METHOD FOR EVALUATING QUALITY OF (3S)-3-(4-(3-(1,4-DIOXASPIRO[4,5]DEC-7-EN-8-YL)BENZYLOXY)PHENYL)HEX-4-INOIC ACID

Abstract

The present invention provides a related substance of (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid, and a method for evaluating the quality of (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en yl)benzyloxy)phenyl)hex-4-inoic acid using the same. According to the present invention, it is possible to evaluate the quality, stability and the like of a pharmaceutical composition comprising (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid using a compound of Chemical Formula 2 or a salt thereof as a reference standard.

Claims

1. A composition for evaluating a pharmaceutical product comprising (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid, comprising a compound of the following Chemical Formula 2 or a salt thereof. ##STR00018##

2. A method for evaluating the quality of a pharmaceutical product comprising (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid, the method comprising evaluating a content of (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid in the pharmaceutical product using a compound of the following Chemical Formula 2 or a salt thereof as a reference standard. ##STR00019##

3. A method for evaluating the quality of a pharmaceutical product comprising (3S)-3-(4-(3-(1,4-dioxaspiro[4,5] dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid, the method comprising evaluating a content of a related substance in the pharmaceutical product using a compound of the following Chemical Formula 2 or a salt thereof as a reference standard. ##STR00020##

4. A method for analyzing a sample comprising (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en-yl)benzyloxy)phenyl)hex-4-inoic acid, the method comprising: a) obtaining data by subjecting a sample to chromatography; and b) comparing the data with the chromatography data of a compound of the following Chemical Formula 2 or a salt thereof. ##STR00021##

5. The method of claim 4, wherein the method comprises: (a) obtaining a chromatogram by subjecting a solution of (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid comprising the compound of Chemical Formula 2 or a salt thereof to high-performance liquid chromatography; and (b) comparing a peak obtained from the chromatogram with a peak produced from the compound of Chemical Formula 2 or a salt thereof.

6. The method of claim 4, wherein the method comprises: (a) obtaining a chromatogram by subjecting a solution of (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid to thin layer chromatography; and (b) comparing a spot obtained from the chromatogram with a spot produced from the compound of Chemical Formula 2 or a salt thereof.

7. A method for measuring a chromatography column retention time for (3S) (4-(3-(1,4-dioxaspiro[4,5]dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid, the method comprising: performing chromatography using a compound of the following Chemical Formula 2 or a salt thereof as a reference standard. ##STR00022##

8. A compound of the following Chemical Formula 2 or a salt thereof: ##STR00023##

Description

DESCRIPTION OF DRAWINGS

[0061] FIG. 1 illustrates IR data of a compound of Chemical Formula 2.

[0062] FIG. 2 illustrates DSC-TGA data of the compound of Chemical Formula 2.

[0063] FIG. 3 illustrates XRD data of the compound of Chemical Formula 2.

[0064] FIG. 4 illustrates a comparison of the HPLC analysis results of a control and a stress sample. In the drawing, blue indicates the control and green indicates the stress sample.

MODES OF THE INVENTION

[0065] Hereinafter, the present invention will be described in more detail through examples, but this is for the purpose of describing the present invention, and the scope of the present invention is not limited by any way by the examples.

EXAMPLES

Example 1: Separation of Related Substance

[0066] About 35 mg of a (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en yl)benzyloxy)phenyl)hex-4-inoic acid-containing substance stored under refrigerated conditions (5° C.±3° C.) for about 2 years was accurately weighed and put into a 50-ml volume flask, a diluent was added to the flask at about ⅔ of the volume, and the resulting mixture was mixed well. Thereafter, a related substance was analyzed using HPLC (Agilent Technologies 1200 Series).

[0067] <HPLC conditions> Operation Parameters

[0068] Column: Kinetex EVO C18, 5 um, 4.6×250 mm

[0069] Column temperature: 25° C.

[0070] Flow rate: 1.0 ml/min

[0071] Injection volume: 5 μl

[0072] Sample concentration: 0.7 mg/ml

[0073] Wavelength: 220 nm

[0074] Mobile Phase A: 0.01 M K.sub.2HPO.sub.4 (pH 7)

[0075] Mobile Phase B: AN

[0076] Diluent: H.sub.2O/AN (80/20)

[0077] Gradient:

TABLE-US-00001 Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 7 80 20 50 30 70 55 30 70 55.10 80 20 62 80 20

[0078] Analysis results are shown below.

