CN110156792A - A kind of pyrimido benzazolyl compounds and its preparation method and application - Google Patents
A kind of pyrimido benzazolyl compounds and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to field of pharmaceutical chemistry technology, and in particular to a kind of pyrimido benzazolyl compounds and its preparation method and application.Application the invention mainly relates to the synthesis of the Benzazole compounds containing pyrimido and its in the field of medicine.Specifically, pyrimido Benzazole compounds of the present invention, the self-replacation of stem cell can be stimulated in vitro and without differentiation, there is application prospect hence for application of the extension stem cell in various therapy fields.
Description
Technical field
The present invention relates to field of pharmaceutical chemistry technology, and in particular to a kind of pyrimido benzazolyl compounds and preparation method thereof and
Using the pyrimido benzazolyl compounds carry out the purposes in amplification in vitro in stem cell, are especially expanded in vitro in liver cell
Purposes in increasing.
Background technique
Two important features of candidate stem cell are that have self-renewal capacity and be divided into the potential of other haemocytes, are pressed
Its source can be divided into marrow, peripheral blood and cord blood stem cell.Hematopoietic stem cell transplantation is that current treatment hematological system is pernicious swollen
Tumor such as acute leukemia, chronic myelocytic leukemia, lymthoma, Huppert's disease, myelodysplastic syndrome etc., it is certain
Hematological system non-malignant tumors, such as aplastic anaemia, the essential therapeutic arsenals of thalassemia.It should be emphasized that
It is that chronic myelocytic leukemia, myelodysplastic syndrome are all that the extremely caused hemopoietic system of candidate stem cell is pernicious swollen
Tumor, although there is many advanced drugs to treat these patients, Allogeneic Hematopoietic Stem Cell Transplantation from genetic level at present
It is still the only treatment means for curing these diseases.
The source of human stem cell of the hematopoietic stem cell transplantation of early stage is mainly marrow, and Bone Marrow Stem Cells Transplantation needs HLA antigen
The matching of body, many patients (especially ethnic group) can not carry out hematopoietic stem cell transplantation due to lacking the HLA to match.
Other than finding the HLA to match difficulty, graft-versus-host reaction (GVHD) is another for hindering Bone Marrow Stem Cells Transplantation
Major obstacle.GVHD has seriously affected success/death rate of marrow hemopoietic stem cells, and the quality of life after transplanting does not make us full yet
Meaning, finding alternative derived from hematopoietic precursor cells is a kind of important solution.
Cord blood is to remain in the blood in placenta and umbilical cord after delivery of baby, ligature of the cord and detachment, is contained in Cord blood
There is the candidate stem cell that can rebuild human body hematopoiesis and immune system.Compared with marrow, containing more than Adult Human Bone Marrow in Cord blood
(hematopoietic stem cells) HSCs and hematopoiesis stimulating factors abundant have Colony forming ability strong, self is more
New and multiplication potentiality is strong, the low feature of cytotoxicity.Umbilical cord blood collection method is easy, to donor and fetus without any injury, HLA
Distribution type be harmonious degree require it is low, be resistant to portion of tissue compatibility antigen difference, the generation of the anti-host disease of Umbilical Cord Blood Transplantation object is general
Rate is very low, immunocyte (T cell, B cell and NK cell) developmental immaturity in Cord blood, and the antigenicity of cell surface is very
Weak, the immune system of patient itself is very low to neoblast recognition capability, the immune row caused so as to avoid HSCs transplanting
Reprimand reaction and hypersensitization reaction.Therefore bleeding of the umbilicus has important prospect and application value as substitution marrow hemopoietic stem cells.
However the total number of nucleated cells and CD34 contained in single cord blood+Number is less, and transplants total karyocyte
Several and CD34+Cell number directly influences the transplanting success rate and death rate, thus single cord blood can only meet children or
The transplanting demand of the lighter people of the scale of construction limits its application in adult patient.Although double Cord blood has solved single part
The low problem of the total karyocyte number of Cord blood, but studies have found that hematopoiesis is carried out using double Cord blood or more cord bloods
Stem cell transplantation will increase the danger that Acute GVHD occurs, and transplanting expense can be made to dramatically increase.It is found in recent years in body
The method of outer effectively amplification umbilical cord blood hematopoietic stem cell becomes hot spot.
The main purpose of research umbilical cord blood hematopoietic stem cell amplification in vitro be find a kind of effective substance can be in vitro
It is induced to carry out self-replacation, postponement or the function of postponing its differentiation.Research in recent years finds the inside and outside factor of various kinds of cell
It can influence self-replacation and the differentiation function of stem cell.It the signal paths such as wherein Wnt, HOX, Notch, Shh, FGF and makes
The duplication of hemocytoblast is related to differentiation function, various kinds of cell element, such as SCF (stem cell factor), TPO
(thrombopoietin),Flt3L(Fms-like tyrosine kinase 3ligand),IL-3,6,11,GM-CSF
(granulocyte-macrophage colony stimulating factor) etc. can stimulate candidate stem cell external
Amplification.However, these Signal Regulation factor, transcription factor, cytokines etc. also result in the differentiation of candidate stem cell.
Therefore, find and find that there is potential potential applicability in clinical practice in the amplification in vitro of candidate stem cell and its transplanting
Noval chemical compound is still the difficult point studied at present and emphasis.
Summary of the invention
The present invention provides new pyrimidine diindyl compound and the like, including its isomers, prodrug, can pharmaceutically connect
The salt or solvate received, the application in terms of the amplification in vitro effect of stem cell (such as cord blood stem cell).
The present invention provides a kind of structural formula I compounds represented:
Wherein, R1Represent hydrogen atom, alkyl, naphthenic base or aromatic radical；R2Represent alkyl, naphthenic base, aromatic radical or benzyl；
R3Hydrogen atom, alkyl, naphthenic base or fragrant ring group are represented, 6 or 7 of pyrimido indole ring are substituted in；R4Represent hydrogen atom,
Alkyl, naphthenic base or aromatic radical；X represents N, S or O.
Further, the R1For hydrogen atom, C1-C6 alkyl, naphthenic base or aromatic radical；Preferably benzyl or substituted benzyl
Base.
Further, the R2For C1-C6 alkyl, naphthenic base, aromatic radical or with X collectively constitute heterocycle, piperidines or
Quinoline；Preferably 2- hydroxypropylamine base, 4- chloro-2-methyl aniline base, 6,7- dimethoxy -1,2,3,4- tetrahydroisoquinoline -2- second
Amido, tetrahydro pyrone -4- amino, 3,4- dimethoxy-phenylethylamine base or 4- hydroxy cyclohexylphenyl amido.
Further, the R3For hydrogen atom, C1-C6 alkyl, naphthenic base, aromatic radical or heterocycle；Preferably 2'- methyl
Tetrazole -5- base.
Further, the R3Replace selected from 6 of pyrimido indole ring or 7；Preferably 7.
Further, the R4For hydrogen atom, C1-C6 alkyl, naphthenic base or aromatic radical, preferably hydrogen atom.
