Heteroditerpenoid compound containing four-membered ring, preparation method and application thereof, and pharmaceutical composition
阅读说明：本技术 一种含四元环的杂二萜类化合物、其制备方法和应用，以及药物组合物 (Heteroditerpenoid compound containing four-membered ring, preparation method and application thereof, and pharmaceutical composition ) 是由 普诺·白玛丹增 颜秉超 周敏 杜雪 李昂 孙汉董 于 2021-01-21 设计创作，主要内容包括：一种含四元环的杂二萜类化合物,涉及药物技术领域,以及它们的制备方法和应用,该杂二萜类化合物的结构新颖,对于T、B淋巴细胞增殖具有抑制作用,对T细胞的抑制作用具有选择性,可广泛应用于制备免疫抑制药物中。该杂二萜类化合物的制备方法不仅操作简单,而且条件温和,可以快速高效地得到上述杂二萜类化合物。一种药物组合物,其包括上述杂二萜类化合物,以及药学上可接受的辅料。其具有较好的免疫抑制活性,可作为免疫抑制剂使用。(A hetero-diterpenoid compound containing a four-membered ring relates to the technical field of medicines, and a preparation method and application thereof, and the hetero-diterpenoid compound has a novel structure, has an inhibition effect on T, B lymphocyte proliferation and a selectivity on the inhibition effect on T cells, and can be widely applied to preparation of immunosuppressive medicines. The preparation method of the hetero-diterpenoid compound is simple to operate, mild in condition and capable of quickly and efficiently obtaining the hetero-diterpenoid compound. A pharmaceutical composition comprises the heteroditerpenoid compound and pharmaceutically acceptable auxiliary materials. It has good immunosuppressive activity, and can be used as immunosuppressant.)
1. The tetratomic ring-containing hetero-diterpenoid compound is characterized by comprising a hetero-diterpenoid compound A and a hetero-diterpenoid compound B, wherein the structural formula of the hetero-diterpenoid compound A is shown in the specification
The structural formula of the heteroditerpenoid compound B is shown in the specification
In the formula, R1Selected from hydrogen, C1-C6 alkyl, C1-C6 substituted alkyl, C1-C6 alkoxy, C1-C6 substituted alkoxy or halogen; r1The number of the (b) can be 1-3, and the binding site is at least one of five unsubstituted sites on a benzene ring; r2、R3Are respectively and independently selected from C1-C6 alkyl or C1-C6 substituted alkyl; r4Selected from hydrogen, C1-C6 alkyl or C1-C6 substituted alkyl; r5Selected from hydrogen, C1-C6 alkyl, C1-C6 substituted alkyl or C1-C6 acyl; r4、R5Not hydrogen at the same time.
2. The four-membered ring hetero diterpenoid-containing compound according to claim 1, wherein R is1Selected from hydrogen, C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkoxy, C1-C4 substituted alkoxy or halogen; r1The number of the (b) can be 1-3, and the binding site is at least one of five unsubstituted sites on a benzene ring; r2、R3Are respectively and independently selected from C1-C5 alkyl or C1-C5 substituted alkyl; r4Selected from hydrogen, C1-C4 alkyl or C1-C4 substituted alkyl; r5Selected from hydrogen, C1-C4 alkyl, C1-C4 substituted alkyl or C1-C4 acyl.
3. The tetramembered ring containing heteroditerpenoid compound according to claim 1, wherein the structural formula of the heteroditerpenoid compound is
4. A method for preparing the tetratomic ring-containing hetero-diterpenoid compound according to any one of claims 1 to 3, which comprises:
oxidizing a compound I and then carrying out deprotection to obtain the heteroditerpenoid compound A;
esterifying or etherifying the heteroditerpenoid compound A to obtain a heteroditerpenoid compound B;
wherein the structural formula of the compound I is
Wherein PG is a hydroxyl protecting group selected from TBDPS, TMS, TES, TBDMS or TIPS.
5. The preparation method according to claim 4, wherein the compound I is obtained by oxidizing the compound II and then esterifying the compound II with an enol compound;
wherein the structural formula of the compound II isThe structural formula of the enol compound is shown in the specification
6. The preparation method according to claim 5, wherein the compound II is obtained by oxidizing the compound III and then coupling with halogenated olefin;
wherein the structural formula of the compound III isThe structural formula of the halogenated olefin is shown in the specification
Wherein X is selected from chlorine, bromine or iodine.
7. The preparation method according to claim 6, wherein the compound III is obtained by performing a hydroboration oxidation after a compound IV undergoes a subunit reaction;
wherein the structural formula of the compound IV is
8. The preparation method according to claim 7, wherein the compound IV is obtained by performing a [2+2] cycloaddition reaction on a raw material V and a raw material VI;
wherein the structural formula of the raw material V isThe structural formula of the raw material VI isIn the formula, R6Selected from C1-C6 alkyl.
9. Use of a heteroditerpenoid according to any one of claims 1 to 3 for the preparation of an immunosuppressive medicament.
10. A pharmaceutical composition comprising the heteroditerpenoid of any one of claims 1 to 3 and a pharmaceutically acceptable excipient.
Technical Field
The invention relates to the technical field of medicines, in particular to a tetratomic ring-containing hetero-diterpenoid compound, a preparation method and application thereof, and a pharmaceutical composition.
Background
Rabdosia plant is one of the important medicinal plant resources in China, wherein rabdosia rubescens is already recorded by Chinese pharmacopoeia, and various plants of the rabdosia plant are widely used as herbal medicines in China, and are used for clearing heat and removing toxicity, resisting inflammation and bacteria, resisting tumors and the like. The plant is rich in diterpene compounds, has various structures, and has activity in resisting tumor, resisting bacteria, and suppressing immunity, such as rubescensin A, eriocalyxin B and early-onset Rabdosia Rubescens (Hemsl.) Hara B with significant anti-tumor activity. Isodon scoparia (Isodon scoparia C.Y.Wu et H.W.Li (Dunn) Kudo) belongs to Rabdosia of Labiatae, is mainly distributed in Yunnan province of China, and is used for treating digestive system inflammation and external infection of people and livestock in folk. Research shows that the plant contains diterpene compounds with novel structures, takes bicyclic diterpene as a main structure type, and is accompanied with a plurality of heteroditerpene compounds. However, there is no good access to these heteroditerpenoids in the prior art, and both the extraction technology from natural products and the artificial synthesis technology are still in need of further development.
