Synthesis, structural elucidation and DNA-dependant protein kinase and antiplatelet studies of 2-amino-[5, 6, 7, 8-mono and 7, 8-di-substituted]-1,3- benzoxazines

Article abstract of DOI:10.1016/j.ejmech.2010.07.066

A number of new 2-amino-[5, 6, 7 and 8]-O-substituted 1,3-benzoxazines, and 2-amino 8-methyl-7-O-substituted-1,3-benzoxazines were synthesized. Thirty one new compounds were tested for their effect on collagen induced platelet aggregation and it was found

Full text of DOI:10.1016/j.ejmech.2010.07.066

European Journal of Medicinal Chemistry 45 (2010) 4934e4946
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structural elucidation and DNA-dependant protein kinase and
antiplatelet studies of 2-amino-[5, 6, 7, 8-mono and 7, 8-di-substituted]-1,
3-benzoxazines
*
Saleh Ihmaid, Jasim Al-Rawi , Christopher Bradley
School of Pharmacy and Applied Science, La Trobe University, P.O. Box 199, Bendigo 3552, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of new 2-amino-[5, 6, 7 and 8]-O-substituted 1,3-benzoxazines, and 2-amino 8-methyl-7-O-
substituted-1,3-benzoxazines were synthesized. Thirty one new compounds were tested for their effect
on collagen induced platelet aggregation and it was found that the most active compounds were
8-methyl-2-morpholin-4-yl-7-(pyridin-3-ylmethoxy)-4H-1,3-benzoxazin-4-one 9f and 8-methyl-
Received 5 February 2010
Received in revised form
23 July 2010
Accepted 29 July 2010
Available online 12 August 2010
2-morpholin-4-yl-7-(pyridin-4-ylmethoxy)-4H-1,3-benzoxazin-4-one 9j with IC50
4 ꢀ 2 M respectively. Inhibition of DNA-PK activity at concentrations of 1.6e4 M were tested for 9
products 5i, 7aee and 9b, 9f and 9j.
¼
2
ꢀ
1.5 and
m
m
Keywords:
Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
2-Amino-O-subst-1,3-benzoxazines
DNA-dependant protein kinase
Antiplatelet
1. Introduction
(PI3K) [12] but also to have activity against platelet phosphodies-
terases [13]. The PI3K inhibitory activity of compound 1 has led to
its use as a lead structure in the development of a DNA-PK inhibitor
with the 2-morpholino substituent found to be important for
DNA-PK inhibition [10,14,15].
The PI3K (phosphoinositide 3-kinase) enzyme family is impor-
tant to numerous intracellular signalling cascades, controlling
numerous physiological processes such as cell growth, prolifera-
tion, adhesion and survival [1]. Therefore, they are expected to have
a significant role in the treatment of diseases such as cancer [2e4],
thrombosis [5] and immunoinflammatory diseases [6,7]. Signifi-
cantly, they are also involved in DNA repair through the PI3K
enzyme, DNA-dependant protein kinase (DNA-PK) [8]. The ability
to repair DNA following radio-therapy or chemotherapy is an
important determinant of the success of the therapy. Hence, drugs
that inhibit this ability have potential clinical applications as
sensitizers to both radiation and cytotoxic drugs which induce
double stranded breaks in the DNA [9]. Griffin et al. [10] previously
Compound 2 (Fig. 1) was shown to have potent antiplatelet
properties against ADP induced aggregation with the IC50 reported
to be 0.85 ꢀ 0.3
mM [16].
Recently [17] we studied the inhibition of ADP and collagen
induced platelet aggregation by fifteen 2-morpholino-substituted
benzoxazines. Products 3 and 7e (Fig. 2) showed potent activity
against ADP induced platelet aggregation (compound 3 IC50 inhi-
bition 6 ꢀ 1.3
induced platelet aggregation (compound
M).
Here we report the synthesis and structure elucidation of
a library of 2-amino-5, 6, 7 and 8-O-sustiuted benzoxazines. Their
DNA-PK inhibitory and antiplatelet activities were also evaluated.
m
M and compound 7e 6 ꢀ 1.4
m
3
M) and collagen
IC50 inhibition
36 ꢀ 3
m
M and compound 7e 6 ꢀ 1.3
m
synthesized selective benzopyranone and pyrimido [2, 1-
a
] iso-
quinolin-4-one as inhibitors of DNA-dependent protein kinase and
demonstrated radiosensitization of human HeLa cervical cancer
cells by these compounds.
Phosphodiesterase inhibitors such as dipyridamole have found
use as antiplatelet drugs especially in combination with aspirin
[11]. 8-Phenyl-2-morpholino chromone (LY294002) 1 (Fig. 1) has
been reported to inhibit not only phosphatidylinositol 3-kinase
2. Results and discussion
2.1. Chemistry
2-Thio-benzoxazines 4 were synthesized from the reaction of
substituted 2-hydroxy aromatic carboxylic acid with freshly
prepared Ph3P(SCN)2 according to the previously reported proce-
dure [18] with no further modification. The physical property, IR, 1H
* Corresponding author. Tel.: þ61 3 54 44 7364; fax: þ61 3 54 44 7476.
E-mail addresses: sihmaid@students.latrobe.edu.au (S. Ihmaid), j.al-rawi@
latrobe.edu.au (J. Al-Rawi), c.bradley@latrobe.edu.au (C. Bradley).
0223-5234/$ e see front matter Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.066

S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
4935
3' X
4'
CH
O
2'
R1
3
R1
HN
X
O
O
O
N
O
N
O
O
S
R
R
O
N
N
NH
O
N
N
O
5
4
O
1
2
X in compounds 5a-n = O, 5o = S and 5p = CH2
Fig. 1. Structures of 2-morpholino-8-phenyl-4H-chromen-4-one 1 and 8-methyl-7-(2-
(4-methylpiperazin-1-yl)ethoxy)-2-morpholino-4H-chromen-4-one 2.
Comp.
R
R1
H
H
Comp.
4i & 5i
4j& 5j
4k& 5k
4l & 5l
R
H
H
5-OH
6-OH
R1
4a & 5a
4b & 5b
4c & 5c
4d & 5d
6-Br
7-F
6-Br
H
Ph
OH
H
NMR and 13C NMR data collected for known products 4a, 4c, 4i, 4m
and 4n were found to agree with previously reported data [18e25]
(Scheme 1). The structures of the new compounds 4b, 4deh, and
4jel were confirmed by the analysis of IR, 1H NMR and 13C NMR
spectroscopy in addition to the microanalysis (see Experimental
section).
2-morpholino, 2-thiomorpholino or 2-piperidino-substituted
benzoxazines 5 were prepared from the reactions of the corre-
sponding 2-thio-benzoxazine 4 with morpholine, thiomorpholine
or piperidine according to the previously reported procedures [17]
(Scheme 1). The physical property, IR, 1H NMR and 13C NMR data
collected for known products 5i, 5m and 5n were found to agree
with previously reported data [17]. The structures of the new
products 5aeh, 5jel and 5o, p were confirmed by the analysis of
their IR, 1H NMR and 13C NMR spectroscopy in addition to the
microanalysis (see Experimental section).
Br
CH3
10
H
HC
9
CH3
4e & 5e
4f & 5f
H
H
4m& 5m
4n& 5n
7-OH
7-OH
H
6
7
O
O
CH CH
2 3
9
10
CH3
CH2 CH3
9
10
4g & 5g
4h & 5h
H
H
5o
5p
7-OH
7-OH
CH3
CH3
O
O
CH3
9
6
5
CH3
9
Scheme 1. Synthesis of 2-morpholino-substituteed1,3-benzoxazines 5 from the reac-
tion of the corresponding 2-thio-1,3-benzoxazines 4 with morpholine in dry dioxane.
Products 6aec were prepared from the reaction of 1,2-dibro-
moethane with 6-, 7- and 8-hydroxy-2-morpholino-1,3-benzox-
azines 5l, 5m and 5j respectively (Scheme 2). Similarly 6def were
prepared from the reaction of 2-chlorobromoethane with 5-, 6- and
8-hydroxy-2-morpholino-1,3-benzoxazines 5k, 5l and 5j respec-
tively (Scheme 2).
Attempting to react compound 5m with the 2-(chloromethyl)-
pyridine hydrochloride according to the previously reported method
[16] was problematic. When compound 5m was heated for 1 h at
60 ꢁC with NaH then the 2-(chloromethyl)-pyridine hydrochloride
added portionwise at 60 ꢁC and stirred for 1 h then neutralized with
NaOH only then oily unidentifiable material resulted. Therefore we
had to modify the above procedure to enable the synthesis of 5-, 6-,
7- and 8-(pyridine-2yl, pyridine-3yl or pyridine-4yl-methyloxy)-2-
The physical property, IR, 1H NMR and 13C NMR data collected
for the known products 6b and 6g were found to agree with
previously reported data [17]. However, the structures of the new
products 6a, 6cef, 6h, and 6i were confirmed by the analysis of
their IR, 1H NMR and 13C NMR spectroscopy in addition to the
microanalysis (see experimental section).
Allowing compounds 6aec and 6gei to react with 1-methyl-
piperazine according to the previously reported procedure [17] gave
products 7aeg (Scheme 2). The physical property, IR, 1H NMR and
13C NMR data collected for the knownproducts 7cand 7ewerefound
to agree with previously reported data [17]. However, the structures
of the new products 7a, 7b, 7d, 7f and 7g were confirmed by the
analysis of their IR,1H NMR and 13C NMR spectroscopy in addition to
the microanalysis (see Experimental section).
The reaction of benzyl bromide with compounds 5jel according
to the previously reported method [17] gave produces 8aec
respectively (Scheme 3). The structures of the products 8aec were
confirmed by the analysis of their IR, 1H NMR and 13C NMR spec-
troscopy in addition to the microanalysis (see Experimental
section).
morpholin-4-yl-4H-1,3-benzoxazin-4-one
compounds
9aek
(Scheme 3). In this procedure a suspension of compounds 5m in dry
acetonitrile and Cs2CO3 was added to the suspension which was then
refluxed for 2 h. Suspensions of 2-(bromomethyl)-pyridine hydro-
bromide, 3-(chloromethyl)- pyridine hydrochloride, or 4-(bromo-
methyl)-pyridine in dry acetonitrile were added drop-wise to the
above reaction mixture then refluxed for a further 30 min. At the
completion of the reaction the work-up gave compounds 9a (yield
91%), 9e (73% yield) and 9i (70% yield). Similarly compounds 9bed,
9feh, and 9j, k were prepared with moderate to good yields
(see experimental section). The structure of the products 9ael was
confirmed by the analysis of their IR, 1H NMR and 13C NMR spec-
troscopy in addition to microanalysis (see Experimental section).
It was noted that the reaction of compound 5k with 1,2-dibro-
moethane did not give the expected 10 (Scheme 4) but gave the Bis
product 11. Product 10 did not form although 1,2-dibromoethane
was used in excess, 10e1 mol of 5k. However, product 11 was
formed in a low yield (<10%) under this condition. Using 1.25 mol
of the 1,2-dibromoethane to 2 mol of 5k gave a good yield (65%) of
product 11. The structure of product 11 was confirmed by the
analysis of IR, 1H and 13C NMR spectroscopy in addition to the
microanalysis (see Experimental section).
CH
O
3
O
O
O
N
R
The formation of the dimer 11 rather than the intermediate 10 is
a result of the fact that the OCH2CH2Br at position 5 in intermediate
10 is ortho to the carbonyl (electron withdrawing by inductive and
resonance) which made the bromine at 2-position of the side chain
of the intermediate 10 (Scheme 4) a better leaving group than the
bromine in the 1,2-dibromoethane molecule. However using
1-bromo-2-chloroethane to react with 5k gave a good yield (75%)
of 6d (Scheme 2).
N
3 R = Cl
7e R =
N
N CH
3
Fig. 2. Structures of 7-(2-chloroethoxy)-8-methyl-2-morpholino-4H-benzo[e][1,3]
oxazin-4-one and 8-methyl-7-(2-(4-methylpiperazin-1-yl)ethoxy)-2-morpholino-
4H-benzo[e][1,3]oxazin-4-one 7e.
3

4936
S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
H
15
C
3
13
N
N
3'
3'
12
10
3'
2'
2'
R
X
A
R
X
Y
2'
X
R
10
9
O
N
O
N
HO
9
O
O
N
B
O
O
N
N
N
O
O
5j-p
6a-i
7a-g
Substitution
position
Comp. Substitution position
R
X
Y
Comp.
7a
R
H
H
H
H
X
O
6a
6b
6c
6d
6e
6f
6
7
8
5
6
8
7
7
H
H
O
O
O
O
O
O
O
S
Br
Br
Br
Cl
Cl
Cl
Br
Br
5
6
7
8
7
7
7
7b
O
H
7c
O
H
7d
O
H
7e
CH3
CH3
CH3
O
H
7f
CH2
S
6g
6h
CH3
CH3
7g
Scheme 2. A is Cs2CO3 with 1,2-dibromoethane or 2-chlorobromoethane. B is 1-methylpiperazine.
3'
3'
2'
R
X
2'
12 11
14 15
O
O
O
N
13
10
9
HO
O
N
O
N
A
N
O
8a-c
5
B
8a subs. at 5- position
8b subs. at 6- position
8c subs. at 8- position
12
13
14
N
10
R
X
9a-c R'=
9d-h R' =
9i-k R' =
R'
O
O
N
15
N
CH
9
2
2
O
N
CH
N
CH
2
9a-k
Comp.
9a
Substitution position
R
H
Comp.
9g
Substitution position
R
-
7
7
8
6
7
7
8
5
7
7
8
9b
CH3
-
9h
H
H
9c
9i
9d
H
9j
CH3
9e
H
9k
-
9f
CH3
Scheme 3. A is Cs2CO3 with benzyl bromide. B is Cs2CO3 with 2-(bromomethyl)-pyridine hydrobromide, 3-(chloromethyl)-pyridine hydrochloride, or 4-(bromomethyl)-pyridine.
Compound 5j R ¼ OH.

