PLK1 degradation inducers with improved cytotoxicity and drug stability

Abstract

The present invention relates to a novel anticancer PLK1 (Polo-like kinase 1) degradation inducer with improved cytotoxicity and drug stability, and to the use thereof. More specifically, the present invention relates to a novel PLK1 degradation inducer PROTAC (proteolysis targeting chimera) technology that simultaneously possesses potent cytotoxicity at the sub-nanomolar level and long-term drug stability. Due to these characteristics, the compounds of the present invention can be utilized in various anticancer applications, including ADC payloads.

Description

Technology Field

[0001] The present invention relates to a novel anticancer PLK1 (Polo-like kinase 1) degradation inducer with improved cytotoxicity and drug stability, and to the use thereof. More specifically, the present invention relates to a novel PLK1 degradation inducer PROTAC (proteolysis targeting chimera) technology that simultaneously possesses potent cytotoxicity at the sub-nanomolar level and long-term drug stability. Due to these characteristics, the compounds of the present invention can be utilized in various anticancer applications, including ADC payloads. Background Technology

[0002] PLK1 (Polo-like kinase 1) is a serine / threonine kinase involved in the G2 / M transition, centrosome maturation, spindle formation, and chromosome segregation of the cell cycle, and is known to be overexpressed in various solid tumors and hematological cancers. Since increased expression or activity of PLK1 is closely associated with the proliferation and survival of cancer cells, PLK1 has been recognized as an important therapeutic target in the field of anticancer drug development (Liu XQ, "Targeting Polo-Like Kinases: A Promising Therapeutic Approach for Cancer Treatment", Translational Oncology (2015). Among these, a strategy to develop a PLK1 degrader as a PROTAC (proteolysis targeting chimera) that induces the removal of the PLK1 protein itself instead of inhibiting the enzymatic activity of PLK1 was disclosed in international patent publications WO2023 / 277583 and WO2024 / 117789, etc.

[0003] Simultaneously improving the cytotoxicity and long-term stability of anticancer PROTAC compounds entails technical challenges that go beyond simply combining elements. This is because, due to their structural complexity, even minute modifications within the moiety of PROTAC compounds can independently exert non-linear effects on target protein resolution, cytotoxicity, and drug stability, respectively.

[0004] Meanwhile, Antibody-Drug Conjugates (ADCs) are attracting attention as anticancer drug platforms capable of achieving maximum efficacy in cancer cells while minimizing unwanted effects in normal cells by enabling the delivery of cancer cell-specific cytotoxic payloads. The antitumor effect of ADCs largely depends on the pharmacological activity of the payload delivered into tumor cells, and the development of novel payloads with diverse mechanisms of action is recently recognized as an important research direction to overcome the limitations of existing cytotoxic drug-based payloads. If a PROTAC-based PLK1 degradation inducer can possess potent cytotoxicity at the sub-nanomolar level and long-term stability in vivo or in lysosome environments, it can be effectively utilized in various anticancer fields, such as ADC payloads.

[0005] Therefore, the development of novel PLK1 degradation inducers that fully maintain PLK1 degradation activity while possessing potent cytotoxicity and long-term stability is a critical technical challenge that can significantly enhance the potential for application in the latest anticancer fields. The problem to be solved

[0006] The objective of the present invention is to provide an improved novel PLK1 (Polo-like kinase 1) proteolytic inducer with potent cytotoxicity at the sub-nanomolar level and long-term drug stability. In particular, the core objective of the present invention is to develop a compound that significantly improves both of these characteristics simultaneously, in the field of PROTACs, where it is difficult to simultaneously achieve enhanced cytotoxicity and enhanced structural stability. Due to these characteristics, the compound of the present invention can be utilized for various anticancer applications, including as a payload for various drug delivery platforms such as ADCs. means of solving the problem

[0007] To solve the above technical problem, the present invention provides a novel PLK1 protein degradation-inducing compound represented by Chemical Formula 1 or by compounds 1 to 44, and uses thereof.

[0008] In one aspect, the present invention provides a PLK1 protein degradation-inducing compound represented by Chemical Formula 1, which will be described later.

[0009] In one aspect, the present invention provides PLK1 protein degradation-inducing compounds represented by compounds 1 to 44.

[0010] The aforementioned PLK1 protein degradation-inducing compound can simultaneously possess sub-nanomolar levels of cytotoxicity and long-term drug stability. Therefore, the PLK1 protein degradation-inducing compound of the present invention can be utilized as an ADC payload.

[0011] In one aspect, the present invention provides an anticancer pharmaceutical composition comprising the above-described PLK1 protein degradation-inducing compound as an active ingredient. Effects of the invention

[0012] The novel PLK1 degradation inducer of the present invention simultaneously achieved potent cytotoxicity at the sub-nanomolar level and long-term drug stability. Along with these characteristics, the compound of the present invention possesses additional properties such as the potential to overcome anticancer drug resistance and induce immunogenic apoptosis, making it highly useful for various anticancer applications, including ADC payloads. Brief explanation of the drawing

[0013] FIG. 1 shows the results of evaluating the cytotoxic efficacy of the compound of the present invention, a comparative example, and a conventional payload drug on H69 cell lines; FIG. 2 shows the results of evaluating the cytotoxic efficacy of the compound of the present invention, a comparative example, and a conventional payload drug on H526 cell lines; FIGS. 3 to 7 show the results of evaluating the ring-opening stability of the compounds of the present invention and comparative example compounds; FIGS. 8 to 12 show the results of evaluating the plasma stability of the compound of the present invention and the compound of the comparative example; FIGS. 13 to 15 show immunogenic cell death (ICD) related indicators of representative compounds of the present invention. Specific details for implementing the invention

[0014] The present invention will be described in more detail below. However, the following description is merely an illustrative explanation to aid in understanding the present invention, and the technical scope of the present invention is not limited thereto. Various modifications, variations, and equivalent embodiments that can be easily derived by a person skilled in the art within the technical spirit and scope of the present invention are included within the scope of the present invention.

[0015] 1. Definition

[0016] In this specification, "PLK1 (Polo-like kinase 1)" refers to a serine / threonine protein kinase involved in cell division processes such as cell cycle progression, centrosome maturation, mitotic spindle formation, chromosome segregation, and cytokinesis. Unless otherwise noted, "inhibition" of PLK1 means reducing the enzymatic or functional activity of the PLK1 protein, and "degradation" of PLK1 means reducing the amount or expression level of the PLK1 protein within the cell.

[0017] In this specification, "PROTAC (Proteolysis Targeting Chimera)" refers to a dual-functional molecule in which a ligand binding to a target protein and a ligand binding to E3 ubiquitin ligase are indirectly linked through a linker. PROTAC can promote ubiquitination and proteasome-mediated degradation of a target protein by inducing proximity between the target protein and E3 ubiquitin ligase.

[0018] In this specification, "degrader" refers to an agent capable of inducing the degradation of a target protein using an intracellular protein degradation mechanism. In one embodiment of this specification, the degrader may be a PLK1 degrader that induces the degradation of the PLK1 protein, and more specifically, may be a PROTAC compound in which a PLK1 binding moiety and an E3 ligase binding moiety are connected through a linker.

[0019] In this specification, "PLK1 degradation inducer" refers to a compound capable of inducing the degradation of PLK1 protein within a cell with PLK1 protein as the target protein. In one embodiment of this specification, the PLK1 degradation inducer may be a PROTAC compound in which a BI-2536 series PLK1 binding moiety and an E3 ligase binding moiety are linked through a linker.

[0020] In this specification, "degradation activity" refers to the ability of a degradation inducer to reduce the amount or expression level of a target protein. Degradation activity includes, for example, a decrease in the expression level of the target protein, an increase in the degradation rate, D max (maximal degradation), DC 50 It can be evaluated using (50% degradation concentration), luciferase assay, western blot, immunoassay, or other appropriate indicators or methods capable of evaluating target protein levels.

[0021] In this specification, "ADC (Antibody-Drug Conjugate)" refers to a conjugate in which an antibody, an antibody fragment, a protein having antigen-binding ability, or a derivative thereof is combined with one or more drug agents, either directly or through a linker. The drug agents may include cytotoxic anticancer agents, enzyme inhibitors, proteolytic agents, or other substances having biological activity, and the ADC can deliver the drug agents to target cells by utilizing the target specificity of the antibody or antigen-binding component.

[0022] In this specification, "ADC payload" refers to a component that acts as a drug agent constituting an ADC and exerts pharmacological activity after being delivered into or released around target cells. The ADC payload may be a component substantially responsible for the therapeutic effect of the ADC and may include cytotoxic anticancer agents, enzyme inhibitors, proteolytic inducers, or other therapeutic agents.

[0023] In this specification, "DAC (Degrader-Antibody Conjugate)" refers to an antibody-drug conjugate in which the ADC payload is a degradation-inducing agent, as a form of ADC. A DAC is a conjugate that combines the target delivery function of an antibody with the target protein removal function of a degradation-inducing agent, and in this specification, DAC may be used as a sub-concept of ADC. In one embodiment of this specification, the payload of the DAC may be a PLK1 degradation-inducing agent.

[0024] In this specification, "cytotoxicity" or "cancer cell proliferation inhibitory activity" refers to an activity that causes a decrease in cell viability, inhibition of proliferation, cell cycle arrest, induction of apoptosis, or loss of other cellular functions. Cytotoxicity or cancer cell proliferation inhibitory activity refers to cell viability, IC50 50 , EC 50 It may be evaluated using apoptosis rate, cell proliferation inhibition rate, or other appropriate biological indicators. Unless otherwise noted, cytotoxicity in this specification includes cytotoxicity to cancer cells.

[0025] In this specification, "sub-nanomolar cytotoxicity" generally refers to IC 50 It refers to cytotoxicity with a value of less than 1 nM. For example, an IC50 of 0.9 nM or less, 0.5 nM or less, 0.25 nM or less, or in the picomolar concentration range. 50Cases indicating values ​​may include, but are not limited to. Unless otherwise noted, sub-nanomolar levels of cytotoxicity are IC50 for the reduction in cancer cell survival or inhibition of proliferation. 50 It can be evaluated based on the value.

[0026] In this specification, "picomolar cytotoxicity" generally refers to IC 50 It refers to cytotoxicity with values ​​in the pM range. For example, IC50 of about 900 pM or less, about 500 pM or less, about 250 pM or less, 100 pM or less, or similar levels. 50 It may include, but is not limited to, cases representing a value.

[0027] In this specification, "drug stability" means the degree to which a compound maintains its original structure, chemical identity, physicochemical properties, or pharmacological activity under specific conditions. Drug stability may be evaluated, for example, through plasma stability, serum stability, chemical stability under human-like conditions, the ring-opening rate of the E3 ligase-binding moiety, stability under acidic or basic conditions, stability under lysosome-like pH conditions, metabolic stability, or other appropriate stability indicators. In one embodiment of this specification, "long-term stability" means that the drug stability of a compound is evaluated under long-term conditions, which may be, for example, 24 hours or more, 72 hours or more, or 168 hours or more. In this specification, long-term stability is distinguished from drug stability evaluated under short-term conditions (e.g., 4 hours or less).

[0028] In this specification, "ring-opening" refers to the phenomenon in which a ring structure within a compound, such as a ring structure contained in a CRBN E3 ligase-binding moiety, opens under chemical or biological conditions. More specifically, in this specification, ring-opening may refer to the phenomenon in which an imide, dihydrouracil, or similar cyclic structure of a CRBN E3 ligase-binding moiety opens. A ring-opening percentage may refer to the proportion of ring-opening products detected after specific conditions and a specific period of time, and the lower the ring-opening percentage, the more structurally stable the compound may be evaluated under those conditions.

[0029] In this specification, "plasma stability" refers to the extent to which a compound remains in its original form over time under plasma conditions. Plasma stability may be evaluated through the retention rate of the compound at a specific point in time, the formation rate of degradation products, half-life, or other appropriate analytical indicators. In this specification, plasma stability may be used as an indicator reflecting the chemical and biological stability of the entire PROTAC molecule as well as the stability of specific substructures.

[0030] In this specification, "ADC payload suitability" refers to the degree to which a specific compound possesses pharmacological, biological, or physicochemical properties suitable for use as an ADC payload. ADC payload suitability can be comprehensively determined by multiple pharmacological, biological, and physicochemical properties in accordance with technical common sense in the field of ADCs. For example, if a compound having anticancer efficacy contains functional groups (amine groups, hydroxyl groups, etc.) that can be chemically linked to an antibody conjugation linker, possesses potent cytotoxicity at the subnanomolar level similar to MMAE or DXd, which are currently used as mainstream ADC payloads, and simultaneously possesses long-term drug stability considering the time required for the ADC drug to circulate in the blood and be introduced into tumor cells, it may be considered for use as an ADC payload in addition to as a monotherapy. In addition, if a drug possesses advantages as a payload drug, such as improved resistance to conventional payload drugs and potential to induce immunogenic apoptosis, or advantages in payload delivery, such as long-term drug stability in plasma or drug stability in a lysosomal acidic environment, it may be evaluated as a drug with ADC payload suitability. In this specification, ADC payload suitability may refer to cases where an individual drug with anticancer efficacy achieves various evaluation criteria suitable for use as an alternative payload drug to conventional ADCs, thereby recognizing new technical significance as an alternative ADC payload in the relevant technical field, and it is not necessarily required to actually manufacture an ADC with said individual drug as a payload.

[0031] Unless otherwise specifically noted, the compounds described in this specification may include not only the compound itself represented by the corresponding structural formula, but also, within a structurally acceptable range, its stereoisomer, optical isomer, geometric isomer, diastereomer, epimer, racemate, rotamer, tautomer, zwitterion, inner salt, N-oxide, and mixtures thereof.

[0032] Additionally, the compounds of this specification may exist in pharmaceutically acceptable salts, solvates, hydrates, co-crystals, crystalline forms, and amorphous forms. The pharmaceutically acceptable salt may include, but is not limited to, acid addition salts with inorganic or organic acids, salts with inorganic or organic bases, metal salts, etc.

[0033] Compounds indicated by a specific stereoconfiguration in this specification are representative examples of compounds having said stereoconfiguration, and individual stereoisomers, optical isomers, and mixtures thereof that can be prepared or separated according to methods known in the art, unless explicitly excluded by the context, may also be understood as embodiments of the compounds described in this specification.

[0034] Additionally, the compounds described in this specification may exist in different polymorphs, hydrates, or solvates depending on the manufacturing process, purification process, or storage conditions, and these forms may also be understood as embodiments of the compounds described in this specification.

[0035] 2. Technical Background and Challenges

[0036] Proteolysis targeting chimera (PROTAC) degradation inducers are attracting attention as a therapeutic modality capable of selectively removing target proteins, and PLK1 (Polo-like kinase 1) degradation inducers can inhibit cancer cell proliferation and induce apoptosis (Applicant's International Publications WO2023 / 277583 and WO2024 / 117789, etc.). However, generally, PROTACs have larger molecular weights and more complex structures compared to small molecule anticancer agents, so optimizing them in terms of cell permeability, solubility, intracellular exposure, and drug stability entails significant technical challenges (Syahputra et al., "PROTAC Delivery Strategies for Overcoming Physicochemical Properties and Physiological Barriers in Targeted Protein Degradation", Pharmaceutics , 2025).

[0037] Below, we specifically describe how prior art has failed to solve the challenge of simultaneously achieving sub-nanomolar levels of cytotoxicity and long-term stability, and explain the technical background explaining how these characteristics achieved by the compound of the present invention directly lead to ADC payload suitability.

[0038] PLK1 (Polo-like kinase 1) is a serine / threonine kinase that regulates the entire mitotic process, including centrosome maturation, mitotic spindle formation, and chromosome separation. It is overexpressed in many cancer types and plays a key role in the proliferation and survival of tumor cells. Due to this biological importance, PLK1 has long been recognized as a promising anticancer target (Strebhardt et al., “Targeting polo-like kinase 1 for cancer therapy”, Nature Reviews Cancer , 2006).

[0039] BI-2536, a representative PLK1 inhibitor and an ATP-competitive inhibitor of the dihydropteridone derivative class, demonstrated potent PLK1 inhibitory activity and excellent preclinical anticancer efficacy; however, limitations such as limited efficacy, hematological toxicity, and a narrow therapeutic range were reported during clinical development (Steegmaier et al., "BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo", Current Biology, 2007; Mross et al., "Phase I Dose Escalation and Pharmacokinetic Study of BI 2536, a Novel Polo-Like Kinase 1 Inhibitor, in Patients With Advanced Solid Tumors", Journal of Clinical Oncology, 2008). Accordingly, a targeted protein degradation (PROTAC) approach that induces the degradation of the PLK1 protein itself, beyond the temporary inhibition of enzyme activity, began to be considered as a new alternative.

[0040] The applicant's prior patents WO2023 / 277583 and WO2024 / 117789 disclose PROTAC-based PLK1 degradation inducers using a BI-2536 derivative family moiety and a cereblon (CRBN) binding moiety. WO2023 / 277583 discloses the target protein degradation efficacy of the PLK1 degradation inducer, and WO2024 / 117789 discloses the cytotoxicity of subsequent PLK1 degradation inducers modified with a linker and a CRBN binding moiety at the tens to hundreds of nanomolar (nM) level. However, the aforementioned prior art does not disclose or suggest the simultaneous achievement of sub-nanomolar cytotoxicity and long-term drug stability.

[0041] Currently, active research is underway regarding the utilization of PROTAC-based degradation inducers as payloads for antibody-drug conjugates (ADCs), in addition to their development as monotherapy drugs. Given the characteristics of the ADC technology field described below, for anticancer PROTACs to possess ADC payload suitability, drug indicators such as enhanced cytotoxicity and drug stability must be simultaneously satisfied.

[0042] Since the amount of drug released by ADCs following receptor-mediated endocytosis is extremely limited, ultra-high activity (sub-nanomolar level) cytotoxicity capable of killing target cancer cells even under conditions where the payload is exposed in minute amounts within the cell is required as a prerequisite. Representative cytotoxic payloads that currently dominate the ADC market, such as MMAE (microtubule inhibitor) and DXd (topoisomerase I inhibitor), also possess potent cytotoxicity at the sub-nanomolar level. In contrast, the cytotoxic efficacy of the PLK1 degradation inducer at levels greater than nanomolar in the prior art (WO2024 / 117789) presents technical limitations for use as a payload for drug delivery platforms such as eADCs (Baah et al., “Antibody-Drug Conjugates—A Tutorial Review”, Molecules , 2021).

[0043] Additionally, if cancer cell resistance, which is one of the causes of frequent clinical failure of the aforementioned MMAE or DXd-based existing ADC therapies, can be overcome, the clinical value may be enhanced (Abelman et al., "Mechanisms of resistance to antibody-drug conjugates", Cancers, 2023). Furthermore, beyond direct tumor-killing effects, the function of inducing immunogenic cell death (ICD), in which dying cancer cells activate surrounding immune cells, can also be positively considered as a function of alternative ADC payloads (Kepp et al., "Immunogenic cell death and bystander killing: expanding the therapeutic potential of modern antibody-drug conjugates", Oncoimmunology , 2025). As such, alternative payloads with mechanisms of action differentiated from existing cytotoxic drug-based payloads can be valued for these additional characteristics in addition to cytotoxicity.

[0044] Meanwhile, to utilize PROTACs as ADC payloads, in addition to efficacy, the challenge of pharmacological stability must inevitably be resolved, ensuring that the payload molecule, connected to the antibody via an antibody-drug conjugate linker, can stably circulate within the body and withstand the process of intracellular release.

[0045] To achieve drug stability as an ADC payload, first, the CRBN binder introduced for the PROTAC mechanism must maintain a structurally stable chemical structure under actual human administration conditions. For example, when using glutarimide or imide structures as CRBN binders, there is a risk of structural deformation due to ring opening upon in vivo exposure. Ring opening can act as a major cause for the loss of binding affinity to CRBN, thereby fundamentally neutralizing target protein degradation activity (Jarusiewicz, et al. "Phenyl dihydrouracil: an alternative cereblon binder for PROTAC design." ACS Medicinal Chemistry letters,2023; Xie, et al. “Development of substituted phenyl dihydrouracil as the novel achiral cereblon ligands for targeted protein degradation.” Journal of Medicinal Chemistry , 2023).

[0046] In addition, ADC payloads must necessarily ensure long-term plasma stability for several hours to several days or more during blood circulation. After an ADC drug is administered into the body, it takes a significant amount of time to remain in the bloodstream until it reaches the target tumor tissue and is internalized into the cell via antigen-receptor binding. If the payload itself degrades prematurely or undergoes structural deformation during blood circulation within this critical timeframe, the amount of the active drug delivered into the tumor cells decreases rapidly, potentially rendering the ADC therapeutic efficacy ineffective. Therefore, ensuring long-term plasma stability is a key challenge for guaranteeing effective in vivo exposure of the payload.

[0047] Meanwhile, since ADCs introduced into tumor cells undergo intracellular processing and ultimately release the payload in an acidic lysosomal environment, the harsh acidic environment of the lysosome acts as a barrier threatening payload stability. If the PROTAC drug is inactivated or degraded by the abundant hydrolytic enzymes and strong acidic conditions inside the lysosome before it can escape into the cytoplasm, it becomes unable to form a ternary complex with the target PLK1. Therefore, stability under the acidic lysosomal environment is a pharmacological evaluation metric that must be satisfied to induce successful payload induction and escape into the cytoplasm and to maintain final cell-based pharmacological activity (Kalim et al., "Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates", Drug design, development and therapy , 2017).

[0048] Based on the industrial background regarding the potential applications of the aforementioned PROTACs, the inventors established a core technical objective to fundamentally redesign the structural characteristics of PLK1 degradation-inducing PROTAC compounds to simultaneously achieve potent cytotoxicity at the sub-nanomolar level and long-term drug stability in the in vivo environment within a single compound. PLK1 degradation inducers possessing these characteristics can simultaneously possess the potential to overcome resistance and induce immunogenic apoptosis due to a mechanism of action differentiated from existing payloads, and can be usefully utilized as payloads for various drug delivery platforms, including ADCs.

[0049] 3. Detailed technical solution of the invention

[0050] In order to solve the aforementioned technical problem, in one aspect, the present invention provides a novel PLK1 protein degradation-inducing compound represented by Formula 1 or compounds 1 to 44.

[0051] The applicant's prior patent WO2024 / 117789 adopts an imide or glutarimide-based CRBN ligand and a rigid linker design as essential components. However, it does not teach a multidimensional structural redesign direction that requires the simultaneous complex application within a single molecule of the adoption of a novel CRBN ligand with a phenyl dihydrouracil (PD) backbone, which is the unique basis of the novel PLK1 degradation inducer of the present invention, the introduction of a specific single substituent on the phenyl ring within the ligand, and the modification of the substituent of the PLK1 binding moiety (BI-2536).

[0052] The unpredictability of the direction of such structural redesign is attributed to the inherent physicochemical properties of the target protein degradation modality. Since the PROTAC compounds to which this invention belongs possess high molecular weights and complex molecular structures compared to conventional small molecule drugs, they structurally fall outside the scope of traditional drug efficacy evaluation criteria such as Lipinski's Rule of Five. Furthermore, because PROTACs must mediate the dynamic formation of a ternary complex between the target protein, the drug, and the E3 ligase, the relationship between the physicochemical anomalies induced by minute structural changes and the activity exhibits thoroughly nonlinear behavior.

[0053] Therefore, attempts to simultaneously modify multiple structural variables, as in the present invention, result in organic ripple effects that are difficult to predict organically regarding PLK1 degradation efficiency, intracellular permeability and behavior, cytotoxicity, and the overall final pharmacological properties. In other words, it is quantitatively impossible to predict in advance from prior art whether the introduction of specific substituents or fine-tuning of binding sites will lead to a dramatic increase in the final anticancer effect, unless this is inferred retrospectively after the effects of the invention are known (Bondeson et al., "Lessons in PROTAC design from selective degradation with a promiscuous warhead", Cell Chemical Biology , 2018).

[0054] In one aspect, the present invention provides a PLK1 protein degradation-inducing compound represented by the following chemical formula 1.

[0055] [Chemical Formula 1]

[0056]

[0057] In the above chemical formula,

[0058] R1 is CH3, OCH3, F, or Cl;

[0059] R2 is H, F, OH, CH3, OCH3, or CN;

[0060] R3 is H, F, OCH3, OH, or OCH2CH2OH;

[0061] X is CH or N.

[0062] In a specific aspect, the above chemical formula 1 includes a sub-chemical formula 1A in which R2 is fixed to H and the remaining substituents are further limited based on the structural commonality of compounds 1-9 or 12.

[0063] [Chemical Formula 1A]

[0064]

[0065] In the above chemical formula,

[0066] R1 is CH3, OCH3, F, or Cl;

[0067] R3 is OCH3 or F;

[0068] X is CH or N.

[0069] Preferably, in the above formula 1A, R1 is CH3 or OCH3, R3 is OCH3 or F, and X can be CH or N, for example, compound 1-5 or 12.

[0070] Compounds 2 and 21 of the prior art (WO2024 / 117789), which can be structurally compared with the group of compounds of Formula 1A of the present invention, are defined herein as Comparative Examples 1 and 2, respectively. Comparative Examples 1 and 2 were never presented or implied within the prior art as starting materials for reaching the structure of the present invention or as a central axis for subsequent optimization, but were merely selected retrospectively for the purpose of comparison to clearly demonstrate the significant effects of the present invention.

[0071] In contrast to Comparative Examples 1 and 2 above, the group of compounds in Formula 1A of the present invention has a complex redesigned structure in which the cereblon (CRBN) binder moiety is completely substituted from the existing PG (Phenyl glutarimide) backbone to the PD (Phenyl dihydrouracil) backbone, a specific single substituent is additionally introduced into the substituted PD structure, and the ethyl group in the core structure of the PLK1 binding moiety (BI-2536) is modified to a methyl group are simultaneously combined within a single molecule.

[0072] Through this multidimensional structural redesign, the compound of Formula 1A of the present invention exhibits significant and potent cytotoxicity at the sub-nanomolar level (Experimental Example 2), while effectively inhibiting the glutarimide ring-opening reaction, which was a chronic limitation of imide-based compounds, and dramatically improving plasma stability (Experimental Example 5), thereby simultaneously achieving a structural and pharmacological qualitative leap required for next-generation ADC payloads.

[0073] In particular, even though Comparative Example 1 shares the same structural redesign directionality as Formula 1A of the present invention, Comparative Example 4, in which the type and position of the substituent on the phenyl ring within the phenyl dihydroracyl were applied differently from the definition of Formula 1A, failed to achieve the unexpected effect (sub-nanomolar level cytotoxicity) of Formula 1A as described above. The comparative experimental results showing that pharmacological activity is neutralized by minute differences in substituents despite similar skeletal directionality demonstrate that the behavior of the PROTAC molecule due to structural changes is extremely unpredictable and that the group of compounds under Formula 1A of the present invention is a product that is clearly unattainable from Comparative Examples 1 and 2.

[0074] In a specific aspect, the above formula 1 comprises a sub-formula 1B in which R1 is fixed to CH3 and the remaining substituents are further limited based on the structural commonality of compounds 10, 11, 13, 14, 17-19, 28-31, preferably compounds 10, 11, 13, 14.

[0075] [Chemical Formula 1B]

[0076]

[0077] In the above chemical formula,

[0078] R2 is F, OH, CH3 or OCH3;

[0079] R3 is H, F, or OCH3;

[0080] X is CH or N.

[0081] In the above chemical formula 1B, R2 may preferably be F or CH3.

[0082] In one embodiment, R2 is F, e.g., compounds 10, 13, 17-19.

[0083] In another embodiment, R2 is CH3, e.g., compound 30 or 31.

[0084] The group of compounds of Formula 1B also includes additional structural diversification, specifically modification of the substituent at the R2 site, in addition to the complex redesigned structure of Formula 1A described above, in comparison to the prior compounds Comparative Examples 1 and 2. Despite the additional expansion of structural variables, the fact that unexpected effects such as potent cytotoxicity at the subnanomolar level (Experimental Example 2) and significant improvement in ring opening inhibition and plasma stability (Experimental Example 5) were consistently maintained and enhanced in the compounds belonging to Formula 1B demonstrates that the group of compounds of Formula 1B is a product that is clearly unattainable from Comparative Examples 1 and 2.

[0085] In a specific aspect, the above chemical formula 1 comprises the following chemical formula 1C, in which R1 is fixed to CH3 and R2 is fixed to a CN group, based on the structural commonality of compounds 21-25.

[0086] [Chemical Formula 1C]

[0087]

[0088] In the above chemical formula,

[0089] R3 is H, F, OCH3, OH, or OCH2CH2OH;

[0090] X is CH or N.

[0091] Preferably, in the above formula 1C, X is CH, and may be, for example, compound 21-25.

[0092] The group of compounds of Formula 1C also includes additional structural diversification, specifically modification of the substituent at the R2 site, in addition to the complex redesigned structure of Formula 1A described above, in comparison to the prior compounds Comparative Examples 1 and 2. Despite the additional expansion of structural variables, the fact that unexpected effects such as potent cytotoxicity at the subnanomolar level (Experimental Example 2) and significant improvement in ring opening inhibition and plasma stability (Experimental Example 5) were consistently maintained and enhanced in the compounds belonging to Formula 1C demonstrates that the group of compounds of Formula 1C is a product that is clearly unattainable from Comparative Examples 1 and 2.

