Diagnostic methods for detecting ecto-5'-nucleotidase (CD73)

Abstract

The present invention relates to radiolabeled and fluorescently labeled compounds and their use as diagnostic methods for detecting ecto-5'-nucleotidase (CD73). The invention is further directed to pharmaceutical compositions comprising said compounds, as well as compounds and pharmaceutical compositions for use in methods of diagnosing diseases associated with increased or decreased CD73 expression and in treating diseases associated with increased CD73 expression.

Description

[Technical field]

[0001] TECHNICAL FIELD OF THEINVENTION The present invention relates to radiolabeled and fluorescently labeled compounds and their use as diagnostic methods for detecting ecto-5'-nucleotidase (CD73). The invention is further directed to pharmaceutical compositions comprising said compounds, as well as to compounds and pharmaceutical compositions for use in methods for diagnosing and treating diseases associated with increased CD73 expression. [Background technology]

[0002] Background technology Ecto-5'-nucleotidase (CD73, eN) is an enzyme that catalyzes the dephosphorylation of extracellular AMP to adenosine. 1 It can be found on the surface of many different cell lines, including endothelial cells, stromal cells, and cells of the immune system, such as lymphocytes and regulatory T cells. Furthermore, CD73 is highly overexpressed on a variety of tumor cells, including bladder, colon, ovarian, melanoma, pancreatic, and breast cancers. 2、3 Stress factors such as hypoxia (HIF-1α), inflammatory factors (TGF-β, IFN, TNF, IL-1β), low pH, low glucose levels, and expression of Wnt / β-catenin are common in the tumor microenvironment and promote the expression of CD73. 4、5 In particular, triple-negative breast cancer lacking the estrogen receptor (ER) has been shown to reduce the expression of CD73. 6 Increased expression of CD73 in pulmonary arterial leukemia patients is associated with poor patient survival 7、8 The extracellular production of adenosine is A 2A and A 2B Activates P1 receptors (P1R) in the presence of receptors such as 3、9 , thereby leading to accelerated tumor growth, metastasis and angiogenesis, and to the infiltration of immune cells into solid tumors and thus suppression of the immune response via activity mediated by the P1 receptor. 10、11 For example, inhibition of CD73 and A 2A Or A 2BSimultaneous antagonism of the receptors leads to improved anticancer activity: antitumor resistance is reduced and immune responses are improved, while metastasis, angiogenesis, and tumor growth are reduced or delayed. 11、12 Therefore, CD73 is a novel and promising target for checkpoint inhibition in cancer immunotherapy. CD73 expression is often altered in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus compared to healthy controls. Therefore, CD73 expression could be a biomarker for these diseases and a biomarker for therapy monitoring.

[0003] Inhibition of CD73 can be achieved using monoclonal antibodies or small molecule inhibitors. For example, MEDI9447, an antibody developed as a treatment for pancreatic cancer, is already in clinical trials. 13 Antibodies often have the disadvantages of being expensive and lacking the ability to penetrate solid tumors. Furthermore, they often do not lead to complete inhibition of enzyme activity. There are also various classes of small molecule inhibitors that have been developed. Sulfonamides 14、15 , polyphenols 16 , and anthraquinone 17、18 shows low inhibitory activity, whereas nucleotide-derived CD73 inhibitors are highly potent 19~22 .

[0004] The Mueller group at the University of Bonn pioneered the development of potent and selective CD73 inhibitors based on the ADP analog AMPCP (AOPCP). 1 This work has led to the discovery of CD73 inhibitors with extremely high, subnanomolar inhibitory potency, high chemical and metabolic stability, and high selectivity. 1~4 2-Chloro- and N-amino acids of the adenine core structure 6 We were able to show that -benzyl substitution leads to potent AMPCP-derived CD73 inhibitors. 2、3 First Promising Study 1~4Following this, structure-activity relationship (SAR) analysis has been expanded to develop further AMPCP derivatives and analogues. Bioisosteric replacements of the adenine ring in combination with appropriate substituents, e.g., shifting or replacing the ring nitrogen atom, or replacing the bicyclic purine with a monocyclic or tricyclic ring system such as a pyrimidine ring, have been well tolerated. 5~10 The result was AB680, which shows a similar substitution pattern to the earlier compound developed by Bhattarai & Mueller. 8 (See Figure 1, Compound III) was developed by Arcus Biosciences 4 AB680 has been selected for clinical trials by Arcus Biosciences, and preliminary results from a Phase II trial in prostate cancer look promising. 11 .

[0005] Published studies of the first fluorescently labeled CD73 inhibitors have shown that 6 The p-position of the -benzyl moiety was shown to be an amenable position for further derivatization, even with bulky substituents. The introduction of a bulky fluorescein residue attached via a linker moiety was well tolerated, leading to potent, selective, and metabolically stable fluorescein-labeled CD73 inhibitors. 12 .

[0006] There is a need for additional fluorescent and radiolabeled CD73 ligands. Summary of the Invention

[0007] In a first aspect, the present invention provides a compound of general formula (I) The present invention relates to a compound of formula (I) or a pharma- ceutical acceptable salt thereof, During the ceremony R a , R b and R c is H, -(C1-C6) alkyl, -(C6-C 10 )aryl, -C(O)(C1-C6)alkyl, -(C1-C6)alkyl(C6-C 10)heteroaryl, -(C6-C 10 ) independently selected from the group consisting of heteroaryl and -C(O)aryl; M1 and M2 are independently selected from the group consisting of H, -OH, and halogen; n is 1 to 6, preferably 1 to 3, more preferably 1; Q is selected from the group consisting of O, S, CH2, NH, preferably O; U is selected from the group consisting of O, S, CH2, (CH2)2, NH, preferably CH2; T is TIFF2025507601000002.tif20128, preferably TIFF2025507601000003.tif20128; V is selected from the group consisting of O, NH, S, CH2; preferably O; R 1 and R 2 are independently selected from the group consisting of H, OH, SH, -O(C1-C6)alkyl, -S(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N3 and halogen, preferably H and -OH; A is TIFF2025507601000004.tif73165, preferably TIFF2025507601000005.tif20128; X is O, S, N, TIFF2025507601000006.tif14128, preferably N; q is 0 to 6, preferably 0 to 4, more preferably 0; Y is -(C6-C 10 )Aryl-, -(CH2) p -, -(CH2) p C(O)-, -(C6-C 10 )Heteroaryl(CH2) p -, -(C6-C 10 )Heteroaryl-, -(CH2) p (C6-C10 )aryl-, and -(C6-C 10 ) arylC(O)-, preferably -(CH2) p - or -(C6-C 10 ) arylC(O)-; Z is TIFF2025507601000007.tif25128, -(CH2) p -, -(C6-C 10 )Aryl -(C6-C 10 )Aryl(CH2)p, -C(O)(CH2) p -, -(C6-C 10 )Heteroaryl(CH2) p -, -(C6-C 10 )Heteroaryl-, -(C6-C 10 ) arylC(O)-, preferably TIFF2025507601000008.tif19128 and -(C6-C 10 )arylC(O)-, where the aryl group is optionally selected from the group consisting of -(C1-C6)alkyl, -(C2-C6)alkynyl, -halogen, -trifluoromethyl, -OH, -SH, -NH2, -SO2NH2, -(C1-C6)alkylOH, -O(C1-C6)alkyl, -SO3H, -(CH2) 1-6 COOH, -COOH, -C(O)NH2, -SO3(C1-C6)alkyl, -(C5-C6)arylCH2C(O)-, -C(O)NH(CH2) o may be substituted with one or more substituents selected from the group consisting of NH; s represents an integer of 1 to 60, preferably 1 to 50, and more preferably 2 to 30; o is an integer from 1 to 8, preferably from 1 to 4, and most preferably 4; p is an integer from 1 to 6, preferably from 1 to 4, and most preferably 4; L is L 1 and L 2 Including L 1 L 2 Connected to L 1 -L 2 and L 1 is absent, -(CH2) q -, -C(O)NH(CH2) p NH-, -(CH2) q (C5-C 10 )Aryl-, -(C1-C 10 )Alkynyl-, -(C6-C 10 )Aryl(CH2) p -, 1-halo-1-vinyl, -(C6-C 10 )Heteroarylenyl (CH2) p -, -(C6-C 10 )heteroaryl-, and -(C6-C 10 ) arylC(O)-, TIFF2025507601000009.tif12128; s represents an integer of 1 to 60, preferably 1 to 50, and more preferably 2 to 30; q is an integer from 1 to 10, preferably from 1 to 6, and most preferably 5; L 2 is non-existent, TIFF2025507601000010.tif43128 (v is 1 to 9, preferably 1 to 7), and TIFF2025507601000011.tif41128 (z is 1 to 6, preferably 1 to 5, more preferably 5) and / or R 3 teeth TIFF2025507601000012.tif12128 (m is an integer between 2 and 10); or TIFF2025507601000013.tif83164; or iii) a fluorophore moiety selected from the group consisting of FITC, fluorescein, NBD, dansyl, squaraine rotaxane, Bodipy FL, Bodipy TR, Bodipy 630 / 650 X, Bodipy 650 / 655 X, Texas Red, Cy5, 1-pyrene, EVOBlue 30, Alexa Fluor 532, Alexa Fluor 488-5, 488-6, or mixtures thereof, Tamra, Tamra 5 / 6-X-SE, Alexa Fluor 488 azide 5 isomer, Alexa Fluor 488 5 isomer, Alexa Fluor 488 5 / 6 mixed isomer, NIR dye 700, NIR dye 800, Janelia Fluor 549 amide, Janelia Fluor 646 amide, and derivatives, analogs, and related fluorophores thereof; or iv) a chelating moiety that is bound to a radioactive metal. (where the chelating moiety is TIFF2025507601000014.tif242170, and Radioactive metals 64 Cu, 68 Ga, 177 Lu, 90 Y, 89 Zr, 211 At, 212 Pb, 188 Rh, 166 Ho, 225 Ac, 99m Tc or 111 In, 123 I, 131 I) selected from the group consisting of; R 4 , R 5 is H, halogen, -(C1-C6) alkyl, -(C1-C6) aryl, -NH2, -N3, -(C1-C6) alkynyl, -(C6-C 10 )Aryl(C1-C6)alkyl, -1-halogen-1-vinyl, (C6-C 10 )Heteroaryl(C1-C6)alkyl-, -(C6-C 10 )heteroaryl, -C(O)(C6-C10 ) Aryl-, -OR 6 , -SR 6 , -NHR 6 , -NR 6 R 7 , -SiR 6 R 7 R 8 , -OC(O)R 6 , -C(O)R 6 , -COOR 6 , -CONR 6 R 7 , -OC(O)NR 6 R 7 , -NR 6 C(O)R 7 , -NR 6 COOR 7 , -NHC(NH2)=NR 6 , -S(O)R 6 , -SO2NR 6 R 7 , -NR 6 SO2R 7 -CN, and -NO2; or R 6 , R 7 , R 8 is H, halogen, -(C1-C6) alkyl, (C1-C6) aryl, -NH2, -N3, -(C1-C6) alkynyl, -(C6-C 10 )Aryl(C1-C6)alkyl, -1-halogen-1-vinyl, -(C1-C6)alkyl(C6-C 10 )heteroaryl, -(C6-C 10 )heteroaryl, -C(O)(C6-C 10 ) aryl independently selected from the group consisting of -, -SOH, -OH, and -SH; R 9 is halogen, (C1-C6) alkyl, -(C1-C6) aryl, -NH2, -N3, -(C1-C6) alkynyl, -(C6-C 10 )aryl(C1-C6)alkylenyl, 1-halogen-1-vinyl, -(C6-C 10 )Heteroaryl(C1-C6)alkyl-, -(C6-C 10 )heteroaryl, -C(O)(C6-C 10) Aryl-, -OR 6 , -SR 6 , -NHR 6 , -NR 6 R 7 , -SiR 6 R 7 R 8 , -OC(O)R 6 , -C(O)R 6 , -COOR 6 , -CONR 6 R 7 , -OC(O)NR 6 R 7 , -NR 6 C(O)R 7 , -NR 6 COOR 7 , -NHC(NH2)=NR 6 , -S(O)R 6 , -SO2NR 6 R 7 , -NR 6 SO2R 7 -CN, -NO2, and -NO3; Here, R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 wherein the aryl or heteroaryl is optionally substituted with one or more substituents selected from the group consisting of -(C1-C6)alkyl, halogen, -trifluoromethyl, -OH, -SH, -NH2, -SO2NH2, -(C1-C6)alkylhydroxy, -(C1-C6)alkoxy, -SO3H, -COO(C1-C6)alkyl, -SO3(C1-C6)alkyl, -C(O)(C5-C6)aryl; R 10 is OH or TIFF2025507601000015.tif8128; However, R 7 is H and A is TIFF2025507601000016.tif20128, R 5 is H, X is N, Y is CH2, Z is phenyl, and L is -C(O)NH(CH2) 1-4NH- and R 9 When is H or Cl, R3 is not fluorescein.

[0008] In a second aspect, the present invention is directed to a pharmaceutical composition comprising a compound as previously defined and at least one pharma- ceutically acceptable carrier.

[0009] In a third aspect, the present invention is directed to a compound or a pharmaceutical composition as described above for use in a method for the diagnosis of a disease associated with increased CD73 expression.

[0010] In a fourth aspect, the present invention is directed to a compound or a pharmaceutical composition as described above for use in the treatment of a disease associated with increased CD73 expression.

[0011] In a fifth aspect, the present invention is directed to a compound or pharmaceutical composition as described above, wherein the disease is selected from the group consisting of cancer and inflammatory diseases, including autoimmune diseases.

[0012] Preferably, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, melanoma, glioma, head and neck cancer and thyroid cancer, or any other solid cancer.

[0013] Preferably, the inflammatory disease is selected from the group consisting of multiple sclerosis, neuroinflammation, Parkinson's disease and rheumatoid arthritis. [Brief description of the drawings]

[0014] [Figure 1] Figure 1: Biodistribution of 18F-PSB-19427(2) after intravenous injection into adult C57BL / 6 WT mice. (A) Representative PET images (maximum intensity projection) obtained 0-90 min after injection of 18F-PSB-19427(2). (B) Time-activity concentration curves of selected regions of interest (n=6 mice, error bars=standard deviation). (C) Final ex vivo gamma counter readings of selected tissue samples after 90 min (n=3) and 4 h (260 min) (n=3) of the experiment. [Diagram 2] Figure 2: In vivo PET images of 18F-PSB-19427 in tumor-bearing mice (subcutaneous MDA-MB-231 xenografts, left and right shoulders). (A) Representative PET images 4 h after injection of 18F-PSB-19427(2) show a significant accumulation of the tracer in the tumor xenografts (white arrows), which was reduced in the blocking study. Left: no blocker, Right: pretreatment with unlabeled compound 2 10 min before tracer injection. (B). Quantitative analysis confirms a significant accumulation of 18F-PSB-19427(2) in the MDA-MB-231 tumor model compared to muscle tissue (C). In the blocking study, quantitative analysis shows a significant reduction in the tumor / muscle ratio in animals blocked with non-radioactive material (D), thereby demonstrating the binding specificity in vivo. Left: [18F]2 radioactivity concentration in muscle and tumor tissues in unblocked mice after 90 and 260 minutes. Right: Tumor / muscle ratio in unblocked and blocked mice after 90 and 260 minutes. [Diagram 3] Figure 3: (A) Representative in vivo PET images 4 h after injection of 18F-PSB-19427 in tumor-bearing mice (subcutaneous AsPC-1 xenografts, left and right shoulders) show a significant accumulation of the tracer in the tumor xenografts (white arrows), which was reduced in the blocking study. (C) Quantitative analysis confirms a significant accumulation of 18F-PSB-19427 in the tumor compared to muscle tissue. Left: no blocker. Right: pretreatment with PSB-12651 (7) 10 min before tracer injection. (B) In the blocking study, quantitative analysis shows a significant decrease in the tumor / muscle ratio in animals blocked with PSB-12651, thereby demonstrating the binding specificity in vivo. Left: radioactivity concentration of [18F]2 in muscle and tumor tissue after 90 and 260 min in unblocked mice. Right: tumor / muscle ratio after 90 and 260 min in unblocked and blocked mice. [Figure 4A]Figure 4: Exemplary PET images of a comparative experiment of [18F]FDG and [18F]PSB-19427 ([18F]2). A. [18F]FDG and [18F]PSB-19427 ([18F]2) labeled mice after 90 min and 4 h. B. Quantitative analysis of [18F]FDG and [18F]PSB-19427 ([18F]2) uptake in MDA-MB-231 tumors and the resulting tumor / muscle ratios of [18F]FDG and [18F]2. Quantitative analysis confirms higher accumulation of 18F-PSB-19427 in the tumor compared to FDG, especially relative to muscle uptake (B). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Diagram 5] Figure 5: External calibration of reference compounds. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description of the Invention definition The term "alkyl" refers to a monoradical of a saturated straight chain or branched hydrocarbon. Preferably, the alkyl group contains 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, or 6 carbon atoms, more preferably 1 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like.

