Novel 1,4-benzoxazine and 1,4-benzodioxine inhibitors of angiogenesis

Article abstract of DOI:10.1016/j.ejmech.2012.10.001

Esters of 1,4-benzoxazine and 1,4-benzodioxine compounds 1 and 10, which combine thrombin inhibitory and GPIIb/IIIa antagonistic activity in one molecule are shown to inhibit endothelial cell migration and tube formation in vitro and angiogenesis in the c

Full text of DOI:10.1016/j.ejmech.2012.10.001

European Journal of Medicinal Chemistry 58 (2012) 160e170
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 1,4-benzoxazine and 1,4-benzodioxine inhibitors of angiogenesis
b
b
d,
a,
**
ꢁ a
ꢀa
ꢀ c
*
ꢀ
Milos Ilic , Janez Ilas , Petra Dunkel , Péter Mátyus , Andrej Bohác , Sandra Liekens , Danijel Kikelj
a
ꢀ
ꢀ
University of Ljubljana, Faculty of Pharmacy, Askerceva 7, SI e 1000 Ljubljana, Slovenia
b
}
Semmelweis University, Department of Organic Chemistry, Hogyes E. u. 7, 1092 Budapest, Hungary
c Comenius University, Faculty of Natural Sciences, Department of Organic Chemistry, Mlynská dolina, 842 15 Bratislava, Slovakia
d Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Esters of 1,4-benzoxazine and 1,4-benzodioxine compounds 1 and 10, which combine thrombin inhib-
itory and GPIIb/IIIa antagonistic activity in one molecule are shown to inhibit endothelial cell migration
and tube formation in vitro and angiogenesis in the chicken chorioallantoic membrane (CAM) assay. The
corresponding carboxylic acids 1 (R2 ¼ H) and 11 were devoid of anti-angiogenic activity, most probably
due to their insufficient entry into the cell. Although thrombin inhibition remains the most probable
explanation for their inhibition of angiogenesis, VEGFR2 kinase assay suggest that other targets such as
VEGFR2 might be involved.
Received 13 April 2012
Received in revised form
24 September 2012
Accepted 1 October 2012
Available online 8 October 2012
Keywords:
Angiogenesis
Ó 2012 Elsevier Masson SAS. All rights reserved.
Thrombin
GPIIb/IIIa antagonist
Vascular endothelial growth factor
1. Introduction
angiogenic activity, which is thought to be mediated by activation of
its protease-activated receptor (PAR-1) which leads to downstream
The association of venous thrombosis and cancer has been
recognized for over 100 years and has a prevalence rate of 10e20%
[1]. A systemic activation of blood coagulation which leads to
increased tendency toward formation of blood clots is frequently
present in cancer patients. Most tumor cells have constitutively
active tissue factor on their surface, capable of generating thrombin
in plasma. The presence of thrombin has been shown in a variety of
tumor types and a clinical study demonstrated that primary
thromboembolism increases the risk of overt cancer diagnosis by
3-fold within 6e12 months after thrombosis [2]. These clinical
observations are in line with animal experiments where thrombin
treatment of B16 melanoma tumors increases dramatically the
number of lung metastases in rats [3]. Malignancy initiates a vicious
cycle in which greater tumor burden supplies more thrombin that
stimulates tumor growth and increases platelet-tumor interaction.
The tumor-promoting effects of thrombin may be related to its pro-
mitogenic signaling events resulting inter alia in the expression of
vascular endothelial growth factor (VEGF) in tumor cells and its
tyrosine kinase receptor VEGFR2 in endothelial cells [4e6].
Thrombin stimulates the migration of tumor cells into the
vasculature and, together with other tumor secreted agents, acti-
vates the endothelial cells and platelets to expose P-selectin.
Weakly activated platelets and endothelial cells bind tumor cells via
P-selectin exposed on their surface inducing weak tethering of
tumor cells to the endothelium and platelets. Finally, a firm binding
of tumor cells to platelets occurs through interaction mediated by
binding of platelet integrin GPIIb/IIIa to tumor integrins via RGD
motif-containing ligands, such as von Willebrand Factor (vWF) and
fibronectin. These events lead to angiogenesis via thrombin-
stimulated synthesis and release of VEGF and other proangiogenic
growth factors from tumor cells and platelets and induction of
VEGFR2 synthesis in endothelial cells. Platelet-tumor aggregates
protect tumor cells from natural killer cells, prolong their survival
in the blood and bind more avidly to subendothelial basement
membranes and matrix. Many tumor cells require platelets for the
development of metastasis and it has been shown that several
tumor cell lines aggregate platelets in vitro [4,5,8]. Targeting the
aberrant growth of blood vessels, a common biological aspect of
anti-angiogenic drugs [9e11], is extensively being explored in
oncology in order to deprive tumors of nutrients normally deliv-
ered by blood flow [12e15]. Recent studies indicate that
Abbreviations: BAEC, bovine aortic endothelial cells; CAM, chick chorioallantoic
membrane; DMF, N,N-dimethylformamide; DCM, dichloromethane; DIAD, diiso-
propyl azodicarboxylate; GPIIb/IIIa, glycoprotein IIb/IIIa; HMEC, human microvas-
cular endothelial cells; MAEC, mouse aortic endothelial cells; MCF-7, a human
breast cancer cell line; VEGFR2, vascular endothelial growth factor receptor 2.
* Corresponding author.
** Corresponding author. Tel.: þ386 1 476 95 61; fax: þ386 1 425 80 31.
E-mail address: danijel.kikelj@ffa.uni-lj.si (D. Kikelj).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.001

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M. Ilic et al. / European Journal of Medicinal Chemistry 58 (2012) 160e170
161
angiogenesis inhibitors, by depriving tumors of oxygen, can have an
unintended effect e promotion of metastasis [16e20].
reduction to amine 14 and further benzylation with benzaldehyde
or 3,5-difluorobenzaldehyde afforded N-benzylamines 15a and
15b. These were acylated with ethyl oxalyl chloride to give N-ethyl
oxalyl derivatives 16a and 16b which were finally hydrolyzed to
afford carboxylic acids 17a and 17b.
Both thrombin and integrin GPIIb/IIIa are thus important players
in angiogenesis and metastasis. The thrombin inhibitor hirudin was
demonstrated to inhibit angiogenesis in a chick chorioallantoic
membrane assay [7] and in some models RGD-containing peptides
were shown to block metastasis [5]. We have recently described
novel potential dual antithrombotic compounds which comprise in
the same molecule both thrombin inhibitory and fibrinogen
receptor (GPIIb/IIIa) antagonistic activity due to highly overlapped
thrombin inhibitor and fibrinogen receptor antagonist pharmaco-
phores [21e23]. Knowing the interplay between cancer and
thrombosis, with thrombin and platelet GPIIb/IIIa receptor as key
players involved in angiogenesis and metastasis, we wanted to
investigate whether our compounds with thrombin inhibitory and
GPIIb/IIIa antagonistic activity are endowed with antiangiogenic
activity. Small-molecule multitarget compounds with antith-
rombotic, antiangiogenic and possible antimetastatic activity
would present an interesting synergistic approach in cancer
therapy which has also been reported for phosphomannopentaose
sulfate (PI-88), a multi-component mixture of phosphomanno-
pentaose and phosphomannotetraose sulfates and related heparan
sulfate mimetics [24]. The sulfated oligosaccharide PI-88 is a potent
antiangiogenic and antimetastatic agent which also inhibits
thrombin but does not aggregate platelets [24,25]. In this paper we
(i) report on the antiangiogenic activity of two series of our mul-
titarget compounds combining in the same molecule highly over-
lapped pharmacophores of thrombin inhibitors and GPIIb/IIIa
antagonists and (ii) seek to establish a rough structureeactivity
relationship. and (iii) discuss a possible mechanism responsible
for their inhibition of angiogenesis.
2.2. Pharmacology
2.2.1. Inhibition of cell proliferation
Several in vitro and in vivo assays have been developed that
recapitulate different steps of the angiogenesis process, including
endothelial cell proliferation, migration and tube formation [28].
We first investigated the anti-proliferative activity of 1,4-
benzoxazine compounds 1a and 1b, nitriles 2a and 2b [1,2,4],tri-
azolo[4,3,b]pyridazines 2c, 1,4-benzodioxines 10 and 11, as well as
compounds 16 and 17 lacking a basic P1 moiety, in two endothelial
cell lines [human microvascular endothelial cells (HMEC-1) and
bovine aortic endothelial cells (BAEC)] [29,30]. The results collected
in Table 1 demonstrate that esters 1a and 1b inhibit the prolifera-
tion of both endothelial cell lines equally well, with IC50 values of
7-N-alkylamino compounds 1b1e1b4 ranging from 1.8 to 4.1 mM
and from 4.6 to 7.9 micromolar for 7-N-acylamino compounds
1b5e1b8. Also the 6-substituted compounds 1a1e1a8 showed
anti-proliferative activity with a trend toward more pronounced
cytostatic activity of 6-N-alkylamino compounds 1a1e1a4 (IC50
ranging from 3.8 to 6.7
mM) versus 6-N-acylamino compounds
1a5e1a8 (IC50 ranging from 6.6 to 17.9
mM). In both endothelial cell
lines the N-acylamino-1,4-benzodioxine compounds (S)-10a and
10b were found to be about 3-fold weaker inhibitors of cell
proliferation than the corresponding 1,4-benzoxazine compounds.
[1,2,4]Triazolo[4,3-b]pyridazine compounds 2c (R2 ¼ Et) lacking
a basic benzamidine moiety inhibited proliferation of BAEC and
2. Results and discussion
HMEC-1 (IC50 values between 33.5 and 46.0 mM; results not shown)
although they were found to be up to 10-fold less potent than
amidines 1a and 1b. The tested 6-and 7-substituted nitriles 2a and
2b, showing a high similarity to amidines 1a and 1b, were either
inactive (i.e. compounds 2a1 and 2a4) or weakly active (i.e.
