Synthesis, structure elucidation, DNA-PK and PI3K and anti-cancer activity of 8- and 6-aryl-substituted-1-3-benzoxazines

Article abstract of DOI:10.1016/j.ejmech.2016.01.042

The synthesis of 6-aryl, 8- aryl, and 8-aryl-6-chloro-2-morpholino-1,3-benzoxazines with potent activity against PI3K and DNA-PK is described. Synthesis of thirty one analogues was facilitated by an improved synthesis of 3-bromo-2-hydroxybenzoic acid 13 by de-sulphonation of 3-bromo-2-hydroxy-5-sulfobenzoic acid 12 en route to 2-methylthio-substituted-benzoxazine intermediates 17-19. From this series, compound 20k (LTURM34) (dibenzo[b,d]thiophen-4-yl) (IC₅₀ = 0.034 μM) was identified as a specific DNA-PK inhibitor, 170 fold more selective for DNA-PK activity compared to PI3K activity. Other compounds of the series show markedly altered selectivity for various PI3K isoforms including compound 20i (8-(naphthalen-1-yl) a potent and quite selective PI3Kδ inhibitor (IC₅₀ = 0.64 μM). Finally, nine compounds were evaluated and showed antiproliferative activity against an NCI panel of cancer cell lines. Compound 20i (8-(naphthalen-1-yl) showed strong anti-proliferative activity against A498 renal cancer cells that warrants further investigation.

Full text of DOI:10.1016/j.ejmech.2016.01.042

Accepted Manuscript
Synthesis, structure elucidation, DNA-PK and PI3K and anti-cancer activity of 8- and
6-aryl-substituted-1-3-benzoxazines
Rick Morrison, Jasim M.A. Al-Rawi, Ian G. Jennings, Philip E. Thompson, Michael J.
Angove
PII:
S0223-5234(16)30042-3
10.1016/j.ejmech.2016.01.042
EJMECH 8332
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 November 2015
Revised Date: 21 January 2016
Accepted Date: 22 January 2016
Please cite this article as: R. Morrison, J.M.A. Al-Rawi, I.G. Jennings, P.E. Thompson, M.J. Angove,
Synthesis, structure elucidation, DNA-PK and PI3K and anti-cancer activity of 8- and 6-aryl-
substituted-1-3-benzoxazines, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.01.042.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis, structure elucidation, DNA-PK and PI3K and some anti-cancer activity of 8-
and 6-aryl-substituted-1-3-benzoxazines.
Rick Morrisona, Jasim M. A. Al-Rawi*a, Ian G. Jenningsb, Philip E.Thompsonb and Michael
J. Angovea.

ACCEPTED MANUSCRIPT
Synthesis, structure elucidation, DNA-PK and PI3K and anti-cancer activity of 8- and
6-aryl-substituted-1-3-benzoxazines.
Rick Morrisona, Jasim M. A. Al-Rawi*a, Ian G. Jenningsb, Philip E.Thompsonb and Michael
J. Angovea.
a Pharmacy and Applied Science, La Trobe Institute for Molecular Science, La Trobe
University, P.O. Box 199 Bendigo VIC 3552, Australia
b Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University,
381 Royal Parade, Parkville VIC 3052, Australia.
Abstract
The synthesis of 6-aryl, 8- aryl, and 8-aryl-6-chloro-2-morpholino-1,3-benzoxazines with
potent activity against PI3K and DNA-PK is described. Synthesis of thirty one analogues was
facilitated by an improved synthesis of 3-bromo-2-hydroxybenzoic acid 13 by de-
sulphonation of 3-bromo-2-hydroxy-5-sulfobenzoic acid 12 en route to 2-methylthio-
substituted-benzoxazine intermediates 17-19.
From this series, compound 20k (LTURM34) (dibenzo[b,d]thiophen-4-yl) (IC50 = 0.034 µM)
was identified as a specific DNA-PK inhibitor, 170 fold more selective for DNA-PK activity
compared to PI3K activity.
Other compounds of the series show markedly altered selectivity for various PI3K isoforms
including compound 20i (8-(naphthalen-1-yl) a potent and quite selective PI3Kδ inhibitor
(IC50 = 0.64 µM).
Finally, nine compounds were evaluated and showed antiproliferative activity against an NCI
panel of cancer cell lines. Compound 20i (8-(naphthalen-1-yl) showed strong anti-
proliferative activity against A498 renal cancer cells that warrants further investigation.

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Keywords: Suzuki coupling synthesis of 8- and 6-aryl-2-morpholino-1,3-benzoxazines,
DNA-PK, PI3K, anti-cancer activity.
____________________________________________________________
* Corresponding author.
Tel.: +61 3 54 44 7364; fax: +61 3 54 44 7476.
E-mail addresses: rmmorrison@students.latrobe.edu.au (R. Morrison), j.al-
rawi@latrobe.edu.au (J. Al-Rawi), Ian.Jennings@monash.edu (I. Jennings),
Philip.Thompson@monash.edu (P. Thompson), m.angove@latrobe.edu.au (M .J. Angove).

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1. Introduction
We have previously reported a range of 2-morpholino-1,3-benzoxazin-4-one compounds that
show moderate to high DNA-PK, PI3K and antiplatelet inhibitory activity [1-5]. These
compounds represent a modified scaffold to the corresponding chromone, quinolone and
pyridopyrimidinone bicyclic compounds which have also been widely applied to those
targets, some of which have undergone extensive clinical and pre-clinical investigation.
Chromone and pyridopyrimidone compounds such as LY294002, the archetypal PI3K family
inhibitor and PI3Kβ inhibitors such as AZD8186 and AZD6482, have been investigated as in
cancer and thrombosis. DNA-PK inhibitors such as the chromone 1a (NU7441), 1b
(KU0060648) and quinolinone 2 (Figure 1) have considerable therapeutic potential as
chemo- and radio-potentiating agents in the therapy of cancer by blocking DNA DSB repair
[6-10].
We recently described the 1,3-benzoxazin-4-one analogue of LY294002 and showed it to
have pan-PI3K and DNA-PK activity [2]. We have also reported on the activity of other
family members, such as 3 which causes sensitization of lung cancer and colon cancer cells to
radiation [11] and 4 (LTUSI122) as an inhibitor of angiogenesis [12].
Based on the cumulative evidence of our previously published work, we hypothesize that the
1,3-benzoxazine nucleus, with its altered electronic structure compared to the other scaffolds
will retain the general binding capabilities for PI3K family members but will show modified
selectivity profiles compared to its isosteric scaffolds and therefore represents an opportunity
to discover more selective ligands at targets of interest. Here we report on the development
of improved syntheses of the class, giving access to a series of compounds that show such
modified selectivity profiles. We identified 20k (LTURM34), a potent DNA-PK inhibitor
with improved selectivity compared to the chromone homologue NU7441 as well as 20i

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(LTURM36), a PI3Kδ selective compound. Both compounds show antiproliferative activity
against a range of cancer cell lines.
Figure 1. Structures of some previously prepared DNA-PK and PI3K inhibitors

