Long-acting prodrugs of entecavir, preparing methods and uses thereof

Abstract

Provided are a long-acting prodrug of Entecavir, preparation method and use thereof, wherein the prodrug of Entecavir has a structure of formula I. The prodrug of Entecavir can be released slowly, sustainably and steady, and converted to active compound of Entecavir to achieve a long-acting effect.

Claims

1. A compound of formula I, or a stereoisomer, or a pharmaceutically acceptable salt thereof, ##STR00029## wherein, R.sub.1 is —C(═O)—X.sub.1—Y.sub.1, and both R.sub.2, and R.sub.3 are H, and wherein either: a) X.sub.1 is selected from O, NH, (CH.sub.2).sub.m, or a chemical bond, and Y.sub.1 is substituted or unsubstituted —NHC(═O)—C.sub.7-30 hydrocarbyl, substituted or unsubstituted —C(═O)NH—C.sub.7-30 hydrocarbyl, wherein each substituted hydrocarbyl is optionally substituted with one or more groups independently selected from oxo (═O), thio (═S), halo, amino, —NHC(═O)R, —C(═O)NHR, —C(═O)R, ester group, cycloalkyl, aryl, or heteroaryl; and wherein m is an integer from 1 to 6, and R is branched or linear, saturated or unsaturated C.sub.1-26 hydrocarbyl, or b) X.sub.1 is a chemical bond, and Y.sub.1 is branched or linear and saturated or unsaturated C.sub.19-29 hydrocarbyl, or Y.sub.1 is selected from one of the following cholane aliphatic groups: ##STR00030## ##STR00031##

2. The compound of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein R.sub.1 is —C(═O)—X.sub.1—Y.sub.1; X.sub.1 is selected from O, (CH.sub.2).sub.m, or a chemical bond; Y.sub.1 is substituted or unsubstituted —NHC(═O)—C.sub.9-29 hydrocarbyl, substituted or unsubstituted —C(═O)NH—C.sub.9-29 hydrocarbyl, and both R.sub.2 and R.sub.3 are H; wherein each substituted hydrocarbyl is optionally substituted with one or more groups independently selected from oxo (═O), thio (═S), F, Cl, amino, —NHC(═O)R, —C(═O)NHR, —C(═O)R, ester group, cycloalkyl, aryl, or heteroaryl; and wherein m is an integer from 1 to 6, and R is branched or linear, saturated or unsaturated C.sub.1-26 hydrocarbyl; or R.sub.1 is —C(═O)—X.sub.1—Y.sub.1; X.sub.1 is selected from O, (CH.sub.2).sub.m, or a chemical bond; Y.sub.1 is substituted or unsubstituted —NHC(═O)—C.sub.9-27 hydrocarbyl, or substituted or unsubstituted —C(═O)NH—C.sub.9-27 hydrocarbyl, and both R.sub.2, and R.sub.3 are H; wherein each substituted hydrocarbyl is optionally substituted with one or more groups independently selected from oxo (═O), thio (═S), F, Cl, amino, —NHC(═O)R, —C(═O)NHR, —C(═O)R, ester group, cycloalkyl, aryl, or heteroaryl; and wherein m is an integer from 1 to 6, and R is branched or linear, saturated or unsaturated C.sub.1-26 hydrocarbyl.

3. The compound of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein R.sub.1 is —C(═O)—X.sub.1—Y.sub.1, X.sub.1 is O or a chemical bond, Y.sub.1 is branched or linear, saturated or unsaturated C.sub.19-25 hydrocarbyl, and both R.sub.2 and R.sub.3 are H.

4. The compound of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein R.sub.1 is CH.sub.3—(CH.sub.2).sub.n—C(═O)NH—(CH.sub.2).sub.m—C(═O)—, n is an integer from 6 to 22, m is an integer from 1 to 6, and both R.sub.2 and R.sub.3 are H; or R.sub.1 is CH.sub.3—(CH.sub.2).sub.n—C(═O)NH—(CH.sub.2).sub.m—C(═O)—, wherein n is an integer from 10 to 20, m is an integer from 1 to 3, and both R.sub.2 and R.sub.3 are H.

5. The compound of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound of formula I is selected from one of the following compounds: ##STR00032## ##STR00033## ##STR00034## ##STR00035##

6. A pharmaceutical composition comprising the compound of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, wherein the pharmaceutical composition is in a form of solution for injection, suspension for injection, or sterile powder for injection.

