ES2359391T3 - FORMULATION OF BORONIC ACID COMPOUNDS. - Google Patents

Abstract

A compound of the formula (1): in which: P is hydrogen or an amino group protection moiety; R is hydrogen or C1-12 alkyl; A is 0, 1, or 2; R1, R2, and R3 are each independently hydrogen, C1-12 alkyl, C3-12 cycloalkyl, C6-14 aryl, or -CH2- R5; R5, in each case, is C6-14 aryl, (C6-14) aryl (C1-12) alkyl, (C1-12) alkyl, (C6-14,) aryl, C3-12 cycloalkyl, heterocyclyl comprising 3 8 atoms, in which one or more atoms is selected from N, O and S, heteroaryl comprising 5 to 14 atoms, in which 1-4 atoms are selected from N, O and S, or -W-R6, in which W is a chalcogen and R6 is C1-12 alkyl; wherein the ring portion of any of said aryl, aralkyl, alkylaryl, cycloalkyl, heterocyclyl, or heteroaryl in R1, R2, R3, or R5 may be optionally substituted; and Z1 and Z2 together form a residue obtained from mannitol, in which the atom attached to boron in each case is an oxygen atom, and in which the compound of formula (1) is optionally lyophilized.

Description

The present invention relates to the formulation of pharmaceutical compounds. More particularly, the invention relates to pharmaceutically acceptable, stable compositions, prepared from boronic acid compounds. Likewise, the invention relates to processes for the preparation of said compositions.

The boronic acid and ester compounds show a variety of pharmaceutically useful biological activities. Shenvi et al., US Pat. No. 4,499,082 (1985), disclose that boronic peptide acids are inhibitors of certain proteolytic enzymes. Kettner and Shenvi, US Pat. No. 5,187,157 (1993); U.S. Patent No. 5,242,904 (1993); and US Pat. No. 5,250,720 (1993), describe a class of boronic peptide acids that inhibit trypsin-like proteases. Kleeman et al., U.S. Pat. No. 5,169,841 (1992), disclose N-terminally modified boronic peptide acids that inhibit the action of renin. Kinder and others, U.S. Pat. No. 5,106,948 (1992), discloses that certain boronic tripeptide acid compounds inhibit the development of cancer cells.

Adams et al., US Pat. No. 5,780,454 (1998), U.S. Pat. No. 6,066,730 (2000), U.S. Pat. No. 6,083,903 (2000), and US Pat. No. 6,297,217 (2001), incorporated herein by reference in its entirety, describes boronic acid and peptide ester compounds useful as proteasome inhibitors. The references also describe the use of boronic acid and ester compounds to reduce the rate of muscle protein degradation, to reduce the activity of NF-κB in a cell, to reduce the rate of degradation of p53 protein in a cell, to inhibit the degradation of cyclin in a cell, to inhibit the development of a cancer cell, to inhibit the presentation of antigen in a cell, to inhibit NF-κB-dependent cell adhesion, and to inhibit HIV replication. Brand et al., WO 98/35691, discloses that proteasome inhibitors, including boronic acid compounds, are useful for the treatment of heart attacks such as those that occur during stroke or myocardial infarction. Elliott et al., WO 99/15183, discloses that proteasome inhibitors are useful for the treatment of inflammatory and autoimmune diseases.

Unfortunately, alkylboronic acids are relatively difficult to obtain purely analytically. Snyder et al., J. Am. Chem. Soc., P. 3611 (1958), state that alkylboronic acid compounds readily form boroxins (anhydrides) under dehydrating conditions. Similarly, alkylboronic acids and their boroxins are frequently sensitive to air. Korcek et al., J. Chem. Soc., Perkin Trans., Vol. 2, p. 242, (1972), states that butylboronic acid is easily oxidized by air to generate 1-butanol and boric acid. These difficulties limit the pharmaceutical utility of boronic acid compounds, complicating the characterization of pharmaceutical agents comprising boronic acid compounds and limiting their storage time.

Accordingly, there is a need in the art for improved formulations of boronic acid compounds. Ideally, such compositions would be conveniently prepared, would show enhanced stability and longer storage time compared to the free boronic acid compound, and would easily release the bioactive boronic acid compound when administered to a subject in need of boronic acid therapy. .

The present invention provides stable, pharmaceutically acceptable compositions prepared from boronic acid compounds. Likewise, the invention provides methods for the preparation of said compositions. The invention provides the discovery that lyophilization of an aqueous mixture comprising a boronic acid compound and a compound having at least two hydroxyl groups produces a stable composition that readily releases the boronic acid compound after dissolution in aqueous medium.

In a first aspect, the invention provides compounds having the formula (1):

image 1

in which: P is hydrogen or an amino group protection moiety; R is hydrogen or C1-12 alkyl;

A is 0, 1, or 2;

R1, R2, and R3 are each independently hydrogen, C1-12 alkyl, C3-12 cycloalkyl, C6-14 aryl, or -CH2R5

;

R5, in each case, is C6-14 aryl, (C6-14) aryl (C1-12) alkyl, (C1-12) alkyl, C6-12, aryl, C3-12 cycloalkyl, heterocyclyl

5 comprising 3 to 8 atoms, in which one or more atoms is selected from N, O and S, heteroaryl comprising 5 to 14 atoms, in which 1-4 atoms are selected from N, O and S, or -W- R6, in which W is a chalcogen and R6 is C1-12 alkyl;

wherein the ring portion of any of said aryl, aralkyl, alkylaryl, cycloalkyl, heterocyclyl, or heteroaryl in R1, R2, R3, or R5 may be optionally substituted; Y

10 Z1 and Z2 together form a residue obtained from mannitol, in which the atom bound to boron in each case is an oxygen atom, and

wherein the compound of formula (1) is optionally lyophilized.

