13-oxidized ingenol derivative and use thereof

Abstract

The present invention relates to a derivative of 13-oxidized ingenol, use thereof in the prevention and/or treatment of a disease associated with proliferation or tumor in a subject, or a cosmetic indication, and use thereof in the prevention and/or treatment of a disease responsive to neutrophil oxidative burst, a disease responsive to a release of IL-8 by keratinocyte, or a disease responsive to induction of necrosis.

Claims

1. A compound of Formula (I), or a pharmaceutically acceptable salt, solvate, prodrug, metabolite or crystal form thereof: ##STR00219## wherein: R.sub.1 is selected from the group consisting of hydrogen and M-C(O)—, wherein M is selected from the group consisting of hydrogen, 1,3-nonadienyl, C.sub.1-C.sub.4 linear alkyl, C.sub.1-C.sub.4 branched alkyl, C.sub.4-C.sub.9 linear alkyl, C.sub.4-C.sub.9 branched alkyl, n-decyl, and n-undecyl; R.sub.2 is Q-C(O)—, wherein Q is selected from the group consisting of methyl, n-propyl, ##STR00220## and R.sub.3 is selected from the group consisting of hydrogen, hydroxyl and X—C(O)—O—, wherein X is selected from the group consisting of methyl, n-propyl, n-pentyl, t-butyl, ##STR00221## or R.sub.1 is M-C(O)—, wherein M is selected from the group consisting of C.sub.1-C.sub.9 linear or branched alkyl and C.sub.2-C.sub.11 linear or branched alkenyl containing 1 or 2 carbon-carbon double bonds; R.sub.2 is selected from the group consisting of hydrogen and Q-C(O)—, wherein Q is selected from the group consisting of methyl, n-propyl, t-butyl, ##STR00222## and R.sub.3 is selected from the group consisting of hydrogen and hydroxyl; or R.sub.1 is M-C(O)—, and M is n-undecyl; R.sub.2 is Q-C(O)—, wherein Q is ##STR00223## and R.sub.3 is selected from the group consisting of hydrogen and X—C(O)—O—, wherein X is selected from the group consisting of methyl, n-propyl, ##STR00224##

2. A compound of Formula (I), or a pharmaceutically acceptable salt, solvate, prodrug, metabolite or crystal form thereof: ##STR00225## wherein: R.sub.1 is selected from the group consisting of hydrogen and M-C(O)—, wherein M is selected from the group consisting of methyl, n-propyl, n-pentyl, n-heptyl, n-nonyl, 1,3-nondienyl and 1,3,5-nontrienyl; R.sub.2 is selected from the group consisting of hydrogen and Q-C(O)—, wherein Q is selected from the group consisting of methyl, n-propyl, n-pentyl, t-butyl, ##STR00226## R.sub.3 is selected from the group consisting of hydrogen and X—C(O)—O—, wherein X is selected from the group consisting of methyl, n-propyl, t-butyl, n-pentyl, ##STR00227## and, when R.sub.1 is M-C(O)—, M is methyl or 1,3,5-nontrienyl, R.sub.3 is X—C(O)—O— and X is ##STR00228## R.sub.2 is not hydrogen; when R.sub.1 is M-C(O)—, M is n-nonyl and R.sub.3 is CH.sub.3—C(O)—O—, R.sub.2 is not ##STR00229## when R.sub.1 is hydrogen and R.sub.3 is (CH.sub.3).sub.3C—C(O)—O—, R.sub.2 is not hydrogen or (CH.sub.3).sub.3C—C(O)—.

3. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, prodrug, metabolite or crystal form thereof, wherein, R.sub.1 is M-C(O)—, and M is n-undecyl; R.sub.2 is Q-C(O)—, wherein Q is selected from the group consisting of methyl, n-propyl, ##STR00230## R.sub.3 is selected from the group consisting of hydrogen, hydroxyl and X—C(O)—O—, wherein X is selected from the group consisting of methyl, n-propyl, t-butyl, ##STR00231##

4. A compound of Formula (I), or a pharmaceutically acceptable salt, solvate, prodrug, metabolite or crystal form thereof: ##STR00232## wherein: R.sub.1 is M-C(O)—, and M is n-undecyl; R.sub.2 is hydrogen; R.sub.3 is X—C(O)—O—, wherein, X is selected from the group consisting of methyl, n-propyl, ##STR00233##

5. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, prodrug, metabolite or crystal form thereof, wherein, R.sub.1 is M-C(O)—, and M is n-undecyl; R.sub.2 is Q-C(O)—, wherein Q is ##STR00234## R.sub.3 is X—C(O)—O—, wherein, X is selected from the group consisting of methyl, n-propyl, ##STR00235##

6. A compound or a pharmaceutically acceptable salt, solvate, prodrug, metabolite or crystal form thereof, wherein the compound is one of the following: TABLE-US-00006 Name of Compounds Structure of Compounds 13-O-decanoyl-ingenol 13-O-(2′E,4′-E-decadienoyl)-ingenol 20-deoxy-13-O-dodecanoyl-ingenol 6,7-epoxy-20-deoxy-ingenol 6,7-epoxy-20-O-(2,3-dimethylbutyryl)-13-O- dodecanoyl-ingenol 3-O-angeloyl-13-hydroxy-ingenol 13-O-acetyl-ingenol 13-O-n-butanoyl-ingenol 13-O-n-hexanoyl-ingenol 13-O-n-octanoyl-ingenol 3-O-angeloyl-13-O-acetyl-ingenol 3-O-acetyl-13-O-dodecanoyl-ingenol 3-O-n-butanoyl-13-O-dodecanoyl-ingenol 20-O-acetyl-13-O-dodecanoyl-ingenol 20-O-n-butanoyl-13-O-dodecanoyl-ingenol 20-O-(2,3-dimethylbutyryl)-13-O-dodecanoyl- ingenol 20-O-angeloyl-13-O-dodecanoyl-ingenol 3,20-O-diangeloyl-13-O-dodecanoyl-ingenol 3-O-angeloyl-13-O-hexanoyl-ingenol 3-O-angeloyl-13-O-hexaoyl-ingenol 3-O-angeloyl-13-O-octanoyl-ingenol 3-O-angeloyl-13-O-decanoyl-ingenol 3-O-(2,3-dimethylbutenoyl)-13-O- dodecanoyl-ingenol 3-O-(3,5-diethylisoxazolyl-4-formyl)-13-O- dodecanoyl-ingenol 20-O-tigloyl-13-O-dodecanoyl-ingenol 3-O-tigloyl-13-O-dodecanoyl-ingenol 20-O-(2,3-dimethylbutenoyl)-13-O-dodecanoyl- ingenol

7. A compound of Formula (II), or a pharmaceutically acceptable salt, solvate, prodrug, metabolite or crystal form thereof: ##STR00263## wherein: R.sub.1 is selected from the group consisting of hydrogen and M-C(O)—, wherein M is selected from the group consisting of hydrogen, 1,3-nonadienyl, C.sub.1-C.sub.4 linear alkyl, C.sub.1-C.sub.4 branched alkyl, C.sub.4-C.sub.9 linear alkyl, C.sub.4-C.sub.9 branched alkyl, n-decyl, and n-undecyl; and R.sub.3 is selected from the group consisting of hydrogen, hydroxyl, and X—C(O)—O—, wherein X is selected from the group consisting of methyl, n-propyl, n-pentyl, t-butyl, ##STR00264## or R.sub.1 is M-C(O)—, wherein M is selected from the group consisting of C.sub.1-C.sub.9 linear or branched alkyl and C.sub.2-C.sub.11 linear or branched alkenyl containing 1 or 2 carbon-carbon double bonds; and R.sub.3 is selected from the group consisting of hydrogen and hydroxyl; or R.sub.1 is M-C(O)—, and M is n-undecyl; and R.sub.3 is selected from the group consisting of hydrogen and X—C(O)—O—, wherein X is selected from the group consisting of methyl, n-propyl, ##STR00265##

8. A pharmaceutical composition comprising the compound or the pharmaceutically acceptable salt, solvate, prodrug, metabolite or crystal form of claim 1, and a pharmaceutically acceptable carrier.

