Oxazolo [4, 3-f] purine compound and application thereof in anticancer activity

Abstract

The invention discloses an oxazolo [4, 3-f] purine compound and application of the oxazolo [4, 3-f] purine compound in anti-cancer activity, and belongs to the technical field of medicinal chemistry. The oxazolo [4, 3-f] purine derivative is synthesized with high chemical selectivity by catalyzing asymmetric [3 + 2] cycloaddition, the anti-tumor activity of the oxazolo [4, 3-f] purine derivative is evaluated, and the compound 20b shows an obvious anti-tumor effect in vivo and has no obvious toxicity. The 5-fluorouracil and cis-platinum compound has strong proliferation inhibition ability in cervical cancer and colon cancer cell lines, IC50 reaches 0.69 [mu] M and 0.51 [mu] M and is obviously better than that of a control drug 5-fluorouracil and cis-platinum (IC50 is 4.48 [mu] M, 11.32 [mu] M, 4.02 [mu] M and 9.43 [mu] M respectively). The results show that 20b can be a novel targeted PPIA anti-cancer drug, and a foundation is laid for further development of novel anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the screening of lead compounds of oxazolo[4,3-f]purine derivatives, specifically to the synthesis of lead compounds of oxazolo[4,3-f]purine and their application in anti-cervical cancer and anti-colorectal cancer activities, belonging to the technical field of medicinal chemistry. Background Art

[0002] Cancer seriously threatens human life and health and is one of the main causes of human death. The research and development of small molecule anti-tumor drugs has always been an important direction in drug development. Artificially synthesized nucleoside compounds are structurally similar to natural nucleoside compounds and can inhibit the replication of viruses or tumor cells after entering the human body to achieve the purpose of anti-virus or anti-tumor.

[0003] People have paid increasing attention to the research and development of nucleoside anti-tumor and anti-viral drugs. Oxazolo[4,3-f]purine derivatives are a unique class of compounds among nucleoside compounds and have unique physical and chemical properties, and have become the focus of the research and development of polycyclic nucleoside drugs.

[0004] At present, there are relatively few studies on the anti-tumor activity of oxazolo[4,3-f]purine derivatives. More and more evidence shows that viral diseases are closely related to the occurrence of tumors. The innovative research on oxazolo[4,3-f]purine derivatives can not only break the international monopoly on nucleoside drugs, but also has important scientific significance and potential application prospects. Summary of the Invention

[0005] In order to provide more nucleoside lead compounds with functional structures, the present invention discloses lead compounds of oxazolo[4,3-f]purine derivatives with anti-cervical cancer and anti-colorectal cancer activities. In terms of the synthesis method: a series of oxazolo[4,3-f]purine derivatives were synthesized through reactions such as purine dearomatization reaction. This type of structure includes three structural types: racemate, 6S,8aS-configuration, and 6R,8aR-configuration.

[0006] The lead compound of a chiral oxazolo[4,3-f]purine derivative described in the present invention has the following general structural formula:

[0007] Wherein, * represents a chiral center, and each general formula structure includes three structures: racemate, 6S,8aS-configuration, and 6R,8aR-configuration.

[0008] Further, in the above technical solution, R 1 , R 2 are each independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, bis-C1-C4 alkylamino; R 3Selected from phenyl, naphthyl, furyl, thienyl or pyridyl biphenyl, and substituents of the foregoing groups; wherein the substituents are selected from one or more of halogen, trifluoromethyl, nitro, C1-C4 alkyl, C1-C4 alkoxy; R 4 and R 5 are each independently selected from C1-C6 carboxylate ester groups; R 6 is selected from hydrogen, C1-C6 alkyl.

[0009] Furthermore, in the above technical solution, the halogen is selected from fluorine, chlorine, bromine or iodine.

[0010] Furthermore, in the above technical solution, the R 1 is selected from hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, bis-C1-C4 alkylamino; R 2 is selected from hydrogen, chlorine; R 3 is ethyl carboxylate, methyl carboxylate, isopropyl carboxylate.

[0011] Furthermore, in the above technical solution, the R 1 and R 2 are both chlorine, R 3 is p-fluorophenyl, R 4 and R 5 are ethyl acetate; R 6 is methyl.

[0012] Furthermore, in the above technical solution, the lead compound is in 6S,8aS-configuration and 6R,8aR-configuration.

[0013] Furthermore, in the above technical solution, for the 6S,8aS-configuration and 6R,8aR-configuration, chiral HPLC is used to separate the racemic compound.

[0014] The second object of the present invention provides the application of the above-mentioned oxazolo[4,3-f]purine derivative lead compound in anti-cervical cancer and anti-colorectal cancer active drugs.

[0015] Furthermore, in the above technical solution, the anti-cervical cancer activity is anti-HeLa activity; the anti-colorectal cancer activity is anti-HCT116 activity.

[0016] The present invention also provides an anti-cervical cancer and anti-colorectal cancer active drug, the active ingredient of which comprises the above-mentioned chiral oxazolo[4,3-f]purine derivative lead compound and its pharmaceutically acceptable salts.

[0017] Furthermore, in the above technical solution, the pharmaceutically acceptable salts include salts formed by acyclic nucleoside compounds and organic acids or inorganic acids.

[0018] Further, in the above technical solution, the organic acid is selected from one or more of malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid or oxalic acid, and the inorganic acid is selected from one or more of phosphoric acid, hydrohalic acid, sulfuric acid or nitric acid.

[0019] Further, in the above technical solution, the anti-cervical cancer activity is the anti-HeLa activity; the anti-colon cancer activity is the anti-HCT116 activity.

[0020] The fourth object of the present invention provides a method for synthesizing the chiral oxazolo[4,3-f]purine derivative of the above structural general formula, comprising the following steps:

[0021] A: For the racemate, the reaction equation is:

[0022]

[0023] Reaction conditions: (a) sodium tert-butoxide, ethyl propiolate, DMF, rt, 6 h. (b) D-Aoxiranes, Ni(ClO4) . 6H2O (20 mol%), N2, chlorobenzene, 50 - 80 °C, 12 h. (c) THF / CH3OH, NaBH4 (6 eq) (Add in baches). (d) Et3N (10.0 eq), DCM, TsCl (12.0 eq).

[0024]

[0025] Reaction conditions: (a) PPh3, NaOAc, HOAc, ethyl propiolate, toluene, 10 °C, 12 h. (b) D-Aoxiranes, Ni(ClO4).6H2O (20 mol%), N2, chlorobenzene, 50 - 80 °C, 12 h.

[0026] B: For the 6S,8aS-configuration and 6R,8aR-configuration, chiral HPLC resolution is used.

[0027] In order to study the anti-tumor activity of the oxazolo[4,3-f]purine derivative, a series of oxazolo[4,3-f]purine derivatives with different substituents at the 2-position / 6-position of the purine and the 1'-position of the sugar ring were synthesized through two-step Michael addition reactions and other reactions.

[0028]

[0029] The experimental results show that the lead compound 20b of oxazolo[4,3-f]purine derivatives exhibits strong ability to inhibit the proliferation of cervical cancer and colon cancer cell lines in vitro, with IC 50 reaching 0.69 μM and 0.51 μM, significantly better than the control drugs 5-fluorouracil (5-FU) and cisplatin (IC 50 being 4.48 μM and 11.32 μM, 4.02 μM and 9.43 μM respectively). The above results provide the basic structural unit of the lead compound of oxazolo[4,3-f]purine derivatives for the design of drugs with anti-cervical cancer and colon cancer activities, and further fine structural modification and transformation can be carried out on this basis.

[0030] Through the research on the synthesis and in vitro and in vivo anti-tumor activity evaluation of oxazolo[4,3-f]purine derivatives, anti-tumor lead compounds or preferred drug molecules with good activity are obtained, providing reasonable synthesis strategies and methods for oxazolo[4,3-f]purine derivative drugs, and laying a good foundation for the development of anti-tumor oxazolo[4,3-f]purine derivative nucleoside drugs with independent intellectual property rights. Description of the Drawings

[0031] Figure 1 It is the figure of the inhibition of HCT116 cell proliferation by compound 20b in Example 5; among them, A is the cell colony experiment, and B is the EdU assay for detecting the uptake of EdU by HCT116 cells within 48 hours.

[0032] Figure 2 It is the figure of the cell cycle and apoptosis experiment in Example 5; among them, A - B is the cell cycle distribution map measured by flow cytometry after treating HCT116 cells with 20b for 48 hours, C - D is the figure of the change in cell cycle-related protein levels by compound 20b, E - F is the figure of the apoptosis rate of HCT116 cells treated with 20b for 48 hours measured by flow cytometry, and G - H is the figure of the change in apoptosis-related protein levels by compound 20b.

[0033] Figure 3Figure showing the anti - proliferation and anti - migration activities induced by silencing PPIA - damaged compound 20b in HCT116 cells in Example 5; where A - B are Western blot analyses of PPIA expression levels, C is the detection of the anti - proliferation activity of 20b on siNC and siPPIA - 3 HCT116 cells using the CCK - 8 method, D - E are cell colony formation assays for detecting the proliferation of HCT116 cells treated with 20b and siPPIA - 3; F - H are evaluations of cell migration by wound healing of HCT116 cells treated with 20b and siPPIA - 3; I is the detection of the effect of 20b on the interaction between PPIA and CD147 by Co - IP; J - K are Western blot analysis diagrams of the expression levels of p - p38, p - JNK, and p - ERK1 / 2 in HCT116 cells treated with 20b and siPPIA - 3.

[0034] Figure 4 Figure showing the interaction between compound 20b and PPIA in Example 5; A - B are Western blot diagrams of PPIA after DARTS assay, C - D are diagrams of compound 20b or CsA binding to PPIA in the CETSA experiment, E is a diagram for determining the KD value between compound 20b and PPIA by SPR analysis, F - G are molecular docking diagrams of compound 20b and PPIA (PDB ID: 4N1R), H - I are Western blot analysis diagrams of the expression levels of p - p38, p - JNK, and p - ERK1 / 2 in HCT116 cells treated with 20b.

[0035] Figure 5 Figure of mouse xenograft tumors in Example 5, where A is the diagram of tumor weight, B is the diagram of body weight, C is the diagram of tumor volume, and D is the diagram of tumor size.

[0036] Figure 6 Figure of H&E staining in Example 5. Detailed implementation methods

[0037] Example 1:

[0038] Representative test operations: All reactions for synthesizing chiral purine - fused ring compounds were carried out in a dry 10 mL reaction tube. The molecular sieve used in the reaction was activated at 500 °C in a muffle furnace for 4 h and used at room temperature after recovery. Solvents used in the experiment: Dry reagents were commercially available ultra - dry solvents, and for those without commercially available dry solvents, they were dried with CaH2. The thin - layer silica gel plate used for separation was GF254 silica gel plate, and the silica gel for column chromatography was 200 - 300 mesh.