TABLE-US-00002 Peak Number Retention Time Area Area % Name 1 6.953 40106 0.0035 2 13.560 50730 0.0044 3 15.907 121406 0.0106 4 16.740 81372 0.0071 5 17.647 72507 0.0063 6 18.127 171612 0.0150 7 18.407 72715 0.0063 8 19.387 450501 0.0393 9 19.613 347157 0.0303 10 19.840 85581 0.0075 11 20.373 121471 0.0106 12 20.740 74147 0.0065 13 21.033 80418 0.0070 14 21.220 195488 0.0171 15 21.827 286792 0.0250 16 22.493 160952 0.0141 17 22.573 289110 0.0252 18 22.967 143309 0.0125 19 23.047 310852 0.0271 20 23.580 2939436 0.2566 ← 21 24.107 111999 0.0098 22 25.167 119138 0.0104 23 25.347 1133145719 98.9289 Chemical Formula 1 24 26.767 872290 0.0762 25 27.980 47225 0.0041 26 31.740 166640 0.0145 27 35.560 4718016 0.4119 28 39.353 137539 0.0120 Totals 1145414228 100.0000

[0079] A substance containing the related substance (RRT: 0.93, Area % 0.26%) analyzed by HPLC was qualitatively analyzed by MS scan and production ion scan using a Q-TOF-LC/MS system. The LC-MS/MS system qualitatively analyzes the substance using an Agilent 1290 Infinity HPLC system and an Agilent 6550 Q-TOF mass spectrometer. Analysis conditions are as follows.

[0080] <HPLC Conditions>

[0081] Column: YMC-Pack Pro C18 column (4.6×150 mm, 5 μm) equipped with Phenomenex SecurityGuard C18 guard column (4×20 mm)

[0082] Mobile Phase A: 0.1% formic acid in H2O/MeOH=60/40

[0083] Mobile Phase B: 0.1% formic acid in acetonitrile/MeOH=60/40

[0084] Flow rate: 1 ml/min (over 34 min)

[0085] Injection volume: 10 μl

[0086] Diluent: H.sub.2O/ACN=10/90

TABLE-US-00003 Time (min) Mobile phase A % Mobile phase B % 0 80% 20%  0-23 80-10% .sup.   20-90% .sup.   23-28 10% 90% 28-29 10-80% .sup.   90-20% .sup.   29-34 80% 20%

[0087] Wavelength: 220 (or 260 nm)

[0088] <Mass Spectrometer Conditions>

[0089] Capillary voltage: 3500 V

[0090] Ionization: Electrospray Ionization, Positive (ESI+)

[0091] Drying gas: 14

[0092] Nebulizer gas: 50

[0093] Sheath gas: 12

[0094] Sheath gas temperature: 400° C.

[0095] Drying gas temperature: 250° C.

[0096] Full scan MS, MS/MS mode: (m/z 100 to 1000)

[0097] An analyte separated in the detection step was set to flow into the mass spectrometer, and in this case, the detected ion of the related substance was qualitatively analyzed by selecting the characteristic ion [M+Na] of the mass spectrum.

[0098] As a result of the analysis, three predicted compounds were derived, and each predicted compound was synthesized and subjected to MS analysis. As a result, it was confirmed that the chemical structure of a related substance represented by RRT 0.93 has the structure of the following Chemical Formula 2.

##STR00012##

(3S)-3-(4-((3-(7-oxaspiro[bicyclo[4.1.0]heptane-3,2′-[1,3]dioxolan]-6-yl)benzyl)oxy)phenyl)hex-4-inoic acid

Example 2. Synthesis of Chemical Formula 2

[0099] The compound of Chemical Formula 2 was synthesized as follows according to the following Reaction Scheme 1.

