JP2005272457A - Pharmaceutical containing cyanopyrrolidine derivative as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical having a new fluorocyanopyrrolidine derivative which exhibits excellent dipeptidylpeptidase IV (DPPIV) inhibitory activity, capable of being used for preventing or treating a disease or a state which is improved by inhibiting DPPIV. <P>SOLUTION: This pharmaceutical contains a (2S,4S)-2-cyano-4-fluoro-1-[(2-hyroxy-1,1-dimethyl)ethylamino]acetylpyrrolidine benzenesulfonic acid salt which exhibits the excellent DPPIV inhibitory activity as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a medicament containing 4-fluoro-2-cyanopyrrolidine derivative benzenesulfonate as an active ingredient.

Dipeptidyl peptidase IV (DPPIV) is a kind of serine protease, widely distributed in tissues and plasma such as kidney and liver, and is involved in the metabolism of various physiologically active peptides.
As a DPPIV inhibitory compound, (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine (Patent Document 1) is known. However, the free form has poor solid stability, and the salts with mineral acids and salts with organic acids shown in this application have poor solid stability, stability under humidification, and are difficult to synthesize. There was a fault, such as having.

International Publication No. WO02 / 38541 Pamphlet

  An object of the present invention is to provide a drug having excellent DPPIV inhibitory activity for preventing or treating a disease or condition that can be improved by inhibiting dipeptidyl peptidase IV, and also having physical properties necessary as a pharmaceutical such as stability Is to provide.

As a result of various studies on 4-fluoro-2-cyanopyrrolidine derivatives in order to achieve the above object, the present inventors have found that a preferable stable compound can be obtained by forming a salt of benzenesulfonic acid. completed.
That is, according to one aspect of the present invention, the present invention provides:

(2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine benzenesulfonate represented by the formula: As a DPPIV inhibitor.
Another aspect of the present invention is (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine-benzenesulfonic acid or a hydrate thereof Is a preventive or therapeutic agent for a disease or condition that can be improved by inhibiting dipeptidyl peptidase IV.

Another aspect of the present invention is (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine-benzenesulfonic acid or a hydrate thereof Is a therapeutic agent for diabetes containing as an active ingredient.
Another aspect of the present invention is (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine-benzenesulfonic acid or a hydrate thereof Is a therapeutic agent for immune diseases containing as an active ingredient.

  The compound according to the present invention can be easily obtained as a high purity and uniform crystal form and has excellent stability under high humidity conditions. Therefore, the adhesion of powder to the machine at the time of production, the decrease in fluidity, and the like are eliminated, and a stable supply of a medicine containing the present compound is enabled. In addition, since the compound according to the present invention is excellent in solid stability, there is no problem with change in appearance, transition of crystal form, etc., and it can withstand harsh conditions during production as a pharmaceutical, It also enables long-term quality retention during addition. With this compound, it has become possible to provide a preventive or therapeutic agent for diabetes, immune diseases and the like having stable and excellent dipeptidyl peptidase IV inhibitory activity.

  The benzenesulfonate according to the present invention is (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] obtained by the method described in WO02 / 38541. Dissolve acetylpyrrolidine in an appropriate solvent, mix benzenesulfonic acid or its hydrate directly or in a dissolved state, and then filter out the precipitate when the subsequent precipitate or poor solvent is added as the target product. Is obtained. This compound is excellent in crystallinity by the above method, and can be easily obtained as a uniform crystal form as compared with other salt compounds (for example, tosylate salt). In addition, a more stable composition could be produced with respect to stability when mixed with a compounding agent that is usually used at the time of pharmaceutical production.

This compound can suppress dipeptidyl peptidase IV in vivo, and thus can enhance insulin action and improve sugar metabolism. Also, it suppresses neuropeptide Y metabolism, T cell activation, It can contribute to inhibition of cancer cell adhesion to the endothelium and prevention of HIV virus entry into lymphocytes.
Accordingly, the present invention relates to diseases or conditions that can be ameliorated by inhibiting dipeptidyl peptidase IV, such as diabetes (particularly type II), immune diseases, arthritis, obesity, osteoporosis, conditions of glucose tolerance, benign Provided is the above medicament for preventing or treating prostate enlargement, skin disease and the like.

Examples of drugs for immune diseases include immunosuppressants in tissue transplantation; for example, cytokine release inhibitors in various autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis (RA), T-cells Drugs useful for the prevention and treatment of AIDS by preventing HIV entry into the body, prevention of metastasis, particularly drugs that prevent metastasis of breast and prostate tumors to the lung, and the like.
The medicament of the present invention can be systemically or locally administered orally or parenterally such as rectal, subcutaneous, intramuscular, intravenous and transdermal.