7. A method of preventing and/or treating hepatitis B disease in a subject in need thereof, comprising administering an effective amount of the compound of claim 1, or a stereoisomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition to the patients in need thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) Some of the examples will be described in detail, with reference to the following figures, wherein like designations denote like members, where:

(2) FIG. 1 shows a change of blood concentration of Entecavir after Entecavir-5′-docosanoate was administered to a beagle;

(3) FIG. 2 shows a change of blood concentration of Entecavir after Entecavir-5′-ursodesoxycholate was administered to a beagle; and

(4) FIG. 3 shows a change of blood concentration of Entecavir after Entecavir-5′-stearoylamidobutanoate was administered to a beagle.

DETAILED DESCRIPTION

(5) The present disclosure will be further described by the following examples. However, the examples are not intended to limit the protection scope of the present disclosure.

Example 1 Preparation of Entecavir-5′-palmitate

(6) ##STR00009##

(7) 690 mg of palmitic acid was added to 5 mL of sulfoxide chloride in a 50 mL single-neck flask, and heated to 78° C. followed by stirring and reacting for 1 to 2 hours. After the reaction, a concentration was performed to dryness under reduced pressure, then 5 mL of DCM was added. Further concentration was performed under reduced pressure while an oil pump was used to vacuumize for 1 hour, and acyl chloride was obtained for later use. 500 mg of Entecavir was added into 10 mL of pyridine, and DMAP in catalytic amount was further added. After the temperatures was lowered to 0° C., the obtained acyl chloride was dropwise added into the reaction flask. Half an hour after the stirring and reaction was started, the temperature was raised to room temperature, and stirring was kept with the reaction overnight.

(8) After the reaction was finished, water and dichloromethane were used for extraction, and saturated sodium bicarbonate and sodium chloride were successively used to wash. Organic phase was dried by anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Column chromatography was performed, wherein dichloromethane and methanol (volume ratio from 20:1 to 10:1) were used for gradient elution, and the elution part at a volume ratio of 10:1 was collected so as to obtain 105 mg of compound 1 of Entecavir-5′-palmitate, with a yield of 11.29%.

(9) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 7.65 (s, 1H), 6.47 (s, 2H), 5.44-5.33 (m, 1H), 5.14 (s, 1H), 5.10 (d, J=2.8 Hz, 2H), 4.63 (s, 1H), 4.18 (m, 3H), 2.74 (s, 1H), 2.32 (m, 3H), 2.15-2.03 (m, 1H), 1.59-1.45 (m, 2H), 1.21 (s, 24H), 0.84 (t, J=6.8 Hz, 3H).

Example 2 Preparation of Entecavir-5′-stearate

(10) ##STR00010##

(11) The process step was performed with reference to the method of Example 1, except that the palmitic acid was replaced with stearic acid. After the reaction product was separated and purified, the compound 2 of Entecavir-5′-stearate was obtained, with a yield of 25.50%.

(12) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 7.66 (s, 1H), 6.47 (s, 2H), 5.43-5.33 (m, 1H), 5.15 (s, 1H), 5.10 (d, J=3.2 Hz, 1H), 4.62 (s, 1H), 4.18 (m, 3H), 2.74 (s, 1H), 2.36-2.24 (m, 3H), 2.13-2.04 (m, 1H), 1.58-1.49 (m, 2H), 1.23 (s, 28H), 0.84 (t, J=6.8 Hz, 3H).

Example 3 Preparation of Entecavir-5′-icosanoate

(13) ##STR00011##

(14) The process step was performed with reference to the method of Example 1, except that the palmitic acid was replaced with icosanoic acid. After the reaction product was separated and purified, the compound 3 of Entecavir-5′-icosanoate was obtained, with a yield of 29.10%.

(15) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 7.66 (s, 1H), 6.47 (s, 2H), 5.38 (m, 1H), 5.15 (s, 1H), 5.08 (d, J=3.2 Hz, 1H), 4.62 (s, 1H), 4.18 (m, 3H), 2.73 (s, 1H), 2.34-2.25 (m, 3H), 2.15-2.01 (m, 1H), 1.63-1.46 (m, 2H), 1.23 (s, 32H), 0.85 (t, J=6.8 Hz, 3H).

Example 4 Preparation of Entecavir-5′-docosanoate

(16) ##STR00012##

(17) The process step was performed with reference to the method of Example 1, except that the palmitic acid was replaced with docosanoic acid. After the reaction product was separated and purified, the compound 4 of Entecavir-5′-docosanoate was obtained, with a yield of 30.50%.