[0039] Synthesis of oxazolo[4,3 - f]purine derivatives of series 12, 13, 14, 15, 17, 19, and 20:

[0040] Into a 10 mL dry reaction tube equipped with a magnetic stir bar, add 0.1 mmol of purine compound 11 or 16 or 18 and 0.2 mmol of epoxide b, then add activated molecular sieve, and then add Ni(ClO4)2-6H2O catalyst (20 mol%). Seal the reaction tube with a rubber stopper, evacuate and backfill with nitrogen three times. Use a syringe to add 2 mL of chlorobenzene to the reaction mixture. React at 80 °C until starting material 11 is completely consumed. Monitor the reaction progress by TLC. After the reaction is complete, obtain the product 12 or 13 or 14 or 15 or 17 or 19 or 20 series of compounds by column chromatography using PE and EA as eluents.

[0041] Add the compound of series 12 or 13 or 14 or 15 (0.1 mmol) to the reaction tube and dissolve it in THF / CH3OH (v / v = 1:1, 1.0 mL). Subsequently, add NaBH4 (10.0 eq) to the reaction mixture all at once. Stir the reaction mixture at room temperature until 12 or 13 or 14 or 15 is completely consumed. Then add saturated aqueous NH4Cl solution (0.5 mL) to the reaction system and extract with ethyl acetate (3 × 5 mL). Combine the organic phases and concentrate under reduced pressure. Purify the mixture by column chromatography (PE / EA = 3 / 1) to obtain products 14a, 14b, 14f.

[0042]

[0043] Reaction conditions: (a) sodium tert-butoxide, ethyl propiolate, DMF, rt, 6 h. (b) D-Aoxiranes, Ni(ClO4)-6H2O (20 mol%), N2, chlorobenzene, 50 - 80 °C, 12 h. (c) THF / CH3OH, NaBH4 (6 eq) (Add in batches). (d) Et3N (10.0 eq), DCM, TsCl (12.0 eq).

[0044]

[0045] Reaction conditions: (a) PPh3, NaOAc, HOAc, ethyl propiolate, toluene, 10 °C, 12 h. (b) D-Aoxiranes, Ni(ClO4).6H2O (20 mol%), N2, chlorobenzene, 50 - 80 °C, 12 h.

[0046] Characterization data of representative compounds are as follows:

[0047] General Procedure for the Synthesis of Compounds. (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate 12a. exemplifies the pattern found in 12a-12i, 13a-13c, 14c-14e, 15a-15l, and 17. To a mixture of ethyl (E)-3-(9H-purin-9-yl)acrylate (218 mg, 1 mM) and diethyl 3-phenyloxirane-2,2-dicarboxylate (317 mg, 1.2 mM) in chlorobenzene (10 mL), Nickel perchlorate hexahydrate (73 mg, 0.2 mM) and (300 mg) was added, and the resulting mixture was stirred at 70℃ under N2 for 12 h. The reaction was detected by TLC, the solvent was removed and purified by flash column chromatography on silica gel (PE / EA=2 / 1) to afford a colorless oil in 43% yield. 1 1H NMR (600 MHz, CDCl3) δ 8.50 (s, 1H), 8.13 (d, J=14.4 Hz, 1H), 7.97 (s, 1H), 7.52 - 7.51 (m, 2H), 7.41 - 7.40 (m, 3H), 6.58 (s, 1H), 6.38 (s, 1H), 5.95 (d, J=14.2 Hz, 1H), 4.24 - 4.15 (m, 3H), 4.13 - 4.09 (m, 1H), 4.06 - 4.01 (m, 2H), 1.28 (t, J=7.2 Hz, 3H), 1.20 (t, J=7.2 Hz, 3H), 1.08 (t, J=7.2 Hz, 3H). 1313C NMR (150 MHz, CDCl3) δ 167.2, 166.6, 165.5, 155.3, 152.9, 136.4, 135.9, 135.2, 129.7, 128.9, 126.7, 101.6, 97.6, 85.9, 83.7, 63.2, 63.1, 60.3, 14.5, 13.8, 13.8. ESI-HRMS (m / z) calcd C 24 H 24 ClFN4O7[M + Na] + , 505.1694; found 505.1699.

[0048] (rac) Diethyl (E)-4-chloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate 12b. It is obtained as a white solid in 38% yield. 1 1H NMR (600 MHz, CDCl3) δ 8.30 (s, 1H), 8.09 (d, J = 14.4 Hz, 1H), 7.56 (s, 2H), 7.39 (s, 3H), 7.09 (s, 1H), 6.42 (s, 1H), 5.92 (d, J = 14.4 Hz, 1H), 4.25 - 4.10 (m, 4H), 4.07 - 3.86 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H), 1.21 (t, J = 7.2 Hz, 3H), 1.11 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.9, 166.85, 165.3, 156.1, 152.6, 139.8, 136.1, 135.6, 129.8, 128.7, 128.5, 126.8, 102.1, 95.3, 86.5, 83.6, 63.4, 63.0, 60.4, 14.5, 13.9, 13.8. ESI-HRMS (m / z) calcd C 24 H 25 ClN4NaO7 (M + Na) + , 539.1304; found, 539.1306.

[0049] (rac)Diethyl (E)-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-4-methoxy-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (12c). It is obtained as a white solid in 41% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 8.12 (d, J = 12.4 Hz, 1H), 7.56 - 7.54 (m, 2H), 7.38 - 7.36 (m, 3H), 6.94 (s, 1H), 6.38 (s, 1H), 5.74 (d, J = 17.2 Hz, 1H), 4.30 - 4.13 (m, 4H), 4.09 - 4.03 (m, 1H), 4.00 (s, 3H), 3.95 - 3.89 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 1.17 (t, J = 7.2 Hz, 3H), 1.11 (t, J = 6.8 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 167.5, 167.3, 165.5, 156.1, 155.2, 152.4, 136.9, 129.4, 128.6, 126.9, 115.8, 99.4, 95.8, 86.8, 84.1, 63.1, 62.7, 60.1, 54.2, 14.6, 13.9, 13.8. ESI-HR MS (m / z) calcd C 25 H 28 N4NaO8 (M + Na) + , 535.1799; found, 535.1797.

[0050] (rac)Diethyl (E)-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-phenyl-4-(propylthio)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (12d). It is obtained as a white solid in 39% yield. 11H NMR (600 MHz, CDCl3) δ 8.35 (s, 1H), 8.10 (d, J = 15.0 Hz, 1H), 7.60 - 7.59 (m, 2H), 7.38 - 7.37 (m, 3H), 7.08 (s, 1H), 6.38 (s, 1H), 5.78 (d, J = 14.4 Hz, 1H), 4.23 - 3.90 (m, 7H), 3.22 (t, J = 7.2 Hz, 2H), 1.71 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H), 1.17 (t, J = 7.2 Hz, 3H), 1.12 (t, J = 6.0 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 167.2, 167.2, 165.3, 152.9, 152.6, 149.9, 136.53, 136.49, 129.6, 129.5, 128.5, 127.8, 127.0, 100.3, 94.8, 86.7, 83.6, 63.2, 62.8, 60.2, 31.5, 23.1, 14.5, 13.84, 13.80, 13.5. ESI - HRMS (m / z) calcd C 24 H 24 BrClN4O7 (M + H) + , 557.2064; found, 557.2068.

[0051] (rac) Diethyl (E)-2,4 - dichloro - 9-(3 - ethoxy - 3 - oxoprop - 1 - en - 1 - yl)-6 - phenyl - 8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8,8 - dicarboxylate (12e). It is obtained as a white solid in 47% yield. 11H NMR (600 MHz, CDCl3) δ 8.01 (d, J = 13.8 Hz, 1H), 7.54 - 7.53 (m, 2H), 7.39 - 7.38 (m, 3H), 7.02 (s, 1H), 6.43 (s, 1H), 5.93 (d, J = 14.4 Hz, 1H), 4.25 - 4.16 (m, 3H), 4.15 - 4.10 (m, 1H), 4.08 - 4.03 (m, 1H), 4.00 - 3.94 (m, 3H), 1.28 (t, J = 7.2 Hz, 3H), 1.25 (s, 2H), 1.20 (t, J = 7.2 Hz, 3H), 1.13 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.7, 166.5, 165.1, 157.6, 152.9, 139.6, 135.8, 134.9, 129.9, 128.7, 127.8, 126.8, 103.5, 95.3, 86.4, 84.4, 63.5, 63.1, 60.6, 29.8, 14.5, 13.82, 13.78. ESI - HRMS (m / z) calcd C 24 H 24 Cl2N4NaO7 (M + Na) + , 573.0914; found, 573.0915.

[0052] (rac) Diethyl (E)-2-chloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-4-methoxy-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (12f). It is obtained as a white solid in 36% yield. 1 1H NMR (600 MHz, CDCl3) δ 7.94 (d, J = 14.4 Hz, 1H), 7.43 - 7.42 (m, 2H), 7.27 - 7.26 (m, 3H), 7.17 (s, 1H), 6.77 (s, 1H), 6.30 (s, 1H), 5.63 (d, J = 13.8 Hz, 1H), 4.13 - 4.05 (m, 4H), 3.98 - 3.93 (m, 1H), 3.91 (s, 3H), 3.86 - 3.79 (m, 1H), 1.17 (t, J = 4.8 Hz, 3H), 1.07 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H). 1313C NMR (150 MHz, CDCl3) δ 167.1, 167.0, 165.3, 156.6, 156.3, 152.7, 136.5, 136.2, 129.5, 128.6, 126.8, 114.8, 100.6, 95.7, 86.7, 84.8, 63.3, 62.8, 60.2, 55.0, 14.5, 13.82, 13.77. ESI-HRMS (m / z) calcd C 25 H 27 ClN4NaO8 (M+Na) + , 569.1410; found, 569.1415.

[0053] (rac) Diethyl (E)-2-chloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-phenyl-4-(propylthio)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (12g). It is obtained as a white solid in 51% yield. 1 1H NMR (600 MHz, CDCl3) δ 7.97 (d, J=14.4 Hz, 1H), 7.52 - 7.50 (m, 2H), 7.31 - 7.30 (m, 3H), 6.95 (s, 1H), 6.35 (s, 1H), 5.74 (d, J=13.8 Hz, 1H), 4.16 - 4.08 (m, 3H), 4.04 - 3.94 (m, 2H), 3.89 - 3.83 (m, 1H), 3.16 - 3.08 (m, 2H), 1.66 - 1.60 (m, 2H), 1.20 (t, J=7.2 Hz, 3H), 1.12 (t, J=7.2 Hz, 3H), 1.07 (t, J=7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.8, 166.5, 164.8, 154.2, 153.2, 151.2, 135.9, 135.5, 129.4, 128.3, 126.67, 126.55, 101.2, 94.5, 86.4, 84.2, 63.0, 62.6, 62.3, 60.0, 31.6, 22.5, 14.2, 13.5 (d, J C-F =9.75 Hz), 13.2. ESI-HRMS (m / z) calcd C 27 H 32 ClN4O7S (M+H) +,591.1675; found,591.1672.