Description

SPECIFIC EMBODIMENTS

(1) Exemplary embodiments for some of the compounds of the invention are provided below to demonstrate the advantageous activity and beneficial technical effects of the compounds of the invention. However, it should be understood that the following experimental schemes are merely illustrative of the present invention and are not intended to limit the scope of the invention. A person skilled in the art can make appropriate modifications or changes to the technical solutions of the present invention with the teachings of the present description without departing from the spirit and scope of the present invention.

(2) Examples wherein the specific conditions are not specified are carried out according to the conventional conditions or conditions suggested by manufacturer. The reagents or instruments used wherein the manufacturers are not indicated, are all commercially available conventional products.

Example 1 Preparation of 13-O-decanoyl-ingenol (Compound 1a), 13-O-(2′E,4′E-decadienoyl)-ingenol (Compound 1b) and 13-O-dodecanoyl-ingenol (Compound 1c), 20-O-deoxy-13-O-dodecanoyl-ingenol (Compound 1d), 6,7-epoxy-20-deoxy-ingenol (Compound 1e), 6,7-epoxy-20-O-(2,3-dimethylbutyryl)-13-O-dodecanoyl-ingenol (Compound 1f)

(3) 20 kg of kansui L. medicinal herbs was cold-soak extracted by 3 to 6 folds amount of 95% ethanol, until complete extraction; the extracting solutions were combined, and concentrated under reduced pressure until an alcohol-free taste to obtain an extract. To the extract, 10 folds amount of a solution of NaOH in methanol (0.5 M) was added, the mixture was stirred for about 2 hours. The mixture was adjusted to pH about 7 with dilute hydrochloric acid, and extracted 3 times with ethyl acetate, and then the ethyl acetate layers were combined, concentrated under reduced pressure to dryness. The extract was purified by normal phase silica gel column chromatography eluted with a gradient of petroleum ether: ethyl acetate (100:1 to 1:2) to obtain fractions rich in 13-oxidized ingenol and fractions rich in deoxy-ingenol, respectively. The fractions rich in 13-oxidized ingenol were purified by reverse-phase silica gel column chromatography, eluted with a gradient of methanol: water (10% methanol to 100% methanol), and the same fractions were combined to obtain 13-O-decanoyl-ingenol (Compound 1a), 13-O-(2′E,4′E-decadienoyl)-ingenol (Compound 1b), 13-O-dodecanoyl-ingenol (Compound 1c), and 6,7-epoxy-20-O-(2,3-dimethylbutyryl)-13-O-dodecanoyl-ingenol (Compound 1f).

(4) The fractions rich in deoxy-ingenol were purified by normal phase silica gel column chromatography eluted with a gradient of petroleum ether:acetone (20:1 to 1:2), and the same fractions were combined to obtain 20-O-deoxy-13-O-dodecanoyl-ingenol (Compound 1d), and 6,7-epoxy-20-deoxy-ingenol (Compound 1e).

(5) The structural formula and hydrogen nuclear magnetic resonance spectrum (.sup.1H-NMR) of the compounds are as follows:

1) 13-O-decanoyl-ingenol (Compound 1a)

(6) ##STR00189##

(7) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.02 (d, 1H), 5.88 (d, 1H), 4.39 (d, 1H), 4.02-4.21 (m, 4H), 3.83 (d, 1H), 3.59 (d, 1H), 3.25 (brs, 1H), 2.73 (brd, 1H), 2.46 (m, 1H), 2.15-2.22 (m, 3H), 1.85 (s, 3H), 1.55 (m, 2H), 1.25-1.29 (m, 13H), 1.22 (s, 3H), 1.07 (s, 3H), 0.96 (d, 3H), 0.88 (t, 3H).

2) 13-O-(2′E,4′E-decadienoyl)-ingenol (Compound 1b)

(8) ##STR00190##

(9) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.20 (m, 1H), 6.12-6.14 (m, 2H), 6.03 (d, 1H), 5.90 (s, 1H), 5.71 (d, 1H), 4.41 (brs, 1H), 4.05-4.22 (m, 4H), 3.84 (brs, 1H), 3.57 (brs, 1H), 3.13 (brs, 1H), 2.76 (d, 1H), 2.58 (brs, 1H), 2.45 (brs, 1H), 2.24 (dd, 1H), 2.15 (m, 2H), 1.86 (s, 3H), 1.42 (m, 2H), 1.24-1.29 (m, 8H), 1.08 (s, 3H), 0.96 (d, 3H), 0.89 (t, 3H).

3) 13-O-dodecanoyl-ingenol (Compound 1c)

(10) ##STR00191##

(11) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.01 (d, 1H), 5.85 (brs, 1H), 4.37 (s, 1H), 4.24 (brs, 1H), 4.17 (d, 1H), 4.08 (d, 1H), 4.05 (dd, 1H), 3.82 (brs, 1H), 2.72 (m, 1H), 2.46 (m, 1H), 2.19 (t, 2H), 2.15 (m, 1H), 1.84 (s, 3H), 1.54 (m, 2H), 1.20-1.29 (m, 20H), 1.06 (s, 3H), 0.94 (d, 3H), 0.87 (t, 3H).

4) 20-deoxy-13-O-dodecanoyl-ingenol (Compound 1d)

(12) ##STR00192##

(13) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 5.94 (s, 1H), 5.70 (d, 1H), 4.40 (brs, 1H), 4.15 (s, 1H), 3.96 (brd, 1H), 3.46 (d, 1H), 2.93 (brd, 1H), 2.66-2.74 (m, 2H), 2.36 (m, 1H), 2.17-2.23 (m, 3H), 1.85 (s, 3H), 1.77 (s, 3H), 1.54 (m, 2H), 1.22-1.24 (m, 17H), 1.21 (s, 3H), 1.05 (s, 3H), 0.96 (d, 3H), 0.87 (t, 3H).

5) 6,7-epoxy-20-deoxy-ingenol (Compound 1e)

(14) ##STR00193##

(15) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 5.96 (d, 1H), 4.28 (s, 1H), 3.26-3.32 (m, 2H), 3.16 (ddd, 1H), 2.26 (m, 1H), 2.16 (m, 1H), 1.82 (s, 3H), 1.70 (m, 1H), 1.41 (s, 3H), 1.18 (s, 3H), 1.07 (s, 3H), 0.93 (d, 3H), 0.86 (m, 1H), 0.69 (m, 1H).

6) 6,7-epoxy-20-O-(2,3-dimethylbutyryl)-13-O-dodecanoyl-ingenol (Compound 1f)

(16) ##STR00194##

(17) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 5.97 (d, 1H), 4.41 (d, 1H), 4.33 (brs, 1H), 4.00 (s, 1H), 3.98 (d, 1H), 3.52 (brd, 1H), 3.46 (d, 1H), 3.11 (dd, 1H), 2.74 (m, 1H), 2.62 (m, 1H), 2.16-2.29 (m, 4H), 1.84-1.91 (m, 4H), 1.55 (m, 2H), 1.18-1.31 (m, 20H), 1.08 (t, 9H), 0.85-0.95 (m, 9H).

Example 2 Preparation of 13-O-dodecanoyl-ingenol acetonide

(18) 13-O-dodecanoyl-ingenol (0.2 mmol) was dissolved in 0.5 mg/mL of a solution of p-toluenesulfonic acid monohydrate in acetone and stirred at 30° C. for 12 h. The reaction solution was concentrated to dryness under reduced pressure, re-dissolved in ethyl acetate, and washed with water and saturated aqueous sodium chloride solution, successively, and then the organic layer was concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether: ethyl acetate=10:1 to 6:4) to obtain 13-O-dodecanoyl-ingenol 3,4,5,20-diacetonide (Compound 2a), 13-O-dodecanoyl-ingenol 5,20-acetonide (Compound 2b), 13-O-dodecanoyl-ingenol 3,4-acetonide (Compound 2c), respectively.

(19) The structural formula and hydrogen nuclear magnetic resonance spectrum (.sup.1H-NMR) of the compounds are as follows:

1) 13-O-dodecanoyl-ingenol 3,4,5,20-diacetonide (Compound 2a)

(20) ##STR00195##

(21) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 5.93 (d, 1H), 5.67 (d, 1H), 4.86 (s, 1H), 4.25 (dd, 1H), 4.10 (d, 1H), 3.93 (s, 1H), 3.54 (brd, 1H), 2.72 (dd, 1H), 2.61 (dt, 1H), 2.21 (t, 2H), 2.10 (dd, 1H), 1.86 (s, 3H), 1.51-1.58 (m, 8H), 1.40 (s, 3H), 1.32 (s, 3H), 1.23-1.30 (m, 17H), 1.21 (s, 3H), 1.06 (s, 3H), 0.98 (d, 3H), 0.87 (t, 3H).

