CN101033238A - Preparation method and use for purine compounds double amino acid ester - Google Patents

Preparation method and use for purine compounds double amino acid ester Download PDF

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CN101033238A
CN101033238A CNA2006100244016A CN200610024401A CN101033238A CN 101033238 A CN101033238 A CN 101033238A CN A2006100244016 A CNA2006100244016 A CN A2006100244016A CN 200610024401 A CN200610024401 A CN 200610024401A CN 101033238 A CN101033238 A CN 101033238A
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ethyl
vitamin
amino
acid
phosphonium mesitoyl
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CN101033238B (en
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杨玉社
付晓钟
李战
嵇汝运
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Shanghai Institute of Materia Medica of CAS
Nanjing Changao Pharmaceutical Science and Technology Co Ltd
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Shanghai Institute of Materia Medica of CAS
Nanjing Changao Pharmaceutical Science and Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Abstract

This invention provides a kind of purine compound diplo-amino acid ester shown as formula (I), and the acceptance of inorganic or organic salt in pharmacology, in which, R1 is amino, R2 is the free L-amino amino acid or with the protection of amino, n is 0 or 1, X is O or S. The invention also provides the preparation method of this compound and its application in medicine for the treatment of viral infectious disease as active materials.

Description

The preparation method of purine compounds double amino acid ester and purposes
Technical field
The present invention relates to pharmaceutical chemistry and anti-virus infection therapeutics field, be specifically related to purine compound and the purposes in the preparation antiviral thereof.
Background technology
Hepatitis B is the disease of a kind of serious harm human health of being caused by hepatitis B virus (HBV).According to statistics, nearly in the world 3.5 hundred million Chronic HBV carrier, annual nearly 1,000,000 people die from the disease that HBV causes.In China 1.2 hundred million people being arranged approximately is hepatitis B virus carrierss, hepatitis B patient 2,800 ten thousand, and 1/3rd people can develop into chronic hepatitis, liver cirrhosis or primary hepatocarcinoma (HCC) in the hepatitis B patient.The main medicine of chronic hepatitis B treatment comprises Interferon, rabbit, ucleosides antiviral and immunomodulator.The ucleosides antiviral is the inhibitor of viral DNA polymerase or reversed transcriptive enzyme, and they can stop the synthetic of viral DNA chain, thereby suppresses virus replication.In the anti-HBV medicine of ucleosides, non-cyclic nucleoside phosphonate compound has vital role, its can be used as nucleosides 5 '-analogue of phosplate, can walk around the restrictive first phosphorylation reaction of speed in the nucleoside analog reactivation process, therefore inhibited equally for the dna virus of the viral thymidine kinase (TK) of can not encoding, has the more antiviral activity of wide spectrum (Erik De Clercq Nature Rev.2002,1,13).In addition because the structure of this compounds is different from the L-nucleoside medicine, thereby and the anti-HBV medicine of L-ucleosides of clinical application such as lamivudine between do not produce cross resistance.(Doo E Gastroenterology 2001，120：1000；Das，K J Virol 2001，75：4771)。Because these characteristics make the acyclonucleosides phosphonic acid ester represent the extremely promising antiviral of a class.
Adefovir dipivoxil (bis (POM) PMEA; be called for short ADV) be 9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 Adefovir (PMEA; Adefovir) two pivaloyl oxygen methyl ester prodrugs; going on the market through the FDA approval in September, 2003, is the anti-HBV medicine of second ucleosides small molecules that goes on the market behind lamivudine.ADV all has very strong antiviral activity (Yuen MF.Expert Opin Pharmacother.2004,5 (11): 2361-7) to hepatitis B virus (HBV), dhbv dna (DHBV) and to the variation hepatitis B virus (YMDD) of lamivudine tolerance in vitro and in vivo.Clinical study shows that this medicine can obviously improve patient's liver histological character, reduce serum HBV-DNA and transaminase level, increase e antigen (HBeAg) transformation efficiency, and the no matter state of HBeAg, genotype, people's species diversity or not existing variant viral to lamivudine resistance all not influence ADV brings into play anti-HBV activity (Anneke KRaney.Expert Opin.Investig Drugs 2003,12 (8): 1281-1295).But during using, ADV also has following major defect: (1) unstable chemcial property, its hydrolysis reaction for the seroenzyme mediation is extremely sensitive, can not effectively increase site of action drug level (Pieter Annaert, Pharmaceutical Research.1997,14 (4): 492-496); (2) nucleic acid of transhipment a part need discharge two normal formaldehyde and trimethylacetic acid (Jae-Taeg Hwang.Drugs of the Future 2004,29 (2) with genotoxic potential; 163-177).A new generation's Adefovir prodrug purpose of design is intended to increase the metabolic stability of medicine, prolongs action time, increases drug bioavailability, reduces drug toxicity simultaneously.
Human peptide transporter (Human peptide transporters) is a kind of integration endochylema membranin that mediation two in the human body, tripeptides absorb, and it belongs to proton according to patience oligopeptides transporter family.Present two kinds of human peptide transporter (PepT1 with transport activity, PepT2) cloned and the function assessment conclusive evidence, wherein PepT1 is called as enteron aisle peptide transporter, it mainly is present on the intestinal epithelial cell pectination film, substrate-the proton of the contrary concentration gradient of the mediation process that cotransports, natural L-amino acid is its suitable transhipment substrate (Isabel Rubio-Aliaga TRENDS inPharmacological Sciences, 2002,23 (9): 434-440).Because illustrating of this mechanism, recently the amino acid ester modifying method is used to antiviral nucleoside derivatives and improves bioavailability and strengthen in the design of drug effect, respectively the bioavailability of former medicine is brought up to 63%, 61% (Birger Brodin.Pharmacology ﹠amp from 6%, 20% as L-valine ester prodrug L-Valaciclovir, the L-Valganciclovir of acyclovir (Acyclovir) and ganciclovir (Ganciclovir); Toxicology.2002,90:285-29; Raymund R Razonable, Expert Rev.Anti-infect.Ther.2004,2 (1): 27-42).Two L-Xie Ansuans of the Penciclovir of MEDIVIR AB company development and the Isoleucine ester derivative all can obviously improve the bioavailability of former medicine and make medicine have potent anti-varicella virus, parotid gland virus and EB-virus function (PerEngelhardt, US 0020188125); The L-L-valine ester derivative BRL 44385 of 9-(3-hydroxypropyl-1-oxygen) guanine of Smithline Beecham company development can be when improving former medicine bioavailability, make medicine have potent restraining effect (Hamden to dna virus (the simple virus 1/2 of the parotid gland, varicella virus, cytomegalovirus, hepatitis B virus etc.), Michael, WO9509855).Above result of study has shown that the amino acid ester modifying method is in the advantage of improving aspect the ucleosides antiviral pharmacokinetics.
Summary of the invention
An object of the present invention is to provide and have antiviral activity particularly the novel purine compounds and the pharmacy acceptable salt thereof of resistance of hepatitis B (HBV) and acquired immune deficiency syndrome (AIDS) (HIV) virus activity.
Another object of the present invention provides above-mentioned have antiviral activity particularly resistance of hepatitis B and the novel purine compounds of HIV virus activity and the preparation method of pharmacy acceptable salt thereof.
A further object of the present invention provide this compounds and salt thereof as active substance in the particularly application in the medicine of the infectious diseases that causes of HBV and HIV virus of preparation treatment disease of viral infection.
The invention provides on purine compounds double amino acid ester shown in the formula (I) and the pharmacology thereof and can accept inorganic or organic salt:
Figure A20061002440100111
In the formula (I),
R 1Be amino;
R 2For amido protecting or free L-type amino acid;
N is 0 or 1;
X is O or S.
The present invention also provides purine compounds double amino acid ester with above-mentioned formula (I) structure and two kinds of preparation methods of salt thereof.
Method one: undertaken by following flow process I
Flow process I
Illustration: bromoethanol a.2-, N, N-Dimethylamino pyridine, dicyclohexylcarbodiimide, 0～25 ℃, 6～24 hours; B. isobutyl chloroformate, N-methylmorpholine, hydrogen sulfide ,-20～-10 ℃, 1～4 hour; C.1,2-ethylene dibromide, sodium hydride ,-20～0 ℃, 1～5 hour; Chloromethyl chlorsulfonic acid ester, 4-n-butyl ammonium hydrogen sulfate, 0～25 ℃, 6～12 hours; D.N, N '-dicyclohexyl-4-morpholinyl-amidine (DCMC), 1,8-diazabicyclo [5,4,0] undecane-7-alkene (DBU) is alkali, 25～90 ℃, 4-48 hour; E. hydrogenchloride saturated/1,4-dioxane or Acetyl Chloride 98Min./methyl alcohol ,-10～25 ℃, 0.5-6 hour.
