CN101022798A - Sulfonyl hydrazines as hypoxia-selective antineoplastic agents - Google Patents

Abstract

Novel phosphate-bearing prodrugs of sulfonyl hydrazines have the formulas (I), (II), (III) and (IV). Pharmaceutical compositions and uses thereof in the treatment of cancer are claimed. The aforementioned prodrugs include enantiomers, stereoisomers and tautomers thereof, as well as pharmaceutically acceptable salts or solvates and metabolites from all stages. The aforementioned prodrugs are preferentially activated in hypoxic tumors and can be given either alone, or in combination with other anticancer agents or with phototheraphy or radiotherapy. Where R = C1-10 alkyl, or C1-10 haloalkyl (preferably containing no more than 5 halogen groups, preferably 2-chloroethyl); R' and R'' are independently C1-10 alkyl, or C5-20 aryl or heteroaryl (preferably methyl); R1 is H, C1-10 alkyl, C1-10 alkoxyl, C5-20 aryl or heteroaryl or C5-20 aroxyl or heteroaroxyl (preferably methyl and ethyl); X is O, NH, or NR (preferably O); Y is (CH2)n , where n = 1, 2, 3, 4 or 5 (preferably n = 2 and 3); or Y = aryl or heteroaryl (preferably para-phenyl); A = CH, or N (preferably CH); and B = CH=CH, O, S, NH, or NR (preferably CH=CH); or pharmaceutically acceptable salts, solvates, polymorphs or metabolites, thereof.

Description

Sulfonyl hydrazines as hypoxia-selective antineoplastic agents

Invention field

The present invention relates to the sulfohydrazide prodrug (SHPs) of presenting of metabolism activation to mammiferous anti-tumor activity.The treatment neoplasia especially comprises method for cancer, is extention of the present invention.

Related application

The application requires the priority of following application: in the U.S. Provisional Application US60/611 of JIUYUE in 2004 application on the 21st, 623; In the U.S. Provisional Application US60/615 of application on October 1st, 2004,419; In the U.S. Provisional Application US60/616 of application on October 6th, 2004,500, any one of these applications all quoted at this as a reference.

Background of invention

The elimination of solid tumor needs some strategies to handle the population that can survive that malignant cell is arranged in the anoxic zones of these tumors.Insufficient and inorganized vascular system is the principal character of the tumor mass that increases rapidly, it causes the oxygenation deficiency, high interstitial pressure, with contain low, dormancy or the growth cycle of oxygen slowly and away from the cell mass of blood supply, therefore in solid tumor insufficient vascular system cause hypoxia and inaccessible drug cell poison level (Hockel, etc. Cancer Res.1991,51:6098).Radiation can not produce enough oxygen-derived free radicals with cause cytotoxicity (Brizel, etc. Radiother Oncol.1999,53:113), be invalid therefore at these regional radiotherapies.And the activity of cell toxicity medicament also can weaken.Therefore, these regional cells cause the recurrence of disease continually.With oxygen dependence sexual cell toxitherapy, as the X-irradiation that produces the oxygen-derived free radicals that destroys cell DNA, and conventional targeting is after the chemotherapy of part oxygen enrichment, that increase rapidly of tumor mass, resistance hypoxic cell part can form once more tumor (Stratford, etc. Anticancer Drug Des.1998,13:519).And hypoxic cell is subjected to promoting to make a variation the optionally domination of environment, and these variations cause tumor to the enhanced gradually phenotype progress value of aggressivity.For example, hypoxia is chosen in the defective cell in apoptosis aspect of p53-mediation, improves aberration rate, rise relates to drug resistance, blood vessel takes place, and the gene of tumor invasion (comprising HIF-1 α), and therefore be attended by the stronger phenotype (Ashur-Fabian etc. of transitivity Pro Natl Acd Sci USA.2004,101:12236).

As the cytotoxic prodrug of hypoxia-selective generally must be that the single electron reductase is (as NADPH: substrate cytochrome (P450) reductase).In the presence of oxygen, the reductive prodrug group (radical) of single electron, redox cycle is got back to the parent prodrug, the release that prevents to activate the development of cascade and have Cytotoxic destruction dna material.Under hypoxia condition, the chemical action that the further reduction of free radical anion changes prodrug with the release that allows the cell toxicant material (Yang, etc. Cancer Res.2003,63:1520).Nitryl aromatic family and nitro-aromatics promptly experience single electron revert to nitro free radical anion (radical anion) (Korbelik, etc. Mutal Res, 1980,78:201).These molecules rapidly and oxygen react and produce parent molecule once more.Yet in the presence of oxygen, their further reduction produce hydroxylamine derivative and finally are the aniline form then.And nitro is a strong electron-withdrawing group, and the azanol base is strong donor residues.This causes the main change of aromatic rings or heterocyclic chemical action, triggers the release of activation cascade and parent drug.

As alkylating agent, a series of novelties 1 that can produce active chlorine ethylization material have been developed recently, 2-two (sulfonyl) hydrazine prodrug (SHPs) (Sartorelli etc., US006,040,338,2000; US005,637,619,1997; US005,256,820,1993; US005,214,068,1993; US005,101,072,1992; US004,849,563,1989; And US004,684,747,1987).Shown anti-tumor activity be since chloroethylation and DNA subsequently crosslinked cause (Shealy etc., J Med Chem.1984,27:664).

1; 2-two (mesyl)-2-(2-chloroethyl)-hydrazine carboxylic acid 1-(4-nitrobenzophenone) ethyl ester (KS119); the lead compound of current SHP series needs the nitroreduction of enzyme to produce alkylation material (alkylatingspecies) 90CE, as shown in fig. 1.Therefore KS119 utilizes hypoxia, the reproducibility environment of solid tumor, therefore between the cell of normal oxygen enrichment tissue and hypoxic tumor cells the available difference of generation (Shyam etc., J Med Chem.1999,42:941; With Seow etc., Proc Natl Acad Sci USA.2005,102:9282).

Yet KS119 is quite insoluble in aqueous solution, even in for the common solution system (as Polyethylene Glycol (PEG) and ethanol) that adapts to the clinical needs of this medicine, do not have enough dissolubility (＜5mg/mL).Therefore, our purpose is the analog of synthetic KS119, makes (a) can improve dissolubility and the stability in the aqueous solution of pH value 3 to 8 in its water; (b) can form the chloroethylation material; It is active (c) to keep hypoxia to select.

For the present invention, we think water miscible and satisfy above-mentioned condition according to The compounds of this invention.The example of such SHP (KS119W) is that reason is as follows at the analog of the KS119 that comprises phosphate (phosphate-containing) shown in Fig. 2:

(a) usually, phosphorous acidic group (phosphate-bearing) analog, the salt form that comprises it should have good water-solubility and in the stability of pH neutral;

(b) biotransformation according to chemical compound of the present invention passes through alkali phosphatase (AP) break oxygen-phosphorus key to form the phenols intermediate, as shown in Figure 2.

(c) biotransformation of 2-nitrophenols intermediate selectivity activation under low-oxygen environment is to produce hydroxylamine derivative or aniline form.

(d) intermediate of above-mentioned amino analog experiences the formation that fracture causes chloroethylation material (90CE) subsequently.Under the low-oxygen environment, only disengaging of 90CE takes place when the reduction of nitro.

(e) chemical compound of the present invention is considered to the prodrug of 90CE, and 90CE has been confirmed to be the alkylating agent of the broad spectrum anticancer of a kind of anti-tumor disease state (comprise, for example, multiple solid tumor).

Goal of the invention

An aspect of invention an object of the present invention is to provide chemical compound, the method for pharmaceutical composition and treatment tumor (cancer that comprises the animal and human).

Another fermentation of invention an object of the present invention is to provide the method for the treatment of tumor, and it utilizes and present good anticancer property and the toxic characteristic of enhanced activity, pharmacokinetics, bioavailability and reduction in low-oxygen environment.

Another purpose of invention provides compositions and the method for the treatment of cancer of the treatment of traditional chemical therapy reagent opposing, and by with other anticarcinogen or with phototherapy and X-ray therapy treatment of cancer with combinations.

In them one or multinomial and/or of the present invention other purpose can be by obtaining rapidly in the following description of the invention.

The invention summary

The present invention relates to chemical compound according to structure I or II:

Wherein R is C _1-10Alkyl, or C _1-10Haloalkyl;

R ' or R " are C _1-10Alkyl, or C _5-20Aryl or heteroaryl;

R ₁Be H; C _1-10Alkyl, C _1-10Alkoxyl;

X is O, NH or NR;

R (P) is the alkyl of phosphorous acidic group, and for example, R (P) is Y ' OPO (OH) ₂, wherein Y is (CH ₂) _n, O (CH ₂) _n, NH (CH ₂) _n, NR (CH ₂) _n, n is 1-5, Y is aryl or heteroaryl;

Ar (N) is the aryl that contains nitro, for example,

Wherein A is CH, CR or N; And B is CH=CH, O, S, NH or NR; Reaching Ar (NP) is phosphorous acidic group and the aryl that contains nitro, for example,

Wherein A is CH, CR or N; And B is CH=CH, O, S, NH or NR; And Y is (CH ₂) _n, O (CH ₂) _n, NH (CH ₂) _n, NR (CH ₂) _n, OCOO (CH ₂) _n, NHCOO (CH ₂) _nN is 1-5, or its officinal salt, solvate, polymorph or metabolite.