In a second aspect, the invention provides a process for preparing a lyophilized compound of the formula (1):

image2

in which: P is hydrogen or an amino group protection moiety; R is hydrogen or C1-12 alkyl; A is 0, 1, or 2;

R1, R2, and R3 are each independently hydrogen, C1-12 alkyl, C3-12 cycloalkyl, C5-14 aryl, or -CH2R5

; R5, in each case, is C6-14 aryl, (C6-14) aryl (C1-12) alkyl, (C1-12) alkyl, (C6-14,) aryl, C3-12 cycloalkyl, heterocyclyl comprising 3 at 8 atoms, in which one or more atoms is selected from N, O and S, heteroaryl comprising 5 to 14 atoms, in which 1-4 atoms are selected from N, O and S, or -W-R6, in that W is a calco25 gene and R6 is alkyl;

wherein the ring portion of any of said aryl, aralkyl, alkylaryl, cycloalkyl, heterocyclyl, or heteroaryl in R1, R2, R3, or R5 it may be optionally substituted; Y Z1 and Z2 have been obtained from a sugar, in which the sugar is mannitol; Understanding the procedure:

30 (a) preparation of a mixture comprising

(i)

Water,

(ii)

a compound of formula (3)

image 1

wherein P, R, A, R1, R2, and R3 are as described above; and Z ’and Z’’OH; Y

(iii) mannitol; Y

(b) lyophilization of the mixture.

Preferred embodiments are set forth in the subclaims.

In a third aspect, the invention provides compositions prepared by the methods of the invention.

The invention provides pharmaceutically acceptable, stable compositions, prepared from boronic acid compounds and processes for the preparation of the compositions. Likewise, the invention provides new boronate ester compounds.

For the purposes of the present invention, the following definitions will be used:

As used in the present invention, the terms "formulate" and "formulation" refer to the preparation of a boronic acid compound in a form suitable for administration to a mammalian subject, preferably a human. Frequently, the formulation of the boronic acid compound comprises the addition of pharmaceutically acceptable excipients, diluents, or carriers. In some embodiments, the formulation of the boronic acid compound comprises the formation of a chemical derivative of the boronic acid compound, preferably the formation of a boronate ester. The term "formulation" refers to any form commonly used for pharmaceutical administration, including solids, liquids, suspensions, creams, and gels. For the purposes of the present invention, the formulation is preferably a lyophilized powder.

As used in the present invention, the term "lyophilized powder" refers to any solid material obtained by lyophilization of an aqueous mixture.

By "stable formulation" is meant any formulation that has sufficient stability to be useful as a pharmaceutical agent. Preferably, the formulation has sufficient stability to allow storage at a convenient temperature, preferably between 0 ° C and 40 ° C, for a reasonable period of time, preferably longer than one month, more preferably longer than three months, even more preferably longer than six months, and most preferably more than one year.

As used in the present invention, the term "boronic acid" refers to any chemical compound comprising a moiety -B (OH). Snyder et al., J. Am. Chem. Soc., P. 3611, (1958), state that alkyl boronic acid compounds easily form oligomeric anhydrides by dehydration of the boronic acid moiety. Accordingly, unless the contrary is obvious from the context, the term "boronic acid" is expressly intended to encompass free boronic acids, oligomeric anhydrides, including but not limited to dimers, trimers, and tetramers, and mixtures. thereof.

As used in the present invention, the term "compound that has at least two hydroxyl groups" refers to any compound that has two or more hydroxyl groups. For the purposes of the present invention, the two hydroxyl groups are preferably separated by at least two connection atoms, preferably from about 2 to about 5 connection atoms, more preferably 2 or 3 connection atoms. The connection atoms may be in a chain or a ring, the chain or ring comprising carbon atoms and, optionally, a heteroatom or heteroatoms, which may be N, S or O. For convenience, the term "dihydroxy compound" may used to refer to a compound having at least two hydroxyl groups, as defined above. Accordingly, as used in the present invention, the term "dihydroxy compound" is not intended to be limited to compounds having only two hydroxyl groups.

As used in the present invention, the term "amino group protection moiety" refers to any group used to derive an amino group, especially an N-terminal amino group of a peptide or amino acid. Such groups include, without limitation, alkyl, acyl, alkoxycarbonyl, aminocarbonyl, and sulfonyl moieties. However, the term "amino group protection moiety" is not intended to be limited to those particular protection groups that are commonly used in organic synthesis, nor are they intended to be limited to groups that are easily cleavable.

The term "chalcogen," as used in the present invention, refers to the elements oxygen or sulfur.

The term "alkyl", as used in the present invention, refers to 20 straight or branched chain aliphatic groups having from 1 to 12 carbon atoms, preferably 1-8 carbon atoms, more preferably 1-6 atoms carbon, and even more preferably 1-4 carbon atoms, which may be optionally substituted with one, two or three substituents. Unless explicitly stated otherwise, the term "alkyl" is understood to include saturated, unsaturated, and partially unsaturated aliphatic groups. When they are particularly intended for unsaturated groups, the terms "alkenyl" or "alkynyl" will be used. When they are only intended for saturated groups, the term "saturated alkyl" will be used. Preferred saturated alkyl groups include, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

The term "cycloalkyl", as used in the present invention, includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons and more preferably 3 to 6 carbons, in which the cycloalkyl group additionally may be optionally substituted. Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

An "aryl" group is a C6-C14 aromatic moiety comprising one to three aromatic rings, which may be optionally substituted. Preferably, the aryl group is a C6-C10 aryl group. Preferred aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl. An "aralkyl" or "arylalkyl" group comprises an aryl group covalently linked to an alkyl group, any of which may independently be optionally substituted or unsubstituted. Preferably, the aralkyl group is (C1-C6) alkyl (C6-C10) aryl, including, without limitation, benzyl, phenethyl, and naphthylinethyl. An "alkaryl" or "alkylaryl" group is an aryl group having one or more alkyl substituents. Examples of alkaryl groups include, without limitation, tolyl, xylyl, mesityl, ethylphenyl, tert-butylphenyl, and methylnaphthyl.

