2,2-dimethyl-3,4-dihydrobenzo[h]chromene-5,6-dione

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The method according to claim 14, wherein the lapachol is reacted with sulfuric acid to provide β-lapachone.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The method according to claim 14, wherein the lapachol is reacted with sulfuric acid to provide β-lapachone.

Conditions: temp:80.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Into a 1,000 ml beaker containing sulfuric acid (300 ml), lapachol (30 g, 0.124 mol) was added in portions slowly over 5 minutes at room temperature while stirring vigorously. After addition, the dark mixture was stirred for an additional 30 min and then poured into ice water (800 g) with manual stirring in a 2,000 ml beaker. The mixture was transferred into a 2,000 ml separatory funnel and extracted twice with toluene (600 ml and 400 ml). The toluene phases were pooled and washed successively with 1% aqueous sodium chloride (800 ml), 1% aqueous sodium bicarbonate (400 ml) and 1% aqueous sodium chloride (800 ml), and then dried with sodium sulfate (50 g). After filtration, the filtrate was evaporated to dryness by Rotovap and then ethanol (300 ml) was added for co-evaporation to completely remove residual toluene. The residue was dissolved in 75% ethanol or absolute ethanol, preferably absolute ethanol (ethanol/water, 3:1, 300 ml) with heating in an 80° C. water bath, then was filtered and cooled to 4° C. slowly. Pure β-lapachone was isolated by filtration, then washed with cold 75% ethanol (4° C., 100 ml), dried under vacuum, packed under Argon atmosphere and stored at −20° C. in the dark. was added in portions slowly over 5 minutes at room temperature After addition the dark mixture was stirred for an additional 30 min stirring in a 2,000 ml beaker The mixture was transferred into a 2,000 ml separatory funnel extracted twice with toluene (600 ml and 400 ml) washed successively with 1% aqueous sodium chloride (800 ml), 1% aqueous sodium bicarbonate (400 ml) and 1% aqueous sodium chloride (800 ml) dried with sodium sulfate (50 g) After filtration the filtrate was evaporated to dryness by Rotovap ethanol (300 ml) was added for co-evaporation to completely remove residual toluene The residue was dissolved in 75% ethanol was filtered cooled to 4° C. slowly

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: β-lapachone, as well as its intermediates, derivatives and analogs thereof (also referred to herein as the “active compounds”), are described in Li, C. J. et al., J Biol. Chem., 1993. Unlike prior art β-lapachone syntheses as illustrated in Scheme 1, the synthesis of β-lapachone, in accordance with the present invention and generally illustrated in Scheme 2, commences with the reaction of 2-hydroxy-1,4-naphthoquinone with 1-bromo-3-methyl-2-butene in the presence of sodium iodide and triethylamine (a weak base) in dimethylsulfoxide (DMSO) to produce lapachol in up to 40% yield, after purification (20 g, up to 40% overall yield). Lapachol is then quantitatively converted to β-lapachone by treatment with sulfuric acid. The β-lapachone is purified by recrystallization from ethanol.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 17.4 g (0.10M) of 2-hydroxy-1,4-naphthoquinone was dissolved in 120 ml of DMSO, and 0.88 g (0.11M) of LiH was gradually added thereto. Here, this should be done with care because hydrogen evolves. The reaction solution was stirred, and after confirming no further production of hydrogen, was additionally stirred for another 30 min. Then, 14.8 g (0.11M) of methallyl bromide (1-bromo-2-methylpropene) and 3.35 g (0.025M) of LiI were gradually added thereto. The reaction solution was heated to 45° C. and then stirred vigorously for 12 hours at that temperature. The reaction solution was cooled below 10° C., and 80 g of ice was first added and 250 ml of water was then added. Thereafter, 25 ml of concentrated HCl was gradually added to maintain the resulting solution at an acidic pH>1. 