WO2025221647A1 - Tricyclic compounds and methods of use thereof - Google Patents

Abstract

The application provides a novel compound and the pharmaceutical composition and formulation thereof, as well as a method of using the compound for the treatment of, inter alia, a condition or disorder related to dysregulation of CDK2, such as a cancer.

Description

TRICYCLIC COMPOUNDS AND METHODS OF USE THEREOF

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 63/635,047, filed on April 17, 2024. The entire teachings of the above application are incorporated herein by reference.

FIELD OF THE APPLICATION

This application relates to new compositions of biologically active compounds that are useful for treating diseases and methods of making such compositions.

BACKGROUND

Cyclin-dependent kinases (CDKs) and related serine/threonine protein kinases are crucial cellular enzymes that regulate cell division and proliferation. CDKs 1-4, 6, 10, and 11 directly control cell cycle progression, while CDKs 5 and 7-9 may indirectly influence it, for instance, through the activation of other CDKs or the regulation of transcription or neuronal functions. Activation of CDK catalytic units occurs upon binding to regulatory subunits called cyclins, followed by phosphorylation. Cyclins, upon expression, regulate different phases of the cell cycle (Gl, S, G2, and M), serving as important checkpoints. Key regulators of cell cycle progression include CDKl/cyclin B, CDK2/cyclin A, CDK2/cyclin E, CDK4/cyclin D, CDK6/cyclin D, and likely other heterodimers.

CDK2 participates in a range of biological activities. CDK2 is a key cell cycle regulator, active from the late Gl -phase and throughout the S-phase. CDK2 is involved in DNA damage response (DDR) through the homologous recombination (HR) pathway. CDK2 also regulates aspects of apoptotic pathways. Cyclin El (CCNE1), cyclin E2 (CCNE2), cyclin Al (CCNA1), and cyclin A2 (CCNA2), along with p21Cipl/Wafl, p27Kipl and p57Kip2 (the cyclin dependent kinase inhibitors of the cyclin-CDK2 complex) are the main regulators of CDK2 activity. In cancer, dysregulation of the binding of CDK2 by cyclin El, E2, Al, or A2 or the activity of the CDK inhibitor proteins may occur (Tadesse et al., Drug Discovery Today, Volume 25, Number 2 Feb. 2020). Overexpression of CDK2/cyclin E is linked to aberrant cell cycle regulation. This complex plays a critical role in regulating the Gl/S transition, histone biosynthesis, and centrosome duplication. Progressive phosphorylation of retinoblastoma (Rb) by CDK4/6/cyclin D and CDK2/cyclin E releases the G1 transcription factors, E2Fs, promoting entry into the S phase. Activation of CDK2/cyclin A during early S-phase allows for phosphorylation of endogenous substrates facilitating DNA replication and activation of E2Fs, promoting S-phase progression (Asghar et al., Nat. Rev. Drug. Discov., 2015; 14(2): 130-146).

CDK2 shows significant over-activation in numerous cancer types, for example CCNE1 or CCNE2 amplified tumors identified occurring in ovarian and breast cancer (Scaltriti et al., Proc. Natl Acad. Sci. USA 108, 3761-3766 (2011); Etemadmoghadam et al., Proc. Natl Acad. Sci. USA 110, 19489-19494 (2013)). Although CDK2 is an appealing target for cancer treatment, most inhibitors designed to target CDK2 act as competitors to ATP, displaying either non-specific effects or high toxicity levels, thus frequently leading to unsuccessful outcomes in clinical trials. No therapeutic agents selectively targeting CDK2 have been FDA approved yet. Hence, there is a pressing need to discover CDK2 inhibitors with novel activity profiles.

SUMMARY OF THE INVENTION

The invention is based on the discovery of the unexpected therapeutic effect of a novel tricyclic compound on treating conditions or disorders related to the dysregulation of CDK2, such as cancers. The compound comprises a core structure of three fused rings. The compound can be represented by Formula (I):

Formula (I), wherein all the variables are as defined below. The compound can be used for treating a variety of cancers. Preferably, the compounds of the invention are selective CDK2 inhibitors, i.e., the compounds have no or low off-target binding with other CDK-family kinases than CDK2, most notably CDK1. Advantages associated with such selectivity include facilitating efficacious dosing and reducing CDK1 -mediated on-target toxicities.

DETAILED DESCRIPTION OF FIGURES

Figure 1. Anti-tumor growth activity in a CCNE amplified (CN=12) gastric PDX model CRT00292. NOG mice were inoculated subcutaneously with tumor fragments and randomized (n=7/group) when tumors reached an average of 150-200 mm³. Animals were dosed daily with XC219 43 (30 and 100 mpk xl4d, i.p.). In the highest dose group 3 animals were moribund or withdrawn from treatment. One-way ANOVA, Dunnett’s multiple comparison test was performed reporting statistical significance of lOOmpk group, p value = 0.0079.

DETAILED DESCRIPTION

Compound

The compounds of the invention are therapeutic inhibitors. In preferred embodiments, the compounds of the invention are selective CDK2 inhibitors. As used herein, the term “selective CDK2 inhibitor” means a compound which selectively inhibits CDK2 over other CDKs and the kinome. Namely, a selective CDK2 inhibitor has no or low activity against other CDKs and the kinome. A selective CDK2 inhibitor's inhibitory activity against CDK2 is more potent in terms of ICso value (e.g., the ICso value is nanomolar) when compared with its inhibitory activity against other CDKs and many other kinases. Potency can be measured using known biochemical assays.

The compounds of the invention are preferably designed to enhance the interaction with CDK2 and the selectivity against other CDKs and the kinome, e.g., CDK1. One or more functional groups can be selectively incorporated into the compounds to target the binding pocket of CDK2. For example, the compounds of the invention can align with the binding pocket of CDK2 to allow for stacking interactions (such as pi stacking, e.g., with Phe80 of CDK2), stabilizing the binding between the compounds and CDK2 over other CDKs and the kinome. As another example, the compounds of the invention can also be designed to form hydrogen bonding with specific atoms within CKD2 (e.g., with Aspl45 and/or Glu51 of CDK2).

In some embodiments, the compounds of the invention are selective against CDK2 versus CDK1. In some such embodiments, compounds show at least 5-fold selectivity for CDK2 versus CDK1. In some such embodiments, compounds show at least 10-fold selectivity for CDK2 versus CDK1. In other embodiments, compounds show at least 20-fold selectivity for CDK2 versus CDK1. In specific embodiments, compounds show at least 30- fold selectivity for CDK2 versus CDK1. In certain embodiments, compounds show at least 40-fold selectivity for CDK2 versus CDK1. In other embodiments, compounds show at least 50-fold selectivity for CDK2 versus CDK1. For example, compounds show 100-fold selectivity for CDK2 versus CDK1. In some embodiments, the compounds of the invention are selective for CDK2 versus CDK4 and/or CDK6. In some such embodiments, compounds show at least 10-fold selectivity for CDK2 versus CDK4 and/or CDK6. In other embodiments, compounds show at least 20-fold selectivity for CDK2 versus CDK4 and/or CDK6. In specific embodiments, compounds show at least 30-fold selectivity for CDK2 versus CDK4 and/or CDK6. In additional embodiments, compounds show more than 30-fold selectivity for CDK2 versus CDK4 and/or CDK6.

In some embodiments, compounds of the invention have the advantage of good metabolic stability. One indicator of good metabolic stability is high microsomal stability. Hepatic metabolism is a predominant route of elimination for small molecule drugs. The clearance of compounds by hepatic metabolism can be assessed in vitro using human liver microsomes (HLMs) or human hepatocytes. Compounds are incubated with HLMs plus appropriate co-factors or human hepatocytes, and compound depletion is measured to determine an in vitro intrinsic clearance (Clint). The Clint is scaled to total body clearance (CL), and a hepatic extraction ratio (ER) is determined by dividing CL to standard human hepatic blood flow. Compounds that have a low hepatic extraction ratio are considered to have good metabolic stability. In some embodiments, a compound of the disclosure has a calculated ER of <0.3, <0.4, <0.5, <0.6. The compound of the invention comprises a tricyclic core structure and is represented by Formula (I):

Formula (I), or a pharmaceutically acceptable salt, a stereoisomer and a mixture of stereoisomers, or a prodrug thereof.

Xi and X2 are each independently selected from O, S, NRY. In some embodiments, Xi is NRY (preferably NH) and X2 is O. In some embodiments, both Xi and X2 are NRY (preferably NH). In some embodiments, Xi is O and X2 is NRY (preferably NH). In some preferred embodiments, both Xi and X2 are O. RY is selected from H, D and substituted or unsubstituted alkyl.

Ri is selected from H, D, halogen, substituted or unsubstituted alkyl (such as -CD3), substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, ORA, CN, NRBRC, NRAC(O)RA, S(O)RA, S(O)₂RA, SO₂NRBRC, SO₃RA, COORA, C(O)RA, and C(O)NRBRC.

Each RA, RB, and Rc is independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl; or RB and Rc can be taken together with the nitrogen to which they are attached to form a substituted or unsubstituted 5, 6, 7, or 8 membered ring.

R2 is selected form H, D, halogen, substituted or unsubstituted alkyl (such as -CD3), substituted or unsubstituted alkenyl. In some embodiments, R2 is halogen, preferably bromine or chlorine, more preferably bromine. In some embodiments, R2 is an unsubstituted alkyl, preferably methyl.

R3 is selected from H, D, OH, a C1-6 alkoxyl, a C1-6 alkyl. Preferably, R3 is selected from H, D and OH, more preferably, OH.

R4 is selected form H, D, halogen, substituted or unsubstituted alkyl (such as -CD3), substituted or unsubstituted alkenyl. In some embodiments R4 is an unsubstituted alkyl, preferably methyl.

Rs and Re are independently selected form hydroxy, substituted or unsubstituted alkyl and substituted or unsubstituted alkoxy. In some cases, Rs is hydroxy or substituted or unsubstituted alkoxy, and Re is a substituted or unsubstituted alkyl. In some cases, Re is hydroxy or substituted or unsubstituted alkoxy, and Rs is a substituted or unsubstituted alkyl.

R7 and Rs are independently selected from hydrogen, hydroxy and substituted or unsubstituted alkoxy. In some cases, R7 is hydroxy or substituted or unsubstituted alkoxy, and Rs is hydrogen. In some cases, Rs is hydroxy or substituted or unsubstituted alkoxy, R7 is hydrogen.

Or, Rs is -ORx, Rx is a substituted or unsubstituted alkyl, ester, amino acid, acyl, amine, or amide. For examples, Rs is -OAc, -OC(O)O-alkyl (such as -OC(O)O-CH3), - OC(O)-alkyl, -O-amino acid. Or, Rs further optionally comprises a linker such as a polyethylene glycol (PEG) (e.g., PEG4-12). For example, Rs is -OC(O)-PEG4-biotin.

Or one of Rs and Re, and one of R7 and Rs (i.e., Rs and R7, Rs and Rs, Re and R7, or Re and Rs), together with the carbon atoms to which they are attached, form a heterocycloalkyl containing at least two oxygen atoms, wherein two oxygen atoms are in a ketal arrangement, wherein the heterocycloalkyl further optionally comprises a fused or spiro ring structure.

In some preferred cases, one of Rs and Re, and one of R7 and Rs (i.e., Rs and R7, Rs and Rs, Re and R7, or Re and Rs), together with the carbon atoms to which they are attached, form a five-membered heterocycloalkyl represented by the structure as shown below: wherein R_q and R_P are each independently a substituted or unsubstituted alkyl; or R_q and R_P together with the intervening atoms form a 4- to 8-membered substituted or unsubstituted cycloalkyl, preferably a 5- or 6-membered unsubstituted cycloalkyl.

R9 is selected from H, D, substituted or unsubstituted alkyl andsubstituted or unsubstituted alkenyl. Preferably, R9 is methyl.

In the compounds of the invention do not include:

In some embodiments, Ri is selected from H, D, halogen, substituted alkyl (such as - CD3), substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted alkoxyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, ORA, CN, NRBRC, NRAC(O)RA, S(O)RA, S(O)₂RA, SO₂NRBRC, SO₃RA, COORA, C(O)RA, and C(O)NRBRC.

In some embodiments, Ri comprises one or more heteroatoms such as N, O, or S; preferably, O or N.

In some embodiments, Ri is a substituted or unsubstituted C1-12 alkyl, for example, a substituted or unsubstituted -(CH₂)_nCH3, a substituted or unsubstituted -(CH₂)_nOH, a substituted or unsubstituted -O(CH₂)_nCH3, a substituted or unsubstituted -(CH₂)_m- O(CH₂)_nCH₃, a substituted or unsubstituted -O(CH₂)_nNRBRc (preferably, -O(CH₂)_nNH₂), a substituted or unsubstituted -(CH₂)_m-O(CH₂)_nNRBRc (preferably, -(CH₂)_m-O(CH₂)_nNH₂), a substituted or unsubstituted -(CH₂)_nNRBRc (preferably, -O(CH₂)_nNH₂), a substituted or unsubstituted -(CH₂)_m-CONRBRc (preferably, -(CH₂)_m-CONH₂); each m or n is independently an integer from 0 to 12, and when both present, the sum of m and n is no greater than 12.

In some embodiments, Ri is a substituted alkyl, preferably a substituted C1-12 alkyl. In some embodiments, Ri is a substituted or unsubstituted 5- to 6-membered aryl (preferably, phenyl), a substituted or unsubstituted 5- to 6-membered heteroaryl, a substituted or unsubstituted 3- to 10-membered cycloalkyl (preferably, a substituted or unsubstituted 3- to 8-membered cycloalkyl; more preferably a substituted or unsubstituted 3- to 6-membered cycloalkyl), or a substituted or unsubstituted 4- to 10-membered heterocycloalkyl (preferably, a substituted or unsubstituted 3- to 8-membered heterocycloalkyl; more preferably a substituted or unsubstituted 4- to 6-membered heterocycloalkyl).

In some embodiments, Ri is substituted or unsubstituted -(CH2)_P-Rz. P is an integer selected from 0 to 6. R_z is ORA, CN, NRBRC, NRAC(O)RA, S(O)RA, S(0)2RA, SC>2NRBRC, SOSRA, COORA, C(O)RA, C(O)NRBRC, a substituted or unsubstituted C3-6 cycloalkyl, a substituted or unsubstituted C4-6 heterocycloalkyl, a substituted or unsubstituted C5-6 aryl, or a substituted or unsubstituted C5-6 heteroaryl.

In some embodiments, Rz is a ring moiety selected from:

wherein each of the ring moieties above is substituted with zero, one, two, three, or four substituents. The substitutes are preferably -OH, a substituted or unsubstituted Ci-4 alkyl (such as -CH₃, -CF₃, -CH2OH), -O-Ci-4 alkyl (such as -OCH₃, -OCH₂CH₃), and -NH-CI-4 alkyl (such as -OCH₃, -OCH2CH?). In some embodiments where Rz is a substituted phenyl, the one or more substituents further optionally comprise halogen (preferably Cl, F, or Br). In some cases, p is 0, and Ri is Rz as defined above.

