CN114890971A - Eriocalyxin B derivative, pharmaceutical composition thereof and application of eriocalyxin B derivative in resisting neocoronary pneumonia - Google Patents

Abstract

The invention discloses a calyxolide B derivative, a pharmaceutical composition thereof, and an application against new coronary pneumonia, and belongs to the technical field of medicines. The calyxolide B derivative of the present invention can inhibit the activity of the new coronavirus (SARS-CoV-2) 3CL ^pro at the enzyme level, inhibit the replication of the SARS-CoV-2 virus at the cellular level, and reduce the SARS-CoV-2 virus in cell culture. ‑CoV‑2 nucleic acid load, which can be used as a drug to treat related diseases. It can be used to prepare a SARS-CoV-2 3CL ^pro inhibitor, and the SARS-CoV-2 3CL ^pro protein is the new coronavirus 3CL protease (main protease); it can be used to prepare a medicine for treating diseases caused by SARS-CoV-2 infection; For the preparation of medicines for the treatment of simple infection, pneumonia, acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock disease; for the preparation and treatment of severe acute respiratory infection, fever, cough , sore throat drugs. The preparation method of the invention is easy to operate, has high yield, and is suitable for industrial production.

Description

Calyxolide B derivatives and their pharmaceutical compositions and their application against new coronary pneumonia

technical field

The invention belongs to the technical field of medicine, and in particular, the invention relates to calyxolide B derivatives and pharmaceutical compositions thereof, as well as their application in the preparation of medicines for treating diseases caused by SARS-CoV-2 infection.

Background technique

The 2019 novel coronavirus (2019-nCoV) is a new strain of coronavirus that has never been found in humans before. On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) announced that the official classification of the 2019 novel coronavirus (2019-nCoV) was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ). SARS-CoV-2 encodes 4 structural and 16 nonstructural proteins, providing multiple targets for identifying potential drug targets. Among nonstructural proteins, SARS-CoV-2 3CL ^pro is an essential protease in the viral life cycle and has no closely related homologous proteins in human cells, making it an attractive drug target.

The 14-hydroxyl-substituted derivatives of calyxolide B have a new type of skeleton and a new structure. There is no report on the structure and resistance to SARS-CoV-2 of calyxolide B derivatives in the existing reports.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide calyxolide B derivatives and pharmaceutical compositions thereof, and their application in the preparation of medicines for the treatment of diseases caused by SARS-CoV-2 infection. In the present invention, it is found through creative research that calyxolide B derivatives have inhibitory activity on SARS-CoV-2 3CL ^pro and inhibit the viral load of SARS-CoV-2 in cells. The disease has potential therapeutic effect.

In order to achieve the above-mentioned purpose of the present invention, the present invention provides the following technical solutions:

The calyxolide B derivative represented by the following structural formula,

According to the described calyxolide B derivative, wherein R is the following functional group:

The invention also provides a method for preparing calyxolide B derivatives. The method takes calyxolide B as a starting material, and obtains calyxolide B derivatives through an esterification reaction.

The method includes the following steps: under the protection of argon, dissolving calyxolide B in 0.1-0.5 mL of dichloromethane, adding EDCI and DMAP, placing the reaction at room temperature for 1-12 hours, and detecting by TLC After the reaction was completed, quenched with saturated NaHCO ₃ , extracted with EtOAc, washed with saturated brine, dried with anhydrous MgSO ₄ , filtered, concentrated, purified by column chromatography or purified by preparative purification, and isolated to obtain the calycolactin B derivative.

The present invention also provides the application of the calyxolide B derivative in the preparation of a SARS-CoV-2 3CL ^pro inhibitor, wherein the SARS-CoV-2 3CL ^pro protein is a novel coronavirus 3CL protease (main protease);

Use in the preparation of medicines for the treatment of diseases caused by SARS-CoV-2 infection;

Application in the preparation of medicines for the treatment of simple infection, pneumonia, acute respiratory tract infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis and septic shock disease;

Application in the preparation of medicines for treating severe acute respiratory tract infection, fever, cough and sore throat.

In addition, the present invention also provides a pharmaceutical composition comprising any one or any combination of the calylactone B derivatives and a pharmaceutically acceptable carrier.

And, the application of the pharmaceutical composition in the preparation of SARS-CoV-2 3CL ^pro inhibitor, the SARS-CoV-2 3CL ^pro protein is the new coronavirus 3CL protease, that is, the main protease.

Application of the pharmaceutical composition in the preparation of a medicine for treating diseases caused by SARS-CoV-2 infection, the diseases are simple infection, pneumonia, acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome , sepsis, septic shock disease.

The preparation method of the pharmaceutical composition is as follows: according to the above-mentioned method for preparing calyxolide B derivatives, the calyxolide B derivatives are first separated and obtained, and then a pharmaceutically acceptable carrier can be added.

When the compound of the present invention is used as a medicine, it can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90% of the compound of the present invention, and the rest are pharmaceutically acceptable, non-toxic and inert pharmaceutically acceptable carriers and/or excipients for humans and animals.

The pharmaceutically acceptable carrier or excipient is one or more solid, semi-solid and liquid diluents, fillers and pharmaceutical preparation adjuvants. The pharmaceutical composition of the present invention is used in a dosage per body weight. The medicament of the present invention can be administered in various forms (liquid preparation, solid preparation, injection, external preparation, spray, compound preparation).

Compared with the prior art, the present invention has the following advantages:

1. The present invention provides a new class of calyxolide B derivatives, which fills the gap in the prior art.

2. The present invention provides a method for preparing calyxolide B derivatives. The method has easily available raw materials, easy operation, high yield, and is suitable for industrial production.

3. The present invention provides a pharmaceutical composition with a calyxolide B derivative as an active ingredient, which provides a new anti-coronavirus drug with a better medicinal effect.

4. The calyxolide B derivatives of the present invention can inhibit the activity of SARS-CoV-2 3CL ^pro at the enzymatic level, inhibit the replication of SARS-CoV-2 virus at the cellular level, and reduce SARS-CoV-2 in cell culture. -2 viral nucleic acid load.

5. The calyxolide B derivatives can be used as medicines for the treatment of related diseases. It can be used in the preparation of SARS-CoV-23CL ^pro inhibitors, and the SARS-CoV-2 3CL ^pro protein is the new coronavirus 3CL protease (main protease); it is used to prepare medicines for the treatment of diseases caused by SARS-CoV-2 infection; For the preparation of medicines for the treatment of simple infection, pneumonia, acute respiratory tract infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock disease; for the preparation and treatment of severe acute respiratory tract infection, fever, Medications for cough and sore throat.

Description of drawings

Fig. 1 The inhibition rate of calyxolide B derivatives on SARS-CoV-2 3CL ^pro at a concentration of 5 μM;

Figure 2 Schematic diagram of the structure of calyxolide B derivatives.

Detailed ways

Below in conjunction with the accompanying drawings, the present invention will be further described with the embodiments of the present invention, but the present invention is not limited in any way. Any transformation or improvement based on the teachings of the present invention shall fall within the protection scope of the present invention.

Example 1

1. First prepare calyxolide B:

Preparation of intermediate L: Compound K was dissolved in acetone (1-100 mL), and Jones reagent (10 mL) was added to react for 15 minutes at 0 °C in an ice bath. After the reaction was detected by TLC, it was quenched with isopropanol. After diluting with water, it was extracted with EtOAc, washed with saturated brine, dried over anhydrous _MgSO4 , filtered, concentrated and purified by column chromatography to obtain an amorphous solid compound L, which was identified as intermediate L by mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance.

Preparation of Intermediate M: Compound L was dissolved in 1,2-dichloroethane (1-100 mL), NaBH(OAc) ₃ (1-50 g) was added under ice bath at 0°C, and then acetic acid was slowly added dropwise (0.1-1 mL) reacted for 10 minutes. After the completion of the reaction was detected by TLC, it was quenched with acetone, diluted with water, extracted with EtOAc, washed with saturated brine, dried over anhydrous MgSO ₄ , filtered, concentrated and purified by column chromatography to obtain amorphous The solid compound was identified as intermediate M by mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance.

Preparation of intermediate N: Compound M was dissolved in (1-100 mL) dichloromethane, and DIPEA (1-50 mL) and TMSCl (1-50 mL) were sequentially added under ice-bath conditions at 0°C, and reacted under ice-bath conditions After 30 minutes it was concentrated under reduced pressure. After concentration, the product was dissolved in acetonitrile, then DBU (1-50 mL) and p-toluenesulfonyl azide (1-50 mL) were added to react for 2 hours, then TBAF (1-50 g) was added dropwise to react for 30 minutes, and TLC was used to detect the completion of the reaction. , diluted with water, extracted with EtOAc, washed with saturated brine, dried over anhydrous _MgSO4 , filtered, concentrated and purified by column chromatography to obtain an amorphous solid compound, which was identified as intermediate N by mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance.

Preparation of intermediate O: Compound N was dissolved in (1-100 mL) dichloromethane, and NaHCO ₃ (1-10 g) and Dess-Martin reagent (1-10 g) were sequentially added under 0°C ice bath conditions, and the The reaction was carried out under ice bath conditions for 10 minutes. After the completion of the reaction was detected by TLC, it was quenched with Na ₂ O ₃ S ₂ , diluted with water, extracted with EtOAc, washed with saturated brine, dried over anhydrous MgSO 4 , filtered, and purified by column chromatography after concentration. , an amorphous solid compound was obtained, and the compound was identified as intermediate O by mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance.

Preparation of calyxolide B: Compound O was dissolved in (1-50 mL) toluene, and the mixture was placed at 110° C. under heating and stirring for sufficient reaction for two hours. After TLC detected the reaction, the reaction was moved to room temperature. After cooling, it was directly concentrated under reduced pressure. After concentration, column chromatography was performed to obtain an amorphous white solid compound. Esterin B.

2. Preparation of calyxolide B derivatives:

Under the protection of argon, calyxolide B (10-20 mg) was dissolved in (0.1-0.5 mL) dichloromethane, EDCI (10-50 mg) and DMAP (10-50 mg) were added, and the reaction was placed in The reaction was carried out at room temperature for 1 to 12 hours. After the reaction was detected by TLC, it was quenched with saturated NaHCO ₃ , extracted with EtOAc, washed with saturated brine, dried with anhydrous MgSO ₄ , filtered, concentrated and purified by column chromatography or by preparative purification to obtain the compound Derivatives 1-63 identified by mass spectrometry, 1D resonance.

Example 2

NMR and mass spectrometry data of calyxolide B derivatives:

Data for 1: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=6.09(s, 1H), 5.67(s, 1H), 5.59(s, 1H), 4.93(d, J=10.8, 1H), 4.87 (d, J=10.8, 1H), 4.52 (dt, J=14.3, 3.6, 1H), 4.06 (d, J=3.7, 1H), 2.88 (dd, J=11.7, 3.3, 1H), 2.49 (dd , J=9.2, 2.6, 1H), 2.48–2.41 (m, 1H), 2.23 (dd, J=13.8, 11.8, 1H), 2.20–2.15 (m, 1H), 2.11 (t, J=7.4, 1H) ), 1.99–1.92 (m, 2H), 1.90 (dd, J=13.9, 3.3, 1H), 1.82–1.73 (m, 1H), 1.68–1.62 (m, 1H), 1.49 (q, J=7.4, 2H), 1.24(s, 3H), 1.21(s, 3H), 0.86(t, J=7.4, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₄ H ₃₀ O ₇ Na ⁺ 430.1884, found 430.1892.