200 ml of CH2Cl2 was added to the reaction mixture which was then shaken vigorously to separate two layers. The aqueous layer was extracted once again with addition of 70 ml of CH2Cl2 and was combined with the previously extracted organic layer. Two materials were confirmed to be formed newly by TLC and were subsequently used without any particular separation process. The organic layer was concentrated by distillation under reduced pressure, dissolved again in xylene and then refluxed for 8 hours. In this process, two materials on TLC were combined into one, thereby obtaining a relatively pure Lapachol derivative. The thus-obtained Lapachol derivative was mixed with 80 ml of sulfuric acid and stirred vigorously at room temperature for 10 min, and 200 g of ice was added thereto to complete the reaction. 80 ml of CH2Cl2 was added to the reaction materials which were then shaken vigorously. Thereafter, a CH2Cl2 layer was separated and washed with 5% NaHCO3. An aqueous layer was extracted once again using 50 ml of CH2Cl2, washed with 5% NaHCO3 and combined with the previously extracted organic layer. The organic layer was dried over MgSO4 and concentrated to give impure β-Lapachone derivative (Compound 4). The thus-obtained β-Lapachone derivative was recrystallized from isopropanol, thereby obtaining 12.21 g of pure Compound 4. the reaction were then shaken vigorously Thereafter, a CH2Cl2 layer was separated washed with 5% NaHCO3 An aqueous layer was extracted once again washed with 5% NaHCO3 The organic layer was dried over MgSO4 concentrated

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The compound 5-(acetyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl N-(tert-butoxycarbonyl)-2-methylalaninate (27) is synthesized as described in scheme 1a using zinc dust (2.0 g, 30.5 mmol), 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (2.0 g, 8.26 mmol), Na2S2O4 (5.75 g, 33.0 mmol), N-(tert-butoxycarbonyl)-2-methylalanine (3.36 g, 16.51 mmol), triethylamine (1.04 mL, 7.43 mmol), HBTU (6.26 g, 16.5 mmol) and DMF (30 mL) for the 1st step. The acetylation step is carried out using zinc dust (1.0 g, 15.3 mmol), thriethylamine (1.04 mL, 7.43 mmol) and acetic anhydride (20 mL). Both the compounds are generated in the reaction. The crude mixture is purified by two consecutive flash column chromatography (SiO2, 1st using a gradient from 100% dichloromethane to 2% EtOAc in dichloromethane and 2nd using a gradient from 10% EtOAc in hexanes to 25% EtOAc in hexanes) to afford pure desired single isomer product (0.405 g, %) as a white solid. 400 MHz 1H NMR (CDCl3) δ: 8.19 (d, J=8.8 Hz, 1H), 7.85 (d, J=7.6 Hz, 1H), 7.50-7.35 (m, 2H), 5.19 (br. s, 1H), 2.67 (t, J=6.6 Hz, 2H), 2.37 (s, 3H), 1.87 (t, J=6.8 Hz, 2H), 1.78 (s, 6H), 1.47 (s, 9H), 1.42 (s, 6H); LCMS: 472 [M+H]. Both the compounds are generated in the reaction The crude mixture is purified by two consecutive flash column chromatography (SiO2 to afford pure desired single isomer product (0.405 g, %) as a white solid

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A mixture of zinc dust (6.0 g, 91.7 mmol), 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (6.0 g, 24.8 mmol), Na2S2O4 (17.26 g, 99.1 mmol), N-(tert-butoxycarbonyl)glycine (8.77 g, 49.6 mmol), triethylamine (3.1 mL, 22.3 mmol), HBTU (18.79 g, 49.6 mmol) and DMF (100 mL) is stirred for 16 hours at room temperature. To the reaction mixture is then added EtOAc (300 mL). The reaction is filtered and the filtrate washed with H2O (4×200 mL). The organic extract is dried with Na2SO4 and concentrated under reduced pressure. The residue is dissolved in acetic anhydride (30 mL) followed by the addition of zinc dust (3.0 g, 45.9 mmol) and thriethylamine (3.35 mL, 24.0 mmol). The reaction is heated at 90° C. with vigorous stirring and held for 2 hours. The reaction is allowed to cool and the solvent removed under reduced pressure. The residue is dissolved in EtOAc (200 mL) and washed with water (2×100 mL). The organic extract is dried with Na2SO4 and concentrated under reduced pressure. The crude product is purified by flash column chromatography (SiO2, 2% EtOAc in dichloromethane) to afford about 60% pure final product. Crystallization of the partly pure solid from EtOAc/Hexane gives the desired product as pure white solid (3.2 g, 31%). M.p.