Pyrrolopyrimidine carboxamides

Abstract

Pyrrolopyrimidine carboxamides and pharmaceutically acceptable salts thereof; pharmaceutical compositions comprising such compounds and salts; use of such compounds and salts to treat or prevent cancers, including PKMYT1-dependent cancers; kits comprising such compounds and salts; and methods for manufacturing such compounds and salts.

Description

PYRROLOPYRIMIDINE CARBOXAMIDES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.63 / 506,165, filed June 5, 2023, and U.S. Provisional Application No. 63 / 594,968, filed November 1, 2023, each of which is incorporated by reference in its entirety for all purposes. FIELD

[0002] The present disclosure relates generally to pyrrolopyrimidine carboxamide compounds and pharmaceutically acceptable salts thereof. The disclosure further relates to pharmaceutical compositions comprising such compounds and salts; use of such compounds and salts to treat or prevent cancers, including cancers having a PKMYT1-dependency because of elevated basal levels of replication stress; kits comprising such compounds and salts; and methods for manufacturing such compounds and salts. BACKGROUND

[0003] The activity of protein kinase CDK1 (also called cell division cycle 2 protein, or CDC2) transitions a cell from the G2 phase of the cell cycle into mitosis (M) where cell division occurs. In response to DNA damage, WEE1 kinase family members WEE1 and PKMYT1 inhibit CDK1 to prevent the cell from dividing 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, in which the genome is duplicated in preparation for cell division, events can occur that result in the stalling of the DNA polymerase and the replication fork. This situation is referred to as replication stress and can result in the generation of DNA damage that leads to a dependency on replication stress response proteins as well as G2 / M cell cycle checkpoint proteins such as ATR, WEE1, and PKMYT1. Forment, J. V. and M. J. O'Connor, "Targeting the replication stress response in cancer," Pharmacol Ther 188: 155-167 (2018). In such cancers with DNA damage resulting from replication stress, whether endogenous or induced by DNA damaging agents or other targeted agents, there is a greater dependency on PKMYT1. Inhibition of PKMYT1 can lead to premature entry into mitosis with unrepaired DNA damage resulting in the induction of cancercell death in mitosis. Chow, J. P. and R. Y. Poon, "The CDK1 inhibitory kinase MYT1 in DNA damage checkpoint recovery," Oncogene 32(40): 4778-4788(2013). Consequently, PKMYT1 inhibitors have the potential to induce cancer cell death, either as a monotherapy in cancers with elevated levels of endogenous or basal replication stress, or in combination with other agents that induce greater levels of replication stress in cancers.

[0005] Targeted pharmacological inhibition of PKMYT1 activity is a presently unexploited therapeutic approach for treating cancers with elevated basal levels of replication stress and there currently are no approved pharmacological agents that inhibit PKMYT1. Accordingly, there is a need for PKMYT1 inhibitors, particularly PKMYT1 inhibitors having pharmacologically appropriate properties (such as selectivity against WEE1, bioavailability, etc.) required for suitable administration to a subject in need of such treatment. The present disclosure addresses this unmet need by providing such compounds together with corresponding pharmaceutical compositions and methods for the treatment or prevention of cancers, including cancers where there is a PKMYT1-dependency because of elevated basal levels of replication stress, either as monotherapy or in combination therapies where such a PKMYT1 dependency can be induced with DNA damaging agents or other targeted therapies such as PARP inhibitors or ATR inhibitors. SUMMARY

[0006] In one aspect, the present disclosure provides compounds having the structure of Formula (1): (1), or a pharmaceutically acceptable salt thereof, wherein: R1is selected from the group consisting of:, , , , and ; R2, R3, R4, R5, R7, R8, R9, R10, and R12are independently selected from the group consisting of halogen and C1-3-alkyl; R6and R11are independently selected from the group consisting of hydrogen and C1-3- alkyl; A is selected from the group consisting of: , , , , , , , and ; R13and R24are independently selected from the group consisting of hydrogen, C1-3-alkyl, and C1-3-alkoxy;R14, R17, R18, R20, R22, and R28are independently selected the group consisting of: (a) hydrogen; (b) halogen; (c) cyano; (d) C1-6-alkyl, wherein the C1-6-alkyl is optionally substituted with one or more substituents independently selected from halogen; (e) -C1-6-alkenyl, wherein the C1-6-alkenyl is optionally substituted with one or more substituents independently selected from halogen; (f) C1-6-alkynyl, wherein the C1-6-alkynyl is optionally substituted with one or more substituents independently selected from halogen; (g) C3-6-cycloalkyl, wherein the C3-6-cycloalkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-6-alkyl, halo-C1-6-alkyl, C1-6-alkoxy, and halo-C1-6-alkoxy; (h) C1-6-alkoxy; wherein the C1-6-alkoxy is optionally substituted with one or more substituents independently selected from halogen; (i) -NR31R32; (j) -S(O)2-C1-6-alkyl, wherein the -S(O)2-C1-6-alkyl is optionally substituted with one or more substituents independently selected from halogen; (k) phenyl, wherein the phenyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-6-alkyl, halo-C1-6-alkyl, C1-6-alkoxy, and halo-C1-6-alkoxy; and (l) heterocyclyl containing a total of 4 to 10 ring atoms, wherein the heterocyclyl ring: (i) is a saturated, partially saturated, or completely unsaturated monocyclic or fused bicyclic ring, (ii) has one or two ring heteroatoms independently selected from nitrogen, oxygen, and sulfur with the remaining ring atoms being carbon, and (iii) is optionally substituted with one or more substituents independently selected from the group consisting of halogen, oxo, C1-6-alkyl, halo-C1-6-alkyl, and C3-6-cycloalkyl; R15, R19, R23, R26, and R30are independently selected from hydrogen, halogen, cyano, C1-3-alkyl, and C1-3-alkoxy; R16is selected from the group consisting of hydrogen, halogen, C1-3-alkyl, halo-C1-3-alkyl, C1-3-alkoxy, and halo-C1-3-alkoxy; R21, R25, R27, and R29are independently selected from hydrogen and -X-R33;X is a bond or C1-3-alkyl; R31and R32are independently selected from hydrogen and C1-6-alkyl; and R33is selected from the group consisting of C1-3-alkyl, C3-6-cycloalkyl, phenyl, and 4- to 6-membered ring heteroaryl having one or two nitrogen ring atoms; wherein the C1-3-alkyl, C3-6- cycloalkyl, phenyl, and 4- to 6-membered ring heteroaryl are optionally substituted with one or more substituents independently selected from the group consisting of halogen and C1-6-alkyl.

[0007] In another aspect, the present disclosure provides compounds wherein the structure of Formula (1) is selected from the group consisting of Formulae (2), (3), (4), (5), (6), (7), (8), and (9) as further defined in this disclosure, and pharmaceutically acceptable salts thereof.

[0008] In another aspect, the present disclosure provides compounds wherein the structure of Formula (1) is selected from the group consisting of Formulae (2-A), (2-B), (2-C), (2-D), and (2- E) as further defined in this disclosure, and pharmaceutically acceptable salts thereof.

[0009] In another aspect, the present disclosure provides compounds wherein the structure of Formula (1) is selected from the group consisting of Formulae (3-A), (3-B), (3-C), (3-D), and (3- E) as further defined in this disclosure, and pharmaceutically acceptable salts thereof.

[0010] In another aspect, the present disclosure provides compounds wherein the structure of Formula (1) is selected from the group consisting of Formulae (4-A), (4-B), (4-C), (4-D), and (4- E): as further defined in this disclosure, and pharmaceutically acceptable salts thereof.

[0011] In another aspect, the present disclosure provides compounds wherein the structure of Formula (1) is selected from the group consisting of Formulae (5-A), (5-B), (5-C), (5-D), and (5- E) as further defined in this disclosure, and pharmaceutically acceptable salts thereof.

[0012] In another aspect, the present disclosure provides compounds wherein the structure of Formula (1) is selected from the group consisting of Formulae (6-A), (6-B), (6-C), (6-D), and (6- E) as further defined in this disclosure, and pharmaceutically acceptable salts thereof.

[0013] In another aspect, the present disclosure provides compounds wherein the structure of Formula (1) is selected from the group consisting of Formulae (7-A), (7-B), (7-C), (7-D), and (7- E) as further defined in this disclosure, and pharmaceutically acceptable salts thereof.

[0014] In another aspect, the present disclosure provides compounds wherein the structure of Formula (1) is selected from the group consisting of Formulae (8-A), (8-B), (8-C), (8-D), and (8- E) as further defined in this disclosure, and pharmaceutically acceptable salts thereof.

[0015] In another aspect, the present disclosure provides compounds wherein the structure of Formula (1) is selected from the group consisting of Formulae (9-A), (9-B), (9-C), (9-D), and (9- E) as further defined in this disclosure, and pharmaceutically acceptable salts thereof.

[0016] In another aspect, the present disclosure provides pharmaceutical compositions comprising a therapeutically-effective amount of a compound having the structure of Formula (1), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0017] In another aspect, the present disclosure provides pharmaceutical compositions comprising therapeutically-effective amounts of a compound having the structure of Formula (1), or a pharmaceutically acceptable salt thereof; a second pharmacological agent; and a pharmaceutically acceptable carrier.

[0018] In another aspect, the present disclosure provides methods for treating or preventing cancers by administering a therapeutically effective amount of a compound having the structure of Formula (1), or pharmaceutically acceptable salt thereof, to a subject in need thereof. In a further aspect, the cancer is a solid tumor cancer. In a still further aspect, the cancer is a hematological cancer. In a still further aspect, the cancer has a PKMYT1-dependency because of elevated levels of replication stress.

[0019] In another aspect, the present disclosure provides compounds having the structure of Formula (1), or pharmaceutically acceptable salts thereof, for use as a medicament for treating or preventing cancers. In a further aspect, the cancer has a PKMYT1-dependency because of elevated levels of replication stress.

[0020] In another aspect, the present disclosure provides use of compounds having the structure of Formula (1), or pharmaceutically acceptable salts thereof, to prepare a medicament for treating or preventing cancers. In a further aspect, the cancer has a PKMYT1-dependency because of elevated levels of replication stress.

[0021] In another aspect, the present disclosure provides kits comprising a compound having the structure of Formula (1), or pharmaceutically acceptable salt thereof.

[0022] In another aspect, the present disclosure provides methods for preparing compounds having the structure of Formula (1), or pharmaceutically acceptable salts thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Fig.1 illustrates WEE1 and PKMYT1 regulation of the mitotic cell.

[0024] Fig.2 illustrates the effect of treatment with a PKMYT1 inhibitor tool compound (GI50) on 16 cancer cell line models having either elevated or low levels of basal replication stress.

[0025] Fig.3 illustrates the replication fork velocities determined for the uterine and breast cancer cell line models of Fig.2 treated with the PKMYT1 inhibitor tool compound.

[0026] Fig.4 illustrates the replication fork velocities determined for the same uterine and breast cancer cell lines of Fig.2 that were treated instead with Example B-1, Isomer 1 (i.e., the eutomer of Compound 1).

[0027] Figure 5 illustrates the dose response curves obtained for Compound 1, Isomer 1 and Compound 137, Isomer 2 in an in vitro hematopoietic stem and progenitor cell assay assessing bone marrow toxicity.

[0028] Fig.6 illustrates the differential expression of phosphorylated proteins in OVCAR3 cells after 24-hour treatment with Compound 137, Isomer 2 or control (adavosertib).

[0029] Fig.7 illustrates (i) the effect of PKMYT1 deletion from SKOV3 cells on CDK1 T14 phosphorylation (left and middle panels) after treatment with Compound 1, Isomer 1, and (ii) the effect of treatment (GI50) with Compound 137, Isomer 2 on PKMYT1 knock out SKOV3 cells (i.e., cells that do not express the drug target PKMYT1) (right panel).

[0030] Fig.8-A illustrates the tolerability in mice of a 300 mg / kg BID dosing regimen with Compound 137, Isomer 2 over a 28-day period.

[0031] Fig.8-B illustrates the tolerability in mice of a 100 mg / kg BID dosing regimen with Compound 137, Isomer 2 in combination with either irinotecan hydrochloride (left panel) or gemcitabine (right panel) over a 28-day period.

[0032] Fig.9 illustrates PKMYT1 substrate (CDK1) phosphorylation (on Thr14) in OVCAR3 tumor bearing SCID mice following dose scheduling with Compound 137, Isomer 2 (twice-daily dosing for 21 days).

[0033] Fig.10 illustrates the in vivo efficacy of Compound 137, Isomer 2 in combination with gemcitabine in a human ovarian OVCAR3 xenograft model.

[0034] Fig.11 illustrates in vivo efficacy of Compound 137, Isomer 2 in combination with irinotecan hydrochloride in a human colorectal SW620 xenograft model.

[0035] Fig.12 illustrates in vitro efficacy of Compound 137, Isomer 2, alone or in combination with 1 nM exatecan, in PKMYT1 wildtype and PKMYT1 knockout SKOV3 cells.

[0036] Fig.13 illustrates the effect on in vivo biomarker expression (phospho-CDK1 Thr14, gH2AX, and phospho-histone H3 Ser10) in a human colorectal SW620 xenograft model after treatment with irinotecan hydrochloride, either alone or in combination with Compound 137, Isomer 2 (100 mg / kg BID).

[0037] Figure 14 is a metabolism scheme showing the dominant routes of biotransformations (oxidation and glucuronidation) observed in vitro of Compound 137, Isomer 2.

[0038] Figure 15 shows the pharmacokinetic profiles in CD-1 mice (total blood plasma concentration) of Compound 137, Isomer 2 following administration either I.V. (0.5 mg / kg, solid line) or P.O. (1 mg / kg, dashed line).

[0039] Figure 16 shows the pharmacokinetic profiles in SCID mice (total blood plasma concentration) of Compound 137, Isomer 2 following administration P.O. at three dose levels: 10 mg / kg (solid line), 30 mg / kg (dashed line), and 100 mg / kg (dotted line).

[0040] Figure 17 shows the pharmacokinetic profiles in Han Wistar rats (total blood plasma concentration) of Compound 137, Isomer 2 following administration I.V. (0.5 mg / kg) to either intact (solid line) or bile-duct cannulated (dashed line) animals.

[0041] Figure 18 shows the pharmacokinetic profiles in Han Wistar rats (total blood plasma concentration) of Compound 137, Isomer 2 following administration P.O. at three dose levels: 10 mg / kg (solid line), 30 mg / kg (dashed line), and 100 mg / kg (dotted line).

[0042] Figure 19 shows a comparison of the pharmacokinetic profiles in SCID mice (total blood plasma concentration) of Compound 137, Isomer 2 following repeat BID oral administration to male SCID mice (n=2) at 100 mg / kg on day 1 (solid line) and day 28 (dashed line).

[0043] Figure 20 shows the pharmacokinetic profiles in SCID mice (total blood plasma concentration) of Compound 137, Isomer 2 following administration P.O. at 300 mg / kg in 5% DMSO, 50% of 20% Captisol, and 45% WFI (pH 3-3.2) (solid line) and in 0.5% HPMC / 0.1% Tween (dashed line).

[0044] For convenience, Compound 1, Isomer 1 is identified in the Figures as AZ1-1 and Compound 137, Isomer 2 is identified in the Figures as AZ137-2.DETAILED DESCRIPTION

[0045] Many embodiments are detailed throughout this disclosure and will be apparent to a reader skilled in the art. The disclosure is not to be interpreted as being limited to any particular embodiment(s) described herein. I. Definitions

[0046] With respect to the embodiments disclosed in this disclosure, the following terms have the meanings set forth below:

[0047] Reference to “a” or “an” means “one or more.” Throughout, the plural and singular should be treated as interchangeable, other than the indication of number.

[0048] Unless the context requires otherwise, the words "comprise" or "comprises" or “comprising" are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that Applicant intends each of those words to be so interpreted in construing this patent, including the claims below.

[0049] The term “cyano” (alone or in combination with another term(s)) means CN.

[0050] The term “halogen” (alone or in combination with another term(s)) means a fluorine radical (which may be depicted as F), chlorine radical (which may be depicted as Cl), bromine radical (which may be depicted as Br), or iodine radical (which may be depicted as I).

[0051] The term “oxo” (alone or in combination with another term(s)) means an oxo radical, and may be depicted as =O.

[0052] The term “alkyl” (alone or in combination with another term(s)) means a straightor branchedchain saturated hydrocarbyl substituent (i.e., a substituent containing only carbon and hydrogen). Alkyl typically contains from 1 to about 20 carbon atoms, more typically from 1 to about 12 carbon atoms, even more typically from 1 to about 8 carbon atoms, and still even more typically from 1 to about 6 carbon atoms. Examples of such substituents include methyl, ethyl, propyl (including npropyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, and tert- butyl), pentyl (including n-pentyl, isoamyl, and 2,2-dimethylpropyl), and hexyl.

[0053] The term “alkenyl” (alone or in combination with another term(s)) means a straight or branched chain saturated hydrocarbyl substituent (i.e., a substituent containing only carbon and hydrogen) containing one or more double bonds in the alkyl chain. Alkenyl typically contains from 2 to about 20 carbon atoms, more typically from 2 to about 12 carbon atoms, even more typically from 2 to about 8 carbon atoms, and still even more typically from 2 to about 6 carbonatoms. Examples of such substituents include ethenyl, propenyl (including 1-propenyl and 2- propenyl), butyl (including 2-butenyl and 3-butenyl), pentenyl, and hexenyl.

[0054] The term “alkynyl” (alone or in combination with another term(s)) means a straight or branched chain saturated hydrocarbyl substituent (i.e., a substituent containing only carbon and hydrogen) containing one or more triple bonds in the alkyl chain. Alkynyl typically contains from 2 to about 20 carbon atoms, more typically from 2 to about 12 carbon atoms, even more typically from 2 to about 8 carbon atoms, and still even more typically from 2 to about 6 carbon atoms. Examples of such substituents include ethynyl, propynyl (including 1-propynyl and 2- propynyl), butynyl (including 1butynyl and 2butynyl), pentynyl (including penta-1,3-diene, 2- methyl-1,3-butadiene, and penta-1,2-diene), and hexynyl.

[0055] The term “cycloalkyl” (alone or in combination with another term(s)) means a saturated carbocyclyl substituent containing from 3 to about 14 carbon ring atoms, more typically from 3 to about 12 carbon ring atoms, and even more typically from 3 to about 8 carbon ring atoms. A cycloalkyl includes a single carbon ring, which typically contains from 3 to 6 carbon ring atoms. Examples of singlering cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0056] The term “alkoxy” (alone or in combination with another term(s)) means an alkylether substituent, i.e., alkyl-O-. Examples of alkoxy include methoxy (CH3-O-), ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy. Thus, for example, the term “alkoxyalkyl” (alone or in combination with another term(s)) means alkyl substituted with alkoxy such as “methoxymethyl” which may be depicted as: .

[0057] The term “heterocyclyl” (alone or in combination with another term(s)) means 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), with the remaining ring atoms being independently selected from the group consisting of carbon, oxygen, nitrogen, and sulfur in stable combinations known to those of skill in the art.

[0058] In some instances, the number of carbon atoms in a substituent (e.g., alkyl, cycloalkyl, etc.) is indicated by the prefix “Cx–y“, wherein x is the minimum and y is the maximum number of carbon atoms in the substituent. Thus, for example, “C1–6alkyl” refers to an alkyl substituentcontaining from 1 to 6 carbon atoms. Illustrating further, C3-6cycloalkyl refers to a cycloalkyl substituent containing from 3 to 6 carbon ring atoms.

