A pharmaceutical composition comprising 5-ethyl-1-phenyl-2-(1H)-pyridone (5-EPP) and its use for the treatment of sepsis are disclosed.

Description of the Invention

TECHNICAL FIELD

This invention relates to a pyridone compound, 5-ethyl-1-phenyl-2-(1H)-pyridone, new processes for its synthesis and purification, pharmaceutical compositions containing it and its use in the treatment of sepsis.

DETAILED DESCRIPTION

A compound, 5-ethyl-1-phenyl-2-(1H)-pyridone and new methods of making it, purifying it, employing it in a pharmaceutical composition for the treatment of sepsis, and using it is herein described.

Although the chemical structure of 5-ethyl-1-phenyl-2-(1H)-pyridone has been named in prior patents and literature, heretofore there has been no known disclosure of an effective way to make and purify this compound.

Effective synthesis methods for 5-ethyl-1-phenyl-2-(1H)-pyridone from a starting compound, 3-ethyl pyridine, are now provided.

A first reaction scheme useful for synthesis of 5-ethyl-1'-phenyl-2-(1H) pyridone is provided in FIG. 1 (see Original Patent).

In said first reaction scheme, we have found that 5-ethyl-1-phenyl-2-(1H)-pyridone can be made from 3-ethyl pyridine (available from Sigma-Aldrich Corporation, St. Louis, Mo.) which is reacted with NaNH.sub.2 in presence of a small amount of oleic acid as a catalyst to speed up the reaction. A general reaction scheme for obtaining 2-amino-5-ethyl pyridine isomer from 3-ethylpyridine is provided in U.S. Pat. No. 5,003,069 to McGill et al, "Chichibabin Reaction," at Example 12. U.S. Pat. No. 5,003,069 is herein incorporated by reference as if fully set forth herein. In this reaction 3-ethyl pyridine (I) is converted to 2-amino-5-ethylpyridine (II) as well as 2-amino-3-ethylpyridine(III).

After repeated distillation, the desired isomer, 2-amino-5-ethyl pyridine (Structure II, above) is obtained.

We have found that 2-amino-5-ethyl pyridine can be converted to the desired product, 5-ethyl-1-phenyl-2-(1H)-pyridone. In the procedure of the invention, 2-amino-5-ethyl pyridine isomer is first oxidized to form 5-ethyl-2-pyridone.

In the preferred oxidation method of the invention, 2-amino-5-ethyl pyridine is dissolved in 20% sulfuric acid (H.sub.2SO.sub.4, wt/vol) and chilled thoroughly in an ice bath. A preferred reactant, sodium nitrite (NaNO.sub.2, wt/vol, 1.2 mole equivalents) solution is then added over 4-5 hours to oxidize 2-amino-5-ethyl pyridine into 5-ethyl-2-pyridone.

After oxidation, Na.sub.2CO.sub.3 (2.0 mole equivalents) is added to the reaction mixture until a pH=8-8.5 is attained, and a two-phase mixture dark yellow upper organic phase and a lower light yellow aqueous phase is obtained.

The aqueous phase is extracted with methylene chloride (CH.sub.2Cl.sub.2) and pooled with the organic phase. After drying, a light yellow solid is obtained which is 5-ethyl-2-pyridone.

A substitution reaction is then conducted in order to substitute a phenyl group on the N of the pyridone ring of 5-ethyl-2-pyridone for the hydrogen. The 5-ethyl-2-pyridone is preferably reacted in the presence of a copper-zinc catalyst and potassium carbonate with a halobenzene. Preferably the halobenzene is selected from iodobenzene and bromobenzene. Most preferably, bromobenzene is utilized. 5-ethyl-1-phenyl-2-(1H)-pyridone is formed and will be in the reaction mixture.

The method of the invention includes purification of the 5-ethyl-1-phenyl-2-(1H)-pyridone from the reaction mixture. After completion of the reaction, as ascertained by thin layer chromatography (TLC), un-reacted bromobenzene was removed by distillation under high vacuum.

The remaining solution was extracted with CH.sub.2Cl.sub.2. The extract was vacuum filtered through a short Celite column to remove any small particles.

The above extract was treated with activated charcoal and boiled for 10-15 minutes, then cooled to room temperature before removing the charcoal by vacuum filtration through a second Celite column.

The volume of filtrate was reduced by rotary evaporation. The resulting oily concentrate was dried at room temperature to yield a dark-brown solid which contained 75% 5-ethyl-1-phenyl-2-(1H)-pyridone.

The dark-brown solid was first dissolved in warm ethyl acetate and then boiled. Hexane was added to the boiling solution and continuously stirred to separate the upper phase (hexane-rich) from the lower phase (ethyl acetate).

The upper phase was poured into a beaker and allowed to cool and settle first, then transferred to a Teflon coated pan.

After evaporation and cooling, white 5-ethyl-1-phenyl-2-(1H)-pyridone crystals formed throughout the pan.

