Ataxia vasodilator and inhibition of Rad3-related protein (ATR)

Abstract

Novel compounds that inhibit ATR protein kinase include the compound of formula (I) disclosed herein, as well as liposome formulations containing ATR protein kinase inhibitor compounds. [Solution] This composition is useful in the treatment of cancer.

Description

[Technical Field]

[0001] Related technologies This application claims priority to U.S. Provisional Patent Application No. 62 / 277,262 filed on 11 January 2016, U.S. Provisional Patent Application No. 62 / 420,258 filed on 10 November 2016, and U.S. Provisional Patent Application No. 62 / 444,172 filed on 9 January 2017, each of which is incorporated herein by reference in whole for all purposes.

[0002] field This disclosure relates to compounds and related methods that inhibit ataxia vasodilator and Rad3-related protein (ATR), including methods and compounds useful for treating cancer. [Background technology]

[0003] Ataxia vasodilator (ATR) and Rad3-associated (ATR) kinases are serine / threonine protein kinases thought to be involved in cellular DNA damage repair processes and cell cycle signaling. ATR kinases, along with ATM ("Ataxia vasodilator mutation") kinases and other proteins, regulate the cellular response to DNA damage, commonly known as the DNA damage response ("DDR"). DDR is thought to stimulate DNA repair, promote survival, and halt cell cycle progression by activating cell cycle checkpoints that provide time for repair. Without DDR, cells would be far more susceptible to DNA damage and would readily die from DNA damage induced by endogenous cellular processes such as DNA replication, or by exogenous DNA damaging agents commonly used in cancer treatment.

[0004] Disruption of ATR function (e.g., due to gene deletion) has been shown to promote cancer cell death both in the absence and in the presence of DNA damaging agents. Mutations in ATR are associated with gastric and endometrial cancers and lead to increased sensitivity to ionizing radiation and loss of cell cycle checkpoints. ATR is essential for somatic cell viability, and ATR deficiency has been shown to result in loss of damage checkpoint response and cell death. See Cortez et al., Science 294:1713-1716 (2001). ATR is also essential for the stability of fragile sites, and in patients with Seckel syndrome, low ATR expression leads to increased chromosomal disruption after replication stress. See Casper et al., Am.J.Hum.Genet 75:654-660 (2004). The replication protein A (RPA) complex recruits ATR and its interacting protein ATRIP to DNA damage sites, and ATR itself mediates the activation of the CHK1 signaling cascade. See Zou et al., Science 300:1542-1548 (2003). ATR, like its related checkpoint kinase ATM, phosphorylates RAD17 upstream in the cascade, which is important for checkpoint signaling in DNA-damaged cells. See Bao et al., Nature 411:969-974 (2001). ATR is thought to be particularly essential in early mammalian embryos for sensing incomplete DNA replication and preventing mitotic cell death. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Cortez et al.,Science 294:1713-1716(2001) [Non-Patent Document 2] Casper et al.,Am.J.Hum.Genet 75:654-660(2004) [Non-Patent Document 3] Zou et al.,Science 300:1542-1548(2003) [Non-Patent Document 4] Bao et al.,Nature 411:969-974(2001) [Overview of the project] [Problems that the invention aims to solve]

[0006] However, while DNA-damaging chemotherapeutic agents and ionizing radiation (IR) therapy have provided early therapeutic advantages for cancer patients, existing therapies have lost their clinical efficacy (e.g., due to tumor cell DNA repair responses). The in vivo effects of ATR inhibitors and DNA damage agents have shown some potential in the selective treatment of cancer, particularly in the treatment of tumor cells lacking G1 checkpoint control (which may be dependent on ATR for survival), compared to normal cells.

[0007] There is still a need for the development of potent and selective therapies to deliver ATR inhibitors for the treatment of cancer, either as monotherapy or as part of a combination therapy (e.g., in combination with chemotherapy and / or radiotherapy). [Means for solving the problem]

[0008] overview The inventors have discovered novel compounds useful for inhibiting ataxia vasodilator and Rad3-related (ATR) kinase, as well as for the treatment of cancer, and liposomal formulations of specific ATR protein kinase inhibitors having desired properties (e.g., extended half-life in the blood circulation and efficacy in treating tumors). The present invention is in part based on the discovery of specific novel compounds for inhibiting ATR protein kinase, and specific liposomal formulations of ATR protein kinase inhibitor compounds exhibiting extended plasma half-lives and enhanced antitumor effects.

[0009] In the first embodiment, a novel compound of formula (I) or a pharmaceutically acceptable salt thereof is useful for the treatment of ataxia vasodilator, inhibition of Rad3-related (ATR) kinase, and cancer:

[0010] [Chemistry]

[0011] In the formula, R is a moiety containing an amine having a pK greater than 7.0 (preferably greater than 8.0, most preferably at least about 9.5). The compound of formula (I) preferably contains, in R, one or more tertiary amine moieties selected to provide the desired ATR inhibition and / or liposome formation and stability properties. In some embodiments, R is preferably a heterocyclic moiety containing a first tertiary substituted nitrogen substituted with an alkylamino moiety containing a second tertiary substituted nitrogen. In particular, the compound of formula (I) has the formula: a which may contain R as the moiety of.

[0012] [Chemistry]

[0013] where A

[0014] is absent or is alkyl (e.g., C1-C4 alkyl (preferably -(CH2)2-)), and R 1 is lower (e.g., C1-C4) alkylamino. In one embodiment, R 1 is (C1-C4 alkyl)-NR 1 R a R b where R a and R b are each independently C1-C4 alkyl, e.g., R1 is -(CH2)2-N(CH3)(CH3). In another embodiment, R 1 is NR a R b where R a and R b are each independently C1-C4 alkyl, e.g., R 1 is -N(CH2CH3)(CH2CH3).

[0015] In another embodiment of formula (I), R is -N(H)(C1-C4 alkyl)-NR a R b And in the formula, R a and R b However, each is independently a C1-C4 alkyl group, or R is -(G)-NR a R b And in the formula, R a and R b However, each is independently a C1-C4 alkyl group, G is a C1-C4 alkyl group, and G may be further substituted with a C1-C4 alkyl group.

[0016] In another embodiment of equation (I), R is:

[0017] [ka]

[0018] It could be the part, In the formula, R c and R d Each of these is independently a C1-C4 alkyl group.

[0019] A preferred example is a liposome containing a compound selected from the group consisting of compounds 1, 2, 3, 4, 5, or 6:

[0020] [ka]

[0021] In a second embodiment, the liposome formulation of the ATR inhibitor compound may comprise one or more liposome-forming lipids (e.g., hydrogenated soy phosphatidylcholine (HSPC)), cholesterol, and polymer-binding lipids (e.g., methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glyceryl (PEG2000-DSG)) in which the compound of formula (I) or other ATR inhibitor compounds (e.g., comparative compound A) are encapsulated with a polyanion (e.g., a polyanionized sugar such as sucrose octasulfate, or a suitable polyanionized polyol). The liposome-forming lipids are preferably selected to provide a desired amount of liposome membrane rigidity while keeping the amount of leakage of the compound of formula (I) from the liposomes sufficiently low. It contains one or more phospholipids having a ratio of quality(s) to cholesterol. The type and amount of polymer-bound lipids can be selected to provide a desirable level of protein binding, liposome stability, and circulation time in the bloodstream. In some examples, liposome vesicles contain HSPC and cholesterol in a molar ratio of 3:2. In particular, liposomes may contain vesicles consisting of HSPC, cholesterol, and PEG2000-DSG in a molar ratio of 3:2:0.15. The compound of formula (I) can be encapsulated within the liposome using a suitable polyanion, such as sucrose octasulfate. In some examples, the liposome encapsulates the compound of formula (I) and sucrose octasulfate in or near the stoichiometric ratio of the compound of formula (I) and sucrose octasulfate.

[0022] One specific example provides a liposome having vesicles formed from HSPC, cholesterol, and PEG2000-DSG in a molar ratio of 3:2:0.15, which encapsulates sucrose octasulfate and compound 5. Another example provides a liposome having vesicles formed from HSPC, cholesterol, and PEG2000-DSG in a molar ratio of 3:2:0.15, which encapsulates sucrose octasulfate and compound 5.

[0023] Another specific example provides a liposome having vesicles formed from HSPC, cholesterol, and PEG2000-DSG in a molar ratio of 3:2:0.15, which encapsulates sucrose octasulfate and compound 6.

[0024] Another specific embodiment provides a liposome having vesicles formed from HSPC, cholesterol, and PEG2000-DSG in a molar ratio of 3:2:0.15, which encapsulates sucrose octasulfate and compound A.

[0025] The ATR inhibitor compounds and / or their liposomal formulations disclosed herein can be used in therapies and therapeutic methods. In some embodiments, the therapy is the treatment of cancer. When used as a therapy, the liposomal composition may be used with one or more other compounds or compositions (e.g., irinotecan such as MM-398).

[0026] [ka]

[0027] It may be used in a therapeutic regimen (in combination with a liposomal formulation). Administration of the liposomal composition with one or more other compounds or compositions may be simultaneous, separate, or sequential. The one or more other compounds or compositions may be further therapeutic agents, for example, further anticancer agents, or compounds designed to mitigate the negative side effects of a therapeutic agent. [Brief explanation of the drawing]