Data for 2: ¹ H NMR (600MHz, Acetone-d ₆ )δ=6.09(s, 1H), 5.68(d, J=1.4, 1H), 5.59(d, J=1.3, 1H), 5.49(s, 1H), 4.96 (d, J=10.8, 1H), 4.91 (d, J=10.8, 1H), 4.58 (dt, J=14.2, 3.7, 1H), 4.02 (d, J=3.6, 1H), 3.33 (s, 1H), 2.88 (dd, J=11.7, 3.3, 1H), 2.48 (dd, J=9.3, 2.3, 1H), 2.47–2.41 (m, 1H), 2.24 (dd, J=13.9, 11.7 ,1H),2.05(s,3H),1.99–1.95(m,1H),1.92(s,1H),1.90(dd,J=13.8,3.4,1H),1.87(s,3H),1.78(s ,1H),1.66(s,1H),1.25(s,3H),1.21(s,3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₅ H ₃₀ O ₇ Na ⁺ 465.1884, found465.1889.

Data for 3: ¹ H NMR (600MHz, Acetone-d ₆ )δ=6.09(s, 1H), 5.67(d, J=1.4, 1H), 5.59(s, 1H), 4.93(d, J=10.8, 1H), 4.87 (d, J=10.8, 1H), 4.52 (dt, J=14.3, 3.7, 1H), 4.04 (d, J=3.7, 1H), 2.88 (dd, J=11.7, 3.3, 1H) , 2.49 (dd, J=9.1, 2.6, 1H), 2.47–2.40 (m, 1H), 2.27–2.09 (m, 3H), 1.99–1.93 (m, 2H), 1.90 (dd, J=13.9, 3.3 ,1H),1.82–1.73(m,1H),1.69–1.61(m,1H),1.51–1.44(m,2H),1.32–1.25(m,8H),1.24(s,3H),1.21(s , 3H), 0.87 (t, J=7.0, 3H); HRESIMS (m/z): [M+Na] ⁺ calcd for C ₂₈ H ₃₈ O ₇ Na ⁺ 509.2510, found 509.2516.

Data for 4: ¹ H NMR (600MHz, Acetone-d ₆ )δ=6.09(s, 1H), 5.67(s, 1H), 5.60(s, 1H), 4.93(d, J=10.8, 1H), 4.87 (d, J=10.8, 1H), 4.52 (dt, J=14.3, 3.5, 1H), 4.06 (d, J=3.6, 1H), 3.33 (s, 1H), 2.89 (dd, J=11.5, 3.1 , 1H), 2.49 (dd, J=9.2, 2.5, 1H), 2.45–2.39 (m, 1H), 2.26–2.08 (m, 3H), 1.98–1.93 (m, 2H), 1.90 (dd, J= 13.9, 3.2, 1H), 1.82–1.73 (m, 1H), 1.68–1.60 (m, 1H), 1.51–1.43 (m, 2H), 1.33–1.24 (m, 10H), 1.24 (s, 3H), 1.21(s, 3H), 0.90–0.85(m, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₉ H ₄₀ O ₇ Na ⁺ 523.2666, found523.2658.

Data for 5: ¹ H NMR (600MHz, Acetone-d ₆ )δ=6.10(s, 1H), 5.63(d, J=1.4, 1H), 5.61(t, J=1.0, 1H), 4.91(d, J=10.9, 1H), 4.86 (d, J=10.8, 1H), 4.52 (dd, J=14.3, 3.6, 1H), 4.11 (d, J=3.5, 1H), 3.35 (s, 1H), 2.89 –2.88(m,1H),2.48(dd,J=9.3,2.4,1H),2.46–2.39(m,1H),2.23(dd,J=13.9,11.8,1H),1.99–1.95(m,1H) ), 1.93 (d, J=14.2, 1H), 1.89 (dd, J=13.8, 3.3, 1H), 1.82–1.72 (m, 1H), 1.67–1.60 (m, 1H), 1.51–1.43 (m, 1H), 1.23(s, 3H), 1.20(s, 3H), 0.86–0.79(m, 3H), 0.76(dt, J=8.8, 4.5, 1H); HRESIMS(m/z): [M+K ] ⁺ calcd for C ₂₄ H ₂₈ O ₇ K ⁺ 467.1467, found 467.1463.

Data for 6: ¹ H NMR (600MHz, Acetone-d ₆ )δ=6.08(s, 1H), 5.64(d, J=1.4, 1H), 5.59(s, 1H), 4.94(d, J=10.8, 1H), 4.88 (d, J=10.8, 1H), 4.56 (dd, J=14.3, 3.7, 1H), 4.13 (dd, J=3.6, 1.0, 1H), 3.32 (s, 1H), 3.02–2.96 (m, 1H), 2.89–2.88 (m, 1H), 2.49 (dd, J=9.2, 2.5, 1H), 2.47–2.40 (m, 1H), 2.23 (dd, J=13.8, 11.7, 1H), 2.13–2.09 (m, 4H), 1.98–1.93 (m, 2H), 1.92–1.87 (m, 2H), 1.84–1.73 (m, 2H), 1.68–1.61 (m, 1H), 1.24 (s, 3H) ), 1.21(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₅ H ₃₀ O ₇ Na ⁺ 465.1884, found 465.1882.

Data for 7: ¹ H NMR (600 MHz, Acetone-d ₆ ) δ=6.11-6.08 (m, 1H), 5.63 (d, J=1.4, 1H), 5.59 (t, J=1.0, 1H), 4.93 ( d, J=10.8, 1H), 4.88 (d, J=10.8, 1H), 4.54 (dd, J=14.3, 3.7, 1H), 4.06 (d, J=3.7, 1H), 3.36–3.30 (m, 1H), 2.88 (dd, J=11.7, 3.3, 1H), 2.60–2.56 (m, 1H), 2.49 (dd, J=9.2, 2.5, 1H), 2.47–2.39 (m, 1H), 2.23 (dd , J=13.9, 11.7, 1H), 2.00–1.92 (m, 2H), 1.90 (dd, J=13.9, 3.3, 1H), 1.83–1.70 (m, 3H), 1.70–1.62 (m, 3H), 1.62–1.55 (m, 2H), 1.55–1.47 (m, 2H), 1.24 (s, 3H), 1.21 (s, 3H); HRESIMS (m/z): [M+Na] ⁺ calcd for C ₂₆ H ₃₂ O ₇ Na ⁺ 479.2040, found 479.2042.

Data for 8: HRESIMS(m/z): ¹ H NMR (600MHz, Acetone-d ₆ )δ=6.62(q, J=2.3, 1H), 6.10(s, 1H), 5.67(d, J=1.4, 1H), 5.60(s, 1H), 4.97(d, J=10.8, 1H), 4.90(d, J=10.8, 1H), 4.58(dd, J=14.2, 3.7, 1H), 4.06(d, J = 3.7, 1H), 3.38 (d, J = 3.9, 1H), 2.89 (dd, J = 11.7, 3.4, 1H), 2.50 (dd, J = 9.1, 2.4, 1H), 2.48–2.36 (m, 5H) ), 2.24 (dd, J=13.9, 11.7, 1H), 2.00–1.93 (m, 2H), 1.93–1.86 (m, 3H), 1.83–1.74 (m, 1H), 1.66 (s, 1H), 1.25 (s, 3H), 1.21 (s, 3H); HRESIMS (m/z): [M+Na] ⁺ calcd for C ₂₆ H ₃₀ O ₇ Na ⁺ 477.1884, found 477.1888.

Data for 9: ¹ H NMR (600MHz, Acetone-d ₆ )δ=6.10(s, 1H), 5.67(d, J=1.4, 1H), 5.61(s, 1H), 4.93(d, J=10.8, 1H), 4.86 (d, J=10.8, 1H), 4.55 (dd, J=14.3, 3.7, 1H), 4.25 (dd, J=8.7, 4.1, 1H), 4.17 (d, J=3.6, 1H) , 3.82–3.75 (m, 2H), 3.38–3.32 (m, 1H), 2.89 (dd, J=11.7, 3.4, 1H), 2.51 (dd, J=9.1, 2.7, 1H), 2.48–2.40 (m , 1H), 2.24 (dd, J=13.9, 11.7, 1H), 2.13–2.09 (m, 1H), 1.99–1.93 (m, 2H), 1.93–1.84 (m, 3H), 1.84–1.74 (m, 2H), 1.69–1.63(m, 1H), 1.24(s, 3H), 1.22(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₅ H ₃₀ O ₈ Na ⁺ 481.1833 ,found 481.1842.

Data for 10: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.81(d,J=1.8,1H),7.10(d,J=3.5,1H),6.65-6.59(m,1H),6.14( s, 1H), 5.85(s, 1H), 5.65(s, 1H), 4.98(d, J=10.8, 1H), 4.92(d, J=10.8, 1H), 4.62(dd, J=14.2, 3.4 ,1H),4.14(d,J＝3.6,1H),3.49(d,J＝3.9,1H),2.91(dd,J＝11.7,3.3,1H),2.55(dd,J＝9.1,2.7,1H) ), 2.53–2.46 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H), 2.02–1.96 (m, 2H), 1.92 (dd, J=13.9, 3.3, 1H), 1.86–1.77 ( m,1H),1.73–1.67(m,1H),1.25(s,3H),1.22(s,3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₅ H ₂₆ O ₈ Na ⁺ 477.1520, found 477.1524.

Data for 11: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=7.86 (dd, J=5.0, 1.3, 1H), 7.66 (dd, J=3.8, 1.3, 1H), 7.18 (dd, J=5.0 ,3.7,1H),6.15(s,1H),5.85(d,J=1.4,1H),5.66(s,1H),5.00(d,J=10.8,1H),4.93(d,J=10.8, 1H), 4.64 (dd, J=14.2, 3.5, 1H), 4.13 (s, 1H), 3.51 (s, 1H), 2.91 (dd, J=11.7, 3.4, 1H), 2.56 (dd, J=9.1 , 2.7, 1H), 2.54–2.46 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H), 2.03–1.97 (m, 2H), 1.92 (dd, J=13.9, 3.4, 1H), 1.87–1.77(m,1H), 1.73–1.67(m,1H), 1.26(s,3H), 1.22(s,3H); HRESIMS(m/z): [M+Na] ⁺ calcdfor C ₂₅ H ₂₆ O ₇ SNa ⁺ 493.1291, found 493.1289.

Data for 12: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.05(s, 1H), 6.71(s, 1H), 6.18(s, 1H), 6.12(s, 1H), 5.79(d, J ＝1.4,1H),5.61(d,J＝1.0,1H),4.99(d,J＝10.8,1H),4.95(d,J＝10.8,1H),4.64(dd,J＝14.2,3.6,1H) ), 4.07 (d, J=3.6, 1.1, 1H), 3.44 (d, J=3.9, 1H), 2.90 (dd, J=11.7, 3.3, 1H), 2.52 (dd, J=9.3, 2.4, 1H) ), 2.50–2.44 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H), 2.02–1.93 (m, 2H), 1.91 (dd, J=13.9, 3.4, 1H), 1.85–1.77 ( m,1H),1.70–1.65(m,1H),1.25(s,3H),1.22(s,3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₅ H ₂₇ NO ₇ Na ⁺ 476.1680, found 476.1677.

