
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00228-6
10.1016/j.tranon.2024.102101
102101
Original Research
Oxazine drug-seed induces paraptosis and apoptosis through reactive oxygen species/JNK pathway in human breast cancer cells
Kim Na Young a1
Dukanya Dukanya b1
Sethi Gautam c1
Girimanchanaika Swamy S b
Yang Jirui d
Nagaraja Omantheswara e
Swamynayaka Ananda e
Vishwanath Divakar b
Venkantesha Keerthikumara e
Basappa Shreeja f
Chinnathambi Arunachalam g
Alharbi Sulaiman Ali g
Madegowda Mahendra e
Sukhorukov Alexey h
Pandey Vijay di
Lobie Peter E. pelobie@sz.tsinghua.edu.cn
dij⁎
Basappa Basappa basappa@chemistry.uni-mysore.ac.in
b⁎
Ahn Kwang Seok ksahn@khu.ac.kr
a1⁎
a Department of Science in Korean Medicine, Kyung Hee University, 24 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea
b Laboratory of Chemical Biology, Department of Studies in Organic Chemistry, University of Mysore, Manasagangotri, Mysuru-570006, India
c Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 16 Medical Drive, 117600, Singapore
d Tsinghua Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen-518055, Guangdong, China
e Department of Studies in Physics, University of Mysore, Manasagangotri, Mysuru-570006, India
f Department of Chemistry, BITS-Pilani Hyderabad Campus, Jawahar Nagar, Medchal-500078, India
g Department of Botany and Microbiology, College of Science, King Saud University, PO Box-2455, Riyadh 11451, Saudi Arabia
h N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect, 47, Moscow, 119991, Russia
i Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen, International Graduate School, Tsinghua University, Shenzhen-518055, Guangdong, China
j Shenzhen Bay Laboratory, Shenzhen 518055, Guangdong, China
⁎ Corresponding authors. pelobie@sz.tsinghua.edu.cnbasappa@chemistry.uni-mysore.ac.inksahn@khu.ac.kr
1 NYK, DD, and GS contributed equally to this work.

18 8 2024
11 2024
18 8 2024
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5 7 2024
11 8 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• To overcome drug-resistant breast cancer, it is essential to develop compounds that induce apoptosis and paraptosis.

• BSO-07 triggered G1 phase arrest, cleaved PARP, inhibited bcl-2, bcl-xL and survivin, and upregulated ATF4, CHOP, and p-JNK expression.

• BSO-07 induced both apoptosis and paraptosis via the reactive oxygen species and JNK pathway in human BC cells.

Small molecule-driven JNK activation has been found to induce apoptosis and paraptosis in cancer cells. Herein pharmacological effects of synthetic oxazine (4aS, 7aS)-3-((4-(4‑chloro-2-fluorophenyl)piperazin-1-yl)methyl)-4-phenyl-4, 4a, 5, 6, 7, 7a-hexahydrocyclopenta[e] [1,2]oxazine (FPPO; BSO-07) on JNK-driven apoptosis and paraptosis has been demonstrated in human breast cancer (BC) MDA-MB231 and MCF-7 cells respectively. BSO-07 imparted significant cytotoxicity in BC cells, induced activation of JNK, and increased intracellular reactive oxygen species (ROS) levels. It also enhanced the expression of apoptosis-associated proteins like PARP, Bax, and phosphorylated p53, while decreasing the levels of Bcl-2, Bcl-xL, and Survivin. Furthermore, the drug altered the expression of proteins linked to paraptosis, such as ATF4 and CHOP. Treatment with N-acetyl-cysteine (antioxidant) or SP600125 (JNK inhibitor) partly reversed the effects of BSO-07 on apoptosis and paraptosis. Advanced in silico bioinformatics, cheminformatics, density Fourier transform and molecular electrostatic potential analysis further demonstrated that BSO-07 induced apoptosis and paraptosis via the ROS/JNK pathway in human BC cells.

Keywords

Oxazine
Apoptosis
Paraptosis
ROS
JNK
Breast cancer
==== Body
pmcIntroduction

Breast cancer (BC) is responsible for approximately 16 % of cancer-related deaths and is the leading cause of cancer mortality among women globally. This disease is highly heterogeneous, comprising various subtypes that exhibit distinct clinical behaviors and treatment responses. The frequent development of chemotherapy resistance in BC often leads to therapeutic failures, highlighting the urgent need for new anticancer agents t#hat can tackle both the heterogeneity and resistance of BC [1,2]. In multi-cellular organisms, apoptosis is a biological process in which unwanted cells are eliminated. Apoptosis is a form of programmed cell death (PCD) induced by external agents. There are five recognized types of cell death: apoptosis, necrosis, autophagy, mitotic catastrophe, and senescence. Apoptosis and autophagy are typically active processes that depend on protein synthesis, while necrosis is often regarded as a passive process [3,4].

In the process of apoptosis, cells undergo distinct morphological changes including rounding up, retraction of pseudopods, reduction in cellular volume, condensation of chromatin, fragmentation of nuclei, and blebbing of the plasma membrane. Subsequently, these cells are typically engulfed by resident phagocytes. Conversely, paraptosis is induced by the expression of insulin-like growth factor receptor I, which leads to cytoplasmic vacuolization and mitochondrial swelling. Importantly, these paraptotic changes cannot be halted by caspase inhibitors or by the overexpression of antiapoptotic Bcl-2-like proteins. The initiation of paraptosis appears to be driven by a signaling cascade that involves mitogen-activated protein kinases [[5], [6], [7]]. Yamaguchi K. and colleagues developed innovative triptycene-peptide hybrids that effectively triggered a form of programmed cell death (PCD) known as paraptosis in Jurkat cells, a type of lymphocyte [8]. Since many cancer cells develop resistance to PCD, the future of cancer therapy could be transformed by compounds that induce both apoptosis and paraptosis. It is crucial to develop such compounds to treat drug-resistant BC cells.

Organic chemistry contributes only about 5 % of the known scaffolds used in contemporary pharmaceuticals. In developing lead compounds, significant focus is placed on privileged scaffold fragments found in natural products and traditional pharmaceutical drugs, particularly those developed for treating BC and other cancer types. The 5,6-dihydro-4H-1,2-oxazine scaffold, penicillazine, was originally isolated from marine fungi. Following this, several natural 1,2-oxazine-based molecules were identified. These molecules are chemically complex to synthesize, which poses challenges in exploring their pharmacological effects. As depicted in Fig. 1A, natural oxazine compounds have been extensively researched and have demonstrated strong anti-oncogenic properties in various tumor models [9].Fig. 1 1,2-oxazines derivatives. (A) The development of 1,2-oxazines from mother nature to laboratory scale up and lead discovery. (B) Synthetic route for the preparation of 1,2-oxazines derivatives.

Fig 1

Oxidative stress is known to play a role in promoting apoptosis. Notably, a traditional Chinese herb, magnolia, contains a component known as honokiol, which not only inhibits growth but also induces apoptosis in various cancer cell lines. At lower concentrations, honokiol can trigger a form of programmed cell death (PCD) that is distinct from apoptosis, characterized by cytoplasmic vacuolization and swelling of the endoplasmic reticulum in K562 cells. This suggests that honokiol's PCD effects encompass both apoptotic and non-apoptotic mechanisms in leukemia cells [10]. Additionally, the biosynthetic pathway of asmaphorazine A, a natural oxazine molecule, involves the oxidation of an alkene bond to an epoxide and the subsequent reaction of this epoxide with neighboring nitrogen. This molecule has also been shown to inhibit nitric oxide production in LPS-stimulated J774.1 cells in a dose-dependent manner [11]. Another natural oxazine compound, (+)-FR900482, has been identified as an anti-tumor agent that acts as a DNA cross-linker. Its unique DNA-protein recognition properties upon interacting with endonucleases make it a promising clinical alternative to mitomycin C [12].

In a study using human tumor cell lines A549, LOVO, HL-60, and 6T-CEM, it was found that Asiaticumines B alkaloid was cytotoxic at a nanomolar concentration. Due to challenges in large-scale synthesis, the pharmacological properties of other oxazines like Flustrarine B and Secuamamine D, which have complex structural syntheses, remained unexplored [13]. Further studies revealed that trichodermamide B was cytotoxic to colorectal cancer cells (HCT-116) at lower micromolar concentrations, while its analogue, trichodermamide A, was not cytotoxic but could induce DNA oxidation [14]. Additionally, Alsmaphorazine A, synthesized through the biogenic oxidation of an alkene bond to an epoxide and subsequent reaction of the nitrogen with the epoxide, effectively inhibited nitric oxide production in LPS-stimulated J774.1 cells in a dose-dependent manner without affecting cell viability [11].

The bioactive fungal natural product, pretrichodermamide A, features transannular disulfide bridges synthesized by an FAD-dependent oxidoreductase. This was discovered through genome mining and gene deletion techniques in Trichoderma hypoxylon [15]. Additionally, the compound 2-ethoxy-4,5-diphenyl-1,3-oxazine-6-one (EDPOO) has demonstrated a protective effect against LPS-induced cell death in rat pheochromocytoma cells by modulating nuclear transcription factors activated by intracellular reactive oxygen species and mediators produced from chemical exposure. EDPOO also enhances γ-GCS levels and antioxidant enzyme activities in a dose-dependent manner [16]. Moreover, the oxazine-based drug WIN 55,212–2 induced apoptosis in U251 cells and triggered ROS-mediated DNA damage through the dysfunction of the VEGF-AKT/FAK signaling pathway. [17]. Fostamatinib, another compound can promote differentiation and inhibit the clonogenic potential of AML cells via the MEK/ERK1/2 pathway and the STAT5A transcription factor [18].

In this study, we have synthesized a new library of oxazines and identified the lead compound FPPO, which effectively induced G1 phase arrest and triggered apoptosis and paraptosis in human BC cells. These responses were found to be systematically mediated through the ROS/JNK pathway for the first time.

Materials and methods

The 1H and 13C NMR spectra were recorded on a Bruker WH-200 and Agilent-NMR-Vnmrs 400 spectrometer in CDCl3 or DMSO‑d6 as solvent, using TMS as an internal standard and chemical shifts are expressed as ppm. Mass and FT-IR spectroscopy were performed using Agilent LC-MS and Perkin Elmer Spectrum-II. The progress of the reaction was monitored by TLC pre-coated silica gel 60 F254 plates and the spots were observed under UV light.

Typical procedure for the synthesis of 1,2-oxazine compounds

To a solution DMF containing various piperazines/piperidines and dried K2CO3 (2.5 equivalence), substituted 3-bromomethyl oxazines was added and the reaction mixture was stirred for the required hour. The completion of the reaction was monitored by TLC. The organic layer was washed with distilled water and dried over sodium sulphate. Ethyl acetate was distilled off under reduced pressure and purified by column chromatography by using ethyl acetate and hexane as an eluent.

3-((4-(4-chlorophenyl)piperazin-1-yl)methyl)−4-(4-methoxyphenyl)−4a,5,6,7,8,8a-hexahydro-4H-benzo[e][1,2]oxazine (3I)

Off White solid; mp: 108–110 °C; 87 % yield; 1H NMR (CDCl3, 400 MHz): δ 7.18 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 4.02 (s, 1H), 3.79 (s, 3H), 3.43 (s, 1H), 3.16–3.06 (m, 5H), 2.92–2.89 (m, 1H), 2.69–2.65 (m, 2H), 2.44–2.41 (m, 2H), 2.08–2.04 (m, 1H), 1,75–1.59 (m, 4H), 1.45–1.40 (m, 1H), 1.34–1.29 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 158.5, 153.7, 149.9, 133.9, 129.3, 128.9, 124.4, 117.1, 114.1, 68.6, 60.1, 55.2, 52.9, 49.1, 43.1, 38.8, 29.3, 27.7, 25.1, 19.9; LCMS (ESI): m/z Calcd. for C26H32ClN3O2 [M + H]+: 454.2261; Found: 454.2251.

