Degraders of fibroblast growth factor receptor 2 (FGFR2)

Abstract

The present invention relates to bispecific compounds, compositions and methods for treating diseases or conditions characterized by or mediated by aberrant fibroblast growth factor receptor 2 (FGFR2) activity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Pursuant to 35 U.S.C. §119(e), this application claims the benefit of priority to U.S. Provisional Application No. 62 / 831,952, filed on April 10, 2019, and U.S. Provisional Application No. 62 / 884,422, filed on August 8, 2019, the entire contents of both applications are incorporated herein by reference.

[0003] Government licensing rights

[0004] This invention was made with government support under Grant No. P50 CA12)003 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] Biliary tract cancer (BTC) accounts for approximately 3% of all gastrointestinal (GI) malignancies and carries a poor prognosis. The 5-year survival rate is less than 15% for all patients, falling to 10% for stage III patients and nearly 0% for stage IV patients, despite the use of the best available chemotherapy. The incidence of BTC has been increasing over the past few decades, primarily due to the rise in intrahepatic cholangiocarcinoma (ICC). Genetic alterations that activate fibroblast growth factor (FGF) signaling are among the most common genomic alterations in ICC, present in more than 20% of tumors, the most common of which includes fusions of fibroblast growth factor receptor 2 (FGFR2) exons 1-18 to multiple partners encoding the dimerization domain, resulting in constitutive FGFR2 kinase activity.

[0006] FGFR plays an important role in many biological processes such as tissue repair, hematopoiesis, bone growth, angiogenesis and regulation of metabolic processes. The emergence of FGFR2 as a therapeutic target has provided new hope for ICC patients. BGJ398, the most advanced FGFR-selective compound in clinical development for ICC, showed efficacy in a phase II trial in patients with advanced refractory ICC with FGFR alterations (fusion, amplification or point mutation). The overall response rate (ORR) was 14.8% (18.8% with FGFR2 fusion alone) and the disease control rate (DCR; i.e., partial response + stable disease) was 5.4% (83.3% with FGFR2 fusion alone). The median progression-free survival was 5.8 months. Although the survival rate of 5.8 months is still unacceptably low, these results are significantly better than historical control data for patients with refractory advanced ICC.

[0007] However, there are many challenges to translating this encouraging clinical signal into long-term clinical benefit. The first challenge is acquired resistance to adenosine triphosphate (ATP)-competitive FGFR inhibitors. The second challenge is the on-target toxicity of inhibiting different members of the FGFR family. Given these challenges, completely new approaches are needed to achieve transformative advances in FGFR targeting. Summary of the invention

[0008] A bispecific compound or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the bispecific compound has a structure represented by formula (I):

[0009] The targeting ligand represents a portion that binds to fibroblast growth factor receptor 2 (FGFR2), the degron represents a portion that binds to E3 ubiquitin ligase, and the linker represents a portion that covalently links the degron and the targeting ligand.

[0010] Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a bispecific compound or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0011] In another aspect of the present invention, a method for preparing the bispecific compound is provided.

[0012] Another aspect of the invention relates to a method of treating a disease or condition characterized by or mediated by aberrant FGFR2 activity, the method comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific compound or a pharmaceutically acceptable salt or stereoisomer thereof.

[0013] Without wishing to be bound by any particular theory of operation, the bispecific compounds of formula (I) (also referred to herein as degradation agents) are believed to promote the degradation of FGFR2 while retaining other FGFR subtypes. By combining pan-FGFR2 ligands with low nanomolar efficacy with E3 ligase binding agents, these bispecific compounds can quickly recruit E3 ligases, and thus promote the degradation of FGFR2. The degradation agent can obtain a higher target selectivity than the expected constitutive binding ligand, thereby greatly reducing the off-target effect.

[0014] Thus, the bispecific compounds of the invention can serve as a new set of chemical tools to knock out FGFR2, exemplifying a broadly applicable approach to obtain degraders that are selective for non-selective binding ligands and may provide effective treatments for FGFR2-mediated diseases and conditions such as cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A-Figure 1C is an immunoblot showing selective knockout of FGFR2. Figure 1A is an immunoblot showing knockdown of FGFR2 in Kato III cells after 16 hours with different concentrations of bispecific compound 6. Figure 1B is an immunoblot showing knockdown of FGFR2 in Kato III cells after 4 hours for bispecific compound 6, BGJ398, control-1, bortezomib, and MLN4924. Figure 1C is an immunoblot showing knockdown of FGFR2 in Kato III cells over a period of 16 hours for bispecific compound 6 at concentrations of 0.1 μM and 0.5 μM.

[0016] Figure 2A-2C is an immunoblot showing degradation of FGFR1 / 3 / 4. Figure 2A is an immunoblot showing knockdown of FGFR1 in cholangiocarcinoma cells (CCLP1) after 16 hours with different concentrations of bispecific compound 6. Figure 2B is an immunoblot showing knockdown of FGFR3 / 4 in hepatocellular carcinoma cells (JHH) after 16 hours with different concentrations of bispecific compound 6. Figure 2C is an immunoblot showing knockdown of FGFR1 in CCLP1 cells for bispecific compound 6 at concentrations of 0.1 μM and 0.5 μM over a period of 16 hours.

[0017] Figure 3A and Figure 3B is a graph showing the cell viability of FGFR2 degrading agents. Figure 3A is a graph showing the cell viability of VHL-based FGFR2 degraders. Figure 3B is a graph showing the cell viability of CRBN-based FGFR2 degraders.

[0018] Figure 4A is an immunoblot showing the degradation of FGFR2 in Kato III cells after 4 hours for different concentrations of bispecific compounds 6 and 7 (negative control).

[0019] Figure 4B Is to show the corresponding IC 50 Figure 3 is a graph of the cell viability of bispecific compounds 6 and 7 (negative control) and BGJ398.

[0020] Figure 5A and Figure 5B is an immunoblot showing degradation of FGFR2. Figure 5A is an immunoblot showing knockdown of FGFR2 in Kato III cells after 6 hours at 1 μM concentration for bispecific compounds 6 and 14-22. Figure 5Bis an immunoblot showing the degradation of FGFR2 in Kato III cells after 4 hours at various concentrations for bispecific compound 20, FIIN2, control-1, bortezomib, and MLN4924.

[0021] Fig. 6A and Figure 6B are immunoblots showing knockdown of FGFR1 and FGFR4, respectively. Fig. 6A is an immunoblot showing knockdown of FGFR1 in CCLP1 cells after 6 hours at 1 μM concentration for bispecific compounds 6 and 14-22. Figure 6B is an immunoblot showing knockdown of FGFR4 in JHH) cells after 6 hours at 1 μM concentration for bispecific compounds 6 and 14-22. DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the subject matter of this article belongs. As used in the specification and the appended claims, unless otherwise indicated, the following terms have the meanings shown to facilitate understanding of the present invention.

[0023] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "a composition" includes a mixture of two or more such compositions, "an inhibitor" includes a mixture of two or more such inhibitors, and so forth.

[0024] Unless otherwise indicated, the term "approximately" means within 10% (eg, within 5%, 2%, or 1%) of the particular value modified by the term "about."

[0025] The transitional term "comprising," which is synonymous with "comprising," "including," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any elements, steps, or ingredients not specified in the claim. The transitional 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."

[0026] With respect to the bispecific compounds of the present invention, to the extent the following terms are used herein to further describe them, the following definitions apply.

[0027] As used herein, the term "alkyl" refers to a saturated straight or branched monovalent hydrocarbon group. In one embodiment, the alkyl group is C1-C 18In other embodiments, the alkyl group is C0-C6, C0-C5, C0-C3, C1-C 12 , C1-C8, C1-C6, C1-C5, C1-C4 or C1-C3 group (wherein C0 alkyl refers to a bond). Examples of alkyl groups include methyl, ethyl, 1-propyl, 2-propyl, 1-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl. In some embodiments, the alkyl group is a C1-C3 alkyl group.

[0028] As used herein, the term "alkoxyl" or "alkoxy" refers to an alkyl group as defined above, to which an oxygen group is attached. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like. An "ether" is a hydrocarbon compound covalently linked by oxygen. Thus, the substituent of an alkyl group that makes an alkyl group an ether is or is similar to an alkoxy group, and can be represented by, for example, one of -O-alkyl, -O-alkenyl, and -O-alkynyl.

[0029] As used herein, the term "halogen" (or "halo" or "halide") refers to fluorine, chlorine, bromine or iodine.

[0030] As used herein, the term "alkylene" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a group, consisting only of carbon and hydrogen, without unsaturated bonds and having 1 to 12 carbon atoms, such as methylene, ethylene, propylene, butylene, etc. The alkylene chain can be connected to the rest of the molecule by a single bond and connected to the group by a single bond. In some embodiments, the alkylene group comprises 1 to 8 carbon atoms (C1-C8 alkylene). In other embodiments, the alkylene group comprises 1 to 5 carbon atoms (C1-C5 alkylene). In other embodiments, the alkylene group comprises 1 to 4 carbon atoms (C1-C4 alkylene). In other embodiments, the alkylene group comprises 1 to 3 carbon atoms (C1-C3 alkylene). In other embodiments, the alkylene group comprises 1 to 2 carbon atoms (C1-C2 alkylene). In other embodiments, the alkylene group comprises one carbon atom (C1 alkylene).

[0031] As used herein, the term "alkynyl" refers to a straight or branched monovalent hydrocarbon radical having at least one carbon-carbon triple bond. 18In other examples, the alkynyl group is a C2-C 12 、C2-C 10 , C2-C8, C2-C6 or C2-C3. Examples include ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl and but-3-ynyl.

[0032] As used herein, the term "cyclic group" refers broadly to any group used alone or as part of a larger moiety, comprising saturated, partially saturated or aromatic ring systems, such as carbocyclic (cycloalkyl, cycloalkenyl), heterocyclic (heterocyclic alkyl, heterocyclic alkenyl), aryl and heteroaryl. A cyclic group may have one or more (e.g., fused) ring systems. Thus, for example, a cyclic group may comprise one or more carbocyclic, heterocyclic, aryl or heteroaryl groups.

[0033] As used herein, the term "carbocycle" (also referred to as "carbocyclyl") refers to a group used alone or as part of a larger portion, which comprises a saturated, partially unsaturated or aromatic ring system having 3 to 20 carbon atoms, alone or as part of a larger portion (e.g., an alkyl carbocyclic group). The term carbocyclyl includes monocyclic, bicyclic, tricyclic, fused, bridged and spirocyclic systems and combinations thereof. In one embodiment, the carbocyclyl includes 3 to 15 carbon atoms (C3-C 15 In one embodiment, the carbocyclyl group comprises 3 to 12 carbon atoms (C3-C 12 In another embodiment, carbocyclyl includes C3-C8, C3-C 10 or C5-C 10 In another embodiment, the carbocyclyl group as a monocyclic ring includes C3-C8, C3-C6 or C5-C6. In some embodiments, the carbocyclyl group as a bicyclic ring includes C ) -C 12 In another embodiment, the carbocyclyl group as a spiro ring system includes C5-C 12Representative examples of monocyclic carbocyclyls include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-enyl, 1-cyclopentyl-2-enyl, 1-cyclopentyl-3-enyl, cyclohexyl, perdeuterated cyclohexyl, 1-cyclohexyl-1-enyl, 1-cyclohexyl-2-enyl, 1-cyclohexyl-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, phenyl, and cyclododecyl; bicyclic carbocyclyls having 1 to 12 ring atoms include [4,3], [4,4], [4,5], [5,5], [5,6], or [6,6] ring systems, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, naphthalene, and bicyclo[3.2.2]nonane. Representative examples of spirocyclic carbocyclic radicals include spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane and spiro[4.5]decane.The term carbocyclic radical includes aromatic ring systems as defined herein.The term carbocyclic radical also includes cycloalkyl rings (e.g., saturated or partially unsaturated single, double or spiral carbocyclic rings).The term carbocyclic group also includes carbocyclic rings fused to one or more (e.g., 1, 2 or 3) different cyclic groups (e.g., aromatic rings or heterocycles), wherein the group or point of attachment is on the carbocyclic ring.

[0034] Thus, the term carbocycle also includes carbocyclylalkyl groups, which, as used herein, refer to groups of the formula -R c - a carbocyclic group, where R c is an alkylene chain. The term carbocycle also includes carbocyclylalkoxy groups, which, as used herein, refer to a carbocyclylalkoxy group of the formula -O-R c - a group bonded to the oxygen atom of a carbocyclic group, wherein R c It is an alkylene chain.

[0035] As used herein, the term "heterocyclyl" refers to a "carbocyclyl", which is used alone or as part of a larger moiety, comprising a saturated, partially unsaturated or aromatic ring system, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms have been replaced by heteroatoms (e.g., O, N, N(O), S, S(O) or S(O)2). The term heterocyclyl includes monocyclic, bicyclic, tricyclic, fused, bridged and spirocyclic systems and combinations thereof. In some embodiments, heterocyclyl refers to a 3 to 15-membered heterocyclyl ring system. In some embodiments, heterocyclyl refers to a 3 to 12-membered heterocyclyl ring system. In some embodiments, heterocyclyl refers to a saturated ring system, such as a 3 to 12-membered saturated heterocyclyl ring system. In some embodiments, heterocyclyl refers to a heteroaryl ring system, such as a 5 to 14-membered heteroaryl ring system. The term heterocyclyl also includes C3-C8 heterocycloalkyl, which is a saturated or partially unsaturated mono-, bi- or spiro ring system containing 3-8 carbons and one or more (1, 2, 3 or 4) heteroatoms.

[0036] In some embodiments, heterocyclic radical includes 3-12 ring atoms, and includes monocyclic, bicyclic, tricyclic and spirocyclic systems, wherein the ring atoms are carbon, and 1-5 ring atoms are heteroatoms, such as nitrogen, sulfur or oxygen. In some embodiments, heterocyclic radical includes 3- to )-membered monocyclic rings with one or more heteroatoms selected from nitrogen, sulfur or oxygen. In some embodiments, heterocyclic radical includes 4- to 6-membered monocyclic rings with one or more heteroatoms selected from nitrogen, sulfur or oxygen. In some embodiments, heterocyclic radical includes 3-membered monocyclic rings. In some embodiments, heterocyclic radical includes 4-membered monocyclic rings. In some embodiments, heterocyclic radical includes 5-6-membered monocyclic rings. In some embodiments, heterocyclic radical includes 0 to 3 double bonds. In any of the foregoing embodiments, heterocyclic radical includes 1, 2, 3 or 4 heteroatoms. Any nitrogen or sulfur heteroatom can be optionally oxidized (e.g., NO, SO, SO2), and any nitrogen heteroatom can be optionally quaternized (e.g., [NR4] + Cl - 、[NR4] + OH -Representative examples of heterocyclic groups include oxirane, aziridinyl, thiiridinyl, azetidinyl, oxetanyl, thienyl, 1,2-bithiphenyl, 1,3-bithiphenyl, pyrrolidinyl, dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydropyranyl, dihydrothienyl, tetrahydrothienyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinanyl, thiazinanyl, thioxanyl, homopiperazinyl, homopiperidinyl, homopiperidinyl), azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl, 1,4-diazepanyl, diazepinyl, thiazepinyl, thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl, thiazolyl, isothiazolyl, 1,1-dioxothiazolinyl, oxazolidinyl, imidazolidinyl, 4,5-dihydrothiopyranyl, ,6,)-tetrahydro[2H]indazolyl, tetrahydrobenzimidazolyl, 4,5,6,)-tetrahydrobenzo[d]imidazolyl, 1,6-dihydroimidazolyl[4,5-d]pyrrolo[2,3-b]pyridinyl, thiazinyl, thienyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidinyl, tetrahydropyrimidinyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, indolyl, thiapyranyl, 2H-pyranyl, 4H-pyranyl, dioxanthenyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithioanthenyl, pyrimidinylnonyl, pyrimidinyldinonyl, pyrimidine-2,4-dione yl, piperazinylnonyl, piperazinyldinyl, pyrazolidinylimidazolyl, 3-azabicyclo[3.1.0]hexyl, 3,6-diazabicyclo[3.1.1]heptyl, 6-azabicyclo[3.1.1]heptyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 2-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]octyl, 2-azabicyclo[2.2.2]octyl, 8-azabicyclo[2.2.2]octyl, )-oxabicyclo[2.2.1]heptane, azaspiro[3.5]nonyl, azaspiro[2.5]octyl, azaspiro[4.5]decyl, 1-azaspiro[4.5]decan-2-yl, azaspiro[5.5] undecyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl, 1,1-dioxane hydropyranyl. Examples of 5-membered heterocycles containing sulfur or oxygen atoms and 1-3 nitrogen atoms are thiazolyl, including thiazol-2-yl and thiazol-2-yl N-oxide, thiadiazolyl, including 1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl, oxazolyl, such as oxazol-2-yl, and oxadiazolyl, such as 1,3,4-oxadiazol-5-yl, and 1,2,4-oxadiazol-5-yl. Examples of 5-membered heterocyclic rings containing 2-4 nitrogen atoms include imidazolyl, such as imidazol-2-yl; triazolyl, such as 1,3,4-triazol-5-yl; 1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl and tetrazolyl, such as 1H-tetrazolyl. Representative examples of benzo-fused 5-membered heterocyclic groups are benzooxazol-2-yl, benzothiazol-2-yl and benzimidazol-2-yl. Examples of 6-membered heterocyclic groups include 1-3 nitrogen atoms and optionally sulfur or oxygen atoms, such as pyridyl, such as pyridin-2-yl, pyridin-3-yl and pyridin-4-yl; pyrimidinyl, such as pyrimidin-2-yl and pyrimidin-4-yl; triazinyl, such as 1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl; pyridazinyl, especially pyridazin-3-yl and pyrazinyl. .

[0037] Pyridine N-oxide and pyridazine N-oxide and pyridyl, pyrimidin-2-yl, pyrimidin-4-yl, pyridazinyl and 1,3,4-triazin-2-yl are other examples of heterocyclic groups. In some embodiments, heterocyclic groups include heterocyclic rings fused to one or more (e.g., 1, 2 or 3) different cyclic groups (e.g., carbocyclic or heterocyclic rings), wherein the radical or point of attachment is on the heterocyclic ring, and in some embodiments, wherein the point of attachment is a heteroatom contained in the heterocyclic ring.

