Synthesis and Antiproliferative Evaluation of Novel Longifolene-Derived Tetralone Derivatives Bearing 1,2,4-Triazole Moiety

Seventeen novel 2-(5-amino-1-(substituted sulfonyl)-1H-1,2,4-triazol-3-ylthio)-6- isopropyl-4,4-dimethyl-3,4-dihydronaphthalen-1(2H)-one compounds were synthesized from the abundant and naturally renewable longifolene and their structures were confirmed by FT-IR, NMR, and ESI-MS. The in vitro cytotoxicity of the synthesized compounds was evaluated by standard MTT assay against five human cancer cell lines, i.e., T-24, MCF-7, HepG2, A549, and HT-29. As a result, compounds 6d, 6g, and 6h exhibited better and more broad-spectrum anticancer activity against almost all the tested cancer cell lines than that of the positive control, 5-FU. Some intriguing structure–activity relationships were found and are discussed herein by theoretical calculation.


Introduction
Tetralin (tetrahydronaphthalene) is a valuable ring for the construction of drugs. This ring structure is found in many naturally occurring plant-derived podophyllotoxin. The chemotherapeutic medications etoposide and teniposide are semisynthetic derivatives of podophyllotoxin, and are used as topoisomerase II inhibitor anticancer drugs [1]. Due to their high toxicity, drug resistance, and the gastrointestinal discomfort associated with their use, lots of structural modifications have been carried out; consequently, some derivatives with better performance, such as NK611, NPF, GL-331, and TOP53, have been discovered and tested in clinical trials [2]. The tetralin ring also exists in the structures of some clinically used anticancer drugs, for example, the anthracycline antibiotics doxorubicin, daunorubicin, epirubicin, and idarubicin [3], and so on. Many studies have been performed on tetralin derivatives for use as efficient antitumor agents [4][5][6][7][8][9].
A tetralin derivative 7-isopropyl-1,1-dimethyl-1,2,3,4-tetrahydronaphthalene can be readily prepared from longifolene, which is a major constituent of the abundant and naturally renewable heavy turpentine oil, a by-product in the production of rosin and turpentine from living Pinus.
According to [27], compound 5 can exist in three tautomeric forms, namely, 1H-, 2H-, and 4Hforms; the 1H-form was preferred. The N-sulfonylation reaction process was monitored by HPLC, and only one new peak was observed, leading us to speculate that compound 5 is in the 1H-form. Then, a N-sulfonylation reaction may occur on the nitrogen atoms of the 1H-site and the amino group. Comparing the 1 H-NMR spectra of compound 5 with the those of the target compounds, it was found Scheme 1. The synthetic route of 6a-6q (Table 1). Reagents and conditions: (a) Nano-crystalline sulfated zirconia catalyst, 180 • C, reflux 1 h; (b) TBHP, CH 3 CN, CuCl 2 , 42 • C; (c) NBS, Et 2 O, NH 4 OAc, rt; (d) 5-amino-3-mercapto-1,2,4-triazole, KOH, EtOH, 80 • C, reflux 4 h; (e) NaHCO 3 , CH 3 CN, a series of different sulfonyl chlorides, 40 • C. According to [27], compound 5 can exist in three tautomeric forms, namely, 1H-, 2H-, and 4H-forms; the 1H-form was preferred. The N-sulfonylation reaction process was monitored by HPLC, and only one new peak was observed, leading us to speculate that compound 5 is in the 1H-form. Then, a N-sulfonylation reaction may occur on the nitrogen atoms of the 1H-site and the amino group. Comparing the 1 H-NMR spectra of compound 5 with the those of the target compounds, it was found that the signals of the amino groups were preserved, meaning that a N-sulfonylation reaction took place on the nitrogen atom of the 1H-site. The result was further confirmed by a nuclear overhauser effect spectroscopy (NOESY) experiment of the target compound 6a. As shown in Figure 1, the hydrogen atoms of the amino group showed a stronger correlation with the hydrogen atoms of the methyl group on the phenylsulfonyl moiety than that of the methyl groups on the tetralone moiety, indicating that the amino group was closer to the phenylsulfonyl group than the methyl groups on the tetralone moiety.
Molecules 2020, 25, x FOR PEER REVIEW 3 of 12 that the signals of the amino groups were preserved, meaning that a N-sulfonylation reaction took place on the nitrogen atom of the 1H-site. The result was further confirmed by a nuclear overhauser effect spectroscopy (NOESY) experiment of the target compound 6a. As shown in Figure 1, the hydrogen atoms of the amino group showed a stronger correlation with the hydrogen atoms of the methyl group on the phenylsulfonyl moiety than that of the methyl groups on the tetralone moiety, indicating that the amino group was closer to the phenylsulfonyl group than the methyl groups on the tetralone moiety. The structures of the target compounds were confirmed by means of FT-IR, 1 H-NMR, 13 C-NMR, and ESI-MS. In the FT-IR spectra, the target compounds exhibited absorption bands at 3100-3465 cm −1 , which were assigned to the N-H stretching vibration. The two strong absorption bands at 3000-2850 cm −1 and 1675 cm −1 were attributed to the stretching vibrations of the saturated C-H and C=O in the tetralone moiety, respectively. The absorption bands at about 1640 cm −1 were due to the vibrations of C=N in the 1,2,4-triazole moiety. The two absorption bands around 1160-1190 cm −1 and 1300-1360 cm −1 , respectively, confirmed the presence of a sulfonyl group. In the 1 H-NMR spectra of 6a-6q, the signals shown at δ 6.0-8.5 ppm were assigned to the aromatic protons in the benzene rings and amino protons in the 1,2,4-triazole moiety. The other protons bonded to the saturated carbons of the tetralone moiety displayed signals in the range of δ 1.2-5.0 ppm. The 13 C-NMR spectra of all the target compounds showed peaks for the carbons of C=O in tetralone moiety at about δ 193.2 ppm, and the other saturated carbons displayed signals in the region of δ 22-50 ppm. For the 1,2,4-triazole moiety, the signals at about δ 170.8 ppm and δ 151.7 ppm were assigned to the carbons of C=N. In addition, the molecular weights of the compounds 2-5 and target compounds 6a-6q were confirmed by the ESI-MS (see supplementary).