2) 13-O-dodecanoyl-ingenol 5,20-acetonide (Compound 2b)

(22) ##STR00196##

(23) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 5.89 (d, 1H), 5.77 (d, 1H), 4.28 (d, 1H), 4.18 (s, 2H), 4.04 (brd, 1H), 3.94 (s, 1H), 3.55 (s, 1H), 2.74 (dd, 1H), 2.59 (dt, 1H), 2.53 (d, 1H), 2.20 (t, 2H), 2.15 (dd, 1H), 1.86 (s, 3H), 1.55 (m, 2H), 1.41 (s, 3H), 1.36 (s, 3H), 1.25 (brs, 17H), 1.23 (s, 3H), 1.07 (s, 3H), 0.95 (d, 3H), 0.87 (t, 3H).

3) 13-O-dodecanoyl-ingenol 3,4-acetonide (Compound 2c)

(24) ##STR00197##

(25) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.09 (d, 1H), 5.75 (d, 1H), 4.77 (s, 1H), 4.10-4.17 (m, 4H), 3.22 (d, 1H), 2.73-2.79 (m, 2H), 2.20 (t, 2H), 1.95 (m, 1H), 1.86 (s, 3H), 1.54 (s, 5H), 1.49 (s, 3H), 1.22-1.31 (m, 20H), 1.08 (s, 3H), 1.01 (d, 3H), 0.88 (t, 3H).

Example 3 Preparation of 3-O-angeloyl-13-hydroxy-ingenol (Compound 3)

(26) ##STR00198##

(27) 13-O-dodecanoyl-ingenol 3,4,5,20-diacetonide (Compound 2a, for which the preparation procedure may refer to Example 2) (0.5 mmol) was dissolved in diethyl ether, and lithium aluminium hydride was added in the condition of ice water bath and stirred for 15 min. The reaction solution was added with a saturated aqueous sodium sulfate solution, and extracted with diethyl ether. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether: ethyl acetate=7:3) to obtain 13-OH-ingenol 3,4,5,20-diacetonide (Compound 31).

(28) Compound 31 (0.4 mmol) was dissolved in methanol, 4 M diluted hydrochloric acid was added and stirred at room temperature for 15 h. The reaction solution was poured into water, adjusted to pH about 7 with sodium hydrogen carbonate solution, and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=3:2) to obtain 13-OH-ingenol (Compound 32).

(29) Compound 32 (0.3 mmol) was dissolved in 0.5 mg/mL of a solution of p-toluenesulfonic acid monohydrate in acetone and stirred at room temperature for 1 h. The reaction solution was concentrated to dryness under reduced pressure, re-dissolved in ethyl acetate, and washed with water and saturated aqueous sodium chloride solution, successively, and then the organic layer was concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=3:1) to obtain 13-OH-ingenol 5,20-acetonide (Compound 33).

(30) Compound 33 (0.2 mmol) was dissolved in acetonitrile, angelic anhydride (0.24 mmol), and cesium carbonate (0.3 mmol) were added, and stirred at room temperature for 24 h. The reaction solution was poured into water, extracted with diethyl ether. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=7:1) to obtain 3-O-angeloyl-13-OH-ingenol 5,20-acetonide (Compound 34).

(31) Compound 34 (0.1 mmol) was dissolved in methanol, 4 M diluted hydrochloric acid was added and stirred at room temperature for 8 h. The reaction solution was poured into water, extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=2:1) to obtain the title compound (Compound 3).

(32) The hydrogen nuclear magnetic resonance spectrum of Compound 3: .sup.1H-NMR (400 MHz, Acetone-d6) δ 6.11 (dq, 1H), 6.00 (d, 1H), 5.93 (brd, 1H), 5.82 (s, 1H), 4.10-4.15 (m, 3H), 3.95 (s, 1H), 2.79 (m, 1H), 2.68 (dd, 1H), 1.97 (m, 3H), 1.86-1.92 (m, 4H), 1.78 (d, 3H), 1.16 (s, 3H), 1.10 (d, 3H), 1.07 (s, 3H), 0.96 (d, 1H).

Example 4 Preparation of 13-O-acetyl-ingenol (Compound 4a)

(33) Method 1:

(34) 50 kg of dry aboveground part of Euphorbia cornigera was cold-soak extracted by 3 to 6 folds amount of 95% ethanol, until complete extraction, the extracting solutions were combined, and concentrated under reduced pressure until an alcohol-free taste to obtain an extract. To the extract, 10 folds amount of a solution of NaOH in methanol (0.5 M) was added, the mixture was stirred for about 2 hours. The mixture was adjusted to pH about 7 with dilute hydrochloric acid, and extracted with ethyl acetate for 3 times, and then the ethyl acetate layers were combined, and concentrated under reduced pressure to dryness. The extract was purified by normal phase silica gel column chromatography eluted with a gradient of petroleum ether: ethyl acetate (100:1 to 1:2) to obtain fractions rich in 13-oxidized ingenol. The fractions were further purified by reverse-phase silica gel column chromatography eluted with a gradient of acetonitrile: water (10% acetonitrile to 100% acetonitrile), and the same fractions were combined to obtain 13-O-acetyl-ingenol (Compound 4a), 13-O-(2′Z,4′E,6′Z-decatrienoyl)-ingenol (Compound 4b) and 13-O-dodecanoyl-ingenol (Compound 1c), respectively.

(35) Method 2:

(36) 13-OH-ingenol 3,4,5,20-diacetonide (Compound 31, for which the preparation procedure may refer to Example 3) (0.4 mmol) was dissolved in anhydrous pyridine, and acetic anhydride (0.5 mmol) was added and stirred at 50° C. for 24 h. The reaction solution was poured into ice water, adjusted to pH about 7 with sodium hydrogen carbonate solution, and extracted with diethyl ether. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether: ethyl acetate=10:1) to obtain 13-O-acetyl-ingenol 3,4,5,20-diacetonide (Compound 41).

(37) Compound 41 (0.25 mmol) was dissolved in methanol, 4 M diluted hydrochloric acid was added and stirred at room temperature for 15 h. The reaction solution was poured into water, adjusted to pH about 7 with sodium hydrogen carbonate solution, and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (chloroform:methanol=94:6) to obtain 13-O-acetyl-ingenol (Compound 4a). The structural formula and hydrogen nuclear magnetic resonance spectrum are as follows:

(38) ##STR00199##

(39) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.01 (d, 1H), 5.84 (d, 1H), 4.37 (d, 1H), 4.23 (s, 1H), 4.03-4.18 (m, 3H), 3.81 (d, 1H), 3.68-3.71 (m, 2H), 2.93 (m, 1H), 2.72 (dd, 1H), 2.47 (m, 1H), 2.15 (dd, 1H), 1.96 (s, 3H), 1.84 (d, 3H), 119-1.24 (m, 4H), 1.08 (s, 3H), 0.95 (d, 3H).

Example 5 Preparation of 13-O-n-butanoyl-ingenol (Compound 5)

(40) ##STR00200##

(41) 13-O-n-butanoyl-ingenol 3,4,5,20-diacetonide (Compound 51) was prepared according to the preparation method of 13-O-acetyl-ingenol 3,4,5,20-diacetonide (Compound 41) (refer to Example 4), except that acetic anhydride was replaced with n-butyric anhydride.

(42) Using Compound 51 as a raw material, the acetonide-protecting group was removed according to the preparation procedure of Compound 4a to obtain 13-O-n-butanoyl-ingenol (Compound 5).

(43) The hydrogen nuclear magnetic resonance spectrum of Compound 5: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.02 (d, 1H), 5.88 (d, 1H), 4.39 (d, 1H), 4.03-4.20 (m, 4H), 3.82 (d, 1H), 3.54 (d, 1H), 3.16 (d, 1H), 2.73 (dd, 1H), 2.58 (m, 1H), 2.45 (m, 1H), 2.14-2.25 (m, 3H), 1.85 (s, 3H), 1.59 (m, 2H), 1.24 (d, 1H), 1.22 (s, 3H), 1.07 (s, 3H), 0.96 (d, 3H), 0.92 (t, 3H).