##STR00013##

<Step 1> Synthesis of (3-(7-oxaspiro[bicyclo[4.1.0]heptane-3,2′-[1,3]dioxolan]-6-yl)phenyl)methanol

[0100] ##STR00014##

[0101] Meta-chloroperoxybenzoic acid (MCPBA) (365.40 mmol) was added to a flask and dissolved in ethyl acetate (450 ml). The internal temperature was cooled to 0° C. while stirring. (3-(1,4-Dioxaspiro[4.5]dec-7-en-8-yl)phenyl)methanol (DS-PMA)(30 g, 121.80 mmol) was dissolved in ethyl acetate (150 ml) and the resulting solution was added thereto at 0° C. After the addition, the mixture was stirred at room temperature and then checked with TLC to confirm that the reaction was completed. The reaction product was washed with a 1 N aqueous sodium hydroxide solution (300 ml×3) and purified water (300 ml×2). The organic layer was dried over anhydrous magnesium sulfate, and then filtered, and concentrated under reduced pressure. The organic layer was separated by silica column chromatography (ethyl acetate:n-hexane=1:2.fwdarw.ethyl acetate:n-hexane=1:1) and concentrated under reduced pressure to obtain a solid compound.

<Step 2> Synthesis of 3-(7-oxaspiro[bicyclo[4.1.0]heptane-3,2′-[1,3]dioxolan]-6-yl)benzyl methanesulfonate

[0102] ##STR00015##

[0103] After (3-(7-oxaspiro[bicyclo[4.1.0]heptane-3,2′-[1,3]dioxolan]-6-yl)phenyl)methanol (4.25 g, 16.20 mmol) was dissolved in ethyl acetate (42.5 ml), triethylamine (3.38 ml, 24.30 mmol) was added thereto at room temperature. The temperature was lowered while stirring for 30 minutes, and mesyl chloride (1.63 ml, 21.06 mmol) was slowly added dropwise thereto at 0° C. The resulting mixture was stirred at 5° C. It was confirmed by TLC that the reaction was completed. Purified water (42.5 ml) was added thereto at 5° C. and the resulting mixture was stirred. The organic layer was extracted, and then washed with purified water (42.5 ml). The organic layer was dried over anhydrous magnesium sulfate (50 g) and filtered to perform the next reaction without concentration.

<Step 3> Synthesis of (3S)-ethyl 3-(4((3-(7-oxaspiro[bicyclo[4.1.0]heptane-3,2′-[1,3]dioxolan]-6-yl)benzyl)oxy)phenyl)hex-4-inoate

[0104] ##STR00016##

[0105] A mesylated substance was calculated at a yield of 100%, and the S.sub.N2 reaction was performed using 5.51 g of the substance.

[0106] 3-(7-oxaspiro[bicyclo[4.1.0]heptane-3,2′-[1,3]dioxolan]-6-yl)benzyl methanesulfonate (5.51 g, 16.20 mmol), (S)-ethyl 3-(4-hydroxyphenyl)hex-4-inoate (3.57 g, 15.39 mmol) was put into a flask, and dissolved in acetonitrile (64 ml). After potassium phosphate tribasic (212.27 g, 32.405 mmol) was added thereto at room temperature, the mixture was stirred for 1 hour and 30 minutes while heating at 74 to 76° C. After it was confirmed by TLC that the reaction was completed, the temperature was lowered to room temperature. Purified water (30 ml) and ethyl acetate (30 ml) were added thereto, the resulting mixture was stirred, and then the organic layer and the aqueous layer were separated. The organic layer was washed twice with a 5% aqueous sodium chloride solution. The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtered organic layer was concentrated under reduced pressure to obtain a solid compound.

<Step 4> Synthesis of (3S)-3-(4-((3-(7-oxaspiro[bicyclo[4.1.0]heptane-3,2′-[1,3]dioxolan]-6-yl)benzyl)oxy)phenyl)hex-4-inoic acid

[0107] ##STR00017##

[0108] The yield was calculated to be 100% at the S.sub.N2 reaction step, and a hydrolysis reaction was performed using 7.11 g of the solid compound.