[0038] Thus, the term heterocycle includes N-heterocyclyl, as used herein, which refers to a heterocyclyl containing at least one nitrogen, wherein the point of attachment of the heterocyclyl to the rest of the molecule is through a nitrogen atom in the heterocyclyl. Representative examples of nitrogen heterocyclyls include 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl. The term heterocycle also includes C-heterocyclyl, as used herein, which refers to a heterocyclyl containing at least one heteroatom, wherein the point of attachment of the heterocyclyl to the rest of the molecule is through a carbon atom in the heterocyclyl. Representative examples of carbon heterocyclyls include 2-morpholinyl, 2- or 3- or 4-piperidinyl, 2-piperazinyl, and 2- or 3-pyrrolidinyl. The term heterocycle also includes heterocyclylalkyl groups, as described above, which refer to heterocyclyls of the formula -R c -heterocyclic group, wherein R c The term heterocycle also includes heterocyclylalkoxy, which, as used herein, refers to a heterocyclic radical of the formula -OR c - a group bonded to the oxygen atom of a heterocyclic group, wherein Rc It is an alkylene chain.

[0039] As used herein, the term "aryl" is used alone or as part of a larger moiety (e.g., "aralkyl", where the terminal carbon atom on the alkyl group is the point of attachment, e.g., benzyl), "aralkyloxy", where the oxygen atom is the point of attachment, or "aryloxyalkyl", where the point of attachment is on the aryl group, refers to a group including a monocyclic, bicyclic or tricyclic carbon ring system, including fused rings, where at least one ring in the system is aromatic. In some embodiments, the aralkyloxy is a phenoxy group. The term "aryl" can be used interchangeably with the term "aryl ring". In one embodiment, the aryl group includes a group having 6-18 carbon atoms. In another embodiment, the aryl group includes a group having 6-10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracenyl, biphenyl, phenanthrenyl, tetraphenyl, 1,2,3,4-tetrahydronaphthyl, 1H-indenyl, 2,3-dihydro-1H-indenyl, naphthyridinyl, etc., which can be substituted or independently substituted by one or more substituents described herein. A special aryl group is phenyl. In some embodiments, aryl groups include an aryl ring fused to one or more (eg, 1, 2, or 3) different cyclic groups (eg, carbocyclic or heterocyclic), wherein the radical or point of attachment is on the aryl ring.

[0040] Thus, the term aryl includes aralkyl groups (eg, benzyl), which, as described above, refer to groups of the formula -R c -aryl radical, wherein R c is an alkylene chain, such as methylene or ethylene. In some embodiments, the aralkyl group is an optionally substituted benzyl group. The term aryl also includes aralkyloxy groups, and aralkyloxy as used herein refers to a alkyl radical of the formula -O-R c - a group bonded to the oxygen atom of an aromatic group, wherein R c is an alkylene chain, such as methylene or ethylene.

[0041] As used herein, the term "heteroaryl", used alone or as part of a larger moiety (e.g., "heteroarylalkyl" (also known as "heteroaralkyl") or "heteroarylalkoxy" (also known as "heteroaralkoxy"), refers to a monocyclic, bicyclic or tricyclic ring system having 5 to 14 ring atoms, wherein at least one ring is aromatic and contains at least one heteroatom. In one embodiment, heteroaryl includes 5-6 membered monocyclic aromatic groups, wherein one or more ring atoms are nitrogen, sulfur or oxygen. Representative examples of heteroaryl groups include thienyl, furanyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidinyl, imidazolyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, tetrazolyl [1,5-b] pyridazinyl, purinyl, deazapurinyl, benzoxazolyl, benzofuranyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzimidazolyl, indolyl, 1,3-thiazol-2-yl, 1,3,4-triazol-5-yl, 1,3-oxazol-2-yl, 1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl, 1 , 3,4-thiadiazol-5-yl, 1H-tetrazol-5-yl, 1,2,3-triazol-5-yl and pyridin-2-yl nitroxides. The term "heteroaryl" also includes groups in which the heteroaryl is fused to one or more cyclic (e.g., carbocyclyl or heterocyclyl) rings, where the radical or point of attachment is on the heteroaryl ring. Non-limiting examples include indolyl, indolizinyl, isoindolyl, benzothiophenyl, benzothiophenyl, methylenedioxyphenyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzodioxazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl , phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolyl, carbazolyl, acridinyl, benzoxazinyl, phenothiazinyl, benzoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and pyrido [2,3-b] -1,4-oxazin-3 (4H) -one. The heteroaryl group can be monocyclic, bicyclic or tricyclic. In some embodiments, the heteroaryl group includes a heteroaryl ring fused to one or more (e.g., 1, 2 or 3) different cyclic groups (e.g., carbocyclic or heterocyclic), wherein the group or point of attachment is on the heteroaryl ring, and in some embodiments, wherein the point of attachment is a heteroatom contained in the heterocyclic ring.

[0042] Thus, the term heteroaryl includes N-heteroaryl, as used herein, which refers to a heteroaryl group as defined above containing at least one nitrogen, wherein the point of attachment of the heteroaryl group to the rest of the molecule is through the nitrogen atom in the heteroaryl group. The term heteroaryl also includes C-heteroaryl groups, as used herein, which refer to heteroaryl groups as defined above, and wherein the point of attachment of the heteroaryl group to the rest of the molecule is through a carbon atom in the heteroaryl group. The term heteroaryl also includes heteroarylalkyl groups, as disclosed above, which refer to groups of the formula -R c-heteroaryl radical, wherein R c is an alkylene chain as defined above. The term heteroaryl also includes heteroaralkoxy (or heteroarylalkoxy) groups, as used herein, which refers to a heteroarylalkyloxy group of the formula -OR c - a group bonded to the oxygen atom of a heteroaryl group, wherein R c is an alkylene group as defined above.

[0043] Any group described herein may be substituted or unsubstituted. As used herein, and to the extent that they are not otherwise defined for any particular group, the term "substituted" refers broadly to all permitted substituents, with the implicit condition that such substitution is determined according to the permitted valence of the substituted atom and the substituent, and that the substitution produces a stable compound, i.e., a compound that does not spontaneously undergo transformation (such as rearrangement, cyclization, elimination, etc.). Representative substituents include halogens, hydroxyls, and any other organic groups containing any number of carbon atoms (e.g., 1-14 carbon atoms), and which may include one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, and nitrogen), grouped in a linear, branched, or cyclic structure.

[0044] Representative examples of substituents may include alkyl, substituted alkyl (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), alkoxy (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), substituted alkoxy (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), haloalkyl (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), F3), alkenyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkenyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), alkynyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkynyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), cyclo (e.g., C3-C 12 , C5-C6), substituted rings (e.g., C3-C 12 , C5-C6), carbocyclic rings (e.g., C3-C 12 , C5-C6), substituted carbocyclic rings (e.g., C3-C 12 , C5-C6), heterocyclic (e.g., C3-C 12 , C5-C6), substituted heterocycles (e.g., C3-C 12, C5-C6), aryl (e.g., benzyl and phenyl), substituted aryl (e.g., substituted benzyl or phenyl), heteroaryl (e.g., pyridyl or pyrimidinyl), substituted heteroaryl (e.g., substituted pyridyl or pyrimidinyl), aralkyl (e.g., benzyl), substituted aralkyl (e.g., substituted benzyl), halogen, hydroxy, aryloxy (e.g., C6-C 12 , C6), substituted aryloxy (e.g., C6-C 12 , C6), alkylthio (e.g., C1-C6), substituted alkylthio (e.g., C1-C6), arylthio (e.g., C6-C 12 , C6), substituted arylthio (e.g., C6-C 12 , C6), cyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, thio, substituted thio, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfinimide, substituted sulfinimide, sulfonamide, substituted sulfonamide, urea, substituted urea, carbamate, substituted carbamate, amino acid and peptidyl.

[0045] The term "binding" relates to the interaction between a targeting ligand and a targeting protein or protein (which is FGFR2 in the present invention), generally refers to an intermolecular interaction, which can be preferential or substantially specific (also referred to herein as "selective"), because the binding of the targeting ligand to other protein entities present in the cell is functionally unimportant. The bispecific compounds of the present invention can preferentially bind to and recruit FGFR2 for targeted degradation.

[0046] The term "binding" when referring to the interaction between a degron and an E3 ubiquitin ligase generally refers to an intermolecular interaction that may or may not exhibit a level of affinity that equals or exceeds the affinity between the targeting ligand and the target protein, but nonetheless, wherein the affinity is sufficient to achieve targeted and selective degradation of the ligase by recruiting the ligase to the target protein.

[0047] In broad terms, the bispecific compound has a structure represented by the following formula:

[0048] The targeting ligand represents a portion that binds to fibroblast growth factor receptor 2 (FGFR2), the degron represents a portion that binds to E3 ubiquitin ligase, and the linker represents a portion that covalently links the degron and the targeting ligand.

[0049] FGFR2 targeting ligands

[0050] In some embodiments, the targeting ligand has a structure represented by Formula (TL-1):

[0051]

[0052] in

[0053] R3 is independently halogen, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted amino, optionally substituted acylamino, carboxyl, acrylamide, optionally substituted carbocyclyl or optionally substituted heterocyclyl; and m is an integer from 0 to 4.

[0054] In some embodiments, R3 is independently methyl, chloro, or methoxy.

[0055] In some embodiments, m is 0.

[0056] In some embodiments, m is 2.

[0057] In some embodiments, m is 4.

[0058] Thus, in some embodiments, the bispecific compounds of the invention have a structure represented by Formula (I-1):

[0059] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0060] In some embodiments, the targeting ligand has a structure represented by Formula TL-1a, TL-1b, TL-1c, TL-1d, or TL-1e:

[0061]

[0062] Thus, in some embodiments, the bispecific compounds of the invention have a structure represented by Formula I-1a, I-1b, I-1c, I-1d, or I-1e:

[0063]

[0064] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0065] In some embodiments, the targeting ligand has a structure represented by Formula (TL-2):

[0066]

[0067] in:

[0068] R 1 is H or optionally substituted alkyl, optionally substituted alkoxy, optionally substituted amino, optionally substituted acylamino, carboxyl, acrylamide, optionally substituted carbocyclic group or optionally substituted heterocyclic group; and n is an integer of 0-4.

[0069] Thus, in some embodiments, the bispecific compounds of the invention have a structure represented by Formula (I-2):

[0070]

[0071] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0072] In some embodiments, the bispecific compound of Formula I-2 is represented by Formula (I-2a):

[0073]

[0074] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0075] In some embodiments, the targeting ligand has a structure represented by Formula (TL-3):

[0076]

[0077] Where R 2 Does not exist or represent

[0078] Thus, in some embodiments, the bispecific compounds of the invention have a structure represented by Formula (I-3a) or (I-3b):

[0079]

[0080] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0081] In some embodiments, the targeting ligand has a structure represented by Formula (TL-4):

[0082]

[0083] Thus, in some embodiments, the bispecific compounds of the invention have a structure represented by Formula (I-4):

[0084] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0085] Other moieties that can be used as FGFR2 targeting ligands are described in U.S. Patent Nos. 8,865, 3) and 9,95), 236 and U.S. Patent Application Publication Nos.: US2014 / 03)8481, US2018 / 0155340, US2016 / 0009)85 and US2015 / 0366866.

[0086] Connectors

[0087] The linker ("L") provides a covalent link between the targeting ligand and the degron. The structure of the linker may not be critical, as long as it does not substantially interfere with the activity of the targeting ligand or the degron.

[0088] In some embodiments, the linker can be an alkylene chain or a divalent alkylene chain, any of which can be replaced by –O–, –S–, –N(R′)–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR′)–, –C(O)N(R′)–, –C(O)N(R′)C(O)–, –C(O)N(R′)C(O)N(R′)–, –N(R′)C(O)–, –N(R′)C(O)N(R′)–, –N(R′)C(O)O–, –OC(O)N(R′)–, –C(NR')–, –N(R’)C(NR’)–, –C(NR’)N(R’)–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O )2–, –S(O)2O–, –N(R’)S(O)2–, –S(O)2N(R’)–, –N(R’)S(O)–, –S(O)N(R’)–, –N(R’)S(O)2N(R’)–, –N(R’)S(O)N(R’)–, C3-C 12 At least one of a carbocyclene, a 3- to 12-membered heterocyclene, a 5- to 12-membered heteroarylene, or any combination thereof, wherein R' is H or a C1-C6 alkyl group, wherein the interrupting group and one or both terminal groups may be the same or different.

[0089] "Carbocyclylene" refers to an optionally substituted divalent carbocyclic group.

[0090] "Heterocyclylene" refers to a divalent heterocyclic group which may be optionally substituted.

[0091] "Heteroarylene" refers to a divalent heteroaryl group which may be optionally substituted.

[0092] Representative examples of linkers suitable for use in the present invention include alkylene chains:

[0093] wherein n is an integer from 1 to 12 (inclusive), for example, 1-12, 1-11, 1-10, 1-9, 1-8, 1-), 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-), 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-), 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-), 4-6, 4-5, 5-10, 5-9, 5-8, 5-), 5-6, 6-10, 6-9, 6-8, 6-), )-10, )-9, )-8, 8-10, 8-9, 9-10 and 1, 2, 3, 4, 5, 6,), 8, 9 and 10, examples of which include:

[0094]

[0095] Alkylene chains terminated in various functional groups (as described above) are exemplified by the following:

[0096]

[0097] Alkylene chains interrupted by various functional groups (as described above) are exemplified by the following:

[0098]

[0099] An alkylene chain interrupted or terminated with a heterocycloalkylene group, e.g.

[0100] Wherein m and n are independently integers of 0-10, examples of which include:

[0101] Examples of alkylene chains interrupted by amide, heterocyclic olefin and / or aryl groups include:

[0102]

[0103] Examples of alkylene chains interrupted by heterocycloolefin and aryl groups and heteroatoms include:

[0104]

[0105] and

[0106] Alkylene chains interrupted and / or terminated by heteroatoms such as nitrogen, oxygen or boron, e.g.

[0107] wherein each n is independently an integer from 1 to 10, for example, 1-9, 1-8, 1-), 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-), 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-), 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-), 4-6, 4-5, 5-10, 5-9, 5-8, 5-), 5-6, 6-10, 6-9, 6-8, 6-),)-10,)-9,)-8, 8-10, 8-9, 9-10 and 1, 2, 3, 4, 5, 6,),), 8, 9 and 10, and R is hydrogen or C1 to C4 alkyl, examples of which are

[0108] In some embodiments, the linker can be a polyethylene glycol chain that can terminate (at one or both ends) in at least one of the following: –S–, –N(R′)–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR′)–, –C(O)N(R′)–, –C(O)N(R′)C(O)–, –C(O)N(R′)C(O)N(R′)–, –N(R′)C(O)–, –N(R′)C(O)N(R′)–, –N(R′)C(O)O–, –OC(O)N(R′) ')–, –C(NR')–, –N(R’)C(NR’)–, –C(NR’)N(R’)–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, – OS(O)2–, –S(O)2O–, –N(R’)S(O)2–, –S(O)2N(R’)–, –N(R’)S(O)–, –S(O)N(R’)–, –N(R’)S(O)2N(R’)–, –N(R’)S(O)N(R’)–, C 3-12 The invention is characterized in that the present invention is interrupted and / or terminated (at one or both ends) by at least one of a carbocyclylene, a 3- to 12-membered heterocyclylene, a 5- to 12-membered heteroarylene, or any combination thereof, wherein R' is H or a C1-C6 alkyl group, wherein one or both end groups may be the same or different.

[0109] Examples of linkers comprising polyethylene glycol chains include:

[0110] Where n is an integer from 2 to 10, examples of which include:

[0111]

[0112] In some embodiments, the polyethylene glycol chain may terminate in a functional group, examples of which are as follows:

[0113]

[0114] In some embodiments, the linker is represented by a structure selected from the group consisting of:

[0115]

[0116] In some embodiments, the bispecific compounds of the invention may include a FGFR2 TL connected to a degron via a PEG linker terminating in a functional group. Representative examples of bispecific compounds include:

[0117]

[0118] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0119] In some embodiments, the bispecific compounds of the invention may include a TL linked to a degron via an alkylene linker, which may be interrupted and / or terminated by a cyclic or acyclic group (e.g., an amide group) or one or more heteroatoms. Representative examples of bispecific compounds include:

[0120]

[0121] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0122] In some embodiments, the bispecific compounds of the invention are represented by any of the following structures (wherein the degron is generically shown):

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0132] Degron

[0133] The ubiquitin-proteasome pathway (UPP) is a key cellular pathway for the regulation of key regulatory proteins and the degradation of misfolded or abnormal proteins. The UPP is central to multiple cellular processes. The covalent attachment of ubiquitin to specific protein substrates is achieved through the action of E3 ubiquitin ligases. These ligases include more than 500 different proteins and are classified into multiple classes based on the structural elements that underlie their E3 functional activity.

[0134] In some embodiments, the degron binds an E3 ubiquitin ligase that is cereblon and is represented by a structure selected from the group consisting of:

[0135]

[0136] in

[0137] Y is NH, NMe or O; and

[0138] Z is NH, O or C≡.

[0139] Thus, in some embodiments, the bispecific compounds of the invention are represented by a general formula selected from the group consisting of:

[0140]

[0141] in

[0142] Y is NH, NMe or O; and

[0143] Z is NH, O or C≡;

[0144] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0145] Other degradation determinants that bind to cereblon and may be suitable for use in the present invention are disclosed in U.S. Patent No. 9,00512 and U.S. Patent Application Publication Nos. 2018 / 001508), 2018 / 0009)9, 2016 / 024324), 2016 / 0235)31, 2016 / 0235)30 and 2016 / 01)6916, and International Patent Publications WO 201) / 19)055, WO 201) / 19)051, WO 201) / 19)036, WO 201) / 19)056 and WO 201) / 19)046.

[0146] In some embodiments, the E3 ubiquitin ligase bound by the degron is the von Hippel-Lindau (VHL) tumor suppressor. See, Iwai, et al., Proc. Nat'l. Acad. Sci. USA 96: 12436-41 (1999).