[0054] (rac)Diethyl (E)-4-amino-2-chloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (12h). It is obtained as a white solid in 45% yield. 1 H NMR (600 MHz, CDCl3) δ 8.04 (d, J = 14.4 Hz, 1H), 7.60 - 7.59 (m, 2H), 7.44 - 7.35 (m, 3H), 6.90 (s, 1H), 6.39 (s, 1H), 5.79 (d, J = 14.4 Hz, 1H), 5.09 (s, 2H), 4.24 - 4.14 (m, 3H), 4.13 - 3.89 (m, 3H), 1.27 (t, J = 7.2 Hz, 3H), 1.21 - 1.12 (m, 6H). 13 C NMR (150 MHz, CDCl3) δ 167.2, 165.4, 159.1, 157.5, 151.3, 141.2, 136.8, 136.4, 129.5, 129.0, 128.5, 127.6, 127.0, 120.1, 111.9, 97.4, 86.6, 84.3, 63.0, 62.8, 60.3, 14.6, 13.0. ESI-HRMS (m / z) calcd C 24 H 27 ClN5O7 (M + H) + ,532.1594; found,532.1596.

[0055] (rac)Diethyl (E)-4-chloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-2-fluoro-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (12i). It is obtained as a white solid in 45% yield. 11H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 14.0 Hz, 1H), 7.56 - 7.54 (m, 2H), 7.40 - 7.38 (m, 3H), 7.02 (s, 1H), 6.48 (s, 1H), 5.95 (d, J = 14.4 Hz, 1H), 4.27 - 4.15 (m, 3H), 4.13 - 4.02 (m, 2H), 4.00 - 3.92 (m, 1H), 1.29 (t, J = 7.2 Hz, 3H), 1.20 (t, J = 6.8 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.8, 166.4, 165.1, 135.9, 134.8, 129.9, 128.7, 126.9, 103.8, 95.7, 86.5, 85.0, 63.6, 63.1, 60.6, 14.5, 13.8. ESI - HRMS (m / z) calcd C 24 H 24 ClFN4NaO7 (M + Na) + , 557.1210; found, 557.1208.

[0056] (rac) Diethyl (E)-2,4 - dichloro - 9-(3 - methoxy - 3 - oxoprop - 1 - en - 1 - yl)-6 - phenyl - 8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8 - carboxylate (13a). It is obtained as a white solid in 31% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 14.4 Hz, 1H), 7.55 - 7.53 (m, 2H), 7.40 - 7.38 (m, 3H), 7.03 (s, 1H), 6.43 (s, 1H), 5.96 (d, J = 14.4 Hz, 1H), 4.23 - 4.11 (m, 2H), 4.09 - 3.94 (m, 2H), 3.74 (s, 3H), 1.20 (t, J = 6.8 Hz, 3H), 1.13 (t, J = 7.6 Hz, 3H). 1313C NMR (100 MHz, CDCl3) δ 166.9, 166.7, 165.1, 157.5, 153.0, 139.9, 135.8, 135.1, 129.9, 128.8, 127.8, 126.8, 103.0, 95.4, 86.5, 84.4, 63.6, 63.1, 51.8, 13.9, 13.8. ESI-HRMS (m / z) calcd C 23 H 22 Cl2N4NaO7 (M+Na) + , 559.0758; found, 559.0750.

[0057] (rac) Diethyl (E)-9-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-2,4-dichloro-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (13b). It is obtained as a white solid in 53% yield. 1 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 14.4 Hz, 1H), 7.55 - 7.51 (m, 2H), 7.40 - 7.38 (m, 3H), 7.02 (s, 1H), 6.42 (s, 1H), 5.85 (d, J = 14.4 Hz, 1H), 4.23 - 3.93 (m, 4H), 1.48 (s, 9H), 1.22 - 1.13 (m, 6H). 13 13C NMR (150 MHz, CDCl3) δ 166.7, 165.8, 165.1, 157.7, 153.0, 139.5, 135.8, 134.2, 129.9, 128.7, 127.7, 126.8, 105.4, 95.3, 86.5, 84.4, 80.8, 63.5, 63.1, 28.3, 13.8, 13.7. ESI-HRMS (m / z) calcd C 26 H 28 Cl2N4NaO7 (M+Na) + , 601.1227; found, 601.1226.

[0058] (rac)Diethyl (E)-9-(3-(benzyloxy)-3-oxoprop-1-en-1-yl)-2,4-dichloro-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8-carboxylate (13c). It is obtained as a white solid in 48% yield. 1 1H NMR (600 MHz, CDCl3) δ 8.09 (d, J = 14.4 Hz, 1H), 7.48 - 7.47 (m, 2H), 7.42 - 7.32 (m, 8H), 6.46 (s, 1H), 5.99 (d, J = 14.4 Hz, 1H), 5.77 (s, 1H), 5.23 (s, 2H), 4.52 - 4.46 (m, 1H), 4.41 - 4.36 (m, 1H), 4.26 - 4.19 (m, 1H), 4.10 - 4.04 (m, 1H), 1.36 (t, J = 7.2 Hz, 3H), 1.25 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 167.2, 166.4, 164.6, 153.1, 145.0, 135.7, 131.4, 131.3, 128.7, 128.5, 128.4, 125.2, 102.4, 95.9, 86.4, 85.1, 66.4, 63.7, 63.6, 29.8, 14.1, 13.8. ESI-HRMS (m / z) calcd C 29 H 26 Cl2N4O7 + (M + H) + , 612.1179; found, 612.1184.

[0059] (rac)Ethyl (E)-3-(2,4-dichloro-8-(hydroxymethyl)-6-phenyl-8,8a-dihydro-6H,9H-oxazolo[4,3-f]purin-9-yl)acrylate (14a). 12e (0.1 mM) was dissolved in THF / CH3OH (v / v, 1:1, 1.0 mL). Then, NaBH4 (6.0 eq) was added in batches. The reaction mixture was stirred at room temperature, the reaction was detected by TLC. Then, the saturated NH4Cl aqueous solution (0.5 mL) was added. The aqueous phase was extracted with EA (3×5 mL) and the combined organic phases were concentrated. The residue was purified by flash column chromatography on silica gel (PE / EA=2 / 1) to afford a colorless oil in 38% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J=14.4 Hz, 1H), 7.60 - 7.58 (m, 2H), 7.46 - 7.42 (m, 3H), 6.25 (s, 1H), 5.75 - 5.71 (m, 2H), 4.25 - 4.18 (m, 3H), 3.75 - 3.64 (m, 2H), 2.0 (s, 1H), 1.30 (t, J=7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.7, 157.7, 153.1, 138.7, 134.9, 129.7, 129.2, 128.9, 126.6, 101.4, 95.8, 82.5, 81.3, 61.8, 60.7, 14.5. ESI-HRMS (m / z) calcd C 19 H 18 Cl2N4NaO4 (M+Na) + , 459.0597;found, 459.0597.

[0060] (rac)Ethyl (E)-3-(2,4-dichloro-8,8-bis(hydroxymethyl)-6-phenyl-8,8a-dihydro-6H,9H-oxazolo[4,3-f]purin-9-yl)acrylate 14b.12e (0.1 mM) was dissolved in THF / CH3OH (v / v, 1:1, 1.0 mL). Then, NaBH4 (10.0 eq) was added in batches. The reaction mixture was stirred at room temperature, the reaction was detected by TLC. Then, the saturated NH4Cl aqueous solution (0.5 mL) was added. The aqueous phase was extracted with EA (3×5 mL) and the combined organic phases were concentrated. The residue was purified by flash column chromatography on silica gel (PE / EA=2 / 1) to afford a colorless oil in 51% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.15 (d, J=14.4 Hz, 1H), 7.57 - 7.53 (m, 2H), 7.46 - 7.39 (m, 3H), 6.51 (s, 1H), 5.90 (s, 1H), 5.74 (d, J=14.4 Hz, 1H), 4.30 - 4.11 (m, 2H), 3.75 - 3.59 (m, 4H), 2.16 (s, 1H), 2.00 (s, 1H), 1.30 (t, J=7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 167.0, 158.4, 151.9, 138.3, 135.9, 129.6, 129.0, 126.6, 101.1, 94.3, 86.8, 82.4, 63.3, 62.6, 60.8, 14.5. ESI-HRMS (m / z) calcd C 20 H 20 Cl2N4O5Na + [M+Na] +,489.0703; found 489.0707.

[0061] (rac)Ethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8-carboxylate (14c). It is obtained as a white solid in 56% yield. 1 H NMR (600 MHz, CDCl3) δ 8.06 (d, J = 12 Hz, 1H), 7.39 - 7.31 (m, 5H), 7.06 (s, 1H), 5.97 (d, J = 12 Hz, 1H), 5.22 (d, J = 6 Hz, 1H), 4.81 (d, J = 6 Hz, 1H), 4.26 - 4.22 (m, 2H), 3.88 - 3.83 (m, 1H), 3.74 - 3.69 (m, 1H), 1.31 (t, J = 6 Hz, 3H), 0.89 (t, J = 6 Hz, 3H). 13 C NMR (150 MHz, CDCl3) δ 167.1, 166.3, 156.6, 153.1, 146.9, 140.6, 135.3, 133.9, 129.0, 128.7, 127.9, 127.3, 104.1, 104.0, 78.8, 67.8, 61.8, 60.7, 14.4, 13.7. ESI-HRMS (m / z) calcd C 22 H 20 Cl2N4NaO7 (M+Na) + ,545.0601; found, 545.0602.

[0062] (rac)Dimethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (14d). It is obtained as a white solid in 42% yield. 11H NMR (600 MHz, CDCl3) δ 8.06 (d, J = 12 Hz, 1H), 7.39 - 7.31 (m, 5H), 7.06 (s, 1H), 5.97 (d, J = 12 Hz, 1H), 5.22 (d, J = 6 Hz, 1H), 4.81 (d, J = 6 Hz, 1H), 4.26 - 4.22 (m, 2H), 3.88 - 3.83 (m, 1H), 3.74 - 3.69 (m, 1H), 1.31 (t, J = 6 Hz, 3H), 0.89 (t, J = 6 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 167.1, 166.3, 156.6, 153.1, 146.9, 140.6, 135.3, 133.9, 129.0, 128.7, 127.9, 127.3, 104.1, 104.0, 78.8, 67.8, 61.8, 60.7, 14.4, 13.7. ESI-HRMS (m / z) calcd C 21 H 20 Cl2N4NaO5 (M + Na) + , 501.0703; found, 50210701.