In some embodiments, Ri is an unsubstituted C2-10 alkyl, a C1-10 alkoxyl, or a substituted alkyl (preferably, a C1-10 substituted alkyl; e.g., a deuterated alkyl) that comprises one or more substitutes selected from -OH, -NH2, -Ci-4 alkyl, -Ci-4 alkoxyl, -Ci-4 alkylamino, a substituted or unsubstituted C₃-6 cycloalkyl, a substituted or unsubstituted C₃-6 heterocycloalkyl, a substituted or unsubstituted C5-6 aryl, or a substituted or unsubstituted C5-6 heteroaryl. As used herein, a deuterated alkyl refers to a deuterated alkyl where from one to the maximum number of hydrogens present are replaced by deuterium. In some preferred embodiments, Ri is selected from -CH₂CH₃, - (CH₂)₂C(O)NH₂, additional preferred embodiments, Ri is selected from yet additional preferred embodiments, Ri is selected from

In other preferred embodiments, Ri is selected from -CH₂O-(CH₂)₂-N(CH₃)₂, -O-CH₂-CH₂F,

In some embodiments, Re and R7 together with the carbon atoms to which they are attached, form a heterocycloalkyl containing two oxygen atoms, wherein two oxygen atoms are in a ketal arrangement, wherein the heterocycloalkyl further optionally comprises a fused or spiro ring structure. Therefore, the compounds of the invention are preferably represented by Formula (Ila) or Formula (lib) as below:

Formula (Ila) and Formula (lib) Ring A formed by Rw and Rv in Formula (Ila) and Ring B formed by Rw and Rv in (lib) each are independently a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl. In some cases, Ring A is a substituted or unsubstituted C3-7 cycloalkyl, or a substituted or unsubstituted C5-6 aryl. In some cases, Ring B is a substituted or unsubstituted C4-7 cycloalkyl, or a substituted or unsubstituted C5-6 aryl. All the other variables are as defined above, including all and preferable embodiments.

In some preferred embodiments, Xi and X2 are both -O-, the compounds of the invention are represented by Formula (III):

Formula (III), wherein every variable is defined as above, including all and preferred embodiments.

In some embodiments, preferred examples of Ri/Re/R? combinations are provided in Table 1. Table 1

Ri Re R7 R1 Re R7

-CH₃ -OH -OH -CH₃ -OH -OCH3

-(CH₂)₃OH -OH -OH -(CH₂)₃OH -OH -OCH3

-OCH3 -OH -OH -OCH3 -OH -OCH3

-(CH₂)₂OCH₃ -OH -OH -(CH₂)₂OCH₃ -OH -OCH3

-OCF3 -OH -OH -OCF3 -OH -OCH3

-(CH₂)₂C(CH₃)₂OH -OH -OH -(CH₂)₂C(CH₃)₂OH -OH -OCH3

-(CH₂)₂CONH₂ -OH -OH -(CH₂)₂CONH₂ -OH -OCH3

In some preferred embodiments, R4, Rs and R9 are methyl, R3 is hydroxyl, R2 and Rs is H. The compounds of the invention are represented by:

Formula (IV). Ri, Re and R7 are defined as above, including all and preferred embodiments.

Each preferred embodiment described herein can be taken in combination with one, any or all other preferred embodiments, as though presented herein in every permutation.

Table 2A and Table 2B provide nonlimiting examples embodying the generic structure of this invention. In some embodiments, the compounds of the invention do not include Compound No. 1 in Table 2 A.

Table 2A, Nonlimiting examples No. Compound No. Compound No. Compound

Table 2B. Nonlimiting Examples

In additional embodiments, the compounds of this invention comprise a compound of Formula (I)-Formula (IV) as described above, an optional linker, and a biologically active moiety, wherein the compound of Formula (I)-Formula (IV) is covalently connected to the biologically active moiety, directly or via the optional linker when the optional linker is present. As used here, the biologically active moiety refers to a moiety that can interact with a biological target (such as a protein, enzyme, or receptor, leading to a physiological or pharmacological effect), and includes, without limitation, amino acid, peptide, vitamer, antibody. The optional linker is a PEG, preferably a PEG4-12. One nonlimiting example embodying this structure is Compound No. 77 in Table 2B.

The Examples depicted can be synthesized by a combination of specific chemical reactions and partially by applying a set of engineered enzymes to perform a specific part of the synthetic route by biosynthetic transformations, as depicted above.

Compositions of the invention can comprise racemic mixtures, pure enantiomers, or an excess of one enantiomer over the other. For example, a composition can comprise an enantiomeric excess of at least 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90%. In one embodiment, the enantiomeric excess is at least 95%.

The compounds of the invention include all enantiomers which may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, as well as their racemic and optically pure forms, and is not limited to those described herein in any of their pharmaceutically acceptable forms, including enantiomers, salts, solvates, polymorphs, solvatomorphs, hydrates, anhydrous and other crystalline forms and combinations thereof. Likewise, all tautomeric forms are intended to be included. The compounds of the invention include all possible deuterated compounds.

Preferably, a pharmaceutical composition comprises a compound of the invention as an R enantiomer in substantially pure form; or, a pharmaceutical composition comprises a compound of the invention as an S enantiomer in substantially pure form; or, a pharmaceutical composition comprises a compound of the invention as enantiomeric mixtures which contain an excess of the R enantiomer or an excess of the S enantiomer. It is particularly preferred that the pharmaceutical composition contains a compound of the invention which is a substantially pure optical isomer. For the avoidance of doubt, a compound of the invention can, if desired, be used in the form of solvates.

In some embodiments, the compounds of the invention are synthesized following schemes analogous to Scheme (I) or Scheme (II) as shown below.

Scheme (I). Exemplary biosynthetic processing for the compounds by introducing various R7 in early precursors.

Scheme (II). Exemplary biosynthetic processing for the compounds by introducing various R1 in early precursors.

In some embodiments, the compounds of this invention can be synthesized by the synthetic routes analogous to these provided in Davison et al., “Genomic Discovery and Structure- Activity Exploration of a Novel Family of Enzyme- Activated Covalent Cyclin- Dependent Kinase Inhibitors,” J. Med. Chem. 2024, 67, 15, 13147-13173.

Formulation of Compositions

The administration of the compounds of the invention may be by any suitable means that results in the therapeutic effects for the targeted conditions or disorders, such as cancer.

The compounds of the invention may be contained in any appropriate amount in any suitable carrier substance and are generally present in amounts totaling 1-99% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intrathecal, epidural, or ocular administration, or by injection, inhalation, or direct contact with the nasal or oral mucosa. Thus, the composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd edition, 2013, ed. L.V. Allen, Pharmaceutical Press, Philadelphia, and Encyclopedia of Pharmaceutical Technology, 4^th Edition, ed. J. Swarbrick, 2013, CRC Press, New York).

The precise amount of compound administered to provide an “effective amount” to the subject will depend on the mode of administration, the type, and severity of the cancer, and on the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. When administered in combination with other therapeutic agents, e.g., when administered in combination with an anti-cancer agent, an “effective amount” of any additional therapeutic agent(s) will depend on the type of drug used. Suitable dosages are known for approved therapeutic agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of a compound of Formula (I) or Formula (la) being used by following, for example, dosages reported in the literature and recommended in the Physician's Desk Reference (57th Ed., 2003).

Each compound may be formulated in a variety of ways that are known in the art. For example, a compound of the invention and a biologically active agent as defined herein may be formulated together or separately. Desirably, a compound of the invention and a biologically active agent are formulated together for their simultaneous or near simultaneous administration. In another embodiment, two or more biologically active agents may be formulated together with a compound of the invention, or separately. Other examples include, but are not limited to, two or more compounds of the invention formulated together, wherein the compounds are formulated together with or without one or more biologically active agents.

The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include but are not limited to kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions.

The kit may be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple patients (“bulk packaging”). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

Controlled Release Formulations

Each compound of the invention, alone or in combination with one or more of the biologically active agents as described herein, can be formulated for controlled release (e.g., sustained or measured) administration, as described in U.S. Patent Application Publication Nos. 2003/0152637 and 2005/0025765, each incorporated herein by reference. For example, a compound of the invention, alone or in combination with one or more of the biologically active agents as described herein, can be incorporated into a capsule or tablet that is administered to the patient.

Any pharmaceutically acceptable vehicle or formulation suitable for local application and/or injection into a site to be treated (e.g., a painful surgical incision, wound, or joint), that is able to provide a sustained release of compound of the invention, alone or in combination with one or more of the biologically active agents as described herein, may be employed to provide for prolonged elimination or alleviation of inflammation, as needed. Controlled release formulations known in the art include specially coated pellets, polymer formulations or matrices for surgical insertion or as sustained release microparticles, e.g., microspheres or microcapsules, for implantation, insertion, infusion or injection, wherein the slow release of the active medicament is brought about through sustained or controlled diffusion out of the matrix and/or selective breakdown of the coating of the preparation or selective breakdown of a polymer matrix. Other formulations or vehicles for controlled, sustained or immediate delivery of an agent to a preferred localized site in a patient include, e.g., suspensions, emulsions, gels, liposomes and any other suitable art known delivery vehicle or formulation acceptable for subcutaneous or intramuscular administration.

A wide variety of biocompatible materials may be utilized as a controlled release carrier to provide the controlled release of a compound of the invention, alone or in combination with one or more biologically active agents, as described herein. Any pharmaceutically acceptable biocompatible polymer known to those skilled in the art may be utilized. It is preferred that the biocompatible controlled release material degrade in vivo within about one year, preferably within about 3 months, more preferably within about two months. More preferably, the controlled release material will degrade significantly within one to three months, with at least 50% of the material degrading into non-toxic residues, which are removed by the body, and 100% of the compound of the invention being released within a time period within about two weeks, preferably within about 2 days to about 7 days. A degradable controlled release material should preferably degrade by hydrolysis, either by surface erosion or bulk erosion, so that release is not only sustained but also provides desirable release rates. However, the pharmacokinetic release profile of these formulations may be first order, zero order, bi- or multi-phasic, to provide the desired reversible local antinociceptive effect over the desired time period.

Suitable biocompatible polymers can be utilized as the controlled release material. The polymeric material may comprise biocompatible, biodegradable polymers, and in certain preferred embodiments, is preferably a copolymer of lactic and glycolic acid. Preferred controlled release materials which are useful in the formulations of the invention include the polyanhydrides, polyesters, co-polymers of lactic acid and glycolic acid (preferably wherein the weight ratio of lactic acid to glycolic acid is no more than 4: 1 i.e., 80% or less lactic acid to 20% or more glycolic acid by weight) and polyorthoesters containing a catalyst or degradation enhancing compound, for example, containing at least 1% by weight anhydride catalyst such as maleic anhydride. Examples of polyesters include polylactic acid, polyglycolic acid and polylactic acid-polyglycolic acid copolymers. Other useful polymers include protein polymers such as collagen, gelatin, fibrin and fibrinogen and polysaccharides such as hyaluronic acid.

The polymeric material may be prepared by any method known to those skilled in the art. For example, where the polymeric material is comprised of a copolymer of lactic and glycolic acid, this copolymer may be prepared by the procedure set forth in U.S. Pat. No. 4,293,539, incorporated herein by reference. Alternatively, copolymers of lactic and glycolic acid may be prepared by any other procedure known to those skilled in the art. Other useful polymers include polylactides, polyglycolides, polyanhydrides, polyorthoesters, polycaprolactones, polyphosphazenes, polyphosphoesters, polysaccharides, proteinaceous polymers, soluble derivatives of polysaccharides, soluble derivatives of proteinaceous polymers, polypeptides, polyesters, and polyorthoesters or mixtures or blends of any of these.

Pharmaceutically acceptable polyanhydrides which are useful in the present invention have a water-labile anhydride linkage. The rate of drug release can be controlled by the particular polyanhydride polymer utilized and its molecular weight. The polysaccharides may be poly-l,4-glucans, e.g., starch glycogen, amylose, amylopectin, and mixtures thereof. The biodegradable hydrophilic or hydrophobic polymer may be a water-soluble derivative of a poly-l,4-glucan, including hydrolyzed amylopectin, derivatives of hydrolyzed amylopectin such as hydroxyethyl starch (HES), hydroxyethyl amylose, dialdehyde starch, and the like. The polyanhydride polymer may be branched or linear.

Examples of polymers which are useful in the present invention include (in addition to homopolymers and copolymers of poly(lactic acid) and/or poly(glycolic acid)) poly[bis(p- carb oxy phenoxy) propane anhydride] (PCPP), poly[bis(p-carboxy)methane anhydride] (PCPM), polyanhydrides of oligomerized unsaturated aliphatic acids, polyanhydride polymers prepared from amino acids which are modified to include an additional carboxylic acid, aromatic polyanhydride compositions, and co-polymers of polyanhydrides with other substances, such as fatty acid terminated polyanhydrides, e.g., polyanhydrides polymerized from monomers of dimers and/or trimers of unsaturated fatty acids or unsaturated aliphatic acids. Polyanhydrides may be prepared in accordance with the methods set forth in U.S. Pat. No. 4,757,128, incorporated herein by reference. Polyorthoester polymers may be prepared, e.g., as set forth in U.S. Pat. No. 4,070,347, incorporated herein by reference. Polyphosphoesters may be prepared and used as set forth in U.S. Pat. Nos. 6,008,318, 6,153,212, 5,952,451, 6,051,576, 6,103,255, 5,176,907 and 5,194,581, each of which is incorporated herein by reference.

Proteinaceous polymers may also be used. Proteinaceous polymers and their soluble derivatives include gelation biodegradable synthetic polypeptides, elastin, alkylated collagen, alkylated elastin, and the like. Biodegradable synthetic polypeptides include poly-(N- hydroxyalkyl)-L-asparagine, poly-(N-hydroxyalkyl)-L-glutamine, copolymers of N- hydroxyalkyl-L-asparagine and N-hydroxyalkyl-L-glutamine with other amino acids. Suggested amino acids include L-alanine, L-lysine, L-phenylalanine, L-valine, L-tyrosine, and the like. In additional embodiments, the controlled release material, which in effect acts as a carrier for a compound of the invention, alone or in combination with one or more biologically active agents as described herein, can further include a bioadhesive polymer such as pectins (polygalacturonic acid), mucopolysaccharides (hyaluronic acid, mucin) or nontoxic lectins or the polymer itself may be bioadhesive, e.g., polyanhydride or polysaccharides such as chitosan.

In embodiments where the biodegradable polymer comprises a gel, one such useful polymer is a thermally gelling polymer, e.g., polyethylene oxide, polypropylene oxide (PEO- PPO) block copolymer such as Pluronic™ F127 from BASF Wyandotte. In such cases, the local anesthetic formulation may be injected via syringe as a free-flowing liquid, which gels rapidly above 30° C. (e.g., when injected into a patient). The gel system then releases a steady dose of a compound of the invention, alone or in combination with one or more biologically active agents as described herein, at the site of administration.

Dosage Forms for Oral Use

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, taste masking agents (such as hydroxypropyl methylcellulose, hydroxypropyl cellulose), and the like. One or more compounds of the invention and one or more biologically active agents, as defined herein, may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned. In one example, a compound of the invention is contained on the inside of the tablet, and the biologically active agent is on the outside of the tablet, such that a substantial portion of the biologically active agent is released prior to the release of the compound of the invention. Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Formulations for oral administration to the mouth may also be provided as a mouthwash, an oral spray, oral rinse solution, oral ointment, or oral gel.

Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.

The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Generally, when administered to a human, the oral dosage of any of the compounds of the combination of the invention will depend on the nature of the compound, and can readily be determined by one skilled in the art. Typically, such dosage is normally about 0.001 mg to 2000 mg per day, desirably about 1 mg to 1000 mg per day, and more desirably about 5 mg to 500 mg per day. Dosages up to 200 mg per day may be necessary.

Administration of each drug in a combination therapy, as described herein, can, independently, be one to four times daily for one day to one year, and may even be for the life of the patient. Chronic, long-term administration will be indicated in many cases.

Parenteral Formulations

Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the compound in the liquid is from about 1 ng/ml to about 10 pg/ml, for example from about 10 ng/ml to about 1 pg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

Topical Formulations

The compositions of the invention, alone or in combination with one or more of the biologically active agents described herein, can also be adapted for topical use with a topical vehicle containing from between 0.0001% and 25% (w/w) or more of active ingredient(s).

In a preferred combination, the active ingredients are preferably each from between 0.0001% to 10% (w/w), more preferably from between 0.0005% to 4% (w/w) active agent. The topical formulation, including but not limited to a cream, gel, or ointment, can be applied one to four times daily, or as needed. Performing the methods described herein, the topical vehicle containing the composition of the invention, or a combination therapy containing a composition of the invention is preferably applied to the site of inflammation on the patient. For example, a cream may be applied to the hands of a patient suffering from arthritic fingers.

The compositions can be formulated using any dermatologically acceptable carrier. Exemplary carriers include a solid carrier, such as alumina, clay, microcrystalline cellulose, silica, or talc; and/or a liquid carrier, such as an alcohol, a glycol, or a water-alcohol/glycol blend. The therapeutic agents may also be administered in liposomal formulations that allow therapeutic agents to enter the skin. Such liposomal formulations are described in U.S. Pat. Nos. 5,169,637; 5,000,958; 5,049,388; 4,975,282; 5,194,266; 5,023,087; 5,688,525; 5,874,104; 5,409,704; 5,552,155; 5,356,633; 5,032,582; 4,994,213; 8,822,537, and PCT Publication No. WO 96/40061. Examples of other appropriate vehicles are described in U.S. Pat. Nos. 4,877,805, 8,822,537, and EP Publication No. 0586106A1. Suitable vehicles of the invention may also include mineral oil, petrolatum, polydecene, stearic acid, isopropyl myristate, polyoxyl 40 stearate, stearyl alcohol, or vegetable oil.

The composition can further include a skin penetrating enhancer, such as those described in “Percutaneous Penetration enhancers”, (eds. Smith E W and Maibach H I. CRC Press 1995). Exemplary skin penetrating enhancers include alkyl (N,N-di substituted amino alkanoate) esters, such as dodecyl 2-(N,N dimethylamino) propionate (DDAIP), which is described in patents U.S. Pat. Nos. 6,083,996 and 6,118,020, which are both incorporated herein by reference; a water-dispersible acid polymer, such as a polyacrylic acid polymer, a carbomer (e.g., Carbopol™ or Carbopol 940P™, available from B. F. Goodrich Company (Akron, Ohio)), copolymers of polyacrylic acid (e.g., PEMULEN™ from B. F. Goodrich Company or Polycarbophil™ from A. H. Robbins, Richmond, Va.; a polysaccharide gum, such as agar gum, alginate, carrageenan gum, ghatti gum, karaya gum, kadaya gum, rhamsan gum, xanthan gum, and galactomannan gum (e.g., guar gum, carob gum, and locust bean gum), as well as other gums known in the art (see for instance, Industrial Gums: Polysaccharides & Their Derivatives, Whistler R. L., BeMiller J. N. (eds.), 3rd Ed. Academic Press (1992) and Davidson, R. L., Handbook of Water-Soluble Gums & Resins, McGraw- Hill, Inc., N.Y. (1980)); or combinations thereof.

Other suitable polymeric skin penetrating enhancers are cellulose derivatives, such as ethyl cellulose, methyl cellulose, hydroxypropyl cellulose. Additionally, known transdermal penetrating enhancers can also be added, if desired. Illustrative are dimethyl sulfoxide (DMSO) and dimethyl acetamide (DMA), 2-pyrrolidone, N,N-diethyl-m-toluamide (DEET), l-dodecylazacycloheptane-2-one (Azone™, a registered trademark of Nelson Research), N,N-dimethylformamide, N-methyl-2-pyrrolidone, calcium thioglycolate and other enhancers such as dioxolanes, cyclic ketones, and their derivatives and so on.

Also illustrative are a group of biodegradable absorption enhancers which are alkyl

N,N-2-(disubstituted amino) alkanoates as described in U.S. Pat. No. 4,980,378 and U.S. Pat. No. 5,082,866, which are both incorporated herein by reference, including: tetradecyl (N,N- dimethylamino) acetate, dodecyl (N,N-dimethylamino) acetate, decyl (N,N-dimethylamino) acetate, octyl (N,N-dimethylamino) acetate, and dodecyl (N,N-diethylamino) acetate.

Particularly preferred skin penetrating enhancers include isopropyl myristate; isopropyl palmitate; dimethyl sulfoxide; decyl methyl sulfoxide; dimethylalanine amide of a medium chain fatty acid; dodecyl 2-(N,N-dimethylamino) propionate or salts thereof, such as its organic (e.g., hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acid addition salts) and inorganic salts (e.g., acetic, benzoic, salicylic, glycolic, succinic, nicotinic, tartaric, maleic, malic, pamoic, methanesulfonic, cyclohexanesulfamic, picric, and lactic acid addition salts), as described in U.S. Pat. No. 6,118,020; and alkyl 2-(N,N-disubstituted amino)- alkanoates, as described in U.S. Pat. No. 4,980,378 and U.S. Pat. No. 5,082,866.

The skin penetrating enhancer in this composition by weight would be in the range of

O.5% to 10% (w/w). The most preferred range would be between 1.0% and 5% (w/w). In another embodiment, the skin penetrating enhancer comprises between 0.5%- 1%, l%-2%, 2%-3%, 3%-4%, or 4%-5%, (w/w) of the composition.

The compositions can be provided in any useful form. For example, the compositions of the invention may be formulated as solutions, emulsions (including microemulsions), suspensions, creams, ointments, foams, lotions, gels, powders, or other typical solid, semisolid, or liquid compositions (e.g., topical sprays) used for application to the skin or other tissues where the compositions may be used. Such compositions may contain other ingredients typically used in such products, such as colorants, fragrances, thickeners (e.g., xanthan gum, a fatty acid, a fatty acid salt or ester, a fatty alcohol, a modified cellulose, a modified mineral material, Krisgel 100™, or a synthetic polymer), antimicrobials, solvents, surfactants, detergents, gelling agents, antioxidants, fillers, dyestuffs, viscosity-controlling agents, preservatives, humectants, emollients (e.g., natural or synthetic oils, hydrocarbon oils, waxes, or silicones), hydration agents, chelating agents, demulcents, solubilizing excipients, adjuvants, dispersants, skin penetrating enhancers, plasticizing agents, preservatives, stabilizers, demulsifiers, wetting agents, sunscreens, emulsifiers, moisturizers, astringents, deodorants, and optionally including anesthetics, anti-itch actives, botanical extracts, conditioning agents, darkening or lightening agents, glitter, humectants, mica, minerals, polyphenols, silicones or derivatives thereof, sunblocks, vitamins, and phytomedicinals.

The compositions can also include other like ingredients to provide additional benefits and improve the feel and/or appearance of the topical formulation. Specific classes of additives commonly use in these formulations include: isopropyl myristate, sorbic acid NF powder, polyethylene glycol, phosphatidylcholine (including mixtures of phosphatidylcholine, such as phospholipon G), Krisgel 100™ distilled water, sodium hydroxide, decyl methyl sulfoxide (as a skin penetrating enhancer), menthol crystals, lavender oil, butylated hydroxytoluene, ethyl di glycol reagent, and 95% percent (190 proof) ethanol.

Formulations for Ophthalmic Administration

The compounds of the invention can also be formulated with an ophthalmically acceptable carrier in sufficient concentration so as to deliver an effective amount of the active compound or compounds to the optic nerve site of the eye. Preferably, the ophthalmic, therapeutic solutions contain one or more of the active compounds in a concentration range of approximately 0.0001% to approximately 5% (weight by volume) and more preferably approximately 0.0005% to approximately 0.1% (weight by volume).

An ophthalmically acceptable carrier does not cause significant irritation to the eye and does not abrogate the pharmacological activity and properties of the charged sodium channel blockers.

Ophthalmically acceptable carriers are generally sterile, essentially free of foreign particles, and generally have a pH in the range of 5-8. Preferably, the pH is as close to the pH of tear fluid (7.4) as possible. Ophthalmically acceptable carriers are, for example, sterile isotonic solutions such as isotonic sodium chloride or boric acid solutions. Such carriers are typically aqueous solutions contain sodium chloride or boric acid. Also useful are phosphate buffered saline (PBS) solutions.

Various preservatives may be used in the ophthalmic preparation. Preferred preservatives include, but are not limited to, benzalkonium potassium, chlorobutanol, thimerosal, phenylmercuric acetate, and phenylmercuric nitrate. Likewise, various preferred vehicles may be used in such ophthalmic preparation. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose and hydroxyethyl cellulose.

Tonicity adjustors may be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, etc., mannitol and glycerin, or any other suitable ophthalmically acceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as the resulting preparation is ophthalmically acceptable. Accordingly, buffers include but are not limited to, acetate buffers, citrate buffers, phosphate buffers, and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed. Ophthalmically acceptable antioxidants can also be included. Antioxidants include but are not limited to sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene.

Formulations for Nasal and Inhalation Administration

The pharmaceutical compositions of the invention can be formulated for nasal or intranasal administration. Formulations suitable for nasal administration, when the carrier is a solid, include a coarse powder having a particle size, for example, in the range of approximately 20 to 500 microns which is administered by rapid inhalation through the nasal passage. When the carrier is a liquid, for example, a nasal spray or as nasal drops, one or more of the formulations can be admixed in an aqueous or oily solution and inhaled or sprayed into the nasal passage.

For administration by inhalation, the active ingredient can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, di chlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount, capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Dry powder compositions for topical delivery to the lung by inhalation may, for example, be presented in capsules and cartridges of, for example, gelatin or blisters of, for example, laminated aluminum foil, for use in an inhaler or insufflator. Powder blend formulations generally contain a powder mix for inhalation of the compound of the invention and a suitable powder base (carrier/diluent/excipient substance) such as mono-, di or ploy- saccharides (e.g. lactose or starch). Use of lactose is preferred. In one embodiment, each capsule or cartridge may contain between about 2 ug to about 100 mg of the compound of formula (I) optionally in combination with another therapeutically active ingredient. In a preferred embodiment, each capsule or cartridge may contain between about 10 ug to about 50 mg of the compound of formula (I) optionally in combination with another therapeutically active ingredient. In another embodiment, each capsule or cartridge may contain between about 20 ug to about 10 mg of the compound of formula (I) optionally in combination with another therapeutically active ingredient. Alternatively, the compound of the invention may be delivered without excipients.

Suitably, the packaging/medicament dispenser is of a type selected from the group consisting of a reservoir dry powder inhaler (RDPI), single use inhaler (capsule or blister inhaler), a multi-dose dry powder inhaler (MDPI), and a metered dose inhaler (MDI).

Solutions or suspensions for use in a pressurized container, pump, spray, atomizer, or nebulizer can be formulated to contain an aqueous medium, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active ingredient(s); a propellant as solvent; and/or a surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

Compositions formulated for nasal or inhalation administration may include one or more taste-masking agents such as flavoring agents, sweeteners, and other strategies, such as sucrose, dextrose, and lactose, carboxylic acids, menthol, amino acids or amino acid derivatives such as arginine, lysine, and monosodium glutamate, and/or synthetic flavor oils and flavoring aromatics and/or natural oils, extracts from plants, leaves, flowers, fruits, etc. and combinations thereof. These may include cinnamon oils, oil of wintergreen, peppermint oils, clover oil, bay oil, anise oil, eucalyptus, vanilla, citrus oil such as lemon oil, orange oil, grape and grapefruit oil, fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, apricot, etc. Additional sweeteners include sucrose, dextrose, aspartame, acesulfame-K, sucralose and saccharin, organic acids (by non-limiting example citric acid and aspartic acid). Such flavors may be present at from about 0.05 to about 4 percent by weight and may be present at lower or higher amounts as a factor of one or more of potency of the effect on flavor, solubility of the flavorant, effects of the flavorant on solubility or other physicochemical or pharmacokinetic properties of other formulation components, or other factors.

Methods of Use

The present application also provides therapeutic methods and uses comprising administering the compounds of the invention, or pharmaceutically acceptable salts thereof, alone or in combination with other therapeutic agents or palliative agents. The compounds disclosed herein inhibit CDK2 and therefore are useful for treating diseases for which CDK2 is dysregulated, such as cancer. The present disclosure provides a method of inhibiting CDK2 in a subject in need thereof, comprising administering to the subject an effective amount of a compound disclosed herein, a pharmaceutically acceptable salt thereof or a pharmaceutical composition disclosed herein.

In some embodiments, the disclosure provides a method of treating a disease or disorder associated with CDK2 in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or Formula (la) or any of the formulas as described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the disease or disorder associated with CDK2 is associated with an amplification of the cyclin El (CCNE1) gene and/or overexpression of CCNE1. In some embodiments, the disease or disorder is cancer.

Subjects “in need of inhibiting CDK2” are those having a disease for which a beneficial therapeutic effect can be achieved by inhibiting CDK2, e.g., a slowing in disease progression, alleviation of one or more symptoms associated with the disease or increasing the longevity of the subject in view of the disease.

In some embodiments, the disclosure provides a method of treating a disease/condition/or cancer associated with or modulated by CDK2, wherein the inhibition of CDK2 is of therapeutic benefit, including but not limited to the treatment of cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of a compound disclosed herein, a pharmaceutically acceptable salt thereof, or pharmaceutical composition disclosed herein.

In another embodiment, the disclosure provides a method of treating a subject with cancer, comprising administering to the subject an effective amount of a compound disclosed herein, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein. In another embodiment, the cancer is characterized by amplification and/or overexpression of CCNE1 or CCNE2.

Accordingly, in some embodiments of the methods, the subject or patient has been previously determined to have an amplification of the cyclin El (CCNE1) gene and/or an expression level of CCNE1 in a biological sample obtained from the subject or patient that is higher than a control expression level of CCNE1.

In another embodiment, the disclosure provides a method for inhibiting growth of tumor (e.g., cancer) cells in vitro. The method includes contacting the tumor (e.g. cancer) cells in vitro with a compound of Formula (I) or Formula (la) or a pharmaceutically acceptable salt thereof. In another embodiment, the present disclosure provides a method for inhibiting growth of tumor (e.g., cancer) cells with CCNE1 amplification and/or overexpression in a subject or a patient. The method includes administering to the subject or patient in need thereof a therapeutically effective amount of a compound of Formula (I) or Formula (la), or a pharmraceutically acceptable salt thereof.

