Conformationally tailored N-[(2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)carbonyl]proline templates as molecular tools for the design of peptidomimetics. Design and synthesis of fibrinogen receptor antagonists.

Article abstract of DOI:10.1039/b400490f

The proline peptide bond was shown by 2D proton NMR studies to exist exclusively in the trans conformation in benzyl (2S)-1-[[(2S)-2-methyl-6-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl]-2-pyrrolidinecarboxylate [(S,S)-11], benzyl (2S)-1-[[(2S

Full text of DOI:10.1039/b400490f

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Conformationally tailored N-[(2-methyl-3-oxo-3,4-dihydro-2H-
1,4-benzoxazin-2-yl)carbonyl]proline templates as molecular
tools for the design of peptidomimetics. Design and synthesis
of fibrinogen receptor antagonists†
a
a
a
b
a
ˇ
Petra Stefanicˇ, Zvone Simoncˇicˇ, Matej Breznik, Janez Plavec, Marko Anderluh,
Elisabeth Addicks,c Athanassios Giannisc and Danijel Kikelj*a
a University of Ljubljana, Faculty of Pharmacy, Asˇkercˇeva 7, 1000 Ljubljana, Slovenia.
E-mail: danijel.kikelj@ffa.uni-lj.si; Fax: ϩ386 1 4258 031; Tel: ϩ386 1 4769 561
b National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
E-mail: janez.plavec@ki.si; Fax: ϩ386 1 4760 300; Tel: ϩ386 1 4760 200
c University of Leipzig, Institute for Organic Chemistry, Johannisallee 29, D-04103, Leipzig,
Germany. E-mail: giannis@chemie.uni-leipzig.de; Fax: ϩ49 341 97 36 599;
Tel: ϩ49 341 9736 527
Received 13th January 2004, Accepted 22nd March 2004
First published as an Advance Article on the web 22nd April 2004
The proline peptide bond was shown by 2D proton NMR studies to exist exclusively in the trans conformation
in benzyl (2S)-1-{[(2S)-2-methyl-6-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl}-2-pyrrolidine-
carboxylate [(S,S)-11], benzyl (2S)-1-{[(2S)-2-methyl-7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl}-
2-pyrrolidinecarboxylate [(S,S)-9], and in the corresponding 6-amino and 7-amino carboxylic acids (S,S)-3 and
(S,S)-4. On the other hand, the diastereomers (R,S)-11 and (R,S)-9 containing an (R) [2-methyl-6/7-nitro-3-oxo-3,4-
dihydro-2H-1,4-benzoxazin-2-yl]carbonyl moiety, and the diastereoisomers (R,S)-3 and (R,S)-4 incorporating an (R)
[6/7-amino-2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl moiety were found to exist as equilibria of
trans (63–83%) and cis (17–37%) isomers. These conformationally defined templates were applied in the construction
of RGD mimetics possessing antagonistic activity at the platelet fibrinogen receptor.
that the acyl moiety derived from (S)-2,6-dimethyl-3-oxo-
Introduction
3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid [(S)-1] in
Bioactive peptides bind to biological macromolecules in specific
acyl-Pro molecules influences the isomerization of the proline
conformations. The exploration of a binding conformation
peptide bond, constraining the ω dihedral angle to the trans
of inherently flexible bioactive peptides is a crucial step in
orientation. For this reason, we considered N-[(2-methyl-3-oxo-
attempts to obtain potent and selective peptidomimetic
therapeutic agents. Restriction of the degrees of conform-
ational freedom of native peptides by designing conform-
ationally constrained analogues which imitate the bioactive
3,4-dihydro-2H-1,4-benzoxazin-2-yl)-2-carbonyl]-(S)-proline
templates as promising, conformationally defined, chiral
peptidomimetic building blocks8 that could be applied, inter
alia, for the design of fibrinogen receptor antagonists.
The fibrinogen receptor is a member of the integrin family of
proteins which are heterodimeric transmembrane glycoproteins.
conformation of the peptide is an important method for the
development of peptidomimetics.1–4
In the field of peptidomimetic drug design a large number of
approaches that reduce the flexibility, and thus the number
of conformations accessible to a peptide molecule, have been
Integrin receptors play an important role in, for example,
platelet aggregation,9–11 angiogenesis,10,12 cell growth and
differentiation,10,13 tumour promotion,9,12,13 cell adhesion,9,10,14
described.2,4 Among them, proline or proline mimetics with
bone resorption9,10,15,16 and immunological processes.10,17
different ring sizes have frequently been introduced into the
Antagonists of the fibrinogen receptor, possessing anti-
sequence of bioactive peptides in order to induce local con-
aggregatory activity, are generally mimetics of the Arg-Gly-Asp
straints.4–7 Proline restricts the local conformational freedom
by locking the Φ angle at about Ϫ60Њ due to restricted rotation
(RGD) sequence in which the distance between the anionic and
cationic centres, which ranges from 13 to 16 Å, is crucial for
about the Nα–Cα bond, included in the pyrrolidine ring.
binding to the receptor.18–20
Additionally, proline exhibits hindered rotation around the
We hypothesized that, in a manner similar to (2S)-1-[(2S)-
2,6-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)carb-
onyl]-2-pyrrolidinecarboxylic acid [(S,S)-2], which exhibits an
exclusively trans proline amide bond, and (2S)-1-[(2R)-2,6-
dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)-carbonyl]-
2-pyrrolidinecarboxylic acid [(R,S)-2], which assumes an
equilibrium of 72% trans and 28% cis conformers in
dimethylsulfoxide solution, 1-[(6-amino-2-methyl-3-oxo-3,4-
Cα–C(O) bond due to the steric interactions of the pyrrolidine
ring and the carbonyl group. As a result of these features,
proline is able to affect the conformation of the preceding
residue. The most interesting conformational characteristic of
proline in peptides is its potential for cis/trans isomerism of the
X-Pro peptide bond, in contrast to other amino acids which
form predominantly trans peptide bonds.2,4
Recently, we have shown that the cis/trans ratio of the proline
dihydro-2H-1,4-benzoxazin-2-yl)carbonyl]-2-pyrrolidinecarb-
peptide bond can be strongly influenced by the chirality of the
oxylic acid [(S,S)-3] and its 7-amino isomer (S,S)-4 also should
acyl residue preceding proline.8 Moreover, we demonstrated
possess trans proline bonds, while simultaneously allowing
easy functionalization at the aromatic ring (Fig. 1). Based on
this assumption, conformationally defined diastereomers of
templates 3 and 4 emerged as promising peptidomimetic
† Electronic supplementary information (ESI) available: experimental
details. See http://www.rsc.org/suppdata/ob/b4/b400490f/
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 1 – 1 5 1 7
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Fig. 1 The evolution of peptidomimetic templates (S,S)-3, (R,S)-3, (S,S)-4 and (R,S)-4.
