CN105669749B

CN105669749B - Antiviral drugs and composition

Info

Publication number: CN105669749B
Application number: CN201610257085.0A
Authority: CN
Inventors: 杨东元; 夏桂民; 韩洁
Original assignee: Hess (xi'an) Biological Technology Co Ltd
Current assignee: Hess (xi'an) Biological Technology Co Ltd
Priority date: 2014-08-14
Filing date: 2014-08-14
Publication date: 2018-03-30
Anticipated expiration: 2034-08-14
Also published as: CN105669749A

Abstract

The present invention relates to antiviral drugs and composition, more particularly to the medicine and its pharmaceutical composition of anti-human HIVvirus blood serum immunity (HIV), more particularly to a kind of medicine as retroviral Protease inhibitors, the retroviral Protease inhibitors have the chemical constitution shown in Formulas I.

Description

Antiviral drugs and composition

The application is the divisional application for the Chinese Patent Application No. 2014103999347 that August in 2014 is submitted on the 14th.

Technical field

The present invention relates to a kind of antiviral drugs and its pharmaceutical composition, and in particular to anti-human HIVvirus blood serum immunity (HIV) medicine and its pharmaceutical composition, should more particularly to a kind of medicine as retroviral Protease inhibitors Retroviral Protease inhibitors have the chemical constitution shown in Formulas I.

Background technology

HIV (HIV) is a kind of to cause AIDS and its pathogenicity retroviruse of relevant diseases.By In HIV discovery, resist AIDS antiviral chemotherapeutic development turns into positive research emphasis.Molecule mesh about AIDS Target research referring to Mitsua et al. document (Science, 1990, pp.1533-154).Hiv protease (HIV PR) and day Door winter amine acyl group protease is considered that AIDS is treated by carat agate (Kramer) et al. (Science 231,1580 (1986)) first The possibility target of method.Hereafter, i.e., widely confirm HIV PR inhibitor when treating AIDS as the potential of effective preparation Purposes, the medical application about HIV PR referring to Tuo Maxili (Tomaselli) et al. document (Chimica, Oggi, 1991 Year May, pp.6-27) and the J.P. not documents (J.Med.Chem.34,2341-2327 (1991)) of (Huff) et al. recklessly.Passing In the transition state of system simulation lucid asparagus amine acyl group protease, hydroxy vinyl, dihydroxy ethylene, aminoethyle alcohol and phosphinic acids electronics Isostere (Bostere) seems there is maximum affinity with HIV PR.Many HIV PR inhibitor is in different cell systems In, antiviral activity is presented in the concentration of nanomole scope, and it is stated that in the patent literature.

Main function of the hiv protease in virus replication is that gag and gag-pol gene outcomes are cracked into virus Structural proteins (matrix, shell, nucleocapsid) and enzyme (protease, integrase, reverse transcriptase) required for maturation, so as to further complete Kind virus structure.Protease inhibitors (PI) is exactly to prevent precursor protein from cracking, and causes the accumulation of no infecting virus particle.

Pharmacophoric conformation research shows, the mechanism of action of this kind of compound be mainly in a manner of hydrogen is strong respectively with protease Asp 25, Cly 27 and the interaction of the residues of Asp 29, body phase interaction is formed with the amino acid residue in proteinase activity group With so as to reach the purpose of protease inhibition activity, and then having checked the duplication of AIDS virus, reached antivirus action.Egg White enzyme inhibitor can play antivirus action into the Various Tissues organ including peripheral blood, thus curative effect is deeply held Long.

At present, have in the hiv protease inhibitor of wide clinical application：Inverase (saquinavir), indenes ground that Wei (indinavir), Ritonavir (ritonavir), Nai Feinawei (nelfinavir), VX-478 (amprenavir) and Lopinavir (lopinavir).There are within 2003 two new protease inhibitors of atazanavir and fosamprenavir to lead to again The examination ＆ approval of U.S. FDA are crossed.The atazanavir researched and developed by Bristol-Myers Squibb companies is that one kind only needs to take daily With brand-new protease inhibitors once.It influences very small, the secondary works such as patient does not suffer from diarrhoea on insulin and fat metabolism With.Bilirubin level can slightly be increased, jaundice etc. occur in few patients, but therefore patient seldom discontinues medication.

Although these protease inhibitors can suppress HIV duplications, internal virus can not be utterly destroyed, can not be prevented The appearance and propagation of internal resistance strain.Not only the quantity of virus is decreased obviously in vivo after protease inhibitors PI applications, simultaneously T helper cell-cd4 cell then increased in lymphocyte.Then these long-term use of protease inhibitors produce obvious Toxic side effect and drug resistance.Disorders of lipid metabolism is most prominent and most complicated in a variety of side effects, and clinical manifestation is for face and outside All fat consumptions, abdomen, the back of the body, breast fat accumulation.There is hypertriglyceridemia and hypercholesterolemia, lactic acid and blood glucose in patient Rise, drug resistance etc. is produced to insulin.Next to that drug resistance, especially cross resistance are cause clinical treatment failure one Individual main cause.Research shows that the mechanism that PI resistances occur is more more complicated than NRIT and NNRTI, often relates to multiple spot gene mutation. In general, the single mutation in protease substrate land, can make the tolerance of virus-drug increase by 10 times.If protease S1 is sub- 82, area valine or 84 isoleucines are substituted, then tolerance can increase to 30 times, if 82 and 84 residue simultaneous mutations, Medication effect can be made to decline 100 times.Make high dose PI can be than the generation using low dosage PI delay drug resistances, with ucleosides The use in conjunction of medicine can also reduce the generation of drug resistance.Also the generation of drug resistance is also due to high virus replication rate, inverse Error of the transcriptase in process of reverse-transcription and the evolution result under the selectively acting of protease inhibitors.Other PI it answers Miscellaneous instructions of taking, angiocardiopathy excessive risk make the patient that it is not suitable for poor compliance, diabetes, angiocardiopathy.

Known with compounds of Formula I is a kind of effective retroviral Protease inhibitors

Its molecular formula：C₃₂H₄₁N₆Na₂O₉P, molecular weight 730.66, its typical English name are properly termed as：t-Butyl 3- isopropyl-3-[(2S,3S)-2-Phosphonooxy-3-(N-quinaldoyl-L-asparaginyl)amino-4- Phenylbutylcarbazate Disodium, typical Chinese name can be described as：3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic oxygen Base -3- (N- quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl tert-butyl carbazate disodium, or it is typical in Literary fame can be described as：3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxides -3- (N- quinoline acyl group-L- lucid asparagus acyl group) amino -4- Phenyl butyl tert-butyl carbazate disodium.

2 of butyl be connected in the structural formula of compound of formula I between phenyl and isopropyl and 3 carbon are hand Property carbon.

Although pharmaceutical work person has stronger ability to be realized in pharmaceutical producing enterprise to bulk drug in Drug's control Effective control；But after material medicine is made into preparation, circulate field, and enter clinical practice in when, it is non- Manufacturing enterprise personnel (including logistics personnel, medical personnel, patient etc.) can weaken significantly to the inherent control ability of preparation.

Therefore it provides a kind of be possessed of good qualities for example with the product for stablizing pharmaceutical characteristic, particularly compound of formula I Bulk drug or the preparation being made from it, it is still those skilled in the art's desirable.

The content of the invention

It is possessed of good qualities for example with the product for stablizing pharmaceutical characteristic, is particularly it is an object of the invention to provide a kind of The bulk drug of compound of formula I or the preparation being made from it.The present inventor is it has been unexpectedly discovered that by by Formulas I chemical combination When impurity Ib in the bulk drug of thing is controlled below certain limit, the bulk drug and conventional pharmaceutic adjuvant particularly carbohydrate group When closing so that pharmaceutical preparation is made, it is non-that there is this kind of pharmaceutical preparation the particularly impurity Ia growth of excellent stability can maintain In often low scope.The present invention is accomplished based on this discovery.

Therefore, first aspect present invention provides a kind of Medicinal crude drug, its active component is with compounds of Formula I：

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, wherein containing more than 97% Formulas I chemical combination Thing, such as containing more than 97.5% compound of formula I, such as contain more than 98% compound of formula I.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, wherein containing 97%~103% Formulas I Compound, such as containing the compound of formula I on 97.5%~102.5%, such as contain 98%~102% compound of formula I.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, wherein also contain (such as trace) conduct Impurity is existing with following formula I a compounds：

Formulas I a compounds are the compounds of the non-Phosphation of compound of formula I, its molecular formula：C₃₂H₄₂N₆O₆, molecular weight 606.72.And the molecular formula of compound of formula I：C₃₂H₄₁N₆Na₂O₉P, molecular weight 730.66.

In the present invention, Formulas I a compounds be also referred to as impurity Ia either can be described as Ia or can be described as Ia impurity or its It is similar to appellation.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, wherein for compound of formula I, formula The content of Ia compounds is less than 2%, is, for example, less than 1.75%, is, for example, less than 1.5%, is, for example, less than 1.25%, is, for example, less than 1.0%, it is, for example, less than 0.75%.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, wherein for compound of formula I, formula The content of Ia compounds is 0.002~2%, for example, 0.002~1.75%, for example, 0.002~1.5%, for example, 0.002 ~1.25%, for example, 0.002~1.0%, for example, 0.002~0.75%.

In the present invention, phrase refers to for involved thing " for compound of formula I, the content of Formulas I a compounds " In material, amount of the Formulas I a compound phases for compound of formula I.If such as wherein including 100mg Formulas I chemical combination in a certain material Thing, wherein also include 0.5mg Formulas I a compounds after measured, then for compound of formula I, the amount of Formulas I a compounds is 0.5%.Also there is similar meaning for similar statement of other impurities phases for the amount of compound of formula I.Above-mentioned " content " can also lead to Cross the present invention【HPLC-A】Method determines to obtain.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I a compound peaks area and compound of formula I peak area is less than 2%, is, for example, less than 1.75%, is, for example, less than 1.5%, it is, for example, less than 1.25%, is, for example, less than 1.0%, is, for example, less than 0.75%.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I a compound peaks area and compound of formula I peak area is 0.002~2%, for example, 0.002~1.75%, example Such as it is 0.002~1.5%, for example, 0.002~1.25%, for example, 0.002~1.0%, for example, 0.002~0.75%.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I a compound peaks areas is more than 50, is greater than 57, is greater than 67, such as greatly In 80,100 are greater than, is greater than 133.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I a compound peaks areas is 50~50000, for example, 57~50000, for example, 67 ~50000, for example, 80~50000, for example, 100~50000, for example, 133~50000.

It has been found that behavioural characteristic is said from pharmacy angle and even can not far away from compound of formula I in Formulas I a compound bodies Receive, therefore (and in pharmaceutical composition of second aspect of the present invention) will in the Medicinal crude drug of first aspect present invention The amount control of Formulas I a compounds is necessary below a certain amount of.Such as it may be referred to control limit of this area typically to impurity Degree requires<1%, such as<0.75%).

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, wherein also contain (such as trace) conduct Impurity is existing with following formula I b compounds：

Formulas I b compounds are the phosphinates of Formulas I a compounds.