[0147] In some embodiments, the VHL-binding degron is represented by any of the following structures:

[0148]

[0149] wherein Y' is a bond, N, O or C;

[0150] wherein Z' is a cyclic group, which in some embodiments is a C5-6 carbocyclic or heterocyclic group, and

[0151] wherein Y" is a bond, CH2, NH, NMe, O or S, or a stereoisomer thereof. In certain embodiments, the heterocyclic group is

[0152] In some embodiments, the bispecific compounds of the invention can be represented by any of the following structures:

[0153] Wherein Y' is a bond, NH, O or CH2,

[0154] Wherein Z' is a cyclic group,

[0155] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0156] In some embodiments, Z' is phenyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, benzofuranyl, benzothienyl, indolyl, quinolyl, or isoquinolyl. In certain embodiments, Z' is

[0157] US Patent Application Publication No. 201) / 0121321 A1 discloses other degrons that bind to VHL and may be suitable for use in the present invention.

[0158] In some embodiments, the E3 ubiquitin ligase bound by the degron is an inhibitor of apoptosis protein (IAP). Representative examples of degrons that bind IAPs and may be suitable for use in the present invention are represented by any of the following structures:

[0159]

[0160]

[0161] Thus, in some embodiments, the bispecific compounds of the invention are represented by any of the following structures:

[0162]

[0163] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0164] Other degrons that bind IAP and may be suitable for use in the present invention are disclosed in International Patent Application Publications WO 20081281)1, WO 2008 / 016893, WO 2014 / 060)68, WO 2014 / 060)6) and WO 15092420.

[0165] Thus, in some embodiments, the bispecific compounds of the invention are represented by any structure resulting from a combination of structures TL-1 to TL-4, L1 to L10, and the structures of the degron described herein, including D1 to D3, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0166] In some embodiments, the bispecific compounds of the invention have the following structure:

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0179] The bispecific compounds of the present invention may be in the form of a free acid or free base or a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of the bispecific compound and is relatively non-toxic, that is, the material can be administered to a subject without causing undesirable biological effects (such as dizziness or stomach discomfort) or interacting in a harmful manner with any component of the composition comprising it. The term "pharmaceutically acceptable salt" refers to the product obtained by reacting the bispecific compound of the present invention with a suitable acid or base. Examples of pharmaceutically acceptable salts of the bispecific compounds of the present invention include those derived from suitable inorganic bases such as lithium, sodium, potassium, calcium, magnesium, iron, copper, aluminum, zinc and manganese salts. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, dextran, sucrose, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, 4-methylbenzenesulfonate or p-toluenesulfonate, etc. Certain bispecific compounds of the present invention can form pharmaceutically acceptable salts with various organic bases such as lysine, arginine, guanidine, diethanolamine or metformin.

[0180] In some embodiments, the bispecific compound is an isotopic derivative in that it has a desired isotopic substitution of at least one atom that is greater than the natural abundance of the isotope, i.e., is enriched. In one embodiment, the bispecific compound includes deuterium or a plurality of deuterium atoms. 2 H)) substitution may offer certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be advantageous in some circumstances.

[0181] The bispecific compounds of the present invention may have at least one chiral center and may therefore be in the form of stereoisomers, which, as used herein, include all isomers of a single compound that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (including enantiomers of the (R-) or (S-) configuration of the compound), mixtures of mirror image isomers of the compound (physical mixtures and racemates or racemic mixtures of enantiomers), geometric (cis / trans or E / Z, R / S) isomers of the compound, and isomers of compounds that have more than one chiral center but are not mirror images of each other (diastereomers). The chiral centers of the compounds may undergo epimerization in vivo; therefore, for these compounds, a compound administered in the (R-) form is considered equivalent to a compound administered in the (S-) form. Therefore, the bispecific compounds of the present invention may be prepared and used in the form of a single isomer, and are substantially free of other isomers, or in the form of a mixture of various isomers, for example, a racemic mixture of stereoisomers.

[0182] Synthesis method

[0183] On the other hand, the present invention relates to a method for preparing a bispecific compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof. In a broad sense, the bispecific compound of the present invention or a pharmaceutically acceptable salt or stereoisomer thereof can be prepared by any known method suitable for preparing chemically related compounds. The bispecific compounds of the present invention will be better understood in conjunction with the synthesis schemes described in the various working examples, which illustrate non-limiting methods by which the bispecific compounds of the present invention can be prepared.

[0184] Pharmaceutical composition

[0185] Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a bispecific compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier. As known in the art, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle suitable for administering the compounds of the present invention to mammals. Suitable carriers may include, for example, liquids (aqueous and non-aqueous, and combinations thereof), solids, encapsulating materials, gases and combinations thereof (e.g., semisolids), and gases, the function of which is to transport or deliver a compound from one organ or part of the body to another organ or another part of the body. The carrier is "acceptable", meaning that it is physiologically inert and compatible with the other ingredients of the preparation, and is harmless to the subject or patient. Depending on the type of preparation, the composition may include one or more pharmaceutically acceptable excipients.

[0186] In broad terms, the bispecific compounds of formula (I) can be formulated into a composition of a given type according to conventional pharmaceutical practices such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and compression processes (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). The type of formulation depends on the mode of administration, which may include enteral (e.g., oral, buccal, sublingual, and rectal), parenteral (e.g., subcutaneous (sc), intravenous (iv), intramuscular (im), and intrasternal injection, or infusion techniques, intraocular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, intradermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation, and inhalation), and topical (e.g., transdermal). In general, the most appropriate route of administration depends on a variety of factors, including, for example, the nature of the medicament (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). For example, parenteral (e.g., intravenous) administration is also advantageous because the compound can be administered relatively quickly, for example, in the case of single-dose treatment and / or acute conditions.

[0187] In some embodiments, the bispecific compounds are formulated for oral or intravenous administration (eg, systemic intravenous injection).

[0188] Thus, the bispecific compounds of the invention can be formulated into solid compositions (e.g., powders, tablets, dispersible particles, capsules, cachets, and suppositories), liquid compositions (e.g., solutions in which the compound is dissolved, suspensions in which solid particles of the compound are dispersed, emulsions, and solutions containing liposomes, micelles, or nanoparticles, syrups, and elixirs); semisolid compositions (e.g., gels, suspensions, and creams); and gases (e.g., propellants for aerosol compositions). The compounds can also be formulated for rapid, intermediate, or extended release.

[0189] Oral solid dosage forms include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with a carrier (such as sodium citrate or dicalcium phosphate) and additional carriers or excipients such as a) fillers or extenders (such as starch, lactose, sucrose, glucose, mannitol and silicic acid), b) binders (such as methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia), c) humectants (such as glycerol), d) disintegrants (such as cross-linked polymers (e.g., Cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium starch glycolate, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate), e) solution retardants (such as paraffin), f) absorption promoters (such as quaternary ammonium compounds), g) wetting agents (such as cetyl alcohol and glycerol monostearate), h) absorbents (such as kaolin and bentonite), and i) lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate) and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also include a buffer. Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or lactose and high molecular weight polyethylene glycols. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells (such as enteric coatings and other coatings). They may further contain opacifiers.

[0190] In some embodiments, the bispecific compounds of the invention can be formulated in hard or soft gelatin capsules. Representative excipients that can be used include pregelatinized starch, magnesium stearate, mannitol, sodium stearyl fumarate, anhydrous lactose, microcrystalline cellulose, and cross-linked sodium carboxymethyl cellulose. The gelatin shell can include gelatin, titanium dioxide, iron oxide, and a colorant.

[0191] Oral liquid dosage forms include solutions, suspensions, emulsions, microemulsions, syrups and elixirs. In addition to the compound, the liquid dosage form may include aqueous or non-aqueous carriers commonly used in the art (depending on the solubility of the compound), such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. Oral compositions may also include excipients, such as wetting agents, suspending agents, coloring agents, sweeteners, flavoring agents and flavoring agents.

[0192] Injectable preparations may include sterile aqueous solutions or oily suspensions. They may be prepared using suitable dispersants or wetting agents and suspending agents according to standard techniques. Sterile injectable preparations may also be sterile injectable solutions, suspensions or emulsions in nontoxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable carriers and solvents include water, Ringer's solution, USP and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspension media. For this purpose, any mild fixed oil may be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids (such as oleic acid) are used to prepare injections. Injectable preparations may be sterilized, for example, by filtering through a bacterial retention filter, or by incorporating a sterilizing agent in the form of a sterile solid composition, which may be dissolved or dispersed in sterile water or other sterile injection media before use. The effect of the compound may be prolonged by slowing its absorption, which may be achieved by using a poorly water-soluble liquid suspension or a crystalline or amorphous material. The extended absorption of the compound from parenteral administration preparations may also be achieved by suspending the compound in an oily vehicle.

[0193] In certain embodiments, the bispecific compound of formula (I) can be applied in a local rather than systemic manner, for example, by injecting the conjugate directly into an organ, usually in the form of a depot preparation or a sustained release preparation. In a specific embodiment, the long-acting preparation is applied by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. The injectable depot form is prepared by forming a microcapsule matrix of the compound in a biodegradable polymer, such as polylactide-polyglycolide, poly (orthoester) and poly (anhydride). The release rate of the compound can be controlled by changing the ratio of the compound to the polymer and the properties of the specific polymer used. The injectable depot preparation is also prepared by encapsulating the compound in a liposome or microemulsion compatible with body tissues. In addition, in other embodiments, the compound is delivered in a targeted drug delivery system, for example, in a liposome coated with an organ-specific antibody. In such an embodiment, the liposome targets the organ and is selectively absorbed by the organ.

[0194] The bispecific compounds of the invention may be formulated for buccal or sublingual administration, examples of which include tablets, lozenges and gels.

[0195] The bispecific compound can be formulated for inhalation administration. Various forms suitable for inhalation administration include aerosols, mists or powders. The pharmaceutical composition can be delivered in an aerosol form from a pressurized package or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In some embodiments, the dosage unit of the pressurized aerosol can be determined by providing a valve to deliver a metered amount. In some embodiments, capsules and cartridges containing gelatin, such as for use in an inhaler or insufflator, can be formulated into a powder mixture containing the compound and a suitable powder base (such as lactose or starch).

[0196] The bispecific compounds of formula (I) may be formulated for topical administration, which as used herein refers to intradermal administration by applying the formulation to the epidermis. These types of compositions are typically in the form of ointments, pastes, creams, lotions, gels, solutions and sprays.

[0197] Representative examples of carriers for preparing compositions for topical application include solvents (e.g., alcohols, polyols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffered solutions (e.g., hypotonic or buffered saline). Creams, for example, can be prepared using saturated or unsaturated fatty acids (e.g., stearic acid, palmitic acid, oleic acid, palmitoleic acid, cetyl alcohol, or oleyl alcohol). Creams can also contain nonionic surfactants, such as polyoxyethylene (40) stearate.

[0198] In some embodiments, topical formulations may also include excipients, an example of which is a penetration enhancer. These agents are capable of transporting pharmacologically active compounds through the stratum corneum and into the epidermis or dermis, preferably with little or no systemic absorption. A variety of compounds have been evaluated for their effectiveness in increasing the rate at which drugs penetrate the skin. See, for example, Percutaneous Penetration Enhancers , Maibach HI and Smith HE (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which surveyed the use and testing of various skin penetration enhancers, and Buyuktimkin et al., Chemical Means of Transdermal Drug PermeationEnhancementi nTransdermal and Topical Drug Delivery Systems, Gosh TK, Pfister WR, Yum S.I. (Eds.), Interpharm Press Inc., Buffalo Grove, Ill. (199). Representative examples of penetration enhancers include triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe gel), ethanol, isopropyl alcohol, octadecylphenyl polyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decyl methyl sulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate and propylene glycol monooleate) and N-methylpyrrolidone.

[0199] Representative examples of other excipients that may be included in topical preparations and other types of preparations (to the extent that they are compatible) include preservatives, antioxidants, humectants, emollients, buffers, solubilizers, skin protectants and surfactants. Suitable preservatives include alcohols, quaternary amines, organic acids, parabens and phenols. Suitable antioxidants include ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherol and chelating agents such as ethylenediaminetetraacetic acid and citric acid. Suitable humectants include glycerol, sorbitol, polyethylene glycol, urea and propylene glycol. Suitable buffers include citric acid, hydrochloric acid and lactic acid buffers. Suitable solubilizers include quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin and polysorbate. Suitable skin protectants include vitamin E oil, allantoin, dimethicone, glycerol, vaseline and zinc oxide.

[0200] Transdermal preparations generally use transdermal application devices and transdermal application patches, wherein the compound is formulated into a lipophilic emulsion or a buffered aqueous solution, dissolved and / or dispersed in a polymer or adhesive. The patch can be configured to deliver a medicament continuously, pulsatilely or on demand. The transdermal delivery of a compound can be accomplished by an iontophoresis patch. A transdermal patch can provide controlled delivery of a compound, wherein the absorption rate is slowed down by using a rate-controlled membrane or by trapping the compound in a polymer matrix or a gel. Absorption enhancers can be used to increase absorption, and examples thereof include absorbable pharmaceutically acceptable solvents that help pass through the skin.

[0201] Ophthalmic preparations include eye drops.

[0202] Formulations for rectal administration include enemas, rectal gels, rectal foams, rectal aerosols and retention enemas, which may contain conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinyl pyrrolidone, polyethylene glycol, etc. Compositions for rectal or vaginal administration may also be formulated as suppositories, which can be prepared by mixing the compound with suitable non-irritating carriers and excipients such as cocoa butter, mixtures of fatty acid glycerides, polyethylene glycol, suppository waxes, and combinations thereof, all of which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the compound.

[0203] dose

[0204] As used herein, the term "therapeutically effective amount" refers to the amount of a bispecific compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof; or a composition comprising a bispecific compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, which is effective in producing the desired therapeutic response in a specific patient suffering from a disease or condition characterized by or mediated by abnormal FGFR2 activity. The term "therapeutically effective amount" therefore includes an amount of a compound of the invention or a pharmaceutically acceptable salt or stereoisomer thereof, which, when administered, can induce a positive change in the disease or condition to be treated (e.g., to selectively inhibit / degrade FGFR2), or is sufficient to prevent the development or progression of a disease or condition, or to alleviate one or more symptoms of the disease or condition being treated in a subject to some extent, or to kill or inhibit the growth of diseased (e.g., neuroblastoma) cells alone, or to reduce the amount of FGFR2 in diseased cells.

[0205] The total daily dose of the bispecific compound and its use can be determined according to standard medical practice, for example, by the attending physician using sound medical judgment. The specific therapeutically effective dose for any particular subject can depend on a variety of factors, including the disease or condition being treated and its severity (e.g., its current state); the subject's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or concurrently with the bispecific compound; and factors well known in the medical field (see, for example, Goodman and Gilman's, Pharmacological Basis of Therapeutics , 10th Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-1)3, 2001).

[0206] The bispecific compounds of formula (I) can be effective over a wide dosage range. In some embodiments, the total daily dose (e.g., for adults) can be in the range of about 0.001 to about 1600 mg, 0.01 to about 1600 mg, 0.01 to about 500 mg, about 0.01 to about 100 mg, about 0.5 to about 100 mg, 1 to about 100-400 mg per day, about 1 to about 50 mg per day, about 5 to about 40 mg per day, and in other embodiments, about 10 to about 30 mg per day. Depending on the number of times the compound is administered per day, a single dose can be formulated to contain the desired dose. For example, a capsule can be formulated with about 1 to about 200 mg of the compound (e.g., 1, 2, 2.5, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, and 200 mg). In some embodiments, depending on the number of times the compound is administered per day, a single dose can be formulated to contain the desired dose.

[0207] How to use

[0208] In some aspects, the invention relates to methods of treating diseases or conditions involving abnormal (e.g., dysfunctional or dysregulated) FGFR2 activity, which requires administering to a subject in need thereof a therapeutically effective amount of a bispecific compound of Formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof.

[0209] It can be said that these diseases or conditions are characterized by abnormal (e.g., loss of function or disorder) FGFR2 activity or mediated by abnormal FGFR2 activity (e.g., relative to a non-pathological state, the level of the protein is increased or functionally abnormal). "Disease" is generally considered to be a subject's health condition, in which the subject cannot maintain homeostasis, and in which if the disease does not improve, the subject's health continues to deteriorate. In contrast, a subject's "disorder" refers to a health condition in which the subject is able to maintain homeostasis, but the subject's health condition is not as good as the health condition when there is no disease. If not treated, the disease may not necessarily lead to a further decline in the animal's health. In some embodiments, the bispecific compounds of formula (I) can be used to treat cell proliferative diseases and disorders (e.g., cancer or benign tumors). As used herein, the term "cell proliferative disease or disorder" refers to a condition characterized by dysregulated or abnormal cell growth or both, including non-cancerous conditions (such as tumors), precancerous conditions, benign tumors, and cancer.

[0210] The term "subject" (or "patient") as used herein includes all members of the animal kingdom susceptible to or suffering from the disease or condition. In some embodiments, the subject is a mammal, e.g., a human or non-human mammal. These methods are also applicable to companion animals, such as dogs and cats, and livestock, such as cattle, horses, sheep, goats, pigs and other domestic and wild animals. A subject who "needs" treatment according to the present invention may "suffer from or be suspected of suffering from" a specific disease or condition, and the subject may have been diagnosed or otherwise present with a sufficient number of risk factors or a sufficient number or combination of signs or symptoms so that a medical professional can diagnose or suspect that the subject suffers from the disease or condition. Therefore, subjects suffering from and suspected of suffering from a specific disease or condition are not necessarily two different groups.

[0211] Exemplary types of non-cancerous (e.g., cell proliferative) diseases or disorders that can be treated with the bispecific compounds of Formula (I) include inflammatory diseases and conditions, autoimmune diseases, neurodegenerative diseases, cardiac diseases, viral diseases, chronic and acute kidney disease or injury, metabolic diseases, and allergic and genetic diseases.

