CN100588653C - Compound with proteasome inhibition function and its preparing method and application - Google Patents
Compound with proteasome inhibition function and its preparing method and application Download PDFInfo
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- CN100588653C CN100588653C CN200610012149A CN200610012149A CN100588653C CN 100588653 C CN100588653 C CN 100588653C CN 200610012149 A CN200610012149 A CN 200610012149A CN 200610012149 A CN200610012149 A CN 200610012149A CN 100588653 C CN100588653 C CN 100588653C
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Abstract
The invention discloses a kind of compound which is characterized by prolease inhibiting function and the preparation method and application. The structure of said compound is as that: X is - C (O)- NH- or -CH(OH)-; R1 and R3 are normal paraffin, branched chain alkane with carbon number being from one to ten, or substituted acrylic acid, cycloalkyl with carbon number being from 3- 6, benzyl, substituted benzyl, heterocyclic substituent group; Pg is amidol protecting group; when X is -C (O)- NH-, R2 is normal paraffin, branched chain alkane with carbon number being from one to ten, or substituted acrylic acid, cycloalkyl with carbon number being from 3- 6, benzyl, substituted benzyl, heterocyclic substituent group; when X is -CH(OH)-, R2 is alkylamino radical or arylamine with 1- 10 carbons.
Description
Technical field
The present invention relates to compound and preparation method thereof and application, particularly relate to compound with proteasome inhibition function and preparation method thereof and application.
Background technology
Proteasome is many catalytic proteins enzyme complex of non-lysosome in a kind of cell of high conservative, is present in the eukaryotic nuclear and in the tenuigenin, the common hydrolysis of this mixture is connected with the albumen of poly ubiquitin chain.The core of 26S proteasome is the hydrolysis unit of a 20S (720kDa), and there is the adjusting mixture of two 19S (890kDa) at two ends.Ubiquitin on the ubiquitin protein is regulated on the mixture cleaved at 19S, albumen enters in the 20S core, and by the avtive spot hydrolysis on the 20S β subunit, these avtive spots comprise: be positioned at two β
5" Chymotrypsin sample site " on the subunit (chymotrypsin-like) tends to cracking substrate after hydrophobic residue; Be positioned at two β
2Site on the subunit is called as " trypsin-like site " (trypsin-like), tends to cracking substrate behind alkaline residue; Be positioned at two β
1Site on the subunit then is called " caspase sample site " (caspase-like), tends to cracking substrate after acidic residues, and is much higher than L-glutamic acid at aspartic acid place hydrolytic activity.
Uiquitin-protease enzyme system system is brought into play the vital role of three aspects in vivo: 1, make inactive amyloid protein precursor be processed into activated protein through degraded, as the activation of transcription factor NF-KB.2, with the accumulative proteolytic degradation to keep protein balance in the body; The process such as differentiation, growth, apoptosis of cell is being controlled in particularly proteic activation of multiple adjusting and degraded in the cell signaling approach.3, optionally remove the albumen of sudden change, damage or false folding.Therefore rely on proteolysis process crucial effect of performance aspect control cell proliferation and necrocytosis of uiquitin-protease enzyme body, and also play an important role in regulating in inflammatory reaction, immunosurveillance, long-term memory acquisition, biorhythm.
Uiquitin-protease enzyme body path and tumour are in close relations: the first, and the degraded of various cyclins and Starch phosphorylase CDC25s etc. is all finished by proteasome.For the tumour cell in the fast breeding, arrestin enzyme body just directly suppresses tumor cell proliferation.The second, pressing down cancer factor p53 is short life albumen, directly is subjected to the degraded of proteasome.Arrestin enzyme body will make the p53 function strengthen, and finally cause apoptosis of tumor cells.The 3rd, transcription factor NF-KB family combines with arrestin I κ B and is inactive state.When proteasome I κ 3 is degraded, NF-κ B promptly is activated, and a series of albumen of expression generation make cell proliferation, differentiation, apoptosis inhibition etc.And NF-κ B still causes the drug-fast one of the main reasons of tumour cell.When proteasome is suppressed, can directly disturb tumor cell proliferation, quicken its apoptosis, increase tumour cell to put, the susceptibility of chemotherapy, and suppress vascularization and metastases.Above-mentioned multiple effect makes proteasome become the new target drone of potential antitumor drug effect.
First enters the proteasome inhibitor medicine Bortezomib (PS-341) of clinical trial in the world, kinds of tumors had obvious therapeutic action, as mammary cancer, colorectal carcinoma, lymphatic cancer, multiple myeloma, ovarian cancer, carcinoma of the pancreas, prostate cancer, flakey cell carcinoma etc., it is antitumour drug safely and effectively, have only side effects such as 30% patient weakness occurs, feels sick, vomiting, appetite stimulator, constipation, thrombopenia, anaemia, peripheral nerve dysfunction, decrement or withdrawal side effects disappear.After the II clinical trial phase finishes, filed an application 4 months in 2003, and promptly quickened examination and approval procedures approval listing, be used for the treatment of intractable multiple myeloma through U.S. FDA.Another proteasome inhibitor in clinical trial is PS-519 at present, has anti-inflammatory action, is used to alleviate the ischemia condition of apoplexy.In addition, the anti-acquired immunodeficiency syndrome drug Ritonavir that FDA has ratified is the hiv protease inhibitor, has confirmed that now this medicine also has selective proteasome and suppresses active, and relevant with its antivirus action.These illustrate that all proteasome is novel, special drug effect target, is indicating that also proteasome inhibitor has challenging potential applicability in clinical practice.Remove as antitumor drug or chemicotherapy sensitizer and use, also may have anti-inflammatory activity, diseases such as treatment autoimmune disease, nerve retrograde affection.
Proteasome is a kind of proteolytic ferment, belongs to the serine/threonine protein enzyme family.Different with other all proteolytic enzyme, the proteolysis site in the proteasome utilizes the terminal Threonine of the N-of β subunit as nucleophilic center.Therefore, existing inhibitor molecules great majority are that one section small peptide can link to each other with the pharmacophore of Threonine reaction with one.Being had an effect by pharmacophore and catalytic site forms reversible or irreversible affixture, and small peptide is responsible for the specific recognition of inhibitor molecules and proteasome.Though proteasome has a plurality of catalytic site, will all not suppress in all sites.In fact, just significantly arrestin degraded of inhibition Chymotrypsin sample site (Chymotrypsin-like site) wherein.Present proteasome inhibitor mainly is divided into two big class, covalent attachment and non-covalent bonded inhibitor.Wherein, covalently bound inhibitor comprises: peptide aldehydes, alpha-keto amide class, peptide boric acid ester class, peptide vinyl sulfone(Remzaol class, natural cyclic peptide TMC-95A-D, be β-lactone compound, tea-polyphenol class material, the epoxy ketone compounds of representative with Lactacystin, the inhibitor of non-covalent tuberculosis comprises that a class is the compound of structure parent nucleus with 2-substituted-amino benzyl statine.
Summary of the invention
What the purpose of this invention is to provide a kind of novel structure has compound of proteasome inhibition function and preparation method thereof.
Compound with proteasome inhibition function provided by the present invention, structure be suc as formula I,
Wherein, X be-C (O)-NH-or-CH (OH)-;
R
1, R
3Be straight chain, branched-chain alkyl or the substituted alkyl of 1 to 10 carbon, 3-6 unit cycloalkyl, benzyl, substituted benzyl, heterocyclic substituting group;
Pg is an amino protecting group;
X is-during C (O)-NH-, and R
2Be straight chain, branched-chain alkyl or the substituted alkyl of 1 to 10 carbon, 3-6 unit cycloalkyl, benzyl, substituted benzyl, heterocyclic substituent;
X is-CH (OH)-time, R
2Be the alkylamino radical of 1 to 10 carbon, aromatic amino.
Here, straight chain, branched-chain alkyl and the substituted alkyl of 1 to 10 carbon are as methyl, ethyl, propyl group, sec.-propyl, normal-butyl, 2-methyl-propyl, fourth-2-base, n-pentyl, methylol, methoxy methylol etc.; Various 3 yuan of-6 yuan of cycloalkyl groups are as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl etc.; Benzyl and various substituted benzyl, as 4-hydroxybenzyl, 4-methoxy-benzyl, 3,4,5-trimethoxy benzyl, 4-nitrobenzyl, menaphthyl etc.; Heterocyclic substituent is as imidazoles-2-base, Pyrrolidine-2-base, indol-3-yl etc.; The alkylamino radical of 1 to 10 carbon is as methylamino, ethylamino-, Propylamino, isopropylamine base, butylamine base, isobutyl amine, amylamine base, cyclopentamine base, cyclohexylamino etc.; Various aromatic aminos, as aniline, 4-anisidine, 3,4,5-trimethoxy-aniline, benzylamine, 4-anisidine, 3,4,5-trimethoxy-aniline etc.; Amino protecting group; as tertbutyloxycarbonyl, the different third oxygen carbonyl of biphenyl, uncle's penta oxygen carbonyl, diisopropyl methoxycarbonyl, ring penta oxygen carbonyl, hexamethylene oxygen carbonyl, piperidines oxygen carbonyl, ethylene oxy carbonyl, carbobenzoxy-(Cbz), to methoxy carbobenzoxy-(Cbz), 3,5-dimethoxy carbobenzoxy-(Cbz), to nitro carbobenzoxy-(Cbz), pyridine-4-methoxycarbonyl, fluorenylmethyloxycarbonyl, formyl radical, ethanoyl, phthaloyl, p-toluenesulfonyl etc.