[0059] The prefix “halo” indicates that the substituent to which the prefix is attached is substituted with one or more independently selected halogen radicals. For example, haloalkyl means an alkyl substituent wherein at least one hydrogen radical is replaced with a halogen radical. Where there is more than one hydrogen replaced with halogens, the halogens may be the identical or different. Examples of haloalkyls include fluoromethyl, difluoromethyl, trifluoromethyl, difluoroethyl, 1,1,1trifluoroethyl, pentafluoroethyl, difluoropropyl, heptafluoropropyl chloromethyl, dichloromethyl, trichloromethyl, difluorochloromethyl, dichlorofluoromethyl, and dichloropropyl.

[0060] A substituent is “substitutable” if it comprises at least one carbon or nitrogen atom that is bonded to one or more hydrogen atoms. Thus, for example, hydrogen, halogen, and cyano do not fall within this definition.

[0061] If a substituent is described as being “substituted,” a non-hydrogen radical is in the place of a hydrogen radical on a carbon or nitrogen of the substituent. Thus, for example, a substituted alkyl substituent is an alkyl substituent wherein at least one non-hydrogen radical is in the place of a hydrogen radical on the alkyl substituent. To illustrate, monofluoroalkyl is alkyl substituted with a fluoro radical, and difluoroalkyl is alkyl substituted with two fluoro radicals. It should be recognized that if there is more than one substitution on a substituent, each nonhydrogen radical may be identical or different (unless otherwise stated).

[0062] If a substituent is described as “optionally substituted”, the substituent may be either (1) not substituted, or (2) substituted. If a carbon of a substituent is described as optionally substituted with one or more of a list of substituents, one or more of the hydrogens on the carbon (to the extent there are any) may separately and / or together be replaced with an independently selected optional substituent. If a nitrogen of a substituent is described as optionally substituted with one or more of a list of substituents, one or more of the hydrogens on the nitrogen (to the extent there are any) may each be replaced with an independently selected optional substituent.

[0063] If substituents are described as being “independently selected” from a group, each substituent is selected independently of the other. Each substituent therefore may be identical to or different from the other substituent(s).

[0064] The term “atropisomers” refers to the stereoisomers resulting from hindered rotation about one or more single bonds, where the energy barrier to rotation is high enough to allow forthe isolation of the conformers. As used in this disclosure, the term “eutomer” refers to the more pharmacologically active conformer of an atropisomer and the term “distomer” refers to the less pharmacologically active conformer of an atropisomer.

[0065] The term “pharmaceutically acceptable” is used adjectivally in this disclosure to mean that the modified noun is appropriate for use as a pharmaceutical product or as a part of a pharmaceutical product. For example, “pharmaceutically acceptable salts” are salts that are suitable for use in mammals, particularly humans, and include salts with an inorganic base, organic base, inorganic acid, organic acid, or basic or acidic amino acid that are suitable for use in mammals, particularly humans.

[0066] A “therapeutically effective amount” of a pharmacological agent is an amount that is sufficient to effect beneficial or desired results, including clinical results, and, as such, will depend upon the situation in which it is being administered. Where the pharmacological agent is being administered to treat cancer, for example, a therapeutically effective amount of the agent is an amount of the agent that is sufficient, either alone or in combination with additional therapies, to provide an anti-cancer effect in a subject as compared to the response obtained without administration of the agent.

[0067] The term “preventing” is readily understood by an ordinarily skilled physician and, with respect to treatment of a particular condition, is intended to have its normal meaning and includes primary prophylaxis to prevent the development of the condition and secondary prophylaxis whereby the condition has already developed and the patient is temporarily or permanently protected against exacerbation or worsening of the disease or the development of new symptoms associated with the condition.

[0068] The term "treating” is readily understood by an ordinarily skilled physician and, with respect to treatment of a particular condition, can include (1) diminishing the extent or cause of the condition being treated, and / or (2) alleviating or ameliorating one or more symptoms associated with that condition. Treatment of cancer, for example, can include stabilizing (i.e., not worsening), delaying, or slowing the spread or progression of the cancer; prolonging survival as compared to expected survival if not receiving treatment; and / or otherwise ameliorating or palliating the cancer or the severity of the cancer, in whole or in part.II. Compounds A. Compounds of Formula (1)

[0069] In one embodiment, the present disclosure provides compounds having the structure of Formula (1): (1), or a pharmaceutically acceptable salt thereof, wherein: R1is selected from the group consisting of: , , , , and ; R2, R3, R4, R5, R7, R8, R9, R10, and R12are independently selected from the group consisting of halogen and C1-3-alkyl; R6and R11are independently selected from the group consisting of hydrogen and C1-3- alkyl; A is selected from the group consisting of: , , ,, , , , and ; R13and R24are independently selected from the group consisting of hydrogen, C1-3-alkyl, and C1-3-alkoxy; R14, R17, R18, R20, R22, and R28are independently selected the group consisting of: (a) hydrogen; (b) halogen; (c) cyano; (d) C1-6-alkyl, wherein the C1-6-alkyl is optionally substituted with one or more substituents independently selected from halogen; (e) -C1-6-alkenyl, wherein the C1-6-alkenyl is optionally substituted with one or more substituents independently selected from halogen; (f) C1-6-alkynyl, wherein the C1-6-alkynyl is optionally substituted with one or more substituents independently selected from halogen; (g) C3-6-cycloalkyl, wherein the C3-6-cycloalkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-6-alkyl, halo-C1-6-alkyl, C1-6-alkoxy, and halo-C1-6-alkoxy; (h) C1-6-alkoxy; wherein the C1-6-alkoxy is optionally substituted with one or more substituents independently selected from halogen; (i) -NR31R32; (j) S(O)2-C1-6-alkyl, wherein the -S(O)2-C1-6-alkyl is optionally substituted with one or more substituents independently selected from halogen;(k) phenyl, wherein the phenyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-6-alkyl, halo-C1-6-alkyl, C1-6-alkoxy, and halo-C1-6-alkoxy; and (l) heterocyclyl containing a total of 4 to 10 ring atoms, wherein the heterocyclyl ring: (i) is a saturated, partially saturated, or completely unsaturated monocyclic or fused bicyclic ring, (ii) has one or two ring heteroatoms independently selected from nitrogen, oxygen, and sulfur with the remaining ring atoms being carbon, and (iii) is optionally substituted with one or more substituents independently selected from the group consisting of halogen, oxo, C1-6-alkyl, halo-C1-6-alkyl, and C3-6-cycloalkyl; R15, R19, R23, R26, and R30are independently selected from hydrogen, halogen, cyano, C1-3-alkyl, and C1-3-alkoxy; R16is selected from the group consisting of hydrogen, halogen, C1-3-alkyl, halo-C1-3-alkyl, C1-3-alkoxy, and halo-C1-3-alkoxy; R21, R25, R27, and R29are independently selected from hydrogen and -X-R33; X is a bond or C1-3-alkyl; R31and R32are independently selected from hydrogen and C1-6-alkyl; and R33is selected from the group consisting of C1-3-alkyl, C3-6-cycloalkyl, phenyl, and 4- to 6-membered ring heteroaryl having one or two nitrogen ring atoms; wherein the C1-3-alkyl, C3-6- cycloalkyl, phenyl, and 4- to 6-membered ring heteroaryl are optionally substituted with one or more substituents independently selected from the group consisting of halogen and C1-6-alkyl.

[0070] In some embodiments, the structure of Formula (1) is selected from the group consisting of Formulae (2), (3), (4), (5), (6), (7), (8), and (9): (2), (3), (4), (5),(6), and (7), (8), and (9); and wherein R1through R30are as defined for the compounds having the structure of Formula (1).

[0071] In some embodiments, the structure of Formula (1) is selected from the group consisting of Formulae (2-A), (2-B), (2-C), (2-D), and (2-E): (2-A), (2-B), (2-C), (2-D), and(2-E); and wherein R2through R15are as defined for the compounds having the structure of Formula (1).

[0072] In some embodiments, the structure of Formula (1) is selected from the group consisting of Formulae (3-A), (3-B), (3-C), (3-D), and (3-E): (3-A), (3-B), (3-C), (3-D), and(3-E); and wherein R2through R12, R16, and R17are as defined for the compounds having the structure of Formula (1).

[0073] In some embodiments, the structure of Formula (1) is selected from the group consisting of Formulae (4-A), (4-B), (4-C), (4-D), and (4-E): (4-A), (4-B), (4-C), (4-D), and(4-E); and wherein R2through R12, R18, and R19are as defined for the compounds having the structure of Formula (1).

[0074] In some embodiments, the structure of Formula (1) is selected from the group consisting of Formulae (5-A), (5-B), (5-C), (5-D), and (5-E): (5-A), (5-B), (5-C), (5-D), and(5-E); and wherein R2through R12and R20are as defined for the compounds having the structure of Formula (1).

[0075] In some embodiments, the structure of Formula (1) is selected from the group consisting of Formulae (6-A), (6-B), (6-C), (6-D), and (6-E): (6-A), (6-B), (6-C), (6-D), and(6-E); and wherein R2through R12, R21, R22, and R23are as defined for the compounds having the structure of Formula (1).

[0076] In some embodiments, the structure of Formula (1) is selected from the group consisting of Formulae (7-A), (7-B), (7-C), (7-D), and (7-E): (7-A), (7-B), (7-C), (7-D), and(7-E); and wherein R2through R12, R24, R25, and R26are as defined for the compounds having the structure of Formula (1).

[0077] In some embodiments, the structure of Formula (1) is selected from the group consisting of Formulae (8-A), (8-B), (8-C), (8-D), and (8-E): (8-A), (8-B), (8-C), (8-D), and(8-E); and wherein R2through R12, R27, and R28are as defined for the compounds having the structure of Formula (1).

[0078] In some embodiments, the structure of Formula (1) is selected from the group consisting of Formulae (9-A), (9-B), (9-C), (9-D), and (9-E): (9-A), (9-B), (9-C), (9-D), and(9-E); and wherein R2through R12, R29, and R30are as defined for the compounds having the structure of Formula (1).

[0079] In some embodiments, the compound having the structure of Formula (1) is an atropisomer. In one aspect, the compound is the eutomer of the atropisomer. In another aspect, the compound is the distomer of the atropisomer. B. Additional Embodiments

[0080] In some embodiments, the present disclosure provides compounds of Formula (1), and pharmaceutically acceptable salts thereof, wherein the compound is selected from the group consisting of:In some embodiments, the compound selected is an atropisomer. In one aspect, the compound selected is the eutomer of the atropisomer.

[0081] 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 structure: (Compound 139), and pharmaceutically acceptable salts thereof. In one aspect, the compound is an atropisomer. In another aspect, the compound selected is the eutomer of the atropisomer.

[0082] 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 structure:(Compound 140), and pharmaceutically acceptable salts thereof. In one aspect, the compound is an atropisomer. In another aspect, the compound selected is the eutomer of the atropisomer.

[0083] 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 structure: (Compound 141), and pharmaceutically acceptable salts thereof. In one aspect, the compound is an atropisomer. In another aspect, the compound selected is the eutomer of the atropisomer.

[0084] In one embodiment, the present disclosure provides a compound that is 6-amino-7-(6- chloro-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5- carboxamide having the structure: (Compound 142),and pharmaceutically acceptable salts thereof. In one aspect, the compound is an atropisomer. In another aspect, the compound selected is the eutomer of the atropisomer.

[0085] In one embodiment, the present disclosure provides a compound that is 6-amino-7- (2,6-dichloro-3-hydroxyphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5- carboxamide having the structure: (Compound 143), and pharmaceutically acceptable salts thereof. In one aspect, the compound is an atropisomer. In another aspect, the compound selected is the eutomer of the atropisomer.

[0086] In one embodiment, the present disclosure provides a compound that is 6-amino-7-(6- bromo-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine- 5-carboxamide having the structure: (Compound 144), and pharmaceutically acceptable salts thereof. In one aspect, the compound is an atropisomer. In another aspect, the compound selected is the eutomer of the atropisomer.

[0087] In one embodiment, the present disclosure provides 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); and pharmaceutically acceptable salts thereof. In one aspect, the compound is an atropisomer. In another aspect, the compound selected is the eutomer of the atropisomer. C. Combination of Embodiments

[0088] Any embodiment of the compounds described in the present disclosure can be combined with any other suitable embodiment described herein to provide additional embodiments. For example, where one embodiment of the compounds of Formula (2) individually or collectively describes possible groups for R13, R14, and / or R15and a separate embodiment describes possible groups for R1, it is understood that these embodiments can be combined to provide an additional embodiment describing the possible groups described for R13, R14, and / or R15together with the possible groups described for R1. In other words, for any of the embodiments of the compounds described in the present disclosure, the R1substituent can be as defined in any of the embodiments of R1described in this disclosure. D. Further Embodiments

[0089] In some embodiments, the compounds of the present disclosure have an IC50value for inhibition of PKMYT1 below about 1 ^M as measured in the Target Engagement Assay described in Example C-2 below. In one aspect, the IC50value is below about 500 nM. In another aspect, the IC50value is below about 250 nM. In another aspect, the IC50value is below about 100 nM. In another aspect, the compounds have a pharmaceutically acceptable Kdvalue for PKMYT1 binding affinity as measured in the SPR Direct Binding Assay described in Example C-3 below.

[0090] In some embodiments, the compounds of the present disclosure have a pharmaceutically acceptable selectivity for PKMYT1 relative to WEE1 as measured in the TargetEngagement Assay described in Example C-2 below. In some embodiments, the compounds are at least about 50 times more selective for PKMYT1 relative to WEE1. In another aspect, the compounds are at least about 100 times more selective for PKMYT1 relative to WEE1. In another aspect, the compounds are at least about 250 times more selective for PKMYT1 relative to WEE1. In another aspect, the compounds are at least about 500 times more selective for PKMYT1 relative to WEE1. In another aspect, the compounds of the present disclosure have an IC50value for inhibition of WEE1 greater than about 1 ^M. In another aspect, the IC50value is greater than about 5 ^M. In another aspect, the IC50value is greater than about 10 ^M.

[0091] In some embodiments, the compounds of the present disclosure have an IC50value for inhibition of CDK1 phosphorylation below about 2 µM as measured in the Functional CDK1 pThr14 AlphaLISA Assay described in Example C-4 below. In one aspect, the IC50value is below about 1 µM. In another aspect, the IC50value is below about 500 nM. In another aspect, the IC50value is below about 250 nM. In another aspect, the IC50value is below about 100 nM.

[0092] In some embodiments, the compounds of the present disclosure inhibit OVCAR3 cellular proliferation as measured in the Cellular Proliferation Assay described in Example C-5 below. In one aspect, the compounds have a GI50value below about 5 ^M. In another aspect, the GI50value is below about 3 ^M. In another aspect, the GI50value is below about 1.5 ^M. E. Salts

[0093] The compounds of the present disclosure may exist in salt form or in non-salt form (i.e., as a free base), and the present disclosure covers both salt forms and non-salt forms. The compounds may form acid addition salts or base addition salts. In general, an acid addition salt can be prepared using various inorganic or organic acids. Such salts can typically be formed by, for example, mixing the compound with an acid (e.g., a stoichiometric amount of an acid) using various methods known in the art. This mixing may occur in water, an organic solvent (e.g., ether, ethyl acetate, ethanol, methanol, isopropanol, or acetonitrile), or an aqueous / organic mixture. In another aspect, the acid addition salts are, for example, trifluoroacetate, formate, acetate or hydrochloric. In general, a base addition salt can be prepared using various inorganic or organic bases, for example an alkali or alkaline earth metal salt such as a sodium, calcium or magnesium salt, or other metal salts, such as potassium or zinc, or an ammonium salt, or a salt with an organic base such as methylamine, dimethylamine, trimethylamine, piperidine or morpholine. The skilled person will be aware of the general principles and techniques ofpreparing pharmaceutical salts, such as those described in, for example, Berge, S., et al., “Pharmaceutical Salts,” J. Pharm. Sci.66, 1 (1977). Examples of pharmaceutically acceptable salts are also described in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002). F. Isomers

[0094] The compounds and salts of the present disclosure may exist in one or more geometrical, optical, enantiomeric, and diastereomeric forms, including, but not limited to, cis- and trans-forms, E- and Z-forms, and R-, S- and meso-forms. Unless otherwise stated a reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof. Where appropriate such isomers can be separated from their mixtures by the application or adaptation of known methods (e.g., chromatographic techniques and recrystallisation techniques). In some embodiments, a single stereoisomer is obtained by isolating it from a mixture of isomers (e.g., a racemate) using, for example, chiral chromatographic separation. In other embodiments, a single stereoisomer is obtained through direct synthesis from, for example, a chiral starting material.

[0095] A particular enantiomer of a compound 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 being in an enantiomeric excess (%ee) of ≥ 90, ≥ 95%, ≥ 96%, ≥ 97, ≥ 98% or ≥ 99%. In one aspect, the single enantiomer is present in an enantiomeric excess (%ee) of ≥ 99%.

[0096] In another embodiment, the present disclosure relates to a pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt thereof, which is a single enantiomer being in an enantiomeric excess (%ee) of ≥ 90, ≥ 95%, ≥ 96%, ≥ 97, ≥ 98% or ≥ 99%, or a pharmaceutically acceptable salt thereof, in association with one or more pharmaceutically acceptable excipients. In one aspect, the single enantiomer is present in an enantiomeric excess (%ee) of ≥ 99%. G. Additional Forms

[0097] The compounds and salts of the present disclosure may exist in various tautomeric forms and the disclosure encompasses all such tautomeric forms. “Tautomers” are structural isomers that exist in equilibrium resulting from the migration of a hydrogen atom.

[0098] The compounds of the present disclosure, and pharmaceutically acceptable salts thereof, may exist as solvates (such as a hydrates) as well as unsolvated forms, and the present disclosure covers all such solvates.

[0099] The compounds of the present disclosure, and pharmaceutically acceptable salts thereof, may exist in crystalline or amorphous form, and the present disclosure covers all such forms.

[0100] Compounds and salts of the present disclosure may be isotopically labeled (or “radio- labeled”). In that instance, one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. The disclosure encompasses isotopically labelled forms of compounds disclosed herein. Examples of isotopes that may be incorporated include2H (also written as “D” for deuterium),3H (also written as “T” for tritium),11C,13C,14C,13N,15N,15O,17O,18O and36Cl. The isotope that is used will depend on the specific application of that radio-labeled derivative. For example, for in vitro receptor labeling and competition assays,3H or14C are often useful. For radio-imaging applications,11C is often useful. In some embodiments, the radionuclide is3H. In some embodiments, the radionuclide is14C. In some embodiments, the radionuclide is11C. H. Intermediates

[0101] In some embodiments, the present disclosure provides additional compounds that are useful as intermediates for preparing the compounds of the present disclosure, and pharmaceutically acceptable salts thereof. III. Methods of Use

[0102] One consequence of cancer cell evolution is often the rewiring of the ability to control cell cycle progression which then leads to more rapid oncogenic expansion to aid oncogenesis. More than 50% of all cancers, and over 90% of cancers such as serous-like endometrial, serous ovarian, and basal-like breast cancers, carry a somatic TP53 mutation that can result in inactivation of the tumor suppressor protein p53 and partial or complete inactivation of the G1 / S cell cycle checkpoint. Under such circumstances, cells enter the replicative S-phase of the cell cycle prematurely, thereby increasing the likelihood of elevated basal replication stress levels, resulting in a greater dependency on the G2 / M cell cycle checkpoint regulated by WEE1 and PKMYT1.

