Process of vitamin K ₂ derivatives preparation

Field of invention

The present invention relates to the process of vitamin K ₂ derivatives preparation.

Vitamins K ₂ play an important role in the blood coagulation cascade and the bones supplementation. Synthetic vitamins K ₂ can be used as the active ingredients of the drug products as well as in the dietary supplements. Background of the invention

Vitamins K ₂ , called menaquinones (MK-n) or phamoquinones are characterized by a menadione structure with a polyisoprenyl side chain at C-3 position consisting of various isoprene unit numbers in the side chain (n=l-13). They are represented by the formula depicted below:

Various biological activity and bioavailability of menaquinones result from the chain length and the number of unsaturated bonds present in that side chain [Chemistry of Natural Compound 2007, 43(3), 277-281].

Vitamin K, as a cofactor of □ -carboxylase, is involved in post-translational □- carboxylation of certain glutamate residues in the precursor proteins PIVKA. Vitamin K is necessary for the biosynthesis and maintenance - at the appropriate level - of coagulation factors II, VII, IX and X, osteocalcin, osteopontin, osteonectin and also calcium binding protein in the kidneys, placenta and lungs. Vitamin K is involved in the coagulation cascade in animals and its presence is essential for the proper synthesis of blood clotting proteins participating in the coagulation homeostasis. It also contributes to strong bones formation, preventing osteoporosis progress. Vitamin K also exerts anti- bacterial, anti-fungal, anti-inflammatory and pain relief activity. Recently, it has been proved that vitamin K ₂ may substantially affect the condition of arterial walls and blood circulation.

Among other vitamins K ₂ , MK-7 form is of special interest.

It is characterized by better bioavailability and efficacy than other vitamins K. It is also characterized by high absorption in the small intestine and sustained presence in blood serum (up to 3 days). Even small daily doses of vitamin MK-7 are sufficient to provide all cells and tissues with vitamin K dependent enzymes and proteins at an appropriate level. On account of participation in calcium metabolism, vitamin MK-7 is indirectly involved in strong bones formation. Unlike vitamin K ₁ , it also influences arterial vessel wall condition.

Vitamin MK-7 structure consists of a naphthalenedione ring (menadione) with an attached alkyl chain comprising seven isoprene units (heptaprenyl), thus it contains seven double bounds of trans configuration. Considering its molecular structure, synthetic vitamin MK-7 could be synthesized from menadione or its protected derivative, menadiol, following one of the strategies mentioned below:

1. attachment of the heptaprenyl chain directly to the menadiol molecule, according to the so called „0 + 7” strategy; 2. attachment of the chain’s shorter fragments to the monoprenyl derivative of menadiol, according to the „1 -i-n + m” strategy; 3. attachment of the hexaprenyl chain to the monoprenyl derivative of menadiol, according to the „1 + 6” strategy.

The US patent No. 4,199,531 discloses the process for the elongation of the side chain of the menadiol derivative comprising from 1 to n terminal activated isoprenyl units in position C-3, accomplished by its stereo- and regio-selective alkylation with an activated side chain precursor consisting of m isoprenyl units. The carbanion generated under basic conditions on the carbon atom adjacent to the arylthio, arylsulfmyl or arylsulfonyl terminal group of one substrate is subsequently alkylated with alkyl halide as the second substrate. Then, in case of the reaction of the monoprenylmenadiol arylsulfonyl derivative with polyisoprenyl halide, the product is subjected to reductive desulfonylation, deprotection of the hydroxyl groups if there is a need thereof, and/or oxidation to afford a menaquinone derivative. According to the specification, alkylation is performed under basic conditions in the presence of such bases as buthyllithium or phenyllithium, under dry conditions; in a solvent such as tetrahydrofurane, ether or 1,2- dimethoxyethane; in the -78°C to 20°C temperature range.

The above mentioned process for the alkylation of the phenylsulfonyl derivative of monoprenylmenadiol using triprenyl halide yielding vitamin MK-4 (according to the “1+3 strategy”) has been described in J. Org. Chem. 2003, 68, 7925. There has also been disclosed a synthesis of the phenylsulfonyl derivative of monoprenyl menadiol dimethoxy-ether (MK-1) from menadiol.

In the International Patent Application WO 2011/117324 a multi-step process for the preparation of polyisoprenyl alcohols and halides comprising chains of various lengths as a result of the Biellmann type reaction has been disclosed. The coupling reaction of the arylsulfonyl or arylthiol polyisoprenyl derivative having p isoprenyl units (p = 0-4) with properly protected (for example with the acetyl groups) primary polyisoprenyl halide having q isoprenyl units (q = 0-4) is carried out in the presence of a non-nucleophilic base. Subsequent removal of the SO ₂ Ar or SAr group under reductive conditions, followed by the deprotection of the hydroxyl group, furnishes the desired product. Example 6 describes the synthesis of pentaprenyl alcohol from diprenyl- alcohol bromide comprising protected acetyl and phenylsulfonyltriprenyl groups. After each step of the process: alkylation, desulfonylation and removal of hydroxyl protecting groups, purification of the product by silica gel flash chromatography is necessary. Polyisoprenyl halides obtained according to this procedure have been used in the vitamin K ₂ synthesis, in particular the vitamin MK-7 synthesis, under Grignard/Kumada or Suzuki conditions, following the “0+7” or “2+5” strategy. WO 2010/03500 discloses the synthesis of vitamin K ₂ that is based on the polyisoprenyl ring attachment to the protected activated menadiol derivative, under Grignard/Kumada or Suzuki conditions, according to the “0+7 strategy”.

In both aforementioned International Patent Applications the activated menadione derivative with carbonyl functions protected with alkyl or benzyl groups as the potential synthetic substrate has been claimed. However, in the preparative examples only methoxy-derivatives of menadiol have been used.

WO 2014/058330 discloses the process for the preparation of MK-7 type vitamin K ₂ by coupling the hexaprenyl chain precursor of all-trans configuration with a menadiol derivative bearing the phenylsulfonyl monoprenyl terminal group and protected in the form of alkoxy-ethers, especially in the form of ethoxy-ether. The said phenylsulfonyl monoprenyl menadiol ethoxy-derivative is used in a crystalline form that significantly improves the process of its purification. In the preferred embodiment, in the reaction under Julia protocol, the hexaprenyl bromide is used bearing a one side chain phenylsulfonyl group. That group is removed together with the second one resulting from the alkyl addition reaction in one step, thus shortening the synthesis without the deterioration of the yield and purity of the final product.

