The invention relates to catalytic, environmentally benign oxidation of betulin leading to synthesis of betulonic and betulinic acid.

State of the art

Betulin has the structure 1 , shown below, and is a naturally occurring penta- cyclic terpene alcohol of lupane family, also known as betulinol and lup- 20(29)-ene-3 ,28-diol. Betulin is found in the bark of some tree species, par- ticularly in the birch (Betula sp.) bark at best amounts up to 40% of the birch outer bark dry weight. In addition to betulin, also minor amounts of betulin derivatives are obtained from tree bark. There are known methods mainly based on extraction for isolation of betulin from bark material.

1

In some applications, poor solubility of betulin causes problems with respect to use and formulation, and accordingly, betulin is converted to its derivatives to improve solubility as well as bioactivity. In recent years it has been found, that especially betulinic acid and betulonic acid seem to possess high bioativi- ties in several applications. This has caused the need to develop feasible and environmental benign catalytic methods for oxidation of betulin to betulinic acid and betulonic acid. In the production of said derivatives, reactivities of the functional groups of betulin, that is, primary and secondary hydroxy groups and the double bond are typically utilized.

Suitability of betulin and the derivatives thereof for medical and cosmetic ap- plications and for industrial chemical applications is known to some extent. Use of betulinic acid in cosmetic applications such as promoters of growth and as components in skin creams is known from WO 0003749. The publication of WO 0174327 discloses the use of betulinic acid in sun creams for prevention of detrimental effects of the UV-light. The publication of WO 07141389 dis- closes the use of betulonic acid in several cosmetic applications and in WO 07141391 is described the use of betulonic acid and some betulin derivatives as antiprotozoal agent. The publication of WO 07141383 discloses the use of betulinic and betulonic acid as antifeedant agent for best management.

Betulinic acid having the structure of 3 shown in the reaction scheme A below may be isolated e.g. from birch (Betula sp.) bark or cork of cork oak (Quercus suber L.) by extraction, and further may be produced by several methods mainly based on direct oxidation of the betulin or birch bark material. The reaction scheme A shows the direct oxidation of the betulin 1 according to US 6,280,778 as Jones oxidation in the presence of chromium(VI) oxide catalyst to give betulonic acid 2, followed by the selective reduction of betulonic acid 2 thus obtained with sodium hydroxide to give betulinic acid 3.

The reaction scheme A:

The problem with this method is that chromium(VI) oxide catalyst is carcinogenic and the reaction cumulates also large amounts of waste. The alternative process for the production of betulinic acid is disclosed in US 5,804,575, comprising an oxidation step where 3-beta-hydroxyl of betulin is protected by acetylation. Isomerization and oxidation of secondary hydroxyl group of betulin is thus prevented. However, protecting the 3-beta-hydroxyl brings a new process stage for this method, since the protective group has to be removed for synthesizing free betulinic acid.

The objects of the invention

The object of the present invention is to get rid of the drawback of the above mentioned prior art:

The first object is to develop environmentally benign method for oxidation bet- ulin to betulinic and/or betulonic acid.

The second object is to develop an industrial-scale oxidation process for oxidation of betulin into betulinic and/or betulonic acid with a good yield and low costs. The third object is to develop an oxidation process of betulin into betulinic and/or betulonic acid in which the product is ready-to-use without excessive purification also for medical and cosmetic applications.

The fourth object is to develop a synthesizing method for betulinic and/or betulonic acid which would require as few process stages as possible.

Description of the invention

The above mentioned objects can be achieved by the method according to claim 1 .

The claimed method relates to preparation of betulonic acid and/or betulinic acid from betulin wherein the method comprises a step of oxidizing betulin first to betulonic aldehyde or betulinic aldehyde with Pd(ll)-catalyst catalyzed oxidation process, in the presence of dioxygen.

Dioxygen means here O ₂ which is present at atmosphere or O ₂ , which has about the same activity as O ₂ present in atmosphere. The general idea behind the invention is to first oxidize betulin in the presence of catalytic amount of palladium catalyst and dioxygen to betulonic aldehyde and possibly also to betulinic aldehyde and then to further oxidize betulonic aldehyde into betulonic acid and further to reduce betulonic acid into betulinic acid. The oxidation of betulin to betulonic aldehyde is a homogeneous palla- dium catalysis process. The palladium catalyst is used in catalytic amount and the palladium-moiety in palladium catalyst is reduced into palladium(O) during the oxidation of betulin to betulonic aldehyde or betulinic aldehyde. Palladium catalyst is regenerated by re-oxidising of palladium(0)-moiety into palladium^ l)-moiety. Additionally, to our surprise, we have found that betulin can be oxidized with Pd(ll) catalyst and air to betulonic aldehyde or betulinic aldehyde and further to betulonic acid or betulinic acid simultaneously without purification, by using additional oxidant in reactions step 2 or 5 of scheme 1 below.

The preparation of betulonic acid and/or betulinic acid from betulin according to above mentioned method, wherein the first step is done by catalytic oxida- tion by using palladium(ll) catalyst, has a high conversion rate and high selectivity.

Due to a highly selective conversion and the possibility to regenerate palladium catalyst the reaction is environmentally benign and prepared betulinic acid or betulonic acid is pure enough to be used for instance in pharmaceutical or cosmetic applications.

According to one embodiment of the invention the betulin is first oxidized with a Pd(ll) catalyst and air to betulonic aldehyde or betulinic aldehyde and the obtained betulonic aldehyde or betulinic aldehyde is oxidized without purifica- tion further to betulonic acid or betulinic acid by using additional oxidation agent. In practice the catalytic oxidation of betulin and oxidation of betulonic aldehyde or betulinic aldehyde to corresponding acids happen simultaneously, in one pot (at the same vessel). Betulonic acid can be reduced to betulinic acid using known reducing methods, for example, by using sodium borohy- dride (NaBH ) -reduction.

