Pyrrolopyrimidinecarboxamide

Abstract

Pyrrolopyrimidine carboxamide and pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising such compounds and salts, use of such compounds and salts for the treatment or prevention of cancer, including PKMYT1-dependent cancer, kits comprising such compounds and salts, and methods for producing such compounds and salts.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 506,165, filed on 5 June 2023, and U.S. Provisional Patent Application No. 63 / 594,968, filed on 1 November 2023, under Section 119(e) of the U.S. Patent Act, which are incorporated by reference in their entirety for all purposes.

[0002] (Field of invention) This disclosure relates, in general, to pyrrolopyrimidine carboxamide compounds and pharmaceutically acceptable salts thereof. This disclosure further relates to pharmaceutical compositions comprising such compounds and salts, the use of such compounds and salts for the treatment or prevention of cancers, including cancers having PKMYT1 dependence due to elevated baseline levels of replication stress, kits comprising such compounds and salts, and methods for producing such compounds and salts. [Background technology]

[0003] The activity of the protein kinase CDK1 (also known as the cell cycle 2 protein or CDC2) causes cells to transition from the G2 phase of the cell cycle to mitosis (M), where cell division occurs. In response to DNA damage, WEE1 and PKMYT1, members of the WEE1 kinase family, inhibit CDK1, preventing cell division until the damaged DNA is repaired (G2 / M DNA damage cell cycle checkpoint arrest).

[0004] During the replication or synthesis (S-) phase of the cell cycle, when the genome is replicated in preparation for cell division, events can occur that stall DNA polymerase and the replication fork. This situation is called replication stress and can lead to the generation of DNA damage that causes dependence on replication stress response proteins and G2 / M cell cycle checkpoint proteins such as ATR, WEE1, and PKMYT1. Forment, JV and MJO'Connor, "Targeting the replication stress response in cancer," Pharmacol Ther 188:155-167 (2018). In such cancers with DNA damage resulting from replication stress, there is a greater dependence on PKMYT1, whether endogenous or induced by DNA damage factors or other targeting agents. Inhibition of PKMYT1 can lead to an early transition to mitosis with unrepaired DNA damage, resulting in the induction of cancer cell death during mitosis. Chow, J.P. and RYPoon, "The CDK1 inhibitory kinase MYT1 in DNA damage checkpoint recovery," Oncogene 32(40):4778-4788(2013). Therefore, PKMYT1 inhibitors have the potential to induce cancer cell death as monotherapy in cancers with elevated levels of endogenous or basal replication stress, or in combination with other agents that induce higher levels of replication stress in cancer.

[0005] Targeted pharmacological inhibition of PKMYT1 is an underexplored therapeutic approach for treating cancers with elevated basal levels of replication stress, and currently, no pharmacological agents that inhibit PKMYT1 are approved. Thus, there is a need for PKMYT1 inhibitors, particularly those having pharmacologically suitable properties (e.g., selectivity for WEE1 and bioavailability) necessary for suitable administration to subjects who require such treatment. The present disclosure addresses this yet unmet need by providing such compounds, along with corresponding pharmaceutical compositions and methods for the treatment or prevention of cancer, including cancers having PKMYT1-dependency resulting from elevated basal levels of replication stress, as a monotherapy or in combination therapies capable of inducing such PKMYT1-dependency with other targeted therapies such as DNA damaging agents or PARP inhibitors or ATR inhibitors. SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides a compound having the structure of formula (1):

[0007]

Chemical formula

[0008]

Chemical formula

[0009] [ka] Selected from the group consisting of; R 13 and R 24 is hydrogen, C 1~3 -alkyl and C 1~3 - Independently selected from the group consisting of alkoxys; R 14 , R 17 , R 18 , R 20 , R 22 , and R 28 teeth, (a) hydrogen; (b) halogens; (c) Cyano; (d)C 1~6 -alkyl, which may optionally be substituted with one or more substituents independently selected from halogens. 1~6 -alkyl; (e)C 1~6 - Alkenyl, which may optionally be substituted with one or more substituents independently selected from halogens. 1~6 -Alkenil; (f)C 1~6 -Alkynyl, which may optionally be substituted with one or more substituents independently selected from halogens. 1~6 -Alkinnir; (g)C 3~6 -Cycloalkyl, optionally containing halogen, C 1~6 -alkyl, halo-C 1~6 -alkyl, C 1~6 -alkoxy and halo-C 1~6 -C may be substituted with one or more substituents independently selected from the group consisting of alkoxys. 3~6 -Cycloalkyl; (h)C 1~6-alkoxy, which may optionally be substituted with one or more substituents independently selected from halogens. 1~6 -alkoxy; (i)-NR 31 R 32 ; (j)-S(O)2-C 1~6 -Alkyl, which may be optionally substituted with one or more substituents independently selected from halogens -S(O)2-C 1~6 -alkyl; (k) Phenyl, optionally a halogen, C 1~6 -alkyl, halo-C 1~6 -alkyl, C 1~6 -alkoxy and halo-C 1~6 - Phenyls that may be substituted with one or more substituents independently selected from the group consisting of alkoxys; (l) A heterocyclyl containing a total of 4 to 10 ring atoms, wherein the heterocyclyl ring is (i) a saturated, partially saturated, or fully unsaturated monocyclic or fused bicyclic ring, (ii) independently having one or two ring heteroatoms selected from nitrogen, oxygen, and sulfur, with the remaining ring atoms being carbon, and (iii) optionally a halogen, oxo, C 1~6 -alkyl, halo-C 1~6 -alkyl and C 3~6 - A heterocycline which may be substituted with one or more substituents independently selected from the group consisting of cycloalkyls. Independently selected from the group consisting of; R 15 , R 19 , R 23 , R 26 , and R 30 Hydrogen, halogen, cyano, C 1~3 -alkyl and C 1~3 - Selected independently of alkoxy; R 16 is hydrogen, halogen, C 1~3 -alkyl, halo-C 1~3 -alkyl, C 1~3 -alkoxy and halo-C 1~3 - Selected from the group consisting of alkoxys; R 21 , R 25 , R 27 , and R 29 is hydrogen and -XR 33 Selected independently of; X is a combination or C 1~3 -It is alkyl; R 31 and R 32 is hydrogen and C 1~6 -Selected independently of alkyl; R 33 C 1~3 -alkyl, C 3~6 - Selected from the group consisting of cycloalkyls, phenyls, and 4- to 6-membered ring heteroaryls having one or two nitrogen ring atoms; C 1~3 -alkyl, C 3~6 -Cycloalkyl, phenyl, and 4- to 6-membered ring heteroaryls are optionally mixed with halogens and C 1~6 The present invention provides compounds which may be substituted with one or more substituents independently selected from the group consisting of alkyl groups.

[0010] In another aspect, the disclosure provides compounds whose structure is selected from the group consisting of formulas (2), (3), (4), (5), (6), (7), (8), and (9) as further defined herein, as well as pharmaceutically acceptable salts thereof.

[0011] In another aspect, the present disclosure provides compounds whose structure of formula (1) is selected from the group consisting of formulas (2-A), (2-B), (2-C), (2-D), and (2-E) as further defined herein, as well as pharmaceutically acceptable salts thereof.

[0012] In another aspect, the present disclosure provides compounds whose structure of formula (1) is selected from the group consisting of formulas (3-A), (3-B), (3-C), (3-D), and (3-E) as further defined herein, as well as pharmaceutically acceptable salts thereof.

[0013] In another aspect, the disclosure provides compounds whose structure is selected from the group consisting of formulas (4-A), (4-B), (4-C), (4-D), and (4-E) as further defined herein, as well as pharmaceutically acceptable salts thereof.

[0014] In another aspect, the present disclosure provides compounds whose structure of formula (1) is selected from the group consisting of formulas (5-A), (5-B), (5-C), (5-D), and (5-E) as further defined herein, as well as pharmaceutically acceptable salts thereof.

[0015] In another aspect, the present disclosure provides compounds whose structure of formula (1) is selected from the group consisting of formulas (6-A), (6-B), (6-C), (6-D), and (6-E) as further defined herein, as well as pharmaceutically acceptable salts thereof.

[0016] In another aspect, the present disclosure provides compounds whose structure of formula (1) is selected from the group consisting of formulas (7-A), (7-B), (7-C), (7-D), and (7-E) as further defined herein, as well as pharmaceutically acceptable salts thereof.

[0017] In another aspect, the present disclosure provides compounds whose structure of formula (1) is selected from the group consisting of formulas (8-A), (8-B), (8-C), (8-D), and (8-E) as further defined herein, as well as pharmaceutically acceptable salts thereof.

[0018] In another aspect, the present disclosure provides compounds whose structure of formula (1) is selected from the group consisting of formulas (9-A), (9-B), (9-C), (9-D), and (9-E) as further defined herein, as well as pharmaceutically acceptable salts thereof.

[0019] In another embodiment, the disclosure provides a pharmaceutical composition comprising a compound having the structure of formula (1) in a therapeutically effective amount or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0020] In another embodiment, the present disclosure provides a pharmaceutical composition comprising a compound having the structure of formula (1) in a therapeutically effective amount or a pharmaceutically acceptable salt thereof, a second pharmacological agent, and a pharmaceutically acceptable carrier.

[0021] In another embodiment, the disclosure provides a method for treating or preventing cancer by administering a therapeutically effective amount of a compound having the structure of formula (1) or a pharmaceutically acceptable salt thereof to a subject in need. In a further embodiment, the cancer is a solid tumor cancer. In a further embodiment, the cancer is a hematological cancer. In a further embodiment, the cancer is PKMYT1 dependent due to an increased level of replication stress.

[0022] In another embodiment, the disclosure provides a compound having the structure of formula (1) or a pharmaceutically acceptable salt thereof for use as a pharmacopoeia for the treatment or prevention of cancer. In a further embodiment, cancer is PKMYT1 dependent due to an increased level of replication stress.

[0023] In another embodiment, the disclosure provides the use of a compound having the structure of formula (1) or a pharmaceutically acceptable salt thereof for preparing a medicament for treating or preventing cancer. In a further embodiment, cancer is PKMYT1 dependent due to an increased level of replication stress.

[0024] In another embodiment, the disclosure provides a kit comprising a compound having the structure of formula (1) or a pharmaceutically acceptable salt thereof.

[0025] In another aspect, the present disclosure provides a method for preparing a compound having the structure of formula (1) or a pharmaceutically acceptable salt thereof. [Brief explanation of the drawing]

[0026] [Figure 1] This figure shows the regulation of WEE1 and PKMYT1 in mitotic cells. [Figure 2]This figure shows the effect (GI50) of treatment with PKMYT1 inhibitor tool compounds on 16 cancer cell line models with either elevated or low levels of basal replication stress. [Figure 3] This figure shows the replication fork velocities obtained for uterine and breast cancer cell line models treated with PKMYT1 inhibitor tool compounds (Figure 2). [Figure 4] Example B-1 shows the replication fork velocities obtained for the same uterine and breast cancer cell lines in Figure 2 that were treated instead with isomer 1 (i.e., the eutom of compound 1). [Figure 5] This figure shows the dose-response curves obtained for compound 1, isomer 1, compound 137, and isomer 2 in an in vitro hematopoietic stem cell and progenitor cell assay to evaluate myelotoxicity. [Figure 6] This figure shows the differential expression of phosphorylated proteins in OVCAR3 cells after 24 hours of treatment with compound 137, isomer 2, or control (adavocertib). [Figure 7] This figure shows (i) the effect of PKMYT1 deletion from SKOV3 cells on CDK1 T14 phosphorylation after treatment with compound 1 and isomer 1 (left and center panels), and (ii) the effect of treatment with compound 137 and isomer 2 on PKMYT1 knockout SKOV3 cells (i.e., cells that do not express the drug target PKMYT1) (GI50) (right panel). [Figure 8-A] This figure shows the tolerability of a 300 mg / kg BID regimen using compound 137 and isomer 2 in mice over a 28-day period. [Figure 8-B] This figure shows the tolerability of a 100 mg / kg BID regimen of compound 137, isomer 2, in combination with either irinotecan hydrochloride (left panel) or gemcitabine (right panel) in mice over a 28-day period. [Figure 9] This figure shows the phosphorylation of the PKMYT1 substrate (CDK1) (at Thr14) in OVCAR3 tumor-bearing SCID mice after a treatment schedule using compound 137 and isomer 2 (administered twice daily for 21 days). [Figure 10] This figure shows the in vivo efficacy of compound 137 and isomer 2 in combination with gemcitabine in a human ovarian OVCAR3 xenograft model. [Figure 11] This figure shows the in vivo efficacy of compound 137 and isomer 2 in combination with irinotecan hydrochloride in a human colorectal SW620 xenograft model. [Figure 12] This figure shows the in vitro efficacy of compound 137 and isomer 2, either alone or in combination with 1nM exatecan, in PKMYT1 wild-type and PKMYT1 knockout SKOV3 cells. [Figure 13] This figure shows the effects on in vivo biomarker expression (phospho-CDK1 Thr14, gH2AX, and phosphohistone H3 Ser10) in a human colorectal SW620 xenograft model after treatment with irinotecan hydrochloride alone or in combination with compound 137 or isomer 2 (100 mg / kg BID). [Figure 14] This figure shows the metabolic scheme illustrating the major pathways of in vitro bioconversion (oxidation and glucuronidation) of compound 137 and isomer 2. [Figure 15] This figure shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in CD-1 mice after administration of IV (0.5 mg / kg, solid line) or PO (1 mg / kg, dashed line). [Figure 16] This figure shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in SCID mice after PO administration at three dose levels: 10 mg / kg (solid line), 30 mg / kg (dashed line), and 100 mg / kg (dotted line). [Figure 17] This figure shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in Han Wistar rats after IV (0.5 mg / kg) administration to either untreated (solid line) or bile duct cannula-treated (dashed line) animals. [Figure 18]This figure shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in Han Wistar rats after PO administration at three dose levels: 10 mg / kg (solid line), 30 mg / kg (dashed line), and 100 mg / kg (dotted line). [Figure 19] This figure compares the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in SCID mice after repeated oral administration of 100 mg / kg to male SCID mice (n=2) on day 1 (solid line) and day 28 (dashed line). [Figure 20] This figure shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in SCID mice after PO administration at 300 mg / kg in 5% DMSO, 50% 20% captisol, and 45% WFI (pH 3-3.2) (solid line), and in 0.5% HPMC / 0.1% Tween (dashed line).

[0027] For convenience, compound 1 and isomer 1 are identified as AZ1-1 in the figure, and compound 137 and isomer 2 are identified as AZ137-2 in the figure. [Modes for carrying out the invention]

[0028] Many embodiments are described in detail throughout this disclosure and will be apparent to those skilled in the art. This disclosure should not be construed as being limited to any specific embodiment described herein.

[0029] I. Definition With respect to the embodiments disclosed herein, the following terms have the meanings set forth below.

[0030] References to "a" or "an" indicate "one or more." Throughout, plural and singular forms should be treated as interchangeable, except when indicating quantity.

[0031] Unless the context requires a different interpretation, the words “comprise,” “comprises,” or “comprising” should be interpreted comprehensively, not exclusively, and the applicant uses these words based on and with a clear understanding that each of these words should be interpreted in this way when interpreting the Patent, including the following claims.

[0032] The term "cyano" (on its own or in combination with another term) means CN.

[0033] The term "halogen" (either alone or in combination with another term) refers to a fluorine radical (which can be represented as F), a chlorine radical (which can be represented as Cl), a bromine radical (which can be represented as Br), or an iodine radical (which can be represented as I).

[0034] The term "oxo" (used alone or in combination with other terms) refers to an oxo radical and can be represented as =O.

[0035] The term "alkyl" (alone or in combination with another term) refers to a linear or branched saturated hydrocarbyl substituent (i.e., a substituent containing only carbon and hydrogen). Alkyl substituents typically contain 1 to about 20 carbon atoms, more typically 1 to about 12 carbon atoms, even more typically 1 to about 8 carbon atoms, and even more typically 1 to about 6 carbon atoms. Examples of such substituents include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, and tert-butyl), pentyl (including n-pentyl, isoamyl, and 2,2-dimethylpropyl), and hexyl.

[0036] The term "alkenyl" (alone or in combination with other terms) refers to a linear or branched saturated hydrocarbyl substituent (i.e., a substituent containing only carbon and hydrogen) that contains one or more double bonds in an alkyl chain. Alkenyls typically contain 2 to about 20 carbon atoms, more typically 2 to about 12 carbon atoms, even more typically 2 to about 8 carbon atoms, and even more typically 2 to about 6 carbon atoms. Examples of such substituents include ethenyl, propenyl (including 1-propenyl and 2-propenyl), butyl (including 2-butenyl and 3-butenyl), pentenyl, and hexenyl.

[0037] The term "alkynyl" (alone or in combination with other terms) refers to a linear or branched saturated hydrocarbyl substituent (i.e., a substituent containing only carbon and hydrogen) that contains one or more triple bonds in an alkyl chain. Alkynnyls typically contain 2 to about 20 carbon atoms, more typically 2 to about 12 carbon atoms, even more typically 2 to about 8 carbon atoms, and even more typically 2 to about 6 carbon atoms. Examples of such substituents include ethynyl, propynyl (including 1-propynyl and 2-propynyl), butynyl (including 1-butynyl and 2-butynyl), pentynyl (including penta-1,3-diene, 2-methyl-1,3-butadiene, and penta-1,2-diene), and hexynyl.

[0038] The term "cycloalkyl" (alone or in combination with other terms) refers to a saturated carbocyclyl substituent containing 3 to approximately 14 carbocyclic atoms, more typically 3 to approximately 12 carbocyclic atoms, and even more typically 3 to approximately 8 carbocyclic atoms. Cycloalkyls typically contain a single carbocyclic ring containing 3 to 6 carbocyclic atoms. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0039] The term "alkoxy" (alone or in combination with another term) refers to an alkyl ether substituent, i.e., alkyl-O-. Examples of alkoxys include methoxy(CH3-O-), ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy. Therefore, for example, the term "alkoxyalkyl" (alone or in combination with another term) refers to an alkyl group substituted with an alkoxy, such as "methoxymethyl," which is...

[0040] [ka] That is the case.

[0041] The term “heterocyclyl” (alone or in combination with other terms) refers to a saturated, partially saturated, or completely unsaturated (i.e., “heteroaryl”) ring structure containing a total of 3 to 14 ring atoms. At least one of the ring atoms is a heteroatom (i.e., oxygen, nitrogen, or sulfur), and the remaining ring atoms are independently selected from the group consisting of carbon, oxygen, nitrogen, and sulfur in stable combinations known to those skilled in the art.

[0042] In some examples, the number of carbon atoms in a substituent (e.g., alkyl, cycloalkyl, etc.) is determined by the prefix "C x~y This is shown by the formula, where x is the minimum number of carbon atoms in the substituent and y is the maximum number. Therefore, for example, "C 1~6 "-alkyl" refers to an alkyl substituent containing 1 to 6 carbon atoms. To explain further, C 3~6 -Cycloalkyl refers to a cycloalkyl substituent containing 3 to 6 carbon ring atoms.

[0043] The prefix "halo" indicates that the substituent to which it is prefixed is substituted with one or more independently selected halogen radicals. For example, haloalkyl means an alkyl substituent in which at least one hydrogen radical is replaced by a halogen radical. If there are two or more hydrogens replaced by halogens, the halogens may be the same or different. Examples of haloalkyls include fluoromethyl, difluoromethyl, trifluoromethyl, difluoroethyl, 1,1,1-trifluoroethyl, pentafluoroethyl, difluoropropyl, heptafluoropropylchloromethyl, dichloromethyl, trichloromethyl, difluorochloromethyl, dichlorofluoromethyl, and dichloropropyl.

[0044] A substituent is "substitutable" if it contains at least one carbon or nitrogen atom bonded to one or more hydrogen atoms. Therefore, for example, hydrogen, halogens, and cyanos are not included in this definition.

[0045] When a substituent is described as "substituted," a non-hydrogen radical is present instead of a hydrogen radical on the carbon or nitrogen of the substituent. Therefore, for example, a substituted alkyl substituent is an alkyl substituent in which at least one non-hydrogen radical is present instead of a hydrogen radical on the alkyl substituent. For example, a monofluoroalkyl is an alkyl substituted with a fluoro radical, and a difluoroalkyl is an alkyl substituted with two fluoro radicals. If there is more than one substitution on the substituent, it should be recognized that (unless otherwise specified) each non-hydrogen radical may be the same or different.

[0046] If a substituent is described as "optionally substituted," it may be either (1) unsubstituted or (2) substituted. If a substituent's carbon is described as being optionally substituted with one or more substituents from the list, one or more hydrogens on the carbon (where available) may be replaced separately and / or together with independently selected optional substituents. If a substituent's nitrogen is described as being optionally substituted with one or more substituents from the list, one or more hydrogens on the nitrogen (where available) may each be replaced with independently selected optional substituents.

[0047] When a substituent is described as being "independently selected" from a group, each substituent is selected independently of the others. Therefore, each substituent may be identical or different from the others.

[0048] The term "atropisomer" refers to a stereoisomer arising from a bound rotation centered on one or more single bonds, where the energy barrier to the rotation is high enough to allow for the isolation of the conformosomers. As used in this disclosure, the term "eutomer" refers to the pharmacologically more active conformosomer of an atropisomer, and the term "distomer" refers to the pharmacologically less active conformosomer of an atropisomer.

[0049] The term “pharmaceutically acceptable” is used adjectivally in this disclosure to mean that the modified noun is suitable for use as a pharmaceutical or as part of a pharmaceutical. For example, “pharmaceutically acceptable salt” is a salt suitable for use in mammals, particularly humans, and includes salts with inorganic bases, organic bases, inorganic acids, organic acids, or basic or acidic amino acids suitable for use in mammals, particularly humans.

[0050] The "therapeutic dose" of a pharmacological agent is the amount sufficient to produce a beneficial or desired outcome, including clinical results, and therefore depends on the circumstances in which it is administered. For example, when a pharmacological agent is administered to treat cancer, the therapeutic dose of the agent is the amount sufficient to provide an anti-cancer effect in the subject, either alone or in combination with additional therapies, compared to the response obtained without the administration of the agent.

[0051] The term “prevent” is intended to be readily understood by a skilled physician and to have its usual meaning in relation to the treatment of a particular condition, and includes primary prevention, which is to prevent the onset of the condition, and secondary prevention, which is to protect the patient temporarily or permanently from exacerbation or worsening of the disease or the onset of new symptoms associated with the condition, once the condition has already developed.

