KR20040111410A - Formulations of anthraquinone derivatives - Google Patents

Abstract

The invention is formulated so that, when dissolved in an aqueous solution, the pH of the solution is in the range of 5 to 9, wherein pK _a is in the form of a salt with a physiologically acceptable acid in the range of -3.0 to 9.0, A compound of formula (I)

Where

A is a C alkylene group having a chain length of 2 or more carbon atoms between NH and N (O) R'R ",

R 'and R "are each independently C _1-4 alkyl, and C _2-4 alkyl selected from hydroxy and C _2-4 di-hydroxyalkyl group, or, R' and R" are together C _2-6 alkylene group Indicates.

Description

Anthraquinone Derivative Formulations {FORMULATIONS OF ANTHRAQUINONE DERIVATIVES}

WO-A-91 / 05824 (National Research Development Corporation) discloses compounds of the formula: which compounds are optionally in the form of physiologically acceptable salts:

Where

R ₁ , R ₂ , R ₃ and R ₄ are each individually selected from hydrogen, X, NH-A-NHR and NH-AN (O) R'R ", wherein X is hydroxy, halogeno, amino, C _1-4 alkoxy or C _2-8 alkanoyloxy, A is a C alkylene group having a chain length of 2 or more carbon atoms between NH and NHR or N (O) R′R ″, R, R ′ and R "are each independently C _1-4 alkyl, and C _2-4 hydroxyalkyl and C _{2-4-dihydroxy} is selected from hydroxy group, a carbon atom bonded to the nitrogen atom in these groups is not bear the hydroxy groups, any carbon atom also is optionally substituted by two hydroxy groups, R 'and R ", together, R' and R" is C _2- to form a heterocyclic group having from 3 to 7 atoms in the ring together with the nitrogen atom to which bond ₆ alkylene group.

Preferred among the compounds of this formula is N-oxide AQ4N, which is generally synthesized by oxidation of AQ4 as follows:

AQ4N is in fact a prodrug and a reverse reaction occurs in vivo resulting in a protonated form of AQ4, an activator due to reductive metabolism in hypoxic cancer cells. The prodrugs are relatively nontoxic when compared to activator AQ4, which is particularly advantageous for administering them as medicaments. However, since AQ4N is not easy to obtain in crystalline form, it needs to be prepared and formulated for administration in salt form.

AQ4N has been reported to date in the dihydrochloride salt form, namely AQ4N.2HCl. See, eg, J. Chem. Soc., Perkin Trans. I, 1999, 2755-2758 (Lee et al.) And WO-A-00 / 05194 (BTG International Limited). However, by investigating the AQ4N.2HCl raw material, a significant amount of impurities referred to as AQMN of the formula, characterized by LCMS, namely 1-amino-4-{[2- (dimethylamino) ethyl] amino} -5,8- The presence of dihydroxyanthraquinones was demonstrated:

This impurity can be formed by the degradation of AQ4N and generally exhibits an undesirably higher level of cytotoxicity than the cytotoxicity of AQ4N. This level of cytotoxicity should be avoided for compounds to be administered in the form of relatively nontoxic prodrugs.

Although decomposition of AQMN occurs with a predominant route, further degradation products of AQ4N under acidic and neutral aqueous solution conditions are the following mono-N-oxides, AQ4M:

We have demonstrated that AQ4N, a dihydrochloride salt in aqueous solution, generally produces a pH of about 2-3. For example, the pH of a 1.4 mmol solution is 2.4. The present inventors have now found that the above problems due to impurity formulations can be reduced or avoided by preparing, formulating and administering the compounds such that when dissolved in solution, the pH of the solution is in the range of 5-9.

The present invention relates to novel formulations of anthraquinone derivatives such as AQ4N, a bis-bioreducing agent of proven value in cancer treatment. This includes novel salt forms of anthraquinone derivatives.

1 shows the pH of the first derivative versus the pH in AQ4N dihydrochloride solution.

2 shows the molar equivalents of NaOH under the same conditions as the pH of the first derivative.

3 shows the increase in AQMN over incubation time of 5 mg / ml solution incubated at 40 ° C. for 14 days.

4 shows the increase in AQMN over the incubation time of a 5 mg / ml solution incubated at 40 ° C. for 63 days.

5 shows the decrease in AQ4N over the incubation time of a 5 mg / ml solution incubated at 40 ° C. for 14 days.

6 shows the decrease in AQ4N over the incubation time of a 5 mg / ml solution incubated at 40 ° C. for 63 days.

7 shows the formalized method used to prepare salts of AQ4N.

8 shows the organic acid used to prepare a salt of AQ4N.

According to the invention, upon dissolution in an aqueous solution, a compound of formula (I) is provided wherein the pH of the solution is in the range of 5-9:

Where

A is a C alkylene group having a chain length of 2 or more carbon atoms between NH and N (O) R'R ", and R 'and R" are each independently a C _1-4 alkyl group, and C _2-4 hydroxyalkyl And C _2-4 dihydroxyalkyl groups, wherein the carbon atoms bonded to the nitrogen atoms in these alkyl groups do not have a hydroxy group, neither carbon atom is substituted by two hydroxy groups, or R 'and R "together, Together with the nitrogen atom to which R 'and R "are bonded represents a C _2-6 alkylene group which forms a heterocyclic group having 3 to 7 atoms in the ring.