[0103] As previously noted, both WEE1 and PKMYT1 inhibit CDK1 to prevent a cell from dividing until damaged DNA is repaired (G2 / M DNA damage cell cycle checkpoint arrest). In contrast to WEE1, however, PKMYT1 is selective for CDK1 relative to CDK2. WEE1 also regulates CDK2 activity that normally prevents aberrant progression of cells through S-phase. Since WEE1 regulates both CDK1 and CDK2, WEE1 inhibition can further increase replication stress (as detected by biomarkers such as phospho-RPA2 (pRPA2) and phospho-Histone H2AX (γH2AX)) in cancers that already have elevated basal levels of replication stress, creating additional DNA damage during 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 the 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 Fig.1.

[0104] Preclinical and clinical data generated with the WEE1 inhibitor adavosertib have identified cancer backgrounds associated with replication stress that are sensitive to WEE1 inhibition. Such cancer backgrounds include those enriched for p53 mutations and / or one or more aberrations in genes and / or their related protein products including, but are 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, uterine serous 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. Although tumor types harboring these deficiencies can be sensitive to monotherapy with WEE1 inhibitors in the clinic, the fact that WEE1 inhibition also generates S-phase replication stress and DNA damage means that certain normal tissues are also affected resulting in gastrointestinal and hematological toxicities. Consequently, monotherapy with WEE1 inhibitors can have a relatively narrow therapeutic window. Liu, J. F., et al., "Phase II Study of the WEE1 Inhibitor Adavosertib in Recurrent Uterine Serous Carcinoma," Journal of Clinical Oncology 39(14): 1531-1539 (2021).

[0105] Since PKMYT1 regulates CDK1 and not CDK2, PKMYT1 inhibitors have the potential to treat cancers with elevated levels of basal replication stress, either as a single agent orin combination with DNA damaging agents or targeted agents that increase the levels of replication stress, but with an improved therapeutic window relative to WEE1 inhibitors. As illustrated in Example C-2, cancer backgrounds associated with replication stress that are sensitive to PKMYT1 inhibition include those enriched for one or more aberrations in genes and / or their related protein products including, but are not limited to, cyclin E1, FBXW7, and / or KRAS. Given the dependency of cancers with elevated levels of replication stress on the G2 / M cell cycle checkpoint, it is hypothesized that further cancer backgrounds associated with replication stress that are sensitive to PKMYT1 inhibition may include those enriched for TP53 mutations and / or one or more aberrations in genes and / or their related protein products including, but are not limited to, cyclin D1, cyclin E1, FBXW7, RB1, INK4a, ARF, CDK4, CDK6, SETD2, KDM4A, LKB1, TSC2, RSP6KA6, MYC, and / or KRAS. Accordingly, PKMYT1 inhibitors that are selective for CDK1 are desirable and are within the scope of this disclosure.

[0106] The compounds of Formula (1), and pharmaceutically acceptable salts thereof, are inhibitors of PKMYT1 activity. In one aspect, the compounds selectively inhibit PKMYT1 activity.

[0107] In some embodiments, the present disclosure provides a method for treating or preventing cancers in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound of Formula (1), or a pharmaceutically acceptable salt thereof. In one aspect, the cancer is a solid tumor cancer. In another aspect, the solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple negative breast cancer), uterine cancer (including uterine serous 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 aspect, the solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple negative breast cancer), uterine cancer (including uterine serous carcinoma and uterine carcinosarcoma), and pancreatic cancer. In another aspect the cancer is a hematological cancer. In another aspect the hematological cancer is B-cell lymphoma.

[0108] In some embodiments, the present disclosure provides a method for treating or preventing cancers in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound of Formula (1), or a pharmaceutically acceptable salt thereof, wherein the cancer is, in whole or in part, a PKMYT1-dependent cancer. In one aspect, thePKMYT1-dependent cancer is a solid tumor cancer. In another aspect, 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 uterine serous 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 aspect, 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 uterine serous carcinoma and uterine carcinosarcoma), and pancreatic cancer. In another aspect the PKMYT1- dependent cancer is a hematological cancer. In another aspect the PKMYT1-dependent hematological cancer is B-cell lymphoma.

[0109] In some embodiments, the present disclosure provides a method for treating or preventing cancers in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound of Formula (1), or a pharmaceutically acceptable salt thereof, wherein the cancer is associated with a higher incidence of genetic or protein aberrations associated with elevated levels of basal replication stress. In one aspect, the cancer is characterized by amplification or overexpression of the gene. In another aspect, the cancer is characterized by one or more deletions and / or mutations in the gene. In another aspect, the cancer is a solid tumor cancer. In another aspect, the solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple negative breast cancer), uterine cancer (including uterine serous 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 aspect, the solid tumor cancer is selected from the group consisting of ovarian cancer, breast cancer (including triple negative breast cancer), uterine cancer (including uterine serous carcinoma and uterine carcinosarcoma), and pancreatic cancer. In another aspect the cancer is a hematological cancer. In another aspect the hematological cancer is B-cell lymphoma.

[0110] In some embodiments, the present disclosure provides a method for treating or preventing cancers in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound of Formula (1), or a pharmaceutically acceptable salt thereof, wherein the cancer is associated with one or more aberrations in a gene, and / or its related proteinproduct, selected from the group consisting of cyclin E1, FBXW7, and / or KRAS. In one aspect, the cancer is associated with one or more aberrations in the cyclin E1 gene (CCNE1) and / or its related protein product. In another aspect, the cancer is characterized by amplification or overexpression of CCNE1. In another aspect, the cancer is associated with one or more aberrations in the FBXW7 gene and / or its related protein product. In another aspect, the cancer is characterized by one or more deletions and / or mutations in the FBXW7 gene. In another aspect, the cancer is associated with one or more aberrations in the KRAS gene and / or its related protein product. In another aspect, the cancer is characterized by one or more deletions and / or mutations in the KRAS gene.

[0111] In some embodiments, the cancer is ovarian cancer. In one aspect, the ovarian cancer is a PKMYT1-dependent ovarian cancer. In another aspect, the cancer is a platinum-sensitive or platinum-resistant ovarian cancer.

[0112] In some embodiments, the cancer is breast cancer. In one aspect, the breast cancer is a PKMYT1-dependent breast cancer. In another aspect, 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 aspect, the breast cancer is a chemotherapy-resistant breast cancer. In another aspect, the breast cancer is a radiotherapy- resistant breast cancer. In another aspect, the breast cancer is an advanced or metastatic breast cancer.

[0113] In some embodiments, the cancer is uterine cancer. In one aspect, the uterine cancer is a PKMYT1-dependent uterine cancer. In another aspect, the uterine cancer is uterine serous carcinoma. In another aspect, the uterine cancer is uterine carcinosarcoma.

[0114] In some embodiments, the cancer is pancreatic cancer. In one aspect, the pancreatic cancer is a PKMYT1-dependent pancreatic cancer.

[0115] In some embodiments, the cancer is lung cancer. In one aspect, the lung cancer is a PKMYT1-dependent lung cancer. In another aspect, the lung cancer is small cell lung cancer (SCLC). In another aspect, the lung cancer is non-small cell lung cancer (NSCLC). In another aspect, the non-small cell lung cancer (NSCLC) is squamous cell carcinoma. In another aspect, the non-small cell lung cancer (NSCLC) is adenocarcinoma. In another aspect, the non-small cell lung cancer (NSCLC) is large-cell carcinoma.

[0116] In some embodiments, the cancer is gastric cancer. In one aspect, the gastric cancer is a PKMYT1-dependent gastric cancer.

[0117] In some embodiments, the cancer is esophageal cancer. In one aspect, the esophageal cancer is a PKMYT1-dependent esophageal cancer.

[0118] In some embodiments, the present disclosure provides a method for treating or preventing a hematological cancer in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, wherein the hematological cancer is selected from the group consisting of non-Hodgkin's lymphoma, leukemia, multiple myeloma (MM), and myelodysplastic syndrome (MDS). In one aspect, the hematological cancer is a PKMYT1-dependent hematological cancer. In another aspect, the hematological cancer is non-Hodgkin's lymphoma (NHL). In another aspect, the 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 aspect, the hematological cancer is leukemia. In another aspect, the 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 aspect, the hematological cancer is multiple myeloma (MM). In another aspect, the hematological cancer is myelodysplastic syndrome (MDS). In another aspect, the hematological cancer is diffuse large B-cell lymphoma (DLBCL).

[0119] In some embodiments, the compound of the present disclosure, or a pharmaceutically acceptable salt thereof, is administered as first line therapy.

[0120] In some embodiments, the compound of the present disclosure, or a pharmaceutically acceptable salt thereof, is administered as second line (or later) therapy.

[0121] In some embodiments, the subject to whom a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, is administered exhibits a partial response (PR) in response to such treatment.

[0122] In some embodiments, the subject to whom a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, is administered exhibits a complete response (CR) in response to such treatment.

[0123] In some embodiments, the subject to whom a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, is administered exhibits an improved progression free survival (PFS) in response to such treatment.

[0124] In some embodiments, the subject to whom a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, is administered exhibits an improved overall survival (OS) in response to such treatment.

[0125] The subject treated typically will be a human or non-human mammal, particularly a human. Suitable subjects can also include domestic or wild animals; companion animals (including dogs, cats, and the like); livestock (including horses, cows and other ruminants, pigs, poultry, rabbits, and the like); primates (including monkeys such as rhesus monkeys, cynomolgus (also known as crab-eating or long-tailed) monkeys, marmosets, tamarins, chimpanzees, macaques, and the like); and rodents (including rats, mice, gerbils, guinea pigs, and the like).

[0126] In some embodiments, the present disclosure provides the compounds of Formula (1), or pharmaceutically acceptable salts thereof, for use as medicaments.

[0127] In some embodiments, the present disclosure provides for the use of the compounds of the Formula (1), or pharmaceutically acceptable salts thereof, for treating or preventing cancers, including PKMYT1-dependent cancers as discussed above.

[0128] In some embodiments, the present disclosure provides for the use of the compounds of the Formula (1), or pharmaceutically acceptable salts thereof, for the manufacture of medicaments for treating or preventing cancer, including PKMYT1-dependent cancers as discussed above. IV. Combination Therapies and Fixed-Dose Combinations

[0129] The compounds and pharmaceutically acceptable salts of the present disclosure may be used in the methods described above as either as single pharmacological agents or in combination with other pharmacological agents or techniques including, for example, DNA damaging agents that increase reliance on the G2 / M checkpoint and targeted therapies that induce higher levels of replication stress. Such combination therapies may be achieved by way of the simultaneous, sequential, or separate dosing of the individual components of the treatment. These combination therapies (and corresponding combination products) employ the compounds and pharmaceutically acceptable salts of the present disclosure within the dosage ranges described in this application and the other pharmacological agent(s), typically within its approved dosage range(s).

[0130] In some embodiments, the present disclosure provides a combination suitable for use in the treatment or prevention of a PKMYT1-dependent cancer, wherein the combinationcomprises a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and radiotherapy.

[0131] In some embodiments, the present disclosure provides a combination suitable for use in the treatment or prevention of a PKMYT1-dependent cancer, wherein the combination comprises a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and chemotherapy. In one aspect, the chemotherapy is induction chemotherapy. In another aspect, the chemotherapy is consolidation chemotherapy. In another aspect, the chemotherapy comprises administration of one or more chemotherapeutics selected from the group consisting of an anthracycline, azacytidine, bleomycin, cisplatin, carboplatin, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, fluorouracil, gemcitabine, idarubicin, mercaptopurine, methotrexate, oxaliplatin, paclitaxel, thioguanine, and / or vincristine.

[0132] In some embodiments, the chemotherapy comprises administration of a nucleoside analog. In one aspect, the nucleoside analog is selected from the group consisting of cytarabine and gemcitabine.

[0133] In some embodiments, the chemotherapy comprises administration of a taxane. In one aspect, the taxane is selected from the group consisting of paclitaxel, docetaxel, and nab- paclitaxel.

[0134] In some embodiments, the chemotherapy comprises administration of a platinum chemotherapeutic. In one aspect, the platinum chemotherapeutic is selected from the group consisting of carboplatin and cisplatin.

[0135] In some embodiments, the chemotherapy further comprises administration of a steroid. In one aspect, the steroid is selected from the group consisting of prednisolone, dexamethasone, and hydrocortisone.

[0136] In some embodiments, the present disclosure provides a combination suitable for use in the treatment or prevention of a PKMYT1-dependent cancer, wherein the combination comprises a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a topoisomerase 1 (TOP1) inhibitor, topoisomerase 2 (TOP2) inhibitor, TOP1-antibody drug conjugate (TOP1-ADC), or TOP2-antibody drug conjugate (TOP2-ADC). In one aspect, the TOP1 inhibitor is selected from the group consisting of irinotecan and topotecan. In one aspect, the TOP1-ADC is trastuzumab deruxtecan. In another aspect, the TOP2 inhibitor is selected from the group consisting of etoposide and teniposide.

[0137] In some embodiments, the present disclosure provides a combination suitable for use in the treatment or prevention of a PKMYT1-dependent cancer, wherein the combination comprises a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a poly ADP ribose polymerase (PARP) inhibitor. In one aspect, 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 aspect, the PARP inhibitor is olaparib. In another aspect, the PARP inhibitor is AZD5305.

[0138] In some embodiments, the present disclosure provides a combination suitable for use in the treatment or prevention of a PKMYT1-dependent cancer, wherein the combination comprises a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and an ataxia telangiectasia mutated and Rad3-related kinase (ATR) inhibitor. In one aspect, the ATR inhibitor is selected from the group consisting of camonsertib (Repare), tuvusertib (Merck), berzosertib (Merck KGaA), ceralasertib (AstraZeneca), and elimusertib (Bayer). In another aspect, the ATR inhibitor is ceralasertib.

[0139] In some embodiments, the present disclosure provides a combination suitable for use in the treatment or prevention of a PKMYT1-dependent cancer, wherein the combination comprises a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a checkpoint kinase 1 (CHK1) inhibitor. In one aspect, the CHK1 inhibitor is selected from the group consisting of bosutinib, crizotinib, dasatinib, erlotinib, gefitinib, lapatinib, pazopanib, ruxolitinib, sunitinib, and vemurafenib. V. Pharmaceutical Compositions

[0140] The compounds of Formula (1), and pharmaceutically acceptable salts thereof, may be administered as pharmaceutical compositions, comprising one or more pharmaceutically acceptable excipients. Therefore, in some embodiments the present disclosure provides pharmaceutical compositions comprising a compound of Formula (1), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0141] The excipient(s) selected for inclusion in a particular composition will 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 the Handbook of Pharmaceutical Excipients, Sixth Edition, Pharmaceutical Press, edited by Rowe, Ray C; Sheskey, Paul J; Quinn, Marian. Pharmaceutically acceptable excipients may function as, forexample, adjuvants, diluents, carriers, stabilisers, flavourings, colorants, fillers, binders, disintegrants, lubricants, glidants, thickening agents and coating agents. As persons skilled in the art will appreciate, certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the composition and what other excipients are present in the composition.

[0142] The compositions may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous or intramuscular dosing), or as a suppository for rectal dosing. Compositions intended for oral use may contain, for example, one or more coloring, sweetening, flavoring and / or preservative agents.

[0143] The total daily dose will necessarily be varied depending upon the subject treated, the route of administration, any therapies being co-administered, and the severity of the illness being treated, and may include single or multiple doses. Specific dosages can be adjusted, for example, depending upon the condition being treated; the age, body weight, general health condition, sex, and diet of the subject; administration routes; dose intervals; excretion rate; and other drugs being co-administered to the subject. The compound of Formula (1), or a pharmaceutically acceptable salt thereof, typically will be administered to a warm-blooded animal at a unit dose within the range 2.5 to 5000 mg / m2body area of the animal, or approximately 0.05 to 100 mg / kg, and this normally provides a therapeutically effective dose.

[0144] In some embodiments, the present 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.

[0145] In some embodiments, the present disclosure provides pharmaceutical compositions for use in the treatment of cancers, comprising a compound of Formula (1), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient. In one aspect, the cancer is a PKMYT1-dependent cancer. In another aspect, the PKMYT1-dependent cancer is a solid tumor cancer. In another aspect, PKMYT1-dependent cancer is a hematological cancer.VI. Kits

[0146] The present disclosure further provides kits comprising a unit dosage form comprising a compound of Formula (1), or a pharmaceutically acceptable salt thereof, contained within a packaging material and a label or package insert which indicates that the unit dosage form can be used for treating one or more of the previously described conditions.

[0147] In some embodiments, the kit comprises a unit dosage form comprising a compound of Formula (1), or a pharmaceutically acceptable salt thereof, contained within a packaging material and a label or package insert which indicates that the pharmaceutical composition can be used for treating a cancer. In one aspect, the cancer is a PKMYT1-dependent cancer. In another aspect, the PKMYT1-dependent cancer is a hematological malignancy. In another aspect, the PKMYT1-dependent cancer is a solid tumor cancer.

[0148] In some embodiments, kit comprises: (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 chemotherapeutics, TOP1 inhibitors, TOP2 inhibitors, TOP1-ADCs, TOP2-ADCs, PARP inhibitors, ATR inhibitors, and CHK1 inhibitors; (c) a container means for containing said first and second dosage forms; and (d) a label or package insert which indicates that the first unit dosage form and second unit dosage form can be used for treating a PKMYT1-dependent cancer. VII. Methods of Preparation

[0149] The present disclosure further provides processes for the preparation of the compounds of Formula (1) and pharmaceutically acceptable salts thereof.

[0150] Schemes 1 to 11 below illustrate synthetic routes to compounds of Formula (1). One of skill in the art will appreciate that these methods are representative and are not inclusive of all possible methods for preparing the compounds of the present disclosure. The RXsubstituents in each Scheme are as defined for the compounds of the present disclosure unless otherwise stated. It is understood that the processes for preparation described in Schemes 1 to 11 can be performed starting from any enantiomer, or a racemic mixture, of intermediate compounds to give compounds of Formula (1) or any stereoisomer of Formula (1). All starting materials are readily available or prepared as described in the Examples.SCHEME 1

[0151] Scheme 1 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (B) (X, Y are halogens like fluoro, chloro, bromo, or iodo) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (D). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C. A compound of formula (D) may be reacted with malononitrile of formula (E) to give a compound of formula (F). The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0152] A compound of formula (F) may be reacted with concentrated sulfuric acid to give a compound of formula (G). The protecting group in the compound of formula (G) may be removed under appropriate reaction conditions to give a compound of Formula (3).SCHEME 2

[0153] Scheme 2 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (H) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (I). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C.

[0154] A compound of formula (I) may be reacted with malononitrile of formula (E) to give a compound of formula J. The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0155] A compound of formula J may be reacted with concentrated sulfuric acid to give a compound of formula (K). The protecting group in the compound of formula (K) may be removed under appropriate reaction conditions to give a compound of Formula (2).SCHEME 3

[0156] Scheme 3 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (L) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (M). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C.

[0157] A compound of formula (M) may be reacted with malononitrile of formula (E) to give a compound of formula (N). The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0158] A compound of formula (N) may be reacted with concentrated sulfuric acid to give a compound of formula (O). The protecting group in the compound of formula (O) may be removed under appropriate reaction conditions to give a compound of Formula (4).SCHEME 4

[0159] Scheme 4 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (P) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (Q). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C.

[0160] A compound of formula (Q) may be reacted with malononitrile of formula (E) to give a compound of formula (R). The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0161] A compound of formula (R) may be reacted with concentrated sulfuric acid to give a compound of formula (S). The protecting group in the compound of formula (S) may be removed under appropriate reaction conditions to give a compound of Formula (5).SCHEME 5

[0162] Scheme 5 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (T) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (U). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C.