[0063] (rac) Dibenzyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (14e). It is obtained as a white solid in 46% yield. 1 1H NMR (600 MHz, CDCl3) δ 7.85 (d, J = 12 Hz, 1H), 7.48 (d, J = 6 Hz, 1H), 7.38 - 7.29 (m, 9H), 7.22 (d, J = 6 Hz, 2H), 7.12 (d, J = 6 Hz, 2H), 7.00 (s, 1H), 6.42 (s, 1H), 5.85 (d, J = 12 Hz, 1H), 5.09 (t, J = 12 Hz, 2H), 4.94 (d, J = 12 Hz, 1H), 4.88 (d, J = 12 Hz, 1H), 4.22 - 4.13 (m, 2H), 1.28 (t, J = 6 Hz, 3H). 1313C NMR (150 MHz, CDCl3) δ 166.5, 166.4, 165.1, 157.4, 152.9, 139.4, 135.8, 134.9, 134.2, 133.5, 129.9, 129.2, 128.9, 128.8, 128.8, 127.6, 126.7, 103.4, 95.4, 86.5, 84.3, 69.1, 68.7, 60.5, 14.5. ESI-HRMS (m / z) calcd C 34 H 29 Cl2N4O7 (M + H) + , 675.1408; found, 675.1415.

[0064] (rac) Ethyl (E)-3-(2,4-dichloro-6-phenyl-8,8-bis((tosyloxy)methyl)-8,8a-dihydro-6H,9H-oxazolo[4,3-f]purin-9-yl)acrylate (14f). It is obtained as a colorless oil in 54% yield. 1 1H NMR (400 MHz, CDCl3) δ 7.90 (d, J = 14.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.44 - 7.38 (m, 4H), 7.33 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 6.39 (s, 1H), 5.64 (d, J = 14.4 Hz, 1H), 5.56 (s, 1H), 4.26 - 4.16 (m, 3H), 4.02 (d, J = 10.8 Hz, 1H), 3.87 (d, J = 11.2 Hz, 1H), 3.69 (d, J = 10.8 Hz, 1H), 2.47 (s, 3H), 2.44 (s, 3H), 1.32 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.2, 157.7, 152.3, 146.0, 138.9, 137.8, 135.1, 131.9, 131.4, 130.3, 130.2, 129.7, 129.0, 128.1, 127.8, 126.3, 102.0, 94.0, 83.3, 83.2, 68.3, 66.9, 60.7, 21.9, 21.9, 14.5. ESI-HRMS (m / z) calcd C 34 H 32Cl2N4NaO7S2(M+Na) + , 797.0880; found, 797.0879.

[0065] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(naphthalen-1-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15a). It is obtained as a white solid in 39% yield. 1 1H NMR (600 MHz, CDCl3) δ 8.05 - 8.01 (m, 2H), 7.88 - 7.85 (m, 3H), 7.62 (d, J = 8.4 Hz, 1H), 7.55 - 7.53 (m, 2H), 7.17 (s, 1H), 6.51 (s, 1H), 5.95 (d, J = 14.4 Hz, 1H), 4.25 - 4.17 (m, 3H), 4.10 - 4.02 (m, 3H), 3.89 - 3.84 (m, 1H), 1.28 (t, J = 4.8 Hz, 3H), 1.14 (q, J = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 166.7, 166.5, 165.1, 157.6, 153.0, 139.7, 134.9, 133.9, 133.1, 132.8, 128.7, 128.5, 127.9, 127.3, 126.9, 126.3, 124.1, 103.6, 95.6, 86.5, 84.5, 63.6, 63.1, 60.6, 14.5, 13.81, 13.77. ESI-HRMS (m / z) calcd C 28 H 26 Cl2N4NaO7(M+Na) + , 623.1071; found, 623.1070.

[0066] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(naphthalen-2-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15b). It is obtained as a white solid in 39% yield. 1 H NMR (600 MHz, CDCl3) δ 8.05 - 8.01 (m, 2H), 7.88 - 7.84 (m, 3H), 7.63 - 7.61 (m, 1H), 7.55 - 7.53 (m, 2H), 7.17 (s, 1H), 6.51 (s, 1H), 5.95 (d, J=14.4 Hz, 1H), 4.25 - 4.17 (m, 3H), 4.10 - 4.02 (m, 2H), 3.89 - 3.84 (m, 1H), 1.28 (t, J=6.6 Hz, 6H), 1.16 - 1.12 (m, 6H). 13 C NMR (150 MHz, CDCl3) δ 166.7, 166.5, 165.1, 157.6, 153.0, 139.7, 134.9, 133.8, 133.1, 132.8, 128.7, 128.5, 127.87, 127.84, 127.3, 126.9, 126.3, 124.1, 103.6, 95.6, 86.5, 84.5, 63.6, 63.1, 60.6, 14.5, 13.81, 13.77. ESI-HRMS (m / z) calcd C 28 H 26 Cl2N4NaO7 (M + Na) + , 623.1071;found, 623.1070.

[0067] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(4-(pyridin-2-yl)phenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15c). It is obtained as a white solid in 26% yield. 11H NMR (400 MHz, CDCl3) δ 8.71 (dd, J = 12.0 Hz, 4.4 Hz, 2H), 8.12 - 8.10 (m, 2H), 7.98 (q, J = 9.2 Hz, 2H), 7.79 - 7.71 (m, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.28 - 7.22 (m, 2H), 6.16 (s, 1H), 5.99 (s, 1H), 4.50 - 4.41 (m, 1H), 4.36 - 4.28 (m, 1H), 3.87 - 3.79 (m, 1H), 3.68 - 3.60 (m, 1H), 1.34 (t, J = 7.2 Hz, 3H), 1.25 (s, 3H), 0.87 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 168.0, 166.7, 157.1, 149.9, 141.4, 139.9, 137.0, 135.9, 135.3, 128.6, 127.6, 127.2, 126.8, 122.5, 121.0, 120.7, 105.3, 87.0, 83.6, 62.7, 62.1, 14.1, 13.6.

[0068] (rac) Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(thiophen-2-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15d). It is obtained as a colorless oil in 37% yield. 1 1H NMR (600 MHz, CDCl3) δ 8.01 (d, J = 14.4 Hz, 1H), 7.33 (dd, J = 4.8 Hz, J = 1.2 Hz, 1H), 7.08 (d, J = 3.6 Hz, 1H), 7.04 (s, 1H), 7.02 (dd, J = 7.2 Hz, J = 3.6 Hz, 1H), 6.17 (s, 1H), 6.05 (d, J = 14.4 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 4.12 (qd, J = 7.2, 1.2 Hz, 2H), 4.05 - 3.94 (m, 2H), 1.33 (t, J = 7.2 Hz, 3H), 1.13 (t, J = 7.2 Hz, 3H), 1.02 (t, J = 7.2 Hz, 3H). 1313C NMR (150 MHz, CDCl3) δ 166.3, 166.3, 163.9, 155.9, 153.0, 140.6, 137.5, 133.8, 127.3, 127.3, 127.1, 126.5, 104.5, 88.6, 64.8, 63.7, 63.0, 60.8, 14.5, 13.9, 13.8. ESI-HRMS (m / z) calcd C 22 H 22 Cl2N4NaO7S (M+Na) + , 579.0478; found, 579.0480.

[0069] (rac) Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(2-fluorophenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15e-1). It is obtained as a white solid in 42% yield. 1 1H NMR (600 MHz, CDCl3) δ 8.02 (d, J = 14.4 Hz, 1H), 7.45 - 7.40 (m, 2H), 7.17 (t, J = 7.2 Hz, 1H), 7.12 (t, J = 10.8 Hz, 1H), 6.94 (s, 1H), 6.51 (s, 1H), 6.12 (d, J = 14.4 Hz, 1H), 4.26 - 4.14 (m, 4H), 4.10 - 4.02 (m, 2H), 1.30 (t, J = 7.2 Hz, 3H), 1.18 (t, J = 7.2 Hz, 3H), 1.13 (t, J = 6.6 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.6, 166.1, 165.4, 162.0, 160.3, 157.6, 153.0, 139.3, 135.0, 132.1 (d, J C-F = 8.7 Hz), 129.0, 128.0, 124.2 (d, J C-F = 3.3 Hz), 123.95, 123.87, 116.4, 116.3, 103.9, 93.8, 86.6, 84.6 (d, J C-F = 3.3 Hz), 63.5, 63.3, 60.6, 14.5, 13.84, 13.78. ESI-HRMS (m / z) calcd C 24 H23 Cl2FN4NaO7(M+Na) + , 591.0820; found, 591.0822.

[0070] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(3-fluorophenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15e-2), It is obtained as a colorless oil in 48% yield. 1 H NMR (400 MHz, CDCl3) δ 8.00 (d, J=14.4 Hz, 1H), 7.40 - 7.33 (m, 2H), 7.28 - 7.25 (m, 1H), 7.12 - 7.07 (m, 1H), 6.99 (s, 1H), 6.40 (s, 1H), 5.93 (d, J=14.4 Hz, 1H), 4.27 - 4.14 (m, 4H), 4.10 - 4.02 (m, 2H), 1.28 (t, J=7.2 Hz, 3H), 1.24 (t, J=7.2 Hz, 3H), 1.13 (t, J=7.2 Hz, 3H). 13 C NMR (150 MHz, CDCl3) δ 166.6, 166.4, 164.9, 163.7, 157.5, 153.2, 139.7, 138.4 (d, J C-F =6.0 Hz), 134.8, 130.5 (d, J C-F =9.0 Hz), 127.5, 122.4 (d, J C-F =4.5 Hz), 116.9 (d, J C-F =21.0 Hz), 114.0 (d, J C-F =22.5 Hz), 103.7, 94.6 86.5, 84.2, 63.6, 63.3, 60.6, 14.5, 13.82, 138.80. ESI-HRMS (m / z) calcd C 24 H 23 Cl2FN4NaO7(M+Na) + , 591.0820; found, 591.0815.

[0071] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(4-fluorophenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15e-3). It is obtained as a white solid in 36% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 14.0 Hz, 1H), 7.55 - 7.52 (m, 2H), 7.08 (t, J = 8.8 Hz, 2H), 6.97 (s, 1H), 6.41 (s, 1H), 5.94 (d, J = 14.0 Hz, 1H), 4.26 - 4.14 (m, 4H), 4.08 - 3.99 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H), 1.23 (t, J = 7.2 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.6, 166.5, 165.0, 157.6, 153.1, 139.7, 134.9, 131.8 (d, J C-F = 3.0 Hz), 129.2 (d, J C-F = 9.0 Hz), 115.7 (d, J C-F = 21.0 Hz), 103.6, 95.0, 86.5, 84.3, 63.6, 63.2, 60.6, 14.5, 13.9, 13.8. ESI-HRMS (m / z) calcd C 24 H 23 Cl2FN4NaO7 (M + Na) + , 591.0820; found, 591.0822.

