THIADIAZOLONE DERIVATIVES AND THEIR USE AS AMPK AGONISTS FOR THE TREATMENT OF DIABETES AND RELATED DISORDERS
20230382930 · 2023-11-30
Assignee
Inventors
Cpc classification
C07F9/65397
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to a compound of formula I, wherein: R.sup.1 is as defined in the specification, or a pharmaceutically acceptable salt or solvate thereof, which compounds are useful in the treatment treatment of a disorder or condition ameliorated by the activation of AMPK, particarly as prodrugs.
##STR00001##
Claims
1. A compound of formula I, ##STR00014## wherein R.sup.1 is selected from the group consisting of —C(O)—C.sub.2H.sub.4—CO.sub.2H and —PO.sub.3H.sub.2, or a pharmaceutically acceptable salt or solvate thereof.
2. The compound according to claim 1, wherein the compound of formula I is: ##STR00015## or a pharmaceutically acceptable salt or solvate thereof.
3. The compound according to claim 1, wherein the compound of formula I is: ##STR00016## or a pharmaceutically acceptable salt or solvate thereof.
4. The compound according to claim 1, wherein the pharmaceutically acceptable salt is an alkali metal salt, an alkaline earth metal salt or a quaternary ammonium salt of the compound of formula I.
5. The compound according to claim 4, wherein the pharmaceutically acceptable salt is a sodium or potassium salt of the compound of formula I.
6. A pharmaceutical formulation comprising a compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.
7. The pharmaceutical formulation according to claim 6, wherein the pharmaceutically acceptable excipient is a basic excipient.
8. The pharmaceutical formulation according to claim 6, wherein the pharmaceutically acceptable excipient is selected from the group consisting of magnesium oxide, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate, magnesium carbonate and calcium carbonate, or any combination thereof.
9. The pharmaceutical formulation according to claim 6, further comprising an enteric coating.
10. (canceled)
11. (canceled)
12. (canceled)
13. A method of treating a disorder or condition ameliorated by the activation of AMPK comprising administering a compound according to claim 1, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.
14. The method according to claim 13, wherein the disorder or condition ameliorated by the activation of AMPK is selected from the group consisting of cardiovascular disease (such as heart failure), diabetic kidney disease, type 2 diabetes, insulin resistance, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, pain, opioid addiction, obesity, cancer, inflammation (including chronic inflammatory diseases), autoimmune diseases, osteoporosis and intestinal diseases.
15. The method according to claim 13, wherein the disorder or condition ameliorated by the activation of AMPK is a condition associated with hyperinsulinemia selected from the group consisting of obesity and cardiovascular disease.
16. The method according to claim 13, wherein the compound is administered orally, subcutaneously or intramuscularly.
17. A process for preparing a compound according to claim 1, wherein the process comprises: (i) reacting a compound of formula V, ##STR00017## with a suitable acid or acid anhydride; or (ii) reacting a compound of formula VI, ##STR00018## with a suitable acid or suitable acid salt.
Description
FIGURES
[0131] The following drawings are provided to illustrate various aspects of the present inventive concept and are not intended to limit the scope of the present invention unless specified herein.
[0132]
[0133]
EXAMPLES
[0134] The present invention is explained in greater detail in the following non-limiting examples.
[0135] The reaction schemes described below are intended to provide a general description of the methodology employed in the preparation of the compounds of the invention. The examples provided herein are offered to illustrate but not limit the compounds of the invention, as well as the preparation of such compounds and intermediates.
[0136] All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents and catalysts utilised to synthesise the compounds of the invention are either commercially available or can be routinely prepared by procedures described in the literature, for example, Houben-Weyl “Science of Synthesis” volumes 1-48, Georg Thieme Verlag, and subsequent versions thereof.
[0137] A reaction may be carried out in the presence of a suitable solvent or diluent or of mixture thereof in a manner known to those skilled in the art of organic synthesis. A reaction may also be carried out, if needed, in the presence of an acid or a base, with cooling or heating, for example in a temperature range from about −30° C. to about 150° C. In some embodiments, a reaction is carried out in a temperature range from about 0° C. to about 100° C., and more particularly, in a temperature range from room temperature to about 80° C., in an open or closed reaction vessel and/or in the atmosphere of an inert gas, for example nitrogen.