Preference is given to said compounds in which the pH of the solution upon dissolution in aqueous solution is in the range of 6 to 8.

Preference is given to said compounds in which the pH of the solution is in said specific range at a concentration of 0.1 to 100 mg / ml upon dissolution in aqueous solution.

The compounds of formula (I) may be used in the form of physiologically acceptable salts which are acid addition salts with inorganic or organic acids. Preferably, the pK _a of _a physiologically acceptable acid is in the range of -3.0 to 9.0, more preferably in the range of 2.0 to 9.0. More preferably, the physiologically acceptable acid has a pK _a in the range of 2.0 to 6.0.

Preferably, the physiologically acceptable acid is selected from the group consisting of tartaric acid, malonic acid, dichloroacetic acid, citric acid, maleic acid, benzenesulfonic acid, pimelic acid and acetic acid.

More preferably, the physiologically acceptable acid has a pK _a in the range of 3.0 to 6.0. Physiologically acceptable acids are especially organic acids, in particular organic mono-, di- or tri-acids, which are in particular selected from the group consisting of tartaric acid, citric acid, pimelic acid and acetic acid.

A in formula (I) may be branched, but this is usually a straight chain alkylene group, ie tetramethylene, in particular trimethylene or especially ethylene.

R 'and R "may also have a branched carbon chain, which is typically an alkyl group or a hydroxy substituted straight chain alkyl group. R' or R" is a monohydroxyalkyl group, which is typically terminally substituted and R ' Or where R "is a dihydroxyalkyl group, this is typically terminated by one hydroxy group. When R 'and R" are alkyl groups, groups of three or in particular two or one carbon atoms are preferred. When R 'and R "are hydroxy substituted alkyl groups, alkyl groups of three carbon atoms, or monohydroxyalkyl groups are preferred, alternatively groups of two carbon atoms are preferred. Preferred individual groups R Examples of 'and R "include CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH ₂ CH ₂ OH, CH ₂ CH ₂ CH ₂ OH, CH (CH ₃ ) CH ₂ OH and CH ₂ CHOHCH ₂ There is OH.

R 'and R "will be more generally the same.

Alternatively, as described, R ′ and R ″ together with the nitrogen atom to which they are attached, heterocyclic group —N (CH ₂ ) _n , ie aziridin-1-yl, an azee, wherein n is 2 to 6 Thidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, and perhydroazin-1-yl, and such as azetidin-1-yl, especially aziridin-1-yl Smaller groups are most noted.

Particularly of particular NH-AN (O) R'R "groups that are of particular interest are NH- (CH ₂ ) ₂ -N (O) (CH ₃ ) C ₂ H ₅ , NH- (CH ₂ ) ₂ -N (O ) (C ₂ H ₅ ) ₂ , NH- (CH ₂ ) ₂ -N (O) (CH ₂ CH ₂ OH) ₂ , NH- (CH ₂ ) ₂ -N (O) (CH ₂ CH ₂ CH ₂ OH ) ₂ , NH- (CH ₂ ) ₂ -N (O) (CH (CH ₃ ) CH ₂ OH) ₂ , NH- (CH ₂ ) ₂ -N (O) (CH ₂ CHOHCH ₂ OH) ₂ and especially NH -(CH ₂ ) ₂ -N (O) (CH ₃ ) ₂ .

Physiologically acceptable salts when simply dissolved in an aqueous solution will generally provide a solution having a pH lower than the desired range. For example, the pH of the acetate salt of AQ4N in a 1.4 mmol aqueous solution is 3.8. Thus, preferably, the compound is formulated in a mixture containing additional components such that upon dissolution in aqueous solution, the pH of the solution is buffered in the range of 5-9.

The buffer is a solvated mixture of salts and acids, which inhibits the pH change when small amounts of acid and base are added to the solution. Suitable buffers include sodium acetate buffer and sodium orthophosphate buffer.

According to a further aspect of the invention there is provided an aqueous solution of the compound of formula (I), wherein the pH of the aqueous solution of the compound of formula (I) is in the range of 5-9.

Certain novel salt forms can be used in the present invention. When they are salts of acids with a higher pK _a (approximately -6) than hydrochloric acid, these novel salt forms are additional ingredients that buffer the pH when dissolved in aqueous solutions, since they are less acidic compared to dihydrochloride salts. It will be more easily formulated as a solid mixture containing.