Above mentioned so called "one one pot oxidation method" comprises following process stages:

(c1 ) oxidizing betulin to betulonic aldehyde in presence of a catalyst and an oxidant, (c2) oxidizing said betulonic aldehyde to betulonic acid with an oxidizing system,

(c3) reducing said betulonic acid to betulinic acid by using reducing agent, or

(d1 ) oxidizing betulin to betulinic aldehyde in presence of a catalyst and an oxidant, (d2) oxidizing said betulinic aldehyde to betulinic acid with an oxidizing system, wherein

- in stage (c1 ) and (d1 ) catalytic oxidation of betulin is performed by a Pd(ll)- catalyst in presence of dioxygen and - catalytic oxidation reaction of betulin to betulonic aldehyde or betulin to betulinic aldehyde in stages (c1 ) or (d1 ) and oxidizing of betulonic aldehyde to betulonic acid (stage c2) or betulinic aldehyde to betulinic acid (stage d2) are performed simultaneously, in the same reaction vessel. Because in this "one pot oxidation method" obtained betulonic aldehyde or betulinic aldehyde is not isolated but used as such, this method has an advantage of being effective and economic method for preparation betulonic or betulinic acid from betulin. This embodiment of the inventive method may be suitable for a large scale conversion of betulin to betulinic acid or betulonic acid.

Neither above mentioned general method for synthesizing betulinic acid or betulonic acid nor above mentioned so called "one pot method", needs functional groups of betulin to be protected with protecting groups. Therefore neither cleavage of the protection groups is needed. Thus, the number of reac- tion steps is reduced.

Homogeneous palladium catalysis process means herein, that the only catalyst is palladium(ll) catalyst without presence of any co-catalyst.

Preparation of betulinic acid and betulonic acid from betulin proceeds mainly according to following reaction scheme 1 .

Reaction scheme 1

The catalytic oxidation of betulin to betulonic aldehyde proceeds via route 1 (to synthesize mainly betulonic aldehyde) or via route 4 (to synthesize mainly betulinic aldehyde). The catalytic oxidation of betulin to betulonic aldehyde or betulinic aldehyde is the first stage when preparing betulinic acid and/or betulonic acid from betulin.

During the catalytic oxidation of betulin to betulonic aldehyde via route 1 it can also be obtained some amount of betulinic aldehyde, which can be oxidized directly to betulinic acid by reaction 5 (via route 5) of reaction scheme 1 . Oxi- dation of betulonic aldehyde into betulonic acid proceeds via route 2 by using appropriate oxidizing system. Betulonic acid is then reduced to betulinic acid by the reaction 3 (via route 3).

The catalytic oxidation of betulin can also proceed via route 4, whereby betulinic aldehyde is obtained. Betulinic aldehyde can be directly oxidized via route 5 to betulinic acid using the same or different oxidation system than in above mentioned oxidation system used in oxidation of betulonic aldehyde into betulonic acid by reaction 2.

The catalytic oxidation reaction from betulin to betulonic aldehyde by reaction 1 or 4 uses molecular dioxygen as the sole oxidant. Primary source of oxygen is air.

In the catalytic oxidation process of betulin using reaction 1 or 4, there can be used a variety of alternative palladium(ll) catalyst. Palladium(ll) catalyst is of the general formula (I):

LnPd(ll) (I) In the palladium catalyst of formula I: n is any integer from 0 to 2, in a case n is 0 ligand L is absent;

L is any suitable ligand containing at least one nitrogen atom (N-ligand), which can be used for chelating palladium metal in palladium(ll) catalyst. Suitable ligands can be found also from references Karimi, B and Zamani, A., Recent advances in the homogeneous palladium-catalyzed aerobioc oxidation of alcohols, J.lran.Chem. Soc, Vol 5, Suppl., October 2008, pp. S1 - S20 or Gli- gorich, M and Sigman, M., recent advances and challenges of palladium(ll)- catalyzed oxidation reactions with molecular oxygen as the sole oxidant, Chem.Commun., 2009, 3854-3867. Especially ligand may be selected from the group consisting of aromatic and aliphatic mono- and bidentate amines and mono- and bidentate heteroarenes. These may chelate palladium and form complexes with palladium(ll) compounds. Ligand L includes bidentate pyridine and bidentate triethylamine and monodentate hydrotalcite. The term "amine" refers herein to an amine group, which is originating to an ammonia group in which one, two or three hydrogen(s) may be substituted with a substituent. The substituent of an amine group is an aryl group in case of aromatic amines and an aliphatic alkyl group in case of aliphatic amines.

Aliphatic alkyl group means herein a substituent having a branched or un- branched saturated hydrocarbon chain from 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms.

Aryl group may be an unsaturated aromatic carbocyclic group or also a het- eroaryl group.

The unsaturated aromatic carbocyclic group has from 3 to 12 carbon atoms and has a single ring or multiple condensed rings, wherein at least one ring is aromatic, whereby said unsaturated aromatic carbocyclic group can be un- substituted or substituted. Preferably unsaturated aromatic carbocyclic group is phenyl. Heteroarene means herein a compound having monocyclic, bicyclic, or tricyclic ring system and containing one, two, or three aromatic rings and containing at least one nitrogen atom in aromatic ring, and which can be unsubstitut- ed or substituted. Preferably heteroarene is pyridine. Heteroaryl is a functional group, which is originating to a heteroarene.

Each symbol R1 or R2 mean a functional group selected independently from the group consisting of amines, aryls, carboxylates, esters, ketones, halogens and nitrates, wherein

The aryl group have the same definition as above, preferably aryl group means phenyl;

Carboxylate means an anion originating from carboxylic acid and which forms salt with palladium(ll) cation, more preferable carboxylate is originating from a lower carboxylic acid such as acetic acid;

Ester means an alkylester, more preferable loweralkylester; Ketone means an alkyl ketone, more preferable loweralkyl ketone or low- eralkyldiketone, preferred loweralkyldiketone is actetylacetonate;

Preferred palladium(ll) catalyst of general formula (I) include following catalysts in a case ligand is not present (n=0): palladium(ll) acetate Pd(OAc)2, phenylpalladium(ll) acetate Pd(C6H ₅ )(O2CCH ₃ ) , palladium(ll) acetylacetonate Pd(acac) ₂ , palladium(ll) nitrate Pd(NO ₃ ) ₂ .