In the present invention, Formulas I b compounds be also referred to as impurity Ib either can be described as Ib or can be described as Ib impurity or its It is similar to appellation.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, wherein for compound of formula I, formula The content of Ib compounds is less than 1%, is, for example, less than 0.75%, is, for example, less than 0.5%, is, for example, less than 0.4%, is, for example, less than 0.3%, it is, for example, less than 0.25%.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, wherein for compound of formula I, formula The content of Ib compounds is 0.002~1%, for example, 0.002~0.75%, for example, 0.002~0.5%, for example, 0.002 ~0.4%, for example, 0.002~0.3%, for example, 0.002~0.25%.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I b compound peaks area and compound of formula I peak area is less than 1%, is, for example, less than 0.75%, is, for example, less than 0.5%, it is, for example, less than 0.4%, is, for example, less than 0.3%, is, for example, less than 0.25%.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I b compound peaks area and compound of formula I peak area is 0.002~1%, for example, 0.002~0.75%, example Such as it is 0.002~0.5%, for example, 0.002~0.4%, for example, 0.002~0.3%, for example, 0.002~0.25%.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I b compound peaks areas is more than 100, is greater than 133, is greater than 200, such as More than 250,333 are greater than, is greater than 400.

The Medicinal crude drug of any embodiment according to a first aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I b compound peaks areas is 100~50000, for example, 133~50000, is, for example, 200~50000, for example, 250~50000, for example, 333~50000, for example, 400~50000.

It has been found that when the first aspect present invention Medicinal crude drug containing a certain amount of Formulas I b compounds is some normal with being used as Advise pharmaceutic adjuvant particularly carbohydrate mix when, wherein impurity Formulas I a compounds can with material storage time extend and occur with The relevant growth trend of Formulas I b compounds contents.That is, when Medicinal crude drug mixes with such pharmaceutic adjuvant, the Medicinal crude drug In Formulas I b compounds when being up to a certain amount of above, the more high then Formulas I a compounds of Formulas I b chemical combination object amount increase bigger；And by the medicine With the Formulas I b compounds control in bulk drug when a certain amount of following, when the Medicinal crude drug mixes with above-mentioned pharmaceutic adjuvant its The growth unobvious of compound of formula la.This is completely unexpected, and has significant pharmacy meaning, because as one Kind of Medicinal crude drug, particularly when being made into the pharmaceutical formulation of oral administration, carbohydrate for example lactose, mannitol, sorbierite, Sucrose etc. is valuable for preparing such oral Preparation；And when the amount of the Formulas I b compounds in the Medicinal crude drug When a certain amount of following, the oral formulations made by it are with the extension of storage time, the Formulas I a compounds as impurity for control Will not significantly it increase, this is for being extremely beneficial to a kind of clinical good preparation of security that provides.But when this is medicinal The higher amount of Formulas I b compounds in bulk drug is ground, and the Medicinal crude drug can not prepare oral formulations together with above-mentioned auxiliary material, This will limit the range of choice of Medicinal crude drug auxiliary material in preparation of preparation significantly, because this kind of auxiliary material is cheap on galenic pharmacy And handy auxiliary material.

According to the present invention, wherein described【HPLC-A】Method is a kind of high performance liquid chromatography, and it can be used for determining simultaneously respectively Formulas I chemical combination in kind of material (including the Medicinal crude drug of the present invention and pharmaceutical composition made of the Medicinal crude drug) Thing, Formulas I a compounds, the content of Formulas I b compound threes.

According to the present invention, it is related to【HPLC-A】Method is specific as follows：

(i) chromatographic condition：

Carried out according to the contained high performance liquid chromatographies of two annex V D of Chinese Pharmacopoeia version in 2010,

Chromatographic column：For the chromatographic column of octadecylsilane chemically bonded silica, [chromatographic column is typical C to stationary phase₁₈Chromatographic column, It easily can buy from commercial channels, such as Diamonsil C₁₈Post, Phenomenex Gemini C₁₈Post etc., at this In the specific experiment of invention hereafter, if not otherwise indicated, Diamonsil C are used₁₈Chromatographic column, its specification can be 250 × 4.6mm, 5 μm],

Flow velocity：1.0ml/min

Mobile phase A：0.01mol/L potassium dihydrogen phosphates (adjust pH to 3.0) with potassium hydroxide solution, Mobile phase B：Second Nitrile,

Linear gradient elution program：

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	80	20
20	20	80
			30	20	80

Detection wavelength：237nm,

Column temperature：25 DEG C,

Sample size：20 μ l,

(ii) liquid is matched somebody with somebody

Test liquid：Taking the test sample of the 100mg containing compound of formula I, [test sample can be the medicinal raw material of compound of formula I Medicine, then now sample weighting amount is about 100mg；The test sample can also be to be prepared using the Medicinal crude drug of compound of formula I for raw material Into pharmaceutical composition such as pharmaceutical preparation, then the amount comprising compound of formula I is about 100mg in the material now weighed], it is accurate It is weighed, put in 100ml measuring bottles, add 50% acetonitrile solution to dissolve and be diluted to scale, shake up, filter if necessary, produce, as confession Test solution (its compounds of formula I concentration about 1000 μ g/ml)；

Contrast solution：Precision is drawn test liquid 1ml and put in 100ml measuring bottles, adds 50% acetonitrile solution to be diluted to scale, shakes It is even；Precision measures solution 10ml and put in 100ml measuring bottles, adds 50% acetonitrile solution to be diluted to scale, shakes up, produce, as right According to solution (its compounds of formula I concentration about 1 μ g/ml)；

Ia solution：Modus ponens Ia compounds 10mg, it is accurately weighed, put in 100ml measuring bottles, add 50% acetonitrile solution to dissolve and dilute Release to scale, shake up (referred to here as Ia liquid, about 100 μ g/ml)；Precision measures solution 10ml and put in 100ml measuring bottles, adds 50% Acetonitrile solution is diluted to scale, shakes up, and produces, as Ia solution (the wherein μ g/ml of Formulas I a compound concentrations about 10)；

Ib solution：Modus ponens Ib compounds 10mg, it is accurately weighed, put in 100ml measuring bottles, add 50% acetonitrile solution to dissolve and dilute Release to scale, shake up (referred to here as Ib liquid, about 100 μ g/ml)；Precision measures solution 10ml and put in 100ml measuring bottles, adds 50% Acetonitrile solution is diluted to scale, shakes up, and produces, as Ib solution (the wherein μ g/ml of Formulas I b compound concentrations about 10)；

System suitability solution：It is accurate respectively to draw test liquid, 100 μ g/ml Ia liquid, 100 μ g/ml each 1ml of Ib liquid, It is placed in same 10ml measuring bottle, adds 50% acetonitrile solution to be diluted to scale, shake up, produces (the μ g/ of 100 μ g/ml+Ia of I 10 ml+Ib 10μg/ml)；

(iii) measure and result calculate：

Precision draws test liquid, contrast solution, Ia solution, Ib solution, each 20 μ L of system suitability solution and is injected separately into liquid Chromatography, chromatogram is recorded respectively；

Peak belongs to and retention time：Formulas I chemical combination is determined according to chromatogram obtained by contrast solution, Ia solution, Ib solution three Thing, Formulas I a compounds, the retention time of Formulas I b compound threes, and determine therefrom that each peak in system suitability solution chromatogram The ownership of each chromatographic peak is (in general, appearance is suitable in chromatogram obtained by the system suitability solution in ownership and test liquid Sequence is followed successively by compound of formula I, Formulas I b compounds, Formulas I a compounds)；

System suitability：In the chromatogram obtained by system suitability solution testing, compound of formula I and Formulas I b chemical combination Separating degree between thing peak is at least 1.5；

In test liquid chromatogram, if detecting Formulas I a compounds, it can be used to lower calculating formula and calculate impurity Ia's Content：

Above-mentioned term " impurity Ia contents (%) " its implication is equivalent to present invention phrase described above " relative to Formulas I chemical combination For thing, the content of Formulas I a compounds ".

In test liquid chromatogram, if detecting Formulas I b compounds, it can be used to lower calculating formula and calculate impurity Ib's Content：

Above-mentioned term " impurity Ib contents (%) " its implication is equivalent to present invention phrase described above " relative to Formulas I chemical combination For thing, the content of Formulas I b compounds ".

In test liquid chromatogram, if detecting Formulas I a compounds, lower calculating formula calculating formula Ia compounds are can be used to The ratio (%) of peak area and compound of formula I peak area：

In test liquid chromatogram, if detecting Formulas I b compounds, lower calculating formula calculating formula Ib compounds are can be used to The ratio (%) of peak area and compound of formula I peak area：

Or in the present invention, " ratio of compound of formula I peak area and Formulas I a compound peaks areas " and " Formulas I can also be used The ratio of compound peaks area and Formulas I b compound peaks areas " expression Ia compounds and Formulas I b compound phases are for compound of formula I Amount.

In test liquid chromatogram, if detecting Formulas I a compounds, lower calculating formula calculating formula Ia compounds are can be used to The ratio of peak area and compound of formula I peak area：

In test liquid chromatogram, if detecting Formulas I b compounds, lower calculating formula calculating formula Ib compounds are can be used to The ratio of peak area and compound of formula I peak area：

In addition, pass through the above【HPLC-A】Method, (it can also be used as by preparing the compound of formula I reference substance of suitable concentration Analysis reference substance, such as its content are more than 99.8%) solution, pass through the compound of formula I content in external standard method test sample (absolute content, i.e., the quality of every gram test sample compounds of formula I, %, w/w).

It is above-mentioned【HPLC-A】" test sample " of method test, this material both can be the medicines provided in the form of compound of formula I Can also be the medicine group being formulated using them as active component and added with other pharmaceutic adjuvants with bulk drug or its crude product Compound, such as pharmaceutical preparation, such as tablet etc., whether Medicinal crude drug or crude product or pharmaceutical composition, in test liquid The amount of the about 100mg containing compound of formula I, impurity Ia and impurity Ib contents are converted to when test sample is weighed in process for preparation Calculate and will not be affected because of the presence that test sample weighs accuracy or other compositions such as pharmaceutic adjuvant.

In addition, in view of analysis method is readily available improved, and Formulas I a compounds, Formulas I b compounds are to be readily available 's.Therefore various materials such as Medicinal crude drug or its crude product are described in the present invention or the pharmaceutical composition that is prepared into by it in Formulas I a compounds, Formulas I b compounds contents when, without needing to limit whether it must shine【HPLC-A】Method tests obtained knot Fruit, particularly in content of the impurities phase in describing test sample for compound of formula I.