The present invention also relates to chemical compound suc as formula I, II, III and IV

R=C wherein _1-10Alkyl, or C _1-10Haloalkyl (preferably contain and be no more than 5 halogen groups, more preferably 2-chloroethyl);

R ' and R " are independent C _1-10Alkyl, or C _5-20Aryl or heteroaryl (preferable methyl);

R ₁Be H, C _1-10Alkyl, C _1-10Alkoxyl, C _5-20Aryl or heteroaryl or C _5-20Aryloxy group or heteroaryloxy (preferable methyl and ethyl);

X is O, NH or NR (preferred O);

Y is (CH ₂) _n, O (CH ₂) _n, NH (CH ₂) _n, NR (CH ₂) _n, OCOO (CH ₂) _n, NHCOO (CH ₂) _nN=1,2,3,4 or 5 (being preferably n=2 and 3); Or Y=aryl or heteroaryl (preferably right-phenyl);

A=CH or N (being preferably CH); With

B=CH=CH, O, S, NH or NR (being preferably CH=CH); Or its officinal salt, solvate, polymorph or metabolite.

Aspect preferred, the present invention relates to chemical compound according to structure I A:

R=C wherein _1-10Alkyl, or C _1-10Haloalkyl; R _i=H; C _1-10Alkyl, C _1-10Alkoxyl; X=O, NH or NR; A=CH, CR or N; And B=CH=CH, O, S, NH or NR or its officinal salt, solvate, polymorph or metabolite.

Above-claimed cpd of the present invention comprises its enantiomer, stereoisomer and tautomer, and officinal salt, solvate, polymorph and from the metabolite in all stages.

Preferred agents in these chemical compounds is the 4-nitrobenzophenone series compound of structure I A, and wherein A is CH; B is CH=CH; X is O; R is CH ₂CH ₂Cl; R ₁Be CH ₃Phosphate group can be free acid or salt (being preferably Tris).Aspect hydrazine-carboxylic acid 1-(4-nitrobenzophenone) ethyl ester (KS119W) especially preferred, the R configuration structure (VNP40541) of enantiomer is for more preferably.

As previously mentioned, according to chemical compound of the present invention and especially preferred compositions is treatment tumor compounds effective extremely according to the present invention.They also present one or more improvement at least, strengthen the antineoplastic activity as comparing with KS119, reduce toxicity, higher water solublity, or more the excellent drug metabolism distributes.

These chemical compounds preferentially activate in hypoxic tumors according to the present invention, and can be individually dosed, or make up with other anticarcinogen or with phototherapy or X-ray therapy.

Chemical compound according to the present invention can be used for treating cancer, and the pharmaceutical composition of many other situations and/or morbid state.Embodiments of the invention can be used as intermediate that synthesizes other chemical compound that presents biologic activity and the standard of determining this chemical compound biologic activity.In some applications, this chemical compound can be used for treating infected by microbes, especially comprises virus, antibacterial and fungal infection.These chemical compounds are included in the above any or multiple chemical compound of disclosed effective dose, randomly with pharmaceutically useful additive, carrier or excipient composition.

Another aspect of the present invention relates to treatment for cancer, comprise its above-described chemical compound of patient's effective dosage of needs, described chemical compound randomly with pharmaceutically useful additive, carrier or excipient composition.The present invention also relates to treat the neoplastic method of mammal, described method comprises the chemical compound to the above-described effective dose of patient's administration of suffering from cancer.To malignant solid tumor, leukemia and lymphadenomatous treatment, comprise that one or more these medicines to patient's administration antitumor effective dose are the preferred embodiments of the invention.Using chemical compound of the present invention also may be effective to the treatment of other different relevant disease states.The method also can be used for comparison test, as the activity of definite related analogs, and is used for determining that patient's cancer is to one or more tests according to the sensitivity of chemical compound of the present invention.

Accompanying drawing and table summary

Fig. 1 represents the KS119 activating mechanism of inferring.

Fig. 2 represent the sample (KS119W) of chemical embodiment and proposed according to the present invention under hypoxia condition their activatory mechanism.

Fig. 3 and the synthetic chemical diagram of 4 expressions according to The compounds of this invention.

Fig. 5 to 7 is illustrated in and relates to the experimental result that proposes according among the activatory the application of the selectivity of hypoxia condition of the present invention.

Fig. 8 to 9 is illustrated in and relates to the experimental result that proposes according among the effectiveness of the preferred embodiments of the invention and toxic the application.

Detailed Description Of The Invention

Following term will use in whole description to describe the present invention.

In whole description, use term " patient " to describe animal, comprise mammal and preferred people, use compositions according to the present invention to provide treatment, comprise prophylactic treatment to them.For the treatment of concrete animal such as people patient's concrete infection, disease or morbid state, the term patient relates to concrete animal.

In whole description, use term " effective dose " to describe according to compound concentrations of the present invention or amount, described concentration or amount are used in the disease of treatment or the disease and produce promising change, disease or disease according to treatment, change can be and alleviate, the decline of cancer or growth of tumor or size, favourable physiology result, minimizing of microbial growth or generation or the like.

Unless otherwise indicated, the term of using in the literary composition " chemical compound " relates to any concrete chemical compound disclosed herein.In contextual use, this term relates generally to individualized compound, but also can relate to stereoisomer and/or optical isomer (comprising racemic mixture), and concrete enantiomer, or the mixture of the enantiomerism enrichment of disclosed chemical compound, and tautomer.

In whole description, use term " neoplasia " to describe to cause neoplasia to form and the pathological process of growth, promptly faster than the normal structure growth by cell amplification, and the abnormal structure of continued growth after the stimulation that is subjected to making new growth to stop.Neoplasia may be different piece of tissue, and it can be optimum (benign tumor) or pernicious (carcinoma).Quote at this, the neoplasia of use term is described all cancerous disease states and is comprised or comprise the pathological process of following pernicious hematogenous, ascites and entity tumor.Term " cancer " and term " tumor " can exchange with term " neoplasia " in this application.

Can use cancer according to combination treatment of the present invention, for example comprise gastric cancer, colon cancer, rectal cancer, hepatocarcinoma, cancer of pancreas, pulmonary carcinoma, breast carcinoma, cervical cancer, carcinoma of uterine body, ovarian cancer, carcinoma of prostate, carcinoma of testis, bladder cancer, renal carcinoma, skin carcinoma, the brain cancer/central nervous system (CNS) cancer, head and neck cancer, laryngocarcinoma, Hodgkin, non-Hodgkin lymphoma, multiple myeloma, acute lymphoblastic leukemia, acute myeloblastic leukemia, Ewing sarcoma, choriocarcinoma, rhabdomyosarcoma, wilms' tumor, neuroblastoma, hairy cell, mouth/pharyngeal cancer, esophageal carcinoma, laryngeal carcinoma, melanoma, kidney and other lymphatic cancer.Tumor treatment is comprised that one or more these medicines to patient's administration antitumor effective dose are the preferred embodiments of the invention.

In whole description, use term " alkyl " to describe alkyl, comprise 1 to 7 carbosilane unit.The alkyl that uses in the present invention comprises the group of straight or branched, as is preferably methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, the tert-butyl group, amyl group, isopentyl, hexyl, cyclohexyl, methyl cyclopropyl and methylcyclohexyl.

Term " aryl " relates to monovalence aromatic radical replacement or unsubstituted, and described aromatic radical has single ring (as phenyl) or a plurality of condensed ring (as naphthyl).Term " aryloxy group " relates to the aryl that connects alkoxyl, is preferably by another group (for example sulfonyl) to link to each other.

Term " heteroaryl " relates to the heteroaromatic cyclic group, contains one or more nitrogen, oxygen or sulphur atom in this ring, for example imidazole radicals, furyl, pyrrole radicals, pyridine radicals, thienyl and indyl.Term " heteroaryloxy " relates to the heteroaryl that connects alkoxyl, is preferably by another group (for example sulfonyl) to link to each other.

In whole description, use term " salt " describe any with according to the consistent salt of the purposes of The compounds of this invention.Wherein when chemical compound is used for the pharmacy indication, comprise treatment for cancer, officinal salt should be represented in term " salt ", consistent with purposes as the chemical compound of medicine.

At preferred fermentation, the present invention relates to chemical compound as structure (IA):

R=C wherein _1-10Alkyl, or C _1-10Haloalkyl; R ₁=H; C _1-10Alkyl, C _1-10Alkoxyl; X=O, NH or NR; A=CH, CR or N; And B=CH=CH, O, S, NH or NR.

Chemical compound according to the present invention comprises enantiomer, stereoisomer and tautomer thereof, and the metabolite in officinal salt, solvate, polymorph and all stages.

The prodrug forms of this compounds represented intermediate, described intermediate it is believed that by the chloroethylation or the mechanism that methylates and present the DNA crosslinking active.The principle of new prodrug design is to change the activated prodrug of enzyme into activatory alkylation material (90CE) by the activation of a series of enzymes and rapid fracture.Finish dephosphorylation to obtain intermediate 1 by alkali phosphatase (AP) activation; By nitroreduction (NR) enzyme catalysis nitroreduction to form intermediate 2; And the generation of phenyl fracture subsequently 90CE, see Fig. 2.

Chemical compound according to the present invention is preferably activation in hypoxic tumors, and can be individually dosed or with other anticarcinogen or with phototherapy or radiotherapy combination.

