Discovery of 4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as potent and orally active PI3K/mTOR dual inhibitors

Accepted Manuscript

Discovery of 4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as potent and

orally active PI3K/mTOR dual inhibitors

Guoyi Yan, Chunlan Pu, Suke Lan, Xinxin Zhong, Meng Zhou, Xueyan Hou, Jie Yang,

Huifang Shan, Lifeng Zhao, Rui Li

PII:

S0223-5234(19)30546-X

DOI:

https://doi.org/10.1016/j.ejmech.2019.06.021

EJMECH 11424

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 11 April 2019

Revised Date: 5 June 2019

Accepted Date: 7 June 2019

Please cite this article as: G. Yan, C. Pu, S. Lan, X. Zhong, M. Zhou, X. Hou, J. Yang, H. Shan, L.

Zhao, R. Li, Discovery of 4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as potent and orally

active PI3K/mTOR dual inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://

doi.org/10.1016/j.ejmech.2019.06.021.

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ACCEPTED MANUSCRIPT

Discovery of 4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives

as potent and orally active PI3K/mTOR dual inhibitors

‡

Guoyi Yan^a,, Chunlan Pu^a,, Suke Lan^a, Xinxin Zhong^a, Meng Zhou^b, Xueyan Hou^c, Jie

Yang^a, Huifang Shan^a, Lifeng Zhao^d,and Rui Li ^a,

* *.

Table of Contents graphic

ACCEPTED MANUSCRIPT

Discovery of 4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives

as potent and orally active PI3K/mTOR dual inhibitors

‡

Guoyi Yan^a,, Chunlan Pu^a,, Suke Lan^a, Xinxin Zhong^a, Meng Zhou^b, Xueyan Hou^c, Jie

Yang^a, Huifang Shan^a, Lifeng Zhao^d,, Rui Li ^a,

* *.

a

State Key Laboratory of Biotherapy, Collaborative Innovation Center of Biotherapy and Cancer

Center, West China Hospital of Sichuan University, Chengdu, 610041, China

^bSichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu 610052, China

^cEngineering Research Center for the Development and Application of Ethnic Medicine and TCM,

Ministry of Education & State Key Laboratory of Functions and Applications of Medicinal Plants,

Guizhou Medical University, Guiyang 550004, Guizhou, China

^dCollege of Pharmacy, Xinxiang Medical University, Xinxiang, Henan 453003, P.R. China

‡

These two authors contribute equally to this work

*Corresponding authors:

Lifeng Zhao*, E-mail: lifengzhao@scu.edu.cn.

Rui Li*, E-mail: lirui@scu.edu.cn;

Notes

The authors declare no competing interest.

ABSTRACT

PI3K/Akt/mTOR signaling pathway plays an important role in cancer cell growth and survival. In

this study, a new class of molecules with skeleton of 4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one

were designed and synthesized targeting this pathway. Bioassays showed that, among all the

molecules, 8d-1 was a pan-class I PI3K/mTOR inhibitor with an IC₅₀of 0.63nM against PI3Kα. In

a wide panel of protein kinases assays, no off-target interactions of 8d-1 were identified. 8d-1 was

orally available, and displayed favorable pharmacokinetic parameters in mice (oral bioavailability

of 24.1%). In addition, 8d-1 demonstrated significant efficiency in Hela/A549 tumor xenograft

models (TGI of 87.7% at dose of 50mg/kg in Hela model) without causing significant weight loss

and toxicity during 30 days treatment. Based on the bioassays, compound 8d-1 could be used as

an anti-cancer drug candidate.

Keywords

PI3K/mTOR; Structural optimization; Dual inhibitor; Cancer treatment

1. INTRODUCTION

PI3K/AKT/mTOR pathway is among the most frequently dysregulated pathways in many human

cancers [1-4]. PI3Ks (Phosphatidylinositol 3-kinases) can be subdivided into four classes

according to their sequence, homology, and substrate preferences: Class I, II and III are lipid

kinases while class IV are Ser/Thr protein kinases [5]. Class IA PI3Ks (PI3Kα, PI3Kβ, and PI3Kδ)

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are heterodimers consisting of a p110 catalytic subunit (p110α, p110β, and p110δ) and a

regulatory subunit, while Class IB subtype (PI3Kγ) is comprised of a catalytic p110γ and a

regulatory p101 subunit [6]. Class I PI3Ks catalyze the phosphorylation of phosphatidylinositol

diphosphate (PIP2) to generate its corresponding triphosphate (PIP3), which activate the

downstream serine-threonine kinase Akt. Subsequently, the phosphorylation of AKT (pAKT) leads

to the activation of the mammalian target of rapamycin (mTOR), a member of the

phosphatidylinositol like kinase (PIKK) family with a high degree of active site similarity with

PI3Ks, which in turn promotes increased protein synthesis and cell growth. Many molecules

targeting this pathway have been reported, compared with individually targeting PI3K or mTOR,

dual inhibition of PI3K and mTOR has been proposed to represent a more effective approach for

cancer therapy [7]. Several molecules with different scaffolds demonstrated dual inhibition

targeting PI3K/mTOR are shown in Figure 1 [8-24].

Figure 1. Examples of PI3K/mTOR dual inhibitors.

Among the mentioned molecules, many scaffolds have been employed as core structure, such as

quinoline in BEZ235, GSK2126458, 16d and 17e; other structures such as

imidazo[1,2-b]pyridazine,

thieno[3,2-d]pyrimidine,

4H-pyrido[1,2-a]pyrimidin-4-one,

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1,3,5-triazine,

3H-[1,2,3]triazolo[4,5-d]pyrimidine,

pyrido[3,2-d]pyrimidine,

and

1,3-dihydro-2H-imidazo[4,5-c][1,5]naphthyridin-2-one were also frequently used in PI3K/mTOR

dual inhibitors design. As can be concluded, many of the core structures are planar structure, to

improve the molecular solubility and get potent inhibitors, here we report a series of

2H-benzo[b][1,4]oxazin-3(4H)-one

derivatives

as

PI3K/mTOR

dual

inhibitors.

2H-benzo[b][1,4]oxazin-3(4H)-one demonstrates extensive biological and pharmacological

activity, such as antimicrobial, anticancer and antithrombotic activities (Figure S1) [25-28], it was

used as core structure in PI3K/mTOR dual inhibitors design; non-planar structure of

2H-benzo[b][1,4]oxazin-3(4H)-one may lead to the improvement of molecular solubility and

potent inhibition. In this core structure, N-4 and C-6 position are attractive positions for

investigation, different fragments were introduced to the two positions for SAR discussion to get

potent inhibitors (Figure 2).

Figure 2. Design and optimization of molecules based on known PI3K/mTOR inhibitors.

On the C-6 position, sulfonamide fragments were employed for its remarkable potency in vitro

and in vivo. Quinoline is another frequently used moiety in PI3K/mTOR inhibitor design, such as

BEZ235, which is a well learned PI3K/mTOR inhibitor [19, 29], quinoline was placed on C-6

position to get better inhibitors. On the N-4 position, chain alkanes have been introduced to get

PI3Kα inhibitors [30], based on the activity of reported molecules in Figure 1, we supposed that

aromatic rings such as benzene or substituted benzene may be more beneficial, meanwhile,

saturated alkanes/cyclanes such as cyclohexyl, isopropyl and cyclopropyl were also introduced for

SAR discussion. 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives were synthesized and the

bioactivities were assayed in vitro and in vivo. Molecules with scaffold of

4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one showed advantages over other compounds, and

among the molecules, 8d-1 with potent anticancer activity was obtained.

2. RESULTS AND DISCUSSION

2.1 Synthetic Chemistry

The chemistry efforts to obtain 8d-1 involved the preparation of a series of analogues, all of

synthetic routes are outlined in the following schemes.

Scheme 1. Synthesis of sulfonamide fragments 2a-2c

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(a) 4-fluorobenzenesulfonyl chloride, DMAP, pyridine, 0 ;

Scheme 1 depicted the synthesis of sulfonamide fragment. 4- fluoro sulfonyl chloride and 1 were

stirred in pyridine overnight with DMAP as catalyst to afford intermediate molecules 2a-2c.

Scheme 2. Synthesis of 6 and 8 series.

(a) Chloroacetyl chloride, NaHCO₃, THF,0-4℃; (b) K₂CO₃, THF, ref; (c) Cu(OAc)₂, Et₃N, THF,

60℃; (d):Suzuki coupling, boronic acid/ester, PdCl₂(dppf), KOAC, dioanxe, 80℃.

In the construction of core structure, firstly, amide condensation reaction was conducted to afford

intermediate 4, subsequently, intramolecular ring forming reaction was conducted to afford

2H-benzo[b][1,4]oxazin-3(4H)-one (compound 5) in 70% yield. Furthermore, 5 was reacted with

substituted benzoic boric acids in THF solutions, copper acetate was employed as catalytic agent,

Et₃N and 4 Å molecular sieve were added and heated over 60℃for 5h, the intermediate of N-

substituted benzoxazine was obtained (compound 7), the yield was about 50%. The target

compounds (6a, 8a-8f) were obtained by the coupling reaction of compound 5/7 and intermediate

molecules 2a-2c, 3-bromoquinoline or 4-bromophenol.

Scheme 3. Synthesis of N-4-alkane substituted derivatives

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(a) Chloroacetyl chloride, K₂CO₃, CH₃CN, 0 -r.t.; (b) 5-bromo-2-chlorophenol, K₂CO₃, CH₃CN,

reflux; (c) Cs₂CO₃, DMF, 150 , microwave; (d) Suzuki coupling, boronic acid/ester, PdCl₂(dppf),

KOAC, dioanxe, 80 .

In the construction of core structure, scheme 3 can be employed when the R is cyclohexyl,

isopropyl and cyclopropyl. Beginning with alcohol amine or cycloalkane amine in ice cold

acetonitrile solution of potassium carbonate, chloroacetyl chloride was added to start amide

condensation reaction, intermediate product 10 was obtained. 5- bromine -2 chlorophenol was

reacted with 10 under reflux condition, to obtain intermediate 11. Intramolecular ring forming

reaction was conducted by microwave reaction to get 12. The target compounds(13a-13c) were

obtained by the coupling reaction.

2.2 PI3K/mTOR inhibition and SAR study of target compounds

The final products were evaluated for their PI3K/mTOR inhibition potency. In this work, three

different kinds of 4- fluoro benzamide sulfonamide fragments and six kinds of substituted benzene

rings were employed in molecular design (Scheme 2). Interestingly, the molecules with

4-fluoro-N-(2-methoxypyridin-3-yl)benzenesulfonamide

or

N-(2-chloro-5-methylpyridin-3-yl)-4-fluorobenzenesulfonamide in C-6 showed good inhibitory

activities both against PI3Kα, PI3Kδ and mTOR among all the compounds (Table 1). In addition,

the activity of molecules with no substitution in N-4 position (6a series) still showed good

inhibitory activities, suggesting the importance of sulfonamide fragment in molecular bioactivity.

To further validate the importance of sulfonamide fragment for the bioactivity of the molecules,

the C-6 position was substituted with 3-quinoline or 4-phenol. The kinases assays turned out that

most compounds demonstrated significant decline in contrary with the molecules with

sulfonamide fragment in C-6 position. This result makes certain the important role of sulfonamide

fragment for the bioactivity of the compounds.

Cyclohexyl, isopropyl and cyclopropyl were introduced to N-4 position to further discuss the SAR.

In contrary with benzene ring in N-4 position (8a series), the introduced cyclohexyl slightly

down-regulated the bioactivities. It can be concluded that the saturated hydrocarbon in N-4

position is not a favorable option. The introduction of isopropyl and cyclopropyl to N-4 position

slightly improved the inhibitory activity in contrary with 6a series, suggesting the size of the

substituent in N-4 is critical for the bioactivity of the compounds. A decrease of bioactivity was

observed in 8f series reference to 8d series, demonstrating the critical influence of the position of

substituent to the bioactivities.