[0093] The above-described chemical formulas 1A, 1B, and 1C present a group of sub-compounds as examples according to structural features for chemical formula 1, and in another embodiment, the compound represented by chemical formula 1 is compound 1-14, compound 17-20, compound 21-25, or compound 28-31.

[0094] In another aspect, the present invention provides a PLK1 protein degradation-inducing compound of any one of compounds 1 to 44. In addition to the compounds incorporated in the aforementioned formula 1 (compounds 1-14, 17-25, 28-31), formulas 1 to 44 include compounds (compounds 15-16, 26-27, 32 or 33-44) that share the characteristic core formula backbone of the novel compounds of the present invention.

[0095] In one embodiment, the present invention provides a PLK1 protein degradation-inducing compound of any one of compounds 1-32.

[0096] In a specific embodiment, the compound is compounds 1-9, 12, 15-16, and corresponds to a novel compound that is non-obvious from Comparative Example 1 or 2 as described above in Formula 1A.

[0097] In a specific embodiment, the compounds are compounds 10, 11, 13, 14, 17-19, 20, 28-31, 32, and correspond to novel compounds that are non-obvious from Comparative Example 1 or 2 as described above in Formula 1B.

[0098] In a specific embodiment, the compound is compounds 21-25, 26, and 27, and corresponds to a novel compound that is non-obvious from Comparative Example 1 or 2 as described above in Formula 1C.

[0099] In one embodiment, the present invention provides a PLK1 proteolytic-inducing compound of any one of compounds 33-44. For example, the compound may be compounds 33, 36, and 43, compounds 35, 40-42, and 44, and compounds 34, 37-39, depending on the type of phenyl substituent in the PD. Compared to Comparative Examples 1 and 2, the compound includes positional isomeric differentiation in which the binding position of the PROTAC linker attached to the phenyl ring in the PD is differently arranged in addition to the structural modification described above in Formula 1A. This change in binding position has distinct non-obviousness in that it completely alters the stereochemical orientation of the molecule, consistently yielding unpredictable effects such as subnanomolar-level potent cytotoxicity (Experimental Example 2) and a significant improvement in ring opening inhibition and plasma stability (Experimental Example 5) in compounds 33-44 compared to Comparative Examples 1 and 2. Furthermore, in the case of Comparative Examples 5 and 6, which share the backbone and linker binding positions of compounds 33-44 but have different substituent orientations, the potent cellular efficacy (sub-nanomolar or less) of the present invention was not achieved (Experimental Example 2). These comparative experimental results, which show that pharmacological activity is neutralized by minute differences in substituents, demonstrate that the behavior of PROTAC molecules due to structural changes is extremely unpredictable.

[0100] 4. Effects and Technical Significance of the Invention

[0101] Based on the PLK1 degradation induction mechanism, the compound of the present invention achieved potent cytotoxicity at the sub-nanomolar level and long-term structural stability as key effects. Furthermore, the potential to overcome resistance to existing ADC payloads and induce immunogenic apoptosis was additionally confirmed, and each effect is described in detail below.

[0102] PLK1 decomposition activity

[0103] As a result of measuring the intracellular PLK1 protein degradation effect of the compounds of the present invention using a luciferase assay, PLK1 degradation efficacy was confirmed in all compounds 1-44 (Experimental Example 1; Table 3). This demonstrates that the compounds of the present invention exert a PLK1 degradation-inducing function as PROTAC compounds.

[0104] Strong cytotoxicity at the sub-nanomolar level

[0105] Because PROTACs require multiple intracellular processes, such as increased molecular weight, decreased cell permeability, ternary complex formation, and dependence on the ubiquitin-proteasome system, their cytotoxicity generally remained in the range of a few to tens of nanomolar even if they exhibited excellent target protein degradation ability. In the cytotoxicity evaluation of the compounds of the present invention targeting two small cell lung cancer cell lines, it was confirmed that they exhibited cytotoxicity with significantly enhanced efficacy compared to the comparative example (Experimental Example 2; Table 4; Figures 1 and 2). As an example to explain these pharmacological characteristics, the present invention [demonstrates] an IC50 of approximately 500 pM or less in Experimental Example 2. 50 Compounds 1-2, 4, 7, 15, 17-20, 27-28, 31-33, 35-36, 39-43 representing numerical values ​​can be provided, preferably with an IC of 200 pM or less. 50 Compounds representing numerical values ​​3, 5, 10, 12-13, 16, 21-26, and 30 can be provided.

[0106] In particular, the PLK1 degradation inducer of the present invention exhibited potent cytotoxicity at subnanomolar or picomolar levels similar to those of microtubule inhibitors (MMAE) and topoisomerase I inhibitors (DXd, Exatecan) used as ADC payload drugs in cytotoxicity experiments targeting cell lines of various solid cancers (small cell lung cancer, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), HER2-positive breast cancer, prostate cancer, gastric cancer) and hematological cancers (multiple myeloma (MM), acute myeloid leukemia (AML), chronic myeloid leukemia (CML)) including small cell lung cancer (Experimental Examples 2 and 3; Tables 4 to 6; Figs. 1 and 2).

[0107] Furthermore, this potent improvement in cytotoxic efficacy as a PROTAC compound with a PLK1 degradation-inducing mechanism goes beyond mere numerical improvement and reaches sub-nanomolar levels of cytotoxicity similar to MMAE or DXd, which are currently used as mainstream payloads in ADCs. This suggests that the compound of the present invention can be utilized as a payload candidate for various drug delivery platforms, such as ADCs, which were difficult for conventional PROTACs to access, and thus has significant technical industrial significance.

[0108] Long-term stability of PROTAC drugs

[0109] Since PROTAC compounds have a high molecular weight and complex molecular structure compared to small molecule drugs, drug stability, which maintains a stable chemical structure for a long period in a biological environment such as plasma, is a major drug indicator criterion independent of cytotoxicity evaluation.

[0110] In the field of PROTACs, the spontaneous ring-opening of the glutarimide structure, which is primarily contained in CRBN binders, corresponds to a physicochemical decay mechanism that permanently loses electrostatic and stereochemical binding ability to CRBN, thereby neutralizing the PROTAC's inherent ternary complex formation and targeted degradation activity. Therefore, the issue of ring-opening under long-term plasma conditions lasting from several hours to several days is a technical challenge that must be resolved for PROTAC drugs. Accordingly, the ring-opening stability of the CRBN binding moiety of the compounds of the present invention was evaluated in a human plasma environment. As a result, it was observed that the control compounds of Comparative Examples 1, 2, and 3, which possess a glutarimide moiety, showed a rapid increase in ring-opening metabolites due to spontaneous hydrolysis at a significant rate as the exposure time to biological conditions increased. On the other hand, the compounds of the embodiments of the present invention showed virtually no ring-opening or remained at an extremely minimal level under the same conditions, confirming that the long-term drug stability of the compounds was improved (Experimental Example 5; Table 10; Figs. 3 to 7).

[0111] In particular, the ring-opening improvement effect of the novel PLK1 degradation inducer identified in the present invention is not limited to evaluation under short-term (4 hours or less) conditions, but also demonstrates significantly improved stability under long-term conditions of 168 hours. This has significant technical significance in terms of ADC payload utilization, considering the in vivo kinetics of ADCs, where a payload conjugated to an antibody requires a considerable blood residence time to circulate through the blood, reach the target tumor tissue, and be introduced into the cell.

[0112] Meanwhile, as a result of evaluating the long-term stability of the compound of the present invention under plasma conditions, it was confirmed that the compound of the present invention exhibited significantly improved drug stability compared to the comparative example under long-term conditions up to 168 hours (Experimental Example 5; Table 11; Figs. 8 to 12). This confirms that the compound of the present invention can remain as a stable drug in plasma beyond the structural stability of the compound, which is ring opening, and exhibit the desired drug properties.

[0113] In particular, regarding the usability of ADC payloads, the ADC payload must maintain structural integrity during the critical circulation time from administration into the blood until it reaches the target tumor tissue and is internalized into the cell. Experimental results showing that the initial concentration was maintained in a plasma environment of up to 168 hours demonstrate that the pharmacological characteristics of the PROTAC, which secures usability as an ADC payload by minimizing the risk of premature degradation or structural deformation during blood circulation in a form conjugated to the ADC by the PLK1 degradation inducer of the present invention, maximize the amount of effective active drug delivered into tumor cells, and ensure effective in vivo exposure, have been secured.

[0114] Simultaneous improvement of cytotoxicity and drug stability

[0115] In PROTAC compounds, pharmacological properties can be independently altered by the complex interaction of target protein binding moiety, E3 ligase binding moiety, linker structure, and ternary complex forming ability; therefore, achieving cytotoxicity and drug stability simultaneously has technical significance distinct from the improvement of individual drug effects.

[0116] Comparative Examples 4, 5, and 6, which contain PD-based CRBN binding moiety similar to the compound of the present invention, showed improved results in terms of ring-opening stability and plasma stability, but failed to achieve the potent sub-nanomolar level cytotoxicity observed in the compound of the present invention (Experimental Example 2; Table 4; Figures 1 and 2). This demonstrates that while structural stability may be improved by merely changing the PG-based moiety in the CRBN binder to a PD-based moiety, potent sub-nanomolar level cytotoxicity cannot be secured.

[0117] In other words, the simultaneous combined effect of enhanced cytotoxicity and stability observed in the compounds of the present invention cannot be explained simply by the substitution of PG-based moiety in the CRBN binder with PD-based moiety. The compounds of the present invention simultaneously achieve enhanced cytotoxicity at the subnanomolar level (Experimental Example 2; Table 4; Figs. 1 and 2) while maintaining excellent structural stability and plasma stability based on PD-based moiety (Experimental Example 5; Tables 10 and 11; Figs. 3 to 12). These results strongly support the fact that the compounds of the present invention correspond to a specific structural combination that achieves both improved stability and improved cytotoxicity, and that the effect is difficult to predict from conventional structural substitutions.

[0118] Drug stability in the lysosomal environment

[0119] For representative compounds 3, 5, and 10 of the present invention, structural stability and relative decomposition (%RD, Relative Decomposition) were evaluated for 24 hours under acidic buffer conditions (pH 5.0) that simulate the acidic environment within the lysosome encountered after endocytosis. As a result, the representative compounds of the present invention all exhibited excellent indicators, with a relative decomposition rate of less than 1% even after 24 hours under acidic conditions, demonstrating that they maintain drug stability against physicochemical structural deformation or inactivation caused by acid stress (Experimental Example 6; Table 12).

[0120] The above results indicate that the ADC payload stability requirement was met, which stipulates that the integrity of the molecular structure must be stably maintained until it escapes from the acidic environment inside the lysosome into the cytoplasm.

[0121] Improvement of cancer resistance to ADC payload drugs

[0122] To measure resistance to drug-resistant cancers, which have been clinically identified as a key limitation of conventional ADC platforms and payload drugs, cytotoxicity was compared against MMAE-resistant non-small cell lung cancer (HCC4006), DXd-resistant ovarian cancer (SK-OV-3), and DS-8201-resistant gastric cancer (NCI-N87) cell lines, which are global standard ADC finished products.

[0123] As a result, it was confirmed that the representative compounds of the present invention (compounds 10, 18) improved resistance and maintained strong cytotoxicity, with the Resistance Index (RI), an indicator of resistance, decreasing compared to the control group not only in cell lines resistant to individual payloads alone but also in resistant carcinomas that exhibit strong refractory resistance to the finished ADC itself (Experimental Example 4; Tables 7 to 9).

[0124] Meanwhile, DXd, used as a comparative compound, is a payload belonging to the topoisomerase I inhibitor class. It acts as a major substrate for BCRP transporters overexpressed on the surface of cancer cells, causing forced efflux out of the cell, which can reduce effective concentrations and induce resistance. Unlike the comparative compound DXd, the representative compounds of the present invention (Compounds 3, 5, 10, 18, 21-23, 25-26) consistently exhibited low IC50s regardless of BCRP inhibitor treatment in BCRP (Breast Cancer Resistance Protein) related evaluations using pancreatic cancer cell line (AsPC-1). 50 It was confirmed that it is relatively less affected by intracellular drug efflux by exhibiting a low resistance rate and a low value (Experimental Example 7; Table 13).

[0125] Furthermore, MMAE acts as a payload in the class of microtubule inhibitors and serves as a major substrate for P-gp (P-glycoprotein, MDR1) transporters expressed on the surface of cancer cells. By being forcibly effluxed out of the cell, it can reduce the effective concentration and induce resistance. In P-gp (MDR1) related evaluations using the colorectal cancer cell line (HCT-15), known as a P-gp-mediated multidrug resistance model, the representative compounds of the present invention (Compounds 17-20, 23, 26, 30-32) exhibited relatively lower IC50 values ​​compared to the comparative compound MMAE, regardless of the presence or absence of P-gp inhibitor treatment. Additionally, the resistance ratios depending on the presence or absence of P-gp inhibitor treatment were generally lower or similar compared to MMAE, confirming that they are relatively less affected by intracellular drug efflux (Experimental Example 8; Table 14).

[0126] The above experimental results suggest technical significance that the compound of the present invention can be utilized as an alternative payload in cancers that have developed resistance to existing payloads by acting through a cell death mechanism different from that of conventional payloads.

[0127] immunogenicity induction potential

[0128] Meanwhile, the potential to induce immunogenic cell death (ICD) was evaluated for the representative compound 10 of the present invention. As a result, the compound of the present invention induced increased exposure to calreticulin (CRT) (Fig. 13), increased extracellular ATP release (Fig. 14), and increased HMGB1 release (Fig. 15) in cancer cells (Experimental Example 9). These indicators are known as representative DAMPs associated with ICD, suggesting that the compound of the present invention may induce immunogenic cell death rather than merely inducing cytotoxicity.

[0129] The above results demonstrate that the novel PLK1 degradation inducer of the present invention is likely to be involved in the activation of immune responses within the tumor microenvironment, going beyond being a drug with simple cytotoxicity, and suggest additional technical applications such as combination therapy with immune checkpoint inhibitors.

[0130] Through the technical effects of the present invention described above, it can be seen that the novel PLK1 degradation inducer of the present invention simultaneously secures potent cytotoxicity at a sub-nanomolar level that surpasses that of PLK1 degradation inducers of the prior art or actual ADC payload drugs, as well as plasma stability and lysosome stability that maintain an initial state without loop opening even under long-term conditions of 7 days. Furthermore, it secures resistance against conventional payload drug-resistant carcinomas and additional immunogenic apoptosis potential. Based on these characteristics, the PLK1 degradation inducer of the present invention can be usefully utilized as an ADC payload and has significant technical significance in suggesting that it can be utilized as a new ADC payload candidate to complement or replace microtubule inhibitors or topoisomerase I inhibitors, which are currently widely used in the ADC field.

[0131] 5. Medicinal uses

[0132] In one embodiment, the present invention provides an anticancer pharmaceutical composition comprising a novel PLK1 degradation inducer as an active ingredient. The cancer includes all malignant tumors in which therapeutic efficacy can be achieved by the abnormal expression, overactivation, or related signaling pathways of the PLK1 protein, or by a mechanism of inducing the degradation of the PLK1 protein. Preferably, the cancer may be one or more selected from the group consisting of small cell lung cancer, non-small cell lung cancer (NSCLC), breast cancer [e.g., triple-negative breast cancer (TNBC) or HER2-positive breast cancer], pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer, and hematological cancer [e.g., multiple myeloma (MM), acute myeloid leukemia (AML), chronic myeloid leukemia (CML)], but is not limited thereto.

[0133] In one embodiment, the cancer may be a drug-resistant cancer that exhibits refractoryness to or has acquired resistance to conventional anticancer chemotherapy, targeted anticancer drugs, or ADC therapy. Specifically, the resistant cancer may be a cancer that has cross-resistance to conventional ADC payload drugs, such as microtubule inhibitors (e.g., MMAE, DM1, DM4, etc.) or topoisomerase I inhibitors (e.g., DXd, SN-38, etc.), or a multi-drug resistant (MDR) cancer caused by the overexpression of ABC transporters including P-gp (MDR1) or BCRP (Breast Cancer Resistance Protein). Furthermore, the resistant cancer includes all post-ADC refractory cancer types that are resistant to the conventional ADC itself (e.g., DS-8201, etc.).

[0134] The pharmaceutical composition of the present invention may be administered in combination with one or more other drugs having conventional anticancer activity. In one embodiment, the combination anticancer agent may be a cytotoxic anticancer agent including a microtubule inhibitor (MMAE, etc.) or a topoisomerase I inhibitor (DXd, etc.), and may exhibit an anticancer synergistic effect through a complementary mechanism with these agents.

[0135] In addition, the compound of the present invention can be used in combination with one or more immuno-oncology drugs that activate the immune system, as it induces potent immunogenic cell death (ICD) within tumor cells to release the immunosuppressed state within the tumor microenvironment (TME). The immuno-oncology drugs may be immune checkpoint inhibitors (ICIs) including PD-1 inhibitors (e.g., pembrolizumab, nivolumab), PD-L1 inhibitors (e.g., atezolizumab), and CTLA-4 inhibitors (e.g., ipilimumab).

[0136] In one embodiment, the PLK1 degradation-inducing compound of the present invention may be used as a payload or functional drug moiety of various target delivery platforms or drug delivery platforms. The target delivery platforms may include antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), small molecule-drug conjugates (SMDCs), ligand-drug conjugates, aptamer-drug conjugates, protein-drug conjugates, polymer-drug conjugates, nanoparticle-based drug delivery systems, or combinations thereof. For example, the compound of the present invention may be organically combined and utilized as a payload of an ADC, DAC, or PDC (Peptide-drug conjugate).

[0137] The compounds of the present invention simultaneously satisfy potent sub-nanomolar-level ADC payload cytotoxicity, ring-opening inhibition, and advanced ADC payload stability under plasma / lysosome conditions through original multidimensional structural redesign. Consequently, ADC payload suitability can be fully exhibited immediately upon release by bypassing transporter-mediated efflux, even under limited exposure conditions where only minute molecules are released into target tumor cells without premature degradation during in vivo circulation. Therefore, the compounds of the present invention can be utilized as key payloads for the development of next-generation target-oriented ADC and DAC conjugate platforms.

[0138] In one embodiment, the ADC or DAC may comprise an antibody or antigen-binding fragment, the PLK1 degradation inducer of the present invention, and a linker connecting the same. The antibody or antigen-binding fragment may recognize an antigen expressed on the surface of a tumor cell, an antigen overexpressed in the tumor microenvironment, or an antigen having a cancer cell-specific or cancer cell-selective expression pattern. For example, the antigen may be, but is not limited to, EGFR, HER2, HER3, TROP2, CEACAM5, c-MET, B7-H3, Nectin-4, MUC1, MUC16, EpCAM, DLL3, CD19, CD22, CD30, CD33, CD37, CD70, CD79b, BCMA, mesothelin, PSMA, FRα, Claudin 18.2, Claudin 6, LIV-1, tissue factor, ROR1, ROR2, AXL, PD-L1, or a combination thereof. In one embodiment, the antibody may target an antigen expressed in lung cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, bladder cancer, biliary tract cancer, liver cancer, blood cancer, or other solid tumors.

[0139] The antibody or antigen-binding fragment may be a full-length antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody, Fab, Fab', F(ab')2Fv, scFv, a single-domain antibody, a nanobody, an antibody-like protein, a fusion protein having antigen-binding ability, or a variant thereof. In one embodiment, the antibody may be in the form of IgG1, IgG2, IgG3, or IgG4, and may be an antibody in which the blood half-life, effector function, Fc receptor binding ability, or conjugation efficiency is controlled through modification of the Fc region.

[0140] In one embodiment, the PLK1 degradation inducer of the present invention may be covalently conjugated to the antibody or antigen-binding fragment via a linker. The linker may be a cleavable linker or a non-cleavable linker. The cleavable linker may be cleaved by the intracellular environment of the target cell, such as lysosomal enzymes, a reducing environment, an acidic pH, or specific enzymatic activity. For example, the cleavable linker may include valine-citrulline (val-cit), valine-alanine (val-ala), phenylalanine-lysine, β-glucuronide, β-galactoside, disulfide, hydrazone, an acid-unstable linker, a peptide linker, a linker cleaved by a protease, or a combination thereof. In another embodiment, the linker may include maleimidocaproyl (MC), SMCC, thioester, alkyl, PEG, alkyl-PEG, triazole, amide, carbamate, urea, or other non-cleavable linkers.

[0141] In one embodiment, the linker may include a self-immolative spacer to allow the PLK1 degradation inducer of the present invention to be released within the cell. The self-immolative spacer may be p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a cyclization-based spacer, a 1,6-removal-based spacer, or a variant thereof. Alternatively, the linker may further include a PEG unit, a sulfonate, a sulfone, a sulfoxide, a tertiary amine, a quaternary ammonium, a carboxylate, a hydroxyl, an amino acid, a peptide, or other hydrophilic portion to adjust water solubility, plasma stability, conjugation efficiency, or drug-to-antibody ratio (DAR).

[0142] In one embodiment, the PLK1 degradation inducer of the present invention may be conjugated to a lysine residue, a cysteine ​​residue, a reduced interchain disulfide bond, an engineered cysteine, a non-natural amino acid, a glycan, an N-terminus, a C-terminus, or an enzymatically introduced reactive functional group of an antibody through an antibody conjugation linker for antibody conjugation. Exemplarily, the conjugation reaction may be carried out by a maleimide-thiol reaction, a haloacetamide-thiol reaction, click chemistry, an azide-alkyne cyclization addition reaction, a modification-promoting cyclization addition reaction, oxime ligation, hydrazone formation, a transglutaminase-mediated conjugation reaction, a sorbase-mediated conjugation reaction, a glycan remodeling-based conjugation reaction, or other bioconjugation reactions.

[0143] In one embodiment, the compound of the present invention may be provided in the form of a compound or a derivative thereof comprising a functional group conjugable to a linker for antibody conjugation. The functional group may include a primary amine, a secondary amine, a tertiary amine, a carboxyl group, a hydroxyl group, a thiol group, an azide group, an alkyne group, a cycloalkyne group, an alkene group, a tetrazine group, a trans-cyclooctene group, an aldehyde group, a ketone group, a hydrazide group, an oxyamine group, a maleimide group, a haloacetamide group, an N-hydroxysuccinimide ester group, or other bioconjugable reactive group. The functional group may be introduced into the linker site of the compound of the present invention, an E3 ligase-binding moiety, a PLK1-binding moiety, or a solvent-exposed site. In one embodiment, the functional group may be introduced at a position that does not substantially impede the PLK1 binding ability, E3 ligazeligase binding ability, ternary complex forming ability, or PLK1 degradation ability of the compound of the present invention.

[0144] In one embodiment, an ADC or DAC carrying the PLK1 degradation inducer of the present invention as a payload may contain, on average, about 1 to about 10 PLK1 degradation inducers of the present invention per antibody. For example, the DAR may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or a range thereof. In one embodiment, the DAR may be about 2 to about 8, about 2 to about 6, or about 3 to about 5. The DAR may be adjusted considering the stability, blood half-life, agglutination, tumor infiltration, payload delivery amount, and toxicity profile of the antibody.

[0145] In one embodiment, the ADC or DAC may bind to a tumor cell expressing a target antigen and then enter the cell via receptor-mediated endocytosis or other cellular entry pathways. After entering the cell, the ADC or DAC may move to an endosome or lysosome and release the PLK1 degradation inducer of the present invention through cleavage of the linker, degradation of the antibody, or chemical transformation by the intracellular environment. The released PLK1 degradation inducer may induce the proximity of the PLK1 protein and E3 ubiquitin ligase within the cancer cell and promote the ubiquitination and proteasome-mediated degradation of PLK1, thereby inhibiting cancer cell proliferation or inducing apoptosis.

[0146] In one embodiment, the PLK1 degradation inducer of the present invention may exist in the cell as a proto-compound or a derivative having pharmacological activity as a release-type payload of an ADC or DAC. In another embodiment, in the case of an ADC or DAC comprising a non-cleavable linker, it may be released in the form of a linker-payload metabolite comprising amino acid or peptide residues after antibody degradation, and such a form may retain PLK1 degradation activity or cancer cell toxicity.

[0147] In one embodiment, an ADC or DAC comprising the PLK1 degradation inducer of the present invention may provide a mechanism of action different from that of conventional cytotoxic payloads. For example, unlike microtubule inhibitors such as MMAE or DM1, topoisomerase inhibitors such as DXd or SN-38, DNA alkylating agents, DNA crosslinking agents, or other traditional cytotoxic payloads, the payload according to the present invention may exhibit antitumor activity through the removal of PLK1 protein. Thus, the ADC or DAC of the present invention may be useful for compensating for resistance to conventional ADC payloads, payload target-related resistance, drug release transporter-related resistance, or reduced efficacy due to changes in intracellular trafficking.

[0148] In one embodiment, an ADC or DAC comprising the PLK1 degradation inducer of the present invention may be administered in combination with or sequentially with a conventional payload-based ADC. For example, the ADC or DAC of the present invention may be used in combination with an ADC comprising a microtubule inhibitor payload, a topoisomerase inhibitor payload, a DNA damage payload, an immune-inducing payload, or other payloads. In another embodiment, the ADC or DAC of the present invention may be used in combination with an immune checkpoint inhibitor, an anticancer chemotherapy agent, a targeted anticancer agent, a radiotherapy agent, a PARP inhibitor, a CDK inhibitor, an EGFR inhibitor, a HER2 inhibitor, an ALK inhibitor, a KRAS inhibitor, or other anticancer therapeutic agent.

[0149] In one embodiment, an ADC or DAC comprising the PLK1 degradation inducer of the present invention may be used for cancer treatment. The cancer may be a cancer associated with increased PLK1 expression or activity, or a cancer expressing a target antigen recognized by the antibody of the ADC or DAC. For example, the cancer may be, but is not limited to, lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, triple-negative breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, liver cancer, biliary tract cancer, head and neck cancer, esophageal cancer, kidney cancer, melanoma, sarcoma, lymphoma, leukemia, multiple myeloma, or other solid or hematological cancers.

[0150] In one embodiment, the ADC or DAC of the present invention may be used to treat cancer that exhibits resistance to conventional ADC payloads, cancer that has relapsed after conventional ADC treatment, cancer with low sensitivity to topoisomerase inhibitor-based payloads, cancer with low sensitivity to microtubule inhibitor-based payloads, or cancer associated with increased expression of drug efflux transporters.

[0151] The present invention is not limited to specific antibodies, antigens, linkers, conjugation modes, DARs, release mechanisms, or cancer types. Those skilled in the art may apply the compounds of the present invention to various ADC or DAC platforms by considering the structure of the PLK1 degradation inducer of the present invention, conjugable functional groups, cleavage characteristics of the linker, antibody targets, and characteristics of the cancer type. Accordingly, the compounds of the present invention can be usefully utilized as novel payloads having a differentiated mechanism of action, specifically PLK1 protein degradation, as alternatives or complements to conventional ADC payload cytotoxic drugs.

[0152] In one embodiment, the compound of the present invention may be applied to various target delivery platforms in addition to antibody conjugates. For example, the compound of the present invention may be included in liposomes, lipid nanoparticles (LNPs), polymer nanoparticles, micelles, cell-derived vesicles, targeting peptide conjugates, or other drug delivery systems.

[0153] In addition, the compound of the present invention can be combined with various targeting technologies to enhance selective delivery to tumor tissue, and such delivery systems can be utilized to more effectively deliver the cytotoxic and PLK1 degradation activities of the compound of the present invention to specific tissues or cells.

[0154] In one embodiment, the compound of the present invention may be used for the prevention, treatment, alleviation, delay, or inhibition of progression of a disease or pathological condition related to the expression, activity, stability, or function of the PLK1 protein. The disease may be cancer, proliferative disease, metastatic disease, recurrent disease, drug-resistant disease, or a disease associated with abnormal cell proliferation. For example, the cancer may be, but is not limited to, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, colorectal cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, renal cancer, glioblastoma, osteosarcoma, leukemia, lymphoma, multiple myeloma, or a combination thereof.

[0155] The compounds of the present invention may be used alone or in combination with other anticancer agents, immunotherapies, cytotoxic drugs, molecular targeted therapies, radiation therapy, or surgical therapy. For example, they may be used in combination with immune checkpoint inhibitors, EGFR inhibitors, KRAS inhibitors, ALK inhibitors, VEGF inhibitors, PARP inhibitors, antibody therapies, or other anticancer agents, but are not limited thereto.

[0156] Furthermore, the compound of the present invention can be used to induce the degradation of PLK1 protein in vivo or in vitro, and can be applied to cells, cell cultures, organoids, tumor tissues, patient-derived samples, or other biological samples. Moreover, it can be utilized for the study of PLK1-related biological mechanisms, the establishment of disease models, drug screening, or other research purposes.

[0157] <Example>

[0158] The compounds of the specific embodiments of the present invention were synthesized according to the following manufacturing method and their structures were analyzed. The analysis conditions are described below, and specific synthesis examples, purification conditions, and structural analysis results for each compound 1 to 44 are described in the corresponding examples.

[0159] Analysis device

[0160] LCMS: Shimadzu LC-20AD & LCMS 2020, Agilent Single Quad system (1260)

[0161] NMR: BRUKER AVANCE II, BRUKER Avance Nanobay 400MHz

[0162] HPLC: Shimadzu model-LC-20AB / AD system, Agilent 1260 II LC

[0163] SFC : SHIMADZU LC-30ADsf

[0164] LCMS Analysis Method

[0165] LCMS data were recorded using the LCMS 2020 of the Shimadzu LC-20AD system equipped with an electron spray ionization device or the Agilent Single Quad system (1260). 0.04% TFA in water (solvent A) and 0.02% TFA in acetonitrile (solvent B) were used as the mobile phase. Alternatively, 0.025% NH3·H2O in water (solvent A) and acetonitrile (solvent B) were used as the mobile phase. The columns used were HALO C18 (3.0*30 mm, 5.0 µm), XPtC-C18 (2.1*30 mm, 5.0 µm), or Kinetex EVO C13 (2.1*30 mm, 5.0 µm).