Primarily act as their anti-tumor activity according to chemical compound of the present invention, comprise their anti entity tumour activity.And these compositionss can also be used for the chemosynthesis of other useful antitumor agent as intermediate, and the effect of described antitumor agent is followed successively by therapeutic agent or other purpose, comprise the standard substance as experiment.

Preferred agents is the 4-nitrobenzophenone series of structure I A in the chemical compound, and wherein A is CH; B is CH=CH; X is O; R is CH ₂CH ₂Cl; R ₁Be CH ₃Phosphate group can be free acid or salt.Aspect hydrazine-carboxylic acid 1-(4-nitrobenzophenone) ethyl ester (KS119W) especially preferred, the R configuration structure (VNP40541) of enantiomer is more more preferred than S configuration structure (VNP40551).

Chemical compound according to the present invention synthesizes by the adjustment of technology well-known in the art and derives from 90CE.Disclose from the synthetic 90CE of the two-step method of 2-ethoxy-hydrazine (referring to, Shyam etc. J Med Chem.1993,36:3496, and J Med Chem.1996,39:796).The analog of this specifically described chemical compound will with above-mentioned general technology and similarly in this area available synthetic method easily synthetic, and need not carry out unnecessary experiment.

By concrete method, for example, shown in Fig. 3,1 of Compound I, 2-two (mesyl)-2-(replacement) hydrazine-carbonic ester (carbonates) (5, R=CH ₂CH ₂Cl) respectively synthetic through the following steps: suitable alpha-alkyl 4-nitro aryl methanol or N-alkyl-N-(4-nitro aryl methyl) amine (3 or 4, R wherein ₁For-CH ₃R ^*Be the blocking group of phosphate group, for example, diethyl or di-t-butyl or 2-trimethyl silyl ethyl (TMSE) group, with phosgene (20% toluene solution) or its equivalent, for example three phosgenes or trichloromethyl chloroformate (referring to, Majer, etc. J Org Chem.1994,59:1937; And Pridgen, etc. J Org Chem.1989,54:3231)) reaction, and with the further original position condensation of 90CE.When using N, N-diisopropylethylamine (DIPEA) remains on 0 ℃ as alkali and with reaction, and when spending the night in anhydrous acetonitrile-dichloromethane solvent, this coupling reaction can obtain high yield.Compound I (6, R=CH ₂CH ₂Cl) hydrazine-amide can be by similar phosgene-coupling approach synthetic (Lin waits US006,855,695,2005).

5 or 6 going-dialkyl group-protection after, can change corresponding phosphoric acid free acid (7 or 8) rapidly into.For example, the going of diethyl ester protect available trimethyl silyl bromide (TMSBr) handle (Matulic-Adamic, etc. J Org Chem.1995,60:2563), the going of di-t-butyl ester protects available trifluoroacetic acid (TFA) to handle (Durgam etc. J Med Chem.2005,48:4919), and the removing the also available TFA of protection US006 such as (, 458,816,2002) Dolye or use BF of two TMSE esters ₃-Et ₂(Tetrahedron Lett.1986 such as Jansson 27:753) handles O.The phosphoric acid free acid form 7 or 8 by flash column chromatography (FCC) purification, for example purification on normal-phase silica gel or reverse phase silica gel, and after lyophilizing, obtain as the required SHP chemical compound of drug substance.

As shown in Figure 4, alpha-alkyl 4-nitro aryl methanol (3) can be adopted the selective reduction of enantiomer and synthesized by corresponding alkylaryl ketone (9).Reducing catalyst can be selected from commercial available reagent, as 2-Me-CB S- azoles borine (oxazaborolidine)/BH ₃(Mathre, etc. J Org Chem..1993,58:2880) or two different loose camphyl chloroboranes (Brown, etc. J Am Chem Soc.1998,110:1539) or boron-(3-pinanyl)-9 bora dicyclo [3.3.1] nonanes (Alpine-borane) (Ramachadran, etc. Tetrahedron:Asymm.5:1061).Also can use asymmetric hydrogenation (J AmChem Soc.1998 such as Ohkuma, the 120:13529 of ketone; With JAm Chem Soc.2004 such as Baar, 126:8216).

N-alkyl-N-(to the nitro aryl methyl) amine (4) can be by corresponding alkylaryl ketone preparation.For example, use sodium borohydride as Reducing agent, 12 obtain corresponding N-aryl methyl-N-methylamine (4) with the methylamine reduction amination separately.

Under temperate condition, the hydroxyl on the aromatic rings can with chloro phosphate reaction with phosphono group-aryl compound that their corresponding dialkyl group protections are provided (promptly 3 or 12).Use the 4-dimethylaminopyridine (DMAP) of phosphite, carbon tetrachloride, DIPEA and catalytic amount reach phenol the selectivity phosphorylation (Silverberg, etc. Tetrahedron Lett.1996,37:771).Usually, phosphorylation can be finished behind reductive amination prior to asymmetric reduction or phosphorylation.The synthesizing of the 4-nitro aryl compound (9 or 11) that is fit to that is used for these reaction designs is being well-known and the chemical technology of the standard of use such as nitrated and propylene acidylate (acrylation) in the art.

After synthetic, crude product is generally by reverse phase silica gel chromatograph and lyophilization purification.Handle KS119W (or VNP40541) with alkaline solution that is fit to or amine and can easily produce separately water soluble salt such as sodium salt, three (hydroxymethyl)-aminomethane (TRIS) salt, triethanolamine salt, triethyl amine salt or lutidines salt.The change of described open chemical synthesis process can be obtained easily by those of ordinary skills, to propose the optionally synthetic route of this chemical compound.

Pharmaceutical composition based on this novel chemical compound comprises the above-claimed cpd for the treatment of disease or disorders such as cancers with the treatment effective dose, randomly, and with pharmaceutically useful additive, carrier or mixed with excipients.

The pharmaceutical dosage form of some chemical compounds can as the prevention do not demonstrate disease or the preventive of disease.

This chemical compound or their derivant can provide with pharmaceutical acceptable salt.As used herein, term " officinal salt " relates to the suitable salt according to reactive compound of the present invention, and described salt keeps the biologic activity of desirable parent compound.The nonrestrictive example of these salt comprises phosphatic sodium salt and potassium salt, and other is as TRIS salt, triethanolamine salt, triethylamine salt, lutidines salt or other is at officinal salt known in the art.

The modification of reactive compound can influence the metabolic rate of dissolubility, pharmacokinetic parameter and active substance, and therefore contrast is provided on delivery of active substances.And modification can influence the active anticancer of chemical compound, in some cases, improves active with respect to parent compound.This can be easily according to this area now in the technology known method by the preparation derivant with detect active anticancer and estimate.

Chemical compound of the present invention can be mixed into the preparation that is used for all route of administration, and described route of administration comprises, for example parenteral and oral administration comprise in intravenous, intramuscular, intraperitoneal, the cheek, percutaneous and suppository form.Parenteral and especially intravenous or intramuscular administration are preferred.

Comprise the chemical compound of above-mentioned treatment cancer and other disease described here and treatment of conditions effective dose based on the pharmaceutical composition of these novel chemical compounds, randomly with pharmaceutically useful additive, carrier and/or mixed with excipients.Those of ordinary skill in the art will understand the treatment effective dose one or more according to chemical compound of the present invention, to change the pharmacokinetics of the infection treated or disease, its severity, the therapeutic scheme that is adopted, used medicine, and the patient who is treated (animal or human).

Aspect pharmacy of the present invention, according to chemical compound of the present invention preferably with pharmaceutically useful carrier mix preparation.Usually, the pharmaceutical composition administration is preferably parenteral, and the dosage form of especially preferred intravenous injection or intramuscular injection, but a lot of preparations are by other parenteral route administration, described parenteral route comprises the by oral route administration as through skin, buccal (buccal), subcutaneous, suppository or other approach.Preparation with Sterile Saline intravenous injection and intramuscular injection is a preferred modes.Certainly, those of ordinary skill in the art can change preparation so that the several formulations of concrete route of administration to be provided in the scope of description instruction, and does not make compositions of the present invention unstable or involve the preparation of their therapeutic activity.Particularly, the modification of chemical compound of the present invention makes their more soluble in water or other carriers, for example, can easily finish by less change (as salt pref etc.), and the described those of ordinary skill in the art of changing into knows.Also can in the prior art scope, change the route of administration and the dosage drug regimen of particular compound, with the pharmacokinetics of adjusting The compounds of this invention to reach maximal effective dose for the patient.

Those skilled in the art will utilize the favourable pharmacokinetic parameter of the present invention's prodrug forms applicatory, send The compounds of this invention makes chemical compound to host's organism or patient's target spot Expected Results maximization.

The scope of the administration of reactive compound can from addition every day frequency be less than once successive administration (intravenous drip), comprise fast injection administration, intravenous injection or intramuscular injection, to the every day of administration several times, and can comprise part, parenteral, intravenous injection, intramuscular injection, subcutaneous, through skin (it can comprise penetration enhancer), buccal and suppository administration, other route of administration comprises for example oral administration.