[0212] Representative examples of specific non-cancerous diseases and disorders include rheumatoid arthritis, alopecia areata, lymphoproliferative conditions, autoimmune blood disorders (e.g., hemolytic anemia, aplastic anemia, anhidrotic ectodermal dysplasia, pure red cell anemia, and idiopathic thrombocytopenia), cholecystitis, acromegaly, rheumatoid spondylitis, osteoarthritis, gout, scleroderma, sepsis, septic shock, dacryoadenitis, protein-associated periodic syndrome (CAPS), endotoxic shock, endometritis, gram-negative sepsis, keratoconjunctivitis sicca, toxic shock syndrome, asthma, adult respiratory syncytial virus (ART), inflammatory bowel disease, ... Distress syndrome, chronic obstructive pulmonary disease, chronic lung inflammation, chronic transplant rejection, hidradenitis suppurativa, inflammatory bowel disease, Crohn's disease, Behcet's syndrome, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis, juvenile diabetes, autoimmune uveoretinitis, autoimmune vasculitis, thyroiditis, Addison's disease, lichen planus, appendicitis, bullous pemphigus, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, myasthenia gravis, immunoglobulin A nephropathy, Hashimoto's disease, Sjögren's syndrome, vitiligo, Wegener's granulomatosis, granulomatous orchitis, autoimmune Oophoritis, sarcoidosis, rheumatic carditis, ankylosing spondylitis, Graves' disease, autoimmune thrombocytopenic purpura, psoriasis, psoriatic arthritis, eczema, dermatitis herpetiformis, ulcerative colitis, pancreatic fibrosis, hepatitis, liver fibrosis, CD14-mediated sepsis, non-CD14-mediated sepsis, acute and chronic kidney disease, irritable bowel syndrome, fever, restenosis, cervicitis, stroke and ischemic injury, nerve damage, acute and chronic pain, allergic rhinitis, allergic conjunctivitis, chronic heart failure, congestive heart failure, acute coronary syndrome, cachexia, malaria, acute and chronic Heart failure, congestive heart failure, acute coronary syndrome, cachexia, malaria, leprosy, leishmaniasis, Lyme disease, Rett's syndrome, acute synovitis, muscle degeneration, bursitis, tendinitis, tenosynovitis, herniated, ruptured or prolapsed disc syndrome, osteosclerosis, sinusitis, thrombosis, silicosis, pulmonary myopathy, bone resorption diseases (such as osteoporosis), fibromyalgia, AIDS and other viral diseases (such as herpes zoster, herpes simplex I or II, influenza virus and cytomegalovirus), type I and II diabetes, obesity, insulin resistance and diabetic retinopathy, 22q11.2-deletion syndrome, Angelman syndrome, Canavan disease, Celiac disease, Charcot-Marie-Tooth disease, color blindness, Cridu chat, Down syndrome, cystic fibrosis, Duchenne muscular dystrophy, hemophilia, Klinefleter syndrome, neurofibromatosis, phenylketonuria, Prader-Willi syndrome, sickle cell disease, Tay-Sachs disease, Turner syndrome, urea cycle disorders, thalassemia, otitis media, pancreatitis, mumps, pericarditis, peritonitis, pharyngitis, pleurisy, phlebitis, pneumonia, uveitis, polymyositis, proctitis, interstitial pulmonary fibrosis, dermatomyositis, atherosclerosis, arteriosclerosis, amyotrophic lateral sclerosis, vascular dementia, varicose veins, vaginitis, depression, and sudden infant death syndrome.

[0213] In some embodiments, the bispecific compounds can be used to treat non-cancerous neurodegenerative diseases and disorders. As used herein, the term "neurodegenerative diseases and disorders" refers to disorders characterized by progressive degeneration or death of nerve cells or both, including movement problems (ataxia) or mental function problems (dementia). Representative examples of such diseases and disorders include Alzheimer's disease (AD) and AD-related dementia, Parkinson's disease (PD) and PD-related dementia, prion diseases, motor neuron disease (MND), Huntington's disease (HD), Pick's syndrome, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), primary progressive aphasia (PPA), amyotrophic lateral sclerosis (ALS), traumatic brain injury (TBI), multiple sclerosis (MS), dementia (e.g., vascular dementia (VaD), Lewy body dementia (LBD), semantic dementia, and frontotemporal dementia (FTD).

[0214] In other embodiments, the method relates to treating a subject having cancer. Broadly speaking, the bispecific compounds of the invention are effective in treating carcinomas (solid tumors, including primary and metastatic tumors), sarcomas, melanomas, and hematological cancers (cancers affecting the blood, including lymphocytes, bone marrow, and / or lymph nodes), such as leukemias, lymphomas, and multiple myeloma. Both adult tumors / cancers and pediatric tumors / cancers are included. Cancers can be vascularized, or not substantially vascularized, or non-vascularized tumors.

[0215] Representative examples of cancer include adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's and AIDS-related lymphomas), appendix cancer, childhood cancers (e.g., childhood cerebellar astrocytoma, childhood brain astrocytoma), basal cell carcinoma, skin cancer (non-melanoma), bile duct cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, brain cancer (e.g., gliomas and glioblastomas (such as brain stem gliomas, gestational trophoblastic tumor gliomas, cerebellar astrocytomas, brain astrocytomas / malignant gliomas, ependymomas, medulloblastomas, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas), breast cancer, bronchial gland cancer, Tumor / Carcinoid, Carcinoid tumor, Nervous system cancer (e.g., CNS cancer, CNS lymphoma), Cervical cancer, Chronic myeloproliferative disorder, Colorectal cancer (e.g., Colon cancer, Rectal cancer), Lymphoid neoplasms, Mycosis fungoides, Sezary syndrome, Endometrial cancer, Esophageal cancer, Extracranial germ cell tumor, Extragonadal germ cell tumor, Extrahepatic bile duct cancer, Eye cancer, Intraocular melanoma, Retinoblastoma, Gallbladder cancer, Gastrointestinal cancer (e.g., Gastric cancer, Small intestine cancer, Gastrointestinal carcinoid, Gastrointestinal stromal tumor (GIST)), Cholangiocarcinoma, Germ cell tumor, Ovarian germ cell tumor, Head and neck cancer, Neuroendocrine tumors, Hodgkin lymphoma, Ann Arbor Stage III and IV childhood non-Hodgkin lymphoma, ROS1-positive refractory non-Hodgkin lymphoma, leukemia, lymphoma, multiple myeloma, hypopharyngeal cancer, intraocular melanoma, eye cancer, islet cell tumor (endocrine pancreas), kidney cancer (e.g., Wilms tumor, renal cell carcinoma), liver cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), ALK-positive anaplastic large lymphoma, ALK-positive advanced malignant solid tumors, Waldenstrom's macroglobulinoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelioma, squamous neck cancer with occult primary metastasis, multiple endocrine neoplasia (MEN), myelodysplasia syndrome, myelodysplastic / myeloproliferative disorders, nasopharyngeal carcinoma, neuroblastoma, oral cancer (e.g., oral cavity, lip, oral cavity, tongue, oropharynx, larynx, pharynx), ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, nasopharyngeal carcinoma, pheochromocytoma, pineoblastoma, metastatic anaplastic thyroid cancer, anaplastic thyroid cancer, papillary thyroid cancer, pituitary tumors, plasmacytoma / multiple myeloma, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine cancer (e.g.,Endometrial cancer, uterine sarcoma, uterine corpus cancer), squamous cell carcinoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, juvenile xanthogranuloma, transitional cell carcinoma of the renal pelvis and ureter and other urinary organs, urethral cancer, gestational trophoblastic tumor, vaginal cancer, vulvar cancer, hepatoblastoma, rhabdoid tumor and Wilms tumor.

[0216] Sarcomas treatable with the compounds of the invention include soft tissue cancers and bone cancers, representative examples of which include osteosarcoma or osteogenic sarcoma (bone) (e.g., Ewing's sarcoma), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelioma or mesothelioma (membranous lining of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (fatty tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), mesenchymal or mixed mesodermal tumors (mixed connective tissue type), and histiocytic sarcoma (immune cancer).

[0217] In some embodiments, the methods of the invention are capable of treating subjects suffering from cell proliferative diseases or disorders of the hematologic, liver, brain, lung, colon, pancreas, prostate, ovary, breast, skin, and endometrium.

[0218] As used herein, "hematologic cell proliferative diseases or disorders" include lymphomas, leukemias, myeloid neoplasms, mast cell tumors, myelodysplasia, benign monoclonal gammopathy, polycythemia vera, chronic myeloid leukemia, idiopathic myeloid metaplasia, and essential thrombocythemia. Thus, representative examples of hematological cancers may include multiple myeloma, lymphoma (including T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and ALK+ anaplastic large cell lymphoma (e.g., a B-cell non-Hodgkin's lymphoma selected from diffuse large B-cell lymphoma (e.g., germinal center B-cell-like diffuse large B-cell lymphoma or activated B-cell-like diffuse large B-cell lymphoma), Burkitt's lymphoma / leukemia, mantle cell lymphoma, mediastinal (thymic) large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, The invention relates to pancreatic cancer, refractory B-cell non-Hodgkin's lymphoma, lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia, metastatic pancreatic cancer, refractory B-cell non-Hodgkin's lymphoma, and relapsed B-cell non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin, e.g., small lymphocytic lymphoma, leukemias (including childhood leukemias), hairy cell leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia (e.g., acute monocytic leukemia), chronic lymphocytic leukemia, small lymphocytic leukemia, chronic myeloid leukemia, chronic myeloid leukemia, and mast cell leukemia, myeloid tumors, and mast cell tumors.

[0219] As used herein, "cell proliferative diseases or disorders of the liver" include all forms of cell proliferative disorders that affect the liver. Cell proliferative disorders of the liver can include liver cancer (e.g., hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and hepatoblastoma), precancerous or precancerous conditions of the liver, benign growths or lesions of the liver, malignant growths or lesions of the liver, and metastatic lesions of tissues and organs in the body other than the liver. Cell proliferative disorders of the liver may include hyperplasia, metaplasia, and dysplasia of the liver.

[0220] As used herein, "cell proliferative diseases or disorders of the brain" include all forms of cell proliferative disorders that affect the brain. Cell proliferative disorders of the brain can include brain cancer (e.g., gliomas, glioblastomas, meningiomas, pituitary adenomas, vestibular schwannomas, and primitive neuroectodermal tumors (medulloblastomas)), precancerous or precancerous conditions of the brain, benign growths or conditions of the brain, malignant growths or lesions of the brain, and metastatic lesions of body tissues and organs other than the brain. Cell proliferative disorders of the brain may include hyperplasia, metaplasia, and dysplasia of the brain.

[0221] As used herein, "cell proliferative diseases or disorders of the lung" include all forms of cell proliferative disorders that affect lung cells. Cell proliferative disorders of the lung include lung cancer, precancerous and precancerous conditions of the lung, benign growths or lesions of the lung, hyperplasia, metaplasia and dysplasia of the lung, and metastatic lesions of body tissues and organs other than the lung. Lung cancer includes all forms of lung cancer, for example, malignant lung tumors, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Lung cancer includes small cell lung cancer ("SLCL"), non-small cell lung cancer ("NSCLC"), adenocarcinoma, small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and mesothelioma. Lung cancer may include "scar cancer", bronchiolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lung cancer also includes lung tumors with histological and ultrastructural heterogeneity (e.g., mixed cell types). In some embodiments, the bispecific compounds of the invention can be used to treat non-metastatic or metastatic lung cancer (eg, NSCLC, ALK-positive NSCLC, NSCLC containing a ROS1 rearrangement, lung adenocarcinoma, and squamous cell lung cancer).

[0222] As used herein, "cell proliferative diseases or disorders of the colon" include all forms of cell proliferative disorders that affect colon cells, including colon cancer, precancerous or premalignant conditions of the colon, adenomatous polyps of the colon, and metachronous lesions of the colon. Colon cancer includes sporadic and hereditary colon cancer, malignant colon tumors, carcinoma in situ, typical and atypical carcinoid tumors, adenocarcinomas, squamous cell carcinomas, and squamous cell carcinomas. Colon cancer may be associated with hereditary syndromes, such as hereditary nonpolyposis colorectal cancer, common adenomatous polyposis, MYH-associated polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome, and juvenile polyposis. Cell proliferative disorders of the colon may also be characterized by hyperplasia, metaplasia, or dysplasia of the colon.

[0223] As used herein, "cell proliferative diseases or disorders of the pancreas" include all forms of cell proliferative disorders that affect pancreatic cells. Cell proliferative disorders of the pancreas may include pancreatic cancer, precancerous or premalignant conditions of the pancreas, hyperplasia of the pancreas, dysplasia of the pancreas, benign growths or lesions of the pancreas, malignant growths or lesions of the pancreas, and metastatic lesions of body tissues and organs other than the pancreas. Pancreatic cancer includes all forms of pancreatic cancer, including ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous adenocarcinoma, osteoclast-like giant cell carcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassified large cell carcinoma, small cell carcinoma, pancreatoblastoma, papillary tumors, mucinous cystadenoma, papillary cystic tumors, and serous cystadenoma, as well as pancreatic tumors with histological and ultrastructural heterogeneity (e.g., mixed cell types).

[0224] As used herein, "cell proliferative diseases or disorders of the prostate" include all forms of cell proliferative disorders that affect the prostate. Cell proliferative disorders of the prostate can include prostate cancer, precancerous or precancerous conditions of the prostate, benign growths or lesions of the prostate, malignant growths or lesions of the prostate, and metastatic lesions of tissues and organs in the body other than the prostate. Cell proliferative diseases of the prostate may include prostatic hyperplasia, metaplasia, and dysplasia.

[0225] As used herein, "cell proliferative diseases or disorders of the ovary" include all forms of cell proliferative disorders that affect ovarian cells. Cell proliferative disorders of the ovary can include precancerous or precancerous conditions of the ovary, benign growths or lesions of the ovary, ovarian cancer, and metastatic lesions of tissues and organs in the body other than the ovary. Cell proliferative disorders of the ovary may include ovarian hyperplasia, metaplasia, and dysplasia.

[0226] As used herein, "cell proliferative diseases or disorders of the breast" include all forms of cell proliferative disorders that affect breast cells. Cell proliferative disorders of the breast can include breast cancer, precancerous or precancerous conditions of the breast, benign growths or lesions of the breast, and metastatic lesions in body tissues and organs other than the breast. Cell proliferative disorders of the breast may include mammary hyperplasia, metaplasia, and dysplasia.

[0227] As used herein, "cell proliferative diseases or disorders of the skin" include all forms of cell proliferative disorders that affect skin cells. Cell proliferative disorders of the skin can include precancerous or precancerous conditions of the skin, benign growths or lesions of the skin, melanoma, malignant melanoma, or other malignant growths or lesions of the skin, and metastatic lesions in body tissues and organs other than the skin. Cell proliferative disorders of the skin may include skin hyperplasia, metaplasia, and dysplasia.

[0228] As used herein, "cell proliferative diseases or disorders of the endometrium" include all forms of cell proliferative disorders that affect endometrial cells. Cell proliferative disorders of the endometrium can include precancerous or precancerous conditions of the endometrium, benign growths or lesions of the endometrium, endometrial cancer, and metastatic lesions of tissues and organs in the body other than the endometrium. Cell proliferative disorders of the endometrium may include hyperplasia, metaplasia, and dysplasia of the endometrium.

[0229] In some embodiments, the disease or condition is liver cancer. In other embodiments, the disease or condition is biliary tract cancer (BTC). In other embodiments, the disease or condition is intrahepatic bile duct carcinoma (ICC) or extrahepatic bile duct carcinoma (ECC).

[0230] The bispecific compound of formula (I) can be applied to patients (e.g., cancer patients) as a monotherapy or by combination therapy, as well as a first-line therapy or a follow-up therapy for patients who are unresponsive to first-line therapy. Therapy can be "first-line", that is, as the initial treatment for patients who have not received a previous anticancer treatment regimen, alone or in combination with other treatments; or "second-line", as a treatment for patients who have undergone a previous anticancer treatment regimen, alone or in combination with other treatments; or as a "third-line", "four-line" and other treatments, alone or in combination with other treatments. Patients who have previously received partial successful treatment but have become intolerant to a particular treatment can also be treated. Therapy can also be given as an adjuvant therapy, that is, to prevent the recurrence of cancer in patients who are currently not detected in the disease or after surgical resection of the tumor. Therefore, in some embodiments, the compound can be applied to patients who have received another therapy (e.g., chemotherapy, radioimmunotherapy, surgical therapy, immunotherapy, radiotherapy, targeted therapy, or any combination thereof).

[0231] The methods of the present invention may require administration of a bispecific compound of Formula (I) or a pharmaceutical composition thereof to a patient in a single dose or multiple doses (e.g., 1, 2, 3, 4, 5, 6, 8, 10, 15, 20 or more doses). For example, the frequency of administration may be from once a day to about once every eight weeks. In some embodiments, the frequency of administration ranges from about once a day for 1, 2, 3, 4, 5 or 6 weeks, and in other embodiments requires a 28-day cycle, which includes 3 weeks (21 days) of daily administration. In other embodiments, the bispecific compound may be administered twice a day (BID) (a total of 5 doses) over the course of two and a half days, or once a day (QD) (a total of 2 doses) over the course of two days. In other embodiments, the bispecific compound may be administered once a day (QD) over five days.

[0232] Combination therapy

[0233] The bispecific compound of formula (I) can be combined or used simultaneously with at least one other active agent (e.g., anticancer agent) or therapy for the treatment of diseases and disorders. The terms "combination" and "simultaneous" herein refer to the co-administration of medicaments, which include substantially simultaneous administration, by the same or separate dosage form, and by the same or different modes of administration, or sequential administration, for example, as part of the same treatment regimen, or by a continuous treatment regimen. Therefore, if continuously administered, at the beginning of administration of the second compound, in some cases, the first of the two compounds can still be detected at an effective concentration at the treatment site. The order and time interval can be determined so that they can work together (e.g., synergistically to provide an increased benefit than when administered in other ways). For example, therapeutic agents can be administered simultaneously or sequentially in any order at different time points; however, if not administered simultaneously, they can be administered within a sufficiently close time to provide a desired therapeutic effect, which can be a synergistic approach. Therefore, these terms are not limited to administering active agents at exactly the same time.