In another embodiment, the disclosure provides a method of treating a subject with cancer, comprising administering to the subject an effective amount of a compound disclosed herein, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein in conjunction with other agents or standard cancer treatments, as described below. As used herein “cancer” refers to any malignant and/or invasive growth or tumor caused by abnormal cell growth. Cancer includes solid tumors named for the type of cells that form them, cancer of blood, bone marrow, or the lymphatic system. Examples of solid tumors include sarcomas and carcinomas. Cancers of the blood include, but are not limited to, leukemia, lymphoma and myeloma. Cancer also includes primary cancer that originates at a specific site in the body, a metastatic cancer that has spread from the place in which it started to other parts of the body, a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a person with a history of previous cancer of a different type from the latter one. In some such embodiments, the cancer is characterized by amplification and/or overexpression of CCNE1 and/or CCNE2.

Cancers to be treated according to the disclosed methods include breast cancer, ovarian cancer, bladder cancer, uterine cancer (e.g., uterine carcinosarcoma), prostate cancer, lung cancer (including NSCLC, SCLC, squamous cell carcinoma (e.g., lung squamous cell carcinoma (LUSC), or adenocarcinoma (e.g., lung adenocarcinoma (LU AD)), esophageal cancer, head and neck cancer, colorectal cancer (e.g., colon cancer, colorectal adenocarcinoma (COADREAD)), kidney cancer (including RCC), liver cancer (including HCC), pancreatic cancer, stomach (i.e., gastric) cancer, urothelial cancer, brain cancers, mesothelioma (MESO), skin cancer (e.g., melanoma), sarcoma, or thyroid cancer, including metastasis (in particular brain metastasis) of all cancers listed. In some embodiments, the cancer is characterized by an overexpression and/or amplification of CCNE1 and/or CCNE2 described herein. In some embodiments of the methods provided herein, the subject is identified as having a cancer characterized by amplification and/or overexpression of CCNE1 and/or CCNE2.

In further embodiments of the methods provided herein, the cancer is breast cancer, ovarian cancer, bladder cancer, uterine cancer, prostate cancer, lung cancer, esophageal cancer, liver cancer, pancreatic cancer or stomach cancer. In some such embodiments, the cancer is characterized by amplification and/or overexpression of CCNE1 and/or CCNE2. In further embodiments, of the methods provided herein, the cancer is selected from the group consisting of ovarian cancer, endometrial cancer, gastric cancer, esophageal cancer, triple negative breast cancer, and lung adenosarcoma. In some embodiments, the cancer is characterized by CCNE1 overexpression and/or amplification. In some embodiments, the cancer has progressed despite platinum treatment.

In some embodiments, the cancer is platinum-resistant and/or platinum-refractory. In some embodiments, the cancer has progressed despite platinum treatment.

In some embodiments, the disease or disorder associated with CDK2 is an adenocarcinoma, carcinoma, or cystadenocarcinoma.

In other embodiments, the cancer is breast cancer, including, e.g., ER-positive/HR- positive, HER2-negative breast cancer; ER-positive/HR-positive, HER2 -positive breast cancer; triple negative breast cancer (TNBC); or inflammatory breast cancer. In some embodiments, the breast cancer is chemotherapy or radiotherapy resistant breast cancer, endocrine resistant breast cancer, trastuzumab resistant breast cancer, or breast cancer demonstrating primary or acquired resistance to CDK4/CDK6 inhibition. In some embodiments, the breast cancer is advanced or metastatic breast cancer. In some embodiments of each of the foregoing, the breast cancer is characterized by amplification and/or overexpression of CCNE1 and/or CCNE2. In some embodiments, the cancer is HR-positive breast cancer. In some embodiments, the breast cancer is ER-positive breast cancer. In some embodiments, the breast cancer is HRpositive, HER2-negative breast cancer. In some embodiments, the breast cancer is ERpositive, IER2-negative breast cancer. In some embodiments, the breast cancer is responsive to treatment with a CDK4/6 inhibitor. In some embodiments, the breast cancer is resistant to treatment with a CDK4/6 inhibitor. In some embodiments, the breast cancer has progressed despite treatment with a CDK4/6 inhibitor. In some embodiments, the CDK4/6 inhibitor is palbociclib. In some embodiments, the breast cancer has progressed despite first treatment with palbociclib and/or fulvestrant and second treatment with abemaciclib and/or fulvestrant. In some embodiments, the method further comprises administering an effective amount of a CDK4/6 inhibitor. In some embodiments, the CDK4/6 inhibitor is selected from palbociclib and ribociclib, or a combination thereof. In some embodiments, the CDK4/6 inhibitor is ribociclib. In some embodiments, the breast cancer has CCNE amplification and/or overexpression. In some embodiments, the breast cancer is triple negative breast cancer.

In some embodiments, the cancer is ovarian cancer. In some such embodiments, the cancer is ovarian cancer characterized by amplification and/or overexpression of CCNE1 and/or CCNE2. In some such embodiments, the cancer is (a) ovarian cancer; (b) characterized by amplification and/or overexpression of cyclin El (CCNE1) or cyclin E2 (CCNE2); or (c) both (a) and (b).

In some embodiments, the compound of the disclosure is administered as first line therapy. In other embodiments, the compound of the disclosure is administered as second (or later) line therapy. In some embodiments, the compound of the disclosure is administered as second (or later) line therapy following treatment with an endocrine therapeutic agent and/or a CDK4/CDK6 inhibitor. In some embodiments, the compound of the disclosure is administered as second (or later) line therapy following treatment with an endocrine therapeutic agent, e.g., an aromatase inhibitor, a SERM or a SERD. In some embodiments, the compound of the disclosure is administered as second (or later) line therapy following treatment with a CDK4/CDK6 inhibitor. In some embodiments, the compound of the disclosure is administered as second (or later) line therapy following treatment with one or more chemotherapy regimens, e.g., including taxanes or platinum agents. In some embodiments, the compound of the disclosure is administered as second (or later) line therapy following treatment with HER2 targeted agents, e.g., trastuzumab. In some embodiments, the disease or disorder associated with CDK2 is N-myc amplified neuroblastoma cells (see Molenaar, et al., Proc Natl Acad Sci USA 106(31): 12968-12973) K-Ras mutant lung cancers (see Hu, S., et al., Mol Cancer Ther, 2015. 14(11): 2576-85, and cancers with FBW7 mutation and CCNE1 overexpression (see Takada, et al., Cancer Res, 2017.77(18): 4881-4893).

In some embodiments, the compounds of the present disclosure can be used to treat sickle cell disease and sickle cell anemia.

Examples of cancers that are treatable using the compounds of the present disclosure include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinona of the fallopian tubes, carcinoma of the endometrium, endometrial cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or urethra, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. The compounds of the present disclosure are also useful for the treatment of metastatic cancers.

In some embodiments, cancers treatable with compounds of the present disclosure include, but are not limited to, melanoma (e.g., metastatic malignant melanoma, BRAF and HSP90 inhibition-resistant melanoma, skin cutaneous melanoma (SKCM), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), squamous cell head and neck cancer (e.g., head and neck squamous cell carcinoma (NHSC), urothelial cancer (e.g., bladder) and cancers with high microsatellite instability (MSIhigh). Additionally, the disclosure includes refractory or recurrent malignancies whose growth may be inhibited using the compounds of the disclosure.

In some embodiments, cancers that are treatable using the compounds of the present disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, renal cancer, hepatic cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, cancers of the head and neck, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., lymphoma, leukemia such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), DLBCL, mantle cell lymphoma, Non-Hodgkin lymphoma (including follicular lymphoma, including relapsed or refractory NHL and recurrent follicular), Hodgkin lymphoma or multiple myeloma) and combinations of said cancers.

In some embodiments, cancers that are treatable using the compounds of the present disclosure include, but are not limited to, cholangiocarcinoma, bile duct cancer, triple negative breast cancer, rhabdomyosarcoma, small cell lung cancer, leiomyosarcoma, hepatocellular carcinoma (e.g., liver hepatocellular carcinoma (LIHC)), Ewing's sarcoma, brain cancer, brain tumor, astrocytoma, neuroblastoma, neurofibroima, basal cell carcinoma, chondrosarcoma, epithelioid sarcoma, eye cancer, Fallopian tube cancer, gastrointestinal cancer, gastrointestinal stromal tumors, hairy cell leukemia, intestinal cancer, islet cell cancer, oral cancer, mouth cancer, throat cancer, laryngeal cancer, lip cancer, mesotheliona, neck cancer, nasal cavity cancer, ocular cancer, ocular melanoma, pelvic cancer, rectal cancer, renal cell carcinoma, salivary gland cancer, sinus cancer, spinal cancer, tongue cancer, tubular carcinoma, urethral cancer, and ureteral cancer.

In some embodiments, cancers treatable with compounds of the present disclosure include Genomic Identification of Significant Targets in Cancer (GISTIC) and pheochromocytoma and paraganglioma (PCPG).

In some embodiments, cancers treatable with compounds of the present disclosure include advanced/relapsed tumors; CCNE1 amplified platinum-resistant or platinum- refractory ovarian cancer; endometrial cancer (with prior platinum therapy) that has progressed following 2 or more lines of therapies; and gastric cancer (with prior platinum therapy) that has progressed following 2 or more lines of therapies; and ER+HER2- BC that has progressed despite CDK4/6i. In some embodiments, cancers treatable with compounds of the present disclosure include Platinum-resistant or platinum-refractory CCNE1 amplified ovarian cancer; CCNE1 amplified endonetrial cancer that has failed 2 or more lines of therapies; CCNE1 amplified advanced/relapsed tumors that do not belong to the other groups; ER+IER2- BC that has progressed despite CDK4/6i; platinum-resistant or platinum- refractory CCNE1 amplified ovarian cancer; and ER+HER2- BC that has progressed despite CDK4/6i.

In some embodiments, diseases and indications that are treatable using the compounds of the present disclosure include, but are not limited to hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.

Exemplary hematological cancers include lymphomas and leukemias such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsed or refractory NHL and recurrent follicular), Hodgkin lymphoma, myeloproliferative diseases (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocytosis (ET)), myelodysplasia syndrome (MDS), T-cell acute lymphoblastic lymphoma (T-ALL) and multiple myeloma (MM).

Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, harmatoma, and teratoma.

Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bronchogenic carcinoma, squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma, alveolar (bronchiolar) carcinoma, bronchial adenoma, chondromatous hamartoma, and mesothelioma. Exemplary gastrointestinal cancers include cancers of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinora, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), and colorectal cancer. Exemplary genitourinary tract cancers include cancers of the kidney (adenocarcinoma, Wilm's tumor [nephroblastoma]), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma (PR AD), sarcoma), and testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma). Exemplary liver cancers include hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma. Exemplary bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumors.

Exemplary nervous system cancers include cancers of the skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, brain lower grade glioma (LGG), ependymoma, germinoma (pinealoma), glioblastoma, glioblastoma multiforme (GBM), oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, and spinal cord (neurofibroma, meningioma, glioma, sarcoma), as well as neuroblastoma and Lhermitte-Duclos disease.

Exemplary gynecological cancers include cancers of the uterus (endometrial carcinoma), cervix (cervical carcinoma, cervical squamous cell carcinoma (CESC), pre-tumor cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), and fallopian tubes (carcinoma).

Exemplary skin cancers include melanoma, basal cell carcinoma, Merkel cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids. In some embodiments, diseases and indications that are treatable using the compounds of the present disclosure include, but are not limited to, sickle cell disease (e.g., sickle cell anemia), triple-negative breast cancer (TNBC), myelodysplastic syndromes, testicular cancer, bile duct cancer, esophageal cancer, and urothelial carcinoma.

Combinations

Compounds of the invention may be administered as single agents or may be administered in combination with other anti-cancer therapeutic agents, in particular standard of care agents appropriate for the particular cancer.

The term “additional anticancer therapeutic agent” as used herein means any one or more therapeutic agent, other than a compound of the disclosure, that is or can be used in the treatment of cancer. In some embodiments, such additional anticancer therapeutic agents include compounds derived from the following classes: mitotic inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, anti-angiogenesis agents, topoisomerase I and II inhibitors, plant alkaloids, hormonal agents and antagonists, growth factor inhibitors, radiation, signal transduction inhibitors, such as inhibitors of protein tyrosine kinases and/or serine/threonine kinases, cell cycle inhibitors, biological response modifiers, enzyme inhibitors, antisense oligonucleotides or oligonucleotide derivatives, cytotoxics, immunooncology agents, and the like.

In some embodiments, the additional anticancer agent is an endocrine agent, such as an aromatase inhibitor, a SERD or a SERM.

In some embodiments, the additional anticancer agent is a PIK3CA inhibitor including, but not limited to, alpelisib (PIQRAY), BEBT-908, BPI-21668, buparlisib, inavolisib, TQB-3525, RLY-2608, miransertib, MEN-1611, LOXO-783, HS-10352, HH- CYH33, gedatolisib, and fimepinostat.

In some embodiments, the additional anticancer agent is an antibody-drug conjugates including, but not limited to, Trastuzumnab deruxtecan (Enhertu), Trastuzumab duocarmazine, Trastuzumab emtansine (Kadcyla), Upifitamab rilsodotin, mirvetuximab soravtansine, Tisotumab vedotin (Tivdak). Praluzatamab ravtansine, Sacituzumab govitecan or Sacituzumab Govitecan-hziy (Trodelvy), Datopotamab deruxtecan, Ladiratuzumab vedotin, Patritumab deruxtecan, STRO-002, MORab-202, DS-6000, Aneturnab, avtansine, XMT-2056, Disitamab Vedotin (RC48-ADC, Aidexi).

In some embodiments, the additional anticancer agent is a PLK1 inhibitor including, but not limited to onvansertib, BI2536, BI6727, GSK461364A, TAK960, rigosertib. In some embodiments, the additional anticancer agent is Estrogen PROTAC (ARV-471, H3B-5942).

In other embodiments, a compound of the disclosure may be administered in combination with a standard of care agent. In some embodiments, a compound of the disclosure may be administered in combination with endocrine therapy, e.g., agents such as letrozole, fulvestrant, tamoxifen, exemestane, or anastrozole. In some embodiments, a compound of the disclosure may be administered in combination with a chemotherapeutic agent, e.g., docetaxel, paclitaxel, cisplatin, carboplatin, capecitabine, gemcitabine, vinorelbine, or liposomal doxorubicin. In other embodiments, a compound of the invention may be administered in combination with an anti-HER2 agent, e.g., trastuzumab or pertuzumab.

In some embodiments, a compound of the disclosure (for example, a compound of Formula (I), Formula (la), Formula (II), Formula (Ila), Formula (III), Formula (Illa), Formula (IVa), Formula (IVb), Formula (IVa-1), Formula (IVb-1), Formula (Va), Formula (Vb), Formula (Vc), Formula (Vd), Formula (Va-1), Formula (Vb-1), Formula (Vc-1), or Formula (Vd-1) or a pharmaceutically acceptable salt thereof may be administered in combination with an effective amount of carboplatin, ribociclib, fulvestrant, or a combination thereof.