[0028] [Figure 1] This is a first chemical reaction scheme useful for the production of the specific compounds disclosed herein. [Figure 2] This is a second chemical reaction scheme useful for the production of the specific compounds disclosed herein. [Figure 3] This is a third chemical reaction scheme useful for the production of the specific compounds disclosed herein. [Figure 4]This is a fourth chemical reaction scheme useful for the production of the specific compounds disclosed herein. [Figure 5] This graph shows the hematopoietic pharmacokinetics of the liposomal ATR inhibitor described in Example 8. [Figure 6] This graph shows the antitumor efficacy of liposomal compound A used in combination with MM-398 in the cervical MS751 xenograft model described in Example 9A. [Figure 7] This graph shows the antitumor efficacy of liposomal compound A used in combination with MM-398 in a cervical C33A xenograft model, as described in Example 9A. [Figure 8] This graph shows the antitumor efficacy of liposomal compound A in combination with MM-398 in a cervical C33A xenograft model according to Example 9A. [Figure 9] This graph shows the tolerability of liposomal compound A in combination with MM-398 in cervical MS751, according to Example 9A. [Figure 10] As described in Example 10, this graph shows the efficacy of liposomal compound 5 used in combination with MM-398 in NCI-H2170 (Figure 10A) and DMS-114 (Figure 10B) mouse xenograft models. [Figure 11] As described in Example 10, this graph shows the Kaplan-Meier survival curves illustrating the efficacy of liposomal compound 5 used in combination with MM-398 in NCI-H2170 (Figure 11A) and DMS-114 (Figure 11B) mouse xenograft models. [Figure 12] This graph shows the tolerability of liposomal compound 5 when used in combination with MM-398 in NCI-H2170 (Figure 12A) and DMS-114 (Figure 12B) mouse xenograft models. [Figure 13] This graph shows the efficacy of liposomal compound 5 used in combination with MM-398 in the Calu-6 (Figure 13A) and COLO-699 (Figure 13B) mouse xenograft models described in Example 11. [Figure 14A]This graph shows cell death induced by in vitro monotherapy with compounds 6 and 5 in a panel of lung cancer cell lines. [Figure 14B] This graph shows the effects of compounds 5 and 6 when used in combination with three chemotherapy agents (carboplatin, gemcitabine, and compound B). [Figure 15] This graph shows the IC50 shift values ​​of the ATR protein kinase inhibitor compound 6 of Example 2 in the presence and absence of various concentrations of compound B, as measured in the Sum190PT cell line (triple-negative breast cancer, TNBC). [Figure 16] Figure 16A is a graph showing the IC50 shift values ​​of the combination therapy with ATR protein kinase inhibitor compound 6 of Example 2, as measured in the MDA-MB-453TNBC cancer cell line. Figure 16B is a graph showing the IC50 shift values ​​of the combination therapy with ATR protein kinase inhibitor compound A, as measured in the MDA-MB-453TNBC cancer cell line. [Figure 17] These are the results of Western blot analysis obtained from DMS-114 lung cancer cells exposed in vitro to either gemcitabine [16 nM] or an ATR inhibitor (compound A or compound 5 [1 uM]), either alone or in combination. [Figure 18A] This study describes cell death and proliferation assays performed in U2OS cells using compound A and gemcitabine at various concentrations. The results are compared with predicted values ​​for cell proliferation and cell death when the two compounds are used together. [Figure 18B] This is a cell death and cell proliferation assay performed in U2OS cells using compound A and gemcitabine at various concentrations. The number of viable cells and apoptotic cells was also measured at the specified concentrations of compound A (1 μM) and gemcitabine (0.04 μM) in U2OS cells. [Figure 19] Compound A was tested alone and in combination with SN38 at set concentrations (1 μM and 0.2 μM, respectively) in several lung cancer cell lines (NCI-H520 and NCI-H596) and U2OS cells, and the cell count was monitored over time. [Figure 20]This graph shows the effects of compound A and SN38 in combination and alone in the cervical cancer cell line MS751. [Figure 21A] This shows the combined efficacy of compound A or compound 5 with gemcitabine. The heatmap shows the combined efficacy of compound A or compound 5 with gemcitabine at various concentrations in U2OS, H358, and A549 cell lines. [Figure 21B] The combined effect of compound A or compound 5 with gemcitabine is shown. Micrographs of cells treated with compound 5 and gemcitabine or compound A and gemcitabine at the specified concentrations are shown. [Figure 21C] The combined efficacy of compound A or compound 5 with gemcitabine is demonstrated. Similarly, proliferation assays of USO2 and H358 cells at specified concentrations are also shown. [Figure 22] The proliferation curves of A549 cells when compound A or compound 5 was used in combination with SN38 (Figure 22A) or alone (Figure 22B) are shown. [Figure 23] The IC50 values ​​(μM) in several cell lines using gemcitabine at set concentrations containing various concentrations of compound A or compound 5 are shown. [Figure 24] This document outlines lung cancer cell lines that respond to compound A or compound 5 in combination with gemcitabine or SN38. [Figure 25] The results of tests on the on-target (ATR) and off-target (ATM) inhibitory abilities of various ATR inhibitors are presented. Inhibition is reported as IC50 at nM (Figure 25A). Further "off-target" kinases are tested similarly using compound A or compound 5 (Figure 25B). [Figure 26A] The results of on-target use of compound A or compound 5 in A549 lung cancer cells are shown. Phosphorylation of CHK1 S345, which is the ATR inhibition reading, was measured by Western blotting. Each compound was used with a constant concentration of gemcitabine. [Figure 26B]The results of on-target use of compound A or compound 5 in A549 lung cancer cells are shown. Phosphorylation of CHK1 S345, which is the ATR inhibition reading, was measured by Western blotting. Each compound was used with a constant concentration of gemcitabine. [Figure 26C] The results of on-target use of compound A or compound 5 in H23 lung cancer cells are shown. Phosphorylation of CHK1 S345, which is the ATR inhibition reading, was measured by Western blotting. Each compound was used with a constant concentration of gemcitabine. [Figure 26D] The results of on-target use of compound A or compound 5 in H23 lung cancer cells are shown. Phosphorylation of CHK1 S345, which is the ATR inhibition reading, was measured by Western blotting. Each compound was used with a constant concentration of gemcitabine. [Figure 26E] The results of on-target use of compound A or compound 5 in DMS-114 cells are shown. Phosphorylation of CHK1 S345, which is the ATR inhibition reading, was measured by Western blotting. Each compound was used with a constant concentration of gemcitabine. [Figure 26F] The results of on-target use of compound A or compound 5 in DMS-114 cells are shown. Phosphorylation of CHK1 S345, which is the ATR inhibition reading, was measured by Western blotting. Each compound was used with a constant concentration of gemcitabine. [Figure 27A] Further on-target analyses for HCC-70 TNBC, MDA-MB-468 TNBC, and DMS-114 cell lines are presented. The set concentration of SN38 is used in conjunction with a range of compound A or compound 5 concentrations. Various on-target activity parameters are tested and measured by Western blotting. [Figure 27B] Further on-target analyses for HCC-70 TNBC, MDA-MB-468 TNBC, and DMS-114 cell lines are presented. The set concentration of SN38 is used in conjunction with a range of compound A or compound 5 concentrations. Various on-target activity parameters are tested and measured by Western blotting. [Figure 28] The cell cycle characteristics of SUM149 cells 24 hours after the addition of SN38 and compound A or compound 5 are shown. [Figure 29] This describes the DMS-114 lung xenograft model used to measure the effect of Ls compound A or Ls compound 5 when used in combination with MM398. A set dose of MM398 (5 mpk) is used along with two different doses of compound A or compound 5 (20 mpk or 80 mpk). The therapeutic effect is assayed by measuring the level of CHK1 S345 phosphorylation. [Figure 30A] This study demonstrates the effect of Ls compound A in the presence of MM398 in the SUM-149 cell line. The therapeutic effect is assayed by measuring the phosphorylation level of RPA2. [Figure 30B] This study demonstrates the effect of Ls compound A in the presence of MM398 in the SUM-149 cell line. Therapeutic efficacy is assayed by measuring the phosphorylation levels of RPA2, DNAPK, CHK1, and γH2AX. [Figure 30C] This study demonstrates the effects of Ls compound A in the presence of MM398 in the SUM-149 cell line. Therapeutic efficacy is assayed by measuring the phosphorylation levels of RPA2, DNAPK, CHK1, and γH2AX. Ls compound 5 is also tested without concomitant use of MM398. [Figure 31] This graph shows the efficacy of liposomal compound 5 when used in combination with MM-398 in a SUM-149 mouse xenograft model. [Figure 32] This graph shows the tolerability of liposomal compound 5 when used in combination with MM-398 in the SUM-149 mouse xenograft model. [Figure 33] These are baseline levels of various PD markers related to DNA damage response pathways, quantified by Western blotting against a panel of cell lines. [Figure 34] This is a schematic diagram illustrating how to calculate the integral score for each well in a dynamic cell viability assay. [Figure 35]This study examines the correlation between basal MRE11 protein expression (quantified by Western blotting) and the integral score, a measure of dynamic cell viability, in lung cancer cell lines exposed to ATR inhibitor compound 5 and / or SN38. [Figure 36] This study examines the correlation between basal ATM protein expression (quantified by Western blotting) and the integral score, a measure of dynamic cell viability, in lung cancer cell lines exposed to ATR inhibitor compound 5 and / or SN38. [Figure 37] This study examines the correlation between basal NBS protein expression (quantified by Western blotting) and the integral score, a measure of dynamic cell viability, in lung cancer cell lines exposed to ATR inhibitor compound 5 and / or SN38. [Figure 38] This study examines the correlation between basal NBS protein expression (quantified by Western blotting) and the integral score, a measure of dynamic cell viability, in p53 dysfunctional lung cancer cell lines exposed to ATR inhibitor compound 5 and / or SN38. [Figure 39] This study examines the correlation between basal NBS protein expression (quantified by Western blotting) and the integral score, a measure of dynamic cell viability, in p53 dysfunctional lung cancer cell lines exposed to ATR inhibitor compound 5 and / or SN38. [Figure 40A] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH1299 after ATR inhibition and / or exposure to SN38. [Figure 40B] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH1299 after ATR inhibition and / or exposure to SN38. [Figure 40C] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH1299 after ATR inhibition and / or exposure to SN38. [Figure 40D] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH1299 after ATR inhibition and / or exposure to SN38. [Figure 40E] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH1299 after ATR inhibition and / or exposure to SN38. [Figure 40F]This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH1299 after ATR inhibition and / or exposure to SN38. [Figure 41A] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH460 after ATR inhibition and / or exposure to SN38. [Figure 41B] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH460 after ATR inhibition and / or exposure to SN38. [Figure 41C] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH460 after ATR inhibition and / or exposure to SN38. [Figure 41D] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH460 after ATR inhibition and / or exposure to SN38. [Figure 41E] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH460 after ATR inhibition and / or exposure to SN38. [Figure 41F] This shows the magnification change in pharmacodynamic markers of the cancer cell line NCIH460 after ATR inhibition and / or exposure to SN38. [Figure 42A] This shows the magnification change in pharmacodynamic markers of the cancer cell line DMS114 after ATR inhibition and / or exposure to SN38. [Figure 42B] This shows the magnification change in pharmacodynamic markers of the cancer cell line DMS114 after ATR inhibition and / or exposure to SN38. [Figure 42C] This shows the magnification change in pharmacodynamic markers of the cancer cell line DMS114 after ATR inhibition and / or exposure to SN38. [Figure 42D] This shows the magnification change in pharmacodynamic markers of the cancer cell line DMS114 after ATR inhibition and / or exposure to SN38. [Figure 42E] This shows the magnification change in pharmacodynamic markers of the cancer cell line DMS114 after ATR inhibition and / or exposure to SN38. [Figure 42F] This shows the magnification change in pharmacodynamic markers of the cancer cell line DMS114 after ATR inhibition and / or exposure to SN38. [Figure 43A] This shows the magnification change in pharmacodynamic markers of the cancer cell line HCC70 after ATR inhibition and / or exposure to SN38. [Figure 43B] This shows the magnification change in pharmacodynamic markers of the cancer cell line HCC70 after ATR inhibition and / or exposure to SN38. [Figure 43C] This shows the magnification change in pharmacodynamic markers of the cancer cell line HCC70 after ATR inhibition and / or exposure to SN38. [Figure 43D] This shows the magnification change in pharmacodynamic markers of the cancer cell line HCC70 after ATR inhibition and / or exposure to SN38. [Figure 43E] This shows the magnification change in pharmacodynamic markers of the cancer cell line HCC70 after ATR inhibition and / or exposure to SN38. [Figure 43F] This shows the magnification change in pharmacodynamic markers of the cancer cell line HCC70 after ATR inhibition and / or exposure to SN38. [Figure 44A] This shows the magnification change in pharmacodynamic markers of the cancer cell line MDAMB468 after ATR inhibition and / or exposure to SN38. [Figure 44B] This shows the magnification change in pharmacodynamic markers of the cancer cell line MDAMB468 after ATR inhibition and / or exposure to SN38. [Figure 44C] This shows the magnification change in pharmacodynamic markers of the cancer cell line MDAMB468 after ATR inhibition and / or exposure to SN38. [Figure 44D] This shows the magnification change in pharmacodynamic markers of the cancer cell line MDAMB468 after ATR inhibition and / or exposure to SN38. [Figure 44E] This shows the magnification change in pharmacodynamic markers of the cancer cell line MDAMB468 after ATR inhibition and / or exposure to SN38. [Figure 44F] This shows the magnification change in pharmacodynamic markers of the cancer cell line MDAMB468 after ATR inhibition and / or exposure to SN38. [Figure 45]Western blot of pharmacodynamic markers in cancer cell line A549 after 6 or 18 hours of exposure to ATR inhibition and / or gemcitabine. [Figure 46] Western blot of pharmacodynamic markers in the cancer cell line NCIH23 after 6 or 18 hours of exposure to ATR inhibition and / or gemcitabine. [Figure 47] Western blot of pharmacodynamic markers in the cancer cell line DMS114 after 6 or 18 hours of exposure to ATR inhibition and / or gemcitabine. [Figure 48] Western blot of pharmacodynamic markers in cancer cell line U20S after 6 or 18 hours of exposure to ATR inhibition and / or gemcitabine. [Figure 49] Western blot of pharmacodynamic markers in cancer cell line NCIH460 after 6 or 18 hours of exposure to ATR inhibition and / or gemcitabine. [Figure 50] Western blot of pharmacodynamic markers in the cancer cell line HCC827 after 6 or 18 hours of exposure to ATR inhibition and / or gemcitabine. [Figure 51] Western blot of pharmacodynamic markers in a colorectal cancer cell line panel after 18 hours of exposure to compound 5 and / or SN38. [Figure 52] This shows the normalized quantification of phosphorylated Chk1 levels in a panel of colorectal cancer cell lines after 18 hours of exposure to compound 5 and / or SN38 (the signal for each cell line is normalized to the signal in the presence of SN38 alone). [Figure 53] This shows the normalized quantification of phosphorylated RPA2 levels in a panel of colorectal cancer cell lines after 18 hours of exposure to compound 5 and / or SN38 (the signal for each cell line is normalized to the signal in the presence of SN38 alone). [Figure 54]These are the normalized quantitative results of γH2AX levels in a panel of colorectal cancer cell lines after 18 hours of exposure to compound 5 and / or SN38 (the signal for each cell line is normalized to the signal in the presence of SN38 alone). [Modes for carrying out the invention]

[0029] Detailed explanation Novel compounds for inhibiting ATR protein kinase, or pharmaceutically acceptable salts thereof, are proposed using formula (I):

[0030] [ka]

[0031] Described by, in the formula, R is a pK greater than 7.0 (preferably greater than 8.0, and most preferably at least about 9.5) selected to provide a plasma half-life of at least about 5 hours in mice. a This is a moiety containing an amine having (obtained according to Example 7). Preferably, R comprises an amine-substituted alkyl moiety having 4 to 12 carbon atoms. R can be selected to consist only of combinations of trisubstituted amines and hydrogenated alkyl groups. R has a pK of at least 7, but most preferably at least about 9.5. a (For example, approximately 9.5-10.5 pK) a The compound further comprises a tertiary alkyl-substituted amine having ). Examples of compounds of formula (I) or pharmaceutically acceptable salts thereof include compounds 1 to 6 (see Examples 1 to 6):

[0032] [ka]

[0033] Compounds of formula (I) preferably contain one or more tertiary amine moieties in R selected to provide desired ATR inhibition and / or liposome formation and stability properties. In some examples, R is a heterocyclic moiety containing a primary tertiary substituted nitrogen substituted with an alkylamino moiety, preferably containing a secondary tertiary substituted nitrogen. In particular, compounds of formula (I) are of formula:

[0034] [ka]

[0035] The formula can include R, which is part of the formula, and A 1 It is either absent or alkyl (e.g., C1-C4 alkyl (preferably -(CH2)2-) and R 1 is a lower (e.g., C1-C4) alkylamino. In one embodiment, R 1 (C1-C4 alkyl)-NR a R b And in the formula, R a and R b Each of these is independently a C1-C4 alkyl, for example, R 1 is -(CH2)2-N(CH3)(CH3). In another embodiment, R 1 is NR a R b And R a and R b Each of these is independently a C1-C4 alkyl, for example, R 1 The expression is -N(CH2CH3)(CH2CH3).