Disclosure of Invention
The first purpose of the invention is to provide a tetratomic ring-containing hetero-diterpenoid compound which has novel structure, wide source and larger medicinal value.
The second objective of the present invention is to provide a method for preparing the tetratomic ring-containing hetero-diterpenoid compound, which has the advantages of simple and convenient operation, mild reaction conditions, and rapid and efficient preparation of the hetero-diterpenoid compound.
The third objective of the invention is to provide an application of the tetratomic ring-containing hetero-diterpenoid compound, which has a certain immunosuppressive activity and can be widely applied to the preparation of immunosuppressive drugs.
The fourth purpose of the present invention is to provide a pharmaceutical composition containing the aforementioned heteroditerpenoid compounds, which has better immunosuppressive activity.
The embodiment of the invention is realized by the following steps:
a tetratomic ring-containing hetero-diterpenoid compound comprises a hetero-diterpenoid compound A and a hetero-diterpenoid compound B, wherein the structural formula of the hetero-diterpenoid compound A is shown in the specification
The structural formula of the heteroditerpenoid compound B is shown in the specification
In the formula, R1Selected from hydrogen, C1-C6 alkyl, C1-C6 substituted alkyl, C1-C6 alkoxy, C1-C6 substituted alkoxy or halogen; r1The number of the (b) can be 1-3, and the binding site is at least one of five unsubstituted sites on a benzene ring; r2、R3Are respectively and independently selected from C1-C6 alkyl or C1-C6 substituted alkyl; r4Selected from hydrogen, C1-C6 alkyl or C1-C6 substituted alkyl; r5Selected from hydrogen, C1-C6 alkyl, C1-C6 substituted alkyl or C1-C6 acyl; r4、R5Not hydrogen at the same time.
A method of making the four-membered ring-containing heteroditerpenoid of claim, comprising:
oxidizing the compound I and then carrying out deprotection to obtain a heteroditerpenoid compound A;
esterifying or etherifying the heteroditerpenoid compound A to obtain a heteroditerpenoid compound B;
wherein the structural formula of the compound I is
Wherein PG is a hydroxyl protecting group selected from TBDPS, TMS, TES, TBDMS or TIPS.
An application of the heteroditerpenoid compound in the preparation of immunosuppressive drugs is provided.
A pharmaceutical composition comprises the heteroditerpenoid compound and pharmaceutically acceptable auxiliary materials.
The embodiment of the invention has the beneficial effects that:
the embodiment of the invention provides a hetero-diterpenoid compound containing a four-membered ring, and a preparation method and application thereof, wherein the hetero-diterpenoid compound has a novel structure, has an inhibition effect on T, B lymphocyte proliferation and a selectivity on the inhibition effect on T cells, and can be widely applied to preparation of immunosuppressive drugs. The preparation method of the hetero-diterpenoid compound is simple to operate, mild in condition and capable of quickly and efficiently obtaining the hetero-diterpenoid compound.
The embodiment of the invention also provides a pharmaceutical composition which comprises the heteroditerpenoid compound and pharmaceutically acceptable auxiliary materials. It has good immunosuppressive activity, and can be used as immunosuppressant.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows a starting material V-a provided in example 1 of the present invention13C NMR spectrum;
FIG. 2 is a diagram of compound IV-a provided in example 1 of the present invention13C NMR spectrum;
FIG. 3 is a diagram of compound III-a provided in example 3 of the present invention13C NMR spectrum;
FIG. 4 is a drawing of an intermediate provided in example 5 of the present invention13C NMR spectrum;
FIG. 5 is a diagram of Compound II-a provided in example 5 of the present invention13C NMR spectrum;
FIG. 6 is a drawing of Compound I-a as provided in example 7 of the present invention13C NMR spectrum;
FIG. 7 shows the preparation of the hetero-diterpenoid compound A-a provided in example 9 of the present invention13C NMR spectrum.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following describes the tetratomic ring-containing hetero-diterpenoid compound, the preparation method and the application thereof, and the pharmaceutical composition of the embodiment of the invention.
The embodiment of the invention provides a tetratomic ring-containing hetero-diterpenoid compound, which comprises a hetero-diterpenoid compound A and a hetero-diterpenoid compound B, wherein the structural formula of the hetero-diterpenoid compound A is shown in the specification
The structural formula of the heteroditerpenoid compound B is shown in the specification
In the formula, R1Selected from hydrogen, C1-C6 alkyl, C1-C6 substituted alkyl, C1-C6 alkoxy, C1-C6 substituted alkoxy or halogen; r1The number of the (b) can be 1-3, and the binding site is at least one of five unsubstituted sites on a benzene ring; r2、R3Are respectively and independently selected from C1-C6 alkyl or C1-C6 substituted alkyl; r4Selected from hydrogen, C1-C6 alkyl or C1-C6 substituted alkyl; r5Selected from hydrogen, C1-C6 alkyl, C1-C6 substituted alkyl or C1-C6 acyl; r4、R5Not hydrogen at the same time.
Wherein, the alkyl group of C1-C6 may be a straight chain alkyl group or a branched chain alkyl group, including but not limited to methyl, ethyl, n-propyl, isopropyl, tert-butyl, etc. The C1-C6 substituted alkyl refers to a C1-C6 alkyl group in which at least one hydrogen atom is substituted by halogen, hydroxyl, alkoxy or aryl. C1 to C6 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy and the like. The C1-C6 substituted alkoxy refers to a group formed by substituting at least one hydrogen atom in C1-C6 alkyl with halogen, hydroxyl, alkoxy or aryl, and includes but is not limited to 2-methoxyethoxy, 3-methoxypropoxy and the like. C3-C8 cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclohexyl, methylcyclohexyl, and the like. C5-C10 aryl includes, but is not limited to, phenyl, pyridyl, naphthyl, and the like. The C5-C10 substituted aryl refers to a C5-C10 aryl group in which at least one hydrogen atom is substituted by halogen, hydroxyl, alkoxy or aryl.