[0234] In some embodiments, the treatment regimen may include administering a bispecific compound of Formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof in combination with one or more other therapeutic agents known to be used to treat a disease or condition (e.g., cancer). The dose of the additional anticancer therapeutic agent may be the same as or even lower than the known or recommended dose. See, Hardman et al., eds., Goodman & Gilman's The Pharmacological Basis Of Basis Of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; Physician's Desk Reference, 60th ed., 2006. For example, anticancer agents that can be used in combination with bispecific compounds are known in the art. See, for example, U.S. Pat. No. 9,101,622 (Section 5.2 therein) and U.S. Pat. No. 9,345,705 B2 (Columns 12-18 therein). Representative examples of additional active agents and treatment regimens include radiation therapy, chemotherapy (e.g., mitotic inhibitors, angiogenesis inhibitors, anti-hormones, autophagy inhibitors, alkylating agents, intercalating antibiotics, growth factor inhibitors, anti-androgens, signal transduction pathway inhibitors, anti-microtubule agents, platinum coordination complexes, HDAC inhibitors, proteasome inhibitors, and topoisomerase inhibitors), immunomodulators, therapeutic antibodies (e.g., monospecific and bispecific antibodies), and CAR-T therapy.

[0235] In some embodiments, the bispecific compounds of formula (I) can be used in combination with other anticancer agents, examples of which include paclitaxel (e.g., ovarian cancer, breast cancer, lung cancer, Kaposi's sarcoma, cervical cancer, and pancreatic cancer), topotecan (e.g., ovarian cancer and lung cancer), irinotecan (e.g., colon cancer and small cell lung cancer), etoposide (e.g., testicular cancer, lung cancer, lymphoma, and non-lymphocytic leukemia), vincristine (e.g., leukemia), folinic acid (e.g., colon cancer), octreotide (e.g., ovarian cancer), daunorubicin (e.g., acute myeloid leukemia), leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), and Kaposi's sarcoma), trastuzumab (e.g., breast cancer, gastric cancer, and esophageal cancer), rituximab (e.g., non-Hodgkin lymphoma), cetuximab (e.g., colorectal cancer, metastatic non-small cell lung cancer, and head and neck cancer), pertuzumab (e.g., metastatic HER2-positive breast cancer), alemtuzumab (e.g., chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), and T-cell lymphoma), panitumumab (e.g., colon cancer), cancer, and colorectal cancer), tamoxifen (e.g., breast cancer), fulvestrant (e.g., breast cancer), letrozole (e.g., breast cancer), exemestane (e.g., breast cancer), azacytidine (e.g., myelodysplastic syndrome), mitomycin C (e.g., gastrointestinal cancer, anal cancer, and breast cancer), dactinomycin (e.g., Wilms tumor, rhabdomyosarcoma, Ewing sarcoma, trophoblastic tumor, testicular cancer, and ovarian cancer), erlotinib (e.g., non-small cell lung cancer and pancreatic cancer), sorafenib (e.g., kidney cancer and liver cancer), temsirolimus (e.g., kidney cancer), bortezomib (e.g., (e.g., multiple myeloma and mantle cell lymphoma), pegaspargase (e.g., acute lymphoblastic leukemia), cabozantinib (e.g., hepatocellular carcinoma, medullary thyroid carcinoma, and renal cell carcinoma), KEYTRUDA (e.g., cervical cancer, gastric cancer, hepatocellular carcinoma, Hodgkin lymphoma, melanoma, Merkel cell carcinoma, non-small cell lung cancer, urothelial carcinoma, and head and neck squamous cell carcinoma), nivolumab (e.g., colorectal cancer, hepatocellular carcinoma, melanoma, non-small cell lung cancer, renal cell carcinoma, small cell lung cancer, and urothelial carcinoma), and regorafenib (e.g., colorectal cancer, gastrointestinal stromal tumors, and hepatocellular carcinoma).

[0236] In some embodiments, the bispecific compound of Formula (I) and the additional (e.g., anticancer) therapeutic agent can be administered less than 5 minutes apart, less than 30 minutes apart, less than 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 0 hours apart, about 0 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 02 hours apart, 02 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. Two or more (eg, anti-cancer) therapeutic agents may be administered during the same patient visit.

[0237] When the active ingredients of the combination are not administered in the same pharmaceutical composition, it is understood that they can be administered to the subject in need in any order. For example, the compounds of the present invention can be administered to the subject in need thereof before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 2 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks before) the administration of the other anticancer therapeutic agent, simultaneously or afterwards (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 2 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks after) the administration of the subject in need thereof. In various aspects, the anticancer therapeutic agent is administered at intervals of 1 minute, 10 minutes, 30 minutes, less than 1 hour, 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 2 hours, 2 hours to 8 hours, 8 hours to 9 hours, 9 hours to 10 hours, 10 hours to 11 hours, 11 hours to 12 hours, no more than 24 hours or no more than 48 hours. In one example, the (e.g., anticancer) therapeutic agent is performed in the same office visit. In another example, the combination anticancer therapeutic agent can be administered at intervals of 1 minute to 24 hours.

[0238] In some embodiments related to cancer treatment, the bispecific compound of formula (I) and another anticancer agent or therapeutic agent are administered in cycles. Cyclic therapy includes administering an anticancer therapeutic agent for a period of time, followed by administering a second anticancer therapeutic agent for a period of time, and repeating this sequential administration (i.e., the cycle) to reduce the development of resistance to one or both anticancer therapies, avoid or reduce the side effects of one or both anticancer therapies, and / or improve the efficacy of therapy. In one example, cyclic therapy includes administering a first anticancer therapeutic agent for a period of time, followed by administering a second anticancer therapeutic agent for a period of time, optionally, then administering a third anticancer therapeutic agent for a period of time, etc., and repeating this sequential administration (i.e., the cycle) to reduce the development of resistance to one of the anticancer agents, avoid or reduce the side effects of one of the anticancer agents, and / or improve the efficacy of the anticancer agent.

[0239] Medical test kit

[0240] The bispecific compounds of the present invention and / or compositions comprising them can be assembled into a kit or pharmaceutical system. The kit or pharmaceutical system according to this aspect of the present invention includes a carrier or packaging, such as a box, carton, tube, etc., in which one or more containers with strict confinement, such as a vial, tube, ampoule or bottle, contain the bispecific compound of formula (I) of the present invention or its pharmaceutical composition. The kit or pharmaceutical system of the present invention may also include printed instructions for using the compounds and compositions.

[0241] These and other aspects of the invention will be further understood upon consideration of the following examples, which are intended to illustrate certain specific embodiments of the invention, but are not intended to limit the scope of the invention, which is defined by the claims.

[0242] Example

[0243] Example 1: Synthesis of thalidomide-based intermediates.

[0244]

[0245] tert-Butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetate

[0246] Tert-butyl bromoacetate (199 mg, 1.02 mmol) was added to a mixture of 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (200 mg, 0.3 mmol) and K2CO3 (304 mg, 2.20 mmol) in 3 mL of DMF at room temperature. The mixture was stirred overnight and then quenched with water. The aqueous mixture was then extracted with 3×5 mL of ethyl acetate, washed with brine, dried over Na2SO4, and concentrated. Purification by silica gel chromatography provided tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)oxy)acetate (245 mg, 0.63 mmol, 86%) as a white crystalline solid. LC / MS m / z calculated as [M+2H–tBu] + 333.1, measured 333.1.

[0247]

[0248] 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid

[0249] Tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetate (140 mg, 0.036 mmol) was dissolved in 1 mL DCM and 1 mL TFA and stirred at room temperature for 1 hour. The solvent was evaporated to give 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid (121 mg, 0.036 mmol, 101%) as a white solid. LC / MS m / z calculated as [M+H] + 333.1, measured 332.).

[0250]

[0251] 2-(2,6-dioxopiperidin-3-yl)-5-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione

[0252] 3-Hydroxy-1,8-naphthalenedicarboxylic anhydride (2.14 g, 10.0 mmol) and 3-aminopiperidine-2,6-dione (1.65 g, 10.0 mmol) were dissolved in THF (40 mL) at room temperature and triethylamine (2.8 mL, 20.0 mmol) was added. The suspension was then refluxed for 5 days, a green precipitate formed within the first 24 hours and eventually turned black. The solvent was evaporated, water was added, the mixture was acidified and stirred for 1 hour. The suspension was then filtered to give the title compound (3.44 g, 9.53 mmol, 95%) as a green solid. LC / MS m / z calculated as [M+H] + 325.1, measured 325.1.

[0253]

[0254] tert-Butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-1H-benzo[de]isoquinolin-5-yl)oxy)acetate

[0255] 2-(2,6-dioxopiperidin-3-yl)-5-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (361 mg, 1.1 mmol) was suspended in 3 mL of DMF, and then K2CO3 (2) (6 mg, 2.0 mmol) and tert-butyl bromoacetate (234 mg, 1.2 mmol) were added. The blue suspension was stirred at room temperature for 4 hours, at which time another 1.0 mmol of tert-butyl bromoacetate was added. After stirring was continued overnight, water was added and the suspension was filtered to give the tert-butyl ester (464 mg, 1.06 mmol, 95%) as a light grey solid. LC / MS m / z calculated as [M+2H–tBu] + 383.1, measured 383.2.

[0256] The ester was then dissolved in DCM (1 mL), TFA (1 mL) was added, and the solution was stirred at room temperature for 2 hours. The solvent was then removed and the product was dried to give the title compound without further purification. LC / MS m / z calculated as [M+H] + 383.08, measured 383.19.

[0257]

[0258] Thalidomide-based intermediates (A–E)

[0259] 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione or 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (1 eq) was dissolved in DMF, treated with K2CO3 (2 eq), the appropriate alkyl bromide linker (1.0 eq) was added, and the mixture was stirred at 50°C overnight. The reaction was quenched with water and extracted with EtOAc. The combined extracts were washed with brine, dried over Na2SO4, then concentrated and purified by silica gel chromatography to afford the protected amine or ester. These intermediates were then dissolved in 1:1 DCM:TFA, stirred at room temperature for 2 hours, then concentrated and dried to afford compounds of Types A–E.

[0260] Example 2: 2-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino}pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)-N-{2-[2-(2-{[3-(2,6-dioxopiperidin-3-yl)-2,4-dioxo-3-azatricyclo[).3.1.0 5 , 13 Synthesis of 1-(12),5-(9),13-pentaenyl)oxy}ethoxy)ethoxy]ethyl}acetamide (1)

[0261]

[0262] tert-Butyl 4-(4-((6-chloropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate

[0263] Tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (2.0 g, .21 mmol) was added to 4,6-dichloropyrimidine (1.61 g, 10.8 mmol) in DIEA (1.88 mL, 10.8 mmol) and isopropanol (15 mL). The purple solution was then stirred at room temperature overnight. The solvent was evaporated and the residue was purified by silica gel chromatography to give the title compound as a maroon solid (2.5 g, .05 mmol, 98%). LC / MS m / z calculated as [M+H] + 390.16, measured 390.30.

[0264]

[0265] tert-Butyl 4-(4-((6-(methylamino)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate

[0266] Tert-butyl 4-(4-((6-chloropyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (1.0 g, 2.6 mmol) was suspended in 1-butanol (20 mL) and DIEA (910 uL, 5.2 mmol) was added followed by methylamine (1.28 mmol, 2 M in THF). The reaction vessel was then sealed and the mixture was heated at 120 °C overnight. The solvent was evaporated to give the title compound without further purification. LC / MS m / z calculated as [M+H] + 385.23, measured 385.0).

[0267]

[0268] tert-Butyl 4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1,3-dimethylureido)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate

[0269] 2,6-Dichloro-3,5-dimethoxyaniline (444 mg, 2.0 mmol) and triphosgene (23 mg, 0.80 mmol) were dissolved in THF. DIEA was slowly added at 0°C, and a white precipitate formed as the reaction slowly warmed to room temperature. After 1 hour, the solvent was evaporated and the crude isocyanate was resuspended in toluene (10 mL). DIEA (1.39 mL, 8.0 mmol) and tert-butyl 4-(4-((6-(methylamino)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (68 mg, 2.0 mmol) were added, and the mixture was stirred at 80°C overnight. The solvent was evaporated and the crude product was purified by silica gel chromatography to give the title compound (828 mg, 1.31 mmol, 66%). LC / MS m / z was calculated as [M+H] + 631.22, measured 631.90.

[0270]

[0271] 1-(2,6-dichloro-3,5-dimethoxyphenyl)-1,3-dimethyl-3-(6-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)urea

[0272] Tert-butyl 4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1,3-dimethylureido)pyrimidin-4-yl)amino)phenyl)piperazine-1-carboxylate (828 mg, 1.31 mmol) was dissolved in DCM (1 mL) and TFA (1 mL) was added. The solution was stirred for 2 hours and the solvent was evaporated. The residue was then dissolved in THF and stirred with saturated aqueous NaHCO3 for 30 minutes. The brown precipitate was then filtered, washed with water and dried to give the title compound (611 mg, 1.0) mmol) as a yellow solid. LC / MS m / z was calculated as [M+H] + 531.1), measured 531.)8.

[0273]

[0274] 2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1,3-dimethylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetic acid

[0275] 1-(2,6-Dichloro-3,5-dimethoxyphenyl)-1,3-dimethyl-3-(6-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)urea (42 mg, 0.09) was dissolved in DMF (1 mL) and treated with K2CO3 (44 mg, 0.32 mmol). Tert-butyl bromoacetate (15 mg, 0.09 mmol) was added and the mixture was stirred at room temperature overnight before water was added followed by extraction with EtOAc. The combined extracts were washed with brine, dried over Na2SO4, and purified by silica gel chromatography to provide the tert-butyl ester. LC / MS m / z calculated as [M+H] + 646.22, measured 646.30.

[0276] Then tert-butyl 2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1,3-dimethylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetate was dissolved in DCM (1 mL) and TFA (1 mL) was added. The solution was stirred for 2 h and the solvent was evaporated to give the title compound (34.4 mg, 0.058 mmol, 4% for two steps). LC / MS m / z calculated as [M+H] + 590.16, measured 590.39.

[0277]

[0278] 2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1,3-dimethylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetic acid (10 mg, 0.01) mmol) was added to a solution of 3-(5-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-1,3-dioxy-2,3-dihydro-1H-phenalen-2-yl)piperidine-2,6-dione TFA (9.) mg, 0.01) mmol) in DIEA (11 mg, 0.085 mmol) and DMF (1 mL). HATU (13 mg, 0.034 mmol) was added and the reaction was stirred for 30 minutes and then purified by HPLC to give the title compound (6.) mg, 0.0059 mmol, 35%). LC / MS m / z calculated as [M+H] + 102).32, measured 102).44.

[0279] Example 3: 2-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino}pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)-N-(3-{[3-(2,6-dioxopiperidin-3-yl)-2,4-dioxo-3-azatricyclo[).3.1.0 5 , 13 Synthesis of tridecan-1(12),5,9(13),10-pentaen-)-yl]oxy}propyl)acetamide (2)

[0280] The synthesis of bispecific compound 2 was similar to that of bispecific compound 1. LC / MS m / z was calculated as [M+H] + 953.28, measured 953.53.

[0281] Example 4: 2-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino}pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)-N-(6-{[3-(2,6-dioxopiperidin-3-yl)-2,4-dioxo-3-azatricyclo[).3.1.0 5 , 13 Synthesis of tridecan-1(12),5,9(13),10-pentaen-)-yl]oxy}hexyl)acetamide (3)

[0282] The synthesis of bispecific compound 3 was similar to that of bispecific compound 1. LC / MS m / z was calculated as [M+H] + 995.33, measured 995.40.

[0283] Example 5: 2-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino}pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)-N-(2-{2-[2-(2-{[3-(2,6-dioxopiperidin-3-yl)-2,4-dioxo-3-azatricyclo[).3.1.0 5 , 13 Synthesis of tridecan-1(12),5,9(13),10-pentaen-)-yl]oxy}ethoxy)ethoxy]ethoxy}ethyl)acetamide (4)

[0284]

[0285] The synthesis of bispecific compound 4 was similar to that of bispecific compound 1. LC / MS m / z was calculated as [M+H] + 10)1.35, measured 10)1.40.

[0286] Example 6: Synthesis of 2-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino}pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)-N-(5-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]oxy}pentyl)acetamide (5)

[0287]

[0288] The synthesis of bispecific compound 5 was similar to that of bispecific compound 1. LC / MS m / z was calculated as [M+H] + 931.30, measured 930.52.

[0289] Example): Synthesis of (2S,4R)-1-[(2S)-2-[2-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino})pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)acetylamino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3)-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (6)

[0290]

[0291] 2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1,3-dimethylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetic acid (5.0 mg, 0.0085 mmol) was added to a solution of (2S,4R)-1-(L-propionyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (4.1 mg, 0.0085 mmol) in DIEA (5.5 mg, 0.043 mmol) and DMF (1 mL). HATU was added and the reaction was stirred for 30 minutes and then purified by HPLC to give the title compound. LC / MS m / z calculated as [M+H] + 1016.3), actual measured 1016.43. 1 H NMR(500MHz,DMSO-d6)δ11.98(s,1H),10.05(s,1H),9.51(s,1H),8.99(d,J=3.9Hz,1H),8.))(d,J=).2Hz,1H),8.40(s,2H ),).55–).35(m,5H),).00(dd,J=11.1,8.8Hz,2H),6.91(s,1H),6.50–6.40(m,2H),5.14(s,1H),4.8)-4.99(m,1H),4.59(d ,J=9.1Hz,1H),4.44(t,J=8.1Hz,2H),4.32(s,1H),4.26–4.02(m,2H),3.95(s,9H),3.)3(s,2H),3.69–3.64(m,2H),3.59(d ,J=10.8Hz,2H),3.12–3.04(m,3H),2.4)(s,3H),2.11–2.00(m,1H),1.89–1.))(m,1H),1.39(d,J=).0Hz,3H),0.98(s,9H).

