CN104628771B - Antiviral drug and pharmaceutical composition thereof - Google Patents

Abstract

The present invention relates to antiviral drugs and pharmaceutical compositions thereof, in particular to drugs against human immunodeficiency virus (HIV) and pharmaceutical compositions thereof, and in particular to a drug that can be used as a retroviral protease inhibitor. The retroviral protease inhibitor has a chemical structure shown in Formula I.

Description

Antiviral drug and pharmaceutical composition thereof

technical field

The present invention relates to an antiviral drug and its pharmaceutical composition, in particular to an anti-human immunodeficiency virus (HIV) drug and its pharmaceutical composition, in particular to a drug that can be used as a retroviral protease inhibitor. The recording virus protease inhibitor has the chemical structure shown in formula I.

Background technique

Human immunodeficiency virus (HIV) is a pathogenic retrovirus that causes AIDS and related diseases. Since the discovery of HIV, the development of antiviral chemotherapy against AIDS has become the focus of active research. For research on the molecular targets of AIDS, see Mitsua et al. (Science, 1990, pp. 1533-154). HIV protease (HIVPR) and aspartyl protease were first considered by Kramer et al. (Science 231, 1580 (1986)) as possible targets for AIDS therapy. Since then, the potential use of HIVPR inhibitors as effective agents has been widely recognized in the treatment of AIDS. For the medical use of HIVPR, refer to the literature of people such as Tomaselli (Chimica, Oggi, May, 1991, pp. 6-27) and the literature of J.P. Hoover (Huff) et al. (J.Med.Chem. 34, 2341-2327 (1991)). Among the transition states that traditionally mimic aspartyl proteases, hydroxyethene, dihydroxyethene, hydroxyethylamine, and the phosphinic acid isostere (Bostere) appear to have the greatest affinity for HIVPR. Many inhibitors of HIVPR have exhibited antiviral activity at concentrations in the nanomolar range in different cellular systems and have been described in the patent literature.

The main role of HIV protease in the viral replication process is to cleave gag and gag-pol gene products into structural proteins (matrix, shell, nucleocapsid) and enzymes (protease, integrase, reverse transcriptase) required for virus maturation, Thereby further perfecting the virus structure. Protease inhibitors (PIs) prevent the cleavage of precursor proteins, leading to the accumulation of non-infectious virus particles.

Pharmacodynamic and conformational studies have shown that the mechanism of action of this type of compound is to interact with the Asp25, Cly27 and Asp29 residues of the protease in the form of hydrogen bonds, and interact with the amino acid residues in the protease active group to form a body, so as to achieve The purpose of inhibiting protease activity, and then inhibit the replication of HIV, to achieve antiviral effect. Protease inhibitors can enter a variety of tissues and organs including peripheral blood to play an antiviral effect, so the curative effect is deep and lasting.

At present, the HIV protease inhibitors that have been widely used in clinical practice include: saquinavir (saquinavir), indinavir (indinavir), ritonavir (ritonavir), nelfinavir (nelfinavir), amprenavir (amprenavir) and lopinavir (lopinavir). In 2003, two new protease inhibitors, atazanavir and fosamprenavir, were approved by the US FDA. Atazanavir, developed by Bristol-Myers Squibb, is a new protease inhibitor that only needs to be taken once a day. It has very little effect on insulin and fat metabolism, and patients have no side effects such as diarrhea. It can slightly increase the bilirubin level, and individual patients may experience jaundice, but patients rarely stop taking the drug because of this.

Although these protease inhibitors can inhibit HIV replication, they cannot completely eliminate the virus in the body, nor can they prevent the emergence and spread of drug-resistant strains in the body. After the application of protease inhibitor PI, not only the number of viruses in the body decreased significantly, but also T helper cells-CD4 cells in lymphocytes increased. Then long-term use of these protease inhibitors produces obvious toxic side effects and drug resistance. Disorders of lipid metabolism are the most prominent and complex side effects, and the clinical manifestations are facial and peripheral fat consumption, and abdominal, back, and chest fat accumulation. Patients have hypertriglyceridemia and hypercholesterolemia, elevated lactic acid and blood sugar, and resistance to insulin. Followed by drug resistance, especially cross-resistance is a major cause of clinical treatment failure. Studies have shown that the mechanism of PI drug resistance is more complex than that of NRIT and NNRTI, often involving multiple gene mutations. In general, a single mutation in the protease substrate binding region can increase the resistance of the virus to drugs by 10 times. If the 82-position valine or 84-position isoleucine of the protease S1 subregion is substituted, the tolerance can be increased to 30 times, and if the 82 and 84 residues are mutated at the same time, the therapeutic effect of the drug can be reduced by 100 times. Using high-dose PI will delay the development of drug resistance than using low-dose PI, and the combined application of nucleoside drugs can also reduce the development of drug resistance. Also, the generation of drug resistance is also due to the high virus replication rate, the error of reverse transcriptase in the process of reverse transcription and the evolution result under the selective action of protease inhibitors. In addition, PI's complicated administration method and high risk of cardiovascular disease make it unsuitable for patients with poor compliance, diabetes and cardiovascular disease.

The compound of formula I is known to be a potent retroviral protease inhibitor

Its molecular formula: C ₃₂ H ₄₁ N ₆ Na ₂ O ₉ P, molecular weight 730.66, its typical English name can be called: t-Butyl3-isopropyl-3-[(2S,3S)-2-Phosphonooxy-3-(N -quinaldoyl-L-asparaginyl)amino-4-phenylbutylcarbazateDisodium, the typical Chinese name can be called: 3-isopropyl-3-[(2S,3S)-2-phosphine carboxyoxy-3-(N-quinoline Acyl-L-asparagyl)amino-4-phenylbutylcarbazinate disodium tert-butyl, or the typical Chinese name can be called: 3-isopropyl-3-[(2S,3S)-2 - Disodium tert-butyl phosphinecarboxy-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenylbutylcarbazate.

In the structural formula of the compound of formula I, the 2- and 3-position carbons of the butyl group connected between the phenyl group and the isopropyl group are both chiral carbons.

Although pharmaceutical workers have a strong ability in drug quality control to realize effective control of raw materials in drug manufacturers; Enterprise personnel (including logistics personnel, medical staff, patients, etc.) will greatly weaken the internal control ability of the preparation.

Therefore, it is still expected by those skilled in the art to provide a product with good quality, such as stable pharmaceutical characteristics, especially the bulk drug of the compound of formula I or a preparation made therefrom.

Contents of the invention

The object of the present invention is to provide a product with good quality, such as stable pharmaceutical characteristics, especially the bulk drug of the compound of formula I or the preparation made therefrom. The present inventors have surprisingly found that by controlling the impurity Ib in the bulk drug of the compound of formula I below a certain range, when the bulk drug is combined with conventional pharmaceutical excipients, especially sugars, to make pharmaceutical preparations, the impurity Ib The pharmaceutical preparation has excellent stability especially the growth of impurity Ia can be maintained in a very low range. The present invention has been accomplished based on this finding.

To this end, the first aspect of the present invention provides a pharmaceutical raw material, the active ingredient of which is the compound of the following formula I:

According to any embodiment of the first aspect of the present invention, the pharmaceutical raw material contains more than 97% of the compound of formula I, such as more than 97.5% of the compound of formula I, for example more than 98% of the compound of formula I.

The pharmaceutical raw material according to any embodiment of the first aspect of the present invention, which contains 97% to 103% of the compound of formula I, for example, contains 97.5% to 102.5% of the compound of formula I, for example contains 98% to 102% of the compound of formula I I compound.

The pharmaceutical raw material according to any embodiment of the first aspect of the present invention, which also contains (for example, a trace amount) the following compound of formula Ia present as an impurity:

The compound of formula Ia is an unphosphorylated compound of the compound of formula I, its molecular formula is: C ₃₂ H ₄₂ N ₆ O ₆ , and its molecular weight is 606.72. The molecular formula of the compound of formula I is: C ₃₂ H ₄₁ N ₆ Na ₂ O ₉ P, and the molecular weight is 730.66.

In the present invention, the compound of formula Ia may also be referred to as impurity Ia or may be referred to as Ia or may be referred to as Ia impurity or other similar titles.

The pharmaceutical raw material according to any embodiment of the first aspect of the present invention, wherein relative to the compound of formula I, the content of the compound of formula Ia is less than 2%, such as less than 1.75%, such as less than 1.5%, such as less than 1.25%, such as Less than 1.0%, such as less than 0.75%.

The pharmaceutical raw material according to any embodiment of the first aspect of the present invention, wherein relative to the compound of formula I, the content of the compound of formula Ia is 0.002-2%, such as 0.002-1.75%, such as 0.002-1.5%, For example, it is 0.002 to 1.25%, for example, it is 0.002 to 1.0%, for example, it is 0.002 to 0.75%.

In the present invention, the phrase "for the compound of formula I, the content of the compound of formula Ia" refers to the amount of the compound of formula Ia relative to the compound of formula I in the material concerned. For example, if a material contains 100 mg of the compound of formula I, and it is determined that 0.5 mg of the compound of formula Ia is contained therein, then for the compound of formula I, the amount of the compound of formula Ia is 0.5%. Similar statements about the amounts of other impurities relative to the compound of formula I have similar meanings. The above "content" can also be measured by the [HPLC-A] method of the present invention.

According to the pharmaceutical raw material according to any embodiment of the first aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ia to the peak area of the compound of formula I is less than 2%, such as less than 1.75% , such as less than 1.5%, such as less than 1.25%, such as less than 1.0%, such as less than 0.75%.

According to the pharmaceutical raw material according to any embodiment of the first aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ia to the peak area of the compound of formula I is 0.002 to 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%.

According to the pharmaceutical raw material according to any embodiment of the first aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ia is greater than 50, for example greater than 57, for example Greater than 67, such as greater than 80, such as greater than 100, such as greater than 133.

According to the pharmaceutical raw material according to any embodiment of the first aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ia is 50 to 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 the in vivo behavior characteristics of the compound of formula Ia are far less than that of the compound of formula I or even unacceptable from a pharmaceutical point of view, so in the pharmaceutical raw material drug of the first aspect of the present invention (and in the pharmaceutical composition of the second aspect of the present invention) It is necessary to control the amount of the compound of formula Ia below a certain amount. For example, reference can be made to the general control limit requirements for impurities in the art (usually <1%, eg <0.75%).

The pharmaceutical raw material according to any embodiment of the first aspect of the present invention, which also contains (for example, a trace amount) the following compound of formula Ib present as an impurity:

The compound of formula Ib is a hypophosphite ester of the compound of formula Ia.

In the present invention, the compound of formula Ib may also be referred to as impurity Ib or may be referred to as Ib or may be referred to as Ib impurity or other similar titles.

The pharmaceutical raw material according to any embodiment of the first aspect of the present invention, wherein relative to the compound of formula I, the content of the compound of formula Ib is less than 1%, such as less than 0.75%, such as less than 0.5%, such as less than 0.4%, such as Less than 0.3%, such as less than 0.25%.