Further, R1The number of (a) may be 1 to 3, and the binding site thereof is at least one of five unsubstituted sites on the benzene ring. That is, there may be a single R on the same phenyl ring1Substituted by radicals, also 2 or 3R1And (4) substituting the group. In the presence of 2 or 3R1When substituted by radicals, each R1The groups may be the same or different groups.
Preferably, R1Selected from hydrogen, C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkoxy, C1-C4 substituted alkoxy or halogen; r1The number of the (b) can be 1-3, and the binding site is at least one of five unsubstituted sites on a benzene ring; r2、R3Are respectively and independently selected from C1-C5 alkyl or C1-C5 substituted alkyl; r4Selected from hydrogen, C1-C4 alkyl or C1-C4 substituted alkyl; r5Selected from hydrogen, C1-C4 alkyl, C1-C4 substituted alkyl or C1-C4 acyl.
More preferably, the structural formula of the heteroditerpenoid compound is shown in the specification
The above structure is also called scopariusin A, which is a natural product separated from stem or leaf of Rabdosia kolomikta.
The embodiment of the invention also provides a preparation method of the compound containing the four-membered ring hetero-diterpenoid, which comprises the following steps:
oxidizing the compound I and then carrying out deprotection to obtain a heteroditerpenoid compound A;
esterifying or etherifying the heteroditerpenoid compound A to obtain a heteroditerpenoid compound B; wherein the structural formula of the compound I is
Wherein PG is a hydroxyl protecting group selected from TBDPS, TMS, TES, TBDMS or TIPS.
The oxidation of the compounds I is effected by means of an oxidizing agent, for example sodium chlorite can be used as oxidizing agent. The oxidant can oxidize aldehyde groups in the compound I into carboxyl groups, and then the protecting groups on the hydroxyl groups are removed to obtain the hetero-diterpenoid compound A. The mass of the oxidant is 0.1-1 times of that of the compound I, the aldehyde group is sufficiently oxidized under the proportion, and the heteroditerpenoid compound A can be obtained with high yield. The reaction is carried out in a mixed solvent of any one of organic solvents and water, and the organic solvent used may be analytically or chromatographically pure t-butanol, chloroform, DMSO, dichloromethane. During the reaction, proper amount of isopentene and sodium dihydrogen phosphate may be added to speed the reaction.
The deprotection of the oxidized compound I can be performed by a conventional hydroxyl protecting group removing method depending on the hydroxyl protecting group, and for example, TBDPS can be performed by removing TBAF (tetrabutylammonium fluoride) in a tetrahydrofuran solution.
The hetero diterpenoid compound B is a derivative of the hetero diterpenoid compound A, and is obtained by esterification or etherification reaction of the hetero diterpenoid compound A, and specific esterification or etherification conditions can be carried out by referring to methods in the prior art, which are not described herein again.
Further, the compound I is obtained by oxidizing the compound II and then esterifying the compound II with an enolic compound;
wherein the structural formula of the compound II isThe structural formula of the enol compound is shown in the specification
The oxidation of the compound II is to oxidize the hydroxyl group of the compound II into an aldehyde group, so as to prevent the self-esterification of two molecules of the compound II in the subsequent esterification process with an enolic compound. Meanwhile, the degree of oxidation needs to be mastered, hydroxyl groups cannot be further oxidized into carboxyl groups, and the oxidized carboxyl groups are prevented from participating in subsequent esterification reaction. Thus, to control the extent of oxidation, a bose-martin oxidation can be employed, which is stopped at the level of aldehyde groups formed by reacting compound II with a commercially available dess-martin reagent. Alternatively, the mass of the dess-martin reagent is 0.1 to 1 times that of the compound II, and the oxidation of the hydroxyl group is sufficient at the above ratio. The compound II after oxidation is subjected to esterification reaction by carboxyl and hydroxyl of an enol compound, and the reaction can be carried out under EDC and DMAP conditions.
Further, the compound II is obtained by oxidizing the compound III and then coupling with halogenated olefin;
wherein the structural formula of the compound III isThe halogenated olefin has the structural formula
Wherein X is selected from chlorine, bromine or iodine.
The oxidation of compound III is catalyzed by a high iodine reagent and ABNO, which may be, for example, iodobenzene diacetate. The higher iodine reagent can efficiently oxidize the hydroxyl group of compound III to the desired carboxyl group. Optionally, the mass of the high-valence iodine reagent is 0.1-1 times of that of the compound III, and under the condition, the oxidation efficiency is better.
The coupling reaction of the oxidized compound III is carried out under the catalysis of a metal catalyst and a ligand, and the metal catalyst can be a palladium reagent, a nickel reagent and the like. For example, nickel diiodide is used as a metal catalyst and 4,4 '-dimethoxy-2, 2' -bipyridine is used as a ligand.
Further, the compound III is obtained by hydroboration oxidation after the reaction of the compound IV and a methylene reaction reagent;
wherein the structural formula of the compound IV is
This reaction is a carburisation reaction by diiodomethane, which adds one carbon atom to the carbonyl site of compound IV. The reaction is carried out under metal catalysis, for example a system consisting of lead dichloride, zinc powder and zirconium tetrachloride. Obtaining hydroxyl after hydroboration and oxidation after the reaction is finished.
Further, the compound IV is obtained by performing cycloaddition reaction on the raw material V and the raw material VI;
wherein the structural formula of the raw material V isThe structural formula of the raw material VI isIn the formula, R6Selected from C1-C6 alkyl.
The raw material V and the raw material VI are both raw materials which can be obtained by simple steps and have wide sources. The application constructs 6/6/4 fused ring skeleton through [2+2] cycloaddition reaction between raw material V and raw material VI, and similar reports in the prior art do not exist. The cycloaddition reaction is to react ketene imine salt formed in situ with the raw material V to generate imine salt, and hydrolyze the imine salt under the condition of neutral medium or alkaline water after the reaction is finished.