2.1. Chemistry
The design and synthesis of 1,4-benzoxazine compounds repre-
sented by general structures 1a and 1b has been described recently
[22,23]. They comprise highly integrated pharmacophores of
thrombin inhibitors (a P1 benzamidine group, a P2 benzoxazine core
and P3 N-carboxymethyl-benzylamino or N-oxalyl-benzylamino
moieties) and GPIIb/IIIa antagonists (a benzamidine moiety sepa-
compound 2b4) with IC50 values in the range of 24.6e49.7
mM.
Compounds 16a and 16b, lacking the basic benzamidino moiety
as well, also displayed no (i.e. compound 16a) or a weak (i.e.
compound 16b) inhibition of HMEC-1 and BAEC proliferation (IC50
values of 89.4 and 59.6 mM respectively), supporting the view that
rated by
a
2-hydroxymethyl-6/7-methylamino-1,4-benzoxazine
in the tested series of compounds a benzamidine moiety is
important for antiproliferative activity. All tested carboxylic acids
series (1a and 1b, 2c: R2 ¼ H; 11a and 11b; 17a and 17b) were
spacer from a carboxylate group). The preparation of nitriles 2a,b
[22,23] and [1,2,4]triazolo[4,3,b]pyridazine analogs 2c [26] has also
been described (Fig. 1). The synthesis of 1,4-benzodioxine analogs
10a,b and 11a,b is presented in Schemes 1 and 2. The reaction of 4-
nitrocatechol (3) with epichlorohydrin (4) in the presence of sodium
hydrogen carbonate in N,N-dimethylformamide according to a pub-
lished procedure [27] afforded (7-nitro-2,3-dihydrobenzo[b][1,4]
dioxin-2-yl)methanol (5b) whereas the reaction of 3 with epichlo-
rohydrin (4) using sodium hydride as a base gave the 6-nitro isomer
5a (Scheme 1). Both nitro isomers were reacted with 4-
hydroxybenzonitrile under Mitsunobu conditions to give ethers 6a
and 6b which were reduced in the next step to amines 7a and 7b
using catalytic hydrogenation over palladium on charcoal. The
amines were benzylated using benzaldehyde and sodium borohy-
dride and the resulting N-benzylamines 8a and 8b acylated with
ethyl oxalyl chloride to give compounds 9a and 9b. They afforded
amidines 10a,b upon Pinner reaction, the ester group of which was
hydrolyzed to the carboxylic acids 11a and 11b (Scheme 2).
devoid of anti-proliferative activity (IC50 > 100
mM; results not
shown), suggesting that cellular uptake, which may be significantly
hampered in zwitterionic compounds, is required for anti-
proliferative activity.
Next, all compounds were evaluated for their capacity to inhibit
the proliferation of two carcinoma cell lines [human cervical
carcinoma cells (HELA) and human breast carcinoma cells (MCF-7)]
(Table 1). The compounds showed comparable anti-proliferative
activity in tumor cells and endothelial cells, the most active
compounds being 6-N-alkylamino compounds 1a1e1a4 and 7-N-
alkylamino compounds 1b1e1b4 with IC50 values between 3.5 and
5.3 mM. These data indicate that the compounds show no selectivity
toward any of the tested cell types.
Several compounds not only inhibited cell growth (i.e. cyto-
static action) but, at a higher concentration, also induced cell
death (i.e. cytotoxic action). In particular, all 1,4-benzoxazine
The preparation of compounds 17a and 17b, lacking the basic
benzamidine moiety is presented in Scheme 3. The 2-(hydrox-
ymethyl)-2H-benzo[b][1,4]oxazine derivative 12 [22] was acety-
lated with acetic anhydride to give ester 13, which upon catalytic
compounds were toxic at 100
shown). Both 6-and 7-N-alkylamino series 1a1e1a4 and 1b1e1b4
were still toxic at 30 M, whereas the respective N-acylamino
series 1a5e1a8 and 1b5e1b8 displayed no toxicity at this
mM after 3 days in culture (not
m

ꢁ
162
M. Ilic et al. / European Journal of Medicinal Chemistry 58 (2012) 160e170
R1
NH
NH
R1
NH2
NH2
CH3
CH3
O
N
X
N
O
O
N
O
O
N
R2O
O
CH3
CH3
X
OR2
1a
1b
R1 = H; 3-F; 4-F; 3,4-difluoro; 3,5-difluoro; R2 = H, Et; X = H, H; O
R1
R1
CN
N
CH3
CH3
N
O
N
O
N
O
O
N
O
O
N
N
R3
R2O
N
EtO
O
CH3
CH3
2c: R1 = H, 4-F; 3,5-difluoro; R2 = H, Et, R3 = H, Me
2a: substitution in position 6; R1 = 3-F; 3,5-difluoro
2b: substitution in position 7; R1 = 4-F; 3,5-difluoro
Fig. 1. 1,4-Benzoxazines 1a, 1b with thrombin inhibitory and GPIIb/IIIa antagonistic activity, intermediary nitriles 2a and 2b and [1,2,4]triazolo[4,3,b]pyridazine analogs 2c.
concentration. In contrast to benzoxazines 1a and 1b the benzo-
dioxine compounds (S)-10a and 10b were not toxic at 100 M (not
shown). These results highlight the contribution of the N-ethyl
oxalyl substituent and 1,4-benzodioxine scaffold to lowering the
toxicity of these compound series.
migration was still present at 3 mM concentration for 1a1, 1a4, 1a5,
1b2, and 1b8 (Fig. 2). The most potent inhibitor of endothelial cell
migration was 1b2, which showed 95%, 78% and 41% inhibition of
m
cell migration at 30, 10, and 3
mM, respectively. Interestingly,
compound 10b of the 7-N-acylaminobenzodioxine series and
compounds 16a and 16b without a basic benzamidine moiety,
which showed only modest anti-proliferative activities, displayed
2.2.2. Inhibition of endothelial cell migration
Endothelial cell migration is an essential step in angiogenesis.
Therefore, compounds with anti-proliferative activity (i.e. 1,4-
benzoxazines of 1a and 1b series, benzodioxine 10b, compounds
16a and 16b) were tested for possible inhibition of endothelial cell
migration in a wound closure assay [28,30]. As shown in Fig. 2,
a clear dose-dependent inhibitory effect was observed for all
compounds. Also here, there was a trend toward more pronounced
inhibition of cell migration by the 6-N-alkylaminobenzoxazine
1a1e1a4 and 7-N-alkylaminobenzoxazine series 1b1e1b4 versus
their acyl counterparts 1a5e1a8 and 1b5e1b8. In particular, the N-
alkyl compounds caused a complete (or nearly complete) inhibition
a potent inhibition of MAEC migration at 30
mM (more than 80%
inhibition) and 10 M (more than 40% inhibition) (Fig. 2).
m
2.2.3. Inhibition of tube formation
One of the most specific tests for angiogenesis is the matrigel
tube formation assay which measures the ability of endothelial
cells to form three-dimensional structures (tubes) [30,31]. Among
the compounds tested at 30 and 10 mM concentration, 6-N-alky-
laminobenzoxazine derivatives 1a1e1a4 as well as 7-N-alkylami-
nobenzoxazine derivatives 1b1e1b4 completely inhibited tube
formation at 30 mM and were weakly active or inactive at 10 mM.
of MAEC migration at 30
m
M and still inhibited wound closure by
The corresponding N-acylamino compounds 1a5e1a8 and 1b5e
1b8 as well as the 7-N-acylaminobenzodioxine derivative 10b,
about 50% at 10 M. A higher than 40% inhibition of MAEC cell
m
O2N
O
a
OH
O
5b
O2N
OH
OH
Cl
O
+
3
4
O
O
OH
b
O2N
5a
Scheme 1. Synthesis of the benzodioxine scaffold. Reagents and conditions: (a) NaHCO3, DMF, 80 ꢀC, 12 h, (b) NaH, DMF, 80 ꢀC, 12 h.

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M. Ilic et al. / European Journal of Medicinal Chemistry 58 (2012) 160e170
163
CN
CN
c
O
O
O
O
O
b
a
O
OH
O
O2N
H2N
O2N
O
7a,b
5a,b
6a,b
CN
CN
O
O
d
O
O
e
O
O
HN
N
O
O
EtO
8a,b
9a,b
NH2
NH
NH2
NH
O
O
O
O
f
O
O
N
N
O
O
O
O
HO
EtO
10a,b
11a,b
Scheme 2. Synthesis of target 1,4-benzoxazine compounds: (a) 4-cyanophenol, PPh3, DIAD, THF, reflux, 48 h; (b) H2, Pd/C, 25 bar, rt, 1 h; (c) benzaldehyde, MeOH, mol. sieves, rt,
12 h, then NaBH4, 1 h; (d) ethyl oxalyl chloride, Et3N, DCM, rt, 2 h; (e) HClg, EtOH, 0 ꢀC, 30 min, rt, 24 h, then CH3COONH4, rt 24 h; (f) 1M LiOH, THF/MeOH, rt, 2 h.
being less toxic than the corresponding N-alkylamino compounds
exhibited a concentration-dependent inhibition of tube formation.
Interestingly, compounds 16a and 16b, lacking a basic benzamidino
series (1a, 1b; R2 ¼ Et) and N-acylamino esters of the of the ben-
zodioxine series ((S)-10a, 10b) were found to be potent inhibitors of
angiogenesis in the CAM assay at 250 nmol/disc, while in the same
experiment the corresponding carboxylic acids (1a, 1b; R2 ¼ H and
11a, 11b) were devoid of angiogenesis inhibiting activity (data not
shown). The observed inhibition of angiogenesis may be attributed
to thrombin inhibition, since compounds 1a, 1b, (S)-10a, and 10b
are all moderate to potent thrombin inhibitors with Ki values in the
moiety also inhibited tube formation at 100 and 30 mM (Fig. 3).
