Synthesis of linear and angular aryl-morpholino-naphth-oxazines, their DNA-PK, PI3K, PDE3A and antiplatelet activity

Article abstract of DOI:10.1016/j.bmcl.2016.10.003

To continue our study of 2-morpholino-benzoxazine based compounds, which show useful activity against PI3K family enzymes or antiplatelet activity, we designed and synthesized a series of linear 6.7-fused, 5,6-angular fused and 7,8-angular fused-aryl-morpholino-naphth-oxazines. The compounds were prepared from substituted 2-hydroxynaphthoic acid to give the corresponding thioxo analogues 8, 9, 15 and 19. The thioxo products were then converted to the morpholino substituted analogue. The aryl group was introduced by Suzuki coupling of bromo precursors. The products were evaluated for activity at PI3K family enzymes and as platelet aggregation inhibitors and compared to reported unsubstituted analogues. The linear 6.7-fused product 13a and 13b were moderated potent but selective PI3Kδ isoform inhibitors (IC₅₀?=?7.7 and 5.61?μM). Good antiplatelet activity was noticed for the angular 7,8-fused compounds 22a, b, k and l with IC₅₀?=?3.0,14.0, 2.0 and 5.0?μM respectively. The antiplatelet activity is independent of PDE3.

Full text of DOI:10.1016/j.bmcl.2016.10.003

Accepted Manuscript
Synthesis of linear and angular aryl-morpholino-naphth-oxazines, their DNA-
PK, PI3K, PDE3A and antiplatelet activity
Rick Morrison, Zhaohua Zheng, Ian G. Jennings, Philip E. Thompson, Jasim
M.A. Al-Rawi
PII:
S0960-894X(16)31031-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.003
BMCL 24306
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 August 2016
1 October 2016
4 October 2016
Please cite this article as: Morrison, R., Zheng, Z., Jennings, I.G., Thompson, P.E., Al-Rawi, J.M.A., Synthesis of
linear and angular aryl-morpholino-naphth-oxazines, their DNA-PK, PI3K, PDE3A and antiplatelet activity,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.003
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Synthesis of linear and angular aryl-morpholino-naphth-oxazines, their DNA-PK,
PI3K, PDE3A and antiplatelet activity
Rick Morrisona, Zhaohua Zhengb, Ian G. Jenningsb, Philip E. Thompsonb and Jasim M. A.
Al-Rawi*a
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.
__________________________________________________________________
* Corresponding author.
Tel.: +61 3 54 44 7364; fax: +61 3 54 44 7476.
E-mail addresses: rmmorrison@students.latrobe.edu.au (R. Morrison),
joyce.zheng@monash.edu (Z. Zheng), Ian.Jennings@monash.edu (I. Jennings),
Philip.Thompson@monash.edu (P. Thompson), j.al-rawi@latrobe.edu.au (J. Al-Rawi),

Abstract
To continue our study of 2-morpholino-benzoxazine based compounds, which show useful
activity against PI3K family enzymes or antiplatelet activity, we designed and synthesized a
series of linear 6.7- fused, 5,6- angular fused and 7,8-angular fused-aryl-morpholino-naphath-
oxazines. The compound were prepared from substituted 2-hydroxynapthoic acid to give the
corresponding thioxo analogues 8, 9, 15 and 19. The thioxo products were then converted to
the morpholino substituted analogue. The aryl group was introduced by Suzuki coupling of
bromo precursors. The products were evaluated for activity at PI3K family enzymes and as
platelet aggregation inhibitors and compared to reported unsubstituted analogues. The linear
6.7- fused product 13a and 13b were moderated potent but selective PI3Kδ isoform inhibitors
(IC50 =7.7 and 5.61 µM). Good antiplatelet activity was noticed for the angular 7,8- fused
compounds 22a, b, k and l with IC50 = 3.0,14.0, 2.0 and 5.0 µM respectively. The antiplatelet
activity is independent of PDE3.
Keywords: 6.7- fused, 5,6- angular fused and 7,8-angular fused –aryl-morpholino-naphath-
oxazines; DNA-PK, PI3K, PDE3A antiplatelet activity

