Preparation method of 9, 10-fractured steroid compound

Abstract

The invention provides a preparation method of a 9, 10-fractured steroid compound. Specifically, the method comprises the following steps: (a) in a liquid reaction system, taking a compound shown as a formula I as a substrate, performing enzymatic reaction in the presence of KshA enzyme and KshB enzyme to obtain a compound shown as a formula II, and further performing ring-opening reaction to obtain a compound shown as a formula III; and (b) separating the compound as shown in the formula III from the reaction system. The invention also provides a corresponding reaction system. The method provided by the invention has the advantages of short steps, wide substrate range and high reaction conversion rate. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for preparing a 9,10-cleavage steroid compound. Background Art

[0002] 9,10-cleaved steroids are an important class of marine steroidal natural products. To date, over 40 such natural products have been reported, primarily from the marine coral Cnidium moniliforme. These steroidal natural products exhibit diverse biological activities, including cytotoxic, antiviral, anti-inflammatory, immunomodulatory, and protein kinase inhibitory properties. Therefore, achieving a simple and efficient unified synthesis of 9,10-cleaved steroids has become a research hotspot for synthetic organic chemists and pharmacologists.

[0003] In 1998, the Sodano group took the lead in realizing the total synthesis of 9,10-fragmented steroids calicoferol E and astrogorgiadiol using a convergent synthesis strategy, with the longest linear steps being 9 or 10. From 2001 to 2011, the Taber group also developed a convergent total synthesis route for the synthesis of 9,10-fragmented steroids astrogorgiadiol, with the longest linear steps being 13. In 2014, the Medard group used sp 2 –sp 3 The Suzuki coupling reaction is the key reaction, and a convergent synthesis strategy is also used to achieve the total synthesis of the 9,10-cleaved steroid astrogorgiadiol, with the longest linear step being 13. Although the longest linear step in the reported convergent total synthetic routes is less than 13 steps, the overall yield is not high. Therefore, the development of a simple and efficient synthetic route for the synthesis of 9,10-cleaved steroids is a top priority.

[0004] As early as 1958, Dodson's group discovered that microorganisms could achieve steroid 9-position hydroxylation, 1- and 2-position dehydrogenation, and B-ring aromatization. Since then, numerous reports have been published on microbial-catalyzed steroid 9-position hydroxylation, but these efforts have been plagued by limitations: limited substrate range, poor regioselectivity, numerous side reactions, and low conversion rates.

[0005] Therefore, there is an urgent need in the art to develop a method for preparing 9,10-fragmented steroid compounds with short steps, a wide substrate range, and high yield. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing a 9,10-cleavage steroid compound with short steps, a wide substrate range and high yield.

[0007] The first aspect of the present invention provides a method for preparing a compound of formula III, comprising the steps of:

[0008] (a) in a liquid reaction system, using a compound of formula I as a substrate, in the presence of KshA and KshB enzymes, conducting an enzymatic reaction to obtain a compound of formula II, which then undergoes a ring-opening reaction to obtain a compound of formula III;

[0009]

[0010] Optionally (b) isolating the compound of formula III from the reaction system;

[0011] Among various

[0012] Indicates single bond / double bond;

[0013] When connecting R1 When it is a single bond, R1 is selected from R5 or

[0014] When connecting R1 When it is a double bond, R1 is O;

[0015] When connecting R4 When it is a single bond, R4 is OH;

[0016] When connecting R4 When it is a double bond, R4 is O;

[0017] R2 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0018] R3 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0019] R5 is selected from:

[0020] R6 is selected from: OH, OZ or C1-C6 alkoxy, wherein Z is a protecting group;

[0021] R7 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0022] R8 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0023] R9 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0024] The substitution refers to substitution by one or more substituent groups selected from the group consisting of halogen or hydroxyl.

[0025] In another preferred embodiment, the method comprises the steps of:

[0026] (a) In a liquid reaction system, an enzymatic reaction is carried out with the compound of formula Ia as a substrate in the presence of KshA and KshB enzymes to form a compound of formula IIa, which then undergoes a ring-opening reaction to obtain a compound of formula IIIa.

[0027] Optionally (b) isolating the compound of formula IIIa from the liquid reaction system;

[0028]

[0029] Among various

[0030] Indicates single bond / double bond;

[0031] When connecting R1 When it is a single bond, R1 is selected from R5 or

[0032] When connecting R1 When it is a double bond, R1 is O;

[0033] When connecting R4 When it is a single bond, R4 is OH;

[0034] When connecting R4 When it is a double bond, R4 is O;

[0035] R2 is selected from: H, OH, C1-C6 alkyl or C1-C6 alkoxy;

[0036] R3 is selected from: H, OH, C1-C6 alkyl or C1-C6 alkoxy;

[0037] R5 is selected from:

[0038] R6 is selected from: OH or OZ, wherein Z is a protecting group.

[0039] In another preferred embodiment, the substrate concentration in the liquid reaction system is 0.05 to 5 mmol / L, preferably 0.1 to 3 mmol / L, more preferably 0.5 to 2 mmol / L, for example 0.8 mmol / L.

[0040] In another preferred embodiment, the KshA enzyme is selected from: KshA1, KshA3, KshA5, KshA N or a combination thereof.

[0041] In another preferred embodiment, the KshB enzyme is selected from: KshB, KshB Nor a combination thereof.

[0042] In another preferred embodiment, the liquid reaction system comprises water and an organic solvent, and optionally, a co-solvent.

[0043] In another preferred embodiment, the liquid reaction system further includes glucose.

[0044] In another preferred embodiment, the organic solvent is selected from: methanol, ethanol, isopropanol, acetone, dimethylformamide, dimethyl sulfoxide or a combination thereof.

