PROCESS FOR PREPARING BETULINIC ACID

FIELD OF THE INVENTION

[0001] The present invention relates to a process for preparing betulinic acid. More specifically, the present invention relates to a process for preparing betulinic acid calling upon the electrochemical oxidation of betulin.

BACKGROUND OF THE INVENTION

[0002] Oxidation is a fundamental transformation in organic synthesis. Numerous oxidation procedures have already been described in the literature. Nevertheless, the direct conversion of primary alcohols to the corresponding aldehyde and carboxylic acid, in particular in the presence of other functional groups such as secondary alcohols, is still associated with problems. The secondary alcohol is commonly protected with a suitable protecting group which must be removed after the oxidation of the primary alcohol is complete.

[0003] Betulin (Ae. betulinol), illustrated below in Formula 1 , is a naturally occurring compound that is isolated from the outer layer of the bark of the white birch tree. Birch bark is a potential source for a variety of organic chemicals useful in pharmaceutical and industrial applications. For example, the bark of white birch, Betula alba, contains betulinic acid (0.025% by weight), betulin (25% by weight), and lup-20(29)-ene-3.beta.,28-dioi. The major interest in betulin lies in the fact that it is a synthetic precursor of triterpenoid compounds having important pharmacological properties. To

obtain these triterpenoid compounds, betulin must be subjected to some chemical reactions.

Formula 1

[0004] U.S. Pat. No. 5,750,578 issued to Carlson et al. on May 12,

1998 discloses that betulin possesses antiviral properties and is useful for treating a mammal afflicted with a herpes virus infection. Betulin has also been reported as possessing anti-inflammatory activity.

[0005] Betulin is a useful starting material for preparing betulinic acid and derivatives thereof, which possess useful pharmacological properties. Pisha, E. et al., (J. M. Nature Medicine, 1 , 1046-1051 , 1995) disclosed betulinic acid as possessing antitumor activity against human melanoma, e.qf., MEL-1 , MEL-2 and MEL-4. Betulinic acid, a pentacyclic triterpene, seems to work by inducing apoptosis in cancer cells. Due to its apparent specificity for melanoma cells, betulinic acid seems to be a more promising anti-cancer substance than drugs like Taxol, the latter being a more general cell poison and not being specific to cancer cells. In fact, the specificity of betulinic acid for melanoma cells is unique in comparison to the specificity of a number of chemotherapy drugs, including camptothecin, ellipticine, mithramycin A, etoposide, vinblastine and vincristine. Additionally,

Fujioka, T. et ai, (J. Nat. Prod., (1994) 57, 243-247) disclosed betulinic acid as possessing anti-HIV activity in H9 lymphocytic cells. Moreover, betulinic acid has been reported as possessing anti-inflammatory activity. The antiinflammatory activity of betulinic acid is, at least in part, due to its capacity to inhibit enzymes involved in leukotriene biosynthesis, including 5-lipoxygenase (Somatsu, S. et a/., Skin and Urology 21:138, 1959 and Inoue, H., et a/,. Chem. Pharm. Bull. 2: 897-901 , 1986).

[0006] U.S. Patent No. 6,943,260 issued to Sauter, M. on

September 13, 2005 discloses a process for obtaining pure crystalline betulinic acid from a methanolic extract of ground plane tree cortex and/or plane tree bark by evaporating the extract down and crystallizing the residue from an alcohol. Before the crystallization step, the residue is washed with a non-polar diluent. The process, however, requires the use of large volumes of solvents, making it less desirable for large scale industrial applications.

[0007] U.S. Patent No. 6,175,035 issued to Draeger, et a/, on

January 16, 2001 discloses a process for obtaining betulinic acid from a powder obtained from the bark of plane trees, by fractional extraction with a solvent of medium polarity such as, for example, dichloromethane, chloroform or diethyl ether, and subsequent recrystallization out of methanol. The process, however, requires the use of very large volumes of solvents, making it less suitable for large scale industrial applications.

[0008] U.S. Pats. No. 5,804,575 and 6,569,842 issued to Pezzuto et a/., on September 8, 1998 and May 27, 2003, respectively, disclose a multi- step process for the synthesis of betulinic acid from betulin. The process, however, is time consuming and requires several protection-deprotection

steps as well as using hazardous and expensive reagents, making it less desirable on a commercial scale.