=177° C.; 400 MHz 1H NMR (CDCl3) δ: 8.21 (d, J=8.4 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H), 7.46 (m, 2H), 5.13 (br. s, 1H), 4.30 (d, J=5.6 Hz, 2H), 2.68 (t, J=6.6 Hz, 2H), 2.38 (s, 3H), 1.87 (t, J=6.6 Hz, 2H), 1.48 (s, 9H), 1.42 (s, 6H); LCMS: 444 [M+H]; Calc. for C24H29NO7: C, 64.94; H, 6.59; N, 3.16; Found C 64.98, H 6.51, N 3.15. The reaction is filtered the filtrate washed with H2O (4×200 mL) The organic extract is dried with Na2SO4 concentrated under reduced pressure The residue is dissolved in acetic anhydride (30 mL) The reaction is heated at 90° C. with vigorous stirring held for 2 hours to cool the solvent removed under reduced pressure The residue is dissolved in EtOAc (200 mL) washed with water (2×100 mL) The organic extract is dried with Na2SO4 concentrated under reduced pressure The crude product is purified by flash column chromatography (SiO2, 2% EtOAc in dichloromethane)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 17.4 g (0.10M) of 2-hydroxy-1,4-naphthoquinone was dissolved in 120 ml of DMSO, and 0.88 g (0.11M) of LiH was gradually added thereto. Here, this should be done with care because hydrogen evolves. The reaction solution was stirred, and after confirming no further production of hydrogen, was additionally stirred for another 30 min. Then, 14.8 g (0.11M) of methallyl bromide (1-bromo-2-methylpropene) and 3.35 g (0.025M) of LiI were gradually added thereto. The reaction solution was heated to 45° C. and then stirred vigorously for 12 hours at that temperature. The reaction solution was cooled below 10° C., and 80 g of ice was first added and 250 ml of water was then added. Thereafter, 25 ml of concentrated HCl was gradually added to maintain the resulting solution at an acidic pH>1.200 ml of CH2Cl2 was added to the reaction mixture which was then shaken vigorously to separate two layers. The aqueous layer was extracted once again with addition of 70 ml of CH2Cl2 and was combined with the previously extracted organic layer. Two materials were confirmed to be formed newly by TLC and were subsequently used without any particular separation process. The organic layer was concentrated by distillation under reduced pressure, dissolved again in xylene and then refluxed for 8 hours. In this process, two materials on TLC were combined into one, thereby obtaining a relatively pure Lapachol derivative. The thus-obtained Lapachol derivative was mixed with 80 ml of sulfuric acid and stirred vigorously at room temperature for 10 min, and 200 g of ice was added thereto to complete the reaction. 80 ml of CH2Cl2 was added to the reaction materials which were then shaken vigorously. Thereafter, a CH2Cl2 layer was separated and washed with 5% NaHCO3. An aqueous layer was extracted once again using 50 ml of CH2Cl2, washed with 5% NaHCO3 and combined with the previously extracted organic layer. The organic layer was dried over MgSO4 and concentrated to give impure β-Lapachone derivative (Compound 4). The thus-obtained β-Lapachone derivative was recrystallized from isopropanol, thereby obtaining 12.21 g of pure Compound 4. the reaction were then shaken vigorously Thereafter, a CH2Cl2 layer was separated washed with 5% NaHCO3 An aqueous layer was extracted once again washed with 5% NaHCO3 The organic layer was dried over MgSO4 concentrated

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (1.21 g, 5 mmol) in benzene (12 ml) is added glycerol (0.92 g, 10 mmol) and p-toluenesulfonic acid monohydrate (0.28 g, 1.5 mmol). The resulting mixture is refluxed for 6 hours. After removal of the solvent, product is purified by column chromatography on silica gel eluting with 30% ethyl acetate in methylene chloride to provide 0.57 g (36% yield) as a light yellow solid, which is a mixture of diasteromers. M.p. 44-46° C.; 400 MHz 1H NMR (CDCl3) δ: 1.41 (s, 6H), 1.73-1.82 (m, 2H), 2.36-2.42 (m, 1H), 2.48-2.54 (m, 1H), 3.72-3.78 (m, 1H), 4.18 (d, J=2.5 Hz, 1H), 4.20-4.29 (m, 1H), 4.40 (t, J=8.0 Hz, 1H), 4.71-4.74 (m, 1H), 4.85 (d, J=8.8 Hz, 1H), 7.38-7.44 (m, 2H), 7.54-7.56 (m, 1H), 7.75-7.78 (m, 1H); LCMS: Calc. for C18H21O5: 217; Found 217. The resulting mixture is refluxed for 6 hours After removal of the solvent, product is purified by column chromatography on silica gel eluting with 30% ethyl acetate in methylene chloride to provide 0.57 g (36% yield) as a light yellow solid, which a mixture of diasteromers