Abbreviations
[0138] Abbreviations as used herein will be known to those skilled in the art. In particular, the following abbreviations may be used herein. [0139] AUC: Area under the concentration-time curve [0140] aq: Aqueous [0141] b.w.: Body weight [0142] C.sub.max: Peak plasma concentration [0143] d: Doublet [0144] DCM: Dichloromethane [0145] DMF: Dimethylformamide [0146] DMAP: 4-Dimethylaminopyridine [0147] DMSO: Dimethylsulfoxide [0148] ESI: Electrospray ionisation [0149] Et.sub.3N: Triethylamine [0150] EtOH: Ethanol [0151] g: Gram [0152] h: Hours [0153] HPLC: High performance liquid chromatography [0154] LC: Liquid chromatography [0155] LCMS: Liquid chromatography—mass spectrometry [0156] LC-MS/MS: Liquid chromatography—(tandem) mass spectrometry [0157] LLOQ: Lower limit of quantification [0158] m: Multiplet [0159] MeOD: Methanol-d.sub.4 [0160] Min: Minutes [0161] mL: Millilitre [0162] MRT: Mean residence time [0163] nBu.sub.3N: Tributylamine [0164] ND: Not detected [0165] NMR: Nuclear magnetic resonance [0166] RT: Room temperature [0167] s: Singlet [0168] T.sub.1/2: Half-life [0169] T.sub.max: Time to reach the peak plasma concentration [0170] THF: Tetrahydrofuran [0171] TLC: Thin-layer chromatography
Instrumentation Conditions
LC Parameters
[0172] Column: Agilent Zorbax SB-C8, 50×4.6 mm, 350 μm [0173] Mobile phase: 5 mM ammonium acetate:methanol with 0.1% formic acid (15:85 v/v) [0174] Separation mode: Isocratic [0175] Flow rate: 0.800 mL/min [0176] Injection volume: 2 μL [0177] Auto sampler temperature: 4° C. [0178] Column oven temperature: 40° C. [0179] LC-MS/MS Shimadzu LCMS-8045 [0180] Source ESI [0181] Polarity Positive [0182] m/z of analyte (Compound 1) 379.800>89.100 [0183] m/z of internal standard (warfarin) 309.100>251.100
[0184] The present invention will be further described by reference to the following examples, which are not intended to limit the scope of the invention.
Example 1: preparation of 4-({(5Z)-5-[(4-chlorobenzoyl)imino]-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4- yl}methoxy)-4-oxobutanoic acid
[0185] ##STR00012##
Compound 1: 4-choro-N-[2-[(4-chlorophenyl)methyl]3-oxo-1,2,4-thiadiazol-5-yl]benzamide
[0186] Compound 1 was prepared in accordance with the procedures described in WO 2011/004162.
Compound 2: 4-choro-N-{2-[(4-chlorophenyl)methyl]-4-(hydroxymethyl)-3-oxo-1,2,4-thiadiazolidin-5-ylidene}-benzamide
[0187] To a stirring solution of Compound 1 (10 g, 0.026 mol) in DMF (200 mL) were added Et.sub.3N (15 mL, 0.105 mol) and 37% formaldehyde (8.5 mL, 0.105 mol). The reaction mixture was stirred at RT for 12 to 14 h.
[0188] The reaction mixture was then concentrated to remove the DMF. The crude material obtained was slurried in water (100 mL) for 30 minutes. The slurry was filtered and dried at ambient temperature to yield Compound 2 as white solid (9.6 g, 90.5% yield).
Compound 3: 4-({(5Z)-5-[(4-chlorobenzoyl)imino]-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4-yl}methoxy)-4- oxobutanoic acid
[0189] To a stirring solution of Compound 2 (10 g, 0.024 mol) in THF (200 mL) were added DMAP (0.44 g, 0.004 mol) and succinic anhydride (7.29 g, 0.072 mol). The reaction mixture was stirred at RT for 12 h.
