CN105859822A - 2-substituted and/or 27-substituted betulinic acid derivatives as well as preparation method and applications thereof - Google Patents
2-substituted and/or 27-substituted betulinic acid derivatives as well as preparation method and applications thereof Download PDFInfo
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Abstract
The invention belongs to the field of medical medicines, and concretely relates to 2-substituted and/or 27-substituted betulinic acid derivatives as well as a preparation method and applications thereof. The structure of 2-substituted and/or 27-substituted betulinic acid derivatives is shown in a formula I. The invention also provides the preparation method and applications of the 2-substituted and/or 27-substituted betulinic acid derivatives. The 2-substituted and/or 27-substituted betulinic acid derivatives have good safety and clear efficacies for resisting cancer, resisting organ fibrosis, regulating immunity, resisting digestive tract inflammation and ulcer, resisting fungus and other respects, and other respects; and the derivatives provide new choices for preparing medicaments for treating the related diseases.
Description
Technical field
The invention belongs to chemical medicine field, be specifically related to 2 and/or 27 substituted betulinic acid derivatives and preparation method thereof
And purposes.
Background technology
Belulinic acid Betulinic acid (betulinic acid) is lupinane type pentacyclic triterpenoid, be distributed widely in Rhamnaceae, Myrtaceae,
In the various plants such as Betulaceae, but universal content is relatively low.Owing to the female ring structure of belulinic acid Betulinic acid is bigger so that its molecular polarity is less,
Fat-soluble bigger.Therefore, belulinic acid Betulinic acid dissolubility in water is less, is soluble in ether, the organic solvent such as chloroform, ethanol.Birch
Wood acid has extremely strong selecting cell toxicity (Pisha E, Chai H, Lee I S, et al.Discovery of to melanoma cells
betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis
[J].Nat Med,1995,1(10):1046-1051)；The primary glioblastoma cells apoptosis rate of induction be significantly larger than vincristine and
Nitroso ureas (Jeremias I, Steiner H H, Benner A, et al.Cell death induction by betulinic acid, ceramide
and TRAIL in primary glioblastoma multiforme cells[J].Acta Neurochir,2006,146(7):721-729)；
With many cytotoxic compound such as doxorubicins, paclitaxel, support pool former times, actinomycin D drug combination, and can have
Synergism, is expected to as synergist, for the chemical combined therapy of tumor, it is to avoid the generation of multidrug resistance, strengthens antitumor
Therapeutic effect (Eder-Czembirek C, Czembirek C, Erovic B M, the et al.Combination of betulinic of medicine
acid with cisplatin-different cytotoxic effects in two head and neck cancer cell lines[J].Oncol Rep,
2005,14(3):667-671)；Can the duplication of HIV in the almost all link of viral lifecycle suppresses H9 lymphocyte
(Mayaux J F,Bousseau A,Pauwels R,et al.Triterpene derivatives that block entry of human
immunodeficiency virus type 1into cells[J].Pro Nat Acad Sci,1994,91(9):3564-3568).Additionally, birch
Wood acid also has the various active such as immunomodulating, antiinflammatory, anti-oxidation stress, antibacterial, parasiticide, malaria and antiulcer (easily
Jin E, Wu Jing, Wen Lixin, etc. the Advance on Pharmacological Activities of belulinic acid Betulinic acid. Chinese herbal medicine, 2014,45 (14): 2118-2124).With
The understanding to belulinic acid Betulinic acid pharmacologically active constantly to deepen, many researcheres are attempted carrying out structural modification with belulinic acid Betulinic acid for parent nucleus, in the hope of
Improve its dissolubility, increase its bioavailability, reduce its toxicity.The structural modification of belulinic acid Betulinic acid is concentrated mainly on three positions:
C-3 position, C-20 position and C-28 position.
Document " belulinic acid Betulinic acid and the progress of derivant thereof " (Li Dan, Zhou Jinpei, Wu Xiaoming. [J]. pharmacy be in progress, 2004,
28 (3): 120-125) in:
(1) transformation to 3 hydroxyls is usually with pyridine solvent, reacts with various ring-type dicarboxylic anhydrides, synthesizes end strips carboxylic acid
The ester of base.The transformation of this type is still the most successfully.As shown in table 1, compound 3,4,5, compared with belulinic acid Betulinic acid, is lived
Property is greatly enhanced.Illustrate that the acyl group of the type may strengthen HIV (human immunodeficiency virus)-resistant activity.To belulinic acid Betulinic acid and the like Betula platyphylla Suk. 3 hydroxyls of element
Other transformation, as changed β position hydroxyl into α position, hydroxyl changes carbonyl into, alcohol changes amide etc. into, mostly causes increased activity.
Illustrating to play hydrogen bond reaction crucial during activity may be relevant to the oxygen of 3 β positions.Wherein, compound 3 (DSB, also known as
YK-FH312), the most noticeable.The result of study of Taisei Kanamoto etc. shows, YK-FH312 may be by making
HIV (human immunodeficiency virus)-resistant activity is realized for virion assembling and (or) virion step of sprouting.