2.2.4. Inhibition of angiogenesis in the chick chorioallantoic
membrane (CAM) assay
The CAM assay is an in vivo test in which potential inhibitors of
angiogenesis are assessed by their effect on normal vascular
development in chick embryos [28e30]. Both 6-and 7-N-alkyla-
mino as well as 6-and 7-N-acylamino esters of the benzoxazine
range of 18 nM to 5.05 mM [23] and inhibition of angiogenesis in
chick chorioallantoic membrane by the thrombin inhibitor hirudin
has been reported [7]. Compounds of the carboxylic acids series
O
O
CH3
CH3
CH3
c or d
O2N
O
N
H2N
O
O2N
O
N
a
OH
O
CH3
O
CH3
b
N
CH3
CH3
CH3
14
12
13
O
O
O
O
OEt
OH
R1
CH3
R1
CH3
R1
H
N
CH3
N
O
N
N
O
O
N
e
OH
f
OH
OH
N
CH3
CH3
CH3
15a R1 = H
15b
16a R1 = H
16b R1 = 3,5-difluoro
17a R1 = H
17b R1 = 3,5-difluoro
R
1 = 3,5-difluoro
Scheme 3. Reagents and conditions: (a) acetic anhydride, 100 ꢀC, 5 h; (b) H2, Pd/C, 25 bar, rt, 1 h; (c) benzaldehyde, MeOH, mol. sieves, rt, 12 h, then NaBH4, 1 h; (d) 3,5-
difluorobenzaldehyde, MeOH, mol. sieves, rt, 12 h, then NaBH4, 1 h; (e) ethyl oxalyl chloride, Et3N, DCM, rt, 2 h; (f) 1M LiOH, THF/MeOH, rt, 2 h.

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M. Ilic et al. / European Journal of Medicinal Chemistry 58 (2012) 160e170
164
Table 1
Antiproliferative activity in HMEC-1, BAEC, HELA and MCF-7 cell lines. Mean ꢁ SD are shown.
.
Comp.
No.
Subst.
Position
R1
X
R2
HMEC-1
IC50 M)
BAEC IC50
M)
HELA IC50
M)
MCF-7
IC50 M)
(
m
(
m
(
m
(m
1a1
1a2
1a3
1a4
1a5
1a6
1a7
1a8
1b1
1b2
1b3
1b4
1b5
1b6
1b7
1b8
2a1
2a4
2b2
2b4
10a(S)
10b
16a
16b
SU5416
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
6
6
7
7
6
7
7
7
e
3-F
4-F
3-F, 4-F
3-F, 5-F
3-F
H,H
H,H
H,H
H,H
O
O
O
O
H,H
H,H
H,H
H,H
O
O
O
O
e
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
e
6.0 ꢁ 0.1
6.0 ꢁ 1.1
4.2 ꢁ 0.4
6.7 ꢁ 0.0
7.7 ꢁ 1.1
8.4 ꢁ 0.1
8.3 ꢁ 0.8
7.8 ꢁ 1.1
2.6 ꢁ 0.1
2.9 ꢁ 0.7
2.5 ꢁ 0.1
1.8 ꢁ 0.1
7.9 ꢁ 0.6
7.6 ꢁ 0.4
6.7 ꢁ 0.4
6.8 ꢁ 0.4
>100
3.9 ꢁ 0.1
3.8 ꢁ 0.5
4.1 ꢁ 0.3
4.0 ꢁ 0.2
18 ꢁ 4
4.6 ꢁ 0.8
4.8 ꢁ 0.9
4.8 ꢁ 0.9
4.5 ꢁ 1.1
20 ꢁ 4
4.2 ꢁ 0.8
4.1 ꢁ 0.6
3.9 ꢁ 0.7
4.5 ꢁ 0.3
6.5 ꢁ 0.5
5.5 ꢁ 0.8
6.3 ꢁ 1.0
5.2 ꢁ 1.1
3.9 ꢁ 0.4
5.3 ꢁ 0.2
4.0 ꢁ 0.3
3.5 ꢁ 0.9
3.8 ꢁ 1.9
6.3 ꢁ 0.6
5.8 ꢁ 0.8
2.9 ꢁ 0.4
>100
4-F
6.6 ꢁ 1.4
7.4 ꢁ 1.6
11 ꢁ 6
14 ꢁ 7
3-F, 4-F
3-F, 5-F
3-F
14 ꢁ 8
6.9 ꢁ 2.7
4.5 ꢁ 0.5
4.0 ꢁ 0.1
4.1 ꢁ 0.2
4.9 ꢁ 0.5
7.7 ꢁ 0.3
14 ꢁ 4
4.1 ꢁ 0.3
2.9 ꢁ 0.8
4.0 ꢁ 0.7
4.1 ꢁ 0.8
6.5 ꢁ 0.1
6.9 ꢁ 0.5
4.9 ꢁ 1.0
4.6 ꢁ 0.3
>100
4-F
3-F, 4-F
3-F, 5-F
3-F
4-F
3-F, 4-F
3-F, 5-F
3-F
3-F, 5-F
4-F
3-F, 5-F
H
H
11 ꢁ 4
6.6 ꢁ 0.5
>100
e
e
>100
>100
>100
>100
>100
>100
>100
>100
e
e
50 ꢁ 7
e
e
49 ꢁ 1
25 ꢁ 9
54 ꢁ 4
e
e
24 ꢁ 1
12 ꢁ 0
19 ꢁ 2
9.5 ꢁ 2.3
8.7 ꢁ 2.7
>100
e
e
22 ꢁ 3
8.5 ꢁ 0.2
>100
8.0 ꢁ 3.1
>100
H
e
e
>100
3-F, 5-F
e
e
e
89 ꢁ 7
60 ꢁ 9
44 ꢁ 6
45 ꢁ 4
e
e
15 ꢁ 9
8.3 ꢁ 2.2
20 ꢁ 6
1.9 ꢁ 1.5
Fig. 2. MAEC wound closure (migration) assay; results are the mean (ꢁSD) of 2e4 independent experiments. All data p < 0.05, except NS (not significant).

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M. Ilic et al. / European Journal of Medicinal Chemistry 58 (2012) 160e170
165
Fig. 3. Tube formation assay in matrigel. Tube formation was evaluated semi-quantitatively by giving a score from 0 to 3. Representative pictures are shown. Results are the mean
(ꢁSD) of 2e4 independent experiments. *p < 0.05.
Fig. 4. Inhibition of angiogenesis in the CAM assay. At day 11, control CAMs are characterized by a network of blood vessels of different size. Only the capillaries (arrows) are formed
during the course of treatment. At 250 and 100 nmol/disc, compounds 1a4 and 10b cause a complete inhibition of angiogenesis and the disappearance (distruction) of the immature
blood vessels, resulting in a nearly avascular CAM. Only major, pre-existing mature blood vessels are not affected by the compounds. At 40 nmol/disc, only nearly destructed,
tortuous blood vessels can be seen with 1a4, whereas 10b still results in an avascular CAM (not shown). The VEGF antagonist SU5416 was toxic at 100 nmol, but inhibited the
formation of new blood vessels at 40 nmol/disc. n ¼ 6e12, p < 0.05. Mean ꢁ SD are shown.

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M. Ilic et al. / European Journal of Medicinal Chemistry 58 (2012) 160e170
166
(1a, 1b; R2 ¼ H and 11a, 11b), which are also thrombin inhibitors,
although generally an order of magnitude less potent than the
corresponding esters [23], were devoid of anti-angiogenic activity
in the CAM assay (data not shown), again confirming that cell
penetration, which is expected to be impaired in zwitterionic
carboxylic acids, is required for biological activity of these
compounds.
visualization with ultraviolet light. Column chromatography was
carried out on silica gel 60 (particle size 240e400 mesh). Melting
points were determined on a Reichert hot stage microscope and are
uncorrected. 1H NMR and 13C NMR spectra were recorded on
a 400 MHz Bruker AVANCE III spectrometer in DMSO-d6 solution
with TMS as the internal standard. The following abbreviations
were used to describe peak patterns wherever appropriate:
br ¼ broad, d ¼ doublet, dd ¼ doublet of doublet, t ¼ triplet,
q ¼ quartet, and m ¼ multiplet. 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 indi-
cated 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 UVeVIS detector (254 nm), using an Eclips Plus C18 column
(4.6 ꢂ 150 mm) at flow rate 1 mL/min. The eluent was a mixture of
0.1% TFA in water (A) and methanol (B). Gradient was 40% B to 80% B
in 15 min. All of the compounds reported in this paper have a purity
>95% (HPLC). Purifications of final esters by reverse phase column
chromatography were performed using a Flash Purification System
ISOLERAÔ. The eluent was a mixture of 0.1% TFA in water (A) and
methanol (B). Gradient was 40% B to 80% B in 30 column volumes.
Two compounds of each series (i.e. 1a4 of the benzoxazine series
and 10b of the benzodioxine series) were selected for further analysis
at various concentrations. Compound 1a4 (Ki (thrombin) ¼ 0.95
mM)
caused a complete inhibition of angiogenesis and the degradation of
immature, existing vessels at 250, 100 and 40 nmol/disc, and
destruction of vessels with bleeding at 20 nmol/disc. Also 10b
(Ki (thrombin) ¼ 0.178
mM) completely abrogated CAM vascularization
at 250 and 100 nmol/disc and vascular destruction at 40 nmol/disc.
Only major, pre-existing mature blood vessels were not affected by
the compounds (Fig. 4). Both compounds were more potent than the
reference compound SU5416, the latter being toxic at 100 nmol/egg,
and reduced angiogenesis by 47% at 40 nmol/disc.
In summary, esters of 1,4-benzoxazine and 1,4-benzodioxine
series were found to be potent inhibitors of angiogenesis in
CAM assay. However, since complete inhibition of angiogenesis was
elicited by potent thrombin inhibitors such as 1b8 (Ki
¼ 18 nM) as well as by weak thrombin inhibitors such as
(thrombin)
1a4 (Ki (thrombin) ¼ 0.95
m
M) [23] a question arises as to whether
4.1.1. 4-((6-Nitro-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)methoxy)
benzonitrile (6a)
4-Cyanophenol (986 mg, 8.28 mmol) and triphenylphosphine
(3.95 g, 15.06 mmol) were added to a solution of (6-nitro-2,3-
other effects, besides thrombin inhibition, might contribute to their
anti-angiogenic activity.