Fig. 2 Synthesis of peptidomimetic templates (S,S)-3, (R,S)-3, (S,S)-4, (R,S)-4, (S,S)-13, (R,S)-13, (S,S)-14 and (R,S)-14.
building blocks for the construction of fibrinogen receptor
antagonists. This would be achieved by coupling them with a
benzamidine fragment which in target inhibitors would provide
a basic moiety at a distance of 13 to 16 Å from the proline
carboxylic group.
In this paper we report the synthesis and NMR conform-
ational analysis of templates 3 and 4, their elaboration to
conformationally tailored fibrinogen receptor antagonists
15–18 and biological evaluation of target compounds support-
ing the rationale of the applied strategy.
pyrrolidinecarboxylates (S,S)-3, (R,S)-3, (S,S)-4, (R,S)-4 and
ethyl (2S)-1-[(2R/S)-(6/7-amino-2-methyl-3-oxo-3,4-dihydro-
2H-1,4-benzoxazin-2-yl)carbonyl]-2-pyrrolidinecarboxylates
(S,S)-13, (R,S)-13, (S,S)-14 and (R,S)-14, respectively (Fig. 2).
The pure diastereomers of amines 13 and 14 were coupled with
4-cyanobenzoyl chloride, whereupon the cyano group of the
resulting intermediates was transformed to an amidine
functionality under the conditions of the Pinner reaction22 to
give the esters (S,S)-15, (R,S)-15, (S,S)-17 and (R,S)-17. After
alkaline hydrolysis, (S,S)-16, (R,S)-16, (S,S)-18 and (R,S)-18
were obtained as potential fibrinogen receptor antagonists
(Fig. 3).
Results and discussion
NMR studies. The conformational study of eight diastereo-
merically pure derivatives of N-[(2-methyl-3-oxo-3,4-dihydro-
2H-1,4-benzoxazin-2-yl)carbonyl]-(S)-proline, i.e. benzyl esters
of 6-nitro derivative [(S,S)-11, (R,S)-11], benzyl esters of 7-
nitro derivative [(S,S)-9, (R,S)-9], and free carboxylates of
6-amino- and 7-amino derivatives [(S,S)-3, (R,S)-3, (S,S)-4,
(R,S)-4], was performed by 2D NMR methods in dimethyl-
sulfoxide. First, the proton NMR spectra were assigned using
DFQ–COSY, HSQC and HMBC experiments in combination
with 13C broad band decoupled and DEPT spectra.
In the proton NMR spectra, the S,S diastereomers of 3,4,9
and 11 exhibited only one set of signals and, in the NOESY
spectra, strong NOEs between δCH2Pro/2-Me and δCH2Pro/
8-H, both evidencing the existence of only the trans conform-
ation of the proline amide bond. For diastereomers of 6- and
7-amino compounds 3 and 4, additional NOEs were observed
Chemistry
Ethyl 2-methyl-7-nitro-3-oxo-3,4-dihydro-2H-benzoxazine-2-
carboxylate (7a) and ethyl 2-methyl-6-nitro-3-oxo-3,4-dihydro-
2H-benzoxazine-2-carboxylate (8a) were synthesized from
2-aminophenols 5 and 6 according to procedures described
previously.21 Alkaline hydrolysis was then performed to
produce the corresponding carboxylic acids 7b and 8b21 which
were subsequently coupled with (S)-proline benzyl ester, using
DPPA as a coupling agent to give 9 and 11, or with (S)-proline
ethyl ester, using EDC and HOBT to give compounds 10 and 12
as diastereomeric mixtures. Column chromatography on silica
gel was used to separate the S,S and R,S diastereomers of 9–12.
These were catalytically hydrogenated using palladium on
activated charcoal to produce (2S)-1-[(2R/S)-(6/7-amino-2-
methyl-3-oxo-3,4-dihydro-2H-1,4-benxazin-2-yl)carbonyl]-2-
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Table 1 The ratio of trans and cis isomers in (R,S)-3, (R,S)-4, (R,S)-9, (R,S)-11, (R,S)-15, (R,S)-16, (R,S)-17 and (R,S)-18a
(R,S)-3
(R,S)-4
(R,S)-9
(R,S)-11
(R,S)-15
(R,S)-16
(R,S)-17
(R,S)-18
Average ratio trans/cis
% trans
% cis
100 : 58
63
37
100 : 54
65
35
100 : 21
83
17
100 : 26
79
21
100 : 48
68
32
100 : 48
68
32
100 : 40
71
29
100 : 35
74
26
1
a Ratios were determined by integration of two sets of well resolved signals of trans and cis isomers in H-NMR spectra in dimethylsulfoxide-d6
solution.
Fig. 3 The synthesis of diastereomers of potential fibrinogen receptor antagonists 15,16,17 and 18.
between the pyrrolidine protons and the protons of the
benzoxazinone ring, indicating that a limited rotation about the
C2–C(O)Pro bond and/or puckering of the pyrrolidine ring
could occur due to long range electrostatic interaction between
the amino and carboxylate groups.
The 2R,2S diastereomers of 3,4,9 and 11 exhibited in their
1H-NMR spectra two sets of signals. The major one exhibited
in its NOE spectrum NOE connectivities between δCH2Pro/
2-Me and δCH2Pro/8-H, attributed to the trans-conformer,
whereas the minor set of signals, based on analogy with the
previous results,8 was assigned to the cis-conformer. The
proportions of the trans- and cis-conformers, deduced as an
average integral ratio of major and minor signals of 2-Me, NH,
α–H, δ–H and OCH2, were found to be 63–83% trans and 17–
37% cis (Table 1). Interestingly, the ratio of the cis proline
amide bond was higher in compounds possessing a free proline
carboxylic group and an aromatic amino group, suggesting that
a weak electrostatic interaction between the carboxylic and
amino group might be responsible for a distortion of the ω
dihedral angle leading to a higher proportion of the cis
conformers.
