Fibroblast Activation Protein (FAP)-Targeted Imaging and Therapy in Cancer and Other Fibrotic and Inflammatory Diseases

Abstract

Fibroblast activation protein (FAP) targeting compounds (eg, conjugates); methods for imaging cancer or fibrosis; and methods for treating inflammatory diseases / disorders and cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 901,792, filed on September 17, 2019, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to certain chemical compounds and groups, such as the development of ligands targeting fibroblast activation protein (fibroblast activation protein, FAP-α). In some cases, the group is connected to a drug or imaging agent. In some cases, such a connected compound (also referred to herein as a "conjugate") is used in a variety of methods, such as treatment and / or imaging of (e.g., FAP positive) cancer, fibrotic diseases and / or inflammatory conditions. In some cases, specifically targeted (e.g., in treatment and / or imaging methods) are cancer-associated fibroblasts (cancer-associatedfibroblast, CAF) and / or activated myofibroblasts (e.g., in cancer and / or other fibrotic and inflammatory diseases). In some cases, the chemical compounds and / or ligands provided herein have good or improved internalization and residence time at tumors and other disease sites. Background Art

[0004] Radiation therapy and chemotherapy are considered for a variety of cancers, fibrotic disorders, and inflammatory diseases. Typically, such treatments are not considered first-line treatments because of the adverse (e.g., systemic) effects that can occur.

[0005] As a result, there is a need for targeted therapies to treat diseases (e.g., cancer, fibrotic diseases, and / or inflammatory diseases) using radiotherapeutic and / or chemotherapeutic agents suitable for targeting diseased cells and tissues with minimal or reduced off-target or systemic effects.

[0006] In some cases, the survival and proliferation of certain tumors depends on the percentage of tumor stroma (TSP). High TSP can be associated with poor long-term patient survival compared to low TSP (>50% vs. ≤50%, respectively). TSP can also be an important prognostic factor for tumor recurrence, growth, and metastasis.

[0007] In some cases, cancer associated fibroblasts (CAF) are abundant in tumor stroma and perform several important functions to promote tumorigenesis. As non-limiting examples, these functions include cytokine secretion and / or extracellular matrix (extracellular matrix, ECM) production and remodeling. In some cases, such effects lead to angiogenesis to promote tumor growth, signal transduction factors to improve chemo-resistance, denser ECM to produce immunosuppressive environment, and enhanced cell movement to directly transfer. In some cases, such processes are similar to the behavior of pathogenic fibroblasts in fibrotic diseases.

[0008] In some cases, the universal marker of CAF is fibroblast activation protein alpha (FAPα). In addition, FAPα is a serine protease on the cell surface of activated fibroblasts (mainly) present in diseased cells and tissues (e.g., diseased cells and tissues in fibrotic diseases, inflammatory diseases and / or cancers (e.g., fibrosis, rheumatoid arthritis, wound healing and cancer)). More than 90% of epithelial cancers show FAPα expression in immunohistochemistry (IHC) staining. Additional FAPα expression was found in subsets of tumor-associated macrophages (TAM) and primary glioma cell cultures. However, the expression of FAPα in most adult tissues is very low or absent. Therefore, since expression is confined to the surface of diseased cells (e.g., cancer), FAPα is identified as a unique receptor for selectively delivering drug therapeutics to tumors through ligand targeting. SUMMARY OF THE INVENTION

[0010] Provided are compounds (eg, conjugates) of Formula X:

[0011] A m -LB (X)

[0012] in:

[0013] A is a fibroblast activation protein alpha (FAPα) ligand (targeting moiety) group (e.g., molecular weight less than 10,000);

[0014] L is a (e.g., bifunctionalized) linker that connects one or more A groups to B (e.g., via a first covalent bond connecting L to A and a second covalent bond connecting L to B);

[0015] B is an imaging agent, a photodynamic therapy agent, a radioimaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an anti-fibrotic agent, or an anti-cancer agent (e.g., an anti-cancer agent effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors) (e.g., a group thereof); and

[0016] m is 1 to 6.

[0017] Also provided are compounds (eg, conjugates) of Formula I:

[0018] ALB (I)

[0019] in:

[0020] A comprises a FAPα ligand (eg, a targeting moiety) (eg, a group thereof);

[0021] L comprises a (e.g. bifunctionalized) linker connecting one or more A groups to B; and

[0022] B comprises an optical imaging agent, a photodynamic therapy agent, a radioimaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an anti-fibrotic agent, or an anti-cancer agent (e.g., an anti-cancer agent effective against cancer cells or cancer-associated fibroblasts, myofibroblasts or other tumor microenvironment factors) (e.g., a group thereof).

[0023] Also provided are methods for imaging cancer or fibrosis in a subject having cancer or fibrosis, the method comprising administering to a subject in need thereof an effective amount of a compound.

[0024] Also provided are methods for treating an inflammatory disease or condition, the method comprising administering to a subject suffering from the inflammatory disease or condition a therapeutically effective amount of a compound.

[0025] There is even provided a method for treating cancer comprising administering to a subject suffering from cancer a therapeutically effective amount of a compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Attached Figure 1 The various embodiments discussed in this document are presented generally by way of example and not by way of limitation.

[0028] Figure 1 Shown is the retrosynthesis of a ligand targeting fibroblast activation protein (FAP).

[0029] Figure 2 Shown are the structures of targeting compounds with targeting ligands.

[0030] Figure 3 Shown are increased binding of increasing concentrations (eg, 50 nM (A), 25 nM (B), 12.5 nM (C), and 6.25 nM (D)) of targeting ligand to fibroblasts with high concentrations of fibroblast activation protein (FAP).

[0031] Figure 4Binding of targeting ligands at a single concentration to fibroblasts with high surface concentrations of FAP is shown after 1 hour (A), 8 hours (B), 24 hours (C), and 48 hours (D) of incubation.

[0032] Figure 5 Binding of targeting ligand to fibroblasts with high surface concentrations of FAP is shown for 1 hour at at least 100-fold excess of competing ligand (eg, A: 25 nM targeting ligand, 2.5 μM competitor; B: 25 nM targeting ligand, 5 μM competitor).

[0033] Figure 6 Binding of targeting ligands to similar fibroblasts without high surface concentrations of FAP is shown (eg, at 100 nM (A) and at 200 nM (B)).

[0034] Figure 7 Binding curves of targeting ligands to fibroblasts with high surface concentrations of FAP (or FAP fibroblasts) are shown.

[0035] Figure 8 Binding curves of targeting ligands to FAP fibroblasts, targeting ligand only (circles) and targeting ligand and competitor (squares), and FAP fibroblasts (triangles) are shown.

[0036] Fig. 9A Imaging results showing in vivo tumor-specific targeting of the targeting ligand 2 to 32 hours after administration to mammals bearing tumors having a FAP-rich environment are shown.

[0037] Fig. 9B Imaging results showing in vivo tumor-specific targeting of the targeting ligand 48 to 122 hours after administration to mammals bearing tumors having a FAP-rich environment are shown.

[0038] Fig. 9C The biodistribution of the targeting ligand in the tumor, heart, liver, lung, spleen, kidney, intestine and stomach after 122 hours after administration to a mammal (MDA-MB-231 xenograft mouse) with a tumor rich in a FAP environment is shown. The black or white ovals or circles in the image highlight the location of the targeting ligand. Darker shading within the ovals or circles indicates a higher concentration of the targeting ligand than lighter shading within the ovals or circles.

[0039] Fig.10Shown are in vivo images of targeting ligands administered to mammals (MDA-MB-231 xenograft mice) with tumors rich in FAP environments, 2 hours and 6 hours later, with (right) and without (left) unlabeled competitors. The black ovals or circles in the images highlight the location of the targeting ligands. Darker shading within the ovals or circles indicates a higher concentration of the targeting ligands than lighter shading within the ovals or circles.

[0040] Fig.11A Shown are in vivo imaging of the targeting ligand 2 to 32 hours after administration to mammals bearing another tumor (KB xenograft mice) with a FAP-rich environment.

[0041] Fig. 11B Shown is in vivo imaging of the targeting ligand 48 to 122 hours after administration to mammals bearing another tumor (KB xenograft mice) with a FAP-rich environment.

[0042] Fig. 11C The biodistribution of the targeting ligand in the tumor, heart, liver, lung, spleen, kidney, intestine and stomach at 122 hours after administration to a mammal with a tumor having a FAP-rich environment is shown. The black ovals or circles in the image highlight the location of the targeting ligand. Darker shading within the ovals or circles indicates a higher concentration of the targeting ligand than lighter shading within the ovals or circles.

[0043] Fig.12 Shown are in vivo images of targeting ligands administered to mammals (KB xenograft mice) with another tumor rich in FAP environment, 2 hours and 6 hours later, with (right) and without (left) unlabeled competitors. The black ovals or circles in the images highlight the location of the targeting ligand. Darker shading within the ovals or circles indicates a higher concentration of the targeting ligand than lighter shading within the ovals or circles.

[0044] Fig.13 The biodistribution of the targeting ligand in the tumor, heart, liver, lung, spleen, kidney, intestine and stomach at time points 2 hours, 4 hours, 6 hours, 6 hours (kidney covered, KC), 15 hours, 24 hours and 122 hours after administration to a mammal with another tumor rich in FAP environment (KB xenograft mouse). The black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. The darker shading adjacent to the arrow or within the ellipse or circle indicates a higher concentration of the targeting ligand than the lighter shading.

[0045] Fig.14AIn vivo imaging of the targeting ligand after administration to mammals bearing another tumor with a FAP-rich environment (FADu xenograft mice M1, M2, M3) 6 hours after injection is shown.

[0046] Fig. 14B The biodistribution of FAP-targeted compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach after 6 hours is shown.

[0047] Fig. 14C Shown is in vivo imaging of a competition experiment between an exemplary FAP-targeting compound and an unlabeled competitor 6 hours after injection into mammals bearing another tumor with a FAP-rich environment (FADu xenograft mice M1, M2, and M3).

[0048] Fig.14D The biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach after 6 hours is shown. The black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. The darker shading adjacent to the arrow or within the ovals or circles indicates a higher concentration of the targeting ligand than the lighter shading.

[0049] Fig.15A Shown is in vivo imaging of the targeting ligand after administration to mammals bearing another tumor with a FAP-rich environment (HT29 xenograft mice, M1, M2, and M3).

[0050] Fig. 15B The biodistribution of FAP-targeted compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach after 6 hours is shown. Fig. 15C Shown is in vivo imaging of a competition experiment between a targeting ligand and an unlabeled competitor following administration.

[0051] Fig. 15C The biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach after 6 hours is shown. The black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. The darker shading adjacent to the arrow or within the ovals or circles indicates a higher concentration of the targeting ligand than the lighter shading.

[0052] Fig.15D The biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach after 6 hours is shown. The black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. In HT29 xenograft mice, the darker shading adjacent to the arrows or within the ovals or circles indicates a higher concentration of the targeting ligand compared to the lighter shading.

[0053] Fig.16A In vivo imaging of targeting ligands following administration to mammals bearing tumors with a FAP-rich environment (KB tumor xenograft mice (eg, M1, M2, and M3)) is shown.

[0054] Fig. 16B Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach is shown.

[0055] Fig. 16C Shown are in vivo imaging of competition experiments between targeting ligands and unlabeled competitors after administration to mammals bearing tumors with a FAP-rich environment (KB tumor xenograft mice (eg, M1, M2, and M3)).

[0056] Fig.16D Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach is shown for competition studies. Black or white arrows, ovals or circles in the images highlight where the targeting ligand is present. Darker shading adjacent to the arrows or within the ovals or circles indicates a higher concentration of the targeting ligand than lighter shading.

[0057] Fig.17A In vivo imaging of targeting ligands following administration to mammals bearing tumors with a FAP-rich environment (MDA-MB-231 tumor xenograft mice (eg, M1, M2, and M3)) is shown.

[0058] Fig. 17B The biodistribution of FAP-targeted compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach is shown.

[0059] Fig. 17C In vivo imaging of a competition experiment between targeting ligand and unlabeled competitor (500 nmol) is shown.

[0060] Fig.17D The biodistribution of FAP-targeted compounds in competition studies in tumors, heart, liver, lung, spleen, kidney, intestine, muscle and stomach is shown. The black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. The darker shading adjacent to the arrow or within the ovals or circles indicates a higher concentration of the targeting ligand than the lighter shading.

[0061] Fig.18A Shown is in vivo imaging of the targeting ligand following administration to mammals bearing another tumor with a FAP-rich environment (U87MG tumor xenograft mice (eg, M1, M2, and M3)).

[0062] Fig.18BThe biodistribution of FAP-targeted compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach is shown.

[0063] Fig. 18C In vivo imaging of a competition experiment between a targeting ligand and an unlabeled competitor is shown.

[0064] Fig.18D Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach in competition studies is shown. Black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. Darker shading adjacent to the arrows or within the ovals or circles indicates a higher concentration of the targeting ligand than lighter shading.

[0065] Fig.19A In vivo imaging of the targeting ligand after administration to mammals bearing another tumor with a FAP-rich environment (PANC1 tumor xenograft mice (eg, M1, M2, and M3)) is shown.

[0066] Fig.19B The biodistribution of FAP-targeted compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach is shown.

[0067] Fig.19C In vivo imaging of competition experiments between targeting ligands provided herein and unlabeled competitors is shown.

[0068] Fig.19D Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach in competition studies is shown. Black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. Darker shading adjacent to the arrows or within the ovals or circles indicates a higher concentration of the targeting ligand than lighter shading.

[0069] Fig. 20A Shown is in vivo imaging of the targeting ligand after administration to mammals bearing another tumor with a FAP-rich environment (4T1 tumor xenograft mice) 2 hours after injection.

[0070] Fig. 20B The biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach after 6 hours is shown. The black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. The darker shading adjacent to the arrow or within the ovals or circles indicates a higher concentration of the targeting ligand than the lighter shading.

[0071] Fig. 20CThe biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach after 6 hours is shown. The black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. In 4T1 xenograft mice, the darker shading adjacent to the arrows or within the ovals or circles indicates a higher concentration of the targeting ligand compared to the lighter shading.

[0072] Fig.21 Shown is a displacement binding curve of the targeting ligand in HEK-FAP cells.

[0073] Fig. 22 Shown are displacement binding curves of targeting ligands in HEK-FAP cells.

[0074] Fig.23 Shown is a Western blot of the phosphorylation level of Akt (protein kinase B) in transforming growth factor (TGF)-β-stimulated human lung fibroblasts after treatment with a phosphoinositide 3-kinase inhibitor (PI3Ki) or a targeted compound provided herein (e.g., at a concentration of 1 nM, 10 nM, or 100 nM).

[0075] Fig.24 Shown are relative changes in the expression of collagen 1A1 mRNA in transforming growth factor (TGF)-β-stimulated human lung fibroblasts following treatment with a phosphoinositide 3-kinase inhibitor (PI3Ki) or a targeted compound provided herein (eg, at a concentration of 1 nM, 10 nM, or 100 nM). DETAILED DESCRIPTION OF THE INVENTION

[0077] definition

[0078] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art of chemistry and biology. In addition, as used in this specification and the appended claims, nouns that are not modified with quantifiers include one / kind and / or more / kinds unless the content clearly indicates otherwise. Thus, for example, when a compound / composition is substituted with an alkyl or aryl group that is not modified with a quantifier, the compound / composition is optionally substituted with at least one alkyl group and / or at least one aryl group. In addition, unless otherwise expressly stated, the term "about" refers to a value for a percentage plus or minus 10% and a range of values ​​for a unit value plus or minus 1.0 units, for example, about 1.0 refers to a range of values ​​from 0.9 to 1.1.

[0079] For therapeutic uses, a "therapeutically effective amount" (or "effective amount") of a compound refers to that amount of the compound in a formulation which, when administered as part of a desired dosage regimen (to a mammal, such as a human), alleviates symptoms, ameliorates symptoms, or slows the onset of a disease condition, according to clinically acceptable standards for the disorder or condition being treated, or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0080] The terms "prophylactic or therapeutic" treatment are art-recognized and include administering one or more compounds of the present disclosure to a patient. If it is administered prior to clinical manifestation of an undesirable condition (e.g., a disease or other undesirable state of the host animal), the treatment is prophylactic (i.e., it protects the host from developing the undesirable condition), while if it is administered after manifestation of the undesirable condition, the treatment is therapeutic (i.e., it is intended to reduce, alleviate, or stabilize an existing undesirable condition or its side effects).

[0081] The terms "patient," "individual," or "subject" refer to a mammal in need of a particular treatment. The patient or subject can be a primate, canine, feline, or equine. The patient or subject can be a bird. The bird can be a domesticated bird, such as a chicken. The bird can be a poultry. The patient or subject can be a human.

[0082] "Oxo" refers to a =0 group.

[0083] "Alkyl" generally refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, such as a group having one to fifteen carbon atoms (e.g., C1-C 15 Unless otherwise indicated, the disclosure provided herein of "alkyl" is intended to include independent recitation of saturated "alkyl". Alkyl groups may contain from one to thirteen carbon atoms (e.g., C1-C 13 An alkyl group may contain one to eight carbon atoms (e.g., a C1-C8 alkyl group). An alkyl group may contain one to five carbon atoms (e.g., a C1-C5 alkyl group). An alkyl group may contain one to four carbon atoms (e.g., a C1-C4 alkyl group). An alkyl group may contain one to three carbon atoms (e.g., a C1-C3 alkyl group). An alkyl group may contain one to two carbon atoms (e.g., a C1-C2 alkyl group). An alkyl group may contain one carbon atom (e.g., a C1 alkyl group). An alkyl group may contain five to fifteen carbon atoms (e.g., a C5-C 15Alkyl). The alkyl group may contain five to eight carbon atoms (e.g., C5-C8 alkyl). The alkyl group may contain two to five carbon atoms (e.g., C2-C5 alkyl). The alkyl group may contain three to five carbon atoms (e.g., C3-C5 alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (isopropyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl group is connected to the rest of the molecule by a single bond.

[0084] "Alkoxy" refers to a group bonded through an oxygen atom of the formula -O-alkyl wherein alkyl is an alkyl chain as defined above.

[0085] "Alkylene" or "alkylene chain" generally refers to a straight or branched divalent alkyl group, e.g., having one to twelve carbon atoms, to which the rest of the molecule is attached, such as methylene, ethylene, propylene, isopropylene, n-butylene, and the like.

[0086] "Aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbons of five to eighteen carbon atoms, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Ring systems from which aryl radicals are derived include, but are not limited to, radicals such as benzene, fluorene, indane, indene, tetralin and naphthalene.

[0087] "Aralkyl" or "aryl-alkyl" refers to a group of the formula -R c An aryl group, wherein R c is an alkylene chain as defined above, for example methylene, ethylene, etc. The alkylene chain part of the aralkyl group is optionally substituted with an alkylene chain as described above.