Method one may further comprise the steps:
(1) preparation of N-tertbutyloxycarbonyl (BOC) L-amino acid ester
N-tertbutyloxycarbonyl L-amino acid and ethylene bromohyrin are in aprotic solvent, at dicyclohexyl carbodiimide (DCC) and N, N dimethylamine yl pyridines (DMAP) exists down, in 0～25 ℃ of reaction 6～24 hours, obtains N-tertbutyloxycarbonyl L-amino acid bromo-ethyl ester; Or
N-tertbutyloxycarbonyl L-amino acid is at isobutyl chloroformate, N-methylmorpholine, hydrogen sulfide reaction 1～4 hour in low temperature-20～-10 ℃, obtain the amino thiocarboxylic acid of N-tertbutyloxycarbonyl L-, and then with 1, the 2-ethylene dibromide is in non-protonic solvent, in the presence of highly basic,, obtain the amino thiocarboxylic acid of N-tertbutyloxycarbonyl L--2-bromo-ethyl ester low temperature-20～0 ℃ reaction 1～5 hour;
Bromochloromethane and chlorsulfonic acid reaction obtain chloromethyl chlorsulfonic acid ester, chloromethyl chlorsulfonic acid ester and N-tertbutyloxycarbonyl L-amino acid are in inert solvent-water two-phase system, at phase-transfer catalyst, 4-n-butyl ammonium hydrogen sulfate exists down, 0～25 ℃ of reaction 6～12 hours, obtain the amino acid whose chloromethyl ester of N-tertbutyloxycarbonyl L-.
(2) amino carbothioic acid ester of various N-tertbutyloxycarbonyl L-amino acid or L-and 9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 is in polar aprotic solvent; with N; N '-dicyclohexyl-4-morpholinyl-amidine (DCMC) or 1; 8-diazabicyclo [5; 4; 0] undecane-7-alkene (DBU) is alkali, 25～90 ℃ of reactions 4-48 hour, obtains product.
(3) product for preparing in the step 2 in the above is in polarity or non-polar solvent, hydrogenchloride saturated 1,4-dioxane or Acetyl Chloride 98Min./methyl alcohol exists down, in-10～25 ℃, reacts and obtains the product compound in 0.5～6 hour.
(4) as required, be prepared into corresponding pharmacy acceptable salt.For example, the purine compound tri hydrochloride is prepared into pharmacy acceptable salt with three times of normal mineral acids such as formation vitriol, phosphoric acid salt, Citrate trianion and maleate etc. such as sulfuric acid, phosphoric acid and organic acid such as citric acid, toxilic acid by method well known in the prior art with free back such as sodium bicarbonate, yellow soda ash.
Method two: undertaken by following flow process II
Flow process II
Figure A20061002440100141
A. oxalyl chloride, N, N '-diethylformamide (DEF), 0～50 ℃, 0.5～4.5 hour; B.N, N-Dimethylamino pyridine (DMAP), dicyclohexylcarbodiimide (DCC), 0～25 ℃, 6～24 hours; C. pyridine, triethylamine, 0～25 ℃, 1～10 hour; D. saturated hydrogenchloride/1,4-dioxane or Acetyl Chloride 98Min./methyl alcohol ,-10～25 ℃, 0.5～6 hour.
Method two may further comprise the steps:
(1) 9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 is in inert solvent, at halogenating agent oxalyl chloride and catalyzer, N; N '-diethylformamide exists down; in 0～50 ℃, reacted, obtain 9-[2-(phosphonium mesitoyl methoxy) ethyl 0.5～4.5 hour] the dichloro-phosphonic acid ester of VITAMIN B4.
(2) N-tertbutyloxycarbonyl L-amino acid and ethylene glycol are in aprotic solvent, and at dicyclohexyl carbodiimide and N, the N dimethylamine yl pyridines exists down, in 0～25 ℃ of reaction 6～24 hours, obtains N-tertbutyloxycarbonyl L-amino acid-hydroxyethyl ester.
(3) 9-[2-(phosphonium mesitoyl methoxy) ethyl] the dichloro-phosphonic acid ester of VITAMIN B4 and N-tertbutyloxycarbonyl L-amino acid-hydroxyethyl ester in inert solvent, in the presence of pyridine and triethylamine,, obtain the product compound 0～25 ℃ of reaction 1～10 hour.
(4) the product compound that obtains in the step (3) above in polarity or non-polar solvent, hydrogenchloride saturated 1, in the presence of 4-dioxane or the Acetyl Chloride 98Min./methyl alcohol,, react and obtained the product compound in 0.5～6 hour in-10～25 ℃.
(5) as required, be prepared into corresponding pharmacy acceptable salt.For example, the purine compound tri hydrochloride is prepared into pharmacy acceptable salt with three times of normal mineral acids such as formation vitriol, phosphoric acid salt, Citrate trianion and maleate etc. such as sulfuric acid, phosphoric acid and organic acid such as citric acid, toxilic acid by method well known in the prior art with free back such as sodium bicarbonate, yellow soda ash.
The present invention further provides Adefovir bisphosphonate compounds shown in the formula (I) and salt thereof in the particularly application on the medicine of the infectious diseases that causes of hepatitis B virus of preparation treatment disease of viral infection.
Acceptable salt can be enumerated and hydrochloric acid particularly on the pharmacology of compound of the present invention, Hydrogen bromide, hydrofluoric acid, sulfuric acid, nitric acid, the salt of mineral acids such as phosphoric acid, with formic acid, acetate, propionic acid, oxalic acid, propanedioic acid, succsinic acid, fumaric acid, toxilic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, picric acid, methylsulfonic acid, organic acid and aspartic acids such as ethyl sulfonic acid, the acid salt of acidic amino acids such as L-glutamic acid, or the salt that forms with alkali, as sodium, potassium, calcium, the salt of mineral alkalis such as aluminium, ammonium salt, methylamine salt, ethylamine salt, ethanolamine salt etc., or and Methionin, arginine, the salt that basic aminoacidss such as ornithine form.
The representative example of compound is as follows shown in the formula of the present invention (I):
(1) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(2) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-acetoxyl group) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(3) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl base sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(4) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-phenyl propionyl sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(5) (2S, 2 ' S)-9-{2-[O, O '-two [(1-tertbutyloxycarbonyl-2-Pyrrolizidine base formyl radical sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(6) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(7) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(8) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl oxygen base) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(9) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(10) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(11) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(12) (2S, 2 ' S)-9-{2-[O, O '-two [(2-glycyl oxygen base) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(13) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylbutyryl base sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(14) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-phenyl propionyl sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(15) (2S, 2 ' S)-9-{2-[O, O '-two [(2-Pyrrolizidine formyl radical sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(16) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-4-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(17) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(18) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylbutyryl oxygen base) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(19) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-4-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(20) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(21) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl oxygen base) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(22) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-phenyl) propionyloxy] ethyl] phosphonium mesitoyl methoxy } ethyl } VITAMIN B4;
(23) (2S, 2 ' S)-9-{2-[O, O '-two [(1-tertbutyloxycarbonyl-2-Pyrrolizidine base methanoyl) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(24) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino propionyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(25) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(26) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylbutyryl oxygen base) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(27) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-phenyl propionyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(28) (2S, 2 ' S)-9-{2-[O, O '-two [(2-Pyrrolizidine base methanoyl) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(29) (2S, 2 ' S)-9-{2-[O, O '-two [(the amino propionyloxy of 2-) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(30) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-4-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride.
The structure of above compound sees Table-1
Figure A20061002440100181
Table-1 compound number and structure
Numbering n X R 1 R 2
1 1 O -NH 2 BOC-L-Ile
2 1 O -NH 2 BOC-Gly
3 1 S -NH 2 BOC-L-Val
4 1 S -NH 2 BOC-L-Phe
5 1 S -NH 2 BOC-L-Pro
6 1 S -NH 2 BOC-L-Leu
7 1 S -NH 2 BOC-L-Ile
8 0 O -NH 2 BOC-L-Val
9 0 O -NH 2 BOC-L-Leu
10 0 O -NH 2 BOC-L-Ile
11 1 O -NH 2 L-Ile
12 1 O -NH 2 Gly
13 1 S -NH 2 L-Val
14 1 S -NH 2 L-Phe
15 1 S -NH 2 L-Pro
16 1 S -NH 2 L-Leu
17 1 S -NH 2 L-Ile
18 0 O -NH 2 L-Val
19 0 O -NH 2 L-Leu
20 0 O -NH 2 L-Ile
21 1 O -NH 2 BOC-L-Val
22 1 O -NH 2 BOC-L-Phe
23 1 O -NH 2 BOC-L-Pro
24 1 O -NH 2 BOC-L-Ala
25 1 O -NH 2 BOC-L-Leu
26 1 O -NH 2 L-Val
27 1 O -NH 2 L-Phe
28 1 O -NH 2 L-Pro
29 1 O -NH 2 L-Ala
30 1 O -NH 2 L-Leu
Acceptable salt has the particularly function of the infectious diseases that causes of HBV and HIV virus of treatment disease of viral infection on the present invention's purine compounds double amino acid ester required for protection and the pharmacology thereof.
Acceptable salt has the activity of the treatment disease of viral infection similar or stronger to the Adefovir of the anti-HBV medicine of ucleosides small molecules in the prior art on the present invention's purine compounds double amino acid ester required for protection and the pharmacology thereof; so these compounds can be used for preparing the medicine of the infectious diseases that novel therapeutic disease of viral infection, particularly HBV and HIV virus causes.
