Design, Synthesis, and Antitumor Activity of a Series of Novel 4-(Aromatic Sulfonyl)-1-oxa-4-azaspiro[4.5]deca-6,9-dien-8-ones

Many sulfonamides show anticancer activity. Based on benzenesulfonylazaspirodienone (HL-X9) identified in our previous work, we optimized the lead compound for better efficacy, thereby synthesizing a series of novel 4-(aromatic sulfonyl)-1-oxa-4-azaspiro[4.5]deca-6,9-dien-8-one derivatives through a key step of metal-catalyzed cascade cyclization. The preliminary antiproliferative tests have shown that the anticancer activities of acetyl-protected mannose-linked sulfonylazaspirodienone derivatives (7i–7l) have been greatly improved. Among them, 7j is the most potent derivative, with IC50 values of 0.17 µM, 0.05 µM, and 0.07 µM for A549, MDA-MB-231, and HeLa cell lines, respectively. Flow cytometry analysis shows that 7j arrests MDA-MB-231 cells in the G2/M phase and has a certain effect on the apoptosis of MDA-MB-231 cells. In addition, the acute toxicity of 7j was lower than that of adriamycin.


Introduction
Sulfonamide is a functional moiety of several types of drugs with a variety of biological activities [1,2]. Although there are no clinically approved sulfonamide anticancer drugs, many reports have shown that many sulfonamides have anticancer activity [3][4][5]. Recently, carbonic anhydrase (CA) isozymes II, IX, and XII have been shown to be involved in tumor formation, sulfonamide-based CA IX and CA XII inhibitor have become a new research topic of anticancer drug development [6][7][8][9]. Many sulfonamides have been identified as low nanomolar CA IX inhibitors, SLC-0111 of which has entered the clinical trial (NCT02215850) (Figure 1). The research also found that CUL4-DCAF15 E3 ubiquitin ligase can be recruited by sulfonamide compounds such as indisulam, tasisulam, and chloroquinoxaline sulfonamide (CQS) (Figure 1) to trigger ubiquitination and degradation of RBM39 protein, resulting in an antitumor effect [10]. Besides, sulfonamides can be selectively concentrated in tumor tissues [11][12][13] and are expected to be useful for the development of tumor-targeting drugs.
Molecules 2020, 25 for many years, and the research on quinone-based anticancer drugs is still ongoing [15,16]. In addition, the inclusion of a spiro ring structure is one of the important strategies for drug development in recent years [17,18]. Some antitumor compounds with a spiro ring moiety such as SAR405838, DS-3032b, and APG-115 are undergoing clinical trials ( Figure 1) [19][20][21]. Spirodienone derivatives of hybridized quinone and spiro scaffolds have a wide range of biological activities [22]. Previously, we have hybridized these pharmacologically active structural elements (sulfonamide, quinone, and spirocyclic moieties) and obtained a series of benzenesulfonylazaspirodienones. [23]. The preliminary screening proved that these compounds have a certain inhibitory effect on cancer cell lines [24]. However, the aqueous solubility is low, and the in vitro activity is mostly at the µM level. At the same time, because the α,β-dienone structure in the spiro compound is a Michael acceptor, it is easy to have Michael's addition reaction with active nucleophilic molecules in vivo, resulting in toxic side effects. To improve the efficacy, the water solubility of the compounds, and improve the Michael acceptor characteristics of α,β-dienone structure in the