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Article

Synthesis and Antitumor Activity of a Series of Novel 1-Oxa-4-azaspiro[4,5]deca-6,9-diene-3,8-dione Derivatives

by
Ze Yang
1,2,
Qiu Zhong
3,
Shilong Zheng
3,
Guangdi Wang
3,* and
Ling He
1,*
1
Key Laboratory of Drug–Targeting and Drug Delivery System of the Education Ministry, Department of Medicinal Chemistry, West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, China
2
Sichuan Engineering Laboratory for Plant–Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, Department of Medicinal Chemistry, West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, China
3
Department of Chemistry, RCMI Cancer Research Center, Xavier University of Louisiana, New Orleans, LA 70125, USA
*
Authors to whom correspondence should be addressed.
Molecules 2019, 24(5), 936; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules24050936
Submission received: 9 February 2019 / Revised: 24 February 2019 / Accepted: 26 February 2019 / Published: 7 March 2019
(This article belongs to the Section Medicinal Chemistry)
Browse Figures
Versions Notes

Abstract

:
A series of novel 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones were designed and synthesized by using 4-aminophenol and α-glycolic acid or lactic acid as starting materials in three or four steps. The key step is the metal-catalyzed oxidative cyclization of the amide to 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones (10a and 10b), the reaction conditions of which are investigated and optimized. The anticancer activity of 17 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione derivatives was evaluated. Preliminary results showed that 15 compounds have moderate to potent activity against human lung cancer A549, human breast cancer MDA-MB-231, and human cervical cancer HeLa cancer cell lines. Among them, compounds 11b and 11h were the most potent against A549 cell line with 0.18 and 0.19 µM of IC50, respectively; compounds 11d, 11h, and 11k showed the most potent cytotoxicity against MDA-MB-231 cell line with 0.08, 0.08, and 0.09 µM of IC50, respectively, while the activities of 11h, 11k, and 12c against HeLa cell line were the most potent with 0.15, 0.14, and 0.14 µM of IC50, respectively. Compound 11h is a promising candidate for further development, which emerged as the most effective compound overall against the three tested cancer cell lines.

Graphical Abstract

1. Introduction

Spirocyclic compounds are an important class of widely distributed natural products [1], such as crotonosine extracted from legumes [2] and brevione O (1) extracted from marine fungi [3]. Many spirocycles exhibit different biological activities including antitumor activities. For example, as shown in Figure 1, spirocyclic oxindole-benzofuro-azepinones (2) have potent antitumor activity comparable to cisplatin [4] and the IC50 value of 3,3′-spirocyclopentene oxindole (3) toward wild-type p53-MDM2 was 3.1 nM [5]. The compounds with a quinone scaffold are another class of interesting natural products. They play an important role in the redox process of organisms due to the special character of the quinones, with potential to become attractive cancer chemotherapy drugs such as cryptotanshinone (CPT, 4), a diterpenoid which exerts antitumor activity through the inhibition of STAT3, [6] and β-lapachone (β-lap, 5), which is a quinone oxidoreductase 1 (NQO1 or NAD(P)H)-dependent antitumor drug [7,8]. Our previous work hybridized the spirocycle and quinone scaffolds to generate a novel series of 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones (6) which showed promising anticancer activities [9,10,11,12]. Herein, we report a new synthetic approach to obtain more diversified 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones for further anticancer activity assessment.