Compounds of the 2-morpholino-1,3-benzoxazine series have shown a range of biological
activities and have in particular shown isosterism with corresponding 2-morpholino-
chromones, for which there is also a long history of biologically active compounds including
those that target the Phosphoinositide 3-kinase (PI3 K) superfamily. While isosteric, these
benzoxazines show a markedly different selectivity profile to the corresponding chromones,
which may result in unique pharmacological properties.[1-3] For example, we recently
showed that the dibenzo[b,d]thiophene analogue LTURM34 shows equivalent inhibitory
potency versus DNA-Dependent Protein Kinase (DNA-PK) to the corresponding chromone,
NU7441 but is much more selective, with relatively poor inhibition of Class I PI3Ks.[4]
In extending our study of these comparative properties we turned our attention to linear and
angular benz-fused versions of these compounds. In the chromone series, 2-morpholino-
benzo-chromenones have also proved to be ATP-competitive DNA-PK inhibitors, including
compounds 2-morpholino-4H-benzo[g]chromen-4-one 1a, 3-morpholino-1H-
benzo[f]chromen-1-one 2a, and 2-morpholino-4H-benzo[h]chromen-4-one 3a. These
compounds show robust DNA-PK inhibition with IC50s of 0.39 µM, 1.25 µM and 0.23 µM
respectively [5].

Figure 1 Structures of some morpholino benzo- and naphth-oxazines or benzo-chromenones
previously prepared as DNA-PK, PI3K and antiplatelet inhibitors
The compounds have also been investigated with respect to Class I PI3K activity, and were
included in Vlahos’ seminal description of LY294002 as a PI3K inhibitor.[6] In addition,
Roma and co-workers reported the anti-platelet activity of 2a, with an IC50 of ~ 300 μM for
collagen-induced platelet aggregation.[7] [8]
We had previously have described the benzoxazine counterparts, 1b, 2b and 3b which
showed comparable anti-DNA-PK inhibition, with IC50s of 6.5, 0.72 and 0.42 µM
respectively and extended this study to 2-morpholino-O-substituted linear or angular naphth-
oxazine compounds such as 4a-c and 5a-b. Compound 4a, 4b, 4c and 5b showed potent
DNA-PK inhibition (IC50 96, 330, 190 and 760 nM respectively) and 4a, 4b and 5b did not

show PI3K α, β, γ, and δ activity at concentrations <100 µM. Only PI3K β activity was
observed for product 5a, R = pyridine-2-ylmethoxy (IC50 = 8.94 µM) [1] [9]. The compounds
were also assessed as inhibitors of collagen-induced platelet aggregation but apart from 5a
(IC50 55 μM) and 4b (IC50 85 μM), the compounds showed only modest inhibitory potency in
these assays (IC50 100 μM or greater).
Carrying on those initial studies, we have extended the synthesis of linear and angular aryl-
morpholino-naphth-oxazine to include compounds bearing additional aryl substituents
derived through Suzuki coupling of the bromo-substituted precursors.
The inhibition of DNA-PK, PI3K, PDE3A and antiplatelet activity of the products were
studied and while, in general, activities were much poorer than the parent compounds, in the
case of platelet aggregation compounds were identified with IC50 values as low as 2 μM.
The linear 10-substituted linear “6,7-fused” substituted-naphth-oxazines 13, angular 8-
substituted “5,6-fused” substituted-naphth-oxazines 17 and 6-substituted angular “7,8”-fused
6-bromo-naphth-oxazines 22 were prepared according to Schemes 1-3 and full experimental
details are reported in the supporting information of this work
Synthesis of 2-morpholino- substituted-naphth-oxazine 13a-c (Scheme 1)
According to scheme 1, 4-bromo-3-hydroxy-2-naphthoic acid 6, was synthesised from the
reaction of bromine in chloroform with the 3-hydroxy-2-naphthoic acid according to the
previously reported method [10,11]. Compound 6 was allowed to react with the freshly
prepared triphenylphosphine thiocyanate according to the previously reported method and
gave the substituted naphth-oxazine 8 [12,13]. The 6-bromo- 2-thioxo-2,3-dihydro-4H-
naphth[1,3]oxazin-4-one 8 was then treated with iodomethane in the presence of NaHCO3

