CN106117304B - Betulic acid derivative - Google Patents
Betulic acid derivative Download PDFInfo
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- CN106117304B CN106117304B CN201610545783.0A CN201610545783A CN106117304B CN 106117304 B CN106117304 B CN 106117304B CN 201610545783 A CN201610545783 A CN 201610545783A CN 106117304 B CN106117304 B CN 106117304B
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Abstract
This application involves a kind of betulic acid derivative and its pharmaceutically acceptable salt, stereoisomer, isotopic label, solvate, polymorph or prodrugs, and the pharmaceutical composition comprising above-mentioned substance and the purposes for treating cancer.
Description
Technical field
This application involves a kind of betulic acid derivative and its pharmaceutically acceptable salt, stereoisomer, isotope marks
Object, solvate, polymorph or prodrug are remembered, including the pharmaceutical composition of above-mentioned substance and the use for treating anti-tumor disease
On the way, belong to field of medicaments.
Background technology
Betulinic acid (or betulic acid, betulinic acid) is lupinane type pentacyclic triterpenoid, is divided extensively
It is distributed in the various plants such as Rhamnaceae, Myrtaceae, Betulaceae, but universal content is relatively low.Due to betulinic acid female ring structure compared with
Greatly so that its molecular polarity is smaller, fat-soluble larger.Therefore, the solubility of betulinic acid in water is smaller, is soluble in ether, chlorine
The organic solvents such as imitative, ethyl alcohol.Betulinic acid to melanoma cells have extremely strong selecting cell toxicity (Pisha E, Chai H,
Lee I S,et al.Discovery of 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)；Use can be combined with many cytotoxic compounds such as Doxorubicin, taxol, support pool former times, actinomycin D
Medicine, and with synergistic effect, be expected to, as synergist, be used for the chemical combined therapy of tumour, avoid the generation of multidrug resistance, increase
Therapeutic effect (Eder-Czembirek C, Czembirek C, Erovic the B M, et of powerful antitumor medicine
al.Combination of betulinic acid with cisplatin-different cytotoxic effects
in two head and neck cancer cell lines[J].Oncol Rep,2005,14(3):667-671)；It can be
The almost all link of viral lifecycle inhibit HIV in H9 lymphocytes duplication (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).In addition, betulinic acid also has immunological regulation, anti-inflammatory, anti-oxidation stress, antibacterial, anti parasitic, anti-malarial and antiulcer
Etc. various actives (Yi Jine, Wu Jing, Wen Lixin wait the Advance on Pharmacological Activities Chinese herbal medicines of betulinic acids, 2014,45 (14):
2118-2124).As the understanding to betulinic acid pharmacological activity is constantly deepened, many researchers attempt using betulinic acid as parent nucleus into
Row structural modification increases its bioavilability in the hope of improving its solubility, reduces its toxicity.To the structural modification master of betulinic acid
Concentrate on three positions：C-3, C-20 and C-28.
In document《The progress of betulinic acid and its derivative》(Li Dan, Zhou Jinpei, Wu Xiao bright [J] pharmacy is in progress,
2004,28 (3):In 120-125)：
(1) it is usually that end band with various cyclic annular diacid anhydride reactants, is synthesized with pyridine solvent to the transformation of 3 hydroxyls
The ester of carboxylic acid group.The transformation of this type is still relatively more successful.As shown in table 1, compound 3,4,5 is living compared with betulinic acid
Property greatly enhances.Illustrate that the acyl group of the type may enhance HIV-resistant activity.To plain 3 hydroxyls of betulinic acid and the like white birch
Other transformations, such as β hydroxyls are changed to α, hydroxyl is changed to carbonyl, alcohol is changed to amide, mostly cause increased activity.Explanation
Playing hydrogen bond reaction crucial when activity may be related to 3 β oxygen.Wherein, compound 3 (DSB, also known as YK-FH312), especially
Its is noticeable.The result of study of Taisei Kanamoto etc. shows that YK-FH312 may be by acting on virion dress
Match and (or) virion budding step realizes HIV-resistant activity.