The terms "heterocycle", "heterocyclic" and "heterocyclyl" refer to any stable ring structure having from about 3 to about 8 atoms, in which one or more atoms are selected from the group consisting of N, O, and S. The nitrogen and sulfur heteroatoms of the heterocyclic moiety may be optionally oxidized, and the nitrogen atoms may optionally be quaternized. The heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom that results in a stable formula. The term "stable compound" or "stable formula" is understood to refer to a compound that is robust enough to survive isolation to a degree of purity useful from a reaction and formulation mixture within an effective therapeutic agent. .

The heterocyclic group may optionally be substituted on carbon in one or more positions with any of the substituents mentioned above. Likewise, the heterocyclic group may independently be substituted on nitrogen with alkyl, aryl, aralkyl, alkylcarbonyl, alkylsulfonyl, arylcarbonyl, arylsulfonyl, alkoxycarbonyl, aralkoxycarbonyl, oxy, or hydroxy, or on sulfur with oxo or lower alkyl. Preferred heterocyclic groups include, without limitation, epoxy, aziridinyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl, thioazolidinyl, oxazolidinyl, oxazolidinonyl, and morpholinyl. Similarly, the heterocyclic group may be fused to an aryl, heteroaryl, or heterocyclic group. Examples of such fused heterocyclics include, without limitation, tetrahydroquinoline and dihydrobenzofuran.

As used in the present invention, the terms "heteroaryl" and "aromatic heterocycle" refer to groups having rings of 5 to 14 atoms, preferably rings of 5, 6, 9, or 10 atoms; having 6, 10, or 14 π electrons shared in a cyclic arrangement; and having, in addition to carbon atoms, from one to four, preferably from one to about three, heteroatoms selected from the group consisting of N, O and S. The heteroaryl group may be optionally substituted on carbon at one or more positions with any of the substituents mentioned above. Preferred heteroaryl groups include, without limitation, thienyl, benzothienyl, furanyl, benzofuranyl, dibenzofuranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, quinoxalinyl, tetrazolyl, oxazolyl, isozolyl, oxazolyl, isozolyl, oxazolyl, isozolyl, oxazolyl, isozolyl, oxazolyl, isoazolyl, oxazolyl, isozolyl, oxazolyl, isoazolyl, oxazolyl, isoazolyl, oxazolyl, isozolyl, oxazolyl, isozolyl, oxazolyl, isozolyl, isozolyl

As used in the present invention, a "substituted" alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group is one having from one to about four, preferably from one to three, more preferably one or two, non-hydrogen substituents. Suitable substituents include, without limitation, halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkylsulfonyl, arenesulfonyl, arenesulfonyl groups , alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido. Preferably, the substituents are independently selected from the groups consisting of C1-C6 alkyl, C3-C8 cycloalkyl, (C1-C6) alkyl (C3-C8) cycloalkyl, C2-C8 alkenyl, C2-C8 alkynyl, cyano , amino, C1-C6 alkylamino, dialkylamino (C1-C6), benzylamino, dibenzylamino, nitro, carboxy, carboalkoxy (C1-C6), trifluoromethyl, halogen, C1-C6 alkoxy, C6-C10 aryl, aryl (C6 -C10) (C1-C6) alkyl, aryl (C6-C10) alkoxy (C1-C6), hydroxy, C1-C6 alkylthio, C6-C10 alkylsulfinyl, C6-C10 alkylsulfonyl, C6-C10 arylthio, arylsulfinyl C6-C10, C6-C10 arylsulfonyl, C6-C10 aryl, (C1C6) alkyl aryl (C6-C10), and haloaryl (C6-C10).

The term "halogen" or "halo" is used in the present invention to refer to chlorine, bromine, fluorine, or iodine.

The term "oxo" refers to an oxygen atom, which forms a carbonyl when bonded to carbon, an N-oxide when bonded to nitrogen, and a sulfoxide or sulfone when bonded to sulfur.

As used in the present invention, the term "acyl" refers to an alkylcarbonyl or arylcarbonyl substituent.

The term "acylamino" refers to an amido group attached to the nitrogen atom. The term "carbamoyl" refers to an amido group attached to the carbonyl carbon atom. The nitrogen atom of a clarino or carbamoyl substituent may be additionally substituted. The term "sulfonamido" refers to a sulfonamido substituent.

attached either to the sulfur atom or to the nitrogen atom. The term "amino" is understood to include NH2, alkylamino, arylamino, and cyclic amino groups. The term "ureido" as used in the present invention refers to a substituted or unsubstituted urea moiety. In a first aspect, the invention provides compounds having the formula (1):

image3

according to claim 1.

As used in the present invention, the term "moiety obtained from mannitol" refers to a moiety formed by the removal of hydrogen atoms from two mannitol hydroxyl groups. The moiety obtained from mannitol can be bound to boron by either of the two mannitol hydroxyl groups. For example in

In various embodiments, the boronate ester forms a ring of 5, 6, 7, 8, or 9 atoms. In some preferred embodiments, the boronate ester forms a ring of 5 or 6 atoms.