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2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of 3-bromo-2-hydroxy-substituted-benzoic acids 9 and 10
We have been able to make useful improvements to previously reported methods [13-15] in
the synthesis of key precursors enabling access to a range of analogues, and have utilized 2D
NMR to fully characterise them. 3-Bromo-5-chloro-2-hydroxy benzoic acid 9 was prepared
by direct bromination of 5-chloro-2-hydroxybenzoic acid using the previously reported
method [13] with slight modification in which the solvent used in the reaction was changed
from acetic acid to chloroform with the yield improved to 96%.
3-Bromo-2-hydroxybenzoic acid 10 was prepared by bromination of 2-hydroxy-5-
sulfobenzoic acid 5 using the previously reported procedure [14] and gave 3-bromo-2-
hydroxy-5-sulfobenzoic acid 8 in a quantitative yield. Heating product 8 with H2SO4 under
microwave conditions gave the de-sulphonated product 10 in good yields (71%).
2.1.2. Synthesis of 8-bromo, 8-bromo-6-chloro and 6- bromo, -2-morpholino-1,3-
benzoxazines 17-19
The reaction of 3-bromo-2-hydroxy-substituted-benzoic acids 7, 9 and 10 with freshly
prepared triphenylphosphine thiocyanate according to the previously reported procedure [15]
gave 8- bromo, 6-chloro-8-bromo and 6-bromo,-1,3-benzoxazines 11, 12 and 13 respectively.
Endeavouring to prepare the 8-bromo-2-morpholino-1,3-benzoxazines 17-20 directly by
boiling 8-bromo-1,3-benzoxazine 11-13 with morpholine according to the previously
reported method [3] unexpectedly gave 2-morpholino-4H-1,3-benzoxazin-4-one (the de-
brominated analogue of products 17-20).

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The de-bromo-hydrogenation reaction could be aided by the presence of excess morpholine
used in the reaction and the dissolved H2S resulting from the formation of 8-bromo-2-
morpholino-1,3-benzoxazine 17-20. The reaction of 8-bromo-1,3-benzoxazine 11 with excess
morpholine in d6-DMSO at 70 °C in the NMR tube was followed by proton NMR. After
15 minutes the 8-bromo-2-morpholino-1,3-benoxazine 17 was formed. Following an
additional 30 minutes the de-bromo-hydrogenation 2-morpholino-1,3-benzoxazine was
observed, and the de-bromo-hydrogenation was completed in ~ 6 hours.
As a result of the de-bromo-hydrogenation in the attempted synthesis of 8-bromo-2-
morpholino-1,3-benzoxazine 17-20 an alternative synthetic route was applied. Synthesis of
the 8-bromo- 2-methylthio-substituted-benzoxazine 14-16 intermediates (which can be
isolated if required) from the reaction of 8- bromo, 8-bromo-6-chloro, and 6-bromo -1,3-
benzoxazines 11, 12 and 13 respectively with iodomethane in the presence of NaHCO3
according to the previously reported method [2]. Morpholine was then allowed to react (in
situ) with the 2-methylthio-substituted-benzoxazines 14-16 and gave 8- bromo-, 8-bromo-6-
chloro- and 6-bromo-2-morpholino-1,3-benzoxazines 17-19.
2.1.3. Synthesis of 6-aryl, 8- aryl, and 8-aryl-6-chloro-2-morpholino-1,3-benzoxazines 20-22
using modified Suzuki coupling reactions
Initial Suzuki coupling reactions were carried out on 6-bromo-2-morpholino-4H-1,3-
benzoxazin-4-one 19 using 1,4-dioxane as a solvent, powdered potassium carbonate,
phenylboronic, acid and tetrakis(triphenylphosphine)palladium(0) as the catalyst. The
reaction was heated for 16 hours at 80 °C in Schlenk flask under nitrogen, following a
previously published procedure [10]. Work-up of the reaction yielded unchanged 6-bromo-2-
morpholino-4H-1,3-benzoxazin-4-one starting material 19. Further reactions under the same
condition were carried out using palladium (II) acetate or

ACCEPTED MANUSCRIPT
[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) as the catalyst.In these cases,
however only starting material was isolated. The above reaction was repeated with the
solvent changed to 3:1 1,4-dioxane/water in order to improve solubility of the potassium
carbonate. These conditions were found to hydrolyse the 2-morpholino-1,3-oxazine ring
resulted in the formation of 5-phenyl-2-hydroxybenzoic acid as product. Changing the
catalyst to
bis(triphenylphosphine)palladium(II) dichloride and lowering the reaction temperature below
60 °C gave the expected products 20-22 (Scheme 1).

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Compound No
Ar
Compound No
Ar
22a
20h, 22i
20a, 21a, 22b
20b, 22c
20i, 22j,
20j, 22k
20c, 22d
20k, 21d, 22l
20d, 22e
22m
20e, 22f
20f, 22g
22n
20l, 21c, 22o
20g, 21b, 22h
Scheme 1. Synthesis of substituted-benzoxazine 11-13, 2-methythio-benzoxazine 14-16, 2-
morpholino-1,3-benzoxazine 17-19 and 6- or 8-aryl substituted-1,3-benzoxazines 20-22 and
reaction conditions: i, 5 in water with bromine vapour passed through gave 8, 6 in CH3Cl
with bromine gave 9. ii, 8 in water and H2SO4 heated in microwave at 400W, 170 °C for 25
minutes. iii, 7, 19¸and 10 in dry DCM with Ph3PSCN. iv, 11-13 with CH3I and NaHCO3 in
water/propanol (50/50). v, 14-16 to solution in step iv morpholine was added at r.t. vi, 18-20
in water/dioxane (30:70) with K2CO3, boronic acid and Pd(PPh3)2Cl2 heated at 50 °C for 16
hours.

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The structures of the new products 11-12, 17-19 and 20-22 were confirmed by the analysis of
their IR, 1H NMR and 13C NMR spectra in addition to the microanalysis/high resolution mass
spectrometry. The assignments of the 1H NMR spectra of compounds 20-22 were achieved
using 1H-1H correlation spectroscopy (COSY) and hydrogen-hydrogen homo decoupling
(HHHD,) while the protonated signals of the 13C NMR spectra were assigned using
heteronuclear single quantum coherence spectroscopy (HSQC)using the assigned proton
NMR spectra.