Fig. 4 Proton numbering used in conformational analysis and in
assigning NMR spectra.
iterative optimization procedure the geometry (expressed by the
phase angle of pseudorotation, P, and the maximum puckering
amplitude, Ψm) of each of the conformers was freely optimized
and the populations of North and South pseudorotamers
were varied to obtain the best fit between experimental and
calculated coupling constants. Agreement between experi-
mental and back-calculated coupling constants was monitored
by calculation of the root-mean-square deviation, which was
found to be below 0.5 Hz. This conformational analysis led to
the conclusion that the pyrrolidine rings are involved in a
conformational equilibrium between a form close to the C2Ј-
exo (P ≈ Ϫ20Њ, Ψm ≈ 65Њ) canonical form and a conformation
between C4Ј-endo and C4Ј-endo-Nδ-exo twist canonical forms
Pyrrolidine ring puckering in trans-(S,S)-3, trans-(S,S)-4,
trans-(S,S)-9, cis/trans-(R,S)-9 and trans-(S,S)-11 was studied
3
by analysis of vicinal JH,H coupling constants, obtained by
3
simulation of individual spectra. The resulting JH,H coupling
constants (presented in Table 1 in the ESI†) (Fig. 4), have been
interpreted in terms of a two-state North ⇔ South pseudo-
rotational equilibrium, using the PSEUROT program.23 In the
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Table 2 IC50 values of diastereomers of compounds 15–18 and of
RGDS as a reference compound for inhibition of fibrinogen binding
to the fibrinogen receptor
suggesting that other interactions, such as hydrogen bonding
and hydrophobic interactions, could contribute to the binding
of diastereomers of 15 and 17 to the fibrinogen receptor.
In conclusion, we have demonstrated that N-[(3,4-dihydro-2-
methyl-3-oxo-2H-1,4-benzoxazin-2-yl)-2-carbonyl]-(S)-proline
templates, in which the cis/trans ratio of the proline peptide
bond can be tailored by the chirality of the acyl residue, can be
used as conformationally defined scaffolds for constructing
fibrinogen receptor antagonists. The conformationally defined
trans-(S,S)-16 and trans-(S,S)-18, which possess a moderate
antagonistic potency at the fibrinogen receptor, verify the
concept of chirality-induced tailoring of the proline peptide
bond conformation as a promising tool in the design of
peptidomimetics.
Compound
R5
Distance/nm
ω/degrees
IC50/µM
RGDS
1.31 0.06
(S,S)-15
(S,S)-16
(R,S)-15
(R,S)-16
Et
H
Et
H
6.79 1.58
0.51 0.43
4.75 0.45
4.10 0.83
1.580
177.3
1.110 (trans)
1.104 (cis)
179.5
5.0
(S,S)-17
(S,S)-18
(R,S)-17
(R,S)-18
Et
H
Et
H
6.48 3.10
6.89 1.43
13.83 2.98
104.22 42.6
1.694
Ϫ174.8
1.208 (trans)
1.475 (cis)
174.2
Ϫ16.4
Experimental
Chemicals from Aldrich Chemical Co., Fluka, Merck, Kemika
and Jannsen were used without further purification. Anhydrous
dichloromethane, N,N-dimethylformamide and triethylamine
were prepared according to standard procedures.25 Analytical
TLC was performed on Merck silica gel 60 F254 plates (0.25
mm) and components were visualized under ultraviolet light.
Column chromatography was carried out on silica gel 60
(particle size 240–400 mesh). Melting points were determined
on a Reichert hot stage microscope and are uncorrected.
1H-NMR spectra were recorded on a Bruker AVANCE DPX300
spectrometer in CDCl3 or DMSO-d6 solution, with TMS as the
internal standard. NMR spectra (DFQ-COSY, HSQC, HMBC,
13C broad band decoupled and DEPT spectra) for conform-
ational analysis were performed on a Varian Unity Inova 600
and a Varian Unity Inova 300, using DMSO-d6 solution with
TMS as the internal standard at 302 K and 298 K. J values are
given in Hz. Conformational analysis of the pyrrolidine moiety
was performed by the computer program PSEUROT26 with the
use of λ electronegativities for the substituents along H–C–C–
H fragments and the six parameter set for the generalized Kar-
plus–type equation.27 The following λ electronegativity values
were used: 0.0 for H, 0.6 for C1Ј, 0.7 for C3Ј, 1.19 for N, 0.42 for
COO and 0.75 for C4Ј. The analysis of 3JHH coupling constants
consists of three standard translation steps. The first step
translates experimental proton–proton coupling constants to
proton–proton torsion angles and is covered by the generalized
Karplus–Altona equation. The second step is the translation
of proton–proton torsion angles into the corresponding
endocyclic torsion angles, and is formulated with the set of
linear equations ΦHH = Aνj ϩ B. ΦHH is the torsion angle
between two vicinal protons and νj is the corresponding
endocyclic torsion angle. The third step of translating endo-
cyclic torsion angles into the pseudorotational parameters is
described by a simple cosine function νj = Ψm cos[P ϩ (j–2)
4π/5], where P is the phase angle of pseudorotation and Ψm is
the maximum puckering amplitude. In the following optimiz-
ation procedure the geometries and populations of North
and South pseudorotamers were varied to obtain the best fit
between experimental and calculated coupling constants. IR
spectra were obtained using a Perkin-Elmer 1600 FT-IR
spectrometer. Microanalyses were performed on a Perkin-
Elmer C, H, N analyzer 240 C. HPLC analyses were performed
on Agilent Technologies HP 1100 instrument with G1365B
UV-VIS detector using a Eurospher C18 column (4.6 × 250
mm). Eluent consisted of 0.1% trifluoroacetic acid in water
(40%) and methanol (60%). Mass spectra were obtained using a
VG-Analytical Autospec Q mass spectrometer. All reported
yields are yields of purified products.