In some embodiments, the additional anticancer agent is an anti-angiogenesis agent, including for example VEGF inhibitors, VEGFR inhibitors, TIE-2 inhibitors, PDGFR inhibitors, angiopoetin inhibitors, PKCb inhibitors, COX-2 (cyclooxygenase II) inhibitors, integrins (alpha-v/beta-3), MMP-2 (matrix-metalloproteinase 2) inhibitors, and MMP-9 (matrix-metalloproteinase 9) inhibitors. Preferred anti-angiogenesis agents include sunitinib (Sutent™), bevacizumab (Avastin™), axitinib (AG 13736), IU 14813 (Pfizer), and AG 13958 (Pfizer). Additional anti-angiogenesis agents include vatalanib (CGP 79787), Sorafenib (Nexavar™), pegaptanib octasodium (Macugen™), vandetanib (Zactima™), PF- 0337210 (Pfizer), SIU 14843 (Pfizer), AZD 2171 (AstraZeneca), ranibizumab (Lucentis™), Neovastat™ (AE 941), tetrathiomolybdata (Coprexa™), AMG 706 (Amgen), VEGF Trap (AVE 0005), CEP 7055 (Sanofi-Aventis), XL 880 (Exelixis), telatinib (BAY 57-9352), and CP-868,596 (Pfizer). Other anti-angiogenesis agents include enzastaurin (LY 317615), midostaurin (CGP 41251), perifosine (KRX 0401), teprenone (Selbex™) and UCN 01 (Kyowa Hakko). Other examples of anti-angiogenesis agents include celecoxib (Celebrex™), parecoxib (Dynastat™), deracoxib (SC 59046), lumiracoxib (Preige™), valdecoxib (Bextra™), rofecoxib (Vioxx™), iguratimod (Careram™), IP 751 (Invedus), SC-58125 (Pharmacia) and etoricoxib (Arcoxia™). Yet further anti-angiogenesis agents include exisulind (Aptosyn™), salsalate (Amigesic™), diflunsal (Dolobid™), ibuprofen (Motrin™), ketoprofen (Orudis™), nabumetone (Relafen™), piroxicam (Feldene™), naproxen (Aleve™, Naprosyn™), diclofenac (Voltaren™), indomethacin (Indocin™), sulindac (Clinoril™), tolmetin (Tolectin™), etodolac (Lodine™), ketorolac (Toradol™), and oxaprozin (Daypro®). Yet further anti-angiogenesis agents include ABT 510 (Abbott), apratastat (TMI 005), AZD 8955 (AstraZeneca), incyclinide (Metastat™), and PCK 3145 (Procyon).

Yet further anti-angiogenesis agents (including VEGFR/PDGFR inhibitors) include, but are not limited to, ponatinib (Iclusig), BT1718, anlotinib, lenvatinib (Lenvima), tivozanib (Fotivda), dovitinib, brolucizumab (Beovu), aflibercept (Eylea), and faricimab. Yet further anti-angiogenesis agents include acitretin (Neotigason™), plitidepsin (Aplidine™), cilengtide (EMD 121974), combretastatin A4 (CA4P), fenretinide (4 HPR), halofuginone (Tempostatin™), Panzem™ (2-methoxyestradiol), PF-03446962 (Pfizer), rebimastat (BMS 275291), catumaxomab (Removab™), lenalidomide (Revlimid™), squalamine (EVIZON™), thalidomide (Thalomid™), Ukraine™ (NSC 631570), Vitaxin™ (MEDI 522), and zoledronic acid (Zometa™).

In other embodiments, the additional anti-cancer agent is a so-called signal transduction inhibitor (e.g., inhibiting how regulatory molecules that govern the fundamental processes of cell growth, differentiation, and survival communicated within the cell). Signal transduction inhibitors include small molecules, antibodies, and antisense molecules. Signal transduction inhibitors include for example kinase inhibitors (e.g., tyrosine kinase inhibitors or serine/threonine kinase inhibitors) and cell cycle inhibitors. More specifically signal transduction inhibitors include, for example, farnesyl protein transferase inhibitors, EGF inhibitor, ErbB-1 (EGFR), ErbB-2, pan erb, IGF1R inhibitors, MEK, c-Kit inhibitors, FLT-3 inhibitors, K-Ras inhibitors, PI3 kinase inhibitors, JAK inhibitors, STAT inhibitors, Raf kinase inhibitors, Akt inhibitors, mTOR inhibitor, P70S6 kinase inhibitors, inhibitors of the WNT pathway and so called multi -targeted kinase inhibitors. Additional examples of signal transduction inhibitors which may be used in conjunction with a compound of the invention and pharmaceutical compositions described herein include BMS 214662 (Bristol-Myers Squibb), lonafarnib (Sarasar™), pelitrexol (AG 2037), matuzumab (EMD 7200), nimotuzumab (TheraCIM h-R3™), panitumumab (Vectibix™), Vandetanib (Zactim™), pazopanib (SB 786034), ALT 110 (Alteris Therapeutics), BIBW 2992 (Boehringer Ingelheim), and Cervene™ (TP 38). Other examples of signal transduction inhibitors include gefitinib (Iressa™), cetuximab (Erbitux™), erlotinib (Tarceva™), trastuzumab (Herceptin™), sunitinib (Sutent™), imatinib (Gleevec™), crizotinib (Pfizer), lorlatinib (Pfizer), dacomitinib (Pfizer), bosutinib (Pfizer), gedatolisib (Pfizer), canertinib (CI 1033), pertuzumab (Omnitarg™), lapatinib (Tycerb™), pelitinib (EKB 569), miltefosine (Miltefosin™), BMS 599626 (Bristol-Myers Squibb), Lapuleucel-T (Neuvenge™) NeuVax™ (E75 cancer vaccine), Osiden™ (IDM 1), mubritinib (TAK-165), CP-724,714 (Pfizer), panitumumab (Vectibix™), ARRY 142886 (Array Biopharm), everolimus (Certican™), zotarolimnus (Endeavor™), tensirolimus (Torisel™), AP 23573 (ARIAD), and VX 680 (Vertex), XL 647 (Exelixis), sorafenib (Nexavar™), LE-AON (Georgetown University), and GL4000 (Globelmmune). Other signal transduction inhibitors include ABT 751 (Abbott), alvocidib (flavopiridol), BMS 387032 (Bristol Myers), EM 1421 (Erimos), indisulam (E 7070), seliciclib (CYC 200), BIO 112 (One Bio), BMS 387032 (Bristol-Myers Squibb), palbociclib (Pfizer), and AG 024322 (Pfizer).

In other embodiments, the additional anti-cancer agent is a so called classical antineoplastic agent. Classical antineoplastic agents include but are not limited to hormonal modulators such as hormonal, anti-hormonal, androgen agonist, androgen antagonist and anti-estrogen therapeutic agents, histone deacetylase (HD AC) inhibitors, DNA methyltransferase inhibitors, silencing agents or gene activating agents, ribonucleases, proteosomics. Topoisomerase I inhibitors, Camptothecin derivatives, Topoisomerase 11 inhibitors, alkylating agents, antimetabolites, poly(ADP-ribose) polymerase- 1 (PARP-1) inhibitor (such as, e.g., talazoparib, olapariv, rucaparib, niraparib, iniparib, veliparib), microtubulin inhibitors, antibiotics, plant derived spindle inhibitors, platinum-coordinated compounds, gene therapeutic agents, antisense oligonucleotides, vascular targeting agents (VTAs), and statins. Examples of classical antineoplastic agents used in combination therapy with a compound of the invention, optionally with one or more other agents include, but are not limited to, glucocorticoids, such as dexamethasone, prednisone, prednisolone, methylprednisolone, hydrocortisone, and progestins such as medroxyprogesterone, megestrol acetate (Megace), mifepristone (RU-486), Selective Estrogen Receptor Modulators (SERMs; such as tamoxifen, raloxifene, lasofoxifene, afimoxifene, arzoxifene, bazedoxifene, fispemifene, ormeloxifene, ospemifene, tesmilifene, toremifene, trilostane and CHF 4227 (Cheisi), Selective Estrogen-Receptor Downregulators (SERD's; such as fulvestrant, LSZ102, G1T48, RAD1901, elacestrant, GDC-9545, giredestrant, SAR439859, amcenestrant, AZD9833, camizestrant, LY3484356, Zn-c5, D-0502), exemestane (Aromasin), anastrozole (Arimidex), atamestane, fadrozole, letrozole (Femara), formestane; gonadotropin-releasing hormone (GnRH; also commonly referred to as luteinizing hormone-releasing hormone [LHRH]) agonists such as buserelin (Suprefact), goserelin (Zoladex), leuprorelin (Lupron), and triptorelin (Trelstar), abarelix (Plenaxis), cyproterone, flutamide (Eulexin), megestrol, nilutamide (Nilandron), and osaterone, dutasteride, epristeride, finasteride, Serenoa repens, PHL 00801, abarelix, goserelin, leuprorelin, triptorelin, bicalutamide: antiandrogen agents, such as enzalutamide, abiraterone acetate, bicalutamide (Casodex); and combinations thereof. Other examples of classical antineoplastic agents used in combination with a compound of the invention include but are not limited to suberolanilide hydroxamic acid (SAHA, Merck Inc./Aton Pharmaceuticals), depsipeptide (FR901228 or FK228), G2M-777, MS-275, pivaloyloxymethyl butyrate and PXD-101; Onconase (ranpirnase), PS-341 (MLN-341), Velcade (bortezomib). 9-aminocamptothecin, belotecan, BN-80915 (Roche), camptothecin, diflomotecan, edotecarin, exatecan (Daiichi), gimatecan, 10-hydroxy camptothecin, irinotecan HC1 (Camptosar), lurtotecan, Orathecin (rubitecan, Supergen), SN-38, topotecan, camptothecin, 10-hydroxy camptothecin, 9-aminocamptothecin, irinotecan, SN-38, edotecarin, topotecan, aclarubicin, adriamycin, amonafide, amrubicin, annamycin, daunorubicin, doxorubicin, elsamitrucin, epirubicin, etoposide, idarubicin, galarubicin, hydroxycarbamide, nemorubicin, novantrone (mitoxantrone), pirarubicin, pixantrone, procarbazine, rebeccamycin, sobuzoxane, tafluposide, valrubicin, Zinecard (dexrazoxane), nitrogen mustard N-oxide, cyclophosphamide, AMD-473, akretamine, AP-5280, apaziquone, brostallicin, bendamustine, busulfan, carboquone, carmustine, chlorambucil, dacarbazine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine, mafosfamide, mechlorethamine, melphalan, mitobronitol, mitolactol, mitomycin C, mitoxatrone, nimustine, ranimustine, temozolomide, thiotepa, and platinum-coordinated alkylating compounds such as cisplatin, Paraplatin (carboplatin), eptaplatin, lobaplatin, nedaplatin, Eloxatin (oxaliplatin, Sanofi), streptozocin, satrplatin, and combinations thereof.

In still other embodiments, the additional anti -cancer agent is a so called dihydrofolate reductase inhibitors (such as methotrexate and NeuTrexin (trimetresate glucuronate)), purine antagonists (such as 6-mercaptopurine riboside, mercaptopurine, 6- thioguanine, cladribine, clofarabine (Clolar), fludarabine, nelarabine, and raltitrexed), pyrimidine antagonists (such as 5-fluomuracil (5-FU), Alimta (premetrexed disodium, LY231514, MT A), capecitabine (Xeloda™), cytosine arabinoside, Gemzar™ (gemcitabine, Eli Lilly), Tegafur (UFT Orzel or Uforal and including TS-1 combination of tegafur, gimestat and otostat), doxifluridine, carmofur, cytarabine (including ocfosfate, phosphate stearate, sustained release and liposomal forms), enocitabine, 5-azacitidine (Vidaza), decitabine, and ethynylcytidine) and other antimetabolites such as eflornithine, hydroxyurea, leucovorin, nolatrexed (Thymitaq), triapine, trimetrexate, N-(5-[N-(3,4-dihydro-2-methyl-4- oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid, AG-014699 (Pfizer Inc.), ABT-472 (Abbott Laboratories), INO-lOOl (Inotek Pharmaceuticals), KU-0687 (KuDOS Pharmaceuticals) and GPI 18180 (Guilford Pharm Inc) and combinations thereof.

Other examples of classical antineoplastic cytotoxic agents include, but are not limited to, Abraxane (Abraxis BioScience, Inc.), Batabulin (Amgen), EPO 906 (Novartis), Vinflunine (Bristol-Myers Squibb Company), actinomycin D, bleomycin, mitomycin C, neocarzinostatin (Zinostatin), vinblastine, vincristine, vindesine, vinorelbine (Navelbine), docetaxel (Taxotere), Ortataxel, paclitaxel (including Taxoprexin a DHA/paciltaxel conjugate), cisplatin, carboplatin, Nedaplatin, oxaliplatin (Eloxatin), Satraplatin, Camptosar, capecitabine (Xeloda), oxaliplatin (Eloxatin), Taxotere alitretinoin, Canfosfamide (Telcyta™), DMXAA (Antisoma), ibandronic acid, L-asparaginase, pegaspargase (Oncaspar™). Efaproxiral (Efaproxyn™ — radiation therapy), bexarotene (Targretin™), Tesmilifene (DPPE-enhances efficacy of cytotoxics), Theratope™ (Biomira), Tretinoin (Vesanoid™), tirapazamine (Trizaone™), motexafin gadolinium (Xcytrin™) Cotara™ (mAb), and NBI-3001 (Protox Therapeutics), polyglutamate-paclitaxel (Xyotax™) and combinations thereof. Further examples of classical antineoplastic agents include, but are not limited to, as Advexin (ING 201), TNFerade (GeneVec, a compound which express TNF alpha in response to radiotherapy), RB94 (Baylor College of Medicine), Genasense (Oblimersen, Genta), Combretastatin A4P (CA4P). Oxi-4503, AVE-8062, ZD-6126, TZT- 1027. Atorvastatin (Lipitor, Pfizer Inc.). Provastatin (Pravachol, Bristol-Myers Squibb), Lovastatin (Mevacor, Merck Inc.), Simvastatin (Zocor, Merck Inc.), Fluvastatin (Lescol, Novartis), Cerivastatin (Baycol, Bayer). Rosuvastatin (Crestor. AstraZeneca), Lovostatin, Niacin (Advicor, Kos Pharmaceuticals), Caduet, Lipitor, torcetrapib, and combinations thereof. In other embodiments, the additional anti-cancer agent is an epigenetic modulator, for example an inhibitor or EZH2, SMARCA4, PBRM1, ARID1 A, ARID2, ARID1B, DNMT3A, TET2, MLL1/2/3, NSD1/2. SETD2, BRD4, D0T1L, HKMTsanti, PRMT1-9, LSD1, UTX, IDH1/2 or BCL6.

In further embodiments, the additional anti-cancer agent is an immunomodulatory agent, such as, but not limited to, an inhibitor of CTLA-4 (e.g., ipilimumab), PD-1 or PD-L1 (e.g., pembrolizumab, nivolumab, avelumab, atezolizumab, durvalumab, cemiplimab, or dosterlimab), LAG-3 (e.g., relatlimab, TIM-3, TIGIT, 4-4BB, 0X40, GITR, CD40, or a CAR-T-cell therapy.