It is evident that the key issue in the process of vitamin MK-7 preparation according to the “1+6” strategy is proper selection of the protecting group of starting menadiol. By “a properly selected group” is meant a group which is stable through the coupling reaction with various polyisoprenyl chains comprising various numbers of isoprene units both under acidic and basic conditions, i.e. in the presence of Lewis acids and/or strong bases. Moreover, this properly selected group should be prone to cleavage from the advanced intermediate of vitamin K 2 in the restoration step of the menaquinone structure under mild conditions and in a high yield. Based on the literature review of the protecting groups known in the art (P. G.

M. Wuts, T. W. Greene, Green’s Protective Groups in Organic Synthesis. 4th ed., 2007, Wiley & Sons) some most promising groups for further process development, e.g., benzyl and lower alkyl groups, have been selected. The experimental works, however, resulted in the failure of the alkylation of menadiol with isopropyl bromide. In turn, alkylation with benzyl bromide gave menadiol protected with a benzyl group in high yield. This group, in turn, was unstable under the conditions of the subsequent coupling reaction with the phenylsulfonyl derivative of isoprenyl bromide.

Despite many synthetic attempts, only the allyl protecting groups met the set goal. The synthesis of vitamins K ₂ starting from menadione protected by the allyl protecting groups has been described in an unpublished International Patent Application PCT/PL2019/000026.

The aim of the present invention was to develop a more efficient process, useful for a larger scale production of the wide range of synthetic all-trans vitamins K ₂ comprising a polyisoprenyl side-chain of various lengths.

The other aim of the present invention was to improve the process of vitamin K ₂ preparation by further limiting the number of synthesis steps as well as to eliminate or reduce troublesome and time-consuming multiple chromatographic purifications of the intermediates and still obtain vitamins K ₂ of high purity and in a good yield.

These aims have been accomplished by means of modifying the process of quinone derivatives’ manufacturing described in the prior art US 4,853,156 patent. The said process comprises reacting the quinone-cyclopentadiene adduct with a polyisoprenyl chain terminated with the leaving group and converting thus obtained compound to the deprotected quinone in the retro-Diels-Alder reaction. As it was mentioned, the process may be useful for manufacturing compounds of the vitamin K series, such as K ₁ , K ₂₍₅₎ , K ₂₍₁₀₎ , etc. However, in the preparative examples only short- chain derivatives of menaquinone are obtained in the all-trans form in a 100% purity, while longer chain derivatives, e.g. prepared from geranylfamesyl bromide or solanesyl bromide, have the HPLC purity of 95.6-98.7% all-trans. Although the synthesis has fewer steps than the process according to Julia protocol (PCT/PL2019/000026), the alkylation product also needs to be chromatographed on a silica gel column before a subsequent deprotection step. Disclosure of the invention c

The invention relates to A process of vitamin K ₂ derivatives preparation, represented by formula (I) wherein n is an integer from 4 to 13, comprising the steps of: providing a polyisoprenyl bromide synthesized from natural or semi-synthetic sources of isoprenyl units, represented by formula (VII) wherein

Y and Z are independently H or -SO ₂ P _h ,

P, q and r are the same or different, and are the integers from 0 to 9, and p + q + r = 4 ÷ 13; the alkylation of the menadione-cyclopentadiene adduct (MDA) of formula (II) in the presence of a strong base, with polyisoprenyl bromide of formula (VII) wherein the substituents have the meaning defined above; to yield menaquinone substituted with a polyisoprenyl chain, of formula (VIII)

(c) optionally, in the case when Y and Z are -SO ₂ P _h , removing the phenylsulfonyl groups from the derivative of formula (VIII) by reductive elimination to yield the menadione derivative of formula (IX) p, q, r have the meaning defined above; (d) removing the cyclopentadiene protection from the menadione derivative of formula (IX), to yield the crude menadione compound of formula (X), m, n and p have the meaning defined above, and Y, Z are H; (e) optionally, purifying the crude menadione derivative of formula (X) to yield pure vitamin K ₂ , characterized in that the menadione derivative of formula (IX) obtained in step (b) or (c) is used in step (d) without purification and the cyclopentadiene protection is removed by heating in an organic solvent in the 60°C to 100°C temperature range. In the preferred embodiment of the invention, the menadione derivative of formula (VIII) is extracted from the post-reaction mixture with a water-immiscible solvent and without purification heated in the same solvent in the 60°C to 100°C temperature range.

In the most preferred embodiment of the invention, the water-immiscible solvent is ester type solvent, especially ethyl acetate.

New intermediates obtained in the process of the invention are also provided.

Detailed description of the invention

In the process of the present invention synthon A represented by formula (II) is the menadione-cyclopentadiene adduct (MDA). The MDA adduct could be synthesized in the reaction of menadione and freshly distilled cyclopentadiene according to the experimental procedure described in the US 4,853,156 and YF Ji, ZM. Zong, XY. Wei, GZ. Tu, L. Xu, LT. He. Improved synthesis of vitamin Kl. Synthetic Commun. 33, 5, 763-772, 2003, in a good yield. Synthon B in the process of the present invention is polyisoprenyl bromide represented by formula (VII) wherein Y and Z are independently H or -SO ₂ P _h , m and n are the same or different, and are both the integers from 0 to 9, while m+n = 4 ÷ 13.

The length of the side chain in the target vitamin K ₂ could be modified by the number of the isoprenyl units of the side chain precursor of formula (VII) derived from the natural or semi-synthetic sources.

The key step in the process of vitamin K ₂ preparation according to the present invention is the coupling of the A and B synthons, accomplished as a result of the nucleophilic addition.

The coupling of the A and B synthons in the alkylation reaction and the subsequent removal of the cyclopentadiene protection results in the formation of the vitamin K ₂ derivative, optionally possessing phenylsulfonyl groups in the side chain. Upon the phenylsulfonyl groups’ removal, vitamin K ₂ of a desired isoprenyl chain length is obtained.

Combining synthon A and synthon B results in the target vitamin K ₂ derivative comprising an even or odd number of the isoprenyl units.

Polyisoprenyl bromide of formula (VII) could be constructed with the use of various isoprenyl compounds of natural origin comprising variable chain lengths, depending on the desired structure of the target vitamin. Thus, by selecting a number of repeating isoprenyl units in the starting synthone B, it is possible to obtain a vitamin K ₂ derivative of a desired polyisoprenyl chain.