Further, the compound are as follows:
2- benzyl -4- (4- trans-hydroxy Cyclohexylamino) -7- (2- methyl -5- tetrazole base) pyrimidine [4,5-b] and Yin
Diindyl；Or
2- benzyl -4- (3,4- dimethoxy benzene ethylamino) -7- (2- methyl -5- tetrazole base) pyrimidine [4,5-b] and Yin
Diindyl；Or
2- benzyl -4- (oxinane -4- amino) -7- (2- methyl -5- tetrazole base) pyrimidine [4,5-b] diindyl；Or
2- benzyl -4- (2- (6,7- dimethoxy -1,2,3,4- tetrahydroisoquinoline) ethylamino) -7- (four nitrogen of 2- methyl -5-
Azoles) pyrimidine [4,5-b] diindyl；Or
2- benzyl -4- (4- chloro-2-methyl aniline) -6- (2- methyl -5- tetrazole) pyrimidine [4,5-b] diindyl；Or
2- benzyl -4- (2- hydroxypropylamino) -6- (2- methyl -5- tetrazole) pyrimidine [4,5-b] diindyl.
In addition, the present invention also provides the preparation methods of the compound, comprising the following steps:
A back flow reaction under the catalysis of zinc bromide by m-nitro formonitrile HCN and sodium azide, synthesis tetrazole ring obtain intermediate
Body 5- (3- nitrobenzophenone) -2H- tetrazole；
B intermediate 5- (3- nitrobenzophenone) -2H- tetrazole obtains intermediate 2- first with iodomethane reaction under alkaline condition
Base -5- (3- nitrobenzophenone) -2H- tetrazole；
C 2- methyl -5- (3- nitrobenzophenone) -2H- tetrazole obtains azanol through hydrazine hydrate reduction, is not required to purify, directly with
Excess acetyl chloride obtains intermediate N acetyl group-N- hydroxy-n-(3- (2- methyl -2H- tetrazole base) aniline；
D N- acetyl group-N- hydroxy-n-(3- (2- methyl -2H- tetrazole base) aniline reacts at low temperature with the third two eyeballs,
Back flow reaction obtains intermediate 2-amino -6- (2- methyl -2H- tetrazole) -1H- indoles -3- formamide under alkaline condition again；
E 2- amino -6- (2- methyl -2H- tetrazole) -1H- indoles -3- formamide and phenyllacetyl chloride react to obtain centre
Body 6- (2- methyl -2H- tetrazole) -2- (2- phenylacetyl amido) -1H- indoles -3- formamide；
F 6- (2- methyl -2H- tetrazole) -2- (2- phenylacetyl amido) -1H- indoles -3- formamide is made in sodium hydroxide
Ring closure reaction is carried out under to prepare intermediate 2- benzyl -4- carbonyl -7- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine is simultaneously
[4,5-b] indoles；
G 2- benzyl -4- carbonyl -7- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and trichlorine
Oxygen phosphorus reaction obtains intermediate 2- benzyl -4- chloro- 7- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles；
H 2- benzyl -4- chloro- 7- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and trans- 4-
Hydroxy cyclohexylphenyl amine reacts to obtain product 2- benzyl -4- (4- trans-hydroxy Cyclohexylamino) -7- (2- methyl -5- tetrazole base) phonetic
Pyridine [4,5-b] diindyl；The chloro- 7- of 2- benzyl -4- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and
3,4- dimethoxy-phenylethylamine reacts to obtain product 2- benzyl -4- (3,4- dimethoxy benzene ethylamino) -7- (2- methyl -5- four
Nitrogen oxazolyl) pyrimidine [4,5-b] diindyl；The chloro- 7- of 2- benzyl -4- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-
B] indoles and oxinane 4- amine reacts to obtain 2- benzyl -4- (oxinane -4- amino) -7- (2- methyl -5- tetrazole base)
Pyrimidine [4,5-b] diindyl；The chloro- 7- of 2- benzyl -4- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles
It reacts to obtain product 2- benzyl -4- (2- (6,7- dimethoxy with 2- (6,7- dimethoxy -1,2,3,4- tetrahydroisoquinoline) ethamine
Base -1,2,3,4- tetrahydroisoquinoline) ethylamino) -7- (2- methyl -5- tetrazole) pyrimidine [4,5-b] diindyl.
Further, the preparation method for the pyrimidine diindyl compound that similar 6- replaces, comprising the following steps:
A back flow reaction under the catalysis of zinc bromide by p-nitrobenzonitfile and sodium azide, synthesis tetrazole ring obtain intermediate
Body 5- (4- nitrobenzophenone) -2H- tetrazole；
B intermediate 5- (4- nitrobenzophenone) -2H- tetrazole obtains intermediate 2- first with iodomethane reaction under alkaline condition
Base -5- (4- nitrobenzophenone) -2H- tetrazole；
C 2- methyl -5- (4- nitrobenzophenone) -2H- tetrazole obtains azanol through hydrazine hydrate reduction, is not required to purify, directly with
Excess acetyl chloride obtains intermediate N acetyl group-N- hydroxy-n-(4- (2- methyl -2H- tetrazole base) aniline；
D N- acetyl group-N- hydroxy-n-(4- (2- methyl -2H- tetrazole base) aniline reacts at low temperature with the third two eyeballs,
Back flow reaction obtains intermediate 2-amino -5- (2- methyl -2H- tetrazole) -1H- indoles -3- formamide under alkaline condition again；
E 2- amino -5- (2- methyl -2H- tetrazole) -1H- indoles -3- formamide and phenyllacetyl chloride react to obtain centre
Body 5- (2- methyl -2H- tetrazole) -2- (2- phenylacetyl amido) -1H- indoles -3- formamide；
F 5- (2- methyl -2H- tetrazole) -2- (2- phenylacetyl amido) -1H- indoles -3- formamide is made in sodium hydroxide
Ring closure reaction is carried out under to prepare intermediate 2- benzyl -4- carbonyl -6- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine is simultaneously
[4,5-b] indoles；
G 2- benzyl -4- carbonyl -6- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and trichlorine
Oxygen phosphorus reaction obtains intermediate 2- benzyl -4- chloro- 6- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles；
H 2- benzyl -4- chloro- 6- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and the chloro- 2- of 4-
Methylaniline reacts to obtain product 2- benzyl -4- (4- chloro-2-methyl aniline) -6- (2- methyl -5- tetrazole) pyrimidine [4,5-b]
Diindyl；The chloro- 6- of 2- benzyl -4- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and 2- hydroxypropylamine
Reaction obtains product 2- benzyl -4- (2- hydroxypropylamino) -6- (2- methyl -5- tetrazole) pyrimidine [4,5-b] diindyl.
The purposes in amplification in vitro is carried out in stem cell in addition, the present invention also provides the compounds.
The present invention is also provided comprising pharmaceutically acceptable carrier and the compound of the present invention or its isomers, prodrug, medicine
At least one pharmaceutical composition of acceptable salt or solvate on.
The compound of the present invention can be used for the amplification in vitro of stem cell and keep its stem cell attribute.