(2) very satisfactory result is not obtained to the transformation of 19 isopropenyls of betulinic acid.Upper substitution, activity at 30
It keeps, but acidic-group substitution is to active apparent harmful；20, carbon is changed to ketone or oxime, and inactivation or activity reduce；19 are changed to acyl
Base or acidic-group can cause to inactivate.Illustrate that the oxygen atom of high electronegativity may change the static behaviour of betulinic acid, makes its cell toxicant
Property reduce.The unique of 19 acquisitions of transformation is successfully dihydrobetulinic acid (Dihydrobetulinic acid) and its derivative.Two
Hydrogen betulinic acid is the fusion inhibitor of HIV and cell membrane.Its AntiHIV1 RT activity is measured using H9 cells as infection model, and IC50 is 12.6 μ
Mol/L, EC50=0.9 μm of ol/L.Using dihydrobetulinic acid as primer, the therapeutic index of the compound 7 obtained after modification is
14000, HIV-resistant activity is about 1000 times higher than its primer.
(3) 28 carboxyls are the modification hot spots of current betulinic acid, and the derivative species modified it are various.In recent years
Research in, with bioactivity be guidance to the structure of modification of 28 carboxyls, gradually concentrate on and carboxylic acid group is transformed into various acyls
Amine, and most of branch terminals all remain carboxylic acid group in these amide derivatives.It now will be more representational
Compound is introduced.As shown in table 2, the amide chain of the analog derivative is bad compared with short-lived activity.R=when amide chain is longer
CONH(CH2)mActivity is significant when COOH, m=7-11, and activity is most strong when m=10；R=CONH (CH2)7CONH(CH2)nActivity is all improved than front m=7,10 when COOH, n=1-4；R=CONH (CH2)7NHCO(CH2)nIt lives when COOH, n=1-3
Property is improved again.Additionally, it is possible to which because amine moiety and adjacent protons interaction play the role of carboxylic acid group on sterically defined, chain
In first CONH, N is upper substituted, and CONH becomes NHCO or NHCONH, all inactivates.Second CONH:1. with small alpha-amido
Sour (amino acid includes the lipophilic moieties of 1-2 methyl) is connected, and activity increases.2. the benzoic acid with ortho position is connected, inactivation；
It is connected with the benzoic acid of meta position, contraposition, IC50 is up to 100nmol/L or so.3. if CONH changes NHCO into, activity just enhances.It should
Foremost in analog derivative is RPR103611, it passes through the rear combination during inhibiting virus and cell membrane fusion, i.e. coating
Relying on the sexual stage inhibits HIV infection (IC50=10nmol/L), is currently the only to prevent HIV from invading by influencing gp41
The non-peptide matters of small molecule are hopeful to be developed into HIV cell membrane fusion agent.The optical isomer IC9564. of PRP103611
(34.) IC90=0.22 ± 0.05 μm ol/L that measurement K NL4-3 are reduced in viral infection shows that very strong AntiHIV1 RT activity is lived
Property.And in the series derivates of IC9564, that more prominent is compound 35 (EC50=0.33 μm of ol/L) and 36 (EC50=
0.46 μm of ol/L), their activity is suitable with IC9564.Studies have shown that IC9564. can strongly block HIV-1 shells to mediate
Film merges, in the duplication for penetrating step blocking HIV.Key substance HIV-1gp120 during this is IC9564 effects
Molecular target.
In document《The three-dimensional quantitative structure activity relationship and molecular docking pattern of 23- HBA derivative antitumor action》
(Zhang Tingting, Bi Yi, Chen Mengmeng etc. [J] Chinese Pharmaceutical Journals, 2014,49 (14):In 1200-1203)：23- HBA
Derivative (structure is as follows) is also pentacyclic triterpenoid, be a kind of betulinic acid isolated in the Chinese bulbul from Chinese medicine seemingly
Object, have with the comparable antitumor activity of Betulinic Acid, but among its mechanism of action is currently studied.Laboratory synthesis early period
A large amount of 23- HBA derivative simultaneously to it has carried out antitumor activity, studies have shown that 28-COOH is to influence
The main portions of such antitumor activity of compound.
Up to the present, certain progress, but C-20 knots have been obtained to the transformation of C-3 hydroxyls and C-28 carboxyls
Structure is modified and transformation still lacks satisfactory result.The betulinic acid derivative of other positions substitution then has not been reported.
Invention content
The present invention provides a kind of betulic acid derivatives, can be used for preparing anti-tumor drug.