The pharmaceutical raw material according to any embodiment of the first aspect of the present invention, wherein relative to the compound of formula I, the content of the compound of formula Ib is 0.002-1%, such as 0.002-0.75%, such as 0.002-0.5%, For example, it is 0.002 to 0.4%, for example, it is 0.002 to 0.3%, for example, it is 0.002 to 0.25%.

According to the pharmaceutical raw material according to any embodiment of the first aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is less than 1%, such as less than 0.75% , such as less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.25%.

According to the pharmaceutical raw material according to any embodiment of the first aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is 0.002 to 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%.

According to the pharmaceutical raw material according to any embodiment of the first aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is greater than 100, for example greater than 133, for example Greater than 200, such as greater than 250, such as greater than 333, such as greater than 400.

According to the pharmaceutical raw material according to any embodiment of the first aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is 100 to 50000, for example, 133 ~50000, for example 200~50000, for example 250~50000, for example 333~50000, for example 400~50000.

It has been found that when the pharmaceutical bulk drug of the first aspect of the present invention containing a certain amount of compound of formula Ib is mixed with certain conventional pharmaceutical excipients, especially sugars, the impurity compound of formula Ia will appear as the storage time of the material prolongs Increasing trend related to the content of the compound of formula Ib. That is, when the pharmaceutical bulk drug is mixed with such pharmaceutical excipients, when the compound of formula Ib in the pharmaceutical bulk drug reaches a certain amount or more, the higher the amount of the compound of formula Ib, the greater the growth of the compound of formula Ia; When the compound of formula Ib in the bulk drug is controlled below a certain amount, the increase of the compound of formula Ia is not obvious when the pharmaceutical bulk drug is mixed with the above-mentioned pharmaceutical excipients. This is completely unexpected and has significant pharmaceutical implications, since sugars such as lactose, mannitol, sorbitol, sucrose etc. are very valuable for the preparation of such oral preparations; and when the amount of the compound of formula Ib in the pharmaceutical bulk drug is controlled below a certain amount, the oral preparations made by it will prolong with the storage time. The compound of formula Ia as an impurity will not increase significantly, which is extremely beneficial for providing a clinical preparation with good safety. But when the amount of the formula Ib compound in the pharmaceutical bulk drug is relatively high, the pharmaceutical bulk drug cannot be prepared with the above-mentioned auxiliary materials for oral preparations, which will greatly limit the range of selection of the pharmaceutical bulk drug in the preparation of auxiliary materials , because such excipients are cheap and easy-to-use excipients in pharmacy.

According to the present invention, wherein said [HPLC-A] method is a kind of high-performance liquid chromatography, it can be used to measure various materials simultaneously (comprising the pharmaceutical bulk drug of the present invention and the medicine that is made by this pharmaceutical bulk drug) The content of the compound of formula I, the compound of formula Ia, and the compound of formula Ib in the composition).

According to the present invention, the relevant [HPLC-A] method is specifically as follows:

(i) Chromatographic conditions:

Carry out according to the high performance liquid chromatographic method contained in two appendices VD of Chinese Pharmacopoeia version in 2010,

Chromatographic column: stationary phase is the chromatographic column of octadecylsilane bonded silica gel [this chromatographic column is a typical C ₁₈ chromatographic column, and it can be purchased from commercial sources easily, such as DiamonsilC ₁₈ post, Phenomenex GeminiC ₁₈ post etc., in In the following specific tests of the present invention, if not otherwise specified, what use is DiamonsilC ₁₈ chromatographic column, and its specification can be 250 * 4.6mm, 5 μ m],

Flow rate: 1.0ml/min,

Mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution (adjust pH to 3.0 with potassium hydroxide solution), mobile phase B: acetonitrile, 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°C,

Injection volume: 20μl,

(ii) Dosing

Need testing solution: get the need testing product that contains formula I compound 100mg [this need testing product can be the medicinal raw material medicine of formula I compound, then this moment weighs about 100mg; The pharmaceutical raw material of the compound is a pharmaceutical composition such as a pharmaceutical preparation prepared from raw materials, then the amount of the compound of formula I contained in the material taken at this time is about 100mg], accurately weighed, put in a 100ml measuring bottle, add 50% The acetonitrile solution was dissolved and diluted to the mark, shaken evenly, and filtered if necessary, to be obtained as the test solution (wherein the concentration of the compound of formula I is about 1000 μg/ml);

Control solution: Accurately draw 1ml of the test solution, put it in a 100ml measuring bottle, add 50% acetonitrile solution to dilute to the mark, and shake well; accurately measure 10ml of the solution, put it in a 100ml measuring bottle, add 50% acetonitrile solution to dilute to the mark, shake well , namely, as a control solution (wherein the concentration of the compound of formula I is about 1 μg/ml);

Ia solution: take 10 mg of formula Ia compound, accurately weighed, put in a 100ml measuring bottle, add 50% acetonitrile solution to dissolve and dilute to the mark, shake up (herein referred to as Ia solution, about 100 μg/ml); accurately measure the Put 10ml of the solution in a 100ml measuring bottle, add 50% acetonitrile solution to dilute to the mark, and shake it up to obtain, as Ia solution (wherein the concentration of the compound of formula Ia is about 10 μg/ml);

Ib solution: get formula Ib compound 10mg, weigh accurately, put in 100ml measuring bottle, add 50% acetonitrile solution to dissolve and dilute to scale, shake up (herein referred to as Ib liquid, about 100 μ g/ml); Accurately measure the Put 10ml of the solution in a 100ml measuring bottle, add 50% acetonitrile solution to dilute to the mark, and shake up to get final product, as Ib solution (wherein the compound concentration of formula Ib is about 10 μg/ml);

System suitability solution: Accurately draw 1ml each of the test solution, 100μg/ml Ia solution, and 100μg/ml Ib solution respectively, put them in the same 10ml measuring bottle, add 50% acetonitrile solution to dilute to the mark, shake well, That is (I100μg/ml+Ia10μg/ml+Ib10μg/ml);

(iii) Determination and calculation of results:

Precisely draw 20 μL each of the test solution, control solution, Ia solution, Ib solution, and system suitability solution into the liquid chromatograph, and record the chromatograms respectively;

Peak assignment and retention time: determine the retention time of the formula I compound, the formula Ia compound, and the formula Ib compound according to the obtained chromatograms of the control solution, the Ia solution, and the Ib solution, and determine the system suitability for each in the chromatogram accordingly. The attribution of the peak and the attribution of each chromatographic peak in the test solution (generally speaking, the order of the peaks in the chromatogram obtained from the system suitability solution is the compound of formula I, the compound of formula Ib, the compound of formula Ia);

System suitability test: in the chromatogram obtained from the system suitability solution test, the resolution between the peaks of the compound of formula I and the compound of formula Ib is at least 1.5;

In the test solution chromatogram, if the compound of formula Ia is detected, the following calculation formula can be used to calculate the content of the impurity Ia:

The meaning of the above term "impurity Ia content (%)" is equivalent to the phrase "the content of the compound of formula Ia relative to the compound of formula I" mentioned above in the present invention.

In the test solution chromatogram, if the compound of formula Ib is detected, the following calculation formula can be used to calculate the content of this impurity Ib:

The meaning of the above-mentioned term "impurity Ib content (%)" is equivalent to the phrase "the content of the compound of formula Ib relative to the compound of formula I" mentioned above in the present invention.

In the test solution chromatogram, if the compound of formula Ia is detected, the following calculation formula can be used to calculate the ratio (%) of the peak area of the compound of formula Ia and the peak area of the compound of formula I:

In the test solution chromatogram, if detect the compound of formula Ib, the ratio (%) of the peak area of the compound of formula Ib and the peak area of the compound of formula I can be calculated by the following calculation formula:

Or, in the present invention, can also use " ratio of formula I compound peak area and formula Ia compound peak area " and " formula I compound peak area and formula Ib compound peak area ratio " represent formula Ia compound and formula Ib compound relative In the amount of formula I compound.

In the test solution chromatogram, if the compound of formula Ia is detected, the following calculation formula can be used to calculate the ratio of the peak area of the compound of formula Ia to the peak area of the compound of formula I:

In the chromatogram of the test solution, if the compound of formula Ib is detected, the following formula can be used to calculate the ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I:

In addition, by the above [HPLC-A] method, it is also possible to measure the formula I in the test product by the external standard method by preparing a solution of a compound reference substance of formula I (as a reference substance for analysis, for example, its content is greater than 99.8%) at a considerable concentration. Content of compound I (absolute content, ie the mass of the compound of formula I per gram of test sample, %, w/w).

The "test article" tested by the above [HPLC-A] method, this material can be a pharmaceutical raw material provided in the form of a compound of formula I or its crude product, or it can use them as active ingredients and add other pharmaceutical ingredients. For pharmaceutical compositions formulated with excipients, such as pharmaceutical preparations, such as tablets, etc., whether it is a pharmaceutical raw material or a crude product or a pharmaceutical composition, it is converted into the containing formula when weighing the test product during the preparation of the test solution. The amount of about 100 mg of compound I is enough, and the calculation of the content of impurity Ia and impurity Ib will not be affected by the weighing accuracy of the test product or the presence of other ingredients such as pharmaceutical excipients.

In addition, in view of the fact that the analysis method is easy to be improved, and the compound of formula Ia and the compound of formula Ib are easy to obtain. Therefore, when describing various materials such as pharmaceutical raw materials or crude products thereof or the content of the formula Ia compound and the formula Ib compound in the pharmaceutical composition prepared from it in the present invention, it is not necessary to limit whether it must be according to [HPLC-A ] The result obtained by the method test, especially when describing the content of impurities in the test product relative to the compound of formula I.

Furthermore, the second aspect of the present invention provides a pharmaceutical composition, which is prepared from the pharmaceutical raw material and pharmaceutical excipients according to any embodiment of the second aspect of the present invention.

The pharmaceutical composition according to any embodiment of the second aspect of the present invention, wherein the compound of formula I accounts for 10-90%, such as 20-80%, such as 30-70%, of the total weight of the pharmaceutical composition.

The pharmaceutical composition according to any embodiment of the second aspect of the present invention, which also contains (for example traces) the following compound of formula Ia present as an impurity:

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

According to the pharmaceutical composition according to any embodiment of the second aspect of the present invention, wherein relative to the compound of formula I, the content of the compound of formula Ia is 0.002-2%, such as 0.002-1.75%, such as 0.002-1.5%, such as It is 0.002 to 1.25%, for example, it is 0.002 to 1.0%, for example, it is 0.002 to 0.75%.

According to the pharmaceutical composition according to any embodiment of the second aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ia to the peak area of the compound of formula I is less than 2%, such as less than 1.75%, For example less than 1.5%, such as less than 1.25%, such as less than 1.0%, such as less than 0.75%.

According to the pharmaceutical composition according to any embodiment of the second aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ia to the peak area of the compound of formula I is 0.002 to 2%, for example 0.002 ~1.75%, such as 0.002-1.5%, such as 0.002-1.25%, such as 0.002-1.0%, such as 0.002-0.75%.

According to the pharmaceutical composition of any embodiment of the second aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ia is greater than 50, such as greater than 57, such as greater than 67, such as greater than 80, such as greater than 100, such as greater than 133.

