Novel potent and selective thrombin inhibitors based on a central 1,4-benzoxazin-3(4H)-one scaffold

Article abstract of DOI:10.1021/jm701622y

Novel thrombin inhibitors with the central 1,4-benzoxazine-3(4H)-one scaffold, benzamidine P₁ arginine side chain mimetic and various P₃ moieties are described. 3-(Benzyl(2-(4-carbamimidoylbenzyl)-4- methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl)amino)-3-oxopropanoic acid (7b), the most potent compound in the series, exhibited a K_i of 2.6 nM in vitro for thrombin and high selectivity against trypsin and factor Xa.

Full text of DOI:10.1021/jm701622y

J. Med. Chem. 2008, 51, 2863–2867
2863
Novel Potent and Selective Thrombin Inhibitors Based on a Central 1,4-Benzoxazin-3(4H)-one
Scaffold4
Janez Ilasˇ, Tihomir Tomasˇic´, and Danijel Kikelj*
UniVersity of Ljubljana, Faculty of Pharmacy, AsˇkercˇeVa 7, 1000 Ljubljana, SloVenia
ReceiVed December 24, 2007
Novel thrombin inhibitors with the central 1,4-benzoxazine-3(4H)-one scaffold, benzamidine P1 arginine
side chain mimetic and various P3 moieties are described. 3-(Benzyl(2-(4-carbamimidoylbenzyl)-4-methyl-
3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl)amino)-3-oxopropanoic acid (7b), the most potent compound in
the series, exhibited a Ki of 2.6 nM in vitro for thrombin and high selectivity against trypsin and factor Xa.
Introduction
Synthesis of the target 1,4-benzoxazine-3(4H)-one thrombin
inhibitors 6a-d and 7a-d was achieved using the chemistry
depicted in Scheme 1. The bromo derivative 111 was synthesized
from 2-amino-5-nitrophenol in four steps involving N-acylation,
cyclization, N-methylation, and bromination, with an overall
yield of 89%. A Wittig reaction of 4-cyanobenzaldehyde and
phosphorus ylide obtained from 1 with triphenylphosphine
proceeded smoothly, yielding 3-oxo-3,4-dihydro-2H-1,4-ben-
zoxazine-2-ylidene derivative 2, mainly as the Z-isomer (chemi-
cal shift of olefinic proton: 7.05 ppm), although a small amount
of E-isomer was also obtained (chemical shift of olefinic proton:
6.84 ppm). The two geometric isomers could be separated by
recrystallization from petroleum ether/ethyl acetate (1:1). Com-
pound 2 (mixture of Z- and E-isomers) was reduced catalytically
at 6 bar to racemic 7-amino-3-oxo-3,4-dihydro-2H-1,4-benzox-
azine-2-yl derivative 3, whereas under less vigorous catalytic
hydrogenation conditions, only the nitro group of 2 was reduced
to give amine 3a. N-Benzylation of 3, employing reductive
amination of benzaldehyde, with sodium triacetoxyborohydride
as reducing agent, afforded secondary amine 4, which was
acylated with ethyl oxalyl chloride, methyl malonyl chloride,
and ethyl succinyl chloride to introduce side chains of different
lengths containing a carboxylic ester moiety, giving 5a-c.
Alternatively, 3 was coupled to 2-benzyl-3-ethoxy-3-oxopro-
panoic acid using EDC/HOBTa coupling strategy to give
compound 5d. Nitriles 5a-d were transformed to the corre-
sponding benzamidines 6a-d under Pinner reaction12 condi-
tions. The esters 6a-d were hydrolyzed to carboxylic acids
7a-d using 1.5 M sodium hydroxide in ethanol solution.