In one embodiment of the invention, synthon A is reacted with synthon B comprising six isoprenyl units, to give the MK-6 type of vitamin K ₂ . In another embodiment of the invention, synthon A is reacted with synthon B comprising seven isoprenyl units, to give the MK-7 type of vitamin K ₂ .

In one embodiment of the invention, a polyisoprenyl halide chain of formula (VII) with an even or odd number of the isoprenyl units may be constructed starting from naturally occurring or semi-synthetic derivatives, such as geraniol, famesol, solanesol, or any combination thereof, by the repeated alkyl addition reactions.

For instance, the processes for the preparation of polyisoprenyl halides from geraniol are known in the art. CN 1218918 C discloses the process for the preparation of all-trans geranyl-geraniol comprising the steps of geranyl acetate oxidation to the mixture of alcohol and aldehyde followed by the reduction of the aldehyde to obtain 8- geranyl-hydroxy-acetate, then the transformation of the said alcohol to geranyl bromoacetate by means of PBr3 in pyridine, and finally its addition to phenylsulfonyl- geraniol with the subsequent removal of the phenylsulfonyl group by means of Li/MeNH ₂ .

The exemplary synthesis of polyisoprenyl halide of formula (VII) from geraniol can be accomplished according to the synthetic approach illustrated in Scheme 1.

SYNTHON B

Scheme 1. Coupling of geraniol fragments In the first step, geraniol is acetylated with the use of AC2O in pyridine and the resulting geranyl acetate is oxidized with the use of selenium dioxide to E.Zi-S-hydroxy- geranyl acetate.

Selenium dioxide (SeCh) mediated incorporation of the oxygen atom at the allylic position is known, for example, from T. Wirth et al., Organoselenium Chemistry, Modem Developments in Organic Synthesis, ed. Springer. According to the present invention, oxidation can be performed using a stoichiometric amount of Se0 ₂ , or, preferably, a catalytic amount of Se02 employing an acid as a co-catalyst, for example, salicylic acid or S1O2, in the presence of a 2-3-fold molar excess of a co-oxidizer, such as tert- butyl peroxide (in water or an organic solvent) or hydrogen peroxide. In another embodiment of the invention the oxidation reaction can be accomplished using SeC>2 in the presence of a molar excess of N- oxide /V-methylmorpholine.

Then, E,E-8-hydroxygeranyl acetate is converted into its phenylsulfonyl derivative (HIA) in a two-step synthesis, via a bromide derivative which is further treated with sodium benzenesulfinate. Phenylsulfonyl derivative of geranyl acetate (HI) is coupled in the alkylation reaction with geranyl bromide (IV) obtained in the reaction of geraniol with one of the commonly used halogenating agents.

Coupling of the polypropenyl chain fragments by way of alkylating proper sulfones is generally known in the art, among others from J. Org. Chem. 2003, 68, 7925; J. Chem. Soc. Perkin I 1981, 761; J. Org. Chem. 2008, 73, 7197; Tetrahedron 2009, 65, 6310. This reaction can be performed in the presence of a strong base, such as potassium tert-butanolate, n-butyllithium, lithium, sodium or potassium bis(trimethylsilyl)amidate, in a polar aprotic solvent.

Phenylsulfonyl polyisoprenyl acetate (V) is then transformed into the polyprenol derivative of formula (VI) comprising one phenylsulfonyl group SO ₂ P _h .

To obtain the polyprenol derivative of formula (VI), the acetyl group is removed upon hydrolysis under basic conditions, while the phenylsulfonyl group is left intact.

The processes of isoprenyl fragments’ coupling and the hydrolysis of phenylsulfonyl polyisoprenyl acetate (V) can be accomplished successively, following isolation and purification of the compound (V). In the preferred embodiment of the invention, however, the steps of the isoprenyl chain fragments’ coupling and further hydrolysis are carried out successively in a “one pot” reaction.

The resulting compound of formula (VI) can be subsequently converted into halide of formula (VII) which can be used as Synthon B in the further synthesis of vitamin K ₂ .

The acetyl and phenylsulfonyl groups can be removed successively or simultaneously.

The methods of removing arylsulfonyl groups of substituted (arylsulfonyl)alkanes are known in the art. They can be removed under different reductive conditions, depending on the molecular structure of the substrate (Y. Liu, Y. Zhang, Org. Prep. Proc. Int. 33 (2001), 372). Among general methods, reduction with alkali metals dissolved in liquid ammonia (for example J. R. Hwu at al., J. Org. Chem. 61 (1996), 1493-1499); reduction with Mg / MeOH or Mg / EtOH+HgCl ₂ (G. H. Lee at al., Tetrahedron Lett. 34 (1993), 4541-2; A. C. Brown, L. A. Carpino, J. Org. Chem. 50 (1985), 1749-50) and also with sodium amalgam in MeOH, buffered with Na ₂ HPO ₄ (B. M. Trost at al., Tetrahedron Lett. 17 (1976), 3477-8) should be mentioned.

In one embodiment of the present invention, acetyl and phenylsulfonyl groups of the compound of formula (V) are removed simultaneously, as a result of reductive elimination with borohydrides of alkali metals, such as sodium or potassium, using complexes of metal (II) dihalides with phenylphosphite type bidentate ligands of the formula [M{Ph ₂ P(CH ₂ )nPPh ₂ }X ₂ ], wherein n=2-5, X=C1 or Br and M=Co, with Ni or Pd as catalysts.

Preferably, metal borohydride is used, unsubstituted or substituted, having up to three substituents selected among Ci-5-alkyl and phenyl, such as lithium triethylborohydride, lithiumt tri-sec-butylborohydride, tri-sec-butyllithium, sodium or potassium, potassium triphenylborohydride. Most preferably, lithium triethyloborohydride in the presence of Pd(dppe)Cl ₂ complex, where dppe represents l,2-bis(diphenylphosphino)ethane or Pd(dppp)Cl ₂ , where dppp represents 1,3- bis(diphenylphosphino)propane. Then, in the reaction of the compound of formula (VI) with a brominating agent, polyisoprenyl halide of formula (VII) substituted with a phenylsulfonyl group in a side chain is obtained in a good yield.

Suitable halogenating agents are, for example, PBr3 or HBr,' converting polyprenol into the corresponding bromide.