[0052] The term “to treat” is readily understood by a normal, experienced physician and, in relation to the treatment of a particular condition, may include (1) reducing the degree or cause of the condition being treated, and / or (2) alleviating or improving one or more symptoms associated with that condition. Treatment of cancer may include, for example, stabilizing (i.e., preventing worsening), delaying, or slowing the spread or progression of cancer, extending survival compared to the survival expected if no treatment is provided, and / or otherwise improving or mitigating cancer or the severity of cancer, in whole or in part.

[0053] II. Compounds A. Compounds of formula (1) In one embodiment, the present disclosure is based on formula (1):

[0054] [ka] A compound having the structure of or a pharmaceutically acceptable salt thereof, wherein the formula is R 1 teeth,

[0055] [ka] selected from the group consisting of; R 2 、R 3 、R 4 、R 5 、R 7 、R 8 、R 9 、R 10 、and R 12 are independently selected from the group consisting of halogen and C 1~3 -alkyl; R 6 and R 11 are independently selected from the group consisting of hydrogen and C 1~3 -alkyl; A is,

[0056]

Chemical formula

[0057] In some embodiments, the structure of formula (1) is selected from the group consisting of formulas (2), (3), (4), (5), (6), (7), (8), and (9):

[0058] [Table 1] (In the formula, R 1 ~R 30(This is defined as for compounds having the structure of formula (1)).

[0059] In some embodiments, the structure of formula (1) is selected from the group consisting of formulas (2-A), (2-B), (2-C), (2-D), and (2-E):

[0060] [Table 2] (In the formula, R 2 ~R 15 (This is defined as for compounds having the structure of formula (1)).

[0061] In some embodiments, the structure of formula (1) is selected from the group consisting of formulas (3-A), (3-B), (3-C), (3-D), and (3-E):

[0062] [Table 3] (In the formula, R 2 ~R 12 , R 16 , and R 17 (This is defined as for compounds having the structure of formula (1)).

[0063] In some embodiments, the structure of formula (1) is selected from the group consisting of formulas (4-A), (4-B), (4-C), (4-D), and (4-E):

[0064] [Table 4] (In the formula, R 2 ~R 12 , R 18 , and R 19 (This is defined as for compounds having the structure of formula (1)).

[0065] In some embodiments, the structure of formula (1) is selected from the group consisting of formulas (5-A), (5-B), (5-C), (5-D), and (5-E):

[0066] [Table 5] (In the formula, R 2 ~R 12 and R 20 (This is defined as for compounds having the structure of formula (1)).

[0067] In some embodiments, the structure of formula (1) is selected from the group consisting of formulas (6-A), (6-B), (6-C), (6-D), and (6-E):

[0068] [Table 6] (In the formula, R 2 ~R 12 , R 21 , R 22 , and R 23 (This is defined as for compounds having the structure of formula (1)).

[0069] In some embodiments, the structure of formula (1) is selected from the group consisting of formulas (7-A), (7-B), (7-C), (7-D), and (7-E):

[0070] [Table 7] (In the formula, R 2 ~R 12 , R 24 , R 25 , and R 26 (This is defined as for compounds having the structure of formula (1)).

[0071] In some embodiments, the structure of formula (1) is selected from the group consisting of formulas (8-A), (8-B), (8-C), (8-D), and (8-E):

[0072] [Table 8] (In the formula, R2 ~R 12 , R 27 , and R 28 (This is defined as for compounds having the structure of formula (1)).

[0073] In some embodiments, the structure of formula (1) is selected from the group consisting of formulas (9-A), (9-B), (9-C), (9-D), and (9-E):

[0074] [Table 9] (In the formula, R 2 ~R 12 , R 29 , and R 30 (This is defined as for compounds having the structure of formula (1)).

[0075] In some embodiments, the compound having the structure of formula (1) is an atropisomer. In one embodiment, the compound is an utemer of an atropisomer. In another embodiment, the compound is a distomer of an atropisomer.

[0076] B. Additional Embodiments In some embodiments, the present disclosure provides compounds of formula (1) and pharmaceutically acceptable salts thereof, the compounds being selected from the group consisting of:

[0077] [Table 10-1]

[0078] [Table 10-2]

[0079] [Table 10-3]

[0080] [Table 10-4]

[0081] Table 10-5

[0082] Table 10-6

[0083] Table 10-7

[0084] Table 10-8

[0085] Table 10-9

[0086] Table 10-10

[0087] Table 10-11

[0088] Table 10-12

[0089] Table 10-13

[0090] Table 10-14

[0091] Table 10-15

[0092] Table 10-16

[0093] Table 10-17

[0094] Table 10-18

[0095] Table 10-19

[0096] Table 10-20

[0097] Table 10-21

[0098] Table 10-22

[0099] Table 10-23

[0100] In some embodiments, the compound selected is an atropisomer. In one aspect, the compound selected is the eutomer of the atropisomer.

[0101] In one embodiment, the present disclosure provides a compound that is 6-amino-4-cyclopropyl-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure:

[0102] [Chemical formula] And their pharmaceutically acceptable salts. In one aspect, the compound is an atropisomer. In another aspect, the compound selected is the eutomer of the atropisomer.

[0103] In one embodiment, the present disclosure provides a compound that is 6-amino-4-(2,2-difluorocyclopropyl)-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure:

[0104] [Chemical formula] And their pharmaceutically acceptable salts. In one aspect, the compound is an atropisomer. In another aspect, the compound selected is the eutomer of the atropisomer.

[0105] In one embodiment, the present disclosure provides a compound that is 6-amino-7-(2-chloro-3-hydroxy-6-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure:

[0106] [Chemical formula] The present invention provides compounds and pharmaceutically acceptable salts thereof. In one embodiment, the compound is an atropisomer. In another embodiment, the selected compound is an utemer of the atropisomer.

[0107] In one embodiment, the present disclosure relates to a compound having the following structure: 6-amino-7-(6-chloro-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide:

[0108] [ka] The present invention provides compounds and pharmaceutically acceptable salts thereof. In one embodiment, the compound is an atropisomer. In another embodiment, the selected compound is an utemer of the atropisomer.

[0109] In one embodiment, the present disclosure relates to a compound having the following structure: 6-amino-7-(2,6-dichloro-3-hydroxyphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide:

[0110] [ka] The present invention provides compounds and pharmaceutically acceptable salts thereof. In one embodiment, the compound is an atropisomer. In another embodiment, the selected compound is an utemer of the atropisomer.

[0111] In one embodiment, the present disclosure relates to a compound having the following structure: 6-amino-7-(6-bromo-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide:

[0112] [ka] The present invention provides compounds and pharmaceutically acceptable salts thereof. In one embodiment, the compound is an atropisomer. In another embodiment, the selected compound is an utemer of the atropisomer.

[0113] In one embodiment, the present disclosure relates to a compound selected from the group consisting of: 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 137); 6-amino-7-(2-chloro-3-hydroxy-6-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 141); 6-amino-7-(6-chloro-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 142); 6-amino-7-(2,6-dichloro-3-hydroxyphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 143); and 6-amino-7-(6-bromo-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 144); The present invention provides pharmaceutically acceptable salts thereof.

[0114] In one embodiment, the compound is an atropisomer. In another embodiment, the selected compound is an utemer of an atropisomer.

[0115] C. Combination of Embodiments Any embodiment of the compounds described herein may be combined with any other preferred embodiment described herein to provide additional embodiments. For example, one embodiment of the compound of formula (2) may be R 13 , R 14 , and / or R 15Possible bases are described individually or collectively, and different embodiments are described as R 1 If you are describing possible bases for R, 13 , R 14 , and / or R 15 The possible groups described for R 1 It is understood that these embodiments may be combined to provide additional embodiments described with regard to the possible groups described. In other words, for any of the embodiments of the compounds described herein, R 1 The substituents are R as described in this disclosure. 1 This may be as defined in any of the embodiments.

[0116] D. Further Embodiments In some embodiments, the compounds disclosed herein exhibit an IC50 of less than approximately 1 μM for PKMYT1 inhibition when measured in the target engagement assay described in Example C-2 below. 50 It has a value. In one embodiment, IC 50 The value is less than approximately 500 nM. In another embodiment, IC 50 The value is less than approximately 250 nM. In another embodiment, IC 50 The value is less than approximately 100 nM. In another embodiment, the compound has a pharmaceutically acceptable K for PKMYT1 binding affinity when measured by the SPR direct binding assay described in Example C-3 below. d It has a value.

[0117] In some embodiments, the compounds of the Disclosure exhibit pharmaceutically acceptable selectivity for PKMYT1 compared to WEE1, as measured by the target engagement assay described in Example C-2 below. In some embodiments, the compounds are at least about 50-fold selective to PKMYT1 compared to WEE1. In another embodiment, the compounds are at least about 100-fold selective to PKMYT1 compared to WEE1. In another embodiment, the compounds are at least about 250-fold selective to PKMYT1 compared to WEE1. In another embodiment, the compounds are at least about 500-fold selective to PKMYT1 compared to WEE1. In another embodiment, the compounds of the Disclosure exhibit IC50 greater than about 1 μM for inhibition of WEE1. 50 It has a value. In another embodiment, IC 50 The value is greater than approximately 5 μM. In another embodiment, IC 50 The value is greater than approximately 10 μM.

[0118] In some embodiments, the compounds of this disclosure inhibit CDK1 phosphorylation with an IC50 of less than approximately 2 μM when measured by the functional CDK1 pThr14 AlphaLISA assay described in Example C-4 below. 50 It has a value. In one embodiment, IC 50 The value is less than approximately 1 μM. In another embodiment, IC 50 The value is less than approximately 500 nM. In another embodiment, IC 50 The value is less than approximately 250 nM. In another embodiment, IC 50 The value is less than approximately 100 nM.

[0119] In some embodiments, the compounds of this disclosure inhibit OVCAR3 cell proliferation as measured by the cell proliferation assay described in Example C-5 below. In one embodiment, the compound is less than 5 μM GI 50 It has a value. In another embodiment, GI 50 The value is less than approximately 3 μM. In another embodiment, GI 50 The value is less than approximately 1.5 μM.

[0120] E. Salt The compounds of this disclosure may exist in salt form or unsalted form (i.e., as a free base), and this disclosure encompasses both salt and unsalted forms. The compounds may form acid addition salts or base addition salts. Generally, acid addition salts can be prepared using a variety of inorganic or organic acids. Such salts can typically be formed by mixing the compound with an acid (e.g., a stoichiometric amount of acid) using, for example, a variety of methods known in the art. This mixing may be carried out in water, an organic solvent (e.g., ether, ethyl acetate, ethanol, methanol, isopropanol, or acetonitrile), or an aqueous / organic mixture. In another embodiment, acid addition salts are, for example, trifluoroacetate, formate, acetate, or hydrochloride salts. Generally, base addition salts can be prepared using salts with a variety of inorganic or organic bases, for example, alkali metal salts or alkaline earth metal salts, e.g., sodium, calcium, or magnesium salts, or other metal salts, e.g., potassium or zinc, or ammonium salts, or organic bases, e.g., methylamine, dimethylamine, trimethylamine, piperidine, or morpholine. Those skilled in the art are familiar with the general principles and techniques for preparing pharmaceutical salts, for example, as described in Berge, S., et al., "Pharmaceutical Salts," J. Pharm. Sci. 66, 1 (1977). Examples of pharmaceutically acceptable salts are also described in Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, Weinheim, Germany, 2002).

[0121] F. Isomers The compounds and salts of this disclosure may exist in one or more geometric, optical, enantiomer, and diastereomer forms, including, but not limited to, cis- and trans- forms, E- and Z- forms, and R-, S- and meso- forms. Unless otherwise specified, references to a particular compound encompass all such isomeric forms, including racemates and other mixtures. If necessary, such isomers may be separated from their mixtures by the application or adaptation of known methods (e.g., chromatographic and recrystallization techniques). In some embodiments, a single stereoisomer is obtained, for example, by isolation from a mixture of isomers (e.g., a racemate) using chiral chromatography separation. In other embodiments, a single stereoisomer is obtained, for example, by direct synthesis from chiral starting materials.

[0122] Certain enantiomers of the compounds described herein may be more active than other enantiomers of the same compound. In one embodiment, the compound or a pharmaceutically acceptable salt thereof is a single enantiomer with an enantiomeric excess (%ee) of 90 or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In one embodiment, the single enantiomer exists with an enantiomeric excess (%ee) of 99% or more.

[0123] In another embodiment, the disclosure relates to a pharmaceutical composition comprising a compound that is a single enantiomer having an enantiomer excess (%ee) of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable excipients. In one embodiment, the single enantiomer is present with an enantiomer excess (%ee) of 99% or more.

[0124] G. Further Forms The compounds and salts of this disclosure may exist in various tautomers, and this disclosure encompasses all such tautomers. A "tautomer" is a structural isomer that exists in equilibrium resulting from the movement of hydrogen atoms.

[0125] The compounds of this disclosure and their pharmaceutically acceptable salts may exist as solvates (such as hydrates) and non-solvated forms, and this disclosure encompasses all such solvates.

[0126] The compounds of this disclosure and their pharmaceutically acceptable salts may exist in crystalline or amorphous forms, and this disclosure encompasses all such forms.

[0127] The compounds and salts of this disclosure may be isotope-labeled (or "radioactively labeled"). In this case, one or more atoms are replaced by atoms having an atomic mass or mass number different from those typically found in nature. This disclosure encompasses isotope-labeled forms of the compounds disclosed herein. Examples of isotopes that may be incorporated include: 2 H (deuterium is also written as "D") 3 H (tritium is also written as "T") 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, and 36 Cl is one example. The isotope used depends on the specific application of its radiolabeled derivative. For example, in in vitro receptor labeling and competitive assays, 3 H or 14 C is often useful. For radioactive imaging applications, 11 C is often useful. In some embodiments, the radionuclide is 3 It is H. In some embodiments, the radionuclide is 14 It is C. In some embodiments, the radionuclide is 11 It is C.

[0128] H. Intermediate In some embodiments, the Disclosure provides additional compounds and pharmaceutically acceptable salts thereof that are useful as intermediates for preparing the compounds of the Disclosure.

[0129] III.How to use One consequence of cancer cell evolution is often a rewriting of the ability to control cell cycle progression, which then supports carcinogenesis by causing more rapid oncogenic growth. More than 50% of all cancers, and more than 90% of cancers such as serous endometrial, serous ovarian, and basal-like breast cancer, harbor somatic TP53 mutations that can cause inactivation of the tumor suppressor protein p53 and partial or complete inactivation of the G1 / S cell cycle checkpoint. Under these circumstances, cells enter the replication S phase of the cell cycle earlier, thereby increasing the likelihood of elevated baseline levels of replication stress and greater dependence on the G2 / M cell cycle checkpoint regulated by WEE1 and PKMYT1.

[0130] As mentioned earlier, both WEE1 and PKMYT1 inhibit CDK1, preventing cell division until damaged DNA is repaired (G2 / M DNA damage cell cycle checkpoint arrest). However, in contrast to WEE1, PKMYT1 is selective for CDK1 compared to CDK2. WEE1 also modulates CDK2 activity, which normally prevents abnormal cell progression throughout the S phase. Because WEE1 modulates both CDK1 and CDK2, WEE1 inhibition can further increase replication stress in cancers where baseline levels of replication stress are already elevated (as detectable by biomarkers such as phospho-RPA2 (pRPA2) and phosphohistone H2AX (γH2AX)), potentially leading to further DNA damage during the S phase. Serra, V., et al., "Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance," Clin Cancer Res 28(20):4536-4550. (2022). This increased replication stress can sometimes be sufficient to kill cancer cells. Lallo, A., et al., "The Combination of the PARP Inhibitor Olaparib and the WEE1 Inhibitor AZD1775 as a New Therapeutic Option for Small Cell Lung Cancer," Clin Cancer Res 24(20):5153-5164 (2018). See Figure 1.

[0131] Preclinical and clinical data generated using the WEE1 inhibitor adavocertib have identified cancer backgrounds associated with replication stress that are sensitive to WEE1 inhibition. Such cancer backgrounds include enrichment of p53 mutations and / or one or more abnormalities in genes and / or their associated protein products, including but not limited to cyclin D1, cyclin E1, FBXW7, RB1, INK4a, ARF, CDK4, CDK6, SETD2, KDM4A, LKB1, TSC2, RSP6KA6, MYC, and / or KRAS. These cancer backgrounds are particularly enriched in tumor types such as ovarian cancer, triple-negative breast cancer, serous uterine carcinoma, uterine carcinosarcoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, colorectal cancer, soft tissue sarcoma, skin cancer, bladder cancer, head and neck cancer, glioma, and B-cell lymphoma. While tumor types with these deficiencies may be sensitive to monotherapy with WEE1 inhibitors in clinical settings, the fact that WEE1 inhibition also induces S-phase replication stress and DNA damage means that certain normal tissues are also affected, leading to gastrointestinal and hematological toxicity. Consequently, monotherapy with WEE1 inhibitors may have a relatively narrow therapeutic concentration range. Liu, JF, et al., "Phase II Study of the WEE1 Inhibitor Adavosertib in Recurrent Uterine Serous Carcinoma," Journal of Clinical Oncology 39(14):1531-1539 (2021).

[0132] Since PKMYT1 modulates CDK1 but not CDK2, PKMYT1 inhibitors have the potential to treat cancers with elevated baseline levels of replication stress, either as monotherapy or in combination with DNA damaging or targeting agents that increase levels of replication stress, but with an improved therapeutic concentration range compared to WEE1 inhibitors. As illustrated in Example C-2, background cancers associated with replication stress that are sensitive to PKMYT1 inhibition include those enriched with one or more abnormalities in genes and / or their associated protein products, including but not limited to cyclin E1, FBXW7, and / or KRAS. Considering the dependence of cancers on elevated levels of replication stress on the G2 / M cell cycle checkpoint, it is hypothesized that the background of further cancers associated with replication stress that are sensitive to PKMYT1 inhibition may include enrichment of TP53 mutations and / or one or more abnormalities in genes and / or their associated protein products, including but not limited to cyclin D1, cyclin E1, FBXW7, RB1, INK4a, ARF, CDK4, CDK6, SETD2, KDM4A, LKB1, TSC2, RSP6KA6, MYC, and / or KRAS. Therefore, PKMYT1 inhibitors selective for CDK1 are desirable and within the scope of this disclosure.

[0133] The compound of formula (1) and its pharmaceutically acceptable salts are inhibitors of PKMYT1 activity. In one embodiment, the compound selectively inhibits PKMYT1 activity.

[0134] In some embodiments, the Disclosure provides a method for treating or preventing cancer in a subject in need by administering a therapeutically effective amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof to the subject. In one embodiment, the cancer is a solid tumor cancer. In another embodiment, the solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple-negative breast cancer), uterine cancer (including serous uterine carcinoma and uterine carcinosarcoma), pancreatic cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, colorectal cancer, soft tissue sarcoma, skin cancer, bladder cancer, head and neck cancer, and glioma. In another embodiment, the solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple-negative breast cancer), uterine cancer (including serous uterine carcinoma and uterine carcinosarcoma), and pancreatic cancer. In another embodiment, the cancer is a hematological cancer. In another embodiment, the hematological cancer is a B-cell lymphoma.

[0135] In some embodiments, the Disclosure provides a method for treating or preventing cancer in a subject requiring such treatment, by administering a therapeutically effective amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof to the subject, wherein the cancer is, in whole or in part, a PKMYT1-dependent cancer. In one embodiment, the PKMYT1-dependent cancer is a solid tumor cancer. In another embodiment, the PKMYT1-dependent solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple-negative breast cancer), uterine cancer (including serous uterine carcinoma and uterine carcinosarcoma), pancreatic cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, colorectal cancer, soft tissue sarcoma, skin cancer, bladder cancer, head and neck cancer, and glioma. In another embodiment, the PKMYT1-dependent solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple-negative breast cancer), uterine cancer (including serous uterine carcinoma and uterine carcinosarcoma), and pancreatic cancer. In yet another embodiment, the PKMYT1-dependent cancer is a hematological cancer. In another aspect, PKMYT1-dependent hematological malignancies are B-cell lymphomas.

[0136] In some embodiments, the Disclosure provides a method for treating or preventing cancer in a subject requiring such treatment, by administering a therapeutically effective amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof to the subject, wherein the cancer is associated with a higher incidence of gene or protein abnormalities associated with elevated baseline levels of replication stress. In one embodiment, the cancer is characterized by gene amplification or overexpression. In another embodiment, the cancer is characterized by one or more deletions and / or mutations in a gene. In another embodiment, the cancer is a solid tumor cancer. In another embodiment, the solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple-negative breast cancer), uterine cancer (including serous uterine carcinoma and uterine carcinosarcoma), pancreatic cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, colorectal cancer, soft tissue sarcoma, skin cancer, bladder cancer, head and neck cancer, and glioma. In another embodiment, solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple-negative breast cancer), uterine cancer (including serous uterine carcinoma and uterine carcinosarcoma), and pancreatic cancer. In another embodiment, cancer is hematological cancer. In another embodiment, hematological cancer is B-cell lymphoma.

[0137] In some embodiments, the Disclosure provides a method for treating or preventing cancer in a subject requiring such treatment, by administering a therapeutically effective dose of a compound of formula (1) or a pharmaceutically acceptable salt thereof to the subject, wherein the cancer is associated with one or more abnormalities in a gene and / or its related protein product selected from the group consisting of cyclin E1, FBXW7, and / or KRAS. In one embodiment, the cancer is associated with one or more abnormalities in the cyclin E1 gene (CCNE1) and / or its related protein product. In another embodiment, the cancer is characterized by amplification or overexpression of CCNE1. In another embodiment, the cancer is associated with one or more abnormalities in the FBXW7 gene and / or its related protein product. In another embodiment, the cancer is characterized by one or more deletions and / or mutations in the FBXW7 gene. In another embodiment, the cancer is associated with one or more abnormalities in the KRAS gene and / or its related protein product. In another embodiment, the cancer is characterized by one or more deletions and / or mutations in the KRAS gene.

[0138] In some embodiments, the cancer is ovarian cancer. In one embodiment, the ovarian cancer is PKMYT1-dependent ovarian cancer. In another embodiment, the cancer is platinum-sensitive or platinum-resistant ovarian cancer.