For preparing according to pharmaceutical composition of the present invention, one or more of treatment effective dose preferably mix with pharmaceutically useful carrier according to the drug regimen technology of the routine for preparing dosage form closely according to chemical compound of the present invention.Carrier can have various ways, depends on the form of administration such as intravenous injection or the required preparation of intramuscular injection.Be fit to use any pharmaceutical media commonly used in the forms of pharmaceutical compositions in preparation.For parenteral formulation, carrier can comprise the D/W (D5W) of aseptic water or sodium-chloride water solution or 5%, and it helps dispersive composition to mix with other, and described composition such as ethanol and other pharmaceutically useful solvent comprise DMSO, and other.Certainly, when using solution and solution to keep aseptic, compositions and carrier also are necessary for aseptic.Injectable suspension can also be prepared, in this case, suitable liquid-carrier, suspending agent etc. can be adopted.

Be used in parenteral, Intradermal, solution or suspension subcutaneous or local application and can comprise following ingredients: sterile diluent such as water for injection, saline solution, D5W solution, expressed oi, Polyethylene Glycol, glycerol, propylene glycol or other synthetic; Antibacterial such as benzyl alcohol or nipagin; Antioxidant such as ascorbic acid or sodium sulfite; Chelating agen such as ethylenediaminetetraacetic acid (EDTA); Buffer agent such as TRIS, acetate, citrate, phosphate, histidine or sodium bicarbonate solution; And the tensile reagent of adjustment such as sodium chloride or glucose.The multi-dose vials that parenteral formulation can be encapsulated in ampoule, disposable syringe or be made by glass or plastics.If be intravenous administration, preferably carrier comprises, for example, and normal saline or phosphate buffered saline (PBS) (PBS).

The pharmaceutical composition of preparation peroral dosage form can use any or multiple pharmaceutical media commonly used.Therefore, for fluid flow port formulation such as suspending agent, elixir or solution, operable suitable carriers and additive comprise water, glycols, oils, alcohols, aromatic, antiseptic, coloring agent etc.For solid orally ingestible such as powder, tablet, capsule and for solid preparation such as suppository, operable suitable carriers and additive comprise starch, sugar carrier (as glucose, mannitol, lactose and relevant carriers), diluent, granule, lubricant, binding agent, disintegrating agent etc.Optionally, tablet or capsule can carry out enteric coating or continue discharging by standard technique.

In one embodiment, reactive compound can prepare with carrier, and described carrier will make chemical compound avoid eliminating fast from health, as controlled release preparation, comprises and implanting and the microencapsulation delivery system.

Can use biodegradable, biocompatible polymer, as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters and polylactic acid.Preparation method for these preparations of those of skill in the art is conspicuous.

The liposome suspension also can be used as pharmaceutically useful carrier.These can be according to those skilled in the art's known method preparation.For example, the preparation Liposomal formulation can be dissolved in the inorganic solvent by the lipid that will be fit to, and with the inorganic solvent evaporation, stays the exsiccant lipid of skim at vessel surface afterwards.Aqueous solution with reactive compound joins in the container then.Stir container with hands then and make lipid feedstocks free and the lipid granule is disperseed, thereby form the liposome suspension from the edge of container.In this one side of the present invention, those of ordinary skill can use other known preparation method.

Can use compounds for treating animal of the present invention, be mammal especially, comprises the mankind, as the patient.Therefore, people, horse, dog, cattle and other animal, and particularly for suffering from the mammal of tumor, and particularly be cancer, or other disease described here, can be by treating according to chemical compound or derivatives thereof of the present invention or its officinal salt to one or more of patient's effective dosage, according to the disease of being treated, above-claimed cpd or derivatives thereof or its officinal salt are optional to be used with pharmaceutically useful carrier or diluent, described carrier or diluent both can use separately, can make up with other known pharmaceutical agents again.This treatment also can or be performed the operation and make up with other conventional treatment of cancer such as radiotherapy.

Reactive compound to be enough being delivered to the treatment effective dose of patient for required indication, and the treatment patient is not caused that the amount of serious toxicity effect is contained in pharmaceutically useful carrier or the diluent.

This chemical compound is the prodrug forms of reaction intermediate.In some pharmaceutical dosage forms, can with this compound-modified be other prodrug forms, to utilize the concrete route of administration of reactive compound.How easily those of ordinary skill in the art will recognize will originally compound-modifiedly be to promote reactive compound to be delivered to patient's target spot by other prodrug forms.Those of ordinary skill also will utilize the favourable pharmacokinetic parameter of applicable prodrug forms, send this chemical compound and arrive patient's target spot so that the neoplasia resisting maximum effect of expected compound.

The amount that is included in the chemical compound in the effective preparation of treatment according to the present invention is the effective dose of treatment cancer.Usually, the scope according to the treatment effective dose of chemical compound of the present invention in the dosage form is generally and is lower than about 0.05mg/kg to about 500mg/kg (patient's that treats body weight), or much more, depend on that used chemical compound, neoplastic kind, the reactive compound of being treated are positioned ability, route of administration and the patient's of the tissue of being treated the pharmacokinetics of chemical compound.Under the situation of treatment cancer, chemical compound preferably with about 0.05mg/kg once to about 250mg/kg or more measure administration.In the patient who is treated, this dosage range produces level concentration in effective blood of reactive compound usually, and level concentration is about 0.01 to about 500 milligrams of every milliliter of blood in the described blood.The duration of treatment can maybe can continue some months or quite grow (several years) for one day or many days, and this depends on the state of the disease for the treatment of.In a more preferred embodiment, administration patient 0.1mg/kg is to this chemical compound of 100mg/kg, and twice to per 14 days of every day, once the duration was that 1 week is to 52 weeks.

The concentration of reactive compound will depend on absorption, distribution, inactivation and the excretion rate of medicine among the patient, and other factors well known by persons skilled in the art.Should be pointed out that to the patient also can change to dosage according to the order of severity of the symptom that will alleviate.It is to be further understood that, for any concrete patient, concrete dosage administrated method should be in time according to the needs and the administration of individuality or instruct the people's of compositions administration professional judgement to adjust, and concentration range scope or the use that does not limit the compositions that proposes only for referencial use as mentioned herein.Active component can single administration, maybe can be divided into a lot of low doses in the different intervals administration.

The material that can not damage the active substance of desirable effect with other or replenish desirable effect according to reactive compound of the present invention mixes, as other anticarcinogen (depending on desirable treatment or target spot in some cases), antibiotic, antifungal agent, anti-inflammatory agent or antiviral compound and other.

These chemical compounds according to the present invention preferably activate in hypoxic tumors, and can be individually dosed or with other anticarcinogen or with phototherapy or radiotherapy combination.

According to chemical compound of the present invention can be individually dosed or with the other medicines combination, especially comprise other chemical compound of the present invention.According to these aspects of the present invention, one or more of treatment effective dose are according at least a other neoplasia resisting/anticarcinogen co-administered of chemical compound of the present invention with the treatment effective dose, described neoplasia resisting/anticarcinogen such as antimetabolite, etoposide, doxorubicin, paclitaxel, vincristine, cyclophosphamide (cyclophosphamide) or ametycin, and multiple other chemical compound, comprise topoisomerase I and topoisomerase II inhibitor, as amycin, hycamtin, camptothecine and Irinotecan, other medicines such as gemcitabine and based on the medicine of camptothecine and cisplatin.In theory, by this chemical compound that the mechanism of destroying DNA works, the chemical compound synergism that will work with mechanism by minimizing or prevention DNA reparation.Therefore, this chemical compound can advantageously make up with any chemical compound that acts on the mechanism of minimizing or prevention DNA reparation, especially comprises the inhibitor of the enzyme that promotes the DNA reparation, as ribonucleotide reductase (RR) inhibitor and O ⁶-alkyl guanine-DNA alkyl-transferase (AGT) inhibitor.Mean by " co-administered ", make the chemical compound of chemical compound of the present invention and co-administered to be detected in the blood flow the patient simultaneously to patient's administration chemical compound of the present invention, no matter these chemical compounds are actually when administration, comprise administration simultaneously.In many cases, this chemical compound produces beyond thought synergism (promptly being not only addition) result with traditional anticarcinogen co-administered.In another embodiment, will according to chemical compound of the present invention simultaneously or in order with antibiotic (combine or non-binding), virus or antibacterial administration.Antibiotic, virus or antibacterial can be carried the gene of enzyme or codase, and described enzyme activates the chemical compound that the present invention describes.Described enzyme includes but not limited to NR.

Be not limited to theoretical method, it is believed that according to chemical compound of the present invention and mainly induce their therapeutic effect in the treatment malignant tumor by function as the chloroethylation agent of hypoxia-selective.

Big volume description the present invention, existing with following illustrate preferred and other embodiment and the specific embodiment of comparison as a reference.Included embodiment and non-limiting scope of the present invention are because its scope is mentioned above and wideer in additional claim.Other chemical compound that does not specifically present in the embodiment of the present application part can according to similar methodology and/or presented be easy to synthetic with synthetic method easily known in the art.Directly according to the synthetic method of detailed synthetic method or reorganization/modification used technology known in the art, those of ordinary skill can easily synthesize the chemical compound that all are mentioned and described, and need not carry out over-drastic experiment simply.

Embodiment

The reagent of all purchases is commercial quality and is not further purified and uses, in case of necessity before use with solvent seasoning and/or distillation.All NMR spectrums ( ¹H, ¹³C reaches ³¹P) on Bruker AC300 spectrum view, measure.Chemical shift is measured with a few millionths (ppm) of relative tetramethylsilane.Coupling constant is with hertz (Hz) record.Flash column chromatography (FCC) carries out with Merck silica gel 60 (230-400 mesh sieve), and reversed-phase column chromatography (RPCC) is with CAT glue (Water, preparation C-18,125 , 55-105 μ m) filling, with milli-Q level deionized water eluting.

