 (d, 1H, J = 8.3 Hz,
Ar–H5), 7.30 (m, 3H, Ar–H6, SO2–Ph–CH3), 7.51 (m, 1H,
Ar–H8), 7.64 (dd, 2H, J = 8.3 Hz, SO2–Ph–CH3), 7.82–8.03
(dd, 4H, J = 7.5 Hz, Ar–H), 8.20 (m, 1H, –CONH–), signals
for amidine protons, carboxylic proton and the lactam proton

46
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
were not observed. MS (FAB) = 609 (MH+-free base). Anal.
CHN C30H32N6O10S × 2 H2O.
5.2.71. Ethyl (2S)-2-{[(4-methylphenyl)sulphonyl]amino}-
3-[2-(7-{[4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)
benzoyl]amino}-2H-1,4-benzoxazine-3(4H)-one-4-yl)
acetyl]aminopropanoate (75)
5.2.68. Ethyl 2-(7-nitro-2H-1,4-benzoxazine-3(4H)-one-
4-il)acetate (72) [12]
Prepared according to the general procedure as in Section
5.2.1.8 from 74 (0.58 g, 1.2 mmol) and 59 (0.255 g,
1.2 mmol). m = 0.60 g (69%). M.p. 258–262 °C. IR (KBr,
cm–1): 3749, 3365, 1790, 1656, 1518, 1400, 1316, 1158,
Prepared according to general procedure as in Section
5.2.1.1 from 7 (3.88 g, 20 mmol) and ethyl bromoacetate
(2.28 ml, 20 mmol). m = 4.68 g (84%). M.p. 116–118 °C. IR
(KBr, cm–1): 3459, 3094, 2996, 1733, 1687, 1603, 1522,
1094, 941, 809, 660. ␣ 20 = –17.8° (c 0.69; 0.05 M NaOH).
͓ ͔
D
1H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.02 (t, 3H,
1
1433, 1346, 1225, 1022, 890, 811, 744. H-NMR (CDCl3,
J = 7.2 Hz, CH2–CH3), 2.37 (s, 3H, Ph–CH3), 3.20 (m, 1H,
N–CH2–CH), 3.39 (m, 1H, N–CH2–CH), 3.81 (q, 2H,
J = 7.2 Hz, O–CH2CH3), 3.94 (m, 1H, CH), 4.45 (s, 2H,
N–CH2–CO), 4.70 (s, 2H, O–CH2–CO), 6.82 (d, 1H,
J = 8.7 Hz, Ar–H5), 7.40 (m, 3H, SO2–Ph–, 2H, Ar–H6), 7.56
(d, 1H, J = 2.3 Hz, Ar–H8), 7.66 (d, 2H, J = 8.3 Hz, SO2–
Ph–), 7.96 (d, 2H, J = 8.7 Hz, CO–Ph–), 8.12 (d, 2H,
J = 8.7 Hz, CO–Ph–), 8.31 (d, 1H, J = 9.0 Hz, NHSO2), 8.38
(t, 1H, J = 6.4 Hz, CO–NH–CH2), 10.41 (s, 1H, –CONH–).
MS (FAB) = 679 (MH+). Anal. CHN C31H30N6O10S ×
1.5 H2O.
300 MHz): d (ppm) 1.31 (t, 3H, J = 7.2 Hz, CH2CH3), 4.24
(m, 2H, CH2CH3), 4.72 (s, 3H, N–CH2), 4.79 (s, 2H,
O–CH2–CO), 6.86 (d, 1H, J = 8.9 Hz, Ar–H5), 7.91 (d, 1H,
J = 2.6 Hz,Ar–H8), 7.96 (dd, 1H, J = 8.9, 2.6 Hz,Ar–H6). MS
(FAB) = 281 (MH+). Anal. CHN C12H12N2O6.
5.2.69. Ethyl (2S)-2-{[(4-methylphenyl)sulphonyl]amino}-
3-[2-(7-nitro-2H-1,4-benzoxazine-3(4H)-one-4-yl)acetyl]-
aminopropanoate (73)
Prepared according to the general procedure as in Section
5.2.1.6 from 72 (4.49 g, 16 mmol). The crude product was
used without further purification according to the general
procedure as in Section 5.2.1.8 with 71 (1.48 g, 4.6 mmol) to
yield 73. m = 0.65 g (7.8%). M.p. 184–185 °C. IR (KBr,
cm–1): 3315, 3254, 2980, 1732, 1667, 1600, 1532, 1397,
1341, 1254, 1165, 1091, 1049, 925, 816, 742, 670.