2.2.5. Radiometric protein kinase assay
dihydrobenzo[b]
[1,4]dioxin-2-yl)methanol
(5a)
(1.59
g,
In order to verify some of the best results obtained by docking to
VEGFR2 variants, compounds 1a4 (R2 ¼ H) and 1a5 were tested for
inhibition of VEGFR-2 kinase in a radiometric protein kinase assay in
which inhibition of poly(Glu,Tyr) 4:1 substrate phosphorylation by
isolated human recombinant VEGFR2 tyrosin kinase was measured
[32]. Both compounds, screened as racemic mixtures, were found to
7.53 mmol) in anhydrous tetrahydrofurane (50 mL). Diisopropyl
azodicarboxylate (DIAD) (3.05 g, 15.06 mmol) dissolved in 10 mL
anhydrous THF was added dropwise at 0 ꢀC, the solution was stirred
afterward for 30 min at 0 ꢀC, and then heated to reflux for 48 h. The
reaction mixture was evaporated in vacuo to dryness and the
residue purified by column chromatography on silica gel (hex-
ane:ethyl acetate ¼ 2:1). A yellow oil obtained was recrystallized
from methanol to give 1.30 g (yield 55%) of pale yellow crystals; mp
be micromolar inhibitors with IC50 values of 22.5
and 80.0
m
M for 1a4 (R2 ¼ H)
m
M for 1a5. On the other side, it has been proven by IC50
determination that compound 1a4 (R2 ¼ H) inhibits VEGFR2 kinase
in concentration depending manner [32]. From these results we can
assume that, besides inhibiting of thrombin, the inhibition of
angiogenesis observed by compounds 1 and 10 in the CAM assay,
could to some extent also be due to slowing down of VEGFR2 activity.
171e174 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d
ppm 4.33 (dd, J ¼ 11.7,
7.3 Hz, 1H, 3-CH2), 4.38 (dd, J ¼ 11.1, 5.6 Hz, 1H, CH2O), 4.45 (dd,
J ¼ 11.1, 3.7 Hz, 1H, CH2O), 4.62 (dd, J ¼ 11.7, 2.5 Hz, 1H, 3-CH2),
0
0
4.72e4.78 (m, 1H, 2-CH), 7.15e7.28 (m, 3H, AreH8, AreH2 , AreH6 ),
0
0
7.79e7.88 (m, 4H, AreH5, AreH7, AreH3 , AreH5 ); 13C NMR
(101 MHz, DMSO-d6): ppm 65.0 (C-3), 66.5 (CH2O), 71.4 (C-2),
d
3. Conclusion
103.4 (C-40), 112.7 (C-7), 115.7 (C-20, C-60), 117.5, 117.6 (C-5, C-8),
119.0 (CN), 134.2 (C-30, C-50), 141.2 (C-6), 142.4, 149.0 (C-4a, C-8a),
161.4 (C-10); HRMS (ESI) m/z calcd for C16H13N2O5 [M þ H]þ
313.0824, found 313.0814; IR (KBr, v, cmꢃ1): 2227, 1603, 1514, 1349,
1256, 1174, 839; HPLC: 100%, tr 16.0 min.
In conclusion, 1,4-benzoxazine and 1,4-benzodioxine
compounds 1 and 10, which combine thrombin inhibitory and
GPIIb/IIIa antagonistic activity in one molecule, were identified as
potent inhibitors of angiogenesis in their ester form. The corre-
sponding carboxylic acids were devoid of antiangiogenic activity,
most probably due to their insufficient entry into the cell. Although
thrombin inhibition remains the most probable explanation for
their inhibition of angiogenesis by compounds 1 and 10, other
targets such as VEGFR2 might be involved. Future experiments
should reveal the exact mechanism of action and potential anti-
tumor and/or anti-metastatic activity of these compounds.
4.1.2. 4-((7-Nitro-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)methoxy)
benzonitrile (6b)
Compound 6b was prepared from 5b (1.59g, 7.53 mmol) and 4-
cyanophenol (986 mg, 8.28 mmol) according to the procedure
described above for the synthesis of 6a; pale yellow crystals; yield
1.00 g (43%); mp 169e172 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d ppm
4.33 (dd, J ¼ 11.7, 7.3 Hz, 1H, 3-CH2), 4.38 (dd, J ¼ 11.1, 5.7 Hz, 1H,
CH2O), 4.45 (dd, J ¼ 11.1, 3.7 Hz, 1H, CH2O), 4.62 (dd, J ¼ 11.7, 2.5 Hz,
1H, 3-CH2), 4.68e4.81 (m, 1H, 2-CH), 7.14e7.27 (m, 3H, AreH50 , Are
4. Experimental section
0
0
0
H2 , AreH6 ), 7.76e7.86 (m, 4H, AreH6, AreH8, AreH3 , AreH5 ); 13
C
4.1. General
NMR (101 MHz, DMSO-d6): d ppm 64.9 (C-3), 66.5 (CH2O), 71.4 (C-
2), 103.4 (C-40), 112.7 (C-6), 115.7 (C-20, C-60), 117.5, 117.6 (C-5, C-8),
119.0 (CN), 134.2 (C-30, C-50), 141.2 (C-7), 142.5, 149.0 (C-4a, C-8a),
161.4 (C-10); HRMS (ESI) m/z calcd for C16H13N2O5 [M þ H]þ
313.0824, found 313.0828; IR (KBr, v, cmꢃ1): 2225, 1600, 1504, 1349,
1251, 820; HPLC: 100%, tr 16.0 min.
Chemicals were obtained from Acros, Aldrich Chemical Co. and
Fluka and used without further purification. The synthesis of
compound (S)-10a is described in ref. [23]b. Analytical TLC was
performed on silica gel Merck 60 F254 plates (0.25 mm), using

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167
4.1.3. 4-((6-(Benzylamino)-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)
methoxy)benzonitrile (8a)
The oily product was purified by column chromatography using
dichloromethane as eluent to obtain 887 mg (93%) of 9a as pale
A mixture of compound 6a (1.10 g, 3.52 mmol) and 10% Pd/C
(110 mg) in methanol (100 mL) was stirred in a hydrogenator
(25 bar) for 1 h at room temperature. The catalyst was filtered off
and the solvent evaporated in vacuo to give amine 7a (890 mg, 90%)
of which was used in the next step without purification. The crude
amine 7a (890 mg, 3.15 mmol), benzaldehyde (334 mg, 3.15 mmol)
yellow amorphous solid; 1H NMR (400 MHz, DMSO-d6):
d ppm 0.93
(t, J ¼ 7.1 Hz, 3H, CH2CH3), 4.02 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 4.13 (dd,
J ¼ 11.6, 7.4 Hz, 1H, 3-CH2), 4.30 (dd, J ¼ 10.9, 5.7 Hz, 1H, CH2O), 4.37
(dd, J ¼ 10.9, 3.7 Hz, 1H, CH2O), 4.44 (dd, J ¼ 11.6, 2.4 Hz, 1H, 3-CH2),
4.49e4.58 (m, 1H, 2-CH), 4.90 (s, 2H, PhCH2), 6.62 (dd, J ¼ 8.6,
2.7 Hz, 1H, AreH7), 6.78 (d, J ¼ 2.7 Hz, 1H, AreH5), 6.89
0
0
and molecular sieves (3 A) were mixed in methanol (50 mL) under
(d, J ¼ 8.6 Hz, 1H, AreH8), 7.15 (d, J ¼ 9.0 Hz, 2H, AreH2 , AreH6 ),
7.18e7.22 (m, 2H, Ph), 7.25e7.37 (m, 3H, Ph), 7.79 (d, J ¼ 9.0 Hz,
ꢂ
Ar atmosphere and the mixture was stirred at room temperature
for 12 h, until the aldimine formation was completed. The reaction
mixture was carefully treated with solid NaBH4 (191 mg,
5.04 mmol) and stirred for additional 1 h. After filtration and
evaporation of solvent in vacuo, a crude residue was dissolved in
dichloromethane (50 mL) and washed successively with saturated
solution of NaHCO3 (3 ꢂ 50 mL) and brine (1 ꢂ 50 mL). The organic
solution was dried over Na2SO4 and the solvent evaporated under
reduced pressure. The oily product was purified by column chro-
matography using dichloromethane as eluent to obtain 563 mg of
8a as pale yellow crystals; yield 43% (from 6a); mp 88e91 ꢀC; 1H
0
0
2H, AreH3 , AreH5 ); 13C NMR (101 MHz, DMSO-d6):
d ppm 13.4
(CH2eCH3), 50.8 (Ph-CH2), 61.3 (CH2eCH3), 64.4 (C-3), 66.6 (CH2O),
71.3 (C-2), 103.3 (C-40), 115.6 (C-20, C-60), 116.2 (C-7), 117.4 (C-5),
119.0 (CN), 120.9 (C-8), 127.5 (C-400), 128.0 (C-200, C-600), 128.6 (C-300,
C-500), 132.3 (C-6), 134.2 (C-30, C-50), 136.1 (C-10), 142.6, 142.7 (C-4a,
C-8a), 161.4, 161.5, 162.4 (COeCOO, COeCOO, C-10); HRMS (ESI) m/z
calcd for C27H25N2O6 [M þ H]þ 473.1713, found 473.1711; IR (KBr, v,
cmꢃ1): 2224, 1605, 1507, 1252, 1175, 1016, 834; HPLC: 100%, tr
17.6 min.
