WO2011098545A1

WO2011098545A1 - Novel 2-methyl-1,4-naphthoquinone derivatives and precursors thereof, having vitamin k activity

Info

Publication number: WO2011098545A1
Application number: PCT/EP2011/052003
Authority: WO
Inventors: Cees Vermeer
Original assignee: Nattopharma Asa
Priority date: 2010-02-10
Filing date: 2011-02-10
Publication date: 2011-08-18

Abstract

New 2-methyl-1,4-naphthoquinone derivatives with vitamin K activity are provided having the general formula (1): wherein R is a aliphatic group with 12 to 50 carbon atoms. Preferably, R is a straight or branched C_12-50 alkylgroup or a straight or branched C_12-50alkenyl group with 1 to 12 unsaturated bonds. Of these, the compound of formula (1) in which R is a straight C₁₄ alkyl group is most preferred. Pharmaceutical and nutraceutical compositions comprising at least one compound of formula (1) or a pharmacologically active precursor there of are also provided.

Description

Novel 2-methyl-1 ,4-naphthoquinone derivatives and precursors thereof,

having vitamin K activity Field of the invention

This invention relates to novel 2-methyl-1 ,4-naphthoquinone derivatives and precursors thereof, having interesting pharmacological properties, in particular vitamin K activity. The invention further relates to the preparation and use of these compounds, especially in pharmaceutical products and feed additives. More in particular, the invention relates to such compounds with a peptide side chain at the 3-position, having interesting vitamin K properties which make them potentially suitable for the treatment and/or prophylaxis of disorders related to bone, cartilage and the cardiovascular system in mammals, in particular humans, animals in the veterinary industry and pets. Background

Vitamin K denotes a group of lipophilic, hydrophobic vitamins needed for the posttranslational modification of certain proteins, mostly required for blood coagulation; the coagulation factors VII, IX, X and pro-thrombin are all vitamin K-dependent. Apart from hemostasis, vitamin K is also associated with various other important biological functions, such as bone metabolism, inhibition of vascular calcification, cellular growth, and early skeletal development. Chemically, all compounds with vitamin K activity are 2-methyl-1 ,4- naphthoquinone derivatives.

The naturally produced vitamins K-i and K₂ are the most important and well- known members of the vitamin K family, which are discussed below in some more detail. There are also three synthetic forms of vitamin K, vitamins K₃, K₄ and K₅, which are used in many areas including the pet food industry (vitamin K₃) and to inhibit fungal growth (vitamin K₅).

Vitamin K-i , also known as phylloquinone or phytomenadione, has a functional naphthoquinone ring and an aliphatic side chain at the 3-position, notably a phytyl side chain (i.e. invariably containing four isoprenoid residues, one of which is unsaturated). It is found in plants, mainly in leafy green vegetables, such as spinach, swiss chard and Brassica (e.g. cabbage, kale, cauliflower, broccoli and Brussels sprouts), and some fruits, such as avocado and kiwifruit.

Vitamin K₂, also known as menaquinone, has a functional naphthoquinone ring and an aliphatic side chain at the 3-position, containing a varying number of unsaturated isoprenoid residues. Generally, menaquinones are designated as MK-n, where n specifies the number of isoprenoids in the side chain. The length of the side-chain governs important physico-chemical properties of these molecules. This is especially relevant for the hydrophobicity/solubility in aqueous media. Menaquinone-4 (MK-4, also known as menatetrenone) is found in meat end eggs, MK-7 is typically found in the Japanese soy product natto, whereas menaquinones with still longer side chains (mainly MK-8, MK-9 and MK-10) are found in dairy products, such as cheese and curd cheese. MK-4 is synthesized by animal tissues, other long chain menaquinones are synthesized by bacteria during fermentation. A major problem is that only a few foods are available that contain these long-chain menaquinones, but only in low dosages.

Previous studies have indicated that MK-7 is the most effective form of vitamin K. Natto is one of the richest food sources of MK-7, but its taste is not appreciated by Western society. For this reason, the active biological compound has been manufactured as enriched oil or as Arabic gum powder. The product MenaQ7™ from NattoPharma ASA, a natural extract derived from natto, contains the most active form of supplemental K vitamins available (MK-7). However, the amount of MK-7 per g product is low (1500 - 2000 ppm), which increases the demand for a synthetic higher menaquinone.