The medicament of the present invention may be in the form of a solid composition, a liquid composition, or other composition, and an optimum one can be selected as necessary, and a pharmaceutically acceptable carrier is blended with the compound. Can be manufactured. Specifically, conventional excipients, extenders, binders, disintegrants, coating agents, sugar coatings, pH adjusters, solubilizers, or aqueous or non-aqueous solvents are added, and by conventional formulation techniques, It can be prepared into tablets, pills, capsules, granules, powders, powders, solutions, emulsions, suspensions, injections, and the like.
Further, the present compound can be formulated by forming an inclusion compound with α, β, γ-cyclodextrin, methylated cyclodextrin or the like.

  The dose of this compound varies depending on the disease, symptoms, body weight, age, sex, route of administration, etc., but is preferably about 1 to about 1000 mg / person / day, more preferably, for oral administration to adults Is about 10 to about 200 mg / person / day, which can be administered once or divided into several times a day.

Hereinafter, the present invention will be described in more detail with reference to Examples, Reference Examples and Test Examples.
Reference example 1
Synthesis of (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine hydrochloride (2S, 4S) -2-cyano-4- To a suspension of fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine (5.00 g) in methanol (75 mL) was added 4M hydrochloric acid (ethyl acetate solution, 6.17 mL) to give a clear solution. It became a solution. Diisopropyl ether (300 mL) was added to this solution and stirred, and the precipitated powder was collected by filtration to give the title compound (5.47 g) as a colorless powder.
Melting point: 197-198 ° C

Reference example 2
Synthesis of (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine methanesulfonate (2S, 4S) -2-cyano- To a suspension of 4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine (0.15 g) in methanol (0.92 mL) was added methanesulfonic acid (0.042 mL) in methanol (0.08 mL). ) When the solution was added, it became a clear solution. This solution was added dropwise to diisopropyl ether (5 mL) with stirring. The precipitated powder was collected by filtration to give the title compound (0.20 g) as a colorless powder.
Melting point: 179-180 ° C

Example 1
Synthesis of (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine benzenesulfonate (1) (2S, 4S) -2 -Cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine 2-amino-2-methyl-1-propanol (0.54 g) in tetrahydrofuran (7.5 mL) and ethanol ( 2.5 ml) was dissolved in a mixed solvent, and (2S, 4S) -1-bromoacetyl-2-cyano-4-fluoropyrrolidine (0.71 g) was added under ice cooling, followed by stirring at room temperature for 1 hour. The precipitated crystals were collected by filtration to give the title compound (0.36 g) as a colorless solid. The filtrate was further purified by silica gel column chromatography (developing solvent; chloroform: methanol: 25% aqueous ammonia = 300: 10: 1) to obtain the title compound (0.22 g).

(2) (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine benzenesulfonate (2S, 4S) -2-cyano -4-Fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine (20 g) was dissolved in methanol (300 mL) with heating to obtain benzenesulfonic acid monohydrate (15.2 g). When methanol (30 mL) solution was added, powder precipitated. Diisopropyl ether (330 mL) was added to the suspension, and the powder was collected by filtration to give the title compound (31.5 g) as a colorless powder.
Melting point: 220-221 ° C

Test example 1 [Weight change test under humidification]
10.0 mg of each specimen was weighed into a microtube (diameter 8 mm, length 50 mm) and left in a desiccator with water so as not to come into contact with water. The desiccator was left at room temperature. The state observation and weight measurement were performed over time, and the change in weight was displayed as a percentage.
<Weight change and state change>

  The hydrochloride salt of Reference Example 1 and the methanesulfonate salt of Reference Example 2 were deliquescent by absorbing moisture, but the benzenesulfonate salt of Example 1 did not change in weight and did not deliquesce.

Test example 2 [solid stability test]
About 1 mg of each sample is weighed precisely, and a screw-opening test for light-shielding (aluminum foil) sealed plug under heating conditions (70 ° C) and light-shielding (aluminum foil) opening under heating / humidification conditions (40 ° C 75% RH) Stored in a tube. The residual ratio of the drug was measured by the following procedure. A 10 ml HPLC mobile phase was added to a test tube after a predetermined storage period and dissolved, followed by quantification by HPLC, and this was compared with the initial value before heating or before warming / humidification to calculate the residual ratio of the drug.

HPLC condition column: CAPCELL PAK UG120, 5μm, φ4.6 × 150mm (SHISEIDO)
Column temperature: 40 ° C
Detection: UV absorptiometer (detection wavelength: 210nm)
Flow rate: 1.0ml / min
Injection volume: 10μl
Mobile phase: water / acetonitrile / phosphoric acid / SDS (700: 300: 1: 2)
<Drug remaining rate>

  The hydrochloride of Reference Example 1 and the methanesulfonate of Reference Example 2 had a drug residual rate of 97% or less under any condition, but the benzenesulfonate of Example 1 was 99% or more under any condition. Met.