5.2.72. Ethyl (2S)-2-{[(4-methylphenyl)sulphonyl]amino}-
3-[2-(7-{[4-(amino(imino)methyl)benzoyl]amino}-2H-1,4-
benzoxazine-3(4H)-one-4-yl)acetyl]aminopropanoate in
the form of acetate (76)
Prepared according to the general procedure as in Section
5.2.1.9 from 75 (0.446 g, 0.65 mmol). m = 0.44 g (97%). M.p.
␣
͓ ͔
2D0= –8.7° (c = 0.41; DMF). 1H-NMR (DMSO-d6,
138–142 °C. IR (KBr, cm–1): 3318, 1734, 1670, 1516, 1406,
300 MHz): d (ppm) 0.99 (t, 3H, J = 7.2 Hz, CH2–CH3), 2.38
(s, 3H, Ph–CH3), 3.19 (m, 1H, N–CH2–CH), 3.38 (m, 1H,
N–CH2–CH), 3.78 (q, 2H, J = 7.2 Hz, O–CH2–CH3), 3.93
(m, 1H, CH), 4.56 (s, 2H, N–CH2–CO), 4.86 (s, 2H, O–CH2–
CO), 7.06 (d, 1H, J = 9.0 Hz, Ar–H5), 7.37 (dd, 2H,
J = 8.3 Hz, –SO2–Ph–), 7.65 (dd, 2H, J = 8.3 Hz, –SO2–Ph–),
7.82 (d, 1H, J = 2.6 Hz,Ar–H8), 7.90 (dd, 1H, J = 9.0, 2.6 Hz,
Ar–H6), 8.29 (d, 1H, J = 9.1 Hz, Ph–NHSO2), 8.45 (t, 1H,
J = 5.7 Hz, –CONH–). MS (FAB) = 521 (MH+). Anal. CHN
C22H24N4O9S.
20
D
1339, 1161, 1091, 1050, 814, 661.
␣
͓ ͔
= +4.2° (c 0.51;
DMF). 1H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.02 (t, 3H,
J = 7.2 Hz, CH2–CH3), 1.79 (s, 3H, CH3COOH), 2.38 (s, 3H,
Ph–CH3), 3.20 (m, 1H, N–CH2–CH), 3.39 (m, 1H, N–CH2–
CH), 3.81 (q, 2H, J = 7.2 Hz, O–CH2CH3), 3.92 (m, 1H, CH),
4.44 (s, 2H, N–CH2–CO), 4.70 (s, 2H, O–CH2–CO), 6.82 (d,
1H, J = 9.0 Hz, Ar–H5), 7.39 (m, 3H, SO2–Ph–, 2H, Ar–H6),
7.57 (d, 1H, J = 2.3 Hz, Ar–H8), 7.67 (d, 2H, J = 8.3 Hz,
SO2–Ph–), 7.94 (d, 2H, J = 8.7 Hz, CO–Ph–), 8.11 (d, 2H,
J = 8.7 Hz, CO–Ph–), 8.5 (t, 1H, J = 6.0 Hz, CO–NH–CH2),
10.44 (s, 1H, –CONH–), signals for amidine protons and the
sulphonamide proton were not observed. MS (FAB) = 637
(MH+-free base). Anal. CHN C31H32N6O8S × CH3COOH ×
2.25 H2O.
5.2.70. Ethyl (2S)-2-{[(4-methylphenyl)sulphonyl]amino}-
3-[2-(7-amino-2H-1,4-benzoxazine-3(4H)-one-4-yl)acetyl]-
aminopropanoate (74)
Prepared according to the general procedure as in Section
5.2.1.3 from 73 (0.50 g, 0.96 mmol). m = 0.46 g (100%). M.p.