Embodiment
The present invention is further elaborated below in conjunction with embodiment, but these embodiment never are any limitation of the invention.Among all embodiment, fusing point is measured with MEL-TEMP fusing point instrument, and thermometer is not proofreaied and correct; 1H-NMR Varian Mercury 400 nuclear magnetic resonance analyser records, chemical shift is represented with δ (ppm); Separate with the undeclared 200-300 order that is of silica gel.
Compound of the present invention can be by two kinds of method preparations.
Prepare compound of the present invention according to method one:
1, with 1, the 3-dioxolane is a starting raw material, presses currently known methods (Nucleoside ﹠amp; Nucleotide 1996,15, and 1771) through some step reaction synthetic intermediate 9-[2-(phosphonium mesitoyl methoxy) ethyls] purine derivative.
2, N-tertbutyloxycarbonyl (BOC) L-amino acid and ethylene bromohyrin are in non-polar solvents such as toluene, methylene dichloride, chloroform, tetrahydrofuran (THF), at N, the N-Dimethylamino pyridine, dicyclohexylcarbodiimide exists down in 0～25 ℃ of reaction 6～24 hours, top condition for the methylene dichloride be solvent in 10～25 ℃ of reactions 12～24 hours, obtain N-tertbutyloxycarbonyl (BOC) L-amino acid-bromo-ethyl ester.
3, N-tertbutyloxycarbonyl L-amino acid and isobutyl chloroformate (IBCF) and hydrogen sulfide obtain the amino thiocarboxylic acid of N-tertbutyloxycarbonyl L-toluene, methylene dichloride, chloroform, tetrahydrofuran (THF) low temperature-30～10 ℃ reaction 1～10 hour.Top condition was that tetrahydrofuran (THF) is a solvent ,-20～10 reactions 2～4 hours.Products therefrom and glycol dibromide are in non-protonic solvents such as toluene, methylene dichloride, chloroform, tetrahydrofuran (THF), in the presence of sodium hydride, sodium tert-butoxide, trimethyl carbinol lithium, sodium ethylate etc. ,-40～10 ℃ of reactions 0.5～10 hour.Optimum reaction condition reacted 2～5 hours in-20～0 ℃ in the presence of sodium hydride for being solvent with the tetrahydrofuran (THF).Obtain the amino thiocarboxylic acid of N-tertbutyloxycarbonyl L--2-bromo-ethyl ester.
4, the reaction of N-tertbutyloxycarbonyl (BOC) L-amino acid and bromochloromethane and chlorsulfonic acid obtains chloromethyl chlorsulfonic acid ester, in inert solvent-water two-phase system (methylene dichloride-water, chloroform-water, dithiocarbonic anhydride-water, tetracol phenixin-water etc.), at phase-transfer catalyst, 4-n-butyl ammonium hydrogen sulfate, tetra-n-butyl ammonium bromide exist down,-20～25 ℃ of reactions 0.5～24 hour, obtain the amino acid whose chloromethyl ester of N-tertbutyloxycarbonyl L-.Optimum reaction condition was phase-transfer catalyst with the 4-n-butyl ammonium hydrogen sulfate for being reaction system with methylene dichloride-water, in 0～15 ℃ of reaction 6～12 hours.
5, the amino thiocarboxylic acid of N-tertbutyloxycarbonyl L-amino acid-bromo-ethyl ester, N-tertbutyloxycarbonyl L--2-bromo-ethyl ester, the amino acid whose chloromethyl ester of N-tertbutyloxycarbonyl L-and 9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 is at N, in dinethylformamide, methyl-sulphoxide, the pyridine isopolarity aprotic solvent, at N, N '-dicyclohexyl-4-morpholinyl-amidine or 1,8-diazabicyclo [5,4,0] undecane-7-alkene exists down, 25～90 ℃ of reactions 4～48 hours, obtain compound 1～10; Optimum reaction condition is for being solvent with N, dinethylformamide, and at N, N '-dicyclohexyl-4-morpholinyl-amidine and 1,8-diazabicyclo [5,4,0] undecane-7-alkene (DBU) exist down, 40～80 ℃ of reactions, and 6～24 hours.
6, compound 1～10 is at ethyl acetate, isopropyl acetate, 1, in the Semi-polarity solvents such as 4-dioxane, hydrogenchloride saturated 1,4-dioxane or Acetyl Chloride 98Min./methyl alcohol exists down, obtains compound 11～20 in 0.5-12 hour in-20～25 ℃ of reactions.Optimum reaction condition is with 1, and the 4-dioxane is a reaction solvent, hydrogenchloride saturated 1, the 4-dioxane exists down in 0～10 ℃ of reaction 3～6 hours.
7, as required, be prepared into corresponding salt.The purine compound tri hydrochloride is prepared into pharmacy acceptable salt with free back such as sodium bicarbonate, yellow soda ash according to a conventional method with three times of normal mineral acids such as formation vitriol, phosphoric acid salt, Citrate trianion and maleate etc. such as sulfuric acid, phosphoric acid and organic acid such as citric acid, toxilic acid.
Prepare compound of the present invention according to method two:
1,9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 is raw material, by with perception method (MarkD.Erion.et al J.Am.Chem.Soc.2004,126,5154-5163) preparation dichloro-9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4.
2, N-tertbutyloxycarbonyl L-amino acid and ethylene glycol are in non-polar solvents such as toluene, methylene dichloride, chloroform, tetrahydrofuran (THF), at N, the N-Dimethylamino pyridine, dicyclohexylcarbodiimide exists down in 0～25 ℃ of reaction 6～24 hours, top condition for the methylene dichloride be solvent in 10～25 ℃ of reactions 12～24 hours, obtain N-tertbutyloxycarbonyl L-amino acid-hydroxyethyl ester.
3,9-[2-(phosphonium mesitoyl methoxy) ethyl] dichloro-of VITAMIN B4 sees acid esters and N-tertbutyloxycarbonyl L-amino acid-hydroxyethyl ester in non-polar solvents such as toluene, methylene dichloride, chloroform, tetrahydrofuran (THF); in the presence of pyridine and triethylamine; 0～25 ℃ of reaction 1～10 hour; optimum reaction condition is for being solvent with the methylene dichloride; at 0～10 ℃, react and obtained compound 21～25 in 2～6 hours.
4, compound 21～25 is 1, in the non-polar solvent such as 4-dioxane, ethyl acetate, isopropyl acetate, methylene dichloride, in the presence of saturated 1 at hydrogenchloride, the 4-dioxane or Acetyl Chloride 98Min./methyl alcohol, in-10～100 ℃, reacted 0.5～6 hour, optimum reaction condition for 1, that the 4-dioxane is a solvent is saturated 1 at hydrogenchloride, under the 4-dioxane condition, obtained compound 26～30 in 2～4 hours 0～10 ℃ of reaction.
5,, be prepared into corresponding salt according to needs.The purine compound tri hydrochloride is prepared into pharmacy acceptable salt with free back such as sodium bicarbonate, yellow soda ash according to a conventional method with three times of normal mineral acids such as formation vitriol, phosphoric acid salt, Citrate trianion and maleate etc. such as sulfuric acid, phosphoric acid and organic acid such as citric acid, toxilic acid.
Embodiment 1:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 1)
1.1 (1S)-and the tertiary butyl-1-[(2-bromine oxethyl) carbonyl]-preparation of 2-methyl butyl carbamate (a)
Figure A20061002440100231
With the N-tert-butoxy-oxo-L-isoleucine (5.00g, 0.02mol), ethylene bromohyrin (2.98g 0.024mol) is dissolved in the 200ml dry methylene chloride, is cooled to 10 ℃ with ice-water-bath, and gradation adds N, the N dimethylamine yl pyridines (2.92g, 0.023mol).Finish, architecture heat preservation stirred 15 minutes.Then slowly drip dicyclohexylcarbodiimide (4.53g, 30ml dichloromethane solution 0.022mol).Finish, temperature of reaction was risen to room temperature reaction 12 hours naturally.Insolubles in the system is filtered, and steaming desolventizes.In the gained resistates, add the 100ml ethyl acetate, made the abundant sedimentation of solid in the system in static 6 hours at low temperature (10 ℃).Filter, filtrate is with 100 * 2ml washing, and the 200ml saturated common salt is washed, anhydrous sodium sulfate drying.Remove solvent under reduced pressure, get crude product 8.65g.With sherwood oil: ethyl acetate=10: 1 is an eluent, and silica gel column chromatography is got colorless oil tertiary butyl 1-[(2-bromine oxethyl) carbonyl]-2-methyl butyl carbamate (a) 4.86g, yield 68.39%.