X is-preparation method of the compound of C (O)-NH-among the formula I, comprises the steps:
1) amino acid that has the tertbutyloxycarbonyl protection of formula II structure is under the effect of peptide condensation reagent, and with N, the reaction of O-dimethyl hydroxylamine hydrochloride obtains the formula III structural compounds;
2) the formula III structural compounds reacts with furans under the organolithium reagent effect, obtains formula IV compound;
3) formula IV compound removes tertbutyloxycarbonyl, obtains formula V compound;
4), obtain the amino acid methyl ester of formula VII structure with the amino acid and the thionyl chloride reaction of formula VI structure;
5) amino acid that has protecting group of formula VIII structure and the amino acid methyl ester of formula VII structure are reacted under the effect of peptide condensation reagent, obtain two peptide prods of formula IX structure; Hydrolysis under this two peptide prods alkaline condition obtains the compound of formula X structure;
6) compound and the formula V compound with formula X structure reacts under the effect of peptide condensation reaction reagent, obtains the compound for-C (O)-NH-of X among the formula I;
Wherein, R
1, R
2, R
3Be straight chain, branched-chain alkyl or the substituted alkyl of 1 to 10 carbon, 3-6 unit cycloalkyl, benzyl, substituted benzyl, heterocyclic substituting group;
Pg is an amino protecting group.
Common peptide condensation reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, I-hydroxybenzotriazole or isobutyl chlorocarbonate.
X is-preparation method of the compound of C (O)-NH-among the formula I, comprises the steps:
1) amino acid that has the tertbutyloxycarbonyl protection of formula II structure is under the effect of peptide condensation reagent, and with N, the reaction of O-dimethyl hydroxylamine hydrochloride obtains the formula III structural compounds;
2) the formula III structural compounds reacts with furans under the organolithium reagent effect, obtains formula IV compound;
3) formula IV compound removes tertbutyloxycarbonyl, obtains formula V compound;
4) amino acid that has protecting group and the methylating reagent reaction with formula VIII structure methylates, and then, reduction obtains the compound of formula XI structure;
5) compound with formula XI structure carries out oxidation with the oxygenant that alcohol can be oxidized to aldehyde, then, and with Ph
3PCHCOOEt reacts, and obtains the compound of formula XII structure; The gained compound is carried out epoxidation with epoxidation reagent, obtain formula XIII compound;
6) formula XIII compound and organic amine are reacted, obtain the compound of XIV structure; Hydrolysis under the compound alkaline condition of XIV structure obtains formula XV compound;
7) formula XV compound and formula V compound be under the effect of peptide condensation reaction reagent, and reaction obtains the compound for-C (O)-NH-of X among the formula I.
Wherein, R
1, R
3Be straight chain, branched-chain alkyl or the substituted alkyl of 1 to 10 carbon, 3-6 unit cycloalkyl, benzyl, substituted benzyl, heterocyclic substituting group;
R
2Be the alkylamino radical of 1 to 10 carbon, aromatic amino;
Pg is an amino protecting group.
Common peptide condensation reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl), I-hydroxybenzotriazole (HOBt) or isobutyl chlorocarbonate.
Another object of the present invention provides the purposes of The compounds of this invention.
The inventor confirms by experiment, The compounds of this invention has the activity and the anti-tumor activity of good arrestin enzyme body, N-[(1 particularly, 1-dimethyl oxyethyl group) carbonyl]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 3-methyl butyl]-L-phenylalanyl amine, the activity of its arrestin enzyme body and anti-tumor activity height, can be used to prepare antitumor drug, be widely used in tumor treatment.
The plan peptide class that the present invention has designed and synthesized a class new texture type has the compound of proteasome inhibition function; i.e. one section small peptide or intend the acyl group furan compound of peptide; show proteasome inhibition activity and antitumor action preferably, wide application value is arranged.
Embodiment
Synthesizing of first part's compound
The preparation of compound of the present invention can be carried out according to following process:
One, preparation intermediate formula V compound
The amino acid (formula II) that will have the tertbutyloxycarbonyl protection is dissolved in a certain amount of methylene dichloride;-13 ℃ add organic amine (as: N-methylmorpholine) and peptide condensation reagent (as: isobutyl chlorocarbonate) down; reacted 5 minutes; add N then; the O-dimethyl hydroxylamine hydrochloride; the temperature of reaction system is elevated to room temperature, continues to stir, the TLC monitoring is finished up to reaction.Organic phase is with pickling (10% aqueous citric acid solution), alkali cleaning (saturated sodium bicarbonate aqueous solution), then with saturated common salt washing, siccative drying (anhydrous sodium sulphate or sal epsom).The filtering siccative, the organic phase evaporated under reduced pressure, the residue oily liquids with column chromatography method separate the formula III compound.
Under-78 ℃, organolithium reagent (as: n-BuLi) is added in the organic solution (as: THF) of furans, rise to 0 ℃ and react half an hour, add the formula III compound then ,-78 ℃ are continued reaction 2.5 hours.Reaction is poured reaction solution into saturated NH after finishing
4Cancellation in the Cl solution is with organic solvent (as: Et
2O) extraction is 3 times, and organic phase washes with water, and saturated NaCl washes, siccative (as: anhydrous sodium sulphate) drying.The filtering siccative, the organic phase evaporated under reduced pressure, the yellow oily liquid that obtains with column chromatography method separate formula IV compound.
Formula IV compound is dissolved in a certain amount of methylene dichloride, adds isopyknic trifluoroacetic acid, stirring at room (2 hours), reaction finishes solvent evaporated, adds small amount of toluene, the evaporated under reduced pressure solvent.Add methylene dichloride dissolving resistates, wash organic phase usefulness siccative (anhydrous sodium sulphate) drying with the NaOH of 0.1N.The filtering siccative, the evaporated under reduced pressure solvent obtains intermediate formula V compound, is directly used in next step reaction without separation.
Two, X is-compound (formula I/A) of C (O)-NH-synthetic among the formula I
The amino acid (formula VI) with any protecting group not is dissolved in the anhydrous methanol, under-10 ℃ to wherein adding thionyl chloride, room temperature reaction 5 hours.Solvent evaporated, remaining solid recrystallization (methanol) gets amino acid methyl ester (formula VII).
The amino acid (formula VIII) and the amino acid methyl ester (formula VII) that will have protecting group are dissolved in DMF, add suitable peptide condensation reaction reagent (N-methylmorpholine+HoBt+EDCHCl).Room temperature reaction spends the night.Add organic solvent (ethyl acetate) dilute reaction solution, organic phase pickling (10% citric acid), alkali cleaning (saturated sodium bicarbonate solution), saturated common salt washing, siccative (anhydrous sodium sulphate and sal epsom) drying.The filtering siccative, the evaporated under reduced pressure solvent, remaining solid recrystallization (ethyl acetate/petroleum ether) gets two peptide prods (formula IX).
Two peptide prods of formula IX structure are dissolved in the organic solvent (THF), add the aqueous solution (1NNaOH) of mineral alkali, room temperature reaction is complete up to the TLC detection reaction.Use mineral acid (1N hydrochloric acid) that the pH value of reaction solution is adjusted between the 2-3 then, with organic solvent (ethyl acetate) extraction, organic phase siccative drying (anhydrous sodium sulphate or sal epsom).The filtering siccative with the solvent evaporated under reduced pressure, obtains product (formula X), is directly used in the next step without separation.
Compounds X and intermediate formula V compound are dissolved among the DMF, add suitable peptide condensation reaction reagent (N-methylmorpholine+HoBt+EDCHCl).Room temperature reaction spends the night.Add organic solvent (ethyl acetate) dilute reaction solution, organic phase pickling (10% citric acid), alkali cleaning (saturated sodium bicarbonate solution), saturated common salt washing, siccative (anhydrous sodium sulphate and sal epsom) drying.The filtering siccative, the evaporated under reduced pressure solvent, the column chromatography rough segmentation of gained crude product, gained solid recrystallization gets product (formula I/A).
Three, X is-compound (formula I/B) of C (O)-NH-synthetic among the formula I
The amino acid (formula VIII) that will have protecting group is dissolved among the DMF, adds mineral alkali (sodium bicarbonate) and methylating reagent (methyl iodide), and room temperature reaction is finished up to the TLC detection reaction.In reaction system, add entry,, merge organic phase, wash with 10% sodium sulfite solution with organic reagent (ethyl acetate) extraction, washing, the saturated common salt washing, siccative (anhydrous sodium sulphate or sal epsom) is washed.The filtering siccative is with the solvent evaporated under reduced pressure.Residual crude product is dissolved in (THF/EtOH=2/3) in the organic solvent, adds reductive agent (NaBH down at 0 ℃
4And CaCl
2), reacted 5 hours.With careful the pouring in 0 ℃ the acid solution (1M citric acid) of solution, with organic solvent (ethyl acetate) extraction, merge organic phase, to wash with alkali lye (saturated sodium bicarbonate solution), saturated common salt is washed, siccative (anhydrous sodium sulphate or sal epsom) drying.The filtering siccative, the evaporated under reduced pressure solvent is with the product (formula XI) of gained crude product with column chromatography method or recrystallization method purifying.
