1. Patent Search
  2. Details

CYCLIC DINUCLEOTIDE ANALOGUE, PHARMACEUTICAL COMPOSITION THEREOF, AND APPLICATION

20220041644 · 2022-02-10

    Inventors

    Cpc classification

    International classification

    Abstract

    A cyclic dinucleotide analogue, a pharmaceutical composition thereof, and application. A cyclic dinucleotide analogue (I), an isomer thereof, a prodrug, a stable isotope derivative, or a pharmaceutically acceptable salt has the following structure. The cyclic dinucleotide analogue can be used as a regulator of a stimulator of interferon genes (STING) and a related signal path thereof, and can effectively treat and/or relieve multiple types of diseases, including but not limited to malignant tumors, inflammations, autoimmune diseases, and infectious diseases. In addition, the STING regulator can also be used as a vaccine adjuvant.

    ##STR00001##

    Claims

    1. A cyclic di-nucleotide analogue (I), an isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof; ##STR00128## wherein each of Z.sub.1, Z.sub.2 is independently O, S, SO.sub.2, CH.sub.2, CF.sub.2 or Se; B.sub.1 is ##STR00129## B.sub.2 is ##STR00130## ##STR00131## ##STR00132## ##STR00133## L and L.sub.1 are each independently a connecting bond or CR.sup.1R.sup.2; L′ and L.sub.1′ are each independently a connecting bond or CR.sup.11R.sup.21; L.sub.2 is O, S or CR.sup.3R.sup.4; L.sub.2′ is O, S or CR.sup.31R.sup.41; X.sup.1 is O, S or CR.sup.5R.sup.6; X.sup.11 is O, S or CR.sup.51R.sup.61; X.sup.2 is O, S or CR.sup.7R.sup.8; and X.sup.21 is O, S or CR.sup.71R.sup.81; X.sup.3 and X.sup.31 are each independently OH, SH or BH.sub.3.sup.−; R and R′ are each independently hydrogen, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl or C.sub.1-6 alkyl; the C.sub.2-6 alkenyl, C.sub.2-6 alkynyl or C.sub.1-6 alkyl is unsubstituted or selectively substituted at any position by 1 to 3 substituents selected from halogen, hydroxyl, amino, azido and cyano; R.sup.1 and R.sup.2 are each independently hydrogen, halogen, cyano, hydroxyl, thiol, amino, azido, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 alkyl, halo-C.sub.1-6 alkyl, halo-C.sub.1-6 alkoxy, halo-C.sub.1-6 alkylthio, C.sub.1-6 alkylamino, OC(O)R.sup.a or OR.sup.a; the C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, or C.sub.1-6 alkyl is unsubstituted or selectively substituted at any position by 1 to 3 substituents selected from halogen, hydroxyl, amino, azido, and cyano; R.sup.11 and R.sup.21 are each independently hydrogen, halogen, cyano, hydroxyl, thiol, amino, azido, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 alkyl, halo-C.sub.1-6 alkyl, halo-C.sub.1-6 alkoxy, halo-C.sub.1-6 alkylthio, C.sub.1-6 alkylamino, OC(O)R.sup.a, or OR.sup.a; the C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, or C.sub.1-6 alkyl is unsubstituted or selectively substituted at any position by 1 to 3 substituents selected from halogen, hydroxyl, amino, azido, and cyano; alternatively, R.sup.1 and R.sup.2 together form carbonyl; alternatively, R.sup.11 and R.sup.21 together form carbonyl; alternatively, R is —CH.sub.2—, R.sup.1 is —O—, R and R.sup.1 are interconnected to form heterocycloalkyl; alternatively, R′ is —CH.sub.2—, R.sup.11 is —O—, R′ and R.sup.11 are interconnected to form heterocycloalkyl; R.sup.3 and R.sup.4 are each independently hydrogen, deuterium, halogen or C.sub.1-6 alkyl; R.sup.31 and R.sup.41 are each independently hydrogen, deuterium, halogen or C.sub.1-6 alkyl; R.sup.5 and R.sup.6 are each independently hydrogen, deuterium, halogen or C.sub.1-6 alkyl; R.sup.51 and R.sup.61 are each independently hydrogen, deuterium, halogen or C.sub.1-6 alkyl; R.sup.7 and R.sup.8 are each independently hydrogen, deuterium, halogen or C.sub.1-6 alkyl; R.sup.71 and R.sup.81 are each independently hydrogen, deuterium, halogen or C.sub.1-6 alkyl; Y and Y.sub.1 are independently CR.sub.E or N; U is CHR.sub.E′ or NR.sub.D′; U.sub.1 is CH or N; V, V.sub.1, V.sub.2 and V.sub.3 are each independently CR.sub.E″ or N; W is O or S; W.sub.1, W.sub.2, W.sub.3 and W.sub.4 are each independently N or CR.sub.F′; each of R.sub.A, R.sub.B, R.sub.C, R.sub.E, R.sub.E′, R.sub.E″, R.sub.F, R.sub.F′ and R.sub.G are independently H, halogen, —CN, —NO.sub.2, —N.sub.3, R.sup.c, —SR.sup.c, —OR.sup.c, —OC(O)R.sup.c, —OC(O)OR.sup.c, —OC(O)NR.sup.bR.sup.c, —C(O)OR.sup.c, —C(O)R.sup.c, —C(O)NR.sup.bR.sup.c, —NR.sup.bR.sup.c, —NR.sup.bC(O)R.sup.c, —N(R.sup.b)C(O)OR.sup.c, —N(R.sup.a)C(O)NR.sup.bR.sup.c, —NR.sup.bS(O).sub.2R.sup.c, —NR.sup.bC(═NH)R.sup.c, —NR.sup.bC(═NR.sup.c)NH.sub.2, —S(O).sub.1-2R.sup.c, —S(O).sub.2NR.sup.bR.sup.c or —NR.sup.aS(O).sub.2NR.sup.bR.sup.c; each of R.sub.D and R.sub.D′ is independently H or R.sup.c; each of R.sup.a and R.sup.b is independently H, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-10 alkyl, halo-C.sub.1-6 alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aryl-C.sub.1-6 alkyl, heteroaryl-C.sub.1-6 alkyl, cycloalkyl-C.sub.1-6 alkyl, or heterocycloalkyl-C.sub.1-6 alkyl; each R.sup.c is independently H, substituted or unsubstituted C.sub.1-10 alkyl, substituted or unsubstituted C.sub.2-8 alkenyl, substituted or unsubstituted C.sub.2-8 alkynyl, substituted or unsubstituted C.sub.3-10 cycloalkyl, substituted or unsubstituted C.sub.6-10 aryl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C.sub.6-10 aryl-C.sub.1-6 alkyl, substituted or unsubstituted C.sub.3-10 cycloalkyl-C.sub.1-6 alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl-C.sub.1-6 alkyl, substituted or unsubstituted 5-10 membered heteroaryl-C.sub.1-6 alkyl; the C.sub.1-10 alkyl, C.sub.2-8 alkenyl, C.sub.2-8 alkynyl, C.sub.3-10 cycloalkyl, C.sub.6-10 aryl, 3-10 membered heterocycloalkyl, 5-10 membered heteroaryl, C.sub.6-10 aryl-C.sub.1-6 alkyl, C.sub.3-10 cycloalkyl-C.sub.1-6 alkyl, 3-10 membered heterocycloalkyl-C.sub.1-6 alkyl, or 5-10 membered heteroaryl-C.sub.1-6 alkyl is unsubstituted or selectively substituted at any position by one or more R.sup.d; each R.sup.d is independently halogen, halo-C.sub.1-6 alkyl, halo-C.sub.1-6 alkoxy, C.sub.1-6 alkyl, —CN, —N.sub.3, —SR.sup.e, —OR.sup.e, —C(O)R.sup.e, —NR.sup.eR.sup.e′, substituted or unsubstituted C.sub.6-10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C.sub.3-10 cycloalkyl, or substituted or unsubstituted 3-10 membered heterocycloalkyl; the C.sub.6-10 aryl, 5-10 membered heteroaryl, C.sub.3-10 cycloalkyl or 3-10 membered heterocycloalkyl is unsubstituted or selectively substituted at any position by one or more substituents selected from halogen, hydroxyl, cyano, amino, C.sub.1-4 alkyl, halo-C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 alkylamino and halo-C.sub.1-4 alkoxy; each of R.sup.e and R.sup.e′ is independently C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-10 alkyl, halo-C.sub.1-6 alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aryl-C.sub.1-6 alkyl, heteroaryl-C.sub.1-6 alkyl, cycloalkyl-C.sub.1-6 alkyl, or heterocycloalkyl-C.sub.1-6 alkyl.

    2. The cyclic di-nucleotide analogue of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein B.sub.1 is any of the following structures: ##STR00134## ##STR00135## ##STR00136## ##STR00137## and/or, B.sub.2 is any of the following structures: ##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142##

    3. The cyclic di-nucleotide analogue of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein L.sub.2 is CH.sub.2; and/or, L.sub.2′ is CH.sub.2; and/or, X.sup.1 is O; and/or, X.sup.11 is O; and/or, X.sup.2 is O; and/or, X.sup.21 is O; and/or, L, L.sub.1, L′ and L.sub.1′ are as defined in the following combinations: 1) L is a connecting bond, L.sub.1 is CR.sup.1R.sup.2, L′ is CR.sup.11R.sup.21, L.sub.1′ is a connecting bond, or 2) L is C R.sup.1R.sup.2, L.sub.1 is a connecting bond, L′ is a connecting bond, L.sub.1′ is CR.sup.11R.sup.21

    4. The cyclic di-nucleotide analogue of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein R and R′ are each independently hydrogen; and/or, R.sup.1 and R.sup.2 are each independently hydrogen, halogen, hydroxyl, or OR.sup.a; and/or, R.sup.11 and R.sup.21 are each independently hydrogen, halogen, hydroxyl, or OR.sup.a; and/or, each R.sup.a is independently C.sub.1-4 alkyl or halo-C.sub.1-4 alkyl.

    5. The cyclic di-nucleotide analogue of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein Z.sub.1 is O; Z.sub.2 is O; B.sub.1 is ##STR00143## B.sub.2 is ##STR00144## L and L.sub.1 are each independently a connecting bond or CR.sup.1R.sup.2; L′ and L.sub.1′ are each independently a connecting bond or CR.sup.11R.sup.21; and, L, L.sub.1, L′ and L.sub.1′ are as defined in the following combinations: 1) L is a connecting bond, L.sub.1 is CR.sup.1R.sup.2, L′ is CR.sup.11R.sup.21, L.sub.1′ is a connecting bond, or 2) L is CR.sup.1R.sup.2, L.sub.1 is a connecting bond, L′ is a connecting bond, L.sub.1′ is CR.sup.11R.sup.21; L.sub.2 is CH.sub.2; L.sub.2′ is CH.sub.2; X.sup.1 is O; X.sup.11 is O; X.sub.2 is O; X.sub.21 is O; X.sup.3 and X.sup.31 are each independently OH or SH; R and R′ are each independently hydrogen; R.sup.1 and R.sup.2 are each independently hydrogen, halogen, hydroxyl or OR.sup.a; R.sup.11 and R.sup.21 are each independently hydrogen, halogen, hydroxyl or OR.sup.a; each R.sup.a is independently C.sub.1-4 alkyl or halo-C.sub.1-4 alkyl.

    6. The cyclic di-nucleotide analogue for formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein Z.sub.1 is O; Z.sub.2 is O; B.sub.1 is ##STR00145## B.sub.2 is ##STR00146## L and L.sub.1 are each independently a connecting bond or CR.sup.1R.sup.2; L′ and L.sub.1′ are each independently a connecting bond or CR.sup.11R.sup.21; and, L, L.sub.1, L′ and L.sub.1′ are as defined in the following combinations: 1) L is a connecting bond, L.sub.1 is CR.sup.1R.sup.2, L′ is CR.sup.11R.sup.21, L.sub.1′ is a connecting bond, or 2) L is CR.sup.1R.sup.2, L.sub.1 is a connecting bond, L′ is a connecting bond, L.sub.1′ is CR.sup.11R.sup.21; L.sub.2 is CH.sub.2; L.sub.2′ is CH.sub.2; X.sup.1 is O; X.sup.11 is O; X.sup.2 is O; X.sup.21 is O; X.sup.3 and X.sup.31 are each independently OH or SH; R and R′ are each independently hydrogen; R.sup.1 and R.sup.2 are each independently hydrogen, halogen, hydroxyl or OR.sup.a; R.sup.11 and R.sup.21 are each independently hydrogen, halogen, hydroxyl or OR.sup.a; each R.sup.a is independently C.sub.1-4 alkyl or halo-C.sub.1-4 alkyl.

    7. The cyclic di-nucleotide analogue of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein, which is shown as the compound of formula (VI) or (VII), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof: ##STR00147## wherein, B.sub.1, B.sub.2, R.sup.2 and R.sup.21 are as defined in claim 1.

    8. The cyclic di-nucleotide analogue of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 7, wherein the stereo-configuration of P is (Sp, Sp), (Sp, Rp), (Rp, Rp) or (Rp, Sp).

    9. The cyclic di-nucleotide analogue of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 7, wherein B.sub.1 is ##STR00148## and/or, B.sub.2 is ##STR00149## in Formula VI, R.sup.2 is —OH; R.sup.21 is —OH; in Formula VII, R.sup.2 is —OH or —OCH.sub.3; R.sup.21 is —OH or F.

    10. The cyclic di-nucleotide analogue of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1, which is any of the following structures: ##STR00150## ##STR00151## ##STR00152## ##STR00153## ##STR00154## ##STR00155## ##STR00156## ##STR00157## ##STR00158## ##STR00159## ##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164## ##STR00165## ##STR00166## ##STR00167## ##STR00168## ##STR00169## ##STR00170## ##STR00171## ##STR00172## ##STR00173## ##STR00174## ##STR00175## ##STR00176## ##STR00177## ##STR00178## ##STR00179## ##STR00180## ##STR00181## ##STR00182## ##STR00183## ##STR00184## ##STR00185## ##STR00186## ##STR00187## ##STR00188## ##STR00189## ##STR00190## ##STR00191## ##STR00192## ##STR00193## ##STR00194##

    11. A pharmaceutical composition comprising a therapeutically effective amount of the compound of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable excipient.

    12. A method of modulating STING in a subject in need thereof, comprising administering the compound of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1 to the subject.

    13. The method according to claim 12, wherein the modulating is activating.

    14. A method of treating, alleviating and/or preventing a STING-mediated disease in a subject in need thereof, comprising administering the compound of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1 to the subject.

    15. The method according to claim 14, wherein the STING-mediated disease is viral infection or other infectious diseases, autoimmune diseases, or malignancies.