[0166] HPLC analysis method

[0167] HPLC was performed using a Shimadzu model-LC-20AB / AD system or an Agilent 1260 II LC, with 0.04% TFA in water (solvent A) and 0.02% TFA in acetonitrile (solvent B) as the mobile phase. The columns used were Kinetex C18 (4.6*50 mm, 5 µm), HALO C18 (3.0*100 mm, 2.7 µm), or Unison UK C18 (4.6*150 mm, 3.0 µm).

[0168] NMR analysis method

[0169] 1 1H NMR spectra were recorded using a BRUKER AVANCE II / 5mm Probe (BBO) or a BRUKER Avance Nanobay 400MHz / 5mm Probe (BBO).

[0170] SFC analysis method

[0171] For SFC, a SHIMADZU LC-30ADsf was used, and CO2 (solvent A) and 0.05% DEA in isopropanol (solvent B) or ethanol and 0.05% DEA in ACN (solvent B) were used as the mobile phase. The columns used were Chiralpak IK-3 50*4.6 mm, 3 µm, Chiralpak AD-3 50*4.6 mm, 3 µm, or (S,S)Whelk-O1 50*4.6 mm, 3.5 µm.

[0172] Example 1. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 1)

[0173]

[0174] Step 1. Synthesis of 3-((3-methyl-4-nitrophenyl)amino)propanoic acid (3)

[0175] 3-methyl-4-nitroaniline (1.00 g, 6.57 mmol) and acrylic acid (568 mg, 7.89 mmol) were dissolved in acetic acid (1.36 mL) and water (6.78 mL) and stirred at 120 °C for 15 hours. LCMS analysis revealed a main peak with the desired mass. Saturated aqueous NaCl solution and EtOAc were added to the mixture, and the organic layer was extracted. The residue was obtained by drying with MgSO4, filtering, and concentrating under reduced pressure. The residue was purified by flash silica gel chromatography (0–25% EtOAc:n-hexane) to obtain the title compound as a yellow solid (1.21 g, 5.40 mmol, yield 82.1%). MS(M+H) + =225.05

[0176] Step 2. Synthesis of methyl 3-((3-methyl-4-nitrophenyl)amino)propanoate (4)

[0177] 3-((3-methyl-4-nitrophenyl)amino)propanoic acid (1.21 g, 5.40 mmol) was dissolved in methanol (27.0 mL). SOCl2 (0.98 mL, 13.5 mmol) was slowly added at 0°C, then the temperature was raised to 80°C and stirred for 30 minutes. LCMS analysis revealed a main peak with the desired mass. A saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The mixture was dried with MgSO4, filtered, and concentrated under reduced pressure to obtain the title compound of the yellow oil (1.10 g, 4.62 mmol, yield 85.6%). MS(M+H) + =239.05

[0178] Step 3. Synthesis of methyl 3-(1-(3-methyl-4-nitrophenyl)ureido)propanoate (5)

[0179] Methyl 3-((3-methyl-4-nitrophenyl)amino)propanoate (1.10 g, 4.62 mmol) was dissolved in acetic acid (23.1 mL). KOCN (3.00 g, 36.9 mmol) was added, the temperature was raised to 80°C, and the mixture was stirred for 6 hours. LCMS analysis revealed a main peak with the desired mass. Saturated aqueous NaCl solution and EtOAc were added to the mixture, and the organic layer was extracted. The residue was dried and filtered with MgSO4, and concentrated under reduced pressure to obtain the residual compound. The residue was purified by flash silica gel chromatography (0–80% EtOAc:n-hexane) to obtain the title compound of the yellow oil (493 mg, 1.75 mmol, yield 38.0%). MS(M+H) + =282.10

[0180] Step 4. Synthesis of 1-(3-methyl-4-nitrophenyl)dihydropyrimidine-2,4(1H,3H)-dione (6)

[0181] Methyl 3-(1-(3-methyl-4-nitrophenyl)ureido)propanoate (493 mg, 1.75 mmol) was dissolved in HCl (5.00 mL) and stirred at room temperature for 15 hours. LCMS analysis revealed a main peak with the desired mass. Saturated aqueous solution of 2N NaOH and EtOAc were added to the mixture, and the organic layer was extracted three times. The mixture was dried with MgSO4, filtered, and concentrated under reduced pressure to obtain the title compound (241 mg, 0.97 mmol, 55.2%) as a yellow solid. MS(M+H) + =250.00

[0182] Step 5. Synthesis of 1-(4-amino-3-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (7)

[0183] 1-(3-methyl-4-nitrophenyl)dihydropyrimidine-2,4(1H,3H)-dione (241 mg, 0.97 mmol) was dissolved in THF (10.0 mL) and methanol (1.00 mL), and then 10% Pd / C (103 mg, 0.01 mmol) was added and stirred for 2 hours while injecting hydrogen gas. LCMS analysis showed a main peak with the desired mass. The solution was filtered through a Celite pad and washed with methanol. The filtrate was concentrated to obtain the title compound, a red, viscous oil (195 mg, 0.89 mmol, yield 91.8%). MS(M+H) + =220.00

[0184] Step 6. Synthesis of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (9)

[0185] 1-(4-amino-3-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (50.0 mg, 0.23 mmol), benzyl ((1r,4r)-4-(4-formylpiperidin-1-yl)cyclohexyl)carbamate (86.4 mg, 0.25 mmol), and acetic acid (39.1 μL, 0.68 mmol) were added to DCM (0.95 mL) and stirred at room temperature for 2 hours. NaBH(OAc)3 (242 mg, 1.14 mmol) was added to the mixture and stirred at room temperature for 1 hour. LCMS analysis revealed a main peak with the desired mass. A saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The mixture was dried with MgSO4, filtered, and concentrated under reduced pressure to obtain the residual compound. The filtrate was dried under reduced pressure and purified by flash silica gel chromatography (0–10% MeOH:DCM) to obtain a white solid title compound (56.7 mg, 0.10 mmol, 45.4% yield). MS(M+H) + =548.20

[0186] Step 7. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-3-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (10)

[0187] Benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (56.7 mg, 0.10 mmol) was dissolved in TFA (1.54 mL, 20.7 mmol) and stirred at 40°C for 4 hours. LCMS analysis showed a main peak with the desired mass. The mixture was concentrated under reduced pressure to obtain the white solid title compound (58.0 mg, crude, 3TFA salt). MS(M+H) + =414.20

[0188] Step 8. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 1)

[0189] 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-3-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (52.3 mg, 0.13 mmol) was dissolved in DMF (0.80 mL), then DIPEA (83.0 uL, 0.43 mmol) was added and stirred for 30 minutes. (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid (40.0 mg, 0.097 mmol), HATU (73.9 mg, 0.19 mmol) and DIPEA (83.0 uL, 0.43 mmol) were dissolved in DMF (0.80 mL) and stirred for 30 minutes. A solution of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid was added to a solution of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-3-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione, and the mixture was stirred at room temperature for 4 hours. It was confirmed by LCMS that all starting materials were consumed, and the main peak of the desired mass was identified. A saturated aqueous solution of NaCl and EtOAc were added to the mixture, the organic layer was extracted, dried and filtered with MgSO4, and concentrated under reduced pressure to obtain the residual compound. The remaining compound was purified by flash silica gel chromatography (0–10% MeOH:DCM) and freeze-dried to obtain the title compound as a beige solid (5.70 mg, 6.67% yield, 91.8% purity). MS(M+H) + =807.30.

[0190] 1 ¹H NMR (400 MHz, DMSO- d 6) δ = 10.18 (s, 1H), 8.42 (d, J = 11.6 Hz, 2H), 8.21 (s, 1H), 8.07 (s, 1H), 7.90 (d, J = 3.2 Hz, 1H), 7.63 (d,J = 3.1 Hz, 1H), 7.47 (t, J = 4.7 Hz, 1H), 7.25 (s, 1H), 6.92 (s, 1H), 6.52 - 6.47 (m, 1H), 4.98 (s, 1H), 4.45 - 4.38 (m, 2H), 4.32 (d, J = 7.3 Hz, 2H), 3.94 (s, 3H), 3.24 (s, 3H), 3.00 (s, 2H), 2.69 - 2.64 (m, 4H), 2.10 - 2.05 (m, 3H), 1.98 - 1.90 (s, 5H), 1.88 - 1.81 (m, 2H), 1.80 - 1.76 (m, 3H), 1.66 - 1.57 (m, 3H), 1.52 - 1.42 (m, 3H), 1.44 - 1.36 (m, 3H), 1.35 - 1.27 (m, 3H), 1.24 (s, 3H), 1.22 - 1.17 (s, 2H).

[0191] Example 2. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methoxyphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 2)

[0192]

[0193] Step 1. Synthesis of 3-((2-methoxy-4-nitrophenyl)amino)propanoic acid (3)

[0194] Acrylic acid (4.29 g, 59.47 mmol, 4.08 mL) was added to a solution of 2-methoxy-4-nitroaniline (5 g, 29.74 mmol) in toluene (15 mL) at 25 °C, and the reaction mixture was stirred at 110 °C for 12 hours. The complete consumption of the sample material and the peak of the target mass (78%) were confirmed by LCMS. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (40 g SepaFlash® silica flash column, 5–33% EtOAc / petroleum ether gradient @ 200 mL / min) to obtain a yellow solid title compound (5.6 g, 23.31 mmol, 78.40% yield). MS(M+H) + =241.0

[0195] Step 2. Synthesis of 1-(2-methoxy-4-nitrophenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0196] Urea (2.80 g, 46.63 mmol, 2.51 mL) was added to a solution of 3-((2-methoxy-4-nitrophenyl)amino)propanoic acid (5.6 g, 23.31 mmol) in AcOH (50 mL), and the reaction mixture was stirred at 120 °C for 12 hours. After confirming the presence of residual material (53%) and the peak of the target mass (27%) by LCMS, the mixture was further stirred at 130 °C for 12 hours. Remaining material (45%) and the peak of the target mass (28%) were confirmed by LCMS. The reaction mixture was concentrated under reduced pressure. The crude product was dissolved in water (100 mL) and extracted with EtOAc (60 mL x 3). The mixed organic phase was dried with Na2SO4 and filtered. The filtrate was concentrated under reduced pressure. The residue was analyzed by flash silica gel chromatography (40 g SepaFlash® silica flash column, 5–55% EtOAc / The yellow solid title compound (568 mg, 2.14 mmol, 9.19% yield) was obtained by purification with a petroleum ether gradient @ 200 mL / min. MS(M+H) + =266.0

[0197] Step 3. Synthesis of 1-(4-amino-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione (5)

[0198] NH4Cl (572.79 mg, 10.71 mmol) and Fe (597.99 mg, 10.71 mmol) were added to a solution of 1-(2-methoxy-4-nitrophenyl)dihydropyrimidine-2,4(1H,3H)-dione (568 mg, 2.14 mmol) in EtOH (20 mL) and H2O (10 mL) at 20 °C, and the reaction mixture was stirred at 80 °C for 12 hours. The complete consumption of the sample material and the peak of the target mass (91%) were confirmed by LCMS. The reaction mixture was filtered. The filter cake was washed with EtOAc (20 mL x 3). The filtrate was concentrated under reduced pressure. The residue was subjected to flash silica gel chromatography (10 g SepaFlash® silica flash column, 33–100% EtOAc / The white solid title compound (358 mg, 1.52 mmol, 71.06% yield) was obtained by purification with a petroleum ether gradient @ 200 mL / min. MS(M+H) + =236.2

[0199] Step 4. Synthesis of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methoxyphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (7)

[0200] AcOH (137.09 mg, 2.28 mmol, 130.68 μL) and MgSO4 (366.36 mg, 3.04 mmol) were added at 25 °C to a solution of 1-(4-amino-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione (358 mg, 1.52 mmol) and benzyl ((1r,4r)-4-(4-formylpiperidin-1-yl)cyclohexyl)carbamate (524.20 mg, 1.52 mmol) in DCM (8 mL) and DMF (2 mL). The reaction mixture was stirred at 25 °C for 16 hours. NaBH(OAc)3 (967.63 mg, 4.57 mmol) was added at 25 °C, and the reaction mixture was stirred at 25 °C for 16 hours. The complete consumption of the sample material and the peak of the target mass (49%) were confirmed by LCMS. The reaction mixture was diluted with H2O (20 mL) and extracted with DCM (15 mL x 3). The mixed organic phase was washed with a saturated aqueous solution of Na2CO3 (15 mL x 3), dried with Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was analyzed by flash silica gel chromatography (10 g SepaFlash® silica flash column, 50–100% EtOAc / The solution was purified with petroleum ether (~20–20% MeOH / DCM gradient @ 200 mL / min) and re-purified with prep-HPLC (Column: Phenomenex Luna C18 150*25 mm*10 µm; Mobile phase: [H2O (0.1% TFA) - ACN]; Gradient: 5% - 35% B 15.0 min). The solution was freeze-dried to obtain the white solid title compound (124 mg, 219.98 μmol, 14.45% yield). MS(M+H) + =564.3

[0201] Step 5. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione (8)

[0202] A mixture of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methoxyphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (124 mg, 219.98 μmol) in TFA (6 mL) was stirred at 40 °C for 13 hours. The complete consumption of the sample material and the peak of the target mass (92%) were confirmed by LCMS. The reaction mixture was concentrated under reduced pressure to obtain the title compound of a yellow oil (120 mg, crude, TFA salt). MS(M+H) + =430.2

[0203] Step 6. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methoxyphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 2)

[0204] HATU (60.99 mg, 160.41 μmol) and DIPEA (226.16 mg, 1.75 mmol, 304.80 μL) were added to a solution of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid (60 mg, 145.82 μmol) in DMF (4 mL) at 25 °C. The reaction mixture was stirred at 25 °C for 10 minutes. 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione (120 mg, 220.76 μmol, TFA salt) was added at 25 °C and stirred for 1 hour at 25 °C. The complete consumption of the sample material and the peak of the target mass (65%) were confirmed by LCMS. The reaction mixture was diluted with H2O (15 mL) and extracted with EtOAc (15 mL x 3). The mixed organic phase was washed with brine (15 mL x 3), dried with Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by prep-HPLC (Column: Phenomenex Luna C18 150*25 mm*10 µm; Mobile phase: [H2O (0.1% TFA) - ACN]; Gradient: 0% - 30% B for 15.0 min). The pH of the solution was adjusted to approximately 8 using a saturated aqueous solution of Na2CO3, and extracted with DCM (20 mL x 3). The mixed organic phase was washed with deionized water (20 mL x 3) and concentrated under reduced pressure. The product was dissolved in a mixed solution (30 mL, ACN : H2O = 1:2) and freeze-hardened to obtain the white solid title compound (56.1 mg, 64.76 μmol, 44.41% yield, 95% purity). MS(M+H) + =823.6

[0205] 1 ¹H NMR (400 MHz, DMSO- d 6) δ = 10.13 (br s, 1H), 8.41 (br d,J = 8.8 Hz, 1H), 8.11 - 7.96 (m, 1H), 7.89 (s, 1H), 7.62 (s, 1H), 7.53 - 7.41 (m, 2H), 6.86 (d, J = 8.4 Hz, 1H), 6.25 (d, J = 1.6 Hz, 1H), 6.14 - 6.05 (m, 1H), 5.77 (br t, J = 5.1 Hz, 1H), 4.49 - 4.36 (m, 1H), 4.36 - 4.27 (m, 1H), 3.93 (s, 3H), 3.80 - 3.61 (m, 4H), 3.49 (br d, J = 15.4 Hz, 2H), 3.23 (s, 3H), 3.00 - 2.76 (m, 4H), 2.72 - 2.59 (m, 2H), 2.34 - 2.24 (m, 1H), 2.22 - 2.06 (m, 2H), 2.05 - 1.87 (m, 4H), 1.85 - 1.68 (m, 8H), 1.62 - 1.46 (m, 3H), 1.39 - 1.28 (m, 4H), 1.22 (br d, J = 6.6 Hz, 3H), 1.17 - 1.05 (m, 3H).

[0206] Example 3. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 3)

[0207]

[0208] Step 1. Synthesis of 3-((4-bromo-2-methylphenyl)amino)propanoic acid (3)

[0209] Acrylic acid (3.87 g, 53.75 mmol, 3.69 mL) was added to a solution of 4-bromo-2-methylaniline (5 g, 26.87 mmol) in toluene (30 mL) at 25 °C, and the reaction mixture was stirred at 110 °C for 12 hours. LCMS confirmed that the sample material was completely consumed and that the peak of the target mass (57%) was present. TLC (SiO2, petroleum ether: EtOAc = 5:1) confirmed that the sample material was completely consumed and that a new main spot had formed. The reaction mixture was concentrated under reduced pressure. The residue was analyzed by flash silica gel chromatography (40 g SepaFlash® silica flash column, 5–25% EtOAc / The title compound of the yellow oil (6.3 g, 24.41 mmol, 90.82% yield) was obtained by purification with a petroleum ether gradient @ 200 mL / min. MS(M+Na) + =281.9

[0210] Step 2. Synthesis of 1-(4-bromo-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0211] Urea (2.93 g, 48.82 mmol, 2.62 mL) was added to a solution of 3-((4-bromo-2-methylphenyl)amino)propanoic acid (6.3 g, 24.41 mmol) in AcOH (40 mL) at 25 °C, and the reaction mixture was stirred at 130 °C for 12 hours. The complete consumption of the sample material and the peak of the target mass (55%) were confirmed by LCMS. The reaction mixture was diluted with H2O (20 mL) and filtered. The filter cake was diluted with H2O (20 mL x 3). The filter cake was dried to obtain the gray solid title compound (4.5 g, 15.89 mmol, 65.12% yield). MS(M+H) + =283.0

[0212] 1 ¹H NMR (400 MHz, DMSO- d 6) δ = 10.38 (s, 1H), 7.52 (d, J = 1.6 Hz, 1H), 7.43 (dd,J = 2.2, 8.4 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 3.82 - 3.72 (m, 1H), 3.49 (td, J = 5.8, 12.0 Hz, 1H), 2.84 - 2.72 (m, 1H), 2.71 - 2.62 (m, 1H), 2.18 (s, 3H)

[0213] Step 3. Synthesis of tert-butyl (4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)carbamate (5)

[0214] XPhos Pd G3 (509.45 mg, 601.87 μmol), 4A MS (2 g), XPhos (286.92 mg, 601.86 μmol), and Cs2CO3 (5.88 g, 18.05 mmol) were added to a solution of 1-(4-bromo-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (1.5 g, 5.30 mmol) and NH2Boc (1.76 g, 15.02 mmol) in dioxane (20 mL) at 25 °C under N2, and the reaction mixture was stirred at 100 °C for 12 hours. The complete consumption of the sample material and the peak of the target mass (33%) were confirmed by LCMS. The reaction mixture was filtered. The filter cake was washed with EtOAc (20 mL x 3). The filtrate was concentrated under reduced pressure. The residue was analyzed by flash silica gel chromatography (20 g SepaFlash® silica flash column, 33–55% EtOAc / The white solid title compound (917 mg, 2.87 mmol, 54.20% yield) was obtained by purification with a petroleum ether gradient @ 200 mL / min. MS(M-56+H) + =264.0

[0215] Step 4. Synthesis of 1-(4-amino-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (6)

[0216] HCl / dioxane (2 M, 20 mL) was added to a solution of tert-butyl (4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)carbamate (917 mg, 2.87 mmol) in DCM (8 mL) at 25 °C, and the reaction mixture was stirred at 25 °C for 12 hours. The complete consumption of the sample material and the peak of the target mass (76%) were confirmed by LCMS. The reaction mixture was concentrated under reduced pressure to obtain the white solid title compound (735 mg, crude, HCl salt). MS(M+H) + =220.1

[0217] Step 5. Synthesis of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (8)

[0218] TEA (356.16 mg, 3.52 mmol, 489.90 μL) and MgSO4 (282.44 mg, 2.35 mmol) were added at 25 °C to a solution of 1-(4-amino-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (0.3 g, 1.17 mmol, HCl salt) and benzyl ((1r,4r)-4-(4-formylpiperidin-1-yl)cyclohexyl)carbamate (444.53 mg, 1.29 mmol) in DCM (10 mL) and DMF (2 mL). The reaction mixture was stirred at 25 °C for 4 hours. NaBH(OAc)3 (745.98 mg, 3.52 mmol) was added at 25 °C, and the reaction mixture was stirred at 25 °C for 12 hours. The complete consumption of the sample material and the peak of the target mass (88%) were confirmed by LCMS. The reaction mixture was diluted with H2O (20 mL) and extracted with DCM (15 mL x 3). The mixed organic phase was washed with a saturated aqueous solution of Na2CO3 (15 mL x 3), dried with Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was analyzed by flash silica gel chromatography (10 g SepaFlash® silica flash column, 50–100% EtOAc / The white solid title compound (604 mg, 1.10 mmol, 94.00% yield) was obtained by purification with petroleum ether (~ 20~20% MeOH / DCM gradient @ 200 mL / min). MS(M+H) + =548.3

[0219] Step 6. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (9)

[0220] A solution of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (604 mg, 1.10 mmol) in TFA (12 mL) was stirred at 40 °C for 13 hours. The complete consumption of the sample material and the peak of the target mass (78%) were confirmed by LCMS. The reaction mixture was concentrated under reduced pressure to obtain the title compound of a yellow oil (582 mg, crude, TFA salt). MS(M+H) + =414.2

[0221] Step 7. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 3)

[0222] HATU (304.96 mg, 802.04 μmol) and DIPEA (1.13 g, 8.75 mmol, 1.52 mL) were added to a solution of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid (0.3 g, 729.12 μmol) in DMF (12 mL) at 25 °C. The reaction mixture was stirred at 25 °C for 10 minutes. 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (582 mg, 1.10 mmol, TFA salt) was added at 25 °C, and the reaction mixture was stirred at 25 °C for 1 hour. The complete consumption of the sample material and the peak of the target mass (53%) were confirmed by LCMS. The reaction mixture was diluted with H2O (30 mL) and extracted with EtOAc (20 mL x 3). The mixed organic phase was washed with brine (20 mL x 3), dried with Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by prep-HPLC (Column: Phenomenex Luna C18 150*25 mm*10 µm; Mobile phase: [H2O (0.1% TFA) - ACN]; Gradient: 0% - 30% B for 15.0 min). The pH of the solution was adjusted to approximately 8 using a saturated aqueous solution of Na2CO3, and extracted with DCM (30 mL x 3). The mixed organic phase was washed with deionized water (30 mL x 3) and concentrated under reduced pressure. The product was dissolved in a mixed solution (40 mL, ACN:H2O = 1:3) and freeze-dried to obtain the white solid title compound (276.8 mg, 336.14 μmol, 46.10% yield, 98% purity). MS(M+H) + =807.5

[0223] 1 ¹H NMR (400 MHz, DMSO- d6) δ = 10.18 (s, 1H), 8.49 - 8.34 (m, 1H), 8.11 - 7.97 (m, 1H), 7.89 (s, 1H), 7.62 (br s, 1H), 7.55 - 7.40 (m, 2H), 6.97 - 6.80 (m, 1H), 6.52 - 6.28 (m, 2H), 5.65 (br s, 1H), 4.49 - 4.36 (m, 1H), 4.36 - 4.27 (m, 1H), 3.93 (s, 3H), 3.79 - 3.67 (m, 1H), 3.66 - 3.56 (m, 1H), 3.23 (br s, 3H), 2.93 - 2.76 (m, 4H), 2.74 - 2.61 (m, 2H), 2.27 (br d, J = 1.2 Hz, 1H), 2.21 - 2.10 (m, 2H), 2.04 (br s, 4H), 1.96 - 1.86 (m, 3H), 1.77 (br s, 8H), 1.64 - 1.55 (m, 2H), 1.53 - 1.45 (m, 1H), 1.41 - 1.28 (m, 4H), 1.22 (br d, J = 6.2 Hz, 3H), 1.19 - 1.02 (m, 3H).

[0224] Example 4. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methoxyphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 4)

[0225]

[0226] Step 1. Synthesis of 3-((4-bromo-3-methoxyphenyl)amino)propanoic acid (3)

[0227] The title compound (1.3 g, 47.9% yield) was obtained by synthesis in a manner similar to Step 1 of Example 3. MS(M+H) + =273.9.

[0228] Step 2. Synthesis of 1-(4-bromo-3-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0229] The title compound (800 mg, 56.4% yield) was obtained by synthesis in a manner similar to Step 2 of Example 3. MS(M+H) + =299.9.

[0230] Step 3. Synthesis of tert-butyl (4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methoxyphenyl)carbamate (6)

[0231] The title compound (110 mg, 49.1% yield) was obtained by synthesis in a manner similar to Step 3 of Example 3. MS(M+Na) + =358.1.

[0232] Step 4. Synthesis of 1-(4-amino-3-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (7)

[0233] The title compound (41 mg, 28.1% yield) was obtained by synthesis in a manner similar to Step 4 of Example 3. MS(M+H) + =236.0.

[0234] Step 5. Synthesis of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methoxyphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (9)

[0235] The title compound (35 mg, 30.6% yield) was obtained by synthesis in a manner similar to Step 5 of Example 3. MS(M+H) + =564.2.

[0236] Step 6. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-3-methoxyphenyl)dihydropyrimidine-2,4(1H,3H)-dione 2,2,2-trifluoroacetate (10)

[0237] The title compound (100 mg, crude) was obtained by synthesis in a manner similar to Step 6 of Example 3. MS(M+H) + =430.1.

[0238] Step 7. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methoxyphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 4)

[0239] A white solid title compound (16 mg, 15.9% yield, 99.3% purity) was obtained by synthesis similar to Step 7 of Example 3. MS(M+H) + =823.4.

[0240] 1 H NMR (400 MHz, DMSO-d6) δ= 10.20 (s, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.90 (s, 1H), 7.62 (s, 1H), 7.48 - 7.47 (m, 2H), 6.78 (s, 1H), 6.70 (d, J = 8.5 Hz, 1H), 6.49 (d, J= 8.5 Hz, 1H), 4.83 (t, J = 5.4 Hz, 1H), 4.43 (t, J = 7.8 Hz, 1H), 4.32 (q, J = 6.7 Hz, 1H), 3.94 (s, 3H), 3.77 (s, 3H), 3.67 (t, J = 6.7 Hz, 2H), 3.24 (s, 3H), 2.97 - 2.96 (m, 2H), 2.83 (d, J = 10.0 Hz, 2H), 2.68 (t, J = 6.7 Hz, 2H), 2.28 (br s, 1H), 2.16 (t, J = 10.7 Hz, 2H), 2.01 - 1.98 (m, 1H), 1.94 - 1.89 (m, 3H), 1.83 - 1.78 (m, 8H), 1.69 (d, J = 12.2 Hz, 2H), 1.62 (br s, 2H), 1.36 (s, 4H), 1.23 (d, J = 6.6 Hz, 3H), 1.14 (d, J = 9.4 Hz, 2H).

[0241] Example 5. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 5)

[0242]

[0243] A beige solid title compound (9.90 mg, 12.8%, 92.5% purity) was obtained by synthesis in a manner similar to Step 7 of Example 3. MS(M+H) + =795.40

[0244] 1 H NMR (400 MHz, DMSO-d6) δ = 10.18 (s, 1H), 8.59 (s, 1H), 8.18 (d, J = 7.7 Hz, 1H), 8.13 (t, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.67 (dd, J = 15.5, 10.7 Hz, 2H), 6.91 (d,J = 8.4 Hz, 1H), 6.47 - 6.38 (m, 2H), 5.77 (s, 1H), 4.32 (dq, J = 13.4, 7.6 Hz, 2H), 3.79 - 3.68 (m, 1H), 3.65 - 3.60 (m, 1H), 3.43 (dd, J = 12.5, 6.3 Hz, 2H), 3.23 (s, 3H), 2.93 (s, 2H), 2.68 (dt, J = 12.7, 6.3 Hz, 3H), 2.06 (s, 3H), 2.00 - 1.83 (q, J = 12.0 Hz, 8H), 1.82 - 1.64 (m, 6H), 1.59 - 1.48 (m, 4H), 1.41 (t, J = 12.1 Hz, 4H), 1.24 (d, J = 4.0 Hz, 3H), 1.22 (s, 2H).

[0245] Example 6. Synthesis of N-((1r,4R)-4-(4-(((2-chloro-4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzamide (Compound 6)

[0246]

[0247] Step 1. Synthesis of 3-((4-bromo-3-chlorophenyl)amino)propanoic acid (3)

[0248] The title compound (770 mg, 57.1% yield) was obtained by synthesis in a manner similar to Step 1 of Example 3. MS(M+H) + =277.9.

[0249] Step 2. Synthesis of 1-(4-bromo-3-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0250] The title compound (260 mg, 31.0% yield) was obtained by synthesis in a manner similar to Step 2 of Example 3. MS(M+H) + =302.9.

[0251] Step 3. Synthesis of tert-butyl(2-chloro-4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)carbamate (6)

[0252] The title compound (23 mg, 6.6% yield) was obtained by synthesis in a manner similar to Step 3 of Example 3. MS(M+Na) + =362.0.