NMR (400 MHz, DMSO-d6):
d
ppm 4.00 (dd, J ¼ 11.4, 7.2 Hz, 1H,
4.1.6. Ethyl 2-(benzyl(3-((4-cyanophenoxy)methyl)-2,3-dihydrobenzo
[b][1,4]dioxin-7-yl)amino)-2-oxoacetate (9b)
3-CH2), 4.18 (d, J ¼ 5.9 Hz, 2H, PhCH2), 4.18e4.31 (m, 3H, 3-CH2,
CH2O), 4.40e4.51 (m, 1H, 2-CH), 5.93 (t, J ¼ 5.9 Hz, 1H, NH), 6.07
(d, J ¼ 2.6 Hz, 1H, AreH5), 6.15 (dd, J ¼ 8.7, 2.6 Hz, 1H, AreH7), 6.62
Compound 9b was prepared from 8b (753 mg, 2.02 mmol) and
ethyl oxalyl chloride (330 mg, 2.42 mmol) according to the proce-
dure described above for the synthesis of 9a; pale yellow solid,
0
0
(d, J ¼ 8.7 Hz, 1H, AreH8), 7.16 (d, J ¼ 9.0 Hz, 2H, AreH2 , AreH6 ),
7.18e7.26 (m, 1H, Ph), 7.27e7.35 (m, 4H, Ph), 7.78 (d, J ¼ 9.0 Hz,
yield 811 mg (85%); 1H NMR (400 MHz, DMSO-d6):
d ppm 0.93
0
0
2H, AreH3 , AreH5 ); 13C NMR (101 MHz, DMSO-d6):
d
ppm 47.0
(t, J ¼ 7.1 Hz, 3H, CH2CH3), 4.01 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 4.11 (dd,
J ¼ 11.6, 7.4 Hz, 1H, 3-CH2), 4.30 (dd, J ¼ 11.1, 5.8 Hz, 1H, CH2O), 4.37
(dd, J ¼ 11.1, 3.7 Hz, 1H, CH2O), 4.44 (dd, J ¼ 11.6, 2.4 Hz, 1H, 3-CH2),
4.56e4.64 (m, 1H, 2-CH), 4.90 (s, 2H, PhCH2), 6.62 (dd, J ¼ 8.6,
2.5 Hz, 1 ¼ , AreH6), 6.78 (d, J ¼ 2.5 Hz, 1H, AreH8), 6.89
(Ph-CH2), 64.2 (C-3), 66.9 (CH2O), 71.4 (C-2), 100.3 (C-5), 103.2 (C-
40), 106.3 (C-7), 115.6 (C-20, C-60), 117.1 (C-8), 119.0 (CN), 126.4
(C-400), 127.1 (C-200, C-600), 128.2 (C-300, C-500), 133.6 (C-8a), 134.2
(C-30, C-50), 140.4 (C-6), 142.7 (C-100), 143.9 (C-4a), 161.6 (C-10);
HRMS (ESI) m/z calcd for C23H21N2O3 [M þ H]þ 373.1552, found
373.1552; IR (KBr, v, cmꢃ1): 2219, 1606, 1504, 1260, 1174, 1045, 830;
HPLC: 98.1%, tr 11.7 min.
0
0
(d, J ¼ 8.6 Hz, 1H, AreH5), 7.15 (d, J ¼ 9.0 Hz, 2H, AreH2 , AreH6 ),
7.18e7.24 (m, 2H, Ph), 7.25e7.38 (m, 3H, Ph), 7.79 (d, J ¼ 9.0 Hz,
0
0
2H, AreH3 , AreH5 ); 13C NMR (DMSO-d6)
d ppm: 13.4 (CH2eCH3),
50.8 (Ph-CH2), 61.3 (CH2eCH3), 64.4 (C-3), 66.6 (CH2O), 71.3 (C-2),
103.3 (C-40), 115.6 (C-20, C-60), 116.1 (C-6), 117.4 (C-5), 119.0 (CN),
120.9 (C-8), 127.5 (C-400), 128.0 (C-200, C-600), 128.6 (C-300, C-500),
132.3 (C-7), 134.2 (C-30, C-50), 136.1 (C-100), 142.6, 142.7 (C-4a, C-8a),
161.4, 161.5, 162.4 (COeCOO, COeCOO, C-10); HRMS (ESI) m/z calcd
for C27H25N2O6 [M þ H]þ 473.1713, found 473.1725; IR (KBr, v,
cmꢃ1): 2225, 1741, 1670, 1606, 1508, 1256, 1172, 1029, 835; HPLC:
100%, tr 17.5 min.
4.1.4. 4-((7-(Benzylamino)-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)
methoxy)benzonitrile (8b)
Starting from 6b (1.10 g, 3.52 mmol) and benzaldehyde (334 mg,
3.15 mmol), compound 8b was prepared according to the proce-
dure described above for the synthesis of 8a; yellow crystals, yield
539 mg (41% from 6b); mp 115e118 ꢀC; 1H NMR (400 MHz, DMSO-
d6):
d
ppm 4.00 (dd, J ¼ 11.5, 7.2 Hz, 1H, 3-CH2), 4.18 (d, J ¼ 5.6 Hz,
2H, PhCH2), 4.20e4.33 (m, 3H, 3-CH2, CH2O), 4.42e4.57 (m, 1H,
2-CH), 5.93 (t, J ¼ 5.6 Hz, 1H, NH), 6.07 (d, J ¼ 2.6 Hz, 1H, AreH8),
6.15 (dd, J ¼ 8.7, 2.6 Hz, 1H, AreH6), 6.62 (d, J ¼ 8.7 Hz, 1H, Are
4.1.7. Ethyl 2-(benzyl(2-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)amino)-2-oxoacetate
trifluoroacetate (10a)
0
0
H5), 7.16 (d, J ¼ 9.0 Hz, 2H, AreH2 , AreH6 ), 7.19e7.25 (m, 1H, Ph),
0
0
7.27e7.38 (m, 4H, Ph), 7.78 (d, J ¼ 9.0 Hz, 2H, AreH3 , AreH5 ); 13
C
Gaseous HCl was slowly introduced over 30 min into a solution
of the nitrile 9a (482 mg, 1.02 mmol) in anhydrous ethanol (30 mL).
The reaction mixture was closed tightly and stirred for 24 h at room
temperature. The solvent was evaporated in vacuo and the residue
washed 3 times with anhydrous diethyl ether. The iminoether
obtained was dissolved in anhydrous EtOH (30 mL), ammonium
acetate (236 mg, 3.06 mmol) was added and the reaction mixture
stirred for 24 h at room temperature. The solvent was evaporated
in vacuo and the crude product purified by gradient reverse
phase column chromatography using methanol/trifluoroacetic
acid (40e80% in 30 min) as eluent. After evaporation of volatiles,
white crystals were precipitated from trifluoroacetic acid, filtered
off and dried to yield 307 mg (50%) of 10a as a white powder; mp
NMR (101 MHz, DMSO-d6):
d ppm 47.0 (Ph-CH2) 64.2 (C-3), 66.7
(CH2O), 71.4 (C-2), 100.3 (C-8), 103.2 (C-800), 106.3 (C-6), 115.6 (C-200,
C-60), 117.1 (C-5), 119.0 (CN), 126.5 (C-400), 127.1 (C-200, C-600), 128.2
(C-300, C-500), 133.6 (C-4a), 134.2 (C-30, C-50), 140.4 (C-7), 142.7
(C-100), 143.9 (C-8a), 161.6 (C-10); HRMS (ESI) m/z calcd for
C
23H21N2O3 [M þ H]þ 373. 1552, found 373.1546; IR (KBr, v, cmꢃ1):
3387, 3050, 2220, 1508, 1262, 1174, 832; HPLC: 98.8%, tr 11.7 min.
4.1.5. Ethyl 2-(benzyl(2-((4-cyanophenoxy)methyl)-2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)amino)-2-oxoacetate (9a)
Ethyl oxalyl chloride (330 mg, 2.42 mmol) was added to a solu-
tion of 8a (753 mg, 2.02 mmol) and triethylamine (245 mg,
2.42 mmol) in dichloromethane (50 mL) and the mixture stirred for
2 h. The solvent was removed under reduced pressure, the residue
dissolved in ethyl acetate (50 mL) and washed successively with
a 10% citric acid solution (3 ꢂ 50 mL), saturated NaHCO3 solution
(3 ꢂ 50 mL) and brine (1 ꢂ 50 mL). The organic phase was dried
over Na2SO4 and the solvent evaporated under reduced pressure.
192e194 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d
ppm 0.93 (t, J ¼ 7.1 Hz,
3H, CH2CH3), 4.02 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 4.14 (dd, J ¼ 11.7,
7.4 Hz, 1H, 3-CH2), 4.32 (dd, J ¼ 11.0, 5.7 Hz, 1H, CH2O), 4.39 (dd,
J ¼ 10.9, 3.6 Hz, 1H, CH2O), 4.46 (dd, J ¼ 11.6, 2.4 Hz, 1H, 3-CH2),
4.57e4.66 (m, 1H, 2-CH), 4.91 (s, 2H, PhCH2), 6.63 (dd, J ¼ 8.6,
2.7 Hz, 1H, AreH8), 6.79 (d, J ¼ 2.7 Hz, 1H, AreH7), 6.89

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M. Ilic et al. / European Journal of Medicinal Chemistry 58 (2012) 160e170
168
0
0
(d, J ¼ 8.6 Hz, 1H, AreH5), 7.16e7.25 (m, 4H, Ph, AreH2 , AreH6 ),
white powder, yield 69 mg (60%); mp 291e294 ꢀC; 1H NMR
(400 MHz, DMSO-d6):
0
0
7.27e7.37 (m, 3H, Ph), 7.83 (d, J ¼ 9.0 Hz, 2H, AreH3 , AreH5 ),
d
ppm 4.13 (dd, J ¼ 11.6, 7.9 Hz, 1H, 3-CH2),
8.92 (s, 2H, NH2), 9.16 (s, 2H, NHþ2 ); 13C NMR (101 MHz, DMSO-
4.34 (dd, J ¼ 10.9, 5.6 Hz, 1H, CH2O), 4.40 (dd, J ¼ 10.9, 3.6 Hz, 1H,
CH2O), 4.48 (dd, J ¼ 11.6, 2.2 Hz, 1H, 3-CH2), 4.55e4.65 (m, 1H,
2-CH), 4.88 (s, 2H, PhCH2), 6.68 (dd, J ¼ 8.6, 2.5 Hz, 1H, AreH6), 6.80
(d, J ¼ 2.5 Hz, 1H, AreH8),06.89 (d, J ¼ 8.6 Hz, 1H, AreH5),07.18e7.37
d6):
d ppm 13.4 (CH2eCH3), 50.8 (Ph-CH2), 61.3 (CH2eCH3), 64.4
(C-3), 66.7 (CH2O), 71.3 (C-2), 114.8 (C-20, C-60), 116.2 (C-7), 117.1
(CF3eCOOH, 1JC,F ¼ 299.3 Hz), 117.4 (C-5), 120.1 (C-40), 120.9 (C-8),
127.6 (C-400),128.0 (C-200, C-600),128.6 (C-300, C-500),130.2 (C-30, C-50),
132.3 (C-6), 136.1 (C-100), 142.6, 142.7 (C-4a, C-8a), 158.7 (CF3e
0
0
(m, 7H, Ph, AreH2 , AreH6 ), 7.83 (d, J ¼ 8.9 Hz, 2H, AreH3 , AreH5 ),
8.91 (s, 2H, NH2), 9.17 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6)
2
COOH, JC,F ¼ 31.5 Hz), 161.5, 162.3, 162.4, 164.5 (COeCOO, COe
d
ppm: 50.6 (Ph-CH2), 64.4 (C-3), 66.8 (CH2O), 71.4 (C-2),114.9 (C-20,
COO, C-10, C(]NH)NH2); HRMS (ESI) m/z calcd for C27H28N3O6
[M þ H]þ 490.1978, found 490.1972; IR (KBr, v, cmꢃ1): 3298, 3122,
1737, 1665, 1506, 1203, 843; HPLC: 100%, tr 12.4 min.