3-((4-(4-chlorophenyl)piperazin-1-yl)methyl)−4-phenyl-4a,5,6,7,8,8a-hexahydro-4H-benzo[e][1,2]oxazine (3II)

Off White solid; mp: 132–134 °C; 91 % yield; IR √max: 1492.96 cm−1 √(C = C), 820.0 cm−1 √(C—Cl); 1H NMR (CDCl3, 400 MHz): δ 7.36–7.32 (m, 2H), 7.28–7.24 (m, 1H), 7.18 (d, J = 8.8 Hz, 4H), 6.81 (d, J = 8.8 Hz, 2H), 4.04 (s, 1H), 3.49 (s, 1H), 3.15–3.06 (m, 5H), 2.94–2.91 (m, 1H), 2.69–2.65 (m, 2H), 2.44–2.40 (m, 2H), 2.08–2.04 (m, 1H), 1.74–1.72 (m, 2H), 1.65–1.62 (m, 2H), 1.47–1.43 (m, 1H), 1.36–1.26 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 153.4, 149.9, 141.8, 128.9, 128.7, 128.3, 126.9, 124.3, 117.1, 68.5, 60.2, 52.8, 49.1, 43.9, 38.7, 29.3, 27.8, 25.1, 19.9; LCMS (ESI): m/z Calcd. for C25H30ClN3O [M + H]+: 424.2155; Found: 424.0968.

4-(4-chlorophenyl)−3-((4-(4-chlorophenyl)piperazin-1-yl)methyl)−6,6-dimethyl-5,6-dihydro-4H-1,2-oxazine (3III)

Off White solid; mp: 138–140 °C; 79 % yield; 1H NMR (CDCl3, 400 MHz): δ 7.31–7.29 (m, 2H), 7.20–7.10 (m, 4H), 6.80 (d, J = 8.8 Hz, 2H), 3.62–3.58 (m, 1H), 3.20–3.0 (m, 4H), 2.88–2.85 (m, 1H), 2.79–2.77 (m, 1H), 2.51–2.50 (m, 2H), 2.31–2.20 (m, 2H), 2.07–2.02 (m, 1H), 196–1.86 (m, 1H), 1.36 (s, 3H), 1.27 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 156.0, 146.9, 139.2, 132.8, 130.0, 128.9, 128.8, 124.5, 117.14, 74.1, 59.6, 52.7, 49.2, 39.8, 37.4, 28.5, 22.9; LCMS (ESI): m/z Calcd. for C23H27Cl2N3O [M + H]+: 432.1609; Found: 432.1613.

3-((4-(4-chlorophenyl)piperazin-1-yl)methyl)−6,6-dimethyl-4-phenyl-5,6-dihydro-4H-1,2- oxazine (3IV)

Off White solid; mp: 118–120 °C; 89 % yield; IR √max: 1495.48cm−1 √(C = C), 1230.95 cm−1 √(C—N), 815.54 cm−1 √(C—Cl); 1H NMR (CDCl3, 400 MHz): δ 7.34–7.32 (m, 2H), 7.27.7.17 (m, 5H), 6.80 (d, J = 8.8 Hz, 2H), 3.65–3.60 (m, 1H), 3.13–3.05 (m, 4H), 2.91–2.88 (m, 1H), 2.79–2.76 (m, 1H), 2.61–2.58 (m, 2H), 2.28–2.25 (m, 2H), 2.09–2.04 (m, 1H), 1.98–1.92 (m, 1H), 1,37 (s,3H), 1.29 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 156.6, 149.9, 140.7, 128.7, 128.9, 128.6, 127.0, 124.4, 117.1, 74.1, 59.6, 52.7, 49.3, 40.0, 38.0, 28.5, 22.9; LCMS (ESI): m/z Calcd. for C23H28ClN3O [M + H]+: 398.1999; Found: 398.1110.

3-((4-(4-chlorophenyl)piperazin-1-yl)methyl)−4-phenyl-4a,5,6,7,8,8a-hexahydro-4H-5,8-methanobenzo[e][1,2]oxazine (3 V)

Off White solid; mp: 148–150 °C; 92 % yield; IR √max: 1494.57 cm−1 √(C = C), 1234.02 cm−1 √(C—N), 823.17 cm−1 √(C—Cl); 1H NMR (CDCl3, 400 MHz): δ 7.34–7.26 (m, 5H), 7.16 (d, J = 8.8 Hz, 2H), 6.76 (d, J = 8.8 Hz, 2H), 3.78 (d, J = 6.4 Hz, 1H), 3.21(d, J = 10.8 Hz, 1H), 2.96–2.84 (m, 6H), 2.53–2.52 (m, 1H), 2.34–2.31 (m, 2H), 2.23–2.13 (m, 4H), 1.98 (s, 1H), 1.61–1.58 (m, 1H), 1.47–1.43 (m, 1H), 1.20–1.01 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 171.7, 149.9, 138.3, 129.9, 128.8, 128.0, 126.9, 124.3, 117.1, 82.8, 57.9, 53.2, 52.2, 49.1, 42.7, 42.2, 40.1, 33.4, 29.0, 24.7; LCMS (ESI): m/z Calcd. for C26H30ClN3O [M + H]+: 436.2155; Found: 436.088.

3-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)−6,6-dimethyl-5,6-dihydro-4H-1,2-oxazin- 4-yl benzoate (3VI)

Off White solid; mp: 128–130 °C; 84 % yield; 1H NMR (CDCl3, 400 MHz): δ 8.06 (d, J = 8.8 Hz, 2H), 7.61–7.59 (m, 1H), 7.51–7.47 (m, 2H), 7.12–7.10 (m, 2H), 6.83–6.81 (m, 1H), 5.74 (t, J = 8 Hz, 1H), 3.48–3.44 (m, 1H), 3.21–3.17 (m, 1H), 2.92–2.83 (m, 4H), 2.67–2.64 (m, 2H), 2.54–2.52 (m, 2H), 2.28–2.23 (m, 1H), 2.07–2.02 (m, 1H), 1.40 (s, 3H), 1.38 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 165.5, 152.9, 151.4, 134.1, 133.5, 129.9, 129.8, 128.7, 127.6, 127.4, 124.6, 118.7, 74.4, 60.4, 59.1, 53.3, 51.2, 36.7, 26.3, 25.4; LCMS (ESI): m/z Calcd. for C24H27Cl2N3O [M + H]+: 476.1507; Found: 476.1513.

4-(4-chlorophenyl)−3-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)−6,6-dimethyl-5,6-dihydro-4H-1,2-oxazine (3VII)

Off White solid; mp: 136–138 °C; 86 % yield; 1H NMR (CDCl3, 400 MHz): δ 7.29 (d, J = 8 Hz, 2H), 7.19–7.09 (m, 4H), 6.95–6.91 (m, 1H), 3.63–3.55 (m, 1H), 2.98–2.89 (m, 4H), 2.84 (d, J = 8 Hz, 2H), 2.62–2.59 (m, 2H), 2.32–2.28 (m, 2H), 2.08–2.02 (m, 1H), 1.92–1.86 (m, 1H), 1.35 (s, 3H), 1.27 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 156.0, 151.1, 139.2, 133.9, 132.7, 129.9, 129.2 128.8, 127.3, 124.5, 118.5, 74.0, 59.6, 52.3, 51.4, 39.9, 37.4, 28.5, 22.9; LCMS (ESI): m/z Calcd. for C23H26Cl3N3O [M + H]+: 466.1219; Found: 465.9870.

3-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)−4-phenyl-4,4a,5,6,7,7a-hexahydrocyclo penta[e][1,2]oxazine (3VIII)

Off White solid; mp: 120–122 °C; 79 % yield; IR √max: 1578.30 cm−1 √(C = C), 1237.95 cm−1 √(C—N), 778.69 cm−1 √(C—Cl); 1H NMR (CDCl3, 400 MHz): δ 7.35–7.32 (m, 2H), 7.28–7.24 (m, 1H), 7.18 (d, J = 8 Hz, 2H), 7.15–7.12 (m, 2H), 6.94–6.92 (m, 1H), 4.03 (s, 1H), 3.49 (s, 1H), 3.11–3.08 (m, 1H), 3.01–2.94 (m, 4H), 2.72–2.69 (m, 2H), 2.47–2.45 (m, 2H), 2.05 (d, J = 8 Hz, 1H), 1.73–1.71 (m, 2H), 1.64–1.61 (m, 2H), 1.27–1.25 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 153.3, 151.2, 141.8, 133.9, 128.7, 128.3, 127.4, 127.3, 126.8, 124.4, 118.5, 68.5, 60.1, 53.1, 51.2, 43.9, 38.7, 29.2, 27.8, 25.1; LCMS (ESI): m/z Calcd. for C24H27Cl2N3O [M + H]+: 444.1609; Found: 444.1614.

3-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)−4-phenyl-4a,5,6,7,8,8a-hexahydro-4H- benzo[e][1,2]oxazine (3IX)

Off White solid; mp: 130–132 °C; 85 % yield; IR √max: 1450.95 cm−1 √(C = C), 1246.62 cm−1 √(C—N), 886.63 cm−1 √(C—Cl); 1H NMR (CDCl3, 400 MHz): δ 7.36–7.32 (m, 2H), 7.28–7.24 (m, 1H), 7.18 (d, J = 8 Hz, 2H), 7.15–7.09 (m, 2H), 6.94–6.92 (m, 1H), 4.03 (s, 1H), 3.49 (s, 1H), 3.24–3.08 (m, 1H), 3.09–2.90 (m, 4H), 2.71–2.69 (m, 2H), 2.48–2.44 (m, 2H), 2.07–2.04 (m, 1H), 1.74–1.68 (m, 2H), 1.65–1.58 (m, 2H), 1.46–1.25 (m, 5H); 13C NMR (CDCl3, 100 MHz): δ 153.4, 151.3, 141.8, 133.9, 128.7, 128.3, 127.4, 127.3, 126.8, 124.4, 118.5, 68.5, 60.2, 53.1, 51.3, 43.9, 43.8, 38.7, 29.3, 27.8, 25.1; LCMS (ESI): m/z Calcd. for C25H29Cl2N3O [M + H]+: 458.1765; Found: 458.0439.

(4R,4aS,5S,8R,8aS)−3-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)−4-phenyl-4a,5,6,7,8,8a-hexahydro-4H-5,8-methanobenzo[e][1,2]oxazine (3X)

Off white; mp: 121–123 °C; 1H NMR (CDCl3, 400 MHz): δ 7.35–7.28 (m, 5H), 7.13–7.11 (m, 2H), 6.91–6.88 (m, 1H), 3.74 (d, J = 8 Hz, 1H), 3.22 (d, J = 8 Hz, 1H), 2.97–2.85 (m, 6H), 2.53–2.52 (m, 1H), 2.38–2.36 (m, 2H), 2.29–2.25 (m, 2H), 2.18–2.14 (m, 2H), 1.99–1.98 (m, 1H), 1.64–1.58 (m, 1H), 1.49–1.42 (m, 1H), 1.20 (d, J = 8 Hz, 2H), 1.13–1.11 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 171.7, 151.3, 138.4, 133.9, 129.9, 128.1, 127.4, 127.3, 126.9, 124.34, 118.5, 58.1, 53.2, 52.5, 51.3,42.7, 42.5, 40.1, 33.4, 29.0, 24.7; MS: m/z: 470.4340 [M + H]+; Molecular formula: C26H29Cl2N3O.

3-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)−6,6-dimethyl-4-phenyl-5,6-dihydro-4H-1,2-oxazine (3XI)

Off white; mp: 128–130 °C; 1H NMR (CDCl3, 400 MHz): δ 7.34–7.30 (m, 2H), 7.25–7.23 (m, 3H), 7.15–7.12 (m, 2H), 6.95–6.93 (m, 1H), 3.00 (s, 4H), 2.93–2.81 (m, 2H), 2.63 (s, 2H), 2.32 (s, 2H), 2.09–2.04 (m, 1H), 1.98–1.91 (m, 1H), 1.36 (s, 3H), 1.29 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 156.6, 151.2 140.8, 134.0, 128.7, 128.6, 127.5, 127.3, 127.0, 124.5, 118.5, 74.1, 59.6, 52.9, 51.4, 40.1, 38.1, 28.5, 22.9; MS: m/z: 432.3860 [M + H]+; Molecular formula: C23H27Cl2N3O.

3-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)−4,6-diphenyl-5,6-dihydro-4H-1,2-oxazine (3XII)

Brown; mp: 82–83 °C; 1H NMR (CDCl3, 400 MHz): δ 7.45–7.28 (m, 10H), 7.16–7.13 (m, 3H), 6.98–6.92 (m, 1H), 3.94 (s, 1H), 3.19–3.15 (m, 1H), 3.05–2.95 (m, 4H), 2.69–2.66 (m, 2H), 2.56–2.47 (m, 2H), 2.41–2.28 (m, 2H), 2.20–2.16 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 158.6, 151.2, 141.6, 139.5, 134.0, 128.9, 128.8, 128.5, 128.4, 128.1, 127.4, 126.6, 126.5, 124.5, 118.5, 78.0, 60.1, 53.1, 51.3, 37.1, 34.3; MS: m/z: 480.4288 [M + H]+; Molecular formula: C27H27Cl2N3O.

