CN114933626B - Ginsenoside Rb 1 Derivatives and uses thereof - Google Patents

Ginsenoside Rb 1 Derivatives and uses thereof Download PDF

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CN114933626B
CN114933626B CN202210449816.7A CN202210449816A CN114933626B CN 114933626 B CN114933626 B CN 114933626B CN 202210449816 A CN202210449816 A CN 202210449816A CN 114933626 B CN114933626 B CN 114933626B
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ginsenoside
formula
boc
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derivative
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CN114933626A (en
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何严萍
张洪彬
郑永唐
谢聪强
周光凤
薛建霞
杨柳萌
张家琪
郑昌博
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Kunming Institute of Zoology of CAS
Yunnan University YNU
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Yunnan University YNU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J17/005—Glycosides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses ginsenoside Rb with a structural formula shown in the formula I 1 The derivative or the pharmaceutically acceptable salt thereof has better effect of inhibiting the Zika virus or dengue virus and low cytotoxicity; the compound has simple preparation method and low cost, and is suitable for industrial production and market popularization and application;

Description

The method comprises the following steps ofGinsenoside Rb 1 Derivatives and uses thereof
Technical Field
The invention belongs to the field of medicines, and in particular relates to ginsenoside Rb 1 Derivatives, preparation method thereof and application thereof in preparing medicines for treating and/or preventing Zika virus and/or dengue fever virus.
Background
Dengue virus (DENV) is an arbovirus transmitted by aedes aegypti and aedes albopictus bites, and belongs to the family of viruses, such as Yellow Fever Virus (YFV), west Nile Virus (WNV), japanese Encephalitis Virus (JEV), and Hepatitis C Virus (HCV). DENV infection can lead to different forms of disease, typical dengue fever without clinical symptoms, dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS), the latter two weighing dengue fever, resulting in death in up to 20% of patients. Dengue fever has been pandemic in many outbreaks worldwide. The prevalence of dengue has increased in the 80 s of the 20 th century. Dengue is currently seen in more than 100 countries and regions of the tropics and subtropics, 40% of the world facing dengue risk, with about 3.9 million people infected each year, and the World Health Organization (WHO) lists dengue to 10 diseases that are serious threatens to human health.
The pathogenic mechanisms of severe dengue are not yet fully elucidated. Each of the 4 serotypes of dengue virus (DENV-1, 2,3, 4) has been found to be capable of eliciting severe dengue fever. After infection with any DENV serotype, the body can only produce specific antibodies against that serotype, but not other serotypes. Secondary infection with DENV of a different serotype can lead to more serious conditions in the body. An effective prophylactic or therapeutic measure must be able to target both of these 4 viral serotypes; thus, dengue control is a great challenge for researchers. In the dengue treatment, no specific medicine is currently marketed, and dengue patients can only relieve clinical symptoms through symptomatic treatment. Ribavirin (ribavirin) is a broad-spectrum nucleoside antiviral drug, has a certain inhibition effect on replication of various RNA and DNA viruses, and has been reported in literature to inhibit replication of dengue viruses, but the exact action mechanism is not clear. At the end of 2015, the first dengue vaccine, denvaxia (CYD-TDV), developed by the samofibard company was registered in the philippines, brazil, mexico, etc. countries for use by residents 9-45 years of epidemic areas, but the safety and effectiveness of this vaccine was still under observation. No anti-DENV drugs are currently in the market, and anti-DENV drugs targeting viruses or host factors are under investigation.
Zika virus was first isolated from rhesus specimens with fever in Uganda in 1947 and was therefore named Zika virus. ZIKV can be transmitted through blood, sex and mother and infant besides by Aedes mosquito bites. Most of clinical symptoms caused by ZIKV infection are light, and mainly are manifested by headache, fever, hypodynamia, rash, conjunctivitis, joint and muscle pain and the like, and can be self-healed after 2-7 days. But may also cause serious damage to the nervous and autoimmune systems. For example: infection of pregnant women with ZIKV may cause congenital developmental deformity of the fetal brain, resulting in microcephaly, and infection of adults with ZIKV may cause guillain-barre syndrome to cause paralysis and even death of the patient. ZIKV initially found sporadic cases of infection only in sub-africa, whereas since 2007 ZIKV was outbreaked and spread to america in oceangoing, and more than hundreds of thousands of cases of infection were reported in america, caribbean and pacific areas in 2015, the number of small-headed malformed infants born in brazil at the same time increased significantly, and has attracted global high attention. The number of ZIKV infections in south america has reached about 200 tens of thousands by 2016. In view of the ever-expanding epidemic scope of ZIKV viruses, the disease burden caused is serious, and the World Health Organization (WHO) announces the ZIKV epidemic as a health emergency of global interest. Today, there are 90 countries and regions where over 36 hundred million people live in ZIKV epidemic areas, which pose a great threat to the health of human beings worldwide, and finding effective antiviral therapies has become a worldwide medical challenge.
Although some vaccines against ZIKV have entered clinical trial stages, long-term effectiveness experiments and approval of vaccines are still long in the market, and the zaka vaccine may cause an antibody-dependent enhancement effect (ADE) of DENV infection, which also greatly hinders development of the zaka vaccine. In recent years, scientists focus on each link of the ZIKV life cycle, find a plurality of potential drug targets, and combine a virtual screening technology and in-vitro experiments to find a plurality of small molecular compounds with ZIKV activity inhibition, so that new breakthrough is expected to be brought to the development of anti-ZIKV drugs, but the research and development of the anti-ZIKV drugs are still far from clinical application. The ZIKV epidemic situation is urgent, and scientific researchers are urgently required to commonly strive to develop specific antiviral drugs.
The natural product is an important source of antiviral drugs, and has the advantages of stable curative effect, small toxic and side effects, difficult drug resistance, difficult virus mutation and the like which are incomparable with western medicines, and attracts attention of a plurality of scientific researchers. However, natural products often have the defects of complex structure, low bioavailability and the like, so that the structure modification is performed on the basis of the known active ingredients of the Chinese herbal medicines to improve the activity and the patentability of the Chinese herbal medicines, and the natural products become an important way for creating new medicines. Notoginseng [ panax notoginseng (Burk.) F.H.Chen ]Commonly known as Stephania sinica Diels, belongs to the genus Panax of the family Araliaceae, and is a famous Chinese medicine of special origin in Yunnan. The main active ingredient of Notoginseng radix is Notoginseng radix saponin (Panax notoginseng saponins, PNS) which is dammarane type tetracyclic triterpene saponin, and is mainly ginsenoside Rb 1 、Rb 2 、Rd、Rg 1 、Rh、Re、Rg 3 And the like, more than 20 monomer saponins. PNS has various effects of regulating immunity, resisting tumor and virus, protecting liver, reducing blood, regulating central nervous system, endocrine and respiratory system, etc., and has been studied more and more in recent years. For example PNS has anti-Coxsackie virus B 3 Activity of Marek's Disease Virus (MDV), enterovirus 71, hepatitis C Virus (HCV), etc., ginsenoside-Rg 3 、Rb 3 Has anti-herpes simplex virus type I (HSV-1) and poliovirus (poliV) activity, and ginsenoside Re can enhance H 3 N 2 Immune response of influenza virus vaccine, ginsenoside R f 、Rg 2 Has anti-HRV effect 3 Active ginsenoside Rb 1 Can inhibit influenza virus H 1 N 1 Infection of (C),Ginsenoside Rb 1 、Rg 1 Can obviously inhibit the activity of Hepatitis A Virus (HAV) in vitro, etc. In the process of searching and structural modification of monomeric saponin with PNS antiviral activity as guiding, after the anti-HCV viral activity of successive PNS, we found that the monomeric ginsenoside Rb in PNS 1 Has very obvious ZIKV and DENV resisting activity after being subjected to amino acid esterification and etherification modification. There has been no report of PNS monomeric saponins or derivatives thereof for use against infection with DENV or ZIKV.