Accordingly, Z1 and Z2 of the compound of formula (1) together form a residue of formula C6H12O6, in which the oxygen atoms of the two deprotonated hydroxyl groups form covalent bonds with boron to form a boronate ester compound.

Preferably, the boronate ester compound has one of the following structures:

image 1

However, structures with larger boronate ester ring sizes are equally possible.

In certain preferred embodiments, the mannitol boronate ester forms a ring of 5 symmetric atoms having the following structure:

image 1

Preferably, the mannitol is of the D configuration, although the L configuration can also be used. In certain particularly preferred embodiments, Z1 and Z2 together form a moiety obtained from D-mannitol. In those embodiments, the boronate ester compound preferably has one of the following structures:

image3

However, structures with larger boronate ester ring sizes are equally possible. In certain particularly preferred embodiments, the boronate ester compound has the following structure:

image4

The residue P of the compound of formula (1) is preferably hydrogen or one of R7-C (O) -, R7-S (O) -, R7-NH = C (O) -, or R7-OC (O ) -, in which R7 is one of alkyl, aryl, alkaryl, or aralkyl, any of which may be optionally substituted, or when Y is R7-C (O) -o R7-S (O) 2-, R7 it can also be a saturated, partially unsaturated, or aromatic heterocycle of 5 to 10 optionally substituted atoms.

5

fifteen

25

35

Four. Five

In certain preferred embodiments, P is one of R7-C (O) -or R7-S (O) 2-, and R7 is a saturated, partially unsaturated, or aromatic heterocycle of 5 to 10 optionally substituted atoms. Preferably, R 7 is an aromatic heterocycle, more preferably pyrazinyl, pyridyl, quinolyl, or quinoxalinyl, or a saturated heterocycle, preferably morpholinyl. In some preferred embodiments, P is (2-pyrazino) carbonyl or (2-pyrazino) sulfonyl.

In some preferred embodiments, R is hydrogen. In some other preferred embodiments, R is alkyl, preferably C1-C6 alkyl, more preferably C1-C4 alkyl, and most preferably methyl or ethyl.

The variable A in the formula (1) can be 0, 1, or 2. Thus, when A is zero, the rest within the parentheses is not present and the boronate ester compound is a dipeptide. Similarly, when A is 1, the rest within the parentheses is present and the compound is a tripeptide. When A is 2, the compound is a tetrapeptide. In certain particularly preferred embodiments, A is zero.

For the purposes of the invention, the terms "peptide", "dipeptide", and "tripeptide" are intended to encompass compounds comprising natural amino acid residues, unnatural amino acid residues, or a combination of natural and unnatural amino acid residues. From formulas (1) - (3), it will be apparent that the terms "peptide", "dipeptide", and "tripeptide" are used in the present invention to refer to compounds in which the carboxylic acid functionality of the amino acid residue C-terminal is replaced by a functionality of boronic acid or boronate ester.

It is preferred that the substituents R1, R2, and R3 in formula (1) are each independently one of hydrogen, C1-C8 alkyl, C3-C10 cycloalkyl, or C6-C10 aryl, or -CH2-R5, wherein each R1, R2, R3, and R5 may be optionally substituted. More preferably, R1, R2, and R3 are each independently one of C1-C8 alkyl or -CH2-R5, and R5 is one of cycloalkyl, aryl, heterocyclyl, heteroaryl, or -W-R6 in which W is chalcogen and R6 is alkyl. Preferably, R5 is one of C6-C10 aryl, (C6-C10) aryl (C1-C6) alkyl, (C1-C6) alkyl (C6-C10) aryl, C3-C10 cycloalkyl, C1-C8 alkoxy, or C1-C8 alkylthio or a heteroaryl ring of 5 to 10 atoms.

In certain particularly preferred applications, the compound of formula (1) is one of:

N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine D-mannitol boronate;

N- (2-quinoline) sulfonyl-L-homophenylalanine-L-leucine D-mannitol boronate;

N- (3-pyridino) carbonyl-L-phenylalanine-L-leucine D-mannitol boronate;

N- (4-morpholino) carbonyl-L-phenylalanine-L-leucine D-mannitol boronate;

N- (4-morpholino) carbonyl-β- (1-naphthyl) -L-alanine-L-leucine D-mannitol boronate;

N- (8-quinoline) sulfonyl-β- (1-naphthyl) -L-alanine-L-leucine D-mannitol boronate;

N- (4-morpholino) carbonyl- (O-benzyl) -L-tyrosine-L-leucine D-mannitol boronate;

N- (4-morpholino) carbonyl-L-tyrosine-L-leucine D-mannitol boronate; or

N- (4-morpholino) carbonyl- [O- (2-pyridylmethyl)] - L-tyrosine-L-leucine D-mannitol boronate.

In some preferred embodiments, the composition further comprises one or more other excipients, carriers, diluents, fillers, salts, buffers, stabilizers, pharmaceutically acceptable solubilizers, and other materials well known in the art. The preparation of pharmaceutically acceptable formulations containing these materials is described, for example, in Remington’s Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, (1990).

The compounds and compositions according to the first aspect of the invention can be prepared by the methods described herein, or by any suitable method for producing the compound or composition. For example, the boronate esters of formula (1) can be prepared from the corresponding boronic acids by lyophilization in the presence of mannitol, as described herein, or, alternatively, can be prepared from another ester of boronate by transesterification. Alternatively, the boronate esters of formula (1) can be prepared by incorporating mannitol at a previous stage in the synthesis.