The reaction of the menadione-cyclopentadiene adduct (MDA) with polyisoprene bromide (VII) comprising different numbers of isoprene units is carried out in an organic solvent under basic conditions, in the presence of a strong base.

Strong bases that could be used in the present invention are alcoholates, alkali metal amides or hydrides.

The preferred base is potassium tert-butoxide, t-BuOK.

The base is used in a molar excess in relation to the starting MDA adduct. Good alkylation results are achieved with the use of more than 5 equivalents of the base in relation to the MDA adduct. The use of t-BuOK in a molar excess generally results in a higher rate of menadione-cyclopentadiene adduct consumption. When t-BuOK amount used in the alkylation of MDA with hexaprenyl bromide in relation to the MDA adduct is below 3 equivalents, the formation of process-related impurities resulting from the deprotection of the adduct (Imp. 1) and alkylation at the carbonyl group (Imp. 2) is observed. Thus, using t-BuOK in the molar excess above 3, more preferably equal or more than 5 allows to avoid the formation of process-related impurities which are hard to remove.

The process of MDA alkylation can be carried out in inert aprotic polar solvents, preferably in THF, at 0-5°C. After the completion of the reaction, usually after 2-5 hours, the crude product is extracted from the post-reaction mixture with an organic solvent.

The preferred organic solvents are the solvents of an ester type, especially ethyl acetate.

The organic extract containing K ₂ vitamin intermediate (VIE) is washed with water, brine, dried over magnesium sulphate, then filtered, and used in the next step of the process without the isolation of the reaction product. Further, the process comprises the removal of the cyclopentadiene protection only or both the cyclopentadiene protection as well as the phenylsulfonyl groups, depending on the structure of polyisoprenyl bromide used in the alkylation reaction.

In one case, when the polyisoprenyl bromide of formula (VII), wherein Y and Z are H is used, the cyclopentadiene protecting group is removed directly after the alkylation reaction and the product’s extraction.

That removal, the so called “retro-Diels-Alder” reaction, is accomplished by heating the organic extract containing K ₂ vitamin intermediate (VIH). After the evaporation of solvents, a crude vitamin K ₂ (IX) is obtained. In the examples of US 4,853,156, efficient removal of the cyclopentadiene protection of menadione is carried out in toluene at reflux, ie. at 110°C. However, such harsh conditions lead to the formation of high levels of further process-related impurities.

Contrary to the disclosure of US 4,853,156, the present Inventors have found out that high temperatures are not only unnecessary to remove the cyclopentadiene protecting group, but, more importantly, lower temperatures allow to obtain crude vitamins K ₂ with a satisfactory impurity profile for further purification.

According to the invention, preferred solvents to be used for an effective deprotection procedure are low boiling solvents in which the deprotection reaction can be carried out at reflux, in the 60°C to 100°C temperature range.

By the term “low boiling solvent” it is meant the solvent boiling in the 60°C to 100°C temperature range, more preferably in the 70°C to 90°C temperature range, such as alcohols, like ethanol, isopropanol, n-propanol or tert-butanol; C1-C5 alkyl esters, like ethyl, n-propyl, isopropyl or amyl acetate, etc. The deprotection reaction can be also carried out in higher boiling solvents, like toluene, at the temperatures below their boiling point, preferably at 80°C.

Whilst not being bound by theory it is thought that menadione substituted with longer polyisoprene chains conjugated to MDA is less thermodynamically stable than unsubstituted MDA substrate. Thus, the cyclopentadiene protection is difficult to remove from the unsubstituted menadione-cyclopentadiene adduct at low temperatures but it requires higher temperature conditions, such as toluene at reflux (110°C). Alternatively,: polyisoprenyl bromide used in the reaction of MDA alkylation, has formula (VII), wherein at least one of Y and Z is -SO ₂ P _h . An alkylation reaction is carried out under the conditions similar to those described above.

Then the cyclopentadiene protecting group and the phenylsulfonyl groups could be removed in any order.

In one embodiment of the invention, phenylsulfonyl groups are removed first with the subsequent removal of the cyclopentadiene protection.

Phenylsulfonyl groups can be removed under reductive elimination conditions, using borohydride of an alkali metal, such as lithium, sodium or potassium, and catalyzed by the complexes of metal (P) dihalides and bidentate ligands of the phenylphosphite type of the formula [M{Ph ₂ P(CH ₂ )nPPh ₂ }X ₂ ], wherein n=2-5, X=C1 or Br and M=Co, Ni or Pd, most preferably, lithium triethyloborohydride with Pd(dppe)Cl ₂ complex, wherein dppe represents l,2-bis(diphenylphosphino)ethane or Pd(dppp)Cl ₂ , wherein dppp represents l,3-bis(diphenylphosphino)propane. While removing the phenylsulfonyl groups, some amount of cyclopentadiene can be unintentionally deprotected, resulting in the reduction of the menadione carbonyl to hydroxylic groups. Therefore, in this case, after the removal of the phenylsulfonyl groups, the compound of formula (IX), wherein Y and Z are H, needs to be additionally subjected to oxidative deetherification to restore the structure of the starting menadione. The oxidation of the phenolic groups to restore the quinone structures is typically accomplished using one of the common oxidizing agents, such as chromium trioxide in acetic acid, sodium dichromate or Fremy’s salt, i.e. potassium nitrosodisulfonate.

In the preferred embodiment of the present invention cerium ammonium nitrate (CAN) is used as the oxidizing agent. CAN is known, for example, from J. Org. Chem. 2003, 68, 7925-27.

Alternatively, in another embodiment of the invention, the phenylsulfonyl groups are removed after the removal of the cyclopentadiene protection. Upon removing the phenylsulfonyl groups and restoring the menadione structure, carried out without isolation of the intermediates, crude vitamin K ₂ is obtained.

In the preferred variant of the process according to the invention, the menadione- cyclopentadiene adduct (MDA) of formula (II) is alkylated, in the presence of a strong base, with polyisoprenyl bromide of formula (VII) wherein Y and Z are H, and p, r and q have the meaning defined above; and the menadione of formula (IX), wherein Y and Z are H, and p, q, r have the meaning defined above; is extracted from the post-reaction mixture with a water-immiscible solvent and cyclopentadiene protection is removed by heating an organic extract in the 60°C to 100°C temperature range without prior purification.