Disclosure of Invention
The invention provides a ginsenoside Rb 1 Derivative or pharmaceutically acceptable salt thereof, ginsenoside Rb 1 The structural formula of the derivative is as follows:
wherein: r is selected from H, C 1 -C 6 Straight or branched alkyl, C 3 -C 6 Cycloalkyl;
R 1 、R 2 or R is 3 Each independently selected from R orn is any integer between 0 and 3;
R 1 selected from H, C 1 -C 6 Straight or branched alkyl, C 3 -C 6 Cycloalkyl, -C 1 -C 6 Straight-chain or branched alkyl-NH of (2) 2 、-C 1 -C 6 Straight-chain or branched alkyl-OH, NH 2 C(＝O)-、C 1 -C 6 Straight-chain or branched alkoxy, C 1 -C 6 Straight-chain or branched alkylthio, C 6 -C 10 Aryl, C of (2) 2 -C 10 Heteroaryl of (a);
R 2 selected from H, t-butoxycarbonyl, or R 2 And R is R 1 Connection structure C 2 -C 6 A heterocycloalkyl group.
Ginsenoside Rb related in the structural formula 1 The specific structure of the derivative is as follows:
another object of the present invention is to provide the above ginsenoside Rb 1 The preparation method of the derivative comprises the steps of preparing ginsenoside Rb in the presence of a solvent and an alkaline catalyst 1 With halogenated hydrocarbons (RX) or amino acidsUniformly mixing according to a certain proportion, stirring and reacting for 0.5-12 hours at the temperature of 0-80 ℃, and separating and purifying a reaction product to obtain the ginsenoside Rb1 derivative, wherein the reaction formula is as follows:
Wherein, ginsenoside Rb 1 Halogenated hydrocarbon (RX, 1) or amino acid2) The molar ratio of (2) is 1:1-36.
The basic catalyst is potassium carbonate (K) 2 CO 3 ) One or more of sodium hydrogen (NaH), N' -Diisopropylcarbodiimide (DIC), 4-Dimethylaminopyridine (DMAP); the solvent is one or more of tetrahydrofuran, toluene, acetonitrile, dichloromethane, N-dimethylformamide and pyridine.
Another object of the present invention is to provide the above ginsenoside Rb 1 Application of derivative or pharmaceutically acceptable salt thereof in preparation of medicinesTreating and/or preventing Zika virus and/or dengue virus.
The components (or active ingredients) of the medicament for treating and/or preventing the Zika virus and/or the dengue virus are ginsenoside Rb 1 The derivatives or pharmaceutically acceptable salts thereof can be added with one or more pharmaceutically acceptable auxiliary materials to improve the drug absorption effect or facilitate the administration, such as capsules or pills, powder, tablets, granules, oral liquid, injection and the like, namely, the preparation is prepared into pharmaceutically suitable use dosage forms; can also be combined with other active ingredients to prepare medicines for treating and/or preventing Zika virus and/or dengue virus.
The invention has the advantages and technical effects that:
1. The preparation method of the compound is simple, and can realize industrial production;
2. the experiment shows that the compound has better inhibition effect on Zika virus and/or dengue virus and low cytotoxicity.
Detailed Description
The present invention is described in detail below by way of examples, but is not meant to be limiting in any way. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the invention without departing from the spirit and scope of the invention.
The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. The starting materials may be obtained commercially, or prepared by methods known in the art, or prepared according to the methods described herein. The structure of the compound is changed into a nuclear magnetic resonance structure 1 H NMR or 13 C NMR) and Mass Spectrometry (MS), wherein the NMR was determined using Bruker AV-400 type nuclear magnetic resonance apparatus with deuterated pyridine (C) 5 D 5 N), TMS is an internal standard.
Example 1: ginsenoside Rb 1 Preparation of methyl ether derivative I-1
Ginsenoside Rb was obtained in a 50mL round bottom flask 1 (200 mg,0.18 mmol) was dissolved in 10mL of DMF, sodium hydride (130 mg,5.41 mmol) was added at room temperature, after stirring for 30min, methyl iodide (337. Mu.L, 5.41 mmol) was added, and the mixture was reacted at room temperature for 6h, pH was adjusted to about 7 with dilute hydrochloric acid, extracted with ethyl acetate, dried and concentrated; separating and purifying by silica gel column chromatography (eluent: dichloromethane: methanol=60:1, v/v), collecting eluent, concentrating and drying to obtain white solid compound I-1 with yield of 70%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.79 (d, j=12.0 hz, 1H), 0.88 (s, 3H), 0.99 (s, 6H), 1.07 (s, 3H), 1.25 (s, 3H), 1.50 (s, 3H), 1.70 (s, 6H), 3.36 (s, 3H), 3.40 (s, 3H), 3.43 (s, 3H), 3.51 (s, 3H), 3.54 (s, 3H), 3.55 (s, 3H), 3.56 (s, 3H), 3.62 (s, 3H), 3.68 (s, 6H), 3.69 (s, 3H), 3.70 (s, 3H), 3.73 (s, 3H), 3.77 (s, 3H), 4.22-4.25 (d, j=12.hz, 1H), 4.60-4.62 (d, j=8.43 (s, 3H), 4.70-1 hz (s, 1H), 3.62 (s, 1H), 3.82-8.8.5 hz, 1H) and other overlaps (s, 3.8.8-0 hz (s, 3H), 3.6H) with the other bands (s, 1H) of the band (s, 1H, 1.8.8-7 (s, 3H) and 1.8.6H; ESI-MS1327.8199[ M+Na ] ] + 。
Example 2: ginsenoside Rb 1 Preparation of an etherified derivative I-2
The preparation method is the same as in example 1, except that bromoethane is used for replacing iodomethane in example 1, the reaction is carried out for 8 hours at room temperature, the product is separated and purified by silica gel column chromatography (eluent: dichloromethane: methanol=60:1), and the eluent is collected, concentrated and dried to obtain a white solid compound I-2, wherein the yield is 78%. 1 H NMR(400M Hz，C 5 D 5 N)δ0.75-0.79(d，J＝12.0Hz，1H)，0.89(s，3H)，0.97(s，6H)，0.99(s，6H)，1.06(s，9H)，1.16(s，18H)，1.17 (s, 6H), 1.18 (s, 9H), 1.25 (s, 3H), 1.70 (s, 3H), 1.81 (s, 6H), 4.23-4.25 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.70-4.73 (d, j=8.0 hz, 1H), 4.81-4.83 (d, j=8.0 hz, 1H), 5.01-5.04 (d, j=8.0 hz, 1H), 5.31-5.34 (t, j=6.0 hz, 1H), overlapping 1.06-2.38 on the mother nucleus and other H overlapping 3.00-4.20 on the sugar ring and other H. ESI-MS1193.7041[ M+H ]] + 。