In some embodiments, the additional anticancer agent is an EGFR inhibitor such as afatinib, osimertinib, lapatinib, erlotinib, dacomitinib, poziotinib, neratinib or gefitinib or an EGFR antibody such as cetuximab, panitumumab, or necitumumab.

Alternatively, a compound of the disclosure, a pharmaceutically acceptable salt thereof or a pharmaceutical composition disclosed herein can be administered in combination with other anti-cancer agents that are not EGFR inhibitors e.g., in combination with MEK, including mutant MEK inhibitors (trametinib, cobimtetinib, binimetinib, selumetinib, refametinib); c-MET, including mutant c-Met inhibitors (savolitinib, cabozantinib, foretinib) and MET antibodies (emibetuzumab); mitotic kinase inhibitors (CDK4/6 inhibitors such as palbociclib, riboci clib, abemacicilb, lerociclib, trilaciclib, dalpiciclib, BPI-16350); anti- angiogenic agents e.g., bevacizumab, nintedanib; apoptosis inducers such as Bcl-2 inhibitors e.g, venetoclax, obatoclax, navitoclax and Mcl-1 inhibitors e.g., AZD-5991, AMG-176, S- 64315; and mTOR inhibitors e.g., rapamycin, temsirolimus, everolimus, ridoforolimus.

A compound of the disclosure, a pharmaceutically acceptable salt thereof or a pharmaceutical composition disclosed herein can also be administered in combination with an effective amount of a second agent selected from the group consisting of palbociclib (e.g., Ibrance®), ribociclib, abemaciclib, tamoxifen, letrozole, olaparib (e.g., Lynparza®), niraparib, carboplatin, cisplatin, paclitaxel, gemcitabine, megestrol acetate, medroxyprogesterone acetate, capecitabine (e.g., Xeloda®), regorafenib (e.g., Stivarga®), afatinib (e.g., Gilotrif®), osimertinib (e.g., Tagrisso®), gefitinib (e.g., Iressa®), erlotinib (e.g., Tarceva®), ramucirumab (e.g., Cyramza®), an EGFR inhibitor, pralsetinib, ABT-263 (navitoclax), MK-1775 (adavosertib), BAY-1895344, berzosertib, ceralasertib, SRA-737, LY2603618 (rabusertib), and trastuzumab (e.g., Herceptin®), or combinations thereof. The EGFR inhibitor may be selected from afatinib, osimertinib, lapatinib, erlotinib, dacomitinib, poziotinib, neratinib, gefitinib JBJ-04-125-02, alflutinib (AST 2818), aumolertinib (formerly almonertinib) (HS10296), BBT-176, BI-4020, BPI-361175, BPI-D0316, CH7233163, gilitertinib, icotinib, JND-3229, lazertinib, nazartinib (EGF 816), avitinib, PCC-0208027, rezivertinib (BPI-7711), TQB3804, zorifertinib (AZ-3759), or DZD9008; an EGFR antibody such as cetuximab, panitumumab, necitumumab, HLX07, JMT101; or a bispecific EGFR and MET antibody (e.g., amivantamab ((JNJ-61186372, JNJ-372)).

Antibody-drug conjugates

In some embodiments, the compounds of this invention can be conjugated to a corresponding target-binding group in a special chemical structure (“the conjugate”). This structure is stable in blood plasma and decomposed into an active pharmaceutical ingredient in a specific biological environment, thereby maximizing therapeutic effects on target cells and minimizing toxic side effects on non-target cells, which for example can be used in the treatment of various malignant tumors. In some cases, while the conjugate is stable in the blood circulation system, it is cleaved after endocytosis by target cells, thereby releasing the compound as the CDK2 inhibitor. In some cases, the corresponding target-binding group is an antibody, and the conjugate is an antibody-drug conjugate (ADC), wherein the compound is conjugated with the antibody, preferably via a linker. In some cases, the compound is the payload of the ADC.

In some embodiments, the invention also provides an antibody-drug conjugate (ADC), wherein the ADC comprises the compound of the invention as described herein, an antibody (preferably, an antibody targeting a cancer cell), and optionally a linker. In some cases, the antibody is selected from the antibodies described herein. In some cases, the targeting cell is a cancer cell as described herein. The linker is a linker used in ADCs for small molecule drugs, including without limitation, cleavable linkers comprising hydrazone bonds (which are acid-sensitive and hydrolyze in the acidic environment of lysosomes or endosomes), disulfide bonds that exploit the higher reductive glutathione concentrations in cancer cells for selective payload release, glucuronide bonds that utilize P-glucuronic acid motifs for hydrophilicity and enzyme-specific cleavage, and peptide bonds that are cleaved by tumor-specific proteases to release the cytotoxic drug. Non-cleavable linkers, such as SMCC (N-succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate), require internalization into tumor cells followed by lysosomal degradation of the antibody to release the active drug, as seen in trastuzumab emtansine (T-DM1). Emerging linker designs focus on improving stability and reducing off-target effects through hydrophilic linkers like P- glucuronic acid derivatives, which reduce aggregation and enhance pharmacokinetics, as well as innovative molecular entities tailored for efficient payload release at targeted sites. The linker is selected from polyethylene glycol (PEG) linkes (such as PEG4), glucuronide linkers, sulfonate linkers, peptide linkers, and emerging designs such as DBCO, tetrazine, TCO, BCN, and cyclopropene linkers.

In some embodiments, the compound is conjugated with the antibody with or without the linker through R7 or Rs with reference to any one of Formula (I)-Formula (V), preferably through R7. For example, the conjugate of the invention with reference to Formula (III) can be represented by the formula shown below: wherein L represents the linker, o is 0 or 1, Ab represents the antibody.

Biomarkers and Pharmacodynamics Markers

The invention further provides predictive markers (e.g., biomarkers and pharmacodynamic markers, e.g., gene copy number, gene sequence, expression levels, or phosphorylation levels) to identify those human subjects having, suspected of having, or at risk of developing a disease or disorder associated with CDK2 for whom administering a CDK2 inhibitor (“a CDK2 inhibitor” as used herein refers to a compound of the disclosure, or a pharmaceutically acceptable salt thereof) is likely to be effective.

CCNE1

In one embodiment, the biomarker is CCNE1. In particular an amplification of the cyclin El (CCNE1) gene and/or an expression level of CCNE1 in a biological sample would indicate that the patient or subject could benefit from administration of a compound of Formula (I) or Formula (la) or a pharmaceutically acceptable salt thereof.

CCNE1 is a cell cycle factor essential for the control of the cell cycle at the Gl/S transition (Ohtsubo et al., 1995, Mol. Cell. Biol. 15:2612-2624). CCNE1 acts as a regulatory subunit of CDK2, interacting with CDK2 to form a serine/threonine kinase holoenzyme complex. The CCNE1 subunit of this holoenzyme complex provides the substrate specificity of the complex (Honda et al., 2005, EMBO 24:452-463). CCNE1 is encoded by the cyclin El (“CCNE1”) gene (GenBank Accession No. NM_001238). The amino acid sequence of human CCNE1 is found at GenBank Accession No. NP_001229/UniProtKB Accession No. P24864).

In one aspect, the present disclosure provides a method of treating a subject having, or at risk of developing, a disease or disorder associated with CDK2, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein, wherein the subject has an amplification of the CCNE1 gene and/or have an expression level of CCNE1 higher than a control expression level of CCNE1. In some embodiments, the disease or disorder associated with CDK2 is cancer.

Also provided herein is a method of treating a patient having an amplified expression level of CCNE1 and suffering from, or at risk of developing, a solid tumor cancer, comprising administering to the patient a therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein.

An amplification of the CCNE1 gene and/or an expression level of CCNE1 that is higher than a control expression level of CCNE1 is indicative/predictive that a human subject having or at risk of developing a disease or disorder associated with CDK2 will respond to a CDK2 inhibitor. In some embodiments, the expression level of CCNE1 may be the level of CCNE1 mRNA. In other embodiments, the expression level of CCNE1 may be the level of CCNE1 protein.

Other Biomarkers

In some embodiments, the contemplated biomarker may be pl6 (also known as cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase 4 inhibitor A, multiple tumor suppressor 1, and pl6-INK4a), which acts as a negative regulator of the proliferation of normal cells by interacting with CDK4 and CDK6. In other embodiments, the contemplated biomarker may be phosphorylation of R^b at the serine corresponding to amino acid position 780. R^b is a regulator of the cell cycle and acts as a tumor suppressor. R^b is activated upon phosphorylation by cyclin D-CDK4/6 at Ser780 and Ser795 and by cyclin E/CDK2 at Ser807 and Ser811.

The contemplated biomarker may also be selected from the group consisting of RBI, RBL1, RBL2, CDKN2A, CDKN1A, CDKN1B, FBXW7, CCNE1, CCNE2, CCNA1, CCNA2, CCND1, CCND2, CCND3, CDK2, CDK3, CDK4, CDK6, CDKN2A, CDNK1A, CDKN1B E2F1, E2F2, E2F3, MYC, MYCL, MYCN, EZH2, ER, HER2, HER3, HPV+, and EGFR.

Biological Samples

Suitable biological samples for the methods described herein include any sample that contains blood or tumor cells obtained or derived from the human subject in need of treatment. For example, a biological sample can contain tumor cells from biopsy from a patient suffering from a solid tumor. A tumor biopsy can be obtained by a variety of means known in the art. Alternatively, a blood sample can be obtained from a patient suffering from a hematological cancer.

A biological sample can be obtained from a human subject having, suspected of having, or at risk of developing, a disease or disorder associated with CDK2. In some embodiments, the disease or disorder associated with CDK2 is a cancer (such as those described supra).

Methods for obtaining and/or storing samples that preserve the activity or integrity of molecules (e.g., nucleic acids or proteins) in the sample are well known to those skilled in the art. For example, a biological sample can be further contacted with one or more additional agents such as buffers and/or inhibitors, including one or more of nuclease, protease, and phosphatase inhibitors, which preserve or minimize changes in the molecules in the sample.

Definitions

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

The term “comprising”, which is used interchangeably with “including”, “containing”, or “characterized by”, is inclusive or open-ended language and does not exclude additional, unrecited elements or method steps.

The phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The application contemplates embodiments of the invention compositions and methods corresponding to the scope of each of these phrases. Thus, a composition or method comprising recited elements or steps contemplates particular embodiments in which the composition or method consists essentially of or consists of those elements or steps.

By “inflammation” is meant any types of inflammation, such those caused by the immune system (immune-mediated inflammation) and any symptom of inflammation, including redness, heat, swelling, pain, and/or loss of function.

The term “pain” is used herein in the broadest sense and refers to all types of pain, including acute and chronic pain, such as nociceptive pain, e.g., somatic pain and visceral pain; inflammatory pain, dysfunctional pain, idiopathic pain, neuropathic pain, e.g., centrally generated pain and peripherally generated pain, migraine, and cancer pain. Pain receptors for tissue injury are located mostly in the skin, musculoskeletal system, or internal organs.

By “patient” it means any animal. In one embodiment, the patient is a human. Other animals that can be treated using the methods, compositions, and kits of the invention include but are not limited to non-human primates (e.g., monkeys, gorillas, chimpanzees), domesticated animals (e.g., horses, pigs, goats, rabbits, sheep, cattle, llamas), and companion animals (e.g., guinea pigs, rats, mice, lizards, snakes, dogs, cats, fish, hamsters, and birds).

Compounds useful in the invention include, but are not limited to, those described herein in any of their pharmaceutically acceptable forms, including isomers such as diastereomers and enantiomers, salts, esters, amides, thioesters, solvates, and polymorphs thereof, as well as racemic mixtures and pure isomers of the compounds described herein. The term “pharmaceutically acceptable salt” represents those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include, but are not limited to acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, isethionate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mesylate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like.

In the generic descriptions of compounds of this invention, the number of atoms of a particular type in a substituent group is generally given as a range, e.g., an alkyl group containing from 1 to 4 carbon atoms or Ci-4 alkyl of C1-C4 alkyl. Reference to such a range is intended to include specific references to groups having each of the integer number of atoms within the specified range. For example, an alkyl group from 1 to 4 carbon atoms includes each of Ci, C2, C3, and C4 alkyls. Other numbers of atoms and other types of atoms may be indicated in a similar manner.

“D” is deuterium.

As used herein, the terms “alkyl” and the prefix “alk-” are inclusive of both straight chain and branched chain groups and of cyclic groups, i.e., cycloalkyl. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 6 ring carbon atoms or 3 to 7 carbon atoms, inclusive. Exemplary cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

By “Ci-4 alkyl” or “C1-C4 alkyl” is meant a branched or unbranched hydrocarbon group having from 1 to 4 carbon atoms. Similarly, a “C1-6 alkyl” or “Ci-Ce” is a branched or unbranched hydrocarbon group having from 1 to 6 carbon atoms. A “C1-3 alkyl” or “C1-C3” is a branched or unbranched hydrocarbon group having from 1 to 3 carbon atoms. An alkyl, including, for example, a Ci-4 alkyl or Ci-6 alkyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, alkylamino, disubstituted amino, quaternary amino, alkylcarboxy, and carboxyl groups. Exemplary substituents also include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide (F, Cl, Br or I), hydroxyl, fluoroalkyl, perfluoralkyl, oxo, amino, alkylamino, disubstituted amino, quaternary amino, amido, ester, alkylcarboxy, alkoxycarbonyl, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, aryl, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Ci-4 alkyls include, without limitation, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, secbutyl, tert-butyl, and cyclobutyl. Ci-6 alkyls include, without limitation, methyl, ethyl, n- propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n- pentyl, n-hexyl, cyclobutyl, cyclopentyl, and cyclohexyl.

An example of a substituted alkyl is a heteroalkyl. By “heteroalkyl” is meant a branched or unbranched alkyl, cycloalkyl, alkenyl, or alkynyl group having one or more heteroatoms in place of the carbon atoms independently selected from the group consisting of N, O, and S. By “C1-7 heteroalkyl” is meant a branched or unbranched alkyl, alkenyl, or alkynyl group having from 1 to 7 carbon atoms in addition to 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyls can include, without limitation, tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. A heteroalkyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members. The heteroalkyl group may be substituted or unsubstituted. Exemplary substituents include alkyl, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide (F, Cl, Br or I), hydroxyl, fluoroalkyl, perfluoralkyl, oxo, amino, alkylamino, disubstituted amino, quaternary amino, amido, ester, alkylcarboxy, alkoxycarbonyl, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, aryl, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Examples of C 1-7 heteroalkyls include, without limitation, methoxymethyl and ethoxy ethyl. Substituted alkyl also include aralkyl.

An alkenyl is a branched or unbranched hydrocarbon group containing one or more double bonds. For example, by “C2-6 alkenyl” or “C2-C6 alkenyl” is meant a branched or unbranched hydrocarbon group containing one or more double bonds and having from 2 to 6 carbon atoms. An alkenyl may optionally include monocyclic or polycyclic rings, in which each ring desirably has from three to six members. The alkenyl group may be substituted or unsubstituted. Exemplary substituents include those described above for alkyl, and specifically include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, alkylamino, disubstituted amino, quaternary amino, alkylcarboxy, and carboxyl groups. C2-6 alkenyls include, without limitation, vinyl, allyl, 2- cyclopropyl-1 -ethenyl, 1 -propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2 -m ethyl- 1 -propenyl, and 2-methyl-2-propenyl.