(18) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 7.66 (s, 1H), 6.41 (s, 2H), 5.38 (t, J=8.9 Hz, 1H), 5.15 (s, 1H), 5.08 (d, J=3.2 Hz, 1H), 4.62 (s, 1H), 4.18 (m, 3H), 2.70 (d, J=23.4 Hz, 1H), 2.38-2.25 (m, 3H), 2.15-2.00 (m, 1H), 1.59-1.45 (m, 2H), 1.22 (s, 36H), 0.85 (t, J=6.7 Hz, 3H).

Example 5 Preparation of Entecavir-5′-ursodesoxycholate

(19) ##STR00013##

(20) 710 mg of ursodesoxycholic acid, 500 mg of Entecavir, 350 mg of EDCI, 0.3 mL DIPEA, and DMAP in catalytic amount were dissolved in 90 mL of DMF in a 250 mL single-neck flask, stirred and reacted for 18 hours. TLC test showed that half of the materials were reacted. Stirring was continued until the reaction finished.

(21) 1 mL of methanol was added for quenching reaction for 1 hour, and water and DCM were added to extract after the reaction. Solid dissoluble in methanol was precipitated and collected. TLC was used to test the solid, organic phase and aqueous phase, and it was shown that the product was mainly in the solid phase. After the solid phase was evaporated to dryness, column chromatography was performed, wherein dichloromethane and methanol (volume ratio from 10:1 to 7:1) were used for gradient elution, and the elution part at a volume ratio of 7:1 was collected so as to obtain 300 mg of compound 5 of Entecavir-5′-ursodesoxycholate, with a yield of 25.53%.

(22) .sup.1H NMR (500 MHz, DMSO-d6) δ 10.59 (s, 1H), 7.66 (s, 1H), 6.41 (s, 2H), 5.37 (t, J=9.1 Hz, 1H), 5.15 (s, 1H), 5.07 (d, J=32 Hz, 1H), 4.61 (s, 1H), 4.43 (d, J=4.6 Hz, 1H), 4.17 (m, 3H), 3.86 (d, J=6.8 Hz, 1H), 3.31-3.23 (m, 2H), 2.73 (s, 1H), 2.42-2.20 (m, 3H), 2.12-2.03 (m, 1H), 1.92 (d, J=11.6 Hz, 1H), 1.88-1.77 (m, 1H), 1.77-1.59 (m, 4H), 1.47-1.34 (m, 18H), 0.89 (d, J=6.5 Hz, 4H), 0.86 (s, 3H), 0.60 (s, 3H).

Example 6 Preparation of Entecavir-5′-oleate

(23) ##STR00014##

(24) The process step was performed with reference to the method of Example 1, except that the palmitic acid was replaced with oleic acid. After the reaction product was separated and purified, the compound 6 of Entecavir-5′-oleate was obtained, with a yield 21.98%.

(25) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 7.64 (s, 1H), 6.70 (s, 2H), 5.44-5.24 (m, 3H), 5.23-5.09 (m, 2H), 4.60 (t, J=2.3 Hz, 1H), 4.25-4.09 (m, 3H), 2.71 (s, 1H), 2.38-2.22 (m, 3H), 2.08 (m, 1H), 1.98 (m, 4H), 1.51 (m, 2H), 1.38-1.11 (m, 20H), 0.84 (t, J=6.9 Hz, 3H).

Example 7 Preparation of Entecavir-5′-naphthylacetate

(26) ##STR00015##

(27) The process step was performed with reference to the method of Example 1, except that the palmitic acid was replaced with naphthylacetic acid. After the reaction product was separated and purified, the compound 7 of Entecavir-5′-naphthylacetate was obtained, with a yield of 18.85%.

(28) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.81 (br, 1H), 7.94 (dt, J=6.7, 2.4 Hz, 2H), 7.85 (dd, J=6.3, 3.3 Hz, 1H), 7.56 (s, 1H), 7.52 (dt, J=6.5, 3.6 Hz, 2H), 7.48-7.42 (m, 2H), 6.65 (s, 2H), 5.33 (t, J=9.3 Hz, 1H), 5.17 (d, J=3.2 Hz, 1H), 5.00 (t, J=2.4 Hz, 1H), 4.50 (t, J=2.5 Hz, 1H), 4.28-4.13 (m, 4H), 4.10 (s, 1H), 2.80-2.69 (m, 1H), 2.14 (td, J=12.0, 11.4, 4.9 Hz, 1H), 2.06-1.90 (m, 1H).