[0163] A compound of formula (U) may be reacted with malononitrile of formula (E) to give a compound of formula (V). The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0164] A compound of formula (V) may be reacted with concentrated sulfuric acid to give a compound of formula (W). The protecting group in the compound of formula (W) may be removed under appropriate reaction conditions to give a compound of formula (A1).

[0165] A compound of formula (A1) may be reacted with boronic acid or ester of formula (B1) to give a compound of Formula (3). The reaction may be catalyzed with a suitable Pd- precatalyst (such as XantPhos Pd G3, XPhos Pd G3, etc.) and ligand (such as XPhos, etc.) in the presence of a base (such as K2CO3, NaOtBu, Cs2CO3, etc.) using a suitable solvent (1,4- dioxane / water mixture, toluene / water mixture, etc.), and at temperatures typically ranging from 60°C to 120°C.

[0166] A compound of formula (A1) may also be reacted with alkyl or aryl amine of formula (C1) to give a compound of Formula (3). The reaction may be catalyzed with a suitable Pd- precatalyst (such as Pd(dba)2, Pd-PEPPSI-iHeptCl, BrettPhos Pd G3, etc.) and ligand (such asXphos, Cphos, BrettPhos, etc.) in the presence of a base (such as LiHMDS, etc.) using a suitable solvent (such as 2-Me THF, 1,4-dioxane, t-BuOH, etc.) and at temperatures typically ranging from 80°C to 120°C.

[0167] A compound of formula (A1) may also be reacted with aryl alcohol of formula (D1) to give a compound of Formula (3). The reaction may be catalyzed with a suitable Pd-precatalyst (such as RockPhos Pd G3, Cphos Pd G3, etc.) and ligand (such as RockPhos, etc.) in the presence of a base (such as NaOtBu, Cs2CO3, K3PO4, etc.) using a suitable solvent (such as t- BuOH, 1,4-dioxane, etc.), and at temperatures typically ranging from 60°C to 120°C. SCHEME 6

[0168] Scheme 6 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (E1) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (F1). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C.

[0169] A compound of formula (F1) may be reacted with malononitrile of formula (E) to give a compound of formula (G1). The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0170] A compound of formula (G1) may be reacted with concentrated sulfuric acid to give a compound of formula (H1). The protecting group in the compound of formula (H1) may be removed under appropriate reaction conditions to give a compound of formula (I1).

[0171] A compound of formula (I1) may be reacted with boronic acid or ester of formula(B1) to give a compound of Formula (2). The reaction may be catalyzed with a suitable Pd- precatalyst (such as XantPhos Pd G3, XPhos Pd G3, etc.) and ligand (such as XPhos, etc.) in the presence of a base (such as K2CO3, NaOtBu, Cs2CO3, etc.) using a suitable solvent (such as 1,4- dioxane / water mixture, toluene / water mixture, etc.), and at temperatures typically ranging from 60°C to 120°C.

[0172] A compound of formula (I1) may also be reacted with alkyl or aryl amine of formula (C1) to give a compound of Formula (2). The reaction may be catalyzed with a suitable Pd- precatalyst (such as Pd(dba)2, Pd-PEPPSI-iHeptCl, BrettPhos Pd G3, etc.) and ligand (such as Xphos, Cphos, BrettPhos, etc.) in the presence of a base (such as LiHMDS, etc.) using a suitable solvent (such as 2-Me THF, 1,4-dioxane, t-BuOH, etc.) and at temperatures typically ranging from 80°C to 120°C.

[0173] A compound of formula (I1) may also be reacted with aryl alcohol of formula (D1) to give a compound of Formula (2). The reaction may be catalyzed with a suitable Pd-precatalyst (such as RockPhos Pd G3, CPhos Pd G3, etc.) and ligand (such as RockPhos, etc.) in the presence of a base (such as NaOtBu, Cs2CO3, K3PO4, etc.) using a suitable solvent (such as t- BuOH, 1,4-dioxane, etc.), and at temperatures typically ranging from 60°C to 120°C. SCHEME 7

[0174] Scheme 7 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (J1) may be reacted with boronic acid or ester of formula (B1) to give a compound of formula (K1). The reaction may be catalyzed with a suitable Pd-precatalyst (such as XantPhos Pd G3, XPhos Pd G3, etc.) and ligand (such as XPhos, etc.) in the presence of a base (such as K2CO3, NaOtBu, Cs2CO3, etc.) using a suitable solvent (1,4-dioxane / water mixture,toluene / water mixture, etc.), and at temperatures typically ranging from 60°C to 120°C.

[0175] A compound of formula (K1) may be reacted with concentrated sulfuric acid to give a compound of formula (L1). The protecting group in the compound of formula (L1) may be removed under appropriate reaction conditions to give a compound of Formula (3). SCHEME 8

[0176] Scheme 8 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (M1) may be reacted with a compound of formula (N1) (X1 is typically a leaving group like iodo, bromo, chloro, or triflate) to give a compound of formula (O1). The reaction may be performed in the presence of a base (typically an inorganic base such as K2CO3, etc.) or catalyzed with a suitable Pd-catalyst (such as BrettPhos Pd G3, etc.) in the presence of a base (such as Cs2CO3, etc.), using a solvent (such as t-BuOH, DMSO, acetone, etc.).

[0177] A compound of formula (O1) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (P1). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C.

[0178] A compound of formula (P1) may be reacted with malononitrile of formula (E) to give a compound of formula (Q1). The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0179] A compound of formula (Q1) may be reacted with concentrated sulfuric acid to give a compound of formula (R1). The protecting group in the compound of formula (R1) may be removed under appropriate reaction conditions to give a compound of Formula (6).SCHEME 9

[0180] Scheme 9 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (S1) may be reacted with a compound of formula (N1) (X1 is typically a leaving group like iodo, bromo, chloro, or triflate) to give a compound of formula (T1). The reaction may be performed in the presence of a base (typically an inorganic base such as K2CO3, etc.) or catalyzed with a suitable Pd-catalyst (such as BrettPhos Pd G3, etc.) in the presence of a base (such as Cs2CO3, etc.), using a solvent (such as t-BuOH, DMSO, acetone, etc.).

[0181] A compound of formula (T1) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (U1). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated. HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C.

[0182] A compound of formula (U1) may be reacted with malononitrile of formula (E) to give a compound of formula (V1). The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0183] A compound of formula (V1) may be reacted with concentrated sulfuric acid to give a compound of formula (W1). The protecting group in the compound of formula (W1) may be removed under appropriate reaction conditions to give a compound of Formula (7).SCHEME 10

[0184] Scheme 10 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (A2) may be reacted with a compound of formula (N1) (X1 is typically a leaving group like iodo, bromo, chloro, or triflate) to give a compound of formula (B2). The reaction may be performed in the presence of a base (typically an inorganic base such as K2CO3, etc.) or catalyzed with a suitable Pd-catalyst (such as BrettPhos Pd G3, etc.) in the presence of a base (such as Cs2CO3, etc.), using a solvent (such as t-BuOH, DMSO, acetone, etc.).

[0185] A compound of formula (B2) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (C2). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C.

[0186] A compound of formula (C2) may be reacted with malononitrile of formula (E) to give a compound of formula (D2). The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0187] A compound of formula (D2) may be reacted with concentrated sulfuric acid to give a compound of formula (E2). The protecting group in the compound of formula (E2) may be removed under appropriate reaction conditions to give a compound of Formula (8).SCHEME 11

[0188] Scheme 11 illustrates synthetic routes to certain compounds of Formula (1). A compound of formula (F2) may be reacted with a compound of formula (N1) (X1 is typically a leaving group like iodo, bromo, chloro, or triflate) to give a compound of formula (G2). The reaction may be performed in the presence of a base (typically an inorganic base such as K2CO3, etc.) or catalyzed with a suitable Pd-catalyst (such as BrettPhos Pd G3, etc.) in the presence of a base (such as Cs2CO3, etc.), using a solvent (such as t-BuOH, DMSO, acetone, etc.).

[0189] A compound of formula (G2) may be reacted with an aryl amine of formula (C) (PG are protecting groups such as OMe, OBn, etc.) to give a compound of formula (H2). The reaction may be performed in the presence of a base (typically an organic base such as DIPEA, etc.) or acid (typically a catalytic amount of concentrated HCl) using a solvent (such as t-BuOH, t-amyl alcohol, DMSO, etc.), and at temperatures typically ranging from 80°C to 120°C.

[0190] A compound of formula (H2) may be reacted with malononitrile of formula (E) to give a compound of formula (I2). The reaction may be catalyzed with a suitable Pd-catalyst (such as PdCl2(dppf), etc.) in the presence of a base (such as NaOtBu, etc.) using a suitable solvent (such as DME, etc.), and at temperatures typically ranging from 80°C to 100°C.

[0191] A compound of formula (I2) may be reacted with concentrated sulfuric acid to give a compound of formula (J2). The protecting group in the compound of formula (J2) may be removed under appropriate reaction conditions to give a compound of Formula (9).

[0192] It should be understood that: (i) the organic reactions described in this disclosure are performed according to laboratory practices known to a person skilled in the art; (ii) some of the reactions described in this disclosure may optionally be performed in different orders than laid out herein; (iii) chiral isomers of compounds in this disclosure can be resolved at any stage in thesynthetic process using chiral resolving agents described in the literature and known to a person skilled in the art, or using chiral chromatography methods described in the literature and known to a person skilled in the art, or as described further in the Examples; (iv) additional and / or other protective groups may optionally be needed in some of the steps described above; and (v) a deprotection step therefore optionally may be performed, using methods described in the literature and known to a person skilled in the art. The protection and deprotection of functional groups are described in “Protective Groups in Organic Synthesis” 3rd Ed, T.W. Greene and P.G.M. Wutz, Wiley-Interscience (1999), which publication is incorporated herein by reference. VIII. Examples

[0193] The following descriptions of experiments, procedures, examples, and intermediates are intended to exemplify embodiments of the disclosure and are in no way intended to be limiting. Other compounds of this disclosure may be prepared using the methods illustrated in these examples, either alone or in combination with techniques generally known in the art. A. General Conditions

[0194] Unless otherwise stated:

[0195] 1H NMR spectra were obtained using a Bruker 300 MHz, 400 MHz or 500 MHz spectrometer at 27 °C unless otherwise noted; chemical shifts are expressed in parts per million (ppm, δ units) and are referenced to the residual mono-1H isotopologue of the solvent (CHCl3: 7.24 ppm; CHDCl2: 5.32 ppm; CD3S(=O)CD2H: 2.49 ppm). Coupling constants are given in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br s (broad singlet). LC-MS was carried out using a Waters UPLC fitted with a Waters SQD mass spectrometer or Shimadzu LC- 20AD LC-20XR LC-30AD with a Shimadzu 2020 mass spectrometer. Reported molecular ions correspond to [M+H]+unless otherwise noted; for molecules with multiple isotopic patterns (Br, Cl, etc.) the reported value is the one obtained for the lowest isotope mass unless otherwise specified.

[0196] Flash chromatography was performed using straight phase flash chromatography on a SP1TMPurification system from BiotageTM, CombiFlash®Rf from ISCO or on Gilson system from Thermo Fisher using normal phase silica FLASH+TM(40M, 25M or 12 M) or SNAPTMKP- Sil Cartridges (340, 100, 50 or 10), Flash Column silica-CS columns from Agela, with C18-flash columns or standard flash chromatography. In general, all solvents used were commerciallyavailable and of analytical grade. Anhydrous solvents were routinely used for reactions. Phase Separators used in the examples are ISOLUTE® Phase Separator columns. The starting materials were obtained from commercial sources or made via literature routes.

[0197] The following abbreviations are used: 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 methanesulfonate; CPhos = 2-Dicyclohexylphosphino-2′,6′-bis(N,N-dimethylamino)biphenyl; CPhos Pd G3 = [(2-Dicyclohexylphosphino-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 = diisopropyl ethylamine; DME = dimethoxyethane; DMSO = dimethylsulfoxide; DMSO-d6= deuterated dimethylsulfoxide; dppf = 1,1'-bis(diphenylphosphino)ferrocene; EA = ethylacetate; HBSS = Hanks' Balanced Salt Solution; HCl = hydrochloric acid; HP = high pressure; H2SO4 = sulfuric acid; IPA = isopropylalcohol; 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 spectrometery; NaHCO3= sodium bicarbonate; NaOtBu = sodium tert-butoxide, NH4HCO3= ammonium bicarbonate; NMR = nuclear magnetic resonance; NaOH = sodium hydroxide; OBn = benzyloxy OMe = methoxy; PBS = Phosphate buffered saline; Pd = palladium; PdCl2(dppf) = 1,1'-bis(di-tert-butylphosphino)ferrocene palladium dichloride; Pd2dba3 = Tris(dibenzylideneacetone)dipalladium (0); Pd-PEPPSI-iHeptCl = dichloro[1,3-bis(2,6-di-4-heptylphenyl)imidazol-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 = retention time; RU = Response units; tBuOH = tert-butanol; TCEP = Tris(2-carboxyethyl)phosphine; Xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; and XPhos = 2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl.

[0198] Examples and Intermediate compounds are named using ChemDraw Professional version 21.0.0 from PerkinElmer. ChemDraw Professional version 21.0.0 generates the names of chemical structures using the Cahn-Ingold-Prelog (CIP) rules for stereochemistry and follows IUPAC rules as closely as possible when generating chemical names. Stereoisomers are differentiated from each other by stereodescriptors cited in names and assigned in accordancewith the CIP rules.

[0199] ChemDraw is optionally using labels in the graphical representation of stereocenters such as ‘&’ and ‘or’ to describe the configuration of the stereochemical centers present in the structure. In general, chemical structures of Examples and Intermediates containing the label ‘&’ at a stereocenter, means the configuration of such Example or Intermediate at that stereocenter is a mixture of both (R) and (S); and a label ‘or’ means the configuration of such Example or Intermediate at the stereocenter is either (R) or (S). Absolute, unspecified, ‘&’, and ‘or’ stereocenters can all be present in a single structure.

[0200] In general for structures of Examples and Intermediates where all of the stereocenters are designated as ‘&’, the structure is named with a “rac-” prefix. For structures of Examples and Intermediates where all of the stereocenters are designated as ‘or’, the structure is named with a “rel-” prefix.

[0201] In general Examples and Intermediate compounds are named using the descriptors (RS) and (SR) to denote general ‘&’ centers for chemical structures with multiple chiral centers where only some are designated as ‘&’. The descriptors (R*) and (S*) are used to denote the general ‘or’ centers for chemical structures with multiple chiral centers where only some are designated as ‘or’. B. Compounds Example B-1: (S)-6-Amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]- pyrimidine-5-carboxamide (Compound 1) Step a) 5-Bromo-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidin-4-amine (Intermediate 3)

[0202] 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 at100 °C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 20% MeOH in DCM, further purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water (0.1% NH4HCO3). Pure fractions were evaporated to dryness to afford 5-bromo-N-(3-methoxy-2,6- dimethylphenyl)-2-methylpyrimidin-4-amine (Intermediate 3, 1.800 g, 77 %) as a yellow solid.1H NMR (DMSO-d6, 300 MHz) δ 1.94 (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. Step b) 6-Amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3-d]- pyrimi-dine-5-carbonitrile (Intermediate 4)

[0203] Sodium t-butoxide (0.597 g, 6.21 mmol) was added slowly 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-methylpyrimidin-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. 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 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford 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.1H NMR (DMSO-d6, 300 MHz) δ 1.71 (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. Step c) (S)-6-Amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3- d]pyrimidine-5-carboxamide (Compound 1)

[0204] 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 added slowly to the stirred solution below 40°C. The mixture was stirred at room temperature for 90 minutes. DL-methionine (1107 mg, 7.42 mmol) was added slowly to the stirred mixture below 40°C. The mixture was stirred for 15 minutes below 40°C. The resulting mixture was then stirred at 40°C for 16 hours. The mixture was cooled to room temperature and added slowly 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 get a precipitate. The precipitation was collected by filtration. The crude product was purified by preparative HPLC Column: XBridge Prep OBD C18 Column, 30*150 mm, 5μm; Mobile Phase A: Water (10 mmol / L NH4HCO3+ 0.05% NH3H2O), Mobile Phase B: ACN; Flow rate: 60 mL / min mL / min; Gradient: 5% B to 30% B in 8 min. Fractions containing the desired compound were evaporated to dryness to afford 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 on a column: LuxCellulose-44.6*50mm, 3μm; Mobile Phase A: CO2, Mobile Phase B: MeOH (0.1% DEA); Flow rate: 4 mL / min; Gradient: isocratic % B. The fractions containing the desired compound were evaporated to dryness to afford (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 (400 MHz, DMSO-d6, 24°C) δ 1.67 (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.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) Intermediate 6: 5-bromo-2-methyl-6-(trifluoromethyl)pyrimidin-4(3H)-one

[0205] Azobisisobutyronitrile (AIBN) (0.184 g, 1.12 mmol) was added to 2-methyl-6- (trifluoromethyl)pyrimidin-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 to 20% EtOAc in DCM. Pure fractions were evaporated to dryness to afford 5-bromo-2-methyl-6-(trifluoromethyl)pyrimidin-4(3H)-one (Intermediate 6, 2.40 g, 83%) as a white solid.1H NMR (300 MHz, DMSO-d6, 25 °C) δ 2.3 (3H, s), 13.46 (1H, s); m / z (ES+) [M+H]+= 257. Intermediate 7: 5-bromo-4-chloro-2-methyl-6-(trifluoromethyl)pyrimidine

[0206] Phosphorus oxychloride (3.58 g, 23.4 mmol) was added to 5-bromo-2-methyl-6- (trifluoromethyl)-pyrimidin-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 to 30% EtOAc in DCM. Pure fractions were evaporated to dryness to afford 5-bromo-4-chloro-2- methyl-6-(trifluoromethyl)pyrimidine (Intermediate 7, 0.75 g, 35%) as a colourless liquid.1H NMR (300 MHz, DMSO-d6, 23°C) δ 2.65 (3H, s).Intermediate 8: 5-bromo-N-(3-methoxy-2,6-dimethylphenyl)-2-methyl-6- (trifluoromethyl)pyrimidin-4-amine

[0207] 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 to 30% EtOAc (containing 50% DCM) in petroleum ether. Pure fractions were evaporated to dryness to afford 5-bromo-N-(3- methoxy-2,6-dimethylphenyl)-2-methyl-6-(trifluoromethyl)pyrimidin-4-amine (Intermediate 8, 780 mg, 75 %) as a colourless liquid.1H NMR (300 MHz,DMSO-d6, 24 °C) δ 1.99 (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. Intermediate 9: 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-4- (trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile

[0208] Sodium t-butoxide (517 mg, 5.38 mmol) was added slowly to a solution of malononitrile (284 mg, 4.31 mmol) in DME (10 mL). The mixture was stirred at r.t. for 30 minutes.5-Bromo-N-(3-methoxy-2,6-dimethylphenyl)-2-methyl-6-(trifluoromethyl)pyrimidin-4- amine (Intermediate 8, 840 mg, 2.15 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (II) (158 mg, 0.220 mmol) was 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 the reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 80% EtOAc (containing 50% DCM) in petroleum ether. Pure fractions were evaporated to dryness to afford 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.1H NMR (400 MHz, DMSO-d6, 24 °C) δ 1.72 (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. Intermediate 10: 6-amino-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-4- (trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide

[0209] 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 r.t. for 1 day. The reaction mixture was poured into water (50 mL), basified with ammonia and then extracted with EtOAc (3 x 50 mL). The organic layer was dried over MgSO4, filtered and evaporated to afford 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.1H NMR (300 MHz, DMSO-d6, 23°C) δ 1.70 (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. (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)

[0210] BBr3in DCM (3.05 mL, 3.05 mmol) 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 r.t. for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 60% MeCN in water modified with 0.1% NH4HCO3. Pure fractions were evaporated to dryness to afford (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.