As the one side of the application, this application provides the compound indicated by Formulas I, its is pharmaceutically acceptable
Salt, stereoisomer, isotopic label, solvate, polymorph or prodrug：
Wherein, R1、R2It is respectively selected from
R3、R4、R5It is respectively selected from-H, halogen, hydroxyl, C1~C6 alkyl, C1~C6 alkoxies, acetoxyl group or acetyl group
At least one of；
X is selected from oxygen, nitrogen, sulphur or carbon atom.
Preferably, R1ForR2For
It is further preferred that X is oxygen atom；
It is further preferred that R4For hydroxyl, R3And R5For-H.
Still more preferably, the compound of formula I structural formula is：
As further aspect of the application, this application provides a kind of pharmaceutical compositions, and it is above-mentioned that it includes the application
Compound of formula I, its pharmaceutically acceptable salt, stereoisomer, isotopic label, solvate, polymorph or prodrug,
And pharmaceutically acceptable carrier.Described pharmaceutical composition includes but not limited to peroral dosage form, parenteral dosage forms, external preparation
Type and forms for rectal administration.In some embodiments, described pharmaceutical composition can be oral tablet, capsule, pill,
Powder, sustained release preparation, solution and suspension are used for sterile solution, suspension or the lotion of parental injection, for external application
Ointment or emulsifiable paste, or the suppository for rectally.In some embodiments, described pharmaceutical composition and at least one are controlled
Agent is treated respectively with independent dosage form combination at combination product, such as kit.
As further aspect of the application, the application provides the compound of formula I, its pharmaceutically acceptable salt, stands
Body isomers, isotopic label, solvate, polymorph or prodrug answering in preparing the drug with antitumor action
With.
As further aspect of the application, the application provides a kind of method for treating tumor disease, and this method includes will
The compound of formula I, its pharmaceutically acceptable salt, stereoisomer, isotopic label, the solvation of therapeutically effective amount
Object, polymorph or prodrug are applied to the individual of thus demand.In some embodiments, the tumor disease includes white blood
Disease, malignant lymphoma, Huppert's disease, gastrointestinal stromal tumor, colon and rectum carcinoma, breast cancer, liver cancer, gastric cancer, ovary
Cancer, uterine cancer, cervical carcinoma, carcinoma of vagina, lung cancer, kidney, prostate cancer, carcinoma of urinary bladder, cancer of pancreas, the cancer of the brain, melanoma etc..
Herein described " pharmaceutically acceptable salt " refers to the life of the free acid and free alkali that remain appointed compound
Object effect, and not salt of ill-effect in terms of biology or other.Salt in the application refers to organic acid/inorganic acid
The acid salt of formation, and the basic salt with organic base/inorganic base formation.
Herein described " solvate " refers to the application compound formed by solvation and solvent molecule
Combination.Such as hydrate, alcohol solvent compound, Methanol Solvate.
Herein described " polymorph " or " polymorph " refer to the application compound existing for different form crystal lattices.
Herein described " isotopic label " refers to the application compound by isotope labelling.Such as the change of the application
The various isotopes that the isotope in object includes H, C, O are closed, such as2H,3H,13C,14C,18O,17O。
Herein described " pharmaceutically acceptable prodrug " refer to the application compound any pharmaceutically acceptable salt,
The salt or other derivatives of ester, ester, the compound of offer the application that can be direct or indirect after being applied to receptor or its
Metabolin with pharmaceutical active or residue.
Herein described " stereoisomer " refers to different caused by atom in molecule spatially arrangement mode difference
Structure body.
Herein described " therapeutically effective amount " refers to taking metapedes to alleviate treated disease or disease to a certain extent
At least one medicament of one or more symptoms of disease or the amount of compound.
Herein described " pharmaceutical composition " refers to optional being mixed at least one pharmaceutically acceptable chemical composition
Bioactive compound, the pharmaceutically acceptable chemical composition include but not limited to carrier, stabilizer, diluent, point
Powder, suspending agent, thickener and/or excipient." carrier " refers to the chemical reagent of relative nontoxic, is contributed to chemical combination
Object is introduced into cell or tissue.
In vitro study of the present invention shows compound of formula I to various tumor cell strains, such as cervical cancer cell lines Hela, people liver
Cancer cell line HepG-2, human hepatoma cell strain BEL7404, human stomach cancer cell line AGS, SMMC-7721 human hepatocarcinoma cell, mouse
Colon cancer cell line C26 all has apparent inhibiting effect.In vivo study shows that compound of formula I can inhibit S180 in Mice Body
Interior growth, inhibiting rate 53.8% is close with chemotherapy Cytoxan, shows it with preferable antitumor activity.
