Chinese Journal of Natural Medicines  2018, Vol. 16Issue (3): 231-240  DOI: 10.1016/S1875-5364(18)30052-9
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WANG Shao-Chi, YANG Yun, LIU Jing, JIANG Ai-Dou, CHU Zhao-Xing, CHEN Si-Ying, GONG Guo-Qing, HE Guang-Wei, XU Yun-Gen, ZHU Qi-Hua. Discovery of novel limonin derivatives as potent anti-inflammatory and analgesic agents[J]. Chinese Journal of Natural Medicines, 2018, 16(3): 231-240.
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Research funding

This work was supported by the National Natural Science Foundation of China (Grant No. 21472242), the National Science and Technology Major Project for "Significant New Drugs Creation" of China (Grant No. 2015ZX09102001), and State Key Laboratory of Natural Medicines, China Pharmaceutical University (No.SKLNMZZCX201406)

Corresponding author

E-mails: gonggq@hotmail.com (GONG Guo-Qing)
E-mails: xu_yungen@hotmail.com (XU Yun-Gen)
E-mails: zhuqihua@vip.126.com (ZHU Qi-Hua)

Article history

Received on: 20-Jun.-2017
Available online: 20 Mar., 2018
Discovery of novel limonin derivatives as potent anti-inflammatory and analgesic agents
WANG Shao-Chi1,2 , YANG Yun2 , LIU Jing1,2 , JIANG Ai-Dou3 , CHU Zhao-Xing4 , CHEN Si-Ying3 , GONG Guo-Qing3 , HE Guang-Wei4 , XU Yun-Gen1,2 , ZHU Qi-Hua1,2     
1 State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;
2 Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China;
3 Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, China;
4 Hefei YiGong Pharmaceutical Co., Ltd., Hefei 230088, China
[Abstract]: Novel series of limonin derivatives (Ⅴ-A-1-Ⅴ-A-8, Ⅴ-B-1-Ⅴ-B-8) were synthesized by adding various tertiary amines onto the C (7)-position of limonin. The synthesized compounds possessed favorable physicochemical property, and the intrinsic solubility of the novel compounds were significantly improved, compared with limonin. Different pharmacological models were used to evaluate the analgesic and anti-inflammatory activities of the target compounds. Compound Ⅴ-A-8 exhibited the strongest in vivo activity among the novel limonin analogs; its analgesic activity was more potent than aspirin and its anti-inflammatory activity was stronger than naproxen under our testing conditions.
[Key words]: Limonin derivatives     Analgesic     Anti-inflammatory    
Introduction

Limonoids are highly oxygenated triterpenoids and mainly found in plants belonging to the Rutaceae and Meliaceae family. Over 300 limonin analogs have been isolated from natural resource [1]. Limonin (Ⅰ-A), nomilin (2) and obacunone (3) (Fig. 1) are the most abundant limonoids existing in citrus species' leaves and fruits and rhizomes [2]. Because limonoids possess various biological effects, such as anti-inflammatory [3-4], analgesic [3-4], antitumor [5-8], anti-mi-crobial [9] and anti-fee-dant [10] activities, the research of these natural products has attracted great interests in medicinal chemistry [11-15].

Figure 1 Structures of Limonin (Ⅰ-A), Nomilin (2) and Obacunone (3)

Limonin (Ⅰ-A) is known as the most common limonoid in the natural environment, which is isolated from navel orange. Since limonin was first identified as analgesic and anti-in-flammatory agent by Matsuda [16-17], various efforts have been put to study its possible clinical application. However, the rigid structure and poor solubility in water (< 0.005 mg·mL-1) of limonin result in low oral bioavailability, discouraging its further pharmacological research [18]. Therefore, structural modification to produce limonin derivatives with favorable physi-coche-mical property has become one of the key research topics.

In order to increase their water solubility, various tertiary amine moieties were introduced onto C(7)-position of limonin or deoxylimonin to afford 16 amide derivatives (Ⅴ-A-1-Ⅴ-A-8, Ⅴ-B-1-Ⅴ-B-8) in the present study. The physiochemical properties of the target compounds were studied. The in vivo analgesic activi-ties were evaluated by acetic acid induced writhing and tail-immersion in mice, and the in vivo anti-inflammatory effects were determined by xylene-induced ear swelling in mice.

Results and Discussion Chemistry

The synthetic routes for target compounds are depicted in Scheme 1. Treatment of limonin (Ⅰ-A) or desoxylimonin (Ⅰ-B) [19] with hydroxylamine hydrochloride yielded exclusively limonin-7-oxime (Ⅱ-A) [20] or desoxylimonin 7-oxime (Ⅱ-B). Intermediate Ⅱ-A was reduced to Ⅲ-A by NaBH3CN. Compound Ⅲ-A was condensed with chloroacetyl chloride or 3-chloropropionyl chloride to give Ⅳ-A-1 or Ⅳ-A-2, which were reacted with the corresponding amine (R1R2NH) to render the target compounds Ⅴ-A-1-Ⅴ-A-8. Compounds Ⅴ-B-1-Ⅴ-B-8 were prepared according to the procedure described for Ⅴ-A-1-Ⅴ-A-8 with good yields. All the target compounds were characterized by 1H/13C NMR and HRMS spectroscopy.

Scheme 1 Reagents and conditions: (a) hydroxylamine hydrochloride, pyridine, anhydrous ethanol, reflux; (b) NaBH3CN, ammonium acetate, titanium trichloride, methyl alcohol, 0 ℃ to room temperature; (c) chloroacetyl chloride or 3-chloropropionyl chloride, DMAP, dichloromethane, 0 ℃ to room temperature; (d) ⅰ: corresponding amine (R1R2NH), potassium carbonate, acetone, 50 ℃; ⅱ: hydrogen chloride, anhydrous ether, anhydrous dichloromethane
Physicochemical properties of the target compounds

The physiochemical properties of the target compounds, including pKa (ionization constants), logD7.4 (partition coefficient at pH 7.4), and aqueous solubility, were studied according to the method of Avdeef and Tsinman on a Gemini Profiler instrument (pION) by the 'gold standard' Av-deef-Bucher potentiometric titration method [21]. The results are summarized in Table 1. A balance between hydrophilicity and lipophilicity is very important for oral absorption. In general, log D7.4 values from -0.5 to 2.0 are considered to be optimal for oral absorption of compounds [22]. The log D7.4 values for Ⅴ-A series compounds, possessing oxygen bridge between C(14) and C(15), were in the range from -0.42 to 1.45, which were more favorable than Ⅴ-B series compounds (log D7.4: from -0.90 to 1.10). In terms of pKa, the un-ionized form of a drug is better absorbed than its ionized counterpart. Since the pH range from duodenal to colon is roughly 5-8 in humans [23], compounds with pKa value greater than 8, like Ⅴ-A-2, Ⅴ-A-3, Ⅴ-A-6 and Ⅴ-A-7, might be better absorbed after oral administration. Besides, the aqueous solubility of the target compounds was much higher than that of limonin and desoxylimonin, the intrinsic solubility of compound Ⅴ-B-8 increased to 1 100.0 mg·mL-1. Of note, the intrinsic solubility of some derivatives was over ten thousand folds greater than that of limonin, but the in vivo activities (Table 2, Table 3, Fig. 2, and Fig. 3) did not show improvement by over one order of magnitude. We reasoned that the intrinsic solubility alone is not able to determine the biological activities, and other factors, like absorption and ligand-receptor interaction, also contribute to their biological activity.