[0016] The term "aryl" in the context of the present invention refers to an aromatic cyclic hydrocarbon monoradical or diradical. For example, for illustration, in general formula (I), Y and Z may be aryl, where aryl is a diradical. Preferably, aryl groups contain 5 to 14 carbon atoms, which can be arranged in one ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl.

[0017] The term "heteroaryl" refers to an aryl group as defined above, in which one or more carbon atoms in the aryl group are replaced with a heteroatom of O, S, or N. Preferably, heteroaryl refers to a 5- or 6-membered aromatic monocyclic ring in which one, two, or three carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Alternatively, heteroaryl refers to an aromatic bicyclic or tricyclic ring system in which one, two, three, four, or five carbon atoms are replaced with the same or different heteroatoms of O, N, or S. For example, by way of illustration, "-(C6-C 10) Heteroaryl" refers to an aromatic ring having 6 to 10 carbon atoms in the ring, where one or more carbon atoms in the aryl group are replaced with a heteroatom of O, S, or N. Preferably, in each ring of the heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4-, and 1,3,5-), benzofuranyl (1- and 2-), indolyl, isoindolyl, benzothienyl (1- and 2-), 1H-indazolyl, benzyl, and the like. Midazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl (1,2,3- and 1,2,4-benzotriazinyl), pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, pyrrolidinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl (1,5-, 1,6-, 1,7-, 1,8-, and 2,6-), cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (1,7-, 1,8-, 1,10-, 3,8-, and 4,7-), phenazinyl, oxazolopyridinyl, isoxazolopyridinyl, pyrrolooxazolyl, and pyrrolopyrrolyl.Exemplary 5- or 6-membered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4- and 1,3,5-), and pyridazinyl.

[0018] The term "alkynyl" refers to a monoradical or diradical of an unsaturated linear or branched hydrocarbon having at least one carbon-carbon triple bond. For example, for illustration, in general formula (I), L may be alkynyl, where alkynyl is a diradical. In general, the maximum number of carbon-carbon triple bonds in an alkynyl group is equal to an integer calculated by dividing the number of carbon atoms in the alkynyl group by 2, and rounding down the result of the division to the next integer if the number of carbon atoms in the alkynyl group is an odd number. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 4, i.e., 1, 2, 3, or 4, more preferably 1 or 2 carbon-carbon triple bonds. Preferably, the alkynyl group contains 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, e.g., 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in preferred embodiments, an alkynyl group contains from 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 (preferably 1, 2, or 3) carbon-carbon triple bonds, more preferably from 2 to 8 carbon atoms and 1, 2, 3, or 4 (preferably 1 or 2) carbon-carbon triple bonds, such as from 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds, or from 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Exemplary alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl, 1-octynyly ... Examples of nonynyl include octynyl, 2-octynyl, 3-octynyl, 4-octynyl, 5-octynyl, 6-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 5-nonynyl, 6-nonynyl, 7-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 3-decynyl, 4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl, 8-decynyl, 9-decynyl, and the like.In the event that an alkynyl group is attached to a nitrogen atom, the triple bond cannot be alpha to the nitrogen atom.

[0019] The term "halogen" means fluoro, chloro, bromo, or iodo, preferably fluoro. In one embodiment, halogen is fluoro, bromo, or iodo. In another embodiment, halogen is bromo.

[0020] "Pharmaceutically acceptable salt" is intended to mean a salt that retains the biological effectiveness of the free acid and base of a particular compound and is not biologically or otherwise objectionable. The compounds of the present invention have sufficiently acidic, sufficiently basic, or both functional groups, and therefore can react with any of a number of inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts. Exemplary pharmaceutically acceptable salts include salts prepared by reacting the compounds of the present invention with mineral or organic acids or inorganic bases, such as sulfate, pyrosulfate, hydrogen sulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate ... The salts include butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate salts.

[0021] As used herein, the term "analog" refers to a member of a group of compounds that have a common core structure and exhibit a common effect, but differ in elemental composition. In particular, an analog is a compound that has the same core structure as another compound, but differs in one or more atoms, functional groups, or substructures, and retains the common properties.

[0022] compound The present invention relates to a compound represented by the general formula (I) The present invention relates to a compound of formula (I) or a pharma- ceutical acceptable salt thereof, R a , R b and R c is H, -(C1-C6) alkyl, -(C6-C 10 )aryl, -C(O)(C1-C6)alkyl, -(C1-C6)alkyl(C6-C 10 )heteroaryl, -(C6-C 10 ) independently selected from the group consisting of heteroaryl and -C(O)aryl, preferably H; M1 and M2 are independently selected from the group consisting of H, -OH and halogen, preferably H; n is 1 to 6, preferably 1 to 3, more preferably 1; Q is selected from the group consisting of O, S, CH2, NH, preferably O; U is selected from the group consisting of O, S, CH2, (CH2)2, NH, preferably CH2; T is TIFF2025507601000018.tif20128, preferably TIFF2025507601000019.tif20128; V is selected from the group consisting of O, NH, S, and CH2; preferably O. R 1 and R 2are independently selected from the group consisting of H, OH, SH, -O(C1-C6)alkyl, -S(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N3 and halogen, preferably H and -OH; In one embodiment, R 1 is OH and R 2 is H. A is TIFF2025507601000020.tif73165, preferably TIFF2025507601000021.tif20128; X is O, S, N, TIFF2025507601000022.tif14128, preferably N; q is 0 to 6, preferably 0 to 4, more preferably 0; Y is -(C6-C 10 )Aryl-, -(CH2) p -, -(CH2) p C(O)-, -(C6-C 10 )Heteroaryl(CH2) p -, -(C6-C 10 )Heteroaryl-, -(CH2) p (C6-C 10 )aryl-, and -(C6-C 10 ) arylC(O)-, preferably -(CH2) p - or -(C6-C 10 ) ArylC(O)-, more preferably -(CH2) p - and; Z is TIFF2025507601000023.tif25128, -(CH2) p -, -(C6-C 10 )Aryl -(C6-C 10 )Aryl(CH2)p, -C(O)(CH2) p -, -(C6-C 10 )Heteroaryl(CH2) p -, -(C6-C 10 )Heteroaryl-, -(C6-C10 ) arylC(O)-, preferably TIFF2025507601000024.tif19128 and -(C6-C 10 )arylC(O)-, where the aryl group is optionally selected from the group consisting of -(C1-C6)alkyl, -(C2-C6)alkynyl, -halogen, -trifluoromethyl, -OH, -SH, -NH2, -SO2NH2, -(C1-C6)alkylOH, -O(C1-C6)alkyl, -SO3H, -(CH2) 1-6 COOH, -COOH, -C(O)NH2, -SO3(C1-C6)alkyl, (C5-C6)arylCH2C(O)-, -C(O)NH(CH2) o may be substituted with one or more substituents selected from the group consisting of NH; o is an integer from 1 to 8, preferably from 1 to 4, and most preferably 4; p is an integer from 1 to 6, preferably from 1 to 4, and most preferably 4; L is L 1 and L 2 Including L 1 L 2 Connected to L 1 -L 2 and L 1 does not exist, -(CH2)q-, -C(O)NH(CH2) p NH-, -(CH2) q (C5-C 10 )Aryl-, -(C1-C 10 )Alkynyl-, -(C6-C 10 )Aryl(CH2) p -, 1-halo-1-vinyl, -(C6-C 10 )Heteroarylenyl (CH2) p -, -(C6-C 10 )heteroaryl-, and -(C6-C 10 ) arylC(O)-, TIFF2025507601000025.tif12128; preferably L is absent or -C(O)NH(CH2) 1-4 NH-; s represents an integer of 1 to 60, preferably 1 to 50, and more preferably 2 to 30; q is an integer from 1 to 10, preferably from 1 to 6, and most preferably 5; L 2 is non-existent, TIFF2025507601000026.tif43128 (v is 1 to 9, preferably 1 to 7; v may be 1, 2, 3, 4, 5, 6 or 7, preferably 1, 4 or 7; TIFF2025507601000027.tif41128 (z is 1 to 6, preferably 1 to 5, more preferably 5) selected from the group consisting of; R 3 teeth TIFF2025507601000028.tif12128 (m is an integer between 2 and 10); or TIFF2025507601000029.tif171170, iii) a fluorophore moiety selected from the group consisting of FITC, fluorescein, NBD, dansyl, squaraine rotaxane, Bodipy FL, Bodipy TR, Bodipy 630 / 650 X, Bodipy 650 / 655 X, Texas Red, Cy5, 1-pyrene, EVOBlue 30, Alexa Fluor 532, Alexa Fluor 488-5, 488-6, or mixtures thereof, Tamra, Tamra 5 / 6-X-SE, Alexa Fluor 488 azide 5 isomer, Alexa Fluor 488 5 isomer, Alexa Fluor 488 5 / 6 mixed isomer, NIR dye 700, NIR dye 800, Janelia Fluor 549 amide, Janelia Fluor 646 amide, and derivatives, analogs, and related fluorophores thereof; or iv) a chelating moiety that is bound to a radioactive metal. (where the chelating moiety is TIFF2025507601000030.tif237170, and Radioactive metals 64 Cu, 68Ga, 177 Lu, 90 Y, 89 Zr, 211 At, 212 Pb, 188 Rh, 166 Ho, 225 Ac, 99m Tc or 111 In, 123 I, 131 I) selected from the group consisting of; Preferably, R 3 CH2-F 18 , TIFF2025507601000031.tif58149 (m is 2 to 4, preferably 2) selected from the group consisting of; R 4 , R 5 is H, halogen, -(C1-C6) alkyl, -(C1-C6) aryl, -NH2, -N3, -(C1-C6) alkynyl, -(C6-C 10 )Aryl(C1-C6)alkyl, -1-halogen-1-vinyl, (C6-C 10 )Heteroaryl(C1-C6)alkyl-, -(C6-C 10 )heteroaryl, -C(O)(C6-C 10 ) Aryl-, -OR 6 , -SR 6 , -NHR 6 , -NR 6 R 7 , -SiR 6 R 7 R 8 , -OC(O)R 6 , -C(O)R 6 , -COOR 6 , -CONR 6 R 7 , -OC(O)NR 6 R 7 , -NR 6 C(O)R 7 , -NR 6 COOR 7 , -NHC(NH2)=NR 6 , -S(O)R 6 , -SO2NR6 R 7 , -NR 6 SO2R 7 is independently selected from the group consisting of -CN, and -NO2; preferably H, -(C1-C6)alkyl or halogen, preferably H or halogen, more preferably halogen; R 6 , R 7 , R 8 is H, halogen, -(C1-C6) alkyl, (C1-C6) aryl, -NH2, -N3, -(C1-C6) alkynyl, -(C6-C 10 )Aryl(C1-C6)alkyl, -1-halogen-1-vinyl, -(C1-C6)alkyl(C6-C 10 )heteroaryl, -(C6-C 10 )heteroaryl, -C(O)(C6-C 10 ) aryl independently selected from the group consisting of -, -SO3H, -OH, and -SH; Preferably R 6 is H, -(C1-C6) alkyl, -(C6-C 10 )Aryl(C1-C6)alkyl, -(C1-C6)alkyl(C6-C 10 ) heteroaryl, Preferably R 7 is H or -(C1-C6)alkyl, preferably H or (C2-C4)alkyl; more preferably (C3)alkyl; Preferably R 8 is H or -(C1-C6)alkyl; X is N; and / or Y is -(C1-C6)alkyl-; and / or R 9 is halogen, (C1-C6) alkyl, -(C1-C6) aryl, -NH2, -N3, -(C1-C6) alkynyl, -(C6-C 10 )aryl(C1-C6)alkylenyl, 1-halogen-1-vinyl, -(C6-C 10 )Heteroaryl(C1-C6)alkyl-, -(C6-C 10 )heteroaryl, -C(O)(C6-C 10) Aryl-, -OR 6 , -SR 6 , -NHR 6 , -NR 6 R 7 , -SiR 6 R 7 R 8 , -OC(O)R 6 , -C(O)R 6 , -COOR 6 , -CONR 6 R 7 , -OC(O)NR 6 R 7 , -NR 6 C(O)R 7 , -NR 6 COOR 7 , -NHC(NH2)=NR 6 , -S(O)R 6 , -SO2NR 6 R 7 , -NR 6 SO2R 7 , -CN, and -NO2, preferably H, halogen, or -(C1-C6)alkyl, more preferably H or halogen; Here, R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 wherein the aryl or heteroaryl is optionally substituted with one or more substituents selected from the group consisting of -(C1-C6)alkyl, halogen, -trifluoromethyl, -OH, -SH, -NH2, -SO2NH2, -(C1-C6)alkylhydroxy, -(C1-C6)alkoxy, -SO3H, -COO(C1-C6)alkyl, -SO3(C1-C6)alkyl, -C(O)(C5-C6)aryl; R 10 is OH or TIFF2025507601000032.tif8128; However, R 7 is H and A is TIFF2025507601000033.tif20128, R 5is H, X is N, Y is CH2, Z is phenyl, and L is -C(O)NH(CH2) 1-4 NH- and R 9 When is H or Cl, R3 is not fluorescein.

[0023] In one embodiment of formula (I), R a , R b and R c is H; and / or M1 and M2 are H; and / or n is 1; and / or Q is O; and / or U is CH2; and / or R 1 is -OH; and / or R 2 is H; and / or A is TIFF2025507601000034.tif20128; and / or R 4 is H, -(C1-C6)alkyl or halogen, preferably H or halogen, more preferably halogen; and / or R 5 is H, and / or R 6 is H, -(C1-C6) alkyl, -(C6-C 10 )Aryl(C1-C6)alkyl, -(C1-C6)alkyl(C6-C 10 ) heteroaryl; and / or R 7 is H or -(C1-C6)alkyl, preferably H or (C2-C4)alkyl; more preferably (C3)alkyl; and / or R 8 is H or -(C1-C6)alkyl; and / or R 9 is H, halogen, or -(C1-C6)alkyl, preferably H or halogen; and / or X is N; and / or Y is -(CH2) p -and / or Z is TIFF2025507601000035.tif19128 or -(C6-C 10 ) arylC(O); and / or L is absent or -C(O)NH(CH2) 1-4 is NH-; and / or R 3 teeth TIFF2025507601000036.tif11128 or fluorescein.