1HNMR(CDCl 3)：4.99(d，J＝8.15Hz，1H)，4.48-4.37(m，2H)，4.28(m，1H)，3.52-3.47(m，2H)，1.87(m，1H)，1.42(s，9H)，1.22-1.17(m，2H)，0.94-0.87(m，6H)；EI-MS(m/e)：323，M+
1.2:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] and ethyl } preparation of VITAMIN B4 (compound 1)
Figure A20061002440100241
Under the room temperature condition; with tertiary butyl 1-[(2-bromine oxethyl) carbonyl]-2-methyl butyl carbamate (a) (0.63g; 1.8mmol); 9-[2-(phosphonium mesitoyl methoxy) ethyl] and VITAMIN B4 (0.10g, 0.36mnol) and N, N '-dicyclohexyl-4-morpholinyl-amidine (0.21g; 0.72mmol) be suspended in the dry N of 10ml; in the dinethylformamide, room temperature reaction 24 hours is then 80 ℃ of reactions 4 hours.With the reaction solvent evaporated under reduced pressure, add the 20ml ethyl acetate in the resistates,-5～0 ℃ of sedimentation 6 hours, filtering insolubles, filtrate are respectively with 1% citric acid, and water and saturated common salt are washed, anhydrous sodium sulfate drying, filter, filtrate concentrates, residue column chromatography for separation (eluent: methylene dichloride: methyl alcohol=25: 1) get white vacuolar membrane shape solid chemical compound 43mg.Productive rate: 15.17%.
1HNMR(CDCl 3)：δ＝8.32(s，1H)，7.96(s，1H)，5.87(brs，2H)，5.21-5.40(m，2H)，4.39(t，J＝5.32Hz，2H)，4.10-4.37(m，10H)，3.93(m，2H)，3.81(d，J＝8.41Hz，2H)，1.84(m，2H)，1.42(s，18H)，1.05-1.26(m，4H)，0.85-0.91(m，12H).EI-MS(m/e)：787，M+
Embodiment 2:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-acetoxyl group) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 2)
2.1: the carbonyl tertiary butyl-1-[(2-bromine oxethyl)] preparation of methyl carbamate (a)
Figure A20061002440100251
With N-tertbutyloxycarbonyl-glycine (3.70g, 0.021mol), ethylene bromohyrin (3.47g 0.028mol) is dissolved in the 200ml dry methylene chloride, is cooled to 10 ℃ with ice-water-bath, and gradation adds N, the N dimethylamine yl pyridines (3.10g, 0.025mol).Finish, architecture heat preservation stirred 15 minutes.Then slowly drip dicyclohexylcarbodiimide (4.53g, 30ml dichloromethane solution 0.022mol).Subsequent process is similar to the synthetic of compound 1.1. get colorless oil (a) 3.21g, yield 54.39%.
2.2:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-acetoxyl group) ethyl] phosphonium mesitoyl methoxy] and ethyl } preparation of VITAMIN B4 (compound 2)
Under the room temperature condition; with (a) (0.99g; 3.55mmol); 9-[2-(phosphonium mesitoyl methoxy) ethyl] and VITAMIN B4 (0.16g, 0.59mnol) and N, N '-dicyclohexyl-4-morpholinyl-amidine (0.33g; 1.13mmol) be suspended in the dry N of 10ml; in the dinethylformamide, room temperature reaction 24 hours, the back was 80 ℃ of reactions 4 hours.With the reaction solvent evaporated under reduced pressure, add the 20ml ethyl acetate in the resistates,-5～0 ℃ of sedimentation 6 hours, filtering insolubles, filtrate are respectively with 1% citric acid, and water and saturated common salt are washed, anhydrous sodium sulfate drying, filter, filtrate concentrates, residue column chromatography for separation (eluent: methylene dichloride: methyl alcohol=25: 1) get bacalite shape compound 52.4mg.Productive rate: 13.15%.
1HNMR(CDCl 3)：δ＝8.26(s，1H)，8.11(s，1H)，5.44(brs，2H)，4.38-4.45(m，2H)，4.23-4.31(m，8H)，3.93-4.01(m，6H)，3.84(d，J＝8.52Hz，2H)，1.43(s，18H).EI-MS(m/e)：675，M+.
Embodiment 3:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl base sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 3)
3.1:(S the preparation of)-2-t-butoxycarbonyl amino-3-methyl list Thiobutyric acid (a)
Figure A20061002440100262
(2.50g 0.0115mol) is dissolved in the 25ml dry tetrahydrofuran, is cooled to-15 ℃ with ice-salt bath with N-tertbutyloxycarbonyl-L-Xie Ansuan, add N-methylmorpholine (11.54g, 0.0575mol) and isobutyl chloroformate (1.74g, 0.0126mol), architecture heat preservation stirring 30 minutes.Temperature remains on below-15 ℃ in reaction system, feeds the self-control hydrogen sulfide, and aeration time continues 1.5-2 hour, and the stink damp bulk absorption is after saturated to the system, and insulation reaction made reaction carry out fully in 2 hours again.Add the 40ml anhydrous diethyl ether, with 0.1M hydrochloric acid regulation system pH to 3, divide and get organic layer, with 2 * 20ml water, the water washing of 2 * 20ml saturated common salt, anhydrous sodium sulfate drying, filter, solvent evaporated gets faint yellow oily thing (S)-2-t-butoxycarbonyl amino-3-methyl list Thiobutyric acid (a) 2.22g, yield 82.85%.
1HNMR(CDCl 3)：5.06(m，1H)，4.24(m，1H)，2.27(m，1H)，1.43(s，9H)，0.93-0.89(m，6H).
3.2:(1S the carbonyl)-tertiary butyl-1-[(2-bromotrifluoromethane sulphur)]-preparation of 2-methyl-propyl carbamate (b)
Figure A20061002440100271
(0.17g 4.29mmol) adds in the 10ml tetrahydrofuran (THF), stirs and is cooled to-15 ℃ with ice-salt bath with sodium hydride, slowly be added dropwise to (S)-2-t-butoxycarbonyl amino-3-methyl list Thiobutyric acid (a) (1.00g, 4.29mmol) the 2ml tetrahydrofuran solution, drip to finish insulation reaction 1.5 hours, room temperature reaction 2 hours, slowly be added dropwise to glycol dibromide (1.60g, 5ml tetrahydrofuran solution 8.58mol), drip and finish system room temperature reaction 12 hours.System is filtered, filtrate is with 2 * 20ml water, the water washing of 2 * 20ml saturated common salt, anhydrous sodium sulfate drying, filter, filtrate concentrates, residue column chromatography for separation (eluent: sherwood oil: ethyl acetate=20: 1), get faint yellow oily thing (S)-2-t-butoxycarbonyl amino-3-methyl list Thiobutyric acid-2-bromo-ethyl ester (b) 0.59g, yield 40.52%.
1HNMR(CDCl 3)：4.95(d，J＝8.95Hz，1H)，4.26(m，1H)，3.42(t，J＝7.34Hz，2H)，3.29(t J＝6.6Hz，2H)2.31-2.25(m，1H)，1.46(s，18H)，0.99(d，J＝6.78Hz，3H)，0.86(d，J＝6.96Hz，3H).
3.3:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl base sulphur) ethyl] phosphonium mesitoyl methoxy] and ethyl } preparation of VITAMIN B4 (compound 3)
Figure A20061002440100281
With (S)-2-t-butoxycarbonyl amino-3-methyl list Thiobutyric acid-2-bromo-ethyl ester (b) (0.44g; 1.3mmol) and 9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 (0.08g; 0.26mmol) and N; N '-dicyclohexyl-4-morpholinyl-amidine (0.15g; 0.26mmol) be suspended in the dry N of 10ml; in the dinethylformamide, room temperature reaction 24 hours, the back was 80 ℃ of reactions 5 hours.With the reaction solvent evaporated under reduced pressure, add the 20ml ethyl acetate in the resistates,-5～0 ℃ of sedimentation, filtering insolubles, filtrate are respectively with 10 * 2ml, 1% citric acid, and water and saturated common salt are washed, anhydrous sodium sulfate drying, filter, filtrate concentrates, residue column chromatography for separation (eluent: methylene dichloride: methyl alcohol=25: 1) get colourless amber shape compound 58mg.Productive rate: 13.09%.
1HNMR(CDCl 3)：δ＝8.27(s，1H)，8.05(s，1H)，6.18(brs，2H)，5.21-5.39(m，2H)，3.97-4.41(m，8H)，3.91(t，J＝4.89Hz，2H)，3.79(d，J＝8.41Hz，2H)，3.04-3.21(m，4H)，2.24(m，2H)，1.42(S，18H)，0.96(d，J＝6.84Hz，6H)，0.83(d，J＝6.65Hz，6H).ESI-MS(m/e)：792.2，(M+H)+.
Embodiment 4:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-phenyl propionyl sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 4)
N-tertbutyloxycarbonyl-L-phenylalanine is a starting raw material; to be similar to the method preparation of embodiment 3; off-white color amber shape compound (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-phenyl propionyl sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl VITAMIN B4 61mg.Productive rate: 12.67%.
1HNMR(CDCl 3)：δ＝8.31(s，1H)，8.01(s，1H)，7.11-7.26(m，10H)，6.12(brs，2H)，5.44-5.37(m，2H)，4.09-4.45(m，8H)，3.81-3.90(m，4H)，3.14-3.31(m，4H)，2.88-3.13(m，4H)，1.42(S，18H)。
Embodiment 5:(2S, 2 ' S)-9-{2-[O, O '-two [(1-tertbutyloxycarbonyl-2-Pyrrolizidine base formyl radical sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 5)
N-tertbutyloxycarbonyl-L-proline(Pro) is a starting raw material; to be similar to the method preparation of embodiment 3; get sundown oily compound (2S; 2 ' S)-9-{2-[O; O '-two [(1-tertbutyloxycarbonyl-2-Pyrrolizidine base formyl radical sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4 55mg, productive rate: 18.61%.