[0211] The racemic mixture was purified by preparative chiral-HPLC on a column: CHIRALPAK IC, 2*25 cm, 5 μm; Mobile Phase A: HEX (0.5% 2M NH3-MeOH), Mobile Phase B: ETOH; Flow rate: 20 mL / min; Gradient: isocratic 7; Wave Length: 220 / 254 nm; RT1(min): 18.873; RT2(min): 22.707; Sample Solvent: EtOH; Injection Volume: 0.4 mL; Number Of Runs: 22. The fractions containing the desired compound were evaporated to dryness to afford 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.1H NMR (400 MHz, DMSO-d6, 23 °C) δ 1.68 (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.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) Intermediate 13: 6-(difluoromethyl)-2-methylpyrimidin-4(3H)-one

[0212] A mixture of sodium ethoxide (3.07 g, 45.2 mmol) in ethanol (50.0 mL) was added to a stirred mixture of ethyl 4,4-difluoro-3-oxobutanoate (Intermediate 11, 5.00 g, 30.1 mmol) and acetimidamide hydrochloride (Intermediate 12, 2.85 g, 30.1 mmol) in toluene (100 mL) at 25 °C。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 to 40% MeOH in water (0.1% NH4HCO3). Pure fractions were evaporated to dryness to afford 6-(difluoromethyl)-2-methylpyrimidin-4(3H)-one (Intermediate 13, 1.8 g, 37%) as a white solid.1H NMR (300 MHz, DMSO-d6, 26 °C) δ 2.33 (3H, s), 6.42 (1H, s), 6.67 (1H, t), 12.77 (1H, s); m / z (ES+) [M+H]+= 161. Intermediate 14: 5-bromo-6-(difluoromethyl)-2-methylpyrimidin-4(3H)-one

[0213] AIBN (0.185 g, 1.12 mmol) was added to 6-(difluoromethyl)-2-methylpyrimidin- 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 to 100% EtOAc (containing 50% DCM) in petroleum ether. Pure fractions were evaporated to dryness to afford 5-bromo-6-(difluoromethyl)-2-methylpyrimidin-4(3H)-one (Intermediate 14, 1.4 g, 52%) as a white solid.1H NMR (500 MHz, DMSO-d6, 25 °C) δ 2.34 (3H, s), 6.99 (1H, t), 13.25 (1H, s); m / z (ES+) [M+H]+= 239.Intermediate 15: 5-bromo-4-chloro-6-(difluoromethyl)-2-methylpyrimidine

[0214] Phosphorus oxychloride (8.34 g, 54.4 mmol) was added to 5-bromo-6- (difluoromethyl)-2-methylpyrimidin-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 to 50% EtOAc in DCM. Pure fractions were evaporated to dryness to afford 5-bromo- 4-chloro-6-(difluoromethyl)-2-methylpyrimidine (Intermediate 15, 1.1 g, 79%) as a colourless liquid.1H NMR (300 MHz, DMSO-d6, 25 °C) δ 2.66 (3H, s), 7.17 (1H, t); m / z (ES+) [M+H]+= 257. Intermediate 16: 5-bromo-6-(difluoromethyl)-N-(3-methoxy-2,6-dimethylphenyl)-2- methylpyrimidin-4-amine

[0215] 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 to 60% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford 5-bromo-6-(difluoromethyl)-N-(3-methoxy-2,6-dimethylphenyl)- 2-methylpyrimidin-4-amine (Intermediate 16, 0.70 g, 48%) as a yellow solid.1H NMR (400 MHz, DMSO-d6, 23°C) δ 1.93 (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. Intermediate 17: 6-amino-4-(difluoromethyl)-7-(3-methoxy-2,6-dimethylphenyl)-2- methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile

[0216] Sodium t-butoxide (452 mg, 4.70 mmol) was added slowly to a solution of malononitrile (248 mg, 3.76 mmol) in DME (10 mL). The mixture was stirred at r.t. for 30 minutes. 5-Bromo-6-(difluoromethyl)-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidin-4- amine (Intermediate 16, 700 mg, 1.88 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]- dichloropalladium(II) (138 mg, 0.190 mmol) was 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 EtOAc (3 x 50 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford yellow solid. The crude product was purified by flash silica chromatography, elution gradient 0 to 60% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford 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 ayellow solid.1H NMR (300 MHz, DMSO-d6, 24 °C) δ 1.76 (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. Intermediate 18: 6-amino-4-(difluoromethyl)-7-(3-methoxy-2,6-dimethylphenyl)-2- methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide

[0217] 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 r.t. for 1 day. The reaction mixture was poured into water (50 mL), basified with ammonia and then extracted with EtOAc (3 x 50 mL). The organic layer was dried over MgSO4, filtered and evaporated to afford 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.1H NMR (300 MHz, DMSO-d6, 24 °C) δ 1.71 (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. (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)

[0218] BBr3 in DCM (3.20 mL, 3.20 mmol) 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 r.t. for 3 hours. The solvent was removed under reduced pressure.The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 60% MeCN in water modified with 0.1% NH4HCO3. Pure fractions were evaporated to dryness to afford (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.

[0219] 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: isocratic 10; Wave Length: 220 / 254 nm; RT1(min):17.882; RT2(min): 24.125; Sample Solvent: EtOH; Injection Volume: 0.4 mL; Number Of Runs: 13) to afford 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.1H NMR (400 MHz, DMSO-d6, 23 °C) δ 1.68 (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. 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) Intermediate 20: 6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidin-4- amine

[0220] Xantphos Pd G3 (4.49 g, 4.73 mmol) was added to 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) in 1,4-dioxane (300 mL) under nitrogen. 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 to 40% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford 6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidin-4-amine (Intermediate 20, 17 g, 66%) as a pale yellow solid.1H NMR (300 MHz, CDCl3, 25 °C) δ 2.11 (3H, s), 2.17 (3H, s), 2.45 (3H, s), 3.84 (3H, s), 3.85 (3H, s), 5.08 (1H, s), 6.72 (1H, s), 6.79 (1H, d), 7.09 (1H, d); m / z (ES+) [M+H]+= 274.Intermediate 21: 5-bromo-6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2- methylpyrimidin-4-amine

[0221] N-Bromosuccinimide (9.77 g, 54.9 mmol) was added to 6-methoxy-N-(3-methoxy- 2,6-dimethylphenyl)-2-methylpyrimidin-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 to 30% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford 5-bromo-6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidin-4-amine (Intermediate 21, 15 g, 78%) as a pale yellow solid.1H NMR (300 MHz, CDCl3, 25 °C) δ 2.10 (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. Intermediate 22: 6-amino-4-methoxy-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H- pyrrolo[2,3-d]pyrimidine-5-carbonitrile

[0222] Sodium t-butoxide (8.19 g, 85.2 mmol) was added slowly to a solution of malononitrile (3.38 g, 51.1 mmol) in DME (100 mL). The mixture was stirred at r.t. for 30 minutes.5-Bromo-6-methoxy-N-(3-methoxy-2,6-dimethylphenyl)-2-methylpyrimidin-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 EtOAc (3 x 150 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford yellow solid. The crude product was purified by flash silica chromatography, elution gradient 0 to 60% MeCN in water (0.1%NH4HCO3). Pure fractions were evaporated to dryness to afford 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.1H NMR (300 MHz, DMSO-d6, 25 °C) δ 1.71 (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. Intermediate 23: 6-amino-4-hydroxy-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H- pyrrolo[2,3-d]pyrimidine-5-carbonitrile

[0223] Lithium chloride (7.54 g, 178 mmol) was added 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 para-toluenesulfonic acid (18.37 g, 106.7 mmol) in DMF (120 mL) at r.t. The resulting mixture was stirred at 100 °C for 3 hours. The reactionmixture was poured into water (750 mL), extracted with EtOAc (5 x 200 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford grey solid. The grey solid washed with water (2 x 75 mL) , filtered and dried to afford clean grey 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 in the next step directly without further purification.1H NMR (300 MHz, DMSO-d6, 25 °C) δ 1.73 (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. Intermediate 24: 6-amino-4-chloro-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H- pyrrolo[2,3-d]pyrimidine-5-carbonitrile

[0224] 6-Amino-4-hydroxy-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H-pyrrolo[2,3- d]pyrimidine-5-carbonitrile (Intermediate 23, 12 g, 37mmol) was added slowly 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), The resulting mixture was basified with saturated NaHCO3, extracted with EtOAc (200 mL). The organic layer was washed with water (100 mL X 2), dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 50% MeCN in water (0.1%NH4HCO3). Pure fractions were evaporated to dryness to afford 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.1H NMR (300 MHz, DMSO-d6, 25 °C) δ 1.72 (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. Intermediate 25: 6-amino-4-chloro-7-(3-methoxy-2,6-dimethylphenyl)-2-methyl-7H- pyrrolo[2,3-d]pyrimidine-5-carboxamide

[0225] 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 in one portion into 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 formed mixture was basified with ammonia, extracted with EtOAc (3 x 100 mL). The organic layer was dried over MgSO4, filtered and evaporated to afford 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 in the next step directly without further purification.1H NMR (300 MHz, DMSO-d6, 24 °C) 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 protons exchanged; m / z (ES+) [M+H]+= 360. Intermediate 26: 6-amino-4-chloro-7-(3-hydroxy-2,6-dimethylphenyl)-2-methyl-7H- pyrrolo[2,3-d]pyrimidine-5-carboxamide

[0226] BBr3in DCM (28.9 mL, 28.9 mmol) 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 formed mixture was basified with NaHCO3, extracted with EtOAc (5 x 100 mL), the organic layer was dried over MgSO4, filtered and evaporated. The crude product was purified by flash C18-flash chromatography, elution gradient 5 to 60% MeCN in water (0.1% NH4HCO3). Pure fractions were evaporated to dryness to afford 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.1H NMR (300 MHz, DMSO-d6, 24 °C) δ 1.68 (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. (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)

[0227] 1,1'-Bis(diphenylphosphino)ferrocenedichloropalladium (II) dichloromethane adduct (0.354 g, 0.430 mmol) was added to 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) in 1,4-dioxane (20 mL) at r.t. under nitrogen. The resulting mixture was stirred at 100 °C for 16 hours. The reaction mixture was poured into water (100 mL), extracted with EtOAc (3 x 50 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford black solid. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 50% MeCN in water (0.1%NH4HCO3). Pure fractions were evaporated to dryness to afford (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.

[0228] The racemic mixture was purified by preparative chiral-HPLC on 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: isocratic 30; Wave Length: 220 / 254 nm; RT1(min): 12.032; RT2(min): 17.364; Sample Solvent: ETOH; Injection Volume: 1.5 mL; Number Of Runs: 3 to afford 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%) as a white solid and rotational isomer 2.1H NMR (400 MHz, DMSO-d6, 22 °C) δ 1.00 (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. C. Biological Data Example C-1: Elevated Basal Replication Stress Causes PKMYT1 Inhibitor Sensitivity A. Methods

[0229] In this study, the PKMYT1 inhibitor concentrations that inhibited cell growth, in vitro, by 50% ( GI50s) were correlated to basal replication stress levels in several cell line models. To establish the GI50values, cells were seeded into plates and exposed to a ½ log 9-point dose response of the PKMYT1 inhibitor. After seven days of PKMYT1 inhibitor exposure, cells were treated with Cell-Titer Glo (Promega), allowing for the number of viable cells in culture to be determined by quantification of adenosine triphosphate (ATP), an indicator of metabolically active cells. Sigmoidal standard curves (data not shown) were established and mean GI50values were interpolated from three (n=3 ±SD) biological replicates.

[0230] GI50values were then correlated to basal replication stress levels for the 16 cell line models identified in Figs.2, 3, and 4. Basal replication stress levels for the uterine cancer and breast cancer cell line models used in the study were determined by DNA combing analysis described in 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. The OVCAR3 and TOV112D FBXW7 knock out (KO) ovarian cancer cell lines express elevated levels of the CCNE1 gene product, cyclin E1 (a biomarker linked to induction of basal replication stress) compared to the SKOV3 and TOV112D ovarian cancer cell lines. See, e.g., Y. P. Kok 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 lineexpresses elevated levels of phosphorylated RPA2 (a biomarker linked to elevated 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). B. Results

[0231] Figure 2 reports the GI50values measured in the 16 cell line models (some having elevated basal replication stress) treated with a PKMYT1 inhibitor tool compound. Figure 3 reports the corresponding median replication fork velocities measured by DNA combing analysis for the uterine cancer and breast cancer cell line models treated with the tool compound. The five uterine cancer and breast cancer cell lines of Figure 2 having the lowest GI50values also had the lowest median replication fork velocities of Figure 3. Figures 2 and 3 collectively show that the 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) and TOV112D FBXW7 KO (cyclin E1 high) ovarian cancer cell lines, and panc0203 (pRPA2 high) pancreatic cancer cell line) were the most sensitive cell lines to PKMYT1 inhibition. In contrast, the cell line models with low basal levels of replication stress were more resistant to PKMYT1 inhibition.

[0232] Median replication fork velocities also were measured by DNA combing analysis for the same uterine cancer and breast cancer cell line models treated instead with the PKMYT1 inhibitor of Example B-1, Isomer 1 (i.e., the eutomer of Compound 1). Figure 4 reports the median replication fork velocities determined for the eutomer of Compound 1. The results were analogous to those obtained with the tool compound and confirm that the uterine cancer and breast cancer cell line models with low basal levels of replication stress were more resistant to PKMYT1 inhibition than the cell line models having elevated levels of replication stress.

[0233] Overall, the data show that cancers with elevated levels of basal replication stress have greater sensitivity to PKMYT1 inhibition. The data also provide evidence confirming that replication stress correlates with cyclin E amplification and with other genetic alterations previously linked to replication stress and the related response to WEE1 inhibition.Example C-2: Target Engagement NanoBRET (Bioluminescence Resonance Energy Transfer) Assay A. NanoBRET Assay (PKMYT1, WEE1, and EPHB3)

[0234] A whole cell-based target engagement assay was used to investigate target engagement with three targets of interest using the NanoBRET technology. Specifically, compounds were tested to assess inhibition of PKMYT1, WEE1, and EPHB3 in engineered HEK293T cell lines.

[0235] HEK293T cell lines overexpressing PKMYT1, WEE1, and EPHB3 were thawed out from cryopreserved vials at 37°C. The thawed cells were resuspended in 10mL of RPM1-1640 supplemented with 10% Fetal calf serum and 1% GlutaMax. Cell lines were seeded using a Multidrop Combi with a small cassette in 384-well low volume plate (Greiner #784080) at 8000 cells / well in 10µL (for PKMYT1 and EPHB3) and 4000 cells / well in 10µL (for WEE1). The plates were incubated at 37°C, 5% CO2for 23 hours.

[0236] After the incubation, media from plates was evacuated using the BlueCat Bluewasher with the GentleSpin step. The tracer probe for each target was dissolved in Opti-MEM at RT and a 10uL volume was dispensed into each well in the plates using the Multidrop Combi with a small cassette. The tracer probes from Promega were dissolved at the concentration of 1µM (K- 10 for PKMYT1 #N2640), 0.33µM (K-4 for EPHB3 #N2520) and 0.13µM (K-10 for WEE1 #N2640). At this stage the plates were protected from light by using dark lids.

[0237] Plates were dosed with compounds immediately after probe addition. Compounds dosed (12-point concentration response starting from a top concentration of 10µM to 0.000000248µM) were added to the wells using an Echo 650 Acoustic Dispenser (Beckman) and plates were placed in incubator maintained at 37°C and 5% CO2and incubated for two hours.

[0238] After the incubation, plates were equilibrated at room temperature for 10 minutes. 5µL of the 3x NanoBRET assay solution was dispensed in each well of the plate using the Multidrop Combi with a small cassette. The plates were incubated at room temperature for 10 minutes (protected from light) and centrifuged at 300G from 1 minute before reading on the PherSTAR FSX (Optic Module: 460 ± 80nm / 610nm-LP; Gain settings: 3600 (A), 2500 (B); Integration time: 0.2 seconds). The acquired raw data for the acceptor and donor channels was loaded into Genedata analyser (Genedata AG) to determine the BRET ratio for the plates.

[0239] IC50values for the compounds tested are reported in Table 1-A.TABLE 1-A1 IC50is reported after a single measurement (n=1) or as an average for multiple measurements (n>1). B. NanoBRET Assay (Kinase Panel)

[0240] Compound 137, Isomer 2 was further profiled (dose response) against selected kinases in a NanoBRET whole-cell target engagement assay conducted at CELLinib GmbH. IC50values for Compound 137, Isomer 2 are reported in Table 1-B.TABLE 1-BExample C-3: PKMYT1 Surface Plasmon Resonance (SPR) Direct Binding Assay A. PKMYT1 Protein Expression and Purification

[0241] Avi-tagged PKMYT1(6His-SSGVDLGT-TEV-AVI-S-PKMYT1(75-362)) was expressed in Escherichia coli BL21(DE3) Tuner cells in Terrific Broth media. Expression was induced at A600 = 1.0 with 50 µM IPTG and expression occurred for 20 hours at 18 °C. Cells were collected and stored at −80 °C. Cell pellets were resuspended in 750 ml per 97g pellet 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). The solution was homogenized and the clarified lysate loaded into a column for nickel affinity purification preequilibrated in buffer A (50 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 mM imidazole)). Protein was eluted in buffer A supplemented with 300 mM imidazole. Fractions containing PKMYT1 were pooled andbuffer exchanged by dialysis into biotinylation buffer (25 mM Hepes pH=7.5, 300 mM potassium glutamate, 0.5 mM TCEP). Biotinylation was achieved using the BirA kit (Avidity) as per manufacturer’s instructions and biotinylation confirmed by mass spectrometry. Size exclusion chromatography was used to further purify the PKMYT1 into Storage buffer (25 mM Hepes pH 7.5, 500 mM NaCl, 0.5 mM TCEP), and fractions containing pure PKMYT1 were pooled and concentrated to 10.2 mg / ml and snap frozen on liquid nitrogen. B. SPR Direct Binding Assay

[0242] All SPR steps were performed on a Biacore 8K Instrument (Cytiva) at a temperature of 25 °C. In the “immobilisation buffer” (50 mM Hepes pH 7.5, 200 mM NaCl, 5 mM MgCl2, 0.05% Tween-20, 1 mM TCEP) a Series S Sensor Chip SA (Cytiva) was functionalized with ~3000 RU biotinylated Avi-tagged PKMYT1 using a 180s injection at 5µl min-1. The surface was conditioned with 10 start-up injections of the “running buffer” (50 mM Hepes pH 7.5, 200 mM NaCl, 5 mM MgCl2, 0.05% Tween-20, 1 mM TCEP, 1% DMSO). A solvent correction step consisting of a 5-point titration of different DMSO containing running buffer solutions (0-2%) were injected at the beginning and the end of the screening run to correct for DMSO deviations. Three blank cycles of running buffer injections were included preceding each compound injection for blank subtraction. A threefold dilution series for each sample was injected sequentially from low to high concentration over the PKMYT1-coupled surfaces in single-cycle kinetics mode, at a flow rate of 30 μl min−1for 120 s, monitoring dissociation for 2500 s.

[0243] All analysis was performed using the Biacore Insight Evaluation Software after applying standard double-referencing and solvent correction. Single-cycle kinetic 1:1 binding model data analysis was applied to determine the on (kon) and off rates (koff) for test compounds. Theoretical Rmax (the maximal feasible SPR signal generated by an interaction between a ligand–analyte pair; presented in response units (RU)) was determined as the (molecular mass of the compound analyte in solution) / (molecular mass of the immobilized target) × (amount of immobilized target captured), and the per cent surface activity was determined as the (experimental Rmax) / (theoretical Rmax).

