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Article

Synthesis of Oleanolic Acid-Dithiocarbamate Conjugates and Evaluation of Their Broad-Spectrum Antitumor Activities

by
Liyao Tang
1,†,
Yan Zhang
1,†,
Jinrun Xu
1,†,
Qingfan Yang
2,
Fukuan Du
1,3,
Xu Wu
1,3,
Mingxing Li
1,3,
Jing Shen
1,3,
Shuai Deng
1,3,
Yueshui Zhao
1,3,
Zhangang Xiao
2,3,* and
Yu Chen
1,3,*
1
Laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou 646000, China
2
Department of Oncology, The Affiliated Hospital of Southwest Medical University, Southwest Medical University, Luzhou 646000, China
3
Cell Therapy & Cell Drugs of Luzhou Key Laboratory, Southwest Medical University, Luzhou 646000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2023, 28(3), 1414; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28031414
Submission received: 4 January 2023 / Revised: 27 January 2023 / Accepted: 30 January 2023 / Published: 2 February 2023
(This article belongs to the Topic Antitumor Activity of Natural Products and Related Compounds)
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Abstract

:
Efficient and mild synthetic routes for bioactive natural product derivatives are of current interest for drug discovery. Herein, on the basis of the pharmacophore hybrid strategy, we report a two-step protocol to obtain a series of structurally novel oleanolic acid (OA)-dithiocarbamate conjugates in mild conditions with high yields. Moreover, biological evaluations indicated that representative compound 3e exhibited the most potent and broad-spectrum antiproliferative effects against Panc1, A549, Hep3B, Huh-7, HT-29, and Hela cells with low cytotoxicity on normal cells. In terms of the IC50 values, these OA-dithiocarbamate conjugates were up to 30-fold more potent than the natural product OA. These compounds may be promising hit compounds for the development of novel anti-cancer drugs.

1. Introduction

Natural products and their derivatives have a long history in cancer therapy and are important for drug development. Efficient and mild synthetic routes for bioactive natural product derivatives are of current interest for drug discovery [1,2,3,4]. Recently, pentacyclic triterpenes have been identified as the main biologically active components in many traditional Chinese medicines [5,6]. Among them, oleanolic acid (OA) is the most abundant and cheap; thus, OA and its derivatives have been widely investigated for their diverse biological activities, including their anti-cancer, anti-inflammatory, anti-HIV, anti-bacterial, anti-diabetic, and anti-hepatotoxic effects, among others [7,8,9,10,11]. Derivatization of OA has yielded a wide variety of novel compounds for anti-cancer investigations (Scheme 1) [11,12,13,14,15]; however, poor pharmacokinetic properties, low cell selectivities, limited bioavailabilities, and synthetic complexity have hindered further clinical application [7]. Therefore, methods for readily accessible modification of OA to enhance its polarity and anti-proliferative activity are urgently required.
Dithiocarbamates are an important class of sulfur-containing organic compounds with a wide range of applications in both academia and industry [16,17,18,19,20,21,22,23,24,25,26,27]. They serve as fungicides and pesticides in agriculture [17,18,19], vulcanization agents in the rubber industry [20], radical chain transfer agents in polymerization [21], effective ligands in coordination chemistry [22], and, last but not least, as biologically important structural motifs in medicinal chemistry (Scheme 2) [23,24,25,26,27].
In recent years, the pharmacophore hybrid strategy has emerged as an essential method for the discovery and modification of lead compounds [28,29,30,31]. Covalently combining two known pharmacophores yields a novel hybrid molecule, which can possess integrated advantages for optimizing certain biological activities and overcoming the deficiencies of a single drug [32,33,34,35]. In view of the high performance of dithiocarbamate derivatives in structural modification, the synthesis of OA-dithiocarbamate conjugates may enhance the polarities and antitumor properties of the reaction products in a readily accessible manner [7,23,24,25,26,27]. The structural modifications of OA have mainly focused on the C-3 hydroxyl and C-28 carboxyl groups (Scheme 1) [7]. The C-28 carboxyl group can easily be esterified by alcohols or amidated by amines; however, the preparation of OA-dithiocarbamate conjugates has not yet been documented in the literature [7,8,9,10,11]. In order to simplify the synthetic route and control the polarity of target molecules, ethylidene was chosen as a linker between OA and dithiocarbamates.

2. Results and Discussion

To establish the optimal reaction conditions, we prepared key intermediate 2, as previously described [36,37]. Under the “standard” conditions, the reaction of 2 with CS2 and pyrrolidine in a one-pot manner afforded the target product 3a in an 80% isolated yield. In the “standard” conditions, 2 equiv. of K3PO4 was shown to be essential to yield the desired product 3a (Entries 1–4, Table 1). Lowering the loading of K3PO4 to 1.5 equiv. led to a decreased yield of 3a (Entry 1, Table 1), while replacement of it by K2HPO4 or Li2CO3 resulted in no desired product (Entries 2–3, Table 1). On the other hand, in the presence of 2 equiv. of K2CO3, product 3a could be isolated with a 62% yield (Entry 4, Table 1). Changing the reaction temperature or using other solvents, such as DMF, CH3CN, and EtOH, did not offer better results (Entries 5–8, Table 1). Lower amounts of CS2 or pyrrolidine resulted in a decreased yield of 3a (Entries 9–10, Table 1).
With the optimal reaction conditions in hand, the substrate scope was subsequently investigated, and the results are compiled in Figure 1. The replacement of the H-atom of the pyrrolidine ring with other substituents, such as methyl, dimethyl, hydroxy, and hydroxymethyl, worked well, affording the corresponding products 3b–3e in 69–85% yields. Among them, hydroxyl containing products were obtained at slightly lower yields. This reaction was also tolerant of fused-ring substrates, such as hexahydroisoindoline and isoindoline, resulting in 3f and 3g with 77% and 90% yields, respectively.
To further enhance the structural diversity of products, various types of piperidine-derived substrates were also examined, and all of them were compatible with the established reaction conditions. First, methyl-, hydroxy-, hydroxymethyl-, hydroxyethyl-, and phenyl-substituted piperidines reacted smoothly to give 3h–3m in 70–88% yields. Then, methyl-, hydroxyethyl-, phenyl-, and aryl-substituted piperazines were also viable substrates, affording 3n–3s in 71–89% yields. Moreover, thiomorpholine was also compatible, leading to the formation of 3t in 72% yield. Gratifyingly, the mild reaction conditions, high yields of products, and good functional group tolerances clearly demonstrated the advantages of our pharmacophore hybrid strategy for the structural modification of OA. The isolated compounds 3a–3t were fully characterized by 1H and 13C NMR spectroscopy as well as high-resolution mass spectrometry (see the Supplementary Information for details).
Having obtained a series of structurally diverse OA-dithiocarbamates, we next performed a systematic biological evaluation to examine whether introducing an extra dithiocarbamate group could improve antitumor activities. These compounds were evaluated by MTT assay against human pancreatic cancer (Panc1), human lung cancer (A549), human hepatoma cell (Hep3B), human hepatoma cell (Huh-7), human colon cancer (HT-29), and human cervical cancer (Hela) cells, with the widely used anticancer drugs fluorouracil, docetaxel, and cisplatin as positive controls (Table 2). Most of the compounds exhibited remarkable antiproliferative activities, and the IC50 values of ten selected compounds were less than 50 µM on certain tumor cell lines. Among them, compounds 3e, 3i, 3j, and 3l were shown to be excellent, with broad-spectrum antitumor activities as well as being up to 30-fold more potent than the natural product OA and the positive controls; this might be ascribed to the introduction of hydroxyl groups. Particularly, compound 3p was also found to be a promising hit compound that was 20-fold more potent than the natural product OA against HT-29 cells. Moreover, the cytotoxicities of compounds 3a-3t were also evaluated in human normal hepatocytes (LO2) to determine whether these compounds preferred killing tumor cells over normal cells. Excitingly, the IC50 value of compound 3e in LO2 cells was 62.8 µM, which was several times higher than that in the tumor cells.

