CN103254195B - 7-amino-tetrazole [1,5-a] pyrimidine-5-alcohol (compound II) and synthetic route thereof - Google Patents

Abstract

The invention discloses new purine analogue 7 aminotetrazole [1,5 a] pyrimidine 5 alcohol (compound II) and synthetic route thereof.Compound II is inhibited to cell division cycle phospho-esterase c DC25 through pharmacological evaluation.Described compound II can, selectivity good, toxicity little antimetabolite high as biological activity, and be widely used in clinic.

Description

7-amino-tetrazole [1,5-a] pyrimidine-5-alcohol (compound II) and synthesis thereof Route

Technical field

The invention belongs to pharmaceutical technology field, relate to a kind of antimetabolite and synthetic route thereof, be specifically related to purine base The basic parent nucleus of base carries out ring etc. and replaces when body, and design has synthesized a new purine analogue and 7-amino-tetrazole [1,5- A] and pyrimidine-5-alcohol (compound II).

Background technology

Purine analogue not only has in the many-side such as antibacterial, antiviral, resisting hypertension, arrhythmia, immunosuppressant Activity.And at anti-tumor aspect, there is the strongest biological activity.Antimetabolite can kill tumor cell and not shadow in theory Ring normal cell, but actually antimetabolite is bred normal structure faster for some and also presented certain toxicity.Therefore, Searching biological activity is high, and selectivity is good, and the antimetabolite that toxicity is little has critically important realistic meaning.

CDC25B (cell devision cycle B) phosphate is in protein-tyrosine-phosphatase family member One class dual specificity phosphatase esterase, it can remove the inhibition phosphorus on cyclin complex CDK-Cyclin Acid, thus promote cell to enter mitosis.Its overexpression in many tumor cells, and think that CDC25B is at least two The potential target spot of oncogene (Raf1, c-Myc), can act on jointly with other oncogene, and CDC25B phosphate Substrate specificity is high and catalyst mechanism is single-minded, is effective cancer therapy drug target spot.

Cancer is a kind of frequently-occurring disease, commonly encountered diseases, at present due to bad life habits, and the impact of the factor such as environmental pollution, The M & M of this disease raises year by year, and human body is lifted one's head and bristled with anger outside first, and each portion tissue and organ are all likely to occur tumor. Though tumor is grown in a certain local of health, but is actually a kind of systemic disease.In some big and medium-sized cities, cancer is to occur Rate raises year by year, and age of onset tends to rejuvenation, and the research and development of cancer drug are put in the first place by World Developed Countries, but cancer one Being directly a global difficult problem, countries in the world are all being found and are being explored various effective means and prevent or treat cancer for many years Disease.

The medicine affecting Nucleic acid claims antimetabolite, their chemical constitution and the required material type of nucleic acid metabolism Seemingly, division and the propagation of cell can be stoped by the metabolism of specificity RNA.Antimetabolite refers to and internal metabolism Thing occurs specific binding, thus impact or the medicine of antagonism metabolic function, generally their chemical constitution and internal nucleic acid Or protein metabolism thing is similar.

At present, antimetabolite mainly has:

1. methotrexate be folic acid antagonist for treating acute leukemia, evident in efficacy.It is also used for treating fine hair Film epithelial cancer, malignant mole, ovarian cancer, tumor of head and neck and digestive tract cancer etc.；

2.6-mercaptopurine is purine antagonistic.Its chemical constitution is similar to hypoxanthine, for purine nucleotides synthesis suppression Agent.In vivo, through the catalysis of hypoxanthine-guanyl enzyme (HGPRT), first 6-MP is transformed into NSC-40774 acid, and the latter stops Inosinic acid is changed into adenylic acid and guanyl, thus disturbs purine metabolism, hinders DNA synthesis, makes tumor cell not breed.6- MP → hypoxanthine-guanyl enzyme → NSC-40774 acid → stop inosinic acid to be changed into adenylic acid and guanyl.To S phase cell And other phase cells are effective, clinic is mainly used in the maintaining treatment of children acute lymphoblastic leukemia, is also used for treating Acute or chronic non-lymphocytic leukemia；

3.5-fluorouracil is pyrimidine antagonistic.5-FU intracellular be transformed into 5-uracil Deoxydization nucleotide after just send out Wave effect.Its Antitumor test is wide, especially preferable to alimentary tract cancer and breast carcinoma curative effect to kinds of tumors.It is clinically used for treatment Esophageal carcinoma, gastric cancer, colon and rectum carcinoma, cancer of pancreas and hepatocarcinoma are it can also be used to treat ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma, bladder Cancer and carcinoma of prostate etc..Often participate in forming several combined treatments, be one of important cancer therapy drug.