Description of the drawings
The HMBC correlation figures of Fig. 1 compounds 1
The NOSEY correlation figures of Fig. 2 compounds 1
The HMBC correlation figures of Fig. 3 compounds 2
The NOSEY correlation figures of Fig. 4 compounds 2
Specific implementation mode
The preparation of 1 compound 1 of embodiment
1. the preparation of ethyl acetate extract
Take vest appropriate, 10 times of amount 95% ethanol percolations extractions, extracting solution obtains medicinal extract after being concentrated under reduced pressure.Medicinal extract adds moisture
It dissipates, uses petroleum ether, ethyl acetate to extract successively, obtain ethyl acetate extract.
2. chromatographic condition
1260 high performance liquid chromatographs of Agilent, chromatographic column：BDS-C 18 (100*4.6mm, 2.4um), flow velocity：
1.0ml/min, column temperature：30 DEG C, Detection wavelength：320nm (DAD), mobile phase：Acetonitrile-methanol (according to the form below gradient elution).
| Time(min) | Acetonitrile (%) | 0.1% formic acid liquid (%) |
| 0 | 2 | 98 |
| 5 | 2 | 98 |
| 55 | 100 | 0 |
3. silica gel column chromatography
(1) dissolution of raw material：(55 DEG C) dissolvings in methanol are added in raw material 200g ethyl acetate extracts, filter, take filtrate.
(2) silica gel mixed sample：180-200 mesh silica gel about 300g is weighed, is slowly added to mix in silica gel by filtrate on water-bath
Sample.
(3) column parameter is filled：Glass column diameter 10cm, filler 180-200 mesh silica gel 1.5kg, chloroform fill column；Eluant, eluent：Three
Chloromethanes → trichlorine chloromethanes：Methanol=15:1 gradient elution；Solvent：Chloroform：Methanol=8:1；TLC：Silica G plate,
The heating colour developing of 10% sulfuric acid ethyl alcohol.
(4) elution process and result：
Pure chloroform fills column, and pure chloroform is eluted to when colour band will be eluted out and changes chloroform into -- methanol
80：1 elution, TLC monitorings merge same section, successively transformation system liquid proportional → 60:1→50:1→40:1→30:1 to white birch
After resin acid elution completely, using high performance liquid chromatograph research and application fraction (chromatographic condition is with " 2. chromatographic condition "), and convert
Ratio 30：1→20：1→15：1, merge the eluate for including 35-40min groups peak.
4.MCI is purified
(1) dissolution of raw material：The minimum amount of alcohol of above-mentioned eluate is dissolved, wet method loading.
(2) column parameter is filled：Glass column diameter 3cm, dress pillar height degree 45cm.
(3) elution process：50% ethanol elution is thin out to wash water color, changes 75% ethanol elution, fraction is per 500ml mono-
Part is collected, and efficient liquid phase monitoring fraction (chromatographic condition is with " 2. chromatographic condition ") simultaneously changes 95% ethanol elution, efficient liquid phase monitoring
(chromatographic condition is with " 2. chromatographic condition "), merges the fraction for including 35-40min groups peak.
(4) dry：Merge 35-40min fraction liquid and be spin-dried for no ethanol flavor, is extracted with ethyl acetate three times, is spin-dried for second
Drying for 24 hours, obtains target phase mixture in vacuum drying chamber after acetoacetic ester.
It is prepared by 5.HPLC
Above-mentioned target phase mixture is dissolved with methanol, and filtering is prepared using preparative high-performance liquid chromatographic, acquisition compound 1,
2 crude products.Chromatographic condition：Detection device：waters；Column model： XB-C18 250×4.6mm 5um；Column
Temperature：35℃；Flow velocity：1ml/min；UV320nm；Mobile phase:- 0.2% phosphoric acid water of acetonitrile (gradient elution), gradient elution program：
Time/min：0→30→35→45→46→58；Acetonitrile/%：53→63→90→90→53→53.