Table 1 pKa, log D7.4, solubility of indicated compounds
Table 2 The analgesic activity by acetic acid induced writhing test in mice of indicated compounds
Table 3 The inflammatory activity by xylene-induced ear swelling test in mice of indicated compounds
Figure 2 Antinociceptive effects of Ⅴ series compounds (100 mg·kg-1), limonin (100 mg·kg-1) and Aspirin (100 mg·kg-1) in the tail-immersion test in mice. Statistical analysis was performed using one-way ANOVA (*P < 0.05, **P < 0.01, *** P < 0.001 vs the respective control). The results are expressed as means ± SEM (n = 8)
Figure 3 Antinociceptive effects of Ⅴ series compounds (100 mg·kg-1), limonin (100 mg·kg-1) and Aspirin (100 mg·kg-1) in the tail-immersion test in mice. Statistical analysis was performed using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001 vs the respective control). The results are expressed as means ± SEM (n = 8)
In vivo analgesic activities of the target compounds

The result of acetic acid-induced writhing test (Table 1) revealed that the analgesic activity of compounds Ⅴ-A-8 and Ⅴ-B-8 were stronger than that of limonin at the same concentration levels. Among series compounds, the analgesic activity of Ⅴ-A-8 and Ⅴ-B-8 was stronger than the rest compounds, indicating that morpholine linked by three carbon atoms might play a critical role in their biological activities. Compound Ⅴ-A-8 (70 mg·kg-1, i.g.) exhibited the most potent analgesic activity among the synthesized compounds, and its inhibition rate (57.35%) of writhings was significantly higher than that of aspirin (200 mg·kg-1, i.g., 40.44% inhibition rate).

In the ear swelling test (Table 3) and acetic acid-induced writhing test (Table 2), the Ⅴ-A series compounds, containing the oxygen bridge between C (14) and C (15), were generally more potent than that of Ⅴ-B series compounds, which was in accordance with our group's previous research [24]. Thus, most of the Ⅴ-A series compounds along with Ⅴ-B-2, Ⅴ-B-4 and Ⅴ-B-5 were subjected in the tail-immersion test in mice. As shown in Figs. 2 and 3, all the tested compounds exhibited analgesic capacity in the tail-immersion test in mice, and their analgesic effects were more potent than that of limonin. The maximal anti-nociceptive response of Ⅴ-A-1-Ⅴ-A-8 was obtained between 30 and 90 min, while compounds Ⅴ-B-2-Ⅴ-B-5 reached maximal anti-nociceptive responses after 60 min of oral administration. In general, most of the Ⅴ-A series compounds demonstrated better analgesic effects than limonin during the tests, but Ⅴ-B-2 and Ⅴ-B-4 showed less or equal potent effect than limonin. These results indicated that the existence of oxygen bridge contributed to the analgesic activity. Besides, compounds with six-member-heterocycle (Ⅴ-A-5, Ⅴ-A-7 and Ⅴ-A-8) were more active than the rest compounds, which were also stronger than aspirin.

In vivo anti-inflammatory tests of the target compounds

All the target compounds were evaluated for their anti-inflammatory activity by ear swelling induced by xylene in mice (Table 3) [25]. In general, several compounds (Ⅴ-A-1, Ⅴ-A-6, Ⅴ-A-7, and Ⅴ-A-8) at the dose of 100 mg·kg-1 exhibited better inflammation inhibitory effects than naproxen (150 mg·kg-1), limonin and desoxylimonin. Among all compounds, Ⅴ-A-8 exhibited the best activity with the inhibitory rate of 58.15%. Interestingly, the anti-inflammatory assay (Table 3) demons-trated that the Ⅴ-A series compounds were more potent than the Ⅴ-B series compounds. These results could be attributed to the existence of the oxygen bridge between C (14) and C (15), which might play an important role in biological activity. Besides, when the linker between C (7)-position of limonin and tertiary nitrogen atom consisted of 4 atoms (-NH-CO-CH2-CH2-), compounds Ⅴ-A-6-Ⅴ-A-8 and Ⅴ-B-6-Ⅴ-B-8 exhibited better anti-inflammatory activity than the rest of the target com-pounds. Thus, the oxygen bridge and 4-atom-length linker may be beneficial for their anti-inflammatory activity.

Conclusion

In the present study, a total of 16 novel limonin derivatives were designed, synthesized, and evaluated for their analgesic and anti-inflammatory activities. A favorable physicochemical property of the new compounds was observed with greatly increased water solubility. And the testing with ear swelling model induced by xylene in mice indicated that 4-atom-length linker between C(7)-position of limonin and tertiary nitrogen atom and existence of oxygen bridge between C(14) and C(15) were beneficial for their anti-infla-mmatory activity. Among the tested compounds, compound Ⅴ-A-8 showed better analgesic and anti-inflammatory activity than the classical NSAIDs (aspirin or naproxen) in the mouse models. In conclusion, compound Ⅴ-A-8 may deserve further biological mechanism study to elucidate how the limonoids and their derivatives exert analgesic and anti-inflammatory activity. Also, compound Ⅴ-A-8 may worth further biological evaluation as a potential candidate for the analgesic and anti-inflammatory drug development.

Experiment protocols Chemistry

Melting points were determined on a melting point apparatus (1101D Mel-TEMP Ⅱ, UK) and were uncorrected. The 1H NMR and 13C NMR spectra were recorded on a 300 MHz or 500 MHz spectrometer (Bruker Avance Ⅲ, Switzerland) using TMS as an internal standard and chemical shifts were given in ppm with tetramethylsilane (TMS). The mass spectra were recorded on a LC-MS 2010 (Shimadzu EI, Japen). The HRMS spectra were acquired on a Q-TOF apparatus (Waters Micros, US). All the solvents and materials used in the present study were purchased from commercial sources and used as received unless otherwise stated.