Data for 13: ¹ H NMR (600MHz, Acetone-d ₆ )δ=8.40(d, J=2.1, 1H), 7.72(d, J=2.0, 1H), 6.16(s, 1H), 5.92(s, 1H), 5.67(s, 1H), 4.97(d, J=10.8, 1H), 4.90(d, J=10.8, 1H), 4.60(dd, J=14.2, 3.7, 1H), 4.19(d, J =3.6,1H),3.52(s,1H),2.92(dd,J=11.7,3.3,1H),2.58(dd,J=9.2,3.0,1H),2.56–2.48(m,1H),2.25( dd, J=13.9, 11.7, 1H), 2.00 (dd, J=12.4, 8.1, 2H), 1.93–1.90 (m, 1H), 1.86–1.78 (m, 1H), 1.75–1.68 (m, 1H) , 1.25(s, 3H), 1.22(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₄ H ₂₅ NO ₈ Na ⁺ 456.1653, found 456.1654.

Data for 14: ¹ H NMR (600MHz, Acetone-d ₆ )δ=9.25(s, 1H), 8.34(s, 1H), 6.16(s, 1H), 5.90(s, 1H), 5.67(s, 1H) ),4.98(d,J=10.8,1H),4.91(d,J=10.9,1H),4.62(dd,J=14.2,3.6,1H),4.20(d,J=3.7,1H),3.54( d, J=3.9, 1H), 2.92 (dd, J=11.7, 3.4, 1H), 2.58 (dd, J=9.0, 2.9, 1H), 2.55–2.48 (m, 1H), 2.26 (dd, J= 13.8, 11.8, 1H), 2.03–1.99 (m, 2H), 1.92 (dd, J=13.8, 3.3, 1H), 1.86–1.78 (m, 1H), 1.71 (ddt, J=15.7, 6.6, 3.0, 1H), 1.25(s, 3H), 1.22(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₄ H ₂₅ NO ₇ SNa ⁺ 472.1424, found472.1428.

Data for 15: ¹ H NMR (600MHz, Acetone-d ₆ )δ=9.50(s, 1H), 6.16(s, 1H), 6.02(d, J=1.4, 1H), 5.68(d, J=1.2, 1H), 5.00 (d, J=10.8, 1H), 4.94 (d, J=10.8, 1H), 4.65 (dd, J=14.2, 3.5, 1H), 4.22 (d, J=3.6, 1H), 3.60 –3.58(m,1H), 2.93(dd,J=11.7,3.4,1H), 2.62–2.60(m,1H), 2.59–2.52(m,1H), 2.26(dd,J=13.8,11.7,1H) ), 2.04–2.01 (m, 2H), 1.93 (dd, J=13.9, 3.4, 1H), 1.88–1.81 (m, 1H), 1.77–1.71 (m, 1H), 1.26 (s, 3H), 1.23 (s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₃ H ₂₄ N ₂ O ₇ SNa ⁺ 495.1196, found 495.1198.

Data for 16: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=6.09(s, 1H), 5.63(s, 1H), 5.59(s, 1H), 4.93(d, J=10.8, 1H), 4.88 (d, J=10.8, 1H), 4.53 (dd, J=14.3, 3.4, 1H), 4.08 (d, J=3.6, 1H), 3.32 (d, J=4.1, 1H), 2.88 (dd, J =11.7,3.3,1H),2.80(s,1H),2.48(dd,J=9.2,2.4,1H),2.47–2.40(m,1H),2.23(dd,J=13.9,11.7,1H), 2.16–2.10 (m, 1H), 1.99–1.92 (m, 2H), 1.90 (dd, J=13.9, 3.3, 1H), 1.82–1.72 (m, 3H), 1.65 (s, 3H), 1.61–1.55 (m,1H), 1.31–1.24(m,3H), 1.24(s,3H), 1.21(s,3H), 1.20–1.13(m,1H); HRESIMS(m/z): [M–H] ^– calcd for C ₂₇ H ₃₃ O ₇ ^– 469.2232, found 469.2233.

Data for 17: ¹ H NMR (600MHz, Acetone-d ₆ )δ=6.82-6.77(m, 1H), 6.10(s, 1H), 5.67(d, J=1.4, 1H), 5.60(s, 1H) ,4.97(d,J＝10.8,1H),4.90(d,J＝10.8,1H),4.58(dd,J＝14.3,3.7,1H),4.06(d,J＝3.7,1H),3.38(d , J=4.1, 1H), 2.88 (dd, J=11.7, 3.4, 1H), 2.50 (dd, J=9.2, 2.3, 1H), 2.47–2.41 (m, 1H), 2.24 (dd, J=13.9 , 11.7, 1H), 2.17–2.12 (m, 2H), 2.08–2.06 (m, 2H), 2.00–1.93 (m, 2H), 1.90 (dd, J=13.9, 3.4, 1H), 1.83–1.74 ( m, 1H), 1.69–1.62 (m, 1H), 1.61–1.51 (m, 4H), 1.25 (s, 3H), 1.21 (s, 3H); HRESIMS (m/z): [M+Na] ⁺ calcd for C ₂₇ H ₃₂ O ₇ Na ⁺ 491.2040, found 491.2041.

Data for 18: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.86-7.82(m, 2H), 7.66-7.62(m, 1H), 7.51-7.47(m, 2H), 6.16(s, 1H) ,5.90(d,J＝1.5,1H),5.66(s,1H),5.02(d,J＝10.8,1H),4.96(d,J＝10.8,1H),4.65(dt,J＝14.3,3.7 ,1H),4.14(d,J=3.5,1H),3.53(s,1H),2.92(dd,J=11.7,3.4,1H),2.57(dd,J=9.1,2.5,1H),2.55– 2.48 (m, 1H), 2.26 (dd, J=13.8, 11.8, 1H), 2.00 (d, J=14.3, 2H), 1.92 (dd, J=13.9, 3.3, 1H), 1.88–1.79 (m, 1H), 1.74–1.68(m, 1H), 1.26(s, 3H), 1.22(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₇ H ₂₈ O ₇ Na ⁺ 478.1727 ,found 478.1726.

Data for 19: ¹ H NMR (800MHz, Acetone-d ₆ )δ=8.80-8.76(m, 2H), 7.69-7.65(m, 2H), 6.18(s, 1H), 5.95(d, J=1.4, 1H), 5.68 (d, J=1.0, 1H), 5.00 (d, J=10.7, 1H), 4.94 (d, J=10.7, 1H), 4.63 (dd, J=14.2, 3.6, 1H), 4.21 (d, J=3.6, 1H), 3.57 (dt, J=4.8, 2.3, 1H), 2.93 (dd, J=11.7, 3.4, 1H), 2.60 (dd, J=9.3, 3.0, 1H), 2.56 –2.50(m,1H),2.26(dd,J=13.9,11.8,1H),2.04–2.02(m,2H),1.93(dd,J=13.9,3.4,1H),1.85–1.84(m,1H) ), 1.74–1.72(m, 1H), 1.26(s, 3H), 1.23(s, 3H); HRESIMS(m/z): [M+H]+calcd for C ₂₆ H ₂₈ NO ₇ +466.1860, found 466.1863.

Data for 20: ¹ H NMR (800MHz, Acetone-d ₆ ) δ=9.01 (d, J=1.5, 1H), 8.84 (d, J=2.4, 1H), 8.75 (dd, J=2.3, 1.5, 1H) ), 6.18(s, 1H), 6.00(d, J=1.4, 1H), 5.69(s, 1H), 5.00(d, J=10.7, 1H), 4.95(d, J=10.8, 1H), 4.64 (dd, J=14.2, 3.6, 1H), 4.24 (d, J=3.5, 1H), 3.58 (d, J=3.1, 1H), 2.93 (dd, J=11.8, 3.4, 1H), 2.61 (d , J=7.7, 1H), 2.57–2.52 (m, 1H), 2.27 (dd, J=13.9, 11.8, 1H), 2.04–2.02 (m, 2H), 1.93 (dd, J=13.9, 3.4, 1H) ), 1.87–1.82(m, 1H), 1.76–1.72(m, 1H), 1.26(s, 3H), 1.23(s, 3H); HRESIMS(m/z): [M+H] ⁺ calcd forC ₂₅ H ₂₇ N ₂ O ₇ ⁺ 467.1813, found 467.1815.

Data for 21: ¹ H NMR (800MHz, Acetone-d ₆ ) δ=9.35(s, 1H), 9.06(s, 2H), 6.19-6.17(m, 1H), 5.98(d, J=1.4, 1H) ,5.68(d,J＝1.0,1H),4.99(d,J＝10.7,1H),4.93(d,J＝10.7,1H),4.64–4.61(m,1H),4.27(s,1H), 3.60 (s, 1H), 2.93 (dd, J=11.8, 3.4, 1H), 2.61 (d, J=5.8, 1H), 2.56–2.51 (m, 1H), 2.26 (dd, J=13.9, 11.7, 1H), 2.04–2.01 (m, 2H), 1.92 (dd, J=13.9, 3.4, 1H), 1.87–1.81 (m, 1H), 1.75–1.71 (m, 1H), 1.25 (s, 3H), 1.22(s,3H); HRESIMS(m/z): [M+H] ⁺ calcd for C ₂₅ H ₂₇ N ₂ O ₇ ⁺ 467.1813, found 467.1817.

Data for 22: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.96-7.84(m, 2H), 7.34-7.18(m, 2H), 6.16(s, 1H), 5.88(s, 1H), 5.66 (s, 1H), 5.01 (d, J=10.8, 1H), 4.95 (d, J=10.7, 1H), 4.68–4.58 (m, 1H), 4.17 (d, J=3.7, 1H), 3.53 ( s, 1H), 2.98 (s, 1H), 2.57 (dd, J=9.2, 2.6, 1H), 2.54–2.46 (m, 1H), 2.25 (dd, J=13.9, 11.8, 1H), 2.00 (d , J=14.3, 2H), 1.91 (dd, J=13.8, 3.3, 1H), 1.88–1.76 (m, 1H), 1.75–1.66 (m, 1H), 1.25 (s, 3H), 1.22 (s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₇ H ₂₇ FO ₇ Na ⁺ 505.1633, found 505.1632.

Data for 23: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.85-7.80(m, 2H), 7.57-7.52(m, 2H), 6.16(s, 1H), 5.90(d, J=1.4, 1H), 5.66(s, 1H), 5.01(d, J=10.8, 1H), 4.94(d, J=10.8, 1H), 4.64(d, J=14.2, 1H), 4.19(s, 1H), 3.53 (s, 1H), 2.94–2.92 (m, 1H), 2.57 (dd, J=9.1, 2.6, 1H), 2.54–2.48 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H) , 2.03–1.98 (m, 2H), 1.91 (dd, J=13.9, 3.3, 1H), 1.87–1.79 (m, 1H), 1.74–1.68 (m, 1H), 1.25 (s, 3H), 1.22 ( s, 3H); HRESIMS(m/z): [M–H] ^– calcd for C ₂₇ H ₂₆ ClO ₇ ^– 497.1373, found 497.1372.