According to the pharmaceutical composition according to any embodiment of the second aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ia is 50 to 50000, for example, 57 to 50000 50000, such as 67-50000, such as 80-50000, such as 100-50000, such as 133-50000.

The pharmaceutical composition according to any embodiment of the second aspect of the present invention, which also contains (for example traces) the following compound of formula Ib present as an impurity:

The pharmaceutical composition according to any embodiment of the second aspect of the present invention, wherein relative to the compound of formula I, the content of the compound of formula Ib is less than 1%, such as less than 0.75%, such as less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.25%.

According to the pharmaceutical composition of any embodiment of the second aspect of the present invention, wherein relative to the compound of formula I, the content of the compound of formula Ib is 0.002-1%, such as 0.002-0.75%, such as 0.002-0.5%, such as It is 0.002 to 0.4%, such as 0.002 to 0.3%, such as 0.002 to 0.25%.

According to the pharmaceutical composition of any embodiment of the second aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is less than 1%, such as less than 0.75%, For example less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.25%.

According to the pharmaceutical composition according to any embodiment of the second aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is 0.002 to 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%.

According to the pharmaceutical composition of any embodiment of the second aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is greater than 100, such as greater than 133, such as greater than 200, such as greater than 250, such as greater than 333, such as greater than 400.

According to the pharmaceutical composition according to any embodiment of the second aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is 100 to 50000, for example, 133 to 50000 50000, for example, 200-50000, for example, 250-50000, for example, 333-50000, for example, 400-50000.

The pharmaceutical composition according to any embodiment of the second aspect of the present invention, wherein the pharmaceutical excipients include sugars selected from the group consisting of lactose, mannitol, sorbitol, sucrose and the like.

The pharmaceutical composition according to any embodiment of the second aspect of the present invention is in the form of tablets, capsules, or granules.

The pharmaceutical composition according to any embodiment of the second aspect of the present invention, which is in the form of a unit dosage pharmaceutical preparation. In one embodiment, each unit dose of the pharmaceutical preparation includes 1-1000 mg of the compound of formula I, for example 10-750 mg of the compound of formula I, for example 20-500 mg of the compound of formula I.

According to the pharmaceutical composition according to any embodiment of the second aspect of the present invention, it is in the form of tablets or capsules, which also optionally includes pharmaceutically conventional diluents (such as but not limited to starch and its derivatives such as pregelatinized starch, modified starch, microcrystalline cellulose, etc.), disintegrants (such as but not limited to crospovidone, sodium carboxymethyl starch, etc.), binders (such as but not limited to PVP, HPMC, etc.), lubricants Agents/glidants (such as but not limited to magnesium stearate, talc, etc.) and the like.

According to the pharmaceutical composition according to any embodiment of the second aspect of the present invention, it is sealed at 40°C and stored in the dark for 5 months, and the concentration of a certain impurity after 5 months of treatment under this condition is measured and calculated relative to 0 months. Content increase percentage, wherein according to [HPLC-A] determination, impurity Ia content increase percentage is lower than 200%, such as lower than 150%, such as lower than 100%, such as lower than 75%, such as lower than 50%. This test method can be referred to as 40°C-5 months test for short in the present invention. The percentage increase in the content of a certain impurity in the term is calculated according to the following formula:

The above calculation formula can be used to calculate the percentage increase of impurity Ia or impurity Ib of various samples before and after treatment at 40°C-5 months. The closer the value is to 0, the smaller the increase of the impurity.

Further, the third aspect of the present invention provides a pharmaceutical composition, which includes the following compound of formula I as an active ingredient

and pharmaceutical excipients.

The pharmaceutical composition according to any embodiment of the third aspect of the present invention, wherein the compound of formula I accounts for 10-90%, such as 20-80%, such as 30-70%, of the total weight of the pharmaceutical composition.

The pharmaceutical composition according to any embodiment of the third aspect of the present invention, which also contains (for example traces) the following compound of formula Ia present as an impurity:

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

According to the pharmaceutical composition according to any embodiment of the third aspect of the present invention, relative to the compound of formula I, the content of the compound of formula Ia is 0.002-2%, such as 0.002-1.75%, such as 0.002-1.5%, such as It is 0.002 to 1.25%, for example, it is 0.002 to 1.0%, for example, it is 0.002 to 0.75%.

According to the pharmaceutical composition according to any embodiment of the third aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ia to the peak area of the compound of formula I is less than 2%, such as less than 1.75%, For example less than 1.5%, such as less than 1.25%, such as less than 1.0%, such as less than 0.75%.

According to the pharmaceutical composition according to any embodiment of the third aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ia to the peak area of the compound of formula I is 0.002 to 2%, for example 0.002 ~1.75%, such as 0.002-1.5%, such as 0.002-1.25%, such as 0.002-1.0%, such as 0.002-0.75%.

According to the pharmaceutical composition of any embodiment of the third aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ia is greater than 50, such as greater than 57, such as greater than 67, such as greater than 80, such as greater than 100, such as greater than 133.

According to the pharmaceutical composition according to any embodiment of the third aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ia is 50 to 50000, for example, 57 to 50000 50000, such as 67-50000, such as 80-50000, such as 100-50000, such as 133-50000.

The pharmaceutical composition according to any embodiment of the third aspect of the present invention, which also contains (for example, a trace amount) the following compound of formula Ib present as an impurity:

The pharmaceutical composition according to any embodiment of the third aspect of the present invention, wherein relative to the compound of formula I, the content of the compound of formula Ib is less than 1%, such as less than 0.75%, such as less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.25%.

According to the pharmaceutical composition according to any embodiment of the third aspect of the present invention, wherein relative to the compound of formula I, the content of the compound of formula Ib is 0.002-1%, such as 0.002-0.75%, such as 0.002-0.5%, such as It is 0.002 to 0.4%, such as 0.002 to 0.3%, such as 0.002 to 0.25%.

According to the pharmaceutical composition according to any embodiment of the third aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is less than 1%, such as less than 0.75%, For example less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.25%.

According to the pharmaceutical composition according to any embodiment of the third aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is 0.002 to 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%.

According to the pharmaceutical composition of any embodiment of the third aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is greater than 100, such as greater than 133, such as greater than 200, such as greater than 250, such as greater than 333, such as greater than 400.

According to the pharmaceutical composition according to any embodiment of the third aspect of the present invention, it is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is 100-50000, for example, 133- 50000, for example, 200-50000, for example, 250-50000, for example, 333-50000, for example, 400-50000.

The pharmaceutical composition according to any embodiment of the third aspect of the present invention, wherein the pharmaceutical excipients include sugars selected from the group consisting of lactose, mannitol, sorbitol, sucrose and the like.

The pharmaceutical composition according to any embodiment of the third aspect of the present invention is in the form of tablets, capsules, or granules.

The pharmaceutical composition according to any embodiment of the third aspect of the present invention, which is in the form of a unit dosage pharmaceutical preparation. In one embodiment, each unit dose of the pharmaceutical preparation includes 1-1000 mg of the compound of formula I, for example 10-750 mg of the compound of formula I, for example 20-500 mg of the compound of formula I.

The pharmaceutical composition according to any embodiment of the third aspect of the present invention, which is in the form of tablets or capsules, which also optionally includes pharmaceutically conventional diluents (such as but not limited to starch and its derivatives such as pregelatinized starch, modified starch, microcrystalline cellulose, etc.), disintegrants (such as but not limited to crospovidone, sodium carboxymethyl starch, etc.), binders (such as but not limited to PVP, HPMC, etc.), lubricants Agents/glidants (such as but not limited to magnesium stearate, talc, etc.) and the like.

According to the pharmaceutical composition according to any embodiment of the third aspect of the present invention, it is sealed at 40°C and stored in the dark for 5 months, and the concentration of a certain impurity after 5 months of treatment under this condition is measured and calculated relative to 0 months. Content increase percentage, wherein according to [HPLC-A] determination, impurity Ia content increase percentage is lower than 200%, such as lower than 150%, such as lower than 100%, such as lower than 75%, such as lower than 50%. This test method can be referred to as 40°C-5 months test for short in the present invention.

The fourth aspect of the present invention provides the following formula Ia compound

In the preparation of pharmaceutical raw materials for detection with the compound of the following formula I as an active ingredient

Or use the compound of formula I as an active ingredient (for example, the application of the pharmaceutical composition made of the pharmaceutical raw material) as a reference product.

According to the application of any embodiment of the fourth aspect of the present invention, wherein the reference substance is determined according to [HPLC-A], the chromatographic purity of the compound of formula Ia is greater than 95%, such as greater than 96%, such as greater than 97%, such as greater than 98% .

According to the application of any embodiment of the fourth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the content of the compound of formula Ia in the pharmaceutical raw material or pharmaceutical composition is relative to the compound of formula I It is less than 2%, such as less than 1.75%, such as less than 1.5%, such as less than 1.25%, such as less than 1.0%, such as less than 0.75%.

According to the application of any embodiment of the fourth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the content of the compound of formula Ia in the pharmaceutical raw material or pharmaceutical composition is relative to the compound of formula I For example, it is 0.002-2%, such as 0.002-1.75%, such as 0.002-1.5%, such as 0.002-1.25%, such as 0.002-1.0%, such as 0.002-0.75%.

According to the application of any embodiment of the fourth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the pharmaceutical raw material or pharmaceutical composition is determined according to the [HPLC-A] method of the present invention, wherein The ratio of the peak area of the compound of formula Ia to the peak area of the compound of formula I is less than 2%, such as less than 1.75%, such as less than 1.5%, such as less than 1.25%, such as less than 1.0%, such as less than 0.75%.

According to the application of any embodiment of the fourth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the pharmaceutical raw material or pharmaceutical composition is determined according to the [HPLC-A] method of the present invention, wherein The ratio of the peak area of the compound of formula Ia to the peak area of the compound of formula I is 0.002 to 2%, for example 0.002 to 1.75%, for example 0.002 to 1.5%, for example 0.002 to 1.25%, for example 0.002 to 1.0%, for example 0.002 ~0.75%.

According to the application of any embodiment of the fourth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the pharmaceutical raw material or pharmaceutical composition is determined according to the [HPLC-A] method of the present invention, wherein The ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ia is greater than 50, such as greater than 57, such as greater than 67, such as greater than 80, such as greater than 100, such as greater than 133.

According to the application of any embodiment of the fourth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the pharmaceutical raw material or pharmaceutical composition is determined according to the [HPLC-A] method of the present invention, wherein The ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ia is 50-50000, such as 57-50000, such as 67-50000, such as 80-50000, such as 100-50000, such as 133-50000.

The fifth aspect of the present invention provides the following formula Ib compound

In the preparation of pharmaceutical raw materials for detection with the compound of the following formula I as an active ingredient

Or use the compound of formula I as an active ingredient (for example, the application of the pharmaceutical composition made of the pharmaceutical raw material) as a reference product.

According to the application of any embodiment of the fifth aspect of the present invention, wherein the reference substance is determined according to [HPLC-A], the chromatographic purity of the compound of formula Ib is greater than 95%, such as greater than 96%, such as greater than 97%, such as greater than 98%.