Example 6 Preparation of 13-O-n-hexanoyl-ingenol (Compound 6)

(44) ##STR00201##

(45) 13-O-n-hexanoyl-ingenol 3,4,5,20-diacetonide (Compound 61) was prepared according to the preparation method of 13-O-acetyl-ingenol 3,4,5,20-diacetonide (Compound 41) (refer to Example 4), except that acetic anhydride was replaced with n-hexanoic anhydride.

(46) Using Compound 61 as a raw material, the acetonide-protecting group was removed according to the preparation method of Compound 4a to obtain 13-O-n-hexanoyl-ingenol (Compound 6).

(47) The hydrogen nuclear magnetic resonance spectrum of Compound 6: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.00 (d, 1H), 5.84 (d, 1H), 4.35 (d, 1H), 4.25 (s, 1H), 4.03-4.18 (m, 3H), 3.85 (d, 1H), 3.76-3.82 (m, 2H), 3.08 (m, 1H), 2.72 (dd, 1H), 2.46 (m, 1H), 2.10-2.22 (m, 3H), 1.84 (s, 3H), 1.55 (m, 2H), 1.23-1.30 (m, 5H), 1.21 (s, 3H), 1.06 (s, 3H), 0.94 (d, 3H), 0.87 (t, 3H).

Example 7 Preparation of 13-O-n-octanoyl-ingenol (Compound 7)

(48) ##STR00202##

(49) 13-O-n-octanoyl-ingenol 3,4,5,20-diacetonide (Compound 71) was prepared according to the preparation method of 13-O-acetyl-ingenol 3,4,5,20-diacetonide (Compound 41) (refer to Example 4), except that acetic anhydride was replaced with n-octanoic anhydride.

(50) Using Compound 71 as a raw material, the acetonide-protecting group was removed according to the preparation method of Compound 4a to obtain 13-O-n-octanoyl-ingenol (Compound 7).

(51) The hydrogen nuclear magnetic resonance spectrum of Compound 7: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.00 (d, 1H), 5.84 (d, 1H), 4.36 (d, 1H), 4.24 (s, 1H), 4.03-4.18 (m, 3H), 3.73-3.82 (m, 3H), 3.00 (brs, 1H), 2.72 (brd, 1H), 2.46 (m, 1H), 2.11-2.21 (m, 3H), 1.84 (s, 3H), 1.54 (m, 2H), 1.22-1.32 (m, 9H), 1.21 (s, 3H), 1.06 (s, 3H), 0.94 (d, 3H), 0.86 (t, 3H).

Example 8 Preparation of 3-O-angeloyl-13-O-acetyl-ingenol (Compound 8)

(52) ##STR00203##

(53) 13-O-acetyl-ingenol (Compound 4a, for which the preparation procedure may refer to Example 4) (0.5 mmol) was dissolved in 0.4 mg/mL of a solution of p-toluenesulfonic acid monohydrate in acetone and stirred at room temperature for 1 h. The reaction solution was concentrated to dryness under reduced pressure, re-dissolved in ethyl acetate, and washed with water and saturated aqueous sodium chloride solution, successively, and then the organic layer was concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether: ethyl acetate=2:1) to obtain 13-O-acetyl-ingenol 5,20-acetonide (Compound 81).

(54) Compound 81 (0.4 mmol) was dissolved in acetonitrile, angelic anhydride (0.6 mmol), and cesium carbonate (0.6 mmol) were added, and stirred at room temperature for 24 h. The reaction solution was poured into water, extracted with diethyl ether. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether: ethyl acetate=8:1) to obtain 3-O-angeloyl-13-O-acetyl-ingenol 5,20-acetonide (Compound 82).

(55) Compound 82 (0.3 mmol) was dissolved in methanol, 4 M diluted hydrochloric acid was added and stirred at room temperature for 12 h. The reaction solution was poured into water and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=4:1) to obtain the title compound (Compound 8).

(56) The hydrogen nuclear magnetic resonance spectrum of Compound 8: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.17 (dq, 1H), 6.01-6.05 (m, 2H), 5.53 (s, 1H), 4.43 (brs, 1H), 4.16 (brs, 2H), 4.07-4.11 (m, 2H), 3.56 (s, 1H), 2.72 (dd, 1H), 2.65 (m, 1H), 2.45 (brs, 1H), 2.19 (m, 1H), 2.01 (dd, 3H), 1.96 (s, 3H), 1.91 (m, 3H), 1.81 (d, 3H), 1.24 (d, 1H), 1.19 (s, 3H), 1.08 (s, 3H), 0.97 (d, 3H).

Example 9 Preparation of 3-O-angeloyl-13-O-dodecanoyl-ingenol (Compound 9)

(57) ##STR00204##

(58) Using 13-O-dodecanoyl-ingenol (Compound 1c, for which the preparation method may refer to Example 1) as raw material, acetonide protection reaction, esterification reaction, and the reaction of removing acetonide-protecting group were carried out in sequence according to the synthesis method of Compound 8 to obtain the title Compound (Compound 9).

(59) The hydrogen nuclear magnetic resonance spectrum of Compound 9: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.17 (dq, 1H), 6.02-6.04 (m, 2H), 5.53 (s, 1H), 4.34 (d, 1H), 4.16 (d, 2H), 4.08-4.11 (m, 2H), 3.54 (s, 1H), 2.73 (dd, 1H), 2.65 (m, 1H), 2.32 (t, 1H), 2.16-2.22 (m, 3H), 2.01 (dd, 3H), 1.91 (s, 3H), 1.81 (s, 3H), 1.60 (s, 2H), 1.55 (m, 2H), 1.22-1.28 (m, 15H), 1.19 (s, 3H), 1.06 (s, 3H), 0.97 (d, 3H), 0.88 (t, 3H).

Example 10 Preparation of 3-O-acetyl-13-O-dodecanoyl-ingenol (Compound 10)

(60) ##STR00205##

(61) Using 13-O-dodecanoyl-ingenol (Compound 1c, for which the preparation method may refer to Example 1) as a starting material, according to the synthesis method in Example 8, 13-O-dodecanoyl-ingenol 5,20-acetonide was synthesized and then angelic anhydride was replaced with acetic anhydride to synthesize 3-O-acetyl-13-O-dodecanoyl-ingenol 5,20-acetonide, and the acetonide-protecting group was removed to obtain the title Compound (Compound 10).

(62) The hydrogen nuclear magnetic resonance spectrum of Compound 10: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.01-6.03 (m, 2H), 5.47 (s, 1H), 4.32 (d, 1H), 3.98-4.16 (m, 4H), 3.58 (s, 1H), 2.71 (dd, 1H), 2.60 (m, 1H), 2.47 (t, 1H), 2.16-2.24 (m, 3H), 2.15 (s, 3H), 1.78 (s, 3H), 1.55 (m, 2H), 1.21-1.33 (m, 17H), 1.19 (s, 3H), 1.06 (s, 3H), 0.97 (d, 3H), 0.87 (t, 3H).

Example 11 Preparation of 3-O-n-butanoyl-13-O-dodecanoyl-ingenol (Compound 11)

(63) ##STR00206##

(64) Using 13-O-dodecanoyl-ingenol (Compound 1c, for which the preparation method may refer Example 1) as a starting material, according to the synthesis method in Example 8, 13-O-dodecanoyl-ingenol 5,20-acetonide was synthesized, and then angelic anhydride was replaced with butyric anhydride to synthesize 3-O-n-butanoyl-13-O-dodecanoyl-ingenol 5,20-acetonide, and the acetonide-protecting group was removed to obtain the title Compound (Compound 11).

(65) The hydrogen nuclear magnetic resonance spectrum of Compound 11: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.01-6.03 (m, 2H), 5.46 (s, 1H), 4.34 (d, 1H), 4.02-4.18 (m, 4H), 3.55 (s, 1H), 2.71 (dd, 1H), 2.60 (m, 1H), 2.47 (m, 1H), 2.38 (dt, 2H), 2.16-2.24 (m, 3H), 1.78 (s, 3H), 1.63-1.72 (m, 2H), 1.55 (m, 2H), 1.20-1.33 (m, 17H), 1.19 (s, 3H), 1.06 (s, 3H), 0.96-1.00 (m, 6H), 0.87 (t, 3H).