EXAMPLE 3 ginsenoside Rb 1 Preparation of amino acid derivative I-3
Ginsenoside Rb was obtained in a 50mL round bottom flask 1 (200 mg,0.18 mmol) was dissolved in 10mL of pyridine, boc-D-valine (313 mg,1.44 mmol) was added at room temperature and stirred for dissolution, then N, N' -diisopropylcarbodiimide (224. Mu.L, 1.44 mmol) was added, TLC was used to monitor the progress of the reaction, after about 2 hours, the reaction was stopped, and after purification by silica gel column chromatography (eluent: dichloromethane: methanol=7:1), the eluent was collected and concentrated to dryness, white solid compound I-3 (R) was obtained f =0.25), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.78 (d, j=12.0 hz, 1H), 0.87 (s, 3H), 0.96 (s, 6H), 0.97 (s, 6H), 1.01 (s, 3H), 1.02 (s, 3H), 1.25 (s, 3H), 1.39 (s, 9H), 1.66 (s, 3H), 1.79 (s, 3H), 4.23-4.25 (d, j=12.0 hz, 1H), 4.61-4.62 (d, j=8.0 hz, 1H), 4.70-4.73 (d, j=8.0 hz, 1H), 4.81-4.84 (d, j=8.0 hz, 1H), 5.02-5.04 (d, j=8.0 hz, 1H), 5.31-5.34 (t, j=6.0 hz, 1H), core and other peaks on the core and other carbohydrate overlaps 1.38-4.40.38 and other peaks on the core and the ring overlaps 40-6.40.7H; ESI-MS 1330.7138[ M+Na ]] + 。
Example 4 ginsenoside Rb 1 Preparation of amino acid derivative I-4
By operating in the same manner as in example 3, TLC monitors the progress of the reaction, after about 2 hours, the reaction is stopped, and the mixture is separated and purified by silica gel column chromatography (eluent: dichloromethane: methanol=12:1), and the eluent is collected, concentrated and dried to obtain white solid compound I-4 (R) f =0.50), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.80 (d, j=12.0 hz, 1H), 0.87 (s, 3H), 0.96 (s, 6H), 0.97 (s, 6H), 0.99 (s, 6H), 1.01 (s, 3H), 1.03 (s, 3H), 1.25 (s, 3H), 1.39 (s, 9H), 1.41 (s, 9H), 1.66 (s, 3H), 1.79 (s, 3H), 4.22-4.25 (d, j=12.0 hz, 1H), 4.60-4.63 (d, j=8.0 hz, 1H), 4.71-4.73 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.02-5.05 (d, j=8.hz, 1H), 5.30-5.33 (t, 6.0hz, 1H), overlap with the parent sugar and other peaks of 1.0.40-0 hz, 1.38, and other peaks of the parent sugar overlap with the parent sugar of 4.0.0-8.0 hz, 1H; ESI-MS 1529.8334[ M+Na ] ] + 。
Example 5 ginsenoside Rb 1 Preparation of amino acid derivative I-5
The procedure was followed in the same manner as in example 3, except that Boc-glycine was used instead of Boc-D-valine in example 3, TLC was used to monitor the progress of the reaction, after about 2 hours, the reaction was stopped, and the mixture was purified by column chromatography on silica gel (eluent: dichloromethane: methanol=7:1), and the eluent was collected and concentrated to dryness to give compound I-5 (R) f =0.25), yield was 29%. 1 H NMR(400M Hz，C 5 D 5 N) 0.75-0.77 (d, J=12.0 Hz, 1H), 0.87 (s, 3H), 0.96 (s, 6H), 0.99 (s, 6H), 1.25 (s, 3H), 1.40 (s, 9H), 1.69 (s, 3H), 1.79 (s, 3H), 4.23-4.26 (d, J=12.0 Hz, 1H), 4.60-4.62 (d, J=8.0 Hz, 1H), 4.70-4.72 (d, J=8.0 Hz, 1H), 4.82-4.85 (d, J=8.0 Hz, 1H), 5.01-5.04 (d, J=8.0 Hz, 1H), 5.31-5.34 (t, J=6.0 Hz, 1H), overlaps 1.06-2.38 on the nucleus and other overlaps 3.00-4.0 Hz,1H on the sugar ring and overlaps 4.0-6.40-6HA peak; ESI-MS 1288.6669[ M+Na ]] + 。
Example 6 ginsenoside Rb 1 Preparation of amino acid derivative I-6
The procedure was followed in the same manner as in example 3, except that Boc-glycine was used instead of Boc-D-valine in example 3, TLC was used to monitor the progress of the reaction, after about 3 hours, the reaction was stopped, and purification was performed by column chromatography on silica gel (eluent: dichloromethane: methanol=12:1), and the eluent was collected and concentrated to dryness to give compound I-6 (R) as a white solid f =0.50), yield was 35%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.79 (d, j=12.0 hz, 1H), 0.88 (s, 3H), 0.96 (s, 6H), 0.99 (s, 6H), 1.25 (s, 3H), 1.40 (s, 18H), 1.41 (s, 9H), 1.69 (s, 3H), 1.79 (s, 3H), 4.21-4.24 (d, j=12.0 hz, 1H), 4.62-4.64 (d, j=8.0 hz, 1H), 4.70-4.73 (d, j=8.0 hz, 1H), 4.82-4.86 (d, j=8.0 hz, 1H), 5.00-5.03 (d, j=8.0 hz, 1H), 5.30-5.34 (t, j=6.hz, 1H), active nuclei and other H overlap 1.06-2.38 on the sugar ring and other peaks overlap 3.01-6.0 hz, 1H; ESI-MS 1580.8319[ M+Na ]] + 。
EXAMPLE 7 ginsenoside Rb 1 Preparation of amino acid derivative I-7
The procedure of example 3 was followed, except that Boc-D-valine was replaced with Boc-L-leucine in example 3, the reaction was stopped after about 2 hours, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=7:1), collected, concentrated and dried to give a white solid compound I-7 (R) f =0.25) yield was 27%. 1 H NMR(400M Hz，C 5 D 5 N)δ0.76-0.78 (d, j=12.0 hz, 1H), 0.87 (s, 3H), 0.92 (s, 6H), 0.98 (s, 6H), 0.99 (s, 6H), 1.25 (s, 3H), 1.40 (s, 9H), 1.69 (s, 3H), 1.79 (s, 3H), 1.85 (m, 2H), 4.21-4.24 (d, j=12.0 hz, 1H), 4.60-4.62 (s, j=8.0 hz, 1H), 4.70-4.72 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.02-5.05 (d, j=8.0 hz, 1H), 5.30-5.34 (t, j=6.0 hz, 1H), 1.06-2.38 overlapping mother nuclei and other H, 3.01-4.28 overlapping sugar rings and other H, 5.76-6.50 overlapping active hydrogen, blunt peaks; ESI-MS 1344.7293[ M+Na ] ] + 。
Example 8 ginsenoside Rb 1 Preparation of amino acid derivative I-8
The procedure was followed in the same manner as in example 3, except that Boc-D-valine was replaced with Boc-L-leucine in example 3, the reaction was stopped after about 3 hours by TLC, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol, 12:1), collected and concentrated to dryness to give compound I-8 (R) f =0.50), yield was 26%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.78 (d, j=12.0 hz, 1H), 0.86 (s, 6H), 0.88 (s, 6H), 0.92 (s, 3H), 0.97 (s, 6H), 1.00 (s, 6H), 1.27 (s, 3H), 1.45 (s, 18H), 1.68 (s, 3H), 1.75 (s, 3H), 3.31-3.34 (d, j=12.0 hz, 1H), parent nucleus and other H overlaps 0.80-2.70, sugar ring and other positions H overlaps 3.82-5.51, active hydrogen overlaps 5.80-6.60, blunt peaks; ESI-MS 1557.8658[ M+Na ]] + 。