Data for 24: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.78-7.73(m, 2H), 7.73-7.68(m, 2H), 6.16(s, 1H), 5.90(s, 1H), 5.66 (s, 1H), 5.00 (d, J=10.8, 1H), 4.94 (d, J=10.8, 1H), 4.63 (dd, J=14.2, 3.6, 1H), 4.16 (d, J=3.6, 1H) ), 3.53(s, 1H), 2.92(dd, J=11.8, 3.3, 1H), 2.57(dd, J=9.1, 2.6, 1H), 2.55–2.48(m, 1H), 2.25(dd, J= 13.9, 11.8, 1H), 2.03–1.98 (m, 2H), 1.92 (dd, J=13.9, 3.3, 1H), 1.87–1.79 (m, 1H), 1.74–1.68 (m, 1H), 1.25 (s ,3H),1.22(s,3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₇ H ₂₇ BrO ₇ Na ⁺ 565.0832, found 565.0826.

Data for 25: ¹ H NMR (600MHz, Acetone-d ₆ )δ=8.02(d, J=8.1, 2H), 7.86(d, J=8.1, 2H), 6.16(s, 1H), 5.94(s, 1H), 5.67(s, 1H), 5.00(d, J=10.7, 1H), 4.94(d, J=10.8, 1H), 4.63(dt, J=14.3, 3.5, 1H), 4.19(d, J = 3.5, 1H), 3.56 (d, J = 3.8, 1H), 2.92 (dd, J = 11.8, 3.3, 1H), 2.58 (d, J = 8.6, 1H), 2.55–2.50 (m, 1H), 2.25 (dd, J=13.8, 11.8, 1H), 2.03–1.99 (m, 2H), 1.91 (dd, J=14.0, 3.3, 1H), 1.86–1.80 (m, 1H), 1.74–1.69 (m, 1H), 1.25(s, 3H), 1.21(s, 3H); HRESIMS(m/z): [M–H] ^– calcd for C ₂₇ H ₂₆ NO ₉ ^– 508.1613, found 508.1613.

Data for 26: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.80-7.77(m, 2H), 7.02-6.98(m, 2H), 6.15(s, 1H), 5.84(d, J=1.4, 1H), 5.65(s, 1H), 5.02(d, J=10.8, 1H), 4.96(d, J=10.8, 1H), 4.65(dd, J=14.2, 3.3, 1H), 4.12(d, J =3.5,1H),3.87(s,3H),3.50(d,J=3.9,1H),2.91(dd,J=11.7,3.3,1H),2.55(dd,J=9.2,2.3,1H), 2.53–2.47 (m, 1H), 2.25 (dd, J=13.9, 11.8, 1H), 2.03–1.96 (m, 2H), 1.91 (dd, J=13.9, 3.3, 1H), 1.86–1.78 (m, 1H),1.72–1.67(m,1H),1.26(s,3H),1.22(s,3H); HRESIMS(m/z):[M–H] ^– calcd for C ₂₈ H ₂₉ O ₈ ^– 493.1868, found 493.1865.

Data for 27: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.72(d, J=7.9, 2H), 7.30(d, J=7.9, 2H), 6.16(s, 1H), 5.87(s, 1H), 5.65 (d, J=1.3, 1H), 5.02 (d, J=10.8, 1H), 4.96 (d, J=10.8, 1H), 4.65 (dd, J=14.3, 3.3, 1H), 4.14 (d, J=3.5, 1H), 3.51 (d, J=3.8, 1H), 2.92 (dd, J=11.8, 3.2, 1H), 2.56 (dd, J=9.1, 2.3, 1H), 2.54–2.48 (m, 1H), 2.39 (s, 3H), 2.25 (dd, J=13.8, 11.8, 1H), 2.03–1.97 (m, 2H), 1.92 (dd, J=13.9, 3.3, 1H), 1.86– 1.80(m,1H),1.73–1.68(m,1H),1.26(s,3H),1.22(s,3H); HRESIMS(m/z): [M–H] ^– calcd for C ₂₈ H ₂₉ O ₇ ^– 477.1919, found 477.1919.

Data for 28: ¹ H NMR (600MHz, Acetone-d ₆ )δ=8.38-8.34(m, 2H), 8.10-8.05(m, 2H), 6.18(s, 1H), 5.97(s, 1H), 5.69 (s, 1H), 5.01 (d, J=10.8, 1H), 4.94 (d, J=10.8, 1H), 4.64 (dt, J=14.2, 3.7, 1H), 4.22 (d, J=3.7, 1H) ), 3.59 (d, J=3.8, 1H), 2.93 (dd, J=11.7, 3.3, 1H), 2.60–2.59 (m, 1H), 2.57–2.50 (m, 1H), 2.26 (dd, J= 13.9, 11.8, 1H), 2.03–2.00 (m, 2H), 1.92 (dd, J=13.9, 3.3, 1H), 1.88–1.80 (m, 1H), 1.76–1.70 (m, 1H), 1.26 (s ,3H),1.22(s,3H); HRESIMS(m/z): [M–H] ^– calcd for C ₂₈ H ₂₆ F ₃ O ₇ ^– 531.1636, found 531.1629.

Data for 29: ¹ H NMR (800MHz, Acetone-d ₆ ) δ=8.43(d, J=5.1, 1H), 7.63(dt, J=5.1, 1.5, 1H), 7.34(s, 1H), 6.18( s, 1H), 5.96 (d, J=1.4, 1H), 5.68 (d, J=1.0, 1H), 4.99 (d, J=10.8, 1H), 4.92 (d, J=10.7, 1H), 4.63 –4.60(m,1H),4.24(d,J=3.6,1H),3.58(s,1H),2.93(dd,J=11.8,3.4,1H),2.62–2.60(m,1H),2.55– 2.51 (m, 1H), 2.26 (dd, J=13.9, 11.7, 1H), 2.04–2.00 (m, 2H), 1.92 (dd, J=13.9, 3.4, 1H), 1.87–1.81 (m, 1H) ,1.76–1.71(m,1H),1.25(s,3H),1.22(s,3H); HRESIMS(m/z): [M–H] ^– calcd for C ₂₆ H ₂₅ FNO ₇ ^– 482.1621, found482. 1623.

Data for 30: ¹ H NMR (800MHz, Acetone-d ₆ ) δ=8.66 (d, J=2.3, 1H), 8.58 (d, J=4.8, 1H), 7.62 (dd, J=6.1, 4.9, 1H) ), 6.15(s, 1H), 5.99(d, J=1.5, 1H), 5.67(d, J=1.0, 1H), 4.98(d, J=10.7, 1H), 4.91(d, J=10.7, 1H), 4.60 (dd, J=14.2, 3.6, 1H), 4.23 (d, J=3.7, 1H), 3.54 (s, 1H), 2.93 (dd, J=11.7, 3.4, 1H), 2.60–2.58 (m, 1H), 2.56–2.50 (m, 1H), 2.26 (dd, J=13.9, 11.8, 1H), 2.01 (dd, J=7.5, 3.5, 2H), 1.92 (dd, J=13.9, 3.4 ,1H),1.86–1.80(m,1H),1.76–1.71(m,1H),1.26(s,3H),1.22(s,3H); HRESIMS(m/z):[M–H] ^– calcd forC ₂₆ H ₂₅ FNO ₇ ^– 482.1621, found 482.1624.

Data for 31: ¹ H NMR (600MHz, Acetone-d ₆ )δ=8.60(d, J=5.0, 1H), 7.68(dd, J=6.6, 1.5, 2H), 6.18(s, 1H), 5.96( d, J=1.5, 1H), 5.68 (s, 1H), 5.62 (d, J=1.7, 1H), 4.98 (d, J=10.8, 1H), 4.91 (d, J=10.8, 1H), 4.61 (dd, J=14.2, 3.4, 1H), 4.25 (d, J=3.6, 1H), 3.58 (t, J=3.7, 1H), 2.93 (dd, J=11.7, 3.3, 1H), 2.64–2.59 (m, 1H), 2.57–2.48 (m, 1H), 2.26 (dd, J=13.9, 11.7, 1H), 2.03–1.99 (m, 1H), 1.92 (dd, J=13.8, 3.3, 1H), 1.84(ddt,J=16.4,8.6,4.5,1H),1.73(ddt,J=15.8,6.6,3.0,1H),1.25(s,3H),1.22(d,J=2.7,3H); HRESIMS( m/z):[M–H] ^– calcd for C ₂₆ H ₂₅ ClNO ₇ ^– 498.1325, found 498.1325.

Data for 32: ¹ H NMR (600MHz, Acetone-d ₆ )δ=8.71(s, 1H), 8.65(d, J=4.9, 1H), 7.57(d, J=4.9, 1H), 6.13(s, 1H), 6.00(s, 1H), 5.65(s, 1H), 4.99(d, J=10.8, 1H), 4.93(d, J=10.8, 1H), 4.61(dd, J=14.2, 3.5, 1H) ), 4.30 (dt, J=3.4, 1.6, 1H), 3.54 (d, J=4.1, 1H), 2.93 (dd, J=11.8, 3.3, 1H), 2.62–2.58 (m, 1H), 2.57– 2.51 (m, 1H), 2.25 (dd, J=13.8, 11.7, 1H), 2.03–1.98 (m, 2H), 1.91 (dd, J=13.8, 3.3, 1H), 1.86–1.79 (m, 1H) , 1.76–1.70(m, 1H), 1.25(s, 3H), 1.22(s, 3H); HRESIMS(m/z): [M–H] ^– calcd for C ₂₆ H ₂₅ ClNO ₇ ^– 498.1325, found498. 1324.

Data for 33: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=8.84 (d, J=2.4, 1H), 8.68 (dd, J=5.4, 2.3, 1H), 7.55 (d, J=4.7, 1H) ),6.13(s,1H),6.00(s,1H),5.65(d,J=2.5,1H),4.99(dd,J=10.8,2.4,1H),4.94(dd,J=10.8,2.5, 1H), 4.64–4.57 (m, 1H), 4.34 (s, 1H), 3.54 (s, 1H), 2.95–2.92 (m, 1H), 2.60 (d, J=7.9, 1H), 2.57–2.51 ( m, 1H), 2.29–2.21 (m, 1H), 2.02–1.97 (m, 2H), 1.91 (dd, J=13.8, 3.2, 1H), 1.86–1.77 (m, 1H), 1.75–1.69 (m ,1H),1.25(s,3H),1.22(s,3H); HRESIMS(m/z): [M+K] ⁺ calcd for C ₂₆ H ₂₆ BrNO ₇ K ⁺ 582.0524, found582.0528.

Data for 34: ¹ H NMR (600 MHz, Acetone-d ₆ ) δ=8.58 (d, J=5.0, 1H), 7.82 (d, J=1.4, 1H), 7.70 (dd, J=5.0, 1.3, 1H) ), 6.17(s, 1H), 5.94(s, 1H), 5.68(s, 1H), 4.98(d, J=10.8, 1H), 4.91(d, J=10.8, 1H), 4.60(dd, J = 14.3, 3.2, 1H), 4.31 (d, J = 3.4, 1H), 3.58–3.55 (m, 1H), 2.95–2.92 (m, 1H), 2.60–2.59 (m, 1H), 2.55–2.48 ( m, 1H), 2.28–2.22 (m, 1H), 2.03–1.99 (m, 2H), 1.90 (dd, J=13.9, 3.3, 1H), 1.85–1.79 (m, 1H), 1.74–1.68 (m ,1H),1.23(s,3H),1.21(s,3H); HRESIMS(m/z): [M+K] ⁺ calcd for C ₂₆ H ₂₆ BrNO ₇ K ⁺ 582.0524, found582.0527.