Table 1. IC₅₀Values for Enzymatic Inhibition of PI3Kα and PI3Kδ

IC₅₀(nM)

Compd

6a -1

R

H

R1

PI3Kα

PI3Kδ

mTOR

500±12

300±11

635±18

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6a -2

6a -3

>1000

92±5

>1000

328±7

115±8

6a -4

6a -5

>1000

8a -1

8a -2

8a -3

102±6

>1000

19±1.1

80±4

>1000

15±0.4

48.6±3

>1000

15.3±0.5

8a -4

8a -5

>1000

8b -1

22±1.3

18±0.9

12.6±0.7

8b -2

8b -3

>1000

2±0.13

>1000

3.2±0.19

14.1±0.52

8b -4

8b -5

>1000

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8c -1

12±0.56

17±0.62

13.8±0.57

8c -2

8c -3

>1000

4.1±0.22

3.7±0.29

19.4±1.2

8c -4

8c -5

>1000

8d-1

8d -2

8d -3

0.63±0.05

>1000

9.2±0.74

>1000

13.85±0.86

>1000

0.43±0.02

3.8±0.12

19.8±1.25

8d -4

8d -5

>1000

8e-1

21±2.15

>1000

19±1.95

>10000

34.9±1.53

>1000

8e -2

8e -3

8e -4

5.1±0.23

>1000

5±0.31

>1000

17.9±0.86

>1000

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8e -5

8f -1

>1000

8f -2

8f -3

>1000

8f -4

8f -5

>1000

13a -1

45.3±2.32

18±0.96

3.6±0.21

13a -2

13a -3

>1000

15.5±0.82

3.8±0.16

4.3±0.13

13a -4

13a -5

>1000

13b -1

>1000

406±16

873±21

13b -2

13b -3

>1000

112±1.16

79±3.32

290.5±3.94

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13b -4

13b -5

>1000

>10000

13c -1

13c -2

131±0.96

>1000

162±1.13

>1000

253±2.31

>1000

13c -3

13c -4

86±2.21

>1000

62±1.32

>1000

267±3.32

>1000

13c -5

>1000

BEZ235

80.5±1.37

80±1.56

1.43±0.06

2.3 In vitro antiproliferative activity

Inhibition of cell proliferation was measured using MTT with human colon cancer cell line

(HCT-116), human lung adenocarcinoma cell line (A549), human breast cancer cell line (MCF-7),

human cervical cancer cell line (Hela), human hepatoma cell line (HepG2), human malignant

melanoma cell line(A375). Generally, the results were correlated with their effects on kinases

activities

(Table

2).

Different

from

kinase

assays,

molecules

with

4-fluoro-N-(2-methoxypyridin-3-yl)benzenesulfonamide in C-6 position always showed better

anti-cancer activities than N-(2-chloro-5-methylpyridin-3-yl)-4-fluorobenzenesulfonamide. The

reason may be that when 4- methoxy was introduced into the sulfonamide fragment, the molecule

demonstrated better permeability than chlorine atom in the same position. Among all the

molecules, compound 8d-1 showed the most potent anti-proliferation activity. The IC₅₀value of

8d-1 against Hela and A549 are 1.22 and 1.35 µM, with the positive agent BEZ235 with IC₅₀

value of 1.17 and 1.08 µM, respectively.

In addition, compound 8d-1 were assessed for toxicities against human normal liver cell line (LO2)

and neuronal cell PC12 by the conventional MTT assay in vitro. The results are given in Figure 3,

in comparation with BEZ235, 8d-1 showed lower toxicities in concentrations from 0.625 µM to

20 µM, demonstrating the safety of 8d-1.

Table 2. Antiproliferative Activities of Various Cell Lines

IC₅₀(µM)

Compd

A549

Hela

7.11±0.16

>40

HCT-116

24.9±0.79

>40

HepG2

6.59±0.21 26.33±0.81 32.27±1.26

>40 20.08±0.82 18.34±0.52

A375

MCF-7

6a-1

8.07±0.21

32.14±0.95

6a -2

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6a -3

6a -4

6a -5

8a-1

>40

15.56±0.45

>40

26.33±0.79

38.15±1.92

>40

3.49±0.12

>40

1.53±0.13 18.81±0.76 14.01±0.33 15.17±0.43 8.87±0.31

8a -2

8a -3

8a -4

8a -5

8b-1

8b -2

8b -3

8b -4

8b -5

8c-1

17.69±0.91

>40

30.28±1.22

>40

14.7±0.82 10.31±0.51 39.82±1.93 35.67±1.74 36.85±1.83

>40

29.04±1.64

8.35±0.62

4.83±0.31 19.24±1.32 17.93±1.23 24.08±1.42 27.56±1.52

31.63±1.79 35.03±2.03 23.89±0.73 27.83±1.88 32.45±1.57 39.81±2.69

21.59±1.39

>40

27.91±1.67 33.93±1.33 30.56±1.24 32.77±1.58

36.08±1.97 32.72±1.63

39.6±2.38 >40

>40

5.87±0.32

>40

4.65±0.23 22.68±0.86 28.27±1.12 12.87±0.81 15.41±0.48

>40 35.62±1.23 >40 36.19±1.86 32.06±1.28

8c -2

8c -3

8c -4

8c -5

8d-1

8d -2

8d -3

8d -4

8d -5

8e-1

30.9±1.52 32.33±1.23 34.32±1.59 39.58±2.21 38.86±2.21 32.64±2.11

>40

26.25±1.08

8.26±0.39

1.35±0.09

1.22±0.06 13.44±0.62 13.08±0.59 18.4±1.17

38.23±1.76 13.16±0.62 28.27±1.37 29.83±1.18 38.52±1.73 32.4±1.17

30.35±1.27 8.32±0.45 30.11±1.12

>40

32.72±1.17 36.03±1.38

>40

17.3±0.74

>40

16.44±0.87 26.11±1.17 10.82±0.49

6.5±0.29

>40

10.45±0.48

>40

36.35±1.76

>40

8e -2

8e -3

8e -4

8e -5

8f-1

>40

22.31±1.19

>40

34.2±1.94

>40

32.64±1.39

>40

38.78±2.37

>40

37.16±2.15

>40

30.2±1.18

>40

11.2±0.55

>40

8f -2

8f -3

8f -4

8f -5

13a-1

>40

32.65±1.77

>40

11.35±0.43 8.24±0.39

2.08±0.06 23.64±0.83

>40

>40 15.33±0.76 26.07±0.94 30.44±1.36

>40

4.92±0.18

2.79±0.09 19.21±0.96 37.19±1.13 9.95±0.36 16.95±0.21

>40 >40 26.03±0.37

5.38±0.18 18.92±0.47 21.48±0.19 25.47±0.28 26.59±0.32

13a -2 29.35±1.32 22.65±1.12 22.58±0.52

13a -3

5.1±0.22

13a -4 24.64±1.26 23.12±1.06 23.19±0.86

>40

39.02±2.35 22.91±1.27

13a -5

13b-1

13b -2

>40

24.64±0.96

6.1±0.29

30.11±1.79

14.5±0.79 10.84±0.72 18.28±0.92 35.9±2.76 23.81±0.99 21.37±0.76

13b -3 37.88±1.78 14.32±0.58

>40

13b -4

13b -5

>40

19.74±0.92

>40

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13c-1

13c -2

13c -3

13c -4

13c -5

3.32±0.13

>40

6.03±0.22 24.32±1.38 14.4±0.56 23.86±0.97 35.19±2.77

>40

4.33±0.24

>40

7.18±0.39 12.41±0.49 8.51±0.42 21.81±0.94 16.74±0.23

>40

29.48±0.59

32.95±0.68

0.45±0.03

>40

32.73±0.75

4.04±0.23

BEZ235 1.08±0.92

1.17±0.11

1.7±0.12

0.58±0.05

Figure 3. Toxicities of 8d-1 and BEZ235 against LO2 (A) and PC12 (B)

The activities of 8d-1 against the PI3Ks and mTOR were assayed (Table 3). The result

demonstrated that 8d-1 is a potent inhibitor against PI3Kα, PI3Kγ, PI3Kδ and mTOR with modest

levels of selectivity against PI3Kβ. This result makes 8d-1 a promising Class I PI3K/mTOR dual

inhibitor.

To characterize the kinase inhibitory activities and selectivity of compound 8d-1, kinase inhibition

profiling assays with a fixed concentration of 100 nM were carried out against a series of 394

kinases through the kinase profiling provided by Reaction Biology Corporation (Figure 4 and

Table S1). Compound 8d-1 displayed almost no inhibitory activity against most of human protein

kinases except for mTOR and DNA-PK, which are members of phosphatidylinositol 3-kinase

related protein kinases (PIKKs) super family [31].

Table 3. Activities of 8d-1 against Class I PI3Ks and mTOR (IC₅₀Values in nM)

Compd

8d-1

PI3Kα

0.63

9.6

PI3Kβ

94.54

11.98

703.9

PI3Kγ

22

PI3Kδ

9.2

mTOR

13.85

5.3

PI-103

BEZ235

64.03

104.2

11

80.5

85.3

1.43

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Figure 4. Kinase binding selectivity for 8d-1 shown on the human kinome dendrogram. The

inhibition rates were determined using the KinaseProfiler of Reaction Biology Corp. The figure

was generated by using an online KinMap program (http://kinhub.org/kinmap/).

Western blot analysis was conducted to evaluate the intracellular PI3K pathway inhibitory

activities of 8d-1. It turned out that 8d-1 could inhibit the PI3K/AKT/mTOR pathway by

dose-dependently decrease the level of phosphorylation of AKT and its downstream target S6 in

Hela cell line (Figure 5).

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Figure 5. Effects of 8d-1 on pAKT, AKT, pS6, and S6.

Plate clone formation assay was conducted to test the influence of the concentration of 8d-1 on the

clone formation of Hela cells. The colony formation rate of Hela cells in the experimental group

decreased significantly compared with the control group, in concentration dependent manner. In

the concentration above 5 µM, the tumor cells almost did not form clones. At the concentration of

1.25 µM, more than half of the clone formation was inhibited in contrary with the negative control

group (Figure 6). This is basically the responding to the MTT results of Hela cells.

Figure 6. Macroscopic images of colonies formed by Hela treated with different concentrations of

8d-1.

2.4 Docking study

To predict the possible binding mode of 8d-1 with PI3Kα and mTOR, docking analysis was

performed by using autodock 4.0 [32]. The X-ray crystal structure of PI3Kα (PDB ID:4L23) [33]

and mTOR (PDB ID:4JT5) [34] was obtained as starting point. As demonstrated in Figure 7, five

hydrogen bond interactions were formed between 8d-1 and PI3Kα. Three hydrogen bonds were

formed between 2H-benzo[b][1,4]oxazin-3(4H)-one and PI3Kα, indicating the advantage of the

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core structure for the bioactivity of molecules. The fluorine of sulfonamide fragment formed a

hydrogen bond with Trp86, another hydrogen bond was formed between the oxygen of 4- methoxy

benzene. In addition, 4- methoxy benzene matched very well with the hydrophobic pocket formed

by Ile153, Ile105 and Met77, that may be the reason why 8d-1 is a potent PI3Kα inhibitor and it

makes 4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one a reasonable skeleton for further investigation

as PI3Kα inhibitors.

The binding mode of 8d-1 with mTOR showed that three hydrogen bonds were formed: two were

formed by Lys2187 with the oxygen atom of N-4 substituted 4-methoxy and the oxygen atom of

sulfonamide structure, respectively; Asp2357 formed a hydrogen bond with the nitrogen atom of

sulfonamide structure. In addition, Lys2187 formed two cation-pi interactions with the benzene

ring of N-4 substituted 4-methoxy and the benzene ring of 4-fluoro benzamide sulfonamide

fragment, respectively. The binding mode indicated that the 4-fluoro benzamide sulfonamide

fragment is a favorable moiety for mTOR inhibitor, and the N-4 substituted 4-methoxy in this

molecule scaffold matched very well with PI3Kα and mTOR makes it useful in PI3Kα/mTOR

dual inhibitor design.

Figure 7. Modeled structure of 8d-1 (green) in PI3Kα (A) and mTOR (B).

2.5 Pharmacokinetic studies

The pharmacokinetic properties of 8d-1 were evaluated in Sprague−Dawley rats. After

intravenous administration at 1 mg/kg and oral administration at 10 mg/kg, blood samples were

taken, and the plasma was detected for the concentrations of 8d-1 by an LC-MS/MS system. As

shown in Table 4, 8d-1 showed a plasma clearance of 8.56 mL·kg^-1·min^-1administered iv in rats,

whereas oral administration in rats gave half-time (T_1/2) of 1.78 h. Acceptable oral bioavailability

of 24.1% was achieved, suggesting that 8d-1 could be further investigated as a novel

PI3Kα/mTOR dual inhibitor.

Table 4: Pharmacokinetic Parameters for 8d-1 in Rats

IV (1 mg/kg)^a

PO (10 mg/kg)^b

AUCinf (ng*h/ml)

4753.35

CL (ml/min/kg)

8.56

Vss (ml/kg)

1199.81

T_max(h)

2.67

C_max(ng/ml)

886.67

T_1/2(h)

1.78

F(%)

24.1

^aVehicle: 5% DMA/10% Solutol HS 15/85 % saline. ^bVehicle: 0.5% CMC-Na.