S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
4937
O
O
3' O
monosubstituted benzoxazines and the most potent of this group
was 9g with 8-(pyridin-3-ylmethoxy)-O-substituent.
However, the 8-methyl-7-O-substituted-amino-benzoxazines
were highly potent in inhibiting collagen induced human platelet
aggregation (Table 1). The high potency compounds were 7e, 9f and
9j respectively.
2'
O
N
N
O
O
O
N
Br
Cs2CO3
+
Br
N
N
N
OH O
O
O
O
Cs2CO3
5k
11
+ 5k
O
As can be seen in Table 1, several of these compounds showed
significant inhibition of collagen induced platelet aggregation with
IC50’s in the low micromolar range. This compares favourably with
the previously prepared 2-aminochromones which are analogues of
our compounds [16]. It is clear from the above that the activity is
dependanton O-substitutions at position 7 with the greatest activity
being found when there is an additional methyl group at position 8.
O
N
N
O
O
Br
10
Intermediate
Scheme 4. The formation of 5,5’-(ethane-1,2-diylbis(oxy))bis(2-morpholino-4H-benzo
[e][1,3]oxazin-4-one) 11
2.3. Inhibition of DNA-PK activity
2.2. Inhibition of platelet aggregation
Compound 12 in (Fig. 3) has previously been shown to have
DNA-PK inhibitory activity [10].
Therefore we used this compound as a lead compound and
tested the oxazine analogues for DNA-PK inhibitory activity (Table 2
and Fig. 4).
Previously we reported the inhibition of ADP and collagen
induced platelet aggregation [17] for a limited number of 2-mor-
pholino 7/8 substituted 1,3-benzoxazines and found that the two
most potent products were 7-[2-(4-methylpiperazin-1-yl)ethoxy]-
It can be seen from Table 2 and Fig. 4 that several of these
compounds displayed significant inhibitory activity. Interestingly
the substitutions at position 7 and at position 8, as with 5i and 7d,
were found to be important both for the inhibition of platelet
aggregation and critical for the inhibition of DNA-PK activity.
Previous work has shown the platelet inhibitory activity of similar
compounds, the 2-aminochromones, to be due to inhibition of
platelet phosphodiesterases though many compounds, such as
quercitin, theophylline and caffeine have also been show to inhibit
both kinases and phosphodiesterases [21,22]. The concentrations
used here are in the same order of magnitude as those found with
previously identified DNA-PK inhibitors [23,24] and future work
will establish IC50’s for each. As similar compounds have been
8-methyl
(IC50 ¼ 6 ꢀ 1.3
lin-4-yl-4H-1,3-benzoxazin-4-one 3 (IC50 ¼ 6 ꢀ 1.3
-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one
M) and 7-(2-Chloroethoxy)-8-methyl-2-morpho-
M).
7e
m
m
We now report the synthesis of a library of 2-aminosubstituted
benzoxazines to establish structure activity relationship data with
regard to the inhibition of collagen induced platelet aggregation.
The in vitro testing was carried out on a total of 31 new 2-amino
substituted benzoxazines to determine their inhibitory effect on
human platelet aggregation induced by collagen.
The 2-morpholino-5-monosubstituted benzoxazines showed
weak inhibition of collagen induced platelet aggregation
(IC50 ꢂ 100
mM see Table 1) whether the substitution was 5-(2-
chloroethoxy) 6d, 5-benzyloxy 8a, 5-[2-(4-methylpiperazin-1-yl)
ethoxy 7a, or 5-(pyridin-3-ylmethoxy) 9h.
shown to be non-toxic in doses up to 30 mM it is hoped that the
compounds synthesized here will have even lower IC50’s than those
thus far described with even less likelihood of toxicity. This will
make them even better candidates for trials as radiosensitizers and
chemosensitizers in combination with radiation and drugs causing
double stranded DNA breaks respectively.
Furthermore 2-morpholino-6-monosubstituted benzoxazines
(6a, 6e, 8b and 9d) showed similar but in some instances more
potent inhibition than their corresponding 5-substituted analogues
(Table 1).
2-Morpholino-7-monosubstituted
benzoxazines
showed
moderate activity in which the most active compound of
this group was 9i with 7-(pyridin-4-ylmethoxy)-O-substituent
(Table 1). Similar activity was observed for 2-morpholino-8-
3. Conclusion
The synthesis of thirty one new 2-amino-[5, 6, 7, 8-mono- and 7,
8-di- substituted]-1,3-benzoxazines compounds 6, 7, 8 and 9 was
achieved. First [5, 6, 7, 8-mono- and 7, 8-di- substituted]-1,3-ben-
zoxazines 4 were synthesized from the reaction of the corresponding
mono- and di- substituted 2-hydroxybenzoic acid with freshly
prepared Ph3P(SCN)2. Then compounds 4 were allowed to react with
sec-amines to give 2-aminosubstituted benzoxazines 5. The
synthesis of the novel compounds 5-, 6-, 7- and 8-(pyridine-2yl, 3yl
Table 1
Inhibition data for 2-morpholino benzoxazines against collagen induced human
platelet aggregation.
Compound
Collagen inhibition
IC50(uM)
Compound
Collagen inhibition
IC50(uM)
5b
5d
5f
5o
5p
6a
6c
6d
6e
6f
6h
6i
7a
7b
7c
7d
80 ꢀ 6
70 ꢀ 2.3
85 ꢀ 6
ꢂ100
7e
7f
6 ꢀ 1.3a
36 ꢀ 2.4
47 ꢀ 1.3
ꢂ100
and
4yl-methyloxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one
7g
8a
8b
8c
9a
9b
9d
9e
9f
9g
9h
9i
9j
9k
9aek were achieved with good to excellent yield by allowing the
reaction of hydroxy substituted-2- morpholino1,3-benzoxazines
ꢂ90
ꢂ100
83 ꢀ 3.4
70 ꢀ 4
ꢂ100
53 ꢀ 4.7
75 ꢀ 8
34 ꢀ 2
ꢂ90
N
CH3
O
87 ꢀ 3
60 ꢀ 2.7
75 ꢀ 4.7
61 ꢀ 5.2
ꢂ100
O
O
O
N
70 ꢀ 6
2 ꢀ 1.5
22 ꢀ 6
ꢂ100
ꢂ86
45 ꢀ 9
4 ꢀ 2
12
ꢂ85a
80 ꢀ 2.3
70 ꢀ 4
Fig. 3. Structure of 8-methyl-2-morpholino-7-(pyridin-2-ylmethoxy)-4H-chromen-4-
one 12.
a
The IC50 of these compounds were taken from Ref. [16].

4938
S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
Table 2
Inhibition of DNA-PK activity data for 2-morpholino benzoxazines. Inhibition was judged to be positive when there was a significant difference between the activity of the
positive control and the activity in the presence of drug, (p < 0.05, n ¼ 3).
R8
O
R7
R6
O
O
N
N
R5
Comp.
R5
H
R6
R7
H
R8
Ph
DNA-PK
Conc.
2.0
Inhibition
5i
H
H
þ
7a
7b
7c
7d
7e
O(CH2)2(4-Me-1-piprazinyl)
H
H
H
H
H
4.0
4.0
4.0
4.0
4.0
e
e
þ
þ
þ
H
H
H
H
O(CH2)2(4-Me-1-piprazinyl)
H
H
H
O(CH2)2(4-Me-1-piprazinyl)
H
O(CH2)2(4-Me-1-piprazinyl)
O(CH2)2(4-Me-1-piprazinyl)
CH3
9b
9f
9j
H
H
H
H
H
H
OCH2(2-pyridyl)
OCH2(3-pyridyl)
OCH2(4-pyridyl)
CH3
CH3
CH3
1.6
4.0
2.0
þ
þ
þ
5jen with 2-(bromomethyl)-pyridinehydrobromide, 3-(chloro
methyl)-pyridine hydrochloride and 4-(bromomethyl)-pyridine
hydrobromide in the presences of Cs2CO3.
The in vitro testing was carried out on a total of 31 new 2-amino
substituted benzoxazines to determine their inhibitory effect on
human platelet aggregation induced by collagen and it was found
that the position and type of O-substitution on the aromatic ring of
the substitute-benzoxazines is important for activity. We found
that the most active compounds have a methyl group at position 8
and a 7-O-substitution.
13C NMR data collected for compounds 4a, 4c and 4i, 4m, 4n were
found to agree with previously reported data in Refs. [18] and [25]
respectively. Similarly the new 2-thio-1,3-benzoxazines 4b, 4deh
and 4jel were prepared.
The substituted 2-hydroxybenzoic acids were purchased from
Aldrich Chemical Company and were used as received except
2,4-dihydroxy-3-methyl benzoic acid which was prepared accord-
ing to the previously reported method [21] by the carboxylation of
2-methylresorcinol.
Inhibitions of DNA-PK activity at concentrations of 1.6e4
were tested for 9 products 5i, 7aee and 9b, 9f and 9j.
m
M
4.2.1. 7-Fluoro-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4b
Compound 4b was prepared according to the previously
reported method [18] from the reaction of 4-fluoro-2-hydroxy-
benzoic acid (0.5 g, 3 mmol) and the freshly prepared triphenyl
phosphine thiocyanogen. The solids collected from the dichloro-
methane and acetone hot-filtrations were combined and recrys-
tallized from toluene to give 4b (65%), mp 202 ꢁC. nmax (KBr) 3117w
4. Experimental
Infrared spectra were obtained using a Perkin Elmer FT-IR
spectrometer. 1H NMR and 13C NMR spectra were obtained using
a Bruker AC 200 NMR spectrometer at 200 and 50 MHz, respec-
tively. All 1H NMR and 13C NMR spectral results are recorded as
and 2937w (NeH),1727s (C]O),1618m (C]C), 1164m (C]S) cmꢃ1
;
1H (DMSO-d6) 13.6 (s, 1H, NH), 8.0 (dd, 1H, J5,6 ¼ 8.4, J5,F ¼ 6.3 Hz,
H-5,), 7.5 (dd,1H, JH8,6 ¼ 2.3 and JH8,F ¼ 9.1 Hz H-8), 7.2 (dt, 1H,
J6,8 ¼ 2.3, JH6,F ¼ 8.9 Hz, H-6); 13C(DMSO-d6) 181.9 (C-2), 166.25 (d,
C-7, JCF ¼ 256.3 Hz), 156.9 (C-4), 156.6 (d, C-8a, JCF ¼ 12.2 Hz), 129.4
(d, C-5 JCF ¼ 12.2 Hz), 114.1 (d, C-6, JCF ¼ 23.2 Hz), 112.7 (d, C-4a,
JCF ¼ 2.4 Hz) 104.0 (d, C-8, JCF ¼ 26.8 Hz); (found C, 48.65; H, 2.06; N,
7.10; C8H4FNO2S, requires C, 48.73; H, 2.04; N, 7.10).
chemical shifts (d) relative to the internal TMS. Microanalysis was
performed by Chemical and Microanalytical Services (CMAS),
Australia. Melting point determinations were carried out using
a Stuart Scientific (SMP3) melting point apparatus and all melting
points are uncorrected.
4.1. Chemistry
4.2.2. 8-Isopropyl-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one
4d
The starting reagents, morpholine, N-methyl piperazine, sodium
hydride, cesium carbonate, benzyl bromide, methyl iodide,1-bromo-
2-chloroethane, 2-(bromomethyl)-pyridinehydrobromide, 3-(chlor-
omethyl)-pyridine chydrochloride or 4-(bromomethyl)-pyridine
hydrobromide were purchased from Aldrich Chemical Company and
were used as received.
Compound 4d was prepared according to the previously
reported method [18] from the reaction of 2-hydroxy-3-isopropyl
benzoic acid (1.5 g, 8 mmol) and the freshly prepared triphenyl
phosphine thiocyanogen. The solid collected from the dichloro-
methane and acetone hot-filtration were combined and recrystal-
lized from cyclohexane to give 4d (75%), mp 160 ꢁC. nmax(KBr)
3197w and 2873w (NeH), 1718s (C]O), 1613m (C]C), 1171m (C]
4.2. Synthesis of 2-thio-benzoxazines 4
S) cmꢃ1
;
1H (CDCl3) 10.1 (s, 1H, NH), 8.0 (dd, 1H, JH7eH6 ¼ 7.5 Hz,
The known 2-thio-1,3-benzoxazines 4a, 4c, 4i, 4m and 4n were
synthesized from the reaction of freshly prepared triphenyl phos-
phine thiocyanogen with the corresponding 2-hydroxy aromatic
acid using the previously reported method [18]. The IR, 1H NMR and
JH7eH8 ¼ 1.3 Hz, H-7), 7.5 (dd, 1H, JH5eH6 ¼ 7.5 Hz, JH5eH7 ¼ 1.3 Hz,
H-5), 7.4 (t, 1H, J ¼ 8 Hz, H-6), 3.6 (q, 2H, J ¼ 7.2 Hz, CH2), 1.3 (s, 6H,
2CH3); 13C (CDCl3) 181.3 (C-2), 158.5 (C-4), 154.0 (C-8a), 137.1 (C-8),
134.4 (C-7), 126.7 (C-6), 125.5 (C-5), 115.2 (C-4a), 27.3 (CH2), 23.0