Data for 35: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.29-7.25(m, 2H), 7.21-7.17(m, 3H), 6.09(s, 1H), 5.67(d, J=1.4, 1H), 5.58(s, 1H), 4.92(d, J=10.8, 1H), 4.86(d, J=10.8, 1H), 4.51(dd, J=14.3, 3.5, 1H), 4.07(d, J = 3.5, 1H), 3.28 (d, J = 4.1, 1H), 2.91–2.88 (m, 1H), 2.82–2.79 (m, 2H), 2.57–2.45 (m, 3H), 2.45–2.40 (m, 1H), 2.23 (dd, J=13.8, 11.7, 1H), 1.98–1.92 (m, 2H), 1.90 (dd, J=13.9, 3.3, 1H), 1.81–1.73 (m, 1H), 1.67–1.62 (m, 1H), 1.24(s, 3H), 1.21(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₉ H ₃₂ O ₇ Na ⁺ 515.2046, found 515.2040.

Data for 36: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=7.70-7.64 (m, 2H), 7.58 (d, J=16.0, 1H), 7.44 (dd, J=5.1, 1.9, 3H), 6.41 (d, J=16.0, 1H), 6.14 (s, 1H), 5.79 (d, J=1.4, 1H), 5.64 (d, J=1.0, 1H), 4.98 (d, J=10.8, 1H) , 4.93 (d, J=10.8, 1H), 4.61 (dd, J=14.3, 3.5, 1H), 4.13 (d, J=3.5, 1H), 3.45–3.40 (m, 1H), 2.90 (dd, J = 11.7, 3.3, 1H), 2.53 (dd, J=9.2, 2.6, 1H), 2.51–2.45 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H), 2.02–1.95 (m, 2H) ), 1.91 (dd, J=13.8, 3.4, 1H), 1.85–1.77 (m, 1H), 1.71–1.65 (m, 1H), 1.26 (s, 3H), 1.22 (s, 3H); /z):[M+Na] ⁺ calcd for C ₂₉ H ₃₀ O ₇ Na ⁺ 513.1893, found 513.1884.

Data for 37: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.52-7.49(m,1H),7.45-7.36(m,5H),6.14(s,1H),5.77(d,J=1.4, 1H), 5.66–5.62 (m, 1H), 5.00 (d, J=10.8, 1H), 4.94 (d, J=10.8, 1H), 4.63 (dd, J=14.2, 3.6, 1H), 4.16 (d , J=3.7, 1H), 3.47 (d, J=3.9, 1H), 2.92 (d, J=3.4, 1H), 2.54 (dd, J=9.1, 2.4, 1H), 2.51–2.45 (m, 1H) ), 2.25 (dd, J=13.8, 11.8, 1H), 2.02–1.99 (m, 2H), 1.99–1.98 (m, 3H), 1.92 (dd, J=13.9, 3.4, 1H), 1.86–1.77 ( m,1H),1.72–1.66(m,1H),1.26(s,3H),1.22(s,3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₃₀ H ₃₂ O ₇ Na ⁺ 527.2040, found 527.2049.

Data for 38: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=7.81-7.74(m, 2H), 7.57(d, J=16.0, 1H), 7.21(t, J=8.7, 2H), 6.37( d, J=16.0, 1H), 6.13 (s, 1H), 5.79 (s, 1H), 5.64 (s, 1H), 4.97 (d, J=10.8, 1H), 4.93 (d, J=10.8, 1H) ), 4.60 (dd, J=14.3, 3.3, 1H), 4.12 (d, J=3.6, 1H), 3.42 (s, 1H), 2.91 (d, J=3.3, 1H), 2.53 (dd, J= 9.2, 2.6, 1H), 2.51–2.44 (m, 1H), 2.25 (dd, J=13.8, 11.8, 1H), 2.01–1.95 (m, 2H), 1.91 (dd, J=13.9, 3.3, 1H) ,1.85–1.76(m,1H),1.71–1.65(m,1H),1.25(s,3H),1.22(s,3H); HRESIMS(m/z): [M+Na] ⁺ calcd forC ₂₉ H ₂₉ FO ₇ Na ⁺ 531.1790, found 531.1793.

Data for 39: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=7.72(d, J=8.4, 2H), 7.56(d, J=16.0, 1H), 7.50-7.43(m, 2H), 6.43( d, J=16.0, 1H), 6.13 (s, 1H), 5.79 (d, J=1.4, 1H), 5.64 (s, 1H), 4.97 (d, J=10.8, 1H), 4.92 (d, J = 10.8, 1H), 4.60 (dd, J = 14.3, 3.2, 1H), 4.13 (d, J = 3.6, 1H), 3.42 (d, J = 4.1, 1H), 2.92–2.90 (m, 1H), 2.53 (dd, J=9.1, 2.6, 1H), 2.51–2.44 (m, 1H), 2.25 (dd, J=13.9, 11.8, 1H), 2.01–1.95 (m, 2H), 1.91 (dd, J= 13.9, 3.3, 1H), 1.84–1.77 (m, 1H), 1.71–1.65 (m, 1H), 1.25 (s, 3H), 1.22 (s, 3H); HRESIMS (m/z): [M+Na ] ⁺ calcd for C ₂₉ H ₂₉ ClO ₇ Na ⁺ 547.1494, found 547.1499.

Data for 40: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.65(d,J=8.6,2H),7.62(d,J=8.6,2H),7.55(d,J=16.0,1H), 6.45(d,J=16.0,1H),6.13(s,1H),5.79(d,J=1.4,1H),5.64(s,1H),4.97(d,J=10.8,1H),4.92(d , J=10.8, 1H), 4.60 (dd, J=14.3, 3.6, 1H), 4.13 (d, J=3.6, 1H), 3.42 (d, J=3.9, 1H), 2.90 (dd, J=11.7 , 3.4, 1H), 2.53 (dd, J=9.2, 2.6, 1H), 2.51–2.44 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H), 2.01–1.95 (m, 2H), 1.91 (dd, J=13.9, 3.3, 1H), 1.84–1.76 (m, 1H), 1.71–1.65 (m, 1H), 1.25 (s, 3H), 1.22 (s, 3H); HRESIMS (m/z ):[M+Na] ⁺ calcd for C ₂₉ H ₂₉ BrO ₇ Na ⁺ 591.0989, found 591.0990.

Data for 41: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=8.31-8.26(m, 2H), 8.01-7.97(m, 2H), 7.68(d, J=16.1, 1H), 6.64(d, J=16.0, 1H), 6.14(s, 1H), 5.82(d, J=1.4, 1H), 5.65(t, J=1.0, 1H), 4.97(d, J=10.8, 1H), 4.92(d , J＝10.8, 1H), 4.60(dd, J＝14.3, 3.7, 1H), 4.14(d, J＝3.7, 1H), 3.44(s, 1H), 2.93–2.90(m, 1H), 2.54( dd, J=9.1, 2.7, 1H), 2.52–2.46 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H), 2.02–1.96 (m, 2H), 1.92 (dd, J=13.9, 3.3, 1H), 1.85–1.77 (m, 1H), 1.72–1.66 (m, 1H), 1.26 (s, 3H), 1.22 (s, 3H); HRESIMS (m/z): [M+Na] ⁺ calcd for C ₂₉ H ₂₉ NO ₉ Na ⁺ 558.1735, found 558.1735.

Data for 42: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.65-7.59(m, 2H), 7.52(d, J=15.9, 1H), 7.00-6.96(m, 2H), 6.23(d, J=15.9, 1H), 6.12(s, 1H), 5.76(d, J=1.4, 1H), 5.62(d, J=1.1, 1H), 4.96(d, J=10.8, 1H), 4.92(d , J=10.8, 1H), 4.60 (dd, J=14.3, 3.6, 1H), 4.11 (d, J=3.6, 1H), 3.84 (s, 3H), 3.39 (d, J=3.9, 1H), 2.91–2.88 (m, 1H), 2.51 (dd, J=9.2, 2.6, 1H), 2.48–2.43 (m, 1H), 2.23 (dd, J=13.9, 11.8, 1H), 2.00–1.93 (m, 2H), 1.92–1.88 (m, 1H), 1.83–1.75 (m, 1H), 1.69–1.64 (m, 1H), 1.24 (s, 3H), 1.21 (s, 3H); HRESIMS (m/z) :[M+Na] ⁺ calcd for C ₃₀ H ₃₂ O ₈ Na ⁺ 543.1995, found 543.1989.

Data for 43: ¹ H NMR (600 MHz, Acetone-d ₆ ) δ=7.58-7.52 (m, 3H), 7.25 (d, J=7.9, 2H), 6.34 (d, J=16.0, 1H), 6.13 ( s, 1H), 5.78 (d, J=1.4, 1H), 5.64 (d, J=1.1, 1H), 4.97 (d, J=10.8, 1H), 4.93 (d, J=10.8, 1H), 4.61 (dd, J=14.3, 3.6, 1H), 4.13 (d, J=3.6, 1H), 3.43–3.40 (m, 1H), 2.92–2.89 (m, 1H), 2.52 (dd, J=9.2, 2.6 ,1H),2.50–2.44(m,1H),2.36(s,3H),2.25(dd,J=13.9,11.8,1H),2.01–1.95(m,2H),1.91(dd,J=13.9, 3.4, 1H), 1.83–1.77 (m, 1H), 1.70–1.65 (m, 1H), 1.25 (s, 3H), 1.22 (s, 3H); HRESIMS (m/z): [M+Na] ⁺ calcd for C ₃₀ H ₃₂ O ₇ Na ⁺ 527.2040, found 527.2035.

Data for 44: ¹ H NMR (600 MHz, Acetone-d ₆ ) δ=7.51 (d, J=15.9, 1H), 7.33 (d, J=2.0, 1H), 7.17 (dd, J=8.3, 2.0, 1H) ), 6.98(d, J=8.3, 1H), 6.28(d, J=15.9, 1H), 6.13(s, 1H), 5.77(d, J=1.3, 1H), 5.64(s, 1H), 4.95 (q, J=10.8, 2H), 4.61 (dd, J=14.3, 3.6, 1H), 4.12 (d, J=3.6, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.40 ( s, 1H), 2.92–2.89 (m, 1H), 2.52 (dd, J=9.1, 2.4, 1H), 2.50–2.44 (m, 1H), 2.24 (dd, J=13.9, 11.8, 1H), 2.01 –1.93(m,2H),1.91(dd,J=13.9,3.3,1H),1.84–1.76(m,1H),1.70–1.65(m,1H),1.25(s,3H),1.22(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₃₁ H ₃₄ O ₉ Na ⁺ 573.2095, found 573.2091.

Data for 45: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.99(s, 1H), 7.64(d, J=1.9, 1H), 7.51(d, J=15.8, 1H), 6.91(d, J=1.9, 1H), 6.16–6.11 (m, 2H), 5.76 (s, 1H), 5.64–5.62 (m, 1H), 4.99–4.90 (m, 2H), 4.59 (dd, J=14.3, 3.6 , 1H), 4.11(d, J=3.4, 1H), 3.42–3.37 (m, 1H), 2.92–2.89 (m, 1H), 2.51 (dd, J=9.3, 2.6, 1H), 2.50–2.44 ( m, 1H), 2.24 (dd, J=13.8, 11.7, 1H), 1.97 (dd, J=17.4, 13.0, 2H), 1.91 (dd, J=13.9, 3.3, 1H), 1.83–1.76 (m, 1H), 1.71–1.65(m, 1H), 1.25(s, 3H), 1.22(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₇ H ₂₈ O ₈ Na ⁺ 503.1686 ,found 503.1676.

