Synthesis and Evaluation of a Series of New Bulleyaconitine A Derivatives as Analgesics

Abstract

As a nonaddictive analgesic widely used in clinics, the LD₅₀ of bulleyaconitine A is just only 0.92 mg/kg, which exhibits obvious toxicity. Therefore, 31 new non-natural C₁₉-diterpenoid alkaloids (2a–w, 2′a–e, 3, 4a, and 4b) were designed and synthesized from bulleyaconitine A to develop nonaddictive analgesics with low toxicity. The chemical structures were characterized by ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry (HRMS) spectra. The analgesic activities were evaluated by a hot plate test in mice. At the dosage of 10 mg/kg, six compounds (2d, 2j, 2k, 2m, 2t, 2w) exhibited good analgesic activities (increased pain threshold >100%) with a long duration. Among them, 2w showed the best analgesic activity and the longest duration. Its pain threshold reached 166.35% in 15 min, peaked at 30 min (182.35%), and remained 82.59% even at 60 min.

Introduction

Pain is one of the most common clinical symptoms. Nearly a third of the world’s population is plagued by pain, especially chronic pain, which seriously interferes with the quality of life and work efficiency of patients. At present, a variety of acute and chronic pains is mostly treated by analgesics like pethidine, morphine, and codeine. The analgesic effects of these drugs are stable, but long-term use is prone to various adverse reactions such as addiction, dependence, tolerability, nausea, vomiting, giddiness, and impaired renal function.¹ These narcotic management drugs are prescribed to treat acute pain, advanced cancer pain, or noncancer chronic pain. However, restricted by addiction, these drugs have strict use regulation. The other category is nonopioid analgesics, such as aspirin, indomethacin, and ibuprofen, which are generally used to treat mild pain. Meanwhile, adverse reactions such as peptic ulcer, bleeding, and cardiac damage could be caused by these drugs. Therefore, finding nonaddictive analgesics with less side effects has become a new trend.

As a medicinal plant, the Aconitium plant of the family Ranunculaceae has been used for hundreds of years.^2,3 The traditional Chinese medicine “caowu” is the dry root of Aconitum kusnezoffii Reichb., which has been widely used in clinics to treat pains, rheumatics, and neurological diseases.^4,5 A large number of chemical and pharmacological studies have pointed out that C₁₈- and C₁₉-diterpenoid alkaloids are the main analgesic active ingredients of aconitine drugs.^6,7 Among them, the diester diterpenoid alkaloids with C8 and C14 ester groups have the strongest analgesic activities.⁸ However, the lipophilic diesterified C₁₉-diterpenoid alkaloids have been identified as the main toxic component of aconitine drugs.⁹ Bulleyaconitine A (BLA, Figure 1), an important C₁₉-diterpenoid alkaloid isolated from Aconitum bulleyanum Diels,¹⁰ displays a range of interesting biological activities including anti-inflammatory, analgesic, and immune-regulating. Its oral tablets and injections were approved by the Chinese State Food and Drug Administration (SFDA) for clinical use in the 1980s to treat rheumatoid arthritis, low back pain, cancerous pain, and chronic pain caused by various reasons.^11,12 As compared with the known analgesics, such as morphine and methadone, BLA induced neither morphine-like tolerance nor addiction. Despite its powerful analgesic effect, toxicity may occur even at small dosages, which can be temporary symptoms of mild panic, nausea, numbness of the tongue, and palpitations. All of these drawbacks limit the widespread use of BLA as a safer and more effective analgesic. Therefore, it is of great significance to study the synthetic transformation of BLA to achieve the purpose of reducing toxicity and preservation effect. The basic structure of BLA is closely related to pharmacology, and the change of side chains could cause great changes in activity and toxicity. It has been reported that the C8-ester group is associated with anti-inflammatory activity,¹³ as well as the C14-ester group is associated with analgesic activity and toxicity.¹⁴ Herein, to find BLA derivatives with more analgesic activity and less toxicity, we designed and synthesized a total of 29 monoesters and two diesters derived from BLA and evaluated their analgesic activity by the hot plate analgesia mice assay.

Figure 1.

Structure of bulleyaconitine A.

Results and Discussion

Chemistry

A series of new bulleyaconitine A mimics with substituents including alkyl, cycloalkyl, phenyl, and heteroaromatic groups have been designed and synthesized to evaluate their analgesic activities. The preparation of these mimics is outlined in Scheme 1.

Scheme 1. Syntheses of Designed Compounds (2a–w, 2′a–e).

All ester groups were removed from bulleyaconitine A by NaOH, giving the precursor 1 in 98% yield. Compound 1 was subjected to esterification with different purchased acyl chlorides 1a–w, which resulted in the formation of the bulleyaconitine A analogues 2a–w. The C₁₈-diterpenoid alkaloid lappaconitine is another analgesic used in clinics in China. Our previous study demonstrated that modification of the 4-O-side-chain (2′-acetamidobenzoate) could obviously decrease the toxicity of lappaconitine.¹⁵ Encouraged by this, we introduced different 2′-acylamidobenzoates to the C14 position of BLA. As shown in Scheme 1, treatment of compound 1 with isatoic anhydride gave 2′-aminobenzoate 3. Under the same esterification condition as described before, 2′a–e were yielded in 30–59% yields. The structures of isolated products 2a–w and 2′a–e were established by their spectral analysis.

It has been reported that diester diterpenoid alkaloids have stronger analgesic activities.¹⁶ To investigate the effects of C8 and C13 acetylation on analgesic activity, the potential monoester derivative 2w was acetylated with acryl chloride to obtain two polyester thienyl derivatives 4a and 4b (Scheme 2).

Scheme 2. Syntheses of Polyesters 4a and 4b.

Biological Activity Evaluation

The analgesic effect and duration of the synthesized compounds (2a–w, 2′a–e, 3, 4a, and 4b) were evaluated by the hot plate analgesia test in mice, using BLA as the positive control. The results (Table 1) showed that some tested compounds (2d, 2j, 2k, 2m, 2t, 2w) exhibited good analgesic activities (increased pain threshold >100%) when the dosage was 10 mg/kg. The newly synthesized compound 2w showed the best analgesic activity and the longest duration. Its pain threshold increased to 166.35% at 15 min and reached the peak at 30 min (182.35%). More surprisingly, the pain threshold remained at 82.59% even at 60 min. Compounds 2d, 2k, 2m, and 2t presented strong analgesic activity. The analgesia duration of these four compounds was the same as that of 2w, and the pain threshold at 60 min could remain at 92.30, 101.45, 107.20, and 82.25%, respectively. Compound 2j had a moderate analgesic effect, while other compounds did not exhibit any activity at the same concentration. Notably, diester 4a and triester 4b, which were predicted to be more active, displayed poor analgesic effect.