Example 8 Preparation of Entecavir-5′-palmitoylaminoacetate

(29) ##STR00016##

(30) Methyl aminoacetate hydrochloride (0.73 g), palmitic acid (1 g), DCC (1.21 g) and DMAP (95 mg) were added into 50 mL single-neck flask. Then 20 mL of THF and 1.51 g of DIPEA were added and stirred at room temperature overnight. Filtrate was collected after filtration, and rotated to dryness. DCM was added to dissolve, and water, dilute HCl, saturated sodium bicarbonate and saturated saline solution were successively used to wash. Anhydrous sodium sulfate was used for drying, followed by filtration and rotation to dryness. Methanol was used for recrystallization, and 0.35 g of methyl palmitoylaminoacetate was obtained.

(31) The 0.35 g of palmitoylaminoacetate was dissolved in 10 mL of THF, and 50 mg of lithium hydrate monohydrate was dissolved in 5 mL of water. The lithium hydrate solution was dropwise added and stirred at room temperature for 2 hours, then the reaction was terminated. Dilute HCl was added to adjust pH to 1˜2 so as to precipitate solid, after the filtration and drying, the palmitoylaminoacetate was obtained.

(32) 88 mg of Entecavir and 100 mg of palmitoylaminoacetate were added into 50 mL single-neck flask, and 10 mL of DMF was added to dissolve. EDCI (612 mg), HOBT (431 mg), and TEA (0.44 mL) were added and stirred at room temperature overnight. After the rotation to dryness, DCM was added to dissolve, and dilute HCl, saturated sodium bicarbonate and saturated saline solution were successively used to wash. Anhydrous sodium sulfate was used for drying, followed by filtration and rotation to dryness. Column chromatography isolation was performed, then 43 mg of compound 8 of Entecavir-5′-palmitoylaminoacetate was obtained with a yield of 23.63%.

(33) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 7.65 (s, 1H), 6.47 (s, 2H), 5.44-5.33 (m, 1H), 5.19-5.05 (in, 2H), 4.63 (s, 1H), 4.18 (dt, J=6.5, 4.0 Hz, 3H), 3.61 (s, 2H), 3.08 (d, J′ 7.3 Hz, 1H), 2.74 (s, 1H), 2.32 (t, J=7.3 Hz, 3H), 2.15-2.03 (m, 1H), 1.59-1.45 (m, 2H), 1.21 (d, J=10.2 Hz, 28H), 0.84 (s, 3H).

Example 9 Preparation of Entecavir-3′-palmitate

(34) ##STR00017##

(35) 1 g of Entecavir was dissolved in 10 mL of pyridine, and 1.4 g of (2.4 eq) TBSCl was added and stirred to react for 2 hours. TLC test showed that the materials basically disappeared, two points appeared. Methanol was added to terminated the reaction, and water and DCM are used to extract. Saturated sodium bicarbonate and saturated sodium chloride were used to wash the organic phase and anhydrous sodium sulfate was used to dry the solution. A concentration was performed to dryness under reduced pressure, and column chromatography isolation was performed to obtain 0.8 g of compound 9.

(36) 167 mg of palmitic acid was added to 1 mL of sulfoxide chloride in 50 mL single-neck flask, and heated to 78° C., After stirring and reaction for 1 to 2 hours, the reaction was finished. A concentration was performed to dryness under reduced pressure, and 5 mL of DCM was added. Further concentration was performed under reduced pressure while an oil puny was used to vacuumize for 1 hour, and acyl chloride was obtained for later use. 200 mg of compound 9 was added into 10 mL of pyridine, and DMAP in catalytic amount was further added. After the temperatures was lowered to 0° C., the obtained acyl chloride was dropwise added into the reaction flask. Half an hour after the stirring and reaction was started, the temperature was raised to room temperature, and stirring was kept with the reaction overnight. After the post-processing reaction was finished, water and dichloromethane were used for extraction, and saturated sodium bicarbonate and sodium chloride were used to wash. Organic phase was dried by anhydrous sodium sulfate, and concentrated to dryness under reduced pressure, so as to obtain 350 mg of compound 10.

(37) 350 mg of compound 10 was dissolved in 5 mL of DCM, and 266 mg of triethylamine trifluoride was added and kept stirring at room temperature overnight. Saturated sodium bicarbonate and saline solution were successively used to wash. The organic phase was dried by anhydrous sodium sulfate and concentrated to dryness under reduced pressure. Column chromatography was performed, wherein dichloromethane and methanol (volume ratio from 30:1 to 10:1) were applied, and the elution part at a volume ratio of 10:1 was collected and concentrated, so as to obtain 130 mg of compound 11 of Entecavir-3′-palmitate, with a yield of 28.00%.

