Synthesis, DNA-PK inhibition, anti-platelet activity studies of 2-(N-substituted-3-aminopyridine)-substituted-1,3-benzoxazines and DNA-PK and PI3K inhibition, homology modelling studies of 2-morpholino-(7,8-di and 8-substituted)-1,3-benzoxazines

Article abstract of DOI:10.1016/j.ejmech.2012.08.035

A number of new 2-(pyridin-3-ylamino)-4H-(substituted) benz[e]-1,3-oxazin- 4-ones were synthesized 10a-g. These were then reacted with the hydro-halogen salt of 2, 3 and 4-(halo-methyl) pyridine in the presence of Cs ₂CO₃ to give eighteen new 2-(N-substituted (pyridin-3-ylmethyl) amino)-substituted-1,3-benzoxazines (compounds 11a-i, 13a-c, and 15a-f). X-ray crystallography was used to confirm that the 2-N-substituted structures 11 and 13 were formed rather than the 3-N-substitution analogues 12 and 14. Eleven of the new compounds were tested for their effect on collagen induced platelet aggregation and it was found that the most active inhibitory compound was 8-methyl-2-(pyridin-3-yl(pyridin-3- ylmethyl)amino)-7-(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15e with an IC₅₀ of 10 ± 2 μM. DNA-dependent protein kinase (DNA-PK) inhibition data for 12 previously prepared 2-morpholino substituted-1,3- benzoxazines (compounds 19-31) were measured and showed high to moderate activity where the most active compound was compound 27 with an IC₅₀ of 0.28 μM. Furthermore DNA-PK inhibition data for six newly prepared 2-(N-substituted (pyridin-3-ylmethyl) amino)-substituted-1,3-benzoxazines (compounds 11b, 13a-b, 15a-b and 15e) and 8-methyl-7-(pyridin-3-ylmethoxy)-3- (pyridin-3-ylmethyl)-2H-benz[e]-1,3-oxazin-2,4(3H)-dione 17d were measured and moderate to low inhibitory activity was observed, with the most active of the compounds in this series being 8-methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl) amino)-7-(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15e with an IC ₅₀ of 2.5 μM. PI3K inhibition studies revealed that compound 27 is highly potent (IC₅₀ for PI3Kα = 0.13 μM, PI3Kβ = 0.14 μM, PI3Kγ = 0.72 μM, PI3Kδ = 2.02 μM). Compound 22 with 7-[2-(4-methylpiperazin-1-yl)ethoxy] group shows greater inhibition of DNA-PK over PI3K. Docking of some 2-morpholino-substituted-1,3-benzoxazine compounds 19-31 within the binding pocket and structure-activity relationships (SAR) analyses were performed with results agreeing well with observed activities.

Full text of DOI:10.1016/j.ejmech.2012.08.035

European Journal of Medicinal Chemistry 57 (2012) 85e101
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, DNA-PK inhibition, anti-platelet activity studies of 2-(N-substituted-3-
aminopyridine)-substituted-1,3-benzoxazines and DNA-PK and PI3K inhibition,
homology modelling studies of 2-morpholino-(7,8-di and 8-substituted)-1,3-
benzoxazines
,
a
a
Saleh K. Ihmaid a, Jasim M.A. Al-Rawi a , Christopher J. Bradley , Michael J. Angove ,
*
Murray N. Robertson b
a School of Pharmacy and Applied Science, La Trobe University, P.O. Box 199, Bendigo, VIC 3552, Australia
b Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK
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-(pyridin-3-ylamino)-4H-(substituted) benz[e]-1,3-oxazin-4-ones were synthesized
10aeg. These were then reacted with the hydro-halogen salt of 2, 3 and 4-(halo-methyl) pyridine in the
presence of Cs2CO3 to give eighteen new 2-(N-substituted (pyridin-3-ylmethyl) amino)-substituted-1,3-
benzoxazines (compounds 11aei, 13aec, and 15aef). X-ray crystallography was used to confirm that the
2-N-substituted structures 11 and 13 were formed rather than the 3-N-substitution analogues 12 and 14.
Eleven of the new compounds were tested for their effect on collagen induced platelet aggregation and it
was found that the most active inhibitory compound was 8-methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl)
Received 23 January 2012
Received in revised form
23 August 2012
Accepted 24 August 2012
Available online 5 September 2012
Keywords:
Synthesis
2-Amino-substituted-1,3-benzoxazines
DNA-PK
amino)-7-(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15e with an IC50 of 10 ꢀ 2
mM. DNA-
dependent protein kinase (DNA-PK) inhibition data for 12 previously prepared 2-morpholino
substituted-1,3-benzoxazines (compounds 19e31) were measured and showed high to moderate
activity where the most active compound was compound 27 with an IC50 of 0.28 mM. Furthermore DNA-
PI3K inhibition
Homology modelling
Anti-platelet activity
PK inhibition data for six newly prepared 2-(N-substituted (pyridin-3-ylmethyl) amino)-substituted-1,3-
benzoxazines (compounds 11b, 13aeb, 15aeb and 15e) and 8-methyl-7-(pyridin-3-ylmethoxy)-3-(pyr-
idin-3-ylmethyl)-2H-benz[e]-1,3-oxazin-2,4(3H)-dione 17d were measured and moderate to low inhib-
itory activity was observed, with the most active of the compounds in this series being 8-methyl-2-
(pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one
with an IC50 of 2.5 M. PI3K inhibition studies revealed that compound 27 is highly potent (IC50 for
PI3K M, PI3K M, PI3K M, PI3K M). Compound 22 with 7-[2-(4-
¼ 0.13
15e
m
a
m
b
¼ 0.14
m
g
¼ 0.72
m
d
¼ 2.02
m
methylpiperazin-1-yl)ethoxy] group shows greater inhibition of DNA-PK over PI3K.
Docking of some 2-morpholino-substituted-1,3-benzoxazine compounds 19e31 within the binding
pocket and structureeactivity relationships (SAR) analyses were performed with results agreeing well
with observed activities.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
Inhibition of DNA-PK has been demonstrated to potentiate the
cytotoxicity of DNA DSB-inducing anticancer therapies [3], and
The DNA-dependent protein kinase (DNA-PK) plays a key role in
the DNA-damage response (DDR) [1], via the non-homologous end-
joining pathway of DNA double-strand break (DSB) repair [2].
there is evidence that DNA-PK is over-expressed in a number of
tumours [2]. The cellular response to DSB formation is an essential
component of normal cell survival following exposure to DNA-
damaging chemicals and ionizing radiation [4]. Structure activity
relationship (SAR) studies around the non-selective PIKK inhibitor
2-morpholino-4H-chromen-4-one (LY294002) 1 (Fig. 1) [5] resul-
ted in the identification of a number of potent DNA-PK inhibitors,
including the thiophen-2-yl 2 [6] and 8-biphenyl derivatives 3 [7]
as well as NU7441 4a (IC50 ¼ 30 nM) [6,8,9]. Elaboration of this
* Corresponding author. Tel.: þ61 3 54 44 7364; fax: þ61 3 54 44 7476.
E-mail addresses: sihmaid@students.latrobe.edu.au (S.K. Ihmaid), j.al-rawi@
latrobe.edu.au (J.M.A. Al-Rawi), c.bradley@latrobe.edu.au (C.J. Bradley),
m.angove@latrobe.edu.au
(M.N. Robertson).
(M.J.
Angove),
murray.robertson@strath.ac.uk
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.035

86
S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
Fig. 1. Structures of some 8-substituted-2-morpholino-4H-chromen-4-one1-3, 8- substituted-2-morpholino-4H-benz[e]-1,3-oxazin-4-one 5 and substituted naphtha-oxazin-one 6
as DNA-PK and PI3K inhibitors.
structure has led to the highly potent water-soluble DNA-PK
inhibitor KU-0060648 4b (IC50 ¼ 5 nM) [10].
The structures of the new products 9aef were confirmed from
the analysis of their IR, 1H NMR and 13C NMR spectra. The 1H and
13C NMR chemical shifts were assigned using the calculated value
starting from the previously reported chemical shifts of 2-thio-1,3-
benzoxazine 8aef [15], and assisted by the calculated values from
the computer package ChemDraw Ultra 12. Compounds 9aef were
used with no further purification in the synthesis of compounds
10aef.
The studies involving compounds 4a and 4b above have
demonstrated activity in vitro as chemo- and radio-potentiators in
a range of human tumour cell lines. Initial in vivo investigations
with these agents have been promising [8,10].
Recently it was found that compound 5 (Fig. 1), inhibits phos-
phatidyl 3-kinases (PI3K), notably of the IA subfamily [11]. The IC50
for 5a is 2.34
compound 5b shows more potent inhibition with an IC50 of 3.41
for p110 , 0.54 M for p110 and 17.78 M for p110
mM for p110b, and 20.5 mM for p110g. However,
mM
2.1.3. Synthesis of substituted 2-(pyridin-3-ylamino)-4H-benz[e]-
1,3-oxazin-4-ones 10aef
a
m
b
m
g.
A major objective of our research is the development of
potent DNA-PK inhibitors suitable for evaluation as chemo- and
radio-sensitizers in the treatment of cancer [12]. Recently we
found that [13] 2-morpholino-7-(pyridin-2-ylmethoxy)-4H-
naphth[2,3-e]-1,3-oxazin-4-one 6 was a potent DNA-PK inhibitor
Synthesis of 2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10a was attempted by refluxing 3-aminopyridine (2 mmol)
(weak primary aromatic base/weak nucleophile) with 2-thio-1,3-
benzoxazine 8a (1 mmol) in dry dioxane for 4 h. Unfortunately
only unreacted materials were recovered (Scheme 1). However,
synthesis with good yields (70e75%) of substituted 2-(pyridin-3-
ylamino)-4H-benz[e]-1,3-oxazin-4-ones 10aef were achieved by
refluxing the corresponding substituted 2-S-methyl-1,3-ben-
zoxazine 9aef in dry dioxane with 3-aminopyridine for 4 h
(Scheme 1).
The structures of the products 10aef were confirmed from the
analysis of their IR, 1H, 13C NMR spectra and elemental analysis. The
1H and 13C NMR chemical shifts were assigned using the calculated
values starting from the previously reported chemical shifts of 2-
thio-1,3-benzoxazine 8aeg [12,15] and assisted by the calculated
values from ChemDraw Ultra 12.
(IC50 ¼ 0.096
mM).
Here we report the synthesis and structural elucidation of
library of new and previously prepared substituted-1,3-
a
benzoxazines. Their DNA-PK, PI3K and platelet inhibitory activi-
ties have also been evaluated. Compounds 19e31 have also been
docked in our newly built homology model of DNA-PK and the SAR
is discussed.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of substituted 2-thio-1,3-benzoxazine 8aef
2.1.4. Synthesis of 2-(pyridin-2, 3 and 4-yl(pyridin-3-ylmethyl)
amino)-substituted-4H-benz[e]-1,3-oxazin-4-ones 11aei, 13aec
and 15aef
The reaction of 2-(pyridin-3-ylamino)-substituted-1,3-benzoxa-
zine 10aec with the hydro-halogen salt of 2, 3 and 4-(halo-methyl)-
pyridine in the presence of Cs2CO3 in acetonitrile led to the
synthesis of 2-(pyridin-2, 3 and 4-yl(pyridin-3-ylmethyl)amino)-
substituted-4H-benz[e]-1,3-oxazin-4-ones 11aei with yields of
55e75% (Scheme 2).
Furthermore, the hydrogen of the hydroxy group at position C-8
in compound 10d and position 7 in compounds 10eef, also
substituted under the same reaction conditions, gave products
13aec (Scheme 3) and 15aef respectively (Scheme 4).
Substituted 2-thio-1,3-benzoxazine 8aef was synthesized from
the reaction of substituted 2-hydroxy benzoic acid 7aef with the
freshly prepared Ph3P(SCN)2 according to the previously reported
procedure [15] with no further modification (Scheme 1). Physical
properties, IR, 1H NMR and 13C NMR collected for known products
8aef were found to agree with previously reported data [15].
2.1.2. Synthesis of substituted 2-S-methyl-1,3-benzoxazines 9aef
2-S-Methyl-1,3-benzoxazines 9aef were synthesized with good
yield (90e95%) from the reaction of substituted 2-thio-1,3-
benzoxazine 8aef with methyl iodide in the presence of NaHCO3
in 1:1 CH3OH/H2O (Scheme 1).