Table 1. Analgesic Activities of Synthesized Compounds on the Hot Plate Analgesia Test in Mice.

		improvement of pain threshold (%)
compounds	dose (mg/kg)	15 min	30 min	45 min	60 min
NS	10	0.00	5.67	6.38	14.18
2a	10	35.20	2.10	0.00	0.00
2b	10	10.50	0.00	0.00	1.60
2c	10	79.20	41.70	25.00	29.20
2d	10	72.30	125.00	125.00	92.30
2e	10	0.00	0.00	0.00	0.00
2f	10	9.55	19.09	0.00	0.00
2g	10	45.04	9.92	0.00	0.00
2h	10	14.98	3.38	2.42	0.00
2i	10	23.54	0.00	0.00	0.00
2j	10	40.06	106.94	76.03	38.17
2k	10	95.18	132.77	131.81	101.45
2l	10	51.09	93.45	93.89	51.09
2m	10	76.80	100.80	168.80	107.20
2n	10	0.00	24.94	0.00	0.00
2o	10	24.04	0.00	0.00	0.00
2p	10	31.60	15.80	26.30	0.00
2q	10	54.10	73.11	20.00	38.36
2r	10	0.00	0.00	0.00	0.00
2s	10	26.61	0.00	0.00	0.00
2t	10	106.27	134.99	121.41	82.25
2u	10	52.60	27.92	58.44	20.78
2v	10	3.97	0.00	0.00	14.29
2w	10	166.35	182.35	165.41	82.59
3	10	63.28	67.23	45.20	44.63
2′a	10	54.12	63.40	51.03	44.33
2′b	10	32.47	54.12	32.47	18.56
2′c	10	23.08	39.39	23.54	20.75
2′d	10	0.00	57.13	123.26	52.20
2′e	10	0.00	7.85	39.43	60.04
4a	10	20.67	41.34	35.20	30.17
4b	10	27.76	36.93	20.22	21.29
bulleyaconitine A	0.35	91.30	73.90	69.60	69.60

Conclusions

To summarize, 31 BLA derivatives were synthesized to find analgesics at least remained activity as BLA but with lower toxicity. At the dosage of 10 mg/kg, six potential analogues (2d, 2j, 2k, 2m, 2t, 2w) showed good improvement of the pain threshold and for a long duration during the hot plate analgesia test in mice. The compound 2w with 5′-chlorothiophene-2′-carboxylate at the C14 position of BLA not only showed good analgesic activity with quick effect but also was effective for the longest duration, even better than the positive control BLA. Another interesting observation was that diester 4a and triester 4b showed poorer analgesic activities than their parent compound monoester 2w. It was reported that diester and triester diterpenoid alkaloids had better analgesic activities than monoesters.¹⁶ However, our finding suggests that the structure–activity relationship of BLA should be further studied.

Experimental Section

Chemistry

Materials and General Method

Bulleyaconitine A was purchased from Wuhan Yuancheng Technology Development Co., Ltd. Unless otherwise specified, the reagents and solvents used in this article are all commercially available analytical or chemical grades and used directly without any purification. The high-resolution mass spectrometry (HRMS) spectrum was determined by a Waters ACQUITY UPLC/Xevo G2-S QTOF mass spectrometer. ¹H NMR spectra were recorded on a Bruker AV 400 nuclear magnetic resonance instrument (400 MHz). Chemical shifts were recorded in parts per million (ppm) relative to tetramethylsilane as the internal standard. Data were reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet–doublet; dt, doublet–triplet; m, multiplet; br, broad), coupling constants (Hz), integration. ¹³C NMR data were collected on a Bruker AV 400 nuclear magnetic resonance instrument (100 MHz) with complete proton decoupling. Chemical shifts were reported in ppm with the tetramethylsilane as the internal standard. Thin-layer chromatography silica gel GF254 and column chromatography silica gel G and H (200–400 mesh) were produced by Qingdao Ocean Chemical Plant.

Preparation of Intermediate 1

BLA (0.75 mmol, 1.0 equiv) was dissolved in a solution of 5% NaOH/methanol (20 mL). The reaction solution was stirred at 50 °C for 30 min. After cooling down to room temperature, the solution was concentrated under reduced pressure. The residue was dissolved in H₂O (15 mL) and extracted with CH₂Cl₂ (15 mL × 3). The organic layers were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the product 1 as a white amorphous powder in 98% yield.

Compound 1, ¹H NMR (400 MHz, CDCl₃) δ 4.10 (d, J = 6.8 Hz, 1H), 3.99 (d, J = 4.8 Hz, 1H), 3.69 (d, J = 8.4 Hz, 1H), 3.43 (d, J = 3.6 Hz, 1H), 3.41 (s, 3H), 3.32 (d, J = 3.6 Hz, 1H), 3.30 (s, 3H), 3.29 (s, 3H), 3.23 (s, 3H), 3.15 (s, 1H), 2.99 (dd, J = 10.8, 6.4 Hz, 1H), 2.64 (d, J = 10.4 Hz, 1H), 2.54–2.45 (m, 3H), 2.34–2.25 (m, 4H), 2.07 (s, 1H), 2.04 (d, J = 7.2 Hz, 1H), 1.94–1.84 (m, 3H), 1.69–1.66 (m, 3H), 1.55–1.39 (m, 2H), 1.29 (d, J = 3.2 Hz, 1H), 1.07 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 85.9, 84.6, 82.4, 80.8, 79.7, 76.9, 72.8, 62.8, 59.3, 57.8, 57.5, 56.4, 53.9, 52.4, 50.6, 50.2, 50.1, 49.5, 42.3, 39.8, 39.5, 36.1, 35.2, 25.9, 13.8; high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) m/z calcd for C₂₅H₄₂NO₇ 468.2956 [M + H]⁺, found 468.2966.

General Preparation of BLA Derivatives 2a–w

Pyridine (6.32 mmol, 30.0 equiv) and acyl chloride (0.47 mmol, 2.2 equiv) were added to a solution of compound 1 (0.21 mmol, 1.0 equiv) in dry dichloromethane (DCM) (5 mL) dropwise at 0 °C. Then, the subsequent reaction solution was stirred at 40 °C for 2 h. A saturated aqueous (aq) solution of NaOH was added to adjust the pH to 10, followed by extracting with DCM (5 mL × 3). The combined organic layer was washed with brine (10 mL), dried over MgSO₄, and evaporated to give compounds 2a–w after purification by column chromatography over silica gel (chloroform–methanol = 20:1–8:1).

Compound 2a, white amorphous powder, yield 30%; ¹H NMR (400 MHz, CDCl₃) δ 4.91 (d, J = 4.8 Hz, 1H), 4.01 (d, J = 6.0 Hz, 2H), 3.67 (d, J = 8.4 Hz, 1H), 3.32 (s, 3H), 3.28 (s, 3H), 3.28 (s, 3H), 3.27–3.23 (m, 1H), 3.22 (s, 3H), 3.08 (s, 1H), 2.99 (dd, J = 10.8, 6.4 Hz, 1H), 2.69 (s, 1H), 2.63–2.40 (m, 6H), 2.40–2.34 (m, 1H), 2.30 (t, J = 7.6 Hz, 3H), 2.13 (dd, J = 16.8, 3.2 Hz, 1H), 2.04–1.88 (m, 5H), 1.64 (dd, J = 14.8, 7.2 Hz, 3H), 1.56–1.46 (m, 1H), 1.27–1.21 (m, 1H) 1.06 (t, J = 6.8 Hz, 3H), 0.95 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 174.5, 85.7, 83.7, 82.6, 81.4, 80.8, 76.9, 73.6, 62.3, 59.3, 58.6, 57.6, 56.4, 53.8, 53.1, 50.3, 49.9, 49.4, 49.3, 42.6, 41.6, 39.5, 37.5, 36.6, 35.3, 26.1, 18.3, 18.3, 13.8; HR-ESI-MS m/z calcd for C₂₉H₄₈NO₈ 538.3374 [M + H]⁺, found 538.3381.

Compound 2b, white amorphous powder, yield 45%; ¹H NMR (400 MHz, CDCl₃) δ 4.90 (d, J = 4.8 Hz, 1H), 4.01 (d, J = 4.0 Hz, 2H), 3.67 (d, J = 8.4 Hz, 1H), 3.32 (s, 3H), 3.28 (s, 6H), 3.22 (s, 3H), 3.08 (s, 1H), 2.99 (dd, J = 10.8, 6.8 Hz, 1H), 2.69 (s, 1H), 2.60–2.41 (m, 6H), 2.39–2.21 (m, 4H), 2.13 (dd, J = 16.8, 3.2 Hz, 1H), 2.03 (d, J = 6.4 Hz, 2H), 2.00–1.87 (m, 3H), 1.80 (s, 1H), 1.68–1.47 (m, 4H), 1.35 (dd, J = 15.2, 7.6 Hz, 2H), 1.06 (t, J = 7.2 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 174.7, 85.7, 83.7, 82.6, 81.4, 80.8, 76.9, 73.6, 62.4, 59.3, 58.6, 57.6, 56.4, 53.8, 53.1, 50.3, 49.9, 49.4, 49.3, 42.6, 41.6, 39.5, 37.5, 35.3, 34.4, 26.9, 26.1, 22.3, 13.8; HR-ESI-MS m/z calcd for C₃₀H₅₀NO₈ [M + H]⁺ 552.3531, found 552.3568.