[0024] In one embodiment, the compound is TIFF2025507601000037.tif138146TIFF2025507601000038.tif183128.

[0025] In a further aspect V is O; Q is O; U is CH2; A is TIFF2025507601000039.tif20128; X is N; Y is CH2; Z is phenyl or TIFF2025507601000040.tif24128; L is absent or -C(O)NH(CH2) p NH-; -C(O)NH(CH2) p p in NH- is 1 to 6, preferably 4 to 6, and most preferably 6; R 3 CH2-F 18 , TIFF2025507601000041.tif58148 (m is 2 to 4, preferably 2) It is.

[0026] Pharmaceutical Compositions The present invention is further directed to pharmaceutical compositions comprising the aforementioned compounds and at least one pharma- ceutically acceptable carrier.

[0027] "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as, but not limited to, water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered orally, water is the preferred carrier. When the pharmaceutical composition is administered intravenously, saline and aqueous dextrose solutions are the preferred carriers. Saline and aqueous dextrose and glycerol solutions are preferably used as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories, with traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions will contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with an appropriate amount of carrier to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0028] The actual dosage level of the compound in the pharmaceutical composition of the present invention may vary for a particular patient, composition, and mode of administration to obtain an amount of active ingredient effective to achieve the desired therapeutic response without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in conjunction with the particular composition used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical art.

[0029] Generally, out of 100% (of the pharmaceutical formulation / composition), the amount of active ingredient (particularly the amount of a compound of the present invention, optionally together with other therapeutically active agents, if present in the pharmaceutical formulation / composition) ranges from about 0.01% to about 99%, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, wherein the remainder is preferably comprised of one or more pharma- ceutically acceptable excipients.

[0030] The amount of active ingredient, e.g., a compound of the invention, in a unit dosage form and / or when administered to an individual or used in therapy may range from about 0.1 mg to about 1000 mg (e.g., about 1 mg to about 500 mg, e.g., about 10 mg to about 200 mg) per unit, administration or treatment. In certain embodiments, an appropriate amount of such an active ingredient may be calculated using the body weight or body surface area of ​​the individual, and may be about 1 mg / Kg to 10 mg / Kg (e.g., about 2 mg / Kg to 5 mg / Kg), or about 1 mg / m 2 ~about 400mg / m 2 (approx. 3mg / m 2 ~about 350mg / m 2 or about 10 mg / m 2 ~about 200mg / m 2 etc.)

[0031] For therapeutic / pharmaceutical formulations, the compositions of the present invention include those suitable for enteral administration (such as oral or rectal) or parenteral administration (such as nasal, topical (including vaginal, buccal and sublingual)). The compositions may conveniently be presented in unit dosage form and may be prepared by any method known in the art of pharmacy. The amount of active ingredient (particularly the amount of the compound of the present invention) that can be combined with a carrier material to produce a pharmaceutical composition (such as a single dosage form) will vary depending on the individual being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect.

[0032] As used herein, the term "enteral administration" and "administered parenterally" refers to the administration of a drug by the stomach and / or intestine. Examples of enteral administration include oral and rectal administration. As used herein, the term "parenteral administration" and "administered parenterally" refers to a mode of administration other than enteral administration, usually by injection or topical application, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraosseous, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intracerebral, intraventricular, subarachnoid, intraspinal, epidural and intrapleural administration (such as by injection and / or infusion) and topical administration (e.g., on the skin, by inhalation, or through mucous membranes (such as oral, sublingual or vaginal)).

[0033] For oral administration, the pharmaceutical compositions of the invention can take the form of tablets or capsules prepared by conventional means with pharma- ceutically acceptable excipients, such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica), disintegrants (e.g., potato starch, sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Liquid preparations for oral administration can be in the form of, for example, solutions, syrups, or suspensions, or can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means using pharma- ceutically acceptable additives, such as suspending agents (e.g., sorbitol, syrups, cellulose derivatives, hydrogenated edible fats), emulsifying agents (e.g., lecithin, acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, fractionated vegetable oils), preservatives (e.g., methyl- or propyl-p-hydroxycarbonate, sorbic acid), and the like. Preparations can also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the pharmaceutical compositions of the present invention.

[0034] The pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0035] For administration by inhalation, the pharmaceutical compositions of the present invention are conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, nitrogen, or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated to contain a powder mix of the pharmaceutical composition of the present invention and a suitable powder base, such as lactose or starch.

[0036] The pharmaceutical compositions of the present invention can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Injectable preparations may be presented in unit dosage form (e.g., ampoules, multi-dose containers) with the addition of preservatives. The pharmaceutical compositions of the present invention can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing or dispersing agents. Alternatively, the agent may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied separately or mixed in unit dosage form, e.g., as a lyophilized powder or water-free concentrate in a sealed container such as an ampule or sachet indicating the quantity of active agent. When the composition is administered by injection, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0037] medical applications The present invention is further directed to a compound or a pharmaceutical composition as described above for use in a method for the diagnosis of a disease associated with increased CD73 expression.

[0038] The present invention is further directed to a compound or agent for use in the treatment of a disease associated with increased CD73 expression.

[0039] The disease may be selected from the group consisting of cancer, and inflammatory diseases.

[0040] The cancer is preferably a solid tumor, more preferably a cancer selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, melanoma, glioma, head and neck cancer and thyroid cancer.

[0041] Preferably, the inflammatory disease is selected from the group consisting of multiple sclerosis and rheumatoid arthritis. EXAMPLES

[0042] Examples of the present invention CD73-PET ligands were synthesized as described below. 18 A F-labeled CD73 antagonist was obtained. Compound 2 is the first PET ligand developed for labeling and imaging of CD73. TIFF2025507601000042.tif36149

[0043] Scheme 1. Radiolabeling of ethynyl-substituted precursor 1 (PSB-19425) by click reaction. 18 F-labeled CD73 inhibitor 2 ([ 18 F]PSB-19427). Scheme 1. Radiolabeling of ethynyl-substituted precursor 1 (PSB-19425) by click reaction. 18 F-labeled CD73 inhibitor 2 ([ 18 F]PSB-19427).

[0044] Scheme 2. 18 F-labeled PET tracer [ 18 F]2([ 18 Synthesis of unlabeled analogue 2 (PSB-19427) of [F]PSB-19427 TIFF2025507601000043.tif83164 α Reagents and conditions: (a) trifluoromethanesulfonic acid, 90000 Pa (0.9 bar), 85 °C → room temperature, 1 hour; (b) methanol, room temperature, 2 days; (c) triethylamine, ethanol, reflux, room temperature, overnight; (d) 1M NaOCH3, methanol, room temperature, overnight; (e) 2 steps (i) methylenebis(phosphonic dichloride), trimethyl phosphate, 0°C, 1 h; (ii) 0.5 M triethylammonium bicarbonate (TEAC) buffer pH 7.4-7.6, 15 min at 0°C, then 1 h at room temperature; (f) NaN3, dimethylformamide (DMF), rt, 24 h; (g) sodium ascorbate, CuSO4, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), tetrahydrofuran (THF) / H2O / tert-butanol (t-BuOH), rt, overnight; (h) 2-fluoroethyl azide, sodium ascorbate, CuSO4, TBTA, DMF / H2O, overnight, rt.

[0045] Synthesis of unlabeled compounds Scheme 3. Alkylation of (4-ethionylphenyl)methanamine α TIFF2025507601000044.tif24128 α Reagents and conditions: 1-bromopropane, methanol, room temperature, 2 days.

[0046] Compound 10 (N-(4-ethynylbenzyl)propan-1-amine) was synthesized by alkylation of compound 9 with 1-bromopropane in methanol.

[0047] Scheme 4. Synthesis of 2,6-dichloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine 2 α TIFF2025507601000045.tif41128 α Reagents and conditions: trifluoromethanesulfonic acid, 90000 Pa (0.9 bar), 85°C → room temperature, 1 hour.

[0048] 2,6-Dichloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine (5) was synthesized starting from 1,2,3,5-tetraacetyl-β-D-ribofuranose (3) and 2,6-dichloropurine (4). The reaction was carried out using previously reported reaction conditions. 13 Both compounds were melted at 85°C and reacted after the addition of trifluoromethanesulfonic acid.

[0049] Scheme 5. Synthesis of (2R,3R,4S,5R)-2-(2-chloro-6-((4-ethynylbenzyl)(propyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol α TIFF2025507601000046.tif52160 α Reagents and conditions: (a) 10, triethylamine, ethanol, reflux, room temperature, overnight; (b) 1 M sodium methoxide solution in methanol, room temperature, overnight.

[0050] 2',3',5'-Tri-O-acetyl-2,6-dichlororibofuranosylpurine (5) was reacted with N-(4-ethynylbenzyl)propan-1-amine (10) in the presence of triethylamine in ethanol under basic conditions. The reaction was carried out at reflux overnight to give the desired product as well as the partially deacetylated derivative of 6. The product was not isolated but was used directly in the next step to give the fully deacetylated product 11 by treatment with 1 M sodium methoxide solution in methanol.

[0051] Scheme 6. Synthesis of ((((2R,3S,4R,5R)-5-(2-chloro-6-((4-ethynylbenzyl)(propyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid α TIFF2025507601000047.tif43134 αReagents and conditions: two steps (i) methylene bis(phosphonic dichloride), trimethyl phosphate, 0° C., 1 h; (ii) 0.5 M TEAC buffer pH 7.4-7.6, 15 min at 0° C., then 1 h at room temperature.

[0052] The phosphonylation reaction and subsequent hydrolysis were carried out according to previously published methods with some modifications. 13、14、15、16 Phosphonylation was carried out by reaction of 11 with methylene bis(phosphonic dichloride) in trimethyl phosphate for 1 h on ice. Final hydrolysis was achieved with freshly prepared 0.5 M TEAC buffer. Extraction with tert-butyl methyl ether (TBME) followed by purification by reversed-phase high-performance liquid chromatography (RP-HPLC) afforded the desired product 1.

[0053] Scheme 7. Synthesis of 1-azido-2-fluoroethane α TIFF2025507601000048.tif18128 α Reagents and conditions: Sodium azide, DMF, 24 hours, room temperature.

[0054] 2-Fluoroethyl-4-toluenesulfonate (7) was dissolved in anhydrous DMF. After the addition of sodium azide, the reaction mixture was stirred at room temperature for 24 hours and monitored by TLC. Since the isolation of 1-azido-2-fluoroethane (8) may cause an explosion, the crude mixture was filtered and used in the following reaction step without further purification.

[0055] Scheme 8. Synthesis of ((((2R,3S,4R,5R)-5-(2-chloro-6-((4-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)benzyl)(propyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid α TIFF2025507601000049.tif42155 αReagents and conditions: 2-fluoroethyl azide, sodium ascorbate, CuSO4, TBTA, THF / H2O / t-BuOH, overnight, room temperature.

[0056] To obtain the desired final product 2, (((((2R,3S,4R,5R)-5-(2-chloro-6-((4-ethynylbenzyl)(propyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid (1) was coupled to 1-azido-2-fluoroethane (8) via azide-alkyne Huisgen cycloaddition using TBTA, copper sulfate and sodium ascorbate in a mixture of THF, HO and t-BuOH. Purification by RP-HPLC afforded the desired product 2 in 18% yield.

[0057] Scheme 9. Synthesis of (2R,3R,4S,5R)-2-(2-chloro-6-((4-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)benzyl)(propyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol α TIFF2025507601000050.tif48139 α Reagents and conditions: 2-fluoroethyl azide (8), sodium ascorbate, CuSO4, TBTA, DMF / H2O, overnight, room temperature.

[0058] (2R,3R,4S,5R)-2-(2-chloro-6-((4-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)benzyl)(propyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (12) was synthesized using azide-alkyne Huisgen cycloaddition reaction conditions. To crude 1-azido-2-fluoroethane (8) dissolved in DMF was added 11 and TBTA. Sodium ascorbate and copper sulfate were dissolved in water and then added to the reaction mixture. The mixture was stirred at ambient temperature overnight. Extraction followed by purification by chromatography on silica gel afforded the desired product 12 (73%).

[0059] [ 18 F]-[((2R,3S,4R,5R)-5-{2-chloro-6-[(4-{1-[2-(fluoro)ethyl]-1H-1,2,3-triazol-4-yl}benzyl)(propyl)amino]-9H-purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-yl)methoxy]methylenebisphosphonic acid ([ 18 F]2) TIFF2025507601000051.tif76128

[0060] Computer-controlled TRACERLab Fx FDG In the synthesizer, aqueous [ 18 Batches of [F]F-ions (3.1-5.2 GBq) were passed through an anion exchange resin (Sep-Pak® Light QMA cartridge preconditioned with carbonate counterions). 18 [F]F-ions were dissolved in 1 M K2CO3 (aqueous, 40 μL), water for injection (WFI, 200 μL), and Kryptofix® 2.2.2 (K 2.2.2 The resin was eluted with a mixture of acetonitrile (800 μL, DNA grade) containing K (K 2.2.2 )[ 18The aqueous solution of [F]F was carefully evaporated to dryness under reduced pressure. A certain amount of the precursor compound 2-azidoethyl 4-methylbenzenesulfonate (20 mg, 83 μmol) in acetonitrile (DNA grade, 500 μL) was added and the mixture was heated at 110 °C for 3 min. Meanwhile, the labeled 1-azido-2-[ 18 [F]fluoroethane was distilled from the reactor into an ice-cold 10 mL flask containing a mixture of 1 (5.0 mg, 8.1 μmol) in DMF (300 μL), CuSO4·5H2O (40 mg, 160 μmol) in HEPES buffer (pH: 5.7, 100 μL) and sodium ascorbate (63 mg, 318 μmol) in HEPES buffer (pH: 5.7, 100 μL). After stirring at 60 °C for 30 min, the mixture was passed through a PTFE sterile filter (0.2 μm). The filter was washed with DMF (500 μL) and then with WFI (500 μL). The combined filtrate and washings were purified by gradient radio-HPLC system A (Method A). Compound [ 18 F]2 product fraction (retention time t R ([ 18 F]2) = 12.2 min) was collected in a flask pre-treated with Sigmacote® and the solution was evaporated to dryness under reduced pressure. The residue was redissolved in WFI / EtOH (1 mL, 9:1 v / v). The product compound [ 18 F]2 was obtained at 119 ± 10 min from the end of radionuclide production with a total radiochemical yield of 21.2 ± 3.0% (cyclotron-derived [ 18 F]F-ion was used for the decay correction, n = 21). 18 [F]2 was isolated with radiochemical purity of >99% and molar radioactivity ranging from 2.3 to 54.6 GBq / μmol. 18 F]2(retention time t R ([ 18 The radiochemical purity and molar radioactivity of [F]2) = 9.4 min) were determined by analytical radio-HPLC B (Method B).

[0061] In vitro stability in mouse and human serum Radioactive ligand [ 18The serum stability of [F]2 was evaluated by incubation in mouse serum at 37°C for up to 90 min. 18 A fixed amount of [F]2 (20 μL, 3.9 MBq) was added to a sample of mouse serum (200 μL) and the mixture was incubated at 37°C. After 10, 30, 60 and 90 min, 20 μL of each sample was removed and quenched with ice-cold acetonitrile (100 μL, DNA grade) followed by centrifugation (3000 rpm) for >5 min. The supernatant was analyzed by analytical radio-HPLC B (Method B). Serum stability tests in human serum were performed similarly.

