Activation of n-oxide immunomodulators by radiotherapy

Abstract

The present disclosure provides methods of treating or inhibiting cancer, reducing its severity, reducing its risk, or inhibiting its metastasis in an individual with a radiation-activated N-oxide TLR7 / 8 agonist.

Description

Technical Field

[0001] This disclosure relates to the biomedical field, and particularly to a method for activating an N-oxide TLR7 / 8 agonist by radiation, and a method for treating or inhibiting cancer, reducing its severity, reducing its risk, or inhibiting its metastasis in an individual. The method includes administering a therapeutically effective amount of the N-oxide TLR7 / 8 agonist to the individual and irradiating the individual with a therapeutically effective amount of radiotherapy. Background Technology

[0002] Therapeutic activation of bone marrow cells to stimulate adaptive immunity in tumors and generate effective cytotoxic T lymphocyte (CTL) responses against tumor neoantigens is crucial in antitumor immunotherapy. Among immunomodulatory molecules, Toll-like receptor (TLR) 7 / 8 agonists are potent activators of bone marrow cells, leading to direct killing of tumor cells by enhancing CTL infiltration. However, patients receiving these agonists systemically have experienced uncontrollable immune-related adverse events (irAEs), including fatal cytokine storms, ultimately resulting in premature discontinuation of clinical trials. While efforts have been made to develop TLR7 / 8 agonists to reduce irAEs, the ideal TLR7 / 8 agonist, which avoids the serious side effects of systemic exposure and is capable of spatiotemporal activation in a tumor-selective manner, remains urgently needed and challenging.

[0003] Over 50% of cancer patients receive radiotherapy. Radiotherapy-activated prodrug (RAP) strategies hold promise for addressing the clinical dilemmas of TLR7 / 8 agonists, as clinically relevant ionizing radiation (X-rays, gamma rays, etc.) is increasingly being developed as a precise perturbation tool in vivo due to its advantages of tumor targeting and deep tissue penetration (up to 15 cm). Furthermore, radiotherapy has received considerable attention as a potential immune stimulus and in combination with tumor immunotherapy; however, remote effects of radiotherapy are uncommon clinically. Therefore, radiotherapy-activated tumor-selective immunotherapy is expected to reduce intertrigoergic reactions (irAEs) and achieve better therapeutic efficacy, and even promote local irradiation for treating distant tumors. However, the concept of radiotherapy-activated agonists has not yet been proposed.

[0004] For decades, much work has focused on the development of synthetic imidazoquinoline (IMQ) derivatives, which act as TLR7 / 8 agonists. In particular, the success of FDA-approved imiquimod (R837) has led to the development of more potent IMQs, such as Resiquimod (R848), which holds the highest potential for cancer treatment on the National Cancer Institute's list. Previous structure-activity relationship studies have shown that the nitrogen atom and C4 amine group on the quinoline group form stable hydrogen bonds in synergy with Asp543 at the interface of R848 to a TLR8 dimer that plays a crucial role in receptor activation. Interestingly, we found that inserting a single oxygen atom onto the quinoline group can block the immunostimulatory activity of R848. Furthermore, the inserted oxygen atom can be efficiently eliminated by X-ray irradiation to release the parent R848. Summary of the Invention

[0005] Optimizing antitumor immunotherapy is crucial, requiring selective activation of bone marrow cells while avoiding immune-related adverse events (irAEs). Toll-like receptor (TLR) 7 / 8 agonists effectively activate myeloid cells but trigger uncontrollable irAEs when used systemically, hindering clinical progress. Clinically relevant ionizing radiation, such as X-rays and gamma rays, is precise and deep-penetrating, and holds promise for selective tumor activation. This disclosure explores the insertion of a single oxygen atom into TLR7 / 8 agonists to block their immunostimulatory activity, which can be selectively reversed by X-ray irradiation. This method provides a solution for reducing irAEs, enhancing radiation immunotherapy, promoting desired remote effects, and potentially revolutionizing cancer treatment.

[0006] In one aspect, this disclosure provides N-oxide compounds of formula I. Upon irradiation, the N-oxide compounds can be reduced to their corresponding non-N-oxide forms.

[0007] In another aspect, this disclosure provides compositions comprising N-oxide compounds having Formula I.

[0008] In another aspect, this disclosure provides the in vivo preparation of a non-N-oxide compound of formula II. The non-N-oxide compound of formula II is provided by reducing the N-oxide compound of formula I in vivo after irradiation.

[0009] In another aspect, this disclosure provides a method for activating N-oxide compounds containing imidazoquinoline groups using radiation. Upon irradiation, the N-oxide compounds can be reduced to the corresponding imidazoquinolines.

[0010] In another aspect, this disclosure provides a method of radiation immunotherapy. The method includes administering a therapeutically effective amount of an N-oxide TLR7 / 8 agonist to an individual and irradiating the individual with a therapeutically effective amount of radiation.

[0011] In another aspect, this disclosure provides the use of N-oxide TLR7 / 8 agonists in the preparation of medicaments for radiation immunotherapy in individuals who are also irradiated. Attached Figure Description

[0012] Figure 1 The following are shown: a and b show serum IFN-γ measurements performed 4 hours after injection of R848 and NO-1 (30 μmol / kg (a) and 60 μmol / kg (b) respectively). For each test group, n=3 C57BL / 6J mice. c and d show serum TNF-α measurements performed 4 hours after injection of R848 and NO-1 (30 μmol / kg (c) and 60 μmol / kg (d) respectively). For each test group, n=3 C57BL / 6J mice. e shows the body weight change measurements of C57BL / 6J mice (n=6 mice for each group) after intravenous administration of 30 μmol / kg R848 and NO-1 respectively. f shows the concentration-dependent TLR7 / 8 activation curves of R848 and NO-1 in RAW-Blue reporter cells 24 hours after culture (n=6 independent samples for each test group).

[0013] Figure 2 The following are shown: a and b show the X-ray induced release of R848 from NO-1 (10 μM) in PBS, as measured by UPLC-MS. The UPLC-MS spectrum of the generated R848 ([R848+H)) is also shown. + Selected ion monitoring signal, m / z=315)(a). For R848, positive ion mode mass spectra are shown, where retention time in a = 2.18 min(b).

[0014] Figure 3The following is shown: a. A schematic diagram of the experimental design. In bh, NO-1 (10 μM) was incubated with live RAW264.7 cells and bone marrow-derived DCs (BMDCs) for 24 hours and irradiated with X-rays (10 Gy), followed by measurement of marker expression, released cytokines, and T cell priming. Cells incubated with R848 (1 μM) were set as a positive control. b and c show CD80 / 86 expression in RAW264.7 (b) and BMDCs (c). d and e show secreted TNF-α (d) and MCP-1 (e) released from BMDCs by CBA analysis. fh, NO-1-treated BMDCs were incubated with 10 μg / mL OVA peptide for 24 hours and then irradiated with OT I CD8 + T cells were co-cultured for 3 days. OT I CD8 analysis was performed. + T cell proliferation (f, g) and IFN-γ production (h). Data are expressed as mean ± sd. For each test group, n=3 independent samples, analysis was performed using a two-tailed unpaired Student's t-test.

[0015] Figure 4 The following are shown: a) Treatment regimen. In bg, MC38 cells were subcutaneously implanted into C57BL / 6J mice, followed by intravenous (iv) injection of NO-1 (30 μmol / kg) and radiotherapy (6 Gy per treatment). b) Time-dependent accumulation of NO-1 in the blood and tumor tissue as detected by UPLC-MS (n=3 mice). ce) Mean tumor growth (c), body weight change curves (d), and survival curves of mice before reaching euthanasia criteria (e) are shown. f) Tumor volume of individual mice is shown. g) Photograph of a representative tumor on day 25.

[0016] Figure 5 The treatment regimen is shown. MC38 cells were subcutaneously implanted into C57BL / 6J mice (n=6 mice per group) on days 0 and 2, followed by intratumoral (it) injection of NO-1 (3 μmol / kg) and radiotherapy (6 Gy per treatment). b shows the mean volume of the primary and distal tumors. Data are expressed as mean ± SD.

[0017] Figure 6 Concentration-dependent TLR7 / 8 activation curves of agonists and corresponding N-oxide prodrugs in RAW-Blue reporter cells after 24 hours of incubation are shown (n=6 independent samples for each test group).

[0018] Figure 7 It shows the result of e -aq A schematic diagram of induced deoxygenation of the N-oxide agonist, and the activation yield of the corresponding N-oxide agonist (10 μM) from PBS after 60 Gy irradiation. Data are expressed as mean ± sd, with n = 3 independent samples for each test group.

[0019] Figure 8 MC38 tumor-bearing mice treated with different doses (20 μg / mouse and 100 μg / mouse) on days 10, 13, and 16 are shown. Body weight (a) and tumor growth (n=6) are measured and shown (b). Serum was collected 24 hours after the first injection. Cytokines in the serum were analyzed by cell counting beads (c and d). e and f show mice carrying MC38 tumors treated intravenously with NO-10 on days 10, 13, and 16. After 124 hours, mice were irradiated with 6 Gy or not. Tumor growth was measured as shown in (e), and survival curves are shown in (f) (n=6).

[0020] Figure 9 shows the use of 2.5 × 10 5 Six C57BL / 6J mice were inoculated with 2.5 × 10⁶ B16F10 cells. One week later, the mice were intravenously injected with 2.5 × 10⁶ cells. 6 B16F10 cells. Tumor-bearing mice were treated with intravenous NO-10 and / or radiation on days 8, 11, and 14. Local tumor volume was measured (a). On day 19, mice were sacrificed, and the number of colonies on the lungs was counted (b and c).

[0021] Figure 10 It shows the use of 2×10 5 4T1-luc2 cells were inoculated into dg Balb / c mice (n=8). Tumor-bearing mice were treated with intravenous NO-10 and / or radiation on days 10, 13, and 17. Lung metastasis was measured by IVIS spectroscopy (a and b). Survival curves are shown in (c). Detailed Implementation

[0022] (I) Compounds

[0023] In one aspect, this disclosure provides N-oxide compounds of formula I. Upon irradiation, the N-oxide compounds can be reduced to their corresponding non-N-oxide forms.

[0024] In some embodiments, this disclosure provides compounds of formula I or pharmaceutically acceptable salts thereof: (I) in, Ring A is a 5-10 aryl group or a heteroaryl group containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur; Ring B is a 5-10 aryl group or a heteroaryl group containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur; Y, Z, and W are independently chosen from groups composed of carbon, nitrogen, oxygen, or sulfur, and Y, Z, and W are optionally independent of each other. Ra, Rb, and Rc are independently selected from the group consisting of branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic hydrocarbon chains having 1 to 10 carbon atoms in their main chain. Each R1 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, optionally substituted acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R1s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. Each R2 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R2s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. R3, R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxy, cyano, cyanoalkyl, carboxyl, alkoxycarbonyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic or optionally substituted spirocyclic, or R3 and R4 combined to form an optionally substituted C3-10 heterocyclic alkyl. The optional substituents of R1, R2, R3, R4, R5, Ra, Rb and Rc are independently selected from optional alkyl, cycloalkyl, cycloalkoxy, halogen, haloalkyl, haloalkoxy, alkoxy, alkoxyalkyl, hydroxyl and carboxyl groups. n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; The condition is that the compound of formula I is not .

[0025] In some embodiments, this disclosure provides compounds of formula I or pharmaceutically acceptable salts thereof: (I) in, Ring A is a 5-10 aryl group or a heteroaryl group containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur; Y, Z, and W are independently chosen from groups composed of carbon, nitrogen, oxygen, or sulfur, and Y, Z, and W are optionally independent of each other. Ra, Rb, and Rc are independently selected from the group consisting of branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic hydrocarbon chains having 1 to 10 carbon atoms in their main chain. Each R1 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, optionally substituted acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R1s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. Each R2 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R2s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxy, cyano, cyanoalkyl, carboxyl, alkoxycarbonyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, or optionally substituted spirocyclic, or R3 and R4 are combined to form an optionally substituted C3-10 heterocyclic alkyl. The optional substituents of R1, R2, R3, R4, Ra, Rb and Rc are independently selected from optional substituted alkyl, cycloalkyl, cycloalkoxy, halogen, haloalkyl, haloalkoxy, alkoxy, alkoxyalkyl, hydroxyl and carboxyl groups. n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; The condition is that the compound of formula I is not .

[0026] In some embodiments, formula I is selected from formulas Ia and Ib or pharmaceutically acceptable salts thereof. (Ia) (Ib) in, X1, X2, X3, and X4 are independently selected from carbon, nitrogen, oxygen, or sulfur; It is either a single bond or a double bond.

[0027] In some embodiments, Formula I is Formula Ib or a pharmaceutically acceptable salt thereof: (Ib) in, X1, X2, and X3 can be independently selected from groups composed of carbon, nitrogen, oxygen, or sulfur, and Y, Z, and W can be independently selected from non-existent. Ra, Rb, and Rc are independently selected from the group consisting of branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic hydrocarbon chains having 1 to 10 carbon atoms in their main chain. Each R1 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, optionally substituted acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R1s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. Each R2 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R2s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxy, cyano, cyanoalkyl, carboxyl, alkoxycarbonyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, or optionally substituted spirocyclic, or R3 and R4 are combined to form an optionally substituted C3-10 heterocyclic alkyl. The optional substituents of R1, R2, R3, R4, Ra, Rb and Rc are independently selected from optional substituted alkyl, cycloalkyl, cycloalkoxy, halogen, haloalkyl, haloalkoxy, alkoxy, alkoxyalkyl, hydroxyl and carboxyl groups. n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; The condition is that the compound of formula I is not .

[0028] In some embodiments, compounds of formula I, Ia, or Ib are provided, wherein, The heteroatoms of rings A, X1, X2, X3, and X4 are independently selected from groups composed of carbon and nitrogen; Y does not exist or is independently selected from the group consisting of oxygen or nitrogen; Z does not exist or is independently selected from groups composed of oxygen or nitrogen; W is absent or is nitrogen; Ra is an optional substitution of C. 1-10 Alkylene; Rb can be independently and freely substituted with -C. 1-10 Alkylene-, optionally substituted C 6-10 Aryl-C 1-10 Alkylene- or optionally substituted -C 1-10 Alkylene-C 6-10 Aryl-C 1-10 The group consisting of alkylene groups; Rc is an optional substitution of -C 1-10 Alkylene-.