2.6 In vitro antiproliferative activity of compound 8d-1

ACCEPTED MANUSCRIPT

The anti-tumor activity in vivo of 8d-1 was conducted based on the promising cellular activity and

pharmacokinetic properties. BALB/c nude mice bearing Hela and A549 xenograft tumors were

treated with 8d-1 by intragastric administration for 30 days. As can be depicted in Figure 8, the

growth of xenograft tumors can be inhibited by 8d-1 in a dose-dependent manner. Tumor growth

inhibitions (TGIs) of 87.7%, 66.6% were observed in the Hela xenograft model at dose of 50, and

20 mg/kg, respectively. For the A549 model, administration of 8d-1 at doses of 40 and 20 mg/kg

caused TGIs of 70.16% and 57.8%, respectively. BZE235 as positive control in this assay is

slightly less active than 8d-1: at dose of 20 mg/kg leading to TGI of 66.5% in Hela model and

56.08% in A549 model. In addition, 8d-1 did not cause significant weight loss and toxicity during

the treatment period in contary with BEZ235 caused little weight loss in this period.

Figure 8. Pharmacodynamic profile of 8d-1 in vivo. Growth inhibitory effect of 8d-1 on

established Hela/A549 xenografts in female BALB/c nude mice (N = 6 per group, mean ± SD).

3. CONCLUSIONS

In present study, a series of 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives were synthesized and

characterized. The discussion of SAR revealed that the benzene or substituted benzene introduced

to N-4 position demonstrated advantages over saturated alkane/cyclanes in bioactivities. Based on

the SAR, 4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one as the skeleton of PI3K/mTOR dual

inhibitors will be meaningful for further investigation.

Among all the molecules, 8d-1 demonstrated potent anti-proliferation effect on human cancer cell

lines Hela and A549 in vitro and in vivo. In contrary with BEZ235, 8d-1 hold a tenuous advantage

in anticancer activity of xenograft model as well as no significant weight loss and toxicity during

the treatment period. The existing data indicating that 8d-1 is a promising drug candidate.

ACCEPTED MANUSCRIPT

4. EXPERIMENTAL SECTION

4.1 chemistry

All solvents and reagents obtained from commercial sources were used without further

purification. Flash column chromatography was performed using silica gel from Qingdao Haiyang.

¹H NMR and ¹³C NMR spectra were recorded on a Bruker AMX 400 spectrometer and were

calibrated using TMS or residual deuterated solvent as an internal reference (CDCl₃: ¹H, δ = 7.26

ppm; ¹³C, δ = 77.16 ppm, DMSO-d₆2.50 ppm, CD₃OD 3.31 ppm). All of the target compounds

were examined by HPLC, and the purity of the biologically tested compounds was ≥ 95%.

4.1.1 Synthesis of intermediate product 5:

To a solution of chloroacetic chloride (4.7 ml, 60 mmol) in 25mL THF was added NaHCO₃(6 g,

72 mmol) and the THF solution of 2- amino -4- bromine phenol (9 g, 48 mmol). The reaction

mixture was stirred for 1 h under the ice bath and monitored by TLC. Upon completion, potassium

carbonate (10 g, 72 mmol) was added and the reaction solution heated to 80°C to start the

next-step reaction for 3h. The reaction was cooled to room temperature, quenched with water, and

extracted with EtOAc. The organic layer was collected and washed with diluted hydrochloric acid,

saturated NaHCO₃and saturated salt solution, dried over anhydrous Na₂SO₄and concentrated in

vacuo. The crude material was purified by column chromatography to afford target molecule (6.9g,

63% yield) as brown solid. HRMS (DART-TOF) calculated for C₈H₆BrNO₂Na [M + Na]⁺m/z

249.9480, 251.9459, found 249.9448, 251.9419.

4.1.2 General Procedure A for the synthesis of sulfonamide fragment

To a solution of 1 (2.57 mmol) in 8mL pyridine at 0°C was added the pyridine solution of 4-

fluoro sulfonyl chloride (4 g, 19.4 mmol) in drops and DMAP (20mg) to start the reaction. The

mixture was warmed slowly to room temperature and stirred overnight. After the completion of

the reaction (monitored by TLC), the reaction was quenched with water (30mL) and filtered. The

filter cake was washed with water and EtOH to obtain white solid which was dissolved in MeOH

(10mL) and added with 3 mL saturated potassium carbonate solution. The mixture was stirred at

room temperature for 6h and monitored by TLC. The reaction was quenched with water and

extracted with EtOAc. The organic layer was collected and washed with water for three times,

dried over anhydrous Na₂SO₄and concentrated in vacuo. The crude material was purified by

column chromatography to afford white solid.

4.1.2.1 N- (5- bromo -2- methoxy pyridine -3- base) -4- fluoro benzene sulfonamide(2a)

56% isolated yield, white solid. HRMS (DART-TOF) calculated for C₁₂H₁₁BrFN₂O₃S [M + H]⁺

m/z 360.9658, 362.9637, found 360.9616, 362.9602.

4.1.2.2 N-(3-bromophenyl)-4-fluorobenzenesulfonamide(2b)

61% isolated yield, white solid. HRMS (DART-TOF) calculated for C₁₂H₁₀BrFNO₂S [M + H]⁺

m/z 329.9600, 331.9579, found 329.9586, 331.9558.

4.1.2.3 N- (5- bromine -2- chlorpyridine -3) -4- fluoro sulfonamide(2c)

48% isolated yield, white solid. HRMS (DART-TOF) calculated for C₁₁H₈BrClFN₂O₂S [M + H]⁺

m/z 364.9162, 366.9142 found 364.9131, 366.9114.

4.1.3 General Procedure B for the Synthesis of intermediate product 7.

To a solution of 5 (350 mg, 1.54 mmol) in 20mL THF was added phenylboronic acid (3.28 mmol),

ACCEPTED MANUSCRIPT

copper acetate (420 mg, 2.31 mmol), Et₃N (0.5 mL, 4.62 mmol) and 4 Å molecular sieve (50mg).

The solution was stirred at 60 °C for 6h. After the completion of the reaction (monitored by TLC),

the reaction solution was concentrated, and added with 20mL water. The reaction was filtered to

remove solids and extracted with EtOAc, the organic layer was collected and washed with

saturated salt solution for three times, dried over anhydrous Na₂SO₄and concentrated in vacuo.

The crude material was purified by column chromatography to afford target product.

4.1.3.1 6- bromine -4- phenyl -2H-1,4- benzoxazine -3 (4H) – ketone (7a)

62% isolated yield, white solid. ¹H NMR (400 MHz, DMSO) δ 7.63 – 7.58 (m, 2H, Ar-H), 7.53 (t,

J = 7.4 Hz, 1H, Ar-H), 7.37 (d, J = 7.1 Hz, 2H, Ar-H), 7.18 (dd, J = 8.5, 2.3 Hz, 1H, Ar-H), 7.06 (d,

J = 8.5 Hz, 1H, Ar-H), 6.31 (d, J = 2.3 Hz, 1H, Ar-H), 4.85 (s, 2H, CH₂). HRMS (DART-TOF)

calculated for C₁₄H₁₀BrNO₂Na [M + Na]⁺m/z 325.9793, 327.9772 found 325.9771, 327.9762.

4.1.3.2 6- bromine -4- (4- chlorophenyl) -2H- benzoxazine -3 (4H) - ketone. (7b)

71% isolated yield, grey solid. ¹H NMR (400 MHz, CDCl₃) δ 7.53 (d, J = 8.7 Hz, 2H, Ar-H), 7.22

(d, J = 8.6 Hz, 2H, Ar-H), 7.11 (dd, J = 8.6, 2.2 Hz, 1H, Ar-H), 6.93 (d, J = 8.6 Hz, 1H, Ar-H), 6.55

(s, 1H, Ar-H), 4.75 (s, 2H, CH₂). HRMS (DART-TOF) calculated for C₁₄H₉BrClNO₂Na [M + Na]⁺

m/z 336.9505, 338.9485 found 336.9501, 338.9481.

4.1.3.3 6- bromine -4- (4- methyl phenyl) -2H- benzoxazine -3 (4H) - ketone. (7c)

53% isolated yield, grey solid. ¹H NMR (400 MHz, CDCl₃) δ 7.35 (d, J = 8.1 Hz, 2H, Ar-H), 7.14

(d, J = 8.2 Hz, 2H, Ar-H), 7.08 (dd, J = 8.5, 2.2 Hz, 1H, Ar-H), 6.91 (d, J = 8.5 Hz, 1H, Ar-H), 6.56

(d, J = 2.2 Hz, 1H, Ar-H), 4.75 (s, 2H, CH₂), 2.44 (s, 3H, CH₃). HRMS (DART-TOF) calculated for

C₁₅H₁₂BrNO₂Na [M + Na]⁺m/z 339.9949, 341.9929, found 339.9931, 341.9906.

4.1.3.4 6- bromine -4- (4- methoxy phenyl) -2H- benzoxazine -3 (4H) - ketone. (7d)

38% isolated yield, grey solid. ¹H NMR (400 MHz, DMSO) δ 7.28 (d, J = 8.9 Hz, 2H, Ar-H), 7.16

(dd, J = 8.5, 2.3 Hz, 1H, Ar-H), 7.12 (d, J = 8.9 Hz, 2H, Ar-H), 7.03 (d, J = 2.3 Hz, 1H, Ar-H), 6.35

(d, J = 2.3 Hz, 1H, Ar-H), 4.82 (s, 2H, CH₂), 3.84 (s, 3H, OCH₃). HRMS (DART-TOF) calculated

for C₁₅H₁₂BrNO₃Na [M + Na]⁺m/z 355.9898, 357.9878, found 355.9876, 357.9855.

4.1.3.5 6- bromine -4- (4- fluoro phenyl) -2H- benzoxazine -3 (4H) - ketone. (7e)

66% isolated yield, grey solid. ¹H NMR (400 MHz, DMSO) δ 7.47 - 7.40 (m, 4H, Ar-H), 7.19 (dd, J

= 7.4, 2.3 Hz, 1H, Ar-H), 7.06 (d, J = 8.5 Hz, 1H, Ar-H), 6.34 (d, J = 2.2 Hz, 1H, Ar-H), 4.84 (s, 2H

CH₂). HRMS (DART-TOF) calculated for C₁₄H₉BrFNO₂Na [M + Na]⁺m/z 343.9698, 345.9678,

,

found 343.9612, 345.9655.

4.1.3.6 6- bromine -4- (3- methoxy phenyl) -2H- benzoxazine -3 (4H) – ketone(7f)

43% isolated yield, grey solid. ¹H NMR (400 MHz, DMSO) δ 7.51 (t, J = 8.1 Hz, 1H, Ar-H), 7.18

(dd, J = 8.5, 2.3 Hz, 1H, Ar-H), 7.11 (dd, J = 8.4, 3.4 Hz, 1H, Ar-H), 7.05 (d, J = 8.5 Hz, 1H, Ar-H),

6.99 – 6.96 (m, 1H, Ar-H), 6.92 (d, J = 8.7 Hz, 1H, Ar-H), 6.36 (d, J = 2.3 Hz, 1H, Ar-H), 4.83 (s,

2H, CH₂), 3.79 (s, 3H, OCH₃). HRMS (DART-TOF) calculated for C₁₅H₁₂BrNO₃Na [M + Na]⁺

m/z 355.9898, 357.9878, found 355.9861, 357.9856.

4.1.4 General Procedure C for the Synthesis of intermediate product 12.

To a solution of 9 (30 mmol) in 25mL THF was added K₂CO₃(6.2 g, 45 mmol), and stirred for 20

min under the ice bath to make it well mixed. Chloroacetyl chloride (3 mL, 36 mmol) was added

in drops and stirred for 3 h under room temperature. The reaction solution was concentrated, water

(100 mL) was added, and extracted with EtOAc. The organic layer was collected and washed with

saturated salt solution for three times, dried over anhydrous Na₂SO₄and concentrated in vacuo.

ACCEPTED MANUSCRIPT

The crude material was purified by column chromatography to afford intermediate product 10.

To a solution of 5- bromine -2- chlorophenol (2.9 g, 14.3 mmol) in 50mL acetonitrile was added

K₂CO₃(2.5 g，17.1 mmol), and stirred for 30 min. intermediate product 10 was added and the

solution was heated to 80°C for 4h. The reaction solution was concentrated, water (100 mL) was

added, and extracted with EtOAc. The organic layer was collected and washed with water and

brine for three times, dried over anhydrous Na₂SO₄and concentrated in vacuo. The crude material

was purified by column chromatography to afford intermediate product 11.

To a solution of intermediate product 11 (0.64 mmol) in 3mL DMF in microwave reacting tube,

Cs₂CO₃(500 mg, 1.53 mmol) was added. The mixture was heated to 150 °C under microwave

irradiation for 60 min and monitored by TLC. The reaction was cooled to room temperature,

quenched with water, and extracted with EtOAc. The organic layer was collected and washed with

water for three times, dried over anhydrous Na₂SO₄and concentrated in vacuo. The crude material

was purified by column chromatography to afford target molecule.