Example 12 Preparation of 20-O-acetyl-13-O-dodecanoyl-ingenol (Compound 12)

(66) ##STR00207##

(67) 13-O-dodecanoyl-ingenol (Compound 1c, for which the preparation method may refer to Example 1) (0.06 mmol) was dissolved in dichloromethane, acetic anhydride (0.06 mmol) and 4-dimethylaminopyridine (0.06 mmol) were added, and stirred at room temperature for 8 h. The reaction solution was poured into water, extracted with diethyl ether. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=4:1) to obtain the title compound (Compound 12).

(68) The hydrogen nuclear magnetic resonance spectrum of Compound 12: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.07 (d, 1H), 5.92 (d, 1H), 4.71 (d, 1H), 4.52 (d, 1H), 4.45 (d, 1H), 4.12 (s, 1H), 4.03 (dd, 1H), 3.69 (d, 1H), 2.91 (d, 1H), 2.73 (dd, 1H), 2.65 (d, 1H), 2.42 (m, 1H), 2.16-2.22 (m, 3H), 2.06 (s, 3H), 1.86 (s, 3H), 1.55 (m, 2H), 1.23-1.29 (m, 17H), 1.22 (s, 3H), 1.07 (s, 3H), 0.97 (d, 3H), 0.88 (t, 3H).

Example 13 Preparation of 20-O-n-butanoyl-13-O-dodecanoyl-ingenol (Compound 13)

(69) ##STR00208##

(70) 13-O-dodecanoyl-ingenol (Compound 1c, for which the preparation method may refer to Example 1) (0.07 mmol) was dissolved in dichloromethane, n-butyric anhydride (0.08 mmol) and 4-dimethylaminopyridine (0.08 mmol) were added, and stirred at room temperature for 8 h. The reaction solution was poured into water, extracted with diethyl ether. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=5:1) to obtain the title compound (Compound 13).

(71) The hydrogen nuclear magnetic resonance spectrum of Compound 13: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.07 (brd, 1H), 5.91 (s, 1H), 4.73 (d, 1H), 4.53 (d, 1H), 4.44 (d, 1H), 4.09 (s, 1H), 4.03 (m, 1H), 3.69 (brd, 1H), 2.95 (d, 1H), 2.73 (brd, 1H), 2.61 (d, 1H), 2.43 (m, 1H), 2.29 (t, 2H), 2.17-2.22 (m, 3H), 1.86 (s, 3H), 1.55 (m, 2H), 1.22-1.29 (m, 19H), 1.21 (s, 3H), 1.07 (s, 3H), 0.86-0.98 (m, 9H).

Example 14 Preparation of 20-O-(2,3-dimethylbutyryl)-13-O-decanoyl-ingenol (Compound 14)

(72) ##STR00209##

(73) 13-O-decanoyl-ingenol (Compound 1a, for which the preparation method may refer to Example 1) (0.06 mmol) was dissolved in dichloromethane, and 2,3-dimethylbutyric acid (0.06 mmol), N,N′-dicyclohexylcarbodiimide (0.08 mmol) and 4-dimethylaminopyridine (0.08 mmol) were added in the condition of ice water bath, then stirred at 20° C. for 32 h. The reaction solution was poured into water, extracted with diethyl ether. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=4:1) to obtain the desired compound (Compound 14).

(74) The hydrogen nuclear magnetic resonance spectrum of Compound 14: .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.08 (d, 1H), 5.89 (d, 1H), 4.72 (d, 1H), 4.53 (d, 1H), 4.43 (s, 1H), 4.03 (dd, 1H), 3.66 (d, 1H), 2.72 (dd, 1H), 2.43 (m, 1H), 2.15-2.28 (m, 4H), 1.89 (m, 1H), 1.84 (s, 3H), 1.55 (m, 2H), 1.23-1.28 (m, 13H), 1.20 (s, 3H), 1.08 (d, 3H), 1.07 (s, 3H), 0.96 (d, 3H), 0.86-0.90 (m, 9H).

Example 15 Preparation of 3,20-O-diangeloyl-13-O-dodecanoyl-ingenol (Compound 15a) and 20-O-angeloyl-13-O-dodecanoyl-ingenol (Compound 15b)

(75) 13-O-dodecanoyl-ingenol (Compound 1c, for which the preparation procedure may refer to Example 1) (0.06 mmol) was dissolved in acetonitrile, angelic anhydride (0.08 mmol) and cesium carbonate (0.08 mmol) were added, and stirred at room temperature for 30 h. The reaction solution was poured into water, extracted with diethyl ether. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=12:1 to 6:1), to obtain 3,20-O-diangeloyl-13-O-dodecanoyl-ingenol (Compound 15a), and 20-O-angeloyl-13-O-dodecanoyl-ingenol (Compound 15b), respectively.

1) 3,20-O-diangeloyl-13-O-dodecanoyl-ingenol (Compound 15a)

(76) ##STR00210##

(77) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.04-6.20 (m, 4H), 5.55 (s, 1H), 4.86 (d, 1H), 4.57 (d, 1H), 4.08 (dd, 1H), 3.92 (d, 1H), 3.77 (d, 1H), 3.53 (s, 1H), 2.72 (dd, 1H), 2.62 (m, 1H), 2.18-2.25 (m, 3H), 2.01 (dd, 3H), 1.90-1.95 (m, 6H), 1.88 (brs, 3H), 1.80 (brs, 3H), 1.56 (m, 2H), 1.24-1.28 (m, 17H), 1.18 (s, 3H), 1.06 (s, 3H), 0.98 (d, 3H), 0.88 (t, 3H).

2) 20-O-angeloyl-13-O-dodecanoyl-ingenol (Compound 15b)

(78) ##STR00211##

(79) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.05-6.09 (m, 2H), 5.91 (d, 1H), 4.79 (d, 1H), 4.62 (d, 1H), 4.43 (d, 1H), 4.04-4.09 (m, 2H), 3.72 (d, 1H), 3.06 (d, 1H), 2.73 (dd, 1H), 2.62 (d, 1H), 2.44 (m, 1H), 2.16-2.22 (m, 3H), 1.94 (m, 3H), 1.85-1.89 (m, 6H), 1.56 (m, 2H), 1.24-1.28 (m, 17H), 1.22 (s, 3H), 1.07 (s, 3H), 0.97 (d, 3H), 0.88 (t, 3H).

Example 16 Preparation of 3-O-angeloyl-13-O-butanoyl-ingenol (Compound 16)

(80) ##STR00212##

(81) Using 13-O-n-butanoyl-ingenol (Compound 5, for which the preparation method may refer to Example 5) as raw material, acetonide protection reaction, esterification reaction, and the reaction of removing acetonide-protecting group were carried out in sequence according to the synthesis method in Example 8 to obtain the title Compound (Compound 16).

(82) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.15 (dq, 1H), 6.00-6.03 (m, 2H), 5.55 (s, 1H), 4.56 (brs, 1H), 4.05-4.17 (m, 4H), 3.61 (s, 1H), 2.61-2.76 (m, 3H), 2.14-2.23 (m, 3H), 1.99 (dd, 3H), 1.89 (t, 3H), 1.79 (d, 3H), 1.58 (m, 2H), 1.22 (d, 1H), 1.18 (s, 3H), 1.05 (s, 3H), 0.96 (d, 3H), 0.91 (t, 3H).

Example 17 Preparation of 3-O-angeloyl-13-O-hexanoyl-ingenol (Compound 17)

(83) ##STR00213##

(84) Using 13-O-n-hexanoyl-ingenol (Compound 6, for which the preparation method may refer to Example 6) as raw material, acetonide protection reaction, esterification reaction, and the reaction of removing acetonide-protecting group were carried out in sequence according to the synthesis method in Example 8 to obtain the title Compound (Compound 17).

(85) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.15 (dq, 1H), 5.98-6.03 (m, 2H), 5.55 (s, 1H), 4.59 (brs, 1H), 4.05-4.18 (m, 4H), 3.62 (s, 1H), 2.59-2.75 (m, 2H), 2.14-2.23 (m, 3H), 1.99 (dd, 3H), 1.89 (t, 3H), 1.80 (s, 3H), 1.55 (m, 2H), 1.19-1.31 (m, 5H), 1.17 (s, 3H), 1.05 (s, 3H), 0.96 (d, 3H), 0.86 (t, 3H).