The higher menaquinones (MK-7 to MK-13) are absorbed in the same way as vitamin K-i , but are efficiently redistributed by the liver, predominantly in HDL and LDL. Since LDL has a long half-life time in the circulation, these menaquinones have much better bioavailability for extra-hepatic tissue uptake compared to vitamin K-i .

The object of the present invention is to provide novel synthetic vitamin K- analogues with the following properties or characteristics:

• High vitamin K-activity

• No cytotoxicity (this is a requirement for the new compounds, as well as for their breakdown products that can be generated in vivo)

· Relatively straightforward preparative methods that allow for upscaling to industrial levels.

The underlying hypothesis is that synthetic molecules with vitamin K-activity may become commercially interesting as drugs, in particular pharmaceutical compositions containing such a molecule as an active ingredient, or as food-additives, especially if the industrial synthesis becomes a challenging alternative to cumbersome biological production routes such as fermentation. Furthermore, chemical synthesis may yield entirely new combinations of K-activity and water-solubility, and this in turn may lead to drug molecules with unprecedented bio-distribution properties. In other words, synthetic molecules with K-activity may become useful as drugs since they can be designed to target for specific tissues or body compartments and thus possess pharmacokinetic properties different from phylloquinone or menaquinones.

As used herein, vitamin K activity is defined as the capability to serve as a cofactor for the endoplasmic enzyme gammaglutamyl carboxylase (GGCX), and thus to promote the formation of gammacarboxy glutamate residues in proteins belonging to the Gla-protein family. Brief description of the drawings

In the figures the results are shown of the expression of cOC and ucOC in osteoblast cells (MG-63) with various 2-methyl-1 ,4-naphthoquinone ("MX") derivatives according to the present invention and precursors thereof.

Figures 1 and 2 show the effect of 50 nM of MX-5, MX-6, MX-7 and menaquin- one 4 (MK-4) on the carboxylation of cOC and ucOC synthesis in osteoblast cells.

Figures 3 and 4 show the effect of 500 of nM MX-8, MX-9, MX-10, MX-1 1 , MX- 12 and MK-4 on the carboxylation of cOC and ucOC synthesis in osteoblast cells.

Figures 5 and 6 show the effect of a lower amount (50 nM) of the same compounds as in Figures 3 and 4 on the carboxylation of cOC and ucOC synthesis in osteoblast cells

Summary of the invention

In one aspect of the present invention, new 2-methyl-1 ,4-naphthoquinone derivatives are provided having the general formula (1 ):

wherein R is a aliphatic group with 12 to 50 carbon atoms.

Preferably, R is a straight or branched C12-50 alkyl group or a straight or branched C12-50 alkenyl group with 1 to 12 unsaturated bonds.

A preferred group of compounds according to the present invention is the group of formula (1 ) wherein R is a straight alkyl group with 12 to 50 carbon atoms, even more preferably 12 to 30 carbon atoms and most preferably 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Of these, the compound of formula (1 ) in which R is a straight C14 alkyl group, also designated with the internal code MX12, is at present most preferred.

X-12 (C-14 tail)

Another preferred group of compounds according to the present invention is the group of formula (1 ) wherein R is an aliphatic group of 3 to 12 isoprenoid residues, more preferably 4 to 9 isoprenoid residues. Most preferably, the isoprenoid residues each have a saturated bond.

The compounds according the present invention including certain precursors thereof show a pharmacological activity similar to vitamin K, in particular in carboxylating the above-mentioned coagulation factors and the activation of the calcification inhibitor Matrix Gla-Protein (MGP), which make them suitable, or potentially suitable, for the treatment and/or prophylaxis of various disorders in mammals, in particular humans, animals in the veterinary industry and pets. These disorders relate primarily to bone and cartilage disorders and disorders of the cardiovascular system, such as osteoporosis, atherosclerosis and osteoarthritis.

Precursors of the compounds of formula (1 ) showing pharmacological activity similar to vitamin K are com

and formula (3):

or pharmaceutically acceptable salts and esters thereof and therefore form part of the present invention.

In another aspect of the present invention pharmaceutical and nutraceutical compositions are provided comprising at least one compound according to the invention as defined above, or a precursor thereof, optionally in conjunction with one or more pharmacological active ingredients, and/or adjuvants and/or additives.

