CN100439387C - Novel cytosine monophosphate medicine precursor - Google Patents

Abstract

Compounds of Formula I, their preparation and uses are described: wherein: M and V are cis to one another and MH is cytarabine; the 5' oxygen of said cytarabine is attached to the phosphorus; V is 4-pyridyl; and pharmaceutically acceptable prodrugs and salts thereof.

Description

Cytosine monophosphate medicine precursor

The application requires No. the 60/423rd, 259, U.S. Provisional Application submitting on October 31st, 2002 and the right of priority of No. the 60/423rd, 211, the U.S. Provisional Application submitted on October 31st, 2002, and it is hereby expressly incorporated by reference in full.

Technical field

The present invention relates to cytosine monophosphate medicine (araCMP) lactide of novel 1,3 propane-1-aryl-diol and its production and use.More particularly, the present invention relates to have the field of cytosine monophosphate medicine (araCMP) lactide of 1,3 propane-1-(4-pyridyl) glycol of cis three-dimensional chemical configuration.

Background technology

Below the description of background technology of the present invention is provided with helping understand the present invention, but be not to constitute or describe prior art of the present invention.The full content of all publications is combined in herein as a reference.

AraC is a kind of analogue of cytosine deoxyriboside, and it is transported in the cell by the nucleosides vehicle, and is become active metabolite araC triphosphate (araC TP) with nucleoside kinase by phosphorylation by nucleosides.It is one of the most successful medicine of treatment acute nonlymphocytic leukemia, but it is invalid for the treatment of hepatocellular carcinoma (" HCC "), this is because the expression level of nucleoside kinase in liver very low (Arner et al.Pharmacol.Ther.67 (2): 155-86, (1995) that need; Ruiz van Haperen et al.Semin.Oncol.22Suppl 11 (4): 35-41 (1995)).Yet kinases keeps highly expression in the toxicity target organ (for example, marrow), and it causes the dose limitation toxicity of being correlated with.The ring-type prodrug of araC is delivered to the liver and the HCC cell of expressing CYP3A4 by the araCTP with greater concn, makes to improve araC and become possibility in the activity of liver.Can reckon with that araC limits cytosine deoxyriboside kinases, the cytosine deoxyriboside deaminase normal and the resistance tumor cell and transports active as sending from bypass of its monophosphate araCMP.Therefore, compare with araC, AraCMP 1, the lactide of ammediol has the enhanced anti-tumor activity in liver, have the toxicity that the outer hemopoietic system of liver is reduced simultaneously, and the bone marrow depression of the dose limitation that it causes in the people being seen is (referring to US 6,312,662).

Because be subjected to outer side effect of (dose limiting) liver of dose limitation or chemotherapeutics can not be discharged into target tissue fully, current methods of treatment is still relatively poor to the curative effect of hepatitis and liver cancer.The limitation of above-described method comprises the filling capability of medicine, preparation and the complicacy of sign and the decrement adjusting of acceptor of binding substances.Therefore, still need a kind ofly will be delivered to the method for liver such as the such medicine of araC.

Description of drawings

Fig. 1 a shows at 0 time point, when giving male NIHSwiss mouse compd A and compd B with the dosage of 100mg/kg CE, and the level of araCTP in liver.

Fig. 1 b shows at 0 time point, when giving male NIHSwiss mouse compd A and compd B with the dosage of 100mg/kg CE, and the level of prodrug in blood plasma.

Fig. 1 c shows at 0 time point, when giving male NIHSwiss mouse compd A and compd B with the dosage of 100mg/kg CE, and the level of araC in blood plasma.

After Fig. 2 a shows and gives compd A, compd B or Compound C by continuous vein (i.v.) injection, the level of araCTP in liver.

Fig. 2 b shows with after compd A or the compd B processing, the dose response of liver araCTP.

Fig. 3 a shows in handle 5 days mouse with dosage 30-1000mg/kg CE by intraperitoneal every day (IP) injection araC, the body weight function of representing with the percentage ratio of initial weight in time.

Fig. 3 b shows in handle 5 days mouse with dosage 30-1000mg/kg CE by intraperitoneal every day (IP) injection Compound C, the body weight function of representing with the percentage ratio of initial weight in time.

Fig. 4 a shows with respect to the saliferous carrier, the hematology terminal point after 5 days with araC or Compound C processing.The bony nodule myelocyte is arranged.

Fig. 4 b shows with respect to saline vehicle, the hematology terminal point after 5 days with araC or Compound C processing.The peripheral blood syncyte (PMN ' s).

Fig. 4 c shows with respect to saline vehicle, the hematology terminal point after 5 days with araC or Compound C processing.Peripheral blood lymphocytes.

Fig. 4 d shows with respect to saline vehicle, the hematology terminal point after 5 days with araC or Compound C processing.Thrombocyte.

Summary of the invention

The present invention relates to cytosine monophosphate medicine (araCMP) lactide of novel 1,3 propane-1-aryl-diol and its production and use.More particularly, the present invention relates to have the field of cytosine monophosphate medicine (araCMP) lactide of 1,3 propane-1-(4-pyridyl) glycol of cis three-dimensional chemical configuration.

One aspect of the present invention relates to compound and medicinal (pharmaceutically) the acceptable drug precursor and the salt of Formula I:

Formula I

Wherein:

M and V be cis each other each other, and MH is a cytosine arabinoside;

5 ' oxygen of cytosine arabinoside links to each other with phosphorus;

V is the 4-pyridyl.

On the other hand, the present invention relates to compound and medicinal acceptable drug precursor and its esters of Formulae II I:

Formulae II I.

Another aspect of the present invention relates to the method for the compound that is used to prepare Formulae II I:

Formulae II I

Wherein:

5 ' oxygen of cytosine arabinoside links to each other with phosphorus, and it comprises the phosphoric acid agent of coupling Formula I V, and the cytosine arabinoside of optional protection;

Formula I V

Wherein, L is selected from the group of being made up of chlorine and 4-nitrophenoxy.

Definition

According to the present invention and as used herein, unless narration is clearly arranged, following term all defines according to following implication.

Term " cis " stereochemical structure refers to the V group on 6 ring structures and the relation of M group position.Following chemical formula shows the cis stereochemical structure.

Another kind of cis (cis) stereochemical structure should be the V and the M of side's indication in the plane.Following chemical formula shows the cis stereochemical structure.

Term " S-configuration ", " S-isomer " and S-prodrug " refer to the absolute configuration S of carbon C '.Following chemical formula shows the S-stereochemical structure.

Term " R-configuration ", " R-isomer " and R-prodrug " refer to the absolute configuration R of carbon C '.Following chemical formula shows the R-stereochemical structure.

Term " enantiomeric excess per-cent (%ee) " refers to optical purity.Equation below it uses obtains:

$\frac{\left[R\right]-\left[S\right]}{\left[R\right]+\left[S\right]}\×100=\%R-\%S$

Wherein, [R] is the amount of R isomer, and [S] is the amount of S isomer.This equation has provided the %ee when R is main isomer.

Term " stereocenter " refers to

Term " d.e. " refers to diastereomeric excess.Equation below it uses obtains:

$\frac{\left[\mathrm{cis}\right]-\left[\mathrm{trans}\right]}{\left[\mathrm{cis}\right]+\left[\mathrm{trans}\right]}\×100=\%\left[\mathrm{cis}\right]-\%\left[\mathrm{trans}\right]$

Term " diastereomer " refers to the compound that has two or more center of asymmetries, this center of asymmetry has identical substituting group and carries out the chemical reaction of same type, wherein non-corresponding isomer has different physical propertys, have the substituting group that occupies different locus, and have different biological properties.

Term " racemic " refers to a kind of compound or mixture, and this compound or mixture are by the molecular composition of the enantiomorph molecular form of equal amts, and these molecules do not have the optics activity.

Term " enantiomorph " refers to any one molecule in a pair of molecule with mirror images of each other relational structure.

Term " halogen " refers to muriate, bromide, iodide or fluorochemical.

Term " alkyl " refers to saturated aliphatic group, comprises straight chain, side chain and cyclic group.Suitable alkyl group comprises methyl, ethyl, sec.-propyl and cyclopropyl.

Term " aryl " refers to the aromatic group with 5-6 ring-type atom.Suitable aromatic yl group comprises phenyl, furyl, pyridyl and thienyl.Aromatic yl group can be substituted.

Term " aryloxy " refers to group aryl-O-.

Term " rudimentary " refers in this article to link to each other with organic radical or compound and limits respectively, as have smaller or equal to 10, preferably smaller or equal to 6,1～4 carbon atom preferably.Such group can be straight chain group, branched group or loop chain group.

Term " optional replacement " or " replacement " comprise the aromatic yl group that is replaced by 1～2 substituting group, are independently selected from low alkyl group, lower aryl and halogen.Preferably, these substituting groups are selected from the group of being made up of halogen.

Term " medicinal acceptable salt " comprises that chemical formula is the salt of the compound of I, and available from the bonded prodrug of compound of the present invention and organic acid or mineral acid or alkali.Suitable acid comprises acetate, hexanodioic acid, Phenylsulfonic acid, (+)-7,7-dimethyl-2-oxygen dicyclo [2.2.1] heptane-1-methylsulfonic acid, citric acid, 1, the 2-ethionic acid, ten disulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glucuronide, urobenzoic acid, hydrochloric acid half oxyacetic acid, HBr, HCl, HI, the 2-ethylenehydrinsulfonic acid, lactic acid, lactobionic acid, toxilic acid, methylsulfonic acid, the first bromic acid, methylsulfuric acid, the 2-naphthene sulfonic acid, nitric acid, oleic acid, 4,4 '-methylene-bis [3-hydroxyl-2-naphthalene monocarboxylic acid], phosphoric acid, galactosan aldehydic acid, stearic acid, succsinic acid, sulfuric acid, sulphosalicylic acid, tannic acid, tartrate, terephthalic acid, and tosic acid.

After term used herein " prodrug " refers in being administered to biosystem, owing to spontaneous chemical reaction, enzyme catalysis chemical reaction and/or metabolic chemistry reaction or various reacting phase in conjunction with any M compound that generates bioactive compounds.The prodrug of standard uses and is connected to functional group (for example, HO-, HS-, HOOC-, the R that is associated with medicine ₂N-) group on is made, and these groups can disconnect in vivo.The prodrug of standard includes but not limited to carboxylicesters; wherein; group is the ester of alkyl, aryl, aralkyl, acyloxy alkyl, alkoxyl group carbonyl oxygen base alkyl and hydroxyl, thiol, amino; in such ester, the group that links to each other is acyl group, alkoxy carbonyl, aminocarboxyl, phosphoric acid ester or sulfuric ester.Shown group only is for example, not limit, and those skilled in the art can prepare other known various prodrugs.Chemical formula is that the prodrug of the compound of I all drops among protection scope of the present invention.Prodrug must carry out the chemical transformation of some form, with the compound of generation biologically active, or the prerequisite of generation bioactive compounds.In some cases, the biological activity that prodrug has is usually less than medicine itself, and by improving effect or the security that oral bioavailability rate, drug effect half life or the like has improved medicine.Bioactive compounds comprises carcinostatic agent and antiviral agent.The certain drug precursor of cytosine arabinoside, for example, N ⁴-acidylate cytosine arabinoside (Wechter et al., J.Med.Chem.19 (8), 1013 (1976)); wherein; the group of acidylate is a palmityl Huo behenolyl, is considered to improve the transportation of lipid solubleness and film, and reduces deamination by the cytidine deaminase.Other groups that are positioned at N4 are considered to such as alkylidene group (that is imido-group).In vivo these groups are removed to produce the 4-amino group of cytosine arabinoside.Expect that similar prodrug can be used for prodrug of the present invention.

Term " cyclic 1 ', 3 '-propane ester ", " cyclic 1,3-propane ester ", " cyclic 1 ', 3 '-alkynes propyl ester " and " cyclic 1,3-alkynes propyl ester ", as follows:

Term " 4-pyridyl ", " pyridin-4-yl " and " 4-pyridyl " refer to following group:

Term " 5 ' oxygen " refers to oxygen as follows:

Term " contains N heteroaryl solvent " and is meant and has 1～3 heteroaryl as the nitrogen-atoms that becomes annular atoms, 4＜pka＜6, and with the non-any mixture that contains N heteroaryl solvent.

Term " heteroaryl that contains N-hydroxyl-nitrogen " refers to that hydroxyl links to each other with nitrogen-atoms contains the N heteroaryl.An example is N-hydroxyl-benzotriazole.

Term " contains the N heteroaryl " and is meant that having 1～3 conduct becomes the nitrogen-atoms of annular atoms and the heteroaryl groups that connects by carbon atom.

Term " electron-withdrawing group " is generally being accepted in the art, refers to substituting group and has the trend that attracts valence electron from adjacent atom, that is, this substituting group is electronegative with respect to adjacent atom.The quantification of electron-withdrawing power provides by Hammett (σ) constant.This known constant all has description in many documents, for example, and J.March, AdvancedOrganic Chemistry, McGraw Hill Book Company, New York, (1997edition) pp.251-259.For electron-donating group, the value of Hammett constant is generally negative value (NH ₂σ _p=-0.66), and electron-withdrawing group is on the occasion of (the σ of nitro _p=0.78), σ _pThe expression para-orientation.The example of electron-withdrawing group comprise nitro, ketone, aldehyde, alkylsulfonyl, trifluoromethyl ,-CN, muriate or the like.

Term " leavings group " refers to the substrate molecule part that is not included in the phosphorus that bonding is provided in the reaction process when reaction breaks.

Term " P450 " refers to cytochrome P-450.The P450 enzyme is present in liver by a large amount of discoveries and other comprise in the tissue of these enzymes.Specific P450 isozyme is responsible for oxidation annular phosphonate of the present invention, so that free phosphonic acid ester or phosphoric acid salt finally are made into.The P450 enzyme is found in mammiferous tissue and cell.

Term " is expressed the tissue of P450 " and is referred in liver and other analogous tissues and the cell, comprises to be found CYP3A4 isozyme or any other P450 isozyme that is used for oxidation ring-type prodrug of the present invention.According to De Waziers et al. (J.Pharm.Exp.Ther., 253,387-394 (1990)), CYP3A4 is positioned at following tissue (determining by immunoblotting and/or enzyme method of masurement):

Tissue The active % of liver

Liver 100

Duodenum 50

Jejunum 30

Ileum 10

Colon＜5

Stomach＜5

Esophagus＜5

The kidney immeasurability

Term " disease in the tissue of expression P450 " refers to multiple disease, trades off in this function of expressing the P450 tissue, can carry out its metabolic function so that these are organized no longer.This will cause the overproduction or the minimizing of biological chemistry final product.These diseases can comprise disease such as primary or secondary liver cancer (for example, HCC), the liver fibrosis or the liver cirrhosis of liver; These diseases can also relate to the disease of liver and relate to the disease of the tissue of expressing P450, can comprise primary or metastatic colorectal cancer, hyperlipemia, diabetes and virus infection and parasitic infection.

Term " cytosine arabinoside of optional protection " refers to 2 ' and 3 ' hydroxyl and the 4-nitrogen base by the cytosine arabinoside of standard protecting group protection.

Term " enhancing " refers to increases or improves specific performance properties.

Term " enrichment " refers to increase by the quantity of reacting the specific isomer that is generated.

Term " gives " to refer to when giving a kind of medicine or approaching another medicine that gives simultaneously simultaneously.Preferably, each other the timed interval in 30 minutes.

Term " therapeutic index " (" TI ") refers to the dosage rate of medicine or prodrug, for the dosage that produces undesirable anti-(raising and/or the pharmacology side effect as dead, the toxic marker of indication), produces treatment and goes up useful reaction.

Term " remission " refers to the seriousness of the elimination or the symptom that palliates a disease.

Term " not cancer stricken " refers to and lacks indication malignant tumour (cancer) and exist or transfer to evidence in its hetero-organization or the organ.

It below is the known medicine of behaviour mentioned in this specification sheets and claims.Abbreviation and common name also are provided simultaneously.

CH ₂CL ₂Methylene dichloride or methene chloride

DCM; Methylene dichloride or methene chloride

(-)-DIP-CL; (-)-β-chlorine di-isopropyl amphene borine

DMAP; 4-dimethylaminopyridine

DMF; Dimethyl formamide

DMPU; 1,3-dimethyl-3,4,5,6-tetrahydrochysene-2 (1H)-pyrimidone

DMSO; Dimethyl sulfoxide (DMSO)

HCl; Hydrochloric acid

KI; Potassiumiodide

MgSO ₄Sal epsom

MTBE; T-butyl methyl ether

NaCl; Sodium-chlor

NaOH; Sodium hydroxide

P450; Cytochrome P450

PyBOP; Phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole Wan Ji Phosphonium

TBDMSCL; TBSCL; TERT-BUTYL DIMETHYL CHLORO SILANE

TBS; TBDMS; T-butyldimethylsilyl

TEA; Triethylamine

THF; Tetrahydrofuran (THF)

TMSCL; Trimethylchlorosilane

5 '-O-is suitable-[4-(4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-2-oxo-2-yl]-cytosine(Cyt)-β-D-arbinofuranose glycosides; 2 (1H)-pyrimidones, 4-amino-1-[5-O-cis-[2-oxidation (oxido)-4-(4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose base]

It below is the known medicine of behaviour mentioned in this specification sheets and claims.Abbreviation and common name also are provided simultaneously.

Cytosine arabinoside; 1-(β-D-arbinofuranose base) cytosine(Cyt); AraC

Summary of the invention

The present invention relates to 5 '-O-suitable-[4-(4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-2-oxo-2-yl]-cytosine(Cyt)-β-D-arbinofuranose glycoside compound and in the application of treatment in the hepatic diseases.In one aspect, hepatic diseases is selected from the group of being made up of virus infection and liver cancer.On the other hand, liver cancer is hepatocellular carcinoma.In second aspect, liver cancer is colorectal carcinoma.In one aspect, 5 '-O-is suitable-[4-(4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-2-oxo-2-yl]-isomer of cytosine(Cyt)-β-D-arbinofuranose glycoside compound is the isomer that carbon C ' has the S configuration.On the other hand, the invention still further relates to be used for synthetic 5 '-O-suitable-[4-(4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-2-oxo-2-yl]-synthetic method of cytosine(Cyt)-β-D-arbinofuranose glycoside compound.Method of the present invention relates to two kinds of cis-isomerides synthetic of araCMP ring-type phosphodiester.On the one hand, the cis-isomeride of cytosine arabinoside phosphodiester is the cis-isomeride that has the cytosine arabinoside phosphodiester of S configuration at C ' carbon.

It is effective that therapy seldom demonstrates in the treatment of liver cancer.In the patient of low ratio, at the tumour profile very clearly and very hour, surgery melts, the method for cryogenic ablation and ethanol injection has demonstrated effect limited in the treatment of liver cancer.Yet most of patient finishes the back tumor recurrence by these therapy for treating.In yet another aspect, the invention still further relates to the tumour of P450 is expressed in prevention in the patient who carried out internal medicine or surgical intervention cancer return.

In another aspect of the present invention, the preferred prodrug of the present invention is used for the treatment of metastatic cancer.In one aspect, metastatic cancer is to be selected from the Secondary cases cancer that is derived from colorectal carcinoma.

The method that is used for the treatment of relapsed cancer

Utilize several different methods to differentiate and monitor liver cell cancer patient, comprise ultrasonic collection of illustrative plates, computerized X-ray axial tomogram (CT), Magnetic resonance imaging, vasography and examination of living tissue.Alpha-fetoprotein (AFP) level also is used in the diagnosis, and may be a useful index of anti-tumor activity, especially for the patient who suffers from high initial AFP level.These technology are useful when definite treatment plan and patient's appropriateness usually.Treatment plan comprises liver homotopic transplantation (OLT), surgical excision, reagent (comprising alcohol), low temperature therapy, intra arterial chemotherapy, arteriopuncture chemoembolization (TACE), systemic chemotherapy, radiotherapy, immunotherapy and hormonotherapy that percutaneous puncture injection is different.Normally can not be diagnosed less than the tumour of 1cm.Patient through surgical method treatment (OLT or surgical excision) may not can after carrying out this therapy demonstrates the observable sign of tumour.Similarly, significantly reduce and seem do not have tumour through the treatment of non-surgical method as the size that the patient of ablation (ethanol, microwave, radio frequency) and TACE may also demonstrate tumour.The patient also can adopt the method for microsphere (with the radiolabeled microglass bead of Yttrium-90) to treat, the particulate of drug delivery vehicle as forming by iron, can utilize external magnets to be positioned on the tumour, the medicine of the chemotherapy agents of direct injection such as gelatinous cis-platinum, target HCC tumour such as Zorubicin and the chemical reagent (as Zorubicin) that in ethanol melts, uses.Other chemotherapy agents equally also can be used as the initial whole body therapeutic of potential to be handled, and comprises anti-angiogenic agent; Thymidylate synthetase inhibitor (as, Nola Qu Te (thymitaq)); The tubulin polymerization inhibitor (as, T67); Various topoisomerase enzyme inhibitors (for example, such as the such medicine of Exatecan) from the tecan class; The pharmaceutical composition that comprises cis-platinum, Interferon, rabbit, Zorubicin (adriamycina) and 5-FU.

All methods of treatment of HCC are all relevant with the high incidence of cancer return.Relapsed cancer may be because one or more former thereby generations.For example, shift in the Secondary cases liver often owing to its size (＜1cm) cause in operation, can not being detected.Patient's prognosis of portal vein or hepatic vein morbidity is not good, and this is because tumour may be inoculated other lobe of the liver.The influence that the size of these small transfers increases and breeds and especially be subject to prodrug of the present invention.Cause second factor of recurrent disease to be since the incomplete excision of primary tumor or melt due to.With the 3rd factor of the environment (liver cirrhosis, virus infection) of liver can be so that liver be in the excessive risk for the primary tumor of " newly ", wherein the primary tumor of " newly " has existed when treatment, but is not detected.