[0088] "Carbocyclyl" or "cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms, including a fused ring system or a bridged ring system, having three to fifteen carbon atoms. Carbocyclyl may contain three to ten carbon atoms. Carbocyclyl may contain five to seven carbon atoms. Carbocyclyl is connected to the rest of the molecule by a single bond. Carbocyclyl or cycloalkyl is saturated (i.e., only contains a single C-C bond) or unsaturated (i.e., contains one or more double bonds or triple bonds). Some examples of saturated cycloalkyl include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Unsaturated carbocyclyl is also referred to as "cycloalkenyl". Some examples of monocyclic cycloalkenyl include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl. Polycyclic carbocyclyls include, for example, adamantyl, norbornyl (ie, bicyclo[2.2.1]heptyl), norbornenyl, decalinyl, 7,7-dimethylbicyclo[2.2.1]heptyl, and the like.

[0089] "Carbocyclylalkyl" refers to a group of the formula -R c -Carbocyclic group, where R c is an alkylene chain as defined above.

[0090] "Halo" or "halogen" refers to a bromo, chloro, fluoro, or iodo substituent.

[0091] "Haloalkyl" refers to an alkyl group as defined above substituted with one or more halo groups as defined above, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.

[0092] The term "heteroalkyl" refers to an alkyl group as defined above, wherein one or more of the backbone carbon atoms of the alkyl group is replaced by a heteroatom (with an appropriate number of substituents or valences, e.g., -CH2- can be replaced by -NH- or -O-). For example, each substituted carbon atom is independently substituted by a heteroatom, e.g., wherein the carbon is substituted by nitrogen, oxygen, selenium or other suitable heteroatoms. In some cases, each substituted carbon atom is independently substituted by oxygen, nitrogen (e.g., -NH-, -N(alkyl)- or -N(aryl)- or with another substituent considered herein) or sulfur (e.g., -S-, -S(=O)- or -S(=O)2-). Heteroalkyl is connected to the rest of the molecule at a carbon atom of the heteroalkyl group. Heteroalkyl is connected to the rest of the molecule at a heteroatom of the heteroalkyl group. Heteroalkyl is C1-C 18 Heteroalkyl. Heteroalkyl is C1-C 12 Heteroalkyl. Heteroalkyl is C1-C6 heteroalkyl. Heteroalkyl is C1-C4 heteroalkyl. Heteroalkyl may include alkoxy, alkoxyalkyl, alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocycloalkyl and heterocycloalkylalkyl as defined herein.

[0093] "Heteroalkylene" refers to a divalent heteroalkyl group as defined above that links one part of the molecule to another part of the molecule.

[0094] "Heterocyclyl" refers to a stable 3 to 18-membered non-aromatic ring group that may contain two to twelve carbon atoms and one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise specifically stated in the specification, the heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which optionally includes an aromatic ring system, a condensed ring system and / or a bridged ring system. The heteroatoms in the heterocyclyl are optionally oxidized. The heterocyclyl is partially or completely saturated. Unless otherwise stated, the disclosure provided herein of "heterocyclyl" is intended to include independent records of heterocyclyls containing aromatic and non-aromatic ring structures. The heterocyclyl is connected to the rest of the molecule through any atom of the ring. Some examples of such heterocyclyls include, but are not limited to, dioxolyl, thienyl [1,3] dithianyl, decahydroisoquinolinyl, imidazolinyl, 1,3-benzodioxolyl, 1,4-benzodioxin ... alkyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cycloheptyl[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, indolyl, isoindolyl, imidazolidinyl, isothiazolidinyl, isothiazolidinyl, oxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidine, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxo-thiomorpholinyl.

[0095] "N-heterocyclyl" or "N-attached heterocyclyl" refers to a heterocyclyl as defined above, containing at least one nitrogen, and wherein the point of attachment of the heterocyclyl to the rest of the molecule is through the nitrogen atom in the heterocyclyl. Some examples of such N-heterocyclyls include, but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.

[0096] "Heteroaryl" refers to a group derived from a 3 to 18-membered aromatic ring radical, which may contain two to seventeen carbon atoms and one to six heteroatoms selected from nitrogen, oxygen and sulfur. The heteroaryl used herein is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is completely unsaturated, i.e., it contains a cyclic, delocalized (4n+2)π-electron system according to the Hückel theory. Heteroaryl includes a fused ring system or a bridged ring system. The heteroatoms in the heteroaryl are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl is connected to the rest of the molecule through any atom of the ring. Some examples of heteroaryl include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, benzofuranyl, benzo oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4] Azinyl, benzonaphthofuranyl, benzo oxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothiophenyl (benzothiophenyl), benzothiophene [3,2-d] pyrimidinyl, benzotriazolyl, benzo [4,6] imidazo [1,2-a] pyridinyl, carbazolyl, cinnolinyl, cyclopenta [d] pyrimidinyl, 6,7-dihydro-5H-cyclopenta [4,5] thieno [2,3-d] pyrimidinyl, 5,6-dihydrobenzo [h] quinazolinyl, 5, 6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclopenta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furano[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, isoquinolinyl, indolizinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, isoquinolinyl, indolizinyl, isothiazolyl, imidazolyl ... oxazolyl, 5,8-methanol-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinone, Oxazolyl, 2-oxoazepanyl, oxazolyl, oxirane, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phen oxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (i.e., thienyl).

[0097] Compounds disclosed herein may include one or more asymmetric centers, and thus produce enantiomers, diastereomers, and other stereoisomeric forms defined as (R)- or (S)- according to absolute stereochemistry. Unless otherwise stated, all stereoisomeric forms of compounds disclosed herein are intended to be considered by the present disclosure. When compounds described herein include olefin double bonds, and unless otherwise stated, it is intended that the present disclosure includes both E and Z geometric isomers (e.g., cis or trans). Similarly, all possible isomers are also intended to be included, as well as racemic and optically pure forms, and all tautomeric forms. The term "geometric isomer" refers to E or Z geometric isomers (e.g., cis or trans) of olefin double bonds. The term "positional isomer" refers to structural isomers around a central ring, such as ortho, meta, and para isomers around a benzene ring.

[0098] The term "linker" as used herein generally refers to a portion of a compound that forms a chemical bond with A (e.g., a binding ligand) and / or B (e.g., a therapeutic agent or an imaging agent). In particular, a "linker" can connect two or more functionalized portions of a molecule to form a compound provided herein. Illustratively, the linker can include atoms selected from C, N, O, S, Si, and P; C, N, O, S, and P; or C, N, O, and S. The linker can connect different functional capabilities of a compound (e.g., a FAP ligand and a PI3K inhibitor). The linker can include several linker groups, such as in the range of about 2 to about 100 atoms in a continuous backbone. The linker can be a releasable linker. The linker can be a non-releasable linker.

[0099] The compound can be a monovalent conjugate (e.g., a compound comprising one binding ligand (as described elsewhere herein, e.g., one FAP binding ligand). The compound can be a bivalent conjugate (e.g., a compound comprising one or more binding ligands (as described elsewhere herein, e.g., one or more FAP binding ligands) conjugated to a therapeutic or imaging agent (e.g., via a linker) (as described elsewhere herein)). The compound can be a multivalent conjugate (e.g., a compound comprising two or more binding ligands (as described elsewhere herein, e.g., two or more FAP binding ligands) conjugated to multi-point linkers).

[0100] A binding ligand (also referred to herein as a targeting ligand or targeting moiety) can be a compound (or a group thereof) that binds to a biomolecule (e.g., a polypeptide (e.g., an enzyme)) that is localized to a specific cell, tissue, organ, etc. The binding ligand can be a fibroblast activation protein (FAP) ligand (or a group thereof). The binding ligand can be a fibroblast activation protein alpha (FAPα) ligand (or a group thereof).

[0101] Therapeutic agent (or its group) can be any entity that can produce the desired physiological response.Therapeutic agent (or its group) can be an antifibrotic agent, an anticancer agent, a chemotherapeutic agent, a radiotherapeutic agent, etc. Therapeutic agent can be a compound (for example, or its group) that is effective against cancer cells or profibrotic cells (for example, cancer-associated fibroblasts, myofibroblasts, etc. (for example, other tumor microenvironment factors)) (for example, effectively eliminate, destroy, reduce (for example, reduce the amount of cancer cells or profibrotic cells) or alleviate the effect of cancer cells or profibrotic cells). Some examples of therapeutic agent (or its group) include but are not limited to photodynamic therapy agents, radiotherapeutic agents, chemotherapeutic agents, antifibrotic agents and anticancer agents. Therapeutic agent provided herein can be a phosphoinositide-3-kinase (PI3K) inhibitor (or its group). Therapeutic agent can be an anticancer agent (or its group). Therapeutic agent can be an antifibrotic agent (or its group). The therapeutic agent may be a compound selected from the following (or a group thereof): a tumor growth factor (TGF) β / Smad inhibitor, a Wnt / β-catenin inhibitor, a kinase inhibitor (e.g., a kinase inhibitor of vascular endothelial growth factor receptor (VEGFR), a kinase inhibitor of fibroblast growth factor receptor (FGFR), a kinase inhibitor of platelet-derived growth factor receptor (PDGFR), a kinase inhibitor of focal adhesion kinase (FAK), or a kinase inhibitor of Rho-associated protein kinase (ROCK)), a Toll-like receptor agonist (TLR), a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor, a collagen synthesis inhibitor, and a phosphoinositide-3-kinase (PI3K) inhibitor. The therapeutic agent may be a phosphoinositide-3-kinase (PI3K) inhibitor (or a group thereof).

[0102] An imaging agent can be a compound (or a group thereof) that emits a detectable signal (e.g., an electromagnetic signal (e.g., a radio signal, a fluorescent signal, a gamma ray) or a mass). Some examples of imaging agents include, but are not limited to, radio imaging agents (e.g., PET imaging agents or SPECT imaging agents), fluorescent imaging agents (e.g., fluorescent dyes), etc.

[0103] Compound

[0104] Provided are compounds (eg, conjugates) of Formula X:

[0105] A m -LB (X)

[0106] in:

[0107] A is a group (e.g., molecular weight less than 10,000) of a fibroblast activation protein alpha (FAPα) ligand (targeting moiety);

[0108] L is a (e.g., bifunctionalized) linker that connects one or more A groups to B (e.g., via a first covalent bond connecting L to A and a second covalent bond connecting L to B);

[0109] B is an optical imaging agent, a photodynamic therapy agent, a radioimaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an anti-fibrotic agent, or an anti-cancer agent (e.g., an anti-cancer agent effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors) (e.g., a group thereof); and

[0110] m is 1 to 6.

[0111] A can be a group of a FAPa ligand having a molecular weight of less than 10,000, 7,500 less than 5,000, less than 2,500, less than 1,000, less than 760, less than 500; about 500 to about 10,000 g / mol, about 1,000 to about 7,500 g / mol, about 750 g / mol to about 1,500 g / mol, about 1000 to about 5,000 g / mol, or about 500 to about 2,500 g / mol.

[0112] m may be 1. m may be 2. m may be 3. m may be 4. m may be 5. m may be 6. m may be 1 to 3, 2 to 4, 1 to 5.

[0113] The present disclosure also relates to compounds (eg, conjugates) of Formula I:

[0114] ALB (I)

[0115] in:

[0116] A comprises a FAPα ligand (eg, a targeting moiety) (eg, a group thereof);

[0117] L comprises a (e.g. bifunctionalized) linker connecting one or more A groups to B; and

[0118] B comprises an optical imaging agent, a photodynamic therapy agent, a radioimaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an anti-fibrotic agent, or an anti-cancer agent (e.g., an anti-cancer agent effective against cancer cells or cancer-associated fibroblasts, myofibroblasts or other tumor microenvironment factors) (e.g., a group thereof).

[0119] The targeting moiety can bind to activated fibroblasts expressing FAPα, and such activated fibroblasts are involved in cancer or inflammatory diseases. The molecular weight of the targeting moiety can be less than 10,000. L can include a bifunctionalized linker. The (e.g., biofunctionalized) linker can form a chemical bond with A and B. L can be a (e.g., bifunctionalized) linker that connects one or more A groups to B (e.g., by a first covalent bond connecting L to A and a second covalent bond connecting L to B). B can include an imaging agent, a radioimaging agent, a photodynamic therapy agent, a chemotherapeutic agent, an antifibrotic agent and / or a radiotherapeutic agent (e.g., a group thereof), wherein B is an anticancer agent that is effective against cancer cells or cancer-associated fibroblasts, myofibroblasts or other tumor microenvironmental factors.

[0120] A may have a structure represented by Formula IA:

[0121]

[0122] in:

[0123] is a functionalized 5- to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocyclic ring, said heterocyclic ring optionally further comprising 1 to 3 heteroatoms selected from oxygen, nitrogen and sulfur;

[0124] Z is a bond, substituted or unsubstituted alkylene (e.g., -CH2-), substituted or unsubstituted amino (e.g., -NH-), -O-, or -S-;

[0125] T is substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O- or -S-;

[0126] R 1 and R 2 Each is independently selected from -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F and 5-tetrazolyl;

[0127] R3 and R 4 Each independently selected from -H, -OH, F, Cl, Br, I, -C 1-6 Alkyl, -OC 1-6 Alkyl and -SC 1-6 alkyl;

[0128] R 5 , R 6 , R 7 and R 8 are each independently selected from H, alkyl and halogen; and

[0129] It is the point of attachment of the FAPα binding ligand (e.g., through the linker L or the imaging agent / therapeutic agent portion B), wherein the point of attachment can be through any carbon atom of the following: a 5- to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocyclic ring or a 1°, 2° amine or having a functionalized alkyl or cycloalkyl motif, as well as stereoisomers thereof and pharmaceutically acceptable salts thereof.

[0130] A may have a structure represented by Formula IB:

[0131]

[0132] T is substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O- or -S-;

[0133] R 1 and R 2 Each is independently selected from -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F and 5-tetrazolyl;

[0134] R 3 and R 4 Each independently selected from -H, -OH, F, Cl, Br, I, -C 1-6 Alkyl, -OC 1-6 Alkyl and -SC 1-6 alkyl;

[0135] R 5 , R 6 , R 7 and R 8 are each independently selected from H, alkyl and halogen; and

[0136] R 9 , R 10 and R 11 Each independently selected from H, -C1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl, F, Cl, Br and I.

[0137] A may have a structure represented by Formula IC:

[0138]

[0139] T is substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O- or -S-;

[0140] R 1 and R 2 Each is independently selected from -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F and 5-tetrazolyl;

[0141] R 3 and R 4 Each independently selected from -H, -OH, F, Cl, Br, I, -C 1-6 Alkyl, -OC 1-6 Alkyl and -SC 1-6 alkyl;

[0142] R 5 , R 6 , R 7 and R 8 are each independently selected from H, alkyl and halogen; and

[0143] R 9 , R 10 and R 11 Each independently selected from H, -C 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl, F, Cl, Br and I.

[0144] A may have a structure represented by the following formula:

[0145]

[0146] wherein T is a substituted or unsubstituted methylene (-CH2-), a substituted or unsubstituted amino (-NH-), -O- or -S-;

[0147] R 1 and R 2Each is independently selected from -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F and 5-tetrazolyl;

[0148] R 3 and R 4 Each independently selected from -H, -OH, F, Cl, Br, I, -C 1-6 Alkyl, -OC 1-6 Alkyl and -SC 1-6 alkyl;

[0149] R 5 , R 6 , R 7 and R 8 are each independently selected from H, alkyl and halogen; and

[0150] R 9 , R 10 and R 11 Each independently selected from H, -C 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl, F, Cl, Br and I.

[0151] A may have a structure represented by formula XA:

[0152]

[0153] in:

[0154] Q is aryl, heteroaryl or heterocyclyl (e.g., containing aromatic and non-aromatic ring structures) (e.g., a 5- to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle optionally further containing 1 to 3 heteroatoms selected from O, N and S);

[0155] Z is a bond, substituted or unsubstituted C1-C3 alkylene (e.g., -CH2-), substituted or unsubstituted heteroalkyl (e.g., 1 to 3 atoms in length), amino (e.g., NH), -O-, or -S-;

[0156] T is substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O-, or -S- (e.g., wherein the substitution of T is C1-C3 alkyl, haloalkyl, or halogen);

[0157] R 1 and R 2Each is independently selected from -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, -CONH2 and 5-tetrazolyl;

[0158] R 3 and R 4 Each independently selected from -H, -OH, F, Cl, Br, I, -C 1-6 Alkyl, -OC 1-6 Alkyl and -SC 1-6 Alkyl; and

[0159] R 5 , R 6 , R 7 and R 8 Each is independently selected from H, alkyl and halogen.

[0160] Q can be connected to L (e.g., L or L1). Q can be an aryl, heteroaryl or heterocyclic group. The heterocyclic group can contain aryl and non-aryl ring structures. Q can be connected to L at the heteroalkyl, alkyl or aryl position of Q. Q can be connected to L at the aryl position of Q. Q can be connected to L through a nitrogen atom (e.g., of L). Q can be connected to L through a triazolyl or amide (e.g., of L). The heteroaryl group can contain aryl and non-aryl ring structures. The heteroaryl or heterocyclic group can contain 1 to 3 heteroatoms selected from O, N and S. The heterocyclic group can contain 1 to 3 heteroatoms selected from O, N and S. Q can be a 5 to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle (e.g., optionally containing aryl and non-aryl ring structures). Q can be an N-connected heterocyclic group (e.g., optionally containing aryl and non-aryl ring structures). Q can be a C6-C9-N-connected heterocyclic group (e.g., optionally containing aryl and non-aryl ring structures). The N-linked heterocyclyl is linked to Z via the N-heterocycloalkyl group. Q may be a (eg, N-linked) isoindolinyl group (eg, wherein N is linked to Z).

[0161] Z can be a bond, a substituted or unsubstituted C1-C3 alkylene, a substituted or unsubstituted heteroalkylene (e.g., 1 to 3 atoms in length), an amino group (e.g., NH), -O-, or -S-. Z can be a bond. Z can be a substituted methylene. Z can be -CH2-. Z can be a substituted ethylene. Z can be an ethylene substituted with oxo. Z can be -C(CO)CH2-. Z can be -CH2CH2-. Z can be a C1-C3 heteroalkylene.

[0162] A may have a structure represented by Formula XB:

[0163]

[0164] in:

[0165] Q is aryl, heteroaryl or heterocyclic (e.g., containing aryl and non-aryl ring structures); (e.g., a 5- to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocyclic ring, the heterocyclic ring optionally further containing 1 to 3 heteroatoms selected from O, N and S),

[0166] T is substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O-, or -S- (e.g., wherein the substitution of T is C1-C3 alkyl, haloalkyl, or halogen);

[0167] J is C(R J )2, where each R J are independently H or alkyl, or two R J Together they form an oxo group;

[0168] R 1 and R 2 Each is independently selected from -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, -CONH2 and 5-tetrazolyl;

[0169] R 3 and R 4 Each independently selected from -H, -OH, F, Cl, Br, I, -C 1-6 Alkyl, -OC 1-6 Alkyl and -SC 1-6 alkyl;

[0170] R 5 , R 6 , R 7 and R 8 are each independently selected from H, alkyl and halogen; and

[0171] R 9 , R 10 and R 11 Each independently selected from H, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl, F, Cl, Br and I.

[0172] J may be linked to L (e.g., L or L1). J may be linked to L via a nitrogen atom. J may be linked to L via a triazole group (e.g., of L) or an amide. J may be C(R J )2, where each R J are independently H or alkyl, or two R J Together they form oxo. J may be C1-C3 alkyl. J may be -CH2-. J may be -CH2CH2-. J may be C=O.