[0009] U.S. Pat. No. 6,867,314 issued to Krasutsky, P.A. on

March 15, 2005 as well as U.S. Pats. No. 6,407,270, 6,271,405 and 6,232,481 issued to Krasutsky, P.A. et al. on June 18, 2002, August 7, 2001 and May 15, 2001 , respectively, and US Patent application No. 2003/0073858 filed in the name of Krasutsky, P.A. et al. and published on April 17, 2003, disclose a further multi-step process for the synthesis of betulinic acid from betulin. The process involves a one-pot conversion of betulin to the mono- protected alcohol derivative which is subsequently converted into betulinic acid following oxidation and deprotection steps. The oxidation of the mono- protected alcohol derivative (unprotected hydroxyl group at C-28) to provide the corresponding carboxylic acid functionality can be effected by a variety of reagents, often involving metal catalysts and requiring the protection of the hydroxyl group at C-3. Moreover, the olefin at C20-C29 is somewhat sensitive to several of these oxidizing reagents, calling for the mildest possible reaction conditions in order to avoid substrate degradation such as by oxidation, isomerization or even transannular product formation. Substrate degradation is particularly prevalent when using reagents such as free oxygen or free radical initiators. Finally, the process is time consuming, making it less desirable on a commercial scale.

[0010] Since the reported literature procedures for the synthetic preparation of betulinic acid are subject to generating unwanted side-products and contaminants resulting from the degradation of the substrates, there remains a need for an improved process for preparing betulinic acid and synthetic precursors thereof that minimizes the number of steps and

minimizes the formation of side-products while providing for good yields of the desired product.

[0011] The present invention seeks to meet these and other needs.

[0012] The present invention refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0013] The present invention relates to an electrochemical process for preparing betulinic acid and synthetic precursors thereof. The method is less time consuming and is more cost effective than known procedures. Furthermore, the method can be easily implemented to meet contemporary industrial production demands as well as meeting safety and environmental constraints.

[0014] The present invention relates to a process for preparing betulinic acid comprising subjecting betulin to an electrochemical oxidation reaction to provide a reaction product wherein a primary alcohol function of betulin is first converted to an aldehyde functionality. The reaction product is then subjected to a further oxidation reaction to convert the aldehyde functionality to a carboxylic acid functionality, thereby forming betulinic acid.

[0015] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating illustrative embodiments of the invention, are given

by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0016] In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.

[0017] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more.

[0018] As used in this specification and claim(s), the words

"comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0019] The term "about" is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value.

[0020] The term "noble metal", as used herein, refers to metals and alloys of the group comprising silver, gold, iridium, osmium, palladium, ruthenium, rhodium and platinum.

[0021] The term "derivative" as used herein, is understood as being a substance which comprises the same basic carbon skeleton and carbon functionality in its structure as a given compound, but can also bear one or more substituents or rings.

[0022] The term "scavenger", as used herein, is understood as being an agent reactive with free radicals.

[0023] Abbreviations: TEMPO: 2,2,6,6-Tetramethyl-i- piperidinyloxy, free radical;

[0024] The present invention relates to an electrochemical process for preparing betulinic acid and synthetic precursors thereof.

[0025] Surprisingly it has been found that betulinic acid can be readily prepared via the electrochemical oxidation of betulin, without prior protection of the hydroxyl groups at C3 and C28, and without the risk of transannular reactions with the olefin at C29-C30 and/or isomerization thereof. In an embodiment of the present invention, the electrochemical oxidation of the C28 hydroxyl group of betulin is carried out using TEMPO as the electrochemical oxidizing agent. In further embodiments of the present invention, the electrochemical oxidation of the C28 hydroxyl group of betulin is carried out using derivatives of TEMPO as the electrochemical oxidizing agent.

[0026] Contrary to the reported literature procedures, the electrochemical oxidation process of the present invention can be advantageously carried out using crude betulin rather than the highly purified diol. Crude betulin (1), which is extracted, for example, from white birch bark, is amenable to the electrochemical oxidation process as described in the present invention without the requirement of prior purification. In an embodiment of the present invention, betulin having a purity of about 65% was electrochemically oxidized to the corresponding aldehyde, which was subsequently converted to betulinic acid. The aldehyde (Formula 2) is readily purified either by elution over a silica column or more simply by crystallisation. In an embodiment of the present invention, trifluorotoluene/chloroform proved to be particularly efficient eluent system.

Formula 2

[0027] The further oxidation of the aldehyde to provide the carboxylic acid functionality of betulinic acid (Formula 3) can, in an embodiment of the present invention, be advantageously achieved using a mild oxidant such as sodium chlorite dissolved f-butanol in the presence of a radical scavenger such as 2-methyl-2-butene, without prior protection of the hydroxyl group at C3 and without the risk of transannular reactions with the olefin at C29-C30 and/or isomerization thereof. Other mild oxidants and scavengers are known in the art, and are within the capacity of a skilled

technician. The overall conversion of betulin to the corresponding aldehyde using the method of the present invention is sensitive to the type of TEMPO reagent and ranges from essentially no conversion to about 70% of recovered aldehyde.