[0045] In another preferred embodiment, the co-solvent includes cyclodextrin.

[0046] In another preferred embodiment, the compound of formula III is selected from:

[0047]

[0048] In another preferred embodiment, the amino acid sequence of KshA1 is shown in SEQ ID NO: 1.

[0049] In another preferred example, the amino acid sequence of KshA3 is shown in SEQ ID NO: 2.

[0050] In another preferred example, the amino acid sequence of KshA5 is shown in SEQ ID NO: 3.

[0051] In another preferred embodiment, the KshA N The amino acid sequence is shown in SEQ ID NO:4.

[0052] In another preferred example, the amino acid sequence of KshB is shown in SEQ ID NO:5.

[0053] In another preferred embodiment, the KshB N The amino acid sequence is shown in SEQ ID NO:6.

[0054] In another preferred embodiment, the KshA enzyme and the KshB enzyme are each independently present in the following manner:

[0055] (i) isolated or purified enzymes;

[0056] (ii) immobilized enzyme;

[0057] (iii) cells of an engineered bacterium expressing KshA enzyme and / or KshB enzyme, a lysate thereof, or a supernatant thereof;

[0058] (iv) viable bacteria of engineered bacteria expressing KshA enzyme and / or KshB enzyme;

[0059] (v) or a combination thereof.

[0060] In another preferred embodiment, the cyclodextrin is selected from: α-cyclodextrin, methyl-β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin or a combination thereof.

[0061] In another preferred embodiment, the concentration of the cosolvent in the reaction system is 0.01 to 0.5 mmol / L, preferably 0.01 to 0.2 mmol / mL, more preferably 0.05 to 0.1 mmol / mL, for example, 0.08 mmol / mL.

[0062] In another preferred embodiment, the molar ratio of the co-solvent to the compound of formula I is 5:1 to 1:5, preferably 3:1 to 1:3, more preferably 2:1 to 1:2, for example 1:1.

[0063] In another preferred embodiment, the molar ratio of glucose to the compound of formula I is 1 to 10:1, preferably 2 to 10:1, and more preferably 4 to 8:1.

[0064] In another preferred embodiment, the volume ratio of the organic solvent to the reaction system is 1:15-30, preferably 1:20-30, and more preferably 1:23-28.

[0065] In another preferred embodiment, the reaction is carried out at 15-40°C, preferably 20-37°C, more preferably 25-35°C.

[0066] In another preferred embodiment, the reaction is carried out for 1 to 5 hours, preferably 1 to 4 hours, more preferably 2 to 3 hours.

[0067] In another preferred embodiment, the yield of the method (the compound of formula I reacts to form the compound of formula III) is ≥85%, preferably ≥90%, and more preferably ≥92%.

[0068] In another preferred embodiment, the molar ratio of the total amount of the KshA enzyme and the KshB enzyme to the compound of formula I is 1:10-500, more preferably 1:20-200, and even more preferably 1:30-100.

[0069] In another preferred embodiment, the ratio of the KshA enzyme to the KshB enzyme is 30:1 to 1:30, preferably 20:1 to 1:20, more preferably 10:1 to 1:10, for example 1:1.

[0070] In another preferred example, the mass ratio of the live bacteria or bacteria to the compound of formula I is 50-200:0.05-1, preferably 70-150:0.1-1, more preferably 80-120:0.5-1, for example 100:0.8.

[0071] In a second aspect, the present invention provides a reaction system comprising:

[0072] (i) a reaction solvent;

[0073] (ii) a substrate, wherein the substrate is a compound represented by formula I;

[0074]

[0075] and (iii) KshA enzyme and KshB enzyme;

[0076] Where,

[0077] Indicates single bond / double bond;

[0078] when When it is a single bond, R1 is selected from R5 or when When it is a double bond, R1 is O;

[0079] R2 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0080] R3 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0081] R5 is selected from:

[0082] R6 is selected from: OH, OZ or substituted or unsubstituted C1-C6 alkoxy, wherein Z is a protecting group;

[0083] R7 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0084] R8 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0085] R9 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0086] Wherein, the substitution refers to substitution by one or more substituents selected from the group consisting of halogen or hydroxyl. In another preferred embodiment, the substrate is a compound represented by Formula Ia;

[0087]

[0088] and (iii) KshA enzyme and KshB enzyme;

[0089] in,

[0090] Indicates single bond / double bond;

[0091] when When it is a single bond, R1 is selected from R5 or when When it is a double bond, R1 is O;

[0092] R2 is selected from: H or OH;

[0093] R3 is selected from: H, α-OH or β-OH

[0094] R5 is selected from:

[0095] R6 is selected from: OH, OAc or OBn.

[0096] In another preferred embodiment, the reaction solvent comprises water and an organic solvent, optionally a co-solvent.

[0097] In another preferred embodiment, the reaction solvent further comprises glucose.

[0098] In a third aspect, the present invention provides a method for preparing a compound of formula III or formula IIIa, comprising the steps of:

[0099]

[0100] The reaction system described in the second aspect of the present invention is used to prepare a compound of formula III or formula IIIa; wherein,

[0101] Indicates single bond / double bond;

[0102] When connecting R1 When it is a single bond, R1 is selected from R5 or

[0103] When connecting R1 When it is a double bond, R1 is O;

[0104] When connecting R4 When it is a single bond, R4 is OH;

[0105] When connecting R4 When it is a double bond, R4 is O;

[0106] R2 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0107] R3 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0108] R5 is selected from:

[0109] R6 is selected from: OH, OZ or C1-C6 alkoxy, wherein Z is a protecting group;

[0110] R7 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0111] R8 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0112] R9 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy;

[0113] The substitution refers to substitution by one or more substituent groups selected from the group consisting of halogen or hydroxyl.