Accordingly, according to a further aspect of the invention there is provided a compound of formula (I), which is in the form of a salt with a physiologically acceptable acid, wherein the pK _a of the salt is in the range of -3.0 to 9.0:

Where

A is a C alkylene group having a chain length of 2 or more carbon atoms between NH and N (O) R'R ", and R 'and R" are each independently a C _1-4 alkyl group, and C _2-4 hydroxy Alkyl and a C _2-4 dihydroxyalkyl group, wherein the carbon atom bonded to the nitrogen atom does not contain a hydroxy group, neither carbon atom is substituted by two hydroxy groups, or R 'and R "together, Together with the nitrogen atom to which R 'and R "are bonded represents a C _2-6 alkylene group which forms a heterocyclic group having 3 to 7 atoms in the ring.

The pK _a of the physiologically acceptable acid is preferably in the range of 2.0 to 9.0, and more preferably, the pK _a of the physiologically acceptable acid is in the range of 2.0 to 6.0.

The salt may generally be formed as an anhydrous salt, which may be more stable as a crystalline solid than the dihydrochloride salt with the water of crystallisation present. This salt will also be easy to obtain in pure form.

The compounds of formula (I) will be used in the form of physiologically acceptable salts which are acid addition salts with organic or inorganic acids. Physiologically acceptable acids having _a pK _a in the range of -3.0 to 9.0 can be obtained from Table 1 below:

Table 1: pK of some common acids _a value

Free acid or base PK at 25 ° C _a Benzenesulfonic Acid -2.50 p-toluenesulfonic acid -1.34 Methanesulfonic acid -1.2 Dichloroacetic acid 1.25 Maleic acid 2.00 (pK _a1 ) Benzenehexacarboxylic acid (melt acid) 2.08 (pK _a1 ) Phosphoric Acid 2.12 (pK _a1 ) Brucin Carbohydrate 2.30 (pK _a1 ) Benzene Pentacarboxylic Acid 2.34 (pK _a1 ) Glycine 2.34 (pK _a1 ) Benzene-1,2,4,5-tetracarboxylic acid (pyromellitic acid) 2.43 (pK _a1 ) Malonic acid 2.85 (pK _a1 ) Phthalic acid 2.90 Salicylic acid 2.98 Benzene-1,2,3-tricarboxylic acid (hememilic acid) 2.98 (pK _a1 ) Tartaric acid 3.02 (pK _a1 ) Fumaric acid 3.03 (pK _a1 ) Glycylglycine 3.06 Glycofentatetra-1,2,3,4-carboxylic acid 3.07 (pK _a1 ) O-phthalic acid 3.10 (pK _a1 ) Citric acid 3.13 (pK _a1 ) Benzene-1,2,4,5-tetracarboxylic acid (pyromellitic acid) 3.13 (pK _a1 ) Benzene-1,3,5-tricarboxylic acid (trimelitic acid) 3.16 (pK _a1 ) Glucuronic acid 3.18 Dimethyl Malonic Acid 3.29 (pK _a1 ) Mandelic acid 3.36 Butane-1,2,3,4-tetracarboxylic acid 3.36 (pK _a1 ) Malic acid 3.40 (pK _a1 ) 1,1-cyclohexanediacetic acid 3.52 (pK _a1 ) 2-Methylpropane-1,2,3-triscarboxylic acid 3.53 (pK _a1 ) Mount Hipur 3.64 Propane-1,2,3-tricarboxylic acid (tricarvalic acid) 3.67 (pK _a1 ) Formic acid 3.75 Gluconic Acid 3.76 3,3-dimethylglutaric acid 3.79 (pK _a1 ) 1,1-cyclopentanediacetic acid 3.82 (pK _a1 )

Itaconic acid 3.84 (pK _a1 ) Lactic acid 3.86 Barbituric acid 3.98 Ascorbic acid 4.10 (pK _a1 ) 2,2-dimethylsuccinic acid 4.11 (pK _a1 ) Succinic acid 4.19 (pK _a1 ) Benzoic acid 4.20 3,6-endomethylene-1,2,3,6-tetrahydrophthalic acid "EMTA" 4.30 (pK _a1 ) 2,2-dimethylglutaric acid 4.31 (pK _a1 ) Pimelic acid 4.48 Acetic acid 4.75 Sorbic acid 4.76 n-butyric acid 4.82 Propionic acid 4.87 Malic acid 5.05 (pK _a2 ) Pyridine 5.23 Hydroxyamine 6.03 Edetic acid 6.16 (pK _a3 ) Carbonic acid 6.35 (pK _a1 ) Citric acid 6.40 (pK _a3 ) Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane "BIS-TRIS" 6.46 Imidazole 7.00 2- (aminoethyl) trimethylammonium chloride "CHOLAMINE" 7.10 Phosphoric Acid 7.21 (pK _a2 ) 2-hydroxyethyliminotris (hydroxymethyl) methane "MONO-TRIS" 7.83 4- (2-hydroxyethyl) -1-piperazinepropane sulfonic acid "EPPS" 8.00

Preferably, the physiologically acceptable acid is selected from the group consisting of tartaric acid, malonic acid, dichloroacetic acid, citric acid, maleic acid, benzenesulfonic acid, pimelic acid and acetic acid.