Test Example 3 [Combination change test with additives]
Serajuddin ATM et al. (J. Pharm. Sci., 88, 696-704, 1999). After weighing the drug substance and additives into a screw test tube in the amount of Formulation A (Drug substance 10 mg, Crystalline cellulose 68 mg, Magnesium stearate 2 mg) or Formulation B (Drug substance 10 mg, Lactose 68 mg, Hardened oil 2 mg), The mixture was mixed for 1 hour with a mix rotor (MIX-ROTAR VMR-5, Seiei Iuchi). Nothing was added to Formulation A, and 16 μl of purified water was added to Formulation B and stirred with a vortex mixer (TOUCH MIXER MT-31, Yamato Kagaku). This was stored at 65 ° C. for 1 week in a sealed cap and in a completely light-shielded state, the content after storage was measured, and the residual rate was calculated.

  The content was determined by the following procedure. Add 10 ml of 50% methanol to the sample after storage, irradiate with ultrasound (using BRANSONIC 5200 from BRANSON) for 30 minutes for dispersion and extraction, shake for 1 hour with a shaker, and add 50 ml of this solution. Transfer to volumetric flask. Wash up with 50% methanol to make up the volume. This was further irradiated with ultrasonic waves for 30 minutes. This solution was filtered through a 0.45 μm membrane filter, 5 ml was collected, and a 10 ml volumetric flask diluted with 50% methanol was used as a sample solution and quantified by HPLC. HPLC conditions are the same as in Test Example 2.

<Drug remaining rate>

In the case of Formulation A, the residual ratio of the drug was 91.6% with the methanesulfonate of Reference Example 2, but the decrease was hardly observed with the benzenesulfonate of Example 1. Even in the case of Formulation B where the residual rate of methanesulfonate was reduced to 74.5%, the residual rate of benzenesulfonate was 90% or more.
It was confirmed that the compound of Example 1 has little deterioration even when a compounding agent used at the time of pharmaceutical production is added, and can provide a stable compounding composition as a drug.

Test Example 4 [Dipeptidyl peptidase IV activity inhibition experiment]
The dipeptidyl peptidase IV (DPPIV) activity inhibition experiment was performed according to the method described in Diabetes, 47, 764-769, 1998. Plasma containing dipeptidyl peptidase IV was prepared by collecting blood from healthy volunteers and centrifuging. The enzyme reaction was performed using a 96-well flat bottom plate in a buffer solution consisting of 25 mM HEPES, 140 mM NaCl, 1% BSA, pH 7.8. 100 μM Gly-Pro-4-methylcoumaryl-7-amide (manufactured by Peptide Institute) solution 25 μl, 133 mM magnesium chloride solution 7.5 μl, sample compound 5 μl were mixed, and then plasma 12 diluted 1/100 times with the above buffer solution .5 μl was added. After reacting for 2 hours at room temperature, 50 μl of 25% acetic acid aqueous solution was added to stop the reaction. The amount of 7-amino-4-methylcoumarin liberated was measured using a fluorescence plate reader (1420 ARVO ™ Multilabel Counter; manufactured by Wallac), and the fluorescence intensity at 460 nm was measured. The fluorescence intensity when the solvent was added and the reaction time was 0 minutes was taken as the blank value, and the value obtained by subtracting the blank value from each measured value was taken as the specific fluorescence intensity. From the obtained specific fluorescence intensity, the dipeptidyl peptidase IV activity inhibition rate (%) was determined by the following equation. As the test compound, a dimethyl sulfoxide solution having a concentration 1000 times higher was prepared and diluted with the above buffer solution. The compound concentration (IC ₅₀ value) showing 50% inhibition was calculated from the inhibition rate of each concentration.

Inhibition rate (%) = A ÷ B × 100
(A = fluorescence intensity when solvent is added-fluorescence intensity when sample compound is added)
(B = fluorescence intensity upon addition of solvent)
As a result of the test, the compound of Example 1 showed an IC ₅₀ value of 5 nM.

According to the present invention, it is possible to provide a preventive or therapeutic agent for diabetes, immune diseases and the like, which are representative of modern diseases, having stable and excellent dipeptidyl peptidase IV inhibitory activity.

Claims (4)

DPPIV containing (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine benzenesulfonate or its hydrate as an active ingredient Inhibitor. (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine benzenesulfonate or a hydrate thereof is contained as an active ingredient, A preventive or therapeutic agent for a disease or condition that can be improved by inhibiting dipeptidyl peptidase IV. Diabetes containing (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine benzenesulfonate or a hydrate thereof as an active ingredient Therapeutic agent. Immune containing (2S, 4S) -2-cyano-4-fluoro-1-[(2-hydroxy-1,1-dimethyl) ethylamino] acetylpyrrolidine benzenesulfonate or a hydrate thereof as an active ingredient Disease treatment agent.