Further, second aspect of the present invention provides a kind of pharmaceutical composition, and it is any by second aspect of the present invention What Medicinal crude drug described in embodiment and pharmaceutic adjuvant were prepared together.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, its compounds of formula I account for the drug regimen The 10~90% of thing gross weight, such as 20~80%, such as 30~70%.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, wherein also contain (such as trace) conduct Impurity is existing with following formula I a compounds：

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, wherein for compound of formula I, formula The content of Ia compounds is less than 2%, is, for example, less than 1.75%, is, for example, less than 1.5%, is, for example, less than 1.25%, is, for example, less than 1.0%, it is, for example, less than 0.75%.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, wherein for compound of formula I, formula The content of Ia compounds is 0.002~2%, for example, 0.002~1.75%, for example, 0.002~1.5%, for example, 0.002 ~1.25%, for example, 0.002~1.0%, for example, 0.002~0.75%.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I a compound peaks area and compound of formula I peak area is less than 2%, is, for example, less than 1.75%, is, for example, less than 1.5%, it is, for example, less than 1.25%, is, for example, less than 1.0%, is, for example, less than 0.75%.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I a compound peaks area and compound of formula I peak area is 0.002~2%, for example, 0.002~1.75%, example Such as it is 0.002~1.5%, for example, 0.002~1.25%, for example, 0.002~1.0%, for example, 0.002~0.75%.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I a compound peaks areas is more than 50, is greater than 57, is greater than 67, such as greatly In 80,100 are greater than, is greater than 133.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I a compound peaks areas is 50~50000, for example, 57~50000, for example, 67 ~50000, for example, 80~50000, for example, 100~50000, for example, 133~50000.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, wherein also contain (such as trace) conduct Impurity is existing with following formula I b compounds：

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, wherein for compound of formula I, formula The content of Ib compounds is less than 1%, is, for example, less than 0.75%, is, for example, less than 0.5%, is, for example, less than 0.4%, is, for example, less than 0.3%, it is, for example, less than 0.25%.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, wherein for compound of formula I, formula The content of Ib compounds is 0.002~1%, for example, 0.002~0.75%, for example, 0.002~0.5%, for example, 0.002 ~0.4%, for example, 0.002~0.3%, for example, 0.002~0.25%.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I b compound peaks area and compound of formula I peak area is less than 1%, is, for example, less than 0.75%, is, for example, less than 0.5%, it is, for example, less than 0.4%, is, for example, less than 0.3%, is, for example, less than 0.25%.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I b compound peaks area and compound of formula I peak area is 0.002~1%, for example, 0.002~0.75%, example Such as it is 0.002~0.5%, for example, 0.002~0.4%, for example, 0.002~0.3%, for example, 0.002~0.25%.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I b compound peaks areas is more than 100, is greater than 133, is greater than 200, such as More than 250,333 are greater than, is greater than 400.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I b compound peaks areas is 100~50000, for example, 133~50000, is, for example, 200~50000, for example, 250~50000, for example, 333~50000, for example, 400~50000.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, wherein described pharmaceutic adjuvant includes being selected from Following carbohydrate：Lactose, mannitol, sorbierite, sucrose etc..

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is in tablet, capsule or granule that it, which is, Form.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is the pharmaceutical preparation shape in unit dose Formula.In one embodiment, each unit dose of the pharmaceutical preparation includes 1~1000mg compound of formula I, such as Compound of formula I including 10~750mg, such as the compound of formula I including 20~500mg.

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, its be in the form of tablet or capsule, Wherein also optionally include the conventional diluent of pharmacy (be such as, but not limited to starch and its derivative such as pregelatinized starch, change Good starch, microcrystalline cellulose etc.), disintegrant (such as but unlimited PVPP, sodium carboxymethyl starch etc.), adhesive (such as But be not limited to PVP, HPMC etc.), lubricants/glidants (such as, but not limited to magnesium stearate, talcum powder etc.) etc..

The pharmaceutical composition of any embodiment according to a second aspect of the present invention, it is sealed under the conditions of 40 DEG C, lucifuge is put Put 5 months, determine and calculate on this condition dispose 5 months after certain impurities phase at 0 month content increase percentage, wherein According to【HPLC-A】Measure, impurity Ia contents increase percentage is less than 200%, such as less than 150%, such as less than 100%, such as Less than 75%, such as less than 50%.The test method may be simply referred to as investigation in 40 DEG C-May in the present invention.Described term is miscellaneous The content increase percentage of matter calculates according to following formula：

Above-mentioned calculating formula can be used for calculating various samples in the forward and backward impurity Ia or impurity Ib increases of experience disposal in 40 DEG C-May Percentage amounts, the value shows that the impurity increment is smaller closer to 0.

Further, third aspect present invention provides a kind of pharmaceutical composition, including as active component as Compounds of Formula I

And pharmaceutic adjuvant.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, its compounds of formula I account for the drug regimen The 10~90% of thing gross weight, such as 20~80%, such as 30~70%.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, wherein also contain (such as trace) conduct Impurity is existing with following formula I a compounds：

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, wherein for compound of formula I, formula The content of Ia compounds is less than 2%, is, for example, less than 1.75%, is, for example, less than 1.5%, is, for example, less than 1.25%, is, for example, less than 1.0%, it is, for example, less than 0.75%.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, wherein for compound of formula I, formula The content of Ia compounds is 0.002~2%, for example, 0.002~1.75%, for example, 0.002~1.5%, for example, 0.002 ~1.25%, for example, 0.002~1.0%, for example, 0.002~0.75%.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I a compound peaks area and compound of formula I peak area is less than 2%, is, for example, less than 1.75%, is, for example, less than 1.5%, it is, for example, less than 1.25%, is, for example, less than 1.0%, is, for example, less than 0.75%.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I a compound peaks area and compound of formula I peak area is 0.002~2%, for example, 0.002~1.75%, example Such as it is 0.002~1.5%, for example, 0.002~1.25%, for example, 0.002~1.0%, for example, 0.002~0.75%.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I a compound peaks areas is more than 50, is greater than 57, is greater than 67, such as greatly In 80,100 are greater than, is greater than 133.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I a compound peaks areas is 50~50000, for example, 57~50000, for example, 67 ~50000, for example, 80~50000, for example, 100~50000, for example, 133~50000.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, wherein also contain (such as trace) conduct Impurity is existing with following formula I b compounds：

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, wherein for compound of formula I, formula The content of Ib compounds is less than 1%, is, for example, less than 0.75%, is, for example, less than 0.5%, is, for example, less than 0.4%, is, for example, less than 0.3%, it is, for example, less than 0.25%.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, wherein for compound of formula I, formula The content of Ib compounds is 0.002~1%, for example, 0.002~0.75%, for example, 0.002~0.5%, for example, 0.002 ~0.4%, for example, 0.002~0.3%, for example, 0.002~0.25%.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I b compound peaks area and compound of formula I peak area is less than 1%, is, for example, less than 0.75%, is, for example, less than 0.5%, it is, for example, less than 0.4%, is, for example, less than 0.3%, is, for example, less than 0.25%.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, Wherein the ratio of Formulas I b compound peaks area and compound of formula I peak area is 0.002~1%, for example, 0.002~0.75%, example Such as it is 0.002~0.5%, for example, 0.002~0.4%, for example, 0.002~0.3%, for example, 0.002~0.25%.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I b compound peaks areas is more than 100, is greater than 133, is greater than 200, such as More than 250,333 are greater than, is greater than 400.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is according to the present invention【HPLC-A】Method determines, The ratio of its compounds of formula I peak area and Formulas I b compound peaks areas is 100~50000, for example, 133~50000, is, for example, 200~50000, for example, 250~50000, for example, 333~50000, for example, 400~50000.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, wherein described pharmaceutic adjuvant includes being selected from Following carbohydrate：Lactose, mannitol, sorbierite, sucrose etc..

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is in tablet, capsule or granule that it, which is, Form.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is the pharmaceutical preparation shape in unit dose Formula.In one embodiment, each unit dose of the pharmaceutical preparation includes 1~1000mg compound of formula I, such as Compound of formula I including 10~750mg, such as the compound of formula I including 20~500mg.

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, its be in the form of tablet or capsule, Wherein also optionally include the conventional diluent of pharmacy (be such as, but not limited to starch and its derivative such as pregelatinized starch, change Good starch, microcrystalline cellulose etc.), disintegrant (such as but unlimited PVPP, sodium carboxymethyl starch etc.), adhesive (such as But be not limited to PVP, HPMC etc.), lubricants/glidants (such as, but not limited to magnesium stearate, talcum powder etc.) etc..

The pharmaceutical composition of any embodiment according to a third aspect of the present invention, it is sealed under the conditions of 40 DEG C, lucifuge is put Put 5 months, determine and calculate on this condition dispose 5 months after certain impurities phase at 0 month content increase percentage, wherein According to【HPLC-A】Measure, impurity Ia contents increase percentage is less than 200%, such as less than 150%, such as less than 100%, such as Less than 75%, such as less than 50%.The test method may be simply referred to as investigation in 40 DEG C-May in the present invention.

Fourth aspect present invention is provided such as following formula I a compounds

It is used to detect the Medicinal crude drug using such as compounds of Formula I as active component preparing

Or using the compound of formula I as (such as made of the Medicinal crude drug) pharmaceutical composition made of active component Reference substance in application.

The application of any embodiment according to a fourth aspect of the present invention, wherein the reference substance shines【HPLC-A】Measure, formula The chromatographic purity of Ia compounds is more than 95%, is greater than 96%, is greater than 97%, is greater than 98%.

The application of any embodiment according to a fourth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is to ensure that the content of the Formulas I a compounds in the Medicinal crude drug or pharmaceutical composition is less than for compound of formula I 2%, it is, for example, less than 1.75%, is, for example, less than 1.5%, be, for example, less than 1.25%, be, for example, less than 1.0%, is, for example, less than 0.75%.

The application of any embodiment according to a fourth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is for compound of formula I to ensure the content of the Formulas I a compounds in the Medicinal crude drug or pharmaceutical composition 0.002~2%, for example, 0.002~1.75%, for example, 0.002~1.5%, for example, 0.002~1.25%, it is, for example, 0.002~1.0%, for example, 0.002~0.75%.

The application of any embodiment according to a fourth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is of the invention to ensure the Medicinal crude drug or pharmaceutical composition photograph【HPLC-A】Method determines, wherein Formulas I a compound peaks area It is less than 2% with the ratio of compound of formula I peak area, is, for example, less than 1.75%, is, for example, less than 1.5%, be, for example, less than 1.25%, such as It is, for example, less than 0.75% less than 1.0%.

The application of any embodiment according to a fourth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is of the invention to ensure the Medicinal crude drug or pharmaceutical composition photograph【HPLC-A】Method determines, wherein Formulas I a compound peaks area Ratio with compound of formula I peak area is 0.002~2%, for example, 0.002~1.75%, for example, 0.002~1.5%, such as For 0.002~1.25%, for example, 0.002~1.0%, for example, 0.002~0.75%.

The application of any embodiment according to a fourth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is of the invention to ensure the Medicinal crude drug or pharmaceutical composition photograph【HPLC-A】Method determines, its compounds of formula I peak area It is more than 50 with the ratio of Formulas I a compound peaks areas, is greater than 57, is greater than 67, be greater than 80, be greater than 100, example Such as larger than 133.

The application of any embodiment according to a fourth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is of the invention to ensure the Medicinal crude drug or pharmaceutical composition photograph【HPLC-A】Method determines, its compounds of formula I peak area Ratio with Formulas I a compound peaks areas is 50~50000, for example, 57~50000, for example, 67~50000, for example, 80~ 50000, for example, 100~50000, for example, 133~50000.

Fifth aspect present invention is provided such as following formula I b compounds

The application of any embodiment according to a fifth aspect of the present invention, wherein the reference substance shines【HPLC-A】Measure, formula Ib compounds chromatographic purity is more than 95%, is greater than 96%, is greater than 97%, is greater than 98%.

The application of any embodiment according to a fifth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is to ensure that the content of the Formulas I b compounds in the Medicinal crude drug or pharmaceutical composition is less than for compound of formula I 1%, it is, for example, less than 0.75%, is, for example, less than 0.5%, be, for example, less than 0.4%, be, for example, less than 0.3%, is, for example, less than 0.25%.

The application of any embodiment according to a fifth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is for compound of formula I to ensure the content of the Formulas I b compounds in the Medicinal crude drug or pharmaceutical composition 0.002~1%, for example, 0.002~0.75%, for example, 0.002~0.5%, for example, 0.002~0.4%, it is, for example, 0.002~0.3%, for example, 0.002~0.25%.