3-((4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)methyl)−6-ethoxy-4-(4-methoxyphenyl)−5,6-dihydro-4H-1,2-oxazine (3XIII)

Off White solid; mp: 100–102 °C; 91 % yield; IR √max: 2935.28 cm−1 √(NH), 1511.54 cm−1 √(C = C), 1239.53 cm−1 √(C—N); 1H NMR (CDCl3, 400 MHz): δ 7.72–7.41 (m, 2H), 7.23–7.21 (m, 2H), 7.13 (d, J = 8 Hz, 2H), 6.81(d, J = 8 Hz, 2H), 5.14 (s, 1H), 3.88–3.83 (m, 1H), 3.78 (s, 3H), 3.73–3.57 (m, 2H), 3.00–2.99 (m, 1H), 2.88–2.85 (m, 2H), 2.68–2.65 (m, 1H), 2.49–2.46 (m, 1H), 2.25–2.20 (m, 1H), 2.16–1.95 (m, 8H), 1.25–1.16 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 160.8, 158.5, 157.6, 133.4, 132.2, 129.8, 122.3,119.1, 114.0, 95.8, 63.3, 60.0, 55.2, 54.1, 51.3, 36.5, 34.1, 32.8,31.1, 15.1; LCMS (ESI): m/z Calcd. for C26H32N4O3 [M + H]+: 449.2552; Found: 449.2612.

3-((4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)methyl)−4,6-diphenyl-5,6-dihydro-4H-1,2-oxazine (3XIV)

Off White solid; mp: 112–114 °C; 88 % yield; IR √max: 2821.58 cm−1 √(NH), 1447.64 cm−1 √(C = C), 1248.74 cm−1 √(C—N); 1H NMR (CDCl3, 400 MHz): δ 10.9 (Br-s, 1H) 7.64–7.49 (m, 2H) 7.44–7.27 (m, 7H), 7.22–7.09 (m, 5H), 4.90–4.78 (m, 1H), 3.86–3.82 (m, 1H), 3.11–3.01 (m, 1H), 2.91–2.71 (m, 3H), 2.62–2.60 (m, 1H), 2.47–2.24 (m, 2H), 2.14–1.19 (m, 6H); 13C NMR (CDCl3, 100 MHz): δ 159.4, 157.9, 156.4, 141.5, 140.2, 139.3, 128.9, 128.8, 128.4, 128.2, 127.2, 126.6, 122.1, 78.2, 73.5, 60.30, 54.5, 52.1, 41.4, 36.9, 34.1, 31.1; LCMS (ESI): m/z Calcd. for C29H30N4O [M + H]+: 451.2497; Found: 451.2535.

(3XV)

Off white solid; mp: 95–99 °C; 1H NMR (CDCl3, 400 MHz): δ 7.37–7.34 (m, 2H), 7.29–7.27 (m, 1H), 7.20–7.18 (m, 2H), 7.03 (d, J = 8 Hz, 2H), 6.85 (t, J = 8 Hz, 1H), 4.04 (s, 1H), 3.50 (s, 1H), 3.11–2.95 (m, 6H), 2.71–2.69 (m, 2H), 2.47–2.45 (m, 2H), 2.06 (d, J = 12 Hz, 1H), 1.74 (m, 2H), 1.64 (d, J = 12 Hz, 2H), 1.45 (d, J = 12 Hz, 2H), 1.40–1.25 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 156.3, 153.4, 141.5, 128.8, 128.4, 126.9, 124.5, 124.4, 119.6, 116.9, 116.7, 68.5, 60.2, 52.9, 50.5, 50.5, 43.9, 38.7, 29.3, 27.8, 25.2, 19.9; MS: m/z: 441.1983 [M + H]+; Molecular formula: C25H29ClFN3O.

(4aS,8aS)−3-((4-(4‑chloro-2-fluorophenyl)piperazin-1-yl)methyl)−4-(4-methoxyphenyl)−4a,5,6,7,8,8a-hexahydro-4H-benzo[e][1,2]oxazine (3XVI)

Off white; mp: 145–149 °C; 1H NMR (CDCl3, 400 MHz): δ 7.09 (d, J = 8 Hz, 2H), 7.04–7.01 (m, 2H), 6.88–6.84 (m, 3H), 4.02 (s, 1H), 3.80 (s, 3H), 3.43 (s, 1H), 3.11–2.92 (m, 6H), 2.69–2.68 (m, 2H), 2.46–2.45 (m, 2H), 2.05 (d, J = 12 Hz, 1H), 1.75–1.59 (m, 4H), 1.44 (d, J = 12 Hz, 1H), 1.33–1.25 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 158.5, 153.7, 138.9, 133.9, 129.3, 124.4, 119.5, 116.9, 116.7, 114.1, 68.4, 60.1, 55.3, 52.9, 50.5, 43.1, 38.8, 29.3, 27.7, 25.1, 19.9; MS: m/z: 471.9946 [M + H]+; Molecular formula: C26H31ClFN3O2.

3-((4-(4‑chloro-2-fluorophenyl)piperazin-1-yl)methyl)−6,6-dimethyl-5,6-dihydro-4H-1,2-oxaz in-4-yl benzoate (3XVII)

Brown; mp: 83–85 °C; 1H NMR (CDCl3, 400 MHz): δ 8.03 (d, J = 4 Hz, 2H), 7.61–7.58 (m, 1H), 7.48–7.45 (m, 2H), 7.00–6.98 (m, 2H), 6.72 (t, J = 8 Hz, 1H), 3.43 (d, J = 8 Hz, 1H), 3.15 (d, J = 8 Hz, 1H), 2.88 (s, 4H), 2.63–2.61 (m, 2H), 2.50–2.48 (m, 2H), 2.25–2.21 (m, 1H), 2.04–2.00 (m, 2H), 1.39 (s, 3H), 1.37 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 165.4, 152.8, 133.5, 129.7, 129.7, 128.5, 124.4, 119.5, 116.9, 116.7, 74.4, 60.2, 58.9, 53.0, 50.3, 36.5, 26.2, 25.3; MS: m/z: 459.9409 [M + H]+; Molecular formula: C24H27ClFN3O3.

(4R,4aS,5S,8R,8aS)−3-((4-(4‑chloro-2-fluorophenyl)piperazin-1-yl)methyl)−4-phenyl-4a,5,6,7,8,8a-hexahydro-4H-5,8-methanobenzo[e][1,2]oxazine (3XVIII)

Off white; mp: 81–83 °C; 1H NMR (CDCl3, 400 MHz): δ 7.35–7.27 (m, 5H), 7.02–6.98 (m, 2H), 6.82–6.77 (m, 1H), 3.73 (d, J = 4 Hz, 1H), 2.96–2.85 (m, 6H), 2.52 d, J = 4 Hz, 1H), 2.37–2.32 (m, 2H), 2.26–2.21 (m, 2H), 2.18–2.13 (m, 2H), 1.99–1.98 (m, 1H), 1.65–1.57 (m, 1H), 1.49–1.41 (m, 1H), 1.25–1.18 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 71.7, 156.5, 153.9, 139.0, 138.9, 138.4, 129.9, 128.0, 126.9, 126.6, 126.5, 124.4, 124.3, 119.5, 119.4, 116.9, 116.6, 82.8, 58.0, 53.2, 52.3, 50.5, 50.4, 42.7, 42.2, 40.1, 33.3, 29.0, 24.7; MS: m/z: 453.9794 [M + H]+; Molecular formula: C26H29ClFN3O.

(4aS,7aS)−3-((4-(4‑chloro-2-fluorophenyl)piperazin-1-yl)methyl)−4-phenyl-4,4a,5,6,7,7a-hexahydrocyclopenta[e][1,2]oxazine (BSO-07) (3XIX)

Off white; mp: 83–84 °C; 1H NMR (CDCl3, 400 MHz): δ 7.27(t, J = 8 Hz, 2H), 7.19 (d, J = 8 Hz, 1H), 7.09–6.99 (m, 3H), 6.88–6.77 (m, 2H), 3.51 (s, 1H), 3.43–3.31 (m, 2H), 3.07 (s, 3H), 2.96 (s, 3H), 2.69 (s, 2H), 2.60 (s, 1H), 2.46 (s, 1H), 2.09–2.03 (m, 2H), 1.80 (s, 1H), 1.26–1.23 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 130.7, 129.6, 128.4, 124.4, 119.6, 116.6, 116.9, 59.3, 53.1, 52.8, 52.5, 50.6, 50.5; MS: m/z: 427.9421 [M + H]+; Molecular formula: C24H27ClFN3O.

3-((4-(4‑chloro-2-fluorophenyl)piperazin-1-yl)methyl)−6,6-dimethyl-4-phenyl-5,6-dihydro-4H-1,2-oxazine (3XX)

Off white; mp: 78–80 °C; 1H NMR (CDCl3, 400 MHz): δ 7.33–7.30 (m, 2H), 7.25–7.21 (m, 3H), 7.02 (s, 1H), 7.00–6.99 (m, 1H), 6.83 (t, J = 8 Hz, 1H), 3.65–3.60 (m, 1H), 3.01–2.96 (m, 4H), 2.90–2.87 (m, 1H), 2.81–2.78 (m, 1H), 2.61–2.59 (m, 2H), 2.30–2.27 (m, 2H), 2.09–2.03 (m, 1H), 1.97–1.90 (m, 1H), 1.35 (s, 3H), 1.27 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 156.6, 154.2, 140.9, 139.0, 128.9, 128.8, 127.2, 124.6, 124.5, 119.7, 119.7, 117.1, 116.8, 74.3, 59.7, 52.9, 50.7, 40.2, 38.2, 28.6, 23.1; MS: m/z: 415.9314 [M + H]+; Molecular formula: C23H27ClFN3O.

3-((4-(4‑chloro-2-fluorophenyl)piperazin-1-yl)methyl)−4-(4-chlorophenyl)−6,6-dimethyl-5,6–dihydro-4H-1,2-oxazine (3XXI)

Off white; mp: 73–76 °C; 1H NMR (CDCl3, 400 MHz): δ 7.32–7.26 (m, 2H), 7.17 (d, J = 8 Hz, 2H), 7.02–7.00 (m, 2H), 6.85–6.80 (m, 1H), 3.67–3.63 (m, 1H), 3.02 (s, 4H), 2.90–2.80 (m, 2H), 2.62–2.61 (m, 2H), 2.33–2.32 (m, 2H), 2.15 (s, 1H), 2.07–2.02 (m, 1H), 1.88 (t, J = 12 Hz, 1H), 1.35 (s, 3H), 1.27 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 156.5, 154.0, 139.2, 138.9, 132.9, 130.0, 128.9, 124.5, 124.4, 119.6, 119.6, 116.9, 116.7, 74.3, 59.5, 52.9, 50.5, 39.9, 37.5, 28.5, 22.9; MS: m/z: 415.9314 [M + H]+; Molecular formula: C23H27ClFN3O.

6,6-dimethyl-4-phenyl-3-((4-(p-tolyl)piperazin-1-yl)methyl)−5,6-dihydro-4H-1,2-oxazine (3XXII)

Off white mp: 81–83 °C; 1H NMR (CDCl3, 400 MHz): δ 8.53–8.51 (m, 2H), 8.26 (d, J = 8 Hz, 1H), 7.75–7.73 (m, 1H), 7.69–7.68 (m, 1H), 7.55–7.50 (m, 2H), 7.36–7.35 (m, 2H), 7.30 (d, J = 8 Hz, 1H), 7.26–7.20 (m, 2H), 6.47 (d, J = 8 Hz, 1H), 5.69 (s, 1H), 4.76–4.74 (m, 2H), 4.41–4.39 (m, 2H), 4.35 (s, 2H), 4.22–4.19 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 165.2, 157.7, 149.9, 132.6, 130.8, 128.8, 123.7, 123.0, 116.7, 90.8, 68.1, 67.1, 63.6, 33.9, 28.9, 23.7, 22.9; MS: m/z: 406.0646 [M + H]+; Molecular formula: C24H31N3O.