[0292] Example 8: Synthesis of (2S,4S)-1-[(2S)-2-[2-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino})pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)acetylamino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3)-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (7)

[0293]

[0294] The synthesis of bispecific compound 7 was similar to that of bispecific compound 6. LC / MS m / z was calculated as [M+H] + 1016.3), actual measured value is 1016.33. 1 H NMR(500MHz,DMSO-d6)δ11.91(s,1H),10.06(s,1H),9.46(s,1H),8.92(s,1 H),8.)3(d,J=9.4Hz,1H),8.45–8.23(m,2H),8.02(s,1H),).45–).26(m,5H ),6.84(dd,J=6.6,2.5Hz,3H),6.39(s,1H),4.83(t,J=).3Hz,1H),4.58(d, J=10.4Hz,1H),4.38(dd,J=8.1,6.4Hz,1H),4.35–4.2)(m,1H),4.20–4.10( m,1H),4.03(d,J=16.3Hz,1H),3.8)(s,6H),3.)3–3.64(m,1H),3.60(s,1H) ,3.51(dd,J=10.4,3.3Hz,1H),3.25(s,3H),3.14(s,1H),2.9)(s,3H),2.3) (d,J=4.4Hz,3H),2.05–1.95(m,1H),1.93–1.)9(m,1H),1.29(d,J=).0Hz,3 H),0.91(s,9H),0.84(t,J=).5Hz,1H),0.80(t,J=).0Hz,1H),0.66(s,1H).

[0295] Example 9: N-(4-{[3-(3,5-dimethoxyphenyl)-)-{[4-(4-{[(3-{[3-(2,6-dioxopiperidin-3-yl)-2,4-dioxo-3-azatricyclo[).3.1.0 5 , 13 Synthesis of 1H,2H,3H,4H-[1,3]diazinyl[4,5-d]pyrimidin-1-yl]methyl}phenyl)acrylamide (8)

[0296]

[0297] N-((2,4-dichloropyrimidin-5-yl)methyl)-3,5-dimethoxyaniline

[0298] 2,4-Dichloro-5-(iodomethyl)pyrimidine (200 mg, 0.69 mmol) and 3,5-dimethoxyaniline (106 mg, 0.69 mmol) were mixed in acetone (3 mL) at room temperature, and K2CO3 (190 mg, 1.38 mmol) was added. The solvent was evaporated and the residue was purified by silica gel chromatography to give the title compound (180 mg, 0.5) mmol, 83%). LC / MS m / z calculated as [M+H] + 313.04, measured 313.9).

[0299]

[0300] 2-Chloro-5-(((3,5-dimethoxyphenyl)amino)methyl)-N-(4-nitrobenzyl)pyrimidin-4-amine

[0301] 4-Aminomethylnitrobenzene (108 mg, 0.5) mmol) was added to N-((2,4-dichloropyrimidin-5-yl)methyl)-3,5-dimethoxyaniline (180 mg, 0.05) mmol) in DIEA (14) mg, 1.14 mmol) and dioxane (3 mL) at room temperature and stirred at 60°C for 20 hours. The solvent was then evaporated and the residue was purified by silica gel chromatography to give the title compound (1) 6 mg, 0.41 mmol, 2%). LC / MS m / z calculated as [M+H] + 430.12, measured 430.1).

[0302]

[0303] 7-Chloro-3-(3,5-dimethoxyphenyl)-1-(4-nitrobenzyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one

[0304] 2-Chloro-5-(((3,5-dimethoxyphenyl)amino)methyl)-N-(4-nitrobenzyl)pyrimidin-4-amine (1) 6 mg, 0.41 mmol) and triphosgene (4) mg, 0.16 mmol) were dissolved in THF (2 mL). Triethylamine was slowly added at room temperature and the reaction was stirred for 1 hour. The mixture was then concentrated and purified by silica gel chromatography to give the title compound (1) 0 mg, 0.3) mmol, 90%). LC / MS m / z calculated as [M+H] + 456.10, measured 456.18.

[0305]

[0306] 1-(4-aminobenzyl)-7-chloro-3-(3,5-dimethoxyphenyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one

[0307] Tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (103 mg, 0.3) mmol) and TFA (84 mg, 0.) 4 mmol) were added to 1-chloro-3-(3,5-dimethoxyphenyl)-1-(4-nitrobenzyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one in s-BuOH. The mixture was then refluxed for 16 h, the solvent was evaporated and the residue was purified by silica gel chromatography to give the title compound (192 mg, 0.28 mmol, ) 6%). LC / MS m / z calculated as [M+H] + 69).30, measured 69).40.

[0308]

[0309] 3-(3,5-dimethoxyphenyl)-1-(4-nitrobenzyl)-7-((4-(piperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one

[0310] 1-(4-Aminobenzyl)-)-chloro-3-(3,5-dimethoxyphenyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (192 mg, 0.28 mmol) was dissolved in DCM (1 mL), TFA (1 mL) was added, and the solution was stirred at room temperature for 1 hour. The solvent was then removed to give the amine. LC / MS m / z calculated as [M+H] + 59).25, measured 59).32.

[0311] The intermediate was then dissolved in DMF (1 mL), treated with K2CO3 (116 mg, 0.84 mmol), and tert-butyl bromoacetate (55 mg, 0.28 mmol) was added. The mixture was stirred at 50°C for 14 h and purified by silica gel chromatography to give the title compound (12) mg, 0.18 mmol, 64% for two steps). LC / MS m / z calculated as [M+H] + )11.32, measured)11.44.

[0312]

[0313] tert-Butyl 2-(4-(4-((8-(4-aminobenzyl)-6-(3,5-dimethoxyphenyl)-7-oxo-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetate

[0314] Tert-butyl 2-(4-(4-((6-(3,5-dimethoxyphenyl)-8-(4-nitrobenzyl)-)-oxo-5,6,),8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetate (12 mg, 0.18 mmol) was dissolved in acetic acid (2 mL) and zinc was added at room temperature. The suspension was stirred for 3 hours, then filtered, concentrated and purified by silica gel chromatography to give the title compound (0) mg, 0.11 mmol, 63%). LC / MS m / z calculated as [M+H] + 681.34, measured 681.40.

[0315]

[0316] tert-Butyl 2-(4-(4-((6-(3,5-dimethoxyphenyl)-7-oxo-8-(4-propionamidobenzyl)-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetate

[0317] Tert-butyl 2-(4-(4-((8-(4-aminobenzyl)-6-(3,5-dimethoxyphenyl)-)-oxo-5,6,),8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetate (20 mg, 0.11 mmol) was dissolved in THF (1 mL) and treated with saturated NaHCO3 solution. At 0°C, propionyl chloride (12 mg, 0.13 mmol) was added, stirred for 5 minutes, water was added, and the mixture was extracted with EtOAc. The combined extracts were washed with brine, dried over Na2SO4 and concentrated to give the title compound (23 mg, 0.031 mmol, 28%). LC / MS m / z calculated as [M+H] + )3).3), Actual measurement) 36.91.

[0318]

[0319] 2-(4-(4-((6-(3,5-dimethoxyphenyl)-7-oxo-8-(4-propionamidobenzyl)-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetic acid

[0320] Tert-butyl 2-(4-(4-(4-((6-(3,5-dimethoxyphenyl)-)-oxo-8-(4-propionamidobenzyl)-5,6,),8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetate (23 mg, 0.031) was dissolved in DCM (1 mL), TFA (1 mL) was added, and the solution was stirred at room temperature for 1 hour. The solvent was then removed to give the title compound. LC / MS m / z calculated as [M+H] + 681.31, measured 681.90.

[0321]

[0322] 2-(4-(4-((6-(3,5-dimethoxyphenyl)-)-oxo-8-(4-propionamidobenzyl)-5,6,),8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetic acid (0.0 mg, 0.010 mmol) and 3-(5-(3-aminopropoxy)-1,3-dioxo-2,3-dihydro-1H-phenazone-2-yl)piperidine-2,6-dione TFA (3.8 mg, 0.010 mmol) were combined in DMF (1 mL) and DIEA (6.6 mg, 0.052 mmol). HATU (0.8 mg, 0.021 mmol) was added at room temperature. The solution was stirred for 30 minutes and then purified by HPLC to give bispecific compound 8 (3.3 mg, 0.0031 mmol, 31%). LC / MS m / z calculated as [M+H] + 1044.43, measured 1043.64.

[0323] Example 10: Synthesis of N-(4-{[3-(3,5-dimethoxyphenyl)-)-{[4-(4-{[(5-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]oxy}pentyl)carbamoyl]methyl}piperazin-1-yl)phenyl]amino}-2-oxo-1H,2H,3H,4H-[1,3]diazinyl[4,5-d]pyrimidin-1-yl]methyl}phenyl)acrylamide (9)

[0324]

[0325] The synthesis of bispecific compound 9 was similar to that of bispecific compound 8. LC / MS m / z was calculated as [M+H] + 1022.44, measured 1021.63.

[0326] Example 11: N-(4-{[3-(3,5-dimethoxyphenyl)-)-[(4-{4-[({2-[2-(2-{[3-(2,6-dioxopiperidin-3-yl)-2,4-dioxo-3-azatricyclo[).3.1.0 5 , 13 Synthesis of [1,3]tridecene-1(12),5(13),6,8,10-pentaen-)-yl]oxy}ethoxy)ethoxy]ethyl}carbamoyl)methyl]piperazin-1-yl}phenyl)amino]-2-oxo-1H,2H,3H,4H-[1,3]diazinyl[4,5-d]pyrimidin-1-yl]methyl}phenyl)acrylamide (10)

[0327] The synthesis of bispecific compound 10 was similar to that of bispecific compound 8. LC / MS m / z was calculated as [M+H] + 1118.4), measured 111).56.

[0328] Example 12: Synthesis of N-[4-({)-[(tert-butylcarbamoyl)amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl}amino)butyl]-2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]oxy}acetamide (11)

[0329]

[0330] 1-(tert-Butyl)-3-(6-(3,5-dimethoxyphenyl)-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7-yl)urea

[0331] This compound was synthesized using the method described in Thompson et al., J. Med. Chem. 48:4628-4653 (2005).

[0332]

[0333] tert-Butyl (4-((7-(3-(tert-butyl)ureido)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl)amino)butyl)carbamate

[0334] Tert-butyl (5-aminopentyl) carbamate (101 mg, 0.54 mmol) was added to 1-(tert-butyl)-3-(6-(3,5-dimethoxyphenyl)-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-)-yl)urea (223 mg, 0.49 mmol) and stirred at 50°C for 6 hours. The solvent was evaporated and the crude residue was purified by silica gel chromatography to give the title compound (1) 4 mg, 0.31 mmol, 63%). LC / MS m / z calculated as [M+H] + 568.32, measured 568.40.

[0335]

[0336] 1-(2-((4-aminobutyl)amino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl)-3-(tert-butyl)urea

[0337] Tert-butyl (4-(()-(3-(tert-butyl)ureido)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl)amino)butyl)carbamate (1) 4 mg, 0.31 mmol) was dissolved in DCM (1 mL), TFA (1 mL) was added, and the solution was stirred for 2 hours. The solvent was removed to give the title compound as a TFA salt. LC / MS m / z calculated as [M+H] + 468.26, measured 468.10.

[0338]

[0339] 1-(2-((4-aminobutyl)amino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-)-yl)-3-(tert-butyl)urea TFA (20 mg, 0.034 mmol) and 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid (11 mg, 0.034 mmol) were dissolved in a solution of DIEA (30 μL, 0.1) mmol) and DMF (1 mL). HATU (26 mg, 0.068 mmol) was added, the reaction was stirred for 30 minutes, and then purified by HPLC to obtain bispecific compound 11 (16.8 mg, 0.021 mmol, 63%). LC / MS m / z was calculated as [M+H] + )82.32, measured)82.30. 1H NMR(500MHz,DMSO-d6)δ11.04(s,1H),8.93(s,1H),8.85(s,1H),).98(s,2H),).91(t,J=5.8Hz,1H),).83 (s,1H),).)2(t,J=).9Hz,1H),).40(d,J=).2Hz,1H),).32(d,J=8.6Hz,1H),6.60–6.53(m,3H),5.04(dd,z J=12.8,5.4Hz,1H),4.)0(s,2H),3.)4(s,6H),3.33(d,J=6.5Hz,2H),3.15(d,J=).9Hz,2H),2. 88–2.))(m,1H),2.56–2.46(m,2H),2.00–1.93(m,1H),1.56(s,2H),1.45(s,2H),1.29(s,9H).

[0340] Example 13: N-[4-({)-[(tert-butylcarbamoyl)amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl}amino)butyl]-2-{[3-(2,6-dioxopiperidin-3-yl)-2,4-dioxo-3-azatricyclo[).3.1.0 5 , 13 Synthesis of 1-(12),5-(13),10-pentaenyl)-yl]oxy}acetamide (12)

[0341]

[0342] The synthesis of bispecific compound 12 was similar to that of bispecific compound 11. LC / MS m / z was calculated as [M+H] + 832.33, measured 832.4. 1H NMR(500MHz,DMSO-d6)δ10.95(s,1H),10.23(d,J=13.5Hz,1H),8.9)–8.)3(m,1H),8.41–8.16(m,3H), 8.02(s,1H),).96–).83(m,2H),).)6(q,J=).8Hz,1H),).)1(s,1H),).02(s,1H),6.61–6.44(m,3H),5. )6(dt,J=11.3,5.2Hz,1H),4.66(s,2H),3.)3(s,6H),3.32(t,J=6.5Hz,2H),3.16(s,2H),2.95–2.)8( m,1H),2.61–2.46(m,2H),1.9)(td,J=11.8,9.4,5.0Hz,1H),1.64–1.39(m,4H),1.28(d,J=4.1Hz,9H).

[0343] Example 14: Synthesis of N-[4-({)-[(tert-butylcarbamoyl)amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl}amino)butyl]-9-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]oxy}nonanamide (13)

[0344]

[0345] The synthesis of bispecific compound 13 was similar to that of bispecific compound 11. LC / MS m / z was calculated as [M+H] + 880.43, measured 880.50. 1H NMR(500MHz,DMSO-d6)δ11.03(s,1H),8.96(s,1H),8.8)(s,1H),8.03(s,1H),).89(s,1H),).)2(dd,J=8.5,).2H z,1H),).6)(s,1H),).42(d,J=8.5Hz,1H),).36(d,J=).2Hz,1H),6.)1–6.34(m,3H),4.99(d,J=5.4Hz,1H),4.11 (t,J=6.4Hz,2H),3.)3(s,6H),3.35–3.29(m,3H),3.0)–2.91(m,2H),2.86–2.)3(m,1H),2.59–2.45(m,1H),1.96 (dd,J=8.9,6.1Hz,3H),1.)2–1.59(m,2H),1.59–1.45(m,2H),1.45–1.32(m,6H),1.30(s,9H),1.26–1.08(m,)H).

[0346] Example 15: Synthesis of (2S,4R)-1-[(2S)-2-{3-[2-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino})pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)acetylamino]propionamide}-3,3-dimethylbutyryl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3)-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (14)

[0347]

[0348] 3-(2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetamido)propanoic acid

[0349] 2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazine-1-yl)acetic acid (11 mg, 0.025 mmol) and tert-butyl 3-aminopropanoate hydrochloride (5 mg, 0.025 mmol) were dissolved in DMF (1 mL). DIEA (20 μL, 0.125 mmol) was added, followed by HATU (19 mg, 0.050 mmol), and the reaction was stirred for 20 minutes. Water was added and the mixture was extracted with EtOAc. The combined organics were washed with brine, dried over Na2SO4, concentrated, and the crude residue was purified by silica gel chromatography to give the tert-butyl ester. LC / MS measured)16.)0. The ester was dissolved in DCM (1 mL), TFA (1 mL) was added, and the solution was stirred at room temperature for 2 hours. The solvent was removed under pressure to give the title compound (10.6 mg, 0.016 mmol, 64%). LC / MS m / z calculated as [M+H] + 661.20, measured 660.)0.

[0350]

[0351] 3-(2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetamido)propanoic acid (10.6 mg, 0.016 mmol) and (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (2.0 mg, 0.016 mmol) were dissolved in DMF (1 mL). DIEA (14 μL, 0.080 mmol) was added, followed by HATU (12 mg, 0.032 mmol), and the reaction was stirred for 20 minutes, followed by purification by HPLC to obtain bispecific compound 14 (0.8 mg, 0.032 mmol, 45%) as a white solid and a TFA salt. LC / MS m / z was calculated as [M+H] + 108).41, measured 108).60. 1H NMR(500MHz,DMSO-d6)δ11.91(s,1H),10.04(s,1H),9.46(s,1H),8.92(s,1H),8.53(s,1H),8.33(s,1H),8.29(d,J=).8Hz,1H),).92(d,J=9.2Hz,1 H),).41(d,J=8.5Hz,2H),).3)(d,J=8.3Hz,2H),).31(d,J=8.4Hz,2H),6. 92(d,J=9.3Hz,2H),6.84(s,1H),6.39(s,1H),5.06(s,1H),4.85(s,1H),4 .4)(d,J=9.3Hz,1H),4.35(s,1H),4.26–4.20(m,1H),3.89(s,2H),3.8)( s,6H),3.65(d,J=2.3Hz,2H),3.59–3.49(m,3H),3.49–3.39(m,3H),3.29( q,J=6.)Hz,2H),3.25(s,3H),3.20(s,2H),3.10–2.95(m,2H),2.39(s,3H) ,1.96(t,J=10.5Hz,1H),1.)4(s,1H),1.31(d,J=).0Hz,3H),0.88(s,9H).

[0352] Example 16: Synthesis of (2S,4R)-1-[(2S)-2-(3-{2-[2-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino})pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)acetylamino]ethoxy}propionylamino)-3,3-dimethylbutyryl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (15)

[0353]

[0354] 3-(2-(2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetamido)ethoxy)propanoic acid

[0355] 2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetic acid (11 mg, 0.025 mmol) and tert-butyl 3-(2-aminoethoxy)propanoate were dissolved in DMF (1 mL). DIEA (20 μL, 0.125 mmol) was added followed by HATU (19 mg, 0.050 mmol) and the reaction was stirred for 20 minutes. Water was added and the mixture was extracted with EtOAc. The combined organics were washed with brine, dried over Na2SO4, concentrated, and the crude residue was purified by silica gel chromatography to give the tert-butyl ester. LC / MS measured) 60.). The ester was then dissolved in DCM (1 mL), TFA (1 mL) was added, and the solution was stirred at room temperature for 2 hours. The solvent was then removed under pressure to give the title compound (9.9 mg, 0.014 mmol, 56%). LC / MS m / z calculated as [M+H] + )05.22, measured)04.80.