Example 18 Preparation of 3-O-angeloyl-13-O-octanoyl-ingenol (Compound 18)

(86) ##STR00214##

(87) Using 13-O-n-octanoyl-ingenol (Compound 7, for which the preparation method may refer to Example 7) as raw material, acetonide protection reaction, esterification reaction, and the reaction of removing acetonide-protecting group were carried out in sequence according to the synthesis method in Example 8 to obtain the title Compound (Compound 18).

(88) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.15 (dq, 1H), 5.98-6.05 (m, 2H), 5.54 (s, 1H), 4.53 (brs, 1H), 4.05-4.19 (m, 4H), 3.60 (s, 1H), 2.58-2.74 (m, 3H), 2.14-2.23 (m, 3H), 1.99 (dd, 3H), 1.90 (t, 3H), 1.80 (s, 3H), 1.54 (m, 2H), 1.20-1.31 (m, 9H), 1.18 (s, 3H), 1.05 (s, 3H), 0.96 (d, 3H), 0.86 (t, 3H).

Example 19 Preparation of 3-O-angeloyl-13-O-decanoyl-ingenol (Compound 19)

(89) ##STR00215##

(90) Using 13-O-decanoyl-ingenol (Compound 1a, for which the preparation method may refer to Example 1) as raw material, acetonide protection reaction, esterification reaction, and the reaction of removing acetonide-protecting group were carried out in sequence according to the synthesis method in Example 8 to obtain the title Compound (Compound 19).

(91) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.14 (q, 1H), 6.00 (brs, 2H), 5.55 (s, 1H), 4.60 (brs, 1H), 4.03-4.19 (m, 4H), 3.63 (s, 1H), 2.58-2.76 (m, 3H), 2.13-2.24 (m, 3H), 1.99 (d, 3H), 1.89 (brs, 3H), 1.79 (s, 3H), 1.54 (m, 2H), 1.19-1.28 (m, 13H), 1.17 (s, 3H), 1.05 (s, 3H), 0.95 (d, 3H), 0.86 (t, 3H).

Example 20 Preparation of 3-O-tigloyl-13-O-dodecanoyl-ingenol (Compound 20)

(92) ##STR00216##

(93) Using Compound 13-O-dodecanoyl-ingenol (Compound 1c, for which the preparation method may refer to Example 1) as a starting material, according to the synthesis method in Example 8, 13-O-dodecanoyl-ingenol 5,20-acetonide was synthesized, and then angelic anhydride was replaced with tiglic anhydride to synthesize 3-O-tigloyl-13-O-dodecanoyl-ingenol 5,20-acetonide, and the reaction of removing acetonide-protecting group were carried out to obtain the title Compound (Compound 20).

(94) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.87 (q, 1H), 6.00 (s, 2H), 5.52 (s, 1H), 4.65 (m, 1H), 4.00-4.18 (m, 4H), 3.61 (s, 1H), 2.96 (m, 1H), 2.71 (dd, 1H), 2.61 (m, 1H), 2.11-2.23 (m, 3H), 1.83 (s, 3H), 1.81 (d, 3H), 1.77 (s, 3H), 1.54 (m, 2H), 1.19-1.33 (m, 17H), 1.16 (s, 3H), 1.04 (s, 3H), 0.97 (d, 3H), 0.86 (t, 3H).

Example 21 Preparation of 3-O-(2,3-dimethylbutenoyl)-13-O-dodecanoyl-ingenol (Compound 21)

(95) ##STR00217##

(96) Using Compound 13-O-dodecanoyl-ingenol (Compound 1c, for which the preparation method may refer to Example 1) as a starting material, according to the synthesis method in Example 8, 13-O-dodecanoyl-ingenol 5,20-acetonide was synthesized, and then angelic anhydride was replaced with 2,3-dimethylbutenoyl chloride to synthesize 3-O-(2,3-dimethylbutenoyl)-13-O-dodecanoyl-ingenol 5,20-acetonide, and the reaction of removing acetonide-protecting group was carried out to obtain the title Compound (Compound 21).

(97) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 5.96-6.05 (m, 2H), 5.50 (s, 1H), 4.50 (d, 1H), 4.02-4.18 (m, 4H), 3.60 (s, 1H), 2.56-2.78 (m, 2H), 2.15-2.24 (m, 3H), 2.04 (d, 3H), 1.86 (s, 3H), 1.84 (s, 3H), 1.79 (d, 3H), 1.54 (m, 2H), 1.19-1.32 (m, 17H), 1.18 (s, 3H), 1.05 (s, 3H), 0.96 (d, 3H), 0.87 (t, 3H).

Example 22 Preparation of 3-O-(3,5-diethylisoxazolyl-4-formyl)-13-O-dodecanoyl-ingenol (Compound 22)

(98) ##STR00218##

(99) 13-O-dodecanoyl-ingenol (Compound 1c, for which the preparation procedure may refer to Example 1) (0.5 mmol) was dissolved in 0.4 mg/mL of a solution of p-toluenesulfonic acid monohydrate in acetone and stirred at room temperature for 1 h. The reaction solution was concentrated to dryness under reduced pressure, re-dissolved in ethyl acetate, and washed with water and saturated aqueous sodium chloride solution, successively, and then the organic layer was concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether: ethyl acetate=4:1) to obtain 13-O-dodecanoyl-ingenol 5,20-acetonide (Compound 221).

(100) Compound 221 (0.1 mmol) was dissolved in dichloromethane, and 3,5-diethylisoxazolyl-4-carboxylic acid (0.15 mmol), N,N′-dicyclohexylcarbodiimide (0.15 mmol), and 4-dimethylaminopyridine (0.15 mmol) were added in the condition of ice water bath, and stirred at 20° C. for 8 h. The reaction solution was poured into water and extracted with diethyl ether, The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=15:1) to obtain 3-O-(3,5-diethylisoxazolyl-4-formyl)-13-O-dodecanoyl-ingenol 5,20-acetonide (Compound 222).

(101) Compound 222 (0.05 mmol) was dissolved in methanol, 4 M diluted hydrochloric acid was added and stirred at room temperature for 12 h. The reaction solution was poured into water and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, and then concentrated to dryness under reduced pressure. The resulting residual was purified by flash chromatography (petroleum ether:acetone=2:1) to obtain the title compound (Compound 22).

(102) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.10 (d, 1H), 6.03 (d, 1H), 5.69 (s, 1H), 4.07-4.22 (m, 4H), 3.78 (brs, 1H), 3.06 (dq, 2H), 2.85 (m, 2H), 2.58-2.74 (m, 2H), 2.26 (dd, 1H), 2.19 (t, 2H), 2.03 (s, 1H), 1.83 (s, 3H), 1.54 (m, 2H), 1.17-1.32 (m, 23H), 1.15 (s, 3H), 1.04 (s, 3H), 0.99 (d, 3H), 0.86 (t, 3H).

Example 23 Test of the Inhibitory Activity of Compounds of the Present Invention Against Proliferation of Tumor Cell Strains

(103) In this example, the inhibitory activity of compounds of the present invention against proliferation of seven human tumor cell lines and mouse B16-F10 melanoma cells was tested in vitro, and the IC.sub.50 value (50% inhibitory concentration) of compounds against each tumor cell line was calculated.

(104) The seven human tumor cell lines are: MCF-7 (human breast cancer cells), LOVO (human colon cancer cells), A549 (human lung cancer cells), BGC-823 (human gastric cancer cells), SMMC-7721 (human liver cancer cells), K562 (human leukemia cells), and HeLa (human cervical cancer cells).

