Claims:CLAIMS
1. A method for making copper alpha-ketoglutarate, the method comprising the following steps:
i) mixing alpha-ketoglutaric acid in water thereby forming a solution;
ii) mixing an alkali metal salt of an acid in water thereby forming a solution;
iii) mixing solution of step (i) with the solution of step (ii) at optimal temperature for optimal amount of time, thereby forming a bis salt of alpha-ketoglutarate;
iv) mixing the bis salt of alpha-ketoglutarate with a copper salt at optimal temperature for optimal amount of time followed by continuous stirring at optimal amount of time, thereby forming copper alpha-ketoglutarate precipitate;
v) keeping the precipitate of step (iv) at optimal temperature to increase precipitation; and
vi) collecting solid dried precipitate of copper alpha-ketoglutarate

2. The method of claim 1 wherein the alpha-ketoglutaric acid and water mixture contains from about 0.92 to about 1.60 weight equivalents of alpha-ketoglutaric acid per one weight equivalent of water.

3. The method of claim 1 wherein alkali metal salt and water mixture contains from about 1.56 to about 3.2 weight equivalents of alkali metal salt per one weight equivalent of water.

4. The method of claim 1 wherein the alkali metal salt is sodium bicarbonate.

5. The method of claim 1 wherein sodium bicarbonate and water mixture contains from about 1.56 to about 3.2 weight equivalents of sodium bicarbonate per one weight equivalent of water.
6. The method of claims 1 wherein the alpha-ketoglutaric acid is mixed with the sodium bicarbonate and water mixture at about 40 °C to about 45 °C.

7. The method of claim 1 wherein the alpha-ketoglutaric acid and the sodium bicarbonate and water mixture is stirred for at least about 12 hours.

8. The method of claim 1 wherein the copper salt is Copper(II) Chloride dihydrate.

9. The method of claim 1 wherein the copper salt is from 0.9 to 3.2 weight equivalents per one weight equivalent of alpha-ketoglutaric acid.

10. The method of claim 1 wherein Copper(II) Chloride dihydrate and disodium salt of alpha-ketoglutaric acid mixture contains from about 0.9 to 3.2 about weight equivalents of Copper(II) Chloride dihydrate per one weight equivalent of disodium salt of alpha-ketoglutaric acid .

11. The method of claims 1 wherein the disodium salt of alpha-ketoglutaric acid is mixed with Copper(II) Chloride dihydrate at about 0 °C to about 5 °C.

12. The method of claims 1 wherein the disodium salt of alpha-ketoglutaric acid is continuously stirred with Copper(II) Chloride dihydrate at about 40 °C to about 45 °C.

13. The method of claims 1 wherein the disodium salt of alpha-ketoglutaric acid is continuously stirred with Copper(II) Chloride dihydrate for about 24 hours.

14. The method of claim 1 wherein the precipitate is kept at about 0 °C to about 5 °C to increase precipitation.

15. A method claim 1 wherein, the method comprises the following steps:
i) mixing alpha-ketoglutaric acid in water thereby forming a solution;
ii) mixing sodium bicarbonate in water thereby forming a solution;
iii) mixing solution of step (i) with the solution of step (ii) at 40 - 45 °C for 12 hrs, thereby forming a disodium salt of alpha-ketoglutarate;
iv) mixing the disodium salt of alpha-ketoglutarate with Copper(II) Chloride dihydrate at 0 – 5 °C followed by continuous stirring at 40 - 45 °C for 24 hours, thereby forming copper alpha-ketoglutarate precipitate;
v) keeping the precipitate of step (iv) at 0 -5 °C to increase precipitation; and
vi) collecting solid dried precipitate of copper alpha-ketoglutarate

Dated this on 25th day of November 2020

Asha P. Hole
(IN/PA 2269)
, Description:FIELD OF THE INVENTION

The present invention relates to the method for synthesis of copper salt of alpha-ketoglutarate (Cu-AKG).

BACKGROUND OF THE INVENTION

Copper (Cu) is a zinc-balancing mineral important in many enzymes as well as in the production of hemoglobin. Further, copper is an integral component of ceruloplasmin, which facilitates iron absorption and transport. Copper is also known to play a role in the functioning of the prostate gland and the activity of the oil glands, helping prevent acne. Nerves and joints also reportedly require copper for healthy functioning. On a more molecular level, copper enzymes regulate various physiologic pathways, such as energy production, iron metabolism, connective tissue maturation, and neurotransmission. The ability of copper to easily accept and donate electrons explains its important role in redox reactions and in scavenging free radicals. Benefits of copper have been explored in treatment and prevention of various related physiological and pathological conditions including cardiovascular, immune system function, osteoporosis, neurodegenerative diseases and so on. In addition to using copper in various forms for treatment of various conditions in human, copper is also known to have utility in treating conditions in animal subjects.