Formula XI compound is dissolved in the methylene dichloride, and adding can be oxidized to alcohol the oxygenant (PySO of aldehyde
3), the TLC monitoring reaction is poured reaction solution in the saturated aqueous common salt of ice after finishing, and with organic solvent (ether) extraction, merges organic phase, washes washing, saturated common salt washing, siccative (anhydrous sodium sulphate or sal epsom) drying with diluted acid (10% citric acid solution).The filtering siccative, at 30 ℃ of following evaporated under reduced pressure solvents, the gained crude product is dissolved in (toluene) in an amount of organic solvent without separation, reflux (80 ℃) certain hour (1 hour).Concentration of reaction solution after reaction is finished, column chromatography for separation or recrystallization obtain product (formula XII).
(formula XII) is dissolved in the methylene dichloride with product, adds epoxidation reagent (m-PCBA), and normal temperature stirs down, finishes up to the TLC monitoring reaction.Use the ether dilute reaction solution, organic phase is washed with 10% S-WAT, alkali cleaning (saturated sodium bicarbonate solution), saturated common salt washing, siccative (anhydrous sodium sulphate or sal epsom) drying.The filtering siccative, the evaporated under reduced pressure solvent, crude product uses column chromatography purifying, obtains product (formula XIII).
Product (formula XIII) is dissolved in (dehydrated alcohol) in the organic solvent, adds primary amine (aniline and benzylamine), (60 ℃ of reacting by heating certain hours, 3 days), finish the evaporated under reduced pressure solvent up to the TLC monitoring reaction, use column chromatography the gained crude product, get product (formula XIV).
Formula XIV compound is dissolved in the organic solvent (THF), adds the aqueous solution (1N NaOH) of mineral alkali, room temperature reaction is complete up to the TLC monitoring reaction.Use mineral acid (1N hydrochloric acid) that the pH value of reaction solution is adjusted between the 2-3 then, with organic solvent (ethyl acetate) extraction, organic phase siccative drying (anhydrous sodium sulphate or sal epsom).The filtering siccative with the solvent evaporated under reduced pressure, obtains product X V, is directly used in the next step without separation.
Compounds X V and formula V compound are dissolved among the DMF, add suitable peptide condensation reaction reagent (N-methylmorpholine+HoBt+EDCHCl).Room temperature reaction spends the night.Add organic solvent (ethyl acetate) dilute reaction solution, organic phase pickling (10% citric acid), alkali cleaning (saturated sodium bicarbonate solution), saturated common salt washing, siccative (anhydrous sodium sulphate and sal epsom) drying.The filtering siccative, the evaporated under reduced pressure solvent, the column chromatography rough segmentation of gained crude product, gained solid recrystallization gets product formula I/B.
Below to describe synthesizing of particular compound:
One, the aminoacyl furan segments is synthetic
1, (S)-2-amino-1-(furans-2-yl)-3-methyl fourth-1-ketone (compound 3a) is synthetic
(S)-1-(N-methoxyl group-N-methyl carboxamide)-2-methyl-propyl t-butyl carbamate (compound 1a) synthetic
With Boc-Val-OH (Xie Ansuan of tertbutyloxycarbonyl protection) (2.17g; 10mmol) be dissolved in the 30mL methylene dichloride, add N-methylmorpholine (NMM, 2.2mL; 22mmol); (1.3mL 11mmol), reacted 5 minutes to add isobutyl chlorocarbonate at-13 ℃; add N then; the O-dimethyl hydroxylamine hydrochloride (1.02g, 11mmol), room temperature reaction 5 hours.After reaction finished, organic phase was with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na SO
4Dry.The filtering siccative, organic phase evaporated under reduced pressure, residue oily liquids ethyl acetate/petroleum ether=1/8-1/5 post branch.Get compound 1a 2.08g (productive rate 80%), colourless transparent oil liquid.
(S)-1-(furans-2-yl)-3-methyl isophthalic acid-oxo fourth-2-aminocarbamic acid tert-butyl ester (compound 2a) synthetic
Under-78 ℃, (the hexane solution 22mL of 1.6M 35mmol) adds furans (2.6mL with n-BuLi, in 50mL THF solution 36mmol), rise to 0 ℃ and react half an hour, add compound 1a (2.60g at-78 ℃ then, THF solution 10mmol) continues reaction 2.5 hours.Reaction is poured reaction solution into saturated NH after finishing
4Et is used in cancellation in the Cl solution
2O extraction 3 times, organic phase washes with water, and saturated NaCl washes, anhydrous sodium sulfate drying.The filtering siccative, organic phase evaporated under reduced pressure, the yellow oily liquid that obtains ethyl acetate/petroleum ether=1/10-1/8 post branch.Get compound 2a 2.42g (productive rate 92%), the brown oily liquids.
1H?NMR(300MHz，CDCl
3)：δ0.84(d，J＝6.9，3H，CH
3)，1.03(d，J＝6.9，3H，CH
3)，1.44(s，9H，Boc)，2.19(m，1H，CH)，4.96(m，1H，CH)，5.30(m，1H，NH)，6.56(dd，J＝1.8Hz，3.6Hz，1H，4’-H)，7.30(d，J＝3.6Hz，1H，3’-H)，7.63(d，J＝1.8Hz，1H，5’-H)。
(S)-2-amino-1-(furans-2-yl)-3-methyl fourth-1-ketone (compound 3a) synthetic
(267mg 1mmol) is dissolved in the 2mL methylene dichloride, adds the 2mL trifluoroacetic acid with compound 2a, stirring at room 2 hours, reaction finishes solvent evaporated, adds small amount of toluene evaporated under reduced pressure solvent once more, add the methylene dichloride dissolved residue, wash anhydrous sodium sulfate drying with 0.1N NaOH.The filtering siccative, the evaporated under reduced pressure solvent gets compound 3a, and dark oil liquid is directly used in next step reaction without separation.
2, (S)-2-amino-1-(furans-2-yl)-4-methylpent-1-ketone (compound 3b) is synthetic
(S)-(N-methoxyl group-N-methyl carboxamide)-3-methyl butyl t-butyl carbamate (compound 1b) synthetic
With Boc-Leu-OH (leucine of tertbutyloxycarbonyl protection) (2.31g; 10mmol) be dissolved in the 30mL methylene dichloride; (2.2mL 22mmol), adds isobutyl chlorocarbonate (1.3mL at-13 ℃ to add N-methylmorpholine; 11mmol); reacted 5 minutes, and added N then, O-dimethyl hydroxylamine hydrochloride (1.02g; 11mmol), room temperature reaction is 5 hours.After reaction finished, organic phase was with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na SO
4Dry.The filtering siccative, the organic phase evaporated under reduced pressure, the residue oily liquids gets compound 1b 1.97g (productive rate 72%), colourless transparent oil liquid with ethyl acetate/petroleum ether=1/8-1/5 post branch.
(S)-1-(furans-2-yl)-4-methyl isophthalic acid-oxo penta-2-aminocarbamic acid tert-butyl ester (compound 2b) synthetic
Under-78 ℃, (the hexane solution 22mL of 1.6M 35mmol) adds furans (2.6mL with n-BuLi, in 50mL THF solution 36mmol), rise to 0 ℃ and react half an hour, add compound 1b (2.74g at-78 ℃ then, THF solution 10mmol) continues reaction 2.5 hours.Reaction is poured reaction solution into saturated NH after finishing
4Et is used in cancellation in the Cl solution
2O extraction 3 times, organic phase washes with water, and saturated NaCl washes, anhydrous sodium sulfate drying.The filtering siccative, organic phase evaporated under reduced pressure, the yellow oily liquid that obtains ethyl acetate/petroleum ether=1/10-1/8 post branch.Get compound 2b 2.24g (productive rate 80%), the brown oily liquids.
1H?NMR(300MHz，CDCl
3)：δ0.93(d，J＝6.6，3H，CH
3)，1.04(d，J＝6.6，3H，CH
3)，1.44(s，9H，Boc)，1.57(m，2H，CH
2)，1.80(m，1H，CH)，5.07(m，1H，CH)，5.22(d，J＝8.4Hz，1H，NH)，6.57(dd，J＝1.8Hz，3.6Hz，1H，4’-H)，7.31(d，J＝3.6Hz，1H，3’-H)，7.63(d，J＝1.8Hz，1H，5’-H)，MS(ESI)，m/e，282.17(M+1)，304.16(M+Na)。
(S)-2-amino-1-(furans-2-yl)-4-methylpent-1-ketone (compound 3b) synthetic
(281mg 1mmol) is dissolved in the 2mL methylene dichloride, adds the 2mL trifluoroacetic acid with compound 2b, stirring at room 2 hours, reaction finishes solvent evaporated, adds small amount of toluene evaporated under reduced pressure solvent once more, add the methylene dichloride dissolved residue, wash anhydrous sodium sulfate drying with 0.1N NaOH.The filtering siccative, the evaporated under reduced pressure solvent gets compound 3b, and dark oil liquid is directly used in next step reaction without separation.