In order to prevent and to postpone relapsed cancer, prodrug of the present invention can be used for before the above-mentioned treatment by expection, during or afterwards.During 1-2, this treatment need give HCC patient a prodrug 1-10 cycle of the present invention, preferably, and 3-6 cycle.One-period need one can delay or the process of prophylaxis of tumours growth in demonstrate effective therapeutic process.In one aspect of the invention, this treatment needed the continuous infusion prodrug 7-14 days, then, and drug withdrawal at least 14 days (one-period).The patient is carried out overtime monitoring.Prodrug causes the cancer eliminating time to increase, improve lifetime and/or quality of life is improved.

Be used for the treatment of leukemic method

Cytosine arabinoside is used for the treatment of various leukemia.Typically, cytosine arabinoside is by with 100-200mg/m ²/ day dosed administration, successive administration up to 7 days after, in several weeks of drug withdrawal, this is owing to the bone marrow depression of cytosine arabinoside inductive causes.Typically, with 3 or the more cycle be used for the treatment of the leukaemic.Also because a variety of causes has used high dosage instructions about how to take medicine (for example, 3gm/m ²).In a word, owing to the tachymetabolism of cytosine arabinoside needs higher levels of drugs, become non-activity metabolite araU so that mainly need araC to deaminize.Think to prolong and send for utilizing cell cycle dependency oncolytic cell agent such as cytosine arabinoside treatment cancer the best.Suppress the ability of DNA synthetic ability or its inhibition archaeal dna polymerase by it, cytosine arabinoside is that part is effective at least, and/or causes the chain termination of the DNA chain of growth after the archaeal dna polymerase catalyzed combination.As DNA synthetic inhibitor, perhaps, the requirement of it being incorporated into DNA has its maximum cytotoxicity in the cell cycle S phase owing to causing araC, and stronger in the activity of S phase anabolism enzyme.Therefore, cellular exposure is directly relevant with killing and wounding of cell during the araC, and this is because permission of longer exposure duration araC is merged in during the phase by S among the DNA of cell of larger proportion when it.

The patient who utilizes higher dosage or araC or long-term treatment is owing to the relevant toxicity of various araC causes on the line.In addition, some patient especially may be in the toxic danger of cytosine arabinoside, for example, and old liver injury patient.Toxicity comprises bone marrow depression, gastrointestinal epithelial ulcer, intrahepatic cholestasis, pancreatitis, cerebellum and cerebral disorder, reaches conjunctivitis.

Prodrug of the present invention is expected to reduce some these dose limitation toxicity.In particular, activate at prodrug that the back produces and the araC that discharges will provide a kind of still less toxic anti-leukocythemia liveness that is used for realizing having in liver.Prodrug of the present invention needn't reach and cause cerebellum and cerebral disorder, the toxic peak value level of conjunctivitis, just can reach the stable status level.This prodrug also provides a kind of medication of cytosine arabinoside, can reduce the side effect relevant with the injection site.The lasting release of cytosine arabinoside from prodrug can change quantitative instructions about how to take medicine, becomes venoclysis or short-term infusion, h inf, intramuscular infusion, oral or the like from continuous infusion.Bone marrow depression can be still relevant with treatment.Number of mechanisms can be used to reduce the active effect of bone marrow depression of prodrug, and comprise drug holiday, bone marrow transplantation or increase myeloid element activated reagent, for example, interleukin-3 (IL-3), GM-CSF, G-CSF, erythropoietin.

The therapeutic index that increases

Multiple toxicity is also almost relevant with all antitumor and anticancer agents.In order to reduce these toxicity in the process of treatment primary and secondary liver cancer, the medicine door artery of directly being offerd medicine its objective is that increasing liver drug exposes sometimes.Because the oncolytic cell drug is usually relevant with pronounced side effects, so topical makes the picked-up of liver higher, thereby reduces liver toxicity outward.In order further to increase the picked-up of liver, sometimes chemoembolization is formed and use with hepatic artery ligation.Similarly, the prodrug among the present invention shows that for the high specific of liver the prodrug method by this novelty minimizes systemic side effects.

And especially primary and secondary liver cancer have resistance for chemotherapy and radiation.Though the mechanism that is used to resist is not also understood fully, it may be owing to the liver gene product that increases causes, and the liver gene product of increase causes the quick metabolism and/or the discharge of chemotherapy agents.In addition, usually relevant with the generation of the metabolism of xenobiotic and cell toxicant intermediate product liver possessed a plurality of protective mechanisms originally, so that be minimized from the damage of these intermediate products.For example, for its hetero-organization, the concentration of gsh is very high in liver cell, so presumably can alkylation protein and the intermediate product of DNA get rid of toxicity by cell internal reaction fast.Therefore thereby liver can have resistance to chemotherapy agents because of above-mentioned these mechanism, need be than the higher concentration of common oncolytic cell reagent concentration to succeed.Higher liver concentration needs higher drug dose, and it can cause the outer toxicity of liver usually.

Prodrug among the present invention can significantly increase the therapeutic index (" TI ") of cytosine arabinoside.In many cases, the TI of increase is that high liver specificity causes.For example because low kinases level, cytosine arabinoside in liver seldom by phosphorylation.Yet kinases but can high expression level (for example, marrow) in the toxicity target organ, and it causes the dose limitation toxicity of being correlated with.Thereby araC ring-type prodrug provides possibility by specifically the araCTP of greater concn being delivered in the liver and HCC cell of expressing CYP3A4 for increasing the curative effect of araC in liver.

The high liver specificity of prodrug splitted shows that prodrug splitted by product still produces at first in liver.Correspondingly, the toxicity relevant with by product is reduced to bottom line, this is because detoxifcation reaction fast takes place by product usually, so just, can eliminate and minimize By-product Toxicology, for example, at by product and compound and/or be present in the reaction that takes place between the protein in the liver cell (for example, gsh and decompose the α that produced, β-unsaturated olefin by prodrug).In addition, the enzyme that is present in the liver also further changes into by product non-toxic chemical (for example, the oxidation of pyridone and sulfation, perhaps reduction of alpha, beta-unsaturated ketone or the like).In addition, be included in reactive group and decompose the α that is produced by prodrug, the intramolecular reaction of cyclization can minimize the toxicity of prodrug between β-undersaturated carbonyl containing compound.

The active clone of cytotoxicity utilization shortage P450 of prodrug is easily estimated (for example, CYP3A4 activity).

With respect to the parent drug of Isodose, can be delivered to liver by bioactive agents and reach higher therapeutic index (TI) higher amount.By prodrug and induce the active reagent Combined Preparation of P450 to realize the increase of the liver level of bioactive agents, for example, CYP3A4 activity (for example, Rifampin, suprarenal gland glucocorticosteroid, phenylethyl barbituric acid, erythromycin).

Improve the method for prodrug stability

The stability of compound is vital for the prodrug of development such as the such cytotoxic drugs of cytosine arabinoside in the biosystem.For example, the enzymatic defect stability that is present in the blood plasma will cause active medicine to decompose in blood plasma, rather than decompose in the target tissue of expressing CYP3A4, and will correspondingly lower the TI of the expection that will pass through the acquisition of prodrug strategy.Similarly,, will cause prodrug not only in blood plasma, and in any organ that this prodrug can be distributed to, decompose, cause the reduction of TI once more if in water-soluble medium, lack intrinsic stability.In addition, deficient in stability also may harm drug development, this be because it make the final formulation of active medicine more unfavorable, particularly, when this prodrug when parenteral uses.In one aspect, the present invention relates to the application of 1-(4-pyridyl)-1, the 3 propylene glycol prodrug of araCMP, its objective is the resistance to overturning of improving the araCMP prodrug.In one aspect, the prodrug of araCMP is suitable-(4-pyridyl) prodrug of araCMP.In yet another aspect, the prodrug of araCMP is that the prodrug of araCMP is suitable-(4-pyridyl) prodrug of araCMP, and wherein carbon C ' has the S configuration.

The stability of prodrug by the monitoring bio fluid (as, blood plasma, with several pH and utilize different buffer reagent solutions buffered) decomposition of Chinese traditional medicine precursor determines.

The drug effect transformation period of improving

Can prolong drug effect transformation period of cytosine arabinoside by the prodrug method of novelty, this be because its can produce medicine one period time length and in some cases drug effect transformation period of prodrug longer.Two times that characteristic can make therapeutic drug levels keep an elongated segment individually, these have caused the improvement of drug effect transformation period.Can prolong the drug effect transformation period by the passage that stops metabolism or elimination to follow parent drug.For some medicines, prodrug of the present invention can stop metabolism or elimination to follow the passage of parent drug, thereby can prolong lifetime in animal body.For example, cytosine arabinoside is the matrix that is used for metabolic enzyme cytidine deaminase, cytidine can be converted into the urine nucleosides, and similarly, cytosine arabinoside is converted into arbinofuranose base-uridylic.Yet the prodrug of araCMP is not the substrate that is used for this kind of enzyme, and the drug effect transformation period of the long cytosine arabinoside that correspondingly causes comparing with the direct administration of cytosine arabinoside.

The ordinary method of eliminating the phosphoric acid salt medicine is by kidney and the transmitting device that can discern anionic compound.From circulation, thoroughly get rid of the phosphoric acid salt mostly just in the several minutes after taking medicine that comprises medicine.Prodrug of the present invention is by removing the eliminating that negative charge slows down medicine, after oxidation and hydrolysis in tissues such as liver.

Formulation

Compound of the present invention with every day about 0.1mg/kg carry out administration to total dose every day of about 100mg/kg, preferably, about 1mg/kg is about 30mg/kg extremely.The preferred dosage scope is about 10mg/kg/ every day.This dosage can give by multiple divided dose as required.

Compound of the present invention when being used in combination with other antiviral agents or the agent of oncolytic cell, can by every day dosage or every day dosage (for example, every day twice) suitably in batches carry out administration.The giving and when giving other antiviral agents or the agent of oncolytic cell or near this time or at different time, to take place of prodrug.Compound of the present invention can multiple medicine instructions about how to take medicine use, and is known as combination therapy or " Mixture in treatment, wherein, plurality of reagents can be by the while administration, at the same time separate administration or with different time separate administration, perhaps administration sequentially at interval.Compound of the present invention can carry out administration after by the therapeutic process of other reagent, can administration during utilizing the therapeutic process of other reagent, as a part of administration of treatment instructions about how to take medicine, perhaps also can be by carrying out administration before other reagent treatments in treatment procedure.

Prodrug of the present invention can be used in combination with other all ingredients, so that further heighten the effect of a treatment and/or reduce the relevant toxicity of cytosine arabinoside.Various combinations are considered to strengthen the effect of treatment.With think that the medicine that can resist cancer effectively combines and can help to treat the liver and prodrug administered not have the metastasis (metastases) that responds of escaping.This reagent comprises the group that is selected from known chemotherapy reagent composition; known chemotherapy reagent comprises DNA synthetic inhibitor (archaeal dna polymerase inhibitor; the inhibitor of denitrogenation pyrimidine passage (for example; thymidylate synthetase inhibitor; two hydrogen vitamin B13 enzyme dehydrogenase inhibitors; the aspartate carbamoyltransferase inhibitor); antifolate (for example; Tetrahydrofolate dehydrogenase; the polyglutamic acid synthetase inhibitors); purine biosynthesis inhibitor (inosine 5 '-monophosphate dehydrogenase inhibitor; glycinamide ribonucleotide transformylase inhibitor; the ribonucleotide diphosphate reductase inhibitor; polyamine biosynthesis inhibitor (for example, S-adenosine-L-methionine(Met) decarboxylase; ornithine decarboxylase; spermidine/spermine N-acetyltransferase); antitumor antibiotic; vegeto-alkali; farnesyl tranfering enzyme inhibitor; platinum base medicine; anti-angiogenic medicaments; tubulin polymerization enzyme inhibitors or the like.

Various known drug categories all are considered suitable for prodrug of the present invention and combine, and comprise Etoposide, camptothecine, ring anthramycin, anthracene pyrazoles, Kang Burui Taka spit of fland analogue, enediyne microbiotic, Taxan.

In one aspect of the invention, Etoposide preferably includes etoposide, Vumon NK-611, GL-331 and azatoxin.In one aspect, Etoposide is etoposide and Vumon.On the other hand, these camptothecine preferably include camptothecine, topotecan, irinotecan (CPT-11), lurtotecan (GI 147211), 9-aminocamptothecin, GG-211, DX-8951F, SKF 107874 and SKF 108025.On the other hand, camptothecine comprises camptothecine, topotecan, irinotecan, lurtotecan, 9-aminocamptothecin.On the other hand, camptothecine comprises topotecan and irinotecan.In one aspect, Taxan comprises taxol, many Xi Tasai and FCE-28161.In yet another aspect, Taxan comprises taxol.In one aspect, Etoposide comprises (S, S) the dioxolane analogue (Bioorg.Med.Chem.Lett.88:1997-2000 (1998)) of Etoposide A-4 and report.In one aspect, the anthracene pyrazoles comprises mitoxantrone, PD 111815 and Losoxanthrone.In one aspect, the ring anthramycin comprises Zorubicin, daunomycin, idarubicin, Perarubicin, Plicamycin, watt ear mycin, dactinomycin and pidorubicin.In yet another aspect, the ring anthramycin is daunomycin, Perarubicin, pidorubicin and idarubicin.On the other hand, the ring anthramycin comprises Perarubicin and daunomycin.In one aspect, the enediyne microbiotic comprises neocarzinostatin, calicheamicin and esperamicin (ten-ring enediyne class).In yet another aspect, the enediyne microbiotic comprises neocarzinostatin, calicheamicin and esperamicin (ten-ring enediyne class).On the other hand, use the destructive medicine of DNA such as alkylating agent (mustargen, aziridine), nitrosourea and metal complex.In one aspect, mitomycin is preferred.In one aspect, platinum complex comprises cis-platinum, S 254, rice Rui's platinum and NSC-241240.In one aspect, alkylating agent comprises endoxan, ifosfamide.In one aspect, nitrosourea comprises carmustine mustargen (BCNU), Temozolomide.Of the present invention preferred aspect, cell cycle dependency inhibitor preferably includes 5-FU, doxifluridine, gemcitabine, CldAdo, fludarabine.In one aspect, antifolate comprises methotrexate.In yet another aspect, the useful oncolytic cell drug that combines with prodrug of the present invention comprises busulfan, tespamin, melphalan, luroteca.

In one aspect, use plays synergistic reagent with cytosine arabinoside.These reagent comprise alkylating agent such as endoxan, ifosfamide, carmustine (BCNU), cis-platinum, mercaptopurine, Tioguanine, methotrexate, 6-Tioguanine and 3-deoxyuridine.

In yet another aspect, prodrug of the present invention combines with known drug promoting cell cycle to S the carrying out of phase, thereby makes them be more vulnerable to the influence of prodrug of the present invention.

On the other hand, the prodrug that the present invention produces can accelerate to promote that with some apoptotic medicine combines, and these medicines comprise the caspase-3 inhibitor.

Combined therapy needs to give the host reagent respectively or simultaneously.In one aspect, prodrug and oncolytic cell drug give simultaneously.Medicine can be identical vehicle or give respectively.Two kinds of medicines can parenterai administration (comprising subcutaneous administration, intramuscular administration, intravenous administration and intradermal administration) or other administrations of, rectal administration oral by comprising, nasal administration, topical, vagina administration and percutaneous dosing.In one aspect, two kinds of reagent can be simultaneously administration in same capsule or independent pill.In yet another aspect, two kinds of reagent administrations at table (preceding on the feed just or feed back).In yet another aspect, the medicine time separately that is combined in gives.In one aspect, except during cycle therapy, medicine gives in time to be separated.In yet another aspect, a kind of medicine is given at the drug holiday for the medicine that is used (partner drug).

Oncolytic cell reagent or Anti-virus agent and compound of the present invention can separately give or give simultaneously.Suitable oncolytic cell reagent comprises busulfan, carbon platinum, cis-platinum, rice Rui platinum, Temozolomide, tespamin, melphalan, ifosfamide, endoxan, Chlorambucil, Zorubicin, daunomycin, pidorubicin, idarubicin, Plicamycin, valrubicin, gengshengmeisu, gemcitabine, floxuridine, Fluracil, mercaptopurine, Tioguanine, Methotrexate, mitomycin, Etoposide, taxol, many western Japanese yews, irinotecan, topotecan, Etoposide, teniposide, S 254, carmustine, doxifluridine, CldAdo, fludarabine, carmustine, mercaptopurine, Tioguanine, azatoxin, camptothecine, lurtotecan, camptothecine, 9-aminocamptothecin, Perarubicin, neocarzinostatin, calicheamicin, esperamicin (a kind of ten-ring enediyne), and luroteca.

For the present invention, in the formulation that comprises medicinal acceptable carrier, adjuvant and vehicle, these compounds can give by variety of way, comprise oral, administered parenterally, by sucking spraying, topical or rectal administration.Term " parenteral (administration) " comprises the subcutaneous injection, intravenous injection, intramuscular injection and the intra-arterial injection that utilize various infusion techniques as used herein.Intra-arterial injection and intravenous injection comprise and pass through catheter drug delivery as used herein.Usually, intravenous injection is preferred.

Medicinal acceptable salt comprises acetate, adipate, benzene sulfonate, bromide, d-camphorsulfonic acid salt, muriate, Citrate trianion, ethanedisulphonate, Estolate, fumarate, gluceptate, gluconate, glucoranate, hippurate, glycolylurea, hydrobromide, hydrochloride, iodide, isethionate, lactic acid salt, lactobionate, maleate, mesylate, methyl bromide, methyl-sulfate, naphthalenesulfonate, nitrate, oleate, palmitate, phosphoric acid salt, polygalacturonate, stearate, succinate, vitriol, sulfosalicylate, tannate, tartrate, terephthalate, tosylate, and triethiodide.

The medicinal compositions that comprises activeconstituents can be that any form that is applicable to administration expection method exists.For example, can be prepared to tablet, lozenge, lozenge, aqueous suspension or oily suspension, can disperse powder or particle, emulsion, hard or soft capsule, syrup or elixir when oral when being used to.The composition that expection is used to orally use can be prepared according to any currently known methods of prior art, these currently known methodss are used to make medicinal compositions, this composition can comprise that one or more comprise sweeting agent, correctives, tinting material and sanitas, so that delicious preparation is provided.Be applicable to that the tablet that comprises activeconstituents with the medicinal acceptable mixed with excipients of non-toxicity of making tablet is acceptable.For example, these vehicle can be inert diluents, as lime carbonate or yellow soda ash, lactose, calcium phosphate or sodium phosphate; Granulating agent and disintegrating agent are as corn starch or Lalgine; Tackiness agent is as starch, gelatin or gum arabic; And lubricant, as Magnesium Stearate, stearic acid or talcum.These tablets can be intectate or can be coated with by known technology that known technology comprises microencapsulation with delay disintegration and absorption in gi tract, thereby secular continuous action is provided.For example, postpone material such as glycerine Monostearate or glycerine distearate separately duration of service, perhaps use with wax.

The formulation that is used to orally use also can be made into hard gelatine capsule, in this capsule, activeconstituents is mixed and the inert solid mixing diluents, for example, calcium phosphate or kaolin, or be soft gelatin capsule, at this capsule activeconstituents is mixed with water and oily medium, as peanut oil, Liquid Paraffin or sweet oil.

Aqueous suspension of the present invention is comprising active material with being applicable in the mixture of making aqueous suspension.This vehicle comprises suspensoid such as Xylo-Mucine, methylcellulose gum, Vltra tears, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and Sudan Gum-arabic, and the phosphatide of dispersion agent or wetting agent such as natural formation (for example, Yelkin TTS), (for example contain oxidation of fatty acids alkene enriched material, polyoxyethylene stearic acid ester), have long chain aliphatic alcohol ethylene oxide enriched material (for example, heptadecane vinyloxy group hexadecanol), contain ethylene oxide enriched material (for example, polyoxyethylene 20 sorbitan monolaurate) derived from the partial ester of lipid acid and hexitan.Aqueous suspension also can comprise one or more sanitass such as ethyl p-hydroxybenzoate or P-hydroxybenzoic acid n-propyl, one or more tinting materials, one or more correctivess and one or more sweeting agents such as sucrose or asccharin.

The oily suspension can be prepared by suspendible activeconstituents in vegetables oil such as peanut oil, sweet oil, sesame oil or Oleum Cocois or mineral oil such as Liquid Paraffin.Oral administration mixed suspension can comprise thickening material such as beeswax, paraffinum durum or hexadecanol.Can will add so that delicious oral preparations to be provided as above-mentioned those sweeting agents and correctives.These compositions can come anticorrosion by adding antioxidant such as xitix.

But be suitable for by add entry prepare the dispersed powders of the present invention of aqueous suspension and particle with the mixture of dispersion agent and wetting agent in activeconstituents is provided.Suitable dispersion agent or wetting agent and suspension agent are shown in above-mentioned example.Other vehicle also may occur as sweeting agent, correctives and tinting material.

Pharmaceutical composition of the present invention also may exist with the form of oil-in-water shape latex.Containing oil phase may be vegetables oil such as sweet oil or peanut oil, mineral oil such as whiteruss or its mixture.Examples of suitable emulsifiers comprises naturally occurring natural gum such as Sudan Gum-arabic and gum tragacanth, naturally occurring phosphatide such as soybean lecithin, or derived from ester or the partial ester such as the sorbitan monooleate of lipid acid and hexitol acid anhydrides, and the enriched material of these partial esters and ethylene oxide such as polyoxyethylene sorbitan monooleate.Emulsion also can comprise sweeting agent and correctives.