    16. A method of regulating the proliferation of T cells or other immune cells in a subject in need thereof, comprising administering the compound of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1 to the subject.

    17. A method of treating and/or alleviating malignancies in a subject in need thereof, comprising administering the compound of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1 to the subject.

    18. A method of improving the effectiveness of vaccine in a subject in need thereof, comprising administering the compound of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1 to the subject.

    19. A combination formulation, comprising the compound of formula (I), the isomer, prodrug, stable isotope derivative or pharmaceutically acceptable salt thereof according to claim 1 and other kinds of therapeutic agents and/or therapeutic methods for the treatment of cancer.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0359] FIG. 1 is the tumor volume change curves of compounds 6-p3 (1 mg/kg, 2 mg/kg, i.t.) and Ref.1 (2 mg/kg, i.t.) in the colon cancer CT26 subcutaneously implanted tumor in mouse.

    DETAILED DESCRIPTION OF THE EMBODIMENT

    [0360] The following Embodiments serve to illustrate the present disclosure, but the Embodiments should not be considered as limiting the scope of the disclosure. Some of the experimental methods of the following Embodiments that are not indicated the specific conditions, can in according with the commonly used reaction conditions and procedures, or in accordance with the product specifications.

    [0361] All the structures of the compounds in the present disclosure were confirmed by Nuclear Magnetic Resonance (H NMR) and/or Mass Spectra (MS).

    [0362] .sup.1H NMR chemical shifts (6) were recorded in ppm (10.sup.−6). NMR Spectra were recorded on Bruker AVANCE-400 spectrometer. The proper solvents were Chloroform-d (CDCl.sub.3), Methanol-d.sub.4 (CD.sub.3OD), and Dimethyl sulfoxide-d.sub.6 (DMSO-d.sub.6), tetramethylsilane as internal standard (TMS).

    [0363] The analytical low-resolution mass spectra (LCMS) were recorded on Agilent 1200 HPLC/6120 using an XBridge C18, 3.0×50 mm, 3μm, column temperature: 35° C.; or recorded on ThermoUltiMate 3000HIPLC/MSQPLUS using an XBridge C18, 3.0×50 mm, 3.5 m, column temperature: 30° C. The gradient elution method 1 of Agilent: 95-5% solvent A.sub.1 and 5-95% solvent B.sub.1 (0-2.0 min), and then 95% solvent B.sub.1 and 5% solvent A.sub.1 (for 1.1 min). Percentage as used herein is volume percentage of the volume of a solvent in the total solvent volume. Solvent A.sub.1: 0.01% aqueous solution of trifluoroacetic acid (TFA); Solvent B.sub.1: 0.01% trifluoroacetic acid acetonitrile solution. Percentage is the volume of a solvent in the total solvent volume. The gradient elution method 2 of Thermo: 95-5% solvent A.sub.2 and 5-95% solvent B.sub.2 (0-2 min), and then 95% solvent B.sub.2 and 5% solvent A.sub.2 (for 1.8 min), Percentage is the volume of a solvent in the total solvent volume. Solvent A.sub.2: 10 mM aqueous solution of ammonium bicarbonate; Solvent B.sub.2: acetonitrile.

    [0364] All the compounds in the present disclosure were separated by preparative high-performance liquid chromatography or flash column chromatography.

    [0365] Preparative high-performance liquid chromatography purification (prep-HPLC) was performed on Shimadzu LC-20 HPLC, chromatographic column: waters xbridge Pre C18, 10 um, 19 mm×250 mm. Separation method 1 (acidic condition): mobile phase A: 0.05% aqueous solution of trifluoroacetic acid, mobile phase B: acetonitrile; elution B was 40%, elution time: 20 min. Separation method 2 (alkali condition): mobile phase A: 10 mmol/L aqueous solution of ammonium bicarbonate, mobile phase B: acetonitrile; the gradient elution B was from 10% to 80%, elution time: 30 min. Separation method 3 (alkali condition): mobile phase A: 10 mmol/L aqueous solution of ammonium bicarbonate, mobile phase B: acetonitrile; the gradient elution B was from 0% to 15%, elution time: 30 min. Separation method 4 (alkali condition): mobile phase A: 10 mmol/L aqueous solution of ammonium bicarbonate, mobile phase B: acetonitrile; the gradient elution B was from 0% to 4%, elution time: 10 min; the gradient elution B was from 4% to 8%, elution time: 15 min. Separation method 5 (alkali condition): mobile phase A: 10 mmol/L aqueous solution of ammonium bicarbonate, mobile phase B: acetonitrile; the gradient elution B was from 0% to 5%, elution time: 10 min; the gradient elution B was from 5% to 10%, elution time: 15 min. Separation method 6 (alkali condition): mobile phase A: 10 mmol/L aqueous solution of ammonium bicarbonate, mobile phase B: acetonitrile; the gradient elution B was from 10% to 30%, elution time: 5 min; the gradient elution B was from 30% to 75%, elution time: 20 min. Separation method 7 (alkali condition): mobile phase A: 10 mmol/L aqueous solution of ammonium bicarbonate, mobile phase B: acetonitrile; the gradient elution B was from 0% to 10%, elution time: 7 min; the gradient elution B was from 10% to 40%, elution time: 18 min. Detection wavelength: 214 nm&254 nm; the flow rate: 15.0 mL/min.

    [0366] Flash column chromatography (flash system/Cheetah™) was performed on Agela Technologies MP200. Normal-phase chromatography column was Flash column Silica-CS (25 g, 40 g, 80 g, 120 g or 330 g), Agela Technologies, Tianjing. Ethyl acetate/petroleum ether or dichloromethane/methanol was chosen as elution system. Reversed-phase chromatography column was C18 column (12 g, 20 g or 40 g), Santai Technologies, Changzhou. Acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L) were chosen as elution system.

    [0367] All the compounds in the present disclosure were analyzed by high-performance liquid chromatography. High-performance liquid chromatography (HPLC) was performed on Waters e2695, 2498 UV/VIS Detector, chromatography column: Waters Xselect CHS C18 (4.6*150 mm) 5 m, mobile phase A: acetonitrile, mobile phase B: acetic acid triethylamine buffer solution which was adjusted pH to 5.0 with acetic acid. Gradient elution of mobile phase B from 95% to 15%, elution time: 30 min. Detection wavelength: 214 nm&254 nm; column temperature: 35° C.

    Embodiment 1: Synthesis of Intermediate 1-8

    [0368] ##STR00094##

    [0369] Step 1: To a suspension of adenosine (50 g, 187 mmol) in acetic acid/sodium acetate buffer solution (pH=4.0, 0.5M, 1 L) was added liquid bromine (60 g, 374 mmol), keep the system temperature below 10° C. After addition, the reaction system was stirred at room temperature for 48 h. The saturated aqueous solution of sodium bisulfate was added into the reaction solution to remove the excess bromine, and then adjusted pH to neutral with an aqueous solution of sodium hydroxide (1M), the reaction solution was stirred for 2 h under ice-water bath. The precipitate was formed and collected by filtration, dried under vacuum to afford intermediate 1-1 (29 g). m/z: [M+H].sup.+ 346.0/348.0.

    [0370] Step 2: To a suspension of intermediate 1-1 (10 g, 28.9 mmol) in methanol (100 mL) was added sodium methanolate (9.36 g, 173 mmol), the reaction system was stirred at reflux for 5 h, methanol was concentrated under reduced pressure. The residue was dissolved in a mixed solvent of methanol/dichloromethane (1/10). The solution was filtered through a Büchner funnel which was covered with a layer of silica gel. The filtrate was concentrated under reduced pressure to afford intermediate 1-2 (3.8 g). m/z: [M+H].sup.+298.2.

    [0371] Step 3: To a solution of intermediate 1-2 (10 g, 336 mmol) in pyridine (40 mL) was added chlorotrimethylsilane (16 mL, 121 mmol) under nitrogen at 0° C. The reaction system was stirred at room temperature for 2 h, benzoyl chloride (9.4 mL, 80.7 mmol) was slowly added to the above reaction system. The resulting solution was stirred at room temperature for overnight, and then ammonium hydroxide solution (25-28%) was added thereto and stirred for 30 min. The solvent was concentrate under reduced pressure. The residue was purified by Flash column chromatography (0-10% methanol/dichloromethane) to afford intermediate 1-3 (7.3 g) as a white solid. m/z: [M+H].sup.+402.2.

    [0372] Step 4: To a solution of intermediate 1-3 (7 g, 17.4 mmol) in anhydrous pyridine (40 mL) was added 4,4′-dimethoxytrityl chloride (DMTrCl, 5.9 g, 17.4 mmol) at 0° C. under nitrogen. The reaction system was stirred at room temperature for 3 h and then quenched by addition of water (1 mL). The solvent was concentrated under reduced pressure. The residue was purified by Flash column chromatography (0-10% methanol/dichloromethane) to afford intermediate 1-4 (7.4 g) as a yellow solid. m/z: [M+H].sup.+704.2.

    [0373] Step 5: To a solution of intermediate 1-4 (1.2 g, 1.70 mmol) in pyridine (5 mL) was added tert-butyldimethylsilyl chloride (TBSCl, 0.31 g, 2.05 mmol) and imidazole (0.29 g, 4.30 mmol) at 0° C. under nitrogen. The reaction system was stirred at room temperature for 16 h, and then cooled with ice-water, diluted with water (10 mL) and ethyl acetate (50 mL), the organic layer washed with brine (50 mL×2), the organic layer was separated and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by Flash column chromatography (0-60% ethyl acetate/petroleum ether) to afford intermediate 1-5 (less polar, 276 mg, white solid) and 1-6 (more polar, 670 mg, off-white solid). Intermediate 1-5: m/z: [M+H].sup.+818.3; TLC R.sub.f=0.42 (DCM/MeOH=15/1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.00 (s, 1H), 8.48 (s, 1H), 8.02 (d, J=7.8 Hz, 2H), 7.62 (t, J=7.4 Hz, 1H), 7.53 (t, J=7.6 Hz, 2H), 7.39 (d, J=7.8 Hz, 2H), 7.29-7.16 (m, 7H), 6.83 (d, J=8.6 Hz, 4H), 5.90 (d, J=5.8 Hz, 1H), 5.13 (d, J=6.0 Hz, 1H), 5.08 (t, J=5.5 Hz, 1H), 4.27 (dd, J=9.6, 5.2 Hz, 1H), 4.07 (d, J=4.0 Hz, 1H), 4.00 (s, 3H), 3.71 (s, 6H), 3.27-2.23 (m, 1H), 3.14-3.10 (m, 1H), 0.74 (s, 9H), 0.10 (d, J=6.6 Hz, 6H). Intermediate 1-6: m/z: [M+H].sup.+818.3; TLC R.sub.f=0.23 (DCM/MeOH=10/1); .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.00 (s, 1H), 8.50 (s, 1H), 8.01 (d, J=7.6 Hz, 2H), 7.62 (m, 1H), 7.52 (m, 2H), 7.32 (d, J=7.5 Hz, 2H), 7.26-7.16 (m, 7H), 6.82 (dd, J=8.8, 2.3 Hz, 4H), 5.83 (d, J=5.2 Hz, 1H), 5.40 (d, J=6.1 Hz, 1H), 5.06 (m, 1H), 4.59 (t, J=4.5 Hz, 1H), 4.06 (s, 3H), 3.71 (s, 6H), 3.35-3.33 (m, 1H), 3.31-3.26 (m, 1H), 3.09-3.01 (m, 1H), 0.85 (s, 9H), 0.08 (d, J=7.8 Hz, 6H).

    [0374] Step 6: To a solution of intermediate 1-6 (400 mg, 0.48 mmol) in pyridine (4 mL) was added diphenyl phosphite (460 mg, 1.92 mmol) and then stirred at room temperature for 30 min. Triethylamine (0.4 mL) and water (0.4 mL) was successively added to the above reaction system and stirred for 30 min. Dicholormethane (5 mL) and aqueous solution of sodium bicarbonate (5 mL, 5%) was successively added to the above reaction system. The organic layer washed with water, and then the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (0-10% methanol/dichloromethane) to afford intermediate 1-7 (triethylamine salt, 600 mg) as a white solid. m/z: [M+H].sup.+882.3.

    [0375] Step 7: To a solution of intermediate 1-7 (2.8 g, 2.85 mmol) in a mixed solvent of dichloromethane (20 mL) and water (0.3 mL) was added a dichloromethane solution of dichloroacetic acid (DCA) (0.6M, 23.7 mL), and then stirred at room temperature for 1 h. To the reaction solution was added pyridine (20 mL), the resulting mixture was stirred at room temperature for 10 min. The solvent was concentrate under reduced pressure to afford intermediate 1-8 (pyridinium salt, curd product). m/z: [M+H].sup.+580.1.

    Embodiment 2: Synthesis of Compounds 1-p1, 1-p2, 1-p3 and 1-p4

    [0376] ##STR00095## ##STR00096## ##STR00097##

    [0377] Step 1: Intermediate 1-8 (2.85 mmol) was dissolved in anhydrous acetonitrile (15 mL) and then the solvent was concentrated under reduced pressure, repeated twice and left 10 mL acetonitrile at last time, 4 A molecular sieve (0.8 g) was added thereto. Compound 1-9 (CASNo: 104992-55-4, 3.3 g, 3.42 mmol) was dissolved in anhydrous acetonitrile (15 mL) and then the solvent was concentrated under reduced pressure, repeated twice and left 5 mL acetonitrile at last time. To the solution of 1-8 in acetonitrile was slowly added the acetonitrile solution of compound 1-9 at 0° C., the reaction system was stirred at room temperature for 0.5 h, ((Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazoline-3-thione (DDTT, 697 mg, 3.42 mmol) was added thereto, and stirred for additional 40 min. The molecular sieve was removed by filtration, and the filtrate was concentrated under reduced pressure to afford compound 1-10 (7.5 g). m/z: [M+H].sup.+1499.3.

    [0378] Step 2: Compound 1-10 (3.7 g, 2.35 mmol) was dissolved in dichloromethane (35 mL) and water (0.7 mL); dichloromethane solution of DCA (0.6 M, 31 mL, 18.8 mmol) was dropped thereto at room temperature. The reaction system was stirred at room temperature for 2 h. Triethylsilane (20 mL) was dropped thereto, and the reaction solution was stirred at room for additional 1 h. Pyridine (10 mL) was dropped thereto, the the reaction solution was concentrated under reduced pressure. The residue was purified by prep-HPLC to afford compound 1-11 (400 mg) as a white solid. m/z: [M+H].sup.+1196.2.