The application of any embodiment according to a fifth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is of the invention to ensure the Medicinal crude drug or pharmaceutical composition photograph【HPLC-A】Method determines, wherein Formulas I b compound peaks area It is less than 1% with the ratio of compound of formula I peak area, is, for example, less than 0.75%, is, for example, less than 0.5%, be, for example, less than 0.4%, such as It is, for example, less than 0.25% less than 0.3%.

The application of any embodiment according to a fifth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is of the invention to ensure the Medicinal crude drug or pharmaceutical composition photograph【HPLC-A】Method determines, wherein Formulas I b compound peaks area Ratio with compound of formula I peak area is 0.002~1%, for example, 0.002~0.75%, for example, 0.002~0.5%, such as For 0.002~0.4%, for example, 0.002~0.3%, for example, 0.002~0.25%.

The application of any embodiment according to a fifth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is of the invention to ensure the Medicinal crude drug or pharmaceutical composition photograph【HPLC-A】Method determines, its compounds of formula I peak area It is more than 100 with the ratio of Formulas I b compound peaks areas, is greater than 133, is greater than 200, be greater than 250, be greater than 333, it is greater than 400.

The application of any embodiment according to a fifth aspect of the present invention, it is used to monitor the Medicinal crude drug or medicine group Compound is of the invention to ensure the Medicinal crude drug or pharmaceutical composition photograph【HPLC-A】Method determines, its compounds of formula I peak area Ratio with Formulas I b compound peaks areas is 100~50000, for example, 133~50000, for example, 200~50000, is, for example, 250~50000, for example, 333~50000, for example, 400~50000.

Further, the method that sixth aspect present invention provides control drug quality, described medicine is with Formulas I chemical combination Thing is the Medicinal crude drug of active component or the pharmaceutical composition being prepared into by the Medicinal crude drug, and this method includes making the medicine Impurity Formulas I b compounds in product are controlled within the specific limits, so that medicine impurity Formulas I a compounds during long-term storage Low growth is presented.

The method of any embodiment according to a sixth aspect of the present invention, wherein the medicine (i.e. Medicinal crude drug or medicine Composition) in for compound of formula I, the contents of Formulas I b compounds is less than 1%, is, for example, less than 0.75%, is, for example, less than 0.5%, it is, for example, less than 0.4%, is, for example, less than 0.3%, is, for example, less than 0.25%.

The method of any embodiment according to a sixth aspect of the present invention, wherein in the medicine relative to compound of formula I and Speech, the content of Formulas I b compounds is 0.002~1%, for example, 0.002~0.75%, for example, 0.002~0.5%, is, for example, 0.002~0.4%, for example, 0.002~0.3%, for example, 0.002~0.25%.

The method of any embodiment according to a sixth aspect of the present invention, wherein the medicine is according to the present invention【HPLC-A】Method is surveyed Fixed, wherein the ratio of Formulas I b compound peaks area and compound of formula I peak area is less than 1%, is, for example, less than 0.75%, is, for example, less than 0.5%, it is, for example, less than 0.4%, is, for example, less than 0.3%, is, for example, less than 0.25%.

The method of any embodiment according to a sixth aspect of the present invention, wherein the medicine is according to the present invention【HPLC-A】Method is surveyed Fixed, wherein the ratio of Formulas I b compound peaks area and compound of formula I peak area is 0.002~1%, for example, 0.002~0.75%, For example, 0.002~0.5%, for example, 0.002~0.4%, for example, 0.002~0.3%, for example, 0.002~0.25%.

The method of any embodiment according to a sixth aspect of the present invention, wherein the medicine is according to the present invention【HPLC-A】Method is surveyed Fixed, the ratio of its compounds of formula I peak area and Formulas I b compound peaks areas is more than 100, is greater than 133, is greater than 200, 250 are greater than, is greater than 333, is greater than 400.

The method of any embodiment according to a sixth aspect of the present invention, wherein the medicine is according to the present invention【HPLC-A】Method is surveyed Fixed, the ratio of its compounds of formula I peak area and Formulas I b compound peaks areas is 100~50000, for example, 133~50000, example Such as it is 200~50000, for example, 250~50000, for example, 333~50000, for example, 400~50000.

The method of any embodiment according to a sixth aspect of the present invention, wherein the medicine is sealed under the conditions of 40 DEG C, kept away Light place 5 months, determine and calculate on this condition dispose 5 months after certain impurities phase at 0 month content increase percentage, Wherein according to【HPLC-A】Measure, impurity Ia contents increase percentage is less than 200%, such as less than 150%, such as less than 100%, Such as less than 75%, such as less than 50%.

The method of any embodiment according to a sixth aspect of the present invention, it is described to make medicine impurity during long-term storage Formulas I a compounds present low growth refer to the medicine sealed under the conditions of 40 DEG C, avoid light place 5 months, determine and calculate Certain impurities phase increases percentage for content at 0 month after being disposed 5 months under the conditions of this, wherein according to【HPLC-A】Measure, impurity Ia contents increase percentage is less than 200%, such as less than 150%, such as less than 100%, such as less than 75%, such as less than 50%.

Further, seventh aspect present invention, which provides, prepares medical raw described in first aspect present invention any embodiment Expect the method for medicine, this method comprises the following steps：

(1) to N- quinoline acyl group-ASPARTIC ACID, 3- isopropyls -3- [(2S, 3S) -2- hydroxyl -3- (Phenylmethoxies Carbonyl) amino-4-phenyl butyl] tert-butyl carbazate, BTA -1- bases oxygen three (dimethylamino) Phosphonium fluorophosphate and DIPEA is added in the agitating solution of the anhydrous dimethyl formamide of I-hydroxybenzotriazole, is stirred at room temperature Make reaction complete, reactant is diluted with ethyl acetate, and with water, 2% potassium acid sulfate, 5% sodium acid carbonate and saturated sodium-chloride water Solution washs, and is dried through anhydrous magnesium sulfate, and solvent is evaporated under reduced pressure, is purified with silica gel column chromatography with ethyl acetate, obtains 3- isopropyls Base -3- [(2S, 3S) -2- hydroxyls -3- (N- quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl] tertiary fourth of carbazic acid Ester (i.e. Formulas I a compounds)

(2) under room temperature, blanket of nitrogen, dicyclohexyl carbodiimide, 3- isopropyls -3- [(2S, 3S) -2- hydroxyls -3- are made (N- quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate (i.e. Formulas I a compounds), anhydrous sodium Phosphoric acid mixes in anhydrous pyridine, after 60 DEG C of stirring reactions, solvent is evaporated under reduced pressure, is handled with sodium bicarbonate aqueous solution, in room Warm lower high degree of agitation l hours, sediment is filtered out, with water washing, with concentrated hydrochloric acid acidified filtrate to about pH1.5；Extracted with ethyl acetate Absorption product is taken, organic matter is dehydrated through anhydrous magnesium sulfate, solvent evaporation, obtains 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic oxygen Base -3- (N- quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate is (i.e.Formulas I b compounds)

(3) 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxide -3- (N- quinoline acyl group-L- lucid asparagus acyl group) ammonia are made Base -4- phenyl butyls] tert-butyl carbazate is (i.e.Formulas I b compounds)Suspension in HMDS is in 120 ± 5 Being stirred at DEG C, which transmits reactant mixture, homogenizes, and adds double (trimethylsilyl) peroxide, then stirs at the temperature disclosed above Mix 1 hour；After reactant mixture is cooled to room temperature, it is evaporated in vacuo to dry；Residue is dissolved in methanol, is evaporated to dryness under reduced pressure, then It is dissolved in 0.1M sodium bicarbonate aqueous solutions, gained mixture is acidified to about pH1.5 with concentrated hydrochloric acid, through sodium chloride saturation, with acetic acid Ethyl ester extracts.The organic phase of merging is dehydrated through anhydrous magnesium sulfate, is evaporated to dryness, and obtains 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylics Epoxide -3- (N- quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate

(4) at room temperature by 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxides -3- (N- quinoline acyl group-L- aspartoyls Base) amino-4-phenyl butyl] tert-butyl carbazate added in sodium bicarbonate aqueous solution, and stirring is until solution becomes clarification；Add different Propyl alcohol, stir mixture, filtering；Gained filtrate is distilled under 55-60 DEG C of vacuum removes solvent；Material is set to be cooled to 25-35 DEG C, Isopropanol to be added, is warming up to 55-60 DEG C, vacuum distillation removes solvent, dries, and gained solid is washed with isopropanol, then at 70-75 DEG C Vacuum under be evaporated to drying, it is 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxides -3- to produce disodium salt compound shown in Formulas I (N- quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl tert-butyl carbazate disodium

(5) purify：Step (4) products therefrom is set to add to heptane：Ethyl acetate=10：2~5 in the mixed solvent, in 70- 1-3 hours are stirred at 75 DEG C, room temperature is cooled to, filters out solid precipitate, divide 2 washings with heptane 20ml, then in 60 DEG C, vacuum Under be evaporated to drying, produce；Optionally according to need repeat this step (5).

Any technical characteristic possessed by any embodiment of either side or the either side of the present invention is equally applicable Any embodiment of other any embodiments or other either sides, as long as they will not be conflicting, certainly mutual Between where applicable, if necessary can individual features be made with appropriate modification.Make to various aspects of the present invention with feature into one below The description of step.

All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary When offering expressed implication and the inconsistent present invention, it is defined by the statement of the present invention.In addition, the various terms that use of the present invention and Phrase has well known to a person skilled in the art general sense, nonetheless, the present invention remain desirable at this to these terms and Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention The implication stated is defined.

It is further described to various aspects of the present invention below.

Some embodiments of the invention provides the Medicinal crude drug using compound of formula I as active component, the Formulas I chemical combination Thing is to form phosphinate by the phosphorylated reaction of Formulas I a compounds, that is, forms Formulas I b compounds, the hypophosphorous acid of Formulas I b compounds Disodium salt is further formed after ester is oxidized, obtains compound of formula I.In preparation process, the introducing of impurity be it is inevitable, Such as impurity Formulas I a compounds or Formulas I b compounds may be incorporated into using compound of formula I as in the bulk drug of active component or It can be incorporated into the pharmaceutical preparation being prepared into by the bulk drug.

Above-mentioned Formulas I a, Formulas I b are incorporated into compound of formula I as impurity, and this is for using compound of formula I as medicinal raw material Medicine and pharmaceutical composition such as pharmaceutical preparation is made into, needs to make great efforts to avoid.

Although chemists can make great efforts to remove known and unknown impurity when preparing compound of formula I, comprehensive Close consider cost-benefit relationship in the case of, micro impurity is difficult to avoid that, and when these trace impurities it is controllable (including They are controllable during as bulk drug, in addition to are also controllable when preparation is made in they) under state when, lead in pharmaceutical field Often still exempt from strong acceptable.However, regrettably, it has been found that, the Formulas I bization in compound of formula I as impurity When compound is higher than a certain amount of limit, the material medicine using such a compound of formula I as active component is auxiliary with conventional formulation Material be particularly some carbohydrates be engaged and during manufactured pharmaceutical composition such as pharmaceutical preparation, over time, the medicine Formulas I a compounds in composition occur significant growth, and the biological property of Formulas I a compounds such as its absorbent properties It is not acceptable that.Therefore, in the material medicine using compound of formula I as active component or with its manufactured medicine group It is extremely advantageous below less than above-mentioned limit by the control of impurity Formulas I b compounds in compound such as pharmaceutical preparation.