Purifying：The compound 1 being prepared, 40 DEG C of 2 crude product are concentrated into no acetonitrile, are extracted with ethyl acetate, acetic acid second
40 DEG C of ester extract is concentrated to dryness to get compound 1,2 sterlings.
6. the discriminating of compound 1
Compound 1 is white powder, is easy to become solution in methanol, ethyl alcohol, acetonitrile etc., not soluble in water；m.p.：201-203℃；IRνmax(KBr)：3389,2947,1702,1604,1584,1513,1450,
1167,832, show that the compound has hydroxyl, carbonyl and benzene ring building blocks；ESI-MS:m/z 779.48[M-H]-,
781.49[M+H]+, 803.44 [M+Na]+, 819.50 [M+K]+, show that the molecular weight of the compound is 780；HRESI-MS：
781.4532[M+H]+(calculated value 781.4522) determines that its molecular formula is C48H60O9。
Hydrocarbon ownership (the DMSO-d of compound 16, δ ppm, J=Hz)
Hydrocarbon ownership (the DMSO-d of compound 16, δ ppm, J=Hz) and (Continued)
1H-NMR (600MHz, DMSO) shows that the compound has 5 methyl signals δH0.74,0.91,0.92,0.93,
1.67 (each 3H, s), wherein δH1.67 be the methyl hydrogen signal being connected to above double bond.In low field part δH7.72 (2H, d,
J=8.4Hz), 7.55 (2H, d, J=8.4Hz), 6.79 (2H, d, J=8.4Hz), 6.76 (2H, d, J=8.4Hz) illustrate point
There are two benzene ring structures in son, and phenyl ring is contraposition substitution, while in δH7.53 (1H, d, J=15.6Hz), 6.82 (1H, d, J
=12.6Hz), 6.39 (1H, d, J=15.6Hz), 5.74 (1H, d, J=12.6Hz) they are above two Cis-trans structures double bonds
Hydrogen signal.δH12.19,10.02,9.87 (1H, s) are the active hydrogen signal in molecule.
In conjunction with13C-NMR (150MHz, DMSO) and 135 ° of DEPT, which shares 48 carbon atom signals, in low field
Part δC177.7,167.1,166.1 be ester carbonyl group carbon signal, δC160.3,159.2,150.5,126.0,125.4, it is quaternary carbon
Signal, δC145.0,143.3,133.2,130.8,116.6,116.2,115.3,114.9, it is tertiary carbon signal peak, δC 110.2
For secondary carbon signal.In conjunction with hydrogen modal data, illustrate the compound there are two to hydroxy cinnamate acyl group, it is normal according to the coupling between them
Number, it can be determined that one of them is transconfiguration, the other is cis-structure.In addition to this, there are one double bonds in molecule.
In 135 ° of DEPT, δC78.5,72.5 be to connect oxygen groups tertiary carbon signal, δC62.5 be to connect oxygen groups secondary carbon signal.In addition to two
To hydroxy cinnamate acyl group, which also remains 30 carbon signals, in conjunction with1H、13C-NMR and DEPT135 ° of comprehensive characteristics are inferred
The compound is C30 type triterpene structures.
The hydrocarbon signal of compound is belonged to by HSQC, two to hydroxy cinnamate acyl group by HMBC correlations come really
It is fixed.δH 4.88(δC72.5, C-2) and δC166.1 (C- γ), δH 4.51(δC62.5, C-27) and δC 167.1(C-γ′)
With correlation；δ simultaneouslyH5.74,6.82 and δC166.1 (C- γ), δH7.53,6.39 and δC167.1 (C- γ ') have phase
It closes, illustrates that cis- to hydroxy cinnamate acyl group is connected with 2 carbon phases of parent nucleus, it is trans- to be and 27 carbon phases of parent nucleus to hydroxy cinnamate acyl group
Even.δH1.67 (H-18), 1.82,1.38 (H-22) and δC177.7 (C-28), 55.5 (C-17) HMBC are related；δH 4.71、
4.58 (H-29), 1.68 (H-30) and δC150.5 (C-20), 46.9 (C-19) are related；δH 0.91(H-23)、0.74(H-24)
With δC78.5 (C-3), 39.5 (C-4) are related.Further illustrate that the compound is that 2-O- is cis- to hydroxy cinnamate acyl group-above
27-O- is trans- to hydroxy cinnamate acyl group betulic acid.