(38) .sup.1H NMR (500 MHz, DMSO-d6) δ10.61 (s, 1H), 7.70 (s, 1H), 6.43 (s, 2H), 5.25-5.26 (m, 2H), 5.16 (s, 1H), 5.00 (t, J=5.0 Hz, 1H), 4.61 (s, 1H), 3.62 (t, J=5.7 Hz, 2H), 2.67 (s, 1H), 2.50 (m, 1H), 2.28 (t, J=7.3) Hz, 2H), 2.19 (m, 1H), 1.58-1.46 (m, 2H), 1.22 (d, J=13.2 Hz, 24H), 0.84 (t, J=6.9 Hz, 3H).

Example 10 Preparation of Entecavir-3′-stearate

(39) ##STR00018##

(40) The process step was performed with reference to the method of Example 9, except that the palmitic acid was replaced with stearic acid. After the reaction product was separated and purified, the compound 13 of Entecavir-3′-stearate was obtained, with a yield of 22.44%.

(41) .sup.1H NMR (500 MHz, DMSO-d6) δ 10.59 (s, 1H), 7.70 (s, 1H), 6.42 (s, 2H), 5.27 (m, 2H), 5.16 (s, 1H), 4.99 (t, J=4.9 Hz, 1H), 4.61 (s, 1H), 3.62 (t, J=5.2 Hz, 2H), 2.67 (s, 1H), 2.50 (m, 1H), 2.29 (d, J=7.0 Hz, 2H), 2.19 (m, 1H), 1.52 (s, 2H), 1.22 (s, 28H), 0.84 (t, J=6.3 Hz, 3H).

Example 11 Preparation of Entecavir-2-dodecylcarbamate

(42) ##STR00019##

(43) 400 mg of Entecavir, 10 mL of pyridine and 0.67 mL of TMSCl were successively added into 50 mL single-neck flask, stirred for 3 hours. TLC test showed that the materials were basically reacted. 0.6 mL of dodecyl chloroformate was added and stirred overnight, then the reaction was finished.

(44) 1 mL of methanol was added for quenching reaction, heated to 45° C. and stirred for reaction for 1 hour. Dichloromethane and water were added to extract after the reaction, and the organic phase was extracted successively by saturated sodium bicarbonate and sodium chloride. The organic phase was dried by anhydrous sodium sulfate and concentrated to dryness. Column chromatography was performed, wherein dichloromethane and methanol (volume ratio from 8:1 to 5:1) were used for gradient elution, and the elution part at a volume ratio of 5:1 was collected and concentrated, so as to obtain 200 mg of compound 14 of Entecavir-2-dodecylcarbamate, with a yield of 18.41%.

(45) .sup.1H NMR (500 MHz, DMSO-d6) δ 11.35 (br, 1H), 7.93 (s, 1H), 5.44 (t, J=8.9 Hz, 1H), 5.10 (s, 1H), 4.93 (s, 1H), 4.86 (s, 1H), 4.53 (s, 1H), 4.26 (s, 1H), 4.15 (t, J=6.3 Hz, 2H), 3.54 (s, 2H), 2.54 (s, 1H), 2.31 (m, 1H), 2.12-2.04 (m, 1H), 1.61 (t, J=6.7 Hz, 2H), 1.32 (d, J=6.5 Hz, 2H), 1.23 (s, 18H), 0.84 (t, J=6.7 Hz, 3H).

Example 12 Preparation of Entecavir-2-dodecylcarbonate

(46) ##STR00020##

(47) 100 mg of Entecavir was added into 25 mL two-neck flask, and 6 mL of pyridine was added under protection of nitrogen at room temperature. 107.6 mg of dodecylchloroformate (dissolved in 2 mL of dichloromethane) (12 equivalent) was dropwise added and stirred at room temperature overnight. The reaction solution was rotated to dryness till no pyridine remained. 10 mL of dichloromethane, 2 mL of isopropanol and 5 mL of sodium bicarbonate were added and stirred for 5 minutes, organic phase was separated. The organic phase was washed with 5 mL of saturated sodium chloride and dried by anhydrous sodium sulfate. TLC was performed to obtain 15.4 mg of white solid, with a yield of 8.72%.

(48) .sup.1H NMR (400 MHz, DMSO-d6) δ10.65 (br, 1H), 7.65 (s, 1H), 6.47 (s, 2H), 5.43-5.33 (m, 1H), 5.16 (s, 1H), 5.12 (d, J=2.8 Hz, 1H), 4.62 (s, 1H), 4.30-4.19 (m, 2H), 4.16 (s, 1H), 4.09 (t, J=6.8 Hz, 2H), 2.77 (s, 1H)), 2.31 (ddd, J=12.6, 10.5, 4.7 Hz, 1H), 2.13-2.03 (m, 1H), 1.59 (p, J=6.8 Hz, 2H), 1.25 (m, 18H), 0.85 (t, J=6.8 Hz, 3H).