according to the previously reported method[14] to give the corresponding 2-methylthio-
substituted-naphth[2,1-e][1,3]oxazine 10. The 2-methylthio-intermediate was reacted in situ
with morpholine and gave 2-morpholino-substituted-naphth-oxazine 12.
Initial attempts at Suzuki coupling with 12 gave poor results. Firstly, employing the
previously reported method [14] for coupling of phenylboronic acid with 12 powdered
potassium carbonate, and tetrakis(triphenylphosphine)palladium(0) as the catalyst in 1,4-
dioxane yielded no reaction after 16 hours at 80 °C in a Schlenk flask under nitrogen. Further
reactions under the same condition were carried out using palladium (II) acetate or [1,1′-
Bis(diphenylphosphino)ferrocene]dichloropalladium(II) as the catalyst were unsuccessful
also. Changing to 3:1 1,4-dioxane/water in order to improve solubility of the potassium
carbonate, gave successful substitution of the bromine of compound 12 however hydrolysis at
C-2 took place with the loss of morpholine and CO2 resulted in the formation 3-hydroxy-4-
phenyl-2-naphth-amide. Changing the catalyst to bis(triphenylphosphine)palladium(II)
dichloride and lowering the reaction temperature to 50 °C gave the expected product but
compound 12 was not completely consumed with 25-50% of unreacted 12 retained, which
could not be removed by recrystallization (ethanol) or column chromatography.

Comp. 7, 9, and 13
Ar
Reaction conditions: i, and viii 6 and 12 in water/dioxane (30:70) with K2CO3, aryl-boronic
acid and Pd(PPh3)2Cl2 heated at 50 °C for 16 hours. ii and iii 6 and 7a-c in dry DCM with
Ph3PSCN. Iv and v the compounds 8 and 9a-c with CH3I and NaHCO3 in water/propanol
(50/50). vi and vii, to solution the intermediate –SMe morpholine was added at r.t.
Scheme 1. Synthesis of linear 6,7-fused substituted-naphth-oxazine 8, 9, 2-methylthio-
substituted-naphth-oxazine 10 and 11, 2-morpholino- substituted-naphth-oxazine 12 and 13
As an alternative route, 6 was dissolved in sodium hydroxide solution in a quartz tube. The
appropriate aryl-boronic acid, powdered potassium carbonate 1,4-dioxane (as a solvent) and
tetrakis(triphenylphosphine)palladium were then added. The mixture was heated for 1 hour at
100 ºC under Microwave condition. After workup 4-Aryl-3-hydroxy-2-naphthoic acid 7a-c
was isolated in high yield (70-98%). Following the general route above, 7a-c were treated
with triphenylphosphine thiocyanate to give 9a-c. Finally, S-methylation followed by the
treatment in situ with morpholine gave 2-morpholino-substituted-naphth-oxazines 13a-c. The

structure of the morpholino products 12 and 13a-c, were elucidated from the analysis of their
IR, 1H, 13CNMR and CHN microanalysis.
Synthesis 8- aryl- 2-morpholino-naphth-oxazine 17a-c (Shame 2) and 6-aryl-2-morpholino-
naphth-oxazine 22a-l (Shame 3)
The synthesis of the angular analogues proceeded more readily and could be achieved with
application of the general Suzuki coupling reaction as the final step under the conditions
described above using bis(triphenylphosphine)palladium(II) dichloride as a catalyst.
As such 6-bromo-2-morpholino-1,3-benzoxazin-4-ones 16 was prepared by the same
sequence as for compound 12 then reacted with aryl-boronic acid and gave the
corresponding 3-morpholino-8-phenyl-1H-naphth[1,2-e][1,3]oxazin-1-ones 17a-e (Scheme 2)
and 8-bromo-3-morpholino-naphth-1,3-oxazines 21 was prepared and gave 2-morpholino-6-
phenyl-4H-naphth[2,1-e][1,3]oxazin-4-ones 22a-l (Scheme 3).
Comp.
Ar
17a
17b
17c
17d
17e
Reaction conditions: i 14 dry DCM with Ph3PSCN ii, 15 in dioxane and morpholine at rt for
16 h iii 16 in water/dioxane (30:70) with K2CO3, boronic acid and Pd(PPh3)2Cl2 heated at 50
°C for 16 hours.
Scheme 2. Synthesis of angular 5,6-fused substituted-naphth-oxazine 15, angular 8-bromo-
2-morpholino-naphth-oxazine 16 and angular 8- aryl- 2-morpholino- naphth-oxazine 17a-c
The structures of the new 6- and 8-aryl-morpholino-naphth-1,3-oxazine analogues 17a-c and
22a-l were confirmed by the analysis of their IR, 1H and 13C NMR spectra in addition to the
high resolution mass spectrometry.