[0114] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. DETAILED DESCRIPTION

[0115] After extensive and in-depth research, numerous experiments, and screening, the present invention unexpectedly developed a biopreparation method for 9,10-cleaved steroid compounds (e.g., compounds of Formula III or Formula V). Specifically, the biopreparation method of the present invention utilizes KshA and KshB enzymes as biocatalysts to efficiently prepare 9,10-cleaved steroid compounds (e.g., compounds of Formula III or Formula V) (yield ≥ 85%), opening the C9–C10 bond of the steroid compound in a single step. This significantly shortens the process, improves production efficiency, and reduces production costs. This is the basis for the completion of the present invention.

[0116] the term

[0117] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0118] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., is understood to include the stated elements or components but does not exclude other elements or components.

[0119] As used herein, the term "calcd" refers to a calculated value. The term "found" refers to an actual value.

[0120] KshA and KshB enzymes

[0121] In the present invention, the KshA enzyme and the KshB enzyme can catalyze the reaction of the compound of formula I to form the compound of formula II, and then the compound of formula II undergoes ring opening to form the compound of formula III, thereby preparing a 9,10-cleaved steroid compound.

[0122]

[0123] In the present invention, the KshA enzyme and KshB enzyme may be wild-type or mutant, and may be isolated or recombinant.

[0124] In another preferred embodiment, the KshA enzyme is selected from: KshA1, KshA3, KshA5, KshA N , or a combination thereof. A typical amino acid sequence of KshA1 is shown in SEQ ID NO: 1. A typical amino acid sequence of KshA3 is shown in SEQ ID NO: 2. A typical amino acid sequence of KshA5 is shown in SEQ ID NO: 3. A typical KshA N The amino acid sequence is shown in SEQ ID NO:4.

[0125] The KshA enzyme of the present invention also includes an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 while maintaining enzyme activity.

[0126] In another preferred embodiment, the KshB enzyme is selected from: KshB, KshB N , or a combination thereof. A typical amino acid sequence of KshB is shown in SEQ ID NO: 5. A typical KshB N The amino acid sequence is shown in SEQ ID NO:6.

[0127] The KshB enzyme of the present invention also includes an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 while maintaining enzyme activity.

[0128] In the present invention, the KshA and KshB enzymes can be used in various forms. For example, resting cells or wet cells expressing the KshA and KshB enzymes of the present invention can be used, or various forms such as crude enzyme solutions, pure enzymes, or crude enzyme powders can be used, or immobilized enzymes can be used.

[0129] Preferably, in order to obtain higher transformation efficiency and reduce costs, a cell suspension or cell lysate of bacteria or fungi containing genes encoding Rieske oxidase KshA and KshB is preferred.

[0130] cosolvent

[0131] As used herein, the term "cosolvent" refers to a compound, an association compound, or a double salt formed between a poorly soluble substance and a third substance added to a solvent to increase the solubility of the poorly soluble substance in the solvent. This third substance is called a cosolvent.

[0132] In the present invention, the substrate solubility is increased by adding a cosolvent to improve the reaction conversion. The optional cosolvent is cyclodextrin.

[0133] 9,10-cleaved steroids

[0134] 9,10-cleaved steroid compounds are an important class of marine steroid natural products. By analyzing the biosynthetic pathway of steroids, it was found that the biosynthetic precursor of 9,10-cleaved steroids is Δ 1(2),4(5) The diketene substrate is then enzymatically hydroxylated at the 9-position of the steroid and aromatized to cleave the C9-C10 bond, yielding the 9,10-cleaved steroid. Based on this, a chemoenzymatic semisynthetic route starting from steroidal raw materials can be developed for the unified synthesis of 9,10-cleaved steroids: first, an enzyme-catalyzed precursor is rapidly prepared by chemical methods, followed by the enzymatic catalysis of the 9,10-cleaved steroid.

[0135] To date, three types of steroid 9-hydroxylases have been discovered: CYP260B1, SptF, and KSH (3-ketosteroid 9α-hydroxylase). Among them, the two-component Rieske non-heme monooxygenase KSH, found in the bacterial cholesterol degradation pathway, can efficiently catalyze the 9-hydroxylation of androstenedione and is an ideal enzyme for the chemoenzymatic synthesis of 9,10-cleaved steroids.

[0136] The main advantages of the present invention include:

[0137] (1) A method for preparing a 9,10-cleavage steroid compound is provided, wherein the reaction conversion rate of the method is ≥85%, preferably 90%.

[0138] (2) The preparation method of the present invention has short steps and a wide range of substrates.

[0139] (3) The preparation method of the present invention involves a biological preparation reaction, which is green, environmentally friendly and economical. It has achieved significant improvement over the existing technology and provides a promising solution to the problem of long reaction steps in the existing production process.

[0140] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0141] Example 1

[0142] 34.4 mL of the clarified lysate of E. coli co-expressing KshA3 and KshB (OD 600 =60), glucose (86 mg, 0.48 mmol, 6.0 equiv) in water (0.8 mL), methyl-β-cyclodextrin (104 mg, 0.08 mmol, 1.0 equiv) in water (0.8 mL), and a methanol solution of compound 1 (0.08 mmol, 1.0 equiv, 2 mM) (4 mL) were reacted at 30°C with shaking at 150 rpm for 2 h. The reaction mixture was extracted with dichloromethane (20 mL x 3). The resulting organic phase was washed with brine and dried over anhydrous Na2SO4. The crude product was concentrated by rotary evaporation and purified by column chromatography to obtain product 3.

[0143]

[0144] Column chromatography gave 3a as a white solid (28.7 mg, 90%).