Compound 2c, white amorphous powder, yield 28%; ¹H NMR (400 MHz, CDCl₃) δ 4.90 (d, J = 5.2 Hz, 1H), 4.01 (d, J = 5.6 Hz, 2H), 3.73–3.64 (m, 2H), 3.32 (s, 3H), 3.28 (s, 3H), 3.28 (s, 3H), 3.21 (s, 3H), 3.08 (s, 1H), 2.99 (dd, J = 10.8, 6.8 Hz, 1H), 2.68 (s, 1H), 2.62–2.40 (m, 6H), 2.38 (m, 1H), 2.31 (t, J = 7.2 Hz, 2H), 2.12 (dd, J = 16.4, 2.8 Hz, 1H), 2.05–1.89 (m, 5H), 1.66–1.55 (m, 3H), 1.55–1.46 (m, 1H), 1.33–1.27 (m, 3H), 1.22 (t, J = 6.8 Hz, 3H), 1.06 (t, J = 7.2 Hz, 3H), 0.88 (t, J = 6.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 174.7, 85.7, 83.7, 82.6, 81.4, 80.8, 76.9, 73.6, 62.3, 59.3, 58.6, 57.6, 56.4, 53.8, 53.1, 50.3, 49.8, 49.4, 49.3, 42.6, 41.6, 39.5, 37.5, 35.3, 34.6, 31.4, 26.1, 24.5, 22.4, 18.5, 13.9; HR-ESI-MS m/z calcd for C₃₁H₅₂NO₈ [M + H]⁺ 566.3687, found 566.3695.

Compound 2d, white amorphous powder, yield 70%; ¹H NMR (400 MHz, CDCl₃) δ 4.91 (d, J = 4.8 Hz, 1H), 4.02 (d, J = 7.6 Hz, 2H), 3.68 (d, J = 8.4 Hz, 1H), 3.33 (s, 3H), 3.29 (s, 6H), 3.27–3.24 (m, 1H), 3.22 (s, 3H), 3.08 (s, 1H), 3.00 (dd, J = 10.4, 6.4 Hz, 1H), 2.67 (s, 1H), 2.62–2.41 (m, 6H), 2.39–2.33 (m, 1H), 2.21 (d, J = 6.8 Hz, 2H), 2.15 (d, J = 2.8 Hz, 1H), 2.13–1.86 (m, 7H), 1.71–1.62 (m, 2H), 1.57–1.46 (m, 1H), 1.07 (t, J = 7.2 Hz, 3H), 0.97 (d, J = 1.6 Hz, 3H), 0.96 (d, J = 1.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 173.9, 85.7, 83.6, 82.6, 81.4, 80.8, 77.5, 73.6, 62.4, 59.4, 58.6, 57.6, 56.5, 53.9, 53.1, 50.3, 49.9, 49.4, 49.4, 43.7, 42.6, 41.7, 39.5, 37.5, 35.3, 26.2, 25.4, 22.6, 22.6, 13.8; HR-ESI-MS m/z calcd for C₃₀H₅₀NO₈ [M + H]⁺ 552.3531, found 552.3548.

Compound 2e, white amorphous powder, yield 49%; ¹H NMR (400 MHz, CDCl₃) δ 4.89 (d, J = 4.8 Hz, 1H), 4.06 (s, 1H), 4.02 (d, J = 6.8 Hz, 1H), 3.67 (d, J = 8.4 Hz, 1H), 3.33 (s, 3H), 3.29 (s, 6H), 3.26–3.25 (m, 1H), 3.22 (s, 3H), 3.08 (s, 1H), 3.00 (dd, J = 10.4, 6.4 Hz, 1H), 2.67 (s, 1H), 2.60–2.57 (m, 1H), 2.56–2.48 (m, 3H), 2.47–2.33 (m, 2H), 2.32–2.21 (m, 1H), 2.15 (dd, J = 16.8, 3.2 Hz, 1H), 2.06–2.04 (m, 1H), 2.03–2.00 (m, 1H), 1.98–1.95 (m, 1H), 1.95–1.88 (m, 1H) 1.73 (s, 1H), 1.66–1.62 (m, 1H), 1.57–1.45 (m, 1H), 1.38–1.24 (m, 2H), 1.22 (s, 9H), 1.07 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 179.3, 85.7, 83.7, 82.6, 81.5, 80.8, 76.8, 73.6, 62.4, 59.4, 58.4, 57.6, 56.5, 53.9, 53.1, 50.3, 49.9, 49.4, 49.2, 42.6, 41.7, 39.5, 38.9, 37.4, 35.3, 27.5, 26.2, 13.8; HR-ESI-MS m/z calcd for C₃₀H₅₀NO₈ [M + H]⁺ 552.3531, found 552.3548.

Compound 2f, white amorphous powder, yield 25%; ¹H NMR (400 MHz, CDCl₃) δ 4.92 (d, J = 4.8 Hz, 1H), 4.06–3.95 (m, 2H), 3.67 (d, J = 8.4 Hz, 1H), 3.31 (s, 3H), 3.28 (s, 6H), 3.22 (s, 3H), 3.05 (s, 1H), 3.00 (dd, J = 10.4, 6.4 Hz, 1H), 2.62–2.40 (m, 7H), 2.36–2.32 (m, 1H), 2.29–2.22 (m, 2H), 2.12 (dd, J = 16.4, 3.2 Hz, 1H), 2.04–1.86 (m, 5H), 1.78 (s, 1H), 1.71–1.43 (m, 6H), 1.31–1.21 (m, 5H), 1.07 (t, J = 7.2 Hz, 3H), 0.92–0.87 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 176.7, 85.7, 83.4, 82.6, 81.1, 80.8, 76.5, 73.6, 62.3, 59.3, 58.3, 57.6, 56.4, 53.9, 53.1, 50.3, 49.9, 49.4, 47.6, 42.6, 41.9, 39.4, 37.3, 35.2, 31.4, 29.6, 26.2, 25.2, 22.8, 14.1, 13.8, 12.0, 11.9; HR-ESI-MS m/z calcd for C₃₃H₅₆NO₈ 594.4000 [M + H]⁺, found 594.4004.

Compound 2g, white amorphous powder, yield 44%; ¹H NMR (400 MHz, CDCl₃) δ 5.29 (s, 1H), 4.72 (d, J = 1.6 Hz, 1H), 4.41 (dd, J = 14.0, 7.2 Hz, 1H), 4.11–4.08 (m, 2H), 3.64 (s, 1H), 3.29 (s, 3H), 3.29 (s, 3H), 3.28 (s, 3H), 3.21 (s, 3H), 2.97 (dd, J = 10.8, 6.8 Hz, 1H), 2.79 (dd, J = 14.8, 6.0 Hz, 1H), 2.65 (d, J = 10.0 Hz, 1H), 2.57–2.41 (m, 4H), 2.36 (t, J = 5.2 Hz, 1H), 2.31–2.21 (m, 2H), 2.05 (d, J = 9.2 Hz, 2H), 1.95–1.83 (m, 3H), 1.69 (d, J = 7.4 Hz, 4H), 1.52–1.44 (m, 4H), 1.07 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 169.6, 87.0, 85.7, 82.4, 80.7, 80.4, 77.6, 72.6, 62.5, 59.3, 58.4, 57.8, 57.4, 56.1, 53.8, 53.0, 52.0, 50.2, 49.4, 49.3, 42.9, 39.9, 39.4, 35.5, 25.8, 21.7, 18.4, 13.8; HR-ESI-MS m/z calcd for C₂₈H₄₅ClNO₈ [M + H]⁺ 558.2828, found 558.2828.