1HNMR(CDCl 3)：δ＝8.35(s，1H)，8.07(s，1H)，5.84(brs，2H)，4.47(m，2H)，4.36-4.24(m，6H)，3.83-3.96(m，4H)，3.38-3.57(m，4H)，3.14-3.26(m，4H)，1.42(S，18H)，2.13-2.24(m，4H)，1.91-2.02(m，4H)，1.43(d，J＝16.67Hz，18H)。
Embodiment 6:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 6)
N-tertbutyloxycarbonyl-L-proline(Pro) is a starting raw material; to be similar to the method preparation of embodiment 3; get white foam shape compound (2S; 2 ' S)-9-{2-[O; O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4 67mg, productive rate: 31.38%.
1HNMR(CDCl 3)：δ＝8.31(s，1H)，7.97(s，1H)，5.97(brs，2H)，5.24-5.41(m，2H)，4.51-4.28(m，8H)，3.91-4.02(m，4H)，3.23-3.41(m，4H)，1.72-1.80(m，4H)，1.07(brs，12H)。
Embodiment 7:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 7)
The N-tert-butoxy-oxo-L-isoleucine is a starting raw material; to be similar to the method preparation of embodiment 3; get off-white color foam-like compound (2S; 2 ' S)-9-{2-[O; O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4; 81mg, productive rate: 35.23%.
1HNMR(CDCl 3)：δ＝8.36(s，1H)，8.04(s，1H)，5.87(brs，2H)，5.18-5.34(m，2H)，4.32-4.42(m，4H)，4.08-4.11(m，4H)，3.92(m，2H)，3.81(d，J＝8.66Hz 2H)，3.02-3.18(m，4H)，1.92-1.99(m，2H)，1.42(s，18H)，1.11-1.17(m，4H)，0.79-0.98(m，12H)。
Embodiment 8:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl oxygen base) methylene radical] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 8)
8.1: chloromethyl chlorsulfonic acid ester (a)
Figure A20061002440100301
(100ml, 1.5mol), (50ml 0.75mol) places in the reaction flask bromochloromethane altogether, slowly reaction system is heated to backflow, reacts 3 hours with chlorsulfonic acid.Reaction is finished, and system is cooled to room temperature, after with in its slow impouring 500g trash ice, treat behind the ice-out with methylene dichloride (400 * 2ml,) extraction, organic layer is told, and anhydrous sodium sulfate drying filters, filtrate is steamed under normal pressure and is desolventized, and 45-50 ℃/9-10mmHg cut is collected in the resistates underpressure distillation.Get product 30.25g, productive rate: 24.4%.
1HNMR(CDCl 3)：5.96(s，2H)
8.2:(1S the carbonyl)-tertiary butyl-1-[(chlorine methoxyl group)]-preparation of 2-methyl-propyl carbamate (b)
With N-tertbutyloxycarbonyl-L-Xie Ansuan (3.00g, 0.0138mol), sodium bicarbonate (4.59g, 0.0546mol) and four-normal-butyl monoammonium sulfate (0.47g, 0.00138mol) in the system as for 50ml water and 50ml methylene dichloride, cryosel is bathed and is cooled to 0 ℃, slowly be added dropwise to chloromethyl chlorsulfonic acid ester (a) (2.78g under the vigorous stirring, 0.0167mol) the 14ml dichloromethane solution, drip to finish, system rises to room temperature naturally and reacted 12 hours.Organic layer is told, and with the washing of 2 * 10ml saturated common salt, anhydrous sodium sulfate drying filters, and filtrate concentrates, residue column chromatography for separation (eluent: sherwood oil: ethyl acetate=15: 1) get colorless oil 2.96g.Productive rate: 80.77%.
1HNMR(CDCl 3)：5.83(d，J＝5.86Hz，1H)，5.58(d，J＝5.37Hz，1H)，4.97(dJ＝8.3Hz，1H)，4.23-4.21(m，1H)，2.15(m，1H)，1.41(s，9H)，0.96(d，J＝6.83Hz，3H)，0.89(d，J＝6.84Hz，3H)。
8.3:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl oxygen base) methylene radical] phosphonium mesitoyl methoxy] and ethyl } preparation of VITAMIN B4 (compound 8)
With 9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 (0.10g; 0.36mmol); tertiary butyl 1-[(chlorine methoxyl group) carbonyl]-2-methyl-propyl carbamate (b) (0.48g; 1.8mmol); N, (0.21g 0.72mmol) is suspended in the 10ml exsiccant N-N-methyl-2-2-pyrrolidone N-N '-dicyclohexyl-4-morpholinyl-amidine; behind the room temperature reaction 24 hours, 80 ℃ were reacted 5 hours.In system impouring 5ml1% citric acid and 5ml ethyl acetate, extraction, tell organic phase, water is continuous with 2 * 5ml ethyl acetate extraction, and ester is also laminated, continuous with 2 * 5ml, 1% citric acid, water and saturated common salt water washing, anhydrous sodium sulfate drying filters, filtrate concentrates, residue column chromatography for separation (eluent: methylene dichloride: methyl alcohol=25: 1) get colourless amber shape thing 55mg.Productive rate: 20.8%.
1HNMR(CDCl 3)：δ＝8.28(s，1H)，7.97(s，1H)，6.18(brs，2H)，5.66-5.72(m，4H)，5.21-5.36(m，2H)，4.41(m，2H)，4.27-4.31(m，2H)，3.91(m，2H)，3.85(d，J＝5.09Hz，2H)，2.13-2.18(m，2H)，1.42(s，18H)，0.96(d，J＝6.32Hz，6H)，0.84(d，J＝6.78Hz，6H)。
Embodiment 9:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 9)
N-tertbutyloxycarbonyl-L-leucine is a starting raw material; to be similar to the method preparation of embodiment 8; get off-white color foam-like compound (2S; 2 ' S)-9-{2-[O; O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4 26mg, productive rate: 9.56%.
1HNMR(CDCl 3)：δ＝8.34(s，1H)，8.06(s，1H)，6.21(brs，2H)，5.76-5.91(m，4H)，5.18-5.29(m，2H)，4.52-4.36(m，4H)，3.94(m，2H)，3.91(d，J＝5.97Hz，2H)，1.71-1.93(m，6H)，1.42(s，18H)，0.91(t，J＝5.5Hz，12H)。
Embodiment 10:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 10)
The N-tert-butoxy-oxo-L-isoleucine is a starting raw material; to be similar to the method preparation of embodiment 8; get white foam shape compound (2S; 2 ' S)-9-{2-[O; O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4 85mg, productive rate: 15.56%.
1HNMR(CDCl 3)：δ＝8.28(s，1H)，7.96(s，1H)，6.09(brs，2H)，5.59-5.72(m，4H)，5.08-5.21(m，2H)，4.38(m，2H)，4.23-4.31(m，2H)，3.89(m，2H)，3.82(d，J＝7.33Hz，2H)，1.84(m，2H)，1.41(s，18H)，1.07-1.16(m，4H)，0.81-0.95(m，12H)。
Embodiment 11:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 11)
Figure A20061002440100331
Compound (0.21g with preparation among the embodiment 1; 0.267mmol) be dissolved in 1ml drying 1; in the 4-dioxane; with the cryosel bath system is cooled to 0 ℃ under the nitrogen protection; stir slowly drip down 15% hydrogenchloride 1,4-dioxane solution (1ml; 4mmol), insulation reaction 1 hour, back room temperature reaction transformed abundant in 3 hours to product.Left standstill 30 minutes, and supernatant liquor in the system was inhaled abandoned, solids is abandoned the solvent suction with the washing of 5 * 2ml ethyl acetate, sedimentation 5 times, gets white foam shape solid 0.176g, productive rate: 95.24% after the resistates thorough drying.
1HNMR(CD 3OD)：δ＝8.43(s，1H)，8.39(s，1H)，4.55(t，J＝5.08Hz，2H)，4.38-4.53(m，4H)4.21-4.36(m，4H)，4.06-4.11(m，2H)，3.98-4.02(m，4H)，1.98-2.01(m，2H)，1.34-1.57(m，4H)，0.96-1.03(m，12H).ESI-MS：588.2(M+H)+。
Embodiment 12:(2S, 2 ' S)-9-{2-[O, O '-two [(2-glycyl oxygen base) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 12)
Compound with preparation among the embodiment 2 is a raw material, to be similar to the method preparation of embodiment 11, gets white powder thing 22mg, productive rate: 87.76%.