89–91 °C. IR (KBr, cm–1): 3364, 2980, 1734, 1670, 1517,
5.2.73. (2S)-2-{[(4-Methylphenyl)sulphonyl]amino}-3-[2-
(7-{[4-(amino(imino)metil)benzoyl]amino}-2H-1,4-
benzoxazine-3(4H)-one-4-yl)acetyl]aminopropanoic acid
in the form of acetate (77)
1430, 1339, 1160, 1091, 1046, 814, 661. ␣ 20 = +18.2° (c
͓ ͔
D
0.73; MeOH). 1H-NMR (DMSO-d6, 300 MHz): d (ppm) 1.01
(t, 3H, J = 7.2 Hz, CH2–CH3), 2.38 (s, 3H, Ph–CH3), 3.17 (m,
1H, N–CH2–CH), 3.40 (m, 1H, N–CH2–CH), 3.79 (m, 2H,
O–CH2–CH3), 3.92 (m, 1H, CH), 4.31 (s, 2H, N–CH2–CO),
4.54 (s, 2H, O–CH2–CO), 5.24 (s, 2H, NH2), 6.20–6.25 (m,
2H, Ar–H6, Ar–H8), 6.50 (d, 1H, J = 8.3 Hz, Ar–H5), 7.38 (d,
2H, J = 8.1 Hz, –SO2–Ph–), 7.65 (d, 2H, J = 8.1 Hz, –SO2–
Ph–), 8.22–8.34 (m, 2H, –NH–O2–, –CONH–). MS (FAB)
= 491 (MH+). Anal. CHN C22H26N4O7S × 2 H2O.
Prepared according to the general procedure as in Section
5.2.1.7 from 76 (0.26 g, 0.38 mmol). m = 0.18 g (71%). M.p.
230–233 °C. IR (KBr, cm–1): 3337, 1665, 1610, 1518, 1407,
1324, 1153, 1091, 868, 812. 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 2.34 (s, 3H, Ph–CH3), 3.81 (m, 1H, CH),
4.37–4.58 (m, 2H, N–CH2–CO), 4.70 (s, 2H, O–CH2–CO),
6.91 (d, 1H, J = 8.7 Hz, Ar–H5), 7.37 (m, 4H, SO2–Ph–, 2H,
Ar–H6, Ar–H8), 7.70 (d, 2H, J = 8.3 Hz, SO2–Ph–), 7.83–

M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
47
7.94 (m, 4H, CO–Ph–), 8.13 (m, 1H, CO–NH–H2), 10.29 (s,
1H, –CONH–), signals for amidine protons, sulphonamide
and carboxylic protons were not observed. MS (FAB) = 609
(MH+-free base). Compound was too hygroscopic for CHN
analysis.
Ar–H8), 7.95–8.14 (dd, 4H, J = 8.7 Hz, Ar–H), 8.01 (s, 1H,
–CONH–), 10.27 (s, 1H, –CONH–). MS (FAB) = 481
(MH+). Anal. CHN C24H24N4O7 × 1 H2O.
5.2.77. Ethyl 5-(7-{[4-(amino(imino)methyl)benzoyl]-
amino}-2H-1,4-benzoxazine-3(4H)-one-4-yl)pentanoate in
the form of acetate (81)
5.2.74. Ethyl 5-(7-nitro-2H-1,4-benzoxazine-3(4H)-one-
4-yl)pentanoate (78)
Prepared according to the general procedure as in Section
5.2.1.9 from 80 (0.300 g, 0.624 mmol). m = 0.234 g (75%).
M.p. 177–179 °C. IR (KBr, cm–1): 2962, 1728, 1665, 1605,
1514, 1408, 1049, 861, 702. 1H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.17 (t, 3H, J = 7.1 Hz, O–CH2–CH3),
1.57 (m, 4H, N–CH2–CH2–CH2–CH2–CO), 1.77 (s, 3H,
CH3COO–), 2.34 (t, 2H, J = 6.4 Hz, N–CH2–CH2–CH2–
CH2–CO), 3.91 (m, 2H, N–CH2–CH2–CH2–CH2–CO), 4.04
(q, 2H, J = 7.1 Hz, O–CH2–CH3), 4.64 (s, 2H, O–CH2–CO),
7.21 (d, 1H, J = 9.0 Hz,Ar–H5), 7.48 (dd, 1H, J = 9.0, 2.3 Hz,
Ar–H6), 7.56 (d, 1H, J = 2.3 Hz, Ar–H8), 7.92–8.11 (dd, 4H,
J = 8.3 Hz, Ar–H), 10.45 (s, 1H, –CONH–), signals for
amidine protons were not observed. MS (FAB) = 439 (MH+-
free base). Anal. CHN C25H30N4O7.
To a stirred suspension of 7 (5.53 g, 28.5 mmol), K2CO3
(9.87 g, 71.3 mmol) and benzyltriethylammonium chloride
(1.30 g, 5.7 mmol) in acetonitrile (150 ml), ethyl
5-bromopentanoate (5.94 ml, 37.05 mmol) was added drop-
wise. The reaction mixture was heated at 75 °C for 48 h. The
precipitated K2CO3 was filtered off and the solvent evapo-
rated. The oily residue was recrystallized from ethanol
(60 ml) to give yellow crystals. m = 7.26 g (79%). M.p.