[0190] After completion (as observed by TLC), the reaction mixture was concentrated to remove the THF. The crude material obtained was diluted with THF (100 mL), stirred for 1 h at RT and filtered to afford Compound 3 as a white solid (7.3 g, 59% yield).
[0191] .sup.1H NMR (300 MHz, MeOD) δ (ppm): 2.61 (m, 4H), 4.72 (s, 2H), 6.10 (s, 2H), 7.29 (dd, 4H), 7.41 (d, 2H), 8.12 (d, 2H).
[0192] LCMS: 510.4 [M+H].
[0193] HPLC: >98% purity at 13.6 minutes.
Example 2: preparation of disodium {(5Z)-5-[(4-chlorobenzoyl)imino]-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4- yl}methyl phosphate
[0194] ##STR00013##
Compound 2: 4-choro-N-{2-[(4-chlorophenyl)methyl]-4-(hydroxymethyl)-3-oxo-1,2,4-thiadiazolidin-5-ylidene}benzamide
[0195] Compound 2 was prepared in accordance with the procedure described in Example 1.
Compound 4: 4-chloro-N-{4-(chloromethyl)-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-5-ylidene}benzamide
[0196] A mixture of Compound 2 (10 g, 0.0243 mol) and thionyl chloride (60 mL) was heated to 75° C. over a period of 3 h.
[0197] After completion (as observed by TLC), the reaction mixture was concentrated and the crude product was washed with diethyl ether (3×100 mL) to afford Compound 4 as a white solid (9.5 g, 90% yield).
Compound 5: disodium {(5Z)-5-[(4-chlorobenzoyl)imino]-2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazolidin-4-yl}methyl phosphate
[0198] To a stirring solution of phosphoric acid (85%, 15.1 g, 0.154 mol) in ethanol (60 mL), tributylamine (83 mL, 0.35 mol) was added at ambient temperature. The solvent was removed by evaporation and the residue was dissolved in DCM (60 mL). The DCM solution was dried over sodium sulphate, filtered and evaporated to afford di-tributyl-ammonium dihydrogen phosphate.
[0199] A mixture of Compound 4 (3.0 g, 0.007 mol) and the above prepared tributyl-ammonium dihydrogen phosphate was taken in DCM (60 mL) and stirred for 10 min. The reaction mixture was distilled off at 40° C. and the residue obtained was heated at 60° C. on a water bath for 10 to 15 minutes. The residue was re-dissolved in DCM (6 mL), the DCM was distilled off and residue obtained was heated at 60° C. on a water bath for 10 to 15 min. The above procedure was repeated for four times.
[0200] After completion (as observed by TLC), the residue was dissolved in DCM (4 mL) and washed with water (3×4 mL). The organic phase was separated, dried over sodium sulphate, filtered and concentrated. The crude product obtained upon evaporation was taken in water and basified (pH 9 to 9.5) with 0.2 M sodium hydroxide. After basification, the mixture was filtered and filtrate was lyophilized to yield Compound 5 as a white solid (0.6 g, 17.5% yield).
[0201] .sup.1H NMR (300 MHz, DMSO-D.sub.6) δ (ppm): 4.78 (s, 2H), 5.55 (s, 2H), 7.40 (dd, 4H), 7.55 (d, 2H), 8.19 (d, 2H).
[0202] LCMS: 490.1 [M+H].
[0203] HPLC: >98% purity at 8.2 minutes.
Example 3—Solubility of Compounds at Different pH
Methods
[0204] Approximately 5 mg of each test compound (Compounds 1 and 5) was weighed in duplicate, and each weighed sample was transferred to a separate centrifuge tube containing 5 mL of the respective buffer. All the tubes were tightly stoppered and agitated at a constant rate at 30±1° C. using an orbital shaker.
[0205] After 24 hours, one tube of each buffer solution was removed from the shaker. 50 μL of the buffer solution from each removed tube was transferred into a 10 mL tube and made up to the mark with diluent. Then 2.5 mL of the supernatant of each tube was transferred into a 50 mL tube and made up to 25 mL.
[0206] The nominal concentration of the solution in each 50 mL tube was 5 μg/mL.
[0207] Linearity solutions of 40 ng/mL to 10000 ng/mL were plotted.
