WO2025247543A1 - 1h-pyrazolo[3,4-d]pyrimidine ribonucleosides with anticancer activity for therapeutic use - Google Patents

Abstract

This disclosure relates to 1H-pyrazolo[3,4-d]pyrimidine ribonucleoside compounds of Formula (I) and pharmaceutically acceptable salts thereof, which activate LACTB and are useful for treating diseases or disorders, such as cancer.

Description

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 1H-PYRAZOLO[3,4-D]PYRIMIDINE RIBONUCLEOSIDES WITH ANTICANCER ACTIVITY FOR THERAPEUTIC USE CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to Czech patent application PV 2024-224, filed on May 30, 2024, which is incorporated by reference in its entirety. TECHNICAL FIELD [0002] This application is directed to 1H-pyrazolo[3,4-d]pyrimidine ribonucleoside compounds which activate beta-lactamase and are useful for treating diseases or disorders, such as cancer. BACKGROUND [0003] Cancer is a multifactorial disease in which cells lose their regulatory properties and begin to grow uncontrollably. Although dozens of antiproliferative drugs exist and are approved, the treatment of many types of leukemia and tumors has a low success rate. In addition, current drugs often have significant side effects. Therefore, there is a need to develop other new types of antitumor agents for therapeutic use. This application describes chemical compounds that show strong cytotoxic effects on cell lines preferentially of tumor origin and in a wide spectrum of diseases, including tumors of epithelial origin. These compounds belong to analogs of purine nucleosides with a modified nucleobase, namely 3,4-disubstituted 1H-pyrazolo[3,4-d]pyrimidine ribonucleosides. [0004] 1H-Pyrazolo[3,4-d]pyrimidine ribonucleosides have been known for decades (Montgomery, J. A.; Clayton, S. J.; Fitzgibbon W. E., Jr.: J. Heterocycl. Chem.1964, 1, 215– 216.), especially for their antiparasitic properties (Bouton, J. et al. J. Med. Chem.2021, 64, 4206–4238; Van Calenbergh, S.; Caljon, G.; Hulpia, F.; Bouton, J. WO 2023/052468; Marr, J. J. J. Cell. Biochem.1983, 22, 187–196), as inhibitors of adenosine kinase (Cottam, H. D. et al. J. Med. Chem.1993, 36, 3424–3430) or as a part of bisphosphonates for inhibition of CD73 (Bowman, Ch. E.; da Silva, R. G.; Pham, A.; Young, S. W. Biochemistry 2019, 58, 3331–3334). From the group of 3,4-disubstituted 1H-pyrazolo[3,4-d]pyrimidin ribonucleosides, the most studied are 8-azaadenosines including the 3-ethynyl derivative A^ethynyl, which is an important Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 intermediate for the synthesis of modified RNA (Zheng, Y.; Beal, P. A. Bioorg. Med. Chem. Lett.2016, 26, 1799–1802; Phelps, K. J. et al. ACS Chem. Biol.2014, 9, 1780–1787). SUMMARY [0005] Without wishing to be bound by theory, the present results provide evidence for therapeutic reactivation of mitochondrial beta-lactamase-like protein (LACTB) tumor suppressor. This was possible through uncovering one of the mechanisms responsible for post- translational downregulation of LACTB in cancer cells and identification of a chemical compound capable of reverting this process. Through the search for a LACTB substrate, it was showed that LACTB can serve as its own substrate; that LACTB possesses autoproteolytic activity. Mechanistically, it was shown that this autoproteolytic activity is enhanced by the mitochondrial small ribosomal subunit protein mS34 (MRPS34), which, through interaction with LACTB, acts as its negative regulator. The LACTB-MRPS34 axis is thrown out of balance by cancer cells, which, through upregulation of MRPS34, achieve efficient down-regulation of LACTB, realized through its own auto-destruction. Furthermore, a nucleoside analogue, PNH733, was identified as being capable of inhibiting LACTB’s autoproteolytic activity. The inhibition of LACTB’s autoproteolytic activity led to an upregulation of LACTB in cancer cells, and subsequently potent therapeutic effects of PNH733 in breast cancer and ovarian cancer models. [0006] In breast cancer, it was shown that while LACTB expression is downregulated in many breast cancer cell lines and tissues, it is never completely absent and genomic databases show no presence of biallelic LACTB deletions in human cells. These characteristics make LACTB a plausible target for activation cancer therapy since cancer cells do not inactivate this protein in its entirety. Instead, cancer cells developed mechanisms to downregulate its levels of expression which, in case of LACTB, happens mostly via non-genetic means. This observation provided an opportunity to design therapeutic approaches for its efficient reactivation in cancer cells. [0007] Since LACTB was shown to have tumor suppressor role in many different types of cancer (such as lung, breast, ovary, pancreas, skin, brain, prostate, and colorectal cancers), the reactivation of LACTB might display therapeutic effect against numerous different cancer types and might complement the cancer types responding to PTEN or p53 reactivation. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0008] Inhibition of autoproteolytic activity of LACTB does not inhibit the catalytic activity of LACTB. This suggests that if LACTB recognizes other as-yet-unknown cellular substrates apart from itself, this part of its physiological biological role will not be affected and the chemical compound inhibiting the autoproteolytic activity of LACTB will not have a detrimental and toxic effects on LACTB’s biology in non-tumorigenic setting. Bioinformatic analyses revealed a negative correlation between MRPS34 and LACTB in several tissues with confirmed tumor suppressor role of LACTB, such as in lung (Xu et al., Exp Ther Med 2022, 23(3): 247) and colorectal cancers (Zeng et al., Oncogene 2018, 37(41): 5534-5551; Zhou et al., Cancer Biother Radiopharm, 2020; and Xu et al., Cancer Manag Res 2020, 12: 5181-5200). This points to the possibility that the compounds targeting LACTB’s autoproteolytic activity or LACTB- MRPS34 axis might be therapeutically beneficial also in cancers arising from these tissues in addition to breast cancers and ovarian cancers. [0009] The nucleoside analogue, PNH733, inhibits the autoproteolytic activity of LACTB thus preventing it from cancer-cell imposed self-degradation. [0010] Provided in the present disclosure are compounds including substituted 1H- pyrazolo[3,4-d]pyrimidine nucleosides having a structure represented by formula (I): wherein R¹ is C1-C5 alkyl, C1-C5 alkoxy, or di(C1-C5)alkylamino; and R² is C1-C5 alkyl, C2-C5 alken-1-yl, C2-C5 alkyn-1-yl, cyano, or halo, or a pharmaceutically acceptable salt thereof. [0011] In some embodiments, the 3,4-disubstituted 1H-pyrazolo[3,4-d]pyrimidine ribonucleosides comprises: (i) 3-ethynyl-4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or (ii) 3-ethynyl-4-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 (iii) 3-ethynyl-4-(N,N-dimethylamino)-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine; or (iv) 4-methoxy-3-methyl-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or (v) 3,4-dimethyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or (vi) 4-N,N-dimethylamino-3-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine; or (vii) 3-iodo-4-methoxy-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or (viii) 4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-3-carbonitril; or (ix) 4-methoxy-3-vinyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine. [0012] In some embodiments, R¹ is C1-C5 alkyl. [0013] In some embodiments, R¹ is C1-C5 alkoxy. [0014] In some embodiments, R¹ is di(C1-C5)alkylamino. [0015] In some embodiments, R¹ is methyl. [0016] In some embodiments, R¹ is methoxy. [0017] In some embodiments, R¹ is N,N-dimethylamino. [0018] In some embodiments of any of the compounds described herein, R² is C1-C5 alkyl. [0019] In some embodiments of any of the compounds described herein, R² is C2-C5 alken-1- yl. [0020] In some embodiments of any of the compounds described herein, R² is cyano. [0021] In some embodiments of any of the compounds described herein, R² is halo. [0022] In some embodiments of any of the compounds described herein, R² is C2-C5 alkyn- 1-yl. [0023] In some embodiments, R² is methyl. [0024] In some embodiments, R² is vinyl. [0025] In some embodiments, R² is ethynyl. [0026] In some embodiments, the compound is selected from: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 . for use as a medicament. [0028] Provided in the present disclosure are compounds of formula (I) for use in inhibition of pathological cell proliferation of tumor/non-tumor/cancer origin and for treatment of tumor/non-tumor/cancer disease associated with cell hyperproliferation. [0029] Provided in the present disclosure are compounds of formula (I) for use in treatment of tumors, cancers, and diseases covering epithelial tumors. [0030] In some embodiments, the tumors, cancers, and diseases comprise cancers expressing high mitochondrial small ribosomal subunit protein mS34 (MRPS34) protein levels. [0031] Provided in the present disclosure are pharmaceutical compositions including a therapeutically effective amount of a compound of formula (I), and one or more pharmaceutically acceptable carriers, excipients, or diluents. [0032] Provided in the present disclosure are pharmaceutical compositions including a therapeutically effective amount of a compound of formula (I) for use in inhibition of pathological cell proliferation of tumor/non-tumor/cancer origin and/or for treatment of tumor/non tumor/cancer disease associated with cell hyperproliferation. [0033] Provided in the present disclosure are pharmaceutical compositions including a therapeutically effective amount of a compound of formula (I), and a pharmaceutically acceptable carrier. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0034] Provided in the present disclosure are methods of activating beta-lactamase-like protein (LACTB) expression and/or activity that include contacting a cell with any of the compounds of formula (I) described herein, or pharmaceutically acceptable salt thereof. [0035] Provided in the present disclosure are methods of reducing or inhibiting LACTB autoproteolytic activity that include contacting a cell with any of the compounds of formula (I) described herein, or pharmaceutically acceptable salt thereof. [0036] Provided in the present disclosure are methods of treating or preventing disease progression in a subject having a disease associated with a loss or inactivation of LACTB expression and/or activity that include administering to the subject a therapeutically effective amount of any of the compounds of formula (I) described herein, or a pharmaceutically acceptable salt, thereby treating or preventing disease progression in the subject. [0037] In some embodiments, the disease associated with a loss of LACTB expression and/or activity is a cancer. [0038] Provided in the present disclosure are methods of treating a cancer in a subject that include: administering to the subject a therapeutically effective amount of any of the compounds of formula (I) described herein, or pharmaceutically acceptable salt thereof, thereby treating the subject. [0039] Provided in the present disclosure are methods of treating a cancer in a subject that include: (a) identifying the cancer as having cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of any of the compounds of formula (I) described herein, or a pharmaceutically acceptable salt thereof, thereby treating the subject. [0040] Provided in the present disclosure are methods of treating a cancer in a subject that include: (a) determining or having determined that the cancer has cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of any of the compounds of formula (I) described herein, or a pharmaceutically acceptable salt thereof, thereby treating the subject. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0041] In some embodiments of any of the methods described herein, the cancer is a multidrug resistant cancer. [0042] In some embodiments, the multidrug resistant cancer is a daunorubicin-resistant cancer or a paclitaxel-resistant cancer. [0043] In some embodiments of any of the methods described herein, the cancer is a metastatic cancer. [0044] In some embodiments of any of the methods described herein, the cancer is an epithelial breast cancer. [0045] In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. [0046] In some embodiments, the cancer is a breast cancer. [0047] In some embodiments, the breast cancer is a triple negative breast cancer. [0048] In some embodiments, the breast cancer is a Her2 negative and estrogen receptor/progesterone receptor positive breast cancer. [0049] In some embodiments, the breast cancer is an inflammatory breast cancer. [0050] In some embodiments of any of the methods described herein, the cancer is a cancer in which LACTB is expressed at low level or has low activity levels, and/or the cancer is p53 negative and/or the cancer is PTEN negative. [0051] In some embodiments of any of the methods described herein, administering the compound inhibits cellular proliferation, cellular invasion, and/or cellular migration. [0052] In some embodiments of any of the methods described herein, the method further includes administering an additional cancer treatment. [0053] In some embodiments, the additional cancer treatment is radiation therapy or chemotherapy. [0054] In some embodiments of any of the methods described herein, the method further includes administering an immunotherapy composition to the subject. [0055] In some embodiments of any of the methods described herein, the compound is administered by intravenous administration or oral administration. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0056] Provided in the present disclosure are methods of selecting a cancer treatment for a subject that include: providing a sample comprising a cancer cell from the subject; detecting the level and/or activity of LACTB in the sample; and selecting a treatment comprising administering any of the compounds of formula (I) described herein, or a pharmaceutically acceptable salt thereof, to the subject if the sample has a reduced level and/or activity of LACTB as compared to a sample comprising a non- cancerous cell from the same subject. [0057] Provided in the present disclosure are methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level of MRPS34 in the sample; and selecting a treatment including administering any of the compounds of formula (I) described herein, or a pharmaceutically acceptable salt thereof, to the subject if the sample has an increased level of MRPS34 as compared to a sample including a non-cancerous cell from the same subject. [0058] Provided in the present disclosure are methods of activating LACTB expression and/or activity that include contacting a cell with PNH733 or pharmaceutically [0059] Provided in the present disclosure are methods of reducing or inhibiting LACTB autoproteolytic activity that include contacting a cell with PNH733 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically [0060] Provided in the present disclosure are methods of treating an epithelial cancer in a subject that include: administering to the subject a therapeutically effective amount of PNH733 or pharmaceutically the subject. [0061] Provided in the present disclosure are methods of treating or preventing disease progression in a subject having a disease associated with a loss or inactivation of LACTB expression and/or activity that include administering to the subject a therapeutically effective amount of PNH733 or a pharmaceutically or preventing disease progression in the subject. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0062] In some embodiments, the disease associated with a loss of LACTB expression and/or activity is a cancer. [0063] Provided in the present disclosure are methods of treating a cancer in a subject that include: administering to the subject a therapeutically effective amount of PNH733 or a pharmaceutically the subject. [0064] Provided in the present disclosure are methods of treating a cancer in a subject that include: (a) identifying the cancer as having cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of PNH733 or a pharmaceutically the subject. [0065] Provided in the present disclosure are methods of treating a cancer in a subject that include: (a) determining or having determined that the cancer has cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of PNH733 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or a pharmaceutically the subject. [0066] In some embodiments of any of the methods described herein, the cancer is a multidrug resistant cancer. [0067] In some embodiments, the multidrug resistant cancer is a daunorubicin-resistant cancer or a paclitaxel-resistant cancer. [0068] In some embodiments of any of the methods described herein, the cancer is a metastatic cancer. [0069] In some embodiments of any of the methods described herein, the cancer is an epithelial breast cancer. [0070] In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. [0071] In some embodiments, the cancer is a breast cancer. [0072] In some embodiments, the breast cancer is a triple negative breast cancer. [0073] In some embodiments, the breast cancer is a Her2 negative and estrogen receptor/progesterone receptor positive breast cancer. [0074] In some embodiments, the breast cancer is an inflammatory breast cancer. [0075] In some embodiments of any of the methods described herein, the cancer is a cancer in which LACTB is expressed at low level or has low activity levels, and/or the cancer is p53 negative and/or the cancer is PTEN negative. [0076] In some embodiments of any of the methods described herein, administering the compound inhibits cellular proliferation, cellular invasion, and/or cellular migration. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0077] In some embodiments of any of the methods described herein, the method further includes administering an additional cancer treatment. [0078] In some embodiments, the additional cancer treatment is radiation therapy or chemotherapy. [0079] In some embodiments of any of the methods described herein, the method further includes administering an immunotherapy composition to the subject. [0080] In some embodiments of any of the methods described herein, the compound is administered by intravenous administration or oral administration. [0081] Provided in the present disclosure are methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level and/or activity of LACTB in the sample; and selecting a treatment including administering PNH733 or a pharmaceutically if the sample has a reduced level and/or activity of LACTB as compared to a sample including a non-cancerous cell from the same subject. [0082] Provided in the present disclosure are methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level of MRPS34 in the sample; and selecting a treatment including administering PNH733 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or a pharmaceutically if the sample has an increased level of MRPS34 as compared to a sample including a non-cancerous cell from the same subject. [0083] Provided in the present disclosure are methods of activating LACTB expression and/or activity that include contacting a cell with MGL060 or pharmaceutically [0084] Provided in the present disclosure are methods of reducing or inhibiting LACTB autoproteolytic activity that include contacting a cell with MGL060 or pharmaceutically [0085] Provided in the present disclosure are methods of treating an epithelial cancer in a subject that include: administering to the subject a therapeutically effective amount of MGL060 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically the subject. [0086] Provided in the present disclosure are of treating or preventing disease progression in a subject having a disease associated with a loss or inactivation of LACTB expression and/or activity that include administering to the subject a therapeutically effective amount of MGL060 or pharmaceutically or preventing disease progression in the subject. [0087] In some embodiments, the disease associated with a loss of LACTB expression and/or activity is a cancer. [0088] Provided in the present disclosure are of treating a cancer in a subject that include: administering to the subject a therapeutically effective amount of MGL060 or pharmaceutically the subject. [0089] Provided in the present disclosure are methods of treating a cancer in a subject that include: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 (a) identifying the cancer as having cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of MGL060 or pharmaceutically the subject. [0090] Provided in the present disclosure are methods of treating a cancer in a subject that include: (a) determining or having determined that the cancer has cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount MGL060 or pharmaceutically the subject. [0091] In some embodiments of any of the methods described herein, the cancer is a multidrug resistant cancer. [0092] In some embodiments, the multidrug resistant cancer is a daunorubicin-resistant cancer or a paclitaxel-resistant cancer. [0093] In some embodiments of any of the methods described herein, the cancer is a metastatic cancer. [0094] In some embodiments of any of the methods described herein, the cancer is an epithelial breast cancer. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0095] In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. [0096] In some embodiments, the cancer is a breast cancer. [0097] In some embodiments, the breast cancer is a triple negative breast cancer. [0098] In some embodiments, the breast cancer is a Her2 negative and estrogen receptor/progesterone receptor positive breast cancer. [0099] In some embodiments, the breast cancer is an inflammatory breast cancer. [0100] In some embodiments of any of the methods described herein, the cancer is a cancer in which LACTB is expressed at low level or has low activity levels, and/or the cancer is p53 negative and/or the cancer is PTEN negative. [0101] In some embodiments of any of the methods described herein, administering the compound inhibits cellular proliferation, cellular invasion, and/or cellular migration. [0102] In some embodiments of any of the methods described herein, the method further includes administering an additional cancer treatment. [0103] In some embodiments, the additional cancer treatment is radiation therapy or chemotherapy. [0104] In some embodiments of any of the methods described herein, the method further includes administering an immunotherapy composition to the subject. [0105] In some embodiments of any of the methods described herein, the compound is administered by intravenous administration or oral administration. [0106] Provided in the present disclosure are methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level and/or activity of LACTB in the sample; and selecting a treatment including administering MGL060 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically if the sample has a reduced level and/or activity of LACTB as compared to a sample including a non-cancerous cell from the same subject. [0107] Provided in the present disclosure are methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level of MRPS34 in the sample; and selecting a treatment including administering MGL060 or pharmaceutically if the sample has an increased level of MRSP34 as compared to a sample including a non-cancerous cell from the same subject. [0108] Provided in the present disclosure are methods of determining the efficacy of a cancer treatment in a subject having a cancer that include: (a) determining the level of MRPS34 or LACTB in a first sample obtained from a subject having the cancer at a first time point; (b) administering to the subject a cancer treatment; (c) providing a second sample obtained from the subject at a second time point after step (b); (d) determining the level of MRPS34 or LACTB a first sample obtained from a subject having the cancer; and Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 (e) identifying the administered cancer treatment as being effective when: (i) the level of MRPS34 is decreased at the second time point as compared to the first time point; or (ii) the level of LACTB is increased at the second time point as compared to the first time point. [0109] In some embodiments, the subject has previously been diagnosed with a cancer. [0110] In some embodiments, the cancer treatment is a compound of formula (I), or a pharmaceutically acceptable salt thereof. [0111] In some embodiments, the cancer treatment is PNH733. [0112] In some embodiments, the cancer treatment is MGL06. [0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. [0114] Other features and advantages of the disclosure will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS [0115] FIG.1 is a picture of Western blots showing protein expression of endogenous LACTB in a panel of breast cancer cells (HS578t, MCF7R, SKBR3 and HCC1806) upon treatment with control dimethyl sulfoxide (DMSO) or PNH733 compound at 10 µM and 30 µM concentration. [0116] FIG.2 are proliferation curves of breast cancer cell lines (HS578t, MCF7R, SKBR3 and HCC1806) upon PNH733 treatment for up to 8 days. p<0.0001. [0117] FIG.3 are dot plots showing growth of tumors formed by injection of MCF7R breast cancer cells (left panels) and HCC1806 breast cancer cells (right panels), upon intravenous administration of vehicle (DMSO) or compound PNH733 (A4). The dot plots represent tumor Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 volume over the time of the treatment (top row), and the tumor weight at the end of the 2 weeks treatment upon isolation from mice (bottom row). n.s. non-significant; ** p<0.01; *** p<0.001; **** p<0.0001. [0118] FIG.4 are dot plots showing growth of tumors formed by injection of OVCAR8 ovarian cancer cells and EFO27 ovarian cancer cell, upon intravenous administration of vehicle (DMSO) and compound PNH733 (A4). [0119] FIG.5 are images of OVCAR8 tumor’s luminescence in mice treated with DMSO (Vehicle) and PNH733 (A4), with respective quantification of luminescence signal, after 1 and 2 weeks of treatment (bottom row). Statistics were calculated using student t-test, n.s. non- significant; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. [0120] FIG.6 are growth curves of control patient-derived tumors and tumors treated with PNH733 and MGL060 compounds for 19 days in ovarian cancer model. [0121] FIG.7 are growth curves of control patient-derived tumors and tumors treated with PNH733 and MGL060 compounds for 19 days in breast cancer model. [0122] FIG.8 are growth curves of control patient-derived tumors and tumors treated with PNH733 and MGL060 compounds for 19 days in pancreatic cancer model. [0123] FIG.9 is a picture of a Western blot showing LACTB-WT protein expression, with and without inhibitor (Z-AAD-CMK), incubated either in PBS or in H₂O for 36 hours at 25ºC. [0124] FIG.10 is a picture of Western blot showing recombinant LACTB-WT protein expression levels following incubation on its own (in H2O containing DMSO solution), with an inhibitor of its activity (Z-AAD-CMK), and with different concentrations (10 µM, 30 µM, 50 µM, 100 µM) of the compound PNH733 for 48 hours at 25 ºC. [0125] FIG.11 is a graph showing enzymatic activity of LACTB against an external probe in the presence or absence of various concentrations of PNH733 (1 µM, 5 µM, 10 µM, 50 µM, and 100 µM). [0126] FIG.12 is a picture of Western blots of LACTB protein expression levels upon overexpression of doxycycline-inducible MRPS34, in a panel of breast cancer cell lines (MCF7R, T47D, HCC1806, HS578t, HCC38) after 1 day, 3 days, and 6 days of doxycycline treatment. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0127] FIG.13 is a picture of immunohistochemistry staining of human breast clinical tissues (n=13) of normal or tumorigenic (grade 1, 2, and 3) origin with LACTB and MRPS34 antibodies. [0128] FIG.14 is a picture of Western blots showing LACTB and LACTB + MRPS34 recombinant protein expression levels, with and without LACTB inhibitor Cells were incubated at 25 ºC at different time points from 18 hours to 48 hours (18 hours, 24 hours, 30 hours, 36 hours, 42 hours, and 48 hours) in H2O. DETAILED DESCRIPTION [0129] Deactivation of tumor suppressors is a crucial step in the development and progression of malignancies. In human cells this can occur by complete loss/inactivation of a particular tumor suppressor, through cellular processes such as gene mutation (Stratton, M.R., et al., The cancer genome. Nature, 2009.458(7239): 719-724; and Greenman, C., et al., Nature, 2007. 446(7132): 153-158), aberrant splicing (Yang, Q., et al., Mol Cell Biol, 2019.11(10): 920-929; and Sciarrillo, R., et al., Drug Resist Updat, 2020.53: 100728), and epigenetic silencing (Esteller, M., F1000 Biol Rep, 2011.3: 9; and Baylin, S.B. and P.A. Jones, Cold Spring Harb Perspect Biol, 2016.8(9)). The complete loss of tumor suppressor is described by the Knudson’s two-hit hypothesis, which stipulates that inactivation of both alleles of a tumor suppressor gene is required to promote tumor progression (Tomlinson, I.P.M., et al., Chromosomes and Cancer, 2002.34(4): 349-353). However, subsequent research revealed the existence of a different class of tumor suppressors termed “haploinsufficient/dose-dependent,” where loss of only one allele of such a tumor suppressor already contributes to the tumorigenic process, which is sometimes manifested through lower expression levels of such proteins (Santarosa, M. and A. Ashworth, Biochim Biophys Acta, 2004.1654(2): 105-122; and Inoue, K. and E.A. Fry, Adv Med Biol., 2017.118: 83-122). Examples of haploinsufficient tumor suppressors include p27Kip1, p53, DMP1, NF1, and PTEN, AML1 and others (Inoue, K. and E.A. Fry, Adv Med Biol., 2017.118: 83-122). These types of tumor suppressors represent attractive therapeutic options for cancer treatment since, in many cases, they are never completely absent from the cell. Instead, they are actively kept at low expression or low activity levels by various regulatory mechanisms devised by cancer cells thus allowing their possible therapeutic reactivation. Recently, beta-lactamase- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 like protein (LACTB) was also characterized as a haploinsufficient mitochondrial tumor suppressor in breast cancer cells (Keckesova, Z., et al., Nature, 2017.543(7647): 681-686). [0130] LACTB is a ubiquitously expressed mitochondrial protein, derived from the bacterial penicillin binding/β-lactamase proteins (PBP-βL) family, which eukaryotic cells acquired during endosymbiosis (Smith, T.S., et al., Genomics, 2001.78(1-2): 12-14; and Peitsaro, N., et al., BMC Evol Biol, 2008.8: 26). Eukaryotic LACTB shares conserved active site motifs with bacterial PBP-βL proteins, including the catalytic serine site -SXXK- (Peitsaro, N., et al., BMC Evol Biol, 2008.8: 26). In bacteria, PBP-βL proteins are involved in peptidoglycan synthesis, which is important for bacterial cell wall biogenesis (Ghuysen, J.-M., International Journal of Antimicrobial Agents, 1997.8: 45-60; and Meroueh, S.O., et al., J. Am. Chem. Soc., 2003.125: 9612–9618). Since eukaryotic cells lack bacterial peptidoglycans, the need for LACTB’s catalytic activity and the identity of its substrate is currently unknown. [0131] LACTB was first described as a tumor suppressor in breast cancer (Keckesova, Z., et al., Nature, 2017.543(7647): 681-686), and follow-up studies extended its tumor suppressor role to other cancer types, such as colorectal cancer (Zeng, K., et al., LACTB, Oncogene, 2018. 37(41): 5534-5551; Zhou, S., et al., Cancer Biother Radiopharm, 2020; Xu, W., et al, Cancer Manag Res, 2020.12: 5181-5200; and Wang, C., et al., Cancer Manag Res, 2020.12: 12185- 12195), hepatocellular carcinoma (Xue, C., et al., Am J Transl Res, 2018.10(12): 4152-4162), glioma (Li, H.T., et al., Oncol Res, 2019.27(4): 423-429), gastric cancer (Yang, F., et al., Oncol Lett, 2021.21(6): 470), melanoma (Ma, Y., et al., Cancer Lett, 2021.506: 67-82; and Liu, J., et al., Int J. Nanomedicine, 2021.16: 7697-7709), lung cancer (Xu, Y., et al., Exp Ther Med, 2022. 23(3): 247), and ovarian cancer (Cutano, V., et al., Life Science Alliance, 2023.6(1): e202201510). However, studies in pancreatic cancer (Xie, J., et al., PLoS One, 2021.16(1): e0245908) and in nasopharyngeal carcinoma (Peng, L.X., et al., Cancer Lett, 2021.498: 165- 177) showed that higher mRNA expression of LACTB was associated with cancer progression, thus identifying LACTB as a possible tumor promoter in these cancer types. LACTB’s tumor suppressor function is realized through inhibition of cellular proliferation, invasion, migration, epithelial-mesenchymal transition (EMT), cancer cell death, and induction of differentiation, through different mechanisms of action in a tissue-specific manner (Keckesova, Z., et al., Nature, 2017.543(7647): 681-686; Zeng, K., et al., LACTB, Oncogene, 2018.37(41): 5534-5551; Xue, C., et al., Am J Transl Res, 2018.10(12): 4152-4162; Li, H.T., et al., Oncol Res, 2019.27(4): Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 423-429; Ma, Y., et al., Cancer Lett, 2021.506: 67-82; and Gonzalez-Morena, J.M., et al., Apoptosis, 2022). Collectively, these studies showed that downregulation/inactivation of LACTB in cancer cells which occurs through both, transcriptional and post-translational mechanisms, is a common mechanism how cancer cells counteract the negative effects of LACTB on their viability. While some information is known about the transcriptional regulation of LACTB (Zeng, K., et al., LACTB, Oncogene, 2018.37(41): 5534-5551; Wang, C., et al., Cancer Manag Res, 2020.12: 12185-12195; Ma, Y., et al., Cancer Lett, 2021.506: 67-82; and Lu, J.B., et al., Arch Biochem Biophys, 2016.590: 64-71), the mechanism that leads to post- translational downregulation of LACTB by cancer cells is currently unknown. [0132] These data provide rationale for further evaluation of post-translational regulation of LACTB in cancer cells, and the design or therapeutic interventions for LACTB based cellular reactivation. This application is directed to this need and others. [0133] To the best of the inventor’s knowledge, this is the first time that a substrate of LACTB was identified as being LACTB itself, and that LACTB possessed autoproteolytic activity. This autoproteolytic activity is important for modulation of the tumor suppressive ability of LACTB. [0134] The autoproteolytic activity of LACTB is hijacked by cancer cells that use it to efficiently downregulate LACTB. Mechanistically, this is achieved through upregulation of the 28S ribosomal protein S34 (MRPS34) protein, which acts as a negative regulator of LACTB. Interaction of MRPS34 and LACTB stimulates LACTB’s autoproteolytic activity and leads to its self-degradation. This effect can be reversed by the nucleoside analogue PNH733, resulting in the upregulation of LACTB levels in cancer cells and subsequent therapeutic effect of PNH733 on cancer growth under in vitro, in vivo and human clinical samples settings. I. Definitions [0135] Throughout the definitions, the term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C1-2, C1-3, C1-4, C1-5, and the like. [0136] As used herein, the term “Cn-m alkyl”, employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 as methyl (Me), ethyl (Et), n-propyl (n-Pr), isopropyl (i-Pr), n-butyl, tert-butyl, isobutyl, sec- butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, and the like. In some embodiments, the alkyl group contains from 1 to 5 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms. [0137] As used herein, “Cn-m alken-1-yl” refers to an alkyl group having one or more double carbon-carbon bonds, including one double carbon-carbon bond at the 1-position, and having n to m carbons. Example alken-1-yl groups include, but are not limited to, vinyl (i.e., ethenyl), n- propen-1-yl, isopropenyl, n-buten-1-yl, and the like. In some embodiments, the alkenyl moiety contains 2 to 5, 2 to 4, or 2 to 3 carbon atoms. [0138] As used herein, “Cn-m alkynyl” refers to an alkyl group having one or more triple carbon-carbon bonds, including one triple carbon-carbon bond at the 1-position, and having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 5, 2 to 4, or 2 to 3 carbon atoms. [0139] As used herein, the term “Cn-m alkoxy”, employed alone or in combination with other terms, refers to a group of formula-O-alkyl, wherein the alkyl group has n to m carbons. Example alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n- propoxy and isopropoxy), butoxy (e.g., n-butoxy and tert-butoxy), and the like. In some embodiments, the alkyl group has 1 to 5, 1 to 4, or 1 to 3 carbon atoms. [0140] As used herein, “halo” refers to F, Cl, Br, or I. In some embodiments, halo is F, Cl, or Br. In some embodiments, halo is F or Cl. In some embodiments, halo is F. In some embodiments, halo is Cl. In some embodiments, halo is Br. In some embodiments, halo is I. [0141] As used herein, the term “di(Cn-m-alkyl)amino” refers to a group of formula - N(alkyl)₂, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group of the dialkylamino independently has 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. [0142] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms. In some embodiments, the compound has the (R)-configuration. In some embodiments, the compound has the (S)- configuration. [0143] Resolution of racemic mixtures of compounds can be carried out by any of numerous methods known in the art. An example method includes fractional recrystallization using a chiral resolving acid which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids such as β-camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include stereoisomerically pure forms of α-methylbenzylamine (e.g., S and R forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and the like. [0144] Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art. [0145] All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g., hydrates and solvates) or can be isolated. [0146] In some embodiments, preparation of compounds can involve the addition of acids or bases to affect, for example, catalysis of a desired reaction or formation of salt forms such as acid addition salts. [0147] In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof. Methods for isolating compounds and their salts are routine in the art. [0148] The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified. [0149] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [0150] As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the claimed compounds of general formula I according to this invention, and which are within reasonable medical judgment suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, and the like, and have an acceptable benefit/risk ratio. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto (e.g., phenol or hydroxyamic acid). Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound, a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred, where practicable. Lists of additional suitable salts can be found, e.g., in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., (1985), which is herein incorporated by reference. [0151] The term "therapeutically effective amount" of a compound of the present invention refers to an amount of the compound or drug that is effective in treating a disease or disorder in a human or mammal. In the case of cancer treatment the "effective amount" refers to the amount that inhibits or reduces proliferation of cancer cells, reduces the primary tumor/cancer size, inhibits (that is, to a certain extent slow down and preferably stop) cancer cell infiltration into peripheral organs, inhibits (that is, to a certain extent slow down and preferably stop) the formation of tumor metastases, inhibits, to a certain extent, tumor growth and/or relieves at least to some extent one or more symptoms associated with tumor or cancer. Whereas the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. [0152] The term "pharmaceutical composition" refers to the formulation of a compound and medium, generally accepted in the art, for the delivery of a biologically active compound to a mammal, e.g., a human. Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients. [0153] The term "pharmaceutically acceptable carrier, diluent or filler" as used herein includes, without limitation, any excipient, carrier, glidant, sweetener, preservative, dye, flavor enhancer, surfactant, dispersing agent, suspending agent, isotonic agent, solvent, or emulsifier that has been approved for use in humans or domestic animals. [0154] The invention further relates to compounds of formula I for use as an active ingredient in a pharmacologically acceptable composition which may be prepared by conventional methods Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 known in the art, e.g., the active ingredient may be in a mixture with pharmaceutically acceptable inert organic and/or inorganic carriers and/or with auxiliaries or, where appropriate, attached to them. [0155] The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (ACN) are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety. [0156] The term “treatment” or “treating,” to the extent it relates to a disease or condition includes inhibiting or ameliorating the disease or condition (e.g., arresting or slowing its development), eliminating the disease or condition (e.g., causing regression or cure of the disease or condition), and/or relieving one or more symptoms of the disease or condition. The term “treat” or “treatment” is used herein to denote delaying the onset of, inhibiting, alleviating the effects of, or prolonging the life of a patient suffering from, a condition, e.g., cancer. The terms “effective amount to treat” and “amount effective to treat”, as used herein refer to an amount or concentration of a compound or composition or treatment described herein, e.g., compounds of formula (I), PNH733, MGL060, utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome. For example, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 effective amounts of any of the compounds described herein (e.g., compounds of formula (I), PNH733, MGL060, or pharmaceutically acceptable salts thereof) for use in the present disclosure include, for example, amounts that inhibit the growth of cancer, e.g., tumors and/or tumor cells, improve or delay tumor growth, improve survival for a subject suffering from or at risk of developing cancer, and improve the outcome of other cancer treatments. As another example, effective amounts of any of the compounds described herein (e.g., compounds of formula (I), PNH733, MGL060, or pharmaceutically acceptable salts thereof) can include amounts that advantageously affect a tumor microenvironment. [0157] In some embodiments, the term “treatment” refers to the administration of a compound or composition according to the present invention to alleviate or eliminate symptoms of a disease or disorder. [0158] The term “patient” or “subject” is used throughout the specification to describe an animal, human or non-human, to whom treatment according to the methods of the present disclosure is provided. Veterinary applications are clearly anticipated by the present disclosure. The term includes, but is not limited to, birds, reptiles, amphibians, and mammals, e.g., humans, other primates, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep, and goats. Preferred subjects are humans, farm animals, and domestic pets such as cats and dogs. [0159] The term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors, oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathological type or stage of invasiveness. Also included are tumors and cancers of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas, which include malignancies such as most breast cancers, colon cancers, renal- cell carcinoma, prostate cancer, and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the esophagus, and cancer of the small intestine. [0160] The term “carcinoma” is a term of art and refers to malignancies of epithelial or endocrine tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumor of mesenchymal tissue origin. [0161] A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to, breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. Metastases develop, e.g., when tumor cells shed from a primary tumor, adhere to vascular endothelium, penetrate into surrounding tissues, and grow to form independent tumors at distinct and often distant sites, separate from a primary tumor. [0162] Also contemplated by the present disclosure is administration of any of the compounds described herein (e.g., compounds of formula (I), PNH733, MGL060, or pharmaceutically acceptable salts thereof) to a subject in conjunction with an additional cancer treatment, e.g., chemotherapy, radiation therapy, immunotherapy, gene therapy, and/or surgery, to treat the cancer. [0163] Chemotherapy can involve administration of any of the following classes of compounds: alkylating agents, antimetabolites, e.g., folate antagonists, purine antagonists, and/or pyrimidine antagonists; paclitaxel, daunorubicin, doxorubicin, cisplatin, etc. Radiation therapy can include the use of ɣ-radiation, neutron beams, and/or radioactive isotopes. [0164] The term “immunotherapy” refers to a therapeutic treatment that involves administering to a subject an agent that modulates the immune system. Examples of immunotherapies include immune checkpoint inhibitors, e.g., PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors. Immunotherapy can be, e.g., an antibody therapy (e.g., a monoclonal antibody, a conjugated antibody) or a cellular immunotherapy (e.g., adoptive T-cell therapy, dendritic cell therapy, natural killer cell therapy). [0165] It is further appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment as if the embodiments were claims written in multiple dependent form. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. [0166] Standard abbreviations and acronyms: Bz benzoyl d doublet Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 dd doublet of doublets ddd doublet of doublets of doublets DMF N,N-dimethylformamide DMSO dimethylsulfoxide dt doublet of triplets EtOH ethanol ESI electrospray ionization HPFC high-performance flash chromatography HPLC high-performance liquid chromatography HR high resolution m multiplet Me methyl MeCN acetonitrile MeOH methanol MS mass spectrometry NMR nuclear magnetic resonance Ph phenyl PPh₃ triphenylphosphine q quartet s singlet SiO2 silica gel t triplet td triplet doublet [0167] The following numbering of compounds is used: 1: R¹ = MeO Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 2: R¹ = Me 3: R¹ = Me2N a: R² = ethynyl b: R² = Me c: R² = I d: R² = CN e: R² = vinyl II. Compounds of Formula (I) [0168] This invention describes 1H-pyrazolo[3,4-d]pyrimidine ribonucleosides appropriately substituted in positions 3 and 4 of the general formula I, which show cytotoxic and cytostatic effects on cell lines of preferentially tumor origin and in a wide spectrum of diseases, including tumors of epithelial origin. [0169] From all the known 1H-pyrazolo[3,4-d]pyrimidine ribonucleosides, the presented compounds fundamentally differ in the combination of substituents in position 3 and 4, which is unique and necessary for their anticancer activity. The main difference lies in the presence of certain groups in position 4 (methoxy, dimethylamino, methyl) with the character of a hydrogen bond acceptor, or an alkyl group without the ability to form a hydrogen bond compared to an amino group as a hydrogen bond donor in compounds of general formula A. [0170] The invention also relates to compounds of the formula I for use as second or other active substances having synergistic effect with other active substances in known drugs, or the administration of the compounds of the formula I together with these drugs. [0171] In one embodiment, the present invention also relates to the use of compounds of formula I as prodrugs or other suitable forms which release the active ingredient in vivo. [0172] The present disclosure relates to compounds including substituted 1H-pyrazolo[3,4- d]pyrimidine nucleosides having a structure represented by formula (I): Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 wherein R¹ is C1-C5 alkyl, C1-C5 alkoxy, or di(C1-C5)alkylamino; and R² is C1-C5 alkyl, C2-C5 alken-1-yl, C2-C5 alkyn-1-yl, cyano, or halo, or a pharmaceutically acceptable salt thereof. [0173] In some embodiments, the 3,4-disubstituted 1H-pyrazolo[3,4-d]pyrimidine ribonucleosides comprises: (i) 3-ethynyl-4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; (ii) 3-ethynyl-4-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; (iii) 3-ethynyl-4-(N,N-dimethylamino)-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine; (iv) 4-methoxy-3-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; (v) 3,4-dimethyl-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; (vi) 4-N,N-dimethylamino-3-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine; (vii) 3-iodo-4-methoxy-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; (viii) 4-methoxy-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-3-carbonitril; or (ix) 4-methoxy-3-vinyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine, or a pharmaceutically acceptable salt thereof. [0174] In some embodiments, R¹ is C1-C4 alkyl, C1-C4 alkoxy, or di(C1-C4)alkylamino. In some embodiments, R¹ is C1-C3 alkyl, C1-C3 alkoxy, or di(C1-C3)alkylamino. In some embodiments, R¹ is C1-C2 alkyl, C1-C2 alkoxy, or di(C1-C2)alkylamino. [0175] In some embodiments, R¹ is methyl, methoxy, or N,N-dimethylamino. [0176] In some embodiments, R¹ is C1-C5 alkyl. In some embodiments, R¹ is C1-C4 alkyl. In some embodiments, R¹ is C1-C3 alkyl. In some embodiments, R¹ is C1-C2 alkyl. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0177] In some embodiments, R¹ is methyl. In some embodiments, R¹ is ethyl. In some embodiments, R¹ is propyl. In some embodiments, R¹ is isopropyl. In some embodiments, R¹ is butyl. In some embodiments, R¹ is pentyl. [0178] In some embodiments, R¹ is C1-C5 alkoxy. In some embodiments, R¹ is C1-C4 alkoxy. In some embodiments, R¹ is C1-C3 alkoxy. In some embodiments, R¹ is C1-C2 alkoxy. [0179] In some embodiments, R¹ is methoxy. In some embodiments, R¹ is ethoxy. In some embodiments, R¹ is propoxy. In some embodiments, R¹ is isopropoxy. In some embodiments, R¹ is butoxy. In some embodiments, R¹ is pentoxy. [0180] In some embodiments, R¹ is di(C1-C5)alkylamino. In some embodiments, R¹ is di(C1-C4)alkylamino. In some embodiments, R¹ is di(C1-C3)alkylamino. In some embodiments, R¹ is di(C1-C2)alkylamino. [0181] In some embodiments, R¹ is N,N-dimethylamino. In some embodiments, R¹ is N,N- diethylamino. In some embodiments, R¹ is N,N-dipropylamino. In some embodiments, R¹ is N,N-diisopropylamino. In some embodiments, R¹ is N,N-dibutylamino. In some embodiments, R¹ is N,N-dipentylamino. [0182] In some embodiments, R² is C1-C4 alkyl, C2-C4 alken-1-yl, C2-C4 alkyn-1-yl, cyano, or halo. In some embodiments, R² is C1-C3 alkyl, C2-C3 alken-1-yl, C2-C3 alkyn-1-yl, cyano, or halo. In some embodiments, R² is C1-C2 alkyl, C2-C3 alken-1-yl, C2-C3 alkyn-1-yl, cyano, or halo. [0183] In some embodiments, R² is methyl, vinyl, ethynyl, cyano, or iodo. [0184] In some embodiments, R² is C1-C5 alkyl. In some embodiments, R² is C1-C4 alkyl. In some embodiments, R² is C1-C3 alkyl. In some embodiments, R² is C1-C2 alkyl. [0185] In some embodiments, R² is methyl. In some embodiments, R² is ethyl. In some embodiments, R² is propyl. In some embodiments, R² is isopropyl. In some embodiments, R² is butyl. In some embodiments, R² is pentyl. [0186] In some embodiments, R² is C2-C5 alken-1-yl. In some embodiments, R² is C2-C4 alken-1-yl. In some embodiments, R² is C2-C3 alken-1-yl. [0187] In some embodiments, R² is vinyl. In some embodiments, R² is propen-1-yl. In some embodiments, R² is buten-1-yl. In some embodiments, R² is penten-1-yl. [0188] In some embodiments, R² is C2-C5 alkyn-1-yl. In some embodiments, R² is C2-C4 alkyn-1-yl. In some embodiments, R² is C2-C3 alkyn-1-yl. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0189] In some embodiments, R² is ethynyl. In some embodiments, R² is propyn-1-yl. In some embodiments, R² is butyn-1-yl. In some embodiments, R² is pentyn-1-yl. [0190] In some embodiments, R² is cyano. [0191] In some embodiments, R² is halo. [0192] In some embodiments, R² is fluoro. In some embodiments, R² is chloro. In some embodiments, R² is bromo. In some embodiments, R² is iodo. [0193] In some embodiments, R¹ is C1-C4 alkyl, C1-C4 alkoxy, or di(C1-C4)alkylamino, and R² is C1-C4 alkyl, C2-C4 alken-1-yl, C2-C4 alkyn-1-yl, cyano, or halo. [0194] In some embodiments, R¹ is C1-C3 alkyl, C1-C3 alkoxy, or di(C1-C3)alkylamino, and R² is C1-C3 alkyl, C2-C3 alken-1-yl, C2-C3 alkyn-1-yl, cyano, or halo. [0195] In some embodiments, R¹ is C1-C2 alkyl, C1-C2 alkoxy, or di(C1-C2)alkylamino, and R² is C1-C2 alkyl, C2-C3 alken-1-yl, C2-C3 alkyn-1-yl, cyano, or halo. [0196] In some embodiments, R¹ is methyl, methoxy, or N,N-dimethylamino, and R² is methyl, vinyl, ethynyl, cyano, or iodo. [0197] In some embodiments, the compound is selected from: , III. PNH733 [0198] The present disclosure further relates to methods of using compound PNH733, and pharmaceutically acceptable salts thereof. [0199] PNH733 is a substituted 7-deazapurine ribonucleoside of formula (see US 9,586,986): Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0200] The IUPAC name of 7-(β-D-ribofuranosyl)-7H- pyrrolo[2,3-d]pyrimidine. IV. MGL060 [0201] The present disclosure further relates to methods of using compound MGL060, and pharmaceutically acceptable salts thereof. [0202] MGL060 is a substituted 7-deazapurine ribonucleoside of formula (see Hinshaw et al., J. Chem. Soc. Perkin1., 1975, 13:1248-1253; WO 2003/061576; and Varaprasad et al., Bioorg. Chem.2007, 35: 25-34): [0203] The IUPAC name of ribofuranosyl)-7H-pyrrolo[2,3- d]pyrimidine-5-carbonitrile. V. Pharmaceutical Compositions [0204] The present disclosure further relates to pharmaceutical compositions including a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents. [0205] Compounds provided herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a mixture of said optical isomer Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 comprising racemic mixtures thereof; PNH733, or a pharmaceutically acceptable salt thereof; or MGL060, or a pharmaceutically acceptable salt thereof) are usually administered in the form of pharmaceutical compositions. Thus, provided herein are also pharmaceutical compositions that comprise one or more of the compounds provided herein or a pharmaceutically acceptable salt, optical isomer, or a mixture of said optical isomer comprising racemic mixtures and one or more pharmaceutically acceptable vehicles selected from carriers, adjuvants, and excipients. The compounds provided herein may be the sole active ingredient or one of the active ingredients of the pharmaceutical compositions. Suitable pharmaceutically acceptable vehicles may include, for example, inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants. Such compositions are prepared in a manner well known in the pharmaceutical art. See, e.g., Remington’s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa.17th Ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc.3rd Ed. (G.S. Banker & C.T. Rhodes, Eds.). [0206] In one aspect, provided herein are pharmaceutical compositions including one or more compound provided herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof; PNH733, or a pharmaceutically acceptable salt thereof; or MGL060, or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical compositions comprise a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt, and a pharmaceutically acceptable excipient or carrier. [0207] The pharmaceutical compositions may be administered in either single or multiple doses. The pharmaceutical compositions may be administered by various methods including, for example, rectal, buccal, intranasal, and transdermal routes. In some embodiments, the pharmaceutical compositions may be administered by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant. [0208] One mode for administration is parenteral, for example, by injection. The forms in which the pharmaceutical compositions described herein may be incorporated for administration by injection include, for example, aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0209] Oral administration may be another route for administration of the compounds provided herein. Administration may be via, for example, capsule or enteric coated tablets. In making the pharmaceutical compositions that include at least one compound provided herein or pharmaceutically acceptable salts, isomer, or a mixture thereof, the active ingredient (such as a compound provided herein) is usually diluted by an excipient and/or enclosed within such a carrier that can be in the form of a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be in the form of a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the pharmaceutical compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders. [0210] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose, Vitamin E-Tocopherol polyethylene glycol succinate (Vitamin E-TPGS), and polyethoxylated castor oil (also known as Cremophor EL or Kolliphor EL), or any combinations thereof. The pharmaceutical compositions can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propylhydroxy-benzoates; sweetening agents; and flavoring agents; or any combinations thereof. [0211] The pharmaceutical compositions that include at least one compound described herein or a pharmaceutically acceptable salt, can be formulated so as to provide quick, sustained, or delayed release of the active ingredient (such as a compound provided herein) after administration to the subject by employing procedures known in the art. Controlled release drug delivery systems for oral administration include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U.S. Patent Nos.3,845,770; 4,326,525; 4,902,514; and 5,616,345. Another formulation for use in the methods of the present disclosure employs transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds provided herein in controlled amounts. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent Nos.5,023,252, 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. [0212] For preparing solid compositions such as tablets, the principal active ingredient may be mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound described herein or a pharmaceutically acceptable salt thereof. When referring to these pre-formulation compositions as homogeneous, the active ingredient may be dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. [0213] The tablets or pills of the compounds described herein may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action, or to protect from the acid conditions of the stomach. For example, the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate. [0214] Pharmaceutical compositions for inhalation or insufflation may include solutions and suspensions in pharmaceutically acceptable, aqueous, or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. In other embodiments, compositions in pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner. [0215] In some embodiments, a compound of Formula (I), or a pharmaceutically acceptable salt thereof; PNH733, or a pharmaceutically acceptable salt thereof; or MGL060, or a Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 pharmaceutically acceptable salt thereof, described herein are provided in the form of pharmaceutical compositions. [0216] Each pharmaceutical composition may comprise one or more pharmaceutically acceptable vehicles selected from carriers, adjuvants, and excipients. [0217] The present disclosure further relates to a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use as a medicament. VI. Methods of Treatment [0218] The present disclosure further relates to a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in inhibition of pathological cell proliferation of tumor/non-tumor/cancer origin and for treatment of tumor/non-tumor/cancer disease associated with cell hyperproliferation. [0219] The present disclosure further relates to a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in treatment of tumor/cancer diseases covering epithelial tumors. In some embodiments, the tumors, cancers, and diseases include cancers expressing high mitochondrial small ribosomal subunit protein mS34 (MRPS34) protein levels. In some embodiments, the cancers expressing high MRPS34 protein levels is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. [0220] The present disclosure further relates to pharmaceutical compositions including a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in inhibition of pathological cell proliferation of tumor/non-tumor/cancer origin and/or for treatment of tumor/non tumor/cancer disease associated with cell hyperproliferation. [0221] The present disclosure further relates to pharmaceutical compositions including a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0222] The present disclosure further relates to methods of activating beta-lactamase expression and/or activity that include contacting a cell with a compound of Formula (I), or a pharmaceutically acceptable salt thereof. [0223] The present disclosure further relates to methods of reducing or inhibiting beta- lactamase autoproteolytic activity that include contacting a cell with a compound of Formula (I), or a pharmaceutically acceptable salt thereof. [0224] The present disclosure further relates to methods of treating or preventing disease progression in a subject having a disease associated with a loss or inactivation of beta-lactamase expression and/or activity that include administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, thereby treating or preventing disease progression in the subject. [0225] In some embodiments, the disease associated with a loss of beta-lactamase expression and/or activity is a cancer. [0226] The present disclosure further relates to methods of treating a cancer in a subject that include: administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, thereby treating the subject. [0227] The present disclosure further relates to methods of treating a cancer in a subject that include: (a) identifying the cancer as having cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of the compound of Formula (I) or pharmaceutically acceptable salt thereof, thereby treating the subject. [0228] Identifying the cancer as having cancer cells that express LACTB at low levels or that have low LACTB activity levels can be performed by any appropriate method. In some embodiments, the step of identifying the cancer in the subject as having cancer cells that express LACTB at low levels or that have low LACTB activity levels includes performing an assay to detect a LACTB protein, or expression, or activity or level in a sample from the subject. In some embodiments, the method further includes obtaining a sample from the subject (e.g., a biopsy sample). An assay can be any appropriate assay. In some embodiments, the assay is selected from the group consisting of sequencing (e.g., next generation sequencing), immunohistochemistry, enzyme-linked immunosorbent assay. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0229] The present disclosure further relates to methods of treating a cancer in a subject that include: (a) determining or having determined that the cancer has cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of the compound of Formula (I) or pharmaceutically acceptable salt thereof, thereby treating the subject. [0230] Determining the cancer as having cancer cells that express LACTB at low levels or that have low LACTB activity levels can be performed by any appropriate method. In some embodiments, the step of identifying the cancer in the subject as having cancer cells that express LACTB at low levels or that have low LACTB activity levels includes performing an assay to detect a LACTB protein, or expression, or activity or level in a sample from the subject. In some embodiments, the method further includes obtaining a sample from the subject (e.g., a biopsy sample). An assay can be any appropriate assay. In some embodiments, the assay is selected from the group consisting of sequencing (e.g., next generation sequencing), immunohistochemistry, enzyme-linked immunosorbent assay. [0231] In some embodiments, the cancer is a multidrug resistant cancer. [0232] In some embodiments, the multidrug resistant cancer is a daunorubicin-resistant cancer or a paclitaxel-resistant cancer. [0233] In some embodiments, the cancer is a metastatic cancer. [0234] In some embodiments, the cancer is an epithelial breast cancer. [0235] In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. [0236] In some embodiments, the cancer is a breast cancer. [0237] In some embodiments, the breast cancer is a triple negative breast cancer. [0238] In some embodiments, the breast cancer is a Her2 negative and estrogen receptor/progesterone receptor positive breast cancer. [0239] In some embodiments, the breast cancer is an inflammatory breast cancer. [0240] In some embodiments, the cancer is a cancer in which LACTB is expressed at low level or has low activity levels, and/or the cancer is p53 negative and/or the cancer is PTEN negative. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0241] In some embodiments, administering the compound inhibits cellular proliferation, cellular invasion, and/or cellular migration. [0242] In some embodiments, the method further comprises administering an additional cancer treatment. [0243] In some embodiments, the additional cancer treatment is radiation therapy or chemotherapy. [0244] In some embodiments, the method further comprises administering an immunotherapy composition to the subject. [0245] In some embodiments, the compound is administered by intravenous administration or oral administration. [0246] The present disclosure further relates to methods of activating LACTB expression and/or activity that include contacting a cell with PNH733 or pharmaceutically acceptable [0247] The present disclosure further relates to methods of reducing or inhibiting LACTB autoproteolytic activity that include contacting a cell with PNH733 or pharmaceutically acceptable [0248] The present disclosure further relates to methods of treating an epithelial cancer in a subject that include: administering to the subject a therapeutically effective amount of PNH733 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically acceptable subject. [0249] The present disclosure further relates to methods of treating or preventing disease progression in a subject having a disease associated with a loss or inactivation of LACTB expression and/or activity that include administering to the subject a therapeutically effective amount of PNH733 or a pharmaceutically or preventing disease progression in the subject. [0250] In some embodiments, the disease associated with a loss of LACTB expression and/or activity is a cancer. [0251] The present disclosure further relates to methods of treating a cancer in a subject that include: administering to the subject a therapeutically effective amount of PNH733