According to the application of any embodiment of the fifth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the content of the compound of formula Ib in the pharmaceutical raw material or pharmaceutical composition is relative to the compound of formula I For example, less than 1%, such as less than 0.75%, such as less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.25%.

According to the application of any embodiment of the fifth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the content of the compound of formula Ib in the pharmaceutical raw material or pharmaceutical composition is relative to the compound of formula I For example, it is 0.002-1%, such as 0.002-0.75%, such as 0.002-0.5%, such as 0.002-0.4%, such as 0.002-0.3%, such as 0.002-0.25%.

According to the application of any embodiment of the fifth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the pharmaceutical raw material or pharmaceutical composition is determined according to the [HPLC-A] method of the present invention, wherein The ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is less than 1%, such as less than 0.75%, such as less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.25%.

According to the application of any embodiment of the fifth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the pharmaceutical raw material or pharmaceutical composition is determined according to the [HPLC-A] method of the present invention, wherein The ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is 0.002 to 1%, such as 0.002 to 0.75%, such as 0.002 to 0.5%, such as 0.002 to 0.4%, such as 0.002 to 0.3%, such as 0.002 ~0.25%.

According to the application of any embodiment of the fifth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the pharmaceutical raw material or pharmaceutical composition is determined according to the [HPLC-A] method of the present invention, wherein The ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is greater than 100, such as greater than 133, such as greater than 200, such as greater than 250, such as greater than 333, such as greater than 400.

According to the application of any embodiment of the fifth aspect of the present invention, it is used to monitor the pharmaceutical raw material or pharmaceutical composition to ensure that the pharmaceutical raw material or pharmaceutical composition is determined according to the [HPLC-A] method of the present invention, wherein The ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is 100-50000, such as 133-50000, such as 200-50000, such as 250-50000, such as 333-50000, such as 400-50000.

Further, the sixth aspect of the present invention provides a method for controlling the quality of pharmaceuticals, wherein the pharmaceuticals are pharmaceutical raw materials with the compound of formula I as the active ingredient or a pharmaceutical composition prepared from the pharmaceutical raw materials, the method comprising: The impurity compound of formula Ib in the medicine is controlled within a certain range, so that the impurity compound of formula Ia in the medicine shows a low increase during long-term storage.

According to the method according to any embodiment of the sixth aspect of the present invention, wherein the content of the compound of formula Ib is less than 1%, such as less than 0.75%, relative to the compound of formula I in the drug (ie, pharmaceutical raw material or pharmaceutical composition) , such as less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.25%.

The method according to any embodiment of the sixth aspect of the present invention, wherein relative to the compound of formula I in the drug, the content of the compound of formula Ib is 0.002-1%, such as 0.002-0.75%, such as 0.002-0.5% , for example 0.002 to 0.4%, for example 0.002 to 0.3%, for example 0.002 to 0.25%.

According to the method of any embodiment of the sixth aspect of the present invention, wherein the drug is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is less than 1%, such as less than 0.75% , such as less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.25%.

According to the method of any embodiment of the sixth aspect of the present invention, wherein the drug is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula Ib to the peak area of the compound of formula I is 0.002 to 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%.

According to the method of any embodiment of the sixth aspect of the present invention, wherein the drug is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is greater than 100, such as greater than 133, such as Greater than 200, such as greater than 250, such as greater than 333, such as greater than 400.

According to the method of any embodiment of the sixth aspect of the present invention, wherein the drug is determined according to the [HPLC-A] method of the present invention, wherein the ratio of the peak area of the compound of formula I to the peak area of the compound of formula Ib is 100 to 50000, for example, 133 ~50000, for example 200~50000, for example 250~50000, for example 333~50000, for example 400~50000.

According to the method according to any embodiment of the sixth aspect of the present invention, wherein the drug is sealed at 40°C and stored away from light for 5 months, and the relative time of a certain impurity after 5 months of treatment under this condition is measured and calculated relative to 0 months The content increase percentage, wherein according to [HPLC-A] determination, the impurity Ia content increase percentage is lower than 200%, for example lower than 150%, for example lower than 100%, for example lower than 75%, for example lower than 50%.

According to the method of any embodiment of the sixth aspect of the present invention, said making the drug show a low increase in the impurity compound of formula Ia during long-term storage means that the drug is sealed at 40°C and placed in the dark for 5 months, Measure and calculate the percentage increase of the content of a certain impurity after 5 months of treatment under this condition relative to that at 0 months, wherein according to [HPLC-A] determination, the percentage increase of the content of impurity Ia is lower than 200%, for example lower than 150%, for example Below 100%, such as below 75%, such as below 50%.

Further, the seventh aspect of the present invention provides a method for preparing the pharmaceutical raw material according to any embodiment of the first aspect of the present invention, the method comprising the following steps:

(1) To N-quinolinoyl-L-aspartic acid, 3-isopropyl-3-[(2S,3S)-2-hydroxyl-3-(phenylmethoxycarbonyl)amino-4- Stirring of tert-butyl phenylbutyl]carbazate, benzotriazol-1-yloxytris(dimethylamino)phosphonium fluorophosphate and 1-hydroxybenzotriazole in anhydrous dimethylformamide Add N,N-diisopropylethylamine to the solution, stir at room temperature to complete the reaction, dilute the reactant with ethyl acetate, and dilute with water, 2% potassium bisulfate, 5% sodium bicarbonate and saturated aqueous sodium chloride Washed, dried over anhydrous magnesium sulfate, evaporated the solvent under reduced pressure, and purified with ethyl acetate by silica gel column chromatography to obtain 3-isopropyl-3-[(2S,3S)-2-hydroxyl-3-(N-quinone Phenoyl-L-aspartoyl) amino-4-phenylbutyl] tert-butyl carbazate (i.e. compound of formula Ia)

(2) At room temperature and under a nitrogen atmosphere, dicyclohexylcarbodiimide, 3-isopropyl-3-[(2S, 3S)-2-hydroxyl-3-(N-quinolinoyl-L-tianmen Naginyl)amino-4-phenylbutyl]carbazate tert-butyl (i.e. compound of formula Ia) and anhydrous phosphorous acid were mixed in anhydrous pyridine, after stirring and reacting at 60°C, the solvent was evaporated under reduced pressure, and carbonic acid Treat with aqueous sodium hydrogen solution, stir vigorously at room temperature for 1 hour, filter out the precipitate, wash with water, acidify the filtrate to about pH 1.5 with concentrated hydrochloric acid; extract and absorb the product with ethyl acetate, dehydrate the organic matter with anhydrous magnesium sulfate, solvent Evaporation gave 3-isopropyl-3-[(2S,3S)-2-phosphinocarboxy-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenylbutyl] Tert-butyl carbazate (i.e. compound of formula Ib)

(3) Make 3-isopropyl-3-[(2S, 3S)-2-phosphinocarboxy-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenylbutyl ] The suspension of tert-butyl carbazate (i.e. the compound of formula Ib) in hexamethyldisilazane was stirred at 120±5°C to make the reaction mixture homogeneous, and bis(trimethylsilyl)peroxide was added and then stirred at the above temperature for 1 hour; after the reaction mixture was cooled to room temperature, it was evaporated to dryness in vacuo; the residue was dissolved in methanol, evaporated to dryness under reduced pressure, and then dissolved in 0.1M aqueous sodium bicarbonate solution, and the resulting mixture was Acidify to about pH 1.5 with concentrated hydrochloric acid, saturate with sodium chloride, and extract with ethyl acetate. The combined organic phases were dehydrated over anhydrous magnesium sulfate and evaporated to dryness to give 3-isopropyl-3-[(2S,3S)-2-phosphinecarboxy-3-(N-quinolinoyl-L-tianmen Butyl)amino-4-phenylbutyl]carbazate tert-butyl

(4) 3-isopropyl-3-[(2S,3S)-2-phosphine carboxyoxy-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenyl Add tert-butyl butyl]carbazate to aqueous sodium bicarbonate solution, stir until the solution becomes clear; add isopropanol, stir the mixture, and filter; the obtained filtrate is distilled to remove the solvent under vacuum at 55-60°C; the material is cooled to 25-35°C, add isopropanol, heat up to 55-60°C, distill off the solvent in vacuum, dry, and wash the obtained solid with isopropanol, then evaporate to dryness under vacuum at 70-75°C to obtain the formula I The disodium salt compound is 3-isopropyl-3-[(2S,3S)-2-phosphine carboxyoxy-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenylbutyl Disodium tert-butyl carbazate

(5) Purification: Add the product obtained in step (4) to a mixed solvent of heptane:ethyl acetate=10:2~5, stir at 70-75°C for 1-3 hours, cool to room temperature, and filter out the solid The precipitate was washed twice with 20 ml of heptane, and then evaporated to dryness at 60° C. under vacuum to obtain the obtained product; this step (5) was optionally repeated as needed.

Any aspect of the present invention or any technical feature of any implementation of this any aspect is also applicable to any other implementation or any implementation of any other aspect, as long as they do not contradict each other, of course When applicable, the corresponding features can be appropriately modified if necessary. Various aspects and features of the present invention are further described below.

All the documents cited in the present invention are incorporated herein by reference in their entirety, and if the meaning expressed in these documents is inconsistent with the present invention, the expression of the present invention shall prevail. In addition, various terms and phrases used in the present invention have common meanings known to those skilled in the art. Even so, the present invention still hopes to make a more detailed description and explanation of these terms and phrases here. The terms and phrases mentioned are as follows: If there is any inconsistency with the known meaning, the meaning expressed in the present invention shall prevail.

Various aspects of the invention are further described below.

Some embodiments of the present invention provide pharmaceutical raw materials with the compound of formula I as the active ingredient. The compound of formula I is phosphorylated to form hypophosphite from the compound of formula Ia, that is, to form the compound of formula Ib. The hypophosphite of the compound of formula Ib is Phosphate ester is oxidized to further form disodium salt to obtain the compound of formula I. In the preparation process, the introduction of impurities is unavoidable, for example, the compound of formula Ia or compound of formula Ib may be introduced into the drug substance with the compound of formula I as the active ingredient or into the pharmaceutical preparation prepared from the drug substance middle.

The above-mentioned formula Ia and formula Ib are introduced into the compound of formula I as impurities, which need to be avoided for using the compound of formula I as a pharmaceutical raw material and making it into a pharmaceutical composition such as a pharmaceutical preparation.

Although chemists will try their best to remove known and unknown impurities when preparing the compound of formula I, under the situation of comprehensive consideration of cost-benefit relationship, trace impurities are unavoidable, and when these trace impurities are in controllable ( They are controllable when they are used as raw materials, and they are also controllable when they are made into preparations), which are generally acceptable in the field of pharmacy. However, it is regrettable that it has been found that when the compound of formula Ib as an impurity in the compound of formula I is higher than a certain amount limit, the raw material drug with the compound of formula I as the active ingredient is not compatible with conventional preparation auxiliary materials, especially certain When a pharmaceutical composition such as a pharmaceutical preparation is prepared in combination with some sugars, the compound of formula Ia in the pharmaceutical composition will increase significantly over time, and the biological properties of the compound of formula Ia such as its absorption Nature is unacceptable. Therefore, it is extremely beneficial to control the impurity compound of formula Ib below the above-mentioned limit in the raw material drug containing the compound of formula I as the active ingredient or the pharmaceutical composition made from it, such as a pharmaceutical preparation.