Compound 2h, white amorphous powder, yield 43%; ¹H NMR (400 MHz, CDCl₃) δ 4.89 (d, J = 4.8 Hz, 1H), 4.00 (d, J = 6.8 Hz, 1H), 3.92 (s, 1H), 3.71–3.64 (m, 2H), 3.61–3.60 (m, 2H), 3.34 (s, 3H), 3.28 (s, 3H), 3.27 (s, 3H), 3.24–3.21 (m, 1H), 3.21 (s, 3H), 3.06 (s, 1H), 2.99 (dd, J = 10.4, 6.8 Hz, 1H), 2.60–2.49 (m, 8H), 2.42–2.40 (m, 2H), 2.30–2.20 (m, 2H), 2.16–1.94 (m, 8H), 1.66–1.63 (m, 1H), 1.57–1.46 (m, 1H), 1.06 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 173.5, 85.6, 83.6, 82.6, 81.5, 80.7, 76.7, 73.6, 62.3, 59.3, 58.6, 57.6, 56.4, 53.8, 53.4, 50.3, 49.8, 49.4, 49.0, 44.1, 42.4, 41.6, 39.4, 37.3, 31.5, 27.6, 26.1, 18.5, 13.8; HR-ESI-MS m/z calcd for C₂₉H₄₇ClNO₈ [M + H]⁺ 572.2985, found 572.2989.

Compound 2i, white amorphous powder, yield 51%; ¹H NMR (400 MHz, CDCl₃) δ 4.92 (d, J = 4.8 Hz, 1H), 4.07 (s, 1H), 4.04 (d, J = 6.8 Hz, 1H), 3.70 (d, J = 8.8 Hz, 1H), 3.38 (s, 3H), 3.32 (s, 6H), 3.25 (s, 3H), 3.11 (s, 1H), 3.02 (dd, J = 10.4, 6.4 Hz, 1H), 2.67–2.60 (m, 2H), 2.59–2.52 (m, 3H), 2.49 (m, 1H), 2.44–2.40 (m, 1H), 2.34–2.23 (m, 1H), 2.17 (dd, J = 16.4, 3.2 Hz, 1H), 2.08–2.04 (m, 2H), 2.03–1.98 (m, 2H), 1.71–1.66 (m, 1H), 1.64–1.59 (m, 1H), 1.46–1.41 (m, 1H), 1.34–1.30 (m, 2H), 1.12–1.08 (m, 3H), 1.08–1.02 (m, 2H), 0.97–0.90 (m, 2H), 0.90–0.82 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 175.7, 85.7, 83.6, 82.6, 81.4, 80.8, 76.7, 73.7, 62.4, 59.4, 58.6, 57.6, 56.5, 53.9, 53.3, 50.3, 49.9, 49.4, 49.1, 42.6, 41.7, 39.5, 37.3, 35.2, 26.1, 13.8, 13.4, 8.9, 8.6; HR-ESI-MS m/z calcd for C₂₉H₄₆NO₈ [M + H]⁺ 536.3218, found 536.3214.

Compound 2j, white amorphous powder, yield 52%; ¹H NMR (400 MHz, CDCl₃) δ 4.89 (d, J = 4.8 Hz, 1H), 4.08 (s, 1H), 4.02 (d, J = 6.8 Hz, 1H), 3.68 (d, J = 8.4 Hz, 1H), 3.32 (s, 3H), 3.29 (s, 6H), 3.27 (s, 1H), 2.25–2.22 (m, 1H), 3.22 (s, 3H), 3.09 (s, 1H), 2.99 (dd, J = 10.4, 6.8 Hz, 1H), 2.74 (s, 1H), 2.63–2.43 (m, 6H), 2.39–2.34 (m, 1H), 2.33–2.24 (m, 2H), 2.14 (dd, J = 16.8, 2.8 Hz, 1H), 2.04 (s, 2H), 2.01 (d, J = 6.8 Hz, 1H), 1.99–1.87 (m, 5H), 1.80–1.71 (m, 3H), 1.70–1.60 (m, 2H), 1.56–1.34 (m, 4H), 1.07 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 176.8, 85.7, 83.8, 82.6, 81.6, 80.8, 76.9, 73.6, 62.3, 59.3, 58.5, 57.6, 56.4, 53.9, 53.1, 50.3, 49.9, 49.4, 49.4, 43.5, 42.7, 41.7, 39.5, 37.5, 35.3, 29.2, 29.1, 26.1, 25.8, 25.6, 25.6, 13.8; HR-ESI-MS m/z calcd for C₃₂H₅₂NO₈ [M + H]⁺ 578.3687, found 578.3688.

Compound 2k, white amorphous powder, yield 53%; ¹H NMR (400 MHz, CDCl₃) δ 6.40 (dd, J = 17.2, 1.2 Hz, 1H), 6.11 (ABq, J = 6.8, 10.8 Hz, 1H), 5.86 (dd, J = 10.4, 1.2 Hz, 1H), 4.98 (d, J = 4.8 Hz, 1H), 4.00 (d, J = 6.8 Hz, 1H), 3.90 (s, 1H), 3.65 (d, J = 8.4 Hz, 1H), 3.33 (s, 3H), 3.28 (s, 3H), 3.27 (s, 3H), 3.25–3.24 (m, 1H) 3.22 (s, 3H), 3.04 (s, 1H), 3.00 (ABq, J = 10.4, 6.4 Hz, 1H), 2.64–2.35 (m, 8H), 2.32–2.18 (m, 1H), 2.14 (dd, J = 16.4, 3.6 Hz, 1H), 2.04–1.99 (m, 4H), 1.96–1.82 (m, 2H), 1.72–1.58 (m, 1H), 1.56–1.46 (m, 1H), 1.06 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.6, 131.6, 128.4, 85.6, 83.4, 82.6, 80.8, 80.7, 76.5, 73.7, 62.3, 59.3, 58.5, 57.6, 56.4, 53.9, 53.5, 50.3, 49.8, 49.4, 48.7, 42.4, 41.7, 39.4, 37.0, 35.2, 26.2, 13.8; HR-ESI-MS m/z calcd for C₂₈H₄₄NO₈ [M + H]⁺ 522.3061, found 522.3060.

Compound 2l, white amorphous powder, yield 51%; ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.26 (m, 5H), 4.92 (d, J = 4.8 Hz, 1H), 3.92 (d, J = 3.2 Hz, 2H), 3.66 (s, 2H), 3.64 (s, 1H), 3.30–3.25 (m, 6H), 3.23 (s, 3H), 3.20 (s, 3H), 3.02–2.91 (m, 2H), 2.64–2.34 (m, 6H), 2.29–2.16 (m, 2H), 2.03–1.88 (m, 6H), 1.84 (dd, J = 16.4, 3.2 Hz, 1H), 1.76–1.56 (m, 3H), 1.55–1.44 (m, 1H), 1.04 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.2, 133.6, 129.7, 129.7, 128.9, 128.9, 127.5, 85.7, 83.5, 82.5, 81.7, 80.8, 76.7, 73.2, 62.2, 59.3, 58.4, 57.6, 56.4, 53.9, 53.4, 50.3, 49.8, 49.4, 49.1, 42.5, 41.9, 41.5, 39.4, 37.3, 35.2, 26.2, 13.8; HR-ESI-MS m/z calcd for C₃₃H₄₈NO₇ [M + H]⁺ 586.3374, found 586.3385.