[0036] In another embodiment of formula (I), R is -N(H)(C1-C4 alkyl)-NR a R b And in the formula, R a and R b Either each is independently a C1-C4 alkyl group, or R is -(G)-NR a R b And in the formula, R a and R bEach of these is independently a C1-C4 alkyl group, and in the formula, G is a C1-C4 alkyl group, and in the formula, G can be further substituted with a C1-C4 alkyl group.

[0037] In another embodiment of equation (I), R is:

[0038] [ka]

[0039] It can be the part, and in the formula, R c and R d Each of these is independently a C1-C4 alkyl group.

[0040] Preferred examples include liposomes containing a compound selected from the group consisting of compounds 1, 2, 3, 4, 5, or 6 described above. In some examples, the compound is compound 5 or compound 6.

[0041] The compound of formula (I) may have the chemical structure of formula (Ia) or a pharmaceutically acceptable salt thereof, where R′ is about 9.5 or greater pK a It is a tertiary alkyl-substituted amine having:

[0042] [ka]

[0043] Examples of compounds of formula (Ia) include compound 5 disclosed herein (e.g., Example 1). In one embodiment of formula (Ia), R′ is NR a R b And R a and R b Each of these is independently a C1-C4 alkyl group.

[0044] Liposome formulations of ATR protein kinase inhibitor compounds (e.g., those described in Example 7) can provide desirable pharmacokinetic properties, such as an improved plasma half-life of 5 hours or more, in the mouse model described in Example 8. Liposomes typically comprise vesicles having one or more lipid bilayers surrounding an aqueous interior. Liposome compositions often contain liposomes in a medium, e.g., an aqueous fluid outside the liposomes. Liposomal lipids may include amphiphilic lipid components that spontaneously form a bilayer membrane, e.g., phospholipids, e.g., phosphatidylcholine, upon contact with an aqueous medium. Liposomes may also contain membrane-binding components, e.g., sterols, e.g., cholesterol. In some cases, liposomes may also contain lipids bound to hydrophilic polymers, e.g., polyethylene glycol (PEG) lipid derivatives, which may reduce the tendency of liposomes to aggregate and may have other beneficial effects.

[0045] The liposome formulation comprises a compound of formula (I) encapsulated with a polyanion (e.g., polyanionized sugar, e.g., sucrose octasulfate, or a suitable polyanionized polyol) in a monolayer vesicle formed from one or more liposome-forming lipids (e.g., hydrogenated soy phosphatidylcholine (HSPC)), cholesterol, and polymer-binding lipids (e.g., methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glyceryl (PEG2000-DSG)). The liposome-forming lipid preferably comprises one or more phospholipids having a phospholipid(s)-to-cholesterol ratio selected to provide a desired amount of liposome membrane rigidity while keeping the amount of leakage of the compound of formula (I) from the liposome sufficiently low.

[0046] Liposomes typically have a size in the micron or submicron range and are well recognized for their ability to hold pharmaceutical substances, including anticancer drugs such as irinotecan, and to alter their pharmaceutical properties in various beneficial ways. Methods for preparing and characterizing pharmaceutical liposome compositions are known in the art (e.g., Lasic D. Liposomes: From physics to applications, Elsevier, Amsterdam 1993; G. Greroriadis (ed.), Liposome Technology, 3 rd See edition, vol.1-3, CRC Press, Boca Raton, 2006; Hong et al., US Pat.8, 147, 867 (the aforementioned document is incorporated herein by reference in its entirety for all purposes).

[0047] In some examples (e.g., Example 7), the ATR protein kinase inhibitor composition may include liposomes containing the ATR protein kinase inhibitor compound encapsulated in liposomes with a polyanion such as a polysulfated sugar (e.g., sucrose octasulfate). Sucrose phosphate, a fully substituted sulfate ester of sucrose, has the following structure in its fully protonated form: [ka] Sucrosofate is also known as sucrose octasulfate or sucrose octasulfate (SOS). Methods for preparing scrosofate in various salt forms, such as ammonium salts, sodium salts, or potassium salts, are well known in the art (e.g., US Pat. 4,990,610, which is incorporated herein by reference in its entirety).

[0048] ATR protein kinase inhibitor liposomes can be prepared in a multi-step process, comprising forming liposomes containing TEA and then filling the liposomes with an ATR protein kinase inhibitor compound (e.g., compound A or compound of formula (I)) as the TEA leaves the liposomes. For example, ATR protein kinase inhibitor liposomes can be prepared by a process comprising (a) preparing liposomes containing triethylamine (TEA) as the triethylammonium salt of sucrose (TEA-SOS), and (b) subsequently contacting the TEA-SOS liposomes with irinotecan under conditions effective in allowing irinotecan to enter the liposomes and a corresponding amount of TEA to leave the liposomes (thus consuming or reducing the resulting concentration gradient of TEA in the liposomes).

[0049] The first step may include forming liposomes containing TEA-sucrose phosphate by hydrating and dispersing liposomal lipids in a solution of TEA-sucrose phosphate. This may involve, for example, dissolving lipids containing HSPC and cholesterol in heated ethanol, and heating the dissolved and heated lipid solution to the transition temperature (T) of the liposomal lipids. mThis can be done by dispersing in an aqueous TEA-sucrosephate solution at a temperature exceeding 5°C, for example, above 60°C. The lipid dispersion can be formed into liposomes having an average size of 75–125 nm (e.g., 80–120 nm, or in some embodiments, 90–115 nm) by extrusion through a track-etched polycarbonate membrane having a specified pore size, for example, 100 nm. The TEA-sucrosephate solution can be obtained containing at least 8 molar equivalents of TEA per molar equivalent of sucrosephate, with a concentration of about 0.40–0.50 N and a pH (e.g., about 6.5) selected to prevent unacceptable degradation of liposomal phospholipids during the dispersion and extrusion steps (e.g., a pH selected to minimize degradation of liposomal phospholipids during these steps). Uncaptured TEA-SOS can then be removed from the liposomal dispersion before drug encapsulation, for example, by dialysis, gel chromatography, ion exchange, or ultrafiltration. The resulting liposomes may contain the ATR protein kinase inhibitor sucrosephate. These ATR inhibitor liposomes can be stabilized by filling the liposomes with enough drug to reduce the amount of TEA in the resulting liposome composition to below a predetermined maximum level after 180 days at 4°C, or to a level that produces lysoPC formation below a predetermined maximum level of lysoPC accumulation rate in the liposome composition during storage in a refrigerator, measured at approximately 4°C, more commonly 5±3°C, for example, mg / mL / month, or by the rate of PC-to-lysoPC conversion (%) over a unit time, for example, mol% lysoPC / month. The TEA, which is exchanged from the liposomes to an external medium during the filling process, is then removed from the liposomes along with any uncaptured ATR inhibitor, usually by any suitable known method (e.g., gel chromatography, dialysis, diafiltration, ion exchange, or ultrafiltration). The liposome external medium can be replaced with an injectable isotonic solution buffered at a desired pH (e.g., an isotonic solution of sodium chloride).

[0050] The antitumor effects of various liposomal formulations containing liposome-encapsulated ATR protein kinase inhibitor compounds were tested in various lung cancer cell lines, including human cervical cancer cell lines (e.g., MS751, C33A, and SiHa cell lines shown in Example 9), lung squamous cell carcinoma cell lines (e.g., NCI-H2170 cell line in Example 10), small cell lung cancer cell lines (e.g., DMS-114 cell line in Example 10), and human Calu-6 and COLO-699 cell lines (Example 11).

[0051] As shown in Figures 6-9 and Example 9, in a mouse xenograft model (Example 9A), the liposomal formulation of ATR inhibitor compound A (Example 7) was tested alone and in combination with the irinotecan liposomal formulation MM398 (Example 9B) against three human cervical cancer cell lines. Compared to control experiments with two of the three cervical cancer cell lines (MS571 and C33A), the liposomal compound A formulation of Example 7 showed a larger tumor volume over time. However, when irinotecan liposomal MM398 (Example 9B) was administered in combination with the compound A liposomal formulation (Example 7), the tumor volume of the three cervical cancer cell lines was significantly suppressed compared to the administration of MM398 alone or liposomal compound A alone.

[0052] As shown in Figures 10A-10B and 11A-11B, liposomal formulations of ATR inhibitor compound 5 of formula (I) and formula (Ia) (compound from Example 1 formulated as liposomes, as described in Example 7) were tested alone and in combination with irinotecan liposomal formulation MM398 (Example 9B) in two lung cancer cell lines of a mouse xenograft model (Example 10). As shown in Figures 10A and 10B, administration of the liposomal formulation of compound 5 reduced tumor volume in each cell line tested compared to the control experiment in Example 10, and the combination of MM398 and the compound 5 liposomal composition from Example 7 significantly reduced tumor volume in the mouse model compared to when either compound was administered independently of the other. Similarly, the Kaplan-Meier survival curves shown in Example 10 (Figures 11A and 11B) demonstrate an increase in survival rate in a mouse lung cancer xenograft study when irinotecan liposome MM398 from Example 9B and compound 5 liposome formulation from Example 7 were administered in combination using two different cell lines.

[0053] As shown in Figures 12A and 12B, the tolerability of various liposomal formulations of ATR protein kinase inhibitor compounds was evaluated in Example 10. As shown in Figure 12A, the decrease in mouse body weight tested in the NCI-H2170 mouse xenograft model was lowest over time with the liposomal formulation of compound 5 (Example 7) compared with irinotecan liposome MM398 (Example 9B), the control, or the combined liposomal formulation of compound 5 and MM398. As shown in Figure 12B, the decrease in mouse body weight tested in the DMS-114 mouse xenograft model was lowest over time with the combined liposomal formulation of compound 5 and MM398 compared with the liposomal formulation of compound 5 (Example 7) or irinotecan liposome MM398 administered independently (Example 9B).

[0054] As shown in Figures 13A and 13B, when irinotecan liposome MM398 (Example 9B) and the liposomal formulation of ATR protein kinase inhibitor compound 5 were administered in combination, the tumor volume was reduced most significantly in both the Calu-6 and COLO699 mouse xenograft models compared to the control, MM398 irinotecan liposome administration alone, administration of compound A liposomal formulation (Example 7), or the combination of MM398 irinotecan liposome (Example 9B) and compound A liposomal formulation (Example 7). [Examples]

[0055] The following examples illustrate several embodiments of the present invention. The following examples and preparations are provided to enable those skilled in the art to better understand and implement these and other embodiments of the present invention. These should be considered merely illustrative and representative, and not limiting the scope of the present invention.

[0056] ATR peptides can be expressed and isolated using various methods known in the literature (see, for example, Unsal-Kacmaz et al, PNAS 99:10, pp. 6673-6678, May 14, 2002; Kumagai et al. Cell 124, pp. 943-955, Mar. 10, 2006; Unsal-Kacmaz et al. Molecular and Cellular Biology, February 2004, pp. 1292-1300; and Hall-Jackson et al. Oncogene 1999, 18, 6707-6713).

[0057] Compound A can be obtained by the method disclosed in WO2010 / 071827A1 (published June 24, 2010), which is incorporated herein by reference by the portion relating to the synthesis and use of Compound II-A-7. The structure of Compound A is as follows:

[0058] [ka]

[0059] Various compounds of formula (I) were prepared as described herein and are summarized in the following table.