The compound of the present invention can be used alone, with other compounds combination of the invention or with it is one or more (excellent
Select one to two kind) other medicines combination.
Detailed description of the invention:
Fig. 1 is compound to CD34+, CD34+CD49f+ cell screening activity；
Fig. 2 is A011 amplifying cells streaming figure；
Fig. 3 is amplification activity of the YYQ16 to CD34+CD38- cell；
Fig. 4 is the composite diagram of the compounds of this invention, in which: a) NaN3, ZnBr2, H2O, refux；b)MeI,K2CO3,
MeCN,reflux；c)N2H4·H2O,Ni,THF；d)CH3COCl,NaHCO3,THF；e)CNCH2CN,Et3N,CHCl3,ice
cool；f)PhCH2COCl,Et3N,1,4-dioxane；g)NaOH,CH3CH2(OH)CH3,reflux；h)POCl3,reflux；
i)R-X,DMF,K2CO3,90℃。
Specific embodiment
The present invention is further described below by way of specific embodiment, the present invention is not limited only to following embodiment.In this hair
In bright range or the contents of the present invention are not being departed from, in spirit and scope, the change that carries out to the present invention is combined or replaced
It changes, will be apparent to the person skilled in the art, and be included within the scope of the present invention.
Embodiment 1. intermediate 5- (3- nitrobenzophenone) tetrazole (B1) synthesis
Take m-nitro formonitrile HCN 30.0g (202.69mmol), sodium azide 14.5g (222.96mmol), zinc bromide 45.7g
Water 500ml is added in 1000ml flask in (202.69mol), and 100 DEG C of back flow reactions are cooled to room temperature afterwards for 24 hours, reaction solution is fallen
Enter in 500ml water, 3N hydrochloric acid 100ml is added, extracted with ethyl acetate (3 × 200ml), collect organic phase, decompression is spin-dried for acetic acid
Ethyl ester obtains white solid, and the NaOH of 800ml 0.25mol/L, stirring is added, and white flock hydrogen is precipitated after first dissolving in solid again
Zinc oxide filters, and filtrate is adjusted to pH=5 with the hydrochloric acid of 3.0mol/L, and a large amount of white solids, dry white B1 product is precipitated
34.8g, yield 90%.1H NMR(DMSO-d6,ppm)δ8.85-8.86(t,1H),8.48-8.51(dt,1H),8.43-8.46
(dq,1H),7.91-7.95(t,1H)。
The synthesis of 2. intermediate 2- methyl -5- (3- nitrobenzophenone) tetrazole (B2) of embodiment
It takes 1 33.0g of intermediate B (172.64mmol) to be dissolved in 150ml DMF, Anhydrous potassium carbonate 28.63g is added
(207.17mmol), iodomethane 29.41g (207,17mmol), 90 DEG C of reaction 1h are cooled to room temperature, reaction solution once buff, will
Reaction solution is slowly dropped into 1L water, and a large amount of white solids are precipitated, and is filtered, and obtains white solid after dry.With ethyl acetate and petroleum
Ether recrystallizes to obtain B2 product 26g, yield 72.2%.1H NMR(DMSO-d6,ppm)δ8.66-8.67(t,1H),8.40-8.42
(dt,1H),8.33-8.35(dt,1H),7.81-7.85(t,1H),4.46(s,3H)。
The synthesis of 3. intermediate N hydroxy-n of embodiment-acetyl group -4- (2- methyl tetrazole -5- base) aniline (B4)
2 30g of intermediate B (146.22mmol) is taken, is dissolved in 350ml tetrahydrofuran at room temperature, NI powder 10.0g, ice is added
Bath is cooled to 0 DEG C, is slowly dropped into hydrazine hydrate 7.3g (143,49mmol) in batches, stirs 30min at 0 DEG C, be to slowly warm up to room temperature
The reaction was continued for 24 hours, and solution becomes light yellow, after TLC monitors raw material disappearance, is cooled to 0 DEG C, 4 equivalent sodium bicarbonate 54g are added,
There is gas generation, stir 30mim, instill chloroacetic chloride 13.77g (175.46mmol) at 0 DEG C, be to slowly warm up to room temperature reaction 2h,
It filters, filter cake washs (3 × 50ml) with tetrahydrofuran and washs, and collects filtrate, and decompression is spin-dried for tetrahydrofuran and obtains yellow solid.Solid
B4 product 28g, yield 84% are obtained with re-crystallizing in ethyl acetate.1H NMR(DMSO-d6,ppm)δ10.83(s,1H),8.43(s,
1H),7.82-7.84(d,2H),7.54-7.58(t,1H),4.45(s,3H),2.28(s,3H)。
The synthesis of 4. intermediate 2-amino -6- of embodiment (2- methyl tetrazole -5-) indoles -3- formamide (B5)
It takes 4 26.0g of intermediate B (111.48mmol) to be dissolved in 500ml chloroform, there is that fraction solids are insoluble, malononitrile is added
7.4g (111.48mmol) is cooled to 0 DEG C, stirs 30mim, is slowly dropped into triethylamine 11.28g (111.48mmol), drips
30min is stirred after finishing, and is to slowly warm up to room temperature reaction 1h, there are a large amount of solids to be precipitated, filters, filter cake (3 × 50ml) chloroform
Washing, filter cake is placed in 500ml flask, is added triethylamine 11.28g (111.48mmol), methanol 200ml, back flow reaction
10h, reaction solution are first clarified and a large amount of pale solids are precipitated afterwards, and filtrate is concentrated under reduced pressure, filters to obtain pale solid B5 product 16g,
Yield 58%.1H NMR(DMSO-d6,ppm)δ10.80(s,1H),7.82(s,1H),7.64-7.68(q,2H),7.03(s,
2H),6.60(s,2H),4.39(s,3H).
The synthesis of embodiment 5. intermediate 2- phenylacetylamino -6- (2- methyl tetrazole -5-) indoles -3- formamide (B6)
It takes B5 14.6g (56.34mmol) to be dissolved in 200mlDMF, is added triethylamine 17.1g (169.03mmol), benzene second
For 24 hours, reaction solution is slowly dropped into 500ml water for acyl chlorides 17.42g (112.69mmol), 37 DEG C of reactions, and it is solid that a large amount of yellow are precipitated
Body filters, dry yellow solid.Solid is placed in 500ml flask, ethyl acetate 200ml is added, flow back 6h, filters
Canescence B6 product 15.0g, yield 70.9%.1H NMR(DMSO-d6,ppm)δ12.23(s,1H),11.76(s,1H),8.29
(s,1H),7.96-8.00(m,1H),7.79-7.81(m,1H),7.26-7.40(m,7H),4.41(s,3H),3.91(s,2H).
