CN104610276B - Benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate and its preparation method and application - Google Patents
Benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate and its preparation method and application Download PDFInfo
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- CN104610276B CN104610276B CN201410619540.8A CN201410619540A CN104610276B CN 104610276 B CN104610276 B CN 104610276B CN 201410619540 A CN201410619540 A CN 201410619540A CN 104610276 B CN104610276 B CN 104610276B
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- thioxanthene
- dicarboxylic acid
- acid anhydride
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
- C09B57/08—Naphthalimide dyes; Phthalimide dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
Abstract
The present invention relates to benzo [k, l] thioxanthene 3,4 dicarboxylic acid anhydride analog derivative uridnine conjugate and its preparation method and application.The present invention is by being built into 3 novel target compounds by benzo [k, l] the thioxanthene 3,4 diformamide parent ring with DNA intercalator character with the uridnine coupling with important biomolecule activity；The present invention is by 3 target compounds of application: first in extracellular molecules level, promoter activated dna sequence to the proto-oncogene c myc of some tumor cell high expressed has carried out the research of selective binding ability；Secondly, result of study shows that three kinds of compounds are respectively provided with certain killing effect to two kinds of tumor cells on a cellular level, and compound 1 is the strongest to tumor cell effect performance, to research and development benzo [k, l] the tangible meaning of Antioncogene targeted drug of thioxanthene 3,4 dicarboxylic acid anhydride analog derivative uridnine couplet mediated c myc gene deregulation.
Description
Technical field
The present invention relates to a kind of novel benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate and
Application in preparing antitumor drug.
Background technology
The precursor 1,8-naphthoyl imide compounds of benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative is a class tool
Have good anti-tumor activity, by the little molecular complex compound of wide research, it is mainly characterized by having conjugate planes virtue
Fragrant structure, has excellent embedded performance to DNA, can be embedded into DNA base to centre, cause the topological structure of DNA to occur
Change, and then affect typeⅡtopoisomerase to the identification of DNA and effect, therefore be often used as DNA intercalator.Additionally, in its structure
There is also coplanar fluorescence chromophoric group, can be as a species specific DNA fluorescent probe after the intercalation of DNA.1,8-naphthoyl
The exploitation of imine compound is from the beginning of 20 century 70s.1973, Hispanic Brana seminar was according to glutarimide
Ring, β-nitro naphthalene nucleus, amino side chain can design and synthesize out a class comprised only one in conjunction with the theoretical basiss of DNA first
Single naphthalimide of naphthalene ring.Research shows, single naphthalimide can be effectively embedding DNA base between thus cause
DNA superhelix is untwisted.Afterwards, in order to increase the binding ability of compound and DNA, Brana starts the knot to naphthalimide
Structure is modified further, such as at the introducing nitro of naphthalimide parent ring, amino, hydroxyl, the tert-butyl group, methoxyl group, chlorine etc.
Substituent group or introduce the substituent group of nitrogen atom or not nitrogen atom on acid imide side chain, synthesized a series of have anti-swollen
The naphthalimide analog derivative of tumor activity.Some single naphthalimide such as amonafide (Amonafide) and mitonafides
And bisnaphthalimides elinafide (Elinafide) and bisnafide (Bisnafide) etc. have been enter into facing (Mitonafide)
Bed I phase or II phase conceptual phase.But these medicines are found to have bone marrow depression, Central neurotoxicity etc. no when Clinical practice
Good reaction, limits the clinical practice of its reality.Therefore, naphthalimide is carried out structural modification and transformation increases targeting, with
Reduce its toxic and side effects, strengthen anti-tumor activity, increase dissolubility and become the study hotspot of this kind of cancer therapy drug.This research makes
With benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride is as precursor structure, and it has bigger conjugate planes, it is intended to increase and DNA,
Especially with G-tetra-serobila DNA selective binding ability.
Nucleoside and deoxynucleoside are the main components of nucleotide, and some nucleoside and deoxynucleoside or derivatives thereof have
Significantly biological activity, such as antiviral, antitumor, antifungal and as the most edible freshener, can indirectly or direct
As drug use, the treatment of multiple disease serves extremely important effect.Uridine is a kind of main of RNA
Nucleoside, 5-fluorouracil is first antimetabolite synthesized according to certain imagination and is the most most widely used
Anti-miazines medicine, the various solid tumors such as digestive tract cancer are had good therapeutic effect, are also treatment breast carcinoma and gastroenteric tumor
First-selected medication, occupies critical role in oncotherapy.5-fluorouracil is the derivant after 5 of uracil are replaced by fluorine,
It in vivo through enzymatic conversion be 5-fluorodeoxyuridine acid, suppression thymidylate synthase and disturb the conjunction of DNA
Become.Additionally, 5-fluorouracil also has certain inhibitory action to the synthesis of RNA.But, the side effect of 5-fluorouracil is relatively big,
Gastrointestinal reaction and the untoward reaction of the bone marrow depression such as leukocyte and thrombocytopenia such as stomachache, diarrhoea can be produced.
Naphthoyl imide compounds and nucleoside compound all have anti-tumor activity, but each of which all exists or many
Or few shortcoming, limit they further application clinically.As naphthalimide compounds can be as the embedding of DNA
Agent, but the water solublity of its parent ring is poor, poor to the DNA selective of different structure, and nucleoside compound has height to DNA
Affinity and structure have hydrophilic group, but but there is certain toxic and side effects.Both being coupled together may
Reaching the effect having complementary advantages, based on this imagination, design has synthesized 3 benzo [k, l] thiophenes with different length connection chain
Ton-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate.Purpose one, increases the targeting of medicine, reduces side effect；Purpose two,
Increase compound and the affinity of DNA, especially G-tetra-serobila DNA；Purpose three, the water solublity increasing compound is beneficial to clinical answering
With.
C-myc belongs to nuclein controlling gene, is one of internal most important proto-oncogene, is also to study at present
For proto-oncogene widely, the whether normal formation on tumor of its transcript and expression has important impact with development, c-myc's
The regulation and control imbalance of unconventionality expression or its expression product is the characteristic mark of all kinds of human tumors of 20%, and the mankind's is multiple pernicious
Tumor, such as breast carcinoma (Breast carcinoma), cervical cancer (Cervical carcinoma), pulmonary carcinoma (Lung
Carcinoma), ovarian cancer (Ovarian carcinoma), osteosarcoma, spongioblastoma (Neuroblastoma), white
Disorders of blood (Leukemia) etc. are all closely related with the unconventionality expression of c-myc.The expression product c-Myc albumen of c-myc is a kind of turning
Record regulatory factor, can regulate and control to account for the expression of the downstream gene of human genome gene number 15%, thus participate in cell widely
Numerous important physiological process such as growth, differentiation, old and feeble, apoptosis and cell cycle regulating.C-Myc albumen is to downstream gene
Express and generally act the effect activated, but some expression controlling the gene that cell grew Sheng shows the effect of suppression, this portion
Gene is divided to account for the 10-25% of all c-myc downstream genes.It is to say, c-Myc albumen can promote the growth of tumor cell, also
The apoptosis of tumor cell can be promoted.In a word, c-myc and expression product c-Myc albumen thereof participate in multiple physiology mistakes that tumor is relevant
Journey, including cell growth, propagation, apoptosis and the formation of tumor cell, deteriorate, migration etc., its abnormal expression or regulation and control imbalance
Oncogenic formation and development further can be led.