Syrup and elixir can utilize sweeting agent such as glycerine, Sorbitol Powder or sucrose preparation.This class formulation also may comprise negative catalyst, sanitas, correctives or tinting material.

Pharmaceutical composition of the present invention can sterile injectable preparation such as sterile injectable moisture or the form that contains suspension exist.This suspension can utilize above-mentioned those suitable dispersion agents or wetting agent and suspensoid to prepare according to known technology.Sterile injectable preparation can also be sterile injectable solution or a suspension in nontoxic parenteral acceptable diluent or solvent, as the solution in 1,3 butylene glycol or make freeze dried powder.Operablely accept carrier or solvent is water, Ge Shi (Ringer) solution and isotonic sodium chlorrde solution.In addition, aseptic expressed oil can be used as solvent or suspensoid medium usually.For this reason, bland any expressed oil be can use, synthetic monoglyceride or triglyceride comprised.In addition, lipid acid such as oleic acid can be used for injectable formulation equally.

The quantity that can combine with solid support material with the activeconstituents that produces one-pack type will change according to the ad hoc fashion of treated host and administration.For example, the slow release formulation that is used for human oral administration can comprise 20 to 2000 μ mol (about 10 to 1000mg) with solid support material suitable and sufficient quantity (can change) blended active material from about 5% to about 95% of total composition.Preferably, the drug prepared combination can provide the dosage that is easy to measure.For example, the aqueous solution that is used for venoclysis should comprise the solution activeconstituents of every milliliter of about 0.05 to 50 μ mol (about 0.025 to 25mg), purpose be can about 30mL/ hour speed carry out the infusion of appropriate volume.

As mentioned above, the formulation of the present invention that is suitable for oral administration can be made isolating unit such as capsule, cachet or tablet, every kind of activeconstituents that all comprises predetermined amount; Make powder or particle; Make solution or at moisture or non-suspension in liquid, aqueous; Perhaps make oil-in-water liquid emulsion or water-in-oil liquid emulsion.Activeconstituents also can give by bolus, electuary or paste.

Tablet can prepare by compression or mold pressing, has one or more ancillary components alternatively.The tablet of compression can prepare by compressing on the machine that is fit to, active ingredient exists with easy liquid form, as powder or particle, alternatively with tackiness agent (for example, polyvinyl pyrrolidone, gelatin, Vltra tears), lubricant, inert diluent, sanitas, disintegrating agent (for example, sodium starch glycolate, crosslinked polyvinyl pyrrolidone, crosslinked sodium carboxymethyl Mierocrystalline cellulose), tensio-active agent or dispersion agent.Molded tablet can be made by molded mixture with the moistening powder compounds of active liquid thinner in suitable machine.These tablets are coated with alternatively or indentation also can be prepared, so that be provided at the slowly-releasing or the controlled release of use therein active ingredient, for example, the Vltra tears of the ratio that has nothing in common with each other is to provide needed releasing curve diagram.These tablets provide enteric coating alternatively, so that partly discharge rather than discharge at the stomach branch at intestines.This is subject to when acidolysis influences at this compound is particularly favourable.

Be suitable for that the formulation of topical comprises lozenge in mouth, be included in the active ingredient in the fragrance matrix, be generally sucrose, Sudan Gum-arabic or gum tragacanth; Pastille is included in the activeconstituents in the inert base, as gelatin and glycerine or sucrose and Sudan Gum-arabic; Mouth-washes comprises the suitable active ingredient in liquid vehicle of activeconstituents.

Being used for the rectal administration formulation can be for having the suppository of suitable matrix, and for example, this matrix comprises cocoa butter or salicylate.

The formulation that is used for vagina administration can except activeconstituents, contain this class carrier that is considered to be fit to for vaginal suppository, tampon, paste, gelinite, paste, foam or aerosol in technology.

Be used for the administered parenterally formulation and comprise moisture and non-moisture isotonic sterile injection solution, can comprise antioxidant, buffer reagent, fungistat and solute, it provides and expects recipient's the isoosmotic formulation of blood; And moisture and non-moisture sterile suspension, can comprise suspensoid and thickening material.These formulations may reside in unitary dose or the multiple doses sealed vessel, for example, and ampoule and phial, and can before will using, under lyophilize (freeze-drying) condition, store, this condition only needs to add sterile liquid carrier, for example is used to the water of injecting.Injection solution and suspension can be by sterilized powder, particle and the tablet of kind are made as previously mentioned.

The formulation that is used for parenteral admin can be by keeping somewhere pump or using bag with the administration of continuous infusion mode by hospital.Continuous infusion comprises the infusion of external pump.These infusions can be undertaken by Hickman or PICC or other suitable devices that gives parenteral or intravenous dosage form.

Preferred unit dose formulations is those dosage or the formulation of unit, sub-doses every day (sub-dose) or its suitable part every day that contain medicine.

Yet, should be understood that the specific dose of level that is used for any particular patient will depend on multiple factor, comprise the activity of used specific compound; Receive treatment individual age, body weight, general health situation, sex and diet; Administration time and approach; Excretion rate; Administered agents before other; And the severity of the specified disease for the treatment of, it is well-known to those skilled in the art.

Compound by the present invention's preparation

Compound and intermediate product by the present invention's preparation are 6 yuan of cyclic phosphoric acid diester that are expressed as the araCMP prodrug of Formula I.

Formula I

Wherein:

M and V be cis each other each other, and MH is a cytosine arabinoside;

5 ' Sauerstoffatom of cytosine arabinoside links to each other with this phosphorus atom;

V is the 4-pyridyl;

And medicinal acceptable drug precursor and its esters.

Another aspect of the present invention is the compound of preparation Formulae II:

Formulae II

Wherein:

MH is the cytosine arabinoside that links to each other with phosphorus in the Formulae II by at the Sauerstoffatom of 5 ' hydroxy position;

V is the 4-pyridyl;

And medicinal acceptable drug precursor and its esters.

Another aspect of the present invention is compound and medicinal acceptable drug precursor and its esters of preparation Formulae II I.

Formulae II I

1.0 phosphorylation agent is synthetic:

Known have multiple preparation 1, a synthetic method of 3-glycol.These methods are divided into two types: 1) racemize type 1-(aryl)-1, ammediol synthetic; 2) rich mapping build 1-(aryl)-1, ammediol synthetic.

1.1 racemize type 1-(aryl)-1, ammediol synthetic:

1, the 3-dihydroxy compound can synthesize by the multiple currently known methods in the document.The enol lithium of the aromatic aldehyde that can utilize replacement by adding alkyl acetates, ester reduction then synthesize racemize type 1-(aryl)-1, ammediol (route A) (Turner, J.Org.Chem.55:4744 (1990)).Perhaps, aryl grignard reagent is added 1-hydroxyl-3-propionic aldehyde and can generate 1-(aryl of replacement)-1, ammediol (route B) equally.This method can change the aryl halide of various replacements into 1-(aryl of replacement)-1, ammediol (Coppi, et al., J.Org.Chem.53:911 (1988)).By 1, the 3-dioxane oneself-4-alkene (1, Heck coupling 3-diox-4-ene), then by reduction and hydrolysis, this aryl halide can also be used for the propylene glycol (Sakamoto, et al., Tetrahedron Lett.33:6845 (1992)) that synthetic 1-replaces.By forming the N-oxide compound, resetting under the diacetyl oxide condition then, the derivative of pyridyl, quinoline, isoquinoline 99.9-3-propyl alcohol can be oxidized to 1-and replace-1,3-glycol (route C) (Yamamoto, et al., Tetrahedron 37:1871 (1981)).By adding vinyl Grignard, reacting by the hydrogen boronation then, multiple aromatic aldehyde also can be converted into 1-and replace-1,3-glycol (route D).

The V=aryl, R=alkyl, R '=benzyl, M=magnesium or lithium, X=halogenide or nothing

1.2 rich mapping build 1-(aryl)-1, ammediol synthetic

The multiple known enantiomorph (Harada, et al., Tetrahedron Lett.28:4843 (1987)) that method that chemical reagent or enzyme reagent separates secondary alcohol can be used to prepare glycol that passes through.It is a kind ofly to prepare the R of beta hydroxy acid or ester or the effective ways of S isomer (Comprehensive Asymmetric Catalysis with high antimer purity that 3-aryl-3-oxo propionic acid of replacing or propyl ester are utilized the transition metal-catalyzed hydrogenation that carries out, Jacobsen, E.N., Pfaltz, A., Yamamoto, H. (Eds), Springer, (1999); Asymmetric Catalysis in organicSynthesis, Noyori, R., John Wiley, (1994)).These beta hydroxy acids or ester products can further reduce and generate the 1-(aryl)-1 of required high ee, ammediol (route A).The acid or the ester substrate that can carry out the β-ketone of high-pressure hydrogenation or hydrogen transfer reactions can prepare by several different methods, for example, under the condition that alkali exists, carry out the polymerization (Chu, et al., J.Het Chem.22:1033 (1985)) of methyl phenyl ketone and methylcarbonate, perhaps by polyisocyanate polyaddition preparation (Tuner, et al., J.Org.Chem.54:4229 (1989)), perhaps utilize aryl halide preparation (Kobayashi, et al., Tetrahedron Lett.27:4745 (1986)).Perhaps, the borane reduction that can carry out enantio-selectivity by the derivative to the derivative of beta-hydroxyethyl aryl ketones or beta-keto acid makes has 1 of high antimer purity, 3-glycol (route B) (Ramachandran, et al., Tetrahedron Lett.38:761 (1997)).In the another kind method, under the condition of the asymmetric epoxy effect of catalysis, commercially available styryl carbinol can be changed into epoxy alcohol.Utilize red aluminium (Red-Al) with the reduction of these epoxies alcohol, can generate and have 1 of high ee, 3-glycol (route C) (Gao, et al., J.Org.Chem.53:4081 (1980)).The aldehyde alcohol polymerization of enantio-selectivity is that another is used for synthetic 1 of the high ee that has, the method of 3-functional compound (oxygenated functionality), this method begins to carry out (route D) (Mukaiyama, Org.React.28:203 (1982)) from aromatic aldehyde.

The V=aryl, R=alkyl or H, R '=-CH ₂OH, CO ₂R

With regard to purpose of the present invention, the intermediate product keto ester is by the 4-acetylpyridine preparation of chemical formula A.C ' is meant the carbon that is arranged in by the prepared final compound methine carbon stereocenter of the present invention.

Chemical formula A

The compound of chemical formula A and dimethyl carbonate are to obtain oxo-lactic acid methyl esters.Stereocenter utilizes the Transhydrogenation of Noyori (Fujii et al., J.Am.Chem.Soc.118 (10): 2521-2 (1996)) to settle.Oxo-lactic acid methyl esters is reduced into the hydroxy ester intermediate product under the situation of ruthenium catalyst (10) existence of rich enantiomorph, it utilizes sodium borohydride further to be reduced into 1 of rich enantiomorph, ammediol, shown in following chemical formula B:

Chemical formula B

The carbon C ' that is arranged in stereocenter establishes at this processing step, and runs through its residue process maintenance enantiomeric excess.

1.3 phosphorylation agent is synthetic

Another aspect of the present invention is the phosphorylation agent of preparation chemical formula C:

Chemical formula C

Wherein:

L is a leavings group, is selected from the group that the aryloxy that replaces by halogen, by 1～3 electron-withdrawing group is formed.

Group L and 4-pyridyl are trans each other.

The compound of chemical formula C is racemic, locates to have the S configuration at carbon C ', perhaps locates to have the R configuration at carbon C '.

The general synthetic method of the phosphorylation agent of chemical formula C is by 1-(4-pyridyl)-1, ammediol and chemical formula Cl ₂The dichloro-phosphoric acid ester of P (O)-L is reacted and is finished.In one aspect, the leavings group L aryloxy that is selected from halogen and replaces by 1～3 electron-withdrawing group.In yet another aspect, leavings group is such as such halogen of chlorine and bromine and aryloxy such as chloro phenoxy group, dichlorophenoxy or the nitro-phenoxy that replaces.In yet another aspect, leavings group is chloro, 4-chlorophenoxy, 3,5-dichlorophenoxy, 2,4 dichloro benzene oxygen base and 4-nitrophenoxy.Dichloro-phosphoric acid ester (wherein, L is an aryloxy) is to synthesize by phenol that will replace and phosphoryl chloride reaction to obtain (Rathore et al., Indian J.Chem B32 (10), 1066 (1993)).

Rich mapping build active phosphorus acidizing reagent utilizes chemistry to be formula L-P (O) Cl by under the condition that exists at alkali ₂The dichloro-phosphoric acid ester to rich mapping build 1-(V)-1, ammediol carries out phosphorylation and synthetic obtain (Ferroni et al.J.Org.Chem.64 (13), 4943 (1999)).On the one hand, the order of interpolation comprises that solution with two pure and mild alkali joins the dichloro-phosphoric acid ester and is dissolved in the solution in the selected solvent.On the other hand, solution and the another kind of solution that comprises the dichloro-phosphoric acid ester that is dissolved in same solvent or the different solvents with two pure and mild alkali joins in a kind of selected solvent simultaneously.On the one hand, glycol solution is joined in the solution of phosphorus reagent again, add alkali then.The typical solvent that is used for the phosphorylation of glycol is a polar aprotic solvent, and the reactivity of it and dichloro-phosphoric acid ester is lower, and can dissolve glycol or dichloro-phosphoric acid ester.On the one hand, the solvent that can carry out phosphorylation reaction is methylene dichloride, THF, acetonitrile, pyridine, tetraalkyl ureas, trialkylphosphate or six alkylphosphonic acid carboxylic acid triamides.On the other hand, these solvents are methylene dichloride, THF, acetonitrile, pyridine, DMPU, DMEU, tetramethyl-urea, tricresyl phosphate methyl ester, hexamethyl-phosphoramide.Should keep low temperature of reaction, especially in the initial stage process of reaction (meeting heat release in this process), thereby the integrity of maintenance reagent.On the one hand, temperature can be lower than room temperature between-20 ℃～10 ℃.On the one hand, can control, make temperature of reaction slowly rise to room temperature, thereby finish the formation of phosphorylation agent heat release.On the other hand, keep temperature-resistant until finishing reaction, to keep the integrity of reagent.Because the chirality characteristic of phosphorus atom, dichloro-phosphoric acid ester will generate the mixture of cis and trans-isomer(ide) with the glycol reaction under above-mentioned reaction conditions, be partial to generate cis-isomeride slightly.The scope of typically suitable/inverse ratio is 50/50～60/40.Cis utilizes the combination of column chromatography and/or crystallization process to separate with trans-isomer(ide).In one aspect of the invention, we find, when the cis-isomeride of the 4-nitrophenoxy phosphorylation agent that will be separated when the salt of 4-nitrophenols heats, surpass in the isolated phosphorylation agent 85% be trans-isomer(ide).In another aspect of this invention, we find, when with the mixture of the cis of isolated 4-nitrophenoxy phosphorylation agent and trans-isomer(ide) when the salt of 4-nitrophenols heats, with the phosphorylation step in the solvent phase that uses with solvent or another solvent in, surpass in the isolated phosphorylation agent 85% be trans-isomer(ide).And we find, need not separate mixture in advance in order to realize enrichmentization.Thereby, the salt of phenol leavings group joined realized in the original reacting mixture that generates the aryloxy phosphorylation agent therein that chemical formula is the enrichmentization of the trans-isomer(ide) of I, it is identical that its ratio and separating mixture at phosphorylation agent carry out the enrichment gained.Similarly, when to chemical formula be the cis of compound of I and trans chlorinated phosphate (phosphorochloridate) etc. molar mixture when heating, have only the trans-isomerism physical efficiency to separate.Phenolate can be preferably trialkylamine, nitrogen heterocyclic ring or sodium reaction and generate by with corresponding phenol and alkali.On the one hand, alkali is triethylamine, diisopropylethylamine, pyridine, DABCO, DBU, sodium hydride or basic metal.On the other hand, alkali is the sodium salt of triethylamine, DBU or phenol.The enrichment step can at room temperature be carried out, but all can heat usually to shorten the reaction times, and preferred range is 40 ℃～70 ℃.Though the transformation of aryloxy phosphorylation agent requires to add the salt of corresponding phenoxide, must not use extra soluble salt hydrochlorate but the transformation of the chloro phosphoric acid ester of the glycol of rich enantiomorph requires, this is because chloro phosphoric acid ester itself that forms and preferred alkali just can generate two normal chlorions.On the one hand, after the phosphorylation of having finished glycol,, thereby cis-isomeride is changed into trans-isomer(ide) fully then to reaction mixture heating (preferred range is 40 ℃～70 ℃).Temperature-resistant when on the other hand, keep adding reagent.

Concerning the glycol by rich enantiomorph prepares the phosphorylation agent of rich enantiomorph, keep the chirality on the carbon atom C ' most important.For 1-(4-pyridyl)-1, ammediol is observed the racemize phenomenon, wherein states reaction conditions before use and separates in the trans phosphorylation agent that obtains, and the initial ee of glycol 98% is brought down below 85%.In one aspect of the invention, the use that has been found that nitrogenous heteroaryl solvent can remain on the ee of trans phosphorylation agent more than 95%.On the one hand, this nitrogenous heteroaryl solvent be optional replacement pyridine, quinoline and pyrazine.On the other hand, this nitrogenous heteroaryl solvent is a pyridine.In another aspect of this invention, have been found that and adding dichloro-phosphoric acid ester or phosphoryl chloride, forming 1-(nitrogenous heteroaryl)-1, the salt of ammediol before adding alkali then, can help prevent the epimerization of C ' carbon atom, and not need to use nitrogenous heteroaryl solvent.On the one hand, 1-(4-pyridyl)-1, the salt of ammediol refer to by with 1-(4-pyridyl)-1, the salt that the organic acid of ammediol and pka＜2 or inorganic acid reaction make.On the other hand, this salt is the salt that the inorganic acid reaction with pka＜1 makes.On the one hand, this salt is hydrochloride and bromate again.Combination by column chromatography and/or crystallization process separates cis with trans-isomer(ide).Yet, have been found that after carrying out the enrichment step, the separation of trans-isomer(ide) is simplified greatly, generated the phosphorylation agent of high purity (＞95% trans-isomer(ide), and ee＞95%).On the one hand, separablely go out trans phosphorylation agent.On the other hand, phosphorylation agent can be remained in the solution, and under the situation of not purifying, be used for nucleosides is carried out phosphorylation.The relative configuration of corresponding phosphorus atom is by comparing ³¹P NMR spectrum is determined.The chemical shift of nearly equator phosphorus oxygen part (trans-isomer(ide)) is than axial isomer (cis-isomeride) (Verkade, et al., J.Org.Chem.42,1549 (1977)) more forward.

2.0araCMP cis-prodrug synthetic

Be cis prodrug synthetic of I for chemical formula, the prodrug part can be introduced when the different steps of building-up process.Because cyclic phosphate is in general all very sensitive for various reaction conditionss, therefore they all are to introduce in stage after leaning on usually.According to the functional group that is present in the compound, can also synthesize by using protected or not protected nucleosides or nucleoside analog.The single stereoisomers of cis prodrug can separate diastereomer by the combination of column chromatography and/or crystallization process and makes, and perhaps can make by the phosphorylation agent enantiomorph specificity (enantiospecific) that adopts rich enantiomorph is synthetic.

2.1 the protection of cytosine arabinoside

Be disclosed in (for example, U.S.3,116,282 in the document; Shen et al., J.Org.Chem.30:835-838 (1965); Hessler, J.Org.Chem.41 (10): 1828-1831 (1976)) various preparations (the Merck Index 11 of the araC in ^ThEdition NO.2790) and resemblance be well-known to those skilled in the art.

Commercial can available from cytosine arabinoside include but are not limited to ShanghaiFreemanInternational Trading Co. (the international freeman in Shanghai trade Co., Ltd), Brantford, Sigma, Fluka, and Sunray Pharmaceuticals.

The phosphorylation process of general protected nucleosides realizes by reacting with the nucleosides of due care and alkali reaction and with the alkoxy compound (alkoxide) that generated and phosphorylation agent.Those skilled in the art can use multiple nucleosides guard method (GreeneT.W., Protective Groups in Organic Chemistry, John Wiley ﹠amp; Sons, New York (1999)) prepares protected nucleosides.The protected mode of nucleosides is to expose 5 '-hydroxyl (to add bound phosphate groups in the above), protected residual hydroxyls all on the nucleosides and other functional groups simultaneously, and these groups may disturb the phosphorylation step, perhaps cause regional isomer phenomenon.On the one hand, selected blocking group has resistibility for highly basic, for example, and ethers and silyl ether.On the other hand, blocking group is MOM ether, MEM ether and the trialkyl silica ether of optional replacement.On the other hand, blocking group is a tertiary butyl dimethyl-silicon ether.In addition, protection can also be sheltered the 4-nitrogen of cytosine arabinoside, thereby has removed any acid proton (acidic proton).On the one hand, the N blocking group of selecting is to be selected from dialkyl group carbonamidine, monoalkyl and dialkyl group imines, single aryl and diaryl imines.On the one hand, the N blocking group is selected from dialkyl group carbonamidine, monoalkyl imines and single aryl imine.On the one hand, the monoalkyl imines is the phenmethyl imines, and single aryl imine is the benzene imines.On the other hand, the N blocking group is the symmetrical dialkyl group carbonamidine that is selected from dimethyl carbonamidine and diethyl carbonamidine.