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or a pharmaceutically acceptable salt thereof, thereby treating the subject. [0252] The present disclosure further relates to methods of treating a cancer in a subject that include: (a) identifying the cancer as having cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of PNH733 or a pharmaceutically acceptable subject. [0253] Identifying the cancer as having cancer cells that express LACTB at low levels or that have low LACTB activity levels can be performed by any appropriate method. In some embodiments, the step of identifying the cancer in the subject as having cancer cells that express LACTB at low levels or that have low LACTB activity levels includes performing an assay to detect a LACTB protein, or expression, or activity or level in a sample from the subject. In some embodiments, the method further includes obtaining a sample from the subject (e.g., a biopsy sample). An assay can be any appropriate assay. In some embodiments, the assay is selected from the group consisting of sequencing (e.g., next generation sequencing), immunohistochemistry, enzyme-linked immunosorbent assay. [0254] The present disclosure further relates to methods of treating a cancer in a subject that include: (a) determining or having determined that the cancer has cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of PNH733

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or a pharmaceutically acceptable subject. [0255] Determining the cancer as having cancer cells that express LACTB at low levels or that have low LACTB activity levels can be performed by any appropriate method. In some embodiments, the step of identifying the cancer in the subject as having cancer cells that express LACTB at low levels or that have low LACTB activity levels includes performing an assay to detect a LACTB protein, or expression, or activity or level in a sample from the subject. In some embodiments, the method further includes obtaining a sample from the subject (e.g., a biopsy sample). An assay can be any appropriate assay. In some embodiments, the assay is selected from the group consisting of sequencing (e.g., next generation sequencing), immunohistochemistry, enzyme-linked immunosorbent assay. [0256] In some embodiments, the cancer is a multidrug resistant cancer. [0257] In some embodiments, the multidrug resistant cancer is a daunorubicin-resistant cancer or a paclitaxel-resistant cancer. [0258] In some embodiments, the cancer is a metastatic cancer. [0259] In some embodiments, the cancer is an epithelial breast cancer. [0260] In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. [0261] In some embodiments, the cancer is a breast cancer. [0262] In some embodiments, the breast cancer is a triple negative breast cancer. [0263] In some embodiments, the breast cancer is a Her2 negative and estrogen receptor/progesterone receptor positive breast cancer. [0264] In some embodiments, the breast cancer is an inflammatory breast cancer. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0265] In some embodiments, the cancer is a cancer in which LACTB is expressed at low level or has low activity levels, and/or the cancer is p53 negative and/or the cancer is PTEN negative. [0266] In some embodiments, administering the compound inhibits cellular proliferation, cellular invasion, and/or cellular migration. [0267] In some embodiments, the method further comprises administering an additional cancer treatment. [0268] In some embodiments, the additional cancer treatment is radiation therapy or chemotherapy. [0269] In some embodiments, the method further comprises administering an immunotherapy composition to the subject. [0270] In some embodiments, the compound is administered by intravenous administration or oral administration. [0271] The present disclosure further relates to methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level and/or activity of LACTB in the sample; and selecting a treatment including administering PNH733 or a pharmaceutically the sample has a reduced level and/or activity of LACTB as compared to a sample including a non-cancerous cell from the same subject. [0272] The present disclosure further relates to methods of activating LACTB expression and/or activity that include contacting a cell with MGL060 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically acceptable [0273] The present disclosure further relates to methods of reducing or inhibiting LACTB autoproteolytic activity that include contacting a cell with MGL060 or pharmaceutically acceptable [0274] The present disclosure further relates to methods of treating an epithelial cancer in a subject that include: administering to the subject a therapeutically effective amount of MGL060 or pharmaceutically acceptable subject. [0275] The present disclosure further relates to methods treating or preventing disease progression in a subject having a disease associated with a loss or inactivation of LACTB expression and/or activity that include administering to the subject a therapeutically effective amount of MGL060 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically acceptable preventing disease progression in the subject. [0276] In some embodiments, the disease associated with a loss of LACTB expression and/or activity is a cancer. [0277] The present disclosure further relates to methods of treating a cancer in a subject that include: administering to the subject a therapeutically effective amount of MGL060 or pharmaceutically acceptable subject. [0278] The present disclosure further relates to methods of treating a cancer in a subject that include: (a) identifying the cancer as having cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of MGL060 or pharmaceutically acceptable subject. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0279] Identifying the cancer as having cancer cells that express LACTB at low levels or that have low LACTB activity levels can be performed by any appropriate method. In some embodiments, the step of identifying the cancer in the subject as having cancer cells that express LACTB at low levels or that have low LACTB activity levels includes performing an assay to detect a LACTB protein, or expression, or activity or level in a sample from the subject. In some embodiments, the method further includes obtaining a sample from the subject (e.g., a biopsy sample). An assay can be any appropriate assay. In some embodiments, the assay is selected from the group consisting of sequencing (e.g., next generation sequencing), immunohistochemistry, enzyme-linked immunosorbent assay. [0280] The present disclosure further relates to methods of treating a cancer in a subject that include: (a) determining or having determined that the cancer has cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount MGL060 or pharmaceutically acceptable subject. [0281] Determining the cancer as having cancer cells that express LACTB at low levels or that have low LACTB activity levels can be performed by any appropriate method. In some embodiments, the step of identifying the cancer in the subject as having cancer cells that express LACTB at low levels or that have low LACTB activity levels includes performing an assay to detect a LACTB protein, or expression, or activity or level in a sample from the subject. In some embodiments, the method further includes obtaining a sample from the subject (e.g., a biopsy sample). An assay can be any appropriate assay. In some embodiments, the assay is selected from the group consisting of sequencing (e.g., next generation sequencing), immunohistochemistry, enzyme-linked immunosorbent assay. [0282] In some embodiments, the cancer is a multidrug resistant cancer. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0283] In some embodiments, the multidrug resistant cancer is a daunorubicin-resistant cancer or a paclitaxel-resistant cancer. [0284] In some embodiments, the cancer is a metastatic cancer. [0285] In some embodiments, the cancer is an epithelial breast cancer. [0286] In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. [0287] In some embodiments, the cancer is a breast cancer. [0288] In some embodiments, the breast cancer is a triple negative breast cancer. [0289] In some embodiments, the breast cancer is a Her2 negative and estrogen receptor/progesterone receptor positive breast cancer. [0290] In some embodiments, the breast cancer is an inflammatory breast cancer. [0291] In some embodiments, the cancer is a cancer in which LACTB is expressed at low level or has low activity levels, and/or the cancer is p53 negative and/or the cancer is PTEN negative. [0292] In some embodiments, administering the compound inhibits cellular proliferation, cellular invasion, and/or cellular migration. [0293] In some embodiments, the method further comprises administering an additional cancer treatment. [0294] In some embodiments, the additional cancer treatment is radiation therapy or chemotherapy. [0295] In some embodiments, the method further comprises administering an immunotherapy composition to the subject. [0296] In some embodiments, the compound is administered by intravenous administration or oral administration. [0297] The present disclosure further relates to methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level and/or activity of LACTB in the sample; and selecting a treatment including administering MGL060 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically acceptable sample has a reduced level and/or activity of LACTB as compared to a sample including a non-cancerous cell from the same subject. VII. Methods of Selecting a Cancer Treatment [0298] The present disclosure further relates to methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level and/or activity of LACTB in the sample; and selecting a treatment including administering a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to the subject if the sample has a reduced level and/or activity of LACTB as compared to a sample including a non-cancerous cell from the same subject. The present disclosure further relates to methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; determining that the sample has a reduced level and/or activity of LACTB as compared to a sample including a non-cancerous cell from the same subject; and selecting a treatment including administering a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to the subject. The present disclosure further relates to methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level of MRPS34 in the sample; and Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 selecting a treatment including administering PNH733 to the subject if the sample has an increased level of MRPS34 as compared to a sample including a non-cancerous cell from the same subject. The present disclosure further relates to methods of selecting a cancer treatment for a subject that include: providing a sample including a cancer cell from the subject; detecting the level of MRPS34 in the sample; and selecting a treatment including administering MGL060 to the subject if the sample has an increased level of MRPS34 as compared to a sample including a non-cancerous cell from the same subject. VIII. Methods of Determining the Efficacy of a Cancer Treatment The present disclosure further relates to methods of determining the efficacy of a cancer treatment in a subject having a cancer that include: (a) determining the level (protein level and/or mRNA level) of MRPS34 or LACTB in a first sample obtained from a subject having the cancer at a first time point; (b) administering to the subject a cancer treatment; (c) providing a second sample obtained from the subject at a second time point after step (b); (d) determining the level (protein level and/or mRNA level) of MRPS34 or LACTB a first sample obtained from a subject having the cancer; and (e) identifying the administered cancer treatment as being effective when: (i) the level (protein level and/or mRNA level) of MRPS34 is decreased at the second time point as compared to the first time point; or (ii) the level (protein level and/or mRNA level) of LACTB is increased at the second time point as compared to the first time point. In some embodiments, the subject has previously been diagnosed with a cancer. In some embodiments, the cancer treatment is any of the compounds disclosed herein (e.g., a compound having the structure of Formula (I), PNH733, or MGL060). In some embodiments, the first sample and the second sample include a liquid biopsy, tissue biopsy, or urine sample. The difference between the first and second time points can be, e.g., between 1 week and 40 weeks, between 1 week and 30 weeks, between 1 week and 20 weeks, between 1 week and 16 weeks, between 1 week and 12 weeks, between 1 week and 8 weeks, between 1 week and 6 weeks, between 1 week and 4 weeks, between 1 week and 2 weeks, between 2 weeks and 16 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 weeks, between 2 weeks and 8 weeks, between 2 weeks and 4 weeks, between 4 weeks and 8 weeks. IX. Administration [0299] The compounds disclosed herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof; PNH733, or a pharmaceutically acceptable salt thereof; or MGL060, or a pharmaceutically acceptable salt thereof) can be administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), transdermal, vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural), and the like. In some embodiments, the administration is oral, intravenous, subcutaneous, or intramuscular. It will be appreciated that the preferred route may vary with, for example, the condition of the recipient. An advantage of certain compounds disclosed herein is that they are orally bioavailable and can be dosed orally. [0300] A compound of the present disclosure may be administered to an individual in accordance with an effective dosing regimen for a desired period of time or duration. [0301] The specific dose level of a compound of the present disclosure for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease in the subject undergoing therapy. For example, a dosage may be expressed as a number of milligrams of a compound described herein per kilogram of the subject’s body weight (mg/kg). Normalizing according to the subject’s body weight is particularly useful when adjusting dosages between subjects of widely disparate size, such as occurs when using the drug in both children and adult humans or when converting an effective dosage in a non-human subject such as dog to a dosage suitable for a human subject. [0302] The dosage or dosing frequency of a compound of the present disclosure may be adjusted over the course of the treatment, based on the judgment of the administering physician. [0303] The compounds provided herein can be administered by any useful route and means, such as by oral or parenteral (e.g., intravenous) administration. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0304] A single dose can be administered hourly, daily, weekly, or monthly. For example, a single dose can be administered once every 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours or once every 24 hours. A single dose can also be administered once every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or once every 7 days. A single dose can also be administered once every 1 week, 2 weeks, 3 weeks, or once every 4 weeks. In certain embodiments, a single dose can be administered once every week. A single dose can also be administered once every month. [0305] Administration may be performed, e.g., at least once (e.g., at least 2-times, at least 3- times, at least 4-times, at least 5-times, at least 6-times, at least 7-times, at least 8-times, at least 9-times, at least 10-times, at least 11-times, at least 12-times, at least 13-times, or at least 14- times) a week. Also contemplated are monthly treatments, e.g., administering at least one per month for at least one month (e.g., at least two, three, four, five, or six or more months, e.g., 12 or more months), and yearly treatments (e.g., administration once year for one or more years). [0306] Administration can be via any art-known means, e.g., intravenous, subcutaneous, intraperitoneal, oral, and/or rectal administration, or any combination of known administration methods. [0307] The frequency of dosage of the compound of the present disclosure will be determined by the needs of the individual patient. EXEMPLARY EMBODIMENTS [0308] Embodiment 1 is substituted 1H-pyrazolo[3,4-d]pyrimidine nucleosides of general formula I wherein R¹ is C1-C5 alkyl, C1-C5 ² group and R is C1-C5 alkyl, C1-C5 alken-1-yl, ethynyl, cyano group, halogen, or C1-C5 alkyn-1-yl and pharmaceutically acceptable salt thereof. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0309] Embodiment 2 is the 1H-pyrazolo[3,4-d]pyrimidine nucleosides of embodiment 1, wherein 3,4-disubstituted 1H-pyrazolo[3,4-d]pyrimidine ribonucleosides of general formula I, being: 3-ethynyl-4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine, 3-ethynyl-4-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine, 3-ethynyl-4-(N,N-dimethylamino)-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine, 4-methoxy-3-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine, 3,4-dimethyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-N,N-dimethyl-3-methyl-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine, 3-iodo-4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-3-carbonitril, or 4-methoxy-3-vinyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine. [0310] Embodiment 3 is the 3,4-disubstituted 1H-pyrazolo[3,4-d]pyrimidine ribonucleosides of general formula I for use as medicaments. [0311] Embodiment 4 is the 3,4-disubstituted 1H-pyrazolo[3,4-d]pyrimidine ribonucleosides of general formula I for use in inhibition of pathological cell proliferation of tumor/non- tumor/cancer origin and for treatment of tumor/non-tumor/cancer disease associated with cell hyperproliferation. [0312] Embodiment 5 is the 3,4-disubstituted 1H-pyrazolo[3,4-d]pyrimidine ribonucleosides of general formula I for use in treatment of tumor/cancer diseases covering epithelial tumors. [0313] Embodiment 6 is the 1H-pyrazolo[3,4-d]pyrimidine nucleosides of embodiment 1 or embodiment 2, wherein a pharmaceutical composition comprising a therapeutically effective amount of a compound of general formula I and one or more pharmaceutically acceptable carriers, excipients/diluents. [0314] Embodiment 7 is the 1H-pyrazolo[3,4-d]pyrimidine nucleosides of embodiment 6 for use in inhibition of pathological cell proliferation of tumor/non-tumor/cancer origin and/or for treatment of tumor/non tumor/cancer disease associated with cell hyperproliferation. [0315] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 EXAMPLES Example 1: Therapeutic Reactivation of LACTB [0316] The reported effect of LACTB on cancer cell differentiation and EMT inhibition and the reported correlation between low LACTB levels and the poor clinical outcome suggested that the LACTB pathway could represent a possible molecular target for future therapeutic tumor suppressor reactivation and differentiation-inducing therapeutic approaches in cancer. Therefore, chemical compounds that can act as positive regulators of the LACTB pathway and that can ultimately lead to the restoration of LACTB levels in cancer cells were evaluated. [0317] For this purpose, the Dual-Luciferase Reporter Assay (Pandolfi and Stecca Methods Mol Biol 2015, 1322: 71-79; Allard and Kopish, “Luciferase Reporter Assays: Powerful, Adaptable Tools for Cell Biology Research 2008, 23-26) which employs two distinct types of luciferases, Firefly Luciferase (FL) and nanoluciferase (Nanoluc) that use different substrates and emit different wavelengths (Nanoluc emission – 460nm; FL emission – 565nm), were set-up and optimized (data not shown). This allowed FL signal to serve as the internal cellular standard and the Nanoluc signal to serve as a readout of expression levels of a protein that was fused to it. A construct where LACTB was fused with Nanoluc protein on its C-terminus was generated, and the LACTB-Nanoluc fusion protein was stably expressed in non-tumorigenic human epithelial HME cells (HME-LACTB-Nanoluc). HME-LACTB-Nanoluc cells were then stably transduced with FL and the follow up Western Blot analysis confirmed the correct expression of both transgenes (data not shown). [0318] Cells were then seeded into 384-wells plates, and incubated for 24-hours with different chemical compounds at 10 μM concentration. Luminescence was measured for both luciferases. The results were normalized to internal firefly luciferase standard. [0319] PNH733, which is a nucleoside analogue 5-ethynyl-4-methoxy-7-(β-D-ribofuranosyl)- 7H-pyrrolo[2,3-d]pyrimidine), was one of the compound hits capable of increasing endogenous LACTB protein levels in human non-tumorigenic epithelial cells. Therefore, PNH733 was added to a panel of breast cancer cell lines to evaluate whether PNH733 could also modulate the endogenous expression levels of LACTB of this panel of breast cancer cell lines. As shown in Fig.1, PNH733, at both 10 μM and 30 μM final concentrations, was able to significantly upregulate endogenous LACTB protein level in a panel of luminal and basal breast cancer cell lines (HS578t, MCF7R, SKBR3 and HCC1806). Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0320] It was determined whether PNH733 could modulate the growth of luminal and basal breast cancer cell lines under in vitro 2D conditions. Indeed, PNH733 was capable of negatively affecting the growth of a wide panel of breast cancer cells to a very significant extent (Fig.2). Example 2: In vivo Testing of PNH733 in Mouse Models of Breast Cancer and Ovarian Cancer [0321] Preclinical in vivo testing of the PNH733 therapeutic potential in mouse xenografts was performed with two breast cancer cell models (basal HCC1806 and luminal MCF7R). Once tumors were formed, the PNH733 compound was injected intravenously at 10 mg/kg for the duration of two weeks and the tumor growth was assessed by tumor measurement. The results of this experiment showed a significant inhibitory effect of PNH733 on tumor growth (volume and weight) for both basal HCC1806 and luminal MCF7 breast cancer models (Fig.3). [0322] Next, the generality of this effect in other types of cancer was examined. Since LACTB was also shown to have a strong tumor suppressive effect in the ovarian cancer setting (Cutano et al., Life Science Alliance 2023, 6(1):e202201510) this type of cancer model was included in our experimental setup. [0323] As was the case in breast cancer, the results showed a strong negative effect of the PNH733 compound on in vivo growth of two aggressive ovarian cancer cell lines (OVCAR8 and EFO27) (Fig.4 and Fig.5). Example 3: In vivo Testing of PNH733 and MGL060 in Patient-Derived xenograft models of Breast Cancer, Ovarian Cancer, and Pancreatic Cancer [0324] Since the experiments in the previous preclinical mouse models showed the efficacy of PNH733 to negatively influence the growth of human cancer cell lines we wanted to examine the therapeutic efficacy of both PNH733 and MGL060 in patient-derived primary human tumors. [0325] Preclinical in vivo testing of the PNH733 therapeutic potential in patient-derived mouse xenografts was performed with three cancer cell models (1 breast cancer model, 1 ovary cancer model, 1 pancreatic cancer model). Once tumors were formed, the PNH733 compound and the MGL060 compound were injected intravenously at 10 mg/kg for the duration of nineteen days and the tumor growth was assessed by tumor measurement. The results of this experiment Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 showed a significant inhibitory effect of PNH733 and MGL060 on tumor growth in all three tested cancer models (Fig.6, Fig.7, and Fig.8). Example 4: Identification of Potential Substrates of LACTB [0326] Eukaryotic LACTB is a serine protease with an unknown substrate. In order to identify the substrate of LACTB, a modified protocol of Proteomic Identification of protease Cleavage Sites (PICS) assay (Schilling et al., PNAS 2009, 106: 18960-18965) was prepared for uncovering substrates of mitochondrial proteases. Mitochondria from MCF7R cells were isolated, and using hypotonic buffers, the intact mitochondrial proteome containing full-length mitochondrial proteins was released. This was followed by blocking the reactive groups within the full-length mitochondrial proteome and addition of wild type recombinant LACTB. Cleavage of its substrate by LACTB revealed new reactive N-termini within the cleaved substrate, which were biotinylated, captured, and analyzed by mass spectrometry. In parallel, the assay was performed under the same conditions in the presence of catalytically inactive S164A LACTB mutant as a negative control. The results identified 15 proteins with a high probability of being the substrate/s of LACTB (data not shown). The recombinant LACTB protein used in the modified PICS assay was isolated from human HEK293T cells and validated by Western blot analysis and by enzymatic activity assay for the correct size, expression, and functional catalytic activity. LACTB-WT was identified as a potential LACTB substrate, but not the catalytically inactive S164A LACTB. Therefore, the possibility of LACTB serving as its own substrate and possessing autoproteolytic activity was investigated. Example 5: Autoproteolytic Activity of LACTB Recombinant LACTB-WT, with and without the inhibitor of its enzymatic activity (Granzyme B inhibitor - Z-AAD-CMK, (Bachovchin et al., Nat Chem Biol 2014, 10(8): 656-663)), was incubated in phosphate-buffered saline (PBS) buffer or in water (H2O) for 36 hours at 25 ºC and the resulting stability of LACTB was examined by Western Blot. The results showed that LACTB-WT, incubated in H₂O was undergoing autoproteolysis as manifested by the appearance of the degradation products of LACTB (Fig.9). This degradation is abolished in the presence of the inhibitor of LACTB’s enzymatic activity. These data showed that LACTB-WT indeed possesses autoproteolytic activity, which is realized through the active catalytic site of LACTB. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 Example 6: Inhibition of LACTB Autoproteolytic Activity by PNH733 [0327] We examined whether mechanistically PNH733 acted through interrupting the LACTB autoproteolysis, thereby leading to restoration of LACTB levels in cancer cells. The recombinant LACTB protein was incubated in the presence of PNH733 at different concentrations (10 µM, 30 µM, 50 µM, or 100 µM) and showed that while in control conditions (LACTB+DMSO) there is an ongoing autoproteolytic activity of LACTB, this activity is abolished in the presence of PNH733 at all tested concentrations and in the presence of the catalytic inhibitor of LACTB (Fig.10). Regarding the LACTB enzymatic activity against an external peptide probe, PNH733 did not significantly affect LACTB’s enzymatic capability at variety of concentrations (1 µM, 5 µM, 10 µM, 50 µM, or 100 µM) (Fig.11). This result suggested that PNH733 did not bind into the catalytic core of LACTB. Example 7: Interaction of LACTB and MRPS34 [0328] Since LACTB was shown by many studies to be efficiently downregulated in cancer cells, these results pointed to the possibility that LACTB might be downregulated by cancer cells through induction of its autoproteolytic activity (Keckesova et al., Nature 2017, 543(7647): 681- 686; Zeng et al., Oncogene 2018, 37(41): 5534-5551; Wang et al., Cancer Manag Res 2020, 12: 12185-12195; Li et al., Oncol Res 2019, 27(4): 423-429; Cutano et al., Life Science Alliance 2023, 6(1): e202201510) . This possible mode of regulation was investigated to uncover the regulator/s of LACTB’s autoproteolytic activity in cancer cells using immunoprecipitation assays. In these experiments, LACTB protein complexes were pulled down from MCF7R cells expressing endogenous LACTB or doxycycline inducible wild type LACTB for 1 day, and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) for the presence of LACTB’s binding partners. The proteins identified through mass spectrometry were then tested for their ability to bind LACTB through secondary immunoprecipitation experiments and subsequent Western Blot analysis. The results of these experiments showed that mitochondrial small ribosomal subunit protein mS34 (MRPS34) was efficiently pulled down through LACTB (data not shown). MRPS34 is a component of the small subunit of the mitochondrial ribosome, which is responsible for synthesizing proteins within the mitochondria. Within mitochondria, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 MRPS34 helps to stabilize the structure of the small ribosomal subunit and is essential for the efficient translation of mitochondrial-encoded genes. [0329] Whether MRPS34 can regulate LACTB levels was examined through overexpression studies. Protein levels of LACTB were monitored upon overexpression of doxycycline-inducible MRPS34, in a panel of breast cancer cell lines (MCF7R, T47D, HCC1806, HS578t, HCC38) after 1 day, 3 days, and 6 days of doxycycline treatment. The results showed a strong inverse relationship in the expression of MRPS34 and LACTB; the increase in the expression levels of MRPS34 led to strong decreases in the expression levels of LACTB (Fig.12). [0330] The inverse relationship in the expression levels of LACTB was further confirmed by immunohistochemistry staining of human breast clinical tissues (n=13) of normal or tumorigenic (grade 1, 2, 3) origin using LACTB and MRPS34 antibodies thus suggesting that MRPS34 might act as a negative regulator of LACTB (Fig.13). Example 8: Acceleration of Autoproteolytic Activity of LACTB by MRPS34 [0331] Since the results showed that LACTB can degrade itself through autoproteolytic activity and that MRPS34 is a negative regulator of LACTB, the possibility whether the MRPS34 exerts its negative regulation of LACTB by enhancing its autoproteolytic activity was investigated. Recombinant LACTB-WT was incubated with and without MRPS34 for different time points, from 18 hours up to 48 hours. MRPS34’s presence accelerated the autoproteolytic activity of LACTB, which was observable at earlier time points (24-hours) of co-incubation (Fig. 14). Of note, it was noticed that the autoproteolytic activity of LACTB in the presence of MRPS34 was more apparent and appeared at earlier time points in the multimeric LACTB forms. In the presence of the inhibitor of the enzymatic activity of LACTB, no autoproteolytic activity was observed at any of the time points (Fig. 14). Example 9: Materials and Methods Cell Culture [0332] Cells were cultivated at 37 ºC in 5% CO₂ humidified atmosphere. Breast cancer cells MCF7R, HEK293T, SKBR3, were grown in Dulbecco’s modified Eagle’s medium (DMEM, Biosera) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% antibiotics (penicillin/streptomycin, Gibco). Breast cancer cells HCC1806, HS578t, T47D, as well as Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 ovarian cancer cells OVCAR8 and EFO27 cells were cultured in Gibco Roswell Park Memorial Institute (RPMI, Sigma-Aldrich) medium supplemented with 10% FBS and 1% penicillin/streptomycin. SUM149 cells were cultured in Ham’s F12 media (Gibco), with 5% FBS, insulin (Sigma Aldrich) and hydrocortisone (Sigma Aldrich). Human mammary epithelial (HME) were grown in a 1:1 mixture of DMEM/Nutrient Mixture F-12 Ham (Sigma-Aldrich) and Mammary Epithelial Cell Growth Basal Medium (MEBM, Lonza) supplemented with Mammary Epithelial Cell Growth Medium BulletKit™ (MEGM™, Lonza). Cell Proliferation Assay [0333] For cell proliferation assessment upon PNH733 compound treatment, cells were seeded in 96-well plates. Once the cells were attached, cells were treated with vehicle (DMSO control) or with PNH733 at a concentration of 10 µM, and proliferation was measured using AlamarBlue (Invitrogen). After the reagent was added (10 μL), plates were incubated 4 h at 37ºC, 5% CO2 in the dark. The absorbance was measured using a Tecan plate reader (Schoeller) at 570 nm using 600 nm as a reference wavelength. After the measurement, the wells were washed with media and the treatment was resumed for a maximum of 9 days depending on the speed of the growth of the cell line. LACTB In Vitro Substrate Assay [0334] 150 ng of recombinant protein was added to a Nunc MicroWell 96-well black, clear optical-bottom plates with polymer base (Sigma-Aldrich) in PBS, together with 1 µL of peptide substrate (100 µM final concentration) - Ac-xxxD-AMC (Enzo Life Sciences and IOCB peptide synthesis facility) at a final volume of 100 μL. Where needed experiments were performed with Granzyme B inhibitor (Sigma-Aldrich, 368050) or PBS was substituted with H₂O. Fluorescence was measured on Spark Multimode Plate Reader (Tecan) for 50- to 180-minutes at 25ºC with a kinetic cycle of 10 minutes, fluorescent top reading of multiple reads per well and with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The peptide substrates contained tetra-peptide sequences with aspartic acids present within the LACTB sequence. As a negative control we used a tetrapeptide ending in alanine instead of aspartic acid: Ac-YGAA- AMC; as a positive control we used a tetrapeptide that has already been described to be cleaved by LACTB: Ac-YVAD-AMC (Keckesova et al., Nature 2017, 543(7647): 681-686). Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 LACTB Autoproteolytic Activity Assay [0335] Purified recombinant LACTB in H2O (distilled and autoclaved) or PBS was incubated at 25ºC, with and without Z-AAD-CMK (Granzyme B inhibitor), which was already described to inhibit LACTB activity (Bachovchin et al., Nat Chem Bio 2014, 10(8): 656-663), for different timepoints. Samples were then processed for western blot analysis and immunoblotted for LACTB. Of note, the exact timing of the occurrence of the autoproteolytic activity depends on the quality and purity of the recombinant LACTB and on the duration of its storage before the onset of the experiment. Therefore, based on these parameters it may occur several hours earlier or later than the average times shown. Western Blot Analysis [0336] Proteins were extracted from cells using RIPA buffer (Sigma) supplemented with protease and phosphatase inhibitors. Approximately 20 μg of protein lysate was loaded into SDS-PAGE 4% to 15% gradient gels (Bio-Rad). Western blots and transfers were done following standard protocols in a wet-transfer system (Bio-Rad) to PVDF membranes (Immobilon-P, Millipore). Next, membranes were blocked for 1 hour with 5% non-fat milk (w/v) in PBS with 0,1% Tween-20 (v/v) followed by blotting with relevant antibodies overnight at 4ºC. Horseradish peroxidase-conjugated secondary antibodies were used (Cell Signaling, 1:5000). Blots were developed using enhanced chemiluminescence (Dura or Femto, Thermo Scientific) and signal was detected using Azure c600 Western blot Imaging system. Immunohistochemistry (IHC) [0337] Commercially obtained human tissues microarray sections (Biomax) were deparaffinized by heating the slides at 60°C for 10 minutes. Next, the slides were washed twice in xylene for 5 minutes, and 1 time in 1:1 xylene:ethanol for 3 minutes. Then slides were hydrated in washes with 100% (twice), 95%, 80%, 75% and 50% ethanol for 3 minutes each wash, followed by wash with 0.85% NaCl, PBS and H₂O for 5 minutes each. Tissues were boiled for 20 minutes in 1x citrate buffer (Sigma-Aldrich). Slides were washed in PBS for 1 min, and then permeabilized with 0,2% Triton X-100 for 20 minutes. The blocking solution was added for 20 min (30% horse serum in PBS, 0,1% Tween®20 at room temperature). Sections Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 were incubated at 4°C overnight with primary antibody. After three washes with PBS, sections were incubated with secondary antibodies (VECTASTAIN ABC-AP) for 1 hour at room temperature, washed three times with PBS, incubated with solution one-step NBT/BCIP. Hematoxylin was used as a counterstain. Slides were dehydrated with 70%, 96%, or 100% ethanol and mounted. IHC images were acquired in optical light microscope (Nikon). In Vivo Therapeutic Treatment with PNH733 [0338] NSG mice were maintained under SPF conditions in individually ventilated cages with controlled temperature (22 ± 2°C) and humidity under a 12 h light/12 h dark cycle and with food and drink ad libitum. Body and weight score condition was accessed twice a week. OVCAR8 and EFO27 ovarian cancer cells transduced with firefly luciferase were resuspended in 15 µL of PBS and Matrigel (Merck) in 1:1 ratio and applied orthotopically and bi-laterally into ovarian fat pad of 20 weeks old female mice in amount of 0.3x10⁶ and 4x10⁶ cells/fat pad, respectively. Two weeks after cell application, mice were treated for 14 days every day with vehicle and PNH733 compound using the following treatment plan: i.v., i.p., i.p., i.v., i.p., i.p., i.p, i.v., i.p., i.p., i.v., i.p., i.p., i.p. During the treatment, in vivo whole-body imaging of mice was performed 3 times on weekly basis using the LagoX machine (Spectral Imaging Instruments) with Aura software. MCF7R and HCC1806 breast cancer cells were resuspended in 30 µl of PBS and Matrigel (Merck) in 1:1 ratio and applied orthotopically and bi-laterally into mammary fat pads of 15-weeks old female mice in amount of 5x10⁵ and 1x10⁵ cells/fat pad, respectively. Two weeks after cell application, mice were treated for 14 days with vehicle and A4 compound using the following treatment plan: i.v., i.p., i.p., i.v., i.p., i.p., i.p, i.v., i.p., i.p., i.v., i.p., i.p., i.p. Tumor dimensions were accessed using a caliper (Mitutoyo) twice a week. Tumor volumes were determined using the formula: width² x length/2. [0339] Collection of samples for histopathology: upon termination of the experiment mice were examined for gross necropsy, tumors were harvested, weighted, and measured followed by fixation in 4% PFA and placed in 70% ethanol for subsequent histological processing and analysis, as described above. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 Statistical Analysis [0340] Data is shown as the average ± standard deviation (SD). Comparative studies between quantitative variables following normal distribution were evaluated using Student’s t-test for paired or unpaired samples according to the experiment. For in vivo experiments differences in tumor growth were analyzed by 2-way ANOVA. Differences were considered significant when p value ≤ 0.05. Statistical analysis was performed using GraphPad Prism 8 software. Example 10: Synthesis of compounds [0341] The target 3-ethynylnucleosides 1a, 2a and 3a were prepared by 5-step synthesis (Scheme 1) starting from 4-chloro-1H-pyrazolo[3,4-d]pyrimidine (4). In the first step, chlorine was substituted with methoxy or dimethylamino group by nucleophilic substitution or by methyl using palladium-catalyzed methylation. Substituted nucleobases 5, 6, 7 were then iodinated into position 3 and glycosylated by 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in presence of BF3·OEt2 to the benzoylated nucleosides 11, 12 and 13. Following Sonogashira coupling with trimethylsilylacetylene in the presence of Pd(PPh₃)₂Cl₂ and CuI gave the desired 3-ethynyl nucleosides 14, 15 and 16. Final treatment with either potassium carbonate in MeOH for 1 hour at r.t. provided the fully deprotected target nucleosides 1a, 2a and 3a. [0342] Scheme 1. Reagents and conditions: a) (for 5): K₂CO₃, MeOH, 70 °C, 1.5 hours; (for 6): AlMe3 (2 M tol), Pd(PPh3)4, dry THF, 80 °C, 1 hour ; (for 7): NH(CH3)2 (2 M in THF), isopropanol, rt, 1 hour; b) (for 8, 10): b) I₂, AgCOOCF₃, DMF, 70 °C, 1.5 hours; (for 9): NIS, DMF, MW reactor: 60 °C, 1 hour; c) 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose, BF₃·OEt₂, MeCN, reflux, 20 minutes; d) trimethylsilylacetylene, Pd(PPh₃)₂Cl₂, CuI, triethylamine, DMF, rt, 1 hour; e) K2CO3, MeOH, rt, 1 hour. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0343] For the preparation of the 3-methyl nucleosides, the 4-chloro-3-methyl-1H- pyrazolo[3,4-d]pyrimidine 17 was employed as the starting material (Scheme 2). The corresponding 4-OMe, 4-Me and 4-NMe2 nucleobases 18, 19, and 20 were prepared following previously described methylation and nucleophilic substitution procedures. Further glycosylations and removal of OBz groups afforded the target nucleosides 1b, 2b, and 3b. [0344] Scheme 2. Reagents and conditions: a) (for 18) K2CO3, MeOH, 70 °C, 1.5 hours; (for 19) AlMe3 (2 M tol), Pd(PPh3)4, dry THF, 80 °C, 1 hour; (for 20): NH(CH3)2 (2 M in THF), isopropanol, rt, 1 hour; b) 1-O-acetyl-2,3,5-tri-O-benzoyl-β-^D-ribofuranose, BF3·OEt2, MeCN, reflux, 20 minutes; c) (for 1b, 2b): K₂CO₃, MeOH, rt, 1 hour; (for 3b): NaOMe (25% wt MeOH), MeOH, rt, 5 min. [0345] Deprotection of benzoyl groups from intermediate nucleoside 11 furnished iodonucleoside 1c, which was then converted to the desired nitrile 1d using palladium-catalyzed cyanation by Zn(CN)₂ in DMF in microwave reactor at 150 °C. The target vinyl nucleoside 1e was synthesized from intermediate 11 by Stille reaction followed by deprotection (Scheme 3).