Have in the hiv protease inhibitor of wide clinical application at present：Inverase (saquinavir), indinavir (indinavir), Ritonavir (ritonavir), Nai Feinawei (nelfinavir), VX-478 (amprenavir) and Lip river That Wei (lopinavir), atazanavir and fosamprenavir.SQV (Saquinavir) be it is a kind of potent and The inhibitor of high selectivity protease, it is different from other nucleotide derivatives, it is not required to directly work by metabolism, partly declines Phase：132h, cerebrospinal fluid are with serum-concentration ratio>90, this product has effect to HIV-1 and HIV-2, and has to virus protease The specificity of height, pair is not acted on then with people's relevant asparagus fern ammonia enzyme closely, experiment in vitro show this be it is a kind of so far The most strong anti HIV-1 virus medicine newly seen, the usual better tolerance of this product, diarrhoea, headache, abdominal distension hyperlipemia are had, fat Dysbolism.That Wei (ritonavir) of benefit is a kind of potent protease inhibitors, and 3~5h of half-life period, can with food with taking Strengthen tolerance, to reduce adverse reaction, it is proposed that gradual dosage, side effect, diarrhoea, tired, concentrated force decline, hyperlipemia. Amprenavir is by suppressing viral, the protease of coding, causes to handle gag and gag-pol incapabilities, produces nonfunctional virus, It is adapted to HIV-1 and HIV-2 infection, disables in the patient to its any composition clinically allergy, side effect, nausea, diarrhoea, Abdominal distension, fash.Indinavir (Indinair) is the inhibitor, effective of another potent protease of meck companies of U.S. exploitation To anti-HIV-1, oral administration biaavailability is good, for the HIV person of former unused Lamivudine or protease inhibitors for treating, This product adds Zidovudine and Lamivudine more only can preferably slow down the progress of disease with two kinds of nucleoside medicines and reduce dead Rate.Atazanavir (ATV) is the currently the only protease inhibitors that need to only take once daily, and now just for III, it is clinical Experimental stage, its major advantage is its powerful antiviral effect, exclusive drug-resistance pattern, and fat metabolism is influenceed most It is small.

The scheme for having new treatment HIV is clinically still expected at present.Compound of formula I provided by the invention is a kind of effective Inverase, it has good oral administration biaavailability, the treatment suitable for HIV and to HIV-1 and HIV-2 There is effect.To give when people uses compound of formula I its typical day dosage as 50~500mg, such as 50~250mg, Such as 50~200mg.

Especially, it is of the invention using compound of formula I as the Medicinal crude drug of active component or the medicine being prepared by it Composition has excellent property.

Embodiment

The present invention can be further described by the following examples, however, the scope of the present invention and unlimited In following embodiments.One of skill in the art, can be with it is understood that on the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out general to the material and test method that are arrived used in experiment And/or specific description.Although for realize many materials used in the object of the invention and operating method be it is known in the art that But the present invention is still described in detail as far as possible herein.

A, compound prepares embodiment part

Embodiment 1：Prepare3- isopropyls-[(2R, 3S) -2- hydroxyls -3- (phenylmethyloxycarbonyl group) amino-4-phenyl fourth Base] tert-butyl carbazate

Step A：Prepare3- isopropyl tert-butyl carbazates

Title compound can pass through Dutta et al. (J.C：S.Perkin/1975,1712-1720) method or following sides It is prepared by method：At room temperature, by 13.2g (0.1mol) tert-butyl carbazates and 6g (0.103mol) acetone and 12.5g The mixture of the 100ml dichloromethane of (0.1mol) anhydrous magnesium sulfate stirs 12 hours, and after drier is filtered to remove, filtrate exists The lower evaporation of decompression is as after being crystallized by hexamethylene, obtaining 16.9g (98% yield) corresponding hydrazone, its fusing point is 104-105 DEG C. Under greenhouse in blanket of nitrogen, added into the suspension of the 100ml anhydrous tetrahydro furans of 2.04g (0.094mol) lithium borohydride 12ml (0.094mol) trim,ethylchlorosilane, after 30 minutes, 13.45g (0.078mol) hydrazone is slowly added at room temperature, and continue Stirring 2 hours.Then, 50ml methanol is carefully added into, and the mixture is evaporated to dryness under reduced pressure, residue is in ether Distributed between (150ml) and water (50ml), organic phase is dry through anhydrous magnesium sulfate and filters, and dry hydrogen chloride is used in combination by filtrate The white solid to be formed is filtered to remove, is washed with a part of fresh ether, is dried to obtain the hydrochloric acid of (10.5g) title compound Salt.Its free alkali form is translated into by the distribution between hexane (150ml) and 20% potassium hydroxide aqueous solution to obtain 8.3g (61%) product.

Step B：Prepare3- isopropyls-[(2R, 3S) -2- hydroxyls -3- (phenylmethyloxycarbonyl group) amino-4-phenyls butyl] Tert-butyl carbazate

At room temperature, by 0.15g (0.41mmol) N-CBZ-L- phenylalanine chlorine in anhydrous dimethyl formamide The mixture of MIBK and 1ml saturation IodineSodium Solutions stirs 15 minutes.0.074g (0.47mmol) 3- isopropyls are added thereto Base tert-butyl carbazate, it is subsequently added into 0.095g (1.13mmol) sodium acid carbonate.After being stirred at room temperature 6 hours, add 0.051g (1.3mmol) sodium borohydrides simultaneously continue to stir other 30 minutes.The solution is diluted to 30ml with ethyl acetate, is used in combination 2% aqueous potassium hydrogen sulfate, water and saturated sodium-chloride water solution washing, are then dried through anhydrous magnesium sulfate.Evaporate under reduced pressure Solvent, and with flash chromatography (silica gel；Hexane/acetic acid ethyl ester 20:5) remaining poplar is purified, obtains title compound, its fusing point is 118-119.5 DEG C, yield 49%；R (A)=0.11；R (B)=0.47；

NMR(CDCl₃) 1.0 (m, 6H, isopropyl CH₃)；1.44 (S, 9H, tert-butyl group CH₃)；2.62 (m, 2H, butyl CH₂- 1)；2.75-3.2 (m, 3H, butyl CH-3, CH₂-4)；3.47 (m, 1H, isopropyl CH)；3.89 (m, 1H, butyl CH-2)；4.44 (wide S, 1H, OH)；4.6 (wide m, 1H, NH)；5.03 (S, 2H, methoxyl group CH₂)；5.3 (wide S, 1H, carbazates NH)；7.23 (m, 10H, fragrant H).

Embodiment 2：Prepare3- isopropyls -3- [(2R, 3S) -2- hydroxyls -3- (N- quinoline acyl group-L- valyls base) amino - 4- phenyl butyls] tert-butyl carbazate

Step A：PrepareN- quinoline acyl group-Valine

At room temperature, by 0.62g (3.6mmol) quinolinic acids and 0.61g in the anhydrous Isosorbide-5-Nitrae-dioxanes of 1ml The mixture of (3.76mmol) 1,1 '-carbonyl dimidazoles stirs 30 minutes.0.43g (3.7mmol) Valine is added thereto With the solution of the 1ml water of 0.155g (3.7mmol) lithium hydroxide, gained mixture is stirred vigorously about 4 hours at room temperature.This is mixed Compound is diluted with water to 10ml, cools down (ice-water bath), is then acidified to pH about 3 with 1N hydrochloric acid, and it is small at 4 DEG C to be placed 2 When.The crystal to be formed with being filtered to remove, it is washed 3 times with 5ml cold water and is dried to obtain under a high vacuum through phosphorus pentoxide 0.75g products.Yield=76%；Fusing point is 134-136 DEG C；

NMR(DMSO-d₆) 1.03 (d, 6H, Val CH₃)；2.3 (m, 1H, Val CH- β)；3.35 (wide S, 1H, OH)；4.49 (q, 1H, Val CH- α)；7.5-8.3 (m, 5H, fragrant H)；8.5-8.76 (m, 2H, fragrant H, NH).

Step B：Prepare3- isopropyls -3- [(2R, 3S) -2- hydroxyls -3- (phenylmethyloxycarbonyl group) amino-4-phenyl fourths Base] tert-butyl carbazate

Under nitrogen atmosphere, added into the cooling solution of the 2ml methanol of the product of 0.113g (0.24mmol) embodiment 1 0.1g 10% palladium/activated carbon, it is subsequently added into 0.1g sodium borohydrides.Reactant is warming up to room temperature and stir 1 hour, then mistake Filter out catalyst, and with a part of fresh methanol rinses.The filtrate of merging is handled and subtracted with 1ml 0.1N aqueous hydrochloric acid solution Pressure evaporation as.Residue 5ml 0.1N potassium hydroxide treatments, product 30ml ether dissolutions.Organic phase saturation chlorine Change sodium water solution washing, and reduction vaporization dry through anhydrous magnesium sulfate obtains 0.0797g (99% yield) step B products, the production Thing is used for next step in the case of no further purification.

Step C：Prepare3- isopropyls -3- [(2R, 3S) -2- hydroxyls -3- (N- quinoline acyl group-L- valyls base) amino -4- Phenyl butyl] tert-butyl carbazate

To the acid of 0.0643g (0.24mmol) from step A, the 0.0797g in 0.5ml anhydrous dimethyl formamides 0.071g is added in the mixture of (0.236mmol) amine and 0.032g (0.24mmol) I-hydroxybenzotriazole from step B (0.24mmol) 1- (3- dimethylaminopropyls) -3- ethylcarbodiimine methiodides.After being stirred overnight at room temperature, mix Compound is diluted to 30ml with acetic acid ethyl ester, successively with water, 5% sodium bicarbonate aqueous solution, 2% aqueous potassium hydrogen sulfate and saturation chlorine Change sodium solution washing, and dried through anhydrous magnesium sulfate.Solvent is evaporated under reduced pressure, with column chromatography (silica gel, hexane/ethyl acetate 3:2) Residue is purified, obtains 0.091g (65% yield) title compound, its fusing point is 186-189 DEG C：Rf (B)=0.19；Rf(C) =0.83；NMR(CDCl₃) 1.0 (m, 12H, Val and isopropyl CH₃)；1.71 (S, 9H, tert-butyl group CH₃)；2.3 (m, 1H, Val CH-β)；2.5-3.27 (m, 3H, butyl CH-3, CH₂)；3.5 (m, 1H, isopropyl CH)；4.31 (m, 2H, Val CH-2, OH)； 5.43 (wide S, 1H, carbazates NH)；6.22 (wide d, 1H, butyl NH)；6.7-8.73 (m, 12H, fragrant H, NH).

Embodiment 3：Prepare3- isopropyls -3- [(2R, 3S) -2- hydroxyls -3- (N- quinoline acyl group-L- lucid asparagus acyl group) ammonia Base -4- phenyl butyls] tert-butyl carbazate

Step A：PrepareN- quinoline acyl group-ASPARTIC ACID

When replacing the Valine in the step A of embodiment 2 with asparatate, obtained with identical method titled Compound, its fusing point are 200-203 DEG C, yield 85%.

NMR(DMSO-d₆) 3.0 (m, 2H, aSn CH₂)；5.0 (m, 1H, aSn CH-2)；6.3 (wide S, 1H, OH)；6.55 (wide 2,1H, NH₂)；7,3 (wide S, 1H, NHz)；7.55-8.6 (m, 6H, fragrant H)；9.22 (d, 1H, NH).