Formula 3

[0028] As illustrated in Scheme 1, betulinic acid (3) is readily prepared in high purity and good yield from naturally occurring betulin (1) via a short two-step process. The process is amenable to contemporary industrial production demands, allowing for the preparation of commercial scale quantities of betulinic acid, as well as meeting safety and environmental constraints. Moreover, the process does not require any preliminary purification of the betulin (1) starting material. Crude betulin (1) can be isolated from white birch bark in significant quantities by extraction processes well known in the art. Such isolated crude betulin (1) is amenable to the electrochemical oxidation process as described in the present invention. In the known literature procedures, the extracted betulin (1) must be purified following a series of recrystallization steps prior to being used in the preparation of betulinic acid (3). In an embodiment of the present invention, betulin having a purity of about 65% was used to prepare betulinic acid (3). Additionally, the process of the present invention does not require any prior protection of the hydroxyl groups at C3 and C28 of betulin (1).

[0029] As illustrated in Scheme 1, betulin (1) dissolved in glacial acetic acid, is electrochemically oxidized to the corresponding aldehyde (2) using TEMPO. The TEMPO reagent is continuously oxidized in situ in the acidic reaction medium. The aldehyde (2) is optionally isolated and purified either by elution over a silica column or more simply by crystallisation. Alternatively, the aldehyde (2) is precipitated from the reaction mixture by the addition of water thereto, followed by washing with water. Taking-up the crude aldehyde (2) in refluxing chloroform comprising activated carbon provides purified aldehyde (2). The aldehyde (2) can be further purified either by crystallisation or elution over silica. In an embodiment of the present invention, the aldehyde (2) is isolated and used without prior purification in the conversion to betulinic acid (3).

[0030] The further oxidation of the aldehyde (2) to betulinic acid

(3) is advantageously achieved using a mild oxidizing agent while in the presence of a scavenger. In an embodiment of the present invention, the oxidation is advantageously achieved using a mild oxidant such as sodium chlorite dissolved f-butanol. A variety of mild oxidizing agents are known in the art, and are within the capacity of a skilled technician. Such mild oxidizing agents should selectively oxidize the aldehyde to the corresponding carboxylic acid while remaining inactive with respect to the secondary alcohol at C3 and the olefin at C29-C30. In light of the present invention it is within the skill of a skilled technician to determine what additional oxidants are appropriate.

[0031] The oxidation of the aldehyde (2) to betulinic acid (3) is essentially quantitative. The acid (3) is readily extracted from the reaction mixture using a solvent such as chloroform and is purified by crystallization from the same solvent. The acid (3) can be isolated as the free acid, or can be isolated as a salt of the acid. Alternatively, the crude betulinic acid is purified by neutralisation in an alcohol solution, redissolved in acetic acid and re-precipitated by water dilution. In light of the present invention it is within the skill of a skilled technician to determine what additional solvents are appropriate

[0032] The electrochemical oxidation reaction can be carried out employing any suitable TEMPO reagent. Suitable TEMPO reagents include TEMPO (i.e. 2,2,6,6-Tetramethyl-i-piperidinyloxy) (A), 4-hydroxy-2,2,6,6-

tetramethyl-1-piperidinyloxy (B), 4-methoxy-2,2,6,6-tetramethyl-1-piperidinyloxy (C), and 4-amino-4-carboxylic acid-2,2,6,6-tetramethyl-1-piperidinyloxy (D). In light of the present invention it is within the skill of a skilled technician to determine what additional TEMPO reagents are appropriate. Structures for these reagents are provided hereinbelow in Scheme 2.

Scheme 2

[0033] The TEMPO reagent is electrochemically oxidized in situ in the absence of oxygen. It was observed that reaction yields of aldehyde, as well as reaction speeds, could be enhanced when the electrochemical oxidation is carried in the presence of transition metals. Suitable metals include Cu, Mn and Co. In light of the present invention it is within the skill of a skilled technician to determine what additional metals are appropriate.

[0034] The electrochemical generation of active TEMPO is carried out in a two-compartment cell equipped with a fritted glass separation. In an embodiment of the present invention the cathode is composed of platinum and the anode is composed of vitreous carbon. Additionally, a saturated calomel reference electrode varying from about +1.0 V to about +2.0 V is used for optimal performances. Alternatively, the anode is composed of an essentially non-oxidizeable metal, non-limiting examples of which are noble metals such as silver, gold, iridium, osmium, palladium, ruthenium, rhodium

and platinum. In light of the present invention it is within the skill of a skilled technician to determine what other electrode systems are appropriate.