The relative configuration of 2,3 bit substituents is determined by NOESY, the δ in NOESY spectrogramsH4.88 (H-2) and δH 3.05
(H-3) related, while δH4.88 (H-2) and δH0.93 (H-25) is related, δH3.05 (H-3) and δH0.74 (H-24) phase
It closes, δH0.74 (H-24) and δH0.93 (H-25) is related.Illustrate that 2,3 bit substituents are α configurations, equally from NOESY collection of illustrative plates
It can be seen that 19 bit substituents are α configurations.
HMBC correlations figure figure related to NOESY is shown in Fig. 1,2 respectively.
In summary structural analysis, the compound identification are that 2 α-O- are cis- to -3 alpha-hydroxy-2 7-O- of hydroxy cinnamate acyl group
It is trans- to hydroxy cinnamate acyl group betulic acid.It is retrieved through scifinder, there are no the relevant report of the compound, determine that it is
New triterpenoid structure.
7. the discriminating of compound 2
Compound 2 is white indefiniteness powder, m.p.202-203 DEG C,(c 0.02, MeOH), ESI-
MS：Anion m/z 779.02 [M-H]-, cation m/z 803.43 [M+Na]+, it is 780 to prompt the molecular weight of the compound；
HRESI-MS provides m/z 781.4530 [M+H]-(calculated value 781.4522), determines that the molecular formula of the compound is
C48H60O9。
Hydrocarbon ownership (the DMSO-d of compound 26, δ ppm, J=Hz)
Hydrocarbon ownership (the DMSO-d of compound 26, δ ppm, J=Hz) and (Continued)
Hydrocarbon ownership (the DMSO-d of compound 26, δ ppm, J=Hz) and (Continued)
13C-NMR modal datas show that the compound has 48 carbon, by1Fragrant hydrogen and alkene the Hydrogen Proton letter that H-NMR modal datas are shown
Number δ 7.51 (2H, d, J=8.4Hz, H-2 ', 6 '), 6.76 (2H, d, J=8.4Hz, H-3 ', 5 '), 7.54 (1H, d, J=
15.6Hz, H- α), 6.32 (1H, d, J=15.6Hz, H- β), 7.65 (2H, d, J=8.4Hz, H-2 ", 6 "), 6.80 (2H, d, J
) ,=8.4Hz, H-3 ", 5 " 6.87 (1H, d, J=12.6Hz, H- α '), 5.78 (1H, d, J=12.6Hz, H- β ') and13C-
18 sp in H NMR spectroscopy2Hydridization carbon absorption signal can speculate that there are 2 propenyl benzene acidic groups, further according to chemical displacement value δ 160.3
With 159.2, thus it is speculated that the contraposition of phenyl ring may respectively have 1 OH substitution, it is known that containing 2 to hydroxy cinnamate acyloxy in compound 2
Structural unit；Again by the coupling constant J=15.6Hz and H- α ' of H- α and H- β and the coupling constant J=12.6Hz of H- β ', explanation
There is the double bond hydrogen of 1 transconfiguration and 1 cis-structure in molecule.
13Remaining 30 carbon signals include 6 × CH in C-NMR spectrums3、11×CH2, 6 × CH, 7 × C, and there is δ 177.8
(C) and δ 150.5 (C), 110.1 (CH2) and 19.5 (CH3) etc. lupane-type triterpenoids characteristic peak, it may be determined that compound 2 is
Betulic acid derivative.It can be seen that by the Structure Comparison with betulic acid, C-2 and C-27 chemical shifts become δ 72.6
(d) with δ 62.5 (t), show C-2 and C-27 aerobic substitutions.In HMBC spectrums (Fig. 1), δH4.90 (H-2) and δC
166.8 (C- γ), δH4.54 (H-27) and δC166.6 (C- γ ') are related；δ simultaneouslyH7.54 (H- α) and δC 166.8(C-
γ), δH6.87 (H- α ') and δC166.6 (C- γ ') are related, illustrate that trans- substituent group is connected to C-2, and cis- are connected to
C-27.Then speculate that compound 2 is that 2-O- is trans- cis- to hydroxy cinnamate acyl group -27-O- to hydroxy cinnamate acyl group betulic acid.
The relative configuration of 2,3 bit substituents is determined by NOESY, the δ in NOESY spectrogramsH4.90 (H-2) and δH 3.11
(H-3) related, while δH4.90 (H-2) and δH0.93 (H-25) is related, δH3.11 (H-3) and δH0.74 (H-24) is related,
δH0.74 (H-24) and δH0.93 (H-25) is related.Illustrate that 2,3 bit substituents are α configurations, it equally can from NOESY collection of illustrative plates
To find out 19 bit substituents for α configurations.
