[go: up one dir, main page]

WO2025240868A1 - Method of using chemically stable keto-amide fibroblast activation protein-targeted conjugates and compositions comprising same to treat fibrosis - Google Patents

Method of using chemically stable keto-amide fibroblast activation protein-targeted conjugates and compositions comprising same to treat fibrosis

Info

Publication number
WO2025240868A1
WO2025240868A1 PCT/US2025/029775 US2025029775W WO2025240868A1 WO 2025240868 A1 WO2025240868 A1 WO 2025240868A1 US 2025029775 W US2025029775 W US 2025029775W WO 2025240868 A1 WO2025240868 A1 WO 2025240868A1
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
prf
pct
group
inhibitor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/029775
Other languages
French (fr)
Inventor
Philip S. Low
Ramesh Mukkamala
Madduri SRINIVASARAO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Purdue Research Foundation
Original Assignee
Purdue Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Purdue Research Foundation filed Critical Purdue Research Foundation
Publication of WO2025240868A1 publication Critical patent/WO2025240868A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/14—Ortho-condensed systems
    • C07D491/147—Ortho-condensed systems the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • Fibroblast activation protein FLAP
  • FAP fibroblast activation protein
  • CAFs cancer-associated fibroblasts
  • myofibroblasts engaged in collagen production. Consequently, there has been an emergence recently of reported FAP-targeted drugs and imaging agents designed for applications in cancer and other fibrotic diseases.
  • FAP9 FAP9
  • FAP9 demonstrates enhanced stability under both acidic and basic conditions, while maintaining binding affinity that is similar to, or better than, that of previously disclosed FAP-targeted ligands.
  • 1 PRF_70572-03 PCT [0006]
  • active agents such as inhibitors of phosphatidylinositol-3-kinase (PI3k) and transforming growth factor ⁇ (TGF ⁇ )
  • PI3k inhibitors of phosphatidylinositol-3-kinase
  • TGF ⁇ transforming growth factor ⁇
  • the method comprises administering to the subject an effective amount of a compound of formula I or II: or wherein A has the structure: , wherein L is a releasable or non-releasable bi-functionalized linker, which binds A and B, or L is a releasable or non-releasable tri-functionalized linker, which binds A, B, and C; B is an active agent effective for the treatment of fibrosis; C is a pharmacokinetic (PK) extender; represents a functionalized 5- to 10-membered, N-containing, aromatic or non-aro , - or bi-cyclic heterocycle, which optionally comprises 1-3 heteroatoms independently selected from O, N, and S, and indicates the point of attachment of A to L or A attaches to L via any carbon atom of the f tionalized 5- to 10-membered, N-containing, aromatic or non-aromatic, mono- or bicyclic heterocycle, an alkyl primary amine, an alky
  • A has the structures of formulae III, formulae IV, formulae V, formulae VI, formulae VII, or formulae VIII: PRF_70572-03 PCT 5 PRF_70572-03 PCT PRF_70572-03 PCT R R6 R5 R4 R 7 8 R 3 R 2 III wherein n [0017] R 1 is selected from the group of substituents consisting of -F, -Cl, -Br, -I, -OH, -CF 3 , -NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, - adamantyl, -aryl and -C 7 -C 20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted; [0018] R2, R3, R4, R
  • R13 is independently selected from the group of substituents consisting of , PRF_70572-03 PCT , wherein any substituent can be optionally substituted.
  • B can be a phosphatidylinositol-3-kinase (PI3k) inhibitor, a dual inhibitor of the PI3k/mTOR signaling pathway.
  • B can be a transforming growth factor ⁇ (TGF ⁇ ) inhibitor, an inhibitor of the TGF ⁇ 1/ALK5/Smad-2, -3 signalling pathway.
  • TGF ⁇ transforming growth factor ⁇
  • B can be, variously, a Rho-kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor; see, e.g., Fig.14), a focal adhesion kinase (FAK) inhibitor (see, e.g., Fig.14), vascular endothelial growth factor receptor (VEGFR) inhibitor (e.g., VEGFR1, VEGFR2, or VEGFR3; see, e.g., Fig.14), or a platelet-derived growth factor receptor (PDGFR) inhibitor (see, e.g., Fig.14).
  • Rho-kinase inhibitor rho-associated protein kinase inhibitor or ROCK inhibitor
  • FK focal adhesion kinase
  • VEGFR vascular endothelial growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • the dual inhibitor can be: 9 PRF_70572-03 PCT .
  • the PI3k inhibitor can have the structure: 10 PRF_70572-03 PCT m the group consisting of: . [0024] . [0025] can have the structure.
  • the TGF ⁇ inhibitor can have the structure: wher e R 1 rand R 2 are each, independently, H, methyl, Cl, Br. O, I, or F; and X is NH, O, or S. [0027] In certain embodiments The TGF ⁇ inhibitor can have the structure: . [0028] In s, L is or comprises a PEG monomer of the formula -(PEG)n- where n is 1-6. One example conjugate having the above TGF ⁇ inhibitor has the structure: .
  • L is O O O O O HN O O HN O O Or HN O HN HN O O HN O S S Or N O HN HN O S S NH Or r H S S NH S S N S Or NH .
  • Another example conjugate having the above TGF ⁇ inhibitor has the structure: .
  • the ROCK inhibitor can have one of the structures: O N N N .
  • the FAK inhibitor can be 13 PRF_70572-03 PCT .
  • the VEGFR inhibitor can have the structure: .
  • the PDGFR inhibitor can have the structure: .
  • L can comprise one or more of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a polylactone, a polymethylmethacrylate, a polyoxymethylene, a heterocycle, or any combination of two or more thereof.
  • PEG polyethylene glycol
  • L can comprise an oligomer of one or more peptidoglycans, glycans, anions, heterocycles, or any combination of two or more thereof.
  • L can comprise at least one diamino butyric acid group, a substituted benzene group, a lysine group, a 2,3-diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or 14 PRF_70572-03 PCT any combination of two or more thereof.
  • L can comprise an ether, a thioether, a tertiary amine, a C1-6 alkyl, piperazine, piperidine, a bicycloheptane, a substituted benzene, or a combination of two or more thereof.
  • L can be or can comprise a moiety of the formula: H O H O O N N O O n N N , . oiety of the formula: mula: ety of the formula: [0037] , e or can comprise a moiety of the formula: 15 PRF_70572-03 PCT .
  • I ise a moiety of the formula: [0039]
  • L can be or can comprise a moiety of the formula: N N O N N N N N O N N O N O , the formula: O PRF_70572-03 PCT .
  • L can be or comprises: 17 PRF_70572-03 PCT O O O HN O O O HN HN O O Or Or HN HN O O HN O HN O S S N S Or Or HN HN O H S NH S S NH S S N S Or NH . alkyl- , sugar-, and peptide-based dual linkers.
  • L is a non-releasable linker.
  • L is covalently bonded to A and B of formula (I) or A, B and C of formula (II).
  • L can be or can comprise a moiety of the formula: S O O O O O O S O CO 2 H .
  • L can be or can comprise a moiety selected from: COOH COOH S 2 8 l, which can be optionally substituted; and z is an integer from 1 to 8.
  • L can be or can comprise: .
  • L can be or can comprise: 18 PRF_70572-03 PCT 2 9b , R 30a , and R 30b is independently H or C 1 - C6 alkyl, which can be optionally substituted (e.g., wherein each of R29a, R29b, R30a, and R30b is C1-alkyl (i.e., methyl)).
  • C can be an albumin binding ligand, a disulfide-stabilized protein scaffold comprising albumin binding domain 035 (ABD035), albumin binding domain Con (i.e., a peptide of a three-helix bundle 45 amino acids in length (ABDCon)), a designed ankyrin repeat protein (DARPin), a disulfide-stabilized Fv fragment (dsFv) of an anti-albumin antibody (e.g., CA645), a nanobody that complexes with human serum albumin (HSA), or a variable new antigen receptor (VNAR) (e.g., E06).
  • C can be or can comprise: 19 PRF_70572-03 PCT .
  • C can be or can comprise: O 2 . , p 21 PRF_70572-03 PCT .
  • C can be or can comprise: .
  • C can be or can comprise (PEG)n, wherein n is an integer 0 to 32, a peptide, a peptidoglycan, or a saccharide.
  • C can be or can comprise: .
  • an autologous antibody in embodiments, C can be or can comprise a hapten selected from rhamnose, an ⁇ -galactosyl moiety, a dinitrophenyl (DNP) moiety, and a trinitrophenyl (TNP) moiety.
  • A can have the structure: 22 PRF_70572-03 PCT . . , 23 PRF_70572-03 PCT [0 [0 24 PRF_70572-03 PCT Me PRF_70572-03 PCT PRF_70572-03 PCT so pov e s a pa aceu ca co pos o co p s g one of the aforementioned A- L-B conjugates (i.e., 29-31, 37, 38, 40, 43, 47-49, and 60-63) and a pharmaceutically acceptable carrier.
  • A- L-B conjugates i.e., 29-31, 37, 38, 40, 43, 47-49, and 60-63
  • FIGURES [0059] Fig.1 shows the docking of FAP9 base ligand with human fibroblast activation protein alpha (FAP ⁇ ) (PDB number: 1Z68).
  • Fig.2 shows the docking of different FAP9 analogues with human FAP alpha (FAP ⁇ ) (PDB number: 1Z68), wherein (A) is FAP8, (B) is with methyl, (C) is with ethyl, (D) is with propyl, (E) is with butyl, and (F) is with fluoro substitution.
  • Fig.3 shows the docking of FAP10 with human FAP ⁇ (PDB number: 1Z68).
  • Fig.4 shows the synthesis of FAP9 with a cysteine linker.
  • Fig.5 shows the synthesis of different activated phosphatidylinositol-3-kinase (PI3k) inhibitors (24, 26, and 28).
  • Fig.6 shows the synthesis of different FAP9-PI3k conjugates (29-31).
  • Fig.7 shows the synthesis of FAP9-PEG3 with a cysteine linker (34).
  • Fig.8 shows the synthesis of FAP9-transforming growth factor ⁇ (FAP9-TGF ⁇ ) conjugates (37-38).
  • Fig.9 shows the synthesis of conjugate (40) with a non-cleavable linker.
  • Fig.10 shows the synthesis of conjugate (43) with a non-cleavable linker.
  • Fig.11 shows the synthesis of different FAP9-PI3k conjugates (47-49) with cathepsin- cleavable linkers.
  • Fig.12 shows the synthesis of different FAP9-TGF ⁇ conjugates (60-63) with cathepsin- cleavable linkers.
  • Fig.13 shows a conjugate with a reductively cleavable linker and a conjugate with an enzymatically cleavable linker.
  • Fig.14 shows examples of ROCK inhibitors, a FAK inhibitor, a PDGFR inhibitor, and VEGFR inhibitors.
  • Fig.15 shows the analysis of pAKt and ⁇ -SMA assay of compound 29 and 40.
  • Fig.18 shows the analysis of pAKt and ⁇ -SMA assay of FAP9-TGF ⁇ conjugate (37).
  • Fig.19 presnets optical imaging of FA-induced renal fibrosis in mice with an FAP- targeted NIR dye (FAP9-S0456).
  • Fig.20 presents an evaluation of FAP9-TGFBR1i for the for treatment of FA-induced renal fibrosis in mice.
  • Fig.21 shows FAP9-FITC internalization in 3T3-mFAP cells.
  • DESCRIPTION [0080] The present disclosure employs chemically stable keto-amide-based ligands that target fibroblast activation protein (FAP ⁇ ). Incorporation of the ligands into conjugates enables the targeted delivery of active agents to subjects with fibrosis (e.g., such as fibrosis of the heart, kidney, bladder, muscle, skin or liver). Advantages of such conjugates include, but are not limited to, increased tumor retention and better signal to background ratios. [0081] In view of the above, provided is a method of treating fibrosis in a subject. As used herein, "subject” means either an animal or human subject.
  • Animal means primates, livestock animals (including, without limitation, cows, horses, sheep, pigs and goats), companion animals (including dogs, cats, rabbits and guinea pigs), and captive wild animals (including those commonly found in a zoo environment).
  • Laboratory animals such as rabbits, mice, rats, guinea pigs and hamsters are also contemplated as they may provide a convenient test system.
  • FAP homologs have been found in zebrafish and amphibians, i.e., two species of the Xenopus genus, the subject, in certain instances, could be a fish or an amphibian.
  • the subject can be a human or a mammal of economic importance and/or social importance to humans, for instance, carnivores other than humans (e.g., cats and dogs), swine (e.g., pigs, hogs, and wild boars), ruminants (e.g., cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), horses, and birds including those kinds of birds that are endangered and kept in zoos, and fowl, more particularly domesticated fowl (e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like) as they are also of economic importance to humans.
  • carnivores other than humans e.g., cats and dogs
  • swine e.g., pigs, hogs, and wild boars
  • ruminants e.g., cattle, oxen, sheep,
  • subject does not denote a particular age. Thus, adult, juvenile and newborn subjects are covered.
  • subject, “ “individual” and “patient” may be used interchangeably herein.
  • the subject is a mammal. In certain embodiments, the subject is a human.
  • the method comprises administering to the subject an effective amount of a compound of formula I or II: , wherein L is a releasable or non-releasable bi-functionalized linker, which binds A and B, or L is a releasable or non-releasable tri-functionalized linker, which binds A, B, and C; B is an active agent effective for the treatment of fibrosis; C is a pharmacokinetic (PK) extender; represents a functionalized 5- to 10-membered, N-containing, aromatic or non-aro mat c, mono- or bi-cyclic heterocycle, which optionally comprises 1-3 heteroatoms independently selected from O, N, and S, and indicates the point of attachment of A to L or A attaches to L via any carbon atom of the f tionalized 5- to 10-membered, N-containing, 30 PRF_70572-03 PCT aromatic or non-aromatic, mono- or bicyclic heterocycle, an alkyl
  • A has the structures of formulae III, formulae IV, formulae V, formulae VI, formulae VII, or formulae VIII: PRF_70572-03 PCT 33 PRF_70572-03 PCT PRF_70572-03 PCT R R6 R5 R4 R 7 8 R 3 R 2 III where [0091] R 1 is selected from the group of substituents consisting of -F, -Cl, -Br, -I, -OH, -CF 3 , - NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, - adamantyl, -aryl and -C 7 -C 20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted; [0092] R2, R3, R4, R5, R6
  • R13 is independently selected from the group of substituents consisting of 35 PRF_70572-03 PCT , PRF_70572-03 PCT O O O O O , O , wherein any substituent can be optionally substituted.
  • B can be a phosphatidylinositol-3-kinase (PI3k) inhibitor, a dual inhibitor of the PI3k/mTOR signaling pathway.
  • B can be a transforming growth factor ⁇ (TGF ⁇ ) inhibitor, an inhibitor of the TGF ⁇ 1/ALK5/Smad-2, -3 signalling pathway.
  • TGF ⁇ transforming growth factor ⁇
  • B can be, variously, a Rho-kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor; see Fig. 14 for example structures), a focal adhesion kinase (FAK) inhibitor (see Fig. 14 for example structure), vascular endothelial growth factor receptor (VEGFR) inhibitor (see Fig. 14 for example structures), or a platelet-derived growth factor receptor (PDGFR) inhibitor (see Fig. 14 for example structure).
  • the dual inhibitor can be: PRF_70572-03 PCT .
  • n em o ment, t e 3 n tor can have the structure: 38 PRF_70572-03 PCT wherein X and Z independently can be -CH2-, -O-, -S-, -SO2-, -NH-, -NHR-, or -CO-, and y can be 1 to 20.
  • TGF- ⁇ 1 Transforming growth factor-beta 1 (TGF- ⁇ 1) is a key mediator in the development and progression of fibrotic diseases, including renal fibrosis.
  • TGF- ⁇ 1 In the canonical TGF- ⁇ 1/ALK5/Smad- 2, -3 signaling pathway, TGF- ⁇ 1 initially binds to its receptor, TGF- ⁇ receptor type II (TGFBR2), which subsequently recruits TGF- ⁇ receptor type I (TGFBR1, also known as activin receptor-like kinase 5 or ALK5). This forms the TGF- ⁇ –TGFBR1–TGFBR2 receptor complex. Upon activation, TGFBR1 phosphorylates downstream effectors Smad2 and Smad3.
  • TGFBR2 TGF- ⁇ receptor type II
  • ALK5 activin receptor-like kinase 5
  • the TGF ⁇ inhibitor has the structure: 39 PRF_70572-03 PCT where 1 ran 2 are eac , n epen enty, , methyl, Cl, Br. O, I, or F; and X is NH, O, or S. [0100] In certain embodiments the TGF ⁇ inhibitor can have the structure: . [0101] In some embodiments, the FAK inhibitor is F 3 C N N O . [0102] The VEGFR inhibitor can have the structure: 40 PRF_70572-03 PCT N N H 2 [0103] In certain embodiments, the PDGFR inhibitor is of the structure: O NH .
  • the ROCK inhibitor can have the structure: [0105] In some embodments t e ROCK n btor as one o te structures: 41 PRF_70572-03 PCT . [0106]
  • the PK extender can be an albumin-binding ligand, a plasma protein-binding ligand, a hapten, or an internalization-inducing peptide.
  • L is or can comprise (the use of “is or can comprise” includes radicals thereof) one or more of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a polylactone, a polymethylmethacrylate, a polyoxymethylene, a heterocycle, or any combination of two or more thereof.
  • L is or can comprise an oligomer of one or more peptidoglycans, glycans, anions, heterocycles, or any combination of two or more thereof.
  • L is or can comprise at least one diamino butyric acid group, a substituted benzene group, a lysine group, a 2,3-diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or any combination of two or more thereof.
  • L is can comprise an ether, a thioether, a tertiary amine, a C 1-6 alkyl, piperazine, piperidine, a bicycloheptane, a substituted benzene, or a combination of two or more thereof.
  • L is or can comprise a moiety of the formula: [0108] , can comprise a moiety of the formula: .
  • [0109] I n embodiments, L is or can comprise a moiety of the formula: 43 PRF_70572-03 PCT [0110]
  • L is or can comprise a moiety of the formula: PRF_70572-03 PCT .
  • L is or can comprise an oxidatively cleavable linker.
  • L is or can comprise an oxime ester.
  • L is or can comprise a hydrazone.
  • L is or can comprise an enzyme-cleavable linker (see Fig.13 for example).
  • Fig.13 further illustrates attachment of a drug moiety to the linker.
  • L is or can comprise a peptide.
  • L is or can comprise a peptidoglycan. In embodiments, L is or comprises: 45 PRF_70572-03 PCT .
  • L is a linker selected from the group consisting of pegylated-, alkyl-, sugar-, and peptide-based dual linkers. In embodiments, L is a non-releasable linker. In embodiments, L is covalently bonded to A and B of formula (I) or A, B and C of formula (II). In embodiments L is or comprises a moiety of the formula: .
  • L is or comprises a moiety selected from: yl, which can be optionally substituted; and z is an integer from 1 to 8.
  • L is or comprises: 46 PRF_70572-03 PCT .
  • L is or comprises: 29b, R30a, and R30b is independently H or C1- C 6 alkyl (e.g., wherein each of R 29a , R 29b , R 30a , and R 30b is C 1 -alkyl (i.e., methyl)), which can be optionally substituted.
  • C is an albumin binding ligand, a disulfide-stabilized protein scaffold comprising albumin binding domain 035 (ABD035), albumin binding domain Con (i.e., a peptide of a three-helix bundle 45 amino acids in length (ABDCon)), a designed ankyrin repeat protein (DARPin), a disulfide-stabilized Fv fragment (dsFv) of an anti-albumin antibody (e.g., CA645), a nanobody that complexes with human serum albumin (HSA), or a variable new antigen receptor (VNAR) (e.g., E06).
  • C is or comprises: 47 PRF_70572-03 PCT .
  • C is or comprises: .
  • C is or comprises (PEG)n, wherein n is an integer 0 to 32, a peptide, a peptidoglycan, or a saccharide.
  • C is or comprises: . [0123] , logous antibody.
  • C is or comprises a hapten selected from rhamnose, an ⁇ -galactosyl moiety, a dinitrophenyl (DNP) moiety, and a trinitrophenyl (TNP) moiety.
  • A has the structure: 50 PRF_70572-03 PCT . .
  • B has th 52 PRF_70572-03 PCT N S S O N N 22 3 5 (TGF ⁇ inhibitor) H F F O OMe N N N e Me PRF_70572-03 PCT 54 PRF_70572-03 PCT PRF_70572-03 PCT . rising one of the aforementioned A- L-B conjugates (i.e., 29-31, 37, 38, 40, 43, 47-49, and 60-63) and a pharmaceutically acceptable carrier.
  • A- L-B conjugates i.e., 29-31, 37, 38, 40, 43, 47-49, and 60-63
  • the conjugates can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as (R)- or (S)-. Unless stated otherwise, it is intended that all stereoisomeric forms of the conjugates are contemplated.
  • the conjugates described herein contain alkene double bonds, and unless specified otherwise, it is intended that this disclosure includes both E and Z geometric isomers (e.g., cis or trans). Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included.
  • geometric isomer refers to E or Z geometric isomers (e.g., cis or trans) of an alkene double bond.
  • positional isomer refers to structural isomers around a central ring, such as ortho-, meta-, and para- isomers around a benzene ring. Further, it is understood that replacement of one or more hydrogen atoms with deuterium can significantly lower the rate of metabolism of a drug and, therefore, increase its half-life.
  • substituted refers to a functional group in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms.
  • a halogen e.g., F, Cl, Br, and I
  • an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters
  • a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups
  • a nitrogen atom in groups such as amines, azides, hydroxylamines, cyano, nitro groups, N-oxides, hydrazides, and enamine
  • Alkyl generally refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as having from one to fifteen carbon atoms (e.g., C 1 - C15 alkyl). "Alkyl” is intended to include independent recitations of a saturated “alkyl, " unless otherwise stated. An alkyl can comprise one to thirteen carbon atoms (e.g., C 1 -C 13 alkyl). An alkyl can comprise one to eight carbon atoms (e.g., C 1 -C 8 alkyl). An alkyl can comprise one to five carbon atoms (e.g., C1-C5 alkyl).
  • An alkyl can comprise one to four carbon atoms (e.g., C1- C 4 alkyl).
  • An alkyl can comprise one to three carbon atoms (e.g., C 1 -C 3 alkyl).
  • An alkyl can comprise one to two carbon atoms (e.g., C 1 -C 2 alkyl).
  • An alkyl can comprise one carbon atom (e.g., C1 alkyl).
  • An alkyl can comprise five to fifteen carbon atoms (e.g., C5-C15 alkyl).
  • An alkyl can comprise five to eight carbon atoms (e.g., C5-C8 alkyl).
  • An alkyl can comprise two to five carbon atoms (e.g., C 2 -C 5 alkyl).
  • An alkyl can comprise three to five carbon atoms (e.g., C 3 -C 5 alkyl).
  • the alkyl group is selected from methyl, ethyl, 1-propyl (n- propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2- methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl).
  • the alkyl is attached to the rest of the molecule by a single bond.
  • Alkoxy refers to a radical bonded through an oxygen atom of the formula –O-alkyl, where alkyl is an alkyl chain as defined above.
  • Alkylene or “alkylene chain” generally refers to a straight or branched divalent alkyl group linking the rest of the molecule to a radical group, such as having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, i-propylene, n-butylene, and the like.
  • Aryl refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and from five to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ⁇ –electron system in accordance with the Hückel theory.
  • the ring systems from which aryl groups are derived include, but are not limited to, benzene, fluorene, indane, indene, tetralin and naphthalene.
  • Alkyl or "aryl-alkyl” refers to a radical of the formula -R c -aryl, where R c is an alkylene chain as defined above, for example, methylene, ethylene, and the like. The alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain.
  • Carbocyclyl or “cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which includes fused or bridged ring systems, having from three to fifteen carbon atoms. A carbocyclyl can comprise three to ten carbon atoms.
  • a carbocyclyl can comprise five to seven carbon atoms.
  • the carbocyclyl is attached to the rest of the molecule by a single bond.
  • Carbocyclyl or cycloalkyl is saturated (i.e., containing single C-C bonds only) or unsaturated (i.e., containing one or more double bonds or triple bonds).
  • saturated cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • An unsaturated carbocyclyl is also referred to as "cycloalkenyl.”
  • monocyclic cycloalkenyls include, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.
  • Polycyclic carbocyclyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like.
  • Carbocyclylalkyl refers to a radical of the formula –R c -carbocyclyl, where R c is an alkylene chain as defined above. 58 PRF_70572-03 PCT [0140] "Halo” or “halogen” refers to a bromo, chloro, fluoro or iodo substituent.
  • Haloalkyl refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, as defined above, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.