Compound
22a
Ar
Compound
22e
Ar
Compound
22i
Ar
22b
22f
22j
22c
22d
22g
22h
22k
22l
Reaction conditions: i, 18 in dry DCM with Ph3PSCN and. ii 19 with CH3I and NaHCO3 in
water/propanol (50/50). iii to solution the intermediate –SMe 20 morpholine was added at r.t
iv 20 in water/dioxane (30:70) with K2CO3, boronic acid and Pd(PPh3)2Cl2 heated at 50 °C
for 16 hours.
Scheme 3. Synthesis of angular 7,8-fused 6-bromo--naphth-oxazine 18, 6-bromo, 2-
methylthio-naphth-oxazine 20, 6-bromo-2-morpholino-naphth-oxazine 21 and 6-aryl-2-
morpholino-naphth-oxazine 22a-l
The assignment of the 1H NMR spectra of compounds 13a-c, 17a-c, and 22a-l were
achieved using 1H-1H correlation spectroscopy (COSY) and hydrogen-hydrogen homo
decoupling (HHHD) while the protonated carbon signals of the 13C NMR spectra were
assigned using heteronuclear single quantum coherence spectroscopy (HSQC)using the
assigned proton NMR spectra.
Biological Activity

The 20 target compounds were evaluated as inhibitors of PI3K, DNA-PK and PDE3 well as
anti-platelet aggregation.
Full data of the different assays are provided in the supporting information, and attention is
focussed on key compounds of interest.
In this series, only the linear benz-fused analogues 13a-c showed > 50% inhibition at 10 μM
or lower. The 2-morpholino-10-phenyl-naphth[2,3-e][1,3]oxazin-4-one 13a showed good
improvement (relative to 1b) in the inhibition of PI3Kα and PI3Kδ isoforms with IC50 43.8
and 7.7 µM respectively. The 10-(4-methoxyphenyl)-analogue 13b was relatively selective
for PI3Kδ with an IC50 of 5.6 µM. These compounds are better inhibitors than the
unsubstituted example 1b, which shows the contribution of the pendant aryl ring in a similar
way to the literature data for LY294002 or the equivalent benzoxazine analogue.[6] The
angular compounds were much poorer PI3K inhibitors, with no compound reaching 50%
inhibition activity of PI3K at 10 µM concentration. The 5,6-fused compounds were especially
poor PI3K inhibitors. While in previous work, PI3Kβ inhibition was observed for the 7,8-
fused product 5a, R = pyridine-2-ylmethoxy (IC50 = 8.94 µM) [9], in this work, where the
aryl substitution was directly attached to the naphth-1,3-oxazine ring activity was lost.
These results are probably readily explained by reference to the crystal structure of
LY294002 [15] and other modelling [16] whereby, the presence of the fused ring in an
angular fashion, plus an additional aryl substituent will clash with the wall of the binding
pocket. The linear fused ring will be more readily accommodated.
The results against DNA-PK were even more dramatic. While we previously reported good to
moderate DNA-PK activity was observed for linear and angular 7,8-fused compounds with
various substitutions, [1] in this series there were no compounds with useful potency. It was
noted, that the assay results for compounds 1b, 2b and 3b gave much higher IC50 values

compared to our previous reports also, but of all the new compounds reported here only 13b
(IC50 = 13.9 μM) showed comparable activity to its unsubstituted counterpart, 1b.
Finally, but perhaps most promisingly, the 7,8-,fused (angular) 6-aryl-substituted
morpholino-naphth[1,3]oxazin-4-one compounds 22a, b, k and l proved to be relatively
potent anti-platelet compounds with 22k giving an IC50 of 2.0 µM. Thus the presence of an
aryl substitution results in a major increase in potency even compared to previously reported
compounds 4a-c and 5a, b. As the inhibition of platelet aggregation by morpholino-
chromones has previously been attributed to PDE3 inhibition,[17] we examined this activity,
but again the series were poor PDE3 inhibitors. There remains a yet to be identified
mechanism by which morpholino-chromones or their isosteres inhibit platelet
aggregation.[18]