S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
4939
acetone hot-filtration were combined and recrystallized from ethyl
acetate to give 4e (78%), mp 192 ꢁC. nmax (KBr) 3181w and 2931w
(NeH), 1698s (C]O), 1621m (C]C), 1180m (C]S) cmꢃ1; 1H (CDCl3)
10.0 (s, 1H, NH), 7.4 (s, 1H, H-5), 7.3 (d, 1H, J ¼ 8 Hz, H-7), 7.2 (d,1H,
J ¼ 8 Hz, H-8), 4.1(q, 2H, J ¼ 5.6 Hz, CH2eO),1.4 (t, 3H, J ¼ 5.6 Hz,
CH3); 13C (d6-DMSO) 181.7 (C-2), 157.5 (C-4), 156.0 (C-6), 149.7 (C-
8a), 124.5 (C-5), 118.0 (C-7), 108.2 (C-4a), 101.6 (C-8), 64.1 (CH2eO),
14.3 (CH3); (found C, 53.84; H, 4.10; N, 6.29; C10H9NO3S, requires C,
53.80; H, 4.06; N, 6.27).
DNA-PK Activity
20000
15000
10000
5000
0
4.2.4. 7-Ethoxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4f
Compound 4f was prepared according to the previously reported
method [18] from the reaction of 4-ethoxy-2-hydroxyl benzoic acid
(1.5 g, 8 mmol) and the freshly prepared triphenyl phosphine thi-
ocyanogen. The solid collected from the dichloromethane and
acetone hot-filtration was combined and recrystallized from ethyl
acetate to give 4f (88%), mp 229 ꢁC. nmax(KBr) 3180w and 2929w
(NeH), 1697s (C]O), 1624m (C]C), 1160m (C]S) cmꢃ1; 1H (CDCl3)
9.9 (s,1H, NH), 8.0 (d,1H J ¼ 8.5 Hz, H-5), 6.9 (dd,1H, JH6eH5 ¼ 8.5 Hz,
JH6eH8 ¼ 2.0 Hz, H-6), 6.7 (d,1H, J ¼ 2.0 Hz, H-8), 4.1(q, 2H, J ¼ 6.8 Hz,
CH2eO),1.4 (t, 3H, J ¼ 6.8 Hz, CH3); 13C (d6-DMSO) 182.2 (C-2), 164.8
(C-7),157.10 (C-8a),157.15 (C-4),128.1 (C-5),114.5 (C-6),108.1 (C-4a),
100.5 (C-8), 64.6 (CH2eO), 14.0 (CH3); (found C, 53.87; H, 4.03; N,
6.30; C10H9NO3S, requires C, 53.80; H, 4.06; N, 6.27).
Drug
DNA-PK Activity
7000
6000
5000
4000
3000
2000
1000
0
4.2.5. 6-Methoxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one
4g
Product 4g was prepared according to the previously reported
method [18] from the reaction of 5-methoxybenzoic acid (1.5 g,
9 mmol) and the freshly prepared triphenyl phosphine thiocya-
nogen. The solids collected from the dichloromethane and acetone
hot-filtrationwere combined and recrystallized from toluene to give
4g (72%), mp 192 ꢁC. nmax (KBr) 3204w and 2942w (NeH), 1703s
(C]O),1621m (C]C),1190m (C]S) cmꢃ1; 1H (d6-DMSO) 13.4 (s,1H,
NH), 7.4 (s, 1H, H-5), 7.4 (m, 2H, H-7, H-8), 3.8 (s, 3H, CH3eO); 13C
(d6-DMSO) 181.7 (C-2), 157.6 (C-4), 156.8 (C-6), 149.9 (C-8a), 124.3
(C-5), 118.0 (C-7), 116.0 (C-4a), 107.2 (C-8), 56.0(CH3eO); (found C,
51.74; H, 3.30; N, 6.53; C9H7NO3S, requires C, 51.67; H, 3.37; N, 6.69).
Drug
DNA-PK Activity
4000
3000
2000
1000
0
4.2.6. 5-Methoxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one
4h
Product 4h was prepared according to the previously reported
method [18] from the reaction of 6-methoxy-2-hydroxy acid (1.5 g,
9 mmol) and the freshly prepared triphenyl phosphine thiocya-
nogen. The solids collected from the dichloromethane and acetone
hot-filtration were combined and recrystallized from toluene to
give 4h (78%), mp 192 ꢁC. nmax (KBr) 3217w and 2937w (NeH), 1702s
(C]O), 1611m (C]C), 1176m (C]S) cmꢃ1 1H (d6-DMSO), 13.21
;
(s, 1H, NH), 7.7 (t, 1H, J ¼ 8.5 Hz, H-7), 7.3 (d, 1H, J ¼ 8.5 Hz, H-6), 7.2
(d,1H, J ¼ 8.5 Hz, H-8), 3.8 (s, 3H, CH3eO); 13C (d6-DMSO) 181.5 (C-2),
159.9 (C-5), 156.6 (C-4), 155.3 (C-8a), 136.8 (C-7), 108.9 (C-6), 104.8
(C-4a), 107.9 (C-8), 56.5(CH3eO); (found C, 51.74; H, 3.30; N, 6.53;
C9H7NO3S, requires C, 51.67; H, 3.37; N, 6.69).
Drug
Fig. 4. DNA-PK inhibitory activity for the compounds in Table 2. Negative control 1 is
no substrate in the presence of double stranded DNA and Negative control 2 is
substrate with no double stranded DNA. Positive control is activity in the presence of
both substrate and double stranded DNA. Results are expressed as counts per minute
þ/ꢃ SE (n ¼ 3).
4.2.7. 8-Hydroxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one
4j
Product 4j was prepared according to the previously reported
method [18] from the reaction of 2,3-Dihydroxybenzoic acid (1.5 g,
98 mmol) and the freshly prepared triphenyl phosphine thiocya-
nogen. The solids collected from the dichloromethane and acetone
hot-filtration were combined and recrystallized from ethyl acetate/
n-hexane to give 4j (87%), mp 265 ꢁC. nmax (KBr) 3411e2942b (OH),
3366w and 3098w (NeH), 1690s (C]O), 1621m (C]C), 1165m
(C]S) cmꢃ1; 1H (DMSO-d6) 13.4 (s, 1H, NH), 10.7 (s, 1H, 8-OH), 7.4
(m, 3H, H-5, 6, 7); 13C (DMSO-d6) 181.7 (C-2),157.8 (C-4),144.6 (C-8/
(CH3); (found C, 59.65; H, 5.06; N, 6.20; C11H11NO2S, requires C,
59.71; H, 5.01; N, 6.33).
4.2.3. 6-Ethoxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4e
Compound 4e was prepared according to the previously repor-
ted method [18] from the reaction of 5-ethoxy-2-hydroxybenzoic
acid (1.5 g, 8 mmol) and the freshly prepared triphenyl phosphine
thiocyanogen. The solids collected from the dichloromethane and

