SYNTHESIS AND IN VITRO ACTIVITY OF D-LACTIC ACID OLIGOMERS

Abstract

This invention describes the synthesis and pharmacologic activity of n=2, n=3, n=4, n=5, and n=6 D-lactic acid oligomers. D-lactic acid dimer has pharmacologic activity and sequesters L-lactate, and the other D-lactic acid oligomers have no activity in vitro, but may have pharmacologic activity in vivo as prodrugs of D-lactic acid dimer.

Claims

1. A method to synthesize n=2, n=3, n=4, n=5, and n=6 D-lactic acid oligomers from methyl D-(+)-lactate.

2. The method of claim 1 where an equivalent, derivative, or analog of methyl D-(+)-lactate including D-lactic acid and D-lactate, are substituted for methyl D-(+)-lactate.

3. A method to treat cancer in a human subject by administering to said subject a prodrug of D-lactic acid dimer comprising an n=3, n=4, n=5, or n=6 D-lactic acid oligomer or a composite comprising n=3, n=4, n=5, and n=6 D-lactic oligomers.

4. The method of claim 3 to treat cancer and pain.

5. The method of claim 3 to treat pain.

Description

DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1-4 show the synthesis of partially protected D-lactic acid dimer from methyl D-(+)-lactate.

[0009] FIGS. 5-7 show the synthesis of D-lactic acid trimer from the partially protected D-lactic acid dimer.

[0010] FIGS. 8-10 show the synthesis of partially protected D-lactic acid tetramer from the protected D-lactic acid trimer.

[0011] FIGS. 10-13 show the synthesis of D-lactic acid pentamer from the partially protected D-lactic acid tetramer.

[0012] FIGS. 14-17 shows the synthesis of D-lactic acid hexamer from the protected D-lactic acid pentamer.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Synthesis of n=2, n=3, n=4, n=5, and n=6 D-Lactic Acid Oligomers

[0014] A solution of methyl D-(+)-lactate (25 g, 241 mmol) and imidazole (21 g, 313 mmol, 1.3 eq.) in CH.sub.2Cl.sub.2 (125 mL) was cooled in an ice-NaCl bath. Tert-butyldiphenylchlorosilane (97%, 66 mL, 253 mmol, 1.05 eq.) was added dropwise over 30 minutes. The resulting slurry was stirred for an additional 3 h after which time analysis by TLC (100% CH.sub.2Cl.sub.2, KMnO.sub.4 staining) indicated the starting alcohol had been completely consumed. The reaction was diluted with CH.sub.2Cl.sub.2 (400 mL) and brine (500 mL). Following phase separation, the aqueous phase was extracted with CH.sub.2Cl.sub.2 (200 mL). The combined extracts were dried (MgSO.sub.4). The drying agent was removed by filtration and the filtrate was concentrated to dryness under reduced pressure giving the silylether as a translucent, pale yellow oily-liquid (82 g, 99%). FIG. 1

[0015] A solution of NaOH (19.3 g, 482 mmol, 2 eq.) in H.sub.2O (250 mL) was added in one portion to a solution of the methyl ester (82 g, 240 mmol) in iPrOH (600 mL). The reaction mixture was stirred for 1.5 h after which time analysis by TLC (100% CH.sub.2Cl.sub.2) indicated essentially complete consumption of the starting ester. The mixture was poured into brine (1200 mL) and extracted with hexane (2300 mL). This extraction was done to remove the slight excess of starting material as well as a few unidentified nonpolar impurities. The aqueous phase was acidified to pH2-3 (universal indicating pH paper) with 12M HCl, and extracted with hexane (2500 mL). The combined extracts were washed with brine (2400 mL) and dried (MgSO.sub.4). The drying agent was removed by filtration and the filtrate was concentrated to dryness under reduced pressure giving the crude acid as a thick, clear pale oil [crystallized after standing 5-6 days at room temperature] (75 g, 95%) which was used in the next step without further purification. FIG. 2

[0016] Diisopropyl azodicarboxylate (94%, 16 mL, 75 mmol, 1.5 eq.) was added dropwise over 20 minutes to an ice bath cooled solution of benzyl (S)-()-lactate (9.0 g, 50 mmol), the TBDPS protected (R)-lactic acid (16.4 g, 50 mmol) and PPh3 (19.7 g, 75 mmol, 1.5 eq.) in Et.sub.2O (1 L). After 30 min, the cooling bath was removed. After an additional 30-60 minutes, analysis of the reaction by TLC (30% EtOAc in hexanes) indicated complete consumption of the benzyl (S)-()-lactate. Silica gel (30 g) was added and the reaction mixture was concentrated to dryness under reduced pressure. Flash column chromatography (RediSepR.sub.f SiO.sub.2 (220 g), 100% hexanes.fwdarw.5% EtOAc in hexanes, monitoring at 220 and 254 nm) gave the fully protected dimer as a transparent oil (16.2 g, 66%). FIG. 3