2. Results and Discussion

2.1. General Route to Novel 1-Oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones

1-Oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione derivatives were prepared as shown in Scheme 1. The condensation of 4-hydroxyphenol (7) and glycolic acid (8a) or lactic acid (8b) using N,N′-dicyclohexylcarbodiimide (DCC) as an activating agent formed 2-hydroxy-N- (4-hydroxyphenyl)acetamide (9a) or 2-hydroxy-N-(4-hydroxyphenyl)propanamide (9b). The amides (9a and 9b) were then oxidized by bis(acetoxy)iodobenzene (PhI(OAc)2) [13,14,15] to cyclize into 1-oxo-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (10a) and 2-methyl-1-oxo-4-azaspiro[4.5] deca-6,9-diene-3,8-dione (10b) under the catalysis of tetrakis(acetonitrile)copper (I) perchlorate (Cu[(CH3CN)4ClO4]). The N-alkylation of 10a and 10b afforded 1-oxa-4-azaspiro[4.5] deca-6,9-diene-3,8-dione derivatives 11a–11k. Among them, 11c and 11j with a terminal alkyne clickly reacted with the azide compounds to afford the triazole derivatives 12a–12d through a one-pot protocol catalyzed by copper (I) generated in situ from copper (II) sulfate and sodium ascorbate [16,17].

2.2. Optimization for the Formation of 1-Oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione from 2-Hydroxy-N-(4-hydroxyphenyl)acetamide

The metal-catalyzed oxidative cyclization for the conversion from 2-hydroxy-N- (4-hydroxyphenyl)acetamide (9a) to 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione is a new reaction and a key step in the synthesis of the compound. We first investigated and optimized its reagents and conditions. The results in Table 1 show that Cu[(CH3CN)4ClO4] [18] and rhodium acetate (Rh2(OAc)4) catalyze the reaction to form 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (10a) with PhI(OAc)2 as an oxidant with yields of 72% and 75%, respectively (Entry 1 and 2), but manganese acetate (Mn(OAc)2), ferrous chloride (FeCl2), and zinc chloride (ZnCl2) failed to catalyze the reactions (Entry 3, 4, and 5). Considering that the metal copper catalyst is more economical and more environmentally friendly, Cu[(CH3CN)4ClO4] was chosen as the reaction catalyst. We chose the oxidant PhI(OAc)2 since it displayed slightly better activity than bis(trifluoroacetoxy) iodobenzene (PIFA) in the reaction (Entry 1 and 6).
Compared to previous work on the synthesis of triazole-spirodiones [8], our current approach shortens the synthesis steps and broadens the range of substituents on nitrogen. For example, in the reaction step converting triazoles to alkanes, a variety of heterocyclic or aromatic rings derivatives can be obtained in fewer synthetic steps. At the same time, R1 can be varied to increase the application range of the substrate.
Next, the effect of different bases was investigated, including potassium carbonate (K2CO3), sodium hydride (NaH), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and trimethylamine (Et3N) on the alkylation of 1-oxa-4-azaspiro[4.5] deca-6,9-diene-3,8-dione (10a) with propargyl bromide. DBU was the best available base for the reaction based on the results in Table 2.
Based on literature reports [19,20,21,22,23,24], the PIDA oxidation step may have proceeded by a mechanism where a copper-conjugated intermediate is formed as illustrated in Scheme 2. In the presence of PhI(OAc)2, 2-hydroxyn-(4-hydroxyphenyl) acetamide is oxidized to quinone and then converted to a carbon cation. At the same time, the hydroxyl group forms a complex with copper, so that the oxygen of hydroxyl can facilitate the attack to the carbon cation, thereby closing the ring to form a spiral ring derivative.

2.3. Antitumor Activity of 1-Oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones

Next, we investigated in vitro antitumor activities of the synthesized 1-oxa-4-azaspiro[4.5] deca-6,9-diene-3,8-dione derivatives toward lung cancer cell line A549, breast cancer cell line MDA-MB-231, and cervical cancer cell line HeLa with bendamustine (a bifunctional alkylating agent) and vorinostat (an HDAC inhibitor) as positive drug controls (Table 3). All the compounds except 12a showed more potent antitumor activity against MDA-BM-231 and HeLa cell lines than bendamustine and vorinostat. Among them, compounds 11b and 11h had IC50 values of 0.18 and 0.19 μM against A549, respectively; the IC50 values of 11d, 11h, and 11k were 0.09, 0.08, and 0.08 μM toward MDA-BM-231, respectively; and the compounds with IC50 value below 0.20 μM against HeLa cells were 11f, 11h, 11k, and 12c. Compound 11h emerged as a promising candidate for further research, showing most antiproliferative efficacy overall against all three cancer cell lines. From the activity test of this series of derivatives, it can be seen that 1-oxa-4-azaspiro[4.5]indole-6,9-diene-3,8-dione is an active essential group. Secondly, from the antitumor data of 10a ≤ 10b, 11c ≤ 11j, 11h ≤ 11k, it can be seen that the activity of 2-CH3 decreased slightly. When the 4-position substituent is p-bromobenzyl, the activity appears to be optimal.