Data for 46: ¹ H NMR (600 MHz, Acetone-d ₆ ) δ=8.65 (d, J=5.2, 2H), 7.64-7.59 (m, 2H), 7.53 (d, J=16.1, 1H), 6.65 ( d, J=16.0, 1H), 6.14 (s, 1H), 5.81 (d, J=1.4, 1H), 5.65 (t, J=1.0, 1H), 4.97 (d, J=10.8, 1H), 4.92 (d, J=10.8, 1H), 4.60 (dd, J=14.3, 3.6, 1H), 4.23–4.18 (m, 1H), 3.44 (s, 1H), 2.94–2.90 (m, 1H), 2.54 ( dd, J=9.1, 2.7, 1H), 2.53–2.44 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H), 2.03–1.95 (m, 2H), 1.91 (dd, J=13.9, 3.3, 1H), 1.86–1.75 (m, 1H), 1.72–1.64 (m, 1H), 1.25 (s, 3H), 1.22 (s, 4H); HRESIMS (m/z): [M+H] ⁺ calcd for C ₂₇ H ₃₀ NO ₇ ⁺ 492.2018, found 492.2017.

Data for 47: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.22-7.15(m, 2H), 7.15-7.09(m, 2H), 6.11(s, 1H), 5.70(s, 1H), 5.61 (s, 1H), 4.98–4.91 (m, 1H), 4.91–4.85 (m, 1H), 4.59–4.52 (m, 1H), 4.18–4.13 (m, 1H), 3.37 (s, 1H), 3.25 –3.18(m,1H), 3.12–3.04(m,4H), 2.92–2.88(m,1H), 2.54–2.49(m,1H), 2.49–2.41(m,1H), 2.28–2.20(m, 1H), 2.00–1.93 (m, 2H), 1.92–1.88 (m, 1H), 1.84–1.74 (m, 1H), 1.69–1.63 (m, 1H), 1.23 (s, 3H), 1.21 (s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₃₀ H ₃₂ O ₇ Na ⁺ 537.1884, found 537.1893.

Data for 48: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.86-7.79(m, 1H), 7.63(d, J=8.4, 1H), 7.57-7.50(m, 2H), 7.38(t, J=7.4, 1H), 6.18 (s, 1H), 5.95 (s, 1H), 5.73–5.67 (m, 1H), 5.01 (d, J=10.8, 1H), 4.95 (d, J=10.7, 1H) ), 4.65 (dd, J=14.2, 3.5, 1H), 4.23 (d, J=3.6, 1H), 3.56 (d, J=3.8, 1H), 2.93 (dd, J=11.8, 3.3, 1H), 2.60–2.57 (m, 1H), 2.56–2.50 (m, 1H), 2.26 (dd, J=13.8, 11.8, 1H), 2.04–1.98 (m, 2H), 1.92 (dd, J=14.0, 3.3, 1H), 1.88–1.79(m, 1H), 1.75–1.69(m, 1H), 1.26(s, 3H), 1.22(s, 3H); HRESIMS(m/z): [M+K] ⁺ calcd forC ₂₉ H ₂₈ O ₈ K ⁺ 543.1416, found 543.1426.

Data for 49: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.69(d,J=8.1,1H),7.48(d,J=8.3,1H),7.31-7.27(m,1H),7.13- 7.08(m, 1H), 7.06(d, J=2.2, 1H), 6.17(s, 1H), 5.92(s, 1H), 5.67(s, 1H), 5.01(d, J=10.8, 1H), 4.97 (d, J=10.8, 1H), 4.65 (dd, J=14.2, 3.6, 1H), 4.14 (dd, J=3.6, 1.5, 1H), 3.55–3.51 (m, 1H), 2.92 (dd, J=11.7, 3.3, 1H), 2.56 (dd, J=9.2, 2.6, 1H), 2.54–2.48 (m, 1H), 2.26 (dd, J=13.9, 11.8, 1H), 2.03–1.97 (m, 2H), 1.92 (dd, J=13.9, 3.3, 1H), 1.87–1.80 (m, 1H), 1.73–1.68 (m, 1H), 1.26 (s, 3H), 1.22 (s, 3H); HRESIMS ( m/z):[M+Na] ⁺ calcd for C ₂₉ H ₂₉ NO ₇ Na ⁺ 526.1836, found 526.1839.

Data for 50: ¹ H NMR (600MHz, Acetone-d ₆ )δ=8.03(d, J=8.1, 1.3, 2H), 8.00(s, 1H), 7.57-7.52(m, 1H), 7.51-7.46( m, 1H), 6.19(s, 1H), 5.92(s, 1H), 5.69(s, 1H), 5.02(d, J=10.8, 1H), 4.94(d, J=10.8, 1H), 4.66( dd, J=14.2, 3.4, 1H), 4.21 (d, J=3.4, 1H), 3.56 (s, 1H), 2.95–2.92 (m, 1H), 2.58 (dd, J=9.2, 2.7, 1H) , 2.56–2.49 (m, 1H), 2.26 (dd, J=13.9, 11.8, 1H), 2.04–1.98 (m, 2H), 1.92 (dd, J=13.9, 3.3, 1H), 1.87–1.79 (m ,1H),1.75–1.69(m,1H),1.26(s,3H),1.22(s,3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₉ H ₂₈ O ₇ SNa ⁺ 543.1448, found 543.1449.

Data for 51: ¹ H NMR (800 MHz, Acetone-d ₆ ) δ=8.56 (d, J=6.9, 1H), 8.22 (s, 1H), 7.56 (d, J=9.2, 1H), 7.38–7.31 ( m, 1H), 6.99(t, J=6.7, 1H), 6.16(s, 1H), 5.90(s, 1H), 5.66(s, 1H), 5.02(d, J=10.8, 1H), 4.98( d, J=10.8, 1H), 4.67 (dd, J=14.2, 3.5, 1H), 4.15 (d, J=3.6, 1H), 3.51 (s, 1H), 2.93–2.91 (m, 1H), 2.56 (dd, J=9.1, 2.5, 1H), 2.54–2.50 (m, 1H), 2.25 (dd, J=13.9, 11.8, 1H), 1.99–1.96 (m, 2H), 1.92 (dd, J=13.9 ,3.4,1H),1.86–1.80(m,1H),1.73–1.69(m,1H),1.26(s,3H),1.22(s,3H); HRESIMS(m/z):[M+Na] ⁺ calcd for C ₂₈ H ₂₈ N ₂ O ₇ Na ⁺ 527.1792, found 527.1789.

Data for 52: ¹ H NMR (600MHz, Acetone-d ₆ )δ=9.08 (dd, J=7.0, 1.8, 1H), 8.80 (dd, J=4.1, 1.8, 1H), 8.31 (s, 1H), 7.30(dd, J=7.0, 4.1, 1H), 6.16(s, 1H), 5.88(s, 1H), 5.65(s, 1H), 5.04(d, J=10.8, 1H), 4.99(d, J = 10.8, 1H), 4.70 (dd, J = 14.3, 3.6, 1H), 4.10 (d, J = 3.4, 1H), 3.52 (s, 1H), 2.93–2.92 (m, 1H), 2.55 (dd, J=9.3, 2.5, 1H), 2.53–2.48 (m, 1H), 2.26 (dd, J=13.8, 11.8, 1H), 2.03–1.96 (m, 2H), 1.92 (dd, J=13.8, 3.4, 1H), 1.87–1.79 (m, 1H), 1.74–1.68 (m, 1H), 1.27 (s, 3H), 1.22 (s, 3H); HRESIMS (m/z): [M+Na] ⁺ calcd for C _27H27N3O7Na ⁺ _528.1741 , _{found 528.1738} .

Data for 53: ¹ H NMR (600MHz, Acetone-d ₆ )δ=8.56(s, 1H), 6.15(s, 1H), 6.01(d, J=1.4, 1H), 5.67(s, 1H), 5.01 (d, J=10.8, 1H), 4.95 (d, J=10.8, 1H), 4.66 (dd, J=14.2, 3.9, 1H), 4.09 (d, J=3.9, 1H), 3.98 (s, 3H) ), 3.53–3.50 (m, 1H), 2.95–2.92 (m, 1H), 2.57 (dd, J=9.2, 3.2, 1H), 2.56–2.51 (m, 1H), 2.26 (dd, J=13.8, 11.8, 1H), 2.03–1.98 (m, 2H), 1.93 (dd, J=13.9, 3.4, 1H), 1.88–1.80 (m, 1H), 1.76–1.70 (m, 1H), 1.28 (s, 3H) ), 1.22(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₆ H ₂₇ N ₅ O ₈ Na ⁺ 560.1752, found 560.1744.

Data for 54: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=8.48-8.45 (m, 1H), 8.05 (d, J=8.2, 1H), 7.99 (dd, J=8.5, 4.3, 2H), 7.85(dd,J＝8.6,1.7,1H),7.68(ddd,J＝8.2,6.8,1.3,1H),7.62(ddd,J＝8.1,6.8,1.3,1H),6.20(s,1H), 5.96(s, 1H), 5.69(t, J=1.0, 1H), 5.05(d, J=10.8, 1H), 5.00(d, J=10.8, 1H), 4.70(dd, J=14.3, 3.6, 1H), 4.20 (d, J=3.5, 1H), 3.58 (d, J=3.9, 1H), 2.94 (dd, J=11.7, 3.3, 1H), 2.60–2.58 (m, 1H), 2.57–2.51 (m, 1H), 2.27 (dd, J=13.8, 11.8, 1H), 2.04–1.99 (m, 2H), 1.93 (dd, J=13.9, 3.4, 1H), 1.89–1.81 (m, 1H), 1.76–1.70(m, 1H), 1.27(s, 3H), 1.22(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₃₁ H ₃₀ O ₇ Na ⁺ 537.1884, found 537.1893.

Data for 55: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.34-7.32(m,1H),7.31-7.25(m,2H),6.99-6.95(m,1H),6.83(d,J= 8.1, 1H), 6.14(s, 1H), 5.79(s, 1H), 5.64(s, 1H), 4.98(d, J=10.8, 1H), 4.91(d, J=10.8, 1H), 4.81( t, J=1.7, 2H), 4.60 (dd, J=14.2, 3.3, 1H), 4.21 (d, J=3.4, 1H), 3.47 (d, J=3.9, 1H), 2.90 (dd, J= 11.7, 3.3, 1H), 2.54 (dd, J=9.1, 2.6, 1H), 2.51–2.45 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H), 2.02–1.97 (m, 2H) , 1.91 (dd, J=13.9, 3.4, 1H), 1.84–1.78 (m, 1H), 1.71–1.66 (m, 1H), 1.25 (s, 3H), 1.22 (s, 3H); HRESIMS (m/ z):[M+H] ⁺ calcd for C ₃₀ H ₃₁ O ₈ ⁺ 519.2013, found 519.2017.

Data for 56: ¹ H NMR (600MHz, Acetone-d ₆ )δ=8.53-8.48(m,1H), 8.16-8.11(m,1H), 8.08-8.04(m,1H), 7.95-7.91(m, 1H), 7.90–7.84 (m, 1H), 7.78–7.72 (m, 1H), 6.19 (s, 1H), 6.03 (s, 1H), 5.70 (s, 1H), 5.08–4.97 (m, 2H) ,4.73–4.66(m,1H),4.20(s,1H),3.61(s,1H),2.97–2.92(m,1H),2.60–2.54(m,2H),2.31–2.23(m,1H) ,2.10–2.08(m,2H),1.96–1.91(m,1H),1.90–1.83(m,1H),1.79–1.72(m,1H),1.28(s,3H),1.23(s,3H) ; HRESIMS(m/z): [M+Na] ⁺ calcd for C ₃₀ H ₂₉ NO ₇ Na ⁺ 538.1836, found 538.1842.

