Patent Application
20080004281
The invention is further illustrated by the following examples.
1,2-Bis(bromomethyl)-3-nitrobenzene: A 1 liter flask was charged with 1,2-dimethyl-3-nitrobenzene (20 g, 0.13 mol), N-bromosuccinimide (50 g, 0.28 mol), azobis(isobutyronitrile) (5 g, 3.0 mmol), and 200 mL of dichloromethane. This was irradiated with a 120 watt floodlamp to affect gentle reflux under nitrogen for 18 hours. The mixture was then cooled and precipitated succinimide was removed by filtration. The filtrate was concentrated and the residue was purified by chromatography on silica (5%-50% CH2Cl2 in hexanes) to give 2.6 g white solid (64%).
Dimethyl-4-nitroindane-2,2-dicarboxylate: To a solution stirred under nitrogen at room temperature, to 5.0 mL methanol in 15.0 mL ether was added 60% sodium hydride (0.84 g, 0.021 mol) in small portions. After the addition was complete, the nearly clear and colorless solution was stirred for 5 minutes. To it was then added 1.3 g dimethyl malonate, giving a slightly cloudy colorless solution. To this was rapidly added a suspension of 3.1 g 1,2-bis(bromomethyl)3-nitrobenzene, which immediate gave a precipitate suspended in a dark green solution. This was removed by filtration and the filtrate was concentrated. The residue was purified on silica (20%-100% CH2Cl2 in hexanes) to give 1.93 g off-white solid (67%).
Methyl-4-nitroindane-2-carboxylate: A mixture of dimethyl-4-nitroindane-2,2-dicarboxylate (4.84 g, 0.0167 mol), lithium chloride (0.84 g, 0.0198 mol), 1.11 mL water, and 18 mL dimethylsulfoxide was heated to 160° C. under nitrogen for two hours. It was then allowed to cool and the dimethylsulfoxide was removed under high vacuum. The residue was purified on silica (10%-100% CH2Cl2 in hexanes) to give 2.5 g white solid (65%).
Methyl-4-aminoindane-2-carboxylate: A mixture of methyl-4-nitroindane-2-carboxylate (2.4 g, 0.11 mol) and 10% palladium on carbon (1.1 g, 0.01 mol) in ethyl acetate (15 mL) was shaken under 55 PSI hydrogen for 1 hour. It was then filtered and the filtrate was concentrated to give 2.07 g white solid (100%).
Methyl 4-chlorosulfonyl-indan-2-carboxylate: A mixture of methyl-4-aminoindane-2-carboxylate (2.5 g, 0.013 mol), 12.5 mL acetonitrile, and 12.5 mL H2O was cooled to −5° C. in an ice-salt bath. To this was added 2.6 mL concentrated HCl (0.014 mol). To this was added dropwise over 20 minutes a solution of 1.0 g sodium nitrite (0.021 mol) in 5 mL water. After the addition was complete the solution was stirred for 20 minutes. It was then transferred to a jacketed addition funnel cooled with ice water. The solution was added dropwise to a solution stirred under nitrogen at 55° C. of 4.2 g potassium thioxanthate (0.026 mol) in 20 mL H2O. As the addition took place, a dark layer rose to the top of the diazonium ion solution which was not added. After the addition was complete the mixture was stirred at 55° C. for 30 minutes, then was allowed to cool and was extracted with 40 mL ethyl acetate. The organic layer was dried (MgSO4) and concentrated. The residue was loaded on 80 mL silica gel which was slurry-packed in hexanes. This was eluted with 100 mL hexanes, then 1%-50% CH2Cl2 in hexanes in 50 mL fractions to give 1.3 g amber oil (33%).
A mixture of 3.6 g of the above compound in 30 mL CCl4 and 10 mL H2O was vigorously stirred and cooled to 3 C. Chlorine gas was bubbled through at such a rate that the temperature stayed below 10° C. After conversion was complete, the phases were separated and the aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (MgSO4) and concentrated to give 4.0 g yellow oil (100%).