1HNMR(CD 3OD)：δ＝8.54(brs，4H)，8.49(s，1H)，8.42(s，1H)，4.44(m，2H)，4.31(m，4H)4.16(m，4H)，3.97(d，J＝7.82Hz，2H)，3.92(m，2H)，3.85(s，4H).EI-MS(m/e)：475M+。
Embodiment 13:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylbutyryl base sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 13)
Figure A20061002440100341
With Acetyl Chloride 98Min. (0.077ml, 1.06mmol), methyl alcohol (0.058ml, 1.36mmol) place the dry ethyl acetate of 1.6ml, system is bathed with cryosel and is cooled to-5～0 ℃, reacts 30 minutes, slowly is added dropwise to the compound (30mg of preparation among the embodiment 3,0.0379mmol) the dry ethyl acetate solution of 1.6ml, drip and finish, insulation reaction 2 hours, the back rises to room temperature reaction naturally and transformed abundant to product in 6 hours.Left standstill 30 minutes, and supernatant liquor in the system was inhaled abandoned, solids is abandoned the solvent suction with the washing of 5 * 2ml ethyl acetate, sedimentation 5 times, gets white semi-solid 13mg, productive rate: 58.03% after the resistates thorough drying.
1HNMR(CD 3OD)：δ＝8.43(s，1H)，8.39(s，1H)，，4.58(t，J＝4.69Hz，2H)，4.14-4.22(m，6H)，3.98-4.02(m，4H)，3.31(t，J＝6.26Hz，4H)，2.26-2.41(m，2H)，1.12(d，J＝6.85，6H)，1.04(d，J＝7.04Hz，6H).ESI-MS：592.1(M+H)+。
Embodiment 14:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-phenyl propionyl sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 14)
Compound with preparation among the embodiment 4 is a raw material, to be similar to the method preparation of embodiment 13, gets white powder thing 14mg, productive rate: 84.46%.
1HNMR(CD 3OD)：δ＝8.38(s，1H)，8.35(s，1H)，7.28-7.38(m，10H)，4.56(m，4H)，4.05-4.18(m，4H)，3.96(t，J＝4.59Hz)，3.91(d，J＝8.25Hz，2H)，3.24(m，4H)，3.13-3.19(m，4H)。
Embodiment 15:(2S, 2 ' S)-9-{2-[O, O '-two [(2-Pyrrolizidine formyl radical sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 15)
Compound with preparation among the embodiment 5 is a raw material, to be similar to the method preparation of embodiment 13, gets off-white color spumescence solid 10mg, productive rate: 41.55%.
1HNMR(CD 3OD)：δ＝8.44(s，1H)，8.40(s，1H)，4.68(m，2H)，4.56(m，2H)，4.17-4.19(m，4H)，3.97-4.01(m，4H)，3.39(t，J＝6.96Hz，4H)，3.33(m，4H)，2.50-2.56(m，2H)，2.07-2.16(m，6H).ESI-MS：588.1(M+H)+。
Embodiment 16:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-4-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 16)
Compound with preparation among the embodiment 6 is a raw material, to be similar to the method preparation of embodiment 13, gets colourless gelatinous semi-solid 7mg, productive rate: 46.34%.
1HNMR(CD 3OD)：δ＝8.42(s，1H)，8.37(s，1H)，4.55(m，2H)，4.18-4.26(m，6H)，4.01(m，4H)，3.31(m，4H)，1.72-1.81(m，4H)，1.02(m，12H).ESI-MS：620.2(M+H)+。
Embodiment 17:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 17)
Compound with preparation among the embodiment 7 is a raw material, to be similar to the method preparation of embodiment 13, gets white fluffy solid 14mg, productive rate: 50.26%.
1HNMR(CD 3OD)：δ＝8.41(s，1H)，8.36(s，1H)，4.56(t，J＝5.12Hz，2H)，4.18-4.25(m，6H)，3.91-4.01(m，4H)，3.24(m，4H)，2.07(m，2H)，1.24-1.61(m，4H)，0.97-1.08(m，12H)ESI-MS：620.2(M+H)+。
Embodiment 18:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylbutyryl oxygen base) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 18)
Compound with preparation among the embodiment 8 is a raw material, to be similar to the method preparation of embodiment 11, gets white foam shape solid 18mg, productive rate, 57.45%.
1HNMR(CD 3OD)：δ＝8.42(s，1H)，8.37(s，1H)，5.65-5.91(m，4H)，4.57(t，J＝4.89Hz，2H)，4.11(m，2H)，4.08(d，J＝8.22Hz，2H)，4.02(t，J＝4.89Hz，2H)，2.14-2.21(m，2H)，1.02-1.16(m，12H).ESI-MS：532.1(M+H)+。
Embodiment 19:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-4-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 19)
Compound with preparation among the embodiment 9 is a raw material, to be similar to the method preparation of embodiment 11, gets white foam shape solid 14mg, productive rate: 90.74%.
1HNMR(CD 3OD)：δ＝8.42(s，1H)，8.38(s，1H)，5.85-5.73(m，4H)，4.56(t，J＝5.13Hz，2H)，4.18(m，2H)，4.09(d，J＝8.43Hz，2H)，4.02(t，J＝4.76Hz，2H)，1.71-1.86(m，6H)，1.01(m，12H)。
Embodiment 20:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 20)
Compound with preparation among the embodiment 10 is a raw material, to be similar to the method preparation of embodiment 11, gets off-white color spumescence solid 15mg, productive rate: 78.26%.
1HNMR(CD 3OD)：δ＝8.41(s，1H)，8.36(s，1H)，5.73-5.89(m，4H)，4.56(t，J＝5.13Hz，2H)，4.18(m，2H)，4.08(d，J＝8.07Hz，2H)，4.01(t，J＝5.14Hz，2H)，+12H)。
Embodiment 21:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl oxygen base) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 21)
21.1:(1S the carbonyl)-tertiary butyl-1-[(2-hydroxy ethoxy)]-preparation of 2-methyl-propyl carbamate (a)
Figure A20061002440100371
With N-tertbutyloxycarbonyl L-Xie Ansuan (0.22g, 1mmol), ethylene glycol (0.06g, 1mmol), place the 15ml dry methylene chloride, be cooled to 10 ℃ with ice-water-bath, gradation adds N, the N dimethylamine yl pyridines (0.122g, 1mmol), architecture heat preservation stirred 15 minutes, slowly be added dropwise to dicyclohexylcarbodiimide (0.22g, 1.1mmol) the 10ml dichloromethane solution, finish, temperature of reaction was risen to room temperature reaction 24 hours naturally.Insolubles in the system is filtered, steaming desolventizes, and resistates is dissolved in the 10ml ethyl acetate, and low temperature (10 ℃) leaves standstill, filter, with 2 * 5ml 1M sal enixum, water and the washing of 5% sodium carbonate solution, anhydrous magnesium sulfate drying filters filtrate respectively, solvent removes under reduced pressure, resistates is with sherwood oil: ethyl acetate=4: 1 is an eluent, and silica gel column chromatography is got colorless oil tertiary butyl 1-[(2-hydroxy ethoxy) carbonyl]-2-methyl-propyl carbamate (a) 0.167g, yield: 76.95%.
1HNMR(CDCl 3)：5.15(d，J＝8.43Hz，1H)，4.20-4.27(m，3H)，3.77(t，J＝4.59Hz，2H)，3.24(brs，1H)1.81(m，1H)，1.41(s，9H)，1.21-1.13(m，2H)，0.85-0.91(m，6H)。
21.2: dichloro-9-[2-(phosphonium mesitoyl methoxy) ethyl] preparation of VITAMIN B4 (b)
With oxalyl chloride (0.54ml; 6.28mmol) slowly be added dropwise to contain 9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 (0.50g; 1.8mmol); N; the N-diethylformamide (0.20ml, in 14ml methylene dichloride 1.9mmol), system refluxed 3 hours; extract reaction solution and use the methyl alcohol cancellation, detect two chloro things to TLC and transform fully.System is chilled to room temperature, and concentrating under reduced pressure is with yellow foam dichloro-9-[2-(phosphonium mesitoyl methoxy) ethyl of remnants] VITAMIN B4 (b) 0.37g, thick yield 66.95%.Gains are dissolved in the 2ml dry methylene chloride, and cryosel is bathed and is cooled to 0 ℃, slowly adds the 0.3ml pyridine, low temperature (preserving standby below 0 ℃).
21.3:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl oxygen base) ethyl] phosphonium mesitoyl methoxy] and ethyl } preparation of VITAMIN B4 (compound 21)
With tertiary butyl 1-[(2-hydroxy ethoxy) carbonyl]-2-methyl-propyl carbamate (a) (0.94g; 3.6mmol); triethylamine (1.50ml; 10.78mmol) be dissolved in the 6ml dry methylene chloride; system is bathed with cryosel and is cooled to 0 ℃, slowly is added dropwise to dichloro-9-[2-(phosphonium mesitoyl methoxy) ethyl] dichloromethane solution of VITAMIN B4 (b), drip and finish; insulation reaction 1 hour, after slowly rose to room temperature reaction 4 hours.Solution is used 3 * 1ml water, 2 * 1ml1% citric acid and saturated common salt water washing respectively, and anhydrous magnesium sulfate drying filters, filtrate concentrates, resistates is with methylene dichloride: methyl alcohol=15: 1 is eluent, and silica gel column chromatography is got faint yellow semi-solid 0.22g, yield: 15.53%.