(2) transformation to 19 isopropenyls of belulinic acid Betulinic acid does not obtains the most gratifying result.Replacing on 30, activity is protected
Hold, but activity is substantially harmful to by acidic-group replacement；20, carbon changes ketone or oxime, inactivation or activity into be reduced；19 change acyl into
Base or acidic-group, can cause inactivation.Illustrate that the oxygen atom of high electronegativity may change the static behaviour of belulinic acid Betulinic acid so that it is cell toxicant
Property reduce.Transforming the unique of 19 acquisitions is successfully dihydrobetulinic acid (Dihydrobetulinic acid) and derivant thereof.Dihydro
Belulinic acid Betulinic acid is the fusion inhibitor of HIV and cell membrane.Its AntiHIV1 RT activity measures with H9 cell as infection model, and IC50 is
12.6 μm ol/L, EC50=0.9 μm ol/L.With dihydrobetulinic acid as primer, modified after the treatment of compound 7 that obtains refer to
Number is 14000, and HIV (human immunodeficiency virus)-resistant activity is higher than its primer about 1000 times.
(3) 28 carboxyls are the modification focuses of current belulinic acid Betulinic acid, it are modified the derivant obtained of a great variety.In recent years
In research, to the structure of modification of 28 carboxyls with biological activity for instructing, gradually concentrate on and carboxylic acid group be transformed into various amide,
And most branch terminals all remains carboxylic acid group in these amide derivatives.Now compare representational compound
Introduced.As shown in table 2, the amide chain of this analog derivative is bad compared with short-lived activity.The when that amide chain being longer
R=CONH (CH2)mWhen COOH, m=7-11, activity is meaningful, and during m=10, activity is the strongest；
R=CONH (CH2)7CONH(CH2)nM=7 before specific activity when COOH, n=1-4,10 all it is improved；R=CONH (CH2)7NHCO(CH2)nWhen COOH, n=1-3, activity is improved again.Additionally, it is possible to because amine moiety and adjacent protons interaction are right
Carboxylic acid group plays sterically defined effect, and in chain, first CONH, N are upper substituted, CONH become NHCO or
NHCONH, inactivates.Second CONH: 1. (this aminoacid comprises the lipotropy of 1-2 methyl with little a-amino acid
Part) it is connected, activity increases.2. it is connected with the benzoic acid at ortho position, inactivation；It is connected with the benzoic acid of meta, para-position, IC50
Up to about 100nmol/L.If 3. CONH changes NHCO into, activity just strengthens.Foremost in this analog derivative it is
RPR103611, it suppresses HIV by the rear combination during suppression virus and cell membrane fusion, i.e. peplos dependency stage
Infect (IC50=10nmol/L), be currently the only by affecting the non-peptide matters of little molecule that gp41 stops HIV to invade,
It is hopeful to be developed into HIV cell membrane fusion agent.The optical isomer IC9564. (34.) of PRP103611 is at viral infection
Reduce IC90=0.22 ± 0.05 μm ol/L measuring K NL4-3, demonstrate the strongest HIV (human immunodeficiency virus)-resistant activity.And at IC9564 be
In row derivant, more prominent is compound 35 (EC50=0.33 μm ol/L) and 36 (EC50=0.46 μm ol/L), they
Activity is suitable with IC9564.Research display, IC9564. can strongly block the film of HIV-1 shell mediation and merge, thus penetrate
Step blocks the duplication of HIV.Key substance HIV-1gp120 during this is the molecular target of IC9564 effect.
At document " 3D-QSAR of 23-HBA derivant antitumor action and molecular docking pattern " (Zhang Ting
Graceful, Bi Yi, Chen Mengmeng etc. [J]. and Chinese Pharmaceutical Journal, 2014,49 (14): 1200-1203) in: 23-HBA derivant
(structure below figure) is also pentacyclic triterpenoid, is a kind of betulinic acid analogue of isolated from the Chinese medicine Radix Pulsatillae,
There is the anti-tumor activity suitable with Betulinic Acid, but among its mechanism of action currently studies.This laboratory early stage has synthesized greatly
Amount 23-HBA derivant and it has been carried out antitumor activity, research shows, 28-COOH is to affect such
The main portions of antitumor activity of compound.
Up to the present, to the transformation of C-3 position hydroxyl and C-28 position carboxyl achieved with certain progress, but the structural modification of C-20 position and
Transformation still lacks gratifying result.The substituted betulinic acid derivative in other position then has no report.
Summary of the invention
The invention provides a kind of 2 and/or 27 substituted betulinic acid derivatives, its structure is as shown in formula I:
Wherein, R1、R2Independently be-H, hydroxyl,
R3、R4、R5Independently be-H, halogen, hydroxyl, C1～C6 alkyl, C1～C6 alkoxyl, acetoxyl group or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
Preferably, R1、R2Independently be-H, hydroxyl,
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
It is further preferred that R1、R2Independently be
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
Further preferred, R1、R2Independently be
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen atom.