The purified white crystals after drying yielded a fine white crystalline powder with a melting point of 57-59.degree. C.

Reverse phase HPLC analysis and NMR revealed the powder contained >99% 5-ethyl-1-phenyl-2-(1H) pyridone.

In a second reaction scheme, a one-step synthesis method for making the compound 5-ethyl-1-phenyl-2-(1H)-pyridone has also been developed. The first reaction scheme discussed above involves a three step process.

We have found that 5-ethyl-1-phenyl-2-(1H)-pyridone can be made from the starting material, 5-ethyl-2-pyridone (J & W Pharlab LLC, Levitton, Pa.). 5-ethyl-2-pyridone is reacted with bromobenzene in the presence of a Cu--Zn catalyst under a blanket of argon to produce 5-ethyl-1-phenyl-2-(1H)-pyridone.

Preparation of Catalyst: A copper-zinc (Cu--Zn) catalyst was prepared immediately prior to the above reaction. Finely divided Zn dust (1.0 mole equivalents) was first washed two to four times with 3% hydrochloric acid until the solution was clear and evolved hydrogen gas. The zinc was then washed twice with 18 M-Ohm water. Next, a 2% CuSO.sub.4 aqueous solution was added to the freshly add-washed Zn dust and the solution mixed via a magnetic stirrer until it became colorless. The colorless supernatant was decanted and a second volume of 2% CuSO.sub.4 solution was added to the Zn dust. This process was repeated several times until the CuSO.sub.4 solution remained blue and the Cu--Zn catalyst became a red powder. Finally, the completed Cu--Zn catalyst was rinsed 3-4 times with water, followed by 2-3 methanol rinses. The resulting dark-red powder was then dried under low vacuum for at least 2 hours at 40-50.degree. C.

Chemical Reaction is as follows: 5-ethyl-2-pyridone (.gtoreq.96%, 1.0 mole equivalents), and potassium carbonate (1.2 mole equivalents) were combined in a 5 liter reaction flask mounted in a heating mantle. Next, the bromobenzene (2.5 mole equivalents) and the Cu--Zn catalyst (0.05 mol equivalents) were added to the flask. A stirring apparatus was attached and the reaction flask flushed with argon gas for several minutes. After adjusting the stirring apparatus, the mixture was heated for 60-90 min. until it refluxed gently under argon for 48-72 hours. Formation of an off-white solid on the side of the flask indicated the start of the reaction. The progress of the reaction was checked by thin layer chromatography (TLC) every 24 hours. When TLC analysis detected no substrate, the stirring apparatus and argon source were removed. After completion of the reaction, un-reacted bromobenzene was removed from the reaction mixture by distillation under high vacuum.

Extraction, filtration and concentration: After allowing the flask to cool for at least 30 min, 0.5-0.6 L of methylene chloride and 10-20 g of Norit were added into the flask and mixed thoroughly. The liquid phase was filtered through a short Celite (diatomaceous earth) column to remove any small particles. This extraction was repeated several times without Norit and the filtrate was pooled in a large flask. The remaining solids left behind were allowed to dry overnight, then pulverized and extracted with CH.sub.2Cl.sub.2. The resulting washings were filtered as before.

The pooled filtrates and CH.sub.2Cl.sub.2 washings of the Celite column were later filtered through a folded filter paper into a rotary distillation flask. Next, the filtrate solution was distilled under vacuum to remove all CH.sub.2Cl.sub.2. The resulting brown viscous solution was then transferred to a Teflon-coated baking pan. The residual viscous solution in the flask was rinsed with ethyl acetate and transferred to the baking pan and then dried overnight or longer at room temperature in the hood to yield a dark-brown solid containing 5-ethyl-1-phenyl-2-(1H)-pyridone (5-EPP).

Re-crystallization and purification: Approximately 100-150 g of crude 5-EPP was dissolved with approximately 100 mL ethyl acetate in a 1000 mL beaker (Beaker-I). Beaker-I was covered with aluminum foil and the contents gently brought to a boil on a hot plate in the hood. After removing the beaker from the heating plate, hexanes were added to the boiling solution while stirring continuously with a Teflon stirring rod. When the upper, hexanes-enriched phase began to separate from the lower phase (ethyl acetate-enriched), all stirring ceased for 5-10 minutes and the solution was allowed to slowly cool and fully separate. The upper phase was clear and had a faint yellow or orange color. The upper phase was gently poured into a second beaker (Beaker-II) and this solution was allowed to settle without stirring for a while (<5 min). As usually happens, a small amount of an oily yellow liquid fell out of solution forming a distinct layer on the bottom of Beaker-II. The upper phase was carefully poured into a Teflon-coated baking pan placed in the hood, leaving the yellow oily contaminant on the bottom of Beaker-II. Beaker-II was rinsed into Beaker-I with ethyl acetate, pooling any residual contents and rinses with the original ethyl acetate solution in Beaker-I. Beaker-I was placed back on the heating plate and the extraction as described above was then repeated. All hexanes-enriched extracts from Beaker-II were transferred into the same baking pan. The process was repeated until the solution volume in Beaker-I was less than 10 mL and became dark and oily. As the hexanes and ethyl acetate solution evaporated from the baking pan, yellow or white crystals of purified 5-EPP formed throughout the pan. A Teflon spatula was used to gently scrape the sides and bottom of the pan and to mix the crystallized contents. Following thorough extraction of the Beaker-I contents, the pan contents were left in the fume hood for 15-30 minutes to allow some of the solvent to evaporate. The pan contents were intermittently mixed and scraped to allow for continuous contact between the crystalline material and the liquid phase. When the liquid level had been reduced by one-third to one-half, the remaining liquid was poured off into a 1 L beaker and the remaining crystals left in the hood to dry for several hours.