Further, the embodiment of the invention also provides an application of the heteroditerpenoid compound in preparation of immunosuppressive drugs. It has inhibitory effect on T, B lymphocyte proliferation, selective effect on T cell inhibition, IC50The value can reach 0.68 μm.
Further, the embodiment of the invention also provides a pharmaceutical composition, which comprises the heteroditerpenoid compound and pharmaceutically acceptable auxiliary materials. It has better immunosuppressive activity. Has better application prospect.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example provides a process for the preparation of compound IV-a having the formula:
the preparation method comprises the following specific steps:
dissolving 0.01-100 g of raw material V-a and VI-a in 1-100 mL of 1, 2-dichloroethane or dichloromethane, adding 0.01-10 mL of organic base, and dropwise adding Tf at-40-25 deg.C2Reacting O (0.01-20 mL) in 1, 2-dichloroethane or dichloromethane at-20-25 ℃ for 1-48 hours, detecting the disappearance of raw materials by using a dot plate, adding 1-200 mL of inorganic alkaline water or water, stirring at room temperature or under heating for 1-10 hours, detecting no more new products by using the dot plate, extracting for 2-6 times by using an equal volume of organic solvent, combining filtrates, concentrating a sample on a rotary evaporator, obtaining a white solid by various purification methods, and identifying the white solid as a compound IV-a (the yield is 79-93%) by mass spectrometry and one-dimensional and two-dimensional nuclear magnetic resonance.
Wherein the organic base is pyridine base such as 2-fluoropyridine, 2-bromopyridine, 2-iodopyridine, 2-chloropyridine, 3-chloropyridine, 2,4, 6-trimethylpyridine, 2, 6-di-tert-butyl-4-methylpyridine, etc. The inorganic base is sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate or potassium bicarbonate. The organic solvent is industrial, analytical or chromatographic pure ethyl acetate, chloroform, dichloromethane or 1, 2-dichloroethane.
Compound IV-a is characterized as follows:
(c ═ 0.19, chloroform);1H NMR(400MHz,CDCl3):δ＝7.76–7.63(m,4H),7.48–7.31(m,6H),6.74(d,J＝8.5Hz,2H),6.70(d,J＝8.5Hz,2H),4.03(s,1H),3.59–3.50(m,1H),3.45–3.35(m,1H),3.28(d,J＝7.3Hz,1H),2.23–2.12(m,1H),2.12–2.05(m,1H),1.99–1.87(m,1H),1.66(d,J＝12.7Hz,1H),1.56–1.32(m,5H),1.21–1.05(m,11H),0.99(dd,J＝13.5,3.6Hz,1H),0.92(s,3H),0.91(s,3H),0.85(s,3H),0.82(s,3H)ppm；13C NMR(126MHz,CDCl3):δ＝212.25,154.88,135.67,135.64,132.96,132.91,130.06,130.05,129.57,128.28,127.91,127.89,120.17,76.53,66.21,57.09,51.29,41.88,39.47,37.88,37.69,34.14,33.49,33.27,30.21,26.66,21.47,20.46,19.60,18.47,18.22,15.19ppm；HRESIMS(m/z):[M+NH4]+calcd for C40H55BrNO2Si+688.3180,found 688.3175.
example 2
This example provides a process for the preparation of compound IV-a having the formula:
the preparation method comprises the following specific steps:
dissolving a raw material V-b (0.01-100 g) and a raw material VI-b in 1-100 mL of 1, 2-dichloroethane or dichloromethane, adding 0.01-10 mL of organic base, and dropwise adding Tf at-40-25 DEG C2Reacting O (0.01-20 mL) in 1, 2-dichloroethane or dichloromethane at-20-25 deg.C for 1-48 hr, detecting the disappearance of raw material, adding 1-200 mL of inorganic alkaline aqueous solution or water, stirring at room temperature or under heatingStirring for 1-10 hours, detecting that new products are not increased by using a dot plate, extracting for 2-6 times by using an equal volume of organic solvent, combining filtrates, concentrating a sample on a rotary evaporator, obtaining a white solid by various purification means, and identifying the white solid as a compound IV-b (the yield is 45-56%) by mass spectrometry and one-dimensional and two-dimensional nuclear magnetic resonance.
Example 3
This example provides a process for the preparation of compound III-a having the formula:
the preparation method comprises the following specific steps:
slowly dropwise adding diiodomethane (0.01-10 mL) into THF (0.5-300 mL) containing lead dichloride (0.01-100 g) and zinc powder (0.01-100 g) at 0 ℃ under stirring, stirring for reaction for 0.5-1 hour, simultaneously adding anhydrous zirconium chloride (0.01-100 g) into another reaction bottle, carefully adding THF (0.5-300 mL), stirring for 0.5-1 hour at room temperature, adding the suspension into the former reaction bottle, continuously stirring for 0.5-2 hours at 0 ℃ until the system becomes dark green, adding a tetrahydrofuran solution of a compound IV-a into the reaction bottle, reacting for 0.5-6 hours at 0 ℃, detecting that the raw material disappears by using a point plate, adding an inorganic alkaline solution or water 1-500 mL for quenching reaction, extracting for 2-6 times by using an isovolumetric organic solvent, combining filtrates, and concentrating on a rotary evaporator to obtain a crude product.
Dissolving the crude product in tetrahydrofuran solution, and slowly adding the solution into prepared Sia at the temperature of-20-5 DEG C2Adding 0.5-time volume of 2-methyl-2-butene into dimethyl sulfide-borane at the temperature of-20 ℃, stirring for 0.5-3 hours at the temperature of 0 ℃, continuously stirring for 1-10 hours until the plate detection raw materials disappear, adding 0.01-20 mL of hydrogen peroxide and 0.01-20 mL of saturated inorganic alkaline water solution, stirring for 0.5-6 hours at room temperature, quenching reaction by using saturated sodium thiosulfate or sodium sulfite (0.01-50 mL) solution until the color is developed by using a potassium iodide test paper, extracting for 2-6 times by using an isovolumetric organic solvent after the color is not changed, combining filtrates, and performing rotary evaporation to obtain the productSamples are concentrated on the instrument, white solids are obtained by various purification means, and the white solids are identified as the compound III-a (the yield is 65-84%) by mass spectrum, one-dimensional nuclear magnetic resonance and two-dimensional nuclear magnetic resonance.