[0017] A suspension of the benzyl ester (16.2 g, 33 mmol) and 10% Pd/C (1.8 g) in EtOAc (100 mL) was stirred overnight under a balloon of H.sub.2 after which time analysis of the reaction mixture by TLC (10% EtOAc in hexanes) indicated complete consumption of starting material. Celite (5 g) was added and, after 5 minutes of stirring, the mixture was filtered through a pad of Celite. The pad was washed with EtOAc (250 mL) and the filtrate was concentrated to dryness under reduced pressure giving the product as a transparent oil (13.2 g, 99%). FIG. 4

[0018] Diisopropyl azodicarboxylate (94%, 9.8 mL, 50 mmol, 1.5 eq.) was added dropwise over 10 minutes to an ice bath cooled solution of benzyl (S)-()-lactate (5.9 g, 33 mmol), the TBDPS protected (R)-lactic acid dimer (13.2 g, 33 mmol) and PPh3 (13.1 g, 50 mmol, 1.5 eq.) in Et.sub.2O (650 mL). After 30 min, the cooling bath was removed. After an additional 30-60 minutes, analysis of the reaction by TLC (30% EtOAc in hexanes) indicated complete consumption of the benzyl (S)-()-lactate. Silica gel (20 g) was added and the reaction mixture was concentrated to dryness under reduced pressure. Flash column chromatography (RediSepR.sub.f SiO.sub.2 (220 g), 100% hexanes.fwdarw.5% EtOAc in hexanes over 30 min, monitoring at 220 and 254 nm) gave the fully protected trimer as a transparent oil (17.4 g, 94%). FIG. 5

[0019] Glacial acetic acid (5.9 mL, 100 mmol, 12.7 eq.) was added to a solution of the silyl protected trimer (4.3 g, 7.6 mmol) in THF (35 mL). A solution of TBAF (1.0 M in THF, 17 mL, 17 mmol, 2.2 eq.) was added slowly and the reaction mixture was stirred at room temperature for approximately 2 h after which time, analysis by TLC (10% EtOAc in hexanes) indicated essentially complete consumption of starting material. The mixture was partitioned between ethyl acetate (150 mL) and brine (150 mL). The organic phase was washed with saturated aqueous NaHCO.sub.3 (2150 mL), aqueous 10% citric acid (150 mL), and brine (150 mL). The organic layer was drained onto silica gel (10 g) and the mixture was concentrated to dryness under reduced pressure. Flash column chromatography (RediSepR.sub.f SiO.sub.2 (40 g), 100% hexanes.fwdarw.40% EtOAc in hexanes, monitoring at 220 and 254 nm and visualizing fractions with KMnO.sub.4 stain) gave the OH-trimer as a clear oil (2.2 g, 89%). FIG. 6

[0020] A suspension of the benzyl ester (2.0 g, 6.2 mmol) and 10% Pd/C (300 mg) in EtOAc (20 mL) was stirred overnight under a balloon of H.sub.2 after which time analysis of the reaction mixture by TLC (30% EtOAc in hexanes) indicated complete consumption of starting material. The mixture was filtered through a pad of Celite. The pad was washed with EtOAc (210 mL) and the filtrate was concentrated to dryness under reduced pressure giving the trimer as a clear oil which crystallized while on the high vacuum line overnight (1.4 g, 97%). FIG. 7

[0021] A suspension of the benzyl ester (17.3 g, 31 mmol) and 10% Pd/C (2 g) in EtOAc (100 mL) was stirred overnight under a balloon of H.sub.2 after which time analysis of the reaction mixture by TLC (5% EtOAc in hexanes) indicated complete consumption of starting material. Celite (5 g) was added and, after 5 minutes of stirring, the mixture was filtered through a pad of Celite. The pad was washed with EtOAc (250 mL) and the filtrate was concentrated to dryness under reduced pressure giving the product as a transparent oil (14.7 g, 100%). FIG. 8

[0022] Diisopropyl azodicarboxylate (98%, 8.1 mL, 41 mmol, 1.5 eq.) was added dropwise over 10 minutes to an ice bath cooled solution of benzyl (S)-()-lactate (4.9 g, 27.1 mmol), the TBDPS protected (R)-lactic acid trimer (12.8 g, 27.1 mmol) and PPh3 (10.7 g, 41 mmol, 1.5 eq.) in Et.sub.2O (500 mL). After 30 min, the cooling bath was removed. After an additional 30-60 minutes, analysis of the reaction by TLC (30% EtOAc in hexanes) indicated complete consumption of the benzyl (S)-()-lactate. Silica gel (20 g) was added and the reaction mixture was concentrated to dryness under reduced pressure. Flash column chromatography (RediSepR.sub.f SiO.sub.2 (220 g), 100% hexanes.fwdarw.10% EtOAc in hexanes over 30 min, monitoring at 220 and 254 nm) gave the fully protected tetramer as a transparent oil (15.6 g, 91%). FIG. 9