C-60), 116.1 (C-8), 117.3 (C-6), 120.1 (C-40), 120.7 (C-5), 127.4 (C-400),
127.8 (C-200, C-600), 128.5 (C-300, C-500), 130.2 (C-30, C-50), 133.0 (C-7),
136.3 (C-100), 142.5, 142.6 (C-4a, C-8a), 162.3, 163.1, 164.2, 164.5
(COeCOO, COeCOO, C-10, C(]NH)NH2); HRMS (ESI) m/z calcd for
4.1.8. Ethyl 2-(benzyl(3-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydrobenzo[b][1,4]dioxin-7-yl)amino)-2-oxoacetate
trifluoroacetate (10b)
C
25H24N3O6 [M þ H]þ 462.1665, found 462.1674; IR (KBr, v, cmꢃ1):
3309, 1609, 1492, 1262, 839; HPLC: 96.2%, tr 9.7 min.
Compound 10b was prepared from nitrile 9b (482 mg,1.02 mmol)
according to procedure described above for the synthesis of 10a;
white powder, yield 335 mg (54%); mp 185e188 ꢀC; 1H NMR
4.1.11. (2,4-Dimethyl-7-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-
2-yl)methyl acetate (13)
Alcohol 12 [22] (2.00 g, 8.4 mmol) was dissolved in acetic
anhydride (50 mL) and heated at 100 ꢀC for 5 h. The excess acetic
anhydride was removed in vacuo to yield 2.02 g (86%) of 13 as
(400 MHz, DMSO-d6):
d
ppm 0.91 (t, J ¼ 7.1 Hz, 3H, CH2CH3), 4.00 (q,
J ¼ 7.1 Hz, 2H, CH2CH3), 4.15 (dd, J ¼ 11.6, 7.2 Hz, 1H, 3-CH2), 4.31 (dd,
J ¼ 11.1, 6.1 Hz,1H, CH2O), 4.39(dd, J ¼ 10.9, 3.4 Hz,1H, CH2O), 4.46(dd,
J ¼ 11.6, 2.2 Hz, 1H, 3-CH2), 4.58e4.64 (m, 1H, 2-CH), 4.90 (s, 2H,
brown oil; 1H NMR (400 MHz, DMSO-d6)
d ppm 1.36 (s, 3H, 2-CH3),
2.09 (s, 3H, COCH3), 2.99 (s, 3H, NCH3), 3.16 (d, J ¼ 12.1 Hz, 1H, 3-H),
3.32 (d, J ¼ 12.1 Hz, 1H, 3-H), 4.15 (d, J ¼ 11.6 Hz, 1H, CH2O), 4.20 (d,
J ¼ 11.6 Hz, 1H, CH2O), 6.92 (d, J ¼ 8.8 Hz, 1H, AreH5), 7.51 (d,
J ¼ 2.6 Hz,1H, AreH8), 7.58 (dd, J ¼ 8.8, 2.6 Hz, 1H, AreH6); 13C NMR
PhCH2), 6.63(dd, J ¼ 8.7, 2.5 Hz,1H, AreH6), 6.80(d, J ¼ 2.5 Hz,1H, Are
0
H8), 6.89 (d, J ¼ 8.7 Hz,1H, AreH5), 7.15e7.038 (m, 7H, Ph, AreH2 , Are
0
0
H6 ), 7.83 (d, J ¼ 8.9 Hz, 2H, AreH3 , AreH5 ), 8.91 (s, 2H, NH2), 9.17 (s,
2H, NHþ2 ); 13C NMR (101 MHz, DMSO-d6)
d
ppm: 13.3 (CH2eCH3), 50.8
(101 MHz, DMSO-d6) d ppm 20.5 (COCH3), 20.6 (2-CH3), 38.0
(Ph-CH2), 61.3 (CH2eCH3), 64.4 (C-3), 66.7 (CH2O), 71.3(C-2),114.8 (C-
20, C-60), 116.2 (C-6), 117.1 (CF3eCOOH, 1JC,F ¼ 299.2 Hz), 117.3 (C-5),
120.1(C-40),120.8(C-8),127.5(C-400),127.9(C-200, C-600),128.6(C-300, C-
500), 130.2 (C-30, C-50), 132.5 (C-7), 136.1 (C-100), 142.5, 142.8 (C-4a, C-
8a), 158.8 (CF3eCOOH, 2JC,F ¼ 31.5 Hz), 161.5, 162.3, 162.4, 164.5 (COe
COO, COeCOO, C-10, C(]NH)NH2); HRMS (ESI) m/z calcd for
(NCH3), 52.8 (C-3), 66.0 (CH2O), 75.4 (C-2), 106.2 (C-8), 114.1 (C-6),
115.7 (C-5), 135.4 (C-7), 141.5 (C-8a), 148.2 (C-4a), 170.0 (CO); HRMS
(ESI) m/z calcd for C13H17N2O5 [M þ H]þ 281.1137, found 281.1132;
HPLC: 97.0%, tr 14.3 min; Anal. (C13H16N2O5): C, H, N.
4.1.12. (7-(Benzylamino)-2,4-dimethyl-3,4-dihydro-2H-benzo[b]
[1,4]oxazin-2-yl)methanol (15a)
Prepared from compound 13 (1.66 g, 5.93 mmol) and benzal-
dehyde (548 mg, 5.16 mmol) following the procedure for the
synthesis of compound 8a; yellow oil, yield 1.23 g (70% from 13); 1H
C
27H28N3O6 [M þ H]þ 490.1978, found 490.1970; IR (KBr, v, cmꢃ1):
3345, 3109, 1742, 1670, 1500, 1197, 843; HPLC: 98.4%, tr 12.7 min.
4.1.9. 2-(Benzyl(2-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)amino)-2-oxoacetic acid (11a)
To a solution of ester 10a (150 mg, 0.25 mmol) in tetrahydro-
furan (3 mL) and methanol (1 mL), 1 M LiOH (1.50 mL, 1.50 mmol)
was added and the mixture was stirred for 1 h at room temperature.
The organic solvents were evaporated under vacuum and the
resulting aqueous solution neutralized with 0.1% trifluoroacetic
acid to precipitate the product which was filtered off and dried to
obtain 72 mg (63%) of 11 as a white powder, mp 290e293 ꢀC; 1H
NMR (400 MHz, DMSO-d6) d ppm 1.16 (s, 3H, 2-CH3), 2.74 (s, 3H,
NCH3), 2.81 (d, J ¼ 11.4 Hz, 1H, 3-H), 3.03 (d, J ¼ 11.4 Hz, 1H, 3-H),
3.27 (dd, J ¼ 10.7, 5.8 Hz, 1H, CH2O), 3.35e3.43 (m, overlapped with
H2O, 1H, CH2O), 4.18 (d, 6.13 Hz, NeCH2), 4.90 (t, J ¼ 5.3 Hz, 1H, OH),
5.56 (t, J ¼ 5.6 Hz, 1H, NH), 5.83 (dd, J ¼ 8.4, 2.3 Hz, 1H, AreH6), 6.03
(d, J ¼ 2.3 Hz, 1H, AreH8), 6.37 (d, J ¼ 8.4 Hz, 1H, AreH5), 7.17e7.40
(m, 5H, Ph); 13C NMR (101 MHz, DMSO-d6)
d ppm 20.9 (2-CH3), 38.3
(NCH3), 47.5 (NCH2), 54.2 (C-3), 65.0 (CH2O), 74.5 (C-2), 97.6 (C-6),
101.8 (C-8), 115.5 (C-5), 126.4 (C-40), 127.2 (C-20, C-60), 128.1 (C-30,
C-50), 134.4, 135.5 (C-4a, C-8a), 140.9, 143.1 (C-7, C-10); HRMS (ESI)
m/z calcd for C18H23N2O2 [M þ H]þ 299.1760, found 299.1755;
HPLC: 97.3%, tr 6.5 min; Anal. (C18H22N2O2 ꢂ 1/4H2O): C, H, N.
