CN106117304A - Betulic acid derivant - Google Patents
Betulic acid derivant Download PDFInfo
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- CN106117304A CN106117304A CN201610545783.0A CN201610545783A CN106117304A CN 106117304 A CN106117304 A CN 106117304A CN 201610545783 A CN201610545783 A CN 201610545783A CN 106117304 A CN106117304 A CN 106117304A
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- 0 *Cc1*(*)cc(CC=CC(**)=O)cc1N Chemical compound *Cc1*(*)cc(CC=CC(**)=O)cc1N 0.000 description 1
- AJPIHXPHXPJAEU-UHFFFAOYSA-N C1[I]2[U]CCC12 Chemical compound C1[I]2[U]CCC12 AJPIHXPHXPJAEU-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
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Abstract
The application relates to a kind of betulic acid derivant and pharmaceutically acceptable salt, stereoisomer, isotopic label, solvate, polymorph or prodrug, and the pharmaceutical composition that comprises above-mentioned substance and for treating the purposes of cancer.
Description
Technical field
The application relates to a kind of betulic acid derivant and pharmaceutically acceptable salt, stereoisomer, isotope mark
Note thing, solvate, polymorph or prodrug, the pharmaceutical composition comprising above-mentioned substance and for treating the use of anti-tumor disease
On the way, field of medicaments is belonged to.
Background technology
Belulinic acid Betulinic acid (or betulic acid, betulinic acid) is lupinane type pentacyclic triterpenoid, extensively divides
It is distributed in the various plants such as Rhamnaceae, Myrtaceae, Betulaceae, but universal content is relatively low.Due to belulinic acid Betulinic acid female ring structure relatively
Greatly so that its molecular polarity is less, fat-soluble bigger.Therefore, belulinic acid Betulinic acid dissolubility in water is less, is soluble in ether, chlorine
The organic solvents such as imitative, ethanol.Belulinic acid Betulinic acid melanoma cells is had 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 compound such as doxorubicins, paclitaxel, support pool former times, actinomycin D
Medicine, and there is synergism, it is expected to as synergist, for the chemical combined therapy of tumor, it is to avoid the generation of multidrug resistance, increases
Therapeutic effect (Eder-Czembirek C, Czembirek C, Erovic B M, the 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)；Can be
Viral lifecycle almost all link suppression H9 lymphocyte in HIV 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).Additionally, belulinic acid Betulinic acid also has immunomodulating, antiinflammatory, anti-oxidation stress, antibacterial, parasiticide, malaria and antiulcer
Etc. various active (Yi Jine, Wu Jing, Wen Lixin, etc. the Advance on Pharmacological Activities of belulinic acid Betulinic acid. Chinese herbal medicine, 2014,45 (14):
2118-2124).Along with constantly deepening the understanding of belulinic acid Betulinic acid pharmacologically active, many researcheres are attempted entering with belulinic acid Betulinic acid for parent nucleus
Row structural modification, in the hope of improving its dissolubility, increases its bioavailability, reduces its toxicity.Structural modification master to belulinic acid Betulinic acid
Concentrate 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
The ester of carboxylic acid group.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.Explanation
Playing 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), especially
It is noticeable.The result of study of Taisei Kanamoto etc. shows, YK-FH312 may be by acting on virion dress
Join and (or) virion step of sprouting realizes HIV (human immunodeficiency virus)-resistant activity.
