COMPOSITIONS OF BIOLOGICALLY ACTIVE MENAQUINOL DERIVATIVES AND METHODS OF TREATMENT

Abstract

The present application discloses, in part, isolated, stable and biologically active menaquinol derivatives and their methods of use for the treatment of various diseases.

Claims

1. A biologically active menaquinol derivative of the formulae: ##STR00040## wherein: each n is independently 7, 8 or 10; and each R.sup.1 and R.sup.2 is independently C.sub.1-C.sub.6alkyl.

2. The menaquinol derivative of claim 1 selected from the group consisting of: ##STR00041##

3. A biologically active menaquinol derivative of the formulae: ##STR00042## ##STR00043## ##STR00044## wherein: m is 1, 2, 3, 4 or 5; each n is independently 7, 8 or 10; and each R.sup.1 and R.sup.2 is independently C.sub.1-C.sub.6alkyl.

4. The menaquinol derivative of claim 3 selected from the group consisting of: ##STR00045## wherein each n is independently 7, 8 or 10.

5. A menaquinol derivative of the formula I, II, III, IV, V, VI, VII or VIII: ##STR00046## ##STR00047## wherein: each D and E is independently − (a negative charge) or H; M is a metal ion or an amine salt selected from the group consisting of Li.sup.+, Na.sup.+, K.sup.+, Mg.sup.2+, Ca.sup.2+, Al.sup.3+ and Zn.sup.2+, and arginine, benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, ethylenediamine, lysine, histidine, meglumine, procaine and triethylamine; X is 0, 1 or 2; Y is 0.5, 1, 2 or 3; Z is 0 or 1; n is 7, 8, 9 or 10; and t is 0, 1, 2 or 3; and the hydrates thereof.

6. The menaquinol derivative of claim 5 that is a dimer salt of the formula: ##STR00048## wherein: each D and E is independently − (a negative charge) or H; M is a metal or a salt selected from the group consisting of Li.sup.+, Na.sup.+, K.sup.+, Mg.sup.2+, Ca.sup.2+, Al.sup.3+ and Zn.sup.2+, and arginine, benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, ethylenediamine, lysine, histidine, meglumine, procaine and triethylamine; X is 0, 1 or 2; Y is 0.5, 1, 2 or 3; Z is 0 or 1; and n is 7, 8, 9 or 10.

7. The menaquinol derivative of claim 5 that is a dimer of the formula: ##STR00049## wherein: n is 7, 8, 9 or 10; and t is 0, 1, 2 or 3.

8. The menaquinol derivative of claim 5 that is a dimer of the formula: ##STR00050## wherein: n is 7, 8, 9 or 10; and t is 0, 1, 2 or 3.

9. The menaquinol derivative of claim 5 that is a dimer of the formula: ##STR00051## wherein: n is 7, 8, 9 or 10; and t is 0, 1, 2 or 3.

10. The menaquinol derivative of claim 5 that is a dimer of the formula: ##STR00052## wherein n is 7, 8, 9 or 10.

11. The menaquinol derivative of claim 5 that is a dimer of the formula: ##STR00053## wherein n is 7, 8, 9 or 10.

12. The menaquinol derivative of claim 5 that is a dimer of the formulae: ##STR00054## wherein n is 7, 8, 9 or 10.

13. The menaquinol derivative of claim 5 that is a salt of the formulae Ia to Ii: ##STR00055## ##STR00056## wherein n is 7, 8, 9 or 10.

14. A pharmaceutical composition comprising a therapeutically effective amount of a menaquinol derivative of any one of claim 1, or a mixture thereof; and a pharmaceutically acceptable excipient, wherein the composition is effective for the treatment of a condition associated with vitamin K selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification.

15. A method for increasing the tissue concentration of menaquinol as a co-factor for gamma glutamate carboxylase (GGCX) for catalyzing the carboxylation of vitamin K dependent proteins that is associated with the treatment or prevention of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification in a patient in need thereof, the method comprising an administration of a therapeutically effective amount of a menaquinol derivative or a pharmaceutical composition comprising an effective amount of a menaquinol derivative of claim 1, or a mixture thereof.

16.-45. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURE

[0116] FIG. 1 is a representation of a chromatogram of menaquinone-7 and its regioisomer shown with a ratio of 3:1, as determined by .sup.1H NMR.

[0117] FIG. 2 is a scheme showing the uremia and dialysis induced oxidation of KH2 functional carboxylation of vitamin K dependent proteins.

[0118] FIG. 3 is graph showing the VKORC I in arbitrary units and specific tissues.

[0119] FIG. 4 is a graph showing the NADPH in arbitrary units and specific tissues.

[0120] FIG. 5 is a graph showing CKD and ESRD patients exhibit a higher percentage of carbonyl proteins compared to normal controls.

EXPERIMENTAL

[0121] The following procedures may be employed for the preparation of the compounds of the present invention. The starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as the Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis, Mo.), or are prepared by methods well known to a person of ordinary skill in the art, following procedures described in such references as Fieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

[0122] In some cases, protective groups may be introduced and finally removed. Suitable protective groups for amino, hydroxy, and carboxy groups are described in Greene et al., Protective Groups in Organic Synthesis, Second Edition, John Wiley and Sons, New York, 1991. Standard organic chemical reactions can be achieved by using a number of different reagents, for examples, as described in Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

Preparation of Menaquinol Derivatives:

[0123] Sodium salt of Menaquinol-7: 4.0 g (6.16 mmol) of MK-7 and 60 ml of ethanol is added into a 250 ml 3-N RBF equipped with a magnetic stirrer, and the resulting mixture was stirred at 0° C. for 1 hour under nitrogen. Sodium hydroxide (0.246 g, 1.0 mole equiv) dissolved in water (15 ml) was added dropwise (or slowly titrated in) to the solution over a period of 15 minutes. The resulting mixture was stirred at 0° C. for another hour and slowly warmed to RT. The solvent was removed by rotoevaporation at 35° C. under reduced pressure to afford the isolated menaquinol compound I, where M=Na.sup.+ (x is 0), Y is 1, D is − and Z is 0, as an off-white powder.

[0124] Sodium salt of Menaquinol-7: 4.0 g (6.16 mmol) of MK-7 and 60 ml of ethanol is added into a 250 ml 3-N RBF equipped with a magnetic stirrer, and the resulting mixture was stirred at 0° C. for 1 hour under nitrogen. Sodium hydroxide (0.495 g, 2.01 mole equiv) dissolved in water (15 ml) was added dropwise (or slowly titrated in) to the solution over a period of 15 minutes. The resulting mixture was stirred at 0° C. for another hour and slowly warmed to RT. The solvent was removed by rotoevaporation at 35° C. under reduced pressure to afford the isolated menaquinol compound I, where M=Na.sup.+ (x is 0), Y is 1, D and E are both − and Z is 0, as an off-white powder.

[0125] Potassium salt of Menaquinol-7: 4.0 g (6.16 mmol) of MK-7 and 60 ml of ethanol is added into a 250 ml 3-N RBF equipped with a magnetic stirrer, and the resulting mixture was stirred at 0° C. for 1 hour under nitrogen. Potassium hydroxide (0.346 g, 1.0 mole equiv) dissolved in water (15 ml) was added dropwise (or slowly titrated in) to the solution over a period of 15 minutes. The resulting mixture was stirred at 0° C. for another hour and slowly warmed to RT. The solvent was removed by rotoevaporation at 35° C. under reduced pressure to afford the isolated menaquinol compound I, where M=K.sup.+ (x is 0), Y is 1, D is − and Z is 0, as an off-white powder.

[0126] Calcium salt of Menaquinol-7: 4.0 g (6.16 mmol) of MK-7 and 60 ml of ethanol is added into a 250 ml 3-N RBF equipped with a magnetic stirrer, and the resulting mixture was stirred at 0° C. for 1 hour under nitrogen. Calcium hydroxide (0.228 g, 0.5 mole equiv) dissolved in water (15 ml) was added dropwise (or titrated in) to the solution over a period of 15 minutes. The resulting mixture was stirred at 0° C. for another hour and slowly warmed to RT. The solvent was removed by rotoevaporation at 35° C. under reduced pressure to afford the isolated menaquinol compound I, where M=Ca+.sup.2 (x is 1), Y is 2, D is − and Z is 0, as an off-white powder.

[0127] Calcium salt of Menaquinol-7: 4.0 g (6.16 mmol) of MK-7 and 60 ml of ethanol is added into a 250 ml 3-N RBF equipped with a magnetic stirrer, and the resulting mixture was stirred at 0° C. for 1 hour under nitrogen. Calcium hydroxide (0.456 g, 1.0 mole equiv) dissolved in water (15 ml) was added dropwise (or titrated in) to the solution over a period of 15 minutes. The resulting mixture was stirred at 0° C. for another hour and slowly warmed to RT. The solvent was removed by rotoevaporation at 35° C. under reduced pressure to afford the isolated menaquinol compound I, where M=Ca+.sup.2 (x is 1), Y is 1, D and E are both − and Z is 1, as an off-white powder.

[0128] The menaquinol derivatives, such as the menaquinol-7 derivatives may be prepared according to the general methods as described below. Such acylated linked compounds may be symmetrical, wherein both hydroxyl groups of the menaquinol are acylated and linked to another menaquinol molecule, or only one of the two hydroxyl groups, either the 5-position or the 8-position, are acylated and linked to another menaquinol molecule, and the other remaining as the free hydroxyl group of the menaquinol.

[0129] Accordingly, the menaquinone, such as MK-7, may be contacted with a metal, such as zinc, in an acid, such as acetic acid or dilute HCl, for a sufficient time under condition to form the corresponding menaquinol intermediate. The menaquinol may be isolated before taking the acylation reaction, or the menaquinol may be acylated in situ with an (di)acid halide (X═Cl, Br, I) such as succinoyl dichloride, or dibromide, an acid anhydride such as succinic anhydride, maleic anhydride, malonic anhydride, or an acylating agent such as an acid anhydride in a solvent to form, under the acidic conditions, an initial mono-ester acid, followed by the corresponding dimer of the formula II, III or IV. The acylation reactions may also be performed directly upon reduction of MK-7 upon treatment with one or more equivalents of succinoyl halide in a solvent and a base such as Et3N, DMAP and di-isopropylethylamine.

[0130] Similarly, the carbonates of the formula IV, V, VI, VII or VIII may be prepared from a carbonylation reaction using a dicarbonate such as dimethylcarbonate (or di-alkyl carbonates), 1,1′-carbonyldiimidazole (CDI), disuccinimidyl carbonate (DSC), a phosgene equivalent such as diphosgene (trichloromethyl chloroformate, CCl.sub.3OC(O)Cl), triphosgene (hexachloromethyl carbonate, or bis(trichloromethyl) carbonate (Cl.sub.3COC(O)OCCl.sub.3) in a solvent to form the corresponding mono- or di-carbonate menaquinol derivative(s).

[0131] To a round-bottom flask is added menaquinol-7 (MK-7, 0.15 g, 0.23 mmol, 1 equiv), zinc powder (0.1 g, 1.5 mmol, 6.5 equiv), and acetic acid (0.2 mL) in methanol (1 mL). The reaction is stirred at room temperature. After the reaction is complete, the reaction is concentrated by exposure to high vacuum to remove all volatiles, and then diluted with pyridine (1 mL). To this mixture is then added the acylating agent such as succinic anhydride (0.5 equiv) and the mixture is allowed to stir at rt until the hydroquinone is consumed. The reaction mixture is then diluted with hexanes and filtrated through Celite. The solution is then washed with a 1M HCl aqueous solution (2×20 mL) and then saturated aqueous Na.sub.2CO.sub.3 solution. The organic layer is dried over anhydrous MgSO.sub.4, filtered and concentrated in vacuo to yield the acylated product. Further purification could be accomplished via recrystallization or column chromatography.

[0132] To a round-bottom flask was added MK-7 (0.15 g, 0.23 mmol, 1 equiv), zinc powder (0.1 g, 1.5 mmol, 6.5 equiv) and pyridine (0.8 mL, 9.9 mmol, 43 equiv) in acetic anhydride (3 mL, 138 equiv). The reaction was stirred for 0.5 h at room temperature (at t=0 h, MK-7 is poorly soluble and the mixture is yellow; after completion, the product is well dispersed and the solution is brown). The reaction was diluted with hexanes (40 mL) and filtrated through Celite. The organic layer was washed in succession with a 1M HCl aqueous solution (2×20 mL) and saturated Na.sub.2CO.sub.3 aqueous solution. The organic layer was dried over anhydrous MgSO.sub.4, filtered and concentrated in vacuo to yield a pale yellow oil (915 mg, 91%). R.sub.f=0.46 (9:1 hexanes/Et.sub.2O). .sup.1H NMR and .sup.13C NMR (500 MHz, CDCl.sub.3) shows that the structure is consistent with the desired product.

[0133] Reduction of menaquinone with Pd/C and hydrogen formate: In a two neck round bottom flask fitted with a condenser, a nitrogen purge tube and a magnetic bar is added 10 mL of methanol and toluene mixture (70:30) and 0.93 g ammonium formate dissolved in 1 mL water. Pd-carbon (10%) 100 mg was added after stirring for 15 min under nitrogen followed by the menaquinone (10 mmol) after about 30 seconds. The mixture is stirred for 4 h at room temperature. The catalyst is removed by filtration through a sintered disk under suction and the filtrate evaporated under reduced pressure to give about 2 g of crude solid product. The residue is extracted with dichloromethane and the extract may be used in the acylation step without further purification or isolation.

[0134] Depending on the reaction conditions and stoichiometry of the acylating reagent relative to the menaquinol, the formation of the di-acylated product, such as II, III, or IV, may be prepared selectively.

##STR00023##

Preparation of Carbonate Dimer VIIIa:

[0135] Step-1: Preparation of acetic acid 4-acetoxy-3-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27 -heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-2-methyl-naphthalen-1-yl ester IXa, where n=7, from 2-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27 -heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-3-methyl-[1,4]naphthoquinone (MK-7), where n=7:

##STR00024##

[0136] To a stirring solution of MK-7 (5.0 g, 7.7 mmol) in pyridine (10 mL, 2V) were added acetic anhydride (75 mL, 15V) and Zn dust (3.2 g, 50 mmol) at room temperature (25-30° C.). The reaction mixture was stirred at RT for 2 h. The reaction completion was monitored by TLC (15% ethyl acetate/hexane). The reaction mixture was filtered through celite and washed with ethyl acetate (10 mL), filtrate was diluted with ethyl acetate (250 mL) and washed with water (2×100 mL), separated organic layer was concentrated and crude obtained was purified by column chromatography (3-4% ethyl acetate/hexane) to yield the compound IXa as a pale yellow liquid (4.2 g, 75%). The H.sup.1 nmr obtained was consistent with the product.

[0137] Step-2: Preparation of Acetic acid 2-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-4-hydroxy-3-methyl-naphthalen-1-yl ester IXb (n=7) from acetic acid 4-acetoxy-3-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-2-methyl-naphthalen-1-yl ester IXa (n=7):

##STR00025##

[0138] To a stirring solution of compound IXa (2.2 g, 3.0 mmol) in methanol (22 mL, 10V) and MTBE (11 mL, 5V) was added tert-butylamine (1.29 mL, 12 mmol) at room temperature (25-30° C.) and stirred for 12-16 at same temperature. The reaction completion was monitored by TLC (10% ethyl acetate/hexane). The reaction mixture was concentrated (to remove methanol), diluted with ethyl acetate (200 mL) and washed with water (2×100 mL). The organic layer was separated and washed with brine solution (50 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (3-4% ethyl acetate/hexane) to yield compound IXb, n=7, as a pale brown thick liquid (0.45 g, 22%). The H.sup.1 nmr obtained was consistent with the product.

[0139] Step-3: Preparation of 4-[({[4-(acetyloxy)-3-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-2-methylnaphthalen-1-yl]oxy}carbonyl)oxy]-2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-3-methylnaphthalen-1-yl acetate, VIIIa (n=7), from acetic acid 2-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-4-hydroxy-3-methyl-naphthalen-1-yl ester IXb (n=7):

##STR00026##

[0140] To a stirring solution of compound IXb (1.0 g, 14.4 mol) in DCM (10 mL, 10 V) was added TEA (0.4 mL, 28.8 mmol) and phosgene (0.42 mL, 8.6 mmol, 0.6 eq.) at room temperature and stirred for 30 min. The reaction completion was monitored by TLC (15% ethyl acetate/hexane). The reaction mixture was diluted with ethyl acetate (200 mL) and washed with water (2×100 mL). The organic layer was separated and washed with brine solution (50 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (10% ethyl acetate/hexane) to yield MK-7-carbonate dimer VIIIa (n=7), as a waxy solid (0.36 g, 40%). The H.sup.1 nmr obtained was consistent with the product.

Preparation of Carbonate VIIIb:

[0141] ##STR00027##

[0142] Preparation of 3-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-4-hydroxy-2-methylnaphthalen-1-yl acetate IXc, (n=7), from 4-hydroxy-2-methylnaphthalen-1-yl acetate:

##STR00028##

[0143] The overall process provides chemo- or regioselective alkylation of the prenoidal side chain (n=7, 8 or 10) to the desired position in >99% selectivity and provides a single isomer. The mono-acetate in step 1, 4-hydroxy-2-methylnaphthalen-1-yl acetate, may be prepared by the reduction and subsequent di-acylation of 2-methyl-1,4-naphthoquinone using Pd/C, acetic anhydride, ethyl acetate and DMAP to form the diacylated quinol, which is then selectively de-acylated in methanol and ter-butylamine to provide the desired product, 4-hydroxy-2-methylnaphthalen-1-yl acetate.

[0144] To a stirring solution of 4-hydroxy-2-methylnaphthalen-1-yl acetate (10.0 g, 46.3 mmol) in toluene (100 mL, 10V) were added heptaprenol (16.0 g, 37.0 mmol, 0.8 eq.) and benzene sulfonic acid (0.64 g, 4.63 mmol) at room temperature (25-30° C.). The reaction mixture was stirred at RT for 16-24 h. The reaction completion was monitored by TLC (15% ethyl acetate/hexane). The reaction mixture was diluted with ethyl acetate (500 mL) and washed with water (2×250 mL). The organic layer was separated and washed with brine solution (200 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (2-3% ethyl acetate/hexane) and crystallized from ethanol to yield IXc (n=7) as an off white solid (4.0 g, 13%).

[0145] Step-1-H-NMR:

[0146] Step-2: Preparation of 4-[({[4-(acetyloxy)-2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-3-methylnaphthalen-1-yl]oxy}carbonyl)oxy]-3-[(2E,6E,10E,14E,18E,22E,26E)-2,6,10,14,18,22,26-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-2-methylnaphthalen-1-yl acetate from 3-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-4-hydroxy-2-methylnaphthalen-1-yl acetate VIIIb (n=7):

##STR00029##

[0147] To a stirring solution of IXc (n=7), (1.0 g, 14.4 mol) in DCM (10 mL, 10 V) was added TEA (0.4 mL, 28.8 mmol) and phosgene (0.42 mL, 8.6 mmol, 0.6 eq.) at room temperature and stirred for 30 min. The reaction completion was monitored by TLC (15% ethyl acetate/hexane). The reaction mixture was diluted with ethyl acetate (200 mL) and washed with water (2×100 mL). The organic layer was separated and washed with brine solution (50 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (10% ethyl acetate/hexane) to yield MK-7-carbonate-2 dimer VIIIb (n=7), as a sticky solid (0.51 g, 50%).

##STR00030##

Preparation of Succinate IX (n=7):

##STR00031##

[0148] Step-1: Preparation of acetic acid 4-acetoxy-3-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-2-methyl-naphthalen-1-yl ester IXa (n=7), from 2-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-3-methyl-[1,4]naphthoquinone (MK-7):

[0149] To a stirring solution of MK-7 (5.0 g, 7.7 mmol) in pyridine (10 mL, 2V) were added acetic anhydride (75 mL, 15V) and Zn dust (3.2 g, 50 mmol) at room temperature (25-30° C.). The reaction mixture was stirred at RT for 2 h. The reaction completion was monitored by TLC (15% ethyl acetate/hexane). The reaction mixture was concentrated and crude obtained was purified by column chromatography (3-4% ethyl acetate/hexane) to yield IXa as a pale yellow liquid (4.2 g, 75%).

##STR00032##

[0150] Step 2: Preparation of acetic acid 2-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-4-hydroxy-3-methyl-naphthalen-1-yl ester IXb, n=7, from acetic acid 4-acetoxy-3-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-2-methyl-naphthalen-1-yl ester IXa, (n=7):

[0151] To a stirring solution of IXa (2.2 g, 3.0 mmol) in methanol (22 mL, 10V) and MTBE (11 mL, 5V) was added tert-butylamine (1.29 mL, 12 mmol) at room temperature (25-30° C.) and stirred for 12-16 at same temperature. The reaction completion was monitored by TLC (10% ethyl acetate/hexane). The reaction mixture was concentrated (to remove methanol), diluted with ethyl acetate (200 mL) and washed with water (2×100 mL). The organic layer was separated and washed with brine solution (50 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (3-4% ethyl acetate/hexane) to yield IXb as a brown liquid (0.45 g, 22%).

##STR00033##

[0152] Step 3: Preparation of 4-{[4-(acetyloxy)-3-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-2-methylnaphthalen-1-yl]oxy}-4-oxobutanoic acid IXc (n=7) from acetic acid 2-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyl-octacosa-2,6,10,14,18,22,26-heptaenyl)-4-hydroxy-3-methyl-naphthalen-1-yl ester IXb (n=7):

[0153] To a stirring solution of IXb (0.25 g, 0.36 mmol) in DCM (2.5 mL, 10V) were added succinic anhydride (0.054 g, 0.54 mmol) and DMAP (0.023 g, 0.18 mmol) at 25-30° C. and stirred for 1-2 at same temperature. The reaction completion was monitored by TLC (80% ethyl acetate/hexane). The reaction mixture was diluted with DCM (100 mL) and washed with water (2×50 mL). The organic layer was separated and washed with brine solution (30 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (40% ethyl acetate/hexane) to yield IXc as an off white low melting solid (0.2 g, 70%).

##STR00034##

[0154] Step 4: Preparation of bis[4-(acetyloxy)-3-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-2-methylnaphthalen-1-yl]butanedioate IX (n=7), from 4-{[4-(acetyloxy)-3-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-2-methylnaphthalen-1-yl]oxy}-4-oxobutanoic acid IXc, n=7:

[0155] To a stirring solution of IXc (n=7), (0.62 g, 0.78 mmol) in DCM (6.2 mL, 10V) was added EDCI (0.176 g, 1.17 mmol) and DMAP (0.09 g, 0.78 mmol) at 0-5° C. and stirred for 10 min at same temperature. To the above reaction mixture IXd (n=7), (0.52 g, 0.78 mmol) in DCM (4.0 mL) was added at 0-5° C. and stirred for 1-2 at 25-30° C. Reaction completion was monitored by TLC (15% ethyl acetate/hexane). The reaction mixture was diluted with ethyl acetate (200 mL) and washed with water (2×100 mL). The organic layer was separated and washed with brine solution (50 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (10% ethyl acetate/hexane) to yield MK-7-succinate-1 dimer IX (n=7), as an off white sticky solid (0.44 g, 38%).

Preparation of MK-7 Succinate Dimer X:

[0156] ##STR00035##

[0157] NOTE: For the above process, step 1 is in common with the preparation of the carbonate, as described above.

[0158] Step 1: Preparation of 3-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-4-hydroxy-2-methylnaphthalen-1-yl acetate IXc, (n=7), from 4-hydroxy-2-methylnaphthalen-1-yl acetate:

##STR00036##

[0159] To a stirring solution of 4-hydroxy-2-methylnaphthalen-1-yl acetate (10.0 g, 46.3 mmol) in toluene (100 mL, 10V) were added heptaprenol (16.0 g, 37.0 mmol, 0.8 eq.) and benzene sulfonic acid (0.64 g, 4.63 mmol) at room temperature (25-30° C.). The reaction mixture was stirred at RT for 16-24 h. The reaction completion was monitored by TLC (15% ethyl acetate/hexane). The reaction mixture was diluted with ethyl acetate (500 mL) and washed with water (2×250 mL). The organic layer was separated and washed with brine solution (200 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (2-3% ethyl acetate/hexane) and crystallized from ethanol to yield IXc (n=7) as an off white solid (4.0 g, 13%).

##STR00037##

[0160] Step 2: Preparation of 4-{[4-(acetyloxy)-2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-3-methylnaphthalen-1-yl]oxy}-4-oxobutanoic acid Xa (n=7), from 3-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-4-hydroxy-2-methylnaphthalen-1-yl acetate IXc (n=7):

[0161] To a stirring solution of IXc (0.25 g, 0.36 mmol) in DCM (2.5 mL, 10V) were added succinic anhydride (0.054 g, 0.54 mmol) and DMAP (0.023 g, 0.18 mmol) at 25-30° C. and stirred for 1-2 at same temperature. The reaction completion was monitored by TLC (80% ethyl acetate/hexane). The reaction mixture was diluted with DCM (100 mL) and washed with water (2×50 mL). The organic layer was separated and washed with brine solution (30 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (40% ethyl acetate/hexane) to yield Xa as an off white low melting solid (0.2 g, 70%).

[0162] Step 3: Preparation of bis[4-(acetyloxy)-2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-3-methylnaphthalen-1-yl]butanedioate X from 4-{[4-(acetyloxy)-2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-3-methylnaphthalen-1-yl]oxy}-4-oxobutanoic acid Xa (n=7):

##STR00038##

[0163] To a stirring solution of Xa, n=7, (0.62 g, 0.78 mmol) in DCM (6.2 mL, 10V) was added EDCI (0.176 g, 1.17 mmol) and DMAP (0.09 g, 0.78 mmol) at 0-5° C. and stirred for 10 min at same temperature. To the above reaction mixture Xb, n=7, (0.52 g, 0.78 mmol) in DCM (4.0 mL) was added at 0-5° C. and stirred for 1-2 at 25-30° C. Reaction completion was monitored by TLC (15% ethyl acetate/hexane). The reaction mixture was diluted with ethyl acetate (200 mL) and washed with water (2×100 mL). The organic layer was separated and washed with brine solution (50 mL) then dried over sodium sulfate and concentrated. Crude product obtained was purified by column chromatography (10% ethyl acetate/hexane) to yield MK-7-succinate dimer X, n=7, as a waxy solid (0.44 g, 38%).

Preparation of MK-7 via α-Allylation and Retro-Diels-Alder Reaction:

[0164] ##STR00039##

[0165] α-Allylation. To a 20 mL oven-dried scintillation vial was added freshly prepared heptaprenyl bromide (1.7 g, 3.05 mmol, 2.44 equiv) and a stir bar. The di-ketone (298 mg, 1.25 mmol, 1.00 equiv) was then added and allowed to disperse with gentle stirring. Sodium t-butoxide (300 mg, 3.13 mmol, 2.50 equiv) was removed from a glove box in a 1-dram vial and added in one addition to the stirring reaction mixture at RT resulting in a red reaction mixture. The vial was then sealed and stirred rapidly (900 RPM) at RT. After 1 h, the reaction was deemed complete by TLC. The entire reaction mixture was then taken up in Et.sub.2O and dried onto Celite. The crude reaction was then loaded onto a packed silica column (25 mm×152 mm) and filtered through the silica using two column volumes of hexanes, 1 column volume of 3.5% Et.sub.2O/hexanes, and four column volumes of 7% Et.sub.2O/hexanes, the latter of which was collected. The organics were then evaporated into a 20 mL scintillation vial resulting in 869 mg of the α-allylated product, as a golden yellow oil which was carried over directly to the retro-Diels-Alder reaction.

[0166] Retro-Diels-Alder Reaction: The vial containing the α-allylated product was then placed under high vacuum (<15 torr pressure) and heated in a 20 mL scintillation aluminum heating block to 85° C. internal temperature neat with no stirring. The reaction was allowed to heat until a constant mass was observed from loss of cyclopentadiene, as well as completion by TLC. The resulting golden oil was then purified by flash chromatography (4% Et.sub.2O/hexanes) and dried under high vacuum resulting in a yellow solid (648 mg, 80% yield over two steps). R.sub.t=0.60 (1:9 Et.sub.2O/hexanes).

Administration of the Compounds in Subjects at Risk for Development of Calciphylaxis:

[0167] This example describes the administration of the compounds of the present application to subjects at risk for development of calciphylaxis, but who have not yet developed the characteristic skin lesions of calciphylaxis. Risk factors to be considered include, but are not limited to, diabetes mellitus, obesity, hemodialysis, and prior treatment with warfarin (Nigwekar et al. (2016) “A Nationally Representative Study of Calcific Uremic Arteriolopathy Risk Factors,” J. Am. Soc. Nephrol. 27(11):3421-9). The administration of these compounds can result in protection of the subjects from skin lesions and a change in certain biomarker levels indicative of the prevention of the development of calciphylaxis.

[0168] Subjects at risk of development of calciphylaxis orally receive a selected compound of the present application at 5 mg, 10 mg, 20 mg, 30 mg, 50 mg or 100 mg once daily for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form is a 5 mg, 10 mg, 20 mg, 50 mg or 100 mg soft-gel capsule. Two 50 mg capsules are be administered once daily to the 100 mg dosage cohort. It should be noted that not all subjects with elevated risk factors for calciphylaxis will develop the characteristic skin lesions of calciphylaxis. The intent of treating with the compound of the present application proactively (prior to a clinical diagnosis of calciphylaxis) is the prevention of lesion appearance. Thus, a drop in frequency of, or elimination of lesion appearances is contemplated to be a successful treatment.

[0169] Several biomarkers can be assessed to determine the efficacy of the compound to be administered at the three dose levels. Exemplary biomarkers include PIVKA-II; uncarboxylated and total Matrix Gla Protein (MGP); uncarboxylated, carboxylated and total osteocalcin protein; uncarboxylated, carboxylated and total Protein C, osteoprotegerin, Fetuin A and hs-CRP. Blood samples are obtained to measure the biomarkers according to the following schedule. Blood sampling can occur during treatment on a weekly or monthly basis. It is contemplated that administration of the disclosed compounds will result in (i) an increase in PIVKA-II, which is indicative of slowing the progression of, arresting, or reversing, calciphylaxis, (ii) a decrease in uncarboxylated MGP, uncarboxylated osteocalcin, and/or uncarboxylated Protein C, which is indicative of slowing the progression of, arresting, or reversing calciphylaxis. Further, pulse wave velocity (PWV) can be measured to assess arterial compliance. Improved vascular compliance will be indicative of slowing the progression of, arresting, or reversing calciphylaxis.

Administration of the Disclosed Compounds of the Application in Subjects Diagnosed with Calciphylaxis:

[0170] This example describes the administration of the disclosed compounds to subjects diagnosed with calciphylaxis. Typical symptoms include presentation of characteristic painful skin lesions (Nigwekar et al. (2015) Calciphylaxis: Risk Factors, Diagnosis, and Treatment. Am. J. Kidney Dis. 66:133-46). Definitive diagnosis of calciphylaxis is achieved via skin biopsy. Further conditions need to be considered for correct diagnosis.

[0171] Subjects diagnosed with calciphylaxis orally receive the disclosed compound at 5 mg, 10 mg, 25 mg, 50 mg or 100 mg once daily for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form is a 5 mg, 10 mg, 25 mg, 50 mg or 100 mg soft-gel capsule. Two 50 mg capsules are administered once daily to the 100 mg dosage cohort.

[0172] The arrest of, or decreases in lesion size and frequency is contemplated to be an indication of successful treatment. The administration of the disclosed compounds according to the foregoing will result in the arrest of, or decrease in lesion size and frequency. Additionally, because calciphylaxis has a considerable mortality risk, increased overall survival time of diagnosed subjects will be an indication of treatment success. Furthermore, the administration of the disclosed compounds according to the foregoing will result in an increased overall survival time of diagnosed subjects.

Administration of the Disclosed Compounds in Subjects with End Stage Renal Disease (ESRD) to Reverse or Slow the Progression of Tissue Calcification:

[0173] This example describes the administration of the disclosed compounds to a subject with ESRD and on stable hemodialysis. The administration of the disclosed compounds will result in a change in aortic compliance (via plethysmography), vascular calcification and certain biomarker levels indicative of slowing the progression of, arresting, or reversing tissue calcification.

[0174] ESRD subjects on stable hemodialysis orally receive the disclosed compounds at 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300, mg, 400 mg or 500 mg once daily for least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form is a 5 mg, 10 mg, 25 mg, 50 mg, 75 mg or 100 mg soft-gel capsule. Two 50 mg capsules are administered once daily to the 100 mg dosage cohort.

[0175] A 50 y.o., 65 kg male patient diagnosed with the typical symptoms associated with moderate calciphylaxis is treated with 100 mg of the compound of the formula I wherein M is Ca.sup.2+, Y is 2 and m is 7, for a period of 8 weeks. After the treatment period, the patient is admitted and evaluated. The patient is found to have a significant change in the examined biomarker levels suggesting about a 10% reduction in vascular calcification, and is also shown to have a 10% reduction in tissue calcification.

[0176] A 65 y.o., 45 kg female patient diagnosed with the typical symptoms associated with moderate calciphylaxis is treated with 20 mg of the compound of the formula I wherein M is Ca.sup.2+, Y is 2 and m is 7, for a period of 10 weeks. After the treatment period, the patient is admitted and evaluated. The patient is found to have a significant change in the examined biomarker levels suggesting about a 20% reduction in vascular calcification, and is also shown to have a 15% reduction in tissue calcification.

[0177] A 55 y.o., 70 kg male patient diagnosed with the typical symptoms associated with moderate calciphylaxis is treated with 50 mg of the compound of the formula I wherein M is K.sup.+, Y is 1 and m is 7, for a period of 3 months. After the treatment period, the patient is admitted and evaluated. The patient is found to have a significant change in the examined biomarker levels suggesting about a 25% reduction in vascular calcification, and is also shown to have a 20% reduction in tissue calcification.

[0178] Coronary arterial calcium scores (CAC) are used to estimate the extent of calcification of thoracic arteries. A high CAC score is indicative of calcification, and treatment has the aim of arresting the long-term increase in CAC score, or reversing it, or slowing the rate of increase.

[0179] Aortic plethysmography also is used to measure arterial compliance, which decreases as calcification increases. Pulse wave velocity (PWV) also is measured to assess arterial compliance. The foregoing measures are useful in estimating the utility of treatments intended to prevent, slow the progression of, arrest or reverse vascular calcification. These measurements are used pre- and post-treatment with the disclosed compounds to assess treatment value.

[0180] Several biomarkers are assessed to determine the efficacy of the disclosed compounds at the three dose levels. Exemplary biomarkers include PIVKA-II; uncarboxylated and total Matrix Gla Protein (MGP); uncarboxylated, carboxylated and total osteocalcin protein; uncarboxylated, carboxylated and total Protein C, and hs-CRP. Blood samples are obtained to measure the biomarkers, most conveniently during patient visits for hemodialysis.

[0181] The administration of the disclosed compounds can result in (i) an increase in PIVKA-II, which is indicative of slowing the progression of, arresting or reversing tissue calcification, (ii) a decrease in uncarboxylated MGP, uncarboxylated osteocalcin, and/or uncarboxylated Protein C, which is indicative of slowing the progression of, arresting or reversing tissue calcification, and/or (iii) a decrease in hs-CRP, which is indicative of slowing the progression of, arresting or reversing tissue calcification and/or reduced inflammation. Following the daily administration of 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg or 500 mg or more, of the disclosed compounds and compositions, at least one of PIVKA-II, under-carboxylated Matrix Gla Protein (MGP), under-carboxylated osteocalcin protein, will show a change indicative of slowing the progression of, arresting or reversing tissue calcification.

[0182] While a number of exemplary embodiments, aspects and variations have been provided herein, those of skill in the art will recognize certain modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations. It is intended that the following claims are interpreted to include all such modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations are within their scope.

[0183] The entire disclosures of all documents cited throughout this application are incorporated herein by reference.

[0184] References: 1) Rachel M. Holden et al. Vitamins K and D Status in Stages 3-5 Chronic Kidney Disease; Clin J Am Soc Nephrol 5: 590-597, 2010. 2) Pilkey, R. M. MD et al. Subclinical Vitamin K Deficiency in Hemodialysis Patients Am J Kidney Dis 49:432-439, 2007. 3) Westhofen P et al. Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function. J Biol Chem 2011; 286: 15085-94. 4) Caspers, M. et al., Two enzymes catalyze vitamin K 2,3-epoxide reductase activity in mouse: VKORC1 is highly expressed in exocrine tissues while VKORC1L1 is highly expressed in brain. Thrombosis Research 135:977-983, 2015. 5) Himmelfarb, J. et al., Plasma protein thiol oxidation and carbonyl formation in chronic renal failure. Kidney International, Vol. 58: 2571-2578 2000. 6) Price, P. A. et al., Discovery of a High Molecular Weight Complex of Calcium, Phosphate, Fetuin, and Matrix-Carboxyglutamic Acid Protein in the Serum of Etidronate-treated Rats. J Biol Chem. 277 (6): 3926-3934, 2002. 7) Pasch, A. et al. Nanoparticle-Based Test Measures Overall Propensity for Calcification in Serum J Am Soc Nephrol 23: 1744-1752, 2012. 8) Nigwekar, S. U. et al. Vitamin K-Dependent Carboxylation of Matrix Gla Protein Influences the Risk of Calciphylaxis. J Am Soc Nephrol 28: 1717-1722, 2017.