At present, the selectivity of the big multipair inhibiting tumour cells of antitumor drug used clinically is not strong, systemic administration poison Property relatively big, and there is immunosuppressive action more.Antimetabolite can kill tumor cell and not affect normal cell in theory, but Actually antimetabolite is bred normal structure faster for some and is also presented certain toxicity.Therefore, biological activity is found Height, selectivity is good, and the antimetabolite that toxicity is little has critically important realistic meaning.

Summary of the invention

It is an object of the invention to provide that a kind of biological activity is high, selection type is good, the antimetabolite that toxicity is little.

The key problem in technology of the present invention is: the basic parent nucleus of purine bases carries out ring etc. and replaces when body, devise tetrazole [1,5-a] pyrimidine parent nucleus, 5,7 introduce amino or hydroxyl, and design has synthesized new purine analogue 7-amino-four nitrogen Azoles [1,5-a] pyrimidine-5-alcohol (compound II).

For achieving the above object, used synthetic route is as follows for the present invention:

Route one:

Route two:

Route three:

Present invention employs synthetic method in detail below:

One, route one:

Compound II synthetic method:

(1) 5-aminotetrazole, malonic acid and chloroform are added in round-bottomed flask, be heated to reflux, treat that solid disperses After Jun Yun, add phosphorus oxychloride, be heated to reflux being stirred overnight.Water bath method solvent, adds frozen water, after being sufficiently stirred for, sucking filtration, To solid.Gained solid is heated with water, backflow, add appropriate ethanol, after solid all dissolves, filtered while hot, filter Liquid stands and is cooled to room temperature, separates out solid, sucking filtration, obtains tetrazole [1,5-a] pyrimidine-5,7-glycol；

(2) by tetrazole [1,5-a] pyrimidine-5,7-glycol and phosphorus oxychloride add in round-bottomed flask, are heated to 100 DEG C After, keep temperature, the lower reaction of stirring 4 hours, obtain the solution of clarification.Decompression distillation is pulpous state to solution, adds trash ice and stirs Mixing, wash with after chloroform extraction, U-Ramin MC is dried, and solvent evaporated obtains compound 5,7-dichloro tetrazole [1,5-a] And pyrimidine；

(3) by 5,7-dichloro tetrazole [1,5-a] pyrimidine join in the round-bottomed flask containing strong aqua ammonia, stir under room temperature Mix 2h；Sucking filtration obtains solid, and water-ethanol recrystallization obtains compound 5-chlorine tetrazole [1,5-a] pyrimidine-7-amino；

(4) NaOH of 5-chlorine tetrazole [1,5-a] pyrimidine-7-amino and 10% is added round-bottomed flask, heating in water bath, Stirring 2h.Cooling, sucking filtration, with dilute HCl acidified filtrate, sucking filtration obtains solid, obtains compound II with water-ethanol recrystallization.

Two, route two:

The synthetic method of compound II:

(1) 5-aminotetrazole and ethyl cyanoacetate are joined in round-bottomed flask, heating, keep temperature 150 DEG C, stirring 2h, with ethanol dilution, forms solid product, sucking filtration, washs with ether；Compound is obtained with DMF and ethyl alcohol recrystallization；

(2) take previous step products therefrom, by thin layer chromatography board, developing solvent (chloroform: methanol=9:1), collect and produce Thing speckle, obtains solid chemical compound；

(3) take step (2) gained solid chemical compound, by thin layer silica gel chromatographic column, by methanol-eluted fractions, collect eluent, Volatilize to obtain compound II.

