CN100494207C - Antiviral agent cidofovir derivatives and intermediates thereof - Google Patents

Abstract

The invention discloses a new derivant and intermediate for antiviral cidofovir, as well as the method for making such derivant, which is taking key intermediate (S)-N1-[(3-trity methoxy-2-ethphosphoric acid methoxy) glycerol alcohol]-cytosine as raw material, to condensation react with Boc-amino acid with the help of condensating agent to get intermediate compound, and then stripping protecting group Boc and trityl, and hydrolyzing phosphate to get new derivant.

Description

Antiviral agent cidofovir novel derivative and intermediate

Technical field

The present invention relates to the antiviral field, more specifically relate to cidofovir novel derivative and intermediate.

Background technology

Virus is to infect the small organism of all biomass cellss, and it can utilize the metabolic system of host cell to carry out parasitism and propagation, produces cytotoxicity or give rise to diseases in animal or human's body.Virus disease is to propagate the infectious diseases the widest, that sickness rate is the highest at present, according to western countries' incomplete statistics, virus disease has reached more than 60% in the sickness rate of whole disease, considerably beyond infectation of bacteria 16%, and in these virus diseases, have in case infected back virus just can exist several years or lifelong existence in vivo, what have then has a carcinogenic possibility.Therefore researching and developing novel antiviral is the target that many medicine workers endeavour.

Cytomegalovirus (Cytomegalovirus, CMV) belong to a kind of of simplexvirus, it is the pathogenic agent of serious harm HUMAN HEALTH, the crowd of all age brackets is PI CMV, and to the newborn infant of congenital infection, the patient of immunodeficiency (as patient AIDS), accept organ transplantation and patient that the immunity such as cancer patients of receiving treatment are suppressed, cmv infection all may be fatal.After infecting CMV, generally be asymptomatic inapparent infection, in case will cause serious disease during acquired immune deficiency syndrome, its clinical manifestation is heating, lethargic sleep, oligoleukocythemia, thrombopenia, hepatitis, pneumonia, encephalitis, gastritis and other systemic infection symptom; The cytomegalovirus disease can influence patient 40%AIDS, and particularly those CD4+ cell numbers patient of being lower than 50/ μ l can show serious symptoms, as chorioretinitis, esophagitis and neurological symptom.Postmortem and clinical study show that the retinitis is the modal performance of patient's AIDS cmv infection, can account for the 75%-80% of CMV disease.Neurological symptom is not general, but stomach and intestine CMV disease can take place patient 5%-10%AIDS.

Because duplicating of virus must rely on host's cell and enzyme system, and can suppress the medicine of virus replication, also may disturb Normocellular metabolism simultaneously.Therefore, require antiviral that the selectivity of height must be arranged, can blocking virus duplicate and don't the cell that do not infect of damage.Virus replication proliferating cycle can be divided into four-stage, promptly adsorbs, invasion, nucleic acid replication and proteinic synthetic.Dissimilar antiviral can act on different steps and play a role.

1) absorption phase: virus is adsorbed on the cell in this process, and invasion then if stop absorption, utilizes antibody to combine with virus antigen, then can reach the purpose of prophylaxis of viral infections.As with gamma-globulin prevention measles and hepatitis A, prevent hepatitis B with the efficient immunoglobulin (Ig) of hepatitis B.

2) the invasion stage: if can stop virus to enter cell effectively in this stage, just can successfully prevent and treat virus infection.Change the cytolemma electric charge as making, stop the invasion cell with amantadine etc.

3) the nucleic acid replication stage: after virus is invaded cell, shelling, replicating nucleic acid, if with nucleoside medicine such as iodoxuridine, ribavirin, vidarabine, acycloguanosine etc., phosphoric acid changes into single phosphoric acid, bisphosphate and triphosphoric acid derivative in vivo, can suppress the needed plurality of enzymes of nucleic acid, blocking virus RNA or DNA duplicate, thereby reach the purpose of treatment virus disease.

4) virus protein is synthetic: virus is removed to copy the genetic material of virus by inducing cell and is made protein and duplicate.It is synthetic that rifomycin, Ftibamzone etc. can suppress virus protein by the arrestin polysaccharase.

An ideal antiviral should be able to be disturbed duplicating of (or prevention) virus effectively, and don't influences the normal cell metabolism.The antiviral of using clinically mainly can be divided into ucleosides, tricyclic amines, tetra-sodium class, polypeptide class etc. by chemical structure at present, wherein, the most species of ucleosides antiviral accounts for 65%, being most widely used, is the important antiviral of a class.Nucleoside medicine mainly acts on the viral nucleic acid duplicate stage.

Cidofovir (Cidofovir, HPMPC) be the open loop nucleotide analog, develop by U.S. Gilead company, the injection cidofovir was gone on the market through drugs approved by FDA in May, 1996, France and Canada is approval use in succession also, and commodity are called Vistide, only for venoclysis, be applicable to treatment AIDS patient's the CMV retinitis, dosage is 375mg/ bottle cidofovir.Its ointment can be used for treating the Genital warts of HIV and the genital herpes of HSV-2, still in clinical experimental study.Cidofovir can suppress viral dna polymerase, people CMV there is very strong restraining effect, other simplexvirus such as herpes simplex I, II C-type virus C, varicella-zoster virus, bleb 6 C-type virus Cs and adenovirus, human papilloma shape virus are also had very strong activity.This medicine not only has the advantage of long half time, and need not a phosphorylation of the first step, and clinical proof ganciclovir persister is still to the cidofovir sensitivity.