(2) transformation to 19 isopropenyls of belulinic acid Betulinic acid does not obtains the most gratifying result.30 replace, activity
Keep, 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.Two
Hydrogen 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 μ
Mol/L, EC50=0.9 μm ol/L.With dihydrobetulinic acid as primer, modified after the therapeutic index of compound 7 that obtains be
14000, 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
Research in, to the structure of modification of 28 carboxyls with biological activity for instructing, gradually concentrate on and carboxylic acid group be transformed into various acyl
Amine, and in these amide derivatives, most branch terminals all remains carboxylic acid group.Now will be more representational
Compound is introduced.As shown in table 2, the amide chain of this analog derivative is bad compared with short-lived activity.The R=when that amide chain being longer
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)nLive when COOH, n=1-3
Property is improved again.Additionally, it is possible to because amine moiety and adjacent protons interact, and carboxylic acid group plays sterically defined effect, chain
In first CONH, N upper substituted, CONH becomes NHCO or NHCONH, all inactivates.Second CONH: 1. with little alpha-amido
Acid (lipophilic moieties that this aminoacid comprises 1-2 methyl) is connected, and activity increases.2. it is connected with the benzoic acid at ortho position, inactivation；
Being connected with the benzoic acid of meta, para-position, IC50 is up to about 100nmol/L.If 3. CONH changes NHCO into, activity just strengthens.Should
Foremost in analog derivative is RPR103611, and it is by the rear combination during suppression virus and cell membrane fusion, i.e. peplos
The dependency stage suppresses HIV (IC50=10nmol/L), is currently the only to stop HIV to invade by affecting gp41
The non-peptide matters of little molecule, is hopeful to be developed into HIV cell membrane fusion agent.The optical isomer IC9564. of PRP103611
(34.) reduce IC90=0.22 ± 0.05 μm ol/L of mensuration K NL4-3 at viral infection, demonstrate that the strongest AntiHIV1 RT activity is lived
Property.And in the series derivates of IC9564, that more prominent is compound 35 (EC50=0.33 μm ol/L) and 36 (EC50=
0.46 μm ol/L), their activity is suitable with IC9564.Research display, IC9564. can strongly block the mediation of HIV-1 shell
Film merges, thus is penetrating the duplication of step blocking-up HIV.Key substance HIV-1gp120 during this is IC9564 effect
Molecular target.
In document " 3D-QSAR of 23-HBA derivant antitumor action and molecular docking pattern "
(Zhang Tingting, Bi Yi, Chen Mengmeng etc. [J]. Chinese Pharmaceutical Journal, 2014,49 (14): 1200-1203) in: 23-HBA
Derivant (structure is as follows) is also pentacyclic triterpenoid, is that a kind of from the Chinese medicine Radix Pulsatillae, the betulinic acid of isolated is seemingly
Thing, has an anti-tumor activity suitable with Betulinic Acid, but among its mechanism of action currently studies.This laboratory early stage synthesizes
Substantial amounts of 23-HBA derivant has also carried out antitumor activity to it, and research shows, 28-COOH is impact
The main portions of such antitumor activity of compound.
Up to the present, the transformation to C-3 position hydroxyl and C-28 position carboxyl is in progress achieved with certain, but the knot of C-20 position
Structure is modified 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 betulic acid derivant, can be used for preparing anti-tumor drug.
As an aspect of the application, this application provides represented by Formulas I compound, it 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 alkoxyl, acetoxyl group or acetyl group
In at least one；
X is selected from oxygen, nitrogen, sulfur or carbon atom.
Preferably, R1ForR2For
It is further preferred that X is oxygen atom；
It is further preferred that R4For hydroxyl, R3And R5For-H.
Further preferred, described compound of formula I structural formula is:
As further aspect of the application, this application provides a kind of pharmaceutical composition, it is above-mentioned that it comprises the application
Compound of formula I, its pharmaceutically acceptable salt, stereoisomer, isotopic label, solvate, polymorph or prodrug,
With 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, slow releasing preparation, solution and suspension, for sterile solution, suspension or the emulsion of parental injection, for external
Ointment or emulsifiable paste, or the suppository for rectally.In some embodiments, described pharmaceutical composition is controlled with at least one
Treat agent and become combination product with independent dosage form combination respectively, such as kit.
As further aspect of the application, the application provides described compound of formula I, its pharmaceutically acceptable salt, stands
Body isomer, isotopic label, solvate, polymorph or prodrug answering in preparation has the medicine of antitumor action
With.
As further aspect of the application, the application provides a kind of method treating tumor disease, the method include by
The described compound of formula I of therapeutically effective amount, its pharmaceutically acceptable salt, stereoisomer, isotopic label, solvation
Thing, polymorph or prodrug are applied to the individuality of thus demand.In some embodiments, described tumor disease includes white blood
Disease, malignant lymphoma, multiple myeloma, gastrointestinal stromal tumor, colon and rectum carcinoma, breast carcinoma, hepatocarcinoma, gastric cancer, ovary
Cancer, uterus carcinoma, cervical cancer, cancer of vagina, pulmonary carcinoma, renal carcinoma, carcinoma of prostate, bladder cancer, cancer of pancreas, the brain cancer, melanoma etc..