The compounds of formula (1 ) according to the present invention can be suitably prepared by a method which comprises reacting the 3-(2-methyl-1 ,4-naphtho- quinone)acetic acid derivative of formula 2):

or a reactive derivative thereof with a primary amine of the formula R-NH₂, wherein R has the same meanings as in formula (1 ), or a reactive derivative thereof, in a manner known per se for the reaction of compounds with similar or identical reactive groups, followed by removing the protective groups of the naphthoquinone structure.

These and other aspects of the present invention will be discussed below in further detail.

Detailed description of the invention

The compounds of formula (1 ) according to the present invention can be made by a peptide coupling reaction of a reactive 3-(2-methyl-1 ,4-naphthoquinone)acetic acid derivative of formula (2 also designated with the internal code MX-7:

with a primary amine of the general formula R-NH₂ or a reactive equivalent thereof in a manner well-known to a person skilled in the art. Generally, the quinone oxygen atoms must be protected and deprotected before and after the coupling reaction, respectively, in order to avoid undesired side reactions.

As used herein, the terms "reactive", "reactive derivative" and "reactive equivalent" are commonly known terms, for example in chemical handbooks or textbooks, which are meant to indicate alternative reactions which are fully within the skill of an average person skilled in the art. For example, acid anhydrides and in particular acyl chlorides (or bromides) are known to be reactive derivatives of carboxylic acids and can be used instead for the formation of, for example, amides.

The inventors reasoned that 3-(2-methyl-1 ,4-naphthoquinone)acetic acid of formula (3), also designated with the internal code MX-6,

(3) (MX-6) would be a versatile synthon, since the COOH group allows for virtually infinite possibilities for further derivatization. For instance, the COOH group can be used in the synthesis of esters of amides, using standard synthetic organic chemistry reaction protocols. However, in performing this route complex mixtures of the desired product and undesired side- products were obtained. The reaction conditions presumably also induced reactions of the quinone moiety.

In contrast, it was found that derivatization of MX-7 appeared to be possible without the difficulties experienced during the derivatization of MX-6. Both MX-7 and MX-6 and their ethyl esters are known from the literature as synthetic intermediates, but no pharmacological properties of these compounds or salts or esters thereof have been described. See Jacobs et al., Tetrahedron 63:2503-2510 (2007); Kestelyn et al., Synthesis 11:1881 -1883 (1999); Maccorquodale et al., J. Biol. Chem., Amer. Soc. for Biochem. and Mol. Biol., Inc. US, 131:357-370 (1939-1 1 -01 ); Kazemekaite et ai, Tetrahedron Letters 45:3551 -3555 (2004).

It has now been found that the precursor compound (2), having the internal code MX-7, can be suitably made according to the following reaction scheme:

It was found that using bromoacetic acid or esters of bromoacetic acid, for example ethyl bromoacetic acid, in combination with pyridine or another heterocyclic aromatic molecule containing nitrogen as one of the heterocyclic atoms, leads under conditions of absolute dryness and elevated temperature, e.g. 40-60°C, to the formation of an ylide. In such an ylide, the heterocyclic nitrogen carries a positive charge, whereas bromine is present as the negatively charged counterion. The ylide is a vulnerable and relatively short-lived structure that must be handled with care. Under certain conditions, such as absolute dryness and a reaction time between 10 min and 6 h, depending on the exact nature of the ylide, usually at elevated temperature, the ylides can be reacted with menadione, and this produces a unique C-C bond formation, between the methylene group that originally carried bromine (in bromoacetic acid or one of the esters derived thereof), and C2 of menadione. This reaction is essentially the key of the synthetic pathway to prepare the compounds of formula (1 ) through the intermediate compounds of formula (2) and/or formula (3).

Subsequently, the resulting menadione derivative is reduced to produce MX-4, for example by standard treatment with tetrabutylammonium iodide. Finally, the ester is hydrolyzed in a manner known per se yielding MX-7 which has the carboxylic acid functionality.

It appeared that this ylide-based synthetic approach gives much better access to MX-7. It will be evident to a person skilled in the art that MX-7 can be derivatized ad infinitum through its COOH functionality. Finally, the molecule must re-oxidized through standard treatment, e.g. with eerie ammonium nitrate.