Results
[0208] The results for the solubility experiments are tabulated in Tables 1 and 2 below.
[0209] The results show that Compound 5 has a higher solubility compared to Compound 1 in basic solutions (i.e. pH>7) as well as in mildly acidic conditions. In particular, Compound 5 is up to 30-times more soluble in basic solution compared to Compound 1.
TABLE-US-00001 TABLE 1 Results of a Two-hour Solubility Study of Compounds 1 and 5 Absolute Solubility: 2 hours incubation Solubility (mg/ml) Buffer Compound 1 Compound 5* HCl Buffer, PH: 1.20 ND ND Acetate Buffer, pH: 4.50 ND 0.015 Phosphate Buffer, pH: 7.40 ND 0.119 Alkaline Borate Buffer, pH: 9.20 0.029 0.720 Ultrapure water ND 0.419 Note: ND indicates not detected, concentration was below LLOQ; *HPLC was analysis performed for Compound 5
TABLE-US-00002 TABLE 2 Results of a Twenty-hour Solubility Study of Compounds 1 and 5 Absolute Solubility: 20 hours incubation Solubility (mg/ml) Buffer Compound 1 Compound 5* HCl Buffer, pH; 1.20 ND ND Acetate Buffer, pH: 4.50 ND 0.015 Phosphate Buffer, pH: 7.40 ND 0.098 Alkaline Borate Buffer, PH: 9.20 0.044 1.239 Ultrapure water ND 0.036 Note: ND indicates not detected, concentration was below LLOQ; *HPLC was analysis performed for Compound 5
Example 4—Stability of compounds
[0210] 5 μM solutions of Compounds 1, 3 and 5 in USP buffer (pH: 7.40) were prepared with 5 mM DMSO stock solutions. The solutions were incubated at 37° C. for 120 minutes, with shaking at 400 rpm using a thermomixer.
[0211] After 0, 15, 30, 60 and 120 minute intervals, aliquots of sufficient volume of the compound solutions were taken, diluted to the final concentration and analysed.
Results
[0212] The results for the stability experiments are tabulated in Table 3 below.
[0213] The results show that Compounds 3 and 5 possess comparable stability to Compound 1 in mildly basic solutions (i.e. pH=7.4).
TABLE-US-00003 TABLE 3 Comparative Stability Data in Phosphate Buffer (pH: 7.40) % remaining compared to 0.0 min Test 0.0 15 30 60 120 Compound min min min min min Compound 1 100.00 97.62 101.31 99.41 106.18 Compound 3 100.00 86.96 93.61 88.74 75.40 Compound 5 100.00 103.92 107.58 107.88 97.30
Example 5—Activation of AMPK
Cultivation and Compound Treatment of INS-1E Insulinoma Cells
[0214] INS-1E cells were cultivated as described in Steneberg et al., JCI Insight. 2018; 3(12):e99114. https://doi.org/10.1172/jci.insight.99114, using 5% instead of 10% fetal bovine serum when plated for treatment.
[0215] Compound 3 was dissolved at 10 mM in DMSO and frozen at −20° C.
[0216] Compound 5 was dissolved at 10 mM in a 50:50 water/DMSO mixture, incubated for 15 minutes at room temperature in ultrasonic bath (VWR ultrasonic cleaner), and stored at room temperature.
[0217] The INS-1E cells were treated in serum free medium with increasing doses of Compounds 3 and 5 for 4 and 16 hours, respectively, in accordance with the methods described in Steneberg et al., JCI Insight. 2018; 3(12):e99114. https://doi.org/10.1172/jci.insight.99114.
Western Blot Analysis
[0218] Western blot analysis of the INS-1E cells was performed as described in Steneberg et. al., JCI Insight. 2018; 3(12):e99114. https://doi.org/10.1172/jci.insight.99114. The cell lysates were passed through a 30-gauge needle, ˜8-times and centrifuged for 10 min at +4° C. at 14000 rpm. Quantified values for AMPKα, and phosphorylated-T172 AMPKα, were normalized toward the quantified values for β-Actin.