S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
87
Compound 7, 8, 9 and 10: a R = R1 = H; b R = CH3, R1 = H; c R = Ph, R1 = H; d R = OH, R1
= H; e R = H, R1 = OH; f R = CH3, R1 = OH
Reaction conditions: (i) (Ph)3P(SCN)2, dry DCM, reflux, (ii) CH3I, NaHCO3, 1:1
CH3OH/H2O, (iii) 3-aminopyridine, dry dioxane, reflux 4 hours.
Scheme 1. Synthesis of benzoxazine 8aef, 2-S-methyl-benzoxazine 9aef and 2-(pyridin-3-yl amino)-benzoxazine 10aef starting from substituted 2-hydroxy benzoic acid 7aeg.
The products from the reaction of compounds 10aec with the
hydro-halogen salts of 2, 3 and 4-(halo-methyl)-pyridine in the
presence of Cs2CO3 were expected to be either 11 or 12 or both,
because of the possible tautomerism which can take place in
compound 10. However, structural determination using X-ray
crystallography for the reaction of compound 10b (R ¼ CH3) with 3-
(chloromethyl)-pyridine hydrochloride under the same conditions,
showed that the only product formed was 11b (Fig. 2).
Compound
R
H
R1
Compound
R
R1
Ph
11a
11f
CH3
H
11b
11c
11d
11e
11g
11h
11i
CH3
Ph
H
CH3
Reaction condition: (i) Hydrohalogen salt of 2, 3 and 4-(halo methyl)-pyridine, Cs2CO3,
acetonitrile, reflux.
Scheme 2. The synthesis of 2-(pyridin-2, 3 and 4-yl(pyridin-3-ylmethyl)amino)-substituted 4H-benz[e]-1,3-oxazin-4-ones 11aei.

88
S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
Reaction condition: (i) Hydrohalogen salt of 2, 3 and 4-(halo methyl)-pyridine, Cs2CO3,
acetonitrile, reflux.
Scheme 3. Synthesis of 2-(pyridin-3-yl(pyridin-4-ylmethyl)amino)-8-(pyridin-2, 3 and 4-yl methoxy)-4H-benz[e]-1,3-oxazin-4-one 13aec.
Furthermore when compound 10e, containing 7-hydroxy group,
(pyridin-2 and 3-ylmethyl)-substituted-2H benz[e]-1,3-oxazin-2,4-
(3H)-dione 17aed with good yields.
was reacted with 3-(chloromethyl)-pyridine hydrochloride under
the same conditions, X-ray crystallography showed that the only
product formed was 15b (Fig. 3).
The fact that compounds 12, 14 and 16 did not form under these
conditions indicates that the NH group attached to position 2 is
more acidic than the N3eH. This is probably the result of the
presence of the electron withdrawing pyridine ring.
The structures of the compounds 11aei, 13aec and 15aef were
confirmed from the analysis of their IR, 1H and 13C NMR spectra and
microanalysis. 1H and 13C NMR chemical shifts were assigned using
the calculated value starting from the parent compounds 10aeg
chemical shifts and assisted using the calculated values from
ChemDraw Ultra 12.
The reaction products might result from the neutralization of
the hydro-halogen salts of 2 and 3-(halo-methyl)pyridine with
Cs2CO3 and the subsequent formation carbonic acid which
decomposes to water and CO2. This available water then hydrolysed
the 2-methylthio group in compounds 9e and 9f to the 2-oxo-
analogue 17aed (R1 ¼ H). These analogues could then de-protonate
either at the phenolic hydroxy group at 7- position, or from the 3-
NH group to generate the nucleophile which attacks the 2 and
3-(halo-methyl)-pyridines to give 17aed.
Similarly, in the reaction of benzyl bromide with 7-hydroxy-8-
methyl-2-methylthio-1,3-benzoxazine 9f water released from the
neutralisation of the 7-hydroxy group with Cs2CO3, which hydro-
lysed the 2-methylthio group and gave the 2-oxo-analogue then
reacted with benzyl bromide to give 17e (Scheme 5).
2.1.5. Synthesis of 7-(pyridin-2, 3 and 4-ylmethoxy)-3-(pyridin-2, 3
and 4-ylmethyl)-substituted-2H benz[e]-1,3-oxazin-2,4-(3H)-dione
17aed
2.2. Biological activity
The reaction of the 7-hydroxy-2-methylthio-substituted-1,3-
benzoxazines 9e and 9f in the presence of cesium carbonate and
the hydro-halogen salts of 2, 3 and 4-(halo-methyl)-pyridine in
acetonitrile did not give the expected product 18 (Scheme 5) but
instead led to the synthesis of 7-(pyridin-2, 3 and 4-ylmethoxy)-3-
2.2.1. Inhibition of platelet aggregation
We previously reported the inhibition of collagen induced
platelet aggregation by a series of 2-amino-1,3-benzoxazines and
showed the most active products to be the 8-methyl-2-morpholin-
Compound
R
H
R1
Compound
R
R1
CH3
15a
15d
H
H
CH3
CH3
15b
15c
15e
15f
Reaction condition: (i) Hydrohalogen salt of 2, 3 and 4-(halo methyl)-pyridine, Cs2CO3,
acetonitrile, reflux.
Scheme 4. Synthesis of 2-(pyridin-3-yl(pyridin-2, 3 and 4-ylmethyl)amino)-7-(pyridin-2, 3 and 4-yl methoxy)-4H-benz[e]-1,3-oxazin-4-one 15aef.

S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
89
aminopyrydine)-substituted-1,3-benzoxazines, compounds 11b,
13aeb, 15aeb, 15e and 8-methyl-7-(pyridin-3-ylmethoxy)-3-
(pyridin-3-ylmethyl)-2H-benz[e]-1,3-oxazine-2,4(3H)-dione
compound 17d.
All compounds were dissolved in DMSO and tested for their
ability to inhibit DNA-PK. This was accomplished using the purified
DNA-PK enzyme (Promega) and the substrate peptide EPPLSQEA-
FADLWKK (Shanghai Research Institute of Chemical Industry
Testing co., Ltd, Shanghai) as previously reported [13]. The IC50
values required for inhibition of DNA-PK are reported in Tables 2
and 3.
The DNA-PK IC50 of 0.3
[e]-1,3-oxazin-4-one19 (Table 2)isthesameasthatfor2-morpholino-
M) [3]. This
mM for 2-morpholino-8-phenyl-4H-benz
8-phenyl-4H-chromen-4-one 1 (LY294002) (IC50 ¼ 0.3
m
was used as a starting point in the modification of the structures to
produce potentially more potent analogue compounds, and the
establishment of a library of compounds now allows us to make some
comments on important structureeactivity relationships.
Firstly, 2-morpholino-8-(substituted) compounds (19, 21, 28, 30
and 31) showed DNA-PK inhibitory activities between 0.3 and
Fig. 2. X-ray crystallography of 8-methyl-2-[pyridin-3-yl(pyridin-2-ylmethyl)amino]-
4H-benz[e]-1,3-oxazin-4-one 11b.
1.7 mM IC50. All of these compounds have substitution at position 8,
indicating the importance of this position irrespective of the
functional group tested.
4-yl-7-(pyridin-3-ylmethoxy)-4H-1,3-benzoxazin-4-one
(IC50 ¼ 2.0 ꢀ 1.5
m
M) and 8-methyl-2-morpholin-4-yl-7-(pyridin-4-
Secondly, compounds substituted at position 7 (23, 25 and 26)
appear to have lower activity compared to substitution at position
8. It is interesting, however, that the addition of a methyl group to
position 8 in structures 23, 25 and 26dresulting in compounds 24,
27 and 29dincreased the activity. This again illustrates the
apparent importance of substitution at position 8. The most
significant effect was for 8-methyl insertion in compound 20
ylmethoxy)-4H-1,3-benzoxazin-4-one (IC50 ¼ 4.0 ꢀ 1.5
mM) [12].
The aim in this work was to synthesise a library of 2-(3-amino-
pyrydine)-substituted-1,3-benzoxazines 10, 2-(N-substituted-3-
aminopyrydine)-substituted-1,3-benzoxazines 11 and 15 and 8-
methyl-7-(pyridin-2 and 3-ylmethoxy)-3-(pyridin-2 and 3-
ylmethyl)-2H-benz[e]-1,3-oxazine-2,4(3H)-dione 17 and establish
structure activity relationships with regard to the inhibition of
collagen induced platelet aggregation.
(IC50 ¼ 6.8
m
M), which gave the 7-(2-(4-methylpiperazin-1-yl)
ethoxy) 8-methyl analogue 22 (IC50 ¼ 0.5
mM).
The 2-(N-substituted-3-aminopyrydine)-substituted-1,3-ben-
zoxazines 15cee showed moderate activity (Table 1) in which the
It is perhaps worth mentioning that DNA-PK activity dimin-
ished if the substitution on the aromatic ring in 2-morphlino-1,3-
benzoxazine was at positions 5 or 6 (as in compounds 32aef,
Fig. 4). The measured DNA-PK IC50 for these compounds was
most active compound of this group was 15e (IC50 ¼ 10 ꢀ 2
mM).
This high activity of compound 15e was further augmented in the
2-morpholino analogue 27a (IC50 ¼ 2 ꢀ 1.5) [12].
previously reported to be more than100
mM [12]. The great
difference in the observed activities certainly highlights the rela-
tive importance of positions 7 and 8 compared to positions 5 or 6.
It is likely that a substituent at position 5 or 6 lies outside the
binding site.
2.2.2. DNA-PK IC50 for the 2-morpholino-substituted-1,3-
benzoxazines
Previously we reported that some of 2-morpholino-substituted-
1,3-benzoxazines displayed DNA-PK inhibitory activity in the
concentration range of 2e4
m
M [12].
2.2.3. DNA-PK IC50 of the 2-(N-substituted (pyridin-3-ylmethyl)
amino) substituted-1,3-benzoxazines
In this work we report the DNA-PK inhibitory activity data for
twelve previously prepared [12] 2-morpholino 1,3-benzoxazines,
compounds 19e31 (Table 2), six new 2-(N-substituted-3-
The replacement of the 2-morpholine group of the substituted-
1,3-oxazine mentioned above with 2-(pyridin-2 and 3-yl(pyridin-
3-ylmethyl)amino) group created a new class of substituted-1,3-
oxazines (Table 3). The DNA-PK IC50 of some of these compounds
showed moderate to low inhibition. 8-methyl-2-(pyridin-3-
yl(pyridin-3-ylmethyl)amino)-4H-benz[e]-1,3-oxazin-4-one
showed low potency (IC50 ¼ 25.2 M) while replacing the 8-methyl
group with an 8-(pyridin-2-ylmethoxy) group, compound 13a,
improved inhibition (IC50 ¼ 7.8 M). However, replacing it with an
8-(pyridin-3-ylmethoxy), compound 13b, slightly decreased the
M).
11b
m
m
activity (IC50 ¼ 13.5
m
The 7-O-substituted analogue 7-(pyridin-2 and 3-ylmethoxy)-
2-((pyridin-2 and 3-ylmethyl)(pyridin-3-yl)amino)-4H-benz[e]-
1,3-oxazin-4-ones 15a and 15b showed moderate activity
(IC50 ¼ 5.7 and 14.8
mM) respectively. However, the most active
compound in this series was the 8-methyl-2-(pyridin-3-yl(pyridin-
3-ylmethyl)amino)-7-(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-
oxazin-4-one 15e (IC50 ¼ 2.5
mM).
Changing the 1,3-oxazine skeleton to a dione as for product 17d
(Table 3) did not cause loss of DNA-PK inhibition and gave
Fig. 3. X-ray crystallography of 2-(pyridin-3-yl(pyridin-2-ylmethyl)amino)-7-(pyridin-
2-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15b.
moderate activity (IC50 ¼ 10
mM).