[0029] In some embodiments, Formula I is Formula I-1, Formula I-2, or Formula I-3, or a pharmaceutically acceptable salt thereof: I-1, I-2, I-3.

[0030] In some embodiments, each R2 is independently selected from the group consisting of hydrogen, alkyl, and optionally substituted carboxyl groups; preferably, each R2 is independently selected from hydrogen, C 1-10 Alkyl, C 1-10 The group consisting of alkyl-O-(CO)-.

[0031] In some embodiments, each R1 is independently selected from hydrogen, hydroxyl, C 1-10 Alkyl-, C 1-10 Alkyl-(CO)-, C 1-10 Alkyl-O-(CO)-, C 1-10 Alkyl-SO2-, C 1-10 Alkyl-NH-C 1-10 Alkyl-(CO)-, C 1-10 Alkyl-O-(CO)-NH-C 1-10 Alkyl-(CO)- or C 6-10 Aryl-C 1-10 Alkyl-O-(CO)-NH-C 1-10 The group consisting of alkyl-(CO)-.

[0032] In some embodiments, R3 and R4 are independently selected from hydrogen, C1-10 alkyl, -F, -Cl, -Br, -I, and halogenated C. 1-10 Alkyl, Halogenated C 1-10 Alkoxy, C 1-10 Alkoxy, hydroxy, cyano, cyano C 1-10 A group composed of alkyl groups.

[0033] In some embodiments, a compound of formula I is provided, wherein the compound is selected from the following compounds or pharmaceutically acceptable salts thereof: .

[0034] In some embodiments, the compounds of Formula I are selected from Table 1 or their pharmaceutically acceptable salts.

[0035] Table 1

[0036] (II) Pharmaceutical Composition

[0037] In another aspect, this disclosure provides pharmaceutical compositions comprising compounds of formula I or pharmaceutically acceptable salts. The compositions further comprise at least one pharmaceutically acceptable diluent, excipient, or inert carrier.

[0038] In some embodiments, the N-oxide compound of Formula I is selected from any one of Formula I, Formula Ia, Formula Ib, Formula I-1, Formula I-2 and Formula I-3 as defined above.

[0039] In one specific variation, the composition is a solid dosage form suitable for oral administration. In another specific variation, the composition is a liquid dosage form suitable for oral administration. In yet another specific variation, the composition is a tablet. In yet another specific variation, the composition is a liquid dosage form suitable for parenteral administration.

[0040] This disclosure also provides pharmaceutical compositions comprising any one of the compounds according to the above embodiments and variations, wherein the compositions are suitable for administration via routes selected from the group consisting of: oral, parenteral, intraperitoneal, intravenous, intra-arterial, percutaneous, sublingual, intramuscular, rectal, buccal, intranasal, liposomally, via inhalation, vaginal, intraocular, via local delivery (e.g., via catheter or stent), subcutaneous, intra-fat, intra-articular, and intrathecal.

[0041] This disclosure also provides a combination of a compound of formula I or a pharmaceutically acceptable salt thereof comprising a therapeutically effective amount of the compound and another therapeutically active agent.

[0042] (III) Preparation of non-N-oxide compounds

[0043] In one aspect, this disclosure provides a method for activating N-oxide compound I by radiation. The N-oxide comprises an imidazoquinoline group, which is reduced to the corresponding imidazoquinoline upon irradiation.

[0044] In another aspect, this disclosure provides for the in vivo preparation of a non-N-oxide compound of formula II. The non-N-oxide compound of formula II is provided by reducing an N-oxide compound of formula I in vivo upon irradiation.

[0045]

[0046] In some embodiments, the N-oxide compound of Formula I is selected from any one of Formula I, Formula Ia, Formula Ib, Formula I-1, Formula I-2 and Formula I-3 as defined above.

[0047]

[0048] In some embodiments, the non-N-oxide compound of formula IIb is provided by reducing the N-oxide compound of formula Ib in vivo after irradiation.

[0049]

[0050] (IV) Treatment methods

[0051] This disclosure also provides a method for treating cancer, comprising administering an N-oxide compound of formula I or a pharmaceutically acceptable salt thereof to a subject in need, and subjecting the subject to radiation after administration of the N-oxide compound.

[0052] This disclosure also provides an N-oxide compound of formula I or a pharmaceutically acceptable salt thereof, used as a medicament for treating cancer in a subject in need, and subjecting the subject to radiation after administration of the N-oxide compound.

[0053] In some embodiments, the N-oxide compound of Formula I is selected from any one of Formula I, Formula Ia, Formula Ib, Formula I-1, Formula I-2 and Formula I-3 as defined above.

[0054] In some embodiments, the subject is irradiated after the application of the compound, preferably 20 minutes, 1 hour, 2 hours, or 4 hours after the application of the compound.

[0055] In some implementations, the cancer is a solid tumor or a non-solid tumor.

[0056] In some embodiments, the cancer is selected from the group consisting of colon cancer, small cell lung cancer, Hodgkin's lymphoma, malignant lymphoma, melanoma, nasopharyngeal carcinoma, head and neck cancer, skin cancer, esophageal cancer, lung cancer, liver cancer, prostate cancer, cervical cancer, thyroid cancer, and breast cancer. In some embodiments, the cancer is a primary cancer and / or a secondary cancer (metastasis).

[0057] In some embodiments, the radiation following the application of the N-oxide compound is radiotherapy. The combination of radiotherapy and chemotherapy utilizing the N-oxide compound of Formula I provides a synergistic therapeutic effect compared to the application of radiotherapy alone, the N-oxide compound of Formula I, or the non-N-oxide compound of Formula II. In some embodiments, the combination of radiotherapy and chemotherapy utilizing the N-oxide compound of Formula I is radiotherapy-activated tumor-selective immunotherapy, which is expected to reduce irAEs and achieve better therapeutic efficacy, and even promote local irradiation for the treatment of distant tumors.

[0058] In some embodiments, the compound is applied in amounts from about 0.005 mg / kg to about 100 mg / kg, for example, in amounts of about 0.005 mg / kg, 0.05 mg / kg, 0.5 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, and 100 mg / kg.

[0059] In some implementations, the compound and radiation are applied every two days, every three days, every four days, every five days, every seven days, every ten days, every two weeks, every three weeks, or every four weeks, and the compound and radiation are applied continuously for at least three, four, five, six, seven, eight, nine, or ten rounds.

[0060] In some embodiments, the compound is administered orally, parenterally, intraperitoneally, intravenously, intra-arterially, transdermally, sublingually, intramuscularly, rectally, buccally, intranasally, via liposomes, via inhalation, via vagina, via eye, via local delivery, subcutaneously, intra-fat, intra-articularly, intrathecally, or intratumorally.

[0061] In some embodiments, the radiation is X-rays or gamma rays.

[0062] In some embodiments, the intensity of the X-rays or gamma rays is less than 60 Gy, less than 50 Gy, less than 40 Gy, less than 30 Gy, less than 20 Gy, less than 10 Gy, less than 8 Gy, less than 6 Gy, or less than 4 Gy.

[0063] (V) Manufacturing items

[0064] In another aspect, this disclosure also provides the use of the N-oxide compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer in a subject of need. Irradiation is administered to the subject following administration of the N-oxide compound.

[0065] In some embodiments, the cancer is a solid tumor or a non-solid tumor. In some embodiments, the cancer is selected from the group consisting of colon cancer, small cell lung cancer, Hodgkin's lymphoma, malignant lymphoma, melanoma, nasopharyngeal carcinoma, head and neck cancer, skin cancer, esophageal cancer, lung cancer, liver cancer, prostate cancer, cervical cancer, thyroid cancer, and breast cancer. In some embodiments, the cancer is a primary cancer and / or a secondary cancer (metastasis).

[0066] (VI) General Definitions

[0067] 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 the claimed subject matter pertains.

[0068] Unless the context otherwise requires, throughout this disclosure and claims, the word “comprising” and its variations (e.g., “including” and “containing”) shall be interpreted as having an open-ended, inclusive meaning, i.e., “including but not limited to”.

[0069] "Aliphatic hydrocarbon chain" refers to a straight or branched hydrocarbon group containing hydrogen and carbon atoms (which are usually linked together in the chain by single, double or triple bonds). Aliphatic hydrocarbon chains have 1-10, 1-8, 1-5 or 1-3 carbon atoms in their main chain and can be interrupted by one or more heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0070] "Alkyl" refers to a straight-chain or branched hydrocarbon group composed of carbon and hydrogen atoms, which is saturated and has 1 to 10 carbon atoms (C20-C40). 1-10 Alkyl group, or 1 to 8 carbon atoms (C 1-8 Alkyl group, or 1 to 6 carbon atoms (C 1-6 Alkyl group, or 1 to 4 carbon atoms (C 1-4 Alkyl group, or 1 to 3 carbon atoms (C 1-3 Alkyl groups, which are attached to the rest of the molecule by single bonds, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), n-pentyl, hexyl, 3-methylhexyl, 2-methylhexyl, etc.

[0071] "alkylene" refers to a straight-chain or branched hydrocarbon group having 1 to 10 carbon atoms, optionally substituted with a substituent selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxygen, hydroxyl, mercapto, optionally alkyl-substituted amino, carboxyl, optionally alkyl-substituted carbamoyl, optionally alkyl-substituted aminosulfonyl, nitro, cyano, halogen, and lower perfluoroalkyl, with multiple degrees of substitution permitted. Examples of "alkylene" as used herein include, but are not limited to, methylene, ethylene, n-propylene, and n-butylene.

[0072] "Alkenyl" refers to a hydrocarbon group selected from straight-chain and branched hydrocarbon groups containing at least one C=C double bond and 2 to 8 (more preferably 2 to 6) carbon atoms. Examples of alkenyl groups (e.g., C2-6 alkenyl groups) include, but are not limited to, vinyl, propenyl, propenyl, 2-methylpropenyl, butenyl, butenyl, butenyl, 3-alenyl, butenyl, 2-methylbutenyl, hexenyl, hexenyl, 2-alenyl, hexenyl, 4-alenyl, and hexa-1,3-dienyl.

[0073] "Alynyl" refers to a hydrocarbon group selected from straight-chain and branched hydrocarbon groups, which contains at least one C≡C triple bond and 2 to 8 (more such as 2 to 6) carbon atoms. Examples of alkynyl groups (e.g., C2-6 alkynyl) include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl (propynyl), 1-butynyl, 2-butynyl, and 3-butynyl.

[0074] "Alkoxy" refers to an oxygen moiety having additional alkyl substituents. The alkoxy group of this disclosure may be an optionally substituted C1-C8 alkoxy group.

[0075] "Cycloalkyl" refers to a hydrocarbon group selected from saturated cyclic hydrocarbon groups, comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups, including fused, bridged, or spirocyclic alkyl groups. For example, cycloalkyl groups may contain 3 to 8 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0076] The term "heterocyclic group" or "heterocyclic alkyl group" refers to a non-aromatic carbocyclic system containing one or two heteroatoms independently selected from N, O, or S and having one or two rings, wherein the rings may be fused, as defined above. Heterocyclic groups also include bicyclic structures, which may be bridged or spirocyclic in nature, wherein each individual ring within the bicyclic group has 3-6 atoms and contains 0, 1, or 2 N atoms. The term "heterocyclic group" includes cyclic esters (i.e., lactones) and cyclic amides (i.e., lactams), and also specifically includes, but is not limited to, epoxy groups, oxocyclic butyl groups, tetrahydrofuranyl, tetrahydropyranyl (i.e., oxalyl), pyranyl, dioxalyl, acridineyl, aziridyl, pyrrolyl, 2,5-dihydro-1H-pyrrolyl, oxazolyl, thiazolyl, piperidinyl, morpholinyl, piperazine, thiomorpholinyl, 1,3-oxazinyl, and 1,3-thiazolyl.

[0077] The term "aryl" used alone or in combination with other terms refers to a 6- to 10-membered carbon-ring aromatic ring, such as phenyl or naphthyl.

[0078] The term "heteroaryl" refers to a 6- to 10-membered monocyclic or bicyclic ring containing at least one heteroatom, such as 1 to 3 heteroatoms selected from N, O, and S. 6-10 Examples of heteroaryl groups include pyridyl, cyclolinyl, pyrazinyl, pyrimidinyl, imidazolyl, imidazopyridyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, thiadiazolyl, tetrazolyl, thiophene, triazinyl, benzothiophene, furanyl, benzofuranyl, benzimidazolyl, indoleyl, isoindoleyl, oxadiazolyl, phthalazinyl, pyrazinyl, pyridazinyl, pyrroleyl, triazolyl, quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridyl, pyrazolopyridyl, benzoxazolyl, pteridinyl, purinyl, and 1-oxa-2,3-diazolyl. 1-Oxa-2,4-diazolyl, 1-Oxa-2,5-diazolyl, 1-Oxa-3,4-diazolyl, 1-Thia-2,3-diazolyl, 1-Thia-2,5-diazolyl, 1-Thia-3,4-diazolyl, furazanyl, benzofurazanyl, benzothiophene, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxolinyl, naphthidyl, furopyridinyl, benzothiazolyl, and indole.

[0079] "Halogen" or "halogen" refers to bromine, chlorine, fluorine, or iodine.

[0080] "Halogenated alkyl" refers to an alkyl group as defined above that is substituted with one or more halogen groups as defined above. Examples of cycloalkyl groups include C1-C8 halogenated alkyl groups, such as trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, etc.

[0081] "Acylamino" refers to any group (such as an acetamide group) formed by removing a hydrogen atom from a nitrogen atom in an organic acid amide. Examples of acylamino groups include R-CONH-R, C... 1-10 Alkyl-CONH-, -CONH-C 1-10 Alkyl, C 1-8 Alkyl-CONH-, -CONH-C 1-8 Alkyl, C 1-6 Alkyl-CONH-, -CONH-C 1-6 Alkyl, C 1-3 Alkyl-CONH-, -CONH-C 1-3 Alkyl groups, etc.

[0082] "Sulfoylalkyl" refers to an alkyl substituent that is further replaced by one or more sulfonyl groups. Examples of sulfonylalkyl groups include C 1-10 Alkyl-SO2-, C 1-8 Alkyl-SO2-, C 1-6 Alkyl-SO2-, C 1-3 Alkyl-SO2-.