3. Materials and Methods

3.1. General Information

All organic solvents were dried and distilled by standard methods prior to use. 1H and 13C NMR spectra were recorded on a Bruker AV II-400 spectrometer (BURKERT, Ingelfingen, Germany) at 400 and 100 MHz, respectively. All chemical shifts were reported in δ units with references to the residual solvent resonances of the deuterated solvents for proton and carbon chemical shifts. High Resolution Mass Spectra (HRMS) were obtained on a Thermo Q Exactive™ Focus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (SCIEX, Framingham, Massachusetts, USA). All other chemicals were purchased from either Aldrich (Sigma-Aldrich, Shanghai, China) or Aladdin Chemical Co. (Aladdin Holdings Group Co., Ltd, Shanghai, China) and used as received, unless otherwise specified.
The optical density at 490 nm of each well was measured using a microplate reader (Molecular devices corporation, Sunnyvale, CA. USA) to calculate the percent of cell viability. The inhibition rates were calculated using GraphPad Prism 7.0 software. The seven tested cell lines were obtained from the laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University.

3.2. Experimental Section of Synthesis

[2-bromoethyl] 3-hydroxy-12-en-28-oic acid (2) [36,37,38] To a mixture of oleanolic acid (913.4 mg, 2.0 mmol), K2CO3 (552.8 mg, 4.0 mmol), and DMF (40 mL), 1, 2-dibromoethane (513 µL, 6.0 mmol) was slowly added at room temperature, and the mixture was then stirred at 40 °C for 4 h. The resulting mixture was cooled to room temperature, then quenched with ice water (50.0 mL), and the insoluble material was removed by a Buchner funnel. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (50 mL × 3). The organic solutions were combined and dried over anhydrous MgSO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200-300 mesh) using petroleum ether and ethyl acetate (15/1 in v/v) as eluents to give 2 (957.6 mg, 85% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 5.30 (s, 1H, H-12), 4.35 (m, 2H, -OCH2C-), 3.49 (t, J = 5.5 Hz, 2H, BrCH2C-), 3.20 (d, J = 6.9 Hz, 1H, H-3), 2.87 (d, J = 12.2 Hz, 1H, H-18), 1.99 (m, 1H, -OH), 1.87 (m, 2H, -CH2), 1.72 (m, 3H, H-22, -CH, -CH2,), 1.62 (m, 6H, 3 × -CH2), 1.54 (m, 3H, H-22, -CH, -CH2), 1.33 (m, 6H, 3 × -CH2), 1.18 (s, 1H, H-9), 1.13 (s, 3H, -CH3), 1.06 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.77 (s, 3H, -CH3), 0.73 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 177.2 (C-28), 143.4 (C-13), 122.6 (C-12), 78.8 (C-3), 63.6 (-CO-), 55.2 (C-5), 47.6 (C-9), 46.8 (C-17), 45.7 (C-19), 41.6 (C-14), 41.2 (C-18), 39.3 (C-8), 38.7 (C-1), 38.4 (C-4), 37.0 (C-10), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 30.7 (C-20), 29.1 (-CBr), 28.2 (C-15), 27.7 (C-23), 27.1 (C-27), 25.9 (C-30), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 18.3 (C-6), 17.0 (C-26), 15.6 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C32H51BrO3 [M+H]+: 563.3100. Found: 563.3054.
[2-((pyrrolidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3a). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), pyrrolidine (1.0 mmol, 82 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. To the resulting mixture another THF solution (4.0 mL) of 2 (0.4 mmol, 225.4 mg) was added dropwise. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (2/1 in v/v) as eluents to give 3a (201.3 mg, 80% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 5.29 (s, 1H, H-12), 4.27 (m, 2H, -OCH2C-), 3.92 (t, J = 6.9 Hz, 2H, -SCH2), 3.66 (t, J = 6.8 Hz, 2H, -NCH2), 3.59 (m, 2H, -NCH2), 3.20 (m, 1H, H-3), 2.87 (dd, J = 13.5, 3.4 Hz, 1H, H-18), 2.10 (m, 1H, -OH), 1.97 (m, 5H, H-22, -CH, 2 × -CH2), 1.87 (m, 2H, -CH2), 1.66 (d, J = 8.4 Hz, 3H, -NCH2CH2CH), 1.59 (m, 5H, -NCH2CH2CH, 2 × -CH2), 1.53 (m, 3H, H-22, -CH, -CH2), 1.35 (m, 6H, 3 × -CH2), 1.16 (d, J = 4.0 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.04 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.77 (s, 3H, -CH3), 0.73 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 191.7 (-CS2), 177.4 (C-28), 143.6 (C-13), 122.5 (C-12), 78.8 (C-3), 62.5 (-CO-), 55.2 (-NCH2), 55.1 (-NCH2), 50.6 (C-5), 47.6 (C-9), 46.7 (C-17), 45.8 (C-19), 41.6 (C-14), 41.2 (C-18), 39.3 (C-8), 38.7 (C-1), 38.5 (C-4), 37.0 (C-10), 35.0 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.2 (C-27), 26.1 (-CH2), 25.9 (C-30), 24.3 (-CH2), 23.7 (C-2), 23.4 (C-11), 22.9 (C-16), 18.3 (C-6), 17.1 (C-26), 15.7 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C37H59NO3S2 [M+H]+: 630.4015. Found: 630.3961.
[2-((2-methylpyrrolidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3b). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 2-methylpyrrolidine (1.0 mmol, 101 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.2 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3b (213.6 mg, 83% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 5.30 (s, 1H, H-12), 4.52 (m, 1H, -OH), 4.26 (m, 2H, -OCH2C-), 3.93 (m, 1H, -NCH), 3.73 (m, 1H, -NCH), 3.44 (m, 2H, -SCH2), 3.21 (m, 1H, H-3), 2.87 (dd, J = 13.7, 3.8 Hz, 1H, H-18), 2.25 (m, 1H, -NCH), 2.02 (m, 4H, 2 × -CH2), 1.81 (m, 3H, H-11, -CH, -CH2), 1.63 (m, 7H,-NCH2CH2, -NCH2CH2, H-22, -CH, -CH2), 1.53 (m, 3H, H-22, -CH, -CH2), 1.42 (m, 2H, -CH2), 1.35 (m, 4H, 2 × -CH2), 1.28 (m, 3H, -CH3), 1.16 (t, J = 4.2 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.04 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.77 (s, 3H, -CH3), 0.74 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 191.7 (-CS2), 177.5 (C-28), 143.6 (C-13), 122.5 (C-12), 78.9 (C-3), 62.6 (-CO-), 61.3 (-NCH2CH3), 58.0(-CH3), 55.2 (C-5), 50.4 (-NCH2), 47.6 (C-9), 46.7 (C-17), 45.8 (C-19), 41.7 (C-14), 41.3 (C-18), 39.4 (C-8), 38.8 (C-1), 38.5 (C-4), 37.0 (C-10), 34.8 (-CS), 33.9 (C-29), 33.1 (C-22), 32.4 (C-21), 31.3 (C-7), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.2 (C-27), 25.9 (C-30), 23.7 (C-2), 22.9 (C-11), 21.6 (C-16), 18.6 (-CH2), 18.3 (C-6), 17.5 (-CH2), 17.1 (C-26), 15.6 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C38H61NO3S2 [M+H]+: 644.4171. Found: 644.4116.