[0062] [ 18 F]2 logD 7.4 Determining Values [ 18 The lipophilicity of [F]2 was determined by Prante et al. 17 The triazole [ 18 [F]2 (approximately 400 kBq) was added to PBS buffer (590 μL, pH 7.4) and octan-1-ol (600 μL). The biphasic mixture was shaken on a vortex mixer at room temperature for 3 min and centrifuged (3000 rpm) for 5 min. The main part of the octanol layer (400 μL) was carefully collected and added to a new tube containing PBS buffer (400 μL, pH 7.4). The biphasic mixture was shaken again for 10 min and centrifuged (3000 rpm) for 5 min. Triplicate samples were prepared and two aliquots (100 μL) of both layers were measured in a g-counter 2480 Wizard2 (Perkin-Elmer, Waltham, USA). The partition coefficient was calculated by dividing cpm (octanol) by cpm (PBS) and expressed as logD 7.4 (exp.).

[0063] Partition coefficient (logD(exp.)) [ 18 F]2 is a hydrophilic compound with a logD(exp.) of -0.12±0.03. The calculated logP (clogP) is 0.22 2 and 0.47 3 In comparison, the [18 The measured solubility of [F]2 was increased by 2.2- and 3.9-fold, respectively. 2 Calculated logP values ​​(clogP) were calculated using ChemBioDraw Ultra 13.0. 3 Calculated logP (clogP) was calculated using ACD / Chemsketch freeware.

[0064] Measurement of plasma protein binding (PPB) was performed by Boergel et al. 24 I followed the procedure in For the quantification of the different analytes, the LC system was coupled with a single quadrupole (SQ) mass spectrometer. UPLC-UV / MS (Agilent, Waldbronn, Germany): Pump: 1260 Bin Pump (G1212B); Degasser: 1260 HiP (G4225A); Column oven: 1290 TCC (G1316C), 30 °C; Autosampler: 1260 HiP ALS (G1367E), 1 μL injection unless otherwise stated; UV / vis detector: 1260 VWD (G1314F); MS source: Multimode source (G1978B); MS detector: 6120 quadrupole (G1978B); MS parameters: Vaporizer temperature: 250 °C; Drying gas: 10 L / min; Nebulizer pressure: 40 psi; Capillary voltage: 3000 V; Fragmenter voltage: 100 V; Drying gas temperature: 350 °C; LC parameters: Precolumn: Chiralpak® HSA HPLC Guard Column (2.0 x 10 mm, particle size 5 μm); Main column: Chiralpak® HSA HPLC Column (2.0 x 50 mm, particle size 5 μm, Daicel, Eschborn, Germany); Temperature: 25°C; Mobile phase: aqueous ammonium acetate (50 mM, pH 7.4) / i-propanol 96:4; Flow rate: 0.3 mL / min; Isocratic. Standard compounds and 2 were dissolved in methanol and retention times were measured by LC-MS. D-glucose was used to measure the dead time of the system (0.75 min). Standard compounds were used to plot k' / (k'+1) against known PPB values ​​(Tables 1, 2). TIFF2025507601000052.tif9128 here k'=retention factor t R = retention time [minutes] t D = Dead time [min]

[0065] Table 1: Measured retention times and known PPB values ​​of standard compounds TIFF2025507601000053.tif100170

[0066] Triple-negative breast cancer (TNBC) is often associated with increased expression of CD73. This not only makes CD73 a good target for cancer immunotherapy, but also provides an opportunity to target CD73 for diagnosis. The use of highly potent nucleotide-derived CD73 inhibitors as diagnostic tools with fluorescent, ultrasound-labeled or radioactive nucleotides may facilitate early localization of primary tumors and metastases in TNBC and improve prognosis.

[0067] Design of fluorescently labeled CD73 inhibitors Schmies et al. recently reported the synthesis of a fluorescein-labeled AOPCP-derived CD73 inhibitor. 6 The p-position of the -benzyl-substituent was found to be suitable for the introduction of a linker bearing a fluorescein moiety. 18 However, initial attempts to synthesize the 2-chloro substituted analogues failed. 18 Additional substitution at the 2-position, for example with a chloro substituent, can lead to improved properties. 15 developed a new strategy to synthesize the desired fluorescently labeled 2-chloro substituted AOPCP derivatives.

[0068] The successful synthesis and pharmacological evaluation of the resulting (((((2R,3S,4R,5R)-5-(2-chloro-6-((4-((6-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamido)hexyl)carbamoyl)benzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid is described below.

[0069] Synthesis of chemically-fluorescently labeled CD73 inhibitors 6-Carboxyfluorescein (13) was coupled to commercially available N-boc-1,6-hexanediamine by amide coupling in DMF using O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HATU) as the coupling reagent and N,N-diisopropylethylamine (DIPEA) as the base to give 14. 19 Subsequent boc deprotection using trifluoroacetic acid (TFA) in dichloromethane (DCM) gave 15. 18 4-(boc-aminobenzyl)benzoic acid was then coupled to the primary amino group of 15, again using HATU and DIPEA, to give 16, followed by boc deprotection using TFA in DCM to give 17. 18、19 In a parallel reaction, commercially available 2,6-dichloro-9-(β-D-ribofuranosyl)purine (18) was phosphonylated with methylene bis(phosphonic dichloride) in trimethyl phosphate and subsequently quenched with aqueous triethylammonium bicarbonate (TEAC) buffer according to an optimized published method. 13、18 RP-HPLC purification afforded the pure product 19. In the final reaction step, nucleophilic substitution of 19 with primary amine 17 in the presence of triethylamine in absolute ethanol under reflux conditions, followed by purification by RP-HPLC afforded the desired final product 20 (23% yield). 18 20 structures, 1 H, 13 C and 31Confirmation by P-NMR spectroscopy and LC / ESI-(UV)MS analysis (positive and negative modes) showed a purity of >95%.

[0070] Scheme 10. Synthetic procedure to obtain compound 20 a TIFF2025507601000054.tif63170 a Reagents and conditions: (a) N-boc-1,6-hexanediamine, HATU, DIPEA, rt, overnight; (b) 6-8% TFA, DCM, rt, 5 h; (c) 4-(boc-aminobenzyl)benzoic acid, HATU, DIPEA, rt, overnight; (d) 6-8% TFA, DCM, rt, 5 h; (e) two-step: (i) methylenebis(phosphonic dichloride), trimethyl phosphate, Ar, 0 °C, 1 h; (ii) TEAC buffer pH 7.4-7.6, rt, 1 h; (f) EtN, anhydrous EtOH, reflux, overnight.

[0071] Scheme 11. Synthesis of tert-butyl (6-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamido)hexyl)carbamate a TIFF2025507601000055.tif33143 a Reagents and conditions: N-boc-1,6-hexanediamine, HATU, DIPEA, room temperature, overnight. 6-Carboxyfluorescein (13) was coupled to commercially available N-boc-1,6-hexanediamine by amide coupling using HATU and DIPEA in DMF to give 14. 19 .

[0072] Scheme 12. Synthesis of N-(6-aminohexyl)-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide a TIFF2025507601000056.tif24130 a Reagents and conditions: 6-8% TFA, DCM, room temperature, 5 h. After the reaction, boc deprotection of 14 with TFA in DCM gave 15. 18 .

[0073] Scheme 13. Synthesis of tert-butyl (4-((6-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamido)hexyl)carbamoyl)benzyl)carbamate a TIFF2025507601000057.tif23136 a Reagents and conditions: 4-(boc-aminobenzyl)benzoic acid, HATU, DIPEA, room temperature, overnight. 4-(boc-aminobenzyl)benzoic acid was then coupled to the primary amine of 15 using HATU and DIPEA in DMF to give 16. 19 .

[0074] Scheme 14. Synthesis of N-(6-(4-(aminomethyl)benzamido)hexyl)-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide a TIFF2025507601000058.tif20136 a Reagents and conditions: 6-8% TFA, DCM, room temperature, 5 h. Subsequent boc deprotection of 16 using TFA in DCM gave 17. 18 .

[0075] Scheme 15. Synthesis of (((((2R,3S,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid a TIFF2025507601000059.tif38128 a Reagents and conditions: Two steps: (i) methylene bis(phosphonic dichloride), trimethyl phosphate, Ar, 0 °C, 1 h; (ii) TEAC buffer pH 7.4-7.6, room temperature, 1 h.

[0076] Commercially available 2,6-dichloro-9-(β-D-ribofuranosyl)purine (18) was phosphonylated with methylene bis(phosphonic dichloride) in trimethyl phosphate, followed by quenching with aqueous TEAC buffer. Both reaction steps were carried out according to optimized published procedures to give 19. 13、18 .

[0077] Scheme 16. Synthesis of ((((2R,3S,4R,5R)-5-(2-chloro-6-((4-((6-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamido)hexyl)carbamoyl)benzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid a TIFF2025507601000060.tif83131 a Reagents and conditions: Et3N, absolute EtOH, reflux, overnight.

[0078] In the final reaction step, nucleophilic substitution of 19 with primary amine 17 in the presence of triethylamine in absolute ethanol under reflux conditions, followed by purification by RP-HPLC, afforded the desired final product 20 (23% yield). 18 .

[0079] Pharmacological evaluation Compound 2b, its precursors 1, and 20 were examined for their CD73 inhibitory potential using tritium-labeled AMP as a substrate. 20 After incubation with CD73, the non-hydrolyzed substrate and inorganic phosphate were precipitated with lanthanum chloride solution, and the precipitate was identified as the reaction product [2,8- 3The CD73 was separated from the solution containing [H] adenosine by filtration through GF / B glass fiber filters (Whatman™ GE Healthcare, Chicago, IL, USA) using a cell harvester (M-48, Brandel, Gaitherburg, MD, USA). The filtrates (approximately 1.8 mL each) were transferred to scintillation vials, scintillation cocktail ULTIMA Gold XR (PerkinElmer, Waltham, MA, USA) was added (5 mL), and radioactivity was quantified by scintillation counting. Preparations of CD73 were: (1) recombinant soluble human CD73; 13 (2) Recombinant soluble rat CD73 21 , both of which were expressed in Spodoptera frugiperda 9 (Sf9) cells, and (3) in MDA-MB-231 cells, a human triple-negative breast cancer cell line that highly expresses CD73. 16 Membrane preparations of 18 , and (4) membrane preparations of the 4T1.2 cell line, a murine CD73-expressing breast cancer cell line. 22 The potency of the compounds was determined by measuring the complete concentration inhibition curve. i The obtained IC values 50 The values ​​were calculated using the Cheng-Prusoff equation. 23 .

[0080] Table 1. Potency of compounds 1, 2 and 20 on different CD73 preparations a . TIFF2025507601000061.tif68142 a [2,8- 3 H]AMP (5 μM) was used as a substrate; the K values ​​for purified recombinant soluble human CD73, native membrane-anchored human CD73 (in MDA-MB-231 cell membrane preparation), and purified recombinant soluble rat CD73 were m Values ​​59 μM, 17 μM and 14.8 μM; nd - not determined.

[0081] Inhibition of human NTPDase The inhibitory activities of CD73 inhibitors 2 and 20 on human NTPDases 1, 2, 3, and 8 were examined using ATP (100 μM) as a substrate according to a previously published procedure. 24 ATP and 2 were incubated with recombinantly expressed human NTPDase for 30 min at 37 °C, and the enzymatic reaction was terminated by heating at 90 °C for 10 min. The amount of enzyme preparation was adjusted to ensure 10–20% substrate conversion. Products were separated by capillary electrophoresis (CE) and individually quantified by diode array detection (DAD) at a wavelength of 260 nm. For inhibition analysis, at least three independent experiments were performed in triplicate each. Inhibition of enzyme activity was calculated relative to a positive control without inhibitor and plotted with GraphPad Prism 8 software (GraphPad software, San Diego, CA, USA) (see Tables 2 and 3 for results).

[0082] ADP receptor effects The interaction of 2 with the Gq protein-coupled ADP-activated human P2Y1 receptor, which was stably expressed in 1231N1 astrocytoma cells, was determined by calcium mobilization assay. 25、26 (See Table 2 for results.) Compound 20 was not tested at the P2Y1 receptor because the fluorescent compound would interfere with the calcium mobilization assay. 12 Interaction with the receptor was examined in a β-arrestin recruitment assay using the galactosidase complementation technique as previously described. 25-27 Human P2Y 12 The receptor was expressed in CHO-PK1 cells (Eurofins DiscoverX, Fremont, Calif., USA). See Tables 2 and 3 for results.

[0083] Table 2: Compound 2 and human NTPDase and human ADP-activated G protein-coupled receptors P2Y1 and P2Y 12 Interaction with TIFF2025507601000062.tif180158 aFor the antagonist test, 2-methylthio-ADP was used as an agonist and its EC 80 It was used at a concentration of 3 μM.

[0084] (Table 3) Compound 20 and human NTPDase and human ADP-activated G protein-coupled receptor P2Y 12 Interaction with TIFF2025507601000063.tif133158 a For the antagonist test, 2-methylthio-ADP was used as an agonist and its EC 80 It was used at a concentration of 3 μM.

[0085] Compound synthesis experiment details General All reagents were obtained commercially from a variety of suppliers (Acros, Aldrich, Fluka, Merck, Sigma, etc.) and used without further purification. Commercially available solvents of specific reagent grade were used without further purification or drying. Reactions were run on silica gel 60 F. 254The fractions were monitored by thin layer chromatography (TLC) using aluminum sheets (Merck) and dichloromethane / methanol mixtures (99:1-3:1) as developing solvent. Column chromatography was performed on silica gel 0.060-0.200 mm, pore size approximately 6 nm. Mass spectra were recorded on an API2000 mass spectrometer (ABSciex) with an ESI source connected to an HPLC HP1100 instrument (Agilent) with an EC50 / 2 Nucleodur C18 Gravity 3 μm column (Macherey-Nagel). Samples were dissolved in H2O / methanol mixtures containing 2 mM ammonium acetate. 8 μl of sample was injected and a flow rate of 0.3 ml / min was applied. Elution was performed with a gradient of water / methanol 90:10-0:100 in 10 min. The column was then flushed with 100% methanol containing 2 mM ammonium acetate for 10 min. Positive total ion scans were observed from 150 to 800 m / z. UV absorption was detected from 190 to 900 nm using a diode array detector (DAD). Purity was determined from 220 to 400 nm. The purity of all test compounds was 95% or higher. High-resolution spectra were recorded using flow injection mode on a micrOTOF-Q mass spectrometer (Bruker) with an ESI source connected to an HPLC Dionex Ultimate 3000 (Thermo Scientific) instrument. Sample solutions were injected and a flow rate of 0.3 ml / min was applied. Elution was performed with acetonitrile containing 0.1% acetic acid or 0.1% formic acid, respectively. Positive or negative full scan MS was observed from 50 to 1000 m / z. Sodium acetate or sodium formate was used as an internal calibrant. 1 H, 13 C and 31 P-NMR spectra were performed on a Bruker AVANCE 500 or Bruker AVANCE III HD 600 MHz spectrometer using DMSO-d6, MeOD-d4, or D2O as solvents. 31 P NMR spectra were recorded at room temperature; orthophosphoric acid (85%) was used as the external standard. Shifts are expressed as 31P NMR spectrum), or relative to the residual protons of the deuterated solvent used as an internal standard ( 1 H, 13 C NMR spectra are given in ppm. Melting points were determined on a Büchi 530 melting point apparatus and are uncorrected. Preparative HPLC was performed on a Knauer Smartline 1050 HPLC system equipped with a Knauer Eurospher-100 C18 column, 250 mm × 20 mm, 10 μm particle size. UV absorption was detected at 254 nm. A freeze dryer (CHRIST ALPHA 1-4 LSC) was used for lyophilization.