Thromboembolic diseases, including stroke, deep vein throm-
bosis, and myocardial infarction, are the leading causes of death
in developed countries.1 The search for low molecular weight
inhibitors of thrombin, the key enzyme in the coagulation
cascade, has been a major goal in the search for novel
antithrombotic agents over the past decade.2 Argatroban was
the first small-molecule thrombin inhibitor introduced to the
market,3 but its widespread use has been hindered by the fact
that it is not orally active. The withdrawal of ximelagatran,4
the first orally available thrombin inhibitor, has shown that there
is not yet a satisfactory thrombin inhibitor on the market. With
ongoing progress in the development of efficient antithrombotic
drugs, focus has also turned to other important enzymes in the
coagulation cascade, mainly factor Xa5 and factor VIIa,6 as well
as to dual inhibitors targeting two coagulation enzymes at the
same time,7 which have not yet resulted in marketable products.
Therefore, thrombin remains an important target in the discovery
of novel antithrombotic compounds.
In our ongoing research directed toward a novel class of
antithrombotic compounds with dual function, possessing in the
same molecule both thrombin inhibitory and fibrinogen receptor
antagonistic activity,8 1,4-benzoxazine-3(4H)-one was found to
be a suitable scaffold for their design. On the basis of its strong
interaction with Asp189 in the thrombin S1 pocket, the benza-
midine group was chosen as arginine mimetic9 for the first
generation of antithrombotic compounds with dual action,
although being aware of its strong basicity. The problem of poor
bioavailability of compounds containing a benzamidine group,
arising from its basicity, can be overcome by a prodrug
approach, as shown in the case of ximelagatran4 and dabigat-
ran.10 In the search for the optimal spacer between the 1,4-
benzoxazin-3(4H)-one and benzamidine moieties to produce
antithrombotic compounds with dual activity, we prepared a
series of compounds incorporating the methylene group con-
necting both moieties that, although devoid of fibrinogen
receptor antagonistic activity, were found to be potent and highly
selective thrombin inhibitors. Here we report on the design,
synthesis, in vitro biological activity, and structure-activity
relationship of this novel class of thrombin inhibitors possessing
a central 1,4-benzoxazin-3(4H)-one core.
Results and Discussion
The in vitro thrombin inhibitory potencies of the synthesized
compounds and their selectivities against factor Xa and trypsin
are presented in Table 1. Our preliminary studies demonstrated
(data not shown) that the best thrombin inhibition was obtained
when the P3 moiety was attached to a 2-substituted 1,4-
benzoxazine-3(4H)-one scaffold at position 7. Introduction of
a methyl group at position 4 additionally contributed to the
hydrophobic interaction in the thrombin S2 pocket.8 Optimiza-
tion of the P3 moiety revealed the beneficial effect of benzylation
of the amino group at position 7 which, to improve potency
and increase solubility, was additionally substituted with acyl
a Abbreviations: ADT, Auto Dock Tools; DMF, N,N-dimethylformamide;
EDC, N-ethyl N′-(3-dimethylaminopropyl)-carbodiimide; ESI, electrospray
ionization; HOBT, 1-hydroxybenzotriazole; THF, tetrahydrofuran; PDB,
Protein Data Bank.
* To whom correspondence should be addressed. Tel.: +386 1 476 95
61. Fax: +386 1 425 80 31. E-mail: danijel.kikelj@ffa.uni-lj.si.
4
Dedicated to Professor Slavko Peèar on the occasion of his 60th
birthday.
10.1021/jm701622y CCC: $40.75
2008 American Chemical Society
Published on Web 04/16/2008

2864 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Brief Articles
Scheme 1a
a Reagents and conditions: (a) 4-cyanobenzaldehyde, PPh3, Et3N, 0 °C, then rt, 24 h; (b) H2 (1 bar), Pd/C, rt, 12 h; (c) H2 (6 bar), Pd/C, rt, 72 h; (d)
benzaldehyde, NaBH(AcO)3, 1,2-dichloroethane, rt, 4 h; (e) acyl chloride, Et3N, CH2Cl2, rt, 1 h; (f) 2-benzyl-3-ethoxy-3-oxopropanoic acid, EDC, HOBt,
DMF, rt, overnight; (g) (i) HCl(g), EtOH, 0 °C, 30 min; (ii) NH4OAc, EtOH, rt, 24 h; (h) NaOH, dioxane/H2O, rt, 4 h.