In order to control the expression of c-Myc albumen, transcriptional level carries out selected gene regulation and control and is one and potential is available for
The strategy selected.So, the gene target treatment of the disease of c-myc gene mediated designed, research and develop for c-myc genetic transcription
Medicine has important value and practical significance.Current research is concentrated mainly on induces c-myc promoter region by little molecule
Form G-tetra-serobila rich in G base sequence and stablize its structure and carry out regulatory transcription level, thus controlling the expression of gene.Research
Show, at upstream-142 to-115, the P1 promoter region base pair of c-myc, there is a super quick element NHE III of nucleic acid1
(Nuclear Hypersensitivity Element III1), NHE III1It it is the sequence rich in guanine of one section of 27 base pair
Row, also referred to as Pu27, it controls the transcription of c-myc gene 85-90%.Owing to Pu27 is rich in guanine, at physiological condition
Under have formed G-tetra-serobila potentiality.Pu27 is various activating transcription factor and the identification of DNA and calmodulin binding domain CaM, when it is formed
After G-tetra-stranded structure, various activating transcription factors and DNA None-identified also combine, thus promote c-myc expression to lower.
About 1,8-naphthalimide analog derivative and the report of anti-tumor activity thereof:
2007, Qian Xuhong seminar designed and synthesized 5-position fat amido and the 5-position substituted naphthoyl of aromatic group is sub-
Amine derivative, wherein has 8 compounds that Hela and P388D1 tumor cell exhibits improvements over the extracorporeal anti-tumor of amonafide
Activity.2009, Wang Chao outstanding person seminar synthesized 6 naphthalimide polyamines conjugates, through mtt assay to K562 cell, MB-231
Cell and prostate gland cancer cell have carried out external activity test, and result shows, most compounds shows and is better than amonafide
Anti tumor activity in vitro, wherein QSG-7701 normal person's liver epithelial cell and BEL-7402 hepatoma carcinoma cell are had by 3 kinds of compounds
There is good selectivity.2012, the design of Lee little six seminars synthesized the naphthalene with different length 4-hydroxyl-alkyl amine side chain
Imide derivative.Utilize they affinitys with Ct-DNA of the technique study such as UV-Vis, fluorescence spectrum and CD, and tentatively comment
Estimate its anti-tumor activity.Wherein, following compounds 1 and 2 shows potential anti-tumor activity, and they are to Bel-7402's
IC50Value is respectively 5.57 and 9.17 μMs.
The retrieval analysis of the background information by being above correlated with the present invention finds: up to now, all researchs be all with
1,8-naphthalimide is the derivant medicine as potential treatment tumor of precursor structure, does not utilize 1, and 8-naphthalimide derives
Thing-uridnine conjugate or benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate regulate and control c-myc gene
Report；1,8-naphthalimide derivative-uridnine conjugate or benzo [k, l] thioxanthene-3,4-dioctyl phthalate anhydride is not the most utilized to spread out
Biology-uridnine conjugate is to c-myc promoter region DNA sequence-specific recognition reaction and the report of suppression apoptosis of tumor cells
Road.
Summary of the invention
For the above-mentioned problems in the prior art, it is an object of the invention to provide a kind of novel benzo [k, l]
Thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate and application thereof, increase compound water solublity and specifically with
The binding ability of DNA, and there is the characteristic to the c myc selective recognition reaction of promoter DNA base sequence；This
Quasi-molecule can cause the cancerous cell of c-myc gene high expression to produce withering in various degree by lowering the expression of c-myc gene
Die, therefore can be as potential gene target medicine for oncotherapy.
Benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate, it is characterised in that include following three kinds
Compound, structural formula is respectively as shown in formula I, formula II, formula III:
。
Described benzo [k, l] thioxanthene-3, the preparation method of 4-dicarboxylic acid anhydride analog derivative-uridnine conjugate, its feature
Be with benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride and 2-aminouridine be raw material, DMF be solvent, DCC be condensing agent, at argon
React 24-26 hour at 35 DEG C under gas shielded, after TLC monitoring reaction terminates, after being cooled down by reactant liquor, cross what ripple removing was formed
N, N-dicyclohexylurea precipitate, then with acetone rinsing, collect filtrate and be also further purified with preparing thin layer chromatography, thin layer chromatography with
Corresponding product tape, as developing solvent, is rinsed as eluant by the mixed liquor of chloroform and methanol with ethanol, collects eluting
Liquid, rotation is evaporated off ethanol and is placed at 30 DEG C vacuum drying and obtains the compound (I) of yellow powder, and above-mentioned thin layer chromatography is used
HF254, the silica gel plate of 20 × 25 cm, thickness is 1.5mm, the chloroform used by thin layer chromatography and the mixeding liquid volume ratio 9 of methanol:
1。
Described benzo [k, l] thioxanthene-3, the preparation method of 4-dicarboxylic acid anhydride analog derivative-uridnine conjugate, its feature
It is first to be reacted with 3-alanine by benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride and obtains benzothioxanthene-3,4-two formyl Asia
Amine-n-propanoic acid, then by benzothioxanthene-3,4-dicarboximide-N-propanoic acid and 2-aminouridine in DMF using DCC as condensation
Agent is reacted 24-26 hour under argon shield at 35 DEG C, after TLC monitoring reaction terminates, reactant liquor cooled and filtered is removed
N, the N-dicyclohexylurea precipitate formed, with acetone rinsing, collects filtrate and is further purified with preparing thin layer chromatography, thin layer color
Compose using the mixed liquor of chloroform and methanol as developing solvent, as eluant, corresponding product tape is rinsed with ethanol, collect
Eluent, rotation is evaporated off ethanol；It is placed at 30 DEG C vacuum drying and obtains the compound (II) of yellow powder, above-mentioned thin layer color
Spectrum HF254, the silica gel plate of 20 × 25 cm, thickness is 1.5mm, the chloroform used by thin layer chromatography and the mixeding liquid volume of methanol
Compare 9:1.
Described benzo [k, l] thioxanthene-3, the preparation method of 4-dicarboxylic acid anhydride analog derivative-uridnine conjugate, its feature
It is to be reacted with 6-Aminocaproic Acid by benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride that to obtain benzothioxanthene-3,4-two formyl sub-
Amine-n-is the sourest, then by benzothioxanthene-3,4-dicarboximide-N-acid and 2-aminouridine in DMF using DCC as condensation
Agent is reacted 24-26 hour under argon shield at 35 DEG C, after TLC monitoring reaction terminates, reactant liquor cooled and filtered is removed
N, the N-dicyclohexylurea precipitate formed, with acetone rinsing, collects filtrate and is further purified with preparing thin layer chromatography, thin layer color
Compose using the mixed liquor of chloroform and methanol as developing solvent, as eluant, corresponding product tape is rinsed with ethanol, collect
Eluent, rotation is evaporated off ethanol；It is placed at 30 DEG C vacuum drying and obtains the compound (III) of yellow powder, above-mentioned thin layer color
Spectrum HF254, the silica gel plate of 20 × 25 cm, thickness is 1.5mm, the chloroform used by thin layer chromatography and the mixeding liquid volume of methanol
Ratio 9:1, above-mentioned urea is also referred to as carbamide.