4940
S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
8a), 144.4 (C-8a/8), 126.2 (C-6), 122.3 (C-7), 115.9 (C-5), 116.6 (C-4a).
(found C, 48.50; H, 2.57; N, 7.26; C8H5NO3S, requires C, 49.23; H,
2.58; N, 7.18).
4.3.2. 6,8-Dibromo-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5c
6,8-Dibromo-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one
4c was allowed to react with morpholine and the solid was
collected and recrystallized from ethanol to give 5c (85%yield), mp
225 ꢁC. nmax(KBr) 3072e2866 (CeH) 1691 (C]O), 1558, (C]N)
4.2.8. 5-Hydroxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one
4k
cmꢃ1; 1H (CDCl3)
d
8.2 (d, 1H, J ¼ 2.3 Hz, H-5), 7.9 (d. 1H, J ¼ 2.3 Hz,
Product 4k was prepared according to the previously reported
method [18] from the reaction of 2,6-dihydroxybenzoic acid (1.5 g,
98 mmol) and the freshly prepared triphenyl phosphine thiocya-
nogen. The solid product was collected from the dichloromethane
and was recrystallized from ethyl acetate/n-hexane to give 4k (83%),
mp 214 ꢁC. nmax (KBr) 3270e2905b(OH), 3267w and 3059w (NeH),
1696s (C]O),1630m (C]C),1191m (C]S) cmꢃ1; 1H (DMSO-d6) 13.6
(s, 1H, NH), 10.6 (s, 1H, 5-OH), 7.7 (t, 1H, J ¼ 8 Hz, H-7), 6.9 (d, 1H,
J ¼ 8 Hz, H-8), 6.85 (d, 1H, J ¼ 8 Hz, H-6); 13C (DMSO-d6) 181.5 (C-2),
161.2 (C-4), 159.2 (C-8a), 155.2 (C-5), 137.4 (C-7), 112.9 (C-6), 106.04
(C-8), 102.12 (C-4a); (found C, 48.50; H, 2.28; N, 7.53; C8H5NO3S,
requires C, 49.23; H, 2.58; N, 7.18).
H-7), 3.8 (bm, 8H, 4ꢄ CH2 of morpholine); 13C (CDCl3)
d 165.2 (C-4),
156.5 (C-2), 150.2 (C-8a), 139.7 (C-7), 130.1 (C-5), 120.2 (C-6), 119.1
(C-8), 110.5 (C-4a), 66.6 (C-30), 45.4 (C-20); (found C, 37.01; H, 2.60;
N, 7.15; C12H10Br2N2O3 requires C, 36.95; H, 2.58; N, 7.18%).
4.3.3. 8-Isopropyl-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5d
8-Isopropyl-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4d
was allowed to react with morpholine and the solid was collected
and recrystallized from ethyl acetate to give 5d (85%yield), mp
155 ꢁC. nmax(KBr) 3182e2876 (CeH), 1695 (C]O), 1592 (C]N) cmꢃ1
;
1H (CDCl3) 7.9 (d, 1H, J ¼ 7.5 Hz C-5), 7.5 (d, 1H, J ¼ 7.5 Hz, H-7), 7.4 (t,
1H, J ¼ 7.5 Hz, H-6), 3.3 (q, 2H, J ¼ 7.0 Hz, CH2), 1.3 (s, 6H, 2CH3), 3.8
(bm, 8H, 4ꢄ CH2 of morpholine); 13C (CDCl3) 167.7 (C-4), 157.1 (C-2),
151.3 (C-8a), 135.4 (C-8), 131.1 (C-7), 125.7 (C-6), 125.5 (C-5), 117.4
(C-4a), 66.5(C-30), 44.7 (C-20), 27.3 (CH2), 22.7 (CH3); (found C, 65.70;
H, 6.65; N, 10.21; C15H18N2O3 requires C, 65.68; H, 6.61; N, 10.21%).
4.2.9. 6-Hydroxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one
4l
Product 4l was prepared according to the previously reported
method [18] from the reaction of 2,5-dihydroxybenzoic acid (1.5 g,
98 mmol) and the freshly prepared triphenyl phosphine thiocya-
nogen. The solids collected from the dichloromethane and acetone
hot-filtration were combined and recrystallized from ethyl acetate/
n-hexane to give 4l (93%), mp 265 ꢁC. nmax (KBr) 3366e2928b (OH),
3366w and 3080w (NeH), 1696s (C]O), 1620m (C]C), 1169m (C]
S) cmꢃ1; 1H (DMSO-d6) 13.4 (s,1H, NH),10.2 (s, 1H, 6-OH), 7.3 (s,1H,
H-5), 7.2 (d, 1H, J ¼ 7 Hz, H-8), 7.2 (d, 1H, J ¼ 7 Hz, H-7); 13C (DMSO-
d6) 181.7 (C-2), 157.5 (C-4), 155.2 (C-6), 148.7 (C-8a), 124.2 (C-7),
117.7 (C-5), 110.2 (C-8), 116.1 (C-4a); (found C, 48.57; H, 2.27; N,
7.26; C8H5NO3S, requires C, 49.23; H, 2.58; N, 7.18).
4.3.4. 6-Ethoxy -2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5e
6-Ethoxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4e was
allowed to react with morpholine and the solid was collected and
recrystallized from ethyl acetate to give 5e (75% yield) as a white
solid, mp 180 ꢁC. nmax(KBr) 2993, 2843 (CeH), 1676 (C]O), 1622s
(C]C),1576s (C]N) cmꢃ1; 1H (CDCl3)
d 7.5 (s,1H, H-5), 7.1 (m, 2H, H-7
and H-8), 4.1 (q, 2H, J ¼ 5.6, CH2eO), 1.4 (t, 3H, J ¼ 5.6, CH3), 3.7 (bm,
8H, 4 ꢄ CH2 of morpholine); 13C (CDCl3) 167.1 (C-4),156.9 (C-2),156.4
(C-6), 147.8 (C-8a), 123.1 (C-7), 116.7 (C-5), 117.7 (C-4a), 108.8 (C-8),
64.2 (CH2eO), 14.7 (CH3), 66.2 (C-30), 44.4 (C-20); (found C, 60.95; H,
6.09; N, 9.98; C13H14N2O4 requires C, 60.86; H, 5.84; N, 10.14%).
4.3. Reaction of 2-thio-1,3-benzoxazines with the amines
4.3.5. 7-Ethoxy -2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5f
7-Ethoxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4f was
allowed to react with morpholine and the solid was collected and
recrystallized from ethyl acetate to give 5f (75% yield) as a white
solid, mp 195 ꢁC. nmax(KBr) 3071, 2855 (CeH), 1668 (C]O), 1627s
The 2-amino-substituted benzoxazines 5aen (Scheme 1) were
synthesized from the reactions of 2-thio-1,3-(substituted) ben-
zoxazines 4aen (Scheme 1) with morpholine, thiomorpholine or
piperidine according to the general procedure A [17] .
(C]C),1597s (C]N) cmꢃ1; 1H (CDCl3)
d
8.0 (d,1H, J ¼ 8.5 Hz, H-5), 6.9
General procedure A: Substituted 2-thioxo-1,3-benzoxazine-4-one
4aen (2.5 mmol) was suspended in 10 mL of 1,4-dioxane in a 25 mL
round-bottomed flask. Morpholine, thiomorpholine or piperidine
(12.5 mmol) was added drop-wise with stirring and the reaction
mixture was heated to reflux for 2 h. At the completion of the reaction
the mixture was evaporated to dryness in vacuo, washed with diethyl
ether before being filtered and recrystallized from a suitable solvent.
The physical data, IR, 1H NMR and 13C NMR collected for known
compounds 5i, 5m, and 5n were found to agree with previously
reported data [17].
(dd, 1H, JH6eH5 ¼ 2.0 Hz, JH6eH8 ¼ 2.3 Hz, H-6), 6.6 (d, 1H, J ¼ 2.3 Hz,
H-8), 4.1 (q, 2H, J ¼ 5.6, CH2eO), 1.4 (t, 3H, J ¼ 5.6, CH3), 3.8 (bm, 8H,
4ꢄ CH2 of morpholine); 13C (CDCl3) 167.0 (C-4), 163.8 (C-2), 156.9
(C-7), 155.3 (C-8a), 129.1 (C-5), 113.9 (C-6), 110.5 (C-4a), 99.9 (C-8),
66.5 (C-30), 64.5 (CH2eO), 44.7 (C-20), 14.8 (CH3) (found C, 60.93; H,
5.90; N,10.10; C13H14N2O4 requires C, 60.86; H, 5.84; N, 10.14%).
4.3.6. 6-Methoxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5g
6-Methoxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4g
was allowed to react with morpholine and the solid was collected
and recrystallized from ethanol to give 5g (75% yield) as a white
solid, mp 160 ꢁC. nmax(KBr) 2963, 2850 (CeH), 1665 (C]O), 1619s
4.3.1. 7-Fluoro-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5b
7-Fluoro-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4b was
allowed to react with morpholine and the solid was collected and
recrystallized from ethanol to give 5b (75% yield), mp 219 ꢁC.
(C]C), 1554s (C]N) cmꢃ1 ;1H (DMSO-d6)
d
7.3 (s, 1H, H-5), 7.3
(m, 2H, H-7,8), 3.8 (s, 3H, 6-OCH3), 3.7 (bm, 8H, 4ꢄ CH2 of mor-
pholine); 13C (DMSO-d6)
d 166.1 (C-2), 156.7 (C-6), 156.6 (C-4), 148.1
nmax(KBr) 3064, 2872 (CeH), 1701s (C]O), 1615m (C]C) cmꢃ1
;
1H
(C-8a), 122.1 (C-7), 117.6 (C-8), 117.6 (C-4a), 108.4 (C-5), 65.8 (C-30),
44.1 (C-20), 55.6 (OeCH3); (found C, 59.51; H, 5.43; N, 10.56;
C13H14N2O4 requires C, 59.54; H, 5.38; N, 10.68%).
(CDCl3)
d
8.1 (dd, 1H, JH-5,H-6 ¼ 8.4, JH-5,F ¼ 6.3 Hz, H-5), 7.1 (dd, 1H,
JH-8,H-6 ¼ 2.3 JH-8,F ¼ 9.1 Hz, H-8), 6.9 (dt, 1H, JH-6,H-8 ¼ 2.3 and JH-6,F
,
JH-6,H-5 ¼ 8.4 Hz, H-6), 3.8 (bm, 8H, 4ꢄ CH2 of morpholine); 13C
(CDCl3)
d
168.6 (C-4), 164.9 (C-7, JCF ¼ 256.3 Hz), 157.0 (C-2), 154.9 (d,
4.3.7. 5-Methoxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5h
5-Methoxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4h
was allowed to react with morpholine and the solid was collected
and recrystallized from ethanol to give 5h (75% yield) as a white
solid, mp 172 ꢁC. nmax(KBr) 2976, 2866 (CeH), 1668 (C]O), 1592s
C-8a, JCF ¼ 12.2 Hz), 130.5 (d, C-5, JCF ¼ 12.2 Hz), 114.2 (d, C-6,
JCF ¼ 23.2 Hz), 113.7 (d, C-4a, JCF ¼ 2.4 Hz), 104.0 (d, C-8,
JCF ¼ 26.8 Hz), 66.5 (C-30), 45.0 (C-20); (found C, 57.70; H, 4.45; N,
11.23; C12H12N2O4 requires C, 57.60; H, 4.43; N, 11.20%).