Embodiment 1-2

The phosphorylation of phenolic compound

Conventional method A.Under the room temperature, to acetonitrile (15mL) the solution adding DMAP (1mmol) and the DIPEA (20mmol) of the phenolic compound (10.0mmol) that is fit to through stirring.Reactant mixture is cooled to-13 ℃.Acetonitrile (5mL) solution that drips chlorine phosphate dialkyl ester (10mmol) is to keep internal temperature less than-10 ℃.Reactant mixture is warming up to 0 ℃, continues then to stir 2 hours, finish with the TLC monitoring reaction.Reactant mixture is concentrated by rotary evaporation, and with dichloromethane and 0.5M KHSO ₄Aqueous solution handle the oily residue.Organic layer is by anhydrous MgSO ₄Drying is filtered then and is concentrated and obtains brown thickness grease.Can use rough dialkyl phosphine carboxyl-aryl compound and be not further purified.

1-(3-diethyl phosphono group-4-nitrobenzophenone) ethanol (13).According to conventional method A, (18.2g, 100mmol) (15.0mL, 100mmol) reaction obtains required product 13 (32.0g, 100%) to R-1-(3-hydroxyl-4-nitrobenzophenone (nitrophaenyl)) ethanol with diethyl chloro-phosphate.

¹H NMR (300MHz, CDCl ₃) δ 1.36 (t, J=7.1Hz, 6H), 1.47 (d, J=6.6Hz, 3H), 3.02 (br s, 1H), 4.25 and 4.27 (q, J=7.1Hz, 4H), 4.91 (q, J=6.6Hz, 4H), 7.26-7.31 (m, 1H), 7.55 (s, 1H), 7.90 (dd, J=8.5,0.8Hz, 1H).

¹³C NMR (75MHz, CDCl ₃) δ 16.1 and 16.2,25.3,65.6 and 65.7,68.8,119.6,122.2,126.0,140.0,143.5 and 143.6,154.4.

³¹P NMR(121MHz，CDCl ₃)δ-6.7。

Conventional method B.With phenolic compound (10mmol), acetonitrile (20mL) solution of DIEA (20mmol) and DMAP (1mmol) places-50 ℃ of baths.In above-mentioned cold soln, add CCl ₄(50mmol) and dialkyl phosphite (10mmol).Reaction solution was at room temperature preserved 2 hours.Remove by rotary evaporation and to desolvate.With 0.5M KHSO ₄Aqueous solution and dichloromethane are handled remaining grease.After the separation, by anhydrous MgSO ₄Dry, evaporation and vacuum drying can use rough dialkyl phosphine carboxyl-aryl compound and not be further purified.

1-(3-diethyl phosphono group-4-nitrobenzophenone) ethanol (13).According to conventional method B, (15.0g, 82.4mmol) (10.6mL, 82.4mmol) reaction obtains required product 13 (27.1g, 100%) to R-1-(3-hydroxyl-4-nitrobenzophenone (nitrophaenyl)) ethanol with chlorine phosphorus carboxylic diethylester.

Embodiment 3

The Ge Liya of benzaldehyde class (Grignard) reaction

Conventional method.At-50 ℃,, in 4-nitrobenzaldehyde (4-nitrobenzaldehydes) anhydrous THF (30mL) solution (10mmol), slowly add Grignard reagent, as ether (25mmol) solution of methylmagnesium-bromide through 45 minutes.In adding the Grignard reagent process, the temperature of reactant mixture remains on below-40 ℃.Reactant mixture can be warming up to room temperature, and stir 3.5 hours.Reactant mixture is cooled to-10 ℃, and with 5% hydrochloric acid (25mL) cancellation.With reactant mixture water (25mL) dilution and (3 * 15mL) extractions of product ethyl acetate.It is 5 that the organic extract water that merges is flushed to pH value, passes through anhydrous Na ₂SO ₄Drying is filtered and is concentrated.With residue fast silica gel chromatogram purification, with the hexane solution eluting of 25% ethyl acetate.Behind evaporation and the vacuum drying, obtain alpha-alkyl 4-Nitrobenzol methanol.

1-(3-hydroxyl-4-nitrobenzophenone) ethanol (14).According to conventional method, (90.0g 539mmol) obtains required red oily product 14 (40.0g, 41%) by 3-hydroxyl-4-nitrobenzaldehyde.

¹H NMR(300MHz，CDCl ₃)δ1.50(d，J＝6.6Hz，3H)，2.06(br s，1H)，4.93(q，J＝6.3Hz，1H)，6.98(dd，J＝8.8，1.9Hz，1H)，7.17(d，J＝1.7Hz，1H)，8.08(d，J＝8.8Hz，1H)，10.6(s，1H)。

¹³C NMR(75MHz，CDCl ₃)δ25.3，69.5，116.3，117.6，125.5，155.5，156.9。

Embodiment 4

The reduction of acetophenones

Conventional method.A solution (1.0M toluene solution, 240mL) adding 1.0M BH to chirality (R or S)-2-methyl-CBS- azoles borine (oxazaborlidine) ₃-THF solution (120mL) is cooled to-50 ℃ with gained solution.Through 4 hours, in above-mentioned solution, slowly add THF/ toluene (200mL/800mL) solution and the 1.0M BH of 3 '-hydroxyl-4 '-nitro-acetophenone (100g) simultaneously ₃-THF solution (1.0L), vigorous stirring simultaneously.Reactant mixture continues to stir 2-3 hour at-50 ℃, finishes by the HPLC monitoring reaction.In reactor, drip acetone (200mL) in-50 ℃ then., reactant mixture is warming up to ambient temperature, and stirred 1.5 hours after 10 minutes in-50 ℃ of stirrings.Rotary evaporation concentrates in 45 ℃ of baths, and residue is with saturated Na ₂CO ₃Aqueous solution (2L) is handled.Mixture was heated 30 minutes at 50 ℃, then cool to room temperature.(TBME 1L) and hexane (1L), stirs mixture 1 hour in room temperature (RT), then separation to add t-butyl methyl ether.Being added dropwise to dense HCL adjust pH to water layer is 6, maintains the temperature at 25 ℃ simultaneously.(3 * 2L) extract this mixture, and concentrated organic facies with ethyl acetate.Obtain brown buttery crude product, and with its recrystallization purifying in hexane.

R-1-(3-hydroxyl-4-nitrobenzophenone) ethanol (15).According to conventional method, by 3-hydroxyl-4-nitro-acetophenone (100g, 0.55mol) obtain required yellow solid product 15 (52g, 51%, 99.3%ee).

Embodiment 5

The reductive amination of benzaldehyde (benzaldehydes)

Conventional method.THF (20mmol) solution that in dichloromethane (10mL) solution of benzaldehyde (10mmol), adds the 2N methylamine.Reaction solution is remained on ambient temperature overnight and filtering precipitate.Filtrate is concentrated and vacuum drying, and the rough grease of gained is dissolved in the methanol (50mL).In above-mentioned solution, add a NaBH in 0 ℃ by part ₄(20mmol), solution is continued to stir 4 hours.After the evaporation, residue is scattered in water (50mL) and the dichloromethane (50mL).With aqueous phase separation, and with dichloromethane (50mL) extraction once.The organic facies that merges is by anhydrous MgSO ₄Drying, filtration, concentrated and vacuum drying.Rough N-benzyl-the N-methylamine is enough pure can be used, and need not to be further purified.

N-(4-diethyl phosphono group benzyl)-N-methylamine (16).According to conventional method, (29.9g 116mmol) obtains 15 (22.3g, 71%) of yellow oily by diethyl phosphono group-benzaldehyde.

¹H NMR (300MHz, CDCl ₃) δ 7.31 (and d, J=8.0Hz, 2H), 7.17 (d, J=8.5Hz, 2H), 4.21 (m, 4H), 3.73 (s, 2H), 2.42 (s, 3H) and 1.34 (t, J=6.9Hz, 6H).

¹³C NMR (75MHz, CDCl ₃) δ 149.4 (d), 135.6,129.3,119.5 (d), 64.2 (d), 54.5,35.1 and 15.7 (d).

³¹P NMR(121MHz，CDCl ₃)δ-5.5。

Embodiment 6-7

The phosgene coupling reaction

Conventional method A.In acetonitrile (40mL) solution of the 90CE (10mmol) through stirring, add phosgene (20% toluene solution, 10mmol) and DIPEA (10mmol).Kept 20 minutes at 0 ℃, in solution, add N-(dialkyl phosphine carboxyl-benzyl)-N-methylamine (10mmol) and DIEA (10mmol).End reaction solution remains on 5 ℃ and spends the night.After the evaporation, water and dichloromethane are handled residue.The organic facies that merges is passed through anhydrous MgSO ₄Drying, filtration and evaporation.Obtain corresponding buttery shielded phosphate ester.