(12S, 12aS)-8-amino-12-(furan-3-yl)-6, 6, 12a-trimethyldo-decahydro-1H, 3H-oxireno[2, 3-d]pyrano[4', 3': 3, 3a]iso-benzofuro[5, 4-f]isochromene-3, 10(9aH)-dione (Ⅲ-A)

Compound Ⅱ-A (1.00 g, 2.06 mmol), HCOONH4 (1.91 g, 24.74 mmol) and NaBH3CN (0.39 g, 6.19 mmol) were dissolved and stirred in 70 mL of methanol (Solution A). 15 mL of TiCl3 in 3% hydrochloric acid was added drop-wise to Solution A in ice-bath. The mixture was then warmed to room temperature. The reaction was monitored by thin-layer chromatography until completion. The reaction solution was diluted with 300 mL of brine, washed with 200 mL of CH2Cl2. The aqueous layer's pH was adjusted to 8-9 with 1 mol·L-1 NaOH, extracted with 300 mL of CH2Cl2 and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure to afford the crude product, which was purified to give Ⅲ-A as white solids (0.61 g, 63% yields, mp 234 ℃ (decompo-sition)). 1H NMR (500 MHz, DMSO-d6) δ: 7.92 (br s, 3H), 7.71 (s, 1H), 7.67 (s, 1H), 6.49 (s, 1H), 5.58 (s, 1H), 4.49 (d, J = 13.2 Hz, 1H), 4.40 (d, J= 12.9 Hz, 1H), 4.10 (s, 1H), 4.03 (d, J = 2.7 Hz, 1H), 3.74 (br s, 1H), 2.60 (dt, J = 16.5, 9.5 Hz, 2H), 2.39 (dd, J = 11.3, 6.9 Hz, 1H), 2.14 (d, J = 12.7 Hz, 1H), 2.10-1.97 (m, 1H), 1.95-1.86 (m, 1H), 1.78 (dd, J = 24.6, 13.0 Hz, 1H), 1.68 (td, J1 = 17.8 Hz, J2 = 8.5 Hz, 2H), 1.20 (s, 3H), 1.18 (s, 3H), 1.02 (s, 3H), 0.97 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 170.49, 167.24, 143.93, 142.36, 120.58, 110.60, 80.23, 79.16, 77.62, 74.88, 65.02, 58.75, 55.96, 55.78, 45.30, 44.84, 41.16, 39.56, 36.04, 30.70, 25.30, 24.06, 21.86, 18.88, 17.33, 15.45. HR-ESI-MS m/z 472.232 9 [M + H]+ (Calcd. for C26H34NO7, 472.233).

(1R, 14aR)-5-amino-1-(furan-3-yl)-7, 7, 14a-trimethyl-1, 4b, 5, 6, 6a, 7, 8a, 9, 12b, 13, 14, 14a-dodecahydro-3H, 10H, 12H-py-rano [4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromene-3, 10-dione (Ⅲ-B)

Compound Ⅲ-B was synthesized by following the procedure described for Ⅲ-A in 72 % yields as white solids, mp 242 ℃ (decomposition). 1H NMR (500 MHz, DMSO-d6) δ: 8.10 (br s, 3H), 7.71 (s, 1H), 7.69 (s, 1H), 6.51 (s, 1H), 6.13 (s, 1H), 5.11 (s, 1H), 4.50 (d, J = 13.2 Hz, 1H), 4.41 (d, J = 12.9 Hz, 1H), 3.96 (br s, 2H), 2.68-2.54 (m, 3H), 2.25-2.11 (m, 1H), 2.07-1.91 (m, 2H), 1.76-1.48 (m, 3H), 1.22 (s, 3H), 1.16 (s, 3H), 1.15 (s, 3H), 1.01 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 171.64, 170.46, 164.62, 143.79, 142.17, 120.65, 116.24, 110.77, 81.46, 80.30, 79.05, 65.53, 54.66, 50.03, 45.78, 43.86, 37.76, 37.59, 35.89, 30.39, 27.41, 25.68, 21.87, 21.65, 17.39, 16.14, 4.24. HR-ESI-MS m/z 456.237 5 [M + H]+ (Calcd. for C26H34NO6, 456.238 1).

2-Chloro-N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetra-methyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano [4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl)acetamide (Ⅳ-A-1)

Compound Ⅲ-A (1.00 g, 2.12 mmol) and catalytic amount of DMAP were dissolved in 50 mL of CH2Cl2 (solution A). 0.18 mL (2.34 mmol) of chloroacetyl chloride was slowly added to Solution A from -5 to 0 ℃. The reaction was warmed to room temperature and stirred for 5 h. Then, the reaction solution was diluted with 150 mL of CH2Cl2, washed with brine, and dried over anhydrous MgSO4. The solvent was evaporated in vacuo and the resulting crude product was purified by chromatography to afford Ⅳ-A-1 as white solids in 84% yields (mp > 250 ℃). 1H NMR (500 MHz, CDCl3) δ: 7.41 (s, 1H), 7.41 (s, 1H), 6.72 (d, J = 8.3 Hz, 1H), 6.31 (s, 1H), 5.54 (s, 1H), 4.55 (d, J = 13.2 Hz, 1H), 4.44 (d, J= 13.0 Hz, 1H), 4.15-4.20 (m, 1H), 4.00 (br s, 1H), 3.83 (s, 1H), 2.93 (dd, J = 16.9, 3.7 Hz, 1H), 2.60 (dd, J = 16.8, 1.7 Hz, 1H), 2.37 (dd, J = 11.8, 5.8 Hz, 1H), 2.14-2.06 (m, 1H), 2.02-1.91 (m, 2H), 1.83-1.67 (m, 2H), 1.55-1.44 (m, 2H), 1.29 (s, 6H), 1.12 (s, 3H), 1.10 (s, 3H). MS (ESI (-) 70V) m/z 546.2 [M-H]-.

3-Chloro-N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetra-methyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano [4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl) propa-namide (Ⅳ-A-2)

Compound Ⅳ-A-2 was synthesized by following the procedure described for Ⅳ-A-1 in 79 % yields as white solids, mp 192 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 7.78 (d, J = 8.7 Hz, 1H), 7.40 (s, 2H), 6.30 (s, 1H), 5.54 (s, 1H), 4.65 (d, J = 13.5 Hz, 1H), 4.54 (d, J= 13.5 Hz, 1H), 4.10 (brs, 1H), 3.99 (s, 1H), 3.91 (s, 1H), 3.05 (dd, J = 14.7, 7.6 Hz, 2H), 2.92 (dd, J = 16.8, 3.6 Hz, 1H), 2.69-2.46 (m, 5H), 2.43-2.32 (m, 1H), 2.11-2.02 (m, 1H), 2.01-1.90 (m, 2H), 1.82-1.63 (m, 2H), 1.55-1.46 (m, 1H), 1.42-1.30 (m, 1H), 1.28 (s, 3H), 1.26 (s, 1H), 1.11 (s, 2H), 1.09 (s, 1H), 1.06 (t, J = 6.9 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ: 171.48, 169.48, 166.75, 143.23, 141.26, 120.16, 109.76, 80.81, 79.36, 77.55, 77.23, 72.59, 65.70, 58.82, 57.58, 56.48, 55.95, 48.85, 46.79, 45.50, 41.86, 39.53, 35.52, 30.75, 26.29, 26.06, 21.69, 19.18, 18.75, 15.00, 12.66. HR-ESI-MS m/z 585.317 7 [M + H]+ (Calcd. for C32H45N2O8, 585.317 9).