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2.2. Biology
The collected class I PI3K and DNA-PK activity of the 31 compounds prepared here are
shown in Table 1. Compounds were initially screened at a concentration of 10µM, and where
> 50% inhibition was observed an IC50 determination was made. Compounds of general
structure 20 showed the strongest potency, showing that neither aryl- nor chloro-substitution
at position 6 is favourable in this class.
Table 1 Chemical Structures, DNA-PK and PI3K % Inhibition activity (IC50 µM) of 8-
aryl, 6-chloro-8-aryl and 6-aryl-2-morpholino-1,3-benzoxazines 20-22
DNA-PK
Inhibition %
at10µ M
PI3Kα
PI3Kβ
PI3Kγ
Inhibition %
at 10 µM
PI3Kδ
Inhibition %
at 10 µM
Inhibition % at Inhibition %
Ar
10 µM
at 10 µM
(IC50 µM )
(IC50 µM )
(IC50 µM )
(IC50 µM )
(IC50 µM )
LY29
4002*
(4.0)
(3.0)
(1.5)
(0.95)
(0.3)
(2.8)
22
34
7
(2.7)
(9.94)
(10.9)
20a
20b
(>10)
16
(~10)
(~10)
13
(3.2)
31
0
(9.0)
4
(15.7)
(63.5)
20c
20d
(~10)
(>10)
26
13
10
24
(>10)
37
(8.81)
(78.4)
20e
20f
(>10)
(3.3)
(3.0)
34
43
16
29
29
32
(4.5)
(4.8)
(7.12)
(9.80)
20g
20h
20i
(3.4)
(5.0)
(6.9)
(0.64)
(12.0)
(20.30)
(0.269)
26
20j

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25
(5.8)
15
27
17
(8.5)
(3.7)
(0.034)
(2.12)
20k
20l
(~10)
16
42
0
0
0
0
(2.60)
( ~10)
(12.3)
(6.05)
21a
21b
35
0
15
59
14
1
16
36
(5.77)
21c
21d
(0.564)
2
7
0
9
0
0
0
1
8
22a
22b
17
10
7
0
0
10
0
0
0
7
11
7
22c
22d
22e
a
a
a
a
-
-
-
-
a
a
a
a
-
-
-
-
8
6
22f
22g
22h
11
36
0
3
a
a
a
a
-
-
-
-
24
a
a
a
a
-
-
-
-
21
4
22i
22j
22k
10
5
0
6
4
0
0
0
12
0
21
0
0
8
22l

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11
3
13
29
20
0
4
3
0
0
0
7
23
0
22m
22n
22o
3
* DNA-PK and PI3K inhibition IC50 takrn from refrences 22
a PI3K inhibition has not been tested
2.2.1. 8-aryl-2-morpholino-1,3-benzoxazines 20
For compounds 20a – l, a number of compounds with a significant and interesting activity
profile were identified. Prominent among these were 20k (LTURM34), which showed potent
inhibition of DNA-PK (IC50 0.034 µM) with excellent selectivity over the Class I PI3K
isoforms (IC50 all > 5 µM). Compound 20j also showed sub-micromolar potency at DNA-PK.
Several compounds also showed potent inhibition of specific PI3K isoforms. Compound 20i
(LTURM36) (8-(naphthalen-1-yl)) showed the most potent PI3Kδ isoform activity of all the
compounds tested with IC50 = 0.64 µM and moderate PI3Kβ isoform inhibition (IC50 = 5.0
µM), giving PI3Kδ specificity of 32 fold compared with the DNA-PK activity.
Compounds 20a –h had potency in the low micromolar range at certain isoforms, and showed
some diverse selectivity profiles. For example, 20a inhibited PI3Kα and PI3Kδ, preferentially
to the other isoforms, while 20c showed a modest preference for PI3Kβ (IC50 = 3.2 µM).
None of the 8-aryl-substituted compounds 20a-l showed substantial PI3K γ isoform activity
at the assayed concentration of 10 µM. (Table 1). Compounds 20f (8-(pyridin-3-yl)) and 20d
(8-(3-aminophenyl)) specifically inhibited the PI3K β isoform with IC50 below or about 10
µM. It is worth noting that these 2 compounds (20f and 20d) displayed poor DNA-PK
potency (IC50 = 78.4 µM and 63.5 µM respectively) (Table 1) compared to the rest of the 8-

ACCEPTED MANUSCRIPT
aryl-substituted compound 20a-l therefore, these two compounds show PI3Kβ isoform
selectivity.
2.2.2. 6-Chloro-8-aryl-2-morpholino-1,3-benzoxazines 21
The inclusion of the chlorine atom at position 6 in 8-aryl-compound 21a-c resulted in
compounds of significantly less potency than there unsubstituted counterparts at PI3Kα
though the activities at PI3Kδ and DNA-PK were less affected. 6-chloro-8-(thiophen-2-yl)-
benzoxazin-4-one 21b (DNA-PK IC50 = 7.1 µM) showed comparable inhibition with the non-
6-cloro-analogue 20g (DNA-PK IC50 = 6.5 µM)) (Tables 1). However, in the case of the 8-
(dibenzo[b,d]thiophen-4-yl analogue 21d the IC50 (DNA-PK IC50 = 0.564 µM) increased by
17 fold compared to the 6-unsubstituted analogue 20k (DNA-PK IC50 = 0.043 µM).
2.2.3. 6-aryl-2-morpholino-1,3-benzoxazines 22a-o
Changing the 8-aryl substitution in compounds 20 to the 6-position, gave the 6-aryl products
22 which were found to show a significant loss of DNA-PK activity. The most active
compound of this series was 22h which showed 24% inhibition at 10 µM (Table 1). 6-Aryl
substituted compounds 22a-o showed no appreciable PI3K inhibition at or below 10 µM.
This may be a consequence of a steric ‘clash’ between the 6-aryl substituent and amino-acid
residues within the ATP-binding site of the kinase. Furthermore, this reinforces that 8-aryl
substitutions are important for PI3K as well as DNA-PK activity.
2.2.4. Cell based assays
Nine of the synthesized compounds were selected and submitted to The National Cancer
Institute, USA, for one dose single concentration (10 µM) anticancer screening. In vitro
screening was carried out against a panel of 60 human tumour cell lines. Results for each of
the tested compounds were reported as reported as % growth inhibition of compound treated

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cells compared to vehicle treated cells [16, 17]. The results are presented in Table-1
(Supplementary materials).
Of the nine compounds assayed, two compounds 20i and 20k showed the most potent and
broad ranging antiproliferative activity (Table 2). 20i showed over 30% inhibition at 9 out of
16 cell lines, including > 90% inhibition of the renal cancer cell line A498. 20k was more
consistently active against the selected cell lines (11 of 16), but at best showed 54%
inhibition against the HOP-92 non-small cell lung cancer line.
Table 2 In vitro anticancer screening results (%GI) of the selected 9 compounds against
NCI60 cell lines at 10 µM concentration.
Cell Line
Percentage Growth Inhibition (%GI)
20a
20e
20g
20h
20i
20j
20k
50
20a
22l
Leukemia
SR
26
8
17
21
27
34
26
19
Non-Small Cell
Lung Cancer
EKVX
HOP-62
HOP-92
NCI-H226
11
5
34
15
2
4
NT
17
14
8
44
21
16
15
40
21
18
19
39
33
18
13
45
27
28
26
53
38
11
5
34
15
5
4
41
11
CNS Cancer
SNB-75
38
19
25
7
32
20
38
31
41
37
38
19
10
12
53
52
Melanoma
UACC-62
45
45
Renal Cancer
786-0
A498
RXF 393
UO-31
9
27
7
0
1
0
7
36
9
11
37
14
23
25
91
38
46
21
27
15
40
27
46
38
51
9
27
7
5
37
0
17
10
20
17
1
Prostate Cancer
PC-3
14
10
17
24
40
23
40
14
39
Breast Cancer
MCF7
MDA-MB-
231/ATCC
T-47D
17
3
9
8
21
0
22
6
10
6
11
18
30
29
17
3
21
12
38
6
12
0
17
20
26
15
20
23
15
18
42
26
38
6
16
19
MDA-MB-468