More preferably, the physiologically acceptable acid has a pK _a in the range of 3.0 to 6.0. Such physiologically acceptable acids are in particular organic acids, in particular organic mono-, di- or tri-acids, which are especially selected from the group consisting of tartaric acid, citric acid, pimelic acid and acetic acid.

A in the compound of formula (I) may be branched, but it is usually a straight chain alkylene group, ie tetramethylene, in particular trimethylene or especially ethylene.

R 'and R "may also have branched carbon chains, but they are typically alkyl groups or hydroxy substituted straight chain alkyl groups. When R' or R" is a monohydroxyalkyl group, it is usually terminated at the end When R 'or R "is a dihydroxyalkyl group, it is usually terminated by one hydroxy group. When R' and R" are alkyl groups, three or in particular two or one carbon atoms If the group is preferred and R 'and R "are hydroxy substituted alkyl groups, the groups of three carbon atoms, or monohydroxyalkyl groups, are preferred, alternatively groups of two carbon atoms are preferred. Examples of groups R ′ and R ″ include CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH ₂ CH ₂ OH, CH ₂ CH ₂ CH ₂ OH, CH (CH ₃ ) CH ₂ OH and CH ₂ CHOHCH ₂ OH.

R 'and R "will be more generally the same.

Alternatively, as described, R ′ and R ″ together with the nitrogen atom to which they are attached, heterocyclic group —N (CH ₂ ) _n , ie, aziridin-1-yl, an azee, wherein n is 2 to 6 Thidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, and perhydroazin-1-yl, and such as azetidin-1-yl, especially aziridin-1-yl Smaller groups are most preferred.

Particularly of particular NH-AN (O) R'R "groups that are of particular interest are NH- (CH ₂ ) ₂ -N (O) (CH ₃ ) C ₂ H ₅ , NH- (CH ₂ ) ₂ -N (O ) (C ₂ H ₅ ) ₂ , NH- (CH ₂ ) ₂ -N (O) (CH ₂ CH ₂ OH) ₂ , NH- (CH ₂ ) ₂ -N (O) (CH ₂ CH ₂ CH ₂ OH ) ₂ , NH- (CH ₂ ) ₂ -N (O) (CH (CH ₃ ) CH ₂ OH) ₂ , NH- (CH ₂ ) ₂ -N (O) (CH ₂ CHOHCH ₂ OH) ₂ and especially NH -(CH ₂ ) ₂ -N (O) (CH ₃ ) ₂ .

Salts with physiologically acceptable acids can be prepared by any conventional means, for example by reaction of an organic base (I) with a suitable inorganic or organic acid, usually by simple mixing in solution. Such acid addition salts are generally crystalline solids that are relatively readily soluble in water, methanol, ethanol and similar solvents. One salt form can also be converted to another by chromatography using a column pretreated with the desired physiologically acceptable acid.

The compounds of formula (I) may be formulated with physiologically acceptable diluents or carriers for use as medicaments for both animals and especially humans in a variety of ways. For example, they can be applied as compositions incorporating liquid diluents or carriers, such as aqueous solutions, suspensions or emulsions, which can often be used in injectable forms for parenteral administration, so they are typically sterile or nonpyrogenic. Can be.

Oral modes of administration may also be used, and although compositions for this purpose may be incorporated into liquid diluents or carriers, it is more convenient to use conventional solid carrier materials such as solids such as starch, lactose, dextrin or magnesium stearate It is common. Such solid compositions may take the form of powders, but already formed types such as tablets, sachets or capsules are more commonly used. Alternatively, more specialized types of formulations include liposomes and nanoparticles.

Other types of administration other than via injection or oral use for both humans and animals include suppositories or pessaries. Another form of pharmaceutical composition is for administration via the cheeks or nose, or alternatively eye drops for administration into the eye, which may typically contain a sterile liquid diluent or carrier. Other formulations for topical administration include lotions, ointments, creams, gels and sprays.

The composition may be formulated in unit dosage form, ie in the form of unit doses, or individual portions containing repeat or subdose of a unit dose.

Although the dose of the compound used will vary depending on the activity of the particular compound and its processing conditions, a dose selected in the range of 25 to 500 mg / m 2 per day, in particular 50 to 300 mg / m 2 per day, may be mentioned as suitable. Higher doses, such as 25 to 750 mg / m 2 daily, or even up to 1200 mg / m 2 daily, in which lower levels of toxic side effects are obtained when using compounds of formula (I). Can be considered. However, such dosing regimens, which can last for several days, should be suitable for the patient to be administered, and the daily dose may be divided into multiple individual doses as needed. Thus, for diseases such as, for example, advanced breast cancer, non-Hodgkin's lymphoma, and liver cancer, repeated administration after a day, such as after 21 days, may be suitable, whereas acute nonlymphocytic leukemia For treatment of 5 consecutive days of treatment may be more suitable. Alternatively, a single dose every few days, for example, once every two or three weeks, may be adopted.