3. Materials and Methods

3.1. Instruments and Reagents

NMR spectra were recorded on a Mercury 400 MHz NMR, Varian, Palo Alto, CA, USA and an 600 MHz NMR, Agilent Technologies Inc. Palo Alto, California, USA(CDCl3 was the solvent and TMS was the internal standard). MS spectra were measured on Bruker Daltonics Data Analysis 3.4 Mass Spectrometer, Bruker, Karlsruhe, Germany and Thermo LTQ Orbitrap-XL Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). A YRT-3 melting point instrument (Tianda Tianfa Company, Tianjin, China) was used for measuring melting points, where the measured temperature was uncorrected. HSGF 254 high-efficiency thin-layer chromatography silica gel plates were purchased from Huiyou Development Co., Yantai, Shangdong, China. Ltd. HSGF 254 thin-layer silica gel (300 mesh ~ 400 mesh) was purchased from Ocean Chemical Plant (Qingdao, Shangdong, China). The reagents used were analytically pure unless otherwise specified, and the solvents used were dried by conventional methods. (NMR spectra (1H and 13C) of all 17 1-oxa-4-azaspiro[4.5] deca-6,9-diene-3,8-dione derivatives are provided in Supplementary Materials).

3.2. Synthesis of 1-Oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones

3.2.1. Synthesis of Compounds 10a–10b

In a 25 mL single-neck round bottom flask and under a nitrogen atmosphere, compounds 9a or 9b (1 mmol), PhI(OAc)2 (2 mmol), and Cu[(CH3CN)4ClO4] (0.05 mmol) were added to dried DCM (10 mL), and the reaction mixture was stirred at room temperature. The reaction was monitored by thin layer chromatography (TLC) (petroleum ether/ethyl acetate = 1:1). After completion of the reaction, the mixture was concentrated under reduced pressure and purified by column chromatography.
1-Oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (10a): 119 mg viscous off-white solid, yield 72%; 1H NMR (600 MHz, CDCl3) δ 7.77 (s, 1H), 6.72 (d, J = 8.8 Hz, 2H), 6.22 (d, J = 8.8 Hz, 2H), 4.36 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 183.9, 172.8, 143.9, 129.6, 83.8, 66.2. HR-MS (ESI) m/z: calcd C8H7NO3 [M + H]+ 166.0504, found 166.0502.
2-Methyl-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (10b): 130 mg viscous off-white solid, yield 73%; 1H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 6.70 (ddd, J = 43.1, 10.0, 3.1 Hz, 2H), 6.23 (ddd, J = 14.6, 10.2, 1.9 Hz, 2H), 4.53 (q, J = 6.7 Hz, 1H), 1.49 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 184.2, 175.2, 145.5, 144.4, 130.2, 129.0, 82.2, 73.3, 18.4. HRMS (ESI) calcd C9H9NO3 [M + H]+ 180.0661, found 180.0658.