[0173] T can be substituted or unsubstituted methylene (e.g., -CH2-), substituted or unsubstituted amino (e.g., -NH-), -O-, or -S-. T can be substituted with C1-C3 alkyl, C1-C3 haloalkyl, or (for methylene) halogen. T can be (-CH2-). T can be substituted with C1-C3 alkyl, haloalkyl, or halogen. T can be unsubstituted.

[0174] R 1 and R 2 Each is independently selected from -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, -CONH2 and 5-tetrazolyl. R 1 and R 2 R may be independently selected from H, -CN, -CHO and -B(OH)2. 1 and R 2 R may be independently selected from H, -CN, -CHO and -CONH2. 1 It can be H. R 2 It can be -CN, -CHO, -B(OH)2 or -CONH2. 1 Can be H and R 2 It can be -CN, -CHO, -B(OH)2 or -CONH2. 1 Can be H and R 2 Can be -CN. 1 Can be H and R 2 Can be -CHO. 1 Can be H and R 2 It can be -B(OH)2. 1 Can be H and R 2 It can be -CONH2.

[0175] R 3 and R 4 can be independently selected from -H, -OH, F, Cl, Br, I, -C 1-6Alkyl, -OC 1-6 Alkyl and -SC 1-6 Alkyl. 3 and R 4 R may be independently -H or -F. 3 Can be H and R 4 Can be -F. R 3 Can be F and R 4 Can be -F.

[0176] R 1 Can be H, R 2 Can be -CN, R 3 Can be H and R 4 Can be -F. R 1 Can be H, R 2 Can be -CN, R 3 Can be F and R 4 Can be -F. R 1 Can be H, R 2 Can be -CHO, R 3 Can be H and R 4 Can be -F. R 1 Can be H, R 2 Can be -CHO, R 3 Can be F and R 4 Can be -F. R 1 Can be H, R 2 It can be -B(OH)2, R 3 Can be H and R 4 Can be -F. R 1 Can be H, R 2 It can be -B(OH)2, R 3 Can be F and R 4 Can be -F. R 1 Can be H, R 2 Can be -CONH2, R 3 Can be H and R 4 Can be -F. R 1 Can be H, R 2 Can be -CONH2, R 3 Can be F and R 4 Can be -F.

[0177] R 5 , R 6 , R 7 and R 8 R may be independently selected from H, alkyl and halogen. 5 , R 6 , R 7 and R8 Each can be H.

[0178] R 9 , R 10 and R 11 can be independently selected from H, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl, F, Cl, Br and I. R 9 , R 10 and R 11 can be independently selected from H, -C 1-6 Haloalkyl, F and Cl. R 9 and R 11 Can be H and R 10 Can be H, -C 1-6 Haloalkyl, F or Cl. R 9 and R 11 Can be H and R 10 It can be H, -CF3, F or Cl. 9 and R 11 Can be H and R 10 It can be -CF3. 9 and R 11 Can be H and R 10 It can be F. R 9 and R 11 Can be H and R 10 It can be Cl. 9 , R 10 and R 11 It can be H.

[0179] A may be linked to L via a nitrogen atom (e.g. of L). A may be linked to L via a triazole group (e.g. of L) or an amide.

[0180] A can be selected from the following:

[0181]

[0182]

[0183] A can be selected from the following:

[0184]

[0185] The binding affinity of A (eg, a FAPa binding ligand) for FAP (eg, FAPa) can be about 1 nM to about 25 nM, such as 1 nM to about 25 nM or about 1 nM to 25 nM.

[0186] L can be a linker, such as any suitable linker. L can be a non-releasable linker. L can be a releasable linker.

[0187] L may comprise one or more joint groups, each of which is independently selected from (sub) alkyl, (sub) heteroalkyl, (sub) heterocycloalkyl, heteroaryl, aryl, alkoxy, thioether, disulfide, carboxylic acid, anhydride, carbonate, carbamate, thioether, sugar and peptide.L may comprise one or more joint groups, each of which is independently selected from polyethylene glycol (PEG), (sub) alkyl, disulfide, amide, carboxylic acid, anhydride, carbonate, ester, carbamate, thioether, triazole, sugar and peptide.L may comprise one or more joint groups, each of which is independently selected from polyethylene glycol (PEG), (sub) alkyl, disulfide, amide, carboxylic acid, anhydride, carbonate, ester, carbamate, thioether, triazole, sugar and peptide.L may comprise one or more joint groups, each of which is independently selected from polyethylene glycol (PEG), (sub) alkyl, disulfide, amide, carboxylic acid, carbonate, ester, phenyl, triazole and carbamate.L may comprise one or more joint groups, each of which is independently selected from polyethylene glycol (PEG), (sub) alkyl, disulfide, amide, carboxylic acid, phenyl, triazole, ester and carbonate. L may comprise one or more joint groups, each of which is independently selected from polyethylene glycol (PEG), (sub) alkyl, disulfide, amide, carboxylic acid, ester and carbonate. L may comprise one or more joint groups, each of which is independently selected from polyethylene glycol (PEG), (sub) alkyl, disulfide and amide. L may comprise one or more joint groups, each of which is independently selected from (sub) alkyl, disulfide and amide. L may comprise one or more joint groups, each of which is independently selected from amide, (sub) alkyl, PEG, phenyl and triazole. L may comprise one or more joint groups, each of which is independently selected from PEG, (sub) alkyl and amide. L may comprise one or more joint groups, each of which is independently selected from (sub) alkyl and amide. L may comprise one or more joint groups, each of which is independently selected from PEG and amide. The joint may comprise one or more triazole joint groups. The joint may comprise one or more disulfide joint groups. The joint may comprise one or more amide joint groups. The joint may comprise one or more PEG joint groups.

[0188] L may comprise one or more releasable groups.

[0189] L may be (covalently) linked to A via an amide linker group. L may be (covalently) linked to B via an amide linker group. L may be (covalently) linked to A and B independently via an amide linker group.

[0190] L may be (covalently) linked to A via a triazole linker group. L may be (covalently) linked to B via a triazole linker group. L may be (covalently) linked to A and B independently via a triazole linker group.

[0191] L may be (covalently) linked to A via a triazole linker group. L may be (covalently) linked to B via an amide linker group. L may be linked to A via a triazole linker group and to B via an amide linker group.

[0192] L may be (covalently) linked to A via an amide linker group. L may be (covalently) linked to B via a carbamate linker group. L may be linked to A via an amide linker group and to B via a carbamate linker group.

[0193] The linker can be a divalent linker (e.g., connecting a single A to a single B). The linker can be a multivalent linker (e.g., connecting two or more A to a single B). The linker can be a releasable linker. The linker can be a non-releasable linker.

[0194] L can be (L 1 ) o -Y-(L2) p ,in:

[0195] Each L 1 It is the first joint;

[0196] Each L 2 It is the second joint;

[0197] Y is the third joint;

[0198] o is an integer from 1 to 5; and

[0199] p is an integer of 1-5.

[0200] L 1 and L 2 Can be the same. 1 and L 2 Can be different. Each L 1 can be connected to the A group (and the Y group). 2 can be connected to the B group (and the Y group). o and m can be the same, for example, 1 to 6, 1 to 3 or 1. p can be 1. o can be 1. p and o can each be 1.

[0201] Each L 1 and L 2 Each L independently comprises oligoethylene glycol (chain), polyethylene glycol (chain), alkyl (chain), oligopeptide (chain) or polypeptide (chain). 1 and L 2 Independently comprises oligoethylene glycol (chain) or polyethylene glycol (chain).

[0202] Each L 1 and L 2 Independently comprises a triazole or an amide.

[0203] Each L1 and L 2 Independently comprises an oligopeptide (chain) or a polypeptide (chain). Each L 1 and L 2 Independently contains peptidoglycan (chain).

[0204] Each L 1 and L 2 Independently comprises oligoproline or oligopiperidine.

[0205] Each L 1 and L 2 The length of the

[0206] o may be an integer from 1 to 5. o may be an integer from 1 to 3. o may be 1.

[0207] p may be an integer from 1 to 5. p may be an integer from 1 to 3. p may be 1.

[0208] Also provided are multivalent conjugates having Formula II:

[0209] (AS) m YLB

[0210] in:

[0211] A is a group (e.g., molecular weight less than 10,000) of a FAPα ligand (targeting moiety);

[0212] S is the spacer (e.g., the length of an arm with a multivalent targeting ligand (e.g., a drug) to reach multiple adjacent FAPs on a target cell);

[0213] Y is a connector;

[0214] L is a (e.g., bifunctionalized) linker that connects one or more A groups to B (e.g., via a first covalent bond connecting L to A and a second covalent bond connecting L to B); and

[0215] B is a group of a fluorescent dye, a photodynamic therapy agent, a radioimaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an anti-fibrotic agent, or an anti-cancer agent (e.g., an anti-cancer agent effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironmental factors); and

[0216] m is 2 to 6.

[0217] The spacer can be an optimal length for the arms of the multivalent drug to reach multiple adjacent FAPs on the target (eg, cancer or profibrotic) cells.

[0218] S may comprise oligoethylene, polyethylene glycol, an alkyl chain, an oligopeptide or a polypeptide. S may be oligoethylene glycol or polyethylene glycol.

[0219] S can be an oligopeptide or a polypeptide.

[0220] S may be peptidoglycan.

[0221] The spacer may be a rigid linker. S may be a rigid linker such as, for example, oligoproline or oligopiperidine.

[0222] The length of S may be at least 15 angstroms The length of S can be up to 200 angstroms The length of S can be 15 to 200 angstroms.

[0223] Y may be a linker that connects multiple arms of a compound (e.g., a conjugate). Y may have a repeating structure. Y may contain a releasable bond. L may contain a disulfide bond. Y may contain at least one citric acid group (or a group thereof). Y may contain one or more triazoles. Y may contain one or more amines. Y may contain one or more amides. Y may have an aromatic core (e.g., an aromatic core or a heteroaryl core). Y may have an (alkylene) core. Y may have an amine core. Y may be N(L 1 )3(For example, where L 1 may be as described elsewhere herein). Y may be 1 (For example, where L 1 Y may be C(L 1 )4(For example, where L 1 may be as described elsewhere herein).

[0224] Y can be combined with a single L 1 Y can be connected with a single L 2 Y can be connected with a single L 1 and a single L 2 Y can be independently connected to each L through an amide bond 1 and L 2 Y can be connected to L.

[0225] Y may be a linker (e.g., a multivalent linker) that connects multiple arms of a compound (e.g., a conjugate). Y may have a repeating structure. Y may include at least one citric acid group (or a group thereof). The linker may have the following structure:

[0226]

[0227] Y can be a linker (e.g., a multivalent linker) that connects multiple arms of a compound (e.g., a conjugate) and can include a linker (e.g., a repeating unit) of the structure:

[0228]

[0229] Y can be a linker that connects multiple arms of a compound (e.g., a conjugate) and can have a citric acid-based linker. Y can be a linker that connects multiple arms of a compound (e.g., a conjugate) (e.g., a multivalent template) and can have a (e.g., citric acid-based) linker of the following structure:

[0230]

[0231] Y can be a linker (e.g., a multivalent linker) that connects multiple arms of a compound (e.g., a conjugate) and can have a (e.g., citric acid-based) linker of the following structure:

[0232]

[0233] Y can be a linker (e.g., a multivalent linker) that connects multiple arms of a compound (e.g., a conjugate) and can have a (e.g., citric acid-based) linker of the following structure:

[0234]

[0235] L may comprise at least one linker group, each linker group being selected from polyethylene glycol (PEG), alkyl, sugar and peptide. The linker may be a dual linker based on polyethylene glycol (PEG) (eg, PEGylated), alkyl, sugar and peptide.

[0236] L may be a non-releasable linker (eg, divalently (eg, covalently) linked to B and A). L may be a releasable linker (eg, divalently (eg, covalently) linked to B and A).

[0237] L, L1, L2 or any combination thereof may comprise one or more linker groups having the following structure:

[0238]

[0239] Wherein n is 0 to 10.

[0240] L may comprise one or more linker groups having the following structure:

[0241]

[0242] Wherein n is 0 to 10.

[0243] L may comprise one or more linker groups having the following structure:

[0244]

[0245] Wherein n is 0 to 10.

[0246] L may comprise one or more linker groups having the following structure:

[0247]

[0248] Where n is 1 to 32.

[0249] L may comprise one or more linker groups having the following structure:

[0250]

[0251] Where n is 1 to 32.

[0252] L may comprise one or more linker groups having the following structure:

[0253]

[0254] in:

[0255] R 12 and R 13 may each independently be H or C1-C6 alkyl; and

[0256] z is an integer of 1-8.

[0257] L may comprise one or more linker groups having the following structure:

[0258]

[0259] in:

[0260] R 12 and R 13 may each independently be H or C1-C6 alkyl; and

[0261] z is an integer of 1-8.

[0262] L, L 1 , L 2 or any combination thereof may comprise one or more linker groups having the following structure:

[0263]

[0264] L may comprise one or more linker groups having the following structure:

[0265]

[0266] L may comprise one or more linker groups having the following structure:

[0267]

[0268] in:

[0269] R 16 is H or C1-C6 alkyl; and

[0270] R 14a , R 14b and R 15a , R 15b They may each independently be H or a C1-C6 alkyl group.

[0271] L can have the following structure:

[0272]

[0273] L may comprise one or more linker groups having the following structure:

[0274]

[0275] Wherein n is 0 to 15.

[0276] B may be linked to L via a carbon atom or a nitrogen atom (e.g., of L). B may be linked to L via a triazole group. B may be linked to L via an oxo group (e.g., an ester). B may be linked to L via an amide (e.g., of L).

[0277] B can be an optical dye (or a group thereof). B can be a fluorescent dye (or a group thereof) (for example, it can be used in fluorescence guided surgery (FGS)). The fluorescent dye can include fluorescent dye groups, each of which is selected from carbocyanine, indolecarbocyanine, oxacarbocyanine, thiocarbocyanine, merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, etc. The fluorescent dye (or its group) can be boron dipyrromethene (BODIPY), CyS, CyS.S, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S7S0, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor7S0, 10AlexaFluor790, Dy677, Dy676, Dy682, Dy7S2, Dy780, DyLightS47, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 7S0, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, ADS832WS, S0456, etc.

[0278] The excitation of the payload can be 600 nanometers (nm) to 1000 nm. The emission of the payload can be 700 nm to 1800 nm.

[0279] B may be a fluorescent dye group (or a group thereof) having the following structure:

[0280]

[0281]

[0282] The compound may be a FAP targeting ligand (or a group thereof) attached to a linker comprising one or more linker groups, each linker group being selected from alkyl, pegylated, and peptidoglycan, wherein the linker may also be attached to a fluorescent dye as described herein.

[0283] An ALB can have the following structure:

[0284]

[0285] in:

[0286] n is an integer from 1 to 5; and

[0287] B is:

[0288]

[0289] An ALB can have the following structure:

[0290]

[0291]

[0292] in:

[0293] n is an integer from 1 to 5; and

[0294] B is:

[0295]

[0296] A compound (eg, a conjugate) may have the following structure:

[0297] Table 1:

[0298]

[0299]

[0300]

[0301]

[0302] The following is a scheme illustrating a method for generating unlabeled (competing) FAP ligands, such as used in the Examples and Figures herein:

[0303]

[0304] The disadvantage of some therapeutic agents (e.g., chemotherapeutic agents or radiotherapeutic agents) is that such agents cannot reach and / or maintain therapeutically effective concentrations of agents at target locations (e.g., in cancer, tumors, or fibrotic tissues) without producing undesirable, toxic and / or lethal (e.g., systemic) effects. General systemic or even local administration of such agents (e.g., which may be pathway-specific, but not target tissue-specific) results in a tendency (liability) of the therapeutic agent to be off-target in some cases, thereby increasing side effects. In some cases, compounds (e.g., such as compounds comprising targeting ligands) that locate a payload (e.g., a therapeutic agent) to a target site (e.g., a tumor or fibrotic tissue) can improve the residence time of the payload at the target site. In some cases, increasing the residence time of the therapeutic payload at the target site helps to increase the concentration of the therapeutic agent and / or be effective, even at low doses or tolerable doses. In some cases, the effective concentration of the active payload is maintained at the target location for a time sufficient to achieve a therapeutic concentration (e.g., at a tolerable dose and / or at a dose if a similarly administered free payload is insufficient to achieve a therapeutic concentration) and / or for a time sufficient to reduce the frequency of dosing (e.g., relative to the time required to administer the free payload). In addition, in some cases, increasing the residence time of the therapeutic payload at the target location means that off-target effects can be reduced (e.g., because the active agent is maintained at the target location). In some cases, the compound facilitates the administration of the active agent or payload and, for example, reduces the frequency of dosing, reduces side effects, or a combination thereof (e.g., relative to the administration of another similar free active agent or payload).

[0305] The targeting ligands provided herein are based on Figure 1 The reverse synthetic scheme is shown in . Figure 1 The retrosynthetic scheme is used to synthesize Figure 2 Compounds shown in. However, any suitable synthesis scheme or process can be used to produce compounds. For example, certain synthesis schemes are shown in the embodiments. For any process, step or compound (e.g., the end product of the scheme, the intermediate of the scheme and / or the reagent of the scheme), all such synthesis schemes are incorporated into the specific embodiments herein.

[0306] The compound targets (e.g., localizes to) a cell expressing a FAP (e.g., FAP5). The compound binds to a FAP (e.g., FAP5) expressed by (e.g., and embedded in the cell membrane of) a cell (e.g., a cancer cell or a profibrotic cell). Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown in the gray shading in FIG, compound 1 (at different concentrations, for example, at 50 nM, 25 nM, 12.5 nM and 6.5 nM ( Figure 3 )) localizes to cells expressing FAP5 (e.g., HT1080-FAP cells). In addition, Compound 1 localizes and internalizes the ligand to the membrane of FAP5-expressing cells (e.g., where FAP5 is located), and maintains localization and internalization (e.g., when administered at a concentration of 12.5 nM) to the cells for at least 1 hour (e.g., 1 hour, 8 hours, 24 hours or longer) (e.g., Figure 4 ).

[0307] Figure 3 Binding of targeting ligands (eg, at 50 nM (A), 25 nM (B), 12.5 nM (C), and 6.25 nM (D)) to (FAPHT1080) cells is shown for 1 hour. Increasing concentrations of targeting ligand show more surface binding. Figure 4 Binding of targeting ligands (e.g., at 12.5 nM) to FAP HT1080 cells is shown for 1 hour (A), 8 hours (B), 24 hours (C), and 48 hours (D). At early time points, compounds (targeting ligands) were observed on the cell surface, and the compounds were internalized into the cells over time. Figure 5 Binding of targeting ligand to FAP HT1080 cells is shown for 1 hour in the presence of at least 100-fold excess of competing ligand (eg, A: 25 nM targeting ligand, 2.5 μM competitor; B: 25 nM targeting ligand, 5 μM competitor). Figure 6 Binding of targeting ligands to non-FAP HT1080 cells is shown (e.g., at 100 nM (A) and 200 nM (B)). At comparable time points, very little compound (targeting ligand) was observed on the surface of such cells after 1 hour (compared to Figures 3 to 5 compared to, Figures 3 to 5 Good surface binding of the compound on cells with high FAP surface concentrations is shown after similar times, even at much lower concentrations).