4.1.4.1 6- bromo -4- isopropyl -2H- benzoxazine -3 (4H) – ketone (12a)

32% isolated yield, grey solid. ¹H NMR (400 MHz, DMSO) δ 7.47 (dd, J = 7.5, 1.4 Hz, 1H, Ar-H),

7.14 (d, J = 1.6 Hz, 1H, Ar-H), 6.99 (d, J = 7.5 Hz, 1H, Ar-H), 5.33 (hept, J = 5.9 Hz, 1H, CH), 4.81 (s,

2H, CH₂), 1.34 (d, J = 5.9 Hz, 6H, CH₃). HRMS (DART-TOF) calculated for C₁₁H₁₃BrNO₂[M +

H]⁺m/z 270.0130, 272.0109, found 270.0115, 272.0101.

4.1.4.2 6- bromo -4- cyclohexyl -2H- benzoxazine -3 (4H) – ketone (12b)

32% isolated yield, grey solid. ¹H NMR (400 MHz, CDCl3) δ 7.47 (dd, J = 7.5, 1.4 Hz, 1H, Ar-H),

7.16 (d, J = 1.6 Hz, 1H, Ar-H), 6.99 (d, J = 7.5 Hz, 1H, Ar-H), 4.81 (s, 2H, CH₂), 4.64 – 4.56 (m, 1H,

CH), 2.08 (dt, J = 7.1, 6.0 Hz, 2H, CH₂), 1.77 (dd, J = 11.1, 5.6 Hz, 2H, CH₂), 1.67 – 1.59 (m, 4H,

CH₂), 1.49 (p, J = 5.7 Hz, 2H, CH₂). HRMS (DART-TOF) calculated for C₁₄H₁₇BrNO₂[M + H]⁺

m/z 310.0443, 312.0422, found 310.0436, 312.0415.

4.1.4.3 6- bromo -4- cyclopropyl -2H- benzoxazine -3 (4H) – ketone (12c)

43% isolated yield, grey solid. ¹H NMR (400 MHz, CDCl3) δ 7.47 (dd, J = 7.5, 1.4 Hz, 1H, Ar-H),

7.22 (d, J = 1.4 Hz, 1H, Ar-H), 6.98 (d, J = 7.5 Hz, 1H, Ar-H), 4.80 (s, 2H, CH₂), 3.62 (p, J = 9.0 Hz,

1H, CH), 1.27 – 0.88 (m, 4H, CH₂). HRMS (DART-TOF) calculated for C₁₁H₁₁BrNO₂[M + H]⁺

m/z 267.9973, 269.9953, found 267.9952, 269.9932.

4.1.5 General Procedure for the Synthesis of target molecules.

To a solution of intermediate product 5/7/12 (0.26 mmol) and 2a-2c, 3-bromoquinoline or

4-bromophenol (0.26 mmol) in 4 mL dioxane, was added PdCl₂(dppf) (14 mg, 0.02 mmol) and

potassium acetate (80 mg, 0.8 mmol). Under N₂atmosphere, the mixture was heated to 80 °C for 5

h. Then the reaction mixture was cooled, diluted with ethyl acetate, washed with brine, dried over

Na₂SO₄, and the solvent removed in vacuo. Purification on silica yielded the desired compounds.

4.1.5.1

4-fluoro-N-(2-methoxy-5-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)benzenesu

lfonamide (6a-1)

43% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.81 (s, 1H, CONH), 10.06 (s, 1H,

SO₂NH), 8.09 (s, 1H, Ar-H), 7.82 (dd, J = 8.8, 5.2 Hz, 2H, Ar-H), 7.71 (s, 1H, Ar-H), 7.40 (t, J = 8.8

Hz, 2H, Ar-H), 7.13 (d, J = 10.4 Hz, 1H, Ar-H), 7.07 – 7.00 (m, 2H, Ar-H), 4.61 (s, 2H, CH₂), 3.67 (s,

3H, OCH₃). ¹³C NMR (101 MHz, DMSO) δ 164.77, 163.6 (d, J_C-F= 250 Hz), 149.56, 148.21, 143.97,

138.30, 136.98, 136.95, 130.89 (d, J_C-F= 9 Hz), 130.40, 128.36, 125.25, 124.27, 117.09, 116.94, 116.77,

ACCEPTED MANUSCRIPT

116.14(d, J_C-F= 23 Hz), 68.15, 53.95. HRMS (DART-TOF) calculated for C₂₀H₁₆FN₃O₅S [M + H]⁺

m/z 429.0795, found 429.0771. Purity 96.3% by HPLC.

4.1.5.2 4-fluoro-N-(3-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)

benzenesulfonamide (6a-2)

32% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.82 (s, 1H, CONH), 10.47 (s, 1H,

SO₂NH), 7.85 (dd, J = 8.8, 5.2 Hz, 2H, Ar-H), 7.40 (t, J = 8.8 Hz, 2H, Ar-H), 7.28 (d, J = 7.3 Hz, 2H,

Ar-H), 7.20 (d, J = 8.0 Hz, 1H, Ar-H), 7.09 – 6.99 (m, 4H, Ar-H), 4.61 (s, 2H, CH₂). ¹³C NMR (101

MHz, DMSO) δ = 165.27, 163.6 (d, J_C-F= 262 Hz), 140.99, 138.89, 136.45, 136.42, 134.47,

130.89 (d, J_C-F= 6.7 Hz), 128.21, 122.51, 121.72, 119.28, 118.23, 117.08 (d, J_C-F= 21 Hz), 116.84,

114.25, 67.26. HRMS (DART-TOF) calculated for C₂₀H₁₅FN₂O₄S [M + H]⁺m/z 398.0737, found

398.0721. Purity 98.0% by HPLC.

4.1.5.3

N-(2-chloro-5-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)-4-fluorobenzenesulf

onamide (6a-3)

1

29% isolated yield, white powder. H NMR (400 MHz, DMSO) δ 10.86 (s, 1H, CONH), 8.97 (s,

1H, SO₂NH), 8.82 (s, 1H, Ar-H), 8.12 – 7.93 (m, 2H, Ar-H), 7.84 (s, 1H, Ar-H), 7.63 – 7.51 (m, 2H

Ar-H), 7.24 (t, J = 8.8 Hz, 1H, Ar-H), 7.12 (d, J = 8.3 Hz, 1H, Ar-H), 7.03 (s, 1H, Ar-H), 4.66 (s,

2H, CH₂). HRMS (DART-TOF) calculated for C₁₉H₁₃ClFN₃O₄S [M + H]⁺m/z 433.0299, found

433.0291. Purity 96.2% by HPLC.

,

4.1.5.4 6-(quinolin-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (6a-4)

23% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ = 10.86 (s, 1H, CONH), 9.14 (d,

J=2.3, 1H, Ar-H), 8.51 (d, J=2.1, 1H, , Ar-H), 8.06 (dd, J=8.0, 3.2, 2H, Ar-H), 7.81 – 7.73 (m, 1H

,

Ar-H), 7.65 (t, J=7.5, 1H, Ar-H), 7.44 (dd, J=8.3, 2.2, 1H, Ar-H), 7.33 (d, J=2.1, 1H, Ar-H), 7.13 (d,

J=8.3, 1H, Ar-H), 4.66 (s, 2H, CH₂). ¹³C NMR (101 MHz, DMSO) δ = 165.21, 149.63, 147.14,

144.01, 132.79, 132.68, 131.89, 129.92, 129.12, 128.80, 128.50, 128.11, 127.56, 122.53, 117.41,

114.83, 67.32, 50.99. HRMS (DART-TOF) calculated for C₁₇H₁₂N₂O₂[M + H]⁺m/z 276.0899,

found 276.0889. Purity 95.5% by HPLC.

4.1.5.5 6-(4-hydroxyphenyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (6a-5)

29% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ = 10.71 (s, 1H, CONH), 9.48 (d,

J=7.3, 1H, Ar-H), 7.34 (t, J=5.7, 2H, Ar-H), 7.10 (dd, J=8.3, 2.2, 1H, Ar-H), 7.05 (d, J=2.1, 1H,

Ar-H), 6.97 (d, J=8.3, 1H, Ar-H), 6.82 (t, J=5.8, 2H, Ar-H), 4.57 (s, 2H, CH₂). HRMS

(DART-TOF) calculated for C₁₄H₁₁NO₃[M + H]⁺m/z 241.0739, found 241.0736. Purity 98.3% by

HPLC.

4.1.5.6

4-fluoro-N-(2-methoxy-5-(3-oxo-4-phenyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)

benzenesulfonamide (8a-1)

41% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, J = 2.3 Hz, 1H, Ar-H),

7.93 (dd, J = 8.9, 5.0 Hz, 3H

7.41 (d, J = 2.3 Hz, 1H Ar-H), 7.34 (d, J = 7.3 Hz, 2H

(d, J = 8.3 Hz, 1H Ar-H), 7.01 (dd, J = 8.3, 2.0 Hz, 1H

4.83 (s, 2H, CH₂), 3.59 (s, 3H, OCH₃). ¹³C NMR (101 MHz, CDCl₃) δ = 167.25, 164.69 (d, J_C-F

,

Ar-H), 7.58 (t, J = 7.4 Hz, 2H

Ar-H), 7.20 (t, J = 8.5 Hz, 3H

Ar-H), 6.54 (d, J = 2.0 Hz, 1H

,

Ar-H), 7.52 (t, J = 7.4 Hz, 1H

Ar-H), 7.13

Ar-H),

, Ar-H),

,

=

262.3 Hz), 164.00, 163.60, 160.70, 146.82, 139.66, 135.48, 131.80 (d, J_C-F= 6.7 Hz), 131.70,

130.24, 129.19, 128.72, 122.54, 117.80, 116.14 (d, J_C-F= 26.7 Hz), 114.99, 68.21, 53.73. HRMS

ACCEPTED MANUSCRIPT

(DART-TOF) calculated for C₂₆H₂₀FN₃O₅S [M + H]⁺m/z 505.1108, found 505.1101. Purity 97.5%

by HPLC.

4.1.5.7

4-fluoro-N-(3-(3-oxo-4-phenyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)

benzenesulfonamide (8a-2)

53% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.71 (dd, J = 9.0, 5.0 Hz, 2H,

Ar-H), 7.57 (t, J = 7.5 Hz, 2H, Ar-H), 7.52 – 7.46 (m, 1H, Ar-H), 7.33 (dd, J = 8.3, 1.2 Hz, 2H,

Ar-H), 7.20 (t, J = 7.9 Hz, 1H, Ar-H), 7.10 (dd, J = 8.4, 1.8 Hz, 3H, Ar-H), 7.06 (s, 1H, Ar-H),

7.05 – 7.03 (m, 1H, Ar-H), 6.92 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H, Ar-H), 6.64 (s, 1H, , Ar-H), 6.57 –

6.51 (m, 1H, Ar-H), 4.81 (s, 2H, CH₂). ¹³C NMR (101 MHz, CDCl₃) δ = 164.14, 144.23(d, J_C-F

=

320.7 Hz), 136.64, 135.61, 134.90, 130.94, 130.15, 129.96 129.96 (d, J_C-F= 9.4 Hz,), 129.80,

129.09, 128.73, 124.01, 122.84, 120.05, 119.76, 117.45, 116.34 (d, J_C-F= 22.7 Hz). 115.45, 68.22.

HRMS (DART-TOF) calculated for C₂₆H₁₉FN₂O₄S [M + H]⁺m/z 474.1050, found 474.1025.

Purity 98.1% by HPLC.

4.1.5.8

N-(2-chloro-5-(3-oxo-4-phenyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)-4-fluorobe

nzenesulfonamide (8a-3)

29% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J = 2.3 Hz, 1H, Ar-H),

7.93 (dd, J = 8.9, 5.0 Hz, 3H

7.41 (d, J = 2.3 Hz, 1H Ar-H), 7.34 (d, J = 7.3 Hz, 2H

(d, J = 8.3 Hz, 1H Ar-H), 7.01 (dd, J = 8.3, 2.0 Hz, 1H

,

Ar-H), 7.58 (t, J = 7.4 Hz, 2H

Ar-H), 7.20 (t, J = 8.5 Hz, 3H

Ar-H), 6.54 (d, J = 2.0 Hz, 1H,

,

Ar-H), 7.52 (t, J = 7.4 Hz, 1H

Ar-H), 7.13

Ar-H),

, Ar-H),

,

4.83 (s, 2H, CH₂). ¹³C NMR (101 MHz, CDCl₃) δ = 165.66 (d, J_C-F= 257 Hz), 145.60, 143.20,

136.12, 135.38, 131.99, 131.39, 130.32, 130.29, 130.14, 129.95 (d, J_C-F= 9.6 Hz), 129.34, 128.70,

127.72, 122.87, 117.97, 116.76 (d, J_C-F= 22.8 Hz). 115.45, 68.16, 29.70. HRMS (DART-TOF)

calculated for C₂₅H₁₇ClFN₃O₄S [M + H]⁺m/z 509.0612, found 509.0611. Purity 96.3% by HPLC.