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a mixture of zinc dust (50.0 g, 0.765 mol) and 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (50.0 g, 0.206 mol) is added DMF (500 mL). The mixture is allowed to stir for 15 min. To the reaction is then added a mixture of N-(tert-butoxycarbonyl) glycine (43.38 g, 0.247 mol) and HBTU (156.5 g, 0.413 mol) in 5 small portions over a period of 3 hours to control the exothermic reaction. The reaction was stirred for 16 hours. Ethyl acetate (EtOAc) (1300 mL) is then added, and the reaction is filtered through celite. The celite is bed washed with EtOAc (200 mL). The combined EtOAc solutions are washed with water (2×1000 mL) followed by saturated NaCl (500 mL). The organic layer is dried over sodium sulfate and concentrated under reduced pressure. The crude residue (103 g) is dissolved in acetic anhydride (150 mL) followed by the addition of zinc dust (13.5 g, 0.206 mol) and triethylamine (25.9 mL, 0.186 mol). The reaction is stirred for 1.15 hours. The solvent (acetic anhydride and triethylamine) is removed under reduced pressure to yield a residue. The residue was dissolved in EtOAc (1000 mL) and washed with water (2×500 mL), 5% sodium bicarbonate (2×500 mL) saturated NaCl (500 mL) and dried over Na2SO4. Concentration of the ethyl acetate solution under reduced pressure yields a yellowish white crude product (103 g) that is purified by crystallization from hexane-ethyl acetate. For crystallization, the crude product is dissolved in refluxing EtOAc (200 mL) and hexane (300 mL) is added. The mixture is allowed to cool to room temperature and stirred for 14 hr. A solid separated out which is collected by filtration and washed with 5% EtOAc in hexane (500 mL). The desired product (38.2 g) is obtained 98% pure with 2% of bis-glycine as a side-product. The 98% pure product (38.2 g) is again crystallized from refluxing EtOAc (165 mL) and hexanes (250 mL). The hot solution is allowed to cool to room temperature and stirred for 15 hr. The white solid obtained is filtered, washed with 5% EtOAc in hexane (400 mL), and dried under high vacuum at 45-50° C. The desired product (34.6 g, 38%) is obtained as a white solid at 99.83% purity based upon HPLC analysis. The 1H NMR is the same as described in Example 5a. To the reaction is then added the exothermic reaction The reaction was stirred for 16 hours the reaction is filtered through celite washed with EtOAc (200 mL) The combined EtOAc solutions are washed with water (2×1000 mL) The organic layer is dried over sodium sulfate concentrated under reduced pressure The crude residue (103 g) is dissolved in acetic anhydride (150 mL) The reaction is stirred for 1.15 hours The solvent (acetic anhydride and triethylamine) is removed under reduced pressure to yield a residue washed with water (2×500 mL), 5% sodium bicarbonate (2×500 mL) saturated NaCl (500 mL) dried over Na2SO4 Concentration of the ethyl acetate solution under reduced pressure yields a yellowish white crude product (103 g) that is purified by crystallization from hexane-ethyl acetate For crystallization the crude product is dissolved in refluxing EtOAc (200 mL) and hexane (300 mL) is added stirred for 14 hr A solid separated out which is collected by filtration washed with 5% EtOAc in hexane (500 mL)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A mixture of zinc dust (6.0 g, 91.7 mmol), 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (6.0 g, 24.8 mmol), Na2S2O4 (17.26 g, 99.1 mmol), N-(tert-butoxycarbonyl)glycine (8.77 g, 49.6 mmol), triethylamine (3.1 ml, 22.3 mmol), HBTU (18.79 g, 49.6 mmol), and DMF (100 ml) is stirred for 16 hours at room temperature. To the reaction mixture is then added ethyl acetate (300 ml). The reaction is filtered and the filtrate washed with H2O (4×200 ml). The organic extract is dried with Na2SO4, and concentrated under reduced pressure to yield a residue. The residue is dissolved in acetic anhydride (30 ml), zinc dust (3.0 g, 45.9 mmol) and triethylamine (3.35 ml, 24.0 mmol) are then added. The mixture is heated to 90° C. with vigorous