In Vitro Assay of Antiproliferative Activity
The in vitro antiproliferative activity of compounds 2, 3, 4, 5, and all 17 target compounds, i.e., 6a-6q, against five human cancer cell lines (T-24 human bladder cancer cell, MCF-7 human breast cancer cell, HepG2 human liver cancer cell, A549 human lung adenocarcinoma drug-resistant cell line, and HT-29 human colon cancer cell line) were evaluated by MTT (methylthiazolytetrazolium) assay. In addition, the potent anticancer drug 5-fluorouracil (5-FU) was used as a positive control in each panel. The in vitro antiproliferative activity test results of compounds 2, 3, 4, 5, and all 17 target compounds 6a-6q are shown in Table 2. The structures of the target compounds were confirmed by means of FT-IR, 1 H-NMR, 13 C-NMR, and ESI-MS. In the FT-IR spectra, the target compounds exhibited absorption bands at 3100-3465 cm −1 , which were assigned to the N-H stretching vibration. The two strong absorption bands at 3000-2850 cm −1 and 1675 cm −1 were attributed to the stretching vibrations of the saturated C-H and C=O in the tetralone moiety, respectively. The absorption bands at about 1640 cm −1 were due to the vibrations of C=N in the 1,2,4-triazole moiety. The two absorption bands around 1160-1190 cm −1 and 1300-1360 cm −1 , respectively, confirmed the presence of a sulfonyl group. In the 1 H-NMR spectra of 6a-6q, the signals shown at δ 6.0-8.5 ppm were assigned to the aromatic protons in the benzene rings and amino protons in the 1,2,4-triazole moiety. The other protons bonded to the saturated carbons of the tetralone moiety displayed signals in the range of δ 1.2-5.0 ppm. The 13 C-NMR spectra of all the target compounds showed peaks for the carbons of C=O in tetralone moiety at about δ 193.2 ppm, and the other saturated carbons displayed signals in the region of δ 22-50 ppm. For the 1,2,4-triazole moiety, the signals at about δ 170.8 ppm and δ 151.7 ppm were assigned to the carbons of C=N. In addition, the molecular weights of the compounds 2-5 and target compounds 6a-6q were confirmed by the ESI-MS (see Supplementary).