Using this synthetic strategy, the following novel compounds were prepared:

MX-9: C-17 tail

MX-10: C-16 tail

MX-1 1 : C-18 tail

Formulation of products and dosages

The pharmaceutical compositions comprising one or more compounds according to the present invention are preferably administered systematically (e.g. orally or parenterally). The most preferred dosage form is an oral dosage form; especially capsules and tablets. The dosage and route of administration will depend on factors such as the age and sex of the individual, the severity and nature of the disorder or disease, and the like, and can easily be determined by a person skilled in the art, usually a physician, or the instructions leaflet of the manufacturer are to be followed which usually accompany the product.

Tablets and capsules can be prepared by any conventional manner known in the art. The capsules can for example be soft or hard gelatin capsules with in addition to the active ingredient comprise an inactive pharmaceutically acceptable adjuvant, for example starch. Tablets may be prepared, for example, by direct compression or by compression of granules using conventional tablet machines. Tablets according to the present invention might in addition to the active ingredient comprise of pharmaceutically acceptable inactive ingredients well known in the art. Such agents can for example be cellulose derivatives and magnesium stearate. Tablets according to the present invention can be coated with a gastric resistant coating, for example cellulose acetate phthalate.

The oral dosage forms, capsules and tablets, according to the present invention, have each a weight between 100 mg and 2 grams. The amount of the active ingredient according to the invention, i.e. one or more compounds of formula (1 ) as defined above, in each tablet or capsule may vary over a wide range, depending inter alia upon factors such as the severity and nature of the disease or disorder, and the condition, sex and age of the patient. The amount of active ingredient in one tablet or capsule is typically between 1 and 500 μg, although higher amounts are also possible. Preferred dosages are between 2 and 300 μς, more preferably 5-50 μς, and most preferably between 10-20 per day.

Adjuvants and/or additives may be added to facilitate administration of the pharmaceutical compositions or to improve their bioavailability, and the like. Such adjuvants and/or additives are well within the skill of the average artisan and need not to be discussed therfore in further detail.

Nutraceutical compositions are preferably in the form of food additives, the various ingredients of which as well as their preparation is also known to a person skilled in the art.

The compositions according to the present invention can optionally comprise other pharmaceutically or nutraceutically active components; for example vitamins like vitamin D or vitamin D derivatives; active drug compounds such as bisphosphonates, typically alendronate; or cardiovascular drugs, such as ACE inhibitors, for example enalapril, angiotensin II receptor antagonists, for example losartan, beta-blockers, for example propranolol, plasma lipid reducing components, such as statins, typically simvastatin, and other drugs. Such combinations are known in the art and a person skilled in the art will have no difficulty in selecting the combination of his choice depending on the intended use and preparing such compositions.

The present invention will now be further described with reference to the following examples and experimental work, which however are not to be construed as limiting the invention in any respect.

EXPERIMENTAL

Synthetic procedures

The preparation of the compounds of formula (1 ) was essentially carried out according to the reaction scheme mentioned below which will be discussed hereinafter in more detail.:

Step 1 : Synthesis of the ylide

The following reagents were transferred into a 100-mL round bottom flask (ambient conditions): Pyridin (10.0 mL), absolute ethanol (15.0 mL), ethyl bromoacetate (10.0 g, 59.88 mmol). The reaction mixture adopted a light-yellow color. After several minutes, the start of a chemical reaction was noticed (increase of the temperature). The flask was closed with a loose stopper, and the reaction mixture was left to stand for 24 h. Then, the reaction mixture was mixed with diethyl ether (100 mL, and the solution was cooled to. Crystals appeared after several hours and were isolated through filtration on a Buchner funnel.

Step 2: Reaction of the ylide with menadione

The pyridium bromide prepared in step 1 (10.0 g, 40.65 mmol) was dissolved in 300 mL of absolute ethanol. The solution was magnetically stirred for 5 min, and triethylamine (5.6 mL, 40.17 mmol) was added. The color of the solution turned yellow. Then, menadione (6.8 g, 39.53 mmol) was added, and stirring was continued for 96 h. The reaction mixture was diluted with dichloromethane (approx. 500 mL), and washed with a diluted solution of hydrochloric acid. The organic layer (bottom) was concentrated (rotary evaporator), leaving a solid material. Part of this material was taken up in toluene. After tic analysis, it was decided to carry out silica gel column chromatography (height 20 cm, diameter 25 mm), using a mixture of ethyl acetate and petroleum ether (ratio 1 : 9) as the eluent. The desired product had Rf = 0.2. Evaporation of the eluent left yellow crystals with melting point 67-69°C.