1HNMR(CDCl 3)：8.36(s，1H)，8.09(s，1H)，6.21(brs，2H)，5.25-5.41(m，2H)，4.45(t，J＝6.25Hz，2H)，4.11-4.36(m，10H)，3.97(t，J＝5.51Hz，2H)，3.82(d，J＝9.35Hz，2H)，2.02-2.18(m，2H)，1.42(s，18H)，0.96(d，J＝6.41Hz，6H)，0.82(d，J＝6.59Hz，6H).EI-MS(m/e)：759，M+。
Embodiment 22:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-phenyl) propionyloxy] ethyl] phosphonium mesitoyl methoxy } ethyl } preparation of VITAMIN B4 (compound 22)
N-tertbutyloxycarbonyl L-phenylalanine is a starting raw material, to be similar to the method preparation of embodiment 21, gets light yellow spumescence solid 43mg, yield: 13.97%.
1HNMR(CDCl 3)：8.31(s，1H)，8.01(s，1H)，7.14-7.26(m，10H)，6.01(brs，2H)，5.37-5.44(m，2H)，4.75(m，2H)，4.21-4.27(m，10H)，3.91(m，2H)，3.81(d，J＝8.80Hz，2H)，2.98-3.13(m，4H)，1.38(s，18H).ESI-MS：856.2(M+H)+。
Embodiment 23:(2S, 2 ' S)-9-{2-[O, O '-two [(1-tertbutyloxycarbonyl-2-Pyrrolizidine base methanoyl) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 23)
N-tertbutyloxycarbonyl L-proline(Pro) is a starting raw material, to be similar to the method preparation of embodiment 21, gets light yellow amber shape thing 31mg, yield: 11.41%.
1HNMR(CDCl 3)：8.34(s，1H)，7.96(s，1H)，5.76-5.84(m，2H)，4.41(t，J＝4.76Hz，2H)，4.21-4.33(m，10H)，3.95(m，2H)，3.79-3.86(m，2H)，3.35-3.53(m，4H)，2.14-2.24(m，4H)，1.91-2.02(m，4H)，1.43(d，J＝17.38Hz，18H).EI-MS(m/e)：755，M+。
Embodiment 24:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino propionyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 24)
N-tertbutyloxycarbonyl L-L-Ala is a starting raw material, to be similar to the method preparation of embodiment 21, gets the semi-solid 44mg of off-white color, yield: 22.4%.
1HNMR(CDCl 3)：8.33(s，1H)，7.98(s，1H)，5.92(brs，2H)，5.40-5.48(m，2H)，4.23-4.42(m，12H)，3.83-3.93(m，4H)，1.37-1.43(m，24H)。
Embodiment 25:(2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } preparation of VITAMIN B4 (compound 25)
N-tertbutyloxycarbonyl L-leucine is a starting raw material, to be similar to the method preparation of embodiment 21, gets light yellow spumescence solid 24mg, yield: 8.47%.
1HNMR(CDCl 3)：8.36(s，1H)，8.04(s，1H)，6.24(brs，2H)，5.25-5.40(m，2H)，4.24-4.42(m，12H)，3.94(m，2H)，3.85(d，J＝8.47Hz，2H)，1.44-1.59(m，6H)，1.42(s，18H)，0.91(t，J＝6.59Hz，12H).EI-MS(m/e)：787，M+。
Embodiment 26:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylbutyryl oxygen base) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 26)
Compound with preparation among the embodiment 21 is a raw material, to be similar to the method preparation of embodiment 11, gets the semi-solid 13mg of off-white color, yield: 61.36%.
1HNMR(CD 3OD)：8.41(s，1H)，8.36(s，1H)，4.54(t，J＝4.76Hz，2H)，4.40-4.47(m，4H)，4.31(m，4H)，3.98-4.02(m，6H)，2.30-2.33(m，2H)，1.04-1.16(m，12H).EI-MS(m/e)：559，M+。
Embodiment 27:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-phenyl propionyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 27)
Compound with preparation among the embodiment 22 is a raw material, to be similar to the method preparation of embodiment 11, gets the semi-solid 0.106g of off-white color, yield: 91.51%.
1HNMR(CD 3OD)：δ＝8.37(s，1H)，8.35(s，1H)，7.25-7.36(m，10H)，4.51(t，J＝5.09Hz，2H)，4.27-4.31(m，6H)，4.22-4.26(m，4H)，3.98-4.01(m，4H)，3.16-3.28(m，4H)，ESI-MS：656.3(M+H)+
Embodiment 28:(2S, 2 ' S)-9-{2-[O, O '-two [(2-Pyrrolizidine base methanoyl) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 28)
Compound with preparation among the embodiment 23 is a raw material, to be similar to the method preparation of embodiment 11, gets the semi-solid 0.106g of off-white color, yield: 91.51%.
1HNMR(CD 3OD)：δ＝8.43(s，1H)，8.39(s，1H)，4.49-4.55(m，4H)，4.52(m，2H)，4.11(m，4H)，3.99-4.01(m，4H)，3.37-3.43(m，4H)，2.41-2.45(m，2H)，2.06-2.19(m，6H)。ESI-MS：556.2(M+H)+。
Embodiment 29:(2S, 2 ' S)-9-{2-[O, O '-two [(the amino propionyloxy of 2-) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 29)
Compound with preparation among the embodiment 24 is a raw material, to be similar to the method preparation of embodiment 11, gets the semi-solid 26mg of off-white color, yield: 77.65%.
1HNMR(CD 3OD)：δ＝8.42(s，1H)，8.38(s，1H)，4.55(t，J＝5.08Hz，2H)，4.43(t，J＝4.41Hz，2H)，4.29-4.32(m，4H)，4.18(q，J＝7.14Hz，2H)，4.01(m，4H)，1.56(d，J＝7.28Hz，6H)。EI-MS(m/e)：503，M+。
Embodiment 30:(2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-4-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. the preparation of tri hydrochloride (compound 30)
Compound with preparation among the embodiment 25 is a raw material, to be similar to the method preparation of embodiment 11, gets light yellow semi-solid 46mg, yield: 81.45%.
1HNMR(CD 3OD)：δ＝8.44(s，1H)，8.41(s，1H)，4.57(m，2H)，4.31(m，4H)，4.11(m，4H)，4.08-4.11(m，4H)，4.02(d，J＝7.42Hz，2H)，1.71-1.85(m，6H)，0.98(t，J＝5.76Hz，12H)，EI-MS(m/e)：587，M+。
Embodiment 31: the extracorporeal antivirus effect determination of activity
1, test method:
(1) cell toxicity test
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay MTT) detection of drugs is to the restraining effect of HepG 2215 growths to adopt the tetrazole reduction method.Get one bottle in HepG 2215 cells, with being prepared into single cell suspension after the trysinization, cell concn to 2 * 104cell/ml is adjusted in the counting back, adds in 96 well culture plates.Place cell culture incubator, 37 ℃, 5%CO2 overnight incubation.Suction goes to add the DMEM substratum (5% foetal calf serum) that contains the different concns medicine behind the supernatant.After acting on 9 days, add the MTT of 10 μ l in each hole and continue cultivation 4 hours.The careful suction goes supernatant, every hole to add 150 μ l DMSO, after the vibration, detects the OD value at 570nm place with microplate reader gently.
(2) to the inhibition test of hepatitis B virus-DNA (HBV-DNA)
A) cell drug is handled
Get one bottle in HepG 2215 cells,, regulate cell concn to 2 * 104cell/ml, add in the 24 porocyte culture plates (1ml/well) with being prepared into single cell suspension after the trysinization.37 ℃, 5%CO2 overnight incubation.Take out 24 porocyte culture plates, add 55 times of weaker concn medicines behind the sucking-off supernatant successively, and the positive control medicine.Continue to cultivate, changed the substratum that contains medicine in per 3 days, and collected in the 9th day each hole supernatant liquor to centrifuge tube-20 ℃ frozen standby.
B) quantitative fluorescent PCR reaction
Serum specimen (100ul) places lysate (1mmol/L Tris-Hcl, pH8.0,10mmol/LNacl, 0.1mmol/L EDTA, 0.5%SDS, 0.8mg/ml Proteinase K) 37 ℃ of hatching 5h, with phenol chloroform and chloroform extracting twice respectively, use ethanol sedimentation more then.Be dissolved in the 50 μ l water in order to making pcr amplification.
The 2ul viral DNA is put into the reaction mixture of 48 μ l, comprises the Gold Taq polymerase of 5mmol/L Mgcl2, every kind of dNTP0.2mmol/L, 2.5U, the UNG enzyme of 0.2U and each 0.4mmol/L of primer, probe 0.15mmol/L.Behind 37 ℃ of 5min, 95 ℃ of sex change 3min30s, 94 ℃ of 20s, 60 ℃ of 40s (fluorescent signal detection) circulate last 4 ℃ of preservations 41 times.Detect used Taqman probe sequence: 5 ' FAM-CCAGCAGCGCCTCCTCCTGC-3 ' TAMARA; Primer sequence: Forward primer:5 '-CCC TCAGGCTCAGGGCATA-3 ', Reverse primer:5 '-CTTCCTGACTGCCGATTGGT-3 '.