[0109] Tetrahydrofuran (36 ml) and methanol (36 ml) were added to (3S)-ethyl 3-(4-((3-(7-oxaspiro[bicyclo[4.1.0]heptane-3,2′-[1,3]dioxolan]-6-yl)benzyl)oxy)phenyl) hex-4-inoate (7.11 g, 14.92 mmol) and the resulting mixture was stirred. Potassium hydroxide (4.92 g, 74.59 mmol) was dissolved in purified water (36 ml), the resulting solution was added thereto, and the resulting mixture was stirred at 30° C. for 30 minutes. After it was confirmed by TLC that the reaction was completed, the resulting product was concentrated under highly reduced pressure. Purified water was added thereto, the resulting mixture was stirred, and then the aqueous layer was washed twice with ethyl acetate. After the internal temperature was cooled to 0 to 5° C., the organic layer was extracted by acidifying with a 2 N aqueous hydrochloric acid solution. The organic layer was washed twice with a 5% aqueous sodium chloride solution, dried using anhydrous magnesium sulfate, and then concentrated under reduced pressure to obtain a compound.

[0110] The synthesized substance was confirmed by HPLC and mass spectrometry, and it was confirmed that the substance had the structure of Chemical Formula 2. In addition, the NMR and IR spectra of this substance were analyzed. The analysis results of 1HNMR, 13CNMR and IR (FIG. 1) are shown below.

[0111] 1H NMR (300 MHz, DMSO-d6): 12.27 (br, 1H), 7.43 (1H), 7.37 (1H), 7.32-7.27 (m, 3H), 6.96 (d, 2H), 5.09 (s, 1H), 3.95-3.81 (m, 5H), 3.12 (d, 1H), 2.61-2.51 (m, 3H), 2.16 (t, 2H), 2.01 (d, 1H), 1.79 (s, 3H), 1.68-1.56 (m, 2H)

[0112] 13C NMR (DMSO d6) 171.85, 157.18, 141.11, 137.27, 133.48, 128.45, 128.29, 126.73, 124.68, 124.39, 114.64, 105.44, 80.58, 78.16, 69.14, 63.84, 63.52, 59.88, 59.27, 42.81, 34.72, 32.70, 27.91, 25.45, 3.14 ppm

[0113] Absorption spectrum (FT-IR, KBr): 2912 cm.sup.−1, 1706.14 cm.sup.−1, 1508.03 cm.sup.−1, 1111.62 cm.sup.−1

[0114] Furthermore, XRD and DSC/TGA were analyzed, and the results are shown in FIGS. 2 and 3, respectively. XRD and DSC/TGA analysis conditions are as follows. It was confirmed that the synthesized substance was amorphous.

[0115] <Xrd Conditions>

[0116] Model: D8 Advance (Bruker)

[0117] Power: 40 mA, 40 kV,

[0118] Time range: 3 min to 45 min (6 deg/min*3)

[0119] <DSC/TGA Conditions>

[0120] Model: TGA Q5000 IR/SDT Q600 (TA)

[0121] Temperature range: 40° C. to 600° C.

[0122] Rate: 20° C.

Example 3: Confirmation of Compound of Chemical Formula 2 as Related Substance

[0123] A stress sample stressed using an oxidant was prepared to confirm the production of the related substance. About 35 mg of a (3S)-3-(4-(3-(1,4-dioxaspiro[4,5]dec-7-en-8-yl)benzyloxy)phenyl)hex-4-inoic acid-containing substance stored under refrigerated conditions (5° C.±3° C.) for about 2 years was accurately weighed and put into a 50-ml volume flask. A diluent was added to the flask at about ⅔ of the volume, and the resulting mixture was mixed well. After 2 ml of a 3% hydrogen peroxide solution was added thereto, the resulting mixture was mixed well and marked with a diluent. The stress sample was left to stand at room temperature, and 24 hours later, the related substance was analyzed using HPLC (Agilent Technologies 1200 Series). The related substance was analyzed under the same conditions as in Example 1. As a result, it was confirmed that the related substance was produced from the stress sample (RRT 0.93, Area 3.83%). A comparison of the HPLC data of both samples using the sample of Example 1 as a control is shown in FIG. 4 (blue: control, green: stress sample). It was confirmed that the production of the related substance of Chemical Formula 2 in the stress sample was increased compared to the control.