Step 3: Reduction to obtain MX-4

A procedure described by R.J. Payne and co-workers was followed. The workers used this procedure to prepare 2-bromo-3-methyl-1 ,4-dimethoxynaphthalene. See Bioorganic Medicinal Chemistry Vol. 12, p 5785-5791 (2004). The product of step 2 (1 .0 g, 3.86 mmol), tetrabutylammonium iodine, tetrahydrofuran, and water were combined in a 100-mL round bottom flask. This solution was added to a solution of sodium dithionite (4.04 g, 23.22 mmol) in water (3.5 mL). The mixture was stirred for 15 min, and then a solution of KOH (4.99 g, 89.00 mmol) in water (5.6 mL) was added carefully. The reaction mixture was stirred for another 5 min, and then dimethylsulfate (4.28 mL, 46.44 mmol) was added. Stirring was continued for 48 h. After a few hours the color changed to light-yellow. The reaction mixture was then extracted with dichloromethane. The organic layer was separated, washed (with water), dried (MgS0₄), filtered, and concentrated. The residual oil was chromatographed on a silica gel column (length: 22 cm, diameter: 25 mm), using a mixture of ethyl acetate and petroleum ether (ratio 1 ; 4) as the eluent. The product (MX-4) had Rf = 0.23.

NMR data: 8.3 and 7.6 ppm (4 aromatic Hs), 4.15 (CH₂ of the ethyl group), 3.83 (coinciding signals of both OCH₃ groups and the CH₂ adjacent to C=0), 2.2 methyl group), 1 .3 (CH₃ of ethyl).

Step 4: Conversion of MX-4 into MX-7 (ester hydrolysis)

This reaction was carried out according to a protocol described by Karlsson and coworkers. See Tetrahedron Letters 48:2497-2499 (2007).

MX-4 (240 mg) was mixed with 5 mL of acetontril and 0.1 mL of water. Triethylamine (252 mg) and LiBr (720 mg) were then added and the resulting mixture was stirred magnetically overnight. Then, the reaction was quenched through addition of glacial acetic acid (4.2 mL). Ethyl acetate (50 mL) was added and the resutling solution was washed with water. The organic layer was separated, dried (MgS0₄), filtered and concentrated. The resulting oil was chromatographed on a silica gel column (height 1 1 cm, diameter 25 mm), using ethyl acetate and petroleum ether (ratio 1 ; 4) as the eluent. The desired product had Rf = 0.1 ,

NMR data: 8.3 and 7.7 ppm (4 aromatic Hs), 3.8 (CH₂ group and the two OCH₃ groups coinciding), 2.2 (CH₃ group). Step 6: Reaction of MX-7 with any primary amine

This reaction is the versatile element in the synthetic procedure. The method is illustrated with the hexadecylamine [H₂N-(CH₂)i5-CI-l3] . In other words, this is the preparation of MX-10 (carrying the C-16 side chain).

MX-7 (216 mg) and hexadecylamine (400 mg) were dissolved in dry dichloromethane (5 ml_). Then Ν,Ν-dicyclohexylcarbodiimide (DCC, 343 mg) was added. Stirring was continued overnight. The reaction mixture was cooled on an ice bath and the dicyclohexylurea was removed by filtration. The filtrate was concentrated and the residue was chromatographed on a silica gel column (height 5 cm, diameter 20 mm), using ethyl acetate and petroleum ether (ratio 1 : 2) as the eluent. The purified product had R_f = 0.22.

This reaction can be executed in principle with any primary amine, as will be clear to a person skilled in the art. The reaction is to be carried out under strict anhydrous conditions.

NMR data: 8.4 and 7.7 ppm (4 aromatic Hs), 3.0 (CH₂ next to NH), 3.8 (both OCH₃ groups), 2.2 (methyl group), 1.5 - 1 .0 (CH₂ groups of the side chain or "tail"), 0.9 terminal CH₃ group of the side chain).