    [0379] Step 3&4: To pyridine (5 mL) was slowly added diphenyl chlorophosphate (DPCP, 1 g, 3.8 mmol) dropwise at −40° C., to the above solution was slowly added anhydrous dichloromenthane solution (5 mL) of compound 1-11 (230 mg, 0.19 mmol) dropwise at −40° C., and then stirred at this temperature for 30 min, and obtained the reaction solution of compound 1-12. To the solution of compound 1-12 was directly added 3H-1,2-benzodithiol-3-one (64 mg, 0.38 mmol) and stirred for 1 h. Water (68 mg, 0.38 mmol) was added thereto and stirred for additional 1 h. The reaction solution was diluted with ethyl acetate and washed with aqueous solution of sodium bicarbonate (2.7%, 30 mL), the organic layer was separated and concentrated to afford compound 1-13. Compound 1-13 was separated by prep-HPLC (separation method 2) to afford 3 isomers: 1-13-p1 (60 mg) as a yellow solid, 1-13-p2 (30 mg) as a white solid, and 1-13-p3 (40 mg) as a white solid.

    [0380] Step 5: To a solution of compound 1-13-p1 (80 mg, 0.066 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL). The reaction system was stirred at room temperature for 0.5 h. The solvent was concentrate under reduced pressure. The residue was dissolved in methanol (4 mL), methanolic hydrochloric acid solution (2M, 4 mL) was added thereto, the reaction solution was stirred at 45° C. for 1 h, and the solvent was concentrate under reduced pressure to afford compound 1-14-p1 (100 mg) as a yellow solid. m/z: [M+H].sup.+ 1143.1.

    [0381] Step 6: To a solution of compound 1-14-p1 (100 mg) in methanol (6 mL) was added ammonium hydroxide solution (6 mL). The reaction system was stirred at 45° C. for overnight.

    [0382] The solvent was concentrate under reduced pressure. The residual liquid was lyophilized to afford compound 1-15-p1 (100 mg, crude compound) as a yellow solid. m/z: [M+H].sup.+ 935.2.

    [0383] Step 7: Compound 1-15-p1 (45 mg) was subjected to azeotropic dehydration three times with anhydrous pyridine (10 mL) and then dissolved in pyridine (2 mL), and then triethylamine (0.66 mL) and triethylamine trihydrofluoride (387 mg) was added thereto under nitrogen. The resulting solution was stirred at 45° C. for 3 h. The solvent was concentrate under reduced pressure. The residue was purified by prep-HPLC (separation method 3) to afford compound 1-p1 (0.34 mg, m/z: [M+H].sup.+ 706.8, HPLC-RT: 8.584 min) and 1-p2 (0.30 mg, m/z: [M+H].sup.+ 706.8, HPLC-RT: 8.662 min), as white solids.

    Synthesis of Compound 1-p3

    [0384] In a same way, to a solution of compound 1-13-p2 (50 mg, 0.041 mmol) in acetonitrile (2.0 mL) was added tert-butylamine (2 mL), and stirred at room temperature for 0.5 h, the solvent was concentrate under reduced pressure. The residue was dissolved in methanol (1 mL), methanolic hydrochloric acid solution (2 mL, 2M) was added thereto, the reaction solution was stirred at 40° C. for 4 h, and then the solvent was concentrate under reduced pressure. The residue was dissolved in methanol (1 mL), ammonium hydroxide solution (1 mL) was added thereto and stirred at 50° C. for 16 h, and then the reaction solution was purged with nitrogen to remove most of the ammonia. The solvent was concentrate under reduced pressure. The residual liquid was lyophilized. The crude compound was subjected to azeotropic dehydration three times with anhydrous pyridine (10 mL) and then dissolved in pyridine (2 mL), triethylamine (0.66 mL) and triethylamine trihydrofluoride (0.36 mL) was added thereto under nitrogen. The resulting solution was stirred at 50° C. for 2 h. The solvent was concentrate under reduced pressure. The residue was purified by prep-HPLC (separation method 3) to afford compound 1-p3 (0.40 mg, m/z: [M+H].sup.+ 706.8, HPLC-RT: 9.789 min) as an off-white solid.

    Synthesis of Compound 1-p4

    [0385] Compound 1-p4 (2.4 mg, m/z: [M+H].sup.+ 706.8, HPLC-RT: 9.960 min) was obtained as a white solid in the same manner as compound 1-p3, by using compound 1-13-p3 (40 mg, 0.033 mmol) as a starting material. .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 8.37 (s, 1H), 8.16 (s, 1H), 7.97 (s, 1H), 5.93 (d, J=7.8 Hz, 1H), 5.74 (d, J=8.0, 1H), 5.35 (m, 1H), 5.14 (m, 1H), 4.70 (m, 1H), 4.59 (s, 1H), 4.18 (s, 1H), 4.15 (m, 1H), 3.85 (m, 2H), 3.53 (m, 1H), 3.06 (m, 1H); .sup.31P NMR (161 MHz, DMSO-d.sub.6+D.sub.2O): δ 58.47, 46.58.

    Embodiment 3: Synthesis of Intermediates 2-8 and 2-9

    [0386] ##STR00098##

    [0387] Step 1: To a solution of 7-aminothiazolo[4,5-d]pyrimidin-2 (3H)-one (refer to J. Med. Chem. 1990, 33, 407-415, compound 28) (6.5 g, 38.7 mmol) and tetraacetylribose (1 g, 46.4 mmol) in acetonitrile (120 mL) was added N,O-bis(trimethylsilyl)acetamide (BSA, 13.6 g, 116 mmol), and then stirred at reflux for 1 h. To the above reaction solution was added trimethylsilyl trifluoromethylsulphonate (TMSOTf, 17.2 g, 77.4 mmol) after cooling to room temperature, and stirred at reflux for additional 48 h. The reaction solution was cooled to room temperature, and saturated aqueous solution of sodium bicarbonate was slowly added thereto, the mixture was extracted with ethyl acetate (150 mL×3), the combined organic layers were washed with water, the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (ethyl acetate/petroleum ether=3/4) to afford intermediate 2-1 (4.3 g) as a light yellow solid. m/z: [M+H].sup.+ 427.0.

    [0388] Step 2: To a solution of intermediate 2-1 (0.5 g, 1.17 mmol) in pyridine (5 mL) was added chlorotrimethylsilane (0.07 mL, 0.58 mmol) at 0° C. under nitrogen, and stirred at for 5 min, to the above reaction solution was added benzoyl chloride (0.32 mL, 2.81 mmol), the reaction system was stirred at room temperature for overnight, and then quenched by addition of water (50 mL). The aqueous layer was extracted with ethyl acetate (50 mL×2), the combined organic layers were washed with water, and the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (ethyl acetate/petroleum ether=9/10) to afford intermediate 2-2 (0.45 g) as a light yellow solid. m/z: [M+H].sup.+ 34.8.

    [0389] Step 3: To a solution of intermediate 2-2 (3 g, 4.7 mmol) in acetonitrile (150 mL) was added aqueous solution of lithium hydroxide (47 mL, 1M), the reaction system was stirred at room temperature for 15 min and then neutralized pH to 6 with hydrochloric acid (2M). The reaction solution was concentrated to ⅓ of the total volume, the solid was precipitated, filtered, the filter cake washed with water for 3 times, and then dried under vacuum to afford intermediate 2-3 (1 g) as a yellow solid. m/z: [M+H].sup.+405.0.

    [0390] Step 4: To a solution of intermediate 2-3 (7.6 g, 18.8 mmol) in pyridine (95 mL) was added DMTrCl (9.5 g, 28.5 mmol) under nitrogen. The reaction system was stirred at room temperature for overnight. The solvent was concentrate under reduced pressure. The residue was purified by Flash column chromatography (methanol/dichloromethane=1/25) to afford intermediate 2-4 (8.3 g) as an off-white solid. m/z: [M+H].sup.+706.8.

    [0391] Step 5: To a solution of intermediate 2-4 (8.3 g, 11.7 mmol) and imidazole (2 g, 29.3 mmol) in pyridine (60 mL) was added TBSCl (2.1 g, 14 mmol) at 0° C. under nitrogen. The reaction system was stirred at room temperature for overnight, and then quenched by addition of water (1 mL) and saturated aqueous solution of sodium bicarbonate (100 mL), the aqueous layer was extracted with ethyl acetate (200 mL×2), the combined organic layers were washed with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, the residue was purified by Flash column chromatography (methanol/dichloromethane=1/50-1/20) to afford intermediate 2-5 (4 g, off-white solid, LCMS-RT (Thermo): 2.813 min) and 2-6 (2 g, off-white solid, LCMS-RT (Thermo): 2.763 min. Intermediate 2-5: m/z: [M+H].sup.+ 821.1; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.71 (s, 1H), 8.66 (s, 1H), 8.05-8.03 (m, 2H), 7.68-7.64 (m, 1H), 7.56-7.52 (m, 2H), 7.43-7.32 (m, 2H), 7.27-7.17 (m, 7H), 6.84-6.82 (m, 4H), 6.05-6.04 (m, 1H), 5.29-5.28 (m, 1H), 4.88-4.84 (m, 1H), 4.53-4.50 (m, 1H), 4.05-3.98 (m, 1H), 3.71 (s, 6H), 3.35-3.27 (m, 1H), 3.08-3.04 (m, 1H), 0.82 (s, 9H), 0.05 (s, 3H), 0.01 (s, 3H); Intermediate 2-6: m/z: [M+H].sup.+ 821.1; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.73 (s, 1H), 8.62 (s, 1H), 8.06-8.04 (m, 2H), 7.68-7.65 (m, 1H), 7.57-7.53 (m, 2H), 7.41-7.36 (m, 2H), 7.27-7.17 (m, 7H), 6.85-6.82 (m, 4H), 6.09-6.08 (m, 1H), 5.00-4.95 (m, 2H), 4.31-4.27 (m, 1H), 4.05-4.03 (m, 1H), 3.72 (s, 6H), 3.23-3.16 (m, 2H), 0.95 (s, 9H), 0.05 (s, 6H).

    [0392] Step 6: To a solution of intermediate 2-5 (0.5 g, 0.61 mmol) in pyridine (5 mL) was added diphenyl phosphite (0.57 g, 1.43 mmol) at 0° C. under nitrogen, the reaction system was stirred for 1 h, and then triethylamine (0.6 mL) and water (0.6 mL) was added thereto. The resulting mixture was stirred at room temperature for 5 min and then diluted with water (50 mL), the aqueous layer was extracted with dicholormethane (30 mL×2), the combined organic layers were washed with water, and the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (methanol/dichloromethane=1/10) to afford intermediate 2-7 (triethylamine salt, 0.6 g) as an off-white solid. m/z: [M+H].sup.+ 884.5.

    [0393] Step 7: To a solution of intermediate 2-7 (0.6 g, 0.61 mmol) in a mixed solvent of dichloromethane (10 mL) and water (1 mL) was added a dichloromethane solution of DCA (0.6 M, 9.1 mL). The reaction mixture was stirred for 0.5 h, and then pyridine (20 mL) was added thereto, the resulting mixture was concentrate under reduced pressure to afford intermediate 2-8 (pyridinium salt, curd product). m/z: [M+H].sup.+ 582.9.

    [0394] Synthesis of intermediate 2-9: intermediate 2-9 (pyridinium salt) was obtained in the same manner as intermediate 2-8, by using intermediate 2-6 as a starting material. m/z: [M+H].sup.+ 583.0.

    Embodiment 4: Synthesis of Compounds 2-p1, 2-p2, 2-p3 and 2-p4

    [0395] ##STR00099## ##STR00100## ##STR00101##

    [0396] Step 1: Intermediate 2-8 (0.68 mmol) and 1-9 (0.81 g, 0.82 mmol) were subjected to azeotropic dehydration twice with anhydrous acetonitrile (10 mL) respectively, and then dissolved in acetonitrile (5 mL) respectively for use. To the solution of intermediate 2-8 in acetonitrile, which contained 4 A molecular sieve, was slowly added the acetonitrile solution of intermediate 1-9 at 0° C. under nitrogen, the resulting mixture was stirred for 1 h. To the above reaction system was added DDTT (0.16 g, 0.79 mmol) and stirred for additional 1 h. The molecular sieve was removed by filtration, and the filtrate was concentrated under reduced pressure to afford compound 2-9 (1 g). m/z: [M+H].sup.+1501.5.

    [0397] Step 2: To the solution of compound 2-9 (0.33 g, 0.22 mmol) in a mixed solvent of dichloromethane (3 mL) and water (0.3 mL) was added dichloromethane solution of DCA (0.6 M, 2.93 mL) under nitrogen. The reaction system was stirred for 0.5 h, and then pyridine (1 mL) was added thereto and concentrated under reduced pressure, and the residue was purified by Flash column chromatography (acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L)=60%) to afford compound 2-10 (0.25 g, pyridinium salt) as a white solid. m/z: [M+H].sup.+ 1199.6.

    [0398] Step 3&4: Compound 2-10 (250 mg, 0.21 mmol) was subjected to azeotropic dehydration three times with pyridine (1 mL) and then dissolved in a mixed solvent of pyridine (2 mL) and dichloromethane (2 mL). 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide (DMOPC) (775 mg, 4.2 mmol) was added thereto, the mixture was stirred at room temperature for 10 min to obtain the reaction solution of compound 2-11. To the above reaction solution of compound 2-11 was directly added water (756 mg, 42 mmol) and 3H-1,2-benzodithiol-3-one (71 mg, 0.42 mmol), and stirred at room temperature for 20 min, aqueous solution of sodium bicarbonate (2.7%, 50 mL) was added thereto. The aqueous layer was extracted with ethyl acetate, and the separated organic layer was concentrated. The residue was purified by Flash column chromatography (acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L)=10-80%) to afford compound 2-12-p1 (30 mg, LCMS-RT(Thermo): 2.147 min), 2-12-p2 (35 mg, LCMS-RT(Thermo): 2.247 min), and 2-12-p3 (50 mg, LCMS-RT(Thermo): 2.327 min), as white solids.

    [0399] Step 5: To a solution of compound 2-12-p1 (30 mg, 0.01 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL), the reaction system was stirred at room temperature for 0.5 h, and then directly concentrated to afford compound 2-13-p1 (30 mg, crude product). m/z: [M+H].sup.+ 1159.9.