[0308] The compound may have a strong (e.g., binding) affinity for FAP (e.g., FAP5) expressing cells. In some cases, this strong binding affinity allows the compound to localize to FAP (e.g., FAP5) expressing cells for an extended period of time (e.g., a period of time sufficient to measure a signal from the compound localized to a tissue of interest (e.g., a tumor or fibrotic tissue) or to deliver a payload (e.g., a therapeutic agent)). The binding affinity of the compound to FAP (e.g., FAP5) expressing cells may be 0.01 nM to 1 μM. For example, the K of Compound 1 in cells expressing FAP5 is 0.01 nM to 1 μM. d 5nM to 15nM ( Figure 7 ). In addition, if Figure 8 As shown, Compound 1 did not bind to HT1080 cells that do not express FAP5 and Compound 1 did not bind to HT1080 cells that express FAP5 in the presence of an unlabeled FAP5 ligand (eg, Compound 8).

[0309] Figure 7 The binding curves of the targeting ligands to HT1080-FAP cells are shown. Figure 8 Binding curves of targeting ligands to HT1080-FAP cells (targeting ligand only (circles) and targeting ligand and competitor (squares)) and HT1080 cells (triangles) are shown.

[0310] The compound can target cancer cells (e.g., or tumors). The compounds provided herein can target tumors with minimal off-target effects. The compound can accumulate rapidly and can maintain its concentration in the tumor for a long time. As shown, the ligand shows good accumulation in the target position (tumor), and shows limited accumulation in the off-target position. As shown in addition, there is almost no accumulation or no accumulation of the compound (FAP ligand conjugate) in non-tumor organs. In particular, as shown, in some cases, high concentrations of the compound can be achieved at the target position (e.g., tumor) for a long time (e.g., up to 5 days or longer), and there is almost no accumulation or no accumulation in the heart, liver, lungs, spleen, stomach or intestine at any time. There is some accumulation in the kidney in a short time, but most of it is cleared during 15 hours (e.g., compared with high concentrations that last for several days at the target position). In some cases, this means that certain treatments that need to be administered once or twice a day to maintain therapeutic concentrations and / or provide therapeutic effects (if such results can be obtained even without intolerable side effects and / or death) without targeting ligands can be administered in a much lower frequency (e.g., once or twice a week) in the form provided herein. In addition, as noted, the compound provided herein is almost free of off-target accumulation, which shows the tolerance of such a compound administered in an effective amount. In some cases, the compound accumulates in the kidney of the object. In some cases, the accumulation of the compound (for example, in the kidney) is up to 6 hours. In some cases, the compound is significantly removed from an organ (for example, kidney) during 24 hours (for example, during 24 hours, during 15 hours, etc.). In some cases, the compound remains positioned to the tumor for at least 1 day (for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, etc.).

[0311] For example, 9A to 20C Compounds (eg, Compound 8) are shown to localize to tumors (eg, in MDA-MB-231 xenograft mice (eg, Figures 9A to 10 and Figures 17A to 17D ), KB xenograft mice (e.g., Figures 11A to 13 D and Figures 16A to 16D ), FADu xenograft mice (e.g., Figures 14A to 14D ), HT29 xenograft mice (e.g., Figures 15A to 15D ), U87MG tumor xenograft mice (e.g., Figures 18A to 18D ), PANC1 xenograft mice (e.g., Figures 19A to 19D ) and 4T1 tumor xenograft mice (e.g., Figures 20A to 20C) in tumors expressing FAP5). The data show that the compounds provided herein are (only) localized to tumors (e.g., in several tumor types) for at least 1 day, 2 days, 3 days, 4 days, 5 days or longer. In addition, although the compound (e.g., compound 8) shows some accumulation in the kidney (e.g., usually starting at 2 hours), the peak accumulation in the kidney is at 6 hours, which is then rapidly reduced and cleared. For example, the significant reduction in kidney exposure is only achieved after 15 hours, and is almost completely cleared during 24 hours. In contrast, the compound is rapidly absorbed (e.g., during 2 hours) and maintained in the tumor for a much longer time than it is present in the kidney (e.g., more than 1 day, 2 days, 3 days, 4 days, 5 days, etc.). In short, these in vivo data provide herein provided conjugates targeting the kidney, allowing the delivery of payloads (e.g., imaging agents or chemotherapeutic agents) for a long time (e.g., several days).

[0312] Fig. 9A and Fig. 9B The targeting ligands provided herein (e.g., at a dose of 10 nmol) are shown to be effective in MDA-MB-231 xenograft mice (tumor size 400 mm 3 ) for 2 hours to 122 hours of in vivo imaging. After 122 hours, biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine and stomach was Fig. 9C The black or white ovals or circles in the image highlight the location of the targeting ligand. Darker shading within the ovals or circles indicates a higher concentration of the targeting ligand than lighter shading within the ovals or circles. More generally, Fig. 9A and Fig. 9B Imaging results showing in vivo tumor-specific targeting of the targeting ligand 2 hours to 122 hours after administration to mammals bearing tumors having a FAP-rich environment are shown. Fig. 9C The biodistribution of the targeting ligand in the tumor, heart, liver, lung, spleen, kidney, intestine and stomach after 122 hours following administration to a mammal with a tumor having a FAP-rich environment is shown. As can be seen, when looking at mammals in general or looking at organs of mammals in particular, there is good tumor targeting (i) for several days and (ii) with little to no compound found elsewhere.

[0313] Fig.10In vivo imaging of the targeting ligand provided herein for 2 to 6 hours on MDA-MB-231 xenograft mice with and without unlabeled competitors is shown. In the study without competitors, 10 nmol of labeled ligands were administered to mice. In the study with competitors, 10 nmol of labeled ligands and 1,000 nmol of unlabeled ligands were administered to mice. For each time point, the leftmost mouse represents mice treated with only the targeting ligand, while the rightmost mouse represents mice treated with the targeting ligand and unlabeled competitors. The black ovals or circles in the image highlight the location of the presence of the targeting ligand. The darker shadows in the ovals or circles indicate that the concentration of the targeting ligand is higher than the lighter shadows in the ovals or circles. As can be seen, good targeting of the tumor location was achieved after 2 and 6 hours in the absence of FAP competitors. In contrast, the presence of FAP competitors limits the tumor targeting of the compound, which indicates that the ability of the compound to target FAP is important in some cases for the compound to be able to target the desired location. Furthermore, in some cases, such information indicates that non-FAP-enriched locations are not expected to achieve disproportionate or undesirable accumulation of the targeted compound.

[0314] Fig.11A and Fig. 11B The targeting ligands provided herein (e.g., at a dose of 5 nmol) are shown to be effective in KB xenograft mice (tumor size 600 mm 3 ) for 2 hours to 122 hours of in vivo imaging. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine and stomach at 122 hours Fig. 11C The black oval or circle in the image highlights the location of the targeting ligand. Darker shadows within the oval or circle indicate a higher concentration of the targeting ligand than lighter shadows within the oval or circle. High concentrations of active agents were observed at the tumor location for 5 days or longer. In contrast, other organs were clearly free of FAP targeting compounds. In addition, these data demonstrate similar effects in different systems, including MDA-MB-231 xenograft mice in Figure 9 and KB xenograft mice in Figure 11.

[0315] Fig.12In vivo imaging of the targeting ligand provided herein for 2 to 6 hours on KB xenograft mice with and without unlabeled competitors is shown. In the study without competitors, 10 nmol of labeled ligands were administered to mice. In the study with competitors, 10 nmol of labeled ligands and 1,000 nmol of unlabeled ligands were administered to mice. For each time point, the leftmost mouse represents mice treated with only the targeting ligand, while the rightmost mouse represents mice treated with the targeting ligand and unlabeled competitors. The black ovals or circles in the image highlight the location of the presence of the targeting ligand. Darker shadows within the ovals or circles indicate a higher concentration of the targeting ligand than lighter shadows within the ovals or circles. As can be seen, good targeting of the tumor location was achieved after 2 and 6 hours in the absence of FAP competitors. In contrast, the presence of FAP competitors limits the tumor targeting of the compound, which indicates that the ability of the compound to target FAP is important in some cases for the compounds provided herein to be able to target the desired location. Furthermore, in some cases, such information indicated that non-FAP-enriched locations were not expected to achieve disproportionate or undesirable accumulation of the targeted compound. Fig.10 MDA-MB-231 xenograft mice and Fig.12 Similar effects were observed in KB xenograft mice.

[0316] Fig.13 The biodistribution of the targeting ligand (injected in the tail vein at a dose of 10 nmol) in tumor, heart, liver, lung, spleen, kidney, intestine and stomach at 2 hours, 4 hours, 6 hours, 6 hours (kidney coverage (KC)), 15 hours, 24 hours and 122 hours (in KB tumor-bearing mice). The black or white arrows, ovals or circles in the images highlight the presence of the targeting ligand. The darker shadows adjacent to the arrows or within the ovals or circles indicate that the concentration of the targeting ligand is higher than that of the lighter shadows.

[0317] Fig.14A In vivo imaging (systemic distribution) of targeting ligands (e.g., at a dose of 5 nmol) on FADu xenograft mice (e.g., M1, M2, and M3) 6 hours after injection is shown. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours is shown. Fig. 14B Shown in. Fig. 14CIn vivo imaging (systemic distribution) of competition experiments between targeting ligand (e.g., at a dose of 5 nmol) and unlabeled competitor 500 nmol 6 hours after injection on FADu xenograft mice (e.g., M1, M2, and M3) is shown. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Fig.14D The black or white arrows, ovals or circles in the image highlight the location of the targeting ligand. The darker shades adjacent to the arrows or within the ovals or circles indicate a higher concentration of the targeting ligand than the lighter shades. Fig.14A and Fig. 14B As can be seen in the Figure 3, FAP-targeted compounds target tumors with almost no off-target accumulation. Fig. 14C and Fig.14D indicating that in the presence of a FAP-targeting competitor, FAP-targeting compounds accumulate less at tumors (e.g., due to competition for FAP), and Fig.14D This suggests that when there is competition for FAP, there are more off-target effects (e.g., in the stomach and kidney).

[0318] In addition, if Fig.13 and Fig. 14B As can be seen in the Figure 2, high concentrations of active agents are seen at the tumor location for 5 days or longer. In contrast, other organs are clearly free of FAP targeting compounds. Some accumulation of active agents is shown in the kidney, but such accumulation is shown to peak at about 6 hours, and a significant decrease in the concentration in the kidney is observed during 15 hours, and it is almost completely cleared during 24 hours. In some cases, the use of a targeting ligand to deliver an active payload helps to deliver the payload well to the target location with good (e.g., almost no or no) off-target effects or side effects. In addition, in some cases, the ability to maintain delivery for several days at the target location with a single administration helps to reduce the frequency of treatment administration, improve patient compliance (e.g., by reducing the requirement for frequency of administration), reduce side effects (e.g., reducing the frequency of administration also reduces off-target / side effects) and / or other benefits.

[0319] Fig.15A In vivo imaging (systemic distribution) of targeting ligand (5 nmol) on HT29 xenograft mice (e.g., M1, M2, and M3) 6 hours after injection is shown. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours is shown. Fig. 15B Shown in. Fig. 15CIn vivo imaging (systemic distribution) of competition experiments between targeting ligand (5 nmol) and unlabeled competitor (500 nmol) are shown on HT29 xenograft mice (e.g., M1, M2, and M3) 6 hours after injection. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Fig.15D The black or white arrows, ovals or circles in the image highlight the location of the targeting ligand. The darker shades adjacent to the arrows or within the ovals or circles indicate a higher concentration of the targeting ligand than the lighter shades. Fig.16A In vivo imaging (systemic distribution) of targeting ligand (5 nmol) on KB tumor xenograft mice (e.g., M1, M2, and M3) 6 hours after injection is shown. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours is shown. Fig. 16B Shown in. Fig. 16C In vivo imaging (systemic distribution) of competition experiments between targeting ligand (5 nmol) and unlabeled competitor (500 nmol) are shown on KB tumor xenograft mice (e.g., M1, M2, and M3) 6 hours after injection. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Fig.16D The black or white arrows, ovals or circles in the image highlight the location of the targeting ligand. The darker shades adjacent to the arrows or within the ovals or circles indicate a higher concentration of the targeting ligand than the lighter shades. Fig.17A In vivo imaging (systemic distribution) of the targeting ligands provided herein (e.g., at a concentration of 5 nmol) in MDA-MB-231 tumor xenograft mice (e.g., M1, M2, and M3) 6 hours after injection is shown. Biodistribution in tumors (e.g., M1, M1 kidney cover (KC), M2, and M3), heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Fig. 17B Shown in. Fig. 17C In vivo imaging (systemic distribution) of a competition experiment between a targeting ligand (e.g., at a concentration of 5 nmol) and an unlabeled competitor (500 nmol) is shown in MDA-MB-231 tumor mice (e.g., M1, M2, and M3) 6 hours after injection. Biodistribution in tumors, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Fig.17D The black or white arrows, ovals or circles in the image highlight the location of the targeting ligand. The darker shades adjacent to the arrows or within the ovals or circles indicate a higher concentration of the targeting ligand than the lighter shades. Fig.18AIn vivo imaging (systemic distribution) of targeting ligand (5 nmol) on U87MG tumor xenograft mice (e.g., M1, M2, and M3) 6 hours after injection is shown. Biodistribution in tumors (e.g., M1, M1 kidney cover (KC), M2, M3 KC, and M3), heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Fig.18B Shown in. Fig. 18C In vivo imaging (systemic distribution) of competition experiments between targeting ligand (5 nmol) and unlabeled competitor (500 nmol) on U87MG tumor-bearing mice (e.g., M1, M2, and M3) 6 hours after injection is shown. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours Fig.18D The black or white arrows, ovals or circles in the image highlight the location of the targeting ligand. The darker shades adjacent to the arrows or within the ovals or circles indicate a higher concentration of the targeting ligand than the lighter shades. Fig.19A In vivo imaging (systemic distribution) of the targeting ligands provided herein (e.g., at a dose of 5 nmol) on PANC1 tumor xenograft mice (e.g., M1, M2, and M3) 6 hours after injection is shown. Biodistribution in tumors (e.g., M1, M1 kidney cover (KC), M2 KC, M2, M3 KC, and M3), heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Fig.18B Shown in. Fig. 18C In vivo imaging (systemic distribution) of competition experiments between targeting ligands (e.g., at a dose of 5 nmol) and unlabeled competitor (500 nmol) are shown on PANC1 tumor-bearing mice (e.g., M1, M2, and M3) 6 hours after injection. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Fig.18D The black or white arrows, ovals or circles in the image highlight the location of the targeting ligand. The darker shades adjacent to the arrows or within the ovals or circles indicate a higher concentration of the targeting ligand than the lighter shades. Fig. 20A Shown are in vivo imaging (whole body distribution) of targeting ligand alone (eg, at a dose of 5 nmol) compared to targeting ligand and unlabeled competitor 2 hours after injection on 4T1 tumor xenograft mice. Fig. 20B In vivo imaging (systemic distribution) of targeting ligand alone (e.g., at a dose of 5 Nmol) versus targeting ligand and unlabeled competitor is shown on 4T1 tumor xenograft mice 6 hours after injection. Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach (e.g., targeted, targeted kidney coverage (KC), and competition) after 6 hours. Fig. 20C The black or white arrows, ovals or circles in the images highlight the location of the presence of the targeting ligand. The darker shades adjacent to the arrows or within the ovals or circles indicate a higher concentration of the targeting ligand than the lighter shades. These results are consistent with the results presented in the other figures, further indicating the consistency of the results across multiple tumor types and / or models.

[0320] in addition, Figures 21 to 22 Several compounds (eg, Compound 21, Compound 1, Compound 5, and Compound 6) were shown to target cells expressing a FAP (eg, FAP5) with very high affinity (eg, 1 nM to 10 nM).

[0321] In addition, combined Figures 9A to 22 , showing that the compound is localized at the target site, Fig.23 and Fig.24 Compounds (e.g., Compound 11) are shown to have efficacy (e.g., reduce fibrotic response) on FAP (e.g., FAP5) expressing cells. For example, compounds (e.g., Compound 11) are effective in reducing pathobiological responses (e.g., reducing phosphorylation of Akt in TGF-β stimulated human lung fibroblasts ( Fig.23 ) and decreased the relative expression of collagen 1A1 mRNA in TGF-β-stimulated human lung fibroblasts ( Fig.24 )) has the same efficacy as PI3Ki alone. Therefore, although a single payload (e.g., PI3Ki) effectively reduces pathogenic biological responses, the compounds provided herein effectively reduce pathogenic biological responses and increase the residence time at the target site (e.g., tumor or fibrotic tissue), ultimately increasing the effective concentration of the payload at the target site (e.g., reducing side effects, reducing dosage, etc.).

[0322] B may be an imaging agent. B may be a radio-imaging agent. B may be a photodynamic therapy agent. B may be a chemotherapeutic agent. B may be an anti-fibrotic agent. B may be a radiotherapeutic agent. B may be an anti-cancer agent. B may be an anti-cancer agent that is effective against cancer cells or cancer-associated fibroblasts, myofibroblasts or other tumor microenvironmental factors.

[0323] B may include a radioactive isotope. The radioactive isotope may be any suitable radioactive isotope. The radioactive isotope may be selected from 99m Tc, 111 In, 18 F. 68 Ga, 124 I. 125 I and 131 I.

[0324] B may contain radioactive isotopes. Radioactive isotopes may be selected from 177 Lu, 90 Y and 211 At.

[0325] B may be a chelating agent, and in the case of a radiotherapeutic nuclide, B may chelate the nuclide.

[0326] B may comprise a radiolabeled prosthetic group (or a group thereof). The radiolabeled prosthetic group may comprise a group selected from 18 F. 124 I. 125 I. 131 I and 211 Radioactive isotope of At.

[0327] AL- has the following structure:

[0328]

[0329]

[0330] wherein n is an integer from 1 to 5.

[0331] B (eg, a radiolabeled prosthetic group (or group thereof)) has the following structure:

[0332]

[0333] in:

[0334] Each X is independently selected from 18 F. 124 I. 125 I. 131 I and 211 Radioactive isotopes of At;

[0335] Each R or R 1 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; and

[0336] Each n is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0337] Representative radiolabeled prosthetic groups (e.g., B) include, but are not limited to:

[0338]

[0339] B may be a chelating group (e.g., a chelating agent (or a group thereof)). Representative chelating groups include, but are not limited to, the following (including their free bases, e.g., where one or more protons (H+) of CO2H(COOH) are removed to form COO-):

[0340]

[0341]

[0342] B may be an antifibrotic agent or a group thereof.

[0343] B may be a PI-3 kinase inhibitor or a group thereof.

[0344] B may be a transforming growth factor β (TGFβ) / Smad inhibitor or a group thereof.

[0345] B can be a Wingless-related integration site (Wnt) / β-catenin inhibitor or a group thereof. B can be a kinase inhibitor of vascular endothelial growth factor receptor (VEGFR1, VEGFR2, VEGFR3), fibroblast growth factor receptor (FGFR1 or FGFR2) or platelet-derived growth factor receptor (PDGFR), or a group thereof.