HIV protease inhibitors that have been widely used clinically include: saquinavir, indinavir, ritonavir, nelfinavir, amprenavir ( amprenavir) and lopinavir (lopinavir), atazanavir and fosamprenavir. Saquinavir is a potent and highly selective protease inhibitor. Unlike other nucleotide derivatives, it does not need to be metabolized and acts directly. The half-life: 13.2h. If it is >90, this product has effects on both HIV-1 and HIV-2, and has a high degree of specificity on viral proteases, but has no effect on aspartase closely related to humans. In vitro experiments show that this is a so far The strongest anti-HIV virus drug that has been seen so far, this product is usually well tolerated, and there will be diarrhea, headache, abdominal distension, hyperlipidemia, and lipodystrophy. Ritonavir is a potent protease inhibitor with a half-life of 3 to 5 hours. Taking it with food can enhance tolerance. To reduce adverse reactions, it is recommended to increase the dose gradually. Side effects, diarrhea, fatigue, and decreased concentration , hyperlipidemia. Amprenavir works by inhibiting the virus, the encoded protease, resulting in an inability to process gag and gag-pol, resulting in non-functional virus, indicated for HIV-1 and HIV-2 infection, contraindicated in patients clinically allergic to any of its components, side effects, nausea , diarrhea, bloating, rash. Indinavir (Indinair) is another potent protease inhibitor developed by meck company in the United States. It is effective against HIV-1 and has good oral bioavailability. For HIV that has not been treated with lamivudine or protease inhibitors before Infected persons, this product plus zidovudine and lamivudine can slow down the progression of the disease and reduce mortality better than using only two nucleoside drugs. Atazanavir (ATV) is currently the only protease inhibitor that only needs to be taken once a day. It is now in its clinical trial phase III. Its main advantages lie in its powerful antiviral effect, unique drug resistance map, and fat metabolism. minimal impact.

At present, there is still hope for a new treatment program for HIV clinically. The compound of formula I provided by the invention is an effective anti-HIV drug, has good oral bioavailability, is suitable for the treatment of HIV infection and has effects on both HIV-1 and HIV-2. When the compound of formula I is administered to humans, its typical daily dosage is 50-500 mg, such as 50-250 mg, such as 50-200 mg.

In particular, the pharmaceutical raw material or pharmaceutical composition prepared from the compound of formula I of the present invention has excellent properties.

detailed description

The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made in the present invention without departing from the spirit and scope of the present invention. The present invention provides general and/or specific descriptions of the materials and test methods used in the tests. While many of the materials and methods of manipulation which are employed for the purposes of the invention are well known in the art, the invention has been described here in as much detail as possible.

A, compound preparation example part

Example 1: Preparation of tert-butyl 3-isopropyl-[(2R,3S)-2-hydroxyl-3-(phenylmethoxycarbonyl)amino-4-phenylbutyl]carbazate

Step A: Preparation of tert-butyl 3-isopropylcarbazinate

The title compound can be prepared by the method of Dutta et al. (J.C: S.Perkin/1975, 1712-1720) or by mixing 13.2 g (0.1 mol) tert-butyl carbazate and 6 g (0.103 mol) of acetone and 12.5g (0.1mol) of anhydrous magnesium sulfate in 100ml of dichloromethane was stirred for 12 hours, after the desiccant was removed by filtration, the filtrate was evaporated to dryness under reduced pressure, and after crystallization from cyclohexane, 16.9g (98% yield) The corresponding hydrazone has a melting point of 104-105°C. In a nitrogen atmosphere under a greenhouse, add 12ml (0.094mol) trimethylchlorosilane to a suspension of 2.04g (0.094mol) lithium borohydride in 100ml anhydrous tetrahydrofuran, and after 30 minutes, slowly add 13.45g (0.078 mol) hydrazone, and stirring was continued for 2 hours. Then, 50ml of methanol was carefully added, and the mixture was evaporated to dryness under reduced pressure, the residue was partitioned between diethyl ether (150ml) and water (50ml), the organic phase was dried over anhydrous magnesium sulfate and filtered, and the dried hydrogen chloride was passed through The filtrate was filtered and the white solid formed was removed by filtration, washed with a portion of fresh ether and dried to give (10.5g) the hydrochloride salt of the title compound. It was converted to its free base form by partitioning between hexanes (150ml) and 20% aqueous potassium hydroxide to give 8.3g (61%) of product.

Step B: Preparation of tert-butyl 3-isopropyl-[(2R,3S)-2-hydroxy-3-(phenylmethoxycarbonyl)amino-4-phenylbutyl]carbazate

A mixture of 0.15 g (0.41 mmol) of N-CBZ-L-phenylalanine chloromethyl ketone and 1 ml of saturated sodium iodide solution in anhydrous dimethylformamide was stirred for 15 minutes at room temperature. Thereto was added 0.074 g (0.47 mmol) of tert-butyl 3-isopropylcarbazinate, followed by 0.095 g (1.13 mmol) of sodium bicarbonate. After stirring at room temperature for 6 hours, 0.051 g (1.3 mmol) of sodium borohydride was added and stirring was continued for a further 30 minutes. The solution was diluted to 30 ml with ethyl acetate, washed with 2% aqueous potassium hydrogensulfate solution, water and saturated aqueous sodium chloride solution, and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residual poplar was purified by flash chromatography (silica gel; hexane/ethyl acetate 20:5) to obtain the title compound with a melting point of 118-119.5° C. and a yield of 49%; R(A)= 0.11; R(B) = 0.47;

NMR (CDCl ₃ ) 1.0 (m, 6H, isopropyl CH ₃ ); 1.44 (S, 9H, tert-butyl CH ₃ ); 2.62 (m, 2H, butyl CH ₂ -1); 2.75-3.2 (m , 3H, butyl CH-3, CH ₂ -4); 3.47 (m, 1H, isopropyl CH); 3.89 (m, 1H, butyl CH-2); 4.44 (broad S, 1H, OH); 4.6 (broad m, 1H, NH); 5.03 (S.2H, methoxy _CH2 ); 5.3 (broad S, 1H, carbazate NH); 7.23 (m.1OH, aryl H).

Example 2: Preparation of 3-isopropyl-3-[(2R, 3S)-2-hydroxyl-3-(N-quinolinoyl-L-valyl)amino-4-phenylbutyl]hydrazine tert-butyl formate

Step A: Preparation of N-quinolinoyl-L-valine

A mixture of 0.62 g (3.6 mmol) of quinolinic acid and 0.61 g (3.76 mmol) of 1,1'-carbonyldiimidazole in 1 ml of dry 1,4-dioxane was stirred for 30 minutes at room temperature. A solution of 0.43 g (3.7 mmol) of L-valine and 0.155 g (3.7 mmol) of lithium hydroxide in 1 ml of water was added thereto, and the resulting mixture was vigorously stirred at room temperature for about 4 hours. The mixture was diluted with water to 10 ml, cooled (ice-water bath), then acidified to pH ca. 3 with 1N hydrochloric acid and allowed to stand at 4°C for 2 hours. The crystals formed were removed by filtration, washed 3 times with 5 ml of cold water and dried over phosphorus pentoxide under high vacuum to give 0.75 g of product. Yield=76%; melting point is 134-136°C;

NMR (DMSO-d ₆ ) 1.03 (d, 6H, ValCH ₃ ); 2.3 (m, 1H, ValCH-β); 3.35 (broad S, 1H, OH); 4.49 (q, 1H, ValCH-α); 7.5 -8.3 (m, 5H, aryl H); 8.5-8.76 (m, 2H, aryl H, NH).

Step B: Preparation of tert-butyl 3-isopropyl-3-[(2R,3S)-2-hydroxy-3-(phenylmethoxycarbonyl)amino-4-phenylbutyl]carbazate

To a cooled solution of 0.113 g (0.24 mmol) of the product of Example 1 in 2 ml of methanol was added 0.1 g of 10% palladium on activated carbon followed by 0.1 g of sodium borohydride under a nitrogen atmosphere. The reaction was allowed to warm to room temperature and stirred for 1 hour, then the catalyst was removed by filtration and washed with a portion of fresh methanol. The combined filtrates were treated with 1 mL of 0.1N aqueous hydrochloric acid and evaporated to dryness under reduced pressure. The residue was treated with 5 mL of 0.1N potassium hydroxide and the product was dissolved in 30 mL of ether. The organic phase was washed with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate and evaporated under reduced pressure to give 0.0797 g (99% yield) of the product of step B, which was used in the next step without further purification.

Step C: Preparation of 3-isopropyl-3-[(2R,3S)-2-hydroxy-3-(N-quinolinoyl-L-valyl)amino-4-phenylbutyl]carbazate tert-butyl ester

To 0.0643 g (0.24 mmol) of the acid from step A, 0.0797 g (0.236 mmol) of the amine from step B and 0.032 g (0.24 mmol) of 1-hydroxybenzotriazole in 0.5 ml of anhydrous dimethylformamide 0.071 g (0.24 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methyl iodide was added to the mixture. After stirring overnight at room temperature, the mixture was diluted to 30 ml with ethyl acetate, washed successively with water, 5% aqueous sodium bicarbonate solution, 2% aqueous potassium hydrogensulfate solution and saturated sodium chloride solution, and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, hexane/ethyl acetate 3:2) to obtain 0.091 g (65% yield) of the title compound with a melting point of 186-189° C.: Rf(B)= 0.19; Rf(C)=0.83; NMR (CDCl ₃ ) 1.0 (m, 12H, Val and isopropyl CH ₃ ); 1.71 (S, 9H, tert-butyl CH ₃ ); 2.3 (m, 1H, ValCH- β); 2.5-3.27 (m, 3H, butyl CH-3, CH ₂ ); 3.5 (m, 1H, isopropyl CH); 4.31 (m, 2H, ValCH-2, OH); 5.43 (broad S , 1H, carbazate NH); 6.22 (broad d, 1H, butyl NH); 6.7-8.73 (m, 12H, aryl H, NH).

Example 3: Preparation of 3-isopropyl-3-[(2R,3S)-2-hydroxyl-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenylbutyl]hydrazine tert-butyl formate

Step A: Preparation of N-quinolinoyl-L-aspartic acid

When aspartic acid was used instead of L-valine in Step A of Example 2, the title compound was obtained in the same manner, with a melting point of 200-203° C. and a yield of 85%.

NMR (DMSO-d ₆ ) 3.0 (m, 2H, aSnCH ₂ ); 5.0 (m, 1H, aSnCH-2); 6.3 (broad S, 1H, OH); 6.55 (broad 2, 1H, NH ₂ ); 7.3 (broad S, 1H, NH ₂ ); 7.55-8.6 (m, 6H, aromatic H); 9.22 (d, 1H, NH).