Data for 57: ¹ H NMR (600MHz, Acetone-d ₆ )δ=9.25(s,1H), 8.21-8.17(m,2H), 8.05-8.01(m,1H), 8.00-7.96(m,1H) ,6.21(s,1H),6.09(s,1H),5.72(s,1H),5.03(d,1H),4.99(d,J=10.8,1H),4.68(dd,J=14.2,3.4, 1H), 4.29 (d, J=3.5, 1H), 3.65 (d, J=3.9, 1H), 2.95 (dd, J=11.8, 3.3, 1H), 2.63 (dd, J=9.1, 3.1, 1H) , 2.60–2.55 (m, 1H), 2.28 (dd, J=13.8, 11.8, 1H), 2.09–2.06 (m, 2H), 1.95–1.92 (m, 1H), 1.88–1.83 (m, 1H), 1.79–1.74(m, 1H), 1.28(s, 3H), 1.23(s, 3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₂₉ H ₂₈ N ₂ O ₇ Na ⁺ 539.1789, found 539.1794.

Data for 58: ¹ H NMR (600 MHz, Acetone-d ₆ ) δ=7.57 (dt, J=7.7, 1.3, 1H), 7.49 (t, J=8.0, 1H), 7.46-7.42 (m, 2H), 7.41 (dd, J=2.6, 1.5, 1H), 7.25 (ddd, J=8.2, 2.6, 1.1, 1H), 7.23–7.20 (m, 1H), 7.07–7.04 (m, 2H), 6.13 (s, 1H), 5.87(s, 1H), 5.63(s, 1H), 5.00(d, J=10.8, 1H), 4.93(d, J=10.8, 1H), 4.63(dd, J=14.3, 3.6, 1H) ), 4.16 (d, J=3.6, 1H), 3.51 (s, 1H), 2.94–2.91 (m, 1H), 2.56 (dd, J=9.2, 2.6, 1H), 2.52–2.46 (m, 1H) , 2.25 (dd, J=13.8, 11.7, 1H), 2.03–1.97 (m, 2H), 1.91 (dd, J=13.9, 3.3, 1H), 1.85–1.79 (m, 1H), 1.72–1.67 (m ,1H),1.25(s,3H),1.21(s,3H); HRESIMS(m/z): [M+Na] ⁺ calcd for C ₃₃ H ₃₂ O ₈ Na ⁺ 579.1989, found 579.1985.

Data for 59: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=8.11 (d, J=5.1, 1H), 7.94 (d, J=7.8, 1H), 7.82 (d, J=7.7, 1H), 7.67 (d, J=7.7, 2H), 7.59 (t, J=7.8, 1H), 7.51 (t, J=7.7, 2H), 7.42 (t, J=7.4, 1H), 6.16 (s, 1H) ,5.92(s,1H),5.67(s,1H),5.03(d,J=10.6,1H),5.01–4.95(m,1H),4.73–4.63(m,1H),4.22(s,1H) ,3.60–3.54(m,1H),2.96–2.92(m,1H),2.61–2.57(m,1H),2.56–2.50(m,1H),2.29–2.22(m,1H),2.03–1.98( m, 2H), 1.95–1.91 (m, 1H), 1.88–1.80 (m, 1H), 1.75–1.69 (m, 1H), 1.26 (s, 3H), 1.22 (s, 3H); HRESIMS (m/ z): [M–H] ^– calcd for C ₃₃ H ₃₁ O ₇ ^– 539.2075, found 539.2084.

Data for 60: ¹ H NMR (600 MHz, Acetone-d ₆ ) δ=8.69 (d, J=4.8, 2H), 7.99–7.95 (m, 2H), 7.92–7.88 (m, 2H), 7.74–7.69 ( m, 2H), 6.18(s, 1H), 5.94(s, 1H), 5.68(s, 1H), 5.03(d, J=10.8, 1H), 4.97(d, J=10.8, 1H), 4.67( dd, J=14.2, 3.6, 1H), 4.18 (d, J=3.6, 1H), 3.57 (d, J=3.9, 1H), 2.93 (dd, J=11.7, 3.3, 1H), 2.59 (dd, J=9.5, 2.9, 1H), 2.56–2.50 (m, 1H), 2.27 (dd, J=13.9, 11.8, 1H), 2.04–2.00 (m, 2H), 1.93 (dd, J=13.9, 3.4, 1H), 1.89–1.81 (m, 1H), 1.76–1.69 (m, 1H), 1.27 (s, 3H), 1.23 (s, 3H); HRESIMS (m/z): [M+H] ⁺ calcd forC ₃₂ H ₃₂ NO ₇ ⁺ 542.2173, found 542.2180.

Data for 61: ¹ H NMR (600MHz, Acetone-d ₆ )δ=7.79-7.74(m, 2H), 7.27(t, J=7.5, 2H), 7.25-7.21(m, 2H), 7.19-7.14( m, 1H), 7.01–6.96(m, 2H), 6.15(s, 1H), 5.84(s, 1H), 5.64(s, 1H), 5.01(d, J=10.8, 1H), 4.96(d, J=10.8, 1H), 4.65 (dd, J=14.3, 3.6, 1H), 4.15–4.06 (m, 3H), 3.50 (s, 1H), 2.91 (dd, J=11.8, 3.4, 1H), 2.70 (t, J=7.1, 2H), 2.56–2.53 (m, 1H), 2.53–2.46 (m, 1H), 2.25 (dd, J=13.9, 11.7, 1H), 2.03–1.96 (m, 2H), 1.94–1.89 (m, 1H), 1.86–1.77 (m, 5H), 1.72–1.67 (m, 1H), 1.26 (s, 3H), 1.22 (s, 3H); HRESIMS (m/z): [M +K] ⁺ calcd for C ₃₇ H ₄₀ O ₈ K ⁺ 651.2355, found 651.2352.

Data for 62: ¹ H NMR (600MHz, Acetone-d ₆ )δ=9.19(s, 1H), 9.10-9.07(m, 2H), 8.18(d, J=2.1, 1H), 8.08-8.05(m, 1H), 7.96–7.91(m, 1H), 7.69(t, J=7.8, 1H), 6.17(s, 1H), 5.93(s, 1H), 5.67(s, 1H), 5.03(d, J= 10.7, 1H), 4.97 (d, J=10.8, 1H), 4.68 (dd, J=14.3, 3.6, 1H), 4.19 (dd, J=3.7, 1.4, 1H), 3.59 (s, 1H), 2.93 (dd, J=11.7, 3.3, 1H), 2.59 (d, J=9.3, 1H), 2.57–2.50 (m, 1H), 2.27 (dd, J=13.9, 11.8, 1H), 2.04–2.00 (m HRESIMS (m/z): [M+Na] ⁺ calcd for C ₃₁ H ₃₀ N ₂ O ₇ Na ⁺ 565.1945, found 565.1936.

Data for 63: ¹ H NMR (600MHz, Acetone-d ₆ ) δ=9.13(d, J=2.6, 1H), 8.80(s, 1H), 8.61(dd, J=4.7, 1.5, 1H), 8.26( ddd, J＝8.4, 2.7, 1.4, 1H), 7.98(s, 1H), 7.56(dd, J＝8.3, 4.7, 1H), 6.15(s, 1H), 5.86(s, 1H), 5.65(s ,1H),5.00(d,J＝10.8,1H),4.95(d,J＝10.8,1H),4.65(dd,J＝14.3,3.2,1H),4.14(d,J＝3.5,1H), 3.50 (s, 1H), 2.91 (dd, J=11.7, 3.4, 1H), 2.56 (dd, J=9.2, 2.5, 1H), 2.54–2.47 (m, 1H), 2.26 (dd, J=13.9, 11.8, 1H), 2.04–1.97 (m, 2H), 1.92 (dd, J=13.9, 3.4, 1H), 1.87–1.78 (m, 1H), 1.73–1.67 (m, 1H), 1.26 (s, 3H) ), 1.22(s, 3H); HRESIMS(m/z): [M+H] ⁺ calcd for C ₂₉ H ₃₀ N ₃ O ₇ ⁺ 532.2078, found 532.2086.

Example 3

SARS-CoV-2 3CL ^pro inhibitor screening process:

1. Preparation of samples. Take an appropriate amount of inhibitor sample to be assayed, and prepare a solution of appropriate concentration with an appropriate solvent such as assay buffer (Assay Buffer) or DMSO. If necessary, it can be prepared into an appropriate concentration gradient for use.

2. Sample determination.

a. According to the number of samples (including relevant controls), prepare an appropriate amount of Assay Reagent with reference to the following table. Note: Due to the high glycerol content of 2019-nCoV M ^pro /3CL ^pro , care should be taken to avoid the loss of suction by the tip and complete pipetting when micro-absorbing. After adding 2019-nCoV M ^pro /3CL ^pro , it is necessary to pay attention to thorough mixing.

1 sample 5 samples 10 samples 20 samples Assay Buffer 92μL 460μL 920μl 1.84ml 2019-nCoV 3CL^pro 1μL 5μL 10μL 20μL

b. Referring to the table below, use a 96-well black plate to set up each group, and add detection reagents and samples in sequence according to the table below. After adding the sample to be tested, mix well. For more reliable detection results, it is recommended to set up 2 replicate wells for each sample.

Note: The sample solvent refers to the solvent used to formulate and dilute the inhibitor to be tested.

c. Quickly add 2 μl of Substrate to each well and mix well. Note: The reaction will start immediately after the addition of Substrate. If the number of wells is large, it is recommended to operate at a low temperature or to use a spray gun to reduce the error caused by the time difference of adding Substrate between wells. The mixing operation can be performed on the culture plate shaker. Carried on.

d. After incubation at 37°C in the dark for 10 minutes, the signal tends to be stable, and the multifunctional enzyme label can be used for fluorescence measurement within 5-20 minutes. The maximum excitation wavelength is 325 nm, and the maximum emission wavelength is 393 nm. When the fluorescence reading is low, the incubation time can also be appropriately extended to 20-30 minutes.

3. Calculation: a. Calculate the average fluorescence value of each sample well and blank control well, which can be recorded as RFU _{blank control} , RFU _{100% enzyme activity control} , RFU _{positive control} and RFU _sample respectively. b. Calculate the percent inhibition for each sample. The calculation formula is as follows: Inhibition rate (%)=(RFU _{100% enzyme activity control} -RFU _sample )/(RFU _{100% enzyme activity control} -RFU _{blank control} ) × 100%; c. For the effective inhibitors found in the detection, by detection The dose-response of the inhibitor allows the calculation of the inhibitor's _IC50 .

4. Results: As shown in Figure 1, the initial screening concentration of all compounds was 5 μM, and the screening results showed that the inhibitory activities of different derivatives ranged from 10% to 99%. A total of 16 compounds exhibited over 80% inhibition, and these compounds with high inhibition rates against SARS-CoV-23CL ^pro were characterized by bromine-containing or 2-halogenated isonicotinoids. _IC50 values were determined for the above 16 compounds. The results are shown in Table 1. Compounds 12, 24, 30 and 47 have a good ability to inhibit SARS-CoV-2 3CL ^pro , and the _IC50 values of the above four compounds are less than 1.0 μM.