Table 1. Inhibitory Potencies and Selectivities of Compounds 6a-d and 7a-d
chains bearing a carboxylic ester or free carboxylic group.
Comparison of low nanomolar thrombin inhibitory potencies
of compounds 6a-c and 7a-c with those of 3 orders of
magnitude higher Ki values of compounds 6d and 7d, demon-

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2865
the 1,4-oxazine ring formed a hydrogen bond with the Gly216
backbone (2.72 Å), which is often regarded as a key interaction
in the thrombin active site.18 The methylene spacer between
benzamidine and benzoxazin-3(4H)-one moiety allows the
optimal positioning of both moieties and enables favorable
interactions between inhibitor and enzyme, thus making an
important contribution to the high potency of this novel
structural type of thrombin inhibitor. Docking studies showed
that also compounds 6a, 6b, and 7a-c bind to the thrombin
active site in an analogous fashion. The same is true for
compounds 6d and 7d, the only difference being that the phenyl
moiety is not optimally bound to the S3 pocket, which accounts
for the much lower thrombin inhibitory potency of these two
compounds.
Figure 1. Highest score hypothetical binding mode of inhibitor (S)-
6b in the thrombin binding site as predicted by AutoDock 3.0.
Conclusion
strate the crucial importance of the proper placement of the P3
benzyl moiety. The benzyl moiety bound to N-7 (in 6a-c and
7a-c) appears to be optimally positioned, whereas the insertion
of two bonds between the benzyl group and the nitrogen atom
(compounds 6d and 7d) reduces interaction with the thrombin
S3 pocket. Neither the length of the N-acyl chain nor the nature
of the carboxylate moiety (ester or carboxylic acid) substantially
affected the activity of the compounds. Thus, the best compound,
7b, with Ki of 2.6 nM, bears an N-malonyl moiety, and
compound 6b, bearing a methylmalonyl moiety attached to the
N-7, has almost the same potency. This demonstrates that the
carboxylate moiety, which is turned to the water environment,
does not interact with the thrombin active site surface. The
shorter ethyloxalyl moiety only slightly reduced thrombin
inhibitory potency; the carboxylic acid 7a exhibited a 4-fold
higher inhibition constant (Ki ) 11 nM), presumably because
of the electrostatic repulsion of the carboxylate group with the
surface of the active site. Thrombin inhibitors 6a-c and 7a-c
possess excellent 535- to 112400-fold selectivity against trypsin
and 181- to 52453-fold selectivity against factor Xa. The best
thrombin inhibitor in the series, compound 7b, was also the
most selective against trypsin and factor Xa, whereas compounds
6d and 7d, with low micromolar thrombin inhibition constants,
showed only 2- to 4-fold selectivity for trypsin and 11- to 22-
fold selectivity for factor Xa. A high selectivity of 7b, bearing
a terminal malonyl moiety, compared to other compounds from
this series, is presumably due to less favorable interaction of
the malonyl carboxylate moiety of 7b with the D pocket of factor
Xa and trypsin, which has been filled many times by basic
residues in the case of factor Xa inhibitors that were not selective
versus trypsin and very selective over thrombin.13–15
Docking studies were performed for compounds 6a-d and
7a-d using AutoDock16 to predict their binding mode in the
thrombin active site. Because the synthesized compounds were
all racemic mixtures, both enantiomers were docked, and it was
found that only the S-isomers bind favorably to the active site
of thrombin. Compound (S)-6b docked into the thrombin active
site is shown in Figure 1. As expected from our previous results,8
it binds with the benzamidine moiety situated in the thrombin
S1 binding pocket. The distance between the C-atoms of the