[0083] As used herein, “pharmaceutically acceptable” means compounds, materials, compositions, and / or dosage forms that are suitable for human and animal tissue contact with reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0084] The phrase "effective amount" refers to the amount of a compound or composition sufficient to significantly and positively alter the symptoms and / or condition to be treated (e.g., provide a positive clinical response). To the extent of the knowledge and expertise of the attending physician, the effective amount of the active ingredient used in a pharmaceutical composition will vary depending on factors such as the specific condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapies, the specific active ingredient used, and the specific pharmaceutically acceptable excipient / carrier used.

[0085] Unless otherwise stated, the term "treatment" as used herein means reversing, alleviating, inhibiting the progression of the disorder or condition to which the term applies, or one or more symptoms of the disorder or condition, delaying its progression, delaying its onset, or preventing the disorder or condition or one or more symptoms of the disorder or condition. Unless otherwise stated, the term "treatment" as used herein means therapeutic actions as defined above. The term "treatment" also includes adjunctive and neoadjunctive treatments for the subject. For the avoidance of doubt, "treatment" as used herein includes curative treatment, palliative treatment, and preventative treatment, as well as the administration of medicines used in such treatments.

[0086] Compounds of Formula I can form stable, pharmaceutically acceptable acid or base salts, and in such cases, the application of the compound as a salt may be appropriate.

[0087] Salts can be formed by conventional methods, such as reacting the product in its free basic form with an equivalent or multiple equivalent of a suitable acid in a solvent or medium in which the salt is insoluble; or reacting it in a solvent such as water, which is removed under vacuum or by freeze-drying; or by exchanging the anion of an existing salt for another anion on a suitable ion exchange resin.

[0088] Compounds of Formula I may have more than one chiral center, and it should be understood that this application covers all individual stereoisomers, enantiomers, and diastereomers, and mixtures thereof. Therefore, it should be understood that, where compounds of Formula I may exist in an optically active or racemic form due to one or more asymmetric carbon atoms, this application includes, in its definition, any such optically active or racemic form having the aforementioned activity. This application covers all such stereoisomers having the activity as defined herein.

[0089] It should also be understood that certain compounds of Formula I and their pharmaceutical salts may exist in both solvated and non-solvated forms, such as hydrated and anhydrous forms. It should be understood that the compounds herein include all such solvated forms. For clarity, this includes solvated (e.g., hydrated) forms of the free form of the compound, as well as solvated (e.g., hydrated) forms of the salts of the compound.

[0090] Formula I, as described herein, is intended to cover all isotopes of its constituent atoms. For example, FI (or hydrogen) includes any isotopic form of hydrogen, including... 1 H, 2 H(D) and 3 H(T); C includes any isotopic form of carbon, including 12 C 13 C and 14 C; O includes any isotopic form of oxygen, including 16 O、 17 O and 18 O; N includes any isotopic form of nitrogen, including 13 N、 14 N and 15 N. It should be understood that this application includes all such isotopic forms.

[0091] Compounds of Formula I or pharmaceutically acceptable salts thereof are typically administered orally in pharmaceutically acceptable dosage forms, in the form of an active ingredient or a pharmaceutically acceptable salt or solvation thereof, or a solvation of such a salt. Depending on the condition being treated and the patient, the composition may be administered at different doses.

[0092] As used herein, the term "synergy" refers to a combination of treatments that is more effective than the sum of the effects of two or more individual therapies. Combination therapies can provide a "synergistic effect" and demonstrate a "synergistic effect," meaning that the effect achieved when the therapies are used together is greater than the sum of the effects produced by using the therapies alone. In some embodiments, when the therapies are used together, the irAE is reduced. The synergistic effect can be assessed by tumor growth inhibition. Specifically, the tumor volume growth trend is inhibited, and preferably, the tumor volume shrinks significantly, or the tumor regresses completely.

[0093] Example

[0094] To make the objectives and technical solutions of this invention clearer, the invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are not intended to limit the scope of the invention. Furthermore, specific experimental methods not mentioned in the following embodiments are performed according to conventional experimental methods.

[0095] I. Materials and Synthesis

[0096] The compounds of the present invention can be prepared using suitable materials according to the procedures described in the following examples. However, the compounds exemplified in the examples are not to be construed as forming the only kind considered to be of the present invention.

[0097] abbreviations

[0098] Throughout the Examples section, the following abbreviations are used to refer to various reagents, substituents, and solvents.

[0099] 1-Isobutylimidazo[4,5-c]quinoline-4-amine (Imiquimod, R837)

[0100] 1-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinoline-1-yl]-2-methyl-prop-2-ol (R848), N-[4-(4-amino-2-ethyl-imidazo[4,5-c]quinoline-1-yl)butyl]methanesulfonamide (852A)

[0101] 1-[4-amino-2-(ethylaminomethyl)imidazo[4,5-c]quinoline-1-yl]-2-methyl-prop-2-ol (Gardiquimod), 2-propylthiazo[4,5-c]quinoline-4-amine (CL075)

[0102] 1-Benzyl-2-butyl-imidazo[4,5-c]quinoline-4-amine (BBIQ)

[0103] 1-(4-aminobutyl)-2-butyl-imidazo[4,5-c]quinoline-4-amine (T785)

[0104] 2-Ethoxymethyl-1H-imidazo[4,5-c]quinoline-4-amine (CL097)

[0105] 1-[[3-(aminomethyl)phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-4-amine (IMQ3)

[0106] 1-[[4-(aminomethyl)phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-4-amine (IMQ4)

[0107] N-[2-[2-[2-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate (IMQ6)

[0108] m-chloroperbenzoic acid (m-CPBA)

[0109] Phosphate-buffered saline (PBS)

[0110] Fetal bovine serum (FBS), penicillin

[0111] Cell Counting Kit-8 (CCK-8)

[0112] Instrument Operation

[0113] X-ray irradiation was performed using an X-ray generator (RS2000 Pro 225, 225 kV, 17.7 mA, Rad Source Technologies, Inc.). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE 400 MHz spectrometer. Signals are expressed in parts per million (ppm), and multiplicity is expressed as singlet (s), broad (b), doublet (d), triplet (t), quartet (q), or multiplet (m). Ultra-high performance liquid chromatography (UPLC-MS) was performed on an ACQUITY UPLC H-Class PLUS instrument equipped with a Waters PDA eλ detector and a Waters SQ detector 2. Preparative HPLC (neutral conditions, column: Waters Xbridge Prep OBD C18) was performed. High-resolution mass spectrometry analysis was performed on a Bruker Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. Fluorescence spectra were measured using an F-7000 spectrophotometer (Hitachi, Japan). Flow cytometry was performed using a BD FACSLyric™ Clinical instrument. In vivo optical images were captured using an IVIS Lumina III in vivo imaging system. Cell absorbance was measured using a TECAN Infinite E Plex instrument.

[0114] Example 1

[0115] 4-Amino-2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinoline 5-oxo Compound (NO-1)

[0116] Five reactions were carried out in parallel. m-CPBA (96.87 mg, 477.13 μmol, 85% purity, 1.5 equivalents) was added to a DCM solution of 1-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinoline-1-yl]-2-methyl-prop-2-ol (100 mg, 318.09 μmol, 1 equivalent) at 0 °C (2 mL). The mixture was stirred at 20 °C for 12 h. The five reactions were combined. The reactants were added to a saturated aqueous solution of sodium thiosulfate (1 mL) and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC to give NO-1 (76.4 mg, 228.88 μmol, 14.39% yield). 1H NMR (400 MHz, DMSO-d6) δ = 8.58 - 8.51 (m, 1H), 8.46 (d, J = 8.2 Hz, 1H), 7.64 (t, J = 7.8 Hz,1H), 7.57 (br d, J = 6.2 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 4.91 (s, 2H), 4.71 (br s, 2H), 3.54 (q, J = 7.0 Hz, 2H), 1.38 - 1.01 (m, 9H).

[0117] Example 2

[0118] 4-Amino-2-ethyl-1-(4-(methanesulfonylamino)butyl)-1H-imidazo[4,5-c]quinoline 5-oxide (Compound NO-2)

[0119] m-CPBA (151.65 mg, 746.97 μmol, 85% purity, 1.5 equivalence) was added to a DCM (3 mL) solution of N-[4-(4-amino-2-ethyl-imidazo[4,5-c]quinoline-1-yl)butyl]methanesulfonamide (180 mg, 497.98 μmol, 1 equivalent) at 0 °C. The mixture was stirred at 20 °C for 12 hours. A saturated aqueous solution of sodium thiosulfate (0.5 mL) was added to the reactants, followed by filtration and concentration under reduced pressure to obtain the residue. The residue was purified by preparative HPLC to give NO₂ (11.3 mg, 28.02 μmol, 5.63% yield). 1H NMR (400 MHz, methanol-d4) δ = 8.52 (d, J = 8.6 Hz,1H), 8.27 (d, J = 8.4 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.66 - 7.59 (m, 1H),4.65 - 4.61 (m, 2H), 3.15 (t, J = 6.6 Hz, 2H), 3.06 (q, J = 7.4 Hz, 2H), 2.91(s, 3H), 2.07 - 1.98 (m, 2H), 1.82 - 1.72 (m, 2H), 1.51 (t, J = 7.4 Hz, 3H).

[0120] Example 3

[0121] 4-Amino-2-(ethylamino)methyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinoline 5- Oxides (compound NO-3)

[0122] Step 1: 2-Methyl-1-[(3-nitro-4-quinolinyl)amino]prop-2-ol

[0123] To a solution of 4-chloro-3-nitro-quinoline (5 g, 23.97 mmol, 1 equivalent) and TEA (1.60 g, 15.81 mmol, 2.20 mL, 0.66 equivalent) in DCM (80 mL), 1-amino-2-methyl-propane-2-ol (2.33 g, 26.18 mmol, 1.09 equivalent) was added, and the mixture was stirred at 20 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to give 2-methyl-1-[(3-nitro-4-quinolinyl)amino]propane-2-ol (6.2 g, crude product).

[0124] Step 2: 1-[(3-amino-4-quinolinyl)amino]-2-methyl-prop-2-ol

[0125] Fe (6.63 g, 118.65 mmol, 5 equivalents) was added to a solution of 2-methyl-1-[(3-nitro-4-quinolinyl)amino]prop-2-ol (6.2 g, 23.73 mmol, 1 equivalent) and NH4Cl (12.69 g, 237.30 mmol, 10 equivalents) in EtOH (60 mL) and H2O (6 mL). The mixture was stirred at 80 °C for 3 hours. The reactants were filtered and concentrated under reduced pressure, then water (200 mL) was added, followed by extraction with ethyl acetate (3 × 200 mL) and drying with anhydrous sodium sulfate. The mixture was filtered and concentrated under reduced pressure to give 1-[(3-amino-4-quinolinyl)amino]-2-methyl-prop-2-ol (5.5 g, crude product).

[0126] Step 3: N-[2-[(3-amino-4-quinolinyl)-(2-hydroxy-2-methylpropyl)amino]-2-oxy-ethyl]-N-ethyl-carbamate tert-butyl ester

[0127] To a DMF (100 mL) solution of 1-[(3-amino-4-quinolinyl)amino]-2-methyl-prop-2-ol (4.4 g, 19.02 mmol, 1 equivalent) and 2-[tert-butoxycarbonyl(ethyl)amino]acetic acid (4.64 g, 22.83 mmol, 1.2 equivalent), DIEA (4.92 g, 38.05 mmol, 6.63 mL, 2 equivalent) and HATU (7.96 g, 20.93 mmol, 1.1 equivalent) were added, and the mixture was stirred at 20 °C for 12 hours. The reaction mixture was then added to water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (3 × 150 mL) and dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain N-[2-[(3-amino-4-quinolinyl)-(2-hydroxy-2-methyl-propyl)amino]-2-oxy-ethyl]-N-ethyl-carbamate tert-butyl ester (7.9 g, crude product).

[0128] Step 4: N-ethyl-N-[[1-(2-hydroxy-2-methyl-propyl)imidazo[4,5-c]quinoline-2-yl]methyl]tert-butyl carbamate

[0129] A solution of N-[2-[(3-amino-4-quinolinyl)-(2-hydroxy-2-methyl-propyl)amino]-2-oxy-ethyl]-N-ethyl-carbamate tert-butyl ester (7.90 g, 18.97 mmol, 1 equivalent) in EtOH (100 mL) was added, followed by stirring of NaOH (2.66 g, 66.38 mmol, 3.5 equivalent) in H₂O (10 mL). The mixture was then stirred at 80 °C for 2 hours. The reactants were added to a saturated aqueous solution of sodium thiosulfate (100 mL) and extracted with dichloromethane (3 × 100 mL). The combined organic layers were washed with a saturated sodium bicarbonate solution (3 × 150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The crude products (1.8 g scale) were combined for purification. The residue was purified by column chromatography to obtain N-ethyl-N-[[1-(2-hydroxy-2-methyl-propyl)imidazo[4,5-c]quinoline-2-yl]methyl]carbamate tert-butyl ester (6.4 g, crude product).

[0130] Step 5: N-ethyl-N-[[1-(2-hydroxy-2-methyl-propyl)-5-oxo-imidazo[4,5-c]quinoline-5-onthium-2-yl]methyl]tert-butyl carbamate

[0131] m-CPBA (4.89 g, 24.09 mmol, 85% purity, 1.5 equivalent) was added fractionally to a DCM (100 mL) solution of N-ethyl-N-[[1-(2-hydroxy-2-methyl-propyl)imidazo[4,5-c]quinoline-2-yl]methyl]carbamate (6.4 g, 16.06 mmol, 1 equivalent) at 0 °C, and the mixture was stirred at 20 °C for 12 h. The reactants were added to a saturated aqueous solution of sodium thiosulfate (100 mL) and extracted with dichloromethane (3 × 100 mL). The combined organic layers were washed with a saturated sodium bicarbonate solution (3 × 150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain N-ethyl-N-[[1-(2-hydroxy-2-methyl-propyl)-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-2-yl]methyl]carbamate tert-butyl ester (4.15 g, 10.01 mmol, 62.34% yield).

[0132] Step 6: N-[[4-amino-1-(2-hydroxy-2-methyl-propyl)imidazo[4,5-c]quinoline-2-yl]methyl]-N-ethyl-carbamate tert-butyl ester

[0133] Eight reactions were carried out in parallel. TosCl (459.95 mg, 2.41 mmol, 2 equivalents) was added to a solution of N-ethyl-N-[[1-(2-hydroxy-2-methyl-propyl)-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-2-yl]methyl]carbamate (500 mg, 1.21 mmol, 1 equivalent) in DCM (15 mL) and NH3·H2O (5 mL), and the mixture was stirred at 20 °C for 12 hours. The eight reactions were combined. Water (50 mL) was added to the reactants, followed by extraction with dichloromethane (3 × 50 mL), drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain N-[[4-amino-1-(2-hydroxy-2-methyl-propyl)imidazo[4,5-c]quinolin-2-yl]methyl]-N-ethyl-carbamate tert-butyl ester (3.77 g, 9.12 mmol, 94.47% yield).