Preferred palladium (II) catalysts of general formula (I), in which ligands are present (n=1 or n= 2) are those, wherein palladium(ll) catalyst is of general formula (IA).

L _n Pd(ll)R ₂ (IA) Whereby the ligand L has the same definition as above and R have the same definition as R1 or R2 above.

The palladium(ll) catalyst with formula (IA) comprises following preferable Pd(ll) complexes: palladium(ll)-hydrotalcite-complexes, palladium(ll) com- pound - pyridine -complexes and palladium(ll) compound-triethylamine (TEA)-complexes. These include following important palladium(ll) catalysts:

(py) ₂ Pd(OAc) ₂ , (py) ₂ Pd(acac) ₂ , (py) ₂ Pd(NO ₃ ) ₂ , (NEt ₃ ) ₂ Pd(OAc) ₂ ,

(NEt ₃ ) ₃ Pd(OAc) ₂ , wherein py means pyridine, Et means ethyl.

The first stage in reaction scheme 1 , catalytic oxidation of betulin by palladi- um(ll) catalyst and air to betulonic aldehyde or betulinic aldehyde, proceeds according to reaction step 1 or 4. It is performed in any suitable inert organic solvent in which at least betulin dissolves.

Inert solvent means herein that the solvent will not react essentially with betulin or its derivatives or reactants used in the methods according to reaction scheme 1 (for example oxidants).

Suitable inert solvents can be selected from the group comprising saturated or unsaturated aliphatic hydrocarbons, branched or unbranched aliphatic hydrocarbons, halogenated aliphatic hydrocarbons, aromatic hydrocarbons, fluorous solvents, chlorinated solvents, ethers, esters, ketones, H ₂ O, or mixtures thereof.

Preferable inert solvent is selected from the group consisting of toluene, xylene, decalin, DMSO, fluorous solvent, perfluoroalkane e.g. perfluorodecaline (can also improve O ₂ solubility), THF, CH ₂ CI ₂ , H ₂ O or mixtures thereof.

Most preferable inert solvents for this system are selected from the group consisting of aliphatic and aromatic hydrocarbons (eg toluene, xylenes, decalin), fluorous- and perfluoroalkanes (e.g. perfluorodecaline), chlorinated solvents (e.g. CH ₂ CI ₂ ) and ethers (eg. tetrahydrofuran).

The second stage for preparation of betulonic acid and/or betulinic acid from betulin proceeds via route 2 or 5 in reaction scheme 1 . Betulonic aldehyde from reaction step 1 (via route 1 ) is oxidized into betulonic acid by reaction step 2 (via route 2) using suitable oxidizing system. Betulinic aldehyde from reaction 4 (via route 4) is oxidized into betulinic acid via route 5 using suitable oxidizing system. If this second reaction stage is done immediately after or simultaneously to first reaction stage (after reaction step 1 or 4), the solvent used therein is preferably such that betulin and betulonic aldehyde and/or betulinic aldehyde will dissolve therein. In this case preferable solvent(s) are the same as above. If the second reaction stage (reaction step 2 or 5) is performed independently from the first reaction stage (reaction step 1 or 4), for instance in a separate vessel, the solvent may be any suitable organic solvent in which betulonic aldehyde or betulinic aldehyde will dissolve and which do not react with reac- tants.

The third stage of preparation of betulonic acid from betulin proceeds via route 3 (with reaction step 3) in reaction scheme 1 . In this reaction step 3, betulonic acid is reduced into betulinic acid by using any suitable reduction agent such as NaBH , LiBH or KBH . The reduction of betulonic acid to betulinic acid according to reaction step 3 can be performed also by using common methods known from the prior art. There are many possible alternatives to be used as oxidizing systems in reaction steps 2 or 5. Such an oxidizing system includes an appropriate oxidizing agent and a possible buffer and possible auxiliary agents.

The used oxidizing agents can preferable be selected from the group consisting of: O2, sodium hypochlorite (NaOCI), sodium chlorite (NaCIO2), potassium chlorite (KCIO2) _, potassium hypochlorite (KOCI), peroxides such as hydrogen peroxide (H2O2), hypervalent iodine reagents (eg. 2-iodoxybenzoic acid (IBX), 2-iodoxybenzenesulfonic (IBS)), Oxone (2KHSO ₅ , KHSO ₄ , K ₂ SO ₄ ) and phthalimides such as /V-hydroxyphthalimide and mixtures thereof. As to other possible oxidizing agents, we refer here to literature of this field. In a case betulinic aldehyde is synthesized by a catalytic oxidation of betulin (via route 4 in reaction scheme 1 ), this can be conversed directly into betulinic acid by oxidizing betulinic aldehyde.

In combination with above mentioned oxidizing agents it is often useful to use suitable buffer in an oxidation system. These buffers may be selected from the group consisting of:

KH ₂ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO _{4 ,} CHES, MES (A/-morpholino)ethanesulfonic acid, TAPS([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine (bis(2-hydroxyethyl)glycine), Tris (tris(hydroxymethyl)methylamine), Tricine (/V-t s(hydroxymethyl)methylglycine), TAPSO (3-[/V- Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid), HEPES (4- 2-hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2- {[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(/V- morpholino)propanesulfonic acid) PIPES (piperazine-/V,/V'-bis(2- ethanesulfonic acid), Cacodylate (dimethylarsinic acid), SSC (saline sodium citrate) and MES (2-(/V-morpholino)ethanesulfonic acid).