[0023] A suspension of the benzyl ester (15.6 g, 24.6 mmol) and 10% Pd/C (2 g) in EtOAc (100 mL) was stirred overnight under a balloon of H.sub.2 after which time analysis of the reaction mixture by TLC (5% EtOAc in hexanes) indicated complete consumption of starting material. Celite (5 g) was added and, after 5 minutes of stirring, the mixture was filtered through a pad of Celite. The pad was washed with EtOAc (250 mL) and the filtrate was concentrated to dryness under reduced pressure giving the product as a transparent oil (13 g, 97%). FIG. 10

[0024] Diisopropyl azodicarboxylate (98%, 7.0 mL, 35.9 mmol, 1.5 eq.) was added dropwise over 10 minutes to an ice bath cooled solution of benzyl (S)-()-lactate (4.3 g, 23.9 mmol), the TBDPS protected (R)-lactic acid tetramer (13 g, 23.9 mmol) and PPh3 (9.4 g, 35.9 mmol, 1.5 eq.) in Et.sub.2O (500 mL). After 30 min, the cooling bath was removed. After an additional 30-60 minutes, analysis of the reaction by TLC (30% EtOAc in hexanes) indicated complete consumption of the benzyl (S)-()-lactate. Silica gel (20 g) was added and the reaction mixture was concentrated to dryness under reduced pressure. Flash column chromatography (RediSepR.sub.f SiO.sub.2 (220 g), 100% hexanes.fwdarw.10% EtOAc in hexanes over 30 min, monitoring at 220 and 254 nm) gave the fully protected pentamer as a transparent oil (13.1 g, 78%). FIG. 11

[0025] Glacial acetic acid (6.4 mL, 112 mmol, 12.7 eq.) was added to a solution of the silyl protected pentamer (6.2 g, 8.8 mmol) in THF (40 mL). A solution of TBAF (1.0 M in THF, 20 mL, 20 mmol, 2.2 eq.) was added slowly and the reaction mixture was stirred at room temperature for approximately 2 h after which time, analysis by TLC (10% EtOAc in hexanes) indicated essentially complete consumption of starting material. The mixture was partitioned between ethyl acetate (150 mL) and brine (150 mL). The organic phase was washed with saturated aqueous NaHCO.sub.3 (2150 mL), aqueous 10% citric acid (150 mL), and brine (150 mL). The organic layer was drained onto silica gel (10 g) and the mixture was concentrated to dryness under reduced pressure. Flash column chromatography (RediSepR.sub.f SiO.sub.2 (40 g), 100% hexanes.fwdarw.40% EtOAc in hexanes, monitoring at 220 and 254 nm and visualizing fractions with KMnO.sub.4 stain) gave the OH-pentamer as a clear oil (3.6 g, 88%). FIG. 12

[0026] A suspension of the benzyl ester (3.5 g, 7.5 mmol) and 10% Pd/C (300 mg) in EtOAc (20 mL) was stirred overnight under a balloon of H.sub.2 after which time analysis of the reaction mixture by TLC (30% EtOAc in hexanes) indicated complete consumption of starting material. The mixture was filtered through a pad of Celite. The pad was washed with EtOAc (210 mL) and the filtrate was concentrated to dryness under reduced pressure giving the pentamer as a clear pale oil (g, 95%). FIG. 13

[0027] A suspension of the benzyl ester (6.4 g, 9 mmol) and 10% Pd/C (0.7 g) in EtOAc (30 mL) was stirred overnight under a balloon of H.sub.2 after which time analysis of the reaction mixture by TLC (5% EtOAc in hexanes) indicated complete consumption of starting material. Celite (5 g) was added and, after 5 minutes of stirring, the mixture was filtered through a pad of Celite. The pad was washed with EtOAc (250 mL) and the filtrate was concentrated to dryness under reduced pressure giving the product as a transparent oil (5.5 g, 100%). FIG. 14

[0028] Diisopropyl azodicarboxylate (98%, 2.6 mL, 13.4 mmol, 1.5 eq.) was added dropwise over 10 minutes to an ice bath cooled solution of benzyl (S)-()-lactate (1.6 g, 8.9 mmol), the TBDPS protected (R)-lactic acid pentamer (5.5 g, 8.9 mmol) and PPh3 (3.5 g, 13.4 mmol, 1.5 eq.) in Et.sub.2O (500 mL). After 30 min, the cooling bath was removed. After an additional 30-60 minutes, analysis of the reaction by TLC (30% EtOAc in hexanes) indicated complete consumption of the benzyl (S)-()-lactate. Silica gel (20 g) was added and the reaction mixture was concentrated to dryness under reduced pressure. Flash column chromatography (RediSepR.sub.f SiO.sub.2 (80 g), 100% hexanes.fwdarw.20% EtOAc in hexanes over 30 min, monitoring at 220 and 254 nm) gave the fully protected hexamer as a transparent oil (6.0 g, 86%). FIG. 15