[0072] (rac)Diethyl (E)-2,4-dichloro-6-(2-chlorophenyl)-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15f-1). It is obtained as a white solid in 43% yield. 11H NMR (600 MHz, CDCl3) δ 8.04 (d, J = 14.4 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.46 - 7.45 (m, 1H), 7.37 (t, J = 6.0 Hz, 1H), 7.31 (t, J = 8.4 Hz, 1H), 7.05 (s, 1H), 6.59 (s, 1H), 6.06 (d, J = 14.4 Hz, 1H), 4.27 - 4.16 (m, 3H), 3.94 - 3.89 (m, 1H), 1.30 (t, J = 7.2 Hz, 3H), 1.15 (t, J = 7.2 Hz, 3H), 1.11 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.5, 166.4, 165.0, 157.7, 153.3, 139.9, 134.9, 134.5, 133.3, 131.4, 130.6, 128.6, 128.0, 127.0, 103.7, 95.1, 86.4, 85.2, 63.6, 63.3, 60.6, 14.5, 13.8, 13.7. ESI - HRMS (m / z) calcd C 24 H 23 Cl3N4NaO7 (M + Na) + , 607.0525; found, 607.0517.

[0073] (rac) Diethyl (E) - 2,4 - dichloro - 6 - (3 - chlorophenyl) - 9 - (3 - ethoxy - 3 - oxoprop - 1 - en - 1 - yl) - 8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8,8 - dicarboxylate (15f - 2). It is obtained as a colorless oil in 41% yield. 1 1H NMR (600 MHz, CDCl3) δ 8.00 (d, J = 14.4 Hz, 1H), 7.55 (s, 1H), 7.45 (d, J = 7.2 Hz, 1H), 7.39 - 7.32 (m, 2H), 6.97 (s, 1H), 6.40 (s, 1H), 5.94 (d, J = 13.8 Hz, 1H), 4.26 - 4.17 (m, 4H), 4.09 - 4.03 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 1313C NMR (100 MHz, CDCl3) δ 166.6, 166.4, 164.9, 164.4, 157.6, 153.2, 137.9, 134.9, 134.8, 130.2, 130.1, 127.5, 127.1, 125.0, 124.8, 103.7, 94.6, 86.5, 84.2, 63.7, 63.4, 62.9, 60.7, 31.3, 14.5, 14.2, 13.9, 13.8, 13.5. ESI-HRMS (m / z) calcd C 24 H 23 Cl3N4NaO7 (M+Na) + , 607.0525; found, 607.0535.

[0074] (rac) Diethyl (E)-2,4-dichloro-6-(4-chlorophenyl)-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15f-3). It is obtained as a white solid in 54% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 14.0 Hz, 1H), 7.50 - 7.48 (m, 2H), 7.38 - 7.36 (m, 2H), 6.95 (s, 1H), 6.40 (s, 1H), 5.95 (d, J = 14.4 Hz, 1H), 4.26 - 4.15 (m, 4H), 4.10 - 4.01 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 6.8 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.6, 166.5, 165.0, 157.6, 153.2, 136.1, 134.9, 134.5, 129.0, 128.3, 127.7, 103.7, 95.0, 86.5, 84.3, 63.6, 63.3, 60.7, 14.5, 13.9, 13.8. ESI-HRMS (m / z) calcd C 24 H 23 Cl3N4NaO7 (M+Na) + , 607.0525; found, 607.0533.

[0075] (rac)Diethyl (E)-2,4-dichloro-6-(3,4-dichlorophenyl)-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15f-4). It is obtained as a white solid in 54% yield. 1 1H NMR (600 MHz, CDCl3) δ 7.99 (d, J = 14.4 Hz, 1H), 7.66 (m, 1H), 7.48 - 7.47 (m, 1H), 7.41 - 7.40 (m, 1H), 6.92 (s, 1H), 6.38 (s, 1H) 5.94 (d, J = 14.4 Hz, 1H), 4.26 - 4.17 (m, 4H), 4.12 - 4.04 (m, 2H), 1.28 (q, J = 6.6 Hz, 6H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.45, 166.38, 164.8, 157.5, 153.4, 139.8, 136.2, 134.8, 134.3, 133.2, 130.9, 129.0, 127.4, 126.1, 103.8, 94.2, 86.6, 84.1, 63.7, 63.5, 60.7, 14.5, 13.9, 13.8. ESI-HRMS (m / z) calcd C 24 H 22 Cl4N4NaO7 (M + Na) + , 641.0135; found, 641.0142.

[0076] (rac)Diethyl (E)-6-(2-bromophenyl)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15g-1). It is obtained as a colorless oil in 41% yield. 11H NMR (600 MHz, CDCl3) δ 8.29 (d, J = 13.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.54 - 7.50 (m, 1H), 7.26 (s, 1H), 6.84 (s, 1H), 6.29 (d, J = 14.4 Hz, 1H), 4.51 - 4.41 (m, 3H), 4.38 - 4.29 (m, 2H), 4.17 - 4.12 (m, 1H), 1.55 (t, J = 7.2 Hz, 3H), 1.40 (t, J = 7.2 Hz, 3H), 1.35 (t, J = 4.8 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.5, 166.4, 165.0, 157.7, 153.3, 140.0, 134.9, 133.9, 131.6, 128.6, 127.7, 123.9, 103.7, 96.5, 86.3, 85.3, 63.6, 63.3, 60.6, 14.5, 13.8, 13.7. ESI-HRMS (m / z) calcd C 24 H 23 BrClN4NaO7 (M+Na) + , 651.0019; found, 651.0018.

[0077] (rac) Diethyl (E)-6-(3-bromophenyl)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15g-2). It is obtained as a colorless oil in 38% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 14.4 Hz, 1H), 7.71 (s, 1H), 7.51 (q, J = 7.2 Hz, 2H), 7.29 - 7.25 (m, 1H), 6.97 (s, 1H), 6.40 (s, 1H), 5.94 (d, J = 14.4 Hz, 1H), 4.24 - 4.16 (m, 4H), 4.10 - 4.02 (m, 2H), 1.28 (q, J = 7.2 Hz, 6H), 1.14 (t, J = 7.2 Hz, 3H). 1313C NMR (100 MHz, CDCl3) δ 166.6, 166.4, 164.9, 157.6, 153.2, 139.8, 138.1, 134.8, 133.0, 130.4, 130.0, 127.5, 125.4, 123.0, 103.7, 94.5, 86.5, 84.2, 63.6, 63.4, 60.7, 14.5, 13.9, 13.8. ESI-HRMS (m / z) calcd C 24 H 24 BrClN4O7 (M + H) + , 629.0200; found, 629.0207.

[0078] (rac) Diethyl (E)-6-(4-bromophenyl)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15g-3). It is obtained as a white solid in 46% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 14.4 Hz, 1H), 7.54 - 7.52 (m, 2H), 7.43 - 7.41 (m, 2H), 6.93 (s, 1H), 6.39 (s, 1H), 5.94 (d, J = 14.4 Hz, 1H), 4.25 - 4.15 (m, 4H), 4.10 - 4.01 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H), 1.23 (t, J = 7.2 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.5, 166.4, 164.9, 157.5, 153.2, 139.7, 135.0, 134.9, 132.0, 128.6, 127.8, 124.3, 103.9, 95.0, 86.5, 84.2, 63.6, 63.3, 60.6, 14.5, 13.9, 13.8. ESI-HRMS (m / z) calcd C 24 H 23 BrClN4NaO7 (M + Na) + , 651.0019; found, 651.0021.

[0079] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(2-iodophenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15h-1). It is obtained as a white solid in 32% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J=14.4 Hz, 1H), 7.92 (d, J=9.2 Hz, 1H), 7.47 - 7.45 (m, 1H), 7.37 (t, J=7.6 Hz, 1H), 7.11 - 7.07 (m, 1H), 6.87 (s, 1H), 6.58 (s, 1H), 6.02 (d, J=14.4 Hz, 1H), 4.28 - 4.13 (m, 4H), 4.11 - 4.03 (m, 2H), 3.91 - 3.83 (m, 1H), 1.28 (t, J=7.2 Hz, 3H), 1.16 - 1.08 (m, 6H). 13 13C NMR (100 MHz, CDCl3) δ 166.4, 166.3, 164.9, 157.8, 153.2, 140.5, 139.9, 137.7, 134.8, 131.5, 128.4, 128.0, 127.8, 103.6, 99.6, 98.1, 86.2, 85.4, 63.5, 63.2, 60.6, 14.5, 13.8, 13.7. ESI-HRMS (m / z) calcd C 24 H 24 Cl2IN4O7 (M + H) + , 677.0061;found, 677.0063.

[0080] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(4-iodophenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15h-2). It is obtained as a white solid in 32% yield. 11H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 14.0 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.30 - 7.28 (m, 2H), 6.90 (s, 1H), 6.38 (s, 1H), 5.93 (d, J = 14.4 Hz, 1H), 4.23 - 4.13 (m, 4H), 4.08 - 3.99 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 7.2 Hz, 3H), 1.12 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.4, 166.3, 164.9, 157.4, 153.0, 140.1, 139.5, 137.8, 135.5, 134.7, 128.6, 127.6, 127.5, 127.3, 103.5, 96.0, 94.9, 86.3, 84.1, 63.5, 63.2, 60.5, 14.4, 13.78, 13.69. ESI - HRMS (m / z) calcd for C 24 18 24 H18Cl2IN4O7 (M + H) + , 677.0061; found, 677.0065.

[0081] (rac) Diethyl (E)-2,4 - dichloro - 9-(3 - ethoxy - 3 - oxoprop - 1 - en - 1 - yl)-6-(2-(trifluoromethyl)phenyl)-8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8,8 - dicarboxylate (15i - 1). It is obtained as a white solid in 43% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 14.4 Hz, 1H), 7.77 - 7.70 (m, 2H), 7.63 - 7.50 (m, 2H), 7.17 (s, 1H), 6.53 (s, 1H), 6.05 (d, J = 14.4 Hz, 1H), 4.31 - 4.05 (m, 5H), 3.97 - 3.89 (m, 1H), 1.29 (t, J = 7.2 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H), 1.10 (t, J = 7.2 Hz, 3H). 1313C NMR(100 MHz, CDCl3) δ 166.5, 166.1, 165.0, 157.7, 153.2, 139.8, 134.9, 134.2, 132.3, 130.3, 129.8, 129.5, 129.1, 129.0, 128.8, 127.8(d, J=5), 127.3, 125.4, 122.6, 103.8, 94.3, 86.4, 85.0, 63.5, 63.3, 60.6, 14.5, 13.8, 13.6. ESI-HRMS(m / z) calcd C 25 H 24 Cl2F3N4O7(M + H) + , 619.0969;found, 619.0972.

[0082] (rac) Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(3-(trifluoromethyl)phenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15i-2). It is obtained as a white solid in 43% yield. 1 1H NMR(400 MHz, CDCl3) δ 8.00(d, J=14.4 Hz, 1H), 7.82(s, 1H), 7.78 - 7.76(m, 1H), 7.68 - 7.66(m, 1H), 7.56 - 7.52(m, 1H), 7.02(s, 1H), 6.40(s, 1H), 5.93(d, J=14.4 Hz, 1H), 4.31 - 4.15(m, 4H), 4.10 - 4.03(m, 1H), 4.02 - 3.95(m, 1H), 1.28(t, J=7.2 Hz, 3H), 1.22(t, J=6.8 Hz, 3H), 1.14(t, J=7.2 Hz, 3H). 13 13C NMR(100 MHz, CDCl3) δ 166.5, 166.4, 164.9, 157.5, 153.3, 139.8, 137.1, 134.8, 131.5, 131.2, 130.2, 129.5, 127.5, 126.8, 126.7, 125.2, 123.7, 123.7, 122.5, 103.7, 94.7, 86.5, 84.1, 63.7, 63.4, 60.6, 14.5, 13.8, 13.7. ESI-HRMS(m / z) calcd C25 H 24 Cl₂F₃N₄O₇(M + H) + ,619.0969; found, 619.0965.