Example 9 ginsenoside Rb 1 Preparation of amino acid derivative I-9
Operating in the same manner as in example 3, the preparation of the compounds of the formula I-3 differs from example 3 only inIn the case where Boc-L-leucine was used instead of Boc-D-valine in example 3, TLC was used to monitor the progress of the reaction, after about 3 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol, 12:1), and the eluent was collected, concentrated and dried to give Compound I-9 (R) as a white solid f =0.45), yield was 26%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.77-0.79 (d, j=12.0 hz, 1H), 0.88 (s, 6H), 0.89 (s, 6H), 0.94 (s, 3H), 0.98 (s, 6H), 1.01 (s, 6H), 1.26 (s, 3H), 1.46 (s, 18H), 1.69 (s, 3H), 1.78 (s, 3H), 3.31-3.34 (d, j=12.0 hz, 1H), parent nucleus and other H overlaps 0.80-2.70, sugar ring and other positions H overlaps 3.82-5.51, active hydrogen overlaps 5.80-6.60, blunt peaks; ESI-MS 1557.8662[ M+Na ]] + 。
Example 10: ginsenoside Rb 1 Preparation of amino acid derivative I-10
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The procedure was followed in the same manner as in example 3 except that Boc-D-valine was used instead of Boc-D-valine in example 3 and TLC was used to monitor the progress of the reaction, after about 2 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=7:1), collected and concentrated to dryness to give compound I-10 (R) as a white solid f =0.25), yield was 27%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.79 (d, j=12.0 hz, 1H), 0.87 (s, 3H), 0.96 (s, 6H), 0.97 (s, 6H), 1.01 (s, 3H), 1.05 (s, 3H), 1.25 (s, 3H), 1.39 (s, 9H), 1.66 (s, 3H), 1.79 (s, 3H), 4.22-4.25 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.70-4.72 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.02-5.04 (d, j=8.0 hz, 1H), 5.31-5.34 (t, j=6.0 hz, 1H), core overlap and others 1.06. 38, overlapping sugar rings and other H with 3.01-4.30, overlapping active hydrogen with 5.80-6.60, and blunt peak; ESI-MS 1330.7133[ M+Na ]] + 。
EXAMPLE 11 ginsenoside Rb 1 Preparation of amino acid derivatives
The procedure was followed in the same manner as in example 3 except that Boc-D-valine was used instead of Boc-D-valine in example 3 and TLC was used to monitor the progress of the reaction, after about 2 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=7:1), collected and concentrated to dryness to give compound I-11 (R) as a white solid f =0.30), yield was 27%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.75-0.78 (d, j=12.0 hz, 1H), 0.87 (s, 3H), 0.96 (s, 6H), 0.97 (s, 6H), 1.01 (s, 3H), 1.02 (s, 3H), 1.25 (s, 3H), 1.39 (s, 9H), 1.68 (s, 3H), 1.79 (s, 3H), 4.23-4.26 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.70-4.72 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.00-5.03 (d, j=8.0 hz, 1H), 5.30-5.34 (t, j=6.0 hz, 1H), 1.06-2.38 overlapping mother nuclei and other H, 3.01-4.20 overlapping sugar rings and other H, 5.80-6.60 overlapping active hydrogen, blunt peaks; ESI-MS 1330.7137[ M+Na ]] + 。
Example 12: ginsenoside Rb 1 Preparation of amino acid derivative I-12
The procedure was followed in the same manner as in example 3 except that Boc-D-valine was used instead of Boc-D-valine in example 3 and TLC was used to monitor the progress of the reaction, after about 3 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=12:1), collected and concentrated to dryness to give compound I-1 as a white solid2(R f =0.50), yield was 27%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.78 (d, j=12.0 hz, 1H), 0.96 (s, 3H), 0.97 (s, 6H), 0.99 (s, 6H), 1.00 (s, 6H), 1.03 (s, 6H), 1.26 (s, 3H), 1.44 (s, 18H), 1.71 (s, 3H), 1.76 (s, 3H), 3.27-3.30 (d, j=8.0 hz, 1H), 4.29-4.31 (d, j=8.0 hz, 1H), 4.47-4.49 (d, j=8.0 hz, 1H), 5.36-5.37 (d, j=4.0 hz, 1H), overlap on the mother nucleus and other H on 0.70-2.70, overlap on the sugar ring and other positions on 3.80-5.20, active hydrogen overlaps 5.76-6.40, blunt peaks; ESI-MS 1529.8344[ M+Na ]] + 。
EXAMPLE 13 ginsenoside Rb 1 Preparation of amino acid derivative I-13
The procedure was followed in the same manner as in example 3 except that Boc-D-valine was used instead of Boc-D-valine in example 3 and TLC was used to monitor the progress of the reaction, after about 3 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=12:1), collected and concentrated to dryness to give compound I-13 (R) as a white solid f =0.45), yield was 25%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.79 (d, j=12.0 hz, 1H), 0.96 (s, 6H), 0.98 (s, 6H), 0.99 (s, 3H), 1.00 (s, 6H), 1.02 (s, 6H), 1.26 (s, 3H), 1.45 (s, 18H), 1.68 (s, 3H), 1.75 (s, 3H), 3.28-3.31 (d, j=8.0 hz, 1H), 4.28-4.31 (d, j=8.0 hz, 1H), 4.47-4.49 (d, j=8.0 hz, 1H), 5.36-5.37 (d, j=4.0 hz, 1H), overlap on the mother nucleus and other H on 0.70-2.70, overlap on the sugar ring and other positions on 3.80-5.20, active hydrogen overlaps 5.76-6.40, blunt peaks; ESI-MS 1529.8341[ M+Na ]] + 。
EXAMPLE 14 ginsenoside Rb 1 Preparation of amino acid derivative I-14
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The procedure was followed in the same manner as in example 3 except that Boc-D-valine in example 3 was replaced with Boc-L-tryptophan and the reaction was stopped after about 3 hours by TLC, and the mixture was purified by column chromatography over silica gel (eluent: dichloromethane: methanol=8:1), collected and concentrated to dryness to give compound I-14 (R) f =0.30), yield was 25%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.77-0.79 (d, j=12.0 hz, 1H), 0.97 (s, 3H), 0.98 (s, 3H), 1.00 (s, 3H), 1.01 (s, 3H), 1.03 (s, 3H), 1.25 (s, 3H), 1.43 (s, 9H), 1.69 (s, 3H), 1.79 (s, 3H), 3.28-3.31 (d, j=8.0 hz, 1H), 4.28-4.31 (d, j=8.0 hz, 1H), 4.47-4.49 (d, j=8.0 hz, 1H), 4.69 (m, 1H), 5.36-5.37 (d, j=4.0 hz, 1H), 6.99-7.60 (m, 5H), 10.69 (s, 1H), overlap on the parent nucleus and other sugar and other peaks on the ring and other positions in the range of 0.70-2.70, and other peaks in the range of 3.80-5.80 overlaps with 40.80H and 40.76H. ESI-MS 1395.7421[ M+H ] ] + 。