In Vitro Assay of Antiproliferative Activity
The in vitro antiproliferative activity of compounds 2, 3, 4, 5, and all 17 target compounds, i.e., 6a-6q, against five human cancer cell lines (T-24 human bladder cancer cell, MCF-7 human breast cancer cell, HepG2 human liver cancer cell, A549 human lung adenocarcinoma drug-resistant cell line, and HT-29 human colon cancer cell line) were evaluated by MTT (methylthiazolytetrazolium) assay. In addition, the potent anticancer drug 5-fluorouracil (5-FU) was used as a positive control in each panel. The in vitro antiproliferative activity test results of compounds 2, 3, 4, 5, and all 17 target compounds 6a-6q are shown in Table 2. Table 2. Antiproliferative activity of the compounds 2, 3, 4, 5, and 6a-6q a.

Compound
IC 50 (µM) The target compounds presented obviously different antineoplastic activity against the five tested cancer cell lines. As shown in Table 2, compounds 6g, 6h, and 6d exhibited excellent and broad-spectrum antitumor activity against almost all the tested cancer cell lines, and compounds 6b, 6c, and 6f showed good activity against A549 and HT-29. All these compounds displayed better or comparable antitumor activity than that of the positive control, 5-FU. Notably, compound 6g had the best antineoplastic activity against MCF-7, with IC 50 values of 4.42 ± 2.93 µM, and compound 6h against A549 with IC 50 values of 9.89 ± 1.77 µM.
As shown in Figure 2a,d,g, a large portion of the HOMO was located on the 1,2,4-triazole ring, the amino group, the sulfur atom of thioether group, and the oxygen atom of carbonyl group for three compounds. However, the LUMO for compounds 6h (R = 3 -NO 2 -4 -Cl) and 6k (R = 3 -NO 2 ) was located on the substituted phenylsulfonyl group moieties (Figure 2b,e), and showed phase reversal. The compound 6j (R = 4 -Cl) had LUMO on the tetralone moiety, showing an obvious difference with compounds 6h and 6k. In addition, molecule charge distribution was also one of the influential factors concerning activity. So, the electrostatic potentials (ESPs) and dipole moments for three compounds were calculated by means described above (Figure 2c,f,i). In comparison to compounds 6k and 6j, 6h showed higher negative electrostatic potential on the end of the R group, and possessed higher dipole moment, i.e., 9.48 D, than compounds 6k, i.e., 9.01 D, and 6j, 5.23 D. Overall, these differences were

General Information
The structures of the synthesized derivatives of tetralin were confirmed by means of 1 H-NMR, 13 C-NMR, FT-IR, and ESI-MS. NMR spectra were recorded in a CDCl3 or DMSO-d6 solvent on a Bruker Avance III HD 600 MHz spectrometer (Bruker Co., Ltd., Zurich, Switzerland). FT-IR spectra were recorded as KBr pellets on Nicolet iS50 FT-IR spectrometer (Thermo Scientific Co., Ltd., Madison, WI., USA). MS spectra were obtained by means of the electrospray ionization (ESI) method on TSQ Quantum Access MAX HPLC-MS instrument (Thermo Scientific Co., Ltd., Waltham, MA, USA). Melting points were determined using a MP420 automatic melting point apparatus (Hanon Instruments Co., Ltd., Jinan, China) and were uncorrected. The GC analysis was conducted on an Agilent 6890 GC (Agilent Technologies Inc., Santa Clara, CA., USA) equipped with column HP-1 (30 m, 0.530 mm, 0.88 µ m) and FID. The HPLC analysis was performed on a Waters 1525 instrument (Waters Co., Ltd., USA) equipped with column SunFire C18 5 µ m (4.6 mm × 150 mm). Longifolene (GC purity 65%) was provided by Wuzhou Pine Chemicals Co., Ltd. Wuzhou, Guangxi, China. Other reagents were provided by commercial suppliers, and were used as received.

Preparation of Catalyst
A catalyst was prepared according to a method described in the literature [31]. After diluting with n-propanol (30% by weight), zirconium was used as the precursor. Sulfuric acid (1.02 mL) was added to the zirconium precursor, and water was then added dropwise under continuous stirring until the formation of gel occurred (4.2 mL, water to propoxide molar ratio = 2.7). The resulting gel was dried at 110 °C for 12 h, followed by calcination at 600 °C for 2 h in a static air atmosphere to yield the sulfated zirconia catalyst.