In the following are given pH-ranges for several above mentioned buffers KH ₂ PO _{4 ,} pH 5.8 - 8, NaH ₂ PO _{4 ,} pH 6 - 7.5 and CHES, pH 8.6-10.

In the following are given pK _a at 20°C ApK _a /°C for several above mentioned buffers : MES 6.15 -0.01 1 , ADA 6.6 -0.01 1 , PIPES 6.8 -0.0085, ACES 6.9 - 0.020, Cholamine chloride 7.1 -0.027, BES 7.15 -0.016, TES 7.5 -0.020,

HEPES 7.55 -0.014, Acetamidoglycine 7.7 - , Tricine 8.15 -0.021 , Glycinamide 8.2 -0.029, Bicine 8.35 -0.018.

In the following are given pK _a at 25°C, (Buffer Range Temperature Effect) , dpH/dT in (1 /K) ** Mol Weight and Full Compound Name for several above mentioned buffers:

TAPS 8.43 7.7-9.1 -0.018 243.3 3- {[tris(hydroxymethyl)methyl]amino}propanesulfonic acid,

Bicine 8.35 7.6-9.0 -0.018 163.2 A/,/V-bis(2-hydroxyethyl)glycine,

Tris 8.06 7.5-9.0 -0.028 121 .14 tris(hydroxymethyl)methylamine, Tricine 8.05 7.4-8.8 -0.021 179.2 A/-tns(hydroxymethyl)methylglycine,

TAPSO 7.635 7.0-8.2 259.3 3-[/V-Tris(hydroxymethyl)methylamino]-2- hydroxypropanesulfonic Acid,

HEPES 7.55 6.8-8.2 -0.014 238.3 4-2-hydroxyethyl-1 - piperazineethanesulfonic acid,

TES 7.40 6.8-8.2 -0.020 229.20 2- {[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid,

MOPS 7.20 6.5-7.9 -0.015 209.3 3-(/V-morpholino)propanesulfonic acid ,

PIPES 6.76 6.1 -7.5 -0.008 302.4 piperazine-/V,/V'-bis(2-ethanesulfonic acid), Cacodylate 6.27 5.0-7.4 138.0 dimethylarsinic acid,

SSC 7.0 6.5-7.5 189.1 saline sodium citrate and

MES 6.15 5.5-6.7 -0.01 1 195.2 2-(/V-morpholino)ethanesulfonic acid.

Above mentioned auxiliary agents for oxidation systems, using routes 2 or 5 in reaction scheme 1 (oxidation of betulonic aldehyde or betulinic aldehyde to corresponding acid), include possible palladium scavenging. These scavenging agents are used especially when betulonic acid is prepared so that reaction steps of 1 and 2 are performed simultaneously or betulinic acid is prepared so, that reaction steps of 4 and 5, in reaction scheme 1 , are performed simultaneously (this is so called one pot oxidation method). When betulinic acid or betulonic acid is synthesized using above described one pot method, betulonic aldehyde or betulinic aldehyde which have been obtained from catalytic oxidation of betulin according to reaction step of 1 or 4 are used as such, that means, without isolating them, before using in subsequent reaction steps 2 and 5. Regenerating palladium(ll) catalyst enhances significantly overall process efficiency of the process according to reaction scheme 1 . Regenerating catalyst is especially important in large scale production. There are many known palladium scavengers, which can be used after catalytic oxidation process via route 1 or 4 for scavenging used palladium(O) compounds. Palladium scavengers which can be used for scavenging used palladium(O) compounds are known from the literature of this field. These suitable palladium(O) scavengers include free, polymer bound, silica supported or functionalized silica scaven- gers. Suitable functional groups in palladium(O) scavengers includes thiol, thiourea, cysteine, dimercaptotriazine (DMT), amine, diamine, triamine, imidazole, triaminetetraacetic Acid (TAAcOH), triaminetetraacetate sodium salt (TAAcONa), for example, but not limited to, 3-(diethylenetriamino)propyl, DL- dithiothreitol, ethylenediaminetriacetic acid acetamide, Ν,Ν,Ν'- trimethylethylenediamine, 6-thionicotinamide, bis-[(diphenylphosphanyl)- methyl]amine, 2-mercaptoethylamine, bipyridine, 3-(ethylenediamino)propyl, 3-mercaptopropyl, 2-(4-(ethylenediamino)benzyl)ethyl, 3-(1 -thioureido)propyl, /V-acetyl-L-cysteine, mercaptophenylaminobut-2-enoate ester, trimercaptotria- zine, triamine tetraacetate and triamine tetracetate sodium salt. In one embodiment of the invention palladium scavenger is /V-acetyl-L- cysteine (used 1 1 -fold excess) or QuadraPure MPA (mercaptophenylamino- but-2-enoate ester).

Above mentioned auxiliary agents for oxidation can also include oxidation preventative agents which will prevent undesirable oxidation of betulin or its derivatives (betulonic aldehyde or betulinic aldehyde).

Oxidation preventative agents are well known in this field and they are usually compounds, which will itself become reversible or irreversible oxidized. A typical oxidation preventative agents include olefins such as 2-methylbutene or more electron-rich reaction partner as sacrificial electrophile scavenger (e.g. resorcinol, 1 ,3-dihydroxybenzene).

Pd catalyst may be bound on a conventional catalyst support such as zeolite, alumina, carbon, or the like. Molecular sieves, preferably 3 A MS, can be optionally used to enhance the rate of the Pd(OAc)2-pyridine catalysed oxidation and to improve catalyst stability by decrasing precipitation of Pd(0).