Herein described " pharmaceutically acceptable salt " refers to remain the life of the free acid of appointed compound and free alkali
Thing effect, and the salt of ill effect is not had in biology or other aspects.Salt in the application refers to organic acid/mineral acid
The acid salt formed, and the basic salt formed with organic base/inorganic base.
Herein described " solvate " refers to the application compound and the solvent molecule formed by solvation
Combination.Such as hydrate, alcohol solvent compound, Methanol Solvate etc..
Herein described " polymorph " or " polymorph " refer to the application compound existed with different form crystal lattices.
Herein described " isotopic label " refers to by isotope-labeled the application compound.The change of such as the application
Isotope in compound includes the various isotopes of H, C, O, as2H,3H,13C,14C,18O,17O。
Herein described " pharmaceutically acceptable prodrug " refer to the application compound any pharmaceutically acceptable salt,
Ester, the salt of ester or other derivants, the compound of offer the application that it can be direct or indirect after using to receptor or its
There is metabolite or the residue of pharmaceutical active.
Herein described " stereoisomer " refers to by different produced by molecule Atom spatially arrangement mode difference
Structure body.
Herein described " therapeutically effective amount " refers to take 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 be mixed with at least one pharmaceutically acceptable chemical composition
Bioactive compound, described pharmaceutically acceptable chemical composition include but not limited to carrier, stabilizer, diluent, point
Powder, suspending agent, thickening agent and/or excipient.Described " carrier " refers to the chemical reagent of relative nontoxic, and it contributes to chemical combination
Thing is incorporated in cell or tissue.
In vitro study of the present invention shows, compound of formula I is to various tumor cell strains, such as cervical cancer cell lines Hela, people liver
JEG-3 HepG-2, human hepatoma cell strain BEL7404, human stomach cancer cell line AGS, SMMC-7721 human hepatocarcinoma cell, mice
Colon cancer cell line C26 is respectively provided with obvious inhibitory action.In vivo study shows, compound of formula I can suppress S180 at Mice Body
Interior growth, suppression ratio is 53.8%, close with chemotherapeutic cyclophosphamide, shows that it has preferable anti-tumor activity.
Accompanying drawing explanation
The relevant figure of the HMBC of Fig. 1 compound 1
The relevant figure of the NOSEY of Fig. 2 compound 1
The relevant figure of the HMBC of Fig. 3 compound 2
The relevant figure of the NOSEY of Fig. 4 compound 2
Detailed description of the invention
The preparation of embodiment 1 compound 1
1. the preparation of ethyl acetate extract
Take waistcoat appropriate, 10 times amount 95% ethanol percolate extraction, obtain extractum after extracting solution concentrating under reduced pressure.Extractum adds moisture
Dissipate, successively with petroleum ether, ethyl acetate extraction, obtain ethyl acetate extract.
2. chromatographic condition
Agilent 1260 high performance liquid chromatograph, chromatographic column: BDS-C 18 (100*4.6mm, 2.4um), flow velocity:
1.0ml/min, column temperature: 30 DEG C, detection wavelength: 320nm (DAD), flow 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) material dissolution: raw material 200g ethyl acetate extract adds (55 DEG C) in methanol and dissolves, sucking filtration takes filtrate.
(2) silica gel mixed sample: weigh 180-200 mesh silica gel about 300g, is slowly added to filtrate in silica gel mix on water-bath
Sample.
(3) dress column parameter: glass column diameter 10cm, filler 180-200 mesh silica gel 1.5kg, chloroform dress post；Eluant: three
Chloromethanes → trichlorine chloromethanes: methanol=15:1 gradient elution；Developing solvent: chloroform: methanol=8:1；TLC: silica gel G plate,
10% sulphuric acid ethanol heating colour developing.