[0145] mp:88.5–92.0℃; TLC(petroleum ether:EtOAc,10 / 1v / v):R f =0.30; [α]

[0146] +28.9 (c 0.60, CHCl3); 1H NMR (500MHz, CDCl3): δ6.97(d,J=8.0Hz,1H),6.68(d,J=2.5Hz,1H),6.59(dd,J =8.0,2.5Hz,1H),2.66(td,J=13.0,5.0Hz,1H),2.50(td,J=14.5,6.5Hz,1H),2. 42(td,J=11.5,5.5Hz,1H),2.40–2.33(m,1H),2.32(ddd,J=15.0,5.0,2.0Hz,1H ),2.25(s,3H),2.16(ddd,J=13.0,7.0,2.0Hz,1H),2.02–1.93(m,1H),1.80–1.7 1(m,1H),1.74–1.66(m,1H),1.72–1.62(m,1H),1.63–1.53(m,2H),1.57–1.48(m ,1H),1.52–1.38(m,1H),1.48–1.38(m,1H),1.38–1.30(m,2H),1.34–1.24(m,1H ),1.25–1.16(m,1H),1.21–1.10(m,1H),1.19–1.06(m,2H),1.08–0.96(m,1H),0 .98(s,3H),0.93(d,J=6.5Hz,3H),0.88(d,J=6.5Hz,3H),0.87(d,J=6.5Hz,3H); 13 C NMR (126MHz, CDCl3): δ213.9,153.8,142.4,131.0,127.8,115.7,112.6,55.2,55.0,50.4,42.8,39.4 ,38.5,38.3,35.9,35.6,31.0,29.0,28.0,27.6,25.1,23.8,22.8,22.5,18.5,18.4,11.5;IR(KBr):ν max =3389,2953,2868,1694,1609,1586,1502,810cm -1 ; HRMS (FI, m / z): calculated for C 27 H 42 O2[M] ·+ 398.3179; found 398.3181.

[0147]

[0148] Column chromatography gave 3b as a white solid (31.4 mg, 92%).

[0149] mp:93.2–96.1℃;TLC(petroleum ether:EtOAc,10 / 1v / v):R f =0.33;[α]+34.0(c0.10,CHCl3); 1 H NMR(500MHz,CDCl3):δ6.97(d,J=8.0Hz,1H),6.68(d,J=2.5Hz,1H),6.59(dd,J=8.0,2.5Hz,1H),2.66(td,J=13.0,5.0Hz,1H),2.50(td,J=14.5,6.5Hz,1H),2.47–2.41(m,1H),2.41–2.35(m,1H),2.37–2.30(m,1H),2.25(s,3H),2.20–2.14(m,1H),2.03–1.95(m,1H),1.80–1.73(m,1H),1.75–1.67(m,1H),1.72–1.64(m,2H),1.63–1.57(m,1H),1.62–1.55(m,1H),1.49–1.37(m,1H),1.48–1.40(m,1H),1.40–1.31(m,1H),1.34–1.25(m,2H),1.27–1.21(m,1H),1.26–1.19(m,1H),1.23–1.15(m,2H),1.10–1.00(m,1H),0.98(s,3H),0.98–0.92(m,1H),0.94(d,J=7.0Hz,3H),0.85(t,J=7.4Hz,3H),0.84(d,J=7.0Hz,3H),0.82(d,J=7.0Hz,3H); 13 C NMR(126MHz,CDCl3):δ213.8,153.8,142.4,131.0,127.9,115.7,112.5,55.2,54.9,50.4,45.8,42.8,38.5,38.3,36.0,33.7,31.0,29.11,29.06,27.6,26.0,25.1,23.0,19.8,19.0,18.6,18.4,12.0,11.5;IR(KBr):ν max =3389,2957,2870,1694,1608,1586,1503,810cm -1 ;HRMS(FI,m / z):calcd for C 29 H 46 O2[M] ·+ 426.3492;found 426.3497.

[0150]

[0151] Column chromatography gave 3c (29.7 mg, 90%) as a yellow oil. [α]+12.9 (c 0.62, CHCl3); 1 H NMR (500MHz, CDCl3): δ6.96(d,J=8.5Hz,1H),6.68(d,J=2.5Hz,1H),6.60(dd,J=8.5,2.5Hz,1H),2 .64(td,J=13.0,5.0Hz,1H),2.50(td,J=14.5,6.5Hz,1H),2.43–2.29(m,3H),2.23(s,3H),1.94–1. 86(m,1H),1.84–1.72(m,3H),1.71–1.49(m,6H),1.49–1.40(m,1H),1.39–1.32(m,2H),1.29(s,3H ),1.28–1.20(m,2H),1.16(s,3H),1.15–1.11(m,2H),0.87(d,J=6.6Hz,3H),0.87(d,J=6.6Hz,3H); 13 C NMR (126MHz, CDCl3): δ213.8,154.2,142.2,131.0,127.4,115.8,112.7,75.2,56.6,55.3,49.9,44.2 ,43.1,39.5,38.9,38.1,31.0,27.9,27.5,26.1,24.6,23.3,22.7,22.6,22.0,18.4,13.1;IR(KBr):ν max =3363,2953,2870,1694,1609,1586,1504,811cm -1 ; HRMS (FI, m / z): calculated for C 27 H 42 O3[M] ·+ 414.3128; found 414.3130.

[0152]

[0153] Column chromatography gave a white solid (24.3 mg, 93%).

[0154] 1H NMR (500MHz, CDCl3): δ6.97 (d, J=8.0Hz, 1H), 6.67 (d, J=2.5Hz, 1H), 6.60 (dd, J= 8.0,2.5Hz,1H),2.66(td,J=13.0,4.5Hz,1H),2.59(t,J=9.0Hz,1H),2.52(td,J =15.0,7.0Hz,1H),2.40(m,3H),2.29(m,1H),2.24(s,3H),2.23–2.18(m,1H),2. 15(s,3H),1.87–1.74(m,5H),1.64–1.54(m,1H),1.49–1.37(m,1H),0.95(s,3H).