On the one hand, the protection of cytosine arabinoside is finished by following four steps.Cytosine arabinoside utilizes Benzoyl chloride to handle to obtain benzoate in DMPU.This ester heats with tertiary butyl dimethylsilyl chlorine, to obtain the mixture of dimethylamino silane based compound and single silyl compound.Crude mixture is handled to decompose ester with the ethanol hydrazine.Water treatment and purification are hydrochloride, 2 ', 3 '-dimethyl silanyl cytosine arabinoside of the single entities that obtains expecting.It is mixed with dimethyl formamide, dimethyl-acetal to generate the shielded cytosine arabinoside carbonamidine shown in chemical formula D:

Chemical formula D

2.2 the phosphorylation of shielded cytosine arabinoside

The generation of the alkoxide of the exposed hydroxyl on the cytosine arabinoside of due care is to realize by the alkali that is dissolved in aprotic solvent, and this solvent does not have susceptibility for alkali, for example THF, dialkyl group and ring carbonamidine, ether, toluene and their mixture.On the one hand, solvent is the mixture of DMF, DMA, DEF, N-crassitude, THF and these solvents.

The phosphorylation of the ring-like and acyclic phosphorylation agent of the use of having used multiple different alkali to carry out nucleosides and non-nucleoside compound.For instance, trialkylamine, for example triethylamine (Roodsari et al., J.Org.Chem.64 (21), 7727 (1999)) or diisopropylethylamine (Meek et al., J.Am.Chem.Soc.110 (7), 2317 (1988)); Nitrogen heterocyclic ring amine, for example, pyridine (Hoefler et al., Tetrahedron 56 (11), 1485 (2000)), N-methyl miaow (Vankayalapati et al., J.Chem.Soc.Perk T 1 14,2187 (2000)), 1,2,4-triazole (Takaku et al., Chem.Lett. (5), 699 (1986)) or imidazoles (Dyatkina et al., Tetrahedron Lett.35 (13), 1961 (1994)); Organo-metallic alkali, tertiary butyl potassium (Postel et al. for example, J.Carbohyd.Chem.19 (2), 171 (2000)), butyllithium (Torneiro et al.J.Org.Chem.62 (18), 6344 (1977)), chlorination tertiary butyl magnesium (Hayakawa et al., Tetrahedron Lett.2820), 2259 (1987)) or LDA (Aleksiuk et al., J.Chem.Soc.Comm. (1), 11 (1993)); Mineral alkali, cesium fluoride (Takaku et al. for example, Nippon KagakuKaishi (10), 1968 (1985)), sodium hydride (Hanaoka et al., Heterocycles 23 (11), 2927 (1985)), sodium iodide (Stromberg et al., J.Nucleos.Nucleot.6 (5), 815 (1987)), iodine (Stromberg et al., J.Nucleos.Nucleot.6 (5), 815 (1987)) or sodium hydroxide (Attanasi et al., Phosphorus Sulfur 35 (1-2), 63 (1988)); Metal, for example copper (Bhatia et al., Tetrahedron Lett.28 (3), 271 (1987)).Yet, when using aforesaid method to attempt to carry out chemical formula, do not observe on the phosphorus chiral centre and react or produce racemization as the coupling (coupling) of the phosphorylation agent of C.Especially, the ring-like phosphorylation agent of not observing used alkali in front and replacement reacts and the corresponding ring-like phosphoric acid ester of high yield ground generation, sodium hydride (Thuong etal. for example, Bull.Soc.Chim.Fr.667 (1974)), pyridine (Ayral-Kaloustian et al., Carbohydr.Res.187 (1991)), butyllithium (Hulst et al., Tetrahedron Lett.1339 (1993)), DBU (Merckling et al., Tetrahedron Lett.2217 (1996)), triethylamine (Hadvary et al., Helv.Chim.Acta, 1986,69 (8), 1862), N-Methylimidazole (Li et al., Tetrahedron Lett.6615 (2001)) or sodium methylate (Gorenstein etal., J.Am.Chem.Soc.5077 (1980)).In one aspect of the invention, have been found that the use of Grignard reagent has promoted phosphorylation, have the epimerization at minimum phosphorus center simultaneously.On the one hand, Grignard reagent is alkyl and aryl grignard reagent.On the other hand, Grignard reagent is chlorination tertiary butyl magnesium and phenylmagnesium chloride.

In another aspect of this invention, alkoxide magnesium is used to generate 5 ' of cytosine arabinoside-alkoxyl magnesium.On the one hand, alkoxyl magnesium is selected from Mg (O-t-Bu) ₂, and Mg (O-iPr) ₂Group.

In another aspect of this invention, Lewis acid can be added in the solution (utilizing aforesaid a kind of alkali to make) of 5 '-alkoxylate magnesium, be used for or exchange the carbocation of alkoxy compound and/or the reactivity of regulating formed alkoxy compound and phosphorylation agent.Lewis acidic example comprises an alkali metal salt, rare-earth salts or transition metal salt.On the one hand, Lewis acid is magnesium salts, calcium salt, cesium salt, aluminium salt or cerium salt.On the other hand, lewis acidic salt is magnesium chloride, magnesium bromide and magnesium iodide.

On the one hand, the synthetic chemistry formula is that the reaction conditions of the compound of I comprises: the first, and utilize Grignard reagent or a kind of other alkali to generate alkoxy compound, add magnesium salts then; The second, be that the phosphorylation agent of C joins in the solution of nucleosides with chemical formula, phosphorylation agent or be solution state (solvent for use is identical with nucleosides solution usually, but needn't be necessarily like this) is perhaps directly with solid-state adding.On the other hand, the solution with alkoxy compound joins in the solution of phosphorylation agent.On the one hand, the temperature that generates alkoxy compound with alkali is selected from scope-78 ℃～40 ℃.On the one hand, range of reaction temperature is-20 ℃～25 ℃.On the other hand, the temperature of phosphorylation step is selected from scope-10 ℃～70 ℃.On the one hand, this temperature is selected from 10 ℃～40 ℃ of scopes again.

Then, use several different methods well-known to those skilled in the art (Greene T.W., Protective Groups in Organic Chemistry, John Wiley ﹠amp; Sons, New York (1999)) a kind of in goes the protected prodrug that is generated as mentioned above to protect step, and to remove all protectiveness groups, used method is suitable for the stability of phosphoric acid ester prodrug equally.On the one hand, if exist, go to protect reagent to comprise and be used for removing silyl fluoride salt, be used for removing the mineral acid or the organic acid of the labile acid blocking group of silyl for example and/or ketal and N blocking group.On the other hand, removing to protect reagent is the moisture TFA solution of TBAF, hydrochloric acid solution.On the other hand, removing to protect reagent is the hydrochloric acid solution.The final prodrug and the separation of all intermediates and purification are finished by the combination of column chromatography and/or crystallization process, to obtain the compound of Formula I.

On the other hand, the invention provides that to be used for the synthetic chemistry formula be the method for individual isomer of the compound of I.Because the existence of the stereocenter of C ' on the cyclic phosphate reagent, this carbon atom has two kinds of different orientations, i.e. R or S.Therefore, trans or R-transconfiguration exists the trans phosphoric acid ester reagent of chemical formula C with 5-, and these two kinds of reagent are enantiomorphs.Thereby, the racemic mixture of and S-trans-isomer(ide) trans by the synthetic generation R-of the phosphorylation agent of racemic diol.In addition because cytosine arabinoside is achirality, therefore with racemize trans-phosphoric acid ester reagent carries out the mixture of cis-prodrug that phosphorylation will generate the diastereomer of two kinds of Formula I to these nucleosides.These compounds can separate by the combination of column chromatography and/or crystallization process.Perhaps, the alkoxy compound of nucleosides is carried out phosphorylation, can generate single cis-prodrug with the trans-phosphoric acid ester reagent of rich enantiomorph.Therefore, with C '-S-trans-reaction of phosphoric acid ester reagent (glycol of being made by chemical formula B) will generate C '-S-cis-prodrug of Formulae II I, and and C '-R-trans-reaction of phosphoric acid ester reagent will generate C '-R-cis-prodrug.

On the other hand, on the basis that cis-phosphorylation agent is compared to the speed of response of the epimerization speed of trans-phosphoric acid ester reagent and cytosine arabinoside and trans-phosphoric acid ester reagent, have been found that cis-phosphorylation agent still can generate the cis-prodrug of cytosine arabinoside.In this one side, cis-phosphorylation agent epimerization is to trans-phosphorylation agent process, simultaneously by forming the leavings group that a spot of prodrug generates trace.Then, cytosine arabinoside and generated in-situ trans-phosphorylation agent reacts and generates cis-prodrug.On the other hand, the reaction of the crude mixture of cytosine arabinoside and phosphorylation agent generates cis-prodrug.On the one hand, the crude mixture of phosphorylation agent enrichment in trans-isomer.On the other hand, employed phosphorylation agent does not carry out the enrichment step.

2.3 the phosphorylation of not protected nucleosides

Alternatively, utilize the reaction conditions of selectivity phosphorylation primary hydroxyl, can synthesize the prodrug of cytosine arabinoside, and need not protect in advance cytosine arabinoside.Utilize acyl chlorides that the nucleosides of for example araC is carried out selectivity 5 '-acidylate known (Gish et al., J.Med.Chem.14,1159 (1971)).Yet, therefore because phosphorylation agent is considered to have stronger reactivity, infer that they will cause relatively poor regioselectivity and lower yield, just as with used solvent reaction in reaction (DMF).In one aspect of the invention, we find that chemical formula is that phosphorylation agent and the reaction of not protected cytosine arabinoside of C generated the 5 '-cis-phosphoric acid ester prodrug of the Formula I of high yield, and has high regioselectivity and stereoselectivity.On the one hand, isolated phosphorylation agent is added in the solution of cytosine arabinoside.On the other hand, cytosine arabinoside can be joined in the solution of phosphorylation agent.Similarly, with S-trans-reaction of phosphoric acid ester reagent (being made by the S-glycol of chemical formula B) generates S-cis-prodrug of Formulae II I, and with R-trans-reaction of phosphoric acid ester reagent will generate R-cis-prodrug.Because not protected nucleosides solvability in common solvent is relatively poor, and the potential reaction activity of solvent and phosphorylation agent, therefore need have high dielectric constant and the solvent lower with the reactivity of phosphorylation agent.The example of this solvent is a tetraalkyl ureas.On the one hand, this solvent is DMPU, tetramethyl-urea, DMEU, trimethyl phosphate or hexamethyl-phosphoramide.

Embodiment

Embodiment 1.3-oxo-3-(pyridine-4-yl)-Pyruvic Acid Methyl ester (1) synthetic

Three mouthfuls of round-bottomed flasks of a 50L are assembled agitator, heat packs and the nitrogen inlet on tops.(5kg 44.6mol), adds other THF (18L) to THF (8L) and the uncle-butanols potassium of packing in bottle then.Along with the rising of temperature add the 4-acetylpyridine (2.5kg, 20.6mol), then add methylcarbonate (3.75L, 44.5mol).After two kinds of compositions all added, the temperature of mixture was higher than 40 ℃.Under situation about not heating, reaction mixture stirring reaction 2.5 hours, in this process, temperature is brought up to medial temperature and is about 55 ℃.With mixture heating up to 57 ℃-60 ℃, continue 3 hours then.This reaction process is monitored by thin-layer chromatography (TLC).Stop heating, make slowly cool overnight (15 hours) of mixture.Then the B of mixture by 45cm filtered.The enolization potassium solid of keto ester is backed in the flask of 50L, and diluted (acetate that in the water of 15L, adds 3L) with acetic acid aqueous solution.This acidic mixture is extracted (1 * 16L, 1 * 12L) with methyl-tert-butyl ether (MTBE).Organic layer is merged, and with aqueous sodium carbonate (Na ₂CO ₃) (1750g is in 12.5L water) washing, with sodium bicarbonate aqueous solution (NaHCO ₃) (8L) and salt solution (8L) saturated.With sal epsom (MgSO ₄) (500g) the dry organic layer that merges spend the night (15h).Dried organic solution is filtered, and remove by the rotary evaporation method and to desolvate, obtain the quality of 6.4kg.After the solvent of approximately removing half, begin to have species precipitate to come out.Resulting suspension cooling and stirring 2 hours under ice bath.Solid collected by filtration, with MTBE (500mL) washing, and in vacuum drying oven 20 ℃ of following dry 15h, provide the light yellow solid of the keto ester 1 of 2425g (yield 60%).

The MTBE mother liquor is concentrated into about 1L.Resulting suspension cooled off in ice bath 1 hour.By solid collected by filtration, and with MTBE (2 * 150ml) flushings, dry in vacuum drying oven, obtain the secondary product of 240g.

The TLC condition: with Merck silica gel 60 plates reaction mixture is monitored, 2.5 * 7,5cm, 250 microns, UV lamp: 1: 2THF/ hexane solvent.The Rf=0.25 of starting raw material; The Rf=0.3 of product

Embodiment 2. (S)-3-hydroxyl-3-(pyridine-4-yl)-Pyruvic Acid Methyl ester (2) synthetic

With three mouthfuls of round-bottomed flasks of 22L, assembling top agitator, thermowell/thermometer, feed hopper (1L) and water cooler (empty).With nitrogen flask is purged, the formic acid of packing into (877g) also cools off with ice bath.With (755g) feed hopper of packing into of triethylamine (TEA), and join lentamente in the formic acid of stirring with the timed interval that surpasses 50min.There is one to have the thermopositive reaction that moderate is fuming, the moderate disappearance of being fuming when reinforced trend terminal point.After reinforced finishing, remove cooling bath, and with (5.0L) diluting reaction product of dimethyl formamide (DMF).Disposable adding keto ester 1 (2648g), the then DMF of the other 0.5L of adding in thermo-negative reaction.Temperature descends about 5 ℃, and at this moment keto ester 1 is insoluble.Flask is equipped with heat packs, and stirred mixture is heated to 16 ℃ gradually so that dissolve all solids.Disposable adding chiral ruthenium catalyst (18.8g), and will stir the mixture at one hour post-heating to 55 ℃.The dark solution that is produced stirred 16 hours down at 55 ℃.Determine with TLC when reaction is finished.With solvent vapourisation under reduced pressure (Buchi R152 rotatory evaporator, under high vacuum condition, heating bath temperature=60 ℃), produce the brown oil of 3574g.Oily matter is dissolved in (10L) in the methylene dichloride and is transferred in 5 gallons the fixed separating funnel.With saturated sodium bicarbonate solution (3.0L) washing dark solution, use methylene dichloride (3.0L) that water is stripped then.With the dichloromethane extract MgSO that merges ₄(300g) drying is filtered, and under reduced pressure concentrates, provide 3362g brown oil (be theoretical 125%, by ¹HNMR analyzes and contains DMF).Present embodiment and following embodiment are carried out ¹H-NMR analyzes and carries out on the VARLANGEMINI-200MHz spectrometer.In the solvent that marks, and chemical shift is at residual solvent with sample dissolution.

TLC condition: reaction mixture is monitored 2.5 * 7.5cm, 250 microns, UV lamp: the dichloromethane solution of 5% methyl alcohol: the Rf=0.44 of starting raw material with Merck silica gel 60 plates; The Rf=0.15 of product.

¹HNMR(CDCl ₃)：δ2.73(d，2H，J＝1.5Hz)，3.73(s，3H)，4.35(s，1H)，5.11-5.19(m，1H)，7.31(d，2H，J＝6.6Hz)，8.53(d，2H，J＝6.0Hz)

E.e=87%S isomer (determining) by HPLC.

The HPLC condition:

Post: Chiralpak AD, 0.46x 25cm; Moving phase=10: 90, ethanol: hexane, degree of grade (isocratic); Flow rate=1.5ml/min; Annotating sample volume=10 μ L UV under 254nm measures; R.t.:R-hydroxy ester=19.9min; S-hydroxy ester=21.7min.

Embodiment 3. (S) (-)-1-(4-pyridine)-1, ammediol synthetic

With 22L four-hole round-bottomed flask assembling top agitator, thermowell/thermometer, feed hopper (2L), condenser and water cooler (sky).Use the nitrogen purging flask, and pack into successively sodium borohydride (419g) and 1-butanols (9.0L).Thick hydroxy ester (2) is dissolved in 1-butanols (1.0L, the cumulative volume of solution are 3.2L).With the hydroxy ester solution of 1/4th (800mL) with in the sodium borohydride slurries that joined stirring in 90 minutes lentamente.Temperature is elevated to 32 ℃ from 19 ℃, and has the gas release of moderate to take place.Mixture was stirred 45 minutes, and top temperature reaches 36 ℃.Second 1/4th hydroxy ester solution was added lentamente with 45 minutes, and temperature is elevated to 52 ℃ from 36 ℃.Mixture was stirred 20 minutes, and top temperature reaches 57 ℃.With ice bath mixture is cooled to 40 ℃, and the 3rd 1/4th hydroxy ester solution was added with 45 minutes.Temperature is elevated to 51 ℃ from 38 ℃ again.Mixture was stirred 20 minutes, and top temperature reaches 57 ℃.Mixture is cooled to 38 ℃, and last 1/4th hydroxy ester solution was added with 25 minutes, significantly heating phenomenon at this moment do not occur.Mixture was stirred 40 minutes, and temperature is 45 ℃, installs flask additional heat packs then, and with 2.5 hours stirred mixture is heated to 80 ℃.Stirred the mixture 3 hours at 80 ℃-85 ℃.In 12 hours time, make mixture be cooled to envrionment temperature gradually.Come monitoring reaction whether to finish with TLC once more.(20wt% 5.2L) finishes the reaction of mixture, and stirs the mixture 10 minutes with wet chemical.Separate different layers, and with wet chemical (20wt%, 2.6L) and sodium chloride solution (15wt%, 1.3L) washing butanols layer mutually.Under reduced pressure remove solvent (BuchiR152 rotatory evaporator, high vacuum, heating bath temperature=60 ℃), produce yellow semisolid.(3L) joins in the evaporative flask with acetonitrile, and the evaporation under reduced pressure solvent.Once more acetonitrile (9L) is joined in the evaporative flask, and stirred (on rotatory evaporator, rotating) slurries 15 minutes down 60 ℃ (heating bath temperature=65 ℃, normal atmosphere).Hot slurries are passed through SiO ₂(C salt or celite, Celite 521) filtering medium (in the 1L acetonitrile 250g is arranged, be pre-filled in as slurries in 24 centimetres the B) filters.With filtrate partial concentration (overhead product of 4L is concentrated) under reduced pressure, and with resulting slurries under normal pressure on rotatory evaporator heating make all solid dissolvings (heating bath temperature=65 ℃).Close heating source, and resulting solution was stirred in rotatory evaporator 16 hours, and progressively be cooled to normal temperature.The mixture that obtains is filtered, and (2 * 200mL) wash, and are dried to constant weight and (at-30Hg, 55 ℃, 4h), obtain light yellow solid with acetonitrile.

Fusing point=98-100 ℃

E.e.=98%; S isomer (determining) by HPLC

HPLC condition: post: Chiralpak AD, 0.46 * 25cm; Moving phase=10: 90, ethanol: hexane, degree of grade; Flow rate=1.5mL/min; Annotating sample volume=10 μ L UV under 254nm measures; R.t.:R-glycol=12.7min; S-glycol=14min.

Embodiment 4. (4S)-(-)-anti--(4-pyridine)-2-(4-nitrophenoxy)-2-oxo-1,3,2-two oxa-phospha cyclohexanes (4) synthetic

In being equipped with the 1L round-bottomed flask of magnetic stirring apparatus and nitrogen inlet, pack into glycol 3 (100g, 0.65mol) and pyridine (500ml).With mixture violent stirring at normal temperatures, dissolve fully up to glycol 3.The solvability of glycol 3 is reduced by TEA.3 mouthfuls of round-bottomed flasks of another 3L are assembled feed hopper and the nitrogen inlet of top agitators, thermopair, 1L.Dichloro-phosphatase 24-nitro phenyl ester (4-nitrophenylphosphorodichloridate) is housed in this container, and (166.4g 0.65mol), puts into ice bath, adds pyridine (500mL) by feed hopper then.The mixture that generates was stirred 15 minutes under the ice bath temperature.

After compound 3 dissolves fully (approximately 0.5h), (190mL 1.36mol), and transfers to the solution of the Huang-brown of slight haze in the feed hopper on the 3L flask, and with the 2 hours 40 minutes cold dichloro acid ester solutions of adding to add TEA.This reaction is heat release, keeps this feed rate so that temperature of reaction is no more than 6 ℃.After reinforced finishing, change ice bath under the room temperature water-bath, and continue to stir 3h.

During this period, three mouthfuls of round-bottomed flasks of another 3L are assembled feed hopper and the nitrogen inlet of top agitators, thermopair, 250mL.Then with the sodium hydride of packing in this flask (15g, 0.38mol) and THF (100mL), and the 4-nitrophenols of in feed hopper, packing into (67.5g, THF 0.49mol) (100mL) solution.Then flask is placed in the ice bath, and lentamente nitrophenols solution is joined in the cold sodium hydride suspension.In adding the process of nitrophenols temperature remain on＜40 ℃.Gas takes place stops to show reinforced finishing.The bright orange suspension that is generated at room temperature continues to stir 1h.