[2-((2,2-dimethylpyrrolidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3c). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 2,2-dimethylpyrrolidine (1.0 mmol, 120 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.9 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). To the resulting solution was added water (15.0 mL), the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3c (223.5 mg, 85% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 5.30 (s, 1H, H-12), 4.26 (m, 2H, -OCH2C-), 3.83 (t, J = 6.8 Hz, 1H, -NCH), 3.56 (m, 2H, -SCH2), 3.21 (m, 1H, -NCH), 2.87 (dd, J = 13.6, 3.7 Hz, 1H, H-18), 2.01 (m, 1H, -OH), 1.96 (m, 2H, -CH2), 1.88 (m, 3H, H-22, -CH, -CH2), 1.73 (s, 6H, 3 × -CH2), 1.63 (m, 8H, 4 × -CH2), 1.55 (m, 3H, -CH3), 1.46 (m, 3H, -CH3), 1.31 (m, 6H, 3 × -CH2), 1.16 (t, J = 5.6 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.04 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.78 (s, 3H, -CH3), 0.73 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 191.4 (-CS2), 177.5 (C-28), 143.7 (C-13), 122.5 (C-12), 79.0 (C-3), 69.2 (-NCCH3CH3), 62.7 (-CO-), 55.2 (-NCH2), 53.8 (C-5), 47.6 (C-9), 46.7 (C-17), 45.8 (C-19), 43.3 (-CH2), 41.7 (C-14), 41.3 (C-18), 39.4 (C-8), 38.8 (C-1), 38.5 (C-4), 37.0 (C-10), 34.7 (-CS), 33.9 (C-29), 33.2 (C-22), 32.8 (C-21), 32.4 (C-7), 30.7 (C-20), 28.2 (C-15), 27.7 (C-23), 27.2 (C-27), 26.1 (-CH2), 25.9 (C-30), 24.8 (-CH2), 23.7 (C-2), 23.5 (C-11), 22.9 (C-16), 22.1 (-CH2), 18.4 (C-6), 17.1 (C-26), 15.7 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C39H63NO3S2 [M+H]+: 658.4328. Found: 658.4303.
[2-((3-hydroxypyrrolidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3d). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 3-hydroxypyrrolidine (1.0 mmol, 81 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 224.7 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (2/1 in v/v) as eluents to give 3d (180.7 mg, 70% yield) as a yellowish gel. 1H NMR (400 MHz, CDCl3): δ 5.31 (s, 1H, H-12), 4.57 (m, 1H, -OH), 4.21 (m, 2H, -OCH2C-), 4.03 (m, 2H, -SCH2), 3.82 (m, 2H, -NCH2), 3.55 (m, 2H, -NCH2), 3.20 (m, 1H, H-3), 2.86 (d, J = 12.2 Hz, 1H, H-18), 2.15 (m, 1H, -OH), 2.05 (m, 2H, -CH2), 1.89 (m, 4H, 2 × -CH2), 1.67 (m, 6H, 3 × -CH2), 1.53 (m, 4H, 2 × -CH2), 1.33 (m, 7H, -CHOH, 3 × -CH2), 1.16 (t, J = 3.8 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.97 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (d, J = 2.3 Hz, 6H, 2 × -CH3), 0.77 (s, 3H, -CH3), 0.73 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 192.5 (-CS2), 177.7 (C-28), 143.5 (C-13), 122.5 (C-12), 79.0 (C-3), 70.7 (-COH), 68.8 (-NCH2CH), 63.2 (-NCH2), 62.5 (-CO-), 55.2 (C-5), 52.9 (-CH2), 48.6 (C-7), 47.6 (C-9), 46.8 (C-17), 45.8 (C-19), 41.7 (C-14), 41.3 (C-18), 39.3 (C-8), 38.7 (C-1), 38.5 (C-4), 37.0 (C-10), 35.0 (-CS), 33.8 (C-29), 32.9 (C-22), 32.4 (C-21), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 25.9 (C-30), 23.7 (C-2), 23.4 (C-11), 22.9 (C-16), 18.3 (C-6), 17.1 (C-26), 15.7 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C37H59NO4S2 [M+H]+: 646.3964. Found: 646.3917.
[2-((3-(hydroxymethyl)pyrrolidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3e). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 3-(hydroxymethyl)pyrrolidine (1.0 mmol, 103 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.8 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (1/1 in v/v) as eluents to give 3e (181.9 mg, 69% yield) as a yellowish gel. 1H NMR (400 MHz, CDCl3): δ 5.30 (t, J = 3.2 Hz, 1H, H-12), 4.25 (m, 2H, -OCH2C-), 3.85 (m, 1H, -OH), 3.68 (m, 2H, -SCH2), 3.54 (m, 3H, -CH2OH, -NCH), 3.21 (m, 1H, H-3), 2.86 (dd, J = 13.7, 3.9 Hz, 1H, H-18), 2.55 (m, 1H, -NCH), 2.22 (m, 1H, -NCH), 2.08 (m, 1H, -NCH), 1.95 (m, 2H, -CH2), 1.84 (m, 3H, -NCH2CH, -CH2), 1.70 (m, 1H, -OH), 1.63 (m, 6H, 2 × -CH3), 1.53 (m, 3H, -NCH2CH, -CH2), 1.42 (m, 3H, H-22, -CH, -CH2), 1.28 (m, 5H, H-22, -CH, 2 × -CH2), 1.21 (s, 1H, -CHCH2OH), 1.17 (t, J = 3.2 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.77 (s, 3H, -CH3), 0.73 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 192.0 (-CS2), 177.6 (C-28), 143.6 (C-13), 122.5 (C-12), 79.0 (C-3), 63.6 (-COH), 62.4 (-CO-), 57.5 (-NCH2CH), 55.2 (-C`OH), 54.5 (-NC`H2CH), 53.1 (-NCH2CH2), 50.1 (C-5), 47.6 (C-9), 46.8 (C-17), 45.8 (C-19), 41.7 (C-14), 41.3 (C-18), 39.5 (-CCH2OH), 39.3 (C-8), 38.7 (C-1), 38.5 (C-4), 37.0 (C-10), 35.0 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 31.5 (-NC`H2CH2), 30.7 (C-20), 30.2 (-NCH2CH2), 28.4 (-C`CH2OH), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 26.6 (-C`CH2OH), 25.9 (C-30), 23.7 (C-2), 23.4 (C-11), 22.9 (C-16), 18.3 (C-6), 17.1 (C-26), 15.7 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C38H61NO4S2 [M+H]+: 660.4120. Found: 660.4069.