[0155] Example 2

[0156] 34.4 mL of the clarified lysate of E. coli co-expressing KshA3 and KshB (OD 600 =60), glucose (86 mg, 0.48 mmol, 6.0 equiv) in water (0.8 mL), γ-cyclodextrin (104 mg, 0.08 mmol, 1.0 equiv) in water (0.8 mL), and compound 1a (0.08 mmol, 1.0 equiv, 2 mM) in methanol (4 mL) were reacted at 28°C with shaking at 200 rpm for 2 h. The reaction mixture was extracted with dichloromethane (20 mL x 3), and the resulting organic phase was washed with brine and dried over anhydrous Na2SO4. The crude product was concentrated by rotary evaporation, and column chromatography afforded 3a (27.7 mg, 87%) as a white solid.

[0157]

[0158] Example 3

[0159] 34.4 mL of the clarified lysate of actinomycetes co-expressing KshA3 and KshB (OD 600 =60), glucose (86 mg, 0.48 mmol, 6.0 equiv) in water (0.8 mL), methyl-β-cyclodextrin (104 mg, 0.08 mmol, 1.0 equiv) in water (0.8 mL), and compound 1a (0.08 mmol, 1.0 equiv, 2 mM) in ethanol (4 mL) were reacted at 30°C with shaking at 200 rpm for 2 h. The reaction mixture was extracted with dichloromethane (20 mL x 3), and the resulting organic phase was washed with brine and dried over anhydrous NaSO. The crude product was concentrated by rotary evaporation, and column chromatography afforded 3a (27.0 mg, 84.8%) as a white solid.

[0160] Example 4

[0161] 34.4 mL of yeast clarified lysate (OD 600 =60), glucose (72 mg, 0.40 mmol, 5.0 equiv) in water (0.8 mL), methyl-β-cyclodextrin (104 mg, 0.08 mmol, 1.0 equiv) in water (0.8 mL), and compound 1a (0.08 mmol, 1.0 equiv, 2 mM) in acetone (4 mL) were reacted at 30°C with shaking at 150 rpm for 3 h. The reaction mixture was extracted with dichloromethane (20 mL x 3), and the resulting organic phase was washed with brine and dried over anhydrous Na2SO4. The crude product was concentrated by rotary evaporation, and column chromatography afforded 3a (22.0 mg, 69.4%) as a white solid.

[0162] Example 5

[0163] 34.4 mL of the clarified lysate of E. coli co-expressing KshA3 and KshB (OD 600 =60), glucose (86 mg, 0.48 mmol, 6.0 equiv) in water (0.8 mL), methyl-β-cyclodextrin (104 mg, 0.08 mmol, 1.0 equiv) in water (0.8 mL), and compound 1a (0.08 mmol, 1.0 equiv, 2 mM) in isopropanol (4 mL) were reacted at 30°C with shaking at 150 rpm for 2 h. The reaction mixture was extracted with dichloromethane (20 mL x 3), and the resulting organic phase was washed with brine and dried over anhydrous Na2SO4. The crude product was concentrated by rotary evaporation, and column chromatography afforded 3a (22.8 mg, 71.7%) as a white solid.

[0164] Example 6

[0165] 34.4 mL of the clarified lysate of E. coli co-expressing KshA3 and KshB (OD 600=60), glucose (86 mg, 0.48 mmol, 6.0 equiv) in water (0.8 mL), 2-hydroxypropyl-β-cyclodextrin (220 mg, 0.16 mmol, 2.0 equiv) in water (0.8 mL), and compound 1a (0.08 mmol, 1.0 equiv, 2 mM) in methanol (4 mL) were reacted at 30°C with shaking at 150 rpm for 2 h. The reaction mixture was extracted with dichloromethane (20 mL x 3), and the resulting organic phase was washed with brine and dried over anhydrous Na2SO4. The crude product was concentrated by rotary evaporation, and column chromatography afforded 3a (28.3 mg, 88.9%) as a white solid.

[0166] Example 7

[0167] 430 mL of clarified E. coli lysate (OD 600 =60), glucose (1.1 g, 6.0 mmol, 6.0 equiv) in water (10 mL), methyl-β-cyclodextrin (1.3 g, 1.0 mmol, 1.0 equiv) in water (0.8 mL), and compound 1d (3.14 mmol, 1.0 equiv, 2 mM) in methanol (50 mL) were stirred at 30°C and 150 rpm for 2 h. The reaction mixture was extracted with dichloromethane, and the resulting organic phase was washed with brine and dried over anhydrous Na2SO4. The crude product was concentrated by rotary evaporation, and column chromatography afforded 3d (1.28 g, 93%) as a white solid.

[0168]

[0169] Column chromatography gave 3d (26.9 mg, 87%) as a colorless oil. [α]+16.1 (c 0.41, CHCl3); 1H NMR (500MHz, CDCl3): δ6.96 (d, J = 8.0 Hz, 1H), 6.67 (d, J = 3.0 Hz, 1H), 6.59 (dd, J = 8.0, 3.0 Hz, 1H), 4.09 (dd,J=11.0,3.5Hz,1H),3.80(dd,J=11.0,7.0Hz,1H),2.65(td,J=12.0,4.5Hz,1H),2.50(td,J=14.5, 6.5Hz,1H),2.45–2.30(m,3H),2.24(s,3H),2.14(ddd,J=12.5,6.5,1.5Hz,1H),2.07(s,3H),1.98–1.9 0(m,1H),1.82–1.55(m,6H),1.54–1.45(m,1H),1.35–1.26(m,2H),1.03(d,J=6.5Hz,3H),0.99(s,3H); 13 C NMR (126MHz, CDCl3): δ213.3,171.6,154.0,142.3,131.0,127.7,115.7,112.6,69.2,54.8,5 1.6,50.4,42.9,38.2,38.1,35.6,31.0,28.5,27.6,25.1,21.0,18.4,17.0,11.6;IR(KBr):ν max =3390,2953,2872,1732,1608,1585,1504,811cm -1 ; HRMS (FI, m / z): calculated for C 24 H 34 O4[M] ·+ 386.2452; found 386.2458.