Cidofovir can be regarded the analogue of single phosphoric acid ganciclovir as.Nucleoside medicine is played a role by triphosphoric acidization by virocyte kinases elder generation coverlet phosphorylation in vivo again, therefore, see that theoretically external phosphorylation modification nucleosides should have antiviral activity, and toxic side effect can not strengthen.There are bibliographical information list phosphoric acid Ara, the anti-HSV effect of single phosphoric acid ACV to be better than Ara, ACV respectively, and water-soluble increase.External phosphorylation ACV walks around body inner virus TK phosphorylation process, so single phosphoric acid ACV effect is better than ACV.But compatible because the high polarity of phosphonate moiety is difficult to the lipotropy of cytolemma, the phosphorylation acycloguanosine does not almost have antiviral activity.Its reason may be: because all above-claimed cpds only produce (if any) limited cytoactive for virocyte system, estimate the ability of their permeate through cell membranes, also be not enough to cause biological effect.

Summary of the invention

The effect power of medicine had both depended on the pharmacodynamic properties of drug molecule, and was also relevant with its pharmacokinetic property.Though many medicines have stronger drug effect, the defective because of such as the pharmacokineticss such as distribution of specific of gastrointestinal absorption, tissue, organ has limited the performance of drug effect.In order to improve these defectives, the prodrug design is a kind of method comparatively commonly used.Harper proposes principle of pro-drug in nineteen fifty-nine, its principle is meant having activity or active higher medicine (former medicine or title parent drug) to be transformed into nonactive or active lower compound, these compounds are in vivo through enzyme or chemical action, discharge former medicine and bring into play pharmacological action.This nonactive or active lower compound is exactly a prodrug, though itself non-activity or activity are lower, has than former medicine better physical, chemistry or pharmacokinetic property.

Purpose of the present invention is to give medicine with useful character by structure of modification, mainly comprises:

1) changes intravital pharmacokinetic property, adjust medicine absorption in vivo and distribution, i.e. bioavailability;

2) improve stability of drug and solvability;

3) reduced toxicity and untoward reaction;

4) raising is to the transhipment and the distribution of privileged site;

5) prolong action time etc.

These ucleosides prodrug designs of analysis-by-synthesis all are the modifications to the nucleoside compound glycosyl part, mainly contain following several types:

1, the glycosyl terminal hydroxyl becomes ester bond with amino acid whose carbon teminal, and improves its oral availability;

2, the glycosyl terminal hydroxyl becomes interior ring with the phosphoric acid side chain of end, to reduce its toxic side effect;

3, the phosphoric acid of glycosyl end and chloro trimethylacetic acid methyl esters are reacted into ester, to improve its fat water distribution system, improve the medicine antiviral activity.

The contriver adopts synthetic respectively earlier glycosyl part intermediate and base portion intermediate, again the two is condensed into key intermediate, gets target compound by key intermediate through further derivation

Glycosyl part intermediate (R)-2,3-contract synthesizing of isopropylidene glycerol-4-methanol alcohol sulfonyl methane ester (5):

With D-N.F,USP MANNITOL (1) is raw material, uses ZnCl in acetone ₂For catalyst reaction gets compound (2), (2) oxidation scission under the sodium periodate effect gets aldehyde (3), and its hydrogenation under the POTASSIUM BOROHYDRIDE effect gets pure (4), and alcohol gets intermediate (5) with the methane sulfonyl chloride effect.

Synthesizing of base portion intermediate benzoyl cytosine (6):

With the pyridine is solvent, cytosine(Cyt) and Benzoyl chloride are at room temperature reacted 45 minutes after, add dilute hydrochloric acid and regulate pH value benzoyl cytosine is separated out, stirring 2h crystallization is complete, filtration under diminished pressure, Glacial acetic acid recrystallization, yield 90%.

Key intermediate (S)-N ¹Synthesizing of-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-cytosine(Cyt) (11):

Reach (R)-2 with benzoyl cytosine (6); 3-contract isopropylidene glycerol-4-methanol alcohol methane sulfonate (5) for raw material under the effect of salt of wormwood, react (7); compound (7) open loop under the concentrated hydrochloric acid effect gets compound (8); with the Triphenyl methane chloride 99 effect primary hydroxyl group is protected again; gained compound (9) under the effect of sodium hydride with tolysulfonyl oxygen methyl acid phosphate diethyl ester react (10), (10) are separated with ammoniacal liquor and are sloughed the benzoyl protecting group and get key intermediate (11).

Synthesizing of target compound:

Target compound is with key intermediate (S)-N1-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-cytosine(Cyt) (11) is a raw material; under the effect of condensing agent, get compound (13A-13E), remove tertbutyloxycarbonyl protecting group, trityl-protecting group and hydrolysis phosphoric acid ester again and get target compound (I-1-I-5) with tertbutyloxycarbonyl-amino acid (12A-12E) condensation.

R is-CH in the formula ₂-CH (CH ₃) ₂,-CH (CH ₃)-C ₂H ₅,-CH ₃,-H ,-CH (CH ₃) ₂

Embodiment

Below by following embodiment the present invention is described in further detail, still, it is limitation of the present invention that following embodiment not can be regarded as.

The contract preparation of isopropylidene glycerol-4-methanol alcohol sulfonyl methane ester (5) of embodiment 1 intermediate (R)-2,3-

1, preparation 1,2-5, two (dioxy isopropylidene) N.F,USP MANNITOL (2) of 6-

40g (0.293mol) zinc chloride, 250mL acetone add 37.5g (0.206mol) N.F,USP MANNITOL (1), and stirring at room 20-24h is dissolved in 45g (0.325mol) salt of wormwood in the 45mL hot water, be added dropwise in the reaction flask, stirred 15-20 minute, and filtered 50ml * 3 water washings, filtrate adds about 15mL strong aqua, concentrate white solid, add less water, with dichloromethane extraction 3 times, anhydrous sodium sulfate drying is directly used in next step reaction.Can get 51.75g white paste solid, yield 97-98% (document yield 87%) as the evaporate to dryness dichloromethane layer.