  • heteroalkyl refers to an alkyl group as defined above in which one or more skeletal carbon atoms of the alkyl are substituted with a heteroatom (with the appropriate number of substituents or valencies – for example, -CH 2 - may be replaced with -NH- or -O-).
  • each substituted carbon atom is independently substituted with a heteroatom, such as wherein the carbon is substituted with a nitrogen, oxygen, selenium, or other suitable heteroatom.
  • a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl.
  • a heteroalkyl is attached to the rest of the molecule at a heteroatom of the heteroalkyl.
  • a heteroalkyl is a C1-C18 heteroalkyl.
  • a heteroalkyl is a C1-C12 heteroalkyl.
  • a heteroalkyl is a C1-C6 heteroalkyl.
  • a heteroalkyl is a C1- C 4 heteroalkyl.
  • Heteroalkyl can include alkoxy, alkoxyalkyl, alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocycloalkyl, and heterocycloalkylalkyl, as defined herein. [0143] "Heteroalkylene" refers to a divalent heteroalkyl group defined above which links one part of the molecule to another part of the molecule.
  • Heterocyclyl refers to a stable 3- to 18-membered non-aromatic ring radical that can comprise two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which optionally includes aromatic, fused, and/or bridged ring systems. The heteroatoms in the heterocyclyl radical are optionally oxidized. The heterocyclyl radical is partially or fully saturated. "Heterocyclyl" is intended to include independent recitations of heterocyclyl comprising aromatic and non- aromatic ring structures, unless otherwise stated.
  • heterocyclyl is attached to the rest of the molecule through any atom of the ring(s).
  • heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 59 PRF_70572-03 PCT 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, indolinyl, isoin
  • N-heterocyclyl or “N-attached heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one nitrogen and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical.
  • N-heterocyclyl radicals include, but are not limited to, 1-morpholinyl, 1- piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.
  • Heteroaryl refers to a radical derived from a 3- to 18-membered aromatic ring radical that can comprise two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur.
  • the heteroaryl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ⁇ –electron system in accordance with the Hückel theory.
  • Heteroaryl includes fused or bridged ring systems.
  • the heteroatom(s) in the heteroaryl radical is optionally oxidized.
  • heteroaryl is attached to the rest of the molecule through any atom of the ring(s).
  • heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazo
  • the compounds and conjugates can be presented as a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to those salts whose counter ions can be used in pharmaceuticals.
  • such salts include, but are not limited to 1) acid addition salts, which can be obtained by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like; or 2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.
  • compositions are well-known to those skilled in the art, and any such pharmaceutically acceptable salt is contemplated in connection with the embodiments described herein.
  • Pharmaceutically acceptable salts can be synthesized from the parent conjugate/compound which contains a basic or acidic moiety by conventional chemical methods. In some instances, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is hereby incorporated by reference. 61 PRF_70572-03 PCT
  • it can be desired to modify the conjugate and/or composition synthesis process to optimize yield at production e.g., when the conjugate is optionally bound to a metal suitable for radio-imaging, radiotherapy, or magnetic resonance imaging).
  • a multiple step process can be utilized to facilitate stability of the conjugate at the pH required for radiolabeling.
  • suitable acid addition salts are formed from acids which form non-toxic salts.
  • Illustrative examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts.
  • suitable base salts are formed from bases which form non-toxic salts.
  • bases include the arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
  • Hemisalts of acids and bases also can be formed, for example, hemisulphate and hemicalcium salts.
  • the formulae include and represent not only all pharmaceutically acceptable salts of the compounds and conjugates, but also include any and all hydrates of the compound formulae or salts thereof where appropriate.
  • solvate means a compound, or a salt thereof, that further includes a stoichiometric or non- stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a “hydrate.”
  • Certain functional groups, such as the hydroxy, amino, and like, can form complexes and/or coordination conjugates with water and/or various solvents. Accordingly, the formulae are to be understood to include and represent those various hydrates and/or solvates. Non-hydrates and/or non-solvates of the compounds and conjugates are also included.
  • the ligands and conjugates can be synthesized in accordance with methods known in the art.
  • the conjugates can be formulated as pharmaceutical compositions comprising the conjugate and a pharmaceutically acceptable carrier.
  • composition generally refers to any product comprising more than one ingredient, including the conjugate.
  • compositions can be prepared from isolated conjugates or from salts, solutions, hydrates, solvates, and other forms of the conjugates.
  • pharmaceutically acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the carrier can be an excipient.
  • the choice of carrier can depend on factors such as the particular mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form.
  • the carrier can be suitable for parenteral administration.
  • Pharmaceutical compositions suitable for the delivery of compounds as described herein and methods for their preparation may0 be found, for example, in Remington: The Science & Practice of Pharmacy, 21st edition (Lippincott Williams & Wilkins, 2005).
  • Pharmaceutically acceptable carriers can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • carriers include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Liquids within which the conjugate can be dispersed include a carrier liquid or an in vivo liquid. By the conjugate being "dispersed" throughout or in a liquid is meant that the conjugate presents as a dispersed phase within the liquid which itself, relative to the conjugate, presents as a continuous liquid medium or phase.
  • liquid in the context of a liquid carrier is intended to mean a vehicle in which the conjugate is dispersed and which is in a liquid state at least at the temperature of intended use.
  • a liquid carrier can be made up of one or more different liquids. Suitable pharmacologically acceptable liquid carriers are described in Martin, Remington's Pharmaceutical Sciences, 18 th Ed., Mack Publishing Co., Easton, PA, (1990), and include, but are not limited to, liquids that are sterilized, such as water and oils, including those of petroleum, animal, vegetable, mineral or synthetic origin, such as peanut oil, soya bean oil, mineral oil, sesame oil, and the like.
  • liquid carriers include methylene glycol, propylene glycol, 63 PRF_70572-03 PCT polyethylene glycol, polypropylene glycol, ethanol, isopropyl alcohol, and benzyl alcohol.
  • Water or soluble saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions.
  • the conjugate can be taken up by a subject in vivo, for example, when the conjugate is administered orally or parenterally.
  • a liquid carrier originally carrying the conjugate can become so dilute in vivo that the surrounding liquid environment throughout which the conjugate is dispersed becomes more representative of an in vivo liquid (i.e., a biological liquid/fluid within the subject) than the original liquid carrier.
  • an in vivo liquid carrier i.e., a biological liquid/fluid within the subject
  • compositions also can be commingled with the conjugate, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • the composition can comprise cremophor, polysorbate, nanoparticles, a polymer, or a hydrogel, for example.
  • the pharmaceutical composition comprises a plurality of conjugates and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, and combinations thereof, that are physiologically compatible.
  • One or more other active agents also can be incorporated into a pharmaceutical composition.
  • a pharmaceutical composition further comprises at least one additional pharmaceutically active agent.
  • the at least one additional pharmaceutically active agent can be an agent useful in the treatment of a cancer.
  • the at least one additional pharmaceutically active agent can be an agent useful for radiotherapy.
  • the at least one additional pharmaceutically active agent can be an agent useful for imaging (e.g., diagnostic imaging).
  • Pharmaceutical compositions can be prepared by combining one or more conjugates with a pharmaceutically acceptable carrier and, optionally, one or more additional ingredients (e.g., pharmaceutically active ingredients).
  • compositions can be administered in pharmaceutically 64 PRF_70572-03 PCT acceptable solutions, which can routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • Compositions can comprise one or more pharmacologically acceptable additives known to those in the art.
  • the liquid carrier may comprise one or more additives such as wetting agents, de-foaming agents, surfactants, buffers, electrolytes, preservatives, colourings, flavourings, and sweeteners.
  • additives such as wetting agents, de-foaming agents, surfactants, buffers, electrolytes, preservatives, colourings, flavourings, and sweeteners.
  • the particular nature of a liquid carrier and any additive (if present) can, in part, depend upon the intended application of the composition.
  • a suitable liquid carrier and additive can be selected for the intended application of the composition.
  • the composition is suitable for administration to a subject for therapeutic applications.
  • suitable for administration is meant that administration of the conjugate/composition to a subject will not result in unacceptable toxicity, including allergenic responses and disease states.
  • an effective amount of the conjugate or composition can be administered to a subject by any mode that delivers the conjugate(s) as desired. Administering a composition can be accomplished by any means known to the skilled artisan.
  • Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection, mucosal (e.g., topical to eye), inhalation, and topical.
  • Colorants and/or flavoring agents can be included.
  • the conjugate can be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
  • Illustrative formats for oral administration include, but are not limited to, tablets, capsules, elixirs, syrups, and the like.
  • a conjugate and/or composition can be administered directly into the blood stream, into muscle, or into an internal organ.
  • suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, intranasal, and subcutaneous.
  • Suitable means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.
  • the compound(s) and/or 65 PRF_70572-03 PCT composition can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Parenteral formulations are typically aqueous or non-aqueous isotonic sterile solutions that can contain carriers or excipients, such as salts, carbohydrates, anti-oxidants, bactericide, solute and/or buffering agents (preferably at a pH of 3–9) which renders the composition isotonic with the blood of the intended subject, but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle, such as sterile, pyrogen-free water.
  • Such compositions can be presented in unit- dose or multi-dose sealed containers, for example, ampoules and vials.
  • a liquid formulation can be adapted for parenteral administration of a conjugate or composition as described herein.
  • the preparation of parenteral formulations under sterile conditions, for example, by lyophilization under sterile conditions, can readily be accomplished using standard pharmaceutical techniques well-known to those skilled in the art.
  • the solubility of a conjugate can be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
  • Formulations for parenteral administration can be formulated for immediate and/or modified release.
  • a conjugate can be administered in a time-release formulation, for example in a composition which includes a slow-release polymer.
  • the conjugate can be prepared with a carrier that will protect it against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a carrier that will protect it against rapid release
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PGLA). Methods for the preparation of such formulations are generally known to those skilled in the art.
  • Sterile injectable solutions can be prepared by incorporating the conjugate(s), alone or in further combination with one or more other active agents, in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the conjugate(s) into a sterile vehicle, which contains a dispersion medium and any additional 66 PRF_70572-03 PCT ingredients of those described above.
  • a sterile vehicle which contains a dispersion medium and any additional 66 PRF_70572-03 PCT ingredients of those described above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying, which yield a powder of the active ingredients plus any additional desired ingredient from a previously sterile-filtered solution thereof, or the ingredients can be sterile-filtered together.
  • the pharmaceutical composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • a conjugate, or a pharmaceutical composition comprising a conjugate can be continuously administered, where appropriate.
  • the therapeutic regimen for the treatment of a disease state can be determined by a person skilled in the art and will typically depend on factors including, but not limited to, the type, size, stage and receptor status of a tumor (e.g., with cancer) in addition to the age, weight and general health of the subject.
  • Another determinative factor can be the risk of developing recurrent disease. For instance, for a subject identified as being at high risk or higher risk or developing recurrent disease, a more aggressive therapeutic regimen can be prescribed as compared to a subject who is deemed at a low or lower risk of developing recurrent disease.
  • a more aggressive therapeutic regimen can be prescribed as compared to a subject that has a less advanced stage of cancer.
  • the terms “treat,” “treatment,” and “treating” refer to any and all uses which remedy a condition or symptom, or otherwise prevent, hinder, retard, abrogate or reverse the onset or progression of cancer or other undesirable symptoms in any way whatsoever.
  • the term “treating,” and the like is to be considered in its broadest possible context. For example, treatment does not necessarily imply that a subject is treated until total recovery or cure.
  • the treatment need not necessarily remedy, prevent, hinder, retard, abrogate or reverse all signs or symptoms, but can remedy, prevent, hinder, retard, abrogate or reverse one or more signs or symptoms.
  • the expressions “effective amount” and "therapeutically effective amount” mean the amount of conjugate when administered to a mammal, in particular a human, in need of such treatment, is sufficient to treat cancer.
  • the precise amount of conjugate to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the subject.
  • administering of the conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, and/or composition to a subject is meant that the conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, or composition is presented such that the conjugate(s) and/or pharmaceutically acceptable salts, hydrates, or solvates thereof can be transferred to the subject.
  • mode of administration There is no particular limitation on the mode of administration, but this will generally be by way of oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intracerebrally, intranasally, intrathecal, and intraspinal), inhalation (including nebulization), topical, rectal and vaginal modes.
  • the conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, and/or composition can also be administered directly into a tumor and/or into tissue adjacent one or more segments of a tumor or administered directly into blood vessels.
  • the conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, and/or composition can be administered in a treatment effective amount.
  • a treatment effective amount includes an amount which, when administered according to the desired dosing regimen, achieves a desired therapeutic effect, including one or more of: alleviating the symptoms of, preventing or delaying the onset of, inhibiting or slowing the progression of, diagnosing, or halting or reversing altogether the onset or progression of a particular condition being treated and/or assessed.
  • Suitable dosage amounts and dosing regimens to achieve this can be determined by the attending physician and can depend on the particular condition being treated, the severity of the condition as well the general age, health and weight of the subject. [0183] Depending upon the route of administration, a wide range of permissible dosages are contemplated. The dosing can occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages of the particulate material per se can lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage.
  • the dosage can be in the range of 1 ⁇ g to 1 g per kg of body weight per dosage, such as 68 PRF_70572-03 PCT is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage can be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage can be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage can be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage. [0184] Conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, and/or compositions hereof can be administered in a single dose or a series of doses.
  • dosages may be single or divided and may be administered according to a wide variety of protocols, including q.d. (once a day), b.i.d. (twice a day), t.i.d. (three times a day), or even every other day, once a week, once a month, once a quarter, and the like.
  • q.d. once a day
  • b.i.d. tilt a day
  • t.i.d. three times a day
  • the effective amounts described herein correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol.
  • an effective amount of any one or a mixture of the compounds described herein can be determined by the attending diagnostician or physician by the use of known techniques and/or by observing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.
  • a use of a conjugate, a pharmaceutically acceptable salt, hydrate, or solvate of the conjugate, or a composition hereof in the manufacture of a medicament for the treatment of a disease in a subject is provided.
  • the conjugate can be any compound or conjugate hereof.
  • the disease in the subject can be cancer.
  • the disease in the subject can be fibrosis.
  • connection between two components does not necessarily mean a direct, unimpeded connection, as a variety of other components may reside between the two components of note. Consequently, a connection does not necessarily mean a direct, unimpeded connection unless otherwise noted.
  • Step ii Dess-Martin periodinane (4.92 g, 11.60 mmol) was added portion-wise to a solution of tert- butyl 4,4-difluoro-2-(hydroxymethyl) pyrrolidine-l-carboxylate (2) (2.5 g, 10.55 mmol) in DCM (30 mL) at 0 °C. After complete addition, the reaction was warmed to room temperature and stirred for 2 hours. Saturated NaHCO 3 was added, and the layers were separated using a phase separator.
  • Triethylamine (2.0 mL) was added dropwise and stirring continued until full conversion (LC-MS), which usually 71 PRF_70572-03 PCT occurred in less than 2 hours.
  • the liquids were evaporated under reduced pressure, and the mixture was redissolved in DCM and washed three times with water. Organic phase was washed with brine and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and the crude residue was purified by using combiflash with hexanes + ethyl acetate as mobile phase to provide the desired ⁇ -hydroxyamide (8).
  • Step-2 72 PRF_70572-03 PCT
  • DMP Dess–Martin periodinane
  • H2O H2O
  • Example 2 Induced fit docking (IFD)
  • IFD Induced fit docking
  • the standard induced fit docking (IFD) protocol in the Schrodinger software package was used to dock the ligands of interest into the binding pocket of FAP. Firstly, a receptor grid box was generated by specifying the amino acid residues in FAP reported being involved in binding interactions.
  • the IFD protocol utilizes the Glide docking protocol to generate up to 20 poses for each ligand which are further refined using the Prime Refinement module.
  • the residues within 5 ⁇ of ligand poses were refined, and the side chains of the residues were optimized.
  • the ligands were re-docked into structures that are within 30.0 kcal/mol of the best structure and within the top 20 structures overall.
  • the standard precision (SP) scoring function was used in the Glide redocking step to get the final docking scores.
  • SP standard precision
  • FAP9 analogues introduction of small alkyl groups, such as methyl, ethyl, propyl or butyl groups, and replacement of hydrogen with fluorine in FAP8 further increased binding interaction docking scores of the resultant analogues, referred to herein as FAP9 analogues.
  • reaction mixture was further transfer to room temperature and continued stirring there 1h-2h, progress of the reaction was monitored by TLC, reaction mixture was evaporated under vacuum and obtained residue was redissolved in CH 2 Cl 2 (100 mL) followed extracted by saturated aqueous sodium bicarbonate solution (50 mL), organic layer was separated and evaporated under vacuum, then obtained crude residue was purified by column chromatography using ethyl acetate hexanes as mobile offered desired compound (3) 850 mg, 95% as white gummy liquid.
  • Step-ii Dess-Martin periodinane (4.92 g, 11.60 mmol) was added portion wise to a solution of tert- butyl (S)-4,4-difluoro-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (3) (2.5 g, 10.55 mmol) in DCM (30 mL) at 0 °C. After complete addition the reaction was warmed to rt and stirred for 2 h. Saturated NaHCO3 was added, and the layers separated using a phase separator.
  • Triethylamine (2.0 mL) was added dropwise, and the stirring continued till the full conversion (LC-MS), usually less than 2 hours.
  • the liquids were evaporated under reduced pressure, the mixture was redissolved in CH 2 Cl 2 and washed 3 times with water.
  • Organic phase was washed with brine, dried over sodium sulfate and the solvent was evaporated under reduced pressure and obtained crude residue was purified by using combiflash with hexanes + ethyl acetate as mobile phase provided desired ⁇ -hydroxyamide (8).
  • Example 4 (Fig.6) Synthesis of compound (18 and 29) To a solution of compound 14 (200.0 mg, 0.297mmol) in CH 2 Cl 2 (5 mL) in different reaction vials were added PyBOP (1.2 eq) + DIPEA (5.0eq) followed by respective amines (compound 16 (1.1 eq)), then continued the stirring at room temperature for an additional 2 hours. Reaction mixture was further diluted with water and extracted into CH 2 Cl 2 (2x30 mL), and combined organic extracts were evaporated under reduced pressure. Obtained crude compounds were purified by combi-flash using methanol+CH2Cl2 system to provide the respective compound 17.
  • Example 5 Synthesis of FAP9-PI3K conjugate (29-31): To a stirred solution of compound 18 (50 mg, 0.144 mmol) in DMF (1.0 mL) in three different reaction vials were added activated PI3K inhibitors (24, 26 and 28) (1.0 eq) and DMAP (1.0 eq) and stirred at rt for 2h under argon atmosphere.
  • reaction mixtures were further diluted with water, followed by extracted into dichloromethane (2x20 mL), then the combined organic extracts dried over anhydrous sodium sulfate, concentrated under vacuum, absorbed in celite, and subjected to reverse phase chromatography using acetonitrile in 20 mM ammonium acetate (pH 7.0) as mobile phase. Lyophilization of the acquired fractions provided the desired final conjugates (29-31) as white solids.
  • Example 6 (Fig.7) Synthesis of compound (34) 78 PRF_70572-03 PCT For the synthesis and purification of activated TGF- ⁇ compounds 36 and FAP9- TGF- ⁇ conjugates (33 and 34) followed the same experimental conditions as described in the scheme-1- 3 using appropriate starting materials.