[0086] Radiochemistry Overall Method The first step of radiosynthesis was performed using an improved PET tracer radiosynthesizer (TRACERLab Fx FDGThe separation and purification of radiolabeled compounds was carried out on a semi-preparative radio-HPLC system A: K-500 and K-501 pumps, K-2000 UV detector (Herbert Knauer GmbH), NaI(TI) Scintibloc 51 SP51 γ-detector (Crismatec) and an ACE 5 AQ column (250 mm × 10 mm). Method A started with a linear gradient of 10% to 90% CH3CN in water (0.1% TFA) in 30 min, held for 5 min, followed by a linear gradient of 90% to 10% CH3CN in water (0.1% TFA) in 5 min, at l = 254 nm and a flow rate of 5.0 mL / min. Radiochemical purity and molar radioactivity were determined using analytical radio-HPLC system B: two Smartline 1000 pumps with a Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar gamma detector (Raytest Isotopenmessgeraete GmbH) and a Nucleosil 100-5 C-18 column (250 mm x 4 mm). Method B started with a linear gradient of 10% to 100% CH3CN in water (0.1% TFA) in 15 min, held for 3 min, followed by a linear gradient of 100% to 10% CH3CN in water (0.1% TFA) in 2 min, at 1 = 254 nm and a flow rate of 1.0 mL / min. Data recorded from both HPLC systems were processed with GINA Star software (Raytest Isotopenmessgeraete GmbH). Aqueous [ 18 [F] fluoride was measured at the RDS 111e cyclotron (CTI-Siemens) 18 O(p,n) 18 97.0% enriched by F nuclear reaction [ 18 O]H2O was produced by irradiation of a water target (2.8 mL) with a 10 MeV proton beam.

[0087] Recombinant soluble CD73 Human soluble CD73 was expressed in Fall Armyworm (Sf9) insect cells and purified as previously described.16 cDNA encoding mature human CD73 (residues 27-549) fused to a C-terminal 6xHis tag (Genbank accession number NM_002526) corresponding to the natural variant T376A (P21589 / VAR_022091, UniProtKB / Swiss-Prot) was ligated into the pAcGP67B vector. For transfection, 1 μl of recombinant vector (1000 ng / μl) mixed with 2.5 μl of baculovirus genomic DNA ProEasy™ (AB vector, CA, USA) was used to transfect Sf9 cells cultured in Insect-XPRESS™ medium (#BE12-730Q, Lonza, Switzerland) supplemented with 10 mg / l gentamicin. The produced soluble enzyme was then concentrated by ultrafiltration on an Amicon® Ulta-15 filter, 10 KDa cutoff (Merck Millipore, MA, USA) and then subjected to metal affinity chromatography (IMAC) purification on a HisPur™ Ni-NTA spin column (#88226, Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol. The purified enzyme was aliquoted and stored at -80°C until further use. Soluble rat CD73 glutathione-S-transferase fusion protein was expressed in Sf9 insect cells using a previously published method. 21 .

[0088] cell culture Human triple-negative breast cancer cells (MDA-MB-231) natively expressing CD73 were cultured in Dulbecco's modified Eagle's medium (DMEM, #:41966, Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS, #:P30-1502, PAN Biotech, Germany) and 100 U / ml penicillin + 100 μg / ml streptomycin (#:P06-07100, PAN Biotech, Germany). Cells were incubated for 72 h at 37 °C with 5% CO2 until they reached confluence (80-90%). Confluent cells were washed with phosphate-buffered saline (PBS) and then detached by incubation with trypsin / EDTA (0.05% / 0.6 mM, #P10-022100, PAN Biotech, Germany) for 5 min. Detached cells were resuspended in medium and split 1:20.

[0089] Preparation of membranes 175cm 2 Confluent cells cultured in a culture flask were detached as described above. 2) at approximately 100 cells / dish and incubated at 37°C, 5% CO2 for 4 days. The medium was discarded, the dishes were washed with 10 ml of PBS and frozen at -20°C. The frozen cells were scraped off the dishes with 1 ml of ice-cold buffer (50 mM Tris, 2 mM EDTA, pH 7.4), collected in a conical tube and then centrifuged at 1000g for 10 min at 4°C. The pellet was then resuspended in buffer (0.5 ml / dish; 25 mM Tris, 1 mM EDTA, 320 mM sucrose, pH 7.4, 1:1000 protease inhibitor cocktail #P8340, Sigma-Aldrich, MO, USA) and homogenized three times for 30 s each (20,500 rpm, Ultraturrax, IKA-Labortechnik, Germany). The homogenate was centrifuged at 1000g for 10 min at 4°C and the supernatant was collected and centrifuged for a further 30 min at 48,000g at 4°C. The resulting pellet was resuspended in wash buffer (0.5 ml / dish) and centrifuged using the same conditions. After three further washing steps, the pellet was resuspended in Tris buffer 50 mM, pH 7.4 (0.1 ml / dish), aliquoted and stored at -80°C until use.

[0090] Enzyme Inhibition Assay Compounds were tested using previously described methods. 20 Stock solutions were prepared in demineralized water and further diluted in reaction buffer (Tris 25 mM, NaCl 140 mM, sodium dihydrogen phosphate 25 mM, pH 7.4). For screening, 10 μl of each test compound was transferred to the respective test tube containing 70 μl of reaction buffer. To determine the concentration-response curve, 10 μl of different dilutions of the test compound were pipetted into the test tubes. A solution or suspension of soluble or membrane-bound CD73 (10 μl per vial, soluble rat CD73: 1.63 ng; soluble human CD73: 0.365 ng; membrane preparation of MDA-MB-231 cells expressing CD73: 7.4 ng of protein) was transferred to all test tubes except the negative control. The substrate [2,8- 3The reaction was started by the addition of [H]AMP (specific activity 7.4 × 108 Bq / mmol (20 mCi / mmol), American Radio-labeled Chemicals, MO, USA, supplied by Hartman Analytic, Germany) in a volume of 10 μl (final concentration 5 μM). After 25 min of incubation at 37 °C in a shaking water bath, the samples were cooled on ice and the reaction was stopped and precipitated by the addition of 500 μl of precipitation buffer (lanthanum chloride, 100 mM in 100 mM sodium acetate, pH 4.0). Samples were kept on ice for at least 30 min until complete precipitation was obtained, and then filtered through GF / B glass fiber filters using a Brandel cell harvester (M-48, Brandel, MD, USA). Reaction vials were washed three times with 400 μl each of cold (4°C) demineralized water, then 5 ml of scintillation cocktail (ULTIMA Gold XR, PerkinElmer, MA, USA) was added and radioactivity was measured using a scintillation counter (Tri-Carb 2900TR, Packard / PerkinElmer). All experiments were performed in duplicate, baseline corrected and normalized to negative and positive controls. Three independent experiments were performed and data were analyzed using Prism-GraphPad 7 (GraphPad Software, La Jolla, USA). Ki values ​​were calculated using the Cheng-Prusoff equation with the following K values ​​(K, rat CD73: 53.0 μM; K, human CD73: 17.0 μM; K, (MDA-MB-231): 14.8 μM). 23 .

[0091] Capillary electrophoresis assay of human NTPDase 1, 2, 3 and 8 Test compounds were tested at ATP substrate concentrations of 50 μM and 100 μM according to previously published procedures. 24Recombinantly expressed human NTPDase was used. The amount of enzyme preparation was adjusted to ensure 10–20% substrate conversion. The reaction buffer contained 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, pH 7.4. Samples were incubated at 37 °C for 30 min and the enzymatic reaction was stopped by heating at 90 °C for 10 min. Samples were cooled on ice and then examined by capillary electrophoresis (CE) with diode array detection (DAD) at a wavelength of 260 nm. For inhibition analysis, at least three independent experiments were performed in triplicate (n = 3). Inhibition of enzyme activity was calculated relative to a positive control without inhibitor and plotted with GraphPad Prism 8 software (GraphPad software, San Diego, CA, USA).

[0092] The analysis was performed using a P / ACE MDQ capillary electrophoresis system (Beckman Instruments, Fullerton, CA, USA). Separation was performed in a polyacrylamide-coated capillary [30 cm (10 cm effective length) × 50 μm (id) × 360 μm (od)]. Before each run, the capillary was washed with background electrolyte (50 mM phosphate buffer, pH 6.5) at 30 psi for 1 min. Samples were electrokinetically injected at the capillary outlet with a voltage of −6 kV for 30 s, and nucleotides were separated at a voltage of −15 kV and a pressure of 0.2 psi. Detection was at a wavelength of 260 nm. Data collection and peak area analysis were performed with P / ACE MDQ software 32 KARAT from Beckman Coulter (Fullerton, CA, USA). Quantification by external standard curves for AMP and ADP and corrected areas under the electrophoretic peaks at 260 nm.

[0093] In vivo experiments Animal models All animal experiments were performed in accordance with the legal requirements of the European Community (Directive 2010 / 63 / EU) and the corresponding German Animal Welfare Law (TierSchG, TierSchVersV) and were approved by the local approval authority (State Office for Nature, Environment and Consumer Protection North Rhine-Westphalia). MDA-MB-231 or AsPC-1 cells were cultured in RPMI supplemented with 10% fetal bovine serum and 100 U / mL penicillin and 100 μg / mL streptomycin. 5 × 10 6 MDA-MB-231 or AsPC-1 cells were injected subcutaneously into the shoulder region of 10–12 week-old NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ, Charles River Laboratories). Two tumors were implanted per mouse and growth was tracked by digital caliper measurements (volume = 1 / 2(length * width)). 2 )).

[0094] In vivo imaging Adult C57bl / 6 (biodistribution studies, 21±1 g) or tumor-bearing NSG mice (24±3 g) were anesthetized with isoflurane / O2 and the tail vein was cannulated using a 27 G needle connected to a 15 cm polyethylene catheter tubing. 18F-PSB-19427 (~450 kBq / g body weight) was injected as a bolus via the tail vein (50 μL compound, flushed with 50 μL saline) followed by PET scanning. For blocking studies, a subgroup of tumor-bearing mice was injected with ~1000-fold excess of unlabeled PSB-19427 10 min prior to radiotracer injection. PET imaging studies were performed using a sub-millimeter high-resolution (0.7 mm full width at half maximum) small animal scanner (32-module quadHIDAC, Oxford Positron Systems Ltd., Oxford, UK) with uniform spatial resolution (<1 mm) over a large cylindrical field (165 mm diameter, 280 mm axial length). List-mode data were acquired for 90 min and reconstructed into dynamic time frames using an iterative reconstruction algorithm. In a subgroup of mice, dynamic measurements were complemented by a later time point PET scan 4 h post-injection. After PET acquisition, the scan bed was transferred to a computed tomography (CT) scanner (Inveon, Siemens Medical Solutions, US) and a CT acquisition with a spatial resolution of 80 μm was performed for each mouse. The reconstructed image datasets were coregistered based on external markers attached to the multimodal scanning bed and the in-house developed image analysis software MEDgical. Three-dimensional volumes of interest (VOIs) were defined over each organ in the CT dataset, transferred to the coregistered PET data and quantitatively analyzed. Regional uptake was calculated as a percentage of the injected dose by dividing the counts per milliliter in the VOI by the total counts in the mouse and multiplying by 100 (%ID / mL).

[0095] In vitro gamma counter measurement After final PET-CT acquisition at 90 or 250 min post-injection, respectively, mice were euthanized by cervical dislocation and necropsy was performed. Ex vivo distribution of radioactivity was analyzed by scintillation counting (Wizard2 gamma counter, Perkin-Elmer Life Science), and radioactivity in each organ was decay-corrected and calculated as %ID per gram of tissue (%ID / g).

[0096] Preparation of triethylammonium bicarbonate buffer A 1M solution of TEAC was prepared by the following procedure. Dry ice was slowly added to a 1M solution of triethylamine in deionized water until the pH value reached approximately 8.4-8.6. The solution was stirred for several hours. 1 .

[0097] TIFF2025507601000064.tif401282,6-Dichloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine (5) The reaction was carried out using previously reported reaction conditions. 13 1,2,3,5-Tetraacetyl-β-D-ribofuranose (5 g, 16.0 mmol, 1.0 equiv.) was melted at 85° C. and 2,6-dichloropurine (3.0 g, 16.0 mmol, 1.0 equiv.) was added with stirring. To catalyze the reaction, trifluoromethanesulfonic acid (70 μl, 0.8 mmol, 0.05 equiv.) was added to the reaction mixture. The reaction mixture was stirred at 85° C. under reduced pressure for 30 min. Analysis by thin layer chromatography (TLC) was performed, which showed the completion of the reaction. The mixture was then cooled to room temperature. The desired product was obtained by recrystallization from absolute ethanol (4.84 g, 69% yield). TIFF2025507601000065.tif38165LC-MS (m / z): Positive mode 447.0 [M+H] + Purity as determined by HPLC-UV (254 nm)-ESI-MS: 88%. Mp: 160° C.

[0098] TIFF2025507601000066.tif16128N-(4-ethynylbenzyl)propan-1-amine (10) Compound 10 (N-(4-ethynylbenzyl)propan-1-amine) was synthesized according to the following reaction conditions: (4-ethynylphenyl)methanamine (1, 0.5 g, 3.8 mmol, 1.0 equiv.) was reacted with 1-bromopropane (0.34 ml, 3.8 mmol, 1.0 equiv.) in methanol at room temperature for 2 days. The desired product precipitated in the reaction mixture and was filtered to give 0.27 g of 10 (46%). TIFF2025507601000067.tif32170LC-MS (m / z): Positive mode 173.9 [M+H] + Purity as determined by HPLC-UV (254 nm)-ESI-MS: 95%.

[0099] TIFF2025507601000068.tif42128(2R,3R,4S,5R)-2-(2-chloro-6-((4-ethynylbenzyl)(propyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (11) 2',3',5'-Tri-O-acetyl-2,6-dichlororibofuranosylpurine (5, 0.34 g, 0.77 mmol, 1.0 equiv.) was suspended in absolute ethanol. Triethylamine (0.2 ml, 1.54 mol, 2.0 equiv.) and N-(4-ethynylbenzyl)propan-1-amine 10 (0.26 g, 1.54 mmol, 2.0 equiv.) were added to the suspension and refluxed overnight. The progress of the reaction was monitored by silica gel TLC (DCM / methanol 9 / 1). After TLC showed the reaction was complete, the solvent was evaporated. Subsequently, 6 was deprotected using 1M sodium methoxide in methanol (10 ml) without further purification. Purification by column chromatography (methanol / DCM 1:19) afforded the desired product as a white solid (0.25 g, 71%). TIFF2025507601000069.tif52170LC-MS (m / z): Positive mode 458.2 [M+H] + Purity as determined by HPLC-UV (254 nm)-ESI-MS: 88%. Mp. 110° C.

[0100] TIFF2025507601000070.tif42128(((((2R,3S,4R,5R)-5-(2-chloro-6-((4-ethynylbenzyl)(propyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid (1) The phosphonylation reaction and subsequent hydrolysis were carried out according to a previously reported method with modified reaction conditions. 13~15、16 (2R,3R,4S,5R)-2-(2-chloro-6-((4-ethynylbenzyl)(propyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (11, 0.1 g, 0.22 mmol, 1.0 equiv.) was dissolved in trimethyl phosphate (2 ml) and stirred at 0-4 °C. A solution of methylene bis(phosphonic dichloride) (0.27 g, 1.09 mmol. 5.0 equiv.) in trimethyl phosphate (3 ml) was added cooled to 0-4 °C. The reaction mixture was stirred at 0-4 °C and samples were taken at 15 min intervals for TLC to control the disappearance of the nucleoside. After 30 min, once the nucleoside was completely reacted, 20 ml of cold 0.5 M aqueous TEAC (pH 7.4-7.6) was added and the solution was stirred at 0 °C for 15 min followed by 1 h at room temperature. Trimethyl phosphate was extracted with 2 × 250 ml of tert-butyl methyl ether and the aqueous layer was lyophilized. The crude product was then purified by RP-HPLC (0-50% MeCN / 50 mM NH4HCO3 buffer in 20 min, 20 ml / min) and the appropriate fractions were pooled. After lyophilization, the product was obtained as a white solid (0.11 g, 89%). TIFF2025507601000071.tif52170LC / ESI-MS (m / z): Positive mode 616.1130 [M+H] + (C 23 H 29 Calculated value for ClN5O9P2 is 616.1129) and negative mode is 614.0991 [MH] - (C 23 H 27 Calculated for ClN5O9P2: 614.0973. Purity as determined by HPLC-UV (254 nm)-ESI-MS: 84%. Mp. 141 °C.