Embodiment 6. intermediate 2- benzyl -7- (2- methyl tetrazole -5- base) -3H-4- oxygen-pyrimidine [4,5] diindyl
(B7) synthesis
It takes 6 16.3g of intermediate B (43.4mmol) to be dissolved in 500ml isopropanol, potassium tert-butoxide 29.2g is added
Reaction solution is concentrated under reduced pressure after disappearing for (260.4mmol), 90 DEG C of reflux 12h, raw material point, 300ml water is added, with the salt of 6mol/L
Acid solution adjusts pH=6, and suction filtration obtains pale solid.The solid is placed in 1L flask, 500ml methanol is added, flow back 8h,
It filters, 3 times repeatedly, filters to obtain white B7 product 11.6g, yield 74.8%.1H NMR(DMSO-d6,ppm)δ12.48(b,1H),
12.40(b,1H),8.07-8.20(m,3H),7.91-7.97(m,1H),7.26-7.41(m,4H),4.43(s,3H),4.04
(s,2H).
Embodiment 7. the chloro- 7- of intermediate 2- benzyl -4- (2- methyl tetrazole -5- base) pyrimidine [4,5] diindyl (B8)
Synthesis
Take 7 6.3g of intermediate B (16.76mmol) in phosphorus oxychloride 100ml in 250ml flask, is added, 110 DEG C are flowed back
Reaction solution is concentrated under reduced pressure into about 20ml, is slowly dropped into 500ml ice water by 12h, and yellow solid is precipitated in stirring, uses 10%NaOH
Solution adjusts pH to 8 or so, filters, is dried to obtain yellow B8 product 6.1g, yield 92%.1H NMR(DMSO-d6,ppm)δ
12.90(s,1H),8.32-8.35(d,1H),8.19(s,1H),8.04-8.07(dd,1H),7.22-7.38(m,5H),4.46
(s,3H),4.30(s,2H).
8. 2- benzyl -4- of embodiment (trans- -4- hydroxy cyclohexylphenyl amino) -7- (2- methyl tetrazole -5- base) pyrimidine [4,
5] synthesis of diindyl (A004)
8 0.4g of intermediate B (1.07mmol) is taken, is dissolved in 10ml DMSO, 0.3g (2.14mmol) Carbon Dioxide is added
Potassium, it is trans- that aminocyclohexanol 0.24g (2.14mmol), 90 DEG C of reaction 8h are after reaction instilled reaction solution in ice water,
Yellow solid is precipitated, filters, dry, yellow solid obtains A004 product 0.31g, yield 63% with recrystallizing methanol.Fusing point:
284.3 DEG C~287.0 DEG C；1H NMR(DMSO-d6, ppm) and δ 12.02 (s, 1H), 8.43-8.45 (d, J=8.2,1H), 8.10
(s, 1H), 7.89-7.91 (dd, J=8.2,1H), 7.40-7.41 (d, J=7.2,2H), 7.30 (m, 2H), 7.21 (m, 1H),
6.73-6.75 (m, 1H), 4.65-4.66 (d, J=4.4,1H), 4.44 (s, 3H), 4.22-4.29 (m, 1H), 4.07 (s, 2H),
1.92-1.94(m,4H),1.55-1.64(m,2H),1.28-1.37(m,2H),1.14-1.21(m,1H).13C NMR(DMSO-
d6,ppm)δ166.46,165.35,157.59,156.71,139.80,136.77,129.73(2C),128.46(2C),
126.45,122.80,122.16,121.80,118.37,109.04,93.97,69.08,49.38,46.22,46.13,35.00
(2C),30.59(2C)；LC/MS-m/z:455.2[M+1]+(exact mass:454.2)。
Embodiment 9. 2- benzyl -4- (3,4- dimethoxy benzene ethylamino) -7- (2- methyl tetrazole -5- base) pyrimidine [4,
5] synthesis of diindyl (A006)
8 0.4g of intermediate B (1.07mmol) is taken, is dissolved in 10mlDMSO, 0.3g (2.14mmol) Carbon Dioxide is added
Reaction solution is instilled ice water after reaction by potassium, 3,4- dimethoxy-phenylethylamine 0.39g (2.14mmol), 90 DEG C of reaction 8h
In, yellow solid is precipitated, filters, dry, yellow solid obtains A006 product 0.24g, yield 42% with re-crystallizing in ethyl acetate.It is molten
Point: 252.4 DEG C~253.2 DEG C；1H NMR(DMSO-d6, ppm) and δ 12.03 (s, 1H), 8.37-8.40 (d, J=8.2,1H),
8.09 (s, 1H), 7.89-7.92 (d, J=8.2,1H), 7.38-7.42 (m, 3H), 7.27-7.31 (m, 2H), 7.19 (m, 1H),
6.84-6.88(m,2H),6.77-6.78(m,1H),4.44(s,3H),4.08(s,2H),3.77-3.82(m,2H),3.69-
3.71(6H,2OMe),2.88-2.91(t,2H).13C NMR(DMSO-d6,ppm)δ166.61,165.34,157.50,
157.08,149.08,147.68,139.90,136.82,132.70,129.58(2C),128.58(2C),126.49,
122.84,121.74,121.01,118.51,112.98,112.44,109.13,94.01,55.99,55.74,46.32,
42.70,35.41；LC/MS-m/z:521.1[M+1]+(exact mass:520.2)。
Pyrimidine [4,5] is simultaneously by embodiment 10. 2- benzyl -4- (oxinane -4- amino) -7- (2- methyl tetrazole -5- base)
The synthesis of indoles (A009)
8 0.4g of intermediate B (1.07mmol) is taken, is dissolved in 10mlDMSO, 0.3g (2.14mmol) Carbon Dioxide is added
Potassium, 4- amino tetrahydro pyran 0.22g (2.14mmol), 90 DEG C of reaction 8h after reaction instill reaction solution in ice water, analysis
Yellow solid out filters, and dry, yellow solid ethyl acetate, recrystallizing methanol obtain A009 product 0.2g, yield 41%.Fusing point:
279.5 DEG C~281.2 DEG C；1H NMR(DMSO-d6, ppm) and δ 12.04 (s, 1H), 8.45-8.47 (d, J=8.2,1H), 8.08
(s, 1H), 7.89-7.91 (dd, J=8.2,1H), 7.37-7.39 (d, J=7.7,2H), 7.27-7.30 (t, J=7.5,2H),
7.18-7.21 (t, J=7.3,1H), 6.88-6.89 (d, J=7.7,1H), 4.46-4.49 (m, 1H), 4.44 (s, 3H), 4.06
(s,2H),3.93-3.95(m,2H),3.41-3.46(m,2H),1.75-1.88(m,4H).13C NMR(DMSO-d6,ppm)δ
166.40,165.34,157.64,156.52,139.76,136.84,129.74(2C),128.49(2C),126.44,
122.92,122.29,121.75,118.39,109.07,94.05,67.01(2C),47.41,46.08,32.87(2C)；LC/
MS-m/z:441.2[M+1]+(exact mass:440.2)。
11. 2- benzyl -4- of embodiment (6,7- dimethoxy -1,2,3,4- tetrahydroisoquinoline -2- ethylamino) -7- (2- first
Base tetrazole -5- base) pyrimidine [4,5] diindyl (A011) synthesis
8 0.4g of intermediate B (1.07mmol) is taken, is dissolved in 10ml DMSO, 0.3g (2.14mmol) Carbon Dioxide is added
Potassium, 2- (6,7- methoxyl group -3,4- dihydro-isoquinoline)-ethylenediamine 0.5g (2.14mmol), 90 DEG C of reaction 8h, after reaction,
Reaction solution is instilled in ice water, faint yellow solid is precipitated, is filtered, dry, faint yellow solid re-crystallizing in ethyl acetate obtains A011 production
Object 0.18g, yield 29%.Fusing point: 240.7 DEG C~243.6 DEG C；1H NMR(DMSO-d6,ppm)δ12.03(s,1H),8.35-
8.37 (d, J=8.2,1H), 8.08 (s, 1H), 7.88-7.90 (d, J=8.2,1H), 7.39-7.41 (d, J=7.2,2H),