Asp189 carboxylate and the amidine group in (S)-6b was 4.0
Å, and two hydrogen bonds linked the amidine hydrogens and
carboxylate oxygen atoms. There was an additional hydrogen
bond between benzamidine and the Gly219 backbone. The
benzamidine moiety of (S)-6b binded in an identical mode to
that of benzamidine itself.17 The 1,4-benzoxazin-3(4H)-one
scaffold was located in the S2 binding pocket and the benzyl
group in the lipophilic S3 binding pocket, with the P3 carboxylate
stretching outward from the thrombin surface. The oxygen of
We have synthesized a new series of highly potent and
selective thrombin inhibitors possessing a central 2,7-disubsti-
tuted 1,4-benzoxazin-3(4H)-one core. Compound 7b, with Ki
of 2.6 nM for thrombin, 112400-fold selectivity for trypsin and
52435-fold selectivity for factor Xa, offers considerable potential
for the development of an orally active prodrug to overcome
unsatisfactory biovailability due to the presence of benzamidine
moiety. The straightforward synthetic approach, using a Wittig-
type reaction followed by reduction of the resulting alkene,
enables stereoselective synthesis and future preparation of
enantiomerically pure thrombin inhibitors of this novel structural
class.
Experimental Section
General. Chemicals were obtained from Acros, Aldrich Chemi-
cal Co., and Fluka and used without further purification. THF was
kept over sodium and distilled immediately prior to use. Analytical
TLC was performed on silica gel Merck 60 F254 plates (0.25 mm),
using visualization with ultraviolet light and ninhydrin. 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 and 13C NMR
spectra were recorded at 300 and 75 MHz respectively on a Bruker
AVANCE DPX300 spectrometer in CDCl3 or DMSO-d6 solution
with TMS as the internal standard. IR spectra were recorded 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 were within (0.4% of
the theoretical values. Mass spectra were obtained using a VG-
Analytical Autospec Q mass spectrometer. HPLC analyses were
performed on an Agilent Technologies HP 1100 instrument with
G1365B UV-vis detector (254 nm), using a Luna C18 column
(4.6 × 250 mm) at flow rate 1 mL/min. The eluant was a mixture
of 0.1% TFA in water (A) and acetonitrile (B). Gradient was 10%
B to 80% B in 30 min.
Biological Evaluation. The ability of the new thrombin inhibitors
to competitively inhibit the enzymatic activities of thrombin, trypsin,
and factor Xa was measured as described previously8 by amidolytic
enzyme assay using chromogenic substrates, and is expressed as
inhibition constant Ki.19 Competitive inhibition of thrombin, factor
Xa, and trypsin by compounds 6a-6d and 7a-7d was established
by the double reciprocal plotting 1/V against 1/[S] for each inhibitor
concentration (Lineweaver-Burk plot).
Values for Ki were calculated according to Cheng and Prusoff20
based on the relation between reaction velocity equations in the
absence and presence of inhibitor, using the relevant Km.21 The
selectivity for thrombin over trypsin and factor Xa, as two closely
related serine proteases, was expressed as the ratios Ki(trypsin)/
Ki(thrombin) and Ki(factor Xa)/Ki(thrombin). Inhibition of binding
of fibrinogen to isolated platelet fibrinogen receptor was measured
as previously described.8

2866 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Brief Articles
4-((4-Methyl-7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-
ylidene)methyl)benzonitrile (2). To a stirred solution of triphe-
nylphosphine (0.756 g, 2.88 mmol) in 80 mL of anhydrous THF
cooled to 0 °C, compound 1 (0.828 g, 2.88 mmol) dissolved in 15
mL of anhydrous THF was added dropwise over 10 min. The
reaction mixture was stirred for 30 min at 0 °C under an atmo-
sphere of argon and left overnight to warm to room temperature.