Compounds of formula (I) are particularly effective in treating cancers that develop in warm-blooded animals, including humans. The compounds are of interest with respect to the treatment of solid tumors such as various types of sarcomas and carcinomas, and also seeded tumors such as leukemia. Of particular interest are the treatment of breast cancer, lung cancer, prostate cancer, pancreatic cancer and esophageal cancer, and treatment of non-Hodgkin's lymphoma and acute nonlymphocytic leukemia. In the treatment of cancer, parenteral and sometimes topical administration is often of particular note. In addition, other anticancer agents, such as cleavage inhibitors, such as vinblastine, provided separately or together in the same composition; Alkylating agents such as cisplatin, carboplatin and cyclophosphamide; Other antimetabolic agents such as 5-fluorouracil, cytosine arabinoside and hydroxyurea; Antibiotics to be inserted, such as adriamycin and bleomycin; Enzymes such as asparaginase; It may be advantageous to use the compounds of formula (I) in combination therapy with topical isomerase inhibitors such as etoposide and biological response modifiers such as interferon. The compounds of formula (I) may also be used in combination therapy with radiation therapy of tumors.

Accordingly, the present invention includes methods for assisting in the regression and alleviation of cancer, including administering to a patient a therapeutically effective amount of a compound of formula (I) as defined above.

In addition to their anticancer use, the compounds of formula (I) are noted for a variety of other pharmaceutical uses in terms of their activity as chelating agents.

Example 1 Instability of AQ4N Dihydrochloride- Demonstration of Physicochemical Properties of AQ4N

To verify the degradation of AQ4N to AQMN, the pH change of the AQ4N dihydrochloride solution was monitored. The pH curves are shown in FIGS. 1 and 2, where FIG. 1 shows clear dissolution at pH 7.7 to pH 9.4, which is identical to the dissolution pattern shown in FIG. 2 at approximately 2 molar equivalents. In particular, a low pH dissolution pattern can be identified which is theoretically assigned to a pH in the range of 4.1 to 4.6, wherein the molar equivalent is 0.95 to 1.15.

Example 2: Demonstrating Cytotoxicity of AQMN

Toxicity of pure samples (99.3%) in the P388 system of AQ4N and AQMN was measured and the results obtained are shown in Table 2 below.

Table 2: AQ4N and AQMN Cytotoxicity Values

compound IC ₅₀ P388 (nM) Relative toxicity (standardized for AQ4N) AQ4N 410 1.0 AQMN 77 5.2

From the data, it can be seen that the cytotoxicity of AQMN is at least five times that of AQ4N cytotoxicity in situ. Since all samples of AQ4N contain a substantial percentage of AQMN that will affect the toxic results, a much higher amount of modifier is required.

Example 3: Instability of AQ4N Dihydrochloride in Solution- Demonstration of Accumulation of AQMN

Degradation of AQ4N was measured using 5 mg / ml AQ4N solution at pH 2.4, 4.5 and 6.8 and the same was done for water, 20 mM sodium acetate buffer and 20 mM sodium orthophosphate buffer, respectively. The main degradation pathway of AQ4N is that it is converted to AQMN. The increase in AQMN concentration of the 5 mg / ml solution incubated at 40 ° C. over a moderate time period (14 days) is shown in FIG. 3.

The degradation rates corresponded to 0.84% (w.r.t. AQ4N), 0.19% (w.r.t. AQ4N) and 0.02% (w.r.t. AQ4N) increases in AQMN content per day under these conditions.

By linear regression and correlation methods using known amounts of AQMN in the materials used as standards, these data represent the accumulation rate of AQMN described in Table 3 below.

Table 3: Accumulation of AQMN

H ₂ O acetate PO ₄ Increase in AQMN per day 0.84% 0.19% 0.02%

Similar trends were observed in the data obtained after 63 days, which is shown in FIG. 4. It can be seen that the rate of accumulation (calculated in the same manner as described above) increases AQMN by 0.6% per month in phosphate buffer.

Example 4: Instability of AQ4N Dihydrochloride in Solution-Demonstration of Degradation of AQ4N

The pH effect on the stability of AQ4N was measured by examining the degree of AQ4N degradation in different solutions. The solution chosen was distilled water, 20 mM sodium acetate buffer (pH = 4) and 20 mM sodium phosphate buffer (pH = 7). After preparing 5 mg / ml AQ4N buffer, the pH was corrected to the desired buffer pH. Final pH was 2.4, 4.5 and 6.8 for distilled water, 20 mM sodium acetate buffer and 20 mM sodium phosphate buffer, respectively. These samples were incubated at 40 ° C. and sampled at equal intervals. After assaying the sample by dilution, HPLC analysis was performed.

After 14 days, an intermediate analysis was performed using the data shown in FIG. 5.

The solution was further incubated at 40 ° C. for a total of 63 days. The final graph is shown in FIG. The value obtained for the AQ4N content was consistent with the amount of AQ4N weighed into the original sample (within the experimental error range).

Example 5: Synthesis of Some Organic Acid Salts of AQ4N

A series of organic acid addition salts of AQ4N were prepared as shown in FIG. 7 and their stability against AQMN formulations was measured.