3, (2S, 3R-) 2-amino-1-(furans-2-yl)-3-methylpent-1-ketone (compound 3c) is synthetic
(1S, 2R)-1-(N-methoxyl group-N-methyl carboxamide)-2-methyl butyl t-butyl carbamate (compound 1c) synthetic
With Boc-Ile-OH (Isoleucine of tertbutyloxycarbonyl protection) (2.31g; 10mmol) be dissolved in the 30mL methylene dichloride; (2.2mL 22mmol), adds isobutyl chlorocarbonate (1.3mL at-13 ℃ to add N-methylmorpholine; 11mmol); reacted 5 minutes, and added N then, O-dimethyl hydroxylamine hydrochloride (1.02g; 11mmol), room temperature reaction is 5 hours.After reaction finished, organic phase was with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na SO
4Dry.The filtering siccative, organic phase evaporated under reduced pressure, residue oily liquids ethyl acetate/petroleum ether=1/8-1/5 post branch.Get compound 1c 1.9g (productive rate 70%), colourless transparent oil liquid.
(2S, 3R)-1-(furans-2-yl)-3-methyl isophthalic acid-oxo penta-2-aminocarbamic acid tert-butyl ester (compound 2c) synthetic
Under-78 ℃, (the hexane solution 22mL of 1.6M 35mmol) adds furans (2.6mL with n-BuLi, in 50mL THF solution 36mmol), rise to 0 ℃ and react half an hour, add compound 1c (2.74g at-78 ℃ then, THF solution 10mmol) continues reaction 2.5 hours.Reaction is poured reaction solution into saturated NH after finishing
4Et is used in cancellation in the Cl solution
2O extraction 3 times, organic phase washes with water, and saturated NaCl washes, anhydrous sodium sulfate drying.The filtering siccative, organic phase evaporated under reduced pressure, the yellow oily liquid that obtains ethyl acetate/petroleum ether=1/10-1/8 post branch.Get compound 2c 2.47g (productive rate 88%), the brown oily liquids.
1H?NMR(300MHz，CDCl
3)：δ0.86(t，3H，CH
3)，0.98(d，3H，CH
3)，1.14(m，2H，CH
2)，1.43(s，9H，Boc)，1.90(m，1H，CH)，4.96(dd，1H，CH)，5.28(d，1H，NH)，6.57(dd，J＝1.8Hz，3.6Hz，1H，4’-H)，7.31(d，J＝3.6Hz，1H，3’-H)，7.63(d，J＝1.8Hz，1H，5’-H)。
(2S, 3R-) 2-amino-1-(furans-2-yl)-3-methylpent-1-ketone (compound 3c) is synthetic
(281mg 1mmol) is dissolved in the 2mL methylene dichloride, adds the 2mL trifluoroacetic acid with compound 2c, stirring at room 2 hours, reaction finishes solvent evaporated, adds small amount of toluene evaporated under reduced pressure solvent once more, add the methylene dichloride dissolved residue, wash anhydrous sodium sulfate drying with 0.1N NaOH.The filtering siccative, the evaporated under reduced pressure solvent gets compound 3c, and dark oil liquid is directly used in next step reaction without separation.
Two, X is-compound (compound 7-15) of C (O)-NH-synthetic among the formula I
1,2-[2-[[(1,1-dimethyl oxyethyl group)-and carbonyl] amino]-3-phenyl-propionic acid amide]-3-phenyl-propionic acid (compound 6a) synthetic
(S) 2-amino-3-phenylpropionic acid methyl esters (compound 4a) is synthetic
Under-10 ℃, with SOCl
2(1.65g is in the anhydrous MeOH solution of 30mL 10mmol), then room temperature reaction 5 hours (5mL) to splash into H-Phe-OH.The reaction finish after with the solution decompression evaporate to dryness, remaining solid MeOH/Et
2The O recrystallization gets compound 4a 1.68g (productive rate 94%), white needle-like crystals, mp 161-163 ℃.
2-[2-[[(1,1-dimethyl oxyethyl group)-and carbonyl] amino]-3-phenyl-propionic acid amide]-3-phenyl-methyl propionate (compound 5a) synthetic
With Boc-Phe-OH (2.65g, 10mmol) and compound 4a (1.79g 10mmol) is dissolved among the DMF, to wherein add NMM (1.6mL, 15mmol), HOBt (2.03g, 15mmol) and EDCHCl (2.88g, 15mmol), mixture room temperature reaction 12 hours.Stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, remaining solid recrystallization from ethyl acetate/petroleum ether after the solvent evaporated gets compound 5a 3.07g (productive rate 72%), white needle-like crystals.mp?118-119℃，
1H?NMR(300MHz，CDCl
3)δ1.40(s，9H，Boc)，3.02-3.06(m，4H，PhCH
2)，3.67(s，3H，CH
3)，4.32(m，1H，CH)，4.79(m，1H，CH)，4.95(m，1H，NH)，6.25(m，1H，NH)，6.96-7.29(m，10H，Ph)。
2-[2-[[(1,1-dimethyl oxyethyl group)-and carbonyl] amino] 3-phenyl-propionic acid amide] 3-phenyl-propionic acid (compound 6a) synthetic
(426mg 1mmol) was dissolved among the 5mL THF, adds the NaOH of 1.1mL 1N, room temperature reaction 10 hours with compound 5a.HCl with 1N is adjusted to 2-3 with the pH value then, uses ethyl acetate extraction 4-5 time, organic phase anhydrous Na SO
4Dry.The filtering siccative, the evaporated under reduced pressure solvent obtains compound 6a, light yellow solid.Product is directly used in the next step without separation.
2,2-[2-[[(1,1-dimethyl oxyethyl group)-and carbonyl] amino]-3-phenyl-propionyl ammonia]-4-methyl-valeric acid (compound 6b) synthetic
(S)-2-amino-4-methylvaleric acid methyl esters (compound 4b) synthetic
Under-10 ℃, with SOCl
2(1.31g is in the anhydrous MeOH solution of 30mL 10mmol), then room temperature reaction 5 hours (5mL) to splash into H-Leu-OH.The reaction finish after with the solution decompression evaporate to dryness, remaining solid MeOH/Et
2The O recrystallization gets compound 4b 1.27g (productive rate 88%), white needle-like crystals, mp 151-152 ℃.
2-[2-[[(1,1-dimethyl oxyethyl group)-and carbonyl] amino] 3-phenyl-propionyl ammonia]-4-methyl-methyl valerate (compound 5b) synthetic
With Boc-Phe-OH (2.65g, 10mmol) and compound 4b (1.45g 10mmol) is dissolved among the DMF, to wherein add NMM (1.6mL, 15mmol), HOBt (2.03g, 15mmol) and EDCHCl (2.88g, 15mmol), mixture room temperature reaction 12 hours.Stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, remaining solid recrystallization from ethyl acetate/petroleum ether after the solvent evaporated gets compound 5b 2.80g (productive rate 70%), white needle-like crystals.mp?106-108℃，
1H?NMR(300MHz，CDCl
3)δ0.89(t，6H，CH
3)，1.41(s，9H，Boc)，1.43-1.60(m，3H，CHand?CH
2)，3.05-3.08(m，2H，PhCH
2)，3.69(s，3H，CH
3)，4.35(m，1H，CH)，4.57(m，1H，CH)，4.98(m，1H，NH)，6.24(m，1H，NH)，7.19-7.29(m，5H，Ph)，MS(ESI)，m/e.393.34(M+1)，415.22(M+Na)。
2-[2-[[(1,1-dimethyl oxyethyl group)-and carbonyl] amino]-3-phenyl-propionyl ammonia]-4-methyl-valeric acid (compound 6b) synthetic
(392mg 1mmol) was dissolved among the 5mL THF, adds the NaOH of 1.1mL 1N, room temperature reaction 10 hours with compound 5b.HCl with 1N is adjusted to 2-3 with the pH value then, uses ethyl acetate extraction 4-5 time, organic phase anhydrous Na SO
4Dry.The filtering siccative, the evaporated under reduced pressure solvent obtains compound 6b, white solid.Product is directly used in the next step without separation.
3,2-[2-[[(1,1-dimethyl oxyethyl group)-and carbonyl] amino]-3-phenyl-propionyl ammonia]-3-methyl-butyric acid (compound 6c) synthetic
(S)-2-amino-3 Methylbutanoic acid methyl esters (compound 4c) synthetic
Under-10 ℃, with SOCl
2(1.17g is in the anhydrous MeOH solution of 30mL 10mmol), then room temperature reaction 5 hours (5mL) to splash into H-Val-OH.The reaction finish after with the solution decompression evaporate to dryness, remaining solid MeOH/Et
2The O recrystallization gets compound 4c 1.17g (productive rate 90%), white bulk crystals, mp 152-154 ℃.