The reaction mixture was then cooled to 0 °C, anhydrous triethy-
lamine (0.80 mL, 5.76 mmol) was added, and the mixture was
stirred for 30 min at 0 °C. 4-Cyanobenzaldehyde (0.325 g, 2.88
mmol) dissolved in 15 mL of THF was added dropwise. The
reaction was allowed to proceed overnight under an argon
atmosphere. The precipitate that formed was filtered off, dissolved
in dichloromethane (250 mL), and washed successively with 10%
citric acid (2 × 50 mL), saturated NaHCO3 solution (2 × 50 mL),
and saturated NaCl solution (1 × 50 mL). The organic phase was
dried over Na2SO4 and filtered, and the solvent was evaporated
under reduced pressure. Compound 2 (0.521 mg) was obtained as
a yellow powder. Recrystallization from petroleum ether/ethyl
acetate yielded pure Z-(2a) and E-(2b) isomers.
room temperature. Solvent was removed under reduced pressure,
and the residue was dissolved in ethyl acetate (150 mL) and washed
successively with saturated NaHCO3 solution (3 × 50 mL) and
brine (1 × 50 mL). The organic phase was dried over Na2SO4,
filtered, and the solvent evaporated under reduced pressure.
Compound 4 was obtained as a yellow powder. The crude product
was purified by column chromatography using dichloromethane/
acetone (30:1) as eluant. Yield: 0.839 g (55.5%), mp 153-154 °C.
MS (EI): m/z (%) 383 (M+, 100). 1H NMR (DMSO-d6, 300 MHz):
3
2
δ 3.05 (dd, 1H, J ) 8.7 Hz, J ) 14.4 Hz, CH-CH2), 3.19-3.24
(m, 4H, N-CH3, CH-CH2), 4.23 (d, 2H, 3J ) 6.0 Hz, Ph-CH2-NH),
4
3
4
4.81 (dd, 1H, J ) 3.9 Hz, J ) 8.7 Hz, 2-H), 6.12 (d, 1H, J )
2.4 Hz, Ar-H8), 6.22 (t, 1H, 3J ) 6.0 Hz, NH), 6.30 (dd, 1H, 4J )
3
3
2.4 Hz, J ) 8.7 Hz, Ar-H6), 6.85 (d, 1H, J ) 8.7 Hz, Ar-H5),
7.23-7.34 (m, 5H, Ph), 7.41 (d, 2H, 3J ) 8.1 Hz, Ar-H2′,H6′), 7.70
(d, 2H, 3J ) 8.1 Hz, Ar-H3′,H5′) ppm. Anal. (C24H21N3O2) C, H, N.
General Procedure for the Synthesis of N-Acyl-4-((7-(benzyl-
amino)-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)meth-
yl)benzonitriles 5a, 5b, and 5c. To a solution of secondary amine
4 (459 mg, 1.20 mmol) in dichloromethane (50 mL) were added
triethylamine (145 mg, 1.44 mmol) and the corresponding acyl
chloride (1.44 mmol), and the mixture was stirred for 2 h at room
temperature. Solvent was removed under reduced pressure, and the
residue was dissolved in ethyl acetate (100 mL) and washed
successively with 10% citric acid (2 × 50 mL), saturated NaHCO3
solution (3 × 50 mL), and brine (1 × 50 mL). The organic solution
was dried over Na2SO4 and filtered, and the solvent was evaporated
under reduced pressure. The crude product was purified by column
chromatography using petroleum ether/ethyl acetate (1:1) as eluant.
Ethyl 2-(Benzyl(2-(4-cyanobenzyl)-4-methyl-3-oxo-3,4-dihydro-
2H-1,4-benzoxazin-7-yl)amino)-2-oxoacetate (5a). Yellow oil, yield:
77.5%. MS (EI): m/z (%) 483 (M+, 41), 91 (100). 1H NMR (CDCl3,
(Z)-4-((4-Methyl-7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-
2-ylidene)methyl)benzonitrile (2a). Yield: 455 mg (49.2%); mp
285-288 °C. MS (EI): m/z (%) 321 (M+, 100). 1H NMR (CDCl3,
300 MHz): δ 3.59 (s, 3H, N-CH3), 7.05 (s, 1H, CdCH), 7.16 (d,
1H, 3J ) 8.7 Hz, Ar-H5), 7.75 (d, 2H, 3J ) 8.4 Hz, Ar-H2′,H6′),
7.93 (d, 2H, 4J ) 8.4 Hz, Ar-H3′,H5′), 8.09 (m, 2H, Ar-H6,
Ar-H8) ppm. Anal. (C17H11N3O4) C, H, N.