[0244] Data for the compounds tested are reported in Table 2. TABLE 21 kon and koff are reported after a single measurement (n=1) or as an average for multiple measurements (n>1). Example C-4: Functional CDK1 pThr14 AlphaLISA Assay

[0245] Compounds were tested to assess their effect on PKMYT1 activity in a cellular context. Specifically, phosphorylation of CDK1 (a PKMYT1 substrate) on Thr14 was used as a specific marker of PKMYT1 activity. Inhibition of PKMYT1 activity by inhibitors decreases levels of pCDk1(Thr14) in the cell.

[0246] Compounds prepared in DMSO were dispensed into low volume white 384-well plates (Greiner 784075) as concentration-response curves.5µL of FUOV-1 cells in Opti-MEM was seeded on top of the assay ready plate at a density of 3000 cells per well using a multidrop combi. This was incubated for 2 hours at 37°C, 5% CO2. The rest of the assay was run using the AlphaLISA Surefire Ultra Human Phospho-CDK1 (Thr14) kit available from Perkin Elmer (ALSU-PCDK1-A10K). All reagents were prepared according to the manufacturer’s recommendations.5µL of 2 x Lysis Buffer containing added protease inhibitors (Roche, #11836170001) was added to the plate and was mixed thoroughly. Acceptor stock and donor stock was then diluted 1 parts mix to 2 parts HBSS.4 µL of acceptor was added using a multidrop Combi and incubated for 1 hour at room temperature. Following this, 4 µL of donor stock was added using a multidrop Combi and the plate was incubated for 18 hours. Alpha counts were measured using a PheraSTAR FSx (BMG Labtech), and the resulting raw data analysed using Genedata analyser (Genedata AG) to derive compound IC50.

[0247] IC50values for the compounds tested are reported in Table 3. TABLE 31 IC50is reported after a single measurement (n=1) or as an average for multiple measurements (n>1). Example C-5: Cellular Proliferation in OVCAR3 and SKOV3 Cell Lines (Hoechst Staining)

[0248] An imaging-based assay was used to investigate changes in cell number. Compounds were tested to assess inhibition of cellular proliferation in a CCNE1-amplified ovarian cancer cell line and a CCNE1-nonamplified SKOV3 ovarian cancer cell line.

[0249] OVCAR3 and SKOV3 cells were cultured in RPMI 1640 medium with 4.5 g / ml Glucose, supplemented with 20% heat in-activated Fetal bovine serum and McCoy’s modified 5A medium supplemented with 10% heat in-activated Fetal bovine serum along with 1% GlutaMax respectively. Both the cell lines were seeded in complete growth medium using a multidrop into 384-well plates (Greiner, 781090) at the density 4000 cells / well in 40 µL / well. The plates (test compound plates + a day 0 control plate) were placed in incubator maintained at 37°C and 5% CO2for about 20 to 22 hours. Day 0 (control plate) was fixed with 4% PFA (v / v), washed 3X in PBS (Biotek 406) and stored at 4°C in 40 ul PBS until further use. A 12-point concentration response starting from a top concentration of 30 µM to 0.00003 uM was added to the test compound plates using an Echo 650 Acoustic Dispenser (Beckman) and then incubated at 37°C and 5% CO2for four days. On Day 4, cells were fixed with 4% PFA (v / v) and stained with Hoechst 33342 (Thermofisher H3570) to a final well dilution of 1 / 10,000 in PBS+1.1% BSA+0.1% TritonX for 1 hour. Cells were washed 3X with PBS and left in 40 ul of PBS. Thestained cells were imaged using Cell Insight cellular imaging and analysis benchtop platform (10X objective). The acquired raw data output for the total cell number was loaded into Genedata analyser (Genedata AG) to determine the rate of proliferation from control plate and test compound dosed plates. The concentration of compound that gives 50% growth inhibition between the day 0 value and the top of the curve of GI50was calculated for all test compounds.

[0250] GI50values for the compounds tested are reported in Table 4. TABLE 41 IC50is reported after a single measurement (n=1) or as an average for multiple measurements (n>1). Example C-6: Kinase Profiling A. General Methods

[0251] Test compounds were profiled against a broad panel of kinases at ThermoFisher Scientific using the SelectScreen Kinase Profiling Services. Each kinase assay used one of the following assay technology protocols: 1) Z-LYTE™ Screening Protocol and Assay Conditions (Revised 23-Jan-2018 version) available at http: / / assets.thermofisher.com / TFS-Assets / BID / Methods-&- Protocols / 20180123_SSBK_Customer_Protocol_and_Assay_Conditions.pdf; 2) Adapta™ Screening Protocol and Assay Conditions (Revised 23-Jan-2018 version) 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 23-Jan-2018 version) available at http: / / assets.thermofisher.com / TFS- Assets / BID / Methods-&- Protocols / 20180123_SSBK_LanthaScreen_Binding_Customer_Protocol_and_Assay_Co nditions.pdf.B. Kinase Selectivity (Single Point Concentration Mode)

[0252] Compound 137, Isomer 2 was profiled against a broad kinase panel at ThermoFisher Scientific. Kinase selectivity was measured in single point (SP) concentration mode: % inhibition at [Compound 137, Isomer 2] = 1 µM at KmATP. Data are reported in Table 5-A. TABLE 5-AC. Kinase Selectivity (Dose Response)

[0253] Compound 137, Isomer 2 was further profiled for dose response against selected kinases at ThermoFisher Scientific. IC50s were determined from a 10-point concentration response (CR) study with top testing concentration of 10 µM. Data are reported in Table 5-B. TABLE 5-BExample C-7: Metabolic Stability Assays A. Assessment Using Human Liver Cytosol (HLC)

[0254] The stability of control (zaleplon and 06-benzylguanine) and 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 removed at 0, 10, 30, 60, 90 and 120 min, quenched 1:5 with ACN containing internal standard. Samples were analyzed for loss of parent compound using LC / MS / MS. Potential hydroxylation metabolites were monitored by adding transitions of Parent+16. Extent of inhibition by raloxifene was derived by comparing the formation of metabolite and or the depletion of parent compound in the presence and absence of inhibitor. B. Assessment Using Human Liver Microsomes (HLM)

[0255] The stability of control (phenacetin, verapamil, diclofenac, imiprimine, benzydamine and metoprolol) and test compounds was evaluated using 1 mg / mL HLM (150 mixed gender donor pool) containing 1 mM NADPH, and 1 µM test compound / control. In short, 222.5 µL of HLM mixture containing 1.12 mg / mL HLM in phosphate buffer pH 7.4 was mixed with 25 µL 10 mM NADPH and pre-warmed at 37°C for 8 minutes prior to addition of 2.5 µL of 100 µM test compound / control prepared in DMSO. Aliquots (20 µL) were removed at 0.5, 5, 10, 15, 20 and 30 minutes and quenched with 100 µL of cold stop solution (100% ACN with 100 nM of alprazolam, caffeine, and tolbutamide as internal standards). Samples were centrifuged for 20 minutes at 4,000 rpm and 4°C and a 40 µL aliquot of supernatant was mixed with 160 µL pure water for LC-MS / MS analysis. Each incubation was conducted in singlicate. Peak areas were determined from extracted ion chromatograms and percent (%) parent remaining was calculated from peak area of test compound / control. C. Assessment Using Human Hepatocytes in Suspension (hHEP)

[00256] The stability of control (phenacetin, verapamil, diclofenac, imipramine, chlorpromazine, and naloxone) and test compounds was evaluated using 1 million viablecells / mL (10 mixed gender donor pool) and 1 µM test compound / control. In short, cryopreserved hepatocytes are thawed in a 37° water bath, centrifuged in thawing medium and prepared in Leibovitz’s 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 a Cellometer Vision and greater than 80% cell viability was required to proceed with the compound incubation. The compound / cell solution (250 µl) was incubated for 2 hours at 37οC and shaken at 900 rpm on an Eppendorf Thermomixer Comfort plate shaker. Samples (20 μl) were taken at 0.5, 5, 15, 30, 45, 60, 80, 100, and 120 minutes and quenched with 100 μl of 100% ice-cold acetonitrile. Samples were shaken at 800 rpm for 2 minutes and centrifuged at 4000 rpm for 20 minutes at 4οC to pellet precipitated protein. The supernatant fraction was diluted 1:5 with deionized water, shaken at 1000 rpm for 2 minutes, and further diluted 1:1 with deionized water. Samples were analyzed by LC-MS / MS. Each incubation was conducted in singlicate. Peak areas were determined from extracted ion chromatograms and percent (%) parent remaining was calculated from peak area of test compound / control. D. Assessment Using Co-Culture Human Hepatocyte Model for Low Clearance

[0257] The stability of the test compound was determined over 72h incubation using human hepatocytes co-cultured with mouse JT3 fibroblast cells (Hμrel). In short, test compound was dissolved in DMSO at 10 mM, then diluted to 2 μM in dosing medium using Echo dispense. All steps were performed at room temperature, while the 2 μM test compound was preheated at 37°C before start of incubation. Prior to assay, maintenance medium was removed from the cells, followed by a washing step with 100 μL of preheated blank dosing medium without serum. Compound incubation was initiated by addition of 50 μL of dosing medium and 50 μL of dosing medium containing 2 μM test compound. The final test concentration was 1 μM. Plates were kept without shaking in the incubator during the experiments, at 37°C in humidified atmosphere containing 95% air and 5% CO2.70 μL aliquots of sample duplicates were taken at 0, 1, 3, 5, 24, 48 and 72 hours and quenched with 70 μL of ice-cold acetonitrile stop solution. Quenched samples were diluted with 140 μL of 0.1% formic acid in water and analyzed on a mass spectrometer. E. Data Analysis for Metabolic Stability Assessments

[0258] The t1 / 2and the CLintof the compounds incubated in the HH, HLM, and HμREL assays as described above were calculated according to the following equations:t1 / 2 (min) = ln(2) / -slope, and Clint(μl / min / 106cells or mg protein) = ln(2)*V / (t1 / 2), where V (μl / x 106cells or mg protein) is the incubation volume (μl) divided by the number of cells (x 106) or microsomal protein content (mg) in the incubation.

[0259] Data for the metabolic stability assessments are reported in Table 6 below. TABLE 6Example C-8: Log D

[0260] The Log D values of test compounds at 10 µM were determined in octanol / PBS pH 7.4 using LC / MS / MS and nicardapine, cyclobenzaprine, and caffeine as control compounds.10 µL of test compound or control was added to a 96-deep well plate followed by addition of 500 µL of saturated octanol and 500 µL of saturated phosphate buffer. The plate was shaken at room temperature for 2h at 2,000 rpm. The samples were centrifuged at room temperature, at 4,000rpm for 30 minutes. 100 µL of sample was removed from the octanol and buffer phase. 5 µL of the octanol sample was mixed with 1:1 H20:ACN containing internal standard and vortexed at 1,000 rpm for 5 minutes (100-fold octanol sample), 50 µL of 100-fold samples were added to 450 µL of 1:1 H2O:ACN containing internal standard and vortexed at 1,000 rpm for 5 min (1,000- fold octanol sample). Serial dilution of 1,000-fold octanol samples to 10,000, 100,000 and 1,000,000-fold was conducted. 50 µL of buffer samples were added to 450 µL of 1:1 H2O:ACN containing internal standard and vortexed at 1,000 rpm for 5 min (10-fold buffer sample), and then further diluted to 100, 1,000 and 10,000 fold with 1:1 H2O:ACN. Samples were analyzed using LC / MS / MS. Log D was calculated as follows:

[0261] Data are reported in Table 7 below. TABLE 7Example C-9: Caco-2 Cell Permeability

[0262] Test compounds were evaluated for gastrointestinal permeability and potential bioavailability in a Caco-2 cell permeability assay. Detailed description of the methodology 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 brief, Caco-2 cells were plated at 6.86 x 105cells / mL and were cultured for 14 to 18 days with culture medium replaced every other day. Test compound (10 μM) was added to the donor well and the appearance in the receiver well measured after 2 hours incubation at 37°C. The rate of compound transport in the apical to basolateral (A-B) direction, was determined from the donor well as apical (A) compartment and the receiver as basolateral (B) compartment. Similarly, the rate of compound transport in the basolateral to apical (B-A) direction, the donor well was the basolateral (B) compartment and the receiver was the apical (A) compartment. Samples were analyzed by liquid chromatography (LC)–mass spectrometry (MS) / MS.

[0263] The permeability coefficient (1 × 10−6cm / s) was calculated using the following equation: Papp=(dCr / dt)×Vr / (A×C0), and the efflux ratio (ER) was calculated using the following equation: Efflux ratio = Papp(B to A) / Papp(A to B), where: dCr / dt is the cumulative concentration of the compound in the receiver chamber as a function of time (in μM / s); Vr is the solution volume in the receiver chamber (0.1 ml on the apical side and 0.3 ml on the basolateral side); A is the surface area for the transport (i.e., 0.11 cm2for the area of the monolayer); and C0is the initial concentration in the donor chamber (in μM).

[0264] Data are reported in Table 8 below.TABLE 8Example C-10: CYP Assays A. CYP Inhibition

[0265] The potential for a test compound to inhibit cytochrome P450 (CYP) isoforms was determined using a 150-donor mixed gender human liver microsomes (HLM) and a cocktail of substrates selective for CYPs 1A2, 2C9, 2C19, 2D6 and 3A at incubation concentrations equivalent to their Km values. Specific inhibitors of these pathways were included as positive controls. Substrates were, Phenacetin (30 µM), diclofenac (10 µM), S-mepheytoin (35 µM), bufuralol (5 µM) and midazolam (3 µM), for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 / 5, respectively. The final concentration of HLMs was 0.2 mg / mL and the final concentrations of test compound were 0, 0.1, 0.3, 1, 3, 10 and 30 µM. The incubation was intiated by addition of 20 µL of 10 mM NADPH in phosphate buffer and quenched 1:1 with ACN containing 3% formic acid and 40 nMol verapamil as internal standard, following five minutes at 37°C. The samples were centrifuged at 4,000 rpm for 30 minutes, placed on ice for 20 min and recentrifuged for an additional 30 min at 4,000 rpm. Supernatant (200 µL) was transferred for analysis of isoform selective metabolite formation. The extent of inhibition was calculated by comparing the remaining activity in the presence of inhibitor to the DMSO onlycontrol using the following equation. The concentration yielding 50% inhibition, IC50, were calculated using non-linear regression analysis in Xlfit.

[0266] The IC50values for Example B-1, Isomer 1 (i.e., the eutomer of Compound 1) and Example B-2, Isomer 2 (i.e., the eutomer of Compound 137) against each of CYPs 1A2, 2C9, 2C19, 2D6, and 3A were greater than 30µM. B. CYP Time Dependent Inhibition

[0267] The potential for test compounds to inhibit Cytochrome P450 (CYP) activity in a time dependent manner was determined using 1 mg / mL of a 150-donor mixed gender human liver microsome pool (HLM), 10 µM test compound, a 30 min preincubation time, and a 10-fold dilution step to measure formation of specific substrates towards, CYPs 1A2, 2C9, 2C19, 2D6 and 3A at 3X their respective Km values. Substrates were, Phenacetin (90 µM), diclofenac (30 µM), S-mepheytoin (105 µM), bufuralol (15 µM), and midazolam (9 µM), for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 / 5, respectively. Specific inhibitors of these pathways were included as positive controls. The primary incubation consisted of 1 mg / mL HLM ± 10 mM NADPH with 10 µM of test compound incubated for 30 min at 37°C. At 30 min a 10 µL aliquot of the pre-incubation solution was transferred to the secondary incubation containing 10 mM NADPH and substrate cocktail. Following 15 min incubation at 37°C the secondary incubation was quenched by addition of 150 µL of cold methanol containing 20 nM verapamil as internal standard. A decrease in the formation of isoform specific metabolites was determined by comparing with and with NADPH control and the % time dependent inhibition (TDI) was calculated with the following equation. A % TDI of > 20 was considered positive and warranting further investigation.The irreversible IC50,representing the concentration where 50% inhibition would be observed, was calculated as follows:

[0268] The % time dependent inhibition (TDI) values calculated for Example B-1, Isomer 1 (i.e., the eutomer of Compound 1) and Example B-2, Isomer 2 (i.e., the eutomer of Compound 137) against each of CYPs 1A2, 2C9, 2C19, 2D6, and 3A were less than 20. C. CYP Induction

[0269] The potential for test compounds to induce drug metabolizing enzymes was evaluated by assessing the Pregnane X Receptor (PXR) activation potential in DPX2 cells. DPX2 cells were purchased from Puracyp and treated according to the vendor protocol. Test compounds, positive control (rifampicin, 1 and 10 µM) and negative control (propranolol, 10 µM) were prepared in DMSO to a final concentration of 0.1%. Test compounds were evaluated at 0.1, 0.3, 1, 3, 10 and 30 µM to enable derivation of EC50and Emaxvalues, corresponding to the concentration resulting in half maximal induction and the maximal induction observed, respectively. Cells were treated with test compound or control for 24h. After 24h dosing media was removed and 1xCellTiter-Fluor™ was added for 30 minutes at 37°C. The fluoresecence was measured using 400 nm excitation and 505 nm emission. The ONE-Glo assay reagent was then added for 5 min and luminescence was determined using a luminometer. The cell viability was determined using the following equation:The normalized luciferase activity was determined by taking the ratios of relative luminescence units (RLU), and relative fluorescence units for three replicates at each concentration and the fold-activation of mRNA levels was determined using the following equation:

[0270] No induction of PXR was observed for Example B-1, Isomer 1 (i.e., the eutomer of Compound 1). Example C-11: Aqueous Solubility

[0271] The thermodynamic solubility of the test compounds was measured in a shake-flask approach starting from 10 mM DMSO solutions. DMSO was evaporated and dried compounds were equilibrated in glass vials in aqueous phosphate buffer (0.1 M, pH 7.4) for 24 hours at 25°C under constant stirring. The portion with the dissolved compound was then separated from the remainder through a double centrifugation with a tip wash in between to remove possible interference of residual dried compound. The solutions were diluted with purified water before quantification using UPLC / MS / MS.

[0272] Data are reported in Table 9 below. TABLE 9Example C-12: Kinase Selectivity (Kinase Affinity Tool Assay)

[0273] A Kinase Affinity Tool (“KAT”) chemical proteomics method based on the Kinobead technology1-2was used for in situ kinase profiling of test compound. KAT is an affinity matrix made up of promiscuous ATP-competitive kinase inhibitors to enrich for a large subset of the native kinome directly from disease / safety relevant lysate. Proteomic RB50 values indicate the selective affinity of the test compound for the enriched kinases.

[0274] Test compound was incubated with a cell lysate mix of four cell lines (K562, MV4- 11, SN-N-BE2, and COLO-205) in a dose-response format for 30 minutes at 4°C. The compound-incubated lysates were then bound to KAT beads for 30 minutes at 4°C. Beads were washed twice with binding buffer followed by two washes with 1X PBS. Proteins bound to beads were reduced, alkylated, and digested overnight with Trypsin / LysC enzyme at 37°C. Digested peptides were eluted with 60% ACN / 0.1% formic acid in water and desalted with in- house made C18 StageTips. Peptides were analyzed by label-free Data Independent Acquisition (DIA) quantitative mass spectrometry method on Orbitrap LUMOS. Proteomics data was analyzed with in-house software Doscheda to generate RB50 values3. RB50 values determined for Compound 137, Isomer 2 are reported in Table 10 and confirm that the compound is highly selective for PKMYT1.