(105) (1) Test Method

(106) IC.sub.50 values of the compounds for A549, BGC-823, LOVO, MCF-7, SMMC-7721, HeLa, and B16-F10 cells were tested. The above cells were routinely cultured in 1640 nutrient solution containing 10% calf serum, with passage for 1 time every 2 days, and the cells in logarithmic growth phase were taken for experiments. After the A549, BGC-823, LOVO, MCF-7, SMMC-7721, HeLa and B16-F10 cells in logarithmic growth phase were subjected to 0.25% trypsin digestion, they were blown into a single cell suspension, and then the cell concentration was adjusted to about 1×10.sup.5.Math.mL.sup.−1 using RPMI 1640 solution containing 10% calf serum. The cells were seeded in a 96-well culture plate at 100 μL per well, and cultured for 24 hours until the cells were attached. K562 cells were taken and centrifuged and diluted into 2×10.sup.5.Math.mL.sup.−1 single cell suspension with RPMI 1640 solution containing 10% calf serum, and inoculated into a 96-well culture plate at 100 μL per well.

(107) 100 μL of sample solution with different concentrations to be tested were added to the above 96-well plate, each concentration was added to 4 wells in parallel, and the cell control group was added with an equal volume of RPMI 1640 medium containing 10% calf serum to make a reaction volume of 200 μL. After incubating in an incubator at 37° C., 5% CO.sub.2 for 72 h, 10 μL of 5 mg.Math.mL.sup.−1 MTT solution was added to each well, and incubated at 37° C. for 4 h. The supernatant was carefully aspirated, 180 μL of DMSO was added to each well followed by shaking, and the absorbance (OD) value was measured at 570 nm on the microplate reader. The inhibition rate was calculated, and regression equation was established with the logarithmic value of the sample concentration and the inhibition rate (IR) to find the IC.sub.50 value of the compound against the cells to be tested.
IR (%)=(1−OD value of the experimental group/OD value of the cell control group)×100%

(108) (2) Test Results

(109) The IC.sub.50 values of compounds of the present invention for inhibition of proliferation of seven human tumor cell lines and mouse B16-F10 melanoma cells are shown in Table 1.

(110) TABLE-US-00002 TABLE 1 IC.sub.50 value of compound for inhibition of proliferation of tumor cell lines (μg/mL) Cell line SMM Compound name and number A549 BGC-823 LOVO K562 C-7721 MCF-7 HeLA B16-F10 20-deoxy-13-O-dodecanoyl-ingenol 26.8 15.4 19.2 6.88 31.5 29.2 — — (Compound 1d) 13-O-decanoyl-ingenol 15.1 15.0 9.77 0.70 19.5 13.8 29.1 — (Compound 1a) 13-O-(2′E,4′E-decadienoyl)-ingenol 12.8 10.6 12.9 8.97 8.01 11.8 — — (Compound 1b) 13-O-dodecanoyl-ingenol 11.1 6.19 8.97 5.51 6.49 6.36 15.1 — (Compound 1c) 6,7-epoxy-20-deoxy-ingenol >200 >200 >200 5.52 >200 >200 — — (Compound 1e) 6,7-epoxy-20-O-(2,3-dimethylbutyryl)- — — — — — — 16.8 — 13-O-dodecanoyl-ingenol (Compound 1f) 13-O-n-octanoyl-ingenol (Compound 7) 29.7 25.1 24.1 1.72 16.0 16.2 29.3 38.4 3-O-acetyl-13-O-dodecanoyl-ingenol 10.7 14.7 9.41 0.22 13.1 16.3 10.9 21.4 (Compound 10) 3-O-n-butanoyl-13-O-dodecanoyl-ingenol 14.3 13.4 8.82 0.10 19.1 18.5 20.3 26.2 (Compound 11) 20-O-acetyl-13-O-dodecanoyl-ingenol 16.0 10.3 6.71 0.06 12.7 12.7 32.3 14.0 (Compound 12) 20-O-n-butanoyl-13-O-dodecanoyl- 17.2 12.4 6.93 0.07 12.5 18.0 24.2 30.5 ingenol (Compound 13) 20-O-angeloyl-13-O-dodecanoyl- 10.0 20.6 15.2 0.001 13.1 23.3 10.2 9.09 ingenol (Compound 15b) 3,20-O-diangeloyl-13-O-dodeanoyl- >100 >100 >100 3.22 26.9 >100 — — ingenol (Compound 15a) 13-O-dodecanoyl-ingenol 5,20-acetonide 15.0 17.8 23.0 0.85 15.2 13.7 — — (Compound 2b) 13-O-dodecanoyl-ingenol 3,4-acetonide 9.59 5.30 10.3 0.42 8.21 8.89 — — (Compound 2c) 3-O-angeloyl-13-O-acetyl-ingenol >50 >100 >50 0.0065 96.6 >50 >100 — (Compound 8) 3-O-angeloyl-13-O-butanoyl-ingenol 46.8 44.1 36.9 4.21 43.5 38.4 61.1 17.0 (Compound 16) 3-O-angeloyl-13-O-hexanol-ingenol 21.7 21.7 16.9 2.93 21.1 18.6 18.6 6.50 (Compound 17) 3-O-angeloyl-13-O-octanoyl-ingenol 13.6 10.1 8.47 1.41 12.4 11.7 12.2 8.43 (Compound 18) 3-O-angeloyl-13-O-decanoyl-ingenol 12.6 14.0 7.90 2.13 13.4 9.30 8.58 9.85 (Compound 19) 3-O-tigloyl-13-O-dodecanoyl-ingenol 13.1 11.0 9.91 0.81 18.4 6.80 7.45 3.33 (Compound 20) 3-O-(2,3-dimethylbutenoyl)-13-O- 11.8 10.4 12.4 2.06 12.8 8.32 15.3 15.1 dodecanoyl-ingenol (Compund 21) 3-O-(3,5-diethylisoxazolyl-4-formyl)-13- 18.3 11.3 11.0 1.14 20.1 10.9 9.59 2.96 O-dodecanoyl-ingenol (Compound 22) 3-O-angeloyl-13-O-dodecanoyl-ingenol 10.8 0.85 10.1 1.87 8.86 10.1 12.5 10.4 (Compound 9) 3-O-(2,3-dimethylbutyryl)-13-O- 11.0 10.4 7.60 0.12 14.5 9.05 24.3 17.8 dodecanol-ingenol 3-O-trimethylacetyl-13-O-dodecanoyl- 17.5 6.81 9.68 <0.20 15.1 8.79 14.2 7.70 ingenol 3-O-hexanoyl-13-O-dodecanoyl-ingenol 18.5 18.3 16.5 <0.20 16.1 14.0 — — 20-O-(2,3-dimethylbutyryl)-13-O- 8.89 11.0 8.36 0.64 10.7 10.1 14.5 16.6 dodecanoyl-ingenol 3-O-angeloyl-ingenol 31.2 43.5 33.4 0.0076 28.2 57.4 49.4 38.1 The results in Table 1 indicate that the derivative of 13-oxidized ingenol can exhibit inhibitory effect on the proliferation of various tumor cells, and some compounds exhibit selective inhibitory effect on leukemia cells.

Example 24 Effects of Compounds on the Neutrophil Oxidative Burst

(111) This example investigated the effect of compound on neutrophil oxidative burst and calculated the efficiency of each compound in stimulating neutrophil oxidative burst.

(112) (1) Test Method

(113) Blood was taken from the abdominal aorta of rat. The neutrophilic granulocytes were isolated by the operational procedure of the rat peripheral blood neutrophil isolation kit. The neutrophilic granulocytes were re-suspended by Hanks, and were plated in a 96-well white plate at 1×10.sup.5 cells/well, 80 μL/well; 1 mM of Luminol was added at 10 μL/well; 10 μL of the sample was added to each well, and the degree of oxidative burst was evaluated by chemiluminescence.
Calculation formula of Efficiency: Efficiency (%)=(C.sub.sample−C.sub.control)/C.sub.control×100%

(114) (2) Test Results

(115) The efficiency of compounds in stimulating neutrophil oxidative burst is shown in Table 2.