4-((7-Amino-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-
yl)methyl)benzonitrile (3). A solution of compound 2 (2.26 g, 7.03
mmol) in DMF (250 mL) was stirred with 10% palladium on
activated charcoal (226 mg) under hydrogen atmosphere at 6 bar
for 3 days at room temperature. The product was isolated by
filtration and the solvent evaporated under reduced pressure. The
residue was dissolved in dichloromethane (100 mL) and washed
successively with 10% citric acid (2 × 50 mL), saturated NaHCO3
solution (2 × 50 mL), and saturated NaCl solution (1 × 50 mL).
The organic phase was dried over Na2SO4, filtered, and the solvent
evaporated under reduced pressure. Compound 3 was obtained as
a light yellow powder. Yield: 1.16 g (56.6%), mp 135-138 °C.
MS (EI): m/z (%) 293 (M+, 100). 1H NMR (DMSO-d6, 300 MHz):
δ 3.08 (dd, 1H, 3J ) 9.0 Hz, 2J ) 14.7 Hz, CH-CH2), 3.20 (s, 3H,
N-CH3), 3.25 (dd, 1H, 3J ) 4.0 Hz, 2J ) 14.7 Hz, CH-CH2), 4.81
3
300 MHz): δ 1.10 (t, 3H, J ) 6.9 Hz, -CH2-CH3), 3.16 (dd, 1H,
3J ) 8.1 Hz, 2J ) 14.4 Hz, CH-CH2), 3.29-3.35 (m, 4H, N-CH3,
3
4
CH-CH2), 4.09 (q, 2H, J ) 6.9 Hz, -CH2-CH3), 4.77 (dd, 1H, J
) 3.9 Hz, 3J ) 8.1 Hz, 2-H), 4.84 (d, 1H, 2J ) 14.6 Hz, Ph-CH2-
2
4
N), 5.03 (d, 1H, J ) 14.6 Hz, Ph-CH2-N), 6.70 (d, 1H, J ) 2.4
Hz, Ar-H8), 6.75 (dd, 1H, J ) 2.4 Hz, J ) 8.7 Hz, Ar-H6),
4
3
6.81 (d, 1H, 3J ) 8.7 Hz, Ar-H5), 7.23-7.33 (m, 7H, Ph,
Ar-H2′,H6′), 7.56 (d, 2H, J ) 8.4 Hz, Ar-H3′,H5′) ppm. Anal.
3
(C28H25N3O5) C, H, N.
General Procedure for the Synthesis of N-Acyl-4-((7-(benzyl-
amino)-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)meth-
yl)benzimidamides 6a-6d. Gaseous HCl was introduced into a
solution of the nitrile 5 (1.00 mmol) in 30 mL of absolute EtOH
(5a and 5c) or MeOH (5b and 5d) for 30 min. The reaction mixture
was closed tightly and stirred at room temperature for 24 h. The
solvent was evaporated and the residue was washed 2-3 times with
diethyl ether. The obtained iminoether was dissolved in anhydrous
EtOH (30 mL), ammonium acetate (0.308 g, 4.00 mmol) was added,
and the reaction mixture was stirred for 2 days. The solvent was
then evaporated to one-third of the starting volume, two drops of
trifluoroacetic acid were added, and the residual solution stored at
4 °C. The precipitated crystals were filtered off and washed with
cold diethyl ether. If the obtained product was not pure, it was
purified by column chromatography using dichloromethane/
methanol (6:1) as eluant.