    [0400] Step 6: To a solution of compound 2-13-p1 (30 mg, crude) in methanol (2 mL) was added ammonium hydroxide solution (2 mL), the reaction system was stirred at 45° C. for overnight in a sealed tube, and then the reaction solution was concentrated in reduced pressure. The residue was subjected to azeotropic dehydration three times with pyridine (2 mL) and then dissolved in pyridine (2 mL). To the above reaction system was added triethylamine (0.35 g, 3.5 mmol) and triethylamine trihydrofluoride (0.28 g, 1.75 mmol) under nitrogen. The resulting mixture was stirred at 45° C. for 6 h. The solvent was concentrate under reduced pressure. The residue was adjust pH to 8 with aqueous solution of ammonium bicarbonate (1M), and then purified by prep-HPLC (separation method 3) to afford compound 2-p1 (di-ammonium salt, 2 mg, m/z: [M+H].sup.+ 723.8, HPLC-RT: 8.116 min) and 2-p2 (di-ammonium salt, 2 mg, m/z: [M+H].sup.+ 723.8, HPLC-RT: 10.121 min), as white solids.

    Synthesis of Compound 2-p3

    [0401] To a solution of compound 2-12-p2 (30 mg, 0.01 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL). The mixture was stirred at room temperature for 0.5 h, and then concentrated under reduced pressure to afford compound 2-13-p2. To a solution of compound 2-13-p2 in methanol (2 mL) was added ammonium hydroxide solution (2 mL), the reaction system was stirred in a sealed tube at 45° C. for overnight, and then the reaction solution was directly concentrated under reduced pressure. The residue was subjected to azeotropic dehydration three times with pyridine (2 mL) and then dissolved in pyridine (2 mL). To the above reaction system was added triethylamine (0.35 g, 3.5 mmol) and triethylamine trihydrofluoride (0.28 g, 1.75 mmol) under nitrogen. The resulting mixture was stirred at 45° C. for 6 h. The solvent was concentrate under reduced pressure. The residue was adjust pH to 8 with aqueous solution of ammonium bicarbonate (1M), and then purified by prep-HPLC (separation method 3) to afford compound 2-p3 (di-ammonium salt, 0.3 mg, m/z: [M+H].sup.+723.7, HPLC-RT: 10.121 min) as a white solid.

    Synthesis of Compound 2-p4

    [0402] Compound 2-p4 (di-ammonium salt, 0.4 mg, m/z: [M+H].sup.+723.7, HPLC-RT: 9.632 min) was obtained as a white solid in the same manner as compound 2-p3, by using compound 2-12-p3 (17 mg, 0.01 mmol) as a starting material.

    Embodiment 5: Synthesis of Compounds 3-p1, 3-p1/3-p2 and 3-p3

    [0403] ##STR00102##

    [0404] Step 1: Compound 3-1 (CAS No.: 129451-95-8) (2.36 g, 2.4 mmol) and Intermediate 2-9 (1.05 g, 2 mmol) were subjected to azeotropic dehydration twice with anhydrous acetonitrile (10 mL) respectively, and then dissolved in acetonitrile (5 mL) respectively for use. To the solution of intermediate 2-9 in acetonitrile, which contained 4 A molecular sieve, was slowly added the acetonitrile solution of compound 3-1 at 0° C. under nitrogen, the resulting mixture was stirred for 1 h, To the above reaction system was added DDTT (0.49 g, 2.4 mmol), and stirred for additional 1 h. The molecular sieve was removed by filtration, and the filtrate was concentrated under reduced pressure to afford compound 3-3 (1.3 g). m/z: [M+H].sup.+1501.5.

    [0405] Step 2: To the solution of compound 3-3 (1 g, 0.66 mmol) in a mixed solvent of dichloromethane (3 mL) and water (0.3 mL) was added dichloromethane solution of DCA (0.6 M, 10 mL). The reaction system was stirred for 0.5 h. Pyridine (3 mL) was added thereto, the mixture was concentrated under reduced pressure. The residue was purified by Flash column chromatography (acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L)=60%) to afford compound 3-4 (390 mg, pyridinium salt) as a white solid. m/z: [M+H].sup.+1199.6.

    [0406] Step 3&4: Compound 3-4 (250 mg, 0.21 mmol) was subjected to azeotropic dehydration three times with pyridine (1 mL) and then dissolved in a mixed solvent of pyridine (2 mL) and dichloromethane (2 mL). To the above reaction system was added DMOPC (775 mg, 4.2 mmol), the mixture was stirred at room temperature for 10 min to obtain the reaction solution of compound 3-5. To the reaction solution of compound 3-5 was added water (756 mg, 42 mmol) and 3H-1,2-benzodithiol-3-one (71 mg, 0.42 mmol) and stirred at room temperature for 20 min, and then aqueous solution of sodium bicarbonate (2.7%, 50 mL) was added thereto, the aqueous layer was extracted with ethyl acetate, and the separated organic layer was concentrated. The residue was purified by Flash column chromatography (acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L)=10-80%) to afford compound 3-6-p1 (50 mg, LCMS-RT(Thermo): 2.143 min), 3-6-p2 (15 mg, LCMS-RT(Thermo): 2.230 min), and 3-6-p3 (18 mg, LCMS-RT(Thermo): 2.320), as white solids.

    [0407] Step 5: To a solution of compound 3-6-p1 (20 mg, 0.02 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL), the reaction system was stirred at room temperature for 0.5 h, and then directly concentrated to afford compound 3-7-p1 (25 mg, crude product). m/z: [M+H].sup.+1159.9.

    [0408] Step 6: To a solution of compound 3-7-p1 (25 mg, crude) in methanol (2 mL) was added ammonium hydroxide solution (2 mL), the reaction system was stirred in a sealed tube at 45° C. for overnight, and then the reaction solution was concentrated in reduced pressure. The residue was subjected to azeotropic dehydration three times with pyridine (2 mL) and then dissolved in pyridine (2 mL). To the above reaction system was added triethylamine (0.35 g, 3.5 mmol) and triethylamine trihydrofluoride (0.28 g, 1.75 mmol) under nitrogen. The resulting mixture was stirred at 50° C. for 6 h. The solvent was concentrate under reduced pressure. The residue was adjust pH to 8 with aqueous solution of ammonium bicarbonate (1M), and then purified by prep-HPLC (separation method 3) to afford compound 3-p1 (di-ammonium salt, 0.7 mg, m/z: [M+H].sup.+ 723.8, HPLC-RT: 9.726 min) and a mixture of 3-p1/3-p2 (di-ammonium salt, 1.1 mg, m/z: [M+H].sup.+ 723.8, HPLC-RT: 9.726 min and 11.161 min).

    Synthesis of Compound 3-p3

    [0409] To a solution of compound 3-6-p3 (50 mg, 0.04 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL). The mixture was stirred at room temperature for 0.5 h, and then concentrated under reduced pressure to afford compound 3-7-p3. To a solution of compound 3-7-p3 in methanol (2 mL) was added ammonium hydroxide solution (2 mL), the reaction system was stirred in a sealed tube at 45° C. for overnight, and then the reaction solution was directly concentrated under reduced pressure. The residue was subjected to azeotropic dehydration three times with pyridine (2 mL) and then dissolved in pyridine (2 mL). To the above reaction system was added triethylamine (0.35 g, 3.5 mmol) and triethylamine trihydrofluoride (0.28 g, 1.75 mmol) under nitrogen. The resulting mixture was stirred at 45° C. for 6 h. The solvent was concentrate under reduced pressure. The residue was adjust pH to 8 with aqueous solution of ammonium bicarbonate (1M), and then purified by prep-HPLC (separation method 3) to afford compound 3-p3 (di-ammonium salt, 2.2 mg, m/z: [M+H].sup.+ 723.7, HPLC-RT: 11.76 min) as a white solid.

    Embodiment 6: Synthesis of Intermediate 4-3

    [0410] ##STR00103## ##STR00104## ##STR00105##

    [0411] Step 1: To a solution of 2′-fluoro-2′-deoxyadenosine (CAS No.: 64183-27-3) (9.7 g, 36.0 mmol) in pyridine (110 mL) was added chlorotrimethylsilane (23.5 g, 216 mmol) at 0° C. under nitrogen. The mixture was stirred at room temperature for 2 h, and then benzoyl chloride (7.6 g, 54 mmol) was added thereto. The resulting mixture was stirred at room temperature for overnight. To the reaction solution was added water (40 mL) and stirred for 1 h, and then ammonium hydroxide solution (40 mL) was added thereto and stirred for additional 2 h. Additional water (40 mL) was added thereto, the mixture was extracted with ethyl acetate (500 mL×2), and the combined organic layers were dried over anhydrous sodium sulfate, and then concentrated to ⅕ of the total volume, filtered, the filter cake was dried under vacuum to afford intermediate 4-1 (10 g) as a white solid. m/z: [M+H].sup.+374.0.

    [0412] Step 2: To a solution of intermediate 4-1 (1.2 g, 3.2 mmol) in pyridine (15 mL) was added DMTrCl (1.6 g, 4.8 mmol) under nitrogen. The reaction system was stirred at room temperature for overnight. To the reaction solution was added water (50 mL), the mixture was extracted with ethyl acetate (40 mL×3), and the combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by Flash column chromatography (2% methanol/dichloromethane) to afford intermediate 4-2 (1.9 g) as a light yellow solid. m/z: [M+H].sup.+676.0.

    [0413] Step 3: To a solution of intermediate 4-2 (1.35 g, 2 mmol) in dichloromethane (10 mL) was added N,N-diisopropylethylamine (1.1 mL, 6 mmol) and 2-cyanoethyl N,N-diisopropylchloro-phosphoramidite (947 mg, 4 mmol) at 0° C. under nitrogen. The resulting mixture was stirred at room temperature for 2 h. To the reaction solution was added water (50 mL) and saturated aqueous solution of sodium bicarbonate (20 mL). The resulting mixture was extracted with ethyl acetate (50 mL×3), and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by Flash column chromatography (3% methanol/dichloromethane) to afford intermediate 4-3 (1.2 g) as a yellow solid. m/z: [M+H].sup.+876.1.

    Embodiment 7: Synthesis of Compounds 4-p1, 4-p2 and 4-p3

    [0414] ##STR00106##

    [0415] Step 1: To a solution of intermediate 4-4 (4-4 was obtained as a mixture of stereoisomers in the same manner as Embodiment 4 steps 1-4, by using intermediates 4-3 and 2-8 as starting materials) (50 mg, 0.05 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL). The resulting mixture was stirred at room temperature for 0.5 h, and then concentrated under reduced pressure. The residue was purified by Flash column chromatography (acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L)=10-80%) to afford compound 4-5 (15 mg) as a white solid. m/z: [M+H].sup.+1047.8.

    [0416] Step 2: To the solution of compound 4-5 (30 mg, 0.03 mmol) in methanol (2 mL) was added ammonium hydroxide solution (2 mL), the reaction solution was stirred in a sealed tube at 45° C. for overnight, and then the solvent was concentrated in reduced pressure. The residue was subjected to azeotropic dehydration three times with anhydrous pyridine (2 mL) and then dissolved in pyridine (1 mL), and then triethylamine (0.91 g, 9 mmol) and triethylamine trihydrofluoride (0.58 g, 3.6 mmol) was added thereto under nitrogen. The resulting mixture was stirred at 50° C. for 6 h. The solvent was concentrate under reduced pressure. The residue was adjusted pH to 8 with ammonium hydroxide solution, and then directly purified by prep-HPLC (separation method 3) to afford compound 4-p1 (di-ammonium salt, 0.76 mg, m/z: [M+H].sup.+725.8, HPLC-RT: 9.04 min), 4-p2 (di-ammonium salt, 0.82 mg, m/z: [M+H].sup.+725.8, HPLC-RT: 10.45 min), and 4-p3 (di-ammonium salt, 0.97 mg, m/z: [M+H].sup.+725.8, HPLC-RT: 10.35 min), as white solids.

    Embodiment 8: Synthesis of Compounds 5-p1, 5-p2, 5-p3 and 5-p4

    [0417] ##STR00107##

    [0418] Step 1: To a solution of compound 5-1-p1 (5-1-p1, 5-2-p2 and 5-3-p3 were obtained in the same manner as Embodiment 4 steps 1-4, by using intermediates 3-2 and 3′-TBDMS—IBU-RG phosphoramidite (CAS No: 1445905-51-0) as starting materials, LCMS-RT(Thermo) of 5-1-p1, 5-2-p2 and 5-3-p3 were 2.11 min, 2.14 min and 2.31 min, respectively) (30 mg, 0.03 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL). The resulting mixture was stirred at room temperature for 0.5 h, and then concentrated to afford compound 5-2-p1 (30 mg, crude product). m/z: [M+H].sup.+1141.6.

    [0419] Step 2: To the solution of compound 5-2-p1 (30 mg, crude) in methanol (2 mL) was added ammonium hydroxide solution (2 mL), the reaction solution was stirred in a sealed tube at 45° C. for overnight, and then the solvent was concentrated in reduced pressure. The residue was subjected to azeotropic dehydration three times with anhydrous pyridine (2 mL) and then dissolved in pyridine (1 mL), and then triethylamine (0.35 g, 3.5 mmol) and triethylamine trihydrofluoride (0.28 g, 1.75 mmol) was added thereto under nitrogen. The resulting mixture was stirred at 50° C. for 6 h. The solvent was concentrate under reduced pressure. The residue was adjusted pH to 8 with ammonium hydroxide solution, and then directly purified by prep-HPLC (separation method 3) to afford compound 5-p1 (di-ammonium salt, 0.89 mg, m/z: [M+H].sup.+ 739.8, HPLC-RT: 6.495 min) as a white solid.

    Synthesis of Compounds 5-p2 and 5-p3

    [0420] Compounds 5-p2 (di-ammonium salt, 13 mg, m/z: [M+H].sup.+ 739.8, HPLC-RT: 10.666 min, .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 8.22 (s, 1H), 8.10 (s, 1H), 5.99 (d, J=7.7 Hz, 1H), 5.85 (d, J=8.5 Hz, 1H), 5.34-5.28 (m, 1H), 5.21-5.25 (m, 1H), 5.12-5.16 (m, 1H), 4.43-4.31 (m, 1H), 4.16-4.12 (m, 1H), 4.08-4.06 (m, 1H), 4.01-3.97 (m, 2H), 3.66 (d, J=11.8 Hz, 1H), 3.59-3.55 (m, 1H); .sup.31P NMR (162 MHz, DMSO-d.sub.6): δ 59.36, 57.52) and 5-p3 (di-ammonium salt, 0.7 mg, m/z: [M+H].sup.+ 739.9, HPLC-RT: 10.663 min) were obtained as white solids in the same manner as compound 5-p1, by using compound 5-1-p2 (76.5 mg, crdue) as a starting material.

    Synthesis of Compound 5-p4

    [0421] Compounds 5-p4 (di-ammonium salt, 1.41 mg, m/z: [M+H].sup.+ 739.9, HPLC-RT: 11.973 min) was obtained as a white solid in the same manner as compound 5-p1, by using compound 5-1-p3 (100 mg, crude) as a starting material.