(P ≈ 245Њ, Ψm ≈ 51Њ). The population of the above two con-
formers of the pyrrolidine rings was found to be approximately
equal at 29Њ C in DMSO-d6.
Biological activity. The biological activity of pure dia-
stereomers of the target compounds 15–18 was evaluated in
the integrin receptor assay.24 The extent of competitive binding
to the fibrinogen and vitronectin receptors in the presence of
chemically derivatized natural agonist (biotinylated fibrinogen)
was determined from the rate of chemiluminescence produced
by horse-radish peroxidase attached to the biotin directed anti-
body. Compounds 15–18 inhibiting the binding of fibrinogen
in the low micromolar range were selective and moderately
potent fibrinogen receptor antagonists, when compared to
therapeutically used tirofiban and eptifibatide. The IC50 values
of diastereomers of compounds 15–18, representing the con-
centration of an antagonist causing a 50% inhibition of binding
of natural agonist fibrinogen, are listed in Table 2. In the
preliminary screening, the estimated IC50 values for the binding
of 15–18 to the vitronectin receptor were higher than 100 µM
and are not presented here.
General discussion. Peptidomimetic compounds trans-(S,S)-
15, trans-(S,S)-16, trans-(S,S)-17 and trans-(S,S)-18 were
designed as conformationally tailored fibrinogen receptor ant-
agonists, their well defined conformation being due to a defined
absolute configuration at C2 and the due existence of a solely
trans proline amide bond. Molecular modelling of compounds
trans-(S,S)-15, trans-(S,S)-16, trans-(S,S)-17 and trans-(S,S)-
18 revealed conformationally highly restricted structures with
a proline ring folded almost perpendicularly over the benz-
oxazinone moiety and the COOR group located at the external
side of a twisted structure (Fig. 1 in ESI). The distances
between C-atoms of the C(NH)NH2 and the proline COOR (R
= H, Et) groups were found to be 1.580 nm for trans-(S,S)-15 or
trans-(S,S)-16 and 1.694 nm for trans-(S,S)-17 or trans-(S,S)-
18. The (R,S)-diastereomers of compounds 15–18 exist as
mixtures of (mainly) trans- and cis isomers. As a consequence
of the different absolute configuration at C2, in trans-(R,S) 15–
18 the distance between the C(NH)NH2 and the proline COOR
(R = H, Et) groups is shorter than in the corresponding trans-
(S,S) isomers (1.110 nm in trans-(R,S)-16 vs. 1.580 nm in
trans-(S,S)-16, 1.208 nm in trans-(R,S)-18 vs. 1.694 nm
in trans-(S,S)-18). A pharmacophore model for antagonistic
activity at the fibrinogen receptor, demands for an antagonist
a slightly cup-shaped conformation of the scaffold with a
distance of 1.3–1.8 nm between the anionic and cationic
moieties.9–11 Thus, the higher potency of trans-(S,S)-16 vs. cis/
trans-(R,S)-16 and trans-(S,S)-18 vs. cis/trans-(R,S)-18 can be
explained on the basis of different distances between the
C(NH)NH2 and COOH groups. The relationship between
potency and distance of the C(NH)NH2 and COOEt groups in
isomers of esters 15 and 17 is, however, not straightforward,
*
*
General procedure for preparing ethyl 2-methyl-(6/7)-nitro-3-
oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylates 7a and 8a
A suspension of potassium fluoride (2.81 g, 48.4 mmol) and
diethyl 2-bromo-2-methyl-malonate (4.92 g, 19.4 mmol) in
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N,N-dimethylformamide (40 mL) was stirred for 15 min at
room temperature. 2-Amino-4/5-nitrophenol (3.00 g, 19.4
mmol) was then added and the reaction mixture heated at 60 ЊC
for 6 h. When the cooled reaction mixture was poured into
ice-cold water (50 g), a precipitate formed, which was filtered
off, washed with water and recrystallized from ethanol (90 mL).
1041, 754 and 697; δH(300 MHz; CDCl3; Me4Si) 1.66 (3H, s,
CH3), 1.73–1.83 (1H, m, J1Ј,2Љ 8.8, J2Ј,2Љ 12.7, J2Љ,3Ј 7.5, J2Љ,3Љ 7.4,
H2Љ), 1.85–1.96 (2H, m, J2Ј,3Ј 6.7, J2Ј,3Љ 6.3, J3Ј,3Љ 12.2, J3Ј,4Ј 7.0, J3Ј,4Љ
6.9, J3Љ,4Ј 6.4, J3Љ,4Љ 6.4, H3Ј, H3Љ), 2.06–2.13 (1H, m, J1Ј,2Ј 5.0, H2Ј),
3.74–3.79 (2H, m, J4Ј,4Љ 10.7, H4Ј, H4Љ), 4.29 (1H, dd, J1Ј,2Ј 5.0,
J1Ј,2Љ 8.8, H1Ј), 4.67 and 4.96 (each 1H, d, J 12.6, CH2Ph), 7.10
(1H, d, J5,6 8.7, H5), 7.33–7.39 (5H, m, Ph), 7.92 (1H, d, J6,8 2.4,
H8) and 7.96 (1H, dd, J6,8 2.4, J5,6 8.7, H6); m/z (EI): 439 (Mϩ,
63%) and 91 (100).
Ethyl 2-methyl-7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-
2-carboxylate 7a
The procedure described yielded 7a (4.05 g, 75%) as violet
crystals, mp 190–195 ЊC (lit.,21 194–195 ЊC); νmax(KBr)/cmϪ1
3087, 2945, 1748, 1695, 1605, 1540, 1340, 1237, 1123, 1011, 825,
743 and 550; δH(300 MHz; CDCl3; Me4Si) 1.21 (3H, t, J 7.2,
CH2CH3), 1.93 (3H, s, CH3), 4.13–4.29 (2H, m, CH2CH3), 6.93
(1H, d, J5,6 8.7, H5), 7.95 (1H, dd, J6,8 2.3, J5,6 8.7, H6) and 7.99
(1H, d, J6,8 2.3, H8); m/z (EI): 280 (Mϩ, 37%) and 207 (100).