  • Example 7 Synthesis of FAP9-TGF ⁇ conjugate (37-38): To a stirred solution of compound 18 (50 mg, 0.144 mmol) or 34 (50 mg, 0.054 mmol) in DMF (1.0 mL) in two different reaction vials were added activated TGF ⁇ inhibitors (36) (1.0 eq) and DMAP (1.0 eq) and stirred at room temperature for two hours under argon atmosphere.
  • reaction mixtures were further diluted with water, followed by extracted into dichloromethane (2x20 mL), then the combined organic extracts dried over anhydrous sodium sulfate, concentrated under vacuum, absorbed in celite, and subjected to reverse phase chromatography using acetonitrile in 20 mM ammonium acetate (pH 7.0) as mobile phase. Lyophilization of the acquired fractions provided the desired final conjugates (37 and 38) as white solids.
  • Example 8 (Fig.9) To a stirred solution of compound 23 (1.0 eq) in DMF (1 mL/100mg) was added NaH ( 2.0 eq), followed by tert-butyl (2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)carbamate (1.0 eq),then stirred the reaction mixture at room temperature for 2-3 hours, progress of the reaction was monitored by LC-MS. Reaction mixture was diluted with water and extracted into ethyl acetate, then evaporate and obtained crude residue was purified by HPLC provided desired compound 39. Then compound 39 was dissolved in CH 2 Cl 2 , TFA was added, and the mixture was stirred at room temperature.
  • Example 9 To a stirred solution of acid (14) in dichloromethane was added PyBOP/DIPEA followed by Val-Cit linkers (44), provided the couple product 45 and which was further reacted with 4- nitrophenyl carbonochloridate gave the activated compound 46.
  • Example 11 Evaluation of biological activity Bleomycin-induced lung fibrosis model (prophetic example)
  • lungs are harvested at 7, 14, and 21 days after bleomycin instillation and assayed as described below.
  • induction of IPF is initiated as described above, and drug (2 ⁇ mol/kg) is intravenously injected every other day beginning on day 10.
  • Lungs are harvested on day 21 and assayed as described below (day 0 was taken as the day of bleomycin administration).
  • Western blot analysis of lung tissue Frozen lungs are lysed in 1 ml of lysis buffer containing a protease inhibitor cocktail using an ULTRA-THURRAX. Lysates are cleared by centrifugation before total protein determination using the BCA protein assay.
  • SDS–polyacrylamide gel electrophoresis and Western blotting are performed following standard procedures. Membranes are blocked and then probed with antibodies directed against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pAkt, and total Akt (for FAP-TGFb inhibitor conjugates, this step is omitted). The membranes are then washed in tris-buffered saline/Tween 20 followed by incubation with horseradish peroxidase (HRP)– conjugated secondary antibodies. Immunoreactive bands are detected by addition of an enhanced chemiluminescence substrate. Hydroxyproline assay 80 PRF_70572-03 PCT Total lung collagen is determined by the analysis of hydroxyproline.
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • pAkt pAkt
  • total Akt for FAP-TGFb inhibitor conjugates
  • the right lung is consistently set aside for this assay. Briefly, harvested right lung is homogenized in PBS (pH 7.4), and digested with 12 N HCl at 120°C for three hours. Citrate/acetate buffer (pH 6.0) and chloramine-T solution are added at room temperature for 20 min, and the samples are incubated with Ehrlich’s solution for 15 min at 65°C. Samples are cooled to room temperature and read at 550 nm. Hydroxyproline standards (Sigma-Aldrich) at concentrations between 0 and 400 ⁇ g/ml are used to construct a standard curve. Histopathological evaluation of pulmonary fibrosis The left lung is inflated and fixed with 10% formalin solution (neutral buffered).
  • mice Lung tissues are embedded in paraffin, and 10- ⁇ m sections are prepared and stained using hematoxylin and eosin and trichrome stain. The severity of bleomycin-induced fibrosis is determined by semiquantitative histopathological scoring at the indicated dates after bleomycin administration.
  • Animal model Eight weeks old male C57BL/6J mice were purchased from The Jackson Laboratory (Strain #:000664). The mice were housed Mice were housed under a 12-h light/dark cycle with free access to standard mouse chow and tap water and allowed one week of acclimatory period. At nine weeks of age, twelve mice were randomly divided into four groups.
  • mice in negative control group were injected intraperitoneally with 500 ⁇ L of 0.3 M sodium bicarbonate solution. All mice in positive control group (fibrotic mice) and two treatment groups were injected intraperitoneally with folic acid at the dose 250 mg/kg dose and at a concentration of 12.5 mg/mL once on day 0. High concentration of folic acid such as the 250 mg/kg used here, is known to induce renal fibrosis within four weeks of administration (Yan 2021). From day 3 of folic acid administration, mice in treatment group one started receiving FAP9-PI3Ki at a dose of 10 nmol/mice (calculated as 400 nmol/kg body weight) administered intravenously every other day.
  • RNA samples were measured using SpectraMax QuickDrop UV-Vis spectrophotometer (Molecular Devices, San Jose, CA) and adjusted accordingly to produce uniform sample concentrations. All the RNA samples were amplified, and the transcript levels of targeted genes were quantified using the iTaqTM Universal SYBR® Green One-Step Kit (Bio-Rad Laboratories, Hercules, CA) following the manufacturer’s protocol, in the CFX Connect Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA). The relative expression levels of collagen type I (col1a1; forward primer 5’- TCCGGCTCCTGCTCCTCTTA-3’ (SEQ ID NO.
  • reverse primer 5’- GTATGCAGCTGACTTCAGGGATGT-3’ (SEQ ID NO. 2)), collagen type 3 (col3a1; forward primer 5’- AATGGTGGCTTTCAGTTCAGCT-3’ (SEQ ID NO. 3); reverse primer 5’- TGTAATGTTCTGGGAGGCCC -3’ (SEQ ID NO.4)) and ⁇ -smooth muscle actin (acta2; forward primer 5’- ACAGCCCTCGCACCCA-3’(SEQ ID NO.
  • reverse primer 5’- GCCACCGATCCAGACAGAGT -3’ (SEQ ID NO.6)) were compared against the housekeeping gene GAPDH (gapdh; forward primer 5’- ACAGCCCTCGCACCCA-3’(SEQ ID NO.7); reverse primer 5’- GCCACCGATCCAGACAGAGT -3’ (SEQ ID NO. 8)), and calculated using the 2 ⁇ Ct method (Yang et al., 2023).
  • the relative gene expression levels between different animal groups were analyzed and their statistical significance were calculated using one-way ANOVA in GraphPad Prism 10. Hydroxyproline assay
  • Pre-weighed kidney samples were digested individually in 6M hydrochloric acid at 100°C for 24 hours.
  • the hydroxyproline content measured for each mouse was divided by their respective kidney weight and expressed as ⁇ g of total hydroxyproline per g of wet kidney mass.
  • the variation in the hydroxyproline levels between different animal groups was analyzed in GraphPad Prism 10 and their statistical relevance were measured through one-way ANOVA. Results Relative gene expressions of fibrotic markers Compared to the negative control mice with a relative gene expression level of single unit, the positive control mice expressed significantly (p ⁇ 0.0001) higher levels of both col1a1 and col3a1 ( Figure 1), thereby indicating an increased collagen production by the folic acid-induced fibrotic mice.
  • Example 12 Western blot analysis of lung tissue using FAP9-TGF ⁇ conjugate (37) PHLF (primary human lung fibroblasts) were seeded in a plate and stimulated by 10 ng/ml TGF ⁇ 1 overnight.
  • the SD rats or mice used in this experiment are about 6-month-old. Animals are anesthetized, and the surface of the left lung is marked by a marker. Radiation is be given with the applicator tube fixed at the mark on the left lung. The dose normalization point is 0.5 cm below the source center, and the irradiation area is a 0.5 cm radius around the mark point. Different groups of animals are given a single increasing dose of irradiation (in the range of 10-90 Gy), and the degree of fibrosis on the lung at the irradiated site is detected each week for four weeks after irradiation. After optimizing the dose for consistent fibrosis, the treatment is started with FAP-targeted antifibrotic agents after the fibrotic stage begins.
  • Example 16 Heart fibrosis (prophetic example) Tissue inhibitor of metalloproteinase-3 knockout (TIMP3 ⁇ / ⁇ ) (C57BL/6) mice will be used as a model for cardiac fibrosis. Angiotensin II (1.5 mg/kg/day) or saline (control) will be delivered to these mice. The treatment with FAP-targeted antifibrotic drugs or saline control will begin on the same day of administering angiotensin II (or saline).
  • Example 17 Bladder fibrosis (prophetic example) Six-week-old Balb/c mice will be injected with 100 mg/kg ketamine for 20 weeks intraperitonially. Saline injected mice will be used as control mice. The treatment with FAP- targeted antifibrotic drugs or saline control will begin at four weeks after administering the first dose of ketamine (or saline). After 20 weeks, the heart will be studied for signs of fibrosis and therapeutic benefit.
  • Example 18 Muscle fibrosis (prophetic example) 85 PRF_70572-03 PCT Five-month-old dystrophic mdx(C57BL/10ScSnDmdmdx/J) mice will be used as a spontaneous model for muscle fibrosis. Treatment with FAP-targeted antifibrotic drugs or saline control will begin at different ages to assess the therapeutic benefit. The muscle tissue will be studied for signs of fibrosis and therapeutic benefit. [0209] Example 19 FAP9-TGFBR1i treatment study in FA-treated mice Methods Cell lines and cell culture. PHLF, 3T3 and HT1080 cells were purchased from ATCC.
  • HT1080-hFAP cells were seeded on glass chamber slides (Thermo Scientific) in complete DMEM medium. Cells were incubated with 50 nM of FAP9-FITC at 37 °C for 1 hour, followed by three washes with PBS containing 2% FBS. Fluorescence imaging was captured and processed using a confocal microscopy (FV 1000, Olympus) with FV10-ASW Olympus software. PRF_70572-03 PCT Mice. Male C57BL/6J mice (weight 20–25 g) were purchased from the Jackson Laboratory and housed under pathogen-free conditions with a 12 h light/dark cycle.
  • mice were injected via tail vein with 5 nmol of FAP-targeted NIR dye conjugate (FAP9-S0456) and imaged at 24 hours post-injection using a Spectral AMI optical imaging system.
  • FAP9-S0456 FAP-targeted NIR dye conjugate
  • Imaging settings included: object height, 1.5; excitation wavelength, 745 nm; emission wavelength, 810 nm; field of view (FOV), 25; binning, 2; f-stop, 2; acquisition time, 5 s.
  • Major organs were collected and imaged for biodistribution analysis.
  • mice Treatment of mice with FAP9-TGFBR1i. Three days following FA injection, mice were randomized based on body weight and treated daily tail vein injection with either 10 nmol of FAP9-TGFBR1i (Compound 37) or PBS (100 ⁇ l/dose). On day 28 post-treatment, mice were euthanized and kidneys were harvested for subsequent analysis. RNA isolation, RT-PCR and qPCR. Mice kidneys were dissected, minced and lysed in 1ml TRIzol reagent (Invitrogen). Total RNA was isolated following the manufacturer’s protocol.
  • RNA concentration and purity was assessed using a Nanodrop spectrophotometer (Thermo Scientific), and integrity was confirmed by agarose gel electrophoresis.1 ⁇ g RNA was used for cNDA synthesis using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), following the manufacturer’s protocol.
  • RT-qPCR was performed using SYBR Green Master Mix (Thermo Fisher Scientific) on a CFX Connect Real-Time PCR Detection System (Bio-Rad). Technical replicates were included for all samples. Primer specificity was validated via melt curve analysis and agarose gel electrophoresis of the amplified product. No nonspecific 87 PRF_70572-03 PCT amplification products were observed.
  • Relative gene expression was analyzed using the 2(-Delta Delta C(T)) method. Primer sequences are listed below.
  • Hydroxyproline assay Freshly isolated mouse kidneys were dissected, weighed and homogenized in 100 ⁇ l H2O per 10 mg tissue, following by the addition of an equal volume of 12N HCl. Samples were hydrolyzed 24 hours at 95°C. After centrifugation at 3,500g for 10 minutes, 300 ⁇ l of supernatant was transferred to new tubes and neutralized with 300 ⁇ L of 6N NaOH. Samples were centrifuged again at 1,000g for 10 minutes.150uL of each sample and hydroxyproline standard were transferred to a 96-well plate.
  • chloramine- T solution 40 mM chloramine-T in 90% citrate/acetate buffer plus 10% isopropanol
  • 100 ⁇ L of Ehrlich’s solution (1.14M DMAB, 70% isopropanol and 30% perchloric acid) was added and incubated at 65°C for 20 minutes.
  • Absorbance was measured at 550 nm using a SpectraMax384 Plus plate reader. A standard curve was generated using the OD values of the hydroxyproline standards. Western blot.
  • Membranes were blocked with 5% non-fat milk and incubated with the following primary antibodies: anti-Collagen I (Invitrogen, PA1-26204, 1:1000), anti-GAPDH (Proteintech 60004-1-Ig, 1:50000), anti-Alpha-smooth muscle actin (cell signaling, #19245, 1:1000), anti-pSMAD2 (cell signaling, #3108, 1:1000) and anti-SMAD2 (cell signaling, #3103, 1:1000).
  • Secondary antibodies used were IRDye 800CW goat anti-rabbit IgG or IRDye® 680RD goat anti-mouse IgG (LI-COR).
  • FIG.19a presents representative optical image showing tissue biodistribution of FAP9- S045624 hours after intravenous administration in with FA-induced kidney fibrosis. Minimal to no retention of FAP9-S0456 was observed in tissues other than the fibrotic kidney (middle) and was absent in healthy mice (left). Uptake in fibrotic kidneys was blocked by excess FAPL (right), demonstrating the specificity of FAP9-S0456 for the fibrotic kidney.
  • FIG.19b presents quantitative analysis of FAP9-S0456 retention in healthy and FA-treated mice.
  • FIG.19c shows the time course of FAP9-S0456 retention in the kidneys of FA-treated mice.
  • FIG.20a presents a schematic representation of the mouse study for induction and treatment in the FA-induced kidney fibrosis model and changes in body weight of healthy mice, FAP9- TGFBR1i-treated or vehicle-treated mice following FA injection.
  • FIG.20b presents qPCR quantification of gene expression (collagen, SMA and FAP) in moue kidneys.
  • FIG.20d shows Western blot for kidney samples of healthy mice, FAP9-TGFBR1i-treated or vehicle-treated mice.
  • FIG.20e shows change in collalgen protein, FAP protein and ⁇ -SMA protein with healthy, vehicle and FAP-TGFBR1i treatment in moue kidneys.
  • Fig.21 shows FAP9-FITC internalization in 3T3-mFAP cells.
  • a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 89 PRF_70572-03 PCT 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range.
  • the statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise.
  • the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.
  • a method of treating fibrosis in a subject comprises administering to the subject an effective amount of a compound of formula I or II: , wherein L is a releasable or non-releasable bi-functionalized linker, which binds A and B, or L is a releasable or non-releasable tri-functionalized linker, which binds A, B, and C; B is an active agent effective for the treatment of fibrosis; C is a pharmacokinetic (PK) extender; represents a functionalized 5- to 10-membered, N-containing, aromatic or non-aro , or bi-cyclic heterocycle, which optionally comprises 1-3 heteroatoms independently selected from O, N, and S, and indicates the point of attachment of A to L or A attaches to L via any carbon atom of the functionalized 5- to 10-membered, N-containing, aromatic or non-aromatic, mono- or bicyclic heterocycle, an alkyl primary amine, an alkyl secondary
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are independently selected from the group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -C 1-6 alkyl, -O-C 1-6 alkyl, and -S-C 1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted;
  • R 9 , R 10 , and R 11 are independently selected from group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br,
  • EE 2 The method of EE 1, wherein A has the structures of formulae III, formulae IV, formulae V, formulae VI, formulae VII, or formulae VIII: PRF_70572-03 PCT 94 PRF_70572-03 PCT PRF_70572-03 PCT R R6 R5 R4 R 7 8 R 3 R 2 III where R 1 is selected from the group of substituents consisting of -F, -Cl, -Br, -I, -OH, -CF 3 , -NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, -adamantyl, -aryl and -C 7 -C 20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted; R2, R3, R4, R5, R6
  • EE 3 The method of EE 1 or 2, wherein R13 is independently selected from the group of substituents consisting of , PRF_70572-03 PCT O O , wherein any substituent can be optionally substituted. [0232] EE 4.
  • B is a phosphatidylinositol-3-kinase (PI3k) inhibitor, a dual inhibitor of the PI3k/mTOR signaling pathway, a transforming growth factor ⁇ (TGF ⁇ ) inhibitor, a Rho-kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor), a focal adhesion kinase (FAK) inhibitor, vascular endothelial growth factor receptor (VEGFR) inhibitor, or a platelet-derived growth factor receptor (PDGFR) inhibitor.
  • PI3k phosphatidylinositol-3-kinase
  • TGF ⁇ transforming growth factor ⁇
  • Rho-kinase inhibitor Rho-associated protein kinase inhibitor or ROCK inhibitor
  • FAK focal adhesion kinase
  • VEGFR vascular endothelial growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • EE 4 wherein the dual inhibitor of the PI3k/mTOR signaling pathway is: 98 PRF_70572-03 PCT .
  • EE 6 The method of EE 4, wherein the PI3k inhibitor has a structure: , wherein X has a structure selected from the group consisting of: 99 PRF_70572-03 PCT .
  • EE 7. The method of EE 4, wherein the PI3k inhibitor has the structure: .
  • EE 8. The method of EE 4, wherein the PI3K inhibitor has the structure: N A , , , , -NH-, -NHR-, Or -CO-, and y can be 1 to 20. [237] EE 9.
  • EE 4 wherein the TGF ⁇ inhibitor has the structure: 100 PRF_70572-03 PCT where 1 ran 2 are eac , n epen ent y, , methyl, Cl, Br. O, I, or F; and X is NH, O, or S.
  • EE 10 The method of EE 9, wherein R1 is F and R2 is methyl.
  • EE 11 The method of EE 9, wherein the TGF ⁇ inhibitor has the structure: . [0240] EE 12.
  • EE 13 wherein the TGF ⁇ inhibitor has the structure: 100 PRF_70572-03 PCT where 1 ran 2 are eac , n epen ent y, , methyl, Cl, Br. O, I, or F; and X is NH, O, or S.
  • EE 10 The method of EE 9, wherein R1
  • the method of EE 11, wherein the conjugate has the structure: . [0244] EE 16.
  • EE 19 The method of EE 4, wherein the VEGFR inhibitor is N H 2 .
  • EE 20 The method of EE 4, wherein the PDGFR inhibitor is 103 PRF_70572-03 PCT .
  • EE 23 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises one or more of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a polylactone, a polymethylmethacrylate, a polyoxymethylene, a heterocycle, or any combination of two or more thereof.
  • EE 23 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an oligomer of one or more peptidoglycans, glycans, anions, heterocycles, or any combination of two or more thereof.
  • EE 24 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an oligomer of one or more peptidoglycans, glycans, anions, heterocycles, or any combination of two or more thereof.
  • EEs 1-11 and 15-21 wherein L is or comprises at least one diamino butyric acid group, a substituted benzene group, a lysine group, a 2,3- diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or any combination of two or more thereof.
  • L is or comprises at least one diamino butyric acid group, a substituted benzene group, a lysine group, a 2,3- diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or any combination of two or more thereof.
  • EEs 1-11 and 15-21 wherein L is or comprises an ether, a thioether, a tertiary amine, a C 1-6 alkyl, piperazine, piperidine, a bicycloheptane, a substituted benzene, or a combination of two or more thereof.
  • EE 26 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 104 PRF_70572-03 PCT [0255] EE 27.
  • L is or comprises a moiety of the formula: w [0256] EE 28.
  • EE 30 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 105 PRF_70572-03 PCT O O H N OH [0258] EE 30.
  • EE 31 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: [0260] EE 32.
  • EE 33 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 106 PRF_70572-03 PCT [0261] EE 33.
  • EE 34 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: [0262] EE 34.
  • EE 35 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 107 PRF_70572-03 PCT . [0264] EE 36.
  • EE 37 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a reductively cleavable linker.
  • EE 38 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an oxidatively cleavable linker.
  • EE 39 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an oxime ester.
  • EE 40 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a hydrazone.
  • EE 41 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an enzyme-cleavable linker.
  • EE 43 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a peptide.
  • EE 44 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a peptidoglycan.
  • EE 45 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a peptidoglycan.
  • EE 46 The method of any one of EEs 1-11 and 15-21, wherein L is: .
  • EE 46 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a linker selected from the group consisting of pegylated-, alkyl-, sugar-, and peptide-based dual linkers.
  • EE 47 The method of any one of EEs 1-11 and 15-21, wherein L is a non-releasable linker.
  • EE 48 The method of any one of EEs 1-11 and 15-21, wherein L is covalently bonded to A and B of formula (I) or A, B and C of formula (II).
  • EE 49 The method of any one of EEs 1-11 and 15-21, wherein L is covalently bonded to A and B of formula (I) or A, B and C of formula (II).
  • EE 50 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 109 PRF_70572-03 PCT S O O O S O O O O O O N CO 2 H [0278] EE 50.
  • L is or comprises a moiety selected from: COOH COOH N S N S 2 8 l, which can be optionally substituted; and z is an integer from 1 to 8.
  • EE 51 The method of any one of EEs 1-11 and 15-21, wherein L is or comprises: . [0280] EE 52.
  • C is an albumin binding ligand, a disulfide-stabilized protein scaffold comprising albumin binding domain 035 (ABD035), albumin binding domain Con (i.e., a peptide of a three-helix bundle 45 amino acids in length (ABDCon)), a designed ankyrin repeat protein (DARPin), a disulfide-stabilized Fv fragment (dsFv) of an anti-albumin antibody, a nanobody that complexes with human serum albumin (HSA), or a variable new antigen receptor (VNAR).
  • C is an albumin binding ligand, a disulfide-stabilized protein scaffold comprising albumin binding domain 035 (ABD035), albumin binding domain Con (i.e., a peptide of a three-helix bundle 45 amino acids in length (ABDCon)), a designed ankyrin repeat protein (DARPin), a disulfide-stabilized Fv fragment (dsFv) of an anti-albumin antibody,
  • EE 55 The method of any one of EEs 1-11 and 16-52, wherein C is or comprises one of: . [0283] EE 55.
  • EE 56 The method of any one of EEs 1-11 and 16-52, wherein C is or comprises: 112 PRF_70572-03 PCT O 2 I .
  • EE 58 The method of any one of EEs 1-11 and 16-52, wherein C is or comprises: 113 PRF_70572-03 PCT .
  • EE 60 The method of any one of EEs 1-11 and 16-52, wherein C is or comprises: .
  • EE 61 The method of any one of EEs 1-11 and 16-52, wherein C is or comprises a hapten bound by an autologous antibody.
  • EE 62 The method of any one of EEs 1-11 and 16-52, wherein C is or comprises a hapten selected from rhamnose, an ⁇ -galactosyl moiety, a dinitrophenyl (DNP) moiety, and a trinitrophenyl (TNP) moiety.
  • EE 63 The method of EE 1, wherein A has the structure: 114 PRF_70572-03 PCT .
  • EE 64 The method of EE 1, wherein A has the structure: .
  • EE 65 The method of EE 1, wherein A has the structure: 115 PRF_70572-03 PCT .
  • EE 66 The method of EE 1, wherein A has the structure: . [0295] EE 67.
  • EE 67 The method of EE 1, wherein A-L-B has the structure: 116 PRF_70572-03 PCT .
  • Me [0296] EE 68 The conjugate having the structure: 117 PRF_70572-03 PCT om e Me M m PRF_70572-03 PCT e 119 PRF_70572-03 PCT or 120 PRF_70572-03 PCT .
  • EE 69 A conjugate having the structure: [0298] EE 70.
  • a pharmaceutical composition comprising a conjugate of EE 68 or EE 69 and a pharmaceutically acceptable carrier. 121