Table 1 Selected Assay Data, Percentage inhibition (IC50), for compounds 1-3b, 13a-c and
22a, b, k, l against PI3K, DNA-PK, PDE3 and antiplatelet aggregation at 10 µM
concentration
Comp.
R
p110 p110 p110 p110
Inhibition % at 10 µM (IC50 µM )
DNA-PK PDE3 Agg.
IC50
N3-LY294002
1b
4.1a
3.4a
1.5a
0.95a
0.3a
(12.5)c
(78.1)
(11.9)
n.d.
n.d
32
40b
H
-25
-10
-4
-10
> 80
> 80
> 80
13a
13b
(43.8) (>50) (>50) (7.7)
(>50) (>50) (>50) (5.6)
6
8
0
3
52
13c
2b
24
n.d
13
18
-4
> 80
> 80
n.d.
(>100)
(38.8)c
0.0
H
4
23
-3
2
1
8
-1
-2
17a
17b
17c
3b
0.0
-5
0.0
-1
-2
0.0
4
-0.0
-1
n.d.
n.d.
8.1
n.d.
(10.0)c
n.d
> 80
H
22
15
12
35
20
10
42
34
17
3
9
22a
22b
22
19
15
26
3.0
14.0
9
22
24
0
22k
24
11
2.0
22l
23
11
12
0
0.0
30
5.0
a Data taken from [2] bData taken from [3] cIC50 Previously reported values for 1b 6.5 μM, 2b 0.72 μM, 3b 0.42
μM [1];

Series of linear 6.7- fused, 5,6- angular fused 7,8-angular fused bromo-morpholino-naphath-
oxazines 12, 16 and 21 were synthesised from the reaction of bromo-substituted 2-hydroxy-
naphthoic acid 6, 14 and 18 respectively with (Ph)3(SCN)2, S-methylation then with
morpholine. The aryl-substituted analogue 17 and 22 were prepared according to Suzuki
coupling general procedure C (Supplementary) from the angular bromo-morpholino-naphth-
oxazine 16 and 21 with the relevant aryl-boronic acid, potassium carbonate in dioxane /water
for 16 h at 50 °C. However adopting this procedure on linear bromo-morpholino-
benzoxazines 12 did not produced the aryl substitute analogue 13 which could be as result of
steric hindered of the 10 position in the linear structure 12. The linear 6.7- fused product 13a
and 13b were moderated potent but selective PI3Kδ isoform inhibitors (IC50 =7.7 and 5.61
µM). Good antiplatelet activity was noticed for the angular 7,8-fused compounds 22a, b, k
and l with IC50 = 3.0, 14.0, 2.0 and 5.0 µM respectively. It was found that the antiplatelet
activity is independent of PDE3. Unlike the benzoxazines analogue the bulky 8- aryl
substitution on the naphtha-oxazines is not tolerated by DNA-PK and PI3K.
Supplementary data
Supplementary data related to this article can be found at
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Graphical Abstract
Synthesis of linear and angular aryl-morpholino-naphth-oxazines, their DNA-PK,
PI3K, PDE3A and antiplatelet activity
Rick Morrisona, Zhaohua Zhengb, Ian G. Jenningsb, Philip E. Thompsonb and Jasim M. A.
Al-Rawi*a

Highlights


Suzuki coupling novel Linear and angular fused-aryl-morpholino-naphath-oxazines
Linear 6.7- fused 13a and 13b selective PI3Kδ isoform inhibitors (IC50 =7.7 and 5.61
µM)

Antiplatelet activity for the angular 7,8- fused 22a, b, k and l with IC50 = 3.0,14.0, 2.0
and 5.0 µM