The crude vitamin K ₂ product (I) obtained in the process of the invention could be isolated, for example by column chromatography, and then it may be purified, for example by high performance liquid chromatography, and/or by crystallization. Preferably, the obtained vitamin K ₂ is crystallized from the mixture of ethyl acetate and ethanol. As confirmed by gas chromatography analysis, the final vitamin K ₂ does not contain cyclopentadiene or the dimer or trimer thereof. These hydrocarbons may be formed from the cyclopentadiene adduct during the deprotection step.

The process for vitamin K ₂ derivatives preparation according to the present invention enables the preparation of different types of vitamin K ₂ using easily available starting compounds, providing a desired all-trans configuration of double bonds that conforms with the configuration of A and B synthons.

In comparison to the prior art methods the present invention provides a simplified and shorter process affording vitamin K ₂ . Regardless of the elimination of the separation and purification steps concerning some intermediates, the described process furnishes vitamins MK-4 to MK-13 of purity which meets the requirements for dietary supplements and active pharmaceutical ingredients.

The present invention is illustrated by the following examples.

Examples

General materials and methods

The ¹ H and ¹³ C NMR spectra were measured using a Bruker AVANCE III HD spectrometer at the 500 MHz transmitter frequency for ¹ H at the temperature of 298 K. The spectra were measured at the temperature of 295 K in the CDCI ₃ solution relatively to the TMS signal as a ¹ H and ¹³ C chemical shift standard. NMR analysis were performed using the results from one and two-dimensional NMR spectroscopy: ¹ H, ¹³ C, HSQC, HMBC and COSY. The ESI-MS spectra were recorded on a PE Biosystems Mariner mass spectrometer. The progress of the reaction was monitored by thin layer chromatography (TLC) with Merck DC-Alufolien Kieselgel 60 F ₂₅₄ . Column chromatography was performed on Merck silica gel 60 (230-400 mesh).

MDA was obtained according to the method described in US 4,854,156.

Example 1.

Step 1: MDA-MK-6

The suspension of t-BuOK (2.5 g, 22.26 mM) in THF (18 mL) was stirred under nitrogen atmosphere and cooled to 0-5°C. The solution of MDA (lg, 4.2 mM) in THF (10 mL) was added drop wise and stirring of the resulting red mixture continued at 0- 5°C for 30 min. Then, the solution of hexaprenyl bromide BrK-6 (2.87 g, 5.87 mM) in

THF (5 mL) was added to that mixture. The reaction was stirred for 2-3 h (TLC monitoring) under argon atmosphere. The reaction was quenched with CH ₃ COOH and water. The product was extracted with AcOEt (3x1 OmL), the organic phase was separated and washed with brine and water. The crude product was used in the next step without purification.

A sample of crude MDA-MK-6 was purified by column chromatography (hexane/ethyl acetate 10: 1 v/v) to afford oily product MDA-MK-6 for the NMR analysis. ¹ H NMR (CDCI3, 500 MHz) d 7.96-7.94 (m, 1H, H-9), 7.89-7.88 (m, 1H, H-6), 7.66-7.64 (m, 2H, H-7, H-8), 6.06-6.04 (m, 2H, H-2”, H-3”), 5.10-5.09 (m, 5H, H-6’, H-10' H-14' H- 18’, H-22’), 4.91-4.90 (m, 1H, H-2’), 3.22-3.20 (brs, 1H, H-4”), 3.14-3.13 (brs, 1H, H-

1”), 2.87-2.86 (m, 1H, H-1), 2.49-2.48 (m, 1H, H-1’), 2.10-1.80 (m, 20H, H-4’, H-5' H-8’, H-9’, H-12’, H-13’, H-16’, H-17’, H-20’, H-21’), 1.90 (m, 1H, H-5”), 1.67 (s, 3H, H-24’), 1.56 (s, 3H, H-1a), 1.49-1.48 (m, 1H, H-5”), 1.67-1.56 (m, 18H, H-25’, H- 26’, H-27’, H-28’, H-29’, H-30’); ¹³ C NMR (CDCI3, 125 MHz) d 202.3 (C-1), 201.6 (C-4), 138.3 (C-3”), 137.4 (C-2”), 137.7 (C-3’), 136.8 (C-5), 135.0 (C-10), 133.8 (C-

7), 133.5 (C-8), 134.5-133.1 (C-7’, C-11’, C-15’, C-19’, C-23’), 126.9 (C-9), 126.1 (C- 6), 124.3-123.8 (C-6' C-10’, C-14’, C-18’, C-22’), 119.8 (C-2’), 60.5 (C-3), 57.2 (C-2), 55.4 (C-1”), 54.1 (C-4”), 43.7 (C-5”), 39.7-39.2 (C-4’, C-8’, C-12’, C-16’, C-20’), 36.4 (C-1’), 26.7-26.2 (C-5’, C-9’, C-13’, C-17’, C-21’), 25.6 (C-24’), 23.6 (C-1a), 17.6-15.9 (C-25\ C-26’, C-27’, C-28’, C-29' C-30’).

When t-BuOK amount used in the alkylation of MDA with hexaprenyl bromide was less than 3 equivalents, impurity IMP 1 was isolated by column chromatography from crude oil as a by-product. Impurity IMP 1 was obtained as oil. Its structure has been identified as: ¹ H NMR (CDCl ₃ , 500 MHz) d 8.13 (d, 1H, H-6), 8.08 (d, 1H, H-9), 7.75 (t, 1H, H-7), 7.68 (t, 1H, H-8), 7.30 (s, 1H, OH), 2.11 (s, 3H, H-1a); ¹³ C NMR (CDC1 ₃ , 125 MHz) d 185.0 (C-4), 181.1 (C-1), 153.1 (C-3), 134.8 (C-7), 132.9 (C-8), 132.8 (C-5), 129.3 (C- 10), 126.7 (C-6), 126.1 (C-9), 120.6 (C-2), 8.6 (C-1a). The other by-product, impurity IMP 2 resulting from the alkylation of MDA with hexaprenyl bromide, was isolated from crude oil by column chromatography. Impurity IMP 2 was obtained as oil. Its structure has been identified as: ¹ H NMR (CDCI3, 500 MHz) d 7.70 (d, 1H, H-6), 7.49-7.48 (m, 1H, H-8), 7.45-7.43 (m, 1H, H-9), 7.23-7.22 (m, 1H, H-7), 6.32-6.31 (m, 1H, H-3”), 6.13-6.10 (m, 1H, H-2”),