Step B：Prepare3- isopropyls -3- [(2R, 3s) -2- hydroxyls -3- (N- quinoline acyl group-L- lucid asparagus acyl group) amino - 4- phenyl butyls] tert-butyl carbazate

To step A product (0.111g；0.386mmol), the step B products (0.13022g of embodiment 2；0.386mmol)、 (the dimethylamino) Phosphonium fluorophosphates (0.205g of BTA -1- bases oxygen three；0.46mmol) and I-hydroxybenzotriazole (0.052g；N, N- diisopropylethylamine are added in the agitating solution of 1ml anhydrous dimethyl formamides 0.384mmol) (0.24ml, 1.38mmol).After stirring 12 hours at room temperature, reactant is diluted to 30ml with ethyl acetate, and with water, 2% Potassium acid sulfate, 5% sodium acid carbonate and saturated sodium-chloride water solution washing, are dried through anhydrous magnesium sulfate.Solvent is evaporated under reduced pressure, uses post Chromatogram (silica gel, ethyl acetate) purification residue obtains 0.152g (65% yield) title product, and its fusing point is 109-114 DEG C； Rf (C)=0.36；Rf (D)=0.37；

NMR(CDCl₃) 1.0 (m, 6H, Val, isopropyl CH₃)；1.42 (S, 9H, tert-butyl group CH₃)；2.5-3.1 (m, 7H, aSn CH₂, butyl CH₂- 1, -4, CH-3)；3.44 (m, 1H, isopropyl CH)；4.21 (m, 1H, butyl CH-2)；4.55 (S, 1H, OH)；4,94 (m, 1H, aSn CH-2)；5.4-6.2 (m, 3H, acid amides)；6.7-8.4 (m, 11H, fragrant H)；9.25 (m, 1H, NH).

Embodiment 4：Prepare2 (R, S) -3 (S) -1,2- epoxy -3- phenylmethyloxycarbonyl group amino-4-phenyl butane

To the solution of the tetrahydrochysene furosemide feeding of the methanol of 30ml 50% of 6g (18mmol) N-CBZ-L- phenylalaninechloromethyl ketones Middle addition 0.68g sodium borohydrides, after stirring 30 minutes at room temperature, mixture is carefully acidified with 1N hydrochloric acid and steamed under reduced pressure It is sent to dry.Residue dchloromethane is washed, and done through anhydrous magnesium sulfate to 50ml with water and saturated sodium-chloride water solution It is dry.Evaporation obtains 6.02g (100%) 2 (R, S) -3 (S) -1- chlorine-2-hydroxyl -3- phenylmethyloxycarbonyl group amino-4-phenyls Butane, it is white solid.It is dissolved in 50ml isopropanols, and the methanol for adding 9ml 2N potassium hydroxide at room temperature is molten Liquid.After stirring 1 hour at room temperature, removal of solvent under reduced pressure, residue distributes between 50ml acetic acid ethyl esters and 20ml water.It is organic Mutually washed, through anhydrous magnesium sulfate drying and be evaporated to dryness, as by red-NCH (3.74ppm with saturated sodium-chloride water solution； And Soviet Union-NCH (4.2 72%)；28%) as relative integral measure, 5.3g (99% yield) is obtained with 2 (S) alloisomerisms Title compound based on body；

NMR(CDCl₃) 2.42-3.17 (m, 5H, butane CH₂- 1, -4, CH-2)；3.74 (m, 0.72H, butane CH-3)；4.2 (m, 0.28H, butane CH-3)；4.73 (wide m, 1H, NH)；5.08 (S, 2H, methoxyl group CH₂)；7.3 (m, 10H, fragrant H).

Embodiment 5：Prepare3- isopropyls -3- [(2S, 3S) -2- hydroxyls -3- (phenylmethyloxycarbonyl group) amino-4-phenyls Butyl] tert-butyl carbazate

Step A：Prepare2 (R) -3 (S) -1,2- epoxy -3- phenylmethyloxycarbonyl group amino-4-phenyl butane

In ammonia atmosphere, 2.6ml is added into the agitating solution of the 50ml anhydrous acetonitriles of 6.02g (40mmol) sodium iodide (22mmol) trim,ethylchlorosilane.After stirring 10 minutes, 2 (R, the S) -3 that 6g (20.1mmol) is mainly red isomers are added (S) -1,2- epoxy -3- phenylmethyloxycarbonyl group amino-4-phenyl butane (embodiment 4), continue to stir other 1 hour.To this 4g (61.2mmol) zinc powder is added in mixture, is subsequently added into 6ml acetic acid.It is small that gained mixture is stirred vigorously about 5 at room temperature When and be filtered to remove solid matter.Filtrate is evaporated in vacuo to doing, and residue is diluted to 75ml with ether, thio with water and 5N Aqueous sodium persulfate solution is washed and dried through anhydrous magnesium sulfate.It is evaporated in vacuo and uses silica gel chromatograph (hexane/ethyl acetate 4:1) Purification, obtains 5.1g (90%) (S) -2- (phenylmethyloxycarbonyl group) amino -1- phenyl butyl- 3- alkene；Rf (A)=0.5；Fusing point =87-88 DEG C (hexane)；

NMR(CDCl₃) 2.87 (d, 2H, buteneCH₂-1)；4.77 (m, 2H, butylene CH₂-4)；5.0 (m, 1H, NCH)； 5.06 (s, 2H, methoxyl group CH₂)；5.18 (wide d, 1H, NH)；5.55-6 (m, 1H, butylene CH-3)；7.19,7.27 (m, s, 5H, 5H, fragrant H)

The material (2.23g；7.93mmol) it is dissolved in 25ml anhydrous methylene chlorides, and 4.5g is added at+4 DEG C (22.1mmol) 85% 3- chloroperoxybenzoic acids.Gained mixture stirs 2 days at the temperature disclosed above, is then diluted to ether 50ml, washed successively with 0 DEG C of 10% sodium sulfite aqueous solution, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution, and It is dried over magnesium sulfate.After evaporation solvent, the crystalline mixture of crude product hexanes/ch, 2.1g (89% yield) is obtained The predominantly title epoxides of Soviet Union's formula stereochemical structure；Fusing point=83-84 DEG C；

NMR(CDCl₃) 2.47 (m, 5H, butylene CH2-1,4, CH-2)；3.74 (m, 0.15H, NCH)；4.2 (m, 0.85H, NCH)；4.53 (wide d, 1H, NH)；5.03 (m, 2H, methoxyl group CH₂)；7.3 (m, 10H, fragrant H)

Step B：Prepare3- isopropyls -3- [(2S, 3S) -2- hydroxyls -3- (phenylmethyloxycarbonyl group) amino-4-phenyl fourths Base] tert-butyl carbazate

In blanket of nitrogen, 2.03g (6.83mmol) steps A product and 1.2g (7.6mmol) 3- isopropyl carbazic acids The mixture of the 8ml isopropanols of the tert-butyl ester stirs 12 hours at 70 ± 5 DEG C.After solvent is evaporated in vacuo, solid residue Recrystallized with hexane, obtain 2.6g (80% yield) title compound, fusing point is 114-115 DEG C；

R_f(A)=0.2；R_f(B)=0.61；NMR(CDCl₃) 0.95 (m, 6H, isopropyl CH₃)；1.42 (s, 9H, the tert-butyl groups CH₃)；2.44 (m, 2H, butyl CH₂-1)；2.94 (m, 3H, butyl CH₂- 4, CH-3)；3.33-3.93 (m, 2H, isopropyl CH, fourth Base CH-2)；4.4 (wide m, 1H, OH)；5.05 (s, 2H, methoxyl group CH₂)；5.33 (wide m, 2H, NH)；7.18,7.27 (m, s, 5H, 5H, fragrant H)

Embodiment 6：Prepare3- isopropyls -3- [(2S, 3S) -2- hydroxyls -3- (N- quinoline acyl group-L- lucid asparagus acyl group) ammonia Base -4- phenyl butyls] tert-butyl carbazate (Formulas I a compounds)

The title compound of the present embodiment is also known as 3- isopropyls -3- [(2S, 3S) -2- hydroxyls -3- (N- quinoline acyl groups-L- Lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate.

3- isopropyls -3- [(2R, 3S) -2- hydroxyl -3- (phenyl for being used in embodiment 3 is replaced with the product of embodiment 5 Methoxycarbonyl) amino-4-phenyl butyl] tert-butyl carbazate is (i.e. instead of the step B of embodiment 2 used in embodiment 3 Product), the title compound of the present embodiment, yield 66% can be prepared with method same as Example 3；Fusing point= 203-204 DEG C (chloroform)；【HPLC-A】Chromatographic purity 98.1%；

R_f(C)=0.36；R_f(D)=0.37；NMR (5%CD₃OD in CDCl₃)；1.0 (m, 6H, isopropyl CH₃)；1.4 (s, 9H, tert-butyl group CH₃)；2.53 (d, 2H, butyl CH₂-1)；2.87 (m, 4H, asn CH₂, butyl CH₂-4)；3.13 (s, 6H, CD₃OH)；3.42 (m, 2H, isopropyl CH, butyl CH-3)；4.0 (m, 1H, butyl CH-2)；4.89 (m, 1H, asn CH- α)； 7.11 (m, 5H, phenyl)；7.41-8.47 (m, 6H, quinaldine acyl group)

Embodiment 7：Prepare3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxide -3- (N- quinoline acyl group-L- aspartoyls Base) amino-4-phenyl butyl] tert-butyl carbazate (Formulas I b compounds)

Under room temperature, blanket of nitrogen, stirring adds 0.28 gram of (1.4 mMs) dicyclohexyl carbodiimide to containing 0.4 gram (0.67 mM) 3- isopropyls -3- [(2S, 3S) -2- hydroxyls -3- (N- quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyls Butyl] tert-butyl carbazate (i.e. Formulas I a compounds) and 0.12 gram of (1.47 mMs) anhydrous sodium phosphate mixture 1.5 millis Rise in anhydrous pyridine.After 60 DEG C are stirred 2 hours, solvent is evaporated under reduced pressure, is handled with 28 milliliters of 0.1ml sodium bicarbonate aqueous solutions, High degree of agitation l hours at room temperature.Sediment is filtered out, with water washing, with concentrated hydrochloric acid acidified filtrate to pH~1.5.With acetic acid second Ester (3x50 milliliters) extracts absorption product, and organic matter is dehydrated through anhydrous magnesium sulfate.Solvent evaporates, and produces 0.42 gram (yield 95%) The colorless solid of title product, Rf (B)=0.62；【HPLC-A】Chromatographic purity 98.2%；

H¹NMR(CDCl₃)；1.08 (m, 6H, isopropyl CH₃）；1.41 (s, the 9H. tert-butyl group CH₃)；2.7-4.8 (m, 14H, asn CH₂, butyl CH₂- Isosorbide-5-Nitrae；CH-2,3；Isopropyl CH；P-OH×2H₂O)；5.12 (m, 1H, asn CH)；5.89 (s, 0.5H, PH)；6.2-8.5 (m, 15.5H, aromatic series, amide NH, 0.5PH)；9.02 (m, 1H, asn NH)；p³¹NMR(CDC1₃)14.99 (J=636Hz).