2-Chloro-N-((8aR, 9R)-9-(furan-3-yl)-1, 1, 8a, 12b-tetra-methyl-4, 11-dioxo-1, 2a, 3, 6b, 7, 8a, 9, 11, 12b, 13, 14, 14a-dodecahydro-4H, 6H, 8H-pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-13-yl)acetamide (Ⅳ-B-1)

Compound Ⅳ-B-1 was synthesized by following the procedure described for Ⅳ-A-1 in 88 % yields as white solids, mp 216 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 7.48 (s, 1H), 7.44 (s, 1H), 6.70 (d, J = 7.9 Hz, 1H), 6.40 (s, 1H), 5.79 (s, 1H), 4.99 (s, 1H), 4.55 (d, J = 13.2 Hz, 1H), 4.46 (d, J= 13.3 Hz, 1H), 4.42 (d, J = 7.7 Hz, 1H), 4.08 (s, 1H), 4.06 (s, 2H), 2.98 -2.85 (m, 1H), 2.56 (d, J = 16.7 Hz, 1H), 2.20 (t, J = 9.8 Hz, 1H), 2.07-1.91 (m, 4H), 1.91-1.81 (m, 1H), 1.64-1.52 (m, 2H), 1.28 (s, 3H), 1.24 (s, 3H), 1.22 (s, 3H), 1.14 (s, 3H). MS (ESI (+) 70V) m/z 554.2 [M + Na]+.

3-Chloro-N-((8aR, 9R)-9-(furan-3-yl)-1, 1, 8a, 12b-tetra-methyl-4, 11-dioxo-1, 2a, 3, 6b, 7, 8a, 9, 11, 12b, 13, 14, 14a-dodecahydro-4H, 6H, 8H-pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-13-yl)propanamide (Ⅳ-B-2)

Compound Ⅳ-B-2 was synthesized by following the procedure described for Ⅳ-A-1 in 85 % yields as white solids, mp 210 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 7.46 (s, 1H), 7.44 (s, 1H), 6.71 (s, 1H), 6.38 (s, 1H), 5.84 (s, 1H), 4.97 (s, 1H), 4.68-4.39 (m, 3H), 4.06 (s, 1H), 3.90-3.75 (m, 1H), 3.75 -3.58 (m, 1H), 2.93 (d, J = 13.9 Hz, 1H), 2.82 (d, J = 13.6 Hz, 1H), 2.70-2.55 (m, 2H), 2.51 (d, J = 16.5 Hz, 1H), 2.36 (t, J = 9.5 Hz, 1H), 2.12 (d, J = 13.7 Hz, 1H), 2.05 -1.73 (m, 5H), 1.23 (s, 3H), 1.20 (s, 3H), 1.18 (s, 3H), 1.10 (s, 3H). MS (ESI (+) 70V) m/z 568.2 [M + Na]+.

2-(Dimethylamino)-N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetramethyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl) acetamide hydrochloride (Ⅴ-A-1)

Dimethylamine dissolved in THF (2.74 mL, 2.74 mmol) and K2CO3 (0.63 g, 4.57 mmol) were mixed in 15 mL of acetone. The mixture was stirred at room temperature for 30 min and then added compound Ⅳ-A-1 (0.50 g, 0.91 mmol). The resulting reaction solution was heated to 60 ℃ and stirred for 5 h. When the reaction completed, the mixture was diluted with 150 mL of CH2Cl2, washed with brine, and dried with anhydrous Na2SO4. The solvent was evaporated and the crude product was purified by column chromatography (MeOH-CH2Cl2, 1 : 50). The purified product was dissolved in anhydrous dichloromethane (3 mL) and treated with a saturated solution of dry hydrogen chloride in diethyl ether. The solid precipitated from the solution was filtered, washed with diethyl ether, and dried to give Ⅴ-A-1 (Yield 61%, white solid, mp > 250 ℃ (decomposition)). 1H NMR (500 MHz, CDCl3) δ: 7.64 (br s, 1H), 7.39 (s, 2H), 6.30 (s, 1H), 5.52 (s, 1H), 4.52 (d, J= 13.2 Hz, 1H), 4.44 (d, J= 13.2 Hz, 1H), 4.19 (td, J = 10.2, 4.7 Hz, 1H), 4.00 (s, 1H), 3.94 (s, 1H), 3.13 (br s, 2H), 2.93-2.89 (m, 1H), 2.59 (d, J = 15.7 Hz, 1H), 2.42 (s, 6H), 2.37 (dd, J = 11.9, 5.9 Hz, 1H), 1.98-1.93 (m, 3H), 1.82-1.65 (m, 2H), 1.56-1.43 (m, 2H), 1.30 (s, 3H), 1.27 (s, 3H), 1.11 (s, 3H), 1.09 (s, 3H). 13C NMR (75 MHz, CDCl3), δ: 169.06, 166.45, 142.73, 140.74, 119.64, 109.25, 80.27, 78.86, 77.19, 71.74, 65.21, 61.85, 58.52, 55.51, 55.13, 46.59, 45.24, 45.03, 41.76, 39.01, 35.05, 30.20, 26.44, 25.92, 21.17, 18.88, 18.34, 14.50. HR-ESI-MS m/z 557.285 4 [M + H]+ (Calcd. for C30H41N2O8, 557.285 7).

2-(Diethylamino)-N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetramethyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl) acetamide hydrochloride (Ⅴ-A-2)

Compound Ⅴ-A-2 was synthesized by following the procedure described for Ⅴ-A-1 in 69 % yields as white solids, mp 220 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 7.78 (d, J = 8.7 Hz, 1H), 7.40 (s, 2H), 6.30 (s, 1H), 5.54 (s, 1H), 4.52 (d, J= 13.1 Hz, 1H), 4.44 (d, J= 13.1 Hz, 1H), , 4.10 (br s, 1H), 3.99 (s, 1H), 3.91 (s, 1H), 3.09 (d, J = 17.6 Hz, 1H), 3.01 (d, J= 17.6 Hz, 1H), 2.92 (dd, J = 16.8, 3.6 Hz, 1H), 2.69-2.46 (m, 2H), 2.43-2.32 (m, 1H), 2.11-2.02 (m, 1H), 2.01-1.90 (m, 2H), 1.82-1.63 (m, 2H), 1.55-1.46 (m, 1H), 1.42-1.30 (m, 1H), 1.28 (s, 1H), 1.26 (s, 1H), 1.11 (s, 6H), 1.09 (s, 6H), 1.06 (t, J = 6.9 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ: 171.48, 169.48, 166.75, 143.23, 141.26, 120.16, 109.76, 80.81, 79.36, 77.55, 77.23, 72.59, 65.70, 58.82, 57.58, 56.48, 55.95, 48.85, 46.79, 45.50, 41.86, 39.53, 35.52, 30.75, 26.29, 26.06, 21.69, 19.18, 18.75, 15.00, 12.66. HR-ESI-MS m/z 585.317 7 [M + H]+ (Calcd. for C32H45N2O8, 585.317 9).