4.45-4.44 (m, 1H, H-1’), 5.55 (t, 1H, H-2’), 5.11-5.09 (m, 5H, H-6’, H-10’, H-14’, H- 18’, H-22’), 4.35-4.32 (m, 1H, H-1’), 3.82-3.81 (m, 1H, H-1”), 3.14-3.12 (m, 1H, H- 4”), 2.05-1.80 (m, 20H, H-4’, H-5’, H-8’, H-9’, H-12’, H-13’, H-16’, H-17’, H-20’, H- 21’), 1.92-1.91 (m, 1H, H-5”), 1.72-1.70 (m, 1H, H-5”), 1.72-1.68 (m, 18H, H-25’, H- 26’, H-27’, H-28’, H-29’, H-30’), 1.61 (s, 3H, H-24’), 1.49 (s, 3H, H-1a); ¹³ C NMR (CDCb, 125 MHz) d 205.6 (C-1), 142.0 (C-4), 141.1 (C-2’), 138.3 (C-3”), 136.6 (C- 2”), 137.7 (C-5), 135.5-134.1 (C-7’, C-1l’, C-15’, C-19’), 134.6 (C-3), 133.5 (C-7), 131.2 (C-23’), 130.7 (C-10), 127.3 (C-8), 126.9 (C-9), 124.3-123.8 (C-6\ C-10’, C-14’, C-18’, C-22’), 122.8 (C-6), 120.2 (C-2’), 69.1 (C-1’), 57.9 (C-2), 47.8 (C-4”), 45.7 (C- 1”), 44.6 (C-5”), 39.7- 39.6 (C-4’, C-8’, C-12’, C-16’, C-20’), 26.7-26.3 (C-5’, C-9’,

C-13’, C-17’, C-21’), 25.7 (C-24’), 25.1 (C-1a), 17.6 (C-25’), 16.6 (C-30’), 16.1-16.0 (C-26’, C-27’, C-28’, C-29’).

Step 2: MK-6

The organic extract of MDA-MK-6 obtained in Step 1 was stirred at reflux (78°C) for about 1-2 h until the starting material was entirely consumed (TLC monitoring). The yellow solution was evaporated to oil and the crude product was purified by column chromatography (hexane/toluene 3:1) yielding MK-6 as yellow oil (1.46 g, 60%). The product was crystallized from 5 mL of ethyl acetate/anhydrous ethanol (1:4, v/v) at - 10°C for 24 h. Crystalline vitamin MK-6 with 98.52% purity (HPLC) was obtained. ¹ H NMR (CDCI ₃ , 500 MHz) d 8.08-8.07 (m, 2H, H-6, H-9), 7.69-7.68 (m, 2H, H-7, H-8), 5.11-5.01 (m, 6H, H-2' H-6’, H-10’, H-14' H-18’, H-22’), 3.38-3.36 (m, 2H, H-1’), 2.19 (s, 3H, H-1 a), 2.05-1.97 (m, 20H, H-4’, H-5’, H-8’, H-9’, H-12’, H-13’, H-16’, H- 17’, H-20’, H-21), 1.79 (s, 3H, H-30’), 1.67 (s, 3H, H-24’), 1.59 (s, 3H, H-25’), 1.67-

1.58 (m, 12H, H-26’, H-27’, H-28’, H-29’); ¹³ C NMR (CDCI ₃ , 125 MHz) d 185.4 (C-1), 184.5 (C-4), 146.1 (C-3), 143.3 (C-2), 137.5 (C-3’), 135.2-134.3 (C-7’, C-11’, C-15’, C- 19’), 133.3 (C-7), 133.2 (C-8), 132.1 (C-5), 132.0 (C-10), 131.2 (C-23’), 126.2 (C-9), 126.1 (C-6), 124.3-123.8 (C-6’, C-10’, C-14’, C-18’, C-22’), 119.0 (C-2’), 39.7-39.6 (C-4', C-8’, C-12’, C-16’, C-20’), 26.7-26.4 (C-5', C-9’, C-13’, C-17’, C-21’), 25.9 (C- 1’), 25.6 (C-24’), 17.6 (C-25’), 16.4 (C-30’), 12.6 (C-1a), 16.4-15.9 ( C-26’, C-27’, C- 28’, C-29’). HR-MS (ESI) calc, for C ₄₁ H ₅₇ O2 (M+H) ⁺ : 581.4359. Found: 581.4365.

Example 2

Step 1: MDA-MK-7 The suspension of t-BuOK (2.23 g, 19.87 mM) in THF (15 mL) was stirred under nitrogen atmosphere and cooled to 0-5°C. Then, the solution of MDA (0.893 g, 3.75 mM) in THF (6 mL) was added dropwise and stirring of the red mixture continued at 0- 5°C for 30 min. After that, the solution of heptaprenyl bromide BrK-7 (6.27 g, 11.2 mM) in THF (12 mL) was added to the mixture. The reaction was stirred for 2 h (TLC monitoring) under argon atmosphere. The reaction was quenched with CH3COOH and water. The product was extracted with AcOEt (3x20mL), the organic phase was separated and washed with brine and water. The crude product was used in the next step without purification.

A small amount of crude MDA-MK-7 was purified by column chromatography (hexane/ethyl acetate 20:1— > 10:1) to obtain oily product MDA-MK-7 for the NMR studies. ¹ H NMR (CDCI ₃ , 500 MHz) d 7.95-7.94 (m, 1H, H-9), 7.89-7.88 (m, 1H, H-6), 7.65-7.64 (m, 2H, H-7, H-8), 6.06-6.04 (m, 2H, H-2”, H-3”), 5.11-5.09 (m, 6H, H-6’, H-10’, H-14’, H-18’, H-22’, H-26’), 4.91-4.90 (m, 1H, H-2’), 3.22-3.20 (brs, 1H, H- 4”), 3.14-3.13 (brs, 1H, H-1”), 2.85-2.84 (m, 1H, H-1’), 2.47-2.46 (m, 1H, H-1’), 2.10- 1.80 (m, 24H, H-4’, H-5’, H-8’, H-9’, H-12’, H-13’, H-16’, H-17’, H-20’, H-2G, C-24’, C-25’), 1.90 (m, 1H, H-5”), 1.68 (s, 3H, H-28’), 1.66 (s, 3H, H-1a), 1.49-1.48 (m, 1H, H-5”), 1.69-1.60 (m, 21H, H-29’, H-30’, H-31’, H-32’, H-33’, H-34’, H-35’); ¹³ C NMR (CDCI3, 125 MHz) d 202.3 (C-1), 201.7 (C-4), 138.3 (C-3”), 137.4 (C-2”), 137.7 (C- 3’), 136.8 (C-5), 135.0 (C-10), 133.8 (C-7), 133.5 (C-8), 134.0-131.2 ( C-7’, C-11’, C-