[0346] B may be a kinase inhibitor of focal adhesion kinase (FAK) or Rho-associated protein kinase (ROCK), or a group thereof.

[0347] B may be a toll-like receptor (TLR) agonist, or a group thereof.

[0348] B may be an inhibitor of NF-κB (nuclear factor kappa-light-chain enhancer of activated B cells), or a group thereof.

[0349] B may be an inhibitor of collagen synthesis, or a group thereof.

[0350] B is linked to L via a hydroxyl group.

[0351] The PI-3 kinase inhibitor (or a group thereof) (eg, a compound or conjugate comprising the PI-3 kinase inhibitor (or a group thereof)) may have the structure of Formula III:

[0352]

[0353] in:

[0354] X is selected from:

[0355]

[0356] X can be a group of B (eg, where the group is on a heteroatom (eg, S, N, or O of X)). B can be linked to L through X (eg, a hydroxyl group of X).

[0357] A compound (eg, a conjugate) may have the following structure:

[0358] Table 2

[0359]

[0360]

[0361] in:

[0362] X is selected from:

[0363]

[0364] The PI-3 kinase inhibitor (or a group thereof) (e.g., a compound or conjugate comprising the PI-3 kinase inhibitor (or a group thereof)) may have the following structure:

[0365]

[0366] A compound (eg, a conjugate) may have the following structure:

[0367] Table 3

[0368]

[0369]

[0370] A compound (eg, a conjugate) may have the following structure:

[0371] Table 4

[0372]

[0373] A compound (eg, a conjugate) may have the following structure:

[0374]

[0375] A compound (eg, a conjugate) may have the following structure:

[0376]

[0377] Treatment

[0378] Also provided is a method for treating an inflammatory disease or condition. The method is used to treat an inflammatory disease or condition by regulating the activity of activated fibroblasts. The method may include administering a compound (e.g., a conjugate) of any formula (e.g., formula (I), formula (IA), formula (IB), formula (IC), formula (II), formula (III), formula (X), formula (XA), formula (XB), Table 2, Table 3, or Table 4) provided herein.

[0379] A method for treating cancer is provided. The method for treating cancer can be by regulating the activity of activated fibroblasts. The method may include any formula (e.g., formula (I), formula (IA), formula (IB), formula (IC), formula (II), formula (III), formula (X), formula (XA), formula (XB), table 2, table 3 or table 4) provided herein The compound (e.g., conjugate). The method may include contacting cancer activated fibroblasts (CAF) (e.g., CAF of cancer patients) with any formula (e.g., formula (I), formula (IA), formula (IB), formula (IC), formula (II), formula (III), formula (X), formula (XA), formula (XB), table 2, table 3 or table 4) provided herein The compound (e.g., conjugate)

[0380] Also provided is a method for treating fibrosis. The method for treating fibrosis can be by regulating the activity of activated fibroblasts. The method may include administering a compound (e.g., a conjugate) of any formula (e.g., formula (I), formula (IA), formula (IB), formula (IC), formula (II), formula (III), formula (X), formula (XA), formula (XB), Table 2, Table 3, or Table 4) provided herein.

[0381] The method may be chemotherapy or radiation therapy.

[0382] Methods for imaging cancer or fibrosis in a subject having cancer or fibrosis are provided.

[0383] Compositions and methods for optical imaging are also provided. The compositions and methods can be used for fluorescence guided surgery. The compositions and methods can be used for radiological imaging.

[0384] The above method includes the following steps: providing a pharmaceutically effective amount of conjugate ALB to a patient in need thereof, wherein A comprises a fibroblast activation protein alpha (FAPα) targeting portion with a molecular weight of less than 10,000; L comprises a bifunctionalized linker that can form a chemical bond with A and B; and B comprises an optical dye (e.g., a fluorescent dye), a photodynamic therapy agent, a radioimaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an anti-fibrotic agent, or an anti-cancer agent that is effective against cancer cells or cancer-associated fibroblasts, myofibroblasts or other tumor microenvironmental factors.

[0385] Pharmaceutical compositions, routes of administration and dosing

[0386] In certain embodiments, the present disclosure relates to pharmaceutical compositions comprising a compound and a pharmaceutically acceptable carrier. In certain embodiments, a pharmaceutical composition comprises a plurality of compounds and a pharmaceutically acceptable carrier.

[0387] In certain embodiments, the pharmaceutical composition further comprises at least one additional pharmaceutically active agent.The at least one additional pharmaceutically active agent may be an agent useful for treating ischemia-reperfusion injury.

[0388] Pharmaceutical compositions can be prepared by combining one or more compounds with a pharmaceutically acceptable carrier and, optionally, one or more additional pharmaceutically active agents.

[0389] As mentioned above, "effective amount" refers to any amount sufficient to achieve the desired biological effect. In conjunction with the teachings provided herein, by selecting and weighing factors such as efficacy, relative bioavailability, patient weight, severity of adverse side effects and mode of administration among a variety of active compounds, an effective preventive or therapeutic treatment regimen can be planned that does not cause significant undesirable toxicity, but is effective for treating a specific subject. The effective amount of any particular application may vary according to such factors as the disease or condition being treated, the specific compound being administered, the size of the object, or the severity of the disease or condition. Those of ordinary skill in the art can empirically determine the effective amount of a specific compound and / or other therapeutic agent without excessive experimentation. A maximum dose may be used, i.e., the highest safe dose according to some medical judgment. Multiple doses per day may be considered to reach the appropriate systemic level of the compound. Appropriate systemic levels may be determined, for example, by measuring the peak or sustained plasma levels of the drug in the patient. "Dose" and "dosage" are used interchangeably herein.

[0390] Typically, for human subjects, the daily oral dose of the compound is about 0.01 mg / kg / day to 1,000 mg / kg / day. An oral dose of 0.5 to 50 mg / kg in one or more administrations per day can produce therapeutic results. Depending on the mode of administration, the dosage can be appropriately adjusted to achieve the desired drug level locally or systemically. For example, intravenous administration can vary from a lower dose of one order of magnitude to several orders of magnitude per day. If the response of the subject is insufficient at such a dose, an even higher dose (or an effectively higher dose achieved by a different, more localized delivery route) can be used until the patient's tolerance allows. Consider multiple doses per day to achieve the appropriate systemic level of the compound.

[0391] For any compound, the therapeutically effective amount can be initially determined from an animal model. The therapeutically effective dose can also be determined from human data of compounds that have been tested in humans and compounds known to exhibit similar pharmacological activity, such as other related active agents. Higher doses may be required for parenteral administration. The dose applied can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is fully within the capabilities of ordinary technicians to adjust the dose to achieve maximum efficacy based on the above methods and other methods as known in the art.

[0392] For clinical use, any compound can be administered in an amount equal to or equivalent to 0.2 to 2,000 milligrams (mg) of compound / kilogram (kg) of subject body weight / day. The compound can be administered in a dose equal to or equivalent to 2 to 2,000 mg of compound / kg of subject body weight / day. The compound can be administered in a dose equal to or equivalent to 20 to 2,000 mg of compound / kg of subject body weight / day. The compound can be administered in a dose equal to or equivalent to 50 to 2,000 mg of compound / kg of subject body weight / day. The compound can be administered in a dose equal to or equivalent to 100 to 2,000 mg of compound / kg of subject body weight / day. The compound can be administered in a dose equal to or equivalent to 200 to 2,000 mg of compound / kg of subject body weight / day. When a precursor or prodrug of a compound is to be administered, it is administered in an amount equal to (i.e., sufficient to) deliver the above amount of the compound.

[0393] The preparation of the compound can be administered to a human subject in a therapeutically effective amount. Typical dosage ranges are from about 0.01 micrograms / kg body weight / day to about 2 mg / kg body weight / day. The drug dosage to be administered may depend on such variables, such as the type and degree of the disease, the overall health of the particular subject, the particular compound administered, the excipient used to prepare the compound, and its route of administration. Routine experiments can be used to optimize the dosage and dosing frequency of any particular compound.

[0394] The compound can be administered at a concentration of about 0.001 microgram / kg to greater than about 500 mg / kg. For example, the concentration can be 0.001 microgram / kg, 0.01 microgram / kg, 0.05 microgram / kg, 0.1 microgram / kg, 0.5 microgram / kg, 1.0 microgram / kg, 10.0 microgram / kg, 50.0 microgram / kg, 100.0 microgram / kg, 500 microgram / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, 20.0 mg / kg, 25.0 mg / kg, 30.0 mg / kg, 35.0 mg / kg, In some embodiments, the present invention relates to an aqueous solution of at least about 1,000 mg / kg, or a combination thereof. The aqueous solution of at least about 1,000 mg / kg, or a combination thereof is preferably at least about 2,000 mg / kg, or a combination thereof. The aqueous solution of at least about 1,000 mg / kg, or a combination thereof is preferably at least about 2,000 mg / kg, or a combination thereof. The aqueous solution of at least about 1,000 mg / kg, or a combination thereof is preferably at least about 2,000 mg / kg, or a combination thereof.

[0395] The compound can be administered at a dosage of about 0.2 mg / kg / day to greater than about 100 mg / kg / day. For example, the dosage can be 0.2 mg / kg / day to 100 mg / kg / day, 0.2 mg / kg / day to 50 mg / kg / day, 0.2 mg / kg / day to 25 mg / kg / day, 0.2 mg / kg / day to 10 mg / kg / day, 0.2 mg / kg / day to 7.5 mg / kg / day, 0.2 mg / kg / day to 5 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day, 0.25 mg / kg / day to 50 mg / kg / day, 0.25 mg / kg / day to 2 ... day to 10mg / kg / day, 0.25mg / kg / day to 7.5mg / kg / day, 0.25mg / kg / day to 5mg / kg / day, 0.5mg / kg / day to 50mg / kg / day, 0.5mg / kg / day to 25mg / kg / day, 0.5mg / kg / day to 20mg / kg / day, 0.5mg / kg / day to 15mg / kg / day, 0.5mg / kg / day to 10mg / kg / day, 0.5mg / kg / day to 7.5mg / kg / day, 0.5mg / kg / day to 5mg / kg / day, 0.75mg / kg / day to 50mg / kg / day, 0.75mg / kg / day to 25mg / kg / day, 0.75mg / kg / day to 20mg / kg / day, 0.75mg / kg / day to 15mg / kg / day, 0.75mg / kg / day to 10mg / kg / day, 0.75mg / kg / day to 7.5mg / kg / day, 0.75mg / kg / day to 5mg / kg / day, 1.0mg / kg / day to 50mg / kg / day, 1.0mg / kg / day to 25mg / kg / day, 1.0mg / kg / day to 20mg / kg / day, 1.0mg / kg / day to 25mg / kg / day In some embodiments, the present invention may be used in combination with any one of the following: 1.0 mg / kg / day to 15 mg / kg / day, 1.0 mg / kg / day to 10 mg / kg / day, 1.0 mg / kg / day to 7.5 mg / kg / day, 1.0 mg / kg / day to 5 mg / kg / day, 2 mg / kg / day to 50 mg / kg / day, 2 mg / kg / day to 25 mg / kg / day, 2 mg / kg / day to 20 mg / kg / day, 2 mg / kg / day to 15 mg / kg / day, 2 mg / kg / day to 10 mg / kg / day, 2 mg / kg / day to 7.5 mg / kg / day, or 2 mg / kg / day to 5 mg / kg / day.

[0396] The compound can be administered at a dosage of about 0.25 mg / kg / day to about 25 mg / kg / day. For example, the dosage can be 0.25 mg / kg / day, 0.5 mg / kg / day, 0.75 mg / kg / day, 1.0 mg / kg / day, 1.25 mg / kg / day, 1.5 mg / kg / day, 1.75 mg / kg / day, 2.0 mg / kg / day, 2.25 mg / kg / day, 2.5 mg / kg / day, 2.75 mg / kg / day, 3.0 mg / kg / day, 3.25 mg / kg / day, 3.5 mg / kg / day, 3.75 mg / kg / day, 4.0 mg / kg / day, 4.25 mg / kg / day, 4.5 mg / kg / day, 4.75 mg / kg / day, 4.0 ...25 mg / kg / day, 4.5 mg / kg / day, 4.75 mg / kg / day, 4.0 mg / kg / day, 4.25 mg / kg / day, 4.5 mg / kg / day, 4.75 mg / kg / day, 4.0 mg / kg / day / kg / day, 4.25mg / kg / day, 4.5mg / kg / day, 4.75mg / kg / day, 5mg / kg / day, 5.5mg / kg / day, 6.0mg / kg / day, 6.5mg / kg / day, 7.0mg / kg / day, 7.5mg / kg / day, 8.0mg / kg / day, 8.5mg / kg / day, 9.0mg / kg / day, 9.5mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, 13mg / kg / day, 14mg / kg / day, 15mg / kg / day, 16mg / kg / day, 17mg / kg / day, 18mg / kg / day, 19mg / kg / day, 20mg / kg / day, 21mg / kg / day, 22mg / kg / day, 23mg / kg / day, 24mg / kg / day, 25mg / kg / day, 26mg / kg / day, 27mg / kg / day, 28mg / kg / day, 29mg / kg / day, 30mg / kg / day, 31mg / kg / day, 32mg / kg / day 50 mg / kg / day, 51 mg / kg / day, 52 mg / kg / day, 53 mg / kg / day, 54 mg / kg / day, 55 mg / kg / day, 56 mg / kg / day, 57 mg / kg / day, 58 mg / kg / day, 59 mg / kg / day, 60 mg / kg / day, 61 mg / kg / day, 62 mg / kg / day, 63 mg / kg / day, 64 mg / kg / day, 65 mg / kg / day, 66 mg / kg / day, 67 mg / kg / day, 68 mg / kg / day, 69 mg / kg / day or 70 mg / kg / day.

[0397] The compound or its precursor can be administered at a concentration of 0.01 micromolar to greater than or equal to 500 micromolar. For example, the dosage can be 0.01 micromolar, 0.02 micromolar, 0.05 micromolar, 0.1 micromolar, 0.15 micromolar, 0.2 micromolar, 0.5 micromolar, 0.7 micromolar, 1.0 micromolar, 3.0 micromolar, 5.0 micromolar, 7.0 micromolar, 10.0 micromolar, 15.0 micromolar, 20.0 micromolar, 25.0 micromolar, 30.0 micromolar, 35.0 micromolar, 4 0.0 micromolar, 45.0 micromolar, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 150.0 micromolar, 200.0 micromolar, 250.0 micromolar, 300.0 micromolar, 350.0 micromolar, 400.0 micromolar, 450.0 micromolar to greater than about 500.0 micromolar or any incremental value thereof. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be encompassed.

[0398] The compound or precursor thereof may be administered at a concentration of 0.10 micrograms / mL to 500.0 micrograms / mL. For example, the concentration can be 0.10 micrograms / mL, 0.50 micrograms / mL, 1 microgram / mL, 2.0 micrograms / mL, 5.0 micrograms / mL, 10.0 micrograms / mL, 20 micrograms / mL, 25 micrograms / mL., 30 micrograms / mL, 35 micrograms / mL, 40 micrograms / mL, 45 micrograms / mL, 50 micrograms / mL, 60.0 micrograms / mL, 70.0 micrograms / mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 150.0 micrograms / mL, 200.0 micrograms / mL, 250.0 micrograms / mL, 250.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 400.0 micrograms / mL, 450.0 micrograms / mL, to greater than about 500.0 micrograms / mL or any incremental value thereof. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be encompassed.

[0399] The preparation can be applied in a pharmaceutically acceptable solution, which can conventionally contain pharmaceutically acceptable concentrations of salt, buffers, preservatives, compatible carriers, excipients and optional other therapeutic ingredients. For treatment, an effective amount of the compound can be applied to the object by any means by which the compound is delivered to the desired surface. The administration of the pharmaceutical composition can be completed by any means known to those skilled in the art. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection (e.g., into a tumor or abscess), mucosal (e.g., ocular surface), inhalation and surface.

[0400] For intravenous and other parenteral administration routes, the compound can be formulated as a lyophilized preparation, a lyophilized preparation of the active compound embedded or encapsulated by liposomes, a lipid complex in an aqueous suspension, or a salt complex. The lyophilized preparation is usually reconstituted in a suitable aqueous solution (e.g., in sterile water or saline) shortly before administration.

[0401] For oral administration, the compound can be easily prepared by combining the active compound with a pharmaceutically acceptable carrier known in the art. Such a carrier enables the compound to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries (slurry), suspensions, etc., for oral ingestion by the object to be treated. The pharmaceutical preparation for oral use can be obtained as a solid excipient, and the resulting mixture is optionally ground, and if desired, the mixture of particles is processed after adding a suitable auxiliary agent to obtain a tablet or dragee core. Suitable excipients are particularly fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If desired, a disintegrant, such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or its salt (such as sodium alginate) can be added. Optionally, oral formulations may also be formulated in saline or buffer (eg, EDTA for neutralization of internal acidic conditions), or may be administered without any carrier.

[0402] Oral dosage forms of the compounds are also contemplated. The compounds may be chemically modified so that oral delivery of the derivatives is effective. Generally speaking, the chemical modification contemplated is the attachment of at least one moiety to the compound itself, wherein the moiety allows (a) inhibition of acid hydrolysis; and (b) uptake from the stomach or intestine into the bloodstream. It is also desirable to improve the overall stability of the compound and increase the in vivo circulation time. Some examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, and polyproline. Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts," In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367 to 383 (1981); Newmark et al., J Appl Biochem 4: 185-189 (1982). Other polymers that may be used are poly-1,3-dioxolane and poly-1,3,6-trioxocane.For pharmaceutical uses, as indicated above, polyethylene glycol moieties are suitable.

[0403] The site of compound release can be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. Formulations that will not dissolve in the stomach but will release the substance elsewhere in the duodenum or intestine are available to those skilled in the art. Release can be by protecting the compound or by releasing the compound outside the gastric environment (e.g., in the intestine) to avoid the deleterious effects of the gastric environment.

[0404] To ensure complete gastric resistance, a coating that is impermeable at at least pH 5.0 is necessary. Some examples of more common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulosephthalate (HPMCP), HPMCP 50, HPMCP55, polyvinyl acetatephthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings can be used as mixed films.

[0405] Coatings or coating mixtures that are not intended to provide protection against the stomach may also be used on tablets. This may include sugar coatings or coatings that make the tablet easier to swallow. Capsules may consist of a hard shell (e.g., gelatin) for delivery of dry therapeutic agents (e.g., powders); for liquid forms, a soft gelatin shell may be used. The shell material for cachets may be thick starch paper or other edible paper. For pills, lozenges, molded tablets, or tablet triturates, moist massing techniques may be used.

[0406] The therapeutic agent may be included in the formulation as finely divided multiparticulates in the form of granules or pellets of about 1 mm particle size. The formulation of the material for capsule administration may also be a powder, a lightly compressed plug or even a tablet. The therapeutic agent may be prepared by compression.