N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetramethyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl)-2-(pyrrolidin-1-yl) acetamide hydrochloride (Ⅴ-A-3)

Compound Ⅴ-A-3 was synthesized by following the procedure described for Ⅴ-A-1 in 62 % yields as white solids, mp 248 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 7.40 (s, 2H), 6.31 (s, 1H), 5.53 (s, 1H), 4.53 (d, J = 13.0 Hz, 1H), 4.46 (d, J = 13.0 Hz, 1H), 4.22 (br s, 1H), 3.99 (s, 1H), 3.93 (s, 1H), 3.73-2.45 (m, 5H), 2.92 (dd, J = 16.7, 3.3 Hz, 1H), 2.60 (d, J = 16.7 Hz, 1H), 2.43-2.30 (m, 1H), 2.19-1.82 (m, 6H), 1.82-1.62 (m, 3H), 1.60-1.43 (m, 2H), 1.29 (s, 3H), 1.27 (s, 3H), 1.11 (s, 3H), 1.08 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 169.02, 166.49, 142.73, 140.73, 119.63, 114.12, 109.25, 80.26, 78.89, 77.26, 76.73, 65.24, 58.73, 55.40, 53.83, 46.82, 45.07, 38.96, 35.08, 30.21, 25.88, 23.67, 21.20, 19.00, 18.43, 14.52. HR-ESI-MS m/z 583.301 2 [M + H]+ (Calcd. for C32H43N2O8, 583.301 4).

N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetramethyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl)-2-(piperidin-1-yl) acetamide hydrochloride (Ⅴ-A-4)

Compound Ⅴ-A-4 was synthesized by following the procedure described for Ⅴ-A-1 in 62 % yields as white solids, mp 248 ℃ (decomposition). 1H NMR (300 MHz, CDCl3) δ: 10.91 (br s, 1H), 9.15 (d, J = 2.8 Hz, 1H), 7.40 (s, 2H), 6.33 (s, 1H), 5.48 (s, 1H), 4.60 (d, J = 12.5 Hz, 1H), 4.51-4.40 (m, 2H), 3.68-3.19 (m, 4H), 2.93 (d, J = 14.0 Hz, 1H), 2.64 (d, J = 16.8 Hz, 1H), 2.31 (d, J = 9.8 Hz, 1H), 2.22-2.04 (m, 2H), 2.01-1.71 (m, 11H), 1.36 (s, 3H), 1.27 (s, 3H), 1.12 (s, 3H), 1.05 (s, 3H). 13C NMR (75 MHz, CDCl3), δ: 169.19, 167.15, 163.32, 142.71, 140.64, 119.48, 109.24, 80.12, 78.95, 77.80, 76.71, 69.13, 65.42, 59.74, 57.43, 55.80, 53.10, 52.71, 48.23, 45.28, 43.32, 38.61, 35.30, 30.11, 29.84, 29.19, 26.30, 22.74, 21.16, 21.02, 19.80, 18.91, 14.45. HR-ESI-MS m/z 597.317 1 [M + H]+ (Calcd. for C33H45N2O8, 597.317 0).

N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetramethyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl)-2-morpholinoa-cetamide hydrochloride (Ⅴ-A-5)

Compound Ⅴ-A-5 was synthesized by following the procedure described for Ⅴ-A-1 in 64 % yields as white solids, mp 248 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 7.55 (br s, 1H), 7.41 (s, 2H), 6.30 (s, 1H), 5.54 (s, 1H), 4.52 (d, J= 13.1 Hz, 1H), 4.48 (d, J= 13.1 Hz, 1H), 4.08 (br s, 1H), 4.00 (s, 1H), 3.93 (s, 1H), 3.75 (s, 4H), 3.03 (s, 2H), 2.92 (dd, J = 16.9, 3.6 Hz, 1H), 2.69-2.44 (m, 5H), 2.39 (dd, J = 11.8, 6.8 Hz, 1H), 2.15-1.90 (m, 3H), 1.84-1.64 (m, 2H), 1.55-1.47 (m, 1H), 1.44-1.32 (m, 1H), 1.28 (s, 3H), 1.25 (s, 3H), 1.14 (s, 3H), 1.12 (s, 3H). 13C NMR (75 MHz, CDCl3), δ: 169.39, 169.22, 166.66, 143.29, 141.30, 120.04, 109.70, 80.83, 79.32, 77.23, 67.21, 65.60, 61.79, 58.66, 57.14, 55.94, 53.91, 46.52, 45.44, 41.64, 39.67, 35.45, 30.76, 26.04, 25.64, 21.73, 19.08, 18.65, 15.21. HR-ESI-MS m/z 599.297 7 [M + H]+ (Calcd. for C32H43N2O9, 599.296 3).

3-(Dimethylamino)-N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetramethyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl) propanamide hydrochloride (Ⅴ-A-6)

Compound Ⅴ-A-6 was synthesized by following the procedure described for Ⅴ-A-1 in 62 % yields as white solids, mp 218 ℃ (decomposition). 1H NMR (300 MHz, CDCl3), δ: 11.04 (br s, 1H), 8.27 (br s, 1H), 7.39 (s, 2H), 6.33 (s, 1H), 5.48 (s, 1H), 4.62 (d, J = 13.4 Hz, 1H), 4.39-4.33 (m, 2H), 4.02 (s, 1H), 3.94 (s, 1H), 3.67-3.35 (m, 2H), 3.13-3.05 (m, 2H), 2.95 (s, 7H), 2.67 (d, J = 16.9 Hz, 1H), 2.29 (d, J = 10.1 Hz, 1H), 1.99-1.65 (m, 6H), 1.56-1.43 (m, 1H), 1.38 (s, 3H), 1.26 (s, 3H), 1.12 (s, 3H), 0.97 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 169.38, 169.01, 167.59, 142.69, 140.60, 119.50, 109.23, 80.11, 78.94, 77.97, 68.49, 65.55, 59.92, 55.37, 53.74, 52.02, 48.55, 45.36, 43.50, 43.16, 43.00, 38.45, 35.39, 30.69, 30.51, 30.05, 26.28, 21.12, 19.97, 19.02, 13.76. HR-ESI-MS m/z 571.302 5 [M + H]+ (Calcd. for C31H43N2O8, 571.301 4).

N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetramethyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl)-3-(piperidin-1-yl) propanamide hydrochloride (Ⅴ-A-7)

Compound Ⅴ-A-7 was synthesized by following the procedure described for Ⅴ-A-1 in 61 % yields as white solids, mp 212 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 10.60 (s, 1H), 8.25 (d, J = 8.9 Hz, 1H), 7.40 (s, 1H), 7.39 (s, 1H), 6.34 (s, 1H), 5.48 (s, 1H), 4.64 (d, J = 13.0 Hz, 1H), 4.38 (s, 1H), 4.34 (d, J = 13.3 Hz, 1H), 3.98 (s, 1H), 3.92 (s, 1H), 3.77-3.59 (m, 2H), 3.48 (dd, J = 14.1, 7.0 Hz, 2H), 3.30-3.02 (m, 2H), 2.99-2.73 (m, 7H), 2.66 (d, J = 16.5 Hz, 1H), 2.29 (d, J = 11.0 Hz, 1H), 2.19-2.04 (m, 2H), 2.00 -1.85 (m, 5H), 1.38 (s, 3H), 1.26 (s, 3H), 1.11 (s, 3H), 0.98 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 169.89, 167.94, 143.12, 141.15, 120.00, 109.76, 80.59, 79.43, 78.35, 69.08, 65.99, 60.37, 55.82, 54.02, 53.63, 53.30, 52.56, 49.02, 45.83, 43.95, 38.94, 35.89, 31.04, 30.51, 30.41, 26.72, 22.79, 21.91, 21.53, 20.45, 19.47, 14.29. HR-ESI-MS m/z 611.332 7 [M + H]+ (Calcd. for C34H47N2O8, 611.332 7).