Step 7. Re-oxidation of the product of step 6

This reaction is carried out according to a procedure published by R.J. Payne and coworkers. See: Bioorganic Medicinal Chemistry Vol. 12, p 5785-5791 (2004). The product of step 6 (200 mg) was dissolved in a mixture of acetonitrile and water (ratio 2 : 1 ; 2.4 ml_). The solution was cooled on an ice bath. Then, a solution of CAN (eerie ammonium nitrate (600 mg in 2 mL acetonitrile-water (1 :1 )) was added carefully. Stirring was continued for 30 min (still in the ice bath). Then, stirring was continued for 30 min at room temperature). The reaction mixture was extracted with dichloromethane, The organic phase was dried (MgS0₄), filtered, and concentrated. The residue was chromatographed on a small-scale silica gel column (height 4 cm, diameter 12 mm), using ethyl acetate and petroleum ether (ratio 1 : 2) as the eluent. The R_f of the desired product was 0.25.

NMR data: 8.1 (NH), 8.0 and 7.6 (4 aromatic Hs), 3.0 (CH₂ next to NH), 2.85 (CH₂ next to C=0), 2.4 (methyl group), 1.5 - 1 .0 (CH₂ groups in the tail), 0.9 terminal CH₃ group of the tail).

Pharmacological activity

The synthetic compounds of the present invention with the internal codes MX-5 to MX-12 were subjected to various tests. First of all, the effect of these compounds on cOC and ucOC synthesis in osteoblastic cells was determined. In these experiments, it was observed that 50 nM concentration of MX-10 or MX-12 had a clear K-activity. Both compounds had approximately the double activity as compared to menaquinone 4 (MK-4).

Furthermore, the compounds MX-9 to MX-12 were tested with respect to their cytotoxicity. These experiments revealed that the compounds were no more cytotoxic than MK-4. In fact, there was a close resemblance with MK-4.

Expression of cOC and ucOC in osteoblastic cells (MG-63)

Experiment 1 : Effect of MX-5, MX-6, MX-7 and MK-4 on the carboxylation of cOC and ucOC synthesis in osteoblastic cells in the presence of warfarin

MG-63 cells were grown to confluency (80%) in the presence of 10% fetal calf serum. The media were then exchanged for serum-free media containing 10 nM 1 ,25(01-1)

2D3 and 10 uM warfarin (to deplete the system from any vitamin K that might be present).

After 24 h of pre-treatment, the media were again exchanged for fresh serum-free media containing 10 nM 1 , 25(OH) 2D3 and 10 uM warfarin and in the presence or absence of 50 nM MX-5-6-7or MK-4. Samples were taken at 0, 8, 24, 48 and 72 hours. Medium aliquots of 10Oul were assayed for ucOC and cOC (Takara, EIA Kit). The results are shown in

Figures 1 and 2.

Conclusions:

1 . The control cells (no MX added) produced uncarboxylated osteocalcin at a regular rate.

Cells supplemented with MK-4 showed significantly less ucOC production, those supplemented with MX compounds showed still lower ucOC production rates, in the order: MX-7 < MX-6 < MX-5 (see Figure 1 ).

2. The control cells (no MX added) produced no measurable amounts of carboxylated osteocalcin, which is consistent with total vitamin K deficiency. Cells supplemented with MK-4 showed cOC production in significant amounts, in the order: MK-4 < MX-5 < MX-6 < MX-7 (see Figure 2).

3. The data for ucOC production and for cOC production are precisely complementary.

4. Taken together, these data demonstrate vitamin K activity of the compounds MX-5, MX-6 and MX-7.

5. Each of the MX compounds has a higher vitamin K activity than MK-4.

6. The increasing vitamin K activity of the various MX compounds suggests that other (longer) side chains may possess even higher vitamin K activity.

Experiment 2: Effect of MX-8, MX-9, MX-10, MX-1 1 , MX-12 and MK-4 on the carboxylation of cOC and ucOC synthesis in osteoblastic cells in the presence of warfarin (at 500 nM dose)

MG-63 was grown to confluency (80%) in the presence of 10% fetal calf serum. The media were then exchanged for serum-free media containing 10 nM 1 , 25(OH) 2D3 and 10 uM warfarin. After 24 h of pre-treatment, the media were again exchanged for fresh serum-free media containing 10 nM 1 , 25(OH) 2D3 and 10 uM warfarin and in the presence or absence of 500 nM of MX-8, MX-9, MX-10, MX-1 1 , MX-12 or MK-4. Samples 5 were taken at 0, 24, 48 and 72 hours. Medium aliquots of 100 ul were assayed for ucOC and cOC (Takara, EIA Kit). The results are shown in Figures 3 and 4.