Above-mentioned 2 and/or 27 substituted betulinic acid derivatives, work as R1ForTime, its structure such as formula II institute
Show:
Wherein, R2For-H, hydroxyl,
R3、R4、R5Independently be-H, halogen, hydroxyl, C1～C6 alkyl, C1～C6 alkoxyl, acetoxyl group or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
Preferably, R2For-H, hydroxyl,
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
It is further preferred that R2For
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
Further preferred, R2For
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen atom.
Above-mentioned 2 and/or 27 substituted betulinic acid derivatives, work as R2ForTime, its structure such as formula III institute
Show:
Wherein, R1For-H, hydroxyl,
R3、R4、R5Independently be-H, halogen, hydroxyl, C1～C6 alkyl, C1～C6 alkoxyl, acetoxyl group or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
Preferably, R1For-H, hydroxyl,
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
It is further preferred that R1For
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
Further preferred, R1For
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen atom.
Present invention also offers the preparation method of above-mentioned 2 and/or 27 substituted betulinic acid derivatives, comprise the following steps:
A, the use 95%wt alcohol steep waistcoat fresh leaf 2 of son～4 times, extract 24h every time, merge leachate and also filter, and filtrate is dense
Extractum is obtained after contracting；Again extractum after ultrasonic dissolution, is extracted with ethyl acetate in 20%wt ethanol water；
After b, the ethyl acetate layer of extraction concentrate, after column chromatography, obtain crude product；Described column chromatography eluent is petroleum ether and acetic acid
The mixed solution of ethyl ester, its volume ratio is followed successively by 20 1,15 1,10 1,51,31,11；Described column chromatography
Developer be 5% vanillin concentrated sulfuric acid solution；
C, above-mentioned crude product 70%wt ethanol and DMF heating for dissolving, be filtered to remove insoluble matter, and filtrate is divided through preparative liquid chromatography
From purification, concentrate drying obtains 2 and/or 27 substituted betulinic acid derivatives.
In the preparation method of above-mentioned 2 and/or 27 substituted betulinic acid derivatives, the waistcoat fresh leaf of son and 95%wt described in step a
The mass ratio of ethanol is 1 10.
In the preparation method of above-mentioned 2 and/or 27 substituted betulinic acid derivatives, extractum described in step a and 20%wt ethanol
The mass volume ratio (kg/L) of aqueous solution is 1 10.
In the preparation method of above-mentioned 2 and/or 27 substituted betulinic acid derivatives, the preparative liquid chromatography described in step c is adopted
Purify with acetic acidacetonitrile system gradient elution；In described acetic acidacetonitrile system, the volume content of acetic acid is that 3%～90% gradient increases.
Present invention also offers above-mentioned 2 and/or 27 substituted betulinic acid derivative pharmaceutically acceptable salts.
Present invention also offers the prodrug of the compounds of this invention, according to the present invention, prodrug is the derivant of above-claimed cpd, they
Self be likely to be of more weak activity or even without activity, but upon administration, in physiological conditions (such as by metabolism,
Solvolysis or other mode) it is converted to corresponding biologically active form.
Present invention also offers above-mentioned 2 and/or 27 pharmaceutically acceptable hydrates of substituted betulinic acid derivative.
The present invention also provides for a kind of pharmaceutical composition, is that above-mentioned 2 and/or 27 substituted belulinic acid Betulinic acid provided by the present invention spread out
Biology adds what the complementary composition of pharmaceutically acceptable was prepared from.2 and/or 27 the substituted birch that the present invention provides
Wood acid derivant structure is as shown in formula I～III.
Present invention also offers above-mentioned 2 and/or 27 substituted betulinic acid derivatives, its salt or hydrate to have in preparation
Purposes in anti-fibrosis medicine.
Present invention also offers above-mentioned 2 and/or 27 substituted betulinic acid derivatives, its salt or hydrate to have in preparation
Purposes in antifungal activity medicine.
Present invention also offers above-mentioned 2 and/or 27 substituted betulinic acid derivatives, its salt or hydrate to have in preparation
Purposes in anti-tumor activity medicine.
Present invention also offers above-mentioned 2 and/or 27 substituted betulinic acid derivatives, its salt or hydrate to have in preparation
Purposes in the medicine of two-way immunoregulation effect.
Present invention also offers above-mentioned 2 and/or 27 substituted betulinic acid derivatives, its salt or hydrate to have in preparation
Purposes in the medicine of anti-alimentary tract inflammation and ulcer.
There is provided 2 of the present invention and/or 27 substituted betulinic acid derivatives are respectively provided with good safety, at antitumor, anti-
The many-sides such as internal organs fibrosis, regulation immunity, anti-alimentary tract inflammation and ulcer, and antifungal have clear and definite drug effect, for preparation
The medicine treating above-mentioned effect relevant disease provides new selection.