15’, C-19’, C-23’, C-27’), 126.9 (C-9), 126.1 (C-6), 124.0-123.8 (C-6’, C-10’, C-14’, C- 18’, C-22’, C-26’), 119.8 (C-2’), 60.5 (C-3), 57.2 (C-2), 55.4 (C-1”), 54.1 (C-4”), 43.7 (C-5”), 39.0-39.2 (C-4’, C-8’, C-12’, C-16’, C-20’, C-24’), 36.4 (C-1’), 26.5-26.2 (C- 5’, C-9’, C-13’, C-17’, C-21’, C-25’), 25.6 (C-28’), 23.6 (C-1a), 17.6-15.8 (C-29’, C- 30’, C-31’, C-32’, C-33’, C-34’, C-35’).

Step 2: MK-7

The organic extract of MDA-MK-7 obtained in Example 3 was stirred at reflux (78°C) 2 h until the starting material was entirely consumed (TLC monitoring). The yellow solution was evaporated to oil and the crude product was purified by column chromatography (hexane, hexane/toluene 3:1 v/v) yielding MK-7 as yellow oil (1.10 g, 45%). The chromatographically purified product was crystallized from ethyl acetate / anhydrous ethanol mixture at -10°C for 24 h. Crystalline vitamin MK-7 with 96.3% purity (HPLC) was obtained. ¹ H NMR (CDC1 ₃ , 500 MHz) d 8.08-8.07 (m, 2H, H-6, H- 9), 7.69-7.68 (m, 2H, H-7, H-8), 5.11-5.10 (m, 7H, H-2’, H-6’, H-10’, H-14’, H-18’, H- 22’, H-26’), 3.38-3.36 (m, 2H, H-1’), 2.19 (s, 3H, H-1a), 2.06-1.97 (m, 24H, H-4’, H-5’, H-8’, H-9’, H-12’, H-13’, H-16’, H-17’, H-20’, H-21’, H-24’, H-25’), 1.79 (s, 3H, H- 35’), 1.68 (s, 3H, H-28’), 1.59-1.58 (m, 18H, H-29’, H-30’, H-31’, H-32’, H-33’, H- 34’); ¹³ G NMR (CDCI3, 125 MHz) d 185.4 (C-1), 184.5 (C-4), 146.1 (C-3), 143.3 (C-2), 137.5 (C-3’), 135.2-134.8 (C-7’, C-11’, C-15’, C-19’, C-23’), 133.3-132.1 (C-5, C-7, C- 8, C-10), 131.2 (C-27’), 126.2-126.1 (C-6, C-9), 124.3-123.8 (C-6’, C-10’, C-14’, C- 18’, C-22’, C-26’), 119.0 (C-2’), 39.7-39.6 (C-4’, C-8’, C-12’, C-16’, C-20’, C-24’), 26.7-26.4 (C-5’, C-9’, C-13’, C-17’, C-21’, C-25’), 25.9 (C-1’), 25.6 (C-28’), 17.6 (C-

29’), 16.4 (C-35’), 16.4-16.0 ( C-30’, C-31’, C-32’, C-33’, C-34’), 12.6 (C-1a).

Example 3

Step 1: MDA-MK-6-SO ₂ Ph

The suspension of t-BuOK (0.94 g, 8.4 mM) in THF (20 mL) was stirred under nitrogen atmosphere and cooled to 0-5°C. The solution of MDA (1 g, 4.2 mM) in THF (10 mL) was added dropwise and stirring of the red mixture continued at 0-5°C for 30 min. Then, the solution of phenylsulfonyl hexaprenyl bromide BrK-6-SO ₂ Ph (3.44 g, 5.46 mM) in THF (10 mL) was added to the mixture. The reaction was stirred for 1 h (TLC monitoring) under argon atmosphere. The reaction was quenched with CH ₃ COOH and water. The product was extracted with AcOEt (3 x 15mL), the organic phase was separated and washed with brine and water. The crude product was purified by column chromatography (hexane/ethyl acetate 10:1 ® 3:1) to obtain oily product MDA-MK-6-SO ₂ Ph (2.93 g, 89 %). ¹ H NMR (CDCI ₃ , 500 MHz) d 7.95-7.94 (m, 1H, H-9), 7.89-7.88 (m, 1H, H-6), 7.81-7.80 (m, 2H, SO ₂ Ph), 7.67-7.65 (m, 2H, H-7, H-8), 7.61-7.60 (m, 1H, SO ₂ Ph), 7.55-7.49 (m, 2H, SO ₂ Ph), 6.04-6.03 (m, 2H, H-2”, H-3”), 5.30-5.01 (m, 3H, H-6’, H-18’, H-22’), 4.91-4.90 (m, 3H, H-2’, H-10’, H-14’), 3.48-3.47 (m, 1H, H- 12’), 3.22-3.20 (brs, 1H, H-4”), 3.14-3.13 (brs, 1H, H-1”), 2.87-2.84 (m, 2H, H-1\ H- 13’), 2.60-2.57 (m, 1H, H-13’), 2.49-2.48 (m, 1H, H-1’), 2.02-1.79 (m, 16H, H-4’, H-5’, H-8’, H-9’, H-16’, H-17’, H-20’, H-21’), 1.90 (m, 1H, H-5”), 1.56 (s, 3H, H-1a), 1.49- 1.48 (m, 1H, H-5”), 1.67-1.56 (m, 21H, H-24’, H-25’, H-26’, H-27’, H-28’, H-29’, H- 30’); ¹³ C NMR (CDCI3, 125 MHz) d 202.2 (C-1), 201.5 (C-4), 138.3 (C-3”), 137.4 (C-

2”), 137.7 (C), 137.5 (SO ₂ Ph), 136.8 (C), 135.0 (C), 133.8 (C), 133.5 (C), 133.2 (SO ₂ Ph), 134.5-133.1 (C), 128.8-128.6 (SO ₂ Ph), 126.9 (C), 126.1 (C), 124.3-123.8 (CH: C-6’, C-10’, C-14’, C-18’, C-22’), 119.8 (C-2’), 60.5 (C-3), 57.2 (C-2), 55.4 (C-1”), 54.0 (C-4”), 43.7 (C-5”), 39.7-38.4 (CH ₂ : C-4’, C-8’, C-12’, C-16’, C-20’), 36.3 (C- 1’), 26.8-26.3 (CH ₂ : C-5’, C-9’, C-13’, C-17’, C-21’), 23.6 (C-1a), 17.6-15.9 (CH ₃ : C-

24’, C-25’, C-26’, C-27’, C-28’, C-29’, C-30’). HR-MS (ESI) calc, for C ₅₂ H ₆₆ O ₄ S (M+Na) ⁺ : 809.4580. Found: 809.4587.