spirodienone, reduce the possibility of addition reaction with active molecules in vivo to reduce toxicity and side effects. Therefore we further optimize the sulfonamide azaspirodienone by introducing the water-soluble 1,2,3-triazole moiety [25,26] through "Click Reaction" [27,28], replacing the benzene ring with a more polar heterocycle, and introducing glucose [29] or mannose [30] to affect the inhibitory effect of tumor growth and tumor targeting and so on ( Figure 2). Herein, we report the synthesis of these newly designed sulfonamide azaspirocyclodienone derivatives and their preliminary antiproliferative activity on cancer cell lines and flow cytometry analysis. Quinone is also a key moiety of many biologically active molecules [14]. Among them, doxorubicin and mitoxantrone ( Figure 1) have been the first-line anticancer drugs for solid tumors for many years, and the research on quinone-based anticancer drugs is still ongoing [15,16]. In addition, the inclusion of a spiro ring structure is one of the important strategies for drug development in recent years [17,18]. Some antitumor compounds with a spiro ring moiety such as SAR405838, DS-3032b, and APG-115 are undergoing clinical trials ( Figure 1) [19][20][21]. Spirodienone derivatives of hybridized quinone and spiro scaffolds have a wide range of biological activities [22].
Previously, we have hybridized these pharmacologically active structural elements (sulfonamide, quinone, and spirocyclic moieties) and obtained a series of benzenesulfonylazaspirodienones. [23]. The preliminary screening proved that these compounds have a certain inhibitory effect on cancer cell lines [24]. However, the aqueous solubility is low, and the in vitro activity is mostly at the µM level. At the same time, because the α,β-dienone structure in the spiro compound is a Michael acceptor, it is easy to have Michael's addition reaction with active nucleophilic molecules in vivo, resulting in toxic side effects. To improve the efficacy, the water solubility of the compounds, and improve the Michael acceptor characteristics of α,β-dienone structure in the spirodienone, reduce the possibility of addition reaction with active molecules in vivo to reduce toxicity and side effects. Therefore we further optimize the sulfonamide azaspirodienone by introducing the water-soluble 1,2,3-triazole moiety [25,26] through "Click Reaction" [27,28], replacing the benzene ring with a more polar heterocycle, and introducing glucose [29] or mannose [30] to affect the inhibitory effect of tumor growth and tumor targeting and so on ( Figure 2). Herein, we report the synthesis of these newly designed sulfonamide azaspirocyclodienone derivatives and their preliminary antiproliferative activity on cancer cell lines and flow cytometry analysis. azaspirodienone by introducing the water-soluble 1,2,3-triazole moiety [25,26] through "Click Reaction" [27,28], replacing the benzene ring with a more polar heterocycle, and introducing glucose [29] or mannose [30] to affect the inhibitory effect of tumor growth and tumor targeting and so on ( Figure 2). Herein, we report the synthesis of these newly designed sulfonamide azaspirocyclodienone derivatives and their preliminary antiproliferative activity on cancer cell lines and flow cytometry analysis.  Figure 2. Design of novel sulfonylazaspirodienone derivatives from 4-tosyl-1-oxa-4-azaspire [4.5] deca-6,9-dien-8-one (HL-X9).