[2-((octahydro-1H-isoindole-2-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3f). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), octahydro-1H-isoindole (1.0 mmol, 115 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.5 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3f (210.4 mg, 77% yield) as a yellowish gel. 1H NMR (400 MHz, CDCl3): δ 5.20 (s, 1H, H-12), 4.18 (m, 2H, -OCH2C-), 3.76 (m, 2H, -SCH2), 3.52 (m, 4H, 2 × -NCH2), 3.10 (m, 1H, H-3), 2.77 (d, J = 11.0 Hz, 1H, H-18), 2.26 (m, 2H, -CH2), 2.09 (m, 1H, -OH), 1.84 (m, 3H, H-22, -CH, -CH2), 1.52 (m, 8H, 4 × -CH2), 1.44 (m, 5H, H-22, -CH, 2 × -CH2), 1.30 (m, 8H, 4 × -CH2), 1.18 (m, 4H, 2 × -CH2), 1.07 (s, 1H, H-9), 1.04 (s, 3H, -CH3), 0.95 (s, 1H, H-5), 0.88 (s, 3H, -CH3), 0.83 (s, 3H, -CH3), 0.80 (s, 6H, 2 × -CH3), 0.67 (s, 3H, -CH3), 0.64 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 192.3 (-CS2), 177.3 (C-28), 143.5 (C-13), 122.5 (C-12), 78.6 (C-3), 62.5 (-CO-), 58.9 (2 × -CH2), 55.2 (C-5), 54.5 (2 × -CH2), 47.5 (C-9), 46.6 (C-17), 45.7 (C-19), 41.6 (C-14), 41.2 (C-18), 39.3 (C-8), 38.7 (C-1), 37.6 (C-4), 36.9 (C-10), 35.8 (-CS), 34.9 (2 × -CH2), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.3 (C-7), 30.6 (C-20), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 26.9 (C-30), 25.9 (C-2), 25.6 (2 × -CH2), 23.6 (C-11), 22.6 (C-16), 18.3 (C-6), 17.1 (C-26), 15.7 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C41H65NO3S2 [M+H]+: 684.4484. Found: 684.4430.
[2-((isoindoline-2-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3g). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), isoindoline (1.0 mmol, 113 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 226.4 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3g (243.9 mg, 90% yield) as a yellowish solid. 1H NMR (400 MHz, CDCl3): δ 7.31 (m, 4H, Ar-H), 5.30 (t, J = 3.3 Hz, 1H, H-12), 5.20 (s, 2H, -OCH2C-), 4.99 (s, 2H, -NCH2), 4.32 (m, 2H, -NCH2), 3.66 (m, 2H, -SCH2), 3.19 (m, 1H, H-3), 2.95 (m, 1H, -OH), 2.88 (dd, J = 13.7, 4.0 Hz, 1H, H-18), 1.85 (m, 2H, -CH2), 1.69 (m, 3H, H-22, -CH, -CH2), 1.54 (m, 6H, 3 × -CH2), 1.42 (m, 3H, H-22, -CH, -CH2), 1.23 (m, 6H, 3 × -CH2), 1.17 (t, J = 4.4 Hz, 1H, H-9), 1.12 (s, 3H, -CH3), 1.04 (s, 1H, H-5), 0.95 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 3H, -CH3), 0.86 (s, 3H, -CH3), 0.74 (s, 3H, -CH3), 0.72 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 192.7 (-CS2), 177.5 (C-28), 143.6 (C-13), 135.2 (Ph), 134.9 (Ph), 128.1 (Ph), 127.9 (Ph), 122.8 (Ph), 122.7 (Ph), 122.5 (C-12), 78.9 (C-3), 62.3 (-CO-), 60.5 (-NCH2), 55.7 (-NCH2), 55.1 (C-5), 47.5 (C-9), 46.7 (C-17), 45.8 (C-19), 41.6 (C-14), 41.3 (C-18), 39.3 (C-8), 38.7 (C-1), 38.4 (C-4), 37.0 (C-10), 35.3 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 25.9 (C-30), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 18.2 (C-6), 17.1 (C-26), 15.5 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C41H59NO3S2 [M+H]+: 678.4015. Found: 678.3996.
[2-((4-methylpiperidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3h). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-methylpiperidine (1.0 mmol, 100 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 224.6 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3h (199.8 mg, 76% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 5.23 (s, 1H, H-12), 4.21 (m, 2H, -OCH2C-), 3.54 (m, 2H, -SCH2), 3.10 (m, 3H, -NCH2, H-3), 2.80 (d, J = 12.2 Hz, 1H, H-18), 1.89 (m, 1H, -OH), 1.80 (m, 2H, -NCH2), 1.70 (m, 3H, H-22, -CH, -CH2), 1.58 (m, 7H, -CHCH3, 3 × -CH2), 1.46 (m, 4H, 2 × -CH2), 1.33 (m, 5H, H-22, -CH, 2 × -CH2), 1.20 (m, 6H, 3 × -CH2), 1.10 (s, 1H, H-9), 1.06 (s, 3H, -CH3), 0.98 (s, 1H, H-5), 0.92 (s, 6H, 2 × -CH3), 0.86 (s, 3H, -CH3), 0.83 (s, 6H, 2 × -CH3), 0.71 (s, 3H, -CH3), 0.67 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 194.8 (-CS2), 177.5 (C-28), 143.7 (C-13), 122.6 (C-12), 79.1 (C-3), 62.5 (-CO-), 55.3 (C-5), 53.6 (-NCH2, -NCH2), 47.7 (C-9), 46.8 (C-17), 45.9 (C-19), 41.8 (C-14), 41.4 (C-18), 39.4 (C-8), 38.8 (C-1), 38.5 (C-4), 37.1 (C-10), 35.8 (-CS), 34.0 (C-29), 33.2 (-NCH2CH2, -NCH2CH2), 33.1 (C-22), 32.8 (C-21), 32.5 (C-7), 31.0 (-CCH3), 30.8 (C-20), 28.2 (C-15), 27.8 (C-23), 27.3 (C-27), 26.0 (C-30), 23.7 (C-2), 23.5 (C-11), 23.0 (C-16), 21.4 (-CH3), 18.4 (C-6), 17.2 (C-26), 15.7 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C39H63NO3S2 [M+H]+: 658.4328. Found: 658.4275.
[2-((4-hydroxypiperidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3i). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-hydroxypiperidine (1.0 mmol, 103.5 mg) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.4 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (1/1 in v/v) as eluents to give 3i (184.6 mg, 70% yield) as a yellow gel. 1H NMR (400 MHz, CDCl3): δ 5.50 (s, 1H, -OH), 5.30 (s, 1H, H-12), 4.60 (s, 1H, -CHOH), 4.28 (m, 2H, -SCH2), 3.61 (m, 2H, -NCH2), 3.21 (m, 1H, H-3), 3.08 (s, 2H, -NCH2), 2.87 (d, J = 11.0 Hz, 1H, H-18), 2.00 (m, 1H, -OH), 1.87 (s, 2H, -CH2), 1.76 (m, 3H, H-22, -CH, -CH2), 1.62 (m, 6H, 3 × -CH2), 1.53 (m, 5H, H-22, -CH, 2 × -CH2), 1.42 (m, 4H, 2 × -CH2), 1.27 (m, 6H, 3 × -CH2), 1.16 (t, J = 3.7 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.04 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.77 (s, 3H, -CH3), 0.74 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 194.6 (-CS2), 177.4 (C-28), 143.6 (C-13), 122.5 (C-12), 78.9 (C-3), 62.4 (-CO-), 55.2 (C-5), 47.6 (C-9), 46.7 (C-17), 45.8 (C-19), 41.7 (C-14), 41.3 (C-18), 39.4 (C-8), 38.8 (C-1), 38.5 (C-4), 37.0 (C-10), 35.7 (-CS), 33.9 (C-29), 33.1 (C-22), 32.8 (C-21), 32.4 (C-7), 31.0 (-COH), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.2 (C-27), 26.9 (-NCH2, -NCH2), 25.9 (C-30), 23.7 (C-2), 23.4 (C-11), 22.9 (C-16), 21.3 (-NCH2CH2, -NCH2CH2), 18.4 (C-6), 17.1 (C-26), 15.6 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C38H61NO4S2 [M+H]+: 660.4120. Found: 660.4080.