Wherein the inorganic base is sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, or potassium bicarbonate. The organic solvent is industrial, analytical or chromatographic pure ethyl acetate, chloroform or dichloromethane.
The characterization of compound III-a is as follows:
(c ═ 0.13, methanol);1H NMR(500MHz,CDCl3):δ＝7.73–7.67(m,4H),7.44–7.38(m,2H),7.38–7.33(m,4H),6.95(d,J＝8.5Hz,2H),6.70(d,J＝8.5Hz,2H),4.00(dd,J＝10.6,7.6Hz,1H),3.91(dd,J＝10.6,7.6Hz,1H),3.36–3.29(m,1H),3.18–3.10(m,1H),2.82(d,J＝6.1Hz,1H),2.70–2.62(m,1H),2.54(t,J＝9.6Hz,1H),2.00–1.92(m,1H),1.81(dd,J＝14.8,3.9Hz,1H),1.72–1.61(m,3H),1.49–1.38(m,4H),1.27–1.18(m,2H),1.10(s,9H),0.98(td,J＝12.5,3.9Hz,1H),0.92(s,3H),0.82(s,3H),0.75(s,3H),0.65(s,3H)；13C NMR(151MHz,CDCl3):δ＝154.24,135.72,135.71,133.99,133.22,129.97,129.96,129.50,127.85,127.83,119.55,65.86,59.52,55.72,52.17,42.80,42.44,40.85,40.10,40.07,37.47,35.40,33.90,33.33,29.76,26.71,22.30,21.43,19.81,19.63,18.71,15.64ppm；HRESIMS(m/z):[M+NH4]+calcd for C41H59BrNO2Si+704.3493,found 704.3489.
example 4
This example provides a process for the preparation of compound III-b, having the formula:
the preparation method comprises the following specific steps:
slowly dropwise adding diiodomethane (0.01-10 mL) into THF (0.5-300 mL) containing lead dichloride (0.01-100 g) and zinc powder (0.01-100 g) at 0 ℃ under stirring, stirring for reaction for 0.5-1 hour, simultaneously adding anhydrous zirconium chloride (0.01-100 g) into another reaction bottle, carefully adding THF (0.5-300 mL), stirring for 0.5-1 hour at room temperature, adding the suspension into the former reaction bottle, continuously stirring for 0.5-2 hours at 0 ℃ until the system becomes dark green, adding a tetrahydrofuran solution of a compound IV-b into the reaction bottle, reacting for 0.5-6 hours at 0 ℃, detecting that the raw material disappears by using a point plate, adding an inorganic alkaline solution or water 1-500 mL for quenching reaction, extracting for 2-6 times by using an isovolumetric organic solvent, combining filtrates, and concentrating on a rotary evaporator to obtain a crude product.
Dissolving the crude product in tetrahydrofuran solution, and slowly adding the solution into prepared Sia at the temperature of-20-5 DEG C2Adding 0.5 volume time of 2-methyl-2-butene into dimethyl sulfide-borane at the temperature of-20 ℃, stirring for 0.5-3 hours at the temperature of 0 ℃, continuously stirring for 1-10 hours until plate detection raw materials disappear, adding hydrogen peroxide (0.01-20 mL) and saturated inorganic alkaline water solution (0.01-20 mL), stirring for 0.5-6 hours at room temperature, quenching reaction by using saturated sodium thiosulfate or sodium sulfite (0.01-50 mL) solution until potassium iodide paper develops color and the color does not change blue, extracting for 2-6 times by using an isovolumetric organic solvent, combining filtrates, concentrating samples on a rotary evaporator, and obtaining white solids by various purification means, wherein the white solids are identified as compounds III-b (the yield is 37-48%) through mass spectrometry and one-dimensional and two-dimensional nuclear magnetic resonance.
Example 5
This example provides a process for the preparation of compound II-a, having the formula:
the preparation method comprises the following specific steps:
dissolving a compound III-a (0.01-10 g) in a dichloromethane solvent (0.02-20 mL) and a pH 6.8 phosphate buffer solution (0.02-20 mL), adding iodobenzene diacetate (0.01-10 g) and a 9-azabicyclo [3.3.1] nonane-N-oxyl radical (ABNO, 0.001-1 g), stirring at room temperature for 1-12 hours, detecting by TLC, removing the raw material, quenching with a saturated sodium bisulfite or sodium sulfite or sodium thiosulfate aqueous solution (1-20 mL), extracting with an equal volume of an organic solvent for 2-6 times, combining filtrates, concentrating the sample on a rotary evaporator, and obtaining a white solid by various purification means, and identifying the compound as an intermediate by mass spectrometry, one-dimensional nuclear magnetic resonance and two-dimensional nuclear magnetic resonance (the yield is 72-84%).
Dissolving the intermediate (0.01-10 g) in a solvent N, N-dimethylformamide or N, N-dimethylacetamide or N-methylpyrrolidone (0.01-40 mL), adding pyridine (0.001-4 mL) and TBSCl (0.01-3 g), stirring for 0.2-2 hours, adding 2-bromo-2-butenol (0.01-3 g), sodium iodide (0.01-3 g), nickel diiodide (0.01-3 g), 4 '-dimethoxy-2, 2' -bipyridine (0.01-3 g), and manganese powder (0.01-3 g), stirring for 1-48 hours at 40-80 ℃ until the plate detects the disappearance of the raw materials, adjusting the pH to less than 3 with a dilute acid solution, extracting for 2-6 times with an isovolumetric organic solvent, combining filtrates, concentrating the sample on a rotary evaporator, and obtaining white solids by various purification means, the compound is identified as compound II-a (yield 68-79%) by mass spectrum, one-dimensional and two-dimensional nuclear magnetic resonance.
Wherein the dilute acid is hydrochloric acid, sulfuric acid, phosphoric acid; the organic solvent is industrial, analytical or chromatographic pure ethyl acetate, chloroform or dichloromethane.