[0060] [Table 1]

[0061] Examples 1, 2, 3, and 6 were prepared by a one-pot Suzuki cross-coupling method using in-situ generated boronic acid esters, as shown in Scheme 1 of Figure 1. As shown in Figure 1, the synthesis of intermediate 3:1-bromo-4-(2-bromoethylsulfonyl)benzene can be obtained as follows.

[0062] [ka]

[0063] To a solution of intermediate 2 (35 g, 133 mmol) in DCM (400 mL), PBr3 (40 g, 146 mmol) was added dropwise at 0°C. The mixture was then stirred overnight at room temperature. Water (15 mL) was added to quench the reaction. The resulting mixture was then washed with water (120 mL) and brine (120 mL). The organic phase was concentrated to obtain 20 g of crude product 3 as a yellow oily substance, which was used in the next step without further purification.

[0064] As shown in Figure 1, the synthesis of intermediate 2 can be carried out as follows:

[0065] [ka]

[0066] To a solution of intermediate 1 (45 g, 194 mmol) in DCM (500 ml), m-CPBA (134 g, 776 mmol) was added in several batches at room temperature. The mixture was then stirred overnight at room temperature. The reaction mixture was filtered, and the solid was washed by adding DCM (500 ml). The filtrate was washed with NaOH (1 M, 300 mL x 3) and brine (300 mL). The organic layer was concentrated to dryness to obtain 36 g of 2 (70%) as a white solid.

[0067] As shown in Figure 1, the synthesis of intermediate 1 can be carried out as follows.

[0068] [ka]

[0069] To a solution of 4-bromobenzenethiol (45 g, 238 mmol) in MeCN (600 ml), K2CO3 (60 g, 476 mmol) and NaI (36 g, 238 mmol) were added. The mixture was stirred at room temperature for 10 minutes. Next, 2-bromoethanol was added dropwise. After the addition, the mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the filtrate was concentrated to dryness. The residue was purified by silica gel column chromatography to obtain 1 (81%) of 45 g as a pale yellow oily substance.

[0070] Block B can be prepared according to Scheme 2 in Figure 2. As shown in Figure 2, the synthesis of Block B can be carried out as follows.

[0071] [ka]

[0072] To a solution of intermediate 6 (6.0 g, 27.4 mmol) in DMSO (30 mL), CDI (8.9 g, 54.8 mmol), DIPEA (3.8 g, 30.1 mmol), and DMAP (0.17 g, 1.37 mmol) were added. The solution was stirred at room temperature for 4 hours. Aniline (2.5 g, 27.4 mmol) was added, and the mixture was stirred at room temperature overnight. Water was added, and the resulting solid was collected by filtration. The crude product was purified by silica gel column chromatography to obtain 2.5 g of block B (31%) as a yellow solid.

[0073] LC-MS (M+1): 293.2; 1 H NMR (400 MHz, DMSO-d6) δ10.28 (s, 1H), 8.42 (s, 1H), 7.78 (d, J = 8.0 Hz, 2H), 7.74 (s, 2H), 7.36 (t, J = 8.0 Hz, 2H), 7.13 (t, J = 7.6 Hz, 1H).

[0074] As shown again in Figure 2, the synthesis of intermediate 6 can be carried out as follows.

[0075] [ka]

[0076] A solution of methyl 3-amino-6-bromopyrazine-2-carboxylate (10.0 g, 43.1 mmol) in MeOH (70 mL) was added to a solution of LiOH (9.0 g, 215 mmol) in water (70 mL). The mixture was stirred at 90°C for 3 hours. The reaction mixture was cooled to room temperature and acidified to pH 4-5 with HCl (2 M). The mixture was filtered to obtain 7.4 g of 6 (79%) as a yellow solid.

[0077] LC-MS (M+1): 218.0; 1H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.59 (br, 2H).

[0078] Example 1: Synthesis of compound 5(3-amino-6-(4-((2-(4-(2-((dimethylamino)ethyl)piperidine-1-yl)ethyl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide

[0079] [ka]

[0080] Exact mass: 536.26; Molecular weight: 536.70; Compound 5; More basic; 143 mg; Yield 8.2%; pK a 10.00.

[0081] To a solution of 2-(1-(2-((4-bromophenyl)sulfonyl)ethyl)piperidine-4-yl)-N,N-dimethylethane-1-amine (block A1) (261 mg, 0.648 mmol) in anhydrous dioxane (3 ml), potassium acetate (191 mg, 1.944 mmol) and bis(pinacolato)diborane (246 mg, 0.971 mmol) were added. The reaction vessel was degassed by repeating the vacuum / nitrogen cycle, and then Pd(dppf)2Cl2 was added. CH2Cl2 (53 mg, 0.0648 mmol) was degassed again, and the reaction mixture was heated under nitrogen at 90°C for 2 hours. The reaction mixture was then cooled to room temperature, 3-amino-6-bromo-N-phenylpyrazine-2-carboxamide (block B) and 2M K2CO3 (1 ml) were added, the mixture was degassed, and the mixture was purged with nitrogen. Pd(PPh3)4 (75 mg, 0.0648 mmol) was added. The reaction mixture was heated at 100°C for 4 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate, washed three times with brine, and the organic layer was dried over Na2SO4. After removing the solvent with a rotary evaporator, a dark oily residue was obtained, which was purified by silica gel column chromatography (Reveleris Flash Chromatography System) using 0-15% methanol in dichloromethane as the eluent. The desired product was obtained as a yellow solid (149 mg, 43% yield). MS (M+H)+ 537; 1H NMR (400MHz, DMSO-d6): δ 10.45 (s, 1H), 9.03 (s, 1H), 8.49 (d, 2H, 6.8Hz), 7.95 (d, 2H, 6.8Hz), 7.88 (s, br, 2H), 7.81 (d, 2H, 8.8Hz), 7.40 (t, 2H, 7.2Hz), 7.18( t, 1H, 7.2Hz), 3.53 (t, 2H, 7.2Hz), 2.64 (d, 2H, 11.6 Hz), 2.55 (t, 2H, 7.2Hz), 2.05 (m, 2H), 1.98 (s, 6H), 1.17 (m, 2H), 1.42 (d, 2H, 12.0Hz), 1.18 (m, 3H), 0.78 (m, 2H).

[0082] High-resolution mass (Thermo Scientific(TM) Q Exactive(TM) hybrid quadrupole Orbitrap mass spectrometer): Calculated C 28 H 36 N6O3S + proton (1.00728) = 537.2642; Theoretical m / z value for a single charged ion: 537.2642; Measured value: 537.2636.

[0083] Example 2: Synthesis of compound 6(3-amino-6-(4-((2-(4-(diethylamino)piperidine-1-yl)ethyl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide

[0084] [ka]

[0085] Exact mass: 536.26; Molecular weight: 536.70; Compound 6; More basic; 53 mg; Yield 11.1%; pK a 9.81.

[0086] Example 2 was prepared in the same manner using Block A2(1-(2-((4-bromophenyl)sulfonyl)ethyl)-N,N-diethylpiperidine-4-amine to obtain a yellow solid (52 mg, 24% yield). MS (M+H)+ 537; 1 H NMR (400MHz, DMSO-d6): δ 10.45 (s, 1H), 9.05 (s, 1H), 8.51 (d, 2H, 8.4Hz), 7.94 (d, 2H, 8.8Hz), 7.87 (s, br, 2H), 7.79 (d, 2H, 8.8Hz), 7.42 (t, 2H, 8.4Hz), 7.18( t, 1H, 7.2Hz), 3.54 (t, 2H, 6.4Hz), 2.65 (d, 2H, 11.2 Hz), 2.56 (t, 2H, 6.4Hz), 2.20 (q, 4H, 6.8 Hz), 1.71 (t, 2, 10.4 Hz), 1.33 (d, 2H, 12.4Hz), 0.85 (qd, 2H, 12.4Hz), 0.74(t, 6H, 7.2Hz).

[0087] 1-(2-((4-bromophenyl)sulfonyl)ethyl)-N,N-diethylpiperidine-4-amine (block A2) was prepared using the same method with the corresponding 4-diethylaminopiperidine. A colorless oil was obtained (661 mg, yield 47%), MS(M+H)+ 403, 405.

[0088] Example 3: Synthesis of Compound 2(3-amino-6-(4-((2-(diethylamino)ethyl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide

[0089] [ka]

[0090] Exact mass: 453.18; Molecular weight: 453.56; Compound 2; Less basic; 98 mg; Yield 7.7%; pK a 7.46.

[0091] The compound of Example 3 was prepared in the same manner using 2-((4-bromophenyl)sulfonyl)-N,N-diethylethane-1-amine from intermediate block A3, yielding a yellow solid (98 mg, yield 11%). MS (M+H)+ 454; 1 H NMR (400MHz, DMSO-d6): δ 10.46 (s, 1H), 9.05 (s, 1H), 8.51 (d, 2H, 6.8Hz), 7.98 (d, 2H, 6.8Hz), 7.88 (s, br, 2H), 7.81 (d, 2H, 8.8Hz), 7.41 (t, 2H, 7.2Hz), 7.17( t, 1H, 7.2Hz), 3.48 (dd, 2H, 6.8Hz), 2.73 (m, 2H), 2.33 (q, 4H, 6.8Hz), 0.81 (t, 6H, 6.8Hz).

[0092] Block A3 (2-((4-bromophenyl)sulfonyl)-N,N-diethylethane-1-amine) was prepared using the corresponding 4-diethylamine in the same manner. A colorless oil was obtained (1.42 g, 73% yield), MS (M+H)+ 320, 322.

[0093] Example 4: Compound 4(3-amino-6-(4-(((2-(dimethylamino)ethyl)-λ 2 Synthesis of azanyl(sulfonyl)phenyl(-N-phenylpyrazine-2-carboxamide)

[0094] [ka]

[0095] Exact mass: 439.16; Molecular weight: 439.51; Compound 4; pK a 8.36.

[0096] Compound 4 can be prepared as shown in Scheme 3 of Figure 3. Block B (150 mg, 0.51 mmol), Block F (153 mg, 0.56 mmol), and Na2CO3 (216 mg, 2.0 mmol) were added to toluene / ethanol / water (2 mL / 2 mL / 2 mL), to which Pd(dppf)Cl2 (30 mg) was added. The mixture was stirred under an argon atmosphere at 75°C for 4 hours. The reaction mixture was concentrated to dryness. The residue was purified by preparative HPLC to obtain 100 mg of TM4 (45%) as a white solid.

[0097] LC-MS (M+1): 441.4; 1 H NMR (400 MHz, CD3OD) δ 8.88 (s, 1H), 8.33 (dd, J = 6.8 Hz, 1.6 Hz, 2H), 8.00 (dd, J = 6.8 Hz, 1.6 Hz, 2H), 7.80 (dd, J = 8.4 Hz, 1.2 Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.19 (t, J = 7.6 Hz, 1H), 3.10 (t, J = 6.4 Hz, 2H), 2.73 (t, J = 6.4 Hz, 2H), 2.46 (s, 6H).

[0098] Again, as shown in Scheme 3 of Figure 3, the synthesis of block F can be carried out as follows.

[0099] [ka]

[0100] To a solution of intermediate 7 (10.0 g, 32.6 mmol) in THF (200 mL), B(i-Pr)3 (30.6 g, 163 mmol) was added under an argon atmosphere at -78 °C. Then, n-BuLi (2.5 M, 65 mL) was added dropwise. The mixture was stirred at -78 °C for 2 hours, then at room temperature for a further 16 hours. Water was added to stop the reaction. The mixture was concentrated to dryness. The residue was purified by preparative HPLC to obtain 5.2 g of block F (59%) as a white solid.

[0101] LC-MS (M+1): 273.4; 1 H NMR (400 MHz, DMSO-d6) δ 7.90-7.45 (m, 4H), 2.91 (s, 2H), 2.69 (t, J = 6.4 Hz, 2H), 2.18 (t, J = 6.8 Hz, 2H), 2.03 (s, 6H).

[0102] Again, as shown in Scheme 3 of Figure 3, the synthesis of intermediate 7 can be carried out as follows.

[0103] [ka]

[0104] To a solution of 4-bromobenzene-1-sulfonyl chloride (20 g, 78.3 mmol) in 300 mL of DCM at 0°C, TEA (22 mL, 158 mmol) was added, followed by N,N′-dimethylethane-1,2-diamine (8.3 g, 94.0 mmol). The resulting solution was stirred at room temperature for 1 hour and then diluted with 300 mL of DCM. The solution was washed with water (200 mL) and brine (200 mL). The organic layer was concentrated to dryness. The residue was purified by silica gel column chromatography to obtain 17.0 g of 7 (71%) as an off-white solid.