In a second aspect, the invention provides a process for the formulation of a boronic acid compound according to claim 14.

In certain preferred embodiments, the mixture comprises one or more co-solvents in addition to water. Preferably, the co-solvent is miscible with water. More preferably, the co-solvent is an alcohol, including, without limitation, ethanol and tert-butanol. The composition of the solvent mixture may vary from about 5% to about 95% v / v alcohol. In some embodiments, the aqueous solvent mixture comprises from about 30% to about 50% alcohol, preferably from about 35% to about 45% alcohol. In certain embodiments, the aqueous solvent mixture comprises about 40% tert-butanol.

In some other embodiments, the aqueous solvent mixture comprises from about 1% to about 15% alcohol, preferably from about 5% to about 10% alcohol. In certain preferred embodiments, the aqueous solvent mixture comprises from about 5% to about 10% ethanol.

Preferably, the compound having at least two hydroxyl groups and the boronic acid compound are present in the mixture in a w / w ratio ranging from about 1: 1 to about 100: 1. In various embodiments, the w / w ratio of dihydroxy compound to boronic acid compound is approximately 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1 , 90: 1, or 100: 1. Other relationships are equally possible.

The aqueous mixture can be prepared by any order of addition. For example, in some embodiments, the dihydroxy compound is added to an aqueous mixture comprising a boronic acid compound. In some other embodiments, the boronic acid compound is added to an aqueous mixture comprising a dihydroxy compound. In still other embodiments, the boronic acid compound and the dihydroxy compound can be added at the same time, or almost at the same time. In some embodiments, it may be advantageous initially to add the boronic acid compound and / or the dihydroxy compound to a solvent mixture containing a higher percentage of co-solvent than desired for the lyophilization step, and then dilute with water.

In some preferred embodiments, the mixture further comprises one or more excipients, carriers, diluents, fillers, salts, buffers, stabilizers, pharmaceutically acceptable solubilizers, and other materials well known in the art. The preparation of pharmaceutically acceptable formulations containing these materials is described, for example, in Remington’s Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, (1990).

After reconstitution in aqueous medium, a balance is established between any boronate ester present in the composition and the corresponding boronic acid. Typically, equilibrium is reached quickly, for example, in 10-15 minutes, after the addition of water. The relative concentrations of boronate ester and boronic acid present in the equilibrium depend on the pH of the solution, temperature, and the ratio of dihydroxy compound to boronic acid compound.

The following examples are intended to further illustrate certain preferred embodiments of the invention.

EXAMPLES

Example 1: Preparation of a lyophilized formulation of N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine boronic acid with D-mannitol

Approximately 40 mg of N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine boronic acid was weighed into a vessel, and 16 ml of tert-butanol was added. The vessel was closed and the suspension was heated at approximately 45 ° C for 5 minutes to complete the dissolution of the compound. Water (24 ml) was added with stirring, followed by 0.4 g of mannitol, added as an excipient, at 1% w / v. The mixture was stirred to complete the dosing and then cooled to room temperature. The solution was filtered through a 0.45 µm nylon membrane. Aliquots of one milliliter were introduced into 5 ml serum bottles. The cracked rubber caps were partially inserted into the bottles and the bottles were placed in a cryo dryer with a storage temperature of -45 ° C. After about 1 hour, vacuum was applied. The storage temperature was allowed to rise gradually to -35 ° C and was maintained at -35 ° C until the ice disappeared from the samples (approximately 40 hours). Then, the storage temperature control was turned off and the storage temperature was allowed to gradually rise to 0 ° C. A secondary drying cycle was carried out by increasing the storage temperature in 3 increments at 25 ° C over a period of 1.5 hours. The storage temperature was maintained at 25 ° C for 2 hours. The samples were sealed under nitrogen and removed from the cryo dryer.

The residual moisture content of the samples was determined by Karl Fischer analysis, using three lyophilized products. The water content was 0.88% by weight.

Rapid atom bombardment (FAB) mass spectral analysis of the lyophilized product showed a strong signal am / z = 531, indicative of the formation of a covalent boronate ester adduct between the N- (2-pyrazine) carbonyl-L- acid Phenylalanine-L-Boronic Leucine and D-Mannitol. As the matrix, glycerol was used, and at m / z = 441 a signal was observed for the glycerol adduct with N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine boronic acid. However, the signal strength at m / z = 441 was very weak compared to the signal at m / z = 531, possibly indicative of the enhanced stability of the D-mannitol adduct.

Example 2: Production-scale preparation of a lyophilized formulation of N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine boronic acid with D-mannitol

5

10

fifteen

twenty

25

30

35

40

Four. Five

In a container for the formation of the clean compound, a solution of 97% tert-butanol / 3% water for injection was prepared by heating the required amount of tert-butanol at 35 ° C and adding water for injection. Approximately 5% of the solution was reserved for use in washing. The solution was cooled to 15-30 ° C, and boronic N- (2-pyrazino) carbonyl-Lphenylalanine-L-leucine acid was added with stirring. The vessel was washed with the tert-butanol / reserved water solution, and the washings were added to the main vessel. The mixture was stirred until the boronic acid compound dissolved completely. Mannitol was added, washing the residual mannitol into the reaction vessel with fresh water for injection. Sufficient water for injection was added to reduce the total alcohol content to 40% v / v. The mixture was stirred until the mannitol dissolved completely. The mixture was filtered through a 0.22 micrometer filter. Within previously sterilized vials, aliquots of the filtered solution were introduced. The vials were sealed with lyophilization caps and placed on the trays of the lyophilizer chamber kept at -45 ° C. After two hours, the vacuum was made in the cryo dryer chamber and the chamber pressure was adjusted to 100-200 micrometers with sterile nitrogen. The freeze dryer chamber trays were heated to -30 ° C using an appropriate speed slope, and kept at that temperature for 10-15 hours. After each product thermocouple marked -33 ° C or hotter, the storage temperature was adjusted to -15 ° C for 7 hours using an appropriate velocity slope and maintained at that temperature for 5 hours. After all product thermocouples recorded the storage temperature, the trays were heated at 0 ° C for a period of at least 7 hours using an appropriate speed slope. When all thermocouples recorded 0 ° C, the trays were heated to 27 ° C and kept at that temperature for 4 hours. At the end of the terminal drying phase, the chamber pressure was restored using sterile nitrogen, and the vials were sealed and removed.