In connection with the present invention, various additives and measurements can be useful to improve process efficiency. These additives include phase transfer catalysts, if a heterogeneous reaction mixture is used in any of the routes 1 , 2, 3, 4 or 5 in reaction scheme 1 . Phase transfer catalysts are used in heterogeneous reaction mixtures to facilitate movement of a reactant from one phase to another. Phase transfer catalysts are also known to sometimes accelerate reaction rates and minimize solvent waste, since the reactions tend to be heterogeneous. Herein used phase transfer catalyst may be selected from the group consisting of: ammonium salts, heterocyclic ammonium salts and phosphonium salts. Preferable phase transfer catalysts can be selected from the following group comprising of: tert-butanol, 1 ,2,3- trimethylimidazolium methyl sulfate, 1 -butyl-2,3-dimethylimidazolium chloride, 1 -butyl-2,3-dimethylimidazolium hexafluorophosphate, 1 -butyl-2,3- dimethylimidazolium tetrafluoroborate, 1 -ethyl-2,3-dimethylimidazolium ethyl sulphate, 1 -methylimidazolium hydrogen sulfate, methyltrioctylammonium thi- osalicylate, tetrabutylammonium heptadecafluorooctanesulfonate, tetrabu- tylammonium nonafluorobutanesulfonate, tetraphenylphosphonium chloride, tributylmethylammonium methyl sulphate, trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)phosphinate, trihexyltetradecylphosphonium bromide, trihexyltetradecylphosphonium chloride, trihexyltetradecylphosphonium dicy- anamide, hexadecyltrimethylammonium bromide, tetrabutylammonium hy- droxide, tetraethylammonium chloride hydrate, tributylhexadecylphosphonium bromide, (-)-/V,/V-dimethylephedrinium bromide, (-)-/V,/V-dimethylephedhnium bromide, (-)-/V-dodecyl-/V-methylephedrinium bromide, benzyltributylammoni- um chloride, hexyltributylphosphonium bromide, methyltrial kyl(C8- C10)ammonium chloride, hexyltributylphosphonium bromide, N- benzylcinchoninium chloride, tetrahexylammonium hydrogensulfate, tetraoctylammonium hydroxide, (1 R,2S)-(-)-/V-methylephedrine, 3-bromo-2- methylpropene, A/-(diphenylmethylene)aminoacetonitrile,

bis(triphenylphosphoranylidene)ammonium chloride, tricaprylylmethylammo- nium chloride, riethylbenzylammonium chloride (TEBAC), tetrabutylammoni- um bromide (TBAB ), tetraethylammonium bromide (TEAB ), tetrabutylammo- nium hydrogensulfate, ethyltriphenylphosphonium iodide, methyltri- phenylphosphonium iodide, ethyltriphenylphosphonium bromide, methyltri- phenylphosphonium bromide, tetramethylammonium hydroxide, methyl trioctyl ammonium hydrogen sulfate (MTOAHS) and dimethyl dioctadecyl ammonium chloride (Arquad 2HT).

Especially following oxidizing systems (oxidizing agent + buffer + possible auxiliary agents + possible additive) has proven to be useful herein: NaCIO ₂ + NaH ₂ PO ₄ + 2-methylbutene + ferf-butanol,

NaCIO2 + NaH ₂ PO ₄ + 2-methylbutene + dimethyldioctadecylammonium chloride (arquad 2HT)

NaCIO2 + NaH ₂ PO ₄ + 2-methylbutene + methyl trioctyl ammonium hydrogen sulfate (MTOAHS) NHPI (N-hydroxyphthalimide) + air and

QuadraPure MPA (mercaptophenylaminobut-2-enoate ester) + air.

These all are suitable for a method, in which routes 1 and 2 or 4 and 5 are performed simultaneously using so called "one pot oxidation method".

Measurements which will facilitate oxygen dissolving into reaction mixture may also be of the advantage in oxidation reactions via route 1 , 2, 4 or 5. Such measurements include for example vigorous stirring of reaction vessel and performing oxidation process under overpressure (up to about 100 atm).

Examples

General procedure for oxidation of betulin with Pd(ll) catalyst includes loading of reactor with inert solvent and connecting air flow and stirring to the system and adding then Pd-catalyst (2-10 mol-% from the amount of betulin) and N- ligand (10-2000 mol-%) and heating the reactor to 50-120 °C. Then betulin is added in small portions within 1 -2 hrs and reaction is allowed to proceed for 5- 15 hrs at 50-120 °C. Then Pd recycling agent, eg. /V-acetyl-L-cysteine (1 1 -fold excess) or QuadraPure MPA (mercaptophenylaminobut-2-enoate ester) is added and adsorbed Pd is allowed to precipitate while cooling the reaction to 5-25 °C and precipitate is removed by filtration. The filtrate contains betulonic aldehyde, crude yield 83-92% based on betulin.

According to this invention betulonic aldehyde can be further oxidized to betulonic acid by adding additional oxidant to filtrate containing betulonic aldehyde and stirring the reaction typically at 5-50 °C for 1 - 25 hrs, depending the type of additional oxidant system. The overall isolated yield of 48-76 % for betu- Ionic acid (purity >90%) based on betulin is obtained.

Examples concerning to step 1 for catalytic oxidation of betulin to betulonic aldehyde

8

This reaction step was performed using palladium (II) catalyst containing Pd(OAc)2 + pyridine or catalyst comprising Pd-hydrotalcite and air as an oxi- dizing agent. Pd(OAc)2 = Palladium(ll) acetate;

Example 1.1

Pd(OAc)2+pyridine+air (W-acetyl-L-cysteine as Pd scavenger)

Betulonic aldehyde. Toluene (500 ml) was added to a 1 I flask equipped with condenser, mechanical stirring and tubing for air bubbling. Palladium(ll) ace- tate (1 .2 g, 6 w-%) and pyridine (5 ml) were added. Temperature was adjusted to 80 °C, air flow to the reaction mixture was adjusted to 1000 ml/min and stirring to 500 rpm. Betulin (20.0 g) was added in small proportions during 1 h. Oxidation reaction was monitored by GC and after 7 hour reaction was fin- ished. /V-acetyl-L-cysteine (8.0 g) was added to scavenge Pd(ll). Reaction mixture was cooled down and stirred under N ₂ flow for 1 h. Residue was filtered with vacuum and washed with toluene (3 20 ml). Obtained filtrate (including betulonic aldehyde 88%) was subsequently used in the next reaction step (2.1 , 2.3 and 2.3) in one pot without further purification. XRF-analysis of dried precipitate: 5.7% Pd (resulting 85% Pd recovery).