[0275] 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 / .TABLE 10Example C-13: Radioligand Binding and Cell-Based Functional Assays (Secondary Pharmacology)

[0276] Secondary pharmacology assays were performed at Eurofins using standard experimental techniques against 80 targets across a broad pharmacological space. Assay details along with experimental protocols are described at https: / / www.eurofinsdiscoveryservices.com. Radioligand binding assays were used to assess the ability of Compound 137, Isomer 2 to interact with a panel of G-protein coupled receptors (GPCRs), ion channels, and transmembrane transporters. Assays measuring substrate turnover or phosphorylation by isolated proteins were used for kinase and enzyme targets, allowing direct determination of the mode of action of thecompounds. Where binding activity was observed at a GPCR target, mode of action was determined using cell based functional assays with secondary messenger read outs. Assays were run in eight points concentration response mode with half log dilutions and IC50, EC50or Kivalues were determined (Cheng, Y., Prusoff, W.H.1973 Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction Biochem Pharmacol 22(23):3099-108).

[0277] Table 11 reports the values obtained for Compound 137, Isomer 2. TABLE 11Example C-14: THP-1 Assay (Cytotoxicity)

[0278] Test compounds were evaluated for compound-induced cytotoxicity in a human monocytic cell line (THP-1). The in vitro assay used the fluorescent signal generated by thereduction of non-fluorescent resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) to the fluorescent resorufin (Alamar blue assay) as a measure of cytotoxicity. Because cellular reduction of resazurin is dependent on a pool of reductase or diaphorase enzymes derived from the mitochondria and cytosol, resazurin can be used as an oxidation-reduction indicator in cell viability assays for mammalian cells (McMillian et al, 2002; O’Brien et al, 2000).

[0279] THP-1 cells were subcultured in suspension in a T175cm2tissue culture flask with medium (RPMI, supplemented with 1% L-glutamine and 10% quantified FBS). The sub-cultures were maintained at 37°C in a 95% humidified atmosphere with 5% CO2and passaged approximately every 2 to 3 days. Cell density was maintained between 50,000 cells / mL and 1 x106cells / mL.

[0280] Test compounds (40 nL) were added to a 1536-well low volume plate (Greiner #782077) at 10, half-log concentrations with a top concentration of either 100µM or 250µM depending on the stock concentration selected. For both, the DMSO concentration was 1%.

[0281] Using a Multidrop Combi Reagent dispenser (Type 836), with a Multidrop standard tube plastic tip dispensing cassette, THP-1 cells were seeded into the plates at a density of 2,000 cells per well in 4µl THP-1 media on slow speed. The plates containing THP-1 cells with compounds / solvent were incubated for 48 hours under standard cell culture conditions (37°C, 5% CO2).

[0282] Following an incubation period of 48 hours, a stock solution of resazurin (113 mg of resazurin in 1000 mL of PBS) was prepared, warmed to 37°C, and vortexed. Using the Multidrop dispenser, with the Multidrop standard cassette, 1µL of stock resazurin solution was added to all wells at high speed to aid mixing. The plates were incubated for two hours under standard cell culture conditions. The plates were then incubated for a further one hour at room temperature, with shaking (700 rpm).

[0283] The plates were then read on a BMG PheraStar reader using an excitation wavelength of 540 nm and an emission wavelength of 580nm. Controls were 100 µM Lapatinib (minimum / inhibitor control) and 1% DMSO (maximum / neutral control). The acquired raw data were loaded into Genedata analyser (Genedata AG) to determine IC50values. The primary (fluorescent) signal for each concentration-effect curve was normalized to vehicle control (0% inhibition) and inhibitor control (100% inhibition). The obtained inhibition values were fitted using four-parameter logistic equation and the IC50values calculated.

[0284] Compound 137, Isomer 2 had an IC50value of 34 µM indicating that the compound has a reduced risk of cytotoxicity in THP-1 cells relative to earlier compounds tested.

[0285] 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. Example C-15: In Vitro Hematopoietic Stem and Progenitor Cell Assay (Bone Marrow Toxicity)

[0286] Test compounds were evaluated to determine their effect on hematopoietic stem and progenitor cell (“HSPC”) proliferation and differentiation. Cryopreserved human bone marrow derived CD34+hematopoietic stem and progenitor cells (Lonza) were defrosted and left to recover overnight in maintenance media (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)), in a humidified incubator at 37°C with 5% CO2. The next day cells were resuspended in the presence of test compound into media capable of supporting erythroid cell differentiation (Preferred Cell Systems, SEC-BFU1-40H), at a concentration of 10,000 cells / ml. Cells (30 µl) were plated into black-walled, clear bottomed 384 well tissue culture plates (Perkin Elmer) with the addition of 30, 10, 3.3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, or 0 µM of test compound. Cells were cultured for 5 days in a humidified incubator at 37°C, with 5% CO2.

[0287] Viability was determined using CellTiter-Glo 2.0 from Promega (using an optimised volume of 3 µl / well), with luminescence detected using an Envision plate reader (Perkin Elmer). Relative Luminescence signal was normalised to percentage of control wells (0 µM compound) in Genedata Screener software (Genedata) or GraphPad Prism 9. The 50% inhibitory concentration (IC50) was determined using a four parameter logistic regression curve fit.

[0288] Results obtained in primary CD34+bone marrow-derived HSPCs induced to differentiate into the erythroid lineage are reported in Table 12 and Figure 5. Table 12 reports the mean IC50values for Compound 1, Isomer 1 and Compound 137, Isomer 2. TABLE 12Figure 5 illustrates the dose response curves obtained for those compounds. Example C-16: Effect on Cell Cycle in Human Ovarian OVCAR3 Cell Line

[0289] An immunoblotting analysis of CCNE1-amplified ovarian OVCAR3 cells was performed to determine whether short (24 hours) treatment with Compound 137, Isomer 2 results in DNA damage response (“DDR”) signaling in the replicative phase of the cell cycle. Phopho- CHK1 (Ser345) and phospho-RPA2 (Ser4 / Ser8) levels, biomarkers considered to be specifically expressed following DNA damage formation during active replication of cells, were assessed as a measure of effect on cell cycle. A. Methods

[0290] OVCAR3 cells were treated with Compound 137, Isomer 2 for 24 hours and then lysed in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitors (Roche), phosphatase inhibitors (Sigma-Aldrich), and benzonase (Merck). After 30 minutes (min) incubation on ice the lysates were cleared by centrifugation at 15,000 rpm 4°C for 20 minutes and the supernatants were kept 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 amounts of whole 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), phospho-Histone 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 antibody: Anti-rabbit IgG, HRP-linked (CST, #7074) and anti-mouse IgG, HRP-linked (CST, #7076). Adavosertib was used as a treatment control. B. Results

[0291] The differential expression of the phospho-proteins in response to treatment with Compound 137, Isomer 2 is shown in Figure 6. Treatment of the OVCAR3 cells with Compound 137, Isomer 2 resulted in a concentration-dependent reduction of phospho-CDK1 Thr14 levels. Phospho-CDK1 Thr14 is the direct substrate of PKMYT1. In addition, expression of phospho- Histone H3 Ser10 increased without affecting expression of the WEE1 substrate phospho-CDK1Tyr15, indicating an increase of the mitotic population due to G2 / M DNA damage cell cycle checkpoint override independent of WEE1 inhibition. At the 24-hour treatment time point, minimal to no phosphorylation of biomarkers (phospho-CHK1, phospho-RPA2) linked to DNA damage formation in the actively replicating cell population was observed. This is consistent with PKMYT1 regulating the activity of CDK1, which is not associated with S-phase cell cycle progression. Overall, the data indicate that treatment of the OVCAR3 cells with Compound 137, Isomer 2 resulted in PKMYT1 target engagement, an increased mitotic population, and minimal to no DNA damage signaling of replicating cells. Example C-17: Cellular Proliferation in PKMYT1 Knock Out SKOV3 Cells

[0292] The potential effect of off-target activity of Compound 137, Isomer 2 and impact on cell viability was evaluated in PKMYT1 knock out (KO) SKOV3 cells. Specifically, loss of PKMYT1 protein expression, reduction of PKMYT1 substrate phosphorylation, and growth inhibition were evaluated in PKMYT1 KO SKOV3 cells. A. Methods

[0293] CRISPR / Cas9 technology was used to delete PKMYT1 kinase from SKOV3 cells and prepare two monoclonal PKMYT1 KO clones (B2 and H2). Parental SKOV3 cells and PKMYT1 KO SKOV3 cells were either left untreated or were treated with Compound 1, Isomer 1 for 24 hours. Cells were then lysed in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitors (Roche, Basel, Switzerland), phosphatase inhibitors (Sigma-Aldrich), and benzonase (Merck). After 30 minutes of incubation on ice the lysates were cleared through centrifugation at 15,000 rpm 4°C for 20 minutes and the supernatants were kept for sample loading. NuPAGE™ LDS Sample Buffer (ThermoFisher Scientific) and NuPAGE™ Sample Reducing Agent (ThermoFisher Scientific) were added to the samples. Equal amounts of whole cell lysates were separated on 4-12% Bis-Tris NuPAGE gels and analyzed by standard immunoblotting. Primary antibody incubation overnight at 4°C: (CDK1 (CST, 9116) pCDK1 Thr14 (Abcam, ab58509), PKMYT1 (Abcam, ab307146) and vinculin (Sigma, V9131). Secondary antibody incubation for two hours at room temperature was performed with anti-rabbit IgG, HRP-linked (CST, 7074) and anti-mouse IgG, HRP-linked (CST, 7076). Quantification was performed using Bio-Rad ChemiDoc, which was also used for visualisation of western blot bands, with quantification manually performed using ImageJ. CDK1 and phospho-CDK1 Thr14 bands were normalised relative to parental bands, and ratios of CDK1 versus phospho-CDK1 Thr14 were calculated.Graphpad Prism was used to visualise data, plotted as mean with SD (n=2).

[0294] In addition, a CellTiter-Glo (CTG) assay was employed to evaluate the effect of Compound 137, Isomer 2 on growth inhibition of the PKMYT1 KO SKOV3 cells. Parental SKOV3 and OVCAR3 cells were included as controls. Cells were seeded at 500 cells per well in a 96-well plate and exposed to Compound 137, Isomer 2 for seven days. After cell attachment on day 0, Compound 137, Isomer 2 was dispensed from compound stocks dissolved in DMSO, in seven titration dilutions with a top concentration of 30 µM. DMSO served as a vehicle control. Plates were incubated at 37ºC, 5% CO2. Cell growth was stopped by adding CTG as per manufacturer’s instructions (Promega, Madison, WI, USA; G7570) and luminescence read on an Envision plate reader (Perkin Elmer). Data analysis was performed by normalization to vehicle treatment. Data were normalized and plotted to respective vehicle-control using Prism GraphPad software and GI50values were derived. B. Results

[0295] The results of the immunoblotting analysis are illustrated in the left and middle panels of Figure 7 and show that PKMYT1 deletion from SKOV3 cells resulted in loss of PKMYT1 protein expression and a significant reduction of phospho-CDK1 Thr14 expression which could not be further reduced by treatment (24 hours) with Compound 1, Isomer 1.

[0296] The GI50values derived from 7-day end-point CTG measurements for the cell lines treated with Compound 137, Isomer 2 are shown in the right panel of Figure 7 and also reported in Table 13. PKMYT1 deletion from SKOV3 resulted in GI50values for Compound 137, Isomer 2 greater than 20 µM. TABLE 13

[0297] Overall, the data indicate that PKMYT1 knock out (KO) SKOV3 cells are resistant to treatment with Compound 137, Isomer 2.Example C-18: In Vivo Tolerability (Mouse Model)

[0298] A tolerability study was conducted to evaluate long-term dosing of Compound 137, Isomer 2 in mice as a monotherapy, as a combination therapy with gemcitabine, and as a combination therapy with irinotecan hydrochloride. A. Monotherapy

[0299] Female SCID mice (18 g+ at time of first procedure) were orally dosed with 300 mg / kg BID of Compound 137, Isomer 2 for 28 days. Compound 137, Isomer 2 was formulated for dosing using a 5% DMSO, 50% of 20% Captisol, 45% water for injection (pH 3-3.2) vehicle. Bodyweights were collected and recorded daily. Clinical observations, which included monitoring of paw and tail swelling, were assessed at least twice daily.300 mg / kg BID dosing achieved a free Cmax for Compound 137, Isomer 2 of approximately 21 µM.

[0300] Figure 8-A illustrates the effect of 300 mg / kg BID monotherapy dosing of Compound 137, Isomer 2 on body weight loss. No significant loss of body weight, paw or tail swelling, or other noticeable adverse events were observed at this monotherapy dose. B. Combination Therapy (Gemcitabine)

[0301] Female SCID mice (18 g+ at time of first procedure) were orally dosed with 100 mg / kg BID of Compound 137, Isomer 2 for a period of 28 days in combination with four weekly intraperitoneal injections of 20 mg / kg of gemcitabine during that period. Bodyweights were recorded daily. Clinical observations, which included monitoring of paw and tail swelling, were collected and assessed at least twice daily.100 mg / kg BID dosing achieved a free Cmaxfor Compound 137, Isomer 2 of approximately 5.7 µM.

[0302] Figure 8-B illustrates the effect of such combination dosing of Compound 137, Isomer 2 and gemcitabine on body weight loss. No significant loss of body weight, paw or tail swelling, or other noticeable adverse events were observed at this combination dose. C. Combination Therapy (Irinotecan)

[0303] Female athymic nude mice (18 g+ at time of first procedure) were orally dosed with 100 mg / kg BID of Compound 137, Isomer 2 for a period of 28 days in combination with four weekly intraperitoneal injections of 50 mg / kg of irinotecan hydrochloride during that period. Bodyweights were collected and recorded daily. Clinical observations, which included monitoring of paw and tail swelling, were assessed at least twice daily.100 mg / kg BID dosing achieved a free Cmaxfor Compound 137, Isomer 2 of approximately 5.7 µM.

[0304] Figure 8-B illustrates the effect of such combination dosing of Compound 137, Isomer 2 and irinotecan hydrochloride on body weight loss. No significant loss of body weight, paw or tail swelling, or other noticeable adverse events were observed at this combination dose. Example C-19: In Vivo PKMYT1 Target Engagement

[0305] An in vivo study in mice was conducted to evaluate PKMYT1 target engagement by Compound 137, Isomer 2. Target engagement was assessed by analysis of phospho-CDK1 (T14) expression in tumor.

[0306] OVCAR3 tumor-bearing female SCID mice were dosed orally with 10 mg / kg BID, 30 mg / kg BID, or 100 mg / kg BID of Compound 137, Isomer 2. Doses were calculated for individual animals on the day of dosing and administered with a 10 mL / kg dosing volume. After 21 days of dosing, 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 orthovanadate, 0.27 M sucrose, 10 mM β-glycerophosphate, 5 mM pyrophosphate, 0.5% Triton X-100), protease inhibitor (Sigma, P8340), phosphatase inhibitors (Sigma, P0044, P5726) and benzonase (Sigma, (E10141:2000) was used to lyse and homogenize (3x 30 seconds, 6.0 m / s) tumor pieces in a fast prep tube with lysing matrix. Lysates were sonicated and supernatants collected. Lysates were transferred and chilled in 2ml assay blocks in fast prep tubes and centrifuged again for 15 minutes for 2100 rpm at 4°C to ensure no debris transferred. Supernatants were then boiled for 5 minutes at 95°C and immunoblotted as described in Example C-16, using phospho- CDK1 Thr14 (Biolegend, 947402), and vinculin (Sigma, V9131) primary, and anti-mouse IgG, HRP-linked (CST, 7076) secondary antibodies. Following detection with G-box (Syngene), bands were quantified with Gene Tools software. Phospho-CDK1 Thr14 signals were normalized to vinculin, geometrical means were normalized to the control group and plotted (n= 5-7 mice per condition ± S.E.M, P* <0.05 is significant (one-way ANOVA)).

[0307] Figure 9 illustrates the relative in vivo expression levels of phospho-CDK1 Thr14 for each dose. The quantified in vivo phospho-CDK1 Thr14 signals after 21 days of dosing with Compound 137, Isomer 2 indicated significant PKMTYT1 kinase inhibition versus treatment with vehicle for all three doses tested. All three doses of Compound 137, Isomer 2 tested following 21 days of dosing achieved ≥IC90inhibition.Example C-20: Anti-Tumor Effect in Human OVCAR3 and SW620 Xenograft Models

[0308] A study was conducted to evaluate the in vivo efficacy of combination therapy with Compound 137, Isomer 2 and the S-phase DNA damaging agents gemcitabine and irinotecan hydrochloride in a human ovarian OVCAR3 model and a human colorectal SW620 xenograft model, respectively. A. Human Ovarian OVCAR3 Cell Line-Derived Xenograft Model

[0309] OVCAR3 cells were implanted onto the right flank of female SCID mice. When tumors reached approximately 200 mm3, the mice were randomized to treatment groups based on tumor volume and treated according to one of the dosing schedules shown in Table 14. TABLE 14After treatment cessation, the gemcitabine combination arms were left to regrow for an additional 35 days. Tumor sizes were measured twice weekly and relative tumor volumes were plotted (geometrical mean (n= 6) mice per treatment arm ± S.E.M).

[00310] Figure 10 shows relative tumor volume over time. Tumor volume measurements at the end of treatment (Day 21) with twice-daily dosing of 100 mg / kg of Compound 137, Isomer 2 resulted in significant tumor growth inhibition (TGI, 77% P < 0.01) of replication stress high OVCAR3 (CCNE-amplified) tumors (see also, Table 14). Twice-daily dosing of 30 mg / kg or 10 mg / kg of Compound 137, Isomer 2 resulted in 12% or 25% tumor growth inhibition, respectively (P > 0.05, non-significant). Both combinations tested outperformed Compound 137, Isomer 2monotherapy and gemcitabine monotherapy. At Day 21, twice-daily dosing of 50 mg / kg of Compound 137, Isomer 2 in combination with 20 mg / kg of gemcitabine administered intraperitoneal once weekly resulted in 62% tumor regression (P < 0.001), and twice-daily dosing of 10 mg / kg of Compound 137, Isomer 2 in combination with 20 mg / kg of gemcitabine administered intraperitoneal once weekly resulted in 31% tumor regression (P < 0.001). Dose- dependent regrowth to 200 mm3tumor volumes, following cessation of treatment in the combination arms, was observed after 11 days (10 mg / kg BID of Compound 137, Isomer 2 + gemcitabine) and 35 days (50 mg / kg BID of Compound 137, Isomer 2 + gemcitabine). These results indicate that more durable tumor regressions can be achieved with Compound 137, Isomer 2 in combination with gemcitabine compared to gemcitabine monotherapy, particularly as Compound 137, Isomer 2 exposure increases. B. Human Colorectal SW620 Cell Line-Derived Xenograft Model

[0311] SW620 cells were implanted onto the right flank of female athymic nude mice. When tumors reached approximately 200 mm3, the mice were randomized to treatment groups based on tumor volume and treated according to one of the dosing schedules shown in Table 15. TABLE 15Tumor sizes were then measured and relative tumor volumes were plotted (geometrical mean (n= 6) mice per treatment arm ± S.E.M).