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2.3 Discussion
The 2-morpholino-1,3-benzoxazine class of compounds represent an isosteric replacement for
the well-studied morpholinochromones of which LY294002 is the best known example. As
an established inhibitor of the PI3K isoform family, adaptation of this structure has been
widely used to modify the potency and selectivity at PI3K isoforms. This includes the
development of NU7441 as a potent, but moderately selective inhibitor of DNA-PK. Our
hypothesis is that the change to a 1,3-benzoxazine scaffold should alter the observed
selectivity profile, taking advantage of the subtle differences of the isoform binding sites. We
achieved an improved synthetic route to these compounds which allowed us to prepare a
series of 31 compounds for evaluation.
2.3.1 Structure Activity Relationships
We were gratified to observe that compound 20k, a direct analogue of NU7441 showed
equivalent inhibitory potency at DNA-PK activity with an IC50 of 0.034 µM. In addition,
compound 20k is much more selective than NU7441, with 170 fold selectivity over the
PI3Kβ isoform, and even more at the other isoforms (Figure 2). Accordingly, the 1,3-
benzoxazine compound 20k is the most selective inhibitor of DNA-PK versus PI3K (Figure 2
and Table 1) reported.

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S
O
S
O
O
CH4
O
O
N
O
N
O
N
N
O
O
LY294002
20k (LTURM34)
1 (NU7441)
IC50/ (
PI3K
PI3K
PI3K
PI3K
DNAꢀPK 1.4
0.042
0.034
Figure 2. PI3K isoforms and DNA-PK IC50 for compound LY294002 and NU7441 taken
from reference [18], NA = less than 30% PI3k inhibition at 10 µM
Additionally, we were able to identify compounds with marked selectivity against the various
isoforms tested. Most notable among these was the naphthyl derivative 20i. It showed itself
to be a quite potent selective inhibitor of PI3Kδ, with sub-micromolar IC50 and 8-fold
preference over PI3Kβ and poor inhibition at other isoforms tested. While less potent, the
other compounds in the series provide interesting SAR that can be used to further probe
specific binding site interaction in the PI3K family.
The gained assay data allows us to consider how these benzoxazine compounds exert a
modified selectivity profile compared to the corresponding morpholinochromones or other
isosteric scaffolds. We have previously used molecular docking studies against DNA-PK
homology models to examine the mode of binding of these benzoxazine structures [2] and
they confirmed the likely isosteric relationship to the corresponding chromones. For example,
the improved selectivity of LTURM34 compared to NU7441, might to be due to changes in
the electronic structure of the benzoxazine nucleus that could govern the strength of the
interaction of the ring carbonyl with the Lys124 of DNA-PK as compared to the equivalent

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interactions against PI3K (eg Lys833 of PI3Kγ). In general terms it appears that,
benzoxazines have poorer affinity for PI3K isoforms than the corresponding chromones, but
that the difference is more marginal in DNA-PK. The differences within the Class I PI3K
isoforms be a more subtle but also could be sufficient to render improved selectivity for one
or other isoform. Note that the chromone equivalent of 20i has been tested against DNA-PK
only (IC50 3ꢁM), [10] so its PI3K activity would be interesting to determine.
Restricting our considerations to the benzoxaine compounds described here, it can be
concluded a) the 8-aryl group substituent has impact on both PI3K/DNA-PK potency and
selectivity; b) the hydrophobicity of the 8-aryl group is not the main factor affecting the
selectivity and potency of PI3K/DNA-PK but rather the functional group type, and c) the
nature of the 8-aryl group did not show the same influence on the PI3K/DNA-PK potencies.
Finally, a series that represented the most interesting compounds from the study were
screened against a panel of cancer cell lines. While a number of compounds inhibited cell
growth by ~ 50% at the chosen 10µM dose, the clear stand out result was the activity of the
8-(naphthalen-1-yl)-4H-benzo[1,3]oxazin-4-one compound 20i against the Renal Cancer line
A498. Noting that 20i was the most potent PI3Kδ isoform inhibitor in the series (IC50 =
0.64ꢁM) the expression of that isoform in the A498 cell line would be worth examining. It is
notable that TGX221, a PI3Kβ inhibitor that also shows potent PI3Kδ inhibition was recently
identified as a potent inhibitor of Renal Cell Carcinoma Cells with both VHL and SETD2
gene mutations of which A498 cells were represented [19]. It will be important to further
evaluate these and other isoform selective inhibitors to identify the precise mechanism for the
observed actions of these compounds.

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3. Conclusion
In this work the synthesis of 3-bromo-2-hydroxybenzoic acid 10 was improved and was used
to synthesise 8-bromo-1,3-benzoxazine 11, 8-bromo-2-morpholino-1,3-benzoxazines 17 and
8-aryl-2-morpholino-1,3-benzoxazines 20.
Modified conditions of Suzuki coupling were used to synthesise the 8-aryl, 8-aryl-6-chloro,
and 6-aryl-2-morpholino-1,3-benzoxazines 20-22, the DNA-PK and PI3K inhibition % and
some IC50 data of 31 compounds were measured. The DNA-PK activity (IC50 = 0.034 µM)
for compound 20k (LTURM34) (dibenzo[b,d]thiophen-4-yl) was 170.6 fold more selective
against the PI3K activity. From this result compound 20k (LTURM34) can be considered as
a specific DNA-PK inhibitor. Furthermore, Compound 20i (8-(naphthalen-1-yl)) was a potent
and selective PI3Kδ inhibitor with which could explain the high growth inhibition (GI = 92%
at 10 µM) against the renal cancer cell line A498.

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4. Experimental
4.1. Chemistry
4.1.1. Analytical techniques
Melting point determinations were carried out using a Stuart Scientific (SMP3) melting point
apparatus and all melting points are uncorrected. Infrared spectra were recorded on an
Agilent Cary 630 FTIR spectrometer fitted with a diamond ATR attachment. 1H NMR and
13C NMR spectra were recorded on a Bruker Avance 300 NMR spectrometer at 300 MHz for
1H and 75 MHz for 13C respectively. 1H COSY and 1H-13C HSQC experiments were
performed using the Bruker topspin 2.1 software package. All 1H and 13C NMR spectral
results are recorded as chemical shifts (δ). Chemical shifts recorded in CDCl3 are relative to
the internal TMS (0 ppm) for 1H spectra and solvent peak (77.0 ppm) for 13C spectra.
Chemical shifts recorded in d6-DMSO are relative to the solvent peak of 2.5 ppm for 1H
spectra and 39.5 ppm for 13C spectra. 1H NMR multiplicities are expressed as singlet (s),
broad singlet (bs) doublet (d), double doublet (dd), triplet (t), double triplet (dt), triple triplet
(tt), quartet (q), and multiplet (m). Microanalysis was performed by Chemical and
Microanalytical Services Pty Ltd in Belmont, Victoria. HRMS analyses were carried out on
an Agilent 6224 TOF LC/MS Mass Spectrometer coupled to an Agilent 1290 Infinity
(Agilent, Palo Alto, CA). All data were acquired and reference mass corrected via a dual‐
spray electrospray ionisation (ESI) source. Acquisition was performed using the Agilent
Mass Hunter Data Acquisition software version B.05.00 Build 5.0.5042.2 and analysis was
performed using Mass Hunter Qualitative Analysis version B.05.00 Build 5.0.519.13.
4.1.2. Synthesis of 3-bromo-2-hydroxy-substituted-benzoic acid 7, 9 and 10 starting material
5-Bromo-2-hydroxybenzoic acid 7 was commercially available and used without further
purification.