    Embodiment 9: Synthesis of Intermediate 6-14

    [0422] ##STR00108## ##STR00109##

    [0423] Step 1: Tetraacetylribose (150 g, 472 mmol) was dissolved in acetone (1 L), and iodine (11.9 g, 47.2 mmol) was added thereto at 0° C. After addition, the reaction system was stirred at room temperature for 12 h. The saturated aqueous solution of sodium bisulfate was added into the reaction solution to remove the excess iodine, and then extracted with ethyl acetate (500 mL×3), and the combined organic layers were washed with brine, the separated organic layer was dried over anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (0-60% ethyl acetate/petroleum ether) to afford intermediate 6-1 (100 g) as a yellow oil. m/z: [M+H].sup.+ 275.0.

    [0424] Step 2: To the suspension of intermediate 6-1 (100 g, 365 mmol) in methanol (100 mL) was added potassium carbonate (150 g, 1.09 mmol). The reaction system was stirred at room temperature for 12 h, and then filtered, the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (0-10% methanol/dichloromethane) to afford intermediate 6-2 (60.3 g) as a white solid. m/z: [M+H].sup.+ 191.0.

    [0425] Step 3: To a solution of intermediate 6-2 (60 g, 316 mmol) in pyridine (300 mL) was added DMTrCl (128 g, 379 mmol) in small portions at 0° C. under nitrogen. The reaction system was stirred at room temperature for 12 h and then quenched by addition of water (100 mL). The solvent was concentrate under reduced pressure. The residue was purified by Flash column chromatography (0-50% petroleum ether/ethyl acetate) to afford intermediate 6-3 (150.3 g) as a white solid.

    [0426] Step 4: To a solution of intermediate 6-3 (150 g, 305 mmol) in DMF (500 mL) was added sodium hydride (18.3 g, 458 mmol, 60%) under nitrogen at 0° C. The reaction system was stirred at 0° C. for 0.5 h. To the reaction system was added iodomethane (56.3 g, 396 mmol) and stirred at room temperature for 3 h. The mixture was diluted with water (100 mL) and ethyl acetate (300 mL×3). The organic layer washed with brine, and then separated and dried over anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure to afford intermediate 6-4 (150 g) as a white solid. m/z: [M+H].sup.+ 507.1.

    [0427] Step 5: To a solution of intermediate 6-4 (150 g, 296 mmol) in dichloromethane (500 mL) was added DCA (344 g, 2.67 mol) at 0° C. The reaction system was stirred at 0° C. for 3 h, and then quenched by addition of saturated aqueous solution of sodium carbonate, the aqueous layer was extracted with dichloromethane (300 mL×3), the combined organic layers were washed with brine (150 mL×2), the separated organic layer was dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by Flash column chromatography (0-10% methanol/dichloromethane) to afford intermediate 6-5 (50.2 g) as a white solid. m/z: [M+H].sup.+205.0.

    [0428] Step 6: To a solution of intermediate 6-5 (50 g, 245 mmol) in dichloromethane (500 mL) was added benzoyl chloride (41.2 g, 294 mmol) at 0° C. The reaction system was stirred at room temperature for 12 h, and then the reaction was quenched by addition of water, the aqueous layer was extracted with dichloromethane (150 mL×3), the combined organic layers were washed with brine (150 mL×2), the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by Flash column chromatography (0-70% petroleum ether/ethyl acetate) to afford intermediate 6-6 (65.3 g) as a white solid. m/z: [M+H].sup.+ 309.0.

    [0429] Step 7: Intermediate 6-6 (65 g, 211 mmol) was dissolved in an aqueous solution of trifluoroacetic acid (150 mL, 80%), the reaction system was stirred at room temperature for 5 h. Most of the solvent was removed under reduced pressure, and then the reaction solution washed with saturated aqueous solution of sodium bicarbonate, the aqueous layer was extracted with dichloromethane (100 mL×3), the organic layer washed with brine (150 mL×2), the separated organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford intermediate 6-7 (50.2 g) as an off-white solid.

    [0430] Step 8: To a solution of intermediate 6-7 (50 g, 187 mmol) in pyridine (150 mL) was slowly added acetic anhydride (114 g, 1.11 mol), the reaction system was stirred at room temperature for 12 h. Most of the solvent was removed under reduced pressure, and then the reaction solution washed with saturated aqueous solution of sodium bicarbonate, the aqueous layer was extracted with ethyl acetate (100 mL×3), the combined organic layers were washed with brine (100 mL×2), the separated organic layer was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (0-70% petroleum ether/ethyl acetate) to afford intermediate 6-8 (60.2 g) as an off-white solid.

    [0431] Step 9: To a solution of 7-aminothiazolo[4,5-d]pyrimidin-2 (3H)-one (6.5 g, 38.7 mmol) and intermediate 6-8 (16.3 g, 46.4 mmol) in acetonitrile (120 mL) was added BSA (13.6 g, 116 mmol), the reaction system was stirred at reflux for 1 h. To the reaction solution was added TMSOTf (17.2 g, 77.4 mmol) after the reaction solution was cooled to room temperature and stirred at reflux for additional 72 h, and then to the reaction solution was slowly added saturated aqueous solution of sodium bicarbonate after the reaction solution was cooled to room temperature, and then the aqueous layer was extracted with ethyl acetate (150 mL×3), the combined organic layers were washed with water, the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (ethyl acetate/petroleum ether=3/4) to afford intermediate 6-9 (5 g) as a light yellow solid. m/z: [M+H].sup.+ 461.0.

    [0432] Step 10: To a solution of intermediate 6-9 (5 g, 10.9 mmol) in pyridine (5 mL) was added benzoyl chloride (5.3 g, 38 mmol) at 0° C. The reaction system was stirred at room temperature for overnight, and then quenched by addition of water (50 mL), the aqueous layer was extracted with dichloromethane (150 mL×3), the combined organic layers were washed with water, the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (petroleum ether/ethyl acetate=1/1) to afford intermediate 6-10 (6.8 g) as a white solid. m/z: [M+H].sup.+ 669.1.

    [0433] Step 11: To a solution of intermediate 6-10 (6.8 g, 10.1 mmol) in a mixed solvent of tetrahydrofuran and methanol (60/20 mL) was added aqueous solution of lithium hydroxide (36 mL, 1M) at 0° C., the reaction system was stirred at 0° C. for 2 h and then neutralized pH to 6 with acetic acid. The reaction solution was concentrated to ⅓ of the total volume, the solid was precipitated, filtered, the filter cake washed with water for 3 times, and then dried under vacuum to afford intermediate 6-11 (3.8 g) as a yellow solid. m/z: [M+H].sup.+419.0.

    [0434] Step 12: To a solution of intermediate 6-11 (3.8 g, 9.09 mmol) in pyridine (60 mL) was added DMTrCl (3.6 g, 10.9 mmol) under nitrogen. The reaction system was stirred at room temperature for overnight. The solvent was concentrate under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether/ethyl acetate=1/1) to afford intermediate 6-12 (6 g) as a light yellow solid.

    [0435] Step 13: To a solution of intermediate 6-12 (5 g, 6.94 mmol) in pyridine (5 mL) was added diphenyl phosphite (4.9 g, 20.8 mmol), the reaction solution was stirred at room temperature for 1 h. Triethylamine (2 mL) and water (1 mL) was successively added thereto. The reaction solution was stirred at room temperature for 5 min, and then diluted with water (50 mL) and extracted with dicholormethane (60 mL×3), the combined organic layers were washed with aqueous solution of sodium bicarbonate (5%), the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (methanol/dichloromethane=1/10) to afford intermediate 6-13 (5.1 g, triethylamine salt) as an off-white solid. m/z: [M+H].sup.+886.3.

    [0436] Step 14: To a solution of intermediate 6-13 (3 g, 3.39 mmol) in dichloromethane (20 mL) was added a dichloromethane solution of DCA (0.6M, 50 mL) at 0° C. The reaction mixture was stirred at room temperature for 1 h and then triethylsilane (1 mL) and pyridine (3 mL) were added thereto, the resulting mixture was stirred at room temperature for additional 10 min, the solvent was concentrated under reduced pressure to afford intermediate 6-14 (2.5 g, pyridinium salt).

    Embodiment 10: Synthesis of Compounds 6-p1, 6-p2, 6-p3 and 6-p4

    [0437] ##STR00110##

    [0438] Step 1: To a solution of compound 6-15 (6-15 was obtained as a mixture of stereoisomers in the same manner as Embodiment 4 steps 1-4, by using intermediates 6-14 and 4-3 as starting materials) (50 mg, 0.05 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL). The resulting mixture was stirred at room temperature for 0.5 h, and then concentrated under reduced pressure to afford compound 6-16 (40 mg). m/z: [M+H].sup.+948.0.

    [0439] Step 2: To a solution of compound 6-16 (40 mg, crude) in methanol (1 mL) was added ammonium hydroxide solution (1 mL), the mixture was stirred in a sealed tube at 50° C. for 5 h, and then cooled to room temperature and quenched by addition of acetic acid, the mixture was lyophilized. The residue was purified by prep-HPLC (separation method 4) to afford compound 6-p1 (di-ammonium salt, 3.6 mg, m/z: [M+H].sup.+740.0, HPLC-RT: 10.814 min), 6-p2 (di-ammonium salt, 3.2 mg, m/z: [M+H].sup.+740.0, HPLC-RT: 11.380 min), 6-p3 (di-ammonium salt, 3.9 mg, m/z: [M+H].sup.+740.0, HPLC-RT: 10.370 min; .sup.1H NMR (400 MHz, D.sub.2O): δ 8.36 (s, 2H), 8.12 (s, 1H), 6.61 (s, 1H), 6.11 (s, 1H), 5.79 (s, 1H), 5.39 (d, J=51.6 Hz, 1H), 4.93-5.02 (m, 1H), 4.36-4.48 (m, 3H), 3.84-4.03 (m, 4H), 3.51 (s, 3H); .sup.31P NMR (162 MHz, D.sub.2O): δ 56.49, 51.13; .sup.19F NMR (162 MHz, D.sub.2O): δ−202.92) and 6-p4 (di-ammonium salt, 2.3 mg, m/z: [M+H].sup.+740.0, HPLC-RT: 11.650 min), as white solids.

    Embodiment 11: Synthesis of Intermediate 7-1

    [0440] ##STR00111##

    [0441] Intermediate 7-1 was obtained as a yellow solid in the same manner as Embodiment 3 intermediate 2-8, by using tetraacetylribose and 5-aminothiazolo[4,5-d]pyrimidine-2,7 (3H,6H)-dione (refer to J. Med. Chem. 1990, 33, 407-415, compound 4) as starting materials. m/z: [M+H].sup.+599.1.

    Embodiment 12: Synthesis of Compounds 7-p1, 7-p2, 7-p3 and 7-p4

    [0442] ##STR00112##

    [0443] Step 1: To a solution of compound 7-2 (7-2 was obtained as a mixture of stereoisomers in the same manner as Embodiment 4 steps 1-4, by using intermediates 7-1 and 4-3 as starting materials) (330 mg, 0.3 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL). The resulting mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The residue was purified by Flash column chromatography (acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L)=0˜60%) to afford compound 7-3 (100 mg) as a white solid. m/z: [M+H].sup.+1063.8.

    [0444] Step 2: To the solution of compound 7-3 (100 mg, 94 mol) in methanol (2 mL) was added ammonium hydroxide solution (2 mL), the reaction solution was stirred in a sealed tube at 55° C. for 5 h, and then the solvent was concentrated in reduced pressure. The residue was subjected to azeotropic dehydration three times with anhydrous pyridine (2 mL) and then dissolved in pyridine (1 mL) and then triethylamine (1 mL) and triethylamine trihydrofluoride (0.5 mL) was added thereto under nitrogen. The resulting mixture was stirred at 50° C. for 3 h. The solvent was concentrate under reduced pressure. The residue was neutralized with ammonium hydroxide solution, and then directly purified by prep-HPLC (separation method 5) to afford compound 7-p1 (di-ammonium salt, 1.29 mg, m/z: [M+H].sup.+741.3, HPLC-RT: 9.058 min), 7-p2 (di-ammonium salt, 4.41 mg, m/z: [M+H].sup.+741.3, HPLC-RT: 9.590 min), 7-p3 (di-ammonium salt, 2.3 mg, m/z: [M+H].sup.+ 741.3, HPLC-RT: 10.438 min) and 7-p4 (di-ammonium salt, 24 mg, m/z: [M+H].sup.+741.7, HPLC-RT: 10.929 min), as white solids.

    Embodiment 13: Synthesis of Compounds 8-p1, 8-p2, 8-p3 and 8-p4

    [0445] ##STR00113##

    [0446] Step 1: To a solution of compound 8-1 (8-1 was obtained as a mixture of stereoisomers in the same manner as Embodiment 4 steps 1-4, by using intermediates 2-9 and 4-3 as starting materials) (618 mg, 0.61 mmol) in acetonitrile (3 mL) was added tert-butylamine (3 mL). The resulting mixture was stirred at room temperature for 1 h, and then concentrated under reduced pressure. The residue was purified by Flash column chromatography (acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L)=10˜80%) to afford compounds 8-2-p1 (67 mg), 8-2-p2 (40 mg), 8-2-p3 (40 mg), and 8-2-p4 (87 mg), as white solids. m/z: [M+H].sup.+1047.7.

    [0447] Step 2: To a solution of compound 8-2-p1 (50 mg, 52 mol) in methanol (1 mL) was added ammonium hydroxide solution (1 mL), the reaction solution was stirred in a sealed tube at 50° C. for 4 h, and then the solvent was concentrated in reduced pressure. The residue was subjected to azeotropic dehydration three times with anhydrous pyridine (1 mL) and then dissolved in pyridine (1 mL) and then triethylamine (1 mL) and triethylamine trihydrofluoride (0.5 mL) was added thereto under nitrogen. The resulting mixture was stirred at 55° C. for 4 h. The solvent was concentrate under reduced pressure. The residue was neutralized with ammonium hydroxide solution, and then directly purified by prep-HPLC (separation method 5) to afford compound 8-p1 (di-ammonium salt, 7 mg, m/z: [M+H].sup.+725.5, HPLC-RT: 11.702 min) as a white solid.

    Synthesis of Compound 8-p2

    [0448] Compound 8-p2 (di-ammonium salt, 1.98 mg, m/z: [M+H].sup.+ 725.6, HPLC-RT: 10.602 min) was obtained as a white solid in the same manner as compound 8-p1, by using compound 8-2-p2 (40 mg, 0.04 mmol) as a starting material.