90
S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
Compound
R
H
R1
Compound
R
R1
CH3
17a
17d
CH3
H
CH3
17b
17c
17e
Reaction condition: (i) Hydro-halogen salt of 2, and 3 (halo methyl) pyridine, or benzyl
Cs2CO3, acetonitrile, reflux.
Scheme 5. Reaction of 7-hydroxy-2-methylthio-8-H/CH3-1,3-benzoxazines 9e and 9f with the hydro-halogen salts of 2, 3 and 4-(halo-methyl)pyridine and benzyl bromide.
Table 1
Inhibition data for 2-amino-benzoxazines against collagen induced human platelet aggregation.
Structure IC50
Ma
m
Structure
IC50 m
Ma
90 ꢀ 2
76 ꢀ 1.5
10a
10c
100 ꢀ 5
65 ꢀ 3
11e
11f

S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
91
Table 1 (continued )
Structure
IC50
m
Ma
Structure
IC50 m
Ma
75 ꢀ 2.5
55 ꢀ 4.5
11i
15c
35 ꢀ 1.5
10 ꢀ 2
15d
15e
85 ꢀ 2.5
75 ꢀ 1.5
17b
17d
85 ꢀ 1.5
17e
a
All values are mean plus or minus standard deviation and are the result of at least three separate experiments.
Table 2
IC50 inhibition of DNA-PK activity data for 2-morpholino 1,3-benzoxazines.
Structureb
IC50
m
Ma
Structure
IC50 m
Ma
0.3
6.8
20
19
(continued on next page)

92
S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
Table 2 (continued )
Structureb
IC50
m
Ma
Structure
IC50 m
Ma
1.7
0.5
22
21
2.7
1.30
23
24
2.5
4.2
26
25
0.28
0.36
27
28
1.20
1.6
29
30
0.62
31
a
r
2 for all curves was greater than 0.9.
Products 19e31 were prepared according to the previously reported procedures [11] and showed correct physical and spectroscopic data.
b

S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
93
Table 3
IC50 inhibition of DNA-PK activity data for 2-amino-pyridyl-substituted-1,3-benzoxazines.
Structure
IC50
m
Ma
Structure
IC50 mM
25.2
7.8
11b
13a
13.5
5.7
15a
13b
14.8
2.5
15b
15e
10
17d
a
r2 for all curves was greater than 0.9.
2.2.4. PI3K IC50 for some 2-morphlino-substituted -1,3-
benzoxazines
previously reported for the analogue compounds 5a and 5b [11]
(Fig. 1).
In Table 4 the PI3K IC50 for compounds 19, 22, 27 were reported
with PI-103 as a positive control. With the exception of 22, the
activities were comparable, and in most instances better than those
It is worth noting that aromatic substitution at position 8 is
important for PI3K activity in the case of compound 19 as well in
compounds 5a and 5b. Furthermore, methyl substitution at posi-
tion 8 in addition to a 7-(pyridin-3-ylmethoxy) substituent gave
much more active compound 27 (Table 4) compared with that of
the 8-aryl substitution (compounds 19, 5a and 5b). However,
replacing the 7-(pyridin-3-ylmethoxy) in 27 with 7-[2-(4-
methylpiperazin-1-yl)ethoxy] (compound 22) diminished the
activity giving an IC50 ꢁ 100
mM.
It is important to note that product 22 with 7-[2-(4-
methylpiperazin-1-yl)ethoxy] group is selective for DNA-PK over
PI3K inhibition compared with the non-selective product 27 with
a 7-(pyridin-3-ylmethoxy) group (see Tables 2 and 4). This may be
because in compound 22 the substitution on the 7 position (4-
methylpiperazin-1-yl ethoxy) is flexible as compared to the
planar aromatic group at position 7 in compound 27.
Fig. 4. Structures of 5 and 6-O-substituted 2-morpholino-1,3-benzoxazines.

94
S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
Table 4
IC50 inhibition of PI3K activity data for 2-amino-1,3-benzoxazines with PI-103 as a positive control.
Structureb
IC50 m
M
Structure
IC50 m
Ma
PI3K
PI3K
PI3K
PI3K
a
4.07
3.36
1.50
0.95
PI3K
PI3K
PI3K
PI3K
a
b
g
d
0.0045
0.009
0.026
b
g
d
0.0083
19
PI-103
PI3K
PI3K
PI3K
PI3K
a
b
g
d
0.13
0.14
0.72
2.02
PI3K
PI3K
b
g
2.34
20.50
27
5a
PI3K
PI3K
PI3K
PI3K
a
b
g
d
ꢁ 100
ꢁ 100
ꢁ 100
ꢁ 100
PI3K
PI3K
PI3K
a
b
g
3.41
0.54
17.78
22
5b
a
IC50
m
M values for compounds 5a and 5b were reported in reference [11].
b
Products 19, 22 and 27 were prepared according to the previously reported procedures [16] and showed correct physical and spectroscopic data.
2.3. Docking protocol (GOLD)
interaction and thus can help to explain the 4 fold drop in activity.
Additionally, the increase in activity from 25 to 27 could be
attributed to the favourable methyl group sticking into a highly
hydrophobic region.
The docked pose of 28 (Fig. 5 D) shows the pyridine nitrogen in
a position where it is able to form a fork of hydrogen bonds with the
backbone NH and side chain OH of Thr182. The less active
compounds 30 and 31 show the potential to pick up only one of
these interactions each.
As previously described a homology model [13] of the catalytic
subunit of human DNA-PK was generated based on the template
protein phosphoinositide 3-kinase (PI3K) [14]. Compounds from
Table 2 were docked in the active site of the homology model using
GOLD 5.0. Side-chain flexibility was set to free for Ser102 and
Lys124, the remaining residues were kept rigid. Fig. 5, A and B
shows compounds 27 and 28 docked respectively with the surface
coloured by atom type. C and D highlight the key residues inter-
acting with 27 and 28. Hydrogen bonds are shown as red dashed
lines. Only the key hydrogens are shown to simplify the view.
As with the related compounds previously docked, the mor-
pholino head of the ligand is buried into the active site with the
oxygen hydrogen bonding to the backbone NH of Leu177. Hydrogen
bonding between the oxazine carbonyl and the side chain of Lys124
is also constantly observed.
3. Conclusion
In this work we developed a procedure used for the synthesis of
eight new 2-(pyridin-3-ylamino)-4H-(substituted benz[e]-1,3-
oxazin-4-one) compounds (10aeg). These were then reacted with
the hydro-halogen salt of 2, 3 and 4-(halo-methyl)pyridine in the
presence of Cs2CO3 to give eighteen new 2-(N-substituted (pyridin-
3-ylmethyl) amino) -substituted-1,3-benzoxazines 11aei, 13aec
and 15aef. X-ray crystallography was used to confirm that the 2-
N-substitution structures 11aei, 13aec and 15aef were formed
rather than 3-N-substitution analogues 14.
The increased potency of 27 and 28 against the related pyridine
analogues from Table 2 can be explained by the docked poses
generated. The 7-substituted pyridinyl-methoxy groups of 23e27
and 29 were found to sit in the space between Ser102 and
Arg104. In this orientation the pyridine ring can
p
-stack with
DNA-PK inhibition data for previously prepared 2-morpholino
substituted-1,3-benzoxazines, compounds 19e31, were measured
Arg104. The docked pose of 27 (Fig. 5C) shows the pyridine nitrogen
in a position where it can further stabilise this pose by hydrogen
bonding with Ser102, direct analogy 24 and 29 don’t show this
and showed high to moderate activity (ca. 0.28e6.80
mM IC50) in
which the most active compound was product 27 with an

S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
95
Fig. 5. Compounds 27 and 28 docked respectively in the binding site of the modelled structure of DNA-PK with surface coloured by atom type.
IC50 ¼ 0.28
(pyridin-3-ylmethyl)amino)-substituted-1,3-benzoxazines
13a,b, 15a,b and 15e and a dione analogue 17d showed moderate to
low (ca. 2.5e25.2 M IC50) inhibitory activity, with the most potent
being 15e with an IC50 ¼ 2.5 M.
m
M. Furthermore, six newly prepared 2-(N-substituted
2-thio-1,3-benzoxazine 8 (1.7 mmol) was added to the reaction
11b,
mixture with stirring and heated to w40 ꢃC. Methyl iodide (1.8 mL)
was then added and allowed to stir at room temperature for 1 h. At
the completion of the reaction, the pH was adjusted to 5e6 using
conc. HCl. The precipitate was collected by filtration; the resulting
residue was washed with water and used without any further
purification.
m
m
Docking of some of the previously prepared compounds 19e31
within the binding pocket and SAR analyses of the poses were
performed and results agreed well with observed activity.
4.2.2. 2-(Methylthio)-4H-benz[e]-1,3-oxazin-4-one 9a
4. Experimental section
2-Thioxo-2,3-dihydro-4H-benz[e]-1,3-oxazin-4-one 8a was
allowed to react with methyl iodide according to general procedure
A. The crude solid was collected to give 9a (90% yield) mp 214e
217 ꢃC. nmax (KBr)/cmꢄ1 2976, 2866 (CeH), 1690 (C]O), 1592 (C]
4.1. Chemistry
Infrared spectra were obtained using a Perkin Elmer FT-IR
1720ꢂ spectrometer. 1H and 13C NMR spectra were obtained
using a Bruker AC 200 NMR spectrometer at 200 and 50 MHz,
respectively. All 1H and 13C NMR spectral results are recorded as
C); 1H NMR (DMSO-d6)
d
7.92 (d, J ¼ 7.5 Hz, 1H, H-5), 7.85 (t,
J ¼ 7.5 Hz, 1H, H-7), 7.42 (t, 1H, J ¼ 7.5 Hz, H-6), 7.00 (d, J ¼ 8 Hz, 1H,
H-8), 2.62 (s, 3H, SCH3); 13C NMR (DMSO-d6)
173.6 (C-4), 162.8 (C-
d
2), 154.9 (C-8a), 135.4 (C-7), 127.0 (C-5), 126.8 (C-6), 117.4 (C-4a),
chemical shifts (
d
) relative to the internal TMS for proton and
116.4 (C-8), 14.0 (SeCH3).
77.0 ppm in CDCl3 solvent and 39.4 ppm in DMSO-d6 solvent 13C
NMR. Microanalysis was performed by Chemical and Micro
Analytical Services (CMAS), Australia. Melting point determinations
were carried out using a Stuart Scientific (SMP3) melting point
apparatus and all melting points are uncorrected.
4.2.3. 8-Methyl-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-one 9b
8-Methyl-2-thioxo-2,3-dihydro-4H-benz[e]-1,3-oxazin-4-one
8b was allowed to react with methyl iodide according to general
procedure A. The crude solid was collected to give 9b (95% yield)
mp 140e142 ꢃC. nmax (KBr)/cmꢄ13060, 2926 (CeH), 1689 (C]O),
4.1.1. Starting materials
1592 (C]C); 1H NMR (DMSO-d6)
d
7.72 (d, J ¼ 7.1 Hz, 1H, H-5), 7.65
(d, J ¼ 7.1 Hz, 1H, H-7), 7.40 (t, J ¼ 7.1 Hz, 1H, H-6), 2.32 (s, 3H, 8-
CH3), 2.62 (s, 3H, SCH3); 13C NMR (DMSO-d6)
173.0 (C-4), 162.8
The starting reagents 3-aminopyridine, sodium hydrogen
carbonate, cesium carbonate, methyl iodide, 2-(bromomethyl)-
pyridine hydrobromide, 3-(chloromethyl)-pyridine hydrochloride
and 4-(bromomethyl)-pyridine hydrobromide were purchased
from Aldrich Chemical Company and were used as received.
d
(C-2), 153.4 (C-8a), 136.2 (C-7), 126.2 (C-5), 125.7 (C-8), 124.4 (C-6),
117.1 (C-4a), 14.5 (CH3), 14.0 (SeCH3).
4.2.4. 2-(Methylthio)-8-phenyl-4H-benz[e]-1,3-oxazin-4-one 9c
8-Phenyl-2-thioxo-2,3-dihydro-4H-benz[e]-1,3-oxazin-4-one 8c
was allowed to react with methyl iodide according to general proce-
dure A. The crude solid was collected to give 9c (90% yield) mp 125 ꢃC.
(KBr)/cmꢄ1 3053, 2928 (CeH), 1687 (C]O), 1589 (C]C); 1H NMR
4.2. Synthesis of 2-methylthio-substituted-1,3-benzoxazin-4-one
9aeg
4.2.1. General procedure A
Sodium hydrogen carbonate (1 g) was suspended in 10 mL of
wateremethanol (50%) solution in 100 mL beaker. The appropriate
(DMSO-d6)
d
7.95 (dd, JH5,H7 ¼1.8 Hz, JH5,H6 ¼ 7.6 Hz,1H, H-5), 7.82 (dd,
JH7,H5 ¼ 1.8 Hz, JH7,H6 ¼ 7.6 Hz, 1H, H-7), 7.62 (dd, JH10,H12 ¼ 1.6 Hz,