The intermediates were characterized as follows:
(c ═ 0.12, chloroform);1H NMR(600MHz,CDCl3):δ＝7.66–7.60(m,4H),7.37–7.31(m,2H),7.32–7.25(m,4H),6.87(d,J＝8.5Hz,2H),6.65(d,J＝8.5Hz,2H),3.44(d,J＝4.9Hz,1H),3.36–3.26(m,2H),3.15(dd,J＝12.3,4.9Hz,1H),2.79(dd,J＝12.3,7.5Hz,1H),1.99–1.92(m,1H),1.83(dd,J＝14.8,2.7Hz,1H),1.74(dd,J＝8.3,4.1Hz,1H),1.69–1.61(m,1H),1.56(dt,J＝12.0,2.4Hz,1H),1.47–1.40(m,2H),1.36–1.25(m,2H),1.12–1.02(m,11H),0.88(td,J＝12.8,3.2Hz,1H),0.78(s,3H),0.72(s,3H),0.68(s,3H),0.54(s,3H)ppm；13C NMR(151MHz,CDCl3):δ＝179.39,154.40,135.70,135.68,133.12,133.09,132.83,130.01,130.00,129.43,127.88,127.85,119.77,57.51,51.18,50.03,43.32,42.79,42.51,42.27,39.69,37.31,34.63,33.59,33.34,30.59,26.69,21.42,21.39,20.44,19.62,18.58,15.35ppm；HRESIMS(m/z):[M+NH4]+calcd for C41H57BrNO3Si+,718.3286,found 718.3283.
compound II-a is characterized as follows:
(c ═ 0.18, chloroform);1H NMR(600MHz,CDCl3):δ＝7.72–7.67(m,4H),7.43–7.39(m,2H),7.37–7.32(m,4H),6.93(d,J＝8.6Hz,2H),6.71(d,J＝8.6Hz,2H),5.44–5.38(m,1H),4.20(dd,J＝12.2,7.5Hz,1H),4.09(dd,J＝12.2,6.2Hz,1H),3.40(d,J＝4.3Hz,1H),3.17(dd,J＝12.3,4.3Hz,1H),2.86(dd,J＝12.3,7.2Hz,1H),2.07(t,J＝7.6Hz,2H),1.91(dd,J＝14.6,2.7Hz,1H),1.73–1.70(m,1H),1.69–1.64(m,4H),1.53–1.46(m,2H),1.42–1.32(m,2H),1.26–1.15(m,2H),1.09(s,11H),1.03–0.96(m,1H),0.87(s,3H),0.79(s,3H),0.76(s,3H),0.59(s,3H)ppm；13C NMR(151MHz,CDCl3):δ＝179.21,154.11,139.94,135.69,133.59,133.19,129.96,129.49,127.84,124.16,119.58,59.46,55.99,50.73,50.20,43.34,43.28,43.22,42.51,40.04,39.77,37.90,33.58,33.33,26.69,24.23,21.51,21.32,20.50,19.62,18.67,16.70,15.22ppm；HRESIMS(m/z):[M+Na]+calcd for C45H60O4SiNa+715.4153,found 715.4154。
example 6
This example provides a process for the preparation of compound II-b, having the formula:
the preparation method comprises the following specific steps:
dissolving a compound III-b (0.01-10 g) in a dichloromethane solvent (0.02-20 mL) and a pH 6.8 phosphate buffer solution (0.02-20 mL), adding iodobenzene diacetate (0.01-10 g) and a 9-azabicyclo [3.3.1] nonane-N-oxyl radical (ABNO, 0.001-1 g), stirring at room temperature for 1-12 hours, detecting by TLC, removing the raw material, quenching with a saturated sodium bisulfite or sodium sulfite or sodium thiosulfate aqueous solution (1-20 mL), extracting with an equal volume of an organic solvent for 2-6 times, combining filtrates, concentrating the sample on a rotary evaporator, and obtaining a white solid by various purification means, and identifying the compound as an intermediate by mass spectrometry, one-dimensional nuclear magnetic resonance and two-dimensional nuclear magnetic resonance (the yield is 65-78%).
Dissolving the intermediate (0.01-10 g) in a solvent N, N-dimethylformamide or N, N-dimethylacetamide or N-methylpyrrolidone (0.01-40 mL), adding pyridine (0.001-4 mL) and TBSCl (0.01-3 g), stirring for 0.2-2 hours, adding 2-bromo-2-butenol (0.01-3 g), sodium iodide (0.01-3 g), nickel diiodide (0.01-3 g), 4 '-dimethoxy-2, 2' -bipyridine (0.01-3 g), and manganese powder (0.01-3 g), stirring for 1-48 hours at 40-80 ℃ until the plate detects the disappearance of the raw materials, adjusting the pH to less than 3 with a dilute acid solution, extracting for 2-6 times with an isovolumetric organic solvent, combining filtrates, concentrating the sample on a rotary evaporator, and obtaining white solids by various purification means, the compound is identified as compound II-b by mass spectrum, one-dimensional and two-dimensional nuclear magnetic resonance (yield 63% -71%).
Example 7
This example provides a process for the preparation of compound I-a, having the formula:
the preparation method comprises the following specific steps:
dissolving the compound II-a (0.01-100 g) in an anhydrous organic solvent (1-100 mL), adding Dess-Martin periodinane (0.01-10 g) to the mixture, stirring the mixture at room temperature for 1-6 hours, when the raw material disappears by point plate detection, adding inorganic salt aqueous solution, extracting for 2-6 times by using an organic solvent with the same volume, combining filtrates, concentrating a sample on a rotary evaporator to obtain a crude product, dissolving the crude product in an anhydrous organic solvent (1-100 mL), adding EDC & HCl (0.01-100 g), (R) - (-) -1-octen-3-ol (0.01-100 g) and 4-DMAP (0.01-100 g), reacting at room temperature for 1-24 hours, when the detection raw material disappears, the sample is concentrated on a rotary evaporator, white solid is obtained by various purification means, and the compound is identified as the compound I-a (the yield is 63-74%) by mass spectrum, one-dimensional and two-dimensional nuclear magnetic resonance.