[0101] TIFF2025507601000072.tif121281-Azido-2-fluoroethane(8) 2-Fluoroethyl-4-toluenesulfonate (7, 0.05 ml, 0.3 mmol, 1.0 equiv) was dissolved in anhydrous DMF (0.5 ml). Sodium azide (0.05 g, 0.9 mmol, 3.0 equiv) was added. The reaction mixture was stirred at room temperature for 24 h and monitored by TLC. The crude mixture was filtered and the filtrate was used without further purification. WARNING: Attempting to isolate pure 1-azido-2-fluoroethane may result in explosion.

[0102] TIFF2025507601000073.tif42128(((((2R,3S,4R,5R)-5-(2-chloro-6-((4-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)benzyl)(propyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid (2) (((((2R,3S,4R,5R)-5-(2-chloro-6-((4-ethynylbenzyl)(propyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid 1 (15.0 mg, 0.02 mmol, 1.0 equiv) was dissolved in THF / HO / t-BuOH (3:1:1, 0.5 ml). Crude 2-fluoroethyl azide 8 (50.0 μl) was added. Additionally, 1 M sodium ascorbate in HO (0.03 ml, 0.03 mmol, 1.2 equiv) was added. Finally, a premixed solution of CuSO4 (1.0 mg, 0.009 mmol, 0.3 equiv.) and TBTA (4.0 mg, 0.003 mmol, 0.3 equiv.) in THF / H2O / t-BuOH (3:1:1, 0.5 ml) was added to the reaction mixture. The reaction mixture was stirred at room temperature in the dark under an argon atmosphere. During the reaction, the color of the reaction mixture changed from bright yellow to a cloudy mint green. After stirring overnight, the reaction mixture was evaporated and directly purified by RP-HPLC (0–70% MeCN / 50 mM NH4HCO3 buffer within 20 min, 20 ml / min). The appropriate fractions were pooled and lyophilized overnight to give the final product as a white solid (0.003 g, 17.5%). TIFF2025507601000074.tif58170LC / ESI-MS (m / z): Positive mode 705.1486 [M+H] + (C 25 H 33 Calculated value of ClFN8O9P2 is 705.1518) and negative mode is 703.1335 [MH] - (C 25 H 31 Calculated value for ClFN8O9P2: 703.1362). Purity as determined by HPLC-UV (254 nm)-ESI-MS: 99%. Mp. 169 °C. Estimated log P 27 3.3, estimated clogP: 0.22.

[0103] (2R,3R,4S,5R)-2-(2-chloro-6-((4-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)benzyl)(propyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (12) TIFF2025507601000075.tif50128 (2R,3R,4S,5R)-2-(2-chloro-6-((4-ethynylbenzyl)(propyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (11, 0.080 g, 0.017 mmol, 1.0 equiv) was dissolved in a solution of 8 in DMF (5 ml). TBTA (0.028 g, 0.052 mmol, 0.3 equiv) was added to the reaction mixture. Copper sulfate (0.008 g, 0.052 mmol, 0.3 equiv) and sodium ascorbate (0.041 g, 0.21 mmol, 1.2 equiv) were premixed in 2 ml of water and then added to the reaction mixture, which was then stirred overnight. After TLC showed the reaction was complete, the reaction mixture was diluted with water and extracted with ethyl acetate, followed by washing with aqueous lithium chloride (10%). The organic phase was dried over sodium sulfate, evaporated, and then purified by normal phase column chromatography (silica gel, DCM / methanol 95 / 5). Appropriate fractions were pooled and the eluent was evaporated to give the desired compound as a colorless solid (yield: 76%, 0.073 g). Mp. 180-183 °C. TIFF2025507601000076.tif71170LC / ESI-MS (m / z): Positive mode 547.40 [M+H] + Purity as determined by HPLC-UV (254 nm)-ESI-MS: 98%. Mp. 180-183°C.

[0104] [ 18 F]-[((2R,3S,4R,5R)-5-{2-chloro-6-[(4-{1-[2-(fluoro)ethyl]-1H-1,2,3-triazol-4-yl}benzyl)(propyl)amino]-9H-purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-yl)methoxy]methylenebisphosphonic acid ([ 18 F]2) TIFF2025507601000077.tif76128 Computer-controlled TRACERLab Fx FDG In the synthesizer, aqueous [ 18 Batches of [F]F-ions (3.1-5.2 GBq) were passed through an anion exchange resin (Sep-Pak® Light QMA cartridge preconditioned with carbonate counterions). 18 [F]F-ions were dissolved in 1 M K2CO3 (aqueous, 40 μL), water for injection (WFI, 200 μL), and Kryptofix® 2.2.2 (K 2.2.2 The resin was eluted with a mixture of acetonitrile (800 μL, DNA grade) containing K (K 2.2.2 )[ 18 The aqueous solution of [F]F was carefully evaporated to dryness under reduced pressure. A certain amount of the precursor compound 2-azidoethyl 4-methylbenzenesulfonate (20 mg, 83 μmol) in acetonitrile (DNA grade, 500 μL) was added and the mixture was heated at 110 °C for 3 min. Meanwhile, the labeled 1-azido-2-[ 18 [F]fluoroethane was distilled from the reactor into an ice-cold 10 mL flask containing a mixture of PSB-19425 (1, 5.0 mg, 8.1 μmol) in DMF (300 μL), CuSO4·5H2O (40 mg, 160 μmol) in HEPES buffer (pH: 5.7, 100 μL) and sodium ascorbate (63 mg, 318 μmol) in HEPES buffer (pH: 5.7, 100 μL). After stirring at 60 °C for 30 min, the mixture was passed through a PTFE sterile filter (0.2 μm). The filter was washed with DMF (500 μL) and then with WFI (500 μL). The combined filtrate and washings were purified by gradient radio-HPLC system A (Method A). Compound [ 18 F]PSB-19427 product fraction (retention time t R ([ 18F]PSB-19427) = 12.2 min) was collected in a flask pre-treated with Sigmacote® and the solution was evaporated to dryness under reduced pressure. The residue was redissolved in water for injection (WFI) / EtOH (1 mL, 9:1 v / v). The product compound [ 18 F]PSB-19427 was synthesized in 119 ± 10 min from the end of radionuclide production with a total radiochemical yield of 21.2 ± 3.0% (cyclotron-derived [ 18 F]F-ion decay correction, n = 20). 18 [F]PSB-19427 was isolated with radiochemical purity of >99% and molar radioactivity ranging from 2.3 to 176.6 GBq / μmol. 18 F]PSB-19427(retention time t R ([ 18 The radiochemical purity and molar radioactivity of [F]PSB-19427) = 9.4 min) were determined by analytical radio-HPLC B (Method B).

[0105] In vitro stability in mouse and human serum Radioactive ligand [ 18 The serum stability of [F]PSB-19427 was evaluated by incubation in mouse serum at 37°C for up to 90 min. 18 A fixed amount of [F]PSB-19427 (20 μL, 3.9 MBq) was added to a sample of mouse serum (200 μL) and the mixture was incubated at 37 °C. After 10, 30, 60 and 90 min, 20 μL of each sample was removed and quenched with ice-cold acetonitrile (100 μL, DNA grade) followed by centrifugation (3000 rpm) for >5 min. The supernatant was analyzed by analytical radio-HPLC B (Method B). Serum stability tests in human serum were performed similarly.

[0106] [ 18 F]PSB-19427 logD 7.4 Determining Values [ 18 The lipophilicity of [F]PSB-19427 was determined by Prante et al. 28 The triazole [ 18[F]PSB-19427 (approximately 400 kBq) was added to PBS buffer (590 μL, pH 7.4) and octan-1-ol (600 μL). The biphasic mixture was shaken on a vortex mixer at room temperature for 3 min and centrifuged (3000 rpm) for 5 min. The main part of the octanol layer (400 μL) was carefully collected and added to a new tube containing PBS buffer (400 μL, pH 7.4). The biphasic mixture was shaken again for 10 min and centrifuged (3000 rpm) for 5 min. Triplicate samples were prepared and two aliquots (100 μL) of both layers were measured in a g-counter 2480 Wizard2 (Perkin-Elmer, Waltham, USA). The partition coefficient was calculated by dividing cpm (octanol) by cpm (PBS) and expressed as logD 7.4 (exp.).

[0107] Partition coefficient (logD(exp.)) [ 18 F]PSB-19427 is a hydrophilic compound with a logD(exp.) of -0.12 ± 0.03. The calculated logP (clogP) is 0.22 13 and 0.47 15 In comparison, [ 18 The measured solubility of [F]PSB-19427 was increased by 2.2- and 3.9-fold, respectively.

[0108] Measurement of plasma protein binding (PPB) was performed by Boergel et al. 13 I followed the procedure in For the quantification of the different analytes, the LC system was coupled with a single quadrupole (SQ) mass spectrometer. UPLC-UV / MS (Agilent, Waldbronn, Germany): Pump: 1260 Bin Pump (G1212B); Degasser: 1260 HiP (G4225A); Column oven: 1290 TCC (G1316C), 30 °C; Autosampler: 1260 HiP ALS (G1367E), 1 μL injection unless otherwise stated; UV / vis detector: 1260 VWD (G1314F); MS source: Multimode source (G1978B); MS detector: 6120 quadrupole (G1978B); MS parameters: Vaporizer temperature: 250 °C; Drying gas: 10 L / min; Nebulizer pressure: 40 psi; Capillary voltage: 3000 V; Fragmenter voltage: 100 V; Drying gas temperature: 350 °C; LC parameters: Precolumn: Chiralpak® HSA HPLC Guard Column (2.0 x 10 mm, particle size 5 μm); Main column: Chiralpak® HSA HPLC Column (2.0 x 50 mm, particle size 5 μm, Daicel, Eschborn, Germany); Temperature: 25°C; Mobile phase: aqueous ammonium acetate (50 mM, pH 7.4) / i-propanol 96:4; Flow rate: 0.3 mL / min; Isocratic. Standard compounds and PSB-19427 were dissolved in methanol and retention times were measured by LC-MS. D-glucose was used to measure the dead time of the system (0.75 min). Standard compounds were used to plot k' / (k'+1) against known PPB values ​​(Table 1, Error! Reference not found). TIFF2025507601000078.tif9128 here k'=retention factor t R = retention time [minutes] t D = Dead time [min]

[0109] Table 2: Measured retention times and known PPB values ​​of standard compounds TIFF2025507601000079.tif100170

[0110] Synthesis of chemically-fluorescently labeled CD73 inhibitors 5(6)-Carboxyfluorescein (21) was coupled to commercially available N-boc-1,6-hexanediamine by amide coupling in DMF using O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HATU) as the coupling reagent and N,N-diisopropylethylamine (DIPEA) as the base to give 22. 19 Subsequent boc deprotection using trifluoroacetic acid (TFA) in dichloromethane (DCM) gave 23. 18 4-(boc-aminobenzyl)benzoic acid was then coupled to the primary amino group of 23, again using HATU and DIPEA, to give 24, followed by boc deprotection using TFA in DCM to give 28. 18、19 .

[0111] Commercially available 2-amino-6-chloro-9-(β-D-ribofuranosyl)-9H-purine (25) was reacted by a non-aqueous diazotization reaction with tert-butyl nitrite (TBN) and trimethylsilyl bromide (TMSBr) in dibromomethane to give 2-bromo-6-chloro-9-(β-D-ribofuranosyl)purine (26). 31、32 .

[0112] 2-Bromo-6-chloro-9-(β-D-ribofuranosyl)purine (26) was then phosphonylated with methylene bis(phosphonic dichloride) in trimethyl phosphate, followed by quenching with aqueous ammonium bicarbonate buffer. 13、18 .

[0113] RP-HPLC purification afforded the pure product 27. In the final reaction step, nucleophilic substitution of 27 with primary amine 28 in the presence of triethylamine in absolute ethanol under reflux conditions, followed by purification by RP-HPLC, afforded the desired final product 20 (8% yield). 18 29 structures, 1 H, 13 C and 31Confirmation by P-NMR spectroscopy and LC / ESI-(UV)MS analysis (positive and negative modes) showed a purity of >95%.

[0114] Scheme 17. Synthetic procedure to obtain compound 20 a TIFF2025507601000080.tif64170 a Reagents and conditions: (a) N-boc-1,6-hexanediamine, HATU, DIPEA, rt, overnight; (b) 6-8% TFA, DCM, rt, 5 h; (c) 4-(boc-aminobenzyl)benzoic acid, HATU, DIPEA, rt, overnight; (d) 6-8% TFA, DCM, rt, 5 h; (e) TBN, TMSBr, rt, overnight; (f) two-step: (i) methylenebis(phosphonic dichloride), trimethyl phosphate, Ar, 0 °C, 1 h; (ii) NH4CO3 buffer, rt, 1 h; (g) Et3N, anhydrous EtOH, reflux, overnight.

[0115] Scheme 18. Synthesis of tert-butyl (5(6)-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamido)hexyl)carbamate a TIFF2025507601000081.tif33143 a Only the 6-position isomer is shown. Reagents and conditions: N-boc-1,6-hexanediamine, HATU, DIPEA, room temperature, overnight. 5(6)-Carboxyfluorescein (21) was coupled to commercially available N-boc-1,6-hexanediamine by amide coupling using HATU and DIPEA in DMF to give 22. 19 .

[0116] Scheme 19. Synthesis of N-(6-aminohexyl)-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide a TIFF2025507601000082.tif30162 aReagents and conditions: 6-8% TFA, DCM, room temperature, 5 h. After the reaction, boc deprotection of 22 with TFA in DCM gave 23. 18 .

[0117] Scheme 10. Synthesis of tert-butyl (4-((6-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamido)hexyl)carbamoyl)benzyl)carbamate a TIFF2025507601000083.tif28162 a Reagents and conditions: 4-(boc-aminobenzyl)benzoic acid, HATU, DIPEA, room temperature, overnight. 4-(boc-aminobenzyl)benzoic acid was then coupled to the primary amine of 23 using HATU and DIPEA in DMF to give 24. 19 .

[0118] Scheme 21. Synthesis of N-(6-(4-(aminomethyl)benzamido)hexyl)-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide a TIFF2025507601000084.tif25170 a Reagents and conditions: 6-8% TFA, DCM, room temperature, 5 h. Subsequent boc deprotection of 24 using TFA in DCM gave 28. 18 .

[0119] Scheme 22. Synthesis of 2-bromo-6-chloro-9-(β-D-ribofuranosyl)-9H-purine a TIFF2025507601000085.tif38128 a Reagents and conditions: TMSBr, TBN, CH2BR2, room temperature under argon atmosphere overnight.

[0120] Commercially available 2-amino-6-chloro-9-(β-D-ribofuranosyl)purine (25) was reacted by a non-aqueous diazotization reaction with tert-butyl nitrite (TBN) and trimethylsilyl bromide (TMSBr) in dibromomethane according to a modified version of a previously published method to give 26. 31、32 .

[0121] Scheme 23. Synthesis of (((((2R,3S,4R,5R)-5-(2-bromo-6-chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid TIFF2025507601000086.tif38128 a Reagents and conditions: Two steps: (i) methylenebis(phosphonic dichloride), trimethyl phosphate, Ar, 0°C, 1 h; (ii) NH4CO3, room temperature, 1 h.