3.2.2. Synthesis of Compounds 11a–11k

In a 10 mL one-necked round bottom flask, compound 3 (0.1 mmol), dry THF (2 mL), and DBU (0.15 mmol) were added, and the mixture was stirred for 10 min in an ice water bath, then the halogenated hydrocarbon (0.12 mmol) was slowly added. The reaction was monitored by chromatography (TLC) (petroleum ether/ethyl acetate = 2:1). After completion of the reaction, a saturated NH4Cl solution was added and extracted with ethyl acetate. The organic layer was separated, dried with MgSO4, and evaporated to obtain crude products which was purified by flash chromatography.
4-Methyl 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11a): 13 mg white solid, yield 74%, mp 112–114 °C; 1H NMR (400 MHz, CDCl3) δ 6.60 (d, J = 10.1 Hz, 2H), 6.36 (d, J = 10.0 Hz, 2H), 4.43 (s, 2H), 2.74 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 183.7, 169.4, 143.4, 131.5, 87.5, 66.6, 25.1. HRMS (ESI) calcd C9H9NO3 [M + H]+ 180.0661, found 180.0658.
4-Ethyl-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11b): 17 mg of white solid, yield 86%, mp 93–96 °C; 1H NMR (400 MHz, CDCl3) δ 6.66–6.60 (m, 2H), 6.37–6.28 (m, 2H), 4.39 (s, 2H), 3.21 (q, J = 7.2 Hz, 2H), 1.16 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 183.9, 169.5, 144.1, 130.9, 87.5, 66.5, 35.1, 14.6. HRMS (ESI) calcd C10H11NO3 [M + H]+ 194.0817, found 194.0815.
4-(Prop-2-yn-1-yl)-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11c): 14 mg of white solid. Yield 67%, mp 155–157 °C; 1H NMR (400 MHz, CDCl3) δ 6.68 (d, J = 10.1 Hz, 2H), 6.35 (d, J = 10.1 Hz, 2H), 4.43 (s, 2H), 3.99 (d, J = 2.5 Hz, 2H), 2.22 (t, J = 2.5 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 183.7, 168.7, 143.1, 131.5, 87.1, 77.3, 73.1, 66.2, 28.8. HRMS (ESI) calcd C11H9NO3 [M + H]+ 203.0582, found 203.0580.
4-(3-Methylbut-2-en-1-yl)-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11d): 17mg brown liquid, yield 74%: 1H NMR (400 MHz, CDCl3) δ 6.62 (d, J = 10.1 Hz, 2H), 6.29 (d, J = 10.1 Hz, 2H), 4.41 (s, 2H), 3.81 (d, J = 7.2 Hz, 2H), 1.65 (s, 3H), 1.53 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 184.1, 169.3, 144.2, 137.4, 130.4, 118.8, 87.2, 66.5, 37.8, 25.5, 18.0. HRMS (ESI) calcd C13H15NO3 [M + H]+ 234.1130, found 234.1128.
4-(But-2-yn-1-yl)-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11e): 17 mg yellow-white solid, yield 76%, mp 77–79 °C; 1H NMR (400 MHz, CDCl3) δ 6.67 (d, J = 10.1 Hz, 2H), 6.34 (d, J = 10.1 Hz, 2H), 4.41 (s, 2H), 3.94 (d, J = 2.3 Hz, 2H), 1.72 (t, J = 2.3 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 184.0, 169.0, 143.5, 131.1, 87.1, 81.2, 72.8, 66.3, 29.3, 3.4. HRMS (ESI) calcd C12H11NO3 [M + H]+ 218.01717, found 218.0815.