[2-((4-(hydroxymethyl)piperidine-1-carbonothioyl)thio)-ethyl] 3-hydroxy-12-en-28-oic acid (3j). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-(hydroxymethyl)piperidine (1.0 mmol, 115.3 mg) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.9 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (2/1 in v/v) as eluents to give 3j (199.3 mg, 74% yield) as a yellow gel. 1H NMR (400 MHz, CDCl3): δ 5.57 (s, 1H, -CHCH2OH), 5.31 (s, 1H, H-12), 4.64 (s, 1H, -CHOH), 4.26 (m, 2H, -OCH2C-), 3.60 (m, 2H, -SCH2), 3.51 (m, 2H, -NCH2), 3.21 (m, 2H, -NCH2), 3.13 (m, 1H, H-3), 2.86 (dd, J = 13.5, 3.7 Hz, 1H, H-18), 1.96 (m, 1H, -OH), 1.91 (m, 2H, -CH2), 1.87 (m, 3H, -OH, -CH2), 1.63 (m, 6H, 3 × -CH2), 1.52 (m, 5H, -CHOH, 2 × -CH2), 1.38 (m, 6H, 3 × -CH2), 1.27 (m, 4H, 2 × -CH2), 1.16 (t, J = 4.4 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.77 (s, 3H, -CH3), 0.73 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 194.8 (-CS2), 177.5 (C-28), 143.6 (C-13), 122.5 (C-12), 78.9 (C-3), 66.5 (-CH2OH), 62.4 (-CO-), 55.2 (C-5), 47.6 (C-9), 46.7 (C-17), 45.8 (C-19), 41.6 (C-14), 41.3 (C-18), 39.3 (C-8), 38.7 (C-1), 38.5 (C-4), 38.4 (-NCH2, -NCH2), 37.0 (C-10), 36.6 (-NCH2CH2, -NCH2CH2), 35.6 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 31.5 (-CCH2OH), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 25.9 (C-30), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 18.3 (C-6), 17.1 (C-26), 15.6 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C39H63NO4S2 [M+H]+: 674.4277. Found: 674.4230.
[2-((2-(2-hydroxyethyl)piperidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3k). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 2-(2-hydroxyethyl)piperidine (1.0 mmol, 128.1 mg) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.6 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (2/1 in v/v) as eluents to give 3k (200.7 mg, 73% yield) as a yellowish gel. 1H NMR (400 MHz, CDCl3): δ 5.93 (m, 1H, -OH), 5.30 (t, J = 3.3 Hz, 1H, H-12), 4.55 (m, 1H, -CHOH), 4.28 (m, 2H, -OCH2C-), 3.62 (m, 3H, -CHOH, -SCH2), 3.39 (m, 1H, H-3), 3.16 (m, 3H, -NCH2, -NCH), 2.87 (dd, J = 13.7, 3.9 Hz, 1H, H-18), 2.14 (m, 1H, -OH), 1.95 (m, 2H, -CH2), 1.87 (m, 3H, -CHCH2CH2OH, H-22, -CH), 1.79 (m, 1H, H-22, -CH), 1.70 (m, 6H, 3 × -CH2), 1.59 (m, 6H, 3 × -CH2), 1.53 (m, 4H, 2 × -CH2), 1.38 (m, 6H, 3 × -CH2), 1.17 (t, J = 4.3 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.77 (s, 3H, -CH3), 0.74 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 196.4 (-CS2), 177.4 (C-28), 143.5 (C-13), 122.5 (C-12), 78.8 (C-3), 58.1 (-CO-), 56.0 (-NCH), 55.2 (C-5), 47.6 (C-9), 46.7 (C-17), 46.0 (C-19), 45.8 (-CH2OH), 41.6 (C-14), 41.3 (C-18), 39.3 (C-8), 38.7 (C-1), 38.5 (C-4), 37.0 (C-10), 35.6 (-CS), 33.8 (C-29), 33.1 (C-22), 32.9 (C-21), 32.7 (C-7), 32.4 (-NCH2), 30.7 (C-20), 29.3 (-CH2CH2OH), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 25.9 (C-30), 25.8 (-NCHCH2), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 19.2 (-NCH2CH2CH2), 18.3 (C-6), 17.1 (C-26), 17.1 (-NCH2CH2), 15.6 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C40H65NO4S2 [M+H]+: 688.4433. Found: 688.4418.
[2-((4-(2-hydroxyethyl)piperidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3l). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-(2-hydroxyethyl)piperidine (1.0 mmol, 130.8 mg) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 226.4 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (2/1 in v/v) as eluents to give 3l (195.2 mg, 71% yield) as a yellowish gel. 1H NMR (400 MHz, CDCl3): δ 5.23 (t, J = 3.3 Hz, 1H, H-12), 4.53 (m, 1H, -OH), 4.21 (m, 2H, -OCH2C-), 3.95 (m, 2H, -SCH2), 3.75 (m, 2H, -NCH2), 3.53 (m, 2H, -NCH2), 3.14 (m, 1H, H-3), 2.79 (dd, J = 13.6, 3.7 Hz, 1H, H-18), 2.07 (m, 2H, -CH2), 1.90 (m, 1H, -OH), 1.81 (m, 3H, -CH2, -NCH2CH2CH), 1.61 (m, 2H, -CH2), 1.56 (m, 6H, 3 × -CH2), 1.45 (m, 4H, 2 × -CH2), 1.25 (m, 6H, 3 × -CH2), 1.10 (t, J = 4.6 Hz, 1H, H-9), 1.06 (s, 3H, -CH3), 0.98 (s, 1H, H-5), 0.91 (s, 3H, -CH3), 0.86 (s, 3H, -CH3), 0.83 (s, 6H, 2 × -CH3), 0.70 (s, 3H, -CH3), 0.67 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 194.5 (-CS2), 177.5 (C-28), 143.5 (C-13), 122.5 (C-12), 77.0 (C-3), 62.4 (-CO-), 60.4 (-CH2OH), 59.6 (-NCH2), 55.2 (C-5), 52.3 (-NCH2), 50.4 (-CH2CH2OH), 47.5 (C-9), 46.7 (C-17), 45.7 (C-19), 41.6 (C-14), 41.2 (C-18), 39.3 (C-8), 38.7 (C-1), 38.5 (C-4), 36.9 (C-10), 35.6 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 31.6 (-NCH2CH2), 30.7 (C-20), 28.1 (C-15), 27.6 (C-23), 27.1 (C-27), 25.9 (C-30), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 21.1 (-CHCH2CH2OH), 18.3 (C-6), 17.1 (C-26), 15.7 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C40H65NO4S2 [M+H]+: 688.4433. Found: 688.4366.
[2-((4-phenylpiperidine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3m). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-phenylpiperidine (1.0 mmol, 164.7 mg) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.2 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3m (253.2 mg, 88% yield) as a yellowish gel. 1H NMR (400 MHz, CDCl3): δ 7.31 (m, 2H, Ar-H), 7.21 (m, 3H, Ar-H), 5.30 (t, J = 3.3 Hz, 1H, H-12), 4.32 (m, 2H, -OCH2C-), 3.63 (m, 2H, -SCH2), 3.20 (m, 3H, H-3, -NCH2), 2.88 (m, 2H, H-18, -NCH2CH2CH), 1.99 (m, 1H, -OH), 1.96 (m, 2H, -CH2), 1.88 (m, 3H, H-22, -CH, -CH2), 1.64 (m, 8H, 2 × -NCH2, 2 × -CH2), 1.51 (m, 3H, H-22, -CH, -CH2), 1.44 (m, 4H, 2 × -NCH2), 1.28 (m, 6H, 3 × -CH2), 1.17 (t, J = 3.6 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.97 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 3H, -CH3), 0.89 (s, 3H, -CH3), 0.76 (s, 3H, -CH3), 0.75 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 195.1 (-CS2), 177.5 (C-28), 144.3 (Ph), 143.6 (C-13), 128.7 (Ph), 126.8 (Ph), 122.6 (C-12), 78.9 (C-3), 62.4 (-CO-), 55.2 (C-5), 47.6 (C-9), 46.8 (C-17), 45.8 (C-19), 42.6 (-NCH2), 41.7 (C-14), 41.3 (C-18), 39.4 (C-8), 38.8 (C-1), 38.5 (C-4), 37.0 (C-10), 35.9 (-CS), 34.7 (-NCH2CH2CH), 33.9 (C-29), 33.2 (C-22), 32.8 (C-21), 32.4 (C-7), 30.7 (C-20), 28.2 (C-15), 27.7 (C-23), 27.2 (C-27), 27.0 (-NCH2CH2), 25.9 (C-30), 25.3 (-NCH2CH2), 23.7 (C-2), 23.5 (C-11), 23.0 (C-16), 18.4 (C-6), 17.2 (C-26), 15.7 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C44H65NO3S2 [M+H]+: 720.4484. Found: 720.4450.