Step B: Preparation of 3-isopropyl-3-[(2R,3s)-2-hydroxy-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenylbutyl]carbazate tert-butyl ester

To the product of Step A (0.111g; 0.386mmol), the product of Step B of Example 2 (0.13022g; 0.386mmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium fluorophosphate (0.205g 0.46mmol) and 1-hydroxybenzotriazole (0.052g; 0.384mmol) in 1ml of anhydrous dimethylformamide stirred solution was added N,N-diisopropylethylamine (0.24ml, 1.38mmol) . After stirring at room temperature for 12 hours, the reaction was diluted to 30 ml with ethyl acetate, washed with water, 2% potassium bisulfate, 5% sodium bicarbonate and saturated aqueous sodium chloride, and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate) to obtain 0.152 g (65% yield) of the title product with a melting point of 109-114° C.; Rf(C)=0.36; Rf(D)= 0.37;

NMR (CDCl ₃ ) 1.0 (m, 6H, Val, isopropyl CH ₃ ); 1.42 (S, 9H, tert-butyl CH ₃ ); 2.5-3.1 (m, 7H, aSnCH ₂ , butyl CH ₂ -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 , aSnCH-2); 5.4-6.2 (m, 3H, amide); 6.7-8.4 (m, 11H, aryl H); 9.25 (m, 1H, NH).

Example 4: Preparation of 2(R,S)-3(S)-1,2-epoxy-3-phenylmethoxycarbonylamino-4-phenylbutane

Add 0.68 g of sodium borohydride to a solution of 6 g (18 mmol) of N-CBZ-L-phenylalanine chloromethyl ketone in 30 ml of 50% methanol in THF, and stir at room temperature for 30 minutes, and the mixture is carefully Acidify with 1N hydrochloric acid and evaporate to dryness under reduced pressure. The residue was diluted to 50 ml with dichloromethane, washed with water and saturated aqueous sodium chloride solution, and dried over anhydrous magnesium sulfate. Evaporation afforded 6.02 g (100%) of 2(R,S)-3(S)-1-chloro-2-hydroxy-3-phenylmethoxycarbonylamino-4-phenylbutane as a white solid . This was dissolved in 50 ml of isopropanol and 9 ml of 2N potassium hydroxide in methanol were added at room temperature. After stirring at room temperature for 1 hour, the solvent was removed under reduced pressure and the residue was partitioned between 50 mL ethyl acetate and 20 mL water. The organic phase was washed with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate and evaporated to dryness, as determined by the relative integration of erythro-NCH (3.74 ppm; 72%) and threo-NCH (4.2; 28%), Obtained 5.3 g (99% yield) of the title compound dominated by the 2(S) stereoisomer;

NHR (CDCl ₃ ) 2.42-3.17 (m, 5H, butane CH ₂ -1, -4, CH-2); 3.74 (m, 0.72H, butane CH-3); 4.2 (m, 0.28H, butane alkane CH-3); 4.73 (broad m, 1H, NH); 5.08 (S, 2H, methoxy _CH2 ); 7.3 (m, 10H, aryl H).

Example 5: Preparation of tert-butyl 3-isopropyl-3-[(2S,3S)-2-hydroxyl-3-(phenylmethoxycarbonyl)amino-4-phenylbutyl]carbazate

Step A: Preparation of 2(R)-3(S)-1,2-epoxy-3-phenylmethoxycarbonylamino-4-phenylbutane

To a stirred solution of 6.02 g (40 mmol) of sodium iodide in 50 ml of anhydrous acetonitrile was added 2.6 ml (22 mmol) of trimethylchlorosilane under an atmosphere of ammonia. After stirring for 10 minutes, 6 g (20.1 mmol) of 2(R,S)-3(S)-1,2-epoxy-3-phenylmethoxycarbonylamino-4-benzene, mainly the erythro isomer, was added Butane (Example 4), stirring was continued for another 1 hour. To this mixture was added 4 g (61.2 mmol) of zinc powder, followed by 6 ml of acetic acid. The resulting mixture was stirred vigorously at room temperature for about 5 hours and filtered to remove solid material. The filtrate was evaporated to dryness in vacuo, the residue was diluted to 75ml with ether, washed with water and 5N aqueous sodium thiosulfate and dried over anhydrous magnesium sulfate. Evaporation in vacuo and purification by silica gel chromatography (hexane/ethyl acetate 4:1) gave 5.1 g (90%) of (S)-2-(phenylmethoxycarbonyl)amino-1-phenylbutyl- 3-ene; Rf(A)=0.5; melting point=87-88°C (hexane);

NMR (CDCl ₃ ) 2.87 (d, 2H, butene CH ₂ -1); 4.77 (m, 2H, butene CH ₂ -4); 5.0 (m, 1H, NCH); 5.06 (s, 2H, methoxy CH ₂ ); 5.18 (broad d, 1H, NH); 5.55 6 (m, 1H, butene CH 3); 7.19, 7.27 (m, s, 5H, 5H, aromatic H).

This substance (2.23 g; 7.93 mmol) was dissolved in 25 ml of anhydrous dichloromethane and 4.5 g (22.1 mmol) of 85% 3-chloroperoxybenzoic acid were added at +4°C. The resulting mixture was stirred at the above temperature for 2 days, then diluted to 50 ml with diethyl ether, washed successively with 0°C 10% aqueous sodium sulfite, saturated aqueous sodium bicarbonate and saturated aqueous sodium chloride, and dried over magnesium sulfate. After evaporation of the solvent, the crude product was crystallized from a mixture of hexane/dichloromethane to give 2.1 g (89% yield) of the title epoxide with predominantly threo stereochemistry; melting point = 83-84 °C;

NMR (CDCl ₃ ) 2.47 (m, 5H, butene CH2-1, 4, CH-2); 3.74 (m, 0.15H, NCH); 4.2 (m, 0.85H, NCH); 4.53 (bd, 1H , NH); 5.03 (m, 2H, methoxy CH ₂ ); 7.3 (m, 10H, aryl H).

Step B: Preparation of tert-butyl 3-isopropyl-3-[(2S,3S)-2-hydroxy-3-(phenylmethoxycarbonyl)amino-4-phenylbutyl]carbazate

A mixture of 2.03 g (6.83 mmol) of the product of step A and 1.2 g (7.6 mmol) of tert-butyl 3-isopropylcarbazinate in 8 ml of isopropanol was stirred at 70±5° C. for 12 hours under a nitrogen atmosphere. After evaporation of the solvent in vacuo, the solid residue was recrystallized from hexane to give 2.6 g (80% yield) of the title compound, mp 114-115 °C;

R _f (A) = 0.2; R _f (B) = 0.61; NMR (CDCl ₃ ) 0.85 (m, 6H, isopropyl CH ₃ ); 1.42 (s, 9H, tert-butyl CH ₃ ); 2.44 (m , 2H, butyl CH ₂ -1); 2.94 (m, 3H, butyl CH ₂ -4, CH-3); 3.33-3.93 (m, 2H, isopropyl CH, butyl CH-2); 4.4 (width m, 1H, OH); 5.05 (s, 2H, methoxy CH ₂ ); 5.33 (width m, 2H, NH); 7.18, 7.27 (m, s, 5H, 5H, aromatic H).

Example 6: Preparation of 3-isopropyl-3-[(2S,3S)-2-hydroxyl-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenylbutyl]hydrazine tert-butyl formate (compound of formula Ia)

The title compound of this example is also known as 3-isopropyl-3-[(2S,3S)-2-hydroxyl-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenylbutyl Base] tert-butyl carbazate.

With the product of Example 5 instead of 3-isopropyl-3-[(2R,3S)-2-hydroxyl-3-(phenylmethoxycarbonyl)amino-4-phenylbutyl used in Example 3 ] tert-butyl carbazate (replacing the product of step B of Example 2 used in Example 3), the title compound of this example can be prepared by the same method as in Example 3, and the yield is 66%; Melting point = 203-204°C (chloroform); [HPLC-A] chromatographic purity 98.1%;

R _f (C) = 0.36; R _f (D) = 0.37: NMR (5% CD ₃ ODlnCDCl ₃ ); 1.0 (m, 6H, isopropyl CH ₃ ); 1.4 (s, 9H, tert-butyl CH ₃ ); 2.53 (d, 2H, butyl CH ₂ -1); 2.87 (m, 4H, asnCH ₂ , butyl CH ₂ -4); 3.13 (s, 6H, CD ₃ OH); 3.42 (m, 2H, isopropyl CH, butyl CH 3); 4.0 (m, 1H, butyl CH 2); 4.89 (m, 1H, asnCH-α); 7.11 (m, 5H, phenyl); 7.41-8.47 ( m, 6H, quinaldinoyl).

Example 7: Preparation of 3-isopropyl-3-[(2S,3S)-2-phosphinecarboxy-3-(N-quinolinoyl-L-aspartoyl)amino-4-phenylbutyl Base] tert-butyl carbazate (compound of formula Ib)

At room temperature under a nitrogen atmosphere, add 0.28 g (1.4 mmol) dicyclohexylcarbodiimide with stirring to 0.4 g (0.67 mmol) 3-isopropyl-3-[(2S,3S)-2-hydroxyl -3-(N-quinolinoyl-L-aspartoyl) amino-4-phenylbutyl] tert-butyl carbazate (i.e. formula Ia compound) and 0.12 grams (1.47 mmol) of anhydrous phosphorous acid mixture of 1.5 mL of anhydrous pyridine. After stirring at 60°C for 2 hours, the solvent was evaporated under reduced pressure, treated with 28 ml of 0.1 ml aqueous sodium bicarbonate solution, and stirred vigorously at room temperature for 1 hour. The precipitate was filtered off, washed with water, and the filtrate was acidified to pH~1.5 with concentrated hydrochloric acid. The absorbed product was extracted with ethyl acetate (3x50 mL), and the organics were dried over anhydrous magnesium sulfate. The solvent was evaporated to yield 0.42 g (yield 95%) of the title product as a colorless solid, Rf(B)=0.62; [HPLC-A] chromatographic purity 98.2%;

H ¹ NMR (CDCl ₃ ); 1.08 (m, 6H, isopropyl CH ₃ ); 1.41 (s, 9H, tert-butyl CH ₃ ); 2.7-4.8 (m, 14H, asnCH ₂ , butyl CH ₂ - 1,4; CH-2,3; Isopropyl CH; P-OH×2H ₂ O); 5.12 (m, 1H, asnCH); 5.89 (s, 0.5H, PH); 6.2-8.5 (m, 15.5 H, aromatic, amide NH, 0.5PH); 9.02 (m, 1H, asnNH); p ³¹ NMR (CDCl ₃ ) 14.99 (J=636Hz).