Example 13 Preparation of Entecavir-5′-hexacosanate

(49) ##STR00021##

(50) The process step was performed with reference to the method of Example 1, except that the palmitic acid was replaced with hexacosanic acid. After the reaction product was separated and purified, the compound 16 of Entecavir-5′-hexacosanate was obtained, with a yield of 21.33%.

(51) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 7.66 (s, 1H), 6.40 (s, 2H), 5.38 (d, J=8.9 Hz, 1H), 5.14 (s, 1H), 5.08 (d, J=3.2 Hz, 1H), 4.62 (s, 1H), 4.18 (m, 3H), 2.73 (d, J=23.4 Hz, 1H), 2.34-2.26 (m, 3H), 2.10-2.05 (m, 1H), 155-1.51 (m, 2H), 1.22 (s, 44H), 0.85 (t, J=6.7 Hz, 3H).

Example 14 Preparation of Entecavir-5′-triacontanate

(52) ##STR00022##

(53) The process step was performed with reference to the method of Example 1, except that the palmitic acid was replaced with triacontanic acid. After the reaction product was separated and purified, the compound 17 of Entecavir-5′-triacontanate was obtained, with a yield of 16.59%.

(54) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 7.65 (s, 1H), 6.40 (s, 2H), 5.37 (t, J=8.9 Hz, 1H), 5.14 (s, 1H), 5.08 (d, J=3.2 Hz, 1H), 4.61 (s, 1H), 4.18 (m, 3H), 2.73 (s, 1H), 2.34-2.26 (in, 3H), 2.10-2.04 (m, 1H)), 1.54-1.51 (m, 2H), 1.22 (s, 52H), 0.85 (t, J=6.7 Hz, 3H).

Example 15 Preparation of Entecavir-5′-chenodeoxycholate

(55) ##STR00023##

(56) The process step was performed with reference to the method of Example 5, except that the ursodesoxycholic acid was replaced with chenodeoxycholic acid. After the reaction product was separated and purified, the compound 18 of Entecavir-5′-chenodeoxycholate was obtained, with a yield of 20.09%.

(57) .sup.1H NMR (500 MHz, DMSO-d6) δ 10.59 (s, 1H), 7.66 (s, 1H), 6.41 (s, 2H), 5.37 (t, J=9.1 Hz, 1H), 5.15 (s, 1H), 5.07 (d, J=3.2 Hz, 1H), 4.61 (s, 1H), 4.43 (d, J=4.6 Hz, 1H), 4.17 (m, 3H), 3.86 (d, J=6.8 Hz, 1H), 3.31-3.23 (m, 2H), 2.73 (s, 1H), 2.42-2.20 (m, 3H), 2.12-2.03 (m, 1H), 1.92 (d, J=11.6 Hz, 1H), 1.88-1.77 (m, 1H), 1.77-1.59 (m, 4H), 1.47-1.34 (m, 18H), 0.89 (d, J=6.5 Hz, 4H), 0.86 (s, 3H), 0.60 (s, 3H).

Example 16 Preparation of Entecavir-5′-hyodeoxycholate

(58) ##STR00024##

(59) The process step was performed with reference to the method of Example 5, except that the ursodesoxycholic acid was replaced with hyodeoxycholic acid. After the reaction product was separated and purified, the compound 19 of Entecavir-5′-hyodeoxycholate was obtained, with a yield of 23.50%.

(60) .sup.1H NMR (500 MHz, DMSO-d6) δ 10.59 (s, 1H), 7.66 (s, 1H), 6.41 (s, 2H), 5.37 (t, J=9.1 Hz, 1H), 5.15 (s, 1H), 5.07 (d, J=3.2 Hz, 1H), 4.61 (s, 1H), 4.23 (d, J=4.6 Hz, 1H), 4.17 (m, 3H), 3.66 (d, J=6.8 Hz, 1H), 3.31-3.23 (m, 2H), 2.73 (s, 1H), 2.12-2.20 (m, 3H), 2.12-2.03 (m, 1H), 1.92 (d, J=11.6 Hz, 1H), 1.88-1.77 (m, 1H), 1.77-1.59 (in, 4H), 1.47-1.34 (m, 18H), 0.89 (d, J=6.5 Hz, 4H), 0.86 (s, 3H), 0.60 (s, 3H).