[0253] Step 4. Synthesis of 1-(4-amino-3-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (7)

[0254] The title compound (44 mg, crude) was obtained by synthesis in a manner similar to Step 4 of Example 3. MS(M+H) + =239.9.

[0255] Step 5. Synthesis of benzyl ((1r,4r)-4-(4-(((2-chloro-4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (9)

[0256] The title compound (30 mg, 33.1% yield) was obtained by synthesis in a manner similar to Step 5 of Example 3. MS(M+H) + =568.10.

[0257] Step 6. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-3-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (10)

[0258] The title compound (43 mg, crude, TFA salt) was obtained by synthesis in a manner similar to Step 6 of Example 3. MS(M+H) + =434.1.

[0259] Step 7. Synthesis of N-((1r,4R)-4-(4-(((2-chloro-4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzamide (Compound 6)

[0260] A white solid title compound (9 mg, 10.7% yield, 95.5% purity) was obtained by synthesis in a manner similar to Step 7 of Example 3. MS(M+H) + =827.2.

[0261] 1 H NMR (400 MHz, DMSO-d6) δ= 10.27 (s, 1H), 8.41 (d, J = 8.3 Hz, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 1.4 Hz, 1H), 7.62 (s, 1H), 7.49 - 7.47 (m, 2H), 7.24 (s, 1H), 7.10 (d, J = 8.6 Hz, 1H), 6.70 (d, J = 8.2 Hz, 1H), 5.34 (t, J = 5.4 Hz, 1H), 4.45 - 4.41 (m, 1H), 4.32 (q, J= 6.7 Hz, 1H), 3.94 (s, 3H), 3.67 (t, J = 6.5 Hz, 2H), 3.24 (s, 3H), 3.07 - 3.03 (m, 2H), 2.84 (d, J = 10.0 Hz, 2H), 2.68 (t, J = 6.2 Hz, 3H), 2.27 (br s, 1H), 2.18 - 2.12 (m, 2H), 2.01 - 1.99 (m, 1H), 1.94 - 1.88 (m, 3H), 1.83 - 1.78 (m, 7H), 1.69 (d, J = 11.1 Hz, 2H), 1.61 (m, 2H), 1.36 (s, 4H), 1.23 (d, J = 5.9 Hz, 3H), 1.20 - 1.14 (m, 2H).

[0262] Example 7. Synthesis of N-((1r,4R)-4-(4-(((3-chloro-4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzamide (Compound 7)

[0263]

[0264] Step 1. Synthesis of 1-(2-chloro-4-nitrophenyl)pyrimidine-2,4(1H,3H)-dione (3)

[0265] 2-chloro-1-fluoro-4-nitrobenzene (2.00 g, 11.4 mmol), uracil (1.53 g, 13.7 mmol), and Cs2CO3 (11.1 g, 34.2 mmol) were dissolved in DMF (22.8 mL) and stirred at 110°C for 15 hours. Saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted three times. The mixture was dried and filtered with MgSO4 and concentrated under reduced pressure to obtain the residual compound. A small amount of the residual compound was dissolved in methanol, and then DCM was added to proceed with solidification. The solid was filtered to obtain the title compound as a brown solid (1.70 g, 6.35 mmol, 55.8% yield).

[0266] 1 H NMR (400 MHz, DMSO-d6) δ = 11.68 (s, 1H), 8.54 (t, J = 2.2 Hz, 1H), 8.35 (dt, J= 8.8, 2.2 Hz, 1H), 7.95 (dd, J = 8.7, 1.8 Hz, 1H), 7.68 (dd, J = 7.9, 1.8 Hz, 1H), 5.79 (dd, J = 7.9, 1.8 Hz, 1H).

[0267] Step 2. Synthesis of 1-(4-amino-2-chlorophenyl)pyrimidine-2,4(1H,3H)-dione (4)

[0268] 1-(2-chloro-4-nitrophenyl)pyrimidine-2,4(1H,3H)-dione (600.0 mg, 2.24 mmol) was dissolved in ethanol (22.4 mL), SnCl2 hydrate (1.52 g, 6.73 mmol) was added, and the mixture was stirred at 80°C for 3 hours. After neutralizing to pH 7 with 2N NaOH, the mixture was filtered with Celite using EtOAc. The residue was treated with a saturated aqueous solution of EtOAc and NaCl, and the organic layer was extracted to obtain the title compound as an orange solid (270 mg, 1.14 mmol, 50.7% yield).

[0269] 1 H NMR (400 MHz, DMSO-d6) δ = 11.39 (s, 1H), 7.51 (dd, J = 8.1, 2.5 Hz, 1H), 7.12 (dt, J = 10.7, 5.2 Hz, 1H), 6.72 (s, 1H), 6.61 - 6.43 (m, 1H), 5.69 (s, 2H), 5.64 - 5.58 (m, 1H).

[0270] Step 3. Synthesis of 1-(4-amino-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (5)

[0271] 1-(4-amino-2-chlorophenyl)pyrimidine-2,4(1H,3H)-dione (200.0 mg, 2.24 mmol) and NiCl2 hydrate (40.0 mg, 0.17 mmol) were dissolved in methanol, and NaBH4 (127 mg, 3.37 mmol) was slowly added at 0°C. The mixture was heated to 40°C and stirred for 4 hours. The mixture was filtered with a Celite filter using methanol, and the filtrate was concentrated. The remaining compound was purified by flash silica gel chromatography (0–100% EtOAc:n-hexane) to obtain a white solid title compound (55.9 mg, 0.23 mmol, 27.7% yield).

[0272] 1 H NMR (400 MHz, MeOD) δ = 7.10 (d, J = 8.5 Hz, 1H), 6.81 (s, 1H), 6.64 (dd, J = 8.5, 2.6 Hz, 1H), 3.74 - 3.65 (m, 2H), 2.83 (q, J = 6.5 Hz, 2H).

[0273] Step 4. Synthesis of benzyl ((1r,4r)-4-(4-(((3-chloro-4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (7)

[0274] 1-(4-amino-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (55.0 mg, 0.16 mmol), benzyl ((1r,4r)-4-(4-formylpiperidin-1-yl)cyclohexyl)carbamate (60.9 mg, 0.18 mmol), and acetic acid (46.0 μL, 0.80 mmol) were added to DCM (1.60 mL) and stirred at room temperature for 1 hour. NaBH(OAc)3 (170.0 mg, 0.80 mmol) was added to the mixture and stirred at room temperature for 1 hour. LCMS analysis revealed a main peak with the desired mass. A saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The mixture was dried with MgSO4, filtered, and concentrated under reduced pressure to obtain the residual compound. The filtrate was dried under reduced pressure and purified by flash silica gel chromatography (0–20% MeOH:DCM) to obtain the white solid title compound (59.3 mg, 0.10 mmol, 65.0% yield). MS(M+H) + =568.20

[0275] Step 5. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (8)

[0276] Benzyl ((1r,4r)-4-(4-(((3-chloro-4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (59.3 mg, 0.10 mmol) was dissolved in TFA (1.55 mL, 20.9 mmol) and stirred at 40°C for 11 hours and 30 minutes. LCMS analysis showed a main peak with the desired mass. The mixture was concentrated under reduced pressure to obtain the white solid title compound (62.2 mg, crude, 3TFA salt). MS(M+H) + =434.10

[0277] Step 6. Synthesis of N-((1r,4R)-4-(4-(((3-chloro-4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzamide (Compound 7)

[0278] 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (51.2 mg, 0.10 mmol) was dissolved in DMF (1.00 mL), then DIPEA (68.5 μL, 0.40 mmol) was added and stirred for 30 minutes. (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid (33.0 mg, 0.08 mmol), HATU (61.0 mg, 0.16 mmol) and DIPEA (68.5 μL, 0.40 mmol) were dissolved in DMF (1.00 mL) and stirred for 30 minutes. A solution of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid was added to a solution of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione, and the mixture was stirred at room temperature for 4 hours. It was confirmed by LCMS that all starting materials were consumed, and the main peak of the desired mass was identified. A saturated aqueous solution of NaCl and EtOAc were added to the mixture, the organic layer was extracted, dried and filtered with MgSO4, and concentrated under reduced pressure to obtain the residual compound. The residual compound was purified by flash silica gel chromatography (0–10% MeOH:DCM) and separated and purified again using prep-HPLC (Column: Agilent 10 Prep-C18 150*30 mm*10 µm; Mobile phase: [H2O (0.1% FA) - ACN (0.1% FA)]; Gradient: 0% - 95% B 50.0 min), after which the solvent was concentrated to obtain the residual compound. The residue was purified by amine column chromatography (0–10% MeOH:DCM) and freeze-dried to obtain the white solid title compound (7.20 mg, 10.5% yield, 96.5% purity). MS(M+H) + =827.20

[0279] 1 H NMR (400 MHz, DMSO-d6) δ = 10.30 (s, 1H), 8.42 (d, J = 5.9 Hz, 1H), 8.02 (d, J = 7.3 Hz, 1H), 7.90 (d, J = 3.2 Hz, 1H), 7.62 (s, 1H), 7.47 (d, J = 3.7 Hz, 2H), 7.09 (d, J = 5.6 Hz, 1H), 6.65 (s, 1H), 6.54 (d, J = 8.7 Hz, 1H), 6.10 (s, 1H), 4.48 - 4.38 (m, 1H), 4.35 - 4.27 (m, 2H), 3.94 (s, 3H), 3.77 - 3.65 (m, 2H), 3.24 (s, 3H), 2.93 - 2.79 (m, 6H), 2.72 - 2.65 (m, 2H), 2.20 - 2.12 (m, 2H), 2.00 (d, J = 9.7 Hz, 4H), 1.90 (s, 2H), 1.83 - 1.69 (m, 8H), 1.62 (s, 2H), 1.36 (s, 2H), 1.25 - 1.23 (m, 3H), 1.18 - 1.11 (m, 2H).

[0280] Example 8. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 8)

[0281]

[0282] Step 1. Synthesis of 3-((4-bromo-3-fluorophenyl)amino)propanoic acid (3)

[0283] The title compound (590 mg, 42.8% yield) was obtained by synthesis in a manner similar to Step 1 of Example 3. MS(M+H) + =261.9.

[0284] Step 2. Synthesis of 1-(4-bromo-3-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (5)

[0285] The title compound (410 mg, 63.4% yield) was obtained by synthesis in a manner similar to Step 2 of Example 3. MS(M+H) +=286.9.

[0286] Step 3. Synthesis of tert-butyl (4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-fluorophenyl)carbamate (7)

[0287] The title compound (80 mg, 17.3% yield) was obtained by synthesis in a manner similar to Step 3 of Example 3. MS(M+Na) + =346.1.

[0288] Step 4. Synthesis of 1-(4-amino-3-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (8)

[0289] The title compound (60 mg, 93.4% yield) was obtained by synthesis in a manner similar to Step 4 of Example 3. MS(M+H) + =224.1.

[0290] Step 5. Synthesis of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (10)

[0291] The title compound (50 mg, 39.2% yield) was obtained by synthesis in a manner similar to Step 5 of Example 3. MS(M+H) + =552.3.

[0292] Step 6. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-3-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione 2,2,2-trifluoroacetate (11)

[0293] The title compound (60 mg, 80.1% yield) was obtained by synthesis in a manner similar to Step 6 of Example 3. MS(M+H) + =418.2.

[0294] Step 7. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 8)

[0295] A beige solid title compound (11 mg, 18.6% yield, 89.3% purity) was obtained by synthesizing in a manner similar to Step 7 of Example 3 MS(M+H). + =811.4.

[0296] 1 H NMR (400 MHz, DMSO-d6) δ = 10.27 (s, 1H), 8.42 (d, J = 8.6 Hz, 1H), 8.09 (br s, 1H), 7.89 (s, 1H), 7.63 (s, 1H), 7.48 - 7.46 (m, 2H), 7.06 (d, J= 13.0 Hz, 1H), 6.93 (d, J = 9.1 Hz, 1H), 6.74 - 6.70 (m, 1H), 5.66 (br s, 1H), 4.43 - 4.39 (m, 1H), 4.33 (q, J = 6.3 Hz, 1H), 3.94 (s, 3H), 3.77 (s, 1H), 3.67 (t, J = 6.6 Hz, 2H), 3.24 (s, 3H), 3.03 (s, 4H), 2.70 - 2.67 (m, 3H), 2.02 - 1.91 (m, 9H), 1.83 - 1.79 (m, 5H), 1.61 (s, 3H), 1.42 - 1.40 (m, 4H), 1.23 (d, J = 6.6 Hz, 5H).

[0297] Example 9. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 9)

[0298]

[0299] Step 1. Synthesis of 3-((4-((tert-butoxycarbonyl)amino)-2-fluorophenyl)amino)propanoic acid (3)

[0300] Acrylic acid (805.84 mg, 11.18 mmol, 766.73 μL) was added to a solution of tert-butyl (4-amino-3-fluorophenyl)carbamate (2.3 g, 10.17 mmol) in toluene (25 mL) at 25 °C, and the reaction mixture was stirred at 110 °C for 12 hours. LCMS confirmed that 45% of the tert-butyl (4-amino-3-fluorophenyl)carbamate remained and a peak of the target mass (25%). Additionally, acrylic acid (366.29 mg, 5.08 mmol, 348.52 μL) was added, and the reaction mixture was further stirred at 110 °C for 12 hours. The presence of tert-butylamino-3-fluorophenyl)carbamate and the formation of three new spots were confirmed by TLC (SiO2, petroleum ether:EtOAc = 2:1). The reaction mixture was concentrated under reduced pressure. The residue was analyzed by flash silica gel chromatography (20 g SepaFlash® silica flash column, 10–40% EtOAc). / The gray solid title compound (856 mg, 2.87 mmol, 28.23% yield) was obtained by purification with a petroleum ether gradient @ 200 mL / min. MS(M+H) + =299.1

[0301] Step 2. Synthesis of N-(4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-fluorophenyl)acetamide (4)

[0302] Urea (344.66 mg, 5.74 mmol, 308.56 μL) was added to a solution of 3-((4-((tert-butoxycarbonyl)amino)-2-fluorophenyl)amino)propanoic acid (856 mg, 2.87 mmol) in AcOH (15 mL) at 25 °C, and the reaction mixture was stirred at 120 °C for 12 hours. The complete consumption of the sample material and the peak of the target mass (66%) were confirmed by LCMS. The reaction mixture was concentrated under reduced pressure. The crude product was dissolved in H2O (20 mL). The pH of the mixed aqueous layer was adjusted to approximately 10 using a saturated aqueous solution of Na2CO3, and extracted with EtOAc (10 mL x 8). The mixed organic phase was dried with Na2SO4 and filtered. The filtrate was concentrated under reduced pressure. Residues by flash silica gel chromatography (10 g SepaFlash® silica flash column, 33–100% EtOAc / The white solid title compound (242 mg, 912.38 μmol, 31.80% yield) was obtained by purification with a petroleum ether gradient @ 200 mL / min. MS(M+H) + =266.4

[0303] Step 3. Synthesis of 1-(4-amino-2-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (5)

[0304] HCl (12 M, 5 mL) was added at 25 °C to a solution of N-(4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-fluorophenyl)acetamide (242 mg, 912.38 μmol) in water (5 mL), and the reaction mixture was stirred at 60 °C for 12 hours. The complete consumption of the sample material and the peak of the target mass (73%) were confirmed by LCMS. The reaction mixture was concentrated under reduced pressure. The residue was analyzed by flash silica gel chromatography (10 g SepaFlash® silica flash column, 33–100% EtOAc / It was purified with a petroleum ether gradient @ 200 mL / min, and a white solid title compound (82 mg, 367.38 μmol, 40.27% yield) was obtained. MS(M+H) + =224.0

[0305] Step 4. Synthesis of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (7)

[0306] AcOH (33.09 mg, 551.07 μmol, 31.55 μL) and MgSO4 (88.44 mg, 734.76 μmol) were added at 25 °C to a solution of 1-(4-amino-2-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (82 mg, 367.38 μmol) and benzyl ((1r,4r)-4-(4-formylpiperidin-1-yl)cyclohexyl)carbamate (126.54 mg, 367.38 μmol) in DCM (4 mL) and DMF (1 mL). The reaction mixture was stirred at 25 °C for 16 hours. NaBH(OAc)3 (233.59 mg, 1.10 mmol) was added at 25 °C, and the reaction mixture was stirred at 25 °C for 16 hours. The complete consumption of the sample material and the peak of the target mass (12%) were confirmed by LCMS. The reaction mixture was diluted with H2O (20 mL) and extracted with DCM (15 mL x 3). The mixed organic phase was washed with a saturated aqueous solution of Na2CO3 (15 mL x 3), dried with Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was analyzed by flash silica gel chromatography (10 g SepaFlash® silica flash column, 50–100% EtOAc / The solution was purified with petroleum ether (~20–20% MeOH / DCM gradient @ 200 mL / min) and re-purified with prep-HPLC (Column: Phenomenex Luna C18 150*25 mm*10 µm; Mobile phase: [H2O (0.1% TFA) - ACN]; Gradient: 8% - 38% B 15.0 min). The solution was freeze-dried to obtain the white solid title compound (21 mg, 38.07 μmol, 10.36% yield). MS(M+H) + =552.3

[0307] Step 5. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (8)

[0308] A mixture of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (21 mg, 38.07 μmol) in TFA (4 mL) was stirred at 40 °C for 13 hours. The complete consumption of the sample material and the peak of the target mass (90%) were confirmed by LCMS. The reaction mixture was concentrated under reduced pressure to obtain the title compound of a yellow oil (21 mg, crude, TFA salt). MS(M+H) + =418.2

[0309] Step 6. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 9)

[0310] HATU (10.17 mg, 26.73 μmol) and DIPEA (37.69 mg, 291.65 μmol, 50.80 μL) were added to a solution of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid (10 mg, 24.30 μmol) in DMF (1 mL) at 25 °C. The reaction mixture was stirred at 25 °C for 10 minutes. 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (19.38 mg, 36.46 μmol, TFA salt) was added at 25 °C, and the reaction mixture was further stirred at 25 °C for 1 hour. The complete consumption of the sample material and the peak of the target mass (44%) were confirmed by LCMS. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL x 3). The mixed organic phase was washed with brine (10 mL x 3), dried with Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by prep-HPLC (Column: Phenomenex Luna C18 150*25 mm*10 µm; Mobile phase: [H2O (0.1% TFA) - ACN]; B%: 3%, isolytic elution mode). The pH of the solution was adjusted to approximately 8 using a saturated aqueous solution of Na2CO3, and extracted with DCM (10 mL x 3). The mixed organic phase was washed with deionized water (10 mL x 3). The mixed organic phase was concentrated under reduced pressure. The product was dissolved in a mixed solution (20 mL, ACN:H2O=1:1) and freeze-dried to obtain the white solid title compound (5.5 mg, 5.83 μmol, 24.00% yield, 86% purity). MS(M+H) + =811.5

[0311] 1 H NMR (400 MHz, DMSO-d6) δ = 10.29 (br s, 1H), 8.41 (d, J= 8.9 Hz, 1H), 8.03 (br d, J = 7.9 Hz, 1H), 7.89 (s, 1H), 7.62 (s, 1H), 7.50 - 7.44 (m, 2H), 7.21 (br t, J = 7.6 Hz, 1H), 7.08 - 6.99 (m, 1H), 6.38 (br d, J = 11.0 Hz, 2H), 6.08 (br t, J = 5.2 Hz, 1H), 4.47 - 4.37 (m, 1H), 4.31 (q, J = 6.7 Hz, 1H), 3.93 (s, 3H), 3.77 - 3.66 (m, 1H), 3.23 (s, 3H), 2.93 - 2.76 (m, 4H), 2.30 - 2.23 (m, 1H), 2.16 (br t, J = 11.0 Hz, 2H), 2.07 - 1.95 (m, 2H), 1.88 (br s, 3H), 1.82 - 1.70 (m, 8H), 1.61 (br d, J = 3.9 Hz, 2H), 1.52 - 1.43 (m, 2H), 1.39 - 1.32 (m, 4H), 1.22 (br d, J = 6.6 Hz, 3H), 1.14 - 1.07 (m, 3H).

[0312] Example 10. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 10)

[0313]

[0314] Step 1. Synthesis of tert-butyl 4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-carboxylate (3)

[0315] 1-(4-bromo-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (400 mg, 1.41 mmol), tert-butyl 4-(aminomethyl)-4-fluoropiperidine-1-carboxylate (427 mg, 1.84 mmol, 1.4 eq), Gphos Pd G6 (133 mg, 0.141 mmol, 0.1 eq), and Cs2CO3 (1.38 g, 4.24 mmol, 3.0 eq) were dissolved in dioxane (4 mL). The temperature was set to 100 oThe temperature was raised to C and stirred for 3 hours. The solution was filtered via Celite and concentrated under reduced pressure. The residue was purified by silica column chromatography (0–10% MeOH:DCM) and dried to obtain the title compound (800 mg, 18% yield). MS(M-100+H) + =335.1.

[0316] Step 2. Synthesis of 1-(4-(((4-fluoropiperidin-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (4)

[0317] tert-butyl 4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-carboxylate (320 mg, 0.736 mmol) was dissolved in DCM (6 mL). 4N HCl / dioxane (3.7 mL, 14.7 mmol, 20 eq) was added, and the mixture was stirred at room temperature for 1 hour. After concentration under reduced pressure, the solution was filtered through DCM to collect the solid, which was then dried to obtain the title compound (190 mg, 70% yield). MS(M+H) + =335.2.

[0318] Step 3. Synthesis of tert-butyl (4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)carbamate (6)

[0319] 1-(4-(((4-fluoropiperidin-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (140 mg, 0.344 mmol), tert-butyl (4-iodocyclohexyl)carbamate (559 mg, 1.72 mmol, 5 eq), and Cs2CO3 (1.12 g, 3.44 mmol, 10 eq) were dissolved in DMF. The temperature was set to 100 o The temperature was raised to C and stirred for 17 hours. Water (20 mL) and EtOAc (50 mL) were added, and the organic layer was extracted, dried with MgSO4, and concentrated under reduced pressure. The residue was purified by silica column chromatography (0–10% MeOH:DCM) and dried to obtain the title compound (800 mg, 18% yield). MS(M+H) + =532.30.

[0320] Step 4. Synthesis of 1-(4-(((1-(4-aminocyclohexyl)-4-fluoropiperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (7)

[0321] tert-butyl (4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)carbamate (10 mg, 0.019 mmol) was dissolved in DCM (1 mL). After adding 4N HCl / dioxane (0.094 ml, 0.376 mmol, 20 eq), the mixture was stirred at room temperature for 30 minutes. After confirming the reaction by LCMS, the solution was concentrated under reduced pressure to obtain the title compound (8 mg, 90% yield). MS(M+H) + =432.2.

[0322] Step 5. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 10)

[0323] (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid (10 mg, 0.0243 mmol), 1-(4-(((1-(4-aminocyclohexyl)-4-fluoropiperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (11.4 mg, 0.0243 mmol), and HATU (11.1 mg, 0.0292 mmol, 1.2 eq) were dissolved in DMF (1 mL). After adding DIPEA (0.0125 mL, 0.0729 mmol, 3.0 eq), the mixture was stirred at room temperature for 13 hours. After confirming the reaction by LCMS, the residual compound was obtained by separating and purifying using prep-HPLC (Column: Agilent 10 Prep-C18 150*30 mm*10 µm; Mobile phase: [H2O (0.1% FA) - ACN (0.1% FA)]; Gradient: 0% - 95% B 42.0 min), followed by solvent concentration. The residue was purified by amine column chromatography (0–5% MeOH:DCM) and freeze-dried to obtain the white solid title compound (1.5 mg, 7.5% yield, 98.41% purity). MS(M+H) + =825.3.

[0324] 1H NMR (400 MHz, DMSO-d6) δ = 10.18 (s, 1H), 8.41 (d, J = 8.9 Hz, 1H), 8.03 (d, J = 7.9 Hz, 0.7H), 7.97 (d, J = 8.9 Hz, 0.3H), 7.90 (s, 1H), 7.62 (s, 1H), 7.50 - 7.47(m, 2H), 6.90 (d, J = 8.4 Hz, 1H), 6.53 - 6.50 (m, 2H), 5.77 - 5.70 (m, 1H), 4.43 (t, J = 8.0 Hz, 1H), 4.32 (q, J = 6.7 Hz, 1H), 3.94 (s, 3H), 3.72(br s, 1H), 3.66 - 3.59 (m, 1H), 3.24 (s, 3H), 2.81 (d, J = 10.1 Hz, 1H), 2.71 - 2.65 (m, 5H), 2.44 - 2.41 (m, 1H), 2.33 (s, 1H), 2.25 - 2.23 (m, 1H), 2.05 (s, 3H), 1.94 - 1.91 (m, 3H), 1.82 - 1.78 (m, 11H), 1.62 (s, 3H), 1.56 - 1.46 (m, 2H), 1.40 - 1.36 (m, 2H), 1.23 (d, J = 6.6 Hz, 3H).

[0325] Example 11. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-hydroxypiperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 11)

[0326]

[0327] Step 1. Synthesis of benzyl (4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-hydroxypiperidin-1-yl)cyclohexyl)carbamate (3)

[0328] 1-(4-(((4-hydroxypiperidin-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (45.0 mg, 0.135 mmol) and benzyl (4-oxocyclohexyl)carbamate (40.2 mg, 0.162 mmol) were dissolved in DMAC (1.32 mL), followed by the addition of acetic acid (31 μL, 0.542 mmol) and stirring at room temperature for 1 hour. NaBH3CN (25.5 mg, 0.406 mmol) was added to the reaction mixture and stirred at room temperature for 4 hours. The consumption of all starting materials was confirmed by LCMS, and the main peak at the desired mass was identified. The mixture was filtered through a Pasteur pipette lined with cotton and washed with ACN (1 mL). After separation and purification of the filtrate using prep-HPLC (Column: Agilent 10 Prep-C18 150*30 mm*10 µm; Mobile phase: [H2O (0.1% FA) - ACN (0.1% FA)]; Gradient: 0% - 95% B 42.0 min), the solvent was concentrated and dried to obtain the title compound, a colorless, viscous oil (29.3 mg, 0.052 mmol, 38.5% yield). MS(M+H) + =564.20

[0329] Step 2. Synthesis of 1-(4-(((1-(4-aminocyclohexyl)-4-hydroxypiperidin-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0330] The title compound as an ivory solid (68.2 mg, 171.8% yield, 3TFA salt, crude) was synthesized in a manner similar to Step 7 of Example 1 to obtain MS(M+H) + =430.15

[0331] Step 3. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-hydroxypiperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 11)

[0332] A white solid title compound (12.0 mg, 32.3% yield, 97.0% purity) was obtained by synthesis in a manner similar to Step 8 of Example 1. MS(M+H) + =823.25

[0333] 1H NMR (400 MHz,DMSO-d6) δ = 10.17 (s, 1H), 8.40 (d, J = 8.3 Hz, 1H), 8.04 - 7.95 (m, 1H), 7.89 (s, 1H), 7.61 (s, 1H), 7.53 - 7.44 (m, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.51 - 6.43 (m, 2H), 5.40 - 5.27 (m, 1H), 4.45 - 4.39 (m, 1H), 4.31 (q, J = 6.8 Hz, 1H), 4.26 - 4.20 (m, 1H), 3.93 (s, 3H), 3.91 - 3.87 (m, 1H), 3.76 - 3.69 (m, 1H), 3.64 - 3.57 (m, 2H), 3.23 (s, 3H), 2.99 - 2.93 (m, 2H), 2.71 - 2.62 (m, 3H), 2.36 - 2.24 (m, 2H), 2.20 - 2.15 (m, 1H), 2.05 (s, 3H), 2.02 - 1.98 (m, 1H), 1.95 - 1.85 (m, 3H), 1.83 - 1.71 (m, 6H), 1.66 - 1.57 (m, 3H), 1.56 - 1.41 (m, 6H), 1.39 - 1.32 (m, 1H), 1.23 (d, J = 6.6 Hz, 3H).