[0407] Both colorants and flavoring agents may be included. For example, the compound may be formulated (eg, by liposome or microsphere encapsulation) and then further included in an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

[0408] The therapeutic agent can be diluted with an inert material or the volume of the therapeutic agent can be increased. These diluents can include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran and starch. Certain inorganic salts can also be used as fillers, including tricalcium phosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0409] Disintegrants may be included in the formulation of the therapeutic agent into a solid dosage form. Materials used as disintegrants include, but are not limited to, starch, including the commercial disintegrant Explotab based on starch. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge, and bentonite may all be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered gum may be used as a disintegrant and a binder, and these may include powdered gum, such as agar, karaya, or tragacanth. Alginic acid and its sodium salt may also be used as a disintegrant.

[0410] Binders can be used to hold the therapeutic agent together to form a hard tablet, and include materials from natural products such as gum arabic, gum tragacanth, starch, and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC), and carboxymethyl cellulose (CMC). Both polyvinyl pyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) can be used to granulate the therapeutic agent in an alcohol solution.

[0411] Anti-frictional agents may be included in the formulation of the therapeutic agent to prevent sticking during formulation. Lubricants may be used as a layer between the therapeutic agent and the die wall, and these may include, but are not limited to: stearic acid (including its magnesium and calcium salts), polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000.

[0412] Glidants may be added which may improve the flow characteristics of the drug during formulation and aid in rearrangement during compression. Glidants may include starch, talc, pyrogenic silica, and hydrated aluminosilicates.

[0413] To help dissolve the therapeutic agent into the aqueous environment, a surfactant may be added as a wetting agent. The surfactant may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents that may be used include benzalkonium chloride and benzethonium chloride. Potential nonionic detergents that may be included in the formulation as surfactants include lauromacrogol 400; polyoxyl 40 stearate; polyoxyethylene hydrogenated castor oil 10, 50, and 60; glyceryl monostearate; polysorbate 40, 60, 65, and 80; sucrose fatty acid esters; methylcellulose and carboxymethylcellulose. These surfactants may be present in the formulation of the compound or its derivatives alone or as a mixture in different proportions.

[0414] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and a plasticizer (e.g., glycerol or sorbitol). Push-fit capsules may contain a mixture of the active ingredient with a filler (e.g., lactose), a binder (e.g., starch), and / or a lubricant (e.g., talc or magnesium stearate), and optionally a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, a stabilizer may be added. Microspheres formulated for oral administration may also be used. Such microspheres are well defined in the art. All formulations for oral administration should be doses suitable for such administration.

[0415] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0416] For topical administration, the compounds can be formulated into solutions, gels, ointments, creams, suspensions, etc. as known in the art. Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection), as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.

[0417] For administration by inhalation, the compound may be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base (e.g., lactose or starch).

[0418] Pulmonary delivery of the compound (or its salt) is also contemplated. The compound is delivered to the lungs of a mammal upon inhalation and crosses the epithelial lining of the lung into the bloodstream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13 (Suppl 5):143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989) (a1-antitrypsin); Smith et al., 1989, J Clin Invest 84:1145-1146 (a-1-proteinase); Oswein et al., 1990, "Aerosolization of Proteins," Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-γ and tumor necrosis factor α) and Platz et al., US Pat. No. 5,284,656 (granulocyte colony stimulating factor; incorporated by reference). Methods and compositions for pulmonary delivery of drugs for systemic effect are described in U.S. Pat. No. 5,451,569, issued September 19, 1995 to Wong et al. (specifically incorporated by reference for its disclosure therein).

[0419] A variety of mechanical devices designed for pulmonary delivery of therapeutic products are contemplated for use, including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

[0420] Nasal delivery of pharmaceutical compositions is also contemplated. Nasal delivery allows the pharmaceutical composition to enter the bloodstream directly after administering the therapeutic product to the nose, without the need for deposition of the product in the lungs. Formulations for nasal delivery include formulations with dextran or cyclodextrin.

[0421] When systemic delivery of the compound is desired, the compound may be formulated for parenteral administration by injection (e.g., by bolus or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multidose containers) with the addition of preservatives. The composition may take the form of a suspension, solution, or emulsion, for example, in an oily or aqueous vehicle, and may contain preparatants, such as suspending agents, stabilizers, and / or dispersants.

[0422] Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compound in water-soluble form. In addition, the suspension of the active compound can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or carriers include fatty oils (e.g., sesame oil), or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides), or liposomes. Aqueous injection suspensions may include substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also include a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a highly concentrated solution.

[0423] Alternatively, the active compound may be in powder form for constitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.

[0424] The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0425] In addition to the formulations described above, the compounds may also be formulated as a depot preparation. Such long-acting preparations may be formulated using suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (for example, as a sparingly soluble salt).

[0426] The pharmaceutical composition may also contain a suitable solid or gel phase carrier or excipient. Some examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers, such as polyethylene glycol.

[0427] Suitable liquid or solid pharmaceutical preparations are in the form of, for example, aqueous or saline solutions for inhalation; microencapsulated, encapsulated, coated on microscopic gold particles, contained in liposomes, atomized (nebulized) aerosols; pellets for implantation into the skin or drying to a sharp object to be scratched into the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with extended release of active compounds, in which the above-mentioned excipients and additives and / or adjuvants are customarily used, such as disintegrants, adhesives, coatings, swelling agents, lubricants, flavoring agents, sweeteners or solubilizers. Pharmaceutical compositions are suitable for a variety of drug delivery systems. For a brief review of drug delivery methods, see Langer R, Science 249: 1527-1533 (1990).

[0428] The compound and optionally one or more other therapeutic agents can be administered as such (pure) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare its pharmaceutically acceptable salt. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. Similarly, such salts can be prepared as alkali metal or alkaline earth salts, such as sodium, potassium or calcium salts of carboxylic acid groups.

[0429] Suitable buffers include: acetic acid and salt (1% to 2% w / v); citric acid and salt (1% to 3% w / v); boric acid and salt (0.5% to 2.5% w / v); and phosphoric acid and salt (0.8% to 2% w / v). Suitable preservatives include benzalkonium chloride (0.003% to 0.03% w / v); chlorobutanol (0.3% to 0.9% w / v); parabens (0.01% to 0.25% w / v) and thimerosal (0.004% to 0.02% w / v).

[0430] The pharmaceutical composition contains an effective amount of a compound as described herein and optionally one or more other therapeutic agents contained in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic organic or inorganic ingredient with which the active ingredient is combined to facilitate application. The components of the pharmaceutical composition may also be mixed with the compound, as well as with each other, in a manner such that there is no interaction that would significantly impair the desired drug efficacy.

[0431] The therapeutic agent (specifically including but not limited to compounds) can be provided as particles. "Particles" used herein means nanoparticles or microparticles (or larger particles in some cases) that can be composed of the compound or other therapeutic agents as described herein in whole or in part. The particles can contain therapeutic agents in a core surrounded by a coating (including but not limited to an enteric coating). The therapeutic agent can also be dispersed throughout the particles. The therapeutic agent can also be adsorbed into the particles. The particles can have release kinetics of any order, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof. In addition to the therapeutic agent, the particles can also include any of those materials conventionally used in the field of pharmacy and medicine, including but not limited to erodible, non-erodible, biodegradable or non-biodegradable materials or combinations thereof. The particles can be microcapsules containing compounds in a solution or semi-solid state. The particles can actually be in any shape.

[0432] Both non-biodegradable and biodegradable polymer materials can be used to prepare particles for delivering therapeutic agents. Such polymers can be natural or synthetic polymers. Polymers are selected based on the time period of the desired release. Particularly interesting bioadhesive polymers are included in Sawhney et al., Macromolecules 26: The bioerodible hydrogel described in 581-587 (1993), which is particularly incorporated herein by reference. These include polyhyaluronic acid, casein, gelatin, gelatin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate) and poly (octadecyl acrylate).

[0433] The therapeutic agent may be included in a controlled release system. The term "controlled release" is intended to refer to any preparation containing a drug, wherein the mode and spectrum of the drug released from the preparation is controlled. This refers to immediate release preparations and non-immediate release preparations, wherein non-immediate release preparations include, but are not limited to, sustained release and delayed release preparations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug preparation that provides a gradual release of the drug over an extended period of time and can result in a substantially constant blood level of the drug over an extended period of time. The term "delayed release" is used in its conventional sense to refer to a drug preparation wherein there is a time delay between the administration of the preparation and the release of the drug therefrom. "Delayed release" may or may not involve the gradual release of the drug over an extended period of time, and therefore may or may not be "sustained release".

[0434] The use of long-term sustained release implants (implant) can be particularly suitable for treating chronic conditions. "Long-term" release used herein means that the implant is constructed and arranged to deliver the active ingredient of the treatment level for at least 7 days, and up to 30 to 60 days. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the above-mentioned release systems.

[0435] Those of ordinary skill in the relevant art will appreciate that other suitable modifications and adaptations of the compositions and methods described herein will readily emerge from the descriptions contained herein, in view of the information known to those of ordinary skill, and may be made without departing from the scope of the present disclosure or any embodiment thereof. Having now described the present disclosure in detail, the present disclosure will be more clearly understood by reference to the following examples, which are included herein for illustrative purposes only and are not intended to limit the present disclosure. Example

[0436] Chemical Examples:

[0437] Embodiment 1:

[0438] Synthesis of trans-2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid:

[0439] Trans-2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid was synthesized according to Scheme 1.

[0440] Solution 1

[0441]

[0442] Step I:

[0443] Benzyl (2S)-N-tert-butoxycarbonylpyroglutamate (Compound 1, 5 grams (g), 15.67 millimoles (mmol)) was dissolved in tetrahydrofuran (THF, 75 milliliters (mL)) with stirring under nitrogen and cooled to -78°C. Lithium bis(trimethylsilyl)amide (LiHMDS) (1.0 M in THF, 34.5 mL, 34.5 mmol) was added dropwise over 5 minutes and stirring was continued for 1 hour. Tert-butyl bromoacetate (4.36 mL, 31.34 mmol) was added dropwise over 5 minutes and stirring was continued at -78°C for another 2 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride solution (100 mL) and extracted into ethyl acetate (2×100 mL). The organic layer was washed with water and saturated aqueous sodium chloride solution and then dried over anhydrous magnesium sulfate (MgSO4). The solvent was removed in vacuo to give a brown oil which was purified by combi flash on silica gel chromatography (eluting with hexanes / EtOAc) to give a mixture of compound 2a (trans-2-benzyl 1-(tert-butyl) (2S,4S)-4-(2-(tert-butoxy)-2-oxoethyl)-5-oxopyrrolidine-1,2-dicarboxylate) and compound 2b (cis-2-benzyl 1-(tert-butyl) (2S,4S)-4-(2-(tert-butoxy)-2-oxoethyl)-5-oxopyrrolidine-1,2-dicarboxylate) (6.2 gm, 89%) as a gummy liquid.

[0444] Step II:

[0445] A mixture of compound 2a / 2b (6 g, 13.85 mmol) was dissolved in CH2Cl2 (90 mL) and cooled to 0°C. DBU (6.2 mL, 41.55 mmol) was added dropwise and the mixture was stirred at 0°C for 30 minutes, and then at room temperature for 24 hours. The reaction mixture was diluted with CH2Cl2 (20 mL) and washed with water (100 mL). The organic layer was dried, filtered and concentrated in vacuo with MgSO4 to give a crude product, which was purified by column chromatography as described in step 1 to give trans-2-benzyl 1-(tert-butyl) (2S, 4S)-4-(2-(tert-butoxy)-2-oxoethyl)-5-oxopyrrolidine-1,2-dicarboxylate (compound 3, 4.5 g, 75%) as a pale yellow liquid.

[0446] Step III:

[0447] To a solution of compound 3 (4 g, 9.23 mmol) in MeOH (40 mL) was added 5% Pd / C (400 mg) under a nitrogen atmosphere. The mixture was stirred vigorously at room temperature under a hydrogen atmosphere for 12 hours. The mixture was filtered through a diatomaceous earth pad and concentrated in vacuo to obtain trans-(2S, 4S)-4-(2-(tert-butoxy)-2-oxoethyl)-1-(tert-butoxycarbonyl)-5-oxopyrrolidine-2-carboxylic acid (compound 4, 2.7 g, 87%), as a white solid.

[0448] Step IV:

[0449] To a stirred solution of compound 4 (2.5 g, 7.28 mmol) in anhydrous DMF (20 mL) was added HATU (3.3 g, 8.7 mmol), DIPEA (3.6 mL, 21.84 mmol) and stirring was continued for 10 min for activation of the acidic functional groups. (S)-4,4-difluoropyrrolidine-2-carboxamide hydrochloride (1.3 g, 8.736 mmol) followed by DIPEA (1.46 mL, 8.73 mmol) was added to the above reaction mixture and stirring was continued for 5 hours at room temperature under nitrogen atmosphere. The reaction mixture was diluted with water (30 mL), brine (30 mL) and extracted into ethyl acetate (2×50 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered and the filtrate was evaporated under reduced pressure and the crude residue obtained was purified by combi flash using DCM / MeOH as mobile phase to afford trans-tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((S)-2-carbamoyl-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (5, 2.4 g, 88%) as a gummy solid.

[0450] Step V:

[0451] Phosphorus oxychloride (POCl3) (1.02 mL, 10.94 mmol) was added to a mixture of compound 5 (2 g, 4.21 mmol) and imidazole (333 milligrams (mg), 4.73 mmol) in pyridine (13 mL) cooled to -20°C under nitrogen. After stirring at -20°C for 30 minutes to 1 hour, the mixture was evaporated to dryness in vacuo. The resulting brown solid was dissolved in CH2Cl2 (40 mL) and washed with 1.0 N aqueous citric acid solution (40 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a crude material as a viscous oil. The crude material was purified by combi flash (eluting with hexanes / EtOAc) to give trans-tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (Compound 6, 1.3 g, 68%) as a white solid.

[0452] Step VI:

[0453] To a solution of compound 6 (1.0 g, 2.1 mmol) in acetonitrile (CH3CN) (5 mL) was added TFA (5 mL) dropwise at 0 ° C. in 5 minutes. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum and crystallized from ethyl acetate (EA) / ether to obtain trans-2-((3S, 5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-3-yl)acetic acid (compound 7, 500 mg, 76%) as a white powder.

[0454] Embodiment 2:

[0455] Synthesis of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate and tert-butyl 4-(aminomethyl)isoindoline-2-carboxylate:

[0456] According to Scheme 2, tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate and tert-butyl 4-(aminomethyl)isoindoline-2-carboxylate were synthesized.

[0457] Solution 2

[0458]

[0459] Step I

[0460] To a stirred solution of isoindoline methyl ester hydrochloride (compound 8, 1.00 g, 5.64 mmol) in DCM (20 mL) was added Boc2O (4.9 mL, 22.59 mmol) at room temperature, followed by dropwise addition of triethylamine (2.9 mL, 22.59 mmol). Stirring was continued for 12 hours, and the reaction mixture was diluted with water (30 mL) and extracted into DCM (2×25 mL). The organic layer was dried over anhydrous MgSO4 and filtered, and the filtrate was evaporated under reduced pressure. The crude residue obtained was purified by combi flash using hexane and ethyl acetate as mobile phases to provide 2-(tert-butyl) 4-methylisoindoline-2,4-dicarboxylate (compound 9, 1.2 g, 92%) as a white, colorless, colloidal liquid. LC-MS (m / z); [M+H] calculated value C 15 H 20 Actual measured value of NO4: 278.13g / mol.

[0461] Step II:

[0462] To a stirred solution of 2-(tert-butyl) 4-methylisoindoline-2,4-dicarboxylate (Compound 9, 1.0 g, 3.61 mmol) in THF (10.0 mL) was added sodium borohydride (1.37 g, 36.101 mmol) in a nitrogen atmosphere at room temperature. Methanol (MeOH, 10 mL) was added dropwise to the stirred mixture over 5 minutes. The reactants were warmed to 55 ° C and stirred for 5 hours. The reaction mixture was cooled to 0 ° C and slowly quenched with saturated aqueous ammonium chloride solution and extracted into EtOAc (60 mL). The organic phase was collected, dried over sodium sulfate and the solvent was distilled to provide a crude residue, which was purified by combi flash to give tert-butyl 4-(hydroxymethyl)isoindoline-2-carboxylate (Compound 10, 700 mg, 70%) as a white colloidal solid. LC-MS (m / z): [M+H]: Calculated value: C 14 H 20 Actual measured value of NO3: 250.14g / mol.

[0463] Step III:

[0464] Tert-butyl 4-(hydroxymethyl)isoindoline-2-carboxylate (Compound 10, 500 mg, 2.00 mmol) was dissolved in DMF (10 mL), followed by the addition of PPh3 (790 mg, 3.01 mmol) and freshly recrystallized NBS (532 mg, 3.01 mmol). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 4 to 5 hours. The reaction mixture was diluted with water (40 mL) and extracted into ethyl acetate (2×25 mL), and the organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated under reduced pressure and purified by combi flask to provide tert-butyl 4-(bromomethyl)isoindoline-2-carboxylate (Compound 11, 450 mg, 72%) as a white solid. LC-MS (m / z): [M+H] Calculated: C 14 H 19 Measured value of BrNO2: 312.05 g / mol.

[0465] Step IV:

[0466] To a stirred solution of tert-butyl 4-(bromomethyl)isoindoline-2-carboxylate (compound 11, 400 mg, 1.286 mmol) in DMF was added NaN3 (420 mg, 6.430 mmol), and stirring was continued at 65°C for 6 hours. The reaction mixture was diluted with water and extracted into ethyl acetate, and the organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated under reduced pressure and purified by combi flask to give tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (compound 12a, 300 mg, 85%) as a colorless colloidal liquid. LC-MS (m / z): [M+H] calcd. C 14 H 19 Actual measured value of N4O2: 274.14 g / mol.

[0467] Step V:

[0468] To a stirred solution of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (compound 12a, 1.0 equiv) was added THF followed by PPh3 (1.5 equiv) and water (3.0 equiv) and stirring was continued at room temperature for 12 h. The reaction mixture was evaporated under reduced pressure and the crude residue obtained was purified by using a combi flask using methanol and dichloromethane as mobile phases to provide tert-butyl 4-(aminomethyl)isoindoline-2-carboxylate (compound 12b, 85%) as a white solid. LC-MS (m / z): [M+H] Calculated value C 14 H 21 Actual measured value of N2O2: 249.15 g / mol.

[0469] Embodiment 3:

[0470] 5-((2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-9,9a-dihydro-3H-xanthene-9-yl)benzoate was synthesized according to Scheme 3.

[0471] Synthesis of 5-((2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-9,9a-dihydro-3H-xanthene-9-yl)benzoate:

[0472] Solution 3

[0473]

[0474] Step I

[0475] To a stirred solution of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (Compound 12a) in DCM was added trifluoroacetic acid (TFA) and stirring was continued for 1 hour. In another flask, trans-2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid (Compound 7) was dissolved in DMF, followed by the addition of HATU (1.3 eq.) and DIPEA (3.0 eq.) for pre-activation of the acidic functional groups. The resulting amine was added to the mixture containing the pre-activated acid, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with water and extracted into ethyl acetate. The organic layer was evaporated and the crude residue obtained was purified by combiflash using methanol and DCM as mobile phase to afford (S)-1-((2S,4S)-4-(2-(4-(azidomethyl)isoindolin-2-yl)-2-oxoethyl)-5-oxopyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonitrile (Compound 13). LC-MS (m / z): Calculated value C 21 H 22 Actual measured value of F2N7O3: 458.17.