The choice for the method of synthesis of the AQ4N salt was limited by the high solubility of the AQ4N free base in polar solvents. Synthesis of malonic acid, citric acid, tartaric acid addition salts of AQ4N was carried out using a selected aqueous organic acid solution (1M), a polar solvent / aqueous mixture such as H ₂ O / CH ₃ CN (1: 1) or H ₂ O using a minimum amount of DMSO. The first attempt was made by adding to an AQ4N free base solution in water. After stirring for 2 hours, the reaction mixture was cooled to 4 ° C. to facilitate precipitation. However, no solids were identified after 7 days. In subsequent work, MeOH was used as a solvent to easily dissolve both AQ4N and all organic acids. In all cases except for the preparation of benzenesulfonic acid, AQ4N was added to a 20-fold excess of organic acid and this mixture was left at room temperature for 14-24 hours and then held at -10 ° C to aid precipitation. If an extremely small amount of solid material or no solid material was found, diethyl ether was added to the mixture to promote precipitation.

Example 6: AQ4N Di-benzenesulfonate

AQ4N (36 mg, 0.081 mmol) was dissolved in MeOH (5 mL) and stirred for 5 minutes. Benzenesulfonic acid (25.66 mg, 0.162 mmol) dissolved in 1 mL of MeOH was added to the stirred solution of AQ4N. The reaction mixture was stirred for 30 minutes and the precipitated solid was separated by filtration. The dark blue solid was dried for 16 hours using phosphorus pentoxide in a dryer under vacuum. Yield: 45 mg, 76%, calcd for C ₃₄ H ₄₀ N ₄ O ₁₂ S: C, 56.04; H, 5.53; N, 7.69. Found: C, 55.80; H, 5. 35; N, 9.30.

Example 7: AQ4N di-dichloroacetate

AQ4N (10 mg, 0.023 mmol) was dissolved in MeOH (1 mL) and stirred for 5 minutes. Dichloroacetic acid (38 μl, 0.46 mmol) was added to the stirred solution of AQ4N. The reaction mixture was stirred at room temperature for 1 hour and then at -10 ° C. for 16 hours. The precipitated solid was separated by filtration. The dark blue solid was dried for 16 hours using phosphorus pentoxide in a dryer under vacuum. Yield: 7.4 mg, 46%, calcd for C ₂₆ H ₃₂ N ₄ O ₁₀ Cl ₄ : C, 44.46; H, 4.59; N, 7.98. Found: C, 44.58; H, 4.61; N, 7.91.

Example 8: AQ4N di-maleate

AQ4N (10 mg, 0.023 mmol) was dissolved in MeOH (1 mL) and stirred for 5 minutes. Maleic acid (53.4 mg, 0.46 mmol) in MeOH (1 mL) was added to the stirred solution of AQ4N. The reaction mixture was stirred at room temperature for 1 hour and then at -10 ° C for 16 hours. The precipitated solid was separated by filtration. The dark blue solid was dried for 16 hours using phosphorus pentoxide in a dryer under vacuum. Yield: 7 mg, 47%, calcd for C ₃₀ H ₃₆ N ₄ 0 ₁₄ : C, 53.24; H, 5. 32; N, 8.28. Found: C, 53.11; H, 5. 46; N, 8.20.

Example 9: AQ4N di-malonate

AQ4N (10 mg, 0.023 mmol) was dissolved in MeOH (1 mL) and stirred for 5 minutes. Malonic acid (47.9 mg, 0.46 mmol) in MeOH (1 mL) was added to the stirred solution of AQ4N. The reaction mixture was stirred at room temperature for 1 hour and then at -10 ° C for 24 hours. The precipitated solid was separated by filtration. The dark blue solid was dried for 16 hours using phosphorus pentoxide in a dryer under vacuum. Yield: 8.2 mg, 55%, calcd for C ₂₈ H ₃₆ N ₄ 0 ₁₄ : C, 51.53; H, 5.56; N, 8.59. Found: C, 51.67; H, 5.56; N, 8.74.

Example 10 AQ4N Di-Tartrate

AQ4N (10 mg, 0.023 mmol) was dissolved in MeOH (1 mL) and stirred for 5 minutes. Tartaric acid (69.1 mg, 0.46 mmol) in MeOH (1 mL) was added to the stirred solution of AQ4N. The reaction mixture was stirred at room temperature for 1 hour and then at -10 ° C for 16 hours. The precipitated solid was separated by filtration. The dark blue solid was dried for 24 hours using phosphorus pentoxide in a dryer under vacuum. Yield: 8.5 mg, 50%, calcd for C ₃₀ H ₄₀ N ₄ 0 ₁₈ : C, 48.39; H, 5.41; N, 7.52. Found: C, 48.34; H, 5.41; N, 7.67.