S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
4941
(C]C),1558s (C]N) cmꢃ1; 1H (DMSO-d6)
d
7.6 (t, 1H, J ¼ 8 Hz, H-7),
J ¼ 8 Hz, H-5), 6.8 (d, 1H, J ¼ 8 Hz, H-6), 3.6 (bm, 4H, 2 x CH2eN of
6.9 (d, 1H, J ¼ 7.5 Hz, H-6), 6.9 (d, 1H, J ¼ 7.5 Hz, H-8), 3.8 (s, 3H,
piperidine), 1.6 (bm, 6H, 30, 40CH2 of piperidine), 2.1 (s, 3H, 8-CH3);
5-OCH3), 3.7 (bm, 8H, 4ꢄ CH2 of morpholine); 13C (DMSO-d6)
13C (d6-DMSO)
d 166.1 (C-4), 160.2 (C-7), 156.2 (C-2), 153.0 (C-8a),
d
163.2 (C-2), 159.1 (C-5), 155.1 (C-4), 154.9 (C-8a), 134.1 (C-7), 108.3
124.8 (C-5), 112.6 (C-6), 109.9 (C-8), 108.8 (C-4a), 7.9 (8-CH3), 44.7
(C-20), 25.2 (C30), 25.1 (C40); (found C, 64.52; H, 6.14; N, 10.64;
C14H16N2O3 requires C, 64.60; H, 6.20; N, 10.76%).
(C-6), 107.6 (C-8), 105.4 (C-4a), 65.4 (C-30), 56.4 (OeCH3), 43.7
(C-20); (found C, 59.51; H, 5.43; N, 10.56; C13H14N2O4 requires C,
59.54; H, 5.38; N, 10.68%).
4.4. Reactions of 2-amino-5, 6, 7 and 8 hydroxy substituted
benzoxazine 5jep with 1,2-dibromoethane and 2-
bromochloroethane
4.3.8. 8-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5j
8-Hydroxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4j
was allowed to react with morpholine and the solid was collected
and recrystallized from ethanol to give 5j (95% yield) as a white
solid, mp 270 ꢁC decomp. nmax(KBr) 2951e2867 (OH), 1655
The 2-Amino-O-substituted benzoxazines 6aei (Scheme 2)
were synthesis from the reactions of 2-amino-5, 6, 7 and 8-hydroxy
benzoxazine 5jep (Scheme 2) with 1,2-dibromoethane or 2-bro-
mochloroethane which produce the corresponding O-substituted
analogues 6aei (Scheme 2) according to the general procedures B
[17].
General Procedure B: A suspension of 5jep (1 mmol) in 10 mL of
dry acetonitrile was treated successively with cesium carbonate
(6.2 mmol) and the appropriate alkyl bromide.
(C]O), 1557 (C]N). 1H (DMSO-d6)
d
10.3 (bs, 1H, 8-OH), 7.3 (d,
1H, J ¼ 8.6 Hz, H-5), 7.1 (m, 2H, H-6, H-7), 3.7 (bm, 8H, 4ꢄ CH2 of
morpholine); 13C (DMSO-d6)
165.5 (C-4), 156.2 (C-8), 144.7
d
(C-2), 142.8 (C-8a), 125.0 (C-7), 120.2 (C-5), 118.1 (C-4a), 116.1
(C-6), 65.4 (C-30), 44.0 (C-20); (found C, 58.03; H, 4.90; N, 11.24;
C12H12N2O4 requires C, 58.06; H, 4.87; N, 11.29%).
4.3.9. 5-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5k
5-Hydroxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4k
was allowed to react with morpholine and the solid was collected
and recrystallized from ethanol to give 5k (85% yield) as a white
solid, mp 183 ꢁC. nmax(KBr) 3064, 2872 (OH), 1656 (C]O), 1561,
[1,2-Dibromoethane or 1-bromo-2-chloroethane] (10 mmol)
then was stirred for 20 h at 80 ꢁC. At the completion of the reaction
the mixture was cooled and the acetonitrile evaporated off in
vacuo. The remaining residue was washed with 4 ꢄ 15 mL of CHCl3
and filtered, and the filtrate was then concentrated in vacuo. The
resulting residue was triturated with diethyl ether to give the crude
solid.
(C]N) cmꢃ1 1H (DMSO-d6)
; d 12.4 (bs, 1H, 5-OH), 7.6 (t, 1H,
J ¼ 8.6 Hz, H-7), 6.9 (d, 1H, J ¼ 8.6 Hz, H-6), 6.8 (d, 1H, J ¼ 8.6 Hz,
H-8), 3.7 (bm, 8H, 4ꢄ CH2 of morpholine); 13C (DMSO-d6)
d
171.5
The physical data, IR, 1H NMR and 13C NMR collected for known
compounds 6b, and 6g were found to agree with previously
reported data in Ref. [17].
(C-4), 159.3 (C-2), 157.0 (C-5), 153.3 (C-8a), 135.6 (C-7), 111.8 (C-6),
105.3 (C-8), 102.4 (C-4a), 65.4 (C-30), 44.4 (C-20); (found C, 57.82; H,
4.73; N, 10.90; C12H12N2O4 requires C, 58.06; H, 4.87; N, 11.29%).
4.4.1. 6-(2-Bromoethoxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-
4-one 6a
4.3.10. 6-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5l
6-Hydroxy-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 4l
was allowed to react with morpholine and the solid was collected
and recrystallized from ethanol to give 5l (95% yield) as a white
solid, mp 280 ꢁC decomp. nmax (KBr) 3060e2857 (OH), 1639
6-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5l was
allowed to react with 1,2-dibromoethane and the solid was
collected and recrystallized from ethanol to give 6a (75% yield) as
a white solid, mp 195 ꢁC decomp. nmax(KBr) 2968, 2860 (CeH), 1669
(C]O), 1558 (C]N) cmꢃ1; 1H (DMSO-d6)
d
9.8 (bs, 1H, 6-OH), 7.2
(C]O), 1619 (C]C), 1561 (C]N) cmꢃ1; 1H (CDCl3)
d 7.5 (s, 1H, H-5),
(s, 1H, H-5), 7.3 (d, 1H, J ¼ 7.2 Hz, H-7), 7.1 (d, 1H, J ¼ 7.3 Hz, H-8),
7.2 (d, 1H, J ¼ 8.6 Hz, H-7), 7.1 (d, 1H, J ¼ 8.6 Hz, H-8), 4.3
3.7 (bm, 8H, 4ꢄ CH2 of morpholine); 13C (DMSO-d6)
d 165.6 (C-4),
(t, J ¼ 5.6 Hz, 2H, H-9), 3.8 (bm, 8H, 4ꢄ CH2 of morpholine), 3.7
156.6 (C-2), 154.7 (C-6), 146.4 (C-8a), 121.9 (C-7), 117.1 (C-5), 117.6
(C-4a), 110.4 (C-8), 65.4 (C-30), 43.9 (C-20); (found C, 58.03; H,
4.90; N, 11.28; C12H12N2O4 requires C, 58.06; H, 4.87; N, 11.29%).
(t, J ¼ 5.6 Hz, 2H, H-10); 13C (CDCl3)
d 166.9 (C-4), 156.9 (C-6), 155.7
(C-2), 148.3 (C-8a), 123.5 (C-7), 116.9 (C-5), 117.9 (C-4a), 109.3 (C-8),
68.5 (C-9), 66.3 (C-30), 44.4 (C-20), 28.9 (C-10); (found C, 47.40; H,
4.30; N, 7.91; C14H15BrN2O4 requires C, 47.34; H, 4.26; N, 7.89%).
4.3.11. 7-Hydroxy-8-methyl-2-thio morpholin-4-yl-4H-
1,3-benzoxazin-4-one 5o
4.4.2. 8-(2-Bromoethoxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-
4-one 6c
8-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5j was
allowed to react with 1,2-dibromoethane and the solid was
collected and recrystallized from ethanol to give 6c (85% yield) as
a white solid, mp 168 ꢁC. nmax(KBr) 2968, 2860 (CeH), 1683 (C]O),
7-Hydroxy-8-methyl-2,3-dihydro-4H-1,3-benzoxazin-4-one 4o
allowed to react with thiomorpholine and the solid was collected
and recrystallized from ethanol to give 5o (81% yield), mp 295 ꢁC
decomp. nmax(KBr) 3058e2925 (OH), 1631s (C]O), 1597s (C]C),
1542s (C]N)cmꢃ1 1H (DMSO-d6)
; d 10.6 (bs, 1H, 7-OH), 7.6 (d, 1H,
J ¼ 8.6 Hz, H-5), 6.8 (d, 1H, J ¼ 8.6 Hz, H-6), 3.9 (bm, 4H, CH2eN of
1625 (C]C),1571 (C]N) cmꢃ1; 1H (CDCl3)
d
7.7 (d,1H, J ¼ 8.6 Hz, H-
thiomorpholine), 2.7 (bm, 4H, CH2eS of thiomorpholine), 2.1 (s, 3H,
7), 7.2 (m, 1H, H-5,7), 3.8 (bm, 8H, 4ꢄ CH2 of morpholine), 4.4 (t, 2H,
8-CH3); 13C (DMSO-d6)
d
165.9 (C-4), 160.3 (C-7), 156.3 (C-2), 153.0
J ¼ 5.6 Hz, H-9), 3.7 (t, 2H, J ¼ 5.6 Hz, H-10); 13C (CDCl3)
d 166.9
(C-8a), 124.9 (C-5), 112.7 (C-6), 110.0 (C-8), 108.8 (C-4a), 46.7 (C-20),
26.2 (C-30), 7.9 (8-CH3); (found C, 56.14; H, 5.08; N, 10.03;
C13H14N2O3S requires C, 56.10; H, 5.07; N, 10.06%).
(C-4), 156.8 (C-8), 145.3 (C-2), 142.8 (C-8a), 125.4 (C-7), 119.6 (C-5),
118.5 (C-4a), 117.3 (C-6), 69.0 (C-9), 66.3 (C-30), 44.5 (C-20), 28.9
(C-10); (found C, 47.31; H, 4.31; N, 7.94; C14H15BrN2O4 requires C,
47.34; H, 4.26; N, 7.89%).
4.3.12. 7-Hydroxy-8-methyl-2-piprdine-4-yl-4H-1,3-benzoxazin-
4-one 5p
4.4.3. 5-(2-Chloroethoxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-4-
one 6d
5-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5k was
allowed to react with 1-bromo-2-chloroethane and the solid was
collected and recrystallized from ethyl acetate/toluene to give 6d
(60% yield), mp 161e163 ꢁC, nmax (KBr) 2968, 2860 (CeH), 1663 (C]
7-Hydroxy-8-methyl-2,3-dihydro-4H-1,3-benzoxazin-4-one 4p
allowed to react with piperidine and the solid was collected and
recrystallized from ethanol to give 5p (60% yield), mp 285 ꢁC
decomp. nmax(KBr) 3056e2858 (OH), 1629s (C]O), 1598s (C]C),
1538s (C]N)cmꢃ1 1H (d6-DMSO)
; d 10.5 (bs, 1H, 7-OH), 7.5 (d, 1H,

4942
S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
O), 1621, 1560 (C]N) cmꢃ1; 1H (CDCl3)
d
7.5 (t, 1H, J ¼ 8.0 Hz, H-7),
4.5. Synthesis of 5-, 6-, 7- and 8-[2-(4-methylpiperazin-1-yl)
ethoxy]-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 7aef
6.9 (d, 1H, J ¼ 8.0 Hz, H-6), 6.8 (d, 1H, J ¼ 8 Hz, H-8), 4.4 (t, 2H,
J ¼ 5.6 Hz, H-9), 3.9 (t, 2H, J ¼ 5.6 Hz, H-10), 3.8 (bm, 8H, 4ꢄ CH2 of
morph); 13C (CDCl3)
d
168.6 (C-4), 158.7 (C-5/C-2), 158.5 (C-2/C-5),
The 1-methylpiperazine was allowed to react with 6aed and
6gei according to the general procedures C.
148.9 (C-8a), 134.0 (C-7), 112.9 (C-6), 109.1 (C-8), 102.3 (C-4a), 70.7
(C-9), 66.4 (C-30), 44.5 (C-20), 41.1 (C-10); (found C, 54.15; H, 4.87; N,
9.08; C14H15ClN2O4 requires C, 54.11; H, 4.87; N, 9.02%).
General procedure C: A suspension of 5-(2-chloroethoxy) ben-
zoxazines 6d and 6, 7 and 8-(2-bromoethoxy) benzoxazine 6aec
and 6gei (1 mmol) in 15 mL of acetonitrile was treated 1-methyl-
piperazine (4 mmol) and heated at 80 ꢁC for 20 h. At the completion
of the reaction the mixture was cooled and the acetonitrile was
evaporated off in vacuo. The resulting residue was taken up in
10 mL of CHCl3 and then poured into.
4.4.4. 6-(2-Chloroethoxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-4-
one 6e
6-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5l was
allowed to react with 1-bromo-2-chloroethane and the solid was
collected and recrystallized from ethyl acetate to give 6e (75%
yield), mp 171e173 ꢁC. nmax (KBr) 2968, 2857 (CeH), 1666 (C]O),
10 mL of 2 M NaOH and 10 mL of saturated NaCl and extracted.
The mixture was extracted with a further 3 ꢄ 10 mL of CHCl3 and
the combined organics layer was dried over Mg2SO4 and the
chloroform was evaporated in vacuo. The resulting residue was
triturated with diethyl ether to give the crude solid.
1624, 1555 (C]N) cmꢃ1; 1H (CDCl3)
d
7.6 (d, 1H, J ¼ 8.0 Hz, H-7), 7.2
(d, H, J ¼ 8 Hz, H-8),7.1(s, 1H, H-5), 4.3 (t, 2H, J ¼ 5.6 Hz, H-9), 3.8
(t, 2H, J ¼ 5.6 Hz, H-10), 3.8 (bm, 8H, 4ꢄ CH2 of morph); 13C (CDCl3)
d
167.6 (C-4), 157.7 (C-6), 156.1 (C-2), 148.9 (C-8a), 123.9 (C-7), 118.3
The physical data, IR, 1H NMR and 13C NMR collected for known
compounds 7c, and 7e were found to agree with previously
reported data in Ref. [17].
(C-4a), 117.3 (C-5), 109.6 (C-8), 69.1 (C-9), 66.7 (C-30), 44.8 (C-20),
42.2 (C-10); (found C, 54.11; H, 4.88; N, 8.96; C14H15ClN2O4 requires
C, 54.11; H, 4.87; N, 9.02%).
4.5.1. 5-[2-(4-Methylpiperazin-1-yl)ethoxy]-2-morpholin-4-yl-4H-
1,3-benzoxazin-4-one 7a
4.4.5. 8-(2-Chloroethoxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-4-
one 6f
8-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5j was
allowed to react with 1-bromo-2-chloroethane and the solid was
collected and recrystallized from ethanol to give 6f (60% yield), mp
165e167 ꢁC. nmax (KBr) 2921, 2865 (CeH), 1685 (C]O), 1627, 1572
5-(2-Chloroethoxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one
6d was allowed to react with 1-methylpiperazine. The crude solid was
collected by filtration and recrystallized from toluene to give 7a (67%
yield) mp 158e161 ꢁC.nmax (KBr) 2939, 2796 (CeH),1663(C]O),1623,
1564 (C]N) cmꢃ1; 1H (CDCl3)
J ¼ 8.0 Hz, H-7), 6.7 (d,1H, J ¼ 8.0 Hz, H-6), 4.2 (t, 2H, J ¼ 5.6 Hz, H-9),
3.9e3.7 (bm, 8H, 4ꢄ CH2 of Morpholine), 2.9 (t, 2H, J ¼ 5.6 Hz, H-10),
2.7e2.6 (bm, 4H, H-11), 2.5e2.4 (bm, 4H, H-12), 2.3 (s, 3H, H-13). 13C
d
8.0 (d, 1H, J ¼ 8.0 Hz, H-8), 7.1 (t, 1H,
(C]N) cmꢃ1
;
1H (CDCl3)
d
7.7 (d, 1H, J ¼ 8.0 Hz, H-7), 7.1 (t, 1H,
J ¼ 8.0 Hz, H-6), 7.3(d,1H, J ¼ 8.0 Hz, H-5), 4.4 (t, 2H, J ¼ 5.6 Hz, H-9),
3.9 (t, 2H, J ¼ 5.6 Hz, H-10), 3.8 (bm, 8H, 4ꢄ CH2 of morph); 13C
(CDCl3)
d
165.5 (C-4), 156.2 (C-8), 157.2 (C-2), 146.2 (C-8a), 125.5
(CDCl3) d 168.3 (C-4), 159.3 (C-5), 156.7 (C-2), 148.7 (C-8a), 135.1
(C-7), 120.1 (C-5), 117.6 (C-6), 118.9 (C-4a), 69.8 (C-9), 66.7 (C-30),
44.7 (C-20), 42.3 (C-10); (found C, 54.08; H, 4.77; N,9.05;
C14H15ClN2O4 requires C, 54.11; H, 4.87; N, 9.02%).
(C-7), 113.5 (C-6), 110.1 (C-8), 100.5 (C-4a), 70.7 (C-9), 66.6 (C-30), 55.0
(C-12), 53.5 (C-11), 45.9 (C-13), 44.4 (C-20), 41.1 (C-10); (found C,
60.80; H, 6.95; N, 15.04; C19H26N4O4 requires C, 60.95; H, 7.00;
N, 14.96%).
4.4.6. 7-(2-Bromoethoxy)-8-methyl-2-thiomorpholin-4-yl-4H-1,3-
benzoxazin-4-one 6h
4.5.2. 6-[2-(4-Methylpiperazin-1-yl)ethoxy]-2-morpholin-4-yl-4H-
1,3-benzoxazin-4-one 7b
7-Hydroxy-8-methyl-2-thiomorpholino-2,3-dihydro-4H-1,3-ben-
zoxazin-4-one 5o was allowed to react with 1,2-dibromoethane. The
crude material was collected and recrystallized from ethanol to give
6h (81% yield), mp 210e211 ꢁC. nmax(KBr) 2972, 2865 (CeH), 1669s
The title compound was obtained from 6-(2-Bromoethoxy)-2-
morpholin-4-yl-4H-1,3-benzoxazin-4-one 6a was allowed to react
with 1-methylpiperazine. The crude solid was collected and
recrystallized from cyclohexane to give 7b (78% yield), mp
160e162 ꢁC. nmax (KBr) 2942, 2796 (CeH), 1667s (C]O), 1575s (C]
(C]O), 1597s (C]C), 1567s (C]N) cmꢃ1 1H (CDCl3)
; d 8.0 (d, 1H,
J ¼ 8.0 Hz, H-5), 6.8 (d,1H, J ¼ 8.0 Hz, H-6), 4.4 (t, 2H, J ¼ 5.6 Hz, H-9),
3.7 (t, 2H, J ¼ 5.6 Hz, H-10), 4.2 (bm, 4H, CH2eN of thiomorpholine),
2.8 (bm, 4H, CH2eS of thiomorpholine), 2.3 (s, 3H, 8-CH3); 13C (CDCl3)
N), 1603s (C]C) cmꢃ1; 1H (CDCl3)
d
7.5 (d, 1H, JH5,H7 ¼ 2.6 H-5), 7.2
(dd,1H,, JH7,H5 ¼ 2.6 Hz, JH7,H8 ¼ 8.8 Hz, H-7), 7.1 (d,1H, J ¼ 8.8 Hz, H-
8), 4.2 (t, 2H, J ¼ 5.6 Hz, H-9), 3.8 (bm, 8H, 4ꢄ CH2 of Morpholine),
2.9 (t, 2H, J ¼ 5.6 Hz, H-10), 2.6 (bm, 4H, H-11), 2.4 (bm, 4H, H-12),
d
165.3 (C-4), 159.6 (C-7), 156.2 (C-2), 152.1 (C-8a), 125.0 (C-5), 112.4
(C-8), 110.6 (C-4a), 109.6 (C-6), 68.5 (C-9), 46.5 (C-30), 30.9 (C-10), 26.0
(C-20), 7.6 (8-CH3); (found C, 46.35; H, 4.20; N, 7.03; C15H17BrN2O3S
requires C, 46.76; H, 4.45; N, 7.27%).
2.3 (s, 3H, H-13); 13C (CDCl3)
d 166.5 (C-4), 157.2 (C-6), 155.7 (C-2),
148.9 (C-8a), 124.5 (C-7), 118.0 (C-4a), 117.1 (C-5), 110.6 (C-8), 68.9
(C-9), 66.6 (C-30), 57.3 (C-10), 55.4 (C-12), 53.0 (C-11), 45.9 (C-13),
43.4 (C-20); (found C, 60.85; H, 7.05; N, 115.03; C19H26N4O4 requires
C, 60.95; H, 7.00; N, 14.96%).
4.4.7. 7-(2-Bromoethoxy)-8-methyl-2-piprdine-4-yl-4H-1,3-
benzoxazin-4-one 6i
7-Hydroxy-8-methyl-2-(piprdine-1-yl)-4H-1,3-benzoxazin-4-one
5p was allowed to react with 1,2-dibromoethane. The crude material
was collected and recrystallized from ethyl acetate to give 6i (86%
yield), mp 190 ꢁC. nmax(KBr) 2945, 2861 (CeH), 1681s (C]O), 1598s
4.5.3. 8-[2-(4-Methylpiperazin-1-yl)ethoxy]-2-morpholin-4-yl-4H-
1,3-benzoxazin-4-one 7d
The title compound was obtained from the reaction of 8-
(2-bromoethoxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 6c
was allowed to react with 1-methylpiperazine. The crude solid was
collected and recrystallized from cyclohexane to give 7d (78%
yield), mp 151e154 ꢁC. nmax (KBr) 2939, 2796 (CeH), 1666 (C]O),
(C]C),1567s (C]N) cmꢃ1; 1H (CDCl3)
d
7.9 (d,1H, J ¼ 8.0 Hz, H-5), 6.8
(d,1H, J ¼ 8.0 Hz, H-6), 4.3 (t, 2H, J ¼ 5.6 Hz, H-9), 3.7 (t, 2H, J ¼ 5.6 Hz,
H-10), 3.8(bm, 3H, CH2eNofpipradine), 2.2(s,3H, 8-CH3),1.7(bm, 6H,
30, 4’CH2 ofpipradine);13C(CDCl3)
d167.9(C-4),160.3(C-7),157.1(C-2),
153.1 (C-8a), 126.3 (C-5), 108.9 (C-6), 113.3 (C-8), 111.4 (C-4a), 45.7 (C-
20), 29.5 (C-10), 26.0 (C30), 24.5 (C40), 68.7 (C-9), 8.5 (8-CH3); (found C,
52.31; H, 5.32; N, 7.69; C16H19BrN2O3 requires C, 52.33; H, 5.21;
N, 7.63%).
1624, 1555 (C]N) cmꢃ1; 1H (CDCl3)
d
7.6 (d, 1H, J ¼ 7.6 Hz, H-5), 7.4
(t, 1H, J ¼ 7.6 Hz, H-6), 6.9 (d, 1H, J ¼ 7.6 Hz, H-7), 4.2 (t, 2H,
J ¼ 5.6 Hz, H-9), 3.8 (bm, 8H, 4ꢄ CH2 of morpholine), 2.9 (t, 2H,
J ¼ 5.6 Hz, H-10), 2.7e2.6 (bm, 4H, H-11), 2.5e2.4 (bm, 4H, H-12),