[0029] Glacial acetic acid (5.6 mL, 98 mmol, 12.7 eq.) was added to a solution of the silyl protected hexamer (6.0 g, 7.7 mmol) in THF (35 mL). A solution of TBAF (1.0 M in THF, 17 mL, 17 mmol, 2.2 eq.) was added slowly and the reaction mixture was stirred at room temperature for approximately 2 h after which time, analysis by TLC (10% EtOAc in hexanes) indicated essentially complete consumption of starting material. The mixture was partitioned between ethyl acetate (150 mL) and brine (150 mL). The organic phase was washed with saturated aqueous NaHCO.sub.3 (2150 mL), aqueous 10% citric acid (150 mL), and brine (150 mL). The organic layer was drained onto silica gel (10 g) and the mixture was concentrated to dryness under reduced pressure. Flash column chromatography (RediSepR.sub.f SiO.sub.2 (40 g), 100% hexanes.fwdarw.40% EtOAc in hexanes, monitoring at 220 and 254 nm and visualizing fractions with KMnO.sub.4 stain) gave the OH-hexamer as a clear oil (3.7 g, 89%). FIG. 16

[0030] A suspension of the benzyl ester (3.4 g, 6.3 mmol) and 10% Pd/C (300 mg) in EtOAc (20 mL) was stirred overnight under a balloon of H.sub.2 after which time analysis of the reaction mixture by TLC (30% EtOAc in hexanes) indicated complete consumption of starting material. The mixture was filtered through a pad of Celite. The pad was washed with EtOAc (210 mL) and the filtrate was concentrated to dryness under reduced pressure giving the hexamer as a clear pale oil that crystallized while on the high vacuum line overnight (2.6 g, 91%). FIG. 17

[0031] Testing D-Lactic Acid Oligomers to Sequester L-Lactate

[0032] Lactic acid concentration was measured by tetrazolium indicator color test strips (Accuvin, LLC, Napa, Calif.) from the reaction of L-lactic acid and nicotine-adenine in the presence of lactate dehydrogenase.

[0033] Samples of D-lactic acid oligomers (n=2, n=3, n=4, n=5, and n=6) were weighed on truncated 100 microliter pipette tips. The tips were then inserted into the pipette and D-lactic acid oligomers were mixed in 40 microliters of 10 mM L-lactic acid. The reactions were carried out at room temperature for 2 minutes, then 20 microliters were applied to L-lactic acid test strips, and the results were read after 2 minutes. (Tables 1 and 2) The molar ratio of D-lactic acid dimer to L-lactic acid was 0.004/0.004-0.006/0.004 for complete sequestration of L-lactic acid.

[0034] Stoichiometry of the L-lactic acid+D-lactic acid dimer stereocomplex reaction

TABLE-US-00001 TABLE 1 Stereocomplex reaction of L-lactic acid + D-lactic acid dimer Lactic acid D-lactic acid dimer Tetrazolium indicator 40 microliters of 10 mM 0 mg greater than 400 mg/L 40 microliters of 10 mM 0.5 mg D-lactic acid dimer 80 mg/L 40 microliters of 10 mM 1.0 mg D-lactic acid dimer no color change 40 microliters of 10 mM 5.0 mg D-lactic acid dimer no color change 40 microliters of 10 mM 7.0 mg D lactic acid dimer no color change

[0035] Reaction of L-lactate with n=3, n=4, n=5, and n=6 D-lactic acid oligomers

TABLE-US-00002 TABLE 2 Reaction of L-lactic acid with n = 3, n = 4, n = 5, and n = 6 D-lactic acid oligomers Lactic acid D-lactic acid oligomer Tetrazolium indicator 40 microliters of 10 mM 5.0 mg D-lactic acid n = 3 greater than 400 mg/L 40 microliters of 10 mM 5.0 mg D-lactic acid n = 4 greater than 400 mg/L 40 microliters of 10 mM 5.0 mg D-lactic acid n = 5 greater than 400 mg/L 40 microliters of 10 mM 5.0 mg D-lactic acid n = 6 greater than 400 mg/L

BENEFITS TO SOCIETY

[0036] In vitro, n=3, n=4, n=5, and n=6 D-lactic oligomers acid did not sequester L-lactate, however it is possible that one or more of these oligomers as a progdrug could be hydrolyzed to D-lactic acid dimer. If this occurs, these oligomers could be more potent medications, and these oligomers could be systemically administered.

REFERENCES

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