[0134] Step 7: N-[[4-amino-1-(2-hydroxy-2-methyl-propyl)-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-2-yl]methyl]-N-ethyl-carbamate tert-butyl ester

[0135] Four reactions were carried out in parallel. To a DCM solution (3 mL) of N-[[4-amino-1-(2-hydroxy-2-methyl-propyl)imidazo[4,5-c]quinoline-2-yl]methyl]-N-ethyl-carbamate tert-butyl ester (250 mg, 604.58 μmol, 1 equivalent), m-CPBA (135.01 mg, 665.04 μmol, 85% purity, 1.1 equivalent) was added, and the mixture was stirred at 0 °C for 1 hour. The crude products (100 mg scale) were combined for post-processing. The four reactions were combined. The reactants were added to a saturated aqueous solution of sodium thiosulfate (20 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with a saturated sodium bicarbonate solution (3 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain N-[[4-amino-1-(2-hydroxy-2-methyl-propyl)-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-2-yl]methyl]-N-ethyl-carbamate tert-butyl ester (275 mg, 640.26 μmol, 26.48% yield).

[0136] Step 8: 1-[4-amino-2-(ethylaminomethyl)-5-oxo-imidazo[4,5-c]quinoline-5-onthiol]-2-methyl-prop-2-ol

[0137] A solution of N-[[4-amino-1-(2-hydroxy-2-methyl-propyl)-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-2-yl]methyl]-N-ethyl-carbamate tert-butyl ester (275 mg, 640.26 μmol, 1 equivalent) in HCl / EtOAc (5 mL) was stirred at 20 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC to obtain NO-3 (103.1 mg, 272.26 μmol, 42.52% yield, HCl). 1 H NMR (400 MHz, methanol-) d 4) δ = 8.55(d, J = 8.1 Hz, 1H), 8.22 - 8.16 (m, 1H), 7.87 (t, J = 7.7 Hz, 1H), 7.74 -7.65 (m, 1H), 4.84 (br s, 4H), 3.38 (q, J = 7.3 Hz, 2H), 1.46 (t, J = 7.3 Hz, 3H), 1.32 (br s, 6H).

[0138] Example 4

[0139] 4-Amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline 5-oxide (compound NO-4)

[0140] Step 1: Quinoline-3,4-diamine

[0141] Fe (7.38 g, 132.16 mmol, 5 equivalents) and NH4Cl (14.14 g, 264.31 mmol, 10 equivalents) were added to a solution of 4-nitroquinoline-3-amine (5 g, 26.43 mmol, 1 equivalent) in EtOH (20 mL) and H2O (2 mL), and the mixture was stirred at 80 °C for 3 h. LCMS showed that most of the starting material was used and the desired ms were detected. The reaction mixture was filtered and the filtrate was concentrated to give the crude product. The reaction mixture was added to water (50 mL) and extracted with EtOAc (200 mL × 3). The combined organic layers were washed with 200 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give quinoline-3,4-diamine (4.9 g, crude product).

[0142] Step 2: 2-ethoxymethyl-1H-imidazo[4,5-c]quinoline

[0143] H₂O was added to a solution of quinoline-3,4-diamine (4.9 g, 30.78 mmol, 1 equivalent) in 2-ethoxyacetic acid (23.55 g, 226.24 mmol, 21.37 mL, 7.35 equivalent). The mixture was stirred at 140 °C for 12 hours. LC-MS showed that most of the starting material was used and the desired ms were detected. The reaction mixture was quenched with a saturated aqueous solution of NaHCO₃. Extraction was performed with EtOAc (500 mL × 3). The organic layer was dried over Na₂SO₄, filtered, and the filtrate was concentrated to give the crude product. The residue was subjected to column chromatography (plate 1, SiO₂, petroleum ether:ethyl acetate = 0:1) to give 2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline (5.1 g, crude product).

[0144] Step 3: 2-[[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]methoxy]ethyl-trimethyl-silane

[0145] NaH (971.48 mg, 24.29 mmol, 60% purity, 1.2 equivalence) was added to a DMF (50 mL) solution of 2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline (4.6 g, 20.24 mmol, 1 equivalent) at 0 °C for 0.5 h. Then, 2-(chloromethoxy)ethyl-trimethylsilane (3.71 g, 22.27 mmol, 3.94 mL, 1.1 equivalent) was added. The mixture was stirred at 25 °C for 0.5 h. LC-MS showed complete initial consumption and detected the desired ms. The reactants were concentrated under reduced pressure, then added to water (100 mL), and extracted with ethyl acetate (3 × 300 mL). The combined organic layers were washed with brine (3 × 500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain 2-[[2-(ethoxymethyl)imidazo[4,5-c]quinoline-1-yl]methoxy]ethyl-trimethyl-silane (6.4 g, crude product).

[0146] Step 4: 2-[[2-(ethoxymethyl)-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl]methoxy]ethyltrimethylsilane

[0147] m-CPBA (5.45 g, 26.85 mmol, 85% purity, 1.5 equivalence) was added to a DCM (70 mL) solution of 2-[[2-(ethoxymethyl)imidazo[4,5-c]quinoline-1-yl]methoxy]ethyl-trimethyl-silane (6.4 g, 17.90 mmol, 1 equivalent) at 0 °C, and the mixture was stirred at 20 °C for 9 h. LC-MS showed complete initial consumption and detected the desired ms. A saturated aqueous solution of sodium thiosulfate (100 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (3 × 300 mL). The combined organic layers were washed with a saturated sodium bicarbonate solution (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, ethyl acetate:methanol = 5:1) to give 2-[[2-(ethoxymethyl)-5-oxo-imidazo[4,5-c]quinoline-5-onth-1-yl]methoxy]ethyl-trimethyl-silane (8 g, crude product).

[0148] Step 5: 2,2,2-Trichloro-N-[2-(ethoxymethyl)-1-(2-trimethylsilylethoxymethyl)imidazo[4,5-c]quinoline-4-yl]acetamide

[0149] 2,2,2-trichloroacetyl isocyanate (1.21 g, 6.43 mmol, 762.29 μL, 1.2 equivalent) was added to a mixture of 2-[[2-(ethoxymethyl)-5-oxo-imidazo[4,5-c]quinoline-5-onthio-1-yl]methoxy]ethyl-trimethyl-silane (2 g, 5.35 mmol, 1 equivalent) in DCM (30 mL) at 0 °C, and the mixture was then stirred at 25 °C for 2 h. LCMS showed that most of the starting material was used and the desired ms were detected. Three batches of the reaction mixture were combined for further processing. The reaction mixture was filtered, and the filtrate was concentrated to give 2,2,2-trichloro-N-[2-(ethoxymethyl)-1-(2-trimethylsilylethoxymethyl)imidazo[4,5-c]quinoline-4-yl]acetamide (8 g, crude product).

[0150] Step 6: 2-(ethoxymethyl)-1-(2-trimethylsilylethoxymethyl)imidazo[4,5-c]quinoline-4-amine

[0151] Sodium methoxide (2.09 g, 11.59 mmol, 30% purity, 1.5 equivalence) was added to a mixture of 2,2,2-trichloro-N-[2-(ethoxymethyl)-1-(2-trimethylsilylethoxymethyl)imidazo[4,5-c]quinoline-4-yl]acetamide (4 g, 7.72 mmol, 1 equivalent) in MeOH (60 mL) at 25 °C, and the mixture was stirred at 25 °C for 1 hour. LCMS showed that the starting material was remaining and the desired ms were detected. The mixture was then stirred at 25 °C for 12 hours. LCMS showed that most of the starting material was used and the desired ms were detected. The reaction mixture was filtered and the filtrate was concentrated to obtain a crude product. The crude product was added to water (30 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with 200 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:0 to 1:1) to obtain 2-(ethoxymethyl)-1-(2-trimethylsilylethoxymethyl)imidazo[4,5-c]quinoline-4-amine (3.4 g, crude product).

[0152] Step 7: 2-(ethoxymethyl)-5-oxo-1-(2-trimethylsilylethoxymethyl)imidazo[4,5-c]quinoline-5-onthium-4-amine

[0153] m-CPBA (1.36 g, 6.71 mmol, 85% purity, 1 equivalent) was added to a mixture of 2-(ethoxymethyl)-1-(2-trimethylsilylethoxymethyl)imidazo[4,5-c]quinoline-4-amine (2.5 g, 6.71 mmol, 1 equivalent) in DCM (30 mL) at 0 °C, and the mixture was stirred at 0 °C for 0.5 h. LCMS showed approximately 38% of the starting material remaining and detected the desired ms (21%). The reaction mixture was quenched with saturated Na₂S₂O₃ aqueous solution (50 mL) and extracted with EtOAc (150 mL × 3). The combined organic layers were washed with 200 mL of saturated NaHCO₃ aqueous solution, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 0:1, then to ethyl acetate: methanol = 1:1) to obtain 2-(ethoxymethyl)-5-oxo-1-(2-trimethylsilylethoxymethyl)imidazo[4,5-c]quinoline-5-on-4-amine (1.8 g, crude product).

[0154] Step 8: 2-(ethoxymethyl)-5-oxo-1H-imidazo[4,5-c]quinoline-5-onthium-4-amine

[0155] A mixture of 2-(ethoxymethyl)-5-oxo-1-(2-trimethylsilylethoxymethyl)imidazo[4,5-c]quinoline-5-onthium-4-amine (500 mg, 1.29 mmol, 1 equivalent) was prepared in EtOAc / HCl (5 mL) at 25 °C, and the mixture was then stirred at 25 °C for 2 hours. LCMS showed that most of the starting material was used and the desired ms were detected. The mixture was filtered, and the filter cake was vacuum dried to give NO-4 (57.2 mg, crude product, HCl). ¹H NMR (400 MHz, methanol-d⁴) δ = 8.28 (d, J = 8.0 Hz, ¹H), 8.11 (d, J = 8.6 Hz, ¹H), 7.87 - 7.81 (m, ¹H), 7.68 - 7.63 (m, ¹H), 4.83 (s, 2H), 3.72 (q, J = 7.0 Hz, 2H), 1.31 (t, J = 7.0 Hz, 3H).

[0156] Example 5

[0157] 4-Amino-2-propylthiazo[4,5-c]quinoline 5-oxide (compound NO-5)

[0158] Step 1: Synthesis of 3-aminoquinoline-4-ol

[0159] TEA (40 g, 395.30 mmol, 55.02 mL, 3.01 equivalents) and Pt / C (2.5 g, 1% purity) were added to a solution of 3-nitroquinoline-4-ol (25 g, 131.47 mmol, 1 equivalent) in MeOH (250 mL) and THF (250 mL). The mixture was stirred at 60 °C for 3.3 min under H2 (1 MPa) (flow chemistry). The mixture was filtered, and the filtrate was concentrated under reduced pressure to give 3-aminoquinoline-4-ol (21 g, crude product).

[0160] Step 2: Synthesis of N-(4-hydroxy-3-quinolinyl)butyramide

[0161] TEA (3.16 g, 31.22 mmol, 1 equivalent) was added to a solution of 3-aminoquinoline-4-ol (5 g, 31.22 mmol, 1 equivalent) in DCM (500 mL) and DMF (50 mL), followed by dropwise addition of butyryl chloride (4.99 g, 46.82 mmol, 4.90 mL, 1.5 equivalent) at 0 °C. The mixture was stirred at 0 °C for 1.5 h. The mixture was concentrated under reduced pressure and then purified by flash chromatography (silica gel, dichloromethane:methanol = 1 / 0 to 20 / 1) to give N-(4-hydroxy-3-quinoline)butyramide (5 g, crude product).

[0162] Step 3: Synthesis of 2-propylthiazo[4,5-c]quinoline

[0163] P2S5 (6.76 g, 30.40 mmol, 1 equivalent) was added to a solution of N-(4-hydroxy-3-quinolinyl)butyramide (7 g, 30.40 mmol, 1 equivalent) in Py (350 mL). The mixture was stirred at 120 °C for 3 hours. The mixture was concentrated under reduced pressure, then added to water (500 mL), and extracted with ethyl acetate (3 × 300 mL). The combined organic layers were dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to give 2-propylthiazo[4,5-c]quinoline (7 g, crude product).

[0164] Step 4: Synthesis of 2-propylthiazo[4,5-c]quinoline

[0165] P2S5 (6.76 g, 30.40 mmol, 1 equivalent) was added to a solution of N-(4-hydroxy-3-quinolinyl)butyramide (7 g, 30.40 mmol, 1 equivalent) in Py (350 mL). The mixture was stirred at 120 °C for 3 hours. The mixture was concentrated under reduced pressure, then added to water (500 mL), and extracted with ethyl acetate (3 × 300 mL). The combined organic layers were dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to give 2-propylthiazo[4,5-c]quinoline (7 g, crude product).

[0166] Step 5: Synthesis of 2-propylthiazo[4,5-c]quinoline-4-amine

[0167] To a DCM solution (5 mL) of 5-oxo-2-propyl-thiazo[4,5-c]quinoline-5-onium (0.3 g, 1.23 mmol, 1 equivalent) was added to TosCl (468.21 mg, 2.46 mmol, 2 equivalents) and NH3·H2O (5 mL). The mixture was stirred at 20 °C for 1 hour. The organic layer was concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (neutral conditions, column: Waters Xbridge BEH C18, 100×30mm×1μm; mobile phase: [H2O(10 mM NH4HCO3)-ACN]; gradient: 43%-73%B, 8.0 min) to obtain 2-propylthiazo[4,5-c]quinoline-4-amine (20.5 mg, 82.26 μmol, 6.70% yield, 97.644% purity).