Chemical Synthesis
The sulfonylazaspirodienone derivatives were prepared according to the synthetic route of Scheme 1. Sulfonylation of chloroethylamine hydrochloride by bromo-substituted aromatic sulfonyl chloride afforded the sulfonamides 3, which were converted to 4 by a Sonogashira coupling reaction. O-Alkylation of phenols with 4 formed the intermediates 5. Rhodium acetate-catalyzed oxidation and concomitant intramolecular amidation of 5 resulted in the formation of 6 by bis(trifluoroacetoxy)iodobenzene under mild conditions [23]. The copper-catalyzed click reaction of 6 with the azides [31,32] produced the derivatives 7. Further, 7 were hydrolyzed to the derivatives 8 with potassium carbonate in methanol.

Inhibition of Tumor Cell Proliferation In Vitro
To determine the cytotoxicity of the synthesized sulfonylazaspirodienones, we used three well-established cancer cell lines that are relevant to the study of in vitro anticancer properties. A549 is one of the most abundant human non-small cell lung cancer that has been widely used in screening anticancer agents. HeLa is a cervical cancer cell line commonly used in in vitro cancer research. MDA-MB-231 is a triple-negative breast cancer cell line that is widely used in cytotoxicity assays. The three cell lines have been shown to be viable cell lines for tumor xenografts in C57BL/6 nude mice and can be subsequently used to examine the in vivo effects of cytotoxicity on cancer.
We used 4-tosyl-1-oxa-4-azaspire [4.5]deca-6,9-dien-8-one (HL-X9) as positive controls to investigate the inhibitory activity of the new sulfonylazaspirodienone derivatives on lung carcinoma A549, breast adenocarcinoma MDA-MB-231, and cervical cancer HeLa cells. The results (Table 1) showed that the replacement of benzene ring of HL-X9 with thiophene and morpholine heterocycle increased the inhibitory activity of 2a and 2e from HL-X9 by 2-4 times on MDA-MB-231 and HeLa cell lines. It was also found that the 7-chloro-substituted (on core structure) derivatives (2c and 7f) were more potent than the 6-chloro-substituted derivatives (2b and 7e) against A549, MDA-MB-231 and HeLa cell lines. The introduction of triazole-bridged acetyl-protected mannose further improved the antiproliferative potency of 7i and 7l from 2e, especially in A549 cell line. Comparison of IC 50 values of 7a, 7f, and 7h with 7i, 7j, 7k, and 7l indicates that the acetyl-protected mannose derivatives are better than the acetyl-protected glucose derivatives for anticancer activity. Undesirably, the activity of the acetyl-deprotected derivatives (8a-h) decreased significantly compared with the corresponding 7a, 7f, 7h, 7i, 7j, 7k, and 7l. The most potent derivative 7j has IC 50 values of 0.17 µM, 0.05 µM, and 0.07 µM against A549, MDA-MB-231, and HeLa cancer cell lines, respectively. 7j, together with 2a, 2c, 2e, 7f, 7i, and 7l are all more effective than HL-X9 in inhibiting all three cancer cell lines. In addition, IC 50 values    We used 4-tosyl-1-oxa-4-azaspire[4.5]deca-6,9-dien-8-one (HL-X9) as positive controls to investigate the inhibitory activity of the new sulfonylazaspirodienone derivatives on lung carcinoma A549, breast adenocarcinoma MDA-MB-231, and cervical cancer HeLa cells. The results (Table 1) showed that the replacement of benzene ring of HL-X9 with thiophene and morpholine heterocycle increased the inhibitory activity of 2a and 2e from HL-X9 by 2-4 times on MDA-MB-231 and HeLa cell lines. It was also found that the 7-chloro-substituted (on core structure) derivatives (2c and 7f) were more potent than the 6-chloro-substituted derivatives (2b and 7e) against A549, MDA-MB-231 and HeLa cell lines. The introduction of triazole-bridged acetyl-protected mannose further improved the antiproliferative potency of 7i and 7l from 2e, especially in A549 cell line. Comparison of IC50 values of 7a, 7f, and 7h with 7i, 7j, 7k, and 7l indicates that the acetyl-protected mannose derivatives are better than the acetyl-protected glucose derivatives for anticancer activity. Undesirably, the activity of the acetyl-deprotected derivatives (8a-h) decreased significantly compared with the corresponding 7a, 7f, 7h, 7i, 7j, 7k, and 7l. The most potent derivative 7j has IC50 values of 0.17 µM, 0.05 µM, and 0.07 µM against A549, MDA-MB-231, and HeLa cancer cell lines, respectively. 7j, together with 2a, 2c, 2e, 7f, 7i, and 7l are all more effective than HL-X9 in inhibiting all three cancer cell lines. In addition, IC50 values of the derivatives 2c, 7d, 7h, 7k, and 7l, together with 7f and 7j, reached a double-digit nanomolar concentration level for the MDA-MB-231 cell line. Derivative 7j is the only compound with an IC50 value of a double-digit nanomolar concentration level against the HeLa cell line. The structure-activity relationship of sulfonylazaspirodienone derivatives is summarized in Scheme 2. Scheme 2. Structure-activity relationship of sulfonylazaspirodienone derivatives.

Cell Cycle Arresting
Next, we explored the effect of compound 7j in the regulation of cell cycle distribution by flow cytometry. Combretastatin A4 (CA4) was used as a positive control. As shown in Figure 3, compound 7j at the concentration of 1 µM was sufficient to arrest MDA-MB-231 cells in the G2/M phase, equivalent to CA4.

Cell Cycle Arresting
Next, we explored the effect of compound 7j in the regulation of cell cycle distribution by flow cytometry. Combretastatin A4 (CA4) was used as a positive control. As shown in Figure 3, compound 7j at the concentration of 1 µM was sufficient to arrest MDA-MB-231 cells in the G2/M phase, equivalent to CA4.

Apoptosis
To explore the potential mechanisms of the antiproliferative effect induced by sulfonylazaspirodienone derivatives, annexin V-FITC and PI staining were used, and flow cytometry (FCM) was performed to quantify cell apoptosis ( Figure 4). MDA-MB-231 cells were cultured with compound 7j at concentrations of 0.1 and 1 µM, respectively. Combretastatin A4 (CA4) was used as a positive control. Treatment with a low concentration (0.1 µM) of 7j altered the number of apoptotic cells compared to DMSO. The apoptosis percentage increased after treatment with higher concentrations (1 µM) of compounds 7j.