[0105] Example 5: Synthesis of compound 3(3-amino-6-(4-((2-(4-methylpiperazine-1-yl)ethyl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide

[0106] [ka]

[0107] Exact mass: 480.19; Molecular weight: 480.59; Compound 3; pK a 7.73.

[0108] The compound of Example 5 can be obtained by scheme 4 shown in Figure 4. Intermediate 5 of scheme 4 can be obtained as follows. [ka]

[0109] Under an argon atmosphere, B(i-Pr)3 (4.7g, 25mmol) was added to a solution of intermediate 4 (1.7g, 5.0mmol) in THF (30mL) at -78°C. Then, n-BuLi (2.5M, 10mL) was added dropwise. The mixture was stirred at -78°C for 2 hours, then at room temperature for a further 16 hours. The reaction was quenched by adding water. The mixture was concentrated to dryness. The residue was purified by preparative HPLC to obtain 300mg of 5 (19%) as a white solid.

[0110] As shown again in Figure 4, the intermediate 4 of scheme 4 can be obtained as follows.

[0111] [ka]

[0112] To a solution of intermediate 3 (20 g, 60 mmol) in MeCN (300 mL), 1-methylpiperazine (9.0 g, 90 mmol) and K2CO3 (16.6 g, 120 mmol) were added. The mixture was stirred overnight at room temperature. The reaction mixture was filtered, and the filtrate was concentrated to dryness. The residue was purified by silica gel column chromatography to obtain 15 g of 4 (71%) as a pale solid.

[0113] Example 6: Synthesis of Compound 1 (3-amino-6-(4-((1-(dimethylamino)propane-2-yl)sulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide

[0114] [ka]

[0115] Exact mass: 439.17; Molecular weight: 439.53; Compound 1; Less basic; 427 mg; Yield 12.9%; pK a 7.04.

[0116] The compound of Example 6 was prepared according to the preparation procedure for Compound 1 described in J.Med.Chem.2011, 54, 2320 (Supplementary Material), except for the use of 1-bromo-4-(2-bromoethylsulfonyl)benzene. A yellow solid was obtained (427 mg, yield 11%). MS (M+H)+ 440; 1 H NMR (400MHz, DMSO-d6): δ 10.46 (s, 1H), 9.05 (s, 1H), 8.52 (d, 2H, 6.8Hz), 7.93 (d, 2H, 6.8Hz), 7.89 (s, br, 2H), 7.82 (d, 2H, 8.0Hz), 7.40 (t, 2H, 7.2Hz), 7.18( t, 1H, 7.2Hz), 3.53 (t, 2H, 7.2Hz), 2.64 (d, 2H, 11.6 Hz), 2.55 (t, 2H, 7.2Hz), 2.05 (m, 2H), 1.98 (s, 6H), 1.17 (m, 2H), 1.42 (d, 2H, 12.0Hz), 1.18 (m, 3H), 0.78 (m, 2H).

[0117] Example 7: Preparation of liposomes containing encapsulated triethylammonium SOS salt and filling of liposomes with ATRi Sucrose octasulfate (equivalent 144.8) is the sodium salt of a sucrose derivative in which all hydroxyl groups form sulfate esters. 60 g of sodium sucrose octasulfate (SOS) was dissolved in 150 ml of deionized water and heated in a 50°C water bath with shaking (swirling). This solution was passed through a column packed with sulfonated polystyrene-divinylbenzene copolymer cation exchange resin beads (Dowex 50Wx8-100-200 mesh, Dow Chemical Co.). This column was pre-equilibrated with a 3-3.6 M aqueous HCl solution to convert the resin into hydrogen, and washed with deionized water until the eluate showed conductivity <1 μS / cm. The eluent was monitored using a conductivity detector. The SOS fraction corresponding to the conductivity peak was collected and immediately titrated to pH 6-6.5 with pure triethylamine (TEA) solution. Residual sodium was analyzed in this solution by potentiometric measurement using a sodium-sensitive electrode, and the SOS concentration was analyzed using a refractometer. A solution containing less than 0.25% residual sodium was diluted with deionized water to a final concentration of 1.1 M of SOS, and then sterile filtered using a Millipore 0.22 μm Steri-Top filter.

[0118] Cholesterol (Chol) was purchased from Avanti Polar Lipids, Alabaster, Ala, USA. Hydrogenated soy phosphocholine (HSPC) and methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glyceryl (PEG2000-DSG) were obtained from Lipoid GmbH, Ludwigshafen, Germany.

[0119] Chol, HSPC, and PEG2000-DSG were simultaneously dissolved in 100% ethanol (200 proof, Sigma catalog number: 459828) at 65°C in a molar ratio of 3:2:0.15. A solution of TEA-SOS (10 times the volume of the added ethanol) was mixed with the lipid solution at 60-65°C and stirred at this temperature until a uniform emulsion suspension of multilayer vesicles was formed. This suspension was extruded three times at 60-65°C using an argon pressure extruder (Lipex Biomembranes) through a five-layer polycarbonate track etching filter (Corning Nuclepore) with a pore size of 100 nm. The resulting monolayer liposomes were rapidly cooled on ice and stored at 4-6°C before use. Phospholipid concentrations were measured by phosphate assay, and particle size was recorded using a Malvern Nanosizer.

[0120] Before drug loading, a TEA-SOS gradient was created by removing excess uncaptured TEA-SOS using gel chromatography (Sepharose CL-4B, Pharmacia). The osmotic pressure of the liposomes was equilibrated using a 50% dextrose solution. The final dextrose concentration was 15%.

[0121] ATR inhibitors were dissolved in a 15% dextrose solution in deionized water by titration with 1M HCl and heating at 45°C, and then filtered through a 0.2 μm NALGENE 13 mm syringe filter. The drug concentration in the solution was detected by HPLC. A stock solution of ATR inhibitors containing 9-10 mg / mL of drug was added to liposomes to achieve a drug / lipid ratio of 800 mg / mmol phospholipid, and the pH was adjusted to 6.5 with 1M Hepes buffer and 0.1N NaOH.

[0122] The liposome-drug mixture was incubated at 65°C for 30 minutes with occasional agitation. The incubation mixture was rapidly cooled and incubated at 0°C for 10 minutes, then allowed to reach ambient temperature. Uncaptured drugs were removed by gel chromatography on Sephadex G-25 (Amersham Pharmacia) eluted with HBS-6.5 buffer (5 mM 2-(4-(2-hydroxyethyl)-piperazino)-ethylsulfonic acid (HEPES), 144 mM NaCl, pH 6.5). The eluted liposome fractions were combined by void volume, sterilized by 0.2 μm filtration, and stored at 4–6°C before use. The liposomes were characterized by lipid concentration, drug concentration, and particle size (Table 2). All ATR inhibitors showed good packing efficacy, with the exception of compound 1, which formed aggregates and precipitated at drug / lipid ratios higher than 400 g / mol.

[0123] [Table 2]

[0124] Example 8: General description of PK testing of liposome ATRi Figure 5 is a graph showing the hemopharmacological effects of liposomal ATR inhibitors. A liposomal formulation of the ATR inhibitor was prepared as described in Example 7. The liposomes were intravenously administered at a dose of 20 mg / kg to three 7-9 week old female CD-1 mice (Charles River) (approximately 25 g body weight). Blood samples were collected in lithium heparin tubes by bleeding from the saphenous vein at 0.08, 1.5, 4, 8, and 24 hours. Plasma was separated from the cell fraction by centrifugation at 10,000 rpm for 5 minutes. The drug was extracted by incubating the plasma sample at -80°C for at least 2 hours in the presence of 200 μl of 1% acidic acid (1% Ac / MeOH) in methanol. Plasma proteins were spun down by centrifugation at 15,000 rpm for 20 minutes. 75 μl of supernatant was transferred to an HPLC vial (Thermo Scientific, catalog number C4011-LV1), and another 75 μl of 1% Ac / MeOH was added. Drug content was analyzed by HPLC, and each sample was measured twice. The data are expressed as the injectable dose (%) plotted against time after injection. As shown in Figure 5, the liposomal formulations of compound 1, compound 3, and compound 2 were unstable in the cardiovascular system. Liposomal compounds 1 and 3 were undetectable by HPLC analysis at 24 hours, and liposomal compound 2 was already undetectable at 8 hours. The two liposomal formulations, compound 6 and compound 5, exceeded 16% of the initial injectable dose at 24 hours, indicating a good lifetime in the cardiovascular system. Table 2 below summarizes the blood PK curves. Liposomal compounds 6 and 5 exhibit the highest plasma half-lives compared to the other liposome examples.

[0125] [Table 3]

[0126] Example 9A: In vivo antitumor efficacy and tolerability of LS compound A prepared using TEA.SOS against mouse cervical cancer xenografts. Figure 6 is a graph showing the antitumor efficacy of liposomal compound A when used in combination with MM-398 in the cervical MS571 xenograft model described in Example 9A.

[0127] Figure 7 is a graph showing the antitumor efficacy of liposomal compound A when used in combination with MM-398 in the cervical C33A xenograft model described in Example 9A.

[0128] Figure 8 is a graph showing the antitumor efficacy of liposomal compound A when used in combination with MM-398 in a cervical C33A xenograft model according to Example 9A.

[0129] Figure 9 is a graph showing the tolerability of liposomal compound A when used in combination with MM-398 in cervical MS751 according to Example 9A.

[0130] The antitumor efficacy of liposomes filled with ATR inhibitor compound A (Ls compound A) in combination with MM-398 (liposomal irinotecan) was tested in human cervical MS751, C33A, and SiHa cell line models. Cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in RPMI medium supplemented with 10% fetal bovine serum, 50 U / mL penicillin G, and 50 μg / mL streptomycin sulfate at 37°C and 5% CO2, according to the supplier's recommendations. NCR nu / nu homozygous athymogenic male nude mice (4-5 weeks old, at least 16 g body weight) were obtained from Charles River. Mice were subcutaneously inoculated into the right flank with a 0.1 mL suspension containing 5 × 10⁶ cells suspended in PBS supplemented with 30% Matrigel. The tumor was 150 mm 3 ~350mm 3Upon reaching a certain size, the animals were assigned to treatment groups according to the following method: The animals were ranked by tumor size and divided into six categories based on tumor size reduction. By randomly selecting one animal from each size category, four treatment groups of 10 animals / group were formed, thereby ensuring that all tumor sizes were represented equally within each treatment group.

[0131] The animals were given four tail vein injections of the following preparations at 7-day intervals: 1) control (HEPES buffered saline pH 6.5); 2) MM-398 at a dose of 2 or 5 mg / kg per injection; 3) liposomal compound A at a dose of 20 or 60 mg / kg per injection; 4) MM-398 followed by injection of liposomal compound A at 24-hour intervals. Injectable liposomes were prepared as described in Example 7. The animals' body weight and tumor size were monitored twice weekly. Tumor progression was monitored twice weekly by palpation and caliper measurement of the tumor along the maximum (length) and minimum (width) axes. Tumor size was expressed using the formula (Geran, RI, et al., 1972 Cancer Chemother. Rep. 3: 1-88): Tumor volume = [(length) × (width)] 2 ] / 2 Measurements were taken twice a week using caliper measurements.

[0132] To assess the toxicity associated with the treatment, animal body weight was measured twice a week. Animals were observed for 60 days after tumor inoculation. Animals in a group were euthanized when the tumor volume reached 10% of the mouse's body weight. The mean tumor volume across all groups was plotted together and compared over time. As shown in Figures 6, 7, and 8, the combination of liposomal ATR inhibitor compound A and MM-398 had a significantly potent antitumor effect compared to MM-398 and liposomal compound A alone in all three xenograft models. Treatment-related toxicity was assessed by the dynamics of animal body weight (Figure 9). No significant toxicity was observed in any group. Animal body weight in all treatment groups was comparable to that of the control group and consistently increased. Therefore, the liposomal formulation of ATR inhibitor compound A showed increased antitumor activity in the tested tumor models without significantly increasing toxicity.