Compound 2m, white amorphous powder, yield 31%; ¹H NMR (400 MHz, CDCl₃) δ 7.89 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 5.13 (d, J = 4.8 Hz, 1H), 4.01 (d, J = 6.8 Hz, 1H), 3.81 (s, 1H), 3.66 (d, J = 8.4 Hz, 1H), 3.38 (s, 3H), 3.36–3.32 (m, 1H), 3.29 (s, 3H), 3.27 (s, 3H), 3.25 (s, 3H), 3.08–2.99 (m, 2H), 2.68–2.42 (m, 7H), 2.33–2.18 (m, 2H), 2.13–2.01 (m, 5H), 1.70–1.60 (m, 2H), 1.60–1.50 (m, 1H), 1.09 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.4, 132.0, 132.0, 131.4, 131.4, 129.2, 128.4, 85.6, 83.4, 82.7, 80.8, 80.7, 76.3, 73.9, 62.4, 59.3, 58.5, 57.7, 56.4, 54.1, 53.9, 50.4, 49.9, 49.4, 48.3, 42.4, 41.9, 39.5, 36.5, 35.2, 29.8, 26.3, 13.8; HR-ESI-MS m/z calcd for C₃₃H₄₇BrNO₈ [M + H]⁺ 664.2480, found 664.2499.

Compound 2n, white amorphous powder, yield 25%; ¹H NMR (400 MHz, CDCl₃) δ 7.23 (dd, J = 4.8, 1.2 Hz, 1H), 6.98 (m, 2H), 4.96 (d, J = 4.8 Hz, 1H), 3.95 (d, J = 6.8 Hz, 1H), 3.90 (s, 1H), 3.88 (s, 1H), 3.66 (d, J = 8.4 Hz, 1H), 3.31 (s, 3H), 3.28 (s, 3H), 3.25 (s, 3H), 3.24–3.22 (m, 1H) 3.21 (s, 3H), 3.05–2.96 (m, 2H), 2.57–2.37 (m, 6H), 2.33–2.30 (m, 1H), 2.20–2.15 (m, 2H), 2.04–1.91 (m, 6H), 1.75–1.60 (m, 2H), 1.56–1.45 (m, 1H), 1.05 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.1, 134.5, 127.5, 127.3, 125.4, 85.7, 83.5, 82.5, 82.0, 80.7, 76.8, 73.3, 62.2, 59.3, 58.5, 57.6, 56.4, 53.9, 53.4, 50.3, 49.8, 49.4, 49.1, 42.5, 41.4, 39.4, 37.3, 35.7, 35.2, 26.2, 13.8; HR-ESI-MS m/z calcd for C₃₁H₄₆NO₈S [M + H]⁺ 592.2939, found 592.2964.

Compound 2o, white amorphous powder, yield 47%; ¹H NMR (400 MHz, CDCl₃) δ 7.89 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 5.14 (d, J = 4.8 Hz, 1H), 4.01 (d, J = 6.8 Hz, 1H), 3.90 (s, 1H), 3.70–3.62 (m, 2H), 3.35 (s, 3H), 3.28 (s, 3H), 3.25 (s, 3H), 3.24 (s, 3H), 3.10–2.97 (m, 2H), 2.63–2.46 (m, 7H), 2.39 (s, 4H), 2.27 (dd, J = 16.4, 4.0 Hz, 2H), 2.05–2.03 (m, 4H), 1.72–1.61 (m, 1H), 1.57–1.49 (m, 1H), 1.26–1.22 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 143.9, 129.8, 129.8, 129.3, 129.3, 127.4, 85.6, 83.3, 82.6, 80.7, 80.4, 76.3, 73.8, 62.4, 59.3, 58.3, 57.6, 56.4, 53.8, 53.6, 50.3, 49.8, 49.4, 48.5, 42.4, 41.9, 39.4, 36.6, 26.2, 21.8, 18.5, 13.7; HR-ESI-MS m/z calcd for C₃₃H₄₈NO₈ [M + H]⁺ 586.3374, found 586.3381.

Compound 2p, white amorphous powder, yield 37%; ¹H NMR (400 MHz, CDCl₃) δ 8.09 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.63–7.56 (m, 2H), 7.46 (t, J = 7.2 Hz, 2H), 7.41–7.37 (m, 1H), 5.19 (d, J = 5.2 Hz, 1H), 4.04 (d, J = 6.8 Hz, 1H), 3.91 (s, 1H), 3.67 (d, J = 8.4 Hz, 1H), 3.40 (s, 3H), 3.36 (dd, J = 8.8, 3.2 Hz, 1H), 3.30 (s, 3H), 3.28 (s, 3H), 3.26 (s, 3H), 3.10–2.97 (m, 2H), 2.70–2.42 (m, 7H), 2.39–2.23 (m, 3H), 2.15–2.01 (m, 4H), 2.00–1.91 (m, 1H), 1.74–1.62 (m, 1H), 1.55 (td, J = 13.6, 3.2 Hz, 1H), 1.31–1.19 (m, 1H), 1.09 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.9, 145.9, 140.0, 130.4, 130.4, 129.1, 129.1, 128.9, 128.3, 127.4, 127.4, 127.3, 127.3, 85.6, 83.4, 82.6, 80.7, 80.6, 76.3, 73.8, 62.4, 59.3, 58.4, 57.7, 56.4, 53.9, 53.8, 50.4, 49.9, 49.4, 48.5, 42.4, 42.0, 39.4, 36.6, 35.2, 26.2, 13.8; HR-ESI-MS m/z calcd for C₃₈H₅₀NO₈ [M + H]⁺ 648.3531, found 648.3524.

Compound 2q, white amorphous powder, yield 27%; ¹H NMR (400 MHz, CDCl₃) δ 8.20 (dd, J = 8.8, 5.6 Hz, 2H), 7.26 (t, J = 8.4 Hz, 2H), 5.28 (d, J = 5.2 Hz, 1H), 4.16 (d, J = 6.4 Hz, 1H), 3.98 (s, 1H), 3.81 (d, J = 8.4 Hz, 1H), 3.54 (s, 3H), 3.50–3.47 (m, 1H), 3.44 (s, 3H), 3.41 (s, 3H), 3.40 (s, 3H), 3.20–3.17 (m, 2H), 2.81–2.57 (m, 7H), 2.41 (dd, J = 16.0, 3.6 Hz, 2H), 2.33 (s, 1H), 2.23–2.19 (m, 4H), 2.13–2.06 (m, 1H), 1.87–1.79 (m, 1H), 1.75–1.65 (m, 1H), 1.24 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1/164.6, 165.9, 132.4, 132.3, 126.4, 115.8, 115.6, 85.4, 83.3, 82.6, 80.6, 80.4, 76.1, 73.8, 62.3, 59.2, 58.3, 57.6, 56.3, 53.9, 53.8, 50.3, 49.6, 49.3, 48.2, 42.2, 41.8, 39.3, 36.4, 35.0, 26.1, 13.7; HR-ESI-MS m/z calcd for C₃₂H₄₅FNO₈ [M + H]⁺ 590.3124, found 590.3119.

Compound 2r, white amorphous powder, yield 39%; ¹H NMR (400 MHz, CDCl₃) δ 7.90 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 5.13 (d, J = 4.8 Hz, 1H), 4.01 (d, J = 6.8 Hz, 1H), 3.81 (s, 1H), 3.66 (d, J = 8.4 Hz, 1H), 3.38 (s, 3H), 3.29 (s, 3H), 3.27 (s, 3H), 3.25 (s, 3H), 3.06–3.02 (m, 2H), 2.65–2.52 (m, 5H), 2.50–2.40 (m, 1H), 2.32–2.15 (m, 2H), 2.12–2.00 (m, 5H), 1.98–1.90 (m, 1H), 1.70–1.52 (m, 5H), 1.09 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.4, 132.0, 132.0, 131.4, 131.4, 129.2, 128.4, 85.6, 83.4, 82.7, 80.8, 80.7, 76.3, 73.9, 62.4, 59.4, 58.5, 57.7, 56.5, 54.1, 53.9, 50.4, 49.9, 49.4, 48.3, 42.4, 41.9, 39.5, 36.5, 35.2, 26.3, 13.8; HR-ESI-MS m/z calcd for C₃₂H₄₅BrNO₈ [M + H]⁺ 650.2323, found 650.2308.