S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
4943
2.3 (s, 3H, H-13); 13C (CDCl3)
d
166.6 (C-4), 158.3 (C-8), 147.7 (C-2),
182 ꢁC. nmax(KBr) 2967, 2852 (CeH), 1669 (C]O), 1621 (C]C), 1558
(C]N) cmꢃ1; 1H (CDCl3)
143.5 (C-8a), 125.0 (C-6), 117.6 (C-4a), 116.5 (C-7), 112.1 (C-5), 67.7
(C-9/C30), 66.6 (C-30/C9), 56.7 (C-10), 55.0 (C-12), 53.5 (C-11), 45.9
(C-13), 44.4 (C-20); (found C, 60.60; H, 6.85; N, 14.90; C19H26N4O4
requires C, 60.95; H, 7.00; N, 14.96%).
d
7.7 (d, 1H, J ¼ 3.4 Hz, H-5), 7.5e7.3 (m,5H,
H-11, H-12 and H-13), 7.3 (dd, 1H, JH-7,H-8 ¼ 9, JH-7H-5 ¼ 3.4 Hz, H-7),
7.2(d, 1H, J ¼ 9 Hz, H-8) 5.1 (s, 2H, H-9), 3.8 (bm, 8H, 4ꢄ CH2 of
morpholine); 13C (d6-DMSO)
d 163.3 (C-4), 154.5 (C-6), 153.5 (C-2),
145.6 (C-8a), 134.6 (C-10), 126.4/125.8/125.6 (C-11/C-12/C-13),
120.6 (C-7), 115.4 (C-7), 111.7 (C-4a), 107.2 (C-8), 67.7 (C-9), 66.3
(C-30), 44.5 (C-20); (found C, 67.40; H, 5.46; N, 8.30; C19H18N2O4
requires C, 67.44; H, 5.36; N, 8.28%).
4.5.4. 7-[2-(4-Methylpiperazin-1-yl)ethoxy]-8-methyl-2-
thiomorpholin-4-yl-4H-1,3-benzoxazin-4-one 7g
The title compound was obtained from 7-(2-bromoethoxy)-
8-methyl-2-thiomorpholin-4-yl-4H-1,3-benzoxazin-4-one 6h was
allowed to react with 1-methylpiperazine. The crude solid was
collected and recrystallized from ethyl acetate/n-hexane to give 7g
(55% yield), mp 175 ꢁC. nmax(KBr) 2932, 2794 (CeH), 1663s (C]O),
4.6.3. 8-(Benzyloxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one
8c
8-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5j was
allowed to react with benzyl bromide and the solid was collected
and recrystallized from ethanol to give 8c (85% yield), mp 173 ꢁC.
nmax(KBr) 2925, 2849 (CeH), 1672 (C]O), 1627 (C]C), 1567 (C]N)
1598s (C]C), 1558s (C]N)cmꢃ1; 1H (CDCl3)
d
7.9 (d, 1H, J ¼ 8.5 Hz,
H-5), 6.9 (d, 1H, J ¼ 8.5 Hz, H-6), 4.2 (bm, 4H, CH2-N of thio-
morpholine), 4.2 (t, 2H, J ¼ 5.6 Hz, H-9), 2.9 (bm, 4H, CH2eS of
thiomorpholine), 2.9 (t, 2H, J ¼ 5.6 Hz, H-10), 2.7 (bm, 4H, H-11), 2.5
(bm, 4H, H-12), 2.3 (s, 3H, H-13). 2.2 (s, 3H, 8-CH3); 13C (CDCl3)
cmꢃ1; 1H (CDCl3)
d
7.7 (dd, 1H, JH-7H-6 ¼ 8.0, JH-7H-5 ¼ 2.0 Hz, H-7),
7.6 (m,5H, H-11, H-12 and H-13), 7.3(t, 1H, J ¼ 8.0 Hz, H-6), 7.2(dd,
d
167.9 (C-4), 161.3 (C-7), 157.1 (C-2), 153.1 (C-8a), 126.6 (C-5), 109.3
1H, JH-5H-6 ¼ 8.0, JH-5H-6 ¼ 2Hz, H-5), 5.2 (s, 2H, H-9), 3.7 (bm, 8H,
(C-6), 112.8 (C-8), 110.9 (C-4a), 46.2 (C-30), 46.1 (C-13), 27.5 (C-20),
8.7 (8-CH3); (found C, 59.04; H, 6.20; N, 13.53; C20H28N4O3S
requires C, 59.38; H, 6.98; N, 13.85%).
4ꢄ CH2 of morpholine); 13C (CDCl3)
d 166.9 (C-4), 156.8 (C-8), 145.3
(C-2), 142.8 (C-8a), 134.6 (C-10), 126.4/125.8/125.6 (C-11/C-12/
C-13), 125.4 (C-6), 119.6 (C-5/C-7), 118.5 (C-4a), 117.3 (C-7/C-5), 69.0
(C-9), 66.3 (C-30), 44.5 (C-20); (found C, 67.36; H, 5.17; N, 8.41;
C19H18N2O4 requires C,67.44; H,5.36; N, 8.28%).
4.5.5. 7-[2-(4-Methylpiperazin-1-yl)ethoxy]-8-methyl-2-piprdine-
4-yl-4H-1,3-benzoxazin-4-one 7f
The title compound was obtained from the reaction of 7-(2-bro-
moethoxy)-8-methyl-2-piprdine-4-yl-4H-1,3-benzoxazin-4-one 6i
with 1-methylpiperazine. The crude product was collected and
recrystallizedfromtoluene/n-hexane to give 7f(65%yield), mp195ꢁC.
nmax (KBr) 2927, 2784 (CeH),1681s(C]O),1598s (C]C),1567s (C]N)
4.7. Synthesis of 5, 6, 7 and 8-(pyridine-2yl, 3yl or 4yl-methyloxy)-
2-morpholin-4-yl-4H-1,3-benzoxazin-4-one compounds 9aek
General Procedure D: A suspension of 2-morpholino-1,3-ben-
zoxazine 5jen (1 mmol) in 10 mL of dry acetonitrile in a 50 ml two
necked round-bottomed flask was mixed with cesium carbonate
(8.5 mmol) and then the mixture was refluxed for 2h. 2-(bromo-
methyl)-pyridinehydrobromide, 3-(chloromethyl)-pyridine hydro-
chloride or 4-(bromomethyl)-pyridine hydrobromide (2 mmol)
was added drop-wise. Upon completion of the addition the reaction
mixture was refluxed for further 30 min. The reaction mixture was
then cooled and the acetonitrile was evaporated off under reduced
pressure. The residue was washed with 4 ꢄ15 mL of CHCl3 and then
filtered. The CHCl3 filtrate was evaporated off under reduced
pressure and the resulting residue was triturated with minimal
amount of diethyl ether to give the crude solid which was recrys-
tallized from an appropriate solvent.
cmꢃ1;1H(CDCl3)
d
7.9(d,1H, J¼ 8.6Hz, H-5), 6.8(d,1H, J¼ 8.6Hz,H-6),
4.3 (t, 2H, J ¼ 5.6, Hz H-9), 3.8 (bm, 4H, 2 ꢄ CH2-N of pipradine), 3.7 (t,
2H, J ¼ 5.6 Hz, H-10), 2.2 (s, 3H, 8-CH3), 1.7 (bm, 6H, 30, 4’CH2 of
pipradine); 13C (CDCl3)
d 167.9 (C-4), 160.3 (C-7), 157.1 (C-2), 153.1 (C-
8a), 126.3 (C-5), 113.3 (C-8), 111.4 (C-4a), 108.9 (C-6), 68.7 (C-9), 45.7
(C-20), 29.5 (C-10), 26.0 (C30), 24.5 (C40), 8.5 (8-CH3); (found C, 52.31;
H, 5.32; N, 7.69; C16H19BrN2O3 requires C, 52.33; H, 5.21; N, 7.63%).
4.6. Reactions of 2-morpholino-5, 6, and 8-hydroxy substituted
benzoxazine 5jel with benzyl bromide
The 2-morpholino-O-benzyloxy-benzoxazines 8aec were syn-
thesized from the reactions of 2-morpholino-5, 6, 7 and 8-hydroxy
benzoxazine 5jel (Scheme 3) with benzyl bromide according to the
general procedure B.
4.7.1. 2-Morpholin-4-yl-7-(pyridin-2-ylmethoxy)-4H-1,3-
benzoxazin-4-one 9a
The 7-hydroxy-2-morpholine-4H-1,3-benzoxazin-4-one 5m was
allowed to react with 2-(bromomethyl)-pyridine hydrobromide as
described in the general method A. The crude material was collected
and recrystallized from toluene to give 9a (91% yield), mp 210 ꢁC.
nmax(KBr) 2978, 2858 (CeH), 1663 (C]O), 1623 (C]C), 1557 (C]N)
4.6.1. 5-(Benzyloxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one
8a
5-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5k was
allowed to react with benzyl bromide and the solid was collected
and recrystallized from ethanol to give 8a (75% yield), mp 162 ꢁC.
nmax(KBr) 3063, 2862 (CeH), 1747 (C]O), 1620 (C]C), 1563 (C]N)
cmꢃ1
;
1H (CDCl3)
d
8.6 (bdd, 1H, J ¼ 5.0 Hz, reduced coupling as
a result of nitrogen quaderpul effect, H-12), 8.0 (d,1H, J ¼ 8.6 Hz, H-5),
7.8 (dt, 1H, JH14eH15eH13 ¼ 7.6 Hz, JH14eH12 ¼ 1.7 Hz, H-14), 7.6 (d, 1H,
J ¼ 7.6 Hz, H-15), 7.3 (t,1H, J ¼ 8.0 Hz, H-13), 7.0 (d,1H, J ¼ 8.0 Hz, H-6),
6.7 (s, 1H, H-8), 5.3 (s, 2H, CH2eO) 3.8 (bm, 8H, 4ꢄ CH2 of morpho-
cmꢃ1; 1H (CDCl3)
d 7.5 (under the invelop Ph proton,1H, H-7), 6.8 (d,
2H, J ¼ Hz, H-8, 6), 7.6 (t,1H, J ¼ Hz, H-7), 7.4e7.3(m, 5H, H-11,12,13),
5.3 (s,2H, H-9), 3.8 (bm, 8H, 4ꢄ CH2 of morpholine); 13C (CDCl3)
d
165.7 (C-4),159.2 (C-5),155.8 (C-2),155.6 (C-8a),137.0 (C-10),134.1
line); 13C (CDCl3)
d 167.1 (C-4), 163.3 (C-7), 157.1 (C-10), 156.2 (C-2),
(C-7), 128.9/128.0/127.1 (C-11/C-12/C-13), 110.9 (C-6), 108.3 (C-8),
108.0 (C-4a), 71.42 (C-9), 66.7 (C-30), 44.6 (C-20); (found C, 67.37; H,
5.27; N, 8.19; C19H18N2O4 requires C, 67.44; H, 5.36; N, 8.28%).
155.4 (C-8a), 149.8 (C-12), 137.4 (C-14), 129.7 (C-5), 123.5 (C-15/C-13),
121.9 (C-13/C-15),114.5 (C-6),111.4 (C-4a),100.8 (C-8), 71.6(C-9); 66.7
(C-30), 44.9 (C-20); (found C, 63.81; H, 5.14; N,12.46; C18H17N3O4
requires C, 63.71; H, 5.05; N, 12.38%).
4.6.2. 6-(Benzyloxy)-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one
8b
6-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5l was
allowed to react with benzyl bromide and the solid was collected
and recrystallized from ethyl acetate to give 8b (75% yield), mp
4.7.2. 8-Methyl-2-morpholin-4-yl-7-(pyridin-2-ylmethoxy)-4H-
1,3-benzoxazin-4-one 9b
The 7-hydroxy-8-methyl-2-morpholine-4H-1,3-benzoxazin-4-
one 5n was allowed to react with 2-(bromomethyl)-pyridine