4.1.5.9 4-phenyl-6-(quinolin-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8a-4)

29% isolated yield, yellow powder. ¹H NMR (400 MHz, CDCl₃) δ = 8.91 (d, J=2.2, 1H, Ar-H),

8.08 (dd, J=9.3, 5.4, 2H, Ar-H), 7.80 (d, J=8.2, 1H, Ar-H), 7.69 (ddd, J=8.4, 6.9, 1.4, 1H, Ar-H),

7.61 – 7.52 (m, 3H, Ar-H), 7.49 (ddd, J=6.3, 3.9, 1.3, 1H, Ar-H), 7.35 (tt, J=6.5, 5.1, 3H, Ar-H),

7.20 (d, J=8.3, 1H, Ar-H), 6.73 (d, J=2.1, 1H, Ar-H), 4.85 (s, 2H, CH₂). ¹³C NMR (101 MHz,

CDCl₃) δ = 164.02, 149.37, 145.09, 135.51, 132.98, 132.88, 132.60, 131.35, 130.24, 129.58,

129.22, 129.05, 128.70, 127.89, 127.20, 123.15, 117.83, 115.78, 68.25. HRMS (DART-TOF)

calculated for C₂₃H₁₆N₂O₂[M + H]⁺m/z 352.1212, found 352.1210. Purity 98.1% by HPLC.

4.1.5.10 6-(4-hydroxyphenyl)-4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one (8a-5)

21% isolated yield, yellow powder. ¹H NMR (400 MHz, CDCl₃) δ = 7.58 – 7.52 (m, 2H, Ar-H),

7.47 (ddd, J=7.4, 3.8, 1.2, 1H, Ar-H), 7.33 (dd, J=5.3, 3.2, 2H, Ar-H), 7.23 – 7.17 (m, 2H, Ar-H),

7.15 (dd, J=8.3, 2.1, 1H, Ar-H), 7.08 (d, J=8.3, 1H, Ar-H), 6.83 – 6.76 (m, 2H, Ar-H), 6.56 (d,

J=2.0, 1H, Ar-H), 4.80 (s, 2H, CH₂). ¹³C NMR (101 MHz, CDCl₃) δ = 164.35, 155.09, 130.08,

128.97, 128.75, 128.07, 122.45, 117.25, 115.62, 115.20, 68.27. HRMS (DART-TOF) calculated

for C₂₀H₁₅NO₃[M + H]⁺m/z 317.1052, found 317.1041. Purity 97.3% by HPLC.

4.1.5.11

N-(5-(4-(4-chlorophenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-methoxypyridin-3-yl

)-4-fluorobenzenesulfonamide (8b-1)

33% isolated yield, yellow powder. ¹H NMR (400 MHz, DMSO) δ 10.04 (s, 1H, SO₂NH), 7.96 (d,

J = 2.3 Hz, 1H, Ar-H), 7.72 (dd, J = 8.9, 5.2 Hz, 2H, Ar-H), 7.69 (d, J = 8.7 Hz, 2H), 7.50 (d, J =

ACCEPTED MANUSCRIPT

2.3 Hz, 1H, Ar-H), 7.47 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H, Ar-H), 7.21 (dd, J = 8.3, 2.0

Hz, 1H, Ar-H), 7.17 (d, J = 8.3 Hz, 1H, Ar-H), 6.42 (d, J = 1.9 Hz, 1H, Ar-H), 4.85 (s, 2H, CH₂),

3.84 (s, 3H, OCH₃) HRMS (DART-TOF) calculated for C₂₆H₁₉ClFN₃O₅S [M + H]⁺m/z 539.0718,

found 539.0713. Purity 95.7% by HPLC.

4.1.5.12

N-(3-(4-(4-chlorophenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)-4-fluorobenzen

esulfonamide (8b-2)

35% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.42 (s, 1H, SO₂NH), 7.73 (dd,

J = 8.9, 5.1 Hz, 2H, Ar-H), 7.69 (d, J = 8.7 Hz, 2H, Ar-H), 7.47 (d, J = 8.7 Hz, 2H, Ar-H), 7.36 (t,

J = 8.8 Hz, 2H, Ar-H), 7.24 (t, J = 7.9 Hz, 1H, Ar-H), 7.17 (d, J = 1.1 Hz, 2H, Ar-H), 7.10 – 7.04

(m, 2H, Ar-H), 7.02 – 6.96 (m, 1H, Ar-H), 6.38 (s, 1H, Ar-H), 4.86 (s, 2H, CH₂). HRMS

(DART-TOF) calculated for C₂₆H₁₈ClFN₂O₄S [M + H]⁺m/z 508.0660, found 508.0645. Purity 98.1%

by HPLC.

4.1.5.13

N-(2-chloro-5-(4-(4-chlorophenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)-

4-fluorobenzenesulfonamide (8b-3)

42% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.06 (s, 1H, SO₂NH), 7.94 (d, J

= 2.3 Hz, 1H, Ar-H), 7.72 (dd, J = 8.9, 5.2 Hz, 2H, Ar-H), 7.69 (d, J = 8.7 Hz, 2H), 7.50 (d, J =

2.3 Hz, 1H, Ar-H), 7.47 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H, Ar-H), 7.21 (dd, J = 8.3, 2.0

Hz, 1H, Ar-H), 7.17 (d, J = 8.3 Hz, 1H, Ar-H), 6.42 (d, J = 1.9 Hz, 1H, Ar-H), 4.85 (s, 2H, CH₂).

HRMS (DART-TOF) calculated for C₂₅H₁₆C_l2FN₃O₄S [M + H]⁺m/z 543.0223, found 543.0221.

Purity 95.9% by HPLC.

4.1.5.14 4-(4-chlorophenyl)-6-(quinolin-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8b-4)

23% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ = 8.92 (d, J=2.3, 1H, Ar-H), 8.08

(dd, J=9.5, 5.4, 2H, Ar-H), 7.83 (dd, J=8.2, 1.0, 1H, Ar-H), 7.70 (ddd, J=8.4, 6.9, 1.4, 1H, Ar-H),

7.61 – 7.49 (m, 2H, Ar-H), 7.35 (dd, J=8.3, 2.1, 1H, Ar-H), 7.33 – 7.31 (m, 1H, Ar-H), 7.31 – 7.28

(m, 1H, Ar-H), 7.21 (d, J=8.3, 1H, Ar-H), 6.73 (d, J=2.1, 1H, Ar-H), 4.84 (s, 2H, CH₂). ¹³C NMR

(101 MHz, CDCl₃) δ = 164.06, 149.44, 147.22, 145.11, 135.15, 133.97, 132.89, 132.72, 131.03,

130.52, 130.08, 129.58, 129.19, 127.91, 127.81, 127.20, 123.43, 118.00, 115.64, 68.21. HRMS

(DART-TOF) calculated for C₂₃H₁₅ClN₂O₂[M + H]⁺m/z 386.0822, found 386.0816. Purity 96.8%

by HPLC.

4.1.5.15 4-(4-chlorophenyl)-6-(4-hydroxyphenyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8b-5)

26% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 9.65 (s, 1H, Ar-H), 7.65 (d, J =

8.7 Hz, 2H, Ar-H), 7.45 (d, J = 8.7 Hz, 2H, Ar-H), 7.17 (ddd, J = 22.2, 11.0, 5.2 Hz, 4H, Ar-H),

6.76 (d, J = 8.6 Hz, 2H, Ar-H), 6.41 (d, J = 2.0 Hz, 1H, Ar-H), 4.83 (s, 2H, CH₂). HRMS

(DART-TOF) calculated for C₂₀H₁₄ClNO₃[M + H]⁺m/z 351.0662, found 351.0639. Purity 99.1%

by HPLC.

4.1.5.16

4-fluoro-N-(2-methoxy-5-(3-oxo-4-(p-tolyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl

)benzenesulfonamide (8c-1)

24% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.81 (d, J = 2.2 Hz, 1H, Ar-H),

7.78 (t, J = 4.2 Hz, 1H, Ar-H), 7.71 (dd, J = 8.9, 5.0 Hz, 2H, Ar-H), 7.37 (d, J = 8.1 Hz, 2H, Ar-H),

7.22 (d, J = 8.3 Hz, 2H, Ar-H), 7.11 (d, J = 1.5 Hz, 2H, Ar-H), 7.06 (t, J = 8.6 Hz, 2H, Ar-H), 6.90

(s, 1H, Ar-H), 6.55 (s, 1H, Ar-H), 4.81 (s, 2H, CH₂), 3.81 (s, 3H, OCH₃), 2.43 (s, 3H, CH₃). ¹³C

ACCEPTED MANUSCRIPT

NMR (101 MHz, CDCl₃) δ 165.4(d, J_C-F= 255 Hz), 164.13, 153.58, 144.74, 139.64, 139.24,

134.93, 134.89, 132.81, 131.85, 131.24, 130.88, 130.08, 129.91 (d, J_C-F= 9.5 Hz), 128.42, 126.08,

122.36, 120.75, 117.59, 116.34 (d, J_C-F= 22.7 Hz), 115.15, 68.22, 53.94, 21.29. HRMS

(DART-TOF) calculated for C₂₇H₂₂FN₃O₅S [M + H]⁺m/z 519.1264, found 519.1243. Purity 98.2%

by HPLC.

4.1.5.17

4-fluoro-N-(3-(3-oxo-4-(p-tolyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)

benzenesulfonamide (8c-2)

29% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.73 (dd, J = 8.9, 5.0 Hz, 1H,

Ar-H), 7.34 (d, J = 8.0 Hz, 1H, Ar-H), 7.19 (t, J = 8.3 Hz, 2H, Ar-H), 7.10 – 7.04 (m, 3H, Ar-H),

6.93 (ddd, J = 7.9, 2.0, 1.1 Hz, 1H, Ar-H), 6.58 – 6.55 (m, 1H, Ar-H), 4.80 (s, 2H, CH₂), 2.41 (s,

3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ = 165.24 (d, J_C-F= 255 Hz), 164.34, 141.65, 139.12,

136.76, 135.02, 134.99, 134.94, 132.82, 130.97, 130.84, 129.98 (d, J_C-F= 9.4 Hz)., 129.74, 128.40,

123.87, 122.79, 119.80, 119.55, 117.36, 116.32 (d, J_C-F= 22.7 Hz,), 115.49, 68.22, 21.29. HRMS

(DART-TOF) calculated for C₂₇H₂₁FN₂O₄S [M + H]⁺m/z 488.1206, found 488.1206. Purity 97.6%

by HPLC.

4.1.5.18

N-(2-chloro-5-(3-oxo-4-(p-tolyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)-4-fluorob

enzenesulfonamide (8c-3)

37% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J = 2.3 Hz, 1H, Ar-H),

7.93 (d, J = 2.3 Hz, 1H, Ar-H), 7.70 (dd, J = 9.0, 4.9 Hz, 2H, Ar-H), 7.39 (d, J = 8.0 Hz, 2H, Ar-H),

7.22 (d, J = 8.3 Hz, 3H, Ar-H), 7.15 (d, J = 1.1 Hz, 2H, Ar-H), 7.12 – 7.06 (m, 2H, Ar-H), 6.56 (t,

J = 1.2 Hz, 1H, Ar-H), 4.84 (s, 2H, CH₂), 2.43 (s, 3H, CH₃). HRMS (DART-TOF) calculated for

C₂₆H₁₉ClFN₃O₄S [M + H]⁺m/z 523.0769, found 523.0761. Purity 97.3% by HPLC.

4.1.5.19 6-(quinolin-3-yl)-4-(p-tolyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8c-4)

34% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ = 8.91 (d, J=2.1, 1H, Ar-H), 8.13

– 8.03 (m, 2H, Ar-H), 7.81 (d, J=8.1, 1H, Ar-H), 7.69 (ddd, J=8.4, 6.9, 1.4, 1H, Ar-H), 7.54 (dt,

J=11.8, 2.4, 1H, Ar-H), 7.40 – 7.29 (m, 3H, Ar-H), 7.25 – 7.16 (m, 3H, Ar-H), 6.75 (d, J=2.1, 1H,

Ar-H), 4.84 (s, 2H, CH₂), 2.44 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ = 164.11, 149.59,

145.03, 139.28, 132.82, 132.73, 132.62, 131.42, 130.94, 129.46, 129.18, 128.36, 127.88, 127.12,

123.03, 117.73, 115.80, 68.24, 21.29. HRMS (DART-TOF) calculated for C₂₄H₁₈N₂O₂[M + H]⁺

m/z 351.0662, found 351.0649. Purity 96.8% by HPLC.

4.1.5.20 6-(4-hydroxyphenyl)-4-(p-tolyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8c-5)

26% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ = 7.34 (d, J=8.2, 2H, Ar-H), 7.24 –

7.17 (m, 3H, Ar-H), 7.13 (dd, J=8.3, 2.0, 1H, Ar-H), 7.07 (d, J=8.3, 1H, Ar-H), 6.80 (d, J=8.5, 2H

,

Ar-H), 6.59 (d, J=2.0, 1H, Ar-H), 4.79 (s, 2H, Ar-H), 2.43 (s, 3H, Ar-H). HRMS (DART-TOF)

calculated for C₂₁H₁₇NO₃[M + H]⁺m/z 331.1208, found 331.1202. Purity 95.3% by HPLC.