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0346] Pd(PPh3)4, DMF, MW reactor, 150 °C, 20 min; c) tributyl(vinyl)tin, Pd(PPh3)4, CuI, triethylamine, DMF, 50 °C, 2 h; d) NaOMe (25% wt MeOH), MeOH, rt, 5 min. Experimental Part: General Procedures and Remarks [0347] Reactions were monitored by thin layer chromatography (TLC) on TLC Silica gel 60F254 (Merck) and detected by UV (254 nm) or by a solution of 4-anisaldehyde in ethanol and 10% sulfuric acid. In reasonable cases, reactions were monitored with an Advion Expression Compact mass spectrometer using electrospray ionization (ESI). NMR spectra were measured on a Bruker Avance 500 MHz spectrometer (500.0 MHz for ¹H, 125.7 MHz for ¹³C) or a Bruker Avance 600 MHz spectrometer (600.1 MHz for ¹H and 150.9 MHz for ¹³C) in DMSO-d⁶, CDCl³ (referenced to the residual solvent signal). Chemical shifts are given in ppm (δ-scale), and coupling constants (J) in Hz. Complete assignment of all NMR signals was performed using a combination of H,H-COSY, H,H-ROESY, H,C-HSQC, and H,C-HMBC experiments. High resolution mass spectra were measured on LTQ Orbitrap XL (Thermo Fisher Scientific). High performance flash chromatography (HPFC) was performed with an ISCO Combiflash Rf system Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 on RediSep Rf Gold Silica Gel Disposable columns. The purity of all final compounds (>95%) was determined by analytical HPLC and by clean NMR spectra. [0348] General procedure for the deprotection of both trimethylsilyl and benzoyl groups (General procedure GP1): [0349] The corresponding nucleoside (1.0 mmol) was dissolved in MeOH (10.0 mL) and K2CO3 (2.0 mmol) was added. The resulting mixture was stirred at rt for 1 hour. Then, volatiles were removed, and the residue was purified by HPFC as specified for each compound. [0350] General procedure for the deprotection of both trimethylsilyl and benzoyl groups (General procedure GP2): [0351] The corresponding nucleoside (1.0 mmol) was dissolved in MeOH (20.0 mL) and NaOMe (25% wt MeOH) (1.0 mL) was added. The resulting mixture was stirred at rt for 5 minutes. Then, volatiles were removed, and the residue was purified by HPFC as specified for each compound. [0352] General procedure for the Sonogashira coupling (General procedure GP3): [0353] The corresponding 7-iodonucleoside (1.0 mmol), the trimethylsilyl acetylene (10.0 mmol), Pd(PPh3)2Cl2 (0.05 mmol) and CuI, (0.1 mmol) were dissolved in dry DMF (4.0 mL) and then triethylamine (3.0 mmol) was added dropwise. The resulting mixture was stirred at rt for 1 hour and then volatiles were removed. The residue was purified by HPFC as specified for each compound. [0354] General procedure for the glycosylation of pyrazolopyrimidines (General procedure GP4): [0355] Published procedure was used (F. Seela, K. Xu Org. Biomol. Chem.2007, 5, 3034– 3045). The corresponding nucleobase (1.0 mmol) and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D- ribofuranose (2.2 mmol) were dissolved in dry MeCN (6.0 mL) in a pressure tube and the solution was heated to reflux. After 15 minutes, BF₃·OEt₂ (2.5 mmol) was added dropwise, and the resulting mixture was heated to reflux for 20 minutes. Then, volatiles were removed, and the residue was purified by HPFC as specified for each compound. [0356] 4-Methoxy-1H-pyrazolo[3,4-d]pyrimidine (5) 4-Chloro-1H-pyrazolo[3,4-d]pyrimidine (5.0 g, 32.35 mmol) was dissolved in MeOH (220.0 mL) in a pressure tube and K2CO3 (8.9 g, 64.70 mmol) was added. The resulting solution was Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 heated to 70 °C for 1.5 hours. Then, volatiles were removed and the crude 5 was used for the next step without further purification. ¹H NMR (400 MHz, DMSO-d6) δ: 4.10 (s, 3H, CH3O), 8.22 (s, 1H, H-5), 8.56 (s, 1H, H-6), 13.99 (bs, 1H, NH). HRMS (ESI+) m/z: calc for C6H7N4O 151.06144; found 151.06138. [0357] 4-Methyl-1H-pyrazolo[3,4-d]pyrimidine (6) Based on a described procedure (P. Perlíková et al. ACS Infect. Dis.2021, 7, 917–926), 4-chloro- 1H-pyrazolo[3,4-d]pyrimidine (1.0 g, 6.47 mmol) was dissolved in dry THF (50 mL) in a pressure tube and Pd(PPh3)4 (224.0 mg, 0.19 mmol) was added. Then, AlMe3 (2M in tol, 6 mL) was added dropwise and the resulting solution was heated to 80 °C for 1 hour. Then, volatiles were removed, and the residue was purified by HPFC (80 g SiO₂; DCM/MeOH, 0 → 4%) to give 6 (330 mg, 38%) as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ: 2.75 (s, 3H, CH3), 8.42 (d, J = 1.3 Hz, 1H, H-5), 8.80 (s, 1H, H-2), 13.96 (bs, 1H, NH). HRMS (ESI+) m/z: calc for C6H7N4135.00652; found 135.06650. [0358] N,N-Dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (7) 4-Chloro-1H-pyrazolo[3,4-d]pyrimidine (3.0 g, 19.41 mmol) was dissolved in isopropanol (40.0 mL) and NH(CH₃)₂ (2 M solution in THF, 120 mL) was added. The resulting solution was stirred at rt for 1 hour. Then, volatiles were removed and the crude 7 was used for the next step without further purification. ¹H NMR (400 MHz, CDCl3) δ: 3.39 (s, 6H, N(CH3)2), 8.02 (s, 1H, H-5), 8.37 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C7H10N5164.09307; found 164.09300. [0359] 3-Iodo-4-methoxy-1H-pyrazolo[3,4-d]pyrimidine (8) Based on a described procedure (Gudmunsson K. S. et al. Nucleosides, Nucleotides and Nucleic Acids 2001, 20, 1823–1830), nucleobase 5 (2.00 g, 13.32 mmol) and silver trifluoroacetate (4.71 g, 21.31 mmol) were dissolved in DMF (20.0 mL) and treated dropwise with solution of I2 (4.06 g, 15.98 mmol) in DMF (20.0 mL). The resulting mixture was heated to 70 °C for 5 hours. Then, it was treated with aq Na₂S₂O₃ (20 mL) and extracted with EtOAc (2 × 20 mL). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The residue was purified by HPFC (120 g SiO2; DCM/EtOAc, 0 → 8%) to give 8 (2.60 g, 71%) Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 4.10 (s, 3H, CH₃O), 8.56 (s, 1H, H-2), 14.29 (bs, 1H, NH). HRMS (ESI+) m/z: calc for C6H6IN4O 276.95808; found 276.95789. [0360] 3-Iodo-4-methyl-1H-pyrazolo[3,4-d]pyrimidine (9) Compound 6 (307.0 mg, 2.29 mmol) was dissolved in dry DMF (4.6 mL) in a MW vial, NIS (617.9 mg, 2.75 mmol) was added, and the resulting solution was heated to 60 °C for 1 hour in the MW reactor. Then, it was treated with aq. Na2S2O3 (5 mL) and extracted with EtOAc (2 × 10 mL). The organic layers were combined, dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The residue was purified by HPFC (24 g SiO2; DCM/MeOH, 0 → 2%) to give 9 (352 mg, 59%) as a beige solid. ¹H NMR (400 MHz, DMSO-d6) δ: 2.90 (s, 3H, CH3), 8.81 (s, 1H, H-2), 14.36 (bs, 1H, NH). HRMS (ESI+) m/z: calc for C₆H₆IN₄260.96317; found 260.96326. [0361] 3-Iodo-N,N-dimethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10) Based on a described procedure (Gudmunsson K. S. et al. Nucleosides, Nucleotides and Nucleic Acids 2001, 20, 1823–1830), nucleobase 7 (3.17 g, 19.40 mmol) and silver trifluoroacetate (6.86 g, 31.04 mmol) were dissolved in DMF (30.0 mL) and treated dropwise with solution of I2 (5.91 g, 23.28 mmol) in DMF (30.0 mL). The resulting mixture was heated to 70 °C for 5 hours. Then, it was treated with aq Na2S2O3 (20 mL) and extracted with EtOAc (2 × 20 mL). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The residue was purified by HPFC (120 g SiO2; DCM/EtOAc, 0 → 5%) to yield 10 (729 mg, 13%) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 3.34 (s, 6H, N(CH₃)₂), 8.22 (s, 1H, H-2), 13.92 (bs, 1H, NH). HRMS (ESI+) m/z: calc for C7H9IN5289.98971; found 289.98966. [0362] 3-Iodo-4-methoxy-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine (11) Following the general procedure GP4, nucleobase 8 (2.00 g, 7.24 mmol) and 1-O-acetyl-2,3,5- tri-O-benzoyl-β-D-ribofuranose (8.04 g, 15.94 mmol) in dry MeCN (44.0 mL) reacted in the presence of BF₃·OEt₂ (2.20 mL, 18.11 mmol). After workup, the residue was purified by HPFC (120 g SiO2; DCM/EtOAc, 0 → 100%) to give 11 (722 mg, 14%) as a beige solid. ¹H NMR (500 MHz, DMSO-d6) δ:4.13 (s, 3H, CH3O); 4.59 (dd, 1H, Jgem = 12.4 Hz, J5´a,4´ = 3.8 Hz, H- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 5´a); 4.63 (dd, 1H, J_gem = 12.4 Hz, J_5´b,4´ = 3.3 Hz, H-5´b); 4.91 (dt, 1H, J_4´,3´ = 6.3 Hz, J_4´,5´a = J4´,5´b = 3.5 Hz, H-4´); 6.14 (dd, 1H, J3´,4´ = 6.3 Hz, J3´,2´ = 5.5 Hz, H-3´); 6.34 (dd, 1H, J2´,3´ = 5.5 Hz, J2´,1´ = 3.4 Hz, H-2´); 6.78 (d, 1H, J1´,2´ = 3.4 Hz, H-1´); 7.43 – 7.50 (m, 4H, H-m-Bz); 7.56 (m, 2H, H-m-Bz); 7.61 – 7.71 (m, 3H, H-p-Bz); 7.91, 7.92 and 8.01 (3×m, 3×2H, H-o-Bz); 8.65 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C₃₂H₂₆IN₄O₈721.07898; found 721.07871. [0363] 3-Iodo-4-methyl-1-(2,3,5-tri-O-benzoyl-β-^D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine (12) Following the general procedure GP4, compound 9 (247.0 mg, 0.95 mmol) and 1-O-acetyl-2,3,5- tri-O-benzoyl-β-D-ribofuranose (1.05 g, 2.09 mmol) in dry MeCN (6.0 mL) reacted in the presence of BF₃OEt₂ (293 µL, 2.37 mmol). After workup, the residue was purified by HPFC (24 g SiO2; DCM/EtOAc, 0 → 100%) to give nucleoside 12 (284 mg, 42%) as a white solid. ¹H NMR (400 MHz, CD3OD) δ: 2.96 (s, 3H, CH3), 4.60 (m, 1H, H-5´a), 4.76 (dd, J = 12.2, 3.6 Hz, 1H, H-5´b), 4.89 (m, 1H, H-4´), 6.27 (t, J = 5.7 Hz, 1H, H-3´), 6.46 (dd, J = 5.4, 3.6 Hz, 1H, H- 2´), 6.85 (d, J = 3.6 Hz, 1H, H-1´), 7.37 – 7.43 (m, 2H, OBz), 7.47 – 7.52 (m, 2H, OBz), 7.56 – 7.63 (m, 4H, OBz), 7.92 – 7.98 (m, 5H, OBz), 8.05 – 8.08 (m, 2H, OBz), 8.79 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C32H26IN4O7705.08407; found 705.08373. [0364] 3-Iodo-4-(N,N-dimethylamino)-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1H- pyrazolo[3,4-d]pyrimidine (13) Following the general procedure GP4, nucleobase 10 (360 mg, 1.24 mmol) and 1-O-acetyl-2,3,5- tri-O-benzoyl-β-D-ribofuranose (1.38 g, 2.74 mmol) in dry MeCN (7.6 mL) reacted in the presence of BF₃·OEt₂ (384 µL, 3.11 mmol). After workup, the residue was purified by HPFC (24 g SiO2; DCM/EtOAc, 0 → 100%) to give 13 (760 mg, 83%) as a beige solid. ¹H NMR (400 MHz, DMSO-d6) δ: 3.37 (s, 6H, N(CH3)2), 4.59 – 4.63 (m, 2H, H-5´), 4.87 (dd, J = 6.2, 3.3 Hz, 1H, H-4´), 6.12 (t, J = 5.7 Hz, 1H, H-3´), 6.32 (dd, J = 5.5, 3.7 Hz, 1H, H-2´), 6.73 (d, J = 3.8 Hz, 1H, H-1´), 7.46 – 7.49 (m, 3H, OBz), 7.54 – 7.59 (m, 2H, OBz), 7.60 – 7.68 (m, 4H, OBz), 7.88 – 7.94 (m, 4H, OBz), 8.03 – 8.07 (m, 2H, OBz), 8.29 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C₃₃H₂₉IN₅O₇734.11062; found 734.11047. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0365] 3-Trimethylsilylethynyl-4-methoxy-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1H- pyrazolo[3,4-d]pyrimidine (14) Following the general procedure GP3, nucleoside 11 (250.0 mg, 0.79 mmol) and trimethylsilylacetylene (589 µL, 4.16 mmol) in DMF (1.4 mL) reacted in the presence of Pd(PPh₃)₂Cl₂ (12.1 mg, 0.02 mmol), CuI, (6.6 mg, 0.04 mmol) and triethylamine (145 µL, 1.04 mmol). After workup, the residue was purified by HPFC (24 g SiO2; EtOAc, 5%) to yield 14 (121 mg, 50%) as a brown oil. ¹H NMR (400 MHz, DMSO-d6) δ: 0.32 (s, 9H, Si(CH3)3), 4.12 (s, 3H, CH₃O), 4.58 – 4.64 (m, 2H, H-5´), 4.92 (dt, J = 6.8, 3.5 Hz, 1H, H-4´), 6.16 (dd, J = 6.6, 5.4 Hz, 1H, H-3´), 6.31 (dd, J = 5.3, 3.1 Hz, 1H, H-2´), 6.83 (d, J = 3.1 Hz, 1H, H-1´), 7.36 – 7.40 (m, 4H, OBz), 7.44 – 7.48 (m, 4H, OBz), 7.59 – 7.64 (m, 2H, OBz), 7.94 – 7.97 (m, 3H, OBz), 8.00 – 8.10 (m, 2H, OBz), 8.68 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C₃₇H₃₅N₄O₈Si 691.22187; found 691.22173. [0366] 3-Trimethylsilylethynyl-4-methyl-1-(2,3,5-tri-O-benzoyl-β-^D-ribofuranosyl)-1H- pyrazolo[3,4-d]pyrimidine (15) Following the general procedure GP3, nucleoside 12 (284.0 mg, 0.40 mmol) and trimethylsilylacetylene (570 µL, 4.03 mmol) in DMF (2.8 mL) reacted in the presence of Pd(PPh₃)₂Cl₂ (14.1 mg, 0.02 mmol), CuI, (7.7 mg, 0.04 mmol) and triethylamine (168 µL, 1.21 mmol). After workup, the residue was purified by HPFC (24 g SiO2; DCM/EtOAc, 0 → 12%) to give 15 (178 mg, 65%) as a beige oil. ¹H NMR (400 MHz, DMSO-d6) δ: 0.34 (s, 9H, Si(CH3)3), 2.88 (s, 3H, CH3), 4.61 (m, 2H, H-5´), 4.93 (dt, J = 6.7, 3.4 Hz, 1H, H-4´), 6.17 (dd, J = 6.5, 5.4 Hz, 1H, H-3´), 6.34 (dd, J = 5.4, 3.2 Hz, 1H, H-2´), 6.89 (d, J = 3.2 Hz, 1H, H-1´), 7.45 – 7.50 (m, 4H, OBz), 7.51 – 7.56 (m, 2H, OBz), 7.63 – 7.70 (m, 3H, OBz), 7.90 – 7.94 (m, 4H, OBz), 7.99 – 8.02 (m, 2H, OBz), 8.94 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C₃₇H₃₅N₄O₇Si 675.22695; found 675.22638. [0367] 3-Trimethylsilylethynyl-4-(N,N-dimethylamino)-1-(2,3,5-tri-O-benzoyl-β-^D- ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine (16) Following the general procedure GP3, nucleoside 13 (720.0 mg, 0.99 mmol) and trimethylsilylacetylene (1.67 mL, 11.84 mmol) in DMF (4.0 mL) reacted in the presence of Pd(PPh₃)₂Cl₂ (34.6 mg, 0.05 mmol), CuI, (18.8 mg, 0.10 mmol) and triethylamine (408 µL, 2.96 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 mmol). After workup, the residue was purified by HPFC (80 g SiO₂; EtOAc, 10%) to give 16 (494 mg, 78%) as a brown oil. ¹H NMR (400 MHz, DMSO-d6) δ: 0.27 (s, 9H, Si(CH3)3), 3.42 (s, 6H, N(CH3)2), 4.58 – 4.63 (m, 2H, H-5´), 4.90 (dt, J = 6.4, 3.5 Hz, 1H, H-4´), 6.10 (t, J = 5.7 Hz, 1H, H-3´), 6.35 (dd, J = 5.5, 3.8 Hz, 1H, H-2´), 6.78 (d, J = 3.8 Hz, 1H, H-1´), 7.46 – 7.56 (m, 6H, OBz), 7.66 (tdt, J = 7.4, 4.8, 1.3 Hz, 3H, OBz), 7.88 – 7.95 (m, 4H, OBz), 8.03 – 8.07 (m, 2H, OBz), 8.31 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C38H38N5O7Si 704.25350; found 704.25365. [0368] 3-Ethynyl-4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine (1a) Following the general procedure GP1, protected nucleoside 14 (276.0 mg, 0.40 mmol) was dissolved in MeOH (5.5 mL) and K₂CO₃ (165.7 mg, 1.20 mmol) was added. After workup, the residue was purified by HPFC (24 g SiO2; DCM/MeOH, 0 → 10%) to give 1a (88 mg, 62%) as a white solid. ¹H NMR (500 MHz, DMSO-d6): 3.44 (dt, 1H, Jgem = 11.7 Hz, J5´a,OH = J5´a,4´ = 5.9 Hz, H-5´a); 3.57 (dt, 1H, Jgem = 11.7 Hz, J5´b,OH = J5´b,4´ = 5.1 Hz, H-5´b); 3.93 (dt, 1H, J4´,5´a = 5.9 Hz, J_4´,3´ = J_4´,5´b = 4.6 Hz, H-4´); 4.13 (s, 3H, CH₃O); 4.21 (q, 1H, J_3´,2´ = J_3´,OH = J_3´,4´ = 5.0 Hz, H-3´); 4.60 (bq, 1H, J2´,OH = 5.7 Hz, J2´,3´ = J2´,1´ = 5.2 Hz, H-2´); 4.68 (s, 1H, C≡CH); 4.78 (t, 1H, JOH, 5´a = JOH,5´b = 5.7 Hz, OH-5´); 5.23 (d, 1H, JOH,3´ = 5.4 Hz, OH-3´); 5.48 (d, 1H, JOH,2´ = 5.8 Hz, OH-2´); 6.20 (d, 1H, J_1´,2´ = 4.7 Hz, H-1´); 8.68 (s, 1H, H-2).¹ HRMS (ESI+) m/z: calc for C13H15N4O5307.10370; found 307.10385. [0369] 3-Ethynyl-4-methyl-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine (2a) Following the general procedure GP1, protected nucleoside 15 (107.0 mg, 0.16 mmol) was dissolved in MeOH (2.2 mL) and K2CO3 (43.8 mg, 0.32 mmol) was added. After workup, the residue was purified by HPFC (12 g SiO₂; DCM/MeOH, 0 → 12%) to give 2a (35 mg, 76%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ: 2.88 (s, 3H, CH₃); 3.44 (dt, 1H, J_gem = 11.8 Hz, J5´a,OH = J5´a,4´ = 5.9 Hz, H-5´a); 3.57 (ddd, 1H, Jgem = 11.8 Hz, J5´b,OH = 5.6 Hz, J5´b,4´ = 4.8 Hz, H-5´b); 3.94 (dt, 1H, J4´,5´a = 5.9 Hz, J4´,5´b = J4´,3´ = 4.6 Hz, H-4´); 4.23 (q, 1H, J3´,2´ = J3´,OH = J_3´,4´ = 5.0 Hz, H-3´); 4.63 (q, = 5.1 Hz, H-2´) JOH,5´b = 5.8 Hz, OH-5´); 4.88 (s, 1H, CH≡C); 5.24 (d, 1H, JOH,3´ = 5.5 Hz, OH-3´); 5.49 (d, 1H, JOH,2´ = 5.7 Hz, OH-2´); 6.24 (d, 1H, J1´,2´ = 4.8 Hz, H-1´); 8.94 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C₁₃H₁₅N₄O₄291.10878; found 291.10886. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0370] 3-Ethynyl-4-(N,N-dimethylamino)-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine (3a) Following the general procedure GP1, protected nucleoside 16 (494.0 mg, 0.71 mmol) was dissolved in MeOH (9.7 mL) and K₂CO₃ (194.0 mg, 1.42 mmol) was added. After workup, the residue was purified by HPFC (12 g SiO2; DCM/MeOH, 0 → 5%) to give 3a (169 mg, 75%) as a white solid. ¹H NMR (500 MHz, DMSO-d6) δ: 3.39 (s, 6H, N(CH3)2); 3.44 – 3.61 (m, 2H, H- 5´); 3.91 (q, 1H, J_4´,5´a = J_4´,5´b = J_4´,3´ = 4.9 Hz, H-4´); 4.19 (bt, 1H, J_3´,2´ = J_3´,4´ = 4.8 Hz, H-3´); 4.56 2´); 4.61 (s, 1H, CH≡C); 4.89 (bs, 1H, OH-5´); 5.24 (bs, 1H, OH-3´); 5.46 (bs, 1H, OH-2´); 6.17 (d, 1H, J1´,2´ = 4.7 Hz, H-1´); 8.28 (s, 1H, H-2). HRMS (ESI+) m/z: calc for [0371] 4-Methoxy-3-methyl-1H-pyrazolo[3,4-d]pyrimidine (18) 4-Chloro-3-methyl-1H-pyrazolo[3,4-d]pyrimidine 17 (300.0 mg, 1.78 mmol) was dissolved in MeOH (12.1 mL) in a pressure tube and K₂CO₃ (491.9 mg, 3.56 mmol) was added. The resulting solution was heated to 70 °C for 2.5 hours. Then, volatiles were removed, and the residue was purified by HPFC (24 g SiO2; DCM/MeOH, 0 → 6%) to give 18 (242 mg, 83%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.51 (s, 3H, CH₃), 4.08 (s, 3H, CH₃O), 8.49 (s, 1H, H-2), 13.52 (bs, 1H, NH). HRMS (ESI+) m/z: calc for C7H8N4O 164.06981; found 164.06922. [0372] 3,4-Dimethyl-1H-pyrazolo[3,4-d]pyrimidine (19) Based on a described procedure (Perlíková P. et al. ACS Infect. Dis.2021, 7, 917–926), 4-chloro- 3-methyl-1H-pyrazolo[3,4-d]pyrimidine 17 (400.0 g, 2.37 mmol) was dissolved in dry THF (25.7 mL) in a pressure tube and Pd(PPh₃)₄ (82.3 mg, 0.25 mmol) was added. Then, AlMe₃ (2 M in tol, 1.8 mL) was added dropwise, and the resulting solution was heated to 80 °C for 1 hour. Then, volatiles were removed, and the residue was purified by HPFC (24 g SiO2; DCM/MeOH, 0 → 10%) to give 19 (215 mg, 61%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.63 (s, 3H, CH3), 2.78 (s, 3H, CH3), 8.72 (s, 1H, H-2), 13.50 (bs, 1H, NH). HRMS (ESI+) m/z: calc for C7H8N4148.07490; found 148.0744. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0373] 4-N,N-Dimethyl-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20) 4-Chloro-3-methyl-1H-pyrazolo[3,4-d]pyrimidine 17 (400.0 mg, 2.37 mmol) was dissolved in isopropanol (4.9 mL) and NH(CH3)2 (2 M solution in THF, 4.9 mL) was added. The resulting solution was stirred at rt for 1 hour. Then, volatiles were removed, and the residue was purified by HPFC (24 g SiO₂; DCM/MeOH, 0 → 4%) to yield 20 (337 mg, 80%) as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ: 2.57 (s, 3H, CH3), 3.25 (s, 6H, N(CH3)2), 8.17 (s, 1H, H-2), 13.14 (bs s, 1H, NH). HRMS (ESI+) m/z: calc for C8H12N5178.10872; found 178.10880. [0374] 4-Methoxy-3-methyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine (21) Following the general procedure GP4, nucleobase 18 (320.0 mg, 1.95 mmol) and 1-O-acetyl- 2,3,5-tri-O-benzoyl-β-D-ribofuranose (2.16 g, 4.29 mmol) in dry MeCN (11.9 mL) reacted in the presence of BF3OEt2 (601 µL, 4.87 mmol). After workup, the residue was purified by HPFC (80 g SiO2; DCM/EtOAc, 0 → 100%) to yield protected nucleoside 21 (462 mg, 39%) as a beige solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.45 (s, 3H, CH₃), 4.10 (s, 3H, CH₃O), 4.55 (dd, J = 12.4, 3.9 Hz, 1H, H-5´a), 4.68 (dd, J = 12.4, 3.3 Hz, 1H, H-5´b), 4.88 (dt, J = 6.5, 3.5 Hz, 1H, H- 4´), 6.18 (t, J = 5.8 Hz, 1H, H-3´), 6.34 (dd, J = 5.4, 3.5 Hz, 1H, H-2´), 6.74 (d, J = 3.5 Hz, 1H, H-1´), 7.43 – 7.50 (m, 4H, OBz), 7.50 – 7.55 (m, 2H, OBz), 7.62 – 7.71 (m, 3H, OBz), 7.90 – 7.97 (m, 4H, OBz), 8.00 – 8.04 (m, 2H, OBz), 8.59 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C33H29N4O8609.19799; found 609.19813. [0375] 3,4-Dimethyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine (22) Following the general procedure GP4, nucleobase 19 (215.0 mg, 1.45 mmol) and 1-O-acetyl- 2,3,5-tri-O-benzoyl-β-D-ribofuranose (1.61 g, 3.63 mmol) in dry MeCN (8.8 mL) reacted in the presence of BF₃·OEt₂ (448 µL, 3.63 mmol). After workup, the residue was purified by HPFC (80 g SiO2; DCM/EtOAc, 0 → 100%) to yield nucleoside 22 (759 mg, 88%) as a beige solid. ¹H NMR (400 MHz, DMSO-d6) δ: 2.57 (s, 3H, CH3), 2.79 (s, 3H, CH3), 4.56 (m, 1H, H-5´a), 4.69 (dd, J = 12.3, 3.2 Hz, 1H, H-5´b), 4.89 (dt, J = 6.6, 3.5 Hz, 1H, H-4´), 6.19 (t, J = 5.7 Hz, 1H, H- 3´), 6.37 (dd, J = 5.4, 3.7 Hz, 1H, H-2´), 6.79 (d, J = 3.7 Hz, 1H, H-1´), 7.45 – 7.56 (m, 6H, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 OBz), 7.62 – 7.70 (m, 3H, OBz), 7.89 – 7.95 (m, 4H, OBz), 8.03 (ddd, J = 8.4, 2.6, 1.3 Hz, 2H, OBz), 8.82 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C33H29N4O7593.20308; found 593.20265. [0376] 4-N,N-Dimethylamino-3-methyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1H- pyrazolo[3,4-d]pyrimidine (23) Following the general procedure GP4, nucleobase 20 (340.0 mg, 1.92 mmol) and 1-O-acetyl- 2,3,5-tri-O-benzoyl-β-D-ribofuranose (2.13 g, 4.22 mmol) in dry MeCN (11.7 mL) reacted in the presence of BF₃OEt₂ (681 µL, 4.79 mmol). After workup, the residue was purified by HPFC (80 g SiO2; DCM/EtOAc, 0 → 100%) to yield protected nucleoside 23 (327 mg, 27%) as a beige solid. ¹H NMR (400 MHz, DMSO-d6) δ: 2.51 (m, 3H, CH3), 3.26 (s, 6H, N(CH3)2), 4.58 (m, 1H, H-5´a), 4.67 (dd, J = 12.3, 3.3 Hz, 1H, H-5´b), 4.86 (dt, J = 5.8, 3.6 Hz, 1H, H-4´), 6.16 (t, J = 5.6 Hz, 1H, H-3´), 6.34 (dd, J = 5.4, 3.9 Hz, 1H, H-2´), 6.71 (d, J = 3.9 Hz, 1H, H-1´), 7.43 – 7.55 (m, 6H, OBz), 7.64 – 7.70 (m, 3H, OBz), 7.90 – 8.06 (m, 4H, OBz), 8.25 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C34H32N5O7622.22962; found 622.22950. [0377] 4-Methoxy-3-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine (1b) Following the general procedure GP1, protected nucleoside 21 (459.0 mg, 0.75 mmol) was dissolved in MeOH (10.5 mL) and K₂CO₃ (208.4 mg, 1.51 mmol) was added. After workup, the residue was purified by HPFC (12 g SiO2; DCM/MeOH, 0 → 10%) to give 1b (48 mg, 21%) as a white solid. ¹H NMR (500 MHz, DMSO-d6) δ: 2.53 (s, 3H, CH3-5); 3.43 (dt, 1H, Jgem = 11.8 Hz, J5´a,OH = J5´a,4´ = 6.0 Hz, H-5´a); 3.56 (ddd, 1H, Jgem = 11.8 Hz, J5´b,OH = 5.5 Hz, J5´b,4´ = 4.7 Hz, H-5´b); 3.90 (dt, 1H, J_4´,5´a = 5.9 Hz, J_4´,3´ = J_4´,5´b = 4.6 Hz, H-4´); 4.10 (s, 3H, CH₃O); 4.20 (q, 1H, J3´,2´ = J3´,OH = J3´,4´ = 5.1 Hz, H-3´); 4.61 (q, 1H, J2´,1´ = J2´,3´ = J2´,OH = 5.2 Hz, H-2´); 4.78 (t, 1H, J_{OH, 5´a} = J_OH,5´b = 5.8 Hz, OH-5´); 5.16 (d, 1H, J_OH,3´ = 5.5 Hz, OH-3´); 5.38 (d, 1H, J_OH,2´ = 6.0 Hz, OH-2´); 6.12 (d, 1H, J_1´,2´ = 4.9 Hz, H-1´); 8.58 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C12H17N4O5297.11935; found 297.11950. [0378] 3,4-Dimethyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine (2b) Following the general procedure GP1, protected nucleoside 22 (750.0 mg, 1.26 mmol) was dissolved in MeOH (20.0 mL) and K2CO3 (396.0 mg, 2.55 mmol) was added. After workup, the residue was purified by HPFC (24 g SiO₂; DCM/MeOH, 0 → 10%) to yield 2b (110 mg, 31%) Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ: 2.65 (s, 3H, CH₃-5); 2.81 (s, 3H, CH₃-4); 3.43 (dt, 1H, Jgem = 11.7 Hz, J5´a,OH = J5´a,4´ = 5.9 Hz, H-5´a); 3.57 (dt, 1H, Jgem = 11.7 Hz, J5´b,OH = J5´b,4´ = 5.2 Hz, H-5´b); 3.90 (dt, 1H, J4´,5´a = 5.9 Hz, J4´,3´ = J4´,5´b = 4.6 Hz, H-4´); 4.21 (q, 1H, J_3´,2´ = J_3´,OH = J_3´,4´ = 5.0 Hz, H-3´); 4.63 (q, 1H, J_2´,1´ = J_2´,3´ = J_2´,OH = 5.3 Hz, H-2´); 4.77 (t, 1H, J_{OH, 5´a} = J_OH,5´b = 5.8 Hz, OH-5´); 5.18 (d, 1H, J_OH,3´ = 5.5 Hz, OH-3´); 5.39 (d, 1H, J_OH,2´ = 6.0 Hz, OH-2´); 6.17 (d, 1H, J1´,2´ = 4.9 Hz, H-1´); 8.81 (s, 1H, H-2).¹³C NMR (125.7 MHz, DMSO- d6): 14.7 (CH3-5); 22.3 (CH3-4); 62.3 (CH2-5´); 70.8 (CH-3´); 72.9 (CH-2´); 85.1 (CH-4´); 87.8 (CH-1´); 113.1 (C-4a); 143.3 (C-5); 153.8 (C-7a); 155.2 (CH-2); 163.7 (C-4). HRMS (ESI+) m/z: calc for C12H17N4O4281.12443; found 281.12447. [0379] 4-N,N-Dimethylamino-3-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine (3b) Following the general procedure GP2, protected nucleoside 23 (327.0 mg, 0.53 mmol) was dissolved in MeOH (10.0 mL) and NaOMe (25% wt MeOH, 0.2 mL) was added. After workup, the residue was purified by HPFC (24 g SiO₂; DCM/MeOH, 0 → 9%) to give 3b (25 mg, 15%) as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ: 2.58 (s, 3H, CH3), 3.27 (s, 6H, N(CH3)2), 3.43 (dd, J = 11.8, 5.6 Hz, 1H, H-5´a), 3.57 (dd, J = 11.7, 4.5 Hz, 1H, H-5´b), 3.88 (dt, J = 5.8, 4.5 Hz, 1H, H-4´), 4.19 (t, J = 4.8 Hz, 1H, H-3´), 4.57 (t, J = 5.0 Hz, 1H, H-2´), 4.83 (br s, 1H, OH-5´), 5.10 (br s, 1H, OH-3´), 5.31 (br s, 1H, OH-2´), 6.12 (d, J = 4.8 Hz, 1H, H-1´), 8.23 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C13H20N5O4310.15098; found 310.15091. [0380] 3-Iodo-4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine (1c) Following the general procedure GP1, protected nucleoside 11 (400.0 mg, 0.55 mmol) was dissolved in MeOH (10.0 mL) and K₂CO₃ (153.5 mg, 1.10 mmol) was added. After workup, the residue was purified by HPFC (24 g SiO₂; DCM/MeOH, 0 → 12%) to give 1c (118 mg, 52%) as a white solid. ¹H NMR (500 MHz, DMSO-d6): 3.43 (dt, J = 11.7, 6.0 Hz, 1H, H-5´a), 3.56 (dt, J = 11.6, 5.2 Hz, 1H, H-5´b), 3.92 (m, 1H, H-4´), 4.13 (s, 3H, CH3O), 4.19 (q, J = 5.0 Hz, 1H, H- 3´), 4.60 (q, J = 5.2 Hz, 1H, H-2´), 4.78 (t, J = 5.7 Hz, 1H, OH-5´), 5.21 (d, J = 5.4 Hz, 1H, OH- 3´), 5.44 (d, J = 5.9 Hz, 1H, OH-2´), 6.13 (d, J = 4.9 Hz, 1H, H-1´), 8.64 (s, 1H, H-2). HRMS (ESI+) m/z: calc for C11H14IN4O5409.00034; found 409.00052. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0381] 4-Methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-3-carbonitril (1d) Based on a described procedure (Di Francesco, M. E. et al. Bioorg. Med. Chem.2012, 20, 4801– 4811), free nucleoside 1c (188.0 mg, 0.29 mmol) was dissolved in dry DMF (4.2 mL) in a microwave vial and Zn(CN)₂ (50.9 mg, 0.43 mmol) and Pd(PPh₃)₄ (33.4 mg, 0.03 mmol) were added. The resulting mixture was heated to 150 °C for 20 minutes in the microwave reactor. Then, volatiles were removed, and the residue was purified by HPFC (24 g SiO2; DCM/CH (1/1)/MeOH, 0 → 8%) to give 1d (50 mg, 56%) as a white solid. ¹H NMR (500 MHz, DMSO- d₆) δ: 3.46 (dt, 1H, J_gem = 11.7 Hz, J_5´a,OH = J_5´a,4´ = 5.8 Hz, H-5´a); 3.58 (ddd, 1H, J_gem = 11.7 Hz, J5´b,OH = 5.5 Hz, J5´b,4´ = 4.6 Hz, H-5´b); 3.97 (dt, 1H, J4´,5´a = 5.7 Hz, J4´,3´ = J4´,5´b = 4.6 Hz, H-4´); 4.20 (s, 3H, CH3O); 4.26 (m, 1H, H-3´); 4.63 (dt, 1H, J2´,OH = 5.8 Hz, J2´,3´ = J2´,1´ = 4.8 Hz, H-2´); 4.78 (t, 1H, J_{OH, 5´a} = J_OH,5´b = 5.7 Hz, OH-5´); 5.30 (d, 1H, J_OH,3´ = 5.6 Hz, OH-3´); 5.57 (d, 1H, JOH,2´ = 5.8 Hz, OH-2´); 6.27 (d, 1H, J1´,2´ = 4.5 Hz, H-1´); 8.81 (s, 1H, H-2). HRMS (ESI+) m/z: calc for 308.09880. [0382] 4-Methoxy-3-vinyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine (24) Based on a described procedure (Thiyagarajan, A. et al. Bioorg. Med. Chem. Lett.2012, 22, 7742–7747), nucleoside 11 (188.0 mg, 0.26 mmol), Pd(PPh₃)₄, (23.7 mg, 0.2 mmol), CuI (9.8 mg, 0.05 mmol) and triethylamine (107 µL, 0.77 mmol) were dissolved in dry DMF (1.0 mL). Then, tributyl(vinyl)tin (750 µL, 2.57 mmol) was added dropwise and the resulting solution was heated to 50 °C for 2 hours. Then, volatiles were removed, and the residue was purified by HPFC (12 g SiO₂; DCM/MeOH, 0 → 1%) to give 24 (88 mg, 55%) as beige oil. ¹H NMR (400 MHz, DMSO-d6) δ: 4.14 (s, 3H, CH3O), 4.56 (dd, J = 12.4, 3.9 Hz, 1H, H-5´a), 4.70 (dd, J = 12.3, 3.3 Hz, 1H, H-5´b), 4.92 (dt, J = 6.7, 3.6 Hz, 1H, H-4´), 5.65 (dd, J = 11.4, 1.6 Hz, 1H, CH=CH₂), 6.24 (dd, J = 6.2, 5.4 Hz, 1H, H-3´), 6.32 – 6.38 (m, 2H, CH=CH₂, H-2´), 6.81 – 6.90 (m, 2H, CH=CH2, H-1´), 7.44 – 7.52 (m, 6H, OBz), 7.63 – 7.70 (m, 3H, OBz), 7.93 (m, 4H, OBz), 7.96 – 8.00 (m, 2H, OBz), 8.64 (s, 1H, H-2). HRMS (ESI+) m/z: calc. for C34H29N8O4 621.19799; found 621.19806. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 [0383] 4-Methoxy-3-vinyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine (1e) Following the general procedure GP2, protected nucleoside 24 (84.0 mg, 0.14 mmol) was dissolved in MeOH (3.9 mL) and NaOMe (25% wt MeOH, 77 µL) was added. After workup, the residue was purified by HPFC (12 g SiO₂; DCM/MeOH, 0 → 10%) to give 1e (17 mg, 41%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ: 3.45 (dt, 1H, J_gem = 11.8 Hz, J_5´a,OH = J_5´a,4´ = 5.9 Hz, H-5´a); 3.59 (dt, 1H, Jgem = 11.8 Hz, J5´b,OH = J5´b,4´ = 5.0 Hz, H-5´b); 3.93 (dt, 1H, J4´,5´a = 5.9 Hz, J4´,3´ = J4´,5´b = 4.6 Hz, H-4´); 4.14 (s, 3H, CH3O); 4.23 (bq, 1H, J3´,2´ = J3´,OH = J_3´,4´ = 5.0 Hz, H-3´); 4.63 (q, 1H, J_2´,OH = J_2´,3´ = J_2´,1´ = 5.1 Hz, H-2´); 4.78 (t, 1H, J_{OH, 5´a} = JOH,5´b = 5.8 Hz, OH-5´); 5.19 (d, 1H, JOH,3´ = 5.6 Hz, OH-3´); 5.44 (bd, OH- 2´); 5.64 (dd, 1H, JCH2a,CH = 11.4 Hz, Jgem = 1.6 Hz, CH=CH2-a); 6.21 (d, 1H, J1´,2´ = 4.6 Hz, H- 1´); 6.37 (dd, 1H, J_CH2b,CH = 17.8 Hz, J_gem = 1.6 Hz, CH=CH₂-b); = 17.8Hz, JCH2a,CH = 11.4 Hz, CH=CH2); 8.64 (s, 1H, H-2). HRMS (ESI+) m/z: calc. for C13H20N5O4310.15098; found 310.15091. HRMS (ESI+) m/z: calc. for C13H17N5O4309.11935; found 309.11960. [0384] In vitro anticancer activity [0385] The target compounds were tested on several immortalized breast cancer cell lines (HMLER YUN, T47D, HCC38, HCC1806, MDA-MB-436, SK-BR-3, CAMA 1, MCF7 Ras Z, SUM149, SUM159, SUM1315, MDA-MB-157 and Hs 578T). [0386] To determine cytotoxicity and anticancer activity of the tested compounds, cells were seeded in 6-well plates in an appropriate amount. After 24 hours, the nutrient medium was changed and a new one was added. The tested substance was added at a final concentration of 10 µmol/l. The cells were incubated with the test substance for 72 hours. The amount of surviving cells was then compared to the control (100% surviving). Cells were passaged at the appropriate ratio. After 24 hours, the test substance was added again at a final concentration of 1 µmol/l. The cells were incubated with the tested compound for another 72 hours (total of 2 times 72 hours). The amount of surviving cells was then compared to the control (100% surviving). [0387] The target compounds showed strong activity against the tumor cell lines of epithelial origin, see Table 1. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 Table 1 1a 1b 1c 1d 1e 2a 2b 3a 3b HMLER YUN ** ** ** ** * n ** *** n.d. HCC 1806 *** *** *** *** *** * *** *** *** SUM 149 *** ** n.d. ** n.d. * ** n.d. n.d. MDA-MB 157 *** *** *** *** *** * *** n.d. ** MDA MB 436 *** *** *** *** *** n * *** n SUM1315 *** * * * * * * n.d. n.d. MCF7 RAS Z ** ** *** ** *** * ** *** * SUM159 *** n n * n n * n.d. n.d. T47D *** *** *** *** *** * *** *** n.d. HCC 38 *** *** *** *** *** * *** *** n.d. SK-BR-3 *** n.d. *** n.d. *** n.d. *** *** *** Hs 578T *** n.d. *** n.d. *** n n n.d. ** CAMA-1 n.d. n.d. *** n.d. ** n n.d. ** n.d. n no cancer cell death * lower cancer cell death ** medium cancer cell death *** high cancer cell death n.d. not determined OTHER EMBODIMENTS [0388] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 CLAIMS 1. A compound having a structure represented by formula (I): wherein R¹ is C1-C5 alkyl, C1-C5 alkoxy, or di(C1-C5)alkylamino; and R² is C1-C5 alkyl, C2-C5 alken-1-yl, C2-C5 alkyn-1-yl, cyano, or halo, or a pharmaceutically acceptable salt thereof. 2. The compound of claim 1, wherein the 3,4-disubstituted 1H-pyrazolo[3,4- d]pyrimidine ribonucleosides comprises: (i) 3-ethynyl-4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or (ii) 3-ethynyl-4-methyl-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or (iii) 3-ethynyl-4-(N,N-dimethylamino)-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine; or (iv) 4-methoxy-3-methyl-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or (v) 3,4-dimethyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or (vi) 4-N,N-dimethylamino-3-methyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4- d]pyrimidine; or (vii) 3-iodo-4-methoxy-1-(β-^D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine; or (viii) 4-methoxy-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-3-carbonitril; or (ix) 4-methoxy-3-vinyl-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine. 3. The compound of claim 1, wherein R¹ is C1-C5 alkyl. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 4. The compound of claim 1, wherein R¹ is C1-C5 alkoxy. 5. The compound of claim 1, wherein R¹ is di(C1-C5)alkylamino. 6. The compound of claim 3, wherein R¹ is methyl. 7. The compound of claim 4, wherein R¹ is methoxy. 8. The compound of claim 5, wherein R¹ is N,N-dimethylamino. 9. The compound of any one of claims 1 or 3-8, wherein R² is C1-C5 alkyl. 