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4.1.2.1. 3-bromo-5-chloro-2-hydroxybenzoic acid 9
Was prepared by bromination of 5-chloro-2-hyroxy benzoic acid 9 using the previously
reported method [13] with some modification in which the solvent used in the reaction was
changed from acetic acid to chloroform with high yield of 96%, mp 224-227 °C (Lit. [13]
231-232 °C). νmax (ATR)/cm-1 3100-2900br (O-H), 1661m (C=O), 1597m (C=C). 1H NMR
(300 MHz, acetone-d6, 300 K) δ 7.85-7.84 (d x 2, 1H, JH4,H6 = 0.9 Hz, H-4 & H-6), 3.67 (bs,
2H, OH x 2 with water). 13C NMR (75 MHz, acetone-d6, 300 K) δ 170.0 (1-COOH), 157.2
(C-2), 137.6 (C-4), 128.6 (C-6), 123.2 (C-5), 114.1 (C-1), 111.0 (C-3).
4.1.2.2. 3-bromo-2-hydroxy benzoic acid 10
Was prepared via the direct bromination of 2-hydroxy-5-sulfobenzoic acid 5 using the
previously reported procedure [14] and gave 3-bromo-2-hydroxy-5-sulfobenzoic acid 9 in a
quantitative yield, mp > 300 °C. νmax (ATR)/cm-1 3484w (O-H), 1655m (C=O), 1595w
(C=C). 1H NMR (300 MHz, d6-DMSO, 340 K) δ 8.04 (d, 1H, JH6,H5 = 2.1 Hz, H-6), 7.92 (d,
1H, JH4,H6 = 2.1 Hz, H-4), 4.60 (bs, 2H, 2 x OH with water). 13C NMR (75 MHz, d6-DMSO,
340 K) δ 171.3 (COOH), 158.0 (C-2), 140.8 (C-5), 135.3 (C-4), 127.0 (C-6), 113.9 (C-1),
109.2 (C-3). Product 9 (10.0 g, 33.6 mmol) was combined in a 250 mL, 10 bar reaction vessel
containing 40 mL of RO water and 20 mL of conc. H2SO4. Microwave irradiation was carried
out in a Milestone StartSynth microwave lab station at 400 W, 5 minutes from room
temperature to 170 °C then an additional 25 minutes at 170-172 °C. To afford the de-
sulphonated product 3-bromo-2-hydroxybenzoic acid 10 (5.20 g, 71% yield), mp 181-184 °C
(Lit. [20] 179-182 °C ). νmax (ATR)/cm-1 3200-2800br (O-H), 1655m (C=O), 1602m (C=C).
1H NMR (300 MHz, acetone-d6, 300 K) δ 8.12 (bs, 2H, OH x 2), 7.88 (dd, 1H, JH6,H5 = 7.8
Hz, JH6,H4 = 1.5 Hz, H-6), 7.78 (dd, 1H, JH4,H5 = 7.8 Hz, JH4,H6 = 1.5 Hz, H-4), 6.88 (t, 1H,
JH5,H4/H6 = 7.8 Hz). 13C NMR (75 MHz, acetone-d6, 300 K) δ 171.1 (COOH), 158.3 (C-2),
138.6 (C-4), 129.4 (C-6), 119.7 (C-5), 113.3 (C-1), 110.1 (C-3).

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4.1.3. General Procedure A: Synthesis of 6-bromo, 8- bromo, and 8-bromo-6-chloro-1,3-
benzoxazines 11-13.
Triphenylphosphine (6 mmol) is added to dry DCM (20 mL) then bromine (6.30 mmol) is
slowly added. Lead thiocyanate (7.50 mmol) was added with dry DCM (40 mL) to the above
mixture according to the previously reported procedure [21].
The freshly prepared triphenylphosphine thiocyante was allowed to react with 3 or 5-bromo,
-2-hydroxy-substituted-benzoic acids 7, 9 and 10 (4 mmol) according to the previously
reported procedure some modification [15]. The reaction mixture was then filtered and the
PbBr2 filter cake was washed with DCM (20 mL). The PbBr2 filter cake was collected and
hot filtered using acetone (≈ 75 mL) to extract any remaining product. Both the DCM and
acetone filtrates were separately evaporated to dryness under reduced pressure and minimal
toluene was used to triturate any oil and gave the 6-bromo, 8- bromo, and 8-bromo-6-chloro-
1,3-benzoxazines 11-13.
4.1.3.1. 8-bromo-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 11
A suspension of 3-bromo-2-hydroxybenzoic acid 10 (0.87 g, 4.0 mmol) in dry DCM (20 mL)
was added to a mixture of Ph3P(SCN)2 which was freshly prepared according to General
Procedure A. The solids recrystallized from acetonitrile and gave product 11 as fine yellow
needles (0.79 g, 78% yield), mp 229-231 °C. νmax (ATR)/cm-1 3195w, 3090w, 2933w (N-H),
1
1687m (C=O), 1602 m (C=C), 1113m (C=S). H NMR (300 MHz, d6-DMSO, 340 K) δ
13.42 (bs, 1H, NH), 8.05 (dd, 1H, JH5,H7 = 7.8 Hz, JH5,H6 = 1.5 Hz, H-5), 7.90 (dd, 1H, JH7,H5
7.8 Hz, JH7,H6 = 1.5 Hz, H-7), 7.35 (t, 1H, JH6,H5 = 7.8 Hz, H-6). 13C NMR (75 MHz, d6-
DMSO, 340 K) δ 181.2 (C-2), 157.0 (C-4), 152.4 (C-8a), 139.4 (C-5), 127.1 (C-6), 126.4 (C-
7), 117.5 (C-4a), 108.5 (C-8). MS [M+H+] (C8H5BrNO2S) calculated 257.9, observed 257.9.
=
4.1.3.2. 8-bromo-6-chloro-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 12