Detailed description of the invention
The preparation method of 2 and/or 27 substituted betulinic acid derivatives shown in formula I, comprises the following steps:
A, the use 95%wt alcohol steep waistcoat fresh leaf 2 of son～4 times, extract 24h every time, merge leachate and also filter, and filtrate is dense
Extractum is obtained after contracting；Again extractum after ultrasonic dissolution, is extracted with ethyl acetate in 20%wt ethanol water；
After b, the ethyl acetate layer of extraction concentrate, after column chromatography, obtain crude product；Described column chromatography eluent is petroleum ether and acetic acid
The mixed solution of ethyl ester, its volume ratio is followed successively by 20 1,15 1,10 1,51,31,11；Described column chromatography
Developer be 5% vanillin concentrated sulfuric acid solution；
C, above-mentioned crude product 70%wt ethanol and DMF heating for dissolving, be filtered to remove insoluble matter, and filtrate is divided through preparative liquid chromatography
From purification, concentrate drying obtains 2 and/or 27 substituted betulinic acid derivatives.
In the preparation method of above-mentioned 2 and/or 27 substituted betulinic acid derivatives, the waistcoat fresh leaf of son and 95%wt described in step a
The mass ratio of ethanol is 1 10.
In the preparation method of above-mentioned 2 and/or 27 substituted betulinic acid derivatives, extractum described in step a and 20%wt ethanol
The mass volume ratio (kg/L) of aqueous solution is 1 10.
In the preparation method of above-mentioned 2 and/or 27 substituted betulinic acid derivatives, the preparative liquid chromatography described in step c is adopted
Purify with 3% acetic acidacetonitrile system gradient elution；In described acetic acidacetonitrile system, the volume content of acetic acid is 3%～90% gradient
Increase.
The preparation of embodiment 1 compound 1
Weigh the waistcoat fresh leaf 4kg of son, 10 times amount 95% ethanol mercerations in three times, extract 24h every time, merge leachate and filter,
Extractum is obtained after filtrate reduced in volume.Take the ethanol water liquid 3L that extractum 300g adds 20%, ultrasonic dissolution, use petroleum ether successively
(1 × 3L), ethyl acetate (3 × 6L) extract, fetch water respectively layer, petroleum ether layer, ethyl acetate layer, upper Liquid Detection, knot
Fruit display target component major part enters ethyl acetate layer, and in water liquid, driftlessness composition, petroleum ether layer have a small amount of target component.
Ethyl acetate layer is concentrated into just has solid to separate out, and pours out while hot and stirs and add a small amount of acetic acid ethyl dissolution to without solid
Grain, takes 100-200 mesh silica gel 500g and mixes sample, silica gel 1.5kg wet method dress post, will mix dry method loading after sample silica gel is dried completely.
Drawing sample respectively to carry out elution requirement and grope, obtain a result, use petrol ether/ethyl acetate system to carry out eluting, TLC is carried out
Monitoring, developer is 5% vanillin concentrated sulfuric acid solution.Initially with petrol ether/ethyl acetate 20:1 eluent 10L recycling elution,
There is an orange-yellow band to be displaced downwardly at bottom 1/3 on chromatographic column, now start to collect with stream parts such as 4L/ bottles and number.1-3 bottle
All immaculates；Start to change eluting ratio to make 15:1 from the 4th bottle；5-6 bottle starts more significantly single-point occur, and point hardens
Fruit is consistent, merges, and upper liquid phase detects, without substantially absorbing under 320nm, and non-targeted composition；7th bottle starts eluting ratio
Example is changed and is made 10:1, is multiple purple points to the 12nd bottle of some plate colour developing, and Liquid Detection is still without substantially absorption, non-targeted section；13-14
Bottle point plate colour developing has yellow point to occur, Liquid Detection is it is observed that two peaks before 20min；Eluting ratio is changed work 5 by the 15th bottle:
1, the purple point of some plate colour developing disappears, and light yellow speckle occurs, Liquid Detection is without substantially absorption, non-targeted section；17th bottle start by
Eluting ratio is changed and is made 3:1, and some plate colour developing is green to be occurred, Liquid Detection is without substantially absorption, non-targeted section；, pale yellow to the 20th bottle
Point, green point disappear, and Liquid Detection is without substantially absorbing respectively, non-targeted section；21st bottle starts, and chromatograms starts appearance
Multiple peaks after 50min, but the peak before and after 30min does not all occur, changes to the 25th bottle of eluting ratio and makees 1:1, some plate colour developing
Green disappearance, Liquid Detection is still the multiple peaks after 50min；With eluting ratio 1:1 to the 27th bottle, Liquid Detection target
Section starts appearance, and to the 32nd bottle, some plate colour developing and Liquid Detection target phase elute the most completely.Merge 27-32 bottle and concentrate,
Methanol rinses pillar, Liquid Detection, and target phase confirms noresidue.Though target phase is separated relatively independently, but 50min
Later several peaks still occur along with target phase, and target phase polarity is relatively big, and pre-upper preparation liquid phase separates.