10. The compound of any one of claims 1 or 3-8, wherein R² is C2-C5 alken-1-yl. 11. The compound of any one of claims 1 or 3-8, wherein R² is cyano. 12. The compound of any one of claims 1 or 3-8, wherein R² is halo. 13. The compound of any one of claims 1 or 3-8, wherein R² is C2-C5 alkyn-1-yl. 14. The compound of claim 9, wherein R² is methyl. 15. The compound of claim 10, wherein R² is vinyl. 16. The compound of claim 13, wherein R² is ethynyl. 17. The compound of claim 1, wherein the compound is selected from: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 . 18. The compound of any one of claims 1-17 for use as a medicament. 19. The compound of any one of claims 1-17 for use in inhibition of pathological cell proliferation of tumor/non-tumor/cancer origin and for treatment of tumor/non-tumor/cancer disease associated with cell hyperproliferation. 20. The compound of any one of claims 1-17 for use in treatment of tumors, cancers, and diseases covering epithelial tumors. 21. The compound of claim 20, wherein the tumors, cancers, and diseases comprise cancers expressing high mitochondrial small ribosomal subunit protein mS34 (MRPS34) protein levels. 22. A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of claims 1-17, and one or more pharmaceutically acceptable carriers, excipients, or diluents. 23. The pharmaceutical composition of claim 20 for use in inhibition of pathological cell proliferation of tumor/non-tumor/cancer origin and/or for treatment of tumor/non tumor/cancer disease associated with cell hyperproliferation. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 24. A pharmaceutical composition comprising a therapeutically effective amount of the compound or pharmaceutically acceptable salt thereof of any one of claims 1-17, and a pharmaceutically acceptable carrier. 25. A method of activating beta-lactamase-like protein (LACTB) expression and/or activity, the method comprising contacting a cell with the compound of any one of claims 1-17 or pharmaceutically acceptable salt thereof. 26. A method of reducing or inhibiting LACTB autoproteolytic activity, the method comprising contacting a cell with the compound of any one of claims 1-17 or pharmaceutically acceptable salt thereof. 27. A method of treating or preventing disease progression in a subject having a disease associated with a loss or inactivation of LACTB expression and/or activity, the method comprising administering to the subject a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof of any one of claims 1-17, thereby treating or preventing disease progression in the subject. 28. The method of claim 27, wherein the disease associated with a loss of LACTB expression and/or activity is a cancer. 29. A method of treating a cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of the compound or pharmaceutically acceptable salt thereof of any one of claims 1-17, thereby treating the subject. 30. A method of treating a cancer in a subject, the method comprising: (a) identifying the cancer as having cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of the compound or pharmaceutically acceptable salt thereof of any one of claims 1-17, thereby treating the subject. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 31. A method of treating a cancer in a subject, the method comprising: (a) determining or having determined that the cancer has cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of the compound or pharmaceutically acceptable salt thereof of any one of claims 1-17, thereby treating the subject. 32. The method of any one of claims 28-31, wherein the cancer is a multidrug resistant cancer. 33. The method of claim 32, wherein the multidrug resistant cancer is a daunorubicin- resistant cancer or a paclitaxel-resistant cancer. 34. The method of any one of claims 28-31, wherein the cancer is a metastatic cancer. 35. The method of any one of claims 28-31, wherein the cancer is an epithelial breast cancer. 36. The method of any one of claims 29-35, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. 37. The method of claim 36, wherein the cancer is a breast cancer. 38. The method of claim 37, wherein the breast cancer is a triple negative breast cancer. 39. The method of claim 37, wherein the breast cancer is a Her2 negative and estrogen receptor/progesterone receptor positive breast cancer. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 40. The method of claim 37, wherein the breast cancer is an inflammatory breast cancer. 41. The method of any one of claims 28-31, wherein the cancer is a cancer in which LACTB is expressed at low level or has low activity levels, and/or the cancer is p53 negative and/or the cancer is PTEN negative. 42. The method of any one of claims 27-41, wherein administering the compound inhibits cellular proliferation, cellular invasion, and/or cellular migration. 43. The method of any one of claims 29-31, further comprising administering an additional cancer treatment. 44. The method of claim 43, wherein the additional cancer treatment is radiation therapy or chemotherapy. 45. The method of any one of claims 29-31, further comprising administering an immunotherapy composition to the subject. 46. The method of any one of claims 27-45, wherein the compound is administered by intravenous administration or oral administration. 47. A method of selecting a cancer treatment for a subject, the method comprising: providing a sample comprising a cancer cell from the subject; detecting the level and/or activity of LACTB in the sample; and selecting a treatment comprising administering any of the compounds of claims 1-17 to the subject if the sample has a reduced level and/or activity of LACTB as compared to a sample comprising a non-cancerous cell from the same subject. 48. A method of selecting a cancer treatment for a subject, the method comprising: providing a sample comprising a cancer cell from the subject; Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 detecting the level of MRPS34 in the sample; and selecting a treatment comprising administering any of the compounds of claims 1-17 to the subject if the sample has an increased level of MRPS34 as compared to a sample comprising a non-cancerous cell from the same subject. 49. A method of activating LACTB expression and/or activity, the method comprising contacting a cell with PNH733 or pharmaceutically 50. A method of reducing or inhibiting LACTB autoproteolytic activity, the method comprising contacting a cell with PNH733 or pharmaceutically 51. A method of treating an epithelial cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of PNH733 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically the subject. 52. A method of treating or preventing disease progression in a subject having a disease associated with a loss or inactivation of LACTB expression and/or activity, the method comprising administering to the subject a therapeutically effective amount of PNH733 or a pharmaceutically or preventing disease progression in the subject. 53. The method of claim 52, wherein the disease associated with a loss of LACTB expression and/or activity is a cancer. 54. A method of treating a cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of PNH733 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or a pharmaceutically the subject. 55. A method of treating a cancer in a subject, the method comprising: (a) identifying the cancer as having cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of PNH733 or a pharmaceutically the subject. 56. A method of treating a cancer in a subject, the method comprising: (a) determining or having determined that the cancer has cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of PNH733

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or a pharmaceutically the subject. 57. The method of any one of claims 53-56, wherein the cancer is a multidrug resistant cancer. 58. The method of claim 57, wherein the multidrug resistant cancer is a daunorubicin- resistant cancer or a paclitaxel-resistant cancer. 59. The method of any one of claims 53-56, wherein the cancer is a metastatic cancer. 60. The method of any one of claims 53-56, wherein the cancer is an epithelial breast cancer. 61. The method of any one of claims 53-60, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. 62. The method of claim 61, wherein the cancer is a breast cancer. 63. The method of claim 62, wherein the breast cancer is a triple negative breast cancer. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 64. The method of claim 62, wherein the breast cancer is a Her2 negative and estrogen receptor/progesterone receptor positive breast cancer. 65. The method of claim 62, wherein the breast cancer is an inflammatory breast cancer. 66. The method of any one of claims 53-56, wherein the cancer is a cancer in which LACTB is expressed at low level or has low activity levels, and/or the cancer is p53 negative and/or the cancer is PTEN negative. 67. The method of any one of claims 52-66, wherein administering the compound inhibits cellular proliferation, cellular invasion, and/or cellular migration. 68. The method of any one of claims 54-56, further comprising administering an additional cancer treatment. 69. The method of claim 68, wherein the additional cancer treatment is radiation therapy or chemotherapy. 70. The method of any one of claims 54-56, further comprising administering an immunotherapy composition to the subject. 71. The method of any one of claims 49-70, wherein the compound is administered by intravenous administration or oral administration. 72. A method of selecting a cancer treatment for a subject, the method comprising: providing a sample comprising a cancer cell from the subject; detecting the level and/or activity of LACTB in the sample; and selecting a treatment comprising administering PNH733 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or a pharmaceutically if the sample has a reduced level and/or activity of LACTB as compared to a sample comprising a non- cancerous cell from the same subject. 73. A method of selecting a cancer treatment for a subject, the method comprising: providing a sample comprising a cancer cell from the subject; detecting the level of MRPS34 in the sample; and selecting a treatment comprising administering PNH733 or a pharmaceutically if the sample has an increased level of MRPS34 as compared to a sample comprising a non-cancerous cell from the same subject. 74. A method of activating LACTB expression and/or activity, the method comprising contacting a cell with MGL060 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically 75. A method of reducing or inhibiting LACTB autoproteolytic activity, the method comprising contacting a cell with MGL060 or pharmaceutically 76. A method of treating an epithelial cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of MGL060 or pharmaceutically the subject. 77. A method of treating or preventing disease progression in a subject having a disease associated with a loss or inactivation of LACTB expression and/or activity, the method comprising administering to the subject a therapeutically effective amount of MGL060 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically or preventing disease progression in the subject. 78. The method of claim 77, wherein the disease associated with a loss of LACTB expression and/or activity is a cancer. 79. A method of treating a cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of MGL060 or pharmaceutically the subject. 80. A method of treating a cancer in a subject, the method comprising: (a) identifying the cancer as having cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount of MGL060 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 or pharmaceutically the subject. 81. A method of treating a cancer in a subject, the method comprising: (a) determining or having determined that the cancer has cancer cells that express LACTB at low levels or have low LACTB activity levels; and (b) administering to the subject a therapeutically effective amount MGL060 or pharmaceutically the subject. 82. The method of any one of claims 78-81, wherein the cancer is a multidrug resistant cancer. 83. The method of claim 82, wherein the multidrug resistant cancer is a daunorubicin- resistant cancer or a paclitaxel-resistant cancer. 84. The method of any one of claims 78-81, wherein the cancer is a metastatic cancer. 85. The method of any one of claims 78-81, wherein the cancer is an epithelial breast cancer. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 86. The method of any one of claims 78-85, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, glioma, gastric cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, bladder cancer, and nasopharyngeal carcinoma. 87. The method of claim 86, wherein the cancer is a breast cancer. 88. The method of claim 87, wherein the breast cancer is a triple negative breast cancer. 89. The method of claim 87, wherein the breast cancer is a Her2 negative and estrogen receptor/progesterone receptor positive breast cancer. 90. The method of claim 87, wherein the breast cancer is an inflammatory breast cancer. 91. The method of any one of claims 78-85, wherein the cancer is a cancer in which LACTB is expressed at low level or has low activity levels, and/or the cancer is p53 negative and/or the cancer is PTEN negative. 92. The method of any one of claims 76-91, wherein administering the compound inhibits cellular proliferation, cellular invasion, and/or cellular migration. 93. The method of any one of claims 79-81, further comprising administering an additional cancer treatment. 94. The method of claim 93, wherein the additional cancer treatment is radiation therapy or chemotherapy. 95. The method of any one of claims 79-81, further comprising administering an immunotherapy composition to the subject. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 96. The method of any one of claims 76-95, wherein the compound is administered by intravenous administration or oral administration. 97. A method of selecting a cancer treatment for a subject, the method comprising: providing a sample comprising a cancer cell from the subject; detecting the level and/or activity of LACTB in the sample; and selecting a treatment comprising administering MGL060 or pharmaceutically if the sample has a reduced level and/or activity of LACTB as compared to a sample comprising a non-cancerous cell from the same subject. 98. A method of selecting a cancer treatment for a subject, the method comprising: providing a sample comprising a cancer cell from the subject; detecting the level of MRPS34 in the sample; and selecting a treatment comprising administering MGL060 or pharmaceutically if the sample has an increased level of MRSP34 as compared to a sample comprising a non-cancerous cell from the same subject. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences 58074-0002WO1 April 8, 2025 99. A method of determining the efficacy of a cancer treatment in a subject having a cancer, the method comprising: (c) determining the level of MRPS34 or LACTB in a first sample obtained from a subject having the cancer at a first time point; (d) administering to the subject a cancer treatment; (c) providing a second sample obtained from the subject at a second time point after step (b); (d) determining the level of MRPS34 or LACTB a first sample obtained from a subject having the cancer; and (e) identifying the administered cancer treatment as being effective when: (i) the level of MRPS34 is decreased at the second time point as compared to the first time point; or (ii) the level of LACTB is increased at the second time point as compared to the first time point. 100. The method of claim 99, wherein the subject has previously been diagnosed with a cancer. 101. The method of claim 99, wherein the cancer treatment is the compound of any one of claims 1-17. 102. The method of claim 99, wherein the cancer treatment is PNH733. 103. The method of claim 99, wherein the cancer treatment is MGL060.