[0133] Example 9B: Production of MM-398 irinotecan liposomes MM398, as used in Example 9A and elsewhere in this specification, is an irinotecan liposome that can be prepared by a multi-step process. First, the lipids are dissolved in heated ethanol. The lipids may include DSPC, cholesterol, and MPEG-2000-DSPE mixed in a molar ratio of 3:2:0.015. Preferably, the liposomes can be encapsulated by encapsulating irinotecansucrose octasulfate (SOS) in vesicles consisting of DSPC, cholesterol, and MPEG-2000-DSPE mixed in a molar ratio of 3:2:0.015. The resulting ethanol-lipid solution is dispersed in an aqueous medium containing substituted amines and polyanions under conditions effective for forming essentially monolayer liposomes of a suitable size (e.g., 80-120 nm) containing substituted amines (ammonium type) and polyanions encapsulated in vesicles formed from the dissolved lipids. Dispersion can be carried out, for example, by mixing an aqueous solution containing a substituted amine and a polyanion with an ethanol-lipid solution at a temperature above the lipid transition temperature, e.g., 60-70°C, and extruding the resulting hydrated lipid suspension (multilayer liposomes) under pressure through a one or more track-etched membrane filter, e.g., made of polycarbonate, having specified pore sizes, e.g., 50 nm, 80 nm, 100 nm, or 200 nm. The substituted amine may be triethylamine (TEA), and the polyanion may be sucrose octasulfate (SOS) mixed in a stoichiometric ratio (e.g., TEA8SOS) at a concentration of about 0.4-0.5 N. Then, all or substantially all uncaptured TEA or SOS is removed (e.g., by gel filtration, dialysis, or ultrafiltration), and the liposomes are then contacted with irinotecan under conditions effective in enabling encapsulation of irinotecan in the liposomes in exchange for any remaining TEA.These conditions may include one or more conditions selected from the group consisting of: adding an osmotic agent (e.g., 5% dextrose) to the liposome external medium to equilibrate the osmotic pressure of the captured TEA-SOS solution and / or prevent liposome rupture due to osmotic pressure during filling; adjusting and / or selecting the pH (e.g., to 6.5) to reduce drug and / or lipid degradation during the filling process; and raising the temperature above the liposomal lipid transition temperature (e.g., up to 60-70°C) to promote intermembrane exchange between TEA and irinotecan. Filling with irinotecan by exchange with TEA across the liposome preferably continues until all or substantially all of the TEA is removed from the liposome, thereby depleting its concentration gradient across the liposome. Preferably, the liposome filling process with irinotecan is continued as long as the gram equivalent ratio of irinotecan to sucrose octasulfate is at least 0.9, at least 0.95, 0.98, 0.99, or 1.0 (or in the range of about 0.9 to 1.0, 0.95 to 1.0, 0.98 to 1.0, or 0.99 to 1.0). Preferably, the liposome filling process with irinotecan is continued until at least 90%, at least 95%, at least 98%, at least 99%, or more of the TEA is removed from inside the liposome. Irinotecan can form irinotecan sucrose phosphate inside the liposome, for example, with irinotecan and sucrose octasulfate in a molar ratio of about 8:1. Next, the remaining extraliposomal irinotecan and TEA are removed using gel (size exclusion) chromatography, dialysis, ion exchange, or ultrafiltration to obtain irinotecan liposomes. The liposomal outer medium is replaced with an injectable, pharmacologically acceptable fluid, such as buffered isotonic saline. Finally, the liposomal composition is sterilized, for example by filtration at 0.2 μm, dispensed into administration vials, labeled, and stored in a refrigerator at 2-8°C until use. The liposomal outer medium can be replaced with a pharmacologically acceptable fluid at the same time as the removal of the remaining extraliposomal irinotecan and TEA.The extraliposome pH of the composition can be adjusted or selected to provide desired storage stability (e.g., reduced intraliposome lysoPC formation during storage for more than 180 days at 4°C) by preparing the composition at, for example, a pH of about 6.5 to 8.0 or any suitable pH value in between (e.g., including 7.0 to 8.0 and 7.25).

[0134] DSPC, cholesterol (Chol), and PEG-DSPE were weighed in amounts corresponding to a molar ratio of 3:2:0.015 (e.g., 1264 mg / 412.5 mg / 22.44 mg). The lipids were dissolved in chloroform / methanol (4 / 1 v / v), thoroughly mixed, and divided into four aliquots (A-D). Each sample was evaporated to dryness at 60°C using a rotary evaporator. Residual chloroform was removed from the lipids by standing under reduced pressure (180 μTor) at room temperature for 12 hours. The dried lipids were dissolved in ethanol at 60°C, and preheated TEA8SOS of an appropriate concentration was added to achieve a final alcohol content of 10% (v / v). The lipid concentration was 75 mM. Liposomes with a typical average diameter of 95–115 nm (measured by quasi-elastic light scattering) were produced by extruding a lipid dispersion 10 times into a 2-layer 0.1 μm polycarbonate membrane (Nucleopore) at approximately 65°C using a Lipex thermobarrel extruder (Northern Lipids, Canada). The pH of the extruded liposomes was adjusted to pH 6.5 with 1N NaOH as needed. The liposomes were purified by a combination of ion exchange chromatography and size exclusion chromatography. First, DOWEX IRA910 resin was treated with 1N NaOH, followed by 3 washes with deionized water, 3 washes with 3N HCl, and then multiple washes with water. Liposomes were passed through the prepared resin, and the conductivity of the eluted fraction was measured using a flow cell conductivity meter (Pharmacia, Upsalla, Sweden). If the conductivity was less than 15 μS / cm, the fraction was considered acceptable for further purification. Next, the liposome eluate was applied to a Sephadex G-75 (Pharmacia) column equilibrated with deionized water, and the conductivity of the recovered liposome fraction was measured (typically less than 1 μS / cm). Intermembrane isotonicity was achieved by adding 40% dextrose solution to a final concentration of 5% (w / w) and buffer (Hepes) from the stock solution (0.5 M, pH 6.5) to a final concentration of 10 mM.

[0135] Considering the water content and impurity levels obtained from the analysis certificates for each batch, a stock solution of irinotecan was prepared by dissolving irinotecan-HCl trihydrate powder in deionized water in 15 mg / mL anhydrous irinotecan-HCl. Drug loading was initiated by adding irinotecan to 500 g / mol liposomal phospholipid and heating in a water bath to 60 ± 0.1°C for 30 minutes. The solution was rapidly cooled by immersion in ice water upon removal from the water bath. Extraliposomal drugs were removed by size exclusion chromatography using a Sephadex G75 column equilibrated and eluted with Hepes buffered saline (10 mM Hepes, 145 mM NaCl, pH 6.5). Samples were analyzed for irinotecan by HPLC and phosphate analysis using the Bartlett method (see phosphate analysis method).

[0136] A preferred example of the storage-stable irinotecan liposomes described herein is the product marketed as ONIVYDE (irinotecan liposome injection). ONIVYDE is a topoisomerase inhibitor formulated for intravenous use, containing irinotecan hydrochloride trihydrate in a liposome dispersion. ONIVYDE demonstrated the need for treatment of metastatic pancreatic adenocarcinoma after disease progression following gemcitabine-based therapy.

[0137] ONIVYDE is a storage-stabilized liposome with a pH of approximately 7.25. The ONIVYDE product contains irinotecan sculosphate, obtained from irinotecan hydrochloride trihydrate starting material, encapsulated in liposomes. The chemical name of irinotecan is (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3′,4′:6,7]-indolidino[1,2-b]quinoline-9-yl-[1,4′-bipiperidine]-1′-carboxylate. The dosage of ONIVYDE can be calculated based on the equivalent amount of irinotecan hydrochloride trihydrate starting material used to prepare the irinotecan liposomes, or based on the amount of irinotecan in the liposomes. Approximately 866 mg of irinotecan is present per gram of irinotecan hydrochloride trihydrate. For example, an 80 mg ONIVYDE dose based on the amount of irinotecan hydrochloride trihydrate starting material actually contains about 0.866 × (80 mg) of irinotecan in the final product (i.e., 80 mg / m³ based on the weight of the irinotecan hydrochloride starting material). 2 The dosage of ONIVYDE is approximately 70 mg / m² in the final product. 2(Equivalent to irinotecan). ONIVYDE is a sterile, white to slightly yellow, opaque, isotonic liposome dispersion. Each 10 mL single-dose vial contains 43 mg of irinotecan free base at a concentration of 4.3 mg / mL. The liposomes are monolayer lipid bilayer vesicles with a diameter of approximately 110 nm, encapsulating an aqueous space containing irinotecan as sucrose octasulfate in a gelled or precipitated state. The vesicles consist of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 6.81 mg / mL, cholesterol 2.22 mg / mL, and methoxy-terminated polyethylene glycol (molecular weight 2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) 0.12 mg / mL. Each mL also contains 4.05 mg / mL of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) as a buffer, and 8.42 mg / mL of sodium chloride as an isotonic reagent. Each ONIVYDE vial contains 43 mg / 10 mL of irinotecan free base as a white to slightly yellow opaque liposome dispersion in a single-dose vial.

[0138] In one example of the present invention, the ONIVYDE unit dosage form is a pharmaceutical composition containing a certain amount of irinotecan encapsulated in liposomes, with a dosage of 80 mg / m². 2 The total amount of irinotecan provided is approximately 70 mg / m², which is equivalent to the amount of irinotecan hydrochloride trihydrate. 2 The present invention provides irinotecan and lyso-PC in amounts less than approximately 20%. The unit dosage form may be an intravenous formulation with a total volume of approximately 500 mL. ONIVYDE is prepared for administration by diluting the isotonic liposome dispersion from the vial as follows: Collect the calculated amount of ONIVYDE from the vial. Dilute ONIVYDE with 500 mL of 5% dextrose injection, USP or 0.9% sodium chloride injection, USP, and mix the diluted solution by gentle inversion; protect the diluted solution from light and administer the diluted solution within 4 hours of preparation if stored at room temperature, or within 24 hours of preparation if stored under refrigerated conditions [2°C~8°C (36°F~46°F)].

[0139] The need for ONIVYDE (irinotecan liposome injection) in combination with 5-fluorouracil and leucovorin for the treatment of patients with metastatic adenocarcinoma of the pancreas progressing after gemcitabine-based therapy is shown. ONIVYDE is administered before the administration of leucovorin and fluorouracil. The recommended dose of ONIVYDE is 70 mg / m2 of irinotecan administered by intravenous infusion over 90 minutes every two weeks. UGT1A1 * The recommended starting dose of ONIVYDE in patients known to be homozygous for 28 alleles is 50 mg / m of irinotecan administered by intravenous infusion over 90 minutes 2 . The dose of ONIVYDE can be increased to 70 mg / m 2 in subsequent cycles as tolerated. There is no recommended dose of ONIVYDE for patients with serum bilirubin values exceeding the upper limit of normal. Dilute ONIVYDE in a solution and infuse it intravenously over 90 minutes.

[0140] As an appropriate treatment regimen, ONIVYDE 70 mg / m 2 is given every two weeks for 46 hours together with leucovorin 400 mg / m 2 (or active leucovorin l of 200 mg / m 2 ) and fluorouracil 2,400 mg / m 2 (ONIVYDE / 5-FU / LV; n = 117), ONIVYDE 100 mg / m 2 is given every three weeks (n = 147), or leucovorin 200 mg / m 2 and fluorouracil 2000 mg / m 2 are given every week for 24 hours for a total of four weeks and then a two-week drug holiday period (5-FU / LV; n = 134).

[0141] Example 10: In vivo antitumor efficacy and tolerance of Ls compound 5 prepared using TEA.SOS against mouse lung cancer xenografts Figures 10A and 10B are graphs showing the efficacy of liposomal compound 5 when used in combination with MM-398 in NCI-H2170 (Figure 10A) or DMS-114 (Figure 10B) mouse xenograft models, as described in Example 10.

[0142] Figures 11A and 11B are graphs showing the Kaplan-Meier survival curves illustrating the efficacy of liposomal compound 5 when used in combination with MM-398 in NCI-H2170 (Figure 11A) and DMS-114 (Figure 11B) mouse xenograft models, as described in Example 10.

[0143] Figures 12A and 12B are graphs showing the tolerability of liposomal compound 5 when used in combination with MM-398 in NCI-H2170 (Figure 12A) or DMS-114 (Figure 12B) mouse xenograft models.

[0144] The antitumor efficacy of liposomes filled with a combination of ATR inhibitor compound 5 and MM-398 (liposomal irinotecan) was tested in human NCI-H2170 (squamous cell carcinoma of the lung) and DMS-114 (small cell lung cancer) lung cell line models.

[0145] Cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in RPMI medium supplemented with 10% fetal bovine serum, 50 U / mL penicillin G, and 50 μg / mL streptomycin sulfate at 37°C under 5% CO2 conditions, as recommended by the supplier. NCR nu / nu homozygous athymogenic male nude mice (4-5 weeks old, at least 16 g body weight) were obtained from Charles River. 5 × 10⁶ cells were suspended in PBS supplemented with 30% Matrigel for the mice. 6 A 0.1 mL suspension containing cells was subcutaneously inoculated into the right flank. The tumor was 150 mm. 3 ~350mm 3When the size reached, the animals were assigned to treatment groups according to the following method. The animals were ranked by tumor size and divided into six categories of reduced tumor size. Four treatment groups consisting of 10 animals / group were formed by randomly selecting one animal from each size category, so that all tumor sizes were equally represented in each treatment group. Four intravenous injections of the following preparations were given to the animals at 7-day intervals: 1) control (HEPES buffered saline pH 6.5); 2) MM-398 at a dose of 5 mg / kg per injection; 3) liposomal compound 5 at a dose of 80 mg / kg per injection; 4) MM-398 followed by an injection of liposomal compound 5 at 24-hour intervals. The liposomes for injection were prepared as described in Example 7. MM-398 is described in Example 9B.