Target phase uses 70% ethanol and a small amount of DMF heating for dissolving, leaches insoluble matter, leaches solid detection driftlessness peak, abandons it,
Filtrate crosses post, purification, uses 3%HAc-ACN system gradient elution purification, collects target peak, and concentrate drying obtains compound 1,
Liquid Detection purity ＞ 97.26%.
Compound 1 is khaki pulverulent solids.HREIMS m/z 779.4173[M-H](cal C48H60O9-H 779.4237)。
1HNMRδH 0.91(Me-23),δH 0.73(Me-24),δH 0.92(Me-25),δH 0.92(Me-26),δH 1.69
(Me-30),δH 4.70(br s,H-29),δH 4.58(br s,H-29),δH6.30 (d, J=18Hz, H-α), δH 7.54(d,J
=18Hz, H-β), δH6.38 (d, J=18Hz, H-α ') and δH7.54 (d, J=18Hz, H-β ').13C-NMRδC 115.1
(d),144.6(d),114.4(d)and 144.0(d),δC 109.7(t),δC 159.8(s),159.6(s),δC 78.1(d),δC
166.6(s)and 166.3(s),δC 177.2(s)。
Embodiment 2 compound 2 and the preparation of compound 3
Use the method similar to preparing compound 1, prepare compound 2 and compound 3.
The embodiment of the present invention 3～9 use cell strain, the source of reagent be: cervical cancer cell lines Hela, human hepatoma cell strain
HepG-2, human lung carcinoma cell line A549, human leukemia cell line K562, human stomach cancer cell line MGC-803, mouse junction cancer
Cell strain C26, fungus truffle are provided by Bao Ke bio tech ltd, Chengdu.Hyclone and corresponding culture medium thereof are purchased from
Hyclone company of the U.S.；MTT, DMSO, tragacanth, pentobarbital sodium, TNBS, phytane (pristane) are purchased from
Sigma company of the U.S.；Cyclophosphamide (CTX) is purchased from Hengrui Medicine Co., Ltd., Jiangsu Prov.；Dexamethasone is purchased from Hainan system
Pharmaceutical factory company limited；Carbon tetrachloride is purchased from Chengdu chemical reagent work；Alanine aminotransferase (ALT) builds up purchased from Nanjing；ELISA、
III procollagen type (PC-III), hyaluronic acid (HA) are magnificent purchased from Wuhan；Laminin lens (LH) is purchased from the western Tang in Shanghai；
Tripterygium glycosides, purchased from Huangshi Feiyun Pharmaceutical Co., Ltd.；Krestin is purchased from upper Haikang boat fungus polysaccharide company limited；4-bis-
Nitre fluorobenzene (DNFB) is purchased from sigma company of the U.S..
The antitumor activity in vitro of embodiment 3 compound 1
Take and be in the various tumor cells of exponential phase (tumor cell line of employing has the most several: cervical cancer cell lines Hela；
Human hepatoma cell strain HepG-2；Human lung carcinoma cell line A549；Human leukemia cell line K562；Human stomach cancer cell line MGC-803；
Mouse colonic cell strain C26), prepare cell suspension, by the corresponding culture medium containing 10% hyclone, cell concentration is adjusted to
1×105After individual/mL cell suspension, cell being inoculated in 96 well culture plates, every hole adds cell suspension 200 μ L, adds respectively next day
Enter finite concentration aseptic compound 1 solution, mix rearmounted 37 DEG C, 5%CO2After incubator cultivates 24h, separate out culture fluid
And wash twice with PBS, then MTT phosphate buffer 20 μ L and the 150 μ L culture medium of 5mg/mL are added to every hole, with
Terminate cultivating after continuing under the conditions of sample to cultivate 4h.2000rpm is centrifuged 5min, then discards the culture fluid cultivated in plate hole, often
Hole adds 150 μ L DMSO, shakes 10min, after making the α-granule of formation fully dissolve, and microplate reader detection light absorption value.Choosing
Selecting mensuration wavelength is 490nm.The computerized compound 1 IC50 to tumor cell, the results are shown in Table 1.
Table 1 compound 1 inhibitory action to kinds of tumor cells
| Cell strain | IC50(μmol/L) |
| Hela | 10.29 |
| HepG-2 | 15.85 |
| A549 | 14.75 |
| K562 | 12.97 |
| MGC-803 | 14.91 |
| C26 | 13.93 |
The above results shows, compound 1 has external tumor-inhibiting action.
Embodiment 4 compound 1 impact on mice bearing S180
Choose the S180 tumor source mice that inoculation 8d is in a good state of health, after skin of abdomen sterilization, extract ascites, with sterile physiological salt
Water is standby by 14 (ascites volume: normal saline volume) suspendible.Male mice in kunming 60,18～20g, by body weight
Stratified random is divided into 5 groups, respectively model control group (0.5% tragacanth), positive controls (cyclophosphamide, CTX),
Compound 1 group, all armpit subcutaneous vaccination 0.2mL aforementioned suspension on the right side of it.After 2h, model control group and drug component do not fill
Stomach gives tested material or suspensoid, once a day, continuous 14 days；Positive controls lumbar injection gives CTX, the next day once,
Totally 7 times.After last is administered, 24h cervical dislocation puts to death mice, peels off tumor mass and weighs, and ((1 experimental group is put down to calculate tumour inhibiting rate
All tumor weight/model control group average tumor weights) * 100%).