[0139] In some embodiments, the cancer is breast cancer. In one embodiment, the breast cancer is PKMYT1-dependent breast cancer. In another embodiment, the breast cancer is selected from the group consisting of hormone receptor-positive (HR+) breast cancer, hormone receptor-negative (HR-) breast cancer, and triple-negative breast cancer. In another embodiment, the breast cancer is chemotherapy-resistant breast cancer. In another embodiment, the breast cancer is radiotherapy-resistant breast cancer. In another embodiment, the breast cancer is advanced or metastatic breast cancer.

[0140] In some embodiments, cancer is uterine cancer. In one embodiment, uterine cancer is PKMYT1-dependent uterine cancer. In another embodiment, uterine cancer is serous uterine carcinoma. In yet another embodiment, uterine cancer is carcinosarcoma.

[0141] In some embodiments, the cancer is pancreatic cancer. In one embodiment, the pancreatic cancer is PKMYT1-dependent pancreatic cancer.

[0142] In some embodiments, the cancer is lung cancer. In one embodiment, the lung cancer is PKMYT1-dependent lung cancer. In another embodiment, the lung cancer is small cell lung cancer (SCLC). In another embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In another embodiment, the non-small cell lung cancer (NSCLC) is squamous cell carcinoma. In another embodiment, the non-small cell lung cancer (NSCLC) is adenocarcinoma. In another embodiment, the non-small cell lung cancer (NSCLC) is large cell carcinoma.

[0143] In some embodiments, the cancer is gastric cancer. In one embodiment, the gastric cancer is PKMYT1-dependent gastric cancer.

[0144] In some embodiments, the cancer is esophageal cancer. In one embodiment, the esophageal cancer is PKMYT1-dependent esophageal cancer.

[0145] In some embodiments, the Disclosure provides a method for treating or preventing hematological cancer in a subject in need by administering a therapeutically effective amount of the Compounds of the Disclosure or a pharmaceutically acceptable salt thereof to the subject, wherein the hematological cancer is selected from the group consisting of non-Hodgkin lymphoma, leukemia, multiple myeloma (MM), and myelodysplastic syndrome (MDS). In one embodiment, the hematological cancer is PKMYT1-dependent hematological cancer. In another embodiment, the hematological cancer is non-Hodgkin's lymphoma (NHL). In another embodiment, non-Hodgkin's lymphoma (NHL) is selected from diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma (MCL), and marginal zone lymphoma. In another embodiment, the hematological cancer is leukemia. In another embodiment, leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). In another embodiment, hematological malignancy is multiple myeloma (MM). In another embodiment, hematological malignancy is myelodysplastic syndrome (MDS). In another embodiment, hematological malignancy is diffuse large B-cell lymphoma (DLBCL).

[0146] In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are administered as first-line therapy.

[0147] In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are administered as a second-line (or subsequent) therapy.

[0148] In some embodiments, subjects administered a therapeutically effective dose of the compound of this disclosure or a pharmaceutically acceptable salt thereof exhibit a partial response (PR) in response to such treatment.

[0149] In some embodiments, subjects administered a therapeutically effective dose of the compound of this disclosure or a pharmaceutically acceptable salt thereof exhibit a complete response (CR) in response to such treatment.

[0150] In some embodiments, subjects administered a therapeutically effective dose of the compound of this disclosure or a pharmaceutically acceptable salt thereof exhibit improved progression-free survival (PFS) in response to such treatment.

[0151] In some embodiments, subjects administered a therapeutically effective dose of the compound of this disclosure or a pharmaceutically acceptable salt thereof exhibit improved overall survival (OS) in response to such treatment.

[0152] The subjects of treatment are usually humans or non-human mammals, especially humans. Suitable subjects may also include domestic animals or wild animals; companion animals (including dogs, cats, etc.); livestock (including horses, cattle, and other ruminants, pigs, poultry, rabbits, etc.); primates (including monkeys, e.g., rhesus macaques, crab-eating macaques (also known as cercognates or long-tailed macaques), marmosets, tamarins, chimpanzees, macaques, etc.); and rodents (including rats, mice, gerbils, guinea pigs, etc.).

[0153] In some embodiments, the present disclosure provides compounds of formula (1) and pharmaceutically acceptable salts thereof for use as pharmaceuticals.

[0154] In some embodiments, the disclosure provides the use of a compound of formula (1) or a pharmaceutically acceptable salt thereof for treating or preventing cancer, including PKMYT1-dependent cancers as discussed above.

[0155] In some embodiments, the disclosure provides the use of a compound of formula (1) or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating or preventing cancer, including PKMYT1-dependent cancers as discussed above.

[0156] IV. Combination therapy and fixed dose combinations The compounds and pharmaceutically acceptable salts of the present disclosure may be used as single pharmacological agents or in combination with other pharmacological agents or techniques, such as DNA damaging agents that increase dependence on the G2 / M checkpoint and targeted therapies that induce higher levels of replication stress, in the manner described above. Such combination therapies may be achieved by administering the individual components of the therapy concurrently, sequentially, or individually. These combination therapies (and corresponding combination products) utilize the compounds and pharmaceutically acceptable salts of the present disclosure within the dosage ranges described in this application, as well as other pharmacological agents, typically within their approved dosage ranges.

[0157] In some embodiments, the present disclosure provides combinations suitable for use in the treatment or prevention of PKMYT1-dependent cancers, comprising the compounds of the present disclosure or pharmaceutically acceptable salts thereof and radiotherapy.

[0158] In some embodiments, the Disclosure provides combinations suitable for use in the treatment or prevention of PKMYT1-dependent cancers, comprising a compound of the Disclosure or a pharmaceutically acceptable salt thereof and a chemotherapy regimen. In one embodiment, the chemotherapy regimen is induction chemotherapy. In another embodiment, the chemotherapy regimen is consolidation chemotherapy. In yet another embodiment, the chemotherapy regimen comprises the administration of one or more chemotherapeutic agents selected from the group consisting of anthracyclines, azacitidine, bleomycin, cisplatin, carboplatin, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, fluorouracil, gemcitabine, idarubicin, mercaptopurine, methotrexate, oxaliplatin, paclitaxel, thioguanine, and / or vincristine.

[0159] In some embodiments, chemotherapy includes the administration of a nucleoside analog. In one embodiment, the nucleoside analog is selected from the group consisting of cytarabine and gemcitabine.

[0160] In some embodiments, chemotherapy includes the administration of a taxane. In one embodiment, the taxane is selected from the group consisting of paclitaxel, docetaxel, and nab-paclitaxel.

[0161] In some embodiments, chemotherapy includes the administration of a platinum-based chemotherapy agent. In one embodiment, the platinum-based chemotherapy agent is selected from the group consisting of carboplatin and cisplatin.

[0162] In some embodiments, chemotherapy further includes the administration of steroids. In one embodiment, the steroid is selected from the group consisting of prednisolone, dexamethasone, and hydrocortisone.

[0163] In some embodiments, the Disclosure provides combinations suitable for use in the treatment or prevention of PKMYT1-dependent cancers, comprising a compound of the Disclosure or a pharmaceutically acceptable salt thereof, and a topoisomerase 1 (TOP1) inhibitor, a topoisomerase 2 (TOP2) inhibitor, a TOP1-antibody drug conjugate (TOP1-ADC), or a TOP2-antibody drug conjugate (TOP2-ADC). In one embodiment, the TOP1 inhibitor is selected from the group consisting of irinotecan and topotecan. In one embodiment, the TOP1-ADC is trastuzumab deruxtecan. In another embodiment, the TOP2 inhibitor is selected from the group consisting of etoposide and teniposide.

[0164] In some embodiments, the Disclosure provides combinations suitable for use in the treatment or prevention of PKMYT1-dependent cancers, comprising a compound of the Disclosure or a pharmaceutically acceptable salt thereof and a poly ADP-ribose polymerase (PARP) inhibitor. In one embodiment, the PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, AZD5305 (CAS No. 2589531-76-8), and AZD9574 (CAS No. 2756333-39-6). In another embodiment, the PARP inhibitor is olaparib. In yet another embodiment, the PARP inhibitor is AZD5305.

[0165] In some embodiments, the Disclosure provides combinations suitable for use in the treatment or prevention of PKMYT1-dependent cancers, comprising a compound of the Disclosure or a pharmaceutically acceptable salt thereof, an ataxia vasodilator mutation, and a Rad3-associated kinase (ATR) inhibitor. In one embodiment, the ATR inhibitor is selected from the group consisting of camon sertib (Repare), tub sertib (Merck), belzo sertib (Merck KGaA), sera sertib (AstraZeneca), and elimusertib (Bayer). In another embodiment, the ATR inhibitor is sera sertib.

[0166] In some embodiments, the Disclosure provides combinations suitable for use in the treatment or prevention of PKMYT1-dependent cancers, comprising a compound of the Disclosure or a pharmaceutically acceptable salt thereof and a checkpoint kinase 1 (CHK1) inhibitor. In one embodiment, the CHK1 inhibitor is selected from the group consisting of bosutinib, crizotinib, dasatinib, erlotinib, gefitinib, lapatinib, pazopanib, ruxolitinib, sunitinib, and vemurafenib.

[0167] V. Pharmaceutical Compositions The compound of formula (1) and its pharmaceutically acceptable salts may be administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. Accordingly, in some embodiments, the present disclosure provides a pharmaceutical composition comprising the compound of formula (1) or its pharmaceutically acceptable salt and at least one pharmaceutically acceptable excipient.

[0168] The excipients selected for inclusion in a particular composition depend on factors such as the mode of administration and the form of the composition provided. Suitable pharmaceutically acceptable excipients are described, for example, in *Handbook of Pharmaceutical Excipients, Sixth Edition, Pharmaceutical Press*, Rowe, Ray C; Sheskey, Paul J; Quinn, Marian. Pharmacoagulable excipients can function, for example, as adjuvants, diluents, carriers, stabilizers, flavoring agents, colorants, fillers, binders, disintegrants, lubricants, flow enhancers, thickeners, and coating agents. As those skilled in the art will understand, a particular pharmaceutically acceptable excipient may perform two or more functions, depending on how much of the excipient is present in the composition and what other excipients are present in the composition.

[0169] The composition may be in a form suitable for oral use (e.g., as tablets, licks, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), a form suitable for topical use (e.g., as creams, ointments, gels, aqueous or oily solutions, or suspensions), a form suitable for administration by inhalation (e.g., as finely ground powders or liquid aerosols), a form suitable for administration by air (e.g., as finely ground powders), a form suitable for parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, or intramuscular administration), or a form suitable for suppositories for rectal administration. Compositions intended for oral use may contain, for example, one or more colorants, sweeteners, flavorings, and / or preservatives.

[0170] The total daily dose will inevitably vary depending on the patient being treated, the route of administration, any co-administered therapies, and the severity of the disease being treated, and may include single or multiple doses. Specific dosages may be adjusted, for example, depending on the condition being treated; the patient's age, weight, overall health, sex, and diet; the route of administration; the interval between doses; the rate of excretion; and other drugs administered concurrently to the patient. The compound of formula (1) or its pharmaceutically acceptable salts is typically 2.5–5000 mg / m². 2 It is administered to warm-blooded animals in units of (body area of ​​the animal) or within the range of approximately 0.05 to 100 mg / kg, which usually provides a therapeutically effective dose.

[0171] In some embodiments, the disclosure provides pharmaceutical compositions for use in therapy, comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0172] In some embodiments, the disclosure provides a pharmaceutical composition for use in the treatment of cancer, comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient. In one embodiment, the cancer is a PKMYT1-dependent cancer. In another embodiment, the PKMYT1-dependent cancer is a solid tumor cancer. In yet another embodiment, the PKMYT1-dependent cancer is a hematological cancer.

[0173] VI. Kit The disclosure further provides a kit comprising a unit dosage form containing a compound of formula (1) or a pharmaceutically acceptable salt thereof contained within a packaging material, and a label or accompanying document indicating that the unit dosage form can be used to treat one or more of the conditions described above.

[0174] In some embodiments, the kit comprises a unit dosage form containing a compound of formula (1) or a pharmaceutically acceptable salt thereof contained within packaging material, and a label or accompanying document indicating that the pharmaceutical composition can be used to treat cancer. In one embodiment, the cancer is a PKMYT1-dependent cancer. In another embodiment, the PKMYT1-dependent cancer is a hematological malignancy. In yet another embodiment, the PKMYT1-dependent cancer is a solid tumor cancer.

[0175] In some embodiments, the kit includes (a) a first unit dosage form comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof; (b) a second unit dosage form comprising a pharmacological agent selected from the group consisting of chemotherapeutic agents, TOP1 inhibitors, TOP2 inhibitors, TOP1-ADCs, TOP2-ADCs, PARP inhibitors, ATR inhibitors, and CHK1 inhibitors; (c) container means for housing the first and second dosage forms; and (d) a label or accompanying information indicating that the first and second unit dosage forms can be used to treat PKMYT1-dependent cancer.

[0176] VII. Preparation method This disclosure further provides processes for preparing compounds of formula (1) and pharmaceutically acceptable salts thereof.

[0177] The following schemes 1 to 11 illustrate the synthetic routes to the compound of formula (1). Those skilled in the art will understand that these methods are representative and do not include all possible methods for preparing the compounds of this disclosure. R in each scheme X The substituents are as defined for the compounds of this disclosure unless otherwise specified. It is understood that the preparation processes described in Schemes 1 to 11 can be carried out to obtain the compound of formula (1) or any stereoisomer of formula (1) starting from any enantiomer or racemic mixture of the intermediate compound. All starting materials are readily available or can be prepared as described in the examples.

[0178] [ka]

[0179] Scheme 1 shows a synthetic route to a specific compound of formula (1). Compound (D) can be obtained by reacting a compound of formula (B) (where X and Y are halogens such as fluoro, chloro, bromo, or iodine) with an arylamine of formula (C) (where PG is a protecting group such as OMe or OBn). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0180] The compound of formula (D) can be reacted with the malononitrile of formula (E) to obtain the compound of formula (F). The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu) and a suitable solvent (such as DME), typically at a temperature in the range of 80°C to 100°C.

[0181] The compound of formula (F) can be reacted with concentrated sulfuric acid to obtain the compound of formula (G). The protecting group in the compound of formula (G) can be removed under appropriate reaction conditions to obtain the compound of formula (3).

[0182] [ka]

[0183] Scheme 2 shows a synthetic route to a specific compound of formula (1). The compound of formula (H) can be reacted with the arylamine of formula (C) (PG is a protecting group such as OMe or OBn) to obtain the compound of formula (I). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0184] The compound of formula (I) can be reacted with the malononitrile of formula (E) to obtain the compound of formula J. The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu), using a suitable solvent (such as DME), and typically at a temperature in the range of 80°C to 100°C.

[0185] The compound of formula J can be reacted with concentrated sulfuric acid to obtain the compound of formula (K). The protecting group in the compound of formula (K) can be removed under appropriate reaction conditions to obtain the compound of formula (2).

[0186] [ka]

[0187] Scheme 3 shows a synthetic route to a specific compound of formula (1). The compound of formula (L) can be reacted with the arylamine of formula (C) (PG is a protecting group such as OMe or OBn) to obtain the compound of formula (M). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0188] The compound of formula (M) can be reacted with the malononitrile of formula (E) to obtain the compound of formula (N). The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu) and a suitable solvent (such as DME), typically at a temperature in the range of 80°C to 100°C.

[0189] The compound of formula (N) can be reacted with concentrated sulfuric acid to obtain the compound of formula (O). The protecting group in the compound of formula (O) can be removed under appropriate reaction conditions to obtain the compound of formula (4).

[0190] [ka]

[0191] Scheme 4 shows a synthetic route to a specific compound of formula (1). The compound of formula (P) can be reacted with the arylamine of formula (C) (PG is a protecting group such as OMe or OBn) to obtain the compound of formula (Q). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0192] The compound of formula (Q) can be reacted with the malononitrile of formula (E) to obtain the compound of formula (R). The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu) and a suitable solvent (such as DME), typically at a temperature in the range of 80°C to 100°C.

[0193] The compound of formula (R) can be reacted with concentrated sulfuric acid to obtain the compound of formula (S). The protecting group in the compound of formula (S) can be removed under appropriate reaction conditions to obtain the compound of formula (5).

[0194] [ka]

[0195] Scheme 5 shows a synthetic route to a specific compound of formula (1). The compound of formula (T) can be reacted with the arylamine of formula (C) (PG is a protecting group such as OMe or OBn) to obtain the compound of formula (U). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0196] The compound of formula (U) can be reacted with the malononitrile of formula (E) to obtain the compound of formula (V). The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu), using a suitable solvent (such as DME), and typically at a temperature in the range of 80°C to 100°C.

[0197] The compound of formula (V) can be reacted with concentrated sulfuric acid to obtain the compound of formula (W). The protecting group in the compound of formula (W) can be removed under appropriate reaction conditions to obtain the compound of formula (A1).

[0198] The compound of formula (A1) can be reacted with the boronic acid or ester of formula (B1) to obtain the compound of formula (3). The reaction can be catalyzed by a suitable Pd-precatalyst (XantPhos Pd G3, XPhos Pd G3, etc.) and ligand (XPhos, etc.) in the presence of a base (K2CO3, NaOtBu, Cs2CO3, etc.) and using a suitable solvent (1,4-dioxane / water mixture, toluene / water mixture, etc.) at a temperature typically in the range of 60°C to 120°C.

[0199] Furthermore, the compound of formula (A1) can be reacted with the alkyl or arylamine of formula (C1) to obtain the compound of formula (3). The reaction can be catalyzed by a suitable Pd-precatalyst (Pd(dba)2, Pd-PEPPSI-iHeptCl, BrettPhos Pd G3, etc.) and ligand (Xphos, Cphos, BrettPhos, etc.) in the presence of a base (LiHMDS, etc.) and using a suitable solvent (2-Me THF, 1,4-dioxane, t-BuOH, etc.) at a temperature typically in the range of 80°C to 120°C.

[0200] Furthermore, the compound of formula (A1) can be reacted with the aryl alcohol of formula (D1) to obtain the compound of formula (3). The reaction can be catalyzed by a suitable Pd-precatalyst (RockPhos Pd G3, Cphos Pd G3, etc.) and ligand (RockPhos, etc.) in the presence of a base (NaOtBu, Cs2CO3, K3PO4, etc.) and a suitable solvent (t-BuOH, 1,4-dioxane, etc.), typically at a temperature in the range of 60°C to 120°C.

[0201] [ka]

[0202] Scheme 6 shows a synthetic route to a specific compound of formula (1). The compound of formula (E1) can be reacted with the arylamine of formula (C) (PG is a protecting group such as OMe or OBn) to obtain the compound of formula (F1). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0203] The compound of formula (F1) can be reacted with the malononitrile of formula (E) to obtain the compound of formula (G1). The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu), using a suitable solvent (such as DME), and typically at a temperature in the range of 80°C to 100°C.

[0204] The compound of formula (G1) can be reacted with concentrated sulfuric acid to obtain the compound of formula (H1). The protecting group in the compound of formula (H1) can be removed under appropriate reaction conditions to obtain the compound of formula (I1).

[0205] Compound (2) can be obtained by reacting the compound of formula (I1) with the boronic acid or ester of formula (B1). The reaction can be catalyzed by a suitable Pd-precatalyst (XantPhos Pd G3, XPhos Pd G3, etc.) and ligand (XPhos, etc.) in the presence of a base (K2CO3, NaOtBu, Cs2CO3, etc.) and using a suitable solvent (e.g., 1,4-dioxane / water mixture, toluene / water mixture, etc.) at a temperature typically in the range of 60°C to 120°C.

[0206] Furthermore, the compound of formula (I1) can be reacted with the alkyl or arylamine of formula (C1) to obtain the compound of formula (2). The reaction can be catalyzed by a suitable Pd-precatalyst (Pd(dba)2, Pd-PEPPSI-iHeptCl, BrettPhos Pd G3, etc.) and ligand (Xphos, Cphos, BrettPhos, etc.) in the presence of a base (LiHMDS, etc.) and using a suitable solvent (2-Me THF, 1,4-dioxane, t-BuOH, etc.) at a temperature typically in the range of 80°C to 120°C.

[0207] Furthermore, the compound of formula (I1) can be reacted with the aryl alcohol of formula (D1) to obtain the compound of formula (2). The reaction can be catalyzed by a suitable Pd-precatalyst (RockPhos Pd G3, CPhos Pd G3, etc.) and ligand (RockPhos, etc.) in the presence of a base (NaOtBu, Cs2CO3, K3PO4, etc.) and a suitable solvent (t-BuOH, 1,4-dioxane, etc.), typically at a temperature in the range of 60°C to 120°C.

[0208] [ka]

[0209] Scheme 7 shows a synthesis route to a specific compound of formula (1). The compound of formula (J1) can be reacted with the boronic acid or ester of formula (B1) to obtain the compound of formula (K1). The reaction can be catalyzed by a suitable Pd-precatalyst (XantPhos Pd G3, XPhos Pd G3, etc.) and ligand (XPhos, etc.) in the presence of a base (K2CO3, NaOtBu, Cs2CO3, etc.) and using a suitable solvent (1,4-dioxane / water mixture, toluene / water mixture, etc.) at a temperature typically in the range of 60°C to 120°C.

[0210] The compound of formula (K1) can be reacted with concentrated sulfuric acid to obtain the compound of formula (L1). The protecting group in the compound of formula (L1) can be removed under appropriate reaction conditions to obtain the compound of formula (3).

[0211] [ka]

[0212] Scheme 8 shows a synthetic route to a specific compound of formula (1). The compound of formula (M1) can be reacted with the compound of formula (N1) (where X1 is typically a leaving group such as iodine, bromo, chloro, or triflate) to obtain the compound of formula (O1). The reaction can be carried out in the presence of a base (typically an inorganic base such as K2CO3), or catalyzed with a suitable Pd catalyst (such as BrettPhos Pd G3) in the presence of a base (such as Cs2CO3) and a solvent (such as t-BuOH, DMSO, or acetone).

[0213] Compound (P1) can be obtained by reacting compound (O1) with arylamine (C) (PG is a protecting group such as OMe or OBn). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0214] The compound of formula (P1) can be reacted with the malononitrile of formula (E) to obtain the compound of formula (Q1). The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu) and a suitable solvent (such as DME), typically at a temperature in the range of 80°C to 100°C.