[0476] Step II:

[0477] Using the same procedure as provided in Example 2, Step V, (S)-1-((2S,4S)-4-(2-(4-(azidomethyl)isoindolin-2-yl)-2-oxoethyl)-5-oxopyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonitrile (Compound 13) was reacted with triphenylphosphine (PPh3), water, and tetrahydrofuran to produce (S)-1-((2S,4S)-4-(2-(4-(aminomethyl)isoindolin-2-yl)-2-oxoethyl)-5-oxopyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonitrile (Compound 14), which was carried on to the next step without purification. (S)-1-((2S,4S)-4-(2-(4-(aminomethyl)isoindolin-2-yl)-2-oxopyrrolidin-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonitrile (Compound 14) was reacted with 4-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-4-oxobutanoic acid, HATU (1.3 equiv), DIPEA (3.0 equiv), and DCM to provide tert-butyl (2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamate (Compound 15).

[0478] Step III

[0479] To a stirred solution of tert-butyl(2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamate (Compound 15) in DCM was added TFA and stirring was continued for 10 min. The reaction mixture was evaporated under reduced pressure and the crude residue obtained was treated with NHS rhodamine (1.0 eq) and DIPEA (2.0 eq) in DMF for 1 h. The reaction mixture was diluted with water and purified by UHPLC (A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile, solvent gradient 5% B to 95% in 60 minutes) to provide 5-((2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-9,9a-dihydro-3H-xanthene-9-yl)benzoate (Compound 16). LC-MS (m / z): [M+H] Calcd. C 52 H56 Measured value of F2N9O9: 988.41 g / mol.

[0480] Embodiment 4:

[0481] N-(9-(2-carboxy-4-((1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18-tetraoxa-2,5-diazaeicosan-20-yl)carbamoyl)phenyl)-6-(dimethylamino)-9,9a-dihydro-3H-xanthene was synthesized according to Schemes 4a and 4b. -3-ylidene)-N-methylmethanammonium and 5-((1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18-tetraoxa-2,5-diazaicosan-20-yl)carbamoyl)-2-(6-hydroxy-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoic acid.

[0482] N-(9-(2-carboxy-4-((1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18-tetraoxa-2,5-diazaeicosan-20-yl)carbamoyl)phenyl)-6-(dimethylamino)-9,9a-dihydro-3H-xanthen-3-ylidene)- Synthesis of N-methylmethanamine and 5-((1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18-tetraoxa-2,5-diazaeicosan-20-yl)carbamoyl)-2-(6-hydroxy-3-oxo-9,9a-dihydro-3H-xanthene-9-yl)benzoic acid:

[0483] Option 4a

[0484]

[0485] Step I

[0486] To a stirred solution of 4-ethynylbenzoic acid (compound 17, 500 mg, 3.424 mmol) in anhydrous DCM (10 mL) was added HATU (1.4 gm, 3.76 mmol) followed by DIPEA (1.7 mL, 10.27 mmol) at room temperature under nitrogen atmosphere and stirring was continued for 10 minutes for activation of the acid. N-Fmoc-ethylenediamine (1.0 g, 3.76 mmol) was added to the reaction mixture and stirring was continued for another 3 hours. The reaction mixture was diluted with DCM (20 mL) and the obtained precipitate was filtered through a Buchner funnel. The resulting white solid was washed again with DCM (2×20 mL) and dried under vacuum for 1 hour to provide (9H-fluoren-9-yl)methyl (2-(4-ethynylbenzamido)ethyl)carbamate (compound 18, 1.2 g, 85%). LRMS-LC / MS (m / z): [M+H] calculated value C 26 H 23 The measured value of N2O3 is 411.16.

[0487] Step II:

[0488] To a mixture of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (compound 12a, 200 mg, 0.729 mmol) and (9H-fluoren-9-yl)methyl(2-(4-ethynylbenzamido)ethyl)carbamate (compound 18, 360 mg, 0.874 mmol) in anhydrous DMF (5.0 mL) was added copper iodide (CuI, 70 mg, 0.364 mmol) followed by DIPEA (0.3 mL, 1.458 mmol). The reaction mixture was stirred at 50° C. under a nitrogen atmosphere for 5 hours, and the mixture was cooled to room temperature, diluted with water (20 mL), and stirred vigorously for 15 minutes. The solid residue formed in the reaction mixture was filtered and washed with water 2×20 mL and dried under vacuum for 1 hour to provide tert-butyl 4-((4-(4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)phenyl)-1H-1,2,3-triazol-1-yl)methyl)isoindoline-2-carboxylate (Compound 19, 480 mg, 96%) as a brown solid which was carried forward to the next step without purification. LRMS-LC / MS (m / z): [M+H] Calcd. C 40 H 41 The measured value of N6O5 is 684.31.

[0489] Step III:

[0490] To a stirred solution of compound tert-butyl 4-((4-(4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)phenyl)-1H-1,2,3-triazol-1-yl)methyl)isoindoline-2-carboxylate (compound 19, 400 mg, 0.584 mmol) in DCM and MeOH (1:0.5 mL) was added (Et)2NH (1 mL) and stirring was continued for 2 hours. The reaction mixture was evaporated under reduced pressure and the crude residue obtained was treated with ether (3×10 mL). Scratching the residue was used to produce a solid. The ether was decanted and the solid obtained was dried under vacuum for 1 hour before proceeding to the next step. The amine compound was dissolved in DCM (1 mL, 1 mmol) and then dissolved in Fmoc-NH(PEG) n NHS ester (1.2 eq., n=3, 5 and 11) was added and then dissolved in DIPEA (2.0 eq.), and the reaction mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. The reaction mixture was evaporated under reduced pressure and the obtained crude residue was purified by combi flash using DCM and MeOH as mobile phase to provide compounds 20a to c in 70% to 80% yield.

[0491] i) LCMS of 20a: LC / MS (m / z): [M+H] calculated value C 51 H 62 N7O 10 Actual measured value: 931.45.

[0492] ii) LCMS of 20b: LC / MS (m / z): [M+H] calculated value C 55 H 69 N7O 12 Actual measured value: 1020.50.

[0493] iii) LCMS of 20c: LC / MS (m / z): [M+H] calculated value C 67 H 93 N7O 18 Actual measured value: 1284.66.

[0494] Step IV:

[0495] At room temperature, TFA (10 equivalents) was added to a stirred solution of compound 20a, 20b or 20c (1.0 equivalents) in DCM (1.0 mL) and continued to stir for 30 minutes. The reaction mixture was evaporated and dried under vacuum. In a separate round-bottomed flask, trans-2-((3S, 5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-3-yl)acetic acid (compound 7, 1.2 equivalents) was dissolved in DMF (0.5 mL), then dissolved in HATU (1.3 equivalents) and DIPEA (5.0 equivalents), and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 10 minutes for activating the acidic functional groups in compound 7. The amine obtained from the Fmoc deprotection of compound 20a to c was dissolved in DCM (1 mL), added to the above-mentioned reaction mixture and continued to stir for another 2 hours. The reaction mixture was diluted with water (15 mL) and extracted into ethyl acetate (2×15 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated. The crude residue obtained was purified by combi flash using DCM and MeOH as mobile phase to give the desired compound (9H-fluoren-9-yl)methyl(1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)- 1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18-tetraoxa-2,5-diazaeicosane-20-yl)carbamate, (9H-fluoren-9-yl)methyl (1-(4-(1-((2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-3 -yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18,21,24-hexaoxa-2,5-diazacosano-26-yl)carbamate and (9H-fluoren-9-yl)methyl (1-(4-(1-((2-(2-((3S,5S)-5-((S)-2- cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18,21,24,27,30,33,36,39,42-dodeca-2,5-diazatetradecane-44-yl)carbamate.

[0496] i) LCMS of compound 21a: LC / MS (m / z): [M+H] calculated value: C 58 H65 F2N 10 O 11 Actual measured value: 1115.47.

[0497] ii) LCMS of compound 21b: LC / MS (m / z): [M+H] calculated value: C 62 H 72 F2N 10 O 13 Actual measured value: 1203.52.

[0498] iii) LCMS of compound 21c: LC / MS (m / z): [M+H] calculated value: C 74 H 97 F2N 10 O 19 Actual measured value: 1467.68.

[0499] Option 4b

[0500]

[0501] Step V:

[0502] Deprotection of the Fmoc functional group in compounds 21a to c was performed following the same procedure as provided in step III of Example 4. The free amine obtained from compound 21a was treated with NHS rhodamine (1.1 eq) or 1.1 eq NHS FITC in DMF in the presence of DIPEA (1.1 eq) for 30 min, followed by purification using UHPLC (A = 20 Mm ammonium acetate buffer (pH = 7), B = acetonitrile, solvent gradient 5% B to 95% in 60 min) to provide quantitative yields of targeted rhodamine compound 22 or FITC (23) conjugates, respectively. LCMS of compound 22: LC / MS (m / z): [M+H] calculated value C 68 H 78 F2N 12 O 13 Found: 1308.57 g / mol. LCMS of compound 23: LC / MS (m / z): [M+H] calculated value C 64 H 67 F2N 10 O 15 Measured value: 1253.47 g / mol.

[0503] Embodiment 5:

[0504] 2-((E)-2-((E)-2-(4-(1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6,21-trioxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6,21-trioxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl) (((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1-yl)-(((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1-yl) -5-Sodium sulfonate.

[0505] 2-((E)-2-((E)-2-(4-(1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6,21-trioxo-9, 1-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1-yl)-1-((2,14,17-tetraoxa-2,5,20-triazatricosan-23-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1-yl) -Synthesis of sodium 5-sulfonate:

[0506] Solution 5

[0507]

[0508] Step I:

[0509] The amine (1.0 equiv.) obtained from the reduction of compound 21a with diethylamine as provided in Example 4, step III, was dissolved in DMF. 3-(4-Hydroxyphenyl)propionic acid (1.2 equiv.), HATU (1.3 equiv.) and DIPEA (3.0 equiv.) were added, and the reaction mixture was stirred at room temperature for 2 to 3 hours and then evaporated under reduced pressure. The crude residue obtained was purified by using UHPLC (A = 20 Mm ammonium acetate buffer (pH = 7), B = acetonitrile, solvent gradient from 5% B to 95% in 60 minutes) to provide the desired compound 24a. LRMS-LC / MS (m / z): [M+H] calculated value C 50 H 59 F2N10 O 10 , measured value: 997.43.

[0510] Step II

[0511] To a stirred solution of compound 24a in anhydrous DMSO was added ClS0456 dye (1.0 eq.) at room temperature under argon atmosphere, followed by Cs2CO3 (5.0 eq.), and stirring was continued for another 3 to 4 hours, and the progress of the reaction was monitored by LCMS. The reaction mixture was diluted with water and purified by using UHPLC (A=20 Mm ammonium acetate buffer (pH=7), B=0 acetonitrile, solvent gradient 5% B to 35% in 60 minutes) to provide 2-((E)-2-((E)-2-(4-(1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl) (methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6,21-trioxo-9,12,14,17-tetraoxa-2,5,20-triazatricosan-23-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1-yl -5-sodium sulfonate (Compound 25a). LC-MS of 25a: LC / MS (m / z): [M+H] calculated value C 90 H 105 F2N 12 Na3O 23 S4:1957.60.

[0512] Embodiment 6:

[0513] 4-((2-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid was synthesized according to Scheme 6.

[0514] Synthesis of 4-((2-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid:

[0515] Solution 6

[0516]

[0517] Step I

[0518] The synthesis of (9H-fluoren-9-yl)methyl(2-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)carbamate (Compound 26) was carried out according to a procedure similar to that provided in Example 4, Step IV (LC / MS (m / z): [M+H] Calcd. C 47 H 44 F2N9O6 found: 868.33). The free amine obtained from the reduction of compound 26 using a similar procedure provided in step III of example 4 was dissolved in DCM followed by the addition of succinic anhydride (1.5 eq.) and DIPEA (2.0 eq.). The solution was stirred at room temperature for 1 hour. The reaction mixture was evaporated and the crude residue obtained was purified by UHPLC (A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile, solvent gradient 5% B to 95% in 60 minutes) to provide 4-((2-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid (compound 27). LC / MS (m / z): [M+H] calcd. 36 H 38 F2N9O7 measured value: 746.28.

[0519] Embodiment 7:

[0520] 2-((E)-2-((E)-2-(4-(1-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)-3,25-dioxo-6,9,12,15,18,21-hexaoxa-2,24-diazaheptacosano-27-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1-yl was synthesized according to Scheme 7. -5-Sodium sulfonate.

[0521] 2-((E)-2-((E)-2-(4-(1-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)-3,25-dioxo-6,9,12,15,18,21-hexaoxa-2,24-diazaheptacosane-27-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1- -Synthesis of sodium 5-sulfonate:

[0522] Solution 7

[0523]

[0524] Step 1:

[0525] To a stirred solution of 3-(4-(tert-butoxy)phenyl)propanoic acid (Compound 28) in anhydrous DMF was added HATU (1.3 eq.) and DIPEA (3.0 eq.), followed by amino-PEG6-tert-butyl ester (1.2 eq.). The reaction mixture was diluted with water and extracted into ethyl acetate (2×20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure. The crude residue was purified by using combi flash using methanol and dichloromethane as mobile phases to provide tert-butyl 3-(2-(2-(3-(4-(tert-butoxy)phenyl)propionamido)ethoxy)ethoxy)propanoate (Compound 29) as a colloidal liquid. LC / MS (m / z): [M+H]: calculated value C 32 H 56 NO 10 Actual measured value: 614.38.

[0526] Step II:

[0527] Tert-butyl 3-(2-(2-(3-(4-(tert-butoxy)phenyl)propionamido)ethoxy)ethoxy)propanoate (Compound 29) was dissolved in DCM. Trifluoroacetic anhydride was added and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated under reduced pressure and the crude residue obtained was dissolved in DMF. DIPEA (5.0 eq.) and HATU (1.3 eq.) were added followed by tert-butyl 4-(aminomethyl)isoindoline-2-carboxylate (Compound 12b, 1.1 eq.). The mixture was stirred for 1 hour. The reaction mixture was diluted with water and extracted into ethyl acetate (2×20 mL), the organic layer was evaporated, and the crude residue was purified by using combi flash using methanol and dichloromethane as mobile phase to provide tert-butyl 4-(27-(4-hydroxyphenyl)-3,25-dioxo-6,9,12,15,18,21-hexaoxa-2,24-diazacosanodecanoyl)isoindoline-2-carboxylate (Compound 30) as a gummy liquid. LC / MS (m / z): [M+H]: Calculated value C 38 H 58 N3O 11 Actual measured value: 732.40.

[0528] Step III and Step IV:

[0529] N-((2-(2-((3S,5R)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1-(3-(4-hydroxyphenyl)propionamido)-3,6,9,12,15,18-hexaoxaheneicosane-21-amide (Compound 31) and 2-((E)-2-((E)-2-(4-(1-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1-(3-(4-hydroxyphenyl)propionamido)-3,6,9,12,15,18-hexaoxaheneicosane-21-amide (Compound 31) (((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1-yl)-(((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1-yl) -5-Sodium sulfonate (Compound 32) was synthesized using procedures similar to those provided in Example 4 Step IV and Example 5 Step II, respectively. LCMS of Compound 31: LC / MS (m / z): [M+H] Calculated value C 45 H 61 F2N6O 12Found: 915.42, LCMS of compound 32: LC / MS (m / z): [M+H] calculated: C 83 H 104 F2N8Na3O 24 S4 measured value: 1831.56.

[0530] Embodiment 8:

[0531] 2-((E)-2-((E)-2-(4-(1-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)-3,19-dioxo- 6,9,12,15-tetraoxa-2,18-diazaheneicosan-21-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1- -5-Sodium sulfonate.

[0532] 2-((E)-2-((E)-2-(4-(1-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)-3,19-dioxo-6,9, 1-(2,15-tetraoxa-2,18-diazaheneicosan-21-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indole-1-yl -Synthesis of sodium 5-sulfonate:

[0533] Solution 8

[0534]

[0535] Step I

[0536] To a mixture of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (compound 12a, 1.0 eq.) and Fmoc propargylamine (1.2 eq.) in anhydrous DMF (5.0 mL) was added CuI (0.5 eq.) followed by DIPEA (2.0 eq.). The reaction mixture was stirred at 50 °C under nitrogen atmosphere for 5 hours, then the reaction mixture was cooled to room temperature, diluted with water (20 mL), and stirred vigorously for 15 minutes. The solid residue formed in the reaction mixture was filtered and washed with water 2×20 mL and dried under vacuum for 1 hour to provide tert-butyl 4-((4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)isoindoline-2-carboxylate (compound 33, 96%) as a solid and it was carried forward to the further step without further purification.

[0537] To the stirring solution of compound 33 (1 mmol) in DCM (5mL), add (Et) 2NH (2mL) and continue to stir 2 hours, and the process of the reaction is monitored by LCMS simultaneously.The reaction mixture is evaporated under reduced pressure, and the thick residue ether obtained is treated.The ether layer is decanted, and the solid compound is dried under vacuum, and is subsequently dissolved in DCM.To the reaction mixture, add Fmoc-NH (PEG4) NHS ester (1.2 equivalents) and DIPEA (3.0 equivalents).The reaction mixture is at room temperature stirred for 1 hour, evaporated under reduced pressure, and the thick residue is purified by using combi flash using methanol+DCM as mobile phase, so that compound 34 is provided.

[0538] Embodiment 9:

[0539] 2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid was synthesized according to Scheme 9.

[0540] Synthesis of 2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid:

[0541] Solution 9

[0542]

[0543] Step I:

[0544] To a stirred solution of (2S,4S)-4-(2-(tert-butoxy)-2-oxoethyl)-1-(tert-butoxycarbonyl)-5-oxopyrrolidine-2-carboxylic acid (compound 4, 1.0 eq.) in anhydrous DMF was added HATU (1.3), DIPEA (3.0 eq.) and stirring was continued for 10 min for activation of the acidic functional groups in 4. 4-cis-Fluoro-L-prolinamide hydrochloride (1.2) and DIPEA (2.0 eq.) were added to the mixture and stirring was continued for 5 h at room temperature under nitrogen atmosphere. The reaction mixture was diluted with water and brine and extracted into ethyl acetate. The combined organic extracts were dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the crude residue obtained was purified by combi flash using DCM / MeOH as mobile phase to afford tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((2S,4S)-2-carbamoyl-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (Compound 38) as a gummy solid.

[0545] Step II:

[0546] To a mixture of tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((2S,4S)-2-carbamoyl-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (Compound 38, 1.0 equiv) and imidazole (1.2 equiv) in pyridine cooled to -20°C under nitrogen was added phosphorus oxychloride (POCl3) (1.0 equiv). After stirring at -20°C for 30 min to 1 h, the mixture was evaporated to dryness in vacuo. The resulting brown solid was dissolved in CH2Cl2 (40 mL) and washed with 1.0 N aqueous citric acid solution (40 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude material as a viscous oil. The crude material was purified by combi flash (eluting with hexanes / EtOAc) to give tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (Compound 39) as a white solid.