Example 15: ginsenoside Rb 1 Preparation of amino acid derivative I-15
The procedure was followed in the same manner as in example 3 except that Boc-D-valine in example 3 was replaced with Boc-L-tryptophan and the reaction was stopped after about 3 hours by TLC, and the mixture was purified by column chromatography over silica gel (eluent: dichloromethane: methanol=8:1), collected and concentrated to dryness to give compound I-15 (R) f =0.25), yield was 25%. 1 H NMR(400M Hz，C 5 D 5 N)δ0.76-0.78(d，J＝12.0Hz，1H)，0.96(s，3H)，0.98(s，3H)，0.99(s，3H)，1.01(s，3H)，1.02(s，3H)，1.26 (s, 3H), 1.43 (s, 9H), 1.69 (s, 3H), 1.78 (s, 3H), 3.29-3.31 (d, j=8.0 hz, 1H), 4.29-4.31 (d, j=8.0 hz, 1H), 4.46-4.49 (d, j=8.0 hz, 1H), 4.69 (m, 1H), 5.35-5.37 (d, j=4.0 hz, 1H), 6.99-7.60 (m, 5H), 10.69 (s, 1H), overlapping 0.70-2.70 on the mother nucleus and other positions H on the sugar ring, 3.80-5.20, overlapping active hydrogens on 5.76-6.50, blunt peaks. ESI-MS 1395.7420[ M+H ]] + 。
EXAMPLE 16 ginsenoside Rb 1 Preparation of amino acid derivative I-16
The procedure was followed in the same manner as in example 3, except that Boc-L-cysteine was used instead of Boc-D-valine in example 3, TLC was used to monitor the progress of the reaction, after about 2 hours, the reaction was stopped, and purification was performed by column chromatography on silica gel (eluent: dichloromethane: methanol=7:1), and the eluent was collected and concentrated to dryness to give compound I-16 (R) as a white solid f =0.30), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.78-0.80 (d, j=12.0 hz, 1H), 0.87 (s, 3H), 0.96 (s, 6H), 0.97 (s, 6H), 1.25 (s, 3H), 1.40 (s, 9H), 1.69 (s, 3H), 1.79 (s, 3H), 4.21-4.24 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.70-4.73 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.02-5.04 (d, j=8.0 hz, 1H), 5.30-5.34 (t, j=6.0 hz, 1H), overlap of active nuclei and other H on the sugar rings and other peaks on the 3.01-4.0 hz, 1.76-40.76 by blunt peaks and the other peaks on the sugar rings. ESI-MS 1312.6719[ M+H ]] + 。
EXAMPLE 17 ginsenoside Rb 1 Preparation of amino acid derivative I-17
Operating in the same manner as in example 3, the preparation of the compound of formula I-3 is different from that of example 3Except that Boc-L-cysteine was used instead of Boc-D-valine in example 3, TLC was used to monitor the progress of the reaction, after about 2 hours, the reaction was stopped, and the mixture was purified by column chromatography on silica gel (eluent: dichloromethane: methanol=7:1), and the eluent was collected and concentrated to dryness to give Compound I-17 (R) as a white solid f =0.25), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.78 (d, j=12.0 hz, 1H), 0.88 (s, 3H), 0.97 (s, 6H), 0.98 (s, 6H), 1.26 (s, 3H), 1.40 (s, 9H), 1.69 (s, 3H), 1.80 (s, 3H), 4.20-4.24 (d, j=12.0 hz, 1H), 4.59-4.61 (d, j=8.0 hz, 1H), 4.71-4.73 (d, j=8.0 hz, 1H), 4.82-4.85 (d, j=8.0 hz, 1H), 5.02-5.04 (d, j=8.0 hz, 1H), 5.31-5.35 (t, j=6.0 hz, 1H), overlap of active nuclei and other H on the sugar rings and other peaks on the 3.01-4.0 hz, 1.76-40.76 by blunt peaks and the rest; ESI-MS 1312.6718[ M+H ] ] + 。
EXAMPLE 18 ginsenoside Rb 1 Preparation of amino acid derivative I-18
The procedure was followed in the same manner as in example 3, except that Boc-L-cysteine was used instead of Boc-D-valine in example 3, TLC was used to monitor the progress of the reaction, after about 3 hours, the reaction was stopped, and purification was performed by column chromatography on silica gel (eluent: dichloromethane: methanol=10:1), and the eluent was collected and concentrated to dryness to give compound I-18 (R) as a white solid f =0.50), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.75-0.79 (d, j=12.0 hz, 1H), 0.86 (s, 3H), 0.95 (s, 6H), 0.96 (s, 6H), 1.25 (s, 3H), 1.40 (s, 18H), 1.68 (s, 3H), 1.78 (s, 3H), 4.22-4.26 (d, j=12.0 hz, 1H), 4.61-4.64 (d, j=8.0 hz, 1H), 4.69-4.72 (d, j=8.0 hz, 1H), 4.82-4.85 (d, j=8.0 hz, 1H), 5.00-5.03 (d, j=8.0 hz, 1H), 5.30-5.34 (t, j=6.0 hz, 1H), overlap of active nuclei and other H on the sugar rings and other peaks on the 3.01-4.0 hz, 1.76-50.76 by blunt peaks;ESI-MS 1515.7334[M+H] + 。
EXAMPLE 19 ginsenoside Rb 1 Preparation of amino acid derivative I-19
The procedure was followed in the same manner as in example 3 except that Boc-D-valine was replaced with Boc-L-threonine in example 3, the reaction was stopped after about 2 hours by TLC, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=7:1), collected and concentrated and dried to give compound I-19 (R) as a white solid f =0.30), yield was 29%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.75-0.78 (d, j=12.0 hz, 1H), 0.87 (s, 3H), 0.97 (s, 6H), 0.98 (s, 6H), 1.25 (s, 3H), 1.40 (s, 9H), 1.69 (s, 3H), 1.80 (s, 3H), 4.21-4.26 (d, j=12.0 hz, 1H), 4.61-4.64 (d, j=8.0 hz, 1H), 4.70-4.73 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.02-5.04 (d, j=8.0 hz, 1H), 5.30-5.34 (t, j=6.0 hz, 1H), overlap of active nuclei and other H on the sugar rings and other peaks on the 3.01-4.0 hz, 1.76-50.76 by blunt peaks; ESI-MS 1310.7103[ M+H ]] + 。
EXAMPLE 20 ginsenoside Rb 1 Preparation of amino acid derivative I-20