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Methods of using keto-amide based FAP-targeted conjugates and compositions comprising same to treat fibrosis.

Description

PRF_70572-03 PCT METHOD OF USING CHEMICALLY STABLE KETO-AMIDE FIBROBLAST ACTIVATION PROTEIN-TARGETED CONJUGATES AND COMPOSITIONS COMPRISING SAME TO TREAT FIBROSIS CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. provisional patent application no.63/648,296, which was filed May 16, 2024, and U.S. provisional patent application no.63/761,300, which was filed February 21, 2025, both of which are hereby incorporated by reference in their entireties. TECHNICAL FIELD [0002] The present disclosure relates to fibroblast activation protein (FAP)-targeted ligands, conjugates comprising same, compositions comprising the conjugates, and methods of targeting active agents in subjects with fibrosis. BACKGROUND [0003] Fibroblast activation protein (FAP) is characterized as a type-II, membrane-bound serine protease responsible for cleaving proline-amino acid peptide bonds. Its expression is notably identified on cancer-associated fibroblasts (CAFs) and myofibroblasts engaged in collagen production. Consequently, there has been an emergence recently of reported FAP-targeted drugs and imaging agents designed for applications in cancer and other fibrotic diseases. [0004] While several small molecule ligands targeting FAP are available, recently disclosed FAP-targeted ligands have encountered challenges related to chemical instability, particularly under acidic or basic conditions and/or under reaction conditions involving heat, resulting in the formation of undesired side products, leading to their accumulation in healthy tissues, such as the liver and spleen; etc. Conjugates comprising such FAP-targeted ligands are plagued by short tumor retention and, consequently, poor signal to background ratios. [0005] To address these issues, a new FAP-targeted ligand, which is referred to as FAP9, has been developed. Notably, FAP9 demonstrates enhanced stability under both acidic and basic conditions, while maintaining binding affinity that is similar to, or better than, that of previously disclosed FAP-targeted ligands. 1 PRF_70572-03 PCT [0006] In view of the foregoing, it is an object of the present disclosure to provide a method of using conjugates comprising FAP9 and compositions comprising same in the targeted delivery of active agents, such as inhibitors of phosphatidylinositol-3-kinase (PI3k) and transforming growth factor β (TGFβ), to treat subjects with fibrosis. This and other objects and advantages will be apparent from the detailed description provided herein. SUMMARY [0007] A method of treating fibrosis in a subject is provided. The method comprises administering to the subject an effective amount of a compound of formula I or II: or wherein A has the structure: , wherein L is a releasable or non-releasable bi-functionalized linker, which binds A and B, or L is a releasable or non-releasable tri-functionalized linker, which binds A, B, and C; B is an active agent effective for the treatment of fibrosis; C is a pharmacokinetic (PK) extender; represents a functionalized 5- to 10-membered, N-containing, aromatic or non-aro , - or bi-cyclic heterocycle, which optionally comprises 1-3 heteroatoms independently selected from O, N, and S, and indicates the point of attachment of A to L or A attaches to L via any carbon atom of the f tionalized 5- to 10-membered, N-containing, aromatic or non-aromatic, mono- or bicyclic heterocycle, an alkyl primary amine, an alkyl secondary amine, a functionalized alkyl amine, or a functionalized cycloalkyl amine; 2 PRF_70572-03 PCT [008] R1 is selected from the group consisting of F, Cl, Br, I, OH, CF3, -NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, Cl-Cl0 alkyl, C3-Cl0 cycloalkyl, adamantyl, aryl, and C7- C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted; [009] R2, R3, R4, R5, R6 and R7 are independently selected from the group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; [0010] R8 is selected from the group of substituents consisting of -H, -D, -OH, =CH2, -CH3, -CH2CH3, -C(H)(CH3)2, -C(CH3)3, and -CH2Ph, wherein any substituent comprising at least two atoms can be optionally substituted; [0011] R9, R10, and R11 are independently selected from group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -N3, -NH=NH-, -N-C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; [0012] R12 is selected from the group of substituents consisting of -H, -D, -F, -C1-C6 alkyl, -C(O)CH3, and -Cl-Cl0 alkyl, wherein any substituent comprising at least two atoms can be optionally substituted; and [0013] R13 is selected from the group of substituents consisting of -H, -D, Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, adamantyl, aryl, and C7-C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted and the aryl in C7-C20 alkyl aryl is: [0014] elected from the group consisting of -H, -D, -halo, and Cl-C4 alkyl, which is optionally substituted, and R14a, R15a, R16a, and R17a are independently selected from the group of substituents consisting of -H, -D, -halo, -Cl-C3 alkyl, -Cl-C3 alkoxy, -CF3, and - C(=O)OR12, wherein R12 is as defined above, the dashed line indicates the point of attachment to the nitrogen of A, and any substituent comprising at least two atoms can be optionally substituted; or 3 PRF_70572-03 PCT R13 is: [0015] ndependently selected from the group of substituents consisting of -H, -D, -OMe, -C1-C3 alkyl, benzyl (Ph-CH2-), and substituted/functionalized benzyls, the dashed line indicates the point of attachment to the nitrogen of A, and any substituent comprising at least two atoms can be optionally substituted; [0016] or a stereoisomer, a pharmaceutically acceptable salt, or a hydrate thereof. In embodiments of the method, A has the structures of formulae III, formulae IV, formulae V, formulae VI, formulae VII, or formulae VIII: PRF_70572-03 PCT 5 PRF_70572-03 PCT PRF_70572-03 PCT R R6 R5 R4 R 7 8 R3 R2 III wherein n [0017] R1 is selected from the group of substituents consisting of -F, -Cl, -Br, -I, -OH, -CF3, -NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, - adamantyl, -aryl and -C7-C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted; [0018] R2, R3, R4, R5, R6 and R7 are independently selected from the group of substituents consisting of -H, -D, - OH, -F, -Cl, -Br, -I, -C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; [0019] R8 is selected from the group of substituents consisting of -H, -D, -OH, =CH2, -CH3, -CH2CH3, -C(H)(CH3)2, -C(CH3)3, -CH2Ph, wherein any substituent comprising at least two atoms can be optionally substituted; [0020] R12 and R13 are independently selected from the group of substituents consisting of -H, -D, -F, -C1-C6 alkyl, -C(O)CH3, and -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, adamantyl, aryl, and C7-C20 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; and [0021] R23-R26 are independently selected from group of substituents consisting of -H, -OH, -F, -Cl, -Br, -I, -CF3, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH2, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, and the structure -X-L-B or -X-L(BC), wherein X is O, S, -NH, -NCH3, or -CH2, wherein any substituent comprising at least two atoms can be optionally independently 7 PRF_70572-03 PCT substituted. In embodiments thereof, R13 is independently selected from the group of substituents consisting of , PRF_70572-03 PCT , wherein any substituent can be optionally substituted. [0022] In certain embodiments, B can be a phosphatidylinositol-3-kinase (PI3k) inhibitor, a dual inhibitor of the PI3k/mTOR signaling pathway. In other embodiments, B can be a transforming growth factor β (TGFβ) inhibitor, an inhibitor of the TGFβ1/ALK5/Smad-2, -3 signalling pathway. In yet other embodiments, B can be, variously, a Rho-kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor; see, e.g., Fig.14), a focal adhesion kinase (FAK) inhibitor (see, e.g., Fig.14), vascular endothelial growth factor receptor (VEGFR) inhibitor (e.g., VEGFR1, VEGFR2, or VEGFR3; see, e.g., Fig.14), or a platelet-derived growth factor receptor (PDGFR) inhibitor (see, e.g., Fig.14). In embodiments where B is a dual inhibitor of the PI3k/mTOR signaling pathway, the dual inhibitor can be: 9 PRF_70572-03 PCT . [0023] In embodiments, the PI3k inhibitor can have the structure: 10 PRF_70572-03 PCT m the group consisting of: . [0024] . [0025] can have the structure. N A PRF_70572-03 PCT wherein X and Z independently can be -CH2-, -O-, -S-, -SO2-, -NH-, -NHR-, or -CO-, and y can be 1 to 20. [0026] In some embodiments, the TGFβ inhibitor can have the structure: wher e R1 rand R2 are each, independently, H, methyl, Cl, Br. O, I, or F; and X is NH, O, or S. [0027] In certain embodiments The TGFβ inhibitor can have the structure: . [0028] In s, L is or comprises a PEG monomer of the formula -(PEG)n- where n is 1-6. One example conjugate having the above TGFβ inhibitor has the structure: . PRF_70572-03 PCT [0029] In other embodiments, L is O O O O HN O O HN O O Or HN O HN HN O O HN O HN HN O S S Or N O HN HN O S S NH Or r H S S NH S S N S Or NH . [0030] Another example conjugate having the above TGFβ inhibitor has the structure: . [0031] In some embodiments the ROCK inhibitor can have one of the structures: O N N N . [0032] In some embodiments, the FAK inhibitor can be 13 PRF_70572-03 PCT . [0033] The VEGFR inhibitor can have the structure: . [0034] In certain embodiments, the PDGFR inhibitor can have the structure: . [0035] -binding ligand, a hapten, or an internalization-inducing peptide. [0036] In embodiments of L, L can comprise one or more of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a polylactone, a polymethylmethacrylate, a polyoxymethylene, a heterocycle, or any combination of two or more thereof. In embodiments, L can comprise an oligomer of one or more peptidoglycans, glycans, anions, heterocycles, or any combination of two or more thereof. In embodiments, L can comprise at least one diamino butyric acid group, a substituted benzene group, a lysine group, a 2,3-diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or 14 PRF_70572-03 PCT any combination of two or more thereof. In embodiments, L can comprise an ether, a thioether, a tertiary amine, a C1-6 alkyl, piperazine, piperidine, a bicycloheptane, a substituted benzene, or a combination of two or more thereof. In embodiments, L can be or can comprise a moiety of the formula: H O H O O N N O O n N N , . oiety of the formula: mula: ety of the formula: [0037] , e or can comprise a moiety of the formula: 15 PRF_70572-03 PCT . [0038] I ise a moiety of the formula: [0039] In embodiments, L can be or can comprise a moiety of the formula: N N O N N N N N O N N O N O , the formula: O PRF_70572-03 PCT . [0040] he formula: . [0041] iety of the formula: . [0042] eavable linker. In embodiments, L is or can comprise an oxidatively cleavable linker. In embodiments, L can be or can comprise an oxime ester. In embodiments, L is or can comprise a hydrazone. In embodiments, L is or can comprise an enzyme-cleavable linker. In embodiments, L can be or can comprise a PEGn, and n = 0-36. In embodiments, L can be or can comprise a peptide. In embodiments, L can be or can comprise a peptidoglycan. In embodiments, L can be or comprises: 17 PRF_70572-03 PCT O O O HN O O O O HN HN O O Or Or HN HN O O HN O HN HN O S S N S Or Or HN HN O H S NH S S NH S S N S Or NH . alkyl- , sugar-, and peptide-based dual linkers. In embodiments, L is a non-releasable linker. In embodiments, L is covalently bonded to A and B of formula (I) or A, B and C of formula (II). In embodiments L can be or can comprise a moiety of the formula: S O O O O O O S O O CO2H . [0044] In embodiments, L can be or can comprise a moiety selected from: COOH COOH S 28 l, which can be optionally substituted; and z is an integer from 1 to 8. In embodiments, L can be or can comprise: . , L can be or can comprise: 18 PRF_70572-03 PCT 29b, R30a, and R30b is independently H or C1- C6 alkyl, which can be optionally substituted (e.g., wherein each of R29a, R29b, R30a, and R30b is C1-alkyl (i.e., methyl)). [0046] In embodiments, C can be an albumin binding ligand, a disulfide-stabilized protein scaffold comprising albumin binding domain 035 (ABD035), albumin binding domain Con (i.e., a peptide of a three-helix bundle 45 amino acids in length (ABDCon)), a designed ankyrin repeat protein (DARPin), a disulfide-stabilized Fv fragment (dsFv) of an anti-albumin antibody (e.g., CA645), a nanobody that complexes with human serum albumin (HSA), or a variable new antigen receptor (VNAR) (e.g., E06). In embodiments, C can be or can comprise: 19 PRF_70572-03 PCT . [0047] In embodiments, C can be or can comprise: PRF_70572-03 PCT wherein, as applicable: each of R12-19 (where applicable) is independently selected from -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, and -SO2F; and each of R20 and R21 is independently selected from -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -O-C1-6 alkyl, -CN, -CHO, -B(OH)2, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, O n -PO3H2, -SO2F, CF3, and . [0048] In embodiments, C can be or can comprise: O2 . , p 21 PRF_70572-03 PCT . [0050] s, C can be or can comprise: . [0051] C can be or can comprise (PEG)n, wherein n is an integer 0 to 32, a peptide, a peptidoglycan, or a saccharide. In embodiments, C can be or can comprise: . [0052] , an autologous antibody. In embodiments, C can be or can comprise a hapten selected from rhamnose, an α-galactosyl moiety, a dinitrophenyl (DNP) moiety, and a trinitrophenyl (TNP) moiety. [0053] In embodiments, A can have the structure: 22 PRF_70572-03 PCT . . , 23 PRF_70572-03 PCT [0 [0 24 PRF_70572-03 PCT Me PRF_70572-03 PCT PRF_70572-03 PCT so pov e s a pa aceu ca co pos o co p s g one of the aforementioned A- L-B conjugates (i.e., 29-31, 37, 38, 40, 43, 47-49, and 60-63) and a pharmaceutically acceptable carrier. 27 PRF_70572-03 PCT FIGURES [0059] Fig.1 shows the docking of FAP9 base ligand with human fibroblast activation protein alpha (FAPα) (PDB number: 1Z68). [0060] Fig.2 shows the docking of different FAP9 analogues with human FAP alpha (FAPα) (PDB number: 1Z68), wherein (A) is FAP8, (B) is with methyl, (C) is with ethyl, (D) is with propyl, (E) is with butyl, and (F) is with fluoro substitution. [0061] Fig.3 shows the docking of FAP10 with human FAPα (PDB number: 1Z68). [0062] Fig.4 shows the synthesis of FAP9 with a cysteine linker. [0063] Fig.5 shows the synthesis of different activated phosphatidylinositol-3-kinase (PI3k) inhibitors (24, 26, and 28). [0064] Fig.6 shows the synthesis of different FAP9-PI3k conjugates (29-31). [0065] Fig.7 shows the synthesis of FAP9-PEG3 with a cysteine linker (34). [0066] Fig.8 shows the synthesis of FAP9-transforming growth factor β (FAP9-TGFβ) conjugates (37-38). [0067] Fig.9 shows the synthesis of conjugate (40) with a non-cleavable linker. [0068] Fig.10 shows the synthesis of conjugate (43) with a non-cleavable linker. [0069] Fig.11 shows the synthesis of different FAP9-PI3k conjugates (47-49) with cathepsin- cleavable linkers. [0070] Fig.12 shows the synthesis of different FAP9-TGFβ conjugates (60-63) with cathepsin- cleavable linkers. [0071] Fig.13 shows a conjugate with a reductively cleavable linker and a conjugate with an enzymatically cleavable linker. [0072] Fig.14 shows examples of ROCK inhibitors, a FAK inhibitor, a PDGFR inhibitor, and VEGFR inhibitors. [0073] Fig.15 shows the analysis of pAKt and α-SMA assay of compound 29 and 40. [0074] Fig.16 shows the relative gene expression levels of col1a1 (Fig.16A), col3a1 (Fig. 16B), and acta2 (Fig.16C) in healthy mice vs. fibrotic mice with no treatment, and fibrotic mice receiving FAP9-PI3Ki conjugate (29) treatment from day 3 and day 14 of folic acid administration, respectively. All data points are expressed as mean ± standard error (n=3). Statistical significances are illustrated as p<0.05 for single asterisk, p<0.01 for double asterisks, 28 PRF_70572-03 PCT p<0.001 for triple asterisks, and p<0.0001 for quadruple asterisks, while ‘ns’ represents no statistical significance. [0075] Fig.17 shows the hydroxyproline content expressed as μg/g of wet kidney mass in healthy mice vs. fibrotic mice with no treatment, and fibrotic mice receiving FAP9-PI3Ki (Conjuate-29) treatment from day 3 and day 14 of folic acid administration, respectively. All data points are expressed as Mean ± Standard error (n=3). Statistical significances are illustrated as p<0.05 for single asterisk, p<0.01 for double asterisks, p<0.001 for triple asterisks and p<0.0001 for quadruple asterisks, while ‘ns’ represents no statistical significance. [0076] Fig.18 shows the analysis of pAKt and α-SMA assay of FAP9-TGFβ conjugate (37). [0077] Fig.19 presnets optical imaging of FA-induced renal fibrosis in mice with an FAP- targeted NIR dye (FAP9-S0456). [0078] Fig.20 presents an evaluation of FAP9-TGFBR1i for the for treatment of FA-induced renal fibrosis in mice. [0079] Fig.21 shows FAP9-FITC internalization in 3T3-mFAP cells. DESCRIPTION [0080] The present disclosure employs chemically stable keto-amide-based ligands that target fibroblast activation protein (FAPα). Incorporation of the ligands into conjugates enables the targeted delivery of active agents to subjects with fibrosis (e.g., such as fibrosis of the heart, kidney, bladder, muscle, skin or liver). Advantages of such conjugates include, but are not limited to, increased tumor retention and better signal to background ratios. [0081] In view of the above, provided is a method of treating fibrosis in a subject. As used herein, "subject" means either an animal or human subject. "Animal" means primates, livestock animals (including, without limitation, cows, horses, sheep, pigs and goats), companion animals (including dogs, cats, rabbits and guinea pigs), and captive wild animals (including those commonly found in a zoo environment). Laboratory animals such as rabbits, mice, rats, guinea pigs and hamsters are also contemplated as they may provide a convenient test system. Given that FAP homologs have been found in zebrafish and amphibians, i.e., two species of the Xenopus genus, the subject, in certain instances, could be a fish or an amphibian. 29 PRF_70572-03 PCT [0082] The subject can be a human or a mammal of economic importance and/or social importance to humans, for instance, carnivores other than humans (e.g., cats and dogs), swine (e.g., pigs, hogs, and wild boars), ruminants (e.g., cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), horses, and birds including those kinds of birds that are endangered and kept in zoos, and fowl, more particularly domesticated fowl (e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like) as they are also of economic importance to humans. [0083] The term "subject" does not denote a particular age. Thus, adult, juvenile and newborn subjects are covered. The terms "subject, " "individual" and "patient" may be used interchangeably herein. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. [0084] The method comprises administering to the subject an effective amount of a compound of formula I or II: , wherein L is a releasable or non-releasable bi-functionalized linker, which binds A and B, or L is a releasable or non-releasable tri-functionalized linker, which binds A, B, and C; B is an active agent effective for the treatment of fibrosis; C is a pharmacokinetic (PK) extender; represents a functionalized 5- to 10-membered, N-containing, aromatic or non-aro mat c, mono- or bi-cyclic heterocycle, which optionally comprises 1-3 heteroatoms independently selected from O, N, and S, and indicates the point of attachment of A to L or A attaches to L via any carbon atom of the f tionalized 5- to 10-membered, N-containing, 30 PRF_70572-03 PCT aromatic or non-aromatic, mono- or bicyclic heterocycle, an alkyl primary amine, an alkyl secondary amine, a functionalized alkyl amine, or a functionalized cycloalkyl amine; [0085] R1 is selected from the group consisting of F, Cl, Br, I, OH, CF3, -NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, Cl-Cl0 alkyl, C3-Cl0 cycloalkyl, adamantyl, aryl, and C7- C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted; [0086] R2, R3, R4, R5, R6 and R7 are independently selected from the group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; [0087] R8 is selected from the group of substituents consisting of -H, -D, -OH, =CH2, -CH3, - CH2CH3, -C(H)(CH3)2, -C(CH3)3, and -CH2Ph, wherein any substituent comprising at least two atoms can be optionally substituted; [0088] R9, R10, and R11 are independently selected from group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -N3, -NH=NH-, -N-C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; [0089] R12 is selected from the group of substituents consisting of -H, -D, -F, -C1-C6 alkyl, -C(O)CH3, and -Cl-Cl0 alkyl, wherein any substituent comprising at least two atoms can be optionally substituted; and [0090] R13 is selected from the group of substituents consisting of -H, -D, Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, adamantyl, aryl, and C7-C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted and the aryl in C7-C20 alkyl aryl is: elected from the group consisting of -H, -D, -halo, and Cl-C4 alkyl, which is optionally substituted, and R14a, R15a, R16a, and R17a are independently selected from the group of substituents consisting of -H, -D, -halo, -Cl-C3 alkyl, -Cl-C3 alkoxy, -CF3, and 31 PRF_70572-03 PCT -C(=O)OR12, wherein R12 is as defined above, the dashed line indicates the point of attachment to the nitrogen of A, and any substituent comprising at least two atoms can be optionally substituted; or R13 is: ependently selected from the group of substituents consisting of -H, -D, -OMe, -C1-C3 alkyl, benzyl (Ph-CH2-), and substituted/functionalized benzyls, the dashed line indicates the point of attachment to the nitrogen of A, and any substituent comprising at least two atoms can be optionally substituted; or a stereoisomer, a pharmaceutically acceptable salt, or a hydrate thereof. In embodiments of the method, A has the structures of formulae III, formulae IV, formulae V, formulae VI, formulae VII, or formulae VIII: PRF_70572-03 PCT 33 PRF_70572-03 PCT PRF_70572-03 PCT R R6 R5 R4 R 7 8 R3 R2 III where [0091] R1 is selected from the group of substituents consisting of -F, -Cl, -Br, -I, -OH, -CF3, - NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, - adamantyl, -aryl and -C7-C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted; [0092] R2, R3, R4, R5, R6 and R7 are independently selected from the group of substituents consisting of -H, -D, - OH, -F, -Cl, -Br, -I, -C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; [0093] R8 is selected from the group of substituents consisting of -H, -D, -OH, =CH2, -CH3, - CH2CH3, -C(H)(CH3)2, -C(CH3)3, -CH2Ph, wherein any substituent comprising at least two atoms can be optionally substituted; [0094] R12 and R13 are independently selected from the group of substituents consisting of -H, - D, -F, -C1-C6 alkyl, -C(O)CH3, and -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, adamantyl, aryl, and C7-C20 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; and [0095] R23-R26 are independently selected from group of substituents consisting of -H, -OH, -F, - Cl, -Br, -I, -CF3, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH2, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1- 6 alkyl, and the structure -X-L-B or -X-L(BC), wherein X is O, S, -NH, -NCH3, or -CH2, wherein any substituent comprising at least two atoms can be optionally independently substituted. In embodiments thereof, R13 is independently selected from the group of substituents consisting of 35 PRF_70572-03 PCT , PRF_70572-03 PCT O O O O O , O , wherein any substituent can be optionally substituted. [0096] In certain embodiments, B can be a phosphatidylinositol-3-kinase (PI3k) inhibitor, a dual inhibitor of the PI3k/mTOR signaling pathway. In other embodiments, B can be a transforming growth factor β (TGFβ) inhibitor, an inhibitor of the TGFβ1/ALK5/Smad-2, -3 signalling pathway. In yet other embodiments, B can be, variously, a Rho-kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor; see Fig. 14 for example structures), a focal adhesion kinase (FAK) inhibitor (see Fig. 14 for example structure), vascular endothelial growth factor receptor (VEGFR) inhibitor (see Fig. 14 for example structures), or a platelet-derived growth factor receptor (PDGFR) inhibitor (see Fig. 14 for example structure). In embodiments where B is a dual inhibitor of the PI3k/mTOR signaling pathway, the dual inhibitor can be: PRF_70572-03 PCT . In embodiments, the PI3k inhibitor as e s ucue: rom the group consisting of: In an emb . [0097] n em o ment, t e 3 n tor can have the structure: 38 PRF_70572-03 PCT wherein X and Z independently can be -CH2-, -O-, -S-, -SO2-, -NH-, -NHR-, or -CO-, and y can be 1 to 20. [0098] Transforming growth factor-beta 1 (TGF-β1) is a key mediator in the development and progression of fibrotic diseases, including renal fibrosis. In the canonical TGF-β1/ALK5/Smad- 2, -3 signaling pathway, TGF-β1 initially binds to its receptor, TGF-β receptor type II (TGFBR2), which subsequently recruits TGF-β receptor type I (TGFBR1, also known as activin receptor-like kinase 5 or ALK5). This forms the TGF-β–TGFBR1–TGFBR2 receptor complex. Upon activation, TGFBR1 phosphorylates downstream effectors Smad2 and Smad3. The phosphorylated Smad2/3 complex then binds to Smad4 and translocates into the nucleus to regulate the transcription of TGF-β1 target profibrotic genes, including α-smooth muscle actin (α-SMA), fibronectin (FN), hydroxyproline and collagen. Notably, TGF-β1–mediated fibrosis is closely associated with the upregulation of TGFBR1 expression in both kidney and lung fibrosis. Various therapeutic strategies have been explored to inhibit TGFBR1 in experimental fibrosis models using small-molecule inhibitors, which have demonstrated promising antifibrotic effects in both in vitro and in vivo studies." [0099] In some embodiments, compounds that inhibit any of the three isoforms are contemplated. In certain embodiments, the TGFβ inhibitor has the structure: 39 PRF_70572-03 PCT where 1 ran 2 are eac , n epen enty, , methyl, Cl, Br. O, I, or F; and X is NH, O, or S. [0100] In certain embodiments The TGFβ inhibitor can have the structure: . [0101] In some embodiments, the FAK inhibitor is F3C N N O . [0102] The VEGFR inhibitor can have the structure: 40 PRF_70572-03 PCT N N H2 [0103] In certain embodiments, the PDGFR inhibitor is of the structure: O NH . [0104] In certain embodiments, the ROCK inhibitor can have the structure: [0105] In some embodments t e ROCK n btor as one o te structures: 41 PRF_70572-03 PCT . [0106] The PK extender can be an albumin-binding ligand, a plasma protein-binding ligand, a hapten, or an internalization-inducing peptide. [0107] In embodiments of L, L is or can comprise (the use of “is or can comprise” includes radicals thereof) one or more of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a polylactone, a polymethylmethacrylate, a polyoxymethylene, a heterocycle, or any combination of two or more thereof. In embodiments, L is or can comprise an oligomer of one or more peptidoglycans, glycans, anions, heterocycles, or any combination of two or more thereof. In embodiments, L is or can comprise at least one diamino butyric acid group, a substituted benzene group, a lysine group, a 2,3-diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or any combination of two or more thereof. In embodiments, L is can comprise an ether, a thioether, a tertiary amine, a C1-6 alkyl, piperazine, piperidine, a bicycloheptane, a substituted benzene, or a combination of two or more thereof. In embodiments, L is or can comprise a moiety of the formula: 42 PRF_70572-03 PCT H O H O O N N O O n N N , of the formula: S O O H , wherein n = 0-10. In an embodiment, L is or can comprise a moiety of the formula: [0108] , can comprise a moiety of the formula: . [0109] I n embodiments, L is or can comprise a moiety of the formula: 43 PRF_70572-03 PCT [0110] In embodiments, L is or can comprise a moiety of the formula: wherein n = 0=10. In embodiments, L is or can comprise a moiety of the formula: PRF_70572-03 PCT . [0111] ormula: . [0112] f the formula: . [00113 able linker (see Fig.13 for example). In embodiments, L is or can comprise an oxidatively cleavable linker. In embodiments, L is or can comprise an oxime ester. In embodiments, L is or can comprise a hydrazone. In embodiments, L is or can comprise an enzyme-cleavable linker (see Fig.13 for example). Fig.13 further illustrates attachment of a drug moiety to the linker. In embodiments, L is or can comprise a PEGn, and n = 0-36. In embodiments, L is or can comprise a peptide. In embodiments, L is or can comprise a peptidoglycan. In embodiments, L is or comprises: 45 PRF_70572-03 PCT . [0114] In embodiments, L is a linker selected from the group consisting of pegylated-, alkyl-, sugar-, and peptide-based dual linkers. In embodiments, L is a non-releasable linker. In embodiments, L is covalently bonded to A and B of formula (I) or A, B and C of formula (II). In embodiments L is or comprises a moiety of the formula: . [0115] In embodiments, L is or comprises a moiety selected from: yl, which can be optionally substituted; and z is an integer from 1 to 8. In embodiments, L is or comprises: 46 PRF_70572-03 PCT . [ L is or comprises: 29b, R30a, and R30b is independently H or C1- C6 alkyl (e.g., wherein each of R29a, R29b, R30a, and R30b is C1-alkyl (i.e., methyl)), which can be optionally substituted. [0117] In embodiments, C is an albumin binding ligand, a disulfide-stabilized protein scaffold comprising albumin binding domain 035 (ABD035), albumin binding domain Con (i.e., a peptide of a three-helix bundle 45 amino acids in length (ABDCon)), a designed ankyrin repeat protein (DARPin), a disulfide-stabilized Fv fragment (dsFv) of an anti-albumin antibody (e.g., CA645), a nanobody that complexes with human serum albumin (HSA), or a variable new antigen receptor (VNAR) (e.g., E06). In embodiments, C is or comprises: 47 PRF_70572-03 PCT . [0118] In embodiments, C is or comprises: PRF_70572-03 PCT wherein, as applicable: each of R12-19 (where applicable) is independently selected from -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, and -SO2F; and each of R20 and R21 is independently selected from -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -O-C1-6 alkyl, -CN, -CHO, -B(OH)2, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, O , p 49 PRF_70572-03 PCT . [0121] s, C is or comprises: . [0122] C is or comprises (PEG)n, wherein n is an integer 0 to 32, a peptide, a peptidoglycan, or a saccharide. In embodiments, C is or comprises: . [0123] , logous antibody. In embodiments, C is or comprises a hapten selected from rhamnose, an α-galactosyl moiety, a dinitrophenyl (DNP) moiety, and a trinitrophenyl (TNP) moiety. In embodiments, A has the structure: 50 PRF_70572-03 PCT . . , 51 PRF_70572-03 PCT [0 [0127] B has th 52 PRF_70572-03 PCT N S S O N N 22 35 (TGF^ inhibitor) H F F O OMe N N N e Me PRF_70572-03 PCT 54 PRF_70572-03 PCT PRF_70572-03 PCT . rising one of the aforementioned A- L-B conjugates (i.e., 29-31, 37, 38, 40, 43, 47-49, and 60-63) and a pharmaceutically acceptable carrier. [0129] The conjugates can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as (R)- or (S)-. Unless stated otherwise, it is intended that all stereoisomeric forms of the conjugates are contemplated. When the conjugates described herein contain alkene double bonds, and unless specified otherwise, it is intended that this disclosure includes both E and Z geometric isomers (e.g., cis or trans). Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included. The term “geometric isomer” refers to E or Z geometric isomers (e.g., cis or trans) of an alkene double bond. The term “positional isomer” refers to structural isomers around a central ring, such as ortho-, meta-, and para- isomers around a benzene ring. Further, it is understood that replacement of one or more hydrogen atoms with deuterium can significantly lower the rate of metabolism of a drug and, therefore, increase its half-life. [0130] The term “substituted” as used herein refers to a functional group in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted 56 PRF_70572-03 PCT onto a molecule. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, azides, hydroxylamines, cyano, nitro groups, N-oxides, hydrazides, and enamines; and other heteroatoms in various other groups. [0131] The term “optionally substituted,” or “optional substituents,” as used herein, means that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent, the substituents may be the same or different. When using the terms “independently,” “independently are,” and “independently selected from” mean that the groups in question may be the same or different. Certain of the herein defined terms may occur more than once in the structure, and upon such occurrence each term shall be defined independently of the other. [0132] "Oxo" refers to the =O radical. [0133] "Alkyl" generally refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as having from one to fifteen carbon atoms (e.g., C1- C15 alkyl). "Alkyl" is intended to include independent recitations of a saturated "alkyl, " unless otherwise stated. An alkyl can comprise one to thirteen carbon atoms (e.g., C1-C13 alkyl). An alkyl can comprise one to eight carbon atoms (e.g., C1-C8 alkyl). An alkyl can comprise one to five carbon atoms (e.g., C1-C5 alkyl). An alkyl can comprise one to four carbon atoms (e.g., C1- C4 alkyl). An alkyl can comprise one to three carbon atoms (e.g., C1-C3 alkyl). An alkyl can comprise one to two carbon atoms (e.g., C1-C2 alkyl). An alkyl can comprise one carbon atom (e.g., C1 alkyl). An alkyl can comprise five to fifteen carbon atoms (e.g., C5-C15 alkyl). An alkyl can comprise five to eight carbon atoms (e.g., C5-C8 alkyl). An alkyl can comprise two to five carbon atoms (e.g., C2-C5 alkyl). An alkyl can comprise three to five carbon atoms (e.g., C3-C5 alkyl). In various embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n- propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2- methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl is attached to the rest of the molecule by a single bond. 57 PRF_70572-03 PCT [0134] "Alkoxy" refers to a radical bonded through an oxygen atom of the formula –O-alkyl, where alkyl is an alkyl chain as defined above. [0135] "Alkylene" or "alkylene chain" generally refers to a straight or branched divalent alkyl group linking the rest of the molecule to a radical group, such as having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, i-propylene, n-butylene, and the like. [0136] "Aryl" refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and from five to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ^–electron system in accordance with the Hückel theory. The ring systems from which aryl groups are derived include, but are not limited to, benzene, fluorene, indane, indene, tetralin and naphthalene. [0137] "Aralkyl" or "aryl-alkyl" refers to a radical of the formula -Rc-aryl, where Rc is an alkylene chain as defined above, for example, methylene, ethylene, and the like. The alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain. [0138] "Carbocyclyl" or "cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which includes fused or bridged ring systems, having from three to fifteen carbon atoms. A carbocyclyl can comprise three to ten carbon atoms. A carbocyclyl can comprise five to seven carbon atoms. The carbocyclyl is attached to the rest of the molecule by a single bond. Carbocyclyl or cycloalkyl is saturated (i.e., containing single C-C bonds only) or unsaturated (i.e., containing one or more double bonds or triple bonds). Examples of saturated cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. An unsaturated carbocyclyl is also referred to as "cycloalkenyl." Examples of monocyclic cycloalkenyls include, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Polycyclic carbocyclyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. [0139] "Carbocyclylalkyl" refers to a radical of the formula –Rc-carbocyclyl, where Rc is an alkylene chain as defined above. 58 PRF_70572-03 PCT [0140] "Halo" or "halogen" refers to a bromo, chloro, fluoro or iodo substituent. [0141] "Haloalkyl" refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, as defined above, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like. [0142] The term "heteroalkyl" refers to an alkyl group as defined above in which one or more skeletal carbon atoms of the alkyl are substituted with a heteroatom (with the appropriate number of substituents or valencies – for example, -CH2- may be replaced with -NH- or -O-). For example, each substituted carbon atom is independently substituted with a heteroatom, such as wherein the carbon is substituted with a nitrogen, oxygen, selenium, or other suitable heteroatom. In some instances, each substituted carbon atom is independently substituted for an oxygen, nitrogen (e.g. -NH-, -N(alkyl)-, or -N(aryl)- or having another substituent contemplated herein), or sulfur (e.g. -S-, -S(=O)-, or -S(=O)2-). A heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. A heteroalkyl is attached to the rest of the molecule at a heteroatom of the heteroalkyl. A heteroalkyl is a C1-C18 heteroalkyl. A heteroalkyl is a C1-C12 heteroalkyl. A heteroalkyl is a C1-C6 heteroalkyl. A heteroalkyl is a C1- C4 heteroalkyl. Heteroalkyl can include alkoxy, alkoxyalkyl, alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocycloalkyl, and heterocycloalkylalkyl, as defined herein. [0143] "Heteroalkylene" refers to a divalent heteroalkyl group defined above which links one part of the molecule to another part of the molecule. [0144] "Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring radical that can comprise two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which optionally includes aromatic, fused, and/or bridged ring systems. The heteroatoms in the heterocyclyl radical are optionally oxidized. The heterocyclyl radical is partially or fully saturated. "Heterocyclyl" is intended to include independent recitations of heterocyclyl comprising aromatic and non- aromatic ring structures, unless otherwise stated. The heterocyclyl is attached to the rest of the molecule through any atom of the ring(s). Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 59 PRF_70572-03 PCT 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, indolinyl, isoindolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. [0145] "N-heterocyclyl" or "N-attached heterocyclyl" refers to a heterocyclyl radical as defined above containing at least one nitrogen and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical. Examples of such N-heterocyclyl radicals include, but are not limited to, 1-morpholinyl, 1- piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl. [0146] "Heteroaryl" refers to a radical derived from a 3- to 18-membered aromatic ring radical that can comprise two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. The heteroaryl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ^–electron system in accordance with the Hückel theory. Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical is optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl is attached to the rest of the molecule through any atom of the ring(s). Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[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]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[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, 60 PRF_70572-03 PCT indazolyl, isoindolyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, 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]pridinyl, and thiophenyl (i.e. thienyl). [0147] The compounds and conjugates can be presented as a pharmaceutically acceptable salt. The term “pharmaceutically acceptable salt” refers to those salts whose counter ions can be used in pharmaceuticals. In various embodiments, such salts include, but are not limited to 1) acid addition salts, which can be obtained by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like; or 2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N- methylglucamine, and the like. Pharmaceutically acceptable salts are well-known to those skilled in the art, and any such pharmaceutically acceptable salt is contemplated in connection with the embodiments described herein. [0148] Pharmaceutically acceptable salts can be synthesized from the parent conjugate/compound which contains a basic or acidic moiety by conventional chemical methods. In some instances, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is hereby incorporated by reference. 61 PRF_70572-03 PCT [0149] In certain embodiments, it can be desired to modify the conjugate and/or composition synthesis process to optimize yield at production (e.g., when the conjugate is optionally bound to a metal suitable for radio-imaging, radiotherapy, or magnetic resonance imaging). For example, a multiple step process can be utilized to facilitate stability of the conjugate at the pH required for radiolabeling. [0150] In various embodiments, suitable acid addition salts are formed from acids which form non-toxic salts. Illustrative examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts. [0151] In various embodiments, suitable base salts are formed from bases which form non-toxic salts. Illustrative examples include the arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases also can be formed, for example, hemisulphate and hemicalcium salts. [0152] In each embodiment hereof, it will be understood that the formulae include and represent not only all pharmaceutically acceptable salts of the compounds and conjugates, but also include any and all hydrates of the compound formulae or salts thereof where appropriate. The term “solvate” means a compound, or a salt thereof, that further includes a stoichiometric or non- stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a “hydrate.” [0153] Certain functional groups, such as the hydroxy, amino, and like, can form complexes and/or coordination conjugates with water and/or various solvents. Accordingly, the formulae are to be understood to include and represent those various hydrates and/or solvates. Non-hydrates and/or non-solvates of the compounds and conjugates are also included. [0154] The ligands and conjugates can be synthesized in accordance with methods known in the art. Methods are also exemplified herein. 62 PRF_70572-03 PCT [0155] The conjugates can be formulated as pharmaceutical compositions comprising the conjugate and a pharmaceutically acceptable carrier. The term "composition" generally refers to any product comprising more than one ingredient, including the conjugate. The compositions can be prepared from isolated conjugates or from salts, solutions, hydrates, solvates, and other forms of the conjugates. [0156] The term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The carrier can be an excipient. The choice of carrier can depend on factors such as the particular mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form. For example, the carrier can be suitable for parenteral administration. Pharmaceutical compositions suitable for the delivery of compounds as described herein and methods for their preparation may0 be found, for example, in Remington: The Science & Practice of Pharmacy, 21st edition (Lippincott Williams & Wilkins, 2005). [0157] Pharmaceutically acceptable carriers can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. 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 glycols. Liquids within which the conjugate can be dispersed include a carrier liquid or an in vivo liquid. By the conjugate being "dispersed" throughout or in a liquid is meant that the conjugate presents as a dispersed phase within the liquid which itself, relative to the conjugate, presents as a continuous liquid medium or phase. The term "liquid" in the context of a liquid carrier is intended to mean a vehicle in which the conjugate is dispersed and which is in a liquid state at least at the temperature of intended use. [0158] A liquid carrier can be made up of one or more different liquids. Suitable pharmacologically acceptable liquid carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA, (1990), and include, but are not limited to, liquids that are sterilized, such as water and oils, including those of petroleum, animal, vegetable, mineral or synthetic origin, such as peanut oil, soya bean oil, mineral oil, sesame oil, and the like. Other liquid carriers include methylene glycol, propylene glycol, 63 PRF_70572-03 PCT polyethylene glycol, polypropylene glycol, ethanol, isopropyl alcohol, and benzyl alcohol. Water or soluble saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions. [0159] In practice, the conjugate can be taken up by a subject in vivo, for example, when the conjugate is administered orally or parenterally. In that case, a liquid carrier originally carrying the conjugate can become so dilute in vivo that the surrounding liquid environment throughout which the conjugate is dispersed becomes more representative of an in vivo liquid (i.e., a biological liquid/fluid within the subject) than the original liquid carrier. For example, once administered parenterally, the conjugate might more aptly be described as being dispersed throughout blood rather than an original liquid carrier. Under those circumstances, it can be convenient to refer to the conjugate as being dispersed throughout an in vivo liquid carrier (i.e., a biological liquid/fluid within the subject). [0160] The components of the compositions also can be commingled with the conjugate, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency. [0161] The composition can comprise cremophor, polysorbate, nanoparticles, a polymer, or a hydrogel, for example. In certain embodiments, the pharmaceutical composition comprises a plurality of conjugates and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, and combinations thereof, that are physiologically compatible. One or more other active agents also can be incorporated into a pharmaceutical composition. [0162] In certain embodiments, a pharmaceutical composition further comprises at least one additional pharmaceutically active agent. The at least one additional pharmaceutically active agent can be an agent useful in the treatment of a cancer. In certain embodiments, the at least one additional pharmaceutically active agent can be an agent useful for radiotherapy. In certain embodiments, the at least one additional pharmaceutically active agent can be an agent useful for imaging (e.g., diagnostic imaging). [0163] Pharmaceutical compositions can be prepared by combining one or more conjugates with a pharmaceutically acceptable carrier and, optionally, one or more additional ingredients (e.g., pharmaceutically active ingredients). The formulations can be administered in pharmaceutically 64 PRF_70572-03 PCT acceptable solutions, which can routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. [0164] Compositions can comprise one or more pharmacologically acceptable additives known to those in the art. For example, the liquid carrier may comprise one or more additives such as wetting agents, de-foaming agents, surfactants, buffers, electrolytes, preservatives, colourings, flavourings, and sweeteners. [0165] The particular nature of a liquid carrier and any additive (if present) can, in part, depend upon the intended application of the composition. A suitable liquid carrier and additive (if present) can be selected for the intended application of the composition. [0166] The composition is suitable for administration to a subject for therapeutic applications. By "suitable" for administration is meant that administration of the conjugate/composition to a subject will not result in unacceptable toxicity, including allergenic responses and disease states. [0167] For use in therapy or treatment, an effective amount of the conjugate or composition can be administered to a subject by any mode that delivers the conjugate(s) as desired. Administering a composition can be accomplished by any means known to the skilled artisan. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection, mucosal (e.g., topical to eye), inhalation, and topical. [0168] Colorants and/or flavoring agents can be included. For example, the conjugate can be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents. [0169] Illustrative formats for oral administration include, but are not limited to, tablets, capsules, elixirs, syrups, and the like. [0170] In certain embodiments, a conjugate and/or composition can be administered directly into the blood stream, into muscle, or into an internal organ. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, intranasal, and subcutaneous. Suitable means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques. Where it is desirable to deliver the compound(s) and/or compositions systemically, the compound(s) and/or 65 PRF_70572-03 PCT composition can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. [0171] Parenteral formulations are typically aqueous or non-aqueous isotonic sterile solutions that can contain carriers or excipients, such as salts, carbohydrates, anti-oxidants, bactericide, solute and/or buffering agents (preferably at a pH of 3–9) which renders the composition isotonic with the blood of the intended subject, but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle, such as sterile, pyrogen-free water. Such compositions can be presented in unit- dose or multi-dose sealed containers, for example, ampoules and vials. [0172] A liquid formulation can be adapted for parenteral administration of a conjugate or composition as described herein. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization under sterile conditions, can readily be accomplished using standard pharmaceutical techniques well-known to those skilled in the art. The solubility of a conjugate can be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents. [0173] Formulations for parenteral administration can be formulated for immediate and/or modified release. A conjugate can be administered in a time-release formulation, for example in a composition which includes a slow-release polymer. The conjugate can be prepared with a carrier that will protect it against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PGLA). Methods for the preparation of such formulations are generally known to those skilled in the art. [0174] Sterile injectable solutions can be prepared by incorporating the conjugate(s), alone or in further combination with one or more other active agents, in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the conjugate(s) into a sterile vehicle, which contains a dispersion medium and any additional 66 PRF_70572-03 PCT ingredients of those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying, which yield a powder of the active ingredients plus any additional desired ingredient from a previously sterile-filtered solution thereof, or the ingredients can be sterile-filtered together. [0175] The pharmaceutical composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. [0176] A conjugate, or a pharmaceutical composition comprising a conjugate, can be continuously administered, where appropriate. [0177] The therapeutic regimen for the treatment of a disease state (i.e., fibrosis) can be determined by a person skilled in the art and will typically depend on factors including, but not limited to, the type, size, stage and receptor status of a tumor (e.g., with cancer) in addition to the age, weight and general health of the subject. Another determinative factor can be the risk of developing recurrent disease. For instance, for a subject identified as being at high risk or higher risk or developing recurrent disease, a more aggressive therapeutic regimen can be prescribed as compared to a subject who is deemed at a low or lower risk of developing recurrent disease. Similarly, for a subject identified as having a more advanced stage of cancer, for example, stage III or IV disease, a more aggressive therapeutic regimen can be prescribed as compared to a subject that has a less advanced stage of cancer. [0178] The terms "treat," "treatment," and "treating" refer to any and all uses which remedy a condition or symptom, or otherwise prevent, hinder, retard, abrogate or reverse the onset or progression of cancer or other undesirable symptoms in any way whatsoever. Thus, the term "treating," and the like, is to be considered in its broadest possible context. For example, treatment does not necessarily imply that a subject is treated until total recovery or cure. In conditions that display or are characterized by multiple signs or symptoms, the treatment need not necessarily remedy, prevent, hinder, retard, abrogate or reverse all signs or symptoms, but can remedy, prevent, hinder, retard, abrogate or reverse one or more signs or symptoms. 67 PRF_70572-03 PCT [0179] The expressions “effective amount” and "therapeutically effective amount" mean the amount of conjugate when administered to a mammal, in particular a human, in need of such treatment, is sufficient to treat cancer. The precise amount of conjugate to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the subject. [0180] "Administration" of the conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, and/or composition to a subject is meant that the conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, or composition is presented such that the conjugate(s) and/or pharmaceutically acceptable salts, hydrates, or solvates thereof can be transferred to the subject. There is no particular limitation on the mode of administration, but this will generally be by way of oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intracerebrally, intranasally, intrathecal, and intraspinal), inhalation (including nebulization), topical, rectal and vaginal modes. The conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, and/or composition can also be administered directly into a tumor and/or into tissue adjacent one or more segments of a tumor or administered directly into blood vessels. [0181] The conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, and/or composition can be administered in a treatment effective amount. A treatment effective amount includes an amount which, when administered according to the desired dosing regimen, achieves a desired therapeutic effect, including one or more of: alleviating the symptoms of, preventing or delaying the onset of, inhibiting or slowing the progression of, diagnosing, or halting or reversing altogether the onset or progression of a particular condition being treated and/or assessed. As used herein, “effective amount” means and encompasses both therapeutically effective amount and treatment or diagnostic effective amount. [0182] Suitable dosage amounts and dosing regimens to achieve this can be determined by the attending physician and can depend on the particular condition being treated, the severity of the condition as well the general age, health and weight of the subject. [0183] Depending upon the route of administration, a wide range of permissible dosages are contemplated. The dosing can occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages of the particulate material per se can lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. The dosage can be in the range of 1 µg to 1 g per kg of body weight per dosage, such as 68 PRF_70572-03 PCT is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage can be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage can be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage can be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage. [0184] Conjugate(s), pharmaceutically acceptable salt, hydrate, or solvate thereof, and/or compositions hereof can be administered in a single dose or a series of doses. For example, dosages may be single or divided and may be administered according to a wide variety of protocols, including q.d. (once a day), b.i.d. (twice a day), t.i.d. (three times a day), or even every other day, once a week, once a month, once a quarter, and the like. In each of these cases it is understood that the effective amounts described herein correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol. [0185] In addition to the illustrative dosages and dosing protocols described herein, an effective amount of any one or a mixture of the compounds described herein can be determined by the attending diagnostician or physician by the use of known techniques and/or by observing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances. [0186] In certain embodiments, a use of a conjugate, a pharmaceutically acceptable salt, hydrate, or solvate of the conjugate, or a composition hereof in the manufacture of a medicament for the treatment of a disease in a subject is provided. The conjugate can be any compound or conjugate hereof. The disease in the subject can be cancer. The disease in the subject can be fibrosis. [0187] Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible. [0188] While the conjugates and pharmaceutical compositions are illustrated and described in 69 PRF_70572-03 PCT detail in the foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only certain embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. [0189] Various techniques and mechanisms will sometimes describe a connection or link between two components. Words such as attached, linked, coupled, connected, and similar terms with their inflectional morphemes are used interchangeably, unless the difference is noted or made otherwise clear from the context. These words and expressions do not necessarily signify direct connections but include connections through mediate components. It should be noted that a connection between two components does not necessarily mean a direct, unimpeded connection, as a variety of other components may reside between the two components of note. Consequently, a connection does not necessarily mean a direct, unimpeded connection unless otherwise noted. EXPERIMENTAL SECTION [0190] The following serves to illustrate the present disclosure. It is not intended to limit the scope of the claimed invention in any way. [00191] Example 1 Synthesis of FAP9 ligand with free amine (14) 1-(tert-butyl) 2-methyl (S)-4,4-difluoro-2-methylpyrrolidine-1,2-dicarboxylate (2): To a stirred solution of acid (500 mg, 1.908 mmol) in dry acetone (5 ml) were added K2CO3 (1.5 eq) and methyl iodide (5 eq) and stirring at