[0083] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(4-(trifluoromethyl)phenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15i-3). It is obtained as a white solid in 28% yield. 1 ¹H NMR (400 MHz, CDCl₃) δ 8.00 (d, J = 14.4 Hz, 1H), 7.68 (dd, J = 14.0 Hz, 8.8 Hz, 4H), 7.00 (s, 1H), 6.40 (s, 1H), 5.95 (d, J = 14.4 Hz, 1H), 4.26 - 4.14 (m, 4H), 4.12 - 4.00 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 7.2 Hz, 3H), 1.15 (t, J = 7.2 Hz, 3H). 13 ¹³C NMR (100 MHz, CDCl₃) δ 166.4, 164.9, 157.6, 153.3, 139.9, 139.7, 134.8, 127.6, 127.4, 125.8, 125.8, 103.8, 94.9, 86.5, 84.2, 63.7, 63.4, 60.7, 14.5, 13.83, 13.81. ESI-HRMS (m / z) calcd C 25 H 24 Cl₂F₃N₄O₇(M + H) + ,619.0969; found, 619.0962.

[0084] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(3-nitrophenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15j-1). It is obtained as a white solid in 44% yield. 11H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 14.4 Hz, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.02 (s, 1H), 6.37 (s, 1H), 5.95 (d, J = 14.4 Hz, 1H), 4.28 - 4.17 (m, 4H), 4.13 - 4.03 (m, 2H), 1.31 - 1.21 (m, 6H), 1.15 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.3, 166.2, 164.8, 157.5, 153.5, 148.6, 139.8, 138.5, 134.8, 132.9, 130.1, 127.4, 124.8, 121.9, 103.9, 94.4, 86.6, 84.0, 63.8, 63.6, 14.5, 13.8. ESI - HRMS (m / z) calcd for C 24 H 23 Cl2N5NaO9 (M + Na) + , 618.0765; found, 618.0763.

[0085] (rac) Diethyl (E) - 2,4 - dichloro - 9 - (3 - ethoxy - 3 - oxoprop - 1 - en - 1 - yl) - 6 - (4 - nitrophenyl) - 8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8,8 - dicarboxylate (15j - 2). It is obtained as a white solid in 44% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 8.8 Hz, 2H), 7.99 (d, J = 14.4 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.00 (s, 1H), 6.38 (s, 1H), 5.96 (d, J = 14.0 Hz, 1H), 4.26 - 4.18 (m, 4H), 4.13 - 4.04 (m, 2H), 1.28 (t, J = 6.8 Hz, 3H), 1.25 (t, J = 5.6 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 1313C NMR (100 MHz, CDCl3) δ 166.3, 166.1, 164.9, 157.5, 153.4, 148.9, 142.8, 139.7, 134.8, 128.0, 127.4, 124.0, 103.9, 94.7, 86.6, 84.0, 63.7, 63.5, 60.7, 14.5, 13.9, 13.8. ESI-HRMS (m / z) calcd C 24 H 24 Cl2N5O9 (M + H) + , 596.0946; found, 596.0944.

[0086] (rac) Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(o-tolyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15k-1). It is obtained as a white solid in 35% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 14.4 Hz, 1H), 7.34 - 7.32 (m, 2H), 7.27 (t, J = 7.6 Hz, 1H), 7.21 - 7.19 (m, 1H), 7.00 (s, 1H), 6.44 (s, 1H), 5.95 (d, J = 14.4 Hz, 1H), 4.25 - 4.13 (m, 4H), 4.11 - 4.03 (m, 1H), 4.02 - 3.94 (m, 1H), 2.37 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.20 (t, J = 6.8 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.8, 166.5, 165.2, 157.6, 152.9, 139.6, 138.6, 135.7, 134.9, 130.5, 128.7, 127.8, 127.4, 123.9, 103.5, 95.4, 86.4, 84.4, 63.5, 63.1, 60.6, 21.6, 14.5, 13.8. ESI-HRMS (m / z) calcd C 25 H 26 Cl2N4NaO7 (M + Na) + , 587.1071; found, 587.1074.

[0087] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(m-tolyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15k-2). It is obtained as a white solid in 35% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J=14.4 Hz, 1H), 7.34 - 7.20 (m, 4H), 7.01 (s, 1H), 6.45 (s, 1H), 5.95 (d, J=14.0 Hz, 1H), 4.26 - 4.10 (m, 4H), 4.08 - 3.94 (m, 2H), 2.34 (s, 3H), 1.29 (t, J=9.2 Hz, 3H), 1.22 (t, J=7.2 Hz, 3H), 1.15 (t, J=7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.8, 166.5, 165.1, 157.6, 152.9, 139.6, 138.6, 135.7, 134.9, 130.6, 128.6, 127.8, 127.4, 123.9, 103.5, 95.4, 86.4, 84.4, 63.5, 63.1, 60.6, 21.6, 14.5, 13.8. ESI-HRMS (m / z) calcd C 25 H 26 Cl2N4NaO7 (M+Na) + , 587.1071;found, 587.1080.

[0088] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(p-tolyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15k-3). It is obtained as a white solid in 45% yield. 11H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 14.4 Hz, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 6.99 (s, 1H), 6.43 (s, 1H), 5.94 (d, J = 14.4 Hz, 1H), 4.25 - 4.12 (m, 4H), 4.08 - 3.97 (m, 2H), 2.36 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.21 (t, J = 7.2 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.8, 166.5, 165.2, 157.6, 152.9, 139.9, 139.6, 135.0, 132.9, 129.4, 127.9, 126.8, 103.5, 95.4, 86.4, 84.4, 63.5, 63.1, 60.6, 21.4, 14.5, 13.84, 13.81. ESI - HRMS (m / z) calcd for C 25 18 26 H14Cl2N4NaO7 (M + Na) + , 587.1071; found, 587.1072.

[0089] (rac) Diethyl (E) - 2,4 - dichloro - 6 - (3,4 - dimethylphenyl) - 9 - (3 - ethoxy - 3 - oxoprop - 1 - en - 1 - yl) - 8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8,8 - dicarboxylate (15k - 4). It is obtained as a white solid in 45% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 14.4 Hz, 1H), 7.29 - 7.24 (m, 2H), 7.15 - 7.09 (m, 2H), 6.99 (s, 1H), 6.44 (s, 1H), 5.95 (d, J = 14.4 Hz, 1H), 4.25 - 4.12 (m, 4H), 4.08 - 3.97 (m, 2H), 2.28 (s, 6H), 1.29 (t, J = 7.2 Hz, 3H), 1.21 (t, J = 6.8 Hz, 3H), 1.14 (t, J = 7.4 Hz, 3H). 1313C NMR (100 MHz, CDCl3) δ 166.8, 166.5, 165.2, 157.6, 152.7, 139.5, 138.5, 137.0, 134.9, 133.2, 129.9, 127.8, 124.1, 103.4, 95.4, 86.3, 84.4, 63.4, 63.0, 60.5, 20.0, 19.7, 14.8, 13.5. ESI-HRMS (m / z) calcd C 26 H 28 Cl2N4NaO7 (M+Na) + , 601.1227; found, 601.1233.

[0090] (rac) Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(3-methoxyphenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (15l-1). It is obtained as an white solid in 38% yield. 1 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 12.0 Hz, 1H), 7.32 - 7.27 (m, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.08 (s, 1H), 7.00 (s, 1H), 6.92 (dd, J = 8.0, 3.2 Hz, 1H), 6.44 (s, 1H), 5.95 (d, J = 14.4 Hz, 1H), 4.26 - 4.13 (m, 4H), 4.10 - 3.98 (m, 2H), 3.82 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 6.8 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.7, 166.5, 165.1, 159.9, 157.5, 153.0, 139.6, 137.3, 134.9, 129.9, 127.7, 118.9, 115.2, 112.6, 103.5, 95.1, 86.5, 84.3, 63.5, 63.2, 60.6, 55.5, 14.5, 13.8. ESI-HRMS (m / z) calcd C 25 H 26 Cl2N4NaO8 (M+Na) +,603.1020; found,603.1016.

[0091] (rac)Diethyl (E)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-(3,4,5-trimethoxyphenyl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate 15l-2. It is obtained as a white solid in 43% yield. 1 H NMR (600 MHz, CDCl3) δ 8.03 (d, J = 13.8 Hz, 1H), 7.06 (s, 1H), 6.58 (s, 2H), 6.04 (d, J = 14.4 Hz, 1H), 5.74 (s, 1H), 6.38 (s, 1H) 4.26 (q, J = 6.6 Hz, 2H), 4.18 - 4.13 (m, 2H), 3.93 - 3.81 (m, 11H), 1.33 (t, J = 7.2 Hz, 3H), 1.17 (t, J = 7.2 Hz, 3H), 0.92 (t, J = 7.2 Hz, 3H). 13 C NMR (150 MHz, CDCl3) δ 166.4, 166.3, 164.7, 156.1, 153.5, 153.0, 140.4, 138.6, 133.9, 130.7, 128.0, 104.9, 104.8, 104.3, 88.8, 69.5, 63.6, 62.8, 61.0, 60.8, 56.5, 14.5, 13.9, 13.7. ESI-HRMS (m / z) calcd C 27 H 30 Cl2N4NaO 10 (M + Na) + ,663.1231; found,663.1230.

[0092] (rac)Diethyl (Z)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-1-yl)-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (17). It is obtained as a white solid in 31% yield. 11H NMR (600 MHz, CDCl3) δ 7.59 - 7.58 (m, 2H), 7.40 - 7.39 (m, 3H), 7.07 (s, 1H), 6.90 (s, 1H), 6.85 (d, J=10.2 Hz 1H), 5.51 (d, J=9.6 Hz, 1H), 4.23 - 4.18 (m, 3H), 4.10 - 3.95 (m, 3H), 1.26 (t, J=7.2 Hz, 3H), 1.16 - 1.14 (m, 6H). 13 13C NMR (150 MHz, CDCl3) δ 166.7, 166.4, 165.8, 165.7, 165.1, 165.0, 158.2, 157.5, 152.9, 152.2, 139.6, 138.2, 136.6, 135.7, 134.9, 129.9, 129.6, 129.5, 128.7, 128.6, 127.8, 127.6, 126.9, 126.8, 107.4, 103.5, 95.8, 95.3, 86.9, 86.4, 84.6, 84.3, 63.5, 63.2, 63.1, 62.6, 60.9, 60.5, 14.4, 14.2, 13.8. ESI - HRMS (m / z) calcd C 24 H 25 Cl2N4O7(M + H) + , 551.1095;found, 551.1093.