[0168] Step 6: Synthesis of 5-oxy-2-propyl-thiazo[4,5-c]quinoline-5-onthium-4-amine

[0169] m-CPBA (1.50 g, 7.40 mmol, 85% purity, 1.8 equivalents) was slowly added to a DCM (10 mL) solution of 2-propylthiazo[4,5-c]quinoline-4-amine (1 g, 4.11 mmol, 1 equivalent) at 20 °C. The mixture was stirred at 20 °C for 2 hours. The reaction mixture was added to a mixture of saturated Na₂SO₃ (20 mL) and saturated NaHCO₃ (20 mL), and then extracted with DCM (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (neutral conditions, column: Waters Xbridge BEH C18, 250×50 mm×10 μm; mobile phase: [H2O(10 mM NH4HCO3)-ACN]; gradient: 25%-65%B, 10.0 min) to obtain 5-oxo-2-propyl-thiazo[4,5-c]quinoline-5-on-4-amine (62.5 mg, 239.90 μmol, 5.84% yield). LCMS m / z 260.0[M+H]+ 1H NMR (400 MHz, DMSO-d6) δ = 8.45 (d, J = 8.4 Hz, 1H), 7.99 (d, J =7.6 Hz, 1H), 7.74 (br t, J = 7.3 Hz, 3H), 7.51 (t, J = 7.3 Hz, 1H), 3.19 (t,J = 7.5 Hz, 2H), 1.93 - 1.85 (m, 2H), 1.03 (t,J = 7.3 Hz, 3H).

[0170] Example 6

[0171] 4-Amino-1-benzyl-2-butyl-1H-imidazo[4,5-c]quinoline 5-oxide (compound NO-6)

[0172] Step 1: 1-Benzyl-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-4-amine

[0173] Four reactions were carried out in parallel. m-CPBA (101.38 mg, 499.36 μmol, 85% purity, 1.5 equivalents) was added to a DCM solution of 1-benzyl-2-butyl-imidazo[4,5-c]quinoline-4-amine (110 mg, 332.90 μmol, 1 equivalent) in 3 mL at 0 °C. The mixture was stirred at 20 °C for 12 h. The four reactions were combined. The reactants were added to a saturated aqueous solution of sodium thiosulfate (1 mL), and then concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC to give NO-6 (53.3 mg, 150.30 μmol, 11.29% yield). 1H NMR (400 MHz, DMSO-d6) δ = 8.50 (d, J =8.6 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.72 - 7.48 (m, 3H), 7.34 - 7.23 (m,4H), 7.04 (br d, J = 7.5 Hz, 2H), 5.92 (s, 2H), 2.94 (br t, J = 7.6 Hz, 2H), 1.71 (quin, J = 7.6 Hz, 2H), 1.37 (sxt, J = 7.4 Hz, 2H), 0.85 (t, J = 7.3 Hz, 3H).

[0174] Example 7

[0175] 4-Amino-1-(4-aminobutyl)-2-butyl-1H-imidazo[4,5-c]quinoline 5-oxide (compound NO-7)

[0176] Step 1: N-[4-(2-Butylimidazo[4,5-c]quinoline-1-yl)butyl]carbamate tert-butyl

[0177] TEA (3.20 g, 31.64 mmol, 4.40 mL, 0.66 equivalents) and N-(4-aminobutyl)carbamate tert-butyl ester (9.93 g, 52.73 mmol, 1.1 equivalents) were added to a DCM (200 mL) solution of 4-chloro-3-nitro-quinoline (10 g, 47.94 mmol, 1 equivalent). The mixture was stirred at 25 °C for 2 h. LCMS showed that the starting material was remaining and detected the desired ms. The mixture was then stirred at 25 °C for 12 h. LCMS showed that most of the starting material was used and detected the desired ms. The reactants were filtered and the filtrate was concentrated under reduced pressure to give N-[4-[(3-nitro-4-quinolinyl)amino]butyl]carbamate tert-butyl ester (17 g, crude product).

[0178] Step 2: N-[4-[(3-amino-4-quinolinyl)amino]butyl]carbamate tert-butyl ester

[0179] Fe (54.23 g, 97.11 mmol, 10% purity, 5 equivalents) and NH4Cl (10.39 g, 194.23 mmol, 10 equivalents) were added to a solution of N-[4-[(3-nitro-4-quinolinyl)amino]butyl]carbamate (7 g, 19.42 mmol, 1 equivalent) in EtOH (500 mL) and H2O (50 mL). The mixture was stirred at 80 °C for 12 h. LCMS showed that the reaction was complete and the desired ms were detected. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was added to water (200 mL) and extracted with EtOAc (500 mL × 3). The combined organic layers were washed with 1000 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography on silica gel (SiO2, plate 1, petroleum ether: ethyl acetate = 0:1) to obtain N-[4-[(3-amino-4-quinolinyl)amino]butyl]carbamate tert-butyl ester (6.4 g, crude product).

[0180] Step 3: N-[4-(2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-1-yl)butyl]carbamate tert-butyl

[0181] To a DMF (100 mL) solution of N-[4-[(3-amino-4-quinolinyl)amino]butyl]carbamate (6.4 g, 19.37 mmol, 1 equivalent) and pentanal (2.00 g, 23.24 mmol, 2.47 mL, 1.2 equivalent), NaOH (116.32 mg, 1.94 mmol, 110.88 μL, 0.1 equivalent) was added. The mixture was stirred at 85 °C for 12 h. LC-MS showed that most of the starting material was used and the desired ms were detected. The reactants were concentrated under reduced pressure, then added to water (150 mL), and extracted with ethyl acetate (3 × 300 mL). The combined organic layers were washed with brine (3 × 500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (plate 1, SiO2, petroleum ether / ethyl acetate = 0 / 1) to obtain N-[4-(2-butylimidazo[4,5-c]quinoline-1-yl)butyl]carbamate tert-butyl ester (5.8 g, crude product).

[0182] Step 4: N-[4-(2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-1-yl)butyl]carbamate tert-butyl

[0183] m-CPBA (4.45 g, 21.94 mmol, 85% purity, 1.5 equivalence) was added to a DCM (100 mL) solution of N-[4-(2-butylimidazo[4,5-c]quinoline-1-yl)butyl]carbamate (5.8 g, 14.63 mmol, 1 equivalent) at 0 °C. The mixture was stirred at 25 °C for 12 h. LC-MS showed the starting material remaining and detected the desired ms. A saturated aqueous solution of sodium thiosulfate (200 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (3 × 600 mL). The combined organic layers were washed with a saturated sodium bicarbonate solution (3 × 500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, ethyl acetate:methanol = 5:1) to obtain N-[4-(2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onth-1-yl)butyl]carbamate (1.98 g, crude product).

[0184] Step 5: N-[4-(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)butyl]carbamate tert-butyl

[0185] TosCl (277.29 mg, 1.45 mmol, 2 equivalents) and NH3·H2O (2 mL) were added to a mixture of N-[4-(2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthio-1-yl)butyl]carbamate (300 mg, 727.23 μmol, 1 equivalent) in DCM (6 mL), and the mixture was stirred at 25 °C for 10 h. LCMS showed approximately 24% of the starting material remaining and detected the desired ms (13%). Six batches of the reaction mixture were combined for further processing. The reaction mixture was added to water (50 mL) and extracted with EtOAc (200 mL × 3). The combined organic layers were washed with 300 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was purified by preparative TLC (petroleum ether: ethyl acetate = 0:1) to obtain N-[4-(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)butyl]carbamate tert-butyl ester (800 mg, crude product).

[0186] Step 6: N-[4-(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)butyl]carbamate tert-butyl

[0187] m-CPBA (246.66 mg, 1.21 mmol, 85% purity, 1 equivalent) was added to a mixture of N-[4-(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)butyl]carbamate (500 mg, 1.21 mmol, 1 equivalent) in DCM (6 mL) at 0 °C, and the mixture was stirred at 0 °C for 0.5 h. LCMS showed approximately 27.4% of the starting material remaining, and the desired ms (6.6%) were detected. The reaction mixture was quenched with saturated Na₂S₂O₃ aqueous solution (10 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with 50 mL of saturated NaHCO₃ aqueous solution, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 0:1, then to ethyl acetate: methanol = 1:1) to obtain N-[4-(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)butyl]carbamate tert-butyl ester (176 mg, crude product).

[0188] Step 7: 1-(4-aminobutyl)-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-4-amine

[0189] N-[4-(4-amino-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl)butyl]carbamate (300 mg, 701.69 μmol, 1 equivalent) was mixed in EtOAc / HCl (2 mL) at 25 °C, and the mixture was then stirred at 25 °C for 0.5 h. LCMS showed that most of the starting material was used up and the desired ms were detected. The reaction mixture was concentrated to obtain the crude product. The crude product was purified by preparative HPLC (HCl conditions; column: Phenomenex luna C18100×40 mm×5 μm; mobile phase: [H2O(0.04%HCl)-ACN]; gradient: 1%-40%B, 8.0 min) to obtain NO-7 (67.3 mg, 297.91 μmol, 42.46% yield). 1H NMR (400 MHz, DMSO-d6) δ = 9.39 (br s,1H), 9.15 (br s, 1H), 8.27 (d, J = 8.1 Hz, 1H), 8.18 (br s, 2H), 8.10 (dd, J= 0.8, 8.6 Hz, 1H), 7.83 - 7.76 (m, 1H), 7.68 - 7.62 (m, 1H), 4.62 (br t, J =7.2 Hz, 2H), 3.06 - 2.93 (m, 2H), 2.87 - 2.75 (m, 2H), 1.94 - 1.70 (m, 6H),1.47 (qd, J = 7.4, 14.9 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H).

[0190] Example 8

[0191] 4-Amino-1-(4-(aminomethyl)benzyl)-2-butyl-7-(methoxycarbonyl)-1H-imidazo[4,5-c]quinoline 5-Oxide Phosphate (Compound NO-8)

[0192] Step 1: Synthesis of methyl 7-bromo-4-chloro-3-nitro-quinoline

[0193] DMF (712.50 mg, 9.75 mmol, 0.75 mL, 5.25e-1 equivalent) was added to a POCl3 (15 mL) solution of 7-bromo-3-nitro-quinoline-4-ol (5 g, 18.58 mmol, 1 equivalent). The solution was stirred at 85 °C for 12 hours. The solution was then filtered to obtain 7-bromo-4-chloro-3-nitro-quinoline (4 g, crude product).

[0194] Step 2: Synthesis of N-[[4-[[(7-bromo-3-nitro-4-quinolinyl)amino]methyl]phenyl]methyl]tert-butyl carbamate

[0195] TEA (1.40 g, 13.84 mmol, 1.93 mL, 1 equivalent) and N-[[4-(aminomethyl)phenyl]methyl]carbamate tert-butyl ester (3.57 g, 15.09 mmol, 1.09 equivalent) were added to a DCM (80 mL) solution of 7-bromo-4-chloro-3-nitro-quinoline (3.98 g, 13.84 mmol, 1 equivalent) at 0 °C. The solution was then stirred at 25 °C for 12 hours. The solution was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain N-[[4-[[(7-bromo-3-nitro-4-quinoline)amino]methyl]phenyl]methyl]carbamate tert-butyl ester (3.5 g, 7.18 mmol, 51.88% yield).

[0196] Step 3: Synthesis of N-[[4-[[(3-amino-7-bromo-4-quinolinyl)amino]methyl]phenyl]methyl]tert-butyl carbamate

[0197] SnCl₂ (3.72 g, 19.64 mmol, 3.3 equivalents) was added to a solution of N-[[4-[[(7-bromo-3-nitro-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate tert-butyl ester (2.9 g, 5.95 mmol, 1 equivalent) in EtOH (72 mL). The mixture was stirred at 65 °C for 3 hours. The reaction mixture was concentrated under reduced pressure and then extracted with ethyl acetate (3 × 50 mL) in a saturated sodium bicarbonate solution (50 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain N-[[4-[[(3-amino-7-bromo-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate tert-butyl ester (3.1 g, crude product).

[0198] Step 4: Synthesis of N-[[4-[(7-bromo-2-butyl-imidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]tert-butyl carbamate

[0199] AcOH (32.83 mg, 546.61 μmol, 31.29 μL, 0.1 equivalent) was added to a DMF (25 mL) solution of pentanal (564.97 mg, 6.56 mmol, 697.49 μL, 1.2 equivalent) and N-[[4-[[(3-amino-7-bromo-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate tert-butyl ester (2.5 g, 5.47 mmol, 1 equivalent), and the mixture was stirred at 85 °C for 15 h. The reaction mixture was added to a saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (3 × 20 mL) and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain N-[[4-[(7-bromo-2-butyl-imidazo[4,5-c]quinolin-1-yl)methyl]phenyl]methyl]carbamate tert-butyl ester (3.5 g, crude product).

[0200] Step 5: Synthesis of methyl 1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-7-carboxylate

[0201] Pd(dppf)Cl2 (265.59 mg, 362.97 μmol, 0.1 equivalent) was added to a MeOH (200 mL) solution of N-[[4-[(7-bromo-2-butyl-imidazo[4,5-c]quinolin-1-yl)methyl]phenyl]methyl]carbamate (1.9 g, 3.63 mmol, 1 equivalent) and TEA (1.84 g, 18.15 mmol, 2.53 mL, 5 equivalent). The suspension was degassed under vacuum and purged several times with CO (50 PSI). It was stirred at 80 °C under CO (50 PSI) for 12 hours. It was then concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 1:1) to obtain methyl 1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-7-carboxylate (2 g, crude product).

[0202] Step 6: Synthesis of methyl 1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-7-carboxylate

[0203] At 0 °C, m-CPBA (1.21 g, 5.97 mmol, 85% purity, 1.5 equivalents) was added to a DCM (40 mL) solution of methyl 1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-7-carboxylate (2 g, 3.98 mmol, 1 equivalent). The mixture was then stirred at 25 °C for 2 hours. The reaction mixture was slowly added to 50 mL of saturated Na₂SO₃, washed with 30 mL of saturated NaHCO₃, and then extracted with DCM (3 × 40 mL). The combined organic layers were washed with brine (3 × 20 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 3 / 1 to 0 / 1) to give methyl 1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-7-carboxylate (1.26 g, 2.43 mmol, 53.09% yield).

[0204] Step 7: Synthesis of methyl 4-amino-1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-7-carboxylate

[0205] To a solution of methyl 1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-7-carboxylate (300 mg, 578.48 μmol, 1 equivalent) in DCM (5 mL) and NH3·H2O (5 mL) was added to TosCl (220.57 mg, 1.16 mmol, 2 equivalents), and the mixture was stirred at 20 °C for 1 hour. Four batches of the reaction mixture were combined for post-treatment. The mixture was extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to obtain methyl 4-amino-1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-7-carboxylate (1.4 g, crude product).