96
S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
JH10,H11 ¼ 8.0 Hz, 1H, H-10), 7.55 (m, 5H, H-6, H-11, H-12), 2.50 (s, 3H,
SeCH3); 13C NMR (DMSO-d6)
173.7 (C-4), 163.1 (C-2), 152.1 (C-8a),
4.3.3. 8-Methyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10b
d
135.8 (C-7), 134.4 (C-9), 129.4 (C-11), 128.6 (C-8), 128.4 (C-10), 128.1
(C-12), 126.7 (C-6), 126.3 (C-5), 118.0 (C-4a), 14.0 (SeCH3).
8-Methyl-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-one 9b was
allowed to react with 3-aminopyridine according to general
procedure B. The crude solid was collected and recrystallized from
ethanol to give 10b (82% yield), mp 235e237 ꢃC decomp. nmax
(KBr) 3254, 2870 (NeH), 1673 (C]O), 1624 (C]C), 1484 (C]N)
4.2.5. 8-Hydroxy-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-one 9d
8-Hydroxy-2-thioxo-2,3-dihydro-4H-benz[e]-1,3-oxazin-4-one
8d was allowed to react with methyl iodide as in general procedure
A. The crude solid was collected to give 9d (95% yield), mp 225 ꢃC.
cmꢄ1 1H NMR (DMSO-d6)
; d 10.60 (s, 1H, NeH), 8.74 (d, Jme-
¼ 2.2 Hz, 1H, H-20), 8.34 (bdd, JH4 ,H5 ¼ 4.5 Hz, JH4 ,H2 ¼ 1.5 Hz,
0
0
0
0
ta
nmax (KBr) 3037e2652 (OeH), 1656 (C]O), 1600 (C]C), cmꢄ1
;
1H
1H, H-40), 8.00 (d, J ¼ 7.5 Hz, 1H, H-5), 7.78 (d, J ¼ 7.5 Hz, 1H, H-7),
5.5 Hz, 1H, H-60), 7.39 (dd, JH5 ,H4
¼
5.0 Hz,
0
0
NMR (DMSO-d6)
d
7.20 (m, 3H, H-5/H-6/H-7); 5.32 (s, 1H, 8-OH),
169.7 (C-4), 162.8 (C-2),
7.58(d,
J
¼
2.62 (s, 3H, SCH3); 13C NMR (DMSO-d6)
d
JH5 ,H6 ¼ 5.0 Hz, 1H, H-50), 7.28 (t, J ¼ 7.5 Hz, 1H, H-6), 2.30 (s, 3H,
0
0
144.6 (C-8a), 140.4 (C-8), 126.2 (C-7), 123.3 (C-6), 120.9 (C-5), 116.6
(C-4a) 13.3 (SeCH3).
8-CH3); 13C NMR (DMSO-d6)
d 163.7 (C-4), 152.7 (C-2), 151.3 (C-
8a), 144.3 (C-20), 142.8 (C-40), 135.1 (C-10), 134.8 (C-7), 128.2 (C-60),
124.6 (C-8), 124.0 (C-6), 123.6 (C-5), 122.6 (C-50), 116.3 (C-4a), 13.5
(CH3); (Found C, 66.31; H, 4.38; N, 16.25; C14H11N3O2, requires C,
66.40; H, 4.38; N, 16.59).
4.2.6. 7-Hydroxy-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-one 9e
7-Hydroxy-2-thioxo-2,3-dihydro-4H-1,3-benz[e]-1,3-oxazin-4-
one 8e was allowed to react with methyl iodide according to general
procedure A. The crude solid was collected to give 9e (95% yield), mp
225 ꢃC. (KBr)/cmꢄ1 3056e2706 (OeH), 1689 (C]O), 1617 (C]C); 1H
4.3.4. 8-Phenyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10c
NMR (DMSO-d6)
H-6), 6.62 (s,1H, H-8), 5.32 (s,1H, 7-OH), 2.56 (s, 3H, SCH3); 13C NMR
(DMSO-d6) 171.8 (C-4), 166.8 (C-7), 162.3 (C-2), 157.0 (C-8a), 128.6
d
7.72 (d, J ¼ 8.6 Hz,1H, H-5), 6.85 (d, J ¼ 8.6 Hz,1H,
2-(Methylthio)-8-phenyl-4H-benz[e]-1,3-oxazin-4-one 9c was
allowed to react with 3-aminopyridine according to general
procedure B. The crude solid was collected and recrystallized from
toluene to give 10c (65% yield), mp 220 ꢃC. nmax (KBr) 3254, 2870
d
(C-5), 116.9 (C-6), 107.5 (C-4a), 101.0 (C-8), 14.0 (SeCH3).
(NeH), 1673 (C]O), 1624 (C]C), 1484 (C]N) cmꢄ1 1H NMR
;
4.2.7. 7-Hydroxy-8-methyl-2-(methylthio)-4H-benz[e]-1,3-oxazin-
4-one 9f
(DMSO-d6)
d
10.60 (s, 1H, NeH), 8.68 (bd, Jmeta ¼ 2.2 Hz, 1H, H-20),
8.34 (bdd, JH4 ,H5 ¼ 4.5 Hz, JH4 ,H2 ¼ 1.5 Hz, 1H, H-40), 8.00 (dd,
0
0
0
0
7-Hydroxy-8-methyl-2-thioxo-2,3-dihydro-4H-benz[e]-1,3-
oxazin-4-one 8f was allowed to react with methyl iodide according
to general procedure A. The crude solid was collected to give 9f
(95% yield) mp 220 ꢃC. (KBr)/cmꢄ1 3300e2800 (OeH), 1629 (C]O),
JH5,H7 ¼ 1.7 Hz, JH5,H6 ¼ 7.7 Hz, 1H, H-5), 7.72e7.12 (m, 9H, phenyl
ring protons, H-50, H-60, H-6 and H-7); 13C NMR (DMSO-d6)
d 163.5
(C-4), 152.3 (C-2), 150.0 (C-8a), 144.3 (C-20), 143.1 (C-40), 134.7 (C-
10), 134.6 (C-7), 134.3 (C-9), 128.8 (C-8), 128.6 (C-11), 128.5 (C-60),
127.7 (C-10), 127.2 (C-12), 125.5 (C-6), 124.5 (C-5), 122.5 (C-50), 117.0
(C-4a); (Found C, 72.13; H, 4.23; N, 13.70; C19H13N3O2, requires C,
72.37; H, 4.16; N, 13.33).
1620 (C]C); 1H NMR (DMSO-d6)
d
7.54(d, J ¼ 8.1 Hz, 1H, H-5),
6.81(d, J ¼ 8.6 Hz, 1H, H-6), 4.72 (s, 1H, 7-OH), 2.55(s, 3H, SCH3),
2.05(s, 3H, 8-CH3); 13C NMR (DMSO-d6)
171.9 (C-4), 163.1 (C-7),
d
162.8 (C-2), 154.7 (C-8a), 125.4 (C-5), 114.8 (C-6), 109.9 (C-8), 108.2
(C-4a), 13.9 (SeCH3), 7.8 (8-CH3).
4.3.5. 8-Hydroxy-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10d
8-Hydroxy-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-one
9d
4.3. Synthesis of substituted 2-(3-aminopyridine)-1,3-benzoxazines
10aeg
was allowed to react with 3-aminopyridine according to general
procedure B. The crude solid was collected and recrystallized from
ethanol to give 10d (72% yield), mp 254 ꢃC decomp. nmax (KBr)
3071e2636 (OeH), 2944, 2740 (NeH), 1686 (C]O), 1626 (C]C),
4.3.1. General procedure B
The appropriate crude 2-(methylthio)-4H-benz[e]-1,3-oxazin-4-
one 9aef (2.5 mmol) was suspended in dry 1,4-dioxane (10 mL) in
a 50 mL round-bottomed flask. 3-aminopyridine (12.5 mmol) was
then added with stirring before heating and reflux for 4 h. At the
completion of the reaction the reaction mixture was evaporated to
dryness under reduced pressure and triturated with minimal
diethyl ether. The resulting solid was collected by vacuum filtration
and recrystallized from an appropriate solvent.
1591 (C]N) cmꢄ1 1H NMR (DMSO-d6)
; d 10.20 (bs, 2H, NeH and
OH), 8.83 (d, Jmeta ¼ 2.2 Hz,1H, H-20), 8.35 (d, JH4 ,H5 ¼ 5.2 Hz,1H, H-
0
0
40), 8.08 (m, 1H, H-60), 7.40e7.30 (m, 2H, H-50, H-5), 7.20e7.10 (m,
2H, H-6, H-7); 13C NMR (DMSO-d6)
d 164.2 (C-4), 153.2 (C-2), 144.4
(C-8a), 144.1 (C-20), 142.6 (C-40), 134.7 (C-10), 127.8 (C-60), 124.2 (C-
7), 122.6 (C-50), 120.1 (C-5), 117.8 (C-4a), 115.7 (C-6), 100.7 (C-8);
(Found C, 61.08; H, 3.68; N, 16.59; C13H9N3O3, requires C, 61.18; H,
3.55; N, 16.46).
4.3.2. 2-(Pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one 10a
2-(Methylthio)-4H-benz[e]-1,3-oxazin-4-one 9a was allowed to
react with 3-aminopyridine according to general procedure B. The
crude solid was collected and recrystallized from toluene to give
10a (70% yield), mp 243e245 ꢃC. nmax (KBr) 3254, 2870 (NeH), 1690
4.3.6. 7-Hydroxy-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10e
7-Hydroxy-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-one
9e
was allowed to react with 3-aminopyridine according to general
procedure B. The crude solid was collected and recrystallized from
methanol to give 10e (80% yield), mp 262 ꢃC decomp. nmax (KBr)
3058e2851 (OeH), 1667s (C]O), 1607m (C]C), 1543s (C]N)
(C]O), 1614 (C]C), 1471 (C]N) cmꢄ1; 1H NMR (DMSO-d6)
d 10.91
(s, 1H, NeH), 8.78 (bd, JH2 ,H4 ¼ 1.80 Hz, 1H, H-20), 8.34 (bd,
0
0
JH4 ,H5 ¼ 5.5 Hz, JH4 ,H2 ¼ 1.8 Hz, 1H, H-4 ), 8.03 (d, J ¼ 7.5 Hz, 1H, H-
cmꢄ1; 1H NMR (DMSO-d6)
d 10.20 (bs, 2H, NeH and OH), 8.74 (bd,
0
0
0
0
0
5),7.98 (d, JH5 ,H6 ¼ 5.0, 1H, H-6 ), 7.75 (t, J ¼ 7.5 Hz, 1H, H-7), 7.43 (t,
1H, Jmeta ¼ 2.2 Hz, H-20), 8.35 (bd, 1H, J ¼ 4.4 Hz, H-40), 8.00 (d, 1H,
J ¼ 5.0 Hz, H-60), 7.80 (d, 1H, J ¼ 10.2 Hz, H-5), 7.40 (m, 1H, H-50),
6.85 (dd, 1H, J6,8 ¼ 3, J5,6 ¼ 10.0 Hz, H-6), 6.65 (d, J ¼ 2.4 Hz, 1H, H-
0
0
J ¼ 7.5 Hz, 1H, H-6), 7.39 (t, J ¼ 5.2 Hz, 1H, H-50) 7.34 (s, 1H, H-8); 13C
NMR (DMSO-d6)
d
163.7 (C-4), 153.0 (C-2/C-8a), 144.3 (C-20), 142.6
(C-40), 134.7 (C-10), 133.9 (C-7), 128.0 (C-60), 126.2 (C-5), 124.7 (C-6),
122.6 (C-50), 116.6 (C-4a), 115.2 (C-8); (Found C, 65.43; H, 3.73; N,
17.50; C13H9N3O2, requires C, 65.27; H, 3.79; N, 17.56).
8); 13C NMR (DMSO-d6)
d 162.9 (C-4), 162.8 (C-7), 154.6 (C-2), 152.1
(C-8a), 144.1 (C-20), 142.6 (C-40), 135.4 (C-10), 127.9 (C-60), 127.8 (C-
5), 122.7 (C-50), 113.6 (C-6), 108.3 (C-4a), 100.7 (C-8); (Found C,