[0093] General Procedure for the Synthesis of Compounds 19a - 19d, 20a - 20d. Exemplified by (rac) Diethyl (E) - 2,4 - dichloro - 9 - (3 - ethoxy - 3 - oxoprop - 1 - en - 1 - yl) - 6 - phenyl - 8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8,8 - dicarboxylate 19a. To a mixture of methyl 2 - (2,6 - dichloro - 9H - purin - 9 - yl)acrylate (252 mg, 1 mM) and diethyl 3 - phenyloxirane - 2,2 - dicarboxylate (325 mg, 1.2 mM) in Chlorobenzene (10 mL), Nickel perchlorate hexahydrate (73 mg, 0.2 mM) and (300 mg) was added, and the resulting mixture was stirred at 80 °C under N2 for 12 h. The reaction was detected by TLC, the solvent was removed and purified by flash column chromatography on silica gel (PE / EA = 2 / 1) to afford a colorless oil (31% yield). 1 1H NMR (400 MHz, CDCl3) δ 7.59 - 7.56 (m, 2H), 7.39 - 7.38 (m, 3H), 6.87 (s, 1H), 6.53 (s, 1H), 6.44 (s, 1H), 6.03 (s, 1H), 4.37 - 4.20 (m, 3H), 4.16 - 4.08 (m, 1H), 4.07 - 3.98 (m, 2H), 1.33 (t, J = 7.2 Hz, 3H), 1.19 (t, J = 9.2 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.2, 166.0, 162.2, 160.7, 152.1, 137.3, 136.8, 132.1, 129.7, 128.6, 128.0, 127.0, 124.2, 96.3, 86.9, 85.0, 63.0, 62.8, 62.4, 14.2, 13.9, 13.8. ESI - HRMS (m / z) calcd for C 24 H 25 Cl2N4O7 (M + H) + , 551.1095; found, 551.1094.

[0094] (rac) Diethyl 2,4 - dichloro - 9 - (3 - ethoxy - 3 - oxoprop - 1 - en - 2 - yl) - 6 - (2 - fluorophenyl) - 8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8,8 - dicarboxylate (19b). It is obtained as a colorless oil in 41% yield. 11H NMR (600 MHz, CDCl3) δ 7.52 - 7.50 (m, 1H), 7.41 - 7.37 (m, 1H), 7.19 (t, J = 12.6 Hz, 1H), 7.10 (t, J = 19.2 Hz, 1H), 6.81 (s, 1H), 6.52 (s, 1H), 6.02 (s, 1H), 4.37 - 4.27 (m, 2H), 4.26 - 4.20 (m, 2H), 4.17 - 4.11 (m, 1H), 4.08 - 4.03 (m, 1H), 1.34 (t, J = 6.6 Hz, 3H), 1.20 (t, J = 7.2 Hz, 3H), 1.12 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 166.4, 165.6, 162.2, 162.0, 161.0, 160.3, 152.0, 136.7, 131.9, 131.82, 131.79 (d, J C-F = 9.0 Hz), 129.59 (d, J C-F = 3.3 Hz), 128.1, 125.6, 124.75, 124.67, 124.18 (d, J C-F = 3.3 Hz), 116.3, 116.2, 94.3, 87.2, 84.88 (d, J C-F = 4.5 Hz), 63.1, 62.9, 62.3, 14.2, 13.9, 13.8. ESI - HRMS (m / z) calcd for C 24 H 24 Cl2FN4O7 (M + H) + , 569.1001; found, 569.1003.

[0095] (rac) Diethyl 2,4 - dichloro - 6 - (2 - chlorophenyl) - 9 - (3 - ethoxy - 3 - oxoprop - 1 - en - 2 - yl) - 8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8,8 - dicarboxylate (19C). It is obtained as a colorless oil in 41% yield. 11H NMR (600 MHz, CDCl3) δ 7.60 - 7.58 (m, 1H), 7.43 - 7.42 (m, 1H), 7.36 - 7.30 (m, 2H), 6.95 (s, 1H), 6.59 (s, 1H), 6.52 (s, 1H), 6.03 (s, 1H), 4.37 - 4.30 (m, 2H), 4.29 - 4.23 (m, 1H), 4.20 - 4.14 (m, 1H), 4.10 - 4.03 (m, 2H), 1.36 (t, J = 7.2 Hz, 3H), 1.18 - 1.14 (m, 6H). 13 13C NMR (150 MHz, CDCl3) δ 166.1, 165.7, 162.3, 161.2, 152.3, 137.3, 134.2, 134.1, 131.9, 131.2, 130.3, 129.4, 128.1, 127.1, 125.6, 95.7, 87.1, 85.3, 63.1, 62.9, 62.4, 14.3, 13.8. ESI-HRMS (m / z) calcd C 24 H 24 Cl3N4O7 (M + H) + , 585.0705; found, 585.0715.

[0096] (rac) Diethyl 6-(2-bromophenyl)-2,4-dichloro-9-(3-ethoxy-3-oxoprop-1-en-2-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (19d). It is obtained as a colorless oil in 53% yield. 1 1H NMR (600 MHz, CDCl3) δ 7.24 - 7.26 (m, 1H), 7.19 - 7.16 (m, 1H), 6.84 (s, 1H), 6.51 (s, 1H), 6.43 (s, 1H), 5.95 (s, 1H), 4.29 - 4.21 (m, 2H), 4.19 - 4.14 (m, 1H), 4.11 - 4.05 (m, 1H), 4.02 - 3.94 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H), 1.09 - 1.06 (m, 6H). 1313C NMR (150 MHz, CDCl3) δ 166.1, 165.7, 162.3, 161.2, 152.3, 137.4, 133.6, 131.4, 129.4, 127.7, 125.6, 97.3, 85.4, 63.1, 62.9, 62.4, 14.3, 13.85, 13.83. ESI-HRMS (m / z) calcd C 24 H 24 BrCl2N4O7 (M + H) + , 629.0200; found, 629.0210.

[0097] (rac) Diethyl 2,4-dichloro-9-(3-methoxy-3-oxoprop-1-en-2-yl)-6-phenyl-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate (20a). It is obtained as a colorless oil in 36% yield. 1 1H NMR (400 MHz, CDCl3) δ 7.59 - 7.57 (m, 2H), 7.41 - 7.38 (m, 3H), 6.86 (s, 1H), 6.51 (s, 1H), 6.44 (s, 1H), 6.04 (s, 1H), 4.28 - 4.20 (m, 1H), 4.17 - 4.09 (m, 1H), 4.06 - 3.99 (m, 2H), 3.88 (s, 3H), 1.19 (t, J = 7.2 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 166.2, 166.0, 162.8, 160.6, 152.1, 137.2, 136.8, 131.8, 129.7, 128.6, 128.0, 127.1, 124.4, 96.4, 86.9, 84.9, 63.0, 62.8, 53.1, 13.9, 13.8. ESI-HRMS (m / z) calcd C 23 H 23 Cl2N4O7 (M + H) + , 537.0938; found, 537.0938.

[0098] (rac)Diethyl 2,4-dichloro-6-(2-fluorophenyl)-9-(3-methoxy-3-oxoprop-1-en-2-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate(20b). It is obtained as a colorless oil in 42% yield. 1 H NMR(600 MHz,CDCl3)δ7.52(t,J=9.0Hz,1H),7.39(q,J=5.4 Hz,1H),7.16(t,J=7.2 Hz,1H),7.12-7.09(m,1H),6.81(s,1H),6.52-6.50(m,2H),6.05(s,1H),4.31-4.20(m,2H),4.17-4.12(m,1H),4.09-4.03(m,1H),3.88(s,3H),1.21(t,J=7.2 Hz,3H),1.12(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl3)δ166.4,165.7,162.7,162.0,160.9,160.3,152.0,136.7,131.83,131.78,131.6,129.61,129.59,128.1,125.9,124.7,124.7,124.2,124.2,116.3,116.2,94.3,87.2,84.79,84.76,63.1,62.9,53.1,13.9,13.8. ESI-HRMS(m / z) calcd C 23 H 22 Cl2FN4O7(M+H) + ,555.0844;found,555.0847.

[0099] (rac)Diethyl 2,4-dichloro-6-(2-chlorophenyl)-9-(3-methoxy-3-oxoprop-1-en-2-yl)-8a,9-dihydro-6H,8H-oxazolo[4,3-f]purine-8,8-dicarboxylate(20c). It is obtained as a colorless oil in 41% yield. 1HNMR(600MHz,CDCl3)δ7.61 - 7.59(m,1H),7.43 - 7.42(m,1H),7.36 - 7.30(m,2H),6.95(s,1H),6.57 - 6.52(m,2H),6.05(s,1H),4.30 - 4.23(m,1H),4.21 - 4.15(m,1H),4.10 - 4.04(m,2H),3.88(s,3H),1.18 - 1.14(m,6H). 13 CNMR(150MHz,CDCl3)δ166.1,165.7,162.8,161.1,152.3,137.3,134.2,134.1,131.6,131.2,130.3,129.3,128.1,127.1,125.8,95.7,87.1,85.2,63.1,62.9,62.7,53.1,29.8,14.2,13.8.ESI - HRMS(m / z)calcd C 23 H 22 Cl3N4O7(M + H) + ,571.0549;found,571.0553.

[0100] (rac)Diethyl 6-(2 - bromophenyl)-2,4 - dichloro - 9-(3 - methoxy - 3 - oxoprop - 1 - en - 2 - yl)-8a,9 - dihydro - 6H,8H - oxazolo[4,3 - f]purine - 8,8 - dicarboxylate(20d).It is obtained as a colorless oil in 41% yield. 1 H NMR(600MHz,CDCl3)δ7.61(t,J=7.2Hz,1H),7.37(t,J=7.2Hz,1H),7.28 - 7.27(m,1H),6.93(s,1H),6.58 - 6.52(m,2H),6.05(s,1H),4.30 - 4.22(m,1H),4.21 - 4.15(m,1H),4.11 - 4.04(m,2H),3.88(s,3H),1.16(dd,J=16.2Hz,7.2Hz,6H). 1313C NMR (150 MHz, CDCl3) δ 166.1, 165.7, 162.8, 161.1, 152.3, 133.5, 131.4, 129.4, 127.7, 125.8, 97.4, 85.2, 63.1, 62.9, 53.1, 13.8. ESI-HR MS (m / z) calcd for C 23 H 22 BrCl2N4O7 (M + H) + , 615.0043; found, 615.0049.

[0101] Example 2:

[0102] For the 6S, 8aS - configuration and 6R, 8aR - configuration, chiral HPLC resolution was used. Using a Shimadzu LC - 20AD CP - HPLC - 09 with a chiralcel OZ - H (0.46 cm I.D. × 25 cm L), 20ba and 20bb were separated; Hexane / EtOH = 50 / 50 (v / v), flow rate 1.0 mL / min, detected at 214 nm UV. Enantiomers 20ba (ee > 98%), 20bb (ee > 98%) (Daicel Chiral Technologies (China) Co., LTD.). In the HPLC trace, the peak with a shorter retention time was 20ba, and the longer peak was 20bb; the specific corresponding structure was confirmed by single - crystal X - ray diffraction.