stirring and held at 90° C. for 2 hours, after which it is allowed to cool and the solvent is removed under reduced pressure to yield a second residue. The second residue is dissolved in ethyl acetate (200 ml) and washed with water (2×100 ml). The organic extract is dried with Na2SO4 and concentrated under reduced pressure to yield unrefined product. The unrefined product is purified by flash column chromatography on silica, eluting with 2% ethyl acetate in dichloromethane, to afford product about 60% pure. The product is further purified by crystallization from ethyl acetate/hexane, which gives the desired product as pure white solid (3.2 g, 31%). M.p.=177° C.; 400 MHz 1H NMR (CDCl3) δ: 8.21 (d, J=8.4 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H), 7.46 (m, 2H), 5.13 (br. s, 1H), 4.30 (d, J=5.6 Hz, 2H), 2.68 (t, J=6.6 Hz, 2H), 2.38 (s, 3H), 1.87 (t, J=6.6 Hz, 2H), 1.48 (s, 9H), 1.42 (s, 6H); LCMS: 444 [M+H]; Calc. for C24H29NO7: C, 64.94; H, 6.59; N, 3.16; Found C, 64.98; H, 6.51; N, 3.15. The reaction is filtered the filtrate washed with H2O (4×200 ml) The organic extract is dried with Na2SO4 concentrated under reduced pressure to yield a residue The mixture is heated to 90° C. with vigorous stirring held at 90° C. for 2 hours to cool the solvent is removed under reduced pressure to yield a second residue washed with water (2×100 ml) The organic extract is dried with Na2SO4 concentrated under reduced pressure to yield unrefined product The unrefined product is purified by flash column chromatography on silica eluting with 2% ethyl acetate in dichloromethane

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution of 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (2.975 g, 12.3 mmoles) in 120 ml of methylene chloride is treated with 6.14 ml of a 1.0 M solution of TiCl4 in methylene chloride. The mixture is stirred at room temperature for 5 minutes, then a solution of 2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl 4-aminobenzoate (5.436 g, 13.5 mmoles) in 60 ml of methylene chloride is introduced followed by triethylamine (10.3 ml, 73.7 mmoles). The mixture is stirred at room temperature for 10 minutes. A second portion of 6.14 ml of a 1.0 M solution of TiCl4 in methylene chloride is added and the mixture is stirred at room temperature for 10 minutes. A third portion of 6.14 ml of a 1.0 M solution of TiCl4 in methylene chloride is added and the mixture is stirred at room temperature for 15 minutes. After stirring with the third aliquot of TiCl4, the mixture is poured onto 300 ml of ice, to form a mixture having a precipitate. The mixture is filtered to remove the precipitate and the organic and aqueous phases are then separated. The aqueous phase is extracted with two portions of 200 ml of methylene chloride. The combined organic extracts are dried with Na2SO4, filtered and concentrated in vacuo, giving a brown oil. Purification on silica gel using a gradient of 0-10% ethyl acetate in methylene chloride provided a brown reddish foam, which is triturated in diethyl ether, yielding 990 mg (13%) of the title compound as a brown solid. M.p.: 163-164° C. 400 MHz 1H NMR (DMSO-d6) δ: 8.13 (d, J=7.3 Hz, 1H), 7.93 (d, J=8.4 Hz, 2H), 7.88 (d, J=7.7 Hz, 2H), 7.82 (d, J=8.1 Hz, 1H), 7.7-7.55 (m, 5H), 7.40 (t, J=7.5 Hz, 2H), 7.30 (t, J=7.5 Hz, 2H), 6.79 (d, J=8.4 Hz, 2H), 4.33 (d, J=7.0 Hz, 2H), 4.3-4.2 (m, 3H), 3.45-3.35 (m, 2H), 2.25 (t, J=6.4 Hz, 2H), 1.79 (t, J=6.4 Hz, 2H), 1.41 (s, 6H). LCMS: 627 [M+H]. Calc. for C39H34N2O6.0.1 water: C, 74.46; H, 5.48; N, 4.46; Found C, 74.48; H, 5.06; N, 4.43. The mixture is stirred at room temperature for 10 minutes the mixture is stirred at room temperature for 10 minutes the mixture is stirred at room temperature for 15 minutes to form a mixture The mixture is filtered to remove the precipitate the organic and aqueous phases are then separated The aqueous phase is extracted with two portions of 200 ml of methylene chloride The combined organic extracts are dried with Na2SO4 filtered concentrated in vacuo giving a brown oil Purification on silica gel using a gradient of 0-10% ethyl acetate in methylene chloride provided a brown reddish foam, which is triturated in diethyl ether