[0206] Step 8: Synthesis of methyl 4-amino-1-[[4-(aminomethyl)phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-7-carboxylate

[0207] A solution of methyl 4-amino-1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-7-carboxylate (80 mg, 154.55 μmol, 1 equivalent) in HCl / EtOAc (3 mL) was stirred for 1 hour at 20 °C. The solution was then filtered to obtain the residue. The residue was purified by preparative HPLC (HCl conditions, column: Phenomenex luna C18 100×40mm×5μm; mobile phase: [H2O(0.04% HCl)-ACN]; gradient: 1%-45% B, 8.0 min) to obtain methyl 4-amino-1-[[4-(aminomethyl)phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-7-carboxylate (17.2 mg, 37.58 μmol, 24.31% yield, 99.183% purity, HCl).

[0208] Step 9: Synthesis of methyl 4-amino-1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-7-carboxylate

[0209] m-CPBA (58.83 mg, 289.79 μmol, 85% purity, 1.5 equivalents) was added to a DCM (5 mL) solution of methyl 4-amino-1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-imidazo[4,5-c]quinoline-7-carboxylate (100 mg, 193.19 μmol, 1 equivalent) at 0 °C. The mixture was then stirred at 25 °C for 1 hour. Ten batches of the reaction mixture were combined for post-treatment. The reaction mixture was slowly added to 50 mL of saturated Na₂SO₃, washed with 50 mL of saturated NaHCO₃, and then extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 3 / 1 to 0 / 1) to give methyl 4-amino-1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-7-carboxylate (350 mg, 655.90 μmol, 33.95% yield).

[0210] Step 10: Synthesis of methyl 4-amino-1-[[4-(aminomethyl)phenyl]methyl]-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-7-carboxylate

[0211] A solution of methyl 4-amino-1-[[4-[(tert-butoxycarbonylamino)methyl]phenyl]methyl]-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-7-carboxylate (0.3 g, 562.20 μmol, 1 equivalent) in HCl / EtOAc (3 mL) was stirred for 1 hour at 20 °C. The solution was filtered to obtain the residue. The residue was purified by preparative HPLC (HCl conditions, column: Phenomenex luna C18 100×40 mm×5 μm; mobile phase: [H2O (0.04% HCl)-ACN]; gradient: 1%-45% B, 8.0 min) to obtain NO-8 (80.4 mg, 164.55 μmol, 29.27% ​​yield, HCl). LCMS m / z 434.2 [M+H]+ 1H NMR (400 MHz, methanol-d4) δ = 8.70 (d, J = 1.1 Hz, 1H), 8.06 (d, J = 8.6Hz, 1H), 7.95 (dd, J = 1.1, 8.5 Hz, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.20 (d, J= 8.0 Hz, 2H), 6.03 (s, 2H), 4.09 (s, 2H), 3.97 (s, 3H), 3.03 (t, J = 7.6 Hz,2H), 1.87 (quin, J = 7.6 Hz, 2H), 1.48 (sxt, J = 7.5 Hz, 2H), 0.95 (t, J =7.4 Hz, 3H).

[0212] Example 9

[0213] 4-Amino-1-(3-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline 5-oxide (compound) Material NO-9)

[0214] Step 1: N-[[3-[[(3-nitro-4-quinolinyl)amino]methyl]phenyl]methyl]tert-butyl carbamate

[0215] N-[[3-(aminomethyl)phenyl]methyl]carbamate tert-butyl ester (11.33 g, 47.94 mmol, 1 equivalent) was added to a DCM (150 mL) solution of 4-chloro-3-nitro-quinoline (10 g, 47.94 mmol, 1 equivalent) and TEA (3.20 g, 31.64 mmol, 4.40 mL, 0.66 equivalent) at 0 °C. The mixture was stirred at 20 °C for 12 hours. The reaction mixture was filtered and concentrated under reduced pressure to give N-[[3-[[(3-nitro-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate tert-butyl ester (39 g, crude product).

[0216] Step 2: N-[[3-[[(3-amino-4-quinolinyl)amino]methyl]phenyl]methyl]tert-butyl carbamate

[0217] Fe (8.89 g, 159.14 mmol, 5 equivalents) and NH4Cl (17.03 g, 318.28 mmol, 10 equivalents) were added to a solution of N-[[3-[[(3-amino-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate (13 g, 31.83 mmol, 1 equivalent) in EtOH (150 mL) and H2O (30 mL). The mixture was stirred at 80 °C for 6 hours. The reaction mixture was filtered and concentrated under reduced pressure to give tert-butyl N-[[3-[[(3-amino-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate (41 g, crude product).

[0218] Step 3: N-[[3-[(2-Butylimidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]tert-butyl carbamate

[0219] AcOH (317.34 mg, 5.28 mmol, 302.52 μL, 0.1 equivalent) was added to a DMF (200 mL) solution of N-[[3-[[(3-amino-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate (20 g, 52.84 mmol, 1 equivalent) and pentanal (5.46 g, 63.41 mmol, 6.74 mL, 1.2 equivalent). The mixture was stirred at 80 °C for 12 h. The mixture was then stirred at 95 °C for another 12 h. The reactants were concentrated under reduced pressure, then added to water (500 mL), and extracted with ethyl acetate (3 × 500 mL). The combined organic layers were washed with brine (3 × 500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain N-[[3-[(2-butylimidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]tert-butyl carbamate (20 g, 44.99 mmol, 42.57% yield).

[0220] Step 4: N-[[3-[(2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]tert-butyl carbamate

[0221] m-CPBA (6.85 g, 33.74 mmol, 85% purity, 1.5 equivalents) was added to a DCM (150 mL) solution of N-[[3-[(2-butylimidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]carbamate (10 g, 22.49 mmol, 1 equivalent) at 0 °C, and the mixture was stirred at 20 °C for 12 h. The reaction mixture was added to a saturated aqueous solution of sodium thiosulfate (200 mL) and then extracted with dichloromethane (5 × 200 mL). The combined organic layers were washed with a saturated sodium bicarbonate solution (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain N-[[3-[(2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]tert-butyl carbamate (9.5 g, crude product).

[0222] Step 5: N-[[3-[(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]tert-butyl carbamate

[0223] Six reactions were carried out in parallel. TosCl (413.94 mg, 2.17 mmol, 2 equivalents) was added to a solution of N-[[3-[(2-butyl-5-oxoimidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]carbamate tert-butyl ester (500 mg, 1.09 mmol, 1 equivalent) and NH3·H2O (3 mL) in DCM (9 mL), and the mixture was stirred at 20 °C for 12 h. The six reactions were combined. The reactants were added to water (50 mL), extracted with dichloromethane (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The crude products (800 mg scale) were combined for purification. The residue was purified by column chromatography to obtain N-[[3-[(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]carbamate tert-butyl ester (2.8 g, 6.09 mmol, 73.65% yield).

[0224] Step 6: N-[[3-[(4-amino-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]tert-butyl carbamate

[0225] Four reactions were carried out in parallel. m-CPBA (121.48 mg, 598.37 μmol, 85% purity, 1.1 equivalent) was added to a DCM solution of N-[[3-[(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]carbamate (250 mg, 543.97 μmol, 1 equivalent) in 3 mL of DCM at 0 °C, and the mixture was stirred at 0 °C for 1 hour. The four reactions were combined. The reactants were added to a saturated aqueous solution of sodium thiosulfate (20 mL) and extracted with dichloromethane (3 × 20 mL). The combined organic layers were washed with a saturated sodium bicarbonate solution (3 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain N-[[3-[(4-amino-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]carbamate tert-butyl ester (340 mg, 714.91 μmol, 32.86% yield).

[0226] Step 7: 1-[[3-(aminomethyl)phenyl]methyl]-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-4-amine

[0227] A solution of N-[[3-[(4-amino-2-butyl-5-oxoimidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]tert-butyl carbamate (340 mg, 714.91 μmol, 1 equivalent) in HCl / EtOAc (5 mL) was stirred for 1 hour at 20 °C. The reaction mixture was concentrated under reduced pressure to obtain a residue. The crude products (50 mg scale) were combined for purification. The residue was purified by preparative HPLC to give NO-9 (204.1 mg, 480.25 μmol, 58.41% yield, HCl). 1H NMR (400 MHz, DMSO-d6) δ = 9.48 (br s, 1H), 9.19 (br s, 1H), 8.53 (br s, 3H), 8.06 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.76 - 7.65 (m, 1H), 7.51- 7.37 (m, 3H), 7.25 (s, 1H), 7.19 - 7.09 (m, 1H), 5.95 (s, 2H), 3.93 (br d,J = 5.0 Hz, 2H), 2.98 (t, J = 7.7 Hz, 2H), 1.77 (quin, J = 7.6 Hz, 2H), 1.41(sxt, J = 7.4 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H).

[0228] Example 10

[0229] 4-Amino-1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline 5-oxide (compound) Material NO-10)

[0230] Step 1: tert-butyl N-[[4-[[(3-nitro-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate

[0231] Two reactions were carried out in parallel. N-[[4-(aminomethyl)phenyl]methyl]carbamate tert-butyl ester (11.33 g, 47.94 mmol, 1 equivalent) was added to a DCM (200 mL) solution of 4-chloro-3-nitro-quinoline (10 g, 47.94 mmol, 1 equivalent) and TEA (3.20 g, 31.64 mmol, 4.40 mL, 0.66 equivalent), and the mixture was stirred at 20 °C for 12 hours. The reactants were filtered and concentrated under reduced pressure to give N-[[4-[[(3-nitro-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate tert-butyl ester (40 g, crude product).

[0232] Step 2: tert-butyl N-[[4-[[(3-amino-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate

[0233] Two reactions were carried out in parallel. Pd / C (2 g, 10% purity) was added to a MeOH (400 mL) solution of N-[[4-[[(3-nitro-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate (20 g, 48.97 mmol, 1 equivalent) under N2. The mixture was stirred at 20 °C for 3 hours under H2 (15 Psi). The two reactions were combined. The reactants were filtered and concentrated under reduced pressure to give N-[[4-[[(3-amino-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate (37 g, crude product).

[0234] Step 3: N-[[4-[(2-Butylimidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]tert-butyl carbamate

[0235] Two reactions were carried out in parallel. AcOH (293.54 mg, 4.89 mmol, 279.83 μL, 0.1 equivalent) was added to a DMF (200 mL) solution of N-[[4-[[(3-amino-4-quinolinyl)amino]methyl]phenyl]methyl]carbamate (18.5 g, 48.88 mmol, 1 equivalent) and pentanal (4.63 g, 53.77 mmol, 5.72 mL, 1.1 equivalent), and the mixture was stirred at 100 °C for 24 hours. The reactants were added to water (800 mL) and then extracted with ethyl acetate (5 × 500 mL). The combined organic layers were washed with brine (3 × 500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain N-[[4-[(2-butylimidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]tert-butyl carbamate (14 g, 31.49 mmol, 32.21% yield).

[0236] Step 4: N-[[4-[(2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]tert-butyl carbamate

[0237] m-CPBA (9.59 g, 47.24 mmol, 85% purity, 1.5 equivalents) was added fractionally to a DCM (200 mL) solution of N-[[4-[(2-butylimidazo[4,5-c]quinolin-1-yl)methyl]phenyl]methyl]carbamate (14 g, 31.49 mmol, 1 equivalent) at 0 °C, and the mixture was stirred at 20 °C for 3 h. The reactants were added to a saturated aqueous solution of sodium thiosulfate (200 mL) and extracted with dichloromethane (3 × 200 mL). The combined organic layers were washed with a saturated sodium bicarbonate solution (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain N-[[4-[(2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]tert-butyl carbamate (9.88 g, 21.45 mmol, 68.12% yield).

[0238] Step 5: N-[[4-[(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]tert-butyl carbamate

[0239] Six reactions were carried out in parallel. TosCl (413.94 mg, 2.17 mmol, 2 equivalents) was added to a solution of N-[[4-[(2-butyl-5-oxoimidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]carbamate (500 mg, 1.09 mmol, 1 equivalent) in DCM (9 mL) and NH3·H2O (3 mL), and the mixture was stirred at 20 °C for 12 h. The six reactions were combined. Water (50 mL) was added to the reactants, followed by extraction with dichloromethane (3 × 50 mL), drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure to obtain the residue. The crude products (380 mg scale) were combined for purification. The residue was purified by column chromatography to obtain N-[[4-[(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]carbamate tert-butyl ester (3 g, 6.53 mmol, 88.92% yield).

[0240] Step 6: N-[[4-[(4-amino-2-butyl-5-oxoimidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]tert-butyl carbamate

[0241] Four reactions were carried out in parallel. m-CPBA (121.48 mg, 598.37 μmol, 85% purity, 1.1 equivalent) was added to a DCM solution of N-[[4-[(4-amino-2-butyl-imidazo[4,5-c]quinoline-1-yl)methyl]phenyl]methyl]carbamate tert-butyl ester (250 mg, 543.97 μmol, 1 equivalent) in 3 mL of DCM at 0 °C, and the mixture was stirred at 0 °C for 1 hour. The four reactions were combined. The reactants were added to a saturated aqueous solution of sodium thiosulfate (20 mL) and extracted with dichloromethane (3 × 20 mL). The combined organic layers were washed with a saturated sodium bicarbonate solution (3 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain N-[[4-[(4-amino-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]carbamate tert-butyl ester (270 mg, 567.73 μmol, 26.09% yield).

[0242] Step 7: 1-[[4-(aminomethyl)phenyl]methyl]-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthium-4-amine

[0243] A solution of N-[[4-[(4-amino-2-butyl-5-oxo-imidazo[4,5-c]quinoline-5-onthiol-1-yl)methyl]phenyl]methyl]tert-butyl carbamate (270 mg, 567.73 μmol, 1 equivalent) in HCl / EtOAc (3 mL) was added, and the mixture was stirred at 20 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the residue. The crude products (55 mg scale) were combined to the purity. The residue was purified by preparative HPLC to give NO-10 (173.9 mg, 421.42 μmol, 62.91% yield, HCl). 1HNMR (400 MHz, DMSO-d6) δ = 9.60 - 9.06 (m, 2H), 8.48 (br s, 3H), 8.06 (d, J =8.5 Hz, 1H), 7.98 (d, J = 8.2 Hz, 1H), 7.68 (t, J = 7.9 Hz, 1H), 7.47 (d, J =8.1 Hz, 2H), 7.40 (t, J = 7.7 Hz, 1H), 7.11 (d, J = 8.1 Hz, 2H), 5.99 (s,2H), 3.95 (br s, 2H), 2.97 (t, J = 7.7 Hz, 2H), 1.74 (quin, J = 7.6 Hz, 2H), 1.39 (sxt, J = 7.4 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0244] Example 11

[0245] 4-Amino-2-(ethoxymethyl)-1-(5,5,11,11-tetramethyl-3,6-dioxy-1-phenyl-2,10-dioxane) (NO-11)-4,7-diazadodecano-12-yl)-1H-imidazo[4,5-c]quinoline 5-oxide

[0246] Step 1: Synthesis of 2-methyl-1-[(3-nitro-4-quinolinyl)amino]prop-2-ol

[0247] TEA (1.60 g, 15.81 mmol, 2.20 mL, 0.66 equivalents) was added to a DCM (75 mL) solution of 4-chloro-3-nitro-quinoline (5 g, 23.97 mmol, 1 equivalent). Then, 1-amino-2-methyl-propan-2-ol (2.33 g, 26.13 mmol, 1.09 equivalents) was added dropwise at 0 °C. The mixture was stirred at 20 °C for 2 hours. The reaction mixture was filtered to give a yellow solid. 2-Methyl-1-[(3-nitro-4-quinolinyl)amino]propan-2-ol (5.3 g, crude product) was obtained.