General Information
The structures of the synthesized derivatives of tetralin were confirmed by means of 1 H-NMR, 13 C-NMR, FT-IR, and ESI-MS. NMR spectra were recorded in a CDCl 3 or DMSO-d 6 solvent on a Bruker Avance III HD 600 MHz spectrometer (Bruker Co., Ltd., Zurich, Switzerland). FT-IR spectra were recorded as KBr pellets on Nicolet iS50 FT-IR spectrometer (Thermo Scientific Co., Ltd., Madison, WI., USA). MS spectra were obtained by means of the electrospray ionization (ESI) method on TSQ Quantum Access MAX HPLC-MS instrument (Thermo Scientific Co., Ltd., Waltham, MA, USA). Melting points were determined using a MP420 automatic melting point apparatus (Hanon Instruments Co., Ltd., Jinan, China) and were uncorrected. The GC analysis was conducted on an Agilent 6890 GC (Agilent Technologies Inc., Santa Clara, CA., USA) equipped with column HP-1 (30 m, 0.530 mm, 0.88 µm) and FID. The HPLC analysis was performed on a Waters 1525 instrument (Waters Co., Ltd., USA) equipped with column SunFire C18 5 µm (4.6 mm × 150 mm). Longifolene (GC purity 65%) was provided by Wuzhou Pine Chemicals Co., Ltd. Wuzhou, Guangxi, China. Other reagents were provided by commercial suppliers, and were used as received.

Preparation of Catalyst
A catalyst was prepared according to a method described in the literature [31]. After diluting with n-propanol (30% by weight), zirconium was used as the precursor. Sulfuric acid (1.02 mL) was added to the zirconium precursor, and water was then added dropwise under continuous stirring until the formation of gel occurred (4.2 mL, water to propoxide molar ratio = 2.7). The resulting gel was dried at 110 • C for 12 h, followed by calcination at 600 • C for 2 h in a static air atmosphere to yield the sulfated zirconia catalyst.

Synthesis of 7-isopropyl-1,1-dimethyl-1,2,3,4-tetrahydronaphthalene (2)
Compound 2 was prepared according to a method described in the literature [32]. Longifolene (10 g, 49 mmol) and the catalyst (1 g) were mixed and stirred for 1h at 180 • C. The reaction mixture was then filtered to separate the catalyst. The filtrate was separated by column chromatography using silica gel and petroleum ether to obtain a colorless liquid, compound 2, at a 54.6% yield. The characterization data of compound 2 was consistent with that reported in the literature.

Synthesis of 6-isopropyl-4,4-dimethyl-3,4-dihydronaphthalen-1(2H)-one (3)
Compound 2 (10 g, 46 mmol), CuCl 2 (0.62 g, 4.6 mmol), and CH 3 CN (20 mL) were mixed and stirred for 30 min at room temperature. Then, the mixed solution of 25 mL TBHP (70% in water, wt%) (185 mmol) and CH 3 CN (25 mL) was added to the reaction system. Afterwards, the reaction mixture was heated to 42 • C and stirred for 24 h. The reaction process was monitored by TLC. When the reaction was completed, the reaction mixture was cooled to room temperature. The unreacted TBHP was destroyed with an appropriate amount of Na 2 S 2 O 3 . Then, the reaction mixture was extracted three times with ethyl acetate. The combined organic phase was washed with saturated sodium chloride and deionized water, respectively. Finally, the organic layer was concentrated and further purified by silica gel column chromatography using EtOAc/petroleum ether (1:60, v/v) as an eluent to obtain a colorless liquid, compound 3. Yield: 69.2%. The characterization data of compound 3 was consistent with that reported in the literature [33].

General Procedure for the Synthesis of the Target Compounds (6a-6q)
Compound 5 (0.40 g, 1.20 mmol) and NaHCO 3 (0.13 g, 1.50 mmol) in dry acetonitrile (10 mL) were mixed and stirred for 15 min at room temperature. Then, a solution of sulfonyl chloride (1.5 mmol) in acetonitrile (10 mL) was slowly added dropwise. Afterwards, the reaction mixture was stirred for 24 h at 40 • C. The reaction was quenched with water (20 mL), and the reaction mixture was extracted three times with CH 2 Cl 2 . The combined organic phase was washed three times with deionized water, dried over anhydrous Na 2 SO 4 , and distilled to remove the CH 2 Cl 2 . The crude product was purified by silica gel column chromatography using EtOAc/petroleum ether (1:5, v/v) as an eluent to afford the target products 6a-6q. Yield: 85%-90%.                   Compounds 6g, 6h, and 6d, with excellent and broad-spectrum antitumor activity against almost all the tested cancer cell lines, were leading compounds for further investigation.