Embodiment 8：Prepare phosphate compound3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxides -3- (N- quinoline acyl group - L- lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate

Make 2 milliliters of HMDS suspension containing 0.4 gram of (0.6 mM) Formulas I b compound at 120 ± 5 DEG C Stirring 45 minutes.Now reactant mixture, which is transmitted, homogenizes.Double (trimethylsilyl) peroxide (the P.G. storehouses of 0.3 milliliter of addition Gloomy (Cookson) et al., J.Organometal, Chem., 1975,99, C31), then stir 1 hour at the temperature disclosed above.Instead After answering mixture to be cooled to room temperature, it is evaporated in vacuo to dry.Residual matter is dissolved in 20 ml methanols, is evaporated to dryness under reduced pressure, and is re-dissolved in 12 In milliliter 0.1M sodium bicarbonate aqueous solutions, gained mixture is acidified to pH~1.5 with concentrated hydrochloric acid, through sodium chloride saturation, with acetic acid Ethyl ester (3x50 milliliters) extracts.The organic phase of merging is dehydrated through anhydrous magnesium sulfate, is evaporated to dryness, and is produced 0.39 gram (yield 96%) The colorless solid of title compound；Rf (B)=0.07；

H¹NMR(CDCl₃)：1.2 (m, 6H, isopropyl CH₃)；1.4 (s, 9H, tert-butyl group CH₃)；2.8-4.2 (m, 8H, asn CH₂, butyl CH₂- Isosorbide-5-Nitrae；CH=3；Isopropyl CH)；4.2-6.4 (m, 5H, asn CH butyl CH-2, NH, POH)；6.5-8.4 (m, 14H, aromatic series, NH, 8.78 (m, 2H, NH)；p³¹NMR(CDCl₃)9.6(s).

Embodiment 9：Prepare compound of formula I (disodium salt)

Operation：Sodium acid carbonate 2.2g (26mmol) is dissolved in reaction bulb in 22ml deionized waters at room temperature, will The product of 10g (14.5mmol) embodiment 8 is added in above-mentioned solution, and stirring 2-3 hours are until solution becomes clarification；Add isopropanol 100ml, stir 20-30min, filtering；Gained filtrate is distilled under 55-60 DEG C of vacuum removes solvent；Material is set to be cooled to 25-35 DEG C, isopropanol 50ml to be added, is warming up to 55-60 DEG C, vacuum distillation removes solvent, dries, and gained solid is washed with isopropanol 50ml, Drying is evaporated under 70-75 DEG C of vacuum again, produces disodium salt compound shown in Formulas I, yield 98%.【HPLC-A】Chromatographically pure Degree 97.2%, impurity Ia content=0.82%, impurity Ib content=0.68%.

Embodiment 91：Prepare compound of formula I (disodium salt)

The product 1g of embodiment 9 is placed in reaction bulb, heptane/ethyl acetate (10/3, v/v) 20ml is added, at 70-75 DEG C Lower stirring 3 hours, is cooled to room temperature, filters out solid precipitate, divides 2 washings with heptane 20ml, then evaporated under 60 DEG C, vacuum To drying, disodium salt compound shown in Formulas I, yield 96% are produced.【HPLC-A】Chromatographic purity 99.3%, impurity Ia contents= 0.26%, impurity Ib content=0.23% (are down to 0.23% from 0.68%, are reduced to 34%).

Embodiment 92：Prepare compound of formula I (disodium salt)

The product 1g of embodiment 91 is placed in reaction bulb, heptane/ethyl acetate (10/2, v/v) 20ml is added, in 70-75 Stirred 1 hour at DEG C, be cooled to room temperature, filter out solid precipitate, divide 2 washings with heptane 20ml, then steamed under 60 DEG C, vacuum Drying is sent to, produces disodium salt compound shown in Formulas I, yield 95%.【HPLC-A】Chromatographic purity 99.5%, impurity Ia contents= 0.09%, impurity Ib content=0.07% (being reduced to 30%).

Embodiment 93：Prepare compound of formula I (disodium salt)

The product 1g of embodiment 92 is placed in reaction bulb, heptane/ethyl acetate (10/5, v/v) 20ml is added, in 70-75 Stirred 2 hours at DEG C, be cooled to room temperature, filter out solid precipitate, divide 2 washings with heptane 20ml, then steamed under 60 DEG C, vacuum Drying is sent to, produces disodium salt compound shown in Formulas I, yield 96%.【HPLC-A】Chromatographic purity 99.6%, impurity Ia contents= 0.025%, impurity Ib content=0.019% (being reduced to 27%).

Embodiment 94：Prepare compound of formula I (disodium salt)

The product 1g of embodiment 93 is placed in reaction bulb, heptane/ethyl acetate (10/4, v/v) 20ml is added, in 70-75 Stirred 2.5 hours at DEG C, be cooled to room temperature, filter out solid precipitate, divide 2 washings with heptane 20ml, then under 60 DEG C, vacuum Drying is evaporated to, produces disodium salt compound shown in Formulas I, yield 94%.【HPLC-A】Chromatographic purity 99.8%, impurity Ia contents =0.008%, impurity Ib content=0.006% (being reduced to 32%).

Embodiment 95：Prepare compound of formula I (disodium salt)

The product 1g of embodiment 94 is placed in reaction bulb, heptane/ethyl acetate (10/3, v/v) 20ml is added, in 70-75 Stirred 2 hours at DEG C, be cooled to room temperature, filter out solid precipitate；Divide 2 washings with heptane 20ml, then steamed under 60 DEG C, vacuum Drying is sent to, produces disodium salt compound shown in Formulas I, yield 96%.【HPLC-A】Chromatographic purity 99.8%, impurity Ia contents= 0.002%, impurity Ib content=0.002% (being reduced to 33%).

It is visible according to the result of above example 91~95, can be with when being handled using heptane/ethyl acetate product Impurity Ia and impurity Ib content are substantially reduced, often handling once can make such impurity be reduced to about 25%~35% respectively, Reduce about 65%~75%.

Embodiment 97：Prepare compound of formula I (disodium salt)

The product 1g of embodiment 9 is placed in reaction bulb, heptane 20ml is added, stirs 3 hours, be cooled at 70-75 DEG C Room temperature, solid precipitate is filtered out, divide 2 washings with heptane 20ml, then drying is evaporated under 60 DEG C, vacuum, produced shown in Formulas I Disodium salt compound, yield 97%.【HPLC-A】Chromatographic purity 97.5%, impurity Ia content=0.74%, impurity Ib contents= 0.61% (two kinds of impurity reduce about 10% respectively).

The use of heptane/ethyl acetate=10/0.5 or 10/1 is molten in addition, in the complementary testing with reference to embodiment 97 When agent is handled, two kinds of impurity reduce about 9~11% respectively.

Embodiment 98：Prepare compound of formula I (disodium salt)

The product 1g of embodiment 9 is placed in reaction bulb, ethyl acetate 20ml is added, is stirred 2 hours at 70-75 DEG C, it is cold But to room temperature, solid precipitate is filtered out, divides 2 washings with heptane 20ml, then drying is evaporated under 60 DEG C, vacuum, produces Formulas I Shown disodium salt compound, yield 97%.【HPLC-A】Chromatographic purity 97.5%, impurity Ia content=0.79%, impurity Ib contain Amount=0.64%.

The use of heptane/ethyl acetate=10/50 or 10/20 is molten in addition, in the complementary testing with reference to embodiment 98 When agent is handled, two kinds of impurity reduce about 7~10% respectively.

It is visible according to the result of above example 97~98, when use heptane or use ethyl acetate or use this When the larger mixed solvent of the two usage variance is handled, it is impossible to impurity Ia and impurity Ib content is substantially dropped Low, often handling once can only make such impurity reduce about 10%.

Embodiment 99：Prepare compound of formula I (disodium salt)

The product free acid of embodiment 8 is handled with the 0.2M sodium bicarbonate solutions of 2 equivalents, changes into corresponding disodium salt, Resulting solution is freeze-dried, and is produced, yield 99%.【HPLC-A】Chromatographic purity 95.6%, impurity Ia content=2.68%, impurity Ib content=1.91%.

The sign of the gained compound of formula I of embodiment 95：

Infrared data such as following table：

Wave number (cm^-1)	Ownership
		3231	N-H
3063	Aromatics C-H
		2978	Aliphatic C-H
1672	C-O
		1528	Acid amides II keys
1250	P-O
		1165,1101	C-N
976,775	Aromatics

NMR：500MHz, Varian Unity Inova FT-NMR instrument, solvent CD3OD；1H and 13C nmr chemicals position Shifting is represented with δ (ppm), is respectively relative to internal standard compound TMS (δ 0.00) and CD3OD (δ 49.00) meters；In CD3OD 1H NMR not Seeing has exchangeable protons；1H NMR datas are gathered in DMSO-d6 at 30 DEG C and 70 DEG C, to observe being total to for exchangeable protons Shake；Carry out gDQCOSY and gHSQC experiments and TOCSY experiments；The NMR ownership displays of sample are as follows：

Note：1 each position is marked with reference to formula atom position above,

HR-MS researchs are carried out using Waters LCT premier XE instrument, and the molecular formula of derivation is C32H41N6Na2O9P, i.e. C32H41N6O9P2Na, TOF data see the table below：

Polarity	Quality	Formula	Error (ppm)	i-Fit	Annotation
						(+ve)	687.2929	C₃₂H₄₄N₆O₉P	3.2	0.4	(alkali+H)⁺
(-ve)	685.2770	C₃₂H₄₂N₆O₉P	2.8	0.2	(alkali+H)^-

B, test example part

Test example 1：The comparative test of Oral drug absorption

Reagent：It is examination with both the gained Formulas I a compounds of embodiment 6 and the gained compound of formula I (disodium salt) of embodiment 91 Medicine,

Animal, packet and administration：Rat, 200~300g of body weight, is divided into two groups (I groups and Ia groups respectively), often by 16 Group 8, two kinds of each dosages of medicine are 50 μm of ol/kg body weight.Medicine is set fully to dissolve 2% in a manner of grinding In the HPMC aqueous solution, gavage gives reagent in a manner of single-dose, 1 hour after 0 hour (before administration), administration, 2 small When, take tail vein within 4 hours, 8 hours, 15 hours.

The preparation of test sample：By with 3000~5000r/min of blood sample rotating speeds of liquaemin anti-freezing centrifugation 5~ After 30min, upper plasma is taken to be transferred in new blank EP pipes；Internal standard benzoic acid solution (0.1mg/ml) 10 μ l are added, are then pressed Blood plasma and precipitation reagent (acetonitrile-methanol=3: 2 mixed liquors and add 2% formic acid) volume ratio is 1: 5 protein precipitation, during precipitation Between 3min, be vortexed mix 45s after, under 5 DEG C of constant temperatures with 10000r/min centrifuge 10min；Upper liquid is transferred to new sky In white EP pipes, dried up in 30~50 DEG C of nitrogen streams, residue is dissolved with 100 μ l50% acetonitrile solutions, is vortexed and is mixed 30~60s Afterwards, 5~15min is centrifuged with 10000~12000r/min under 0~10 DEG C of constant temperature, it is need testing solution to take supernatant； The another Formulas I a compound standards solution and compound of formula I standard liquid that suitable concentration is prepared with 50% acetonitrile solution.According to the present invention 【HPLC-A】Determine the concentration of the Formulas I a compounds and compound of formula I in blood plasma.