[0146] The body weight and tumor size of the animals were monitored twice a week. The progression of the tumors was monitored twice a week by palpation of the tumors and caliper measurements along the maximum (length) and minimum (width) axes. The tumor size was measured twice a week by caliper measurement using the formula: Tumor volume = [(length) × (width) 2 / 2 and was measured twice a week by caliper measurement.

[0147] To evaluate the toxicity associated with the treatment, the body weight of the animals was measured twice a week. When the tumors within the group reached 10% of the mouse body weight, the animals within the group were euthanized. The average tumor volume across the group was plotted together and compared over time.

[0148] As shown in Figures 10A and 10B, and Figures 11A and 11B, in both lung xenograft models, the liposomal ATR inhibitor compound 5 significantly improved the antitumor efficacy of MM-398. The combined treatment of liposomal ATR inhibitor compound 5 and MM-398 did not affect the body weight of the animals (Figures 12A and 12B).

[0149] Example 11: Comparison of the in vivo antitumor efficacy of liposomal inhibitor Ls compound 5 and LS compound A in combination with MM-398 against mouse lung cancer xenografts Figures 13A and 13B are graphs showing the efficacy of liposomal compound 5 when combined with MM-398 in Calu-6 (Figure 13A) or COLO-699 (Figure 13B) mouse xenograft models. For example, the data in Figures 13A and 13B show that the liposomal ATR inhibitor compound 5 significantly improved the antitumor efficacy of MM-398 in both models, while compound A formulated in liposomes was active only in the COLO-699 xenograft model.

[0150] The antitumor effect of liposomes filled with a combination of ATR inhibitor compound 5 and MM-398 (liposomal irinotecan) was compared with the liposomal formulation of compound A in xenograft models of human Calu-6 and COLO-699 lung cell lines.

[0151] Cells were obtained from the American Type Culture Collection (Rockville, MD) and grown at 37 °C and 5% CO2 in RPMI medium supplemented with 10% fetal bovine serum, 50 U / mL penicillin G, and 50 μg / mL streptomycin sulfate according to the supplier's recommendations. NCR nu / nu homozygous athymic male nude mice (4 - 5 weeks old, body weight at least 16 g) were obtained from Charles River. The mice were subcutaneously inoculated in the right flank with 0.1 mL of a suspension containing 5 × 106 cells suspended in PBS supplemented with 30% Matrigel. Tumors were 150 mm 3 ~350 mm 3Upon reaching the specified size, the animals were assigned to treatment groups according to the following method. The animals were ranked by tumor size and divided into six categories based on tumor size reduction. Four treatment groups of 10 animals / group were formed by randomly selecting one animal from each size category, thereby representing all tumor sizes equally within each treatment group. The animals were given four tail vein injections of the following preparations at 7-day intervals: 1) control (HEPES buffered saline pH 6.5); 2) MM-398 at a dose of 10 or 20 mg / kg per injection; 3) liposomal compound 5 at 80 mg / kg per injection; 4) liposomal compound A at 80 mg / kg per injection; 5) MM-398 followed by liposomal compound 5 at 24-hour intervals; 6) MM-398 followed by liposomal compound A at 24-hour intervals. The liposomes for injection were prepared as described in Example 7.

[0152] The animals' body weight and tumor size were monitored twice a week. Tumor progression was monitored twice a week by palpation and caliper measurement of the tumor along the maximum (length) and minimum (width) axes. Tumor size was calculated using the formula: Tumor volume = [(length) × (width)] 2 ] / 2 Measurements were taken twice a week using caliper measurements.

[0153] To assess the toxicity associated with the treatment, animal body weight was measured twice a week. Animals in a group were euthanized when the tumor volume reached 10% of the mouse's body weight. The average tumor volume across the entire group was plotted together and compared over time.

[0154] Example 12: Screening test for combination therapy with free-release drugs Figure 14A is a graph showing the degree of cell killing by in vitro monotherapy with compound 6 and compound 5 in a panel of lung cancer cell lines. Compound 5 showed higher efficacy than compound 6, and IC 50The IC5 is approximately 3 (=100.5) times lower. Figure 14B is a graph showing the effects of compound 5 and compound 6 when used in combination with three chemotherapeutic agents (carboplatin, gemcitabine, and compound B). This figure shows the IC5 of combinations of chemotherapeutic agents with 1 μg / ml of compound 6 or compound 5. 50 The results were compared using log(μM). In all cytotoxic agents tested and in all cell lines except one, the addition of compound 5 to the chemotherapeutic agent was more potent (lower IC5) than compound 6. 50 ) was.

[0155] Figure 15 shows the IC of the combination (Example 2 + Compound B) in the Sum190PT cell line (TNBC). 50 This is a graph showing the shift.

[0156] Figures 16A and 16B are graphs showing a comparison of the IC50 shifts of the compound from Example 2 and compound B, and the combined use of compound A and compound B, in the MDA-MB-453 cell line (TNBC).

[0157] Culture / treatment conditions The CELLTITER-GLO luminescent cell viability assay (Promega) was performed in vitro using Corning catalog number 3707 384-well White Clear bottom plates to test efficacy. Cells were plated in a 384-well format (1000 cells / well) and incubated at 37°C for 24 hours. At 24 hours, a single therapeutic agent was added, followed by incubation at 37°C for another 24 hours. At 48 hours, the drug was removed from the medium, washed with PBS, and fresh medium was added. The cells were then incubated at 37°C for 72 hours. For combination therapy, cells were exposed to carboplatin or compound B or gemcitabine for 24 hours, after which the chemotherapeutic agent was removed, and the cells were exposed to a second compound (ATR inhibitor) for another 24 hours. The cells were then cultured in fresh medium for a further 48 hours. [ka] At 120 hours, the culture medium was removed and CELLTITER-GLO (CTG) reagent was added (in a 1:1 ratio with PBS). The plate was read using an illuminometer (Envision Multilabel reader).

[0158] Data analysis: Data was analyzed using an in-house algorithm developed with Matlab (Mathworks, Natick MA). In summary, the mean CTG emission values ​​were calculated for four replicated wells. Outlier detection was performed by calculating the coefficient of variation (CV > 20%), and outliers were excluded from the mean. CTG values ​​were normalized based on the control untreated well. Drug concentrations in micromolar concentrations (μM) were logarithmically transformed and fitted to a four-parameter logistic curve.

[0159]

number

[0160] (In the formula, C: drug concentration, y: normalized CTG value, a: upper asymptote (representing maximum cell killing), b: lower asymptote (limited to 0.8-1.2), IC50, slope: slope of the logistic curve.)

[0161] Data quality control was performed to ensure that the concentration range was optimal according to the following rules: (1) If more than 70% of cells are killed at the lowest concentration, the concentration range is considered too potent. (2) If less than 30% of cells are killed at the highest concentration, the concentration range is considered too low, or the cell line is too resistant. Furthermore, R 2 The suitability of the goodness of fit is evaluated using R 2 If the value is <0.9, flag it as non-compliant.

[0162] Statistical analysis was performed using JMP (SAS Institute Inc., NC), and a p-value of < 0.05 was considered statistically significant.

[0163] Example 13: Measurement of ATR activity Using the linear enzyme concentration in the Eurofins ATR / ATRIP HTRF assay with GST-labeled full-length p53 as the substrate, the EC for the supplied compound set against the kinase ATR / ATRIP(h) was determined. 50 Measurements were performed.

[0164] The activity of ATR / ATRIP(h) at ATP concentrations within a Km value of 15 μM was measured with nine concentrations of compounds by a single-log dilution starting from 10 μM. The ATR / ATRIP phosphorylation of p53 at Ser15 was measured via the formation of an energy transfer complex consisting of a europium-labeled anti-phospho Ser15 p53 antibody and an anti-GST-d2 antibody. All data points were performed in duplicate using DMSO controls and EDTA blanks.

[0165] ATR / ATRIP(h) was pre-diluted in 25 mM HEPES pH 8.0, 0.01% Brij-35, 1% glycerol, 5 mM DTT, 1 mg / mL BSA and assayed in 25 mM HEPES pH 8.0, 0.01% Brij-35, 1% glycerol, 10 mM MnCl2 using 30 nM GST, cMyc p53-(hu,FL) as the substrate. The reaction was initiated by adding ATP until a final concentration of 10 μM was reached.

[0166] Individual replicates were expressed as a percentage (%) of the DMSO positive control activity. The mean activity (% control) for each inhibitor concentration was plotted against the inhibitor concentration and the EC 50 values were measured using GRAPHPAD PRISM.

[0167] Data represented as % control activity were plotted against inhibitor concentration and fit to a four-parameter logistic using GRAPHPAD PRISM. The graph for each compound is shown together with the data in the accompanying Excel report. A summary of the potencies of the compounds is shown below.

[0168]

Table 4

[0169] Example 14: Determination of ATM activity Estimated IC 50 The values ​​are as follows (obtained using STANDARD KINASEPROFILER):

[0170] [Table 5]

[0171] Example 15: Detection of total ATR and phospho-ATR by Western blot analysis As shown in Figure 17: Lung cancer cells DMS-114 were exposed in vitro to gemcitabine [16 nM] or either a single or combined ATR inhibitor (compound A or compound 5 [1 μM]). After 1, 3, 6, and 24 hours, total cell protein lysates were prepared using 2% SDS-containing cell lysis buffer, and all samples were collected for Western blotting analysis and stored at -80°C until performed. The following proteins and phosphoproteins of interest were detected by antibodies purchased from Cell Signaling Technology (see Methods and Materials below): total ATR and phosphorylated ATR (S428), phospho-CHK1 (S317 and S345), γH2AX, and β-actin.

[0172] Results in Figure 17: Total ATR and phospho-ATR signaling were potently reduced by the addition of either of the two ATR inhibitors (compound A and compound 5), but not by the control or gemcitabine monotherapy (e.g., compare lanes 1 and 2 at 24 hours with lanes 3, 4 or 5, 6). In response to gemcitabine, phospho-CHK1 (S317 and S345) signaling was significantly increased over 24 hours. The addition of either ATR inhibitor (compound A or compound 5) inactivates the CHK1 phosphorylation signaling. The reduction in ATR protein and downstream CHK1 signaling by both inhibitors confirms specific on-target effects of both molecules. More importantly, our hypothesis is that the loss of a cell cycle checkpoint caused by CHK1 phosphorylation signaling, which should lead to cell cycle arrest and DNA damage repair, induces cell death due to the accumulation of DNA damage. The increase in yH2AX signaling can be considered a surrogate for measuring increased toxicity and the accumulation of DNA damage.

[0173] cell culture U2OS and all other cells were grown in 10% fetal bovine serum (FBS) and antibiotic-supplemented (RPMI). NucLight erythrocyte strains suitable for imaging and slow-motion imaging were generated using lentiviral particle, infection, and puromycin selection protocols as recommended by Essen Bioscience Inc.

[0174] Antibodies and Western blots All target and phosphorus-specific antibodies were purchased from Cell Signaling and / or Epitomics and used at a 1:1000 dilution. A list of all antibodies is shown in Table 6. Standard antibody-based Western blotting and immunohistochemistry protocols were used. Briefly, whole cell lysates for Western blotting were collected using a 2% SDS-based cell lysis buffer. Secondary antibodies and staining protocols were purchased and followed from LiCor Biosciences and / or BD Bioscience.

[0175] [Table 6]

[0176] Whole cell lysis protocol Cells were grown and treated on a 6 cm dish scale. To harvest the cells, the medium was removed and rapidly replaced with ice-cold PBS. The PBS was then replaced with 250 μL of 2% SDS lysis buffer. Five minutes after incubation, the lysed cells were scraped off and packed onto a cell lysate homogenizer microcentrifugation spin column (QIAshredder, Qiagen, catalog number 79656). The filtrate was then packed onto a 0.2 μm centrifugal filter column (Nanosep MF 0.2_m, Pall, catalog number ODM02C34). The lysates were then stored at -80°C.

[0177] The lysis buffer containing 2% SDS (Table 7) was modified according to Steven et al., “Protein microarrays for multiplex analysis of signal transduction pathways,” Nat Med 10, no.12 (December 2004):1390-1396.