After judging that by HPLC glycol (3)-dichloro acid esters reaction is finished, black suspension is carried out vacuum filtration.Wash glassware and filter cake (TEA-HCl) with THF (100mL), and filtrate and the washings that merges is poured in the orange 4-p-nitrophenol sodium suspension.Monitor suitable/trans balance then with mixture heating up to the 40 ℃ lasting 4h that obtains, and by HPLC.Turn off heat packs and stirring reaction at room temperature.Crude product mixture is passed through filtering medium, SiO ₂(521,0.5 inches of C salt) filters, and with dichloromethane rinse glassware and the C salt of 200mL.The filtrate and the washings (oil diffusion pump) 30-35 ℃ in rotatory evaporator, reducing pressure that merge is concentrated down.Resulting heavy-gravity, black spumescence tar are dissolved in 1.0M aqueous hydrochloric acid (1.5L) and the ethyl acetate (800mL), and transfer to phase-splitting in the separating funnel of 4L.Ethyl acetate layer washs with the 1.0M aqueous hydrochloric acid of other 300mL in batches.Methylene dichloride (750mL) is joined in the water layer of merging, and this mixture of vigorous stirring neutralizes this mixture to make it pH value between 7 to 8 carefully with sodium bicarbonate solid simultaneously.Solution is transferred in the separating funnel of 4L once more, and makes each layer separately.Water layer extracts with methylene dichloride (750mL), and with the organic layer that merges with dried over mgso, filter and be concentrated into drying regime.Placing Virahol (200mL) to stir resulting exsiccant residue becomes slurries, obtains the phosphoric acid ester 4 of 190g after filtration.

Raw phosphoric acid ester 4 is dissolved in the methylene dichloride (600mL), in the presence of 10g activated carbon (Darco), stirred 10 minutes, and through SiO ₂(521,0.5 inches of Celite) filters.Wash flask and C salt with methylene dichloride (150mL), and filtrate and the washings that merges is concentrated into drying regime once more.The solid abrasive that obtains is become powder, and become slurries in Virahol (250mL), stirring under 50 ℃.With the slurries cool to room temperature, be cooled to the ice bath temperature subsequently and be used for filtering after 1 hour.With solid 55 ℃ in vacuum drying oven dry 16 hours, obtain light brown (beige) solid of the phosphoric acid ester 4 of 154.6g (70%).

m.p.＝140-142℃

Specific rotation=-80.350 ⁰(c=1.0, MeOH)

HPLC condition: post: Chiralpak AD, 0.46x25cm; Moving phase=50: 50,2-propyl alcohol: hexane, degree of grade; Flow rate=1.0mL/min; Annotating sample volume=10 μ L UV under 254nm measures; R.t.=6.6min., 99+% cis

ee＝99+％

¹HNMR(DMSO-d6)：δ2.23-2.29(m，2H)，4.56-4.71(m，2H)，5.88-5.95(m，1H)，7.44(d，2Hh，J＝5.8Hz)，7.59(d，2H，J＝9.2Hz)，8.34(d，2H，J＝9.4Hz)，8.63(d，2H，J＝5.8Hz)

Synthesizing of embodiment 5.5 '-O-benzoyl cytosine arabinoside (5)

Four-hole round-bottomed flask assembling top agitator, thermopair and nitrogen inlet with 12L.Cytosine arabinoside is housed in this container, and (500g, 2.06mol) and DMPU (1L), it forms heavy-gravity but the solution that can stir.(temperature is elevated to 52 ℃ for 617mL, 2.47mol) disposable adding with HCl De dioxane solution.Mixture was stirred 2 hours, and be cooled to 27 ℃.The adding Benzoyl chloride (578g, 4.11mol), and the 22h that at room temperature stirs the mixture, and temperature rises to 34 ℃ in preceding 30 minutes.After 2.5h, temperature drops to 25 ℃.Add entry (2.5L) and stirred the mixture 30 minutes, simultaneous temperature is elevated to 37 ℃.With each layer separation that obtains, and with methylene dichloride (2 * 1L) extract water layer.With aqueous hydrochloric acid (2 * 500mL) extractions of the organic layer that merges with 10% (vol/vol).The water layer that merges is packed in the flask of 12L, water (1.5L) dilution, and in ice bath, cool off.By the aqueous sodium hydroxide solution (850 milliliters) that adds 30% (wt/wt) the pH value is adjusted to 10.In N-process, be elevated to 21 ℃ from 14 ℃ 30 minutes interpolation phase temperature.In the pH value is to begin to occur throw out at 3 o'clock.In the time of in being kept at ice bath, the thickness suspension that obtains was stirred 8 hours.With solid collection in the B of 24cm.Filter cake water (2L) washing, and in funnel, spend the night and make it the part drying.The solid matter that obtains is descended dry 18h, 5 '-oxygen-benzoyl cytosine arabinoside (5) of output 659g (productive rate 92%) for 70 ℃ at vacuum drying oven.When the water-content that records with karl fischer method is too high, will carry out recrystallization to this material.Pack in the flask of 12L compound (5), methyl alcohol (5L) and ethanol (3L), this mixture becomes sticky thick after stirring.Mixture heating up to refluxing, is generated limpid yellow solution.Mixture is distilled up to the distillate of collecting 2L under normal pressure.Add ethanol (2L), and continue distillation up to the distillate of collecting more than 1 liter.When still-process finished, temperature was 79 ℃.Close heating, and mixture is cooled to 21 ℃, stirs 19h.Solid collected by filtration with ethanol (1L) washing, 70 ℃ of vacuum drying ovens dry 36 hours down, produces the white crystalline solid of the compound 5 of 508g (71%).

HPLC condition: post: Zorbax Eclipse XDB-C8; The damping fluid of the acetonitrile of solvent orange 2 A=5% in the 20mM sodium phosphate; The acetonitrile solution of solvent B=80%; Gradient; Flow rate=1.0mL/min; Annotating sample volume=10 μ L UV under 270nm measures; R.t.=4.3min.

Embodiment 6.5 '-O-benzoyl-2 ', 3 '-two-O-TBS-cytosine arabinoside (6) synthetic

Four-hole round-bottomed flask assembling top agitator, thermopair, condenser and nitrogen inlet with 12L.In flask, be equipped with pyridine (703mL, 8.64mol), DMF (1L) and compound (5).Mixture was stirred 5 minutes.Compound (5) is joined in the solvent to promote dissolving.Add tert-butyl dimethyl chlorination silicon (tert-Butyldimethylsilylchloride) (TBSCl), then add DMF (500mL).With mixture heating up to 93 ℃ ± 1 ℃, continue 44h, during monitor with HPLC every now and then.Mixture is cooled to 51 ℃, and adds methyl alcohol (250mL) so that unreacted TBSCl quenching.After adding entry (2L), mixture is stirred 4.5h.After continuing again to stir 40 minutes, mixture is extracted with ethyl acetate (2L).Aqueous hydrochloric acid with 10% (2x2L), 7% NaHCO ₃The aqueous solution (2x2L) and the organic layer above 10% NaCl (2L) washing.Organic layer MgSO ₄Dry also filtration.In rotatory evaporator, remove and desolvate, obtain heavy-gravity, the crude compound of dark oil thing (6), weight is 1.3kg.This material is used in the subsequent reaction, and need not be further purified.

HPLC condition: post: Zorbax Eclipse XDB-C8; The damping fluid of the acetonitrile of solvent orange 2 A=5% in the 20mM sodium phosphate; The acetonitrile solution of solvent B=80%; Gradient; Flow rate=1.0mL/min; Annotating sample volume=10 μ L UV under 270nm measures; R.t. two-silyl=10.1min; R.t. single silyl=6.9min.

Synthesizing of embodiment 7.2 ', 3 '-two-O-TBS-cytarabine hydrochloride (7)

The condenser, thermopair and the heat packs that have nitrogen bubble with the four-hole round-bottomed flask assembling top agitator of 12L, at the top.In flask, pack into and be dissolved in the solution of the crude compound (6) in ethanol (2.3L) and the hydrazine (185g, 5.76 moles).Monitor with mixture heating up to 80 ℃ lasting 15h and with HPLC.Remove heat packs, mixture was cooled to 35 ℃ with 2 hours.The solution of black poured into 15% NH ₄In the Cl aqueous solution (4L), and extract with ethyl acetate (4L).NH with 15% ₄The Cl aqueous solution (2L) washing organic phase makes it to be evaporated to heavy-gravity oily matter then in rotatory evaporator.Residue is dissolved in the acetonitrile (3L), and is transferred in the big separating funnel.HCl water (3L) solution of adding 10%, and stirred the mixture 5 minutes.The turbid solution that obtains is extracted with hexane (2x3L).After with hexane extraction, two phase transformations are clear.Water/the acetonitrile layer that contains compound (7) of lower floor is concentrated on rotatory evaporator, up to the distillate of collecting 3L.Add entry (3L) and continue distillation, till removing the other solvent of about 500mL.After removing the 2L solvent, begin to occur throw out, after stopping distillation, still be the heavy-gravity slurries simultaneously.With 24 centimetres B solid collected by filtration, and with acetonitrile solution (2 * 1L) washing leaching cakes of 5%vol/vol.The solid (2.6kg) of humidity is transferred in the flask of 12L, and stirred 45 minutes with 5% acetonitrile solution (6L).(lentamente) solid collected by filtration, water (1L) washing, and, obtain the product of 644g (yield 88%) at 65 ℃ of following dry 46h of vacuum drying oven.With HPLC and ¹HNMR (DMSO-d ₆) measure purity: starting material are packed in the flask of 12L.Add ethyl acetate (4L), and mixture heating up is extremely refluxed, keep then refluxing 1 hour at the interval that surpasses 1 hour.Mixture was cooled to 25 ℃ with 3 hours.Collect the solid (7) that obtains by filtering,, and, obtain white solid (335g, 46%) at 65 ℃ of following dry 24h of vacuum drying oven with the ethyl acetate washing.

With mother liquor with saturated NaHCO ₃Solution neutralization, and phase-splitting.With salt water washing organic phase, use MgSO ₄Drying, and in rotatory evaporator, evaporate, the heavy-gravity brown oil of 283g obtained.This material with silica gel (1.1kg) chromatography, with 2% MeOH/ methylene dichloride, is followed the MeOH/ methylene dichloride elution with 15% earlier.The part that will contain clean product merges, and solvent evaporation is fallen.The residue that obtains, 2 ', 3 '-two-oxygen-TBS-cytosine arabinoside free alkali, dried overnight under vacuum obtains amber amorphous solid (171g).

HPLC condition: post: Zorbax Eclipse XDB-C8; The damping fluid of the acetonitrile of solvent orange 2 A=5% in the 20mM sodium phosphate; The acetonitrile solution of solvent B=80%; Gradient; Flow rate=1.0mL/min; Annotating sample volume=10 μ L UV under 270nm measures; R.t.=7.4min.

Embodiment 8.2 ', 3 '-two-O-TBS-cytosine arabinoside-N, N-dimethyl carbonamidine (8) synthetic

In being equipped with 12 L4 mouth round-bottomed flasks of top agitator, pack into compound (7) (333g, 0.66mol) and toluene (2L).Add NaHCO ₃(110g, the 1.31mol) solution in water (2L), and vigorous stirring mixture 15 minutes.In flask, still have a large amount of solids, so add ethyl acetate (2L).In 5 minutes, be that all substances are all dissolved under 8 the condition in the pH value.Phase-splitting, and with 10% the NaCl aqueous solution (1.5

L) the washing organic phase is used MgSO ₄Dry also filtration.In rotatory evaporator, remove and desolvate, obtain the free alkali 398g (theoretical amount 130%) of white foam shape.

In the free alkali that in the rotatory evaporator flask, prepares above 2 ', 3 '-two-O-TBS-cytosine arabinoside free alkali (being obtained by chromatography in embodiment 7) joined.Add toluene (2L), and mixture heating up to 50 ℃ is continued 1 hour, obtain limpid brown solution.Solution is transferred in 4 mouthfuls of flasks of 12L.With toluene (400mL) washing rotary evaporation flask (rotovap flask).Adding DMF dimethyl-acetal (158g, 1.32mol), and the 21h that under 20 ℃, stirs the mixture, come reaction is monitored by TLC simultaneously.Solvent is removed in rotatory evaporator, obtained heavy-gravity oily matter.Add hexane (1L), under vacuum, remove then.With this material under 60 ℃ in rotatory evaporator dry 2h, but still be the oily matter of viscosity.Hexane (2L) and methylene dichloride (2L) are joined in the residue, and under 50 ℃, stir the mixture, obtain limpid brown solution.Mixture is distilled under normal pressure, until the distillment of collecting 2 liters.Mixture is cooled off slightly, and the remaining solvent of vaporising under vacuum.With residue dry 15h in rotatory evaporator, obtain hard foam dress material.This material is scraped off from flask walls, and the free-pouring solid that will obtain dry 24h under 25 ℃ of vacuum, obtain the brown solid of 527.6g (98%) compound 8.

TLC condition: reaction mixture is monitored 2.5 * 7.5cm, 250 microns, UV lamp: the dichloromethane solution of 10% methyl alcohol: the Rf=0.4 of starting raw material with Merck silica gel 60 plates; The Rf=0.7 of product.

¹H NMR(DMSO-d ₆)：δ-0.27(s，3H)，-0.02(s，3H)，.0.11(s，6H)，0.74(s，9H)，0.88(s，9H)，3.02(s，3H)，3.16(s，3H)，3.51-3.69(m，2H)，3.80-3.85(m，1H)，4.05-4.08(m，2H)，4.95-5.03(m，1H)，5.93-6.01(m，2H)，7.67(d，1H，J＝7.2Hz)，8.62(s，1H)。

Embodiment 9. 2 (1H)-pyrimidone, 4-amino-1-[5-O[(2R, 4S)-2-oxidation-4-(4-pyridyl)-1,3,2-dioxane phosphoric acid ester-2-yl]-β-D-arbinofuranose base] (9) synthetic

Four-hole round-bottomed flask assembling top agitator, thermowell/thermopair, feed hopper (1L) and cooling bath with 12L.The flask nitrogen purging, and the protected nucleosides 8 of packing into (250g, 0.47mol) and THF (2.5L).Solution through stirring is cooled to 4 ℃ (ice baths), and with added lentamente in 1 hour chlorination tert-butyl magnesium solution (617mL, 0.62mol), maintenance temperature≤8 ℃.Behind reinforced finishing, solution is stirred 1.25h under the ice bath temperature.Disposable adding phosphoric acid ester reagent (4) (262g, 0.78mo l) is also removed cooling bath.The mixture that obtains was at room temperature stirred 16 hours.(20wt% 2.5L) makes the reaction quenching, and dilutes with ethyl acetate (2.5L) with ammonium chloride solution.Mixture was stirred 5 minutes so that all residues dissolve, and layering.With ethyl acetate (1.1L) strip aqueous, and, use MgSO with the organic phase that sodium chloride solution (15wt%, 1.6 liters) washing merges ₄(260g) drying is filtered and is under reduced pressure concentrated, and obtains 526g darkorange soup compound, it is carried out HPLC analyze (referring to following HPLC condition A).

With three mouthfuls of round-bottomed flasks assembling top agitators, the thermowell/thermometer of 12L, have the condenser and the heat packs of bottom drain valve/water outlet.In flask, pack into the methanol solution (2.5L) of thick soup compound and HCl-dioxane solution (790mL, 3.16mol).To be heated to 50 ℃ through the orange solution that stirs, and under 50 ℃-55 ℃, stir and used HPLC (referring to following HPLC condition B) monitoring in 16 hours simultaneously.The vapourisation under reduced pressure solvent obtains the orange tar of heavy-gravity.To evaporate the agitator/bearing assembly on flask (10L) assembling top, tar will be distributed between water (800mL) and ethyl acetate (800mL).Add sodium bicarbonate solid and stir to emit to weaken and with the every crowd of 2-5g, and the pH value of water is 7 (wide pH value test paper) up to gas.Layering, and use ethyl acetate (800mL) that water is extracted once more.Water is filtered, and under reduced pressure come vaporize water (finishing the ethanol of 4 * 400mL) by component distillation,, obtain the brown oil/solid mixtures of 445 grams along with water is substituted by ethanol gradually by continuous interpolation of alcoholic acid with ethanol.Add ethanol (700mL), and stirred (top agitator) soup compound 2 hours at normal temperatures.

9.1 the mixture that obtains is filtered, and with ethanol (2 * 50mL) wash the solid of collecting, and be dried to constant weight (30 inches Hg, 55 ℃, 2h), obtain the beige solid of 199g, wherein comprise the NaCl of unknown quantity.Solid matter is transferred in the round-bottomed flask that 1L is equipped with magnetic stirring apparatus.Add entry (200mL), and at normal temperatures mixture is stirred 16h.Mixture is filtered, and with solid (9) water collected (2 * 25mL) wash and be dried to constant weight (30 inches Hg, 50 ℃, 16h).The beige powder (9) of regenerant (Recovery)=109g comprises about 10% NaCl.

Under reduced pressure concentrate 9.2 will come from top ethanol filtrate, obtain the brown tar of heavy-gravity.Tar is dissolved in the ethanol (200mL) of heating, and the viscous solution that obtains is stirred 16h at normal temperatures.Formed throw out.Mixture is filtered, and with ethanol (2 * 25mL) wash the solid of collecting (9), and be dried to constant weight (30 inches Hg, 55 ℃, 16h).The Off-white solid of regenerant=6.22g.

Product (9) from present embodiment separates from two independent workflows (9.1 and 9.2).

9.32 (1H)-pyrimidone, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4-(4-pyridyl)-1,3,2-dioxane phosphoric acid ester-2-yl]-β-D-arbinofuranose base] purifying of (9)

With three mouthfuls of round-bottomed flask assembling top agitators of 2L, and the crude compound from each independent workflow (9) and the water (650mL) of packing into and merging.Stir soup compound, and add concentrated hydrochloric acid in batches until solid solid dissolving (needing 34mL, pH=3 wide pH value test paper).Add initial 20mL hydrochloric acid rapidly, and rest parts is added with 5 * 2mL and 4 * 1mL in batches.Orange solution is filtered and joins in the flask once more.Add solid sodium bicarbonate (36g altogether) with every crowd of 2-3g is 6-7 (wide pH value test paper) up to the pH of mixture value in batches.Stir the mixture to emit to weaken until gas.Mixture is stirred 2h at normal temperatures to be filtered then.Water (the solid that 2 * 25mL) washings are collected, and be dried to constant weight (30 inches Hg, 55 ℃, 16h), obtain 2 (1H)-pyrimidones of 133g, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4-(4-pyridyl)-1,3,2-dioxane phosphoric acid ester-2-yl]-β-D-arbinofuranose base] Huang-light brown particulate solid (39.7%).

The C that calculates ₁₇H ₂₁N ₄O ₈The ultimate analysis of P: C, 46.09; H, 4.85; N, 12.65.Record: C, 45.88; H, 4.72; N, 12.58.

HPLC condition: post: the polyphone of following two posts connects; Agilent, Zorbax EclipseXDB-C8,4.6x 250mm, 5 μ m; The 20mM sodium phosphate buffer of solvent orange 2 A=in 11% acetonitrile/water; 50% acetonitrile of solvent B=in deionized water; Anti-phase; Flow rate=1.0mL/min; Annotating sample volume=10 μ L UV under 270nm measures; Column temperature=30 ℃; R.t.=13.4min (S isomer); R.t.=14.1min (R isomer)

¹H NMR(DMSO-d ₆)：δ2.15-2.27(m，2H)，3.90-3.97(m，3H)，4.24-4.58(m，4H)，5.58(d，1H，J＝7.4Hz)，5.62-5.65(m，2H)，5.71-5.79(m，1H)，6.10(d，1H，J＝3.6Hz)，7.08(s，1H)，7.13(s，1H)，7.42(d，2H，J＝5.8Hz)，7.48(d，1H，J＝7.4Hz)，8.59(d，2H，J＝6.0Hz)。

HPLC condition A:

Post: Chiralpak AD, 0.46x25cm; Moving phase=10: 90, ethanol: hexane, degree of grade; Flow rate=1.5mL/min; Annotating sample volume=10 μ L UV under 254nm measures; R.t.=18.9min.

HPLC condition B:

Post: Bondclone 10, C 18,300x 3.9mm; Solvent orange 2 A is 10% acetonitrile (pH 6.2) in the potassium phosphate buffer of 20mM, and solvent B is an acetonitrile; Gradient; Flow rate=1.4mL/min; Annotating sample volume=10 μ L UV under 260nm measures; The r.t.=18.9 branch.

Synthesizing of embodiment 10. chiral ruthenium catalyst (10)

Three mouthfuls of round-bottomed flask assembling top agitators, thermowell/thermometer with 3L.Use the nitrogen purging flask, and the ruthenium complex of packing into (32.0g), (1S, 2S)-(+)-N-ptoluene-sulfonyl-1,2-diphenylethylene diamines (38.24g) and Virahol (1.0L).(30.0mL) joins in the slurries of stirring with triethylamine, and is heated to 80 ℃ with 1h.Orange mixture is stirred 1h at 80 ℃, remove heat packs then, and will stir the mixture with being cooled to normal temperature in 45 minutes.The vapourisation under reduced pressure solvent obtains the darkorange solid.Add methyl alcohol (320mL), and the slurries that stir are warming up to 50 ℃.Under 50 ℃, stirred the mixture 15 minutes, and be cooled to normal temperature gradually with time of 30 minutes then.Under the ice bath temperature,, filter then mixture restir 30 minutes.(solid that 2 * 15mL) washings are collected, (30 inches Hg, 1h), obtain the orange solids catalyzer (productive rate 80%) of 53.1g by 55 ℃ to be dried to constant weight then with methyl alcohol.