room temperature continued overnight. The reaction mixture was filtered, filtrate was evaporated under reduced pressure, and crude compound was purified by combi flash using ethyl acetate and hexane as mobile phase to obtain the desired compound (2) as a white solid. Synthesis of tert-butyl (S)-4,4-difluoro-2-formyl-2-methylpyrrolidine-1-carboxylate (3): To a stirred solution of 1-(tert-butyl) 2-methyl (S)-4,4-difluoro-2-methylpyrrolidine-1,2- dicarboxylate (1g, 3.77 mmol) in THF (10 mL) at 0 oC was added LiBH4 (1.1 eq, 4M in THF) slowly dropwise. The reaction mixture was transferred to room temperature and stirring continued 70 PRF_70572-03 PCT for 1 hour. The reaction mixture was evaporated under vacuum, and the obtained residue was redissolved in CH2Cl2 (100 mL) and extracted with saturated aqueous sodium bicarbonate solution (50 mL). The organic layer was separated and evaporated under vacuum, and crude residue was purified by column chromatography using ethyl acetate hexanes as mobile offered to obtain the desired compound (2) (850 mg, 95% as white gummy liquid). Step ii Dess-Martin periodinane (4.92 g, 11.60 mmol) was added portion-wise to a solution of tert- butyl 4,4-difluoro-2-(hydroxymethyl) pyrrolidine-l-carboxylate (2) (2.5 g, 10.55 mmol) in DCM (30 mL) at 0 °C. After complete addition, the reaction was warmed to room temperature and stirred for 2 hours. Saturated NaHCO3 was added, and the layers were separated using a phase separator. The DCM was removed in vacuo to give a clear oil, which was purified by combi flash using ethyl acetate and hexanes as mobile phase to provide the desired aldehyde (3) (2.25g, 90%) as white solid. (Note: During the purification process of the aldehyde, it was noted that the compound remained undetected by ELSD. Consequently, all peaks were gathered, leading to the desired compound solidifying on the walls of the test tubes.) Synthesis of 4-(isocyanomethyl)-1,2-dimethoxybenzene (5): To a stirred solution of compound 4 (0.5g, 2.56mmol) in dry DCM (1.5 mL) at 0 oC was added Et3N (5.0 eq, 12.82 mmol, 1.68 mL), followed by POCl3 (1.5 eq, 3.84 mmol, 0.24 mL). The reaction was allowed to continue at the same temperature for 1 hour, and progress of the reaction was monitored by TLC. After completion of starting materials as indicated on TLC plates, the reaction mixture was diluted with DCM, absorbed on a silica-gel cartridge, and purified using ethylacetate and hexanes as mobile phase to provide the desired compound (5) (750mg, 81%) as a light yellow liquid, which was slowly converted to light yellow solid upon storage. Synthesis of compound (9): A mixture of tert-butyl (S)-4,4-difluoro-2-formyl-2-methylpyrrolidine-1-carboxylate (4) (1 eq. 200 mg, 0.851 mmol), N-protected glycine (1 eq, 161mg, 0.851mmol) and isocyanide (1 eq, 162 mg, 0.851 mmol.) was dissolved in anhydrous DCM and stirred for 4 hours. After complete conversion (LC-MS) of starting materials, trifluoroacetic acid (2.0 mL) was added, and the mixture was stirred for 1 hour. The volatiles were evaporated under reduced pressure. The oily residue was redissolved in anhydrous DCM and cooled down to 0° C with an ice bath. Triethylamine (2.0 mL) was added dropwise and stirring continued until full conversion (LC-MS), which usually 71 PRF_70572-03 PCT occurred in less than 2 hours. The liquids were evaporated under reduced pressure, and the mixture was redissolved in DCM and washed three times with water. Organic phase was washed with brine and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and the crude residue was purified by using combiflash with hexanes + ethyl acetate as mobile phase to provide the desired α-hydroxyamide (8). The product was then used in the next step by dissolving in MeOH +AcOH (1:1) and adding 10%Pd-C (100 mg for 1g of starting material), then stirring under hydrogen atmosphere for 6 hours. The reaction mixture was filtered thorough celite pad, and the filtrate was evaporated under reduced pressure. The crude residue was azeotrope with EtOH and then purified by combiflash using MeOH +DCM to give the amine 9 as a brown-colored solid. Synthesis of compound (11): To a solution of compound 10 (500 mg, 2.64mmol) in DMF (10. mL) were added Cs2CO3 (2.65 gm, 7.93 mmol) and then tertiary butyl bromo acetate (7.93 mmol). The reaction mixture was stirred at 55 oC for 4 hours. Then KOH (7.93 mmol) and H2O (5.0 mL) were added to the same reaction mixture and stirring continued for an additional 2 hours. Progress of the reaction was monitored by LC-MS, the reaction mixture was carefully neutralized with 1N HCl, and the residue was purified by reversephase combi flash using ammonium acetate buffer (20 mM; pH = 7.0) and acetonitrile as mobile phase to obtain the desired acid 10 as a white solid (650 mg; 81%). Synthesis of compound (12a): Step-1 To a stirred solution of compound 12 (200 mg, 0.66 mmol) in anhydrous CH2Cl2 (10.0 mL) was added PyBOP (411 mg, 0.792 mmol) and DIPEA (0.22 mL, 1.32 mmol), and stirring continued for 10 minutes. Amine 9 (0.66 mmol) was added to the above reaction mixture and stirring continued for an additional 2 hours. The reaction mixture was diluted with water and then extracted into DCM (2x20 mL). The combined organic extracts were dried over anhydrous sodium sulphate and filtered. The filtrate was evaporated under reduced pressure to obtain a crude residue, which was purified by combiflash using MeOH+DCM as mobile phase to provide the compound 12a as a white solid. Step-2 72 PRF_70572-03 PCT To a stirred solution of compound 12a (500 mg, 0.728 mmol) in DCM (10 mL) were added Dess–Martin periodinane (DMP) (3.0 eq, 2.186 mmol) and H2O (10. Eq). Stirring continued at room temperature for six hours, and the progress of the reaction was monitored by LCMS. The reaction mixture was further diluted with saturated sodium bicarbonate solution, followed by extraction into dichloromethane (DCM). The combined organic extracts were evaporated under reduced pressure, and the crude residue was purified by combiflash using methanol + dichloromethane to provide the desired keto compound (12b) as a white solid. Synthesis of compound (14): To a solution of compound 12b (200.0 mg, 0.297mmol) in DCM (5 mL) was added TFA (2.0 mL). The mixture was stirred at room temperature for two hours. The reaction mixture was evaporated under reduced pressure and dried under vacuum. The crude residue was redissolved in DCM (10.0 mL), followed by the addition of PyBOP (1.2 eq) + DIPEA (5.0eq). After 10 minutes of stirring, BocNH(PEG)3NH2 (1.1 eq) was added, and stirring continued for an additional two hours. Work- up and purification followed the same procedure as described in the synthesis of compound 12a to provide compound 13 as a white solid. Then compound 13 was redissolved in DCM, treated with TFA, and evaporated under reduced pressure to give the targeted amine compound 14 as a gummy solid, which was utilized for further steps without purification. [0192] Example 2 Induced fit docking (IFD) The standard induced fit docking (IFD) protocol in the Schrodinger software package was used to dock the ligands of interest into the binding pocket of FAP. Firstly, a receptor grid box was generated by specifying the amino acid residues in FAP reported being involved in binding interactions. The IFD protocol utilizes the Glide docking protocol to generate up to 20 poses for each ligand which are further refined using the Prime Refinement module. The residues within 5Å of ligand poses were refined, and the side chains of the residues were optimized. Upon refinement of the binding site after initial docking, the ligands were re-docked into structures that are within 30.0 kcal/mol of the best structure and within the top 20 structures overall. The standard precision (SP) scoring function was used in the Glide redocking step to get the final docking scores. 73 PRF_70572-03 PCT As seen in Figs. 1-3, introduction of small alkyl groups, such as methyl, ethyl, propyl or butyl groups, and replacement of hydrogen with fluorine in FAP8 further increased binding interaction docking scores of the resultant analogues, referred to herein as FAP9 analogues. [0193] Example 3 Synthesis of FAP9 with cysteine linker (Fig.4) 1-(tert-butyl) 2-methyl (S)-4,4-difluoro-2-methylpyrrolidine-1,2-dicarboxylate (2): To a stirred solution of acid (500 mg, 1.908 mmol) in dry acetone (5 ml) was added K2CO3 (1.5 eq) and methyl iodide (5 eq) and continued the stirring at room temperature for overnight, reaction mixture filtered, and filtrate was evaporated under reduced pressure, and obtained crude compound was purified by using combi flash using ethyl acetate and hexane as mobile provided the desired compound 2 as white solid. Synthesis of tert-butyl (S)-4,4-difluoro-2-formyl-2-methylpyrrolidine-1-carboxylate (3): To a stirred solution of 1-(tert-butyl) 2-methyl (S)-4,4-difluoro-2-methylpyrrolidine-1,2- dicarboxylate (1g, 3.77 mmol) in THF (10 mL) at 0 oC was added LiBH4 (1.1 eq, 4M in THF) slowly dropwise. The reaction mixture was further transfer to room temperature and continued stirring there 1h-2h, progress of the reaction was monitored by TLC, reaction mixture was evaporated under vacuum and obtained residue was redissolved in CH2Cl2 (100 mL) followed extracted by saturated aqueous sodium bicarbonate solution (50 mL), organic layer was separated and evaporated under vacuum, then obtained crude residue was purified by column chromatography using ethyl acetate hexanes as mobile offered desired compound (3) 850 mg, 95% as white gummy liquid. Step-ii: Dess-Martin periodinane (4.92 g, 11.60 mmol) was added portion wise to a solution of tert- butyl (S)-4,4-difluoro-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (3) (2.5 g, 10.55 mmol) in DCM (30 mL) at 0 °C. After complete addition the reaction was warmed to rt and stirred for 2 h. Saturated NaHCO3 was added, and the layers separated using a phase separator. The DCM was removed in vacuo to give a clear oil which was purified by combi flash using ethyl acetate and hexanes as mobile phase provided the desired aldehyde 4 (2.25g, 90%) as white solid. (Note: during purification of aldehyde, it has been observed that, compound was not detected by ELSD 74 PRF_70572-03 PCT so, make sure to collect all the peaks when purifying it, after purification we could see the formation of white solid on test tubes in 30 minutes. Synthesis of 4-(isocyanomethyl)-1,2-dimethoxybenzene (6): To a stirred solution of compound 5 (0.5g, 2.56mmol) in dry DCM (1.5 mL) at 0 oC was added Et3N (5.0 eq, 12.82 mmol, 1.68 mL), followed by POCl3 (1.5 eq, 3.84 mmol, 0.24 mL) and continued the reaction mixture at same temperature for 1h, progress of the reaction was monitored by TLC, after completion of starting materials as indicated on TLC plates, reaction mixture was further diluted with DCM then absorbed on silica-gel cartridge and purified using ethylacetae+hexanes as mobile phase provided the desired compound 6 (750mg, 81%) as light yellow liquid and it was slowly converted to light yellow solid upon storage at -20 oC. Synthesis of compound (8): To a mixture of N-Boc-L-prolinal (1 eq.200 mg, 0.851 mmol), N-protected glycine (1 eq, 161mg, 0.851mmol) and isocyanide (1 eq, 162 mg, 0.851 mmol.) were dissolved in anhydrous CH2Cl2 and stirred for 4 hours. After complete conversion (LC-MS) of starting materials, trifluoroacetic acid (2.0 mL) was added and the mixture was stirred for 1 hour. The volatiles were evaporated under reduced pressure. The oily residue was redissolved in anhydrous CH2Cl2 and cooled down to 0° C with an ice bath. Triethylamine (2.0 mL) was added dropwise, and the stirring continued till the full conversion (LC-MS), usually less than 2 hours. The liquids were evaporated under reduced pressure, the mixture was redissolved in CH2Cl2 and washed 3 times with water. Organic phase was washed with brine, dried over sodium sulfate and the solvent was evaporated under reduced pressure and obtained crude residue was purified by using combiflash with hexanes + ethyl acetate as mobile phase provided desired α-hydroxyamide (8). The product was then used in the next step by dissolving in MeOH +AcOH (1:1) and added 10%Pd-C (100 mg for 1g of starting material), then stirred under hydrogen atmosphere for 6h, the reaction mixture was filtered thorough celite pad and filtrate was evaporated under reduced pressure and obtained crude residue was azeotrope with EtOH then purified by combiflash using MeOH +CH2Cl2 gave the amine 9 as orange color solid. Synthesis of compound (11): To a solution of compound 10 (500 mg, 2.64mmol) in DMF (10. mL) was added Cs2CO3 (2.65 gm, 7.93 mmol) then tertiary-butyl bromo acetate (7.93 mmol), the stirred the reaction mixture at 55 oC, for 4h, then (KOH, 7.93 mmol) +H2O (5.0 mL) were added to the same reaction 75 PRF_70572-03 PCT mixture and continued stirring there for additional 2h, progress of the reaction was monitored by LC-MS, reaction mixture carefully neutralized with 1N HCl, the purified by using reversphase combi flash using (20 mM, pH= 7.0 ammonium acetate buffer) and acetonitrile as mobile phase gave desired acid 11 as white solid (650 mg 81%) upon lypolization. Synthesis of compound (12): To a stirred solution of compound 11 (200 mg, 0.66 mmol) in anhydrous CH2Cl2 (10.0 mL) were added PyBOP (411 mg, 0.792 mmol) and DIPEA (0.22 mL, 1.32 mmol), then continued stirring there for 10 minutes, amine 9 (0.66 mmol) was added to the above reaction mixture and continued stirring there for additional 2h. reaction mixture was diluted with water, then extracted into CH2Cl2 (2x20 mL), the combined organic extracts were dried over anhydrous sodium sulphate, filtered and filtrate was evaporated under reduced pressure, then obtained crude residue was purified by combiflash using MeOH+CH2Cl2 as mobile phase provided the compound 12 as white solid. Synthesis of compound (13): To a stirred solution of compound 12 (1.0 eq) dissolved in CH2Cl2 followed by DMP (3.0 eq) the water (5.0. eq) was added and stirred at room temperature overnight. Rection mixture was further diluted with saturated with sodium bicarbonate solution and extracted into CH2Cl2 (2x30 mL), the combined organic extracts were dried over anhydrous sodium sulphate, filtered and filtrate was evaporated under reduced pressure, then the obtained crude residue was purified by combiflash using MeOH +CH2Cl2 as mobile phase provided the desired keto compound 13 as white solid. Synthesis of compound (14): To a stirred solution of compound 13 (200 mg, 0.237 mmol) in CH2Cl2 (5.0 mL) was added TFA (2 ml) and stirred at for 2h, reaction mixture was evaporated under reduced pressure and obtained crude reduce was solidify with diethyl ether, filtered, and obtained solid was dried and used in further steps without purification or purification by using combiflash with methanol in dichloromethane as mobile phase provide FAP9 base ligand (14) as white solid. Synthesis of compound (16): To a stirred solution of compound 15 (1.0eq) in CH2Cl2 (10 mL for 1g of starting material) was added tert-butyl (Z)-N,N'-diisopropylcarbamimidate (5.0 eq) and continued the stirring at rt for 18h, progress of the reaction was monitored by LCMS. Reaction mixture was further diluted 76 PRF_70572-03 PCT with water and extracted into CH2Cl2 and evaporated under reduced pressure. The obtained crude residue was purified using combi-flash to provide the compound 16 as gummy liquid. [0194] Example 4 (Fig.6) Synthesis of compound (18 and 29) To a solution of compound 14 (200.0 mg, 0.297mmol) in CH2Cl2 (5 mL) in different reaction vials were added PyBOP (1.2 eq) + DIPEA (5.0eq) followed by respective amines (compound 16 (1.1 eq)), then continued the stirring at room temperature for an additional 2 hours. Reaction mixture was further diluted with water and extracted into CH2Cl2 (2x30 mL), and combined organic extracts were evaporated under reduced pressure. Obtained crude compounds were purified by combi-flash using methanol+CH2Cl2 system to provide the respective compound 17. Then the Compound 17 (50 mg, 0.049 mmol) was dissolved in CH2Cl2 (1.0 mL), TFA (1.0 mL) was added, and the mixture was allowed to stir for 6 hours, followed by the addition of TIPS (100 μL) and further stirring for one hour. Upon complete deprotection of the starting material as confirmed by LC-MS, the reaction mixture was concentrated under vacuum, diluted with minimal amount of dichloromethane, absorbed on celite, and subjected to reverse phase column chromatography using acetonitrile in 20 mM ammonium acetate (pH 5.0) as mobile phase. Lyophilization of the acquired fractions afforded compound 18 as white solid. Synthesis of compound (21) Compound 19 (1.0 mL, 11.054 mmol) was dissolved in CH3CN (8.0 mL) and cooled to 0°C, followed by dropwise addition of compound 20 dissolved in CH3CN (5.0 mL), and allowed to stir for 30 minutes at 0°C. 2-mercaptopyridine (0.9 eq) dissolved in CH3CN (20.0 mL) was added dropwise to the above reaction mixture and refluxed for two hours. The appearance of white precipitates in the reaction mixture confirmed the formation of the product. Following reflux, the reaction mixture was cooled to 0°C, stirred for 1h, and filtered, to afford compound 21 as white solid. Synthesis of compound (22) Compound 21 (500 mg, 2.66 mmol) was dissolved in CH2Cl2 (5.0 mL) along with TEA (1.0 eq) and added dropwise to a solution of triphosgene (0.33 eq) in CH2Cl2 (5.0 mL) cooled at 0°C. The reaction mixture was stirred for 1.5h, followed by dropwise addition of a solution of 77 PRF_70572-03 PCT hydroxybenzotriazole (1.0 eq) in CH2Cl2 (10.0 mL) and TEA (1.0 eq), and further allowed to stir at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane (50.0 mL), washed twice with distilled water (100 mL X 2) and brine (100 mL), the combined organic layer dried over anhydrous sodium sulfate and concentrated under vacuum. The crude extract was purified through column chromatography using ethyl acetate in hexane as mobile phase to obtain compound 22 as white solid. Synthesis of compounds (24, 26 and 28) To a stirred solution compound 22 in three different reaction vials (50 mg, 0.095 mmol) in DMF (1.0 mL) were added respective PI3K inhibitors (23, 25 and 27) (1.0 eq) and DIPEA (1.0 eq), stirred at room temperature for two hours. Completion of the reaction was determined by LC- MS. The reaction mixture was added with distilled water and dichloromethane, organic layer was collected and dried over anhydrous sodium sulfate, concentrated under vacuum, absorbed in celite and subjected to reverse phase chromatography using acetonitrile in 20 mM ammonium acetate (pH 7.0) as mobile phase. Lyophilization of the acquired fractions afforded activated PI3K inhibitors (24, 26 and 28) as white solids. [0195] Example 5 (Fig.6) Synthesis of FAP9-PI3K conjugate (29-31): To a stirred solution of compound 18 (50 mg, 0.144 mmol) in DMF (1.0 mL) in three different reaction vials were added activated PI3K inhibitors (24, 26 and 28) (1.0 eq) and DMAP (1.0 eq) and stirred at rt for 2h under argon atmosphere. Upon completion of the reactions as determined by LC-MS, the reaction mixtures were further diluted with water, followed by extracted into dichloromethane (2x20 mL), then the combined organic extracts dried over anhydrous sodium sulfate, concentrated under vacuum, absorbed in celite, and subjected to reverse phase chromatography using acetonitrile in 20 mM ammonium acetate (pH 7.0) as mobile phase. Lyophilization of the acquired fractions provided the desired final conjugates (29-31) as white solids. [0196] Example 6 (Fig.7) Synthesis of compound (34) 78 PRF_70572-03 PCT For the synthesis and purification of activated TGF-β compounds 36 and FAP9- TGF-β conjugates (33 and 34) followed the same experimental conditions as described in the scheme-1- 3 using appropriate starting materials. [0197] Example 7 (Fig.8) Synthesis of FAP9-TGFβ conjugate (37-38): To a stirred solution of compound 18 (50 mg, 0.144 mmol) or 34 (50 mg, 0.054 mmol) in DMF (1.0 mL) in two different reaction vials were added activated TGFβ inhibitors (36) (1.0 eq) and DMAP (1.0 eq) and stirred at room temperature for two hours under argon atmosphere. Upon completion of the reactions as determined by LC-MS, the reaction mixtures were further diluted with water, followed by extracted into dichloromethane (2x20 mL), then the combined organic extracts dried over anhydrous sodium sulfate, concentrated under vacuum, absorbed in celite, and subjected to reverse phase chromatography using acetonitrile in 20 mM ammonium acetate (pH 7.0) as mobile phase. Lyophilization of the acquired fractions provided the desired final conjugates (37 and 38) as white solids. [0198] Example 8 (Fig.9) To a stirred solution of compound 23 (1.0 eq) in DMF (1 mL/100mg) was added NaH ( 2.0 eq), followed by tert-butyl (2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)carbamate (1.0 eq),then stirred the reaction mixture at room temperature for 2-3 hours, progress of the reaction was monitored by LC-MS. Reaction mixture was diluted with water and extracted into ethyl acetate, then evaporate and obtained crude residue was purified by HPLC provided desired compound 39. Then compound 39 was dissolved in CH2Cl2, TFA was added, and the mixture was stirred at room temperature. The reaction mixture was evaporated under reduced pressure, and the obtained crude compound was reacted with acid (4) using PyBOP/DIPEA in CH2Cl2 to provide the compound 40 as white solid. [0199] Example 9 (Fig.10) To a stirred solution of acid (14) in dichloromethane was added PyBOP/DIPEA followed by Val-Cit linkers (44), provided the couple product 45 and which was further reacted with 4- nitrophenyl carbonochloridate gave the activated compound 46. Then the reaction of compound 79 PRF_70572-03 PCT 46 with different PI3K inhibitors in DMF/DIPEA condition provided their corresponding FAP9- PI3K conjugates (47-49) with cathepsin cleavable linkers. [0200] Example 10 (Fig.11) Similarly, the FAP9-TGFβ conjugates (50-53) were synthesized by reaction of compound 46 with different TGFβ inhibitors. [0201] Example 11 (Figs.15-17) Evaluation of biological activity Bleomycin-induced lung fibrosis model (prophetic example) Eight- to 10-week-old C57BL/6-NCrl (strain code: 027) male mice (Charles River) are anesthetized (mixture of xylazine/ketamine) and then injected intratracheally with freshly prepared bleomycin sulfate (0.75 U/kg) (Cayman Chemicals, catalog no.13877) in sterile PBS (volume is varied between 88 and 108 ml depending on the body weight). Control mice are injected with 50 μl of sterile PBS. Body weights are monitored throughout each study. To quantitate FAP expression and fibrosis during longitudinal studies, lungs are harvested at 7, 14, and 21 days after bleomycin instillation and assayed as described below. For therapy studies, induction of IPF is initiated as described above, and drug (2 μmol/kg) is intravenously injected every other day beginning on day 10. Lungs are harvested on day 21 and assayed as described below (day 0 was taken as the day of bleomycin administration). Western blot analysis of lung tissue Frozen lungs are lysed in 1 ml of lysis buffer containing a protease inhibitor cocktail using an ULTRA-THURRAX. Lysates are cleared by centrifugation before total protein determination using the BCA protein assay. SDS–polyacrylamide gel electrophoresis and Western blotting are performed following standard procedures. Membranes are blocked and then probed with antibodies directed against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pAkt, and total Akt (for FAP-TGFb inhibitor conjugates, this step is omitted). The membranes are then washed in tris-buffered saline/Tween 20 followed by incubation with horseradish peroxidase (HRP)– conjugated secondary antibodies. Immunoreactive bands are detected by addition of an enhanced chemiluminescence substrate. Hydroxyproline assay 80 PRF_70572-03 PCT Total lung collagen is determined by the analysis of hydroxyproline. The right lung is consistently set aside for this assay. Briefly, harvested right lung is homogenized in PBS (pH 7.4), and digested with 12 N HCl at 120°C for three hours. Citrate/acetate buffer (pH 6.0) and chloramine-T solution are added at room temperature for 20 min, and the samples are incubated with Ehrlich’s solution for 15 min at 65°C. Samples are cooled to room temperature and read at 550 nm. Hydroxyproline standards (Sigma-Aldrich) at concentrations between 0 and 400 μg/ml are used to construct a standard curve. Histopathological evaluation of pulmonary fibrosis The left lung is inflated and fixed with 10% formalin solution (neutral buffered). Lung tissues are embedded in paraffin, and 10-μm sections are prepared and stained using hematoxylin and eosin and trichrome stain. The severity of bleomycin-induced fibrosis is determined by semiquantitative histopathological scoring at the indicated dates after bleomycin administration. Animal model Eight weeks old male C57BL/6J mice were purchased from The Jackson Laboratory (Strain #:000664). The mice were housed Mice were housed under a 12-h light/dark cycle with free access to standard mouse chow and tap water and allowed one week of acclimatory period. At nine weeks of age, twelve mice were randomly divided into four groups. Mice in negative control group (healthy mice) were injected intraperitoneally with 500 μL of 0.3 M sodium bicarbonate solution. All mice in positive control group (fibrotic mice) and two treatment groups were injected intraperitoneally with folic acid at the dose 250 mg/kg dose and at a concentration of 12.5 mg/mL once on day 0. High concentration of folic acid such as the 250 mg/kg used here, is known to induce renal fibrosis within four weeks of administration (Yan 2021). From day 3 of folic acid administration, mice in treatment group one started receiving FAP9-PI3Ki at a dose of 10 nmol/mice (calculated as 400 nmol/kg body weight) administered intravenously every other day. For treatment group 2, the same treatment of FAP9-PI3Ki started from day 14 of folic acid administration, administered every other day. Both treatments continued until day 28 of folic acid administration. At the end of the four-week study period, mice in all four groups were sacrificed and their kidneys were harvested and assayed as described below. Gene expression analysis using Real-Time qPCR Pre-weighed kidney samples (<50 mg) were homogenized, and total RNA was collected from the homogenized samples using Quick-RNA™ MiniPrep Kit (Zymo Research, Irvine, CA) 81 PRF_70572-03 PCT following the manufacturer’s protocol. The initial concentrations of the RNA samples were measured using SpectraMax QuickDrop UV-Vis spectrophotometer (Molecular Devices, San Jose, CA) and adjusted accordingly to produce uniform sample concentrations. All the RNA samples were amplified, and the transcript levels of targeted genes were quantified using the iTaq™ Universal SYBR® Green One-Step Kit (Bio-Rad Laboratories, Hercules, CA) following the manufacturer’s protocol, in the CFX Connect Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA). The relative expression levels of collagen type I (col1a1; forward primer 5’- TCCGGCTCCTGCTCCTCTTA-3’ (SEQ ID NO. 1); reverse primer 5’- GTATGCAGCTGACTTCAGGGATGT-3’ (SEQ ID NO. 2)), collagen type 3 (col3a1; forward primer 5’- AATGGTGGCTTTCAGTTCAGCT-3’ (SEQ ID NO. 3); reverse primer 5’- TGTAATGTTCTGGGAGGCCC -3’ (SEQ ID NO.4)) and α-smooth muscle actin (acta2; forward primer 5’- ACAGCCCTCGCACCCA-3’(SEQ ID NO. 5); reverse primer 5’- GCCACCGATCCAGACAGAGT -3’ (SEQ ID NO.6)) were compared against the housekeeping gene GAPDH (gapdh; forward primer 5’- ACAGCCCTCGCACCCA-3’(SEQ ID NO.7); reverse primer 5’- GCCACCGATCCAGACAGAGT -3’ (SEQ ID NO. 8)), and calculated using the 2−ΔΔCt method (Yang et al., 2023). The relative gene expression levels between different animal groups were analyzed and their statistical significance were calculated using one-way ANOVA in GraphPad Prism 10. Hydroxyproline assay Pre-weighed kidney samples were digested individually in 6M hydrochloric acid at 100°C for 24 hours. Following the digestion, pH of the samples was adjusted between 5.0 to 7.0 using 6M sodium hydroxide. Stock hydroxyproline solution of 100 μg/mL was prepared and used to produce different concentrations of hydroxyproline standards: 50, 40, 30, 20, 15, 10, 5, and 1 μg/ml.250 μL of all standards, sample or blank (distilled water) was individually added with 125 μL of freshly prepared 0.06 M chloramine T solution and vortexed and incubated for 20 minutes at room temperature. Afterwards, 125 μL of 3.15M perchloric acid was added to each mixture, vortexed, and incubated for 5 minutes at room temperature. Lastly, 125 μL of freshly prepared p- dimethylaminobenzaldehyde solution in methoxyethanol (0.2 g/mL) was added to each mixture, vortexed, and incubated at 60°C for 20 minutes.200 μL from each mixture was loaded on a 96- well plate and their respective absorbance values were measured at 557 nm in the BioTek Synergy Neo2 Reader (Agilent Technologies, Santa Clara, CA) (Błyszczuk et al., 2019). A standard curve 82 PRF_70572-03 PCT was prepared and respective levels of hydroxyproline were calculated for each sample and adjusted with dilution factors to obtain the total hydroxyproline content of individual samples. The hydroxyproline content measured for each mouse was divided by their respective kidney weight and expressed as μg of