[0035] The electrochemical oxidation process of the present invention can be carried but in any suitable solvent and at any suitable temperature that allows for the oxidation of the primary alcohol (C-28) to the corresponding aldehyde. Specifically, the oxidation can be carried out at temperatures ranging from about 2O ⁰ C to about 6O ⁰ C. Particular solvents for use in the present invention will typically dissolve both betulin (1) and the corresponding aldehyde (2), and will not interfere with the oxidation process. Moreover, such solvents will typically be acidic in nature, generating a pH ranging from about 0.0 to about 5.0, and more particularly from about 1.5 to about 2.5. Yet moreover, such solvents must possess good electrical conductivity. The electrical conductivity may be further enhanced by the addition of electrolytes, non-limiting examples of which are ammonium acetate, sodium acetate and potassium acetate. In light of the present invention it is within the skill of a skilled technician to determine what other electrolytes are appropriate.

[0036] Preparation ofbetulinic acid (3). To a solution of betulin (1)

(25 mg; 0.0564 mmol) in glacial acetic acid (30 ml) also comprising ammonium hydroxide (1.7 g), was added 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO; 195 mg; 1.25 mmol). The TEMPO reagent is electrochemically oxidized in situ at 5O ⁰ C using a two-compartment electrolytic cell equipped with a fritted glass separation. Specifically, the betulin (1) solution is placed in the anodic compartment (comprising a vitreous carbon (vc) anode) of the two- compartment electrolytic cell. The cathodic compartment (comprising a platinum cathode) of the two-compartment electrolytic cell is filled with a glacial acetic acid/ ammonium hydroxide solution. A saturated calomel

reference electrode varying from about +1.0 V to about +2.0 V is used for optimal performances. A potential of one (1) volt is then applied to the anode. The anodic solution is sampled every 30 minutes and is analyzed by LC/MS. Following a reaction period of about 3 hours, betulin (1) is no longer detected in the reaction mixture (about 200 to 300 coulombs have been spent in the system). The content of the anodic compartment is transferred into a beaker and treated with water (200 ml), resulting in the precipitation of the crude aldehyde (2). The crude aldehyde (2) is subsequently filtered and rinsed with water (3 washings). The acidic aqueous phase retains any unreacted TEMPO reagent. The filtrate is subsequently taken-up in methanol. The organic solution is dried using sodium sulfate, filtered and finally the methanol was removed under vacuum. The crude aldehyde (2) is purified by elution over a silica gel column using a minimal volume of chloroform to provide pure aldehyde (2) in 70% yield (purity in excess of 98% as determined by NMR experiments). The aldehyde (2) is thus readily prepared via the electrochemical oxidation of betulin (1), without prior protection of the hydroxyl groups at C3 and C28 and without the risk of transannular reactions with the olefin at C29-C30 and/or isomerization thereof.

[0037] The further oxidation of the aldehyde (2) to betulinic acid

(3) is essentially quantitative and is advantageously achieved using a mild oxidizing agent. The oxidation is advantageously carried using a mild oxidant such as sodium chlorite dissolved f-butanol as described by BaI et al. (Tetrahedron 39, 2091, 1981). This procedure selectively oxidizes the aldehyde to the corresponding carboxylic acid while remaining ^" inactive with respect to the secondary alcohol at C3 and the olefin at C29-C30. The crude acid (3) is readily taken up in an alcohol such as ethanol or methanol and converted into its sodium salt using an excess sodium hydroxide. The insoluble sodium salt is filtered off and taken up in glacial acetic acid. The addition of water to the acetic acid solution precipitates the betulinic acid (3)

which is subsequently filtered. The purified acid, following washing with water and vacuum drying has a purity in excess of 95% as determined by HPLC analysis.

[0038] The yield of aldehyde, depending on the reaction conditions (Ae. type of TEMPO reagent, temperature, presence or not of metals, type of anode, voltage, electrolyte concentration and betulin concentration) is reported herein below in Table 1.

[0039] It is to be understood that the invention is not limited in its application to the details of construction and parts as described hereinabove. The invention is capable of other embodiments and of being practiced in various ways. It is also understood that the phraseology or terminology used herein is for the purpose of description and not limitation. Hence, although the present invention has been described hereinabove by way of illustrative embodiments thereof, it can be modified, without departing from the spirit, scope and nature of the subject invention as defined in the appended claims.

Table 1