[2-((4-methylpiperazine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3n). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-methylpiperazine (1.0 mmol, 112 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.0 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (1/4 in v/v) as eluents to give 3n (234.3 mg, 89% yield) as a yellowish gel. 1H NMR (400 MHz, CDCl3): 5.30 (t, J = 3.3 Hz, 1H, H-12), 4.36 (s, 2H, -OCH2C-), 4.28 (m, 2H, -NCH2), 3.96 (s, 2H, -NCH2), 3.61 (m, 2H, -SCH2), 3.21 (m, 1H, H-3), 2.87 (dd, J = 13.6, 4.0 Hz, 1H, H-18), 2.50 (s, 4H, 2 × -NCH2), 2.34 (s, 3H, -CH3), 1.97 (m, 1H, -OH), 1.87 (m, 2H, -CH2), 1.64 (m, 6H, 3 × -CH2), 1.53 (m, 3H, H-22, -CH, -CH2), 1.40 (m, 3H, H-22, -CH, -CH2), 1.27 (m, 6H, 3 × -CH2), 1.16 (t, J = 4.3 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.78 (s, 3H, -CH3), 0.73 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 195.9 (-CS2), 177.4 (C-28), 143.6 (C-13), 122.5 (C-12), 78.9 (C-3), 62.3 (-CO-), 55.2 (C-5), 54.4 (-NCH2), 47.6 (C-9), 46.7 (C-17), 46.4 (-CH2CH2), 45.8 (C-19), 45.6 (-NCH3), 41.7 (C-14), 41.3 (C-18), 39.3 (C-8), 38.7 (C-1), 38.4 (C-4), 37.0 (C-10), 35.6 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.2 (C-27), 25.9 (C-30), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 18.3 (C-6), 17.1 (C-26), 15.6 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C38H62N2O3S2 [M+H]+: 659.4280. Found: 659.4239.
[2-((4-(2-hydroxyethyl)piperazine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3o). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-(2-hydroxyethyl)piperazine (1.0 mmol, 123 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 224.9 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (1/5 in v/v) as eluents to give 3o (195.5 mg, 71% yield) as a yellowish gel. 1H NMR (400 MHz, CDCl3): δ 5.30 (t, J = 3.1 Hz, 1H, H-12), 4.33 (s, 1H, -NCH), 4.28 (m, 2H, -OCH2C-), 3.99 (s, 1H, -NCH), 3.69 (m, 2H, -NCH2), 3.61 (m, 2H, -SCH2), 3.21 (m, 1H, H-3), 2.86 (dd, J = 13.7, 4.1 Hz, 1H, H-18), 2.63 (t, J = 5.2 Hz, 4H, 2 × -NCH2), 2.61 (s, 1H, -OH), 1.97 (m, 1H, -OH), 1.88 (m, 2H, -CH2), 1.66 (m, 2H, -CH2), 1.60 (m, 5H, H-22, -CH, 2 × -CH2), 1.53 (m, 4H, 2 × -CH2), 1.43 (m, 3H, H-22, -CH, -CH2), 1.33 (m, 4H, 2 × -CH2), 1.27 (m, 4H, 2 × -CH2), 1.16 (t, J = 4.4 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.77 (s, 3H, -CH3), 0.73 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 196.1 (-CS2), 177.5 (C-28), 143.6 (C-13), 122.5 (C-12), 79.0 (C-3), 62.3 (-CO-), 59.1 (-NCH2CH2OH), 57.9 (-NCH2CH2OH), 55.2 (C-5), 52.3 (-NCH2CH2), 47.6 (C-9), 46.8 (C-17), 45.8 (C-19), 41.7 (C-14), 41.3 (C-18), 39.4 (C-8), 38.8 (C-1), 38.5 (C-4), 37.0 (C-10), 35.7 (-CS), 33.9 (C-29), 33.1 (C-22), 32.4 (C-21), 31.5 (C-7), 30.7(-NCH2CH2), 30.2 (C-20), 28.1 (C-15), 27.7 (C-23), 27.2 (C-27), 25.9 (C-30), 23.7 (C-2), 23.4 (C-11), 22.9 (C-16), 18.4 (C-6), 17.1 (C-26), 15.6 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C39H64N2O4S2 [M+H]+: 689.4386. Found: 689.4336.
[2-((4-phenylpiperazine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3p). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-phenylpiperazine (1.0 mmol, 150 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 226.7 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (1/5 in v/v) as eluents to give 3p (247.8 mg, 86% yield) as a yellowish solid. 1H NMR (400 MHz, CDCl3): δ 7.30 (m, 2H, Ar-H), 6.93 (m, 3H, Ar-H), 5.30 (t, J = 3.2 Hz, 1H, H-12), 4.49 (s, 2H, -OCH2C-), 4.29 (m, 2H, -NCH2), 4.10 (m, 2H, -NCH2), 3.64 (m, 2H, -SCH2), 3.30 (t, J = 4.9 Hz, 4H, 2 × -NCH2), 3.20 (m, 1H, H-3), 2.87 (dd, J = 13.6, 3.9 Hz, 1H, H-18), 1.97 (m, 1H, -OH), 1.88 (m, 2H, -CH2), 1.67 (m, 4H, 2 × -CH2), 1.58 (m, 4H, 2 × -CH2), 1.51 (m, 3H, H-22, -CH, -CH2), 1.35 (m, 7H, H-22, -CH, 3 × -CH2), 1.17 (t, J = 4.0 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.97 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 3H, -CH3), 0.89 (s, 3H, -CH3), 0.76 (s, 3H, -CH3), 0.74 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 196.1 (-CS2), 177.4 (Ph), 150.2 (Ph), 143.6 (C-13), 129.3 (Ph), 122.5 (C-12), 120.6 (Ph), 116.3 (Ph), 78.9 (C-3), 62.2 (-CO-), 55.2 (C-5), 48.7 (-NCH2), 48.2 (-NCH2), 47.6 (C-9), 46.7 (C-17), 45.8 (C-19), 41.6 (C-14), 41.3 (C-18), 39.3 (C-8), 38.7 (C-1), 38.4 (C-4), 37.0 (C-10), 35.6 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 25.9 (C-30), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 18.3 (C-6), 17.1 (C-26), 15.6 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C43H64N2O3S2 [M+H]+: 721.4437. Found: 721.4411.
[2-((4-(o-tolyl)piperazine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3q). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-(o-tolyl)piperazine (1.0 mmol, 177.9 mg) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.9 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3q (246.7 mg, 84% yield) as a yellowish gel. 1H NMR (400 MHz, CDCl3): δ 7.17 (t, J = 7.9 Hz, 1H, Ar-H), 6.73 (m, 3H, Ar-H), 5.30 (t, J = 3.1 Hz, 1H, H-12), 4.46 (s, 2H, -OCH2C-), 4.27 (m, 2H, -NCH2), 4.10 (s, 2H, -NCH2), 3.64 (m, 2H, -SCH2), 3.28 (t, J = 5.0 Hz, 4H, 2 × -NCH2), 3.18 (m, 1H, H-3), 2.87 (dd, J = 13.6, 3.8 Hz, 1H, H-18), 2.32 (s, 3H, -CH3), 1.97 (m, 1H, -OH), 1.87 (m, 2H, -CH2), 1.69 (m, 4H, 2 × -CH2), 1.58 (m, 5H, H-22, -CH, 2 × -CH2), 1.51 (m, 3H, H-22, -CH, -CH2), 1.35 (m, 6H, 3 × -CH2), 1.17 (t, J = 3.8 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.97 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 3H, -CH3), 0.89 (s, 3H, -CH3), 0.76 (s, 3H, -CH3), 0.74 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 195.9 (-CS2), 177.4 (C-28), 150.2 (Ph), 143.5 (C-13), 139.0 (Ph), 129.1 (Ph), 122.5 (C-12), 121.5 (Ph), 117.1 (Ph), 113.4 (Ph), 78.8 (C-3), 62.3 (-CO-), 55.2 (C-5), 48.8 (-NCH2), 47.6 (C-9), 46.7 (C-17), 45.8 (C-19), 41.6 (C-14), 41.3 (C-18), 39.3 (C-8), 38.7 (C-1), 38.4 (C-4), 37.0 (C-10), 35.6 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 26.9 (-NCH2CH2), 25.9 (C-30), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 21.8 (-CH3), 18.3 (C-6), 17.1 (C-26), 15.6 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C44H66N2O3S2 [M+H]+: 735.4593. Found: 735.4540.