total hydroxyproline per g of wet kidney mass. The variation in the hydroxyproline levels between different animal groups was analyzed in GraphPad Prism 10 and their statistical relevance were measured through one-way ANOVA. Results Relative gene expressions of fibrotic markers Compared to the negative control mice with a relative gene expression level of single unit, the positive control mice expressed significantly (p<0.0001) higher levels of both col1a1 and col3a1 (Figure 1), thereby indicating an increased collagen production by the folic acid-induced fibrotic mice. However, with the treatment of FAP9-PI3Ki conjugate from day 3 of folic acid administration, there was significant suppression (p<0.0001) of the col1a1 and col3a1 expression, compared to those of non-treated fibrotic mice. Moreover, the treatment of fibrotic mice with FAP9-PI3Ki conjugate from day 14 of the folic acid administration also suppressed the col1a1 and col3a1 expression, although to a lesser extent. A similar trend of suppression of genetic expression was also observed acta2, between the healthy mice, the fibrotic mice and mice treated with FAP9- PI3Ki at different time points, representing an overexpression of α-smooth muscle actin in fibrotic mice and subsequent suppression of the overexpression in treated mice, respectively. This is indicative of the fact that treatment with FAP9-PI3Ki could significantly diminish myofibroblast (activated fibroblasts) population in fibrotic mice, thereby reducing the profibrotic mechanisms at tissue level. Change in hydroxyproline content A significant increase of hydroxyproline level, a major component of the collagen helical structure, was observed in fibrotic mice compared to that of healthy mice (p<0.0001). This, in turn, implies an extensive secretion and accumulation of collagen in the extracellular matrix of kidney samples from fibrotic mice. Evidently, treatment of the fibrotic mice with FAP9-PI3Ki from day 3 of folic acid administration exhibited significant (p<0.0001) reduction in the hydroxyproline content compared to that of the fibrotic mice (Figure 2). A decrease in hydroxyproline level was also observed in the fibrotic mice treated from day 14 of folic acid administration, but to a lesser 83 PRF_70572-03 PCT extent. Yet, the difference was statistically significant (p<0.001) to imply that a later administration of the drug could still prevent the buildup of collagen in folic acid administered fibrotic mice. [0202] Example 12 (Fig.18) Western blot analysis of lung tissue using FAP9-TGFβ conjugate (37) PHLF (primary human lung fibroblasts) were seeded in a plate and stimulated by 10 ng/ml TGFβ1 overnight. Cells in two wells were then incubated with 100nM of FAP9-s-s-TGFBR1i or free TGFBR1i for 2 hours. The drug-containing medium was then replaced with drug-free medium containing 10ng/mL of TGFβ1 and incubated 24 hours. Another set of two wells were incubated with 100nM of FAP9-s-s-TGFBR1i or free TGFBR1i without washing steps for 24 hours. Cells were collected for analysis of p-Smad2/Smad2, ^^-SMA and GAPDH expression via western blot. [0203] Example 13 Radiation-induced fibrosis (prophetic example) Establishment and treatment of the rat model for radiation-induced lung fibrosis. The SD rats or mice used in this experiment are about 6-month-old. Animals are anesthetized, and the surface of the left lung is marked by a marker. Radiation is be given with the applicator tube fixed at the mark on the left lung. The dose normalization point is 0.5 cm below the source center, and the irradiation area is a 0.5 cm radius around the mark point. Different groups of animals are given a single increasing dose of irradiation (in the range of 10-90 Gy), and the degree of fibrosis on the lung at the irradiated site is detected each week for four weeks after irradiation. After optimizing the dose for consistent fibrosis, the treatment is started with FAP-targeted antifibrotic agents after the fibrotic stage begins. Hydroxyproline is measured to know the extent of fibrosis. A similar approach is used to assess fibrosis of other organs. [0204] Example 14 Liver fibrosis (prophetic example) Male C57BL∕6 mice (aged 6–8 weeks) are divided into three groups (n=12). To induce liver fibrosis, the mice are injected with 5 µl/g of 20% CCl4 intraperitoneally, two times a week for 8 weeks. Beginning on the day of the CCl4 injection, the mice in the treatment group were treated with FAP-targeted antifibrotic agents. The control group are treated with saline. All the mice are 84 PRF_70572-03 PCT sacrificed at different time points following the final CCl4 injection at 8 weeks, using CO2 asphyxia. Lungs will be analyzed for signs of fibrosis and therapeutic benefit. [0205] Example 15 Skin fibrosis (prophetic example) Male C57BL∕6 mice (aged 6–8 weeks) are divided into three groups (n=12). To induce skin fibrosis, the mice are injected with bleomycin subcutaneous. Beginning on the day of the first bleomycin (1 mg/ml in saline) injection, the mice in the treatment group were treated with FAP- targeted antifibrotic agents. The control group are treated with saline. All the mice are sacrificed at different time points following the final bleomycin injection at 4 weeks, using CO2 asphyxia. Skin tissue will be analyzed for signs of fibrosis and therapeutic benefit. [0206] Example 16 Heart fibrosis (prophetic example) Tissue inhibitor of metalloproteinase-3 knockout (TIMP3−/−) (C57BL/6) mice will be used as a model for cardiac fibrosis. Angiotensin II (1.5 mg/kg/day) or saline (control) will be delivered to these mice. The treatment with FAP-targeted antifibrotic drugs or saline control will begin on the same day of administering angiotensin II (or saline). After four weeks, the hearts will be studied for signs of fibrosis and therapeutic benefit. [0207] Example 17 Bladder fibrosis (prophetic example) Six-week-old Balb/c mice will be injected with 100 mg/kg ketamine for 20 weeks intraperitonially. Saline injected mice will be used as control mice. The treatment with FAP- targeted antifibrotic drugs or saline control will begin at four weeks after administering the first dose of ketamine (or saline). After 20 weeks, the heart will be studied for signs of fibrosis and therapeutic benefit. [0208] Example 18 Muscle fibrosis (prophetic example) 85 PRF_70572-03 PCT Five-month-old dystrophic mdx(C57BL/10ScSnDmdmdx/J) mice will be used as a spontaneous model for muscle fibrosis. Treatment with FAP-targeted antifibrotic drugs or saline control will begin at different ages to assess the therapeutic benefit. The muscle tissue will be studied for signs of fibrosis and therapeutic benefit. [0209] Example 19 FAP9-TGFBR1i treatment study in FA-treated mice Methods Cell lines and cell culture. PHLF, 3T3 and HT1080 cells were purchased from ATCC. Cells were cultured in DMEM (Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% Penicillin/Streptomycin (Gibco). All cell lines were tested for mycoplasma contamination using the PCR Mycoplasma Detection Kit (Sigma). After thawing, cell lines were passaged three times before use in experiments and maintained for no more than 30 passages. All cells were incubated at 37 °C in a humidified incubator containing 5% CO2. Live cell imaging of FAP9-FITC internalization. To assess binding and internalization of FAP9- FITC, 3T3-mFAP, HT1080-hFAP cells were seeded on glass chamber slides (Thermo Scientific) in complete DMEM medium. Cells were incubated with 50 nM of FAP9-FITC at 37 °C for 1 hour, followed by three washes with PBS containing 2% FBS. Fluorescence imaging was captured and processed using a confocal microscopy (FV 1000, Olympus) with FV10-ASW Olympus software. PRF_70572-03 PCT Mice. Male C57BL/6J mice (weight 20–25 g) were purchased from the Jackson Laboratory and housed under pathogen-free conditions with a 12 h light/dark cycle. Freshwater and standard rodent chow were freely available. Mice were acclimated for one week prior to experimental procedures. All animal studies were conducted with approval from the Purdue Animal Care and Use Committee (PACUC), in compliance with NIH guidelines. Folic acid (FA)-induced renal fibrosis model. Eight-week-old male mice received a single intraperitoneal injection of folic acid (250mg/kg, Sigma-Aldrich, #7876) dissolved in 0.3M sodium bicarbonate. Control mice injected sodium bicarbonate alone (vehicle). Mice body weights were monitored daily throughout the study. In vivo fluorescence imaging. To visualize FAP-targeted compound distribution, mice were injected via tail vein with 5 nmol of FAP-targeted NIR dye conjugate (FAP9-S0456) and imaged at 24 hours post-injection using a Spectral AMI optical imaging system. For competition studies, on day11 post-FA injection, a 100-fold molar excess of FAP ligand was co-administered with FAP9-S0456. Imaging settings included: object height, 1.5; excitation wavelength, 745 nm; emission wavelength, 810 nm; field of view (FOV), 25; binning, 2; f-stop, 2; acquisition time, 5 s. Major organs were collected and imaged for biodistribution analysis. Treatment of mice with FAP9-TGFBR1i. Three days following FA injection, mice were randomized based on body weight and treated daily tail vein injection with either 10 nmol of FAP9-TGFBR1i (Compound 37) or PBS (100μl/dose). On day 28 post-treatment, mice were euthanized and kidneys were harvested for subsequent analysis. RNA isolation, RT-PCR and qPCR. Mice kidneys were dissected, minced and lysed in 1ml TRIzol reagent (Invitrogen). Total RNA was isolated following the manufacturer’s protocol. RNA concentration and purity was assessed using a Nanodrop spectrophotometer (Thermo Scientific), and integrity was confirmed by agarose gel electrophoresis.1µg RNA was used for cNDA synthesis using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), following the manufacturer’s protocol. RT-qPCR was performed using SYBR Green Master Mix (Thermo Fisher Scientific) on a CFX Connect Real-Time PCR Detection System (Bio-Rad). Technical replicates were included for all samples. Primer specificity was validated via melt curve analysis and agarose gel electrophoresis of the amplified product. No nonspecific 87 PRF_70572-03 PCT amplification products were observed. Relative gene expression was analyzed using the 2(-Delta Delta C(T)) method. Primer sequences are listed below. Hydroxyproline assay. Freshly isolated mouse kidneys were dissected, weighed and homogenized in 100 μl H2O per 10 mg tissue, following by the addition of an equal volume of 12N HCl. Samples were hydrolyzed 24 hours at 95°C. After centrifugation at 3,500g for 10 minutes, 300μl of supernatant was transferred to new tubes and neutralized with 300 μL of 6N NaOH. Samples were centrifuged again at 1,000g for 10 minutes.150uL of each sample and hydroxyproline standard were transferred to a 96-well plate. Subsequently, 100uL of chloramine- T solution (40 mM chloramine-T in 90% citrate/acetate buffer plus 10% isopropanol) was added to each well, and incubated at room temperature for 20 minutes. Then, 100 μL of Ehrlich’s solution (1.14M DMAB, 70% isopropanol and 30% perchloric acid) was added and incubated at 65°C for 20 minutes. Absorbance was measured at 550 nm using a SpectraMax384 Plus plate reader. A standard curve was generated using the OD values of the hydroxyproline standards. Western blot. Cells were seeded in 6-well plates or harvested mice kidney tissues were lysed in RIPA buffer (Thermo Scientific) containing protease inhibitor cocktail (sigma). Total protein concentration was determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher). Equal amount of protein (20 μg) were resolved by SDS-PAGE gel (6-10%) and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk and incubated with the following primary antibodies: anti-Collagen I (Invitrogen, PA1-26204, 1:1000), anti-GAPDH (Proteintech 60004-1-Ig, 1:50000), anti-Alpha-smooth muscle actin (cell signaling, #19245, 1:1000), anti-pSMAD2 (cell signaling, #3108, 1:1000) and anti-SMAD2 (cell signaling, #3103, 1:1000). Secondary antibodies used were IRDye 800CW goat anti-rabbit IgG or IRDye® 680RD goat anti-mouse IgG (LI-COR). Blots were visualized using the LI-COR Odyssey CLx imaging system and quantified with ImageStudio Lite software (LI-COR). Statistical analysis. Statistical analyses were performed with GraphPad Prism 6.0. Comparisons between two groups were analyzed using unpaired two-tailed t-test. Multiple group comparisons were performed using one-way or two-way analysis of variance (ANOVA). Statistical significance was defined as follows: *P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001. 88 PRF_70572-03 PCT Optical imaging of FA-induced renal fibrosis in mice with an FAP-targeted NIR dye (FAP9- S0456). FIG.19a presents representative optical image showing tissue biodistribution of FAP9- S045624 hours after intravenous administration in with FA-induced kidney fibrosis. Minimal to no retention of FAP9-S0456 was observed in tissues other than the fibrotic kidney (middle) and was absent in healthy mice (left). Uptake in fibrotic kidneys was blocked by excess FAPL (right), demonstrating the specificity of FAP9-S0456 for the fibrotic kidney. FIG.19b presents quantitative analysis of FAP9-S0456 retention in healthy and FA-treated mice. FIG.19c shows the time course of FAP9-S0456 retention in the kidneys of FA-treated mice. Data were analyzed using one-way ANOVA(**P < 0.01, ***P < 0.001, ****P < 0.0001). [0220] Evaluation of FAP9-TGFBR1i for the for treatment of FA-induced renal fibrosis in mice. FIG.20a presents a schematic representation of the mouse study for induction and treatment in the FA-induced kidney fibrosis model and changes in body weight of healthy mice, FAP9- TGFBR1i-treated or vehicle-treated mice following FA injection. FIG.20b presents qPCR quantification of gene expression (collagen, SMA and FAP) in moue kidneys. FIG.20c presents kidneys which were subjected to hydrolyzed and analyzed for hydroxyproline quantification as a measure of collagen content (healthy, n=4; other groups, n=5). Significance among groups was analyzed using one way ANOVA(*P < 0.05, **P < 0.01, ***P < 0.001). FIG.20d shows Western blot for kidney samples of healthy mice, FAP9-TGFBR1i-treated or vehicle-treated mice. FIG.20e shows change in collalgen protein, FAP protein and α-SMA protein with healthy, vehicle and FAP-TGFBR1i treatment in moue kidneys. Fig.21 shows FAP9-FITC internalization in 3T3-mFAP cells. [0221] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. [0222] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 89 PRF_70572-03 PCT 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise. [0223] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. [0224] Any use of section headings and subheadings is solely for ease of reference and is not intended to limit any disclosure made in one section to that section only; rather, any disclosure made under one section heading or subheading is intended to constitute a disclosure under each and every other section heading or subheading. [0225] Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims. [0226] The terms and expressions, which have been employed, are used as terms of description and not of limitation. In this regard, where certain terms are defined and are described or discussed elsewhere, the definitions and all descriptions and discussions are intended to be attributed to such terms. There also is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. [0227] Further, all publications and patents mentioned herein are incorporated by reference in their entireties for all purposes. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls. 90 PRF_70572-03 PCT Enumerated Embodiments (EE) [0228] The following list of enumerated embodiments presents claims with multiply dependent claims depending from multiply dependent claims for presentation in those jurisdictions where such dependencies are allowed as well as additional claims, which may be presented during the examination of the application or any divisional or continuation thereof. [0229] EE 1. A method of treating fibrosis in a subject, which method comprises administering to the subject an effective amount of a compound of formula I or II: , wherein L is a releasable or non-releasable bi-functionalized linker, which binds A and B, or L is a releasable or non-releasable tri-functionalized linker, which binds A, B, and C; B is an active agent effective for the treatment of fibrosis; C is a pharmacokinetic (PK) extender; represents a functionalized 5- to 10-membered, N-containing, aromatic or non-aro , or bi-cyclic heterocycle, which optionally comprises 1-3 heteroatoms independently selected from O, N, and S, and indicates the point of attachment of A to L or A attaches to L via any carbon atom of the functionalized 5- to 10-membered, N-containing, aromatic or non-aromatic, mono- or bicyclic heterocycle, an alkyl primary amine, an alkyl secondary amine, a functionalized alkyl amine, or a functionalized cycloalkyl amine; 91 PRF_70572-03 PCT R1 is selected from the group consisting of F, Cl, Br, I, OH, CF3, -NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, Cl-Cl0 alkyl, C3-Cl0 cycloalkyl, adamantyl, aryl, and C7- C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted. R2, R3, R4, R5, R6 and R7 are independently selected from the group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; R8 is selected from the group of substituents consisting of -H, -D, -OH, =CH2, -CH3, -CH2CH3, -C(H)(CH3)2, -C(CH3)3, and -CH2Ph, wherein any substituent comprising at least two atoms can be optionally substituted; R9, R10, and R11 are independently selected from group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -N3, -NH=NH-, -N-C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; R12 is selected from the group of substituents consisting of -H, -D, -F, -C1-C6 alkyl, -C(O)CH3, and -Cl-Cl0 alkyl, wherein any substituent comprising at least two atoms can be optionally substituted; and R13 is selected from the group of substituents consisting of -H, -D, Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, adamantyl, aryl, and C7-C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted and the aryl in C7-C20 alkyl aryl is: elected from the group consisting of -H, -D, -halo, and Cl-C4 alkyl, which is optionally substituted, and R14a, R15a, R16a, and R17a are independently selected from the group of substituents consisting of -H, -D, -halo, -Cl-C3 alkyl, -Cl-C3 alkoxy, -CF3, and -C(=O)OR12, wherein R12 is as defined above, the dashed line indicates the point of attachment to the nitrogen of A, and any substituent comprising at least two atoms can be optionally substituted; or R13 is: 92 PRF_70572-03 PCT pendently selected from the group of substituents consisting of -H, -D, -OMe, -C1-C3 alkyl, benzyl (Ph-CH2-), and substituted/functionalized benzyls, the dashed line indicates the point of attachment to the nitrogen of A, and any substituent comprising at least two atoms can be optionally substituted; or a stereoisomer, a pharmaceutically acceptable salt, or a hydrate thereof, whereupon the subject is treated for fibrosis. [0230] EE 2. The method of EE 1, wherein A has the structures of formulae III, formulae IV, formulae V, formulae VI, formulae VII, or formulae VIII: PRF_70572-03 PCT 94 PRF_70572-03 PCT PRF_70572-03 PCT R R6 R5 R4 R 7 8 R3 R2 III where R1 is selected from the group of substituents consisting of -F, -Cl, -Br, -I, -OH, -CF3, -NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, -adamantyl, -aryl and -C7-C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted; R2, R3, R4, R5, R6 and R7 are independently selected from the group of substituents consisting of -H, -D, - OH, -F, -Cl, -Br, -I, -C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; R8 is selected from the group of substituents consisting of -H, -D, -OH, =CH2, -CH3, -CH2CH3, -C(H)(CH3)2, -C(CH3)3, -CH2Ph, wherein any substituent comprising at least two atoms can be optionally substituted; R12 and R13 are independently selected from the group of substituents consisting of -H, -D, -F, -C1-C6 alkyl, -C(O)CH3, and -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, adamantyl, aryl, and C7-C20 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; and R23-R26 are independently selected from group of substituents consisting of -H, -OH, -F, -Cl, -Br, -I, -CF3, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH2, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, and the structure -X-L-B or -X-L(BC), wherein X is O, S, -NH, -NCH3, or -CH2, wherein any substituent comprising at least two atoms can be optionally independently substituted. 96 PRF_70572-03 PCT [0231] EE 3. The method of EE 1 or 2, wherein R13 is independently selected from the group of substituents consisting of , PRF_70572-03 PCT O O , wherein any substituent can be optionally substituted. [0232] EE 4. The method of any one of EEs 1-3, wherein B is a phosphatidylinositol-3-kinase (PI3k) inhibitor, a dual inhibitor of the PI3k/mTOR signaling pathway, a transforming growth factor β (TGFβ) inhibitor, a Rho-kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor), a focal adhesion kinase (FAK) inhibitor, vascular endothelial growth factor receptor (VEGFR) inhibitor, or a platelet-derived growth factor receptor (PDGFR) inhibitor. [0233] EE 5. The method of EE 4, wherein the dual inhibitor of the PI3k/mTOR signaling pathway is: 98 PRF_70572-03 PCT . [0234] EE 6. The method of EE 4, wherein the PI3k inhibitor has a structure: , wherein X has a structure selected from the group consisting of: 99 PRF_70572-03 PCT . [0235] EE 7. The method of EE 4, wherein the PI3k inhibitor has the structure: . [0236] EE 8. The method of EE 4, wherein the PI3K inhibitor has the structure: N A , , , , -NH-, -NHR-, Or -CO-, and y can be 1 to 20. [237] EE 9. The method of EE 4, wherein the TGFβ inhibitor has the structure: 100 PRF_70572-03 PCT where 1 ran 2 are eac , n epen ent y, , methyl, Cl, Br. O, I, or F; and X is NH, O, or S. [0238] EE 10. The method of EE 9, wherein R1 is F and R2 is methyl. [0239] EE 11. The method of EE 9, wherein the TGFβ inhibitor has the structure: . [0240] EE 12. The method of EE 11 where L is or comprises a PEG monomer of the formula -(PEG)n- where n is 1-6. [0241] EE 13. The method of EE 11, wherein the conjugate has the structure: . PRF_70572-03 PCT [0242] EE 14. The method of EE 11, wherein L is O O O HN O O O O Or HN HN O Or H O HN HN O O N O HN HN O HN S S N S S NH S Or Or HN O NH S N S Or NH [0243] EE 15. The method of EE 11, wherein the conjugate has the structure: . [0244] EE 16. The method of EE 4, wherein the TGFβ inhibitor has the structure: O N N N . [0245] EE 17. The method of EE 4, wherein the ROCK inhibitor has the structure: 102 PRF_70572-03 PCT , [0246] EE 18. The method of EE 4, wherein the FAK inhibitor is F3C N N O . [0247] EE 19. The method of EE 4, wherein the VEGFR inhibitor is N H2 . [0248] EE 20. The method of EE 4, wherein the PDGFR inhibitor is 103 PRF_70572-03 PCT . [0249] additionally comprises a PK extender selected from an albumin-binding ligand, a plasma protein-binding ligand, a hapten, or an internalization-inducing peptide. [0250] EE 22. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises one or more of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a polylactone, a polymethylmethacrylate, a polyoxymethylene, a heterocycle, or any combination of two or more thereof. [0251] EE 23. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an oligomer of one or more peptidoglycans, glycans, anions, heterocycles, or any combination of two or more thereof. [0252] EE 24. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises at least one diamino butyric acid group, a substituted benzene group, a lysine group, a 2,3- diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or any combination of two or more thereof. [0253] EE 25. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an ether, a thioether, a tertiary amine, a C1-6 alkyl, piperazine, piperidine, a bicycloheptane, a substituted benzene, or a combination of two or more thereof. [0254] EE 26. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 104 PRF_70572-03 PCT [0255] EE 27. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: w [0256] EE 28. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a , wherein n = 0-10. [0257] EE 29. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 105 PRF_70572-03 PCT O O H N OH [0258] EE 30. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: . [0259] EE 31. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: [0260] EE 32. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 106 PRF_70572-03 PCT [0261] EE 33. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: . [0262] EE 34. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: [0263] EE 35. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 107 PRF_70572-03 PCT . [0264] EE 36. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: . [0265] EE 37. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a reductively cleavable linker. [0266] EE 38. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an oxidatively cleavable linker. [0267] EE 39. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an oxime ester. [0268] EE 40. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a hydrazone. [0269] EE 41. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises an enzyme-cleavable linker. [0270] EE 42. The method of any one of EE 1-11 and 15-21, wherein L is or comprises a PEGn, and n = 0-36. 108 PRF_70572-03 PCT [0271] EE 43. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a peptide. [0272] EE 44. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a peptidoglycan. [0273] EE 45. The method of any one of EEs 1-11 and 15-21, wherein L is: . [0274] EE 46. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a linker selected from the group consisting of pegylated-, alkyl-, sugar-, and peptide-based dual linkers. [0275] EE 47. The method of any one of EEs 1-11 and 15-21, wherein L is a non-releasable linker. [0276] EE 48. The method of any one of EEs 1-11 and 15-21, wherein L is covalently bonded to A and B of formula (I) or A, B and C of formula (II). [0277] EE 49. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 109 PRF_70572-03 PCT S O O O S O O O O O O N CO2H [0278] EE 50. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises a moiety selected from: COOH COOH N S N S 28 l, which can be optionally substituted; and z is an integer from 1 to 8. [0279] EE 51. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises: . [0280] EE 52. The method of any one of EEs 1-11 and 15-21, wherein L is or comprises: , 29b, R30a, and R30b is independently H or C1- C6 alkyl, which can be optionally substituted (e.g., wherein each of R29a, R29b, R30a, and R30b is C1-alkyl (i.e., methyl)). 110 PRF_70572-03 PCT [0281] EE 53. The method of any one of EEs 1-11 and 16-52, wherein C is an albumin binding ligand, a disulfide-stabilized protein scaffold comprising albumin binding domain 035 (ABD035), albumin binding domain Con (i.e., a peptide of a three-helix bundle 45 amino acids in length (ABDCon)), a designed ankyrin repeat protein (DARPin), a disulfide-stabilized Fv fragment (dsFv) of an anti-albumin antibody, a nanobody that complexes with human serum albumin (HSA), or a variable new antigen receptor (VNAR). [0282] EE 54. The method of any one of EEs 1-11 and 16-52, wherein C is or comprises one of: . [0283] EE 55. The method of any one of EEs 1-11 and 16-52, wherein C is or comprises: 111 PRF_70572-03 PCT wherein, as applicable: each of R12-19 (where applicable) is independently selected from -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, and -SO2F; and each of R20 and R21 is independently selected from -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -O-C1-6 alkyl, -CN, -CHO, -B(OH)2, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, CF3, and . [0284] EE 56. The method of any one of EEs 1-11 and 16-52, wherein C is or comprises: 112 PRF_70572-03 PCT O2 I . [0286] EE 58. The method of any one of EEs 1-11 and 16-52, wherein C is or comprises: 113 PRF_70572-03 PCT . [0287] f any one of EEs 1-11 and 16-52, wherein C is or comprises (PEG)n, wherein n is an integer 0 to 32, a peptide, a peptidoglycan, or a saccharide. [0288] EE 60. The method of any one of EEs 1-11 and 16-52, wherein C is or comprises: . [0289] EE 61. The method of any one of EEs 1-11 and 16-52, wherein C is or comprises a hapten bound by an autologous antibody. [0290] EE 62. The method of any one of EEs 1-11 and 16-52, wherein C is or comprises a hapten selected from rhamnose, an α-galactosyl moiety, a dinitrophenyl (DNP) moiety, and a trinitrophenyl (TNP) moiety. [0291] EE 63. The method of EE 1, wherein A has the structure: 114 PRF_70572-03 PCT . [0292] EE 64. The method of EE 1, wherein A has the structure: . [0293] EE 65. The method of EE 1, wherein A has the structure: 115 PRF_70572-03 PCT . [0294] EE 66. The method of EE 1, wherein A has the structure: . [0295] EE 67. The method of EE 1, wherein A-L-B has the structure: 116 PRF_70572-03 PCT . Me [0296] EE 68. A conjugate having the structure: 117 PRF_70572-03 PCT om e Me M m PRF_70572-03 PCT e 119 PRF_70572-03 PCT or 120 PRF_70572-03 PCT . [0297] EE 69. A conjugate having the structure: [0298] EE 70. A pharmaceutical composition comprising a conjugate of EE 68 or EE 69 and a pharmaceutically acceptable carrier. 121