(E)-4-(3,7-Dimethyloctyl-2,6-dien-1-yl)-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11f): 21 mg of a brown liquid, yield 70%; 1H NMR (400 MHz, CDCl3) δ 6.60 (d, J = 10.0 Hz, 2H), 6.26 (d, J = 10.0 Hz, 2H), 5.06 (s, 1H), 5.01 (t, J = 6.6 Hz, 1H), 4.40 (s, 2H), 3.82 (d, J = 7.1 Hz, 2H), 2.04–1.99 (m, 2H)), 1.95 (d, J = 7.4 Hz, 2H), 1.67 (s, 3H), 1.58 (s, 3H), 1.51 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 184.1, 169.3, 144.2, 140.8, 131.9, 130.5, 123.6, 118.7, 87.2, 66.6, 39.3, 37.7, 26.0, 25.7, 17.7, 16.4. HRMS (ESI) calcd C18H23NO3 [M + H]+ 302.1756, found 302.1755.
4-(4-Methylbenzyl)-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11g): 22 mg viscous white solid, yield 81%, mp 76–78 °C; 1H NMR (400 MHz, CDCl3) δ 7.06 (d, J = 1.8 Hz, 4H), 6.48–6.39 (m, 2H), 6.17–6.11 (m, 2H), 4.46 (s, 2H), 4.35 (s, 2H), 2.31 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 184.0, 169.7, 143.8, 138.0, 133.1, 130.6, 129.3, 128.6, 87.4, 66.4, 43.5, 21.1. HRMS (ESI) calcd C16H15NO3 [M + Na]+ 292.0950, found 292.0944.
4-(4-Bromobenzyl)-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11h): 25 mg of light yellow liquid, yield 75%, mp 90–93 °C; 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.1 Hz, 2H), 6.43 (d, J = 9.9 Hz, 2H), 6.18 (d, J = 9.9 Hz, 2H), 4.47 (s, 2H), 4.33 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 183.8, 169.9, 143.5, 135.2, 131.9, 130.9, 130.3, 122.3, 87.3, 66.3, 43.1. HRMS (ESI) calcd C15H12BrNO3 [M + Na]+ 355.9898, found 355.9892.
4-(4-Nitrobenzyl)-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11i): 11 mg viscous off-white solid, yield 36%; 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.3 Hz, 2H), 6.59 (d, J = 9.8 Hz, 2H), 6.20 (d, J = 9.8 Hz, 2H), 5.42 (s, 2H), 4.67 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 185.1, 170.7, 145.1, 142.0, 128.6, 128.4, 124.0, 119.2, 97.8, 68.7, 31.6. HRMS (ESI) calcd C15H12N2O5 [M + H]+ 301.0824, found 301.0822.
2-Methyl-4-(prop-2-yn-1-yl)-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11j): 13 mg of off-white solid, yield 60%, mp 153–155 °C; 1H NMR (400 MHz, CDCl3) δ 6.68 (dd, J = 10.0, 2.8 Hz, 1H), 6.61 (dd, J = 10.0, 2.8 Hz, 1H), 6.34 (dd, J = 14.8, 10.1 Hz, 2H), 4.56 (q, J = 6.7 Hz, 1H), 4.05–3.91 (m, 3H), 1.51 (d, J = 6.7 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 183.9, 171.3, 144.6, 143.3, 131.9, 130.8, 85.5, 77.4, 73.1, 73.1, 28.9, 18.5. HRMS (ESI) calcd C12H11NO3 [M + H]+ 218.0817, found 218.0817.
4-(4-Bromobenzyl)-2-methyl-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (11k): 23 mg viscous yellow-white solid, yield 67%; 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 8.3 Hz, 2H), 7.05 (d, J = 8.2 Hz, 2H), 6.44 (dd, J = 10.0, 3.0 Hz, 1H), 6.33 (dd, J = 10.0, 3.0 Hz, 1H), 6.26–6.05 (m, 2H), 4.60 (q, J = 6.7 Hz, 1H), 4.41–4.23 (m, 2H), 1.53 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 183.9, 172.2, 145.1, 143.7, 135.4, 131.9, 131.4, 130.3, 130.2, 122.2, 85.7, 73.2, 43.3, 18.7. HRMS (ESI) calcd C16H15BrNO3 [M + H]+ 301.0824, found 301.0822.