Table 2 compound 1 impact on mice bearing S180
| Group | Number of animals (only) | Dosage | Tumor weight (g) | Tumour inhibiting rate (%) |
| Model comparison | 12 | —— | 1.32±0.47 | —— |
| Positive control (CTX) | 9 | 40mg/kg | 0.41±0.28** | 68.9 |
| Compound 1 | 12 | 40mg/kg | 0.61±0.25** | 53.8 |
Compare with model control group, * P ＜ 0.05, * * P ＜ 0.01.
Test result indicate that, compound 1 gives 400mg/kg gavage, can suppress S180 growth in Mice Body, have relatively
Good anti-tumor activity.
Embodiment 5 compound 1 is on the impact of experimental colitis of rats caused by 2,4,6-trinitrotoluene sulfonic acid (TNBS)
SD rat 72, is divided into 6 groups by body weight stratified random, respectively Sham-operated control group (0.5% tragacanth), mould
Type matched group (0.5% tragacanth), positive controls (dexamethasone), compound 1 one-tenth are grouped.After animal fasting 24h with
Pentobarbital sodium is anaesthetized, and in addition to normal saline group, with TNBS and 40% ethanol coloclysis, replicates experimental colitis model；False
Operative control group is only with normal saline enema.6h after modeling, gives tested material.It is administered 5d, tail venous blood sampling row leukocyte
Counting.6d anaesthetizes with urethane, and after abdominal aortic blood, de-cervical vertebra puts to death animal, upwards intercepts 9cm colon from anus,
In ice bath, cut off enteric cavity along mesentery edge, rinse content, measure ulcer area, calculate ulcer area percentage ratio；Colon claims
Scraping colonic mucosa after Chong, ELISA measures tumor necrosis factor (TNF-α) concentration.
Table 3 compound 1 impact on experimental colitis of rats caused by TNBS
Compare with model control group, * P ＜ 0.05, * * P ＜ 0.01.
Result is as shown in table 3, and experimental colitis of rats caused by TNBS may occur in which inflammatory cell increase, inflammatory cytokine water
Flat rising and colon surface ulcer.Compound 1 can significantly inhibit leukocyte and the rising of important pro-inflammatory cytokine TNF-α, reduces and bursts
Infections face is formed, and has preferable resistive connection enteritis effect.
Embodiment 6 compound 1 impact on Liver Fibrosis
SD rat 60 (200-240g, male), by body weight stratified random be divided into blank group (0.5% tragacanth),
Model control group (0.5% tragacanth), positive controls (dexamethasone, 1mg/kg), compound 1 one-tenth packet (40mg/kg).
In addition to blank group, all by 1mL/kg subcutaneous injection 40% carbon tetrachloride vegetable oil solution, 2 times a week, continuous March,
Give the ethanol water of high lipid food and 5% simultaneously.Gavage gives tested material, every day 1 time, continuous March.Administration terminates
Rear next day abdominal aortic blood, separated plasma, measure alanine aminotransferase (ALT), III procollagen type (PC-III), hyalomitome
Acid (HA) and Laminin lens (LH) level；Put to death animal subsequently, take liver and carry out pathological examination.
Results of serum biochemical detection such as table 4.
Table 4 compound 1 is on experimental liver fibrosis in rats and the impact of blood parameters
| Group | LT(U/L) | PC-Ⅲ(ug/L) | HA(ug/L) | LN(ug/L) |
| Normal control | 51±6** | 8.3±1.5** | 118±22** | 10±3** |
| Model comparison | 605±129 | 40.5±7.8 | 347±75 | 61±15 |
| Positive control (dexamethasone) | 358±92** | 20.4±4.9** | 300±82 | 55±17 |
| Compound 1 | 372±146** | 20.5±5.1** | 225±69** | 40±17* |
Compare with model control group, * P < 0.05, * * P < 0.01.
The result of table 4 shows, compared with model group, compound 1 is to Carbon Tetrachloride Induced Fibrotic Rat Liver hepatic injury caused by carbon tetrachloride
Certain protective effect, each index is had to be obviously improved.
Pathological examination shows, model group rats hepatocyte hydropic degeneration is obvious, has obvious hepatic necrosis and steatosis,
Show as obvious hepatic fibrosis；1 group of hepatocyte hydropic degeneration of compound and steatosis degree relatively model group substantially reduce, prompting
It has good inhibitory action to hepatic fibrosis.