Example 3: Reconstitution of N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine boronic acid

The lyophilized formulation of N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine boronic acid with D-mannitol was prepared as described in Example 1. A sample was reconstituted with 2 ml of water. The solution was completed in 1-2 minutes of stirring. The complete solution was transferred to a volumetric flask, diluted, and analyzed by HPLC to determine the content of N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine boronic acid. The total drug content was 1.09 mg. A second sample was reconstituted with 1 ml of propylene glycol: EtOH: H2O,

40:10:50 The solution was completed with 1 minute of stirring. The total drug content was 1.11 mg.

The lyophilized formulation was also reconstituted with 0.9% w / v saline. The lyophilized material easily dissolved at concentrations of up to 6 mg / ml. In contrast, solid N- (2-pyrazino) carbonyl-L-phenylalanine-Lleucine boronic acid was not soluble in 0.9% w / v saline at a concentration of 1 mg / ml.

In order to ensure that the free boronic N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine acid had been rapidly released after reconstitution of the lyophilized formulation in aqueous solution, the lyophilized formulation was dissolved in pure DMSO and assayed to determine the inhibition of the chymotrypsin-like activity of the 20S proteasome as described in US Pat. No. 5,780,454. Proteasome inhibition can only be observed if hydrolysis under test conditions is rapid. The observed Ki value of 0.3 nM is equivalent to that observed for the free boronic N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine acid, indicating the complete and rapid hydrolysis of the low D-mannitol adduct The test conditions.

Example 4: HPLC analysis of N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine boronic acid

System Parameters:

Column:

Adsorbosphere-HS-C18, 5µ, 250 x 4.6 mm

Mobile phase:

65:35 methanol / water containing 0.1% TFA

Flow rate:

1.0 ml / min

Detection / Sensitivity:

PDA and UV at 255 nm, 0.1 aufs

Injection volume:

25 µl

Internal standard solution:

0.18 mg / ml diphenylamine in methanol

Sample preparation:

0.5-1.5 mg portions weighed with exactness of the sample or reference standard

they were dissolved in 2.00 ml of the internal standard solution. Chromatographic parameters

Sample

Internal pattern

Holding time

8.4 min 18.9 min

Capacity factor, k ’

2.0 5.8

image5

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40

(Cont.)

Relay rel., Α

0.34

Resolution, Rs = ∆T / ΣW½

15.1

∆Ty ΣW½ are, respectively, the differences in retention times and the sum of the half-width of the sample peaks and the internal standard. The minor variation of the mobile phase is admitted to achieve results similar to the previous ones.

Example 5: Stability of formulations

Solid N- (2-pyrazino) carbonyl-L-phenylalanine-L-lecucin acid boronic acid

Boronic N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine acid is prepared as described in US Pat. No. 5,780,454. The product was obtained in the form of a white amorphous powder. The product was stable for more than 2 years when stored at -20 ° C, as determined by HPLC analysis (purity> 97%). When stored at 2-8 ° C, the product was not stable for more than 3-6 months.

Liquid N- (2-pyrazino) carbonyl-L-phenylalanine-L-lecucine acid boronic acid

A sterile formulation (0.5 mg / ml) of N- (2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid in 0.9% w / v saline, 2% v / ethanol was prepared v and 0.1% w / v ascorbic acid. When stored at 2-8 ° C, the liquid formulation was not stable for more than 6 months, as determined by HPLC analysis.

Boron of N- (2-pyrazino) carbonyl-L-phenylalanine-L-lecucine D-mannitol lyophilized

The lyophilized product was prepared according to Example 1 and stored at 5 ° C, at room temperature, 37 ° C, and at 50 ° C. Stability was monitored for approximately 18 months by periodic reconstitution of a sample and analysis of the total contents of the bottle by HPLC. During this period of time, there was no loss of drug in the freeze-dried product stored at any temperature and there was no evidence of product degradation peaks in the HPLC chromatograms.

Solution of reconstituted N- (2-pyrazino) carbonyl-L-phenylalanine-L-lecucine boronic acid

The lyophilized product was prepared according to Example 1, and the samples (2.5 mg / vial) were reconstituted with 2.5 ml of 0.9% sterile saline solution w / v. The solution was completed in 10 seconds and provided a clear, colorless solution containing 1 mg / ml of N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine boronic acid. The solution showed no signs of degradation when stored at room temperature (23 ° C) for 43 hours. No special care was taken to protect the solution from light.