Example 17 Preparation of Entecavir-5′-deoxycholate

(61) ##STR00025##

(62) The process step was performed with reference to the method of Example 5, except that the ursodesoxycholic acid was replaced with deoxycholic acid. After the reaction product was separated and purified, the compound 20 of Entecavir-5′-deoxycholate was obtained, with a yield of 17.95%.

(63) .sup.1H NMR (500 MHz, DMSO-d6) δ 10.59 (s, 1H), 7.66 (s, 1H), 6.41 (s, 2H), 5.37 (t, J=9.1 Hz, 1H), 5.15 (s, 1H), 5.07 (d, J=3.2 Hz, 1H), 4.61 (s, 1H), 4.37 (d, J=4.6 Hz, 1H), 4.17 (m, 3H), 3.80 (d, J=6.8 Hz, 1H), 3.31-3.23 (m, 2H), 2.73 (s, 1H), 2.42-2.20 (in, 3H), 2.12-2.03 (m, 1H), 1.92 (d, J=11.6 Hz, 1H), 1.88-1.77 (m, 1H), 1.77-1.59 (m, 4H), 1.57-1.34 (m, 18H), 0.89 (d, J=6.5 Hz, 4H), 0.86 (s, 3H), 0.68 (s, 3H).

Example 18 Preparation of Entecavir-5′-lauroylamidobutanoate

(64) ##STR00026##

(65) The process step was performed with reference to the method of Example 8 by taking lauroylamidobutanoic acid as the material, and the compound 21 of Entecavir-5′-lauroylamidobutanoate was obtained, with a yield of 27.38%.

(66) .sup.1H NMR (500 MHz, DMSO-d6) δ10.56 (s, 1H), 7.75 (t, J=5.5 Hz, 1H), 7.65 (s, 1H), 6.37 (s, 2H), 5.38 (t, J=9.0 Hz, 1H), 5.14 (s, 1H), 5.07 (d, J=3.0 Hz, 1H), 4.60 (s, 1H), 4.18 (m, 3H), 3.06 (m, 2H), 2.75 (br, 1H), 2.34 (t, 0.1=7.5 Hz, 2H), 2.30 (m, 1H), 2.07 (m, 1H), 2.03 (t, J=7.5 Hz, 2H), 1.66 (m, 2H), 1.46 (m, 2H), 1.23 (s, 16H), 0.85 (t, J=7.0 Hz, 3H).

Example 19 Preparation of Entecavir-5′-stearoylamidobutanoate

(67) ##STR00027##

(68) The process step was performed with reference to the method of Example 8 by taking stearoylamidobutanoic acid as the material, and the compound 22 of Entecavir-5′-stearoylamidobutanoate was obtained, with a yield of 32.01%.

(69) .sup.1H NMR (500 MHz, DMSO-d6) δ 10.55 (s, 1H), 7.77 (t, J=5.5 Hz, 1H), 7.66 (s, 1H), 6.37 (s, 2H), 5.38 (t, J=9.0 Hz, 1H), 5.15 (s, 1H), 5.07 (d, J=3.0 Hz, 1H), 4.61 (s, 1H), 4.18 (m, 3H), 3.06 (m, 2H), 2.74 (br, 1H), 2.34 (t, J=7.5 Hz, 2H), 2.30 (m, 1H), 2.07 (m, 1H), 2.03 (t, J=7.5 Hz, 2H), 1.66 (m, 2H), 1.46 (m, 2H), 1.23 (s, 28H), 0.85 (t, J=7.0 Hz, 3H).

Example 20 Preparation of Entecavir-5′-icosanamidobutanoate

(70) ##STR00028##

(71) The process step was performed with reference to the method of Example 8 by taking icosanamidobutanoic acid as the material, and the compound 23 of Entecavir-5′-icosanamidobutanoate was obtained, with a yield of 35.96%.

(72) .sup.1H NMR (500 MHz, DMSO-d6) δ 10.55 (s, 1H), 7.76 (t, J=5.5 Hz, 1H), 7.67 (s, 1H), 6.37 (s, 2H), 5.36 (t, J=9.0 Hz, 1H), 5.15 (s, 1H), 5.07 (d, J=3.0 Hz, 1H), 4.62 (s, 1H), 4.18 (m, 3H), 3.04 (m, 2H), 2.74 (br, 1H), 2.35 (t, J=7.5 Hz, 2H), 2.30 (m, 1H), 2.05 (m, 1H), 2.02 (t, J=7.5 Hz, 2H), 1.66 (m, 2H), 1.46 (m, 2H), 1.23 (s, 32H), 0.85 (t, J=7.0 Hz, 3H).