4.1.5.21

4-fluoro-N-(2-methoxy-5-(4-(4-methoxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]

oxazin-6-yl)pyridin-3-yl)benzenesulfonamide (8d-1)

31% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.06 (s, 1H, SO₂NH), 7.92 (d, J

= 2.3 Hz, 1H, Ar-H), 7.73 (dd, J = 8.9, 5.2 Hz, 2H, Ar-H), 7.50 (d, J = 2.3 Hz, 1H, Ar-H), 7.38 (d,

J = 8.8 Hz, 2H, Ar-H), 7.34 (d, J = 9.0 Hz, 2H, Ar-H), 7.21 (dd, J = 8.3, 2.0 Hz, 1H, Ar-H), 7.17 (d,

J = 0.8 Hz, 2H, Ar-H), 7.15 (s, 1H, Ar-H), 6.42 (d, J = 1.9 Hz, 1H, Ar-H), 4.85 (s, 2H, CH₂), 3.84

(s, 3H, OCH₃), 3.66 (s, 3H, OCH₃). ¹³C NMR (101 MHz, DMSO) δ 164.72(d, J_C-F= 250 Hz),

164.45, 159.69, 156.13, 144.70, 140.06, 137.24, 131.95, 131.38, 130.57, 130.04 (d, J_C-F= 9 Hz),

ACCEPTED MANUSCRIPT

129.64, 129.35, 128.44, 122.08, 121.62, 117.81, 116.64(d, J_C-F= 22 Hz), 115.64, 114.42, 68.12,

55.86, 53.85. HRMS (DART-TOF) calculated for C₂₇H₂₂FN₃O₆S [M + H]⁺m/z 535.1213, found

535.1201. Purity 98.9% by HPLC.

4.1.5.22

4-fluoro-N-(3-(4-(4-methoxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)benz

enesulfonamide (8d-2)

39% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.42 (s, 1H, SO₂NH), 7.72 (dd,

J = 8.9, 5.1 Hz, 2H, Ar-H), 7.38 – 7.33 (m, 2H, Ar-H), 7.31 (d, J = 6.9 Hz, 2H, Ar-H), 7.23 (t, J =

7.9 Hz, 1H, Ar-H), 7.18 – 7.13 (m, 4H, Ar-H), 7.08 (t, J = 1.8 Hz, 1H, Ar-H), 7.03 (d, J = 7.8 Hz,

1H, Ar-H), 6.98 (dd, J = 8.0, 1.3 Hz, 1H, Ar-H), 6.41 (s, 1H, Ar-H), 4.84 (s, 2H, CH₂), 3.83 (s, 3H,

OCH₃). ¹³C NMR (101 MHz, CDCl₃) δ = 165.25(d, J_C-F= 254 Hz), 163.98, 159.77, 144.76,

141.65, 136.73, 135.02, 134.98, 134.91, 131.14, 129.97 (d, J_C-F= 9.4 Hz), 129.77, 127.91, 123.90,

122.74, 119.86, 119.58, 117.36, 116.34 (d, J_C-F= 22.7 Hz), 115.43, 68.22, 55.54. HRMS

(DART-TOF) calculated for C₂₇H₂₁FN₂O₅S [M + H]⁺m/z 504.1155, found 504.1142. Purity 97.3%

by HPLC.

4.1.5.23

N-(2-chloro-5-(4-(4-methoxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl

)-4-fluorobenzenesulfonamide (8d-3)

22% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.53 (s, 1H, SO₂NH), 8.44 (d, J

= 2.3 Hz, 1H, Ar-H), 8.19 (d, J = 2.3 Hz, 1H, Ar-H), 7.73 (dd, J = 8.9, 5.2 Hz, 2H, Ar-H), 7.50 (d,

J = 2.3 Hz, 1H, Ar-H), 7.38 (d, J = 8.8 Hz, 2H, Ar-H), 7.34 (d, J = 9.0 Hz, 2H, Ar-H), 7.21 (dd, J =

8.3, 2.0 Hz, 1H, Ar-H), 7.17 (d, J = 0.8 Hz, 2H, Ar-H), 7.15 (s, 1H, Ar-H), 6.42 (d, J = 1.9 Hz, 1H,

Ar-H), 4.85 (s, 2H, CH₂), 3.86 (s, 3H, OCH₃). HRMS (DART-TOF) calculated for

C₂₆H₁₉ClFN₃O₅S [M + H]⁺m/z 539.0718, found 539.0703. Purity 97.8% by HPLC.

4.1.5.24 4-(4-methoxyphenyl)-6-(quinolin-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8d-4)

23% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.92 (d, J = 2.1 Hz, 1H, Ar-H),

8.09 (d, J = 8.4 Hz, 2H, Ar-H), 7.82 (d, J = 8.1 Hz, 1H, Ar-H), 7.73 – 7.67 (m, 1H, Ar-H), 7.56 (t,

J = 7.6 Hz, 1H, Ar-H), 7.33 (dd, J = 8.3, 2.1 Hz, 1H, Ar-H), 7.28 – 7.24 (m, 2H, Ar-H), 7.19 (d, J

= 8.3 Hz, 1H, Ar-H), 7.06 (d, J = 8.9 Hz, 2H, Ar-H), 6.76 (d, J = 2.0 Hz, 1H, Ar-H), 4.85 (s, 2H,

CH₂), 3.86 (s, 3H, OCH₃).¹³C NMR (101 MHz, CDCl₃) δ = 164.32, 159.88, 149.42, 146.99, 145.05,

133.00, 132.95, 132.55, 131.58, 129.72, 129.58, 129.01, 127.91, 127.87, 127.78, 127.20, 123.04,

117.76, 115.74, 115.52, 68.24, 55.51. HRMS (DART-TOF) calculated for C₂₄H₁₈N₂O₃[M + H]⁺m/z

382.1317, found 382.1309. Purity 97.6% by HPLC.

4.1.5.25 6-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8d-5)

27% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.22 (t, J = 8.5 Hz, 4H, Ar-H),

7.14 (dd, J = 8.3, 2.1 Hz, 1H, Ar-H), 7.06 (t, J = 8.7 Hz, 3H, Ar-H), 6.80 (d, J = 8.7 Hz, 2H, Ar-H),

6.59 (d, J = 2.0 Hz, 1H, Ar-H), 4.79 (s, 2H, CH₂), 3.87 (s, 3H, OCH₃). ¹³C NMR (101 MHz, CDCl₃)

δ = 164.63, 159.69, 155.13, 143.96, 135.75, 131.03, 129.77, 128.08, 122.32, 117.18, 115.62, 115.37,

115.14, 68.27, 55.53. HRMS (DART-TOF) calculated for C₂₁H₁₇NO₄[M + H]⁺m/z 347.1158,

found 347.1143. Purity 96.3% by HPLC.

4.1.5.26

4-fluoro-N-(5-(4-(4-fluorophenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-methoxypyri

din-3-yl)benzenesulfonamide (8e-1)

ACCEPTED MANUSCRIPT

22% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.79 (dd, J = 6.6, 2.2 Hz,

2

H, Ar-H), 7.73 (dd, J = 8.9, 5.0 Hz,

Hz, H, Ar-H), 7.13 (d, J = 1.7 Hz, H, Ar-H), 7.08 (t, J = 8.5 Hz,

6.51 (s, 1H, Ar-H), 4.81 (s, , CH₂), 3.82 (s,

, OCH₃). ¹³C NMR (101 MHz, CDCl₃) δ

65.40 (d, J_C-F= 256.3 Hz), 164.08, 153.75, 144.80, 139.74, 134.99, 134.96, 134.55 (d, J_C-F= 106

Hz), 132.13, 130.82, 130.44, 130.13, 129.96, 129.84 (d, J_C-F= 9.5 Hz), 126.34, 122.80(d, J_C-F

2

H, Ar-H), 7.55 (d, J = 8.6 Hz,

2

H, Ar-H), 7.29 (d, J = 8.6

2

2H, Ar-H), 6.91 (s,

1

H, Ar-H),

2

H

3H

1

=

13.5 Hz), 120.78, 117.84, 116.38 (d, J_C-F= 22.7 Hz), 115.01, 68.19, 53.99. HRMS

(DART-TOF) calculated for C₂₆H₁₉F₂N₃O₅S [M + H]⁺m/z 523.1013, found 523.1011.

Purity 99.3% by HPLC.

4.1.5.27

4-fluoro-N-(3-(4-(4-fluorophenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)benzen

esulfonamide (8e-2)

26% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 7.75 (dd, J = 8.8, 5.1 Hz, 2H,

Ar-H), 7.69 (d, J = 8.6 Hz, 2H, Ar-H), 7.48 (d, J = 8.6 Hz, 2H, Ar-H), 7.35 (t, J = 8.8 Hz, 2H,

Ar-H), 7.24 (t, J = 7.9 Hz, 1H, Ar-H), 7.18 (s, 2H, Ar-H), 7.10 (s, 1H, Ar-H), 7.06 (d, J = 7.9 Hz,

1H, Ar-H), 7.01 (d, J = 8.0 Hz, 1H, Ar-H), 6.41 (s, 1H, Ar-H), 4.87 (s, 2H, CH₂). ¹³C NMR (101

MHz, DMSO) δ 164.73 (d, J_C-F= 250 Hz), 164.40, 144.80, 139.80 (d, J_C-F= 188.7 Hz), 136.39,

136.36, 135.03, 134.61, 133.90, 131.47, 131.28, 130.56, 130.36, 130.06 (d, J_C-F= 9.6 Hz), 122.47

(d, J_C-F= 13.5 Hz), 119.13, 117.81 (d, J_C-F= 9.4 Hz), 116.89 (d, J_C-F= 22.8 Hz), 114.53, 58.26.

HRMS (DART-TOF) calculated for C₂₆H₁₈F₂N₂O₄S [M + H]⁺m/z 492.0955, found 492.0949.

Purity 98.3% by HPLC.

4.1.5.28

N-(2-chloro-5-(4-(4-fluorophenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)-4

-fluorobenzenesulfonamide (8e-3)

23% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J = 2.2 Hz, 1H, Ar-H),

8.07 – 8.01 (m, 1H, Ar-H), 7.95 (d, J = 2.1 Hz, 1H, Ar-H), 7.72 (dd, J = 8.9, 4.9 Hz, 2H, Ar-H),

7.57 (d, J = 8.6 Hz, 1H, Ar-H), 7.30 (dt, J = 11.7, 4.2 Hz, 3H, Ar-H), 7.19 – 7.16 (m, 2H, Ar-H),

7.11 (t, J = 8.5 Hz, 2H, Ar-H), 6.53 (d, J = 2.8 Hz, 1H, Ar-H), 4.84 (s, 2H, CH₂). HRMS

(DART-TOF) calculated for C₂₅H₁₆ClF₂N₃O₄S [M + H]⁺m/z 527.0518, found 527.0516. Purity

97.3% by HPLC.

4.1.5.29 4-(4-fluorophenyl)-6-(quinolin-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8e-4)

32% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.92 (d, J = 2.2 Hz, 1H, Ar-H),

8.07 (dd, J = 9.2, 5.4 Hz, 2H, Ar-H), 7.82 (d, J = 8.2 Hz, 1H, Ar-H), 7.73 – 7.66 (m, 1H, Ar-H),

7.59 – 7.51 (m, 3H, Ar-H), 7.39 – 7.28 (m, 3H, Ar-H), 7.20 (d, J = 8.3 Hz, 1H, Ar-H), 6.73 (d, J =

2.0 Hz, 1H, Ar-H), 4.84 (s, 2H, CH₂). ¹³C NMR (101 MHz, CDCl₃) δ 164.07, 149.49, 146.19 (d,

J_C-F= 220.2 Hz), 135.14, 133.98, 132.84, 132.81 (d, J_C-F= 20 Hz), 131.02, 130.52, 130.09, 129.55,

129.23, 127.91, 127.80, 127.18, 123.43, 117.99, 117.34 (d, J_C-F= 23.0 Hz), 115.64, 68.21. HRMS

(DART-TOF) calculated for C₂₃H₁₅FN₂O₂[M + H]⁺m/z 370.1118, found 370.1116. Purity 98.7%

by HPLC.