[0334] Example 12. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-[1,4'-bipiperidin]-1'-yl)-3-methoxybenzamide (Compound 12)

[0335]

[0336] Step 1. Synthesis of tert-butyl 4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-carboxylate (3)

[0337] 1-(4-bromo-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (500 mg, 1.77 mmol), tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (757 mg, 3.53 mmol), tBu Xphos Pd G3 (140 mg, 0.177 mmol), and tBuOK (595 mg, 5.30 mmol) were dissolved in 1,4-dioxane (3.28 mL) and stirred at 80°C for 4 hours. The mixture was filtered using Celite and washed with EtOAc. Water was added to the filtrate and mixed, after which the organic layer was extracted. The mixture was dried and filtered with MgSO4 and concentrated under reduced pressure to obtain the residual compounds. The residue was purified by flash silica gel chromatography (0–60% EtOAc:n-hexane) to obtain the yellow solid title compound (455 mg, 1.09 mmol, 61.9% yield). MS(M-Boc+H) + =317.10

[0338] Step 2. Synthesis of 1-(2-methyl-4-((piperidine-4-ylmethyl)amino)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0339] tert-butyl 4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-carboxylate (455 mg, 1.09 mmol) was dissolved in DCM (10.3 mL), 4 M HCl / dioxane (8.19 mL, 32.8 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated, methanol (1 mL) was added to the resulting solid, and the mixture was filtered and dried to obtain the title compound of the beige solid (293 mg, 0.830 mmol, 76.0% yield, 2 HCl salt). MS(M+H) + =317.10

[0340] Step 3. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-[1,4'-bipiperidine]-1'-yl)-3-methoxybenzamide (Compound 12)

[0341] 1-(2-methyl-4-((piperidin-4-ylmethyl)amino)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (73.0 mg, 0.207 mmol, 2HCl salt) was dissolved in DMAC (1.0 mL), then TEA (0.192 mL, 1.38 mmol) was added and stirred at room temperature for 30 minutes. (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxy-N-(4-oxopiperidine-1-yl)benzamide (70 mg, 0.138 mmol) and acetic acid (0.118 mL, 2.07 mmol) were added to the reaction mixture and stirred at room temperature for 3 hours. NaBH3CN (43.3 mg, 0.690 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 17 hours. LCMS confirmed that all starting materials had been consumed, and the main peak at the desired mass was identified. The mixture was filtered through Celite, and the residue was obtained by separating and purifying it using prep-HPLC (Column: Agilent 10 Prep-C18 150*30 mm*10 µm; Mobile phase: [H2O (0.1% FA) - ACN (0.1% FA)]; Gradient: 0% - 95% B for 50.0 min). The solvent was then concentrated to obtain the residue. The residue was purified by amine column chromatography (0–10% MeOH:DCM) and freeze-dried to obtain the white solid title compound (9.80 mg, 8.44% yield, 95.98% purity). MS(M+H) + =808.40

[0342] 1 H NMR (400 MHz, DMSO-d6) δ = 10.17 (s, 1H), 9.26 (s, 1H) 8.42 (d, J = 8.9 Hz, 1H), 7.89 (s, 1H), 7.63 (s, 1H), 7.41 - 7.40 (m, 2H), 6.90 (d, J= 8.4 Hz, 1H), 6.42 - 6.38 (m, 2H), 5.66 (br s, 1H), 4.46 - 4.38 (m, 1H), 4.32 (q, J = 6.7 Hz, 1H), 3.93 (s, 3H), 3.75 - 3.57 (m, 2H), 3.24 (s, 3H), 3.04 (d, J = 10.1 Hz, 2H), 2.96 - 2.94 (m, 2H), 2.89 (d, J = 5.5 Hz, 2H), 2.81 - 2.75 (m, 2H), 2.71 - 2.65 (m, 2H), 2.37 - 2.31 (m, 1H), 2.22 - 2.16 (m, 2H), 2.05 (s, 3H), 2.02 - 2.01 (m, 1H), 1.94 - 1.92 (m, 1H), 1.78 - 1.77 (m, 8H), 1.64 - 1.60 (m, 5H), 1.23 (d, J = 6.7 Hz, 4H), 1.05 (t, J = 7.21 Hz, 1H)

[0343] Example 13. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoro-[1,4'-bipiperidine]-1'-yl)-3-methoxybenzamide (Compound 13)

[0344]

[0345] Step 1. Synthesis of tert-butyl 4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-carboxylate (3)

[0346] The title compound (320 mg, 52.1% yield) was obtained by synthesis in a manner similar to Step 1 of Example 10. MS(M+Na) + =457.10.

[0347] Step 2. Synthesis of 1-(4-(((4-fluoropiperidin-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (4)

[0348] The title compound (190 mg, 69.6% yield) was obtained by synthesis in a manner similar to Step 2 of Example 10. MS(M+H) + =335.2.

[0349] Step 3. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoro-[1,4'-bipiperidine]-1'-yl)-3-methoxybenzamide (Compound 13)

[0350] A white solid title compound (11 mg, 15.5% yield, 96.2% purity) was obtained by synthesis in a manner similar to Step 3 of Example 12. MS(M+H) + =826.4.

[0351] 1 H NMR (400 MHz, DMSO-d6) δ= 10.18 (s, 1H), 9.25 (s, 1H), 8.42 (d, J = 8.4 Hz, 1H), 7.89 (s, 1H), 7.62 (s, 1H), 7.41 - 7.40 (m, 2H), 6.9 (d, J = 7.9 Hz, 1H), 6.54 - 6.50 (m, 2H), 5.74 - 5.71 (m, 1H), 4.46 - 4.40 (m, 1H), 4.32 (q, J = 6.7 Hz, 1H), 3.94 (s, 3H), 3.63 - 3.59 (m, 2H), 3.44 - 3.43 (m, 1H), 3.24 (s, 3H), 3.04 (d, J = 10.1 Hz, 2H), 2.82 - 2.77 (m, 2H), 2.71 - 2.68 (m, 2H), 2.67 - 2.61 (m, 2H), 2.39 (t, J = 10.4 Hz, 2H), 2.33 - 2.29 (m, 2H), 2.05 (s, 3H), 1.94 - 1.92 (m, 1H), 1.83 - 1.74 (m, 10H), 1.61 (s, 5H), 1.24 (d, J = 6.5 Hz, 3H).

[0352] Example 14. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-hydroxy-[1,4'-bipiperidin]-1'-yl)-3-methoxybenzamide (Compound 14)

[0353]

[0354] Step 1. Synthesis of tert-butyl 4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-hydroxypiperidine-1-carboxylate (3)

[0355] A pale yellow, sticky solid title compound (60.5 mg, 53.3% yield) was obtained by synthesis in a manner similar to Step 1 of Example 10. MS(M+Na) +=455.10

[0356] Step 2. Synthesis of 1-(4-(((4-hydroxypiperidin-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0357] A white solid title compound (41.1 mg, HCl salt, crude) was obtained by synthesis in a manner similar to Step 2 of Example 10. MS(M+H) + =333.20

[0358] Step 3. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-hydroxy-[1,4'-bipiperidin]-1'-yl)-3-methoxybenzamide (Compound 14)

[0359] A white solid title compound (4.4 mg, 9.68% yield, 90.6% purity) was obtained by synthesis in a manner similar to Step 3 of Example 12. MS(M+H) + =824.40

[0360] 1 H NMR (400 MHz, DMSO-d6) δ = 10.17 (s, 1H), 9.23 (s, 1H), 8.41 (d, J = 8.8 Hz, 1H), 7.89 (s, 1H), 7.62 (s, 1H), 7.43 - 7.38 (m, 2H), 6.89 (d, J = 8.3 Hz, 1H), 6.47 (d, J = 10.5 Hz, 2H), 5.34 - 5.29 (m, 1H), 4.46 - 4.39 (m, 1H), 4.34 - 4.28 (m, 1H), 4.24 (s, 1H), 3.93 (s, 3H), 3.64 - 3.59 (m, 1H), 3.23 (s, 3H), 3.05 - 3.00 (m, 2H), 2.95 (d, J = 5.9 Hz, 2H), 2.82 - 2.75 (m, 3H), 2.69 - 2.45 (m, 2H), 2.26 - 2.22 (m, 1H), 2.05 (s, 3H), 2.02 - 1.95 (m, 1H), 1.94 - 1.88 (m, 1H), 1.83 - 1.72 (m, 7H), 1.65 - 1.56 (m, 8H), 1.54 (s, 3H), 1.23 (d, J= 6.7 Hz, 3H).

[0361] Example 15. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-2-methoxybenzamide (Compound 15)

[0362]

[0363] A beige solid title compound (5.20 mg, 6.58%, 89.3% purity) was obtained by synthesis similar to Step 7 of Example 3. MS(M+H) + =807.40

[0364] 1 H NMR (400 MHz, DMSO-d6) δ = 10.18 (s, 1H), 9.36 (s, 1H), 7.91 (s, 1H), 7.78 - 7.73 (m, 1H), 7.72 - 7.68 (m, 1H), 7.65 (s, 1H), 7.43 (d, J = 8.9 Hz, 1H), 7.35 (s, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.44 (s, 1H), 5.75 (s, 1H), 4.62 - 4.50 (m, 1H), 4.32 (d, J = 7.4 Hz, 1H), 3.88 (s, 3H), 3.71 (s, 1H), 3.61 (s, 1H), 3.44 (d, J = 13.1 Hz, 2H), 3.24 (s, 3H), 2.95 - 2.85 (m, 2H), 2.68 (s, 3H), 2.06 (s, 3H), 2.02 - 1.87 (m, 8H), 1.81 - 1.65 (m, 6H), 1.63 - 1.50 (m, 4H), 1.41 (d, J = 39.0 Hz, 4H), 1.24 (s, 3H), 1.22 - 1.19 (m, 2H).

[0365] Example 16. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(6-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)spiro[3,3]heptane-2-yl)-3-methoxybenzamide (Compound 16)

[0366]

[0367] Step 1. Synthesis of tert-butyl 4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-carboxylate (3)

[0368] The title compound (455 mg, 61.9% yield) was obtained by synthesis in a manner similar to Step 1 of Example 12. MS(M+Na) + =439.10

[0369] Step 2. Synthesis of 1-(2-methyl-4-((piperidine-4-ylmethyl)amino)phenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (4)

[0370] The title compound (293 mg, crude) was obtained by synthesis in a manner similar to Step 2 of Example 12. MS(M+H) + =317.10

[0371] Step 3. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(6-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)spiro[3,3]heptane-2-yl)-3-methoxybenzamide (Compound 16)

[0372] A white solid title compound (6.50 mg, 7.02% yield, 87.1% purity) was obtained by synthesis in a manner similar to Step 3 of Example 12. MS(M+H) + =819.40

[0373] 1 H NMR (400 MHz, DMSO-d6) δ = 10.16 (s, 1H), 8.40 (d, J = 8.88 Hz, 1H), 8.35 (d, J = 7.52 Hz, 1H), 7.89 (s, 1H), 7.61 (s, 1H), 7.47 - 7.45 (m, 2H), 6.88 (d, J = 8.32 Hz, 1H), 6.40 - 6.36 (m, 2H), 5.64 (t, J = 5.32 Hz, 1H), 4.44 - 4.38 (m, 1H), 4.35 - 4.29 (m, 2H), 3.93 (s, 3H), 3.63 - 3.57 (m, 1H), 3.46 - 3.40 (m, 2H), 3.23 (s, 3H), 2.86 (t, J= 5.90 Hz, 2H), 2.78 - 2.73 (m, 3H), 2.69 - 2.61 (m, 3H), 2.35 - 2.32 (m, 2H), 2.23 - 2.17 (m, 2H), 2.04 - 2.00 (m, 5H), 1.93 - 1.90 (m, 1H), 1.83 - 1.70 (m, 7H), 1.61 (brs, 4H), 1.49 (brs, 1H), 1.23 - 1.21 (m, 3H), 1.17 - 1.11 (m, 2H)

[0374] Example 17. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 17)

[0375]

[0376] Step 1. Synthesis of benzyl ((1r,4r)-4-(4-fluoro-4-(hydroxymethyl)piperidine-1-yl)cyclohexyl)carbamate (3)

[0377] Benzyl (4-oxocyclohexyl)carbamate (10 g, 40.4 mmol) and (4-fluoropiperidin-4-yl)methanol hydrochloride (7.55 g, 44.5 mmol) were dissolved in DCM (50 mL) and DMF (50 mL). After adding TEA (22.5 mL, 162 mmol), the mixture was stirred at room temperature for 1 hour. After adding acetic acid (18.5 mL, 324 mmol), the temperature was raised to 60 o The temperature was raised to C and stirred for 1 hour. After lowering the temperature to room temperature, STAB (25.7 g, 121 mmol) was added, and the mixture was stirred at room temperature for 16 hours. A saturated aqueous solution of NaHCO3 and EtOAc were added, and the organic layer was extracted. The mixture was dried and filtered with MgSO4, and concentrated under reduced pressure. The remaining compound was purified by silica gel chromatography (0–20% MeOH, DCM) to obtain a solid compound. ACN was added to the solid compound and heated to completely dissolve the solid; then, the temperature was slowly lowered to room temperature while stirring to induce solidification. The solid was filtered and dried to obtain a white solid title compound (1.20 g, 3.29 mmol, 8.14% yield).

[0378] 1H NMR (400 MHz, DMSO-d6) δ = 7.39 - 7.29 (m, 5H), 7.16 (d, J = 7.8 Hz, 1H), 4.99 (s, 2H), 4.87 (t, J = 5.7 Hz, 1H), 3.39 (d, J = 5.44 Hz, 1H), 3.26 - 3.17 (m, 1H), 2.61 - 2.58 (m, 2H), 2.42 - 2.37 (m, 2H), 2.26 - 2.20 (m, 1H), 1.85 - 1.82 (m, 2H), 1.76 - 1.58 (m, 6H), 1.57 - 1.48 (m, 1H), 1.33 - 1.13 (m, 4H).

[0379] Step 2. Synthesis of benzyl ((1r,4r)-4-(4-fluoro-4-formylpiperidin-1-yl)cyclohexyl)carbamate (4)

[0380] Benzyl ((1r,4r)-4-(4-fluoro-4-(hydroxymethyl)piperidine-1-yl)cyclohexyl)carbamate (180 mg, 0.494 mmol) and DMP (0.314 g, 0.741 mmol) were dissolved in DCM (3 mL). The mixture was stirred at room temperature for 20 hours. A saturated aqueous solution of NaHCO3 and EtOAc were added, and the organic layer was extracted. The remaining compounds were concentrated by drying with MgSO4 and filtration under reduced pressure. The title compound (220 mg, crude) was obtained. MS(M+H+MeOH) + =395.20

[0381] Step 3. Synthesis of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)carbamate (6)

[0382] Benzyl ((1r,4r)-4-(4-fluoro-4-formylpiperidin-1-yl)cyclohexyl)carbamate (170 mg, 0.469 mmol) and 1-(4-amino-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (132 mg, 0.516 mmol) were dissolved in DCM (3 mL) and DMF (1 mL). TEA (0.13 mL, 0.938 mmol) was added and stirred at room temperature for 20 minutes. Acetic acid (0.134 mL, 2.35 mmol) was added and stirred at room temperature for 1 hour. STAB (398 mg, 1.88 mmol) was added and stirred at room temperature for 13 hours. A saturated aqueous solution of NaHCO3 and EtOAc were added, and the organic layer was extracted. The residual compound was concentrated by drying with MgSO4 and filtration under reduced pressure. The residual compound was purified by silica gel chromatography (0–20% MeOH, DCM) to obtain the title compound (50 mg, 0.084 mmol, 18% yield). MS(M+H) + =566.30

[0383] Step 4. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)-4-fluoropiperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (7)

[0384] Benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidin-1-yl)cyclohexyl)carbamate (50 mg, 0.0884 mmol) was dissolved in THF (2 mL). After adding Pd / C (94 mg, 0.088 mmol), the solution was switched with a H2 (g) balloon and stirred at room temperature for 1 hour. The solution was filtered using Celite, and the filtrate was concentrated under reduced pressure to obtain the title compound (24 mg, 63% yield). MS(M+H) + =432.20.

[0385] Step 5. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 17)

[0386] (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-fluorobenzoic acid (20 mg, 0.050 mmol), 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)-4-fluoropiperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (22 mg, 0.050 mol), and HATU (23 mg, 0.060 mmol) were dissolved in DMF (2 mL), then DIPEA (26 μL, 0.150 mmol) was added and stirred at room temperature for 16 hours. The title compound (4 mg, 91% purity, 9% yield) was obtained by purification using silica gel chromatography (0–20% MeOH, DCM). MS(M+H) + =813.32

[0387] 1 H NMR (400 MHz, DMSO-d6) δ = 10.18 (s, 1H), 8.57 (s, 1H), 8.12 (t, J = 8.3 Hz, 2H), 7.86 (s, 1H), 7.72 - 7.61 (m, 2H), 6.90 (d, J = 8.4 Hz, 1H), 6.56 - 6.47 (m, 2H), 5.72 (t, J = 6.3 Hz, 1H), 4.40 - 4.27 (m, 2H), 3.71 (s, 1H), 3.66 - 3.57 (m, 1H), 3.44 (dt, J = 12.3, 6.2 Hz, 1H), 3.23 (s, 3H), 3.20 - 3.19 (m, 1H), 2.68 (q, J = 7.9 Hz, 4H), 2.42 (d, J = 10.9 Hz, 2H), 2.33 - 2.32 (m, 1H), 2.05 (s, 3H), 1.80 (m, 14H), 1.57 - 1.46 (m, 3H), 1.36 (t, J = 9.2 Hz, 4H), 1.22 (d, J = 6.8 Hz, 3H).

[0388] Example 18. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 18)

[0389]

[0390] Step 1. Synthesis of benzyl ((1r,4r)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)carbamate (2)

[0391] A white solid title compound (25 mg, 0.044 mmol, 12.6% yield) was obtained by synthesis in a manner similar to Step 3 of Example 17. MS(M+H) + = 566.30

[0392] Step 2. Synthesis of 1-(4-(((1-((1r,4r)-4-aminocyclohexyl)-4-fluoropiperidine-4-yl)methyl)amino)-3-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (3)

[0393] The white solid title compound (17 mg, 0.039 mmol, 89.1% yield) was synthesized in a manner similar to Step 4 of Example 17. MS(M+H) + = 432.20

[0394] Step 3. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 18)

[0395] A white solid title compound (4.4 mg, 13.5% yield, 88.95% purity) was obtained by synthesis similar to Step 5 of Example 17. MS(M+H) + = 813.14

[0396] 1 H NMR (400 MHz, DMSO) δ = 10.18 (s, 1H), 8.58 (s, 1H), 8.12 (t, J = 8.3 Hz, 2H), 7.86 (s, 1H), 7.72 - 7.62 (m, 2H), 6.92 (d, J = 4.0 Hz, 2H), 6.67 (d, J = 9.0 Hz, 1H), 6.52 (s, 1H), 4.38 - 4.25 (m, 2H), 3.70 (s, 1H), 3.64 (t, J = 6.7 Hz, 2H), 3.22 (s, 3H), 2.66 (t, J= 6.6 Hz, 3H), 2.10 (s, 3H), 2.00 - 1.84 (m, 7H), 1.82 - 1.62 (m, 7H), 1.56 - 1.46 (m, 3H), 1.37 (s, 3H), 1.27 - 1.17 (m, 8H), 0.84 (t, J = 6.4 Hz, 1H).

[0397] Example 19. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)benzamide (Compound 19).

[0398]

[0399] A white solid title compound (9.5 mg, yield 25.1%, purity 93.98%) was obtained by synthesis in a manner similar to Step 5 of Example 17. MS(M+H) + = 795.40

[0400] 1 H NMR (400 MHz, DMSO) δ = 10.18 (s, 1H), 9.33 (s, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.89 (s, 1H), 7.81 - 7.70 (m, 4H), 6.91 (dq, J = 4.4, 2.6 Hz, 2H), 6.68 - 6.62 (m, 1H), 6.52 (t, J = 6.4 Hz, 1H), 4.49 (p, J = 8.5 Hz, 1H), 4.31 (q, J = 6.7 Hz, 1H), 3.70 (s, 1H), 3.64 (t, J = 6.7 Hz, 2H), 3.28 (s, 1H), 3.23 (s, 3H), 2.66 (t, J = 6.7 Hz, 3H), 2.43 (t, J = 11.0 Hz, 2H), 2.30 (s, 2H), 2.10 (s, 3H), 2.03 (s, 1H), 1.98 - 1.92 (m, 1H), 1.88 (s, 2H), 1.83 - 1.70 (m, 8H), 1.68 - 1.51 (m, 4H), 1.35 (t, J= 9.2 Hz, 4H), 1.25 - 1.19 (m, 4H).

[0401] Example 20. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methoxyphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 20).

[0402]

[0403] A white solid title compound (2.6 mg, 95.9% purity, 20.1% yield) was obtained by synthesis similar to Step 5 of Example 17. MS(M+H) + =829.35

[0404] 1 H NMR (400 MHz, DMSO) δ = 10.21 (s, 1H), 8.57 (s, 1H), 8.12 (t, J = 8.3 Hz, 2H), 7.86 (s, 1H), 7.67 (dd, J = 15.3, 10.7 Hz, 2H), 6.81 (d, J = 2.2 Hz, 1H), 6.73 - 6.61 (m, 2H), 4.80 (t, J = 6.5 Hz, 1H), 4.33 (dq, J = 20.6, 7.5 Hz, 2H), 3.79 (s, 3H), 3.68 (t, J = 6.7 Hz, 3H), 3.23 (s, 3H), 2.68 (t, J = 6.6 Hz, 4H), 2.47 - 2.39 (m, 3H), 2.04 - 1.60 (m, 12H), 1.56 - 1.43 (m, 3H), 1.40 - 1.30 (m, 3H), 1.29 - 1.17 (m, 7H).

[0405] Example 21. Synthesis of N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzamide (Compound 21)

[0406]

[0407] Step 1. Synthesis of 4-(hydroxymethyl)piperidine-4-carbonitrile hydrochloride (2)

[0408] tert-butyl 4-cyano-4-(hydroxymethyl)piperidine-1-carboxylate (2 g, 8.32 mmol) was dissolved in DCM (33.2 ml). 4N HCl / dioxane (62.4 ml) was added. The mixture was stirred at room temperature for 16 hours. After confirmation by LCMS and TLC (DCM:MeOH = 10:1), the resulting solid was filtered and washed with DCM. A white solid title compound (1.38 g, 94.1% yield, crude) was obtained. MS(M+H) + =141.10

[0409] Step 2. Synthesis of benzyl ((1r,4r)-4-(4-cyano-4-(hydroxymethyl)piperidine-1-yl)cyclohexyl)carbamate (3)

[0410] 4-(hydroxymethyl)piperidine-4-carbonitrile hydrochloride (1.33 g, 7.55 mmol) and benzyl (4-oxocyclohexyl)carbamate (1.6 g, 6.86 mmol) were dissolved in DCM (16.3 ml) / DMF (5.4 ml). TEA (2.9 ml, 20.6 mmol) was added and stirred at room temperature for 30 minutes. Acetic acid (2.0 ml, 34.3 mmol) was added and stirred at room temperature for 16 hours. NaBH(OAc)3 (4.36 g, 20.6 mmol) was added and stirred at room temperature for 4 hours. After confirming the reaction by LCMS and TLC (DCM:MeOH=5:1), a saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The mixture was dried with Na2SO4, filtered, and concentrated under reduced pressure. The residue was washed with DCM. A white solid title compound (392 mg, 1.06 mmol, 15.4% yield) was obtained. MS(M+H) + =372.20

[0411] Step 3. Synthesis of benzyl ((1r,4r)-4-(4-cyano-4-formylpiperidin-1-yl)cyclohexyl)carbamate (4)

[0412] Benzyl ((1r,4r)-4-(4-cyano-4-(hydroxymethyl)piperidin-1-yl)cyclohexyl)carbamate (338 mg, 0.910 mmol) was dissolved in DCM (33.2 ml). DMP (579 mg, 1.36 mmol) was added. The mixture was stirred at room temperature for 16 hours. After confirmation by LCMS and TLC (DCM:MeOH=5:1), a saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The mixture was dried with Na2SO4, filtered, and concentrated under reduced pressure. A pink solid title compound (384 mg, crude) was obtained. MS(M+MeOH+H) + =402.20

[0413] Step 4. Synthesis of benzyl ((1r,4r)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (5)

[0414] 1-(4-amino-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (164 mg, 0.641 mmol) and benzyl ((1r,4r)-4-(4-cyano-4-formylpiperidin-1-yl)cyclohexyl)carbamate (158 mg, 0.428 mmol) were dissolved in DMA (4.3 ml). TEA (0.3 ml, 2.14 mmol) was added and stirred at room temperature for 30 minutes. Acetic acid (0.25 ml, 4.28 mmol) was added and stirred at room temperature for 16 hours. NaBH3CN (134 mg, 2.14 mmol) was added and stirred at room temperature for 4 hours. After confirming the reaction by LCMS and TLC (DCM:MeOH = 5:1), a saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The mixture was dried with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0–20% DCM:MeOH) to obtain the white solid title compound (86 mg, 0.15 mmol, 35.1% yield). MS(M+H) + =573.30

[0415] Step 5. Synthesis of 1-((1r,4r)-4-aminocyclohexyl)-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-4-carbonitrile (6)

[0416] A white solid title compound (21 mg, 33% yield) was obtained by synthesis in a manner similar to Step 4 of Example 17. MS(M+H) + =439.20

[0417] Step 6. Synthesis of N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzamide (Compound 21)

[0418] A white solid title compound (17.5 mg, 42.5% yield, 94.93% purity) was obtained by synthesis in a manner similar to Step 5 of Example 17. MS(M+H) + =832.30

[0419] 1 H NMR (400 MHz, DMSO) δ = 10.18 (s, 1H), 8.41 (d, J = 8.9 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.89 (s, 1H), 7.62 (s, 1H), 7.47 (dt, J = 3.6, 1.8 Hz, 2H), 6.92 (d, J = 8.4 Hz, 1H), 6.63 - 6.53 (m, 2H), 5.92 (t, J = 6.7 Hz, 1H), 4.42 (q, J = 8.2 Hz, 1H), 4.32 (q, J = 6.7 Hz, 1H), 3.94 (s, 3H), 3.73 (s, 1H), 3.63 (ddd, J = 12.4, 8.8, 5.8 Hz, 1H), 3.44 (dt, J = 12.5, 6.2 Hz, 1H), 3.23 (s, 3H), 2.89 (d, J = 11.4 Hz, 2H), 2.66 (ddd, J = 16.9, 9.6, 6.1 Hz, 2H), 2.38 (t, J = 11.5 Hz, 2H), 2.33 (t, J = 1.9 Hz, 1H), 2.06 (s, 3H), 2.03 - 1.68 (m, 13H), 1.67 - 1.44 (m, 5H), 1.39 (t, J = 11.2 Hz, 4H), 1.23 (d, J = 6.7 Hz, 3H).

[0420] Example 22. Synthesis of N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-fluorobenzamide (Compound 22)

[0421]

[0422] A white solid title compound (4.9 mg, 94.2% purity, 17.9% yield) was obtained by synthesis similar to Step 5 of Example 17. MS(M+H) + =820.40

[0423] 1 H NMR (400 MHz, DMSO) δ = 10.19 (s, 1H), 8.58 (s, 1H), 8.16 - 8.07 (m, 2H), 7.86 (s, 1H), 7.73 - 7.62 (m, 2H), 6.93 (d, J = 8.4 Hz, 1H), 6.63 - 6.54 (m, 2H), 5.92 (t, J = 6.7 Hz, 1H), 4.32 (dq, J = 13.4, 7.5 Hz, 2H), 3.71 (s, 1H), 3.64 (ddd, J = 13.9, 8.7, 5.8 Hz, 1H), 3.45 (dt, J = 12.5, 6.2 Hz, 1H), 3.23 (s, 3H), 2.89 (d, J = 11.2 Hz, 2H), 2.77 - 2.60 (m, 2H), 2.37 (t, J = 10.5 Hz, 3H), 2.07 (s, 3H), 2.00 - 1.86 (m, 7H), 1.84 - 1.76 (m, 4H), 1.72 - 1.66 (m, 2H), 1.62 - 1.49 (m, 4H), 1.38 (p, J = 11.0 Hz, 4H), 1.22 (d, J = 6.7 Hz, 4H).

[0424] Example 23. Synthesis of N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-fluorobenzamide (Compound 23)

[0425]

[0426] Step 1. Synthesis of benzyl ((1r,4r)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (3)

[0427] A white solid title compound (71 mg, 21% yield) was obtained by synthesis similar to Step 3 of Example 17. MS(M+H) + =573.25

[0428] Step 2. Synthesis of 1-((1r,4r)-4-aminocyclohexyl)-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-4-carbonitrile (4)

[0429] A clear oil title compound (33 mg, 60.7% yield) was obtained by synthesis in a manner similar to Step 4 of Example 17. MS(M+H) + =439.20

[0430] Step 3. Synthesis of N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-fluorobenzamide (Compound 23)

[0431] A white solid title compound (13 mg, 88.2% purity, 37.2% yield) was obtained by synthesizing in a manner similar to Step 5 of Example 17. MS(M+H) + =820.30

[0432] 1 H NMR (400 MHz, DMSO-d6) δ = 10.19 (s, 1H), 8.58 (s, 1H), 8.12 (t, J = 7.9 Hz, 2H), 7.86 (s, 1H), 7.67 (dd, J = 14.4, 10.5 Hz, 2H), 6.93 (s, 2H), 6.80 (d, J = 9.4 Hz, 1H), 5.05 (t, J = 6.8 Hz, 1H), 4.32 (dq, J = 13.5, 7.4 Hz, 2H), 3.71 (s, 1H), 3.65 (t, J = 6.7 Hz, 2H), 3.23 (s, 3H), 2.88 (d, J = 11.2 Hz, 2H), 2.67 (t, J = 6.7 Hz, 2H), 2.41 - 2.32 (m, 3H), 2.12 (s, 3H), 1.89 (t, J = 13.6 Hz, 6H), 1.82 - 1.73 (m, 4H), 1.68 (s, 2H), 1.61 (d, J= 11.8 Hz, 2H), 1.56 - 1.45 (m, 3H), 1.36 (t, J = 9.1 Hz, 4H), 1.22 (d, J = 6.8 Hz, 4H).