[2-((4-(m-tolyl)piperazine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3r). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-(m-tolyl)piperazine (1.0 mmol, 174 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 226.1 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3r (235.0 mg, 80% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 7.16 (t, J = 7.9 Hz, 1H, Ar-H), 6.72 (m, 3H, Ar-H), 5.30 (s, 1H, H-12), 4.40 (s, 2H, -OCH2C-), 4.28 (m, 2H, -NCH2), 4.11 (m, 2H, -NCH2), 3.60 (m, 2H, -SCH2), 3.27 (m, 4H, 2 × -NCH2), 3.18 (m, 1H, H-3), 2.87 (dd, J = 13.5, 3.5 Hz, 1H, H-18), 2.32 (s, 3H, -CH3), 1.95 (m, 1H, -OH), 1.86 (m, 2H, -CH2), 1.62 (m, 7H, H-22, -CH, 3 × -CH2), 1.51 (m, 3H, H-22, -CH, -CH2), 1.42 (m, 2H, -CH2), 1.32 (m, 6H, 3 × -CH2), 1.17 (t, J = 3.1 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.04 (s, 1H, H-5), 0.96 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 3H, -CH3), 0.89 (s, 3H, -CH3), 0.75 (s, 3H, -CH3), 0.74 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 195.9 (-CS2), 177.4 (C-28), 150.2 (Ph), 143.5 (C-13), 139.0 (Ph), 129.1 (Ph), 122.5 (C-12), 121.5 (Ph), 117.1 (Ph), 113.4 (Ph), 78.8 (C-3), 62.3 (-CO-), 55.2 (C-5), 48.8 (-NCH2), 47.6 (C-9), 46.7 (C-17), 45.8 (C-19), 41.6 (C-14), 41.3 (C-18), 39.3 (C-8), 38.7 (C-1), 38.4 (C-4), 37.0 (C-10), 35.6 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 26.9 (-NCH2CH2), 25.9 (C-30), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 21.8 (-CH3), 18.3 (C-6), 17.1 (C-26), 15.6 (C-24), 15.4 (C-25). HRMS (ESI): m/z calculated for C44H66N2O3S2 [M+H]+: 735.4593. Found: 735.4537.
[2-((4-(p-tolyl)piperazine-1-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3s). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), 4-(p-tolyl)piperazine (1.0 mmol, 177.1 mg) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.8 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3s (249.6 mg, 85% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 7.10 (d, J = 8.3 Hz, 2H, Ar-H), 6.84 (d, J = 8.5 Hz, 2H, Ar-H), 5.30 (t, J = 3.3 Hz, 1H, H-12), 4.48 (s, 2H, -NCH2), 4.30 (m, 2H, -OCH2C-), 4.10 (s, 2H, -NCH2), 3.64 (m, 2H, -SCH2), 3.23 (m, 4H, 2× -NCH2), 3.19 (d, J = 4.8 Hz, 1H, H-3), 2.87 (dd, J = 13.7, 4.0 Hz, 1H, H-18), 2.28 (s, 3H, -CH3), 1.97 (m, 1H, -OH), 1.87 (m, 2H, 2× -CH2), 1.65 (m, 6H, 3× -CH2), 1.50 (m, 4H, 2× -CH2), 1.40 (m, 4H, 2× -CH2), 1.29 (m, 4H, 2× -CH2) 1.17 (t, J = 4.1 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.05 (s, 1H, H-5), 0.97 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 3H, -CH3), 0.89 (s, 3H, -CH3), 0.76 (s, 3H, -CH3), 0.74 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 196.0 (-CS2), 177.4 (C-28), 148.1 (Ph), 143.6 (C-13), 130.3 (Ph), 129.8 (Ph), 122.5 (C-12), 116.8 (Ph), 78.9 (C-3), 62.3 (-CO-), 55.2 (C-5), 49.4 (-NCH2), 47.6 (C-9), 46.7 (C-17), 45.8 (C-19), 41.7 (C-14), 41.5 (C-18), 41.3 (-NCH2CH2), 39.3 (C-8), 38.7 (C-1), 38.4 (C-4), 37.0 (C-10), 35.6 (-CS), 33.8 (C-29), 33.1 (C-22), 32.7 (C-21), 32.4 (C-7), 30.7 (C-20), 28.1 (C-15), 27.7 (C-23), 27.1 (C-27), 25.9 (C-30), 23.6 (C-2), 23.4 (C-11), 22.9 (C-16), 20.5 (-CH3), 18.3 (C-6), 17.1 (C-26), 15.6 (C-24), 15.3 (C-25). HRMS (ESI): m/z calculated for C44H66N2O3S2 [M+H]+: 735.4593. Found: 735.4562.
[2-((thiomorpholine-4-carbonothioyl)thio)ethyl] 3-hydroxy-12-en-28-oic acid (3t). To a mixture of CS2 (1.8 mmol, 108 µL), anhydrous K3PO4 (0.8 mmol, 169.1 mg), and THF (8.0 mL), thiomorpholine (1.0 mmol, 94 µL) was slowly added at 0 °C, and the reaction mixture was then stirred at 0 °C for 0.5 h. Another THF solution (4.0 mL) of 2 (0.4 mmol, 225.1 mg) was added dropwise to the resulting mixture. The reaction mixture was stirred for 12 h at room temperature, then quenched with ice water (15.0 mL), and the insoluble material was removed by a Buchner funnel. After removal of the solvent, the residue was dissolved in ethyl acetate (15.0 mL). Water (15.0 mL) was added to the resulting solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15.0 mL × 2). The organic solutions were combined and dried over anhydrous Na2SO4. After removal of the solvent, the residue was submitted to column chromatography on silica gel (200–300 mesh) using petroleum ether and ethyl acetate (10/1 in v/v) as eluents to give 3t (190.4 mg, 72% yield) as a white gel. 1H NMR (400 MHz, CDCl3): δ 5.29 (s, 1H, H-12), 4.61 (s, 2H, -OCH2C-), 4.26 (m, 2H, -NCH2), 3.61 (m, 2H, -SCH2), 3.21 (m, 1H, H-3), 2.86 (dd, J = 13.5, 3.6 Hz, 1H, H-18), 2.75 (m, 4H, 2 × -SCH2), 1.97 (m, 1H, -OH), 1.88 (m, 2H, -CH2), 1.63 (m, 6H, 3 × -CH2), 1.53 (m, 3H, H-22, -CH, -CH2), 1.42 (m, 4H, 2 × -CH2), 1.35 (m, 3H, H-22, -CH, -CH2), 1.27 (m, 4H, 2 × -CH2), 1.16 (t, J = 3.6 Hz, 1H, H-9), 1.13 (s, 3H, -CH3), 1.04 (s, 1H, H-5), 0.98 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.90 (s, 6H, 2 × -CH3), 0.78 (s, 3H, -CH3), 0.73 (s, 3H, -CH3). 13C NMR (100 MHz, CDCl3): δ 195.9 (-CS2), 177.5 (C-28), 143.6 (C-13), 122.6 (C-12), 79.0 (C-3), 62.2 (-CO-), 55.3 (C-5), 47.7 (C-9), 46.8 (C-17), 45.8 (C-19), 41.7 (C-14), 41.3 (C-18), 39.4 (C-8), 38.8 (C-1), 38.5 (C-4), 37.1 (C-10), 35.8 (-CS), 33.9 (C-29), 33.2 (C-22), 32.8 (C-21), 32.5 (C-7), 30.8 (C-20), 28.2 (C-15), 27.8 (C-23), 27.2 (C-27), 27.0 (-NCH2), 25.9 (C-30), 23.7 (C-2), 23.5 (C-11), 23.0 (C-16), 18.4 (C-6), 17.2 (C-26), 15.7 (C-24), 15.4 (C-25), 14.2 (-SCH2). HRMS (ESI): m/z calculated for C37H59NO3S3 [M+H]+: 662.3735. Found: 662.3673.