4-[cis-2,6-Dimethyl-4-(4-trifluoromethoxy-phenyl)-piperazine-1-sulfonyl]-indan-2-methyl ester: A mixture of methyl 4-chlorosulfonyl-indan-2-carboxylate (2.13 g, 0.0078 mol) obtained from Step 6, cis-3,5-dimethyl-1-(4-trifluoromethoxy-phenyl)-piperidine (3.0 g, 0.0109 mol) obtained from Example 51, 20 mL acetonitrile, and 3.0 g K2CO3 (0.0217 mol) was heated to 60° C. under nitrogen with stirring for 20 hours. It was then filtered and the filtrate was concentrated. The residue was purified by chromatography on silica (5%-50% EtOAc in hexanes) to give 2.64 g viscous yellow oil (66%).
4-[cis-2,6-Dimethyl-4-(4-trifluoromethoxy-phenyl)-piperazine-1-sulfonyl]-indan-2-carboxylic acid: To a solution of 4-[cis-2,6-dimethyl-4-(4-trifluoromethoxy-phenyl)-piperazine-1-sulfonyl]-indan-2-methyl ester (2.64 g, 0.0052 mol) in the minimum amount of THF (ca 15 mL) was added a solution of 0.14 g LiOH (0.0057 mol) in the minimum amount of water (ca 2.5 mL). This was capped and stirred at room temperature for 12 hours. Examination by HPLC showed the reaction was 85% complete so an additional 0.020 g LiOH (0.125 eq total) was added and stirring was continued for 3 hours. It was then concentrated to remove THF and partitioned between EtOAc and water. The aqueous layer was treated with 0.54 mL conc. HCl. It was then extracted with ethyl acetate. The organic layer was dried (MgSO4) and concentrated to give 2.38 g yellow amorphous solid (93%).
A single enantiomer of Compound 1 was obtained by chiral HPLC (chiralpak ASH 0.46×15 cm Hex/IPA 94:6 (v/v) with 0.1% TFA, flow rate 1 ml/min) separation from the racemate. LCMS 497.1 (M−1)−1.
cis-3,5-Dimethyl-1-(4-trifluoromethoxy-benzyl)-piperazine: To a solution of 4-(trifluoromethoxy)-benzaldehyde (776 uL, 4.38 mmol) in methylene chloride (30 mL) was added cis-2,6-dimethyl piperazine (1.0 g, 8.77 mmol). After 1 hour sodium triacetoxy borohydride (2.45 g, 8.77 mmol) was added to the mixture. The solution was stirred at room temperature for an additional 4 hours. The reaction was concentrated in vacuo, diluted with ethyl acetate and extracted with 1N HCl (2×50 mL). The aqueous layer was then neutralized with NaOH and extracted with ethyl acetate (3×50 mL). The organic layer was dried (Na2SO4) and concentrated to provide cis-3,5-dimethyl-1-(4-trifluoromethoxy-benzyl)-piperazine (1.01 g, 80%). 1H NMR (400 MHz, CD3OD) δ 7.42 (d, 2H), 7.23 (d, 2H), 3.54 (s, 2H), 2.98-2.88 (m, 2H), 2.82-2.74 (m, 2H), 1.69 (t, 2H), 1.05 (d, 6H); LCMS 289.5 (M+1)+.
4-[cis-2,6-Dimethyl-4-(4-trifluoromethoxy-benzyl)-piperazine-1-sulfonyl]-indan-2-carboxylic acid: The compound 4-[cis-2,6-dimethyl-4-(4-trifluoromethoxyl-benzyl)-piperazine-1-sulfonyl]-indan-2-carboxylic acid was synthesized according to the procedure for Compound 1 using cis-3,5-dimethyl-1-(4-trifluoromethoxy-benzyl)-piperazine. 1H NMR (400 MHz, CD3OD) δ 7.74-7.64 (m, 4H), 7.47 (d, 1H), 7.39-7.28 (m, 2H), 4.42 (s, 2H), 4.21-2.18 (m, 2H), 3.50-3.34 (m, 5H), 3.33-3.19 (m, 4H), 1.56 (d, 6H); LCMS 497.5 (M+1)+.
The product from Compound 2 Step 1 and the product from Compound 1 Step 5 were reacted using the conditions outlined in Compound 1 Step 6 to yield the racemic methyl ester. Chiral separation using OJ-H, 25% methanol in CO2 (100 bar), 5 mL/min followed by hydrolysis using conditions outlined in Compound 1 Step 7 provided a single enantiomer of 4-(cis-2,6-dimethyl-4-(3-trifluoromethoxy)benzyl)piperazin-1-ylsulfonyl)-2,3-dihydro-1H-indene-2-carboxylic acid. 1H NMR (400 MHz, CD3OD) δ 7.66 (d, 1H), 7.46 (d, 1H), 7.41 (d, 2H), 7.36-7.30 (m, 1H), 7.19 (d, 2H), 4.08-3.99 (m, 1H), 3.94-3.8 (m, 1H), 3.56-3.49 (m, 2H), 3.43 (s, 2H), 3.40-3.22 (m, 3H), 2.57 (t, 2H), 2.09-1.92 (m, 2H), 1.56 (d, 6H); LCMS 513.5 (M+1)+.