94–95 °C. IR (KBr, cm–1): 3425, 3088, 1731, 1701, 1598,
1
1506, 1343, 1241, 1091, 900, 742. H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.16 (t, 3H, J = 7.0 Hz, O–CH2–CH3),
1.58 (m, 4H, N–CH2–CH2–CH2–CO), 2.34 (m, 2H, N–CH2–
CH2–CH2–CH2–CO), 3.98 (m, 2H, N–CH2–CH2–CH2–
CH2–CO), 4.03 (q, 2H, J = 7.0 Hz, O–CH2–CH3), 4.80 (s,
2H, O–CH2–CO), 7.44 (d, 1H, J = 9.1 Hz, Ar–H5), 7.81 (d,
1H, J = 2.6 Hz,Ar–H8), 7.94 (dd, 2H, J = 9.1, 2.6 Hz,Ar–H6).
MS (EI) = 322 (M+). Anal. CHN C15H18N2O6.
5.2.78. 5-(7-{[4-(Amino(imino)methyl)benzoyl]amino}-2H-
1,4-benzoxazine-3(4H)-one-4-yl)pentanoic acid in
the form of acetate (82)
Prepared according to the general procedure as in Section
5.2.1.7 from 80 (0.250 g, 0.553 mmol). The crude product
was used in the next step (general procedure as in Section
5.2.1.9) without further purification. m = 0.267 g (89%). M.p.
226–231 °C. IR (KBr, cm–1): 2936, 1730, 1668, 1640, 1514,
5.2.75. Ethyl 5-(7-amino-2H-1,4-benzoxazine-3(4H)-one-
4-yl)pentanoate (79)
Prepared according to the general procedure as in Section
5.2.1.3 from 78 (7.00 g, 21.7 mmol). m = 6.20 g (98%). M.p.
73–75 °C. IR (KBr, cm–1): 3325, 2938, 1728, 1658, 1515,
1423, 1313, 1196, 1046, 846, 802, 654, 587. 1H-NMR
(DMSO-d6, 300 MHz): d (ppm) 1.26 (t, 3H, J = 7.2 Hz,
O–CH2–CH3), 1.71 (m, 4H, N–CH2–CH2–CH2–CH2–CO),
2.36 (m, 2H, N–CH2–CH2–CH2–CH2–CO), 3.93 (m, 2H,
N–CH2–CH2–CH2–CH2–CO), 4.14 (q, 2H, J = 7.2 Hz,
O–CH2–CH3), 4.55 (s, 2H, O–CH2–CO), 6.50–6.53 (m, 2H,
Ar–H8, Ar–H6), 6.81 (d, 1H, J = 9.1 Hz, Ar–H5). MS (EI)
= 292 (M+). Anal. CHN C15H20N2O4.
1
1411, 1327, 1098, 1054, 861, 703. H-NMR (DMSO-d6,
300 MHz): d (ppm) 1.56 (m, 4H, N–CH2–CH2–CH2–CH2–
CO), 1.83 (s, 3H, CH3COO–), 2.14 (t, 2H, J = 8.3 Hz,
N–CH2–CH2–CH2–CH2–CO), 2.73 (s, 3H, CH3–N,N-
DMF*), 2.89 (s, 3H, CH3–N,N-DMF*), 3.91 (m, 2H,
N–CH2–CH2–CH2–CH2–CO), 4.64 (s, 2H, O–CH2–CO),
7.22 (d, 1H, J = 9.0 Hz,Ar–H5), 7.48 (dd, 1H, J = 9.0, 2.3 Hz,
Ar–H6), 7.52 (d, 1H, J = 2.3 Hz, Ar–H8), 7.92–8.09 (dd, 4H,
J = 8.3 Hz, Ar–H), 10.47 (s, 1H, –NHCO–), signals for
amidine protons and the amide proton from N,N-DMF were
not observed. Compound precipitated as pale yellowish crys-
tals with one equivalent of N,N-DMF, which was character-
ized by NMR and CHN analysis. MS (FAB) = 411 (MH+-
free base). Anal. calcd. for C23H26N4O7 × 1.5 H2O × N,N-
DMF.
5.2.76. Ethyl 5-(7-{[4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
3-yl)benzoyl]amino}-2H-1,4-benzoxazine-3(4H)-one-4-yl)-
pentanoate (80)
Prepared according to the general procedure as in Section
5.2.1.8 from 79 (0.70 g, 2.39 mmol) and 59 (0.542 g,
2.64 mmol). The crude product was recrystallized from etha-
nol (40 ml) to give pale yellow crystals. m = 0.77 g (67%).