Example 8: Preparation of phosphate compound 3-isopropyl-3-[(2S,3S)-2-phosphine carboxyoxy-3-(N-quinolinoyl-L-aspartoyl)amino-4-benzene tert-Butyl]carbazate

A suspension of 0.4 g (0.6 mmol) of the compound of formula Ib in 2 ml of hexamethyldisilazane was stirred at 120±5° C. for 45 minutes. At this point the reaction mixture became homogeneous. 0.3 ml of bis(trimethylsilyl)peroxide (P.G. Cookson et al., J. Organometal, Chem., 1975, 99, C31) was added, followed by stirring at the above temperature for 1 hour. After the reaction mixture was cooled to room temperature, it was evaporated to dryness in vacuo. The residue was dissolved in 20 ml of methanol, evaporated to dryness under reduced pressure, and then dissolved in 12 ml of 0.1M aqueous sodium bicarbonate solution. The resulting mixture was acidified with concentrated hydrochloric acid to pH ~ 1.5, saturated with sodium chloride, and dissolved in ethyl acetate ( 3x50ml) extraction. The combined organic phases were dehydrated over anhydrous magnesium sulfate and evaporated to dryness to yield 0.39 g (96% yield) of the title compound as a colorless solid; Rf(B)=0.07;

H ¹ NMR (CDCl ₃ ): 1.2 (m, 6H, isopropyl CH ₃ ); 1.4 (s, 9H, tert-butyl CH ₃ ); 2.8-4.2 (m, 8H, asnCH ₂ , butyl CH ₂ - 1,4; CH=3; isopropyl CH); 4.2-6.4 (m, 5H, asnCH butyl CH-2, NH, POH); 6.5-8.4 (m, 14H, aromatic system, NH, 8.78 (m , 2H, NH); p ³¹ NMR (CDCl ₃ ) 9.6(s).

Embodiment 9: preparation formula I compound (disodium salt)

Operation: Dissolve 2.2g (26mmol) of sodium bicarbonate in 22ml of deionized water in a reaction flask at room temperature, add 10g (14.5mmol) of the product of Example 8 to the above solution, and stir for 2-3 hours until the solution becomes clear Add 100ml of isopropanol, stir for 20-30min, and filter; distill the obtained filtrate to remove the solvent under vacuum at 55-60°C; cool the material to 25-35°C, add 50ml of isopropanol, heat up to 55-60°C, vacuum The solvent was distilled off, dried, and the obtained solid was washed with 50 ml of isopropanol, and then evaporated to dryness under vacuum at 70-75°C to obtain the disodium salt compound shown in formula I with a yield of 98%. [HPLC-A] Chromatographic purity 97.2%, impurity Ia content=0.82%, impurity Ib content=0.68%.

Example 91: Preparation of Compound of Formula I (Disodium Salt)

Put 1 g of the product of Example 9 in a reaction flask, add 20 ml of heptane/ethyl acetate (10/3, v/v), stir at 70-75°C for 3 hours, cool to room temperature, and filter out the solid precipitate. Wash twice with 20ml of heptane, and then evaporate to dryness at 60°C under vacuum to obtain the disodium salt compound shown in formula I with a yield of 96%. [HPLC-A] Chromatographic purity 99.3%, impurity Ia content=0.26%, impurity Ib content=0.23% (from 0.68% to 0.23%, down to 34%).

Example 92: Preparation of Compound of Formula I (Disodium Salt)

Put 1 g of the product of Example 91 in a reaction flask, add 20 ml of heptane/ethyl acetate (10/2, v/v), stir at 70-75°C for 1 hour, cool to room temperature, and filter out the solid precipitate. Wash twice with 20ml of heptane, and then evaporate to dryness at 60°C under vacuum to obtain the disodium salt compound shown in formula I with a yield of 95%. [HPLC-A] Chromatographic purity 99.5%, impurity Ia content = 0.09%, impurity Ib content = 0.07% (reduced to 30%).

Example 93: Preparation of Compound of Formula I (Disodium Salt)

Put 1 g of the product of Example 92 in a reaction flask, add 20 ml of heptane/ethyl acetate (10/5, v/v), stir at 70-75°C for 2 hours, cool to room temperature, and filter out the solid precipitate. Wash twice with 20ml of heptane, and then evaporate to dryness at 60°C under vacuum to obtain the disodium salt compound shown in formula I with a yield of 96%. [HPLC-A] Chromatographic purity 99.6%, impurity Ia content=0.025%, impurity Ib content=0.019% (reduced to 27%).

Example 94: Preparation of Compound of Formula I (Disodium Salt)

Put 1 g of the product of Example 93 in a reaction flask, add 20 ml of heptane/ethyl acetate (10/4, v/v), stir at 70-75°C for 2.5 hours, cool to room temperature, and filter out the solid precipitate. Wash twice with 20ml of heptane, and then evaporate to dryness at 60°C under vacuum to obtain the disodium salt compound shown in formula I with a yield of 94%. [HPLC-A] Chromatographic purity 99.8%, impurity Ia content = 0.008%, impurity Ib content = 0.006% (reduced to 32%).

Example 95: Preparation of Compound of Formula I (Disodium Salt)

Put 1 g of the product of Example 94 in a reaction flask, add 20 ml of heptane/ethyl acetate (10/3, v/v), stir at 70-75°C for 2 hours, cool to room temperature, and filter out the solid precipitate; Wash twice with 20ml of heptane, and then evaporate to dryness at 60°C under vacuum to obtain the disodium salt compound shown in formula I with a yield of 96%. [HPLC-A] Chromatographic purity 99.8%, impurity Ia content = 0.002%, impurity Ib content = 0.002% (reduced to 33%).

According to the results of the above Examples 91-95, it can be seen that when the product is treated with heptane/ethyl acetate, the content of impurity Ia and impurity Ib can be greatly reduced, and each treatment can reduce these impurities to about 25% respectively ~ 35%, that is, a reduction of about 65% to 75%.

Example 97: Preparation of Compound of Formula I (Disodium Salt)

Put 1 g of the product of Example 9 in a reaction flask, add 20 ml of heptane, stir at 70-75 °C for 3 hours, cool to room temperature, filter out the solid precipitate, wash it twice with 20 ml of heptane, and then heat it at 60 °C , and evaporated to dryness under vacuum to obtain the disodium salt compound shown in formula I with a yield of 97%. [HPLC-A] The chromatographic purity is 97.5%, the content of impurity Ia=0.74%, the content of impurity Ib=0.61% (the two impurities are respectively reduced by about 10%).

In addition, in the supplementary test with reference to Example 97, when using heptane/ethyl acetate=10/0.5 or 10/1 as the solvent treatment, the two impurities were respectively reduced by about 9-11%.

Example 98: Preparation of Compound of Formula I (Disodium Salt)

Put 1 g of the product of Example 9 in a reaction flask, add 20 ml of ethyl acetate, stir at 70-75 ° C for 2 hours, cool to room temperature, filter out the solid precipitate, wash it twice with 20 ml of heptane, and then °C and evaporated to dryness under vacuum to obtain the disodium salt compound shown in formula I with a yield of 97%. [HPLC-A] Chromatographic purity 97.5%, impurity Ia content=0.79%, impurity Ib content=0.64%.

In addition, in the supplementary test with reference to Example 98, when using heptane/ethyl acetate=10/50 or 10/20 as the solvent treatment, the two impurities were respectively reduced by about 7-10%.

According to the results of Examples 97 to 98 above, it can be seen that when using heptane, or using ethyl acetate, or using a mixed solvent with a large difference in the amount of the two, the content of impurity Ia and impurity Ib cannot be effectively obtained. Each treatment can only reduce such impurities by about 10%.

Example 99: Preparation of Compound of Formula I (Disodium Salt)

The free acid of the product of Example 8 was treated with 2 equivalents of 0.2M sodium bicarbonate solution to convert it into the corresponding disodium salt, and the resulting solution was freeze-dried to obtain the obtained solution with a yield of 99%. [HPLC-A] Chromatographic purity 95.6%, impurity Ia content=2.68%, impurity Ib content=1.91%.

The characterization of the compound of formula I obtained in embodiment 95:

The infrared data is as follows:

Wave number (cm ¹ ) attribution 3231 N-H 3063 Aromatic C-H 2978 Aliphatic C-H 1672 C-O 1528 Amide II bond 1250 P-O 1165.1101 C-N 976,775 aromatic

NMR: 500MHz, VarianUnityInovaFT-NMR instrument, the solvent is CD3OD; 1H and 13CNMR chemical shifts are expressed in δ (ppm), relative to the internal standard TMS (δ0.00) and CD3OD (δ49.00) respectively; not in CD3OD1HNMR See that there are exchangeable protons; 1HNMR data were collected in DMSO-d6 at 30°C and 70°C to observe the resonance of the exchangeable protons; gDQCOSY and gHSQC tests and TOCSY tests were performed; the NMR assignment of the sample is shown below:

Note: 1 each position is marked with reference to the atomic position of the above structural formula,

The HR-MS research was carried out using WatersLCTpremierXE instrument, the deduced molecular formula is C32H41N6N6Na2O9P, that is, C32H41N6O9P·2Na, and the TOF data is shown in the table below:

polarity quality Mode error(ppm) i-Fit note (+ve) 687.2929 C ₃₂ H ₄₄ N ₆ O ₉ P 3.2 0.4 (alkali+H) ⁺ (-ve) 685.2770 C ₃₂ H ₄₂ N ₆ O ₉ P 2.8 0.2 (Alkali+H) ^-

B. Test case part

Test Example 1: Comparative Test of Drug Oral Absorption

Reagent: the compound of formula Ia obtained in Example 6 and the compound of formula I (disodium salt) obtained in Example 91 are both reagents,

Animals, grouping, and administration: rats, body weight 200～300g, 16 rats, equally divided into two groups (respectively Group I and Group Ia), 8 rats in each group, each administration dose of the two drugs is 50 μmol/ kg body weight. The drug was fully dissolved in 2% HPMC aqueous solution in the form of grinding, and the test drug was administered by intragastric administration in a single administration, respectively at 0 hour (before administration), 1 hour, 2 hours, 4 hours, and 1 hour after administration. Blood from the tail vein was collected at 8 hours and 15 hours.

Preparation of test samples: centrifuge the blood sample anticoagulated with heparin sodium at 3000-5000r/min for 5-30min, then transfer the upper layer of plasma to a new blank EP tube; add 10μl of internal standard benzoic acid solution (0.1mg/ml) , and then precipitate protein according to the volume ratio of plasma to precipitation reagent (acetonitrile-methanol=3:2 mixed solution and add 2% formic acid) as 1:5, precipitation time 3min, vortex mixing for 45s, and at 5°C under constant temperature Centrifuge at 10,000r/min for 10min; transfer the supernatant to a new blank EP tube, blow dry at 30-50°C with nitrogen flow, dissolve the residue in 100μl of 50% acetonitrile solution, vortex and mix for 30-60s, and store at 0-10°C Centrifuge at 10,000 to 12,000 r/min for 5 to 15 minutes at a constant temperature, and take the supernatant as the test solution; in addition, use 50% acetonitrile solution to prepare a standard solution of the compound of formula Ia and a standard solution of the compound of formula I at an appropriate concentration. According to the present invention [HPLC-A], the concentration of the compound of formula Ia and the compound of formula I in blood plasma is measured.