3.2.3. Synthesis of Compounds 12a–12d

To an anhydrous DMSO (1 mL) solution of NaN3 (0.5 mmol) was added organic halide (0.5 mmol) and the mixture was stirred overnight. Water (2–3 mL) was added, followed by solid sodium ascorbate (0.05 mol), the alkyne 11c or 11j (0.5 mmol), and aqueous CuSO4 solution (1mL, 1 M). The mixture was stirred for 3–12 h until the starting material disappeared, then more water was added slowly until the product precipitated completely from the solution. The product was collected by filtration, washed with water, and dried in air.
(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(4-((3,8-dioxo-1-oxa-4-azaspiro[4.5]deca-6,9-dien-4yl)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (12a): 31 mg of white solid, yield 54%, mp 183–185 °C; 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 6.53 (ddd, J = 34.7, 10.1, 3.2 Hz, 2H), 6.26 (ddd, J = 62.1, 10.1, 2.2 Hz, 2H), 5.81 (d, J = 9.2 Hz, 1H), 5.42 (t, J = 9.5 Hz, 1H), 5.26 (dt, J = 29.9, 9.6 Hz, 2H), 4.57 (d, J = 15.7 Hz, 1H), 4.45 (s, 2H), 4.40–4.29 (m, 2H), 4.19–4.12 (m, 1H), 3.99 (ddd, J = 10.2, 5.0, 2.2 Hz, 1H), 2.10 (s, 3H)), 2.06 (s, 3H), 2.03 (s, 3H), 1.89 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 183.7, 170.5, 169.8, 169.3, 168.9, 143.5, 143.4, 143.1, 131.6, 130.9, 121.6, 87.3, 85.9, 75.2, 72.3, 70.6, 67.6, 66.2, 61.4, 35.0, 20.7, 20.5, 20.5, 20.1. HRMS (ESI) calcd C25H28N4O12 [M + H]+ 577.1782, found 577.1776.
4-((1-Ethyl-1H-1,2,3-triazol-4-yl)methyl)-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (12b): 16 mg, yellow-white viscous liquid, yield 58%; 1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 8.4 Hz, 2H), 7.46 (s, 1H), 7.12 (d, J = 8.4 Hz, 2H), 6.50 (ddd, J = 17.9, 10.0, 3.1 Hz, 2H), 6.28 (dd, J = 10.0, 2.1 Hz, 1H), 6.19 (dd, J = 10.0, 2.1 Hz, 1H), 4.54 (q, J = 6.7 Hz, 1H), 4.41 (s, 2H), 1.48 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 183.8, 169.8, 143.4, 142.3, 131.2, 122.5, 87.5, 66.3, 45.4, 35.2, 15.4. HRMS (ESI) calcd C13H14N4O3 [M + H]+ 275.1144, found 275.1140.
4-((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-methyl-1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (12c): 21 mg of yellow-white viscous liquid, yield 49%; 1H NMR (400 MHz, CDCl3) δ 7.53–7.49 (m, 2H), 7.46 (s, 1H), 7.12 (d, J = 8.4 Hz, 2H), 6.55–6.45 (m, 2H), 6.28 (dd, J = 10.0, 2.1 Hz, 1H), 6.19 (dd, J = 10.0, 2.1 Hz, 1H), 4.54 (q, J = 6.7 Hz, 1H), 4.41 (s, 2H), 1.48 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 183.9, 172.1, 144. 8,143.5, 142.9, 133.3, 132.4, 131.7, 130.5, 129.7, 123.1, 123.1, 85.8, 73.2, 35.3, 18.6. HRMS (ESI) calcd C19H17BrN4O3 [M + Na]+ 451.0382, found 451.0378.
(2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(4-((2-methyl-3,8-dioxo-1-oxa-4-azaspiro[4.5]deca-6,9-dien-4-yl) methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (12d): 27 mg of white solid, yield 47%, mp 86–90 °C; 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 6.62–6.05 (m, 4H), 5.81 (d, J = 9.2 Hz, 1H), 5.41 (td, J = 9.5, 1.9 Hz, 1H), 5.31–5.19 (m, 2H), 4.58 (t, J = 15.7 Hz, 2H), 4.35–4.25 (m, 2H), 4.14 (d, J = 12.4 Hz, 1H), 4.03–3.95 (m, 1H), 2.09 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H), 1.87 (s, 3H), 1.51 (t, J = 6.8 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 183.8 (2C), 172.09, 172.1, 170.5, 169.8 (2C), 169.3 (169.32), 169.3 (169.29), 168.9, 168.8, 144.9, 144.6, 143.7, 143.6, 143.5, 143.3, 132.1, 131.3, 131.0, 130.2, 121.65(2C), 85.9(85.88), 85.9(85.85), 85.8, 85.7, 75.2, 75.0, 73.2, 73.1, 72.2 (2C), 70.7, 70.6, 69.2, 67.6 (2C), 61.4, 59.1, 35.2 (2C), 20.7 (2C), 20.5 (20.49), 20.5 (20.46), 20.1 (20.13), 20.1 (20.11), 18.6, 18.4, 16.5 (2C). HRMS (ESI) calcd for C26H30N4O12 [M + H]+ 591.1938, found 591.1933.