Step 2: MDA-MK-6

The solution of MDA-MK-6-SO ₂ P _h (1 g, 1.3 mM) in THF (10 mL) was cooled to 0°C, then 1M LiEt3BH (8.9 mL, 8.9 mM) was added to the mixture. Next, Pd(dppe)Cl ₂ catalyst (264 mg, 0.39 mM) was added and the suspension was stirred at 0°C. The reaction was monitored by TLC. After 20 min. the reaction was quenched with saturated NH4CI (10 mL) and water (10 mL). The product was extracted with AcOEt (2 x 15 mL) and the organic phase was washed with brine. The organic extract of MDA-MK-6 was used in the next step without purification.

Step 3: MK-6

The organic extract of MDA-MK-6 (1.3 mM) obtained in Step 1 was stirred at reflux (78 °C) for lh until the starting material was entirely consumed (TLC monitoring). The resulting yellow solution was evaporated to oil. The crude oil was dissolved in acetonitrile/DCM (4 mL, 1:1 v/v) and cooled to 0°C. The solution of CAN (2.1 g, 3.9 mM) in the mixture of acetonitrile/H ₂ O (1.2 mL, 5:1 v/v) was added dropwise within 15 min at this temperature. The yellow solution was stirred for lh. Next, water (10 mL) and DCM (10 mL) were added to the reaction mixture. The organic phase was separated, washed with brine and evaporated to dryness. The crude product was purified by column chromatography (hexane/toluene 10:1—» 3:1) yielding MK-6 as yellow oil (498 mg, 66%). NMR consistent with Example 1.

Example 4

Step 1: MK-6-SO ₂ Ph

MK-6-SO ₂ Ph

The solution of MDA-MK-6-SO ₂ P _h (0.5 g, 0.63 mM) in AcOEt (10 mL) was stirred at reflux (78°C) for 2h until the starting material was entirely consumed (TLC monitoring). The yellow solution was evaporated to give oil and the crude product was purified by column chromatography (hexane/toluene 10:1→ 3:1) yielding MK-6- SO ₂ P _h as yellow oil (0.382 g, 84%). ¾ NMR (CDCI ₃ , 500 MHz) d 8.09-8.07 (m, 2H, H-9, H-6), 7.81-7.79 (m, 2H, SO ₂ Ph), 7.70-7.69 (m, 2H, H-7, H-8), 7.60-7.59 (m, 1H, SO ₂ Ph), 7.50-7.49 (m, 2H, SO ₂ Ph), 5.06-4.87 (m, 6H, H-2’, H-6’, H-10’, H-14’, H-18’, H-22’), 3.47-3.46 (m, 1H, H-12’), 3.44-3.37 (m, 2H, H-1’), 2.82-2.77 (m, 1H, H-13’), 2.78-2.77 (m, 1H, H-13’), 2.18 (s, 3H, H-1a), 2.01-1.78 (m, 16H, H-4’, H-5’, H-8’, H-

9’, H-16’, H-17’, H-20’, H-21’), 1.67-1.56 (m, 21H, H-24’, H-25’, H-26’, H-27’, H-28’, H-29’, H-30’); ¹³ C NMR (CDCI ₃ , 125 MHz) d 185.4 (C-1), 184.5 (C-4), 146.0 (C-3),

143.3 (C-2), 138.3 (C-15’), 138.0 (SO ₂ Ph), 137.4 (C-3’), 135.7 (C-10’), 135.1 (C-7’),

134.4 (C-19’), 133.3-133.2 (C-7, C-8), 133.2-133.1 (SO ₂ Ph), 132.1-132.0 (C-5, C-10), 131.3 (C-23’), 128.8-128.6 (SO ₂ Ph), 126.4 (C-11’), 126.3 (C-6), 126.2 (C-9), 124.3 (C-

22’), 124.2 (C-6’), 123.8 (C-18’), 119.1 (C-2’), 118.7 (C-14’), 74.0 (C-12’), 39.7-39.6 (C-5’, C-17’, C-21’), 26.7-26.0 (C-4’, C-8’, C-16’, C-20’), 26.0 (C-1’), 25.7 (C-24’), 23.9 (C-13’), 17.6 (C-25’), 16.4-15.8 (C-26’, C-29’, C-30’), 13.6 (C-28’), 12.6 (C-1a). Step 2: MK-6

The solution of MK-6-SO ₂ Ph (200 mg, 0.28 mM) in toluen (5 mL) was cooled to 0°C, and 1M LiEt ₃ BH (2.8 mL, 2.8 mM) was added to the reaction mixture. Then Pd(dppe)Cl ₂ (96 mg, 0.17 mM) was added and the suspension was stirred at 0°C. The reaction was monitored by TLC. After 20 min. the reaction was quenched with saturated NH4CI (10 mL) and water (20 mL). The product was extracted with AcOEt (2 x 10 mL), the organic phase was washed with brine. The organic extract was evaporated to dryness. The crude oil was dissolved in DCM (5 mL) and cooled to 0°C. The solution of CAN (370 mg, 0.7 mM) in acetonitrile/H ₂ O) (3 mL, 5:1 v/v) was added dropwise within 10 min at this temperature. The yellow solution was stirred for lh. Then, water (10 mL) and DCM (10 mL) were added to the reaction mixture. The organic phase was separated, washed with brine and evaporated to dryness. The crude product was purified by column chromatography (hexane, hexane/toluene 3:1 — >1: 1) yielding MK-6 as yellow oil (33 mg, 20%). NMR consistent with Example 1.