[0433] Example 24. Synthesis of N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-(2-hydroxyethoxy)benzamide (Compound 24)

[0434]

[0435] A white solid title compound (3.5 mg, 95.2% purity, 12.1% yield) was obtained by synthesis similar to Step 5 of Example 17. MS(M+H) + =862.35

[0436] 1 H NMR (400 MHz, DMSO-d6) δ = 10.19 (s, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.06 - 7.97 (m, 2H), 7.90 (s, 1H), 7.51 - 7.42 (m, 2H), 6.97 - 6.89 (m, 2H), 6.80 (d, J = 9.6 Hz, 1H), 5.17 (s, 1H), 5.05 (t, J = 6.6 Hz, 1H), 4.43 - 4.40 (m, 1H), 4.33 (q, J = 6.7 Hz, 1H), 4.12 (t, J = 4.6 Hz, 2H), 3.86 - 3.59 (m, 5H), 3.42 (d, J = 6.7 Hz, 1H), 3.24 (s, 3H), 2.89 (d, J = 11.3 Hz, 2H), 2.69 - 2.63 (m, 2H), 2.42 - 2.29 (m, 2H), 2.13 (s, 3H), 2.09 - 1.98 (m, 2H), 1.92 - 1.76 (m, 12H), 1.62 - 1.56 (m, 5H), 1.37 (t, J = 9.3 Hz, 3H), 1.24 (d, J = 6.5 Hz, 3H).

[0437] Example 25. Synthesis of N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-hydroxybenzamide (Compound 25)

[0438]

[0439] Step 1. Synthesis of benzyl ((1r,4r)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (5)

[0440] 1-(4-amino-3-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (50.0 mg, 0.196 mmol, HCl salt) was dissolved in ACN (1.0 mL) and MeOH (0.5 mL), then TEA (54.5 μL, 0.391 mmol) was added and stirred at room temperature for 10 minutes. Benzyl ((1r,4r)-4-(4-cyano-4-formylpiperidin-1-yl)cyclohexyl)carbamate (144 mg, 0.391 mmol), acetic acid (112 mL, 1.96 mmol), and TFA (14.5 μL, 0.196 mmol) were added to the reaction mixture and stirred at room temperature for 17 hours. NaBH3CN (24.6 mg, 0.391 mmol) was added to the reaction mixture and stirred at room temperature for 12 hours. A saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The residue was obtained by drying with MgSO4, filtering, and concentrating under reduced pressure. The residue was purified by flash silica gel chromatography (0–10% MeOH:DCM) to obtain the title compound (112.0 mg, 0.200 mmol, 100.0% yield) as a light brown, viscous solid. MS(M+H) + =573.30

[0441] Step 2 . Synthesis of 1-((1r,4r)-4-aminocyclohexyl)-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-4-carbonitrile (6)

[0442] Benzyl ((1r,4r)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (112.0 mg, 0.200 mmol) was dissolved in TFA (5.97 mL, 80.3 mmol) and stirred at 40°C for 17 hours. LCMS analysis showed a main peak with the desired mass. The mixture was concentrated under reduced pressure to obtain the title compound of a brown, sticky oil (249.0 mg, crude, 3TFA salt). MS(M+H) + =439.20

[0443] Step 3. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-hydroxybenzoic acid (7)

[0444] (R)-2-chloro-8-cyclopentyl-5,7-dimethyl-7,8-dihydropteridine-6(5H)-one (200 mg, 0.712 mmol) and 4-amino-3-hydroxybenzoic acid (175 mg, 1.14 mmol) were dissolved in 1,4-dioxane (6 mL), after which a mixture of 12 M HCl (0.237 mL) and water (3 mL) was added and stirred at 100 °C for 17 hours. The desired main peak appeared in the LCMS. The mixture was cooled to room temperature and stirred for 1 hour, and the resulting solid was filtered. The filtered solid was washed with water and EtOAc and then freeze-dried to obtain the title compound as a light gray solid (202 mg, 0.508 mmol, 71.3% yield). MS(M+H) + =398.10

[0445] Step 4. Synthesis of N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-hydroxybenzamide (Compound 25)

[0446] (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-hydroxybenzoic acid (24.0 mg, 0.060 mmol) and 1-((1r,4r)-4-aminocyclohexyl)-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-4-carbonitrile (3TFA salt, 50.0 mg, 0.068 mmol) were dissolved in DMF (0.8 mL), after which EDCI (23.2 mg, 0.121 mmol), HOBt (18.5 mg, 0.121 mmol), and DIPEA (103.0 µL, 0.604 mmol) were added, and for 17 hours The mixture was stirred. It was confirmed by LCMS that all starting material had been consumed, and the main peak at the desired mass was identified. The solvent was concentrated after separation and purification using prep-HPLC (Column: Agilent 10 Prep-C18 150*30 mm*10 µm; Mobile phase: [H2O (0.1% FA) - ACN (0.1% FA)]; Gradient: 0% - 95% B for 50.0 min). The residue was further purified by amine silica gel chromatography (0–15% MeOH:DCM) and freeze-dried to obtain the white solid title compound (2.90 mg, 5.44% yield, 92.7% purity). MS(M+H) + =818.40

[0447] 1 H NMR (400 MHz, DMSO) δ = 10.20 (s, 1H), 8.16 (d, J = 8.4 Hz, 1H), 8.00 - 7.79 (m, 3H), 7.35 - 7.22 (m, 2H), 6.94 (s, 2H), 6.80 (d, J = 9.4 Hz, 1H), 5.06 - 4.99 (m, 2H), 4.46 - 4.41 (m, 1H), 4.32 (q, J = 6.8 Hz, 1H), 3.65 (t, J = 6.7 Hz, 3H), 3.42 (d,J = 6.8 Hz, 2H), 3.23 (s, 3H), 2.88 (d, J = 11.6 Hz, 2H), 2.72 - 2.63 (m, 3H), 2.43 - 2.25 (m, 4H), 2.13 (s, 3H), 2.05 - 1.84 (m, 6H), 1.83 - 1.73 (m, 5H), 1.68 - 1.54 (m, 4H), 1.40 - 1.31 (m, 3H), 1.24 (d, J = 6.6 Hz, 3H).

[0448] Example 26. N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-

[0449] Synthesis of dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-2-fluorobenzamide (Compound 26)

[0450]

[0451] A white solid title compound (0.7 mg, 95.2% purity, 3.25% yield) was obtained by synthesis similar to Step 5 of Example 17. MS(M+H) + =820.30

[0452] 1 H NMR (400 MHz, DMSO-d6) δ = 10.19 (s, 1H), 9.56 (s, 1H), 7.92 - 7.89 (m, 2H), 7.74 - 7.71 (m, 1H), 7.52 - 7.47 (m, 1H), 7.43 - 7.41 (m, 1H), 6.94 - 6.93 (m, 2H), 6.81 - 6.79 (m, 1H), 5.05 (t, J = 6.7 Hz, 1H), 4.54 - 4.46 (m, 1H), 4.36 - 4.30 (m, 1H), 3.65 (t, J= 6.7 Hz, 3H), 3.24 (s, 3H), 2.88 - 2.86 (m, 1H), 2.68 - 2.67 (m, 2H), 2.34 - 2.33 (m, 2H), 2.12 (s, 3H), 1.98 - 1.89 (m, 10H), 1.77 - 1.72 (m, 7H), 1.63 - 1.57 (m, 4H), 1.37 - 1.30 (m, 3H), 1.24 (d, J = 6.6 Hz, 3H).

[0453] Example 27. Synthesis of N-((1r,4R)-4-(4-cyano-4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclohexyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)benzamide (Compound 27)

[0454]

[0455] A white solid title compound (1.6 mg, 88.0% purity, 4.28% yield) was obtained by synthesis similar to Step 5 of Example 17. MS(M+H) + =816.40

[0456] 1 H NMR (400 MHz, DMSO-d6) δ = 10.19 (s, 1H), 9.38 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.89 (s, 1H), 7.82 - 7.80 (m, 2H), 7.76 - 7.72 (m, 2H) 6.96 - 6.91 (m, 2H), 6.80 (d, J = 9.3 Hz, 1H), 5.05 (t, J = 6.7 Hz, 1H), 4.38 - 4.25 (m, 2H), 3.65 (t, J = 6.7 Hz, 2H), 3.42 (d, J = 7.1 Hz, 1H), 3.23 (s, 3H), 2.89 (d, J = 11.4 Hz, 2H), 2.39 (d, J= 11.5 Hz, 3H), 2.34 - 2.33 (m, 2H), 2.12 (s, 3H), 1.98 - 1.94 (m, 4H), 1.93 - 1.89 (m, 4H), 1.80 - 1.78 (m, 4H), 1.68 - 1.67 (m, 3H), 1.62 - 1.60 (m, 2H), 1.44 - 1.42 (m, 2H), 1.39 - 1.35 (m, 5H), 1.24 (d, J = 6.6 Hz, 3H).

[0457] Example 28. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-methoxypiperidin-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 28)

[0458]

[0459] Step 1. Synthesis of benzyl (4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-methoxypiperidin-1-yl)cyclohexyl)carbamate (3)

[0460] The title compound of a light brown oil (112.1 mg, 21.3% yield) was obtained by synthesis in a manner similar to Step 3 of Example 17. MS(M+H) + =578.30

[0461] Step 2. Synthesis of 1-(4-(((1-(4-aminocyclohexyl)-4-methoxypiperidin-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione(4)

[0462] The title compound as a light pink solid (59.3 mg, 68.9% yield) was synthesized in a manner similar to Step 4 of Example 17. MS(M+H) + =444.20

[0463] Step 3. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-methoxypiperidin-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 28)

[0464] A pale pink solid title compound (8.9 mg, 93.3% purity, 7.5% yield) was obtained by synthesis similar to Step 5 of Example 17. MS(M+H) + =825.40

[0465] 1 H NMR (400 MHz, DMSO) δ = 10.18 (s, 1H), 8.58 - 8.57 (d, J= 1.4 Hz, 1H), 8.14 - 8.10 (m, 2H), 7.86 (s, 1H), 7.71-7.64 (m, 2H), 6.91 - 6.89 (d, J = 8.3 Hz, 1H), 6.53 - 6.50 (m, 2H), 5.18 - 5.15 (m, 1H), 4.37 - 4.27 (m, 2H), 3.70 (brs, 1H), 3.66 - 3.59 (m, 1H), 3.48 - 3.41 (m, 1H), 3.23 (s, 3H), 3.11 (s, 3H), 3.03 - 3.01 (d, J = 5.1 Hz, 2H), 2.71 - 2.65 (m, 2H), 2.57 - 2.54 (m, 2H), 2.44 - 2.42 (m, 2H), 2.27 (brs, 1H), 2.06 (s, 3H) 1.98 - 1.95 (m, 1H), 1.90 - 1.89 (m, 2H), 1.82 - 1.78 (m, 5H), 1.75 - 1.68 (m, 2H), 1.54 - 1.48 (m, 4H), 1.37 - 1.32 (t, J = 9.7 Hz, 4H), 1.24 - 1.22 (m, 5H).

[0466] Example 29. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)-4-methoxypiperidin-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 29).

[0467]

[0468] Step 1. Synthesis of (4-methoxypiperidin-4-yl)methanol hydrochloride (2)

[0469] A white solid title compound (1.38 g, 90% yield, crude) was obtained by synthesis in a manner similar to Step 1 of Example 21. MS(M+H) + = 146.15

[0470] Step 2. Synthesis of benzyl (4-(4-(hydroxymethyl)-4-methoxypiperidin-1-yl)cyclohexyl)carbamate (3)

[0471] The title compound as a yellow liquid (1.37 g, 3.64 mmol, 66.1% yield) was synthesized in a manner similar to Step 2 of Example 21. MS(M+H) + =377.20

[0472] Step 3. Synthesis of benzyl (4-(4-formyl-4-methoxypiperidin-1-yl)cyclohexyl)carbamate (4)

[0473] A brown liquid title compound (2.62 g, crude) was obtained by synthesis in a manner similar to Step 3 of Example 21. MS(M+MeOH+H) + =407.20

[0474] Step 4. Synthesis of benzyl (4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)-4-methoxypiperidin-1-yl)cyclohexyl)carbamate (5)

[0475] A white solid title compound (50 mg, 0.087 mmol, yield 13%) was obtained by synthesis in a manner similar to Step 4 of Example 21. MS(M+H) + =578.30

[0476] Step 5. Synthesis of 1-(4-(((1-(4-aminocyclohexyl)-4-methoxypiperidin-4-yl)methyl)amino)-3-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (6)

[0477] The white solid title compound (28 mg, 0.063 mmol, yield 72.9%) was synthesized in a manner similar to Step 7 of Example 1. MS(M+H) + =444.30

[0478] Step 6. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)-4-methoxypiperidin-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 29)

[0479] A white solid title compound (4.6 mg, yield 7.95%, purity 90.01%) was obtained by synthesis in a manner similar to Step 7 of Example 21. MS(M+H) + =825.40

[0480] 1 H NMR (400 MHz, DMSO) δ = 10.18 (s, 1H), 8.56 (s, 1H), 8.13 - 8.07 (m, 2H), 7.85 (s, 1H), 7.70-7.63 (m, 2H), 6.97 - 6.95 (m, 2H), 6.59 - 6.57 (m, 2H), 4.36 - 4.26 (m, 2H), 4.02-4.01 (m, 1H), 3.70 - 3.62 (m, 2H), 3.32 (s, 4H), 3.12 - 3.08 (m, 5H), 2.66 (t, J= 6.8 Hz, 3H), 2.55 (brs, 1H), 2.33 - 2.28 (m, 2H), 2.08 (s, 3H), 1.90 - 1.87 (m, 3H), 1.83 - 1.77 (m, 5H), 1.70 - 1.67 (m, 2H), 1.55 - 1.50 (m, 5H), 1.34 (t, 8.4 Hz, 3H), 1.23 - 1.20 (m, 5H).

[0481] Example 30. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-methylpiperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 30).

[0482]

[0483] Step 1. Synthesis of (4-methylpiperidine-4-yl)methanol hydrochloride (2)

[0484] tert-butyl 4-(hydroxymethyl)-4-methylpiperidine-1-carboxylate (2 g, 8.72 mmol) was dissolved in DCM (10 mL). After adding 4N HCl (11 mL, 43.6 mmol), the mixture was stirred at room temperature for 18 hours. The solid was filtered and dried to obtain the title compound (2 g, crude). MS(M+H) + =130.20

[0485] Step 2. Synthesis of benzyl (4-(4-(hydroxymethyl)-4-methylpiperidine-1-yl)cyclohexyl)carbamate (4)

[0486] A white solid title compound (2.2 g, 60% yield) was obtained by synthesis in a manner similar to Step 1 of Example 17. MS(M+H) + =361.20

[0487] Step 3. Synthesis of benzyl (4-(4-formyl-4-methylpiperidin-1-yl)cyclohexyl)carbamate (5)

[0488] A white solid title compound (1.9 g, 89% yield) was obtained by synthesis in a manner similar to Step 2 of Example 17. MS(M+H) + =359.20

[0489] Step 4. Synthesis of benzyl (4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-methylpiperidine-1-yl)cyclohexyl)carbamate (7)

[0490] A white solid title compound (600 mg, 38.0% yield, crude) was obtained by synthesis in a manner similar to Step 3 of Example 17. MS(M+H) +=562.30.

[0491] Step 5. Synthesis of 1-(4-(((1-(4-aminocyclohexyl)-4-methylpiperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (8)

[0492] A white solid title compound (60 mg, 38% yield) was obtained by synthesis in a manner similar to Step 4 of Example 17. MS(M+H) + =428.30

[0493] Step 6. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-methylpiperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 30)

[0494] (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid (25 mg, 0.06 mmol), 1-(4-(((1-(4-aminocyclohexyl)-4-methylpiperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (34 mg, 0.08 mmol), and HATU (35 mg, 0.09 mmol) were dissolved in DCM (2 mL) and DMF (0.5 mL). After adding DIPEA (32 μL, 0.18 mmol), the mixture was stirred at room temperature for 2 hours. The organic layer was extracted with a saturated aqueous solution of NaHCO3 and EtOAc, dried with MgSO4, and concentrated under reduced pressure. The filtrate was purified by silica gel chromatography (0–20% MeOH, DCM) followed by amine chromatography (0–5% MeOH, DCM) to obtain the white solid title compound (7 mg, 95.7% purity, 13.4% yield). MS(M+H) + =821.40

[0495] 1 H NMR (400 MHz, DMSO-d6) δ = 10.17 (s, 1H), 8.41 (d, J = 8.8 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.90 (s, 1H), 7.62 (s, 1H), 7.48 (d, J = 5.9 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.53 - 6.45 (m, 2H), 5.44 (t,J = 6 Hz, 1H), 4.42 (p, J = 8.1 Hz, 1H), 4.32 (q, J = 6.7 Hz, 1H), 3.94 (s, 3H), 3.72 (s, 1H), 3.66 - 3.58 (m, 1H), 3.44 (dt, J = 12.5, 6.3 Hz, 2H), 3.24 (s, 3H), 2.86 (d, J = 5.8 Hz, 2H), 2.72 - 2.64 (m, 2H), 2.61 - 2.54 (m, 2H), 2.43 (t, J = 10.2 Hz, 2H), 2.26 (s, 1H), 2.05 (s, 3H), 2.03 - 1.97 (m, 1H), 1.95 - 1.87 (m, 3H), 1.86 - 1.74 (m, 5H), 1.62 (s, 2H), 1.55 - 1.47 (m, 2H), 1.41 - 1.29 (m, 6H), 1.23 (d, J = 6.7 Hz, 3H), 0.94 (s, 3H).

[0496] Example 31. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-methylpiperidine-1-yl)cyclohexyl)-3-fluorobenzamide (Compound 31)

[0497]

[0498] A white solid title compound (5 mg, 93% purity, 9% yield) was obtained by synthesis similar to Step 6 of Example 30. MS(M+H) + =809.4

[0499] 1 H NMR (400 MHz, DMSO-d6) δ = 10.17 (s, 1H), 8.57 (s, 1H), 8.12 (t, J = 8.6 Hz, 2H), 7.86 (s, 1H), 7.67 (dd, J = 14.7, 10.7 Hz, 2H), 6.88 (d, J = 8.4 Hz, 1H), 6.52 - 6.42 (m, 2H), 5.44 (t, J= 5.6 Hz, 1H), 4.32 (dq, J = 13.6, 7.5 Hz, 2H), 3.70 (s, 1H), 3.65 - 3.57 (m, 1H), 3.44 (dt, J = 12.4, 6.2 Hz, 2H), 3.23 (s, 3H), 2.86 (d, J = 5.8 Hz, 2H), 2.68 (q, J = 7.7 Hz, 2H), 2.57 (s, 2H), 2.43 (t, J = 9.8 Hz, 2H), 2.26 (s, 1H), 2.05 (s, 3H), 2.00 - 1.72 (m, 8H), 1.68 (s, 2H), 1.58 - 1.43 (m, 4H), 1.39 - 1.27 (m, 5H), 1.22 (d, J = 6.7 Hz, 3H), 0.94 (s, 3H).

[0500] Example 32. Synthesis of 5-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((4-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-3-methylphenyl)amino)methyl)-4-fluoropiperidine-1-yl)cyclohexyl)-4-methoxypicolinamide (Compound 32)

[0501]

[0502] A white solid title compound (1.6 mg, 96.8% purity, 5.7% yield) was obtained by synthesis in a manner similar to Step 5 of Example 17. MS(M+H) + =826.35

[0503] 1 H NMR (400 MHz, DMSO) δ = 10.18 (s, 1H), 9.25 (s, 1H), 8.26 (d, J = 8.6 Hz, 1H), 7.84 (d, J = 23.5 Hz, 2H), 7.64 (s, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.57 - 6.42 (m, 2H), 5.71 (t, J = 6.4 Hz, 1H), 4.42 - 4.25 (m, 2H), 3.99 (s, 3H), 3.72 (s, 1H), 3.61 (q, J= 13.0 Hz, 1H), 3.48 - 3.39 (m, 1H), 3.24 (s, 3H), 3.20 (d, J = 6.0 Hz, 1H), 2.74 - 2.62 (m, 4H), 2.45 - 2.27 (m, 4H), 2.05 (s, 3H), 1.96 - 1.60 (m, 10H), 1.59 - 1.50 (m, 2H), 1.41 (q, J = 13.8 Hz, 4H), 1.29 - 1.13 (m, 7H).

[0504] Example 33. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)-[1,4'-bipiperidin]-1'-yl)-3-methoxybenzamide (Compound 33)

[0505]

[0506] Step 1. Synthesis of 3-((3-bromophenyl)amino)propanoic acid (3)

[0507] 3-bromoaniline (500 mg, 2.91 mmol) and acrylic acid (314 mg, 4.36 mmol) were dissolved in toluene (14.5 mL) and stirred at 100 °C for 15 hours. LCMS analysis revealed a main peak with the desired mass. Saturated aqueous solution of NaCl and EtOAc were added to the mixture, and the organic layer was extracted. The residue was obtained by drying with MgSO4, filtering, and concentrating under reduced pressure. The residue was purified by flash silica gel chromatography (0–25% EtOAc:n-hexane) to obtain the title compound of a brown, sticky oil (540 mg, 2.21 mmol, 76.1% yield). MS(M+2) + =244.00.

[0508] Step 2. Synthesis of 1-(3-bromophenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0509] 3-((3-bromophenyl)amino)propanoic acid (540 mg, 2.21 mmol) and urea (1.33 g, 22.1 mmol) were dissolved in acetic acid (3.00 mL) and stirred at 120 °C for 15 hours. LCMS analysis revealed a main peak with the desired mass. A saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The residue was obtained by drying with MgSO4, filtering, and concentrating under reduced pressure. The residue was purified by flash silica gel chromatography (0–20% EtOAc:n-hexane) to obtain the white solid title compound (323 mg, 1.20 mmol, yield 54.3%). MS(M+H) + =268.90

[0510] Step 3. Synthesis of tert-butyl 4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-)phenyl)amino)methyl)piperidine-1-carboxylate (6)

[0511] 1-(3-bromophenyl)dihydropyrimidine-2,4(1H,3H)-dione (100 mg, 0.37 mmol), tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (95.6 mg, 0.45 mmol), Gphos Pd G6 TES (35.1 mg, 0.037 mmol), and Cs2CO3 (242 mg, 0.74 mmol) were dissolved in dioxane (1 mL) and stirred at 80°C for 3 hours. The mixture was filtered with Celite using EtOAc and dried under reduced pressure. The residue was purified by flash silica gel chromatography (0–40% EtOAc:n-hexane) to obtain the title compound of the yellow oil (37.7 mg, 0.094 mmol, yield 25.2%). MS(M-100+H) + =303.20

[0512] Step 4. Synthesis of 1-(3-((piperidine-4-ylmethyl)amino)phenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (7)

[0513] tert-butyl 4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-)phenyl)amino)methyl)piperidine-1-carboxylate (37.7 mg, 0.094 mmol) was dissolved in DCM (0.47 mL), 4N HCl / dioxane (0.23 mL, 0.94 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Methanol (3 mL) was added to dissolve the mixture, and ether (10 mL) was added. The resulting solid was filtered to obtain the yellow solid title compound (45.0 mg, 131%, 2HCl salt). MS(M+H) + =303.15

[0514] Step 5. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)-[1,4'-bipiperidine]-1'-yl)-3-methoxybenzamide (Compound 33)

[0515] 1-(3-((piperidin-4-ylmethyl)amino)phenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (45.0 mg, 0.13 mmol, 2HCl salt) was dissolved in DMAC (1.2 mL), then TEA (46.8 μL, 0.335 mmol) was added and stirred at room temperature for 10 minutes. (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxy-N-(4-oxopiperidine-1-yl)benzamide (35 mg, 0.067 mmol) and acetic acid (38.4 μL, 0.67 mmol) were added to the reaction mixture and stirred at room temperature for 30 minutes. NaBH3CN (25.3 mg, 0.40 mmol) was added to the reaction mixture and stirred at room temperature for 17 hours. LCMS confirmed that all starting materials had been consumed and identified the main peak at the desired mass. The mixture was filtered through Celite, separated and purified using prep-HPLC (Column: Agilent 10 Prep-C18 150 × 30 mm × 10 µm; Mobile phase: [H2O (0.1% FA) - ACN (0.1% FA)]; Gradient: 0% - 95% B for 50.0 min), and the solvent was concentrated to obtain the residual compound. The residue was purified by amine column chromatography (0–10% MeOH:DCM) and freeze-dried to obtain the white solid title compound (12 mg, 21.3% yield, 94.4% purity). MS(M+H) + =794.40.

[0516] 1 H NMR (400 MHz, DMSO-d6) δ = 10.24 (s, 1H), 9.24 (s, 1H), 8.41 (d, J = 8.4 Hz, 1H), 7.89 (s, 1H), 7.63 (s, 1H), 7.40 (s, 2H), 7.05 (t, J = 7.8 Hz, 1H), 6.49 (s, 1H), 6.44 (t, J= 7.9 Hz, 2H), 5.72 (s, 1H), 4.42 (s, 1H), 4.32 (d, J = 6.8 Hz, 1H), 3.93 (s, 3H), 3.23 (s, 3H), 3.03 (d, J = 9.5 Hz, 3H), 2.92 - 2.85 (m, 4H), 2.81 - 2.72 (m, 4H), 2.67 (t, J = 6.6 Hz, 4H), 2.28 - 2.16 (m, 3H), 2.15 - 2.06 (m, 2H), 2.04 - 1.87 (m, 1H), 1.84 - 1.70 (m, 5H), 1.75 (m, 2H), 1.67 - 1.54 (m, 3H), 1.23 (s, 3H), 1.19 - 1.14 (m, 1H)

[0517] Example 34. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-4-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 34)

[0518]

[0519] Step 1. Synthesis of 3-((2-fluoro-5-nitrophenyl)amino)propanoic acid (3)

[0520] After dissolving 2-fluoro-5-nitroaniline (2.08 g, 13.3 mmol) and acrylic acid (1.85 mL, 27.0 mmol) in acetic acid (19.4 mL) and water (2.77 mL), 110 o The mixture was stirred at C for 24 hours. After lowering the temperature to room temperature, water (60 mL) and EtOAc (30 mL) were added to the mixture, and the organic layer was extracted. EtOAc (30 mL) was added to the aqueous layer to extract the organic layer once more, and the mixture was dried and filtered with MgSO4. The remaining compound was then concentrated under reduced pressure. DCM (10 mL) and EtOAc (5 mL) were added to form a suspension, and n-hexane (15 mL) was added to filter the resulting solid. The solid was dried, and the filtrate was purified by flash silica gel chromatography (5% MeOH:DCM gradient) to obtain the yellow solid title compound (1.90 g, 8.31 mmol, yield 62.3%). MS(M+H) + =229.00

[0521] Step 2. Synthesis of 3-((5-amino-2-fluorophenyl)amino)propanoic acid (4)

[0522] 3-((2-fluoro-5-nitrophenyl)amino)propanoic acid (1.63 g, 7.14 mmol) was dissolved in methanol (35.8 mL), and then 10% Pd / C (0.106 g, 0.100 mmol) was added. The mixture was stirred for 17 hours in a hydrogen gas atmosphere using a hydrogen gas-filled balloon. It was filtered through a Celite pad and washed with DCM (50 mL). The filtrate was concentrated and dried to obtain the black, sticky oily title compound (1.44 g, 7.25 mmol, 101.0% yield, crude). MS(M+H) + =199.10

[0523] Step 3. Synthesis of 3-((5-(((benzyloxy)carbonyl)amino)-2-fluorophenyl)amino)propanoic acid (5)

[0524] 3-((5-amino-2-fluorophenyl)amino)propanoic acid (443 mg, 2.23 mmol) was dissolved in THF, and DIPEA (1.53 mL, 8.93 mmol) was added. Using an ice bath, the temperature of the reaction solution was 0 o After lowering the temperature, benzyl chloroformate (0.319 mL, 2.23 mmol) was slowly added, and the mixture was stirred at room temperature for 6 hours. As the starting material did not dissolve well, DIPEA (1 mL) was added, and the mixture was stirred at room temperature for 2.5 hours. To remove insoluble substances, the solution was filtered through a Celite pad and washed with THF (10 mL). EtOAc (20 mL) and water (10 mL) were added to the residue, the pH was adjusted to approximately 5 using 1N HCl, and the aqueous layer was extracted three times with EtOAc (20 mL). The organic layer was extracted, dried and filtered with MgSO4, concentrated under reduced pressure, and dried to obtain the title compound as a brown, sticky solid (511 mg, 1.54 mmol, 68.8% yield). MS(M+H) + =333.05

[0525] Step 4. Synthesis of benzyl (3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-4-fluorophenyl)carbamate (6)

[0526] 3-((5-(((benzyloxy)carbonyl)amino)-2-fluorophenyl)amino)propanoic acid (511 mg, 1.54 mmol), urea (1.85 g, 30.7 mmol) in acetic acid (7 mL) 120 o The mixture was stirred at C for 13 hours. LCMS analysis revealed a main peak with the desired mass. An aqueous solution of NaHCO3 was slowly added to adjust the pH to approximately 8, followed by three extractions with EtOAc (20 mL). The extracted organic layer was dried and filtered with MgSO4, and concentrated under reduced pressure to obtain the residual compound. The residue was purified by flash silica gel chromatography (0–70% EtOAc:n-hexane gradient) to obtain the title compound as an ivory solid (84.9 mg, 0.238 mmol, 15.5% yield). MS (2M+Na) + =737.05

[0527] Step 5. Synthesis of 1-(5-amino-2-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (7)

[0528] Benzyl (3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-4-fluorophenyl)carbamate (84.9 mg, 0.238 mmol) was dissolved in methanol (8 mL), DCM (5 mL), and DMF (50 µL), after which 10% Pd / C (37.9 mg, 0.0356 mmol) was added. The mixture was stirred for 2 hours and 20 minutes in a hydrogen gas atmosphere using a balloon filled with hydrogen gas. The solution was filtered through a Celite pad and washed with DCM (10 mL) and methanol (10 mL). The filtrate was concentrated, and DCM (2 mL) was added to disperse the solid, followed by the addition of n-hexane (5 mL) to filter the resulting solid. The filtered solid was dried to obtain the gray solid title compound (38.5 mg, 0.172 mmol, 72.6% yield).