4944
S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
hydrobromide as described in the general method A. The crude
material was collected and recrystallized from toluene to give 9b
(88% yield), mp 195 ꢁC. nmax(KBr) 2952, 2861 (CeH), 1672 (C]O),
(CDCl3) d 167.0 (C-4), 163.2 (C-7), 157.1 (C-2), 155.4 (C-8a), 150.2
(C-11), 149.3 (C-13), 135.6 (C-15), 131.6 (C-10), 129.7 (C-5), 124.0
(C-14), 114.0 (C-6), 111.5 (C-4a), 100.9 (C-8), 68.4 (C-9), 66.6 (C-30),
44.6 (C-20); (found C, 63.66; H, 5.04; N,12.31; C18H17N3O4 requires C,
63.71; H, 5.05; N, 12.38%).
1626 (C]C), 1563 (C]N) cmꢃ1; 1H (CDCl3)
d
8.6 (bdd,1H, J ¼ 5 Hz,
reduced coupling as a result of nitrogen quader ball effect, H-12),
7.9 (d, 1H, J ¼ 8.7 Hz, H-5), 7.7 (dt, 1H, JH14eH15eH13 ¼ 7.6 Hz,
JH14eH12 ¼ 1.7 Hz, H-14), 7.5 (d, 1H, J ¼ 7.6 Hz, H-15), 7.3 (t, 1H,
J ¼ 7.6 Hz, H-13), 6.9 (d, 1H, J ¼ 8.7 Hz, H-6), 5.3 (s, 2H, CH2O) 3.8
4.7.6. 8-Methyl-2-morpholin-4-yl-7-(pyridin-3-ylmethoxy)-4H-
1,3-benzoxazin-4-one 9f
(bm, 8H, 4ꢄ CH2 of morpholine), 2.3 (s, 3H, H-8); 13C (CDCl3)
d
167.6
The 7-hydroxy-8-methyl-2-morpholine-4H-1,3-benzoxazin-4-
one 5n was allowed to react with 3-(Picolyl chloride)-pyridine
hydrochloride as described in the general method A. The crude
material was collected and recrystallized from toluene to give 9f
(75% yield), mp 230 ꢁC decomp. nmax(KBr) 2967, 2866 (CeH), 1668
(C-4), 160.8 (C-7), 157.3 (C-2), 156.6 (C-10), 153.0 (C-8a), 149.7
(C-14), 137.3 (C-12), 135.5 (C-15), 126.6 (C-5), 121.5 (C-13), 113.0 (C-
4a), 111.2 (C-8), 109.7 (C-6), 71.6 (C-9), 66.6 (C-30), 44.6(C-20), 8.5
(CH3); (found C, 64.57; H, 5.40; N, 11.77; C19H19N3O4 requires C,
64.58; H, 5.42; N, 11.89%). 1H (CDCl3)
d
8.7 (s,1H, H-11), 8.6 (bdd, 1H,
(C]O), 1620 (C]C), 1553 (C]N) cmꢃ1 1H (CDCl3)
; d 8.7 (d, 1H,
JH13eH14 ¼ reduced coupling as a result).
J ¼ 2.2 Hz, H-11), 8.6 (b, dd, 1H, JH13eH14 ¼ 5.0 Hz, reduced coupling
as a result of nitrogen quadruple effect, JH13eH11 ¼ 2.0 Hz, H-13), 8.0
(d, 1H, J ¼ 8.7 Hz, H-5), 7.8 (d, 1H, J ¼ 8.2 Hz, H-15), 7.3 (dd, 1H,
JH14eH13 ¼ 8.2 Hz, JH14eH15 ¼ 7.3 Hz, H-14), 6.9 (d, 1H, J ¼ 8.7 Hz, H-
6), 5.2 (s, 2H, CH2O) 3.8 (bm, 8H, 4ꢄ CH2 of morpholine), 2.2 (s, 3H,
4.7.3. 2-Morpholin-4-yl-8-(pyridin-2-ylmethoxy)-4H-1,3-
benzoxazin-4-one 9c
The 8-hydroxy-2-morpholine-4H-1,3-benzoxazin-4-one 5j was
allowed to react with 2-(bromoethyl)-pyridine hydrobromide as
described in the general method A. The crude material was
collected and recrystallized from toluene to give 9g (86% yield), mp
198 ꢁC. nmax(KBr)3056, 2899 (CeH), 1674 (C]O), 1629 (C]C), 1578
H-8); 13C (CDCl3)
d 167.6 (C-4), 160.7 (C-7), 157.3 (C-2), 153.0 (C-8a),
150.1 (C-11), 149.2 (C-13), 135.5 (C-15), 132.1 (C-10), 126.7 (C-14),
124.0 (C-5), 113.2 (C-4a), 111.4 (C-8), 109.4 (C-6), 68.6 (C-9), 66.6 (C-
30), 44.7 (C-20), 8.73 (CH3); (found C, 64.57; H, 5.40; N, 11.77;
C19H19N3O4 requires C, 64.58; H, 5.42; N, 11.89%).
(C]N) cmꢃ1; 1H (CDCl3)
d
8.6 (bdd, 1H, J ¼ 5 Hz, reduced coupling
as a result of nitrogen quader ball effect, H-12), 7.7(d, 1H, J ¼ 8.6 Hz,
H-7), 7.8 (d, 1H, J ¼ 7.3 Hz, H-15), 7.3 (dd, 1H, JH14eH15 ¼ 8.2 Hz,
JH14eH15 ¼ 7.3 Hz, H-14), 7.3 (d, 1H, J ¼ 8.6 Hz, H-5), 6.9 (t, 1H,
J ¼ 8.6 Hz, H-6), 5.3 (s, 2H, CH2O) 3.8 (bm, 8H, 4ꢄ CH2 of mor-
4.7.7. 2-Morpholin-4-yl-8-(pyridin-3-ylmethoxy)-4H-1,3-
benzoxazin-4-one 9g
The 8-hydroxy-2-morpholine-4H-1,3-benzoxazin-4-one 5j
was allowed to react with 3-(chloromethyl)-pyridine hydrochlo-
ride as described in the general method A. The crude material was
collected and recrystallized from toluene to give 9g (80% yield),
mp 189 ꢁC. nmax(KBr) 2967, 2870 (CeH), 1677 (C]O), 1626 (C]C),
1558 (C]N) cm of nitrogen quader ball effect 4.9 Hz,
JH13eH11 ¼ 2.0 Hz, H-13, 8.0 (d, 1H, J ¼ 8.6 Hz, H-7), 7.8 (d, 1H,
J ¼ 7.3 Hz, H-15), 7.3 (dd, 1H, JH14eH13 ¼ 8.2 Hz, JH14eH15 ¼ 7.3 Hz,
H-14), 6.9 (t, 1H, J ¼ 8.6 Hz, H-6), 7.3 (d, 1H, J ¼ 8.6 Hz, H-5), 5.2 (s,
pholine); 13C (CDCl3)
d 167.2 (C-4), 156.9 (C-8), 150.3 (C-2), 149.9
(C-12), 146.1 (C-10), 144.1 (C-8a), 137.3 (C-14), 125.5 (C-15), 123.5
(C-7), 121.5 (C-13), 119.7 (C-5), 118.8 (C-4a), 117.8 (C-6), 72.3 (C-9),
66.7 (C-30), 45.6 (C-20); (found C, 63.57; H, 5.10; N,12.31; C18H17N3O4
requires C, 63.71; H, 5.05; N, 12.38%).
4.7.4. 2-Morpholin-4-yl-6-(pyridin-3-ylmethoxy)-4H-1,3-
benzoxazin-4-one 9d
The 6-hydroxy-2-morpholine-4H-1,3-benzoxazin-4-one 5l was
allowed to react with 3-(chloromethyl)-pyridine hydrochloride as
described in the general method A. The crude material was
collected and recrystallized from toluene to give 9d (90% yield), mp
decomp 268 ꢁC. nmax(KBr) 2960, 2861 (CeH), 1673 (C]O), 1623
2H, CH2O) 3.7 (bm, 8H, 4ꢄ CH2 of morpholine); 13C (CDCl3)
d 167.2
(C-4), 156.9 (C-8), 150.3 (C-2), 149.3 (C-12), 146.0 (C-10), 144.2 (C-
8a), 135.5 (C-14), 132.0 (C-15), 125.5 (C-7), 124.0 (C-13), 119.9
(C-5), 118.9 (C-4a), 117.5 (C-6), 69.3 (C-9), 66.7 (C-30), 44.6 (C-20);
(found C, 63.57; H, 5.10; N,12.31; C18H17N3O4 requires C, 63.71; H,
5.05; N, 12.38%).
(C]C), 1555 (C]N) cmꢃ1 1H (CDCl3)
; d 8.7 (s, 1H, H-11), 8.6 (bdd,
1H, J ¼ 3.9 Hz, reduced coupling as a result of nitrogen quader ball
effect H-13), 7.8 (d, 1H, J ¼ 8.0 Hz, H-5), 7.7 (d, 1H, J ¼ 7.2 Hz, H-15),
7.3 (dd, 1H, JH14eH15 ¼ 8.2 Hz, JH14eH15 ¼ 7.3 Hz, H-14),, 7.3 (d, 1H,
J ¼ 8.6 Hz, H-7), 7.2 (d, 1H, J ¼ 8.6 Hz, H-8) 5.1 (s, 2H, CH2O) 3.8 (bm,
4.7.8. 2-Morpholin-4-yl-5-(pyridin-3-ylmethoxy)-4H-1,3-
benzoxazin-4-one 9h
The 6-hydroxy-2-morpholine-4H-1,3-benzoxazin-4-one 5k was
allowed to react with 3-(chloromethyl)-pyridine hydrochloride as
described in the general method A. The crude material was
collected and recrystallized from toluene to give 9h (90% yield), mp
185 ꢁC nmax(KBr) 2970, 2860 (CeH), 1650 (C]O), 1619 (C]C), 1561
8H, 4ꢄ CH2 of morpholine); 13C (CDCl3)
d 167.3 (C-4), 157.3 (C-6),
156.2 (C-2), 148.6 (C-8a), 150.0 (C-11), 149.4 (C-13), 135.8 (C-15),
132.0 (C-10),123.9 (C-7),123.8 (C-14),118.3 (C-4a),117.3 (C-5),109.7
(C-8), 68.6 (C-9), 66.7 (C-30), 44.6 (C-20); (found C, 63.57; H, 5.10;
N,12.31; C18H17N3O4 requires C, 63.71; H, 5.05; N, 12.38%).
(C]N) cmꢃ1; 1H (CDCl3)
d
8.7 (s,1H, H-11), 8.6 (bdd, 1H, J ¼ 3.9 Hz,
reduced coupling as a result of nitrogen quaderpul effect H-13), 8.2
(d, 1H, J ¼ 8.0 Hz, H-15), 7.3 (dd, 1H, JH14eH13 ¼ 8.2 Hz,
JH14eH15 ¼ 7.3 Hz, H-14), 6.9 (d, 1H, J ¼ 8.6 Hz, H-6), 7.5 (t, 1H,
J ¼ 8.4 Hz, H-7), 6.8 (d, 1H, J ¼ 8.6 Hz, H-8) 5.3 (s, 2H, CH2O) 3.8 (bm,
4.7.5. 2-Morpholin-4-yl-7-(pyridin-3-ylmethoxy)-4H-1,3-
benzoxazin-4-one 9e
The 7-hydroxy-2-morpholine-4H-1,3-benzoxazin-4-one 5m
was allowed to react with 3-(chloromethyl)-pyridine hydrochloride
as described in the general method A. The crude material was
collected and recrystallized from toluene to give 9e (73% yield), mp
185 ꢁC. nmax(KBr) 2978, 2858 (CeH), 1670 (C]O), 1627 (C]C), 1554
8H, 4ꢄ CH2 of morpholine); 13C (CDCl3)
d 165.4 (C-4), 158.7 (C-2),
155.9 (C-5/C-8a), 155.8 (C-8a/C-5), 149.5 (C-11), 148.3 (C-13), 135.6
(C-15), 132.7 (C-10), 134.3 (C-7), 124.2 (C-14), 111.0 (C-6), 108.9 (C-
8), 107.9 (C-4a), 69.2 (C-9), 66.6 (C-30), 44.7 (C-20); (found C, 63.80;
H, 5.19; N, 12.41; C18H17N3O4 requires C, 63.71; H, 5.05; N, 12.38%).
; d 8.7 (s,1H, H-11), 8.6 (bdd, 1H,
(C]N) cmꢃ1 1H (CDCl3)
JH13eH14 ¼ reduced coupling as a result of nitrogen quader ball
effect 8.0 Hz, H-13), 8.0 (d, 1H, J ¼ 8.6 Hz, H-5), 7.8 (d, 1H, J ¼ 8.2 Hz,
H-15), 7.3 (dd, 1H, JH14eH13 ¼ 8.2 Hz, JH14eH15 ¼ 7.3 Hz, H-14), 6.9
(dd, 1H, JH6eH5 ¼ 8.6 Hz, JH6eH8 ¼ 2.5 Hz, H-6), 6.7 (d, 1H, J ¼ 2.5 Hz
H-8), 5.1 (s, 2H, CH2O) 3.8 (bm, 8H, 4ꢄ CH2 of morpholine); 13C
4.7.9. 2-Morpholin-4-yl-7-(pyridin-4-ylmethoxy)-4H-1,3-
benzoxazin-4-one 9i
The 7-hydroxy-2-morpholine-4H-1,3-benzoxazin-4-one 5m was
allowed to react with 4-(bromomethyl)-pyridine hydrobromide as