(4) elution process and result:
Pure chloroform dress post, pure chloroform is eluted to change chloroform into when colour band will be eluted out--methanol
80:1 eluting, TLC monitoring merges same section, and transformation system liquid proportional → 60:1 → 50:1 → 40:1 → 30:1 is to Betula platyphylla Suk. successively
After fat acid eluting is complete, uses high performance liquid chromatograph research and application stream part (chromatographic condition is with " 2. chromatographic condition "), and convert
Ratio 30:1 → 20:1 → 15:1, merges the eluate comprising 35-40min group peak.
4.MCI purification
(1) material dissolution: above-mentioned eluate is dissolved by minimum amount of alcohol, wet method loading.
(2) dress column parameter: glass column diameter 3cm, fills post height 45cm.
(3) elution process: 50% ethanol elution is thin out to wash water color, changes 75% ethanol elution, flows the every 500ml of part mono-
Part is collected, and efficient liquid phase monitoring stream part (chromatographic condition is with " 2. chromatographic condition ") also changes 95% ethanol elution, the monitoring of efficient liquid phase
(chromatographic condition is with " 2. chromatographic condition "), merges the stream part comprising 35-40min group peak.
(4) it is dried: merging 35-40min flows part liquid and is spin-dried for without ethanol taste, is extracted with ethyl acetate three times, is spin-dried for second
In vacuum drying oven, it is dried 24h after acetoacetic ester, obtains target phase mixture.
Prepared by 5.HPLC
Above-mentioned target phase mixture dissolves with methanol, filters, and uses preparative high-performance liquid chromatographic to prepare, it is thus achieved that compound 1,
2 crude products.Chromatographic condition: detection equipment: waters；Pillar model: XB-C18 250×4.6mm 5um；Column temperature:
35℃；Flow velocity: 1ml/min；UV320nm；Flowing phase: acetonitrile-0.2% phosphoric acid water (gradient elution), gradient elution program: time
Between/min:0 → 30 → 35 → 45 → 46 → 58；Acetonitrile/%:53 → 63 → 90 → 90 → 53 → 53.
Purification: compound 1,2 crude product 40 DEG C prepared is concentrated into without acetonitrile, is extracted with ethyl acetate, acetic acid second
Ester extract 40 DEG C is concentrated to dryness, and obtains compound 1,2 sterling.
6. the discriminating of compound 1
Compound 1 is white powder, is easy to become solution in methanol, ethanol, acetonitrile etc., water insoluble；M.p.:201-203 DEG C；IRνmax(KBr): 3389,2947,1702,1604,1584,1513,1450,
1167,832, show that this 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 this compound is 780；HRESI-MS:
781.4532[M+H]+(value of calculation 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 this compound has 5 methyl signals δH0.74,0.91,0.92,0.93,
1.67 (each 3H, s), wherein δH1.67 for being connected to the methyl hydrogen signal 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
Son has two benzene ring structures, and phenyl ring is para-orientation, simultaneously at 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), be above two Cis-trans structures double bonds
Hydrogen signal.δH12.19,10.02,9.87 (1H s) is the active hydrogen signal in molecule.
In conjunction with13C-NMR (150MHz, DMSO) and DEPT 135 °, this compound has 48 carbon atom signals, in low field
Part δC177.7,167.1,166.1 is ester carbonyl group carbon signal, δC160.3,159.2,150.5,126.0,125.4, for quaternary carbon
Signal, δC145.0,143.3,133.2,130.8,116.6,116.2,115.3,114.9, for tertiary carbon signal peak, δC 110.2
For secondary carbon signal.In conjunction with hydrogen modal data, illustrate this compound have two to hydroxy cinnamate acyl group, normal according to the coupling between them
Number, it can be determined that one of them is transconfiguration, and another is cis-structure.In addition, molecule also has a double bond.?
In DEPT 135 °, δC78.5,72.5 is to connect oxygen groups tertiary carbon signal, δC62.5 is to connect oxygen groups secondary carbon signal.Except two
To hydroxy cinnamate acyl group, this compound also remains 30 carbon signals, in conjunction with1H、13C-NMR and DEPT135 ° of comprehensive characteristics, infers
This compound is C30 type triterpene structure.