Embodiment 7 compound 1 therapeutical effect on mouse experiment lupus erythematosus and the impact on cellular immunization
KM mice 72, is divided into 6 groups by body weight stratified random, respectively suspensoid matched group (0.5% tragacanth),
Model control group (0.5% tragacanth), positive controls (tripterygium glycosides), compound 1 one-tenth are grouped.Except suspensoid compares
Group is outer all by 0.5mL/ lumbar injection norphytane (pristane), and gavage gives medicine or suspensoid, every day 1 time, continuous 30d.
24h eye socket blood sampling after last medicine, 4 DEG C of frozen centrifugation separation serum, ELISA measures Anti-hCG action level in serum.
Separately take a collection of animal to be grouped equally and duplicating model, injection pristane after the 24th day, in addition to suspensoid matched group, each group
Animal lumbar injection 5% chicken red blood cell normal saline suspension 0.2mL/ only, and continues to be administered and continues to injection pristane the 30th
Day.After last is administered, 24h takes blood 20 μ L in eye socket, adds in 1mL normal saline, is separately added into 4% chicken red blood cell subsequently raw
Reason saline suspension 0.5ml and 10% guinea pig serum 0.5mL, hatch 0.5h, 3000rpm in 37 DEG C after mixing and be centrifuged 10min, take
Supernatant 1mL, adds 3mL Dou Shi liquid, 540nm colorimetric.
Table 5 compound 1 impact on experimental lupus erythematosus mice
Compare with model control group, * P ＜ 0.05, * * P ＜ 0.01.
Result is as shown in table 5, and compound 1 can effectively reduce dsDNA antibody horizontal in experimental lupus erythematosus mice serum, carries
Show that it can be used for lupus erythematosus zhiliao.Lupus erythematosus can behave as the abnormal rising of humoral immunization, this experiment display model animal haemolysis
Element level is significantly higher than intact animal, and compound 1 can effectively reduce this abnormal hemolysin level raised, and points out these compounds
Abnormal immune hyperfunctioning is significantly inhibited effect.
Embodiment 8 compound 1 impact (ear swelling method caused by 2,4-dinitro fluorobenzene) on hypoimmunity mice specific immune function
KM mice 60, is divided into 6 groups by body weight stratified random, respectively suspensoid matched group (0.5% tragacanth),
Model control group (0.5% tragacanth), positive controls (krestin), compound 1 one-tenth are grouped.Gavage gives investigational agent
Thing or suspensoid, every day 1 time, continuous 14d.In addition to suspensoid matched group, be administered the 8th, 10,12d press 25mg/kg abdominal cavity
Injection gives cyclophosphamide normal saline solution, causes immunocompromised.It is administered 9d with 1%2,4-dinitro fluorobenzene (DNFB)
Solution (taking 100mgDNFB to join in the acetone-vegetable oil mixt of 11 and mix, be settled to 10mL) 25 μ L smear
Mouse web portion.1h after being administered to 13d, takes 10 μ L 1%DNFB solution and is applied in the left ear of mice；Smear rear 24h, i.e. end
1h after secondary administration, cervical dislocation execution animal, auricle is weighed, and calculates ear swelling degree.
Table 6 compound 1 is on the impact of ear swelling caused by hypoimmunity mice DNFB
| Group | Dosage | Ear swelling degree (mg) |
| Suspensoid compares | —— | 12.36±1.89** |
| Model comparison | —— | 4.74±0.58 |
| Positive control (krestin) | 400mg/kg | 6.91±1.25** |
| Compound 1 | 40mg/kg | 7.05±1.33** |
Compare with model control group, * P ＜ 0.05, * * P ＜ 0.01.
Result is as shown in table 6, and compound 1 can improve ear thickness caused by the DNFB of hypoimmunity mice, points out it to exempting from
The cellular immune function of the low animal of epidemic disease has potentiation, has the effect preferably strengthening the immunity of immunocompromised animal specificity.
The In Vitro Anti mycologic test of embodiment 9 compound 1
Weigh Compound 12mg, dissolves with 5% aqueous isopropanol and is formulated into 10mL, and 0.22 μm membrane filtration is degerming.Take 1mL
Degerming above-mentioned solution, joins after 9mL melts and is cooled in the PDA culture medium of about 50 DEG C, fully shake up, and pours into rapidly straight
Footpath is in the culture dish of 6cm, stands, makes the PDA culture plate containing 20 μ g/mL test-compounds.Use same volume
5% aqueous isopropanol make blank PDA culture plate.
The fungus truffle inoculum cut is moved into above-mentioned pastille PDA culture plate, 25 DEG C of constant temperature culture, treats matched group bacterium colony
During close to culture dish edge, measure the colony diameter on all culture plates by decussation method, after correction, calculate bacteriostasis rate.
Table 7 compound 1 inhibition to various funguses
| Candida albicans | Trichophyton | Alpha fungus | Trichophyton verrucosum | Hormodendrum pedrosei | |
| Bacteriostasis rate (%) | 90.2±12.3 | 82.1±11.8 | 51.2±7.9 | 63.4±8.9 | 75.2±11.7 |
Result is as shown in table 7, and compound 1 has obvious inhibitory action to tested all kinds of funguses.Owing to test fungal is
Common causative fungus, has obvious representativeness, and this result prompting compound 1 has stronger antifungal activity, can be used for anti-true
The preparation of mushroom medicine.