[0215] The compound of formula (Q1) can be reacted with concentrated sulfuric acid to obtain the compound of formula (R1). The protecting group in the compound of formula (R1) can be removed under appropriate reaction conditions to obtain the compound of formula (6).

[0216] [ka]

[0217] Scheme 9 shows a synthetic route to a specific compound of formula (1). The compound of formula (S1) can be reacted with the compound of formula (N1) (where X1 is typically a leaving group such as iodine, bromo, chloro, or triflate) to obtain the compound of formula (T1). The reaction can be carried out in the presence of a base (typically an inorganic base such as K2CO3), or catalyzed with a suitable Pd catalyst (such as BrettPhos Pd G3) in the presence of a base (such as Cs2CO3) and a solvent (such as t-BuOH, DMSO, or acetone).

[0218] The compound of formula (T1) can be reacted with the arylamine of formula (C) (where PG is a protecting group such as OMe or OBn) to obtain the compound of formula (U1). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0219] The compound of formula (U1) can be reacted with the malononitrile of formula (E) to obtain the compound of formula (V1). The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu), using a suitable solvent (such as DME), and typically at a temperature in the range of 80°C to 100°C.

[0220] The compound of formula (V1) can be reacted with concentrated sulfuric acid to obtain the compound of formula (W1). The protecting group in the compound of formula (W1) can be removed under appropriate reaction conditions to obtain the compound of formula (7).

[0221] [ka]

[0222] Scheme 10 shows a synthetic route to a specific compound of formula (1). The compound of formula (A2) can be reacted with the compound of formula (N1) (where X1 is typically a leaving group such as iodine, bromo, chloro, or triflate) to obtain the compound of formula (B2). The reaction can be carried out in the presence of a base (typically an inorganic base such as K2CO3), or catalyzed with a suitable Pd catalyst (such as BrettPhos Pd G3) in the presence of a base (such as Cs2CO3) and a solvent (such as t-BuOH, DMSO, or acetone).

[0223] The compound of formula (B2) can be reacted with the arylamine of formula (C) (PG is a protecting group such as OMe or OBn) to obtain the compound of formula (C2). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0224] The compound of formula (C2) can be reacted with the malononitrile of formula (E) to obtain the compound of formula (D2). The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu), using a suitable solvent (such as DME), and typically at a temperature in the range of 80°C to 100°C.

[0225] The compound of formula (D2) can be reacted with concentrated sulfuric acid to obtain the compound of formula (E2). The protecting group in the compound of formula (E2) can be removed under appropriate reaction conditions to obtain the compound of formula (8).

[0226] [ka]

[0227] Scheme 11 shows a synthetic route to a specific compound of formula (1). The compound of formula (F2) can be reacted with the compound of formula (N1) (where X1 is typically a leaving group such as iodine, bromo, chloro, or triflate) to obtain the compound of formula (G2). The reaction can be carried out in the presence of a base (typically an inorganic base such as K2CO3), or catalyzed with a suitable Pd catalyst (such as BrettPhos Pd G3) in the presence of a base (such as Cs2CO3) and a solvent (such as t-BuOH, DMSO, or acetone).

[0228] The compound of formula (G2) can be reacted with the arylamine of formula (C) (PG is a protecting group such as OMe or OBn) to obtain the compound of formula (H2). The reaction can be carried out in the presence of a base (typically an organic base such as DIPEA) or an acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, or DMSO) at a temperature typically in the range of 80°C to 120°C.

[0229] The compound of formula (H2) can be reacted with the malononitrile of formula (E) to obtain the compound of formula (I2). The reaction can be catalyzed with a suitable Pd reagent (such as PdCl2(dppf)) in the presence of a base (such as NaOtBu) and a suitable solvent (such as DME), typically at a temperature in the range of 80°C to 100°C.

[0230] The compound of formula (I2) can be reacted with concentrated sulfuric acid to obtain the compound of formula (J2). The protecting group in the compound of formula (J2) can be removed under appropriate reaction conditions to obtain the compound of formula (9).

[0231] It should be understood that (i) the organic reactions described herein are carried out in accordance with laboratory practices known to those skilled in the art; (ii) some of the reactions described herein may optionally be carried out in a different order than those described herein; (iii) chiral isomers of the compounds of this disclosure may be separated at any stage of the synthetic process using chiral resolving agents described in the literature and known to those skilled in the art, or using chiral chromatography methods described in the literature and known to those skilled in the art, or as further described in the Examples; (iv) optionally, additional and / or other protecting groups may be required in some of the steps described herein; (v) therefore, optionally, the deprotection step may be carried out using methods described in the literature and known to those skilled in the art. Protection and deprotection of functional groups are described in "Protective Groups in Organic Synthesis" 3rd Ed, TW Greene and PGMWutz, Wiley-Interscience (1999), which is incorporated herein by reference.

[0232] VIII. Examples The following descriptions of experiments, procedures, examples, and intermediates are intended to illustrate, and not limit, embodiments of the present disclosure. Other compounds of the present disclosure may be prepared using the methods shown in these examples either alone or in combination with techniques generally known in the art.

[0233] A. General conditions Unless otherwise specified, 1 ¹H NMR spectra were obtained at 27°C using a Bruker 300 MHz, 400 MHz, or 500 MHz spectrometer unless otherwise specified; chemical shifts are expressed in parts per million (ppm, in δ units) and solvent residues. 1The reference values ​​are based on H isotopologs (CHCl3: 7.24 ppm; CHDCl2: 5.32 ppm; CD3S(=O)CD2H: 2.49 ppm). Binding constants are given in Hertz (Hz). Splitting patterns represent apparent multiplicity and are specified as s (singular), d (double), t (tripular), q (quadular), m (multiple), and br s (broad single). LC-MS was performed using a Waters UPLC equipped with a Waters SQD mass spectrometer, or a Shimadzu LC-20AD, LC-20XR, or LC-30AD equipped with a Shimadzu 2020 mass spectrometer. Unless otherwise specified, the reported molecular ions are [M+H]. + This corresponds to; for molecules with multiple isotopic patterns (such as Br and Cl), the reported values ​​are obtained at the lowest isotopic mass unless otherwise specified.

[0234] Flash chromatography was performed using a Biotage® SP1® purification system, an ISCO CombiFlash® Rf system, or a Thermo Fisher Gilson system, using normal-phase silica FLASH+® (40M, 25M, or 12M) or SNAP® KP-Sil cartridges (340, 100, 50, or 10), or an Agela Flash Column silica-CS column in conjunction with a C18 flash column, for either straight-phase or standard flash chromatography. In general, all solvents used were commercially available analytical solvents. Anhydrous solvents were those commonly used for the reaction. The phase separator used in the examples was an ISOLUTE® Phase Separator column. Starting materials were obtained from commercial sources or prepared through literature channels.

[0235] Use the following abbreviations: ACN = Acetonitrile; BrettPhos = 2-(dicyclohexylphosphino)3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl; BrettPhos Pd G3 = [(2-di-cyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-amino-1,1'-biphenyl)] palladium(II) methanesulfonate; CPhos = 2-dicyclohexylphosphino-2',6'-bis(N,N-dimethylamino)biphenyl; CPhos Pd G3 = [(2-dicyclohexylphosphin-2',6'-bis(N,N-dimethylamino)-1,1'-biphenyl)-2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate; BSA = Bovine serum albumin; Cs2CO3 = Cesium carbonate; DCM = Dichloromethane; DEA = Diethylamine, DIPEA = Diisopropylethylamine; DME = Dimethoxyethane; DMSO = Dimethyl sulfoxide; DMSO-d6 = Deuterated Dimethyl Sulfoxide; dppf = 1,1'-bis(diphenylphosphin)ferrocene; EA = ethyl acetate; HBSS = Hanks equilibrium salt solution; HCl = hydrochloric acid; HP = High pressure; H2SO4 = sulfuric acid; IPA = Isopropyl alcohol; IPTG = Isopropyl β-D-1-thiogalactopyranoside; K2HPO4 = potassium hydrogen phosphate; K3PO4 = potassium phosphate; LC = Liquid Chromatography; LiHMDS = Lithium bis(trimethylsilyl)amide; 2-Me THF = 2-methyltetrahydrofuran; MeCN = Acetonitrile; MeOH = methanol; MgSO4 = Magnesium sulfate; MS=mass spectrometry; NaHCO3 = sodium bicarbonate; NaOtBu = sodium tert-butoxide, NH4HCO3 = Ammonium bicarbonate; NMR=nuclear magnetic resonance; NaOH = sodium hydroxide; OBn = benzyl oxy OMe = Methoxy; PBS = Phosphate-buffered saline; Pd = Palladium; PdCl2(dppf) = 1,1'-bis(di-tert-butylphosphin)ferrocenepalladium dichloride; Pd2dba3 = Tris(dibenzylideneacetone)dipalladium(0); Pd-PEPPSI-iHeptCl=Dichloro[1,3-bis(2,6-di-4-heptylphenyl)imidazole-2-ylidene](3-chloropyridyl)palladium(II); PMSF = Phenylmethylsulfonyl fluoride; PFA = Paraformaldehyde; RockPhos Pd G3 = [(2-di-tert-butylphosphino-3-methoxy-6-methyl-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2-aminobiphenyl)]-palladium(II) methanesulfonate; Rt or RT = holding time; RU = Response Unit; tBuOH = tert-butanol; TCEP = Tris(2-carboxyethyl)phosphine; Xantphos = 4,5-bis(diphenylphosphin)-9,9-dimethylxanthene; and XPhos=2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl.

[0236] The examples and intermediate compounds are named using PerkinElmer's ChemDraw Professional version 21.0.0. ChemDraw Professional version 21.0.0 generates chemical structure names using the Cahn-Ingold-Prelog (CIP) rules for stereochemistry and adheres as strictly as possible to IUPAC rules when generating chemical names. Stereoisomers are distinguished from each other by stereodescriptors cited in the names and assigned according to the CIP rules.

[0237] ChemDraw optionally uses stereocenter labels such as "&" and "or" to indicate the stereochemical configurations of stereocenters present in a structure. Generally, the chemical structure of an example or intermediate containing the label "&" at a stereocenter means that the configuration of such example or intermediate at that stereocenter is a mixture of both (R) and (S), while the label "or" means that the configuration of such example or intermediate at that stereocenter is either (R) or (S). All unspecified absolute stereocenters "&" and "or" can exist within a single structure.

[0238] In general, for embodiments and intermediate structures where all stereocenters are specified as "&", the structure is named using the prefix "rac-". For embodiments and intermediate structures where all stereocenters are specified as "or", the structure is named using the prefix "rel-".

[0239] Generally, examples and intermediate compounds are named using descriptors (RS) and (SR) to indicate the general "&" center of a chemical structure having multiple chiral centers, some of which are designated as "&". * ) and (S * ) is used to indicate the general "or" center of a chemical structure that has multiple chiral centers, some of which are indicated as "or".

[0240] B. Compound Example B-1: (S)-6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound 1)

[0241] [ka]

[0242] Step a) 5-bromo-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine (intermediate 3) Concentrated HCl (0.055 mL, 1.81 mmol) was added to 5-bromo-4-chloro-2-methylpyrimidine (intermediate 1, 1.5 g, 7.23 mmol) and 3-methoxy-2,6-dimethylaniline (intermediate 2, 1.093 g, 7.23 mmol) in tBuOH (20 mL). The resulting mixture was stirred at 100°C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography with an elution gradient of 0-20% MeOH in DCM, and further purified by flash C18-flash chromatography with an elution gradient of 0-100% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 5-bromo-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine (intermediate 3, 1.800 g, 77%) as a yellow solid. 1 H NMR(DMSO-d6,300MHz)δ1.94(3H,s),2.04(3H,s),2.38(3H,s),3.80(3H,s),6.93(1H,d),7.13(1H,d),8.81(1H,s),10.06(1H,br s);m / z(ES + )[M+H] + =322.

[0243] Step b) 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 4) Sodium t-butoxide (0.597 g, 6.21 mmol) was slowly added to a solution of malononitrile (0.431 g, 6.52 mmol) in DME (15 mL). The mixture was stirred at 23°C for 30 minutes. 5-bromo-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine (intermediate 3, 1.000 g, 3.10 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.227 g, 0.31 mmol) were added to the mixture under nitrogen gas. The resulting mixture was stirred at 90°C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient: 0-10% MeOH in DCM. The pure fraction was evaporated to dryness to obtain 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 4, 0.670 g, 70.2%) as a red solid. 1 H NMR(DMSO-d6,300MHz)δ1.71(3H,s),1.80(3H,s),2.44(3H,s),3.85(3H,s),7.11(1H,d),7.27(3H,m),8.49(1H,s);m / z(ES + )[M+H] + = 308.

[0244] Step c) (S)-6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 1)

[0245] [ka]

[0246] Methanesulfonic acid (4521 mg, 47.05 mmol) was added dropwise to a solution of H2SO4 (7.5 mL, 140.71 mmol) in water (0.5 mL). The mixture was stirred at 23°C for 5 minutes. 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 4, 570 mg, 1.85 mmol) was slowly added to the stirred solution at below 40°C. The mixture was stirred at room temperature for 90 minutes. DL-methionine (1107 mg, 7.42 mmol) was slowly added to the stirred mixture at below 40°C. The mixture was stirred at below 40°C for 15 minutes. The resulting mixture was then stirred at 40°C for 16 hours. The mixture was cooled to room temperature and slowly added to an aqueous solution of K2HPO4 (0.7 g) and NaOH (3.4 g) in water (30 mL). EA (50 mL) was added, and the mixture was stirred for 5 minutes to obtain a precipitate. The precipitate was collected by filtration. The crude product was purified as follows: Preparative HPLC column: XBridge Prep OBD C18 column, 30 × 150 mm, 5 μm; Mobile phase A: Water (10 mmol / L NH4HCO3 + 0.05% NH3H2O), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 5%B to 30%B over 8 minutes. The fraction containing the desired compound was evaporated to dryness to obtain 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide as a white solid. The racemic mixture was purified by preparative chiral HPLC using a LuxCellulose column (44.6 × 50 mm, 3 μm); mobile phase A: CO2, mobile phase B: MeOH (0.1% DEA); flow rate: 4 mL / min; gradient: uniform concentration %B). The fraction containing the desired compound was evaporated to dryness to obtain (S)-6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Example B-1, isomer 1, 105 mg, 17.12%) as a white solid and (R)-6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Example B-1, isomer 2, 106 mg, 17.47%) as a white solid. 1H NMR(400MHz,DMSO-d6,24℃)δ1.67(3H,s),1.76(3H,s),2.44(3H,s),6.84(2H ,s),6.94(1H,d),7.05(2H,s),7.08(1H,d),8.88(1H,s),9.67(1H,s);m / z(ES + )[M+H] + =312.

[0247] Example B-2: (S)-or (R)-6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 137)

[0248] [ka]

[0249] Intermediate 6: 5-bromo-2-methyl-6-(trifluoromethyl)pyrimidine-4(3H)-one Azobisisobutyronitrile (AIBN) (0.184 g, 1.12 mmol) was added to 2-methyl-6-(trifluoromethyl)pyrimidine-4(3H)-one (intermediate 5, 2.00 g, 11.2 mmol) and NBS (2.398 g, 13.47 mmol) in MeCN (50 mL). The resulting mixture was stirred at 80°C for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient: 0-20% ethyl phosphate in DCM. The pure fraction was evaporated to dryness to obtain 5-bromo-2-methyl-6-(trifluoromethyl)pyrimidine-4(3H)-one (intermediate 6, 2.40 g, 83%) as a white solid. 1 H NMR(300MHz,DMSO-d6,25℃)δ2.3(3H,s),13.46(1H,s);m / z(ES + )[M+H] + =257.

[0250] Intermediate 7: 5-bromo-4-chloro-2-methyl-6-(trifluoromethyl)pyrimidine Phosphorus oxychloride (3.58 g, 23.4 mmol) was added to 5-bromo-2-methyl-6-(trifluoromethyl)pyrimidine-4(3H)-one (intermediate 6, 2.00 g, 7.78 mmol) in MeCN (20 mL). The resulting solution was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient: 0-30% ethyl phosphate in DCM. The pure fraction was evaporated to dryness to obtain 5-bromo-4-chloro-2-methyl-6-(trifluoromethyl)pyrimidine (intermediate 7, 0.75 g, 35%) as a colorless liquid. 1 H NMR (300MHz, DMSO-d6, 23℃) δ2.65 (3H, s).

[0251] Intermediate 8: 5-bromo-N-(3-methoxy-2,6-dimethylphenyl)-2-methyl-6-(trifluoromethyl)pyrimidine-4-amine 3-Methoxy-2,6-dimethylaniline (intermediate 2, 601 mg, 3.98 mmol) was added to 5-bromo-4-chloro-2-methyl-6-(trifluoromethyl)pyrimidine (intermediate 7, 730 mg, 2.65 mmol). The resulting mixture was stirred at 140°C for 16 hours. The reaction mixture was purified by flash silica chromatography, elution gradient: 0-30% ethyl phosphate (containing 50% DCM) in petroleum ether. The pure fraction was evaporated to dryness to obtain 5-bromo-N-(3-methoxy-2,6-dimethylphenyl)-2-methyl-6-(trifluoromethyl)pyrimidine-4-amine (intermediate 8, 780 mg, 75%) as a colorless liquid. 1 H NMR(300MHz,DMSO-d6,24℃)δ1.99(6H,d),2.26(3H,s),3.80(3H,s),6.89(1H,d),7.11(1H,d),9.11(1H,s);m / z(ES + )[M+H] + =390.

[0252] Intermediate 9: 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile Sodium t-butoxide (517 mg, 5.38 mmol) was slowly added to a solution of malononitrile (284 mg, 4.31 mmol) in DME (10 mL). The mixture was stirred at room temperature for 30 minutes. 5-bromo-N-(3-methoxy-2,6-dimethylphenyl)-2-methyl-6-(trifluoromethyl)pyrimidine-4-amine (intermediate 8, 840 mg, 2.15 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (158 mg, 0.220 mmol) were added to the mixture. The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was filtered. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient: 0-80% ethylethanol (containing 50% DCM) in petroleum ether. The pure fraction was evaporated to dryness to obtain 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 9, 650 mg, 80%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6,24℃)δ1.72(3H,s),1.81(3H,s),2.52(3H,s),3.86(3H,s),7.14(1H,d),7.28(1H,d),7.80(2H,s);m / z(ES + )[M+H] + =376.

[0253] Intermediate 10: 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 9, 630 mg, 1.68 mmol) was added to sulfuric acid (6 mL). The resulting solution was stirred at room temperature for 1 day. The reaction mixture was poured into water (50 mL), basicized with ammonia, and then extracted with ethylacetal (3 × 50 mL). The organic layer was dried over MgSO4, filtered, and evaporated to obtain crude 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (intermediate 10, 650 mg, 98%) as a yellow solid, which was used without further purification. 1 H NMR(300MHz,DMSO-d6,23℃)δ1.70(3H,s),1.80(3H,s),2.47(3H,s),3.85(3H,s),6.76(2H,s),6.94(2H,s),7.12(1H,d),7.26(1H,d);m / z(ES + )[M+H] + =394.

[0254] (S)-or (R)-6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 137)

[0255] [ka]

[0256] BBr3 (3.05 mL, 3.05 mmol) in DCM was added dropwise to 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (intermediate 10, 300 mg, 0.760 mmol) in DCM (5 mL) at 0°C. The resulting mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by C18-flash chromatography using an elution gradient of 0.1% NH4HCO3 in water with 0-60% MeCN. The pure fraction was evaporated to dryness to obtain (rac)-6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (210 mg, 73%) as a white solid.

[0257] The racemic mixture was purified by preparative chiral HPLC using a CHIRALPAK IC column (2 × 25 cm, 5 μm), mobile phase A: HEX (0.5% 2M NH3-MeOH), mobile phase B: ETOH, flow rate: 20 mL / min, gradient: uniform concentration 7, wavelength: 220 / 254 nm, RT1 (min): 18.873, RT2 (min): 22.707, sample solvent: EtOH, injection volume: 0.4 mL, and 22 runs. The fraction containing the desired compound was evaporated to dryness to obtain rotational isomer 1 and rotational isomer 2, (S)- or (R)-6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Example B-2, 167 mg, 34%) as a white solid. 1 H NMR(400MHz,DMSO-d6,23℃)δ1.68(3H,s),1.77(3H,s),2.49(3H,s),6.75(2H,s),6.90-7.00(3H,m),7.10(1H,d),9.64(1H,s);m / z(ES + )[M+H] + =380.

[0258] Example B-3: (S)-or (R)-6-amino-4-(difluoromethyl)-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound 138)

[0259] [ka]

[0260] Intermediate 13: 6-(difluoromethyl)-2-methylpyrimidine-4(3H)-one A mixture of sodium ethoxide (3.07 g, 45.2 mmol) in ethanol (50.0 mL) was added at 25°C to a stirred mixture of ethyl 4,4-difluoro-3-oxobutanoate (intermediate 11, 5.00 g, 30.1 mmol) and acetimidoamide hydrochloride (intermediate 12, 2.85 g, 30.1 mmol) in toluene (100 mL). The resulting mixture was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient: 0-40% MeOH in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 6-(difluoromethyl)-2-methylpyrimidine-4(3H)-one (intermediate 13, 1.8 g, 37%) as a white solid. 1 H NMR(300MHz,DMSO-d6,26℃)δ2.33(3H,s),6.42(1H,s),6.67(1H,t),12.77(1H,s);m / z(ES + )[M+H] + = 161.

[0261] Intermediate 14: 5-bromo-6-(difluoromethyl)-2-methylpyrimidine-4(3H)-one AIBN (0.185 g, 1.12 mmol) was added to 6-(difluoromethyl)-2-methylpyrimidine-4(3H)-one (intermediate 13, 1.80 g, 11.2 mmol) and NBS (1.20 g, 6.74 mmol) in MeCN (50 mL). The resulting mixture was stirred at 80°C for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient: 0-100% SiO (containing 50% DCM) in petroleum ether. The pure fraction was evaporated to dryness to obtain 5-bromo-6-(difluoromethyl)-2-methylpyrimidine-4(3H)-one (intermediate 14, 1.4 g, 52%) as a white solid. 1 H NMR(500MHz,DMSO-d6,25℃)δ2.34(3H,s),6.99(1H,t),13.25(1H,s);m / z(ES + )[M+H] + =239.