(4aS,8aS)−4-phenyl-3-((4-(p-tolyl)piperazin-1-yl)methyl)−4a,5,6,7,8,8a-hexahydro-4H-benzo[e][1,2]oxazine (3XXIII)

Brown; mp: 101–102 °C; 1H NMR (CDCl3, 400 MHz): δ 7.37–7.34 (m, 2H), 7.30–7.28 (m, 1H), 7.21–7.19 (m, 2H), 7.07(d, J = 8 Hz, 2H), 6.84 (d, J = 8 Hz, 2H), 4.05 (s, 1H), 3.62 (s, 1H), 3.18–3.15 (m, 4H), 2.99 (d, J = 12 Hz, 1H), 2.76 (s, 2H), 2.53 (s, 2H), 2.28 (s, 3H), 2.10–2.06 (m, 1H), 1.76–1.74 (m, 2H), 1.70–1.64 (m, 2H), 1.49–1.45 (m, 1H), 1.41–1.28 (m, 4H); 13C NMR (CDCl3, 100 MHz): δ 153.2, 149.2, 141.8, 129.7, 129.4, 128.9, 128.5, 127.1, 116.6, 68.9, 60.2, 53.1, 49.6, 44.2, 38.9, 29.4, 27.9, 25.3, 20.5, 20.1; MS: m/z: 403.5597 [M + H]+; Molecular formula: C26H33N3O.

(4R,4aS,5S,8R,8aS)−4-phenyl-3-((4-(p-tolyl)piperazin-1-yl)methyl)−4a,5,6,7,8,8a-hexahy-dro-4H-5,8-methanobenzo[e][1,2]oxazine (3XXIV)

Light yellow; mp: 90–94 °C; 1H NMR (CDCl3, 400 MHz): δ 7.35–7.32 (m, 2H), 7.29–7.27 (m, 3H), 7.06 (d, J = 8 Hz, 2H), 6.80 (d, J = 8 Hz, 2H), 3.76 (d, J = 8 Hz, 1H), 3.23 (d, J = 8 Hz, 1H), 2.97–2.88 (m, 5H), 2.55–2.54 (m, 1H), 2.38–2.36 (m, 2H), 2.27 (s, 3H), 2.19–2.15 (m, 2H), 2.00–1.99 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 171.8, 149.1, 138.3, 130.0, 129.6, 129.2, 128.1, 127.0, 116.3, 82.9, 57.9, 53.2, 52.4, 49.7, 42.8, 42.2, 40.1, 33.4, 29.0, 24.8, 20.4; MS: m/z: 415.5704 [M + H]+; Molecular formula: C27H33N3O.

4-(4-chlorophenyl)−6,6-dimethyl-3-((4-(p-tolyl)piperazin-1-yl)methyl)−5,6-dihydro-4H-1,2- oxazine (3XXV)

Brownish yellow; mp: 103–106 °C; 1H NMR (CDCl3, 400 MHz): δ 7.30 (d, J = 8 Hz, 2H), 7.18 (d, J = 4 Hz, 2H), 6.82 (d, J = 8 Hz, 2H), 3.70 (s, 1H), 3.11 (s, 4H), 2,92–2.81 (m, 2H), 2.65 (s, 2H), 2.35–2.34 (m, 2H), 2.26 (s, 3H), 2.08–2.04 (m, 1H), 1.92–1.87 (m, 1H), 1.37 (s, 3H), 1.29 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 148.9, 139.2, 132.9, 130.1, 129.7, 129.6, 129.5, 128.9, 116.5, 74.4, 59.5, 52.9, 49.7, 39.8, 37.5, 28.5, 22.9, 20.45; MS: m/z: 411.9675 [M + H]+; Molecular formula:C24H30ClN3O.

3-((4-(3,4-difluorophenyl)piperazin-1-yl)methyl)−6,6-dimethyl-4-phenyl-5,6-dihydro-4H-1,2-oxazine (3XXVI)

Brown; mp: 73–76 °C; 1H NMR (CDCl3, 400 MHz): δ 7.34 (t, J = 8 Hz, 2H), 7.27 (d, J = 8 Hz, 1H), 7.18 (d, J = 4 Hz, 2H), 7.14–7.12 (m, 2H), 6.95–6.93 (m, 1H), 4.03 (s, 2H), 3.60–3.52 (m, 1H), 3.15–2.99 (m, 5H), 2.75 (s, 2H), 2.51 (s, 2H), 2.08–2.04 (m, 1H), 1.74–1.72 (m, 3H), 1.64–1.58 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 134.1, 128.8, 128.4, 127.5, 127.4, 126.9, 124.6, 118.6, 68.6, 60.1, 53.2, 51.1, 44.1, 38.8, 29.3, 27.9, 25.2, 19.9; MS: m/z: 399.4768 [M + H]+; Molecular formula: C23H27F2N3O.

(4aS,8aS)−3-((4-(3,4-difluorophenyl)piperazin-1-yl)methyl)−4-phenyl-4a,5,6,7,8,8a-hexah- ydro-4H-benzo[e][1,2]oxazine (3XXVII)

Off white mp: 80–83 °C; 1H NMR (CDCl3, 400 MHz): δ 7.33–7.26 (m, 4H), 7.18 (d, J = 8 Hz, 1H), 7.04–6.97 (m, 1H), 6.70–6.65 (m, 1H), 6.57–6.55 (m, 1H), 4.03 (s, 1H), 3.80–3.76 (m, 1H), 3.26–3.22 (m, 5H), 2.89 (s, 2H), 2.69 (s, 2H), 2.05 (d, J = 12 Hz, 2H), 1.76–1.56 (m, 6H), 1.46–1.43 (m, 2H); 13C NMR (CDCl3, 100 MHz): δ 151.7, 141.2, 128.9, 128.4, 127.1, 117.3, 117.2, 111.8, 105.8, 105.6, 69.1, 59.5, 52.6, 48.6, 44.3, 38.6, 29.1, 27.8, 24.9, 20.0; MS: m/z: 425.5141 [M + H]+; Molecular formula: C25H29F2N3O.

3-((4-(3,4-difluorophenyl)piperazin-1-yl)methyl)−6,6-dimethyl-5,6-dihydro-4H-1,2-oxazin- 4-yl benzoate (3XXVIII)

Pale yellow; mp: 85–88 °C; 1H NMR (CDCl3, 400 MHz): δ 8.03 (d, J = 8 Hz, 2H), 7.59 (t, J = 8 Hz, 1H), 7.46 (t, J = 8 Hz, 2H), 7.01–6.94 (m, 1H), 6.62–6.56 (m, 1H), 6.49–6.47 (m, 1H), 5.73–5.70 (m, 1H), 3.42 (d, J = 12 Hz, 1H), 3.13 (d, J = 12 Hz, 1H), 2.94–2.92 (m, 4H), 2.62–2.57 (m, 2H), 2.50–2.46 (m, 2H), 2.26–2.20 (m, 1H), 2.04–1.99 (m, 1H), 1.39–1.37 (s, 6H); 13C NMR (CDCl3, 100 MHz): δ 165.3, 152.7, 148.4, 148.3, 133.7, 133.4, 129.7, 129.6, 128.6, 117.1, 116.9, 111.5, 105.3, 105.1, 74.4, 60.2, 58.9, 52.9, 49.3, 36.5, 26.2, 25.3; MS: m/z: 406.0646 [M + H]+; Molecular formula: C24H27F2N3O3.

1-(((4aS,8aS)−4-phenyl-4a,5,6,7,8,8a-hexahydro-4H-benzo[e][1,2]oxazin-3-yl)methyl)−1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde (3XXIX)

Brownish yellow; mp: 84–86 °C; 1H NMR (CDCl3, 400 MHz): δ 10.05 (s, 1H), 8.65 (s, 1H), 8.28 (s, 1H), 7.29–7.26 (m, 1H), 7.20–7.12 (m, 3H), 6.93 (d, J = 8 Hz, 2H), 6.55–6.54 (m, 1H), 5.13–5.09 (m, 1H), 4.96–4.92 (m, 1H), 4.03 (s, 1H), 2.89 (s, 1H), 2.05–2.01 (m, 1H), 1.64–1.55 (m, 3H), 1.38–1.13 (m, 6H); 13C NMR (CDCl3, 100 MHz): δ 191.1, 151.6, 150.0, 146.4, 140.3, 130.2, 129.9, 128.6, 127.8, 127.1, 125.6, 120.2, 102.6, 68.8, 47.4, 44.8, 38.4, 29.1, 27.5, 24.9, 19.8; MS: m/z: 373.4476 [M + H]+; Molecular formula: C23H23N3O2.

1-(((4R,4aS,5S,8R,8aS)−4-phenyl-4a,5,6,7,8,8a-hexahydro-4H-5,8-methanobenzo[e][1,2]oxazin-3-yl)methyl)−1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde (3XXX)

Off white; mp: 181–184 °C; 1H NMR (CDCl3, 400 MHz): δ 10.04 (s, 1H), 8.51 (s, 1H), 8.27–8.26 (m, 1H), 7.17 (s, 1H), 7.00–6.90 (m, 5H), 6.58–6.57 (m, 1H), 5.05–4.90 (m, 2H), 3.79–3.78 (m, 1H), 3.12 (d, J = 12 Hz, 1H), 2.54–2.53 (m, 1H), 2.19–2.16 (m, 1H), 2.09–2.08 (m, 1H), 1.88 (s, 1H), 1.61–1.58 (m, 1H), 1.42–1.40 (m, 1H), 1.19–1.17 (m, 2H), 1.08–1.07 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 191.2, 170.7, 146.2, 136.2, 130.0, 129.2, 128.5, 128.5, 127.3, 125.5, 120.1, 102.8, 83.1, 53.2, 45.3, 42.3, 42.2, 40.1, 33.4, 28.9, 24.6; MS: m/z: 385.4583 [M + H]+; Molecular formula: C24H23N3O2.

1-((6,6-dimethyl-4-phenyl-5,6-dihydro-4H-1,2-oxazin-3-yl)methyl)−1H-pyrrolo[2,3-b]pyri- dine-5-carbaldehyde (3XXXI)

Brown; mp: 56–60 °C; 1H NMR (CDCl3, 400 MHz): δ 10.07 (s, 1H), 8.63 (s, 1H), 8.33 (s, 1H), 7.21–7.15 (m, 4H), 6.97–6.95 (m, 2H), 6.57 (d, J = 4 Hz, 1H), 5.05 (d, J = 12 Hz, 1H), 4.74 (d, J = 16 Hz, 1H), 3.24–3.19 (m, 1H), 2.03–1.98 (m, 1H), 1.93–1.86 (m, 1H), 1.34 (s, 3H), 1.19 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 191.2, 154.8, 149.9, 146.3, 138.9, 130.1, 130.1, 128.9, 128.1, 127.4, 125.6, 120.3, 102.4, 74.9, 46.7, 40.8, 38.3, 28.3, 22.7; MS: m/z: 347.4103 [M + H]+; Molecular formula: C21H21N3O2.

1-((4-(4-chlorophenyl)−6,6-dimethyl-5,6-dihydro-4H-1,2-oxazin-3-yl)methyl)−1H-pyrrolo [2,3-b]pyridine-5-carbaldehyde (3XXXII)

Off white; mp: 112–114 °C; 1H NMR (CDCl3, 400 MHz): δ 10.07 (s, 1H), 8.61 (s, 1H), 8.33 (s, 1H), 7.20 (s, 1H), 7.10 (d, J = 8 Hz, 2H), 6.85 (d, J = 8 Hz, 2H), 6.59–6.58 (m, 1H), 5.01 (d, J = 12 Hz, 2H), 4.78 (d, J = 12 Hz, 1H), 3.23–3,19 (m, 1H), 2.00–1.96 (m, 1H), 1.86–1.81 (m, 1H), 1.34 (s, 3H), 1.19 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 191.2, 154.3, 149.8, 146.3, 137.2, 133.3, 130.2, 129.8, 129.4, 128.9, 125.7, 120.3, 102.7, 75.1, 46.5, 40.6, 37.8, 28.3, 22.7; MS: m/z: 381.8554 [M + H]+; Molecular formula: C21H20ClN3O2.

3-((5-formyl-1H-pyrrolo[2,3-b]pyridin-1-yl)methyl)−6,6-dimethyl-5,6-dihydro-4H-1,2-oxa- zin-4-yl benzoate (3XXXIII)

Off white; mp: 138 °C; 1H NMR (CDCl3, 400 MHz): δ 9.90 (s, 1H), 8.33 (s, 1H), 8.22 (s, 1H), 7.69 (d, J = 8 Hz, 2H), 7.53 (t, J = 8 Hz, 1H), 7.39–7.32 (m, 3H), 6.62–6.61 (m, 1H), 5.43 (d, J = 12 Hz, 1H), 5.30–5.27 (m, 1H), 5.18–5.14 (m, 1H), 2.19–2.14 (m, 1H), 1.96–1.91 (m, 1H), 1.33–1.32 (s, 6H); 13C NMR (CDCl3, 100 MHz): δ 190.9, 165.0, 151.3, 150.1, 146.7, 133.4, 130.3, 129.6, 129.5, 129.1, 128.4, 125.9, 120.4, 103.2, 75.2, 60.3, 45.6, 36.6, 26.2, 25.1; MS: m/z: 391.4198 [M + H]+; Molecular formula: C22H21N3O4.