Table 1 The median effective inhibitory concentration (IC ₅₀ ) of compounds against SARS-CoV-2 3CL ^pro

NO. IC₅₀[μM] NO. IC₅₀[μM] 7 1.794±0.099 27 1.662±0.200 8 1.253±0.158 30 0.523±0.093 12 0.463±0.013 32 1.115±0.001 16 1.128±0.322 34 2.427±0.065 17 1.932±0.146 36 1.731±0.197 18 1.535±0.310 40 2.304±0.457 twenty four 0.477±0.012 47 0.824±0.121 25 1.498±0.144 55 1.093±0.021

Example 4

Anti-SARS-CoV-2 experimental process:

1. Determination of Drugs and Compounds

The samples to be tested were dissolved in DMSO. The positive control was Remdesivir, which was purchased from AA BLOCKS and dissolved in DMSO. The concentration of the storage solution of the samples to be tested is 10 mM, and the storage conditions are: 4°C.

2. Reagents and Solutions

(1) Reagents

Probe One-Step qRT-PCR Kit为TOYOBO公司产品。引物和探针购自宝日医生物技术有限公司，N-F：5’GGGGAACTTCTCCTGCTAGAAT 3’、N-R：5’CAGACATTTTGCTCTCAAGCTG 3’、Probe:5’TTGCTGCTGCTTGACAGATT 3’。CCK-8 (cell counting kit-8) is a product of Biyuntian Company. DMEM high glucose medium, fetal bovine serum, 100×Penicillin-Streptomycin (10,000Units/ml penicillin, 10000μg/ml streptomycin) are products of Gibco Company. MTT (3,(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and DMF (N,N'-Dimethyl formamine) were purchased from Sigma Company. EasyPure Viral RNA Kit is a product of Quanxingjin Biotechnology Co., Ltd.

Probe One-Step qRT-PCR Kit is a product of TOYOBO Company. Primers and probes were purchased from Baori Doctor Biotechnology Co., Ltd., NF: 5'GGGGAACTTCTCCTGCTAGAAT 3', NR: 5'CAGACATTTTGCTCTCAAGCTG 3', Probe: 5'TTGCTGCTGCTTGACAGATT 3'.

(2) Culture medium

DMEM high glucose medium, containing 10% fetal bovine serum, 100 Units/mL penicillin, 100 μg/mL streptomycin.

3. Cells and viruses

African green monkey kidney epithelial cell line Vero E6, human alveolar epithelial cell line HPA EpiC were purchased from ATCC, and SARS-CoV-2 virus strain 107 was from Guangdong CDC. All cells and viruses were grown in DMEM complete medium with 10% fetal bovine serum. SARS-CoV-2 was prepared by conventional methods, and the TCID ₅₀ of the virus was titrated and calculated. After aliquoting the virus stock solution, store it at –80°C. Cells and viruses were cryopreserved and recovered by conventional methods.

4. SARS-CoV-2 infectivity titration

Vero E6 cells were seeded into 96-well plates at 4×10 ⁴ cells/well in the P3 laboratory, cultured overnight at 37°C, 5% CO ₂ , and transferred to the P3 laboratory for use when the monolayer cells grew to about 70%. The SARS-COV-2 stock solution was diluted 10-fold in a medium containing 3% fetal bovine serum, for a total of 10 gradients. Aspirate the medium in the cell culture plate, add 100 μl of virus dilution to each well, 10 replicate wells for each gradient, and set 10 control wells at the same time. Culture in a 37°C, 5% CO ₂ incubator, observe the cytopathic effect under a microscope the next day, record the number of positive wells for each gradient, and count the number of cytopathic wells when the number of lesioned wells no longer increases (3-7 days). The TCID ₅₀ (50% Tissue Culture InfectionDose) of the virus was calculated according to the Reed & Muench method.

5. Toxicity experiments on HPA EpiC cells

8×10 ⁵ /mL HPA EpiC cells were seeded into a 96-well plate in a volume of 100 μL, and cultured overnight at 37° C., 5% CO 2 . When the monolayer cells grow to about 70%, different concentrations of the drug to be tested are added, 100 μl/well, and three replicate wells are set. At the same time, control wells without drug were set up and cultured at 37℃, 5% _CO2 for 3 days. Cytotoxicity was detected by MTT colorimetry. The OD value was measured by the ELx800 microplate reader, the measurement wavelength was 570 nm, and the reference wavelength was 630 nm. The CC50 value ( ₅₀ % Cytotoxic Concentration) was calculated, that is, the concentration of the drug that is toxic to 50% of the normal human alveolar epithelial cell line HPA EpiC.

6. Inhibition experiment of SARS-CoV-2 replication in HPA EpiC cells

200 μL/well of 8×10 ⁵ /mL HPA EpiC cells were seeded on a 48-well cell culture plate, and cultured overnight at 37° C., 5% CO 2 . When the monolayer cells grow to about 70%, they are transferred to the P3 laboratory for use.

Probe One-Step qRT-PCR Kit试剂进行RT-qPCR定量，Graphpad作图计算得到EC₅₀，EC₅₀(50％Effective Concentration)为抑制细胞病变效应形成50％时的药物浓度。After aspirating the culture medium, 100 μL/well of the serially diluted drug was added, and then SARS-CoV-2 with MOI=1 was added, 100 μL/well. At the same time, control wells of normal cells without drug and virus-infected wells were set. Remdesivir was the positive drug control. After incubating for 1 h at 37°C in 5% CO ₂ , the supernatant was washed with PBS, and then the medium containing the same concentration of drugs was added. After culturing for 48 hours at 37°C and 5% CO ₂ , the viral RNA in the supernatant was extracted using the EasyPure Viral RNA Kit.

Probe One-Step qRT-PCR Kit reagent was used for RT-qPCR quantification, and EC ₅₀ was calculated by Graphpad plotting. EC ₅₀ (50%Effective Concentration) was the drug concentration that inhibited the formation of cytopathic effect by 50%.

7. Toxicity experiment on Vero E6 cells

4×10 ⁵ /mL Vero E6 cells were seeded into a 96-well plate in a volume of 100 μL, and cultured overnight at 37° C., 5% CO 2 . When the monolayer cells grow to about 70%, different concentrations of the drug to be tested are added, 100 μl/well, and three replicate wells are set. At the same time, control wells without drug were set up and cultured at 37℃, 5% _CO2 for 3 days. Cytotoxicity was detected by MTT colorimetry. The OD value was measured by the ELx800 microplate reader, the measurement wavelength was 570 nm, and the reference wavelength was 630 nm. The CC50 value ( ₅₀ % Cytotoxic Concentration) was calculated, that is, the concentration of the drug that was toxic to 50% of the normal African green monkey kidney epithelial cell line Vero E6.

8. Inhibition experiment of SARS-CoV-2 replication in Vero E6 cells

200 μL/well of 4×10 ⁵ /mL Vero E6 cells were seeded on a 48-well cell culture plate, and cultured overnight at 37° C., 5% CO 2 . When the monolayer cells grow to about 70%, they are transferred to the P3 laboratory for use.

Probe One-Step qRT-PCR Kit试剂进行RT-qPCR定量，Graphpad作图计算得到EC₅₀，EC₅₀(50％Effective Concentration)为抑制细胞病变效应形成50％时的药物浓度。After aspirating the culture medium, add the serially diluted drugs at 100 μL/well, and then add SARS-CoV-2 with MOI=0.1, 100 μL/well. At the same time, control wells of normal cells without drug and virus-infected wells were set. Remdesivir was the positive drug control. After incubating for 1 h at 37°C in 5% CO ₂ , the supernatant was washed with PBS, and then the medium containing the same concentration of drugs was added. After culturing for 48 hours at 37°C and 5% CO ₂ , the viral RNA in the supernatant was extracted using the EasyPure Viral RNA Kit.

Probe One-Step qRT-PCR Kit reagent was used for RT-qPCR quantification, and EC ₅₀ was calculated by Graphpad plotting. EC ₅₀ (50%Effective Concentration) was the drug concentration that inhibited the formation of cytopathic effect by 50%.

9. Results

As shown in Table 2, in terms of EC ₅₀ value, compounds 32 and 47 are stronger than the positive control drug Remdesivir, and compound 47 is even stronger than Remdesivir in terms of EC ₅₀ value or therapeutic index value. , the EC ₅₀ value reached 19±1 nM, and the TI value was greater than 1000. The above results show that this type of structural modifier can be used as an anti-new coronavirus molecule for further development.

Table 2 Inhibition experiments on SARS-CoV-2 virus infection (HPA cells, Vero E6 cells)

Formulation Example

In the following formulation examples, conventional reagents are selected and the formulations are prepared according to the existing conventional methods. This application example only reflects the preparation of the calyxolide B derivatives of the present invention into different formulations, and no specific reagents and operations are made. Specific restrictions.

1. After dissolving any one or any combination of the calyxolactone B derivatives with DMSO, add water for injection according to a conventional method, fine filtration, potting and sterilizing to make an injection, and the concentration of the injection is 0.5 ~5 mg/mL.

2. After dissolving any one or any combination of the pilosin B derivatives with DMSO, dissolve it in sterile water for injection, stir to dissolve it, filter it with a sterile suction filtration funnel, and then sterilize the solution. Filtered, packed in ampoules, freeze-dried at low temperature and aseptically fused to obtain powder injection.

3. Add any one or any combination of the calycilactone B derivatives to the excipient in a ratio of 9:1 by mass to the excipient to prepare a powder.

4. Add any one or any combination of the calycolactin B derivatives into excipients in a ratio of 5:1 by mass ratio to the excipients, and granulate and press into tablets.

5. Any one or any combination of calyxolide B derivatives is prepared into an oral liquid according to a conventional oral liquid preparation method.

6. Add any one or any combination of calyxolide B derivatives into excipients at a mass ratio of 5:1 to the excipients to prepare capsules.

7. Add any one or any combination of the calyxolide B derivatives into the excipient in a ratio of 5:1 by mass to the excipient to prepare a granule.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (8)

1. The eriocalyxin B derivative has a structural formula shown in the specification,

wherein R is the following functional group:

2. use of the eriocalyxin B derivative of claim 1 in preparation of SARS-CoV-23 CL ^pro Application of inhibitor in SARS-CoV-23 CL ^pro The protein is new coronavirus 3CL protease, namely main protease.

3. Use of the eriocalyxin B derivative of claim 1 in the preparation of a medicament for the treatment of a disease caused by SARS-CoV-2 infection.

4. Use of a eriocalyxin B derivative according to claim 1 in the manufacture of a medicament for the treatment of simple infection, pneumonia, acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock.

5. Use of the eriocalyxin B derivative of claim 1 in the preparation of a medicament for the treatment of severe acute respiratory infections, fever, cough, pharyngalgia.

6. A pharmaceutical composition comprising any one or any combination of the eriocalyxin B derivatives of claim 1 and a pharmaceutically acceptable carrier.

7. Use of the pharmaceutical composition of claim 7 in preparing SARS-CoV-23 CL ^pro Application of inhibitor in SARS-CoV-23 CL ^pro The protein is new coronavirus 3CL protease, namely main protease.

8. Use of the pharmaceutical composition of claim 7 for the manufacture of a medicament for the treatment of a disease caused by SARS-CoV-2 infection, said disease comprising simple infection, pneumonia, acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock.

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