7.27-7.31 (d, J=7.4,3H), 7.18-7.22 (t, J=7.3,1H), 6.63-6.66 (m, 2H), 4.43 (s, 3H), 4.08
(s,2H),3.80-3.85(m,2H),3.69-3.70(6H,2OMe),3.58(s,2H),2.71-2.76(m,6H)；13C NMR
(DMSO-d6,ppm)δ166.61,165.33,157.48,157.20,147.60,147.34,139.86,136.83,129.62
(2C),128.57(2C),127.11,126.47,126.39,122.82,121.84,121.75,118.51,112.26,
110.41,109.14,93.97,57.27,55.95,55.91,55.72,51.12,46.29,38.33,28.70；LC/MS-m/
z:576.4[M+1]+(exact mass:575.3)。
The synthesis of 12. 5- of embodiment (4- nitrobenzophenone) tetrazole (Q1)
It takes p-nitrobenzonitfile 10.0g (67.52mmol), sodium azide 4.8g (74.27mmol), zinc bromide 15.2g
Water 150ml is added in the round-bottomed flask of 250ml in (67.52mmol), and 100 DEG C are down to room temperature after back flow reaction 24 hours, will be anti-
It answers liquid to pour into the beaker of 500ml, the hydrochloric acid 100ml of 3N is added, extracted with ethyl acetate (3x100ml), collect ethyl acetate
Phase, reduced pressure remove ethyl acetate, obtain white solid, white solid is dissolved in the NaOH solution of 675ml 0.25mol/L,
White flock zinc hydroxide is precipitated in stirring 30 minutes again after solid is entirely molten, and suction filtration removes zinc hydroxide, and filtrate is used
A large amount of white solids are precipitated in the hydrochloric acid solution tune pH=5 of 3.0mol/L, filter dry that intermediate Q1 product 11.6g, yield are
90%, fusing point: 206 DEG C.1H NMR (DMSO, ppm): δ 8.43 (t, 1H), 8.41 (t, 1H), 8.29 (t, 1H), 8.27 (t,
1H)。
The synthesis of 13. 2- methyl -5- (4- nitrobenzophenone) tetrazole (Q2) of embodiment
It takes intermediate Q1 5.0g (26.16mmol) to be dissolved in 50ml anhydrous DMF, Anhydrous potassium carbonate 4.3g is added at room temperature
(31.39mmol), iodomethane 4.5g (31.39mmol), 90 DEG C are cooled to room temperature after reaction 1 hour, instill reaction solution is slow
In the water of 300ml, a large amount of white solids are precipitated, filter, dry white solid, by white solid ethyl acetate and petroleum ether
Recrystallize to obtain portion of product 1.8g, by remaining impure product through column chromatography for separation, mobile phase be (ethyl acetate: petroleum ether=
1:3), total Q2 product 2.5g, yield 46%, fusing point: 165 DEG C are obtained.1H NMR (DMSO, ppm): δ 8.36 (q, 4H), 4.47
(s,3H)。
14. 4- of embodiment (2- methyl tetrazole -5- base)-N- hydroxy-n-acetyl group-aniline (Q4) synthesis
Intermediate Q2 15.0g (73.11mmol) is taken to be dissolved in the tetrahydrofuran of 300ml at room temperature, solid is substantially all molten
Solution is added nickel powder 5.0g, is cooled to 0 DEG C, delays the hydrazine hydrate 9.0ml (157.41mmol) of instillation 85%, stirs 30 points at 0 DEG C
Clock, being to slowly warm up to room temperature, the reaction was continued 24 hours, after TLC detection raw material completely disappears, is cooled to 0 DEG C, sodium bicarbonate is added
25.5g (0.30mol) has gas generation, delays at stirring 30 minutes, 0 DEG C and instill chloroacetic chloride 5.2ml (73.11mmol), slowly rise
Reaction 2 hours is warmed to room temperature, is filtered, filter cake is washed with tetrahydrofuran (3x50ml), merging filtrate, and tetrahydro furan is removed in reduced pressure
Mutter to obtain yellow solid, solid ethyl acetate and petroleum ether recrystallize to obtain product 6.3g, remaining not pure products through column chromatography for separation,
Mobile phase is (ethyl acetate: petroleum ether=1:1.5 → 1:1 → ethyl acetate), there are Q4 product 15.3g, and yield 84% melts
Point: 141 DEG C.1H NMR (DMSO, ppm): δ 10.82 (s, 1H), 8.05 (d, 2H), 7.87 (d, 2H), 4.42 (s, 3H), 2.27
(s,3H)。
The synthesis of embodiment 15. 2- amino -5- (2- methyl tetrazole -5-) indoles -3- formamide (Q5)
Intermediate Q4 10.2g (43.65mmol) is taken to be dissolved in the chloroform of 300ml, fraction solids are insoluble, are added the third two
Eyeball 2.9g (43.65mmol) is cooled to 0 DEG C, delays after stirring 30 minutes and instill triethylamine 4.4g (43.65mmol), be added dropwise
After continue stirring 30 minutes, be to slowly warm up to room temperature reaction 1 hour, a large amount of solids be precipitated, filter, filter cake washed with 50ml chloroform
It washs, in 250ml flask, will be added triethylamine 4.4g (43.65mmol), methanol 200ml after filtration cakes torrefaction, back flow reaction 10 is small
When, a large amount of solids are precipitated after first becoming clarification in reaction solution, filter to obtain pale solid 8.1g, are precipitated again after concentrating the filtrate to 50ml
White solid filters to obtain white solid, and merging solid Q5 twice is 9.3g, yield 78%.1H NMR(DMSO,ppm):δ
10.85 (s, 1H), 8.15 (s, 1H), 7.59-7.61 (d, 1H), 7.25-7.27 (d, 1H), 6.89 (s, 2H), 5.58 (s, 2H),
4.41(s,3H)。
The synthesis of embodiment 16. 2- phenylacetylamino -5- (2- methyl tetrazole -5-) indoles -3- formamide (Q6)
It takes intermediate Q5 4.3g (16.59mmol) to be dissolved in 240ml Isosorbide-5-Nitrae-dioxane, triethylamine 5.0g is added
(49.77mmol), phenyllacetyl chloride 4.4ml (33.18mmol), 37 DEG C react 24 hours, reaction solution is concentrated into it is thick, then
It pours into 400ml water, a large amount of yellow solids is precipitated, filter, it is dry, yellow solid 6.3g is obtained, by the solid in 250ml flask
In, ethyl acetate 150ml is added, flows back 4 hours, filters to obtain white solid Q6 product 5.3g, yield 82%.1H NMR
(DMSO, ppm): δ 12.19 (s, 1H), 11.57 (s, 1H), 8.45 (s, 1H), 7.78 (d, 1H), 7.57 (d, 1H), 7.40 (m,
4H), 7.28 (m, 3H), 4.42 (s, 3H), 3.91 (s, 3H).