Embodiment 11. utilizes synthetic (4S)-(-)-(S)-(-) of the hydrochloride of glycol-(4-pyridyl)-2-(4-nitrophenoxy)-2-oxo-1,3,2-two oxa-phospha cyclohexanes (4)

With 1 L3 mouth round-bottomed flask make-up machinery agitator, feed hopper, thermometer and nitrogen inlet.S-(-)-1-(the pyridin-4-yl)-propane-1 of in flask, packing into, the 3-glycol (25g, 163.4mmol) and ethyl acetate (250mL), and with the suspension that obtains (43mL, 176mmol) time of solution 15min slowly handles with 4N hydrochloric acid De diox.At room temperature stir after the 30min, under positive nitrogen gas stream, add dichloro-phosphatase 24-nitro phenyl ester solid (41.81g, 163.4 moles) as quickly as possible.By means of dry ice-propanone cooling bath the internal temperature of reaction mixture is adjusted to-10 ℃.Add triethylamine (57.76g, 79mL, ethyl acetate 572mmol) (100mL) solution, and keep temperature of reaction＜-5 ℃.Triethylamine solution is reinforced finish after 30 minutes, cooling bath is removed, and stirred reaction mixture 1h at room temperature.Filter reaction mixture is so that remove triethylamine-hydrochloride, and (3 * 30mL) washing triethylamine-hydrochlorides have only slight absorption until filtrate demonstrating with ethyl acetate.Under reduced pressure the filtrate evaporation is dropped to 150-175mL until volume.With the 4-nitrophenols (7.5g, 54.3mmol) and triethylamine (9mL) be added in the spissated solution, and at room temperature the orange reaction mixture that obtains is stirred 24h.By filter collecting formed solid in the reaction mixture, with ethyl acetate (2 * 25mL) and methyl-tert-butyl ether (25mL) washing, and under 55 ℃ vacuum drying, obtain the needed product of 31.96g (58.4%).The analytical data of this product is as among the embodiment 4.

Embodiment 12:(4S)-(-)-and anti--(4-pyridyl)-2-chloro-2-oxo-1,3,2-two oxa-phospha cyclohexanes (11) synthetic

Pack in the 250ml of the oven dry that magnetic stirring bar is housed round-bottomed flask S-(-) 1-(4-pyridyl)-1 of 3.49g, ammediol adds the acetonitrile of 60ml subsequently.This non-homogeneous mixture at room temperature stirred 15 minutes, handled 5 minutes with the 4M HCl De dioxane solution of 5.7ml then.After stirring 1h under the room temperature, by the POCl of the disposable adding 2.16mL of syringe ₃Reaction mixture is handled.In another narrow-necked bottle of 25ml, the DABCO of 2.68g is dissolved in the acetonitrile of 15ml under nitrogen protection, and was transferred in the reaction mixture with 5 minutes by syringe or feed hopper.In the reinforced endpoint observation of DABCO solution to slightly heat release (+10 ℃).Make reaction mixture at room temperature stir 1h, mixture still is heterogeneous in whipping process.Take out the small amount of sample of reaction mixture and use exsiccant nitrogen gas stream rapid evaporation, and residue is dissolved in DMSO-d ₆In so that be transported to ¹In the H-NMR spectrometer.

¹H NMR (DMSO d ₆, Varian Gemini 200MHZ): C '-proton: trans-isomer 5.85-5.75 (d, 1H).

Embodiment 13.2 (1H)-pyrimidone, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4-(4-pyridyl)-1,3, the own 2-yl of 2-two oxa-phosphorus heterocycles]-β-D-Sudan Gum-arabic furyl glycosyl] (9) synthetic

Method A:

Cytosine arabinoside-the HCl of 3.35g and the DMPU of 6.0ml pack in the 250ml of the oven dry that magnetic stirring bar is housed round-bottomed flask.The reaction mixture direct filtration that will come from embodiment 12 in this flask, and with acetonitrile (1 * 15mL) washs DABCO-HCl salt fast.Under the condition that vacuumizes, in rotatory evaporator, volatile matter is removed (heating bath temperature＜35 ℃).Residual oily matter is temporarily preserved under high vacuum, and at room temperature stirred 48h.With the MeOH reaction mixture of 100mL, and at room temperature stir 2h.With the methanol solution of the NaOMe of 25wt% the pH value of reaction mixture was adjusted to for 7.0 (approximately needing use 13mL).Reaction mixture is muddy in this process.Carry out HPLC and detect the integrity that is used for guaranteeing to react collection of illustrative plates.Reaction mixture is evaporated to drying, and the methylene dichloride of residue and 50mL was stirred under room temperature 30 minutes.By filtering collecting precipitation, with (1 * 20mL) METHYLENE CHLORIDE is washed, and is transferred to again in this flask, and the methylene dichloride with 50mL stirred 15 minutes and filtered once more.The ethanol of this solid and 200mL is stirred 1-2h, filter and (2 * 10mL) wash with ethanol.Filtrate is evaporated to drying, obtains the white solid of 4.9g.This solid is dissolved in the water of 10mL, and at room temperature stirs and spend the night, the solid by filtration that obtains is collected, (2 * 3mL) washings are also dry in vacuum drying oven, obtain the compound 9 (26%) of 1.38g for water.

Method B

Steps A: cytarabine hydrochloride synthetic

With cytosine arabinoside (500g, 2.06mol) and methyl alcohol (2.0L) pack in the 3 mouthfuls of flasks of 5L that are equipped with top agitator and thermopair.Suspension is cooled to 2 ℃.Be blown into HCl gas, obtain very heavy-gravity mixture, heat release is warming up to 25 ℃.With methyl alcohol (0.5L) resulting suspension is diluted, so that stir.The HCl gas that adds 108g (2.96mol) altogether.Mixture is stirred 4h, solid collected by filtration then down at 20 ℃.(3 * 250mL) wash, and dry under 70 ℃ in vacuum drying oven, obtain the cotton-shaped cytarabine hydrochloride of 555g, are white solid with MTBE with this solid.

Step B:5 '-O-is suitable-[4-(S)-(pyridin-4-yl)-1,3,2-two oxa-phosphorus hexamethylenes-2-oxo-2-yl]-cytosine(Cyt)-β-D-arbinofuranose glycosides synthetic

Cylindrical reactor assembling top agitator, thermopair and 2 feed hoppers (60mL and 125mL) with 1L strap clamp cover.Reactor is with nitrogen purging and add DMPU (188

mL，195.8g)。Liquid cooling through stirring is arrived-16 ℃ (Julabo F32 water cooler/circulator).

Glycol solution: with 250mL round-bottomed flask assembling magnetic stirring bar and thermopair.In this flask, add S-(-)-1-(pyridin-4-yl)-1, ammediol (50.0g).Then under nitrogen atmosphere, with DMPU (62.5mL, 64.5g) and pyridine (25.8g) place flask.Content through stirring is heated to 40 ℃ (water-baths), stirs down at 40-42 ℃ then, until all solids is dissolved (10 minutes).Then resulting dark orange solution is cooled to 22 ℃, be encased in then in the feed hopper of 125mL (volume=127mL).

POCl ₃Solution: acetonitrile (22.9g) and phosphoryl chloride (50.0g) are added in the erlenmeyer flask of 125mL.After mixing fully, colourless solution is transferred in the 60mL feed hopper (volume=60mL).

The time of above-mentioned two kinds of solution with 2.6h is joined in the reactor of 1L simultaneously, with temperature maintenance at-11 ℃.After reinforced finishing, stir dark orange viscous soln, with temperature maintenance at-11 ℃--17 ℃ of 1h.Take out the reaction solution sample, and whether finish (hydrolysis of partial reaction thing forms cyclic phosphoric acid, is analyzed by HPLC then) by the HPLC detection reaction.Add cytarabine hydrochloride (60.9g).The mixture that obtains is heated to 5 ℃ with 1h, and stirs 87h at 4-6 ℃.HPLC is carried out in adhesive reaction liquid sampling every day that obtains to be analyzed.So that temperature maintenance slowly uses NaOH (13%wt/vol) solution to make reaction soln termination reaction through stirring in the speed below 20 ℃, reach 5.0 (the NaOH solution that need 290mL) until the pH of solution value.(450mL) joins in the biphasic mixture with methylene dichloride, and stirs 30 minutes at 15-20 ℃.Stop stirring, and mixture was left standstill 30 minutes.Lower floor's organic layer is separated, and upper aqueous layer is with (annotating 5) more than twice of methylene dichloride (450mL stirred 30 minutes, the left standstill 30 minutes) extracting.Reactor is fixed a pH meter, and NaOH solution (13%wt/vol) was added in 10 minutes, make pH value to 7.0 (the NaOH solution that needs 68mL).Refrigerant (5 ℃) is introduced chuck be lower than 20 ℃ to keep temperature.The solution that obtains is at room temperature stirred 20h, be cooled to 5 ℃ of 5h (annotating 6) then.The mixture that obtains is filtered, and with the solid water collected (2 * 100mL) washings, and be dried to constant weight (30in.Hg, 60 ℃, 18h).The dark yellow fine-particulate solid of regenerant=46.6g (yield 48%).

Be used for chloro phosphoric acid ester synthetic HPLC:

Post: Zorbax Eclipse XDB-C8,4.6 * 250mm, 5 μ m particle diameters; Solvent orange 2 A=be dissolved in the 20mM sodium phosphate buffer of 11% acetonitrile/water, solvent B=50% acetonitrile is dissolved in (was 100%A to 100%B at 15 minutes inside gradients) in the deionized water; Flow velocity=1.0mL/min; Annotate sample volume=10 μ L, UV detects under 250nm, column temperature=30..r.t＝4.3min。

Be used for 5 '-O-suitable-HPLC of [4-(S)-(pyridin-4-yl)-1,3,2-two oxa-phosphorus hexamethylenes-2-oxo-2-yl]-cytosine(Cyt)-β-D-arbinofuranose glycosides:

Post: Inertsil ODS-3,4.6 * 150mm, 3 μ m particle diameters; Solvent orange 2 A=the be dissolved in 20mM ammonium phosphate damping fluid of 5% acetonitrile/water; Solvent B=acetonitrile (gradient (time minute/%B in %A): 0/0,30/10,40/40,40.1/0,50/0); Flow velocity=1.0mL/min; Annotate sample volume=50 μ L UV under 210nm and detect, column temperature=30 ℃ ± 5 ℃.r.t.＝15.7min。

Step C: to 5 '-O-suitable-[4-(S)-pyridin-4-yl]-1,3,2-two oxa-phosphorus hexamethylenes-2-oxo-2-yl]-purifying of cytosine(Cyt)-β-D-arbinofuranose glycosides

Flow process 1: the 5 '-O-that packs in the there-necked flask that top agitator, thermometer, feed hopper and pH meter are housed of 1L is suitable-[4-(S)-(pyridin-4-yl)-1,3,2-two oxa-phosphorus hexamethylenes-2-oxo-2-yl]-cytosine(Cyt)-β-D-arbinofuranose glycosides crude product (80g, 0.18mol) and deionized water (256mL).The pH value of this mixture is 5.16.In at least 10 minutes, dropwise add 3.0M (60.6mL, sulfuric acid 0.18mol).With 10 ℃ cooling baths so that temperature maintenance between 19-22 ℃.Obtain little mixed yellow liquid.Solution filters (diameter 47mm) via the nylon film filter of 0.45 μ m.Water (40mL) washing flask and strainer.Filtrate and washings are put back in the 1L flask, and NaOH (155mL) and the 3M sulfuric acid of adding 3M are transferred to 6.5 with pH.In the pH value is to begin to observe precipitation at 5.1 o'clock to form.Mixture is stirred 2.5h, then solid collected by filtration.(2 * 8mL) clean flask and strainer to water, and in vacuum drying oven dried overnight (30in.Hg, 60 ℃, 18h), obtain 5 ' of 73.4g-O-suitable-[4-(S)-(pyridin-4-yl)]-1,3,2-two oxa-phosphorus hexamethylenes-2-oxo-2-yl]-cytosine(Cyt)-β-coarse light yellow solid of D-arbinofuranose glycosides.

Step 2: the 5 '-O-that packs in 250mL is equipped with the there-necked flask of top agitator, thermopair, feed hopper and pH meter is suitable-[4-(S)-(pyridin-4-yl)-1,3,2-two oxa-phosphorus hexamethylenes-2-oxo-2-yl]-cytosine(Cyt)-β-D-arbinofuranose glycosides crude product (16g, 36.3mmol) and deionized water (50mL).Dropwise add 3.0M aqueous sulfuric acid (12mL, 36.3mmol) to pH be 2.5, holding temperature is lower than 22 ℃.In at least 20 minutes, add methyl alcohol (160mL), obtain white precipitate.Suspension is stirred 1.5h, solid collected by filtration then down at 20 ℃.(2 * 25mL) clean this solid, and (30in.Hg, 1.5h), obtains the vitriol of 18.89g by 60 ℃ in the vacuum drying oven drying with methyl alcohol.

Solid is added 250mL to be equipped with in the there-necked flask of top agitator and pH meter.Add entry (180mL), and mixture was stirred 10 minutes, make all solid matter dissolvings (pH=2.7).(0.25g 1.81mmol), and stirs mixture 5 minutes to add a hypophosphite monohydrate sodium dihydrogen.Solution is filtered by C salt.Filtrate is put back in the flask, and the NaOH aqueous solution that adds 13% (wt/vol) to pH be 7.1.Suspension is stirred 3h in 20 ℃.Pass through solid collected by filtration, wash (2 * 15mL) with water, and at vacuum drying oven drying (30in.Hg, 60 ℃, 16h) to constant weight, the 5 '-O-that obtains 13.55g (yield 85%) is suitable-the milky white granules shape solid of [4-(S)-(pyridin-4-yl)-1,3,2-two oxa-phosphorus hexamethylenes-2-oxo-2-yl]-cytosine(Cyt)-β-D-arbinofuranose glycosides.

Synthesizing of embodiment 14:R-(+)-1-(4-pyridyl)-1,3 propylene glycol

R-(+)-1-(4-pyridyl)-1, ammediol utilizes the enantiomorph of the catalyzer 10 among the embodiment 10 as shown in the embodiment 3

Fusing point=98-100 ℃

E.e.=98%R isomer (detecting) by HPLC.

HPLC condition: post: Chirapak AD, 0.46 * 25cm; The ethanol of moving phase=10: 90: hexane, degree of grade; Flow velocity=1.5mL/min; Annotating sample volume=10 μ L UV under 254nm detects; R.t.:R glycol=12.7min; S glycol=14min

Embodiment 15. (4R)-(+)-(4-pyridyl)-2-(4-nitrophenoxy)-2-oxo-1,3,2-two oxa-phospha cyclohexanes (12) synthetic

0 ℃ stir down 4-nitrophenyl dichloro phosphoric acid ester (9.2g, THF 36mmol) (100mL) solution, with 30 minutes slowly the adding pyridine (7.9mL, 98mmol).Reaction mixture was stirred 5 minutes at 0 ℃, and slowly join R-(+)-(4-pyridyl)-1 down and in 1.5 hours at 0 ℃, and ammediol solution (95.8%ee, 5g, 32.7mmol) and THF (300mL) solution of triethylamine (15.6mL, 114mmo l).Reaction mixture is warming up to room temperature, and stirred 2.5 hours.(18.17g 131mmol) also should heterogeneous orange reaction mixture heat 4.5 hours at 40 ℃ to add the 4-p-nitrophenol sodium.Reaction mixture is cooled to 0 ℃,, and makes each layer separation with saturated aqueous ammonium chloride (250mL) termination reaction.With saturated ammonium chloride solution (200ml) washing organic layer, and with water washings methylene dichloride (100mL) reversed phase extraction that merges.Use the NaOH aqueous solution (200mL * 4) of 0.3N to wash the organic extract that merges, use dried over mgso then.Filtered solution is concentrated under reduced pressure, and the oily matter that obtains is to leave standstill (standing) crystalline.This yellow solid is recrystallization in the 2-of 100ml propyl alcohol, and the white solid that obtains expecting is anti--phosphoric acid salt (12) (95%ee, [α] _D ²⁰+ 74.2 (c 1.0, MeOH).

TLC condition: 250 microns silica gel plate (Unitplate); Propyl alcohol/the hexane of moving phase=3/2; Glycol: rf=0.2, trans-phosphoric acid ester: rf=0.6, cis-phosphoric acid ester: rf=0.5.

The HPLC condition that is used for suitable/anti-isomerization reaction: post=Zorbax Rx-C18 (4.6x250mm); The pH value of the acetonitrile of moving phase=35%/65% is 7.95 20mM phosphate buffered saline buffer; Flow rate=0.5mL/min; Detect=UV detects under 250nm; Retention time: cis-isomer=9.39 minute, trans-isomer=10.11 minute.

Be used for the HPLC condition that ee measures: post=Chiral Pak AD; 2-propyl alcohol/the hexane of moving phase=1: 1; Flow rate=1.0mL/min; UV detects under the detection=254nm; Retention time in minute: trans-phosphoric acid salt=7.02.

Embodiment 16.2 (1H)-pyrimidone, 4-amino-1-[5-O-[(2S, 4R)-2-oxidation-4-(4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] (13) synthetic

16.1 phosphorylation step

At room temperature, ((2.4g is in THF 4.55mmol) (40ml) solution 6mmol) slowly to be added to compound 8 through stirring for the THF solution of 1M, 6ml with t-BuMgCl solution.After 30 minutes, with R-trans-(1.84g 5.5mmol) joins in the reaction mixture phosphoric acid ester 12, and at room temperature stirs 16 hours.Reactant is cooled to 0 ℃, comes termination reaction by adding saturated aqueous ammonium chloride, and with ethyl acetate extracting (50mL * 2).With the organic layer dried over mgso that merges, filter and under reduced pressure, concentrate.Thick product is carried out purifying by silica gel column chromatography, use the acetone elution, obtain the prodrug (2.19g, 66%, mp:142.0-145.0 ℃) of needed protected white solid state.

TLC condition: 250 microns silica gel plate; Moving phase=acetone, trans-phosphoric acid ester; Rf=0.6, protected ara-C:rf=0.2.

16.2. protective reaction step

Above-mentioned protected prodrug is dissolved among 70% the TFA (20mL), will this solution be heated to 60 ℃ and continues 16 hours, and under reduced pressure, remove and desolvate through stirring.In residue, add methyl alcohol (30mL), and mixture is transferred to slight alkalinity with yellow soda ash.With the solution dried over mgso of muddiness, filter.Under reduced pressure, remove solvent, and thick product is carried out purifying by silica gel column chromatography, and, obtain needed oyster white solid-state drug precursor (1.06g, 80%, 98%de, mp:178.0-180.0 ℃) with methyl alcohol-acetone (1: 1) elution.

TLC condition: 250 microns silica gel plate; Moving phase=acetone-methyl alcohol (1: 1), protected ara-C:rf=0.7, product rf=0.3.

HPLC condition: post=Zorbax Eclipse x DB-C84.6 * 150mm; Moving phase=the be dissolved in phosphate buffered saline buffer of the 20mM of 5% acetonitrile-water; Flow rate=1.0mL/min; UV detects under the detection=210nm; Retention time in minute: product=10.60.

Embodiment 17:2 (1H)-pyrimidone, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl] β-D-arbinofuranose glycosides base] (+)-camphorsulfonic acid ester (salt) synthetic

With (+)-camphorsulfonic acid (232mg, 1mmol) be added to 2 (1H)-pyrimidones, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] (compound 9,400mg is in methyl alcohol 0.91mmol) (8mL) suspension.The colourless transparent solution that obtains is at room temperature stirred 2h, concentrate under the reduced pressure then.Residual white foam body is ground with ethyl acetate, and under reduced pressure, concentrate.Residue is suspended in the ethyl acetate, and is heated to backflow.Cool off after 1 hour, solid collected by filtration, with ethyl acetate washing, and in 50 ℃ of vacuum drying ovens dried overnight, obtain the titled reference compound (562mg) of white solid state.m.p.193℃。The C that calculates ₁₇H ₂₁N ₄O ₈PC ₁₀H ₁₆O ₄SH ₂O ultimate analysis (RobertsonMicrolit): C, 46.95; H, 5.69; N, 8.11.Record: C, 47.22; H, 5.51; N, 7.81.

Embodiment 18:2 (1H)-pyrimidone, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] maleic acid ester (salt) synthetic

Adopt the identical reaction process of embodiment 17, use toxilic acid (1.1eq).

m.p.140℃。The C that calculates ₁₇H ₂₁N ₄O ₈PC ₄H ₄O ₄.H ₂O0.5C ₄H ₈O ₂Ultimate analysis (Robertson Microlit): C, 44.67; H, 5.05; N, 9.06.Record: C, 44.58; H, 4.61; N, 8.48.

Embodiment 19:2 (1H)-pyrimidone, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base-] hydrated sulfate synthetic

With sulfuric acid (89mg, 0.91mmol) be added to 2 (1H)-pyrimidones, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] (compound 9,400mg is in methyl alcohol 0.91mmol) (8mL) suspension.Free-pouring solid becomes sticky, and is bonded on the flask walls.Mixture was refluxed 15 minutes, be cooled to room temperature, solid comes off from flask walls.After at room temperature stirring 3h, collect white free-pouring solid, use methanol wash, and, obtain titled reference compound (428mg) in 20 ℃ of vacuum-dryings by filtering.

m.p.158℃。The C that calculates ₁₇H ₂₁N ₄O ₈PH ₂SO ₄2H ₂The ultimate analysis of O (Robertson Microlit): C, 35.54; H, 4.74; N, 9.75; Record: C, 35.50; H, 4.73; N, 9.51.