3
2
(dd, 1H, J ) 4.0 Hz, J ) 9.0, Hz 2-H), 5.04 (s, 2H, NH2), 6.14
(d, 1H, 4J ) 2.3 Hz, Ar-H8), 6.27 (dd, 1H, 4J ) 2.3 Hz, 3J ) 8.5
Hz, Ar-H6), 6.82 (d, 1H, 3J ) 8.5 Hz, Ar-H5), 7.48 (d, 2H, 3J )
8.2 Hz, Ar-H2′,H6′), 7.77 (d, 2H, 3J ) 8.2 Hz, Ar-H3′,H5′) ppm.
Anal. (C17H15N3O2) C, H, N.
(E)-4-((7-Amino-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-
2-ylidene)methyl)benzonitrile (3a). A solution of 2b (0.226 g, 0.70
mmol) in 100 mL of dioxane/ethanol (1:1) mixture in the presence
of 10% palladium on activated charcoal (26 mg) was stirred under
a hydrogen atmosphere at 1 bar pressure overnight at room
temperature. The catalyst was filtered off and the solvent evaporated
in vacuo. The residue was dissolved in 100 mL of dichloromethane
and washed successively with 10% citric acid (2 × 50 mL),
saturated NaHCO3 solution (2 × 50 mL), and saturated NaCl
solution (1 × 50 mL). The organic phase was dried over Na2SO4
and filtered, and the solvent was evaporated under reduced pressure.
Compound 3a was obtained as orange crystals. Yield: (184 mg)
90.6%, mp 259-261 °C. MS (EI): m/z (%) 291 (M+, 100). 1H
NMR (DMSO-d6, 300 MHz): δ 3.35 (s, 3H, N-CH3), 5.20 (s, 2H,
Ethyl 2-(Benzyl(2-(4-carbamimidoylbenzyl)-4-methyl-3-oxo-3,4-
dihydro-2H-1,4-benzoxazin-7-yl)amino)-2-oxoacetate Trifluoroac-
etate (6a). White thick oil, yield: 50.8%. MS (FAB): m/z (%) 501
1
(MH+, 80), 69 (100). H NMR (DMSO-d6, 300 MHz): δ 0.94 (t,
3
4
NH2), 6.40 (dd, 1H, J ) 8.7 Hz, J ) 2.3 Hz, Ar-H6), 6.55 (d,
3H, 3J ) 6.9 Hz, -CH2-CH3), 3.07 (dd, 1H, 3J ) 8.7 Hz, 4J ) 14.4
Hz, CH-CH2), 3.25-3.40 (m, 4H, N-CH3, CH-CH2), 4.01 (q, 2H,
1H, 4J ) 2.3 Hz, Ar-H8), 6.82 (s, 1H, CdCH-), 6.95 (d, 1H, 3J )
8.7 Hz, Ar-H5), 7.84 (d, 2H, J ) 8.4 Hz, Ar-H3′,H5′), 7.77 (d,
3
2
3J ) 6.9 Hz, -CH2-CH3), 4.88 (d, 1H, J ) 15.0 Hz, Ph-CH2-N),
2H, J ) 8.4 Hz, Ar-H2′,H6′) ppm. Anal. (C17H13N3O2) C, H, N.