Alpha-ketoglutarate (AKG) is involved in the Krebs cycle: it is a nitrogen acceptor, and therefore an amine acceptor, generating glutamine by means of transamination or amination reactions through glutamate-dehydrogenase in perivenous hepatocytes. The amidation of glutamate to glutamine by means of glutamine-synthetase takes place in several tissues and organs, such as muscles, lungs, liver and brain. Alpha-ketoglutarate is typically used for kidney disease; intestinal and stomach disorders, including bacterial infections; liver problems; cataracts; and recurring yeast infection.

AKG has been widely used in animals and humans as a feed additive and medicine. In animal industry, AKG could effectively improve growth performance, nitrogen utilization, immunity, bone development, intestinal mucosal injury, and oxidative system. In humans, AKG is extensively used in trauma, aged diseases, postoperative recovery, and other nutritional diseases. To facilitate this, salts of AKG have been reported in the prior. Typically, these include alpha-ketoglutarate in conjugation with calcium, ornithine, sodium, and so on. However, copper alpha-ketoglutarate has not yet been reported in the literature.

The present invention, reports and describes a method for the preparation copper alpha-ketoglutarate which can be subsequently formulated into an appropriate target composition suitable for the target therapy.

SUMMARY OF THE INVENTION
In view of the foregoing, the object of the invention is to provide a method for the preparation of copper alpha-ketoglutarate (Cu-AKG).

In one embodiment of the invention is a method for making copper alpha-ketoglutarate, the method comprising the following steps:
i) mixing alpha-ketoglutaric acid in water thereby forming a solution;
ii) mixing an alkali metal salt of an acid in water thereby forming a solution;
iii) mixing solution of step (i) with the solution of step (ii) at optimal temperature for optimal amount of time, thereby forming a bis salt of alpha-ketoglutarate;
iv) mixing the bis salt of alpha-ketoglutarate with a copper salt at optimal temperature for optimal amount of time followed by continuous stirring at optimal amount of time, thereby forming copper alpha-ketoglutarate precipitate;
v) keeping the precipitate of step (iv) at optimal temperature to increase precipitation; and
vi) collecting solid dried precipitate of copper alpha-ketoglutarate

In one embodiment of the invention, the alpha-ketoglutaric acid and water mixture contains from about 0.92 to about 1.60 weight equivalents of alpha-ketoglutaric acid per one weight equivalent of water.

In one embodiment of the invention, alkali metal salt and water mixture contains from about 1.56 to about 3.2 weight equivalents of alkali metal salt per one weight equivalent of water.

In one embodiment of the invention, the alkali metal salt is sodium bicarbonate.

In one embodiment of the invention, sodium bicarbonate and water mixture contains from about 1.56 to about 3.2 weight equivalents of sodium bicarbonate per one weight equivalent of water.

In one embodiment of the invention, alpha-ketoglutaric acid is mixed with the sodium bicarbonate and water mixture at about 40 °C to about 45 °C.

In one embodiment of the invention, the alpha-ketoglutaric acid and the sodium bicarbonate and water mixture is stirred for at least about 12 hours.

In one embodiment of the invention, the copper salt is Copper(II) Chloride dihydrate.

In one embodiment of the invention, copper salt is from 0.90 to 3.2 weight equivalents per one weight equivalent of alpha-ketoglutaric acid.

In one embodiment of the invention, Copper(II) Chloride dihydrate and disodium salt of alpha-ketoglutaric acid mixture contains from about 0.90 to 3.2 about weight equivalents of Copper(II) Chloride dihydrate per one weight equivalent of disodium salt of alpha-ketoglutaric acid.

In one embodiment of the invention, disodium salt of alpha-ketoglutaric acid is mixed with Copper(II) Chloride dihydrate at about 0 °C to about 5 °C.

In one embodiment of the invention, disodium salt of alpha-ketoglutaric acid is continuously stirred with Copper(II) Chloride dihydrate at about 40 °C to about 45 °C.

In one embodiment of the invention, disodium salt of alpha-ketoglutaric acid is continuously stirred with Copper(II) Chloride dihydrate for about 24 hours.