GC analysis of filtrate: Betulin < 0.1 %, betulonic aldehyde 88%, betulinic aldehyde 2%, others 10%.

¹ H NMR (300 MHz, CDCI ₃ ): 0.92 (s, 3H), 0.94 (s, 3H), 0.97 (s, 3H), 1 .01 (s, 3H), 1 .06 (s, 3H), 1 .69 (s, 3H), 2.43 (m, 2H), 2.86 (m, 1 H), 4.62 (s, 1 H), 4.75 (s, 1 H), 9.66 (s, 1 H)

¹³ C NMR (75 MHz, CDCI ₃ ): 14.1 , 15.6, 15.9, 18.9, 19.5, 21 .0, 21 .2, 25.4, 26.5, 28.7, 29.1 , 29.8, 33.1 , 33.5, 34.1 , 36.8, 38.7, 39.6, 40.7, 42.5, 47.3, 47.4, 47.9, 49.7, 54.9, 59.2, 1 10.2, 149.6, 206.5, 218.0

Example 1.2

Pd(OAc)2+pyridine+air (No Pd scavenger)

Betulonic aldehyde. Toluene (30 ml) was added to a 100 ml flask equipped with a condenser, mechanical stirrer and tubing for air bubbling. Palladium(ll) acetate (26 mg, 5 w-%) and pyridine (0.1 ml) were added. Temperature was adjusted to 80 °C, air flow to 50 ml/min and stirring to 500 rpm. Betulin (0.50 g) was added. Oxidation reaction was monitored by GC and after 7 hour reaction was finished. Obtained solution (including betulonic aldehyde 88%) was subsequently used in the next reaction step in one pot without further purification. Examples relating to step 2 for preparing betulonic acid

Oxidation of betulonic aldehyde to betulonic acid using various oxida- tion systems

Example 2.1

2-methylbutene+NaCI02+NaH ₂ P0 ₄ + ferf-butanol

Betulonic acid. Terf-butanol (170 ml) and 2-methyl-2-butene (30 ml) were added to the filtrate obtained from example 1 (including 20 g of betulonic aldehyde, purity 85-90%) under N ₂ flow. The oxidation system comprising NaCIO ₂ (17.0 g) and NaH ₂ PO ₄ ^* H ₂ O (25.2 g) in water (200 ml) were added during 0.5 h and stirring was continued at room temperature under N ₂ for 15 h. Organic and aqueous phases were separated and organic phase was washed with water (200 ml). Aqueous NaOH (2.7 g in 40 ml of water) was added to organic phase and stirred for 0.5 h. Half of the solvent was evaporated off under vacuum and residue was stirred at room temperature for 1 h. Precipitated betulonic acid Na-salt was filtered, washed with toluene (4 χ 20 ml) and dried overnight at vacuum to yield betulonic acid Na-salt (16.6 g). Betulonic acid Na-salt was subsequently treated aqueous HCI solution (140 ml water and 60 ml of 10% HCI) and stirred for 2.5 h. Precipitate was filtered and washed with water (5 30 ml), dried in vacuum oven at 60 °C overnight to obtain betulonic acid 14.9 g (73% from betulin).

GC: betulonic acid 92%, betulinic acid 2%, others 6%. ¹ H NMR (300 MHz, CDCI ₃ ): 0.93 (s, 3H), 0.98 (s, 3H), 0.99 (s, 3H), 1 .02 (s, 6H), 1 .07 (s, 3H), 1 .70 (s, 3H), 1 .96 (m, 3H), 2.27 (m, 2H), 2.44 (m, 2H), 3.01 (m, 1 H), 4.62 (s, 1 H), 4.74 (s, 1 H).

¹³ C NMR (75 MHz, CDCI ₃ ) 14.6, 15.8, 15.9, 19.3, 19.6, 21 .0, 21 .3, 25.5, 26.6, 29.6, 30.5, 32.1 , 33.6, 34.1 , 36.9, 37.0, 38.5, 39.6, 40.6, 42.5, 46.9, 47.3, 49.1 , 49.8, 54.9, 56.3, 109.7, 150.3, 181 .9, 218.4

NaCIO2 = Sodium chlorite

NaH ₂ PO ₄ = Sodium dihydrogen phosphate

Example 2.2. 2-methylbutene+NaCI0 ₂ +NaH ₂ P0 ₄ +MTOAHS

Betulonic acid. Methyl trioctyl ammonium hydrogen sulphate (MTOAHS) (40 mg) and 2-methyl-2-butene (1 ml) were added to the filtrate obtained from example 1 .1 (including 1 .0 g of betulonic aldehyde, purity 85-90%) under N ₂ flow. Oxidation system comprising NaCIO2 (0.5 g) and NaH ₂ PO ₄ ^* H ₂ O 0.2 M in water (20 ml) was added and stirring was continued at room temperature, under N ₂ for 22 h. Organic and aqueous phases were separated and organic phase was washed with water (20 ml). Aqueous NaOH 1 .7 M (4 ml) was added to organic phase and stirred for 0.5 h. Precipitated betulonic acid Na-salt was filtered, washed with toluene (10 ml) and dried in a vacuum oven at 80 °C. Betulonic acid Na-salt was subsequently treated with acetic acid (0.4 ml) in MeOH (8 ml) and stirred for 15 min. Water (15 ml) was added and precipitated betulonic acid was filtered and washed with water (4 x 10 ml), dried in the vacuum oven at 80 °C overnight. Isolated yield 0.34 g, GC purity analysis: betulonic acid 88%. Example 2.3.