2-[2-[[(1,1-dimethyl oxyethyl group)-and carbonyl] amino]-3-phenyl-propionyl ammonia]-3-methyl-methyl-butyrate (compound 5c) synthetic
With Boc-Phe-OH (2.65g, 10mmol) and compound 4c (1.31g 10mmol) is dissolved among the DMF, to wherein add NMM (1.6mL, 15mmol), HOBt (2.03g, 15mmol) and EDCHCl (2.88g, 15mmol), mixture room temperature reaction 12 hours.Stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, remaining solid recrystallization from ethyl acetate/petroleum ether after the solvent evaporated gets compound 5c 3.09g (productive rate 82%), and mp:122-123 ℃,
1H?NMR(300MHz，CDCl
3)δ0.82-0.94(m，6H，CH
3)，1.40(s，9H，Boc)，2.10(m，1H，CH)，3.05-3.07(m，2H，PhCH
2)，3.69(s，3H，CH
3)，4.43(m，1H，CH)，4.75(m，1H，CH)，5.04(m，1H，NH)，6.40(m，1H，NH)，7.15-7.32(m，5H，Ph)。
2-[2-[[(1,1-dimethyl oxyethyl group)-and carbonyl] amino]-3-phenyl-propionyl ammonia]-3-methyl-butyric acid (compound 6c) synthetic
(378mg 1mmol) was dissolved among the 5mL THF, adds the NaOH of 1.1mL 1N, room temperature reaction 10 hours with compound 5c.HCl with 1N is adjusted to 2-3 with the pH value then, uses ethyl acetate extraction 4-5 time, organic phase anhydrous Na SO
4Dry.The filtering siccative, the evaporated under reduced pressure solvent obtains compound 6c, white solid.Product is directly used in the next step without separation.
4, carbonyl N-[(1,1-dimethyl oxyethyl group)]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl-propyl]-L-valine amide (compound 7) synthetic
With compound 6c (364mg, 1mmol) (167mg 1mmol) is dissolved among the 5mL DMF, adds NMM (0.16mL with compound 3a, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, with ethyl acetate/petroleum ether=1/2 post branch, the product that coupled columns divides the back to obtain carries out recrystallization after the solvent evaporated, gets 200mg compound 7 (productive rate 39%).White solid, mp, 196-197 ℃.
1H?NMR(500MHz，CDCl
3)δ0.78-0.98(m，12H，CH
3)，1.40(s，9H，Boc)，1.96-2.11(m，2H，CH)，3.04-3.15(m，2H，CH
2Ph)，4.27-4.34(m，2H，CH)，5.25-5.28(m，1H，CH)，6.50(d，1H，NH)，6.56(dd，J＝1.8Hz，3.6Hz，4’-H)，6.62(m，1H，NH)，6.75(m，1H)，7.18-7.30(m，5H，Ph)，7.34(d，J＝3.6Hz，3’-H)，7.64(d，J＝1.8Hz，5’-H).MS(ESI)，m/e，514.25(M+1)，536.23(M+Na)。
5, carbonyl N-[(1,1-dimethyl oxyethyl group)]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl-propyl]-L-leucyl amine (compound 8) synthetic
With compound 6b (378mg, 1mmol) (167mg 1mmol) is dissolved among the 5mL DMF, adds NMM (0.16mL with compound 3a, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, with ethyl acetate/petroleum ether=1/2 post branch, the product that coupled columns divides the back to obtain carries out recrystallization after the solvent evaporated, gets 226mg compound 8 (productive rate 43%).White solid, mp, 165-166 ℃,
1H?NMR(300MHz，CDCl
3)：δ0.85(d，9H，CH
3)，0.98(d，3H，CH
3)，1.38(s，9H，Boc)，1.45-1.56(m，2H，CH
2)，2.24(m，1H，CH)，2.87-3.08(m，2H，CH
2Ph)，4.41(m，1H，CH)，4.52(m，1H，CH)，5.16(m，1H，CH)，5.26(m，1H，NH)，6.56(dd，J＝1.5Hz，3.6Hz，1H，4’-H)，6.65(m，1H，NH)，6.92(m，1H，NH)，7.18-7.27(m，5H，Ph)，7.38(d，J＝3.6Hz，1H，3’-H)，7.64(d，J＝1.5Hz，1H，5’-H).MS(ESI)，m/e，528.26(M+1)，550.24(M+Na)。
6, carbonyl N-[(1,1-dimethyl oxyethyl group)]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl-propyl]-L-phenylalanyl amine (compound 9) synthetic
With compound 6a (412mg, 1mmol) (167mg 1mmol) is dissolved among the 5mL DMF, adds NMM (0.16mL with compound 3a, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, with ethyl acetate/petroleum ether=1/2 post branch, the product that coupled columns divides the back to obtain carries out recrystallization after the solvent evaporated, gets 168mg compound 9 (productive rate 30%).White solid, mp, 170-172 ℃,
1H?NMR(300MHz，CDCl
3)：δ0.79(d，3H，CH
3)，0.92(d，3H，CH
3)，1.37(s，9H，Boc)，2.13-2.19(m，1H，CH)，2.87-3.05(m，4H，PhCH
2)，4.75(m，1H，CH)，4.42(m，1H，CH)，5.15(m，1H，CH)，5.18(m，1H，NH)，6.57(dd，J＝1.5Hz，3.6Hz，1H，4’-H)，6.75(m，1H，NH)，6.85(m，1H，NH)，7.01-7.26(m，10H，Ph)，7.37(d，J＝3.6Hz，1H，3’-H)，7.64(d，J＝1.5Hz，1H，5’-H).MS(ESI)，m/e，562.26(M+1)，584.24(M+Na)。
7, carbonyl N-[(1,1-dimethyl oxyethyl group)]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 3-methyl butyl]-L-valine amide (compound 10) synthetic
With compound 6c (364mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF, adds NMM (0.16mL with compound 3b, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, with ethyl acetate/petroleum ether=1/2 post branch, the product that coupled columns divides the back to obtain carries out recrystallization after the solvent evaporated, gets 300mg compound 10 (productive rate 57%).White solid, mp, 149-151 ℃.
1H?NMR(500MHz，CDCl
3)δ0.83-0.87(m，9H，CH
3)，0.90-0.97(m，3H，CH
3)，1.01-1.12(m，2H，CH
2)，1.39(s，9H，Boc)，1.97-2.08(m，2H，CH)，3.04-3.14(m，2H，CH
2Ph)，4.32(m，1H，CH)，4.39(m?1H，CH)，5.15(m，1H，CH)，5.28(m，1H，NH)，6.57(dd，J＝1.8Hz，3.6Hz，4’-H)，6.64(m，1H，NH)，6.77(m，1H，NH)，7.22-7.33(m，5H，Ph)，7.35(d，J＝3.6Hz，3’-H)，7.63(d，J＝1.8Hz，5’-H).MS(ESI)，m/e，528.19(M+1)，550.17(M+Na)。
8, carbonyl N-[(1,1-dimethyl oxyethyl group)]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 3-methyl butyl]-L-leucyl amine (compound 11) synthetic
With compound 6b (378mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF, adds NMM (0.16mL with compound 3b, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, with ethyl acetate/petroleum ether=1/2 post branch, the product that coupled columns divides the back to obtain carries out recrystallization after the solvent evaporated, gets 211mg compound 11 (productive rate 39%).White solid, mp, 151-153 ℃.
1H?NMR(300MHz，CDCl
3)δ0.79-0.99(m，12H，CH
3)，1.30(s，9H，Boc)，1.37-1.55(m，4H，CH
2)，2.01(m，1H，CH)，3.05(m，2H，PhCH
2)，4.51(m，2H，CH)，5.21(m，1H，CH)，5.29(m，1H，NH)，6.53(dd，J＝1.8Hz，3.6Hz，4’-H)，6.80(m，1H，NH)，6.99(m，1H)，7.17-7.27(m，5H，Ph)，7.38(d，J＝3.6Hz，3’-H)，7.63(d，J＝1.8Hz，5’-H)；MS(ESI)，m/e，542.27(M+1)，564.26(M+Na)。
9, carbonyl N-[(1,1-dimethyl oxyethyl group)]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 3-methyl butyl]-L-phenylalanyl amine (compound 12) synthetic
With compound 6a (412mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF, adds NMM (0.16mL with compound 3b, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, with ethyl acetate/petroleum ether=1/2 post branch, the product that coupled columns divides the back to obtain carries out recrystallization after the solvent evaporated, gets 201mg compound 12 (productive rate 35%).White solid, mp, 145-147 ℃,
1H?NMR(300MHz，CDCl
3)：δ0.85(t，3H，CH
3)，0.96(t，3H，CH
3)，1.55(m，2H，CH
2)，2.03(m，1H，CH)，2.91-3.06(m，4H，CH
2Ph)，4.31(m，1H，CH)，4.63(m，1H，CH)，4.89(m，1H，CH)，5.25(m，1H，NH)，6.42(m?1H，NH)，6.56(dd，J＝1.5Hz，3.6Hz，1H，4’-H)，7.05(m，2H，NH)，7.15-7.28(m，10H，Ph)，7.31(d，J＝3.6Hz，1H，3’-H)，7.63(d，J＝1.5Hz，1H，5’-H).MS(ESI)，m/e，576.32(M+1)，598.30(M+Na)。
10, carbonyl N-[(1,1-dimethyl oxyethyl group)]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl butyl]-L-valine amide (compound 13) synthetic
With compound 6c (364mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF, adds NMM (0.16mL with compound 3c, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, with ethyl acetate/petroleum ether=1/2 post branch, the product that coupled columns divides the back to obtain carries out recrystallization after the solvent evaporated, gets 258mg compound 13 (productive rate 49%).White solid, mp, 181-182 ℃.