3.3. Cell Culture and Antiproliferative Assays

Human non-small cell lung carcinoma (A549), human breast adenocarcinoma (MDA-MB-231), and human cervix carcinoma (HeLa) cell lines (ATCC, Manassas, VA, USA) were grown in DMEM supplemented with 115 units/mL of penicillin G, 115 μg/mL of streptomycin, and 10% fetal bovine serum (all from Life Technologies, Grand Island, NY, USA). Cells were seeded in 96-well plates (5 × 103 cells/well) containing 50 μL growth medium for 24 h. After medium removal, 100 μL fresh medium containing individual compounds and both bendamustine and vorinostat controls at different concentrations was added to each well and incubated at 37 °C for 72 h. After 24 h of culture, the cells were supplemented with 50 μL of compounds, bendamustine, or vorinostat dissolved in DMSO (less than 0.25% in each preparation). After 72 h of incubation, 20 μL of resazurin was added for 2 h before recording fluorescence at 560 nm (excitation) and 590 nm (emission) using a VICTOR microtiter plate fluorometer (Perkin-Elmer, Waltham, MA, USA). The IC50 was defined as the compound concentration required to inhibit cell proliferation by 50% in comparison with cells treated with the maximum amount of DMSO (0.25%) which was considered as 100% viability.

4. Conclusions

From 4-aminophenol and α-glycolic acid or lactic acid, a series of novel 1-oxa-4- azaspiro[4.5]deca-6,9-diene-3,8-dione derivatives were synthesized through a newly developed approach including a key intramolecular metal-catalyzed oxidative cyclization. The preliminary antitumor activity of 17 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione derivatives was investigated, and the results indicate that all the compounds showed moderate to potent antitumor activity against A549, MDA-BM-231, and HeLa cell lines. The most potent compound, 11h, has IC50 values of 0.19, 0.08, and 0.15 µM against A549, MDA-BM-231, and HeLa cell lines, respectively, which is a promising candidate for further research.

Supplementary Materials

The NMR spectra (1H and 13C) of all 17 1-oxa-4-azaspiro[4.5] deca-6,9-diene-3,8-dione derivatives 10a–10b, 11a–11k, and 12a–12d are available online.

Author Contributions

Conception and design of the work: L.H., G.W., S.Z.; the acquisition, analysis, and interpretation of data: Z.Y., Q.Z., S.Z., G.W., and L.H.; writing and proofread of the paper: L.H., Z.Y., G.W., and S.Z.; all authors finally reviewed and approved the manuscript.

Funding

This work was funded by Sichuan University-Lu Zhou Strategic Cooperation Projects (2017 CDLZ-S34) (L. He) and in part supported by NIH RCMI program at Xavier University of Louisiana through Grant (2G12MD007595) (G. Wang).