    Synthesis of Compound 8-p3

    [0449] Compound 8-p3 (di-ammonium salt, 1.1 mg, m/z: [M+H].sup.+726.1, HPLC-RT: 10.556 min) was obtained as a white solid in the same manner as compound 8-p1, by using compound 8-2-p3 (40 mg, 0.04 mmol) as a starting material.

    Synthesis of Compound 8-p4

    [0450] Compound 8-p4 (di-ammonium salt, 7.5 mg, m/z: [M+H].sup.+725.6, HPLC-RT: 12.102 min) was obtained as a white solid in the same manner as compound 8-p1, by using compound 8-2-p4 (80 mg, 0.08 mmol) as a starting material.

    Embodiment 14: Synthesis of Intermediates 9-4 and 9-5

    [0451] ##STR00114##

    [0452] Step 1: The solution of isocarbostyril (25 g, 172 mmol), tetraacetylribose (137 g, 431 mmol) and BSA (105 g, 517 mmol) in anhydrous acetonitrile was stirred at reflux for 1 h, and then TMSOTf (62 mL, 344 mmol) was added thereto after the reaction solution was cooled to room temperature, the mixture was stirred at reflux for additional 5 h, saturated aqueous solution of sodium bicarbonate was slowly added thereto to adjusted pH to about 7 after the reaction solution was cooled to room temperature, the aqueous layer was extracted with ethyl acetate (150 mL×3), the combined organic layers were washed with water, the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (petroleum ether/ethyl acetate=2/1) to afford intermediate 9-1 (41.1 g) as an orange oil. m/z: [M+H].sup.+404.0.

    [0453] Step 2: To a solution of intermediate 9-1 (41.1 g, 102 mmol) in acetonitrile (300 mL) was added aqueous solution of lithium hydroxide (510 mL, 1M), and then the reaction system was stirred at room temperature for 1.5 h. An amount of white solid was precipitated, filtered, the filter cake washed with water for 3 times, and then dried under vacuum to afford intermediate 9-2 (18.5 g) as a white solid. m/z: [M+H].sup.+278.0.

    [0454] Step 3: Intermediate 9-2 (17.5 g, 63.1 mmol) was subjected to azeotropic dehydration three times with anhydrous pyridine and then dissolved in pyridine (100 mL) under nitrogen. To the above reaction solution was added pyridine solution of DMTrCl (22.5 g, 66.3 mmol, 50 mL) at 0° C. The resulting mixture was stirred at this temperature for 3 h. The reaction was quenched by addition of water (50 mL), the aqueous layer was extracted with ethyl acetate (100 mL×2), and the combined organic layers were concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether/ethyl acetate=1/1) to afford intermediate 9-3 (32.6 g) as an off-white solid. m/z: [M+Na].sup.+ 602.0.

    [0455] Step 4: Intermediate 9-3 (12 g, 20.7 mmol) and imidazole (4.9 g, 72.5 mmol) were subjected to azeotropic dehydration three times with anhydrous pyridine and then dissolved in pyridine (50 mL) under nitrogen, TBSCl (3.4 g, 22.8 mmol) was added thereto at 0° C. The reaction system was stirred at room temperature for overnight, and water (50 mL) was added thereto, the aqueous layer was extracted with ethyl acetate (100 mL×2), the combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered and concentrated, the residue was purified by Flash column chromatography (petroleum ether/ethyl acetate=10/1-6/1) to afford intermediates 9-4 (4.3 g, off-white solid, less polar) 9-5 (3.5 g, off-white solid, more polar). 9-4: m/z: [M+Na].sup.+716.0; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.28 (d, J=8.4 Hz, 1H), 7.71 (t, J=8.0 Hz, 2H), 7.65 (d, J=8.0 Hz, 1H), 7.53-7.45 (m, 3H), 7.36-7.32 (m, 6H), 7.26 (t, J=8.0 Hz, 1H), 6.93 (d, J=8.4 Hz, 4H), 6.46 (d, J=8.0 Hz, 1H), 6.29 (d, J=4.0 Hz, 1H), 5.14 (d, J=6.0 Hz, 1H), 4.28-4.26 (m, 1H), 4.20-4.16 (m, 1H), 4.14-4.20 (m, 1H), 4.05-4.00 (m, 1H), 3.74 (s, 6H), 3.37-3.35 (m, 1H), 0.82 (m, 9H), 0.03 (s, 3H), 0.01 (s, 3H); 9-5: m/z: [M+Na].sup.+716.0; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.27 (d, J=8.0 Hz, 1H), 7.75-7.70 (m, 2H), 7.66-7.64 (m, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.45-7.43 (m, 2H), 7.35-7.25 (m, 7H), 6.92 (d, J=8.4 Hz, 4H), 6.50 (d, J=7.6 Hz, 1H), 6.27 (d, J=3.6 Hz, 1H), 5.39 (d, J=6.0 Hz, 1H), 4.28-4.26 (m, 1H), 4.05-4.00 (m, 2H), 3.74 (s, 6H), 3.46-3.44 (m, 1H), 3.22-3.18 (m, 1H), 0.78 (m, 9H), 0.03 (s, 3H), 0.01 (s, 3H).

    Embodiment 15: Synthesis of Compounds 9-p1, 9-p2 and 9-p3

    [0456] ##STR00115## ##STR00116##

    [0457] Step 1: To a solution of intermediate 9-5 (3.1 g, 4.47 mmol) in pyridine (20 mL) was added diphenyl phosphite (3.1 g, 13.4 mmol) at 0° C. under nitrogen, the reaction system was stirred for 0.5 h, and then triethylamine (3 mL) and water (3 mL) was added thereto. The resulting mixture was stirred at room temperature for 5 min and then diluted with water (50 mL), extracted with propan-2-ol/chloroform (30 mL×2), the combined organic layers were washed with water, the separated organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (dichloromethane/methanol/triethylamine=100/5/1) to afford compound 9-6 (4.5 g, triethylamine salt) as an off-white solid. m/z: [M−H].sup.−756.0.

    [0458] Step 2: To a solution of compound 9-6 (3 g, 3.49 mmol) in a mixed solvent of dichloromethane (28 mL) and water (1 mL) was added dichloromethane solution of DCA (0.6 M, 46.5 mL). The mixture was stirred at room temperature for 30 min, to the the reaction solution was added triethylsilane (28 mL) and stirred for additional 1 h. When the color of the reaction solution changes from brown to colorless, pyridine (28 mL) was added thereto, the solvent was concentrated under reduced pressure to afford compound 9-7 (3 g, pyridinium salt, curd product). m/z: [M+H].sup.+445.0.

    [0459] Step 3&4: Compound 9-7 (3 g, crude) was dissolved in anhydrous acetonitrile (15 mL) and then concentrated under reduced pressure, repeated three times. The residue was dissolved in acetonitrile (50 mL), and 4 A molecular sieve (1 g) was added thereto. 3′-TBDMS-ibu-rG Phosphoramidite (CAS No.: 1445905-51-0, 3.4 g, 3.49 mmol) was dissolved in anhydrous acetonitrile (15 mL) and concentrated under reduced pressure, repeated three times. The residue was dissolved in acetonitrile (20 mL), and 4 A molecular sieve (2 g) was added thereto. To a solution of 9-7 in acetonitrile was slowly added the acetonitrile solution of 3′-TBDMS-ibu-rG Phosphoramidite at 0° C., the resulting mixture was stirred at room temperature for 0.5 h, and then DDTT (697 mg, 3.42 mmol) was added thereto and stirred at room temperature for additional 3 h. The molecular sieve was removed by filtration. To the mixtue was added water (1 mL) and then slowly added DCA (3.6 g, 27.9 mmol) dropwise, the resulting mixture was stirred at room temperature for 2 h, triethylsilane (28 mL) was added thereto and stirred for additional 1 h, pyridine (28 mL) was added thereto, and the reaction solution was concentrated under reduced pressure. The residue was purified by Flash column chromatography (acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L)=50%) to afford compound 9-10 (680 mg) as a white solid. m/z: [M+H].sup.+1053.9.

    [0460] Step 5: To a solution of compound 9-10 (200 mg, 0.19 mmol) in anhydrous pyridine (8 mL) was added DMOPC (0.7 g, 3.8 mmol) for one charge, the mixture was stirred at room temperature for 0.5 h, to the above reaction solution was added 3H-1,2-benzodithiol-3-one (38 mg, 0.23 mmol), and then stirred at room temperature for 0.5 h, the reaction was quenched by addition of aqueous solution of sodium bicarbonate (2.7%). The mixture was extracted with ethyl acetate (50 mL×2), and the combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (separation method 5) to afford compound 9-11-p1 (13.5 mg, LCMS-RT (Thermo): 2.023 min), 9-11-p2 (30.2 mg, LCMS-RT (Thermo): 2.157 min), and 9-11-p3 (38.0 mg, LCMS-RT (Thermo): 2.300 min), as white solids. m/z: [M+H].sup.+1067.9.

    [0461] Step 6: Compound 9-11-p1 (10 mg, 0.094 mmol) was dissolved in methylamine ethanol solution (1 mL, 30%) and stirred at room temperature for 1 h, and then concentrated under reduced pressure, the residue was dissolved in pyridine (0.5 mL), triethylamine (0.5 mL) and triethylamine trihydrofluoride (0.25 mL) was added thereto, the reaction system was stirred at 50° C. for 5 h and then concentrated under reduced pressure. The residue was diluted with methanol, and then ammonium hydroxide solution was slowly dropped to adjusted pH to 8-9. The resulting mixture was purified by prep-HPLC (separation method 5) to afford compound 9-p1 (4.07 mg, HPLC-RT: 11.311 min; m/z: [M+H].sup.+716.8; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.26 (d, J=6.0 Hz, 1H), 8.19 (s, 1H), 7.73 (t, J=7.6 Hz, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.53 (t, J=8.0 Hz, 1H), 7.42 (d, J=7.6 Hz, 1H), 6.69 (d, J=7.6 Hz, 1H), 6.51-6.47 (m, 2H), 6.22 (d, J=4.0 Hz, 1H), 5.90-5.82 (m, 2H), 5.52-5.51 (m, 1H), 5.08-5.06 (m, 1H), 4.95-4.90 (m, 1H), 4.88-4.86 (m, 1H), 4.65-4.61 (m, 1H), 4.57-4.55 (m, 1H), 4.40-4.36 (m, 2H), 4.25-4.19 (m, 2H), 4.10-3.99 (m, 2H), 3.94-3.87 (m, 2H); .sup.31P NMR (162 MHz, DMSO-d.sub.6): δ 56.49, 54.10) a as white solid.

    Synthesis of Compounds 9-p2

    [0462] Compound 9-p2 (4.45 mg, HPLC-RT: 11.389 min; m/z: [M+H].sup.+706.8; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.26 (d, J=8.0 Hz, 1H), 8.02 (s, 1H), 7.74 (t, J=7.2 Hz, 1H), 7.67 (d, J=7.6 Hz, 1H), 7.53 (t, J=7.6 Hz, 1H), 7.37 (d, J=7.6 Hz, 1H), 6.91-6.34 (m, 4H), 5.87-5.85 (m, 1H), 5.37-5.19 (m, 2H), 4.38-4.37 (m, 1H), 4.21-4.19 (m, 1H), 4.11-4.05 (m, 2H), 3.74-3.70 (m, 1H), 3.01-2.99 (m, 8H); .sup.31P NMR (162 MHz, DMSO-d.sub.6): δ 59.18, 56.65, 54.32, 47.74) was obtained as a white solid in the same manner as compound 9-p1, by using compound 9-11-p2 as a starting material and purified by prep-HPLC (separation method 5).

    Synthesis of Compounds 9-p3

    [0463] Compound 9-p3 (7.0 mg, HPLC-RT: 10.912 min; m/z: [M+H].sup.+ 716.8; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.26 (d, J=8.0 Hz, 1H), 7.97 (s, 1H), 7.73 (t, J=7.6 Hz, 1H), 7.65 (d, J=7.6 Hz, 1H), 7.53 (t, J=7.6 Hz, 1H), 7.44 (d, J=7.6 Hz, 1H), 6.62 (d, J=8.0 Hz, 1H), 6.56-6.52 (m, 1H), 6.33 (d, J=6.8 Hz, 1H), 5.86 (d, J=8.4 Hz, 1H), 5.27-5.23 (m, 1H), 5.06-5.04 (m, 2H), 4.46-4.40 (m, 2H), 4.22-4.18 (m, 1H), 4.12-4.08 (m, 2H), 3.96-3.92 (m, 2H), 3.78-3.75 (m, 1H), 3.01-2.95 (m, 4H); .sup.31P NMR (162 MHz, DMSO-d.sub.6): δ 57.77, 50.27) was obtained as a white solid in the same manner as compound 9-p1, by using compound 9-11-p3 as a starting material, and purified by prep-HPLC (separation method 5).

    Embodiment 16: Synthesis of Compounds 10-p1, 10-p2 and 10-p3

    Synthesis of Intermediates 10-4 and 10-5

    [0464] ##STR00117##

    [0465] Intermediates 10-4 (2.0 g, more polar) and 10-5 (3.4 g, less polar) were obtained as white solids in the same manner as compounds 9-4 and 9-5, by using quinazolin-4 (3H)-one as a starting material. m/z: [M+H].sup.+ 695.0; 10-4: .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.50 (s, 1H), 8.16 (d, J=8.0 Hz, 2H), 7.86-7.82 (m, 1H), 7.67 (d, J=8.0 Hz, 2H), 7.58-7.54 (m, 1H), 7.40 (d, J=8.0 Hz, 2H), 7.31-7.22 (m, 7H), 6.89-6.87 (m, 4H), 6.09 (d, J=4.0 Hz, 1H), 5.10 (d, J=8.0 Hz, 1H), 4.38-4.36 (m, 1H), 4.13-4.09 (m, 2H), 3.72 (s, 6H), 0.81 (s, 9H), 0.01-0.00 (s, 6H); 10-5: .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.53 (s, 1H), 8.15 (d, J=8.0 Hz, 1H), 7.86-7.82 (m, 1H), 7.67 (d, J=8.0 Hz, 1H), 7.58-7.54 (m, 1H), 7.41-7.39 (m, 2H), 7.30-7.19 (m, 7H), 6.85 (d, J=8.0 Hz, 4H), 6.06-6.05 (m, 1H), 5.36 (d, J=8.0 Hz, 1H), 4.31-4.26 (m, 2H), 4.02-4.00 (m, 1H), 3.71 (s, 6H), 0.79 (s, 9H), 0.02-0.00 (s, 6H).