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A suspension of 3-bromo-5-chloro-2-hydroxybenzoic acid 6 (1.00 g, 4.0 mmol) in dry DCM
(20 mL) was added to a mixture of Ph3P(SCN)2 which was freshly prepared according to
General Procedure A. The solids recrystallized from acetonitrile and gave product 12 as fine
yellow needles (0.92 g, 78% yield), mp 249-252 °C. νmax (ATR)/cm-1 3127w, 2073w, 1940w
(N-H), 1714m (C=O), 1698w (C=C), 1162m (C=S). 1H NMR (300 MHz, d6-DMSO, 340 K)
δ 8.18 (d, 1H, JH7,H5 = 2.4 Hz, H-7), 7.85 (d, 1H JH5,H7 = 2.4 Hz, H-5). 13C NMR (75 MHz, d6-
DMSO, 340 K) δ 180.7 (C-2), 156.2 (C-4), 151.5 (C-8a), 138.3 (C-7), 130.4 (C-6), 125.4 (C-
5), 118.5 (C-4a), 110.0 (C-8). MS [M-H+] (C8H2BrClNO2S) calculated 289.8, observed
289.7.
4.1.3.3. 6-bromo-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 13
5-Bromo-2-hydroxybenzoic acid 7 (0.86 g, 4.0 mmol) was added freshly prepared
Ph3P(SCN)2 according to General Procedure A. The solids recrystallized from ethyl acetate to
afford 13 as a bright yellow solid (0.84 g, 82%), mp 246-249 °C (Lit. [22] 248 °C). νmax
(ATR)/cm-1 3070w, 2913w (N-H), 1678s (C=O), 1603m (C=C), 1166s (C=S). 1H NMR (300
MHz, d6-DMSO, 340 K) δ 7.98 (d, 1H, JH5,H7 = 1.8 Hz, H-5), 7.94 (dd, 1H, JH7,H8 = 8.7 Hz,
JH7,H5 = 2.1 Hz, H-7), 7.42 (d, 1H, JH8,H7 = 8.7 Hz, H-8). 13C NMR (75 MHz, d6-DMSO, 340
K) δ 181.6 (C-2), 156.5 (C-4), 154.5 (C-8a), 139.0 (C-7), 128.8 (C-5), 118.9 (C-8), 117.8 (C-
6), 117.6 (C-4a).
4.1.4. General Procedure B: Synthesis of 6-bromo, 8- bromo, and 8-bromo-6-chloro-2-
morpholino-1,3-benzoxazines 17-19
Sodium hydrogen carbonate (1.0 g, 11.90 mmol) was suspended in a 50 mL beaker
containing 20 mL of 1:1 RO water and 2-propanol. The appropriate 6- or 8-bromo-2-thio-1,3-
benzoxazine 11-13 (2.0 mmol) was added, the reaction mixture was warmed on a hotplate to
≈ 60 ºC and then removed from heat and allowed to cool to room temperature with stirring.
Iodomethane (0.5 mL, 8.0 mmol) was added drop-wise and allowed to stir at room

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temperature for 30 minutes or until a thick precipitation had formed. Morpholine (1.0 mL,
11.48 mmol) was added and the reaction mixture was stirred for an additional 3 hours. At the
end of the reaction the solid was then collected by vacuum filtration washed with RO water
(~30 mL), the crude solid then recrystallized from an appropriate solvent.
4.1.4.1. 8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17
8-bromo-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 11 (0.52 g) was allowed to react
with iodomethane and morpholine according to general procedure B. The crude material was
collected and recrystallized from abs. ethanol to give 17 as white crystals (0.53 g, 85% yield),
mp 232-235 °C. νmax (ATR)/cm-1 2978w, 2960w, 2858w (C-C), 1671m (C=O), 1590m (C=C)
1
1547s (C=N). H NMR (300 MHz, d6-DMSO, 340 K) δ 7.93 (dd, 1H, JH7,H6 = 7.8 Hz, JH7,H5
= 1.5 Hz, H-7), 7.85 (dd, 1H, JH5,H6 = 7.8 Hz, JH5,H7 = 1.5 Hz, H-5), 7.31 (t, 1H, JH6,H5 = 7.8
Hz, H-6), 3.73 (bs, 8H, 4 x CH2 of morpholine). 13C NMR (75 MHz, d6-DMSO, 340 K) δ
164.6 (C-4), 156.2 (C-2), 150.3 (C-8a), 137.1 (C-7), 126.5 (C-6), 126.2 (C-5), 119.1 (C-4a),
108.7 (C-8) 65.5 (C-3’), 44.5 (C-2’). MS [M+H+] (C12H12BrN2O3) calculated 311.0,
observed 310.9.
4.1.4.2. 8-bromo-6-chloro-2-morpholino-4H-1,3-benzoxazin-4-one 18
6-bromo-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 12 (0.59 g) was allowed to react
with iodomethane and morpholine according to general procedure B. The crude material was
collected and recrystallized from abs. ethanol to afford pure 18 as an off-white solid (0.53 g,
76% yield) mp 242-245 °C. νmax (ATR)/cm-1 3076w, 2924w, 2865w (C-H), 1588m (C=O),
1592s (C=C), 1667s (C=N). 1H NMR (300 MHz, d6-DMSO, 340 K) δ 8.06 (d, 1H, JH7,H5
=
1.2 Hz, H-7), 7.79 (d, 1H, JH5,H7 = 1.2 Hz, H-5), 3.73 (bs, 8H, 4 x CH2 of morpholine). 13C
NMR (75 MHz, d6-DMSO, 340 K) δ 163.6 (C-4), 156.1 (C-2), 149.5 (C-8a), 136.2 (C-7),

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130.0 (C-6), 125.5 (C-5), 119.8 (C-4a), 110.2 (C-8), 65.4 (C-3’), 44.6 (C-2’). MS [M+H+]
(C12H11BrClN2O3) calculated 345.0, observed 344.9.
4.1.4.3. 6-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 19
6-bromo-2-thioxo-2,3-dihydro-4H-1,3-benzoxazin-4-one 13 (0.52 g) was allowed to react
with iodomethane then morpholine according to general procedure B. The crude material was
collected and recrystallized from toluene and gave product 19 as pure white crystals (0.45 g,
73%), mp 192-195 °C. νmax (ATR)/cm-1 3065w, 2967w, 2912w (C-H), 1666m (C=O), 1611m
(C=C), 1549m (C=N). 1H NMR (300 MHz, d6-DMSO, 340 K) δ 7.93 (d, 1H, JH5,H7 = 2.4 Hz,
H-5), 7.83 (dd, 1H, JH7,H8 = 8.7 Hz, JH7,H5 = 2.4 Hz, H-7), 7.39 (d, 1H, JH8,H7 = 8.7 Hz, H-8)
3.73-3.68 (bm, 8H, 4 x CH2 of morpholine). 13C NMR (75 MHz, d6-DMSO, 340 K) δ 164.0
(C-4), 156.5 (C-2), 152.6 (C-8a), 136.8 (C-7), 128.9 (C-5), 119.1 (C-4a), 118.7 (C-8), 117.4
(C-6), 65.5 (C-3’), 44.4 (C-2’). MS [M+H+] (C12H12BrN2O3) calculated 311.0, observed
310.9.
4.1.5. General Procedure C: Synthesis of 6-aryl, 8- aryl, or 8-aryl-6-chloro-2-morpholino-
1,3-benzoxazines 20-22(Suzuki coupling)
6-bromo,8-bromo or 6-chloro-8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17, 18 or 19
(0.48 mmol) was combined with powdered potassium carbonate (200 mg, 1.44 mmol),
bis(triphenylphosphine)palladium(II) dichloride (3 mg, 0.04 mmol) and the appropriate
arylboronic acid (0.52 mmol) in a 25 mL Schlenk flask containing 1,4-dioxane (7 mL) and
RO water (3 ml) under nitrogen. The reaction mixture was heated at 50 °C for 16 h with
stirring before being cooled, evaporated to dryness under reduced pressure, 10 mL of RO
water was added. Extracted with chloroform (3 x 10 mL), the chloroform was dried using
anhydrous magnesium sulphate and evaporated under reduced pressure. Recrystallization
from an appropriate solvent yields pure 6- or 8- aryl-substituted-2-morpholino-4H-1,3-
benzoxazin-4-ones compounds 20-22.