17. 2- benzyl -6- of embodiment (2- methyl tetrazole -5- base) -3H-4- oxygen-pyrimidine [4,5] diindyl (Q7)
Synthesis
It takes intermediate Q6 5.0g (13.31mmol) to be dissolved in the isopropanol of 125ml, the sodium hydroxide of 6.25mol/L is added
Solution 15.7ml (97.83mmol), 90 DEG C are flowed back 8 hours, and solution is become cloudy by clarifying, and reaction solution is concentrated by raw material after disappearing
It is thick, water 300ml is added, with the hydrochloric acid solution tune pH=6 of 6.0mol/L, filters to obtain pale solid, dry solid
Methanol 200ml is added by the solid in 250ml flask in 4.78g, flows back 6 hours, filters to obtain white solid Q7 product 1.6g,
Yield is 35%.1H NMR (DMSO, ppm): δ 12.4-12.5 (d, 2H), 8.58 (s, 1H), 8.01 (s, 1H), 7.25-7.57
(m, 6H), 4.43 (s, 3H), 4.04 (s, 2H).
Embodiment 18. the chloro- 6- of intermediate 2- benzyl -4- (2- methyl tetrazole -5- base) pyrimidine [4,5] diindyl (Q8)
Synthesis
It takes intermediate Q7 1.5g (4.20mmol) in 100ml flask, phosphorus oxychloride 60ml, 110 DEG C of back flow reactions is added
12 hours, reaction solution is concentrated into 10ml, then by the slow ice water for instilling 200ml of concentrate, yellow solid is precipitated, filters,
Vacuum drying 6 hours is cooled to room temperature by crude product with after ethyl acetate backflow 3 hours of 50ml and filters to obtain pale solid
Q8 product 1.4g, yield 89%.1H NMR (DMSO, ppm): δ 12.83 (s, 1H), 8.74 (s, 1H), 8.18 (m, 1H),
7.63 (d, 1H), 7.30-7.34 (m, 4H), 7.21 (m, 1H), 4.41 (s, 3H), 4.26 (s, 2H).
19. 2- benzyl -4- of embodiment (2- methyl -4- chloroanilino) -6- (2- methyl tetrazole -5- base) pyrimidine [4,5]
The synthesis of diindyl (YYQ16)
It takes intermediate Q8 0.4g (1.07mmol) to be dissolved in the anhydrous DMF of 6.0ml, it is anhydrous that 0.3g (2.14mmol) is added
Potassium carbonate, 4- chloro-2-methyl aniline 0.30g (2.14mmol), 90 DEG C are reacted 8 hours, after reaction, by the slow instillation of reaction solution
In ice water, solid is precipitated, filters, dry, crude product re-crystallizing in ethyl acetate obtains YYQ16 product 0.27g, yield 52%.1H
NMR (DMSO, ppm): δ 12.26 (s, 1H), 9.13 (s, 1H), 8.81 (s, 1H), 8.07 (d, 1H), 7.59 (d, 1H), 7.37-
7.41 (m, 2H), 7.25-7.29 (m, 5H), 7.19 (m, 1H), 4.43 (s, 3H), 3.97 (s, 2H), 2,12 (s, 3H)；13C NMR
(DMSO, ppm): δ 166.1,165.7,158.0,156.0,139.5,138.4,137.63,137.56,130.3,130.0,
129.6,128.9,128.5,126.5,126.3,123.7,120.7,120.2,119.7,112.0,95.2,45.8,18.5.
Embodiment 20. 2- benzyl -4- (2- hydroxypropylamino) -6- (2- methyl tetrazole -5- base) pyrimidine [4,5] and Yin
The synthesis of diindyl (YYQ25)
It takes intermediate Q8 0.6g (1.60mmol) to be dissolved in the anhydrous DMF of 6.0ml, it is anhydrous that 0.44g (3.20mmol) is added
Potassium carbonate, 1- amino -2- propyl alcohol 0.24g (3.2mmol), 90 DEG C are reacted 8 hours, after reaction, by the slow instillation ice of reaction solution
In water, solid is precipitated, filters, dry, crude product re-crystallizing in ethyl acetate obtains YYQ25 product 0.35g, yield 53.0%.1H
NMR (DMSO, ppm): δ 12.07 (s, 1H), 8.96 (s.1H), 8.03 (d, 1H), 7.55 (d, 1H), 7.45 (t, 1H), 7.38
(d, 2H), 7.29 (t, 2H), 7.19 (t, 1H), 4.98 (d, 1H), 4.45 (s, 3H), 4.04 (s, 2H), 3.98 (m, 1H), 3.69
(m, 1H), 3.47 (m, 1H), 1.11 (d, 3H)；13C NMR (DMSO, ppm): δ 166.2,165.8,157.33,157.27,
139.8,138.0,129.6,128.6,126.5,123.2,120.6,119.57,119.51,111.9,94.1,65.9,48.5,
46.2 21.8.
Test example one, compound screen activity to cord blood stem cell amplification in vitro
1.1, bleeding of the umbilicus CD34+Cell magnetic bead sorting
It being provided according to ethics, multipara agrees to after obtaining Cord blood, most of red blood cell is settled using hydroxyethyl starch,
The remaining red blood cell of 2 cracking of ACK erythrocyte cracked liquid point is reused, Cord blood karyocyte is obtained.Exempt from after washing with CD34
Epidemic disease magnetic bead is incubated for 30min, is during which softly vortexed and avoids cell agglomerating.After incubation, washing cell washes away unbonded magnetic bead,
Using by the magnetic pole with magnetic field, then negative ingredient is because of the not suction of magnetic force after 30 μm of primary cell strainer filtering cell masses
Subsidiary liquid stream enters negative at being in charge of, and has CD34+The cell of surface antigen is because of the magnetic bead with antibody and cellular antigens
In conjunction with being stayed in magnetic pole by magnetic-adsorption.After whole cell suspensions cross column, with more flushing columns of sterile PBE, delay is washed away
Negative cells only leave positive cell.Magnetic pole is removed from magnetic field later, appropriate culture medium is added, the rubber plug being equipped with magnetic pole
The positive cell of delay is got, bleeding of the umbilicus CD34 is obtained+Cell is counted, is identified, the next-step operations such as bed board.