    ##STR00118##

    [0466] Synthesis of compounds 10-11-p1, 10-11-p2 and 10-11-p3: compounds 10-11-p1 (7.0 mg, LCMS-RT (Thermo): 2.020 min), 10-11-p2 (26 mg, LCMS-RT (Thermo): 2.170 min) and 10-11-p3 (19 mg, LCMS-RT (Thermo): 2.350 min) were obtained as white solids in the same manner as Embodiment 15 steps 1-5, by using compound 10-5 as a starting material, and purified by prep-HPLC (separation method 6). m/z: [M+H].sup.+1068.9.

    Synthesis of Compound 10-p1

    [0467] To compound 10-11-p1 (7.0 mg, 0.006 mmol) was added methylamine ethanol solution (1 mL, 30%), the reaction system was stirred at room temperature for 1 h and then concentrated under reduced pressure, the residue was dissolved in anhydrous pyridine (0.5 mL), triethylamine (0.5 mL) and triethylamine trihydrofluoride (0.25 mL) was added thereto under nitrogen, the reaction solution was stirred at 50° C. for 1 h. The solvent was concentrated under reduced pressure, the residue was diluted with methanol, and then ammonium hydroxide solution was slowly dropped to adjusted pH to 8-9. The mixture was purified by prep-HPLC (separation method 4) to afford compound 10-p1 (0.73 mg, HPLC-RT: 10.671 min, m/z: [M+H].sup.+ 717.8) as a white solid.

    Synthesis of Compound 10-p2

    [0468] Compound 10-p2 (7.10 mg, HPLC-RT: 10.826 min; m/z: [M+H].sup.+ 717.8; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.41 (s, 1H), 8.25-8.20 (m, 1H), 8.03 (s, 1H), 7.89-7.85 (m, 1H), 7.72-6.69 (m, 1H), 7.61-7.57 (m, 1H), 7.61-7.57 (m, 1H), 6.52-6.48 (m, 2H), 6.07-6.00 (m, 1H), 5.91-5.79 (m, 1H), 0.56 (s, 1H), 5.34-5.30 (m, 1H), 5.20-5.19 (m, 1H), 5.09-5.01 (m, 1H), 4.70-4.64 (m, 1H), 4.36-4.33 (m, 1H), 4.27-4.18 (m, 1H), 4.14-4.05 (m, 2H), 3.95-3.70 (m, 3H), 1.24 (s, 2H)) was obtained as a white solid in the same manner as compound 10-p1, by using compound 10-11-p2 as a starting material, and purified by prep-HPLC (separation method 4).

    [0469] Synthesis of Compound 10-p3:

    [0470] Compound 10-p3 (5.46 mg, HPLC-RT: 10.925 min; m/z: [M+H].sup.+ 717.8; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.44 (s, 1H), 8.22-8.20 (m, 1H), 7.99 (s, 1H), 7.89-7.85 (m, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.61-7.57 (m, 1H), 6.56 (s, 2H), 6.09 (d, J=4.0 Hz, 2H), 5.83 (d, J=8.0 Hz, 1H), 5.34-5.32 (m, 1H), 5.26 (s, 1H), 5.08 (m, 1H), 4.71-4.68 (m, 1H), 4.46-4.45 (m, 1H), 4.25 (m, 1H), 4.14-3.95 (m, 5H), 3.79-3.76 (m, 1H), 1.24 (s, 2H).) was obtained as a white solid in the same manner as compound 10-p1, by using compound 10-11-p3 as a starting material, and purified by prep-HPLC (separation method 4).

    Embodiment 17: Synthesis of Compounds 11-p1, 11-p2, 11-p3 and 11-p4

    Synthesis of Intermediates 11-4 and 11-5

    [0471] ##STR00119##

    [0472] Synthesis of compounds 11-4 and 11-5: compounds 11-4 (650 mg, more polar) and 11-5 (610 mg, less polar) were obtained as white solids in the same manner as compounds 9-4 and 9-5, by using pyrido[2,3-d]pyrimidin-4-one as a starting material. m/z: [M+H].sup.+696.0; 11-4: .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.97 (d, J=4.0 Hz, 1H), 8.74 (s, 1H), 8.56 (d, J=8.0 Hz, 1H), 7.60-7.57 (m, 1H), 7.42 (d, J=8.0 Hz, 2H), 7.31-7.20 (m, 1H), 6.88 (d, J=12.0 Hz, 4H), 6.02 (s, 1H), 5.40 (d, J=4.0 Hz, 1H), 4.30-4.29 (m, 2H), 4.01-3.98 (m, 1H), 3.71 (s, 6H), 3.40-3.37 (m, 1H), 3.21-3.18 (m, 1H), 0.74 (s, 9H), 0.00 (s, 6H); 11-5: .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.94 (d, J=4.0 Hz, 1H), 8.67 (s, 1H), 8.55 (d, J=8.0 Hz, 1H), 7.56-7.53 (m, 1H), 7.41 (d, J=8.0 Hz, 2H), 7.29-7.25 (m, 1H), 6.86 (d, J=8.0 Hz, 4H), 6.00 (s, 1H), 5.13 (d, J=4.0 Hz, 1H), 4.13-4.08 (m, 2H), 4.00-3.98 (m, 1H), 3.39 (s, 6H), 3.36-3.32 (m, 1H), 3.27-3.25 (m, 1H), 0.79 (s, 9H), 0.00 (s, 6H).

    ##STR00120##

    [0473] Synthesis of compounds 11-11-p1, 11-11-p2, 11-11-p3 and 11-11-p4: compounds 11-11-p1 (27 mg), 11-11-p2 (15 mg), 11-11-p3 (19 mg) and 11-11-p4 (36 mg) were obtained as white solids in the same manner as Embodiment 15 steps 1-5, by using compound 11-5 as a starting material, and purified by prep-HPLC (separation method 6).

    Synthesis of Compound 11-p1

    [0474] To compound 11-11-p1 (20 mg, 0.019 mmol) was added methylamine ethanol solution (2 mL, 30%), the reaction system was stirred at room temperature for 10 h and then concentrated under reduced pressure, the residue was dissolved in anhydrous pyridine (0.5 mL), and triethylamine (0.5 mL) and triethylamine trihydrofluoride (0.25 mL) were added thereto under nitrogen, the reaction solution was stirred at 50° C. for 1 h. The solvent was concentrated under reduced pressure. The residue was diluted with methanol, and then ammonium hydroxide solution was slowly added to dropped pH to 8-9. The mixture was purified by prep-HPLC (separation method 7) to afford compound 11-p1 (2.45 mg, HPLC-RT: 8.963 min, m/z: [M+H].sup.+ 718.8) as a white solid.

    Synthesis of Compound 11-p2

    [0475] Compound 11-p2 (3.07 mg, HPLC-RT: 8.527 min, m/z: [M+H].sup.+ 718.7) was obtained as a white solid in the same manner as compound 11-p1, by using compound 11-11-p2 as a starting material, and purified by prep-HPLC (separation method 5).

    Synthesis of Compound 11-p3

    [0476] Compound 11-p3 (1.28 mg, HPLC-RT: 9.103 min, m/z: [M+H].sup.+ 719.1) was obtained as a white solid in the same manner as compound 11-p1, by using compound 11-11-p3 as a starting material, and purified by prep-HPLC (separation method 5).

    Synthesis of Compound 11-p4

    [0477] Compound 11-p4 (3.03 mg, HPLC-RT: 9.403 min, m/z: [M+H].sup.+ 718.6) was obtained as a white solid in the same manner as compound 11-p1, by using compound 11-11-p4 as a starting material, and purified by prep-HPLC (separation method 5).

    Embodiment 18: Synthesis of Intermediate 12-1

    [0478] ##STR00121##

    [0479] Step 1: 2,6-dichloronicotinonitrile (25 g, 145 mmol) and ammonium hydroxide solution (250 mL) was charged into a sealed tube. The reaction system was stirred at 120° C. for 48 h and then cooled to 10° C., the solid was filtered and washed with cold water, the the filter cake was dried under vacuum to afford 2,6-diaminonicotinonitrile (12.5 g) as a light yellow solid. m/z: [M+H].sup.+135.0.

    [0480] Step 2: To a solution of 2,6-diaminonicotinonitrile (12.5 g, 93.2 mmol) in acetic acid (120 mL) was slowly added concentrated sulfuric acid (3 mL), the reaction system was stirred at reflux for 9 h, and then cooled to room temperature and concentrated under reduced pressure. The residue was triturated with ammonium hydroxide solution, the solid was filtered, the filter cake washed with cold water, and dried under vacuum to afford 7-aminopyrido[2,3-d]pyrimidin-4 (3H)-one (15.1 g) as a light yellow solid. m/z: [M+H].sup.+163.0.

    [0481] Step 3: 7-aminopyrido[2,3-d]pyrimidin-4 (3H)-one (15.1 g, 93.1 mmol) was dissolved in pyridine (1.5 L), isobutyryl chloride (29.7 g, 279 mmol) was slowly added thereto, the reaction system was stirred at room temperature for 18 h and then water (1.5 L) was added thereto, the reaction solution was extracted with chloroform/propan-2-ol (1.5 L), the organic layer was concentrated under reduced pressure. The residue was purified by Flash column chromatography (0-6.2% methanol/dichloromethane) to afford compound 12-1 (11.9 g) as a white solid. m/z: [M+H].sup.+233.1.

    Embodiment 19: Synthesis of Compounds 12-p1, 12-p2, 12-p3 and 12-p4

    Synthesis of intermediates 12-4 and 12-5

    [0482] ##STR00122##

    [0483] Intermediates 12-4 (1.73 g, more polar) and 12-5 (1.1 g, less polar) were obtained as white solids in the same manner as compounds 9-4 and 9-5, by using compound 12-1 as a starting material. m/z: [M+H].sup.+ 781.0; 12-4: .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.15 (s, 1H), 8.70 (s, 1H), 8.52 (d, J=8.8 Hz, 1H), 8.33 (d, J=8.8 Hz, 1H), 7.43 (d, J=7.6 Hz, 2H), 7.32 (d, J=7.6 Hz, 2H), 7.29 (d, J=8.4 Hz, 4H), 6.89 (d, J=8.4 Hz, 4H), 6.03 (d, J=2.8 Hz, 1H), 5.40 (d, J=6.0 Hz, 1H), 4.30 (t, J=3.2 Hz, 1H), 4.05-4.02 (m, 2H), 3.73 (s, 6H), 3.40-3.36 (m, 1H), 3.25-3.22 (m, 1H), 2.83-2.79 (m, 1H), 1.12 (s, 3H), 1.11 (s, 3H), 0.77 (s, 9H), 0.03 (s, 3H), −0.04 (s, 3H); 12-5: .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.13 (s, 1H), 8.67 (s, 1H), 8.54 (d, J=8.8 Hz, 1H), 8.31 (d, J=8.8 Hz, 1H), 7.45 (d, J=7.6 Hz, 2H), 7.33 (d, J=7.6 Hz, 2H), 7.31 (d, J=8.4 Hz, 4H), 6.90 (d, J=8.4 Hz, 4H), 6.04 (d, J=2.8 Hz, 1H), 5.16 (d, J=6.0 Hz, 1H), 4.40 (t, J=3.2 Hz, 1H), 4.17-4.12 (m, 2H), 3.74 (s, 6H), 3.43-3.40 (m, 1H), 3.30-3.27 (m, 1H), 2.82-2.79 (m, 1H), 1.13 (s, 3H), 1.11 (s, 3H), 0.85 (s, 9H), 0.05 (s, 6H).

    ##STR00123##

    [0484] Synthesis of compounds 12-11-p1, 12-11-p2, 12-11-p3 and 12-11-p4: compounds 12-11-p1 (25 mg), 12-11-p2 (7 mg), 12-11-p3 (40 mg) and 12-11-p4 (32 mg) were obtained as white solids in the same manner as Embodiment 15 steps 1-5, by using compound 12-5 as a starting material, and purified by prep-HPLC (separation method 6).

    Synthesis of Compound 12-p1

    [0485] To compound 12-11-p1 (25 mg, 19 μmol) was added methylamine ethanol solution (2 mL, 30%), the reaction system was stirred at room temperature for 3 h and then concentrated under reduced pressure, the residue was dissolved in anhydrous pyridine (0.5 mL), triethylamine (0.5 mL) and triethylamine trihydrofluoride (0.25 mL) was added thereto under nitrogen, the reaction solution was stirred at 50° C. for 1 h. The solvent was concentrated under reduced pressure. The residue was diluted with methanol, and then ammonium hydroxide solution was slowly dropped to adjusted pH to 8-9. The mixture was purified by prep-HPLC (separation method 4) to afford compound 12-p1 (0.9 mg, HPLC-RT: 8.891 min, m/z: [M+H].sup.+ 733.7) as a white solid.

    Synthesis of Compound 12-p2

    [0486] Compound 12-p2 (0.5 mg, HPLC-RT: 8.571 min, m/z: [M+H].sup.+ 733.7) was obtained as a white solid in the same manner as compound 12-p1, by using compound 12-11-p2 as a starting material, and purified by prep-HPLC (separation method 4).

    Synthesis of Compound 12-p3

    [0487] Compound 12-p3 (1.5 mg, HPLC-RT: 10.020 min, m/z: [M+H].sup.+ 733.8) was obtained as a white solid in the same manner as compound 12-p1, by using compound 12-11-p3 as a starting material, and purified by prep-HPLC (separation method 4).

    Synthesis of Compound 12-p4

    [0488] Compound 12-p4 (3.07 mg, HPLC-RT: 9.468 min, m/z: [M+H].sup.+ 733.8) was obtained as a white solid in the same manner as compound 12-p1, by using compound 12-11-p4 as a starting material, and purified by prep-HPLC (separation method 4)

    Embodiment 20: Synthesis of Intermediates 13-4 and 13-5

    [0489] ##STR00124##

    [0490] Synthesis of imidazo[1,2-c]pyrimidin-5 (6H)-one: The suspension of cytosine (18 g, 0.16 mol), aqueous solution of chloroacetaldehyde (63.6 g, 0.32 mol, 40%) and sodium acetate (32.8 g, 0.40 mol) in water (180 mL) was stirred at 80° C. for 3 h, and then the reaction system was cooled to 10° C., the solid was filtered and washed with cold water, the filter cake was dried under vacuum to afford imidazo[1,2-c]pyrimidin-5 (6H)-one (17.8 g) as a brown solid. m/z: [M+H].sup.+ 136.0.

    [0491] Step 1: To a suspension of imidazo[1,2-c]pyrimidin-5 (6H)-one (17.8 g, 0.13 mmol), tetraacetylribose (46.1 g, 0.14 mmol) and BSA (39.7 g, 0.19 mmol) in acetonitrile (200 mL) was added TMSOTf (43.3 g, 0.19 mol) dropwise at 0° C., the reaction system was stirred at 60° C. for 3 h and then concentrated under reduced pressure to afford compound 13-1 (100 g, crude product) as a brown oil. m/z: [M+H].sup.+ 394.0.