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4.1.5.1. 8-(4-methoxyphenyl)-2-morpholino-4H-1,3-benzoxazin-4-one 20a
8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17 (150 mg) was allowed to react with 4-
methoxyphenylboronic acid (79 mg) according to general procedure C. Recrystallization
from 2-propanol gave 20a as a creamy white solid (111 mg, 68% yield), mp 169-172 °C. νmax
(ATR)/cm-1 2967w, 2914w, 2856w (C-H), 1664m (C=O), 1618m (C=C), 1551s (C=N). 1H
NMR (300 MHz, d6-DMSO, 340 K) δ 7.85 (dd, 1H, JH5,H6 = 7.8 Hz, JH5,H7 = 1.8 Hz, H-5),
7.66 (dd, 1H, JH7,H6 = 7.8 Hz, JH7,H5 = 1.8 Hz, H-7), 7.54 (d, 2H, JH10,H11 = 8.7 Hz, H-10 & H-
14), 7.41 (t, 1H, JH6,H5 = 7.8 Hz, H-6), 7.37 (d, 2H, JH11,H10 = 8.7 Hz, H-11 & H-13), 3.80 (s,
3H, O-CH3), 3.66-3.59 (bm, 8H, 4 x CH2 of morpholine). 13C NMR (75 MHz, d6-DMSO, 340
K) δ 165.6 (C-4), 159.5 (C-2), 156.8 (C-8a), 150.4 (C-12), 134.4 (C-7), 130.5 (C-10 & C-14),
128.9/127.3 (C-8/C-9), 125.5 (C-5), 125.4 (C-6), 117.8 (C-4a), 114.2 (C-11 & C-13), 65.4
(C-3’), 55.4 (O-CH3), 44.4 (C-2’). HRMS (C19H18N2O4) calculated 338.1267, observed
338.1262.
4.1.5.2. 8-(4-chlorophenyl)-2-morpholino-4H-1,3-benzoxazin-4-one 20b
8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17 (150 mg) was allowed to react with 4-
chlorophenylboronic acid (81 mg) according to general procedure C. Recrystallization from
2-propanol gave 20b as a pale yellow (89 mg, 54% yield), mp 205-209 °C. νmax (ATR)/cm-1
2971w, 2868w (C-H), 1671m (C=O), 1621m (C=C), 1561s (C=N). 1H NMR (300 MHz, d6-
DMSO, 340 K) δ 7.91 (dd, 1H, JH5,H6 = 7.8 Hz, JH5,H7 = 1.5 Hz, H-5), 7.71 (dd, 1H, JH7,H6
7.5 Hz, JH7,H5 = 1.8 Hz, H-7), 7.64 (d, 2H, JH10,H11 = 8.7 Hz, H-10 & H-14), 7.53 (d, 2H,
=
JH11,H10 = 8.7 Hz, H-11 & H-13), 7.45 (t, 1H, JH6,H5 = 7.5 Hz, H-6), 3.67-3.57 (bm, 8H, 4 x
CH2 of morpholine). 13C NMR (75 MHz, d6-DMSO, 340 K) δ 165.4 (C-4), 156.7 (C-2),
150.3 (C-8a), 134.6 (C-7), 134.0/133.3 (C-9/C-12), 131.1 (C-10 & C-14), 128.7 (C-11 & C-
13), 127.9 (C-8), 126.5 (C-5), 125.5 (C-6), 117.9 (C-4a), 65.4 (C-3’), 44.4 (C-2’). HRMS
(C18H15ClN2O3) calculated 342.0771, observed 341.0765

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4.1.5.3. 8-[4-(hydroxymethyl)phenyl]-2-morpholino-4H-1,3-benzoxazin-4-one 20c
8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17(150 mg) was allowed to react with 4-
(hydroxymethyl)phenylboronic acid (79 mg) according to general procedure C.
Recrystallization from 2-propanol gave 20c as an off white solid (102 mg, 63% yield), mp
178-181 °C. νmax (ATR)/cm-1 3382w (O-H), 2965w, 2917w, 2860w (C-H), 1670m (C=O),
1620m (C=C), 1560s (C=N). 1H NMR (300 MHz, d6-DMSO, 340 K) δ 7.88 (d, 1H, JH7,H6
7.8 Hz, H-7), 7.69 (d, 1H, JH5,H6 = 7.8 Hz, H-5), 7.56 (d, 2H, JH11,H10 = 8.1 Hz, H-11 & H-13),
7.44 (t, 1H, JH6,H5 = 7.2 Hz, H-5), 7.43 (d, 2H, JH10,H11 = 8.1 Hz, H-10 & H-14), 5.03 (t, 1H,
=
JH16,H15 = 5.4 Hz, H-16), 4.55 (d, 1H, JH15,H16 = 5.4 Hz, H-15), 3.65-3.58 (bm, 8H, 4 x CH2 of
morpholine). 13C NMR (75 MHz, d6-DMSO, 340 K) δ 165.5 (C-4), 156.8 (C-2), 150.4 (C-
8a), 142.8 (C-12), 134.6 (C-7), 133.4 (C-9), 129.1 (C-8), 128.9 (C-10 & C-14), 126.6 (C-11
& C-13), 125.9 (C-6), 125.4 (C-5), 117.8 (C-4a), 65.4 (C-3’), 62.8 (C-15), 44.4 (C-2’).
HRMS (C19H18N2O4) calculated 338.1267, observed 338.1265.
4.1.5.4. 8-(3-aminophenyl)-2-morpholino-4H-1,3-benzoxazin-4-one 20d
8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17(150 mg) was allowed to react with 3-
aminobenzeneboronic acid (93 mg) according to general procedure C. Recrystallization from
toluene gave 20d as a light brown powder 76 mg, 48% yield), mp 225-228 °C. νmax
(ATR)/cm-1 3456m, 3364m (N-H), 3249w, 2989w, 2861w (C-H), 1665m (C=O), 1617m
(C=C), 1561s (C=N). 1H NMR (300 MHz, d6-DMSO, 340 K) δ 7.86 (dd, 1H, JH5,H6 = 7.8 Hz,
JH5,H7 = 1.5 Hz, H-5), 7.64 (dd, 1H, JH7,H6 = 7.8 Hz, JH7,H5 = 1.5 Hz, H-7), 7.41 (t, 1H, JH6,H5
=
7.8 Hz, H-6), 7.11 (t, 1H, JH13,H14 = 7.8 Hz, H-13), 6.78 (d, 1H, JH10,H12 = 1.8 Hz, H-10), 6.72
(d, 1H, JH14,H13 = 7.8 Hz, H-14), 6.62 (dd, 1H, JH12,H13 = 7.8 Hz, JH12,H10 = 1.5 Hz, H-12), 4.98
(s, 2H, 11-NH2), 3.66-3.61 (bm, 8H, 4 x CH2 of morpholine). 13C NMR (75 MHz, d6-DMSO,
340 K) δ 165.6 (C-4), 156.8 (C-2), 150.3 (C-8a), 148.8 (C-11), 135.6 (C-9), 134.3 (C-7),
130.0 (C-8), 129.0 (C-13), 125.7 (C-5), 125.4 (C-6), 117.8 (C-4a), 116.9 (C-14), 114.8 (C-