Described benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate is in preparing antitumor drug
Application.
Described benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate is in preparation treatment cervix uteri
Cancer, Non-small cell lung carcinoma medicine in application.
Described benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate is in preparing antitumor drug
Application, it is characterised in that the antitumor drug with c-myc gene as target.
The reaction equation of the present invention is as follows:
Wherein, formula IV is benzothioxanthene-3, the structural formula of 4-dicarboxylic acid anhydride, and formula (V) is the structure of 2-aminouridine
Formula, in formula II or (III) as n=2, for compound formula II；As n=5, for compound formula III, in substituted amino acid
N is 2 or 5.
By using above-mentioned technology, compared with prior art, beneficial effects of the present invention is as follows:
1) present invention will be by having benzo [k, l] thioxanthene-3,4-diformamide parent ring and the tool of DNA intercalator character
The uridnine coupling having important biomolecule activity is built into 3 novel benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-urine
Glycosides conjugate is target compound, all certifications of the structure of each compound；
2) c myc of some tumor cell high expressed is started by the present invention by 3 target compounds of application
Sub-activated dna sequence has carried out the research of the selective binding ability of extracellular molecules level, electrophoretic mobility experiment (EMSA),
Ultraviolet melting experiment (UV-melting) and circular dichroism spectra (CD) result show: three kinds of compounds have different from target DNA sequence
Specific binding capacity；
3) present invention applies three kinds of compounds tumor cell Hela(cervical cancer to two Expression of Plant Height c-myc genes)
With A549(adenocarcinoma of lung) cell, with Normal Lung cell (HLF-a) for reference to having carried out the research of active anticancer, application is the thinnest
Born of the same parents analyze (RTCA) technical research apoptosis situation, and result of study shows: two kinds of tumor cells are respectively provided with by three kinds of compounds
Certain killing effect, to normal person's pneumonocyte (HLF-a) almost without effect, and tumor cell effect is showed by compound 1
The strongest, IC50Respectively: 2.26 μMs (A549) and 1.96 μMs (Hela), to research and development benzo [k, l] thioxanthene-3,4-dioctyl phthalate anhydride
The tangible meaning of Antioncogene targeted drug of derivant-uridnine couplet mediated c-myc gene deregulation.
4) the application compound 1 tumor cell Hela(cervical cancer to two Expression of Plant Height c-myc genes in the present invention) and
A549(adenocarcinoma of lung) cell carried out c-myc mRNA by RT-PCR and Western Blot technology respectively and transcribed and c-Myc egg
White expression study, result shows: compound 1 transcribes two kinds of tumor cell c-myc mRNA and the expression of c-Myc albumen is equal
Having certain inhibitory action, this type of benzo [k, l] thioxanthene-3 are described, 4-dicarboxylic acid anhydride analog derivative-uridnine conjugate is to pass through
The downward of mediation c-myc gene expression reaches antitumor action.
Accompanying drawing explanation
Fig. 1 is the transformation of the super quick element III 1 regional DNA structure of the c-myc gene nucleic acid impact on c-myc genetic transcription
Mechanism；In figure, NHE III 1 is the super quick element III 1 of nucleic acid；Hn RNP K is heterogeneity ribonucleoprotein in core
Special member in (heterogeneous-nuclear ribonucleoprotein, hnRNP) family, is a kind of many merits
Can albumen；CNBP is nucleus binding-protein；TBP is tributyl phosphate；RNA pol II is RNA polymerase II；
Fig. 2 is benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate and c-myc gene promoter area
The native polyacrylamide gel electrophoresis figure that sequence interacts on the gene level of extracellular；In figure: swimming lane 1:Marker；
Swimming lane 2: the single stranded DNA rich in guanine in corresponding double-stranded DNA；Swimming lane 3: the list rich in guanine in corresponding double-stranded DNA
Chain DNA and compound mole ratio 1:30；Swimming lane 4: the single stranded DNA rich in cytosine in corresponding double-stranded DNA；Swimming lane 5: corresponding
The single stranded DNA rich in cytosine in double-stranded DNA and compound mole ratio 1:30；Swimming lane 6-12: corresponding double-stranded DNA and chemical combination
Thing mole ratio 1:0(swimming lane 6), 1:1(swimming lane 7), 1:5(swimming lane 8) and, 1:10(swimming lane 9), 1:15(swimming lane 10) and, 1:20(swimming lane
11), 1:30(swimming lane 12)；Swimming lane 13: the double-stranded DNA of corresponding mispairing；Swimming lane 14: the double-stranded DNA of corresponding mispairing rubs with compound
You compare 1:30 by number.The effect (Fig. 2 (1)) of (a) compound 1 and DNA.The effect (Fig. 2 (2)) of (b) compound 2 and DNA.C () is changed
The effect (Fig. 2 (3)) of compound 3 and DNA；
Fig. 3 is benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate and c-myc gene promoter area
The melting curve of sequence effect.The melting curve (Fig. 3 (1)) of (a) compound 1 and corresponding double-stranded DNA, [DNA]=2 μM, [change
Compound 1]=0,10,40 μMs.The melting curve (Fig. 3 (2)) of (b) compound 2 and corresponding double-stranded DNA, [DNA]=2 μM,
[compound 1]=0,10,40 μM.The melting curve (Fig. 3 (3)) of (c) compound 3 and corresponding double-stranded DNA, [DNA]=2 μ
M, [compound 1]=0,10,40 μM.(d) three compounds respectively with melting curve (Fig. 3 of corresponding mismatched dsdna
(4)), DNA and compound mole ratio are 1:20；
Fig. 4-1 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 1 is rich with c-myc
Containing the circular dichroism spectrogram of guanine double-stranded DNA effect, [DNA]=2 μM, [compound]=0,2,4,8,16,32,