4.1.5.30 4-(4-fluorophenyl)-6-(4-hydroxyphenyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8e-5)

24% isolated yield, white powder. ¹H NMR (400 MHz, MeOD) δ 7.60 (d, J = 8.7 Hz, 1H, Ar-H),

7.37 (d, J = 8.7 Hz, 2H, Ar-H), 7.20 (dd, J = 8.4, 2.1 Hz, 1H, Ar-H), 7.16 (d, J = 8.7 Hz, 1H, Ar-H),

7.08 (d, J = 8.4 Hz, 1H, Ar-H), 6.75 (dd, J = 8.8, 2.3 Hz, 1H, Ar-H), 6.53 (d, J = 2.0 Hz, 1H, Ar-H),

4.78 (s, 1H, CH₂). HRMS (DART-TOF) calculated for C₂₀H₁₄FNO₃[M + H]⁺m/z 335.0958, found

ACCEPTED MANUSCRIPT

335.0939. Purity 98.5% by HPLC.

4.1.5.31 4-fluoro-N-(2-methoxy-5-(4-(3-methoxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]

oxazin-6-yl)pyridin-3-yl)benzenesulfonamide (8f-1)

28% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ = 8.15 (d, J=2.2, 1H, Ar-H), 7.94

(dd, J=8.8, 5.0, 4H, Ar-H), 7.48 (t, J=8.1, 1H, Ar-H), 7.44 (d, J=2.2, 1H, Ar-H), 7.20 (t, J=8.5, 4H,

Ar-H), 7.12 (d, J=8.3, 1H, Ar-H), 7.03 (ddd, J=16.9, 8.3, 2.1, 2H, Ar-H), 6.91 (d, J=7.8, 1H, Ar-H),

6.86 (s, 1H, Ar-H), 6.58 (d, J=1.8, 1H, Ar-H), 4.82 (s, 2H, CH₂), 3.83 (d, J=10.0, 3H, OCH₃), 3.59

(s, 3H, OCH₃). ¹³C NMR (101 MHz, CDCl₃) δ = 165.98 (d, J_C-F= 257.6 Hz), 164.70, 163.91,

161.02, 160.70, 146.86, 144.85, 139.70, 136.49, 135.49, 135.45, 131.76 (d, J_C-F= 9.7 Hz,), 131.15,

130.94, 130.89, 130.32, 122.55, 120.71, 117.75, 117.68, 116.14 (d, J_C-F= 22.8 Hz), 115.05, 115.02,

114.36, 68.18, 55.51, 53.72. HRMS (DART-TOF) calculated for C₂₇H₂₂FN₃O₆S [M + H]⁺m/z

535.1213, found 535.1211. Purity 97.8% by HPLC.

4.1.5.32

4-fluoro-N-(3-(4-(3-methoxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)benz

enesulfonamide (8f-2)

1

33% isolated yield, white powder. H NMR (400 MHz, CDCl₃) δ 7.73 (dd, J = 8.8, 5.0 Hz, 2H,

Ar-H), 7.45 (t, J = 8.1 Hz, 1H, Ar-H), 7.18 (t, J = 7.8 Hz, 1H, Ar-H), 7.12 – 6.98 (m, 7H, Ar-H),

6.96 – 6.89 (m, 2H, Ar-H), 6.85 (t, J = 2.1 Hz, 1H, Ar-H), 6.57 (d, J = 1.4 Hz, 1H, Ar-H), 4.81 (s,

2H, CH₂), 3.80 (s, 3H, OCH₃). ¹³C NMR (101 MHz, CDCl₃) δ = 165.21 (d, J_C-F= 254 Hz), 164.26,

160.95, 144.67, 141.50, 136.88, 136.59, 135.02, 134.99, 130.86, 130.73, 129.99 (d, J_C-F= 9.4 Hz),

129.75, 123.73, 122.92, 120.78, 119.76, 119.39, 117.39, 116.34 (d, J_C-F= 22.7 Hz), 115.50, 115.07,

114.29, 68.19, 55.51. HRMS (DART-TOF) calculated for C₂₇H₂₁FN₂O₅S [M + H]⁺m/z 504.1155,

found 504.1149. Purity 97.9% by HPLC.

4.1.5.33

N-(2-chloro-5-(4-(3-methoxyphenyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl

)-4-fluorobenzenesulfonamide (8f-3)

23% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.56 (s, 1H, Ar-H), 8.60 (d, J =

2.3 Hz, 1H, Ar-H), 7.96 (dd, J = 9.0, 5.0 Hz, 2H, Ar-H), 7.69 (d, J = 2.3 Hz, 1H, Ar-H), 7.61 –

7.47 (m, 3H, Ar-H), 7.35 (dd, J = 8.3, 2.1 Hz, 1H, Ar-H), 7.26 (d, J = 8.3 Hz, 1H, Ar-H), 7.10 (dd,

J = 8.7, 2.9 Hz, 1H, Ar-H), 7.01 (t, J = 2.1 Hz, 1H, Ar-H), 6.96 (d, J = 10.3 Hz, 1H, Ar-H), 6.62 (d,

J = 2.1 Hz, 1H, Ar-H), 4.90 (s, 2H, CH₂), 3.78 (s, 3H, OCH₃). HRMS (DART-TOF) calculated for

C₂₅H₁₇ClF₂N₃O₄S [M + H]⁺m/z 528.0596, found 528.0579. Purity 98.5% by HPLC.

4.1.5.34 4-(3-methoxyphenyl)-6-(quinolin-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8f-4)

1

27% isolated yield, white powder. H NMR (400 MHz, CDCl₃) δ 8.93 (s, 1H, Ar-H), 8.23 – 8.08

(m, 2H, Ar-H), 7.84 (d, J = 8.1 Hz, 1H, Ar-H), 7.72 (t, J = 7.4 Hz, 1H, Ar-H), 7.58 (t, J = 7.5 Hz,

1H, Ar-H), 7.47 (t, J = 8.1 Hz, 1H, Ar-H), 7.34 (dd, J = 8.3, 2.0 Hz, 1H, Ar-H), 7.20 (d, J = 8.3 Hz,

1H, Ar-H), 7.03 (dd, J = 8.4, 2.4 Hz, 1H, Ar-H), 6.96 – 6.92 (m, 1H, Ar-H), 6.88 (t, J = 2.1 Hz, 1H,

Ar-H), 6.77 (d, J = 2.0 Hz, 1H, Ar-H), 4.85 (s, 2H, CH₂), 3.83 (s, 3H, OCH₃). ¹³C NMR (101 MHz,

CDCl₃) δ = 163.91, 161.05, 145.09, 136.47, 131.28, 130.96, 129.92, 127.93, 127.46, 123.18,

120.68, 117.85, 115.77, 115.00, 114.42, 68.21, 55.49. HRMS (DART-TOF) calculated for

C₂₄H₁₈N₂O₃[M + H]⁺m/z 382.1317, found 382.1309. Purity 97.5% by HPLC.

4.1.5.35 6-(4-hydroxyphenyl)-4-(3-methoxyphenyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (8f-5)

33% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 9.69 (s, 1H, Ar-H), 7.49 (t, J =

8.1 Hz, 1H, Ar-H), 7.20 – 7.07 (m, 5H, Ar-H), 7.00 – 6.92 (m, 2H, Ar-H), 6.75 (d, J = 8.6 Hz, 2H,

ACCEPTED MANUSCRIPT

Ar-H), 6.43 (d, J = 2.0 Hz, 1H, Ar-H), 4.82 (s, 2H, CH₂), 3.78 (s, 3H, OCH₃). HRMS (DART-TOF)

calculated for C₂₀H₁₅FNO₃[M + H]⁺m/z 336.1036, found 336.1029. Purity 97.3% by HPLC.

4.1.5.36

4-fluoro-N-(5-(4-isopropyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-methoxypyridin-3-

yl)benzenesulfonamide (13a-1)

22% isolated yield, white powder. 1H NMR (400 MHz, DMSO) δ 10.05 (s, 1H, SO₂NH), 8.27 (d, J =

2.3 Hz, 1H, Ar-H), 7.81 (dd, J = 9.0, 5.2 Hz, 2H, Ar-H), 7.79 (d, J = 2.3 Hz, 1H, Ar-H), 7.42 (t, J = 8.9

Hz, 2H, Ar-H), 7.36 (d, J = 1.9 Hz, 1H, Ar-H), 7.21 (dd, J = 8.3, 1.9 Hz, 1H, Ar-H), 7.12 (d, J = 8.3

Hz, 1H, Ar-H), 4.70 (dt, J = 13.8, 6.8 Hz, 1H, CH), 4.55 (s, 2H, CH₂), 3.66 (s, 3H, OCH₃), 1.50 (d, J =

6.9 Hz, 6H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ = 165.78, 165.4 (d, J_C-F= 254 Hz), 154.00, 146.34,

140.02, 135.18, 135.15, 132.00, 130.43, 129.93 (d, J_C-F= 9.5 Hz), 129.83, 126.94, 122.24, 120.83,

117.96, 116.38 (d, J_C-F= 22.7 Hz), 114.66, 68.63, 54.00, 47.52, 19.98. HRMS (DART-TOF)

calculated for C₂₃H₂₂FN₃O₅S [M + H]⁺m/z 471.1264, found 471.1246. Purity 97.2% by HPLC.

4.1.5.37

4-fluoro-N-(3-(4-isopropyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)

benzenesulfonamide (13a-2)

1

33% isolated yield, white powder. H NMR (400 MHz, CDCl₃) δ 7.86 (dd, J = 8.8, 5.0 Hz, 2H,

Ar-H), 7.66 (s, 1H, Ar-H), 7.35 – 7.25 (m, 3H, Ar-H), 7.11 (dt, J = 7.8, 5.7 Hz, 4H, Ar-H), 7.04 (d,

J = 8.3 Hz, 1H, Ar-H), 4.76 (tt, J = 9.1, 4.5 Hz, 1H, CH), 4.53 (s, 2H, CH₂), 1.57 (d, J = 7.0 Hz,

6H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ = 166.06, 165.3 (d, J_C-F= 254 Hz), 146.34, 141.91,

137.04, 135.15, 135.12, 135.07, 130.09, 129.96 (d, J_C-F= 6.7 Hz), 129.57, 124.01, 122.62, 120.22,

120.02, 117.74, 116.39 (d, J_C-F= 22.6 Hz), 114.98, 68.63, 47.68, 19.98. HRMS (DART-TOF)

calculated for C₂₃H₂₁FN₂O₄S [M + H]⁺m/z 440.1206, found 440.1205. Purity 96.3% by HPLC.

4.1.5.38

N-(2-chloro-5-(4-isopropyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)-4-fluoro

benzenesulfonamide (13a-3)

25% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.31 (d, J = 1.5 Hz, 1H, Ar-H),

8.13 (d, J = 1.4 Hz, 1H, Ar-H), 7.82 (dd, J = 8.8, 4.9 Hz, 2H, Ar-H), 7.21 – 7.10 (m, 4H, Ar-H),

7.03 (s, 1H, Ar-H), 4.81 (dt, J = 13.8, 6.9 Hz, 1H, CH), 4.56 (s, 2H, CH₂) 1.60 (d, J = 7.0 Hz, 6H,

CH₃). ¹³C NMR (101 MHz, CDCl₃) δ = 165.74 (d, J_C-F= 256 Hz), 165.56, 147.25, 143.54, 141.03,

136.57, 134.70, 130.43, 130.39, 130.15, 130.00, 129.51 (d, J_C-F= 9 Hz), 122.64, 118.30, 116.84 (d,

J_C-F= 23 Hz), 114.88, 68.59, 47.63, 20.00. HRMS (DART-TOF) calculated for C₂₂H₁₉ClFN₃O₄S

[M + H]⁺m/z 475.0769, found 475.0757. Purity 99.3% by HPLC.

4.1.5.39 4-isopropyl-6-(quinolin-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (13a-4)

1

22% isolated yield, white powder. H NMR (400 MHz, DMSO) δ = 9.14 (d, J=2.3, 1H, Ar-H),

8.51 (d, J=2.1, 1H, , Ar-H), 8.06 (dd, J=8.0, 3.2, 2H, Ar-H), 7.81 – 7.73 (m, 1H, Ar-H), 7.65 (t,

J=7.5, 1H, Ar-H), 7.44 (dd, J=8.3, 2.2, 1H, Ar-H), 7.33 (d, J=2.1, 1H, Ar-H), 7.13 (d, J=8.3, 1H

,

Ar-H), 4.76 (tt, J = 9.1, 4.5 Hz, 1H, CH), 4.66 (s, 2H, CH₂), 1.57 (d, J = 7.0 Hz, 6H, CH₃). HRMS

(DART-TOF) calculated for C₂₀H₁₈N₂O₂[M + H]⁺m/z 318.1368, found 318.1356. Purity 98.3%

by HPLC.