N-((12S, 12aS)-12-(furan-3-yl)-6, 6, 8a, 12a-tetramethyl-3, 10-dioxotetradecahydro-1H, 3H-oxireno[2, 3-d]pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochromen-8-yl)-3-morpholino-pro-panamide hydrochloride (Ⅴ-A-8)

Compound Ⅴ-A-8 was synthesized by following the procedure described for Ⅴ-A-1 in 58 % yields as white solids, mp > 250 ℃. 1H NMR (500 MHz, CDCl3) δ: 11.56 (s, 1H), 8.07 (s, 1H), 7.39 (s, 2H), 6.33 (s, 1H), 5.47 (s, 1H), 4.63 (d, J = 11.7 Hz, 1H), 4.36 (s, 2H), 4.20-4.00 (m, 4H), 3.99 (s, 1H), 3.92 (s, 1H), 3.70-3.50 (m, 3H), 3.47 (s, 1H), 3.21 (s, 3H), 2.91 (d, J = 16.2 Hz, 1H), 2.67 (d, J = 16.5 Hz, 1H), 2.48 (s, 4H), 2.30 (d, J = 10.4 Hz, 1H), 1.90-1.76 (m, 3H), 1.37 (s, 3H), 1.26 (s, 3H), 1.11 (s, 3H), 0.96 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 169.93, 169.43, 167.93, 143.16, 141.14, 119.94, 109.72, 80.57, 79.41, 78.38, 69.15, 66.00, 63.72, 60.30, 55.85, 53.54, 52.55, 48.97, 45.83, 43.89, 38.92, 35.89, 30.94, 30.52, 30.08, 26.79, 21.57, 20.42, 19.43, 14.27. HR-ESI-MS m/z 613.311 9 [M + H]+ (Calcd. for C33H45N2O9, 613.312).

2-(Dimethylamino)-N-((8aR, 9R)-9-(furan-3-yl)-1, 1, 8a, 12b-tetramethyl-4, 11-dioxo-1, 2a, 3, 6b, 7, 8a, 9, 11, 12b, 13, 14, 14a-dodecahydro-4H, 6H, 8H-pyrano[4', 3': 3, 3a]isobenzo-furo[5, 4-f]isochromen-13-yl)acetamide hydrochloride (Ⅴ-B-1)

Compound Ⅴ-B-1 was synthesized by following the procedure described for Ⅴ-A-1 in 66 % yields as white solids, mp > 250 ℃. 1H NMR (500 MHz, CDCl3) δ: 12.23 (s, 1H), 9.29 (s, 1H), 7.49 (s, 1H), 7.43 (s, 1H), 6.41 (s, 1H), 5.82 (s, 1H), 4.96 (s, 1H), 4.59 (s, 1H), 4.50 (d, J = 8.1 Hz, 2H), 4.32 (s, 1H), 3.83 (s, 1H), 3.76 (s, 1H), 3.48 (d, J = 6.8 Hz, 1H), 2.94 (s, 4H), 2.89 (s, 4H), 2.70 (d, J = 13.9 Hz, 1H), 2.53 (d, J = 16.5 Hz, 1H), 2.17 (s, 1H), 2.00 (s, 1H), 1.90 (s, 2H), 1.35 (s, 3H), 1.32 (s, 3H), 1.26 (s, 3H), 1.14 (s, 3H). 13C NMR (75 MHz, DMSO-d6), δ: 174.22, 170.14, 164.65, 163.13, 143.30, 141.67, 120.15, 112.57, 110.24, 80.96, 79.92, 78.52, 65.38, 57.57, 51.74, 50.93, 45.39, 44.62, 42.94, 42.44, 37.96, 37.37, 35.51, 29.63, 26.85, 25.20, 24.16, 21.47, 17.38, 15.94. HR-ESI-MS m/z 541.290 9 [M + H]+ (Calcd. for C30H41N2O7, 541.290 8).

2-(Diethylamino)-N-((8aR, 9R)-9-(furan-3-yl)-1, 1, 8a, 12b-te--tramethyl-4, 11-dioxo-1, 2a, 3, 6b, 7, 8a, 9, 11, 12b, 13, 14, 14a-dodecahydro-4H, 6H, 8H-pyrano[4', 3': 3, 3a]isobenzo-furo[5, 4-f]isochromen-13-yl)acetamide hydrochloride (Ⅴ-B-2)

Compound Ⅴ-B-2 was synthesized by following the procedure described for Ⅴ-A-1 in 77 % yields as white solids, mp 172 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 11.20 (br s, 1H), 9.32 (d, J = 9.8 Hz, 1H), 7.48 (s, 1H), 7.44 (s, 1H), 6.41 (s, 1H), 5.84 (s, 1H), 4.96 (s, 1H), 4.59 (d, J = 9.4 Hz, 1H), 4.49 (q, J = 13.7 Hz, 2H), 4.34 (s, 1H), 4.08 (d, J = 15.1 Hz, 1H), 3.84 (d, J = 15.4 Hz, 1H), 3.61-3.44 (m, 1H), 3.42-3.14 (m, 3H), 2.99-2.83 (m, 2H), 2.76 (d, J = 13.9 Hz, 1H), 2.53 (d, J = 16.7 Hz, 1H), 1.99 (t, J = 14.1 Hz, 1H), 1.90 (d, J = 7.3 Hz, 2H), 1.62-1.49 (m, 3H), 1.43 (t, J = 7.1 Hz, 3H), 1.38 (t, J = 7.1 Hz, 3H), 1.33 (s, 3H), 1.30 (s, 3H), 1.24 (s, 3H), 1.13 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 173.59, 169.85, 165.18, 162.87, 143.19, 141.33, 119.70, 113.63, 109.92, 82.27, 80.44, 78.68, 66.13, 52.25, 51.63, 49.02, 48.77, 45.62, 45.03, 38.39, 38.24, 35.38, 29.69, 27.97, 25.97, 24.83, 21.58, 17.85, 17.40, 10.01. HR-ESI-MS m/z 569.321 7 [M + H]+ (Calcd. for C32H45N2O7, 569.322 1).