Wherein. The inorganic salt is sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium chloride, and ammonium chloride; the anhydrous organic solvent is chloroform, DMSO, or dichloromethane.
The characterization of compound I-a is as follows:
(c ═ 0.18, chloroform);1H NMR(500MHz,CDCl3):δ＝9.96(d,J＝8.1Hz,1H),7.72–7.68(m,4H),7.44–7.38(m,2H),7.37–7.32(m,4H),6.91(d,J＝8.5Hz,2H),6.71(d,J＝8.5Hz,2H),5.78(d,J＝8.1Hz,1H),5.72(ddd,J＝17.3,10.5,7.3Hz,1H),5.25(d,J＝17.3Hz,1H),5.18(d,J＝10.5Hz,1H),5.14(dd,J＝13.3,7.3Hz,1H),3.44(d,J＝5.6Hz,1H),3.23(dd,J＝12.0,5.6Hz,1H),2.80(dd,J＝12.0,8.2Hz,1H),2.21–2.13(m,1H),2.09(dd,J＝12.6,3.8Hz,1H),2.06(s,3H),1.80–1.72(m,2H),1.68–1.49(m,6H),1.45–1.39(m,1H),1.38–1.33(m,1H),1.33–1.20(m,7H),1.15(dd,J＝13.5,3.9Hz,2H),1.09(s,9H),1.00–0.93(m,1H),0.85(t,J＝6.4Hz,3H),0.84(s,3H),0.79(s,3H),0.73(s,3H),0.65(s,3H)ppm；13C NMR(126MHz,CDCl3):δ＝191.50,173.69,165.15,154.33,136.78,135.69,133.14,133.11,130.00,129.76,127.85,126.61,119.55,117.93,76.24,58.59,52.28,49.84,44.69,42.38,42.27,42.19,41.64,39.69,37.49,34.33,33.66,33.31,31.74,26.68,24.71,23.85,22.64,21.61,21.45,20.50,19.61,18.70,18.06,15.29,14.12ppm；HRESIMS(m/z):[M+K]+calcd for C53H72O4SiK+839.4831,found 839.4847。
example 8
This example provides a process for the preparation of compound I-b, having the formula:
the preparation method comprises the following specific steps:
dissolving the compound II-b (0.01-100 g) in an anhydrous organic solvent (1-100 mL), adding Dess-Martin periodinane (0.01-10 g) into the anhydrous organic solvent, stirring the mixture at room temperature for 1-6 hours, when the raw material disappears by point plate detection, adding inorganic salt aqueous solution, extracting for 2-6 times by using an organic solvent with the same volume, combining filtrates, concentrating a sample on a rotary evaporator to obtain a crude product, dissolving the crude product in an anhydrous organic solvent (1-100 mL), adding EDC & HCl (0.01-100 g), (R) - (-) -1-octen-3-ol (0.01-100 g) and 4-DMAP (0.01-100 g), reacting at room temperature for 1-24 hours, when the detection of the spot plate shows that the raw material disappears, the sample is concentrated on a rotary evaporator, white solid is obtained by various purification means, and the compound is identified as the compound I-b by mass spectrum, one-dimensional nuclear magnetic resonance and two-dimensional nuclear magnetic resonance (the yield is 59-70%).
Example 9
This example provides a method for preparing a heteroditerpenoid a-a, which has the following reaction formula:
the preparation method comprises the following specific steps:
dissolving a compound I-a (0.01-100 g) in an organic solvent (1-100 mL), adding isoamylene (1-100 mL), sodium dihydrogen phosphate (0.01-10 g) and sodium chlorite (0.01-10 g) aqueous solution (1-100 mL), stirring at room temperature for 0.5-24 hours until a spot plate detection raw material disappears, adding acetaldehyde (0.1-100 mL), stirring, adding TBAF (tetrabutylammonium fluoride, 0.01-100 g) tetrahydrofuran solution, stirring at room temperature for 1-24 hours until a spot plate detection new product does not increase, concentrating a sample on a rotary evaporator, obtaining a white solid by various purification means, and identifying the compound as a heteroperpene compound A-a (yield is 63-70%) by mass spectrometry, one-dimensional nuclear magnetic resonance and two-dimensional nuclear magnetic resonance.
Wherein the organic solvent is tert-butanol, chloroform, DMSO, or dichloromethane.
The heteroditerpenoid compounds A-a were characterized as follows:
(c ═ 0.19, methanol);1H NMR(400MHz,pyridine-d5):δ＝7.39(d,J＝8.4Hz,2H),7.24(d,J＝8.4Hz,2H),6.20(s,1H),5.95(ddd,J＝17.3,10.4,7.0Hz,1H),5.52–5.45(m,1H),5.42(d,J＝17.3Hz,1H),5.25(d,J＝10.4Hz,1H),3.94(d,J＝5.8Hz,1H),3.65(dd,J＝11.8,5.8Hz,1H),2.99(dd,J＝11.8,8.4Hz,1H),2.43(s,3H),2.43–2.36(m,2H),2.08–1.97(m,2H),1.86–1.74(m,2H),1.74–1.60(m,3H),1.60–1.50(m,1H),1.50–1.21(m,11H),1.07(s,3H),1.06–1.00(m,1H),0.96(s,3H),0.89(s,3H),0.85(t,J＝6.8Hz,3H),0.77(s,3H)ppm；13C NMR data see Table S3；HRESIMS(m/z):[M+Na]+calcd for C37H54O5Na+601.3863,found 601.3866。
example 10
This example provides a method for preparing a heteroditerpenoid A-b, which has the following reaction formula:
the preparation method comprises the following specific steps:
dissolving a compound I-b (0.01-100 g) in an organic solvent (1-100 mL), adding isoamylene (1-100 mL), sodium dihydrogen phosphate (0.01-10 g) and sodium chlorite (0.01-10 g) aqueous solution (1-100 mL), stirring at room temperature for 0.5-24 hours until a spot plate detection raw material disappears, adding acetaldehyde (0.1-100 mL), stirring, adding TBAF (tetrabutylammonium fluoride, 0.01-100 g) tetrahydrofuran solution, stirring at room temperature for 1-24 hours until a spot plate detection new product does not increase, concentrating a sample on a rotary evaporator, obtaining a white solid by various purification means, and identifying the compound as a heteroperpene compound A-b (yield 54-62%) by mass spectrometry, one-dimensional nuclear magnetic resonance and two-dimensional nuclear magnetic resonance.