[0103] Example 3:

[0104] A series of racemic oxazolo[4,3 - f]purine derivatives were synthesized by the above method in the present invention, and the cell activities of these compounds against tumor cells were tested by the CCK8 method. 4 × 10 3 cells per well in a 96 - well plate. The test compound or the positive control 5 - FU was diluted to 100 mmol / L with cell - grade dimethyl sulfoxide for use. The cells in the culture flask were digested, centrifuged, mixed well and then counted and plated in the 96 - well plate. The test compound was diluted with the culture medium and mixed well. The drug and HeLa cells and HCT116 cells were co - incubated for 48 h respectively. 110 μL of the culture medium (containing 10 μL of CCK8) was added to each well for incubation, and the OD value was recorded at 450 nm, and analyzed with GraphPad Prism 8 software.

[0105] The anti - tumor activity results of the oxazolo[4,3 - f]purine derivatives are as follows:

[0106]

[0107]

[0108]

[0109] a The anti - proliferative activity of individual compounds against tumor cells was determined by the CCK8 assay. The data are the average values measured in triplicate. b HCT116: Human colon cancer cells.

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] Example 4

[0119] The in vitro and in vivo anti - tumor activities of synthetic oxazolo[4,3 - f]purine derivatives were evaluated, the most optimized compounds were selected, and the structure - activity relationship was summarized. Compound 20b with good anti - tumor activity was selected; two compounds with 6S,8aS - configuration and 6R,8aR - configuration corresponding to 20b were synthesized, and their anti - tumor activities were studied by CCK8.

[0120]

[0121] It was found that compound 20b had the best anti - tumor activity against colon cancer cells. Then, human cancer cell lines such as MCF - 7, SW480, CT26, Panc - 1, and HepG - 2 were selected to test whether compound 20b had inhibitory effects on other cancer cells. As shown in the following table, compounds 20b, 20c, and 20d all had certain anti - tumor activities against these cancer cells, but their anti - tumor activities against cervical cancer and colon cancer cells were not as good as that of compound 20b. Among them: human breast cancer cells (MCF - 7), human liver cancer cells (SK - hep - 1), human colon cancer cells (SW480), mouse colon cancer cells (CT 26), human pancreatic cancer cells (Panc - 1), human liver cancer cells (HepG - 2). The experimental results showed that among the racemates, compound 20b showed strong inhibitory ability on the proliferation of cervical cancer cell line cells (HeLa) and colon cancer cell line cells (HCT116) in vitro, IC 50They were 0.69 μM and 0.51 μM respectively. The IC of the control drugs 5-fluorouracil (5-FU) and cisplatin against HeLa and HCT116 cells 50 were 4.48 μM and 11.32 μM, 4.02 μM and 9.43 μM respectively. In the chiral structure, the 6S,8aS-configuration and 6R,8aR-configuration chiral oxazolo[4,3-f]purine derivatives had comparable anti-tumor activities to the racemate 20b.

[0122]

[0123] The inhibitory effects of compound 20b on MCF-7, SW480, CT26, Panc-1, HepG-2

[0124]

[0125]

[0126] a The anti-proliferative activities of individual compounds against tumor cells were determined by the CCK8 assay. The data were the average values measured in triplicate. b MCF-7 human breast cancer cells; c SW480 human colon cancer cells; d CT26 mouse colon cancer cells; e Panc-1 human pancreatic cancer cells; f HepG-2 human liver cancer cells.

[0127] Example 5

[0128] Cell colony formation assay: HCT116 cells were seeded in 6-well plates (1.5×10 3 cells / well). Different concentrations of compound 20b were added to the wells and incubated for 48 h. The medium was changed every 72 h. After 14 d, the cells were fixed with methanol for 30 min, stained with 0.1% crystal violet staining solution at room temperature for 30 min, washed 3 times with PBS, and air-dried. Then photos were taken. It Figure 1 could be seen that the number of formed cell colonies was inhibited with the increase of the concentration of 20b, indicating that compound 20b could inhibit the formation of cancer cell colonies in a dose-dependent manner and had good anti-tumor activity.

[0129] Cell cycle assay: Cells were seeded in 6-well plates (5×105 Cells / well). The next day, the compound 20b at the specified concentration was added to the wells and incubated for 48 h at 37 °C in 5% CO2. Cells were harvested and frozen overnight in 70% ethanol. Then, the cells were stained with propidium iodide (PI) dye for 15 min in the dark, and the cell DNA content was detected by flow cytometry (BD Bioscience). The experimental results showed that compound 20b could induce a slight cell cycle arrest at the G2 / M phase ( Figure 2 ). Therefore, compound 20b has a certain inhibitory effect on cell division and can arrest cancer cells at the G2 / M phase.

[0130] Cell apoptosis assay: HCT116 cells were seeded in 6-well plates (8×10 5 cells / well). The next day, the compound 20b at the specified concentration was added to the wells and incubated for 48 h at 37 °C in 5% CO2. Cells were harvested and stained with Annexin V-FITC and PI for 15 min at room temperature. Apoptotic cells were analyzed by flow cytometry (BD Bioscience). The results showed that as the concentration of 20b increased, the total number of induced apoptotic cells also increased, indicating that compound 20b could induce cell apoptosis in a concentration-dependent manner ( Figure 2 ).

[0131] Effect on cell invasion: The transwell chambers (24-well) (Corning, USA) were coated with Matrigel (BD Biosciences, USA), and 1×10 5 cells were seeded in the upper chamber in serum-free medium and treated with the specified dose of compound 20b. Complete medium was placed in the lower chamber. After 24 h, the cells that penetrated the pores were stained with 0.1% crystal violet solution and observed with a Leica dil fluorescence microscope (Leica, Germany). Under normal conditions, cells would pass through the membrane formed by Matrigel and transfer to the lower chamber, but after interference with 20b, the number of HCT116 cells passing through Matrigel decreased. This indicated that 20b could inhibit the invasion of HCT116 cells in a concentration gradient manner. It was verified by cell scratch assay and transwell assay that 20b inhibited the migration and invasion of HCT116 cells in a concentration-dependent manner ( Figure 3 ).

[0132] Target PPIA validation experiment: Through molecular docking, drug affinity responsive target stability assay (DARTS), cellular thermal shift assay (CETSA), and small interfering RNA (siRNA) experiments. The results showed that compound 20b targeted PPIA and inhibited its phosphorylation ( Figure 4 ).

[0133] Xenograft tumor experiment: All animal experiments and experimental procedures were approved by the Animal Ethics Committee of the College of Chemistry and Chemical Engineering, Henan Normal University, China. CT26 cells (ATCC, Rockville, MD, USA) were used for tumor allograft experiments. Specific pathogen-free (SPF) BALB / c mice (male, 22 ± 2 g, Changzhou Cavens Laboratory Animal Co., Ltd., China) were randomly divided into 7 experimental groups (n = 6): control group, positive control 5-FU (45 mg / kg), and 20b (15 mg / kg and 45 mg / kg). Mice were subcutaneously injected with CT26 cells (1×10 6 per mouse). The diameter of the transplanted tumors in mice was measured with a vernier caliper. When the tumor grew to approximately 120 mm 3 , 20b (15 mg / kg or 45 mg / kg), 5-FU (45 mg / kg), or the control solvent (0.2 mL) was intraperitoneally injected daily for 14 consecutive days. The antitumor activity of the compounds was evaluated by measuring the tumor diameter. The tumor diameter and body weight were measured every 2 days. After 15 days, the tumor tissues were surgically removed, weighed, and photographed. The tumor volume (TV) was calculated using the formula V = 1 / 2 × a × b 2 , where a is the length and b is the width. The samples were used for histological examination. The tumor growth inhibition rate TGI (%) was calculated using the formula TGI = (1 - Tw / Cw) × 100%, where Tw and Cw are the average tumor weights of the treatment group and the model group on the 15th day, respectively. Compound 20b showed a significant dose-dependent inhibitory effect on both tumor volume and tumor weight. There was no significant decrease in body weight after treatment with 20b( Figure 5 ), and H&E staining showed( Figure 6 ) that there was no obvious damage to the organs after administration, and the tumor showed obvious necrosis. Compound 20b had no significant toxic effect on body weight, indicating the safety of 20b( Figure 5 ). These results indicate that compound 20b is a promising antitumor drug worthy of further study.

[0134] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the description in the specification only illustrate the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention will have various changes and improvements. Any structural changes to oxazolo[4,3-f]purine derivatives fall within the scope of protection of the present invention.

Claims

1. An oxazolo[4,3-f]purine compound, characterized in that: The general structure is as follows: Wherein: * represents a chiral center, and each general structure includes a racemate, a 6S, 8aS-configuration or a 6R, 8aR-configuration; R 1 , R 2 Each is independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, di-C1-C4 alkylamino; R 3 is selected from phenyl, naphthyl, furanyl, thienyl or pyridylbiphenyl, and substituents of the aforementioned groups; wherein the substituent is selected from one or more of halogen, trifluoromethyl, nitro, C1-C4 alkyl, C1-C4 alkoxy; R 4 , R 5 Each is independently selected from a carboxylic acid C1-C6 ester group; R 6 Selected from hydrogen, C1-C6 alkyl.

2. The oxazolo[4,3-f]purine compound according to claim 1, characterized in that: R 1 , R 2 All are chlorine, R 3 is p-fluorophenyl, R 4 , R 5 is ethyl carboxylate; R 6 It is methyl.

3. The oxazolo[4,3-f]purine compound according to claim 2, characterized in that: The compound has a 6S, 8aS-configuration or a 6R, 8aR-configuration.

4. Use of the oxazolo[4,3-f]purine compound according to any one of claims 1 to 3 in the preparation of an anti-cervical cancer or anti-colon cancer active drug.

5. The use of the chiral oxazolo[4,3-f]purine compound according to claim 5 in the preparation of anti-cervical cancer and anti-colon cancer active drugs, characterized in that: The anti-cervical cancer activity is anti-HeLa activity; the anti-colon cancer activity is anti-HCT116 activity.

6. An anti-cervical cancer or anti-colon cancer active drug, characterized in that: The active ingredients thereof include the compound according to any one of claims 1 to 3 and its corresponding pharmaceutically acceptable salt.

7. The anti-cervical cancer or anti-colon cancer active drug according to claim 6, characterized in that: The pharmaceutically acceptable salts include salts formed between acyclic nucleoside compounds and organic or inorganic acids.

8. The anti-cervical cancer or anti-colon cancer active drug according to claim 7, characterized in that: The organic acid is selected from one or more of malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid or oxalic acid, and the inorganic acid is selected from one or more of phosphoric acid, hydrohalic acid, sulfuric acid or nitric acid.

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