[0122] 2-Bromo-6-chloro-9-(β-D-ribofuranosyl)-9H-purine (26) was phosphonylated with methylene bis(phosphonic dichloride) in trimethyl phosphate, followed by quenching with aqueous TEAC buffer. Both reaction steps were carried out according to optimized published procedures to give 27. 13 .

[0123] Scheme 24. Synthesis of ((((2R,3S,4R,5R)-5-(2-chloro-6-((4-((6-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamido)hexyl)carbamoyl)benzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid a TIFF2025507601000087.tif83131 a Reagents and conditions: Et3N, absolute EtOH, reflux, overnight.

[0124] In the final reaction step, nucleophilic substitution of 27 with primary amine 28 in the presence of triethylamine in absolute ethanol under reflux conditions, followed by purification by RP-HPLC, afforded the desired final product 29 (8% yield). 18 .

[0125] Scheme 25. Synthesis of BODIPY-labeled CD73 inhibitors a TIFF2025507601000088.tif147168 α Reagents and conditions: (a) 5-norbornene-2-carboxylic acid, HATU, DIPEA, DCM, rt, overnight; (b) 6-8% TFA, DCM, rt, overnight; (c) trifluoromethanesulfonic acid, 90000 Pa (0.9 bar), 85°C → rt, 1 h; (d) 2 steps: (i) 4-(aminomethyl)benzoic acid, triethylamine, ethanol, 90°C, overnight; (ii) 2N in MeOH NH3, rt, overnight; (e) HATU, DIPEA, DMF, rt, overnight; (f) 2 step: (i) methylenebis(phosphonic dichloride), trimethyl phosphate, Ar, 0°C, 1 h; (ii) NH4CO3 buffer, rt, 1 h; (g) 2 step: (i) Zn(OTf)2, ACN, hydrazine hydrate, 60°C, 24 h, rt, 24 h; (ii) NaNO2, HCl; (h) HATU, DIPEA, DMF, rt, overnight; (i) DMF, rt, overnight.

[0126] Scheme 26. Synthesis of 2,6-dichloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine α TIFF2025507601000089.tif42128 α Reagents and conditions: trifluoromethanesulfonic acid, 90000 Pa (0.9 bar), 85°C → room temperature, 1 hour.

[0127] 2,6-Dichloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine (36) was synthesized starting from 1,2,3,5-tetraacetyl-β-D-ribofuranose (34) and 2,6-dichloropurine (35). The reaction was carried out using previously reported reaction conditions. 13 Both compounds were melted at 85°C and reacted after the addition of trifluoromethanesulfonic acid.

[0128] Scheme 27. Synthesis of 4-(((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)methyl)benzoic acid α TIFF2025507601000090.tif35145 α Reagents and conditions: Two steps: (i) 4-(aminomethyl)benzoic acid, triethylamine, ethanol, 90° C., overnight; (ii) 2N NH 3 in MeOH, room temperature, overnight.

[0129] 2',3',5'-Tri-O-acetyl-2,6-dichloropurine riboside (36) was reacted with 4-(aminomethyl)benzoic acid (37) in the presence of triethylamine under basic conditions. The mixture was then refluxed at 90°C overnight. 18 After TLC showed the reaction was complete, the volatiles were evaporated, 2N NH3 in MeOH was added to the crude product, and the mixture was stirred at room temperature overnight. After evaporation of the solvent, absolute ethanol was added to the flask and heated to 100 °C to recrystallize the unreacted 4-(aminomethyl)benzoic acid. After filtration, the volatiles were evaporated, and 38 could be crystallized from methanol.

[0130] Scheme 28. Synthesis of tert-butyl (6-(bicyclo[2.2.1]hept-5-ene-2-carboxamido)hexyl)carbamate α TIFF2025507601000091.tif32158 a Reagents and conditions: 6-8% TFA, DCM, room temperature, overnight.

[0131] A mixture of endo and exo isomers of 5-norbornene-2-carboxylic acid (31) was coupled to commercially available N-boc-1,6-hexanediamine (30) by amide coupling using HATU and DIPEA in DCM to give 32. 19 .

[0132] Scheme 29. Synthesis of N-(6-aminohexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide α TIFF2025507601000092.tif19159 a Reagents and conditions: 6-8% TFA, DCM, room temperature, 5 h. After the reaction, boc deprotection of 32 with TFA in DCM gave 33.

[0133] Scheme 30. Synthesis of (1S,4S)-N-(6-(4-(((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)methyl)benzamido)hexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide α TIFF2025507601000093.tif34158 a Reagents and conditions: N-(6-aminohexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide, HATU, DIPEA, DMF, room temperature, overnight.

[0134] N-(6-aminohexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (33) was coupled to 4-(((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)methyl)benzoic acid (38) via an amide coupling reaction to give 39. 19 .

[0135] Scheme 31. Synthesis of ((((2R,3S,4R,5R)-5-(6-((4-((6-((1R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxamido)hexyl)carbamoyl)benzyl)amino)-2-chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid α TIFF2025507601000094.tif30160 a Reagents and conditions: Two steps: (i) methylenebis(phosphonic dichloride), trimethyl phosphate, Ar, 0°C, 1 h; (ii) NH4CO3, room temperature, 1 h.

[0136] (1S,4S)-N-(6-(4-(((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)methyl)benzamido)hexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (39) was phosphonylated with methylene bis(phosphonic dichloride) in trimethyl phosphate, followed by quenching with aqueous ammonium bicarbonate buffer. Both reaction steps were carried out according to optimized published procedures to give 40. 13、18 .

[0137] Scheme 32. Synthesis of 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzoic acid α TIFF2025507601000095.tif26128 a Reagents and conditions: Two steps: (i) Zn(OTf)2, ACN, hydrazine hydrate, 60 °C, 24 h, room temperature, 24 h; (ii) NaNO2, HCl.

[0138] 4-Cyanobenzoic acid (41), Zn(OTf)2, and acetonitrile were added to a solution of 80 wt% hydrazine hydrate and stirred at 60 °C for 24 h and then at room temperature for another 24 h. Aqueous NaNO2 was then added slowly, followed by concentrated HCl until a pH of 3 was reached and gas evolution ceased, to give 42. 33 .

[0139] Scheme 33. N-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 Synthesis of -dipyrrolo[1,2-c:2',1'-f][1,3,2]dioazaborin-10-yl)butyl)-4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzamide α TIFF2025507601000096.tif90159 a Reagents and conditions: HATU, DIPEA, DMF, room temperature, overnight.

[0140] 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzoic acid (42) was reacted with 4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-10-yl)butan-1-amine (43) was coupled via an amide coupling reaction to give 44.

[0141] Scheme 34. (((((2R,3S,4R,5R)-5-(2-chloro-6-((4-((6-((4aS,5S,8S)-1-(4-((4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4Synthesis of -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine-10-yl)butyl)carbamoyl)phenyl)-4-methyl-2,4a,5,6,7,8-hexahydro-5,8-methanophthalazine-6-carboxamido)hexyl)carbamoyl)benzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid α TIFF2025507601000097.tif69170 a Reagents and conditions: DMF, room temperature, overnight.

[0142] ((((2R,3S,4R,5R)-5-(6-((4-((6-((1R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxamido)hexyl)carbamoyl)benzyl)amino)-2-chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid (40) was reacted with N-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-10-yl)butyl)-4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzamide (44) via inverse electron demand Diels-Alder reaction (IEDDA) to give 45 as a mixture of endo / exo and 1,4- and 2,5-dihydropyridazine isomers.

[0143] Scheme 35. Synthesis of BODIPY-TR-labeled CD73 inhibitors TIFF2025507601000098.tif132170 αReagents and conditions: (a) 5-norbornene-2-carboxylic acid, HATU, DIPEA, DCM, rt, overnight; (b) 6-8% TFA, DCM, rt, overnight; (c) 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid, HATU, DIPEA, DMF, rt, overnight; (d): TBN, TMSBr, rt, overnight; (e) 2 steps: (i) methylenebis(phosphonic dichloride), phosphoric acid (ii) NH4CO3 buffer, rt, 1 h; (f) 6-8% TFA, DCM, rt, overnight; (g) Et3N, anhydrous EtOH, reflux, overnight; (h) 2-step: (i) Zn(OTf)2, ACN, hydrazine hydrate, 60 °C, 24 h, rt, 24 h; (ii) NaNO2, HCl; (i) HATU, DIPEA, DMF, rt, overnight; (j) DMF, rt, overnight.

[0144] Scheme 36. Synthesis of 2-bromo-6-chloro-9-(β-D-ribofuranosyl)-9H-purine a TIFF2025507601000099.tif38128 a Reagents and conditions: TMSBr, TBN, CH2Br2, room temperature under argon atmosphere overnight.

[0145] Commercially available 2-amino-6-chloro-9-(β-D-ribofuranosyl)purine (52) was reacted by a non-aqueous diazotization reaction using tert-butyl nitrite (TBN) and trimethylsilyl bromide (TMSBr) in dibromomethane according to a modified version of a previously published method to give 53. 31、32 .

[0146] Scheme 37. Synthesis of (((((2R,3S,4R,5R)-5-(2-bromo-6-chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid TIFF2025507601000100.tif38128 aReagents and conditions: Two steps: (i) methylenebis(phosphonic dichloride), trimethyl phosphate, Ar, 0°C, 1 h; (ii) NH4CO3, room temperature, 1 h.

[0147] 2-Bromo-6-chloro-9-(β-D-ribofuranosyl)-9H-purine (53) was phosphonylated with methylene bis(phosphonic dichloride) in trimethyl phosphate, followed by quenching with aqueous TEAC buffer. Both reaction steps were carried out according to optimized published procedures to give 54. 13、18 .

[0148] Scheme 38. Synthesis of tert-butyl (6-(bicyclo[2.2.1]hept-5-ene-2-carboxamido)hexyl)carbamate α TIFF2025507601000101.tif33158 a Reagents and conditions: 6-8% TFA, DCM, room temperature, overnight.

[0149] A mixture of endo and exo isomers of 5-norbornene-2-carboxylic acid (47) was coupled to commercially available N-boc-1,6-hexanediamine (46) by amide coupling using HATU and DIPEA in DCM to give 48.

[0150] Scheme 39. Synthesis of N-(6-aminohexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide α TIFF2025507601000102.tif19159 a Reagents and conditions: 6-8% TFA, DCM, room temperature, 5 h. After the reaction, boc deprotection of 48 with TFA in DCM gave 49.

[0151] Scheme 40. Synthesis of tert-butyl (4-((6-((1R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxamido)hexyl)carbamoyl)benzyl)carbamate α TIFF2025507601000103.tif36157 a Reagents and conditions: 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid, HATU, DIPEA, DMF, room temperature, overnight.

[0152] 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid (50) was coupled to 49 via amide coupling using HATU and DIPEA in DMF to give 51.

[0153] Scheme 41. Synthesis of (1R,4R)-N-(6-(4-(aminomethyl)benzamido)hexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide α TIFF2025507601000104.tif19159 a Reagents and conditions: 6-8% TFA, DCM, room temperature, 5 h. After the reaction, boc deprotection of 51 with TFA in DCM gave 55.

[0154] Scheme 42. Synthesis of ((((2R,3S,4R,5R)-5-(6-((4-((6-(bicyclo[2.2.1]hept-5-ene-2-carboxamido)hexyl)carbamoyl)benzyl)amino)-2-bromo-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid a TIFF2025507601000105.tif50155 a Reagents and conditions: Et3N, absolute EtOH, reflux, overnight.

[0155] In the reaction step, nucleophilic substitution of 54 with primary amine 55 in the presence of triethylamine in absolute ethanol under reflux conditions, followed by purification by RP-HPLC, afforded the desired final product 56. 18 .

[0156] Scheme 43. Synthesis of (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine α TIFF2025507601000106.tif27128 a Reagents and conditions: Two steps: (i) Zn(OTf)2, ACN, hydrazine hydrate, 60 °C, 24 h, room temperature, 24 h; (ii) NaNO2, HCl.

[0157] 4-(Aminomethyl)benzonitrile (57), Zn(OTf)2, and acetonitrile were added to a solution of 80 wt% hydrazine hydrate and stirred at 60 °C for 24 h and then at room temperature for another 24 h. Aqueous NaNO2 was then added slowly, followed by concentrated HCl until a pH of 3 was reached and gas evolution ceased, to give 59. 33 .

[0158] Scheme 44. 2-(4-(5,5-difluoro-7-(thiophen-2-yl)-5H-4λ 4 ,5λ 4 Synthesis of -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)phenoxy)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)acetamide α TIFF2025507601000107.tif75160 a Reagents and conditions: HATU, DIPEA, DMF, room temperature, overnight.

[0159] 59, 2-(4-(5,5-difluoro-7-(thiophen-2-yl)-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)phenoxy)acetic acid (58-BODIPY-TR) was coupled via an amide coupling reaction to give 60.

[0160] Scheme 45. Synthesis of ((((2R,3S,4R,5R)-5-(6-((4-((6-((1S,4S)-bicyclo[2.2.1]hept-5-ene-2-carboxamido)hexyl)carbamoyl)benzyl)amino)-2-bromo-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid α TIFF2025507601000108.tif120144 a Reagents and conditions: DMF, room temperature, overnight.

[0161] 56 was coupled to 60 via an inverse electron demand Diels-Alder reaction (IEDDA) to give 61 as a mixture of endo / exo and 1,4- and 2,5-dihydropyridazine isomers.

[0162] Scheme 46. Synthesis of cyanine-labeled CD73 inhibitors. TIFF2025507601000109.tif158170 α Reagents and conditions: (a) 5-norbornene-2-carboxylic acid, HATU, DIPEA, DCM, rt, overnight; (b) 6-8% TFA, DCM, rt, overnight; (c) trifluoromethanesulfonic acid, 90000 Pa (0.9 bar), 85°C → rt, 1 h; (d) 2 steps: (i) 4-(aminomethyl)benzoic acid, triethylamine, ethanol, 90°C, overnight; (ii) 2N in MeOH NH3, rt, overnight; (e) HATU, DIPEA, DMF, rt, overnight; (f) 2 step: (i) methylenebis(phosphonic dichloride), trimethyl phosphate, Ar, 0°C, 1 h; (ii) NH4CO3 buffer, rt, 1 h; (g) 2 step: (i) Zn(OTf)2, ACN, hydrazine hydrate, 60°C, 24 h, rt, 24 h; (ii) NaNO2, HCl; (h) HATU, DIPEA, DMF, rt, overnight; (i) DMF, rt, overnight.

[0163] Scheme 47. Synthesis of 2,6-dichloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine α TIFF2025507601000110.tif42128 α Reagents and conditions: trifluoromethanesulfonic acid, 90000 Pa (0.9 bar), 85°C → room temperature, 1 hour.

[0164] 2,6-Dichloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine (68) was synthesized starting from 1,2,3,5-tetraacetyl-β-D-ribofuranose (67) and 2,6-dichloropurine (66). The reaction was carried out using previously reported reaction conditions. 13 Both compounds were melted at 85°C and reacted after the addition of trifluoromethanesulfonic acid.

[0165] Scheme 48. Synthesis of 4-(((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)methyl)benzoic acid α TIFF2025507601000111.tif35145 α Reagents and conditions: Two steps: (i) 4-(aminomethyl)benzoic acid, triethylamine, ethanol, 90° C., overnight; (ii) 2N NH 3 in MeOH, room temperature, overnight.