M.p. 225–229 °C. IR (KBr, cm–1): 3301, 2987, 1805, 1773,
1730, 1686, 1648, 1587, 1514, 1408, 1280, 1179, 1046, 940,
865, 815, 755, 665. 1H-NMR (DMSO-d6, 300 MHz):
d (ppm) 1.17 (t, 3H, J = 7.0 Hz, O–CH2–CH3), 1.58 (m, 4H,
N–CH2–CH2–CH2–CH2–CO), 2.34 (t, 2H, J = 6.8 Hz,
N–CH2–CH2–CH2–CH2–CO), 3.91 (m, 2H, N–CH2–CH2–
CH2–CH2–CO), 4.04 (q, 2H, J = 7.0 Hz, O–CH2–CH3), 4.65
(s, 2H, O–CH2–CO), 7.21 (d, 1H, J = 9.1 Hz, Ar–H5), 7.48
(dd, 1H, J = 9.1, 2.3 Hz, Ar–H6), 7.55 (d, 1H, J = 2.3 Hz,
5.3. Inhibition of ADP-induced platelet aggregation assay
[34]
Venous blood was collected from apparently healthy vol-
unteers who were using no medication, and centrifuged at
room temperature for 15 min at 1000 × g to give platelet-rich
plasma (PRP). The remaining blood was further centrifuged
for 10 min at 2000 × g to give platelet-poor plasma (PPP). In
PRP the number of platelets was adjusted to 250 25 ×
106/ml by dilution with PPP. Platelet aggregation was mea-

48
M. Anderluh et al. / European Journal of Medicinal Chemistry 40 (2005) 25–49
sured at 37 °C by light transmittance in an automatic blood
coagulation analyzer (Behring Coagulation Timer, Behring
Diagnostics GmbH). A 15 µl of test compounds at various
concentrations were added to 135 µl of PRP and after 5 min
aggregation was initiated by adding 15 µl ADP at a final
concentration of 20 µM. Results were expressed relative to
platelet aggregation with no test compound added. The con-
centration of the test compound which reduced platelet ag-
gregation by 50% (IC50) was determined from the dose–
response curve [35]. Final IC50 values were determined as
the average of determinations conducted on plasma samples
from at least two blood donors. The average standard error
for determinations was 20%. RGDS and tirofiban (Aggr-
astat®, Merck & Co., Inc.) were included as controls, with
IC50 260 52 µM and 0.0075 0.0011 µM, respectively (the
results are in accordance with those previously reported).
were fitted to the sigmoid model and IC50 values were calcu-
lated from the dose–response curve (OriginPro, OriginLab®,
Version 7.5).
Acknowledgments
The authors gratefully acknowledge the assistance of Dr.
Ralph Mazitschek and the personnel in the Institut für Orga-
nische Chemie at Universität Karlsruhe, Germany in per-
forming the in vitro assays. We thank Dr. Bogdan Kralj and
Dr. Dušan Žigon for MS analyses and Ms.Tatjana Stipanovic´
for microanalyses. We thank Dr. Roger Pain and prof. Dr.
Slavko Pecˇar for careful reading of the manuscript and many
useful suggestions.
5.4. Inhibition of in vitro binding of fibrinogen to isolated
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The binding affinities of the synthesized compounds to
integrins aIIbb3 and aVb3 were characterized by a solid-phase
competitive displacement assay. Human fibrinogen (100 mg)
was dissolved in aqueous NaCl (0.3 M, 5 ml) at 30 °C and
then further diluted with 0.1 M NaHCO3(aq) to a final concen-
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was incubated for 90 min at 30 °C and dialyzed for 3 h at RT
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5400 rpm and Tween 20 (0.005%) was added (stock solu-
tion). Human integrins (10 µl of aIIbb3 or 5 µl of aVb3) were
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Tween 20). The potential antagonists were serially diluted
with buffer and solutions added (50 µl/well) at the same time
as biotinylated fibrinogen (50 µl/well, 1:10 dilution of stock
solution in buffer 2) to each well. The plates were incubated
for 2 h at RT and then washed twice with buffer 3. In each
well, peroxidase-conjugated antibiotin goat antibody
(100 µl/well, 1:1000 dilution of purchased solution in buffer
3 + 0.1% of BSA) was added and incubated for another hour.
The microtiter plates were washed with buffer 3 three times.
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AG) multimode research reader. Positive controls received
no inhibitors while negative controls received no ligands.
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