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.
Molecules 24 00936 g001 550
Figure 1. Biologically active spirocyclic and quinone derivatives.
Figure 1. Biologically active spirocyclic and quinone derivatives.
Molecules 24 00936 g001
Molecules 24 00936 sch001 550
Scheme 1. Synthesis of novel 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones.
Scheme 1. Synthesis of novel 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones.
Molecules 24 00936 sch001
Molecules 24 00936 sch002 550
Scheme 2. Proposed copper-catalyzed oxidation mechanism.
Scheme 2. Proposed copper-catalyzed oxidation mechanism.
Molecules 24 00936 sch002
Table
Table 1. Optimization of the reaction conditions for the formation of compound 10a.
Table 1. Optimization of the reaction conditions for the formation of compound 10a.
EntryOxidizing Agent
(2 eq)		Metal Catalyst
(0.05 eq)		Yield (%)
1PhI(OAc)2Cu[(CH3CN)4ClO4]72
2PhI(OAc)2Rh2(OAc)475
3PhI(OAc)2Mn(OAc)2trace
4PhI(OAc)2FeCl2trace
5PhI(OAc)2ZnCl2trace
6PIFACu[(CH3CN)4ClO4]68
7PhI(OAc)2Notrace
Table
Table 2. Optimization of the reaction conditions for compound 11c.
Table 2. Optimization of the reaction conditions for compound 11c.
EntryBase (1.5 eq)Temperature (°C)Yield (%)
1K2CO325trace
2NaH050
3DBU0-2567
4DABCO80trace
5Et3N25trace
6No25no reaction
Table
Table 3. In vitro antitumor activities of 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones against cancer cell lines.
Table 3. In vitro antitumor activities of 1-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-diones against cancer cell lines.
CompoundIC50 (μM)
A549MDA-BM-231HeLa
10a0.23 ± 0.150.28 ± 0.070.26 ± 0.12
10b0.52 ± 0.270.18 ± 0.081.34 ± 0.28
11a0.34 ± 0.090.23 ± 0.060.47 ± 0.13
11b0.18 ± 0.010.12 ± 0.060.27 ± 0.15
11c0.37 ± 0.110.13 ± 0.070.22 ± 0.05
11d0.72 ± 0.090.09 ± 0.020.20 ± 0.05
11e0.29 ± 0.100.15 ± 0.040.31 ± 0.16
11f0.24 ± 0.080.11 ± 0.050.19 ± 0.12
11g0.26 ± 0.140.10 ± 0.030.20 ± 0.07
11h0.19 ± 0.030.08 ± 0.020.15 ± 0.02
11i0.81 ± 0.130.21 ± 0.110.27 ± 0.14
11j0.39 ± 0.140.17 ± 0.060.60 ± 0.12
11k0.26 ± 0.080.08 ± 0.0040.14 ± 0.07
12a>104.75 ± 0.90>10
12b0.76 ± 0.100.27 ± 0.040.41 ± 0.04
12c0.90 ± 0.030.31 ± 0.010.14 ± 0.1
12d5.31 ± 1.010.31 ± 0.081.65 ± 0.46
bendamustine-13.28 ± 0.53>20
vorinostat-3.62 ± 0.184.52 ± 0.27
± standard deviation (SD) of triplicate experiments.

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Yang, Z.; Zhong, Q.; Zheng, S.; Wang, G.; He, L. Synthesis and Antitumor Activity of a Series of Novel 1-Oxa-4-azaspiro[4,5]deca-6,9-diene-3,8-dione Derivatives. Molecules 2019, 24, 936. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules24050936

AMA Style

Yang Z, Zhong Q, Zheng S, Wang G, He L. Synthesis and Antitumor Activity of a Series of Novel 1-Oxa-4-azaspiro[4,5]deca-6,9-diene-3,8-dione Derivatives. Molecules. 2019; 24(5):936. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules24050936

Chicago/Turabian Style

Yang, Ze, Qiu Zhong, Shilong Zheng, Guangdi Wang, and Ling He. 2019. "Synthesis and Antitumor Activity of a Series of Novel 1-Oxa-4-azaspiro[4,5]deca-6,9-diene-3,8-dione Derivatives" Molecules 24, no. 5: 936. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules24050936

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