Conventional method B.In 0 ℃ of toluene (30mmol) solution, add DIPEA (12mmol) to 20% phosgene.Acetonitrile (15mL) solution that slowly adds 1-(3-diethyl phosphono group-4-nitrobenzophenone) ethanol (10mmol).At room temperature reactant mixture was stirred 2 hours, concentrate then.Residue is dissolved in the acetonitrile (15mL), and adding DIPEA (15mmol) and 90CE (10mmol) also is cooled to below 20 ℃.Mixture at room temperature stirred 2 hours.Behind the evaporating solvent, handle residue with water and dichloromethane.HCL solution (35mL) flushing organic facies with 1% is by anhydrous MgSO ₄Drying, and be evaporated to dried.With fast silica gel chromatogram (with the hexane solution eluting of 40-50% ethyl acetate) purification residue, concentrate with high vacuum dry and obtain corresponding buttery shielded phosphate ester.

1,2-two (mesyl)-2-(2-chloroethyl) hydrazine-carboxylic acid 1-(3-diethyl phosphono group-4-nitrobenzophenone) ethyl ester (16).According to conventional method B, (114.3g 358mmol) obtains 16 (151.2g, 71%) by 1-(3-diethyl phosphono group-4-nitrobenzophenone) ethanol.

¹H NMR (300MHz, CDCl ₃) δ 1.30-1.42 (m, 6H), 1.69 and 1.70 (d, J=6.6Hz, 3H), 3.16 and 3.21 (s, 3H), 3.46 and 3.47 (s, 3H), 3.65-3.75 (m, 2H), and 3.80-3.92 (m, 1H), 4.00-4.10 (m, 1H), 4.20-4.35 (m, 4H), 5.95 and 5.96 (q, J=6.6Hz, 1H), 7.34 and 7.36 (d, J=8.2Hz, 1H), 7.60 and 7.65 (s, 1H), 7.96 and 7.97 (d, J=8.5Hz, 1H).

³¹P NMR(121MHz，CDCl ₃)δ-6.7。

Embodiment 8

The formation of phosphoric acid free acid (phosphate free acid).

Conventional method.Handle phosphoric acid (phosphate) dichloromethane (60mL) solution (10mmol) that diethyl is separately protected with excessive TMSBr (100mmol), spend the night in 5 ℃.Evaporation reaches dry in a vacuum, obtains the rough phosphoric acid free acid of vitreous solid.In crude compound (10mmol), add entry (about 30mL).Stirred suspension is 2 hours at ambient temperature, and the water that adds minimum then dissolves it fully.RPCC purification of aqueous solutions with the deionized water solution of 10% acetonitrile.This part with UV or ³¹P NMR and combine detection thereof.After the lyophilizing, obtain the white or the cream-coloured phosphate free acid powder of purification.

1,2-two (mesyl)-2-(2-chloroethyl) hydrazine-carboxylic acid 1-(3-dihydro phosphono group-4-nitrobenzophenone) ethyl ester (17).According to conventional method, with 1, (73.9g 124mmol) goes protection to 2-two (mesyl)-2-(2-chloroethyl) hydrazine-carboxylic acid 1-(3-diethyl phosphono group-4-nitrobenzophenone) ethyl ester, obtains white powder 17 (52.8g, 80%).

¹H NMR (300MHz, DMSO-d ₆) δ 1.58 and 1.59 (d, J=6.1Hz, 3H), 3.25 and 3.29 (s, 3H), 3.54 and 3.55 (s, 3H), 3.65-3.83 (m, 2H), and 3.85-4.00 (m, 2H), 5.97 and 5.99 (q, J=6.1Hz, 1H), 7.42 (d, J=8.2Hz, 1H), 7.60 and 7.65 (s, 1H), 7.96 and 7.97 (d, J=8.4Hz, 1H).

³¹P NMR(121MHz，DMSO-d ₆)δ-5.6。

Embodiment 9

Phosphatic formation

Conventional method.KS119W (200mg) solution and stoichiometric alkali dissolution stirred 1 hour in water (2.0mL) and at 20 ℃; With this solution lyophilizing 20 hours; Then the gained solid is analyzed with NMR and HPLC.

The sodium salt (18) of KS119W.NaHCO with 5% ₃Solution (210mL) slowly joins the KS119W of stirring, and (66.1g in the solution of methanol 122.4mmol) (70mL) and water (400mL), is 4.0-4.5 up to pH value.(impurity that decomposes is removed with ether (200mL) flushing in 2 * 500mL) flushing backs with dichloromethane with reactant mixture.In below 30 ℃ aqueous portion being concentrated.Residue is dissolved in acetone (200mL) and under firmly stirring, it is slowly being joined in cold (10 ℃) ether (2.0L).The gained serosity stirred 1 hour down at 0 ℃, filtered, and with ether (200mL) flushing, used hexane (200mL) flushing then, and dry, obtained light yellow solid 18 (109.7g, 86%).

¹H NMR (300MHz, D ₂O) δ 1.37 and 1.38 (d, J=6.6Hz, 3H), 2.91 and 3.00 (s, 3H), 3.24 and 3.25 (s, 3H), 3.30-3.50 (m, 2H), and 3.55-3.75 (m, 2H), 5.69 and 5.70 (q, J=6.6Hz, 1H), 7.06 (d, J=8.1Hz, 1H), 7.24 and 7.25 (s, 1H), 7.65 (d, J=8.4Hz, 1H).

¹³C NMR(75MHz，DMSO-d ₆)δ22.5，41.2，41.3，41.8，41.9，42.5，42.6，52.9，53.1，77.1，77.2，118.6，118.8，120.0，120.2，124.9，141.5，145.8，145.9，147.7，147.8，151.4。

³¹P NMR(121MHz，DMSO-d ₆)δ-4.09。

Embodiment 10

The mensuration of dissolubility and stability in the aqueous solution

By adding the incremental change medicine in the 2.0mL water in vial, visually measure the dissolubility of KS119W (or VNP40541).Under the room temperature, the jolting on Glas Col gyroscope of this bottle is dissolved fully up to medicine.Extra quantitative medicine is added, and this bottle of jolting, up to dissolving fully.Continue this process, up to no longer including medicine dissolution.The dissolubility that obtains KS119W (or VNP40541) is greater than 400mg/mL.The aqueous solution of KS119W (or VNP40541) is light yellow.

By adding excess drug in the vial of the 2.0mL water that contains, similarly measure the dissolubility of new synthetic KS119W salt.Under the room temperature, the jolting 24 hours on Glas Col gyroscope of this bottle.The suspension that will contain undissolved medicine is centrifugal, the careful separation supernatant, and with HPLC analysis drug level.Measured from Mg (OH) ₂, NaOH, KOH, BET and TRIS the water of KS119W salt in dissolubility (mg/mL) be respectively: 51.2,67.2,71.0,58.7 and 70.5.

The stability of research VNP40541.(20mg/mL) is dissolved in the citric acid of 20mM with sample, and titration pH value to 2.0, and 3.0,4.0,5.0,6.0 and 7.0.Catalysis contrast for being used for buffer does not exist under the citric acid, and also medicine being titrated to pH value is 2.0,5.0 and 6.0.In 40 ℃ with sample preservation 3 hours, 24 hours and 3 days, and preserved 24 hours, 3 days in room temperature.Above after each time point finishes, sample is positioned over-15 ℃ cold closet.The control sample of each goods (preparention) also is positioned in the cold closet.By each sample of HPLC replicate analysis,, measure each drug concentrations at different time points.As shown in following table 1, the result clearly illustrates that at 40 ℃ and observes tangible degraded after 24 hours.The existence of citric acid is influence degraded significantly not.

The existence of table 1.pH value and citrate buffer is to the influence of the short-term stability of VNP40541.

The HPLC measurement result is represented with respect to the control sample that is stored in-15 ℃.

pH	Citric acid		24 hours room temperatures		3 hours 40 ℃		24 hours 40 ℃
									Final pH value	Measure	Final pH value	Measure	Final pH value	Measure
			2.0 2.0 3.0 4.0 5.0 5.0 6.0 6.0 7.0	20mM does not have 20mM 20mM 20mM and does not have 20mM and do not have 20mM	2.00 2.04 3.07 4.08 5.03 5.12 6.17 6.09 7.07	99.3％ 99.4％ 99.3％ 100.1％ 99.4％ 99.7％ 98.6％ 100.0％ 99.7％	2.00 2.04 3.07 4.07 5.04 5.14 6.16 6.10 7.06	98.8％ 98.9％ 98.9％ 99.8％ 98.8％ 99.4％ 99.0％ 99.3％ 99.6％							2.02 2.06 3.06 4.09 5.06 5.10 6.10 6.01 6.96	92.3％ 91.7％ 91.3％ 92.2％ 92.2％ 92.5％ 93.8％ 93.8％ 94.0％

Embodiment 11

Aerobic/hypoxic cell survival research

By a rubber slab that inserts No. 13 (inflow) pins and No. 18 (outflow) pins, use 5%CO ₂, variable concentrations the mixture of oxygen, regulate the balance of described mixture with nitrogen, setting up different low-oxygen environments, EMT6 in the vial (glass milk dilution bottles) of diluted milk or the ventilation of CHO (parental generation of transfection or human-cytochrome P-450 reductase) cell 2 hours will be seeded in.Injectable drug and do not destroy hypoxia then.After two hours, collecting cell also places the product clonogenic assay to detect the survival part, compares with untreated matched group.

For analyzed in vitro, must in above-mentioned AP-catalytic reaction shown in Figure 2, change KS119W into KS119OH (1), because the parent drug of phosphorylation can not pass through cell membrane.In the indicated in vitro study of this part, all use KS119OH below.