General procedure for preparing ethyl (2S )-1-{[(2R/S )-2-methyl-
(6/7)-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carb-
onyl}-2-pyrrolidine-carboxylates (S,S )-10, (R,S )-10, (S,S )-12
and (R,S )-12
2-Methyl-(6/7)-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-
carboxylic acid 7b or 8b (2.52 g, 10.0 mmol) and ethyl (2S)-2-
pyrrolidinecarboxylate hydrochloride (1.80 g, 10.0 mmol) were
dissolved in anhydrous N,N-dimethylformamide (20 mL).
1-Hydroxybenzotriazole (1.62 g, 12.0 mmol) was added and the
pH adjusted to 8 with N-methylmorpholine before adding EDC
(2.59 g, 13.5 mmol). The reaction mixture was stirred overnight
at room temperature. The solvent was evaporated under
reduced pressure and the oily residue dissolved in dichloro-
methane (150 mL), washed with 10% citric acid (2 × 40 mL)
and saturated sodium hydrogen carbonate solution (2 × 40
mL). The organic solution was dried over sodium sulfate and
the solvent evaporated under reduced pressure to yield an oily
product. The separation of the diastereomers was achieved by
column chromatography on silica gel with diethyl ether as an
eluant.
General procedure for preparing 2-methyl-(6/7)-nitro-3-oxo-3,4-
dihydro-2H-1,4-benzoxazine-2-carboxylic acids 7b and 8b
Lithium hydroxide (0.389 g, 16.3 mmol) was added to a
solution of ethyl 2-methyl-(6/7)-nitro-3-oxo-3,4-dihydro-2H-
1,4-benzoxazine-2-carboxylate 7a or 7b (3.5 g, 12.5 mmol) in
a mixture of methanol and water (50 mL, 50% v/v) and the
mixture was stirred for 12 h. The methanol was evaporated
under reduced pressure and the aqueous phase washed with
diethyl ether (3 × 30 mL), acidified with 1 M HCl to pH 2–3 and
extracted with ethyl acetate (5 × 30 mL). The combined organic
fractions were dried with sodium sulfate and the solvent
evaporated under reduced pressure to produce a crystalline
product.
Ethyl (2S )-1-{[(2S )-2-methyl-7-nitro-3-oxo-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]carbonyl}-2-pyrrolidinecarboxylate (S,S )-10
2-Methyl-7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-
carboxylic acid 7b
The procedure described yielded (S,S)-10 (0.302 g, 8%) as pale
yellow crystals, mp 137–139 ЊC; [α]2D0 Ϫ37.9 (c 0.100 in MeOH);
Found: C, 53.0; H, 5.5; N, 10.55. C17H19N3O7 × 1/2H2O
requires: C, 52.85; H, 5.2; N, 10.9%; νmax (KBr)/cmϪ1 3088,
2944, 1750, 1696, 1605, 1537, 1340, 1237, 1124, 878, 826 and
744; δH(300 MHz; DMSO-d6; Me4Si) 1.16 (3H, t, J 7.0,
CH2CH3), 1.68–1.78 (1H, m, H2Љ), 1.71 (3H, s, CH3), 1.81–1.99
(2H, m, H3Ј, H3Љ), 2.03–2.15 (1H, m, H2Ј), 3.74–3.82 (2H, m, H4Ј,
H4Љ), 4.01–4.12 (2H, m, CH2CH3), 4.19 (1H, dd, J1 8.7, J2 4.9,
H1Ј), 7.11 (1H, d, J5,6 9.4, H5), 7.96–8.00 (2H, m, H6, H8) and
11.54 (1H, s, NH ); m/z (FAB): 378 (MHϩ,100%), 362 (10), 304
(30), 207 (22), 142 (45) and 70 (51).
The procedure described yielded 7b (3.06 g, 97%) as violet
crystals, mp 180–184 ЊC (lit.,21 183–185 ЊC); νmax (KBr)/cmϪ1
3494, 3098, 1698, 1606, 1538, 1342, 1250, 1136, 832 and 745;
δH(300 MHz; CDCl3; Me4Si) 1.94 (3H, s, CH3), 6.91 (1H, d, J5,6
8.7, H5), 7.92 (1H, dd, J6,8 2.3, J5,6 8.7, H6) and 7.99 (1H, d, J6,8
2.3, H8); m/z (EI): 252 (Mϩ, 53%) and 208 (100).
General procedure for preparing benzyl (2S )-1-{[(2R/S )-2-
methyl-(6/7)-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-
yl]carbonyl}-2-pyrrolidinecarboxylates (S,S )-9, (R,S )-9,
(S,S )-11 and (R,S )-11
2-Methyl-(6/7)-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-
carboxylic acid 7b or 8b (0.86 g, 3.40 mmol) and benzyl (2S)-2-
pyrrolidinecarboxylate hydrochloride (0.83 g, 3.40 mmol) were
dissolved in anhydrous N,N-dimethylformamide (20 mL) and
DPPA (0.92 mL, 4.30 mmol) and triethylamine (1.2 mL, 8.60
mmol) were added at 0 ЊC. The reaction mixture was stirred first
for 1 h at 0 ЊC and then for 60 h at room temperature. Ethyl
acetate (50 mL) was then added and the reaction mixture
washed with 10% citric acid (5 × 20 mL), water (3 × 20 mL), a
saturated solution of sodium hydrogen carbonate (3 × 20 mL),
water (3 × 20 mL) and a saturated solution of sodium chloride
(2 × 20 mL). The organic solution was dried over sodium
sulfate and the solvent evaporated under reduced pressure to
give a crystalline product. Separation of the diastereomers was
achieved by column chromatography on silica gel with diethyl
ether as eluant.
General procedure for preparing (2S )-1-{[(2R/S )-(6/7)-amino-2-
methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl}-2-
pyrrolidinecarboxylic acids (S,S )-3, (R,S )-3, (S,S )-4 and
(R,S )-4
The solution of benzyl (2S)-1-{[(2R/S)-2-methyl-(6/7)-nitro-
3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]-carbonyl}-2-pyrro-
lidinecarboxylate (S,S)-9, (R,S)-9, (S,S)-11 or (R,S)-11
(0.15 g, 0.341 mmol) in anhydrous methanol was catalytically
hydrogenated in the presence of palladium on activated
charcoal (10% by weight) by hydrogen bubbling into the
reaction mixture for 2 hours followed by stirring the reaction
mixture under a hydrogen atmosphere for 12 hours. The
catalyst was filtered off and the solvent evaporated under
reduced pressure to give a crystalline product.