S. Ihmaid et al. / European Journal of Medicinal Chemistry 45 (2010) 4934e4946
4945
described in the general method A. The crude material was collected
4.9. Platelet aggregometry
and recrystallized from toluene to give 9i (70% yield), mp decomp
203 ꢁC. nmax(KBr) 2978, 2860 (CeH), 1670 (C]O), 1626 (C]C), 1560
Venous blood was collected from apparently healthy, drug free
volunteers into trisodium citrate 22.0 g/L. Ethics approval was
obtained from La Trobe University Human Ethics Committee (HEC
approval No 07-127). Platelet aggregation was determined by the
optical method in a two-channel platelet aggregometer (Chrono-
Log) using the previously reported protocol [17].
(C]N) cmꢃ1; 1H (CDCl3)
d
8.6 (d,2H, J ¼ 8.6 Hz, H-12 and H-14), 7.3
(d, 2H, J ¼ 8.0 Hz, H-11 and H-15), 8.1 (d, 1H, J ¼ 8.0 Hz, H-5), 6.8 (d,
1H, J ¼ 8.0 Hz, H-6), 6.7 (s, 1H, H-8), 5.2 (s, 2H, CH2O) 3.8 (bm, 8H,
4ꢄ CH2 of morpholine); 13C (CDCl3)
d 167.0 (C-4), 163.0 (C-7), 157.1
(C-2), 155.4 (C-8a), 150.6 (C-11), 149.2 (C-13), 145.2 (C-10), 135.5
(C-15), 129.8 (C-5), 126.8 (C-14), 114.0 (C-6), 111.9 (C-4a), 100.9 (C-8),
69.0 (C-9), 66.6 (C-30), 44.9 (C-20); (found C, 63.66; H, 5.04; N,12.31;
C18H17N3O4 requires C, 63.71; H, 5.05; N, 12.38%).
4.10. DNA-PK inhibition assay
DNA-PK was measured using the SignaTECTÒ DNA-dependent
4.7.10. 8-Methyl-2-morpholin-4-yl-7-(pyridin-4-ylmethoxy)-4H-
1,3-benzoxazin-4-one 9j
protein kinase assay system (Promega). Briefly, a reaction mix was
prepared consisting of 2.5
stranded DNA, 5.0
l 5ꢄ reaction buffer, 2.5
substrate, 0.2 l BSA (10 mg/ml), 5.0 l [
32P] ATP mix (0.05 mCi),
l DNA-dependant protein kinase (10 units, Promega) containing
0.1 mg/ml BSA and deionised water to a final volume of 25 l. This
was incubated at 30 ꢁC for 6 min prior to the reaction being stopped
ml activation buffer containing double
The 7-hydroxy-8-methyl-2-morpholine-4H-1,3-benzoxazin-4-
one 5n was allowed to react with 4-(bromomethyl)-pyridine
hydrobromide as described in the general method A. The crude
material was collected and recrystallized from toluene to give 9j (90%
yield), mp 215 ꢁC decomp. nmax(KBr) 2952, 2861 (CeH), 1672 (C]O),
m
ml biotinylated peptide
m
m g
5
m
m
1624 (C]C), 1572 (C]N) cmꢃ1
;
1H (CDCl3)
d
8.6 (d, 1H, J ¼ 5.3 Hz,
by the addition of 12.5 ml of termination buffer. 10 ml from each
reaction was spotted onto squares of SAM2Ò biotin capture
membrane and membranes washed thoroughly as per manufac-
turer’s instructions and bound reaction product assayed by scin-
tillation counting (Wallac 1450 Microbeta plus). This reaction mix
represented the positive control.
H-12 and H-14), 7.3 (d, 1H, J ¼ 5.0 Hz, H-11 and H-15), 7.9 (d, 1H,
J ¼ 8.7 Hz, H-5), 6.9 (d, 1H, J ¼ 8.7 Hz, H-6), 5.2 (s, 2H, CH2O) 3.8 (bm,
8H, 4ꢄ CH2 of morpholine), 2.3 (s, 3H, H-8); 13C (CDCl3)
d 167.6 (C-4),
160.5 (C-7),157.3 (C-2),152.5 (C-8a),150.6 (C-12 and 14),145.6 (C-10),
126.8 (C-5),121.7 (C-11 and C-15),113.2 (C-4a),111.6 (C-8),109.4 (C-6),
69.0 (C-9), 66.7 (C-30), 44.7 (C-20), 8.7 (CH3); (found C, 64.70; H, 5.49;
N, 11.69; C19H19N3O4 requires C, 64.58; H, 5.42; N, 11.89%).
To test for inhibition 1
substituted for 1 l of deionised water in the above mix (final conc.
1.6e4.0 M). Negative controls consisting of reaction mix without
ml of each compound in DMSO was
m
m
4.7.11. 2-Morpholin-4-yl-8-(pyridin-4-ylmethoxy)-4H-1,3-
benzoxazin-4-one 9k
substrate plus activator (negative control 1) and reaction mix
without activator plus substrate (negative control 2) were included
in every run.
Calculation of percentage inhibition was by the following:
(A ꢃ B)/B ꢄ 100 where A was the cpm for the positive control and B
was the cpm for the reaction plus compound.
The 8-hydroxy-2-morpholine-4H-1,3-benzoxazin-4-one 5j was
allowed to react with 4-(bromomethyl)-pyridine hydrobromide as
described in the general method A. The crude material was
collected and recrystallized from toluene to give 9k (80% yield), mp
180 ꢁC nmax(KBr) 2960, 2861 (CeH), 1675 (C]O), 1626 (C]C), 1559
(C]N) cmꢃ1; 1H (CDCl3)
d
8.6 (d,2H, J ¼ 8.6 Hz, H-12 and H-14), 8.3
Acknowledgements
(d, 2H, J ¼ 8.0 Hz, H-11 and H-15), 8.0 (d,1H, J ¼ 8.6 Hz, H-7), 7.3e7.1
(m, 2H, H-6 and H-7), 5.2 (s, 2H, CH2O) 3.8 (bm, 8H, 4ꢄ CH2 of
The authors gratefully acknowledge the financial support of La
Trobe University.
morpholine). 13C (CDCl3)
d 167.1 (C-4), 156.8 (C-2), 150.5 (C-12 and
C-14), 145.8 (C-10/C-8), 145.5 (C-8/C-10), 144.5 (C-8a), 125.4 (C-6),
121.6 (C-11 and C-15), 119.8 (C-7), 118.8 (C-4a), 117.3 (C-5), 69.6
(C-9), 66.6 (C-30), 44.6 (C-20); (found C, 63.50; H, 5.07; N,12.40;
C18H17N3O4 requires C, 63.71; H, 5.05; N, 12.38%).
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5-Hydroxy-2-morpholin-4-yl-4H-1,3-benzoxazin-4-one 5k
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