1-(((4aS,8aS)−4-(4-methoxyphenyl)−4a,5,6,7,8,8a-hexahydro-4H-benzo[e][1,2]oxazin-3-yl) methyl)−7-methyl-1H-indole-3-carbaldehyde (3XXXIV)

Off white; mp: 58 °C; 1H NMR (CDCl3, 400 MHz): δ 9.85 (s, 1H), 8.15 (d, J = 8 Hz, 1H), 7.22 (s, 1H), 7.16 (t, J = 8 Hz, 1H), 7.00 (d, J = 8 Hz, 1H), 6.92 (d, J = 8 Hz, 2H), 6.85 (d, J = 8 Hz, 1H), 5.16–5.12 (m, 1H), 5.05–5.01 (m, 1H), 4.04 (s, 1H), 3.81 (s, 3H), 2.52 (s, 3H), 2.43 (s, 1H), 2.06 (d, J = 12 Hz, 1H), 1.65–1.54 (m, 4H), 1.42 (d, J = 12 Hz, 1H), 1.33–1.22 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 184.7, 159.2, 152.7, 140.7, 136.4, 132.2, 129.2, 127.6, 126.5, 123.4, 121.9, 120.2, 118.7, 114.7, 69.1, 55.5, 51.9, 43.2, 38.9, 29.2, 27.4, 25.0, 19.9, 19.7; MS: m/z: 416.5121 [M + H]+; Molecular formula: C26H28N2O3.

3-((5-formyl-1H-pyrrolo[2,3-b]pyridin-1-yl)methyl)−6,6-dimethyl-5,6-dihydro-4H-1,2-oxa- zin-4-yl benzoate (3XXXV)

Light yellow; mp: 104–106 °C; 1H NMR (CDCl3, 400 MHz): δ 9.78 (s, 1H), 8.06 (d, J = 8 Hz, 1H), 7.79 (d, J = 8 Hz, 2H), 7.61–7.56 (m, 2H), 7.41 (t, J = 8 Hz, 2H), 7.08 (t, J = 6 Hz, 1H), 6.95 (d, J = 8 Hz, 1H), 5.34 (s, 1H), 5.28–5.25 (m, 2H), 2.67 (s, 3H), 2.21–2.16 (m, 1H), 2.01–1.97 (m, 1H), 1.36 (s, 3H), 1.31 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 184.6, 165.1, 150.7, 140.2, 136.4, 133.9, 129.6, 128.8, 128.6, 127.6, 126.6, 123.4, 121.4, 120.4, 118.9, 75.1, 60.1, 50.9, 36.4, 26.4, 24.9, 19.9; MS: m/z: 391.4198 [M + H]+; Molecular formula: C22H21N3O4.

1-((4,6-diphenyl-5,6-dihydro-4H-1,2-oxazin-3-yl)methyl)−7-methyl-1H-indole-3-carbaldehyde (3XXXVI)

Light yellow; mp: 151–153 °C; 1H NMR (CDCl3, 400 MHz): δ 9.82 (s, 1H), 8.17 (t, J = 6 Hz, 1H), 7.37 (s, 2H), 7.31 (d, 4H), 7.18–7.14 (m, 3H), 7.10 (d, J = 8 Hz, 2H), 5.23–5.19 (m, 1H), 5.07–5.05 (m, 1H), 4.93–4.90 (m, 1H), 4.84–4.81 (m, 1H), 3.43–3.38 (m, 1H), 3.09 (d, J = 4 Hz, 1H), 2.50 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 184.5, 156.7, 140.2, 138.4, 129.4, 128.6, 128.5, 128.2, 128.1, 127.9, 127.5, 127.4, 126.6, 126.5, 126.4, 126.3, 123.3, 121.8, 120.1, 118.6, 78.4, 73.5, 51.6, 36.8, 33.9, 19.6; MS: m/z: 408.4917 [M + H]+; Molecular formula: C27H24N2O2.

1-((4-(4-chlorophenyl)−6,6-dimethyl-5,6-dihydro-4H-1,2-oxazin-3-yl)methyl)−7-methyl-1H-indole-3-carbaldehyde (3XXXVII)

Off white; mp: 164–168 °C; 1H NMR (CDCl3, 400 MHz): δ 9.84 (s, 1H), 8.13 (d, J = 8 Hz, 1H), 7.30 (d, J = 8 Hz, 2H), 7.16–7.13 (m, 2H), 6.98–6.91 (m, 3H), 5.04–4.94 (m, 2H), 2.97–2.92 (m, 1H), 2.39 (s, 3H), 2.02–1.97 (m, 1H), 2.39 (s, 3H), 2.02–1.97 (m, 1H), 1.79 (t, J = 14 Hz, 1H), 1.33 (s, 3H), 1.12 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 184.6, 154.4, 140.4, 137.8, 136.4, 134.0, 129.7, 127.6, 126.4, 123.5, 121.9, 120.1, 118.6, 75.2, 51.6, 40.9, 36.8, 28.3, 22.8, 19.5; MS: m/z: 394.8939 [M + H]+; Molecular formula: C23H23ClN2O2.

N1-((4,6-diphenyl-5,6-dihydro-4H-1,2-oxazin-3-yl)methyl)benzene-1,4-diamine (3XXXV III)

Brownish black; mp: 120–122 °C; 1H NMR (CDCl3, 400 MHz): δ 7.34 (t, J = 7.2 Hz, 1H), 7.30–7.21 (m, 1H), 7.15 (d, J = 7.0 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.46 (s, 1H), 4.00 (s, 1H), 3.82 (d, J = 15.4 Hz, 1H), 3.66 (d, J = 15.4 Hz, 1H), 3.19 (s, 1H), 2.03 (d, J = 13.4 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ: 153.8, 141.5, 140.2, 129.1, 128.5, 128.3, 128.2, 127.2, 122.83, 114.9, 68.9, 48.3, 44.6, 39.1, 29.4, 27.7, 25.1, 20.1; MS: m/z: 357.4482 [M + H]+; Molecular formula: C23H23N3O.

N-((4,6-diphenyl-5,6-dihydro-4H-1,2-oxazin-3-yl)methyl)−5-(4-methoxyphenyl)−1,3,4-thi- adiazol-2-amine (3XXXIX)

Pale yellow; mp: 60–63 °C; 1H NMR (CDCl3, 400 MHz): δ 7.73–7.61 (m, 2H), 7.45–7.39 (m, 1H), 7.34–7.22 (m, 4H), 7.19–7.11 (m, 3H), 7.06 (d, J = 8 Hz, 1H), 6.99–6.95 (m, 1H), 6.93–6.87 (m, 2H), 4.72–4.65 (m, 1H), 4.10–4.05 (m, 1H), 3.86–3.83 (m, 3H), 3.40 (d, J = 8 Hz, 1H), 3.27–3.16 (m, 2H), 2.08–2.04 (m, 2H); 13C NMR (CDCl3, 100 MHz): δ 160.8, 151.6, 150.9, 140.8, 140.2, 129.1, 128.8, 128.6, 128.4, 128.0, 127.9, 127.2, 127.2, 126.9, 122.2, 114.5, 114.2, 55.4, 29.2, 29.1, 27.7, 27.6, 25.0, 24.9, 19.9, 19.8; MS: m/z: 456.5594 [M + H]+; Molecular formula: C26H24N4O2S.

Reagents

RPMI-1640 medium was purchased from Thermo Scientific HyClone (Waltham, MA, USA). 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT), DMSO, SP600125, N-Acetyl-l-cysteine, 2′,7′-Dichlorofluorescein diacetate, and tetramethylrhod amine ethyl ester (TMRE) were purchased from Sigma (St. Louis, MO). Anti-PARP, anti-LC3, anti-Cyclin D1, anti-p-p53, anti-ATF4, anti-CHOP, anti-p-JNK, and anti-JNK antibodies were from Cell Signaling Technology (Massachusetts, USA). Anti-Alix, anti-β-actin, anti-Bcl-2, anti-Bcl-xL, anti-IAP-1, anti-Survivin, and anti-Bax antibodies were obtained from Santa Cruz Biotechnology (Texas, USA).

Cell lines and culture conditions

Human BC MDA-MB231 (CVCL_0062) and MCF-7 (CVCL_0031) cells were obtained from Korean Cell Line Bank (KCLB, Seoul, South Korea) and MCF-10A cell line (CVCL_0598) were purchased by ATCC (American Type Culture Collection, Manassas, VA, USA). Human BC cell lines were cultured with RPMI-1640 medium containing 10 % inactivated FBS and 1 % P/S. Human breast epithelial MCF-10A cells were cultured with DMEM-F12 medium. The DMEM-F12 medium were containing 0.5 µg/mL, 10 µg/mL Insulin, 5 % horse serum, and 1 % P/S. the cells were incubated at 37 °C in 5 % CO2 atmosphere. At 80∼95 % confluence, MDA-MB231 and MCF-7 cells were sub-cultured using 0.25 % Trypsin/EDTA and MCF-10A cells were using 0.05 % Trypsin/EDTA. All cell lines were authenticated and certified by KCLB and ATCC and routinely checked for mycoplasma contamination.

MTT assay

MTT assay was conducted for analyzing the cytotoxic effect of BSO-07. 1 × 104 cells/well of MDA-MB231, MCF-7, and MCF-10A cells were seeded on 96 well plates for overnight. These cells were treated with 0–10–30–50–100 µM of BSO-07 for 24 h and then 30 µL/well of 2 mg/mL MTT solution was treated for 2 h. After 2 h, 100 µL/well of MTT lysis buffer was treated for overnight. By using VARIOSKAN LUX (Thermo Fisher, Waltham, MA), the absorbance was measured as previously mentioned [19].

Cellular morphology

The cells (3 × 105 cells/well) were seeded on 12 well plate. Before the treatment with BSO-07, the cells morphology was observed and after the drug treatment for 24 h, cellular morphology was again detected by microscope [20].

Live and dead assay

To evaluate cytotoxicity, Live and Dead assay kit was used. The BSO-07-treated BC cells were treated with 5 µM calcein-AM and 5 µM ethidium bromide homodimer dye for 30 min at 37 °C and then the cover glasses were sealed with the mounting solution [21]. The stained cells were detected by confocal microscope (Olympus FluoView FV1000, Tokyo, Japan).

Cell cycle analysis

BSO-07 (0–50–100 µM) treated MDA-MB231 and MCF-7 cells were harvested by Trypsin/EDTA. The harvested cells were fixed with 70 % EtOH for overnight at 4 °C. After fixing, the cells were incubated with 1 mg/mL RNases A for 1 h at 37 °C. After the RNase A treatment, propidium iodide (PI) was added, and the cells were analyzed using a BD Accuri™ C6 Plus Flow Cytometer (BD Bioscience, NJ) [22].

Western blot analysis

For examining the expression patterns of p-JNK and JNK, BC cells were treated with BSO-07 for 3 h. For all other proteins, cells were treated for 24 h. After treatment, the cells were collected, lysed, and then subjected to Western blot analysis [23].

Reverse transcription polymerase chain reaction (RT-PCR)

The cells were treated with BSO-07 for 24 h. RNA was extracted using Transzol Up reagent (TransGen Biotech Co., Beijing, China). Chloroform, isopropanol, and ethanol were utilized to purify the RNA. The extracted mRNA was then converted into cDNA. mRNA levels of Bcl-2, Bcl-xL, and Survivin in the BSO-07-treated BC cells were assessed, using GAPDH as the control [24].

Annexin/PI staining assay

The BSO-07-treated cells were collected by using centrifugation at 4 °C. The collected cells were then stained with FITC-labeled Annexin V antibodies and propidium iodide (PI) for 15 min at room temperature, shielded from light. The staining was subsequently analyzed using a BD Accuri™ C6 Plus Flow Cytometer (BD Bioscience, Franklin Lakes, NJ) [25].

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining

The treated cells were fixed with 4 % PFA for 20 min and then treated with 0.2 % Triton X-100 for 10 min. TUNEL staining was conducted according to the manufacturers’ protocols. The stained samples were detected and analyzed by BD Accuri™ C6 Plus Flow Cytometer (BD Bioscience, Franklin Lakes, NJ) [26].