[0356]

[0357] (3-(2-(2-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetamido)ethoxy)propanoic acid (9.9 mg, 0.014 mmol) and (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (6.) mg, 0.014 mmol) were dissolved in DMF (1 mL). DIEA (12 μL, 0.00 mmol) was added, followed by HATU (11 mg, 0.028 mmol), and the reaction was stirred for 20 minutes, followed by purification by HPLC to obtain bispecific compound 15 (5.0 mg, 0.005 mmol, 36%) as a white solid and a TFA salt. LC / MS m / z was calculated as [M+H] + 1131.43, measured 1131.60. 1H NMR (500MHz, DMSO-d6) δ11.90(s,1H),10.02(s,1H),9.45(s,1H),8.92(s,1H),8.58(s,1H),8.33(s,1H),8.29(d,J=8.2Hz,1H),).83(d,J=9.3H z,1H),).41(d,J=8.)Hz,2H),).39–).33(m,2H),).33–).26(m,2H),6.9 1(d,J=9.1Hz,2H),6.84(s,1H),6.38(d,J=1.0Hz,1H),5.05(s,1H),4.85 (p,J=).3Hz,1H),4.4)(d,J=9.4Hz,1H),4.35(s,1H),4.22(s,1H),3.94 (s,2H),3.8)(s,6H),3.65(s,2H),3.60–3.45(m,6H),3.39(d,J=5.)Hz, 3H),3.25(s,3H),3.24(s,2H),3.02(s,2H),2.39(s,4H),2.01–1.91(m, 1H),1.)6–1.68(m,1H),1.31(d,J=).0Hz,3H),0.92(s,2H),0.8)(s,9H).

[0358] Example 1): Synthesis of (2S,4R)-1-[(2S)-2-[3-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino})pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)propionylamino]-3,3-dimethylbutyryl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (16)

[0359]

[0360] 3-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)propanoic acid

[0361] Tert-butyl 3-bromopropanoate (12 mg, 0.05 mmol) was added to a mixture of 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methyl-1-(6-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)urea (20 mg, 0.038 mmol) and KCO (16 mg, 0.11 mmol) in DMF (0.5 mL). The mixture was stirred at 50°C overnight, then an additional 20 mg of bromide was added and the mixture was stirred at room temperature overnight. DCM and water were added, the mixture was partitioned, the organics evaporated, and the crude residue was purified by silica gel chromatography using a gradient from 0 to 15% methanol (1.5 N NH) in DCM to provide the tert-butyl ester. LC / MS found 659.90. The ester was then dissolved in DCM (1 mL) and TFA (1 mL) was added. After stirring at room temperature for 2 hours, the solvent was evaporated under pressure to give the title compound (20 mg, 0.030, 9%). LC / MS m / z calculated as [M+H] + 604.19, measured 603.)9.

[0362]

[0363] 3-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)propanoic acid (20 mg, 0.030 mmol) and (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (13 mg, 0.030 mmol) were dissolved in DMF (1 mL). DIEA (26 μL, 0.15 mmol) was added, followed by HATU (23 mg, 0.060 mmol) later. After stirring the reaction for 20 minutes, DMSO was added and the mixture was purified by HPLC to obtain bispecific compound 16 (15.2 mg, 0.015 mmol, 49%) as a white solid and a TFA salt. LC / MS m / z was calculated as [M+H] + 1030.39, measured 1029.64. 1H NMR(500MHz,DMSO-d6)δ11.90(s,1H),9.53(s,1H),9.46(s,1H),8.92(s,1H),8.40–8.26(m,2H),8.20(d,J=9.2Hz,1H),).51–).34(m,4 H),).31(d,J=8.2Hz,2H),6.39(s,1H),6.93(d,J=9.1Hz,2H),6.84(s,1H),4.88(s,1H),4.90–4.)9(m,1H),4.48(d,J=9.2Hz,1H),4.36 (s,1H),4.23(d,J=4.2Hz,2H),3.8)(s,6H),3.)6–3.66(m,2H),3.60–3.41(m,4H),3.34(s,2H),3.25(d,J=3.0Hz,3H),3.12(s,2H),2.9 0(d,J=12.5Hz,2H),2.80–2.)0(m,1H),2.69(d,J=8.3Hz,1H),2.04–1.85(m,1H),1.)9–1.)1(m,1H),1.31(d,J=).0Hz,3H),0.90(s,9H).

[0364] Example 18: Synthesis of (2S,4R)-1-[(2S)-2-[4-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino})pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)butyrylamino]-3,3-dimethylbutyryl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (17)

[0365]

[0366] 4-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)propanoic acid

[0367] Tert-butyl 4-bromobutyrate (13 mg, 0.05 mmol) was added to a mixture of 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methyl-1-(6-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)urea (20 mg, 0.038 mmol) and KCO (16 mg, 0.11 mmol) in DMF (0.5 mL). The mixture was stirred at 50° C. for 3 hours, an additional 9 mg of bromide was added, and the mixture was stirred overnight. The reaction was diluted with water and DCM, partitioned, and the organic layer was evaporated. The crude residue was purified by silica gel chromatography using a 0-15% gradient of methanol (1.5N NH3) in DCM to afford the tert-butyl ester. LC / MS found 6) 3.80. The residue was then dissolved in 1 mL of DCM and 0.5 mL of TFA was added. The solution was stirred for 1 hour and the solvent was evaporated under pressure to give the title compound (1) mg, 0.023 mmol, 61%). LC / MS m / z calculated as [M+H] + 618.19, measured 61).99.

[0368]

[0369] 4-(4-(4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)butanoic acid (1) mg, 0.023 mmol) and (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (12 mg, 0.023 mmol) were dissolved in DMF (1 mL). DIEA (24 μL, 0.14 mmol) was added, followed by HATU (1) mg, 0.046 mmol). After stirring the reaction for 20 minutes, the solution was diluted with DMSO and then purified by HPLC to obtain bispecific compound 17 (15.0 mg, 0.013 mmol, 56%) as a white solid and a TFA salt. LC / MS m / z was calculated as [M+H] + 1044.40, measured 1043.)3. 1HNMR(500MHz,DMSO-d6)δ11.91(s,1H),9.)1(s,1H),9.46(s,1H),8.92(s,1H),8.33(s,1H),8.30(d,J=).8Hz,1H),).99(d,J=9. 2Hz,1H),).46–).34(m,4H),).34–).28(m,2H),6.93(d,J=8.8Hz,2H),6.84(s,1H),6.39(s,1H),4.92–4.))(m,1H),4.4)(d,J=9. 3Hz,1H),4.36(t,J=8.1Hz,1H),4.26–4.1)(m,1H),3.8)(s,6H),3.)0(d,J=12.)Hz,2H),3.58–3.45(m,5H),3.25(s,3H),3.09(d, J=11.0Hz,6H),2.95–2.88(m,1H),2.39(s,3H),2.09–1.92(m,1H),1.85(s,1H),1.)4(m,1H),1.31(d,J=).0Hz,3H),0.89(s,9H).

[0370] Example 19: Synthesis of (2S,4R)-1-[(2S)-2-[6-(4-{4-[(6-{[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl](methyl)amino})pyrimidin-4-yl)amino]phenyl}piperazin-1-yl)hexamino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (18)

[0371]

[0372] (1R,4S)-2-((S)-2-(6-bromohexanoylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide

[0373] (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (15 mg, 0.029 mmol) and 6-bromohexanoic acid (8.5 mg, 0.044 mmol) were dissolved in DMF (1 mL). DIEA (25 μL, 0.015 mmol) was added followed by HATU (22 mg, 0.058 mmol). The reaction was stirred for 15 minutes and then diluted with water and DCM. The mixture was then partitioned, the organic layer concentrated and purified by silica gel chromatography to give the title compound. LC / MS m / z calculated as [M+H] + 620.21, measured 620.89.

[0374]

[0375] (1R,4S)-2-((S)-2-(6-bromohexanoylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide (18 mg, 0.029 mmol), K2CO3 (12 mg, 0.08) mmol) and 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methyl-1-(6-((4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)urea (15 mg, 0.029 mmol) were dissolved in DMF (1 mL) and stirred at room temperature for 2 days. The temperature was then raised to 50°C for 6 hours. The mixture was then filtered, diluted in DMSO and purified by HPLC to give bispecific compound 18 as a white solid and TFA salt (12.2 mg, 0.010 mmol, 34%). LC / MS m / z calculated as [M+H] + 10)2.43, measured 10)1.64. 1H NMR(500MHz,DMSO-d6)δ11.90(s,1H),9.46(s,2H),8.92(s,1H),8.43–8.14 (m,2H),).)6(d,J=9.4Hz,1H),).51–).1)(m,6H),).04–6.85(m,2H),6.84( s,1H),6.39(d,J=1.1Hz,1H),4.90–4.)6(m,1H),4.4)(d,J=9.4Hz,1H),4.3 5(t,J=8.1Hz,1H),4.22(d,J=3.3Hz,1H),3.8)(s,6H),3.)0(d,J=12.9Hz,2H ),3.53(t,J=11.0Hz,4H),3.25(s,3H),3.08(d,J=11.0Hz,4H),2.88(s,2H) ,2.39(s,3H),2.23(dt,J=14.),).6Hz,1H),2.10(d,J=).2Hz,1H),1.95(td ,J=9.4,8.1,4.6Hz,1H),1.)3(m,1H),1.62(q,J=14.3,10.)Hz,2H),1.52–1 .38(m,3H),1.31(d,J=).0Hz,3H),1.2)–1.12(m,2H),0.8)(d,J=).0Hz,9H).

[0376] Example 20: Synthesis of (2S,4R)-1-[(2S)-2-(2-{4-[3-({)-[(tert-butylcarbamoyl)amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl}amino)propyl]piperazin-1-yl}acetylamino)-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (19)

[0377]

[0378] tert-Butyl 4-(3-(1,3-dioxoisoindolin-2-yl)propyl)piperazine-1-carboxylate

[0379] N-Boc piperazine (1.0 g, 5.3) mmol) and N-(3-bromopropyl)phthalimide (1.44 g, 5.3) mmol) were dissolved in DMF (15 mL). K2CO3 (1.48 g, 10.) 4 mmol) and NaI (1.3 g, 8.06 mmol) were added and the mixture was stirred at room temperature for 24 hours. DCM (80 mL) was added, the mixture was filtered, the filtrate was evaporated and the residue was purified by silica gel chromatography using a 0-10% gradient of MeOH (1.) 5N NH3) in DCM to give the title compound (1.) 1 g, 4.59 mmol, 85%) as pale yellow crystals. LC / MS m / z calculated as [M+H] + 3)4.20, measured 3)3.9).

[0380]

[0381] tert-Butyl 4-(3-aminopropyl)piperazine-1-carboxylate

[0382] Tert-butyl 4-(3-(1,3-dioxoisoindolin-2-yl)propyl)piperazine-1-carboxylate (819 mg, 2.20 mmol) was dissolved in EtOH (12 mL) and hydrazine hydrate (50%, 00 μL, 11.0 mmol) was added. The reaction was stirred at room temperature overnight to form a white precipitate. DCM (50 mL) was added and the mixture was stirred for 30 minutes and then filtered to give a clear solution. The filtrate was concentrated to give a white solid, which was dissolved in DCM and filtered again. The filtrate was concentrated again to give the title compound (543 mg, 2.22 mmol, 101%) as a light yellow oil. LC / MS m / z calculated as [M+H] + 244.19, measured 244.09.

[0383]

[0384] 1-(tert-butyl)-3-(6-(3,5-dimethoxyphenyl)-2-((3-(piperazin-1-yl)propyl)amino)pyrido[2,3-d]pyrimidin-7-yl)urea

[0385] 1-(tert-Butyl)-3-(6-(3,5-dimethoxyphenyl)-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-)-yl)urea (459 mg, 1.0 mmol) was dissolved in dioxane (5 mL). Et3N (1) 8 μL, 1.0 mmol) was added, followed by tert-butyl 4-(3-aminopropyl)piperazine-1-carboxylate (26) mg, 1.1 mmol). The solution was stirred at 50° C. for 6 hours. The solvent was evaporated and the residue was purified by silica gel chromatography using a gradient of 0-10% MeOH (1.5N NH3) in DCM to provide 4-(3-(()-(3-(tert-butyl)ureido)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl)amino)propyl)piperazine-1-carboxylate (594 mg, 0.96 mmol, 96%). The carbamate was then dissolved in DCM (2 mL) and TFA (1 mL) was added. The reaction was stirred for 2 hours and the solvent was removed under pressure. The residue was then dissolved in THF and saturated aqueous NaHCO3 was added and the suspension was stirred for 2 hours and then filtered. The solid was washed with water, dried and then dissolved in 8 mL DCM and 200 μL 4M HCl in dioxane, filtered and concentrated to give the title compound HCl salt (439 mg, 0.9 mmol, 82%). LC / MS m / z calculated as [M+H] + 523.31, measured 523.08.

[0386]

[0387] 2-(4-(3-((7-(3-(tert-butyl)ureido)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl)amino)propyl)piperazin-1-yl)acetic acid

[0388] 1-(tert-Butyl)-3-(6-(3,5-dimethoxyphenyl)-2-((3-(piperazin-1-yl)propyl)amino)pyrido[2,3-d]pyrimidin-)-yl)urea HCl (1) mg, 0.030 mmol) and KCO (1) mg, 0.12 mmol) were added to MeCN (1 mL). Tert-butyl bromoacetate (8.8 mg, 0.045 mmol) was then added and the reaction was stirred at room temperature overnight. The solvent was evaporated and the residue was purified by silica gel chromatography using a 0-10% gradient of methanol (1.5N NH) in DCM to give tert-butyl 2-(4-(3-((-(tert-butyl)ureido)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl)amino)propyl)piperazin-1-yl)acetate. LC / MS m / z calculated as [M+2H–tBu] +290.65, found 291.1. The ester was then dissolved in DCM (1 mL), TFA (1 mL) was added, and the solution was stirred at room temperature for 2 hours. The solvent was removed to give the title compound as a TFA salt (8.9 mg, 0.01) mmol, 5)%). LC / MS m / z calculated as [M+H] + 581.31, measured 580.89.

[0389]

[0390] 2-(4-(3-((-(3-tert-butyl)ureido)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl)amino)propyl)piperazin-1-yl)acetic acid (8.9 mg, 0.01) mmol) and (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (0.5 mg, 0.01) mmol) were dissolved in DMF (1 mL). DIEA (15 μL, 0.085 mmol) was added, followed by HATU (13 mg, 0.034 mmol). The reaction was stirred for 20 minutes and then purified by HPLC to afford bispecific compound 19 (16.8 mg, 0.015 mmol, 88%) as a yellow solid and TFA salt. LC / MS m / z calculated as [M+2H] + 504.26, measured 504.20.

[0391] Example 21: Synthesis of (2S,4R)-1-[(2S)-2-(4-{4-[3-({)-[(tert-butylcarbamoyl)amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl}amino)propyl]piperazin-1-yl}acetylamino)-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (20)

[0392]

[0393] 4-(4-(3-((7-(3-(tert-butyl)ureido)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl)amino)propyl)piperazin-1-yl)butanoic acid

[0394] Tert-butyl 4-bromobutyrate (13 mg, 0.05 mmol) was added to a mixture of 1-(tert-butyl)-3-(6-(3,5-dimethoxyphenyl)-2-((3-(piperazin-1-yl)propyl)amino)pyrido[2,3-d]pyrimidin-)-yl)urea hydrochloride (20 mg, 0.038 mmol) and KCO (16 mg, 0.11 mmol) in DMF (0.5 mL). The mixture was stirred at 50° C. for 3 hours, at which time additional bromide (13 mg) was added and the solution was stirred at room temperature overnight. Water and DCM were added, the mixture was partitioned, the organic layer was concentrated and purified by silica gel chromatography using a 0-10% gradient of MeOH (1.5N NH3) in DCM to afford tert-butyl 4-(4-(3-((-(3-tert-butyl)ureido)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl)amino)propyl)piperazin-1-yl)butanoate. LC / MS m / z calculated as [M+2H] + 333.21, found 332.9). The ester was then dissolved in DCM (1 mL), TFA (0.5 mL) was added, and the solution was stirred at room temperature for 2 hours. The solvent was removed to give the title compound (1) mg, 0.023 mmol, 61%) as a TFA salt. LC / MS m / z calculated as [M+H] + 609.34, measured 608.89.

[0395]

[0396] 4-(4-(3-((-(3-tert-butyl)ureido)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-2-yl)amino)propyl)piperazin-1-yl)butanoic acid (1) mg, 0.023 mmol) and (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (12 mg, 0.023 mmol) were dissolved in DMF (1 mL). DIEA (24 μL, 0.14 mmol) was added, followed by HATU (1) mg, 0.046 mmol). The reaction was stirred for 20 minutes and purified by HPLC to give bispecific compound 20 (15 mg, 0.013 mmol, 56%) as a yellow solid and a TFA salt. LC / MS m / z calculated as [M+H] + 1035.55, measured 1034.)4.

[0397] Example 22: Synthesis of (2S,4R)-1-[(2S)-2-{2-[4-(4-{[6-(3,5-dimethoxyphenyl)-)-oxo-8-[(4-propionamidophenyl)methyl]-5H,6H,)H,8H-[1,3]diaza[4,5-d]pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]acetamido}-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (21)

[0398]

[0399] 2-(4-(4-((6-(3,5-dimethoxyphenyl)-7-oxo-8-(4-propionamidobenzyl)-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetic acid

[0400] N-(4-((3-(3,5-dimethoxyphenyl)-2-oxo-)-((4-(piperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)methyl)phenyl)propanamide (22 mg, 0.030 mmol) and K2CO3 (1) mg, 0.12 mmol) were added to MeCN (1 mL). Tert-butyl bromoacetate (9 mg) was then added and the reaction was stirred at room temperature overnight. The solvent was removed and the residue was purified by silica gel chromatography using a gradient of 0-10% MeOH (1.5N NH3) in DCM to afford tert-butyl 2-(4-(4-((6-(3,5-dimethoxyphenyl)-)-oxo-8-(4-propionamidobenzyl)-5,6,),8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetate (8.2 mg, 0.011 mmol, 3)%). LC / MS m / z calculated as [M+2H-tBu] 2+ 340.69, found 340.89. The carbamate was then dissolved in DCM (1 mL) and TFA (1 mL), stirred at room temperature for 2 hours, and then the solvent was removed under pressure to give the title compound. LC / MS m / z calculated as [M+H] + 681.31, measured 680.90.