[0166] 2',3',5'-Tri-O-acetyl-2,6-dichloropurine riboside (68) was reacted with 4-(aminomethyl)benzoic acid (69) in the presence of triethylamine under basic conditions. The mixture was then refluxed at 90°C overnight. 18 After TLC showed the reaction was complete, the volatiles were evaporated, 2N NH3 in MeOH was added to the crude product, and the mixture was stirred at room temperature overnight. After evaporation of the solvent, absolute ethanol was added to the flask and heated to 100 °C to recrystallize the unreacted 4-(aminomethyl)benzoic acid. After filtration, the volatiles were evaporated, and 70 could be crystallized from methanol.

[0167] Scheme 49. Synthesis of tert-butyl (6-(bicyclo[2.2.1]hept-5-ene-2-carboxamido)hexyl)carbamate α TIFF2025507601000112.tif32158 a Reagents and conditions: 6-8% TFA, DCM, room temperature, overnight.

[0168] A mixture of endo and exo isomers of 5-norbornene-2-carboxylic acid (63) was coupled to commercially available N-boc-1,6-hexanediamine (62) by amide coupling using HATU and DIPEA in DCM to give 64.

[0169] Scheme 50. Synthesis of N-(6-aminohexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide α TIFF2025507601000113.tif19159 a Reagents and conditions: 6-8% TFA, DCM, room temperature, 5 h. After the reaction, boc deprotection of 64 with TFA in DCM gave 65.

[0170] Scheme 51. Synthesis of (1S,4S)-N-(6-(4-(((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)methyl)benzamido)hexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide α TIFF2025507601000114.tif34158 a Reagents and conditions: N-(6-aminohexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide, HATU, DIPEA, DMF, room temperature, overnight.

[0171] N-(6-aminohexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (65) was coupled to 4-(((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)methyl)benzoic acid (70) via an amide coupling reaction to give 71.

[0172] Scheme 52. Synthesis of ((((2R,3S,4R,5R)-5-(6-((4-((6-((1R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxamido)hexyl)carbamoyl)benzyl)amino)-2-chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid α TIFF2025507601000115.tif30160 a Reagents and conditions: Two steps: (i) methylenebis(phosphonic dichloride), trimethyl phosphate, Ar, 0°C, 1 h; (ii) NH4CO3, room temperature, 1 h.

[0173] (1S,4S)-N-(6-(4-(((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)methyl)benzamido)hexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (71) was phosphonylated with methylene bis(phosphonic dichloride) in trimethyl phosphate, followed by quenching with aqueous ammonium bicarbonate buffer. Both reaction steps were carried out according to optimized published procedures to give 72. 13、18 .

[0174] Scheme 53. Synthesis of (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine α TIFF2025507601000116.tif27128 aReagents and conditions: Two steps: (i) Zn(OTf)2, ACN, hydrazine hydrate, 60 °C, 24 h, room temperature, 24 h; (ii) NaNO2, HCl.

[0175] 4-(aminomethyl)benzonitrile (73), Zn(OTf)2, and acetonitrile were added to a solution of 80 wt% hydrazine hydrate and stirred at 60 °C for 24 h and then at room temperature for another 24 h. Aqueous NaNO2 was then added slowly, followed by concentrated HCl until a pH of 3 was reached and gas evolution ceased, to give 74. 33 .

[0176] Scheme 54. Synthesis of 4-(2-((1E,3E,5E,7E)-7-(1,1-dimethyl-3-(8-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-8-oxooctyl)-1,3-dihydro-2H-benzo[e]indol-2-ylidene)hepta-1,3,5-trien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)butane-1-sulfonate α TIFF2025507601000117.tif75162 a Reagents and conditions: HATU, DIPEA, DMF, room temperature, overnight.

[0177] 74 was coupled to 4-(2-((1E,3E,5E,7E)-7-(3-(7-carboxyheptyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)hepta-1,3,5-trien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)butane-1-sulfonate (75-Cy7.5(monoSO3)) via an amide coupling reaction to give 76.

[0178] Scheme 55. 4-(2-((1E,3E,5E,7E)-7-(3-(8-((4-((4aR,5R,8R)-6-((6-(4-(((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphonomethyl)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)methyl)benzamido)hexyl)carbamoyl Synthesis of (4-methyl-2,4a,5,6,7,8-hexahydro-5,8-methanophthalazin-1-yl)benzyl)amino)-8-oxooctyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)hepta-1,3,5-trien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)butane-1-sulfonate TIFF2025507601000118.tif138165 a Reagents and conditions: DMF, room temperature, overnight.

[0179] 72 was coupled to 76 via inverse electron demand Diels-Alder reaction (IEDDA) to give 78 as a mixture of endo / exo and 1,4- and 2,5-dihydropyridazine isomers.

[0180] References TIFF2025507601000119.tif218170TIFF2025507601000120.tif235170TIFF2025507601 000121.tif211170TIFF2025507601000122.tif243170TIFF2025507601000123.tif43165

Claims

1. A compound of general formula (I), or a pharmaceutically acceptable salt thereof: During the ceremony R a , R b and R c is H, -(C 1 -C 6 ) alkyl, -(C 6 -C 10 ) aryl, -C(O)(C 1 -C 6 ) alkyl, -(C 1 -C 6 ) Alkyl(C 6 -C 10 ) heteroaryl, -(C 6 -C 10 ) independently selected from the group consisting of heteroaryl and —C(O)aryl; M 1 and M 2 are independently selected from the group consisting of H, —OH, and halogen; n is 1 to 6, or 1 to 3, or 1; Q is O, S, CH 2 , NH, or is O; U is O, S, CH 2 , (CH 2 ) 2 , NH, or CH 2 and T is or and V is O, NH, S, CH 2 or is O; R 1 and R 2 are H, OH, SH, -O(C 1 -C 6 ) alkyl, -S(C 1 -C 6 ) alkyl, -NH 2 , -NH(C 1 -C 6 ) alkyl, -N 3 and halogen, or independently selected from the group consisting of H and —OH; A is or and X is O, S, N, or N, wherein q is 0 to 6, or 0 to 4, or 0; Y is -(C 6 -C 10 ) aryl-, -(CH 2 ) p -, -(CH 2 ) p C(O)-, -(C 6 -C 10 )Heteroaryl(CH 2 ) p -, -(C 6 -C 10 ) heteroaryl-, -(CH 2 ) p (C 6 -C 10 ) aryl-, and -(C 6 -C 10 )arylC(O)— or —(CH 2 ) p - or -(C 6 -C 10 ) arylC(O)—; Z is ;CH 2 ) p -, -(C 6 -C 10 ) aryl-, -(C 6 -C 10 )Aryl(CH 2 ) p -, -C(O)(CH 2 ) p -, -(C 6 -C 10 )Heteroaryl(CH 2 ) p -, -(C 6 -C 10 ) heteroaryl-, and -(C 6 -C 10 ) arylC(O)—; or and -(C 6 -C 10 )arylC(O)—, wherein optionally the aryl group is selected from the group consisting of —(C 1 -C 6 ) alkyl, -(C 2 -C 6 ) Alkynyl, -halogen, -trifluoromethyl, -OH, -SH, -NH 2 , -SO 2 NH 2 , -(C 1 -C 6 ) alkylOH, -O(C 1 -C 6 ) alkyl, -SO 3 H, -(CH 2 ) 1-6 COOH, -COOH, -C(O)NH 2 , -SO 3 (C 1 -C 6 ) alkyl, -(C 5 -C 6 )ArylCH 2 C(O)-, -C(O)NH(CH 2 ) o NH 2 may be substituted with one or more substituents selected from the group consisting of: o is an integer from 1 to 8, or 1 to 4, or 4; p is an integer from 1 to 6, or 1 to 4, or 4; L is L 1 and L 2 Including L 1 is L 2 Connected to L 1 -L 2 and Here, L 1 is absent, -(CH 2 )q-, -C(O)NH(CH 2 ) p NH-, -(CH 2 ) q (C 5 -C 10 ) aryl-, -(C 1 -C 10 ) alkynyl-, -(C 6 -C 10 )Aryl(CH 2 ) p -, 1-halo-1-vinyl, -(C 6 -C 10 )Heteroaryl(CH 2 ) p -, -(C 6 -C 10 ) heteroaryl-, -(C 6 -C 10 ) arylC(O)-, and selected from the group consisting of: s is an integer from 1 to 60, or from 1 to 50, or from 2 to 30; q is an integer from 1 to 10, or from 1 to 6, or 5; and L 2 is non-existent, (v is 1 to 9, or 1 to 7); and (z is 1 to 6, or 1 to 5, or 5) selected from the group consisting of: R 3 teeth (m is an integer from 2 to 10); or ； iii) a fluorophore moiety selected from the group consisting of FITC, fluorescein, NBD, dansyl, squaraine rotaxane, Bodipy FL, Bodipy TR, Bodipy 630 / 650 X, Bodipy 650 / 655 X, Texas Red, y5, 1-pyrene, EVOBlue 30, Alexa Fluor 532, Alexa Fluor 488-5, 488-6, or mixtures thereof, Tamra, Tamra 5 / 6-X-SE, Alexa Fluor 488 azide 5 isomer, Alexa Fluor 488 5 isomer, Alexa Fluor 488 5 / 6 mixed isomer, NIR dye 700, NIR dye 800, Janelia Fluor 549 amide, Janelia Fluor 646 amide, and derivatives, analogs, and related fluorophores thereof; or iv) a chelating moiety bound to a radiometal (where the chelating moiety is and Radioactive metals 64 Cu, 68 Ga, 177 Lu, 90 Y, 89 Zr, 211 At, 212 Pb, 188 Rh, 166 Ho, 225 Ac, 99m Tc or 111 In, 123 I, 131 I) selected from the group consisting of: R 4 , R 5 is H, halogen, -(C 1 -C 6 ) alkyl, -(C 1 -C 6 ) Aryl, -NH 2 , -N 3 , -(C 1 -C 6 ) alkynyl, -(C 6 -C 10 )Aryl(C 1 -C 6 ) alkyl, -1-halogen-1-vinyl, (C 6 -C 10 ) Heteroaryl (C 1 -C 6 ) alkyl-, -(C 6 -C 10 ) heteroaryl, —C(O)(C 6 -C 10 ) Aryl-, -OR 6 , -SR 6 , -NHR 6 , -NR 6 R 7 , -SiR 6 R 7 R 8 , -OC(O)R 6 , -C(O)R 6 , -COOR 6 , -CONR 6 R 7 , -OC(O)NR 6 R 7 , -NR 6 C(O)R 7 , -NR 6 COOR 7 , -NHC(NH 2 )=NR 6 , -S(O)R 6 , -SO 2 NR 6 R 7 , -NR 6 SO 2 R 7 , -CN, and -NO 2 or R 6 , R 7 , R 8 is H, halogen, -(C 1 -C 6 ) alkyl, (C 1 -C 6 ) Aryl, -NH 2 , -N 3 , -(C 1 -C 6 ) alkynyl, -(C 6 -C 10 )Aryl(C 1 -C 6 ) alkyl, -1-halogen-1-vinyl, -(C 1 -C 6 ) Alkyl(C 6 -C 10 ) heteroaryl, -(C 6 -C 10 ) heteroaryl, —C(O)(C 6 -C 10 ) Aryl-, -SO 3 independently selected from the group consisting of H, -OH, and -SH; R 9 is a halogen, (C 1 -C 6 ) alkyl, -(C 1 -C 6 ) Aryl, -NH 2 , -N 3 , -(C 1 -C 6 ) alkynyl, -(C 6 -C 10 )Aryl(C 1 -C 6 ) alkylenyl, 1-halogen-1-vinyl, -(C 6 -C 10 ) Heteroaryl (C 1 -C 6 ) alkyl-, -(C 6 -C 10 ) heteroaryl, —C(O)(C 6 -C 10 ) Aryl-, -OR 6 , -SR 6 , -NHR 6 , -NR 6 R 7 , -SiR 6 R 7 R 8 , -OC(O)R 6 , -C(O)R 6 , -COOR 6 , -CONR 6 R 7 , -OC(O)NR 6 R 7 , -NR 6 C(O)R 7 , -NR 6 COOR 7 , -NHC(NH 2 )=NR 6 , -S(O)R 6 , -SO 2 NR 6 R 7 , -NR 6 SO 2 R 7 , -CN, and -NO 2 selected from the group consisting of: where R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 The aryl or heteroaryl of -(C 1 -C 6 )Alkyl, halogen, -trifluoromethyl, -OH, -SH, -NH 2 , -SO 2 NH 2 , -(C 1 -C 6 ) alkylhydroxy, -(C 1 -C 6 )Alkoxy, -SO 3 H, -COO(C 1 -C 6 ) alkyl, -SO 3 (C 1 -C 6 ) alkyl, -C(O)(C 5 -C 6 ) optionally substituted with one or more substituents selected from the group consisting of aryl; R 10 is OH or and However, R 7 is H and A is and R 5 is H, X is N, and Y is CH 2 where Z is phenyl and L is -C(O)NH(CH 2 ) 1-4 NH- and R 9 is H or Cl, R 3 is not fluorescein.

2. R a , R b and R c is H; and / or M 1 and M 2 is H; and / or n is 1; and / or Q is O; and / or U is CH 2 and / or R 1 is —OH; and / or R 2 is H; and / or A is and / or R 4 is H, -(C 1 -C 6 ) alkyl or halogen; and / or R 5 is H, and / or R 6 is H, -(C 1 -C 6 ) alkyl, -(C 6 -C 10 )Aryl(C 1 -C 6 ) alkyl, -(C 1 -C 6 ) Alkyl(C 6 -C 10 ) heteroaryl; and / or R 7 is H or -(C 1 -C 6 ) alkyl, or (C 2 -C 4 ) alkyl; or (C 3 ) alkyl, and / or R 8 is H or -(C 1 -C 6 ) alkyl; and / or R 9 is H, halogen, or -(C 1 -C 6 ) alkyl; and / or X is N; and / or Y is -(CH 2 ) p -and / or Z is , -(C 6 -C 10 ) arylC(O); and / or L 1 is absent or -C(O)NH(CH 2 ) 1-4 is NH-; and / or L 2 is non-existent, (v is 1 to 9, or 1 to 7); and (z is 1 to 6, or 1 to 5, or 5) and / or R 3 but or fluorescein, The compound of claim 1.

3. V is O; Q is O; U is CH 2 and A is and X is N; Y is CH 2 and Z is phenyl or and L 1 is absent or -C(O)NH(CH 2 ) p NH-; -C(O)NH(CH 2 ) p p in NH- is 1 to 6, or 4 to 6, or 6; L 2 is non-existent, (v is 1 to 9, or 1 to 7); and (z is 1 to 6, or 1 to 5, or 5) and / or R 3 is CH 2 -F 18 , (m is 2 to 4, or 2) That is, The compound of claim 1.

4. The compound of claim 1 selected from the group consisting of:

5. The compound of claim 1 selected from the group consisting of:

6. R 3 2. The compound of claim 1, wherein is not fluorescein.

7. 10. A pharmaceutical composition comprising the compound of claim 1 and at least one pharmaceutically acceptable carrier.

8. 8. The pharmaceutical composition of claim 7, for use in a method for diagnosing a disease associated with increased CD73 expression.

9. 8. The pharmaceutical composition of claim 7, for use in treating a disease associated with increased CD73 expression.

10. 10. The pharmaceutical composition of claim 8 or 9, wherein the disease is selected from the group consisting of cancer or solid tumors, and inflammatory diseases.

11. 11. The pharmaceutical composition of claim 10, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, melanoma, glioma, head and neck cancer, and thyroid cancer.

12. 11. The pharmaceutical composition of claim 10, wherein the inflammatory disease is selected from the group consisting of multiple sclerosis and rheumatoid arthritis.

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