48, 64 μM；
Fig. 4-2 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 2 is rich with c-myc
Containing the circular dichroism spectrogram of guanine double-stranded DNA effect, [DNA]=2 μM, [compound]=0,2,4,8,16,32,
48, 64 μM；
Fig. 4-3 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 2 is rich with c-myc
Containing the circular dichroism spectrogram of guanine double-stranded DNA effect, [DNA]=2 μM, [compound]=0,2,4,8,16,32,
48, 64 μM；
Fig. 5-1 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 1 is rich with c-myc
Circular dichroism spectrogram containing guanine single stranded DNA effect；[DNA]=2 μM, [compound 1]=0,2,4,8,16,32,64,80 μ
M；
Fig. 5-2 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 2 is rich with c-myc
Circular dichroism spectrogram containing guanine single stranded DNA effect；[DNA]=2 μM, [compound 2]=0,2,4,8,16,32,64,80 μ
M；
Fig. 5-3 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 3 is rich with c-myc
Circular dichroism spectrogram containing guanine single stranded DNA effect；[DNA]=2 μM, [compound 3]=0,2,4,8,16,32,64,80 μ
M；
Fig. 6-1 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 1 is non-to A549(
Small cell lung cancer) the growth inhibited curve of cell, this curve is the meansigma methods in multiple hole；
Fig. 6-2 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 2 is non-to A549(
Small cell lung cancer) the growth inhibited curve of cell, this curve is the meansigma methods in multiple hole；
Fig. 6-3 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 3 is non-to A549(
Small cell lung cancer) the growth inhibited curve of cell, this curve is the meansigma methods in multiple hole；
Fig. 7-1 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 1 is to Hela(
Cervical cancer) the growth inhibited curve of cell, this curve is the meansigma methods in multiple hole；
Fig. 7-2 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 2 is to Hela(
Cervical cancer) the growth inhibited curve of cell, this curve is the meansigma methods in multiple hole；
Fig. 7-3 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 3 is to Hela(
Cervical cancer) the growth inhibited curve of cell, this curve is the meansigma methods in multiple hole；
Fig. 8-1 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 1 is to Normal Lung
The growth inhibited curve of cell (HLF-a), this curve is the meansigma methods in multiple hole；
Fig. 8-2 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 2 is to Normal Lung
The growth inhibited curve of cell (HLF-a), this curve is the meansigma methods in multiple hole；
Fig. 8-3 is that benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate compound 3 is to Normal Lung
The growth inhibited curve of cell (HLF-a), this curve is the meansigma methods in multiple hole；
Fig. 9 is benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate 1 and two kind of tumor cell
The c-myc mRNA that (A549, Hela) and normal cell (HLF-a) act on transcribes and c-Myc protein expression；[compound]=
0, 1, 5, 10 μM.A the c-myc mRNA of () compound 1 and A549 cytosis transcribes (Fig. 9 (1)).(b) compound 1
With the c-myc mRNA of Hela cytosis transcribe (Fig. 9 (2)).(c) compound 1 and the c-Myc albumen of A549 cytosis
Expression (Fig. 9 (3)).The expression (Fig. 9 (4)) of the c-Myc albumen of (d) compound 1 and Hela cytosis.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described, but protection scope of the present invention is not limited to that:
Embodiment 1: the preparation of compound (I)
By 30.43mg(0.1mmol) benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride and the 2-of 24.32mg (0.1mmol)
Aminouridine joins in the DMF of 1.0mL, under argon shield and 25.79mg(0.1mmol, excessive 25% mol)
DCC(dicyclohexylcarbodiimide) in the presence of this solution stirred at 35.5 DEG C reaction 25.5 h, TLC monitor reaction process
(chloroform: methanol=9:1, v/v) Rf=0.55, then, natural cooling reactant liquor, precipitate N, N-bis-hexamethylene that will produce
Base urea element filters out, and uses a small amount of acetone rinsing, by gained filtrate with preparing thin layer chromatography (HF254, the silica gel of 20 × 25 cm
Plate, 1.5mm is thick) purification, the chloroform using volume ratio as 9:1: product tape, as eluant, is rinsed by methanol with ethanol, receive
Collection eluent also removes ethanol with Rotary Evaporators and is placed in vacuum drying oven and is dried to obtain Huang under conditions of less than 30 DEG C
Color powder 21.36mg, productivity 40.34%.1H NMR(600 MHz, DMSO-d6): δ 8.48(d, J5”,6”= 8.4, 1H,
5 "-H), 8.327(d, J1”,2”=7.8,1H, 2 "-H), 7.9(d, J5,6 =7.8,1H, 6-H), 7.77(d,
J6”,5”=7.8,1H, 6 "-H), 7.60(d, J7”,8”=7.8,1H, 7 "-H), 7.48-7.55(m, 3H, 8 "-H,
9 "-H, 10 "-H), 6.58 (d, J1’,2’=3.6,1H, 1 '-H), 5.68(d, J5’,6’=6,2H, 5 '-H),
5.63(d, J2’,1’=6,1H, 2 '-H), 5.49(t, J3’,4’=5.4,1H, 3 '-OH), 4.49(t, J5’,4’=
5.4,1H, 5 '-OH), 4.39-4.43(m, 1H, 4 '-H), 4.33-4.36(m, 1H, 3 '-H), 3.74-3.77(m,
1H, 5 '-H)；FAB-Mass: m/z calculated for C27H19N3O7S: 529.09; found: 530[M+H]+。
Embodiment 2: the preparation of compound (II)
1) synthesis of intermediate benzo [k, l] thioxanthene-3,4-dicarboximide-N-propanoic acid
By 35.80mg(0.085mmol) benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride and 8.91mg (0.1mmol)
3-alanine is dissolved in the DMF of 1.2mL under argon shield, be then stirred at reflux 6h, TLC monitoring reaction process (chloroform:
Methanol=9:1, v/v) Rf=0.65.Remove DMF with Rotary Evaporators at 60 DEG C, the residue of gained is dissolved in (chloroform: first
Alcohol=8:1, v/v) mixed solvent in, then carry out silica gel column chromatography, respectively using chloroform: methanol=15:1,14:1,10:1 as
Eluant, is finally recovered and obtains yellow powder 15mg, and productivity is 73%.1H NMR(600 MHz, DMSO-d6): δ 8.43(d,
J5,6 =8.4,1H, 5-H), 8.42(d, J1,2 =8.4,1H, 2-H), 7.68-7.71(m, 2H, 1-H, 6-H),
7.57(d, J7,8=7.2,1H, 7-H), 7.47-7.52(m, 3H, 8-H, 9-H, 10-H), 4.24(t, Jα,β=
7.8,2H, β-H), 2.68(t, Jβ,α=7.8,2H, α-H).