Compound 2s, white amorphous powder, yield 35%; ¹H NMR (400 MHz, CDCl₃) δ 8.14 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 5.05 (d, J = 4.8 Hz, 1H), 4.10 (d, J = 6.4 Hz, 1H), 3.90 (s, 1H), 3.57 (d, J = 8.4 Hz, 1H), 3.48 (s, 3H), 3.33 (s, 3H), 3.30 (s, 3H), 3.28 (s, 3H), 3.18 (s, 1H), 3.04 (s, 3H), 2.79–2.65 (m, 1H), 2.58 (t, J = 5.6 Hz, 1H), 2.42–2.40 (m, 2H), 2.29–2.15 (m, 3H), 2.04 (d, J = 5.2 Hz, 2H), 1.44–1.38 (m, 1H), 1.30–1.27 (m, 4H), 1.26–1.20 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 134.0, 132.3, 132.3, 130.4, 130.4, 118.0, 116.4, 82.9, 81.7, 80.0, 79.1, 77.3, 75.2, 73.7, 62.4, 59.2, 58.7, 58.2, 56.0, 53.9, 50.4, 49.6, 42.3, 41.0, 38.5, 35.9, 31.9, 29.7, 22.7, 18.8, 14.1, 13.7; HR-ESI-MS m/z calcd for C₃₃H₄₅N₂O₈ [M + H]⁺ 597.3170, found 597.3166.

Compound 2t, white amorphous powder, yield 51%; ¹H NMR (400 MHz, CDCl₃) δ 8.89 (d, J = 8.4 Hz, 1H), 8.23–8.16 (m, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.64–7.47 (m, 3H), 5.26 (d, J = 5.2 Hz, 1H), 4.10–3.97 (m, 2H), 3.66 (d, J = 8.4 Hz, 1H), 3.37 (s, 3H), 3.31 (s, 1H), 3.29 (s, 3H), 3.26 (s, 3H), 3.25 (s, 3H), 3.12 (s, 1H), 3.03 (dd, J = 10.4, 6.4 Hz, 1H), 2.73–2.38 (m, 8H), 2.36–2.25 (m, 2H), 2.16–1.88 (m, 6H), 1.73–1.63 (m, 1H), 1.54 (dt, J = 13.6, 6.8 Hz, 1H), 1.08 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.9, 133.8, 133.4, 131.3, 130.1, 128.5, 127.9, 127.2, 126.4, 125.7, 124.6, 85.6, 83.5, 82.5, 81.2, 80.7, 76.7, 73.5, 62.3, 59.2, 58.3, 57.5, 56.3, 53.8, 53.4, 50.2, 49.8, 49.3, 48.9, 42.5, 41.6, 39.4, 37.0, 35.1, 26.1, 13.7; HR-ESI-MS m/z calcd for C₃₆H₄₈NO₈ [M + H]⁺ 622.3374, found 622.3372.

Compound 2u, white amorphous powder, yield 51%; ¹H NMR (400 MHz, CDCl₃) δ 7.55 (dd, J = 1.6, 0.4 Hz, 1H), 7.19 (dd, J = 3.6, 0.8 Hz, 1H), 6.50 (dd, J = 3.6, 1.6 Hz, 1H), 5.10 (d, J = 4.8 Hz, 1H), 4.03 (d, J = 6.8 Hz, 1H), 3.94 (s, 1H), 3.66 (d, J = 8.4 Hz, 1H), 3.29 (s, 1H), 3.28 (s, 3H), 3.27 (s, 3H), 3.25 (s, 3H), 3.22 (s, 3H), 3.08 (s, 1H), 3.03–2.96 (m, 2H), 2.62–2.40 (m, 7H), 2.33–2.17 (m, 2H), 2.07–2.02 (m, 4H), 1.92 (s, 2H), 1.68–1.63 (m, 1H), 1.57–1.46 (m, 1H), 1.06 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 158.8, 146.7, 144.6, 118.7, 112.2, 85.7, 83.4, 82.6, 81.7, 80.7, 76.9, 73.7, 62.3, 59.3, 58.3, 57.6, 56.4, 53.8, 53.3, 50.3, 49.9, 49.4, 49.3, 42.4, 41.7, 39.4, 37.2, 35.2, 26.2, 13.8; HR-ESI-MS m/z calcd for C₃₀H₄₄NO₉ [M + H]⁺ 562.3011, found 562.3018.

Compound 2v, white amorphous powder, yield 49%; ¹H NMR (400 MHz, CDCl₃) δ 7.82–7.78 (m, 1H), 7.55 (dd, J = 4.8, 0.8 Hz, 1H), 7.14–7.08 (m, 1H), 5.15 (d, J = 4.8 Hz, 1H), 4.02 (d, J = 6.8 Hz, 1H), 3.88 (s, 1H), 3.66 (d, J = 8.4 Hz, 1H), 3.32 (s, 3H), 3.29 (s, 1H), 3.28 (s, 3H), 3.27 (s, 3H), 3.23 (s, 3H), 3.08–2.95 (m, 2H), 2.64–2.41 (m, 8H), 2.29–2.24 (m, 2H), 2.10–2.00 (m, 4H), 1.94–1.93 (m, 1H), 1.71–1.60 (m, 1H), 1.53 (dt, J = 14.0, 3.2 Hz, 1H), 1.07 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 162.3, 134.0, 133.4, 132.8, 128.1, 85.6, 83.2, 82.6, 81.0, 80.7, 76.4, 73.8, 62.3, 59.3, 58.2, 57.6, 56.4, 53.8, 53.6, 50.3, 49.8, 49.4, 48.7, 42.4, 41.8, 39.4, 36.7, 35.1, 26.2, 13.8; HR-ESI-MS m/z calcd for C₃₀H₄₄NO₈S [M + H]⁺ 578.2782, found 578.2812.

Compound 2w, white amorphous powder, yield 57%; ¹H NMR (400 MHz, CDCl₃) δ 7.59 (d, J = 4.0 Hz, 1H), 6.93 (d, J = 4.0 Hz, 1H), 5.10 (d, J = 4.8 Hz, 1H), 4.00 (d, J = 6.8 Hz, 1H), 3.81 (s, 1H), 3.66 (d, J = 8.8 Hz, 1H), 3.36 (s, 3H), 3.29 (s, 3H), 3.28 (s, 3H), 3.23 (s, 3H), 3.06–2.95 (m, 2H), 2.65–2.40 (m, 8H), 2.28–2.17 (m, 3H), 2.06–2.02 (m, 4H), 1.97–1.89 (m, 1H), 1.74 (s, 1H), 1.70–1.63 (m, 1H), 1.55 (dt, J = 14.0, 3.6 Hz, 1H), 1.08 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 161.3, 137.8, 133.6, 131.6, 127.6, 100.1, 85.6, 83.2, 82.6, 81.1, 80.7, 76.3, 73.8, 62.3, 59.3, 58.3, 57.7, 56.4, 53.9, 50.4, 49.8, 49.4, 48.5, 42.3, 41.8, 39.4, 36.5, 35.2, 26.3, 13.8; HR-ESI-MS m/z calcd for C₃₀H₄₃ClNO₈S [M + H]⁺ 612.2392, found 612.2390.

Preparation of Compound 3

A suspension of compound 1 (0.1 mmol, 1.0 equiv), isatoic anhydride (0.1 mmol, 1.0 equiv), and DMAP (0.003 mmol, 0.03 equiv) in dimethylformamide (DMF) (2 mL) was stirred at 120 °C under argon for 7 h. After cooling down to room temperature, the mixture was added to saturated NaHCO₃ aq (5 mL) and extracted with excess ethyl acetate (5 mL × 3). The combined organic phase was washed with water (10 mL) and brine (10 mL) and dried over Na₂SO₄. The solvent was removed, and crude residue was purified by silica gel column chromatography by dichloromethane–methanol (20:1–8:1) to obtain the desired compound 3 as a white amorphous powder, with yield 63%.