S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
97
61.14; H, 3.04; N, 16.49; C13H9N3O3, requires C, 61.18; H, 3.55; N,
16.46).
collected and recrystallized from toluene to give 11b (70% yield),
mp 255e257 ꢃC decomp. nmax (KBr) 3064, 2930 (CeH), 1674 (C]O),
1627 (C]C), 1563 (C]N) cmꢄ1 1H NMR (DMSO-d6)
; d 8.62 (bd,
4.3.7. 7-Hydroxy-8-methyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-
oxazin-4-one 10f
Jmeta ¼ 2.2 Hz, 1H, H-20), 8.52 (d, J ¼ 5 Hz, 1H, H-90), 8.49 (bdd,
0
0
0
0
0
JH4 ,H5 ¼ 4.5 Hz, JH4 ,H2 ¼ 1.5 Hz, 1H, H-4 ), 8.17 (d, J ¼ 7.5 Hz, 1H, H-
7-Hydroxy-8-methyl-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-
one 9f was allowed to react with 3-aminopyridine according to
general procedure B. The crude solid was collected and recrystal-
lized from methanol to give 10f (70% yield), mp 278 ꢃC decomp.
5), 7.6 (m, 1H, H-110), 7.70 (d, J ¼ 7.5 Hz, 1H, H-7), 7.49 (m, 2H, H-100,
H-120),7.40 (dd, JH5 ,H4 ¼ 9.0 Hz, JH5 ,H6 ¼ 5.0 Hz, 1H, H-50), 7.35 (d,
J ¼ 7.5 Hz, 1H, H-60), 7.22 (t, J ¼ 7.5 Hz, 1H, H-6), 5.30 (s, 2H, CH2eN),
0
0
0
0
1.90 (s, 3H, 8-CH3); 13C NMR (DMSO-d6)
d 168.1 (C-4), 158.4 (C-2),
nmax 3300e2860 (OeH), 1688 (C]O), 1583 (C]N) cmꢄ1
;
1H NMR
156.6 (C-90, C-20, C- 40)153.0 (C-8a), 150.0 (C-120), 149.2 (C-100),
145.1 (C-70), 137.2 (C-10), 136.6(C-110), 135.3 (C-7), 126.1 (C-60), 125.8
(C-8), 124.5 (C-50, C-6), 123.5 (C-5), 116.3 (C-4a), 54.2 (CH2eN), 14.9
(CH3); (Found C, 70.03; H, 4.90; N, 16.99; C20H16N4O2, requires C,
69.76; H, 4.68; N, 16.27).
(DMSO-d6)
d
10.20 (bs, 2H, NeH and OH), 8.71 (bd, Jmeta ¼ 2.2 Hz,
1H, H-20), 8.34 (bd JH4 ,H5
¼
4.5 Hz, 1H, H-40), 7.96 (d,
0
0
JH5 ,H6 ¼ 7.5 Hz, 1H, H-50), 7.65 (d, J ¼ 9.0 Hz, 1H, H-5), 7.39 (m, 1H,
0
0
H-60), 6.86 (d, J ¼ 9.6 Hz, 1H, H-6), 2.10 (s, 3H, 8-CH3); 13C NMR
(DMSO-d6) d 163.5 (C-4), 160.3 (C-7), 152.7 (C-2), 151.8 (C-8a), 144.0
(C-20), 142.8 (C-40), 135.9 (C-10), 128.1 (C-60), 124.3 (C-5), 122.5 (C-
50), 112.3 (C-6), 109.7 (C-8), 108.3 (C-4a), 6.7 (8-CH3); (Found C,
62.25; H, 4.10; N, 15.59; C14H11N3O3, requires C, 62.45; H, 4.12; N,
15.61).
4.4.4. 8-Phenyl-2-[pyridin-3-yl(pyridin-2-ylmethyl)amino]-4H-
benz[e]-1,3-oxazin-4-one 11c
8-Phenyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one
10c was allowed to react with 2-(bromomethyl)pyridine hydro-
bromide according to general procedure C. The crude solid was
collected and recrystallized from toluene to give 11c (70% yield),
mp 265e267 ꢃC decomp. nmax (KBr) 3064, 2920 (CeH), 1670 (C]
4.4. Reaction of 2-(pyridin-3-ylamino)-substituted-benz-1,3-
oxazin-4-one 10 with 2-(bromomethyl)pyridine hydrobromide, 3-
(chloromethyl) pyridine hydrochloride and 4-(bromomethyl)-
pyridine hydrobromide to produce compounds 11 and 13
O), 1627 (C]C), 1565 (C]N) cmꢄ1; 1H NMR (DMSO-d6)
d 8.65 (bd,
Jmeta ¼ 2.2 Hz, 1H, H-20), 8.57(d, J ¼ 5 Hz, 1H, H-90), 8.49 (bdd,
JH4 ,H5 ¼ 4.5 Hz, JH4 ,H2 ¼ 1.5 Hz, 1H, H-40), 7.82 (d, J ¼ 7.5 Hz, 1H,
H-5), 7.6 (m, 1H, H-110), 7.72 (d, J ¼ 7.5 Hz, 1H, H-7), 7.49 (m, 2H,
0
0
0
0
4.4.1. General procedure C
H-100, H-120), 7.40 (dd, JH5 ,H4 ¼ 9.0 Hz, JH5 ,H6 ¼ 5.0 Hz, 1H, H-50),
7.30e7.10 (m, 6H, H-60, and, 5H for 8-Ph), 6.80 (t, J ¼ 7.5 Hz, 1H, H-
0
0
0
0
A suspension of 2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-
4-one 10aec (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 refluxed for 2 h. 2-(bromomethyl)pyridine hydro-
bromide, 3-(chloromethyl)pyridine hydrochloride or 4-(bromo-
methyl)-pyridine hydrobromide (2 mmol) in acetonitrile (10 mL)
was added portion-wise. Upon completion of the addition, the
reaction mixture was refluxed for a further 30 min then evaporated
off under reduced pressure. The residue was washed with
4 ꢂ 15 mL of CHCl3 and then filtered. The CHCl3 filtrate was evap-
orated off under reduced pressure and the resulting residue was
triturated with minimal amount of diethyl ether to give the crude
solid which was recrystallized from an appropriate solvent.
6), 5.30 (s, 2H, CH2eN); 13C NMR (DMSO-d6)
d 166.1 (C-4), 158.4
(C-2), 153.0 (C-8a), 156.6 (C-90, C-20, C-40), 150.8 (C-120), 150.0 (C-
100), 145.1 (C-70), 137.2 (C-10), 136.0 (C-110), 135.3 (C-7), 134.7 (C-
9), 128.8 (C-10), 128.30(C-11), 127.7 (C-12), 126.1 (C-60), 125.8 (C-
8), 124.5 (C-50, C-6), 123.5 (C-5), 116.3 (C-4a), 54.2 (CH2eN);
(Found C, 73.82; H, 4.35; N, 13.95; C25H18N4O2, requires C,
73.88; H, 4.46; N, 13.78).
4.4.5. 2-[Pyridin-3-yl(pyridin-3-ylmethyl)amino]-4H-benz[e]-1,3-
oxazin-4-one 11d
2-(Pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one 10a was
allowed to react with 3-(chloromethyl)pyridine hydrochloride
according to general procedure C. The crude solid was collected
and recrystallized from toluene to give 11d (75% yield), mp 220 ꢃC.
nmax (KBr) 3067, 2927 (CeH), 1680 (C]O), 1630 (C]C), 1554 (C]
4.4.2. 2-[Pyridin-3-yl(pyridin-2-ylmethyl)amino]-4H-benz[e]-1,3-
oxazin-4-one 11a
2-(Pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one 10a was
allowed to react with 2-(bromomethyl)pyridine hydrobromide
according to general procedure C. The crude solid was collected and
recrystallized from toluene to give 11a (70% yield), mp 235e237 ꢃC.
nmax (KBr) 3164, 2930 (CeH), 1684 (C]O), 1620 (C]C), 1551 (C]N)
N) cmꢄ1; 1H NMR (CDCl3)
d
8.65 (bd, Jmeta ¼ 2.2 Hz, 1H, H-20), 8.56
(bs, 1H, H-80), 8.54 (bd, JH4 ,H5 ¼ 4.5 Hz, Hz, 1H, H-40), 8.47 (d,
J ¼ 5.4 Hz, 1H, H-100), 7.90e7.70 (m, 3H, H-5, H-60, H-120), 7.50e
7.40 (m, 2H, H-50, H-6), 7.32 (t, J ¼ 4.5 Hz, 1H, H-110), 7.27 (t,
J ¼ 7.5 Hz, 1H, H-7), 6.72 (d, J ¼ 7.5 Hz, 1H, H-8), 5.30 (s, 2H, CH2e
0
0
cmꢄ1; 1H NMR (DMSO-d6)
d
8.70 (bd, Jmeta ¼ 2.2 Hz, 1H, H-20), 8.52
(d, J ¼ 5 Hz,1H, H-90), 8.30 (bdd, JH4 ,H5 ¼ 7.5 Hz, JH4 ,H2 ¼ 2.0 Hz,1H,
H-40), 8.14 (d, J ¼ 7.5 Hz, 1H, H-5), 7.60 (m, 1H, H-110), 7.70 (t,
J ¼ 7.5 Hz, 1H, H-7), 7.49 (m, 2H,0 H-100, H-120), 7.42 (dd,
N); 13C NMR (CDCl3)
d 165.0 (C-4), 155.2 (C-2), 151.5 (C-8a), 148.6
0
0
0
0
(C-80), 148.2 (C-100), 148.0 (C-20), 147.7 (C-40), 136.7 (C-70), 137.2 (C-
10), 136.0 (C-110), 135.3 (C-7), 126.1 (C-60), 124.5 (C-8), 124.5 (C-6,
C-5), 123.5 (C-50), 116.8 (C-4a), 115.2 (C-8), 53.7 (CH2eN); (Found C,
69.45; H, 4.76; N, 17.05; C19H14N4O2, requires C, 69.08; H, 4.27;
N, 16.96).
0
0
0
0
JH5 ,H4 ¼ 9.0 Hz, JH5 ,H6 ¼ 5.0 Hz, 1H, H-5 ), 7.33 (d, J ¼ 7.5 Hz, 1H, H-
60), 7.23 (t, J ¼ 7.5 Hz, 1H, H-6), 6.94 (d, J ¼ 7.5 Hz, 1H, H-8), 5.32 (s,
2H, CH2eN); 13C NMR (CDCl3)
d 165.1 (C-4), 156.4 (C-2), 153.8 (C-
8a), 150.8 (C-100, C-120), 148.0 (C-20), 140.2 (C-40), 145.1 (C-70), 136.0
(C-10), 135.2 (C-110), 134.3 (C-7), 126.1 (C-60), 126.2 (C-5), 124.7 (C-
6), 122.6 (C-50), 116.6 (C-4a), 115.2 (C-8), 54.2 (CH2eN); (Found C,
69.33; H, 4.90; N, 16.99; C19H14N4O2, requires C, 69.08; H, 4.27; N,
16.96).
4.4.6. 8-Methyl-2-[pyridin-3-yl(pyridin-3-ylmethyl)amino]-4H-
benz[e]-1,3-oxazin-4-one 11e
8-Methyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one
10b was allowed to react with 3-(chloromethyl)pyridine hydro-
chloride according to general procedure C. The crude solid was
collected and recrystallized from toluene to give 11e (65% yield),
mp 198e200 ꢃC. nmax (KBr) 3067, 2919 (CeH), 1672 (C]O), 1630
4.4.3. 8-Methyl-2-[pyridin-3-yl(pyridin-2-ylmethyl)amino]-4H-
benz[e]-1,3-oxazin-4-one 11b
8-Methyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one
10b was allowed to react with 2-(bromomethyl)pyridine hydro-
bromide according to general procedure C. The crude solid was
(C]C), 1564 (C]N) cmꢄ1 1H NMR (CDCl3)
; d 8.65 (bd, Jme-
¼ 2.2 Hz, 1H, H-20), 8.56 (bs, 1H, H-80), 8.54 (d, JH4 ,H5 ¼ 4.5 Hz,
0
0
ta
Hz, 1H, H-40), 8.47 (d, J ¼ 5.4 Hz, 1H, H-100), 7.90e7.70 (m, 3H, H-5,