An alkynyl is a branched or unbranched hydrocarbon group containing one or more triple bonds. For example, by “C2-6 alkynyl” or “C2-C6 alkynyl” is meant a branched or unbranched hydrocarbon group containing one or more triple bonds and having from 2 to 6 carbon atoms. An alkynyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has five or six members. The alkynyl group may be substituted or unsubstituted. Exemplary substituents those described above for alkyl, and specifically include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, alkylamino, disubstituted amino, quaternary amino, alkylcarboxy, and carboxyl groups. C2-e alkynyls include, without limitation, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.

By “heterocyclyl,” “heterocyclic,” or “heterocycloalkyl” is meant a stable monocyclic or polycyclic (including a bicyclic or a tricyclic) heterocyclic ring which is saturated, partially unsaturated or unsaturated (including heteroaryl or aromatic), and which consists of 2 or more carbon atoms and 1, 2, 3, 4 or more heteroatoms independently selected from N, O, and S and including any bicyclic or polycyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring, heteroaryl, cycloalkyl or heterocycloalkyl. A “3- to 6- membered heterocycloalkyl” is mean to refer to a heterocyclic ring having 3 to 6 ring atoms wherein at least one ring atom is a heteroatom selected from N, O, and S. Similarly, a “3- to 10- membered heterocycloalkyl” is mean to refer to a heterocyclic ring having 3 to 10 ring atoms wherein at least one ring atom is a heteroatom selected from N, O, and S. In certain aspects, the heterocyclyl is a 3- to 15-membered ring system, a 3- to 12- membered ring system, or a 3- to 9-membered ring system. By “C2-6 heterocyclyl” is meant a stable 5- to 7-membered monocyclic or 7- to 14-membered bicyclic heterocyclic ring which is saturated, partially unsaturated or unsaturated (including heteroaryl or aromatic), and which consists of 2 to 6 carbon atoms and 1, 2, 3 or 4 heteroatoms independently selected from N, O, and S and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring, heteroaryl, cycloalkyl or heterocycloalkyl. The heterocyclyl or heteroaryl group may be substituted or unsubstituted. Exemplary substituents include substituted or unsubstituted alkyl, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, alkylamino, disubstituted amino, quaternary amino, alkylcarboxy, oxo, and carboxyl groups. The nitrogen and sulfur heteroatoms may optionally be oxidized. The heterocyclic ring may be covalently attached via any heteroatom or carbon atom which results in a stable structure, e.g., an imidazolinyl ring may be linked at either of the ring-carbon atom positions or at the nitrogen atom. A nitrogen atom in the heterocycle can be quaternized. Preferably when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to one another. Heterocycles include, without limitation, IH-indazole, 2- pyrrolidonyl, 2H,6H-l,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl, 4aH- carbazole, 4H-quinolizinyl, 6H-l,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH- carbazolyl, b-carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-

1.5.2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, IH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,

1.2.3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinylperimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothi azole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-l,2,5-thiadiazinyl, 1,2,3- thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thi enothiazolyl, thienooxazolyl, thi enoimidazolyl, thiophenyl, triazinyl, 1,2,3- triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, xanthenyl, P-lactam, y-lactam and 5- lactam. Preferred 5 to 10 membered heterocycles include, but are not limited to, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, benzofuranyl, benzothiofuranyl, indolyl, benzimidazolyl, IH-indazolyl, oxazolidinyl, isoxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, quinolinyl, and isoquinolinyl. Preferred 5 to 6 membered heterocycles include, without limitation, pyridinyl, quinolinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, piperazinyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, and tetrazolyl. Preferred substituents include phenyl, methyl, ethyl, propyl, butyl, chloro, bromo, fluoro, iodo and oxo.

By “aryl” is meant an aromatic group having a ring system comprised of carbon atoms with conjugated TI electrons (e.g., phenyl). A “C6-C12 aryl” or “Ce-Cio aryl” is an aryl group that has from 6 to 12 carbon atoms or 6 to 10 carbon atoms, respectively. Aryl groups may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has five or six members. A bicyclic or tricyclic ring system can be fused (e.g., naphthyl) or not (e.g., biphenyl). The aryl group may be substituted or unsubstituted. Exemplary substituents include substituted or unsubstituted alkyl, hydroxyl, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl, alkylcarboxy, amino, alkylamino, monosubstituted amino, disubstituted amino, and quaternary amino groups. A preferred aryl group is phenyl. By “heteroaryl” it is meant an aromatic ring group having a ring system comprised of hetero atoms (such as N, O, S) and carbon atoms with conjugated TI electrons (e.g., pyridine, pyrimidine, triazine). A “5- to 6- membered heteroaryl” refers to a heteroaryl having 5 to 6 ring atoms with conjugated TI electrons wherein at least one ring atom is a heteroatom selected from N, O, and S. Similarly, a “5- to 12- membered heteroaryl” refers to a heteroaryl having 5 to 12 ring atoms with conjugated TI electrons wherein at least one ring atom is a heteroatom selected from N, O, and S. The heteroaryl groups can include monocyclic, bicyclic, or tricyclic rings, with each ring typically having five or six members. Bicyclic or tricyclic ring systems within heteroaryls can be fused (e.g., quinoxaline) or not. Heteroaryl groups may be substituted or unsubstituted, with possible substituents including various functional groups such as substituted or unsubstituted alkyl, hydroxyl, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl, alkylcarboxy, amino, alkylamino, monosubstituted amino, disubstituted amino, and quaternary amino groups. An example of a preferred heteroaryl group is a phenyl group with heteroatoms replacing one or more carbon atoms in the ring.

By “aralkyl” is meant a substituted or unsubstituted alkyl that is substituted by a substituted or unsubstituted aryl (including, for example, (e.g., benzyl, phenethyl, or 3,4- di chi orophenethy 1) .

By “C7-14 aralkyl” is meant an alkyl substituted by an aryl group (e.g., benzyl, phenethyl, or 3, 4-di chlorophenethyl) having from 7 to 14 carbon atoms.

By “halide” or “halogen” is meant bromine, chlorine, iodine, or fluorine. By “fluoroalkyl” is meant an alkyl group that is substituted with a fluorine atom. By “alkylcarboxy” is meant a chemical moiety with the formula — (R) — COOH, wherein R is selected from C1-7 alkyl, C2-7 alkenyl, C2-? alkynyl, C2-6 heterocyclyl, Ce-n aryl, C7-14 aralkyl, C3-10 heterocycloalkyl, or C 1-7 heteroalkyl.

By “alkoxy” is meant a chemical substituent of the formula — OR, wherein R is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl or R can be selected from C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, C2-6 heterocyclyl, Ce-n aryl, C7-14 aralkyl, C3-10 heterocycloalkyl, or C 1-7 heteroalkyl.

By “aryloxy” is meant a chemical substituent of the formula — OR, wherein R is a Cell aryl group. By “alkylthio” is meant a chemical substituent of the formula — SR, wherein R is selected from C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, C2-6 heterocyclyl, Ce-n aryl, C7-14 aralkyl, C3-10 heterocycloalkyl, or C 1-7 heteroalkyl.

By “arylthio” is meant a chemical substituent of the formula — SR, wherein R is a C6-12 aryl group.

By “spiro ring structure” it means a moiety that comprises at least two molecular rings with one common atom.

By “charged moiety” is meant a moiety which gains a proton at physiological pH thereby becoming positively charged (e.g., ammonium, guanidinium, or amidinium) or a moiety that includes a net formal positive charge without protonation (e.g., quaternary ammonium). The charged moiety may be either permanently charged or transiently charged.

By “therapeutically effective amount” or “effective amount” means an amount sufficient to produce a desired result, for example, the reduction or elimination of any symptoms in a patient (e.g., a human) suffering from an inflammatory-related disease or disorder.

The compounds of the present invention, including salts of the compounds, can exist in unsolvated forms as well as solvated forms, including hydrated forms and unhydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Nonlimiting examples of hydrates include monohydrates, dihydrates, hemihydrates, etc. In certain aspects, the compound is a hemihydrate. Nonlimiting examples of solvates include ethanol solvates, acetone solvates, etc. The compounds of the invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for uses contemplated by the present invention and are intended to be within the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.

Examples

Preparation of Precursors

Preparation of precursor XC120 (Ri is CH3) to be used in subsequent biosynthetic transformations to prepare the compounds (Yoshino et al., OrgLett. 2012 Aug 17;14(16):4290-2. doi: 10.1021/ol3019727; Huang et al., The Journal of Organic Chemistry 2017 82 (13), 6604-6614, DOI: 10.1021/acs.joc.7b00622.):

Preparation of precursor Br-XC120 (Ri is Br) to be used in subsequent biosynthetic transformations to prepare the compounds:

Additional method for the preparation of precursors to be used in subsequent biosynthetic transformations to prepare the compounds:

Additional method for the preparation of precursors to be used in subsequent biosynthetic transformations to prepare the compounds (Key steps inverted: a. Ullmann / Buchwald coupling; b. lactonization) (See Takeshi et al., Journal of the American Chemical Society 1995 117 (43), 10757-10758, DOI: 10.1021/ja00148a024; Deraeve et al., Journal of the American Chemical Society 2012 134 (17), 7384-7391, DOI: 10.1021/j a211305j):

These 2 steps can be interconverted

Preparation of the Compounds

An example of biosynthetic processing to obtain cis-diols and trans-diols which are further transformed in subsequent synthesis: Chemical transformations of diols into ketals by reaction with ketones (e.g. acetone or cyclohexanone) are known techniques in the field of chemical synthesis. Dong, et al., ACS Omega 2018 3 (5), 4974-4985, DOI: 10.1021/acsomega.8b00159. Additional exemplary transformations for compounds of the invention are provided below.

Alternatively, the precursors undergo chemical transformations of diols into alkoxy derivatives by reaction with alkyl-bromides, alkyl-iodides, and any other type of alkylating reagent under conditions.

cis-Diol trans-Diol

Synthesis of Exemplary Compound

Compound No. 15 was synthesized according to the synthetic routes below.

General Scheme for the synthesis of 4-((6-bromo-2,4-dihydroxy-3-methylbenzoyl)oxy)-2- hydroxy-3, 6-dimethylbenzoic acid; Step-1: Synthesis of methoxymethyl 2,4-bis(benzyloxy)-6-bromo-3-methylbenzoate;

(eLNB ID G0060-1858349 &1870831)

To the stirred solution of methoxymethyl 6-bromo-2,4-dihydroxy-3-toluate (40 g, 1.0 eq., 137.4 mmol) in DMF (400 mL) was added Potassium carbonate (136.6 g, 3.0 eq., 412 mmol) under nitrogen atmosphere at room temperature. Then, the Benzyl bromide (32.6 mL, 2.0 eq., 274 mmol) was added dropwise and the resulting reaction mixture was stirred at 50 °C for 16 h. Progress of the reaction was monitored by TLC and LCMS. After complete consumption of starting material, the reaction mass was quenched with ice cold water. The precipitated solid was filtered and dried to get methoxymethyl 2,4-bis(benzyloxy)-6-bromo-3-methylbenzoate (48 g, 75%) as brown solid.

Brown solid; LCMS m/z = 471.02 [M+H]⁺; 'H NMR (400 MHz, DMSO-tL) 3 ppm 7.49 - 7.36 (m, 10H), 7.23 (s, 1H), 5.38 (s, 2H), 5.22 (s, 2H), 4.88 (s, 2H), 3.39 (s, 3H), 2.12 (s, 3H).

Step-2: Synthesis of 2,4-bis(benzyloxy)-6-bromo-3-methylbenzoic acid;

(eLNB ID G0060-1858358 &1874478)

To the stirred solution of methoxymethyl 2,4-bis(benzyloxy)-6-bromo-3-methylbenzoate (48 g, 1.0 eq., 55.2 mmol) in DCM (400 mL) was added 4M HCI in Dioxane (400 mL) dropwise under nitrogen atmosphere at 0 °C and the reaction mixture was allowed to stir at room temperature for 1 h. Progress of the reaction was monitored by TLC and LCMS. After complete consumption of starting material, the reaction mixture was concentrated under reduced pressure to get crude residue. The obtained crude residue was quenched with water and extracted with 10% Methanol in DCM. Combined organic layer were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to obtained crude compound. The obtained crude compound was triturated by pentane to get 2,4- bis(benzyloxy)-6-bromo-3-methylbenzoic acid (42 g, 92%) as brown solid.

Brown solid; LCMS m/z = 426.99 [M-H]’; 'H NMR (400 MHz, DMSO-d6) δ ppm 13.42 (br s, 1H), 7.47 - 7.32 (m, 10H), 7.17 (s, 1H), 5.36 (s, 2H), 4.82 (s, 2H), 2.10 (s, 3H).

To the stirred solution of 2,4-bis(benzyloxy)-6-bromo-3-methylbenzoic acid (25 g, 1.0 eq., 91.8 mmol) and 2,6-bis(benzyloxy)-4-bromo-3-toluic acid (41.2 g, 1.1 eq., 96.6 mmol) in DCM (270 mL) was added DCC (14.8 g, 1.5 eq., 138.2 mmol) under nitrogen atmosphere at room temperature. Then, DMAP (2.92 g, 0.5 eq., 45.2 mmol) was added and the resulting reaction mixture was stirred at room temperature for 16 h. Progress of the reaction was monitored by TLC and LCMS. After complete consumption of starting material, the reaction mixture was filtered through sintered funnel and washed with DCM. The filtrate was washed with water and organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to obtained crude compound. The obtained crude compound was triturated with methanol to get benzyl l-[2,4-bis(benzyloxy)-6-bromo-3-toluoxy]-3-hydroxy-2,5-4- xylenecarboxylate (30.8 g, 51%) as white solid.

White solid; LCMS m/z = 678.95 [M-H]’; 'H NMR (400 MHz, DMSO-tL) 3 ppm 10.43 (br s, 1H), 7.48 - 7.34 (m, 15H), 7.31 (s, 1H), 6.37 (s, 1H), 5.36 (s, 2H), 5.25 (s, 2H), 4.95 (s, 2H), 2.18 (s, 3H), 2.16 (s, 3H), 1.95 (s, 3H). Step-4: Synthesis of 4-((6-bromo-2,4-dihydroxy-3-methylbenzoyl)oxy)-2-hydroxy-3,6- dimethylbenzoi

To the solution of benzyl l-[2,4-bis(benzyloxy)-6-bromo-3-toluoxy]-3-hydroxy-2,5-4- xylenecarboxylate (30.8 g, 1.0 eq., 45.2 mmol) in ethyl acetate (1.5 L) was degassed for 10 min. Further, Pd/C (15.4 g, w/2, 50% wet) was added and the reaction mixture was hydrogenated under balloon pressure for 16 h at room temperature. The progress of the reaction was monitored by TLC. After complete consumption of starting material; the reaction mixture was filtered through celite bed and washed with ethyl acetate. The filtrate was concentrated under reduced pressure to obtained crude compound, which was purified by PREP HPLC purification to get 4-(6-bromo-2,4-dihydroxy-3-toluoxy)-2-hydroxy-3,6- xylenecarboxylic acid (8.7 g, 47%) as white solid.

