Monosulfat azapeptide derivative-containing pharmaceutical composition inhibiting hiv protease


(57) Abstract:

Described is a new compound of General formula I

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representing crystalline salt monosulfat azapeptide derivative having a high rate of solubility in water, significantly improved oral bioavailability when introducing animals compared with the free base. Described pharmaceutical composition, comprising as active principle a compound of formula I. 2 C. p. F.-ly, 5 Il.


1. The scope of the invention

The invention provides a new crystalline Sol - monosulfat azapeptide inhibitor of HIV protease formula

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which exhibits unexpectedly superior properties - solubility/dissolution - in comparison with other salts and significantly improved bioavailability when ingested by animals compared with the free base. Thus, monosulfat applicable for pharmaceutical dosage forms of the above protease inhibitor, particularly for oral dosage forms.

2. Art

Published PCT patent application WO 97/40029 reveals some azapeptide HIV. One of the substances included within the scope of WO 97/40029, is a compound having the structural formula

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and chemical name dimethyl ether [3S-(3R*,8'R*,9'R*,12R*)]-3,12-bis(1,1-dimethylethyl)-8-hydroxy-4,11-dioxo-9-(phenylmethyl)-6-[[4-(2-pyridinyl)-phenylmethyl] -2,5,6,10,13-pentaazamacrocyclic acid and evaluated as a possible inhibitor of the second generation HIV protease.

WO 97/40029 describes azapeptide derivatives, such as compound I, in the form of bases, as well as various farmatsevticheskii acceptable salt accession. Although a number of organic and inorganic acids mentioned as a possible salt-forming agent, including sulfuric acid, there is no mention monosulfata, which is the subject of this invention.


This invention provides a Sol - monosulfat of compound I, having the following formula

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The invention is illustrated in the graphs showing:

Fig. 1 - dependence of the solubility of salts, resulting in accession acids, the pH.

Fig.IIa - physical stability monosulfata.

Fig.IIb - physical stability of hydrochloric.

The connection I revealed above, is a weak organic base with a solubility in water of less than 1 µg/ml at 24 3oC. Crystalline free base in the form of a suspension in water or oil has low bioavailability when administered orally to animals, perhaps because of its extremely low solubility in these media.

For the development of pharmaceutical formulations, in particular oral dosage forms, the active ingredient must have sufficient bioavailability when administered orally. Since compound I in the form of a free base has no bioavailability, the authors present invention was investigated salt accession acids. A number of commonly used salts, resulting in accession acids, such as hydrochloride, bansilalpet, methanesulfonate, p-toluensulfonate, phosphate, nitrate, 1,2-etandisulfonat, isethionate and sulfate, were evaluated, in addition to monosulfata according to this invention. All of these salts in crystalline form exhibit reduced solubility in water (1-3 mg/ml or less at 243oC) compared with monosulfata, the solubility of which in the same conditions is approximately 4-5 mg/ml

In the solid is, perhaps due to their dissociation with the formation of the free base. In most cases, these transformations were accompanied by the formation of gel. Unlike the other aforementioned acids additional proton monosulfata prevents the conversion into the free base, which, as mentioned above, is very poorly soluble in water and has poor bioavailability when administered orally. Unusual solubility monosulfata in water detail below.

In General, the conversion of salts in the unionised form and Vice versa can be explained on the basis of theory of solubility depending on pH. The solubility of the free base in water was determined as a function of pH at 243oAnd it is shown below in Fig.1. the pH at which the compound has the highest solubility, denoted pHmaxand, as found, equal to about 1.2. In the literature it was reported that in the case of weakly basic compounds at pH>pHmaxthe equilibrium solid phase aqueous suspension of the compound is a free base. At pH<pH maxthe equilibrium solid compound is converted to the corresponding salt. The term "equilibrium solid phase" refers to the undissolved or excessive twerdok weak base is balanced in the water in the quantity exceeding the limit of its solubility (i.e., suspension of the salt in the water), the pH value of the resulting suspension may be reduced on both sides of the pHmaxdepending, among other factors, on the strength of the acid. When the value of the resulting pH is greater than pHmaxsuspended solid is converted into the free base.