Compound 3, ¹H NMR (400 MHz, CDCl₃) δ 7.85 (dd, J = 8.4, 1.6 Hz, 1H), 7.30–7.14 (m, 1H), 6.78–6.53 (m, 2H), 5.66 (s, 2H), 5.22–5.05 (m, 1H), 4.01 (d, J = 6.8 Hz, 1H), 3.78 (s, 1H), 3.72–3.60 (m, 2H), 3.38 (s, 3H), 3.28 (s, 3H), 3.25 (s, 3H), 3.24 (s, 3H), 3.08–2.96 (m, 2H), 2.67–2.41 (m, 8H), 2.29–2.24 (m, 2H), 2.05–2.00 (m, 4H), 1.98–1.88 (m, 1H), 1.68–1.62 (m, 1H), 1.58–1.45 (m, 1H), 1.22 (t, J = 6.8 Hz, 2H), 1.08 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 168.1, 150.2, 134.4, 131.8, 117.2, 116.9, 111.2, 85.7, 83.3, 82.5, 80.7, 79.5, 76.0, 73.4, 62.5, 59.3, 58.2, 57.6, 56.4, 53.8, 53.7, 50.3, 49.9, 49.4, 48.5, 42.4, 41.6, 39.4, 36.4, 35.1, 26.2, 13.8; HR-ESI-MS m/z calcd for C₃₂H₄₇N₂O₈ [M + H]⁺ 587.3327, found 587.3330.

Preparation of Compounds 2′a–e

Compound 3 (0.17 mmol, 1.0 equiv) and pyridine (6.32 mmol, 30 equiv) were dissolved in dichloromethane (5 mL), and acyl chloride (0.47 mmol, 2.2 equiv) was added dropwise to the solution at 0 °C. The reaction was carried out at 40 °C for 2 h. A saturated aqueous solution of NaOH was added to adjust the pH to 10, followed by extracting with DCM (7 mL × 3). The combined organic layer was washed with brine (10 mL), dried over MgSO₄, and concentrated. The residue was separated by silica gel column chromatography, eluting with dichloromethane–methanol (20:1–8:1), to yield compounds 2′a–e.

Compound 2′a, white amorphous powder, yield 54%; ¹H NMR (400 MHz, CDCl₃) δ 10.97 (s, 1H), 8.73 (d, J = 8.4 Hz, 1H), 8.08 (dd, J = 8.4, 2.0 Hz, 1H), 7.57–7.53 (m, 1H), 7.17–6.98 (m, 1H), 5.17 (d, J = 5.2 Hz, 1H), 4.04 (d, J = 6.8 Hz, 1H), 3.70 (d, J = 8.4 Hz, 1H), 3.43 (s, 3H), 3.38 (dd, J = 8.8, 3.6 Hz, 1H), 3.31 (s, 3H), 3.28 (d, J = 4.0 Hz, 6H), 3.09–3.05 (m, 2H), 2.74–2.48 (m, 6H), 2.44 (t, J = 7.6 Hz, 2H), 2.35–2.25 (m, 2H), 2.12–2.03 (m, 5H), 1.98 (s, 1H), 1.85–1.75 (m, 3H), 1.69 (dt, J = 13.6, 4.4 Hz, 1H), 1.63–1.54 (m, 1H), 1.43 (s, 1H), 1.30 (s, 1H), 1.11 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.2, 168.4, 141.8, 134.8, 131.0, 122.6, 120.5, 115.0, 85.4, 83.0, 82.6, 80.6, 80.2, 76.0, 73.7, 62.3, 59.2, 58.2, 57.6, 56.3, 54.1, 53.7, 50.3, 49.6, 49.3, 47.9, 42.2, 41.8, 40.6, 39.3, 36.1, 31.6, 26.2, 19.0, 13.8, 13.7; HR-ESI-MS m/z calcd for C₃₆H₅₃N₂O₉ [M + H]⁺ 657.3746, found 657.3740.

Compound 2′b, white amorphous powder, yield 56%; ¹H NMR (400 MHz, CDCl₃) δ 10.95 (s, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.05 (dd, J = 8.0, 1.6 Hz, 1H), 7.56–7.45 (m, 1H), 7.10–7.00 (m, 1H), 5.15 (d, J = 5.2 Hz, 1H), 4.03 (d, J = 6.8 Hz, 1H), 3.76 (s, 1H), 3.68 (d, J = 8.4 Hz, 1H) 3.40 (s, 3H), 3.36 (dd, J = 9.2, 4.0 Hz, 1H), 3.29 (s, 3H), 3.27 (s, 3H), 3.26 (s, 3H), 3.08–2.97 (m, 2H), 2.73–2.47 (m, 6H), 2.47–2.39 (m, 2H), 2.33–2.21 (m, 2H), 2.13–2.01 (m, 5H), 1.78–1.48 (m, 6H), 1.45–1.34 (m, 3H), 1.09 (t, J = 7.2 Hz, 3H), 0.95 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.5, 168.5, 141.9, 134.9, 131.1, 122.7, 120.6, 115.1, 85.5, 83.1, 82.7, 80.7, 80.3, 76.1, 73.8, 62.4, 59.3, 58.3, 57.8, 56.4, 54.2, 53.9, 50.4, 49.8, 49.4, 48.1, 42.3, 41.9, 39.4, 38.6, 36.2, 35.2, 27.7, 26.3, 22.5, 14.0, 13.8; HR-ESI-MS m/z calcd for C₃₇H₅₅N₂O₉ [M + H]⁺ 671.3902, found 671.3900.

Compound 2′c, white amorphous powder, yield 59%; ¹H NMR (400 MHz, CDCl₃) δ 10.97 (s, 1H), 8.74 (d, J = 8.4 Hz, 1H), 8.08 (dd, J = 8.0, 1.6 Hz, 1H), 7.57–7.53 (m, 1H), 7.15–7.02 (m, 1H), 5.17 (d, J = 4.8 Hz, 1H), 4.04 (d, J = 6.8 Hz, 1H), 3.70 (d, J = 8.4 Hz, 1H), 3.43 (s, 3H), 3.38 (dd, J = 9.2, 4.0 Hz, 1H), 3.32 (s, 3H), 3.28 (s, 3H), 3.27 (s, 3H), 3.11–3.00 (m, 2H), 2.73–2.41 (m, 9H), 2.33–2.28 (dd, J = 16.0, 4.0 Hz, 2H), 2.16–2.03 (m, 5H), 2.01–1.98 (m, 1H), 1.83–1.53 (m, 5H), 1.43–1.35 (m, 4H), 1.11 (t, J = 6.8 Hz, 3H), 0.95–0.91 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.4, 168.4, 141.8, 134.8, 131.0, 122.5, 120.5, 115.0, 85.4, 83.0, 82.6, 80.6, 80.2, 76.0, 73.7, 62.3, 59.2, 58.2, 57.6, 56.3, 54.0, 53.7, 50.3, 49.7, 49.3, 48.0, 42.2, 41.8, 39.3, 38.7, 36.1, 35.1, 31.4, 26.2, 25.2, 22.4, 14.0, 13.7; HR-ESI-MS m/z calcd for C₃₈H₅₇N₂O₉ [M + H]⁺ 685.4059, found 685.4053.