1H NMR (500MHz, CDCl
3) δ 0.79-0.87 (m, 9H, CH
3), 1.00 (d, 3H, CH
3), 1.38 (s, 9H, Boc), 2.08-2.11 (m, 1H, CH), 2.21-2.28 (m, 1H, CH), 3.04-3.14 (m, 2H, CH
2Ph), and 4.32-4.35 (m, 2H, CH), 5.02 (m, 1H, CH), 5.25 (t, 1H, NH), 6.48 (d, 1H, NH), 6.57 (dd, J=1.8Hz, 3.6Hz, 4 '-H), 6.75 (m, 1H, NH), 7.18-7.26 (m, 5H, Ph), 7.33 (d, J=3.6Hz, 3 '-H), 7.63 (d, J=1.8Hz, 5 '-H) .MS (ESI), m/e, 528.26 (M 10), 550.25 (M+Na).
11, carbonyl N-[(1,1-dimethyl oxyethyl group)]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl butyl]-L-leucyl amine (compound 14) synthetic
With compound 6b (378mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF, adds NMM (0.16mL with compound 3c, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, with ethyl acetate/petroleum ether=1/2 post branch, the product that coupled columns divides the back to obtain carries out recrystallization after the solvent evaporated, gets 200mg compound 14 (productive rate 37%).White solid, mp, 164-165 ℃.
1H?NMR(500MHz，CDCl
3)δ0.81-0.85(m，12H，CH
3)，1.12-1.23(m，1H，CH)，1.29(s，9H，Boc)，1.33-1.44(m，2H，CH
2)，1.45-1.58(m，2H，CH
2)，1.87-1.92(m，1H，CH)，2.68-2.74(m，1H，CH
2Ph)，2.92-2.95(m，1H，CH
2Ph)，4.15(m，1H，CH)，4.43(m，1H，CH)，4.88(t，1H，CH)，6.72(dd，J＝1.5Hz，3.5Hz，4’-H)，6.91(d，1H，NH)，7.16-7.30(m，5H，Ph)，7.55(d，J＝3.5Hz，3’-H)，7.91(d，1H，NH)，8.02(d，J＝1.5Hz，5’-H)，8.24(d，1H，NH).MS(ESI)，m/e，542.27(M+1)，564.26(M+Na)。
12, carbonyl N-[(1,1-dimethyl oxyethyl group)]-L-phenylalanyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl butyl]-L-phenylalanyl amine (compound 15) synthetic
With compound 6a (412mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF, adds NMM (0.16mL with compound 3c, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, and organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.The filtering siccative, with ethyl acetate/petroleum ether=1/2 post branch, the product that coupled columns divides the back to obtain carries out recrystallization after the solvent evaporated, gets 258mg compound 15 (productive rate 45%).White solid, mp, 190-192 ℃,
1H?NMR(300MHz，CDCl
3)：δ0.81(t，3H，CH
3)，0.88(d，3H，CH
3)，0.95(m，2H，CH2)，1.88(m，1H，CH)，2.85-3.06(m，4H，CH
2Ph)，4.38(m，1H，CH)，4.65(m，1H，CH)，4.97(m，1H，CH)，5.14(m，1H，NH)，6.51(m，1H，NH)，6.57(dd，J＝1.5Hz，3.6Hz，1H，4’-H)，6.58(m，1H，NH)，7.03-7.28(m，10H，Ph)，7.33(d，J＝3.6Hz，1H，3’-H)，7.65(d，J＝1.5Hz，1H，5’-H).MS(ESI)，m/e，576.25(M+1)，598.24(M+Na)。
Three, X is-compound (compound 23-28) of C (O)-NH-synthetic among the formula I
1, (2R, 3S, 4S)-2-(benzyl ammonia)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-5-phenylpentanoic acid (compound 21) synthetic
(S)-1-hydroxyl-3-phenyl third-2-aminocarbamic acid tert-butyl ester (compound 16) synthetic
(2.65g 10mmol) is dissolved among the 20mL DMF, adds NaHCO with Boc-Phe-OH
3(1.68g, 20mmol) and CH
3I (1mL, 16mmol), stirring at room 15 hours.In solution, add entry, use ethyl acetate extraction 3 times, organic phase 10%NaSO
3Wash, washing, saturated NaCl washes, MgSO
4Dry.The filtering siccative, the evaporated under reduced pressure solvent, the gained crude product is directly used in the next step without separation.With Boc-Phe-OCH
3(2.79g 10mmol) is dissolved in the mixing solutions of THF/EtOH (16mL/25mL), and 0 ℃ adds CaCl down
2(2.22g, 20mmol) and NaBH
4(1.52g, 40mmol).Slowly rise to room temperature, reacted 5 hours.Solution carefully poured in 0 ℃ the 1M citric acid, with ethyl acetate extraction three times, the saturated NaHCO of organic phase
3Wash, saturated NaCl washes, anhydrous Na SO
4Dry.The filtering siccative, with the organic phase evaporate to dryness, the remaining solid recrystallization from ethyl acetate/petroleum ether gets 2.31g compound 16 (productive rate 92%).White needle-like crystals, mp:92-94 ℃,
1H?NMR(300MHz，CDCl
3)δ1.41(s，9H，Boc)，2.52(br?s，1H，OH)，3.51-3.67(m，2H，CH
2)，3.82(s，1H，CH)，4.83(s，1H，NH)，7.20-7.32(m，5H，Ph)。
(S, E)-4-(ethoxycarbonyl)-1-phenyl fourth-3-alkene-2-aminocarbamic acid tert-butyl ester (compound 17) synthetic
(2.50g 10mmol) is dissolved in the 30mL methylene dichloride, adds Et with compound 16
3(4.2mL, 30mmol) ,-10 ℃ add PySO down to N
3(4.77g, 30mL DMSO solution 30mmol), room temperature reaction 30 minutes.Reaction solution poured in 0 ℃ the saturated NaCl solution, with 0 ℃ Et
2O extraction 3 times, organic phase with 10% lemon pickling is once washed once, and saturated NaCl solution is washed once anhydrous sodium sulfate drying.The filtering siccative, solvent evaporated obtains oily liquids, is directly used in next step reaction without separation.The oily liquids that previous step is obtained is dissolved in toluene, adds Ph
3(3.48g, 10mmol), 80 ℃ were reacted 1 hour PCHCOOEt.Reaction is directly mixed the sample upper prop after finishing.Ethyl acetate/petroleum ether=1/10 gets 2.87g compound 17 (productive rate 90%) to 1/8 separation.White needle-like crystals, mp:67-69 ℃,
1H?NMR(300MHz，CDCl
3)δ1.25(t，J＝7.2Hz，3H，CH
3)，1.39(s，9H，Boc)，2.89(m，2H，PhCH
2)，4.18(q，J＝7.2，2H，CH
2)，4.51(br?s，1H，CH)，4.61(br.s，1H，NH)，5.86(d，J＝15.6Hz，1H，CH)，6.90(dd，J＝15.6Hz，5.1Hz，1H，CH)，7.16-7.34(m，5H，Ph).MS(ESI)，m/e，320.18(M+1)，342.16(M+Na)。
(S)-1-((2R, 3S)-3-(ethoxycarbonyl) epoxy second-2-yl)-2-styroyl t-butyl carbamate (compound 18) synthetic
(319mg 1mmol) is dissolved in 10mL CH with compound 17
2Cl
2In, (920mg 4mmol), reacted 5 days to add m-CPBA under the room temperature.Organic phase Et after reaction finishes
2The O dilution is with the 10%Na of ice
2SO
3Wash saturated NaHCO
3Wash, saturated NaCl solution is washed anhydrous Na SO
4Dry.The filtering siccative, solvent evaporated, column chromatography for separation (ethyl acetate/petroleum ether=1/8 is to 1/6) gets 190mg compound 18 (productive rate 57%).White solid, mp:52-53 ℃,
1H?NMR(300MHz，CDCl
3)δ1.28(t，J＝7.5Hz，3H，CH
3)，1.39(s，9H，Boc)，2.82-3.00(m，2H，PhCH
2)，3.26(d，1H，CH)，3.30(dd，1H，CH)，4.21(t，J＝7.5Hz，CH
2)，4.25(m，1H，CH)，4.50(d，1H，NH)，7.20-7.33(m，5H，Ph).MS(ESI)，m/e，336.18(M+1)，358.16(M+Na)，374.14(M+K)。
(2S, 3R, 4R)-4-(ethoxycarbonyl)-4-(benzyl amino)-3-hydroxyl-1-phenyl fourth-2-aminocarbamic acid tert-butyl ester (compound 19) synthetic
(335mg 1mmol) is dissolved among the anhydrous EtOH of 5mL, and (0.22mL, 2mmol), 60 ℃ were reacted 2 days to add benzylamine with compound 18.Add silica gel mixed sample after reaction finishes, column chromatography for separation (ethyl acetate/petroleum ether=1/5) gets 386mg compound 19 (productive rate 87.5%).Colourless oil liquid.