¹H NMR(CDCl ₃)：δ 3.68(dd，H-1，H-6)，3.27(m，4H，H-2，H-3，H-4，H-5)

2, preparation (S)-2, the 3-isopropylidene glycerol-4-methanol aldehyde (3) that contracts

51.75g (0.197mol) 1,2-5, the dichloromethane solution of two (dioxy isopropylidene) N.F,USP MANNITOL (2) of 6-adds the sodium periodate of 50g (0.233mol) porphyrize, and stirring at room drips 2mL saturated sodium bicarbonate liquid, stirring reaction 24h.Filter, room temperature concentrates, and gets the faint yellow oily solid of 50.11g.Yield 92% (the document yield is 91%).

¹HNMR(CDCl ₃)：δ 4.20-4.05(m，1H，CH)，3.88-3.76(m，1H，CH ₂O)，3.73-3.53(m，1H，CH ₂O)，1.50-1.33(m，6H，2CH ₃)

3, preparation (S)-2, the 3-isopropylidene glycerol-4-methanol alcohol (4) that contracts

50.11 (0.385mol) (S)-2, the 3-isopropylidene glycerol-4-methanol aldehyde (3) that contracts is dissolved in the 200mL methyl alcohol, adds 25g (0.462mol) POTASSIUM BOROHYDRIDE, stirred overnight at room temperature, reaction 24h concentrates evaporate to dryness, add 5mL ammoniacal liquor, 30ml water adds methylene dichloride and stirs, tell methylene dichloride, water layer washs with methylene dichloride 50mL * 3, combined dichloromethane, anhydrous sodium sulfate drying, concentrate evaporate to dryness, get the faint yellow oily thing of 45.79g, yield 90% (document yield 91%).

¹H NMR(CDCl ₃)：δ 4.20-4.05(m，1H，CH)，4.02-3.97(t，J＝5Hz，2H CH ₂OH)，3.88-3.76(m，1H，1fromCH ₂O)，3.73-3.53(m，1H，1from CH ₂O)，1.50-1.34(m，6H，2CH ₃)

4, preparation (R)-2, the 3-isopropylidene glycerol-4-methanol alcohol sulfonyl methane ester (5) that contracts

19g (0.142mol) (S)-2; the 3-isopropylidene glycerol-4-methanol alcohol (4) that contracts is dissolved in the 100mL methylene dichloride; cryosel is bathed cooling; under nitrogen protection, drip the 30mL triethylamine; splash into 16.454g (o.142mol) methane sulfonyl chloride again and be dissolved in the solution of 30mL methylene dichloride; after cryosel is bathed and is stirred 1.5h under the cooling, deicing salt bath, stirring at room 4h; add 40mL water; tell methylene dichloride, water layer dichloromethane extraction, combined dichloromethane; the difference water; saturated sodium bicarbonate liquid; the saturated common salt washing; anhydrous sodium sulfate drying, concentrating under reduced pressure get the faint yellow oily thing of 28.76g, yield 91% (the document yield is 93%).(developping agent is a sherwood oil: ethyl acetate=2:1) can get pure white oily matter by column chromatography.[α] _D ²⁰-4.21(C＝5.5，MeOH)[lit[α] _D ²⁰-4.34(C＝5.21，MeOH)]

¹H NMR(CDCl ₃)：δ 4.40-3.67(m，5H，H-1’，H-2’，H-3’)，3.14(s，3H，SO ₂CH ₃)，1.50-1.23(m，6H，2CH ₃)

The preparation of embodiment 2 intermediate benzoyl cytosine (6)

3g (0.027mol) cytosine(Cyt) adds the 300ml pyridine, drips the 37.5mL Benzoyl chloride again, stirring at room, drip off half an hour approximately, drip and finish, stirred 45 minutes, be added dropwise to 2N hydrochloric acid, stirring at room 2h, suction filtration, solid 5% dissolution of sodium hydroxide splashes into 2N hydrochloric acid again, and refrigerator is placed and spent the night, suction filtration, vacuum-drying gets the 5.8g white solid, mp〉300 ℃ (document mp〉300 ℃), yield 95% (the document yield is 89%).

¹H NMR(d ₆-DMSO)：δ 11.17(s，1H，NH)，11.27(s，1H，NH)，8.18(d，J＝7Hz，1H，H-6)，7.96(d，J＝7Hz，2H，2×BzH)，7.85(d，J＝6Hz，1H，H-5)，7.76-7.46(m，3H，3×BzH)

Embodiment 3 intermediates (S)-N1-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-preparation of cytosine(Cyt) (11)

1, (S)-N ¹-[2, the 3-O-isopropylidene-2 that contracts, 3-(dihydroxyl) glyceryl alcohol]-N ⁴The preparation of-benzoyl cytosine (7)

The salt of wormwood of method 1: the benzoyl cytosine of porphyrize (6) 6g (0.0276mol) and 5.4g (0.0390mol) porphyrize, add anhydrous N, dinethylformamide (DMF) 120mL, oil bath is heated to 90 ℃, and slowly Dropwise 5 .2g (0.0267mol) is (R)-2, the 3-isopropylidene glycerol-4-methanol alcohol sulfonyl methane ester (5) that contracts is dissolved in the solution of 50mLDMF, about 1h drips off, and reacts completely suction filtration, the DMF washing precipitation, filtrate decompression concentrates, and adds 300mL methylene dichloride, stir about 2h in the gained solid, filter, with the washed with dichloromethane precipitation, filtrate decompression concentrates, and gets faint yellow oily thing, column chromatography for separation gets white solid 3.2g, yield 33% (the document yield is 34%).Mp:194-196 ℃ (document Mp:195-197 ℃)

The benzoyl cytosine (6) of method 2:6g (0.0276mol) porphyrize and 5.4g (0.0390) salt of wormwood add 120mlDMF, Dropwise 5 .2g (0.0267mol) (R)-2, the contract DMF liquid of isopropylidene glycerol-4-methanol alcohol sulfonyl methane ester (5) of 3-, microwave reaction 10 minutes, suction filtration, the DMF washing precipitation, filtrate decompression concentrates, and adds 300ml methylene dichloride, stir about 2h in the gained solid, filter, with the washed with dichloromethane precipitation, filtrate decompression concentrates, and gets faint yellow oily thing, column chromatography for separation gets white solid 3g, yield 33%.