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The procedure was as in example 3, except that Boc-L-threonine was used instead of Boc-D-valine in example 3, and after TLC was used to monitor the progress of the reaction, the reaction was stopped and, after about 2h, the reaction was purified by column chromatography over silica gel (eluent: dichloromethane: methanol=7:1) Separating and purifying, collecting eluate, concentrating, and drying to obtain white solid compound I-20 (R) f =0.25), yield was 29%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.79 (d, j=12.0 hz, 1H), 0.87 (s, 3H), 0.95 (s, 6H), 0.97 (s, 6H), 1.26 (s, 3H), 1.40 (s, 9H), 1.68 (s, 3H), 1.78 (s, 3H), 4.22-4.25 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.69-4.72 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.02-5.05 (d, j=8.0 hz, 1H), 5.30-5.34 (t, j=6.0 hz, 1H), overlap of active nuclei and other H on the sugar rings and other peaks on the 3.01-4.0 hz, 1.76-40.76 by blunt peaks and the other peaks on the sugar rings; ESI-MS 1310.7103[ M+H ] ] + 。
Example 21: ginsenoside Rb 1 Preparation of amino acid derivative I-21
The procedure was followed in the same manner as in example 3, except that Boc-D-valine in example 3 was replaced with Boc-L-histidine, the reaction progress was monitored by TLC and after about 3 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=8:1), collected and concentrated to dryness to give compound I-21 (R) as a white solid f =0.30), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.78 (d, j=12.0 hz, 1H), 0.88 (s, 3H), 0.96 (s, 6H), 0.97 (s, 6H), 1.26 (s, 3H), 1.40 (s, 9H), 1.69 (s, 3H), 1.81 (s, 3H), 4.21-4.25 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.70-4.72 (d, j=8.0 hz, 1H), 4.84-4.86 (d, j=8.0 hz, 1H), 5.00-5.03 (d, j=8.0 hz, 1H), 5.31-5.34 (t, j=6.0 hz, 1H), 7.32 (m, 1H), 8.45 (m, 1H), 1.06-2.38 overlapping mother nuclei and other H, 3.01-4.20 overlapping sugar rings and other H, 5.76-6.40 overlapping active hydrogen, blunt peaks; ESI-MS 1346.7216[ M+H ]] + 。
Example 22: ginsenoside Rb 1 Preparation of amino acid derivative I-22
The procedure was followed in the same manner as in example 3, except that Boc-D-valine in example 3 was replaced with Boc-L-histidine, the reaction progress was monitored by TLC and after about 3 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=8:1), collected and concentrated to dryness to give compound I-22 (R) as a white solid f =0.25), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.77-0.79 (d, j=12.0 hz, 1H), 0.85 (s, 3H), 0.95 (s, 6H), 0.97 (s, 6H), 1.27 (s, 3H), 1.40 (s, 9H), 1.70 (s, 3H), 1.80 (s, 3H), 4.23-4.26 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.70-4.73 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.03-5.05 (d, j=8.0 hz, 1H), 5.31-5.34 (t, j=6.0 hz, 1H), 7.32-7.33 (m, 1H), 8.44-8.45 (m, 1H), overlap with the other peaks and the other peaks on the blunt edges of the circles of 1.0.0 hz,1H, 1.8.0H, 3.0H, 3-0H, 3.0H, and other peaks and the other peaks; ESI-MS 1346.7216[ M+H ]] + 。
EXAMPLE 23 ginsenoside Rb 1 Preparation of amino acid derivative I-23
The procedure was followed in the same manner as in example 3 except that Boc-D-valine in example 3 was replaced with Boc-L-proline and TLC was used to monitor the progress of the reaction, after about 3 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=7:1), collected and concentrated to dryness to give compound I-23 (R) as a white solid f =0.30), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N)δ0.77-0.80(d，J＝12.0Hz，1H)，0.86(s，3H)，0.95(s，6H)，0.97(s，6H)，1.25(s，3H)，1.40(s，9H)，1.70(s，3H)1.78 (s, 3H), 1.77-2.02 (m, 4H), 4.21-4.25 (d, j=12.0 hz, 1H), 4.59-4.62 (d, j=8.0 hz, 1H), 4.70-4.72 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.00-5.04 (d, j=8.0 hz, 1H), 5.31-5.35 (t, j=6.0 hz, 1H), parent nucleus and other H overlaps 1.06-2.56, sugar ring and other H overlaps 3.01-4.20, active hydrogen overlaps 5.76-6.40, blunt peaks; ESI-MS 1306.7155[ M+H ] ] + 。
Example 24: ginsenoside Rb 1 Preparation of amino acid derivative I-24
The procedure was followed in the same manner as in example 3 except that Boc-D-valine in example 3 was replaced with Boc-L-proline and TLC was used to monitor the progress of the reaction, after about 3 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=7:1), collected and concentrated to dryness to give compound I-24 (R) as a white solid f =0.25), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.75-0.78 (d, j=12.0 hz, 1H), 0.88 (s, 3H), 0.96 (s, 6H), 0.97 (s, 6H), 1.27 (s, 3H), 1.41 (s, 9H), 1.67 (s, 3H), 1.78 (s, 3H), 1.77-2.09 (m, 4H), 4.21-4.25 (d, j=12.0 hz, 1H), 4.60-4.63 (d, j=8.0 hz, 1H), 4.69-4.72 (d, j=8.0 hz, 1H), 4.82-4.85 (d, j=8.0 hz, 1H), 5.01-5.04 (d, j=8.0 hz, 1H), 5.30-5.34 (t, j=6.hz, 1H), parent nucleus and other H overlaps 1.60-4.60 and other carbohydrate overlaps 3.0H and 3.01-4.0 hz and 1H overlaps 3.40-6.0H; ESI-MS 1306.7154[ M+H ]] + 。
EXAMPLE 25 ginsenoside Rb 1 Preparation of amino acid derivative I-25
Operating in the same manner as in example 3, the preparation of the compound of formula I-3 of example 3 is not The same procedures were repeated except that Boc-D-valine was replaced with Boc-L-lysine in example 3, TLC was used to monitor the progress of the reaction, after about 3 hours, the reaction was stopped, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=7:1), and the eluent was collected and concentrated to dryness to give Compound I-25 (R) as a white solid f =0.30), yield was 27%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.77-0.79 (d, j=12.0 hz, 1H), 0.86 (s, 3H), 0.95 (s, 6H), 0.97 (s, 6H), 1.26 (s, 3H), 1.29 (m, 2H), 1.41 (s, 9H), 1.55 (m, 2H), 1.69 (s, 3H), 1.79 (s, 3H), 1.88 (m, 2H), 2.65 (m, 2H), 4.22-4.25 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.71-4.73 (d, j=8.0 hz, 1H), 4.83-4.85 (d, j=8.0 hz, 1H), 5.01-5.04 (d, j=8.hz, 1H), 5.31-5.34 (t, 6 hz, 1.0 hz), and other active peaks overlapping the band of 1.0H and the band of 1.0H, 4.20-0H; ESI-MS 1337.7577[ M+H ]] + 。
EXAMPLE 26 ginsenoside Rb 1 Preparation of amino acid derivative I-26