4.1.5.40 6-(4-hydroxyphenyl)-4-isopropyl-2H-benzo[b][1,4]oxazin-3(4H)-one (13a-5)

34% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ = 9.14 (s, 1H, Ar-H), 8.24 (d, J=2.1,

1H, Ar-H), 8.16 (d, J=8.4, 1H, Ar-H), 7.90 (d, J=8.1, 1H, Ar-H), 7.74 (t, J=7.6, 1H, Ar-H), 7.60 (t,

J=7.5, 1H, Ar-H), 7.43 (d, J=1.8, 1H, Ar-H), 7.33 (dd, J=8.2, 1.8, 1H, Ar-H), 7.16 (d, J=8.2, 1H

,

Ar-H), 4.91 – 4.77 (m, 1H, CH), 4.56 (s, 2H, CH₂), 1.62 (d, J=7.0, 6H). ¹³C NMR (101 MHz, CDCl₃) δ

ACCEPTED MANUSCRIPT

= 165.74, 149.61, 147.27, 146.65, 133.33, 133.01, 132.80, 130.08, 129.56, 129.25, 127.93, 127.25,

122.96, 118.13, 115.23, 68.67, 47.75, 20.01. HRMS (DART-TOF) calculated for C₁₇H₁₇NO₃[M +

H]⁺m/z 283.1208, found 283.1205. Purity 96.3% by HPLC.

4.1.5.41

N-(5-(4-cyclohexyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-methoxypyridin-3-yl)-4-flu

orobenzenesulfonamide (13b-1)

31% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 2.2 Hz, 1H, Ar-H),

7.96 (d, J = 2.1 Hz, 1H, Ar-H), 7.83 (dd, J = 8.8, 5.0 Hz, 2H, Ar-H), 7.18 – 7.04 (m, 4H, Ar-H),

7.00 (s, 1H, Ar-H, Ar-H), 4.52 (s, 2H, CH₂), 4.27 (ddd, J = 15.5, 9.7, 6.1 Hz, 1H, CH), 3.88 (s, 3H,

OCH₃), 2.37 (tt, J = 12.6, 6.5 Hz, 2H, CH₂), 1.89 (dd, J = 18.5, 15.3 Hz, 4H, CH₂), 1.73 (d, J =

12.3 Hz, 1H, CH₂), 1.49 – 1.34 (m, 2H, CH₂). ¹³C NMR (101 MHz, CDCl₃) δ = 165.99, 165.44 (d,

J_C-F= 255 Hz), 153.86, 146.50, 139.83, 135.20, 135.17, 131.90, 130.46, 130.43, 129.89 (d, J_C-F= 9.5

Hz), 126.72, 122.21, 120.91, 117.93, 116.53, 116.41 (d, J_C-F= 22.7 Hz), 68.80, 56.95, 54.05, 29.65,

26.42, 25.35. HRMS (DART-TOF) calculated for C₂₆H₂₆FN₃O₅S [M + H]⁺m/z 511.1577, found

511.1559. Purity 98.3% by HPLC.

4.1.5.42

N-(3-(4-cyclohexyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)-4-fluorobenzenesulfo

namide (13b-2)

28% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.85 (dd, J = 8.9, 5.0 Hz, 2H,

Ar-H), 7.34 – 7.27 (m, 4H, Ar-H), 7.16 – 7.08 (m, 3H, Ar-H), 7.05 (t, J = 7.4 Hz, 2H, Ar-H), 4.52

(s, 2H, CH₂), 4.29 – 4.16 (m, 1H, CH), 2.37 (tt, J = 12.7, 6.5 Hz, 2H, CH₂), 1.99 – 1.79 (m, 4H,

CH₂), 1.71 (d, J = 12.6 Hz, 1H, CH₂), 1.48 – 1.20 (m, 3H, CH₂). ¹³C NMR (101 MHz, CDCl₃) δ =

166.15, 165.3 (d, J_C-F= 255 Hz), 146.51, 142.02, 136.96, 135.20, 135.17, 134.97, 130.23, 130.04,

129.96 (d, J_C-F= 2.5 Hz), 124.00, 122.57, 120.04, 119.94, 117.70, 116.43 (d, J_C-F= 22.7 Hz),

115.12, 68.81, 57.06, 29.61, 26.39, 25.37. HRMS (DART-TOF) calculated for C₂₆H₂₅FN₂O₄S [M

+ H]⁺m/z 480.1519, found 480.1503. Purity 98.3% by HPLC.

4.1.5.43

N-(2-chloro-5-(4-cyclohexyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)-4-fluor

obenzenesulfonamide (13b-3)

36% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.31 (d, J = 1.5 Hz, 1H, Ar-H),

8.13 (d, J = 1.4 Hz, 1H, Ar-H), 7.82 (dd, J = 8.8, 4.9 Hz, 2H, Ar-H), 7.21 – 7.10 (m, 4H, Ar-H),

7.03 (s, 1H, Ar-H), 4.56 (s, 2H, CH₂) 4.29 – 4.16 (m, 1H, CH), 2.37 (tt, J = 12.7, 6.5 Hz, 2H, CH₂),

1.99 – 1.79 (m, 4H, CH₂), 1.71 (d, J = 12.6 Hz, 1H, CH₂), 1.48 – 1.20 (m, 3H, CH₂). HRMS

(DART-TOF) calculated for C₂₅H₂₃ClFN₃O₄S [M + H]⁺m/z 515.1082, found 515.1065. Purity

97.3% by HPLC.

4.1.5.44 4-cyclohexyl-6-(quinolin-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (13b-4)

1

22% isolated yield, white powder. H NMR (400 MHz, DMSO) δ = 9.14 (d, J=2.3, 1H, Ar-H),

8.51 (d, J=2.1, 1H, , Ar-H), 8.06 (dd, J=8.0, 3.2, 2H, Ar-H), 7.81 – 7.73 (m, 1H, Ar-H), 7.65 (t,

J=7.5, 1H, Ar-H), 7.44 (dd, J=8.3, 2.2, 1H, Ar-H), 7.33 (d, J=2.1, 1H, Ar-H), 7.13 (d, J=8.3, 1H

,

Ar-H), 4.56 (s, 2H, CH₂) 4.29 – 4.16 (m, 1H, CH), 2.37 (tt, J = 12.7, 6.5 Hz, 2H, CH₂), 1.99 – 1.79

(m, 4H, CH₂), 1.71 (d, J = 12.6 Hz, 1H, CH₂), 1.48 – 1.20 (m, 3H, CH₂). HRMS (DART-TOF)

calculated for C₂₃H₂₂N₂O₂[M + H]⁺m/z 358.1681, found 358.1663. Purity 99.3% by HPLC.

4.1.5.45 4-cyclohexyl-6-(4-hydroxyphenyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (13b-5)

36% isolated yield, white powder. ¹H NMR (400 MHz, CDCl₃) δ = 9.14 (d, J=2.1, 1H, Ar-H), 8.24 (d,

ACCEPTED MANUSCRIPT

J=1.9, 1H, Ar-H), 8.15 (d, J=8.4, 1H, Ar-H), 7.91 (d, J=8.1, 1H, Ar-H), 7.80 – 7.71 (m, 1H, Ar-H),

7.66 – 7.57 (m, 1H, Ar-H), 7.45 (d, J=1.7, 1H, Ar-H), 7.32 (dd, J=8.2, 1.9, 1H, Ar-H), 7.15 (d, J=8.2,

1H, Ar-H), 4.55 (s, 2H, CH₂), 4.28 (ddd, J=12.3, 8.0, 3.4, 1H, CH), 2.42 (qd, J=12.7, 3.7, 2H, CH₂),

1.91 (dd, J=15.4, 6.7, 4H, CH₂), 1.73 (d, J=12.5, 1H, CH₂), 1.50 – 1.21 (m, 3H, CH₂). ¹³C NMR (101

MHz, CDCl₃) δ = 165.96, 149.65, 147.30, 146.79, 133.34, 132.96, 132.77, 130.63, 129.55, 129.28,

127.96, 127.23, 122.98, 118.08, 115.41, 68.83, 57.11, 29.62, 26.40, 25.38. HRMS (DART-TOF)

calculated for C₂₀H₂₁NO₃[M + H]⁺m/z 323.1521, found 323.1509. Purity 98.1% by HPLC.

4.1.5.46

N-(5-(4-cyclopropyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-methoxypyridin-3-yl)-4-fl

uorobenzenesulfonamide (13c-1)

23% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.06 (s, 1H, SO₂NH), 8.25 (d, J =

2.3 Hz, 1H, Ar-H), 7.83 (dd, J = 8.9, 5.2 Hz, 2H, Ar-H), 7.78 (d, J = 2.3 Hz, 1H, Ar-H), 7.50 (d, J =

2.1 Hz, 1H, Ar-H), 7.42 (t, J = 8.9 Hz, 2H, Ar-H), 7.22 (dd, J = 8.3, 2.1 Hz, 1H, Ar-H), 7.07 (d, J = 8.3

Hz, 1H, Ar-H), 4.62 (s, 2H, CH₂), 3.68 (s, 3H, OCH₃), 2.87 – 2.80 (m, 1H, CH), 1.15 – 1.09 (m, 2H,

CH2), 0.71 – 0.61 (m, 2H, CH₂). HRMS (DART-TOF) calculated for C₂₃H₂₀FN₃O₅S [M + H]⁺m/z

469.1108, found 469.1105. Purity 95.3% by HPLC.

4.1.5.47

N-(3-(4-cyclopropyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)phenyl)-4-fluorobenzenesulf

onamide (13c-2)

27% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.46 (s, 1H, SO₂NH), 7.86 (dd,

J = 8.9, 5.1 Hz, 2H, Ar-H), 7.47 – 7.39 (m, 3H, Ar-H), 7.37 – 7.27 (m, 3H, Ar-H), 7.14 (dd, J = 8.3,

2.1 Hz, 1H, Ar-H), 7.10 – 7.01 (m, 2H, Ar-H), 4.62 (s, 2H, CH₂), 2.86 – 2.77 (m, 1H, CH), 1.15 –

1.06 (m, 2H, CH₂), 0.72 – 0.61 (m, 2H, CH₂).

¹³C NMR (101 MHz, CDCl₃) δ = 167.33, 165.3 (d,

J_C-F= 255 Hz), 145.40, 141.97, 136.80, 135.17, 134.91, 130.45, 130.06 (d, J_C-F= 9.4 Hz), 129.93,

124.19, 122.52, 120.37, 120.30, 117.28, 116.41 (d, J_C-F= 22.7 Hz), 115.07, 68.44, 29.70, 24.17,

9.16. HRMS (DART-TOF) calculated for C₂₃H₁₉FN₂O₄S [M + H]⁺m/z 438.1050, found 438.1039.

Purity 98.6% by HPLC.

4.1.5.48

N-(2-chloro-5-(4-cyclopropyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyridin-3-yl)-4-fluo

robenzenesulfonamide (13c-3)

31% isolated yield, white powder. ¹H NMR (400 MHz, DMSO) δ 10.06 (s, 1H, SO₂NH), 8.25 (d,

J = 2.3 Hz, 1H, Ar-H), 7.83 (dd, J = 8.9, 5.2 Hz, 2H, Ar-H), 7.78 (d, J = 2.3 Hz, 1H, Ar-H), 7.50 (d,

J = 2.1 Hz, 1H, Ar-H), 7.42 (t, J = 8.9 Hz, 2H, Ar-H), 7.22 (dd, J = 8.3, 2.1 Hz, 1H, Ar-H), 7.07 (d,

J = 8.3 Hz, 1H, Ar-H), 4.62 (s, 2H, CH₂), 2.87 – 2.80 (m, 1H, CH), 1.15 – 1.09 (m, 2H, CH2),

0.71 – 0.61 (m, 2H, CH₂). HRMS (DART-TOF) calculated for C₂₂H₁₇ClFN₃O₄S [M + H]⁺m/z

473.0612, found 473.0601. Purity 97.9% by HPLC.

4.1.5.49 4-cyclopropyl-6-(quinolin-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (13c-4)

1

21% isolated yield, white powder. H NMR (400 MHz, DMSO) δ = 9.14 (d, J=2.3, 1H, Ar-H),

8.51 (d, J=2.1, 1H, , Ar-H), 8.06 (dd, J=8.0, 3.2, 2H, Ar-H), 7.81 – 7.73 (m, 1H, Ar-H), 7.65 (t,

J=7.5, 1H, Ar-H), 7.44 (dd, J=8.3, 2.2, 1H, Ar-H), 7.33 (d, J=2.1, 1H, Ar-H), 7.13 (d, J=8.3, 1H

,

Ar-H), 4.62 (s, 2H, CH₂), 2.87 – 2.80 (m, 1H, CH), 1.15 – 1.09 (m, 2H, CH2), 0.71 – 0.61 (m, 2H,

CH₂). HRMS (DART-TOF) calculated for C₂₀H₁₆N₂O₂[M + H]⁺m/z 316.1212, found 316.1206.

Purity 98.5% by HPLC.

4.1.5.50 4-cyclopropyl-6-(4-hydroxyphenyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (13c-5)

Article Doi

Discovery of 4-phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as potent and orally active PI3K/mTOR dual inhibitors

DOI: 10.1016/j.ejmech.2019.06.021

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Article abstract of DOI:10.1016/j.ejmech.2019.06.021

Full text of DOI:10.1016/j.ejmech.2019.06.021

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