(2S )-1-{[(2S )-7-Amino-2-methyl-3-oxo-3,4-dihydro-2H-1,4-
benzoxazin-2-yl]carbonyl}-2-pyrrolidinecarboxylic acid (S,S )-4
Benzyl (2S )-1-{[(2S )-2-methyl-7-nitro-3-oxo-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]carbonyl}-2-pyrrolidinecarboxylate (S,S )-9
The procedure described yielded (S,S)-4 (0.079 g, 73%) as pink
crystals, mp 171–173 ЊC; [α]2D0 Ϫ52.3 (c 0.435 in MeOH);
νmax(KBr)/cmϪ1 3446, 1684, 1652, 1559, 1519, 1456, 1260
and 1176; δH(300 MHz; DMSO-d6; Me4Si) 1.54 (3H, s, CH3),
1.71–1.78 (1H, m, J1Ј,2Љ 8.7, J2Ј,2Љ Ϫ12.7, J2Љ,3Ј 7.6, J2Љ,3Љ 7.0, H2Љ),
The procedure described yielded (S,S)-9 (0.30 g, 20%) as yellow
crystals, mp 89–95 ЊC; [α]2D0 Ϫ43.8 (c 0.513 in MeOH); Found:
C, 60.25; H, 4.7; N, 9.5. C22H21N3O7 requires C, 60.1; H, 4.8, N,
9.6%; νmax(KBr)/cmϪ1 3240, 1726, 1640, 1531, 1448, 1325, 1156,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 1 – 1 5 1 7
1515

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1.79–1.98 (2H, m, J2Ј,3Ј 6.7, J2Ј,3Љ 6.2, J3Ј,3Љ Ϫ12.4, J3Ј,4Ј 6.6, J3Ј,4Љ
6.3, J3Љ,4Ј 6.8, J3Љ,4Љ 7.0, H3Ј, H3Љ), 2.01–2.10 (1H, m, J1Ј,2Ј 4.8, H2Ј),
2.62 (2H, s, NH2), 3.61–3.74 (2H, m, J4Ј,4Љ Ϫ10.4, H4Ј, H4Љ), 4.14
(1H, dd, J1Ј,2Ј 4.8, J1Ј,2Љ 8.7, H1Ј), 6.21 (1H, dd, J6,8 2.2, J5,6 8.3,
H6), 6.26 (1H, d, J6,8 2.2, H8), 6.58 (1H, d, J5,6 8.3, H5) and 10.40
(1H, s, NH ); m/z (EI): 319 (Mϩ, 35%) and 178 (100). HR-MS
Found: 319.117300, C15H17N3O5 requires: 319.116821.
thin layer chromatography using n-BuOH/H2O/AcOH (5 : 4 : 1)
as eluent.
Ethyl (2S )-1-({(2S )-7-[(4-cyanobenzoyl)amino]-2-methyl-3-oxo-
3,4-dihydro-2H-1,4-benzoxazin-2-yl}-carbonyl)-2-pyrrolidine-
carboxylate
The procedure described yielded 0.943 g (99%) of yellow
crystals, mp 120–124 ЊC; [α]2D0 Ϫ88.5 (c 0.100 in MeOH); Found:
C, 63.2; H, 5.2; N, 11.5, C25H24N4O6 requires: C, 63.0; H, 5.1; N,
11.7%; νmax(KBr)/cmϪ1 3280, 2981, 2231, 1700, 1517, 1374,
1178, 1019, 856 and 760; δH(300 MHz; DMSO-d6; Me4Si) 1.18
(t, 3H, J 7.2, CH2CH3), 1.63 (s, 3H, CH3), 1.72–1.79 (m, 1H,
H2Љ), 1.83–1.98 (m, 2H, H3Ј, H3Љ), 2.07–2.14 (m, 1H, H2Ј), 3.71–
3.82 (m, 2H, H4Ј, H4Љ), 4.02–4.12 (m, 2H, CH2CH3), 4.21 (dd,
1H, J1Ј,2Ј 4.7, J1Ј,2Љ 8.9, H1Ј), 6.90 (d, 1H, J5,6 8.5, H5), 7.39 (dd,
1H, J5,6 8.5, J6,8 2.3, H6), 7.59 (d, 1H, J6,8 2.3, H8), 8.02 (d, 2H,
J 8.7, 4-CN-Ar-H), 8.09 (d, 2H, J 8.7, 4-CN-Ar-H), 10.44 (s,
1H, NH ) and 10.89 (s, 1H, 4-CN-ArCONH-); m/z (FAB): 477
(MHϩ, 100%), 403 (14), 306 (50), 154 (53), 142 (63), 71 (61) and
57 (87).
General procedure for preparing ethyl (2S )-1-{[(2R/S )-(6/7)-
amino-2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]-
carbonyl}-2-pyrrolidine-carboxylates (S,S )-13, (R,S )-13, (S,S )-
14 and (R,S )-14
The solution of ethyl (2S)-1-{[(2R/S)-2-methyl-(6/7)-nitro-3-
oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl}-2-pyrrol-
idinecarboxylate (S,S)-10, (R,S)-10, (S,S)-12 or (R,S)-12
(0.377 g, 1.00 mmol) in anhydrous ethanol (40 mL) was
catalytically hydrogenated in the presence of palladium on
activated charcoal (10% by weight) by stirring the reaction
mixture under hydrogen atmosphere overnight. The catalyst
was filtered off and the solvent evaporated under reduced
pressure to give a crystalline product.