[0262] Intermediate 15: 5-bromo-4-chloro-6-(difluoromethyl)-2-methylpyrimidine Phosphorus oxychloride (8.34 g, 54.4 mmol) was added to 5-bromo-6-(difluoromethyl)-2-methylpyrimidine-4(3H)-one (intermediate 14, 1.30 g, 5.44 mmol) in MeCN (30 mL). The resulting solution was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient: 0-50% ethyl phosphate in DCM. The pure fraction was evaporated to dryness to obtain 5-bromo-4-chloro-6-(difluoromethyl)-2-methylpyrimidine (intermediate 15, 1.1 g, 79%) as a colorless liquid. 1 H NMR(300MHz,DMSO-d6,25℃)δ2.66(3H,s),7.17(1H,t);m / z(ES + )[M+H] + =257.

[0263] Intermediate 16: 5-bromo-6-(difluoromethyl)-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine 5-Bromo-4-chloro-6-(difluoromethyl)-2-methylpyrimidine (intermediate 15, 1.00 g, 3.88 mmol) was added to 3-methoxy-2,6-dimethylaniline (intermediate 2, 0.705 g, 4.66 mmol). The resulting mixture was stirred at 140°C for 16 hours. The crude product was purified by flash silica chromatography, elution gradient: 0-60% ethyl phosphate in petroleum ether. The pure fraction was evaporated to dryness to obtain 5-bromo-6-(difluoromethyl)-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine (intermediate 16, 0.70 g, 48%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6,23℃)δ1.93(3H,s),2.02(3H,s),2.24(3H,s),3.78(3H,s),6.87(1H,d),7.02(1H,t),7.09(1H,d),8.89(1H,s);m / z(ES + )[M+H] + =372.

[0264] Intermediate 17: 6-amino-4-(difluoromethyl)-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile Sodium t-butoxide (452 ​​mg, 4.70 mmol) was slowly added to a solution of malononitrile (248 mg, 3.76 mmol) in DME (10 mL). The mixture was stirred at room temperature for 30 minutes. 5-bromo-6-(difluoromethyl)-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine (intermediate 16, 700 mg, 1.88 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) (138 mg, 0.190 mmol) were added to the mixture. The resulting mixture was stirred at 80°C for 2 hours. The reaction mixture was poured into saturated NaHCO3 (50 mL), extracted with siRNA (3 × 50 mL), the organic layer was dried over Na2SO4, filtered, and evaporated to obtain a yellow solid. The crude product was purified by flash silica chromatography with an elution gradient of 0-60% ethyl phosphate in petroleum ether. The pure fraction was evaporated to dryness to obtain 6-amino-4-(difluoromethyl)-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 17, 570 mg, 85%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6,24℃)δ1.76(6H,d),2.48(3H,s),3.85(3H,s),7.04(1H,s),7.12(1H,d),7.26(1H,d),7.59(2H,s);m / z(ES + )[M+H] + = 358.

[0265] Intermediate 18: 6-amino-4-(difluoromethyl)-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide 6-amino-4-(difluoromethyl)-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 17, 520 mg, 1.46 mmol) was added to sulfuric acid (6 mL). The resulting solution was stirred at room temperature for 1 day. The reaction mixture was poured into water (50 mL), basicized with ammonia, and then extracted with ethyl acetate (3 × 50 mL). The organic layer was dried over MgSO4, filtered, and evaporated to obtain crude 6-amino-4-(difluoromethyl)-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (intermediate 18, 500 mg, 92%) as a yellow solid, which was used without further purification. 1 H NMR(300MHz,DMSO-d6,24℃)δ1.71(3H,s),1.81(3H,s),2.48(3H,s),3.87(3H ,s),6.68(2H,s),7.05(2H,s),7.13(1H,d),7.28(1H,d),7.73(1H,s);m / z(ES + )[M+H] + =376.

[0266] (S)-or (R)-6-amino-4-(difluoromethyl)-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 138)

[0267] [ka]

[0268] BBr3 (3.20 mL, 3.20 mmol) in DCM was added dropwise to 6-amino-4-(difluoromethyl)-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (intermediate 18, 300 mg, 0.80 mmol) in DCM (2 mL) at 0°C. The resulting mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C1-8 flash chromatography, elution gradient: 0.1% NH4HCO3 modified water with 0-60% MeCN. The pure fraction was evaporated to dryness to obtain (rac)-6-amino-4-(difluoromethyl)-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (90 mg, 31%) as a white solid.

[0269] The racemic mixture was purified by preparative chiral HPLC (column: CHIRALPAK IE, 2 × 25 cm, 5 μm; mobile phase A: Hex (0.5% 2M NH3-MeOH), mobile phase B: EtOH; flow rate: 20 mL / min; gradient: uniform concentration 10; wavelength: 220 / 254 nm; RT1 (min): 17.882; RT2 (min): 24.125; sample solvent: EtOH; injection volume: 0.4 mL; number of runs: 13) to obtain rotational isomer 1 and rotational isomer 2, (S) or (R)-6-amino-4-(difluoromethyl)-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Example B-3, 17.40 mg, 17.40%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6,23℃)δ1.68(3H,s),1.77(3H,s),2.49(3H,s),6.65(2H,s),6.96(1H,d),7.00-7.14(3H,m),7.74(1H,t),9.63(1H,s);m / z(ES + )[M+H] + =362.

[0270] Example B-4: (S)-or (R)-6-amino-4-cyclopropyl-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound 139)

[0271] [ka]

[0272] Intermediate 20: 6-Methoxy-N-(3-Methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine Xantphos Pd G3 (4.49 g, 4.73 mmol) was added under nitrogen to 1,4-dioxane (300 mL) containing 4-chloro-6-methoxy-2-methylpyrimidine (intermediate 19, 15.0 g, 94.6 mmol), 3-methoxy-2,6-dimethylaniline (intermediate 2, 15.73 g, 104.1 mmol), and Cs2CO3 (92.0 g, 284 mmol). The resulting mixture was stirred at 100°C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient: 0-40% SiO in petroleum ether. The pure fraction was evaporated to dryness to obtain 6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine (intermediate 20, 17 g, 66%) as a pale yellow solid. 1 H NMR(300MHz, CDCl3, 25℃) δ2.11(3H,s),2.17(3H,s),2.45(3H,s),3.84(3H, m / z(ES + )[M+H] + = 274.

[0273] Intermediate 21: 5-bromo-6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine N-bromosuccinimide (9.77 g, 54.9 mmol) was added to 6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine (intermediate 20, 15.0 g, 54.9 mmol) in DCM (150 mL) at 25°C. The resulting solution was stirred at 0°C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient: 0-30% ethyl phosphate in petroleum ether. The pure fraction was evaporated to dryness to obtain 5-bromo-6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine (intermediate 21, 15 g, 78%) as a pale yellow solid. 1 H NMR(300MHz,CDCl3,25℃)δ2.10(3H,s),2.17(3H,s),2.34(3H,s),3.86(3H,s),4.03(3H,s),6.54(1H,s),6.79(1H,d),7.08(1H,d);m / z(ES + )[M+H] + =352.

[0274] Intermediate 22: 6-amino-4-methoxy-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile Sodium t-butoxide (8.19 g, 85.2 mmol) was slowly added to a solution of malononitrile (3.38 g, 51.1 mmol) in DME (100 mL). The mixture was stirred at room temperature for 30 minutes. 5-bromo-6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidine-4-amine (intermediate 21, 15.0 g, 42.6 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (3.12 g, 4.26 mmol) were added to the mixture. The resulting mixture was stirred at 100 °C for 2 hours. The reaction mixture was poured into saturated NaHCO3 (100 mL), extracted with ELISA (3 × 150 mL), the organic layer was dried over Na2SO4, filtered, and evaporated to obtain a yellow solid. The crude product was purified by flash silica chromatography, elution gradient: 0-60% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 6-amino-4-methoxy-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 22, 12 g, 84%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6,25℃)δ1.71(3H,s),1.80(3H,s),2.38(3H,s),3.85(3H,s),4.01(3H,s),6.88(2H,s),7.10(1H,d),7.24(1H,d);m / z(ES + )[M+H] + = 338.

[0275] Intermediate 23: 6-amino-4-hydroxy-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile Lithium chloride (7.54 g, 178 mmol) was added at room temperature to 6-amino-4-methoxy-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 22, 12.0 g, 35.6 mmol) and p-toluenesulfonic acid (18.37 g, 106.7 mmol) in DMF (120 mL). The resulting mixture was stirred at 100 °C for 3 hours. The reaction mixture was poured into water (750 mL), extracted with siRNA (5 × 200 mL), the organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain a gray solid. The gray solid was washed with water (2 × 75 mL), filtered, and dried to obtain a clean gray solid, 6-amino-4-hydroxy-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 23, 9.00 g, 78%). The product was used directly in the next step without further purification. 1 H NMR(300MHz,DMSO-d6,25℃)δ1.73(3H,s),1.83(3H,s),2.17(3H,s),3.83(3H,s),6.40(2H,s),7.07(1H,d),7.21(1H,d),12.12(1H,s);m / z(ES + )[M+H] + = 324.

[0276] Intermediate 24: 6-amino-4-chloro-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 6-amino-4-hydroxy-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 23, 12 g, 37 mmol) was slowly added to POCl3 (60 mL) at 0°C. The resulting solution was stirred at 100°C for 2 hours. The solvent was removed under reduced pressure. The residue was poured into ice water (200 mL), and the resulting mixture was basicized with saturated NaHCO3 and extracted with siRNA (200 mL). The organic layer was washed with water (100 mL x 2), dried over Na2SO4, filtered, and evaporated to obtain the crude product. The crude product was purified by flash C18-flash chromatography, elution gradient: 0-50% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 6-amino-4-chloro-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 24, 9.00 g, 71%) as a white solid. 1 H NMR(300MHz,DMSO-d6,25℃)δ1.72(3H,s),1.81(3H,s),2.43(3H,s),3.85(3H,s),7.12(1H,d),7.26(1H,d),7.50(2H,s);m / z(ES + )[M+H] + =342.

[0277] Intermediate 25: 6-amino-4-chloro-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide 6-amino-4-chloro-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (intermediate 24, 4.00 g, 11.70 mmol) was added all at once to H2SO4 (3 mL) at 25°C. The resulting mixture was stirred at 25°C for 2 hours. The reaction mixture was poured into ice water (75 mL). The resulting mixture was basicized with ammonia and extracted with siRNA (3 × 100 mL). The organic layer was dried over MgSO4, filtered, and evaporated to obtain crude 6-amino-4-chloro-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (intermediate 25, 2.60 g, 62%) as a pale yellow solid. The product was used directly in the next step without further purification. 1 ¹H NMR (300MHz, DMSO-d6, 24℃) δ 1.71(3H,s), 1.81(3H,s), 2.42(3H,s), 3.86(3H,s), 7.13(1H,d), 7.28(3H,m), two proton exchanges; m / z (ES + )[M+H] + =360.

[0278] Intermediate 26: 6-amino-4-chloro-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide BBr3 (28.9 mL, 28.9 mmol) in DCM was added dropwise to 6-amino-4-chloro-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (intermediate 25, 2.60 g, 7.23 mmol) in DCM (11 mL) at -5°C. The resulting mixture was stirred at 0°C for 1 hour. The reaction mixture was poured into ice water (50 mL). The resulting mixture was basicized with NaHCO3, extracted with ELISA (5 × 100 mL), and the organic layer was dried over MgSO4, filtered, and evaporated. The crude product was purified by flash C18 flash chromatography, elution gradient: 5-60% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 6-amino-4-chloro-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (intermediate 26, 1.90 g, 76%) as a white solid. 1 H NMR(300MHz,DMSO-d6,24℃)δ1.68(3H,s),1.76(3H,s),2.42(3H,d),5.76(1H,d),6.95(1H,d),7.09(2H,d),7.24(2H,s),9.64(1H,s);m / z(ES + )[M+H] + =346.

[0279] (S)-or (R)-6-amino-4-cyclopropyl-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound 139)

[0280] [ka]

[0281] 1,1'-Bis(diphenylphosphino)ferrocenedichloropalladium(II) dichloromethane adduct (0.354 g, 0.430 mmol) was added to 1,4-dioxane (20 mL) containing 6-amino-4-chloro-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (intermediate 26, 1.00 g, 2.89 mmol), cyclopropylboronic acid (0.373 g, 4.34 mmol), and K2CO3 (1.20 g, 8.68 mmol), under nitrogen at room temperature. The resulting mixture was stirred at 100°C for 16 hours. The reaction mixture was poured into water (100 mL), extracted with siRNA (3 × 50 mL), the organic layer was dried over Na2SO4, filtered, and evaporated to obtain a black solid. The crude product was purified by flash C18-flash chromatography, elution gradient: 0-50% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain (rac)-6-amino-4-cyclopropyl-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (14 mg, 1.4%) as a white solid.

[0282] The racemic mixture was purified by preparative chiral HPLC using a column: Lux 5um Cellulose-4, 2.12 × 25 cm, 5 μm; mobile phase A: Hex (0.5% 2M NH3-MeOH), mobile phase B: EtOH; flow rate: 220 mL / min; gradient: uniform concentration 30; wavelength: 220 / 254 nm; RT1 (min): 12.032; RT2 (min): 17.364; sample solvent: ETOH; injection volume: 1.5 mL; number of runs: 3 to obtain rotational isomer 1, (S) or (R)-6-amino-4-cyclopropyl-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Example B-4), 2.77 mg, 21%) and rotational isomer 2 as white solids. 1H NMR(400MHz,DMSO-d6,22℃)δ1.00(2H,dq),1.18(2H,p),1.67(3H,s),1.76(3H,s),2.34 (3H,s),2.77(1H,tt),6.48(2H,s),6.80-6.96(3H,m),7.07(1H,d),9.59(1H,s);m / z(ES + )[M+H] + =352.

[0283] C. Biological data Example C-1: Increased basal replication stress induces PKMYT1 inhibitor sensitivity A. Method In this study, the concentration of a PKMYT1 inhibitor that inhibits cell proliferation by 50% in vitro (GI) 50 ) correlated with baseline levels of replication stress in several cell line models. GI 50 To establish the values, cells were seeded on plates and exposed to a 1 / 2 log9 point dose response of a PKMYT1 inhibitor. After 7 days of exposure to the PKMYT1 inhibitor, cells were treated with Cell-Titer Glo (Promega), which allowed for the determination of the number of viable cells in culture by quantifying adenosine triphosphate (ATP), an indicator of metabolically active cells. An S-shaped standard curve (data not shown) was established, and the mean GI was determined. 50 The values ​​were interpolated from three (n=3±SD) biological copies.

[0284] Next, GI 50The values ​​were correlated with the baseline levels of replication stress for the 16 cell line models identified in Figures 2, 3, and 4. The baseline levels of replication stress for the uterine and breast cancer cell line models used in this study were determined by DNA combing analysis as described in Young, LA, et al. (2019). "Differential Activity of ATR and WEE1 Inhibitors in a Highly Sensitive Subpopulation of DLBCL Linked to Replication Stress." Cancer Research 79(14):3762-3775. OVCAR3 and TOV112D FBXW7 knockout (KO) ovarian cancer cell lines express elevated levels of cyclin E1, a CCNE1 gene product (a biomarker associated with the induction of baseline replication stress), compared to SKOV3 and TOV112D ovarian cancer cell lines. For example, see YPKok et al (2020). "Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors." Oncogenesis Vol.9 Issue 10 Pages 88. The Panc0203 pancreatic cancer cell line expresses elevated levels of phosphorylated RPA2 (a biomarker associated with increased basal replication stress), while the pancreatic cancer KP4 cell line does not. Serra, V., et al., "Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance," Clin Cancer Res 28(20):4536-4550. (2022).

[0285] B. Results Figure 2 shows the GI measured in 16 cell line models (some with elevated basal replication stress) treated with PKMYT1 inhibitor tool compounds. 50 The values ​​are reported. Figure 3 reports the corresponding median replication fork rates measured by DNA combing analysis for uterine and breast cancer cell line models treated with the tool compound. Lowest GI 50 The five uterine and breast cancer cell lines in Figure 2 that possessed the values ​​also had the lowest median mean replication fork rate in Figure 3. Figures 2 and 3 together show that cell line models with elevated levels of basal replication stress (KRAS and CCNE1-amplified uterine cancer cell lines, CCNE1-amplified breast cancer cell lines, OVCAR3 (CCNE1-amplified), TOV112D FBXW7 KO (high cyclin E1) ovarian cancer cell line, and panc0203 (high pRPA2) pancreatic cancer cell line) were the most sensitive to PKMYT1 inhibition. In contrast, cell line models with lower levels of basal replication stress were more resistant to PKMYT1 inhibition.

[0286] The median replication fork rate was also measured by DNA combing analysis for the same uterine and breast cancer cell line models treated instead with the PKMYT1 inhibitor of isomer 1 (i.e., the utomer of compound 1) in Example B-1. Figure 4 reports the median replication fork rate obtained for the utomer of compound 1. The results were similar to those obtained with the tool compound, confirming that uterine and breast cancer cell line models with lower baseline levels of replication stress were more resistant to PKMYT1 inhibition than cell line models with elevated levels of replication stress.

[0287] Overall, the data indicate that cancers with elevated levels of basal replication stress are more sensitive to PKMYT1 inhibition. The data also provide evidence that replication stress correlates with cyclin E amplification, as well as with other genetic alterations already associated with replication stress and with associated responses to WEE1 inhibition.

[0288] Example C-2: Target Engagement NanoBRET (Bioluminescence Resonance Energy Transfer) Assay A. NanoBRET assay (PKMYT1, WEE1, and EPHB3) Using a whole-cell-based target engagement assay with NanoBRET technology, we investigated target engagement with three target groups. Specifically, we tested compounds to evaluate the inhibition of PKMYT1, WEE1, and EPHB3 in genetically engineered HEK293T cell lines.

[0289] HEK293T cell lines overexpressing PKMYT1, WEE1, and EPHB3 were thawed at 37°C from cryopreserved vials. The thawed cells were resuspended in 10 mL of RPM1-1640 supplemented with 10% fetal bovine serum and 1% GlutaMax. The cell lines were seeded into 384-well low-volume plates (Greiner #784080) using a Multidrop Combi with a small cassette at a rate of 8000 cells / well per 10 μL (for PKMYT1 and EPHB3) and 4000 cells / well per 10 μL (for WEE1). The plates were incubated at 37°C in 5% CO2 for 23 hours.

[0290] After incubation, the culture medium was removed from the plate using a BlueCat Bluewasher in the GentleSpin process. Tracer probes for each target were dissolved in Opti-MEM at room temperature, and 10 μL volumes were dispensed into each well of the plate using a Multidrop Combi with a small cassette. Promega tracer probes were dissolved at concentrations of 1 μM (K-10 for PKMYT1#N2640), 0.33 μM (K-4 for EPHB3#N2520), and 0.13 μM (K-10 for WEE1#N2640). At this stage, the plate was protected from light by using a dark-colored lid.

[0291] The compounds were added to the plate immediately after probe addition. The added compounds (12 concentration responses from a maximum concentration of 10 μM down to 0.000000248 μM) were added to the wells using an Echo 650 Acoustic Dispenser (Beckman), and the plates were incubated for 2 hours in an incubator maintained at 37°C and 5% CO2.

[0292] After incubation, the plates were equilibrated at room temperature for 10 minutes. 5 μL of 3×NanoBRET assay solution was dispensed into each well of the plate using a Multidrop Combi with a small cassette. The plates were incubated at room temperature for 10 minutes (protected from light), centrifuged at 300 G for 1 minute, and read using a PherSTAR FSX (optical module: 460±80 nm / 610 nm-LP; gain settings: 3600 (A), 2500 (B); integration time: 0.2 seconds). The raw data obtained for acceptor and donor channels was loaded into a Geneda analyzer (Genedata AG) to determine the BRET ratio of the plates.

[0293] IC of the tested compound 50 Report the values ​​in Table 1-A.

[0294] [Table 11-1]

[0295] [Table 11-2] 1 I C 50 This is reported after a single measurement (n=1) or as the average value of multiple measurements (n>1).

[0296] B. NanoBRET assay (kinase panel) Compound 137 and isomer 2 were further profiled against selected kinases in a NanoBRET whole-cell target engagement assay performed at CELLinib GmbH (dose response). IC of Compound 137 and isomer 2 50 Report the values ​​in Table 1-B.

[0297] [Table 12]

[0298] Example C-3: PKMYT1 surface plasmon resonance (SPR) direct coupling assay A. Expression and purification of PKMYT1 protein Avi-tagged PKMYT1 (6His-SSGVDLGT-TEV-AVI-S-PKMYT1(75-362)) was expressed in Escherichia coli BL21(DE3) Tuner cells in Terrific Broth medium. Expression was induced with 50 μM IPTG at A600 = 1.0 and expressed at 18°C ​​for 20 hours. Cells were harvested and stored at -80°C. The cell pellet was resuspended in 750 mL of PKMYT1 lysis buffer (50 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP, DNase (0.1 μg / mL), 0.1 mM PMSF) per 97 g of pellet. The solution was homogenized, and the clarified lysate was loaded onto a nickel affinity purification column pre-equilibriumized with Buffer A (50 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 mM imidazole). The protein was eluted with Buffer A supplemented with 300 mM imidazole. The fraction containing PKMYT1 was pooled, and the buffer was changed by dialysis to biotinylation buffer (25 mM Hepes pH 7.5, 300 mM potassium glutamate, 0.5 mM TCEP). Biotinylation was performed using the BirA kit (Avidity) according to the manufacturer's instructions, and biotinylation was confirmed by mass spectrometry. Using size exclusion chromatography, PKMYT1 was further purified in storage buffer (25 mM Hepes pH 7.5, 500 mM NaCl, 0.5 mM TCEP). The fraction containing pure PKMYT1 was pooled, concentrated to 10.2 mg / mL, and rapidly frozen in liquid nitrogen.