¹H NMR(d ₆-DMSO)：δ 7.98-7.95(d，J＝6Hz，1H，H-6)，7.83-7.76(d，J＝14Hz，1H，H-5)，7.67-7.26(m，5H，5×BzH)，4.51-4.45(m，1H，H-2’)，4.21-4.08(m，2H，H-1’andH-3’)，3.88-3.84(dd，J＝7，14Hz，1H，H-1’)，3.82-3.00(m，1H，H-3’)，1.43(m，3H，CH ₃)，1.34(m，3H，CH ₃)

2, (S)-N ¹-[(2, the 3-dihydroxyl) glyceryl alcohol]-N ⁴The preparation of-benzoyl cytosine (8)

3g (9.11mmol) (S)-N ¹-[2, the 3-O-isopropylidene-2 that contracts, 3-(dihydroxyl) glyceryl alcohol]-N ⁴-benzoyl cytosine (7) adding 20ml methyl alcohol, dropping 3mL concentrated hydrochloric acid stirs 2h, and refrigerator is placed and is spent the night, and filters, and filter residue is washed with cold methanol, gets white solid, and vacuum-drying gets the 2.57g white solid, and yield is 92%.Mp:190-192 ℃ (document Mp:193-195 ℃)

¹H NMR(d ₆-DMSO)：δ 11.17(s，1H，NH)，11.27(s，1H，NH)，8.18(d，J＝7Hz，1H，H-6)，7.96(d，J＝7Hz，2H，2×BzH)，7.85(d，J＝6Hz，1H，H-5)，7.46-7.76(m，3H，3×BzH)，4.67-4.58(m，1H，H-2’)，4.20-4.08(m，2H，H-1’and H-3’)，3.91-3.84(dd，J＝7，14Hz，1H，H-1’)，3.29-3.27(dd，J＝6，9Hz，1H，H-3’)

3, (S)-N ¹-[(3-three benzyloxies-2-hydroxyl) glyceryl alcohol]-N ⁴The preparation of-benzoyl cytosine (9)

1g (3.4mmol) (S)-N ¹-[(2, the 3-dihydroxyl) glyceryl alcohol]-N ⁴-benzoyl cytosine (8), 1.059g (3.8mmol) triphenylmethyl chloride, 21mg (0.16mmol) DMAP add the 30mL pyridine, and back flow reaction 5h removes pyridine under reduced pressure, adding 40mL sodium bicarbonate is full to close liquid, and dichloromethane extraction, dichloromethane layer are used saturated sodium bicarbonate liquid respectively, the saturated common salt water washing, anhydrous sodium sulfate drying concentrates, and gets the faint yellow oily thing of thickness, add sherwood oil, stir 3h, filter, get 2g off-white color solid.Yield is 84% (the document yield is 82%).Mp:123-126 ℃ (document Mp:122-123 ℃)

¹H NMR(d ₆-DMSO)：δ 11.17(s，1H，NH)，11.27(s，1H，NH)，8.18(d，J＝7Hz，1H，H-6)，7.96(d，J＝7Hz，2H，2×BzH)，7.85(d，J＝6Hz，1H，H-5)，7.46-7.76(m，19H，3×BzH，15×CPh ₃)，4.67-4.58(m，1H，H-2’)，4.38-4.08(m，2H，H-1’andH-3’)，3.91-3.84(dd，J＝7，14Hz，1H，H-1’)，3.70-3.63(dd，J＝6，9Hz，1H，H-3’)

4, (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴The preparation of-benzoyl cytosine (10)

1.13g (2.01mmol) (S)-N ¹-[(3-three benzyloxies-2-hydroxyl) glyceryl alcohol]-N ⁴-benzoyl cytosine (9) adds 30mLDMF and 15mL sherwood oil, and cryosel is bathed cooling, and nitrogen protection adds 18mg (0.75mmol) sodium hydride down; insulation reaction 2h; add 0.95g (2.9mmol) diethyl [(tosic acid) methyl] phosphoric acid ester (14) again, stirring at room reaction 2 days adds entry; ethyl acetate extraction; ethyl acetate layer saturated common salt water washing, anhydrous sodium sulfate drying, concentrating under reduced pressure; get the 1.63g yellow oil, be directly used in next step reaction.

¹HNMR(d ₆-DMSO)：δ 7.80-7.75(d，J＝4Hz，1H，H-6)，7.37-7.31(d，J＝2Hz，1H，H-5)，7.30-7.20(m，20H，5×BzH，15×CPh ₃)，4.20-4.05(m，5H，H-1’，H-2’，H-3’)，2.93-2.88(d，J-10，2H，OCH ₂P)，2.44(s，4H，4×CH ₂)，1.37-1.24(m，6H，2×CH ₃)

5, (S)-N ¹The preparation of-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-cytosine(Cyt) (11)

1.63g (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴-benzoyl cytosine (10) adds 100mL methyl alcohol, feeds ammonia to saturated, stirring at room 8h, and evaporated under reduced pressure gets the 1.38g yellow oil, is directly used in next step reaction.