3.3. Preliminary Biological Study

The in vitro cytotoxic activities of the compounds were evaluated by MTT assay against Panc1, A549, Hep3B, Huh-7, HT-29, Hela, LO2. Cell lines were obtained from the Laboratory of Molecular Pharmacology, Southwest Medical University. Briefly, different tumor cells grew in DMEM medium except for A549, which used 1640 medium. Cells ((3–5) × 103 cells/well) were harvested at the log phase of growth and seeded in 96-well plates. After 24 h incubation at 37 °C in 5% CO2 to allow cell attachment, cultures were exposed to various concentrations of the isolated compounds for 48 h. Finally, the MTT solution was added. Plates were further incubated for 4 h at 37 °C after adding 150 µL/well of DMSO and shaking for 10 min on the shaker platform. The plates were read in a 96-well plate reader at 490 nm wavelength. The results were expressed as IC50 values, and were defined as the concentration at which 50% survival of cells was obtained. Fluorouracil, docetaxel, and cisplatin were co-assayed as positive controls.

4. Conclusions

In summary, we have synthesized a series of OA-dithiocarbamate derivatives in a two-step protocol at room temperature, offering a readily accessible synthetic route to obtain novel OA derivatives in high yields. Moreover, some of the compounds were shown to be promising hit compounds, with remarkably improved broad-spectrum antiproliferative activities compared to the natural product OA. Mechanistic insights of their activities on certain tumor cell lines are currently underway in our laboratory.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/molecules28031414/s1. Figures S1–S42: 1H and 13C NMR spectrum of 2, 3a–t; Figures S43–S63: HRMS spectrum of 2, 3a–t.

Author Contributions

Y.C., Z.X. and Y.Z. (Yueshui Zhao). conceptualized and designed this article. L.T., Y.Z. (Yan Zhang) and J.X. conducted the experiments and wrote the manuscript. Q.Y., F.D., X.W., M.L., J.S. and S.D. provided critical comments and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81972643), SCU-Luzhou Platform Construction of Scientific and Technological Innovation (Grant No. 2022CDLZ-20), and the Research Fund of Southwest Medical University (Grant No. 2021ZKQN106).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Molecules 28 01414 sch001 550
Scheme 1. Oleanolic Acid and Its Derivatization for Antitumor Medicinal Chemistry [11,12,13,14,15].
Scheme 1. Oleanolic Acid and Its Derivatization for Antitumor Medicinal Chemistry [11,12,13,14,15].
Molecules 28 01414 sch001
Molecules 28 01414 sch002 550
Scheme 2. Pharmaceutically Important Dithiocarbamates [23,24,25,26,27].
Scheme 2. Pharmaceutically Important Dithiocarbamates [23,24,25,26,27].
Molecules 28 01414 sch002
Molecules 28 01414 g001 550
Figure 1. Synthesis of OA-Dithiocarbamate Derivatives.
Figure 1. Synthesis of OA-Dithiocarbamate Derivatives.
Molecules 28 01414 g001
Table
Table 1. Optimization of Reaction Conditions.
Table 1. Optimization of Reaction Conditions.
Molecules 28 01414 i001
EntryBase (equiv.)SolventIsolated Yield of 3a (%)
1K3PO4 (1.5)THF72
2K2HPO4 (2.0)THF0
3Li2CO3 (2.0)THF0
4K2CO3 (2.0)THF62
5K3PO4 (2.0)DMF23
6K3PO4 (2.0)CH3CN19
7K3PO4 (2.0)EtOH34
8 aK3PO4 (2.0)THF78
9 bK3PO4 (2.0)THF64
10 cK3PO4 (2.0)THF70
Variations from the “standard” conditions. a Reaction temperature was raised to 60 °C. b CS2 was used in 3.0 equiv. instead of 4.5 equiv. c Pyrrolidine was used in 1.5 equiv. instead of 2.0 equiv.
Table
Table 2. In Vitro Cytotoxicity Data of OA and Its Derivatives.
Table 2. In Vitro Cytotoxicity Data of OA and Its Derivatives.
CompoundIC50 a (µM)
Panc1A549Hep3BHuh-7HT-29HelaLO2
OA>200>200>200>200>200>200140.1
5-fluorouracil160.0125.3152.2140.093.3130.4108.9
Docetaxel>200>200172.2104.1>200135.0139.7
Cisplatin>200>200>200>200>200142.5>200
3a>20092.1>200144.9ND b89.4113.4
3b130.0135.8>200>200100.377.1136.0
3c>20064.3ND>200>200133.7ND
3d15.742.526.364.6~18.311.934.1
3e13.128.815.229.9~17.67.062.8
3fNDNDNDNDNDNDND
3g185.2>200>200>200121.3>200>200
3h>200ND176.596.9106.7106.2>200
3i16.924.4~18.770.6~18.47.830.3
3j13.533.616.949.47.610.925.2
3k59.0>200>200106.7>20049.8>200
3l13.434.314.739.98.3NDND
3m>200>200160.1>20081.8131.2>200
3n39.534.5124.3132.862.248.676.9
3o31.132.635.822.927.536.424.1
3p14.625.213.959.110.3ND20.6
3q>200NDND183.1187.4>200ND
3r>200>200161.2>200>200192.0>200
3s>200ND>200102.1ND115.4ND
3t>20040.364.572.9ND175.4114.7
a Concentration inhibiting 50% of cell growth for 48 h exposure period of tested samples. b ND, not determined.
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MDPI and ACS Style

Tang, L.; Zhang, Y.; Xu, J.; Yang, Q.; Du, F.; Wu, X.; Li, M.; Shen, J.; Deng, S.; Zhao, Y.; et al. Synthesis of Oleanolic Acid-Dithiocarbamate Conjugates and Evaluation of Their Broad-Spectrum Antitumor Activities. Molecules 2023, 28, 1414. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28031414

AMA Style

Tang L, Zhang Y, Xu J, Yang Q, Du F, Wu X, Li M, Shen J, Deng S, Zhao Y, et al. Synthesis of Oleanolic Acid-Dithiocarbamate Conjugates and Evaluation of Their Broad-Spectrum Antitumor Activities. Molecules. 2023; 28(3):1414. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28031414

Chicago/Turabian Style

Tang, Liyao, Yan Zhang, Jinrun Xu, Qingfan Yang, Fukuan Du, Xu Wu, Mingxing Li, Jing Shen, Shuai Deng, Yueshui Zhao, and et al. 2023. "Synthesis of Oleanolic Acid-Dithiocarbamate Conjugates and Evaluation of Their Broad-Spectrum Antitumor Activities" Molecules 28, no. 3: 1414. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28031414

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