[0299] B.SPR direct binding assay All SPR steps were performed at 25°C in a Biacore 8K Instrument (Cytiva). 5 μL of the immobilization buffer (50 mM Hepes pH 7.5, 200 mM NaCl, 5 mM MgCl2, 0.05% Tween-20, 1 mM TCEP) was used. -1Using a 180-second injection, Series S sensor tips SA (Cytiva) were functionalized with approximately 3000 RU of biotinylated Avi-tagged PKMYT1. The surface was conditioned by 10 start injections of "running buffer" (50 mM Hepes pH 7.5, 200 mM NaCl, 5 mM MgCl2, 0.05% Tween-20, 1 mM TCEP, 1% DMSO). To correct for DMSO deviation, a solvent correction step consisting of 5-stop titrations was performed, injecting running buffer solutions (0-2%) containing different DMSOs at the start and end of the screening. Three blank cycles were included for blank subtraction, injecting running buffer before injecting each compound. A 3-fold dilution series of each sample was administered in 30 μL in a single-cycle dynamic mode. -1 The PKMYT1 binding solution was injected sequentially from low to high concentrations onto the PKMYT1 binding surface at a flow rate of 120 seconds, and dissociation was monitored for 2500 seconds.

[0300] After applying standard dual-reference and solvent corrections, all analyses were performed using Biacore Insight Evaluation Software. Single-cycle dynamic 1:1 coupling model data analysis was applied to determine the on-rate (k) for the test compound. on ) and off speed (k off The theoretical Rmax (the maximum feasible SPR signal produced by the interaction between ligand-analyte pairs; expressed in response units (RUs)) was calculated as (molecular weight of compound analyte in solution) / (molecular weight of immobilized target) × (amount of immobilized target captured), and the surface activity rate was calculated as (experimental Rmax) / (theoretical Rmax).

[0301] The data for the tested compounds are reported in Table 2.

[0302] [Table 13] 1 k on and k offThis is reported after a single measurement (n=1) or as the average value of multiple measurements (n>1).

[0303] Example C-4: Functional CDK1 pThr14 AlphaLISA assay Compounds were tested to evaluate their effects on PKMYT1 activity under cellular conditions. Specifically, phosphorylation of CDK1 (a PKMYT1 substrate) on Thr14 was used as a specific marker for PKMYT1 activity. Inhibition of PKMYT1 activity by inhibitors reduces the level of pCDk1 (Thr14) in cells.

[0304] Compounds prepared in DMSO were dispensed into low-volume white 384-well plates (Greiner 784075) as concentration-response curves. 5 μL of FUOV-1 cells in Opti-MEM were seeded onto assay preparation plates at a density of 3000 cells / well using a multidrop Combi. These were incubated at 37°C and 5% CO2 for 2 hours. The remainder of the assay was performed using the AlphaLISA Surefire Ultra Human Phospho-CDK1 (Thr14) kit (ALSU-PCDK1-A10K) available from Perkin Elmer. All reagents were prepared according to the manufacturer's recommendations. 5 μL of 2× lysis buffer containing the added protease inhibitor (Roche, #11836170001) was added to the plates and thoroughly mixed. The acceptor and donor stocks were then diluted with 1 part mix to 2 parts HBSS. 4 μL of acceptor was added using a multidrop Combi and incubated at room temperature for 1 hour. Then, 4 μL of donor stock was added using a multidrop Combi and the plate was incubated for 18 hours. Alpha counts were measured using PheraSTAR FSx (BMG Labtech), and the resulting raw data was analyzed using Geneda analyzer (Genedata AG) to determine the compound IC- 50 I obtained it.

[0305] IC of the tested compound 50The values ​​are reported in Table 3.

[0306] [Table 14-1]

[0307] [Table 14-2] 1 I C 50 This is reported after a single measurement (n=1) or as the average value of multiple measurements (n>1).

[0308] Example C-5: Cell proliferation in OVCAR3 and SKOV3 cell lines (Hoechst staining) Imaging-based assays were used to investigate changes in cell number. Compounds were tested to evaluate the inhibition of cell proliferation in CCNE1-amplified ovarian cancer cell lines and CCNE1-non-amplified SKOV3 ovarian cancer cell lines.

[0309] OVCAR3 cells and SKOV3 cells were cultured in RPMI 1640 medium containing 4.5 g / mL glucose supplemented with 20% heat-inactivated fetal bovine serum, and in McCoy's modified 5A medium supplemented with 10% heat-inactivated fetal bovine serum and 1% GlutaMax, respectively. Both cell lines were seeded using multidrop in complete growth medium in 384-well plates (Greiner, 781090) at a density of 4000 cells / well in 40 μL / well. The plates (test compound plate + day 0 control plate) were placed in an incubator maintained at 37°C and 5% CO2 for approximately 20-22 hours. The day 0 (control plate) was fixed with 4% PFA (v / v), washed three times in PBS (Biotek 406), and then stored at 4°C in 40 μL of PBS until use. Using an Echo 650 Acoustic Dispenser (Beckman), 12 concentration responses were added to the test compound plate, starting from a maximum concentration of 30 μM down to 0.00003 μM, and then incubated at 37°C and 5% CO2 for 4 days. On day 4, cells were fixed with 4% PFA (v / v) and stained with Hoechst 33342 (Thermofisher H3570) for 1 hour in PBS + 1.1% BSA + 0.1% TritonX up to a final well dilution of 1 / 10,000. The cells were washed three times with PBS and left to stand in 40 μL of PBS. Stained cells were imaged using the Cell Insight Cell Imaging and Analysis Benchtop Platform (10× objective lens). The raw data output obtained for the total cell count was loaded into a Geneda Analyzer (Genedata AG) to determine the growth rate from the control plate and the plates containing the test compound. The values ​​for day 0 and GI were also determined. 50 The concentration of the compound that provides a 50% growth inhibition between the peak of the curve and the peak of the curve was calculated for all test compounds.

[0310] The GI of the tested compound 50 The values ​​are reported in Table 4.

[0311] [Table 15] 1 I C 50 This is reported after a single measurement (n=1) or as the average value of multiple measurements (n>1).

[0312] Example C-6: Kinase profiling A. General method The test compounds were profiled against a broad kinase panel using ThermoFisher Scientific's SelectScreen Kinase Profiling Services. Each kinase assay used one of the following assay technique protocols: 1) The Z-LYTE® Screening Protocol and Assay Conditions (revised January 23, 2018), available at http: / / assets.thermofisher.com / TFS-Assets / BID / Methods-&-Protocols / 20180123_SSBK_Customer_Protocol_and_Assay_Conditions.pdf; 2) The Adapta® Screening Protocol and Assay Conditions (revised January 23, 2018), available at http: / / assets.thermofisher.com / TFS-Assets / BID / Methods-&-Protocols / 20180123_SSBK_Adapta_Customer_Protocol_and_Assay_Conditions.pdf; or 3) LanthaScreen® EU Kinase Binding Assay Screening Protocol and Assay Conditions (revised January 23, 2018), available at http: / / assets.thermofisher.com / TFS-Assets / BID / Methods-&-Protocols / 20180123_SSBK_LanthaScreen_Binding_Customer_Protocol_and_Assay_Conditions.pdf.

[0313] B. Kinase selectivity (single-point enrichment mode) Compound 137 and isomer 2 were profiled against a broad kinase panel using ThermoFisher Scientific. Kinase selectivity was measured in single-point (SP) concentration mode: K mATP Inhibition percentage at [Compound 137, isomer 2] = 1 μM. Data are reported in Table 5-A.

[0314] [Table 16-1]

[0315] [Table 16-2]

[0316] [Table 16-3]

[0317] [Table 16-4]

[0318] [Table 16-5]

[0319] [Table 16-6]

[0320] [Table 16-7]

[0321] [Table 16-8]

[0322] [Table 16-9]

[0323] C. Kinase selectivity (dose response) Compound 137 and isomer 2 were further profiled for dose response to selected kinases using ThermoFisher Scientific. IC was obtained from a 10-point concentration response (CR) study using the highest test concentration of 10 μM. 50 The data was obtained and is reported in Table 5-B.

[0324] [Table 17]

[0325] Example C-7: Metabolic Stability Assay A. Evaluation using human liver cytosol (HLC) The stability of controls (zalepron and 06-benzylguanine) and the test compounds was evaluated at 1 μM using 2.5 mg / mL human liver cytosol in the presence and absence of 3 μM raloxifene in 0.1 M phosphate buffer pH 7.4 at 37°C. Aliquots were taken at 0, 10, 30, 60, 90, and 120 minutes and quenched with ACN containing an internal standard in a 1:5 ratio. Samples were analyzed for parental compound loss by LC / MS / MS. Potential hydroxylated metabolites were monitored by adding a Parent+16 transition. The degree of inhibition by raloxifene was derived by comparing metabolite formation and / or parental compound depletion in the presence and absence of the inhibitor.

[0326] B. Evaluation using human liver microsomes (HLM) The stability of the control (phenacetin, verapamil, diclofenac, imiprimin, benzydamine, and metoprolol) and the test compounds was evaluated using 1 mg / mL HLM (150 mixed-sex donor pools) containing 1 mM NADPH and 1 μM test compound / control. Briefly, 222.5 μL of an HLM mixture containing 1.12 mg / mL of HLM in phosphate buffer pH 7.4 was mixed with 25 μL of 10 mM NADPH, preheated at 37°C for 8 minutes, and then 2.5 μL of 100 μM test compound / control prepared in DMSO was added. Aliquots (20 μL) were taken at 0.5, 5, 10, 15, 20, and 30 minutes and quenched with 100 μL of cold stop solution (100% ACN containing 100 nM alprazolam, caffeine, and tolbutamide as internal standards). The samples were centrifuged at 4,000 rpm and 4°C for 20 minutes, and 40 μL aliquots of the supernatant were mixed with 160 μL of pure water for LC-MS / MS analysis. Each incubation was performed once. Peak areas were determined from the extracted ion chromatograms, and the percentage of parent compound remaining was calculated from the peak areas of the test compound / control.

[0327] C. Evaluation using human hepatocytes (hHEP) in suspension The stability of controls (phenacetin, verapamil, diclofenac, imipramine, chlorpromazine, and naloxone) and test compounds was evaluated using 1 million viable cells / mL (a mixed-sex donor pool of 10 individuals) and 1 μM of the test compound / control. Briefly, cryopreserved hepatocytes were thawed in a 37°C water bath, centrifuged in thawing medium, and prepared in Leibowitz L-15 medium (pH 7.4) to contain 1 million viable hepatocytes / mL and a final compound concentration of 1 μM. Cell viability was determined using Cellometer Vision, and a cell viability of over 80% was required to proceed to compound incubation. Compound / cell solution (250 μL) was incubated at 37°C for 2 hours and shaken at 900 rpm on an Eppendorf Thermomixer Comfort plate shaker. Samples (20 μL) were collected at 0.5, 5, 15, 30, 45, 60, 80, 100, and 120 minutes and quenched with 100 μL of 100% ice-cold acetonitrile. The samples were shaken at 800 rpm for 2 minutes and centrifuged at 4000 rpm for 20 minutes at 4°C to pelletize the precipitated protein. The supernatant fraction was diluted 1:5 in deionized water, shaken at 1000 rpm for 2 minutes, and further diluted 1:1 with deionized water. The samples were analyzed by LC-MS / MS. Each incubation was performed once. Peak areas were determined from the extracted ion chromatograms, and the parent compound retention rate (%) was calculated from the peak areas of the test compound / control.

[0328] D. Evaluation of low clearance using a co-cultured human hepatocyte model The stability of the test compound was determined over a 72-hour incubation period using human hepatocytes co-cultured with mouse JT3 fibroblasts (Hμrel). Briefly, the test compound was dissolved in DMSO at 10 mM and then diluted to 2 μM in the administration medium using an Echo dispenser. All steps were performed at room temperature, except that the 2 μM test compound was preheated to 37°C before the start of incubation. Before the assay, the maintenance medium was removed from the cells, followed by a washing step with 100 μL of preheated serum-free blank administration medium. The incubation of the compound was initiated by adding 50 μL of administration medium and 50 μL of administration medium containing 2 μM of the test compound. The final test concentration was 1 μM. During the experiment, the plate was maintained in an incubator at 37°C without shaking in a humid atmosphere containing 95% air and 5% CO2. 70 μL aliquots of the double-repeated sample were taken at 0, 1, 3, 5, 24, 48, and 72 hours and quenched with 70 μL of ice-cold acetonitrile stop solution. The quenched samples were diluted with 140 μL of 0.1% formic acid in water and analyzed by mass spectrometer.

[0329] E. Data analysis for metabolic stability assessment As described above, the t of the compounds incubated with HH, HLM, and HμREL assays 1 / 2 and CL int This was calculated using the following formula: t 1 / 2 (minutes) = ln(2) / - slope, and Cl int (μL / min / 10 6 (individual cell or mg protein) = ln(2) * V / (t 1 / 2 ) (In the formula, V(μL / ×10 6 Individual cells or mg of protein) are the number of cells in incubation (×10 6 ) or incubation volume (μL) divided by the microsomal protein content (mg).

[0330] The data for evaluating metabolic stability are reported in Table 6 below.

[0331] [Table 18-1]

[0332] [Table 18-2]

[0333] Example C-8: LogD The LogD values ​​of 10 μM test compounds were determined by LC / MS / MS, and by nicadapine, cyclobenzaprine, and caffeine as control compounds in octanol / PBS pH 7.4. 10 μL of the test compound or control was added to a 96-well deep plate, followed by 500 μL of saturated octanol and 500 μL of saturated phosphate buffer. The plate was shaken at 2,000 rpm for 2 hours at room temperature. The samples were centrifuged at 4,000 rpm for 30 minutes at room temperature. 100 μL of the sample was removed from the octanol and buffer phases. 5 μL of octanol sample was mixed with an internal standard containing 1:1 H2O:ACN and vortexed at 1,000 rpm for 5 minutes (100-fold octanol sample). 50 μL of the 100-fold sample was added to an internal standard containing 450 μL of 1:1 H2O:ACN and vortexed at 1,000 rpm for 5 minutes (1,000-fold octanol sample). The 1,000-fold octanol sample was serially diluted 10,000, 100,000, and 1,000,000 times. 50 μL of the buffer sample was added to an internal standard containing 450 μL of 1:1 H2O:ACN and vortexed at 1,000 rpm for 5 minutes (10-fold buffer sample), and then further diluted 100, 1,000, and 10,000 times with 1:1 H2O:ACN. The samples were analyzed using LC / MS / MS.

[0334] LogD was calculated as follows:

[0335]

number

[0336] The data is reported in Table 7 below.

[0337] [Table 19-1]

[0338] [Table 19-2]

[0339] Example C-9: Caco-2 cell permeability The test compounds were evaluated for gastrointestinal permeability and potential bioavailability using a Caco-2 cell permeability assay. A detailed description of this method was previously published in "Evaluation of the Disconnect between Hepatocyte and Microsome Intrinsic Clearance and In Vitro In Vivo Extrapolation Performance"; Williamson Beth, Harlfinger Steffanie, McGinnity F. Dermot; Drug Metab Dispos 48:1137-1146, November 2020. In summary, Caco-2 cells were subjected to a 6.86 × 10⁶ Caco-2 cell permeability assay. 5 Cells were seeded at individual cells / mL and cultured for 14-18 days with the culture medium changed every other day. The test compound (10 μM) was added to the donor well, and its appearance in the receiver well was measured after incubation at 37°C for 2 hours. The rate of compound transport from the apical side to the bottom (AB) side was determined from the donor well as the apical (A) compartment and the receiver as the bottom (B) compartment. Similarly, the rate of compound transport from the bottom (BA) side was determined with the donor well as the bottom (B) compartment and the receiver as the apical (A) compartment. The samples were analyzed by liquid chromatography-mass spectrometry (MS) / MS.

[0340] Transmittance coefficient (1 × 10 -6 The formula for cm / s is as follows: P app =(dC r / dt)×V r (A × C 0) Calculated using The emission ratio (ER) is calculated using the following formula: Emission ratio=P app (B→A) / P app (A→B) Calculated using (In the formula, dC r / dt is the cumulative concentration of the compound in the receiver chamber as a function of time (μM / s), V r This is the solution volume in the receiver chamber (0.1 mL at the top and 0.3 mL at the bottom). A has a surface area for transport (i.e., a single layer area of ​​0.11 cm²). 2 ) and C0 is the initial concentration (μM) in the donor chamber.

[0341] The data is reported in Table 8 below.

[0342] [Table 20]

[0343] Example C-10: CYP assay A. CYP inhibition The potential of test compounds to inhibit cytochrome P450 (CYP) isoforms was determined using sex-mixed human liver microsomes (HLMs) from 150 donors, as well as cocktails of substrates selective for CYP 1A2, 2C9, 2C19, 2D6, and 3A, at incubation concentrations equal to their Km values. Specific inhibitors of these pathways were included as positive controls. The substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 / 5 were phenacetin (30 μM), diclofenac (10 μM), S-mephenytoin (35 μM), buflarol (5 μM), and midazolam (3 μM), respectively. The final concentration of HLM was 0.2 mg / mL, and the final concentrations of the test compounds were 0, 0.1, 0.3, 1, 3, 10, and 30 μM. Incubation was initiated by adding 20 μL of 10 mM NADPH in phosphate buffer and quenched at 37°C for 5 minutes with ACN containing 3% formic acid and 40 nMol verapamil as an internal standard in a 1:1 ratio. The sample was centrifuged at 4,000 rpm for 30 minutes, placed on ice for 20 minutes, and recentrifuged at 4,000 rpm for another 30 minutes. The supernatant (200 μL) was transferred to analyze isoform-selective metabolite formation. The degree of inhibition was calculated by comparing the residual activity in the presence of the inhibitor with a control in DMSO alone using the following formula: IC20 is the concentration at which 50% inhibition occurs. 50 This was calculated using nonlinear regression analysis in Xlfit.

[0344]

number

[0345] ICs of Example B-1, isomer 1 (i.e., eutomer of compound 1) and Example B-2, isomer 2 (i.e., eutomer of compound 137) for CYP 1A2, 2C9, 2C19, 2D6, and 3A, respectively. 50 The value was greater than 30 μM.

[0346] B.CYP time-dependent inhibition The time-dependent inhibitory potential of test compounds on cytochrome P450 (CYP) activity was determined using a 1 mg / mL mixed-sex human liver microsome pool (HLM) from 150 donors, 10 μM of the test compound, a 30-minute pre-incubation time, and a 10-fold dilution step. Specific substrate formation for CYP1A2, 2C9, 2C19, 2D6, and 3A was measured at three times their respective Km values. The substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 / 5 were phenacetin (90 μM), diclofenac (30 μM), S-mephenytoin (105 μM), buflalol (15 μM), and midazolam (9 μM), respectively. Specific inhibitors of these pathways were included as positive controls. The primary incubation consisted of 1 mg / mL HLM ± 10 mM NADPH along with 10 μM test compound incubated at 37°C for 30 minutes. After 30 minutes, 10 μL aliquots of the pre-incubation solution were transferred to a secondary incubation containing 10 mM NADPH and a substrate cocktail. After incubation at 37°C for 15 minutes, the secondary incubation was quenched by adding 150 μL of cold methanol containing 20 nM verapamil as an internal standard. The reduction in isoform-specific metabolite formation was determined by comparison with the NADPH control, and the time-dependent inhibition percentage (TDI) was calculated using the following formula. A TDI of over 20% was considered positive and warranted further investigation.

[0347]

number

[0348] Irreversible IC represents the concentration at which 50% inhibition is observed. 50 We calculated it as follows:

[0349]

number

[0350] The time-dependent inhibition percentage (TDI) values ​​calculated for Example B-1, isomer 1 (i.e., eutomer of compound 1) and Example B-2, isomer 2 (i.e., eutomer of compound 137) for each of the CYP cells 1A2, 2C9, 2C19, 2D6, and 3A were less than 20.

[0351] C.CYP induction The potential of the test compounds to induce drug-metabolizing enzymes was evaluated by assessing their potential for pregnane X receptor (PXR) activation in DPX2 cells. DPX2 cells were purchased from Puracyp and processed according to the supplier's protocol. The test compounds, positive controls (rifampicin, 1 and 10 μM), and negative control (propranolol, 10 μM) were prepared to a final concentration of 0.1% with DMSO. The test compounds were evaluated at 0.1, 0.3, 1, 3, 10, and 30 μM, with the EC values ​​corresponding to the concentrations that yielded the observed maximum half-dose induction and maximum induction, respectively. 50 and E max The values ​​were derived. Cells were treated with the test compound or control for 24 hours. After 24 hours, the administration medium was removed and 1×CellTiter-Fluor® was added at 37°C for 30 minutes. Fluorescence was measured using 400 nm excitation and 505 nm emission. Then, the ONE-Glo assay reagent was added for 5 minutes and emission was measured using a light meter. Cell viability was determined using the following formula:

[0352]

number

[0353] Normalized luciferase activity was determined by taking the ratio of relative luminescence units (RLU) to relative fluorescence units for three replicate experiments at each concentration, and the mRNA-level activation factor was calculated using the following formula:

[0354]

number

[0355] In Example B-1, isomer 1 (i.e., the eutomaire of compound 1), no PXR induction was observed.

[0356] Example C-11: Water solubility The thermodynamic solubility of the test compound was measured using the shaking flask method, starting with a 10 mM DMSO solution. After evaporating the DMSO, the dried compound was equilibrated in a phosphate buffer solution (0.1 M, pH 7.4) in a glass vial at 25°C under constant stirring for 24 hours. The dissolved compound was then separated from the remainder by double centrifugation with a tip wash in between, removing any potential interference from residual dried compounds. The solution was diluted with purified water before quantification using UPLC / MS / MS.

[0357] The data is reported in Table 9 below.

[0358] [Table 21-1]

[0359] [Table 21-2]

[0360] Example C-12: Kinase selectivity (kinase affinity tool assay) Kinobead technology 1、2 A kinase affinity tool ("KAT") chemopromics method based on [a specific algorithm] was used for in-situ kinase profiling of the test compound. KAT is an affinity matrix composed of indiscriminate ATP-competitive kinase inhibitors for enriching a large subset of native quinomes directly from disease / safety-related lysates. The proteome RB50 value indicates the selective affinity of the test compound to the enriched kinase.

[0361] The test compound was incubated with a mixture of cell lysates from four cell lines (K562, MV4-11, SN-N-BE2, and COLO-205) in a dose-response format at 4°C for 30 minutes. The lysates incubated with the compound were then conjugated to KAT beads at 4°C for 30 minutes. The beads were washed twice with conjugation buffer, followed by two washes with 1×PBS. The proteins conjugated to the beads were reduced, alkylated, and digested overnight at 37°C using trypsin / LysC enzyme. The digested peptides were eluted in water with 60% ACN / 0.1% formic acid and desalted using in-house developed C18 StageTips. The peptides were analyzed by label-free independent analysis (DIA) quantitative mass spectrometry using Orbitrap LUMOS. Proteomics data were analyzed using in-house software Doscheda to generate RB50 values. 3 The RB50 values ​​obtained for compound 137 and isomer 2 are reported in Table 10, confirming that the compounds are highly selective for PKMYT1.