Being belonged to the hydrocarbon signal of compound by HSQC, two relevant next true by HMBC to hydroxy cinnamate acyl group
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′)
Have relevant；δ simultaneouslyH5.74,6.82 and δC166.1 (C-γ), δH7.53,6.39 and δC167.1 (C-γ ') have phase
Close, illustrate that cis is to be connected with 2 carbon of parent nucleus to hydroxy cinnamate acyl group, trans to hydroxy cinnamate acyl group be and 27 carbon phases of parent nucleus
Even.δH1.67 (H-18), 1.82,1.38 (H-22) and δC177.7 (C-28), 55.5 (C-17) HMBC are relevant；δH 4.71、
4.58 (H-29), 1.68 (H-30) and δC150.5 (C-20), 46.9 (C-19) are correlated with；δH 0.91(H-23)、0.74(H-24)
With δC78.5 (C-3), 39.5 (C-4) are relevant.Illustrate further above this compound be 2-O-cis to hydroxy cinnamate acyl group-
27-O-is trans to hydroxy cinnamate acyl group betulic acid.
The relative configuration of prosposition substituent group is to be determined by NOESY, δ in NOESY spectrogramH4.88 (H-2) and δH 3.05
(H-3) relevant, δ simultaneouslyH4.88 (H-2) and δH0.93 (H-25) is correlated with, δH3.05 (H-3) and δH0.74 (H-24) phase
Close, δH0.74 (H-24) and δH0.93 (H-25) is correlated with.Illustrate that prosposition substituent group is α configuration, equally from NOESY collection of illustrative plates
Can be seen that 19 bit substituents are α configuration.
HMBC is relevant scheme figure relevant with NOESY see respectively Fig. 1,2.
Comprehensive above structural analysis, this compound identification is that 2 α-O-are cis to hydroxy cinnamate acyl group-3 alpha-hydroxy-2 7-O-
Trans to hydroxy cinnamate acyl group betulic acid.Retrieve through scifinder, there are no the relevant report of this 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]+, the molecular weight pointing out this compound is 780；
HRESI-MS provides m/z 781.4530 [M+H]-(calculated value is 781.4522), determines that the molecular formula of this 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 data shows that this compound has 48 carbon, by1Fragrant hydrogen and alkene Hydrogen Proton that H-NMR modal data shows are believed
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 2 propenyl benzene acidic groups of existence, further according to chemical displacement value δ 160.3
With 159.2, thus it is speculated that the para-position of phenyl ring may be respectively arranged with 1 OH and replace, it is known that containing 2 to hydroxy cinnamate acyloxy in compound 2
Construction unit；Again by coupling constant J=15.6Hz of H-α and H-β and H-α ' and coupling constant J=12.6Hz of H-β ', explanation
Molecule has the double bond hydrogen of 1 transconfiguration and 1 cis-structure.
13In C-NMR spectrum, remaining 30 carbon signals include 6 × CH3、11×CH2, 6 × CH, 7 × C, and δ 177.8 occurs
And δ 150.5 (C), 110.1 (CH (C)2) and 19.5 (CH3) etc. the characteristic peak of lupane-type triterpenoids, it may be determined that compound 2 is
Betulic acid derivant.Be can be seen that by the Structure Comparison with betulic acid, the chemical shift of C-2 and C-27 position becomes δ 72.6
D () and δ 62.5 (t), show that C-2 position and C-27 position aerobic replace.In HMBC composes (Fig. 1), δH4.90 (H-2) and δC
166.8 (C-γ), δH4.54 (H-27) and δC166.6 (C-γ ') are correlated with；δ simultaneouslyH7.54 (H-α) and δC 166.8(C-
γ), δH6.87 (H-α ') and δC166.6 (C-γ ') are correlated with, and illustrate that trans substituent group is connected to C-2 position, and cis being connected to
C-27 position.Then speculate that compound 2 is that 2-O-is trans cis to hydroxy cinnamate acyl group betulic acid to hydroxy cinnamate acyl group-27-O-.