[0170] Example 8

[0171] 34.4 mL of KshA3 mutant KshA3 was added to a 250 mL conical flask. N251A Clarified lysate of E. coli co-expressing KshB (OD 600=60), glucose (86 mg, 0.48 mmol, 6.0 equiv) in water (0.8 mL), methyl-β-cyclodextrin (104 mg, 0.08 mmol, 1.0 equiv) in water (0.8 mL), and compound 1a (0.08 mmol, 1.0 equiv, 2 mM) in methanol (4 mL) were reacted at 30°C with shaking at 150 rpm for 2 h. The reaction mixture was extracted with dichloromethane (20 mL x 3), and the resulting organic phase was washed with brine and dried over anhydrous Na2SO4. The crude product was concentrated by rotary evaporation, and column chromatography afforded 3a (13.2 mg, 41.4%) as a white solid.

[0172] Example 9

[0173] 34.4 mL of the clarified lysate of E. coli co-expressing KshA3 and KshB (OD 600 =60), glucose (86 mg, 0.48 mmol, 6.0 equiv) in water (0.8 mL), methyl-β-cyclodextrin (104 mg, 0.08 mmol, 1.0 equiv) in water (0.8 mL), and a methanol solution of compound 4 (0.08 mmol, 1.0 equiv, 2 mM) (4 mL) were reacted at 30°C with shaking at 150 rpm for 2 h. The reaction mixture was extracted with dichloromethane (20 mL x 3), and the resulting organic phase was washed with brine and dried over anhydrous Na2SO4. The crude product was concentrated by rotary evaporation and purified by column chromatography to afford product 5.

[0174]

[0175] Column chromatography gave a white solid (9.8 mg, 31%, 75% recovery yield).

[0176] mp:187.5–189.2℃; TLC(petroleum ether:EtOAc,10 / 1v / v):R f =0.18;[α]+95.0(c 0.10,CHCl3); 1H NMR (500MHz, CDCl3): δ5.86 (d, J=1.5Hz, 1H), 2.52–2.37 (m, 4H), 2.30 (dd, J=15.1, 4.9Hz, 1H), 1.95–1.82(m,3H),1.81–1.68(m,2H),1.65–1.58(m,2H),1.52–1.48(m,2H),1.47–1.36(m,4H), 1.36–1.32(m,2H),1.32(s,3H),1.30–1.26(m,1H),1.22–1.17(m,1H),1.16–1.05(m,4H),1.04 –0.97(m,1H),0.91(d,J=6.5Hz,3H),0.87(d,J=6.6Hz,3H),0.86(d,J=6.6Hz,3H),0.72(s,3H); 13 C NMR (126MHz, CDCl3): δ199.1,169.1,126.6,76.3,55.8,49.5,44.3,42.2,39.5,37.4,36.1,35.7,3 5.0,34.0,31.8,28.4,28.1,28.0,26.7,25.4,24.1,23.7,22.8,22.5,19.9,18.6,11.0;IR(KBr):ν max =3440,2929,2865,1660,1463,1373,1280cm -1 ; HRMS (FI, m / z): calculated for C 27 H 44 O2[M] ·+ 400.3336; found400.3334.

[0177]

[0178] Column chromatography gave 5b as a white solid (5.3 mg, 21%, 62% recovery yield).

[0179] 1H NMR (500MHz, CD3OH): δ5.94(d,J=1.5Hz,1H),4.03(d,J=11.2Hz,1H),3.89(d,J=11.2Hz,1H),2.72(ddd,J=16.5,13.0,6.1Hz,1H),2.6 2–2.52(m,1H),2.49–2.30(m,4H),2.20(td,J=11.6,4.2Hz,1H),2.13–2.04(m,2H),1.99–1.82(m,3H),1.76–1.53(m,6H),0.93(s,3H); 13 C NMR (126MHz, CD3OD): δ221.9,201.6,168.9,127.4,76.3,65.0,50.2,47.3,44.1,38.0,35.2,34.4,32.4,26.8(2C),25.2,24.2,21.0,11.9.

[0180] Example 10

[0181] 34.4 mL of the clarified lysate of E. coli co-expressing KshA5 and KshB (OD 600 =60), glucose (86 mg, 0.48 mmol, 6.0 equiv) in water (0.8 mL), methyl-β-cyclodextrin (104 mg, 0.08 mmol, 1.0 equiv) in water (0.8 mL), and compound 4 (0.08 mmol, 1.0 equiv, 2 mM) in methanol (4 mL) were reacted at 30°C with shaking at 150 rpm for 2 h. The reaction mixture was extracted with dichloromethane (20 mL x 3). The resulting organic phase was washed with brine and dried over anhydrous Na2SO4. The crude product was concentrated by rotary evaporation and purified by column chromatography to afford 5. 5a was obtained as a white solid (21.7 mg, 64%, 90% recovery yield); 5b was obtained as a white solid (27.2 mg, 80%); and 5c was obtained as a white solid (31.9 mg, 94%).