32% HCl is added to a solution of sodium nitrite in water and acetonitrile at 0° C. The solution is cooled to −5° C. and a solution of (R,S)-4-amino-indan-2-carboxylic acid methyl ester hydrochloride in water, acetonitrile, and 32% HCl is added, keeping the temperature between −7 and −10° C. The resulting cold diazonium solution is added to a solution of potassium ethylxanthogenate, in water and acetonitrile, at 60° C. After heating at 60° C., the mixture is cooled to room temperature and extracted from dichloromethane. The organic solution is charged into the reactor and concentrated under reduced pressure. Dichloromethane and water are added, the mixture cooled to 5° C., and chlorine gas passed through the mixture. The organic solution is separated and the aqueous solution is extracted from dichloromethane. The combined organic solution is dried over magnesium sulfate and concentrated under reduced pressure to afford (R,S)-4-chlorosulfonyl-indan-2-carboxylic acid. HPLC may be used to monitor the reaction.
Potassium carbonate is added to a mixture of cis-3,5-dimethyl-1-(4-trifluoromethoxy-phenyl)-piperazine hydrochloride in dichloromethane and water. After stirring at room temperature, the organic phase is collected and the aqueous layer extracted from dichloromethane. The combined organic solution is charged into the reactor and concentrated under reduced pressure, followed by the addition of acetonitrile and potassium carbonate. A solution of (R,S)-4-chlorosulfonyl-indan-2-carboxylic acid, in acetonitrile, is added to the reaction mixture. After heating at 50° C., the reaction mixture is cooled to 20° C. The mixture is transferred into a 200 L movable agitation feed tank, which is charged with Celite, and the suspension is stirred. The suspension is filtered, filter cake washed with acetonitrile, and the filtrate is concentrated under reduced pressure, cooled to 0-5° C., and 32% HCl added. Following further concentration and filtration, the filtrate is concentrated to give an oil which is purified by silica gel chromatography and recrystallized from isopropanol to give the product (R,S)-4-[cis-2,6-dimethyl-4-(4-trifluoromethoxy-phenyl)-piperazine-1-sulfonyl]-indan-2-carboxylic acid methyl ester (>95% by HPLC).
Simulated moving bed (SMB) chromatography was used to separate the S- and R-enantiomers of (R,S)-4-[cis-2,6-dimethyl-4-(4-trifluoromethoxy-phenyl)-piperazine-1-sulfonyl]-indan-2-carboxylic acid methyl ester. The SMB method uses a Chiralpak AS column and nheptane/isopropanol (1:1 v/v) to yield the S-enantiomer, (S)-4-[cis-2,6-dimethyl-4-(4-trifluoromethoxy-phenyl)-piperazine-1-sulfonyl]-indan-2-carboxylic acid methyl ester (>99.0% by chiral HPLC).
To a solution of (S)-4-[cis-2,6-dimethyl-4-(4-trifluoromethoxy-phenyl)-piperazine-1-sulfonyl]-indan-2-carboxylic acid methyl ester, in THF, is added a solution of lithium hydroxide in water, which is stirred at 20° C. and concentrated under reduced pressure. The reaction mixture is cooled to 9° C., neutralized with 32% HCl, and extracted from toluene. Water is removed from the organic solution by azeotropic distillation. Following distillation, the organic solution is cooled to ambient temperature and transferred to a feeding vessel. The reactor is charged with p-toluenesulfonic acid in toluene and water is removed by azeotropic distillation. The solution is cooled to 60° C., followed by the addition of the organic solution from the feeding vessel. The mixture is stirred at 83° C., then cooled to 10° C. to induce crystallization. The product suspension is filtered, the filter cake rinsed with heptane, and dried on a rotovap, at 40° C., to afford (S)-4-[cis-2,6-dimethyl-4-(4-trifluoromethoxy-phenyl)-piperazine-1-sulfonyl]-indan-2-carboxylic acid tosylate. 1HNMR δ 1.60 (d), 1.62 (d), 2.33 (s), 3.23 (m), 3.49 (m), 3.39 (m), 4.05 (m), 4.49 (m), 3.40 (dd), 3.23 (dd), 7.14 (d), 7.14 (d), 7.09 (d), 7.09 (d), 7.59 (d), 7.59 (d), 7.71 (d), 7.26, dd, 7.57 (d), 7.57 (d), 7.40 (d).