1.2, bleeding of the umbilicus CD34+Cell culture
The CD34 obtained after magnetic bead sorting+Cell, through counting after with hematopoietic stem cell expansion special culture media (IMDM+
9500 candidate stem cell serum substitute+100ng/mL SCF+100ng/mL Flt3L+50ng/mL TPO+1% of 10%BIT
It is dual anti-) mixing, make cell density are as follows: 1 × 105/mL, the drug after the dilution for designing concentration is added are spread after mixing into suspension
Dedicated 24 orifice plate of cell, every 3 multiple holes of concentration, is put into 37 DEG C, CO2Cultivated in constant incubator, the next day half amount change liquid to keep
The best vegetative state of cell collects cell after 7 days, carry out surface antibody dyeing, uses CD34 in this experiment+CD49f+As
HSC index, the ratio of flow cytometer analysis cell colony after dyeing.
1.3, compound screens activity to cord blood stem cell amplification in vitro
The CD34 sub-elected+Cell mixes (9500 hematopoiesis of IMDM+10%BIT with HSC amplification culture medium after counting
Stem cell serum substitute+100ng/mL SCF+100ng/mL Flt3L+50ng/mL TPO+1% is dual anti-).By having for drug
Effect concentration and toxicity are divided into 3 primary dcreening operation gradients 100nM, 500nM, 1000nM.Drug concentration is adjusted, by working solution and cell suspension
Mixing is to obtain the agent-cell suspension of aimed concn.At 37 DEG C, CO2It is thin using streaming after being cultivated 7 days in constant incubator
Born of the same parents' art analyzes living cells, CD34+、CD34+CD49f+The ratio of cell.It is as shown in Figure 1 to obtain result.
1.4, test result:
Fig. 1 is compound to CD34+, CD34+CD49f+ cell screening activity.Wherein: 01-A011 pairs of compound A-40 of (A)
Stem cell CD 34+, the amplification in vitro of CD34+CD49f+ cell activity.The selection result selects A004, A006, A009, and A011 is to live
Property compound.(B) toxicity screening of the active medicine to cell.Compound A-40 06 is significant to cytotoxicity as the result is shown, compound
A011 shows toxicity in 1000nM, and other compounds are in 1000nM without display toxicity.(C) drug A004, A009, A011 are most
Good Valid concentration screening.The optimum effective concentration of compound A-40 04 and A009 are in 400nM or more as the result is shown, and compound
The optimum effective concentration of A011 is in 750nM or less.
Test example two, 11 amplifying cells streaming figure of compound A-40
2.1, using the A011 of effective concentration according to above-mentioned cell culture processes, at 37 DEG C, 5%CO2Under the conditions of culture point
Cord blood CD 34+cell of choosing collects cell after 7 days, uses the expression feelings of flow cytomery cell CD34, CD49f
Condition, uses Flowjo vx.0.7 software data processing later, and FSC/SSC circle door irises out the complete living cells part of form, Zhi Hougen
CD34 is irised out according to negative control+And CD34+CD49f+Antigen presentation situation.Test result is as shown in Figure 2.
2.2, test result:
Fig. 2 is A011 amplifying cells streaming figure.Wherein: the DMSO that joined same amount is control group, and A011 group is A011
To the antigen presentation situation of CD34+ and CD34+CD49f+.A011 group is to complete to cell in 100nM (test concentrations) as the result is shown
Completely without toxicity, 49.2% is increased to from the 17.6% of control group to the expanding effect of CD34+, it is bis- to CD34+ and CD34+CD49f+
The expanding effect of index is increased to 27.1% from the 5.83% of control group.
Test example three, compound YYQ16 are to cord blood CD 34+CD38-Cell expansion ex vivo activity
3.1, the bleeding of the umbilicus CD34 of immunological magnetic bead sorting+Cell, according to the cultural method of above-mentioned offer, with amplification culture medium and
Cell concentration is adjusted after combination of cytokines mixing, and after adding drug to be measured according to gradient, is with 100,000 cells/wells
Standard is spread in 96 orifice plates, at 37 DEG C, 5%CO2Under the conditions of cultivate 7 days, the next day change liquid.After cultivation cycle, every hole is added
10uL CCK-8 (comes from colleague, Japan), is incubated for 4h in the incubator, then puts plate and mix gently on the oscillator 3 minutes,
To ensure that color is uniformly distributed.Finally, using the absorbance at microplate reader measurement 450nm, absorbance height embodies viable count
Mesh.Data are analyzed using GraphPad Prism v 7.0.
3.2, test result:
Test result is as shown in Figure 3.Fig. 3 is amplification activity of the YYQ16 to CD34+CD38- cell.(A) YYQ16 is to cell
Toxicity detection.YYQ16 has no obvious cytotoxicity within 100nM as the result is shown.(B) stream data of YYQ6.As a result it shows
Showing and is added after YYQ16 drug, target cell numbers increase, and 50nM/L dosage group is best, and amplification rate is 3.15 times of control group,
The result of 100nM or more can be understood as cytotoxicity and cause the cell colony death of non-stem cell that stem cell is caused to be able to richness
Collection expands optium concentration in 25-100nM.The ratio of CD34+CD38- cell is normalized in DMSO group, is obtained at 50nM,
YYQ16 is best to preventing candidate stem cell differentiation from having the effect of.
Test example four, compound are to cord blood stem cell amplification in vitro activity
4.1, using cell amplification cultivation method ibid, the bleeding of the umbilicus CD34 of these drugs amplification immunological magnetic bead sorting is added+Cell, at 37 DEG C, 5%CO2Under the conditions of cultivate 7 days, the next day change liquid, collect cell, Flow cytometry CD34+CD38-'s
Ratio, and the data compared with being normalized with DMSO control group use the recurrence of GraphPad Prism v7.0 software bent
Wire module analyzes the EC of drug50。
4.2, test result:
Test result is as shown in table 1.
1. drug of table is to CD34+CD38-Cell expansion ex vivo efficiency, optium concentration and EC50Value
Wherein: ND:not determined is not measured.
Claims (10)
1. a kind of structural formula I compound represented:
Wherein, R1Represent hydrogen atom, alkyl, naphthenic base or aromatic radical；R2Represent alkyl, naphthenic base, aromatic radical or benzyl；R3Generation
Table hydrogen atom, alkyl, naphthenic base or fragrant ring group, are substituted in 6 or 7 of pyrimido indole ring；R4Represent hydrogen atom, alkane
Base, naphthenic base or aromatic radical；X represents N, S or O.
2. compound according to claim 1, which is characterized in that the R1For hydrogen atom, C1-C6 alkyl, naphthenic base or virtue
Perfume base；Preferably benzyl or substituted benzyl.
3. compound according to claim 1, which is characterized in that the R2For C1-C6 alkyl, naphthenic base, aromatic radical or and
X collectively constitutes heterocycle, piperidines or morpholine；Preferably 2- hydroxypropylamine base, 4- chloro-2-methyl aniline base, 6,7- dimethoxy-
1,2,3,4- tetrahydroisoquinoline -2- ethylamino-, tetrahydro pyrone -4- amino, 3,4- dimethoxy-phenylethylamine base or 4- hydroxyl ring
Hexylamine base.
4. compound according to claim 1, which is characterized in that the R3For hydrogen atom, C1-C6 alkyl, naphthenic base, virtue
Perfume base or heterocycle；Preferably 2'- methyl tetrazole -5- base.