ER stress assay

2 × 104 cells/well of cells were seeded on 8 well chamber slide and allowed to settle overnight. The following day, the cells were treated with BSO-07 (ranging from 0 to 100 µM) for 24 h. Subsequently, the cells were treated with 1 µM ER tracker for 25 min at 37 °C, followed by staining with DAPI (diluted 1:500) for 15 min at 37 °C. After washing twice with PBS, the stained cells were examined using a confocal microscope.

Mitochondrial membrane potential assay

Mitochondrial membrane potential was measured with tetramethyl rhodamine ethyl ester (TMRE). The cells treated for 24 h were incubated with 50 nM of TMRE for 30 min at 37 °C and then the flow cytometry was used for analysis [27].

GSH/GSSG assay

The glutathione levels and cellular H2O2 levels were measured by ROS-Glo™ H2O2 Assay and GSH/GSSG-Glo Assay (Promega). These assays were conducted according to the instructions provided by the manufacturer [28].

ROS detection by using H2DCF-DA

The cells were treated with drugs for 12 h and then harvested. For ROS detection, they were incubated with 10 µM of the cell-permeable fluorescent probe 2′,7′-dichlorofluorescin diacetate (H2DCF-DA) for 30 min at 37 °C. Following incubation, ROS levels were analyzed using flow cytometry.

DFT in silico DFT calculations

The molecular structure of the synthesized compound was drawn using GaussView software. The molecular geometry optimization of the compound was carried out by employing the density functional theory at B3LYP level and 6–31G+(d,p) basis set by using Gaussian 09 software package.

Structure preparation and molecular docking analysis of JNK3-BSO-07

There are several missing regions in JNK3′s available structure. We have modelled those regions to generate an all-atom structure of JNK3 from a high-resolution crystal structure (PDB ID: 1JNK). In the first step, we reconstructed the structure of the JNK3 kinase using the intrinsically disordered regions of the crystal structure that was available (i.e., from residues 212 to 216 and 374 to 382). The obtained model was then applied to the corrected crystal coordinate using the MODELLER [29] program.

The binding pose of BSO-07 inside the binding pocket of the receptor JNK3 was investigated using AutoDock 4.2 [30]. It was used to perform a standard docking procedure for a rigid protein and a flexible ligand. Before docking simulations, the protein was prepared by removing crystalline water, adding polar hydrogen atoms, and updating the missing residues. A grid box (80 Å × 80 Å × 80 Å) with a spacing of 0.375 Å was created in the crystal structure and was centered on the ligand's mass center. Prior to docking, Autogrid 4 was used to generate energy grid maps for all potential ligand atom types. The docking method was initially demonstrated to be feasible by docking the original ligand (ANP) into the binding pocket. Later, JNK3′s inhibitor BSO-07 was cross-docked at its binding pocket and checked with the co-crystal ligand position. The docking modes were investigated using the Maestro (Schrödinger Inc.) and ChimerX software [31].

Molecular dynamics simulations

Simulations of apo and ATP-bound JNK3 kinase were performed in this study to evaluate the conformational stability of identified hit compound at the JNK3 binding domain. Molecular dynamics simulations are used to analysed the structural and dynamic changes throughout protein-ligand interactions (Desmond, v2020.4). The orthorhombic simple point charge (TIP3P) water models are used to solve complex structures. The system was found to have a cut-off for Lennard-Jones interactions of 10 Å. on periodic boundary conditions. A time step of 2.0 fs was used during the integration steps. Using the Nose-Hoover thermostat and Martyna-Tobias Klein protocols, the temperature and pressure of the systems were set to 300 K and 1.01325 bar, respectively. Before the production run, the system was minimized and equilibrated. To analyse the trajectory, root mean square deviation (RMSD), and root mean square fluctuations, MD simulations were run for 500 ns for both apo and BSO-07 bound complex.

Statistical analysis

All values are represented as the mean ± SD. For statistical significance, one-way analysis of variance (ANOVA) test was conducted for ROS detection, Live and Dead assay, and Annexin/PI staining assay in BSO-07/NAC treated BC cells. Except those experiment, student unpaired t-test was performed for the statistical significance.

Results

Chemical synthesis of newer 1,2-oxazines

Previously, we had reported the synthesis of 1,2-oxazines, which were effective in inhibiting COX2 but not COX1, thereby suggesting their potential as COX2-specific anti-inflammatory agents [32]. Additionally, we have synthesized and studied anticancer properties of 1,2-oxazine-based lead compounds that induced apoptosis and DNA fragmentation by targeting NF-κB signaling in hepatocellular carcinoma cells [33]. We herein synthesized a huge library of novel 1,2-oxazines by reacting 3-bromomethyl-tagged 1,2-oxazines with piperazines or piperidine bases bearing a key fragment of drug-like molecules via SN2 reaction in DMF solvent under basic conditions. After completion of the reaction, the molecules were obtained by crystallization procedures or column chromatography techniques, and characterized by advanced spectroscopic techniques (Fig. 1B).

Cytotoxicity studies of newer 1,2-oxazines against human BC cells

Oxazines fused with tocotrienols at the gamma and delta positions demonstrated anticancer activity by inhibiting mammary tumor growth in syngeneic mice, surpassing the effectiveness of the parent tocotrienols. This anticancer effect was linked to a reduction in cell proliferation by targeting kinase enzymes [34]. Therefore, we initially screened thirty-nine 1,2-oxazines for their anti-proliferative activities against MCF-7 cells using MTT assay. MCF-7 cell lines were treated with compounds dissolved in DMSO in a dose-dependent manner for 72 h. Tamoxifen drug was used as positive control. Among the tested 1,2-oxazines, 3XIV, 3XIX (BSO-07), 3XXV, 3XXXIV, 3XXXVII, and 3XXXIX compounds displayed significant cytotoxic effects with an IC50 values of 17.31, 24.81, 17.40, 21.0, 12.15, and 24.38 µM (Supplementary Table S1, and Figure S1) respectively. The reference compounds Tamoxifen inhibited MCF-7 cell proliferation with IC50 value of 3.50 µM (Supplementary Table S1). Additionally, the effect of BSO-07 was further investigated on MDA-MB231 cells, which showed a significant decrease in cell proliferation in a concentration-dependent manner (Fig. 2A), whereas proliferation in MCF-10A cells remained unaffected by BSO-07 (Fig. 2B). Specifically, at 100 µM, cell viability was about 59 % for MDA-MB231 and about 42 % for MCF-7 cells, while the viability of normal breast MCF-10A cells was around 71 %, indicating lower cytotoxicity of BSO-07 towards normal cells compared to BC cells. As shown in Fig. 2C, BSO-07 altered the morphology of BC cell lines and reduced their viability, whereas the normal breast cell line MCF-10A appeared intact, highlighting the specificity of BSO-07 towards BC cells.Fig. 2 BSO-07 exhibited cytotoxic effects by inducing apoptosis and paraptosis.(A) Structure of BSO-07. (B) Cell viability was measuring by MTT assay in BSO-07-treated breast cell lines. MDA-MB231, MCF-7, and MCF-10A cells were treated with BSO-07 (0–10–30–50–100 µM) for 24 h. ⁎⁎⁎p < 0.001 and *p < 0.05 vs. non-treated (NT) cells. (C) MDA-MB231, MCF-7, and MCF-10A cells were treated with 0–100 µM of BSO-07 for 24 h. Cellular morphology was detected with microscope at 0 h and 24 h treatment. (D) 0–50–100 µM of BSO-07 was treated for 24 h and Live and Dead assay was performed. ⁎⁎⁎p < 0.001 vs. non-treated (NT) cells. (E) BSO-07 (0–50–100 µM) was incubated with both cell lines for 24 h and cell cycle analysis was performed. (F) MDA-MB231 and MCF-7 cells was treated BSO-07 (0–10–30–50–100 µM) for 24 h and 100 µM of BSO-07 for the indicated time intervals. Western Blot analysis was performed to measure protein levels.

Fig 2

BSO-07 induced programmed cell death (PCD) in human BC cells

Since oxazines are known to trigger PCD, we assessed whether BSO-07 could induce PCD using Calcein AM and EthD-1 assays. Calcein AM, a non-fluorescent compound, is converted by cellular esterases in live cells into a substance that emits green fluorescence [35]. Our findings from the treatment of MDA-MB231 and MCF-7 cells with BSO-07 showed that it effectively induced PCD in a dose-dependent manner, with the proportion of PCD increasing significantly following BSO-07 treatment (Fig. 2D).

BSO-07 induced cell cycle arrest, apoptosis and paraptosis

We further investigated the impact of BSO-07 on cell cycle progression to analyze the distribution of human BC cells across different stages of the cell cycle. Our results revealed a progressive increase in the G0/G1 phase cell population in MDA-MB231 cells and a significant increase in the Sub G1 phase population in MCF-7 cells (Fig. 2E). These findings suggest that BSO-07 induces PCD primarily through apoptosis in these cell lines. Additionally, we explored other potential mechanisms underlying the cytotoxic effects of BSO-07. Apoptosis, a well-known form of PCD, often features cleaved PARP as a major marker in particular [36]. Paraptosis, identified by endoplasmic reticulum (ER) stress and swelling, along with diminished mitochondrial membrane potential, represents a distinct type of cell death, with Alix serving as a specific indicator [37]. LC3, widely recognized as a marker of autophagy, plays a role in a process that, while not inherently lethal, can lead to cell death when excessively activated [23,38]. According to the data presented in Fig. 2F, BSO-07 promoted cleaved PARP and reduced Alix levels in a concentration- and time-dependent manner, while the levels of LC3 proteins remained unchanged. This indicates that BSO-07 triggers apoptosis and paraptosis, but not autophagy, in MDA-MB231 and MCF-7 cells.

BSO-07 down-regulated oncogenic proteins and up-regulated apoptotic proteins

We examined the effect of BSO-07 on the modulation of oncogenic and apoptotic proteins. Our findings showed that BSO-07 suppressed the expression of Bcl-2, Bcl-xL, IAP-1, Survivin, and Cyclin D1, as illustrated in Fig. 3A. Additionally, the compound increased the expression of Bax and p-p53, shown in Fig. 3B. The effects of BSO-07 were observed not only at the protein level but also at the mRNA level, where it reduced the expression of Bcl-2, Bcl-xL, and Survivin (Fig. 3C).Fig. 3 BSO-07 modulated apoptotic proteins and induced apoptosis. (A and B) The cells were treated with BSO-07 for 24 h, then harvested and lysed for Western Blot analysis. (C) The cells were incubated with BSO-07 (0–10–30–50–100 µM) for 24 h, followed by cDNA synthesis and RT-PCR to assess mRNA levels, using GAPDH as a control (D) BSO-07 was treated 0–50–100 µM for 24 h and then the Annexin/PI staining analysis was conducted to measure apoptotic cell rates. (E) MDA-MB231 and MCF-7 cells were treated with 0–50–100 µM of BSO-07 for 24 h and then TUNEL staining assay was conducted. ⁎⁎⁎p < 0.001 vs. non-treated (NT) cells.

Fig 3

BSO-07 induced late apoptotic cells and DNA damage

Following the analysis of factors involved in apoptosis, we conducted Annexin/PI staining to quantify the percentage of apoptotic cells [25,39]. According to the results of Fig. 3D, in both cancer cell lines, late apoptotic cells increased from 1.2 % to 8.0 % and from 2.5 % to 10.5 %. Additionally, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, a commonly used method for detecting apoptotic cells, indicates that apoptosis involves DNA damage, which TUNEL staining can identify by labeling the dUTP nick ends. Treatment with BSO-07 resulted in an increase of apoptotic cells from 1.6 % to 19.7 % in MDA-MB231 cells and from 2 % to 23.3 % in MCF-7 cells, as shown in Fig. 3E.

BSO-07 induced ATF4/CHOP pathway of ER stress response signals

Because paraptosis induces ER stress [40], we examined the protein levels of the ATF4/CHOP pathway, which are markers of ER stress [41]. BSO-07 up-regulated the levels of both ATF4 and CHOP (Fig. 4A). Additionally, ER tracker staining revealed a reduction in ER staining in cells treated with BSO-07 compared to the untreated (NT) group, as shown in Fig. 4. These findings suggest that BSO-07 triggered ER stress via ATF4 and CHOP.Fig. 4 BSO-07 induced proteins related in ER stress signals and ROS levels.(A) The cells were incubated with the indicated concentration of BSO-07 for 24 h. β-actin was used for the control of the Western blots. (B) The cells were seeded on 8 well chamber slide and BSO-07 was treated for 24 h. ER tracker (1 µM) was treated for 25 min and DAPI (1:500) was stained for 15 min. The stained cells were analyzed by confocal microscope. (C) Cells were treated with 0–50–100 µM of BSO-07 for 24 h and then incubated with TMRE (50 nM) for 30 min at 37 °C. The treated cells were detected by flow cytometry. (D) Both cells were treated with 0–100 µM of BSO-07 for 24 h and then GSH/GSSG assay was conducted. The luminescence was detected by luminescence readers. (E) ROS levels were measured by using flow cytometry. The indicated concentrations of BSO-07-treated cells were exposed to H2DCFH-DA for 30 min at 37 °C. ⁎⁎⁎p < 0.001 and ⁎⁎p < 0.01 vs. non-treated (NT) cells.