The relative configuration of prosposition substituent group is to be determined by NOESY, δ in NOESY spectrogramH4.90 (H-2) and δH 3.11
(H-3) relevant, δ simultaneouslyH4.90 (H-2) and δH0.93 (H-25) is correlated with, δH3.11 (H-3) and δH0.74 (H-24) is correlated with,
δH0.74 (H-24) and δH0.93 (H-25) is correlated with.Illustrate that prosposition substituent group is α configuration, equally can from NOESY collection of illustrative plates
To find out that 19 bit substituents are for α configuration.
HMBC is relevant scheme figure relevant with NOESY see respectively Fig. 3,4.
Comprehensive above analysis, it is trans cis right to hydroxy cinnamate acyl group-3 alpha-hydroxy-2 7-O-that compound 2 is accredited as 2 α-O-
Hydroxy cinnamate acyl group betulic acid.Retrieve through scifinder, there are no the relevant report of this compound, determine that compound 2 is new
Triterpenoid structure.
Embodiment 2 compound 1,2 anti-tumor activity test
The present embodiment 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 colonic cell strain C26
Being thered is provided by Bao Ke bio tech ltd, Chengdu, human stomach cancer cell line AGS is purchased from Shanghai cell research institute of the Chinese Academy of Sciences.
Hyclone and corresponding culture medium thereof are purchased from Hyclone company of the U.S.；MTT, DMSO, tragacanth, pentobarbital sodium, TNBS,
Phytane (pristane) is purchased from sigma company 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
Build up；ELISA, III procollagen type (PC-III), hyaluronic acid (HA) are magnificent purchased from Wuhan；Laminin lens (LH) is purchased from Shanghai
Western Tang；Tripterygium glycosides, purchased from Huangshi Feiyun Pharmaceutical Co., Ltd.；Krestin is purchased from the limited public affairs of upper Haikang boat fungus polysaccharide
Department；4-dinitro fluorobenzene (DNFB) is purchased from sigma company of the U.S..
1, antitumor activity in vitro
Take and be in the various tumor cells of exponential phase (tumor cell line of employing has the most 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), prepare cell suspension, will be thin by the corresponding culture medium containing 10% hyclone
Born of the same parents' 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, secondary
Day is separately added into the aseptic compound of finite concentration 1,2 solution, mixes rearmounted 37 DEG C, 5%CO2After incubator cultivates 24h, separate out
Culture fluid also washes twice with PBS, then adds MTT phosphate buffer 20 μ L and the 150 μ L culture medium of 5mg/mL 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, every hole
Add 150 μ L DMSO, shake 10min, after making the α-granule of formation fully dissolve, microplate reader detection light absorption value.Select to measure
Wavelength is 490nm.The IC50 of 1,2 pairs of tumor cells of computerized compound, the results are shown in Table 1.
The inhibitory action of 1,2 pairs of kinds of tumor cells of table 1 compound
The above results shows, compound 1,2 has external tumor-inhibiting action, and effect is significantly better than chemotherapeutic 5-fluorine and urinates phonetic
Pyridine.
2, the 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 aseptic life
Reason saline 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), change
Compound 1 group, all armpit subcutaneous vaccination 0.2mL aforementioned suspension on the right side of it.Model control group and the other gavage of drug component after 2h
Give tested material or suspensoid, once a day, continuous 14 days；Positive controls lumbar injection gives CTX, the next day once, totally 7
Secondary.Last be administered after 24h cervical dislocation put to death mice, peel off tumor mass weigh, and calculate tumour inhibiting rate ((1-experimental group average tumor weight/
Model control group average tumor weight) * 100%), the results are shown in Table 2.
Table 2 compound 1 on the impact of 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 | 0.47±0.25** | 64.4 |
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
Preferably anti-tumor activity.