Research conducted, in particular, methanesulfonate hydrochloride and free base, confirmed the above General conclusions, known from the literature. These salts in quantities exceeding their solubility, was balanced in water at 243oC for at least 24 hours. the pH of the suspension after equilibrium was 2,10,1, higher than pHmax. Undissolved solid particles in these suspensions were separated, dried in the air and identified. Thermal and elemental analysis of the undissolved solids from these compounds were identified as the free base. This result was expected on the basis of the curve of the pH-solubility shown in the above graph (Fig.1), and from literature data.

When excess monosulfata reached equilibrium in water, in the solid phase in equilibrium with rastvoreniem, although pH (1,90,2) suspensions was higher pHmaxand compare with the pH of the suspensions described above salts - methansulfonate and hydrochloride. Solid phase after at least 24 hours of equilibration identified by elemental analysis as hydrated form 2:1 salt of the free base and sulfuric acid (called sulfate). This behavior monosulfata is unexpected on the basis of theory dependence of the pH-solubility.

When, in turn, excess sulfate balanced in the water, in solid balanced with the solution phase was changed. Undissolved particles suspensie was separated, dried in the air and identified. Thermal and elemental analysis of this undissolved solid phase was similar to the analysis of the free base, although the conversion of sulfate in the free state was not as straightforward as in the case of methansulfonate and hydrochloride. From the pharmaceutical point of view, the tendency of salts to transform into a free state in the aquatic environment is not desirable because of the low bioavailability of the free base by oral administration. Therefore, monosulfat, because of their unique behavior in aqueous solution, provides an unexpected advantage.

Elevated properties solubility/dissolution monosulfata lead to increased oral bioavailability in animals compared with the free base. It was found that the absolute oral bioavailability monosulfata approximately 20% of dogs with the introduction of "unstated" solids (not in part) in a gelatin capsule. Compared to the crystalline free base has a minimal oral bioavailability in dogs.

In addition to optimal solubility other desirable property dosage forms salts is physical stability. The term "physical stability" shows on the ability of salt to preserve their crystalline structure (including, if any, crystallization solvents) storage conditions/loads. Notable izmeneniya, junk. Sol - monosulfat - showed good physical stability when stored at 40oC/75% relative humidity (RH) within 9 months, as shown in Fig. IIa. Differential scanning calorimetry showed no visible changes in thermal properties of the stressed sample (sample under load) salt - monosulfata compared with the sample without load (store at 2-8oWith in a closed container). On the other hand, methanesulfonate, hydrochloride and sulfate showed significant changes in thermal properties when stored at 40oC/75% RH for two weeks, as shown in Fig.IIb, c and d. Though the difference in the physical stability of the salts is not unusual propensity specific salt to form the solvate (or crystalline modification) and its ability to hold the crystallization solvent (physical stability of the crystalline modifications) storage conditions/loads cannot be predicted in advance.

Monosulfat can be obtained by reaction of a solution of free base of compound I with sulfuric acid in solvents such as acetonitrile, isopropanol, ethanol or acetone, and then extract thus obtained is ti and high stability, Sol-monosulfat suitable for therapeutic forms of compound I for oral administration. The following examples illustrate the specific formulations for oral administration.

Sol-monosulfat and its formulation is used, as described in WO 97/40029 for the treatment of diseases caused by viruses, especially retroviruses, such as HIV virus.


Example 1

Preparation of salt - monosulfata from ethanol

In a three-neck round bottom flask, equipped with a high stirrer and addition funnel, is added under stirring 15,013 g (0,0213 mol) of free base of compound I and 113 ml of ethanol samples 200. To this suspension is added dropwise within 90 seconds of 1.28 ml of concentrated sulfuric acid. After adding sulfuric acid to obtain a solution of a transparent amber color. The solution is filtered through a fine filter 1 Whatman paper and washed with 5 ml ethanol sample 200. To the solution was added 58 ml of heptane and 37.5 mg (0.25 weight. %) crystal seed of the compounds of formula II and then added 55 ml of heptane. The resulting mixture is stirred for 6 hours at 300 rpm the resulting crystalline suspension is filtered and washed with 50 ml smkoe monosulfate salt (output to 88.4 mol. %) having the above formula II.