[0182]

[0183] Column chromatography gave 5c as a white solid (10.2 mg, 30%, 82% recovery yield). [α]+254.2 (c 0.10, CHCl3); 1H NMR (500MHz, CDCl3): δ5.88(d,J=1.0Hz,1H),5.08(d,J=17.5Hz,1H),4.70(d,J=1 7.5Hz,1H),3.42(d,J=12.0Hz,1H),2.86–2.77(m,1H),2.77–2.67(m,1H),2.54–2. 45(m,3H),2.42–2.28(m,2H),2.25–2.18(m,1H),2.17(s,3H),2.16–2.10(m,2H),1 .92–1.82(m,1H),1.75–1.62(m,3H),1.48(s,3H),1.48–1.38(m,1H),0.69(s,3H); 13 C NMR (126MHz, CDCl3): δ208.0,204.2,199.2,170.6,166.4,127.5,89.0,79.4,67.5,51.0 ,45.9,44.0,43.7,38.4,35.2,33.8,31.4,28.2,24.7,23.0,20.5,18.9,15.0;IR(KBr):ν max =3416,2952,2937,1754,1737,1714,1660,1621cm -1 ; HRMS (ESI, m / z): calculated for C 23 H 31 O7[M+H] + 419.2064; found 419.2069.

[0184] Example 11

[0185] In a 25 mL reaction vial, compound 3a (50.0 mg, 0.13 mmol, 1.0 equiv) was dissolved in 2.5 mL of methanol. NaBH₄ (9.5 mg, 0.25 mmol, 2.0 equiv) was added and stirred at room temperature for 30 min. The reaction was quenched with 1 mL of water, the methanol was concentrated by rotary evaporation, and 2 mL of water was added. The reaction mixture was extracted with dichloromethane (3 × 20 mL). The resulting organic phase was washed with brine and dried over anhydrous Na₂SO₄. The crude product was concentrated by rotary evaporation and purified by column chromatography to afford 3f (13.9 mg, 28%) and 3g (25.2 mg, 50%).

[0186]

[0187] 3f:TLC(petroleum ether:EtOAc,3:1 v / v):R f=0.56;[α]-3.2(c 1.34,CHCl3); 1 H NMR(400 MHz,CDCl3)δ6.98(d,J=8.4 Hz,1H),6.65(d,J=2.4 Hz,1H),6.57(dd,J=8.4,2.8 Hz,1H),4.04(brs,1H),2.75–2.65(m,1H),2.47–2.37(m,1H),2.22(s,3H),1.84–1.77(m,1H),1.79–1.73(m,1H),1.76–1.68(m,2H),1.60–1.52(m,1H),1.57–1.49(m,2H),1.54–1.47(m,1H),1.54–1.45(m,1H),1.52–1.46(m,1H),1.49–1.43(m,1H),1.40–1.34(m,1H),1.34–1.30(m,1H),1.33–1.31(m,1H),1.24–1.17(m,1H),1.19–1.13(m,1H),1.16–1.10(m,1H),1.14–1.07(m,2H),1.12–1.04(m,1H),1.02–0.95(m,1H),0.92(d,J=6.4 Hz,3H),0.69(s,3H),0.86(d,J=6.8 Hz,3H),0.86(d,J=6.8 Hz,3H); 13 C NMR(101 MHz,CDCl3)δ153.9,142.8,131.2,128.0,115.6,112.6,67.4,56.3,47.9,43.0,41.1,39.6,36.3,35.9,34.3,31.0,30.4,30.2,28.2,27.9,24.6,23.9,22.9,22.7,18.8,18.5,11.2;IR(KBr):ν max =3343,2952,2924,1609,1587,1463,1261,1081,807 cm -1 ;HRMS(DART,m / z):calcdC 27 H 48 NO2 for[M+NH4] + 418.3680,found 418.3679.

[0188] 3g:TLC(petroleum ether:EtOAc,3:1 v / v):R f= 0.37; [α] + 37.9 (c 2.52, MeOH); 1 H NMR (400 MHz, CDCl3) δ 6.96 (d, J = 8.0 Hz, 1H), 6.66 (d, J = 2.8 Hz, 1H), 6.57 (dd, J = 8.4, 3.2 Hz, 1H), 3.44 (td, J = 10.8, 4.8 Hz, 1H), 2.56 (td, J = 13.6, 5.2 Hz, 1H), 2.51 (td, J = 13.2, 5.6 Hz, 1H), 2.22 (s, 3H), 1.95–1.87 (m, 1H), 1.95–1.85 (m, 1H), 1.86–1.78 (m, 1H), 1.77–1.68 (m, 1H), 1.71–1.63 (m, 1H), 1.67–1.59 (m, 1H), 1.64–1.54 (m, 1H), 1.56–1.46 (m, 1H), 1.52–1.42 (m, 1H), 1.43–1.33 (m, 1H), 1.39–1.27 (m, 1H), 1.36–1.30 (m, 1H), 1.36–1.29 (m, 1H), 1.28–1.16 (m, 1H), 1.27–1.20 (m, 1H), 1.26–1.20 (m, 1H), 1.18–1.09 (m, 1H), 1.15–1.06 (m, 1H), 1.15–1.05 (m, 2H), 1.04–0.94 (m, 1H), 0.91 (d, J = 6.4 Hz, 3H), 0.873 (d, J = 6.4 Hz, 3H), 0.869 (d, J = 6.8 Hz, 3H), 0.75 (s, 3H); 13 C NMR (101 MHz, CDCl3) δ 154.1, 143.0, 131.1, 127.7, 115.8, 112.7, 74.4, 55.8, 52.8, 43.4, 43.1, 39.6, 37.8, 36.2, 35.8, 32.3, 30.2, 29.6, 28.9, 28.2, 24.4, 23.9, 23.0, 22.7, 18.8, 18.6, 12.1; IR (KBr): ν max = 3590, 2955, 2885, 1770, 1747, 1462, 1099, 835, 779 cm -1 ; HRMS (ESI, m / z): calcd for C 27 H 48 NO2 [M+NH4] + 418.3680, found 418.3682.