In one embodiment of the invention, precipitate is kept at about 0 °C to about 5 °C to increase precipitation.

Accordingly, in one embodiment of the invention, is the method that comprises the following steps:
i) mixing alpha-ketoglutaric acid in water thereby forming a solution;
ii) mixing sodium bicarbonate in water thereby forming a solution;
iii) mixing solution of step (i) with the solution of step (ii) at 40 - 45 °C for 12 hrs, thereby forming a disodium salt of alpha-ketoglutarate;
iv) mixing the disodium salt of alpha-ketoglutarate with Copper(II) Chloride dihydrate at 0 - 5 °C followed by continuous stirring at 40 - 45 °C for 24 hours, thereby forming copper alpha-ketoglutarate precipitate;
v) keeping the precipitate of step (iv) at 0 -5 °C to increase precipitation; and
vi) collecting solid dried precipitate of copper alpha-ketoglutarate

DESCRIPTION OF THE DRAWINGS
For a more complete understanding of the invention, reference should now be made to the embodiments illustrated in greater detail in the accompanying drawings and described by way of embodiments of the invention.
Figure 1: Illustrate FT-IR test data for copper alpha-ketoglutarate dehydrate.
Figure 2: Illustrate Mass spectrometry test data for copper alpha-ketoglutarate dihydrate wherein m/z = 169.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the method for synthesizing copper alpha-ketoglutarate.

To clarify the above and other purposes, features, and advantages of this invention, specific embodiment of this invention is especially listed and described in detail with the examples as follows. The principal and mode of operation of this invention have been described and illustrated in its embodiment. At the outset, a person skilled in the art will appreciate that this invention may be practiced otherwise than is specifically described and illustrated. The invention should not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention. Also, in the following description of the invention, certain terminology may be used for the purpose of reference only, and is not intended to be limiting.

Definitions and Use of Terms
As used in this specification and in the claims which follow, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an ingredient" includes mixtures of ingredients; reference to "an active pharmaceutical agent" includes more than one active pharmaceutical agent, and the like. "Treating" or "treatment" of a disease includes (1) preventing the disease from occurring in an animal that may be predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e. arresting its development, or (3) relieving the disease, i.e. causing regression of the disease.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value and/or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Central to this invention is the object to prepare Cu-AKG. It should be noted that the synthesis of Cu-AKG involves two major steps – firstly, preparation of a bis salt of alpha-ketoglutarate; and secondly, reacting the bis salt of alpha-ketoglutarate with copper salt to form copper alpha-ketoglutarate. It should also be noted that to achieve these two steps, a series of sequential steps with careful calibration of reactive agents, reagents, process parameters such as time, temperature and so on are required and have been described herein. It is to be understood that all weight equivalents of components, reactants, reagents, solvents, and the like, described herein are a description of the corresponding mole equivalents, each of which is obtained from the molecular weight of such components, reactants, reagents, solvents, and the like. In addition, it is to be understood that certain weight equivalents, such as weight equivalents of specific alkali metal salts to water can be converted to the corresponding weight equivalents of alternative alkali metal salts to water by accounting for the different molecular weights of the specific alkali metal salts and the alternative alkali metal salts. It is also to be understood that the same conversion between specific weight equivalents of alpha-ketoglutaric acid to water, or weight equivalents of alpha-ketoglutaric acid to alkali metal salts, or weight equivalents of copper salts to water, or weight equivalents of copper salts to alpha-ketoglutaric acid to alternatives, respectively, by accounting for the different molecular weights in each case.

The invention sets out a chemical method that is used for preparing copper salt of alpha-ketoglutarate i.e. Cu-AKG. Firstly, sodium bicarbonate dissolved in water was allowed to be added slowly in different portions into alpha-ketoglutaruic acid which was pre-dissolved in water at 0-5°C. The mixture was further stirred at 40 - 45°C for 12 hours followed by cooling it to room temperature. Next, Copper (II) Chloride Dihydrate was added gradually into the alpha-ketoglutaric acid disodium salt solution at 0 - 5°C. The mixture was allowed to come to room temperature and subjected to heat at 40 - 45°C for 24 hours. After 24 hours, the reaction mixture temperature was reduced to room temperature and the precipitate was kept at 0 - 5°C in ice bath to increase precipitation. Then, precipitate was filtered by using Buchner funnel under vacuum filtration. The solid particles were repeatedly washed with cold water to remove water miscible impurities. The solid was dried in an oven at 50°C for 3 hours and kept at 2 - 8°C in refrigerator. The copper alpha-ketoglutarate dihydrate, thus yielded, was collected and confirmed for the presence of Cu2+ ion.
The following examples broadly illustrate the nature of this invention and the manner in which it is to be performed without limiting the nature and scope of the invention. Examples 1 and 2 are comparative examples useful to demonstrate that the process for the preparation of copper alpha-ketoglutarate results from optimal combination of chosen process parameters, excipients and amount thereof. The present inventors report that in addition to time and temperature parameters, optimal proportions of AKG, alkali metal salt and copper salt critically govern the final yield. Example 1 and 2 illustrate the process for synthesizing copper alpha-ketoglutarate on a scale of 10 gm and 50 gm respectively. Comparative preparations on different scales exhibit yields in a similar range.