Example 11: AQ4N Di-citrate

AQ4N (10 mg, 0.023 mmol) was dissolved in MeOH (1 mL) and stirred for 5 minutes. Citric acid (88.4 mg, 0.46 mmol) in MeOH (1 mL) was added to the stirred solution of AQ4N. The reaction mixture was stirred at room temperature for 1 hour and then at -10 ° C for 16 hours. Ether (2 mL) was added to the reaction mixture, which was left at −10 ° C. for an additional 3 hours. The precipitated solid was separated by filtration. The dark blue solid was dried for 24 hours using phosphorus pentoxide in a dryer under vacuum. Yield: 7.1 mg, 37%, calcd for C ₃₄ H ₄₄ N ₄ O ₂₀ : C, 49.28; H, 5. 10; N, 6.65. Found: C, 49.59; H, 5.09; N, 6.64.

Example 12 AQ4N Di-lactate

AQ4N (10 mg, 0.023 mmol) was dissolved in MeOH (1 mL) and stirred for 5 minutes. Lactic acid (35 μl, 0.46 mmol) was added to the stirred solution of AQ4N. The reaction mixture was stirred at room temperature for 1 hour and then at -10 ° C for 16 hours. Ether (2 mL) was added to the reaction mixture, which was left at −10 ° C. for an additional 3 hours. The precipitated solid was separated by filtration. The dark blue solid was dried for 24 hours using phosphorus pentoxide in a dryer under vacuum. Yield: 4.5 mg, 31%, calcd for C ₂₈ H ₄₀ N ₄ O ₁₂ : C, 53.84; H, 6. 45; N, 8.97. Found: C, 51.54; H, 6. 24; N, 8.89.

Example 13: AQ4N Di-Pimelate

AQ4N (10 mg, 0.023 mmol) was dissolved in MeOH (1 mL) and stirred for 5 minutes. Pimelic acid (73.7 mg, 0.46 mmol) in MeOH (1 mL) was added to the stirred solution of AQ4N. The reaction mixture was stirred at room temperature for 1 hour and then at −10 ° C. for 14 hours. The precipitated solid was separated by filtration. The dark blue solid was dried for 24 hours using phosphorus pentoxide in a dryer under vacuum. Yield: 8.2 mg, 47%, calcd for C ₃₆ H ₅₂ N ₄ 0 ₁₄ : C, 56.80; H, 6. 80; N, 7.34. Found: C, 56.49; H, 6. 41; N, 8.46.

Example 14 AQ4N Di-Acetate

AQ4N (10 mg, 0.158 mmol) was dissolved in MeOH (1 mL) and stirred for 5 minutes. Acetic acid (183 μl, 3.16 mmol) in MeOH (1 mL) was added to a stirred solution of AQ4N. The reaction mixture was stirred at room temperature for 1 hour and then at -10 ° C. for 18 hours. Ether (3 mL) was added to the reaction mixture, which was left at −10 ° C. for an additional 3 hours. The precipitated solid was separated by filtration. The dark blue solid was dried for 24 hours using phosphorus pentoxide in a dryer under vacuum. Yield: 68 mg, 76%, calcd for C ₂₆ H ₃₆ N ₄ O ₁₀ : C, 55.31; H, 6. 43; N, 9.92. Found: C, 55.28; H, 6.57; N, 9.91.

Example 15 pH Measurement of AQ4N Organic Acid Salts in Aqueous Solution at 37 ° C

The stock solution of the individual AQ4N salt was accurately weighed to about 1 mg, which was diluted with water to give a solution of 1.4 mM. The solution was stirred for 3 minutes using Fisons WhirlMixer ^™ (Roborosa, Leicestershire, UK). Prepare all AQ4N salt solutions and measure the pH value of this solution on the same day using a digital pH meter (calibrated at 21 ± 1 ° C. using standard buffers pH 7.00 and 4.00 from BDH, UK) It was.

Table 4 and FIG. 7 below describe the nine organic acids used to prepare the AQ4N salts in order of increasing pK _a1 values.

Table 4: pK of acids used to prepare salts of AQ4N _a1 And pH of the resulting aqueous solution

Acids Used to Prepare AQ4N Dichloride pK _a1 pH ^a Hydrochloric acid ～ -6 2.4 Benzenesulfonic Acid -2.5 2.5 Dichloroacetic acid 1.25 2.4 Maleic acid 2 2.6 Malonic acid 2.85 2.8 Tartaric acid 3.02 2.8 Citric acid 3.13 3.3 DL-lactic acid 3.86 3.5 Pimelic acid 4.48 3.8 Acetic acid 4.75 3.8

^a : measured pH of AQ4N phosphate solution (1.4 mM)

The pK _a data are described in “IUPAC Handbook of Pharmaceutical Salts, Properties, selection and Use”, P. Heinrich Stahl, Camille G. Wermuth (Eds) VHCA VerlagHelvitica Chimica Acta, Zurich & Wiley-VCH, Weinheim (joint publ.) 2002 Is an excerpt from "

PK _a for hydrochloric acid was also included for comparison purposes. The organic acids used to prepare the AQ4N salts in this study are as follows:

Mono-acid: benzenesulfonic acid, dichloroacetic acid, lactic acid, acetic acid

ㆍ Di-acid: maleic acid, malonic acid, tartaric acid, pimelic acid

Tree-acid: citric acid

Since the pK _a of these acids indicates the degree of ionization of each acid moiety, this will reflect these acids measured by pH. From Table 4 it can be seen that the measured pH of the AQ4N salt solution (1.4 mM) is low at pH 2.4 and 2.5 for benzenesulfonate and dichloroacetate salts and pH 3.8 for acetate and pimelate salts, respectively.