Claims (16)
1.2 and/or 27 substituted betulinic acid derivatives, its structure is as shown in formula I:
Wherein, R1、R2Independently be-H, hydroxyl,
R3、R4、R5Independently be-H, halogen, hydroxyl, C1～C6 alkyl, C1～C6 alkoxyl, acetoxyl group or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
2 and/or 27 substituted betulinic acid derivatives the most according to claim 1, it is characterised in that:
R1、R2Independently be-H, hydroxyl,
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
2 and/or 27 substituted betulinic acid derivatives the most according to claim 2, it is characterised in that:
R1、R2Independently be
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
2 and/or 27 substituted betulinic acid derivatives the most according to claim 3, it is characterised in that:
R1、R2Independently be
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen atom.
2 and/or 27 substituted betulinic acid derivatives the most according to claim 1, it is characterised in that: work as R1ForTime, its structure is as shown in formula II:
Wherein, R2For-H, hydroxyl,
R3、R4、R5Independently be-H, halogen, hydroxyl, C1～C6 alkyl, C1～C6 alkoxyl, acetoxyl group or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
2 and/or 27 substituted betulinic acid derivatives the most according to claim 5, it is characterised in that:
R2For-H, hydroxyl,
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom；
It is further preferred that R2For
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom；
Further preferred, R2For
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen atom.
2 and/or 27 substituted betulinic acid derivatives the most according to claim 1, it is characterised in that: work as R2ForTime, its structure is as shown in formula III:
Wherein, R1For-H, hydroxyl,
R3、R4、R5Independently be-H, halogen, hydroxyl, C1～C6 alkyl, C1～C6 alkoxyl, acetoxyl group or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom.
2 and/or 27 substituted betulinic acid derivatives the most according to claim 7, it is characterised in that:
R1For-H, hydroxyl,
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom；
It is further preferred that R1For
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen, nitrogen, sulfur or carbon atom；
Further preferred, R1For
R3、R4、R5Independently be-H ,-F ,-Cl ,-Br, hydroxyl, C1～C4 alkyl, C1～C4 alkoxyl, acetoxyl group
Or acetyl group；
X is oxygen atom.
9. 2 and/or 27 substituted betulinic acid derivative pharmaceutically acceptable salts described in any one of claim 1～8.
10. 2 and/or 27 pharmaceutically acceptable hydrations of substituted betulinic acid derivative described in any one of claim 1～8
Thing.
11. 1 kinds of pharmaceutical compositions, are to be derived by described in any one of claim 1～8 2 and/or 27 substituted belulinic acid Betulinic acid
Thing adds what the complementary composition of pharmaceutically acceptable was prepared from.
Described in 2 and/or 27 substituted betulinic acid derivatives, claim 10 described in 12. any one of claim 1～8
Salt or claim 11 described in hydrate preparation there is the purposes in anti-fibrosis medicine.
Described in 2 and/or 27 substituted betulinic acid derivatives, claim 10 described in 13. any one of claim 1～8
Salt or claim 11 described in hydrate preparation there is the purposes in antifungal activity medicine.
Described in 2 and/or 27 substituted betulinic acid derivatives, claim 10 described in 14. any one of claim 1～8
Salt or claim 11 described in hydrate preparation there is the purposes in anti-tumor activity medicine.
Described in 2 and/or 27 substituted betulinic acid derivatives, claim 10 described in 15. any one of claim 1～8
Salt or claim 11 described in hydrate purposes in preparation has the medicine of two-way immunoregulation effect.
Described in 2 and/or 27 substituted betulinic acid derivatives, claim 10 described in 16. any one of claim 1～8
Salt or claim 11 described in hydrate purposes in preparation has the medicine of anti-alimentary tract inflammation and ulcer.
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| CN106117304A (en) * | 2016-07-12 | 2016-11-16 | 四川省中医药科学院 | Betulic acid derivant |
| CN106188211A (en) * | 2016-07-12 | 2016-12-07 | 四川省中医药科学院 | Betulic acid derivant and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102690317A (en) * | 2012-06-13 | 2012-09-26 | 东北林业大学 | Derivant of 30-halogenated betulinic acid and preparation method and application thereof |
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| CN102690317A (en) * | 2012-06-13 | 2012-09-26 | 东北林业大学 | Derivant of 30-halogenated betulinic acid and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106117304A (en) * | 2016-07-12 | 2016-11-16 | 四川省中医药科学院 | Betulic acid derivant |
| CN106188211A (en) * | 2016-07-12 | 2016-12-07 | 四川省中医药科学院 | Betulic acid derivant and application thereof |
| CN106117304B (en) * | 2016-07-12 | 2018-09-18 | 四川省中医药科学院 | Betulic acid derivative |
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