Example 11
This example provides a tablet of a pharmaceutical composition, which is prepared by adding excipient to the heteroditerpene compound a-a prepared in example 9 at a weight ratio of 1:1 to the excipient, and tabletting.
Example 12
This example provides a capsule of a pharmaceutical composition, which is prepared by taking the diterpenoid compound a-a prepared in example 9 and making into a capsule according to a conventional capsule preparation method.
Example 13
This example provides a tablet of a pharmaceutical composition, which is prepared by mixing the heteroditerpenoid A-a prepared in example 9 with appropriate amounts of starch, corn steep liquor, and magnesium stearate and compressing into tablets.
Example 14
This example provides a capsule of a pharmaceutical composition, which is prepared by mixing 100mg of the heteroditerpenoid compound a-a prepared in example 9 with an appropriate amount of starch and magnesium stearate, sieving, uniformly mixing in a suitable container, and encapsulating the resulting mixture in a hard gelatin capsule.
Example 15
This example provides a nasal spray of a pharmaceutical composition, which is prepared from 80mg of the heteroditerpenoid A-a prepared in example 9, 8mg of sodium oxide, 1mg of EDTA1mg, 1mg of uranium phosphate buffer (pH 6.5), 10mg of polysorbate, and 2mL of double distilled water. Mix with stirring until completely dissolved to give a solution. The solution was filtered on a sterile filter, bottled and partitioned according to the appropriate dose.
Example 16
This example provides a dripping pill of pharmaceutical composition, which is prepared from the diterpenoid compound A-a 1g and polyethylene glycol 60009 g as prepared in example 9. After slightly heating and dissolving, adding the molten polyethylene glycol solution with the amount of the prescription (60 ℃ water bath heat preservation), stirring and mixing uniformly until ethanol is volatilized completely, standing in 60 ℃ water bath for heat preservation for 30 minutes, transferring the uniformly mixed molten polyethylene glycol solution with bubbles removed completely into a liquid storage barrel, controlling the dropping speed under the condition of 80-85 ℃ heat preservation, dropping the uniformly mixed molten polyethylene glycol solution into condensate dropwise until the completely condensed polyethylene glycol solution is completely condensed, pouring off the condensate, collecting dropping pills, draining, removing the condensate on the pills by using filter paper, and placing in a silica gel dryer or naturally drying.
Test examples
Taking the heteroditerpenoid compound A-a prepared in the example 9 for carrying out an immunosuppressive activity test, wherein the specific method comprises the following steps:
the MTT method detects the cytotoxicity of the compound on mouse spleen lymphocytes: taking separated Balb/c mouse spleen, preparing single cell suspension according to 8 multiplied by 10 mouse spleen lymphocyte suspension5The preparation method comprises the steps of inoculating the vaccine/well to a 96-well plate, adding screening drugs with different concentrations, taking an immunosuppressant cyclosporine A (CsA) as a positive control drug, and arranging a corresponding solvent control (cell control) and a culture solution background control (blank control) in addition, wherein the total volume is 200 mu L. 37 ℃ and 5% CO2The culture was carried out in an incubator for 48 hours. MTT solution was added at a concentration of 5mg/ml 4 hours before the end of the culture. When the incubation was completed, the supernatant was aspirated off, 200. mu.L of DMSO was added to each well to dissolve the purple crystals, and the OD value was measured at 570nM in a microplate reader.
3Detecting the proliferation inhibition activity of the compound on mouse spleen T |, B lymphocytes by an H-TdR incorporation method: mouse spleen lymphocyte suspension 5X 105Per well was inoculated in a 96 well plate, ConA (final concentration 1. mu.g/ml) or LPS (final concentration 10. mu.g/ml) and various concentrations of compounds were added, the immunosuppressant cyclosporin A (CsA) was used as a positive control drug, and corresponding ConA-free, LPS cell control wells and stimulated drug-free control wells were set in a total volume of 200. mu.L. 37 ℃ and 5% CO2The culture was carried out in an incubator for 48 hours. At 8 hours before the end of the incubation, 25. mu.L of the solution was added to each well3H-Thymidine nucleotides (10. mu. Ci/ml). The culture was continued until the end of the experiment. The cells were collected on a glass fiber membrane using a cell collector, and the cell-DNA-doped cells were read in a Beta counter (Microbeta Trilux, Perkinelmer) after addition of scintillation fluid3The amount of H-TdR, expressed as cpm value, represents the proliferation of the cells. The results of the experiment are shown in table 1.
TABLE 1 results of the immunocompetence test (CsA as positive control)
As shown in Table 1, the compound heteroditerpenoid A-a shows a certain immunosuppressive activity, and has an inhibitory effect on T lymphocyte proliferation and IC under the condition of non-cytotoxic activity concentration500.68 μm (CsA is positive control), and has inhibitory effect on B lymphocyte proliferation, IC5013.81 μm (CsA is positive control). The compound has immunosuppressive activity, has selectivity on T lymphocyte proliferation inhibition, and has application prospect in immunosuppressive drugs.
In conclusion, the embodiment of the invention provides the hetero-diterpenoid compound containing the four-membered ring, the preparation method and the application thereof, the hetero-diterpenoid compound has a novel structure, has an inhibiting effect on T lymphocyte proliferation, and can be widely applied to the preparation of immunosuppressive drugs. The preparation method of the hetero-diterpenoid compound is simple to operate, mild in condition and capable of quickly and efficiently obtaining the hetero-diterpenoid compound.
The embodiment of the invention also provides a pharmaceutical composition which comprises the heteroditerpenoid compound and pharmaceutically acceptable auxiliary materials. It has good immunosuppressive activity, and can be used as immunosuppressant.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.