In terms of testing result, two groups of animals are only able to detect Formulas I a compounds and can't detect compound of formula I, and reason exists Accepted way of doing sth Ia compounds are metabolized after compound of formula I is absorbed, therefore each group is using Formulas I a compounds as calculating target.For each group Animal, the blood concentration average value at each time point is calculated, and calculated below the plasma concentration curve between 0~15 hour Product AUC, it is calculated as follows the relative AUC of Formulas I a compound groups：

=[Formulas I a compound group AUC ÷ compound of formula I groups AUC] × 100

This shows that the intestines and stomach absorbent properties of two kinds of compounds are closer with respect to AUC closer to 100%, if this is relative AUC is less than the intestines and stomach absorption difference of the absorptance compound of formula I of 100% Formulas I a compound, if this is more than with respect to AUC The intestines and stomach good absorbing of the absorptance compound of formula I of 100% Formulas I a compound.As a result show, in the mouth that above-mentioned rat is carried out In clothes administration, the relative AUC of Formulas I a compound groups is only 13.6%, shows the oral absorption of Formulas I a compounds well below Formulas I Compound.In the experiment of supplement, tested, tied with reference to the above method using rabbit as animal (each 6 animals of administration group) The relative AUC of fruit Formulas I a compound groups is only 9.3%.In the experiment of supplement, using beasle dog as animal (each administration group 3 Animal) tested with reference to the above method, as a result the relative AUC of Formulas I a compound groups is only 11.2%.

It can be seen that using compound of formula I as in the Medicinal crude drug of active component or its manufactured preparation, compound of formula I It is unfavorable for maintaining the dosage of medicine to change into Formulas I a compounds.

Test example 2：The stability of bulk drug

Directly take the corresponding material for preparing of the present invention, or using a certain amount of Formulas I a compounds (product of embodiment 6) and/ Or Formulas I b compounds (product of embodiment 7) assemble what is obtained with compound of formula I (product of embodiment 95) by being fully ground to mix The mixing of different impurities content assembles thing.

These materials are made to be sealed with vial, then sealing, avoid light place 5 months under the conditions of 40 DEG C, use【HPLC-A】Survey Determine each sample and calculate dispose 5 months on this condition after certain impurities phase increase percentage, and Formulas I for content at 0 month Compound phase is for remaining percentage (content of compound of formula I divided by compound of formula I at 0 month during May in material at 0 month Content multiplied by with 100% gained percentage).As a result it is as follows：

Note：* used when initial composition refers to 0 month in material【HPLC-A】Various compounds contains in the material that measure obtains Amount, its moderate purity are the purity of the area normalization method of compound of formula I, and Formulas I a compounds contents are to shine【HPLC-A】The phase of measure For the percentage amounts of compound of formula I, Formulas I b compounds contents are to shine【HPLC-A】The percentage relative to compound of formula I of measure Amount.

The result from table, Medicinal crude drug of the invention are stable, even if being wherein contaminated with a certain amount of impurity Ia and/or impurity Ib.

Test example 3：The stability of bulk drug and the composition of sugar

By 30 samples in test example 2 or assemble the sugar (lactose) of thing and equivalent respectively and be fully ground uniformly, formed with The composition of sugar.These compositions are made to be sealed with vial, then sealing, avoid light place 5 months under the conditions of 40 DEG C, use【HPLC- A】Certain impurities phase increases percentage for content at 0 month after measure each sample and calculating are disposed 5 months on this condition, and Compound of formula I is relative to remaining percentage (content of compound of formula I divided by Formulas I chemical combination at 0 month during May in material at 0 month The content of thing is multiplied by with 100% gained percentage).As a result it is as follows：

From result above, the composition being prepared into using different material with sugar, wherein impurity Ib contents significantly affect Impurity Ia growth and the decline of active component in composition, particularly when Ib contents>When 0.5%, this raw material and sugared institute Composition is obtained under the hot conditions that simulation keeps sample for a long time after processing, impurity Ia therein can be dramatically increased, and increase degree It is proportionate with Ib contents, similarly the residual content of active component also can be reduced correspondingly.But completely it is unaccountable be, No matter impurity Ib contents are high or low in raw material, and itself shows as impurity Ia is but presented during long-term storage Show increase.

In addition, according to the method for this test example 3, the difference is that lactose dosage therein to be changed to 0.2 times or 20 of bulk drug Times, experiment above is also carried out, as a result shows composition made of different material medicine in " I remnants/% " " Ia increases/% ", " Ib Basically identical with upper table result in terms of increase/% " threes, i.e., lactose yield can in the range of 0.2~20 times of active component The above results are presented.

Test example 31：The stability of bulk drug and the composition of sugar

With reference to the method for test example 3, different is only that lactose therein is replaced with into sucrose.As a result show substantially with The identical result of test example 3, such as No.21 compositions I remnants/% of preparation is 91.8%, Ia increases/% is 273%, Ib increases Add/% be 61%；In another example the No.22 compositions I remnants/% prepared is<85%th, Ia increases/% is>500%th, Ib increases Add/% be 63%.

Test example 32：The stability of bulk drug and the composition of sugar

With reference to the method for test example 3, different is only that lactose therein is replaced with into mannitol.As a result show substantially With the identical result of test example 3, such as No.21 compositions I remnants/% of preparation is 91.1%, Ia increases/% is 253%, Ib Increase/% is 67%；In another example the No.22 compositions I remnants/% prepared is<85%th, Ia increases/% is>500%th, Ib increases Add/% be 65%.

Test example 33：The stability of bulk drug and the composition of sugar

With reference to the method for test example 3, different is only that lactose therein is replaced with into sorbierite.As a result show substantially With the identical result of test example 3, such as No.21 compositions I remnants/% of preparation is 91.4%, Ia increases/% is 271%, Ib Increase/% is 63%；In another example the No.22 compositions I remnants/% prepared is<85%th, Ia increases/% is>500%th, Ib increases Add/% be 62%.

Result above shows that there is the carbohydrate commonly used in the Medicinal crude drug and pharmacy of different impurities Ib contents to coordinate When preparing pharmaceutical preparation, visibly different increment can be presented because of the difference of impurity Ib contents in raw material in wherein impurity Ia.To the greatest extent This phenomenon is managed not in bulk drug middle discovery in itself, but will limit Medicinal crude drug significantly when impurity Ib contents are higher and make Auxiliary material range of choice during agent is prepared, because these above-mentioned carbohydrates are to be commonly used on galenic pharmacy, be cheap, superior performance medicinal auxiliary Material, the problem of other extensive such as cost, tablet will likely be triggered when using other pharmaceutic adjuvants instead without using these carbohydrates Energy, range of choice etc..

Test example 4：The stability of pharmaceutical composition (tablet) prepared by bulk drug

Using 30 samples in test example 2 or thing is assembled as Medicinal crude drug, piece is prepared with following tablet formulation respectively Agent：Medicinal crude drug 15mg, starch 15mg, lactose 100mg, gelatin 1mg, magnesium stearate 1mg.Preparation method：Gelatin is made of water Solution is as adhesive；Bulk drug, starch, lactose are sufficiently mixed uniformly, with adhesive softwood, wet granular processed, dried；Will Dry particl is well mixed with magnesium stearate, tabletting, every 15mg containing Medicinal crude drug.These tablets are made to be sealed with vial, then Sealing, avoid light place 5 months under the conditions of 40 DEG C, are used【HPLC-A】Determine each sample and calculate and dispose 5 months on this condition Certain impurities phase increases percentage for content at 0 month afterwards, and compound of formula I is relative to remaining percentage at 0 month.As a result Show that such as I remnants/% of No.21 tablets is 91.8%, Ia increases/% is substantially with the identical result of test example 3 292%th, Ib increases/% is 65%；In another example the I remnants of No.22 tablets/% is<85%th, Ia increases/% is>500%th, Ib increases Add/% be 69%.

Industrial applicability

The invention provides antiviral drugs and its pharmaceutical composition, and it can be as retroviral Protease inhibitors Medicine, the retroviral Protease inhibitors have the chemical constitution shown in Formulas I.

Claims

1. the method using compounds of Formula I as the Medicinal crude drug of active component is prepared,

,

This method comprises the following steps：

(1) to N- quinoline acyl group-ASPARTIC ACID, 3- isopropyls -3- [(2S, 3S) -2- hydroxyls -3- (Phenylmethoxy carbonyls Base) amino-4-phenyl butyl] tert-butyl carbazate, BTA -1- bases oxygen three (dimethylamino) Phosphonium fluorophosphate and 1- DIPEA is added in the agitating solution of the anhydrous dimethyl formamide of hydroxybenzotriazole, being stirred at room temperature makes Reaction is complete, and reactant is diluted with ethyl acetate, and with water, 2% potassium acid sulfate, 5% sodium acid carbonate and saturated sodium-chloride water solution Washing, is dried through anhydrous magnesium sulfate, and solvent is evaporated under reduced pressure, is purified with silica gel column chromatography with ethyl acetate, obtains 3- isopropyls -3- [(2S, 3S) -2- hydroxyls -3- (N- quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate, i.e., Formulas I a compounds

；

(2) under room temperature, blanket of nitrogen, dicyclohexyl carbodiimide, 3- isopropyls -3- [(2S, 3S) -2- hydroxyl -3- (N- are made Quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate is that Formulas I a compounds, anhydrous phosphorous acid exist Mixed in anhydrous pyridine, after 60 °C of stirring reactions, solvent is evaporated under reduced pressure, is handled with sodium bicarbonate aqueous solution, swashed at room temperature Strong stirring l hours, sediment is filtered out, with water washing, with concentrated hydrochloric acid acidified filtrate to pH1.5；Extracted with ethyl acetate and absorb production Thing, organic matter are dehydrated through anhydrous magnesium sulfate, solvent evaporation, obtain 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxide -3- (N- Quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate, i.e. Formulas I b compounds

；

(3) 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxide -3- (N- quinoline acyl group-L- lucid asparagus acyl group) amino -4- are made Phenyl butyl] tert-butyl carbazate is that suspension of the Formulas I b compounds in HMDS stirs at 120 ± 5 DEG C Transmit reactant mixture to homogenize, add double (trimethylsilyl) peroxide, then stir 1 hour at the temperature disclosed above； After reactant mixture is cooled to room temperature, it is evaporated in vacuo to dry；Residue is dissolved in methanol, is evaporated to dryness under reduced pressure, and is re-dissolved in 0.1M In sodium bicarbonate aqueous solution, gained mixture is acidified to pH1.5 with concentrated hydrochloric acid, through sodium chloride saturation, is extracted with ethyl acetate；Close And organic phase be dehydrated through anhydrous magnesium sulfate, be evaporated to dryness, obtain 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxide -3- (N- Quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate

；

(4) at room temperature by 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxides -3- (N- quinoline acyl group-L- lucid asparagus acyl group) ammonia Base -4- phenyl butyls] tert-butyl carbazate added in sodium bicarbonate aqueous solution, and stirring is until solution becomes clarification；Add isopropanol, Stir mixture, filtering；Gained filtrate is distilled under 55-60 °C of vacuum removes solvent；Material is set to be cooled to 25-35 °C, addition Isopropanol, is warming up to 55-60 °C, and vacuum distillation remove solvent, and drying, gained solid is washed with isopropanol, then true at 70-75 °C Drying is evaporated under sky, it is 3- isopropyls -3- [(2S, 3S) -2- phosphine carboxylic epoxide -3- (N- to produce disodium salt compound shown in Formulas I Quinoline acyl group-L- lucid asparagus acyl group) amino-4-phenyl butyl tert-butyl carbazate disodium

；

(5) purify：Step (4) products therefrom is set to add to heptane：Ethyl acetate=10：4 in the mixed solvent, stirred under 70-75 °C 1-3 hours are mixed, are cooled to room temperature, filter out solid precipitate, divide 2 washings with heptane 20ml, then be evaporated under 60 °C, vacuum Dry, produce；Optionally according to need repeat this step (5).