98
S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
H-60, H-120), 7.50e7.40 (m, 2H, H-50, H-6), 7.32 (t, J ¼ 4.5 Hz, 1H,
H-110), 7.27 (d, J ¼ 7.5 Hz, 1H, H-7), 5.30 (s, 2H, CH2eN), 1.98 (s, 3H,
4.4.10. 8-Phenyl-2-[pyridin-3-yl(pyridin-4-ylmethyl)amino]-4H-
benz[e]-1,3-oxazin-4-one 11i
8-CH3); 13C NMR (CDCl3)
d
165.0 (C-4), 157.2 (C-2), 151.5 (C-8a),
8-Phenyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one
10c was allowed to react 4-(bromomethyl)pyridine hydrobromide
according to general procedure C. The crude solid was collected and
recrystallized from toluene to give 11i (60% yield), mp 265 ꢃC
decomp. nmax (KBr) 3056, 2918 (CeH), 1669 (C]O), 1627 (C]C),
148.6 (C-80), 148.2 (C-100), 148.0 (C-20), 147.7 (C-40), 136.7 (C-70),
137.2 (C-10), 136.0 (C-110), 135.3 (C-7), 126.1 (C-60), 124.5 (C-8),
124.5 (C-6, C-5), 123.5 (C-50), 116.8 (C-4a), 50.7 (CH2eN), 12.8
(CH3); (Found C, 69.57; H, 4.81; N, 16.09; C20H16N4O2, requires C,
69.76; H, 4.68; N, 16.27).
1567 (C]N) cmꢄ1; 1H NMR (DMSO-d6)
d
8.56 (bd, Jmeta ¼ 2.2 Hz,1H,
H-20), 8.52 (d, J ¼ 5 Hz, 2H, H-90, H-110), 8.49 (bdd, JH4 ,H5 ¼ 4.5 Hz,
0
0
JH4 ,H2 ¼ 1.5 Hz, 1H, H-40), 8.17 (d, J ¼ 7.5 Hz, 1H, H-5), 7.58 (d,
J ¼ 5 Hz, 2H, H-80, H-120), 7.44 (d, J ¼ 7.5 Hz, 1H, H-7), 7.30e7.10 (m,
8H, H-50, H-60, H-6, and 5H of the 8-aryl group), 5.30 (s, 2H, CH2e
0
0
4.4.7. 8-Phenyl-2-[pyridin-3-yl(pyridin-3-ylmethyl)amino]-4H-
benz[e]-1,3-oxazin-4-one 11f
8-Phenyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one
10c was allowed to react with 3-(chloromethyl)pyridine hydro-
chloride according to general procedure C. The crude solid was
collected and recrystallized from toluene to give 11f (53% yield), mp
257e259 ꢃC. nmax (KBr) 3067, 2919 (CeH), 1672 (C]O), 1624 (C]C),
N); 13C NMR (DMSO-d6)
d 166.7 (C-4), 157.7 (C-2), 150.5(CeC-8a),
149.1 (C-80, C-110), 148.0 (C-20), 147.9 (C-40), 144.2 (C-70), 136.5 (C-
10), 135.1 (C-7), 135.0 (C-60), 134.7 (C-9), 128.8 (C-10, C-14), 128.30
(C11, C-13), 127.7 (C-12), 127.1 (C-50), 125.9 (C-6), 125.0 (C-60), 123.9
(C-5), 117.8 (C-4a), 54.3 (CH2eN); (Found C, 73.72; H, 4.46; N, 13.83;
C25H18N4O2, requires C, 73.88; H, 4.46; N, 13.78).
1464 (C]N) cmꢄ1; 1H NMR (CDCl3)
d
8.66 (bd, Jmeta ¼ 2.2 Hz, 1H, H-
20), 8.58(bs, 1H, H-80), 8.52 (bd, JH4 ,H5 ¼ 4.5 Hz, Hz, 1H, H-40), 8.46
(d, J ¼ 5.4 Hz, 1H, H-100), 7.90e7.70 (m, 3H, H-5, H-60, H-120), 7.60e
7.20 (m, 9H, H-50, H-110-H-6, H-7, and 5H for 8-Ph), 5.30 (s, 2H,
0
0
4.4.11. 8-(Pyridin-2-ylmethoxy)-2-((pyridin-2-ylmethyl)(pyridin-
3-yl)amino)-4H benz[e][1,3]oxazin-4-one 13a
CH2eN); 13C NMR (CDCl3)
d 165.0 (C-4), 156.2 (C-2), 150.5 (C-8a),
149.1 (C-90. 110), 143.0 (C-20), 142.7 (C-100), 140.7 (C-10), 138.2 (C-40),
137.8 (C-9), 137.5 (C-7), 136.0 (C-110), 135.3 (C-70), 134.1 (C-120),
133.1 (C-60), 129.1 (C-11), 128.9 (C-8), 128.5 (C-10), 128.1 (C-12),
125.9 (C-6), 125.3 (C-5), 122.5 (C-50), 120.5 (C-6), 117.4 (C-4a), 53.7
(CH2eN); (Found C, 73.80; H, 4.60; N,13.93; C25H18N4O2, requires C,
73.88; H, 4.46; N, 13.78).
8-Hydroxy-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10d was allowed to react with 2-(bromomethyl)pyridine
hydrobromide according to general procedure C. The crude solid
was collected and recrystallized from toluene to give 13a (60%
yield), mp 275 ꢃC decomp. nmax (KBr) 3061, 2975(CeH), 1680 (C]
O), 1624 (C]C), 1582 (C]N) cmꢄ1; 1H NMR (DMSO-d6)
d 8.74 (bd,
Jmeta ¼ 2.2 Hz, 1H, H-20), 8.60e8.50 (m, 3H, H-90, H-170), 7.91 (d,
0
0
0
0
4.4.8. 2-[Pyridin-3-yl(pyridin-4-ylmethyl)amino]-4H-benz[e]-1,3-
oxazin-4-one 11g
J ¼ 7.5 Hz, 1H, H-5), 7.83 (dd, JH4 ,H5 ¼ 4.5 Hz, JH4 ,H2 ¼ 1.5 Hz, 1H, H-
40), 7.70e7.60 (m, 3H, H-110, H-180, H-200), 7.50e7.40 (m, 2H, H-50,
H-60), 7.30e7.10 (m, 3H, C-190, H-100, H-120), 6.80 (m, 2H, H-6, H-7),
2-(Pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one 10a was
allowed to react with 4-(bromomethyl)pyridine hydrobromide
according to general procedure C. The crude solid was collected and
recrystallized from toluene to give 11g (60% yield), mp 234 ꢃC. nmax
(KBr) 3067, 2927 (CeH), 1680 (C]O), 1630 (C]C), 1554 (C]N)
5.32 (s, 2H, CH2eO), 5.20 (s, 2H, CH2eN); 13C (DMSO-d6)
d 165.2 (C-
4), 154.0 (C-70), 155.6 (C-150), 152.2 (C-2), 150.7 (C-8), 149.8 (C-90/C-
170), 149.2 (C-20), 140.5 (C-10), 137.3 (C-110/C-190, 60), 126.2 (C-5),
124.3 (C-50, 4a), 123.2 (C-80/C-200), 121.4 (C-100/C-180), 113.7 (C-8),
111.4 (C-7), 110.0 (C-6), 71.6 (OeCH2), 56.4 (NeCH2); (Found C,
68.57; H, 4.21; N, 16.45; C25H19N5O3, requires C, 68.64; H, 4.38; N,
16.01).
cmꢄ1; 1H NMR (DMSO-d6)
d
8.56 (bd, Jmeta ¼ 2.2 Hz, 1H, H-20), 8.52
(d, J ¼ 5 Hz, 2H, H-90, H-110), 8.49 (bdd, JH4 ,H5 ¼ 4.5 Hz,
0
0
JH4 ,H2 ¼ 1.5 Hz, 1H, H-40), 8.17 (d, J ¼ 7.5 Hz, 1H, H-5), 7.58 (d,
J ¼ 5 Hz, 2H, H-80, H-120), 7.44 (t, J ¼ 7.5 Hz, 1H, H-7), 7.40 (m, 1H, H-
50), 7.32 (d, J ¼ 5 Hz, 1H, H-60), 7.20 (t, J ¼ 7.5 Hz, 1H, H-6), 6.70 (d,
J ¼ 7.5 Hz, 1H, H-8), 5.30 (s, 2H, CH2eN); 13C NMR (DMSO-d6)
0
0
4.4.12. 2-(Pyridin-3-yl(pyridin-3-ylmethyl)amino)-8-(pyridin-3-
ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 13b
d
165.0 (C-4), 155.2 (C-2), 151.5 (C-8a), 148.6 (C-90, 110), 148.2 (C-20),
8-Hydroxy-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10d was allowed to react with 3-(chloromethyl)pyridine
hydrochloride according to general procedure C. The crude solid
was collected and recrystallized from toluene to give 13b (55%
yield), mp 295 ꢃC decomp. nmax (KBr) 3158, 2970 (CeH), 1683 (C]
148.0 (C-40), 147.7 (C-40), 144.7 (C-70), 137.2 (C-10), 135.3 (C-7), 126.1
(C-50, 60), 124.5 (C-5/C-8), 123.5 (C-8, C-120), 116.8 (C-4a), 53.7
(CH2eN); (Found C, 69.45; H, 4.76; N, 17.05; C19H14N4O2, requires C,
69.08; H, 4.27; N, 16.96).
O), 1624 (C]C), 1550 (C]N) cmꢄ1; 1H NMR (DMSO-d6)
d 8.60e8.40
4.4.9. 8-Methyl-2-[pyridin-3-yl(pyridin-4-ylmethyl)amino]-4H-
benz[e]-1,3-oxazin-4-one 11h
(m, 4H, H-20, H-80, H-100, H-160), 8.14 (d, J ¼ 5.5 Hz, 1H, H-180), 7.90e
7.72 (m, 2H, H-40, H-200), 7.50e7.30 (m, 5H, H-50, H-60, H-110, H-120,
H-190), 7.00e6.90 (m, 2H, H-5, H-6), 6.55 (d, J ¼ 7.5 Hz, 1H, H-7),
8-Methyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-one
10b was allowed to react with 4-(bromomethyl)pyridine hydro-
bromide according to General Procedure C. The crude solid was
collected and recrystallized from toluene to give 11h (68% yield),
mp 210 ꢃC. nmax (KBr) 3056, 2918 (CeH), 1669 (C]O), 1627 (C]C),
5.24 (s, 2H, CH2eO), 5.12 (s, 2H, CH2eN); 13C NMR (CDCl3)
d 166.5
(C-4), 157.9 (C-2), 155.7 (C-8a), 150.4 (C-160, C-20), 150.3 (C-80), 149.8
(C-100, C-180), 149.6 (C-8), 147.5 (C-40, C-70), 136.9 (C-10), 136.8 (C-
120), 135.5 (C-60), 131.7 (C-150, C-200), 124.5 (C-5), 124.2 (C-7), 124.1
(C-50), 123.9 (C-110, C-190), 114.7 (C-6), 112.1 (C-4a), 68.8 (OeCH2),
53.2 (NeCH2); (Found C, 68.77; H, 4.48; N, 16.10; C25H19N5O3,
requires C, 68.64; H, 4.38; N, 16.01).
1567 (C]N) cmꢄ1; 1H NMR (DMSO-d6)
d
8.56 (bd, Jmeta ¼ 2.2 Hz,1H,
H-20), 8.52 (d, J ¼ 5 Hz, 2H, H-90, H-110), 8.49 (bdd, JH4 ,H5 ¼ 4.5 Hz,
0
0
JH4 ,H2 ¼ 1.5 Hz, 1H, H-40), 8.17 (d, J ¼ 7.5 Hz, 1H, H-5), 7.58 (d,
J ¼ 5 Hz, 2H, H-80, H-120), 7.44 (d, J ¼ 7.5 Hz,1H, H-7), 7.40 (m,1H, H-
50), 7.32 (d, J ¼ 5 Hz, 1H, H-60), 7.20 (t, J ¼ 7.5 Hz, 1H, H-6), 5.30 (s,
0
0
4.4.13. 2-(Pyridin-3-yl(pyridin-4-ylmethyl)amino)-8-(pyridin-4-
ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 13c
2H, CH2eN), 1.90 (s, 3H, 8-CH3); 13C NMR (DMSO-d6)
d 167.3 (C-4),
158.1 (C-2), 152.7 (C-8a), 150.9 (C-90, C-110), 149.9 (C-20), 149.5 (C-
40), 144.9 (C-70), 137.2 (C-10), 135.2 (C-7), 126.9 (C-60) 126.0 (C-50, C-
6), 125.9 (C-8), 124.3 (C-5), 123.5 (C-80, C-120), 118.1 (C-4a), 54.3
(CH2eN), 14.7 (CH3); (Found C, 69.57; H, 4.81; N,16.09; C20H16N4O2,
requires C, 69.76; H, 4.68; N, 16.27).
8-Hydroxy-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10d was allowed to react with 4-(bromomethyl)pyridine
hydrobromide according to general procedure C. The crude solid
was collected and recrystallized from toluene to give 13c (61%
yield), mp 265e267 ꢃC decomp. nmax (KBr) 3062, 2943(CeH), 1672