Ethyl (2S )-1-{[(2S )-7-({4-[amino(imino)methyl]benzoyl}amino)-
2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl}-
2-pyrrolidinecarboxylate acetate (S,S )-15
Ethyl (2S )-1-{[(2S )-7-amino-2-methyl-3-oxo-3,4-dihydro-2H-
1,4-benzoxazin-2-yl]carbonyl}-2-pyrrolidinecarboxylate (S,S )-
13
The procedure described yielded (S,S)-15 (0.238 g, 43%) as
yellow crystals, mp 195–200 ЊC; [α]2D0 Ϫ91.6 (c 0.100 in MeOH);
Found: C, 56.5; H, 5.9; N, 12.1, C27H31N5O8 × H2O requires: C,
56.7; H, 5.8; N, 12.3%; νmax(KBr)/cmϪ1 3416, 1646, 1558, 1519,
1416, 1180, 1019 and 654; δH(300 MHz; DMSO-d6; Me4Si) 1.18
(t, 3H, J 7.2, CH2CH3), 1.63 (s, 3H, CH3), 1.79 (s, 3H, CH3-
COOH), 1.70–1.98 (m, 3H, H2Љ, H3Ј, H3Љ), 2.05–2.15 (m, 1H,
H2Ј), 3.69–3.82 (m, 2H, H4Ј, H4Љ), 4.03–4.15 (m, 2H, CH2CH3),
4.21 (dd, 1H, J1Ј,2Ј 4.9, J1Ј,2Љ 8.7, H1Ј), 6.92 (d, 1H, J5,6 8.4, H5),
7.43 (dd, 1H, J5,6 8.4, J6,8 2.2, H6), 7.63 (d, 1H, J6,8 2.2, H8), 7.93
(d, 2H, J 8.3, 4–CN–Ar–H), 8.11 (d, 2H, J 8.3, 4–CN–Ar–H),
and 10.52 (s, 1H, NH ); [C(NH)NH3ϩ gives together with H2O a
broad signal at 3.0–4.2 ppm; a signal of 4-CN-ArCONH- was
not detected]; m/z (FAB): 494 (MHϩ, 50%), 413 (7), 329 (19),
176 (84), 154 (76), 136 (68), 71 (81) and 55 (100).
The procedure described yielded (S,S)-13 (0.337 g, 97%) as
white crystals, mp 114–117 ЊC; [α]2D0 Ϫ55.8 (c 0.100 in MeOH);
Found: C, 58.5; H, 6.3; N, 11.8. C17H21N3O5 reqiures: C, 58.8;
H, 6.1; N, 12.1%; νmax(KBr)/cmϪ1 3362, 2982, 1732, 1687, 1635,
1520, 1375, 1180, 1023, 808 and 619; δH(300 MHz; DMSO-d6;
Me4Si) 1.18 (t, 3H, J 7.2, CH2CH3), 1.54 (s, 3H, CH3), 1.72–
1.94 (m, 3H, H2Љ, H3Ј, H3Љ), 2.05–2.14 (m, 1H, H2Ј), 3.64–3.77 (m,
2H, H4Ј, H4Љ), 4.04–4.11 (m, 2H, CH2CH3), 4.21 (dd, 1H, J1Ј,2Ј
5.1, J1Ј,2Љ 8.5, H1Ј), 4.93 (s, 2H, NH2), 6.21 (dd, 1H, J6,8 2.3, J5,6
8.3, H6), 6.28 (d, 1H, J6,8 2.3, H8), 6.58 (d, 1H, J5,6 8.3, H5) and
10.42 (s, 1H, NH ); m/z (FAB): 348 (MHϩ, 100%), 274 (8), 205
(12), 177 (57), 154 (28), 142 (38) and 136 (23).
General procedure for preparing ethyl (2S )-1-{[(2R/S )-6/7-
({4-[amino(imino)methyl]benzoyl}-amino)-2-methyl-3-oxo-3,4-
dihydro-2H-1,4-benzoxazin-2-yl]carbonyl}-2-pyrrolidinecarb-
oxylate acetates (S,S )-15, (R,S )-15, (S,S )-17 and (R,S )-17
General procedure for preparing (2S )-1-{[(2R/S )-(6/7)-
({4-[amino(imino)methyl]benzoyl}amino)-2-methyl-3-oxo-3,4-
dihydro-2H-1,4-benzoxazin-2-yl]-carbonyl}-2-pyrrolidinecarb-
oxylic acid acetates (S,S )-16, (R,S )-16, (S,S )-18 and (R,S )-18
To a stirred solution of the appropriate ethyl (2S)-1-[(2R/S)-
((6/7)-amino-2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-
2-yl)carbonyl]-2-pyrrolidinecarboxylate (S,S)-13, (R,S)-13,
(S,S)-14 or (R,S)-14 (0.695 g, 2.00 mmol) and triethylamine
(0.3 mL, 2.20 mmol) in anhydrous dichloromethane (30 mL)
was added 4-cyanobenzoyl chloride (0.397 g, 2.4 mmol) in small
portions at 0 ЊC. After the addition was complete, stirring was
continued for four hours at room temperature. The reaction
mixture was washed with 10% citric acid (2 × 50 mL) and
saturated NaHCO3 solution (2 × 50 mL), dried with sodium
sulfate, filtered and the solvent evaporated under reduced
pressure to give the corresponding ethyl (2S)-1-({(2R/S)-(6/7)-
[(4-cyanobenzoyl)amino]-2-methyl-3-oxo-3,4-dihydro-2H-1,4-
benzoxazin-2-yl}carbonyl)-2-pyrrolidinecarboxylate.
Ethyl(2S)-1-{[(2R/S)-(6/7)-({4-[amino(imino)methyl]benzoyl}-
amino)-2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]-
carbonyl}-2-pyrrolidinecarboxylate acetate (S,S)-15, (R,S)-
15, (S,S)-17 or (R,S)-17 (0.278 g, 0.5 mmol) was dissolved in
ethanol (10 mL), 2 M NaOH solution (0.5 mL) was added and
the reaction mixture stirred at room temperature overnight. The
solvent was evaporated under reduced pressure to half the
volume, AcOH (2–3 equivalents) was added and the solution
cooled at 4 ЊC to form a crystalline precipitate.