From the test results, the two groups of animals can only detect the compound of formula Ia but not the compound of formula I. The reason is that the compound of formula I is metabolized into the compound of formula Ia after being absorbed, so each group uses the compound of formula Ia as the calculation target. For each group of animals, calculate the mean blood drug concentration at each time point, and calculate the area under the blood drug concentration curve AUC between 0 and 15 hours, the relative AUC of the formula Ia compound group is calculated by the following formula:

=[AUC value of compound group of formula Ia ÷ AUC value of compound group of formula I]×100

The closer the relative AUC to 100% indicates that the gastrointestinal absorption properties of the two compounds are closer, if the relative AUC is less than 100%, the absorption of the compound of formula Ia is poorer than that of the compound of formula I, if the relative AUC is greater than 100 % then the absorption of the compound of formula Ia is better than that of the compound of formula I from the gastrointestinal tract. The results showed that the relative AUC of the formula Ia compound group was only 13.6% in the oral administration to the above rats, indicating that the oral absorption of the formula Ia compound was much lower than that of the formula I compound. In the supplementary test, rabbits were used as animals (6 animals in each administration group) to carry out the test according to the above method, and the result was that the relative AUC of the compound group of formula Ia was only 9.3%. In the supplementary test, beagle dogs were used as animals (3 animals in each administration group) according to the above method, and the relative AUC of the compound group of formula Ia was only 11.2%.

It can be seen that in the pharmaceutical raw materials or preparations made of the compound of formula I as the active ingredient, the conversion of the compound of formula I into the compound of formula Ia is unfavorable for maintaining the dosage of the drug.

Test Example 2: Stability of API

Directly get the corresponding material prepared by the present invention, or use a certain amount of Formula Ia compound (Example 6 product) and/or Formula Ib compound (Example 7 product) and Formula I compound (Example 95 product) by fully grinding and mixing Mixed formulations with different impurity contents.

Seal these materials with glass bottles, and then seal them at 40°C and store them in the dark for 5 months. Use [HPLC-A] to measure each sample and calculate the relative time of a certain impurity after 5 months of treatment under this condition relative to 0 months. The percentage increase of the content, and the residual percentage of the compound of formula I relative to 0 months (that is, the content of the compound of formula I in May in the material is divided by the content of the compound of formula I in 0 months and then multiplied by 100% to obtain the percentage). The result is as follows:

Note: *Initial composition refers to the content of various compounds in the material measured by [HPLC-A] at 0 months in the material, wherein the purity is the purity of the area normalization method of the compound of formula I, and the content of the compound of formula Ia is according to 【HPLC-A】measured relative to the percentage of the compound of formula I, the content of the compound of formula Ib is the percentage measured according to 【HPLC-A】 relative to the compound of formula I.

As can be seen from the results in the table, the pharmaceutical bulk drug of the present invention is stable, even if a certain amount of impurity Ia and/or impurity Ib is mixed therein.

Test Example 3: Stability of Composition of API and Sugar

The 30 samples or compositions in Test Example 2 were fully ground with the same amount of sugar (lactose) to form a sugar-containing composition. These compositions are sealed in glass bottles, and then sealed at 40°C and stored in the dark for 5 months, and each sample is measured by [HPLC-A] and calculated after being treated for 5 months under this condition. The percentage increase of the content of the time, and the residual percentage of the formula I compound relative to the 0 month (that is, the content of the formula I compound in the May in the material is divided by the content of the formula I compound in the 0 month and then multiplied by 100% to obtain the percentage). The result is as follows:

As can be seen from the above results, using the compositions prepared from different raw materials and sugar, wherein the impurity Ib content significantly affects the growth of impurity Ia and the decline of active ingredients in the composition, especially when the Ib content＞0.5%, this raw material and sugar After the obtained composition is processed under the high temperature condition of simulating long-term storage samples, the impurity Ia therein will increase significantly, and the degree of increase is positively correlated with the content of Ib, and the residual content of active ingredients will also be correspondingly reduced. However, it is completely inexplicable that no matter whether the content of impurity Ib in the raw material is high or low, it does not show an increase in the same way as impurity Ia during long-term storage.

In addition, according to the method of this test example 3, the difference is that the amount of lactose wherein is changed into 0.2 times or 20 times of the crude drug, and the above test is also carried out. %", "Ia increase/%" and "Ib increase/%" are basically consistent with the results in the above table, that is, the amount of lactose in the range of 0.2 to 20 times the active ingredient will show the above results.

Test Example 31: Stability of Composition of API and Sugar

Referring to the method of Test Example 3, the only difference is that lactose is replaced by sucrose. The result shows substantially the same result with Test Example 3, for example the No.21 composition I residual/% of preparation is 91.8%, Ia increase/% is 273%, Ib increase/% is 61%; .22 Composition I remaining/% is <85%, Ia increase/% is >500%, Ib increase/% is 63%.

Test Example 32: Stability of Composition of API and Sugar

Referring to the method of Test Example 3, the difference is only that lactose is replaced by mannitol. The result shows substantially the same result with Test Example 3, for example the No.21 composition I residual/% of preparation is 91.1%, Ia increase/% is 253%, Ib increase/% is 67%; .22 Composition I residual/% is <85%, Ia increase/% is >500%, Ib increase/% is 65%.

Test Example 33: Stability of Composition of API and Sugar

Referring to the method of Test Example 3, the only difference is that lactose is replaced by sorbitol. The result shows substantially the same result with Test Example 3, for example the No.21 composition I residual/% of preparation is 91.4%, Ia increase/% is 271%, Ib increase/% is 63%; .22 Composition I residual/% is <85%, Ia increase/% is >500%, Ib increase/% is 62%.

The above results show that when pharmaceutical raw materials with different impurity Ib contents are combined with sugars commonly used in pharmacy to prepare pharmaceutical preparations, impurity Ia will show significantly different increases due to the difference in impurity Ib content in the raw materials. Although this phenomenon is not found in the raw material drug itself, when the impurity Ib content is high, the range of adjuvant materials for pharmaceutical raw material drug preparation will be greatly limited, because the above-mentioned sugars are common, cheap, and For pharmaceutical excipients with superior performance, when using other pharmaceutical excipients instead of these sugars, it may cause other extensive problems such as cost, tablet performance, selection range, etc.

Test example 4: the stability of the pharmaceutical composition (tablet) prepared by crude drug

Taking 30 samples or compositions in Test Example 2 as medicinal raw materials respectively, tablets were prepared with the following tablet formula: medicinal raw materials 15 mg, starch 15 mg, lactose 100 mg, gelatin 1 mg, magnesium stearate 1 mg . Preparation method: make a solution of gelatin with water as a binder; fully mix the raw material drug, starch, and lactose, use the binder to make a soft material, make wet granules, and dry; mix the dry granules with magnesium stearate evenly, Compressed tablets, each containing 15mg of pharmaceutical raw material. These tablets were sealed in glass bottles, and then sealed at 40°C and stored in the dark for 5 months, and each sample was measured by [HPLC-A] and calculated after being treated for 5 months under this condition. The content increase percentage at 0 month, and the residual percentage of the compound of formula I relative to 0 month. Result shows substantially the same result with Test Example 3, for example the I remaining/% of No.21 tablet is 91.8%, Ia increases/% is 292%, Ib increases/% is 65%; Another example No.22 tablet The I residual/% of the agent was <85%, the Ia increase/% was >500%, and the Ib increase/% was 69%.

Industry Applicability

The invention provides antiviral drugs and pharmaceutical compositions thereof, which can be used as drugs for retroviral protease inhibitors, and the retroviral protease inhibitors have the chemical structure shown in formula I.

Claims (1)

1. preparing active component is the method with the medicinal raw material medicine of following formula I compound,

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The method comprises the following steps:

(1) to N-quinoline acyl group-ASPARTIC ACID, 3-isopropyl-3-[(2S, 3S)-2-hydroxyl-3-(phenyl methoxycarbonyl) amino-4-phenyl butyl] tert-butyl carbazate, BTA-1-base oxygen three (adds N in the agitating solution of the anhydrous dimethyl formamide of dimethylamino) Phosphonium fluorophosphate and I-hydroxybenzotriazole, N-diisopropylethylamine, at room temperature stir and make to react completely, reactant dilutes with ethyl acetate, and water, 2% potassium acid sulfate, 5% sodium acid carbonate and saturated sodium-chloride water solution washing, through anhydrous magnesium sulfate drying, solvent evaporated under reduced pressure, purify with ethyl acetate with silica gel column chromatography, obtain 3-isopropyl-3-[(2S, 3S)-2-hydroxyl-3-(N-quinoline acyl group-L-lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate, with following formula Ia compound:

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(2) under room temperature, blanket of nitrogen, make dicyclohexyl carbodiimide, 3-isopropyl-3-[(2S, 3S)-2-hydroxyl-3-(N-quinoline acyl group-L-lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate is that formula Ia compound, anhydrous sodium phosphoric acid mix in anhydrous pyridine, after 60 ° of C stirring reactions, solvent evaporated under reduced pressure, with sodium bicarbonate aqueous solution processing, high degree of agitation l hour under room temperature, leach sediment, with water washing, with concentrated hydrochloric acid acidifying filtrate to pH1.5; With ethyl acetate extraction absorption product; organic matter dewaters through anhydrous magnesium sulfate; solvent evaporation; obtain 3-isopropyl-3-[(2S; 3S)-2-time phosphine carboxylic oxygen base-3-(N-quinoline acyl group-L-lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate, with following formula Ib compound:

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(3) make 3-isopropyl-3-[(2S, 3S)-2-time phosphine carboxylic oxygen base-3-(N-quinoline acyl group-L-lucid asparagus acyl group) amino-4-phenyl butyl] tert-butyl carbazate is that the suspension of formula Ib compound in HMDS stirs and reactant mixture transmitted homogenize at 120 ± 5 DEG C, add two (three silicyls) peroxide, then under said temperature, stir 1 hour; Reactant mixture is cooled to after room temperature, and vacuum evaporation is to dry; Residue is dissolved in methyl alcohol, and reduction vaporization is extremely dry, then is dissolved in 0.1M sodium bicarbonate aqueous solution, and gained mixture is acidified to pH1.5 with concentrated hydrochloric acid, saturated through sodium chloride, extracts with ethyl acetate; The organic phase merging is dewatered through anhydrous magnesium sulfate; be evaporated to dry; obtain 3-isopropyl-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 following formula: compound

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(4) at room temperature by 3-isopropyl-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 adds in sodium bicarbonate aqueous solution, stirs until solution becomes clarification; Add isopropyl alcohol, stir the mixture, filter; Gained filtrate is distilled and is removed solvent under 55-60 ° of C vacuum; Make material be cooled to 25-35 ° of C; add isopropyl alcohol; be warming up to 55-60 ° of C; vacuum distillation desolventizes, dry, gained solid washed with isopropyl alcohol; under the vacuum of 70-75 ° of C, be evaporated to again dry; obtain 3-isopropyl-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, with the compound of disodium salt shown in following formula I:

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(5) purifying: make step (4) products therefrom add to heptane: in the mixed solvent of ethyl acetate=10:2 ~ 5, under 70-75 ° of C, stir 1-3 hour, be cooled to room temperature, leach solid precipitate, divide 2 washings with heptane 20ml, under 60 ° of C, vacuum, be evaporated to again dryly, obtain final product; Optionally repeat as required this step (5).