Fig 4

BSO-07 triggered mitochondrial membrane potential depolarization and cellular ROS production

Next, we investigated whether BSO-07 induced ROS, because it is reported that ROS could mediate both apoptosis and paraptosis [[42], [43], [44]]. Mitochondrial membrane potential (MMP) depolarization can also be induced by ROS [45]. Our findings, as shown in Fig. 4C, indicate that BSO-07 caused a reduction in MMP levels. Additionally, BSO-07 triggered an increase in ROS in both cell lines. Furthermore, we observed a decrease in GSH levels and an increase in GSSG levels, resulting in a lowered GSH/GSSG ratio after BSO-07 treatment (Fig. 4D). For ROS detection, we employed H2DCF-DA, and the ROS levels increased in a concentration-dependent manner, as depicted in Fig. 4E.

Plausible conversion of BSO-07 in situ under ROS condition

Since BSO-07 increased the ROS in BC cells, we hypothesized a radical mechanism involving oxazines that may generate intermediate radicals (Fig. 5A). The free radicals generated by BSO-07 could increase ROS levels in the cancer cells and consequently reduce the GSH/GSSG ratio.Fig. 5 BSO-07 induced apoptosis and paraptosis activations through ROS production. (A) Plausible mechanism for the generation of free radicals in BSO-07. (B) Both cells were treated with 100 µM of BSO-07 or 3 mM of NAC for 12 h. The ROS levels were detected by flow cytometry. (C and D) MDA-MB231 and MCF-7 cells were treated 100 µM of BSO-07 or 3 mM of NAC for 24 h and then Western Blot analysis was performed. (E) Both cells were seeded on 8 well chamber slide for overnight and then incubated with 100 µM of BSO-07 or 3 mM of NAC for 24 h. Live and Dead assay was conducted. (F) The cells were treated 100 µM of BSO-07 and 3 mM of NAC for 24 h and then Annexin/PI staining analysis was performed. ⁎⁎⁎p < 0.001, ⁎⁎p < 0.01, and * p < 0.05 vs. BSO-07-treated cells.

Fig 5

NAC reduced the BSO-07-induced apoptosis and paraptosis activation

We confirmed the role of ROS in apoptosis and paraptosis using the antioxidant N-acetylcysteine (NAC). Initially, we found that 3 mM NAC could inhibit the ROS induced by BSO-07, as depicted in Fig. 5B. The presence of NAC diminished the expression of apoptotic proteins such as cleaved PARP and p-p53, compared to treatment with BSO-07 alone (Fig. 5C). Similarly, the expression of paraptotic proteins like ATF and CHOP was reduced with NAC treatment (Fig. 5D). The combined treatment with BSO-07 and NAC reduced the percentage of dead cells from approximately 50 % to 35 % and from about 70 % to 40 % in each cell line, respectively, compared to cells treated solely with BSO-07 (Fig. 5E). This trend was also observed in Annexin/PI staining analysis, shown in Fig. 5F. These findings suggest that BSO-07 induces apoptosis and paraptosis through ROS-mediated pathways.

BSO-07 was found to induce apoptosis and paraptosis independently

We investigated whether BSO-07′s induction of apoptosis and paraptosis are interdependent processes. Z-DEVD-FMK is an inhibitor of apoptosis [46] and cycloheximide (CHX) is a blocker of paraptosis [47]. Z-DEVD-FMK attenuated BSO-07-induced activity of cleaved PARP, but did not affect the paraptosis markers, Alix and ATF4 (Fig. 6A). Conversely, CHX decreased ATF4 expression and counteracted the reduction in Alix activity caused by BSO-07, while leaving cleaved PARP expression unchanged (Fig. 6B). These findings suggest that BSO-07 triggers apoptosis and paraptosis through independent pathways.Fig. 6 BSO-07 induced apoptosis and paraptosis independently via JNK pathway.(A) MDA-MB231 cells were treated with Z-DEVD-FMK (50 µM) and BSO-07 (100 µM) for 24 h and then Western Blot analysis was performed. (B) MDA-MB231 cells were incubated with CHX (15 µM) and BSO-07 (100 µM) for 24 h and cells were harvested and lysed. Western Blot analysis was done. (C) MDA-MB231 and MCF-7 cells were incubated with the indicated concentration of BSO-07 for 3 h and Western Blot analysis was done. (D) Both cells were treated with SP600125 (5 µM) or BSO-07 (100 µM) for 3 h. The protein expression patterns were detected by performing Western Blot analysis. (E) The cells were seeded on 96 well plates for overnight and treated with 5 µM of SP600125 or 100 µM of BSO-07 for 24 h. After 24 h, MTT solution was added for 2 h and then MTT lysis buffer was treated for overnight. MTT assay was conducted to measure cell viability. ⁎⁎⁎p < 0.001 and * p < 0.05 vs. BSO-07-treated cells. (F) MDA-MB231 and MCF-7 cells were treated with 100 µM of BSO-07 or 5 µM of SP600125 for 24 h and the Western Blot analysis was conducted.

Fig 6

BSO-07 induced apoptosis and paraptosis through JNK pathway

It is established that the JNK signaling pathway plays a role in activating both apoptosis and paraptosis [48,49]. Our in silico analysis indicated that BSO-07 targets the JNK pathway, prompting us to examine its impact on JNK signaling in BC cells. As shown in Fig. 6C, BSO-07 up-regulated p-JNK expression, while JNK expression did not change. When BSO-07 was administered in combination with SP600125, a JNK inhibitor, this effect was partially reversed (Fig. 6D). The cell viability was also slightly improved when SP600125 was used alongside BSO-07 (Fig. 6E). Furthermore, we explored the effects of the JNK inhibitor on apoptotic and paraptotic proteins. The JNK inhibitor reduced the expression of cleaved PARP, which had been elevated by BSO-07, and increased the expression of Alix, which BSO-07 had decreased (Fig. 6F).

Electron density of BSO-07 was centralized at oxazine ring

The optimized structure of the compound BSO-07 has been used to calculate the molecular electrostatic potential (MEP), highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital energy (LUMO). An electron donating and receiving ability of a molecule can be determined by its HOMO and LUMO energy. These molecular orbitals play vital role in electronic, quantum chemistry, and pharmaceutical studies as well as provide the information of biological mechanism [50,51]. The frontier molecular orbital's (FMO) energy gap indicates the stability and chemical reactivity of the structure. The FMO also aids in predicting the most reactive position of a studied molecule. The calculated energy value of HOMO and LUMO orbitals are −6.323 and −0.925 eV, respectively. The FMO's energy gap (∆ELUMO—HOMO) of the mentioned organic molecule was found to be 5.397 eV. The lower HOMO and LUMO energy gap values indicate the studied molecule has a high chemical and biological activity. The FMO distribution of the compound was showed in Fig. 7A. The Global chemical reactivity descriptors (GCRD) parameters of the molecule such as chemical softness (S), chemical hardness (η), ionization potential (I), electron affinity (A), chemical potential (µ), electronegativity (χ) and electrophilicity index (ψ) were calculated with the help of energy of HOMO and LUMO orbitals (supplementary Table S2) [52]. Electrophilicity index (ψ = 2.433 eV) of a molecule provides information about binding ability of the compound to biomolecules [53]. The higher electrophilicity index of the mentioned molecule suggested that it can bind to biomolecules better and can therefore act as an electrophilic species. Accordingly, the molecule studied has a low value of chemical hardness (η = 2.698 eV) but a high negative chemical potential (µ = −3.624 eV), meaning it is a soft molecule with a high polarizability. The HOMO orbitals have mostly localized on (4‑chloro-2-fluorophenyl)piperazine moiety and partially located on oxygen and nitrogen atoms (Fig. 7A). While, LUMO orbitals were located on the phenyl ring and partially located on oxazine moiety. The molecular electrostatic potential (MEP) is used to determine the relative reactivity positions in a species for nucleophilic and electrophilic attacks. The electrostatic potential surface mapped of the studied compound is given in Fig. 7B. The color code of the compound lies in the range of −5.439 × 10−2 to +5.439 × 10−2. Red and blue color in the MEP structure point to more electron rich and electron poor region, respectively. In the MEP, the negative potential regions are localized over the electronegative atoms oxygen and nitrogen atoms in the hexahydrocyclopenta-oxazine moiety and the positive potential regions are localized over the hydrogen atoms. As a result, the more negative electronegative potential and positive electrostatic potential sites are more likely to attract nucleophilic and electrophilic species.Fig. 7 (A) The HOMO and LUMO of molecule BSO-07 obtained by the DFT/B3LYP/6–31+G(d,p) method. (B) Molecular electrostatic potential surfaces of compound BSO-07. (C) Three-dimensional structure of JNK3 in the open state and the zoomed view of the binding pose of the cocrystallized ligand-ANP (orange) and the BSO-07 (blue) inside the active site pocket. (D) 2D interaction diagram representing the key interactions of the BSO-07 inside the active site of JNK3.

Fig 7

3.14. In silico mode-of-action analysis of BSO-07

The central dogma of molecular pharmacology, in an analogous fashion to that of structural biology, is this: structure causes effect. Given this, it is commonly accepted, and with good reason, that “similar” ligands will bind to the same, or similar, targets. Indeed, much of the early, pregenomic work in protein classification was on the basis of joint biochemical or phenotypic activity profiles in the presence of a molecular probe. A wide variety of ligand-based target prediction software is now available online in order to understand the mode-of-action of oxazine BSO-07 towards the inhibition of human BC cells, the in silico mode-of-action analysis was performed using CHEMBL platform [54]. The study design includes the preparation of smile format of BSO-07, and was added into the searching engine of CHEMBL, which yielded 3,933,641 results in which the similar compounds of around 2,331,700, 15,072 targets, 1,498,681 assays, 85,431 documents, 2000 cells, and 757 tissues. The analysis of the results sheet identified JNK as a target to BSO-07 with above threshold ranking, revealed that the in silico mode-of-action was predicted to be JNK pathway of inhibition of human BC cells by BSO-07.

In silico binding studies of BSO-07 towards JNK co-crystal structure

The docking study of the synthesized compound BSO-07 was employed to determine the inhibitor effect against the receptor. AutoDock4.2 was used to simulate the docking of BSO-07 into the active site of JNK3. The docked binding evaluations of co-crystallized ANP ligand in the active site of JNK3 were examined, and all the essential interactions achieved by the co-crystallized ligand structure (PDB: 1JNK) were counterproductive for the further docking investigations with BSO-07. It shows that the docking procedure was appropriate for the docking investigation [55] (Fig. 7C). ANP and BSO-07 have shown binding energies at the active site of −6.15 and −9.22 kcal/mol, respectively. Docking simulations revealed that the ligand strongly interacts with the receptor's active site residues. Through a halogen bond, the chlorine of the fluoro-benzene ring interacts with VAL74 and GLN75 (Fig. 7D). Hydrophobic interactions have also been found in the active site contributing to the stability of the complex (Fig. S2).

Docking simulations were also performed for nine known JNK3 inhibitors: AS601245, AS602801, AV7, BI-78D3, GINSENOSIDE-RG1, JNK INHIBITOR IX, JNK-IN-1, JNK-IN-8, SP600125, and CC-930 [56]. The binding energies and key hydrogen bond interactions with active site residues are summarized in Table 1. The binding energies (in kcal/mol) are −8.5, −8.9, −9.8, −8.2, −8.9, −8.8, −9.6, −10.5, and −8.7, respectively. The hydrogen bond interactions were found to be consistent with the active site residues similar to those observed for BSO-07 and co-crystal ligand, indicating similar binding features.Table 1 Binding Energies and Key Hydrogen Bond Interactions of BSO-07 and Known JNK3 Inhibitors with Active Site Residuesss. Binding energies are reported in kcal/mol, highlighting the strong interactions and comparable binding features between BSO-07 and established JNK3 inhibitors.