Characteristic properties monosulfate salt

Elemental analysis. Calculated for C38H52N6ABOUT 1,0 H2SO4,%: 56,84; N IS 6.78; N 10,37; S 3,99.

Found,%: C 56,72; N. Of 6.65; N 10,41; S 3,83.

So pl. 195oC.

H20=0,28% (KF).

Example 2

Preparation of salt - monosulfata from acetone

5M H2S04(charged 8.52 ml, 42,6 mm) is added dropwise to a suspension of the free base of the compounds of formula I (30.0 g, 42,6 mm) in acetone (213 ml), stir with a mechanical stirrer and placed in an oil bath with a temperature of 50oC. Almost immediately get a clear solution. To the solution add seed crystals of the free base of the compounds of formula II. After two minutes, a precipitate, which becomes a paste. The mixture is stirred for one hour at 50oC, 30 minutes at 25oC and for 2 hours at 0oC. the Solution is filtered and the first filtrate is used to transfer remaining in the flask, the material in the funnel for filtering. The product is washed with acetone, then with heptane and dried in vacuum overnight to obtain 31,48 g (corrected yield 92%) monosulfate with the UB>ABOUT71,0 H2SO40,2 N2O%: 56,59; N TO 6.80; N 10,42; S 3,98; N2About 0,45.

Found,%: C 56,66; N Is 6.78; N 10,50; S 4,20; H2O 0,45 (KF).

Example 3

Preparation of salt - monosulfata from isopropanol

Aqueous sulfuric acid (5.0 M, 0,20 ml, 1 mm) are added to a suspension of the free base of the compounds of formula I (0,704 g, 1 mm) in isopropanol (4.0 ml), cooled in an ice bath. The ice bath removed and the mixture is stirred at room temperature. The suspension is dissolved within 15 minutes. To the solution add seed crystals, as in the above Examples 1 or 2, and stirred for 5 hours. The solid precipitate is filtered off and the first filtrate is used to transfer remaining in the flask, the material in the funnel for filtering. The product is washed with heptane and dried in vacuum overnight to obtain 0,752 g of crystalline monosulfate salt of formula II, yield 90%, so pl. 160-190oWith decomp.

Elemental analysis. Calculated for C38H52N6ABOUT71,0 H2SO42,0 H2O%: 54,40; N 6,97; N 10,02; S 3,82; N2About 4,29.

Found,%: C 54,25; N. OF 6.73; N 10,02; S TO 3.67; N20 4,53 (KF).

Radiograph of the crystals obtained from isopropanol, other than rentenaar what Allami Type II. Crystals of Type I, apparently, are anhydrous/desolvation crystalline substance, whereas the crystals of Type II are gidratirovannuyu, hygroscopic crystalline form.

Example 4

Preparation of encapsulated formulations of salt - monosulfata

A. Capsule (50 and 200 mg equivalent of free base)

Capsules are intended for oral administration, when this capsule is an opaque hard gelatin capsule gray size 0 containing monosulfat formula II, formula which is a combination of moist granules with lactose, crosspovidone and magnesium stearate.

Century Capsules (100 mg equivalent of free base)

Capsules are intended for oral administration, when this capsule is an opaque hard gelatin capsule gray size 0 containing monosulfat formula II, suspended in Gelucire 44/14. Gelucire 44/14 is an unsaturated poliglecaprone the glycerides consisting of mono-, di - and triglycerides and mono - and diesters of fatty acids and polyethylene glycol. Capsules are prepared by melting Gelucire 44/14 in 45-70oWith the subsequent addition of salt - monosulfata p is P CLASS="ptx2">

1. Monosulfat azapeptide derivative having the formula

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2. The pharmaceutical composition inhibiting HIV protease containing compound under item 1 and a pharmaceutically acceptable carrier.


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(4) -NH< / BR>
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