Acute Toxicity
The acute toxicity of compound HL-X9 and 7j were evaluated preliminarily in Kun Ming mice. All animals did not lose weight after drug treatment. Intraperitoneal injection of 30 mg/kg 7j or 60 mg/kg HL-X9 did not cause death within 14 days. The mortality after administration of 80 mg/kg 7j and 73 mg/kg HL-X9 was 40% and 10%, respectively, and the toxicity was lower than that of adriamycin (LD50 = 10.7 mg/kg i.p., Hazardous Substances Data Bank) [33]. The tolerance of female mice to compound 7j was poor, and the mortality rate of female mice was 4 times higher than that of male mice. Different doses of compound HL-X9 and 7j did not cause weight loss. Except that the weight gain of female mice was affected by compound 7j, the weight gain of other groups was similar to that of the control group ( Figure 5).

Apoptosis
To explore the potential mechanisms of the antiproliferative effect induced by sulfonylazaspirodienone derivatives, annexin V-FITC and PI staining were used, and flow cytometry (FCM) was performed to quantify cell apoptosis ( Figure 4). MDA-MB-231 cells were cultured with compound 7j at concentrations of 0.1 and 1 µM, respectively. Combretastatin A4 (CA4) was used as a positive control. Treatment with a low concentration (0.1 µM) of 7j altered the number of apoptotic cells compared to DMSO. The apoptosis percentage increased after treatment with higher concentrations (1 µM) of compounds 7j.

Apoptosis
To explore the potential mechanisms of the antiproliferative effect induced by sulfonylazaspirodienone derivatives, annexin V-FITC and PI staining were used, and flow cytometry (FCM) was performed to quantify cell apoptosis (Figure 4)

Acute Toxicity
The acute toxicity of compound HL-X9 and 7j were evaluated preliminarily in Kun Ming mice. All animals did not lose weight after drug treatment. Intraperitoneal injection of 30 mg/kg 7j or 60 mg/kg HL-X9 did not cause death within 14 days. The mortality after administration of 80 mg/kg 7j and 73 mg/kg HL-X9 was 40% and 10%, respectively, and the toxicity was lower than that of adriamycin (LD50 = 10.7 mg/kg i.p., Hazardous Substances Data Bank) [33]. The tolerance of female mice to compound 7j was poor, and the mortality rate of female mice was 4 times higher than that of male mice. Different doses of compound HL-X9 and 7j did not cause weight loss. Except that the weight gain of female mice was affected by compound 7j, the weight gain of other groups was similar to that of the control group ( Figure 5).

Acute Toxicity
The acute toxicity of compound HL-X9 and 7j were evaluated preliminarily in Kun Ming mice. All animals did not lose weight after drug treatment. Intraperitoneal injection of 30 mg/kg 7j or 60 mg/kg HL-X9 did not cause death within 14 days. The mortality after administration of 80 mg/kg 7j and 73 mg/kg HL-X9 was 40% and 10%, respectively, and the toxicity was lower than that of adriamycin (LD 50 = 10.7 mg/kg i.p., Hazardous Substances Data Bank) [33]. The tolerance of female mice to compound 7j was poor, and the mortality rate of female mice was 4 times higher than that of male mice. Different doses of compound HL-X9 and 7j did not cause weight loss. Except that the weight gain of female mice was affected by compound 7j, the weight gain of other groups was similar to that of the control group ( Figure 5). In addition, the main tissues (heart, liver, spleen, lung, and kidney) of mice in each group were stained with Hematoxylin and Eosin (H&E). The results showed that compared with the negative control group, no obvious damage was found in the sections of compound HL-X9(73 mg/kg) in all tissues; the sections of compound 7j(80 mg/kg) showed some damage in the liver and kidney, and no obvious damage was found in other tissues. In particular, the liver and kidney damage was more serious in female mice ( Figure 6).  In addition, the main tissues (heart, liver, spleen, lung, and kidney) of mice in each group were stained with Hematoxylin and Eosin (H&E). The results showed that compared with the negative control group, no obvious damage was found in the sections of compound HL-X9 (73 mg/kg) in all tissues; the sections of compound 7j (80 mg/kg) showed some damage in the liver and kidney, and no obvious damage was found in other tissues. In particular, the liver and kidney damage was more serious in female mice ( Figure 6). In addition, the main tissues (heart, liver, spleen, lung, and kidney) of mice in each group were stained with Hematoxylin and Eosin (H&E). The results showed that compared with the negative control group, no obvious damage was found in the sections of compound HL-X9(73 mg/kg) in all tissues; the sections of compound 7j(80 mg/kg) showed some damage in the liver and kidney, and no obvious damage was found in other tissues. In particular, the liver and kidney damage was more serious in female mice ( Figure 6).