NMR (400 MHz, DMSO-d6):
d
ppm 4.14 (dd, J ¼ 11.6, 7.6 Hz, 1H,
3-CH2), 4.33 (dd, J ¼ 11.0, 5.6 Hz,1H, CH2O), 4.40 (dd, J ¼ 11.0, 3.7 Hz,
1H, CH2O), 4.46 (dd, J ¼ 11.6, 2.3 Hz, 1H, 3-CH2), 4.57e4.66 (m, 1H,
2-CH), 4.88 (s, 2H, PhCH2), 6.67 (dd, J ¼ 8.6, 2.5 Hz, 1H, AreH7), 6.80
(d, J ¼ 2.5 Hz, 1H, AreH5), 6.89 (d, J ¼ 8.6 Hz, 1H, AreH8), 7.18e7.24
0
0
(m, 4H, Ph, AreH2 ,0 AreH6 ), 7.27e7.35 (m, 3H, Ph), 7.83 (d,
4.1.13. (7-((3,5-Difluorobenzyl)amino)-2,4-dimethyl-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-yl)methanol (15b)
0
J ¼ 8.9 Hz, 2H, AreH3 , AreH5 ), 9.04 (s, 2H, NH2), 9.17 (s, 1H, NH);
13C NMR (101 MHz, DMSO-d6)
d
ppm: 49.6 (Ph-CH2), 64.4 (C-3),
Prepared from compound 13 (1.71 g, 6.10 mmol) and 3,5-
difluorobenzaldehyde (693 mg, 4.88 mmol) following the proce-
dure for the synthesis of compound 8a; yellow oil, yield 1.19 g (58%
66.7 (CH2O), 71.0 (C-2), 114.7 (C-20, C-60), 115.9 (C-7), 116.6 (C-5),
120.2 (C-40), 120.6 (C-8), 127.0 (C-400), 127.6 (C-200, C-600), 128.3
(C-300, C-500), 129.8 (C-30, C-50), 132.3 (C-6), 137.7 (C-100), 141.4,
142.0 (C-4a, C-8a), 162.0, 163.2, 164.0, 164.6 (COeCOO, COeCOO,
C-10, C(]NH)NH2); HRMS (ESI) m/z calcd for C25H24N3O6
[M þ H]þ 462.1665, found 462.1677; IR (KBr, v, cmꢃ1): 3368, 1609,
1503, 1255, 844; HPLC: 96.1%, tr 9.5 min.
from 13); 1H NMR (400 MHz, DMSO-d6)
d ppm 1.16 (s, 3H, 2-CH3),
2.75 (s, 3H, NCH3), 2.82 (d, J ¼ 11.36 Hz, 1H, 3-H), 3.04 (d,
J ¼ 11.36 Hz, 1H, 3-H), 3.28 (dd, J ¼ 10.59, 5.16 Hz, 1H, CH2O), 3.42e
3.36 (m, 1H, CH2O), 4.22 (d, 6.13 Hz, NeCH2), 4.91 (t, J ¼ 5.3 Hz, 1H,
OH), 5.48 (t, J ¼ 5.6 Hz, 1H, NH), 5.79 (dd, J ¼ 8.38, 1.79 Hz, 1H, Are
H6), 6.01 (d, J ¼ 1.68 Hz, 1H AreH8), 6.38 (d, J ¼ 8.40 Hz, 1H, AreH5),
4.1.10. 2-(Benzyl(3-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydrobenzo[b][1,4]dioxin-7-yl)amino)-2-oxoacetic acid (11b)
Compound 11b was prepared from 10b (150 mg, 0.25 mmol)
according to procedure described above for the synthesis of 11a;
6.99e7.12 (m, 3H, 3AreH); 13C NMR (101 MHz, DMSO-d6)
d ppm
20.9 (2-CH3), 38.2 (NCH3), 46.6 (CH2N), 54.2 (C-3), 64.9 (CH2O), 74.5
2
(C-2), 97.7 (C-6), 101.7 (C-8), 101.7 (t, JCeF ¼ 25.9 Hz, C-40), 109.5
2
4
(dd, JCeF ¼ 24.7 Hz, JCeF ¼ 6.3 Hz, C-20, C-60), 115.6 (C-5), 134.7,

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M. Ilic et al. / European Journal of Medicinal Chemistry 58 (2012) 160e170
169
3
135.6, 142.4 (C-7, C-4a, C-8a), 146.6 (t, JCeF ¼ 8.2 Hz, C-10), 162.4
(C-10, C-4a, C-8a), 163.3, 164.4 (COeCOO, COeCOO); HRMS (ESI) m/z
calcd for C20H23N2O5 [M þ H]þ 371.1607, found 371.1594; HPLC:
100%, tr 11.3 min; Anal. (C20H22N2O5): C, H, N.
(dd, 1JCeF ¼ 245.8 Hz, JCeF ¼ 13.1 Hz, C-30, C-50); HRMS (ESI) m/z
3
calcd for C18H21N2O2F2 [M þ H]þ 335.1571, found 335.1558; HPLC:
96.9%, tr 7.8 min; Anal. (C18H20N2O2F2): C, H, N.
4.1.17. 2-((3,5-Difluorobenzyl)(2-(hydroxymethyl)-2,4-dimethyl-3,4-
dihydro-2H-benzo[b][1,4]oxazin-7-yl)amino)-2-oxoacetic acid (17b)
Compound 17b was prepared by hydrolysis of ester 16b
(434 mg, 1 mmol) with 1 M LiOH (6 mL) in a mixture of tetrahy-
drofuran and methanol according to the procedure for the
synthesis of compound 11a to obtain 292 mg (72%) of 17b as brown
4.1.14. Ethyl 2-(benzyl(2-(hydroxymethyl)-2,4-dimethyl-3,4-
dihydro-2H-benzo[b][1,4]oxazin-7-yl)amino)-2-oxoacetate (16a)
Compound 16a was prepared from 15a (483 mg, 1.62 mmol) and
ethyl oxalyl chloride (221 mg, 1.62 mmol) according to the proce-
dure described above for the synthesis of 9a; yellow oil, yield
458 mg (71%); 1H NMR (400 MHz, DMSO-d6)
d
ppm 0.89 (t,
oil; 1H NMR (400 MHz, DMSO-d6)
d ppm 1.19 (s, 3H, 2-CH3), 2.83
J ¼ 7.1 Hz, 3H, CH2CH3), 1.17 (s, 3H, 2-CH3), 2.82 (s, 3H, NCH3), 2.89
(d, J ¼ 11.7 Hz, 1H, 3-H), 3.11 (d, J ¼ 11.7 Hz, 1H, 3-H), 3.29 (dd,
J ¼ 10.8, 5.9 Hz, 1H, CH2O), 3.33e3.43 (m, overlapped with H2O, 1H,
CH2O), 3.97 (q, J ¼ 7.1 Hz, 1H, CH2CH3), 4.85 (s, 2H, CH2N), 5.04 (t,
J ¼ 5.7 Hz, 1H, OH), 6.48 (d, J ¼ 2.2 Hz, 1H, AreH8), 6.51 (dd, J ¼ 8.5,
2.2 Hz, 1H,0AreH6), 6.60 (d, J ¼ 8.5 Hz,1H, AreH5), 7.19 (d, J ¼ 7.4 Hz,
(s, 3H, NCH3), 2.90 (d, J ¼ 11.7 Hz, 1H, 3-H), 3.12 (d, J ¼ 11.7 Hz, 1H,
3-H), 3.31 (d, J ¼ 10.8 Hz, 1H, CH2O), 3.41 (d, J ¼ 10.8 Hz, 1H, CH2O),
4.86 (s, 2H, CH2N), 5.05 (bs, 1H, OH), 6.53e6.66 (m, 3H, AreH8, Are
0
0
H6, AreH5), 6.92 (d, J ¼ 6.6 Hz, 2H, AreH2 , AreH6 ), 7.04e7.18
0
(m, 1H, AreH4 ); 13C NMR (101 MHz, DMSO-d6)
d ppm 20.8
(2-CH3), 38.1 (NCH3), 50.1 (CH2N), 53.2 (C-3), 65.0 (CH2O), 76.0
0
0
2H, AreH2 , AreH6 ), 7.24e7.30 (m, 1H, AreH4 ), 7.33 (m, J ¼ 7.4 Hz,
(C-2), 102.9 (t, 2JCeF ¼ 25.8 Hz, C-40), 110.7 (dd, 2JCeF ¼ 18.6 Hz, 4JCe
0
0
2H, AreH3 , AreH5 ); 13C NMR (101 MHz, DMSO-d6)
d
ppm 13.4
¼ 6.8 Hz, C-20, C-60),111.5 (C-5), 114.1 (C-8),119.7 (C-6),129.0 (C-7),
F
3
(CH2CH3), 20.7 (2-CH3), 38.1 (NCH3), 50.9 (CH2N), 53.2 (C-3), 61.0
(CH2CH3), 64.7 (CH2O), 75.8 (C-2), 111.3 (C-5), 114.5 (C-8), 120.0
(C-6), 127.4 (C-40), 127.8 (C-20, C-60), 128.5 (C-30, C-50), 128.7 (C-7),
135.5, 136.4, 142.5 (C-10, C-4a, C-8a), 161.8, 162.7 (COeCOO, COe
135.4 (C-8a), 141.4 (t, JCeF ¼ 8.9 Hz, C-10), 142.6 (C-4a), 162.4 (dd,
1JCeF ¼ 246.5 Hz, 3JCeF ¼ 13.2 Hz, C-30, C-50), 163.4 (COeCOO), 164, 2
(COeCOO); HRMS (ESI) m/z calcd for C20H21N2O5F2 [M þ H]þ
407.1419, found 407.1414; HPLC: 93.0%, tr 12.4 min; Anal.
(C20H20N2O5F2): C, H, N.
COO); HRMS (ESI) m/z calcd for C22H27N2O5 [M
399.1920, found 399.1916; HPLC: 96.8%, tr 14.5 min; Anal.
(C22H26N2O5): C, H, N.
þ
H]þ
4.1.18. Cell cultures
Bovine aortic endothelial cells (BAEC) were kindly provided by
Prof. M. Presta (Brescia, Italy). Human cervical carcinoma (HELA)
and human breast carcinoma (MCF-7) cells were obtained from the
American Type Culture Collection (ATCC, Middlesex, UK). The cells
were grown in Dulbecco’s modified minimum essential medium
(DMEM, Life Technologies, Inc., Rockville, MD) supplemented with
10 mM Hepes (Life Technologies, Inc., Rockville, MD) and 10% fetal
bovine serum (FBS, Harlan Sera-Lab Ltd., Loughborough, UK).
Human microvascular endothelial cells (HMEC-1) were obtained
from the Centers of Disease Control (CDC, Atlanta, GA) and grown in
EGM-2 medium with supplements and growth factors (Lonza,
Verviers, Belgium).