¹H NMR(d ₆-DMSO)：δ 11.17(s，1H，NH)，7.99-7.77(d，J＝4Hz，1H，H-6)，7.76-7.75(d，J＝2Hz，1H，H-5)，7.37-7.20(m，15H，15×TrH)，4.20-4.05(m，5H，H-1’，H-2’，H-3’)，2.93-2.88(d，J-10，2H，OCH ₂P)，2.44(s，4H，4×CH ₂)，1.37-1.24(m，6H，CH ₃)

The preparation of embodiment 4 carbonyl dimidazoles CDI (15)

12g (0.176mol) imidazoles is dissolved in the 60mL methylene dichloride, drip other 5g (0.0168mol) triphosgene and be dissolved in the solution of 25mL methylene dichloride, cryosel is bathed cooling and is stirred 1h, stirring at room 2h down, filter, with methylene dichloride filter wash cake, dichloromethane layer is evaporated to 20mL, adds the 100mL sherwood oil, refrigerator is placed and is spent the night, there are a large amount of white solids to generate, filter, get the 8.05g white solid.Mp:113-116 ℃ (document 116-200 ℃), yield 80% (the document yield is 82%).

The preparation of embodiment 5 cidofovir-leucine (I-1)

1, the preparation of tertbutyloxycarbonyl-leucine (12A)

2g (0.0152mOl) leucine is dissolved in the 10.1mL water, 25% solution that adds 728mg (0.0182mol) sodium hydroxide, drip the 20.2mL tetrahydrofuran (THF) liquid of 3.32g (0.0152mol) uncle fourth oxygen formic anhydride, be heated to 41 ℃, insulation reaction 8h, concentrate evaporate to dryness, add 3.51g (0.0183mol) citric acid and less water, ethyl acetate extraction, ethyl acetate layer is water, saturated common salt water washing respectively, anhydrous sodium sulfate drying, evaporated under reduced pressure, get the 3.24g white solid, yield 92%, Mp:82-83 ℃ (document 82-85 ℃).

2, (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴The preparation of-tertbutyloxycarbonyl-leucine cytosine(Cyt) (13A)

2g (8.65mmol) tertbutyloxycarbonyl-leucine (12A) and 506mg (8.65mmol) carbonyl dimidazoles add 50mLDMF, 2h is stirred in the cryosel cooling down, add 1.504g (2.37mmol) (S)-N1-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-cytosine(Cyt) (11), be heated to 80 ℃, reaction 24h, add entry, ethyl acetate extraction, ethyl acetate layer is water, saturated common salt water washing respectively, anhydrous sodium sulfate drying, evaporated under reduced pressure gets the faint yellow aqueous thing of 2.28g.The gained crude product is directly used in next step reaction.

¹H NMR(CDCl ₃)：δ 7.81-7.76(d，J＝4，1H，H-6)，7.63-7.50(m，15H，3×BzH)，7.33-7.32(d，J＝8，1H，H-5)，5.29(d，J＝8，1H，H-1’)，4.88-4.67(t，J＝6.14，1H，H-2’)，4.62-4.58(d，J＝4，16，2H，H-3’)，3.37-3.06(m，4H，H-3’’，H-4’’，H-5’’)，3.06(s，2H，CH ₂P)，2.93(m，4H，2×CH ₂)，1.07-0.83(m，16H，CH ₃)

3, the preparation of cidofovir-leucine (I-1)

2g (2.48mmol) (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴-tertbutyloxycarbonyl-leucine cytosine(Cyt) (13A) adds the 50mL acetonitrile; nitrogen protection adds 5.21g (31.38mmol) potassiumiodide down; be heated to 60 ℃, drip the acetonitrile liquid of 4mL (31.38mmol) trimethylchlorosilane, insulation reaction 2 days; concentrating under reduced pressure; add 50mL water, stirring at room 2h, extremely faint yellow with methylene dichloride wash water layer; water liquid concentrating under reduced pressure gets the 0.70g faint yellow solid.Two step total recoverys are 72%.[α] _D ²⁰-102.3(H ₂O)

¹HNMR(d ₆-DMSO)：δ 7.34-7.12(d，J＝8Hz，1H，H-6)，7.31-7.28(d，J＝6Hz，1H，NH)，6.98(s，2H，NH ₂)，5.64-5.53(m，1H，H-5)，4.29-3.60(m，7H，H-1’，H-2’，H-3’，OCH ₂P)，2.51-2.50(d，J＝2Hz，1H，H-3’’)，2.33-2.30(dd，J＝2Hz，2Hz，2H，H-4’’)，2.29(s，1H，H-5’’)，1.31-1.28(d，J＝6Hz，3H，3×CH ₃)，1.23-1.13(d，J＝10Hz，3H，3×CH ₃)

MS(m/e)：393(M ⁺+1)

IR(Vmax：cm ^-1，KBr)：3342，1655，1432

The preparation of embodiment 6 cidofovir-Isoleucine (I-2)

1, the preparation of tertbutyloxycarbonyl-Isoleucine (12B)

2g (0.0152mol) Isoleucine is dissolved in the 10.1mL water, 25% solution that adds 728mg (0.0182mol) sodium hydroxide, the 20.2mL tetrahydrofuran (THF) liquid of dropping 3.32g (0.0152mol) uncle fourth oxygen formic anhydride is heated to 41 ℃, insulation reaction 8h, concentrate evaporate to dryness, add 3.51g (0.0183mol) citric acid and less water, ethyl acetate extraction, ethyl acetate layer is water, saturated common salt water washing respectively, anhydrous sodium sulfate drying, evaporated under reduced pressure gets the 2.95g white solid.Yield is 83%, Mp:56-58 ℃ (document 58-20 ℃).