2) synthesis of target product compound (II)
By 37.53mg(0.1mmol) benzo [k, l] thioxanthene-3,4-dicarboximide-N-propanoic acid and 24.32mg
(0.1mmol) 2-aminouridine is dissolved completely in the DMF of 2.0mL, adds 25.79mg(0.1mmol under argon shield,
Excess 25% mol) DCC and 16.20 mg(0.12mmol) and HOBt(1-hydroxyl benzotriazole), 35 DEG C stir 24 h,
TLC monitors reaction process (chloroform: methanol=5:1, v/v) Rf=0.52.Then reactant liquor is cooled to 100 DEG C and adds 0.2mL
Hot water.After said mixture is cooled to room temperature, the precipitation formed is filtered and uses a small amount of water to rinse, is subsequently placed in vacuum drying
Case is dried.The solid of dry gained is re-dissolved in chloroform, purifies with silica gel column chromatography, with chloroform: methanol=9:1 makees
For eluant.Collect eluent and obtain yellow powder 22.58mg, productivity 37.6% with Rotary Evaporators removing eluant.1H
NMR(600 MHz, DMSO-d6): δ 8.44(d, J5”,6”=8.4,1H, 5 "-H), 8.26(d, J1”,2”= 8.4, 1H,
2 "-H), 8.01(d, J2’,3’=8.4,1H, 2 '-NH), 7.89(d, J6,5 =8.4,1H, 6-H), 7.69(d,
J6”,5”=7.8,1H, 6 "-H), 7.57(d, J7”,6”=6.6,1H, 7 "-H), 7.46-7.53(m, 3H, 8 "-H,
9 "-H, 10 "-H), 5.92(d, J1’,2’=8.4,1H, 1 '-H), 5.68(d, J5,6 =6,1H, 5-H), 5.63
(d, J3’,4’=4.8,1H, 3 '-OH), 5.18(d, J5’,4’=5.4,1H, 5 '-OH), 4.49-4.53(m, 1H,
2 '-H), 4.14-4.22(m, 2H, β-H), 4.08-4.10(m, 1H, 3 '-H), 3.915-3.917(m, 1H, 4 '-
H), 3.54-3.60(m, 2H, 5 '-H), 2.50-2.55(m, 2H, α-H)；FAB-Mass: m/z calculated
for C30H24N4O8S: 600.13; found: 491[M-C4H4N2O2]+。
Embodiment 3: the preparation of compound (III)
The synthetic route of compound (III), with compound 2, replaces 3-alanine with 6-Aminocaproic Acid, obtains yellow powder
36mg, productivity 56.0%.1HNMR(600 MHz, DMSO-d6): δ 8.47(d, J5”,6”=7.2,1H, 5 "-H), 8.29
(d, J1”,2”=7.8,1H, 2 "-H), 7.88(d, J6,5 =8.4,1H, 6-H), 7.70-7.74(m, 3H, 1 "-H,
6 "-H), 7.58(d, J7”,8”=7.8,1H, 7 "-H), 7.47-7.53(m, 3H, 8 "-H, 9 "-H, 10 "-H),
5.88(d, J1’,2’=9,1H, 1 '-H), 5.67(d, J5,6 =7.2,1H, 5-H), 5.17(d, J5’,4’= 5.4,
1H, 5 '-OH), 4.48-4.52(m, 1H, 2 '-H), 4.05-4.07(m, 1H, 3 '-H), 3.97-3.99 (m, 1H,
ε-H), 3.915-3.917(m, 1H, 4 '-H), 3.55-3.60(m, 2H, 5 '-H), 2.13-2.15(m, 2H, α-
H), 1.56-1.61(m, 2H, δ-H), 1.46-1.51(m, 2H, β-H) and, 1.23-1.29(m, 2H, γ-H)；FAB-
Mass: m/z calculated for C33H30N4O8S: 642.18; found: 643[M+H]+。
In following example, compound 1, compound 2, compound 3 refer to the compound (I) in embodiment 1-3, chemical combination respectively
Thing (II), compound (III).
Raw material DNA used in present specification illustrates (Fig. 2-Fig. 5): 1. refer to rich in bird rich in guanine single stranded DNA
Purine (G) comprises 27 super quick element NHE III black italicized items of 1(of base nucleic acid) c-myc promoter region single-stranded DNA sequence:
5-GGGCGCTTATGGGGAGGGTGGGGAGGGTGGGGAAGGTGGGGAG-3’；2. rich in the strand of cytosine
DNA (rich in cytosine (C)):
5-CTCCCCACCTTCCCCACCCTCCCCACCCTCCCCATAAGCGCCC-3 ' 3. double-stranded DNA refer to 1. with 2.
Formed comprise 27 super quick element NHE III black italicized items of 1(of base nucleic acid rich in guanine (G)) c-myc promoter region
Double chain DNA sequence (c-myc is rich in guanine double-stranded DNA):
5’-GGGCGCTTATGGGGAGGGTGGGGAGGGTGGGGAAGGTGGGGAG-3’
3’-CCCGCGAATACCCCTCCCACCCCTCCCACCCCTTCCACCCCTC-5’
4. mismatched dsdna refers to mispairing (the mark black italicized bases) DNA sequence (be called for short) corresponding with 3. double-stranded DNA:
5’-GCGCGCTTATCGCGAGCGTCGCGAGCGTCGCGAACGTCGCGAG-3’
3’-CGCGCGAATAGCGCTCGCAGCGCTCGCAGCGCTTGCAGCGCTC -5’
In a specific embodiment, embodiment 4,5, in 6 DNA used without specified otherwise with defined herein
DNA is consistent, and above DNA is purchased from Shanghai Sheng Gong biological engineering company limited.
Embodiment 4: the Native-that 3 compounds that the present invention prepares interact with c-myc gene promoter region sequence
PAGE analyzes
1) preparation of electrophoresis Sample: the final volume of each electrophoresis Sample is 10 μ L, in sample final concentration of 1 μM of DNA,
The content of DMSO is 2%, according to the proportionate relationship of DNA Yu compound, is configured to the solution of variable concentrations.All samples has been prepared
Cheng Hou, with metal bath degeneration 10 min at 95 DEG C, after naturally cooling to room temperature, hatches 24 hours in 4 DEG C of refrigerators, stand-by；
2) preparation (16% Native-PAGE, table 1) of 16% non-denaturing polyacrylamide gel: be used for investigating compound
With c-myc promoter region DNA sequence interaction property.
Table 1 16% non-denaturing polyacrylamide gel formula
3) Native-PAGE deposition condition: 100V prerunning 30min.Take the sample 1.5 μ L addition that step 1) prepares
6 × Loading buffer of 0.5 μ L makes electrophoresis Sample, joins in loading wells with micropipettor.Use constant voltage mode,
Arranging voltage is 100 V, and temperature is 4 DEG C, and electrophoresis time is generally 8-9h.After electrophoresis terminates, under glue is peeled off from offset plate
After coming and cleaning with deionized water, with SYBR Gold nucleic acid dye, (1 × tbe buffer liquid of the SYBR Gold 20mL of 2 μ L is dilute
Release) dyeing 30min, deionized water rinsing 2 times, it is then placed within gel imaging instrument observing and imaging, result is shown in Fig. 2.
The interpretation of result of glue from Fig. 2: three compounds have necessarily with double-strand and the single stranded DNA rich in guanine base
The effect of degree, this effect is concentration dependent；But to the not mispairing double-strand rich in guanine and the list rich in cytosine
Chain DNA acts on hardly.These results illustrate: compound selectively and is especially enriched in the double-strand of guanine rich in guanine
DNA produces stronger specific effect.
Embodiment 5: compound is to c-myc gene promoter region sequence thermal stability analysis
By UV-melting technical research compound and c-myc gene promoter region sequence interaction ability in the present invention
And the impact on DNA heat stability.
1) preparation of sample: the final volume of sample is 600 μ L, contains in buffer by sample final concentration of 2 μMs of DNA
10mM Tris-HCl, 0.1 mM EDTA and 150 mM KCl, according to the proportionate relationship of DNA Yu compound, be configured to different dense
The solution of degree.After all samples has been prepared, with metal bath degeneration 10min at 95 DEG C, after naturally cooling to room temperature, in 4 DEG C
Refrigerator hatches 24 hours；
2) Tm pH-value determination pH: proceed in quartz colorimetric utensil by above-mentioned sample, is placed in and is equipped with temperature control system (Shimadzu S-1700)
Ultraviolet spectrometer (Shimadzu UV-2550) in measure absorbance.Measuring wavelength and be set to 260nm, initial temperature is set to 10 DEG C, eventually
Only temperature is set to 95 DEG C, and heating rate is set to 1 DEG C/min, and each test sample balances 10min in initial temperature, and result is shown in figure
3；
3), after test terminates, the T of each sample is calculated by ultraviolet spectrometer data handling systemmValue, as shown in table 2, table 2
It it is the dissolving of three kinds of benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate 1-3 and c-myc DNA effect
Temperature (Tm value).