Results
[0219] The results for Western blot analysis are tabulated in Table 3 below and are shown graphically in
[0220] The results show that Compounds 3 and 5 increase phosphorylated-T172 AMPK in a dose-dependent manner in cultured INS-1E cells. Compounds 3 and 5 are therefore agonists of AMPK.
TABLE-US-00004 TABLE 4 The ratio between p-T172 AMPK and non-phosphorylated AMPK Compound Time Control 2.5 μM 5 μM 10 μM 3 (0.1% DMSO) 4 h 1.00 1.62 1.88 2.95 5 (0.05% DMSO) 16 h 1.00 1.29 1.62 3.21
Example 6—Single Dose Oral Pharmacokinetic Study of Compounds 1 and 3 in Sprague Dawley Rats
Formulation of Compound 1
[0221] 10.0 mL of 2% w/v methyl cellulose solution in phosphate buffer (pH 7.5) was added to a 100 mL conical flask along with 1 g of 2 mm glass beads. The solution was vigorously stirred on a magnetic stirrer. 100 mg of Compound 1 was slowly added to the solution, and the solution was vigorously stirred for approximately 1h. The pH of the formulation was measured to be 7.49. Each formulation was freshly prepared prior to administration to the animals. The final concentration of Compound 1 in of the formulation was 10 mg/mL. The formulation was administered at 5 mL/kg body weight.
Formulation of Compound 3
[0222] 10.0 mL of 2% w/v methyl cellulose solution in phosphate buffer (pH 7.5) was added to a 100 mL of conical flask together with 1 g of 2 mm glass beads. The solution was vigorously stirred on a magnetic stirrer. 134 mg of Compound 3 was slowly added to the solution, and the solution was vigorously stirred for approximately 1 h. The pH of the formulation was measured to be 7.44. Each formulation was freshly prepared prior to administration to the animals. The final concentration of Compound 3 in the formulation was 13.4 mg/mL, which is equivalent to 10 mg/mL of Compound 1. The formulation was administered at 5 mL/kg body weight.
Dose Selection and Justification for Selection
[0223] The doses of 50 mg/kg and 67 mg/kg b.w. for Compound 1 and Compound 3, respectively, were selected for comparison of equimolar doses.
Dose Administration
[0224] Adult healthy male Sprague Dawley rats aged 9 to 11 weeks were used for experimentation after a minimum three days of acclimatisation. Fed state animals were administered the formulation of Compound 1 or Compound 3 orally via gavage at a dose of 50 mg/kg or 67 mg/kg body weight, respectively.
Blood Sampling
[0225] Under mild isoflurane anesthesia, blood specimens were collected by retro-orbital puncture method using capillary tubes into pre-labeled tubes containing anticoagulant (K.sub.2EDTA: 2 mg/mL blood) during the next 72 hours post-dosing, as detailed in Table 6. For blood sampling at multiple time points, the right and left eyes were used alternatively. Collected blood specimens were centrifuged at 6000 rpm, 4° C. for 10 minutes and the plasma samples were separated and stored at −80° C. until analysis.
Results
[0226] The results for the single dose oral pharmacokinetic study in rats are tabulated in Tables 4 and 5 below and are shown graphically in
[0227] The results show that there is a surprising two-fold increase in the systemic exposure of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide when Compound 3 is administered compared to Compound 1. Thus, the systemic exposure of 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide is increased by administration of a compound of the invention.