1H?NMR(300MHz，CDCl
3)δ1.23(t，J＝7.2Hz，3H，CH
3)，1.37(s，9H，Boc)，2.58(br?s，1H，OH)，2.82-2.93(m，2H，PhCH
2)，3.31(d，J＝8.4Hz，1H，CH)，3.59and?3.78(AB，2H，PhCH
2NH)3.70(d，J＝7.8Hz，1H，PhCH
2NH)，4.04(q，J＝7.8Hz，1H，CH)，4.18(q，J＝7.2Hz，2H，CH
2)，4.80(d，J＝9.6Hz，1H，NH)，7.17-7.33(m，10H，Ph).MS(ESI)，m/e，443.24(M+1)，465.25(M+Na).
(2R, 3S, 4S)-2-(benzyl ammonia)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-5-phenylpentanoic acid (compound 21) synthetic
(442mg 1mmol) was dissolved among the 5mL THF, adds the NaOH of 1.1mL 1N, room temperature reaction 10 hours with compound 19.HCl with 1N is adjusted to 2-3 with the pH value then, uses ethyl acetate extraction 4-5 time, organic phase anhydrous Na SO
4Dry.The filtering siccative, evaporated under reduced pressure solvent, gained crude product compound 21 are directly used in the next step without separation.
2, (2R, 3S, 4S)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-2-(phenylamino)-5-phenylpentanoic acid (compound 22) synthetic
(2S, 3R, 4R)-4-(ethoxycarbonyl)-3-hydroxyl-1-phenyl-4-(phenylamino) fourth-2-aminocarbamic acid tert-butyl ester (compound 20) synthetic
(335mg 1mmol) is dissolved among the anhydrous EtOH of 5mL, and (0.18mL, 2mmol), 60 ℃ were reacted 2 days to add aniline with compound 18.Add silica gel mixed sample after reaction finishes, column chromatography for separation (ethyl acetate/petroleum ether=1/4) gets 257mg compound 20 (productive rate 60%).Yellow bulk crystals, mp:78-80 ℃,
1H?NMR(300MHz，CDCl
3)δ，1.18(t，3H，CH
3)，1.40(s，9H，Boc)，2.95(m，2H，CH
2Ph)，3.78(m，1H，CH)，3.90(m，1H，CH)，4.05(m，1H，CH)，4.15(m，1H，NH)，4.18(q，2H，CH
2)，4.95(m，1H，NH)，6.62(d，2H，Ph)，6.74(t，1H，Ph)，7.12(t，2H，Ph)，7.10-7.26(m，5H，Ph).MS?ESI)，m/e，429(M+1)，451(M+Na)。
(2R, 3S, 4S)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-2-(phenylamino)-5-phenylpentanoic acid (compound 22) synthetic
(428mg 1mmol) was dissolved among the 5mL THF, adds the NaOH of 1.1mL 1N, room temperature reaction 10 hours with compound 20.HCl with 1N is adjusted to 2-3 with the pH value then, uses ethyl acetate extraction 4-5 time, organic phase anhydrous Na SO
4Dry.The filtering siccative, evaporated under reduced pressure solvent, gained crude product compound 22 are directly used in the next step without separation.
3, (2R, 3S, 4S)-2-(benzyl ammonia)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-5-phenyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl-propyl]-valeramide (compound 23) synthetic
With compound 21 (414mg, 1mmol) (167mg 1mmol) is dissolved among the 5mL DMF with compound 3a, add NMM (0.16mL, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.Filtering siccative, evaporated under reduced pressure solvent, thick product use column chromatography (ethyl acetate/petroleum ether=1/5-1/3), the product that coupled columns divides the back to obtain carries out recrystallization, 141mg compound 23 (productive rate 25%).White crystal, mp:147 ℃-148 ℃.
1H?NMR(300MHz，CDCl
3)δ0.79(d，J＝6.9Hz，3H，CH
3)0.96(d，J＝6.9Hz，3H，CH
3)，1.42(s，9H，Boc)，1.7(br?s，1H，OH)，2.23(m，1H，CH)，2.88-2.97(m，2H，PhCH
2)，3.22(d，J＝9.6Hz，1H，CH)，3.52and?3.72(AB，2H，PhCH
2)，3.66(d，1H，PhCH
2NH)，4.04(m，1H，CH)，5.17(d，J＝19.8Hz，1H，CH)，5.22(dd，J＝5.4Hz，9.3Hz，1H，CH)，5.27(s，1H，NH)，6.56(dd，J＝1.5Hz，3.6Hz，1H，4’-H)，7.16-7.38(m，10H，Ph)，7.36(d，J＝3.6Hz，1H，3’-H)，7.61(d，J＝1.5Hz，1H，5’-H)，8.43(d，1H，NH)，MS(ESI)，m/e：564.25(M+1)，586.24(M+Na)。
4, (2R, 3S, 4S)-2-(benzyl ammonia)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-5-phenyl-N-[1-[(furans-2-yl)-carbonyl]-the 3-methyl butyl]-valeramide (compound 24) synthetic
With compound 21 (414mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF with compound 3b, add NMM (0.16mL, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.Filtering siccative, evaporated under reduced pressure solvent, thick product use column chromatography (ethyl acetate/petroleum ether=1/5-1/3), the product that coupled columns divides the back to obtain carries out recrystallization, 340mg compound 24 (productive rate 60%).White crystal, mp:77-78 ℃,
1HNMR(300MHz，CDCl
3)δ0.88(d，3H，CH
3)，1.00(d，3H，CH
3)，1.11-1.25(m，2H，CH
2)，1.39(s，9H，Boc)，1.58(m，1H，CH)，1.71(br?s，1H，OH)，2.88-2.97(m，2H，CH
2Ph)，3.24(d，1H，CH)，3.52and?3.72(AB，2H，PhCH
2)，3.68(d，1H，PhCH
2NH)，4.05(m，1H，CH)，5.15(d，1H，CH)，5.27(m，1H，CH)，5.31(m，1H，NH)，6.56(dd，J＝1.8Hz，3.6Hz，1H，4’-H)，7.18-7.38(m，10H，Ph)，7.36(d，J＝3.6Hz，1H，3’-H)，7.62(d，J＝1.8Hz，1H，5’-H)，8.40(d，1H，NH)，MS(ESI)，m/e，578.33(M+1)，600.32(M+Na)。
5, (2R, 3S, 4S)-2-(benzyl ammonia)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-5-phenyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl butyl]-valeramide (compound 25) synthetic
With compound 21 (414mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF with compound 3c, add NMM (0.16mL, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.Filtering siccative, evaporated under reduced pressure solvent, thick product use column chromatography (ethyl acetate/petroleum ether=1/5-1/3), the product that coupled columns divides the back to obtain carries out recrystallization, 174mg compound 25 (productive rate 30%).White crystal, mp:67-69 ℃,
1H?NMR(500MHz，CDCl
3)δ，0.82(t，3H，CH
3)，0.92(d，3H，CH
3)，1.25-1.37(m，2H，CH
2)，1.94(m，1H，CH)，2.89-2.99(m，2H，CH
2Ph)，3.21(d，1H，CH)，3.51and?3.72(AB，2H，PhCH
2)，3.67(d，1H，PhNH)，4.04(q，1H，CH)，5.18(m，1H，CH)，5.21(m，1H，CH)，5.23(m，1H，NH)，6.56(dd，J＝1.5Hz，3.0Hz，1H，4’-H)，7.17-7.36(m，10H，Ph)，7.36(d，J＝3.0Hz，1H，3’-H)，7.62(d，J＝1.5Hz，1H，5’-H)，8.38(d，1H，NH).MS(ESI)，m/e：578.33(M+1)，600.32(M+Na)。
6, (2R, 3S, 4S)-2-(phenylamino)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-5-phenyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl-propyl]-valeramide (compound 26) synthetic
With compound 22 (400mg, 1mmol) (167mg 1mmol) is dissolved among the 5mL DMF with compound 3a, add NMM (0.16mL, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.Filtering siccative, evaporated under reduced pressure solvent, thick product use column chromatography (ethyl acetate/petroleum ether=1/3-1/2), the product that coupled columns divides the back to obtain carries out recrystallization, 137mg compound 26 (productive rate 25%).White crystal, mp, 181-182 ℃,
1H?NMR(300MHz，CDCl
3)：δ0.73(d，3H，CH
3)，0.95(d，3H，CH
3)，1.31(s，9H，Boc)，2.20(m，1H，CH)，2.84-2.88(m，2H，PhCH
2)，3.76(m，1H，CH)，3.92(m，1H，CH)，4.01(m，1H，CH)，5.04(m，1H，CH)，5.23(m，1H，NH)，6.55(dd，J＝1.5Hz，3.6Hz，1H，4’-H)，6.57(d，2H，PhNH)，6.62(t，1H，PhNH)，7.06-7.26(m，7H，Ph)，7.26(m，1H，NH)，7.29(d，J＝3.6Hz，1H，3’-H)，7.60(d，J＝1.5Hz，1H，5’-H)，7.80(d，1H，NH).MS(ESI)，m/e，550.27(M+1)，572.25(M+Na)。
7, (2R, 3S, 4S)-2-(phenylamino)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-5-phenyl-N-[1-[(furans-2-yl)-carbonyl]-the 3-methyl butyl]-valeramide (compound 27) synthetic
With compound 22 (400mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF with compound 3b, add NMM (0.16mL, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.Filtering siccative, evaporated under reduced pressure solvent, thick product use column chromatography (ethyl acetate/petroleum ether=1/3-1/2), the product that coupled columns divides the back to obtain carries out recrystallization, 146mg compound 27 (productive rate 26%).White crystal, mp, 206-208 ℃,
1H?NMR(300MHz，CDCl
3)：δ0.86(d，3H，CH
3)，0.99(d，3H，CH
3)，1.32(s，9H，Boc)，1.50-1.53(m，2H，CH
2)，1.55-1.61(m，1H，CH)，2.82-2.87(m，2H，PhCH