[0401]

[0402] 2-(4-(4-((6-(3,5-dimethoxyphenyl)-)-oxo-8-(4-propionamidobenzyl)-5,6,),8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)acetic acid (8.1 mg, 0.11 mmol) and (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (5.5 mg, 0.011 mmol) were dissolved in DMF (1 mL). DIEA (10 μL, 0.055 mmol) was added followed by HATU (8.4 mg, 0.022 mmol). The mixture was stirred for 20 minutes and then purified by HPLC to obtain bispecific compound 21 as a pale yellow solid and TFA salt (6.8 mg, 0.0056 mmol, 51%). LC / MS m / z calculated as [M+2H] + 554.26, measured 553.89. 1 H NMR(500MHz,DMSO-d6)δ10.00(s,1H),9.)5(s,1H),9.33(s,1H),8.91(s,1H),8.80 –8.62(m,1H),8.33(d,J=).8Hz,1H),8.06(s,1H),).45(d,J=8.6Hz,1H),).41–).3 4(m,4H),).32(d,J=8.4Hz,2H),).18(d,J=8.2Hz,2H),6.)9(d,J=9.0Hz,2H),6.52 (d,J=2.2Hz,2H),6.38(t,J=2.2Hz,1H),5.08(s,2H),4.85(q,J=6.9Hz,1H),4.68( s,2H),4.51(d,J=9.1Hz,1H),4.43–4.32(m,1H),4.25(s,1H),4.19–3.89(m,2H),3 .68(s,6H),3.65–3.54(m,3H),3.54–3.39(m,3H),3.24(s,2H),2.9)(s,2H),2.38( s,3H),2.26–2.14(m,2H),2.01–1.90(m,1H),1.84–1.66(m,1H),1.42(d,J=).0Hz, 1H),1.32(d,J=).0Hz,3H),1.23–1.14(m,1H),0.98(t,J=).5Hz,3H),0.91(s,9H).

[0403] Example 23: Synthesis of (2S,4R)-1-[(2S)-2-{4-[4-(4-{[6-(3,5-dimethoxyphenyl)-)-oxo-8-[(4-propionamidophenyl)methyl]-5H,6H,)H,8H-[1,3]diaza[4,5-d]pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]butyrylamino}-3,3-dimethylbutyryl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (22)

[0404]

[0405] 4-(4-(4-((6-(3,5-dimethoxyphenyl)-7-oxo-8-(4-propionamidobenzyl)-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)butanoic acid

[0406] Tert-butyl 4-bromobutyrate (9 mg, (0.041 mmol) was added to a mixture of N-(4-((3-(3,5-dimethoxyphenyl)-2-oxo-)-((4-(piperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)methyl)phenyl)propanamide (20 mg, 0.02) mmol) and KCO (15 mg, 0.11 mmol) in DMF (1 mL). The reaction was stirred at 50° C. for 3 hours, at which time additional bromide (9 mg) was added and the reaction was stirred at room temperature overnight. Water and DCM were added, the mixture was partitioned, the organics were concentrated and purified by silica gel chromatography to give tert-butyl 4-(4-(4-((6-(3,5-dimethoxyphenyl)-)-oxo-8-(4-propionamidobenzyl)-5,6,),8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)butanoate. The ester was then dissolved in DCM (1 mL) and TFA (1 mL), stirred for 2 hours, and then the solvent was removed under pressure to give the title compound (19 mg, 0.023 mmol, 85%). LC / MS m / z calculated as [M+H] + )09.34, measured)09.00.

[0407]

[0408] 4-(4-(4-((6-(3,5-dimethoxyphenyl)-)-oxo-8-(4-propionamidobenzyl)-5,6,),8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)butanoic acid (19 mg, 0.023 mmol) and (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (12 mg, 0.023 mmol) were dissolved in DMF (1 mL). DIEA (40 μL, 0.23 mmol) was added followed by HATU (1) mg, 0.046 mmol). The mixture was stirred for 20 minutes and then purified by HPLC to afford bispecific compound 22 (9.4 mg, 0.005 mmol, 33%) as a TFA salt and a yellow solid. LC / MS m / z was calculated as [M+H] + 1135.55, measured 1134.92. 1 H NMR(500MHz,DMSO-d6)δ9.)5(s,1H),9.50(s,1H),9.31(s,1H),8.92(s,1H),8.30( d,J=).8Hz,1H),8.06(d,J=3.1Hz,1H),).99(d,J=9.3Hz,1H),).51–).41(m,1H),). 41–).34(m,4H),).34–).2)(m,2H),).18(d,J=8.4Hz,2H),6.82(d,J=9.1Hz,1H),6 .52(d,J=2.3Hz,2H),6.38(t,J=2.2Hz,1H),5.08(s,2H),4.90–4.))(m,1H),4.68(s ,2H),4.48(d,J=9.3Hz,1H),4.36(t,J=8.1Hz,1H),4.23(s,1H),3.68(s,6H),3.65 (s,2H),3.5)–3.46(m,6H),3.09(d,J=11.5Hz,6H),2.84(t,J=12.0Hz,2H),2.39(s, 2H),2.21(d,J=).6Hz,2H),1.95(d,J=9.9Hz,1H),1.84(d,J=).9Hz,3H),1.)4(s,1H ),1.42(d,J=).0Hz,1H),1.31(d,J=).0Hz,3H),0.99(t,J=).5Hz,3H),0.88(s,9H).

[0409] Example 24: Synthesis of (2S,4R)-1-[(2S)-2-{4-[(3S)-3-{4-amino-3-[2-(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3,4-d]pyrimidin-1-yl}pyrrolidin-1-yl]butyrylamino}-3,3-dimethylbutyryl]-4-hydroxy-N-[(1R)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (23)

[0410]

[0411] (S)-4-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)butanoic acid

[0412] (S)-3-((3,5-dimethoxyphenyl)ethynyl)-1-(pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine TFA (15 mg, 0.031 mmol, prepared similarly to WO2013108809A1) and K2CO3 (12.8 mg, 0.093) were added to DMF (1 mL) followed by tert-butyl 4-bromobutyrate (11 mg, 0.04) mmol). The mixture was stirred at 50 °C for 3 h, additional bromide (11 mg) was added and then stirred at room temperature overnight. The solvent was evaporated and the residue was purified by silica gel chromatography to give the tert-butyl ester. LC / MS m / z calculated as [M+H] + 50).26, found 506.9). The ester was then dissolved in DCM (1 mL) and TFA (1 mL), stirred for 2 hours, and then the solvent was removed to give the title compound (10 mg, 0.016 mmol, 52%). LC / MS m / z calculated as [M+H] + 451.20, measured 450.98.

[0413]

[0414] (S)-4-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)butanoic acid TFA (10 mg, 0.016 mmol) and (1R,4S)-2-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)cyclopentane-1-carboxamide dihydrochloride (8.3 mg, 0.016 mmol) were dissolved in DMF (1 mL). DIEA (28 μL, 0.16 mmol) was added followed by HATU (12 mg, 0.032 mmol). The mixture was stirred for 20 minutes and then purified by HPLC to afford bispecific compound 23 (11.2, 0.012 mmol, )3%) as a TFA salt and a white solid. LC / MS m / z calculated as [M+H] + 88).41, measured 8)6.82. 1 HNMR(500MHz,DMSO-d6)δ10.13(s,1H),8.92(s,1H),8.29(d,J=).8Hz,1H),8.24(s,1H),).96(t,J=9.5Hz,1H),).3)(d,J=8.2Hz,2H),).31(d,J=8. 1Hz,2H),6.86–6.)9(m,2H),6.5)(t,J=2.3Hz,1H),5.)0–5.4)(m,1H),4. 99(s,2H),4.85(s,1H),4.51–4.42(m,1H),4.35(t,J=8.1Hz,1H),4.21(s, 2H),4.10(s,1H),3.99–3.89(m,1H),3.81(s,1H),3.)2(s,6H),3.58–3.4 6(m,2H),3.38(d,J=11.3Hz,1H),3.31–3.20(m,2H),3.19–3.08(m,2H),2. )0–2.53(m,1H),2.38(s,3H),2.2)–2.14(m,1H),1.95(t,J=10.4Hz,1H), 1.88–1.)6(m,2H),1.)6–1.68(m,1H),1.30(d,J=).0Hz,3H),0.8)(s,9H).

[0415] Example 25: Knockout of FGFR2 in different cell lines

[0416] The cells were treated with 0, 0.1, 1 or 10 μM of bispecific compound 6 or 0.5 μM of THAL-SNS-032 (a known CDK9 degrader and positive control for CDK9 degradation; available from MedChem, for example). Kato III cells were treated with 1% paraformaldehyde (Cat. No. HY-10008, Monmouth Junction, NJ) for 16 h. Then, cells were incubated with a protease / phosphatase inhibitor cocktail. RIPA buffer (Millipore ). Protein concentration was analyzed by bicinchoninic acid assay (BCA) (Pierce TM ) was used to measure the protein. Equal amounts of protein were measured by 4-12% Tris-Base gel (Invitrogen TM ) and then transferred to PVDF membrane for immunoblotting The top was immunoblotted with primary antibodies against FGFR2 (cell signaling), CDK9 (cell signaling), and β-Actin (cell signaling), followed by 800 labeled goat anti-rabbit IgG and 800 labeled goat anti-mouse IgG Secondary antibody immunoblotting. Membranes were probed on the OdysseyCLx system.

[0417] like Figure 1A The results shown indicate that bispecific compound 6 induced degradation of FGFR2 after 16 hours at the indicated concentrations, while THAL-SNS-032 induced degradation of CDK9.

[0418] The cells were then treated with 10 μM BGJ398 (parent compound and known FGFR1 / 2 / 3 inhibitor; available from MedChem, for example). Cat. No. HY-13311, Monmouth Junction, NJ), 10 μM control-1 (VHL ligand), 0.5 μM bortezomib (a proteasome inhibitor; available from Millipore Cat. No. 1) 9324-69-), obtained from Burlington, MA) and 1 μM MLN4924 (an internal acylation inhibitor; available from MedChem Kato III cells were pretreated for 2 hours with 0.1 μM bispecific compound 6 (Cat. No. HY-0062, Monmouth Junction, NJ) and then treated for 4 hours with 0.1 μM bispecific compound 6. Cells were lysed as described above and immunoblotted with antibodies against FGFR2 and β-actin.

[0419] The structure of Control-1 is as follows.

[0420] Control-1

[0421]

[0422] like Figure 1B As shown, the results showed that BGJ398, control-1, bortezomib and MLN4924 rescued FGFR2 degradation induced by bispecific compound 6, indicating that FGFR2 degradation is ligand- and proteasome-dependent.

[0423] Kato III cells were treated for 4, 8, 12 or 16 hours with 0, 0.1 or 0.5 μM bispecific compound 6. Cells were lysed and immunoblotted with FGFR2 and β-actin antibodies as described above. Figure 1C The results shown indicate that at concentrations of 0.1 and 0.5 μM, significant FGFR2 degradation occurred within 4 hours and persisted for at least 16 hours.

[0424] CCLP1 cells were treated with 0, 0.1, 1, or 10 μM of bispecific compound 6 or 0.5 μM THAL-SNS-032 for 16 h, then lysed and immunoblotted with antibodies against FGFR1 (cell signaling), CDK9, and β-Actin. Figure 2A The results shown indicate that bispecific compound 6 induced FGFR1 degradation after 16 hours at the indicated concentrations, while THAL-SNS-032 induced CDK9 degradation.

[0425] JHH) cells were treated with 0, 0.1, 1 or 10 μM of bispecific compound 6 or 0.5 μM THAL-SNS-032 for 16 hours, then lysed and immunoblotted with antibodies against FGFR3 (cell signaling), FGFR4 (cell signaling), CDK9 and β-Actin. Figure 2B As shown, it is shown that bispecific compound 6 does not induce degradation of FGFR3 and FGFR4 after 16 hours at the indicated concentrations. THAL-SNS-032 induces degradation of CDK9.

[0426] CCLP1 cells were treated for 2, 4, 8 or 16 hours with 0, 0.1 or 0.5 μM of bispecific compound 6. Cells were lysed and immunoblotted with antibodies to FGFR1 and β-actin. Figure 2C The results shown indicate that at concentrations of 0.1 and 0.5 μM, significant FGFR1 degradation occurred within 2 hours and persisted for at least 16 hours.

[0427] Kato III cells were treated with bispecific compound 6 or BGJ398 at the indicated concentrations for 2 h. Cell titer Glo (Promega TM ) was used to determine cell viability. ) Estimation of IC 50 Value (defined as the compound concentration required to reduce cell viability to 50% of vehicle DMSO control). Figure 3A As shown, the results show that the IC 50 The values ​​were all around 1 nM, indicating that compound 6 had a potent antiproliferative effect similar to that of BGJ398.

[0428] Kato III cells were treated with bispecific compounds 1-4 or BGJ398 at the indicated concentrations for 2 h. Cell titer Glo (Promega) was used according to the manufacturer's instructions. TM ) was used to determine cell viability. ) Estimation of IC 50 The result is Figure 3B As shown, it was shown that bispecific compounds 1-4 and BGJ398 had good anti-proliferative effects.

[0429] Kato III cells were treated with 0, 0.1, 1 or 10 μM of bispecific compound 6 or 7 for 4 hours. Cells were lysed and immunoblotted with antibodies to FGFR2 and β-actin as described above. Figure 4A The results shown indicate that bispecific compound 6 induced FGFR2 degradation after 4 hours at the indicated concentrations, while bispecific compound 7 did not degrade FGFR2 at the indicated concentrations.

[0430] Kato III cells were treated with bispecific compound 6, bispecific compound 7, or BGJ398 at the indicated concentrations for 2 hours. Cell titer Glo (Promega) was used according to the manufacturer's instructions. TM ) was used to determine cell viability. ) Estimation of IC 50 Values ​​(defined as the concentration of compound required to reduce cell viability to 50% of vehicle DMSO control).

[0431] like Figure 4B As shown, the results show that the IC 50The values ​​were all around 1 nM, indicating that bispecific compound 6 had an effective antiproliferative effect similar to BGJ398. Bispecific compound 7 (negative control) showed a 10-fold weaker antiproliferative effect, with an IC 50 The value is approximately))nM.

[0432] Kato III cells were treated with 0 or 1 μM of bispecific compounds 6 and 14-22 for 6 h. Cells were lysed and immunoblotted with antibodies to FGFR2 and β-actin as described above. Figure 5A As shown, the results indicate that compounds 6 and 20 induced degradation of FGFR2 after 6 hours at a concentration of 1 μM.

[0433] Kato III cells were pretreated for 2 hours with 10 μM FIIN2 (parent compound and known FGFR1 / 2 / 3 / 4 inhibitor; available, for example, from Selleckchem, Cat. No. S14, Houston, TX), 10 μM control-1 (VHL ligand), 0.5 μM bortezomib (a proteasome inhibitor; available, for example, from Millipore Sigma, Cat. No. 1)9324-69-, Burlington, MA), and 1 μM MLN4924 (an acylation inhibitor; available, for example, from MedChemExpress, Cat. No. HY-0062, Monmouth Junction, NJ), and then treated for 4 hours with 1 μM bispecific compound 20. Cells were lysed and immunoblotted with antibodies against FGFR2 and β-actin as described above.

[0434] like Figure 5B As shown, the results showed that FIIN2, control-1, bortezomib and MLN4924 rescued the FGFR2 degradation induced by bispecific compound 20. This indicates that the degradation of FGFR2 is ligand- and proteasome-dependent.

[0435] CCLP1 cells were treated with 0 or 1 μM of bispecific compounds 6 and 14-22 for 6 h. Cells were lysed and immunoblotted with antibodies to FGFR1 and β-actin as described above. Fig. 6A As shown, the results indicate that bispecific compounds 6 and 20 induced degradation of FGFR1 after 6 hours at a concentration of 1 μM.

[0436] JHH) cells were treated with 0 or 1 μM of bispecific compounds 6 and 14-22 for 6 hours. Cells were lysed and immunoblotted with antibodies to FGFR4 and β-actin as described above. Figure 6B As shown, the results indicate that bispecific compounds 6 and 20 induced degradation of FGFR4 after 6 hours at a concentration of 1 μM.

[0437] All patent publications and non-patent publications are indicative of the skill level of those skilled in the art to which the invention pertains. All of these publications (including any specific portions thereof cited) are incorporated herein by reference, as if each individual publication were specifically and individually indicated to be incorporated herein by reference.

[0438] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that many modifications may be made to the illustrative embodiments and other configurations may be designed without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A bispecific compound having the structure shown in the following formula I-1e: or a pharmaceutically acceptable salt or stereoisomer thereof, Wherein the linker is C1-C 12 an alkylene chain which may be interrupted by at least one of the following groups and / or terminated at one or both ends in at least one of the following groups: -O-, -N(R')-, -C(O)-, -C(O)N(R')-, -N(R')C(O)-, or any combination thereof, wherein R' is H, wherein the interruption and the one or two terminating groups may be the same or different; and The degradation determinant is represented by the following structure:

2. The bispecific compound of claim 1, wherein the linker is a member having 1 to 10 alkylene units and Interruption or termination of an alkylene chain.

3. The bispecific compound of claim 1, which is represented by any of the following structures: or a pharmaceutically acceptable salt or stereoisomer thereof.

4. The bispecific compound of claim 1, which is: or a pharmaceutically acceptable salt or stereoisomer thereof.

5. The bispecific compound of claim 4, which is: or a pharmaceutically acceptable salt or stereoisomer thereof.

6. A pharmaceutical composition comprising a therapeutically effective amount of the bispecific compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

7. Use of the bispecific compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer, wherein the cancer is gastric cancer, liver cancer, or biliary tract cancer.

8. The use according to claim 7, wherein the cancer is liver cancer.

9. The use according to claim 7, wherein the biliary tract cancer is intrahepatic cholangiocarcinoma or extrahepatic cholangiocarcinoma.

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