HMBC correlations figure figure related to NOESY is shown in Fig. 3,4 respectively.
In summary it analyzes, it is trans- cis- right to -3 alpha-hydroxy-2 7-O- of hydroxy cinnamate acyl group that compound 2 is accredited as 2 α-O-
Hydroxy cinnamate acyl group betulic acid.It is retrieved through scifinder, there are no the relevant report of the compound, determine that compound 2 is new
Triterpenoid structure.
2 compound 1 of embodiment, 2 anti-tumor activity tests
The source of cell strain, reagent that the present embodiment uses for：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
It is provided by the Chengdu bio tech ltd Bao Ke, human stomach cancer cell line AGS is purchased from Shanghai cell research institute of the Chinese Academy of Sciences.
Fetal calf serum and its corresponding culture medium are purchased from Hyclone companies of the U.S.；MTT, DMSO, tragacanth, yellow Jackets, TNBS,
Phytane (pristane) is purchased from sigma companies of the U.S.；Cyclophosphamide (CTX) is purchased from Hengrui Medicine Co., Ltd., Jiangsu Prov.；Ground
Sai meter Song is purchased from Hainan Pharmaceutical Factory Co., Ltd.；Carbon tetrachloride is purchased from Chengdu chemical reagent work；Alanine aminotransferase (ALT) is purchased from Nanjing
It builds up；ELISA, III procollagen type (PC- III), hyaluronic acid (HA) are magnificent purchased from Wuhan；Laminin lens (LH) are purchased from Shanghai
Western Tang；Tripterygium glycosides are purchased from Huangshi Feiyun Pharmaceutical Co., Ltd.；Coriolan is purchased from the upper limited public affairs of Haikang boat fungi polysaccharide
Department；4- dinitros fluorobenzene (DNFB) is purchased from sigma companies of the U.S..
1, antitumor activity in vitro
Taking the various tumour cells in exponential phase, (tumor cell line of use has following several：Cervical cancer cell
Strain Hela；Human hepatoma cell strain HepG-2；Human hepatoma cell strain BEL7404；Human stomach cancer cell line AGS；Human hepatoma cell strain
SMMC7721；Mouse colonic cell strain C26), cell suspension is prepared, it will be thin with the corresponding culture medium containing 10% fetal calf serum
Born of the same parents' concentration is adjusted to 1 × 105After a/mL cell suspensions, by cell inoculation in 96 well culture plates, cell suspension 200 μ L are added per hole, it is secondary
Day is separately added into the sterile compound 1 of a certain concentration, 2 solution, 37 DEG C of mixing postposition, 5%CO2After being cultivated for 24 hours in incubator, it is precipitated
Culture solution is simultaneously washed twice with PBS, then the 20 μ L of MTT phosphate buffers and 150 μ L culture mediums of 5mg/mL are added to every hole, together
Continue to terminate culture after cultivating 4h under the conditions of sample.2000rpm centrifuges 5min, then discards the culture solution in cultivation plate hole, per hole
It is added 150 μ L DMSO, shakes 10min, make to be formed after formazans particle fully dissolve, microplate reader detection light absorption value.Selection measures
Wavelength is 490nm.The IC50 for calculating 1,2 pair of tumour cell of compound, the results are shown in Table 1.
The inhibiting effect of 11,2 pair of kinds of tumor cells of compound of table
The above results show that compound 1,2 has external tumor-inhibiting action, and significant effect is phonetic better than chemotherapeutic 5- fluorine urine
Pyridine.
2, to the influence of mice bearing S180
The S180 knurl source mouse that inoculation 8d is in a good state of health are chosen, ascites are extracted after skin of abdomen disinfection, with sterile life
It manages brine and presses 1 ︰, 4 (ascites volumes：Physiological saline volume) be suspended it is spare.Male mice in kunming 60,18~20g, by weight
Stratified random is divided into 5 groups, respectively model control group (0.5% tragacanth), positive controls (cyclophosphamide, CTX), change
1 group of object is closed, armpit inoculates 0.2mL aforementioned suspensions on the right side of it.Model control group and medicine group distinguish gavage after 2h
Tested material or suspension are given, once a day, continuous 14 days；CTX is given in positive controls intraperitoneal injection, the next day it is primary, totally 7
It is secondary.After the last administration for 24 hours cervical dislocation put to death mouse, stripping tumor mass weigh, and calculate tumour inhibiting rate ((1- experimental groups average knurl weight/
Model control group average knurl weight) * 100%), the results are shown in Table 2.