[0248] Step 2: Synthesis of 1-[(3-amino-4-quinolinyl)amino]-2-methyl-prop-2-ol

[0249] Pd / C (0.43 g, 10% purity) was added to a MeOH (90 mL) solution of 2-methyl-1-[(3-nitro-4-quinolinyl)amino]prop-2-ol (4.3 g, 16.46 mmol, 1 equivalent). The suspension was degassed under vacuum and purged several times with H2 (15 PSI). The mixture was stirred at 20 °C under H2 (15 PSI) for 5 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give 1-[(3-amino-4-quinolinyl)amino]-2-methyl-prop-2-ol (3.8 g, crude product).

[0250] Step 3: Synthesis of 1-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-2-methyl-prop-2-ol

[0251] A solution of 1-[(3-amino-4-quinolinyl)amino]-2-methyl-prop-2-ol (3.8 g, 16.43 mmol, 1 equivalent) in 2-ethoxyacetic acid (12.51 g, 120.21 mmol, 11.36 mL, 7.32 equivalent) was stirred for 12 hours at 120 °C. The reaction mixture was slowly added to a saturated sodium bicarbonate solution (200 mL), and then extracted with ethyl acetate (3 × 200 mL). The combined organic layers were washed with brine (3 × 100 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to give 1-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-2-methyl-prop-2-ol (4.9 g, 16.37 mmol, 99.63% yield).

[0252] Step 4: Synthesis of N-[2-[2-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethyl]tert-butyl carbamate

[0253] NaH (785.66 mg, 19.64 mmol, 60% purity, 1.2 equivalents) was added to a DMF (50 mL) solution of 1-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-2-methyl-prop-2-ol (4.9 g, 16.37 mmol, 1 equivalent) at 0 °C. The solution was stirred at 0 °C for 1 h, then at 20 °C for 0.5 h, yielding a yellow solution. Tert-butyl 2,2-oxy-oxazolidine-3-carboxylate (3.65 g, 16.37 mmol, 1 equivalent) was added to this solution at 0 °C, and the resulting solution was stirred at 20 °C for 12 h. The solution was then added to 100 mL of saturated NH4Cl and extracted with ethyl acetate (3 × 500 mL). The combined organic layers were washed with brine (3 × 500 mL) and dried over anhydrous sodium sulfate. The residue was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (neutral conditions, column: Phenomenex lunac18 250 mm × 100 mm × 10 μm; mobile phase: [H2O (0.2% FA)-ACN]; gradient: 10%-37% B, 20.0 min) to obtain N-[2-[2-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethyl]tert-butyl carbamate (5 g, 11.30 mmol, 69.03% yield).

[0254] Step 5: Synthesis of 2-[2-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethylamine

[0255] MeOH (5 mL) was added to a solution of N-[2-[2-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethyl]carbamate tert-butyl ester (2 g, 4.52 mmol, 1 equivalent) in HCl / EtOH (20 mL, 4 M). The solution was stirred at 20 °C for 1 hour. The solution was then concentrated under reduced pressure to give 2-[2-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethylamine (1.7 g, crude product).

[0256] Step 6: Synthesis of 2-[2-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethylamine

[0257] HATU (2.27 g, 5.96 mmol, 1.2 equivalent) was added to a solution of 2-(benzyloxycarbonylamino)-2-methylpropionic acid (1.18 g, 4.96 mmol, 1 equivalent), 2-[2-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethoxy]ethylamine (1.7 g, 4.96 mmol, 1 equivalent), and DIEA (1.92 g, 14.89 mmol, 2.59 mL, 3 equivalent) in DMF (35 mL). The mixture was stirred at 20 °C for 12 hours. The reaction mixture was added to water (100 mL) and then extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 3 / 1 to 0 / 1) to obtain N-[2-[2-[2-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate (3 g, crude product).

[0258] Step 7: Synthesis of N-[2-[2-[2-[2-(ethoxymethyl)-5-oxoimidazo[4,5-c]quinoline-5-onthiol-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate

[0259] m-CPBA (1.41 g, 6.94 mmol, 85% purity, 1.3 equivalent) was slowly added to a DCM (30 mL) solution of N-[2-[2-[2-[2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate (3 g, 5.34 mmol, 1 equivalent) at 0 °C. The mixture was stirred at 20 °C for 1 hour. The reaction mixture was added to saturated Na₂SO₃ (30 mL) and then extracted with DCM (3 × 30 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure to obtain N-[2-[2-[2-[2-(ethoxymethyl)-5-oxo-imidazo[4,5-c]quinoline-5-on-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate (3 g, crude product).

[0260] Step 8: Synthesis of N-[2-[2-[2-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate benzyl ester

[0261] To a solution of N-[2-[2-[2-[2-(ethoxymethyl)-5-oxoimidazo[4,5-c]quinoline-5-onthiol-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate (0.5 g, 865.55 μmol, 1 equivalent) in DCM (8 mL) was added with TosCl (330.03 mg, 1.73 mmol, 2 equivalents) and NH3·H2O (8 mL). The mixture was stirred at 20 °C for 1 hour. The organic layer was concentrated under reduced pressure to obtain the residue. The residue was purified by flash chromatography (silica gel, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain a brown gel. The brown gel-like substance was purified by preparative HPLC (neutral conditions, column: Waters Xbridge BEH C18 100×30mm×10μm; mobile phase: [H2O(10 mMNH4HCO3)-ACN]; gradient: 30%-60% B, 8.0 min) to obtain N-[2-[2-[2-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate (18.8 mg, 32.60 μmol, 18.80% yield, 100.0% purity).

[0262] Step 9: Synthesis of N-[2-[2-[2-[4-amino-2-(ethoxymethyl)-5-oxoimidazo[4,5-c]quinoline-5-onthiol-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate

[0263] m-CPBA (52.81 mg, 260.11 μmol, 85% purity, 1.5 equivalents) was slowly added to a DCM (5 mL) solution of N-[2-[2-[2-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-1,1-dimethyl-ethoxy]ethylamino]-1,1-dimethyl-2-oxy-ethyl]carbamate (0.1 g, 173.40 μmol, 1 equivalent) at 0 °C, and the mixture was stirred at 0 °C for 0.5 h. Six batches of the reaction mixture were combined for post-treatment. The reaction mixture was slowly added to 20 mL of saturated Na₂SO₃, washed with 10 mL of saturated NaHCO₃, and then extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine (3 × 25 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (neutral conditions, column: Waters Xbridge Prep OBD C18 150×40mm×10μm; mobile phase: [H2O(10mM NH4HCO3)-ACN]; gradient: 25%-45% B, 8 min) to obtain crude product 1. Crude product 1 was then purified by preparative HPLC (FA conditions, column: Phenomenex luna C18 100×40mm×5μm; mobile phase: [H2O(0.2% FA)-ACN]; gradient: 20%-50% B, 8 min) to obtain NO-11 (26.1 mg, 44.04 μmol, 4.23% yield). LCMS m / z 593.3 [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ = 8.55 (d, J = 8.6 Hz, 1H), 8.49 (br d, J = 8.3 Hz, 1H), 8.35 (s, 1H), 7.69 - 7.49 (m,3H), 7.47 (br t, J = 7.5 Hz, 1H), 7.38 - 7.26 (m, 6H), 7.05 (br s, 1H), 4.96(s, 2H), 3.54 (q, J = 6.9 Hz, 2H), 3.48 - 3.22 (m, 4H), 3.13 (br s, 2H), 2.88(br d, J = 3.7 Hz, 2H), 1.21 (s, 6H), 1.14 (br t, J= 6.9 Hz, 9H).

[0264] II. Biological Assessment

[0265] Cell Culture

[0266] MC38, B16F10, and RAW 264.7 cells were purchased from Procell. RAW-Blue reporter cells and QUANTI-Blue solution were purchased from Invivogen. MC38, B16, RAW 264.7, and RAW-Blue reporter cells were cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum (FBS), penicillin (100 units / mL), and streptomycin (100 μg / mL). All these cells were cultured in a 5% CO2 incubator at 37°C, with the medium changed every 2–3 days. After reaching 80% confluence, the cells were treated with trypsin or a cell scraper and then seeded overnight in culture dishes or 96-well plates for further experiments.

[0267] RAW-Blue reporter cells were used at 5 × 10 4 Cells were seeded at a density of 100 cells / well in 96-well plates. Cells were treated with a series of agonist concentrations for 24 hours. Then, 20 μL of supernatant was extracted from each well and mixed with 180 μL of QUANTI-Blue solution. Absorbance was measured using a microplate reader.

[0268] Bone marrow cells were cultured for 6 days in the presence of rmGM-CSF (20 ng / ml, Biolegend) and then stimulated with NO₂⁻ for 24 hours in the absence of radiation or with 10 Gy radiation to generate BMDCs. After culture, cells were sorted and the surface markers CD80 / 86 were analyzed by flow cytometry (BD Bioscience). The supernatant was collected and measured by CBA (BD).

[0269] Ot I CD8 isolated from the spleen using magnetic beads (stem cells) + T cells were loaded with 2.5 μM CFSE and then co-cultured with antigen- and agonist-treated DCs at an 8:1 ratio. After 72 hours of incubation, T cells were analyzed by flow cytometry to assess CFSE dilution after staining with anti-mouse CD8α-APC and anti-mouse CD3-BV421. The supernatant was collected and IFNγ was measured by CBA.

[0270] animal models

[0271] MC38 and B16 (5×10) 5MC38 cells were subcutaneously injected into the right flank of 6-8 week old C57BL / 6J mice. After tumor formation, mice were randomly assigned to treatment groups and administered PBS or 30 μmol / kg NO-1 (intravenous injection) and / or radiation (6 Gy, for a total of 3 times) every 3 days. For the distal model, MC38 (5 × 10⁻⁶ cells) were injected into the right flank of 6-8 week old C57BL / 6J mice. 5 MC38 cells were subcutaneously injected into the right flank. Two days later, MC38 (2.5 × 10⁻⁶) cells were... 5 Cells were subcutaneously injected into the left flank, followed by intratumoral treatment with 3 μmol / kg NO₁ and irradiation focused solely on the right side of the tumor. Tumor volume was measured twice weekly and calculated as length × width × height / 2.

[0272] Activation of N-oxide compounds by radiation

[0273] X-ray irradiation was provided by an X-ray generator (RS2000 Pro 225, 225 kV, 17.7 mA, Rad Source Technologies, Inc.). Test tubes were irradiated with X-rays at a total dose of 0 to 60 Gy at a rate of 5 Gy / min. Cells were then irradiated with X-rays at a rate of 1.6 Gy / min with a total dose of 10 Gy. Implanted tumors were locally irradiated using custom-made mouse clamps. The tumors were locally irradiated with a dose of 6 Gy at a rate of 1 Gy / min, while the rest of the body was shielded with 5 mm thick lead.

[0274] For in vivo activation by agonists via radiotherapy, MC38 and B16 cells (5 × 10⁻⁶) were activated. 5 Subcutaneous injection was administered into the right flank of 6-8 week old C57BL / 6J mice. After tumor establishment, mice were randomly assigned to treatment groups, receiving PBS or 30 μmol / kg NO-1 and / or radiation (6 Gy, for a total of 3 times) every 3 days. For the distal model, MC38 (5 × 10⁻⁶) was injected into the right flank of mice. 5 ) cells were subcutaneously injected into the right flank and abdomen. Two days later, MC38 (2.5×10) cells were injected into the abdomen. 5 The cells were subcutaneously injected into the left flank, followed by intratumoral treatment with 3 μmol / kg NO-1 and radiation focused only on the right tumor.

[0275] Biological distribution analysis

[0276] MC38 tumor-bearing mice were injected intravenously with NO-1 (30 μmol / kg). The mice were then sacrificed at predetermined time points after injection, tumor tissue was collected, wet-weighed, and the NO-1 concentration was measured by UPLC.

[0277] Statistical analysis

[0278] Statistical analysis was performed using GraphPad Prism 6. Statistical comparisons were analyzed using two-way ANOVA.

[0279] Example 12: Immunostimulation studies of R848 and NO-1

[0280] Serum IFN-γ measurements were taken 4 hours after injection of R848 and NO-1 (30 μmol / kg (a) and 60 μmol / kg (b), respectively). For each test group, n = 3 C57BL / 6J mice. The test results are shown in Figure 1 a and Figure 1 b. Serum TNF-α measurements were taken 4 hours after injection of R848 and NO-1 (30 μmol / kg (c) and 60 μmol / kg (d), respectively). For each test group, n = 3 C57BL / 6J mice. The test results are shown in Figure 1 c and Figure 1 d. Measurement of body weight changes in C57BL / 6J mice (n = 6 mice per group) after intravenous administration of 30 μmol / kg of R848 and NO-1, respectively. The test results are shown in Figure 1 c and Figure 1 e. Concentration-dependent TLR7 / 8 activation curves of R848 and NO-1 were studied in RAW-blue reporter cells after 24-hour incubation (n = 6 independent samples for each test group). The test results are shown in Figure 1 f.

[0281] Example 13: Radiation-induced reduction study of NO-1 in aqueous solution and biological environment

[0282] X-ray-induced release of R848 from NO-1 (10 μM) in PBS was determined by UPLC-MS. The UPLC-MS spectrum of the generated R848 ([R848 + H] + selected ion monitoring signal, m / z = 315). The positive ion mode mass spectrum of R848 is shown, retention time = 2.18 minutes. The test results are shown in Figure 2 a and Figure 2 b.