2)，3.72(m，1H，CH)，3.97(m，1H，CH)，4.00(m，1H，CH)，5.02(m，1H，CH)，5.35(m，1H，NH)，6.56(dd，J＝1.5Hz，3.6Hz，1H，4’-H)，6.62(d，2H，PhNH)，6.78(t，1H，PhNH)，7.05(m，1H，NH)，7.10-7.31(m，7H，Ph)，7.61(d，J＝3.6Hz，1H，3’-H)，7.72(d，J＝1.5Hz，1H，5’-H)，7.75(d，1H，NH).MS(ESI)，m/e，564.29(M+1)，586.49(M+Na)。
8, (2R, 3S, 4S)-and 2-(phenylamino)-4-[[(1,1-dimethyl oxyethyl group)-carbonyl] amino]-3-hydroxyl-5-phenyl-N-[1-[(furans-2-yl)-carbonyl]-the 2-methyl butyl]-valeramide (compound 28) and synthetic
With compound 22 (400mg, 1mmol) (181mg 1mmol) is dissolved among the 5mL DMF with compound 3c, add NMM (0.16mL, 1.5mmol), HOBt (203mg, 1.5mmol) and EDCHCl (288mg, 1.5mmol), room temperature reaction 12-24 hour (TLC monitoring), stopped reaction, reaction solution dilutes with ethyl acetate, organic phase is with 10% lemon pickling, saturated NaHCO
3Wash, saturated NaCl washes, anhydrous Na
2SO
4Dry.Filtering siccative, evaporated under reduced pressure solvent, thick product use column chromatography (ethyl acetate/petroleum ether=1/3-1/2), the product that coupled columns divides the back to obtain carries out recrystallization, 135mg compound 28 (productive rate 24%).White crystal, mp, 189-190 ℃,
1H?NMR(300MHz，CDCl
3)：δ0.78(t，3H，CH
3)，0.90(d，3H，CH
3)，1.25(m，2H，CH
2)，1.32(s，9H，Boc)，1.93(m，1H，CH)，2.84-2.88(m，2H，PhCH
2)，3.75(m，1H，CH)，3.95(m，1H，CH)，3.99(m，1H，CH)，5.02(m，1H，CH)，5.22(m，1H，NH)，6.57(dd，J＝1.5Hz，3.6Hz，1H，4’-H)，6.63(d，2H，PhNH)，6.79(t，1H，PhNH)，7.06(m，1H，NH)，7.10-7.31(m，7H，Ph)，7.60(d，J＝3.6Hz，1H，3’-H)，7.73(d，J＝1.5Hz，1H，5’-H)，7.76(d，1H，NH).MS(ESI)，m/e，564.29(M+1)，586.26(M+Na)。
Second section compound biological activity determination
1, proteasome suppresses active
Measure the rotten albumen sample activity (ChT-L) of proteasome with fluorescent polypeptide, polypeptide structure is Suc-Leu-leu-Val-Tyr-AMC (Suc represents succinyl, and AMC represents 7-acid amides-4-methylcoumarin, available from SIGMA reagent company).In the Tris-HCl of 100 μ l20mM (pH is 7.8), 1 μ g to be hatched 1 hour jointly from the 20S proteasome of HRBC purifying and the testing compound and the 100 μ M fluorescent polypeptides of various concentration, temperature is 37 ℃.Under excitation/emission wavelength 380/440nm, measure the fluorescence that 7-acid amides-4-methylcoumarin discharges with spectrophotofluorometer (Fluostar OPTIMA, BMG Germany).Using knownly has the positive contrast of inhibiting MG-132 to proteasome ChT-L activity, and 0.1%DMSO is a solvent control.0.5 the inhibiting rate of μ M MG132 is 78%.With reference to the solvent control value, calculate the inhibiting rate of testing compound.The experiment triplicate.Part of compounds result such as table 1:
Table 1 The compounds of this invention suppresses active to proteasome
2, anti-tumour cell proliferative effect:
Suppress the effect of tumor cell proliferation with standard based on the colorimetric method for determining testing compound of MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-phenylbenzene bromination tetrazole) or SRB (sulphonyl rhodamine B).Use MTT and SRB to carry out painted respectively to suspension and adherent cell.Different clones are planted on the 96 hole microtest plates, add 104 attached cell HepG2 (Bel7402) in each hole, and 105 suspension cell HL-60 (people's acute myeloid leukemia clone) prepare 5 blocks of plates respectively.After 18 to 24 hours, the cell that is exponential form growth contacts 48 hours with the testing compound (1-100 μ M) that concentration raises successively, with 0.1%DMSO as solvent control.Add PBS (phosphate buffered saline) solution of 5mg/ml MTT or 0.4% SRB (being dissolved in 1% acetic acid) 20 μ l then.After hatching 4 hours, abandon substratum,, add 200 μ l acid isopropyls alcohol or 1% acetic acid then at air drying, with the dissolving crystal, under 570nm and 540nm, measure MTT or the painted optical density of SRB respectively with the multiple detection readout instrument of FLUOstar OPTIMA microtest plate (BMG Germany).Collect data and processing data with computer at last.Experiment repeats 3 times.
The result: the anti-human liver cancer cytoactive of compound 12 is IC
50=34.2 μ M, anti-people's acute myeloid leukemia cytoactive is IC
50=37.07 μ M.
Claims (4)
1, the compound of formula I structure,
(formula I)
Wherein, Boc is a tertbutyloxycarbonyl.
2, the preparation method of the described compound of claim 1 comprises the steps:
1) amino acid that has the tertbutyloxycarbonyl protection of formula II structure is under the effect of peptide condensation reagent, and with N, the reaction of O-dimethyl hydroxylamine hydrochloride obtains the formula III structural compounds;
2) the formula III structural compounds reacts with furans under the organolithium reagent effect, obtains formula IV compound;
3) formula IV compound removes tertbutyloxycarbonyl, obtains formula V compound;
4) amino acid with formula VI structure is dissolved in the anhydrous methanol, under-10 ℃ to wherein adding thionyl chloride, room temperature reaction 5 hours, solvent evaporated, remaining solid methanol recrystallization obtains the amino acid methyl ester of formula VII structure;
5) amino acid that has protecting group of formula VIII structure and the amino acid methyl ester of formula VII structure are reacted under the effect of peptide condensation reagent, obtain two peptide prods of formula IX structure; Hydrolysis under this two peptide prods alkaline condition obtains the compound of formula X structure;
6) compound and the formula V compound with formula X structure reacts under the effect of peptide condensation reaction reagent, obtains the compound shown in the formula I;
(formula II) (formula III) (formula IV) (formula V) (formula VI)
(formula VII) (formula VIII) (formula IX) (formula X)
Wherein, R
1Be the 2-methyl-propyl; R
2, R
3Be benzyl; Pg is a tertbutyloxycarbonyl.
3, preparation method according to claim 2 is characterized in that: described peptide condensation reagent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, I-hydroxybenzotriazole or isobutyl chlorocarbonate.
4, the application of the described compound of claim 1 in the preparation antitumor drug.
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Non-Patent Citations (6)
| Title |
|---|
| Modeling of the binding mode of a non-covalentinhibitor of the20S proteasome. Application tostructure-based analoguedesign. Furet P, et al.Bioorganic & Medicinal Chemistry Letters,Vol.11 No.10. 2001 |
| Modeling of the binding mode of a non-covalentinhibitor of the20S proteasome. Application tostructure-based analoguedesign. Furet P, et al.Bioorganic & Medicinal Chemistry Letters,Vol.11 No.10. 2001 * |
| Proteasome inhibitors: from research tools to drug candidates. Kisselev A.F., Goldberg A.L.Chemistry & Biology,Vol.8 No.8. 2001 |
| Proteasome inhibitors: from research tools to drug candidates. Kisselev A.F., Goldberg A.L.Chemistry & Biology,Vol.8 No.8. 2001 * |
| The synthesis of two furan-based analogues of the α',β'-epoxy ketone proteasome inhibitor eponemycin. Bennacer B, et al.Eur. J. Org. Chem.,Vol.2003 No.23. 2003 |
| The synthesis of two furan-based analogues of the α',β'-epoxy ketone proteasome inhibitor eponemycin. Bennacer B, et al.Eur. J. Org. Chem.,Vol.2003 No.23. 2003 * |
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