[0547] Step III:

[0548] To a solution of tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (Compound 39, 1 mmol) in CH3CN (5 mL) was added TFA (5 mL) dropwise at 0°C over 5 minutes. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum and crystallized from EA / ether to give 2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-3-yl)acetic acid (Compound 40) as a white powder. LC / MS (m / z): Calculated value C 12 H 15 The measured value of FN3O4 is 284.10.

[0549] Embodiment 10:

[0550] 4-((2-(4-(1-((2-(2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid was synthesized according to Scheme 10.

[0551] Synthesis of 4-((2-(4-(1-((2-(2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid:

[0552] Solution 10

[0553]

[0554] TFA (10 equivalents) was added to a stirred solution of compound 19 (1.0 equivalents) in DMF (1.0 mL) at room temperature, and stirring was continued for 30 minutes. The reaction mixture was evaporated and dried under vacuum. In a separate round-bottomed flask, compound 40 (1.2 equivalents) was dissolved in DMF (0.5 mL), then dissolved in HATU (1.3 equivalents) and DIPEA (5.0 equivalents), and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 10 minutes for activating the acidic functional groups in compound 40. The amine obtained from compound 19 was dissolved in DMF (1 mL) and added to the above reaction mixture and continued to stir for another 2 hours. The reaction mixture was diluted with water (15 mL) and extracted into ethyl acetate (2 × 15 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated, and the crude residue obtained was purified by combi flash using DCM and MeOH as mobile phase to obtain the desired compound 41. LCMS of compound 41: LC / MS (m / z): [M+H] calculated value C 47 H 45 FN9O6 found: 850.34. LCMS of compound 42: LC / MS (m / z): [M+H] calculated value C 36 H 40 The measured value of FN9O7 is 728.29 g / mol.

[0555] Embodiment 11:

[0556] 5-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)benzoate was synthesized according to Scheme 11.

[0557] Synthesis of 5-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)benzoate:

[0558] Solution 11

[0559]

[0560] Step I

[0561] LiOH (172.8 mg, 7.2 mmol, 10 equiv) was added to a stirred solution of 2-(tert-butyl) 4-methylisoindoline-2,4-dicarboxylate (Compound 8, 200 mg, 0.72 mmol) in MeOH / THF / H2O (0.2 mL / 0.6 mL / 0.2 mL). The mixture was stirred for 5 hours. The solvent was removed under reduced pressure, and the mixture was dissolved in water (1 mL), and the pH was adjusted to 7 with citric acid (1 M). The product was extracted with EA (3 mL x 3). The organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure to give 2-(tert-butyloxycarbonyl)isoindoline-4-carboxylic acid (Compound 51), which was used in the next step without additional purification.

[0562] Step II

[0563] HATU (456 mg, 1.2 mmol, 1.2 eq) and DIPEA (258 mg, 2 mmol, 2.0 eq) were added to a solution of 2-(tert-butoxycarbonyl)isoindoline-4-carboxylic acid (Compound 51, 263 mg, 1.0 mmol, 1.0 eq) in DMF (5 mL). The mixture was stirred for 10 minutes, and then (9H-fluoren-9-yl)methyl(2-aminoethyl)carbamate hydrochloride (350.9 mg, 1.1 mmol, 1.1 eq) was added. The mixture was diluted with ethyl acetate (2 mL) and washed with H2O (2 mL×3). The organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure. The remaining oil was purified by combi flash chromatography using hexanes / ethyl acetate as eluent to provide tert-butyl 4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)isoindoline-2-carboxylate (Compound 52) in 352.5 mg yield as a white solid.

[0564] Step III

[0565] TFA (10 equivalents) was added to a stirred solution of compound 52 (1.0 equivalents) in DMF (1.0 mL) at room temperature, and stirring was continued for 30 minutes. The reaction mixture was evaporated and dried under vacuum. In a separate round-bottomed flask, acidic compound 7 (1.2 equivalents) was dissolved in DMF (0.5 mL), then dissolved in HATU (1.3 equivalents) and DIPEA (5.0 equivalents), and the reaction mixture was stirred at room temperature for 10 minutes under a nitrogen atmosphere for activating the acidic functional groups in compound 7. The amine obtained from compound 52 was dissolved in DMF (1 mL), added to the above reaction mixture, and stirring was continued for another 2 hours. The reaction mixture was diluted with water (15 mL) and extracted into ethyl acetate (2 × 15 mL). The organic extract was dried over anhydrous sodium sulfate. The extract was concentrated and the crude residue obtained was purified by combiflash using DCM and MeOH as mobile phases to obtain the desired compound 53. (Et)2NH was added to a solution of compound 53 in DCM and stirred at room temperature for 1 hour, evaporated under reduced pressure, and the crude residue was treated with ether, dried, and carried forward to the next step without purification.

[0566] N-(2-aminoethyl)-2-(2-((3S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamide (10.0 mg, 0.02 mmol, 1.0 eq) was dissolved in DMF (1 mL) and then rhodamine-NHS (12.9 mg, 0.024 mmol, 1.2 eq) was added followed by DIPEA (3.87 mg, 0.03 mmol, 1.5 eq). The mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water and purified by using UHPLC (A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile, solvent gradient 5% B to 95% in 60 minutes) to provide 5-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)benzoate (Compound 54). LC / MS (m / z): [M+H] Calcd. C 48 H 46 F2N8O8, measured value 901.3g / mol.

[0567] Embodiment 12:

[0568] 5-((2-(3-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)propionamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)benzoate was synthesized according to Scheme 12.

[0569] Synthesis of 5-((2-(3-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)propionamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)benzoate:

[0570] Solution 12

[0571]

[0572] Step I

[0573] To a solution of tert-butyl 4-bromoisoindoline-2-carboxylate (Compound 55) (297 mg, 1 mmol, 1.0 eq.) in DMF (1 mL) was added benzyl acrylate (486 mg, 3 mmol, 3.0 eq.), Pd(OAc)2 (22.3 mg, 0.1 mmol, 0.1 eq.), P(o-Tol)3 (60.8 mg, 0.2 mmol, 0.2 eq.) and DIPEA (387 mg, 3.0 mmol, 3.0 eq.) under argon atmosphere. The resulting mixture was heated at 100°C for 8 hours. After completion, the reaction was quenched with water (3 mL). The aqueous layer was extracted with EA (10 mL×3), and the organic phases were combined and dried over sodium sulfate. The organic layer was concentrated under reduced pressure and the residue was purified by combi flash chromatography using hexane / ethyl acetate as eluent to yield 148 mg of tert-butyl (E)-4-(3-(benzyloxy)-3-oxopropyl-1-en-1-yl)isoindoline-2-carboxylate (Compound 56) as a light yellow oil without additional purification. Compound 56 was hydrogenated using Pd / C in MeOH under a hydrogen atmosphere for 8 hours to provide 3-(2-(tert-butoxycarbonyl)isoindolin-4-yl)propanoic acid (Compound 57). LC / MS of 57: LC / MS (m / z) [M+H] Calcd. C 16 H 22 The actual measured value of NO4 is 292.15.

[0574] Compounds 58 to 61 were synthesized in a similar manner to that provided in Example 11 to produce 5-((2-(3-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)propionamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)benzoate. LC / MS (m / z) of Compound 61: [M+H] Calcd. C 50 H 51 F2N8O8, measured value 929.3g / mol.

[0575] Example 13

[0576] N-(4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)-2,3,7,7a-tetrahydro-1H-isoindole-4-carboxamido)ethyl)amino)-4-oxobutanoyl)-S-((2-((((5-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinolin-4-yl)pyridin-2-yl)methoxy)carbonyl)oxy)ethyl)thio)cysteine ​​was synthesized according to Scheme 13.

[0577] Synthesis of N-(4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)-2,3,7,7a-tetrahydro-1H-isoindole-4-carboxamido)ethyl)amino)-4-oxobutanoyl)-S-((2-((((5-(6-(5-((2,4-difluorophenyl)sulfonylamino)-6-methoxypyridin-3-yl)quinolin-4-yl)pyridin-2-yl)methoxy)carbonyl)oxy)ethyl)thio)cysteine:

[0578] Solution 13

[0579]

[0580] N-(2-aminoethyl)-2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamide (compound 53, 97.6 mg, 0.2 mmol, 1.0 equiv) was dissolved in DCM (2 mL) and then succinic anhydride (24 mg, 0.24 mmol, 1.2 equiv) was added followed by DIPEA (51.6 mg, 0.4 mmol, 2 equiv). The resulting mixture was maintained for 8 hours. The solvent was removed under reduced pressure. The residue was purified by preparative RP-HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = acetonitrile, solvent gradient 5% B to 55% B over 60 min] to yield 4-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)amino)-4-oxobutanoic acid (Compound 71). LC / MS (m / z): [M+H] exact mass: 588.21 g / mol. 4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)amino)-4-oxobutanoic acid (Compound 71, 117.6 mg, 0.2 mmol, 1.0 equiv) was introduced into solid phase peptide coupling conditions with HATU (91.2 mg, 0.24 mmol, 1.2 equiv) and DIPEA (51.6 mg, 0.4 mmol, 2.0 equiv) using H-Cys(Trt)-2-Cl-Trt (1.2 equiv). The final product was cleaved from the resin using a mixed solution of TFA:water:TIPS:ethanedithiol (95%:2.5%:2.5%:2.5%). The crude compound was precipitated in diethyl ether to give (4-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)amino)-4-oxobutanoyl)cysteine ​​(Compound 72). LC / MS (m / z): [M+H] Exact mass: 691.22.(5-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinolin-4-yl)pyridin-2-yl)methyl(2-mercaptoethyl)carbonate (7.46 mg, 0.01 mmol) and (4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)amino)-4-oxobutanoyl)cysteine ​​(Compound 72, 6.91 mg, 0.01 mmol) were dissolved in DMF (1 mL) and stirred. After the reaction was complete, the crude product was purified by preparative RP-HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = acetonitrile, solvent gradient 5% B to 75% B over 60 min] to give N-(4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)-2,3,7,7a-tetrahydro-1H-isoindole-4-carboxamido)ethyl)amino)-4-oxobutanoyl)-S-((2-((((5-(6-(5-((2,4-difluorophenyl)sulfonylamino)-6-methoxypyridin-3-yl)quinolin-4-yl)pyridin-2-yl)methoxy)carbonyl)oxy)ethyl)thio)cysteine ​​(Compound 73). LRMS-LCMS (m / z): C. 60 H 58 F4N 11 O 14 [M+H]+ calculated for S3: 1328.3; found: 1328.2).

[0581] Cell culture

[0582] FaDu, HT29, MDA-MB231, KB, 4T1PANC1, U87MG, LaNCap and HEK-FAP and HT1080-FAP cells transfected by people FAP were cultivated in a culture medium consisting of RPMI 1640, DMEM and EMEM, 10% FBS, 1% penicillin-streptomycin, 1% 2mM glutamine at 37°C. The cells used in this study were started by thawing the frozen vials of the mother liquor (master stock) preserved from the original cell line purchased from ATCC. All experiments were carried out in 2 to 5 generations after the cells were thawed. Mycoplasma test was not performed on any cell line.

[0583] Animal husbandry

[0584] Female athymic nu / nu mice aged 5 to 6 weeks were purchased from Harlan Laboratories and allowed ad libitum access to normal rodent chow and water. Animals were maintained on a standard 12-hour light-dark cycle. All animal procedures were approved by the Purdue Animal Care and Use Committee.

[0585] Confocal binding studies of FAP targeting ligands

[0586] Method 1: HT1080-FAP cells (1000000 cells / well) were seeded in 4-well confocal plates. Cells were allowed to grow as a monolayer at 37°C for 24 hours and incubated with different concentrations of the conjugate ranging from 3.0 nM (lowest) to 25 nM (highest) in 1% FBS in PBS at 37°C for 1 hour. Cells were washed with 1% FBS (3×500 μL) and cells were placed in 500 μl of 1% FBS and images were acquired using confocal microscopy. Again, PBS in the cells was replaced with growth medium and the cells were incubated at 37°C for another 8 to 48 hours. Images acquired at different concentrations of the compound at 37°C are shown in Figure 3 As shown in the figure, the images at different time points are Figure 4 , and the image with 100-fold excess of competing ligand is shown in Figure 5 Shown in.

[0587] Method 2: HT1080-FAP cells (100,000) transfected with human FAP were plated on 4-well confocal plates and incubated with different concentrations (50nM, 25nM, 12.5nM, 6.25nM, 3.125nM and 1.65nM) of the compound at 37°C for 1 hour. Unbound fluorescence was removed by washing the cells 3 times with culture medium, and the fluorescence of bound cells was imaged using an Olympus confocal microscope. The experiment was performed in triplicate.

[0588] Binding assay

[0589] Method 1: HT1080-FAP cells (200,000 cells / well) were seeded in 24-well plates. Cells were allowed to grow as a monolayer for 24 hours and incubated with different concentrations of rhodamine conjugates targeting FAP in the presence or absence of excess competing ligand (ligand without dye). After incubation at 4°C for 1 hour, cells were washed 3 times with PBS to remove unbound fluorescence. Cells were then dissolved in 1% SDS and the fluorescence of bound cells was measured using a Neo2 plate reader. Results are shown in Table 1. Figure 6 Shown in.

[0590] Method 2: 100,000 HT1080-hFAP and HT1080 cells were seeded in amine-coated 24-well plates to ensure cell adhesion. After monolayer formation, cells were incubated with different concentrations of compounds in the presence or absence of excess unlabeled ligand. After 1 hour of incubation, cells were washed 3 times with medium to remove unbound fluorescence and dissolved in 1% SDS. The RT-PCR reaction was performed using a RT-PCR setup. and The fluorescence of bound cells was measured using a fluorescence spectrophotometer (NeO2 plate reader). The fluorescence of bound cells was plotted against different concentrations and the apparent K was determined by using a single-point binding (hyperbolic) curve fit in GraphPad prism7. d The experiments were performed in triplicate.

[0591] Ex Vivo Fluorescence Imaging and Biodistribution:

[0592] Use 5×10 6 Female nu / nu athymic (5-6 weeks old) mice were injected subcutaneously with KB, MDA-MB231, HT29, U87MG, FaDu, PANC1 (with 20% Matrigel) and BalbC mice were injected with 4T1 cells in 0.1 mL sterile PBS. Tumors were allowed to grow to approximately 250 to 600 mm 3 , and then the imaging study was started. 5 nmol to 10 nmol of compound was injected intravenously (via the tail vein) per tumor-bearing mouse in the presence or absence of 10 to 500-fold excess of unlabeled ligand. Whole-body images were acquired using the AMI instrument for all tumors at two different time points (2 hours and 6 hours after injection), followed by euthanasia using CO2 asphyxiation. After whole-body imaging, the organs of interest were harvested and imaged to quantify fluorescence accumulation. Image acquisition parameters were as follows: i) lamp level - high; ii) excitation - 745 nm; iii) emission - 810; iv) binning (M) 4M; (v) aperture level (f-stop) - 4; (vi) FOV - 12.5; (vii) acquisition time, 5 seconds; (viii) power 55. Images were acquired at 2 hours, 6 hours, 8 hours, 24 hours, 32 hours, 48 ​​hours, 72 hours, 96 hours and / or 122 hours.

[0593] Western blot analysis:

[0594] After 24 hours of co-incubation, the supernatant was removed and the cells were washed with phosphate-buffered saline (PBS). The cells were harvested and lysed for Western blot analysis. 10% sodium dodecyl sulfate polyacrylamide gel was used and the samples were analyzed by gel electrophoresis and then blocked. The nitrocellulose membrane was then incubated with antibodies to detect phosphorylated Akt^Ser473 and α-actin, and the signals were visualized by the Odyssey CLx imaging system ( Fig.23 ).

[0595] Quantitative PCR

[0596] RNA was extracted using the Quick-RNA Microprep kit based on the manufacturer's instructions (Zymo Research; catalog number R1050). The extracted RNA was incubated with DNase I provided by the kit at room temperature for 15 minutes. RNA was reverse transcribed by a high-capacity cDNA reverse transcription kit according to the manufacturer's instructions (Thermo Fisher; catalog number 4368814). The cDNA was then mixed with Cyber-green supermix and primers for human collagen 1a1 and human alpha smooth muscle actin to perform quantitative PCR to analyze the expression of these two pro-fibrotic markers ( Fig.24 ).

Claims

1. A compound represented by the structure of formula (X): A m -L-B (X) in: A is a group of fibroblast activation protein alpha (FAPα) ligand, which comprises the structure of formula (XB): in: T is an unsubstituted methylene group; J is C(R J )2, where each R J H independently, or two R J Together they form an oxo group; R 1 and R 2 Each is independently -H, -CN, -B(OH)2, -CHO or -CONH2; R 3 and R 4 each independently -H or F; R 5 , R 6 , R 7 and R 8 are each independently H; and R 9 , R 10 and R 11 Each is independently H, Cl, F or C1-C3 haloalkyl; L is a linker that connects one or more A groups to B; wherein L comprises one or more linker groups having one of the following structures: in: R 16 is H or C1-C6 alkyl; and R 14a , R 14b and R 15a , R 15b Each independently is H or C1-C6 alkyl; Where n is 1 to 32; B is a group of: an optical dye, an antifibrotic agent, or an anticancer agent; and m is 1.

2. The compound of claim 1, wherein A is selected from the following:

3. The compound of claim 1 or claim 2, wherein L has the following structure:

4. A compound according to claim 1 or claim 2, wherein i) the compound is an imaging agent and B is a group of a fluorescent dye; ii) the compound is a chemotherapeutic agent and B is a group of an anti-fibrotic agent or an anti-cancer agent; or iii) B is a group of a phosphoinositide 3-kinase (PI3K) inhibitor.

5. The compound according to claim 1 or claim 2, wherein i) B has a radical structure of a compound represented by the following formula: in: X is selected from the following: or ii) B is a group of a compound having the structure:

6. A compound having the following structure: or The following structure: Or the following structure:

7. A pharmaceutical composition comprising the compound of claim 1, 2, 3, 4, 5 or 6 and a pharmaceutically acceptable carrier.

8. Use of a compound of claim 1, 2, 3, 4, 5 or 6 for the preparation of a medicament for treating a cancer expressing fibroblast activation protein alpha (FAPα) in a subject in need thereof, wherein the medicament is formulated for administering to the subject a therapeutically effective amount of a compound of claim 1, 2, 3, 4, 5 or 6, wherein the cancer is optionally selected from fibrosarcoma, breast cancer, colorectal cancer, glioblastoma, squamous cell carcinoma, pancreatic cancer and cervical cancer.

9. Use of a compound of claim 1, 2, 3, 4, 5 or 6 for the preparation of a medicament for treating fibrosis in a subject in need thereof, wherein the fibrosis comprises cells expressing fibroblast activation protein alpha (FAPα) and wherein the medicament is formulated for administering to the subject a therapeutically effective amount of a compound of claim 1, 2, 3, 4, 5 or 6.

10. A conjugate comprising a compound of claim 1, 2, 3, 4, 5 or 6 bound to fibroblast activation protein (FAP) expressed by a cell.

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