4.1.15. Ethyl 2-((3,5-difluorobenzyl)(2-(hydroxymethyl)-2,4-
dimethyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)amino)-2-
oxoacetate (16b)
Compound 16b was prepared from 15b (592 mg,1.77 mmol) and
ethyl oxalyl chloride (242 mg, 1.77 mmol) according to the proce-
dure described above for the synthesis of 9a; yellow oil, yield
529 mg (69%); 1H NMR (400 MHz, DMSO-d6)
d ppm 0.90 (t,
J ¼ 7.1 Hz, 3H, CH2CH3), 1.18 (s, 3H, 2-CH3), 2.83 (s, 3H, NCH3), 2.90
(d, J ¼ 11.7 Hz, 1H, 3-H), 3.12 (d, J ¼ 11.7 Hz, 1H, 3-H), 3.30 (dd,
J ¼ 10.8, 4.1 Hz, 1H, CH2O), 3.40 (dd, J ¼ 10.8, 4.1 Hz, 1H, CH2O), 3.99
(d, J ¼ 7.1 Hz, 1H, CH2CH3), 4.88 (s, 2H, CH2N), 5.04 (t, J ¼ 5.7 Hz, 1H,
OH), 6.52e6.57 (m, 2H, AreH80 , AreH6), 6.63 (d, J ¼ 8.3 Hz, 1H, Are
0
0
H5), 6.87e6.94 (m, 2H, AreH2 , AreH6 ), 7.13e7.22 (m, 1H, AreH4 );
4.1.19. Cell proliferation assays
13C NMR (101 MHz, DMSO-d6)
d
ppm 13.4 (CH2CH3), 20.7 (2-CH3),
Cells (HMEC-1, BAEC, HELA or MCF-7) were seeded in 48-well
plates at 10,000 cells per cm2. After 16 h, the cells were incubated
in fresh medium in the presence of the test compounds, as indi-
cated in the Results section. On day 4, (BAEC, HELA, MCF-7) or day 7
(HMEC-1) cells were trypsinized and counted by means of a Coulter
counter (Analis, Belgium). For each compound, the IC50 value was
determined. This is the concentration of compound that causes 50%
inhibition of cell proliferation.
38.1 (NCH3), 50.2 (CH2N), 53.2 (C-3), 61.2 (CH2CH3), 64.7 (CH2O),
75.8 (C-2), 103.0 (t, 2JCeF ¼ 25.8 Hz, C-40), 110.9 (dd, 2JCeF ¼ 18.6 Hz,
4JCeF ¼ 6.8 Hz, C-20, C-60), 111.4 (C-5), 114.2 (C-8), 119.8 (C-6), 128.4
(C-7), 135.7 (C-8a), 141.1 (t, 3JCeF ¼ 9.1 Hz, C-10), 142.6 (C-4a), 161.9
3
(COeCOO), 162.3 (dd, 1JCeF ¼ 246.7 Hz, JCeF ¼ 13.2 Hz, C-30, C-50),
162.5 (COeCOO); HRMS (ESI) m/z calcd for C22H25N2O5F2 [M þ H]þ
435.1732, found 435.1738; HPLC: 97.6%, tr 15.6 min; Anal.
(C22H24N2O5F2): C, H, N.
4.1.20. Cell migration assay
4.1.16. 2-(Benzyl(2-(hydroxymethyl)-2,4-dimethyl-3,4-dihydro-2H-
benzo[b][1,4]oxazin-7-yl)amino)-2-oxoacetic acid (17a)
Compound 17a was prepared by hydrolysis of ester 16a (398 mg,
1 mmol) with 1 M LiOH (6 mL) in a mixture of tetrahydrofuran and
methanol according to the procedure for the synthesis of
compound 11a to obtain 296 mg (78%) of 17a as brown oil; 1H NMR
Wounds were created in confluent MAEC monolayers with a 1.0-
mm wide micropipette tip. Then, cells were incubated in fresh
medium in the presence of the test compounds. After 5 h, the
wounds were photographed and the width of the wound was
calculated from digital pictures. For statistical analyses, the p values
were determined using the Student t test, and p values < 0.05 were
considered significant.
(400 MHz, DMSO-d6)
d ppm 1.18 (s, 3H, 2-CH3), 2.82 (s, 3H, NCH3),
2.89 (d, J ¼ 11.6 Hz, 1H, 3-H), 3.11 (d, J ¼ 11.6 Hz, 1H, 3-H), 3.30 (d,
J ¼ 10.8 Hz, 1H, CH2O), 3.40 (d, J ¼ 10.8 Hz, 1H, CH2O), 4.83 (s, 2H,
CH2N), 5.04 (bs, 1H, OH), 6.48e6.64 (m, 3H, AreH8, AreH6, AreH50),
4.1.21. Tube formation assay
Wells of a 96-well plate were coated with 60 ml matrigel (10 mg/
0
0
7.20 (d, J ¼ 7.5 Hz, 2H, AreH20 , AreH60 ), 7.23e7.29 (m, 1H, AreH4 ),
7.32 (d, J ¼ 7.5 Hz, 2H, AreH3 , AreH5 ); 13C NMR (101 MHz, DMSO-
mL, BD Biosciences, Heidelberg, Germany) at 4 ꢀC. After gelatini-
zation at 37 ꢀC during 30 min, HMEC-1 (60,000 cells) were seeded
d6)
d
ppm 20.8 (2-CH3), 38.1 (NCH3), 50.8 (CH2N), 53.2 (C-3), 64.8
on top of the matrigel in 200 ml DMEM containing the test
compounds. After 4e6 h of incubation, the cells were photographed
and tube formation was quantified by giving a score from 0 (no
(CH2O), 75.9 (C-2), 111.4 (C-5), 114.3 (C-8), 119.9 (C-6), 127.3 (C-40),
127.7 (C-20, C-60), 128.4 (C-30, C-50), 129.3 (C-7), 135.3, 136.7, 142.6

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170
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tubes) to 3 (maximal tube formation, comparable to non-treated
control cultures). Statistical analysis was performed by using the
Student t test, and p values < 0.05 were considered significant.
4.1.22. CAM assay
Fertilized eggs were incubated for 3 days at 37 ꢀC when 3 mL of
albumen was removed (to detach the shell from the developing
CAM) and a window was opened on the eggshell exposing the CAM.
The window was covered with cellophane tape and the eggs were
returned to the incubator until day 9 when the compounds were
applied. The compounds were placed on sterile plastic discs (Ø
8 mm), which were allowed to dry under sterile conditions. A
solution of cortisone acetate (100 lg/disc, Sigma, St. Louis, MO) was
added to all discs in order to prevent an inflammatory response. A
loaded and dried control disc was placed on the CAM approxi-
mately 1 cm away from the disc containing the test compound(s).
Next, the windows were covered and the eggs further incubated
until day 11 when the area around the discs was cut-off and pho-
tographed. Next 2 concentric circles were positioned on the digi-
talized pictures and all vessels intersecting these circles were
counted. Statistical analysis was performed by using the Student t
test, and p values < 0.05 were considered significant.
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4.1.23. In vitro VEGFR2 kinase assay
A radiometric protein kinase assay (33PanQinaseÒ Activity
Assay) was used for measuring the kinase activity of the VEGFR2
protein kinase. VEGFR2 tyrosine kinase was expressed in Sf9 insect
cells as human recombinant GST-fusion protein. The kinase was
purified by affinity chromatography using GSH-agarose. The purity
of the kinase was checked by SDS-PAGE/silver staining and the
identity of the kinase was verified by mass spectroscopy. The IC50
profile of two compounds 1a4 and 1a5 (both in racemic form as
trifluoroacetate salts) was determined. IC50 values were measured
by testing 10 concentrations (1 ꢂ 10ꢃ4 M to 3 ꢂ 10ꢃ9 M) of each
compound. The measurements have been performed by ProQinase
company in singlicate [32].
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dual function. Discovery of 1,4-benzoxazin-3(4H)-one derivatives possessing
thrombin inhibitory and fibrinogen receptor antagonistic activities, J. Med.
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ꢀ
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[22] J. Ilas, Z. Jakopin, T. Borstnar, M. Stegnar, D. Kikelj, 3,4-Dihydro-2H-1,4-
benzoxazine derivatives combining thrombin inhibitory and glycoprotein
IIb/IIIa receptor antagonistic activity as
a novel class of antithrombotic
compounds with dual function, J. Med. Chem. 51 (2008) 5617e5629.
ꢁ
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[23] (a) M. Ilic, D. Kikelj, J. Ilas, Fluorinated dual antithrombotic compounds based
Acknowledgments
on 1,4-benzoxazine scaffold, Eur. J. Med. Chem. 50 (2012) 255e263;
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(b) M. Ilic, P. Dunkel, J. Ilas, E. Chabielska, A. Zakrzeska, P. Mátyus, D. Kikelj,
Enantiomers of potential dual antithrombotic compounds based on 2,3-
dihydro-1,4-benzodioxine scaffold, Submitted for publication.
This work was supported by Slovenian Research Agency Grant
No. P1-208, Slovene Hungarian bilateral project BI-HU/09-10-006,
COST Action CM0602 - Inhibitors of Angiogenesis: design, synthesis
and biological exploitation, and the Flemish grant FWO (G.
0486.08). Financial support by project VEGA 2/0112/10 is gratefully
[24] L.M. Khachigian, C.R. Parish, Phosphomannopentaose sulfate (PI-88): heparan
sulfate mimetic with clinical potential in multiple vascular pathologies, Car-
diovasc. Drug Rev. 22 (2004) 1e6.
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T. Karoli, K. Davis, E. Copeman, A. Gautam, PI-88 and novel heparan sulfate
mimetics inhibit angiogenesis, Semin. Thromb. Hemost. 33 (2007) 557e568.
ꢀ
ꢁ
acknowledged. Milos Ilic thanks Ad Futura Fundation for scholar-
ship. The authors wish to thank Mrs. Eef Meyen for dedicated
technical help. Péter Mátyus and Petra Dunkel are grateful for
a support provided by the National Development Agency, Hungary
(TÁMOP-4.2.1/B-09/1/KMR-2010-0001).
ꢁ
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[26] M. Ilic, J. Ilas, S. Liekens, P. Mátyus, D. Kikelj, Synthesis and antiproliferative
activity of 2-(([1,2,4]triazolo[4,3-b]- pyridazin-6-yloxy)methyl)-2,4-dimethyl-
3,4-dihydro-2H-benzo[b][1,4]oxazine derivatives, ARKIVOC (2011) 298e311.
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