[0283] Example 14: Immunostimulation studies of R848 and NO-1 in reporter cell lines after radiation

[0284] Figure 3 a shows a schematic diagram of the experimental design. In Figure 3In b-3h, NO-1 (10 μM) was incubated with live RAW 264.7 cells and bone marrow-derived DCs (BMDCs) for 24 hours and irradiated with X-rays (10 Gy). Marker expression, released cytokines, and T-cell sensitization were then measured. Cells incubated with R848 (1 μM) served as a positive control. Figure 3 CD80 / 86 expression in RAW264.7 (3b) and BMDC (3c) in b-3c. Figure 3 In d-3e, secreted TNF-α (3d) and MCP-1 (3e) released from BMDC were analyzed by CBA. Figure 3 In f-3h, BMDCs treated with NO-1 were incubated with 10 μg / mL OVA peptide for 24 hours, and then inoculated with OT I CD8 + T cells were co-cultured for 3 days. OT I CD8 analysis was performed. + T cell proliferation (3f, 3g) and IFN-γ production (3h). Data are expressed as mean ± SD. For each test group, n=3 independent samples, analysis was performed using a two-tailed unpaired Student's t-test.

[0285] Example 15: Radiotherapy-activated NO-1 inhibits tumor growth in MC38 cells via intravenous (iv) injection.

[0286] Figure 4 a shows the treatment plan. Figure 4 In b-4g, MC38 cells were subcutaneously implanted into C57BL / 6J mice, followed by intravenous (iv) injection of NO-1 (30 μmol / kg) and radiotherapy (6 Gy per treatment). Figure 4 Figure b shows the time-dependent accumulation of NO-1 in the blood, and tumor tissue (n=3 mice) was detected by UPLC-MS. Figure 4 Figure c-4e shows the average tumor growth (c), body weight change curves (d), and mouse survival curves (e) before reaching the euthanasia criteria. Figure 4 The tumor volume of an individual mouse is shown in f. Figure 4 The image shows a photograph of a representative tumor on day 25.

[0287] Example 16: Radiotherapy-activated NO-1 inhibits tumor growth in MC38 cells via intratumoral (it) injection

[0288] Figure 5The treatment regimen is illustrated in Figure a. MC38 cells were subcutaneously implanted into C57BL / 6J mice (n=6 mice per group) on days 0 and 2, followed by intratumoral injection of NO-1 (3 μmol / kg) and radiotherapy (6 Gy per treatment). Figure 5 Figure b shows the mean volume of the primary and distal tumors. Data are expressed as mean ± SD.

[0289] Example 17: TLR7 / 8 activation study of agonists and corresponding N-oxide prodrugs in RAW-Blue reporter cells

[0290] Figure 6 The concentration-dependent TLR7 / 8 activation curves of the agonist and the corresponding N-oxide prodrug in RAW-Blue reporter cells after 24 hours of culture are shown (n=6 independent samples for each test group). EC50 between N-oxide and the corresponding deoxygenated structure is also shown. 50 The comparisons are summarized in Table 2.

[0291] Table 2

[0292] Example 18: Study on the agonist activation yield of the corresponding N-oxide agonist after irradiation

[0293] Figure 7 It shows the result of e - aq A schematic diagram of induced deoxygenation of the N-oxide agonist, and the activation yield of the corresponding N-oxide agonist (10 μM) from PBS after 60 Gy irradiation. Data are expressed as mean ± sd, with n = 3 independent samples for each test group.

[0294] Example 19: Radiotherapy-activated NO-10 inhibits tumor growth in MC38 cells via intravenous (iv) injection.

[0295] Figure 8 MC38 tumor-bearing mice treated with different doses (20 μg / mouse and 100 μg / mouse) on days 10, 13, and 16 are shown. Body weight (a) and tumor growth (n=6) are measured and shown (b). Serum was collected 24 hours after the first injection. Cytokines in the serum were analyzed by cell counting beads (c and d). e and f show mice carrying MC38 tumors treated intravenously with NO-10 on days 10, 13, and 16. After 124 hours, mice were irradiated with 6 Gy or not. Tumor growth was measured as shown in (e), and survival curves are shown in (f) (n=6).

[0296] Example 20: Radiotherapy-activated NO-10 inhibits lung tumor growth and metastasis in B16F10 cells via intravenous (iv) injection.

[0297] Figure 9 shows the effect of using 2.5 × 10 5 Six C57BL / 6J mice were inoculated with 2.5 × 10⁶ B16F10 cells. One week later, the mice were intravenously injected with 2.5 × 10⁶ cells. 6 B16F10 cells. Tumor-bearing mice were treated with intravenous NO-10 and / or radiation on days 8, 11, and 14. Local tumor volume was measured (a). On day 19, mice were sacrificed, and the number of colonies on the lungs was counted (b and c).

[0298] Example 21: Radiotherapy-activated NO-10 inhibits lung tumor growth and metastasis in 4T1 cells via intravenous (iv) injection.

[0299] Figure 10 It shows the use of 2×10 5 4T1-luc2 cells were inoculated into dg Balb / c mice (n=8). Tumor-bearing mice were treated with intravenous NO-10 and / or radiation on days 10, 13, and 17. Lung metastasis was measured by IVIS spectroscopy (a and b). Survival curves are shown in (c).

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof: (I) in, Ring A is a 5-10 aryl group or contains 1 to 4 5-10 heteroaryl groups independently selected from nitrogen, oxygen or sulfur; Ring B is a 5-10 aryl group or a 5-10 heteroaryl group containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur; Y, Z, and W are independently chosen from groups composed of carbon, nitrogen, oxygen, or sulfur, and Y, Z, and W are optionally independent of each other. Ra, Rb, and Rc are independently selected from the group consisting of branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic hydrocarbon chains having 1 to 10 carbon atoms in their main chain. Each R1 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, optionally substituted acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R1s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. Each R2 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R2s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. R3, R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxy, cyano, cyanoalkyl, carboxyl, alkoxycarbonyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic or optionally substituted spirocyclic, or R3 and R4 combined to form an optionally substituted C3-10 heterocyclic alkyl. The optional substituents of R1, R2, R3, R4, R5, Ra, Rb and Rc are independently selected from optional alkyl, cycloalkyl, cycloalkoxy, halogen, haloalkyl, haloalkoxy, alkoxy, alkoxyalkyl, hydroxyl and carboxyl groups. n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; The condition is that the compound of formula I is not .

2. The compound of claim 1, wherein formula I is one of the following compounds or a pharmaceutically acceptable salt thereof: (I) in, Ring A is a 5-10 aryl group or contains 1 to 4 5-10 heteroaryl groups independently selected from nitrogen, oxygen or sulfur; Y, Z, and W are independently chosen from groups composed of carbon, nitrogen, oxygen, or sulfur, and Y, Z, and W are optionally independent of each other. Ra, Rb, and Rc are independently selected from the group consisting of branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic hydrocarbon chains having 1 to 10 carbon atoms in their main chain. Each R1 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, optionally substituted acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R1s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. Each R2 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R2s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxy, cyano, cyanoalkyl, carboxyl, alkoxycarbonyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, or optionally substituted spirocyclic, or R3 and R4 are combined to form an optionally substituted C3-10 heterocyclic alkyl. The optional substituents of R1, R2, R3, R4, Ra, Rb and Rc are independently selected from optional substituted alkyl, cycloalkyl, cycloalkoxy, halogen, haloalkyl, haloalkoxy, alkoxy, alkoxyalkyl, hydroxyl and carboxyl groups. n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; The condition is that the compound of formula I is not .

3. The compound of claim 2, wherein formula I is selected from formula Ia and formula Ib or a pharmaceutically acceptable salt thereof. (It) (Ib) in, X1, X2, X3, and X4 are independently selected from carbon, nitrogen, oxygen, or sulfur; It is either a single bond or a double bond.

4. The compound of claim 3, wherein formula I is formula Ib or a pharmaceutically acceptable salt thereof: (One) in, X1, X2, and X3 can be independently selected from groups composed of carbon, nitrogen, oxygen, or sulfur, and Y, Z, and W can be independently selected from non-existent. Ra, Rb, and Rc are independently selected from the group consisting of branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic hydrocarbon chains having 1 to 10 carbon atoms in their main chain. Each R1 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, optionally substituted acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R1s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. Each R2 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxyl, cyano, cyanoalkyl, optionally substituted carboxyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyalkynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, optionally substituted spirocyclic, optionally substituted heterospirocyclic or optionally substituted spiroheterocyclic, or two R2s combined to form optionally substituted cycloalkyl, heterocyclic, aryl or heteroaryl. R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, halogen, haloalkyl, haloalkoxy, alkoxy, hydroxy, cyano, cyanoalkyl, carboxyl, alkoxycarbonyl, acylamino, aminocarbonyl, hydroxyalkyl, hydroxyynyl, alkoxyalkyl, aminoalkyl, aminocarbonylalkyl, sulfonylalkyl, aminosulfonylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted bicyclic heterocyclic, optionally substituted bridged optionally substituted heterocyclic, or optionally substituted spirocyclic, or R3 and R4 are combined to form an optionally substituted C3-10 heterocyclic alkyl. The optional substituents of R1, R2, R3, R4, Ra, Rb and Rc are independently selected from optional substituted alkyl, cycloalkyl, cycloalkoxy, halogen, haloalkyl, haloalkoxy, alkoxy, alkoxyalkyl, hydroxyl and carboxyl groups. n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; The condition is that the compound of formula I is not .

5. The compound according to any one of claims 1 to 4, wherein The heteroatoms of rings A, X1, X2, X3, and X4 are independently selected from groups composed of carbon and nitrogen; Y does not exist or is independently selected from the group consisting of oxygen or nitrogen; Z does not exist or is independently selected from groups composed of oxygen or nitrogen; W is absent or is nitrogen; Ra is an optional substitution of C. 1-10 Alkylene; Rb can be independently and freely substituted with -C. 1-10 Alkylene-, optionally substituted C 6-10 Aryl-C 1-10 Alkylene- or optionally substituted -C 1-10 Alkylene-C 6-10 Aryl-C 1-10 The group consisting of alkylene groups; Rc is an optional substitution of -C 1-10 Alkylene-.

6. The compound according to any one of claims 1 to 5, wherein formula I is formula I-1, formula I-2 or formula I-3 or a pharmaceutically acceptable salt thereof: I-1， I-2， I-3。 7. The compound according to any one of claims 1 to 6, wherein each R2 is independently selected from the group consisting of hydrogen, alkyl, and optionally substituted carboxyl groups; preferably, each R2 is independently selected from hydrogen, C 1-10 Alkyl, C 1-10 The group consisting of alkyl-O-(CO)-.

8. The compound according to any one of claims 1 to 7, wherein each R1 is independently selected from hydrogen, hydroxyl, C 1-10 Alkyl-, C 1-10 Alkyl-(CO)-, C 1-10 Alkyl-O-(CO)-, C 1-10 Alkyl-SO2-, C 1-10 Alkyl-NH-C 1-10 Alkyl-(CO)-, C 1-10 Alkyl-O-(CO)-NH-C 1-10 Alkyl-(CO)- or C 6-10 Aryl-C 1-10 Alkyl-O-(CO)-NH-C 1-10 The group consisting of alkyl-(CO)-.

9. The compound according to any one of claims 1 to 8, wherein R3 and R4 are independently selected from hydrogen, C 1-10 Alkyl, -F, -Cl, -Br, -I, Halogenated C 1-10 Alkyl, Halogenated C 1-10 Alkoxy, C 1-10 Alkoxy, hydroxy, cyano, cyano C 1-10 A group composed of alkyl groups.

10. The compound of claim 1, wherein the compound is selected from the group consisting of compounds or pharmaceutically acceptable salts thereof: 。 11. A pharmaceutical composition comprising the compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable diluent, excipient or inert carrier.

12. A method for the in vivo preparation of a non-N-oxide compound of formula II, wherein the compound of formula I according to any one of claims 1-10 or a pharmaceutically acceptable salt thereof is reduced to the non-N-oxide compound by radiation in a subject in need. 。 13. Use of any compound of claims 1-10 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer in subjects in need; or A method of treating cancer, comprising administering to a subject in need a therapeutically effective amount of any one of claims 1-10, or a pharmaceutically acceptable salt thereof; or The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, is used as a medicament for treating cancer in subjects in need.

14. The use, method, or compound of claim 13, wherein the subject is irradiated after administration of the compound, preferably 20 minutes, 1 hour, 2 hours, or 4 hours after administration of the compound.

15. The use, method, or compound of claim 13 or 14, wherein the cancer is a solid cancer or a non-solid tumor, preferably selected from the group consisting of colon cancer, small cell lung cancer, Hodgkin's lymphoma, malignant lymphoma, melanoma, nasopharyngeal carcinoma, head and neck cancer, skin cancer, esophageal cancer, lung cancer, liver cancer, prostate cancer, cervical cancer, thyroid cancer, and breast cancer, preferably the cancer is a primary cancer and / or a secondary cancer.

16. The use, method, or compound according to any one of claims 13 to 15, wherein the compound is applied in an amount of about 0.005 mg / kg to about 100 mg / kg, for example, in amounts of about 0.005 mg / kg, 0.05 mg / kg, 0.5 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or 100 mg / kg.

17. The use, method, or compound according to any one of claims 13 to 16, wherein the compound and the radiation are applied once every two days, once every three days, once every four days, once every five days, once every seven days, once every ten days, once every two weeks, once every three weeks, or once every four weeks, and the compound and the radiation are applied continuously for at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, at least 9 rounds, or at least 10 rounds.

18. The use, method or compound of any one of claims 13 to 17, wherein the compound is administered orally, parenterally, intraperitoneally, intravenously, intra-arterially, percutaneously, sublingually, intramuscularly, rectally, buccally, intranasally, via liposomes, via inhalation, via vagina, via eye, via local delivery, subcutaneously, intra-fat, intra-articularly, intrathecally, or intratumorally.

19. The use, method, or compound according to any one of claims 13 to 18, wherein the radiation is X-rays or gamma rays.

20. The use, method, or compound of claim 19, wherein the intensity of the X-rays or gamma rays is less than 60 Gy, less than 50 Gy, less than 40 Gy, less than 30 Gy, less than 20 Gy, less than 10 Gy, less than 8 Gy, less than 6 Gy, or less than 4 Gy.

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