2. test clone:
HepG 2215 cells draw from university of Fudan University molecule virus laboratory.This cell strain is that the recombinant plasmid transfection recipient cell HepG2 with 2 full genes of HBV DNA from beginning to end forms, can be at external stable secretion HBsAg, and HBeAg and complete Dane particle can also produce a large amount of replicative intermediate (RI).Cell cultures is in the DMEM that contains 10% foetal calf serum.
3. positive controls is the two pivalates (Adefovir Dipivoxil) of Adefovir.
4, each compound suppresses EC to HBV-DNA in the HepG2.2.15 cell 50, cytotoxicity CC 50, and effect selectivity index SI value sees Table-2.
Table-2 test compounds are to the inhibition effect of HBV-DNA in the HepG2.2.15 cell
Compound number aEC 50(Mm) bCC 50(μM) cSI
1 5.87 269 45.93
5 0.25 818 3176.47
6 6.63 3583 540.57
7 11.59 2303 198.96
11 0.095 6636 69523.81
13 0.752 28712 38167.17
14 0.208 10984 52727.27
15 0.096 795 8203.12
16 0.946 18939 20000.00
17 0.211 3409 16129.03
18 0.304 3378 11094.52
19 0.0655 6318 96416.18
20 0.34 8560 25111.11
21 270 454 1.68
22 93.18 217 2.33
23 5.87 19393 3303.23
26 1.31 2587 1963.21
27 0.31 2590 8142.85
28 13.33 6242 468.18
29 12.57 1840 146.38
30 1.00 1128 1128
Adefovir 0.517 540 1044.48
A.EC 50: medium effective concentration b.CC 50: half cytotoxicity concentration c .SI: effect selectivity index (CC 50/ EC 50)
Similar or more excellent to the two pivalates of positive control Adefovir in anti-HBV activity and effect selectivity index by table-2 claimed compounds of the present invention as can be known, this illustrates that they have the better therapeutic index.

Claims (8)

1, a class is by acceptable salt on purine compounds double amino acid ester shown in the following formula (I) and the pharmacology thereof:
Figure A2006100244010002C1
Wherein,
R 1Be amino;
R 2For amido protecting or free L-type amino acid;
N is 0 or 1;
X is O or S.
2, according to acceptable salt on the purine compounds double amino acid ester of claim 1 and the pharmacology thereof, it is characterized in that described purine compounds double amino acid ester is selected from following compound:
(1) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(2) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-acetoxyl group) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(3) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl base sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(4) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-phenyl propionyl sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(5) (2S, 2 ' S)-9-{2-[O, O '-two [(1-tertbutyloxycarbonyl-2-Pyrrolizidine base formyl radical sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(6) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(7) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(8) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl oxygen base) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(9) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(10) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(11) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(12) (2S, 2 ' S)-9-{2-[O, O '-two [(2-glycyl oxygen base) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(13) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylbutyryl base sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(14) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-phenyl propionyl sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(15) (2S, 2 ' S)-9-{2-[O, O '-two [(2-Pyrrolizidine formyl radical sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(16) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-4-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(17) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylpent acyl group sulphur) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(18) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylbutyryl oxygen base) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(19) (2S, 2 ' S)-9-{2-[O, O '-two [(the amino 4-methylpent of 2-acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(20) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylpent acyloxy) methylene radical] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(21) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-methylbutyryl oxygen base) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(22) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-3-phenyl) propionyloxy] ethyl] phosphonium mesitoyl methoxy } ethyl } VITAMIN B4;
(23) (2S, 2 ' S)-9-{2-[O, O '-two [(1-tertbutyloxycarbonyl-2-Pyrrolizidine base methanoyl) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(24) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino propionyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(25) (2S, 2 ' S)-9-{2-[O, O '-two [(2-t-butoxycarbonyl amino-4-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4;
(26) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-methylbutyryl oxygen base) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(27) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-3-phenyl propionyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(28) (2S, 2 ' S)-9-{2-[O, O '-two [(2-Pyrrolizidine base methanoyl) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(29) (2S, 2 ' S)-9-{2-[O, O '-two [(the amino propionyloxy of 2-) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride;
(30) (2S, 2 ' S)-9-{2-[O, O '-two [(2-amino-4-methylpent acyloxy) ethyl] phosphonium mesitoyl methoxy] ethyl } VITAMIN B4. tri hydrochloride.
3, according to acceptable salt on the purine compounds double amino acid ester of claim 1 and the pharmacology thereof, it is characterized in that acceptable salt is on the described pharmacology: the inorganic acid salt that this purine compounds double amino acid ester and hydrochloric acid, Hydrogen bromide, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid form; Organic acid salt with formic acid, acetate, propionic acid, oxalic acid, propanedioic acid, succsinic acid, fumaric acid, toxilic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, picric acid, methylsulfonic acid or ethyl sulfonic acid formation; The acid salt that forms with the acidic amino acid of aspartic acid or L-glutamic acid; Or the salt that forms with the mineral alkali of sodium, potassium, calcium or aluminium; Ammonium salt, methylamine salt, ethylamine salt, ethanolamine salt; Or the salt that forms with the basic aminoacids of Methionin, arginine, ornithine.
4, a kind of method for preparing the purine compounds double amino acid ester of claim 1 may further comprise the steps:
(1) N-tertbutyloxycarbonyl L-amino acid and ethylene bromohyrin are in aprotic solvent, and at dicyclohexyl carbodiimide and N, there is reaction down in the N dimethylamine yl pyridines, obtains N-tertbutyloxycarbonyl L-amino acid bromo-ethyl ester; Or
N-tertbutyloxycarbonyl L-amino acid and isobutyl chloroformate, N-methylmorpholine and stink damp precursor reactant, obtain the amino thiocarboxylic acid of N-tertbutyloxycarbonyl L-, and then with 1, the 2-ethylene dibromide is in non-protonic solvent, reaction in the presence of highly basic obtains the amino thiocarboxylic acid of N-tertbutyloxycarbonyl L--2-bromo-ethyl ester;
Chloromethyl chlorsulfonic acid ester and N-tertbutyloxycarbonyl L-amino acid are in two-phase system, and reaction obtains the amino acid whose chloromethyl ester of N-tertbutyloxycarbonyl L-in the presence of the phase-transfer catalyst 4-n-butyl ammonium hydrogen sulfate;
(2) the amino carbothioic acid ester of various N-tertbutyloxycarbonyl L-amino acid or L-respectively with 9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 is in polar aprotic solvent, with N, N '-dicyclohexyl-4-morpholinyl-amidine or 1,8-diazabicyclo [5,4,0] undecane-7-alkene is alkali reaction, obtains product;
(3) above in the step (2) products therefrom in polarity or non-polar solvent, hydrogenchloride saturated 1,4-dioxane or Acetyl Chloride 98Min./methyl alcohol exists down that reaction obtains product;
(4) as required, according to ordinary method the product that obtains in the step (3) is prepared into corresponding pharmacy acceptable salt.
5, a kind of method for preparing the compound of claim 1 may further comprise the steps:
(1) 9-[2-(phosphonium mesitoyl methoxy) ethyl] VITAMIN B4 is in inert solvent, at halogenating agent oxalyl chloride and catalyst n, there is reaction down in N '-diethylformamide, obtains 9-[2-(phosphonium mesitoyl methoxy) ethyl] the dichloro-phosphonic acid ester of VITAMIN B4;
(2) N-tertbutyloxycarbonyl L-amino acid and ethylene glycol are in aprotic solvent, and at dicyclohexyl carbodiimide and N, there is reaction down in the N dimethylamine yl pyridines, obtains N-tertbutyloxycarbonyl L-amino acid-hydroxyethyl ester;
(3) 9-[2-(phosphonium mesitoyl methoxy) ethyl] the dichloro-phosphonic acid ester of VITAMIN B4 and N-tertbutyloxycarbonyl L-amino acid-hydroxyethyl ester are in inert solvent, and reaction obtains product in the presence of pyridine and triethylamine;
(4) product that obtains in the step (3) is in polarity or non-polar solvent, hydrogenchloride saturated 1,4-dioxane or Acetyl Chloride 98Min./methyl alcohol exists down that reaction obtains corresponding compounds;
(5) as required, according to ordinary method the product that obtains in the top step is prepared into corresponding pharmacy acceptable salt.
6, a kind of pharmaceutical composition comprises acceptable salt on the purine compounds double amino acid ester of at least a claim 1 to 3 as effective constituent and the pharmacology thereof, and described compound can be in conjunction with acceptable vehicle or carrier at least a pharmacology.
7, be used for the treatment of application in the medicine of disease of viral infection according to the pharmaceutical composition of claim 6 in preparation.
8, according to the application of claim 7, it is characterized in that the infectious diseases of described disease of viral infection for causing by hepatitis B virus and hiv virus.
CN2006100244016A 2006-03-06 2006-03-06 Preparation method and use for purine compounds double amino acid ester Expired - Fee Related CN101033238B (en)

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