5. compound according to claim 1, which is characterized in that the R3Replace selected from 6 of pyrimido indole ring or
7.
6. compound according to claim 1, which is characterized in that the R4For hydrogen atom, C1-C6 alkyl, naphthenic base or virtue
Perfume base.
7. described in any item compounds according to claim 1~6, which is characterized in that the compound are as follows:
2- benzyl -4- (4- trans-hydroxy Cyclohexylamino) -7- (2- methyl -5- tetrazole base) pyrimidine [4,5-b] diindyl；Or
2- benzyl -4- (3,4- dimethoxy benzene ethylamino) -7- (2- methyl -5- tetrazole base) pyrimidine [4,5-b] diindyl；Or
2- benzyl -4- (oxinane -4- amino) -7- (2- methyl -5- tetrazole base) pyrimidine [4,5-b] diindyl；Or
2- benzyl -4- (2- (6,7- dimethoxy -1,2,3,4- tetrahydroisoquinoline) ethylamino) -7- (2- methyl -5- tetrazole)
Pyrimidine [4,5-b] diindyl；Or
2- benzyl -4- (4- chloro-2-methyl aniline) -6- (2- methyl -5- tetrazole) pyrimidine [4,5-b] diindyl；Or
2- benzyl -4- (2- hydroxypropylamino) -6- (2- methyl -5- tetrazole) pyrimidine [4,5-b] diindyl.
8. the preparation method of compound according to claim 7, which comprises the following steps:
A back flow reaction under the catalysis of zinc bromide by m-nitro formonitrile HCN and sodium azide, synthesis tetrazole ring obtain intermediate 5-
(3- nitrobenzophenone) -2H- tetrazole；
B intermediate 5- (3- nitrobenzophenone) -2H- tetrazole obtains intermediate 2- methyl-with iodomethane reaction under alkaline condition
5- (3- nitrobenzophenone) -2H- tetrazole；
C 2- methyl -5- (3- nitrobenzophenone) -2H- tetrazole obtains azanol through hydrazine hydrate reduction, is not required to purify, directly and acetyl
Chlorine reacts to obtain intermediate N acetyl group-N- hydroxy-n-(3- (2- methyl -2H- tetrazole base) aniline；
D N- acetyl group-N- hydroxy-n-(3- (2- methyl -2H- tetrazole base) aniline reacts at low temperature with the third two eyeballs, then
Back flow reaction obtains intermediate 2-amino -6- (2- methyl -2H- tetrazole) -1H- indoles -3- formamide under alkaline condition；
E 2- amino -6- (2- methyl -2H- tetrazole) -1H- indoles -3- formamide reacts to obtain intermediate 6- with phenyllacetyl chloride
(2- methyl -2H- tetrazole) -2- (2- phenylacetyl amido) -1H- indoles -3- formamide；
F 6- (2- methyl -2H- tetrazole) -2- (2- phenylacetyl amido) -1H- indoles -3- formamide is under sodium hydroxide effect
It carries out ring closure reaction and prepares intermediate 2- benzyl -4- carbonyl -7- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-
B] indoles；
G 2- benzyl -4- carbonyl -7- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and phosphorus oxychloride
Reaction obtains intermediate 2- benzyl -4- chloro- 7- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles；
H 2- benzyl -4- chloro- 7- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and trans- 4- hydroxyl
Cyclohexylamine react to obtain product 2- benzyl -4- (4- trans-hydroxy Cyclohexylamino) -7- (2- methyl -5- tetrazole base) pyrimidine [4,
5-b] diindyl；The chloro- 7- of 2- benzyl -4- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and 3,4- bis-
Methoxyphenethylamine reacts to obtain product 2- benzyl -4- (3,4- dimethoxy benzene ethylamino) -7- (2- methyl -5- tetrazole base)
Pyrimidine [4,5-b] diindyl；The chloro- 7- of 2- benzyl -4- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles
With oxinane 4- amine react to obtain 2- benzyl -4- (oxinane -4- amino) -7- (2- methyl -5- tetrazole base) pyrimidine [4,
5-b] diindyl；The chloro- 7- of 2- benzyl -4- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and 2- (6,
7- dimethoxy -1,2,3,4- tetrahydroisoquinoline) ethamine react to obtain product 2- benzyl -4- (2- (dimethoxy -1,2 6,7-,
3,4- tetrahydroisoquinoline) ethylamino) -7- (2- methyl -5- tetrazole) pyrimidine [4,5-b] diindyl.
9. the preparation method of compound according to claim 7, which comprises the following steps:
A back flow reaction under the catalysis of zinc bromide by p-nitrobenzonitfile and sodium azide, synthesis tetrazole ring obtain intermediate 5-
(4- nitrobenzophenone) -2H- tetrazole；
B intermediate 5- (4- nitrobenzophenone) -2H- tetrazole obtains intermediate 2- methyl-with iodomethane reaction under alkaline condition
5- (4- nitrobenzophenone) -2H- tetrazole；
C 2- methyl -5- (4- nitrobenzophenone) -2H- tetrazole obtains azanol through hydrazine hydrate reduction, is not required to purify, directly and acetyl
Chlorine reacts to obtain intermediate N acetyl group-N- hydroxy-n-(4- (2- methyl -2H- tetrazole base) aniline；
D N- acetyl group-N- hydroxy-n-(4- (2- methyl -2H- tetrazole base) aniline reacts at low temperature with the third two eyeballs, then
Back flow reaction obtains intermediate 2-amino -5- (2- methyl -2H- tetrazole) -1H- indoles -3- formamide under alkaline condition；
E 2- amino -5- (2- methyl -2H- tetrazole) -1H- indoles -3- formamide reacts to obtain intermediate 5- with phenyllacetyl chloride
(2- methyl -2H- tetrazole) -2- (2- phenylacetyl amido) -1H- indoles -3- formamide；
F 5- (2- methyl -2H- tetrazole) -2- (2- phenylacetyl amido) -1H- indoles -3- formamide is under sodium hydroxide effect
It carries out ring closure reaction and prepares intermediate 2- benzyl -4- carbonyl -6- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-
B] indoles；
G 2- benzyl -4- carbonyl -6- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and phosphorus oxychloride
Reaction obtains intermediate 2- benzyl -4- chloro- 6- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles；
H 2- benzyl -4- chloro- 6- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine simultaneously [4,5-b] indoles and 4- chloro-2-methyl
Aniline reaction obtains product 2- benzyl -4- (4- chloro-2-methyl aniline) -6- (2- methyl -5- tetrazole) pyrimidine [4,5-b] and Yin
Diindyl；Simultaneously [4,5-b] indoles and the reaction of 2- hydroxypropylamine of the chloro- 6- of 2- benzyl -4- (2- methyl-tetrazole base) -3,9- dilzydro-pyrimidine
Obtain product 2- benzyl -4- (2- hydroxypropylamino) -6- (2- methyl -5- tetrazole) pyrimidine [4,5-b] diindyl.
10. described in any item compounds carry out the purposes in amplification in vitro in stem cell according to claim 1~7.
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