3, the compound 1 impact on lotus people hepatocarcinoma SMMC-7721 nude mice
BALB/c nude mice 24, is only inoculated human hepatoma cell strain SMMC-7721 cell suspension by 0.2ml/ in oxter, left side,
About 3.3 × 106/ml of cell density.The every 3 days major diameters (a) by vernier caliper measurement tumor and minor axis (b), calculate tumor body
Long-pending (V=0.5ab2), weigh in simultaneously, treat that gross tumor volume is more than 100mm3Proceed by administration.By gross tumor volume stratified random
It is divided into 3 groups, respectively model control group, positive controls (cyclophosphamide, CTX, 40mg/kg), 1 group of (40mg/ of compound
kg).Being grouped and started the same day to be administered, compound 1 group and model control group give corresponding tested material or suspendible by 10ml/kg gavage
Agent, every day 1 time, continuous 14d；Positive controls subcutaneous injection gives CTX normal saline solution, the next day 1 time, totally 7 times.15d
De-cervical vertebra puts to death animal, peels off tumor, weighs, and measures volume, calculates tumour inhibiting rate ((1-administration group average tumor weight/model
Matched group average tumor weight) × 100%) and inhibition rate of tumor growth ((1-administration group mean tumour volume/model control group
Mean tumour volume) × 100%), the results are shown in Table 3.
Table 3 compound 1 on the impact of lotus SMMC-7721 mice (N=8,)
Compare with model control group, * P ＜ 0.05, * * P ＜ 0.01.
Test result indicate that, compound 1 is given by 40mg/kg gavage, and humanized's tumor SMMC-7721 can be suppressed nude mice
Internal growth, shows preferable anti-tumor activity.
Claims (9)
1. the compound that represented by Formulas I, its 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 alkoxyl, acetoxyl group or acetyl group and appoints
Meaning one；
X is selected from oxygen, nitrogen, sulfur or carbon atom.
2. compound, its pharmaceutically acceptable salt, stereoisomer, isotopic label, solvent as claimed in claim 1
Compound, polymorph or prodrug, it is characterised in that
R1ForR2For
3. compound, its pharmaceutically acceptable salt, stereoisomer, isotopic label, solvent as claimed in claim 1
Compound, polymorph or prodrug, it is characterised in that described X is oxygen atom.
4. compound, its pharmaceutically acceptable salt, stereoisomer, isotopic label, solvent as claimed in claim 1
Compound, polymorph or prodrug, it is characterised in that R4For hydroxyl, R3And R5For-H.
5. compound 1 and pharmaceutically acceptable salt, isotopic label, solvate, polymorph or prodrug:
6. a pharmaceutical composition, it comprises the compound described in any one of Claims 1 to 5 or it is pharmaceutically acceptable
Salt, stereoisomer, isotopic label, solvate, polymorph, prodrug and pharmaceutically acceptable carrier.
7. pharmaceutical composition as claimed in claim 6, it is characterised in that described pharmaceutical composition is tablet, capsule, ball
Agent, powder, slow releasing preparation, solution, suspension, injection, ointment, emulsifiable paste or suppository.
8. compound as described in any one of Claims 1 to 5, its pharmaceutically acceptable salt, stereoisomer, isotope mark
Note thing, solvate, polymorph or prodrug application in preparation has the medicine of antitumaous effect.
Apply the most as claimed in claim 8, it is characterised in that described cancer includes leukemia, malignant lymphoma, multiple bone
Myeloma, gastrointestinal stromal tumor, colon and rectum carcinoma, breast carcinoma, hepatocarcinoma, gastric cancer, ovarian cancer, uterus carcinoma, cervical cancer, cancer of vagina,
Pulmonary carcinoma, renal carcinoma, carcinoma of prostate, bladder cancer, cancer of pancreas, the brain cancer 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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Non-Patent Citations (2)
| Title |
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| BINA SHAHEEN SIDDIQUI ET AL.: "Flavonoid and cardenolide glycosides and a pentacyclic triterpene from the leaves ofNerium oleanderand evaluation of cytotoxicity", 《PHYTOCHEMISTRY》 * |
| 武蕊娟 等: "马甲子的化学成分研究", 《中草药》 * |
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Effective date of registration: 20191008 Address after: 641000 No. 456, Anji street, Neijiang economic and Technological Development Zone, Sichuan Patentee after: Sichuan zitonggong pharmaceutical Limited by Share Ltd Address before: 610041, No. four, 51 South Renmin Road, Chengdu, Sichuan, Wuhou District Patentee before: Sichuan Academy of Chinese Medicine Sciences |