Embodiment 20:2 (1H)-pyrimidone, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] hydrochloride synthetic

Hydrochloric acid soln (228 μ L with 1N, 0.23mmol) join 2 (1H)-pyrimidones are housed, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] (compound 9,100.6mg is in flask 0.228mmol).Water (5mL) and methyl alcohol (5mL) are joined in this partly soluble solid, then mixture is carried out supersound process.The syringe-type strainer of this colourless transparent solution by 0.45 μ m filtered, with this strainer of washed with methanol.Under reduced pressure, the filtrate that merges is concentrated.Make residue with acetonitrile (2 * 10mL) azeotropic.With residue be dissolved in acetonitrile and methanol mixture (1/1,10mL), and be concentrated into drying.This white powder is dry under 20 ℃ of vacuum, obtain inscribing the compound of stating (78mg).

m.p.＞200℃。The C that calculates ₁₇H ₂₁N ₄O ₈PHClH ₂The ultimate analysis of O (Robertson Microlit): C, 42.03; H, 4.77; N, 11.53; Cl, 7.30.Record: C, 41.70; H, 4.84; N, 11.68; Cl, 7.45.

Embodiment 21:2 (1H)-pyrimidone, 4-amino-1-[5-O-[(2R, 4S)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] L-tartrate synthetic

Adopt the reaction process identical, the L-aqueous tartaric acid solution of use 0.1N with embodiment 20.

m.p.＞200℃。The C that calculates ₁₇H ₂₁N ₄O ₈PC ₄H ₆O ₆H ₂O0.1C ₂H ₃The ultimate analysis of N (Robertson Microlit): C=41.57; H, 4.82; N=9.38; Record: C=41.32; H=5.03; N=9.69.

Embodiment 22: racemic anti--4-(4-pyridyl)-2-(4-nitrophenoxy)-2-oxo-1,3,2-two oxa-phospha cyclohexanes synthetic

With racemic 1-(4-pyridyl)-1, ammediol (4g, 26.1mmol) and triethylamine (12mL, THF solution 86mmol) are added to 4-nitrophenyl dichloro phosphoric acid ester, and (7.35g is in THF solution 28.7mmol).After at room temperature stirring was spent the night, (10g 71.8mmol), then heated reaction mixture 4 hours at 50 ℃ to add the 4-p-nitrophenol sodium.With cooled reaction mixture saturated ammonium chloride solution termination reaction, and with ethyl acetate (3x) extracting.The organic extract that merges is washed with saturated nacl aqueous solution, and use dried over sodium sulfate.Filtered solution is concentrated under reduced pressure, and the residue that obtains is carried out purifying (silica gel, the methylene dichloride/ethanol with 95/5) by column chromatography.

¹H NMR (CDCl ₃, Varian Demini 200MHz): C '-proton: cis-isomer 5.6-5.8 (m, 1H); Trans-isomer 5.5-5.69 (m, 1H).

TLC condition: 250 microns silica gel plate; The acetone/hexane of moving phase=3/2; Glycol: rf=0.2, trans-phosphoric acid ester: rf=0.6, cis-phosphoric acid ester rf=0.5.

The HPLC analysis condition that is used for cis/trans-isomerization reaction: post=ZorbaxRx-C18 (4.6x250mm); The 20mM phosphate buffered saline buffer of the acetonitrile of moving phase=35%/65%pH value 7.95; Flow rate=0.5mL/min; Detect=UV detects under 210nm; Retention time in minute: cis-isomer=9.39 minute, trans-isomer=10.11 minute.

Embodiment 232 (1H)-pyrimidone, 4-amino-1-[5-O-is suitable-[2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] synthetic

23.1. phosphorylation step

As embodiment 16.1., use racemic anti--4-(4-pyridyl)-2-(4-nitrophenoxy)-2-oxo-1,3,2-two oxa-phospha cyclohexanes.

TLC condition: 250 microns silica gel plate; Moving phase=acetone, trans-phosphoric acid ester: rf=0.6, protected ara-C:rf=0.2.

23.2. go to protect step

As embodiment 16.2.

TLC condition: 250 microns silica gel plate; Moving phase=acetone/methanol (1: 1), protected ara-C:rf=0.7, product: rf=0.3.

The purposes embodiment of method of the present invention comprises following each embodiment.Should understand that these embodiment are exemplary, and method of the present invention is not restricted to these embodiment.

For clear and concise purpose, below among the embodiment, 2 (1H)-pyrimidones, 4-amino-1-[5-O-(2R, 4S)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl] (9) be known as compd A, 2 (1H)-pyrimidones, 4-amino-1-[5-O-[(2R, 4R)-2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] (13) be known as compd B, and from 2 (1H)-pyrimidones of embodiment 23,4-amino-1-[5-O-is suitable-[2-oxidation-4 (4-pyridyl)-1,3,2-two oxa-phosphorus heterocycles oneself-the 2-yl]-β-D-arbinofuranose glycosides base] be known as Compound C.

Biology embodiment

Embodiment A: people's hepatomicrosome enzymatic reaction kinetics

Measurement activates into araCMP with compd A with compd B by people's hepatomicrosome, compares with the activation with human liver organization.Utilize the pharmacology inhibitor to estimate the specificity of CYP3A4.

Method:

Compd A and compd B are with the mixture that the contains 2mg/mL people's liver microsomes (ex vivo technique (IVT) of carrying out incubation 5 minutes, products catalogue #X00821, lot number #RQX, the other pond of the mixcibility of 50 donors), 100mM potassium phosphate buffer (Sigma, products catalogue #P3786, lot number #62H0619), the NADPH (Calbiochem of pH7.4 and 1mM, products catalogue #481973, lot number #B38806).Under the condition that does not have NADPH, reaction mixture 700rpm, 37 ℃ of following pre-incubations 2 minutes, causes by the NADPH that adds the 1mM final concentration in Eppendorf Thermal Mixer 5436 then.After 5 minutes, this reaction utilizes the methyl alcohol of 112.5 μ L to be terminated, and sample 14, under the 000rpm centrifugal 10 minutes, utilizes the medium heating to be evaporated to drying 150 μ L supernatant liquors in the Eppendorf Eppendorf centrifuge.Sample is resuspended in (10mM potassiumphosphate, Fisher, products catalogue #P286-1 in the ion pair damping fluid of 60 μ l, lot number #9152328A, 50mM TBAH, Aldrich, products catalogue #17,878-0, lot number #07030KO, 85% phosphoric acid in water with about 3.3mL/L transfers to 4.5 with pH, Chempure, products catalogue #831-621, lot number #M224KBRR), carried out vortex, supersound process about 30 minutes, and 14,000rpm rotated about 10 seconds down.

In order to measure the activation of araCMP, by reversed-phase HPLC (Hewlett Packard1100) analytic sample.Each sample of 50 μ l injected have the anti-guard post of Alltech C-18EPS (products catalogue #32607, and in the Agilent C18 ZorbaxSB-AQ reversed-phase column of 7.5mm * 4.6mm) (products catalogue #883975-914,5 μ m, 4.6mm * 150mm).Under 4 ℃ of 40 ℃ of column temperatures and sample temperatures, with the flow velocity of 1mL/min, with ion pair damping fluid (with reference to the above-mentioned) sample of packing into.AraCMP was carried out elution equably about 7 minutes, then about 2 minutes with methanol wash.Monitor effluent by ultraviolet radiation absorption at 272nm.

By utilizing the cyclamycin (TAO of 0.1 μ M to 100 μ M; SigmaT-6514, lot number #81K1655) or the KETOKONAZOL (KTZ of 0.01 μ M to 10 μ M; Research Biochemicals International, products catalogue (catalog) #K 105, lot number #SJG-597A) pre-incubation suppresses research.TAO and KTZ dissolve in methyl alcohol, therefore, comprise that all samples of control group comprises the methyl alcohol that final concentration is 1% (v/v).For the inhibition research that utilizes TAO, under the situation that does not have substrate, utilize TAO with microsome 37 ℃ of following pre-incubations 30 minutes.Reaction (100 μ l volume) is initiated by adding substrate: this substrate is the fresh aliquots containig of the NADPH of the compd B of the compd A of 1000 μ M or 1000 μ M and 1mM.KETOKONAZOL does not need this pre-incubation process, and therefore above-mentioned reaction is undertaken by when adding substrate KETOKONAZOL being added reaction mixture.

Kinetic parameter is represented (n=2 or 3 independently experiments) with mean value ± standard deviation.The Michaelis-Menten equation, speed=V _Max[S]/[S]+K _m, be used to be used to that (SPSS, enzyme kinetics module Inc.) is calculated V from Sigma Plot _MaxAnd K _mInner clearance rate is passed through V _MaxDivided by K _mAnd obtain.Be used to the IC that suppresses to study ₅₀Value is by having IC ₅₀Half maximum method of interpolation of value is determined to detect this IC ₅₀Value is represented (n=2 or 3 independently experiments) with mean value ± standard deviation.K _iDetermine K by Cheng and Prusoff equation _i=IC ₅₀/ 1+[substrate]/K _m

The result:

Utilize people's hepatomicrosome lot number (lot) #RQX (table 1), compd A has the inside clearance rate higher 2.6 times than compd B.

The kinetic parameter of table 1. activating compounds A and compd B in people's hepatomicrosome

Table 1

	Compd A	Compd B
			The concentration of test, mM	0.25，0.5，0.75，1	0.5，1，3，6
V max，nmol/min/mg	0.11±0.02	0.13±0.02
			K m，mM	1.08±0.27	3.13±0.51
Crin spy (Clint), μ L/min/mg	0.11±0.02	0.042±0.003

By the activation of TAO initiation compd A and compd B, be used for the IC of compd A and compd B simultaneously ₅₀Value is respectively 0.9 ± 0.1 μ M and 0.7 ± 0.4 μ M (table 2).Under the TAO concentration of 100 μ M, observe the inhibition fully (99%) of compd A.The highest TAO concentration with the compd B test is 10 μ M, and it causes 73 ± 2% inhibition.The K of TAO _iValue is similar for compd A and compd B, is respectively 0.5 ± 0.1 μ M and 0.5 ± 0.3 μ M.

Identical with the situation of TAO, KTZ has suppressed the activation of compd A and compd B.IC ₅₀Value is lower than those IC of TAO ₅₀Value: for compd A and compd B, be 0.2 ± 0.1 μ M (table 2).Equally, under the KTZ of 10 μ M, for compd A and compd B, observe 96 ± 3% and 89 ± 1% maximum restraining effect respectively.For compd A, K _iValue is 0.1 ± 0.03 μ M, and for compd B, K _iValue is 0.1 ± 0.05 μ M.

Table 2TAO and KTZ suppress the activation of people's hepatomicrosome

Compound	The concentration (μ M) of test	Inhibitor	IC50(μM)	Ki(μM)	% is suppressed under the peak concentration of inhibitor of test
						Compd A
	1000	TAO	0.9±0.1	0.5±0.1	Under the 100 μ M be 99% (under 10 μ M, being 94%)
						Compd B	1000	TAO	0.7±0.4	0.5±0.3	Under 10 μ M, be 73 ± 2%
Compd A
		1000	KTZ	0.2±0.1	0.1±0.03	Under 10 μ M, be 96 ± 3%
Compd B
		1000	KTZ	0.2±0.1	0.1±0.05	Under 10 μ M, be 89 ± 1%

The result:

By people's hepatomicrosome compd A is activated into CMP with compd B.Compd A activates with the speed higher 2.6 times than compd B.The prodrug activation that KTZ and TAO cause has shown that it is to regulate by CYP3A4.

Embodiment B: the level of single dose liver

After mouse being carried out the administration of intraperitoneal (i.p.) infusion, the activation of drug disposition precursor is measured.

Method:

Will be normally not the male Swiss mouse of fasting (body weight is 25-35g, Harlan SpragueDawley, Indianapolis, IN) use respectively compd A and compd B with 189 and 193mg/kg (is the araC of 100mg/kg corresponding to molar equivalent) carry out intraperitoneal injection.Specific period after injection, come bloodletting with mouse anesthesia and by cardiac puncture.Cut off whole liver, and quick-frozen is in liquid nitrogen, (BrinkmannInstruments, Westbury NY) carry out homogenizing in 10% (v/v) perchloric acid (PCA) of 3 volumes to utilize Polytron homogenizer PT 10/35.Under 2500xg, after centrifugal 5 minutes, the supernatant liquor of 1mL is used the KOH/3M KHCO of the 3M of 0.3mL ₃Neutralize, and carry out thorough mixing.Sample is carried out centrifugal 5 minutes then, and resulting supernatant liquor handles with periodate, its objective is remove in the originality ribonucleotide, otherwise it may disturb the detection of araCTP.For this reason, with the tissue extract of 100 μ l and 0.5M sodium periodate (Al drichChemical Co., the Milwaukee of 4 μ l, WI, lot#08009BU) and the 1.8M methylamine of 10 μ l (Aldrich lot#04526DQ PH5.5), at room temperature carried out incubation 30 minutes.By adding the 1M L-rhamnosyl termination reaction (Aldrich lot#06801JS) of 2 μ l.Resulting sample is analyzed by following HPLC.

In order to measure marrow araCTP, the medullary space of marrow sample from femur is flushed to the preweighted Eppendorf pipe with 1.2ml salt solution.Centrifugal 20-30 after second (the Eppendorf microcentrifuge, 14,000rpm) and remove supernatant liquor, the 3%PCA (v/v) of 12 volumes is joined in the little group of medullary cell.Then sample is carried out vortex, dissolve fully, and carried out as mentioned above centrifugal 10 minutes until this little group.1M KOH/1MKHCO with 30 μ L ₃The supernatant liquor that 90 μ l are extracted is neutralized to pH 7-8, and then carries out centrifugal.As mentioned above, resulting supernatant liquor being carried out periodate handles.

(5 μ m, the ion exchange phase HPLC of 4.6 * 250mm) posts (Hewlett Packard 1050) measures the level of liver and marrow araCTP by utilizing Whatman Partisil 5SAX.At pH 3.5 and contain under 6% ethanol, sample (50 μ L) is expelled on the post in the 1M ammonium phosphate damping fluid of 70% 10mM ammonium phosphate damping fluid and 30%.With nucleoside triphosphate with the flow velocity of 1.25mL/min from the post elution that utilizes linear gradient to 1M ammonium phosphate pH3.5/6% ethanol damping fluid.AraCTP is detected by uv-absorbing (280nm), and carried out elution usually 12 to 13 minutes.By following steps production standard curve: before neutralization, the araCTP of known quantity is added PCA extract from contrast liver or marrow, and correspondingly prepare the HPLC sample.

In order to obtain blood plasma, with blood transport in the micro-collection tube of heparinization, and in the Eppendorf Eppendorf centrifuge, 14, under the 000rpm, centrifugal 2 minutes.Resulting blood plasma is collected, placed on the dry ice, then store down at-20 ℃.On the same day of analyzing, protein precipitates by the 1mL acetonitrile being added in the 100 μ L blood plasma.After centrifugal 10 minutes (the Eppendorf Eppendorf centrifuge, 14,000rpm), supernatant liquor is removed, and in Savant SpeedVac Plus SC110A, carried out drying.With these samples with 110

μ L moving phase damping fluid duplicates (20mM KH ₂PO ₄, pH4.5), carried out supersound process 5 minutes, and carried out centrifugal 30 seconds.(5 μ m, the reversed-phase HPLC (Hewlett Packard 1090) of 4.6mm * 250mm) is analyzed by being equipped with the AlltechC18 post with supernatant liquor.After 50 μ L samples were injected the moving phase damping fluid, acetonitrile concentration increased to 10% in 10 minutes, increased to 50% then in 15 minutes.The elution time of araC and prodrug is respectively about 3 minutes and 19 minutes.AraC and prodrug detect by absorbing under 280nm, and the typical curve that obtains by the spike plasma sample with above-mentioned processing carries out quantitatively.

The result represents with the standard error of mean value ± this mean value.Measure ANOVA by multiple and carry out data analysis, then, be used for the Tukey HSD post-hoc test of comparing at single time point place in due course.P value less than 0.05 has been considered to significance,statistical.Dotted line among the figure shows the LOQ value (limiting the quantity of) of utilizing given HPLC method.

The result:

Compd A and compd B generate high-caliber araCTP in liver (Fig. 1 a) injected back 60 minutes, for compd A and compd B, reached peak value 69.89 ± 6.37 and 27.00 ± 4.04 nmole/grams respectively.Compd A produces the araCTP level of the liver higher than compd B obvious (p＜0.05), except 4 hours time point.For compd A and compd B, drug plasma precursor level is similar (p＜0.01; Fig. 1 b).In giving the mouse of compd A the araC level of blood plasma higher (for 0.5,1,2 hour, p＜0.01; Fig. 1 c), the araCTP level of liver has more dependency than the prodrug level of blood plasma.This araC that shows blood plasma may be the level that comes from the araCTP of liver, rather than comes from the degraded of blood plasma Chinese traditional medicine precursor.In all samples of the liver target that demonstrates, the araCTP of marrow is equal to or less than quantitative level (3nmol/g).

Conclusion:

When by the administration of intraperitoneal (i.p.) fast infusion, compd A and compd B produce the araCTP of conspicuous level in liver.Compd A produces about 2 times of araCTP of the liver be higher than compd B.In the marrow of handling with one of two kinds of compounds, do not detect AraCTP.For two kinds of prodrugs, drug plasma precursor level is similar, and still the araC level of the blood plasma of usefulness compd A is high about 2 times.Based on the dependency of the araCTP level of the araC level of blood plasma and liver, infer that the araC that blood plasma may generate araC and araC by the araCTP dephosphorylation and expose from liver cell and return the blood plasma and produced by liver araCTP.

Embodiment C: the araCTP of liver when sending by continuous venoclysis

Become the keeping of araCTP level of araCTP and liver to test to the activation (activation) of prodrug in many rats, these mouse are used for the instrument that medicine sends by installation and carry out the vein continuous infusion.

Method:

Male Simonsen Albino rat (Sprague Dawley, derive from SimonsenLaboratories, Inc., Gilroy GA), contains 150mg ketamine (" Vetamine " by intraperitoneal injection 0.25ml, 100mg/ml, Phoenix Scientific, Inc.St.Joseph, MD), the xylazine (100mg/ml of 10mg, Butler company, Columbus, OH) and 5mg morphine (15mg/ml, Marsam Pharmaceuticals, Inc., Cherry, Hill, narcotic mixture NJ) is anaesthetized.To be full of the blunt PE-50 conduits of brinish (Intramedic, Becton Dickinson, Sparks MD) is inserted in the jugular vein, take out between the scapula, and with the CI system (Lomir Biomedical, Inc., the Malone that limit rotation, NY and Harvard Apparatus, Inc., South Natick MA) connects.Pipeline is filled and is sealed with heparinized saline, and 1-7 angel animal gets well.The animal that installs instruments carries out infusion according to following proposal.

Compd A: with 200mg/kg/24 hour araC (CE) equivalent infusion compd A 2,4,8,16,24 or 48 hours (the n=4-5/ group is except that n=1 in 48 hours groups).

Compd B: with 200mg/kg/24 hour CE infusion compd A 2,4,8,16,24 or 48 hours (n=3-5/ group).In addition, compd A is with identical dosage infusion 4 hours (n=3).

Compound C: with 200mg/kg/24 hour CE infusion compd A 2,4,8,16,24 or 48 hours (n=3-5/ group).

Dose response: with 144 and 200mg/kg/24 hour CE infusion compd A 4 hours (n=4-5/ group).With 50,100 and 200mg/kg/24 hour CE infusion compd A 24 hours (n=3-5/ group).

Behind the infusion during the difference, animal is anaesthetized by the above-mentioned narcotic mixture of injection in the conduit.Blood sample is by utilizing No. 23 (gauge, specification) syringe needle cardiac puncture samplings, and this syringe needle is connected with the 3cc syringe of heparin coating.Utilizing freezing clamp (freeze-clamp) that the liver sample is cut off also quick-frozen carries out in liquid nitrogen and as described in Embodiment B.As described in Embodiment B, the liver araCTP level of sample is analyzed.As described in Embodiment B, blood plasma and marrow sample are collected, and the araC level of drug plasma precursor, blood plasma and the araC level of marrow are analyzed.

The result:

With 200mg/kg/24 hour continuous infusion compd A, in the time of 4 hours, can reach the araCTP level (12.84 ± 1.50 nmole/gram) of stable state liver and remain and to keep mean value 13 ± 1 nmole/grams in 44 hours.For all three compounds, in the scope of 30-60 μ M, in the treatment in 48 hours, the level of drug plasma precursor is similar.Compd B is compared with compd A with Compound C, and the araCTP that produces in liver is few.These researchs are all independently carried out; Thereby, in order to control the variation in the possible test, give a treated animal in the compd B group with compd A.The horizontal infusion of the liver araC of these animals is after 4 hours, with the identical time point of research in 48 hours of compd A obtain similar, the drug plasma precursor level of two kinds of medicines is consistent, and it shows that the difference of the araC level of liver is not because the pharmacokinetics fine difference of two kinds of medicines causes.In all samples, the araC level of marrow is lower than quantitative level (3nmol/g).

The horizontal dose response of the araCTP of liver is by in 24 hours time, with 100 or 50mg/kg/24 speed at one hour rating infusion compd B, or in 4 hours with 144mg/kg/24 hour, the infusion compd B.Shown in Fig. 2 b, the araC level of liver all has dosage susceptibility for two kinds of prodrugs.

Conclusion:

Continuous infusion compd A, B or C keep the araCTP level in the liver.Under condition with similar drug plasma precursor level, to compare with compd B or Compound C, the araC higher level that gives the compd A liver shows that difference is because the difference of active rate causes, rather than because the difference of pharmacokinetics.

Embodiment D: liver target effect

Compare by araCTP and the active metabolite in other organs, send the liver specificity of araCTP in in-vitro measurements by CYP3A activatory prodrug liver.Especially, estimate the liver target effect with respect to marrow, this is because marrow is the toxicity target organ of cytosine arabinoside.