The procedure was followed in the same manner as in example 3 except that Boc-D-valine was replaced with Boc-L-lysine in example 3, the reaction was stopped after about 3 hours by TLC, and the mixture was purified by column chromatography on silica gel (eluent: dichloromethane: methanol=7:1), collected and concentrated to dryness to give compound I-26 (R) f =0.25), yield was 27%. 1 H NMR(400M Hz，C 5 D 5 N)δ0.76-0.78(d，J＝12.0Hz，1H)，0.87(s，3H)，0.96(s，6H)，0.97(s，6H)，1.26(s，3H)，1.28(m，2H)，1.40(s，9H)，1.55(m，2H)，1.70(s，3H)，1.80(s，3H)，1.88(m，2H)，2.65(m，2H)，4.22-4.26(d，J＝12.0Hz，1H)，4.60-4.62(d，J＝8.0Hz，1H)，4.70-4.72(d，J＝8.0Hz，1H)，4.82-4.84(d，J＝8.0Hz，1H)，5.03-5.06(d，J＝8.0Hz，1H)，5.31-5.35(t，J＝6.0Hz，1H)，The mother nucleus and other H are overlapped with 1.06-2.55, the sugar ring and other H are overlapped with 3.01-4.20, the active hydrogen is overlapped with 5.76-6.40, and the peak is blunt; ESI-MS 1337.7576[ M+H ]] + 。
EXAMPLE 27 ginsenoside Rb 1 Preparation of amino acid derivative I-27
The procedure was followed in the same manner as in example 3, except that Boc-D-valine in example 3 was replaced with Boc-L-asparagine, the reaction was stopped after about 3 hours by TLC monitoring the progress of the reaction, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol=7:1), collected and concentrated and dried to give compound I-27 (R) f =0.25), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.79 (d, j=12.0 hz, 1H), 0.87 (s, 3H), 0.96 (s, 6H), 0.97 (s, 6H), 1.25 (s, 3H), 1.41 (s, 9H), 1.68 (s, 3H), 1.79 (s, 3H), 2.65-2.86 (m, 2H), 4.22-4.25 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.70-4.72 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.0 hz, 1H), 5.02-5.04 (d, j=8.0 hz, 1H), 5.31-5.34 (t, j=6.0 hz, 1H), 8.9 (s, 2H), 1.06-2.38 overlapping mother nuclei and other H, 3.01-4.20 overlapping sugar rings and other H, 5.76-6.50 overlapping active hydrogen, blunt peaks; ESI-MS 1323.7056[ M+H ] ] + 。
EXAMPLE 28 ginsenoside Rb 1 Preparation of amino acid derivative I-28
The procedure was followed in the same manner as in example 3, except that Boc-D-valine in example 3 was replaced with Boc-D-cyclohexylalanine, the reaction was stopped after about 3.5 hours, and the mixture was purified by column chromatography on silica gel (eluent: dichloromethane: methanol=9:1), collected and washedConcentrating and drying the removed solution to obtain a white solid compound I-28 (R) f =0.30), yield was 28%. 1 H NMR(400M Hz，C 5 D 5 N) delta 0.76-0.78 (d, j=12.0 hz, 1H), 0.88 (s, 3H), 0.95 (s, 6H), 0.97-1.10 (m, 5H), 1.25 (s, 3H), 1.41 (s, 9H), 1.44-1.56 (m, 5H), 1.59-1.68 (m, 1H), 1.68 (s, 3H), 1.79 (s, 3H), 1.87 (m, 2H), 2.65-2.86 (m, 2H), 4.22-4.25 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 4.70-4.72 (d, j=8.0 hz, 1H), 4.82-4.84 (d, j=8.02-5.04 (d, 1.0 hz), 1.34-5 hz, 1.8.8 hz), 1.58-5.8 hz, 1H) and other peaks superimposed on the band (s, 1H), 2.65-2.86 (m, 2H), 4.22-4.25 (d, j=12.0 hz, 1H), 4.60-4.62 (d, j=8.0 hz, 1H), 1.8.8.0 hz, 1.8.7, 1H; ESI-MS1362.7780[ M+H ]] + 。
Effect example 1: anti-ZIKV activity test
Ribavirin (Ribavirin) is selected as a positive control drug, vero cells are experimental cells, and ZIKV SZ-WIV (GenBank: KU 963796) strain is an experimental strain. Based on the results of the cytotoxicity test and the plaque inhibition test, read was used&The Muench method calculated the Concentration of Compound (CC) that inhibited 50% of viral replication in the sample 50 ) The effective concentration of the sample to inhibit 50% of cell growth (CC 50 ) And pass through formula ti=cc 50 /EC 50 The selectivity index TI value is calculated. The specific method is described as follows:
vero cytotoxicity assay: toxicity of the compounds to cells was determined using the MTT method. Vero cells were seeded in 96-well plates (4X 10) 4 Individual/well), at 37 ℃, 5% co 2 Culturing in an incubator overnight. After the cells grew into a monolayer, the culture supernatant was discarded, a medium containing a gradient of the diluted compound was added, 3 duplicate wells were set for each concentration, and a control group containing no compound was set. After 4 days of culture, 20 mu L of 5mg/mL MTT is added into each well, incubation is carried out for 4 hours at 37 ℃, 100 mu L of supernatant is discarded, 100 mu L of 12% SDS-50% DMF solution is added, incubation is carried out at 37 ℃ overnight, after the formazan crystals are completely dissolved, a Bio-TEK microplate reader is selected for detecting OD value, the measuring wavelength is 570nm, and the reference wavelength is 630nm; calculating half of the cytotoxic concentration CC 50 I.e. the drug concentration at which 50% of Vero cells are toxic Degree.
Plaque inhibition assay: according to the experimental result of the compound on cytotoxicity, the concentration of the compound is diluted to a concentration which is nontoxic to cells. Screening was performed by classical plaque method: vero cells were seeded in 12-well plates (3X 10) 5 Individual/well), constant temperature at 37 ℃, 5% co 2 Culturing in an incubator overnight. After the cells grew into a monolayer, the culture supernatant was discarded, washed 1 time with PBS, and after adsorption with ZIKV virus (MOI. Apprxeq.0.5) for 2 hours, 2% low melting point agarose DMEM (4% FBS) medium containing gradient dilution compounds was added, 37℃and 5% CO 2 After 4 days of incubation, 4% paraformaldehyde was used for 15min, washed, stained with 0.8% crystal violet for 10min, and picture taken with an enzyme-linked fluorescence spot analyzer (CTL, immunospot S6 Universal) and plaques were counted. Dose response curves were plotted from the number of plaques, and half-effective concentrations EC were calculated 50 I.e. the drug concentration at which the inhibition rate of plaque formation is 50% after ZIKV infection of Vero cells.