[0529] 1 H NMR (400 MHz, DMSO-d6) δ = 10.36 (s, 1H), 6.91(t, J = 9.5 Hz, 1H), 6.50 - 6.46 (m, 2H), 5.04 (s, 2H), 3.62 (t,J = 6.6 Hz, 2H), 2.66 (t, J = 6.5 Hz, 2H)

[0530] Step 6. Synthesis of benzyl ((1r,4r)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-4-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (9)

[0531] A colorless, viscous solid title compound (22.4 mg, 23.9% yield) was obtained by synthesis in a manner similar to Step 6 of Example 1. MS(M+H) + =552.20

[0532] Step 7. Synthesis of 1-(5-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (10)

[0533] A brown sticky oil title compound (45.0 mg, 146% yield, 3TFA salt, crude) was obtained by synthesis in a manner similar to Step 7 of Example 1. MS(M+H) + =418.10

[0534] Step 8. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-4-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 34)

[0535] A white solid title compound (8.90 mg, 30.9% yield, 89.1% purity) was obtained by synthesis similar to Step 8 of Example 1. MS(M+H) + =811.30

[0536] 1 H NMR (400 MHz, DMSO-d6) δ = 10.40 (s, 1H), 8.43 (d, J = 8.5 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.91 (s, 1H), 7.64 (s, 1H), 7.50 - 7.48 (m, 2H), 6.99 (t, J= 9.2 Hz, 1H), 6.54 - 6.51 (m, 2H), 5.66 - 5.65 (m, 1H), 4.47 - 4.42 (m, 1H), 4.36 - 4.31 (m, 1H), 3.95 (s, 3H), 3.72 - 3.66 (m, 3H), 3.25 (s, 3H), 2.87 - 2.85 (m, 3H), 2.72 - 2.69 (m, 2H), 2.38 - 2.35 (m, 1H), 2.20 - 2.15 (m, 2H), 2.03 - 2.01 (m, 1H), 1.97 - 1.89 (m, 3H), 1.80- 1.75 (m, 8H), 1.68 - 1.59 (m, 2H), 1.50 - 1.49 (m, 1H), 1.43 - 1.33(m, 4H), 1.25 - 1.16 (m,6H)

[0537] Example 35. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 35)

[0538]

[0539] Step 1. Synthesis of 3-((3-bromo-2-methylphenyl)amino)propanoic acid (3)

[0540] 3-Bromo-2-methylaniline (500 mg, 2.69 mmol) and acrylic acid (290 mg, 4.03 mmol) were dissolved in toluene (13.4 mL) and stirred at 100 °C for 15 hours. LCMS analysis revealed a main peak with the desired mass. Saturated aqueous solution of NaCl and EtOAc were added to the mixture, and the organic layer was extracted. The mixture was dried and filtered with MgSO4, and concentrated under reduced pressure to obtain the residual compound. The residual compound was solidified with DCM and filtered. A white solid title compound (410 mg, 1.59 mmol, 59.1% yield) was obtained. MS(M+2) + =259.05

[0541] Step 2. Synthesis of 1-(3-bromo-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0542] 3-((3-bromo-2-methylphenyl)amino)propanoic acid (490 mg, 1.90 mmol) and urea (1.14 g, 19.0 mmol) were dissolved in acetic acid (2.57 mL) and stirred at 120 °C for 3 days. LCMS analysis revealed a main peak with the desired mass. Saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The residue was obtained by drying with MgSO4, filtering, and concentrating under reduced pressure. The residue was purified by flash silica gel chromatography (0–50% EtOAc:n-hexane) to obtain the white solid title compound (267 mg, 0.94 mmol, 49.6% yield). MS(M+2) + =284.95

[0543] Step 3. Synthesis of tert-butyl (3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)carbamate (6)

[0544] 1-(3-bromo-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (267 mg, 0.94 mmol), tert-butyl carbamate (221 mg, 1.88 mmol), tBu Xphos Pd G3 (75 mg, 0.094 mmol), and t-BuOK (317 mg, 2.83 mmol) were dissolved in dioxane (1.75 mL) and stirred at 100°C for 3 hours. LCMS analysis revealed a main peak with the desired mass. The mixture was filtered with Celite using EtOAc and dried under reduced pressure. The residue was purified by flash silica gel chromatography (0–10% MeOH:DCM) to obtain the title compound as a beige solid (70.6 mg, 0.22 mmol, 23.5% yield). MS (2M+Na) + =661.30

[0545] Step 4. Synthesis of 1-(3-amino-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (7)

[0546] tert-butyl (3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)carbamate (70.6 mg, 0.22 mmol) was dissolved in DCM (0.87 mL), 4N HCl / dioxane (1.11 mL, 4.42 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The white solid title compound (48.2 mg, 0.19 mmol, 85.3%, 2HCl salt) was obtained by concentration under reduced pressure. MS(M+H) + =220.05

[0547] Step 5. Synthesis of benzyl ((1r,4r)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (9)

[0548] 1-(3-amino-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (80.1 mg, 0.20 mmol) and TEA (78.8 µL, 0.57 mmol) were dissolved in DCM (0.78 mL) and stirred at room temperature for 10 minutes. Acetic acid (53.9 µL, 0.94 mmol) and benzyl ((1r,4r)-4-(4-formylpiperidin-1-yl)cyclohexyl)carbamate (77.9 mg, 0.24 mmol) were added to the mixture and stirred at room temperature for 2 hours. NaBH(OAc)3 (200 mg, 0.94 mmol) was added to the mixture and stirred at room temperature for 5 hours. LCMS analysis showed a main peak with the desired mass. Saturated aqueous solution of NaHCO3 and EtOAc were added to the mixture, and the organic layer was extracted. The mixture was dried with MgSO4, filtered, and concentrated under reduced pressure to obtain the residual compound. The filtrate was dried under reduced pressure and purified by flash silica gel chromatography (0–10% MeOH:DCM) to obtain the clear oil title compound (33.5 mg, 0.061 mmol, 32.4% yield). MS(M+H) + =548.30

[0549] Step 6. Synthesis of 1-(3-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (10)

[0550] Benzyl ((1r,4r)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (32.6 mg, 0.060 mmol) was dissolved in TFA (0.88 mL, 11.9 mmol) and stirred at 40°C for 7 hours. LCMS analysis showed a main peak with the desired mass. The mixture was concentrated under reduced pressure to obtain the white solid title compound (33.0 mg, crude, 3TFA salt). MS(M+H) + =414.25

[0551] Step 7. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 35)

[0552] 1-(3-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (32.7 mg, 0.080 mmol) was dissolved in DMF (0.50 mL), and DIPEA (50.0 uL, 0.61 mmol) was added and stirred for 30 minutes. (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid (25 mg, 0.061 mmol), HATU (34.7 mg, 0.091 mmol) and DIPEA (50.0 uL, 0.61 mmol) were dissolved in DMF (0.50 mL) and stirred for 30 minutes. A solution of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzoic acid was added to a solution of 1-(3-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione), and the mixture was stirred at room temperature for 4 hours. It was confirmed by LCMS that all starting materials were consumed, and the main peak of the desired mass was identified. A saturated aqueous solution of NaCl and EtOAc were added to the mixture, the organic layer was extracted, dried and filtered with MgSO4, and concentrated under reduced pressure to obtain the residual compound. The remaining compounds were purified by flash silica gel chromatography (0–10% MeOH:DCM) and freeze-dried to obtain the title compound as a beige solid (16.0 mg, 31.0% yield, 95.0% purity). MS(M+H) + =807.40.

[0553] 1 H NMR (400 MHz, DMSO-d6) δ = 10.24 (s, 1H), 8.42 (d, J = 8.7 Hz, 1H), 8.05 (s, 1H), 7.90 (s, 1H), 7.63 (s, 1H), 7.47 (s, 2H), 7.04 (t, J= 7.9 Hz, 1H), 6.49 (d, J = 7.5 Hz, 2H), 5.02 (s, 1H), 4.46 - 4.38 (m, 1H), 4.33 (d, J = 6.8 Hz, 1H), 3.95 (, 3H), 3.78 - 3.64 (m, 3H), 3.24 (s, 3H), 3.03 - 2.92 (m, 4H), 2.75 (d, J = 8.6 Hz, 2H), 2.70 - 2.61 (m, 2H), 2.04 - 1.97 (s, 2H), 1.92 (s, 8H), 1.84 - 1.73 (m, 5H), 1.66 - 1.56 (m, 3H), 1.45 - 1.31 (m, 4H), 1.23 (d, J = 6.6 Hz, 6H).

[0554] Example 36. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 36)

[0555]

[0556] Step 1. Synthesis of 3-((3-bromophenyl)amino)propanoic acid (3)

[0557] The title compound (0.590 g, 2.42 mmol, 41.6% yield) was obtained by synthesis in a manner similar to Step 1 of Example 35. MS(M+H) + =243.9.

[0558] Step 2. Synthesis of 1-(3-bromophenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0559] The title compound (300 mg, 46.1% yield) was obtained by synthesis in a manner similar to Step 2 of Example 35. MS(M+2) + =269.0.

[0560] Step 3. Synthesis of tert-butyl (3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)carbamate (6)

[0561] The title compound (290 mg, 88.1% yield) was obtained by synthesis in a manner similar to Step 3 of Example 35. MS(M+Na) + =328.10.

[0562] Step 4. Synthesis of 1-(3-aminophenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (7)

[0563] The title compound (150 mg, 65.3% yield) was obtained by synthesis in a manner similar to Step 4 of Example 35. MS(M+H) + =206.1.

[0564] Step 5. Synthesis of benzyl ((1r,4r)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (9)

[0565] The title compound (230 mg, 69.4% yield) was obtained by synthesis in a manner similar to Step 5 of Example 35. MS(M+H) + =534.3.

[0566] Step 6. Synthesis of 1-(3-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (10)

[0567] The title compound (60 mg, 80.1% yield) was obtained by synthesis in a manner similar to Step 2 of Example 28. MS(M+H) + =400.3.

[0568] Step 7. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 36)

[0569] A white solid title compound (10 mg, 10.5% yield, 91.4% purity) was obtained by synthesis in a manner similar to Step 7 of Example 35. MS(M+H) + =793.4.

[0570] 1 H NMR (400 MHz, DMSO-d6) δ= 10.25 (s, 1H), 8.42 (d, J = 8.7 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.90 (s, 1H), 7.62 (s, 1H), 7.48 - 7.47 (m, 2H), 7.05 (t, J = 7.98 Hz, 1H), 6.50 (s, 1H), 6.44 (t, J = 7.3 Hz, 2H), 5.73 (t, J = 5.1 Hz, 1H), 4.45 - 4.41 (m, 1H), 4.32 (q, J = 6.4 Hz, 1H), 3.94 (s, 3H), 3.71 (t, J= 6.5 Hz, 2H), 3.24 (s, 3H), 2.87 - 2.83 (m, 5H), 2.67 (t, J = 6.5 Hz, 2H), 2.30 - 2.28 (m, 1H), 2.16 (t, J = 10.8 Hz, 2H), 2.03 - 1.99 (m, 1H), 1.94 - 1.89 (m, 3H), 1.81 - 1.73 (m, 9H), 1.67 - 1.62 (m, 2H), 1.39 - 1.34 (m, 4H), 1.24 (d, J = 6.6 Hz, 3H), 1.16 (d, J = 9.9 Hz, 2H).

[0571] Example 37. Synthesis of N-((1r,4R)-4-(4-(((3-chloro-5-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzamide (Compound 37)

[0572]

[0573] Step 1. Synthesis of benzyl(3-amino-5-chlorophenyl)carbamate (2)

[0574] The title compound of the yellow oil (1.28 g, 78.9% yield) was obtained by synthesis in a manner similar to Step 3 of Example 34. MS(M+H) + =277.00.

[0575] Step 2. Synthesis of 3-((3-(((benzyloxy)carbonyl)amino)-5-chlorophenyl)amino)propanoic acid (3)

[0576] The title compound of the brown oil (908.1 mg, 56.3% yield) was obtained by synthesis in a manner similar to Step 1 of Example 34. MS(M+H) + =349.10.

[0577] Step 3. Synthesis of benzyl (3-chloro-5-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)carbamate (4)

[0578] A yellow solid title compound (154.3 mg, 35.0% yield) was obtained by synthesis similar to Step 4 of Example 34. MS(M+H2O) + =391.00.

[0579] Step 4. Synthesis of 1-(3-amino-5-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (5)

[0580] The title compound of the yellow oil (214.8 mg, 238% yield, TFA salt, crude) was obtained by synthesis in a manner similar to Step 7 of Example 1. MS(M+H) + =240.10.

[0581] Step 5. Synthesis of benzyl ((1r,4r)-4-(4-(((3-chloro-5-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (7)

[0582] The title compound as a pale yellow solid (196.4 mg, 91.8% yield) was synthesized in a manner similar to Step 6 of Example 1. MS(M+H) + =568.20.

[0583] Step 6. Synthesis of 1-(3-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-5-chlorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (8)

[0584] The title compound of the brown oil (375.6 mg, 198% yield, TFA salt, crude) was obtained by synthesis in a manner similar to Step 5 of Example 2. MS(M+H) + =434.10.

[0585] Step 7. Synthesis of N-((1r,4R)-4-(4-(((3-chloro-5-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzamide (Compound 37)

[0586] The title compound as an ivory solid (11.4 mg, 97.2% purity, 3.07% yield) was synthesized in a manner similar to Step 8 of Example 1. MS(M+H) + =827.30.

[0587] 1 H NMR (400 MHz, DMSO-d6)δ = 10.33 (s, 1H), 8.42 (d, J = 8.90 Hz, 1H), 8.03 (d, J = 7.73 Hz, 1H), 7.90 (s, 1H), 7.62 (s, 1H), 7.48 - 7.47 (m, 2H), 6.51 - 6.50 (m, 1H), 6.47 - 6.46 (m, 2H), 6.10 (br s, 1H), 4.43 (t, J = 8.04 Hz, 1H), 4.33 (q, J = 6.74 Hz, 1H), 3.94 (s, 3H), 3.72 (t, J= 6.66 Hz, 3H), 3.24 (s, 3H), 2.90 - 2.87 (m, 4H), 2.68 (t, J = 6.65 Hz, 2H), 2.29 - 2.18 (m, 3H), 1.92 - 1.91 (m, 3H), 1.82 - 1.78 (m, 8H), 1.63 - 1.61 (m, 2H), 1.52 - 1.46 (m, 1H), 1.40 - 1.35 (m, 4H), 1.24 - 1.18 (m, 6H)

[0588] Example 38. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-5-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 38)

[0589]

[0590] Step 1. Synthesis of methyl 3-((3-bromo-5-fluorophenyl)amino)propanoate (3)

[0591] The title compound of the brown oil (1.30 g, 69.4% yield) was obtained by synthesis in a manner similar to Step 1 of Example 1. MS(M+H) + =276.90.

[0592] Step 2. Synthesis of methyl 3-(1-(3-bromo-5-fluorophenyl)ureido)propanoate (4)

[0593] The title compound of the yellow oil (349.2 mg, 23.2% yield) was obtained by synthesis in a manner similar to Step 3 of Example 1. MS(M+H) + =319.95.

[0594] Step 3. Synthesis of 1-(3-bromo-5-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (5)

[0595] A white solid title compound (80.2 mg, 25.5% yield) was obtained by synthesizing in a manner similar to Step 4 of Example 1.

[0596] 1 H NMR (400 MHz, DMSO-d6) δ = 10.52 (s, 1H), 7.47 - 7.42 (m, 2H), 7.32 - 7.29 (m, 1H), 3.82 (t, J = 6.57 Hz, 2H), 2.71 (t, J = 13.17 Hz, 2H)

[0597] Step 4. Synthesis of tert-butyl (3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-5-fluorophenyl)carbamate (6)

[0598] The title compound of the yellow oil (79.5 mg, 88.0% yield) was obtained by synthesis in a manner similar to Step 3 of Example 35. MS(2M+Na) + =669.20

[0599] Step 5. Synthesis of 1-(3-amino-5-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (7)

[0600] A white solid title compound (64.0 mg, 100% yield, hydrochloride, crude) was obtained by synthesis similar to Step 4 of Example 35. MS(M+H) + =224.10.

[0601] Step 6. Synthesis of benzyl ((1r,4r)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-5-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (9)

[0602] The title compound as a pale yellow solid (15.7 mg, 11.5% yield) was synthesized in a manner similar to Step 5 of Example 35. MS(M+H) + =552.20

[0603] Step 7. Synthesis of 1-(3-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-5-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (10)

[0604] The title compound as a pale yellow solid (29.1 mg, 192% yield, TFA salt, crude) was synthesized in a manner similar to Step 6 of Example 35. MS(M+H) + =418.20

[0605] Step 8. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-5-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 38)

[0606] A white solid title compound (5.9 mg, 95.2% purity, 19.1% yield) was obtained by synthesis similar to Step 7 of Example 35. MS(M+H) + =811.30

[0607] 1 H NMR (400 MHz, DMSO-d6)δ = 10.32 (s, 1H), 8.41 (d, J = 8.87 Hz, 1H), 8.02 (d, J= 7.71 Hz, 1H), 7.90 (s, 1H), 7.62 (s, 1H), 7.48 - 7.47 (m, 2H), 6.36 (s, 1H), 6.28 (d, J = 10.20 Hz, 1H), 6.22 (d, J = 11.95 Hz, 1H), 6.09 (s, 1H), 4.43 (t, J = 8.06 Hz, 1H), 4.32 (q, J = 6.55 Hz, 1H), 3.9 (s, 3H), 3.72 (t, J = 6.58 Hz, 3H), 3.24 (s, 3H), 2.88 (br s, 4H), 2.67 (t, J = 6.51 Hz, 2H), 2.19 (br s, 3H), 1.94 - 1.90 (m, 3H), 1.82 - 1.73 (m, 8H), 1.65 - 1.59 (m, 2H), 1.50 - 1.44 (m, 1H), 1.39 - 1.35 (m, 4H), 1.24 - 1.23 (m, 6H)

[0608] Example 39. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 39).

[0609]

[0610] Step 1. Synthesis of 3-((3-bromo-2-fluorophenyl)amino)propanoic acid (3)

[0611] A brown solid title compound (1.85 g, 7.06 mmol, 63.9%) was obtained by synthesis in a manner similar to Step 1 of Example 35. MS(M+H) + =261.90

[0612] Step 2. Synthesis of 1-(3-bromo-2-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0613] The white solid title compound (572.0 mg, 1.99 mmol, 52.2%) was synthesized in a manner similar to Step 2 of Example 35. MS(M+H) + =286.90

[0614] Step 3. Synthesis of tert-butyl (3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-fluorophenyl)carbamate (6)

[0615] The white solid title compound (193.0 mg, 0.60 mmol, 42.8%) was synthesized in a manner similar to Step 3 of Example 35. MS(M+Na) + =346.00

[0616] Step 4. Synthesis of 1-(3-amino-2-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (7)

[0617] A white solid title compound (146 mg, 0.56 mmol, 94.3%, 2HCl salt) was obtained by synthesis similar to Step 4 of Example 35. MS(M+H) + =224.00

[0618] Step 5. Synthesis of benzyl ((1r,4r)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (9)

[0619] The white solid title compound (27.4 mg, 0.05 mmol, 21.4%) was synthesized in a manner similar to Step 5 of Example 35. MS(M+H) + =552.20

[0620] Step 6. Synthesis of 1-(3-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-fluorophenyl)dihydropyrimidine-2,4(1H,3H)-dione (10)

[0621] The title compound of the yellow oil (32.0 mg, 0.06 mmol, crude, 3TFA salt) was obtained by synthesis in a manner similar to Step 6 of Example 35. MS(M+H) + =418.10

[0622] Step 7. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-fluorophenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 39)

[0623] A yellow solid title compound (1.90 mg, 3.54%, 91.9% purity) was obtained by synthesis similar to Step 7 of Example 35. MS(M+H) + =811.30

[0624] Example 40. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)-[1,4'-bipiperidin]-1'-yl)-3-methoxybenzamide (Compound 40)

[0625]

[0626] Step 1. Synthesis of 3-((3-bromo-2-methylphenyl)amino)propanoic acid (3)

[0627] The title compound (1.61 g, 58.1% yield) was obtained by synthesis in a manner similar to Step 1 of Example 33. MS(M+2) + =259.90

[0628] Step 2. Synthesis of 1-(3-bromo-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0629] The title compound (944 mg, 53.4% ​​yield) was obtained by synthesis in a manner similar to Step 2 of Example 33. MS(M+2) + =284.95

[0630] Step 3. Synthesis of tert-butyl 4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)piperidine-1-carboxylate (6)

[0631] 1-(3-bromo-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (500 mg, 1.77 mmol), tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (757 mg, 3.53 mmol), tBu Xphos Pd G3 (140 mg, 0.18 mmol), and tBuOK (595 mg, 5.30 mmol) were dissolved in dioxane (3 mL) and stirred at 90°C for 6 hours. The mixture was filtered with Celite using EtOAc and dried under reduced pressure. The residue was purified by flash silica gel chromatography (0–100% EtOAc:n-hexane) to obtain the title compound (221 mg, 30.0% yield). MS(M+Na) + =439.10

[0632] Step 4. Synthesis of 1-(2-methyl-3-((piperidine-4-ylmethyl)amino)phenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride (7)

[0633] The title compound (212 mg, crude) was obtained by synthesis in a manner similar to Step 4 of Example 33. MS(M+H) + =317.10

[0634] Step 5. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-2-methylphenyl)amino)methyl)-[1,4'-bipiperidine]-1'-yl)-3-methoxybenzamide (Compound 40)

[0635] A white solid title compound (6.60 mg, 8.01% yield, 96.5% purity) was obtained by synthesis similar to Step 5 of Example 33. MS(M+H) + =808.30

[0636] 1 H NMR (400 MHz, DMSO-d6) δ = 10.23 (s, 1H), 9.23 (s, 1H), 8.40 (d, J = 8.88 Hz, 1H), 7.89 (s, 1H), 7.61 (s, 1H), 7.41-7.39 (m, 2H), 7.02 (t, J = 7.90 Hz, 1H), 6.47 (d, J = 7.92 Hz, 2H), 4.96 (t, J = 5.28 Hz, 1H), 4.43 - 4.37 (m, 1H), 4.31 (q, J = 6.65 Hz, 1H), 3.92 (s, 3H), 3.72 - 3.65 (m, 1H), 3.45 - 3.42 (m, 1H), 3.23 (s, 3H), 3.04 - 2.95 (m, 4H), 2.90 - 2.87 (m, 2H), 2.80 - 2.71 (m, 3H), 2.68 - 2.61 (m, 1H), 2.24 - 2.21 (m, 1H), 2.10 (t, J = 10.94 Hz, 2H), 2.04 - 1.98 (m, 2H), 1.90 (s, 3H), 1.77 - 1.75 (m, 7H), 1.59 (brs, 4H), 1.23 - 1.15 (m, 7H)

[0637] Example 41. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-5-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 41)

[0638]

[0639] Step 1. Synthesis of 3-((3-bromo-5-methylphenyl)amino)propanoic acid (3)

[0640] A yellow solid title compound (1.70 g, 61.3% yield) was obtained by synthesis similar to Step 1 of Example 35. MS(M+H) + =258.90.

[0641] Step 2. Synthesis of 1-(3-bromo-5-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (4)

[0642] A white solid title compound (777.5 mg, 41.7% yield) was obtained by synthesizing in a manner similar to Step 2 of Example 35.

[0643] 1 H NMR (400 MHz, DMSO-d6) δ = 10.42 (s, 1H), 7.38 (s, 1H), 7.29 (s, 1H), 7.17 (s, 1H), 3.78 (t, J = 6.60 Hz, 2H), 2.70 (t, J = 6.59, 2H), 2.32 (s, 3H).

[0644] Step 3. Synthesis of tert-butyl (3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-5-methylphenyl)carbamate (5)

[0645] The title compound (133.5 mg, 59.2% yield) as a light grayish-brown solid was synthesized in a manner similar to Step 3 of Example 35. MS(2M+Na) + =661.20.

[0646] Step 4. Synthesis of 1-(3-amino-5-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (6)

[0647] The title compound as an ivory solid (107.6 mg, 101% yield, hydrochloride, crude) was synthesized in a manner similar to Step 4 of Example 35.

[0648] 1 H NMR (400 MHz, DMSO-d6) δ = 10.37 (s, 1H), 6.86 (s, 2H), 6.73 (s, 1H), 3.75 (t, J = 6.64 Hz, 2H), 2.70 (t, J = 6.65 Hz, 2H), 2.28 (s, 3H).

[0649] Step 5. Synthesis of benzyl ((1r,4r)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-5-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (8)

[0650] The title compound as an ivory solid (153.7 mg, 57.2% yield) was synthesized in a manner similar to Step 5 of Example 35. MS(M+H) + =548.25.

[0651] Step 6. Synthesis of 1-(3-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-5-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (9)

[0652] The title compound (344.9 mg, 233% yield, TFA salt, crude) as a light grayish-brown solid was synthesized in a manner similar to Step 6 of Example 35. MS(M+H) + =414.20.

[0653] Step 7. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-5-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 41)

[0654] A light grayish-brown solid title compound (3.0 mg, 93.0% purity, 2.8% yield) was obtained by synthesis similar to Step 7 of Example 35. MS(M+H) + =807.40.

[0655] 1 H NMR (400 MHz, , DMSO-d6)δ = 10.22 (s, 1H), 8.41 (d, J = 8.92 Hz, 1H), 8.02 (d, J = 7.60 Hz, 1H), 7.90 (s, 1H), 7.62 (s, 1H), 7.49 - 7.47 (m, 2H), 6.31 - 6.27 (m, 3H), 5.63 (br s, 1H), 4.43 (t, J = 7.81 Hz, 1H), 4.32 (q, J = 6.69 Hz, 1H), 3.94 (s, 3H), 3.68 (t, J = 6.64Hz, 3H), 3.24 (s, 3H), 2.86 (br s, 4H), 2.66 (t, J = 13.06 Hz, 2H), 2.18 (s, 3H), 2.03 - 1.98 (m, 1H), 1.94 - 1.91 (m, 3H), 1.82 - 1.74 (m, 3H), 1.62 (s, 10H), 1.51 - 1.45 (m, 2H), 1.37 (t, J = 9.57 Hz, 1H), 1.24 - 1.22 (m, 7H)

[0656] Example 42. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-4-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 42)

[0657]

[0658] Step 1. Synthesis of 3-((2-methyl-5-nitrophenyl)amino)propanoic acid (2)

[0659] A yellow solid title compound (1.84 g, 60.2% yield) was obtained by synthesizing in a manner similar to Step 1 of Example 1.

[0660] 1 H NMR (400 MHz, DMSO-d6) δ = 12.28 (br s, 1H), 7.44(dd, J = 2.0, 8.0 Hz, 1H), 7.30 - 7.26 (m, 2H), 5.59 (br s, 1H), 3.43 (t, J = 6.8 Hz, 2H), 2.62 (t, J = 6.8 Hz, 2H), 2.21 (s, 3H),

[0661] Step 2. Synthesis of methyl 3-((2-methyl-5-nitrophenyl)amino)propanoate (3)

[0662] An orange sticky oil title compound (0.806 g, 99.6% yield) was obtained by synthesis in a manner similar to Step 2 of Example 1. MS(M+H) + =239.05

[0663] Step 3. Synthesis of methyl 3-(1-(2-methyl-5-nitrophenyl)ureido)propanoate (4)

[0664] An orange sticky oil title compound (0.570 g, crude) was obtained by synthesis in a manner similar to Step 3 of Example 1. MS(M+H) + =282.10

[0665] Step 4. Synthesis of 1-(2-methyl-5-nitrophenyl)dihydropyrimidine-2,4(1H,3H)-dione (5)

[0666] A yellow solid title compound (0.321 g, crude) was obtained by synthesizing in a manner similar to Step 4 of Example 1.

[0667] 1 H NMR (400 MHz, DMSO-d6) δ = 10.46 (s, 1H), 8.23 ​​(s, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.57 (d, J= 8.5 Hz, 1H), 3.95 - 3.83 (m, 1H), 3.57 - 3.53 (m, 1H), 2.80 - 2.74 (m, 2H), 1.97 (s, 3H)

[0668] Step 5. Synthesis of 1-(5-amino-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (6)

[0669] A light gray solid title compound (0.276 g, 97.6% yield) was obtained by synthesizing in a manner similar to Step 5 of Example 1.