Claims

PRF_70572-03 PCT WHAT IS CLAIMED IS: 1. A method of treating fibrosis in a subject, which method comprises administering to the subject an effective amount of a compound of formula I or II: or wherein A has the structure: , wherein L is a releasable or non-releasable bi-functionalized linker, which binds A and B, or L is a releasable or non-releasable tri-functionalized linker, which binds A, B, and C; B is an active agent effective for the treatment of fibrosis; C is a pharmacokinetic (PK) extender; represents a functionalized 5- to 10-membered, N-containing, aromatic or non-ar or bi-cyclic heterocycle, which optionally comprises 1-3 heteroatoms independently selected from O, N, and S, and indicates the point of attachment of A to L or A attaches to L via any carbon atom of the f tionalized 5- to 10-membered, N-containing, aromatic or non-aromatic, mono- or bicyclic heterocycle, an alkyl primary amine, an alkyl secondary amine, a functionalized alkyl amine, or a functionalized cycloalkyl amine; R1 is selected from the group consisting of F, Cl, Br, I, OH, CF3, -NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, Cl-Cl0 alkyl, C3-Cl0 cycloalkyl, adamantyl, aryl, and C7- C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted. R2, R3, R4, R5, R6 and R7 are independently selected from the group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; 122 PRF_70572-03 PCT R8 is selected from the group of substituents consisting of -H, -D, -OH, =CH2, -CH3, -CH2CH3, -C(H)(CH3)2, -C(CH3)3, and -CH2Ph, wherein any substituent comprising at least two atoms can be optionally substituted; R9, R10, and R11 are independently selected from group of substituents consisting of -H, -D, -OH, -F, -Cl, -Br, -I, -NO2, -SO3H, -SO2NH2, -N3, -NH=NH-, -N-C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; R12 is selected from the group of substituents consisting of -H, -D, -F, -C1-C6 alkyl, -C(O)CH3, and -Cl-Cl0 alkyl, wherein any substituent comprising at least two atoms can be optionally substituted; and R13 is selected from the group of substituents consisting of -H, -D, Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, adamantyl, aryl, and C7-C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted and the aryl in C7-C20 alkyl aryl is: elected from the group consisting of -H, -D, -halo, and Cl-C4 alkyl, which is optionally substituted, and R14a, R15a, R16a, and R17a are independently selected from the group of substituents consisting of -H, -D, -halo, -Cl-C3 alkyl, -Cl-C3 alkoxy, -CF3, and -C(=O)OR12, wherein R12 is as defined above, the dashed line indicates the point of attachment to the nitrogen of A, and any substituent comprising at least two atoms can be optionally substituted; or R13 is: , , , , pendently selected from the group of substituents consisting of -H, -D, -OMe, -C1-C3 alkyl, benzyl (Ph-CH2-), and 123 PRF_70572-03 PCT substituted/functionalized benzyls, the dashed line indicates the point of attachment to the nitrogen of A, and any substituent comprising at least two atoms can be optionally substituted; or a stereoisomer, a pharmaceutically acceptable salt, or a hydrate thereof, whereupon the subject is treated for fibrosis. 2. The method of claim 1, wherein A has the structures of formulae III, formulae IV, formulae V, formulae VI, formulae VII, or formulae VIII: 124 PRF_70572-03 PCT 125 PRF_70572-03 PCT PRF_70572-03 PCT R R6 R5 R4 R 7 8 R3 R2 III where R1 is selected from the group of substituents consisting of -F, -Cl, -Br, -I, -OH, -CF3, -NO2, -NH2, -N-C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, -adamantyl, -aryl and -C7-C20 alkyl aryl, wherein any substituent comprising at least two atoms can be optionally substituted; R2, R3, R4, R5, R6 and R7 are independently selected from the group of substituents consisting of -H, -D, - OH, -F, -Cl, -Br, -I, -C1-6 alkyl, -O-C1-6 alkyl, and -S-C1-6 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; R8 is selected from the group of substituents consisting of -H, -D, -OH, =CH2, -CH3, -CH2CH3, -C(H)(CH3)2, -C(CH3)3, -CH2Ph, wherein any substituent comprising at least two atoms can be optionally substituted; R12 and R13 are independently selected from the group of substituents consisting of -H, -D, -F, -C1-C6 alkyl, -C(O)CH3, and -Cl-Cl0 alkyl, -C3-Cl0 cycloalkyl, adamantyl, aryl, and C7-C20 alkyl, wherein any substituent comprising at least two atoms can be optionally independently substituted; and R23-R26 are independently selected from group of substituents consisting of -H, -OH, -F, -Cl, -Br, -I, -CF3, -NO2, -SO3H, -SO2NH2, -NH2, -N3, -NH=NH2, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, and the structure -X-L-B or -X-L(BC), wherein X is O, S, -NH, -NCH3, or -CH2, wherein any substituent comprising at least two atoms can be optionally independently substituted. 127 PRF_70572-03 PCT 3. The method of claim 1 or 2, wherein R13 is independently selected from the group of substituents consisting of , PRF_70572-03 PCT O O , wherein any substituent can be optionally substituted. 4. The method of any one of claims 1-3, wherein B is a phosphatidylinositol-3-kinase (PI3k) inhibitor, a dual inhibitor of the PI3k/mTOR signaling pathway, a transforming growth factor β (TGFβ) inhibitor, a Rho-kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor), a focal adhesion kinase (FAK) inhibitor, vascular endothelial growth factor receptor (VEGFR) inhibitor, or a platelet-derived growth factor receptor (PDGFR) inhibitor. 5. The method of claim 4, wherein the dual inhibitor of the PI3k/mTOR signaling pathway is: 129 PRF_70572-03 PCT . 6. The method of claim 4, wherein the PI3k inhibitor has a structure: , wherein X has a structure selected from the group consisting of: 130 PRF_70572-03 PCT . 7. The method of claim 4, wherein the PI3k inhibitor has the structure: . 8. The method of claim 4, wherein the PI3K inhibitor has the structure: N A , , , , -NH-, -NHR-, Or -CO-, and y can be 1 to 20. 9. The method of claim 4, wherein the TGFβ inhibitor has the structure: 131 PRF_70572-03 PCT where 1 ran 2 are eac , n epen ent y, , methyl, Cl, Br. O, I, or F; and X is NH, O, or S. 10. The method of claim 9, wherein R1 is F and R2 is methyl. 11. The method of claim 9, wherein the TGFβ inhibitor has the structure: . 12. The method of claim 11 where L is or comprises a PEG monomer of the formula -(PEG)n- where n is 1-6. 13. The method of claim 11, wherein the conjugate has the structure: . PRF_70572-03 PCT 14. The method of claim 11, wherein L is O O O HN O O O O Or HN HN O H O HN HN O O N O HN S S Or HN O N S S NH S Or Or HN HN O NH S N S Or NH 15. The method of claim 11, wherein the conjugate has the structure: . 16. The method of claim 4, wherein the TGFβ inhibitor has the structure: O N N N . 17. The method of claim 4, wherein the ROCK inhibitor has the structure: 133 PRF_70572-03 PCT , 18. The method of claim 4, wherein the FAK inhibitor is F3C N N O . 19. The method of claim 4, wherein the VEGFR inhibitor is N H2 . 20. The method of claim 4, wherein the PDGFR inhibitor is 134 PRF_70572-03 PCT . dditionally comprises a PK extender selected from an albumin-binding ligand, a plasma protein-binding ligand, a hapten, or an internalization-inducing peptide. 22. The method of any one of claims 1-11 and 15-21, wherein L is or comprises one or more of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a polylactone, a polymethylmethacrylate, a polyoxymethylene, a heterocycle, or any combination of two or more thereof. 23. The method of any one of claims 1-11 and 15-21, wherein L is or comprises an oligomer of one or more peptidoglycans, glycans, anions, heterocycles, or any combination of two or more thereof. 24. The method of any one of claims 1-11 and 15-21, wherein L is or comprises at least one diamino butyric acid group, a substituted benzene group, a lysine group, a 2,3- diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or any combination of two or more thereof. 25. The method of any one of claims 1-11 and 15-21, wherein L is or comprises an ether, a thioether, a tertiary amine, a C1-6 alkyl, piperazine, piperidine, a bicycloheptane, a substituted benzene, or a combination of two or more thereof. 26. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 135 PRF_70572-03 PCT 27. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: w 28. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety , wherein n = 0-10. 29. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 136 PRF_70572-03 PCT O O H N OH 30. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: . 31. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 32. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 137 PRF_70572-03 PCT 33. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: . 34. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 35. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 138 PRF_70572-03 PCT . 36. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: . 37. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a reductively cleavable linker. 38. The method of any one of claims 1-11 and 15-21, wherein L is or comprises an oxidatively cleavable linker. 39. The method of any one of claims 1-11 and 15-21, wherein L is or comprises an oxime ester. 40. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a hydrazone. 41. The method of any one of claims 1-11 and 15-21, wherein L is or comprises an enzyme-cleavable linker. 42. The method of any one of claim 1-11 and 15-21, wherein L is or comprises a PEGn, and n = 0-36. 139 PRF_70572-03 PCT 43. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a peptide. 44. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a peptidoglycan. 45. The method of any one of claims 1-11 and 15-21, wherein L is: . 46. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a linker selected from the group consisting of pegylated-, alkyl-, sugar-, and peptide-based dual linkers. 47. The method of any one of claims 1-11 and 15-21, wherein L is a non-releasable linker. 48. The method of any one of claims 1-11 and 15-21, wherein L is covalently bonded to A and B of formula (I) or A, B and C of formula (II). 49. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety of the formula: 140 PRF_70572-03 PCT S O O O S O O O O O O N CO2H 50. The method of any one of claims 1-11 and 15-21, wherein L is or comprises a moiety selected from: COOH COOH N S N S 28 l, which can be optionally substituted; and z is an integer from 1 to 8. 51. The method of any one of claims 1-11 and 15-21, wherein L is or comprises: . 52. The method of any one of claims 1-11 and 15-21, wherein L is or comprises: , 29b, R30a, and R30b is independently H or C1- C6 alkyl, which can be optionally substituted (e.g., wherein each of R29a, R29b, R30a, and R30b is C1-alkyl (i.e., methyl)). 141 PRF_70572-03 PCT 53. The method of any one of claims 1-11, wherein C is an albumin binding ligand, a disulfide-stabilized protein scaffold comprising albumin binding domain 035 (ABD035), albumin binding domain Con (i.e., a peptide of a three-helix bundle 45 amino acids in length (ABDCon)), a designed ankyrin repeat protein (DARPin), a disulfide-stabilized Fv fragment (dsFv) of an anti-albumin antibody, a nanobody that complexes with human serum albumin (HSA), or a variable new antigen receptor (VNAR). 54. The method of any one of claims 1-11, wherein C is or comprises: . 55. The method of any one of claims 1-11, wherein C is or comprises: 142 PRF_70572-03 PCT wherein, as applicable: each of R12-19 (where applicable) is independently selected from -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, and -SO2F; and each of R20 and R21 is independently selected from -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -O-C1-6 alkyl, -CN, -CHO, -B(OH)2, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, CF3, and . 56. The method of any one of claims 1-11, wherein C is or comprises: 143 PRF_70572-03 PCT NO2 57. The method of any one of claims 1-11, wherein C is or comprises: I . 58. The method of any one of claims 1-11, wherein C is or comprises: 144 PRF_70572-03 PCT . 59. The method of any one of claims 1-11, wherein C is or comprises (PEG)n, wherein n is an integer 0 to 32, a peptide, a peptidoglycan, or a saccharide. 60. The method of any one of claims 1-11, wherein C is or comprises: . 61. The method of any one of claims 1-11, wherein C is or comprises a hapten bound by an autologous antibody. 62. The method of any one of claims 1-11, wherein C is or comprises a hapten selected from rhamnose, an α-galactosyl moiety, a dinitrophenyl (DNP) moiety, and a trinitrophenyl (TNP) moiety. 63. The method of claim 1, wherein A has the structure: 145 PRF_70572-03 PCT . 64. The method of claim 1, wherein A has the structure: . 65. The method of claim 1, wherein A has the structure: 146 PRF_70572-03 PCT . 66. The method of claim 1, wherein A has the structure: . 67. The method of claim 1, wherein A-L-B has the structure: 147 PRF_70572-03 PCT . OMe 68. A conjugate having the structure: 148 PRF_70572-03 PCT om e Me M m PRF_70572-03 PCT e 150 PRF_70572-03 PCT or 151 PRF_70572-03 PCT . 69. A conjugate having the structure: 70. A pharmaceutical composition comprising a conjugate of claim 68 or claim 69 and a pharmaceutically acceptable carrier. 152
PCT/US2025/029775 2024-05-16 2025-05-16 Method of using chemically stable keto-amide fibroblast activation protein-targeted conjugates and compositions comprising same to treat fibrosis Pending WO2025240868A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202463648296P 2024-05-16 2024-05-16
US63/648,296 2024-05-16
US202563761300P 2025-02-21 2025-02-21
US63/761,300 2025-02-21

Publications (1)

Publication Number Publication Date
WO2025240868A1 true WO2025240868A1 (en) 2025-11-20

Family

ID=97720809

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/029775 Pending WO2025240868A1 (en) 2024-05-16 2025-05-16 Method of using chemically stable keto-amide fibroblast activation protein-targeted conjugates and compositions comprising same to treat fibrosis

Country Status (1)

Country Link
WO (1) WO2025240868A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120115866A1 (en) * 2006-09-15 2012-05-10 Tyrogenex, Inc. Kinase inhibitor compounds
US20220001037A1 (en) * 2018-10-17 2022-01-06 Purdue Research Foundation Fibroblast activation protein (fap) targeted imaging and therapy in fibrosis
US20230192647A1 (en) * 2020-03-30 2023-06-22 Ustav Organicke Chemie A Biochemie Av Cr, V. V. I. Compounds for inhibition of fibroblast activation protein
WO2024102956A1 (en) * 2022-11-09 2024-05-16 Purdue Research Foundation Keto-amide-based fibroblast activation protein-targeted ligand linked to an imaging or therapeutic agent, compositions and methods of use
WO2025102002A1 (en) * 2023-11-10 2025-05-15 Purdue Research Foundation Keto-amide-based fibroblast activation protein-targeted ligand linked to a pi3k inhibitor, compositions and methods of use

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120115866A1 (en) * 2006-09-15 2012-05-10 Tyrogenex, Inc. Kinase inhibitor compounds
US20220001037A1 (en) * 2018-10-17 2022-01-06 Purdue Research Foundation Fibroblast activation protein (fap) targeted imaging and therapy in fibrosis
US20230192647A1 (en) * 2020-03-30 2023-06-22 Ustav Organicke Chemie A Biochemie Av Cr, V. V. I. Compounds for inhibition of fibroblast activation protein
WO2024102956A1 (en) * 2022-11-09 2024-05-16 Purdue Research Foundation Keto-amide-based fibroblast activation protein-targeted ligand linked to an imaging or therapeutic agent, compositions and methods of use
WO2025102002A1 (en) * 2023-11-10 2025-05-15 Purdue Research Foundation Keto-amide-based fibroblast activation protein-targeted ligand linked to a pi3k inhibitor, compositions and methods of use

Similar Documents

Publication Publication Date Title
KR101383228B1 (en) A3 adenosine receptor allosteric modulators
TWI525092B (en) Tetrahydrocarboline derivatives
AU2013225938B2 (en) Pyrazol-1-yl benzene sulfonamides as CCR9 antagonists
JP6755922B2 (en) New compounds, their preparation and their use
AU2019233207B2 (en) Heteroaryl compounds as kinase inhibitor
JP2021534248A (en) 3-aryloxyl-3-5-membered heteroaryl-propylamine compound and its use
JP2021508318A (en) Tubulin inhibitor
KR20180094938A (en) Combination of opioid receptor ligand and cytochrome P450 inhibitor
WO2012022121A1 (en) Fused ring compound for use as mineralocorticoid receptor antagonist
WO2008001886A1 (en) Aurora inhibitor
TW200819434A (en) 1-(het)aryl-3-[hetaryl-piperidin-4-yl]-thioureas as modulators of the EP2 receptor
TWI324066B (en) A pharmaceutical composition for inhibiting cell migration induced by an angiogenic factor
CN106795143B (en) Substituted indole compounds and methods and uses thereof
CN104163813A (en) Substituted indole compounds and methods and uses thereof
TW201908319A (en) Method of preparing and using a PDE9 inhibitor
CA3075813A1 (en) Small molecule inhibition of transcription factor sall4 and uses thereof
JP2024099790A (en) Vasodilator and use thereof
WO2019178383A1 (en) Inhibition of bmp signaling, compounds, compositions and uses thereof
EP3470404A1 (en) Five-membered heterocyclic derivative, method for producing same, and pharmaceutical composition comprising same
CN107880038B (en) [1,2,4] triazolo [1,5-a ] pyridine compound and preparation method and medical application thereof
JP2022532746A (en) 3-aryloxyl-3-5-membered heteroaryl-propylamine compound and its crystal form and use
WO2025240868A1 (en) Method of using chemically stable keto-amide fibroblast activation protein-targeted conjugates and compositions comprising same to treat fibrosis
JP2023535692A (en) Entero-degradable co-drugs, preparation and use thereof
US12358911B2 (en) Heterocyclic comipound as CDK-HDAC dual pathway inhibitor
CN107820495A (en) Target the new E PHA4 inhibitor of EPHA4 ligand binding domain