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10), 114.0 (C-12), 65.5 (C-3’), 44.4 (C-2’). HRMS (C18H17N3O3) calculated 323.1270,
observed 323.1270.
4.1.5.5. 4-[2-morpholino-4-oxo-4H-1,3-benzoxazin-8-yl]benzamide 20e
8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17(150 mg) was allowed to react with 4-
aminocarbonylphenylboronic acid (86 mg) according to general procedure C.
Recrystallization from 2-propanol gave 20e as an off white solid (105 mg, 63% yield), mp
244-247 °C. νmax (ATR)/cm-1 3361w (N-H), 3169w, 2869w (C-H), 1656m (C=O), 1621m
(C=C), 1548s (C=N). 1H NMR (300 MHz, d6-DMSO, 340 K) δ 7.97 (d, 2H, JH11,H10 = 8.4 Hz,
H-11 & H-13), 7.93 (dd, 1H, JH5,H6 = 7.8 Hz, JH5,H7 = 1.5 Hz, H-5), 7.74 (dd, 1H, JH7,H6 = 7.5
Hz, JH7,H5 = 1.5 Hz, H-7), 7.69 (d, 2H, JH10,H11 = 8.4 Hz, H-10 & H-14), 7.46 (t, 1H, JH6,H7
=
7.5 Hz, H-6) 3.66-3.58 (bm, 8H, 4 x CH2 of morpholine), 3.09 (bs, 2H, 15-NH2). 13C NMR
(75 MHz, d6-DMSO, 340 K) δ 167.6 (C-4), 165.3 (C-15), 156.7 (C-2), 150.4 (C-8a), 137.8
(C-9), 134.7 (C-7), 134.2 (C-12), 129.1 (C-10 & C-14), 128.4 (C-8), 127.8 (C-11 & C-13),
126.6 (C-5), 125.5 (C-6), 117.9 (C-4a), 65.5 (C-3’), 44.4 (C-2’).HRMS (C18H17N3O3)
calculated 351.1219, observed 351.1219.
4.1.5.6. 2-morpholino-8-(pyridin-3-yl)-4H-1,3-benzoxazin-4-one 20f
8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17 (150 mg) was allowed to react with 3-
pyridinylboronic acid (64 mg) according to general procedure C. Recrystallization from 2-
propanol gave 20f as a light brown powder (46 mg, 31% yield), mp 182-185 °C. νmax
(ATR)/cm-1 3028w, 2962w, 2857w (C-H), 1670m (C=O), 1619m (C=C), 1551s (C=N). 1H
NMR (300 MHz, d6-DMSO, 340 K) δ 8.81 (d, 1H, JH10,H12 = 1.8 Hz, H-10), 8.61 (dd, 1H,
JH12,H13 = 4.8 Hz, JH12,H10 = 1.5 Hz, H-12), 8.43 (dd, 1H, JH14,H13 = 7.8 Hz, JH14,H12 = 1.8 Hz,
H-14), 7.95 (dd, 1H, JH5,H6 = 7.8 Hz, JH5,H7 = 1.8 Hz, H-5), 7.77 (dd, 1H, JH7,H6 = 7.8 Hz,
JH7,H5 = 1.8 Hz, H-7), 7.50 (t, 1H, JH13,H14 = 7.2 Hz, H-13), 7.48 (t, 1H, JH6,H5 = 7.8 Hz, H-6),

ACCEPTED MANUSCRIPT
3.66-3.56 (bm, 8H, 4 x CH2 of morpholine). 13C NMR (75 MHz, d6-DMSO, 340 K) δ 165.2
(C-4), 156.6 (C-2), 150.6 (C-8a), 149.6 (C-10), 149.3 (C-12), 136.7 (C-14), 134.7 (C-7),
131.0 (C-9), 126.8 (C-5), 126.0 (C-8), 125.6 (C-6), 123.6 (C-13), 117.9 (C-4a), 65.4 (C-3’),
44.4 (C-2’). HRMS (C17H15N3O3) calculated 309.1113, observed 309.1111.
4.1.5.7. 2-morpholino-8-(thiophen-2-yl)-4H-1,3-benzoxazin-4-one 20g
8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17(150 mg) was allowed to react with 2-
thienylboronic acid (67 mg) according to general procedure C. Recrystallization from 2-
propanol gave 20g as a light brown solid (57 mg, 38% yield), mp 141-144 °C. νmax
(ATR)/cm-1 3080w, 2963w, 2860w (C-H), 1671m (C=O), 1619m (C=C), 1567s (C=N). 1H
NMR (300 MHz, d6-DMSO, 340 K) δ 7.88 (m, 2H, H-5 & H-7), 7.82 (dd, 1H, JH11,H12 = 5.1
Hz, JH11,H13 = 0.9 Hz, H-11), 7.55 (dd, 1H, JH13,H12 = 3.6 Hz, JH13,H11 = 0.9 Hz, H-13), 7.42 (t,
1H, JH6,H7 = 7.8 Hz, H-6), 7.20 (m,1H, JH12,H11 = 5.1 Hz, JH13,H12 = 3.6 Hz, H-12), 3.72 (m,
8H, 4 x CH2 of morpholine). 13C NMR (75 MHz, d6-DMSO, 340 K) δ 165.1 (C-4), 156.8 (C-
2), 149.8 (C-8a), 135.6 (C-9), 133.8 (C-7), 127.9, 127.8, 127.7 (C-11, C-12 & C-13), 126.2
(C-6), 125.6 (C-5), 122.3 (C-8), 118.2 (C-4a), 65.5 (C-3’), 44.8 (C-2’). HRMS
(C16H14N2O3S) calculated 314.0725, observed 314.0721. HRMS (C16H14N2O3S) calculated
314.0725, observed 314.0721.
4.1.5.8. 2-morpholino-8-(thiophen-3-yl)-4H-1,3-benzoxazin-4-one 20h
8-bromo-2-morpholino-4H-1,3-benzoxazin-4-one 17 (150 mg) was allowed to react with 3-
thienylboronic acid (67 mg) according to general procedure C. Recrystallization from 2-
propanol gave 20h as fine light brown needles (71 mg, 47% yield), mp 202-205 °C. νmax
(ATR)/cm-1 3082w, 2985w, 2861w (C-H), 1661m (C=O), 1619m (C=C), 1543s (C=N). 1H
NMR (300 MHz, d6-DMSO, 340 K) δ 7.86 (dd, 1H, JH5,H6 = 7.8 Hz, JH5,H7 = 1.8 Hz, H-5),
7.83 (dd, 1H, JH10,H12 = 3.0 Hz, JH10,H13 = 2.7 Hz, H-10), 7.80 (dd, 1H, JH7,H6 = 7.8 Hz, JH7,H5
=