Example 1
Preparation of copper alpha-ketoglutarate (10g scale)
In one illustrative embodiment of the inventions, described herein is the method for making copper alpha-ketoglutarate. Alpha-ketoglutaric acid (10 g, 68.442 mmol, 1 equiv.) was dissolved into 30 mL of water in an oven-dried magnetic bar charged 500 mL round bottom flask at 0-5 °C using ice-water bath by stirring for 5 minutes. NaHCO3 (11.5 g, 136.884 mmol, 2 equiv.) was dissolved in 200 mL water. This solution was allowed to add slowly in different portions into alpha-ketoglutaric acid solution at 0-5 °C by stirring continuously. Further, the solutions were continuously stirred at 40-45 °C for 12 hours and subsequently cooled down to room temperature. Copper(II) Chloride Dihydrate (11.668 g, 68.442 mmol, 1 equiv.) was added slowly into the alpha-ketoglutaric acid Disodium salt solution at 0-5 °C under continuous stirring. The mixture were allowed to come at room temperature and subjected to heat at 40-45 °C with continuous stirring for 24 hours. The resultant precipitate was kept at 0-5 °C in ice bath to increase precipitation. The final precipitate was collected and washed repeatedly with 250 mL water. The solid Copper alpha-ketoglutarate dihydrate was dried in an oven at 50 °C for 3 hours and kept at 2-8 °C in refrigerator to give about 37% yield.

Example 2
Preparation of copper alpha-ketoglutarate (50g scale)
In one illustrative embodiment of the inventions, described herein is the method for making copper alpha-ketoglutarate. Alpha-Ketoglutaric acid (50 g, 342.21 mmol, 1 equiv.) was dissolved into 150 mL of water in an oven-dried magnetic bar charged 2000 mL round bottom flask at 0-5 °C using ice-water bath by stirring for 5 minutes. NaHCO3 (57.50 g, 684.42 mmol, 2 equiv) was dissolved in 1000 mL water. This solution was allowed to add slowly in different portions into alpha-ketoglutaric acid solution at 0-5 °C by stirring continuously. Further, the solutions were continuously stirred at 40-45 °C for 12 hours and subsequently cooled down to room temperature. Copper(II) Chloride Dihydrate (58.34 g, 342.21 mmol, 1 equiv.) was added slowly into the alpha-ketoglutaric acid Disodium salt solution at 0-5 °C under continuous stirring. The mixture were allowed to come at room temperature and subjected to heat at 40-45 °C with continuous stirring for 24 hours. The resultant precipitate was kept at 0-5 °C in ice bath to increase precipitation. The final precipitate was collected and washed repeatedly with 500 mL water. The solid Copper alpha-ketoglutarate dihydrate was dried in an oven at 50 °C for 3 hours and kept at 2-8 °C in refrigerator to give about 41% yield.

The copper alpha-ketoglutarate thus obtained was evaluated and characterized using standard tests. FT-IR test exhibited results consistent with the structure (Fig. 1). Further, mass spectrometry test results exhibited m/z value of 169, also consistent with the structure (Fig. 2).

Cu-AKG prepared in this manner shall be used as an active ingredient for the treatment and prevention of related conditions in humans and animals. This can be done via incorporating Cu-AKG as an active ingredient or as a co-therapeutic agent in an appropriate formulation or composition. Various routes of administration such as oral, parenteral, etc., are used for delivery of copper formulation. It should be noted that compositions comprising Cu-AKG may be used in various forms. Oral administration formulations, but not limited to, in form of tablets, capsules, or granulate in bags which may be suitable for administering to the subject. Cu-AKG may also formulated as, but not limited to, injectable, intravenous, intraperitoneal, topical, nasal, aerosols or sprays for such as solutions, suspensions or emulsions, nanoparticle compositions.