Example 16: Determination of Purity of AQ4N Organic Acid Salts in Aqueous Solution at 37 ° C

Chromatographic separation of AQ4N salt solution was performed on a HiChrom HIRAB (250 mm x 4.6 mm, 5 μm) column (HiChrom, Agilent, Reading, UK) embedded in a flat fluid column and a Plattron Column Heater at 24 ° C. , P / N HIRPB-6294). The fluidized phase used for the gradient separation of AQ4N salts was solvent system A [5% acetonitrile and 95% ammonium formate buffer (0.05 M, pH 3.6)] and solvent system B [50% acetonitrile and 50% ammonium formate (0.05 M , pH 3.6)]. The pH was adjusted to 3.6 using formic acid. The gradient shifted from 20% fluid bed A to 95% fluid bed B over 55 minutes. For all separations, a flow rate of 1.3 ml / min was applied. Data was collected using λ = 612 nm and processed using Waters Millennium ^{32 ™} software.

Freshly prepared solution (1.4 mM) of the prepared AQ4N salt was chromatographed within 30 minutes of dissolution time. From all AQ4N salts, mono-N-oxide AQ4M or AQMN was less than 1% and AQ4N was predominant (greater than 99%).

Claims (18)

When dissolved in an aqueous solution, the compound of formula (I) characterized in that it is formulated so that the pH of the solution is in the range of 5-9: Where A is a C alkylene group having a chain length of 2 or more carbon atoms between NH and N (O) R'R ", R 'and R "are carbon atoms bonded to each individually is selected from C _1-4 alkyl, and C _2-4 hydroxyalkyl and C _2-4 di-hydroxyalkyl group, the nitrogen atoms in these alkyl groups bear the hydroxy groups And neither carbon atom is substituted by two hydroxy groups, or R 'and R "together with the nitrogen atom to which R' and R" are bonded form a heterocyclic group having 3 to 7 atoms in the ring; C _2-6 alkylene group is represented. The compound of claim 1, wherein upon dissolution in an aqueous solution, the pH of the solution is formulated to be in the range of 6 to 8. 8. The compound of claim 1 or 2, wherein pK _a is used in the form of a salt with a physiologically acceptable acid in the range of -3.0 to 9.0. A compound of formula (I) wherein pK _a is in the form of a salt with a physiologically acceptable acid in the range of -3.0 to 9.0: Where A is a C alkylene group having a chain length of 2 or more carbon atoms between NH and N (O) R'R ", R 'and R "are carbon atoms bonded to each individually is selected from C _1-4 alkyl, and C _2-4 hydroxyalkyl and C _2-4 di-hydroxyalkyl group, the nitrogen atoms in these alkyl groups bear the hydroxy groups And neither carbon atom is substituted by two hydroxy groups, or R 'and R "together with the nitrogen atom to which R' and R" are bonded form a heterocyclic group having 3 to 7 atoms in the ring; C _2-6 alkylene group is represented. The compound according to claim 3 or 4, wherein the physiologically acceptable acid has a pK _a in the range of 2.0 to 9.0. A compound according to claim 5, wherein the physiologically acceptable acid has a pK _a in the range of 2.0 to 6.0. 7. The compound of claim 6, wherein the physiologically acceptable acid has a pK _a in the range of 3.0 to 6.0. 4. A compound according to claim 3, wherein the physiologically acceptable acid is an organic mono-acid, di-acid or tri-acid. The compound according to claim 3 or 4, wherein the physiologically acceptable acid is selected from the group consisting of tartaric acid, malonic acid, dichloroacetic acid, citric acid, maleic acid, benzenesulfonic acid, pimelic acid and acetic acid. 10. A compound according to any one of claims 1 to 9, wherein A is a straight chain alkylene group. The compound of any one of claims 1 to 10, wherein A is ethylene. 12. A compound according to any one of claims 1 to 11, wherein R 'and R "are straight chain alkyl groups or hydroxy substituted alkyl groups. 13. A compound according to claim 12, wherein R 'and R "are each CH ₃ or CH ₂ CH ₃ . The compound of claim 13, wherein each group of formula NH-AN (O) R′R ″ is a group of formula NH— (CH ₂ ) ₂ —N (O) (CH ₃ ) ₂ . The compound according to any one of claims 1 to 14, characterized in that when dissolved in an aqueous solution, it is formulated with a mixture containing additional components which buffer the solution so that the pH is in the range of 5-9. An aqueous solution of a compound according to any one of claims 1 to 15, characterized in that the pH of the solution is in the range of 5-9. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 14 together with a physiologically acceptable diluent or carrier. A compound of formula (I) according to any one of claims 1 to 15 for use in therapy.