Total fill weight of each capsule is 300 mg, not including capsule weight. Target compound dosage as free base is 1, 5, or 20 mg per capsule, thereby allowing for a range of dosages to be conveniently administered to a patient. Several exemplary dosages are set forth below in Table 1.
The capsules above may be made by the following methods.
Compound 1A is passed between two sieve screens to produce a consistent particle size in the range of 53 to 250 μm. Particle size and XRPD in-process control (IPC) testing are performed, for information only, to profile the particle size distribution and evaluate the solid-state characteristics of Compound 1A following the sieve step. An appropriate amount of Compound 1A is weighed for each batch. The excipients are passed through a 250 μm sieve, collected, and weighed. Approximately half of the excipients are added to the container for blending. Compound 1A is added to the container, followed by the remaining half of the excipients, such that Compound 1A is sandwiched between the excipients. The blend is then mixed until uniform. Blend uniformity IPC (in-process control) testing is performed after blending by sampling 3 points within the container (top, middle, and bottom) and testing each sample for potency. A test result of 95-105% of target, with an RSD of 5%, must be achieved before the process can continue. Additional blend time is allowed to achieve a uniform blend if the IPC test results are not within the specified range. Upon acceptable blend uniformity results, a measured aliquot of the Compound 1A stock formulation is separated to manufacture the lower strengths. This aliquot is removed from the manufacturing area. Magnesium stearate is passed through a 75 μm sieve, collected, weighed, added to the blender as a lubricant, and mixed until dispersed. The final blend is weighed and reconciled. The Coni-Snap capsules (Swedish orange) are opened and the body of the capsule is placed on a Profill tray (holding 100 units) and the cap is placed on the corresponding tray. Blended materials are then flood fed into the body of the capsules using a spatula. The trays may be tamped to settle the blend in each capsule to assure uniform target fill weight. The capsules are then sealed by combining the filled bodies with the caps. Fill weight uniformity IPC testing is performed following encapsulation. Thirteen capsules are removed from a tray of 100 and weighed. This test result must be within the target fill weight (376±15 mg, including capsule weight) to pass. If the target fill weight specification is not met, the entire tray of 100 is weight checked and capsules not meeting weight specification are rejected. Following successful fill weight check, capsules are inspected, de-dusted, reconciled, and placed into a suitable in-process storage container.
Compound 1A stock formulation aliquot can be used to manufacture the lower strengths using serial dilutions. The excipients are passed through a 250 μm sieve, collected, and weighed. Approximately half of the excipients are added to the container for blending. Compound 1A stock formulation aliquot is added to the container, followed by the remaining half of the excipients, such that Compound 1A stock formulation aliquot is sandwiched between the excipients. The blend is then mixed until uniform. Blend uniformity IPC testing is performed after blending by sampling 3 points within the container (top, middle, and bottom) and testing each sample for potency. A test result of 95-105% of target, with an RSD of 5%, must be achieved before the process can continue. Additional blend time is allowed to achieve a uniform blend if the IPC test results are not within the specified range. Upon acceptable blend uniformity results, an aliquot of Compound 1A stock formulation can be removed to manufacture the 1 mg strength. This aliquot is removed from the manufacturing area.
Magnesium stearate is passed through a 75 μm sieve, collected, weighed, and added to the blender as a lubricant and mixed until dispersed. The final blend is weighed and reconciled. The Coni-Snap capsules (5 mg, dark green or 1 mg, white) are opened and the body of the capsule is placed on a Profill tray (holding 100 units) and the cap is placed on the corresponding tray. Blended materials are then flood fed into the body of the capsules using a spatula. The trays may be tamped to settle the blend in each capsule to assure uniform target fill weight. The capsules are then sealed by combining the filled bodies with the caps. Fill weight uniformity IPC testing is performed following encapsulation. Thirteen capsules are removed from a tray of 100 and weighed. This test result must be within the target fill weight (376±15 mg, including capsule weight) to pass. If the target fill weight specification is not met, the entire tray of 100 is weight checked and capsules not meeting weight specification are rejected. Following successful fill weight check, capsules are inspected, de-dusted, reconciled, and placed into a suitable in-process storage container.