3
4.99 (m, 2H, Ph-CH2-N, CH-CH2), 6.74 (d, 1H, 4J ) 2.4 Hz,
4
3
4-((7-(Benzylamino)-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzox-
azin-2-yl)methyl)benzonitrile (4). Acetic acid (0.23 mL, 3.94 mmol)
and benzaldehyde (400 mg, 3.94 mmol) were added to a stirred
solution of amine 3 (1.16 g, 3.94 mmol) in 1,2-dichloroethane (150
mL) under an argon atmosphere. After 20 min, NaBH(OAc)3 (1.25
g, 5.92 mmol) was added, and the mixture was stirred overnight at
Ar-H8), 6.87 (dd, 1H, J ) 2.4 Hz, J ) 8.7 Hz, Ar-H6), 7.14
(d, 1H, 3J ) 8.7 Hz, Ar-H5), 7.18-7.36 (m, 5H, Ph), 7.44 (d, 2H,
3J ) 8.4 Hz, Ar-H2′,H6′), 7.75 (d, 2H, 3J ) 8.4 Hz, Ar-H3′,H5′),
9.15 (br s, 2H, amidino-H), 9.28 (br s, 2H, amidino-H) ppm. HPLC:
97.2%, tr ) 18.78 min. Anal. (C28H28N4O5 × CF3COOH × 5/2
H2O) C, H, N.

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2867
(7) Kranjc, A.; Kikelj, D.; Peterlin-Masˇicˇ, L. Recent advances in the
discovery of tissue factor/factor VIIa inhibitors and dual inhibitors of
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General Procedure for Alkaline Hydrolysis of Alkyl Esters
6a-d. To a solution of the corresponding ester 6a-d (0.18 mmol)
in 50 mL of ethanol/water (1:1) mixture (50 mL), NaOH (27 mg,
0.675 mmol) was added and the reaction mixture was stirred at
room temperature for 6 h. The ethanol was evaporated and the
resulting aqueous solution neutralized with 1 M HCl, until the
product started to precipitate. If the product was not pure, it was
purified by column chromatography using dichloromethane/
methanol (3:1) as eluant.
ˇ
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2-(Benzyl(2-(4-carbamimidoylbenzyl)-4-methyl-3-oxo-3,4-dihy-
dro-2H-1,4-benzoxazin-7-yl)amino)-2-oxoacetic Acid (7a). White
powder, yield: 64.3%, mp 229-231 °C. MS (ESI): m/z (%) 473.25
(MH+, 50), 457.27 (100). 1H NMR (DMSO-d6, 300 MHz): δ
3.05-3.25 (m, 5H, N-CH3, -CH-CH2), 4.74 (d, 1H, 2J ) 15.0 Hz,
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3(4H)-ones and 2-bromo-2H-1,4-benzoxazin-3(4H)-ones. HelV. Chim.
Acta, in press.
2
Ph-CH2-N), 4.84 (d, 1H, J ) 15.0 Hz, Ph-CH2-N), 5.01 (m, 1H,
(12) Roger, R.; Neilson, D. G. The chemistry of imidates. Chem. ReV. 1961,
2-H), 6.64-6.74 (m, 3H, Ar-H8, Ar-H6, Ar-H5), 7.14-7.32 (m,
7H, Ph, Ar-H2′,H6′), 7.64 (d, 2H, 3J ) 8.4 Hz, Ar-H3′,H5′), 9.39
(br s, 2H, amidino-H), 9.69 (br s, 2H, amidino-H) ppm. HPLC:
98.0%, tr ) 14.87 min. Anal. (C26H24N4O5) HRMS.
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Takayanagi, M.; Takenaka, K.; Suzuki, N.; Oono, S.; Kataoka, N.;
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Shima, Y.; Tashiro, K.; Yamamoto, H.; Yoshimoto, R.; Iwata, S.; Tsuji,
T.;Sakurai,K.;Shoji,M.Rationaldesign,synthesis,andstructure-activity
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Acknowledgment. This work was supported financially by
the Slovenian Research Agency (Grant No. P1-0208). The
authors thank Professor Mojca Stegnar for providing us
thrombin, factor Xa, trypsin, and chromogenic substrate and
Professor Roger Pain for critical reading of the manuscript.
Supporting Information Available: Experimental procedures and
characterization of intermediate and target compounds; IR spectra, 13
C
spectra of representative compounds, elemental analyses (C, H, N)
and HRMS results; and molecular docking protocol. This material is
available free of charge via the Internet at http://pubs.acs.org.
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