(116) TABLE-US-00003 TABLE 2 Efficiency of compounds in stimulating neutrophil oxidative burst Efficiency (%) Compound name and number 3 μg/mL 0.1 μg/mL 3-O-angeloy1-13-OH-ingenol (Compound 3) 65.0 — 13-O-decanoyl-ingenol (Compound 1a) 65.2 — 13-O-n-octanoyl-ingenol (Compound 7) 45.0 — 20-O-(2,3-dimethylbutyryl)-13-O-decanoyl- 62.8 — ingenol (Compound 14) 3-O-angeloy1-13-O-dodecanoyl-ingenol 77.0 — (Compound 9) 3-O-n-butanoyl-13-O-dodecanoyl-ingenol 33.5 — (Compound 11) 20-O-acetyl-13-O-dodecanoyl-ingenol 88.3 — (Compound 12) 20-O-n-butanoyl-13-O-dodecanoyl-ingenol 62.4 — (Compound 13) 13-O-dodecanoyl-ingenol 3,4-acetonide 64.6 — (Compound 2c) 3-O-angeloyl-13-O-butanoyl-ingenol 80.1 — (Compound 16) 3-O-angeloyl-13-O-hexanoyl-ingenol — 82.1 (Compound 17) 3-O-angeloyl-13-O-octanoyl-ingenol — 69.1 (Compound 18) 3-O-angeloyl-13-O-decanoyl-ingenol — 43.5 (Compound 19) 3-O-tigloyl-13-O-dodecanoyl-ingenol 43.8 — (Compound 20) 3-O-(2,3-dimethylbutenoyl)-13-O-dodecanoyl- 34.8 — ingenol (Compound 21) 3-O-(3,5-diethylisoxazolyl-4-formyl)-13-O- — 124 dodecanoyl-ingenol (Compound 22) 20-O-(2,3-dimethylbutyryl)-13-O-dodecanoyl- 64.5 — ingenol 3-O-trimethylacetyl-13-O-dodecanoyl-ingenol — 205 3-O-acetyl-13-O-dodecanoyl-ingenol — 111 (Compound 10) 20-O-angeloyl-13-O-dodecanoyl-ingenol 360 — (Compound 15b) 13-O-n-hexanoyl-ingenol (Compound 6) 52.2 — 3-O-(2,3-dimethylbutyryl)-13-O-dodecanoyl- — 71.4 ingenol 3-O-hexanoyl-13-O-dodecanoyl-ingenol — 173 3-O-angeloyl-ingenol 51.4 —

(117) As can be seen from Table 2, the derivative of 13-oxidized ingenol has a stimulating effect on neutrophil oxidative burst, and the efficiency of most of the compounds is higher than that of 3-O-angeloyl-ingenol.

Example 25 Effect of Compounds on Release of IL-8 by Human Keratinocytes (HaCaT)

(118) This Example investigated the effect of compound on release of IL-8 by human keratinocytes (HaCaT) and calculated the efficiency of each compound in stimulating the release of IL-8 by human keratinocytes (HaCaT).

(119) (1) Test Method

(120) Hacat cells were cultured in MEM+10% FBS under the conditions of 37° C., 5% CO.sub.2, and when the cells reached 80-95% fusion, they were inoculated into a 96-well culture plate (1×10.sup.4 cells/well) at 100 μL/well, and incubated for 18-24 h. The supernatant was discarded and the cells were incubated for 3-6 h in 1% FBS medium. Different concentrations of the sample solution of compound to be tested were added to each well, and the cell control group was added with an equal volume of 1% FBS medium at 10 μL/well, and incubated for 24 h. The supernatant was taken and tested according to the operation sequence of the IL-8 Elisa kit.
Calculation formula of Efficiency: Efficiency (%)=(C.sub.sample−C.sub.control)/C.sub.control×100%

(121) (2) Test Results

(122) The efficiency of compound in stimulating the release of IL-8 by human keratinocytes (HaCaT) is shown in Table 3.

(123) TABLE-US-00004 TABLE 3 Efficiency of compound in stimulating the release of IL-8 by HaCaT cells Efficiency Compound name and number 1 μg/mL 0.1 μg/mL 3-O-angeloyl-13-OH-ingenol (Compound 3) 23.8 — 3-O-angeloyl-13-O-acetyl-ingenol 38.3 — (Compound 8) 20-O-(2,3-dimethylbutyryl)-13-O-decanoyl- 22.6 — ingenol (Compound 14) 3-O-angeloy1-13-O-dodecanoyl-ingenol 23.5 — (Compound 9) 3-O-acetyl-13-O-dodecanoyl-ingenol — 79.5 (Compound 10) 20-O-acetyl-13-O-dodecanoyl-ingenol 37.0 — (Compound 12) 20-O-n-butanoyl-13-O-dodecanoyl-ingenol — 30.3 (Compound 13) 3-O-angeloyl-13-O-butanoyl-ingenol — 84.6 (Compound 16) 3-O-angeloyl-13-O-hexanoyl-ingenol — 121 (Compound 17) 3-O-angeloyl-13-O-octanoyl-ingenol — 29.8 (Compound 18) 3-O-angeloyl-13-O-decanoyl-ingenol — 144 (Compound 19) 3-O-tigloyl-13-O-dodecanoyl-ingenol — 109 (Compound 20) 3-O-(2,3-dimethylbutenoyl)-13-O-dodecanoyl- — 127 ingenol (Compound 21) 3-O-(3,5-diethylisoxazolyl-4-formyl)-13-O- — 144 dodecanoyl-ingenol (Compound 22) 3-O-trimethylacetyl-13-O-dodecanoyl-ingenol — 83.7 3-O-(2,3-dimethylbutyryl)-13-O-dodecanoyl- — 50 ingenol 3-O-hexanoyl-13-O-dodecanoyl-ingenol — 66.0 3-O-angeloyl-ingenol 41.4 31.9

(124) As can be seen from Table 3, the derivative of 13-oxidized ingenol has a stimulating effect on release of IL-8 by HaCaT cells, and the efficiency is comparable to that of 3-O-angeloyl-ingenol, or higher than 3-O-angeloyl-ingenol.

Example 26 Evaluation of Antitumor Activity of Compounds in B16-F0 Mouse Melanoma Model

(125) This Example investigated the antitumor activity of compounds in B16-F0 mouse melanoma model.

(126) (1) Test Method

(127) In vitro passage of B16-F0 tumor source was inoculated subcutaneously into female C57BL/6J mice, and passage was carried out after tumor formation. After passage, the subcutaneous tumor tissue of the tumor-bearing mice was taken and ground with 3 times amount of physiological saline to form a tumor cell suspension, and 0.2 mL/mouse of which was inoculated subcutaneously into the axilla of forelimb of mice, which were used as the tumor-source mice. Female C57BL/6J mice were taken and their hair in an area of 4 cm.sup.2 on the neck and back was shaven. After anesthesia with diethyl ether, B16 tumor source was inoculated intradermally at 0.05 mL/mouse. After 3 days of inoculation, the skin was continuously administered for 3 days, once per day, 20 μL/mouse each time, and in the test, a solvent group as a negative control group was included. On the 19th day of the experiment, the experiment was terminated. After the mice were euthanized, the intradermally transplanted tumors were completely separated and excised, and the tumor was weighed to calculate the tumor inhibition rate.
Calculation formula of tumor inhibition rate: tumor inhibition rate (%)=(W.sub.model group−W.sub.administration group)/W.sub.model group×100%

(128) The tumor growths of the experimental group and the solvent control group were compared to evaluate the antitumor activity of each compound, and the compound with P<0.05 was considered to be effective. The tumor inhibition rate of the compounds is shown in Table 4.

(129) TABLE-US-00005 TABLE 4 Tumor inhibition rate of compounds in B16-F0 mouse melanoma model Tumor- inhibition Compound name and number rate 3-O-angeloyl-13-O-acetyl-ingenol (Compound 8) 90.3% 3-O-angeloyl-13-O-butanoyl-ingenol (Compound 16) 89.6% 3-O-angeloyl-13-O-hexanoyl-ingenol (Compound 17) 68.8% 3-O-angeloyl-13-O-octanoyl-ingenol (Compound 18) 59.0% 3-O-angeloyl-13-O-decanoyl-ingenol (Compound 19) 65.7% 3-O-(3,5-diethylisoxazolyl-4-formyl)-13-O-dodecanoyl- 39.5% ingenol (Compound 22) 3-O-angeloyl-13-O-dodecanoyl-ingenol (Compound 9) 71.8% 3-O-(2,3-dimethylbutenoyl)-13-O-dodecanoyl-ingenol 64.1% (Compound 21) 3-O-trimethylacetyl-13-O-dodecanoyl-ingenol 47.4% 20-O-acetyl-13-O-dodecanoyl-ingenol 40.7%

(130) Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that: according to all teachings that have been disclosed, various modifications and substitutions can be made to those details, all of which are within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.