All compounds listed as Examples above are known PPARδ modulators. The activity of these and other compounds as PPARδ modulators is demonstrated in PCT/US2004/010737, filed on Apr. 7, 2004; PCT/US2004/010889, filed on Apr. 7, 2004; PCT/US2004/010970, filed on Apr. 7, 2004; U.S. application Ser. No. 10/820,647, filed Apr. 7, 2004; PCT/US2004/043031, filed Dec. 20, 2004; PCT/US2005/011751 filed Apr. 7, 2005; U.S. application Ser. No. 11/102,356 filed Apr. 7, 2005; and U.S. application Ser. No. 11/258,463, filed Oct. 25, 2005; the contents of all of which are hereby incorporated by reference as if written herein in their entirities.
Rhesus macaques share great genetic similarity with humans, and can naturally develop the same metabolic disorders having equivalent phenotypic hallmarks as those found in humans. (Hansen, B C: Presentation at the IBC Second International Conference, 5-4-2004). For example, both humans and nonhuman primates can develop metabolic syndrome, a suspected prodrome to diabetes mellitus, cardiovascular disease and other life-threatening conditions.
Metabolic syndrome (alternately known by metabolic syndrome X, multiple risk factor syndrome, insulin resistance syndrome, plurimetabolic syndrome, diabesity, etc.) can be defined in both humans and non-human primates by measured increases in plasma LDL, triglycerides (TG), and plasma glucose, and a decrease in plasma HDL. In humans, metabolic syndrome is defined by the World Health Organization (WHO) as insulin resistance plus 2 of the following criteria: plasma TG ≧150 mg/dL, HDL <35 mg/dL (M); <40 mg/dL (W), body mass index (BMI) >30, elevated blood pressure (BP) ≧140/90 or drug Rx, and urinary albumin >20 mg/min. Similar, alternative measures are defined by the National Cholesterol Education Program (NCEP). Comparable indices exist for the diagnosis of metabolic syndrome, and indeed, for may other disorders, in nonhuman primates (Hansen et al.). The similarities between humans and non-human primates such as rhesus macaques make the macaques ideal models for studying the biological mechanisms of metabolic and cardiovascular diseases and for identifying in preclinical trials candidates for pharmaceutical treatments for these disorders.
In the present study, n=6 male obese rhesus macaques were selected for the study and prepared with 4 weeks of diet run-in (week −4 to week 0). At week 0, blood samples were taken in order to determine baseline plasma levels of CRP were measured prior to beginning dosage with Compound 1 as tosylate salt. Dosage began on the same day at 6 mg/kg/day in divided doses, given with food, and continued for four weeks. Dose was subsequently escalated at 4-week intervals to 12 mg/kg/d, 24 mg/kg/d, and finally 48 mg/kg/day. Plasma samples were taken at the close of each 4-week regimen, and a final sample was taken after 4 weeks of washout following discontinuation of dosing.
Quantitative measurements of plasma CRP in each sample taken were made by LabCorp, Inc. (Laboratory Corporation of America) by a multi-calibrator based high sensitivity wide ranged method using a immunoturbidometric assay. The limit of quantitation for the assay was 0.05 mg/dL and the highest point on the calibrator was 32 mg/dL. Mean values as percent change from baseline, and standard deviation as percent change from baseline, were calculated across the group and are shown in Table 2. Mean values for weeks 8-16 fell below the limit of quantiatation, and no data could therefore be reported for the standard deviation.
Table 3 shows that plasma CRP was significantly reduced in hyperlipidemic rhesus macaques over 16 weeks of dose-escalating treatment with Compound 1. Most interestingly, by week 8, CRP levels were below detectable levels (LOQ=0.05 ng/mL) and remained undetectable throughout the rest of the dosing regimen. During the washout period, the CRP levels reverted back to the pre-treatment levels implying that these changes are treatment-related.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
This application claims the benefit of U.S. Provisional Application No. 60/817,571, filed Jun. 28, 2006, the disclosure of which is hereby incorporated by reference as if written herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60817571 | Jun 2006 | US |