2, (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴-tertbutyloxycarbonyl-Isoleucine cytosine(Cyt) (13B)

2g (8.65mmol) tertbutyloxycarbonyl-Isoleucine (12B) and 506mg (8.65mmol) carbonyl dimidazoles add 50mLDMF, 2h is stirred in the cryosel cooling down, add 1.405g (2.37mmol) (S)-N1-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-cytosine(Cyt) (11), be heated to 80 ℃, reaction 24h, add water, ethyl acetate extraction, ethyl acetate layer is water, saturated common salt water washing respectively, anhydrous sodium sulfate drying, evaporated under reduced pressure gets the faint yellow aqueous thing of 2.43g.Product is directly used in next step reaction.

¹H NMR(CDCl ₃)::δ 7.81-7.76(d，J＝4，1H，H-6)，7.63-7.55(m，15H，3×BzH)，7.37-7.32(d，J＝8，1H，H-5)，5.29(d，J＝8Hz，1H，H-1’)，4.88-4.67(t，J＝6，14Hz，1H，H-2’)，4.62-4.58(d，J＝4，16Hz，2H，H-3’)，3.25-3.06(m，4H，H-3’’，H-4’’，H-5’’)，2.95(s，2H，CH ₂P)，2.83-2.56(m，4H，2×CH ₂)，1.15-0.93(m，16H，CH ₃)

3, the preparation of cidofovir-Isoleucine (I-2)

2g (2.48mmol) (S)-N ₁-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ₄-tertbutyloxycarbonyl-Isoleucine cytosine(Cyt) (13B) adds the 50mL acetonitrile; nitrogen protection adds 5.21g (31.38mmol) potassiumiodide down; be heated to 60 ℃, drip the acetonitrile liquid of 4mL (31.38mmol) trimethylchlorosilane, reaction insulation reaction 2 days; concentrating under reduced pressure; add 50mL water, stirring at room 2h, extremely faint yellow with methylene dichloride wash water layer; water liquid concentrating under reduced pressure gets the 0.62g faint yellow solid.Two step total recoverys are: 67%.[α] _D ²⁰-121.3 (H ₂O)

¹H NMR(d ₆-DMSO)：δ 11.93(s，1H，NH)，7.60(s，1H，H-6)，7.07(s，1H，H-5)，6.80(s，1H，NH)，4.27-3.25(m，7H，H-1’，H-2’，H-3’，O CH ₂P)，3.14-3.10(d，1H，J＝8Hz，H-3’’)，2.90-2.68(m，3H，H-4’’，H-5’’)，1.31-1.27(d，J＝10Hz，3H，CH ₃)，1.15-0.99(dd，J＝4Hz，6Hz，3H，CH ₃)

MS(m/e)：392(M ⁺)，323，167

IR(Vmax：cm ^-1，KBr)：3440，1644，1462，1161

The preparation of embodiment 7 cidofovir-L-Ala (I-3)

1, the preparation of tertbutyloxycarbonyl-L-Ala (12C)

2g (0.0224mol) L-Ala is dissolved in the 15mL water, 25% solution that adds 1.076g (0.0269mol) sodium hydroxide, drip the 30mL tetrahydrofuran (THF) liquid of 4.9g (0.0225mol) uncle fourth oxygen formic anhydride, be heated to 41 ℃, insulation reaction 8h, concentrate evaporate to dryness, add 5.176g (0.0269mol) citric acid and less water, ethyl acetate extraction, ethyl acetate layer is water, saturated common salt water washing respectively, anhydrous sodium sulfate drying, evaporated under reduced pressure, get the 3.905g white solid, yield 92%, Mp:79-81 ℃ (document 80-82 ℃)

2, (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴-tertbutyloxycarbonyl-L-Ala cytosine(Cyt) (13C)

2g (10.5mmol) tertbutyloxycarbonyl-L-Ala (12C) and 1.419g (10.5mmol) carbonyl dimidazoles add 50mLDMF, and 2h is stirred in the cryosel cooling down, add 4.30g (3.20mmol) (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-cytosine(Cyt) (11) is heated to 80 ℃, reaction 24h, add entry, ethyl acetate extraction, ethyl acetate layer is water, saturated common salt water washing respectively, anhydrous sodium sulfate drying, evaporated under reduced pressure gets the faint yellow aqueous thing of 2.48g.Crude product is directly used in next step reaction.

¹H NMR(CDCl ₃)：：δ 7.77-7.75(d，J＝4，1H，H-6)，7.68-7.57(m，15H，3×BzH)，7.35-7.31(d，J＝8，1H，H-5)，4.96-4.02(m，5H，H-1’，H-2’，H-3’)，3.45(s，2H，CH ₂P)，2.93-2.76(m，4H，CH ₂)，2.85(m，1H，CH)，1.34(m，9H，CH ₃)

3, the preparation of cidofovir-L-Ala (I-3)

2g (2.61mmol) (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴-tertbutyloxycarbonyl-L-Ala cytosine(Cyt) (13C) adds the 34mL acetonitrile; nitrogen protection adds 4.81g (28.9mmol) potassiumiodide down; be heated to 60 ℃, drip the acetonitrile liquid of 4mL (28.9mmol) trimethylchlorosilane, reaction insulation reaction 2 days; concentrating under reduced pressure; add 50mL water, stirring at room 2h, extremely faint yellow with methylene dichloride wash water layer; water liquid concentrating under reduced pressure gets the 0.283g faint yellow solid.Two step total recoverys are 78%.