(2S )-1-{[(2S )-7-({4-[amino(imino)methyl]benzoyl}-amino)-2-
methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl]carbonyl}-
2-pyrrolidinecarboxylic acid acetate (S,S )-16
To a solution of the respective ethyl (2S)-1-({(2R/S)-6/7-
[(4-cyanobenzoyl)amino]-2-methyl-3-oxo-3,4-dihydro-2H-1,4-
benzoxazin-2-yl}carbonyl)-2-pyrrolidine-carboxylate (0.476 g,
1.00 mmol) in anhydrous ethanol (20 mL) was introduced
gaseous hydrogen chloride at 0 ЊC for 30 minutes. The reaction
mixture was stirred overnight at room temperature. The solvent
was evaporated under reduced pressure and washed 3–4 times
with diethyl ether to give a crystalline product. This was dissol-
ved in anhydrous ethanol (15 mL), ammonium acetate (0.23 g,
3.00 mmol) was added, and the reaction mixture was stirred for
2–3 days at room temperature. The solvent was evaporated
under reduced pressure and the product purified by preparative
The procedure described yielded (S,S)-16 (0.137 g, 52%) as pale
20
yellow crystals, mp > 300 ЊC; [α] Ϫ6.4 (c 0.100 in MeOH);
546
νmax(KBr)/cmϪ1 3446, 1638, 1560, 1412, 1282, 1021, 814 and
644; δH(300 MHz; DMSO-d6; Me4Si) 1.49 (s, 3H, CH3), 1.86 (s,
3H, CH3COOH), 1.57–1.94 (m, 3H, H3Ј, H3Љ, H2Љ), 4.18 (br s,
1H, H1Ј), 6.82 (d, 1H, J5,6 8.6, H5), 7.10–7.13 (m, 1H, H6), 7.54
(br s, 1H, H8), 7.68–7.77 (m, 4H, 4–CN–Ar–H) and 10.46 (br s,
1H, NH ); [C(NH)NH3ϩ gives together with H2O a broad signal
at 3.0–4.2 ppm]; signals of 4-CN-ArCONH- and COOH
were not detected; signals for H2Ј, H4Ј, H4Љ are under the corre-
sponding signal for water]; m/z (FAB): 466 (MHϩ, 1%), 391 (7),
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 5 1 1 – 1 5 1 7
1516

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307 (22), 289 (12), 154 (100), 136 (76), 107 (28), 71 (46) and 55
(50); HPLC: peak area > 95%.
anionic and cationic moieties in trans-(S,S)-16, trans-(R,S)-16,
cis-(R,S)-16, trans-(S,S)-18, trans-(R,S)-18 and cis-(R,S)-18
For data of other compounds see ESI†.
were measured and compared to the known pharmacophore.
The receptor binding assay
Abbreviations
Biotinylation of fibrinogen. 5 mL of 0.3 M NaCl solution was
added to human fibrinogen (100 mg) and incubated at 30 ЊC
until complete dissolution. 300 µL of the fibrinogen solution
and 600 µL of 1 M NaHCO3 were diluted with bidistilled water
to obtain a solution with a final fibrinogen concentration of
1 mg mLϪ1. (ϩ)-Biotin N-succinimidyl ester solution (2 mL)
in N,N-dimethylformamide (1 mg per 1 mL) was added and
incubated for 1.5 h at 30 ЊC. After dialysis of the fibrinogen
solution against Tris buffer (20 mM Tris, 150 mM NaCl, pH =
7.4), the solution was centrifuged for 5 min at 5000 rpm. The
supernatant was stabilised with Tween 20 (0.005%) and stored
at 4 ЊC.
EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride; HOBT: 1-hydroxybenzotriazole hydrate; DPPA:
diphenylphosphoryl azide; NMM: N-methylmorpholine;
RGDS: arginyl-glycyl-aspartyl-serine.
Acknowledgements
The authors wish to thank Professor Roger Pain for critical
reading of the manuscript.
References
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Solid-phase receptor binding assay. Integrin (fibrinogen
receptor (1 µg mLϪ1) or vitronectin receptor (0.2 µg mLϪ1)) in
Tris buffer solution (20 mM Tris, 150 mM NaCl, 1 mM CaCl2,
1 mM MgCl2, 1 mM MnCl2, pH = 4) was applied on a Lumitrac
600 96-well microtiter plate (100 µL per well) and incubated at
4 ЊC overnight. The integrin solution was poured away and the
free binding sites blocked with 1% BSA Tris buffer solution
(200 µL per well) for 1 hour at room temperature, followed
by two-times washing with Tris buffer containing 0.01% Tween
20. 100 µL of different dilutions of the potential integrin
antagonists (200 µM, 60 µM, 20 µM, 6 µM, 2 µM, 0.6 µM, 0.2
µM, 0.06 µM and 0.02 µM in Tris) were applied, followed by
100 µL biotinylated fibrinogen solution and 2h incubation
at room temperature. The plates were washed twice with Tris
buffer containing 0.01% Tween 20. 100 µL biotin directed
horseradish peroxidase conjugated goat antibodies (Calbio-
chem; 1 : 1000 dilution in Tris buffer containing 0.1% BSA)
were added to each well and the plate incubated for 1 hour at
room temperature. After washing the plate three times with Tris
buffer containing 0.01% Tween 20, the chemiluminescence
reagent (50 µL per well) was added and within 15 minutes
the light emission was measured with Tecan GENios using
Magellan (Version 3.0) software. The chemiluminescence inten-
sity data was fitted to a dose-response curve, from which the
concentration of the antagonists causing 50% inhibition of
fibrinogen binding (the IC50 value) was obtained. The data were
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ˇ
Molecular modelling. The structures of trans-(S,S)-16, trans-
(R,S)-16, cis-(R,S)-16, trans-(S,S)-18, trans-(R,S)-18 and
cis-(R,S)-18 were drawn in ISIS Draw 2.3. The structures were
transferred to HyperChemTM (Release 5.01 for Windows,
Hypercube, Inc. 1996) and the ‘add hydrogens and model build’
function was carried out, followed by geometry optimisation
using the Molecular mechanics force field method and Polak-
Ribiere (Conjugate gradient) minimisation algorithm (RMS
gradient of 5 exp(-5) kcal/(Amol)). The obtained minimized/
low-energy conformations were compared to the data obtained
from NOESY experiments. Additionally, the stereochemistry
of benzoxazinone C-2 and proline C-δ and the geometry of
proline amide bond were checked. The distances between the
21 D. Kikelj, E. Suhadolc, U. Urleb and U. Zbontar, J. Heterocycl.
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