Table 2. c-myc DNA T under compound 1-3 presence or absencemValue
As shown in table 2, UV-melting tests it can be seen that compound can improve the thermally-stabilised of c-myc double-stranded DNA
Property, and this action effect is certain concentration dependent, and three kinds of compounds have identical effect trend, can be necessarily
The T of DNA is increased in degreemValue.Wherein the action effect of compound (I) is best, makes TmThe amplitude that value increases is maximum；And for mistake
The c-myc double-stranded DNA joined and the strand rich in cytosine, compound on it almost without impact, TmThe change of value is the most fairly small, several
It is negligible.It can be said that bright, compound is to be selectively applied to the c-myc double-stranded DNA rich in guanine.
Embodiment 6: the circular dichroism spectra experiment that compound and c-myc gene promoter region sequence interact
By circular dichroism spectra (CD) technical research compound and c-myc gene promoter region sequence interaction energy in the present invention
Power and essence.Parallel type G-tetra-serobila has positive absorption maximum near 260-265nm, and 240-245nm has negative maximum suction
Receive；G-tetra-serobila of antiparallel type has positive maximum absorption near 290-295nm, and 260nm has negative absorption maximum；Mixing
The G-tetra-serobila configuration of formula then includes just absorbing and acromion near 265 nm of feature near 290nm.Typical B structure
The CD spectrum signature of type DNA: have a positive absworption peak at 270-280nm corresponding to the packed structures of nucleic acid base, and
The negative absworption peak double-spiral structure corresponding to nucleic acid is had at 248-255nm.
A () compound and c-myc gene promoter region sequence are rich in guanine base double-stranded DNA effect
1) preparation of samples: DNA is become by the buffer preparation containing potassium ion the sample of (2 μMs, 600 μ L), carries out degeneration
Renaturation process (in thermostat water bath, it is to slowly warm up to 95 DEG C, degeneration 10 minutes, it is naturally cooling to room temperature, is positioned over 4 DEG C of ice
Case hatches 24 hours).Respectively benzo [k, the l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative of the present invention-uridnine conjugate is used
DMSO is configured to the solution for standby of 1.2mM；
2) sample is proceeded in the cuvette that optical path length is 5mm, be placed in J-815 circular dichroism spectrometer (JASCO) and survey
Amount, arranging scanning wavelength scope is 600-220nm, and scanning speed is 100nm/min, and slit width is 5.0nm.At test process
In every the set time drip the above-mentioned compound solution prepared, every 5min pipettor blows and stirs once, puts into instrument after 15min
Measure.DNA is respectively as follows: 1/0,1/1,1/2,1/4,1/8,1/16,1/24,1/32,0/10 with the ratio of compound.Each sample
Scan three times, average；
The CD experimental result shown such as Fig. 4-1, Fig. 4-2 and Fig. 4-3 can be seen that compound can be induced to a certain extent
The formation of hybrid G-tetra-serobila, thus the disperse conditions of streaking explained in gel electrophoresis experiment is exactly that this structure is made
Become.In Fig. 4-1, compound (I) and target DNA effect are relatively strong, target DNA after progressively dripping compound, double-stranded DNA characteristic peak
At 275nm, posivtive spike gradually weakens, and occurs posivtive spike at 290nm and 260nm, and antiparallel or hybrid and run-in index G-have been described
Four serobila DNA produce；Further, stronger in 425-550nm scope ICD, these information explanation compound (I) has induces more by force target
Double-strand c-myc DNA forms the ability of G-tetra-serobila and has strong adhesion therewith.Other two kinds of compound 2-3 and target double-strand c-
Myc DNA there is also similar effect in various degree.But from CD spectrum, also can show that final mixture exists a certain amount of double
Chain DNA；Especially, compound (III) is more weak with target double-strand c-myc DNA effect, and DNA is still with Double helix or strand shape for major part
Formula exists, and there is part G-tetra-stranded structure simultaneously, is in the state of a kind of mixture.Owing to compound itself has chirality,
Also there is the CD of self, and show certain ICD(induction CD), can affirm that compound and DNA there occurs effect, but have
Body is any binding mode, thus can not immediately arrive at, and must be studied checking further by other method.
B () compound and c-myc gene promoter region sequence are rich in guanine base single stranded DNA effect
Sample be c-myc gene promoter region sequence rich in guanine base single stranded DNA, preparation of samples and assay method such as
(a).Compound concentration: 0 μM, 2 μMs, 4 μMs, 8 μMs, 16 μMs, 32 μMs, 48 μMs, 64 μMs, 80 μMs, result see Fig. 5-1,
Fig. 5-2 and Fig. 5-3 shows, Fig. 5-1, Fig. 5-2 and Fig. 5-3 show and show, three compounds are all not so good as corresponding richness to target DNA effect
Strong containing guanine base double-stranded DNA, illustrate that compound is good to double-stranded DNA selectivity.Compound (I) is to single stranded DNA effect still
It is better than compound (II) and compound (III), and inducing DNA produces a certain amount of antiparallel or hybrid G-tetra-serobila DNA knot
Structure, at 300-350nm and 425-550nm, produces stronger CD posivtive spike, comes from ICD and the CD of compound itself, compound
(II) worst with target DNA effect.
Embodiment 7: the anti tumor activity in vitro of compound
(a) compound growth inhibited to A549 (nonsmall-cell lung cancer) cell
Material: (non-small cell lung cancer cell is purchased from Microbe Inst., Chinese Academy of Sciences's strain culturing preservation A549 cell
The heart, Beijing), 1640 culture medium, 0.25% pancreatin.
RTCA experiment (experiment is repeated in three holes): set the initial experiment condition of real-time cell analyser.By E-Plate 96
Orifice plate is placed on the cytoanalyze in constant incubator, scanning background value.100 μ L are added in the every hole of E-Plate 96 orifice plate
Cell suspension, makes the cell number in each hole be about 10000, is placed at 37 DEG C and hatches 30min.Afterwards by E-Plate
96 orifice plates are put back to and are scanned on real-time cell analyser i.e. can get continuous print cell index curve.Treat that tumor is thin after about 24 h
Born of the same parents enter exponential phase, suspend scanning, take off E-Plate 96 orifice plate, add 5 μ of variable concentrations in each hole of experimental group
L target compound, its Concentraton gradient is 0.5 μM, 1.0 μMs, 2.0 μMs, 4.0 μMs, 8 μMs.Cell culture fluid is reference, and 2%DMSO makees
For contrast experiment.
As shown in Fig. 6-1, Fig. 6-2 and Fig. 6-3 show, the exercising result of A549 cell is shown by three compounds: this kind ofization
Compound to the growth inhibited of A549 cell all in concentration dependent.Compound (I) is just thin to A549 when concentration is 0.5 μM
Born of the same parents show certain inhibitory action, and along with compound concentration is increasing, inhibitory action is stronger, to Cmax 8 μ
The growth of A549 cell has been completely inhibit during M.Compound (II) is when concentration is 0.5 μM, and its cytotoxicity is not
It is clearly.Along with compound concentration increases to 1.0 μMs, compound starts A549 cells show is gone out certain growth inhibited
Effect.Along with the increase of compound concentration, the growth inhibited effect of cell is the most gradually strengthened by it.Compound (III) in concentration is
Just A549 cells show is gone out obvious inhibitory action when 0.5 μM.But, along with the continuation of compound concentration increases, it is to carefully
The increasing degree of born of the same parents' inhibitory action is not so good as compound (I) and compound (II).
(b) compound growth inhibited to Hela (cervical cancer) cell
Experiment material, method is with (a).Experimental result is as shown in Figure 7.
Fig. 7-1, Fig. 7-2 and Fig. 7-3 show and show, to Hela cell, (cervical cancer cell is purchased from section of China to three compounds
Institute of Micro-biology of institute strain culturing preservation center, Beijing) cytotoxic effect result show: compound is to Hela(cervix uteri
Cancer) growth inhibited of cell is all in concentration dependent.Compound (I) to the action effect of Hela cell clearly,
Just showing inhibition significantly when concentration is 0.5 μM, during to Cmax 8 μMs, the growth of Hela cell is subject to the most completely
Suppression.When compound (II) concentration is 0.5 μM, the growth of Hela cell starts suppression situation occur, and concentration continues to increase, and presses down
Make of strengthening the most further.Compound (III) starts the growth inhibitory effect to cell occur from 0.5 μM of concentration, to maximum dense
When spending 8 μMs, cells show is gone out strong growth inhibitory effect.
(c) compound growth inhibited to normal HLF-a (human pneumonocyte) cell
Experiment material, method is with (a).Its compound concentration gradient is 1.0 μMs, 5.0 μMs, 10 μMs, 20 μMs, 40 μMs.Experiment
Shown in result such as Fig. 8-1, Fig. 8-2 and Fig. 8-3 show.
To normal HLF-a cell, (human pneumonocyte is purchased from Microbe Inst., Chinese Academy of Sciences's spawn culture and protects three compounds
Center, Tibetan, Beijing) cytotoxic effect result show: except compound (I) when concentration 20 μMs and 40 μMs to HLF-a cell
Having outside faint effect, the growth to HLF-a cell in the concentration range investigated of the compound of all investigations is produced hardly
Raw inhibition, although maximum experimental concentration has reached 40 μMs.Illustrate three kinds of compounds to normal cell almost without effect.
By the real-time cell apoptosis data to two kinds of tumor cells (A549, Hela) and Normal Lung cell (HLF-a)
Analysis, draw three kinds of compounds IC to three kinds of experimental cell effects50Value (as shown in table 3).
Table 3 is three kinds of benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate 1-3 and two kinds of tumors
The IC that cell (A549, Hela) and normal cell (HLF-a) act on50Value.
IC from table 350Value explanation compound (I) has stronger inhibition, compound (II), change to two kinds of tumor cells
Compound (III) has certain inhibition to two kinds of tumor cells, and three kinds of compounds are dense in experiment to normal pneumonocyte HLF-a
Almost no effect under degree.
Embodiment 8: tumor cell c-myc mRNA is transcribed and the impact of c-Myc protein expression by compound 1
In order to prove that this compounds is to produce the tumor cell of high expressed c-myc by lowering c-myc gene expression
Raw inhibitory action, research completes compound (I) and tumor cell c-myc mRNA is transcribed and c-Myc protein expression
The experiment of impact.
(a) A549 (nonsmall-cell lung cancer) and Hela (cervical cancer) cell c-myc in the presence of compound (I)
MRNA transcribes
Material: A549 (nonsmall-cell lung cancer) cell Hela (cervical cancer) cell, in RPMI 1640 culture medium
(containing 10% hyclone, 100 U/mL penicillins and 100 U/mL streptomycins), 37 DEG C, the CO of 5%2 , train under saturated humidity
Support.
Method: illustrate to carry out by the technology of RT-PCR kit.The most in vitro cultivate purpose tumor cell Hela and
A549 cell, when its growth conditions to be seen is good, through digestion, resuspended, counting, with 2 × 105Cell/ hole is inoculated in 12 holes
Plate, the concentration arranging compound 1 is: 0 μM, 1 μM, 5 μMs, 10 μMs.After adding medicine 24h, after collecting compound effects
Tumor cell, extracts tumor cell total serum IgE.Extraction process uses the RNeasy mini kit test kit of QIAGEN company, by skill
Art requires operation.
Primer sequence used in PCR experiment is (being synthesized by Shanghai Sheng Gong company):
C-myc upstream 5 '-AGAGA AGCTG GCCTC CTACC-3 '
Downstream 5 '-CGTCG AGGAG AGCAG AGAAT-3 '
β-actin upstream 5 '-AGC GGG AAA TCG TGC GTG ACA-3 '
Downstream 5 '-GTG GAC TTG GGA GAG GAC TGG-3 '
(1) by following composition preparation PCR reactant liquor, total system 25 μ L.
(2) PCR reaction condition is as follows:
1) degeneration: 95 DEG C, 2 min
2) circulation: 94 DEG C, 15 sec；62 DEG C, 30 sec；72 DEG C, 1 min；30 cycles
3) extend: 72 DEG C, 10 min
4) preserve: 4 DEG C.
After PCR reaction terminates, the PCR reactant liquor respectively taking 5 μ L carries out agarose gel electrophoresis, and uses gel ultraviolet imagery
System is taken pictures preservation.Experimental result such as Fig. 9 (1) (A549 cell) and (2) (Hela cell).Result shows: compared with matched group,
The suppression that the c-myc mRNA of tumor cell is transcribed by compound 1 is concentration dependent, when concentration reaches 10 μMs, produces
Raw stronger inhibitory action.
(b) A549 (nonsmall-cell lung cancer) and Hela (cervical cancer) cell c-Myc in the presence of compound (I)
The expression of albumen
(1) tumor cell proteins extracts
1) A549 cell and Hela cell are cultivated, with 5 × 105Cell/ hole is inoculated in 6 orifice plates.The change of preparation variable concentrations
Compound (I), concentration is respectively 0 μM, 1 μM, 5 μMs, 10 μMs.After 6 orifice plate inner cells grow about 20h, put into variable concentrations compound,
With reference to adding equal-volume culture fluid；
2) after compound effects 24 hours, the tumor cell that different factor processes is collected；
3) adding 3mL PBS cell precipitation, 1000rpm is centrifuged 5min, abandoning supernatant；
4) add appropriate cell pyrolysis liquid, add the protease inhibitor of final concentration of 1 mM immediately, crack on ice
30min；
5) 4 DEG C of 12000 rpm is centrifuged 5min, collects supernatant (being total protein of cell), and BCA method measures protein concentration ,-
80 DEG C save backup.
(2) protein immunoblot (Western bloting)
1) according to BCA quantitative result, adjust each sample volume and make its loading Tot Prot consistent (10-100 μ g).
2) according to 4:1 ratio by protein sample and mixing, protein sample and 5 × albumen loading of protein content will have been surveyed
Buffer mixes with the ratio of 4:1,95 DEG C of metal bath heating 5min, makes the abundant degeneration of albumen.
3) in loading hole, it is carefully added into sample and Marker, switches on power, carry out electrophoresis.
4) transferring film.According to the Protein Marker added, confirm required size protein band position on gel, and
After take desirable proteins bar out of gel and cut.Follow negative electrode-support pad-filter paper-gel-NC film-filter paper-support pad-anode
Sequentially, make " sandwich " structure of electricity transferring film, confirm anode and cathode position, be placed in electrophoresis tank, add 1 × transferring film buffering
Liquid, under condition of ice bath, constant current mode 250 mA transferring film 1 h.
5) close.After transferring film, take out and turn the film having albumen, use in the TBST solution containing 5% defatted milk powder and shake
Bed jog 1 h.
6) combine one to resist.Closing terminates, and the film closed is dipped in TBST(Tris-HCl buffer salt solution and one is non-
The mixed liquor of ionic detergent (Tween)) solution slightly rinses, make and close space, add one and resist, be put in 4 ° of C refrigerators
Overnight.
7) combine two to resist.Take out and combine an anti-film, TBST solution rinses 5 min × 3 time.By two anti-1:10000
Dilution, take out film be dipped in two anti-in.1 h is combined under room temperature.TBST solution rinsing 5min × 3 time are reused after end.
8) pressing test kit explanation preparation nitrite ion ECL, A liquid and B liquid join in 1mL pure water in each 50 μ L of 1:1 ratio, mixed
Even.NC film is taken out, nitrite ion dropping is acted on about 2min under the surface of film, room temperature condition.Then use preservative film by film
Environmental sealing, puts in X-ray shadow box, uses adhesive tape to fix.
9) in dark place, take film and be quickly placed in magazine, expose 1min, 2min, 5min and 10 min respectively, operated
Journey is wanted rapidly.
10) development.Use 1 × film development liquid of new preparation, take out film in magazine, be dipped in developer solution, to appear
Substantially after band, proceed to clear water slightly rinses, be finally immersed in fixative solution fixing, again wash, dry up.
Test result such as Fig. 9 (3) (A549 cell) and (4) (Hela cell).Result explanation as shown in Figure 9: with matched group
Comparing, the protein expression level of the c-Myc gene of tumor cell presents reduction in various degree, presents dose-dependent pass
System, compound (I) has bigger impact to the protein expression of A549 cell c-Myc.Experimental result is basic with the result of RT-PCR
Identical.
These two description of tests above: the suppression of compound on tumor cell lowers c-myc gene from compound
MRNA and the expression of albumen.
Claims (7)
1. benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate, it is characterised in that selected from following three kinds of changes
Compound, structural formula is respectively as shown in formula I, formula II, formula III:
。
2. the system of benzo as claimed in claim 1 [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate
Preparation Method, it is characterised in that with benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride and 2-aminouridine be raw material, DMF be solvent, DCC
For condensing agent, react 24-26 hour at 35 DEG C under argon shield, after TLC monitoring reaction terminates, mistake after reactant liquor is cooled down
Filter N, the N-dicyclohexylurea precipitate of formation, then with acetone rinsing, collect filtrate the purest with preparing thin layer chromatography
Changing, corresponding product tape, using the mixed liquor of chloroform and methanol as developing solvent, is rinsed as eluant by thin layer chromatography with ethanol
Getting off, collect eluent, rotation is evaporated off ethanol and is placed at 30 DEG C vacuum drying and obtains the compound (I) of yellow powder.
3. the system of benzo as claimed in claim 1 [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate
Preparation Method, it is characterised in that first by benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride and 3-alanine react obtain benzothioxanthene-
3,4-dicarboximide-N-propanoic acid, then by benzothioxanthene-3,4-dicarboximide-N-propanoic acid and 2-aminouridine are in DMF
React 24-26 hour at 35 DEG C under argon shield using DCC as condensing agent, after TLC monitoring reaction terminates, reactant liquor is cold
It is filtered to remove the N of formation, N-dicyclohexylurea precipitate the most afterwards, with acetone rinsing, collects filtrate and enter one with preparing thin layer chromatography
Step purification, thin layer chromatography is using the mixed liquor of chloroform and methanol as developing solvent, with ethanol as eluant by corresponding product tape
Flushing is got off, and collects eluent, and rotation is evaporated off ethanol；It is placed at 30 DEG C vacuum drying and obtains the compound of yellow powder
(II).
4. the system of benzo as claimed in claim 1 [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate
Preparation Method, it is characterised in that by benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride reacts with 6-Aminocaproic Acid and obtains benzothioxanthene-3,
4-dicarboximide-N-is the sourest, then by benzothioxanthene-3,4-dicarboximide-N-acid and 2-aminouridine in DMF with
DCC reacts 24-26 hour under argon shield as condensing agent at 35 DEG C, after TLC monitoring reaction terminates, reactant liquor is cold
It is filtered to remove the N of formation, N-dicyclohexylurea precipitate the most afterwards, with acetone rinsing, collects filtrate and enter one with preparing thin layer chromatography
Step purification, thin layer chromatography is using the mixed liquor of chloroform and methanol as developing solvent, with ethanol as eluant by corresponding product tape
Flushing is got off, and collects eluent, and rotation is evaporated off ethanol；It is placed at 30 DEG C vacuum drying and obtains the compound of yellow powder
(III).
5. benzo as claimed in claim 1 [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate is in system
Application in standby antitumor drug.
6. benzo as claimed in claim 1 [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate is in system
Application in the standby medicine treating cervical cancer, Non-small cell lung carcinoma.
7. benzo [k, l] thioxanthene-3,4-dicarboxylic acid anhydride analog derivative-uridnine conjugate as claimed in claim 5 is anti-in preparation
Application in tumour medicine, it is characterised in that the antitumor drug with c-myc gene as target.
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| US4115555A (en) * | 1976-09-29 | 1978-09-19 | E. R. Squibb & Sons, Inc. | 2-Substituted-1H-benz[de]-isoquinoline-1,3(2H)-diones |
| CN101279967A (en) * | 2008-05-29 | 2008-10-08 | 武汉远大制药集团有限公司 | Medicinal composition of trimethyl xanthone-4-acetic acid for treating cancer and use thereof |
| EP1280764B1 (en) * | 2000-03-24 | 2010-11-24 | Methylgene, Inc. | Inhibitors of histone deacetylase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US4115555A (en) * | 1976-09-29 | 1978-09-19 | E. R. Squibb & Sons, Inc. | 2-Substituted-1H-benz[de]-isoquinoline-1,3(2H)-diones |
| EP1280764B1 (en) * | 2000-03-24 | 2010-11-24 | Methylgene, Inc. | Inhibitors of histone deacetylase |
| CN101279967A (en) * | 2008-05-29 | 2008-10-08 | 武汉远大制药集团有限公司 | Medicinal composition of trimethyl xanthone-4-acetic acid for treating cancer and use thereof |
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