1H NMR(CDCl ₃)：δ 7.81-7.76(d，J＝4Hz，1H，H-6)，7.37-7.33(d，J＝8Hz，1H，H-5)，4.20-4.06(m，5H，H-1’，2×H-2’，H-3’)，2.44(s，2H，OCH ₂P)，2.15(s，1H，CH)，1.42-1.26(m，3H，CH ₃)

MS(m/e)：352(M ⁺+1)，269，127

IR(Vmax：cm ^-1，KBr)：3464，1620，1462，1192

The preparation of embodiment 8 cidofovir-glycine (I-4)

1, the preparation of tertbutyloxycarbonyl-glycine (12D)

2g (0.0266mol) glycine is dissolved in the 18mL water, 25% solution that adds 1.27g (0.0317mol) sodium hydroxide, the 30mL tetrahydrofuran (THF) liquid of Dropwise 5 .814g (0.0266mol) uncle fourth oxygen formic anhydride, be heated to 41 ℃, insulation reaction 8h, concentrate evaporate to dryness, add 5.8144g (0.0302mol) citric acid and less water, ethyl acetate extraction, ethyl acetate layer is water, saturated common salt water washing respectively, anhydrous sodium sulfate drying, evaporated under reduced pressure, get the 4.128g white solid, yield 88.5%, mp:85-86 ℃ (document 87-88 ℃)

2, (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴-tertbutyloxycarbonyl-glycine cytosine(Cyt) (13D)

2g (11.4mmol) tertbutyloxycarbonyl-glycine (12D) and 2.281g (11.4mmol) carbonyl dimidazoles add 50mLDMF, and 2h is stirred in the cryosel cooling down, add 6.914g (3.81mmol) (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-cytosine(Cyt) (11) is heated to 80 ℃, reaction 24h, add entry, ethyl acetate extraction, ethyl acetate layer is water, saturated common salt water washing respectively, anhydrous sodium sulfate drying, evaporated under reduced pressure gets the faint yellow aqueous thing of 2.77g.Crude product is directly used in next step reaction.

¹H NMR(CDCl ₃)：：δ 7.92(d，J＝4Hz，1H，H-6)，7.77-7.59(m，15H，3×BzH)，7.42(d，J＝8Hz，1H，H-5)，4.63-4.32(m，5H，H-1’，H-2’，H-3’)，3.52(s，2H，CH ₂P)，2.87-2.63(m，4H，2×CH2)，2.13(s，2H，CH ₂)1.07-0.83(m，6H，CH ₃)

3, the preparation of cidofovir-glycine (I-4)

2g (2.66mmol) (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴-tertbutyloxycarbonyl-glycine cytosine(Cyt) (13D) adds the 50mL acetonitrile; nitrogen protection adds 5.0g (34.27mmol) potassiumiodide down, is heated to 60 ℃, drips the acetonitrile liquid of 3.77ml (34.73mmol) trimethylchlorosilane; reaction insulation reaction 2 days; be cooled to room temperature, concentrating under reduced pressure adds 34mL water; stirring at room 2h; to faint yellow, water liquid concentrating under reduced pressure gets the 0.67g faint yellow solid with methylene dichloride wash water layer.Two step total recoverys are 75%.

¹H NMR(CDCl ₃)：δ 7.81-7.76(d，J＝4Hz，1H，H-6)，737-7.32(d，J＝8Hz，1H，H-5)，4.20-4.06(m，5H，H-1’，2×H-2’，H-3’)，2.35(s，2H，OCH ₂P)，2.15(s，2H，CH ₂)

MS(m/e)：339，323，208，197，181

IR(Vmax：cm ^-1，KBr)：3520，1648，1462，1161

The preparation of embodiment 9 cidofovir-Xie Ansuan (I-5)

1, the preparation of tertbutyloxycarbonyl-Xie Ansuan (12E)

2g (0.0171mol) leucine is dissolved in the 11.4mL water, 25% solution that adds 820mg (0.0205mol) sodium hydroxide, drip the 22.8ml tetrahydrofuran (THF) liquid of 3.728g (0.0171mol) uncle fourth oxygen formic anhydride, be heated to 41 ℃ of tertbutyloxycarbonyl-leucines (15), insulation reaction 8h, concentrate evaporate to dryness, add 3.94g (0.0205mol) citric acid and less water, ethyl acetate extraction, ethyl acetate layer is water respectively, the saturated common salt water washing, anhydrous sodium sulfate drying, evaporated under reduced pressure, get the 3.154g white solid, yield is 84-85%, Mp:77-79 ℃ (document 78-79 ℃)

2, (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴-tertbutyloxycarbonyl-Xie Ansuan cytosine(Cyt) (13E)

2g (9.21mmol) tertbutyloxycarbonyl-Xie Ansuan (12E) and 1.245g (9.21mmol) carbonyl dimidazoles add 50ml DMF, and 2h is stirred in the cryosel cooling down, add 3.773g (3.068mmol) (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-cytosine(Cyt) (11) is heated to 80 ℃, reaction 24h, add entry, ethyl acetate extraction, ethyl acetate layer is water, saturated common salt water washing respectively, anhydrous sodium sulfate drying, evaporated under reduced pressure gets the faint yellow aqueous thing of 2.39g.Product is directly used in next step reaction.