S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
99
(C]O), 1624 (C]C), 1553 (C]N) cmꢄ1
;
1H NMR (DMSO-d6)
d
8.75
155.7 (C-8a), 150.4 (C-150, C-20), 150.3 (C-90, 110), 149.8 (C-170, C-190),
149.6 (C-70), 147.5 (C-10, C-40), 136.9 (C-160, 200), 135.5 (C-60), 131.7
(C-5), 124.5 (C-50), 123.9 (C-80, C-120), 114.7 (C-6), 112.1 (C-4a), 101.3
(C-8), 68.8 (OeCH2), 53.2 (NeCH2); (Found C, 68.77; H, 4.48; N,
16.10; C25H19N5O3, requires C, 68.64; H, 4.38; N, 16.01).
(bd, Jmeta ¼ 2.2 Hz, 1H, H-20), 8.60e8.50 (m, 4H, H-90, H-120, H-170,
H-190), 7.96(d, J ¼ 7.5 Hz, 1H, H-5), 7.81 (m, 1H, H-40), 7.70e7.60 (m,
2H, H-160, H-200), 7.52 (d, J ¼ 7.5 Hz, 1H, H-60), 7.40e7.30 (m, 2H, H-
80, H-130), 7.20e7.10 (m, 2H, H-50, H-7), 6.93 (d, J ¼ 7.5 Hz, 1H, H-6),
5.43 (s, 2H, CH2eO), 5.35 (s, 2H, CH2eN); 13C NMR (CDCl3)
d 167.8
(C-4), 158.4 (C-2), 155.7 (C-8a),153.4 (C-20), 149.8 (C-90, C-110, C-170,
C-190), 149.3 (C-8), 144.9 (C-70), 142.3(C-15, C-40), 137.3 (C-10), 135.6
(C-60), 126.8 (C-50), 124.3 (C-80, C-120), 123.3 (C-7, C-160, C-200),
1121.5 (C-5), 111.5 (C-4a), 110.1 (C-6), 71.6 (OeCH2), 56.6 (NeCH2);
(Found C, 68.86; H, 4.50; N, 16.11; C25H19N5O3, requires C, 68.64;
H, 4.38; N, 16.01).
4.4.17. 8-Methyl-7-(pyridin-2-ylmethoxy)-2-((pyridin-2-
ylmethyl)(pyridin-3-yl)amino)-4H-benz[e]-1,3-oxazin-4-one 15d
7-Hydroxy-8-methyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-
oxazin-4-one 10f was allowed to react with 2-(bromomethyl)
pyridine hydrobromide according to general procedure C. The
crude solid was collected and recrystallized from toluene to give
15d (62% yield), mp 257e260 ꢃC decomp. nmax (KBr) 3061, 2975(Ce
4.4.14. 7-(Pyridin-2-ylmethoxy)-2-((pyridin-2-ylmethyl)(pyridin-
3-yl)amino)-4H-benzo[e][1,3]oxazin-4-one 15a
H), 1672 (C]O), 1624 (C]C), 1557 (C]N) cmꢄ1 1H NMR (CDCl3)
;
d
8.72 (bd,1H, Jmeta ¼ 2.2 Hz, H-20), 8.60e8.50 (m, 3H, H-90, H-170, H-
190), 7.92 (d, 1H, J ¼ 5.5 Hz, H-200), 7.82 (bdd, 1H, JH4 ,H5 ¼ 4.5 Hz,
0
0
7-Hydroxy-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10e was allowed to react with 2-(bromomethyl)pyridine
hydrobromide according to general procedure C. The crude solid
was collected and recrystallized from toluene to give 15a (42%
yield), mp 267 ꢃC decomp. nmax (KBr) 3065, 2972(CeH), 1674 (C]
JH4 ,H2 ¼ 1.5 Hz, H-40), 7.70e7.60 (m, 2H, C-5, H-110), 7.50e7.40 (m,
3H, H-50, H-100, H-180), 7.30e7.10 (m, 2H, H-60, H-120), 6.80 (d, 1H,
J ¼ 7.5 Hz, H-6), 5.30 (s, 2H, CH2eO), 5.20 (s, 2H, CH2eN), 2.31 (s, 3H,
0
0
8-CH3); 13C NMR (CDCl3)
d 165.2 (C-4), 161.0 (C-7), 157.0 (C-2), 155.6
O), 1621 (C]C), 1554 (C]N) cmꢄ1; 1H NMR (CDCl3)
d
8.72 (bd, 1H,
(C-8a), 152.2 (C-70), 150.7 (C-150), 149.8 (C-90, C-170), 149.2 (C-20),
140.5 (C-10, C-110), 137.3 (C-40, C-60, 190), 126.2 (C-5), 124.3 (C-50),
123.2 (C-120, C-200), 121.4 (C-100, C-180), 113.7 (C-8), 111.4 (C-4a),
110.0 (C-6), 71.6 (OeCH2), 56.4 (NeCH2), 6.7 (8-CH3); (Found C,
69.57; H, 4.21; N, 15.69; C26H21N5O3, requires C, 69.17; H, 4.69; N,
15.51).
Jmeta ¼ 2.2 Hz, H-20), 8.60e8.50 (m, 3H, H-90, H-170, H-190), 7.92 (d,
1H, J ¼ 5.5 Hz, H-200), 7.82 (bdd,1H, JH4 ,H5 ¼ 4.5 Hz, JH4 ,H2 ¼ 1.5 Hz,
H-40), 7.70e7.60 (m, 2H, C-5, H-110), 7.50e7.40 (m, 3H, H-50, H-100,
H-180), 7.30e7.10 (m, 2H, H-60, H-120), 7.20 (d, Jmeta ¼ 1.8 Hz, 1H, H-
8), 6.80 (d, 1H, J ¼ 7.5 Hz, H-6), 5.30 (s, 2H, CH2eO), 5.20 (s, 2H,
0
0
0
0
CH2eN); 13C NMR (CDCl3)
d 165.2 (C-4), 163.0 (C-7), 157.0 (C-2),
156.6 (C-8a), 152.2 (C-70), 150.7 (C-150), 149.8 (C-90, C-170), 149.2 (C-
20), 140.5 (C-10, C-110), 137.3 (C-40, C-60, 190), 131.2 (C-5), 124.3 (C-50),
123.2 (C-120, C-200), 121.4 (C-100, C-180), 111.4 (C-4a), 110.0 (C-6),
101.3 (C-8), 71.6 (OeCH2), 56.4 (NeCH2)); (Found C, 69.57; H, 4.21;
N, 15.69; C26H21N5O3, requires C, 69.17; H, 4.69; N, 15.51.
4.4.18. 8-Methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-
(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15e
7-Hydroxy-8-methyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-
oxazin-4-one 10f was allowed to react with 3- (chloromethyl)
pyridine hydrochloride according to general procedure C. The crude
solid was collected and recrystallized from toluene to give 15e (55%
yield), mp 264e267 ꢃC decomp. nmax (KBr) 3053, 2923(CeH), 1665
4.4.15. 2-(Pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-(pyridin-3-
ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15b
(C]O), 1624 (C]C), 1550 (C]N) cmꢄ1; 1H NMR (DMSO-d6)
d 8.70e
7-Hydroxy-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10e was allowed to react with 3-(chloromethyl)pyridine
hydrochloride according to general procedure C. The crude solid
was collected and recrystallized from toluene to give 15b (65%
yield), mp 235e237 ꢃC decomp. nmax (KBr) 3050, 2925(CeH), 1663
8.50 (m, 6H, H-20, H-40, H-80, H-100, H-160, H-180), 8.00e7.90 (m, 3H,
H-50, H-120, H-200), 7.82 (d, J ¼ 7.5 Hz,1H, H-5), 7.60e7.40 (m, 3H, H-
60, H-110, H-190), 7.25 (d, J ¼ 7.5 Hz, 1H, H-6), 5.40 (s, 2H, CH2eO),
5.30 (s, 2H, CH2eN), 2.23 (s, 3H, 8-CH3); 13C NMR (CDCl3)
d 164.8
(C-4), 160.2 (C-7), 159.7 (C-2), 156.9 (C-8a), 148.4 (C-160), 148.1 (C-
80), 148.0 (C-20), 147.9 (C-100, C-180), 147.8 (C-40), 147.2 (C-10), 136.2
(C-70), 135.4 (C-120), 134.9 (C-150), 134.8 (C-60), 134.5 (C-200), 124.7
(C-5), 123.2 (C-50), 122.9 (C-110, C-190), 112.6 (C-8), 110.1 (C-4a),
109.5 (C-6), 67.7 (OeCH2), 50.9 (NeCH2), 6.7 (8-CH3); (Found
C,69.26; H, 4.58; N, 15.60; C26H21N5O3, requires C, 69.17; H, 4.69; N,
15.51).
(C]O), 1624 (C]C), 1552 (C]N) cmꢄ1; 1H NMR (DMSO-d6)
d 8.70e
8.50 (m, 6H, H-20, H-40, H-80, H-100, H-160, H-180), 8.0e7.9 (m, 3H, H-
50, H-120, H-200), 7.82 (d, J ¼ 7.5 Hz, 1H, H-5), 7.60e7.40 (m, 3H, H-60,
H-110, H-190), 7.00 (dd, J ¼ 7.5 and 2.3 Hz,1H, H-6), 6.55 (d, J ¼ 2.3 Hz,
1H, H-8), 5.40 (s, 2H, CH2eO), 5.30 (s, 2H, CH2eN); 13C NMR (CDCl3)
d
164.8 (C-4), 163.2 (C-7), 159.7 (C-2), 156.0 (C-8a), 148.4 (C-160),
148.1 (C-80),148.0 (C-20),147.9 (C-100, C-180),147.8 (C-40),147.2 (C-10),
136.2 (C-70), 135.4 (C-120), 134.9 (C-150), 134.8 (C-60), 134.5 (C-200),
131.7 (C-5),123.2 (C-50),122.9 (C-110, C-190),112.6 (C-8),112.1 (C-4a),
113.5 (C-6), 67.7 (OeCH2), 50.9 (NeCH2); (Found C, 68.77; H, 4.48; N,
16.10; C25H19N5O3, requires C, 68.64; H, 4.38; N, 16.01).
4.4.19. 8-Methyl-2-(pyridin-3-yl(pyridin-4-ylmethyl)amino)-7-
(pyridin-4-ylmethoxy)-4H-benzo[e][1,3]oxazin-4-one 15f
7-Hydroxy-8-methyl-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-
oxazin-4-one 10f was allowed to react with 4-(bromomethyl)
pyridine hydrobromide according to general procedure C. The
crude solid was collected and recrystallized from toluene to give
15f (61% yield), mp 285 ꢃC decomp. nmax (KBr) 3062, 2943(CeH),
4.4.16. 2-(Pyridin-3-yl(pyridin-4-ylmethyl)amino)-7-(pyridin-4-
ylmethoxy)-4H-benz[e][1,3]oxazin-4-one15c
7-Hydroxy-2-(pyridin-3-ylamino)-4H-benz[e]-1,3-oxazin-4-
one 10e was allowed to react with 4-(bromomethyl)pyridine
hydrobromide according to general procedure C. The crude solid
was collected and recrystallized from toluene to give 15c (45%
yield), mp 277e280 ꢃC decomp. nmax (KBr) 3058, 2988 (CeH), 1671
1672 (C]O), 1624 (C]C), 1553 (C]N) cmꢄ1 1H NMR (DMSO-d6)
;
d
8.75 (bd, Jmeta ¼ 2.2 Hz,1H, H-20), 8.60e8.50 (m, 4H, H-90, H-110, H-
170, H-190), 7.92 (d, J ¼ 5.5 Hz, 1H, H-5), 7.82 (bdd, bdd, 1H,
JH4 ,H5 ¼ 4.5 Hz, JH4 ,H2 ¼ 1.5 Hz, H-40), 7.70e7.60 (m, 2H, H-160, H-
200), 7.52 (d, J ¼ 5.5 Hz, 1H, H-60), 7.40e7.30 (m, 2H, H-80, H-120),
7.20e7.10 (m, 1H, H-50), 6.92 (d, J ¼ 7.5 Hz, 1H, H-6), 5.40 (s, 2H,
CH2eO), 5.30 (s, 2H, CH2eN) 2.23 (s, 3H, 8-CH3); 13C NMR (CDCl3)
0
0
0
0
(C]O), 1628 (C]C), 1559 (C]N) cmꢄ1 1H NMR (CDCl3)
; d 8.60e
8.40 (m, 6H, H-20, H-40, H-80, H-11, H-170, H-190), 8.15 (d,
J ¼ 7.5 Hz,1H, H-5), 7.90e7.70 (d, J ¼ 5.4 Hz, 2H, H-160, H-200), 7.50e
7.30 (m, 4H, H-50, H-60, H-80, H-120), 7.00 (dd, J ¼ 7.5 and 2.3 Hz, 1H,
H-6), 6.55 (d, J ¼ 2.3 Hz, 1H, H-8), 5.23 (s, 2H, CH2eO), 5.12 (s, 2H,
d
167.8 (C-4), 161.0 (C-7), 158.4 (C-2), 156.7 (C-8a), 155.8 (C-70), 154.1
(C-150), 153.4 (C-10), 149.8 (C-90, C-110, C-170, C-190), 149.3 (C-20),
137.3 (C-40), 135.6 (C-60), 126.8 (C-5), 124.3 (C-80, C-120), 123.3 (C-
160, C-200), 1121.5 (C-50), 113.5 (C-8), 111.5 (C-4a), 110.1 (C-6), 71.6
CH2eN); 13C NMR (CDCl3)
d 166.5 (C-4), 163.7 (C-7), 157.9 (C-2),

100
S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
(OeCH2), 56.6 (NeCH2), 8.3 (8-CH3); (Found C, 69.57; H, 4.31; N,
15.69; C26H21N5O3, requires C, 69.17; H, 4.69; N, 15.51).
and recrystallized from toluene to give 17c (65% yield), mp 205 ꢃC.
nmax (KBr)/cmꢄ1 3089, 2960 (CeH), 1754 (C]O), 1695 (C]O), 1560
(C]C),1565 (C]N); 1H NMR (CDCl3) 8.60e8.40 (m, 4H, H-20, H-40,
d
4.5. Reaction 7-hydroxy-2-methylthio-substiuted-1,3-benzoxazines
with hydro-halogen salt of 2, 3-(halo-methyl)pyridine and benzyl
bromide
H-80, H-100), 8.12 (d, J ¼ 7.5 Hz, 1H, H-5), 7.90e7.70 (m, 2H, H-60, H-
120), 7.52e7.33 (m, 2H, H-50, H-110), 7.00e6.90 (dd, JH6,H5 ¼ 7.5 Hz,
JH6,H8 ¼ 2.2 Hz, 1H, H-6), 6.53 (d, J ¼ 2.3 Hz, 1H, H-8), 5.24 (s, 2H, Oe
CH2), 5.10 (s, 2H, NeCH2); 13C NMR (CDCl3)
d 166.5 (C-4), 163.7 (C-
4.5.1. General procedure D
7), 157.9 (C-2), 155.7 (C-8a), 150.4 (C-20, C-80), 149.8 (C-40, C-100),
149.6 (C-10), 147.5 (C-70), 136.9 (C-120), 131.7 (C-60), 124.5 (C-5),
123.9 (C-50, C-110), 114.7 (C-6), 112.1 (C-4a), 101.3 (C-8), 68.8 (Oe
CH2), 53.2 (NeCH2); (Found C, 66.43; H, 4.30; N, 11.69;
C20H15N3O4, requires C, 66.48; H, 4.18; N, 11.63).
A suspension of 2-(methylthio)-4H-benz[e]-1,3-oxazin-4-one
9e and 9f (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 refluxed for 2 h. 2-(Bromomethyl) pyridine
hydrobromide, 3-(chloromethyl)pyridine hydrochloride or benzyl
bromide (2 mmol) in acetonitrile was added drop wise. Upon
completion of the addition, the reaction mixture was refluxed for
a further 30 min before solvent was evaporated off under reduced
pressure. The residue was washed with 4 ꢂ 15 mL of CHCl3 and then
filtered. The CHCl3 filtrate was evaporated off under reduced
pressure and the resulting residue was triturated with a minimal
amount of diethyl ether to give the crude solid which was recrys-
tallized from an appropriate solvent.
4.5.5. 8-Methyl-7-(pyridin-3-ylmethoxy)-3-(pyridin-3-ylmethyl)-
2H-benz[e]-1,3-oxazine-2,4(3H)-dione 17d
7-Hydroxy-8-methyl-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-
one 9f was allowed to react with 3-(chloromethyl)pyridine
hydrochloride according to general procedure D. The crude solid
was collected and recrystallized from toluene to give 17d (55%
yield), mp 255e258 ꢃC decomp. nmax (KBr)/cmꢄ1 3053, 2923(CeH),
1665 (C]O),1624 (C]C),1550 (C]N); 1H NMR (CDCl3)
d 8.70e8.50
(m, 4H, H-20, H-40, H-80, H-100), 8.00e7.90 (m, 2H, H-60, H-120), 7.82
(d, J ¼ 7.5 Hz, 1H, H-5), 7.60e7.40 (m, 2H, H-50, H-110), 7.25 (d,
J ¼ 7.5 Hz,1H, H-6), 5.42 (s, 2H, CH2eO), 5.34 (s, 2H, CH2eN), 2.43 (s,
4.5.2. 7-(Pyridin-2-ylmethoxy)-3-(pyridin-2-ylmethyl)-2H-benz
[e]-1,3-oxazine-2,4(3H)-dione 17a
7-Hydroxy-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-one
9e
3H, 8-CH3); 13C NMR (CDCl3)
d 164.8 (C-4), 160.2 (C-7), 159.7 (C-2),
was allowed to react with 2-(bromomethyl)pyridine hydrobromide
according to general procedure D. The crude solid was collected and
recrystallized from toluene to give (60% yield), mp 230 ꢃC. nmax
(KBr) 3079, 2951 (CeH), 1756 (C]O), 1693 (C]O), 1592 (C]C),
156.9 (C-8a), 148.4 (C-20), 148.1 (C-80), 147.9 (C-40, C-100), 136.2 (C-
70), 135.4 (C-120), 134.9 (C-130), 134.5 (C-60), 124.7 (C-5), 122.9 (C-50,
C-110), 112.6 (C-8), 110.1 (C-4a), 109.5 (C-6), 71.8 (C-9), 46.9 (C-10),
8.8 (8-CH3); (Found C, 67.08; H, 4.63; N, 11.15; C21H17N3O4, requires
C, 67.19; H, 4.56; N, 11.19).
1571 (C]N) cmꢄ1; 1H NMR (CDCl3)
d
8.62 (d, J ¼ 5.0 Hz, 1H, H-30),
8.54 (d, J ¼ 5.0 Hz, 1H, H-90), 8.00 (d, J ¼ 8.6 Hz, 1H, H-5), 7.80e7.60
(m, 2H, H-50, H-110), 7.52 (d, J ¼ 8.6 Hz, 1H, H-60), 7.30e7.20 (m, 2H,
H-100, H-120), 7.10 (m, 1H, H-40), 7.00 (dd, JH6,H8 ¼ 2.4 Hz,
JH6,H5 ¼ 8.6 Hz, 1H, H-6), 6.83 (d, J ¼ 2.2 Hz, 1H, H-8), 5.42 (s, 2H, 7-
4.5.6. 3-Benzyl-7-(benzyloxy)-8-methyl-2H-benz[e]-1,3-oxazine-
2,4(3H)-dione 17e
7-Hydroxy-8-methyl-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-
one 9f was allowed to react with benzyl bromide according to
general procedure D. The crude solid was collected and recrystal-
lized from toluene to give (67% yield), mp 182e185 ꢃC. nmax (KBr)
3063, 2967 (CeH), 1751s (C]O), 1687s (C]O), 1596m (C]C), 1497s
OCH2), 5.34 (s, 2H, 3-NCH2); 13C NMR (CDCl3)
d 165.3 (C-4, C-2),
160.8 (C-7), 155.9 (C-10), 155.1 (C-70), 154.8 (C-8a), 150.5 (C-30/C-90),
137.5 (C-110), 137.1 (C-50), 130.2 (C-5), 123.6 (C-60), 122.9 (C-120)
122.1 (C-100), 121.9 (C-40), 114.6 (C-6), 108.0 (C-4a), 101.8 (C-8), 71.9
(7-OCH2), 46.9 (3-NCH2); (Found C, 66.43; H, 4.30; N, 11.69;
C20H15N3O4, requires C, 66.48; H, 4.18; N, 11.63).
(C]N) cmꢄ1; 1H NMR (CDCl3)
d
7.91 (d, J ¼ 8.6 Hz, 1H, H-5), 7.50e
7.20 (m, 10H, ArH), 6.90 (d, J ¼ 8.6 Hz, 1H, H-6), 5.20 (s, 4H, H-9/H-
10), 2.30 (s, 3H, 8-CH3); 13C NMR (CDCl3)
163.0 (C-4), 161.1 (C-2),
d
4.5.3. 8-Methyl-7-(pyridin-2-ylmethoxy)-3-(pyridin-2-ylmethyl)-
2H-benz[e]-1,3-oxazine-2,4(3H)-dione 17b
152.3 (C-7),149.1 (C-8a),136.5 (C-10),136.3 (C-70),129.6 (C-20),129.2
(C-30), 129.0 (C-40), 128.8 (C-80), 128.4 (C-5), 127.6 (C-90), 127.2 (C-
100), 114.6 (C-8a), 109.6 (C-6), 107.9 (C-4a), 71.1 (C-9), 45.9 (C-10),
8.7 (8-CH3); (Found C, 74.05; H, 5.16; N, 3.67; C23H19NO4 requires C,
73.98; H, 5.13; N, 3.75).
7-Hydroxy-8-methyl-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-
one 9f was allowed to react with 2-(Bromomethyl)pyridine
hydrobromide according to general procedure D. The crude solid
was collected and recrystallized from toluene to give (55% yield),
mp 192e194 ꢃC. nmax (KBr) 3067, 2925 (CeH), 1762s (C]O), 1698s
4.6. Biological activity assays
(C]O), 1594m (C]C), 1570s (C]N) cmꢄ1 1H NMR (CDCl3)
; d 8.62
(d, J ¼ 5.0 Hz, 1H, H-30), 8.54 (d, J ¼ 5.0 Hz, 1H, H-90), 8.02 (d, 1H,
J ¼ 8.6 Hz, H-5), 7.80e7.60 (m, 2H, H-50, H-110), 7.54 (d, 1H,
J ¼ 8.6 Hz, H-60), 7.30e7.20 (m, 2H, H-100, H-120), 7.15 (m, 1H, H-40),
6.90 (d, J ¼ 8.6 Hz, 1H, H-6), 5.32 (s, 4H, 7-OCH2 and 3-NCH2), 2.43
4.6.1. Platelet aggregometry
Venous blood was collected from ostensibly healthy, drug free
volunteers into trisodium citrate 22.0 g/L. Ethics approval was ob-
tained 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 [16].
(s, 3H, 8-CH3); 13C NMR (CDCl3)
d 162.7 (C-4), 161.1 (C-2), 156.5 (C-
7), 155.4 (C-8a), 152.5 (C-10), 150.0 (C-30), 149.9 (C-90), 149.3 (C-70),
137.4 (C-110), 136.9 (C-50), 127.5 (C-5), 123.5 (C-60), 122.8 (C-120)
122.1 (C-100),121.6 (C-40),114.6 (C-8a),109.6 (C-6),108.0 (C-4a), 71.8
(7-OCH2), 46.9 (3-NCH2), 8.8 (8-CH3); (Found C, 67.08; H, 4.63; N,
11.15; C21H17N3O4, requires C, 67.19; H, 4.56; N, 11.19).
4.6.2. DNA-PK inhibition assay
All compounds were dissolved in DMSO and tested for their
ability to inhibit DNA-PK. This was accomplished using the purified
DNA-PK enzyme (Promega) and the substrate peptide EPPLSQEA-
FADLWKK (Shanghai Research Institute of Chemical Industry Testing
co., Ltd, Shanghai) as previously reported [13]. Assays were done in
triplicate and IC50’s were calculated using the Graphpad Prism curve
fitting software (V5). r [2] for all curves was greater than 0.9.
4.5.4. 7-(Pyridin-3-ylmethoxy)-3-(pyridin-3-ylmethyl)-2H-benz
[e]-1,3-oxazine-2,4(3H)-dione 17c
7-Hydroxy-2-(methylthio)-4H-benz[e]-1,3-oxazin-4-one
9e
was allowed to react with 3-(chloromethyl)pyridine hydrochloride
according to the general procedure D. The crude solid was collected

S.K. Ihmaid et al. / European Journal of Medicinal Chemistry 57 (2012) 85e101
101
potent chromen-4-one inhibitors of the DNA-dependent protein kinase (DNA-
PK) using a small-molecule library approach, J. Med. Chem. 48 (2005) 7829e
7846.
4.6.3. PI-3K inhibition assay
The PI3K assays were performed by Reaction Biology Corpora-
tion, One Great Valley Parkway, Suite 2 Malvern, PA 19355 USA.
All compounds were dissolved in DMSO and tested for their
ability to inhibit PI3K.
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M. Frigerio, N.J. Curtin, K. Menear, C. Richardson, G.C.M. Smith, R.J. Griffin, 8-
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G.C.M. Smith, Identification of a highly potent and selective DNA-dependent
protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone
libraries, Bioorg. Med. Chem. Lett. 14 (2004) 6083e6087.
[10] K. Saravanan, M. Albertella, C. Cano, N.J. Curtin, M. Frigerio, B.T. Golding,
K. Haggerty, I.R. Hardcastle, M. Hummersone, K. Menear, D.R. Newell,
T. Rennison, C. Richardson, L. Rigoreau, S. Rodriguez-Aristegui, G.C.M. Smith,
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Compounds were tested in a 10-dose IC50 with 4-fold serial
dilution starting at 100
The control compound, PI-103, was tested in a 10-dose IC50
with 3-fold serial dilution starting at 10 M. Reactions were carried
out at 10 M ATP using the HTRF assay format.
mM.
m
m
Acknowledgements
WewouldliketothankDrJonathanWhiteAssociateProfessorand
Reader, School of Chemistry, The University of Melbourne, Victoria,
3010, Australia for the measurement and interpretation of X-ray
crystal structures of compounds 11b and 15b. We would also like to
thank the careful and helpful comments given by the reviewers
which significantly improved the presentation of this manuscript.
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Derivatives as Phosphoinositide 3-Kinase Inhibitors. WO 2011/2011012883,
P.C.T. Int., 3 Feb 2011.
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dependent protein kinase and anti-platelet studies of 2-amino-[5,6,7,8-
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