[0670] 1 H NMR (400 MHz, DMSO-d6) δ = 10.22 (s, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.44 (d, J = 8.0 Hz, 1H), 6.40 (s, 1H), 4.95 (s, 2H), 3.69 - 3.62 (m, 1H), 3.48 - 3.42 (m, 1H), 2.74 - 2.50 (m, 2H), 2.00 (s, 3H)

[0671] Step 6. Synthesis of benzyl ((1r,4r)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-4-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)carbamate (8)

[0672] An ivory-colored solid title compound (95.2 mg, 66.3% yield) was obtained by synthesis in a manner similar to Step 6 of Example 1. MS(M+H) + =548.20

[0673] Step 7. Synthesis of 1-(5-(((1-((1r,4r)-4-aminocyclohexyl)piperidine-4-yl)methyl)amino)-2-methylphenyl)dihydropyrimidine-2,4(1H,3H)-dione (9)

[0674] A brown sticky oil title compound (130 mg, 179% yield, 3TFA salt, crude) was obtained by synthesis in a manner similar to Step 7 of Example 1. MS(M+H) + =414.20

[0675] Step 8. Synthesis of 4-(((R)-8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-4-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-3-methoxybenzamide (Compound 42)

[0676] A white solid title compound (8.50 mg, 13.0% yield, 89.9% purity) was obtained by synthesis similar to Step 8 of Example 1. MS(M+H) + =807.30

[0677] 1 H NMR (400 MHz, DMSO-d6) δ = 10.23 (s, 1H), 8.41 (d, J = 8.5 Hz, 1H), 8.01(d, J = 7.1 Hz, 1H), 7.89 (s, 1H), 7.61 (s, 1H), 7.46 (s, 2H), 6.93 (d, J = 8.2 Hz, 1H), 6.47 - 6.41 (m, 2H), 5.52 - 5.50 (m, 1H), 4.45 - 4.40 (m, 1H), 4.31 (q, J = 6.4 Hz, 1H), 4.03 - 3.98 (m, 1H), 3.93 (s, 3H), 3.75 - 3.67 (m, 2H), 3.23 (s, 3H), 2.84 - 2.83 (m, 3H), 2.72 - 2.65 (m, 2H), 2.32 - 2.27 (m, 2H), 2.17 - 2.12 (m, 2H), 2.00 (s, 3H), 1.93 - 1.87 (m, 3H), 1.78 - 1.66 (m, 8H), 1.61 - 1.53 (m, 2H), 1.50 - 1.45 (m, 1H), 1.38 - 1.35 (m, 4H), 1.23 - 1.12 (m,6H)

[0678] Example 43. Synthesis of (R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-N-(6-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)phenyl)amino)methyl)piperidine-1-yl)spiro[3,3]heptane-2-yl)-3-methoxybenzamide (Compound 43)

[0679]

[0680] A white solid title compound (3.60 mg, 8.19%, 88.3% purity) was obtained by synthesis similar to Step 5 of Example 33. MS(M+H) + =805.30

[0681] 1 H NMR (400 MHz, DMSO-d6) δ = 10.25 (s, 1H), 8.42 (d, J= 9.0 Hz, 1H), 8.38 (s, 1H), 7.90 (s, 1H), 7.62 (s, 1H), 7.47 (s, 2H), 7.05 (s, 1H), 6.50 (s, 1H), 6.46 - 6.40 (m, 2H), 5.73 (s, 1H), 4.45 - 4.38 (m, 1H), 4.33 (d, J = 8.2 Hz, 2H), 3.94 (s, 3H), 3.70 (d, J = 6.7 Hz, 2H), 3.24 (s, 3H), 2.89 - 2.83 (m, 2H), 2.80 - 2.71 (m, 2H), 2.67 (d, J = 6.9 Hz, 4H), 2.26 - 1.93 (m, 8H), 1.82 - 1.73 (m, 6H), 1.67 - 1.58 (m, 4H), 1.52 - 1.42 (m, 2H), 1.24 (d, J = 6.8 Hz, 5H)

[0682] Example 44. Synthesis of N-((1r,4R)-4-(4-(((3-(2,4-dioxotetrahydropyrimidine-1(2H)-yl)-4-methylphenyl)amino)methyl)piperidine-1-yl)cyclohexyl)-4-(((R)-8-isopropyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridine-2-yl)amino)-3-methoxybenzamide (Compound 44)

[0683]

[0684] A white solid title compound (9.40 mg, 44.1% yield, 95.0% purity) was obtained by synthesis similar to Step 8 of Example 1. MS(M+H) + =781.40

[0685] 1 H NMR (400 MHz, DMSO) δ = 10.22 (s, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.88 (s, 1H), 7.60 (s, 1H), 7.51 - 7.43 (m, 2H), 6.92 (d, J = 8.3 Hz, 1H), 6.48 - 6.42 (m, 1H), 6.40 (d, J = 2.3 Hz, 1H), 5.50 (d, J= 5.5 Hz, 1H), 4.65 - 4.57 (m, 1H), 4.35 (q, J = 6.5 Hz, 1H), 3.93 (s, 3H), 3.77 - 3.67 (m, 2H), 3.49 - 3.41 (m, 1H), 3.22 (s, 3H), 2.93 - 2.75 (m, 4H), 2.74 - 2.61 (m, 2H), 2.36 - 2.23 (m, 2H), 2.14 (t, J = 11.0 Hz, 2H), 1.99 (s, 3H), 1.92 - 1.85 (m, 2H), 1.82 - 1.67 (m, 4H), 1.62 - 1.57 (m, 1H), 1.53 - 1.40 (m, 2H), 1.39 - 1.26 (m, 7H), 1.23 (d, J = 6.8 Hz, 3H), 1.17 - 1.05 (m, 2H).

[0686] The structures of the above-described example compounds 1-44 are summarized in the following table.

[0687] [Table 1]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696] The comparative examples listed in the table below can be manufactured by referring to, for example, International Patent Publication WO2024 / 117789 for Comparative Examples 1 and 2, International Patent Publication WO2025 / 239662 for Comparative Examples 4-6 for Comparative Examples 4-6.

[0697] [Table 2]

[0698]

[0699] <Experimental Example>

[0700] Experimental Example 1. Evaluation of PLK1 protein degradation ability

[0701] Preparation and culture of HeLa LgBiT(Plk1-HiBiT KI) cell line

[0702] A cell line capable of stable expression was constructed by transfecting HeLa cell lines with an LgBiT vector. Subsequently, gRNA and a donor were designed to express the HiBiT amino acid sequence following the C-terminal of the Plk1 gene within the cells, and these were introduced into the cells along with a CRISPR / Cas9 expression vector. Cells that had completed knock-in were selected, subcultured, and used in the experiment.

[0703] For cell counting, a Thermo cell counter (Catalog # AMQAX1000) and a 0.4% trypan blue solution were used.

[0704] For cell culture, DMEM (Gibco, Cat. No. 11995-065; Lot. No. 3202862), FBS (Gibco, Cat. No. 16000-044; Lot. No. U3318512P), Penicillin / Streptomycin (PS) (Gibco, Cat. No. 15140-122; Lot. No. 268394), 150 mm cell culture dish (SPL, Cat. No. 20150), 96-well white plate (SPL, Cat. No. 30196), PBS pH 7.4 (Gibco, Cat. No. 10010-023; Lot. No. 3320100), TrypLE TMExpress (Gibco, Cat. No. 12605-010; Lot. No. 3259235), a counting chamber (Hemocytometer) (Hirschmann, Cat. No. 8100204), and 0.4% trypan blue solution (DYNEBIO, Cat. No. CBT3710; Lot. No. 20250717) were used.

[0705] Compound treatment and luciferase assay method of the present invention

[0706] The compound of the example was completely dissolved in DMSO (Sigma-Aldrich, Cat. No. D2438, Lot. No. RNBN8491) and used in the experiment.

[0707] For HeLa LgBiT (Plk1-HiBiT KI), compounds were treated following thymidine blocking and release, and the procedure is as follows. Thymidine (Sigma-Aldrich, Cat. No. T9250-5G, WXBD0181V) was completely dissolved in distilled water for the experiment. For thymidine blocking, the sample was incubated for 24 hours after treatment with 2 mM thymidine. For release and chemical treatment, the medium was suctioned and washed with PBS. TrypLE TM Added and incubated in a 37°C CO2 incubator for 5 minutes. Added complete media and counted the neutralized cells using a counter. 3.3 x 10⁶ in each well of a 96-well white plate 4 Dog cells were seeded with a total medium volume of 150 µl and incubated in a CO2 incubator for 2 hours.

[0708] A mixture was prepared by adding 2 µL of 100X Endurazine (Promega, Cat. No. N2572; Lot. No. 0000577987) and 48 µL of complete media to each well, and 50 µL was added to each well. After incubating in a CO2 incubator for an additional 2 hours, the compounds were treated to a concentration of 300 nM or 30 nM. Luminescence was tracked in real-time using a plate reader (BMG Labtech, CLARIOstar Plus). The luminescence values ​​were calculated 9 hours after the treatment. The results are shown below.

[0709] [Table 3]

[0710]

[0711] Experimental Example 2. Evaluation of cytotoxicity with comparative example compounds

[0712] Cell line culture

[0713] Lung cancer-derived cell lines NCI-H69 (hereinafter H69) and NCI-H526 (hereinafter H526) were obtained from the Korea Cell Line Bank and used. For cell culture, RPMI 1640 medium (Gibco, Cat. No. 22400-089; Lot. No. 3256035), Eagle's Minimum Essential Medium (EMEM) (ATCC, Cat. No. 30-2003, Lot. No. 8250620A), RPMI 1640 Medium (ATCC modification) (ATCC, Cat. No. A10491-01, Lot. No. 3062369) were mixed with FBS (Gibco, Cat. No. 16000-044; Lot. No. U3318512P), penicillin / streptomycin (PS) (Gibco, Cat. No. 15140-122; Lot. No. 268394), or MEM Non-Essential Amino Acids Solution (100X) (Gibco, Cat. It was used in combination with No. 11140-050; Lot. No. 3259261), Sodium Pyruvate (100 mM) (Gibco, Cat. No. 11360-070; Lot. No. 3149807). A 75T cell culture flask (SPL, Cat. No. 71075), a 175T cell culture flask (SPL, Cat. No. 71175), a 100 mm cell culture dish (SPL, Cat. No. 20100), a 96-well cell culture plate (SPL, Cat. No. 30096), a counting chamber (Hemocytometer) (Hirschmann, Cat. No. 8100204), and a 0.4% trypan blue solution (DYNEBIO, Cat. No. CBT3710; Lot. No. 20250717) were used.

[0714] Compound treatment of the present invention

[0715] Comparative example, general ADC payload drugs (MMAE, DXd, Exatecan) and example compounds were completely dissolved in DMSO (Sigma-Aldrich, Cat. No. D2438, Lot. No. RNBN8491) and used in the experiment, and compared with Monomethyl auristatin E (MMAE) (MedChemExpress, Cat. No. HY-15162; Lot. No. 545511) and DXd (MedChemExpress, Cat. No. HY-13631D; Lot. No. 268112).

[0716] Each cell line was directly compared under substantially identical assay conditions using the same cell lines (NCI-H69 and NCI-H526) used in the cytotoxicity evaluation of the closest prior art, International Patent Publication WO2024 / 117789. Specifically, each cell line was placed in 96-well cell culture plates at a rate of 2 x 10⁶ per well. 3 , 4 x 10 3 , 5 x 10 3 , 6 x 10 3 , 7 x 10 3 , 8 x 10 3 , 2 x 10 4 , 3 x 10 4 , 5 x 10 4 , 1 x 10 5 Each cell was seeded to the extent of a dog, and the volume of the medium in each well was adjusted to 150 µl.

[0717] The intracellular treatment concentration of the compound of the present invention was varied for each compound of the example, and treatment was performed at different concentrations by diluting the maximum concentration by 1 / 3. After treating each well with the compound so that the total volume of the solution was 200 µl, the cells were cultured in a CO2 incubator (Thermo Fisher Science, Cat. No. 4111) for 5 days.

[0718] cytotoxicity test

[0719] After completing incubation, 20 µl of EZ-Cytox (DOGEN, Cat. NO. EZ-3000, Lot. No. DGB26M12) was added to each well of the 96-well plate, and the plates were incubated for 4 hours in an incubator at 37°C under 5% CO2 conditions. After incubation, the absorbance of the samples was measured using a plate reader (BMG Labtech, CLARIOstar Plus) set to 450 nm; the plates were shaken inside the reader for 3 minutes prior to measurement. The final measurements were organized into an Excel file, graphs were displayed using the Prism-GraphPad program (ver. 10), and the IC50, representing the cytotoxic effect on each cell line, was calculated. 50 The value was measured. The result is shown in Figure 1.

[0720] [Table 4]

[0721]

[0722] Experimental Example 3. Evaluation of cytotoxicity by various cancer types

[0723] Cytotoxicity was measured in the same manner for various solid tumor-derived cancer cell lines (SCLC: Small Cell Lung Cancer, TNBC: Triple-Negative Breast Cancer, BC: Breast Cancer, PC: Prostate Cancer, GC: Gastric Cancer; each cell line was obtained from the Korean Cell Line Bank or ATCC) to determine IC 50 The values ​​(nM) are shown in the table below.

[0724] [Table 5]

[0725]

[0726] Cytotoxicity was measured in the same manner for various blood cancer-derived cell lines (MM: Multiple Myeloma, AMoL: Acute Monocytic Leukemia, CML: Chronic Myeloid Leukemia) to determine IC 50 The values ​​(nM) are shown in the table below.

[0727] [Table 6]

[0728]

[0729] Experimental Example 4. Evaluation of Cytotoxicity of ADC Payloads on Drug-Resistant Cancers

[0730] For Compounds 10 and 18, which are the representative compounds of the present invention, we evaluated whether they maintain cell proliferation inhibitory activity even in cancer cell lines resistant to existing ADC payloads or ADC drugs. To this end, using parental cell lines and drug-resistant cell lines derived therefrom, the IC50 values ​​of each compound were evaluated. 50 and the resistance index (RI) was calculated.

[0731] The cell lines used for evaluation were ovarian cancer cell lines SK-OV-3 and DXd-resistant SK-OV-3 cell lines, lung cancer cell lines HCC4006 and MMAE-resistant HCC4006 cell lines, and gastric cancer cell lines NCI-N87 and DS-8201-resistant NCI-N87 cell lines. SK-OV-3 lineage cells were cultured in McCoy's 5A medium, while HCC4006 and NCI-N87 lineage cells were cultured in RPMI-1640 medium. FBS and penicillin / streptomycin were added to each medium. To maintain resistance, resistant cell lines were subcultured in media containing DXd, MMAE, or DS-8201, respectively, and an assay medium of the same composition without drugs was used for the evaluation of cell proliferation inhibitory activity.

[0732] DXd, compound 10, and compound 18 were evaluated in SK-OV-3 and DXd-resistant SK-OV-3 cell lines, and MMAE, compound 10, and compound 18 were evaluated in HCC4006 and MMAE-resistant HCC4006 cell lines. Additionally, DS-8201, compound 10, and compound 18 were evaluated in NCI-N87 and DS-8201-resistant NCI-N87 cell lines. Staurosporine was used as a positive control.

[0733] Each cell line was seeded into 96-well cell culture plates. SK-OV-3 cells were seeded at a density of 400 cells per well, DXd-resistant SK-OV-3 cells at 1,200 cells per well, HCC4006 and MMAE-resistant HCC4006 cells at 600 cells per well, and NCI-N87 and DS-8201-resistant NCI-N87 cells at 5,000 cells per well. After seeding the cells, each test compound was treated, and the cells were cultured for 7 days at 37°C under 5% CO2 conditions.

[0734] Each compound was treated by serially diluting it from a DMSO stock solution and then diluting it with analytical medium. Concentration-response curves were constructed for each compound by serially diluting it nine times in four-fold azeotropic ratios from the highest treatment concentration. Staurosporine, DXd, Compound 10, and Compound 18 started at a highest concentration of 3,000 nM, while MMAE and DS-8201 started at a highest concentration of 100 nM.

[0735] After the end of culture, cell viability was measured using a luminescence-based cell viability assay. IC50 in parent cell lines for the same compound. 50 IC in resistant cell lines relative to value 50 The ratio of the values ​​was calculated as the resistance index (RI).

[0736] A value closer to 1 indicates that the compound maintains cell proliferation inhibitory activity similar to that of the parent cell line in resistant cell lines, and a higher value indicates increased resistance to the compound in the resistant cell lines.

[0737] The IC of each compound measured 50 The values ​​and RI values ​​are shown in the table below.

[0738] [Table 7]

[0739]

[0740] [Table 8]

[0741]

[0742] [Table 9]

[0743]

[0744] Experimental Example 5. Evaluation of CRBN Binder Ring Opening Reactivity and Plasma Drug Stability

[0745] To evaluate the ring-opening stability of the ring structure of the CRBN binding moiety contained in the example compounds and comparative example compounds of the present invention following hydrolysis in plasma, the time-dependent retention rate (% Remaining) and ring opening rate (% Ring Opening) in human plasma were quantitatively analyzed by LC-MS / MS.

[0746] Reagent and Sample Preparation

[0747] A 500 μM stock solution was prepared by completely dissolving the compound of the example of the present invention in DMSO (Sigma-Aldrich, Cat. No. D2438). DMSO alone was used as a negative control. Human plasma (Human Plasma, Innovative Research, Cat. No. IPLANAH 100ML) was used as the plasma reagent. Solifenacin (Sigma-Aldrich, Cat. No. SML 2141) was prepared to a concentration of 25 ng / mL by dissolving it in methanol and used as the positive mode internal standard. Methanol (Honeywell, Cat. No. AH230-4), acetonitrile (Honeywell, Cat. No. AH015-4), ammonium formate (Sigma-Aldrich, Cat. No. 70221-25G-F), and formic acid (Sigma-Aldrich, Cat. No. F0507) were used for protein precipitation and mobile phase preparation. A liquid chromatograph (Agilent 1290 Infinity II) and a triple quadrupole mass spectrometer (AB Sciex, Qtrap 5500 Plus) were used for LC-MS / MS analysis.

[0748] Experimental Procedures and Conditions

[0749] To stabilize the plasma, human plasma stored at -20°C was slowly thawed in a 37°C water bath prior to the experiment. 495 μL of the thawed plasma was dispensed into microtubes and pre-stabilized in a shaking incubator (37°C, 200 rpm) for 5 minutes. All tests were performed with n = 3.

[0750] Compound processing and sampling

[0751] 5 μL of 500 μM stock solution was added to stabilized plasma to achieve a final test substance concentration of 5 μM (final volume 500 μL). Immediately after addition, 20 μL samples were collected at 0, 4, 24, 72, and 168 hours while maintaining conditions of 37°C and 200 rpm, and stored at -70°C.

[0752] After slowly thawing the stored sample at room temperature, 100 μL of internal standard (Solifenacin / Methanol, 25 ng / mL) was added and the protein was precipitated by vortexing for 30 seconds. Subsequently, the sample was centrifuged at 12,700 rpm at 4°C for 5 minutes, and 50 μL of the supernatant was dispensed into an LC-MS / MS vial for analysis.

[0753] Next, the pre-treated sample was analyzed by LC-MS / MS to calculate the peak area ratio (peak area of ​​test substance / peak area of ​​internal standard substance) of the test substance and the ring-open form.

[0754] The ring opening rate (%Ring Opening) was calculated according to the following formula using the peak area ratio at each time point. The results are as shown in the table below.

[0755] % Ring Opening = [Hourly ring-open form peak area ratio / (Hourly test substance peak area ratio + Hourly ring-open form peak area ratio)] × 100

[0756] [Table 10]

[0757]

[0758]

[0759] In addition, the remaining rate (% Remaining) was calculated according to the following formula using the peak area ratio at each time point.

[0760] % Remaining = (Ratio of test substance peak area by hour / Ratio of test substance peak area at hour 0) × 100

[0761] The survival rate is calculated as the relative survival rate compared to the 0-hour point, and values ​​exceeding 100% may be obtained due to measurement variability. This indicates that the compound remains structurally stable under the measurement conditions. The experimental results are as described in the table below.

[0762] [Table 11]

[0763]

[0764]

[0765] As a result, the compound of the embodiment of the present invention was very stable in human plasma for 168 hours, and the ring opening rate was also maintained at a low level.

[0766] Experimental Example 6. Evaluation of drug stability in a lysosomal environment

[0767] The solution stability of the compounds of the examples of the present invention (Compounds 3, 5, and 10) in a pH buffer corresponding to physiological conditions was evaluated. Stability was confirmed by quantitatively analyzing the change in purity of the compounds over time using HPLC under histidine buffer conditions (pH 5.0).

[0768] A 10 mM stock solution was prepared by completely dissolving each example compound in DMSO. Subsequently, 20 μL of each stock solution was added to 1,500 μL of the corresponding buffer and diluted 76-fold to prepare a test solution with a final DMSO concentration of approximately 1.3% and a target analysis concentration of approximately 132 μM.

[0769] Stability Test Conditions (Stability Parameters)

[0770] - Test temperature: Room Temperature and 37℃

[0771] - Measurement Timepoint: Initial (0 hours) and 24 hours

[0772] - Test concentration: Approximately 0.10–0.11 mg / mL (132 μM)

[0773] - Analysis Diluent: DMSO : Water = 9 : 1 (v / v), 2x dilution

[0774] Analysis and Results

[0775] Test solution was collected at each measurement time point (0 hours, 24 hours), diluted 2-fold with an analytical diluent (DMSO : purified water = 9 : 1, v / v), and analyzed by HPLC.

[0776] The solution stability of each compound was evaluated based on the main peak area and the associated degradation peak area measured in HPLC analysis. The degree of degradation after 24 hours was calculated as %RD (relative degradation). %RD was used as an indicator reflecting the degree of degradation products generated or the decrease in purity after 24 hours compared to the initial time point. In this experiment, a %RD value of less than 1.0 was evaluated as indicating that the compound was stable under those conditions.

[0777] The above results are shown in the table below.

[0778] [Table 12]

[0779]

[0780] Experimental Example 7. Evaluation of BCRP-mediated efflux resistance

[0781] Cell line culture

[0782] AsPC-1, a cell line derived from pancreatic cancer, was obtained from the Korea Cell Line Bank.

[0783] For cell culture, RPMI 1640 medium (Gibco, Cat. No. 22400-089; Lot. No. 3256035) was mixed with FBS (Gibco, Cat. No. 16000-044; Lot. No. U3318512P) and penicillin / streptomycin (PS) (Gibco, Cat. No. 15140-122; Lot. No. 268394). A 100 mm cell culture dish (SPL, Cat. No. 20100), a 96-well cell culture plate (SPL, Cat. No. 30096), a counting chamber (hemocytometer) (Hirschmann, Cat. No. 8100204), and 0.4% trypan blue solution (DYNEBIO, Cat. No. CBT3710; Lot. No. 20250717) were used.

[0784] Compound treatment of the present invention

[0785] The control payload drug (DXd) and the example compound were completely dissolved in DMSO (Sigma-Aldrich Cat. No. D2438, Lot. No. RNBN8491) and used in the experiment, and compared with DXd (MedChemExpress, Cat. No. HY-13631D; Lot. No. 268112).

[0786] Cell lines in a 96-well cell culture plate, 6 x 10⁶ per well 3 Seeding was performed to the extent of 100, and the volume of the medium in each well was adjusted to 150 µl.

[0787] Ko143 hydrate (Sigma-Aldrich, Cat. No. K2144, Lot. No. 214898), a BCRP inhibitor, was treated to a final concentration of 1 μM, and the intracellular treatment concentration of the compound of the present invention was varied for each compound of the example, with treatments performed at different concentrations by diluting the maximum concentration by 1 / 3. After treating each well with the compound to a total solution volume of 200 μL, the cells were cultured in a CO2 incubator (Thermo Fisher Science, Cat. No. 4111) for 5 days.

[0788] cytotoxicity test

[0789] After completing incubation, 20 µl of EZ-Cytox (DOGEN, Cat. NO. EZ-3000, Lot. No. DGB26M12) was added to each well of the 96-well plate, and the plates were incubated for 4 hours in an incubator at 37°C under 5% CO2 conditions. After incubation was complete, the absorbance of the samples was measured using a plate reader (BMG Labtech, CLARIOstar Plus) set to a wavelength of 450 nm; the plates were shaken inside the plate reader for 3 minutes prior to measurement. The final measurements were organized into an Excel file, graphs were displayed using the Prism-GraphPad program (ver. 10), and IC 50 The value was measured.

[0790] To evaluate the effect of BCRP-mediated drug efflux, IC5 under conditions without BCRP inhibitor treatment 50 IC at the value and processed conditions 50 The values ​​were calculated for each, and the ratio was determined. Since a higher ratio value can be interpreted as a significant increase in cytotoxicity caused by BCRP inhibitor treatment, it was assessed that the cytotoxicity of the corresponding compound is relatively significantly affected by BCRP-mediated drug efflux.

[0791] The results are shown in the table below.

[0792] [Table 13]

[0793]

[0794] Experimental Example 8. Evaluation of P-gp (P-glycoprotein)-mediated drug efflux resistance

[0795] Cell line culture

[0796] HCT-15, a colorectal cancer-derived cell line, was obtained from the Korea Cell Line Bank.

[0797] For cell culture, RPMI 1640 medium (Gibco, Cat. No. 22400-089; Lot. No. 3256035) was mixed with FBS (Gibco, Cat. No. 16000-044; Lot. No. U3318512P) and penicillin / streptomycin (PS) (Gibco, Cat. No. 15140-122; Lot. No. 268394). A 100 mm cell culture dish (SPL, Cat. No. 20100), a 96-well cell culture plate (SPL, Cat. No. 30096), a counting chamber (Hemocytometer) (Hirschmann, Cat. No. 8100204), and 0.4% trypan blue solution (DYNEBIO, Cat. No. CBT3710; Lot. No. 20250717) were used.

[0798] Compound treatment of the present invention

[0799] The compound of the example was completely dissolved in DMSO (Sigma-Aldrich Cat. No. D2438, Lot. No. RNBN8491) and used in the experiment, and compared with Monomethyl auristatin E (MMAE) (MedChemExpress, Cat. No. HY-15162; Lot. No. 545511).

[0800] Cell lines in a 96-well cell culture plate at 3.5 x 10⁶ per well 3 Seeding was performed to the extent of 100, and the volume of the medium in each well was adjusted to 150 µl.

[0801] The P-gp inhibitor tariquidar (MedChemExpress Cat. No. HY-10550, Lot. No. 24109) was treated to a final concentration of 1 μM, and the intracellular treatment concentration of the compound of the present invention was varied for each compound of the example, with treatments performed at different concentrations by diluting the maximum concentration by 1 / 3. After treating each well with the compound to a total solution volume of 200 μL, the cells were cultured in a CO2 incubator (Thermo Fisher Science, Cat. No. 4111) for 5 days.

[0802] Cytotoxicity test

[0803] After completing incubation, 20 µl of EZ-Cytox (DOGEN, Cat. NO. EZ-3000, Lot. No. DGB26M12) was added to each well of the 96-well plate, and the plates were incubated for 4 hours in an incubator at 37°C under 5% CO2 conditions. After incubation was complete, the absorbance of the samples was measured using a plate reader (BMG Labtech, CLARIOstar Plus) set to a wavelength of 450 nm; the plates were shaken inside the plate reader for 3 minutes prior to measurement. The final measurements were organized into an Excel file, graphs were displayed using the Prism-GraphPad program (ver. 10), and IC 50 The value was measured.

[0804] To evaluate the effect of P-gp-mediated drug efflux, IC50 under conditions without P-gp inhibitor treatment 50 IC at the value and processed conditions 50 The values ​​were calculated for each, and the ratio was determined. Since a higher ratio value can be interpreted as a significant increase in cytotoxicity caused by P-gp inhibitor treatment, it was assessed that the cytotoxicity of the corresponding compound is relatively significantly affected by P-gp-mediated drug efflux.

[0805] The results are shown in the table below.

[0806] [Table 14]

[0807]

[0808] Experimental Example 9. Evaluation of ICD (Immunogenic Cell Death) Induction Efficacy

[0809] Compound 10, the representative compound of the present invention, was evaluated to determine whether it induces immunogenic apoptosis (ICD) related indicators in cancer cells. As ICD-related indicators, cell surface calreticulin (CRT) exposure, extracellular ATP release, and HMGB1 release were measured.

[0810] After culturing HCT-116 colorectal cancer cell lines, they were treated with a DMSO control, the comparative drug DXd, and Compound 10 of the present invention, respectively. For the CRT exposure analysis, cell surface CRT was stained with a FITC-labeled CRT detection reagent under non-permeable conditions after compound treatment, viable cells were identified using 7-AAD, and the proportion of CRT-positive cells or fluorescence intensity was measured by flow cytometry. For the extracellular ATP release analysis, the luminescence signal corresponding to extracellular ATP after compound treatment was measured in a time-dependent manner using the Realtime-Glo Extracellular ATP assay. For the HMGB1 release analysis, HMGB1 in the culture supernatant after compound treatment was quantified using a Lumit®-based immunoassay and corrected using the cell viability signal of the same well. As a result, Compound 10 of the present invention was confirmed to increase ICD-related indicators in HCT-116 cells, including cell surface CRT exposure (Fig. 13), extracellular ATP release (Fig. 14), and HMGB1 release (Fig. 15).

Claims

Claim 1 PLK1 protein degradation-inducing compound represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula, R1 is CH3, OCH3, F or Cl; R2 is H, F, OH, CH3, OCH3 or CN; R3 is H, F, OCH3, OH or OCH2CH2OH; and X is CH or N. Claim 2 PLK1 protein degradation-inducing compound that is any one of the following compounds 1 to 44: Claim 3 A PLK1 protein degradation-inducing compound having subnanomolar levels of cytotoxicity and long-term drug stability, according to claim 1 or 2. Claim 4 In claim 3, a PLK1 protein degradation-inducing compound that can be utilized as an ADC payload. Claim 5 An anticancer pharmaceutical composition comprising the PLK1 protein degradation-inducing compound of claim 1 or 2 as an active ingredient.

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