[0189] Effect Example

[0190] The antibacterial activity of the secosteroid compounds was tested against vancomycin-resistant Enterococcus faecalis (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative bacteria Pseudomonas aeruginosa (PA).

[0191] The specific procedure is as follows: VRE and PA strains were cultured in BHI broth for 16 hours, and MRSA strains were cultured in LB broth under anaerobes for 16 hours. Secosteroid compounds were dissolved in dimethyl sulfoxide to prepare stock solutions (10.24 mg / mL). In a 96-well microplate incubator, the compound stock solution was diluted into 150 μL of BHI or LB, depending on the strain, to a final concentration of 1024 to 2 μg / mL. Then, 150 μL of the test strain was added to each well of the microplate. The plates were then incubated at 37°C for 20 hours. The MIC was determined by visual inspection of the microplates. The lowest concentration of 9,10-steroid that eliminated cell growth in the well (in μg / mL) was reported as the MIC value.

[0192] The results are shown in Table 1 . Low concentrations of 9,10-secoesteroid compounds exhibited anti-VRE activity.

[0193] Table 1

[0194]

[0195] discuss

[0196] The kinetic analysis of the conversion of 1a to the ring-opening product 3a catalyzed by KshA3 / KshB revealed a kcat of 6.36 min -1 , Km is 1.69x 10 2 μM, kcat / Km is 3.76x 10 -2 μM -1 min -1 The kinetic analysis of the conversion of 4a to 5a showed that its kcat was 3.49 min -1 , Km is 1.31x 10 2 μM, kcat / Km is 2.66x10 -2 μM -1 min -1 .

[0197] Substrates 1a and 4a differ only in the presence of a double bond at C1-C2. Double-bonded substrates 1a to 3a undergo a two-step reaction, C9-hydroxylation and B-ring opening, while double-bond-free substrates 4a to 5a undergo a single-step hydroxylation reaction. The catalytic efficiencies (kcat / Km) are comparable between the two, indicating that KshA / KshB primarily perform the C9-hydroxylation step in the conversion of 1a to 3a to 2a, with the subsequent ring-opening step from 2a to 3a occurring spontaneously.

[0198] Therefore, the C1-C2 double bond structure is extremely important for the formation of 9,10-opened steroid compounds.

[0199] Table 2 Sequence Listing

[0200]

[0201]

[0202] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for preparing a compound of formula III, characterized in that: Including steps: (a) in a liquid reaction system, using a compound of formula I as a substrate, in the presence of KshA and KshB enzymes, conducting an enzymatic reaction to obtain a compound of formula II, which then undergoes a ring-opening reaction to obtain a compound of formula III; Optionally (b) isolating the compound of formula III from the reaction system; Among various Indicates single bond / double bond; When connecting R1 When it is a single bond, R1 is selected from R5 or When connecting R1 When it is a double bond, R1 is O; When connecting R4 When it is a single bond, R4 is OH; When connecting R4 When it is a double bond, R4 is O; R2 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R3 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R5 is selected from: R6 is selected from: OH, OZ or C1-C6 alkoxy, wherein Z is a protecting group; R7 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R8 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R9 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; The substitution refers to substitution by one or more substituent groups selected from the group consisting of halogen or hydroxyl.

2. The method according to claim 1, wherein The KshA enzyme is selected from the group consisting of: KshA1, KshA3, KshA5, KshA N or a combination thereof.

3. The method according to claim 1, wherein The KshB enzyme is selected from: KshB, KshB N or a combination thereof.

4. The method according to claim 1, wherein The liquid reaction system comprises water and an organic solvent, and optionally, a co-solvent.

5. The method according to claim 4, wherein The organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, acetone, dimethylformamide, dimethyl sulfoxide, or a combination thereof.

6. The method according to claim 4, wherein The cosolvent includes cyclodextrin.

7. The method according to claim 1, wherein The substrate concentration in the liquid reaction system is 0.05-5 mmol / L.

8. A reaction system, characterized in that The reaction system comprises: (i) a reaction solvent; (ii) a substrate, wherein the substrate is a compound represented by formula I; and (iii) KshA enzyme and KshB enzyme; Where, Indicates single bond / double bond; when When it is a single bond, R1 is selected from R5 or when When it is a double bond, R1 is O; R2 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R3 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R5 is selected from: R6 is selected from: OH, OZ or substituted or unsubstituted C1-C6 alkoxy, wherein Z is a protecting group; R7 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R8 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R9 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; The substitution refers to substitution by one or more substituent groups selected from the group consisting of halogen or hydroxyl.

9. The reaction system according to claim 8, wherein The reaction solvent comprises water and an organic solvent, optionally a co-solvent.

10. A method for preparing a compound of formula III or formula IIIa, characterized in that: Including steps: Using the reaction system as claimed in claim 8 or 9, thereby preparing the compound of formula III or formula IIIa; Among various Indicates single bond / double bond; When connecting R1 When it is a single bond, R1 is selected from R5 or When connecting R1 When it is a double bond, R1 is O; When connecting R4 When it is a single bond, R4 is OH; When connecting R4 When it is a double bond, R4 is O; R2 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R3 is selected from: H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R5 is selected from: R6 is selected from: OH, OZ or C1-C6 alkoxy, wherein Z is a protecting group; R7 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R8 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; R9 is selected from H, OH, substituted or unsubstituted C1-C6 alkyl or C1-C6 alkoxy; in, The substitution refers to substitution by one or more substituents selected from the group consisting of halogen or hydroxy.

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