2 compound 1 of table to mice bearing S180 influence ()
| Group | Number of animals (only) | Dosage | Knurl 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 | 0.47±0.25** | 64.4 |
Compared with model control group, * P ＜ 0.05, * * P ＜ 0.01.
The experimental results showed that compound 1 gives 400mg/kg gavages, growths of the S180 in Mice Body is can inhibit, is had
Preferable antitumor activity.
3, influence of the compound 1 to lotus human liver cancer SMMC-7721 nude mices
BALB/c nude mices 24 are only inoculated with human hepatoma cell strain SMMC-7721 cell suspensions by 0.2ml/ in left side oxter,
About 3.3 × 106/ml of cell density.Every 3 days major diameters (a) with vernier caliper measurement tumour and minor axis (b) calculate tumour body
Product (V=0.5ab2), it weighs in simultaneously, waits for that gross tumor volume is more than 100mm3Proceed by administration.By gross tumor volume stratified random
It is divided into 3 groups, respectively 1 group of model control group, positive controls (cyclophosphamide, CTX, 40mg/kg), compound (40mg/
kg).The grouping same day starts to be administered, and 1 group of compound and model control group give corresponding tested material or suspension by 10ml/kg gavages
Agent, one time a day, continuous 14d；CTX normal saline solutions are given in positive controls hypodermic injection, the next day 1 time, totally 7 times.15d
Cervical dislocation puts to death animal, removes tumour, weighs, and measures volume, calculates tumour inhibiting rate ((1- administration groups average tumor weight/model
Control group average tumor weight) × 100%) and inhibition rate of tumor growth ((1- administration groups mean tumour volume/model control group
Mean tumour volume) × 100%), it the results are shown in Table 3.
3 compound 1 of table to lotus SMMC-7721 mouse influence (N=8,)
Compared with model control group, * P ＜ 0.05, * * P ＜ 0.01.
The experimental results showed that compound 1 is given by 40mg/kg gavages, humanized's tumour SMMC-7721 is can inhibit in nude mice
Internal growth shows preferable antitumor activity.
Claims (5)
1. compound 1 or its pharmaceutically acceptable salt, isotopic label or polymorph：
2. a kind of pharmaceutical composition, it includes compound described in claim 1 or its pharmaceutically acceptable salt, isotope marks
Remember object, polymorph and pharmaceutically acceptable carrier.
3. pharmaceutical composition as claimed in claim 2, which is characterized in that described pharmaceutical composition is tablet, capsule, ball
Agent, powder, sustained release preparation, solution, suspension, injection, ointment, emulsifiable paste or suppository.
4. prepared by compound as described in claim 1, its pharmaceutically acceptable salt, isotopic label or polymorph
Application in drug with antitumaous effect.
5. application as claimed in claim 4, which is characterized in that the cancer includes leukaemia, malignant lymphoma, multiple bone
Myeloma, gastrointestinal stromal tumor, colon and rectum carcinoma, breast cancer, liver cancer, gastric cancer, oophoroma, uterine cancer, cervical carcinoma, carcinoma of vagina,
Lung cancer, kidney, prostate cancer, carcinoma of urinary bladder, cancer of pancreas, the cancer of the brain or melanoma.
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| CN105859822A (en) * | 2015-01-19 | 2016-08-17 | 四川省中医药科学院 | 2-substituted and/or 27-substituted betulinic acid derivatives as well as preparation method and applications thereof |
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| CN105859822A (en) * | 2015-01-19 | 2016-08-17 | 四川省中医药科学院 | 2-substituted and/or 27-substituted betulinic acid derivatives as well as preparation method and applications thereof |
Non-Patent Citations (2)
| Title |
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| Flavonoid and cardenolide glycosides and a pentacyclic triterpene from the leaves ofNerium oleanderand evaluation of cytotoxicity;Bina Shaheen Siddiqui et al.;《Phytochemistry》;20120124;第77卷;第238-244页 * |
| 马甲子的化学成分研究;武蕊娟 等;《中草药》;20151031;第46卷(第19期);第2837页3.2和图1 * |
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