The use of the terms "a" and "one" and "the" and similar references in the context of the description of the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, except otherwise indicated in the present invention or clearly contradicted by the context. The terms "comprises", "has", "includes", and "contains" should be considered as open terms (ie, they mean "includes, but not limited to,") unless otherwise indicated. The mention of ranges of values in the present invention are merely intended to serve as an abbreviated method of individually referring to each separate value that falls within the range, unless otherwise indicated in the present invention, and each separate value is incorporated into the specification as if cited individually in the present invention. All the procedures described in the present invention can be carried out in any suitable order unless otherwise indicated in the present invention or otherwise clearly contradicted by the context. The use of any and all of the examples, or of the language of the examples (for example, "as") provided in the present invention, is merely intended to better illuminate the invention and does not pose a limitation on the scope of the invention, unless otherwise claimed. No expression in the specification should be considered as indicative of any element not claimed as essential for the practice of the invention.

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Claims (33)

1. A compound of the formula (1): image 1 in which: P is hydrogen or an amino group protection moiety; R is hydrogen or C1-12 alkyl; A is 0, 1, or 2; R1, R2, and R3 are each independently hydrogen, C1-12 alkyl, C3-12 cycloalkyl, C6-14 aryl, or -CH2 R5; R5, in each case, is C6-14 aryl, (C6-14) aryl (C1-12) alkyl, (C1-12) alkyl, (C6-14,) aryl, C3-12 cycloalkyl, heterocyclyl comprising 3 at 8 atoms, in which one or more atoms is selected from N, O and S, heteroaryl comprising 5 to 14 atoms, in which 1-4 atoms are selected from N, O and S, or -W-R6, in that W is a calcogen and R6 is C1-12 alkyl; wherein the ring portion of any of said aryl, aralkyl, alkylaryl, cycloalkyl, heterocyclyl, or heteroaryl in R1, R2, R3, or R5 may be optionally substituted; Y Z1 and Z2 together form a residue obtained from mannitol, in which the atom bound to boron in each case is an oxygen atom, and wherein the compound of formula (1) is optionally lyophilized.

2.

The compound of claim 1, wherein A is 0.

3.

The compound of claim 1, wherein

R1, R2, and R3 are each independently hydrogen, C1-8 alkyl, C3-10 cycloalkyl, C6-10 aryl, or -CH2R5 ; R5 , in each case, is C6-C10 aryl, (C6-C10) aryl (C1-C6) alkyl, (C1-C6) alkyl, (C6-C10) aryl, C3-C10 cycloalkyl, C1-C8 alkoxy, or C1-C8 alkylthio; wherein the ring portion of any of said aryl, aralkyl, alkaryl, cycloalkyl, heterocyclyl, or heteroaryl groups of R1, R2, R3, or R5 may be optionally substituted.

Four.

The compound of claim 1, wherein P is R7-C (O) -, R7-S (O) 2-, R7-NH-C (O) -, or R7-O-C (O) -;

wherein R7 is C1-2 alkyl, C6-14 aryl, (C1-12) alkyl, C6-14 aryl, or (C6-14) aryl (C1-12) alkyl, any of which may be optionally substituted, or when P is R7-C (O) -o R7-S (O) 2-, R7 may also be a saturated, partially saturated, or aromatic heterocycle of 5 to 10 optionally substituted atoms, wherein 1- 4 atoms are selected from N, O and S.

5.

The compound of claim 4, wherein A is zero; R is hydrogen or C1-C8 alkyl; Y R3 is C1-C6 alkyl.

6.

The compound of claim 4, wherein P is R7-C (O) -or R7-S (O) 2-, and R7 is an aromatic heterocycle.

7.

The compound of claim 6, wherein P is (2-pyrazino) carbonyl.

8.

The compound of claim 6, wherein P is (2-pyrazino) sulfonyl.

9.

The compound of claim 1, wherein said compound is a mannitol ester of: 12

5 10 fifteen twenty 30 35 Boronic acid N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine; Boronic N- (2-quinoline) sulfonyl-L-homophenylalanine-L-leucine acid; Boronic N- (3-pyridino) carbonyl-L-phenylalanine-L-leucine acid; Boronic acid N- (4-morpholino) carbonyl-L-phenylalanine-L-leucine; N- (4-morpholino) carbonyl-β- (1-naphthyl) -L-alanine-L-leucine boronic acid; N- (8-quinoline) sulfonyl-β- (1-naphthyl) -L-alanine-L-leucine boronic acid; N- (4-morpholino) carbonyl- (O-benzyl) -L-tyrosine-L-leucine boronic acid; Boronic acid N- (4-morpholino) carbonyl-L-tyrosine-L-leucine; or N- (4-morpholino) carbonyl- [O- (2-pyridylmethyl)] - L-tyrosine-L-leucine boronic acid.

10.

The compound of claim 1, wherein said compound is: N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine D-mannitol boronate; N- (2-quinoline) sulfonyl-L-homophenylalanine-L-leucine D-mannitol boronate; N- (3-pyridino) carbonyl-L-phenylalanine-L-leucine D-mannitol boronate; N- (4-morpholino) carbonyl-L-phenylalanine-L-leucine D-mannitol boronate; N- (4-morpholino) carbonyl-β- (1-naphthyl) -L-alanine-L-leucine D-mannitol boronate; N- (8-quinoline) sulfonyl-β- (1-naphthyl) -L-alanine-L-leucine D-mannitol boronate; N- (4-morpholino) carbonyl- (O-benzyl) -L-tyrosine-L-leucine D-mannitol boronate; N- (4-morpholino) carbonyl-L-tyrosine-L-leucine D-mannitol boronate; or N- (4-morpholino) carbonyl- [O- (2-pyridylmethyl)] - L-tyrosine-L-leucine D-mannitol boronate.

eleven.

The compound of claim 1, wherein the mannitol is D-mannitol.