Example 21 Pharmacokinetic Test of Entecavir-5′-docosanoate, Entecavir-5′-ursodesoxycholate and Entecavir-5′-stearoylamidobutanoate in the Body of Beagle

(73) Three conventional male beagles were used in the test, and they were forbidden to eat for 12 hours before administering drugs, and drank water freely meanwhile. Entecavir-5′-docosanoate (prepared in Example 4), Entecavir-5′-ursodesoxycholate (prepared in Example 5) and entecavir-5′-stearoylamidobutanoate (prepared in Example 19) were used to prepare a suspension, and were intramuscularly injected in a dosage of 0.508 mg/kg, 0.552 mg/kg, and 0.533 mg/kg respectively. Before drug administration and 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours, 48 hours, 96 hours, 144 hours, 192 hours, 240 hours, 288 hours, and 360 hours after drug administration, 1.0 mL whole blood of the beagle was collected from venous vein in front legs each time and centrifuged in an heparinized centrifuge tube at 6000 rpm for 10 minutes, to separate blood plasma, and the blood plasma was preserved at −80° C. to be measured.

(74) Treatment of blood plasma sample: 100 μL of blood plasma sample was added to 20 μL of methanol-water (V:V, 1:1) and 20 μL of interior label (2 μg/mL apigenin), and mixed through vortex. 200 μL of methanol was added and mixed through vortex, followed by being centrifuged under a centrifugal force of 14,000 g for 30 minutes. 10 μL of supernate was collected for analysis. The results are shown in Table 1, Table 2, and Table 3, as well as FIG. 1, FIG. 2 and FIG. 3.

(75) TABLE-US-00001 TABLE 1 BLOOD DRUG CONCENTRATION OF ENTECAVIR AFTER ENTECAVIR-5′-BEHENATE (0.508 mg/kg) WAS INTRAMUSCULARLY INJECTED INTO THE BEAGLE CONCENTRATION t(h) ng/ml 0.5 ND 1 0.18 2 0.3 4 2.07 8 6.6 12 15.6 18 639 24 3.19  48 (2 days) 0.52  96 (4 days) 1.08 144 (6 days) 0.76 192 (8 days) 1.03  240 (10 days) 1.26

(76) TABLE-US-00002 TABLE 2 BLOOD DRUG CONCENTRATION OF ENTECAVIR AFTER ENTECAVIR-5′-URSODESOXYCHOLATE (0.552 mg/kg) WAS INTRAMUSCULARLY INJECTED INTO THE BEAGLE CONCENTRATION TIME ng/ml 1 0.997 2 1.04 4 1.00 8 2.31 12 0.785 24 1.44 48 (2 days) 1.86 96 (4 days) 1.25 144 (6 days)  1.49 192 (8 days)  1.03 240 (10 days) 1.3 288 (12 days) 1.4 360 (15 days) 1.21

(77) TABLE-US-00003 TABLE 3 BLOOD DRUG CONCENTRATION OF ENTECAVIR AFTER ENTECAVIR-5′-STEAROYLAMINOBUTYRATE (0.533 mg/kg) WAS INTRAMUSCULARLY INJECTED INTO THE BEAGLE CONCENTRATION TIME ng/ml 1 0.653 2 1.24 4 1.55 8 2.78 12 3.12 24 3.25 48 (2 days) 2.86  96 (four days) 2.33 144 (6 days)  1.87 192 (8 days)  1.65 240 (10 days) 1.24 288 (12 days) 1.48 360 (15 days) 1.27

(78) Test results: the compound of the present disclosure can be effective in a long term in the body of animals. Particularly, injectable suspensions of Entecavir-5′-docosanoate, Entecavir-5′-ursodesoxycholate and entecavir-5′-stearoylamidobutanoate can be released sustainably and steady after being intramuscularly injected to the beagle, thus to achieve a long-acting effect.

(79) The compound of the present disclosure is suitable for preparing long-acting suspensions. It can be effective in a long term in the body due to proper solubility and dissolving speed, and is safe and reliable.

(80) Although the present invention has been disclosed in the form of preferred embodiments and variations thereon, it will be understood that numerous additional modifications and variations could be made thereto without departing from the scope of the invention.

(81) For the sake of clarity, it is to be understood that the use of ‘a’ or ‘an’ throughout this application does not exclude a plurality, and ‘comprising’ does not exclude other steps or elements.