[0362] References 1.Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors Nat.Biotechnol.2007,25,1035-44 https: / / www.nature.com / articles / nbt1328. 2.Optimized chemical proteomics assay for kinase inhibitor profiling J.Proteome Res.2015,14,1574-86 https: / / pubs.acs.org / doi / 10.1021 / pr5012608. 3.DOSCHEDA:a web application for interactive chemoproteomics data analysis.PeerJ Computer Science 2017 https: / / peerj.com / articles / cs-129 / .

[0363] [Table 22-1]

[0364] [Table 22-2]

[0365] [Table 22-3]

[0366] [Table 22-4]

[0367] [Table 22-5]

[0368] [Table 22-6]

[0369] [Table 22-7]

[0370] [Table 22-8]

[0371] Example C-13: Radioactive ligand binding and cell-based functional assay (Secondary pharmacology) Secondary pharmacological assays were performed in Eurofins using standard experimental techniques against 80 targets across a broad pharmacological space. Details of the assays, along with the experimental protocols, are published in Eurofins at https: / / www.eurofinsdiscoveryservices.com. Radioligand binding assays were used to evaluate the ability of compound 137 and isomer 2 to interact with a panel of G protein-coupled receptors (GPCRs), ion channels, and transmembrane transporters. For kinase and enzyme targets, assays measuring substrate turnover or phosphorylation by isolated proteins were used, allowing for direct determination of the compound's mechanism of action. Where binding activity was observed with GPCR targets, the mechanism of action was determined using cell-based functional assays with secondary messenger readings. The assays were performed in 8-point concentration-response mode using semi-logarithmic dilutions, and IC50 was measured. 50 , EC 50 , or K i The value was calculated (Cheng, Y., Prusoff, WH1973 Relationship between the inhibition constant (K I )and the concentration of inhibitor which causes 50 per cent inhibition(IC 50 ) of an enzymatic reaction Biochem Pharmacol 22(23):3099-108).

[0372] Table 11 reports the values ​​obtained for compound 137 and isomer 2.

[0373] [Table 23-1]

[0374] [Table 23-2]

[0375] Example C-14: THP-1 assay (cytotoxicity) The test compounds were evaluated for compound-induced cytotoxicity in the human monocyte cell line (THP-1). In in vitro assays, the fluorescence signal generated by the reduction of non-fluorescent resazurin (7-hydroxy-3H-phenoxazine-3-one 10-oxide) to fluorescent resorphin was used as a measure of cytotoxicity (Alamar blue assay). Since the cellular reduction of resazurin depends on the pool of reductase or diaphorase enzymes derived from mitochondria and cytosol, resazurin can be used as a redox index in cell viability assays of mammalian cells (McMillian et al, 2002; O'Brien et al, 2000).

[0376] THP-1 cells were incubated in a medium (supplemented with RPMI, 1% L-glutamine, and 10% quantified FBS) in a T175cm container. 2 The cells were subcultured in suspension in tissue culture flasks. The subcultures were maintained at 37°C in a 95% humidified atmosphere containing 5% CO2, and subcultured approximately every 2-3 days. The cell density was 50,000 cells / mL ~ 1 × 10⁶ 6 The cell / mL ratio was maintained.

[0377] Depending on the selected stock concentration, the test compound (40 nL) was added to a 1536-well low-volume plate (Greiner #782077) at 10 semi-logarithmic concentrations, with the highest concentration being either 100 μM or 250 μM. In all cases, the DMSO concentration was 1%.

[0378] THP-1 cells were seeded at a low rate into plates at a density of 2,000 cells / well in 4 μL of THP-1 medium using a Multidrop Combi Reagent dispenser (Type 836) equipped with a Multidrop standard tube plastic tip dispensing cassette. Plates containing THP-1 cells with the compound / solvent were incubated for 48 hours under standard cell culture conditions (37°C, 5% CO2).

[0379] After a 48-hour incubation period, a stock solution of resazurin (113 mg of resazurin in 1000 mL of PBS) was prepared, warmed to 37°C, and vortexed. Using a Multidrop dispenser equipped with a Multidrop standard cassette, 1 μL of the stock solution of resazurin was added to each well at high speed to aid mixing. The plates were incubated for 2 hours under standard cell culture conditions. The plates were then incubated for a further 1 hour at room temperature with shaking (700 rpm).

[0380] Next, the plates were read using a BMG PheraStar reader with an excitation wavelength of 540 nm and an emission wavelength of 580 nm. The controls were 100 μM lapatinib (minimum / inhibitor control) and 1% DMSO (maximum / neutral control). The obtained raw data was loaded into a Geneda analyzer (Genedata AG) and IC50 was performed. 50 The values ​​were determined. The primary (fluorescence) signal of each concentration-effect curve was normalized relative to the vehicle control (0% inhibition) and the inhibitor control (100% inhibition). The obtained inhibition values ​​were fitted using the four-parameter logistic equation, and the IC was calculated. 50 The value was calculated.

[0381] Compound 137, isomer 2, showed a 34 μM IC50. 50 The presence of this value indicates that this compound has a reduced risk of cytotoxicity in THP-1 cells compared to previously tested compounds.

[0382] References 1. McMillian, MK et al (2002). An improved resazurin-based cytotoxicity assay for hepatic cells. Cell Biol. & Toxicol., 18:157-173. 2. O'Brien, J et al (2000). Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell toxicity. Eur J Biochem., 267:5421-5426.

[0383] Example C-15: In vitro hematopoietic stem cell and progenitor cell assay (myelotoxicity) The test compounds were evaluated to determine their effects on the proliferation and differentiation of hematopoietic stem cells and progenitor cells ("HSPCs"). Cryopreserved human bone marrow-derived CD34 + Hematopoietic stem cells and progenitor cells (Lonza) were thawed and allowed to recover overnight in a humidified incubator at 37°C with 5% CO2 in maintenance medium (StemSpan SFEM II (Stem Cell Technologies) containing 25 ng / mL SCF, 50 ng / mL TPO, and 50 ng / mL Flt3-L human recombinant protein (all Peprotech)). The following day, the cells were resuspended at a concentration of 10,000 cells / mL in a medium capable of supporting erythroid cell differentiation (Preferred Cell Systems, SEC-BFU1-40H) in the presence of the test compound. Cells (30 μL) were plated onto 384-well tissue culture plates (Perkin Elmer) with black walls and clear bottoms, supplemented with 30, 10, 3.3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, or 0 μM of the test compound. The cells were cultured for 5 days in a humidified incubator at 37°C containing 5% CO2.

[0384] Viability was determined using a Promega CellTiter-Glo 2.0 (with an optimized volume of 3 μL / well), and luminescence was detected using an Envision plate reader (Perkin Elmer). Relative luminescence signals were normalized to the percentage of the control well (0 μM compound) using Geneda Screener software (Genedata) or GraphPad Prism 9. A 4-parameter logistic regression curve fit was used to determine the 50% inhibitory concentration (IC1). 50 ) was sought.

[0385] Primary CD34 cells induced to differentiate into red blood cell lineage + The results obtained from bone marrow-derived HSPCs are reported in Table 12 and Figure 5. Table 12 shows the average IC50 for compound 1, isomer 1, compound 137, and isomer 2. 50 Report the value.

[0386] [Table 24]

[0387] Figure 5 shows the dose-response curves obtained for these compounds.

[0388] Example C-16: Effects on the cell cycle in human ovarian OVCAR3 cell lines Immunoblotting analysis was performed on CCNE1-amplified ovarian OVCAR3 cells to determine whether short-term (24-hour) treatment with compound 137 and isomer 2 induced DNA damage response ("DDR") signaling during the replication phase of the cell cycle. The levels of phospho-CHK1 (Ser345) and phospho-RPA2 (Ser4 / Ser8), biomarkers thought to be specifically expressed after DNA damage formation during active cell replication, were evaluated as measures of the effect on the cell cycle.

[0389] A. Method OVCAR3 cells were treated with compound 137 and isomer 2 for 24 hours, and then lysed in RIPA buffer (Sigma-Aldrich) supplemented with a protease inhibitor (Roche), a phosphatase inhibitor (Sigma-Aldrich), and benzonase (Merck). After incubation on ice for 30 minutes, the lysates were clarified by centrifugation at 15,000 rpm at 4°C for 20 minutes, and the supernatant was retained for sample loading. NuPAGE® LDS sample buffer (ThermoFisher Scientific) and NuPAGE® sample reducing agent (ThermoFisher Scientific) were added to the samples and heated to 95°C (5 minutes). Equal volumes of total cell lysates were separated on 4-12% Bis-Tris NuPAGE gels and analyzed by standard immunoblotting. Primary antibodies: H2AX (Abcam, ab20669), gH2AX (Cell Signaling Technology (CST), 2577), histone H3 (CST, 3638), phosphohistone H3 Ser10 (CST, 9701), RPA2- (Abcam, ab2175), phospho-RPA2 Ser4 / Ser8 (Bethyl Labs, A300-245A), CDK1 (CST, 9116), phospho-CDK1 Thr14 (Abcam, ab58509 or BioLegend, 947402), phospho-CDK1 Tyr15 (Abcam, ab275958), CHK1 (CST, #2360), phospho-CHK1 Ser345 (CST, 2348), PKMYT1 (Abcam, ab307146), and vinculin (Sigma, V9131). Secondary antibodies: anti-rabbit IgG, HRP-conjugated CST, #7074, and anti-mouse IgG, HRP-conjugated CST, #7076). Adavocertib was used as a treatment control.

[0390] B. Results Figure 6 shows the differential expression of phosphoproteins in response to treatment with compound 137 and isomer 2. Treatment of OVCAR3 cells with compound 137 and isomer 2 resulted in a concentration-dependent decrease in phospho-CDK1 Thr14 levels. Phospho-CDK1 Thr14 is a direct substrate of PKMYT1. Furthermore, the expression of phospho-histone H3 Ser10 increased without affecting the expression of phospho-CDK1 Tyr15, a WEE1 substrate, indicating that the increase in the mitotic population due to G2 / M DNA damage cell cycle checkpoint override is unrelated to WEE1 inhibition. At 24 hours after treatment, phosphorylation of biomarkers associated with DNA damage formation (phospho-CHK1, phospho-RPA2) was minimal or absent in the actively replicating cell population. This is consistent with PKMYT1 regulating CDK1 activity unrelated to S-phase cell cycle progression. Overall, the data indicate that treatment of OVCAR3 cells with compound 137 and isomer 2 resulted in PKMYT1 target engagement, an increase in the mitotic population, and minimal or no DNA damage signaling in replicating cells.

[0391] Example C-17: Cell proliferation in PKMYT1 knockout SKOV3 cells The potential off-target activities and effects on cell viability of compound 137 and isomer 2 were evaluated in PKMYT1 knockout (KO) SKOV3 cells. Specifically, loss of PKMYT1 protein expression, decreased phosphorylation of PKMYT1 substrates, and inhibition of cell proliferation were assessed in PKMYT1 KO SKOV3 cells.

[0392] A. Method Using CRISPR / Cas9 technology, PKMYT1 kinase was deleted from SKOV3 cells to prepare two monoclonal PKMYT1 knockout clones (B2 and H2). Parental SKOV3 cells and PKMYT1 knockout SKOV3 cells were left untreated or treated with compound 1 and isomer 1 for 24 hours. The cells were then lysed in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitors (Roche, Basel, Switzerland), phosphatase inhibitor (Sigma-Aldrich), and benzonase (Merck). After incubation on ice for 30 minutes, the lysates were clarified by centrifugation at 15,000 rpm at 4°C for 20 minutes, and the supernatant was retained for sample loading. NuPAGE® LDS sample buffer (ThermoFisher Scientific) and NuPAGE® sample reducing agent (ThermoFisher Scientific) were added to the samples. Equal volumes of whole cell lysates were separated on 4–12% Bis-Tris NuPAGE gels and analyzed by standard immunoblotting. Primary antibody incubation was performed overnight at 4°C with CDK1 (CST, 9116), pCDK1 Thr14 (Abcam, ab58509), PKMYT1 (Abcam, ab307146), and vinculin (Sigma, V9131). Secondary antibody incubation was performed at room temperature for 2 hours using anti-rabbit IgG, HRP-conjugated (CST, 7074) and anti-mouse IgG, HRP-conjugated (CST, 7076). Quantification was performed using Bio-Rad ChemiDoc, which is also used for visualizing Western blot bands, and manual quantification was performed using ImageJ. The CDK1 and phospho-CDK1 Thr14 bands were normalized relative to the parent band, and the CDK1 to phospho-CDK1 Thr14 ratio was calculated. The data was visualized using Graphpad Prism and plotted as the mean with standard deviation (n=2).

[0393] Furthermore, the effects of compound 137 and isomer 2 on inhibiting the proliferation of PKMYT1 KO SKOV3 cells were evaluated using the CellTiter-Glo (CTG) assay. Parental SKOV3 and OVCAR3 cells were included as controls. Cells were seeded at 500 cells / well in 96-well plates and exposed to compound 137 and isomer 2 for 7 days. After cell attachment on day 0, compound 137 and isomer 2 were dispensed from the compound stock dissolved in DMSO into seven titration dilutions with the highest concentration of 30 μM. DMSO served as the vehicle control. Plates were incubated at 37°C in 5% CO2. Cell proliferation was stopped by adding CTG according to the manufacturer's instructions (Promega, Madison, WI, USA; G7570), and luminescence was read using an Envision plate reader (Perkin Elmer). Data analysis was performed by normalizing for vehicle treatment. The data was normalized and plotted against each vehicle control using Prism GraphPad software, and the GI 50 The value was derived.

[0394] B. Results The results of immunoblotting analysis are shown in the left and center panels of Figure 7, which indicate that deletion of PKMYT1 from SKOV3 cells resulted in loss of PKMYT1 protein expression and a significant decrease in phospho-CDK1 Thr14 expression, which could not be further reduced by treatment with compound 1 and isomer 1 (for 24 hours).

[0395] GI obtained from 7-day endpoint CTG measurements of cell lines treated with compound 137 and isomer 2 50 The values ​​are shown in the right panel of Figure 7 and also reported in Table 13. As a result of deleting PKMYT1 from SKOV3, compound 137 and isomer 2 showed GI values ​​exceeding 20 μM. 50 A value was obtained.

[0396] [Table 25]

[0397] Overall, the data indicate that PKMYT1 knockout (KO) SKOV3 cells are resistant to treatment with compound 137 and isomer 2.

[0398] Example C-18: In vivo tolerability (mouse model) Tolerability studies were conducted to evaluate the long-term administration of compound 137 and isomer 2 in mice as monotherapy, in combination with gemcitabine, and in combination with irinotecan hydrochloride.

[0399] A. Monotherapy Female SCID mice (18g+ at the time of the initial procedure) were orally administered compound 137, isomer 2 at 300 mg / kg BID for 28 days. Compound 137, isomer 2 was formulated for administration using a vehicle of 5% DMSO, 50% 20% captisol, and 45% sterile water for injection (pH 3-3.2). Body weight was collected and recorded daily. Clinical findings, including monitoring of foot and tail swelling, were assessed at least twice daily. 300 mg / kg BID administration resulted in approximately 21 μM of free C137, isomer 2. max This was achieved.

[0400] Figure 8-A shows the effect of compound 137 and isomer 2 at 300 mg / kg BID monotherapy on weight loss. At this monotherapy dose, no significant weight loss, swelling of the legs or tail, or other significant adverse events were observed.

[0401] B. Combination therapy (gemcitabine) Female SCID mice (18g+ at the time of the initial procedure) were orally administered compound 137, isomer 2 at a dose of 100 mg / kg BID in combination with intraperitoneal injection of 20 mg / kg gemcitabine four times a week for 28 days. Body weight was recorded daily. Clinical findings, including monitoring of foot and tail swelling, were collected and evaluated at least twice daily. The 100 mg / kg BID dose resulted in approximately 5.7 μM of free C for compound 137, isomer 2. max This was achieved.

[0402] Figure 8-B shows the effect of such combined administration of compound 137, isomer 2, and gemcitabine on weight loss. No significant weight loss, swelling of the legs or tail, or other significant adverse events were observed with this combined dose.

[0403] C. Combination therapy (irinotecan) Female athymic nude mice (18g+ at the time of the initial procedure) were orally administered compound 137, isomer 2 at a dose of 100 mg / kg BID in combination with intraperitoneal injection of 50 mg / kg irinotecan hydrochloride four times a week for 28 days. Body weight was collected and recorded daily. Clinical findings, including monitoring of foot and tail swelling, were assessed at least twice daily. The 100 mg / kg BID dose resulted in approximately 5.7 μM of free C137, isomer 2. max This was achieved.

[0404] Figure 8-B shows the effect of such concomitant administration of compound 137, isomer 2, and irinotecan hydrochloride on weight loss. No significant weight loss, swelling of the feet or tail, or other significant adverse events were observed with this concomitant dose.

[0405] Example C-19: In vivo PKMYT1 target engagement In vivo studies were conducted in mice to evaluate PKMYT1 target engagement by compound 137 and isomer 2. Target engagement was assessed by analyzing phospho-CDK1(T14) expression in tumors.

[0406] Female SCID mice bearing OVCAR3 tumors were orally administered compound 137 and isomer 2 at doses of 10 mg / kg BID, 30 mg / kg BID, or 100 mg / kg BID. The dose was calculated for each individual animal on the day of administration and administered at a dose volume of 10 mL / kg. After 21 days of administration, tumors were collected, processed, and analyzed by immunoblotting: lysis buffer (25 mM Tris / HCl, 3 mM EDTA, 3 mM EGTA, 50 mM NaF, 2 mM ortho-vanadate, 0.27 M sucrose, 10 mM β-glycerophosphate, 5 mM pyrophosphate, 0.5% Triton X-100), protease inhibitor (Sigma, P8340), phosphatase inhibitor (Sigma, P0044, P5726), and benzoase (Sigma, (E1014) Tumor fragments were lysed and homogenized in a rapid preparation tube containing the lysis matrix (3 × 30 seconds, 6.0 m / s) using 1:2000. The lysate was sonicated and the supernatant was collected. The lysate was transferred and cooled in a 2 mL assay block in the rapid preparation tube, and centrifuged again at 2100 rpm for 15 minutes at 4°C to ensure that no debris moved. The supernatant was then 95°C. o The samples were boiled in 13C for 5 minutes and immunoblotted as described in Example C-16 using phospho-CDK1 Thr14 (Biolegend, 947402) and vinculin (Sigma, V9131) primary antibodies, and anti-mouse IgG, HRP-conjugated (CST, 7076) secondary antibody. After detection with G-box (Syngene), the bands were quantified using Gene Tools software. The phospho-CDK1 Thr14 signal was normalized to vinculin, and the geometric mean was normalized to the control group and plotted (n=5-7 mice per condition ± SEM, P * <0.05 is statistically significant (one-way ANOVA).

[0407] Figure 9 shows the relative in vivo expression levels of phospho-CDK1 Thr14 for each dose. Quantified in vivo phosphorylated CDK1 Thr14 signaling after 21 days of administration of compound 137 and isomer 2 showed significant PKMTYT1 kinase inhibition compared to vehicle treatment for all three doses tested. All three doses of compound 137 and isomer 2 tested after 21 days of administration showed IC50. 90 The above inhibition was achieved.

[0408] Example C-20: Antitumor effect in human OVCAR3 and SW620 xenograft models In human ovarian OVCAR3 models and human colorectal SW620 xenograft models, studies were conducted to evaluate the in vivo efficacy of combination therapy using compound 137, isomer 2, and the S-phase DNA damaging agents gemcitabine and irinotecan hydrochloride, respectively.

[0409] A. Xenotransplantation model derived from human ovarian OVCAR3 cell line OVCAR3 cells were transplanted into the right flank of female SCID mice. The tumor was approximately 200 mm. 3 When the tumor volume was reached, mice were randomized to a treatment group based on tumor volume and treated according to one of the dosing schedules shown in Table 14.

[0410] [Table 26]

[0411] After discontinuing the treatment, the gemcitabine-treated arms were left undisturbed for another 35 days to allow for regrowth. Tumor size was measured twice weekly, and relative tumor volume was plotted (geometric mean of mice per treated arm (n=6) ± SEM).

[0412] Figure 10 shows the relative tumor volume over time. Tumor volume measurements at the end of treatment (day 21) with 100 mg / kg of compound 137 and isomer 2 twice daily showed significant tumor growth inhibition (TGI, 77% P<0.01) in replication-stressed high OVCAR3 (CCNE-amplified) tumors (see also Table 14). Administration of 30 mg / kg or 10 mg / kg of compound 137 and isomer 2 twice daily resulted in 12% and 25% tumor growth inhibition, respectively (P>0.05, not significant). All tested combinations were superior to compound 137 and isomer 2 monotherapy and gemcitabine monotherapy. On day 21, administration of 50 mg / kg of compound 137, isomer 2, twice daily in combination with weekly intraperitoneal administration of 20 mg / kg gemcitabine resulted in a 62% tumor regression (P<0.001). Administration of 10 mg / kg of compound 137, isomer 2, twice daily in combination with weekly intraperitoneal administration of 20 mg / kg gemcitabine resulted in a 31% tumor regression (P<0.001). After cessation of processing in the combined arm, 200 mm 3 Dose-dependent regrowth of tumor volume was observed at 11 days (compound 137, isomer 2 + gemcitabine at 10 mg / kg BID) and 35 days (compound 137, isomer 2 + gemcitabine at 50 mg / kg BID). These results indicate that compound 137, isomer 2, combined with gemcitabine, can achieve more durable tumor regression compared to gemcitabine monotherapy, particularly as exposure to compound 137, isomer 2 increases.

[0413] B. Xenograft model derived from the human colorectal SW620 cell line SW620 cells were transplanted into the right flank of female athymic nude mice. The tumor was approximately 200 mm. 3 When the tumor volume was reached, mice were randomized to a treatment group based on tumor volume and treated according to one of the dosing schedules shown in Table 15.

[0414] [Table 27]

[0415] Next, tumor size was measured and relative tumor volume was plotted (geometric mean of mice per processing arm (n=6) ± SEM).