2-methylbutene+NaCI0 ₂ +NaH ₂ P0 ₄ +TBAHS

Betulonic acid. TBAHS (0.04 g) (tetrabutylammonium hydrogensulphate) and 2-methyl-2-butene (3 ml) were added to the filtrate obtained from example 1 .1 (including 1 .0 g of betulonic aldehyde, purity 85-90%) under N ₂ flow.

NaCIO ₂ (0.8 g) and NaH ₂ PO ₄ *H ₂ O 0.3 M in water (20 ml) were added and stirring was continued at room temperature, under N ₂ for 16 h. GC purity analysis: betulonic aldehyde 32%, betulonic acid 20%, others 48%. Example 2.4.

2-methylbutene+NaCI02+NaH ₂ P0 ₄ +Arquad 2HT

Betulonic acid. Arquad 2HT (0.12 g) (DiaSkyidimethyiammonium acetate) and 2-methyl-2-butene (3 ml) were added to the filtrate obtained from example 1 .1 (including 1 .0 g of betulonic aldehyde, purity 85-90%) under N ₂ flow. Oxidation system NaCIO ₂ (0.8 g) and NaH ₂ PO ₄ *H ₂ O 0.3 M in water (25 ml) was added and stirring was continued at room temperature under N ₂ for 24 h. Organic and aqueous phases were separated and organic phase was washed with water (15 ml). Aqueous NaOH 1 .7 M (4 ml) was added to organic phase and stirred for 1 .5 h. Precipitated betulonic acid Na-salt was filtered, washed with toluene (10 ml) and dried. Betulonic acid Na-salt was subsequently treated with acetic acid (0.4 ml) in MeOH (8 ml) and stirred for 15 min. Water (15 ml) was added and precipitate of betulonic acid was filtered and washed with water (4 x 10 ml), dried in vacuum oven at 80 °C overnight. Isolated yield 0.58 g, GC purity analysis: betulonic acid 83%. Example 2.5.

NHPI+air

Filtrate obtained from example 1 .1 (including 1 .0 g betulonic aldehyde, purity 85-90%) was cooled down to 0-5 °C under air flow of 50 ml/min. Methanol (0.2 ml) and oxidation system comprising N-hydroxyphthalimide (8.5 mg, NHPI) were added. The mixture was stirred at 0-5 °C for 2.5 hours after which NaOH 1 .7 M in water (0.9 ml) was added and the mixture was stirred at 0-5 °C for 1 hour. Formed precipitate betulonic acid Na-salt was filtered and washed with toluene (10 ml). Betulonic acid Na-salt was dissolved in methanol (5 ml) at 50 °C. Acetic acid (0.2 ml) was added and the mixture was stirred at 50 °C for 10 min. Water (20 ml) was added and the mixture was cooled to 5- 10 °C. The precipitate was filtered, washed with water and dried to obtain bet- ulonic acid (0.26 mg). GC purity analysis: 86% betulonic acid.

Example 2.6

MPA+air Reaction mixture (including 0.5 g of betulonic aldehyde, purity 85-90%) obtained from example 1 .1 .1 . was cooled to room temperature and Quadrapure MPA (0.18 g) (Pd-scavenger) was added and reaction mixture was bubbled with air (50 ml/min) and stirred for 18 h. Precipitate was filtered with vacuum and washed with toluene (3 20 ml). Aqueous NaOH 1 .7 M (3 ml) was added and mixture was stirred for 45 min. Precipitated betulonic acid Na-salt was filtered, washed with toluene (10 ml) and dried. Betulonic acid Na-salt was subsequently treated with acetic acid (0.4 ml) in MeOH (8 ml) and stirred for 30 min. Water (15 ml) was added and precipitated betulonic acid was filtered and washed with water (4 x 10 ml), dried in vacuum oven at 80 °C overnight. Isolated yield 0.49 g, GC purity analysis: betulonic acid 87%.

Example relating to step 3 for preparing betulinic acid

Example 3.1. Reduction of betulonic acid to betulinic acid with NaBH

Betulinic acid. To a solution of water (130 ml), NaOH (26 ml, 5%) and 2- propanol (120 ml) was added betulonic acid (13.0 g, purity 94%). NaBH ₄ (1 .2 g) was added and reaction mixture was stirred for 3 hour in ice bath. Alkaline solution was acidified by adding aqueous HCI solution (130 ml, 10%). Precipi- tate betulinic acid was filtered, washed with water (4 ^χ 50 ml) and dried in vacuum oven to yield betulinic acid 12.2 g (94%). GC: Betulinic acid 92%, betulonic acid < 0.1 %, others 8%. To purify betulinic acid (92%) to very high purity (>99%), betulinic acid (92%) (5 g) was dissolved to refluxing ethanol and allowed to crystallize at 4 °C over night. Formed crystals were filtrated and dried in vacuum oven. Yield 82%, GC purity >99%.

¹ H NMR (300 MHz, CDCI ₃ ) 0.75 (s, 3H), 0.82 (s, 3H), 0.93 (s, 3H), 0.96 (s, 6H), 0.97 (s, 3H), 1 .69 (s, 3H), 1 .97 (m, 2H), 2.28 (m, 2H), 3.01 (m, 1 H), 3.19 (dd, J = 5.5, 10.7 Hz, 1 H), 4.60 (s, 1 H), 4.74 (s, 1 H);

¹³ C NMR (75 MHz, DMSO) 14.4, 15.7, 15.8, 16.0, 18.0, 19.0, 20.5, 25.1 , 27.2, 28.1 , 29.2, 30.1 , 31 .7, 34.0, 36.4, 36.7, 37.6, 38.3, 38.5, 40.3, 42.0, 46.6, 48.6, 50.0, 54.9, 55.4, 76.8, 109.7, 150.3, 177.3

NaBH = Sodium borohydride

Example relating to step 4 for preparing betulinic aldehyde Example 4.1

Pd(OAc)2+ triethyl amine (TEA) +air (W-acetyl-L-cysteine as Pd scavenger) Catalytic oxidation of betulin to betulinic aldehyde. Mixture of betulin (2,00 g), Pd(OAc) ₂ (120 mg), TEA triethylamine (500 mg) in THF (120 ml) was bubbled with air flow 500 ml/min with stirring at room temperature for 13 hour. Obtained solution mixture (including betulinic aldehyde 25%) was subsequently used in the next reaction step in one pot without further purification. GC: Betulin 58%, betulinic aldehyde 25%, others 17%. ¹ H NMR (500 MHz, CDCI ₃ ): Aldehyde 45%.