Virus yield reduction experiments: vero cells were seeded in 24-well plates at 1.5X10 per well 5 Individual cells, at 37 ℃, 5% co 2 Culturing in an incubator overnight. After the cells grow into a monolayer, the culture supernatant is discarded, ZIKV virus (MOI approximately equal to 1) is added to adsorb the 2h discarded virus liquid, PBS is used for washing 3 times, and then a compound containing gradient dilution is added, wherein the temperature is 37 ℃ and the concentration is 5% CO 2 Culturing in an incubator for 72 hours, collecting virus supernatant and extracting RNA in the virus supernatant by using a kit Roche High Pure Viral RNA Kit; real-time fluorescent quantitative PCR was performed under a Quantum studio5 quantitative PCR system using quantitative PCR kit RNA-directTM Realtime PCR Master Mix and TaqMan probes; calculating EC of the medicine according to inhibition rate of ZIKV RNA replication by different concentrations of compounds 50 。
Firstly, the concentration of the compound is diluted to a concentration which is nontoxic to cells, and screening is carried out by a classical plaque method, and the result shows that the compound prepared by the embodiment has obvious inhibition effect on ZIKV, can inhibit the formation of ZIKV plaques by 100% at a primary screening concentration of 20 mu M, and further anti-ZIKV activity researches by RT-PCR and plaque method show that the target compound has stronger anti-ZIKV activity, the inhibition activity of which is improved by 20-65 times compared with that of positive control Ribavirin (Ribavirin), has lower cytotoxicity, and can be used as an anti-ZIKV drug candidate for further research and development.
TABLE 2
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Effect example 2: anti-DENV activity assay
Ribavirin (Ribavirin) is selected as a positive control drug, vero cells are experimental cells, and a DENV-II D01090 (GenBank: KY 882458) strain is an experimental strain. Based on the results of cytotoxicity and cytopathic inhibition experiments, read was used &The Muench method calculated the effective concentration of the sample to inhibit 50% of viral replication (EC 50 ) The effective concentration of the sample to inhibit 50% of cell growth (CC 50 ) And pass through formula ti=cc 50 /EC 50 The selectivity index TI value is calculated. The specific method is described as follows:
plaque inhibition assay: the concentration of the compound was diluted to 20. Mu.M and verified by classical plaque assay by plating Vero cells on a 12 well plate 3X 10 5 After 24h incubation, DENV-2 virus (MOI=0.5) was added to adsorb 2-4 h, and DMEM medium containing 1% low melting agarose and 2% FBS at different drug concentrations was added to the culture medium at 37℃and 5% CO 2 After 5 days of culture, observing the number of plaques, fixing 4% paraformaldehyde for 15min, blowing off agar blocks, staining 0.8% crystal violet for 10min, performing picture acquisition by using an enzyme-linked fluorescence spot analyzer (CTL, immunospot S6 Universal), counting plaques, and calculating half-effective drug concentration EC 50 。
Vero cytotoxicity assay: toxicity of the compound on cells was measured by MTT method, and Vero cells were assayed at 3X 10 5 Inoculating the cells/well into 96-well culture plate at 37deg.C with 5% CO 2 Culturing for 24h. After the cells grow into a monolayer, the culture supernatant is discarded, and a gradient dilution test compound is addedDMEM medium was set with 3 duplicate wells per concentration and normal cell control. After 5 days of incubation, 20. Mu.L of 5mg/mL MTT was added to each well and incubated at 37℃for 4 hours, 100. Mu.L of the supernatant was discarded, 100. Mu.L of 12% SDS-50% DMF solution was added and incubated overnight at 37 ℃; after the crystallization is completely dissolved, shaking and mixing are carried out, and a Bio-TEK enzyme-labeled instrument is selected for detecting the OD value (detection wavelength is 570nm and reference wavelength is 630 nm). And drawing a dose response curve according to experimental results, and calculating the concentration of the half cytotoxicity.
The results show that the compounds prepared in the above examples can achieve 100% inhibition of DENV-II at 50. Mu.M, and that the effective compounds are re-screened by plaque method to obtain their ECs 50 The values, as shown in Table 3, are significantly better than the positive control Ribavirin (Ribavirin) for the DENV-II inhibitory activity and have lower cytotoxicity. The therapeutic index (Therapeutic index, TI) is the half-toxicity concentration CC of the compound on cells 50 And half effective compound concentration EC against virus 50 The ratio of (2) represents the safety of the compound, the greater the value the safer. The compounds in the table 3 have obvious inhibition effect on the replication of DENV, and the therapeutic index of the compounds is 2-10 times higher than that of positive control ribavirin, so the compounds can be used as anti-DENV drug candidates for development and application;
TABLE 3 Table 3
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As shown in tables 2 and 3, the compounds I-1, I-3, I-7, I-8, I-10, I-12, I-28, etc. showed strong inhibition activity on ZIKV and DENV, and EC thereof 50 The value is obviously superior to positive control ribavirin, and can be developed and applied as a drug candidate for resisting ZIKV or DENV infection.
Applicants state that the present invention describes the ginsenoside Rb by the above examples 1 Derivatives, their preparation and use, but the invention is not limited thereto The above embodiments do not mean that the present invention must be practiced with the above embodiments. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims (6)

1. Ginsenoside Rb with structural formula shown in formula I 1 A derivative or a pharmaceutically acceptable salt thereof:
wherein: r is selected from H;
R 1 、R 2 or R is 3 Each independently selected from R orn is any integer between 0 and 3;
R 1 selected from H, C 1 -C 6 Straight or branched alkyl, C 3 -C 6 Cycloalkyl, -C 1 -C 6 Straight-chain or branched alkyl-NH of (2) 2 、-C 1 -C 6 Straight-chain or branched alkyl-OH, C 1 -C 6 Straight-chain or branched alkylthio, C 6 -C 10 Aryl, C of (2) 2 -C 10 Heteroaryl of (a);
R 2 selected from H, t-butoxycarbonyl, or R 2 And R is R 1 Connection structure C 2 -C 6 A heterocycloalkyl group;
R、R 1 、R 2 、R 3 h is not selected at the same time.
2. Ginsenoside Rb with structural formula shown in formula I 1 A derivative or a pharmaceutically acceptable salt thereof:
3. ginsenoside Rb of claim 1 or 2 1 The preparation method of the derivative is characterized in that: ginsenoside Rb in the presence of solvent and alkaline catalyst 1 Uniformly mixing with halohydrocarbon shown in formula (1) or amino acid shown in formula (2), stirring at 0-80deg.C for 0.5-12 hr, and separating and purifying the reaction product to obtain ginsenoside Rb 1 The derivative has the following reaction formula:
4. a method of preparation according to claim 3, characterized in that: ginsenoside Rb 1 The molar ratio of the halohydrocarbon shown in the formula (1) or the amino acid shown in the formula (2) is 1:1-36.
5. A method of preparation according to claim 3, characterized in that: the alkaline catalyst is one or more of potassium carbonate, sodium hydrogen, N' -diisopropylcarbodiimide and 4-dimethylaminopyridine; the solvent is one or more of tetrahydrofuran, toluene, acetonitrile, dichloromethane, N-dimethylformamide and pyridine.
6. Ginsenoside Rb of claim 1 1 Use of a derivative or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prophylaxis of zika virus and/or dengue virus.
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