Three, route three:

The synthetic method of compound II:

(1) 5-aminotetrazole, malonic acid are added in round-bottomed flask, add phosphorus oxychloride, be heated to 100 DEG C stirred Night.Water bath method solvent, adds frozen water, after being sufficiently stirred for, sucking filtration, obtains solid chemical compound；

(2) take 5-chlorine tetrazole [1,5-a] pyrimidine-7-amino that previous step obtains and 10% NaOH add round bottom Flask, heating in water bath, stirs 2h；Cooling, sucking filtration, with dilute HCl acidified filtrate, sucking filtration obtains solid, must change with water-ethanol recrystallization Compound II.

Accompanying drawing illustrates:

The nmr spectrum of Fig. 1 compound II¹HNMR。

Detailed description of the invention

1, the preparation (route one) of 7-amino-tetrazole [1,5-a] pyrimidine-5-alcohol (compound II)

(1) 3g5-aminotetrazole, 3.7g malonic acid and 45mL chloroform are added in round-bottomed flask, are heated to reflux, After solid is uniformly dispersed, adds 10.8g phosphorus oxychloride, be heated to reflux being stirred overnight.Water bath method solvent, adds frozen water, fills After dividing stirring, sucking filtration, obtain solid.Gained solid is heated with water, backflow, add appropriate ethanol, treat that solid is the most molten Xie Hou, filtered while hot, filtrate stands and is cooled to room temperature, separates out solid, sucking filtration, obtain compounds I.4.3g, m.p. > 300 DEG C, produce Rate 79.63%, elementary analysis C₄H₃N₅O₂.Measured value: C31.06, H2.12, N45.61；Value of calculation: C31.38, H1.98, N45.74。

(2) 4g tetrazole [1,5-a] pyrimidine-5,7-glycol (i.e. compound I) and 20mL phosphorus oxychloride are added round bottom In flask, after being heated to 100 DEG C, keep temperature, the lower reaction of stirring 4 hours, obtain the solution of clarification.Decompression distillation to solution in Pulpous state, adds trash ice and stirs, and washes with after chloroform extraction, and U-Ramin MC is dried, and solvent evaporated obtains compound 5,7- Dichloro tetrazole [1,5-a] pyrimidine 2.6g, m.p.32-34 DEG C, productivity 52.42%, elementary analysis C₄H₁Cl₂N₅Measured value: C25.36, H0.62, N36.71；Value of calculation: C25.29, H0.53, N36.86.

(3) by 5g5,7-dichloro tetrazole [1,5-a] pyrimidine join in the round-bottomed flask containing 100ml strong aqua ammonia, 2h is stirred under room temperature.Sucking filtration obtains solid, and water-ethanol recrystallization obtains compound 5-chlorine tetrazole [1,5-a] pyrimidine-7-amino 2.9g, m.p.22-24 DEG C, productivity 64.61%, elementary analysis C₄H₃ClN₆Measured value: C28.31, H1.64, N49.36；Value of calculation: C28.17, H1.77, N49.27.

(4) NaOH of 0.5g5-chlorine tetrazole [1,5-a] pyrimidine-7-amino and 10mL10% is added round-bottomed flask, water Bath heating, stirs 2h.Cooling, sucking filtration, with dilute HCl acidified filtrate, sucking filtration obtains solid, obtains compound with water-ethanol recrystallization II0.3g, m.p. > 300 DEG C, productivity 67.25%, elementary analysis C₄H₄N₆O measured value: C31.64, H2.48, N56.31；Value of calculation: C31.58, H2.65, N55.25.The gross production rate of compound II is 18.14%.

2, the preparation (route two) of 7-amino-tetrazole [1,5-a] pyrimidine-5-alcohol (compound II)

(1) 1g5-aminotetrazole and 5.1g ethyl cyanoacetate are joined in round-bottomed flask, heating, keep temperature 150 DEG C, stir 2h, with ethanol dilution, form solid product, sucking filtration, wash with ether.Compound is obtained with DMF and ethyl alcohol recrystallization 1.2g, productivity 67.61%.

(2) take 5g previous step products therefrom, by thin layer chromatography board, developing solvent (chloroform: methanol=9:1), collect Product spot, obtains solid chemical compound.