Conclusions:

7. The control cells (no MX added) produced uncarboxylated osteocalcin at a regular rate. 10 Cells supplemented with 500 nM of MK-4, MX-10 or MX-12 showed no ucOC production at all (lines are superposed), those supplemented with MX-8, MX-9 and MX- 1 1 compounds showed still residual ucOC production rates (see Figure 4).

8. The control cells (no MX added) produced no measurable amounts of carboxylated osteocalcin, which is consistent with total vitamin K deficiency. Cells supplemented

15 with MK-4, MX-10 or MX-12 showed cOC production in significant amounts (lines superposed), those supplemented with MX-8, MX-9 or MX-1 1 hardly produced cOC (see Figure 3).

9. The data for ucOC production and for cOC production are precisely complementary.

10. Taken together, these data demonstrate vitamin K activity of the compounds MX-10 20 and MX-12.

1 1 . Lower dosages should be tested in a separate experiment.

Experiment 3: Effect of MX-8, MX-9, MX-10, MX-1 1 , MX-12 and MK-4 on the carboxylation of cOC and ucOC synthesis in osteoblastic cells in the presence of warfarin (at 50 nM 25 dose)

MG-63 was grown to confluency (80%) in the presence of 10% fetal calf serum. The media were then exchanged for serum-free media containing 10 nM 1 , 25(OH) 2D3 and 10 uM warfarin. After 24 h of pre-treatment, the media were again exchanged for fresh serum-free media containing 10 nM 1 ,25(01-1) 2D3 and 10 uM warfarin and in the 30 presence or absence of 50 nM of MX-8, MX-9, MX-10, MX-1 1 , MX-12 or MK-4. Samples were taken at 0, 24, 48 and 72 hours. Medium aliquots of 100 ul were assayed for ucOC and cOC (Takara, EIA Kit). The results are shown in Figures 5 and 6.

Conclusions:

35 12. The control cells (no MX added) produced uncarboxylated osteocalcin at a regular rate.

Cells supplemented with 50 nM of MK-4, MX-10 or MX-12 showed very little ucOC production at all (MK-4 and MX-12 are superposed), those supplemented with MX-8, MX-9 and MX-1 1 compounds showed still residual ucOC production rates (see Figure 6).

13. The control cells (no MX added) produced no measurable amounts of carboxylated osteocalcin, which is consistent with total vitamin K deficiency. Cells supplemented with MK-4, MX-10 or MX-12 showed cOC production in significant amounts with a 2- fold higher efficacy for MX-10 and a 2.5-fold higher efficacy for MX-12. Cells supplemented with MX-8, MX-9 or MX-1 1 hardly produced cOC (see Figure 5).

14. The data for ucOC production and for cOC production are precisely complementary.

15. Taken together, these data demonstrate vitamin K activity of the compounds MX-10 and MX-12 and are suggestive for an even 2-fold higher efficacy of MX-10 and MX-12 than the well-known form of vitamin K₂, menatetrenone (MK-4).

16. In none of the experiments cytotoxicity was observed for any of the MX compounds

17. Cytotoxicity was also tested in fibroblasts, vascular smooth muscle cells and endothelial cells, but no toxicity was observed.

Based on these data in vivo, experiments were designed to test the biological activity of MX-12.