Method:

Tissue distribution research is carried out in Embodiment B and described mouse of C and rat.In these researchs, liver, blood plasma and marrow sample are to obtain in ptomatopsia, with the evaluating liver targeting.Utilize parent compound, cytosine arabinoside carries out above-mentioned similar research, to show the effectiveness as liver target reagent.

The result represents with the standard error of mean value ± this mean value.In the time of suitable, the final time point from 0 time point to research calculates AUC.For every kind of compound, use the AUC of the AUC of liver araCTP, or the AUC of liver araCTP calculates liver target action index (LTI) divided by the araCTP (marrow LTI) of marrow divided by blood plasma araC (blood plasma LTI).With regard to continuous infusion studies, with stable state araCTP level value divided by steady state blood plasma araC level value.

The result:

As described in Embodiment B, when giving mouse or rat, can in liver, detect high-caliber araCTP with compd A, compd B, Compound C.Table 3 has been summed up the peak level and the AUC of intraperitoneal (i.p.) fast infusion research.In all research, marrow araCTP is equal to or less than quantitatively (3nmol/g), shows to have good liver target effect.On the contrary, if the cytosine arabinoside administration in an identical manner of 100mg/kg then can detect higher levels of araCTP in marrow.There is not evidence to show that marrow directly becomes araCMP with compound activating; The activation that any potential araCTP may come from the araC that absorbs in the blood plasma and changed into araCTP by phosphoric acid in marrow (activates, activation).The araC that is detected in the blood plasma of these animals neutralizes all researchs appears relevant with liver araCTP level.As shown in table 3, after intraperitoneal gave compd A or compd B, the blood plasma araC AUC value of mouse and rat was at 3-22 μ M ^*Hour scope in.By obtaining the liver target action index (LTI) that the liver araCTP that exposes at experimental session and the ratio of blood plasma araC are estimated, the targeting that shows liver is 19.2-27 times with respect to peripheral exposure.These values are higher 100 times than the LTI of cytosine arabinoside.When compd A, compd B, Compound C are sent by continuous venoclysis, steady-state value can be carried out similar comparison (table 4).The scope of LTI is in 5-＞12 in the above-mentioned research, and its LTI than cytosine arabinoside is high 100 times.

Table 3: the external Mouse Liver targeting of the 4-pyridyl of cytosine arabinoside (compd A, compd B) hepatic targeting drug precursor.These compounds carry out administration with 100mg (cytosine arabinoside equivalent)/kg by the Fast IP injection

Compound		Liver araCTP was at 1 hour peak value (nmol/g)	Liver araCTP AUC 0-4 hour (nmol/g ＊hr)	Drug plasma precursor AUC 0.5-4 hour (nmol/g ＊hr)	Blood plasma araC AUC 0-4 hour (μM ＊hr)	LTI (liver/blood plasma) (nmol/g/ μ M)
							Compd A	Mouse	96	225	138	8.3	27
Compd A	Mouse	70	149	156	7.6	19.5
							Compd B	Mouse	27	68	102	3.5	19.6
Compd A	Rat	267	442	147.1	21.9	19.2
							AraC	Mouse	7.6	＜19.2	N/A	121.2	＜0.2

Table 4: cytosine arabinoside hepatic targeting drug precursor 4-pyridyl (compd A, compd B or Compound C) rat continuous infusion.Prodrug is with 200mg (cytosine arabinoside equivalent)/continuous venoclysis of kg/day. ^*AraC is with the 2000mg/ka/day administration

Compound (research)	Liver was at 4 hours araCTP (nmol/g)	Liver was at 24 hours araCTP (nmol/g)	Drug plasma precursor stable state (μ M)	Blood plasma araC stable state (μ M)	At 24 hours LTI (liver/blood plasma) (nmol/g/ μ M)
						Compd A	12.8	12.0	40-50	～1.6	7.5
Compd B	6.4	5.9	25-35	＜0.5	＞12
						Compound C	9.9	7.8	35-60	～1.5	5
AraC *	At 2 hours was 2.3	＜2.26	N/A	～300	＜0.01

Conclusion:

Compd A, compd B, Compound C can make araCTP target liver effectively, can be about when peritoneal injection 20 times of ground reduce peripheral exposure, when continuous intravenous infusion administration, can reduce peripheral exposure in about 10 times of ground.Targeting shows and is higher than araC 100-1000 raising doubly.

Embodiment E: aqueous stability

The aqueous stability of compound is measured with the function of pH and buffer concentration.

Method:

Compd A and compd B are dissolved in the water with 500 μ M (0.23mg/mL), as liquid storage.To do incubation in the bath insulation can (products catalogue #11-718-2) in Fisher Scientific under 37 ℃ through the solution (50 μ M) of dilution, and collect a sample in per 24 hours, and store down in-80 ℃.Plasma sample (100 μ L) the acetonitrile quenching of 1mL.After collecting all plasma samples, sample is thawed, with 14, centrifugal 10 minutes of 000rpm speed is removed the 1mL supernatant liquor in the Eppendorf Eppendorf centrifuge, utilizes medium heating (about 37 ℃) to evaporate 2 or 3 hours to dry.Before the HPLC sample introduction, sample is resuspended in the moving phase damping fluid of 120 μ L.

Sample is analyzed through HP1090 or HP 1100 (Hewlett Packard) HPLC; what use is C-18Alltech Econosphere post; 150mm * 4.6mm (products catalogue #70065), and C-18Alltech Econosphere guard column, 7.5mm * 4.6mm (products catalogue #96121).Sample utilizes the elution of methyl alcohol gradient liquid: 5 minutes is 0%, and being increased to 10%, 20 minute in 10 minutes then and being 30%, 25 minute is 60%.Next time before the sample introduction, make post balance 10 minutes in 0% methyl alcohol.Flow velocity is 1mL/ minute, and column temperature is 40 ℃.The prodrug elution is monitored by the 280nmUV absorption in the time of about 19.5 minutes.For the prodrug under the aqueous conditions, can adopt above-mentioned identical method, but methyl alcohol gradient liquid is: be increased to 30% when being increased to 10%, 20 minute when being 0%, 10 minute in 5 minutes, about 20 minutes of prodrug elution.

The result:

Compd A and compd B are all very stable in the aqueous solution, in the time of in the potassiumphosphate (Pi) of 10mM, pH7.4 and in the Citrate trianion of 10mM, pH5.0, and t ₉₀＞6 days.The stability of compd A in 100mM Pi, pH7.4 is equally＞6 days, but the stability of compd B in sort buffer liquid is poor slightly, t ₉₀It is 4 days.These two kinds of compounds all descend t in the Citrate trianion of 100mM, the stability in the pH5.0 solution ₉₀Be respectively 2 days and 1.7 days.

Conclusion:

4-pyridyl prodrug stable fairly good, but in having the low pH solution of high buffer intensity, these compound stabilities are poor slightly.

Embodiment F: solvability

Under multiple condition, measure the solvability of compd A and compd B, with the possible formulation (formulation, prescription) that is identified for vivo medicine-feeding.

Method:

The solvability of compd A and compd B is estimated in 0.5M Citrate trianion, pH7.0 and 0.5M Citrate trianion, the pH4.0 damping fluid at 0.5M Pi, pH8.0.For in these three buffer systems each, the saturated solution of compd A and B prepares as follows.30mg compd A and 150mg compd B target body are transferred in the clean vial of independent 4cc (vial), in each bottle, add the 1.0mL damping fluid respectively.With the sealing of final bottle, in 25 ℃ of vibration balances at least 24 hours.When cultivate finishing, the nylon injection filter (syringe filter) by 0.45 μ m removes by filter not melt into branch.The sample that obtains is analyzed through the described HPLC method of embodiment E.

The result:

The solubleness of compd A is respectively: be for being 45.8mg/mL in the damping fluid of 2.6mg/mL, pH 4.0 in the damping fluid of pH 8 in the damping fluid of 2.9mg/mL, pH7.0.The solubleness of compd B is respectively: be to be 9.0mg/mL in the damping fluid of 10.2mg/mL, pH 7.0 in the damping fluid of pH 8, and, in the damping fluid of pH 4.0 94.6mg/mL.

Conclusion:

Compd A is relative with compd B to be dissolved in the neutral pH solution, and the solubleness of compd B is higher than compd A.When preparing under pH≤4 conditions, the solubleness of two kinds of compounds increases greatly.

Embodiment G: plasma stability

Measure compd A, compd B and the stability of Compound C in blood plasma, surpass 6 days, whether stable in vivo to determine these compounds.

Method:

For (for example containing heparin, anti-coagulant) male and female coexistence (Male-andfemale-pooled) human plasma (Bioreclamation Inc., Hickville, NY products catalogue #HMPLNAHP, lot number #BRH02236), the pH of all blood plasma samples is all 8.3.Compd A and compd B are soluble in water with 500 μ M (0.23mg/mL), as liquid storage, in plasma sample, be diluted to 50 μ M, and doing incubation in the bath insulation can (products catalogue #11-718-2) in Fisher Scientific under 37 ℃, collected a sample in per 24 hours, and in-80 ℃ of storages.Plasma sample (100 μ L) the acetonitrile quenching of 1mL.After collecting all plasma samples, sample is thawed, in the Eppendorf Eppendorf centrifuge with 14, centrifugal 10 minutes of the speed of 000rpm.Remove the supernatant liquor of 1mL, utilize medium heating (about 37 ℃) to evaporate 2 or 3 hours to dry.Sample is resuspended in the moving phase damping fluid of 120 μ L, carries out HPLC then and detects.For plasma sample, with 40 μ L or 50 μ L samples and moving phase damping fluid, pH is that the potassium phosphate buffer (FisherScientific, products catalogue #P261-1, different lot numbers) of 6.2 20mM injects together as the described post of embodiment E.Every other HPLC analyzes as described in the embodiment E.

The result:

The t1/2 of all samples in 37 ℃ human plasma is 6 ± 1/2 days.

Conclusion:

The significant reduction takes place in compd A, compd B and Compound C transformation period＞1 in human plasma day in vivo as expection.

Embodiment H: rat acute toxicity

Come tolerance and the security of assessing compound A under 2000mg/kg/24 hour dosage by 24 hours continuous intravenous injections.

Method:

Male and female Simonsen rat is injected continuously as installing instruments as described in the Embodiment C.Treat the same day, rat is hooked on the lasting infusion tether (constant infusiontether), make it freely obtain water and food.Utilize 2.08ml/kg/ hour flow velocity, give three male and three female rats the compd A of 2000mg/kg/24 hour dosage.Compd A is dissolved in the water with the concentration of 42.5mg/mL, with 1M HCl pH is transferred to 4.NaCl with 5M is transferred to about 300mOsm with osmolarity.In contrast, two male rats phase in the contemporaneously infusion pH be 4 physiological saline.Signs of periodic observation rat and activity, and attention per their behavior of 1～3 hour record during working days.

By the flow velocity of Fluotec 3 atomizers 5% isoflurane (Abbot Laboratories, NDC 0074-3292-02), 100% oxygen delivery is arrived in the chamber (chamber), with above-mentioned Animal Anesthesia with 1.5～2 liters/minute.No. 23 pins with linking to each other with the 3cc syringe that scribbles heparin obtain blood sample by the cardiac puncture method in the intravenous injection process.The blood sample of each animal is divided in and contains K ₂The micro tube of-EDTA (Becton Dickinson, 36/5974) microtubule (the Becton Dickinson that, has gel serum separator (gel separator for serum), 36/5956) and contain in the microtubule (Becton Dickinson, 36/5958) that is useful on the Lithium heparinate of collecting blood plasma.If possible, from bladder, remove urine by No. 23 pins that link to each other with the 3cc syringe.Tissue sample is according to following order collection: liver, kidney, small intestine, bladder and marrow.The small intestine sample is divided into three equal parts, represents duodenum, jejunum, ileum respectively; Manually remove fecal matter with blunt tweezers with slight sliding pressure (gentlesliding pressure).For each tissue, a part of sample places 10% formalin buffer, to carry out histology.In order to have measured the bony nodule myelocyte, with PBS (the w/o Ca of 1ml ⁺⁺Or Mg ⁺⁺) flushing femur medullary space obtain the marrow sample, strictness is smashed to pieces, vortex is with isolated cell.

In order to carry out cell counting, the bone marrow irrigation liquid that blood of handling through EDTA or the PBS of 50 μ L suspended comprises 0.19mg/mL Viola crystallina (Sigma#C-1658 with 450 μ L or 950 μ L respectively, in the nuclear staining solution of 1M acetic acid solution lot number 112H3660), under room temperature, cultivated at least 5 minutes then.Through behind the short period of time vortex, the aliquots containig of 10 μ L said mixtures is placed on the hemocytometer, use Nikon Optiphot-2 microscopic counting.Monocyte (lymphocyte and monocyte) number and the polymorphonuclear leukocyte number (PMN, comprise neutrophilia, have a liking for eosin, basophilic granulocyte) of counting in the blood sample under the 40x object lens.Counting has the bony nodule myelocyte under the 10x object lens.

(San Diego CA), carries out hemanalysis, comprises analyzing red corpuscle and platelet count, H﹠H to deliver to LabCorp through the blood sample of EDTA processing.Carry out the serum chemistry analysis at LabCorp equally.To be numbered through the tissue sample of handling mouse, (Mountain View, CA) the painted tissue slice of preparation Hematorylin/eosin is so that histopathological analysis to deliver to Comparative Biosciences then in formalin.

The result:

The laboratory animal of observing drug treating group, vehicle treated group or untreated control group draws, the compd A that each treated animal can tolerate high doses, and do not have significant behavior difference.Dissect and find, all stomaches through dabbling animal all are empty, do not observe other differences.Do not survey before the injection or body weight and the feed situation of the animal in the injection process.

Blood, marrow, tissue slice can be according to a plurality of parameter evaluations.One is to be exposed to potential liver toxicity or the overt toxicity relevant with formulation behind the compd A of high density.As previously mentioned, after handling in 24 hours, do not observe tangible toxicity.All serum chemistry and hematologic parameter are all in normal range.Compare with disclosed document, have only a few sample to produce tangible outlier, but itself and untreated control group do not have difference or do not observe xicity related.In the serum parameters that all have been estimated, have only serum triglyceride to exceed normal range.

Surveying blood mononuclear cell, PMN and marrow number is the potential acute effect of testing on the hematology.We are expected in the short-term processing and do not have significantly or evident difference.In fact, the monocyte of untreated fish group, vehicle treated group and drug treating group and PMN cell do not record that there were significant differences.The bony nodule myelocyte number that has of the animal of vehicle treated and drug treating does not have the significant difference of opinion yet.

The tissue sample that is taken at liver, kidney, small intestine (duodenum, jejunum, ileum) and bladder there is no significant histopathology and finds through pathology detection in all samples.

Conclusion:

When continuous (at least 24 hours) high dosage venoclysis compd A, rat can tolerate compd A well.

Example I: 5 days safety pharmacologies of mouse

By the repeated doses research in 5 days of normal male rat, come the security of assessing compound C with respect to parent compound araC.

Method:

AraC is available from Sigma Chemical Co. (St.Louis, MO, products catalogue #C1768, lot number 39H5962).Compound C and araC all are dissolved in the aseptic physiological saline.Use the medication preparation liquid storage of weighing in advance every day.Except maximum concentration, all concentration all can make by adding extra physiological saline dilution storage liquid.The medicine of usefulness does not freeze in refrigerator at once, and uses in 24 hours.All medicines are with cytosine arabinoside molar equivalent (CE) administration.

Male NIH Swiss Webster mouse (body weight 25～33g, Harlan SpragueDawley, Indianapolis, IN) in the time of 0～4 day, intraperitoneal (i.p.) injection Compound C, araC or carrier every other day carries out after animal is gently circulated 3～4 hours of (vivarium light cycle) beginning greatly.The dosage of Compound C is 1000,300,100 and 30mg/kg nucleosides equivalent/sky (equal 1848,554,185 and 55.4mg/kg/ days), and the araC dosage is 100,30,10 and 3mg/kg/ days.Carrier is a salt solution.In the time of 0～4 day, weigh before the compound administration, get in the time of 5 days and weigh before killing.All mouse are bloodletting under (after 23～25 hours of last i.p. dosage) halothane anesthesia in the time of the 5th day, and takes off neck and get extremely.Take from the hematochemistry parameter and the hematologic parameter of the blood of each animal as analysis as described in the embodiment H.Cut the hepatic tissue sample, be fixed in 10% the neutral buffered formalin, the PBS that does not have calcium and magnesium with 1mL washes right femur and obtains marrow, and as described in embodiment H karyocyte is counted.

(LabCorp, San Diego CA) carry out analysis of Hematology Changes, comprise red corpuscle and platelet count, and hemochrome and hematocrit detect to deliver to LabCorp through the blood sample of EDTA processing.Carry out the chemical analysis of serum simultaneously at LabCorp.To taking from the tissue sample numbering of utilizing the mouse that carrier or 100mg/kg araC or 1000mg/kg CE Compound C handled, and (MountainView CA) carries out Hematorylin/eosin dyeing and histopathological analysis to deliver to Comparative Biosciences in formalin.Being numbered of histopathology sample: #1-7, Compound C, 1000mg/kg/ days; #33-40, araC, 100mg/kg/ days; #65-72, saline vehicle.

The result represents that with mean+/-standard error the number of animals of every kind of dosage group is 5～8.For selected hematologic parameter, represent with the per-cent (%) of the mean value of vehicle group.(one-way ANOVA) carries out statistical analysis with the unit variance analysis, and carries out the difference between Dunnett ' s T check (post-hoc test) compare group and variant dosage group.The P value can be thought significance statistically less than 0.05.

The result:

Mouse is injected the last time and gets mouse extremely after 24 hours with the continuous intraperitoneal injection Compound C of different dosage and araC 5 days.Subordinate act and total appearance, obvious disease does not appear in experiment mice.And the maximum dose level group of araC occurred gradually with statistical significance on weight loss (Fig. 3 a, 100mg/kg/ days), Compound C is not significant to be renderd a service, even under the maximum dose level situation (1000mg/kg/ days nucleosides equivalents) (Fig. 3 b).

With 30 with during 100mg/kg/ days dosed administrations, araC has reduced karyocyte number, cycle P MN number, monocyte number and platelet count (Fig. 4) significantly.Under the maximum dose level situation of araC, medullary cell content is reduced to through 11% of the animal of vehicle treated.Compound C reduces situation and then needs 30 times dosage (Fig. 4 a) if this karyocyte number occurs.Compare with araC, the Compound C under any dosage can not have influence on PMN, monocyte or thrombocyte (Fig. 4 b～4d).

AraC and Compound C can both remarkably influenced red blood corpuscle parameters, differ 10 times but influence dosage.Under the dosage of two maximum dose levels 30 and 100mg/kg/ days, AraC has reduced RBC number, hematocrit and hemochrome significantly.Under 10mg/kg/ days araC dosage, the slight change takes place in hematocrit.Compound C need under high 10 times dosage could similarity degree these parameters that influences.

The chemical analysis of serum shows that most of analyte is not subjected to the influence of any compound.Specifically, liver function indexes (bilirubin, aspartic transaminase and alanine aminotransferase) and vehicle treated group, araC treatment group or the Compound C animal of handling is similar.Only the alkaline phosphatase of the araC of high dosage group significantly descends.The albumin and the BUN of all treatment group do not see change.The sarkosine and the glucose of araC maximum dose level treatment group (100mg/kg/ days) have reduced.The glucose of araC 30mg/kg/ days treatment group and Compound C 1000mg/kg/ days treatment group reduces.It is the unique serum chemistry parameter that can observe variation that the dextrose equivalent value of Compound C treatment group changes.

Liver, kidney, the small intestine sample of the formalin fixed of the mouse that the pathologist of Comparative Biosciences handles the CE Compound C of taking from the araC that utilizes carrier or 100mg/kg or 1000mg/kg are estimated, and do not find to exist in any sample the relevant histopathology of toxicity to change.

Conclusion:

According to hematology terminal points (endpoint) such as bony nodule myelocyte, periphery PMN and monocyte being arranged as can be known, under the condition of 5 days repeat administrations, the security of Compound C is more than or equal to 30 times of araC.Compound C or its parent compound araC do not observe tangible hepatotoxicity.

Claims (10)

1. a Formulae II I compound or its pharmacy acceptable salt:

Formulae II I.

2. the compound of Formulae II I or its pharmacy acceptable salt are used for the treatment of application in the medicine of disease of tissue of animal expression P450 in preparation, and described Formulae II I is:

Formulae II I.

3. application according to claim 2, wherein, the disease in the tissue of described expression P450 is selected from the group by liver cancer, colorectal carcinoma and liver infection group and viral infection group one-tenth.

4. application according to claim 3, wherein, the disease in the tissue of described expression P450 is a hepatocellular carcinoma.

5. application according to claim 3, wherein, the disease in the tissue of described expression P450 is a colorectal carcinoma.

6. the compound of Formulae II I or its pharmacy acceptable salt are used for preventing the application of the medicine of described cancer return in preparation after the cancer of the tissue of the expression P450 of animal is carried out medical treatment or surgical intervention, and described Formulae II I is:

Formulae II I.

7. application according to claim 6, wherein, described animal is not cancered animal.

8. application according to claim 6, wherein, described animal is the animal that cancer symptoms is alleviated, and described medicine is used to prevent further developing of described cancer.

9. the compound of Formulae II I or its pharmacy acceptable salt are used for improving the application of the medicine of cytosine arabinoside therapeutic index in preparation, and described Formulae II I is:

Formulae II I.

10. pharmaceutical composition comprises compound or its pharmacy acceptable salt of the Formulae II I of medicinal significant quantity, and pharmaceutically acceptable vehicle, and described Formulae II I is:

Formulae II I.

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