Example relating to steps 1 and 4 for manufacturing mixture of betulinic aldehyde and betulonic aldehyde

Example 5.1

Pd(OAc) ₂ +pyridine+air (MP1 167) (N-acetyl-L-cystine as Pd scavenger)

Betulinic aldehyde and betulonic aldehyde. Toluene (1000 ml) was added to a 2 I flask equipped with a condenser, mechanical stirrer and tubing for air bubbling. Palladium(ll) acetate (2.43 g, 6 w-%) and pyridine (10 ml) were added. Temperature was adjusted to 80 °C, air flow to the reaction mixture was adjusted to 1000 ml/min and stirring to 500 rpm. Betulin (40.5 g) was added in small proportions during 1 h. Oxidation reaction was continued for 1 1 hour. N- acetyl-L-cystine (16.0 g) was added to scavenge Pd(ll). Reaction mixture was cooled down and stirred under N ₂ flow for 1 h. Residue was filtered with vacuum and washed with toluene (3 ^* 40 ml). Obtained filtrate (including betulonic aldehyde and betulinic aldehyde 80% together) was subsequently used in the next reaction step (5) in one pot without further purification; Example 5.2

Formation of Pd(ll)-hydrotalcite. Mixture of Pd(OAc)2 (378 mg), pyridine (333 mg) and hydrotalcite (10.0 g) in toluene (100 ml) was stirred at 80 °C for 1 hour. The mixture was cooled and formed Pd(ll)-hydrotalcite precipitate (Pd- content 0.16 mmol/g) was separated by filtering, washed with diethyl ether (2x20 ml) and dried under vacuum at 25 °C.

Pd(ll) -hydrotalcite + pyridine + air

Betulinic aldehyde and betulonic aldehyde. Toluene (50 ml) was added to a 250 ml flask equipped with a condenser, mechanical stirrer and tubing for air bubbling. Pd(ll)-hydrotalcite (0.7 g, 140 w-%) and pyridine (0.04 ml) were added. Temperature was adjusted to 80 °C, air flow to 50 ml/min and stirring to 700 rpm. Betulin (0.5 g) was added. Oxidation reaction was monitored by GC and after 7 hours reaction was finished. Reaction mixture was cooled down to room temperature, precipitate was filtered and washed with toluene (2x20 ml). Solvent was evaporated in vacuum and dried in a vacuum oven over night to yield mixture of betulin oxidation products.

GC: Betulin 1 %, betulinic aldehyde 21 %, betulonic aldehyde 28%, others 50%.

Example relating to steps 2 and 5 for manufacturing mixture of betulinic acid and betulonic acid Example 6

2-methylbutene+NaCI02+NaH ₂ P0 ₄ + tert-butanol

Betulinic acid and betulonic acid. Tert-butanol (340 ml) and 2-methyl-2- butene (60 ml) were added to the filtrate obtained from example 4.2 (including 40 g mixture of betulinic aldehyde and betulonic aldehyde, aldehyde content 80%) under N ₂ flow. The oxidation system comprising NaCIO2 (34.0 g) and NaH ₂ PO ₄ *H ₂ O (50 g) in water (400 ml) was added during 0.5 h and stirring was continued at room temperature under N ₂ for 18 h. Organic and aqueous phases were separated and organic phase was washed with water (400 ml). Aqueous NaOH (5.4 g in 40 ml of water) was added to organic phase and stirred for 0.5 h. Half of the solvent were evaporated off under vacuum and residue was stirred at room temperature for 1 h. Precipitated mixture of betulinic acid and betulonic acid Na-salt was filtered, washed with toluene (4 * 40 ml) and dried over night at vacuum to yield mixture of betulinic acid and betulonic acid Na-salt. Mixture of betulinic acid and betulonic acid Na-salt was subsequently treated with aqueous HCI solution (280 ml water and 120 ml of 10% HCI) and stirred for 2,5 h. Precipitate was filtered and washed with water (5 *60 ml), dried in vacuum oven at 60 °C over night to obtain mixture of betu- linic acid and betulonic acid 17.8 g (44% from betulin).

GC: betulonic acid 60%, betulinic acid 27%, others 13%. Table 1. Results of betulin oxidation. Oxidation products of betulin. Route refers to reaction step 1 ,2, 3, 4 or 5 in above mentioned reaction scheme 1 .

Route/ Oxidant Betulinic Betulonic Betulonic Betulinic aldehyde aldehyde acid acid Catalyst

1 . Pd(OAc) ₂ + pyriAir 2 92

dine

1 . Pd(ll)-hydrotalcite Air 21 28

2. NHPI Air 48

2. QuadraPure MPA Air 62

2. NaCIO ₂ + 68

NaH ₂ PO ₄ + 2- methyl butene +

arquad 2HT

2. NaCIO ₂ + 4 76 2 NaH ₂ PO ₄ + 2- methylbutene +

arquad 2HT + tert- butanol

3. NaBH reduction 94

4. Pd(OAc) ₂ + TEA 25

4. Pd(OAc) ₂ + pyri25 55

dine

5. NaCIO ₂ + 60 27 NaH ₂ PO ₄ + 2- methylbutene +

arquad 2HT + tert-