N-((8aR, 9R)-9-(furan-3-yl)-1, 1, 8a, 12b-tetramethyl-4, 11-dioxo-1, 2a, 3, 6b, 7, 8a, 9, 11, 12b, 13, 14, 14a-dodecahydro-4H, 6H, 8H-pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochro-men-13-yl)-2-(pyrrolidin-1-yl)acetamide hydrochloride (Ⅴ-B-3)

Compound Ⅴ-B-3 was synthesized by following the procedure described for Ⅴ-A-1 in 71 % yields as white solids, mp 244 ℃ (decomposition). 1H NMR (300 MHz, DMSO-d6) δ: 10.35 (br s, 1H), 9.25 (d, J = 9.9 Hz, 1H), 7.71 (s, 1H), 7.70 (d, J = 1.6 Hz, 1H), 6.51 (s, 1H), 5.64 (s, 1H), 5.08 (s, 1H), 4.54 (q, J = 13.7 Hz, 2H), 4.41 (d, J = 9.8 Hz, 1H), 4.25 -4.02 (m, 2H), 3.91 (dd, J = 15.4, 7.3 Hz, 1H), 3.65 -3.52 (m, 2H), 3.12-2.78 (m, 2H), 2.75-2.52 (m, 4H), 2.14 (t, J = 13.5 Hz, 1H), 2.06-1.80 (m, 5H), 1.80-1.46 (m, 2H), 1.38 (d, J = 13.0 Hz, 1H), 1.18 (s, 6H), 1.15 (s, 3H), 1.00 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 174.07, 170.13, 164.62, 163.53, 143.31, 141.68, 120.15, 112.63, 110.25, 80.95, 79.93, 78.62, 65.32, 54.97, 53.54, 53.48, 51.66, 51.11, 45.40, 44.62, 38.15, 37.38, 35.54, 29.69, 26.86, 25.18, 24.17, 22.60, 22.46, 21.46, 17.39, 15.93. HR-ESI-MS m/z 567.306 2 [M + H]+ (Calcd. for C32H43N2O7, 567.306 5).

N-((8aR, 9R)-9-(furan-3-yl)-1, 1, 8a, 12b-tetramethyl-4, 11-dioxo-1, 2a, 3, 6b, 7, 8a, 9, 11, 12b, 13, 14, 14a-dodecahy-dro-4H, 6H, 8H-pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]iso-chromen-13-yl)-2-(piperidin-1-yl)acetamide hydrochloride (Ⅴ-B-4)

Compound Ⅴ-B-4 was synthesized by following the procedure described for Ⅴ-A-1 in 66 % yields as white solids, mp 248 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 11.63 (br s, 1H), 9.47 (d, J = 9.5 Hz, 1H), 7.46 (s, 1H), 7.43 (s, 1H), 6.39 (s, 1H), 5.79 (s, 1H), 4.96 (s, 1H), 4.58 (d, J = 8.9 Hz, 1H), 4.50 (q, J = 13.4 Hz, 2H), 4.34 (s, 1H), 3.83 (d, J = 11.1 Hz, 1H), 3.63 (d, J = 9.9 Hz, 1H), 3.43 (d, J = 10.2 Hz, 2H), 3.06 (d, J = 9.9 Hz, 1H), 2.90 (dd, J = 12.4, 7.0 Hz, 3H), 2.73 (d, J = 13.6 Hz, 1H), 2.54 (d, J = 16.8 Hz, 1H), 2.16-1.85 (m, 8H), 1.64-1.43 (m, 4H), 1.36 (s, 3H), 1.31 (s, 3H), 1.25 (s, 3H), 1.13 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 173.66, 169.85, 165.12, 161.89, 143.17, 141.32, 119.67, 113.75, 109.88, 82.26, 80.32, 78.73, 66.16, 59.34, 53.71, 53.37, 52.97, 51.93, 45.61, 44.90, 38.47, 38.36, 35.27, 29.81, 27.94, 25.88, 24.72, 22.93, 21.62, 21.43, 18.09, 17.42. HR-ESI-MS m/z 581.322 3 [M + H]+ (Calcd. for C30H45N2O7, 581.322 1).

N-((8aR, 9R)-9-(furan-3-yl)-1, 1, 8a, 12b-tetramethyl-4, 11-dioxo-1, 2a, 3, 6b, 7, 8a, 9, 11, 12b, 13, 14, 14a-dodecahydro-4H, 6H, 8H-pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochro-men-13-yl)-2-morpholinoacetamide hydrochloride (Ⅴ-B-5)

Compound Ⅴ-B-5 was synthesized by following the procedure described for Ⅴ-A-1 in 71 % yields as white solids, mp 240 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 12.54 (br s, 1H), 9.34 (d, J = 9.7 Hz, 1H), 7.48 (s, 1H), 7.44 (s, 1H), 6.40 (s, 1H), 5.76 (s, 1H), 4.96 (s, 1H), 4.57 (d, J = 9.4 Hz, 1H), 4.55-4.43 (m, 2H), 4.30 (s, 1H), 4.12-3.94 (m, 4H), 3.86 (d, J = 13.9 Hz, 1H), 3.72 (d, J = 13.8 Hz, 1H), 3.38 (s, 1H), 3.31 (d, J = 11.7 Hz, 3H), 2.91 (dd, J = 16.9, 3.1 Hz, 1H), 2.84 (t, J = 9.8 Hz, 1H), 2.66 (d, J = 14.4 Hz, 1H), 2.54 (d, J = 16.9 Hz, 1H), 2.00 (t, J = 14.1 Hz, 1H), 1.96-1.86 (m, 2H), 1.68 (s, 2H), 1.62 (d, J = 14.2 Hz, 1H), 1.58-1.47 (m, 2H), 1.34 (s, 3H), 1.29 (s, 3H), 1.26 (s, 3H), 1.13 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 174.21, 170.12, 164.69, 162.65, 143.30, 141.68, 120.14, 112.58, 110.26, 80.95, 79.94, 78.59, 65.33, 62.78, 56.39, 51.78, 51.07, 45.40, 44.62, 38.11, 37.40, 35.52, 29.65, 26.82, 25.17, 24.15, 21.46, 17.39, 15.92. HR-ESI-MS m/z 583.301 0 [M + H]+ (Calcd. for C32H43N2O8, 583.301 4).