Procedure for the Synthesis of Compound 1
To a round-bottom flask were added 2-phenoxy ethylamine derivative (1.2 mmol) and sulfonyl chloride (1 mmol), then dichloromethane (3 mL) and triethylamine (1.5 mmol) were added at 0 • C. The mixture was stirred at room temperature and was monitored by TLC. Upon completion, the reaction solution was washed with saturated aqueous NaCl and dried with Na 2 SO 4 . The solvent was removed under vacuum, and the residue was purified by column chromatography on silica gel with hexane-ethyl acetate (3:1 to 2:1) as eluent to afford the desired product 1.  133.4, 132.3, 132.1, 128.7, 127.5, 122.9, 113.9, 112.5, 67.7, 42.9.

General Procedure for the Synthesis of Compound 5a-h
To a round-bottom flask were added 2-chloroethylamine hydrochloride (1.2 mmol) and 5-bromothiophene-2-sulfonyl chloride (261 mg, 1 mmol), then dichloromethane (3 mL) and triethylamine (2.5 mmol) were added at 0 • C. The mixture was stirred at room temperature and was monitored by TLC. Upon completion, the reaction solution was washed with 2 N HCl and saturated aqueous NaCl and dried with Na 2 SO4. The solvent was evaporated to give the desired compounds 5-bromo-N-(2-chloroethyl)thiophene-2-sulfonamide (3, 289 mg, 95% yield) as a yellowish solid. 1  To an oven-dried round-bottom flask were added THF (10 mL) and phenol derivatives (2 mmol). The solution was cooled to 0 • C and NaH (5 mmol) was added in portions. The mixture was stirred for 2 h at room temperature, and then the solvent was removed under vacuum. DMF (5 mL), 4 (1 mmol), and Benzyltriethylammonium chloride(TEBA) (0.3 mmol) were added successively to the residue. The mixture was stirred at 50 • C and was monitored by TLC. Upon completion, water was added, and the reaction was extracted twice with CH 2 Cl 2 . The combined organic layer was washed with saturated aqueous NaCl and dried with Na 2 SO 4 . The solvent was removed under vacuum, and the residue was purified by column chromatography on silica gel with hexane-ethyl acetate (4:1) as eluent to afford compounds 5a-h.

Procedure for the Synthesis of Compound 5i
To a round-bottom flask were added 2-phenoxy ethylamine(1.2 mmol) and 2-chloropyridine-5-sulfonyl chloride (1 mmol), then dichloromethane (3 mL) and triethylamine (1.5 mmol) were added at 0 • C. The mixture was stirred at room temperature and was monitored by TLC. Upon completion, the reaction solution was washed with saturated aqueous NaCl and dried with Na 2 SO4. The solvent was removed under vacuum, and the residue was purified by column chromatography on silica gel with hexane-ethyl acetate as eluent to afford the desired product 1g (75% yield) as a white solid. 1  To an oven-dried reaction tube were added PdCl 2 (PPh 3 ) (0.1 mmol), CuI (0.2 mmol), and compound 1g (1 mmol) under N 2 . Then THF (2 mL), Et 3 N (1.5 mmol), and trimethylsilylacetylene (1.25 mmol) were added successively by syringe. The mixture was stirred at room temperature and was monitored by TLC. Upon completion, the reaction solution was condensed and purified by column chromatography on silica gel with hexane -ethyl acetate (2:1) as eluent to afford the desired 5i.

General Procedure for the Synthesis of Compound 7
To a round-bottom flask were added compound 6 (0.025 mmol), azide (0.03 mmol), sodium l-ascorbate (0.0075 mmol), CuSO 4 ·5H 2 O (0.005 mmol), DMSO (1 mL), and H 2 O (5 mL). The mixture was stirred for 3 h at room temperature and was monitored by TLC. Upon completion, ethyl acetate was added, and the mixture was washed with saturated aqueous NaCl and dried with Na 2 SO 4 . The solvent was removed under vacuum, and the residue was purified by column chromatography on silica gel with hexane-ethyl acetate (1:2) as eluent to afford compound 7.