Table 1Reported Inhibitors	Binding Energy (kcal/mol)	H-bond Interaction	Interacting Residues	
BSO-7 (Present)	−9.2	–	–	
AS601245	−8.5	1	THR-103	
AS602801	−8.9	2	SER-217
GLY-215	
AV7	−9.8	2	SER-193
MET-149	
BI-78D3	−8.2	3	LYS-106
ALA-211
ARG-383	
GINESENOSIDE-RG1	−8.9	2	SER-217
THR-103	
JNK INHIBITOR IX JNK3	−8.8	–	–	
JNK-IN-1	−9.6	2	ARG-107
ASN-186	
JNK-IN-8	−10.5	3	LYS-22
ARG-107
ALA-211	
SP600125	−8.7	–	–	
CC-930	−9.3	–	–	

To further investigate the stability and dynamic behaviour of the JNK3 protein and its complex with the synthesized compound BSO-07, 500 ns molecular dynamics (MD) simulations were performed for both the apo and the JNK3-BSO-07 complex [57]. The root mean square deviation (RMSD) plot (Fig. S3), for the apo demonstrated remarkable stability, with RMSD values remaining around 2.1 Å throughout the simulation. This indicates that the JNK3 protein maintains a stable conformation in its unbound state. In contrast, the RMSD for the JNK3-BSO-07 complex exhibited an initial increase from 1.8 Å to 3 Å during the first 150 ns, suggesting that the binding of BSO-07 induces conformational adjustments within the protein. Following this period of structural rearrangement, the RMSD stabilized around 2.9 Å for the remaining 350 ns of the simulation, indicating that the complex reached a new equilibrium state and maintained a stable conformation. This higher RMSD value, compared to the apo, suggests that while BSO-07 binding leads to significant structural changes, and these alterations are stable over time.

The Root Mean Square Fluctuation (RMSF) plot (Fig. S4) data provides crucial insights into the flexibility and dynamic behaviour of specific residues in the JNK3 protein and its complex with the synthesized compound BSO-07. The analysis reveals that most residues exhibit similar fluctuations in both the apo and the complex, except in three key regions: the G-loop (residues 71–78), the activation loop (residues 217–226), and the common docking domain (residues 367–370). In the apo, the G-loop exhibits an RMSF of approximately 1.6 Å, indicating relatively low flexibility. However, in the JNK3-BSO-07 complex, the RMSF of the G-loop increases to 3.3 Å, suggesting enhanced flexibility upon ligand binding. The G-loop covers the ATP-binding pocket, and its increased flexibility could impact the accessibility and dynamics of the active site, potentially affecting the kinase's catalytic activity.

The activation loop, critical for the regulation of kinase activity, shows an RMSF of around 3.1 Å in the apo, indicating moderate flexibility. In the complex, the RMSF of the activation loop increases to 4.8 Å, indicating higher flexibility. This enhanced flexibility suggests that BSO-07 binding may influence the regulatory mechanisms of JNK3, possibly affecting its activation state and phosphorylation dynamics. Conversely, the common docking domain (residues 359 to 372) in the apo shows an RMSF of 4 Å, indicating high flexibility. In the complex, the RMSF decreases to 2.1 Å, indicating reduced flexibility. The docking domain is involved in interactions with other MAPKs and scaffold proteins, and its reduced flexibility in the complex suggests that BSO-07 binding stabilizes this region, potentially influencing protein-protein interactions and signalling pathways involving JNK3.

The Protein-ligand contacts plot (Fig. S5) further elucidates the interactions between BSO-07 and JNK3 during the simulations. The ligand engages in several hydrophobic interactions with residues ILE70, ALA91, LEU148, VAL196, and LEU206. These interactions are crucial for maintaining the stability and proper positioning of the ligand within the binding pocket. Additionally, water bridge interactions are observed with MET149, ASN152, and GLN155, indicating the involvement of solvent molecules in mediating protein-ligand interactions. Notably, hydrogen bond interactions with ASN152 and GLN155 play a prominent role in stabilizing the complex, as hydrogen bonds are key determinants of binding specificity and affinity. The combination of these hydrophobic, water-bridge, and hydrogen bond interactions underscores the multifaceted nature of the binding mechanism and highlights the importance of these residues in facilitating effective binding and stabilization of the JNK3-BSO-07 complex. These interactions collectively contribute to the observed changes in RMSF and overall structural dynamics, reinforcing the potential of BSO-07 as a potent inhibitor of JNK3.

The Radius of gyration (Rg) plot (Fig. S6) of the JNK3-BSO-07 complex provides insights into the compactness and stability of the protein-ligand complex during the 500 ns molecular dynamics simulations. The plot indicates that the Rg value fluctuates within a specific range, suggesting that the overall structure of the JNK3-BSO-07 complex remains stable throughout the simulation period. A stable Rg value, with minor fluctuations, implies that the protein maintains its structural integrity and does not undergo significant unfolding or large conformational changes upon ligand binding. Comparatively, analyzing the Rg of the JNK3-BSO-07 complex alongside the RMSF data reveals that specific regions, such as the G-loop and the activation loop, exhibit increased flexibility upon ligand binding. This increased flexibility is reflected in the RMSF plot, where the G-loop and activation loop show higher RMSF values in the complex than the apo. The hydrophobic interactions, water bridge interactions, and hydrogen bond interactions contribute to the stability of the complex. These interactions are crucial for maintaining the structural integrity of the binding pocket and ensuring effective inhibition of JNK3 by BSO-07.

Overall, the Rg plot, in conjunction with the RMSF data, underscores the significant impact of BSO-07 binding on the structural dynamics of JNK3. The ligand-induced conformational changes and stabilization effects highlight the potential of BSO-07 as a potent JNK3 inhibitor, providing valuable insights into its molecular mechanisms of action.

Discussion

Chemical synthesis utilizing 1,2-oxazines has demonstrated anti-oncogenic effects across various cancer models. For instance, the pharmacological inhibition of SYK by R406 diminished the leukemic stem cell (LSC) compartment in vitro and reduced the viability of LSCs, characterized by a decrease in ROS levels [18]. Chronic administration of Efavirenz (EFV) induces hepatic cell death, activates caspase-3, and promotes ROS formation. Moreover, inhibiting the cytochrome P450-dependent metabolism of EFV with 1-aminobenzotriazole substantially reduced ROS production and cell death. Furthermore, treating primary human hepatocytes with EFV and its oxidized derivative, 8-OHEFV, led to the phosphorylation of JNK and its substrate c-Jun [58]. Additionally, active tetrahydro-1,4-oxazine derivatives were found to inhibit lipid peroxidation and reduce triglycerides, total cholesterol, and LDL-cholesterol in hyperlipidemic rats, suggesting their potential as scaffolds for designing anti-atherosclerosis agents [59].

Staurosporine, a broad-spectrum apoptosis inducer, activates caspase-3, significantly reduces cellular glutathione levels, and increases ROS in HeLa cells. Notably, pre-treatment with N-acetylcysteine failed to prevent glutathione depletion, ROS generation, and caspase-3 activation, suggesting that cellular redox status plays a crucial role in the apoptotic pathway leading to caspase-3 activation by staurosporine [60]. Additionally, at concentrations above 50 nM, staurosporine induced apoptosis in human granulosa cell tumor cells, while at concentrations below 50 nM, it caused these normally globular cells to assume a flattened, epithelioid-like morphology. Phosphorylation-specific Western blotting and protein kinase assays showed that staurosporine inhibits p38 phosphorylation and activates JNK, which are involved in the regulation of granulosa cell differentiation [61]. In our lab, we previously developed two oxy-aza-spirocompounds, DMBO and CIMO, which exhibited anticancer effects in metastatic murine liver cancer cells and human BC cells, respectively [62,63]. Isolation of Crinum Asiatic mines B alkaloids was found to be cytotoxic against human tumor cell lines A549, LOVO, HL-60, and 6T-CEM at nanomolar concentrations [13]. However, the pharmacological potential of Flustrarine B and Secuamamine D alkaloids remains unexplored due to their limited availability on a large scale.

BC is the most prevalent malignant tumor among women, with its incidence and mortality rates rising annually, significantly impacting women's health [64]. Several risk factors contribute to BC, including genetic mutations, dense breast tissue, obesity, and biological age [65]. The MAPK pathway is crucial in BC, regulating key cellular functions such as differentiation, proliferation, apoptosis, as well as survival [66], and plays a dual role in both tumor development and tumor cell death, thus acting as a double-edged sword [67]. Apoptosis, a type of PCD, commonly targets cancer cells but also presents challenges with drug resistance in BC treatments. Paraptosis, another form of PCD, offers a potential solution to overcome this resistance. The development of BSO-07, which targets both types of cell death, could yield significant therapeutic outcomes [5].

In this study, chemical substances that can activate the JNK pathway in BC to induce paraptosis and apoptosis were synthesized, and their pharmacological effects were explored. Furthermore, in silico analyses, including bioinformatics, cheminformatics, density Fourier transform, and molecular electrostatic potential analysis, confirmed that BSO-07 triggers apoptosis and paraptosis via the ROS/JNK pathway in human BC cells. It is anticipated that the anticancer potential of BSO-07 in BC could extend from in vitro studies to in vivo experiments and clinical trials.

BSO-07 was found to stimulate ROS and activate the JNK pathway. Activation of JNK resulted in the downregulation of Bcl-2 and Bcl-xL and upregulation of Bax and phosphorylated p53, alongside inducing cleaved PARP. These changes promote apoptosis in human BC cells. Additionally, JNK activation suppressed Alix proteins, which typically inhibit paraptosis, and enhanced ATF4/CHOP signaling associated with ER stress signaling, thus inducing paraptosis in human BC cells, as shown in Fig. 8. However, our study also determined that apoptosis and paraptosis occur independently, as evidenced by the distinct effects observed when using specific inhibitors for each process. Overall, while the findings demonstrated the potential of BSO-07 to induce apoptosis and paraptosis in BC cells through JNK activation, there are notable limitations that must be addressed. Primarily, the research was confined to in vitro experiments, which do not fully replicate the complex biological interactions occurring in living organisms. The absence of in vivo studies in orthotopic or patient-derived xenograft (PDX) models means that the pharmacodynamics, and overall therapeutic efficacy of BSO-07 in a natural tumor environment have not been evaluated. Additionally, the detailed molecular mechanisms underlying the drug's interaction with JNK and other cellular pathways were not thoroughly explored, leaving potential aspects of its mechanism of action unclear. These gaps underscore the necessity for further research to validate the findings and assess the clinical relevance of BSO-07 as a treatment for BC.Fig. 8 Graphical representation of apoptosis and paraptosis mediated by JNK pathway after BSO-07 treatment in human BC cells.

Fig 8

CRediT authorship contribution statement

Na Young Kim: Writing – original draft, Validation, Methodology, Investigation, Conceptualization. Dukanya Dukanya: Methodology, Investigation, Formal analysis. Gautam Sethi: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing. Swamy S Girimanchanaika: Formal analysis, Data curation. Jirui Yang: Resources, Methodology. Omantheswara Nagaraja: Formal analysis, Data curation. Ananda Swamynayaka: Validation, Data curation. Divakar Vishwanath: Visualization, Validation. Keerthikumara Venkantesha: Software, Resources. Shreeja Basappa: Data curation. Arunachalam Chinnathambi: Data curation, Formal analysis, Software. Sulaiman Ali Alharbi: Software, Validation. Mahendra Madegowda: Formal analysis. Alexey Sukhorukov: Software. Vijay Pandey: Formal analysis. Peter E. Lobie: Writing – original draft, Resources, Project administration. Basappa Basappa: Writing – original draft, Visualization, Validation, Conceptualization. Kwang Seok Ahn: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Appendix Supplementary materials

Image, application 1

Image, application 2

Acknowledgement

This work was supported by DBT-NER, and Vision Group on Science and Technology (CESEM), Government of Karnataka. This work was supported by a 10.13039/501100003725 National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2021R1I1A2060024 ). This project was supported by Researchers Supporting Project number (RSP2024R383 ), King Saud University, Riyadh, Saudi Arabia. This work was also supported by the Shenzhen Key Laboratory of Innovative Oncotherapeutics (ZDSYS20200820165400003 ) (Shenzhen Science and Technology Innovation Commission), China; Shenzhen Development and Reform Commission Subject Construction Project ([2017]1434), China; Overseas Research Cooperation Project (HW2020008 ) (Tsinghua Shenzhen International Graduate School), China; Tsinghua University Stable Funding Key Project (WDZC20200821150704001 ); the Shenzhen Bay Laboratory (21310031 ), China and TBSI Faculty Start-up Funds, China. Thanks DST PhD Fellowship from KSTePS, Karnataka, for providing fellowship to D.V.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102101.
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