[0178] [Table 7]

[0179] Example 16: Extensive kinase panel screening (359) Of the 359 entries, the main hits were: ALK, ARK, c-MER, CLK1, DYRK, GSK3a, GSK3b, FLT2, FLT3, MLK1, SIK2, TNIK, and YSK4.

[0180] [Table 8]

[0181] While the present invention is described in relation to its specific embodiments, further modifications are possible, and it should be understood that this application is generally intended to encompass any modifications, uses, or adaptations of the invention, including any deviations from the disclosure that are applicable to the essential features described herein and are within the scope of known or customary practices in the art to which the invention belongs, in accordance with the principles of the invention.

[0182] Example 17: Data using Compound A as a comparative substance Cell death and proliferation assays were performed in U2OS cells using compound A and gemcitabine at various concentrations. The results were compared with predictions of cell proliferation and cell death by the combination of the two compounds (Figure 18A). The number of viable cells and apoptotic cells was also measured in U2OS cells at the specified concentrations of compound A (1 μM) and gemcitabine (0.04 μM) (Figure 18B). The results indicate that the combination of compound A and gemcitabine is superior in promoting cell death compared to either compound alone.

[0183] Compound A was also tested alone and in combination with SN38 at set concentrations (1 μM and 0.2 μM, respectively) in several lung cancer cell lines (NCI-H520 and NCI-H596) and U2OS cells, and cell counts were monitored over time. The results showed that the combination of compound A and SN38 was more effective than either compound A or SN38 alone in maintaining or decreasing cell counts over time (Figure 19).

[0184] In vitro proliferation assays were also performed using the cervical cancer cell line MS-751, with compound A and SN38 used in combination and individually. The in vitro assays showed a decrease in cell number over time when compound A and SN38 were used in combination, compared to when either compound was used alone (Figure 20).

[0185] Example 18: Comparison of Compound A and Compound 5 in combination with gemcitabine or SN38 In vitro cell death and proliferation assays were performed using various concentrations of compound A or compound 5 in combination with gemcitabine in U2OS, H358, and A549 cell lines. The results show the concentrations of each compound and gemcitabine required to induce cell death (Figure 21A). The set concentrations of compound 5 and gemcitabine or compound A and gemcitabine were similarly tested in USO2 and H358 cells. In all cases, relative cell proliferation was reduced by combination therapy compared to drug alone (Figures 21B-C).

[0186] Compound A or Compound 5 was administered in combination with SN38 within A549 cells. Cell proliferation was measured over time and within a certain concentration range. The growth curve at the optimal compound / SN38 concentration is highlighted (Figure 22A). Relative proliferation of A549 cells was measured using either Compound A alone or Compound 5 alone within a certain concentration range (Figure 22B).

[0187] IC50 values ​​were measured in several cell lines using gemcitabine at a set concentration along with various concentrations of compound A or compound 5 (Figure 23).

[0188] Figure 24 shows an overview of lung cancer cell lines that respond to compound A or compound 5 in combination with gemcitabine or SN38.

[0189] Example 19: Comparison of compound A and compound 5 in on-target and off-target conditions. Various ATR inhibitors were tested for their ability to inhibit both ATR (on-target) and ATM (off-target). Inhibition was reported as IC50 in nM (Figure 25A). Further "off-target" kinases were similarly tested using compound A or compound 5 (Figure 25B).

[0190] On-target studies were conducted using compound A or compound 5 in A549 lung cancer cells (Figure 26A-B), H23 lung cancer cells (Figure 26C-D), and DMS-114 cells (Figure 26E-F). CHK1 S345 phosphorylation, an ATR inhibition reading, was measured by Western blotting. Each compound was used in the same manner with a constant concentration of gemcitabine.

[0191] Further on-target studies were performed on HCC-70 TNBC, MDA-MB-468 TNBC, and DMS-114 cell lines. The SN38 setting concentration was used with a range of compound A or compound 5 concentrations. Various on-target activity parameters were tested and measured by Western blotting (Figure 27A-B).

[0192] The cell cycle characteristics of SUM149 cells were determined 24 hours after the addition of SN38 and compound A or compound 5 (Figure 28). When the drugs were administered concomitantly, a greater proportion of cells arrested in the G2 phase compared to the control.

[0193] Example 20: Comparison of liposome formulations of compound A and compound 5 used in combination with MM398 The effects of Ls compound A or Ls compound 5 in combination with MM398 were measured using a DMS-114 lung xenograft model. A set dose of MM398 (5 mpk) was used along with two different doses of compound A or compound 5 (20 mpk or 80 mpk). Therapeutic effects were assayed by measuring the level of CHK1 S345 phosphorylation (Figure 29).

[0194] The effects of Ls compound A in the presence of MM398 were also tested in the SUM-149 cell line. Therapeutic effects were assayed by measuring the phosphorylation levels of RPA2, DNAPK, CHK1, and γH2AX. Ls compound 5 was also tested without the use of MM398 (Figures 30A-C).

[0195] Example 21: In vivo antitumor efficacy and tolerability of Ls compound 5 prepared using TEA.SOS against xenografts of three types of negative breast cancer in mice. The antitumor efficacy of liposomes filled with ATR inhibitor compound 5 in combination with MM-398 (liposomal irinotecan) was tested in a human SUM-149 (triple-negative breast cancer) cell line model.

[0196] Cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in RPMI medium supplemented with 10% fetal bovine serum, 50 U / mL penicillin G, and 50 μg / mL streptomycin sulfate at 37°C under 5% CO2 conditions, according to the supplier's recommendations. NCR nu / nu homozygous athymogenic male nude mice (4-5 weeks old, at least 16 g body weight) were obtained from Charles River. Mice were subcutaneously inoculated into the right flank with a 0.1 mL suspension containing 107 cells suspended in PBS supplemented with 30% Matrigel. When the tumors reached a size of 150 mm³–350 mm³, the animals were assigned to treatment groups according to the following method: Animals were ranked by tumor size and divided into six categories of reduced tumor size. Four treatment groups of 10 animals / group were formed by randomly selecting one animal from each size category, thereby representing all tumor sizes equally within each treatment group. The animals were given five tail vein injections of the following preparations at 7-day intervals: 1) control (HEPES buffered saline pH 6.5); 2) MM-398 at a dose of 5 mg / kg per injection; 3) liposomal compound 5 at a dose of 80 mg / kg per injection; 4) MM-398 followed by injection of liposomal compound 5 at 24-hour intervals; 5) MM-398 followed by injection of unencapsulated free ATR inhibitor compound A. The liposomes for injection were prepared as described in Example 10.

[0197] The animals' body weight and tumor size were monitored twice a week. Tumor progression was monitored twice a week by palpation and caliper measurement of the tumor along the maximum (length) and minimum (width) axes. Tumor size was calculated using the formula: Tumor volume = [(length) × (width)] 2 ] / 2 Measurements were taken twice a week using caliper measurements.

[0198] To assess the toxicity associated with the treatment, animal body weight was measured twice a week. Animals in a group were euthanized when the tumor volume reached 10% of the mouse's body weight. The average tumor volume across the entire group was plotted together and compared over time.

[0199] As shown in Figure 31, liposomal ATR inhibitor compound 5 significantly enhanced the antitumor efficacy of MM-398 in a xenograft model. Combination therapy with liposomal compound A and MM-398 did not affect the animals' body weight (Figure 32).

[0200] Example 22: Comparison of cell line characteristics with Incucyte data A panel of fluorescently labeled cell lines was characterized for various proteins involved in the DNA damage pathway. Cancer cell parental lines were transduced with NucLight Red lentivirus and selected with puromycin. Basal protein levels were measured and quantified by Western blotting. PD marker signals were normalized relative to β-actin signals during quantification. Protein levels correlated with the cell line's "integral score," a measure of in vitro cellular response to compound 5 and chemotherapy combination treatment. The integral score can be calculated as follows:

[0201] As shown in Figures 33 and 34, cell lines were seeded in 96-well plates, and each well was exposed to a compound 5 administration matrix and chemotherapy for 4 days. Dynamic cell viability was measured using the Incucyte assay. For each drug combination in the dose matrix, a dose combination-specific integrated score was calculated by summing the area under the normalized cell growth curve. A cell line-specific integrated score was calculated by averaging the four maximum scores on the dose matrix.

[0202] As shown in Figures 35 and 36, in lung cancer cell lines exposed to compound 5 and SN38, both basal MRE11 protein levels and basal ATM levels were observed to be significantly correlated with the cell line's integrated score. In lung cancer cells with a p53 dysfunction background and exposed to compound 5 and SN38, the integrated score correlated with NBS, MRE11, and RAD50 protein levels. "Dysfunctional p53" cells are those with non-zero basal p53 protein levels (in most cells, p53 levels can only be seen in Western blot after treatment). (See Figures 37, 38, and 39).

[0203] Example 23: Signal transduction experiment: Compound 5 vs. Compound A: Six different PD markers used in combination with SN38 As shown in Figures 40-44: Cancer cell lines were exposed to various doses of compound 5 or compound A in combination with SN38, and then pharmacodynamic markers pChk1, pRPA2, pATR, pDNAPK, pChk2, and γH2AX were quantified by Western blotting. HCC70, DMS114, MDAMB468, NCIH1299, and NCIH460 cells were seeded in 12-well plates, incubated overnight, and then exposed to SN38 and / or ATR inhibitors. 24 hours after drug exposure, cells were lysed for Western blotting. PD marker signals were normalized to β-actin signals during quantification.

[0204] Example 24: Signal transduction experiment: Compound 5 vs. Compound A: Three different PD markers in combination with gemcitabine As shown in Figures 45-50, six fluorescently labeled cell lines were characterized for various proteins involved in the DNA damage pathway after exposure to ATR inhibitors and / or gemcitabine. Cancer cell parent lines were transduced with NucLight Red lentivirus and selected with puromycin. After exposing the cancer cell lines to various doses of compound 5 or compound A in combination with 16 nM gemcitabine, pharmacodynamic markers pChk1, pATR, and γH2AX were quantified by Western blotting. A549, NCIH23, DMS114, U20S, HCC827, and NCIH460 cells were seeded in 12-well plates, incubated overnight, and then exposed to SN38 and / or ATR inhibitors. Cells were lysed for Western blotting 6 or 18 hours after drug exposure. PD marker signals were normalized relative to β-actin signals during quantification.

Claims

1. ATR protein kinase inhibitor compounds of formula (Ia): 【Chemistry 1】 (In the formula, R' is an alkyl-amino moiety containing a tertiary alkyl-substituted amine having a pKa of 9.5 or higher.) or a pharmaceutically acceptable salt thereof.

2. Compound of formula 5: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

3. Compound of formula 6: 【Transformation 3】 or a pharmaceutically acceptable salt thereof.

4. A liposome composition comprising an ATR protein kinase inhibitor compound, or a pharmaceutically acceptable salt thereof, encapsulated in liposomes, and having a plasma half-life of at least about 5 hours in mice, as measured according to Example 8.

5. The liposome composition according to claim 4, wherein the ATR protein kinase inhibitor compound is compound A.

6. The ATR protein kinase inhibitor compound is a compound of formula (I): 【Chemistry 4】 (wherein R is greater than 7.0 (preferably greater than 8.0, most preferably at least about 9.5) pK ａ (This is a portion containing an amine that has the properties of...) The liposome composition according to claim 4, or a pharmaceutically acceptable salt thereof.

7. The ATR protein kinase inhibitor compounds are a group consisting of the following: 【Transformation 5】 The liposome composition according to claim 4, wherein the compound is selected from the above.

8. The ATR protein kinase inhibitor compounds are a group consisting of the following: 【Transformation 6】 The liposome composition according to claim 4, wherein the compound is selected from the above.

9. The composition according to any one of claims 4 to 8, wherein the liposome comprises hydrogenated soy phosphatidylcholine (HSPC) and cholesterol.

10. The composition according to claim 9, wherein the liposome further comprises PEG(2000)-distearoylglycerol (PEG-DSG).

11. The composition according to claim 10, wherein the liposomes contain HSPC, cholesterol, and PEG-DSG in a molar ratio of about 3:2:0.

15.

12. a. Forming liposomes that encapsulate sucrose octasulfate encapsulated in vesicles containing phospholipids and cholesterol; and b. The composition according to any one of claims 4 to 11, obtained by a process comprising the step of contacting the liposomes of step (a) with the ATR protein kinase inhibitor under conditions effective for filling the liposomes with the ATR protein kinase inhibitor.

13. The composition according to claim 12, wherein the ammonium sulfate is present at a concentration of about 1.1 M before contacting the liposomes with the ATR protein kinase inhibitor compound.

14. The liposome composition according to claim 4, wherein the ATR protein kinase inhibitor compound is compound A.

15. The liposome composition according to claim 4, wherein the ATR protein kinase inhibitor compound is compound 1.

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