Reflecting normal air (21% oxygen) or containing under the atmospheric oxygenation level of severe hypoxia of 0.1% oxygen, EMT6 molluscum contagiosum adenocarcinoma cell is exposed under the graded concentration of KS119 or KS119OH.As shown in Figure 5, the result shows under aerobic condition two medicines non-activity in fact, when drug level has very little cytotoxicity to the EMT6 cell during up to 25 μ M.Under 0.1% oxygen, two medicines of 10 μ M drug level all show the effective cytotoxic effect near the cell killing of 5 size order.

Similarly, in graded concentration 0.05% to 21% scope of oxygen, with KS119 and the KS119-OH of EMT6 cellular exposure, to measure the influence of oxygen concentration to pharmaceutically active in 10 or 25 μ M.The result is lower than in oxygen concentration and has showed tangible pharmaceutically active under 10% oxygen as shown in Figure 6, measures sizable activity in the solid tumor under oxygen concentration (shade post).

And, R and the S enantiomer of KS119W (VNP40541 and VNP40551) is converted into corresponding dephosphorylation form, with external aerobic/the hypoxic cell experiment in research.About rendeing a service and the two difference of aerobic/hypoxia, the result shows that R-KS119OH and S-KS119OH are closely similar to EMT6 cells in vitro cytotoxic activity and parent raceme medicine.

As shown in figs. 1 and 2, KS119OH (1) can produce the azanol of spontaneous released dna alkanisation and cytotoxic substance 90CE or the intermediate of aniline (2) in coverlet electron reduction enzyme (as cytochrome P-450 reductase) reduction under the hypoxia condition as KS119.For show that KS119W (raceme or two kinds of enantiomers) can be activated by cytochrome P-450 reductase under hypoxia, the Chinese hamster ovary celI of reaching of transfection being expressed this reductase is exposed to medicine, and with this cell strain to these medicines the parnet strain of sensitivity and the untransfected of the enzyme of expressing low foundation level compare.As shown in table 2, the result only shows that under hypoxia for all 3 kinds of medicines, cytochrome P-450 reductase probably is equal to the strain of ground sensitization Chinese hamster ovary celI.As KS119, cytochrome P-450 reductase can activate KS119OH under hypoxia condition.

The expression of table 2. cytochrome P-450 reductase is to R, S and the Cytotoxic influence of raceme KS119OH

	Survival part in 0.1% oxygen
				Medicine (10 μ M)	VNP40541	VNP40551	KS119W
CHO-SCS-II	0.04737	0.1188	0.3336
				CHO-450red	0.001368	0.002553	0.009444
					Airborne survival part
Medicine (10 μ M)	VNP40541	VNP40551	KS119W
				CHO-SCS-II	1.086	1.148	1.185
CHO-450red	0.9277	1.015	0.8791

Embodiment 13

The assessment of anti tumor activity in vitro

The antitumor action of assessment aforementioned prodrugs in entity and liquid tumor model, this model comprises B16-F10 Mus melanoma, HTB177 people's lung cancer model, DLD1 human colon carcinoma, EMT-6 molluscum contagiosum adenocarcinoma, L1210 murine leukemia, lymphoma, carcinoma of prostate, cancer of pancreas and head and neck cancer.Vein, oral and intraperitoneal are with the dosed administration prodrug of 10mg/kg to 2000mg/kg; Their administration has different dosage regimens, as every day 4 times, every day 1 time or up to every several days of 60 dosage once.With the subcutaneous implantation of tumor cell to be measured mice, tumor cell is implanted back (the 0th day) immediately with its random packet.Give the PBS of injection (ip) 0.1mL in the mouse peritoneum or the fast injection agent of medicine.According to the dosage regimen treatment that designs.Measure the three-dimensional dimension of tumor once in a week with formula L * H * W/2, wherein L, H and W represent length and width and height respectively.As it is definite with the animal performance to lose weight, and these drug toxicitys in mice are slight.

The effectiveness of research KS119 in Mus tumor model and people's xenograft models.With EMT-6 molluscum contagiosum adenocarcinoma cell (3 * 10 ⁵Individual cell/mice) subcutaneous being implanted in the Balb/c mice.H460 human lung carcinoma cell, HT29 human colon cancer cell and SHP77 human lung carcinoma cell are implanted nu/nu CD-1 mice and Scid/Beige mice (7 * 10 respectively as the entity xenograft ⁷Individual cell/mice).After the implantation, arrive 200mm in tumor length to 150 ³Size after, the beginning treat with KS119.KS119 is prepared in the solvent that contains Liquid Macrogol (PEG), ethanol, citric acid and ascorbic acid.

By every day be 80 and the KS119 peritoneal injection administration of 100mg/kg with dosage, be illustrated in figure 7 as the result of representative studies.Data show that KS119 produces the edge rather than statistical significance to institute's remarkable antitumor effect of the tumor model of survey to some extent.KS119 survey tumor growth to some extent suppression ratio be 30 to 50%.Compare with the vehicle Control group, suppression ratio is significant (p＜0.05).

To wait the KS119W of molar dose and the effectiveness of KS119 in EMT-6 tumor and H460 tumor model, to contrast.With 0.25M sodium bicarbonate solution preparation KS119W.When tumor reaches 150 to 200mm ³The time begin treatment, continue the most nearly 2 weeks.Every day by peritoneal injection administration mice dosage be 80 and the KS119 of 100mg/kg and dosage be 97.6 and the KS119W of 121.3mg/kg.Shown in the embodiment of H460 tumor (Fig. 8), KS119 and KS119W suppress the growth of mouse tumor.Compare with the KS119 that waits molar dose, KS119W is obviously more effective and produce better anti-tumor activity.Final gross tumor volume and matched group reduce 78% and 60% more respectively in 121.6mg/kg * 7 dosage for the treatment of with KS119W and 97.2mg/kg * 10 dosage groups; Yet, only reduce 40% in the 100mg/kg of the KS119 of molar doses such as usefulness treatment and 80mg/kg dosage group.The toxicity of KS119 and KS119W is dose dependent.Be the toxicity of assessment treatment, body weight and peripheral blood cells number are detected in the treatment back.Mice with two kinds of Drug therapys of above-mentioned dosage does not have tangible hematotoxicity or serious losing weight.

For being that clinical development is selected chemical compound preferably in the future liberally, obtain the maximum tolerated dose (MTD) of VNP40541 (R type) and VNP40551 (S type), and the result shows in table 3 from the enantiomer of two kinds of KS119W.

Table 3. is the MTD result of two kinds of enantiomers relatively

Mice	Nude CD-1	BAL B/c	Scid/Beige
				VNP40541	140mpk×10	100mpk×5	110mpk×8
VNP40551	80mpk×10	85mpk×5	85mpk×8

As shown in table 4, assessment VNP40541 and VNP40551 in three sample tumor models of mice.Therefore, they have demonstrated quite similar effectiveness and treatment window, yet VNP40541 has shown low toxicity significantly, and especially observable edema is less.

The effectiveness of table 4. enantiomer and treatment window

	The H460/Nude mice		The EMT6/BLAB-c mice		SHP77/Scid-Beige
							Dosage (mpk)	Suppression ratio (%)	Dosage (mpk)	Suppression ratio (%)	Dosage (mpk)	Suppression ratio (%)
	VNP40541	80-140	52-80	85-115	58-69	110							83
VNP40551							40-80	53-80	55-85	52-78	85	82

The CTX that is lower than the KS119W of MTD and non-toxic dosage by using dosage makes up EMT-6 molluscum contagiosum adenocarcinoma and the H460 people's pulmonary carcinoma of anti-CD-1nude mice, assesses the combined therapy of KS119W and cyclophosphamide (CTX).Allow tumor growth to about 200mm ³Size.In the H460 tumor model, the KS119 of the 180mg/kg of animals received 4 dosage and the 240mg/kg of 1 dosage or to accept the each dosage of each animal be the CTX of 100mg/kg, the described medicine of injection in 15,22,27,34 and 43 days posterior peritoneums after tumor is implanted.In combination research, administration CTX after administration KS119W is two hours.As shown in Figure 9, cause 78% tumor growth inhibition separately with KS119W, and CTX only there is 54% inhibition.KS119W and CTX combined therapy cause 90% tumor suppression.The inhibition of comparing combined therapy with all matched groups is to have statistical significance, and the p value is respectively 0.003,0.015 and 0.038.The additional toxicity that is used in combination dosage is manageable; Separately cause 4.5% maximum net to lose weight, and use same dose and the CTX that is used in combination weekly 4 dosage of 100mg/kg cause 8.6% lose weight, but do not have death with the KS119W of 180mg/kg qw * 4 dosage.In the EMT-6 tumor model, also observe the degree that similar tumor growth suppresses.The KS119W treatment of 7 dosage of 97mg/kg causes 59% tumor control rate with every day, and CTX is with the dosage of 100mg/kg, and one time 4 dosage of peritoneal injection produces negligible influence to mice EMT-6 tumor growth weekly.When animal is used KS119 and CTX combined therapy, observe obvious antineoplastic.KS119W and CTX combination cause 91.8% EMT-6 tumor growth inhibition.These results show that medicine KS119W and CTX combination have extra antitumor action.

The explanation and the embodiment that those skilled in the art will appreciate that the front are enforcement explanation of the present invention, but are not limited only to this.Can carry out various variations, and not deviate from the spirit and scope of the present invention that following claim limits at this detailed description.