12.

A compound according to claim 1, which is N- (2-pyrazino) carbonyl-L-phenylalanine-Lleucine D-mannitol boronate.

13.

A lyophilized compound according to any one of claims 1 to 8, 10 and 12.

14.

A process for preparing a lyophilized compound of the formula (1):

image2 in which: P is hydrogen or an amino group protection moiety; R is hydrogen or C1-12 alkyl; A is 0, 1, or 2; R1, R2, and R3 are each independently hydrogen, C1-12 alkyl, C3-12 cycloalkyl, C6-14 aryl, or -CH2 R5; R5, in each case, is C6-14 aryl, (C6-14) aryl (C1-12) alkyl, C1-12 alkyl (C6-14) aryl, C3-12 cycloalkyl, heterocyclyl comprising 3 to 8 atoms, in which one or more atoms is selected from N, O and S, heteroaryl comprising 5 to 14 atoms, in which 1-4 atoms are selected from N, O and S, or -W-R6, in which W is chalcogen and R6 is alkyl; wherein the ring portion of any of said aryl, aralkyl, alkylaryl, cycloalkyl, heterocyclyl, or heteroaryl in R1, R2, R3, or R5 it may be optionally substituted; Y Z1 and Z2 have been obtained from a sugar, in which the sugar is mannitol; Understanding the procedure: (a) preparation of a mixture comprising

(i)

Water,

(ii)

a compound of formula (3)

image 1 wherein P, R, A, R1, R2, and R3 are as described above; and 10 Z’yZ’’OH; and (iii) mannitol; Y (b) lyophilization of the mixture. 15. The method of claim 14, wherein A is zero; R is hydrogen or C1-C8 alkyl; and R3 is C1-C6 alkyl. 16. The method of claim 14, wherein R1, R2, and R3 are each independently hydrogen, C1-C8 alkyl, C3-C10 cycloalkyl, C6-C10 aryl, or CH2-R5; R5, in each case, is C6-C10 aryl, (C6-C10) aryl (C1-C6) alkyl, (C1-C6) alkyl, C6-C10 aryl, C3-C10 cycloalkyl, C1 alkoxy -C8, or C1-C8 alkyl; wherein the ring portion of any of said aryl, aralkyl, alkaryl, cycloalkyl, heterocyclyl, or heteroaryl groups of R1, R2, R3, or R5 may be optionally substituted. 17. The method of claim 14, wherein P is R7-C (O) -, R7-S (O) 2-, R7-NH-C (O) -, or R7-O-C (O) -; Wherein R7 is C1-2 alkyl, C6-14 aryl, (C1-12) alkyl, C6-14 aryl, or (C6-14) aryl (C1-12) alkyl, any of which may be optionally substituted, or when P is R7-C (O) -or R7-S (O) 2-, R7 may also be a saturated, partially saturated, or aromatic heterocycle of 5 to 10 optionally substituted atoms, in which 1 -4 atoms are selected from N, O and S. 18. The method of claim 17, wherein P is R7-C (O) -or R7-S (O) 2-and R7 is an aromatic heterocycle. 19. The process of claim 18, wherein P is (2-pyrazino) carbonyl.

twenty.

The process of claim 18, wherein P is (2-pyrazino) sulfonyl.

twenty-one.

The process of claim 14, wherein the compound of formula (3) is: Boronic acid N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine; Boronic N- (2-quinoline) sulfonyl-L-homophenylalanine-L-leucine acid;

N- (3-Pyridino) carbonyl-L-phenylalanine-L-leucine boronic acid; Boronic acid N- (4-morpholino) carbonyl-L-phenylalanine-L-leucine; N- (4-morpholino) carbonyl-β- (1-naphthyl) -L-alanine-L-leucine boronic acid; N- (8-quinoline) sulfonyl-β- (1-naphthyl) -L-alanine-L-leucine boronic acid; N- (4-morpholino) carbonyl- (O-benzyl) -L-tyrosine-L-leucine boronic acid; Boronic acid N- (4-morpholino) carbonyl-L-tyrosine-L-leucine; or N- (4-morpholino) carbonyl- [O- (2-pyridylmethyl)] - L-tyrosine-L-leucine boronic acid.

22

The process of claim 21, wherein the compound of formula (3) is N- (2-pyrazino) carbonyl-Lphenylalanine-L-leucine boronic acid.

2. 3.

The process of claim 14, wherein the compound of formula (1) is N- (2-pyrazino) carbonyl-L-phenylalanine-L-leucine D-mannitol boronate.

24.

The process of claim 14, wherein the residue obtained from mannitol and the compound of formula (3) are present in a ratio of at least 1: 1.

25.

The process of claim 14, wherein the residue obtained from mannitol and the compound of formula (3) are present in a ratio of at least 5: 1.

26.

The process of claim 14, wherein the mixture further comprises a water miscible solvent.

27.

The process of claim 26, wherein the water miscible solvent is an alcohol.

28.

The process of claim 27, wherein the alcohol is tert-butanol.

29.

The process of claim 14, wherein the mannitol is D-mannitol.

30

The process of claim 14, further comprising (c) reconstitution of the lyophilized mixture with a pharmaceutically acceptable carrier.

31.

A composition comprising (i) the compound of claim 1 and (ii) a pharmaceutically acceptable carrier.

32

A composition comprising (i) the compound of the formula (1) prepared according to the process of claim 14 and (ii) a pharmaceutically acceptable carrier.

33.

A lyophilized cake comprising the compound of claim 1.

3. 4.

A lyophilized cake comprising the compound of the formula (1) prepared according to the process of claim 14.