[0312] Figure 11 shows relative tumor volume over time. Twice-daily dosing of 100 mg / kg of Compound 137, Isomer 2 did not result in tumor growth inhibition of SW620 tumors. Combination benefit and tumor regression (18%, P<0.01) was achieved, however, with twice- daily dosing of 100 mg / kg of Compound 137, Isomer 2 in combination with once weekly intraperitoneal dosing of, 50 mg / kg irinotecan hydrochloride, a topoisomerase 1 inhibitor (TOP1i). Combination benefit was observed but less pronounced when Compound 137, Isomer 2 was dosed twice daily at 10 mg / kg in combination with irinotecan hydrochloride. These results appear to indicate that higher Compound 137, Isomer 2 exposure is needed to drive combinationbenefit and regression in this SW620 model (Geometrical mean ± S.E.M., P <0.01 (**), P <0.001(***) are significant (one-sided t-test with unequal variance). Example C-21: In Vitro Efficacy in Combination with Exatecan

[0313] An in vitro assay was carried out to assess whether treatment of PKMYT1 KO isogenic SKOV3 cells (previously described in Example C-17) with Compound 137, Isomer 2 could further sensitize those cells to exatecan.

[0314] In vitro efficacy of a combination of exatecan and Compound 137, Isomer 2 was assessed in a 6x6 concentration compound combination assay. PKMYT1 wildtype SKOV3 cells and PKMYT1 knock out (KO) SKOV3 cells were seeded and left to attach for 24 hours in 384 well plates and then dosed (ECHO acoustic liquid handler) with a range of exatecan and Compound 137, Isomer 2 concentrations, as single agents and in combination. After continuous drug exposure for seven days, cells were treated with CellTiter-Glo® (CTG) to enable measurement of cellular metabolic activity of the treated cells. The metabolic rate, generally considered to correlate with cell survival, was measured as chemiluminescence and raw data was derived for every concentration of both drugs.

[0315] Figure 12 illustrates the effect of treatment with exatecan (1 nM) in combination with a concentration range of Compound 137, Isomer 2 on PKMYT1 wildtype SKOV3 cells and PKMYT1 KO SKOV3 cells (mean (n=3) ± SD). Treatment of the PKMYT1 KO cells with exatecan (1 nM) resulted in maximum drug activity (Emax) that did not improve further with the addition of Compound 137, Isomer 2. Comparison of the combination activity in PKMYT1 wildtype SKOV3 cells to the combination activity in PKMYT1 knockout cells suggests that higher inhibitory concentrations (3-10 µM) of Compound 137, Isomer 2 can achieve more pronounced in vitro efficacy for the combination. Such high inhibitory concentrations of Compound 137, Isomer 2 can be achieved in vivo based on comparison with the 100 mg / kg free Cmax. Example C-22: Pharmacodynamic Effect in a Human Colorectal SW620 Xenograft Model

[0316] In vivo monotherapy with irinotecan generally induces DNA damage in the replicative S-phase of the cell cycle, G2 / M checkpoint activation, and PKMYT1 dependence. A study was conducted 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 assessed biomarkers for PKMYT1 target engagement (phospho-CDK1 Thr14),DNA damage formation (gH2AX), and changes in the relative mitotic population (phospho- histone H3 Ser10) following treatment. These biomarkers were selected to evaluate whether Compound 137, Isomer 2 coverage was sufficient to override the G2 / M DNA damage cell cycle checkpoint following administration of irinotecan hydrochloride.

[0317] Human colorectal SW620 cells were implanted onto the right flank of female athymic nude mice. When tumors reached approximately 200 mm3, the mice were randomized to treatment groups based on tumor volume and treated with: (i) vehicle administered orally, (ii) one intraperitoneal dose of irinotecan hydrochloride (50 mg / kg), (iii) twice-daily oral doses (100 mg / kg) of Compound 137, Isomer 2 for three days, or (iv) one intraperitoneal dose of irinotecan hydrochloride (50 mg / kg) plus twice-daily oral doses (100 mg / kg) of Compound 137, Isomer 2 for three days. On the third day at 6 hours after the last dose of Compound 137, Isomer 2, 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), phospho-Histone H3 Ser10 (CST, 9701), gH2AX (Cell Signalling Technology (CST) and vinculin (Sigma, V9131), and anti-rabbit IgG, HRP-linked (CST, 7074) and anti-mouse IgG, HRP-linked (CST, 7076) secondary antibodies. Following detection with G-box (Syngene), bands were quantified with Gene Tools software. Phospho-CDK1 Thr14 signals were normalized to CDK1 and vinculin, gH2AX was normalized to vinculin, and phospho-histone H3 Ser10 was normalized to histone H3, and vinculin. Geometrical means were normalized to the control group and plotted (n= 3-6 mice per condition ± S.E.M, P* <0.05 is significant (one-way ANOVA)).

[0318] Results are shown in Figure 13. Twice-daily oral dosing (100 mg / kg) of Compound 137, Isomer 2 for three days in absence or presence of irinotecan hydrochloride resulted in PKMYT1 target engagement (Phospho-CDK1 Thr14). Intraperitoneal dosing of irinotecan hydrochloride (50 mg / kg) resulted in DNA damage formation (gH2AX) that was not further potentiated by the addition of Compound 137, Isomer 2. Irinotecan hydrochloride monotherapy resulted in a reduced mitotic population (phospho-histone H3 Ser10), a finding consistent with activation of the G2 / M DNA damage cell cycle checkpoint following treatment with a TOP1i inhibitor. The combination of irinotecan hydrochloride and Compound 137, Isomer 2, however, did not result in a reduced mitotic population suggesting that Compound 137, Isomer 2 overrides the G2 / M cell cycle checkpoint induced by irinotecan hydrochloride treatment resulting in premature mitotic entry. These results show the PKMYT1 dependency of tumors in the presence of exogenous DNA damaging agents (including TOP1 inhibitors) that activate the G2 / M cellcycle checkpoint. Example C-23: In Vitro Biotransformation in Hepatocytes of Multiple Species

[0319] Compound 137, Isomer 2 was incubated (5 µM) with mouse, rat, dog, minipig, and human hepatocytes (1 million cells / ml) at 37 ºC for 180 minutes. Incubations were stopped by addition of organic solvent, and samples centrifuged to separate soluble and insoluble components, with the supernatant stored at -20 ºC until required for analysis. A semi-quantitative analysis of the dominant biotransformations occurring to the compound in each of the incubations was performed, with liquid chromatographic separation followed by both UV and MS detection of the eluate. Metabolite abundance was calculated as a percentage of the sum of all compound-related UV peak areas extracted at 330-350 nm in the post-incubation (time = 180 mins) samples of each species. All metabolites with UV abundance >1% observed in human hepatocyte incubations were aligned across species. Table 16 summarizes the in vitro human metabolites of Compound 137, Isomer 2 present across species. M = Major (>10%), m = minor (>1% but ≤10%), t = trace (≤1%), t* = detected by MS response only, ND = Not Detected. TABLE 16Figure 14 is a metabolism scheme showing the dominant routes of biotransformations observed in vitro of Compound 137, Isomer 2: oxidation and glucuronidation. Example C-24: In Vivo Pharmacokinetic Studies in the CD-1 Mouse

[0320] The pharmacokinetic profile of Compound 137, Isomer 2 was characterised in vivo using CD-1 mice following either a single intravenous bolus dose or a single oral bolus dose to allow the calculation of key pharmacokinetic properties.

[0321] Animal experiments were conducted in accordance with the relevant welfare policies and procedures of the organisation conducting the study. Male CD-1 mice, weighing ~20-30 g were housed in polycarbonate animal cages with absorbent rodent bedding, and allowed to acclimatize, with ad libitum access to a standard rodent chow food and sterile water. An ambient temperature of 20-25 °C and humidity of 40-70% was maintained, alongside alternating 12-hour light / dark cycles, interrupted only by study-related events (e.g., sampling). The compound wasformulated for administration as an aqueous solution containing 5% dimethylsulfoxide, 95% sulfobutylether-β-cyclodextrin (30% w / v) in purified water for injection (WFI), at a dose level of either 0.5 mg / kg (I.V.) or 1.0 mg / kg (P.O.) animal bodyweight, and dose volume of 2.0 mL / kg (I.V.) or 4.0 mL / kg (P.O.) animal bodyweight. Blood samples (~0.02 mL / timepoint) were collected from the dorsal metatarsal vein into tubes containing EDTA-K2 anticoagulant, and placed on wet ice (4 °C) before centrifugation (4000 g, 5 minutes, 4ºC) to obtain plasma, and then stored (-75 ºC + / - 15 °C) prior to further analysis. Following intravenous administration (time=0), samples were collected at 2, 10, 30, 60, 120, 240, 360, 480, 720, and 1440 minute timepoints. Following oral administration (time=0), samples were collected at 5, 15, 30, 60, 120, 240, 360, 480, 720, and 1440 minute timepoints. The total concentration of the compound was measured in blood plasma samples using a routine liquid chromatography-mass spectrometry method; blank plasma samples and were prepared and analysed in a similar manner. Key pharmacokinetic parameters were calculated using WinNonlin (PhoenixTM). Table 17 provides summary of key pharmacokinetic parameters derived following administration of Compound 137, Isomer 2 to male CD-1 mice (n=2) at 0.5 mg / kg (I.V.) or 1 mg / kg (P.O.). TABLE 17Figure 15 shows the pharmacokinetic profiles in male CD-1 mice (total blood plasma concentration) of Compound 137, Isomer 2 following administration either I.V. (0.5 mg / kg, solid line) or P.O. (1 mg / kg, dashed line). Example C-25: In Vivo Pharmacokinetic Studies in the SCID Mouse

[0322] Pharmacokinetic profiles of Compound 137, Isomer 2 in female SCID mice were obtained following oral administration to establish the short-term tolerability, and confirmed the extent and duration of compound exposure. Animal husbandry, dosing, sample collection, and subsequent analytical work to determine total plasma concentrations were conducted in a similar manner as previously described In Example C-24, with minor adjustments. Compound 137,Isomer 2, formulated in 5% DMSO, 50% of 20% Captisol, and 45% purified water for injection (WFI) (pH 3-3.2), was dosed at three dose levels (10 mg / kg, 30 mg / kg, and 100 mg / kg) and a dose volume of 10 mL / kg. Plasma samples were collected from the lateral tail vein at multiple timepoints (30 / 60 / 120 / 240 / 420 / 1440 minutes) following administration. Table 18 provides a summary of pharmacokinetic parameters derived following oral administration of Compound 137, Isomer 2 to female SCID mice (n=3) at three dose levels: 10 mg / kg, 30 mg / kg, and 100 mg / kg. TABLE 18Figure 16 shows the pharmacokinetic profiles in female SCID mice (total blood plasma concentration) of Compound 137, Isomer 2 following administration P.O. at three dose levels: 10 mg / kg (solid line), 30 mg / kg (dashed line), and 100 mg / kg (dotted line). Example C-26: In Vivo Pharmacokinetic Studies in The Intact and Bile-Duct-Cannulated Han Wistar Rat

[0323] The pharmacokinetic profile of Compound 137, Isomer 2 was characterized in vivo using bile-duct-cannulated Han Wistar rat following an I.V. bolus dose to determine the contribution of enterohepatic circulation to key pharmacokinetic properties.

[00324] Animal experiments were conducted in accordance with the relevant welfare policies and procedures of the organization conducting the study. Male Han Wistar rats – either intact or following routine surgery to cannulate the bile duct – weighing ~200-300 g were housed in polycarbonate animal cages with absorbent rodent bedding, and allowed to acclimatize, with ad libitum access to a standard rodent chow food and sterile water. An ambient temperature of 20- 25ºC and humidity of 40-70% was maintained, alongside alternating 12-hour light / dark cycles, interrupted only by study-related events (e.g., sampling). Compound 137, Isomer 2 was formulated for administration as an aqueous solution containing 5% dimethylsulfoxide, 95% sulfobutylether-β-cyclodextrin (30% w / v) in purified water for injection (WFI), at a dose level of 0.5 mg / kg (I.V.) animal bodyweight, and dose volume of 1.0 ml / kg animal bodyweight. Bloodsamples (~0.20 mL / timepoint) were collected from the jugular vein into tubes containing EDTA-K2 anticoagulant, and placed on wet ice (4°C) before centrifugation (4000 g, 5 minutes, 4 ºC) to obtain plasma, and then stored (-75 ºC + / - 15 ºC) prior to further analysis. Following intravenous administration (time=0), samples were collected at multiple timepoints (intact rats: 2, 10, 30, 60, 120, 240, 360, 480, 720, and 1440 minute timepoints; BDC rats: 5, 15, 30, 60, 120, 240, 420, and 1440 minute timepoints) Compound 137, Isomer 2 was measured in blood plasma samples using a routine liquid chromatography-mass spectrometry method; blank plasma samples and were prepared and analysed in a similar manner. Key pharmacokinetic parameters were calculated using WinNonlin (PhoenixTM). Table 19 provides a summary of pharmacokinetic parameters derived following I.V. administration of Compound 137, Isomer 2 to male intact or bile-duct-cannulated (BDC) Han Wistar rats (n=3) at 0.5 mg / kg. TABLE 19Figure 17 shows the pharmacokinetic profiles in male Han Wistar rats (total blood plasma concentration) of Compound 137, Isomer 2 following administration I.V. (0.5 mg / kg) to either intact (solid line) or bile-duct cannulated (dashed line) animals. Example C-27: In Vivo Pharmacokinetic Studies in the Han Wistar Rat at High Dose

[0325] Pharmacokinetic profiles of Compound 137, Isomer 2 in male Han Wistar rats were obtained following oral administration to establish the short-term tolerability, and confirm the extent and duration of compound exposure at three dose levels (10 mg / kg, 30 mg / kg, and 100 mg / kg) and a dose volume of 10 mL / kg. Animal husbandry, dosing, sample collection, and subsequent analytical work to determine total plasma concentrations were conducted in a similar manner as previously described In Example C-24, with minor adjustments. Plasma samples were collected from the lateral tail vein at multiple timepoints (15 / 30 / 60 / 120 / 240 / 360 / 1440 minutes) following administration. Table 20 provides a summary of the pharmacokinetic parameters derived following oral administration of Compound 137, Isomer 2 to male HanWistar rats (n=3) at three dose levels: 10 mg / kg, 30 mg / kg, and 100 mg / kg. TABLE 20Figure 18 shows the pharmacokinetic profiles in male Han Wistar rats (total blood plasma concentration) of Compound 137, Isomer 2 following administration P.O. at three dose levels: 10 mg / kg (solid line), 30 mg / kg (dashed line), and 100 mg / kg (dotted line). Example C-28: In Vivo Pharmacokinetic Studies at High Dose to Assess Substance Tolerability and Effect of Repeat Dosing

[0326] Pharmacokinetic profiles of Compound 137, Isomer 2 in female SCID mice were obtained following repeated daily oral administration to establish tolerability over 28 days, and confirm the extent and duration of compound exposure. Animal husbandry, dosing, sample collection, and subsequent analytical work to determine total plasma concentrations were conducted in a similar manner as previously described in Example C-24, with minor adjustments. Animals were dosed twice daily (8 hours apart) and a dose volume of 10 mL / kg. Plasma samples were collected at multiple timepoints (30 / 12 / 360 / 720 / 1440 minutes) following the first dose on day 1 and day 28. Figure 19 shows a comparison of the pharmacokinetic profiles (total blood plasma concentration) of Compound 137, Isomer 2 following repeat BID oral administration (vertical dotted lines) to female SCID mice (n=2) at 100 mg / kg on day 1 (solid line), and day 28 (dashed line). Example C-29: In Vivo Pharmacokinetic Studies Comparing Substance Formulations in the SCID Mouse at High Dose

[0327] Pharmacokinetic profiles of Compound 137, Isomer 2 in SCID mice were obtained following oral administration to establish the short-term tolerability at high dose, and confirm the extent and duration of compound exposure. Animal husbandry, dosing, sample collection, and subsequent analytical work to determine total plasma concentrations were conducted in a similar manner as previously described in Example C-24, with minor adjustments. Compound 137, Isomer 2 formulated in either 5% DMSO, 50% of 20% Captisol, and 45% purified water forinjection (WFI), pH 3-3.2), or 0.5% HPMC / 0.1% Tween was dosed at 300 mg / kg and a dose volume of 10 mL / kg. Plasma samples were collected at multiple timepoints (30 / 12 / 360 / 720 / 1440 minutes) following administration. Table 21 provides a summary of pharmacokinetic parameters derived following oral administration of Compound 137, Isomer 2 formulated in either 5% DMSO, 50% of 20% Captisol, and 45% WFI (pH 3-3.2), or 0.5% HPMC / 0.1% Tween 80 to female SCID mice at 300 mg / kg. TABLE 21Figure 20 shows the pharmacokinetic profiles in female SCID mice (total blood plasma concentration) of Compound 137, Isomer 2 following administration P.O. at 300 mg / kg in 5% DMSO, 50% of 20% Captisol, and 45% WFI (pH 3-3.2) (solid line), or 0.5% HPMC / 0.1% Tween (dashed line). * * *

[0328] Although specific embodiments and examples have been described above, these embodiments and examples are only illustrative and do not limit the scope of the disclosure. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the disclosure in its broader aspects as defined in the following claims. For example, any embodiment described herein can be combined with any other suitable embodiment described herein to provide additional embodiments.

[0329] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and understood as being modified in all instances by the term “about.” These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the present teachings of the present disclosure. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

[0330] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the present disclosure encompassesnot only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the present disclosure encompasses not only the main group, but also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion or disclaimer of one or more of any of the group members in the claimed disclosure.

[0331] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof as well as the individual values making up the range, particularly integer values. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. For example, the range C(1-6), includes the subranges C(2-6), C(3-6), C(3-5), C(4-6), etc., as well as C1(methyl), C2(ethyl), C3(propyl), C4(butyl), C5(pentyl) and C6(hexyl) individually. As will also be understood by one skilled in the art, all language such as “up to,” “at least,” “greater than,” “less than,” “more than,“ “or more” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. In the same manner, all ratios disclosed herein also include all subratios falling within the broader ratio.

[0332] Reference to a “step” in this disclosure is used for convenience purposes only and does not categorize, define, or limit the disclosure as set forth herein. Bibliography 1. Forment, J. V. and M. J. O’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

What is claimed is:

1. 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 having the structure: (Compound 137); 6-amino-7-(2-chloro-3-hydroxy-6-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H- pyrrolo[2,3-d]pyrimidine-5-carboxamide having the structure: (Compound 141); 6-amino-7-(6-chloro-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H- pyrrolo[2,3-d]pyrimidine-5-carboxamide having the structure: (Compound 142); 6-amino-7-(2,6-dichloro-3-hydroxyphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3- d]pyrimidine-5-carboxamide having the structure:(Compound 143); and 6-amino-7-(6-bromo-3-hydroxy-2-methylphenyl)-2-methyl-4-(trifluoromethyl)-7H- pyrrolo[2,3-d]pyrimidine-5-carboxamide having the structure: (Compound 144); or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1 that is 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2- methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the structure: (Compound 137); or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1 that is 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2- methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the structure: (Compound 137).

4. The salt of claim 1 that is a pharmaceutically acceptable salt of 6-amino-7-(3-hydroxy- 2,6-dimethylphenyl)-2-methyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide having the structure: (Compound 137).

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

6. The compound of claim 5, or a pharmaceutically acceptable salt thereof, wherein the atropisomer is the eutomer of the atropisomer.

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

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

9. The method of claim 8, wherein the is a cancer is a solid tumor cancer.

10. The method of claim 8, wherein the is a cancer is a hematological cancer.

11. The method of 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, uterine serous carcinoma, soft tissue sarcoma, uterine cancer, skin cancer, bladder cancer, head and neck cancer, glioma, and B-cell lymphoma.

12. The method of any of claims 8 to 11, wherein the cancer has a PKMYT1-dependency because of elevated basal levels of replication stress.

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