Claims (52)

1. according to the chemical compound of formula I, II, III and IV:

R=C wherein _1-10Alkyl, or C _1-10Haloalkyl (preferably contain be no more than 5 halogen groups, be preferably the 2-chloroethyl);

R ' and R " are C independently _1-10Alkyl, or C _5-20Aryl or heteroaryl (preferable methyl);

R ₁Be H, C _1-10Alkyl, C _1-10Alkoxyl, C _5-20Aryl or heteroaryl or C _5-20Aryloxy group or heteroaryloxy (being preferably methyl and ethyl);

X is O, NH or NR (being preferably O);

Y is (CH ₂) _n, O (CH ₂) _n, NH (CH ₂) _n, NR (CH ₂) _n, OCOO (CH ₂) _n, NHCOO (CH ₂) _n

N is 1,2,3,4 or 5 (preferred n=2 and 3); Or Y is aryl or heteroaryl (being preferably phenyl);

A is CH or N (being preferably CH); And

B is CH=CH, O, S, NH or NR (being preferably CH=CH); Or its officinal salt, solvate, polymorph or metabolite.

2. according to the chemical compound of the claim 1 of structure (IA):

Structure I A

Wherein R is C _1-10Alkyl, or C _1-10Haloalkyl; R ₁Be H, C _1-10Alkyl, C _1-10Alkoxyl; X is O, NH or NR; A is CH, CR or N; And B is CH=CH, O, S, NH or NR; Or its officinal salt, solvate, polymorph or metabolite.

3. according to the chemical compound of claim 1 or 2, wherein R is-CH ₃Or-CH ₂CH ₂Cl.

4. according to claim 1,2 or 3 chemical compound, wherein R is-CH ₂CH ₂Cl.

5. according to the chemical compound of claim 1 or 2, wherein R1 is selected from H, C ₁-C ₁₀Alkyl or C ₁-C ₁₀Alkoxyl, wherein alkyl comprises the hexyl of methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, n-pentyl, isopentyl, n-hexyl, isohesyl or replacement.

6. according to each chemical compound among the claim 1-5, wherein C ₁-C ₁₀Alkyl or C ₁-C ₁₀Alkoxyl is-CH ₃Or-OCH ₃

7. according to the chemical compound of claim 6, C wherein ₁-C ₁₀Alkyl or C ₁-C ₁₀Alkoxyl is-CH ₃

8. according to each chemical compound among the claim 1-6, wherein X is selected from O, NH or NR.

9. chemical compound according to Claim 8, wherein X is selected from O.

10. according to each chemical compound among the claim 1-10, wherein A is selected from CH, CR or N.

11. according to the chemical compound of claim 10, wherein A is selected from CH.

12. according to each chemical compound among the claim 1-11, wherein B is selected from CH=CH, O, S, NH or NR.

13. according to the chemical compound of claim 12, wherein B is CH=CH.

14. enantiomer, stereoisomer or tautomer according to each chemical compound in claim 1-13 or 52.

15. according to the chemical compound of claim 1 or 2, wherein said phosphate group is free acid or officinal salt or solvate.

16. metabolite according to each chemical compound in claim 1-15 or 52.

17. pharmaceutical composition comprises the chemical compound according to claim 1 or 52 that is used for the treatment of neoplastic effective dose, randomly with pharmaceutically useful additive, carrier or excipient composition.

18. pharmaceutical composition comprises the chemical compound according to claim 2 for the treatment of neoplastic effective dose, randomly with pharmaceutically useful additive, carrier or excipient composition.

19. according to the compositions of claim 17 or 18, wherein R is-CH ₃Or-CH ₂CH ₂Cl.

20. according to each compositions among the claim 17-19, wherein R is-CH ₂CH ₂Cl.

21. according to each compositions among the claim 17-20, wherein R ₁Be selected from H, C ₁-C ₁₀Alkyl or C ₁-C ₁₀Alkoxyl, wherein alkyl comprises the hexyl of methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, n-pentyl, isopentyl, n-hexyl, isohesyl or replacement.

22. according to the compositions of claim 21, wherein C ₁-C ₁₀Alkyl or C ₁-C ₁₀Alkoxyl is-CH ₃Or-OCH ₃

23. according to the compositions of claim 22, wherein C ₁-C ₁₀Alkyl or C ₁-C ₁₀Alkoxyl is-CH ₃

24. according to each compositions among the claim 17-23, wherein X is selected from O, NH or NR.

25. according to the compositions of claim 24, wherein X is O.

26. according to each compositions among the claim 17-26, wherein A is selected from CH, CR or N.

27. according to the compositions of claim 26, wherein A is CH.

28. according to each compositions among the claim 17-27, wherein B is selected from CH=CH, O, S, NH or NR.

29. according to the compositions of claim 28, wherein B is CH=CH.

30. compositions comprises enantiomer, stereoisomer or tautomer according to each chemical compound in claim 17-30 or 52.

31. according to each compositions in claim 17-30 and 52, wherein said phosphate group is free acid or its officinal salt or solvate.

32. according to each compositions in claim 17-31 and 52, wherein said chemical compound is the metabolite according to the structure I chemical compound.

33. in the patient of needs treatments, treat method for cancer for one kind, comprise to described patient's effective dosage according to claim 1-16 or 52 in each chemical compound, randomly with pharmaceutically useful additive, carrier or excipient composition.

34. in the patient of needs treatment, treat method for cancer, comprise chemical compound, for one kind randomly with pharmaceutically useful additive, carrier or excipient composition according to claim 2 to described patient's effective dosage.

35. according to the method for claim 34, wherein R is-CH ₃Or-CH ₂CH ₂Cl.

36. according to the method for claim 35, wherein R is-CH ₂CH ₂Cl.

37. according to the method for claim 34, wherein R ₁Be selected from H, C ₁-C ₁₀Alkyl or C ₁-C ₁₀Alkoxyl, wherein alkyl comprises the hexyl of methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, n-pentyl, isopentyl, n-hexyl, isohesyl or replacement.

38. according to the method for claim 37, wherein C ₁-C ₁₀Alkyl or C ₁-C ₁₀Alkoxyl is-CH ₃Or-OCH ₃

39. according to the method for claim 38, wherein C ₁-C ₁₀Alkyl or C ₁-C ₁₀Alkoxyl is-CH ₃

40. according to the method for claim 34, wherein X is O, NH or NR.

41. according to the method for claim 40, wherein X is O.

42. according to the method for claim 34, wherein A is CH, CR or N.

43. according to the method for claim 42, wherein A is CH.

44. according to the method for claim 34, wherein B is CH=CH, O, S, NH or NR.

45. according to the method for claim 44, wherein B is CH=CH.

46. according to the method for claim 34, wherein said chemical compound comprises enantiomer, stereoisomer or the tautomer of each chemical compound in claim 1-16 or 52.

47. according to the method for claim 34, wherein said phosphate group is free acid or its officinal salt or solvate.

48. according to the method for claim 34, wherein said chemical compound is the metabolite according to the chemical compound of structure I.

49. according to the method for claim 34, wherein said chemical compound can be individually dosed or with other anticarcinogen combination medicine-feeding or with phototherapy or radiotherapy combination medicine-feeding.

50. according to the method for claim 34, wherein said cancer is a gastric cancer, colon cancer, rectal cancer, hepatocarcinoma, cancer of pancreas, pulmonary carcinoma, breast carcinoma, cervical cancer, carcinoma of uterine body, ovarian cancer, carcinoma of prostate, carcinoma of testis, bladder cancer, renal carcinoma, the brain cancer/central nervous system's cancer, head and neck cancer, laryngocarcinoma, Hodgkin, non-Hodgkin lymphoma, multiple myeloma, melanoma, acute lymphoblastic leukemia, acute myeloblastic leukemia, Ewing sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, wilms' tumor, neuroblastoma, hairy cell, or derive from mouth/pharynx, the cancer of esophagus or larynx.

51. be used for the treatment of purposes in the medicine of cancer in preparation according to each chemical compound in claim 1-16 or 52.

52. chemical compound according to structure I or II:

Structure I structure I I

Wherein R is C _1-10Alkyl or C _1-10Haloalkyl;

R ' and R " are C _1-10Alkyl, or C _5-20Aryl or heteroaryl;

R ₁Be H; C _1-10Alkyl, C _1-10Alkoxyl;

X is O, NH or NR;

R (P) is the alkyl of phosphorous acidic group, and for example, R (P) is Y ' OPO (OH) ₂, wherein Y is (CH ₂) _n, O (CH ₂) _n, NH (CH ₂) _n, NP (CH ₂) _n, n is 1-5, Y is aryl or heteroaryl;

Ar (N) is the aryl that contains nitro, for example,

Ar(N)＝

Wherein A is CH, CR or N; And B is CH=CH, O, S, NH or NR; Reaching Ar (NP) is phosphorous acidic group and the aryl that contains nitro, for example,

Ar(NP)＝

Wherein A is CH, CR or N; And B is CH=CH, O, S, NH or NR; And Y is (CH ₂) _n, O (CH ₂) _n, NH (CH ₂) _n, NR (CH ₂) _n, OCOO (CH ₂) _n, NHCOO (CH ₂) _nN is 1-5; Or its officinal salt, solvate, polymorph or metabolite.

Images