(3) 1.8g(2 is taken) gained solid chemical compound, by 80g thin layer silica gel chromatographic column, by methanol-eluted fractions, collect 150- The eluent of 225mL, volatilizes to obtain compound II.The gross production rate of compound II is 30.24%.

3, the preparation (route three) of 7-amino-tetrazole [1,5-a] pyrimidine-5-alcohol (compound II)

(1) 3g5-aminotetrazole, 3.7g malonic acid are added in round-bottomed flask, add 20mL phosphorus oxychloride, be heated to 100 DEG C are stirred overnight.Water bath method solvent, adds frozen water, after being sufficiently stirred for, sucking filtration, obtains solid chemical compound 5.8g, productivity 86.58%。

(2) 5-chlorine tetrazole [1,5-a] pyrimidine-7-amino that the previous step of 0.5g obtains and 10mL10% are taken NaOH adds round-bottomed flask, heating in water bath, stirs 2h.Cooling, sucking filtration, with dilute HCl acidified filtrate, sucking filtration obtains solid, by water-second Alcohol recrystallization obtains compound II0.3g, m.p. > 300 DEG C, productivity 67.25%.Compound II gross production rate is 58.22%.

4, tetrazole [1,5-a] pyrimidine-5,7-glycol (compound I) and 7-amino-tetrazole [1,5-a] pyrimidine- The test report that cell division cycle phospho-esterase c DC25B is suppressed by 5-alcohol (compound II)

Experimental technique:

Application escherichia expression system express cell division cycle phospho-esterase c DC25B catalyst structure domain, melts with GST Hop protein form is present in supernatant, through GSH-affinity column purification after cell cracking, it is thus achieved that the CDC25B albumen that activity GST merges.

Using fluorogenic substrate OMFP, observe the suppression to the activity of recombinase of the different compound, OMFP hydrolyzate OMF exists The detectable fluorescence signal that wavelength is 530nm can be launched after 485nm excitation, thus observed the activity change of enzyme And the suppression situation that compound is to it.

First the increment (unit: mO.D./min) of unit interval optical absorption intensity in the calculating enzyme initial velocity phase, represents with this The initial velocity of enzyme, then calculates the sample suppression ratio (%Inhibition) to enzymatic activity according to below equation I.Formula I:

$\%\mathrm{Inhibiton}=\frac{V_{\mathrm{DMSO}}-V_{\mathrm{sample}}}{V_{\mathrm{DMSO}}}\×100\%$

Wherein V_SampleRepresent dosing group initial velocity, V_DMSORepresent the initial velocity of DMSO group (i.e. not dosing group).

The group compound concentration that Preliminary screening selects be 20 μ g/ml(crude extracts be 100l μ g/ml), 3 multiple holes are set, just The screening suppression ratio sample more than or equal to 50% continues to measure its IC₅₀Value.

IC₅₀Value is that suppression ratio logarithm value X to sample concentration carries out Nonlinear Quasi by formula 2 and is calculated.Formula 2:

$\%\mathrm{Inhibition}=\mathrm{Bottom}+\frac{\mathrm{Top}-\mathrm{Bottom}}{1+{10}^{(\mathrm{LogIC}50-X)}\×h}$

Wherein h represents Hill coefficient,

Table 1 compound I and the II suppression ratio to cell division cycle phosphate cdc25B

ID	Sample	Concentration μ g/ml	Type	Result (%)	Remarks
						1	Compound II	20	%Inhibition	99.92	Effectively

Part that the present invention does not relate to is the most same as the prior art maybe can use prior art to be realized.

Claims (2)

1. the synthetic method of a compound II:

The synthetic method of compound II the most according to claim 1, concrete synthetic method is:

(1) 5-aminotetrazole and ethyl cyanoacetate are joined in round-bottomed flask, heating, keep temperature 150 DEG C, stir 2h, With ethanol dilution, form solid product, sucking filtration, wash with ether；Compound is obtained with DMF and ethyl alcohol recrystallization；

(2) take previous step products therefrom, by thin layer chromatography board, developing solvent chloroform: methanol=9:1, collect product speckle Point, obtains solid chemical compound；

(3) take step (2) gained solid chemical compound, by thin layer silica gel chromatographic column, by methanol-eluted fractions, collect eluent, volatilize Obtain compound II.