¹H NMR(CDCl ₃)：δ 7.72-7.70(d，J＝4Hz，1H，H-6)，7.53-7.42(m，15H，3×BzH)，7.21(d，J＝8Hz，1H，H-5)，4.20-3.76(m，5H，H-1’，H-2’，H-3’)，3.27(s，2H，CH ₂P)，2.76-2.57(m，4H，2×CH ₂)，2.43-2.31(m，2H，2CH)，1.07-0.83(m，12H，4CH ₃)

3, the preparation of cidofovir-Xie Ansuan (I-5)

2g (2.55mmol) (S)-N ¹-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) glyceryl alcohol]-N ⁴-tertbutyloxycarbonyl-Xie Ansuan cytosine(Cyt) (13E) adds the 50mL acetonitrile; nitrogen protection adds 3.939g (23.72mmol) potassiumiodide down; be heated to 60 ℃, drip the acetonitrile liquid of 3mL (23.72mmol) trimethylchlorosilane, reaction insulation reaction 2 days; concentrating under reduced pressure; add 50mL water, stirring at room 2h, extremely faint yellow with methylene dichloride wash water layer; water liquid concentrating under reduced pressure gets the 0.220g faint yellow solid.Two step total recoverys are 78%.

¹HNMR(CDCl ₃)：δ 8.05-7.95(d，J＝10Hz，1H，H-6)，7.54(s，1H，H-5)，3.36-2.87(m，5H，H-1’，H-2’，H-3’)，1.73-1.17(m，4H，OCH ₂P，CH，CHNH ₂)，1.17-0.87(m，6H，6×CH ₃)

Embodiment 10 vitro enzyme hydrolysising experiments (liver homogenate experiment)

1, experiment purpose:

Primary requirement to prodrug is that prodrug can discharge former medicine in vivo, so whether design liver homogenate experiment can discharge former medicine cidofovir and time of releasing roughly thereof to measure prodrug in liver enzyme.

2, experiment material:

Experimental animal: the bull healthy mice, the about 30g of body weight is provided by Sichuan University's West China Experimental Animal Center.

Test drug: all target compounds all take by weighing about 10mg and are dissolved in the distilled water, promptly use.

Instrument: liver homogenate device, whizzer

3, experimental technique:

Get one of bull small white mouse, weigh, take out its liver, about 1.2g that weighs, dividing equally is two parts, a cidofovir standard control that adds, another part adds the prodrug sample.Sample is got about 10mg be dissolved in the 3ml distilled water, with liver mixing homogenate, about 30 minutes, add in the centrifuge tube, centrifugal 2 minutes, get supernatant liquid, add the methyl alcohol that doubles its amount, there is precipitation to generate, centrifugal 2 minutes, get supernatant liquid, with standard control point plate, thin-layer developing (developping agent: propyl carbinol: water: Glacial acetic acid=12:10:1), develop the color with iodine.

4, experimental result:

The Rf value of serial target compound of table 1 and cidofovir thin-layer developing

	Contrast	Liver homogenate 15 minutes	Liver homogenate 30 minutes
				Cidofovir	Rf＝0.6	Rf＝0.6	Rf＝0.6
I-1	Rf＝0.4	Rf＝0.6 Rf＝0.4	Rf＝0.6
				I-2	Rf＝0.4	Rf＝0.6 Rf＝0.4	Rf＝0.6
I-3	Rf＝0.4	Rf＝0.6 Rf＝0.4	Rf＝0.6
				I-4	Rf＝0.4	Rf＝0.6 Rf＝0.4	Rf＝0.6
I-5	Rf＝0.4	Rf＝0.6 Rf＝0.4	Rf＝0.6

Experimental result shows, 5 compounds of this series all can disconnect in the kind under the effect of vitro enzyme and discharge former medicine in 30 minutes fully, reached purpose of the present invention.

Embodiment 11 measures oral administration biaavailability

To be equivalent to 25 milligrams of/kilogram cidofovir and target compound feeds to Long Evans mouse with gavage.Continued 24 hours and collected in 48 hours the urine of ultrafiltration post dose, analyze with anti-phase highly pressurised liquid chromatography.The oral administration biaavailability of cidofovir and target compound is represented with the percentage ratio of the cidofovir dosage of discharge in the urine.

The urine recovery degree of target compound (dosage percentage ratio %)

Cidofovir 25

I-1 63

I-2 50

I-3 55

I-4 60

I-5 57

Claims (5)

1, cidofovir derivative or its pharmacy acceptable salt of a kind of following formula (I) expression:

R is-CH in the formula ₂-CH (CH ₃) ₂,-CH (CH ₃)-C ₂H ₅,-CH ₃,-H ,-CH (CH ₃) ₂

2, the method for the cidofovir derivative of a kind of preparation following formula (I) expression: this method comprises by following formula (II)

And formula (III) reaction, R is-CH in the formula ₂-CH (CH ₃) ₂,-CH (CH ₃)-C ₂H ₅,-CH ₃,-H ,-CH (CH ₃) ₂

Generate (IV), R is-CH in the formula ₂-CH (CH ₃) ₂,-CH (CH ₃)-C ₂H ₅,-CH ₃,-H ,-CH (CH ₃) ₂

In the presence of (CH3) 3SiCl/KI, remove t-butoxycarbonyl protecting group, trityl-protecting group and hydrolysis phosphoric acid ester reaction production (I);

R is-CH in the formula ₂-CH (CH ₃) ₂,-CH (CH ₃)-C ₂H ₅,-CH ₃,-H ,-CH (CH ₃) ₂

3. pharmaceutical composition, this pharmaceutical composition comprise cidofovir derivative or its pharmacy acceptable salt by following formula (I) expression as the pharmacy effective dose of activeconstituents, also comprise pharmaceutically acceptable carrier or thinner;

R is-CH in the formula ₂-CH (CH ₃) ₂,-CH (CH ₃)-C ₂H ₅,-CH ₃,-H ,-CH (CH ₃) ₂

4, glyceryl alcohol a kind of intermediate (S)-N1-[(3-three benzyloxies-2-ethyl phosphonic acid methoxyl group) as shown in the formula (IV)]-cytosine(Cyt);

5, the preparation method of formula as claimed in claim 4 (IV), it is rapid to comprise following reaction cloth: