Menaquinol compositions and methods of treatment

Abstract

The present application discloses methods for the efficient preparation of high purity compounds of the Formula I, and their methods of use. ##STR00001##

Claims

1. A menaquinone derivative of the Formula 30, 31 or 32: ##STR00029## wherein PEG-OMe is poly(ethylene glycol) monomethylether-2000.

2. The menaquinone derivative of claim 1 of the Formula 30: ##STR00030##

3. The menaquinone derivative of claim 1 of the Formula 31: ##STR00031## wherein PEG-OMe is poly(ethylene glycol) monomethylether-2000.

4. The menaquinone derivative of claim 1 of the Formula 32: ##STR00032## wherein PEG-OMe is poly(ethylene glycol) monomethylether-2000.

5. A method of treating, preventing, slowing the progression of, arresting, and/or reversing calciphylaxis in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising substantially pure menaquinol compound of the Formula 30, 31 or 32: ##STR00033## wherein PEG-OMe is poly(ethylene glycol) monomethylether-2000; and a pharmaceutically acceptable excipient, to prevent, slow the progression of, arrest, or reverse calciphylaxis.

6. The method of claim 5, wherein the mammal has distal calciphylaxis and/or central calciphylaxis.

7. The method of claim 5, wherein the mammal has diabetes, chronic kidney disease or end stage renal disease.

8. A method of treating calciphylaxis in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising substantially pure menaquinol compound of the Formula the Formula 30, 31 or 32: ##STR00034## wherein PEG-OMe is poly(ethylene glycol) monomethylether-2000; and a pharmaceutically acceptable excipient, to treat calciphylaxis.

9. The method of claim 8, wherein the mammal has distal calciphylaxis and/or central calciphylaxis.

10. The method of claim 8, wherein the mammal has diabetes, chronic kidney disease or end stage renal disease.

Description

BRIEF DESCRIPTION OF THE FIGURE

(1) 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.

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

(3) FIG. 3 is graph showing the VKORC1 in arbitrary units and specific tissues.

(4) FIG. 4 is a graph showing the NADPH in arbitrary units and specific tissues.

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

EXPERIMENTAL

(6) 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.

(7) 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.

(8) Preparation of Menaquinol-7:

(9) ##STR00020##

(10) Preparation of Menaquinol-7: Use of a Farnesylfarnesol-Derived Alkyne and Its Subsequent Ni-Catalyzed Coupling/Reduction Reaction:

(11) ##STR00021##

(12) Preparation of farnesylfarnesol-derived alkyne: As described for the farnesol-derived alkyne synthesis, farnesylfarnesol (10 g,) was converted to the corresponding chloride using PCl.sub.3/DMF conditions. Crude chloride (10.4 g made) was obtained in 100% yield and it was sufficiently pure by .sup.1H NMR. The crude chloride (8.9 g) was treated with dilithiopropyne and the resulting crude alkyne was purified on a Biotage chromatography instrument. The alkyne was obtained as a colorless oil, 6.2 g in 69% isolated yield. Q-NMR analysis indicated that the purity of the alkyne as 94.0 wt %. This product was used as it is for the next step without further purifications (no plug filtration nor distillation). Some residual alkyne was retained in the column and was flashed out with a stronger solvent (10% EtOAc/hexane) to afford 1.6 g (18%) of additional alkyne product as a yellow oil.

(13) ##STR00022##

(14) Ni-catalyzed coupling: Me.sub.3Al (2 M in toluene, 1.5 mL, 3.0 mmol, 1.5 equiv) was added to Cp.sub.2ZrCl.sub.2 (29 mg, 0.10 mmol, 5 mol %) at 0° C. To this solution was added alkyne (898 mg, 2 mmol, 1 equiv) at 0° C. After stirring at 0° C. for 1 h, TLC indicated most of the alkyne was consumed. GC/MS assay was also used to monitor this conversion process. The mixture was gently vacuumed using high vacuum pump at ambient temperature to remove excess Me.sub.3Al and some toluene. When the mixture became viscous, vacuum was stopped, leaving some residual toluene. If all of the toluene was removed by vacuum, an exothermic reaction to about 35° C. was observed. The residue was cooled to −20° C. and THF (2 mL) was added to the mixture. To this solution was added a THF (2 mL) solution of naphthoquinone (440 mg, 2.00 mmol, 1 equiv, lot #AP56079013-006-01). The syringe was rinsed with additional THF (1 mL) and it was added to the mixture. In a separate flask, NiCl.sub.2(PPh.sub.3).sub.2 (39 mg, 0.06 mmol, 3 mol %) was suspended in THF (1 mL). To this was added n-BuLi (2.1 M in hexane, 57 μL, 0.12 mmol, 6 mol %) and the resulting light yellow solution was stirred for 1 min before it was transferred to the above vinylalane and naphthoquinone mixture at −20° C. The resulting dark mixture was stirred at −20° C. for 3 h. The reaction was monitored by TLC where no change was observed after being stirred for 1 h at −20° C. The mixture was carefully quenched by the addition of cold water and 0.2 N HCl solution and then extracted with MTBE. The crude oil (1.1 g) was monitored by .sup.1H NMR and it indicated that the ratio of desired isomer and the regioisomer was 96:4. Crude material was purified via Biotage chromatography to give 840 mg of menaquinone-7 in 65% isolated yield along with recovered naphthoquinone (150 mg, 34%). The product was isolated by chromatography and the ratio of the isomers was 96:4 by .sup.1H NMR.

(15) Synthesis of MK-7 Using Reduced Amounts of Me.sub.3Al:

(16) Me.sub.3Al (2 M in toluene, 1.2 mL, 2.4 mmol, 1.2 equiv) was added to Cp.sub.2ZrCl.sub.2 (29 mg, 0.10 mmol, 5 mol %) at 0° C. To this solution was added the alkyne (898 mg, 2 mmol, 1 equiv) at 0° C. After stirring at 0° C. for 1 h, TLC indicated most of the alkyne was consumed. GC/MS assay was used to monitor the reaction progress. The mixture was gently placed under vacuum using high vacuum pump at ambient temperature in order to remove excess Me.sub.3Al and some toluene. When the mixture became viscous, vacuum was stopped, leaving some residual toluene. (It was noted that if substantially all of the toluene was removed by vacuum, an exothermic reaction to about 35° C. was observed). The residue was cooled to −20° C. and THF (2 mL) was added to the mixture. To this solution was added a THF (2 mL) solution of naphthoquinone (440 mg, 2.0 mmol, 1 equiv, lot #AP56079013-006-01). The syringe was rinsed with additional THF (1 mL) and it was added to the mixture. In a separate flask, NiCl.sub.2(PPh.sub.3).sub.2 (39 mg, 0.06 mmol, 3 mol %) was suspended in THF (1 mL). To this was added n-BuLi (2.1 M in hexane, 57 μL, 0.12 mmol, 6 mol %) and the resulting light yellow solution was stirred for 1 min before it was transferred to the above vinylalane and naphthoquinone mixture at −20° C. The resulting dark mixture was stirred at −20° C. for 1 h. .sup.1H NMR of the crude mixture indicates that the ratio of MK-7 and the regioisomer was 93:7. .sup.1H NMR also indicated that the formation of terminal olefin was less compared to the crude mixture that used 1.5 equiv of Me.sub.3Al. It was purified by Biotage chromatography to give 0.97 g of pure MK-7 in 75% isolated yield. 52 mg of unreacted naphthoquinone (12% yield) was recovered. .sup.1H NMR indicated that the ratio of MK-7 and regioisomer was unchanged.

(17) Synthesis of MK-7 with Reduced Me.sub.3Al:

(18) Me.sub.3Al (2 M in toluene, 1.2 mL, 2.4 mmol, 1.2 equiv) was added to Cp.sub.2ZrCl.sub.2 (29 mg, 0.10 mmol, 5 mol %) at 0° C. To this solution was added alkyne (900 mg, 2 mmol, 1 equiv) at 0° C. After stirring at 0° C. for 1 h, TLC indicated most of the alkyne was consumed. GC/MS assay was also used to monitor the reaction progress.

(19) The mixture was cooled to −20° C. To this was added THF (2 mL) and a THF (2 mL) solution of naphthoquinone (440 mg, 2.00 mmol, 1 equiv, lot #AP56079013-006-01). The syringe was rinsed with additional THF (1 mL) and it was added to the mixture. In a separate flask, Ni(0) catalyst was prepared as above, and it was transferred to above vinylalane and naphthoquinone mixture at −20° C. The resulting dark mixture was stirred at −20° C. for 1 h. .sup.1H NMR analysis of the crude mixture indicated that the coupling reaction was nearly completed and the ratio of MK-7 and the regioisomer was 93:7. Comparing all the crude .sup.1H NMR overlaid spectra in the above descriptions, this reaction provided the cleanest conversion. The mixture was purified on Biotage chromatography to give 1.02 g of pure MK-7 in 78% isolated yield, with a recovery of 64 mg (15%) of unreacted naphthoquinone. This process demonstrated that the removal of Me.sub.3Al and toluene from the carboalumination mixture prior to the subsequent Negishi coupling reaction is not necessary.

(20) Recrystallization of Menaquinone-7:

(21) Chromatographed material (0.98 g, ratio of menaquinone-7 and the regioisomer was 93:7) was recrystallized using various solvents (toluene, dichloromethane, hexanes, heptanes, THF, methanol, ethanol, propanol, iso-propanol, MTBE, MEK, DMF and their various mixtures of binary and ternary solvent mixtures) in different ratios did not provide a significant improvement of the regioisomeric ratio. However, the chromatographed menaquinone-7 was recrystallized from EtOAc/EtOH (1:5) provided 0.66 g (67%) of clean menaquinone-7. HPLC indicates that the ratio of MK-7 and regioisomer was 99.8:0.2. Mother liquor was concentrated to give 0.26 g (26%) of oil and regioisomer was enriched, showing the effectiveness of the crystallization solvent mixture to optimize the isolation of the desired isomer.

(22) Large Scale Reactions:

(23) The present reaction was performed using 1.2 equiv of Me.sub.3Al with no vacuuming before the coupling reaction. Farnesylfarnesol (50 g) was chlorinated using the standard method noted above, and a modified workup procedure was used. PCl.sub.3 (7.2 mL, 82 mmol, 0.7 equiv) was carefully added to DMF (240 mL) at 10° C. and vigorously stirred for 30 min. To this was added a DMF (30 mL) solution of farnesylfarnesol (50 g, 117 mmol) using an addition funnel. The addition funnel was rinsed with additional DMF (30 mL) and the DMF rinse was added to the mixture. The resulting orange suspension was stirred for 1 h at 10° C. and stirred for 1 h at ambient temperature. Because the DMF solution of farnesylfarnesol was a very thick and viscous solution, the preparation of the farnesylfarnesol solution may also be prepared in n-heptane to provide a less viscous solution for processing and transfer at a large scale.

(24) The reaction was monitored by LCMS until the farnesylfarnesol was consumed. The product was extracted with n-heptane, dried over MgSO.sub.4 and concentrated under reduced pressure (20 mm Hg, bath temp 38° C.). Some residual n-heptane was allowed to remain in the chloride mixture to reduce the evaporation and loss of the chloride. The mixture was obtained in 62.5 g of clear oil. .sup.1H NMR indicated 49.6 g (95%) of desired chloride and 12.9 g of n-heptane. This mixture was used as it was for the next reaction without further purifications.

(25) Preparation of the Alkyne:

(26) Dilithiopropyne was prepared in the same manner as described above. Accordingly, instead of charging all the n-BuLi solution before propyne gas was introduced, half of n-BuLi was added. After excess propyne gas was introduced, the mixture was stirred at ambient temperature to allow the excess propyne gas to evaporate. To the resulting propyne acetylide was added another one equivalent of n-BuLi to form dilithiopropyne. After addition of the above crude chloride (49.6 g) and the reaction was quenched, a 1:1 mixture of desired alkyne and unreacted chloride was obtained. It is noted that excess propyne gas remained in the THF solution so that the preparation of dilithiopropyne was incomplete. The chloride decomposes in GCMS and gave multiple peaks. The chloride and the alkyne appeared at the same retention time. The material was re-subjected to the dilithio-alkyne displacement.

(27) Accordingly, the dilithiopropyne was prepared again, but a scale was placed in the hood to weigh and monitor the weight of gaseous propyne when it was introduced to the n-BuLi solution. To the resulting dilithiopropyne was added the above mixture of alkyne and chloride in several portions while monitoring the reaction aliquot by .sup.1H NMR, until all chloride converted to the alkyne. The resulting crude yellow oil (60 g) was monitored by .sup.1H NMR and shown to provide an essentially pure desired alkyne and n-heptane. GCMS data indicated that, in addition to the alkyne, the n-butyl adduct (m/z 466.84), which was derived from the excess n-BuLi displacement of the chloride. The crude alkyne was passed through a gravity grade silica-gel plug and the filter cake was rinsed with 5% MTBE/n-heptane. After evaporation of the filtrate, 56.4 g of clear oil (theoretical yield: 49.8 g) was obtained. .sup.1H NMR indicated it was a mixture of desired alkyne and n-heptane and the purity was 76 wt %. This material was used as is for the carboalumination without further purification.

(28) The reaction using the above impure alkyne was slower compared to the previously made, purer alkyne. This batch of alkyne required 3 to 4 hours to complete compared to within 1 h with more thoroughly chromatographed and pure alkyne.

(29) Reaction Using Distilled and Pure Farnesylfarnesol:

(30) 100 mg of the above distilled residue of farnesylfarnesol was used for the coupling reaction using the above cited standard procedure. The reaction was completed within 15 min. It was noted that the reason for the slower conversion observed above was the presence of the volatiles (likely n-heptane) that were removed to provide pure farnesulfarnesol by applying vacuum and heat, as noted above. The n-Butyl adduct did not interfere and the presence of n-Bu adduct was not the cause of slower conversion. Fractions were monitored by GCMS and the fractions that contained pure alkyne peak were collected. 6 g of n-butyl adduct and 29 g of alkyne was obtained. Since the alkyne is not volatile, it was vacuum dried at 50° C. by rotary evaporator (10-12 mmHg) for 30 min. This material was pure by GCMS, and .sup.1H NMR indicated a small impurity that may be a dimer of the chloride resulting from an attachment of a carbon-carbon bond. The Q-NMR suggested that the alkyne was 68 wt % pure.

(31) Preparation of Menaquinone-7 Using Pure Alkyne:

(32) A 10 g reaction scale was performed using the starting alkyne containing some unknown impurities. Accordingly, 10 g of alkyne (14.7 g of impure alkyne as is, 61 wt %, 20.0 mmol) was treated with 1.2 equiv of Me.sub.3Al in toluene in the presence of Cp.sub.2ZrCl.sub.2 (5 mol %) at 0° C. GCMS indicated that the reaction was completed within 1 h. The subsequent coupling reaction was carried out using 1 equiv of naphthoquinone (4.4 g, 20 mmol) at −20° C. for 1 h. A reaction aliquot was monitored by .sup.1H NMR and indicated naphthoquinone was nearly consumed. .sup.1H NMR and HPLC both indicated that the ratio of menaquinone and its regioisomer was 94:6. The reaction mixture was quenched and extracted with n-heptane. The organic layer was passed through a silica-gel plug and the plug was rinsed with 5% EtOAc/n-heptane. Filtrates were concentrated to give crude oil (21.3 g) which was recrystallized from EtOAc (20 mL) and EtOH (100 mL) to provide 15.7 g of solid (theoretical yield: 14.5 g). HPLC indicated that the ratio of desired product to the regioisomer was 97:3. Q-NMR showed a purity of 72 wt %. This product contains 11.3 g (78%) of menaquinone-7. The solid was stirred in n-BuOH (25 mL) at 0° C. and it was filtered. Filter cake was rinsed with cold n-BuOH and the filter cake was dried in vacuo to afforded 9 g of pure material in 62% overall isolated yield. HPLC indicated that the ratio of menaquinone and regioisomer was 99.8:0.2, with a purity of >99.6% by HPLC using an external standard. HPLC Conditions: Column: Thermo Acclaim C30, 250×2.1 mm, 3 μm (Part #078664); Column temperature: 15° C.; Mobile phase: 2% water in methanol; Diluent: 90% IPA in THF; Detector: UV 248 nm, 234 nm; Flow rate: 0.4 mL/min; Injection volume: 4 μL Running time: 50 min.

(33) Reduction of MK-7 Followed by Esterification of Menaquinol-7:

(34) The menaquinol compounds and derivatives, such as menaquinol-7 compounds and derivatives, may be prepared according to the general scheme as described below. Such acylated compounds may be symmetrical, wherein both hydroxyl groups of the menaquinol are acylated, or only one of the two hydroxyl groups, either the 5-position or the 8-position, are acylated, and the other remaining as the hydroxyl group of the menaquinol.

(35) Accordingly, the menaquinone, such as MK-7, may be contacted with a metal, such as zinc, and an acid, such as acetic acid or dilute HCl, in a protic solvent, such as methanol or ethanol, 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 acid halide (X=Cl, Br, I) or an acylating agent such as an acid anhydride in a solvent, to form the corresponding mono- or di-acylated menaquinol derivative. In one variation of the method, the acid anhydride may be a symmetrical or an unsymmetrical or mixed acid anhydride, to form the corresponding mono- or diacylated menaquinol.

(36) ##STR00023##

(37) To a round-bottom flask is added 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 (2.2 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 diacylated product. Further purification could be accomplished via recrystallization or column chromatography.

(38) 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 (500 MHz, CDCl.sub.3) δ 7.74-7.63 (m, 2H), 7.50-7.43 (m, 2H), 5.19-5.01 (m, 7H), 3.42 (s, 2H), 2.49 (s, 3H), 2.47 (s, 3H), 2.25 (s, 3H), 2.16-1.89 (m, 23H), 1.79 (s, 3H), 1.69 (s, 3H), 1.61 (s, 17H), 1.59 (s, 17H), 1.55 (s, 5H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 169.5, 169.1, 142.7, 142.4, 136.4, 135.3, 135.0, 135.0, 135.0, 135.0, 131.3, 130.4, 127.1, 126.4, 126.4, 126.3, 126.3, 124.5, 124.4, 124.4, 124.3, 124.0, 121.5, 121.3, 121.2, 39.9, 39.9, 39.8, 39.7, 29.8, 27.2, 26.9, 26.8, 26.8, 26.8, 26.7, 25.8, 20.8, 20.7, 17.8, 16.5, 16.2, 16.1, 16.1, 13.2.

(39) Reduction of menaquinone: 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.

(40) ##STR00024##

(41) Depending on the reaction conditions and stoichiometry of the acylating reagent relative to the menaquinol, the formation of the mono acylated product, such as 30 and 31, or the diacylated product 32, may be prepared selectively. Accordingly, an excess of the acylation reagent will drive the reaction toward the formation of the diacylated product 32; whereas the use of less than one equivalent of the acylating reagent under the appropriate conditions will provide the mono-acylated product.

(42) ##STR00025##
The preparation or coupling reaction with water soluble PEG groups:

(43) The formation of MPEG derivatives of menaquinol leads to water solubilization of the menaquinol compounds. In one variation, the MPEG derivatives may be prepared with an initial treatment with an anhydride, such as succinic anhydride, to form the ester acid derivative. The ester acid derivative may be isolated, and then treated with an MPEG compound to form the diesters.

(44) To a solution of poly(ethylene glycol) monomethylether-2000 (15 g, 7.5 mmol) and succinic anhydride (1.5 g, 15 mmol) in tolune (7.5 mL) and Et.sub.3N (0.53 mL, 3.75 mmol) is added at RT, and the reaction mixture is stirred at about 60° C. for abut 8 hours until the reaction was complete. Water (15 mL) is added to the reaction mixture and the mixture is extracted with DCM (3×25 mL). The combined organic layers were washed with 1 N HCl (3×50 mL) and then with brine (2×30 mL) and then dried over anhydrous Na.sub.2SO.sub.4. The solution is concentrated under rotoevaporation to afford the poly(ethylene glycol) monomethyl ether-2000 succinate in about 99% yield (15.6 g) as a solid.

(45) Preparation of activated PEGylated Succinic Acid: Poly(ethylene glycol) monoethyl ether-2000 succinate (2.1 g, 1 mmol) is dissolved in DCM (10 mL) and cooled to 0° C. N-Hydroxysuccinimide (0.14 g, 1.2 mmol) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDCI, 0.25 g, 1.3 mmol) is directly added in succession to the reaction mixture as solids. The resulting solution is stirred at RT for about 12 hours. Water (15 mL) is added to the reaction mixture and the product is extracted with DCM (3×20 mL). The combined organic layers are washed with water (3×20 mL), brine (2×20 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated via rotoevaporation to provide the product (2.17 g, 99%) as an off white, waxy solid.

(46) NaH (0.026 g, 0.65 mmol, 60% suspension in mineral oil) is added to a stirred solution of menaquinol (0.6 mmol) in THF (5 mL) at 0° C. After the addition, the reaction mixture is stirred at 20° C. for about 1 hour. A solution of the poly(ethylene glycol) monomethyl ether-2000 succinate (0.50 mmol) in THF (50 mL) is added to the mixture at 0° C., and the reaction is stirred for 30 minutes. The mixture is stirred for another 8 hours at RT. The reaction is cooled to 0° C. and saturated aqueous NH.sub.4Cl (15 mL) is added and then extracted with DCM (3×20 mL). The combined organic layers are washed with water (2×20 mL) and brine (2×15 mL), and the organics are reduced under rotoevaporation to provide a yellow liquid, which is purified by flash column chromatography on silica gel using DCM and 1:20 MeOH/DCM gradient to provide the product in 65% yield.

(47) Preparation of Menaquinol Compounds I:

(48) The preparation of the diesters or mono-esters of the compound of the Formula I may be performed using standard acetylation of quinones known in the art. For example, the quinone may be treated with a symmetrical anhydride, where R.sup.a and R.sup.b are the same group, or with an asymmetrical anhydride, where R.sup.a and R.sup.b are different groups. In one embodiment of the symmetrical or asymmetrical anhydride, the acyl group R.sup.a—C(O)— is selected from the group consisting of:

(49) ##STR00026## ##STR00027##
and R.sup.b may be the same as R.sup.a or R.sup.b may be selected from —CH.sub.3 or —CH.sub.2CH.sub.3; wherein P.sup.1 and P.sup.2 are each independently a protecting group such as —CH.sub.2C.sub.6H.sub.5, —THP (tetrahydropyranyl) or P.sup.1 and P.sup.2 together with the oxygen to which they are attached form a cyclic acetonide, benzyl acetal or p-methoxy-benzyl acetal; P.sup.3 is a hydroxyl protecting group such as —THP, acetyl, benzoyl, β-methoxyethoxymethyl ether (MEM), dimethoxytrityl, methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv) and trityl (Tr); and each R.sup.3 and R.sup.4 is independently H, —CH.sub.3, —CH.sub.2CH.sub.3 and —CH.sub.2C.sub.6H.sub.5; and the acetylation may be performed with the addition of a base such as Cs.sub.2CO.sub.3, CsHCO.sub.3, LiCO.sub.3, Na.sub.2CO.sub.3, K.sub.2CO.sub.3, KHCO.sub.3, NaOAc and NaHCO.sub.3. The acylation may be performed in a solvent such as THF, Me-THF, toluene and ethyl acetate.

(50) In another embodiment, the preparation of the diesters or mono-esters of menaquinol-7 of Formula I (R.sup.1=R.sup.2=H) may be performed using standard acetylation of quinones using an acid halide as known in the art, where the halide is —Cl, —Br or —I along with a base selected from the group consisting of Cs.sub.2CO.sub.3, CsHCO.sub.3, CsOH, LiCO.sub.3, Na.sub.2CO.sub.3, K.sub.2CO.sub.3, KHCO.sub.3, NaOAc, NaHCO.sub.3, or an organic amine base as disclosed herein. For example, in one embodiment of the symmetrical or unsymmetrical anhydride, the acyl group R.sup.a—C(O)— is selected from the group consisting of the above residues including 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27 and 28.

(51) ##STR00028##

(52) Preparation of a Menaquinol I (R.sup.1 and R.sup.2 are as defined herein):

(53) Menaquinone-7 (VIa, 1.00 g, 1.54 mmol), the symmetrical anhydride where R.sup.a and R.sup.b are both the acyl group 15 (1.67 g, 3.08 mmol, MW=544 g/ml). NaOAc (0.154 g, 1.88 mmol) and Zn powder (0.35 g, 5.44 mmol) are added together in a 50 mL RBF equipped with a stir bar and heated to about 140° C. for about 1 hour. The resulting mixture was cooled to RT and THF (45 mL) is added. Et.sub.2N (20 mL) is added and the resulting mixture is stirred for another 30 minutes, and then heptane (60 mL) is added. The resulting mixture is filtered using a buchner funnel and filter paper, and the filtered cake is washed with heptane (2×15 mL). The solvents in the combined filtrate is removed under rotoevaporation at a water bath of about 35° C. and the resulting oil is purified by flash column chromatography (heptane:EtOAc in gradient) to provide 41.9 g (about 50%) yield of the menaquinol I, where R.sup.1 and R.sup.2 are both the acyl group 15.

(54) Preparation of a Menaquinol I (R.sup.1=R.sup.2=H):

(55) Menaquinone-7 (VIa, 1.00 g, 1.54 mmol), the symmetrical anhydride where R.sup.a and R.sup.b are both the acyl group 15 (1.67 g, 3.08 mmol, MW=544 g/ml). NaOAc (0.154 g, 1.88 mmol) and Zn powder (0.35 g, 5.44 mmol) are added together in a 50 mL RBF equipped with a stir bar and heated to about 140° C. for about 1 hour. The resulting mixture was cooled to RT and THF (45 mL) is added. Et.sub.2N (20 mL) is added and the resulting mixture is stirred for another 30 minutes, and then heptane (60 mL) is added. The resulting mixture is filtered using a buchner funnel and filter paper, and the filtered cake is washed with heptane (2×15 mL). The solvents in the combined filtrate is removed under rotoevaporation at a water bath of about 35° C. and the resulting oil is purified by flash column chromatography (heptane:EtOAc in gradient) to provide 41.9 g (about 50%) yield of the menaquinol I, where R.sup.1 and R.sup.2 are both the acyl group 15.

(56) Administration of the Compounds of the Present Application (the Disclosed Compounds) in Subjects at Risk for Development of Calciphylaxis:

(57) 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.

(58) Subjects at risk of development of calciphylaxis orally receive a selected compound of the present application at 5 mg, 10 mg, 25 mg or 50 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 or 25 mg soft-gel capsule. Two 25 mg capsules are be administered once daily to the 50 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.

(59) 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.

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

(61) 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.

(62) Subjects diagnosed with calciphylaxis orally receive the disclosed compound at 5 mg, 10 mg, 25 mg or 50 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 or 25 mg soft-gel capsule. Two 25 mg capsules are administered once daily to the 50 mg dosage cohort.

(63) 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.

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

(65) 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.

(66) ESRD subjects on stable hemodialysis orally receive the disclosed compounds at 5 mg, 10 mg, 25 mg or 50 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 or 25 mg soft-gel capsule. Two 25 mg capsules are administered once daily to the 50 mg dosage cohort.

(67) A 50 y.o., 65 kg male patient diagnosed with the typical symptoms associated with moderate calciphylaxis is treated with 10 mg of the compound of the formula I wherein m is 7, R.sup.1 the compound of the formula 24, R.sup.2 is H and R.sup.3 and R.sup.4 are both —CH.sub.3, for a period of 8 weeks. After the treatment period, the patient is admitted and evaluated. The patient was 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.

(68) A 65 y.o., 45 kg female patient diagnosed with the typical symptoms associated with moderate calciphylaxis is treated with 10 mg of the compound of the formula I wherein m is 7, R.sup.1 the compound of the formula 25, R.sup.2 is H and R.sup.3 and R.sup.4 are both —CH.sub.3, for a period of 10 weeks. After the treatment period, the patient is admitted and evaluated. The patient was 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.

(69) A 55 y.o., 70 kg male 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 7, R.sup.1 the compound of the formula 26, R.sup.2 is H and R.sup.3 and R.sup.4 are both —CH.sub.3, for a period of 3 months. After the treatment period, the patient is admitted and evaluated. The patient was 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.

(70) 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.

(71) 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.

(72) Further, 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.

(73) 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 or 50 mg 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.

(74) 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.

(75) The entire disclosures of all documents cited throughout this application are incorporated herein by reference.

REFERENCES

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