Yields exceeding 90% have been achieved after one round of re-crystallization. When the 5-EPP is not sufficiently pure after one round of re-crystallization as judged by color of 5-EPP, visible oily contaminants, or TLC analysis, then the re-crystallization process can be repeated. Yield of this process ranged from 65-80%. After the purified solid was dried overnight at room temperature, the described process yielded a fine white crystalline powder with melting point of 55-59.degree. C. Reverse phase high-performance liquid chromatography (HPLC) analysis and nuclear magnetic resonance (NMR) confirmed this material contained >99% 5-ethyl-1-phenyl-2-(1H)-pyridone of the structure shown in FIG. 3 (see Original Patent).

It has been found that 5-ethyl-1-phenyl-2-(1H)-pyridone ("5-EPP") may be administered to mammals for treatment of sepsis. Sepsis is a medical condition caused by infection of the blood or tissues of a mammal by microorganisms such as bacteria, viruses, or fungi. Septicemia is a term often used to indicate rampant infection of the body, detectable in the bloodstream. If left untreated, sepsis can lead to serious complications or death.

5-ethyl-1-phenyl-2-(1H)-pyridone can be administered to a mammal in need of treatment for sepsis or infection due to microorganisms. Appropriate routes of administration are by injection (intravenous (i.v), intraperitoneal (i.p.), intramuscular (i.m.)), orally or by any other route which allows the compound to enter the bloodstream. Preferably, it is administered i.v., i.p. or orally. The 5-ethyl-1-phenyl-2-(1H) pyridone is preferably formulated in a pharmaceutical carrier appropriate for the route of administration. Preferably, a pharmaceutical carrier for 5-ethyl-1-phenyl-2-(1H)-pyridone for injection is selected from isotonic saline and propylene glycol and combinations thereof. Most preferably, 5-EPP is added to isotonic saline at a concentration of 20 mg/mL or less to form a colorless composition at room temperature.

It has been found that an effective amount of 5-ethyl-1'-phenyl-2-(1H)-pyridone to treat sepsis ranges from 5 mg/kg body weight to about 200 mg/kg body weight per day, preferably 10 mg/kg body weight to 40 mg/kg body weight per day. Most preferred for non-oral routes of administration is about 10-25 mg/kg body weight per day The effective amount can be given once per day or in multiple doses. In a preferred embodiment, a single dose is given by iv infusion once per day. The effective amount has been found to block the lethal effects of septic shock in mammals.

The compound, 5-ethyl-1-phenyl-2-(1H)-pyridone, may also be provided in an oral total dose form either in tablets or capsules (or otherwise in combination with a pharmaceutical carrier which is suitable for oral administration to an animal) to provide a dose ranging from 5 mg/kg body weight to 200 mg/kg body weight per day, preferably 10 mg/kg body weight to 80 mg/kg body weight per day and most preferred 20 to 50 mg/kg body weight per day. Preferably, the oral dosage is administered in divided dosages. Most preferably, the oral dosage is administered in three (3) daily doses at intervals of 8 hours.

The sepsis syndrome is a complex illness that results from a systemic host response to a variety of insults, particularly infection, which is manifested by varying degrees of hypotension, coagulopathy, and multi-organ dysfunction. Pathophysiologic events occur in early and late phase responses. Early responses in sepsis are characterized by the release of a number of proinflammatory mediators including tumor necrosis factor-alpha, interleukine-6 and interleukine-12 (Dinarello C A. Chest 2000; 118:503-508). Methods for evaluating the protective effects of 5-ethyl-1-phenyl-2-(1H)-pyridone (5-EPP) against multi-organ dysfunction, and the serum levels of proinflammatory cytokines and the mortality in several animal models of sepsis are described. It is herein disclosed that 5-EPP can be used as a treatment agent in mammals for septic shock.
 

Claim 1 of 12 Claims

1. A method of treating sepsis, consisting of administering an aqueous solution containing an effective amount of 5-ethyl-1-phenyl-2-(1H)-pyridone as a sole therapeutic agent, dissolved in a carrier, by injection into a mammal in need of such treatment, wherein said effective amount is about 5 mg/kg body weight to 100 mg/kg body weight.
 

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