    [0492] Step 2: Compound 13-1 (45 g, crude) was dissolved in methanol (100 mL), ammonia in methanol (81.7 mL, 7M) was slowly added thereto, the reaction system was stirred at room temperature for overnight, the solid was filtered and washed with methanol, the filter cake was dried under vacuum to afford compound 13-2 (8 g) as a brown solid. m/z: [M+H].sup.+ 268.0.

    [0493] Step 3: Compound 13-2 (12.5 g, 5.76 mmol) was subjected to azeotropic dehydration three times with anhydrous pyridine (20 mL) and then dissolved in pyridine (100 mL). To the above reaction solution was added DMTrCl (19.52 g, 5.76 mmol) in small portions at 0-5° C. under nitrogen. The reaction system was stirred at room temperature for overnight, the solvent was concentrated under reduced pressure, and the residue was diluted with chloroform (100 mL). The organic layer washed with saturated aqueous solution of sodium bicarbonate (30 mL×2), the organic layer was separated and dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash column chromatography (methanol/dichloromethane=0-3%) to afford compound 13-3 (16.65 g) as a brown solid. m/z: [M+Na].sup.+570.0.

    [0494] Step 4: Compound 13-3 (5.38 g, 9.44 mmol) was subjected to azeotropic dehydration three times with anhydrous pyridine (20 mL) and then dissolved in pyridine (60 mL). To the mixture was added TBSCl (2.13 g, 14.2 mmol) and imidazole (2.25 g, 33.1 mmol) at 0° C. under nitrogen. The reaction system was stirred at room temperature for overnight, the solvent was concentrated under reduced pressure, and the residue was diluted with chloroform (100 mL). The organic layer washed with saturated aqueous solution of sodium bicarbonate (30 mL×2), the separated organic layer was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (0-3% methanol/dichloromethane) to afford compounds 13-4 (1.32 g, less polar) and 13-5 (3.68 g, more polar), as white foam solids. m/z: [M+H].sup.+684.0; 13-4: .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.88 (d, J=5.6 Hz, 1H), 7.82 (s, 1H), 7.76 (s, 1H), 7.40-7.26 (m, 9H), 6.92-6.90 (m, 4H), 6.68 (d, J=6.0 Hz, 1H), 5.90 (d, J=9.2 Hz, 1H), 5.33 (d, J=4.8 Hz, 1H), 4.50 (t, J=4.4 Hz, 1H), 4.15-4.14 (m, 2H), 3.74 (s, 6H), 3.28-3.26 (m, 2H), 0.77 (s, 9H), 0.02 (s, 3H), −0.12 (s, 3H). 13-5: .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.92 (d, J=6.0 Hz, 1H), 7.78 (s, 2H), 7.36-7.21 (m, 9H), 6.88-6.86 (m, 4H), 6.68 (d, J=8.8 Hz, 1H), 5.81 (d, J=4.8 Hz, 1H), 5.63 (d, J=6.0 Hz, 1H), 4.40-4.35 (m, 2H), 4.27 (t, J=4.8 Hz, 1H), 4.06-4.04 (m, 1H), 3.74 (s, 6H), 3.20-3.16 (m, 1H), 0.80 (s, 9H), 0.06 (s, 3H), −0.01 (s, 3H).

    Embodiment 21: Synthesis of Compounds 13-p1, 13-p2, 13-p3 and 13-p4

    [0495] ##STR00125##

    [0496] Synthesis of compounds 1.sup.3-11-p1, 13-11-p2, 13-11-p3 and 13-11-p4: compounds 13-11-p1 (55 mg, LCMS-RT (Thermo): 1.874, 1.944 min), 13-11-p2 (25 mg, LCMS-RT (Thermo): 2.030 min), and 13-11-p3 (11 mg, LCMS-RT (Thermo): 2.170 min) were obtained as white solids in the same manner as Embodiment 15 steps 1-5, by using compound 13-5 as a starting material, and purified by Flash column chromatography (acetonitrile/aqueous solution of ammonium bicarbonate (10 mmol/L)=40%). m/z: [M+H].sup.+1057.9.

    Synthesis of Compounds 13-p1 and 13-p2

    [0497] Compound 13-11-p1 (43 mg, 0.04 mmol) was dissolved in methylamine ethanol solution (3 mL, 30%) and stirred at room temperature for 3 h, and then concentrated under reduced pressure, the residue was dissolved in anhydrous pyridine (0.5 mL), triethylamine (0.5 mL) and triethylamine trihydrofluoride (0.25 mL) was added thereto under nitrogen, the reaction solution was stirred at 50° C. for 3 h and then concentrated under reduced pressure. The residue was diluted with methanol and ammonium hydroxide solution was slowly dropped to adjusted pH to 8-9. The mixture was purified by prep-HPLC (separation method 5) to afford compounds 13-p1 (2.28 mg, HPLC-RT: 6.048 min, m/z: [M+H].sup.+706.8) and 13-p2 (0.68 mg, HPLC-RT: 7.781 min, m/z: [M+H].sup.+706.8), as white solids.

    Synthesis of Compound 13-p3

    [0498] Compound 13-p3 (3.82 mg, HPLC-RT: 6.445 min, m/z: [M+H].sup.+706.8) was obtained as a white solid in the same manner as compounds 13-p1/13-p2, by using compound 13-11-p2 as a starting material, and purified by prep-HPLC (separation method 4).

    Synthesis of Compound 13-p4

    [0499] Compound 13-p4 (4.03 mg, HPLC-RT: 10.395 min, m/z: [M+H].sup.+706.8) was obtained as a white solid in the same manner as compounds 13-p1/13-p2, by using compound 13-11-p3 as a starting material, and purified by prep-HPLC (separation method 5).

    Embodiment 22: Synthesis of Compounds 14-p1, 14-p2, 14-p3 and 14-p4

    Synthesis of intermediates 14-4 and 14-5

    [0500] ##STR00126##

    [0501] Intermediates 14-4 (1.3 g, more polar) and 14-5 (0.56 g, less polar) were obtained as white solids in the same manner as compounds 13-4 and 13-5, by using imidazo[5,1-f][1,2,4]triazin-4 (3H)-one (CAS No.: 865444-76-4) as a starting material. m/z: [M+H].sup.+685.0.

    ##STR00127##

    [0502] Synthesis of compounds 14-11-p1, 14-11-p2, 14-11-p3 and 14-11-p4: compounds 14-11-p1 (5 mg, LCMS-RT (Thermo): 1.850 min), 14-11-p2 (17 mg, LCMS-RT (Thermo): 1.880 min), 14-11-p3 (22 mg, LCMS-RT (Thermo): 2.020 min) and 14-11-p4 (18 mg, LCMS-RT (Thermo): 2.140 min) were obtained as white solids in the same manner as Embodiment 15 steps 1-5, by using compound 14-5 as a starting material, and purified by prep-HPLC (separation method 7). m/z: [M+H].sup.+1059.0.

    Synthesis of Compound 14-p1

    [0503] Compound 14-11-p1 (5.0 mg, 0.05 mmol) was dissolved in methylamine ethanol solution (3.0 mL, 30%), the mixture was stirred at room temperature for 3 h and then concentrated under reduced pressure, the residue was dissolved in anhydrous pyridine (0.5 mL), triethylamine (0.5 mL) and triethylamine trihydrofluoride (0.3 mL) was added thereto under nitrogen, the reaction solution was stirred at 50° C. for 2 h. The solvent was concentrated under reduced pressure. The residue was diluted with methanol and then ammonium hydroxide solution was slowly dropped to adjusted pH to 8-9. The mixture was purified by prep-HPLC (separation method 5) to afford compound 14-p1 (3.65 mg, HPLC-RT: 1.914 min, m/z: [M+H].sup.+ 707.5) as a white solid.

    Synthesis of Compound 14-p2

    [0504] Compound 14-p2 (17.5 mg, HPLC-RT: 3.123 min, m/z: [M+H].sup.+ 707.5) was obtained as a white solid in the same manner as compound 14-p1, by using compound 14-11-p2 as a starting material, and purified by prep-HPLC (separation method 4).

    Synthesis of Compound 14-p3

    [0505] Compound 14-p3 (1.04 mg, HPLC-RT: 3.193 min, m/z: [M+H].sup.+ 707.5) was obtained as a white solid in the same manner as compound 14-p1, by using compound 14-11-p3 as a starting material, and purified by prep-HPLC (separation method 4).

    Synthesis of Compound 14-p4

    [0506] Compound 14-p4 (1.04 mg, HPLC-RT: 36.036 min, m/z: [M+H].sup.+ 707.5) was obtained as a white solid in the same manner as compound 14-p1, by using compound 14-11-p4 as a starting material, and purified by prep-HPLC (separation method 4).

    EMBODIMENTS OF BIOASSAYS

    Embodiment 1: Type 1 IFN Activation Assay

    [0507] Plated THP-1 dual cells (Invivogen) as 100000 cells/well in a 96 well-plate, and then phorbol 12-myristate 13-acetate (PMA) was added and the final concentration of PMA was 30 ng/mL. After 24 h incubation, cells were washed with fresh medium for twice, and 3-folds dilution compounds were added to appropriate wells. The compounds were diluted by PB buffer (50 mM HEPES, 100 mM KCl, 3 mM MgCl.sub.2, 0.1 mM DTT, 85 mM sucrose, 1 mM ATP, 0.1 mM GTP, 0.2% o bovine serum albumin and 5 μg/ml digitonin) and the top dose was 10 μM, minimum dose was 0.0015μM. The cells were incubated for 30 min, and then washed with fresh medium for twice again. Add fresh medium, and the cells were incubated for additional 24 h. After incubation, 10 ul supermant and 50 μL QUANTI-Luc (Invivogen) was add to a 96 well-plate, fluorescence was read with TECAN. The express level of IFN was proportional to the fluorescence intensity. Drew the curve with Graphpad Prism and analyse EC.sub.50 of the compounds.

    TABLE-US-00001 Compound No. EC.sub.50 (μM) 1-p1   >10 1-p2   >10 1-p3   >10 1-p4 25.615 2-p1 45.755 2-p2   >100 2-p3 97.411 2-p4  2.077 4-p1  6.834 4-p2  0.625 4-p3  0.417 5-p1   >10 5-p2 51.981 5-p3 58.238 5-p4  3.223 6-p1  2.273 6-p2  1.063 6-p3  0.429 6-p4  0.311 7-p1  3.464 7-p2  40.62 7-p3  0.275 7-p4  2.307 8-p1   >100 8-p2   >100 8-p3  99.53 8-p4  38.80 9-p1   >100 9-p2   >100 9-p3  4.865 10-p1  70.69 10-p2  118.8 10-p3  0.494 11-p1  60.81 11-p2  9.805 11-p3  17.50 11-p4  0.421 12-p1   >100 12-p2  19.01 12-p3  8.628 12-p4  0.565 13-p1  188.1 13-p2  351.6 13-p3  176.0 13-p4  4.991 14-p1   >100 14-p2   >100 14-p3  40.35 14-p4  21.52 Ref. 1  2.942

    Embodiment 2: STING IFNβ Secretion Assay

    [0508] Plated 40 uL THP-1 cells (ATCC) as 16000 cells/well in a 96 well-plate (Corning, 3596). 3-folds dilution compounds were added to appropriate wells. The compounds were diluted with buffer (RPMI1640+2 mM L-glutamine+1× non-essential amino acids+1 mM sodium pyruvate+0.5% Fetal bovine serum), and the top dose of the compound was 100 μM, minimum dose was 1.23 μM. After 5 h incubation, 2 L supernant was added to 384-well plate (Greiner, Cat: 784075) and the secretion of IFN-β was detected with AllphaLISA IFN-β kit(PerkinElmer, Cat:AL577C). 1× buffer was prepared to dilute the receptor and donor, and then 4 μL 20 μg/mL Anti-pIFNβ AlphaLISA receptor was added to every well. After 30 mins incubation at room temperature, 4 μL 2 nM biotinylated Anti-pIFNβ antibody was added to a 384 well-plate, incubated overnight at 4° C. 10 μL 40 g/mL Streptavidin (SA) donor which was diluted with 1×buffer was added, and then incubated at room temperature for 30 min. Fluorescence was read with TECAN. The expressed level of IFN-β was proportional to the fluorescence intensity. Drew the curve with Graphpad Prism and analysed EC.sub.50 of the compounds.

    TABLE-US-00002 Compound No. EC.sub.50 (μM) 2-p4 47.64 4-p2 36.22 4-p3 94.39 6-p2 58.09 6-p3 9.84 6-p4 9.40

    Embodiment 3: Efficacy Study in a CT-26 Colon Tumor Xenograft Model in Mice

    [0509] Cell Culture: The CT26 colon tumor cells (ATCC) were maintained in vitro as a monolayer culture in RPMI1640 medium supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO.sub.2 in air. The tumor cells were routinely passaged twice per week by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

    [0510] Animals: BALB/c nude mice, 6-8 weeks, 19-22 g, animal supplier: Beijing Vital River Laboratory Animal Technology Co., Ltd.

    [0511] 4 groups were set up as follows:

    TABLE-US-00003 Group No. Treatment Dose Dosing Route Actual Schedule 1 8 Vehicle — i.t. Q3D × 3 doses 2 8 Ref. 1 2 mg/kg i.t. Q3D × 3 doses 3 8 Compound 6-p3 1 mg/kg i.t. Q3D × 3 doses 4 8 Compound 6-p3 2 mg/kg i.t. Q3D × 3 doses

    [0512] Note: i.t.: intra-tumor injection, Q3D: once every 3 days

    [0513] Experiment method: Each mouse was inoculated subcutaneously with CT-26 tumor cells (1×10.sup.5) in 0.1 mL over right flank region. The growth of tumor was observed regularly, when the tumor volume reached 150 mm.sup.3, mice were randomized based on tumor volume and body weight, and treated with schedule. The weight and tumor size of mice were measured 2-3 times a week during the whole experiment.


    Tumor volumes(mm.sup.3)=0.5×(long diameters of the tumorxshort diameters of the tumor.sup.2).  Tumor size formula:

    [0514] The tumor growth curves of different 4 groups are shown in FIG. 1. The result indicated that compared with the positive control Ref. 1, the compound of the disclosure can show better efficacy on the subcutaneous CT-26 colon tumor model in mice.

    [0515] Note: Ref. 1 used in embodiments of bioassays 1 and 3 was MLRR-CDA (ammonia salt), CASNo: 1638750-96-5, the synthesis method could refer to PCT patent application WO2014/189805A1 compound 22.