TABLE-US-00005 TABLE 5 Mean Plasma Pharmacokinetic Parameters Following Administration of Compounds 1 and 3 Compound No. 1 3 Dose (mg/kg b.w.) 50 67 (equivalent to 50 of Compound 1) C.sub.max (μg/ml) 19.60 ± 5.54 40.80 ± 5.66 T.sub.max (h) 7.33 ± 1.16 7.33 ± 1.16 AUC.sub.last (h*μg/mL) 449.40 ± 157.75 847.34 ± 99.98 AUC.sub.inf (h*μg/mL) 458.31 ± 154.77 880.35 ± 91.70 AUC.sub.extrap (%) 2.33 ± 1.74 3.80 ± 3.53 T.sub.1/2 (h) 8.42 ± 1.47 9.59 ± 2.96 MRT.sub.last (h) 14.78 ± 0.68 14.66 ± 1.45
TABLE-US-00006 TABLE 6 Plasma Concentration of Compounds 1 and 3 Plasma concentration of Compound 1 (μg/mL) Compound 1 Compound 3 Time (h) Mean SD Mean SC 0.00 0.00 0.00 0.00 0.00 0.25 0.62 0.62 1.13 0.13 0.50 1.98 0.59 5.53 0.73 1.00 4.24 1.84 9.51 0.74 2.00 7.99 4.49 16.17 1.53 4.00 14.01 5.73 23.83 4.56 6.00 17.00 5.30 35.17 4.85 8.00 19.33 5.33 38.83 8.82 24.00 9.44 4.43 16.03 1.82 48.00 0.72 0.17 2.12 1.30 72.00 0.00 0.00 0.00 0.00 Lower limit of quantification of Compound 1 0.5 μg/ml
Example 7—Single Dose Oral Pharmacokinetic Study of Compound 5 in Sprague Dawley Rats
Formulation of Compound 5
[0228] 6.0 mL of 2% w/v methyl cellulose solution in phosphate buffer (pH 7.5) was added to a 100 mL of conical flask together with 1 g of 2 mm glass beads. The solution was vigorously stirred on a magnetic stirrer. 77 mg of Compound 5 was slowly added to the solution, and the solution was vigorously stirred for approximately 1 h. The pH of the formulation was measured to be 7.43. The formulation was freshly prepared prior to administration to the animals.
Dose Administration
[0229] Adult healthy male Sprague Dawley rats aged 9 to 11 weeks were used for experimentation after a minimum three days of acclimatisation. Fed state animals were administered the formulation of Compound 5 orally via gavage at a dose of 64 mg/kg body weight (5 mL,/kg dose volume).
Blood Sampling
[0230] Under mild isoflurane anesthesia, blood specimens were collected by retro-orbital puncture method using capillary tubes into pre-labeled tubes containing anticoagulant (K.sub.2EDTA: 2 mg/mL blood) during the next 72 hours post-dosing. For blood sampling at multiple time points, the right and left eyes were used alternatively. Collected blood specimens were centrifuged at 6000 rpm, 4° C. for 10 minutes and the plasma samples were separated and stored at −80° C. until analysis.
Results
[0231] The results for the single dose oral pharmacokinetic study in rats are tabulated in Tables 7 and 8 below. In Table 7, the C.sub.max, T.sub.max, AUC.sub.last, AUC.sub.inf, AUC.sub.extrap, T.sub.1/2 and MRT.sub.last values are for 4-chloro-N-[2-[(4-chlorophenyl)methyl]-3-oxo-1,2,4-thiadiazol-5-yl]benzamide (Compound 1), which was detected following the administration of Compound 5. Thus, Compound 5 acts as a prodrug of Compound 1.
TABLE-US-00007 TABLE 7 Mean Plasma Pharmacokinetic Parameters Following Administration of Compound 5 Compound No. 5 Dose (mg/kg b.w.) 64 C.sub.max (μg/mL) 30.77 ± 9.68 T.sub.max (h) 6.00 ± 0.0 AUC.sub.last (h*μg/mL) 612.21 ± 376.22 AUC.sub.inf (h*μg/mL) 688.74 ± 315.05 AUC.sub.extrap (%) 15.22 ± 14.63 T.sub.1/2 (h) 9.09 ± 2.87 MRT.sub.last (h) 11.72 ± 3.69
TABLE-US-00008 TABLE 8 Plasma Concentration of Compound 1 Following Administration of Compound 5 Plasma concentration of Compound 1 (μg/mL) Compound 5 Time (h) Mean SD 0.00 0.00 0.00 0.25 0.84 0.20 0.50 2.39 0.50 1.00 4.78 1.42 2.00 11.09 2.17 4.00 23.97 10.19 6.00 30.77 9.68 8.00 29.23 10.80 24.00 13.84 10.73 48.00 0.23 0.40 72.00 0.00 0.00 Lower limit of quantification 0.5 μg/ml of Compound 1