Characterization of MX-12 in an animal model

Study design

In the first week of the study, all 24 rats (12 male + 12 female WKY rats) received a vitamin K-deficient control diet (½ vitamin K-deficient standard rodent food + ½ dried rice). After this run-in period, 4 control rats (2 M/2 F) were sacrificed, and blood and several organs were collected (t = 0; baseline). The remaining 20 rats (10 M/10 F) were randomly divided over the following diet groups; control diet (n = 8; 4 M/4 F), low MX-12 diet (n = 4; 2 M/2 F), or high MX-12 diet (n = 8; 4 M/4 F). The rats on the control diet were sacrificed after 2 (n = 4; short term effect) and 5 days (n = 4; long term effect). In line, both short-term (n = 4) and long-term (n = 4) effects were investigated in the high MX-12 group (5 μg MX-12/g of food; enriched vitamin K-deficient rodent food). Only long-term effects were measured after feeding the low MX-12 diet (0.5 μg MX-12/g of food; enriched vitamin K-deficient standard food). Again, blood was sampled and some organs were removed. t = day -7

n = 24 WKY rats

control diet (vitamin K deficient) t = day 0 (baseline)

t n = 4 (controls) t = day 0 t = day 0 t = day 0

n = 8 n = 4 n = 8

control diet control diet + low MX-12 control diet + high MX-12

(0.5 (5 μ§ Β diet) t = day 2 t = day 2

t n = 4 (controls) ^† n = 4 (high MX-12) t = day 5 t = day 5 t = day 5

t n = 4 (controls) ^† n = 4 (low MX-12) ^† n = 4 (high MX-12)

Measurements

• Vitamin K activity

Plasma prothrombin levels were measured to investigate the effects coagulation.

• Tissue distribution

MX-12 levels are measured in the following tissues; aorta, liver, and kidney.

Results

Vitamin K activity - male rats

Plasma prothrombin levels (as compared to normal plasma expressed as 100%) significantly decreased by 40% in the male WKY rats after feeding the vitamin K- deficient control diet for one week (baseline). Supplementing the low dose of MX-12 for 5 days significantly increased plasma prothrombin levels back to normal (plasma prothrombin level: 103 ± 4%). Administration of the high dose of MX-12 for only 2 days significantly increased plasma prothrombin activity back to normal (plasma prothrombin level: 107 ± 1 %). Supplementation for 3 extra days with this high dose of MX-12 had no additional effect (plasma prothrombin level: 106 ± 3%). Taken together, these results indicate that MX-12 has high vitamin K activity. Plasma prothrombin Baseline

Start ("normal") t = 0 days t = 2 days t = 5 days

Control (no MX-12) 97 ± 2 60 ± 4 67 ± 4 53 ± 4

Low level of MX-12 103 ± 4

High level of MX-12 107 ± 1 106 ± 3

Vitamin K activity - female rats

Female rats did not reach a vitamin K-deficient state (no change in plasma prothrombin levels; results not shown) after one week of feeding the vitamin K-deficient control diet (½ vitamin K-deficient standard rodent food + ½ dried rice). It is known, however, that estrogen can cause - when vitamin K is low - a more rapid and efficient production of prothrombin (Matschiner and Taggart). This may be an explanation for the lack of effect in the female rats. Final remarks

These results demonstrate that MX-12 has significant vitamin K activity. Further studies are needed to compare its in vivo bioactivity with vitamin K-i, menaquinone- 4 (MK-4) and menaquinone-7 (MK-7).

Claims

1 . A 2-methyl-1 ,4-naphthoquinone derivative having the general formula (1 ):

wherein R is an aliphatic group with 12 to 50 carbon atoms.

2. A compound according to claim 1 , wherein R is a straight or branched C12-50 alkyl group or a straight or branched C12-50 alkenyl group with 1 to 12 unsaturated bonds.

3. A compound according to claim 1 or 2, wherein R is a straight alkyl group with 12 to 50 carbon atoms.

4. A compound according to claim 3, wherein R has 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.

5. Th compound according to claim 4, wherein R has 14 carbon atoms.

6. A pharmaceutical or nutraceutical composition having vitamin K activity, which comprises as an active ingredient one or more compounds as defined in any one of claims 1 to 5 or a precursor thereof, optionally in conjunction with one or more adjuvants and/or additives.

7. A composition according to claim 6, wherein said precursor with vitamin activity is a compound of formula (2):

or formula (3):

or a pharmaceutically acceptable salt or ester thereof.

A method of preparing a compound of formula (1 ) as defined in claim 1 which prises reacting a 3-(2-methyl-1 ,4-naphthoquinone)acetic acid derivative of formula (2):

or a reactive derivative thereof with a primary amine of the formula R-NH₂, wherein R has the same meanings as in formula (1 ), or a reactive derivative thereof, followed by removing the protective groups of the naphthoquinone structure.