White solid; LCMS m/z = 409.02 [M-H]'; 'H NMR (400 MHz, DMSO-tfc) 3 ppm 13.99 -

12.45 (br m, 2H), 10.32 (s, 1H), 9.88 (s, 1H), 6.72 (s, 1H), 6.61 (s, 1H), 2.48 (s, 3H), 2.07 (s, 3H), 1.99 (s, 3H). ent-example 15 example 15 Compound No. 15 (i.e., Example 15) exhibited a purity of 54.4% and an EC50 value of 62.8 nM in Kinobead assay after being corrected for purity.

Biological experiments

Lead Optimization and SAR. We were intrigued by the observation that 2 shows 25-fold enhanced biochemical inhibition of CDK2/cyclin E relative to the co-isolated GEM 1, despite only a single difference between the two compounds, specifically the configuration at the C7 stereocenter. To ascertain the impact of the stereochemical configuration on the activity, we characterized our panel of stereoisomer analogs. To confirm biological activity we tested compounds in parallel in live cells employing NanoBRET alongside in vitro enzyme assays; these data corroborated each other well. Compound 2 showed 21 -fold greater biochemical inhibition to its C6 epimer 28, and for the epimerized scaffold the C7 diastereomer 28 was superior to 27, showing the same trend as 2 and 1. Compounds 24 and 29 (enantiomers of 1 and 27, respectively) lost significant activity against CDK2/cyclin E, and displayed non-covalent binding properties. The C6/C7-simplified analog set was also profiled, however complete removal of substituents at C6 and C7 (compound 38) led to substantial loss of activity on CDK2/cyclin E, which was partially restored upon reintroduction of both a methyl group at C6 and hydroxyl at C7 (39). Epimerization at C7 in 40 provided a further boost to activity, achieving a similar level of cellular inhibition to 1, but still 6-fold lower than 2.

In aggregate, these observations suggest that both the presence of substituents and the natural stereochemical configuration is crucial for the covalent inhibition of human CDK2/cyclin E, likely determining the proximity and orientation of the vinylogous anhydride to the catalytic lysine. Furthermore, the range of activities across the chemical series demonstrates that this unusual motif is not an indiscriminate electrophile, but rather a tunable warhead.

The CDK inhibitory profiles on the semisynthetic derivatives of 2 are shown in Table 3. The C7 OH tolerated diverse functionality, such as acetyl, glycine, methylcarbamate and a linked biotin moiety, but there was a universal loss of activity. The R2 position was also synthetically tractable, and we were able to establish an SAR trend according to size across compounds 41-45. The smaller R2 substituents are more active: biochemically H > F, Me, Cl > Br » Et; and in the NanoBRET cellular context H, F > Cl > Me > Br » Et. It is also worthy of note that larger substituents R2 = Cl (43) and Br (44) show increased selectivity for CDK2 over CDK7 relative to smaller substituents H and F (2 and 41, respectively).

Table 3. SAR of semisynthetic analogs

Rev = Reversible. Enzyme and NanoBRET activity assays were assessed on the active CDK/cyclin holoenzyme as follows: CDK2/cyclin El, CDKl/cyclin Bl, CDK7/cyclin H/MATl. Mean values calculated from at least two independent replicates for enzyme assays and NanoBRET The chlorinated analog 43 (LifeMine code XC219) was selected for additional profiling as it retained potent biochemical and cellular inhibition of CDK2 while demonstrating improved selectivity against CDK7. We acquired an X-ray crystal structure of 43 in complex with CDK2/cyclin E, which showed that its binding mode matches that of 2, with the C3 phenol binding the hinge and formation of a covalent bond from Cl 1 to Lys33. The R2 vector projects towards Val64 of CDK2, which rotates to accommodate the Cl atom of 43. There is a point-change of the corresponding residue to Ile75 in CDK7, and we propose that the larger residue cannot accommodate the R2 = Cl, affording selectivity for CDK2 over CDK7. Selectivity Across the Kinome and Proteome. The vinylogous anhydride of 2 reacts with the active site lysine of CDK2 (Lys33) to form a covalent bond. This residue is highly conserved within the protein kinase family, which raises the question of the selectivity of 2 not only for CDKs but for the entire kinome.³⁴ Furthermore, the vinylogous anhydride of 2 clearly shows conditional reactivity with CDKs, but its electrophilic nature could give rise to off-target reactivity. To assess the selectivity of 2 across the human kinome, we tested 2 in a competitive Kinobead pull-down assay.³⁵ The assay revealed remarkably high overall selectivity, with the primary human targets of 2 being the CMGC family of proline-directed serine/threonine kinases, which contains 21 CDK family members. A small number of additional kinase targets were identified in the CK1, CAMK, and TKL classes. A broader competitive chemoproteomic screen using the biotinylated probe 50 in lysates treated with 25 pM of 2 detected no additional non-kinase targets.

We also performed an analogous Kinobead experiment in S. cerevisiae lysate to profile the fungal kinase targets of 2, revealing potent (ECso=31 nM) and selective engagement of yeast PHO85, consistent with the genomic target prediction based on the fungal Pho85 ETaG in the ros BGC. Compound 2 engaged yeast PHO85 as the top ranking target out of 81 detected kinases in the cell lysate, demonstrating > 100-fold higher potency relative to nearly all other detected kinases, including fungal CDK orthologs CDC28, KIN28, and SGV 1.

We profiled a diverse selection of synthetic analogs in the human Kinobead assay. The improved selectivity of 43 versus 2 was recapitulated in the Kinobead assay, and extended to the larger CDK family CDK5, 7, 12, and 13, and importantly the kinome in general. The transcriptional regulators CDK7, 12, and 13 are known to have a narrow therapeutic window, while reduced inhibition of CDK5 offers more favorable neurological safety.³⁶ Beyond the CDK family we also noted improved selectivity on kinases such as MAP3K1 1 and CSKN1E. The C7 acetylated analog 47 led to a slight shift of activity favoring the CMGC kinase CLK2, and the CAMK kinase AURKC. The simplified analog 38 showed barely detectable CDK2 activity but was highly selective for a therapeutically valued target, CK1 kinase CSNK1 Al,³⁷ albeit with weak activity.

Pharmacokinetics. The in vivo pharmacokinetic profiles of 2 and 43 are reported in Table 4. 2 shows high clearance and low oral bioavailability in mouse and rat. In contrast, 43 shows low clearance and good oral bioavailability in both rodent species. Here, a single atom change afforded a significant improvement in the PK profile, and we attribute this to the improved microsomal stability observed in vitro.

Brain and plasma exposures were evaluated over a 24 h period in mice administered with a single dose of either 2 or 43, at 30 mpk. Interestingly, 2 showed exceptionally high levels of brain exposure (AUC blood-to-plasma ratio of 0.68), and 43 significantly reduced brain exposures (AUC b:p ratio = 0.0003).

High-dose intraperitoneal (i.p.) mouse PK was conducted to determine tolerability. Although animals treated with a single 50 mpk dose of 2 presented with acute body weight loss within 3 days, animals treated with 43 tolerated 7 days of daily dosing at 100 mpk with no adverse observations noted. We attributed the improved tolerability to the improved selectivity profile.

Table 4. Pharmacokinetic and select in vitro ADME parameters of Compounds 2 and 43.

In Vivo Efficacy. Given the favorable selectivity, PK and tolerability profiles, 43 was advanced to an efficacy study to evaluate this novel mechanism of CDK2 inhibition in a gastric Patient Derived Xenotransplant (PDX) model. We selected a rapidly proliferating PDX, CRT00292 gastric mouse model harboring a high CCNE1 amplification (CN = 12), which is a predictive biomarker of response to CDK2 inhibition. Animals with established subcutaneous tumor fragments were dosed daily i.p. for 14 days, resulting in tumor growth inhibition (TGI) measured relative to the vehicle control over the duration of the study. At 14 days 43 showed 44% and 80% TGI at 30 and 100 mpk, respectively, demonstrating a dosedependent response and a remarkable level of TGI at the high dose in this aggressive PDX model.

Figure 1 showed anti-tumor growth activity in a CCNE amplified (CN=12) gastric PDX model CRT00292. NOG mice were inoculated subcutaneously with tumor fragments and randomized (n=7/group) when tumors reached an average of 150-200 mm³. Animals were dosed daily with XC219 43 (30 and 100 mpk xl4d, i.p.). In the highest dose group 3 animals were moribund or withdrawn from treatment. One-way ANOVA, Dunnett’s multiple comparison test was performed reporting statistical significance of lOOmpk group, p value = 0.0079.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. It will also be understood that none of the embodiments described herein are mutually exclusive and may be combined in various ways without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A compound represented by Formula (I):

Formula (I), or a pharmaceutically acceptable salt, a stereoisomer and a mixture of stereoisomers, or a prodrug thereof;

Xi and X₂ are each independently selected from O, S, NRY; wherein RY is selected from H, D and a substituted or unsubstituted alkyl;

Ri is selected from H, D, halogen, substituted or unsubstituted alkyl (such as -CD3), substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkoxyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, ORA, CN, NRBRC, NRAC(O)RA, S(O)RA, S(O)₂RA, SO₂NRBRC, SO₃RA, COORA, C(O)RA, and C(O)NRBRC_;

Each RA, RB, and Rc is independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl; or RB and Rc can be taken together with the nitrogen to which they are attached to form a substituted or unsubstituted 5, 6, 7, or 8 membered ring;

R₂ and R4 are each independently selected form H, D, halogen, a substituted or unsubstituted alkyl (such as a deuterated alkyl, such as -CD3), a substituted or unsubstituted alkenyl;

R3 is selected from H, D, OH, a C1-6 alkoxyl, and a C1-6 alkyl; Rs and Re are independently selected form hydroxy, a substituted or unsubstituted alkyl, and a substituted or unsubstituted alkoxy.

R7 is selected from H, -OH, and a substituted or unsubstituted alkoxy;

Rs is selected from H, -OH, and a substituted or unsubstituted alkoxy (for example Rs is -ORx, wherein Rxis a substituted or unsubstituted alkyl, ester, amino acid, acyl, amine, or amide);

Or one of Rs and Re, and one of R7 and Rs, together with the carbon atoms to which they are attached, form a heterocycloalkyl containing at least two oxygen atoms, wherein two oxygen atoms are in a ketal arrangement, wherein the heterocycloalkyl further optionally comprises a fused or spiro ring structure; and

R9 is selected from H, D, a substituted or unsubstituted alkyl, and a substituted or unsubstituted alkenyl;

Wherein the compound does not comprise:

2. The compound of claim 1, wherein Xi and X2 are both O.

3. The compound of claim 1 or 2, wherein Ri is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkoxyl, a substituted or unsubstituted 5- to 6-membered aryl, a substituted or unsubstituted 5- to 6-membered heteroaryl, a substituted or unsubstituted 3- to 10-membered cycloalkyl, or a substituted or unsubstituted 4- to 10-membered heterocycloalkyl.

4. The compound of claim 1 or 2, wherein Ri is a substituted alkyl, a substituted or unsubstituted alkoxyl, a substituted or unsubstituted 5- to 6-membered aryl, a substituted or unsubstituted 5- to 6-membered heteroaryl, a substituted or unsubstituted 3- to 10-membered cycloalkyl, or a substituted or unsubstituted 4- to 10-membered heterocycloalkyl.

5. The compound of claim 1 or 2, Ri is a substituted or unsubstituted -(CH2)_P-Rz; P is an integer selected from 0 to 6; Rz is ORA, CN, NRBRC, NRAC(O)RA, S(O)RA, S(O)2RA, SO2NRBRC, SOSRA, COORA, C(O)RA, C(O)NRBRC, a substituted or unsubstituted C3-6 cycloalkyl, a substituted or unsubstituted C4-6 heterocycloalkyl, a substituted or unsubstituted C5-6 aryl, or a substituted or unsubstituted C5-6 heteroaryl.

6. The compound of claim 1 or 2, Ri is selected from -CH2CH3, - (CH2)2C(O)NH2,

-(CH₂)₃OCH3, -(CH₂)₃OH, -CH₂O-(CH₂)2-N(CH3)2, -0-CH2-CH2F, -N(CH₃)-CH₂-

7. The compound of any one of the preceding claims, wherein R2 is H, halogen (preferably F, Br or Cl, more preferably Br or Cl, most preferably Br), or an unsubstituted alkyl (preferably methyl, ethylene).

8. The compound of any one of the preceding claims, wherein R3 is selected from H, D and OH, more preferably, OH.

9. The compound of any one of the preceding claims, wherein R3 is OH.

10. The compound of any one of the preceding claims, R4 is an unsubstituted alkyl, preferably methyl.

11. The compound of any one of the preceding claims, wherein one of Rs and Re is -OH or a substituted or unsubstituted alkoxy; and the other is a substituted or unsubstituted alkyl.

12. The compound of any one of the preceding claims, wherein one of R7 and Rs is -OH, and a substituted or unsubstituted alkoxy.

13. The compound of any one of the preceding claims, wherein Re and R7 together with the intervening atoms form a ring structure, a spiro ring structure, or a fused ring structure.

14. The compound of claim 1, wherein the compound is represented by Formula (Ila) or Formula (lib) :

Formula (Ila) and Formula (lib); wherein Ring A formed by Rw and Rv in Formula (Ila) and Ring B formed by Rw and Rv in (lib) each are independently a substituted or unsubstituted cycloalkyl (preferably 3- to 6- membered cycloalkyl), a substituted or unsubstituted aryl (preferably 5- to 6- membered aryl), a substituted or unsubstituted heterocycloalkyl (preferably 4- to 6- membered heterocycloalkyl), or a substituted or unsubstituted heteroaryl (preferably 5- to 6- membered heteroaryl).

15. The compound of any one of claims 1-11, wherein Ri, Re and R7 is selected from these combinations provided in Table 1.

16. The compound of any one of the preceding claims, wherein Rs is -CH3.

17. The compound of any one of the preceding claims, wherein Rs is H.

18. The compound of claim 1, wherein the compound is any one of these in Table 2A, provided that the compound is Compound No. 1.

19. The compound of claim 1, wherein the compound is any one of these in Table 2B.

20. A pharmaceutical composition comprising the compound of any one of the preceding claims or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

21. The composition of claim 20, wherein said composition is formulated for oral, intravenous, intramuscular, rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, inhalation, vaginal, intrathecal, epidural, or ocular administration.

22. A method for treating a disease or disorder related to the dysregulation of a cyclin- dependent kinase (CDK) in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the compound of claims 1-19.

23. The method of claim 22, wherein the CDK is CDK2.

24. The method of claim 22 or 23, wherein the disease or disorder is a cancer.

25. A method of inhibiting CDK2, wherein the method comprises administering to the subject a therapeutically effective amount of the compound of claims 1-19.

26. The method of claim 25, wherein the compound exhibits at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, or about 5-fold selectivity for CDK2 versus CDK1 in terms of ICso value.

27. The method of claim 26, wherein the compound exhibits at least 10-fold selectivity for CDK2 versus CDK1 in terms of IC50 value.

28. The method of claim 25, wherein the compound exhibits at least 1.5-fold, about 2- fold, about 3-fold, about 4-fold, or about 5-fold selectivity for CDK2 versus CDK7 in terms of ICso value.