3-(Dimethylamino)-N-((8aR, 9R)-9-(furan-3-yl)-1, 1, 8a, 12b-tetramethyl-4, 11-dioxo-1, 2a, 3, 6b, 7, 8a, 9, 11, 12b, 13, 14, 14a-dodecahydro-4H, 6H, 8H-pyrano[4', 3': 3, 3a]isobenzo-furo[5, 4-f]isochromen-13-yl)propanamide hydrochloride (Ⅴ-B-6)

Compound Ⅴ-B-6 was synthesized by following the procedure described for Ⅴ-A-1 in 72 % yields as white solids, mp 218 ℃ (decomposition). 1H NMR (300 MHz, CDCl3) δ: 10.93 (s, 1H), 8.89 (d, J = 10.0 Hz, 1H), 7.49 (s, 1H), 7.45 (s, 1H), 6.42 (s, 1H), 5.84 (s, 1H), 4.97 (s, 1H), 4.70-4.46 (m, 3H), 4.38 (s, 1H), 3.40 (s, 2H), 3.19-3.03 (m, 1H), 2.98-2.81 (m, 9H), 2.75 (d, J = 13.6 Hz, 1H), 2.57 (d, J = 16.4 Hz, 1H), 2.08-1.96 (m, 3H), 1.68 -1.48 (m, 3H), 1.36 (s, 3H), 1.34 (s, 3H), 1.25 (s, 3H), 1.15 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 173.53, 169.57, 168.29, 165.26, 142.68, 140.86, 119.18, 113.22, 109.42, 81.90, 80.05, 78.23, 65.65, 54.16, 51.62, 51.33, 45.10, 44.82, 42.99, 42.67, 38.06, 37.85, 34.94, 30.66, 29.44, 29.20, 27.17, 25.29, 24.11, 21.08, 17.29, 16.90. HR-ESI-MS m/z 555.307 7 [M + H]+ (Calcd. for C31H43N2O7, 555.306 5).

N-((1R, 14aR)-1-(furan-3-yl)-4b, 7, 7, 14a-tetramethyl-3, 10-dioxo-3, 4b, 5, 6, 6a, 7, 8a, 9, 12b, 13, 14, 14a-dodecahydro-1H, 10H, 12H-pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochro-men-5-yl)-3-(piperidin-1-yl)propanamide hydrochloride (Ⅴ-B-7)

Compound Ⅴ-B-7 was synthesized by following the procedure described for Ⅴ-A-1 in 61 % yields as white solids, mp 228 ℃ (decomposition). 1H NMR (300 MHz, CDCl3) δ: 10.40 (br s, 1H), 8.97 (br s, 1H), 7.46 (s, 1H), 7.43 (s, 1H), 6.39 (s, 1H), 5.82 (s, 1H), 4.95 (s, 1H), 4.64-4.45 (m, 3H), 4.35 (s, 1H), 3.58-3.03 (m, 5H), 2.88-2.72 m, 6H), 2.65-2.40 (m, 5H), 2.11-1.74 (m, 9H), 1.34 (s, 3H), 1.32 (s, 3H), 1.22 (s, 3H), 1.12 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 173.51, 169.59, 168.87, 165.18, 142.65, 140.83, 119.22, 113.19, 109.44, 81.88, 80.03, 78.19, 65.70, 53.47, 53.21, 52.86, 51.59, 51.29, 45.09, 44.84, 38.03, 37.79, 34.92, 29.99, 29.44, 29.19, 27.19, 25.32, 24.09, 22.50, 21.36, 21.09, 17.36, 16.91. HR-ESI-MS m/z 595.338 4 [M + H]+ (Calcd. for C34H47N2O7, 595.337 8).

N-((1R, 14aR)-1-(furan-3-yl)-4b, 7, 7, 14a-tetramethyl-3, 10-dioxo-3, 4b, 5, 6, 6a, 7, 8a, 9, 12b, 13, 14, 14a-dodecahydro-1H, 10H, 12H-pyrano[4', 3': 3, 3a]isobenzofuro[5, 4-f]isochro-men-5-yl)-3-morpholinopropanamide hydrochloride (Ⅴ-B-8)

Compound Ⅴ-B-8 was synthesized by following the procedure described for Ⅴ-A-1 in 80 % yields as white solids, mp 230 ℃ (decomposition). 1H NMR (500 MHz, CDCl3) δ: 11.40 (s, 1H), 8.78 (d, J = 9.3 Hz, 1H), 7.47 (s, 1H), 7.43 (s, 1H), 6.40 (s, 1H), 5.79 (s, 1H), 4.95 (s, 1H), 4.67-4.40 (m, 3H), 4.30 (s, 1H), 4.07-4.01 (m, 4H), 3.52-3.36 (m, 3H), 3.29 (s, 1H), 3.04 (d, J = 9.2 Hz, 3H), 2.95-2.75 (m, 3H), 2.68 (d, J = 14.0 Hz, 1H), 2.53 (d, J = 16.8 Hz, 1H), 2.05-1.84 (m, 3H), 1.60 (d, J = 14.0 Hz, 1H), 1.56-1.44 (m, 2H), 1.33 (s, 3H), 1.29 (s, 3H), 1.22 (s, 3H), 1.12 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 173.93, 169.98, 168.90, 143.18, 141.36, 119.63, 113.74, 109.90, 82.39, 80.55, 78.80, 66.06, 63.91, 63.76, 53.78, 52.40, 52.22, 51.95, 51.90, 45.59, 45.24, 38.54, 38.47, 35.41, 30.11, 29.94, 27.67, 25.74, 24.56, 21.55, 17.78, 17.38. HR-ESI-MS m/z 597.316 4 [M + H]+ (Calcd. for C33H45N2O8, 597.317 0).

In vivo analgesic tests using acetic acid-induced abdominal writhing model in mice

The test was performed as described by Collier and Fontenele et al [26]. Nociception was induced by an intraperi-toneal (i.p.) injection of 0.6% acetic acid solution (10 mL·kg-1). The mice were orally treated with the tested compounds (70 mg·kg-1), limonin (70 mg·kg-1), and aspirin (200 mg·kg-1), 30 min after the acetic acid was injected. The control group received vehicle (0.5% CMC-Na). Immediately after the injection of acetic acid, each animal was placed in an individual box (24 cm × 11 cm × 10 cm) and observed for 15 min. The number of writhing and stretching was recorded and the anti-nociceptive activity was expressed as percentage change from writhing controls.

In vivo analgesic effects by tail-immersion test in mice

The animals were divided into groups of eight animals each. To control (CMC 0.5%), aspirin (100 mg·kg-1), and limonin derivatives (100 mg·kg-1) were given via orally administration. Time for tail withdrawal from the water was measured before and after drug treatment in intervals of 30, 60, 90, and 120 min by immersing the tail tips (1-2 cm) of the mice in water bath thermostatically maintained at temperature of 55 ± 1 ℃ with a cut-off time of immersion at 10 s. The actual flick response of mice was measured by stop watch and the results were compared with that of the control group.

In vivo anti-inflammatory tests in mouse ear swelling model

The model of ear swelling induced by xylene in mice was used to evaluate the anti-inflammatory activities of the target compounds. The limonin and their derivatives (100 mg·kg-1, i.g.) with naproxen (150 mg·kg-1, i.g.) as positive controls were suspended in 0.5 % CMC-Na. The vehicle control group was given 0.5 % CMC-Na with the same method. At 90 min after drug administration, 25 μL of xylene was applied to anterior and posterior surfaces of right ear lobe of the mice. Left ear was untreated and used as control. At 30 min after xylene application, the mice were sacrificed. Circular sections on both ears were taken using a cork borer with a diameter of 8 mm and weighed. Degree of swelling caused by the xylene was measured based on the weight of left ear without stimulus.

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