[0416] Figure 11 shows the relative tumor volume over time. Administration of compound 137 and isomer 2 at 100 mg / kg twice daily did not inhibit tumor growth in SW620 tumors. However, when compound 137 and isomer 2 were administered twice daily at 100 mg / kg in combination with weekly intraperitoneal administration of irinotecan hydrochloride, a topoisomerase 1 inhibitor (TOP1i), a combination therapy benefit and tumor regression (18%, P<0.01) were achieved. When compound 137 and isomer 2 were administered twice daily at 10 mg / kg in combination with irinotecan hydrochloride, a combination therapy benefit was observed, but it was not as pronounced. These results suggest that in this SW620 model, greater exposure to compound 137 and isomer 2 is necessary to promote combination therapy benefits and regression (geometric mean ± SEM, P<0.01). ** ), P<0.001( *** The result is significant (one-sided t-test with unequal variances).

[0417] Example C-21: In vitro efficacy in combination with exatecan An in vitro assay was performed to evaluate whether treatment of PKMYT1 KO isogenic SKOV3 cells (already described in Example C-17) with compound 137 and isomer 2 could further sensitize those cells to exatecan.

[0418] The in vitro efficacy of the combination of exatecan and compound 137, isomer 2, was evaluated using a 6x6 concentration compound combination assay. PKMYT1 wild-type SKOV3 cells and PKMYT1 knockout (KO) SKOV3 cells were seeded and allowed to adhere in 384-well plates for 24 hours. Then, exatecan and compound 137, isomer 2, were administered as monotherapy and in combination within a range of concentrations (ECHO acoustic liquid handler). After 7 consecutive days of drug exposure, the cells were treated with CellTiter-Glo® (CTG) to enable measurement of cellular metabolic activity in the treated cells. Metabolic rates, which are generally considered to correlate with cell viability, were measured as chemiluminescence, and raw data were obtained for all concentrations of both drugs.

[0419] Figure 12 shows the effects of treatment of PKMYT1 wild-type SKOV3 cells and PKMYT1 KO SKOV3 cells with exatecan (1 nM) in combination with compound 137 and isomer 2 within a certain concentration range (mean (n=3) ± SD). Treatment of PKMYT1 KO cells with exatecan (1 nM) resulted in maximum drug activity (E max The result obtained was not further improved by the addition of compound 137, isomer 2. Comparison of combined activity in PKMYT1 wild-type SKOV3 cells and combined activity in PKMYT1 knockout cells suggests that higher inhibitory concentrations (3-10 μM) of compound 137, isomer 2 may achieve more significant in vitro efficacy for the combination. Such high inhibitory concentrations of compound 137, isomer 2 were found in 100 mg / kg of free C max Based on comparisons, this can be achieved in vivo.

[0420] Example C-22: Pharmacodynamic effects in a human colorectal SW620 xenograft model. In vivo monotherapy with irinotecan generally induces DNA damage during the replication S phase of the cell cycle, activation of the G2 / M checkpoint, and PKMYT1 dependence. This study aimed to evaluate the in vivo pharmacodynamic response to combination therapy with irinotecan hydrochloride and compound 137, isomer 2, in a human colorectal SW620 xenograft model. The study evaluated biomarkers for post-treatment PKMYT1 target engagement (phospho-CDK1 Thr14), DNA damage formation (gH2AX), and changes in the relative mitotic population (phospho-histone H3 Ser10). These biomarkers were selected to assess whether the applicability of compound 137, isomer 2, was sufficient to inactivate the G2 / M DNA damage cell cycle checkpoint after irinotecan hydrochloride administration.

[0421] Human colorectal SW620 cells were transplanted into the right flank of female athymic nude mice. The tumor was approximately 200 mm. 3When tumor volume was reached, mice were randomized to a treatment group based on tumor volume and treated with (i) oral administration of the vehicle, (ii) a single intraperitoneal administration of irinotecan hydrochloride (50 mg / kg), (iii) oral administration of compound 137 and isomer 2 twice daily for 3 days (100 mg / kg), or (iv) a single intraperitoneal administration of irinotecan hydrochloride (50 mg / kg) plus oral administration of compound 137 and isomer 2 twice daily for 3 days (100 mg / kg). Six hours after the last dose of compound 137 and isomer 2 on day 3, the tumors were collected and subsequently processed for immunoblotting as described in Example C-19 using phospho-CDK1 Thr14 (Biolegend, 947402), CDK1 (CST, 9116), histone H3 (CST, 3638), phosphohistone H3 Ser10 (CST, 9701), gH2AX (Cell Signalling Technology (CST)), and vinculin (Sigma, V9131), as well as anti-rabbit IgG, HRP-conjugated (CST, 7074) and anti-mouse IgG, HRP-conjugated (CST, 7076) secondary antibodies. After detection with G-box (Syngene), the bands were quantified using Gene Tools software. The phospho-CDK1 Thr14 signal was normalized to CDK1 and vinculin, the gH2AX signal was normalized to vinculin, and the phosphohistone H3 signal was normalized to vinculin. Ser10 was normalized relative to histone H3 and vinculin. The geometric mean was normalized relative to the control group and plotted (n=3-6 mice per condition ± SEM, P * <0.05 is statistically significant (one-way ANOVA).

[0422] The results are shown in Figure 13. Oral administration of compound 137 and isomer 2 twice daily (100 mg / kg) over 3 days, either in the absence or in the presence of irinotecan hydrochloride, resulted in PKMYT1 target engagement (phospho-CDK1 Thr14). Intraperitoneal administration of irinotecan hydrochloride (50 mg / kg) resulted in DNA damage formation (gH2AX), which was not further enhanced by the addition of compound 137 and isomer 2. Irinotecan hydrochloride monotherapy reduced the mitotic population (phosphohistone H3 Ser10), a finding consistent with the activation of the G2 / M DNA damage cell cycle checkpoint after treatment with TOP1i inhibitors. However, the combination of irinotecan hydrochloride and compound 137, isomer 2, did not reduce the mitotic population, suggesting that compound 137, isomer 2, deactivates the G2 / M cell cycle checkpoint induced by irinotecan hydrochloride treatment, leading to the initiation of immature mitosis. These results demonstrate the PKMYT1 dependence of tumors in the presence of exogenous DNA damaging agents (including TOP1 inhibitors) that activate the G2 / M cell cycle checkpoint.

[0423] Example C-23: In vitro in vivo transformation in multiple types of hepatocytes Compound 137 and isomer 2 were incubated with mouse, rat, dog, miniature pig, and human hepatocytes (1 million cells / mL) at 37°C for 180 minutes (5 μM). Incubation was stopped by adding an organic solvent, and the samples were centrifuged to separate soluble and insoluble components. The supernatant was stored at -20°C until required for analysis. Semi-quantitative analysis of major in vivo changes in the compound during each incubation was performed using both UV and MS detection of the eluate following liquid chromatography separation. The abundance of metabolites was calculated as the percentage of the sum of the UV peak areas of all compounds extracted at 330–350 nm in the samples after each incubation (time = 180 minutes). All metabolites with a UV abundance of more than 1% observed in the human hepatocyte incubation were aligned across species. Table 16 summarizes the in vitro human metabolites of compound 137 and isomer 2 present across species. M = large amount (more than 10%), m = small amount (more than 1% but less than 10%), t = trace amount (less than 1%), t * =Detected only by MS response, ND = Not detected

[0424] [Table 28]

[0425] Figure 14 shows the metabolic scheme illustrating the major pathways of biotransformation observed in vitro for compound 137 and isomer 2: oxidation and glucuronidation.

[0426] Example C-24: In vivo pharmacokinetic study in CD-1 mice The pharmacokinetic profiles of compound 137 and isomer 2 were characterized in vivo using CD-1 mice after either a single intravenous bolus or a single oral bolus to enable the calculation of key pharmacokinetic properties.

[0427] Animal experiments were conducted in accordance with the relevant welfare policies and procedures of the research organization. Male CD-1 mice weighing approximately 20-30 g were housed in polycarbonate animal cages equipped with absorbent rodent bedding and acclimatized with free access to standard rodent solid feed and sterile water. Ambient temperature of 20-25°C and humidity of 40-70% were maintained with alternating 12-hour light / dark cycles interrupted only by research-related events (e.g., sampling). For administration, the compound was formulated as an aqueous solution containing 5% dimethyl sulfoxide and 95% sulfobutyl ether-β-cyclodextrin (30% w / v) in water for injection (WFI) at a dose level of either 0.5 mg / kg (IV) or 1.0 mg / kg (PO) (animal body weight) and an administration volume of either 2.0 mL / kg (IV) or 4.0 mL / kg (PO) (animal body weight). Blood samples (approximately 0.02 mL / time point) were collected from the dorsal metatarsal vein into a tube containing EDTA-K2 anticoagulant, placed on moist ice (4°C), and then centrifuged (4000 g, 5 min, 4°C) to obtain plasma, which was then stored (-75°C+ / -15°C) before further analysis. Samples were taken at 2, 10, 30, 60, 120, 240, 360, 480, 720, and 1440 minutes after intravenous administration (time=0). Samples were taken at 5, 15, 30, 60, 120, 240, 360, 480, 720, and 1440 minutes after oral administration (time=0). The total concentration of the compound was measured in the plasma samples using standard liquid chromatography-mass spectrometry, and blank plasma samples were prepared and analyzed in the same manner. The main pharmacokinetic parameters were calculated using WinNonlin (Phoenix®). Table 17 summarizes the key pharmacokinetic parameters obtained after administering compound 137 and isomer 2 to male CD-1 mice (n=2) at 0.5 mg / kg (IV) or 1 mg / kg (PO).

[0428] [Table 29]

[0429] Figure 15 shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in male CD-1 mice after administration of either IV (0.5 mg / kg, solid line) or PO (1 mg / kg, dashed line).

[0430] Example C-25: In vivo pharmacokinetic study in SCID mice Pharmacokinetic profiles of compound 137 and isomer 2 were obtained in female SCID mice after oral administration to establish short-term tolerability and confirm the degree and duration of compound exposure. Animal rearing, administration, sample collection, and subsequent analysis to determine total plasma concentrations were carried out in the same manner as previously described in Example C-24, with only minor adjustments. Compound 137 and isomer 2, formulated in 5% DMSO, 50% 20% captisol, and 45% water for injection (WFI) (pH 3-3.2), were administered at three dose levels (10 mg / kg, 30 mg / kg, and 100 mg / kg) and a dosing volume of 10 mL / kg. Plasma samples were collected from the side tail vein at multiple time points after administration (30 / 60 / 120 / 240 / 420 / 1440 minutes). Table 18 summarizes the pharmacokinetic parameters obtained after oral administration of compound 137 and isomer 2 to female SCID mice (n=3) at three dose levels: 10 mg / kg, 30 mg / kg, and 100 mg / kg.

[0431] [Table 30]

[0432] Figure 16 shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in female SCID mice after PO administration at three dose levels: 10 mg / kg (solid line), 30 mg / kg (dashed line), and 100 mg / kg (dotted line).

[0433] Example C-26: In vivo pharmacokinetic study in untreated and biliary cannula-treated Han Wistar rats The pharmacokinetic profiles of compound 137 and isomer 2 were characterized in vivo after IV bolus administration in Han Wistar rats treated with bile duct cannula, and the contribution of enterohepatic circulation to important pharmacokinetic properties was determined.

[0434] Animal experiments were conducted in accordance with the relevant welfare policies and procedures of the research organization. Male Han Wistar rats weighing approximately 200-300 g (either untreated or after routine bile duct cannula insertion) were housed in polycarbonate animal cages equipped with absorbent rodent bedding and acclimatized with free access to standard rodent solid feed and sterile water. Ambient temperature of 20-25°C and humidity of 40-70% were maintained with alternating 12-hour light / dark cycles interrupted only by research-related events (e.g., sampling). For administration, compound 137 and isomer 2 were formulated as an aqueous solution containing 5% dimethyl sulfoxide and 95% sulfobutyl ether-β-cyclodextrin (30% w / v) in water for injection (WFI) at a dose level of 0.5 mg / kg (animal body weight) and an administration volume of 1.0 mL / kg (animal body weight). Blood samples (approximately 0.20 mL / time point) were collected from the jugular vein into a tube containing EDTA-K2 anticoagulant, placed on moist ice (4°C), and then centrifuged (4000 g, 5 min, 4°C) to obtain plasma, which was then stored (-75°C+ / -15°C) before further analysis. Samples were taken at multiple time points (untreated rats: 2, 10, 30, 60, 120, 240, 360, 480, 720, and 1440 minutes after intravenous administration (time = 0); BDC rats: 5, 15, 30, 60, 120, 240, 420, and 1440 minutes). Compound 137 and isomer 2 were measured in plasma samples using standard liquid chromatography-mass spectrometry, and blank plasma samples were prepared and analyzed in the same manner. Key pharmacokinetic parameters were calculated using WinNonlin (Phoenix®). Table 19 summarizes the pharmacokinetic parameters obtained after intravenous administration of compound 137 and isomer 2 at a dose of 0.5 mg / kg to male untreated or biliary cannula-treated (BDC) Han Wistar rats (n=3).

[0435] [Table 31]

[0436] Figure 17 shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in male Han Wistar rats after IV (0.5 mg / kg) administration to either untreated (solid line) or bile duct cannula-treated (dashed line) animals.

[0437] Example C-27: In vivo pharmacokinetic study in Han Wistar rats at high doses After oral administration, pharmacokinetic profiles of compound 137 and isomer 2 were obtained in male Han Wistar rats to establish short-term tolerability and to confirm the degree and duration of compound exposure at three dose levels (10 mg / kg, 30 mg / kg, and 100 mg / kg) and a 10 mL / kg administration volume. Animal rearing, administration, sample collection, and subsequent analysis to determine total plasma concentration were carried out in the same manner as previously described in Example C-24, with only minor adjustments. Plasma samples were collected from the caudal vein at multiple time points after administration (15 / 30 / 60 / 120 / 240 / 360 / 1440 minutes). Table 20 summarizes the pharmacokinetic parameters obtained after oral administration of compound 137 and isomer 2 to male Han Wistar rats (n=3) at three dose levels: 10 mg / kg, 30 mg / kg, and 100 mg / kg.

[0438] [Table 32]

[0439] Figure 18 shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in male Han Wistar rats after PO administration at three dose levels: 10 mg / kg (solid line), 30 mg / kg (dashed line), and 100 mg / kg (dotted line).

[0440] Example C-28: In vivo pharmacokinetic study at high doses to evaluate the tolerability and effects of repeated administration of a substance. After repeated daily oral administration, pharmacokinetic profiles of compound 137 and isomer 2 were obtained in female SCID mice to establish tolerability over 28 days and to confirm the degree and duration of compound exposure. Animal rearing, administration, sample collection, and subsequent analysis to determine total plasma concentration were carried out in the same manner as previously described in Example C-24, with only minor adjustments. Animals were administered 10 mL / kg twice daily (8-hour intervals). Plasma samples were collected at multiple time points (30 / 12 / 360 / 720 / 1440 minutes) after the first administration on day 1 and day 28. Figure 19 shows a comparison of the pharmacokinetic profiles (total plasma concentration) of compound 137 and isomer 2 after repeated BID oral administration (vertical dotted line) at 100 mg / kg to female SCID mice (n=2) on day 1 (solid line) and day 28 (dashed line).

[0441] Example C-29: In vivo pharmacokinetic study comparing substance formulations in SCID mice at high doses. After oral administration, pharmacokinetic profiles of compound 137 and isomer 2 were obtained in SCID mice to establish short-term tolerability at high doses and to confirm the degree and duration of compound exposure. Animal rearing, administration, sample collection, and subsequent analysis to determine total plasma concentrations were carried out in the same manner as previously described in Example C-24, with only minor adjustments. Compound 137 and isomer 2, formulated in either 5% DMSO, 50% 20% captisol, and 45% purified water for injection (WFI, pH 3-3.2), or 0.5% HPMC / 0.1% Tween, were administered at dose volumes of 300 mg / kg and 10 mL / kg. Plasma samples were collected at multiple time points (30 / 12 / 360 / 720 / 1440 minutes) after administration. Table 21 summarizes the pharmacokinetic parameters obtained after oral administration of compound 137 and isomer 2, formulated in either 5% DMSO, 50% 20% captisol, and 45% WFI (pH 3-3.2), or 0.5% HPMC / 0.1% Tween 80, to female SCID mice at a dose of 300 mg / kg.

[0442] [Table 33]

[0443] Figure 20 shows the pharmacokinetic profiles (total plasma concentrations) of compound 137 and isomer 2 in female SCID mice after PO administration at 300 mg / kg in 5% DMSO, 50% 20% captisol, and 45% WFI (pH 3-3.2) (solid line) or 0.5% HPMC / 0.1% Tween (dashed line).

[0444] While specific embodiments and examples have been described above, these embodiments and examples are presented as examples only and are not intended to limit the scope of the invention. Modifications and changes can be made according to those skilled in the art without departing from this disclosure in its broader embodiments as defined in the following claims. For example, any embodiment described herein can be combined with any other preferred embodiment described herein to provide additional embodiments.

[0445] As will be understood by those skilled in the art, all numbers, including those representing the amount of components, properties such as molecular weight, and reaction conditions, are approximations and in all cases are understood to be modified by the term "approximately." These values ​​may vary depending on the desired properties that those skilled in the art seek to obtain by utilizing the teachings of this disclosure. It will also be understood that such values ​​inherently include variability that inevitably arises from the standard deviation found in each of their test measurements.

[0446] Those skilled in the art will readily recognize that, when members are grouped together in a common manner, as in the Markush groups, this disclosure encompasses not only the entire enumerated group as a whole, but also each member of the group individually, and all possible subgroups of the principal group. In addition, for all purposes, this disclosure encompasses not only the principal group, but also principal groups in which one or more of the members of the group are absent. This disclosure also assumes the express exclusion or waiver of one or more of the members of the group in the claimed disclosure.

[0447] As will be understood by those skilled in the art, for any and all purposes, particularly with regard to providing written explanations, all scopes disclosed herein also include any and all possible sub-scopes and combinations thereof, as well as the individual values ​​constituting the scopes, in particular integer values. Any enumerated scope can be readily recognized as fully explaining and enabling that the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can readily be decomposed into a lower third, a middle third, an upper third, etc. For example, scope C (1~6) is a subrange C (2~6) , C (3~6) , C (3~5) , C (4~6) These include, as well as C1 (methyl), C2 (ethyl), C3 (propyl), C4 (butyl), C5 (pentyl), and C6 (hexyl) individually. Also, as will be understood by those skilled in the art, all words such as “up to,” “at least,” “greater than,” “less than,” “greater than,” and “or greater than” include the listed numbers and refer to a range that can be subsequently broken down into subranges, as discussed above. Similarly, all ratios disclosed herein also include all sub-ratios that fall within a broader ratio.

[0448] References to “processes” in this disclosure are for convenience only and do not classify, define, or limit this disclosure as described herein.

[0449] List of References 1. Forment, JV and MJO'Connor (2018). "Targeting the replication stress response in cancer." Pharmacol Ther 188:155-167. 2.Chow,J.P.and R.Y.Poon(2013).「The CDK1 inhibitory kinase MYT1 in DNA damage checkpoint recovery.」Oncogene 32(40):4778-4788。 3.Serra,V.,et al.(2022).「Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition,Overcoming PARP Inhibitor Resistance.」Clin Cancer Res 28(20):4536-4550。 4.Lallo,A.,et al.(2018).「The Combination of the PARP Inhibitor Olaparib and the WEE1 Inhibitor AZD1775 as a New Therapeutic Option for Small Cell Lung Cancer.」Clin Cancer Res 24(20):5153-5164。 5.Chen,X.,et al.(2018).「Cyclin E Overexpression Sensitizes Triple-Negative Breast Cancer to Wee1 Kinase Inhibition.」Clinical Cancer Research 24(24):6594-6610。 6.Young,L.A.,et al.(2019).「Differential Activity of ATR and WEE1 Inhibitors in a Highly Sensitive Subpopulation of DLBCL Linked to Replication Stress.」Cancer Research 79(14):3762-3775。 7.Richer,A.L.,et al.(2017).「WEE1 Kinase Inhibitor AZD1775 Has Preclinical Efficacy in LKB1-Deficient Non-Small Cell Lung Cancer.」Cancer Research 77(17):4663-4672。 8.Bo Mi Ku et al.(2017).「Mutational status of TP53 defines the efficacy of Wee1 inhibitor AZD1775 in KRAS-mutant non-small cell lung cancer.」Oncotarget 8(40)::67526-67537。 9.Pfister,Sophia X.,et al.(2015).「Inhibiting WEE1 Selectively Kills Histone H3K36me3-Deficient Cancers by dNTP Starvation.」Cancer Cell 28(5):557-568。 10.Liu,J.F.,et al.(2021).「Phase II Study of the WEE1 Inhibitor Adavosertib in Recurrent Uterine Serous Carcinoma.」Journal of Clinical Oncology 39(14):1531-1539。

Claims

1. It is a compound, 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure: 【Chemistry 1】 6-amino-7-(2-chloro-3-hydroxy-6-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure: 【Chemistry 2】 6-amino-7-(6-chloro-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure: 【Transformation 3】 6-amino-7-(2,6-dichloro-3-hydroxyphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure: 【Chemistry 4】 6-amino-7-(6-bromo-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure: 【Transformation 5】 A compound selected from the group consisting of the above, or a pharmaceutically acceptable salt thereof.

2. 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure: 【Transformation 6】 The composition according to claim 1, or a pharmaceutically acceptable salt thereof.

3. 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure: 【Transformation 7】 The compound according to claim 1.

4. 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the following structure: 【Transformation 8】 The salt according to claim 1, which is a pharmaceutically acceptable salt of the product.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein the compound is an atrop isomer.

6. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, wherein the atrop isomer is the eutomer of the atrop isomer.

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

8. A method for treating or preventing cancer in a subject who is suffering from or susceptible to cancer, comprising administering to the subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof described in any one of claims 1 to 6.

9. The method according to claim 8, wherein the cancer is a solid tumor carcinoma.

10. The method according to claim 8, wherein the cancer is a blood cancer.

11. The method according to claim 8, wherein the cancer is selected from the group consisting of ovarian cancer, triple-negative breast cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, colorectal cancer, serous uterine carcinoma, soft tissue sarcoma, uterine cancer, skin cancer, bladder cancer, head and neck cancer, glioma, and B-cell lymphoma.

12. The method according to any one of claims 8 to 11, wherein the cancer is PKMYT1 dependent due to an increase in the baseline level of replication stress.

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