Compound 2′d, white amorphous powder, yield 30%; ¹H NMR (400 MHz, CDCl₃) δ 11.96 (s, 1H), 8.86 (d, J = 8.4 Hz, 1H), 8.13 (dd, J = 8.0, 1.2 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.70–7.50 (m, 3H), 7.14 (d, J = 7.2 Hz, 1H), 5.18 (d, J = 4.8 Hz, 1H), 4.01 (d, J = 6.8 Hz, 1H), 3.75 (s, 1H), 3.68 (d, J = 8.4 Hz, 1H), 3.42 (s, 3H), 3.41–3.33 (m, 2H), 3.28 (s, 3H), 3.26 (s, 3H), 3.25 (s, 3H), 3.08–3.02 (m, 2H), 2.70–2.41 (m, 9H), 2.32–2.27 (m, 2H), 2.24–2.18 (m, 1H), 2.13–2.07 (m, 3H), 2.02 (s, 2H), 1.96 (s, 1H), 1.80–1.48 (m, 5H), 1.09 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 168.8, 164.7, 141.7, 134.9, 133.7, 132.1, 132.1, 131.3, 129.0, 129.0, 126.9, 123.1, 120.5, 115.4, 85.5, 83.0, 82.6, 80.6, 80.2, 75.9, 73.7, 62.3, 59.2, 58.2, 57.6, 56.3, 54.2, 51.2, 50.3, 49.3, 47.8, 42.2, 41.8, 39.3, 36.0, 35.1, 22.8, 13.7; HR-ESI-MS m/z calcd for C₃₈H₅₇N₂O₉ [M + H]⁺ 769.2694, found 769.2690.

Compound 2′e, white amorphous powder, yield 35%; ¹H NMR (400 MHz, CDCl₃) δ 12.11 (s, 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.22–8.05 (m, 3H), 7.80 (d, J = 8.4 Hz, 2H), 7.66–7.54 (m, 1H), 7.20–7.10 (m, 1H), 5.15 (d, J = 5.2 Hz, 1H), 4.00 (d, J = 6.8 Hz, 1H), 3.75 (s, 1H), 3.69–3.67 (m, 1H), 3.42 (s, 3H), 3.37 (dd, J = 9.2, 4.0 Hz, 1H), 3.27 (s, 3H), 3.25 (s, 3H), 3.24 (s, 3H), 3.08–3.00 (m, 2H), 2.73–2.39 (m, 7H), 2.29 (dd, J = 16.0, 4.0 Hz, 2H), 2.08 (t, J = 6.8 Hz, 3H), 2.01 (s, 2H), 1.99–1.91 (m, 1H), 1.85 (s, 1H), 1.67–1.54 (m, 2H), 1.08 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 168.9, 163.7, 141.4, 138.7, 135.1, 132.8, 132.8, 131.5, 128.2, 128.2, 123.6, 120.5, 118.2, 115.7, 115.5, 85.5, 83.1, 82.7, 80.6, 80.4, 75.9, 73.8, 62.4, 59.3, 58.3, 57.7, 56.4, 54.4, 53.8, 50.3, 49.7, 49.4, 47.7, 42.2, 41.8, 39.4, 36.1, 35.2, 26.3, 13.7; HR-ESI-MS m/z calcd for C₄₀H₅₀N₃O₉ [M + H]⁺ 716.3542, found 716.3557.

Preparation of Compounds 4a and 4b

Compound 2w (0.5 mmol) and pyridine (15 mmol, 30 equiv) were dissolved in dichloromethane (10 mL). Acetyl chloride (1 mmol, 2 equiv) was added dropwise to the solution at 0 °C. Then, the reaction mixture was stirred at 35 °C for 30 min. A saturated aqueous solution of NaOH was added to adjust the pH to 10, followed by extracting with DCM (15 mL × 3). The combined organic layer was washed with brine, dried over MgSO₄, and concentrated. The crude residue was separated by silica gel column chromatography, eluting with dichloromethane–methanol (20:1–8:1), to separate compounds 4a and 4b.

Compound 4a, white amorphous powder, yield 35%; ¹H NMR (400 MHz, CDCl₃) δ 7.65 (d, J = 4.0 Hz, 1H), 6.96 (d, J = 4.0 Hz, 1H), 5.47 (d, J = 5.2 Hz, 1H), 4.04–3.98 (m, 2H), 3.69 (d, J = 8.8 Hz, 1H), 3.31 (s, 3H), 3.30 (s, 3H), 3.29 (s, 3H), 3.25 (s, 3H), 3.15–2.98 (m, 3H), 2.67–2.40 (m, 6H), 2.32–2.23 (m, 2H), 2.19 (s, 1H), 2.10–2.04 (m, 4H), 2.03 (s, 3H), 1.79–1.63 (m, 3H), 1.60–1.53 (m, 1H), 1.11 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.5, 160.4, 137.9, 133.7, 131.7, 127.5, 85.3, 83.6, 82.8, 80.7, 79.4, 77.8, 73.7, 62.2, 59.3, 58.0, 57.7, 56.2, 53.9, 53.7, 50.5, 49.7, 49.3, 46.8, 42.9, 42.3, 39.4, 35.7, 35.2, 26.2, 21.7, 13.8; HR-ESI-MS m/z calcd for C₃₂H₄₅ClNO₉S [M + H]⁺ 654.2498, found 654.2496.

Compound 4b, white amorphous powder, yield 61%; ¹H NMR (400 MHz, CDCl₃) δ 7.57 (d, J = 4.0 Hz, 1H), 6.89 (d, J = 4.0 Hz, 1H), 5.01 (d, J = 5.2 Hz, 1H), 3.94–3.89 (m, 2H), 3.59 (d, J = 8.4 Hz, 1H), 3.37–3.33 (m, 1H), 3.30 (s, 3H), 3.23 (s, 3H), 3.18 (s, 3H), 3.13 (s, 3H), 3.07 (d, J = 8.4 Hz, 1H), 3.00–2.85 (m, 4H), 2.78–2.70 (m, 1H), 2.60–2.32 (m, 5H), 2.30–2.16 (m, 1H), 2.05 (d, J = 6.4 Hz, 2H), 1.98 (s, 3H), 1.90–1.80 (s, 2H), 1.62–1.51 (m, 2H), 1.46 (s, 3H), 1.04 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.2, 169.6, 160.8, 137.9, 133.4, 132.1, 127.4, 85.5, 84.7, 83.3, 82.0, 80.3, 79.8, 77.6, 61.7, 59.1, 57.9, 56.0, 53.6, 50.3, 49.2, 49.1, 49.0, 43.9, 41.6, 39.5, 39.1, 35.5, 35.0, 26.3, 21.7, 21.4, 13.5; HR-ESI-MS m/z calcd for C₃₄H₄₇ClNO₁₀S [M + H]⁺ 696.2604, found 696.2580.

Biology

Materials

All test samples were administered by subcutaneous injection. All experimental samples were dissolved in dimethyl sulfoxide (DMSO) or HCl (0.1 M) and diluted to a certain concentration with distilled water. There were at least 10 animals in each group, making sure there are at least six available data in each group. The 10 animals in each group were randomly assigned and labeled according to body weight, and the animals in each group were controlled to receive the same experimental treatment. The blank group was injected with the same volume of saline as the test group for the control.

Animals

SPF female Kunming mice (20 ± 2g) were purchased from Chengdu Dashuo Animal Co., Ltd. (license number: SCXK (Sichuan) 2015-030). Experimental animals were kept in the animal room of the School of Southwest Jiaotong University. The temperature was kept at 25 ± 2 °C, the humidity and light were kept constant, and there was free access to food and water. All experimental animals were reared adaptively for 2 days before experiments. The experiments were performed strictly in accordance with current laboratory animal care guidelines and ethical guidelines for experimental research.

Hot Plate Analgesia Test in Mice

The mice were moved into the laboratory 1 h before the test to allow them to adapt to the experimental environment. The mice were placed on a 55 °C smart hot plate instrument to record the time when the hind feet were licked. Each group was tested twice, with an interval of 15 min to prevent burns. The mean time between the two calculations was the basal pain threshold. After subcutaneous injection of the test drug according to body weight (0.1 mL/10 g), the time of licking the hind feet of the mouse was recorded every 15 min and recorded four times to calculate the percentage increase in the pain threshold. The cutoff latency was 30 s to prevent hurt. The analgesic effect in each mouse at each time point was calculated as the pain threshold by the following equation: 100 × (latency_{after treatment} – latency_{before treatment})/(30 s – latency_{before treatment}).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02944.

• ¹H and ¹³C NMR spectra for all of the new compounds (PDF)

The authors declare no competing financial interest.